U.S. patent application number 12/060632 was filed with the patent office on 2010-11-04 for diagnostic test for west nile virus.
Invention is credited to Pei-Yong Shi, Susan J. Wong.
Application Number | 20100279400 12/060632 |
Document ID | / |
Family ID | 32233512 |
Filed Date | 2010-11-04 |
United States Patent
Application |
20100279400 |
Kind Code |
A1 |
Wong; Susan J. ; et
al. |
November 4, 2010 |
DIAGNOSTIC TEST FOR WEST NILE VIRUS
Abstract
The present invention provides a rapid and sensitive method for
the detection of a West Nile virus (WNV), Japanese encephalitis
virus (JEV), St. Louis encephalitis virus (SLEV) and Dengue virus
(DENV) and antibodies directed against thereof involving contacting
a biological specimen suspected of being infected with WNV, JE, SLE
or DEN with a substantially purified and isolated WNV E
glycoprotein or subfragment thereof having a native conformation
wherein the E glycoprotein or subfragment thereof has a reactivity
with antibodies against WNV and a cross-reactivity with antibodies
against JEV, SLEV and DENV. The instant invention further provides
a rapid, sensitive, and consistent method for the specific
detection of WNV by employing diagnostic assays having the antigen
NS5 which is specifically reactive with anti-WNV antibodies but not
cross-reactive with antibodies against other flaviviruses such as
JEV, SLEV, or DENV. The present invention also provides a rapid,
sensitive, and consistent method for the specific detection of DENV
by employing diagnostic assays having the antigen NS5 which is
specifically reactive with anti-DENV antibodies but do not
cross-react with antibodies against other flaviviruses such as JEV,
SLEV, or WNV. Further, the DENV NS5 antigens are serospecific and
do not cross react with antibodies to other DENV strains. Thus, the
method of the present invention provides a manner by which to
discriminate infections by each DENV strain. Further, diagnostic
kits for carrying out the methods are provided. The methods and
kits for carrying out the methods of the invention are rapid and
require as little as 10 minutes to detect a result. The invention
also provides monoclonal antibodies against WNV NS5 and DENV NS5
antigen and their use in detecting WNV and DENV infections in a
biological sample.
Inventors: |
Wong; Susan J.; (Albany,
NY) ; Shi; Pei-Yong; (Albany, NY) |
Correspondence
Address: |
FROMMER LAWRENCE & HAUG
745 FIFTH AVENUE- 10TH FL.
NEW YORK
NY
10151
US
|
Family ID: |
32233512 |
Appl. No.: |
12/060632 |
Filed: |
April 1, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10839550 |
May 5, 2004 |
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12060632 |
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10699550 |
Oct 31, 2003 |
7351547 |
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10839550 |
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60422755 |
Oct 31, 2002 |
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60476513 |
Jun 6, 2003 |
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Current U.S.
Class: |
435/339 ;
530/387.3; 530/388.3 |
Current CPC
Class: |
Y02A 50/53 20180101;
C12Q 1/04 20130101; G01N 33/56983 20130101; Y02A 50/30
20180101 |
Class at
Publication: |
435/339 ;
530/388.3; 530/387.3 |
International
Class: |
C12N 5/16 20060101
C12N005/16; C07K 16/08 20060101 C07K016/08 |
Claims
1. An isolated monoclonal antibody, or antigen binding fragment
thereof, wherein the monoclonal antibody is specific for WNV NS5
and not detectably cross-reactive with NS5 of a flavivirus other
than WNV.
2. A hybridoma cell line which produces a monoclonal antibody
wherein the monoclonal antibody is specific for WNV NS5 and not
detectably cross-reactive with NS5 of a flavivirus other than
WNV.
3-70. (canceled)
71. The monoclonal antibody according to claim 1 wherein the
antibody is humanized.
72. The monoclonal antibody according to claim 1 wherein the
antibody is equinized.
73-92. (canceled)
Description
REFERENCE TO RELATED APPLICATIONS
[0001] The present application is filed as a continuation-in-part
of U.S. application Ser. No. 10/699,550, filed Oct. 31, 2003, which
claims priority to U.S. Provisional Application Nos. 60/422,755,
filed Oct. 31, 2002 and 60/476,513, filed Jun. 6, 2003. Reference
is also made to PCT application PCT/US03/34823, filed Oct. 31,
2003, which also claims priority to U.S. Provisional Application
Nos. 60/422,755, filed Oct. 31, 2002 and 60/476,513, filed Jun. 6,
2003. Reference is also made to: PCT application PCT/US02/09036,
filed on Mar. 11, 2002 and published as WO 02/072036 on Sep. 19,
2002, which claims priority to U.S. Provisional Application No.
60/275,025, filed Mar. 12, 2001, and U.S. Provisional Application
No. 60/281,947, filed Apr. 5, 2001; and reference is made to U.S.
Provisional Application No. 60/402,860, filed Aug. 8, 2002, the
disclosures of which are hereby incorporated by reference in their
entireties. Each of the documents cited herein (herein cited
documents), and each of the documents cited in each of the herein
cited documents, together with any manufacturer's specifications,
data sheets, descriptions, product literature, instructions and the
like for any products mentioned herein or in herein cited documents
or in documents cited in herein cited documents, is hereby
incorporated herein by reference. None of the documents
incorporated by reference into this text is admitted to be prior
art with respect to the present invention, but, documents
incorporated by reference into this text may be employed in the
practice of the invention.
FIELD OF THE INVENTION
[0002] The instant invention relates generally to the field of
diagnostic assays for the detection of viruses, infectious
organisms, antibodies, and autoimmune diseases. More in particular,
this invention relates to compositions and methods for diagnosing a
flavivirus infection. Even more in particular, this invention
relates to the use of isolated and/or purified polypeptides of West
Nile virus (WNV), which includes recombinant, synthetic and fusion
proteins comprising the polypeptides or a nucleic acid molecule
encoding a WNV polypeptide, whereby the WNV polypeptide is
substantially pure and of authentic conformation and is reactive
with antibodies against WNV and strongly cross-reactive with
antibodies against one or more members of the genus Flavivirus,
advantageously Japanese encephalitis virus (JEV), St. Louis
encephalitis virus (SLEV), and Dengue virus (DENV); and are useful
to detect a flavivirus infection or exposure in a subject capable
of being infected by a flavivirus or capable of mounting an immune
response (e.g. production of antibodies) against a flavivirus or a
flavivirus antigen, without needing to specify as to which
flavivirus is the source of infection or exposure, e.g., to rapidly
determine whether a subject has a flavivirus infection or has been
exposed to a flavivirus.
[0003] The instant invention is further directed to a novel method
for the rapid detection of antibodies to a flavivirus or an antigen
thereof using a microsphere immunoassay under conditions that
provide enhanced reaction kinetics to provide a more
cost-effective, rapid, and sensitive approach to detecting
antibodies in a biological specimen against a substantially pure
WNV polypeptide of authentic conformation which reacts with
antibodies against WNV and strongly cross-reacts with antibodies
against a flavivirus, advantageously JEV, SLEV, and/or DENV. In one
embodiment, the instant invention further provides a method to
detect a recent or acute infection utilizing an immunodepletion
step to remove a subpopulation of antibodies, such as IgG or IgM
antibodies, raised against the WNV polypeptide of the instant
invention.
[0004] Also within the scope of this invention are diagnostic kits
comprising reagents and including an isolated and/or substantially
purified WNV polypeptide of authentic conformation or a nucleic
acid encoding the said WNV polypeptide, for the detection of a
recent, current or prior flavivirus infection or exposure to a
flavivirus antigen or polypeptide in a subject susceptible
thereto.
[0005] The present invention also relates to the use of isolated
and/or purified nonstructural polypeptides of WNV, which includes
recombinant, synthetic and fusion proteins comprising the
nonstructural polypeptides or a nucleic acid molecule encoding a
WNV nonstructural polypeptide, whereby the WNV nonstructural (NS)
polypeptide is substantially pure and of authentic conformation and
is reactive with WNV antibodies with specificity wherein the WNV NS
polypeptide is not substantially cross-reactive with antibodies
against one or more members of the genus Flavivirus, such as, for
example, JEV, SLEV, or DENV.
[0006] The nonstructural polypeptides of the present invention are
useful for specifically detecting a WNV infection or exposure in a
subject capable of being infected by a WNV or capable of mounting
an immune response (e.g. production of antibodies) against a WNV or
a WNV antigen in a time-efficient manner, i.e., to rapidly
determine whether a subject has a WNV infection or has been exposed
to a WNV.
[0007] The nonstructural polypeptides of WNV which are reactive
with WNV antibodies with specificity but are not substantially
cross-reactive with antibodies against another Flavivirus, such as,
for example, JEV, SLEV, or DENV, may also be used to identify
recently acquired WNV infections within a period of up to
approximately a year or less post-infection. Also within the scope
of the invention is the use of the nonstructural polypeptides of
WNV to discriminate between vaccination with a killed virus vaccine
and a natural infection with WNV. For example, such an application
of the present invention can be used to determine which members of
a population of horses are vaccinated and which are infected or
carriers of WNV.
[0008] The present invention also relates to the use of isolated
and/or purified nonstructural (NS) polypeptides of the four known
strains of DENV, namely, DENV-1, DENV-2, DENV-3, and DENV-4, (a) to
rapidly detect a DENV infection with specificity as to the which
strain is the source of infection, (b) to rapidly discriminate
between past DENV infections and current DENV infections, and (c)
to discriminate between a general flavivirus infection and a DENV
infection. The isolated and/or purified NS polypeptides of the
invention include recombinant, synthetic and fusion polypeptides
comprising the DENV NS polypeptides or a nucleic acid molecule
encoding the DENV NS polypeptide, whereby the DENV NS polypeptides
may be substantially pure and of authentic conformation.
[0009] For the purposes of this invention, a "serospecific DENV"
refers to a single strain of DENV, namely DENV-1, DENV-2, DENV-3,
or DENV-4. Further, a "serospecific" protein or antigen is such
that the protein/antigen has been obtained from a specific strain
DENV, namely a protein/antigen obtained from DENV-1, DENV-2,
DENV-3, or DENV-4.
[0010] DENV NS polypeptides of a first particular strain show
specificity for antibodies raised against the same first DENV
strain and are not cross-reactive with antibodies against other
DENV strains. For example, NS of DENV-1 will show specificity to
anti-DENV-1 sera, but will not be reactive with sera raised against
DENV-2, -3, or -4. In addition, like WNV NS proteins, the DENV NS
polypeptides are not substantially cross-reactive with antibodies
against one or more members of the genus Flavivirus, such as, for
example, JEV, SLEV, or WNV. Thus, the DENV NS can be used to
discriminate between a general flavivirus infection and a DENV
infection. In addition, since the antibodies to DENV NS proteins
are not persistent, the DENV NS proteins can be used to detect
recently acquired infections or current infections.
[0011] The present invention also contemplates diagnostic kits
comprising reagents and including an isolated and/or substantially
purified WNV nonstructural polypeptide of authentic conformation or
a nucleic acid encoding the said WNV nonstructural polypeptide, for
the detection of a recent or current WNV infection or exposure to a
WNV antigen or polypeptide in a subject susceptible thereto.
BACKGROUND OF THE INVENTION
[0012] In the summer of 1999, an outbreak of encephalitis in humans
that was associated with mosquitoes occurred in New York City (CDC,
MMWR, 48, pp. 845-9 (1999); CDC, MMWR, 48, pp. 944-6 (1999); D. S.
Asnis et al., Clin Infect Dis, 30, pp. 413-8 (2000)). At
approximately the same time, American crows began dying in the
Northeastern United States, many in Fairfield County, Conn. Two
reports in December of 1999 demonstrated that these outbreaks in
birds and humans were actually due to WNV virus transmitted by
mosquitoes (R. S. Lanciotti et al., Science, 286, pp. 2333-7
(1999); J. F. Anderson et al., Science, 286, pp. 2331-3 (1999)). It
is clear from these reports that WNV was the cause of the 1999
outbreak of fatal encephalitis in the Northeastern United States.
This is the first reported appearance of WNV in the Western
Hemisphere.
[0013] Future outbreaks of WNV in the United States are a new and
important public health concern. To date, the only method for
preventing WNV infection is spraying large geographic areas with
insecticide to kill mosquito vectors. Spraying is difficult,
potentially toxic to humans, requires repeated applications and is
incompletely effective. There is no known vaccine for use in humans
against WNV.
[0014] WNV is a member of the family Flaviviridae, genus Flavivirus
belonging to the Japanese Encephalitis antigenic complexes of
viruses. This sero-complex includes JEV, SLEV, Alfuy, Koutango,
Kunjin, Cacipacore, Yaounde, and Murray Valley Encephalitis
viruses. This Flaviviridae family also includes the Tick-borne
encephalitis virus (TBEV), Dengue virus (including the four strains
of: DENV-1, DENV-2, DENV-3, and DENV-4), and the family prototype,
Yellow Fever virus (YFV). WNV infections generally have mild
symptoms, although infections can be fatal in elderly and
immunocompromised patients. Typical symptoms of mild WNV infections
include fever, headache, body aches, rash and swollen lymph glands.
Severe disease with encephalitis is typically found in elderly
patients (D. S. Asnis et al., supra). For the most part, treatment
of a subject having a flavivirus infection is a symptomatic
treatment, i.e. the general symptoms of a flavivirus infection are
treated, such that for initial treatment, mere knowledge of the
infection being a flavivirus infection may be sufficient. However,
in certain other cases rapid and accurate diagnosis of the specific
flavivirus, particularly WNV, is critical such that the most
appropriate treatment can be initiated.
[0015] Moreover, with respect to the blood supply (e.g., donor
blood to be supplied to patients), and donor organs (e.g., organs
to be supplied to patients), there is a need to rapidly determine
whether the blood or organs are contaminated by a flavivirus, e.g.,
determine whether the donor suffers from a flavivirus infection,
without needing to know specifically which flavivirus is the source
of infection. Conversely, there is also a need for rapid and
accurate detection of a specific flavivirus such as WNV since it
may be important in some cases to delimit the spread of WNV through
the blood supply.
[0016] Flavivirus infections are a global public health problem (C.
G. Hayes, in The Arboviruses: Epidemiology and Ecology, T. P.
Monath, ed., CRC, Boca Raton, Fla., vol. 5, chap. 49 (1989); M. J.
Cardosa, Br Med Bull, 54, pp. 395-405 (1998); Z. Hubalek and J.
Halouzka, Emerg Infect Dis, 5, pp. 643-50 (1999)) with about half
of the flaviviruses causing human diseases. These viruses are
normally maintained in a natural cycle between mosquito vectors and
birds, where humans and horses are considered dead-end hosts.
Birds, including the American crow, Corvus brachyrhynchos, can
serve as non-human reservoirs for the virus. In the case of WNV,
the virus is transmitted to man by mosquitoes, which in the
Northeastern United States are primarily of the genera Culex and
Aedes, in particular C. pipiens and A. vexans.
[0017] Flaviviruses are the most significant group of
arthropod-transmitted viruses in terms of global morbidity and
mortality. An estimated one hundred million cases coupled with the
lack of sustained mosquito control measures, has distributed the
mosquito vectors of flaviviruses throughout the tropics,
subtropics, and some temperate areas. As a result, over half the
world's population is at risk for flaviviral infection. Further,
modern jet travel and human migration have raised the potential for
global spread of these pathogens. Thus, in certain cases, early and
rapid detection of a flavivirus infection or of exposure to a
flavivirus antigen, without needing to be specific as to which
flavivirus, is important. Conversely, it may also be critical to
accurately and confidently know the identity of the specific
flavivirus causing the infection.
[0018] The WNV, like other flaviviruses, is enveloped by host cell
membrane and contains the three structural proteins capsid (C),
membrane (M), and envelope glycoprotein (E glycoprotein). The E
glycoprotein and M proteins are found on the surface of the virion
where they are anchored in the membrane. Mature E glycoprotein is
glycosylated, whereas M is not, although its precursor, prM, is a
glycoprotein. In other flaviviruses, E glycoprotein is the largest
structural protein and contains functional domains responsible for
cell surface attachment and intraendosomal fusion activities. In
some flaviviruses, E glycoprotein has been reported to be a major
target of the host immune system during a natural infection.
[0019] In general, the flavivirus genome which is replicated in the
cytoplasm of the infected cell is a single positive-stranded RNA of
approximately 10,500 nucleotides containing short 5' and 3'
untranslated regions, a single long open reading frame (ORF), a 5'
cap, and a nonpolyadenylated 3' terminus. The flavivirus genome
encodes a single polyprotein which is co- and post-translationally
processed by viral and cellular proteases into the three structural
proteins, C (capsid), prM/M (premembrane/membrane), and envelope (E
glycoprotein) and seven nonstructural proteins, NS1 (nonstructural
protein 1), NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (T. J. Chambers et
al., Ann Rev Microbiol, 44, pp. 649-88 (1990)).
[0020] With respect to post-translational processing of the
polyprotein, the sites of proteolytic cleavage in the YFV, which is
likely to be predictive of the sites of cleavage in all
flaviviruses, have been determined by comparing the nucleotide
sequence and the amino terminal sequences of the viral proteins.
Subsequent to initial processing of the polyprotein, prM is
converted to M during virus release (G. Wengler at al., J Virol,
63, pp. 2521-6 (1989)), and anchored C is processed during virus
maturation (Nowak et al., Virology, 156, pp. 127-37 (1987)). In
some flaviviruses, the E glycoprotein is the major virus antigen
involved in virus neutralization by specific antibodies (Martin D.
A., et al. 2002, Clin Diagn Lab Immunol. 9:544-9).
[0021] The complete or partial genomes of a number of WNV isolates
from the outbreak in the Northeastern United States have been
sequenced. The complete sequence of WNV isolated from a dead
Chilean flamingo (WN-NY99) at the Bronx Zoo was deposited in
GenBank.TM. (accession number AF196835) (R. S. Lanciotti et al.,
supra). The genome of a WNV isolate from human victims of the New
York outbreak (WNV-NY1999) was sequenced and deposited as
GenBank.TM. accession number AF202541 (X-Y. Jia et al., The Lancet,
354, pp. 1971-2 (1999)). Partial sequences of isolates from two
species of mosquito, a crow and a hawk from Connecticut are
deposited as GenBank.TM. accession numbers AF206517-AF206520,
respectively (J. F. Anderson et al., supra). Comparative
phylogenetic analysis of the NY sequences with previously reported
WNV sequences indicated a high degree of sequence similarity
between the NY isolates and two isolates from Romania and one from
Israel (J. F. Anderson et al., supra; X.-Y. Jia et al., supra; R.
S. Lanciotti et al., supra). Furthermore, PCT WO 02/072056 relates
to the WNV E glycoprotein and its use in diagnostics of WNV
infections. Importantly, the referenced PCT does not at any time
recognize that this antigen is strongly cross-reactive among
flaviviruses, such as, JEV, SLEV, and DENV; rather, this PCT
publication attempts to advance the proposition that the WNV E
glycoprotein is specific for WNV and hence useful to diagnose or
detect only WNV or to immunize or vaccinate against only WNV,
contrary to the herein inventor's discoveries.
[0022] While flaviviruses such as JEV, SLEV, and DENV exhibit
similar structural features and components, the individual viruses
are significantly different at both the sequence and antigenic
levels. Indeed, antigenic distinctions have been used to define
four different strains within just the DENV subgroup of the
flaviviruses. Infection of an individual with one DENV strain does
not provide long-term immunity against the other strains and
secondary infections with heterologous strains are becoming
increasingly prevalent as multiple strains co-circulate in a
geographic area. Such secondary infections indicate that
vaccination or prior infection with any one flavivirus may not to
provide generalized protection against other flaviviruses.
[0023] Serodiagnosis of WNV and other flavivirus infections
currently requires a series of enzyme-linked immunosorbant assays
(ELISA) and viral plaque reduction neutralization (PRN) tests.
Specifically, the recommended assays for the identification of WNV
infection of humans are the immunoglobulin M (IgM) antibody capture
enzyme linked immunosorbent assay (MAC ELISA), the IgG ELISA
(Martin, D. A., 2000, J. Clin. Microbiol. 38:1823-1826; Johnson, A.
J., 2000, J. Clin. Microbiol. 38:1827-1831), detection of
antibodies in cerebrospinal fluid or serum using a plaque assay
(PRN test), isolation of the virus, and RT-PCR. Most public health
laboratories in the United States are performing these assays
according to protocols recommended by the Centers of Disease
Control and Prevention (CDC).
[0024] However, the currently available ELISA assays, while not
precisely specific for WNV, do not provide for a general diagnostic
assay for flavivirus infections (or exposure thereto) with other
members of the JEV serogroup (including JEV and SLEV) and DENV
because the cross reactivity of the assay to other flaviviruses is
unreliable and inconsistent. Further, the currently used ELISA
assays according to the CDC do not provide rapid results. Separate
assays are currently used to properly and reliably diagnose
flavivirus infections other than WNV, such as, JEV, SLEV, and DENV
and there is no available assay to reliably, consistently and
rapidly detect a flavivirus infection, especially WNV, JEV, SLEV,
or DENV. Accordingly, an antigen that is strongly cross-reactive to
antibodies against JEV serogroup flaviviruses, especially JEV,
SLEV, and DENV for use in a rapid diagnostic assay providing rapid
results thereof would be an advance in the art since it would
enable a general flavivirus detection assay when knowledge of the
specific flavivirus is not necessarily needed. Further, in addition
to the current assays that are used to diagnose specific flavivirus
infections, antigens for use in new rapid diagnostic assay
procedures for the specific diagnosis of a specific flavivirus,
such as WNV, that are more accurate, reliable, and sensitive than
those currently available would be an important advance in the
art.
[0025] When rapid, accurate, and sensitive detection of a
flavivirus is desired wherein knowledge of the specific flavivirus
is not required, an antigen with strong cross-reactivity between
flaviviruses is desirable. Further, the antigens currently known in
the art lack a sufficient cross-reactivity to allow for reliable,
consistent, and accurate testing of a flavivirus infection. One
reason limiting the cross-reactivity of current assays in the art,
such as, the CDC ELISA assay for the detection of WNV, may relate
to the impurity of the antigens used in the assays. The assays used
in the art for the detection of WNV and other flaviviruses
typically utilize somewhat impure antigens that are contaminated
with proportionally high levels of cellular protein and other
macromolecules as a result of the purification process. In some
cases, the concentration of contaminating protein, such as bovine
serum albumin, is greater than the concentration of the antigen
being prepared. These impurities can cause a significant reduction
in the sensitivity of a given assay (i.e., higher levels of
background signals relative to true detection signals) in detecting
antibodies against a virus or pathogen of interest from a
biological sample. For example, as a control reaction aimed at
determining the relative level of background inherent with a given
supplied antigen, a separate test of the tissue culture supernatant
from which the antigen was obtained may be required. Thus, an
antigen that is substantially pure, i.e., one that is not
contaminated with unwanted protein or other macromolecules, would
be useful for screening for flavivirus infections or exposure
thereto since it would provide for a more sensitive diagnosis.
[0026] Further, the antigens currently used in the art for the
detection of flaviviruses typically are damaged with respect to
their three-dimensional structure. For example, damage may occur at
specific protein domains or epitopes. Such structural damage is
usually introduced during antigen purification and/or isolation
wherein the antigen is often treated under harsh and/or destructive
conditions that result in damage to an antigen's three-dimensional
form. For example, the antigens currently prepared in the art may
be treated with the chemical, polyethylene glycol ("PEG") to help
carry out the precipitation of the antigen from solution for the
purpose of increasing its concentration. This process can be
harmful to a given antigen and may introduce irreversible damage to
its structure. Additionally during purification, the antigens can
be extracted using acetone. However, acetone extraction can lead to
full and/or partial denaturation of the antigen, which, in turn,
can result in an antigen having lost its authentic and/or native
conformation. Further still, the extent, predictability,
reliability, and consistency of cross-reactivity of an antigen is
typically greater in the case of an antigen having a authentic
and/or native conformation. Thus, it would be useful to have a WNV
polypeptide (i.e., antigen) that is of authentic conformation to
allow for a stronger, more predictable, more reliable and more
consistent cross-reactivity to other flaviviruses, especially, JEV,
SLEV and DENV.
[0027] In contrast, in situations where cross-reactivity to
multiple flaviviruses is undesirable, it would also be an
improvement in the art to have available one or more polypeptides
(i.e., antigens) that could be used to specifically detect
antibodies against a specific flavivirus infection, especially WNV,
more accurately, reliably, and rapidly without cross-reactivity
with antibodies against other Flaviviruses, such as, for example,
JEV, SLEV, or DENV. In other word, in addition to the utility of a
polypeptide for the general detection of a flavivirus infection
without regard as to which flavivirus, it would also be useful to
detect a specific flavivirus infection, such as a WNV infection,
using a specific antigen such that the detection of the WNV
infection is more reliable, more rapid, and more accurate than
currently known methods.
[0028] A number of serologic assays are routinely used for
laboratory diagnosis of flavivirus infections: IgM antibody capture
enzyme immunoassay (MAC-ELISA), indirect IgG ELISA, indirect
fluorescent antibody assay (IFAT), hemagglutination inhibition
(HIT), and serum dilution cross-species plaque reduction
neutralization tests (PRNTs)--each varying markedly in sensitivity,
technical difficulty, turn-around time, and clinical utility of the
results.
[0029] A specific example of a current assay method to detect a
flavivirus infection is an assay available from the CDC for the
detection of a WNV infection using the COS-1 WNV recombinant
antigen (NRA) (Davis, B. S. et al., 2001, J. Virology
75:4040-4047). This antigen can be used in an ELISA procedure to
test biological samples for antibodies against WNV. Positive ELISA
results are typically confirmed by plaque reduction neutralization
(PRN) tests performed in a biosafety level 3 facility. Although
this combination of assays is highly sensitive and specific, it
requires several days to weeks and specialized facilities to
perform the complete panel of tests. For example, the recommended
ELISA procedure considered separately takes two to three working
days to complete, as overnight incubations are deemed necessary to
enhance sensitivity (Martin, D. A., 2000, J. Clin. Microbiol.
38:1823-1826; Johnson, A. J., 2000, J. Clin. Microbiol.
38:1827-1831). Accordingly, the assays currently used in the art to
test for WNV and/or other flavivirus infections, such as the
COS-1-based assay, are slow and do not provide rapid results. Thus,
an assay for determining the presence of a flavivirus infection
that is more rapid than those currently available in the art would
be useful.
[0030] Flavivirus infections occur globally and represent a
continued health problem around the world. For example, WNV has
recently reached the United States and presently constitutes a
growing concern for health officials. Accordingly, surveillance is
rapidly becoming more common and widespread. As a result of the
increased surveillance, the demand by local health departments and
state public health laboratories for critical serologic reagents is
far exceeding the supply. Further, the current assays used for WNV
detection, such as the CDC-recommended assays mentioned above, are
slow and inefficient and do not meet the growing needs of the
general health community to rapidly identify infections and to
effectively assess WNV epidemiology. Further, current assay methods
for detecting WNV are not sufficient to meet growing needs for
quicker, more efficient and more sensitive analysis of blood and
organ supplies for flavivirus contamination, such as, contamination
by WNV and other flavivirus species, including, JEV, SLEV, and
DENV. Further still, there exists a need for new diagnostic assays
directed to the detection of flavivirus infections in animals to
facilitate the handling of infected animal populations or animal
populations at-risk of infection. Such diagnostic assays could be
in the form of a general flavivirus test wherein knowledge of the
specific flavivirus in not required or of a specific flavivirus
test, such as a specific test for WNV.
[0031] Accordingly, there is a need in the art for methods and kits
for improved diagnostic assays for the detection of infections by
WNV and other flaviviruses in animals and humans, including JEV,
SLEV, and DENV, that are more rapid, cost-effective, efficient and
sensitive than the current diagnostic assays available in the art.
Further, there is a need for a single assay to enable the general
detection of a flavivirus, especially WNV, JEV, SLEV, and DENV,
without the requirement of knowing exactly which species is the
source of infection. Further still, there is a need in the art for
an efficient, sensitive and cost-effective high-throughput
diagnostic assay for large-scale detection of flaviviruses to allow
for public health control over flaviviral diseases, for example,
improving the study of flaviviral-disease epidemiology or improving
the ability to analyze blood and organ supplies. In addition to the
need for a general flavivirus detection method, there also exists a
need for improved methods and reagent, such as monoclonal
antibodies against flavivirus antigens, to enable the rapid and
reliable detection of a specific flavivirus, such as WNV and DENV,
without substantial cross-reaction to other flaviviruses with
respect to both diagnostic methods and use in flavivirus
pathogenesis basic research. Moreover, with respect to DENV, there
exists a further need for a method to reliably and rapidly
discriminate between the different strains of DENV, including
DENV-1, DENV-2, DENV-3, and DENV-4. Further still, a need exists
for a method that is capable also of discriminating reliably
between a recent and/or current flavivirus infection and a previous
infection of a flavivirus in a manner that is either specific or
nonspecific as to the flavivirus of the infection.
OBJECTS AND SUMMARY OF THE INVENTION
[0032] It is an object of the present invention to provide an
improved method for the diagnosis of a flavivirus infection,
especially WNV, JEV, SLEV, and DENV, which is more sensitive,
easier to use, and less expensive than methods used in the prior
art; and further, wherein the improved method enables one to
determine whether there is a flavivirus infection, for instance,
infection by any of WNV, JEV, SLEV, or DENV, by a single assay.
[0033] Another object of the present invention is to reduce the
window between initial infection by a flavivirus, especially WNV,
JEV, SLEV, and DENV, and initial detection of IgM and/or IgG
antibodies against the infective flavivirus by providing a more
sensitive and rapid assay which can separately determine IgG and
IgM antibody levels.
[0034] Yet another object of the present invention is to
significantly reduce the time it takes to diagnose a flavivirus
infection by providing a novel method for the rapid detection of a
flavivirus using a microsphere immunoassay and conditions that
enhance the reaction kinetics.
[0035] Another object of the present invention is to significantly
reduce the time it takes to diagnose a WNV infection by providing a
novel method for the rapid and specific detection of WNV using a
microsphere immunoassay and a WNV nonstructural antigen, such as
NS5, which is reactive with antibodies against WNV with specificity
but which does not significantly cross-react with antibodies
against other flaviviruses.
[0036] Yet another object of the present invention is to
significantly reduce the time it takes to diagnose a flavivirus
infection by providing a novel method for the rapid and specific
detection of flavivirus, such as, but not limited to WNV and DENV,
using a microsphere immunoassay and a flavivirus nonstructural
antigen, especially NS5, which is reactive with antibodies against
a specific type of flavivirus, such as WNV or DENV, with
specificity but which does not significantly cross-react with
antibodies against other flaviviruses.
[0037] Still another object of the present invention is to
significantly reduce the time it takes to diagnose a flavivirus
infection by providing a novel method for the rapid and specific
detection of flavivirus, such as, but not limited to WNV and DENV,
using an immunochromatographic (also known as "lateral flow test"
or "membrane strip test") and a flavivirus nonstructural antigen,
especially NS5, which is reactive with antibodies against a
specific type of flavivirus, such as WNV or DENV, with specificity
but which does not significantly cross-react with antibodies
against other flaviviruses.
[0038] A further object of the present invention is to
significantly reduce the time it takes to diagnose a DENV-1
infection by providing a novel method for the rapid and specific
detection of DENV-1 using a microsphere immunoassay and a DENV-1
nonstructural antigen, such as NS5, which is reactive with
antibodies against DENV-1 with specificity but which does not
significantly cross-react with antibodies against other DENV
strains, including DENV-2, DENV-3, and DENV-4 or other
flaviviruses.
[0039] A still further object of the present invention is to
significantly reduce the time it takes to diagnose a DENV-2
infection by providing a novel method for the rapid and specific
detection of DENV-2 using a microsphere immunoassay and a DENV-2
nonstructural antigen, such as NS5, which is reactive with
antibodies against DENV-2 with specificity but which does not
significantly cross-react with antibodies against other DENV
strains, including DENV-1, DENV-3, and DENV-4 or other
flaviviruses.
[0040] Yet another object of the present invention is to
significantly reduce the time it takes to diagnose a DENV-3
infection by providing a novel method for the rapid and specific
detection of DENV-3 using a microsphere immunoassay and a DENV-3
nonstructural antigen, such as NS5, which is reactive with
antibodies against DENV-3 with specificity but which does not
significantly cross-react with antibodies against other DENV
strains, including DENV-1, DENV-2, and DENV-4 or other
flaviviruses.
[0041] Still another object of the present invention is to
significantly reduce the time it takes to diagnose a DENV-4
infection by providing a novel method for the rapid and specific
detection of DENV-4 using a microsphere immunoassay and a DENV-4
nonstructural antigen, such as NS5, which is reactive with
antibodies against DENV-4 with specificity but which does not
significantly cross-react with antibodies against other DENV
strains, including DENV-1, DENV-2, and DENV-3 or other
flaviviruses.
[0042] Another object of the present invention is to permit the
broad application of the WNV E glycoprotein to the non-specific
detection of flaviviruses, such as WNV, DENV, JEV, and SLEV through
the inventor's own discovery that a substantially purified WNV E
glycoprotein having an authentic conformation is reactive with
antibodies against WNV and strongly, reliably, predictably and
consistently cross-reactive with antibodies against various other
flaviviruses, especially DENV, JEV, and SLEV.
[0043] Still another object of the present invention is to provide
a novel method to detect a recent or ongoing infection by WNV or a
flavivirus, especially JEV, SLEV, and DENV, utilizing a microsphere
immunoassay in combination with an immunodepletion step to remove
IgG antibodies to enable the specific detection of IgM antibodies
against the WNV E glycoprotein, which would be indicative of a
likely recent or ongoing infection.
[0044] Yet another object of the present invention is to provide a
novel method to detect a protective immune response to an infection
by WNV or a flavivirus, especially JEV, SLEV, and DENV, utilizing a
microsphere immunoassay in combination with an immunodepletion step
to remove IgM antibodies to enable the specific detection of IgG
antibodies against the WNV E glycoprotein, which would be
indicative of a protective immune response.
[0045] A further object of the present invention is to provide a
flavivirus antigen, especially, WNV E glycoprotein, WNV NS protein,
such as NS5, or DENV NS proteins, such as NS5, coupled to a
microsphere to be used in an immunoassay to detect anti-flavivirus
antibodies in a biological specimen wherein the coupled antigen is
highly stable over time such that 90% or more of the antigen's
reactivity is preserved following 3 months or more of storage.
[0046] Yet another object of the present invention is to provide a
WNV nonstructural antigen, especially, the NS5 antigen, a
nonstructural protein that forms a key enzyme in flavivirus RNA
replication, that is reactive with WNV antibodies with specificity
but which does not significantly cross-react with antibodies
against other flaviviruses.
[0047] Yet another object of the present invention is to provide a
DENV nonstructural antigen from a specific strain of DENV,
especially, the NS5 antigen, a nonstructural protein that forms a
key enzyme in flavivirus RNA replication, that is reactive with
DENV antibodies raised to the same DENV strain with specificity but
which does not significantly cross-react with antibodies against
other DENV strains or other flaviviruses.
[0048] Still a further object of the present invention is to
provide a WNV nonstructural antigen, especially, the NS5 antigen,
for use in a rapid diagnostic test to specifically detect WNV
infection in humans and animals without any significant
cross-reactivity with other Flavivirus infections.
[0049] Another object of the present invention is to provide a DENV
nonstructural antigen of a specific DENV strain, especially, the
NS5 antigen, for use in a rapid diagnostic test to specifically
detect an infection in animals, especially humans and monkeys (e.g.
chimpanzees), by the same specific DENV strain without any
significant cross-reactivity with other DENV strains or other
flaviviruses.
[0050] Another object of the present invention is to provide a
flavivirus nonstructural antigen, especially, the WNV NS5 antigen
and DENV NS5 antigens of each strain, for the use in a rapid
diagnostic test to discriminate between vaccination with a killed
virus vaccine and a natural infection with the flavivirus,
especially WNV or DENV.
[0051] Yet another object of the instant invention is to provide an
assay utilizing a flavivirus nonstructural antigen, especially the
WNV NS5 antigen and DENV NS5 antigens of each strain, to reliably
discriminate an infection with a specific flavivirus, especially
WNV or DENV, and infections of other Flaviviruses, such as, for
example JEV or SLEV.
[0052] Yet a further object of the present invention is to
significantly reduce the time it takes to diagnose a WNV infection
by providing a novel method for the detection of a WNV infection
using a microsphere immunoassay and conditions that enhance the
reaction kinetics.
[0053] Another object of the present invention is to provide a
novel method to detect a recent or ongoing infection in humans and
animals, including but not limited to birds, mice, and horses, by a
flavivirus, especially WNV.
[0054] Yet another object of the present invention is to provide a
WNV antigen, especially WNV NS5 antigen, coupled to a microsphere
to (i) reliably discriminate between WNV infections and infections
of other flaviviruses such as DENV or SLEV; (ii) differentiate
between vaccination with inactivated flavivirus and natural WNV
infection; and (iii) indicate recent infections in animals,
including in particular, humans, birds, horses, cats and dogs.
[0055] Still another object of the present invention is to provide
a DENV antigen, especially DENV NS5 antigen from one of the four
known strains of DENV, namely, DENV-1, DENV-2, DENV-3, and DENV-4,
coupled to a microsphere (a) to rapidly detect a DENV infection
with specificity as to the which strain is the source of infection,
(b) to rapidly discriminate between past DENV infections and
current DENV infections, and (c) to discriminate between a general
flavivirus infection and a DENV infection.
[0056] Another object of the present invention is to provide a
method of using a NS5-based immunoassay, especially WNV NS5, to
determine whether animals, in particular, humans, birds, horses,
cats and dogs, who have previously been vaccinated with a
killed-flavivirus vaccine also have sustained new exposure to a
flavivirus, especially WNV.
[0057] Still another object of the present invention is to provide
a sensitive, reproducible, rapid, and inexpensive diagnostic assay
to detect the presence of antibodies to WNV in a sample using the
WNV nonstructural protein NS5 antigen as a probe.
[0058] The present invention endeavors to address the need in the
art for a more rapid, efficient, cost effective and sensitive
diagnostic assay for detecting WNV and/or other flavivirus
infections in subjects suspected of carrying a WNV and/or
flavivirus infection, such as subjects with encephalitis,
meningitis, or fever of unknown origin. More in particular, this
invention provides compositions and methods using purified WNV
polypeptides, fragments or derivatives thereof for the rapid
specific detection of an infection by WNV or the rapid detection of
an infection by a flavivirus, advantageously, WNV, JEV, SLEV, and
DENV, without needing to be specific as to the flavivirus.
[0059] Moreover, and as herein demonstrated, the present invention
relates to a novel use for the WNV E glycoprotein as an antigen to
be used for the detection of antibodies against certain species of
flaviviruses relevant to human disease, such as, WNV, JEV, SLEV,
DENV, using a single assay to take the place of a multitude of
assays currently used in the art for the detection of these
flaviviruses. Thus, by the present invention, one can determine
whether there is a flavivirus infection, for instance, infection by
any of WNV, JEV, SLEV, or DENV, by a single assay. The inventor has
discovered that a substantially purified WNV E glycoprotein antigen
having a substantially authentic conformation is reliably,
consistently, predictably, and strongly cross-reactive to
antibodies against any of WNV, JEV, SLEV, and DENV, and is
therefore useful to broadly assay or test for flavivirus infection,
non-specifically, e.g., in subjects, donors, blood, organs, etc. In
contrast, antigens currently available in the art for the detection
of DENV, SLEV, JEV, and WNV infections are often concentrated by
polyethylene glycol and/or extracted with acetone, treatments which
are likely to alter the structural domains of a given antigen.
[0060] Another aspect of the present invention relates to a novel
use for the WNV nonstructural protein, NS5 or a specific antigenic
determinant or specific epitope thereof, as an antigen for the
specific detection of antibodies against WNV. Importantly, the NS5
antigen is not cross-reactive to other Flaviviruses, such as, for
example, JEV, SLEV, or DENV. Thus, in accordance with the present
aspect of the invention, one can consistently, reliably, and
accurately determine whether there is a WNV infection with the
confidence and assurance that the detection signal is not due to
cross-reactivity with other flaviviruses.
[0061] In one aspect of the invention, it has been discovered that
a substantially purified WNV NS5 antigen is reliably, consistently,
predictably, and strongly reactive to antibodies against a WNV
without having substantial cross-reactivity with other
flaviviruses, such as, for example, JEV, SLEV, and DENV. Therefore,
NS5 antigen is useful to specifically assay or test for WNV
infection, e.g., in subjects, donors, blood, organs, etc. In
contrast, current serologic diagnoses of WNV infection is based on
detection of antibodies against viral structural proteins, such as
the E protein. Although, the cross-reactivity of the E protein
among flaviviruses, as also discovered by the instant inventors, is
certainly advantageous with respect to its use as a rapid
diagnostic for detecting a general flavivirus infection when
knowing the identity of the flavivirus is not critical, it would
also be desirable to have a rapid test that could confidently,
accurately, and correctly identify a WNV infection with specificity
and without cross-reactivity with other types of flaviviruses.
[0062] The methods currently available in the art are neither
optimized for the detection of a general flavivirus infection nor
are they optimized for specific detection of a particular
flavivirus, such as WNV or DENV. For example, many of the currently
available antigens are highly, but inconsistently, cross-reactive
with multiple flaviviruses. Thus, the positive sera or spinal
fluids detected by current methods must be verified by
cross-species plaque reduction neutralization tests to exclude the
possibility of infection with cross-reactive viruses such as SLEV
and DENV. Further, these confirmatory plaque reduction tests have
to be performed in level 3 biocontainment for many flaviviruses,
which substantially lengthens the overall time required for a
definitive serologic test. Thus, in contrast to the current methods
used in the art, the advantages of the instant invention relate to,
inter alia, the increased efficiency, speed, reliability and
predictability of the specific detection of a WNV infection without
significant cross-reactivity to other flaviviruses.
[0063] In certain embodiments, the WNV polypeptides of the instant
invention are derived from WNV isolates from the Northeastern
United States, in particular from isolate 2741 (GenBank accession
No. AF206518; see FIG. 37a-d) or from WNV 3356 from kidney of a New
York crow used in the infectious cDNA clone developed by Dr.
Pei-Yong Shi, GenBank accession no. AF404756 (see FIG. 38a-d) (Shi
et al., 2002. Infectious cDNA Clone of the epidemic West Nile Virus
from New York City, J. Virology 76:5847-5856.) More in particular,
the WNV isolates of the present invention contain a WNV E
glycoprotein (e.g. encoded by nucleotide positions 949-2451 of
GenBank accession No. AF206518 of FIG. 37a-d) or an immunogenic
fragment thereof or alternatively a WNV NS5 nonstructural protein
(e.g. encoded by nucleotide positions 7681-10395 of GenBank
accession No. AF404756 shown in FIG. 38a-d) or an immunogenic
fragment thereof. This invention further provides methods for the
production and isolation of said WNV polypeptides, such as E
glycoprotein or NS5 protein, preferably by either recombinant or
synthetic production methods, especially for use in flavivirus or
WNV assays, respectively. One of ordinary skill in the art will
certainly appreciate that the methods of the instant invention
could be applied to corresponding proteins from other flaviviruses,
especially DENV, and are not meant to be particularly limited to
the E glycoprotein and NS proteins of WNV or DENV. For example, the
present invention contemplates the use of DENV NS5 antigen from any
known strain, including DENV-1, -2, -3, and -4. In particular, the
invention relates to the use of NS5 of DENV-1 "WestPac", encoded by
nucleotide positions 7574-10270 of GenBank accession No. U88535
(see FIG. 39a-d) and NS5 of DENV-2 "New Guinea", encoded by
nucleotide positions 7570-10269 of GenBank accession No. AF038403
(see FIG. 40a-d).
[0064] In a further embodiment, the instant invention provides a
novel method of a microsphere immunoassay comprising microspheres
that are coupled to substantially purified WNV E glycoprotein in an
authentic conformation for use in detecting antibodies in a
biological sample (e.g., bodily fluid, blood, serum, plasma,
saliva, tears, feces, semen, mucous, tissue, tissue homogenate,
cellular extract, or spinal fluid, inter alia) against
flaviviruses, especially WNV, JEV, SLEV, and DENV. In this
embodiment, a biological specimen, for example, bodily fluid,
blood, serum, plasma, saliva, tears, feces, semen, mucous, tissue,
tissue homogenate, cellular extract, or spinal fluid, inter alia,
is contacted with microspheres coupled to WNV E glycoprotein which
is strongly, reliably, predictably and consistently cross-reactive
to antibodies against any of WNV, JEV, SLEV, and DENV under
conditions sufficient to form a complex between the WNV E
glycoprotein and any antibodies capable of recognizing and
specifically binding thereto. The bound antibodies are then
detected using a detection reagent, such as a secondary antibody
coupled to a detectable fluorescent tag or to an enzyme, such as
horseradish peroxidase.
[0065] In another embodiment, the biological sample (e.g., bodily
fluid, blood, serum, plasma, saliva, tears, feces, semen, mucous,
tissue, tissue homogenate, cellular extract, or spinal fluid inter
alia) may first be immunodepleted in order to remove or block a
functional site of a specific antibody population, such as an IgM
or IgG antibody population. Immunodepletion can be carried out by
contacting the biological sample with a second antibody against the
specific antibody subpopulation to be removed to form an insoluble
complex, comprising the second antibody and antibody subpopulation
to be removed, that can be subsequently removed by a separation
process, such as centrifugation.
[0066] Accordingly, the instant invention can be used to
specifically detect a recent or ongoing infection, for example,
following IgG removal, or to specifically detect a protective
immune response, for example, following IgM removal.
[0067] In another embodiment, the present invention provides a
novel method relating to a microsphere immunoassay comprising
microspheres that are coupled to substantially purified WNV NS5
antigen for use in detecting in a biological sample (e.g., bodily
fluid, blood, serum, plasma, saliva, tears, feces, semen, mucous,
tissue, tissue homogenate, cellular extract, or spinal fluid, inter
alia) antibodies specific to WNV wherein the NS5 antigen is not
substantially cross-reactive with antibodies against other
Flaviviruses, including WNV, JEV, SLEV, and DENV. In this
embodiment, a biological specimen, for example, blood, plasma,
serum, or spinal fluid, is contacted with the microspheres coupled
to NS5 antigen which is strongly, reliably, predictably,
consistently, and specifically reactive to antibodies against a WNV
yet is not substantially cross-reactive against antibodies to other
Flaviviruses, such as JEV, SLEV, and DENV. Subsequently, conditions
are provided that allow for a complex to form between the NS5
antigen and anti-WNV antibodies capable of recognizing and
specifically binding thereto. The bound antibodies are then
detected using a detection reagent, such as a secondary antibody
coupled to a detectable fluorescent tag or to an enzyme, such as
horseradish peroxidase.
[0068] Also within the scope of this invention are diagnostic kits
and methods for detecting antibodies against WNV and other
flaviviruses, especially JEV, SLEV, and DENV, characterized by the
compositions of the present invention comprising at least one
isolated and substantially purified polypeptide comprising a WNV E
glycoprotein or an immunogenic fragment/derivative thereof in an
authentic conformation whereby the WNV E glycoprotein or the
immunogenic fragment/derivative thereof is reactive with antibodies
against WNV and strongly, reliably, predictably and consistently
cross-reactive with antibodies against flaviviruses, especially
JEV, SLEV, and DENV. The cross-reactivity of the WNV E glycoprotein
or immunogenic fragment/derivative thereof, is the inventor's own
discovery which permits the broad application of the WNV E
glycoprotein to the non-specific detection of flaviviruses, such as
WNV, DENV, JEV, and SLEV. Prior to the instant invention, one of
ordinary skill in the art would not have unequivocally and/or
reliably known WNV E glycoprotein having an authentic conformation
strongly cross-reacts with antibodies against various flaviviruses,
in addition to antibodies against WNV.
[0069] In one embodiment, the diagnostic kits alternatively
comprise at least one isolated and substantially purified
polypeptide comprising a WNV NS5 antigen or an immunogenic
fragment/derivative thereof whereby the WNV NS5 antigen or the
immunogenic fragment/derivative thereof, especially of humans,
birds, horses, cats, dogs, any animal or mammal, is specifically,
strongly, reliably, predictably and consistently reactive with
antibodies against WNV but is not substantially or detectably
cross-reactive with antibodies against other flaviviruses, such as
JEV, SLEV, and DENV. The specificity of the WNV NS5 antigen towards
WNV antibodies and the lack of cross-reactivity of NS5 with
antibodies against other Flaviviruses permits the application of
the WNV NS5 to the detection method of WNV as taught by the present
application. As it is used herein, the phrase "detectably
cross-reactive" is meant to refer to an antigen-antibody
interaction that can be substantiated by measuring or detecting a
binding complex formed from the interaction between the antigen and
antibody. Thus, the recitation "not substantially or detectably
cross-reactive" is meant to exclude antigen-antibody interactions
that are non-specific, i.e. background "noise".
[0070] The diagnostic kits and methods according to the present
invention are also useful for detecting a protective immune
response to WNV infection or infection by various flaviviruses,
especially JEV, SLEV, and DENV. Further, the methods of the instant
invention are also useful in monitoring the course of immunization
against WNV and various flaviviruses. In patients previously
inoculated with the vaccines against WNV or various flaviviruses,
the detection means and methods disclosed herein are also useful
for determining if booster inoculations are appropriate. The
neutralizing antibodies which develop are primarily IgG antibodies,
which are readily detectable in the microsphere immunoassay format
of the present invention.
[0071] In an embodiment, the instant invention relates to a novel
method for detecting a non-specific flavivirus infection,
especially WNV, DENV, JEV, or SLEV, comprising the step of
contacting a biological sample from a subject suspected of having
said infection with an isolated and substantially purified
polypeptide comprising a WNV E glycoprotein or an immunogenic
fragment/derivative thereof having an authentic conformation
wherein the E glycoprotein or the immunogenic fragment/derivative
thereof is reactive with antibodies against WNV and strongly,
reliably, predictably and consistently cross-reactive with
antibodies against a flavivirus, especially JEV, SLEV, and
DENV.
[0072] In yet another embodiment, the present invention relates to
a method for detecting a protective immune response in a subject
comprising the step of contacting a biological sample from said
subject with an isolated and substantially purified polypeptide
comprising a WNV E glycoprotein or an immunogenic fragment thereof
having an authentic conformation wherein the E glycoprotein or the
immunogenic fragment thereof is reactive with protective antibodies
against WNV and strongly, reliably, predictably and consistently
cross-reactive with protective antibodies against a flavivirus,
especially JEV, SLEV, and DENV.
[0073] Also within the scope of the present invention is a method
for detecting a first antibody to a flavivirus from a biological
specimen of a subject suspected of being infected by said
flavivirus comprising the steps of contacting the biological
specimen with a substantially purified WNV E glycoprotein or an
immunogenic fragment/derivative thereof having an authentic
conformation under conditions to form a complex between the WNV E
glycoprotein and the first antibody, if present, that recognizes
and binds the WNV E glycoprotein followed by detecting the first
antibody of said complex, wherein the WNV E glycoprotein is
reactive to an antibody against a WNV and strongly, reliably,
predictably and consistently cross-reactive to an antibody against
a flavivirus, especially JEV, DENV, and SLEV.
[0074] The instant invention further relates to a method for
rapidly detecting a recent or ongoing flavivirus infection using a
microsphere immunoassay to detect an IgM antibody against a
flavivirus in a biological specimen by first contacting the
biological specimen with anti-IgG antibodies to form IgG immune
complexes followed by the removal of the IgG complexes to form a
biological specimen comprising IgM antibodies and lacking IgG
antibodies. Next, the biological specimen is contacted with a
microsphere comprising a substantially purified WNV E glycoprotein
antigen or immunogenic fragment/derivative thereof having an
authentic conformation to form a microsphere mixture under
conditions sufficient to form a binding complex between the WNV E
glycoprotein antigen and a IgM antibody whereby the WNV E
glycoprotein antigen is reactive to antibodies against WNV and
strongly, reliably, predictably and consistently cross-reactive to
antibodies against a flavivirus, especially JEV, DENV, and SLEV.
Next, the microsphere mixture is contacted with a detection reagent
capable of detecting a IgM antibody. Finally, the detection reagent
is detected wherein detecting the detection reagent indicates a
recent or ongoing flavivirus infection.
[0075] Also within the scope of the invention is a diagnostic kit
comprising at least one isolated and purified polypeptide
comprising a WNV NS5 protein or an immunogenic fragment thereof
having an native conformation or non-denatured structure whereby
the NS5 protein or the immunogenic fragment thereof is specifically
reactive with antibodies against WNV but not detectably
cross-reactive with antibodies against a flavivirus other than WNV.
The invention also provides a method for detecting a WNV infection
in a subject suspected of having said infection comprising the
steps of (a) contacting a biological sample (e.g., bodily fluid,
blood, serum, plasma, saliva, tears, feces, semen, mucous, tissue,
tissue homogenate, cellular extract, or spinal fluid, inter alia)
from the subject with an isolated and substantially purified
polypeptide comprising a WNV NS5 protein or an immunogenic fragment
thereof having a native conformation or non-denatured structure
whereby the NS5 protein or the immunogenic fragment thereof is
specifically reactive with anti-WNV antibodies but not detectably
cross-reactive with antibodies against a flavivirus other than WNV,
and (b) detecting anti-WNV antibodies that have reacted with the
WNV NS5 protein, wherein detection of the anti-WNV antibodies
indicates a WNV infection.
[0076] The present invention further relates to methods for
detecting a protective immune response in a subject comprising the
step of contacting a biological sample from said subject with an
isolated and substantially purified polypeptide comprising a WNV
NS5 protein or an immunogenic fragment thereof whereby the WNV NS5
protein or the immunogenic fragment thereof having a native
conformation or non-denatured structure is specifically reactive
with protective antibodies against WNV with no detectable
cross-reactivity with protective antibodies against a flavivirus
other than WNV. While antibodies to NS5 would not neutralize
against infection, they could be effective in rapidly decreasing
the spread of the infection.
[0077] Also within the scope of the present invention is a method
for detecting a first antibody to a WNV from a biological specimen
of a subject suspected of being infected by said WNV comprising the
steps of: (a) contacting the biological specimen with a
substantially pure WNV NS5 protein or an immunogenic fragment
thereof having a native conformation and non-denatured structure
under conditions to form a complex between the NS5 protein and the
first antibody, if present, that recognizes and binds the NS5
protein, (b) detecting the first antibody of said complex, wherein
the NS5 protein is not detectably cross-reactive to an antibody
against a flavivirus other than a WNV.
[0078] The invention further relates to a method for rapidly
detecting an anti-WNV antibody comprising the steps of: (a)
contacting a biological sample (e.g., bodily fluid, blood, serum,
plasma, saliva, tears, feces, semen, mucous, tissue, tissue
homogenate, cellular extract, or spinal fluid, inter alia) with a
microsphere suspension, each microsphere coupled to a substantially
pure WNV NS5 protein having a native conformation or non-denatured
structure whereby each NS5 protein is specifically reactive to
antibodies against WNV but not detectably cross-reactive with
antibodies against a flavivirus other than WNV, (b) incubating the
microsphere suspension under conditions sufficient to increase
reaction kinetics to promote the binding of an anti-WNV antibody to
the NS5 proteins, (c) contacting the microsphere suspension with a
detection reagent capable of detecting an anti-WNV antibody, (d)
detecting the detection reagent, wherein detection of the detection
reagent indicates the presence an anti-WNV in the biological
sample.
[0079] The instant invention also contemplates a method for the
detection of a WNV infection in a biological specimen comprising
the steps of: (a) obtaining a suspension of microspheres each
coupled to a substantially pure WNV NS5 protein having a native
conformation or non-denatured structure wherein the WNV NS5 protein
is specifically reactive with anti-WNV antibodies but not
detectably cross-reactive with antibodies against a flavivirus; (b)
performing a microsphere immunoassay; (c) obtaining a result
indicating either the presence or absence of an anti-WNV antibody,
wherein the presence of an anti-WNV antibody indicates a WNV
infection.
[0080] In another embodiment, the present invention relates to a
method for discriminating between whether (1) a host has an ongoing
WNV infection or (2) a host has been vaccinated with a
killed-flavivirus vaccine wherein the host in the case of (1) has
both anti-E glycoprotein antibodies and anti-NS5 antibodies but in
the case of (2) has anti-E glycoprotein but not anti-NS5 antibodies
comprising the steps of: (a) carrying out a first reaction
comprising the steps of (i) contacting a biological sample (e.g.,
bodily fluid, blood, serum, plasma, saliva, tears, feces, semen,
mucous, tissue, tissue homogenate, cellular extract, or spinal
fluid, inter alia) from the host with a first detection reagent for
the detection of anti-E glycoprotein antibodies, (ii) detecting
said first detection reagent to provide either a positive or
negative signal wherein a positive signal indicates the presence of
anti-E glycoprotein antibodies and a negative signal indicates the
absence of anti-E glycoprotein antibodies; (b) carrying out a
second reaction comprising the steps of (i) contacting a biological
sample from the host with a second detection reagent for the
detection of anti-NS5 antibodies, (ii) detecting said second
detection reagent to provide either a positive or negative signal
wherein a positive signal indicates the presence of anti-NS5
antibodies and a negative signal indicates the absence of anti-NS5
antibodies; and (c) comparing the results of the first and second
reactions wherein the following may be true: (i) a positive signal
for anti-E glycoprotein antibody and a positive signal for anti-NS5
antibody indicates that the host has an ongoing WNV infection and
(ii) a positive signal for anti-E glycoprotein antibody and a
negative signal for anti-NS5 antibody indicates that the host does
not have an ongoing WNV infection but may have been vaccinated with
a killed-flavivirus vaccine.
[0081] In yet another embodiment, the instant invention relates to
a method for detecting a recent or ongoing WNV infection in a host
comprising the steps of: (a) carrying out a first reaction
comprising the steps of (i) contacting a biological sample from the
host with a first detection reagent for the detection of anti-E
glycoprotein antibodies, (ii) detecting said first detection
reagent to provide either a positive or negative signal wherein a
positive signal indicates the presence of anti-E glycoprotein
antibodies and a negative signal indicates the absence of anti-E
glycoprotein antibodies; (b) carrying out a second reaction
comprising the steps of (i) contacting a biological sample from the
host with a second detection reagent for the detection of anti-NS5
antibodies, (ii) detecting said second detection reagent to provide
either a positive or negative signal wherein a positive signal
indicates the presence of anti-NS5 antibodies and a negative signal
indicates the absence of anti-NS5 antibodies; and (c) comparing the
results of the first and second reactions wherein the following may
be true: (i) a positive signal for anti-E glycoprotein antibody and
a positive signal for anti-NS5 antibody indicates that the host has
a recent or ongoing WNV infection and (ii) a positive signal for
anti-E glycoprotein antibody but a negative signal for anti-NS5
antibody indicates that the host does not have an recent or ongoing
WNV infection.
[0082] The present invention also endeavors to address the need in
the art for a more rapid, efficient, cost effective and sensitive
diagnostic assay for detecting DENV infections in subjects
suspected of carrying a DENV infection. More in particular, this
invention provides compositions and methods using purified DENV
polypeptides, fragments or derivatives thereof for the rapid
specific detection of an infection by DENV, advantageously where
the different strains, namely DENV-1, DENV-2, DENV-3, and DENV-4
can be discriminated.
[0083] Another aspect of the present invention relates to a novel
use for the DENV nonstructural protein, NS5 or a specific antigenic
determinant or specific epitope thereof, as an antigen for the
specific detection of antibodies against DENV. Importantly, the NS5
antigen is not cross-reactive to other flaviviruses, such as, for
example, JEV, SLEV, or DENV. Also, the NS5 antigen shows
specificity with antibodies to the particular strain (also referred
to as "strain"), namely DENV-1, DENV-2, DENV-3, or DENV-4, from
which is sourced from and is not measurably cross-reactive with the
remaining DENV strains. Thus, in accordance with the present aspect
of the invention, one can consistently, reliably, and accurately
determine whether there is a DENV infection and the identity of the
specific strain thereof with the confidence and assurance that the
detection signal is not due to cross-reactivity with other
flaviviruses or to other DENV strains.
[0084] In one aspect of the invention, it has been discovered that
a substantially purified DENV NS5 antigen is reliably,
consistently, predictably, and strongly reactive to antibodies
against a DENV without having substantial cross-reactivity with
other flaviviruses, such as, for example, JEV, SLEV, and WNV.
Therefore, DENV NS5 antigen is useful to specifically assay or test
for DENV infection, e.g., in subjects, donors, blood, organs, etc.
In contrast, current serologic diagnoses of DENV infection is based
on detection of antibodies against viral structural proteins, such
as the E protein. Although, the cross-reactivity of the E protein
among flaviviruses, as also discovered by the instant inventors, is
certainly advantageous with respect to its use as a rapid
diagnostic for detecting a general flavivirus infection when
knowing the identity of the flavivirus is not critical, it would
also be desirable to have a rapid test that could confidently,
accurately, and correctly identify a WNV infection with specificity
and without cross-reactivity with other types of flaviviruses.
[0085] In another embodiment, the present invention provides a
novel method relating to a microsphere immunoassay comprising
microspheres that are coupled to substantially purified DENV NS5
antigen for use in detecting in a biological sample (e.g., bodily
fluid, blood, serum, plasma, saliva, tears, feces, semen, mucous,
tissue, tissue homogenate, cellular extract, or spinal fluid, inter
alia) antibodies specific to DENV wherein the NS5 antigen is not
substantially cross-reactive with antibodies against other
flaviviruses, including WNV, JEV, and SLEV. In this embodiment, a
biological specimen, for example, blood, plasma, serum, or spinal
fluid, is contacted with microspheres coupled to DENV NS5 antigen
which is strongly, reliably, predictably, consistently, and
specifically reactive to antibodies against its specific
corresponding DENV strain yet is not substantially cross-reactive
against antibodies to other flaviviruses, such as WNV, JEV, and
SLEV. Subsequently, conditions are provided that allow for a
complex to form between the NS5 antigen and anti-DENV antibodies
capable of recognizing and specifically binding thereto. The bound
antibodies are then detected using a detection reagent, such as a
secondary antibody coupled to a detectable fluorescent tag or to an
enzyme, such as horseradish peroxidase.
[0086] In one embodiment, the diagnostic kits of the invention
alternatively comprise at least one isolated and substantially
purified polypeptide comprising a DENV NS5 antigen of a specific
strain thereof or an immunogenic fragment/derivative thereof
whereby the DENV NS5 antigen or the immunogenic fragment/derivative
thereof, especially of humans or birds, is specifically, strongly,
reliably, predictably and consistently reactive with antibodies
against DENV but is not substantially or detectably cross-reactive
with antibodies against other flaviviruses, such as JEV, SLEV, and
WNV. Further, the NS5 antigen is specific as to the particular DENV
strain isolated therefrom and is not cross-reactive to the
remaining DENV strains. The specificity of the DENV NS5 antigen
towards DENV antibodies and the lack of cross-reactivity of DENV
NS5 with antibodies against other flaviviruses (and to other DENV
strains) permits the application of the DENV NS5 to the detection
method as taught by the present invention. As it is used herein,
the phrase "detectably cross-reactive" is meant to refer to an
antigen-antibody interaction that can be substantiated by measuring
or detecting a binding complex formed from the interaction between
the antigen and antibody. Thus, the recitation "not substantially
or detectably cross-reactive" is meant to exclude antigen-antibody
interactions that are non-specific, i.e. background "noise".
[0087] The present invention further relates to methods for
detecting a protective DENV immune response in a subject comprising
the step of contacting a biological sample from said subject with
an isolated and substantially purified polypeptide comprising a
DENV NS5 protein or an immunogenic fragment thereof whereby the
DENV NS5 protein or the immunogenic fragment thereof having a
native conformation or non-denatured structure is specifically
reactive with protective antibodies against DENV with no detectable
cross-reactivity with protective antibodies against a flavivirus
other than DENV. While antibodies to DENV NS5 would not neutralize
against infection, they could be effective in rapidly decreasing
the spread of the infection.
[0088] Also within the scope of the present invention is a method
for detecting a first antibody to a DENV from a biological specimen
of a subject suspected of being infected by said DENV comprising
the steps of: (a) contacting the biological specimen with a
substantially pure DENV NS5 protein or an immunogenic fragment
thereof having a native conformation and non-denatured structure
under conditions to form a complex between the NS5 protein and the
first antibody, if present, that recognizes and binds the NS5
protein, (b) detecting the first antibody of said complex, wherein
the NS5 protein is not detectably cross-reactive to an antibody
against a flavivirus other than DENV. Further, a DENV NS5 protein
isolated from a specific strain of DENV will be specific for
antibodies to that DENV strain and not cross-reactive to antibodies
against the remaining strains of DENV.
[0089] The invention further relates to a method for rapidly
detecting an anti-DENV antibody comprising the steps of: (a)
contacting a biological sample (e.g., bodily fluid, blood, serum,
plasma, saliva, tears, feces, semen, mucous, tissue, tissue
homogenate, cellular extract, or spinal fluid, inter alia) with a
microsphere suspension, each microsphere coupled to a substantially
pure DENV NS5 protein having a native conformation or non-denatured
structure whereby each DENV NS5 protein is specifically reactive to
antibodies against DENV but not detectably cross-reactive with
antibodies against a flavivirus other than DENV, (b) incubating the
microsphere suspension under conditions sufficient to increase
reaction kinetics to promote the binding of an anti-DENV antibody
to the NS5 proteins, (c) contacting the microsphere suspension with
a detection reagent capable of detecting an anti-DENV antibody, (d)
detecting the detection reagent, wherein detection of the detection
reagent indicates the presence an anti-DENV in the biological
sample.
[0090] The instant invention also contemplates a method for the
detection of a DENV infection in a biological specimen comprising
the steps of: (a) obtaining a suspension of microspheres each
coupled to a substantially pure DENV NS5 protein having a native
conformation or non-denatured structure wherein the DENV NS5
protein is specifically reactive with anti-DENV antibodies but not
detectably cross-reactive with antibodies against a flavivirus; (b)
performing a microsphere immunoassay; (c) obtaining a result
indicating either the presence or absence of an anti-DENV antibody,
wherein the presence of an anti-DENV antibody indicates a DENV
infection. Further, a DENV NS5 protein isolated from a specific
strain of DENV will be specific for antibodies to that DENV strain
and not cross-reactive to antibodies against the remaining strains
of DENV. It will be appreciated that four DENV strains are known,
namely, DENV-1, DENV-2, DENV-3, and DENV-4.
[0091] The present invention is further directed to an isolated
monoclonal antibody, or antigen binding fragment thereof, wherein
the monoclonal antibody is specific for WNV NS5 and not detectably
cross-reactive with NS5 of a flavivirus other than WNV.
[0092] Also within the scope of the invention is hybridoma cell
lines which produce monoclonal antibodies that are specific for WNV
NS5 but are not detectably cross-reactive with NS5 of another
flavivirus, e.g. a flavivirus other than WNV.
[0093] The invention also relates to a method for detecting the
presence of a WNV infection in a biological sample suspected of
having a WNV infection having the steps of: (a) providing the
biological sample that may be infected with WNV; (b) contacting the
biological sample with an isolated monoclonal antibody, or antigen
binding fragment thereof, wherein the monoclonal antibody is
specific for WNV NS5 and not detectably cross-reactive with NS5
from flaviviruses other than WNV; (c) incubating under conditions
sufficient for a complex to form between WNV NS5 and the monoclonal
antibody; and (d) assaying for the presence of the complex; wherein
the detection of said complex indicates the presence of a WNV
infection.
[0094] The invention also contemplates a method for detecting the
presence of a WNV infection in a biological sample suspected of
having a WNV infection comprising the steps of: (a) providing said
biological sample having IgM antibodies and suspected of having a
WNV infection; (b) contacting the biological sample with a
suspension of microspheres; (c) capturing the IgM antibodies,
including anti-WNV IgM antibodies if present, on the surface of the
microspheres; (d) contacting the microspheres with purified WNV NS5
under conditions sufficient form a first complex comprising the WNV
NS5 and the anti-WNV IgM antibodies bound to the micropheres; (e)
contacting the microspheres with anti-WNV NS5 monoclonal antibody
under conditions sufficient to form a second complex comprising the
bound WNV NS5 of said first complex and the monoclonal antibody,
said monoclonal antibody being specific for WNV NS5 and not
detectably cross-reactive with NS5 of a flavivirus other than WNV;
and (f) assaying for the presence of the second complex, wherein
the detection of said complex indicates the presence of a WNV
infection.
[0095] In yet another aspect of the present invention, an isolated
monoclonal antibody, or antigen binding fragment thereof, is
provided wherein the monoclonal antibody is specific for DENV NS5
but not detectably cross-reactive with NS5 of another flavivirus,
e.g. not cross-reactive with a flavivirus other than DENV. The
monoclonal antibodies can be type-specific in that they are
specific to only a single type of DENV, namely, DENV-1, DENV-2,
DENV-3, or DENV-4, and are not cross-reactive thereto. Thus, an
anti-WNV DENV-1 monoclonal antibody would be specifically reactive
against WNV DENV-1 but not detectably reactive against WNV DENV-2,
-3, or -4. Likewise, an anti-WNV DENV-2 monoclonal antibody would
be specifically reactive against WNV DENV-2 but not detectably
reactive against WNV DENV-1, -3, or -4. Similarly, an anti-WNV
DENV-3 monoclonal antibody would be specifically reactive against
WNV DENV-3 but not detectably reactive against WNV DENV-1, -2, or
-4. In the same fashion, an anti-WNV DENV-4 monoclonal antibody
would be specifically reactive against WNV DENV-4 but not
detectably reactive against WNV DENV-1, -2, or -3.
[0096] The present also relates to hybridoma cell lines which
produce monoclonal antibodies which are specific for DENV NS5 but
are not detectably cross-reactive with NS5 antigens derived from
other flaviviruses other than DENV. Moreover, and as mentioned
above, the DENV NS5 monoclonal antibodies are type-specific and do
not detectably cross-react among DENV-1, -2, -3, and -4.
[0097] In yet another aspect of the present invention, a method is
provided for detecting the presence of a DENV infection in a
biological sample suspected of having a DENV infection comprising
the steps of: (a) providing the biological sample; (b) contacting
the biological sample with an isolated monoclonal antibody, or
antigen binding fragment thereof, wherein the monoclonal antibody
is specific for DENV NS5 and not detectably cross-reactive with NS5
from flaviviruses other than DENV; (c) incubating under conditions
sufficient for a complex to form between DENV NS5 and the
monoclonal antibody; and (d) assaying for the presence of said
complex; wherein the detection of said complex indicates the
presence of a DENV infection.
[0098] In yet a further aspect of the instant invention, a method
is provided for detecting the presence of a DENV infection in a
biological sample suspected of having a DENV infection comprising
the steps of: (a) providing said biological sample having IgM
antibodies and suspected of having a DENV infection; (b) contacting
the biological sample with a suspension of microspheres; (c)
capturing the IgM antibodies, including anti-DENV IgM antibodies if
present, on the surface of the microspheres; (d) contacting the
microspheres with purified DENV NS5 under conditions sufficient
form a first complex comprising the DENV NS5 and the anti-DENV IgM
antibodies bound to the micropheres; (e) contacting the
microspheres with anti-DENV NS5 monoclonal antibody under
conditions sufficient to form a second complex comprising the bound
DENV NS5 of said first complex and the monoclonal antibody, said
monoclonal antibody being specific for DENV NS5 and not detectably
cross-reactive with NS5 of a flavivirus other than DENV; and (f)
assaying for the presence of the second complex; wherein the
detection of said complex indicates the presence of a DENV
infection.
[0099] Throughout this specification and claims, the word
"comprise," or variations such as "comprises" or "comprising," will
be understood to imply the inclusion of a stated integer or group
of integers but not the exclusion of any other integer or group of
integers.
[0100] These and other embodiments are disclosed or are obvious
from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0101] The following Detailed Description, given by way of example,
but not intended to limit the invention to specific embodiments
described, may be understood in conjunction with the accompanying
Figures, incorporated herein by reference.
[0102] FIG. 1 is the amino acid sequence of the WNV-288-301
fragment (peptide 1).
[0103] FIG. 2 is the amino acid sequence of the random 288-301
fragment (peptide 2).
[0104] FIG. 3 is the amino acid sequence of the WNV-121-139 peptide
(peptide 3).
[0105] FIG. 4 is a diagrammatic representation of the 71 kDa Tr-env
fusion protein. Tr, thioredoxin domain; EK, enterokinase cleavage
site; WNV, 55 kDa full length sequence of WNV envelope
glycoprotein; V5, V5 epitopes; His, 3 kDa six histidine-tag
sequence; 1, location of WNE-288-301 fragment; 3, location of
WNE-121-139 fragment.
[0106] FIG. 5 is a Coomassie-blue stained SDS-PAGE gel showing
purified, recombinant TR-env fusion protein.
[0107] FIG. 6 depicts the utility of mice as an experimental model
organism for WNV infection and further demonstrates that the
purified Tr-env protein is able to elicit a protective antibody
response. C3H mice were immunized with Tr-env protein (upper line),
or Tr control protein (lower line) and challenged with WNV. Five
mice were in each group.
[0108] FIG. 7 shows the results of an ELISA demonstrating the
specificity of antibodies generated following inoculation of mice
with purified Tr-env protein. Ova, ovalbumin; Ova-random,
ovalbumin-conjugated random-288-301 peptide (SEQ ID NO: 5);
Ova-281, ovalbumin-conjugated WNE-288-301 peptide (SEQ ID NO: 4).
100, 1000, and 6000 represent serum dilutions of 1:100, 1:1000 and
1:6000.
[0109] FIG. 8 shows the results of the WNV E microsphere
immunoassay testing serum dilutions ranging from 1:25 to 1:6400.
The graph shows linear responses for selected (P) positive sera.
The graph show that 1:100 dilution of serum provides near maximal
MFI. Since 1:25 dilution of serum were shown to be inhibitory in
other experiments, 1:100 was chosen as the best screening dilution
for subsequent experiments.
[0110] FIG. 9 shows the results of a stability analysis of the WNV
E glycoprotein coated microspheres over time at the temperatures of
25.degree. C., 37.degree. C. and 50.degree. C. The plot shows the
Maximum Fluorescence Intensity (MFI) versus time at each given
temperature. Antigen was shown to be stable on beads when stored at
4.degree. C. for over four months. Curves in the plots of FIG. 9
are likely due to voltage fluctuation affecting the energy output
of the lasers.
[0111] FIG. 10A demonstrates the greater sensitivity of the WNV E
glycoprotein microsphere immunoassay (WNV E MI) over ELISA testing
in detecting antibodies against WNV in two of three sera from WNV
patients. The figure further shows that WNV E MI detected no WNV
antibodies in 7 sera, which was consistent with the negative PRN
test results for the same 7 sera. Similarly, WNV E MI identified
WNV antibodies in the three WNV sera that had positive PRN test
results (PRN titers of 160 and 320).
[0112] FIG. 10B demonstrates the ability of the WNV E glycoprotein
microsphere immunoassay (WNV E MI) to detect antibodies against
SLEV in sera of four of six SLEV patients. The P/N value of the WNV
E MI results were substantially higher than ELISA P/N values for
sera from 2 patients. Two sera from SLEV patients were missed by
the microsphere assay, which were collected on day 0 and day 2
after onset.
[0113] FIG. 10C demonstrates the greater sensitivity of the WNV E
glycoprotein microsphere immunoassay (WNV E MI) over ELISA testing
in detecting antibodies against DENV in three sera from Dengue
patients. The results show that the WNV E MI identified correctly
the three sera from Dengue fever patients with much higher P/N
values (range 15.00 to 55.23) than the traditional ELISA tests with
an IgG P/N range of 4.95 and IgM P/N range from 2.98 to 8.67.
[0114] FIGS. 11A and 11B show scatter plots comparing the P/N
values between the WNV E glycoprotein microsphere immunoassay using
the polyvalent (IgG/IgM/IgA) detection reagent and either the WN
IgG ELISA (A) or the WN IgM ELISA (B) with trendline according to
Example 12.
[0115] FIG. 12, as outlined in Example 11, shows two plots
comparing the P/N values measured by either standard ELISA methods
(A) or the WNV E microsphere immunoassay (B). Serum was obtained
sequentially over time at 3 days prior to infection with WNV and
then 2, 18, 72 and 260 days post-infection. The serum in 11B was
untreated or treated with antibodies to remove either the IgM or
the IgG antibody subpopulations. These immunodepleted serum samples
were tested using the microsphere immunoassay. The microsphere
immunoassay shows that, unlike the IgM and IgG ELISAs, there is a
greater IgM P/N than a IgG P/N for early serum samples, which may
indicate that the patient had an active or recent infection.
[0116] FIG. 13, according to Example 13, shows the P/N value as
determined by carrying out the WNV E glycoprotein microsphere
immunoassay on sera from twelve persons having received a
flavivirus vaccine (and four sera from non-vaccinated persons). The
sera was either immunodepleted of IgM antibodies (B) or the sera
was not depleted of IgM antibodies (A). The assay demonstrated that
the microsphere immunoassay could detect antibodies to JEV, as well
as WNV, SLEV, and DENV.
[0117] FIG. 14, according to Example 9, shows the results of
testing different sera from patients with different viral
infections, bacterial infections or autoimmune diseases by the WNV
E glycoprotein microsphere immunoassay (A). The results demonstrate
that the immunoassay performs well given that only sera from
patients with syphilis had a high frequency of falsely positive
test results with the microsphere immunoassay. Graphical
representation of the data is shown in (B).
[0118] FIG. 15 shows the results of WNV-E microsphere immunoassay
in comparison to results of the MAC ELISA test on spinal fluids of
patients with diagnosed encephalitis due to flavivirus infection.
Confirmation of diagnosis was by plaque reduction neutralization
tests including WNV, DENV, and SLEV.
[0119] FIG. 16 shows the results of testing seven pairs of serum
along with same-day collected spinal fluid specimens from seven
patients using the recombinant WNV-E microsphere immunoassay along
with both the polyvalent antibody reagent and the "IgM" serum
(anti-IgG treated serum). The seven patients were chosen on the
basis of having been tested positive for WNV by either an IgM
and/or an IgG ELISA using the reagents and protocol recommended by
the CDC. The data are presented in the table shows both the MFI and
the P/N values. The results show that the WNV-E assay has a greater
sensitivity than the standard ELISA since 5 patients who were shown
to test negative for a WNV infection by MAC ELISA were shown to be
strongly positive by the WNV-E assay.
[0120] FIG. 17 shows a schematic of a lateral-flow or strip test
for use in rapid detection of a flavivirus infection or rapid
specific detection of a WNV infection according to the instant
invention. See Detailed Description for further details.
[0121] FIG. 18 shows the median fluorescence intensity (MFI) data
for mouse sera tested by the microsphere immunoassay (MIA) using
WNV E glycoprotein (column 1), WNV NS3 antigen (column 2), and WNV
NS5 antigen (column 3) as detected with goat antimouse polyvalent
conjugate. The data demonstrates that WNV NS5 (column 3) is
equivalent to superior to WNV E glycoprotein (column 1) as an
antigen to detect WNV infection in mice.
[0122] FIG. 19 shows MFI data for human sera tested by MIA using
WNV E glycoprotein, WNV NS3 antigen, and WNV NS5 antigen. While the
negative range for normal non-infected subjects was higher, the
overall MFI for infected patients was 2.5 to 3 fold higher than the
MFI signal to the WNV E glycoprotein.
[0123] FIG. 20 demonstrates that WNV NS5 can be used to
discriminate between infection by DENV and WNV. The data shows that
all the sera from the DENV patients examined were highly reactive
(positive) to the WNV E glycoprotein in the MIA, but conversely
each were negative to the WNV NS5 antigen. All the convalescent
dengue sera were positive and 11 of 17 acute sera were positive by
MIA. Data were fully concordant with Dengue ELISA and
Hemagglutination inhibition results. It is likely that the
polymerase structures of the DENV and the WNV are significantly
different. DENV polymerase did not induce antibodies that
recognized the WNV NS5 antigen.
[0124] FIG. 21 demonstrates that NS5 can be used to discriminate
between vaccination and active infection. Sera from employees who
received JEV vaccine (a series of three shots), who developed
neutralizing antibodies, each developed an increase in antibody to
the envelope protein. However, the sera of the JEV recipients were
all non-reactive to the NS5 antigen. This result is intuitive since
the polymerase (NS5 protein) would not be expressed by the killed
virus of the JEV killed-virus vaccine. The data also demonstrates
that NS5 is more specific as a reagent in immunoassay than the WNV
E glycoprotein since one of ten sera from HIV-infected patients was
positive to NS5 and each remaining sera sample including the
negative control were negative to NS5.
[0125] FIG. 22 demonstrates that antibodies to NS5 disappear before
antibodies to WNV E glycoprotein. Since the level of anti-NS5 drops
prior to the levels of anti-E antibody, NS5 likely is a useful
marker to indicate recent WNV infections.
[0126] FIG. 23 (A) shows WNV genome structure. Three recombinant
proteins, E, NS3, and NS5 used, are shaded. (B and C) Purified
NTPase/helicase domain of NS3 and full-length NS5 were analyzed on
SDS-PAGE stained with Coomassie Blue. (D) ATPase activity of the
recombinant NTPase/helicase domain of WNV NS3. In the presence of
recombinant NS3, [.alpha.-.sup.32P]ATP was hydrolyzed to
[.alpha.-.sup.32P]ADP and phosphate (lane 2). No ATP is hydrolyzed
in the absence of NS3 (lane 1). (E) RdRp ("RNA-Dependent RNA
Polymerase") activity of the recombinant NS5. The RdRp activity of
NS5 was assayed using a WNV subgenomic RNA transcript (890-nt in
length) containing a large deletion from nucleotide 269 to 10408.
The reactions (RXT) were labeled with [.alpha.-.sup.32P]UTP, and
the products of double-stranded RNA (a replicative 2.times. form)
and single-stranded RNA (1.times. form) were analyzed on a
denaturing polyacrylamide gel followed by autoradiography (lane 1).
A .sup.32P-labeled template RNA was loaded as a size control (lane
2).
[0127] FIG. 24 shows the results of microsphere immunoassays (MIA)
using recombinant proteins of WNV NS5 (A), NS3 (B), and E (C).
Median fluorescence intensity (MFI) of each WNV patient serum is
plotted against days post-symptom onset. Dashed lines indicate
assay cut-off levels. X in (B) indicates samples not tested. (D) A
time course of reactivity to NS5 and E protein for sera collected
from a patient infected with WNV. MFI from NS5- and E-based assays
are indicated by solid and dashed lines, respectively. The cut-off
values of the assays are correspondingly indicated.
[0128] FIG. 25 shows the specificity of a NS5-based MIA as
demonstrated by challenging 120 sera from patients with various
infections, autoimmune conditions, JEV vaccination, YFV
vaccination, or good health.
[0129] FIG. 26 shows the cross-reactivity of WNV NS5 and E protein
with dengue patient sera. The data indicates that only 8.8% of the
total dengue patient sera samples showed a cross-reaction with the
WNV NS5 antigen as compared to 71% with WNV E glycoprotein.
[0130] FIG. 27 shows the cross-reactivity of WNV NS5 and E protein
with St. Louis encephalitis patient sera. The data indicates that
only 5% of the total St. Louis encephalitis patient sera samples
showed a cross-reaction with the WNV NS5 antigen as compared to 28%
with WNV E glycoprotein.
[0131] FIG. 28 shows a comparison of MIA values measured for wild
bird sera samples using the NS5 antigen as compared to the E
glycoprotein.
[0132] FIG. 29 shows a comparison of MIA values in sera from humans
who received the live-attenuated virus vaccine Yellow Fever
vaccine. The data show that 10 out of 19 sera were cross-reactive
(above the MIA cutoff value) to the WNV E glycoprotein whereas only
1 out of 19 sera were cross-reactive (above the MIA cutoff value)
to the WNV NS5 protein. The data indicate that the recipients of
Yellow Fever vaccine are negative in assays using WNV NS5 protein.
Accordingly, the data demonstrate that the WNV NS5 is useful for
discriminating between sera of humans vaccinated with Yellow Fever
vaccine and sera of humans infected with WNV.
[0133] FIG. 30 (A) shows a comparison of MIA values measured for
various horse sera samples tested against WNV E glycoprotein, WNV
NS5 antigen, and WNV NS3 antigen. (B) shows a of a multiplex assay
comparing the MIA values of various horse sera tested with WNV E
glycoprotein, WNV NS5, or WNV NS3 protein.
[0134] FIG. 31 shows rWNV-E MIA analysis of serially diluted serum
specimens. Sera from patients with West Nile infection (closed
symbols), and negative control human sera (open symbols) were
serially diluted and evaluated in the rWNV-E MIA using polyvalent
detector antibody. Results are reported as median fluorescent
intensity per 100 microspheres (MFI).
[0135] FIG. 32 shows rWNV-E MIA and ELISA analysis of anti-WN virus
antibodies in sequential serum specimens from a patient infected
with WN virus. A. Unadsorbed sera were evaluated in the rWNV-E MIA
using polyvalent detector antibody (polyvalent). Sera adsorbed with
anti-IgG (IgG adsorbed) or anti-IgM (IgM adsorbed) were evaluated
in the rWNV-E MIA using polyvalent detector antibody. The IgM
adsorbed sera were also analyzed in the rWNV-E MIA with anti-IgM
detector antibody (M conjugate). B. The results with the MAC-ELISA
and indirect IgG ELISA are compared on sequential sera.
[0136] FIG. 33 shows a retrospective parallel WNV-E MIA and WN
virus IgG ELISA analysis of sera from patients with suspected viral
encephalitis. Dashed lines indicate P/N cut-off values for a
positive result. n=702; r.sup.2=0.60; slope=1.68.
[0137] FIG. 34 shows the results of an E protein based microsphere
immunoassay (MIA). The assay tested a coded serum panel revealing
that the rWNV-E MIA detects human antibodies elicited by SLEV and
DENV. FIG. 34 is a tabular form of the data shown in FIGS. 10 A, B,
and C.
[0138] FIG. 35 shows the results of an E protein based microsphere
immunoassay (MIA) on sera from patients with various viral
infections, bacterial infections, or autoimmune diseases were
tested in the rWNV-E MIA. This shows the same data as FIG. 14A.
[0139] FIG. 36 compares the results of an E protein based
microsphere immunoassay (MIA) on human cerebral spinal fluid
samples from patients infected with WNV, DENV, or an unknown
flavivirus. Serum samples shown here include those shown in FIG.
15. The data in FIG. 15 is a subset of the data shown in FIG.
36.
[0140] FIG. 37 shows the nucleotide sequence of GenBank accession
No. AF206518 comprising the genome sequence of WNV isolate 2741
(SEQ ID NO.1).
[0141] FIG. 38 shows the nucleotide sequence of GenBank accession
No. AF404756 comprising the genome sequence of WNV isolate 3356
(SEQ ID NO.2).
[0142] FIG. 39 shows the nucleotide sequence of GenBank accession
No. U88535 comprising the genome sequence of DENV-1 isolate
"WestPac" (SEQ ID NO.3).
[0143] FIG. 40 shows the nucleotide sequence of GenBank accession
No. AF038403 comprising the genome sequence of DENV-2 isolate "New
Guinea" (SEQ ID NO.4)
[0144] FIG. 41 shows the nucleotide sequence for nucleotide
positions 982-1494 (SEQ ID NO:5) of GenBank accession No. AF206518
(WNV isolate 2741) corresponding to the amino acid sequence of WNV
E glycoprotein.
[0145] FIG. 42 shows the amino acid sequence of WNV E glycoprotein
(SEQ ID NO.6) corresponding to nucleotide sequence positions
982-1494 of GenBank accession No. AF206518 (WNV isolate 2741).
[0146] FIG. 43 shows the nucleotide sequence for nucleotide
positions 7681-10395 (SEQ ID NO:7) of GenBank accession No.
AF404756 (WNV isolate 3356) corresponding to the amino acid
sequence of WNV NS5.
[0147] FIG. 44 shows the amino acid sequence of WNV NS5 (SEQ ID
NO.8) corresponding to nucleotide sequence positions 7681-10395 of
GenBank accession no. AF404756 (WNV isolate 3356).
[0148] FIG. 45 shows the nucleotide sequence of nucleotide
positions 7574-10270 (SEQ ID NO:9) of GenBank accession No. U88535
(DENV-1 isolate "WestPac") corresponding to the amino acid sequence
of DENV-1 NS5.
[0149] FIG. 46 shows the amino acid sequence of DENV-1 NS5 (SEQ ID
NO.10) corresponding to nucleotide sequence positions 7574-10270 of
GenBank accession No. U88535 (DENV isolate "WestPac").
[0150] FIG. 47 shows the nucleotide sequence for nucleotide
positions 7570-10269 (SEQ ID NO:11) of GenBank accession No.
AF038403 (DENV-2 isolate "New Guinea") corresponding to the amino
acid sequence of DENV-2 NS5.
[0151] FIG. 48 shows the amino acid sequence of DENV-2 NS5 (SEQ ID
NO.12) corresponding to nucleotide sequence positions 7570-10269 of
GenBank accession No. AF038403 (DENV isolate "New Guinea").
[0152] FIG. 49 shows the amino acid sequence of DENV-3 NS5 (SEQ ID
NO.13) of GenBank accession No. AY099336 (DENV isolate
"D3/HAMTSSA-SRI/2000/1266")
[0153] FIG. 50 shows the amino acid sequence of DENV-4 NS5 (SEQ ID
NO.14) of GenBank accession No. AF326825 (DENV isolate
"814669")
[0154] FIG. 51(a) shows the results of a microsphere immunoassay to
test for the presence and relative quantity of anti-NS5 monoclonal
antibodies expressed from 37 distinct hybridomas. The relative
level of NS3 was also measured and is shown. Bolded hybridoma clone
ID numbers indicate those hybridomas having a positive detection of
anti-NS5 monoclonal antibodies. The two highest producing clones
are indicated as #215 and #270.
[0155] FIG. 51(b) shows the results of a microsphere immunoassay to
test for the presence and relative quantity of anti-NS5 monoclonal
antibodies formed in ascites fluid of mice injected with hybridoma
clones #215 and #270. The relative level of NS3 was also measured
and is shown.
DETAILED DESCRIPTION OF THE INVENTION
[0156] The subject invention relates to compositions and methods
for diagnosing an infection by a flavivirus, especially WNV, JEV,
SLEV, or DENV, in a subject suspected of carrying said infection
that are more rapid, efficient, cost effective and sensitive than
the methods and compositions currently available in the art. More
in particular, this invention relates to the use of an isolated
and/or substantially purified polypeptide of WNV, in particular,
WNV E glycoprotein, which includes recombinant, synthetic and
fusion proteins comprising the polypeptides, subfragments or
derivatives thereof, or a nucleic acid molecule encoding a WN
polypeptide or subfragment thereof, whereby the WNV polypeptide is
of authentic conformation and is reactive to antibodies against WNV
and strongly, reliably, predictably and consistently cross-reactive
with antibodies against a flavivirus, advantageously, JEV, SLEV,
and/or DENV.
[0157] Moreover, the present invention relates to a novel use for
the WNV E glycoprotein as an antigen for the detection of
antibodies against a flavivirus. The inventors have discovered that
a substantially purified WNV E glycoprotein antigen having an
authentic conformation is strongly, reliably, predictably and
consistently cross-reactive among WNV, JEV, SLEV, and DENV, and is
therefore useful to broadly assay or test for flavivirus infection,
non-specifically, e.g., in subjects, blood donors, organ donors,
blood, organs, etc. Accordingly, by the present invention, one can
determine whether there is a recent or past flavivirus infection,
for instance, infection by any of WNV, JEV, SLEV, or DENV, by a
single assay therein providing a faster, simpler, more cost
effective approach to broadly assaying for an infection by a
flavivirus when the exact identity of the flavivirus is not
required.
[0158] Another aspect of the present invention includes
compositions and methods for consistently and reliably diagnosing
an infection specifically by WNV that are more rapid, efficient,
cost effective and sensitive than the methods and compositions
currently available in the art. More in particular, this aspect of
the invention relates to the use of an isolated and/or
substantially purified nonstructural polypeptide of WNV, in
particular, NS5, which includes recombinant, synthetic and fusion
proteins comprising the polypeptides, subfragments or derivatives
thereof, or a nucleic acid molecule encoding a nonstructural
polypeptide, in particular, NS5, or subfragment thereof, whereby
the WNV nonstructural polypeptide is of authentic conformation and
is reactive to WNV antibodies with specificity without having
substantial cross-reactivity to antibodies against other
Flaviviruses, such as JEV, SLEV, and/or DENV.
[0159] Moreover, the present aspect of the invention relates to a
novel use for the WNV NS5 nonstructural protein as an antigen for
the specific detection of antibodies against WNV without
substantial cross-reactivity to antibodies against other
Flaviviruses, such as JEV, SLEV, and/or DENV. It has been
discovered that the WNV NS5 nonstructural protein is strongly,
reliably, predictably and consistently reactive to WNV antibodies
with high specificity without cross-reactivity with antibodies
against other Flaviviruses, such as JEV, SLEV, and DENV, and is
therefore useful to assay or test for WNV infection with
specificity, e.g., in subjects, blood donors, organ donors, blood,
organs, etc. In addition, the present aspect of the invention
relates to compostions and methods for differentiating between
vaccination with inactivated flavivirus and natural WNV infection.
It has been recognized by the inventors that only replicative
viruses produce NS proteins. Thus, inactivated flavivirus vaccines
do not produce NS proteins since they do not replicate.
Accordingly, the WNV NS5 protein can be used according to the
instant invention to discriminate between vaccination with inactive
flavivirus and a natural WNV infection. Moreover, the present
aspect of the invention relates to compositions and methods for
indicating the timing of WNV infection and can be used to
discriminate between recent and remote WNV infections.
[0160] Yet another aspect of the present invention includes
compositions and methods for consistent and reliable diagnosis of
an infection by DENV that are more rapid, efficient, cost effective
and sensitive than the methods and compositions currently available
in the art. More in particular, this aspect of the invention
relates to the use of an isolated and/or substantially purified
nonstructural polypeptide of DENV, in particular, NS5, which
includes recombinant, synthetic and fusion proteins comprising the
polypeptides, subfragments or derivatives thereof, or a nucleic
acid molecule encoding a nonstructural polypeptide, in particular,
NS5, or subfragment thereof, whereby the DENV nonstructural
polypeptide is of authentic conformation and is reactive to DENV
antibodies with specificity without having substantial
cross-reactivity to antibodies against other flaviviruses, such as
JEV, SLEV, and/or DENV. Further, the DENV NS5 protein of the
present invention when isolated from a specific strain of DENV will
be specific for antibodies to that same DENV strain and will not be
cross-reactive to antibodies against the remaining strains of DENV.
It will be appreciated to one of ordinary skill in the art that
DENV is comprised of four serologically distinct type, including
DENV-1, DENV-2, DENV-3, and DENV-4. Thus, a NS5 of DENV-1 will be
specific to antibodies against DENV-1, and not detetectably
cross-reactive with antibodies to DENV-2, -3, or -4.
[0161] Moreover, the present aspect of the invention relates to a
novel use for the DENV NS5 nonstructural protein as an antigen for
the specific detection of antibodies against DENV without
substantial cross-reactivity to antibodies against other
flaviviruses, such as JEV, SLEV, and/or DENV. It has been
discovered that the DENV NS5 nonstructural protein is strongly,
reliably, predictably and consistently reactive to DENV antibodies
with high specificity without cross-reactivity with antibodies
against other flaviviruses, such as JEV, SLEV, and WNV, and is
therefore useful to assay or test for DENV infection with
specificity, e.g., in subjects, blood donors, organ donors, blood,
organs, etc. In addition, the present aspect of the invention
relates to compostions and methods for differentiating between
vaccination with inactivated flavivirus and natural WNV infection.
One of skill in the art will appreciate that only replicating
viruses produce NS proteins. Thus, inactivated (or "heat-killed")
flavivirus vaccines do not produce NS proteins since they do not
replicate. Accordingly, the DENV NS5 protein can be used according
to the instant invention to discriminate between vaccination with
inactive flavivirus and a natural WNV infection. Moreover, the
present aspect of the invention relates to compositions and methods
for indicating the timing of DENV infection and can be used to
discriminate between recent and remote DENV infections since the
antibody response to NS proteins is not sustained. Similarly, one
of ordinary skill in the art would appreciate that the methods and
compositions of the present invention, in particular, the
nonstructural flavivirus antigens, including but not limited to WNV
NS5 or DENV NS5, could be used to discriminate a vaccination with a
recombinant or subunit flavivirus vaccine, such as a recombinant or
subunit WNV, DENV, SLEV, or JEV vaccine, from a recent or ongoing
infection with a flavivirus, including but not limited to WNV,
DENV, SLEV, and JEV.
[0162] In another embodiment, the methods and compositions of the
present invention, especially flavivirus nonstructural proteins
such as NS5, can be used to distinguish recent or ongoing
infections of a flavivirus, such as WNV, DENV, SLEV, or JEV, in a
susceptible animal, such as a human, horse, cat, dog, or bird,
inter alia, from vaccination by an in vivo flavivirus vaccine,
which provides flavivirus structural proteins to an animal to be
vaccinated by direct expression to the protein within the animal.
It will be appreciated that an in vivo vaccine comprises a DNA
molecule coding for one or more structural immunogenic viral
polypeptides or portions thereof that are directly administered to
a host, such as a human, horse, cat, dog, or bird, inter alia, that
is to be vaccinated.
[0163] One of ordinary skill in the art would certainly appreciate
the numerous benefits of the instant invention, which in one aspect
provides a novel method for the broad detection of a flavivirus
infection, such as an infection with WNV, JEV, SLEV, and/or DENV,
in light of a hypothetical scenario wherein knowledge of the
identity of the flavivirus would not be immediately required, but a
rapid identification of a putative flaviviral infection would be
critical. Such a scenario could involve a patient arriving at a
hospital in a geographical region having recently experienced cases
of flavivirus infections in patients, such as an infection with
WNV, JEV, SLEV, or DENV, wherein the patient arrives with typical
flavivirus-like symptoms, such as headache, sudden fever, malaise,
and swollen glands. The treating physician would want to know
immediately whether or not the symptoms are due to a flavivirus
infection, but immediate identification of the flavivirus species
or strain is not initially critical. The instant invention provides
a rapid diagnostic assay to broadly detect a flavivirus infection,
such as an infection with WNV, JEV, SLEV, or DENV, which in one
embodiment, can be completed in under 3 hours. Thus, providing a
rapid result in the diagnosis of a flavivirus infection is clearly
an advantage of the instant invention. Another aspect of the
instant invention relates to a novel method for the detection of
antibodies to a flavivirus using enhanced reaction kinetics and a
microsphere immunoassay that together drastically reduce the time
it takes for diagnosis of a WNV infection or a general flavivirus
infection from 3 days and up to 3 weeks, to less than 3 hours.
[0164] Also within the scope of the instant invention are
diagnostic kits and methods for detecting antibodies against a
flavivirus, especially WNV, JEV, SLEV, and/or DENV, characterized
by the compositions of the present invention comprising a
substantially pure WNV polypeptide, fragments, or derivatives
thereof, such as WNV E glycoprotein, that are reactive with
antibodies against WNV and strongly, reliably, predictably and
consistently cross-reactive with antibodies against a flavivirus
wherein the WNV polypeptide has an authentic conformation. These
diagnostic kits and methods for detecting antibodies against a
flavivirus are also useful for detecting a protective immune
response to a flavivirus infection, especially by WNV, JEV, SLEV,
and/or DENV.
[0165] Further, the methods of the instant invention are also
useful in monitoring the course of immunization against a
flavivirus, especially, WNV, JEV, SLEV, and/or DENV. In patients
previously inoculated with the vaccines against a flavivirus, the
detection means and methods disclosed herein are also useful for
determining if booster inoculations are appropriate.
[0166] It will be appreciated by those having ordinary skill in the
art based on the teachings and examples set forth herein that the
methods of the present invention represent a more reliable,
consistent, effective and time efficient approach to detecting in
biological samples, including those from humans and animals such as
wild or domestic birds, and mammals such as horses, cats or dogs,
antibodies to WNV, DENV and other flaviviruses. It will be
understood that the various proteins isolated from the various
flaviviruses of the invention, especially WNV and DENV, can be used
as antigens in the methods of the instant invention to detect in a
specific or nonspecific manner, flaviviruses, especially WNV and
DENV.
[0167] It will be appreciated that the WNV E glycoprotein can be
used as an antigen to in the method of the instant invention to
detect antibodies against certain species of flaviviruses relevant
to human disease, such as, WNV, JEV, SLEV, DENV, using a single
assay to take the place of a multitude of assays currently used in
the art for the detection of these flaviviruses. Thus, by the
present invention, one can determine whether there is a flavivirus
infection, for instance, infection by any of WNV, JEV, SLEV, or
DENV, by a single assay. It has been discovered here that a
substantially purified WNV E glycoprotein antigen having a
substantially authentic conformation is reliably, consistently,
predictably, and strongly cross-reactive to antibodies against any
of WNV, JEV, SLEV, and DENV, and is therefore useful to broadly
assay or test for flavivirus infection, non-specifically, e.g., in
subjects, donors, blood, organs, etc. In contrast, antigens
currently available in the art for the detection of DENV, SLEV,
JEV, and WNV infections are often concentrated by polyethylene
glycol and/or extracted with acetone, treatments which are likely
to alter the structural domains of a given antigen.
[0168] It will further be appreciated that a WNV nonstructural
protein, especially NS5, or a specific antigenic determinant or
specific epitope thereof, can be used as an antigen for the
specific detection of antibodies against WNV in the method of the
instant invention. Importantly, the NS5 antigen is not
cross-reactive to other flaviviruses, such as, for example, JEV,
SLEV, or DENV. Thus, in accordance with the present aspect of the
invention, one can consistently, reliably, and accurately determine
whether there is a WNV infection with the confidence and assurance
that the detection signal is not due to cross-reactivity with other
flaviviruses.
[0169] It will also be appreciated that the substantially purified
DENV NS5 antigen of the present invention is reliably,
consistently, predictably, and strongly reactive to antibodies
against a DENV without having substantial cross-reactivity with
other flaviviruses, such as, for example, JEV, SLEV, and WNV.
Therefore, DENV NS5 antigen will be useful to specifically assay or
test for DENV infection, e.g., in subjects, donors, blood, organs,
etc. In contrast, current serologic diagnoses of DENV infection is
based on detection of antibodies against viral structural proteins,
such as the E protein. Although, the cross-reactivity of the E
protein among flaviviruses, as also discovered by the instant
inventors, is certainly advantageous with respect to its use as a
rapid diagnostic for detecting a general flavivirus infection when
knowing the identity of the flavivirus is not critical, it would
also be desirable to have a rapid test that could confidently,
accurately, and correctly identify a DENV infection with
specificity and without cross-reactivity with other types of
flaviviruses. Further, the DENV NS5 protein of the present
invention when isolated from a specific strain of DENV will be
specific for antibodies to that same DENV strain and will not be
cross-reactive to antibodies against the remaining strains of
DENV.
[0170] As one of ordinary skill in the art will understand, prior
to the present invention, a number of serologic assays were
routinely used for laboratory diagnosis of flavivirus infections,
including infections of WNV, DENV, JEV, and SLEV: IgM antibody
capture enzyme immunoassay (MAC-ELISA), indirect IgG ELISA,
indirect fluorescent antibody assay (IFAT), hemagglutination
inhibition (HIT), and serum dilution cross-species plaque reduction
neutralization tests (PRNTs). As described below, these assays vary
markedly in sensitivity, technical difficulty, turn-around time,
and clinical utility of the results.
[0171] IgM Antibody Capture ELISA
[0172] IgM response to WNV infection occurs earlier than that of
IgG, and is often used to indicate recent infections of WNV (G.
Tardei et al.). MAC-ELISA is typically performed to detect the IgM
level in serum samples, as described by Beaty and coworkers (Martin
et al.) with modifications (Martin et al.). Briefly, goat
anti-human IgM (PerImmune Inc., Rockville, Md.) is used as a
capture antibody, and is coated onto 96-well flat-bottom plates.
After blocking of the plates with nonfat dry milk, diluted human
sera are reacted with the anti-human IgM. Viral antigens, either
sucrose-acetone extracts of infected suckling mouse brains [32, 33]
or recombinant viral structural proteins (B. Davis et al.), are
added to the plates. Flavivirus group-reactive SLE monoclonal
antibody 6B6C-1 (conjugated to horseradish peroxidase) (Roehrig et
al.) is then reacted with the immobilized viral antigen. After
addition of substrate 3,3'5,5' tetra methyl benzidine (TMB-ELISA;
Neogen, Lexington, Ky.), reactions are measured using a microplate
reader at an absorbance of 450 nm. A ratio of 2.0 or greater of the
absorbance of the test serum over the absorbance of the negative
control serum is usually considered positive. Serial dilutions of
positive sera can be evaluated. The maximum dilution that exhibits
positive signal is the titer for the serum. The titer of the
MAC-ELISA can be compared with the titers of HI and PRN tests. It
should be noted that, in the CDC MAC-ELISA, all serum samples are
tested on control antigen in addition to viral antigen, to reduce
the number of false-positive results due to non-specific binding of
the serum or other factors. Any "positive" test result
(ratio>2.0) is deemed un-interpretable if the mean absorbance of
the test specimen reacted on viral antigen is <2.times. the mean
absorbance of the test specimen reacted on the control antigen.
These samples require testing with an alternate method, generally
PRNT.
[0173] MAC-ELISA has replaced complement fixation and HIT in most
public health laboratories, because of its superior sensitivity in
diagnosing early acute infections of flaviviruses. The MAC-ELISA
was reported to be more sensitive than PCR-based assay for spinal
fluid and blood specimens from patients with symptomatic infection
(D Nash et al.). The MAC-ELISA is a more appropriate assay to use
for symptomatic patients, because the virus is cleared so
efficiently by the immune system in healthy individuals, that it is
not usually possible to detect viral nucleic acid once clinical
symptoms are observed. Since the 1999 outbreak of WNV in New York,
the Centers for Disease Control and Prevention (CDC) have
extensively trained public health laboratories to perform this
complex 2-day assay. MAC-ELISA is currently the most widely used
diagnostic test for acute WNV encephalitis in the United
States.
[0174] Initially, the ELISA antigen was a sucrose-acetone extract
of WNV-infected suckling mouse brain (Martin et al.). The antigen
was later replaced by unpurified E and prM proteins secreted from a
plasmid-transformed COS cell line (B Davis et al.). The cell
line-derived antigens are concentrated by precipitation with
polyethylene glycol followed by lyophilization. Several companies
have purchased licenses to commercialize the noninfectious
recombinant antigens (NRA), including Abbott Laboratories (Abbott
Park, Ill.), Focus Technologies (Cypress, Calif.), GenBio (San
Diego, Calif.), Hennesy Research Associates (Shawnee, Kans.),
Immucor (Atlanta, Ga.), InBIOS (Seattle, Wash.), RMZ Biotech
Corporation (Baltimore, Md.), and Rapid Medical Diagnostic
Corporation (Miami, Fla.). These products are sold as
analyte-specific reagents (ASR) and, as such, must be validated by
the user. The FDA recently approved the enzyme immunoassay kit for
diagnostic testing from PanBio (Baltimore, Md.).
[0175] The IgM response to WNV infection is rapid; most sera and
spinal fluids are positive in the MAC-ELISA within 8 days of
symptom onset (Roehrig et al.). IgM detected by MAC-ELISA in spinal
fluids is directly indicative of recent infection (B. J. Beaty et
al.). This is because the detected IgM is synthesized by
lymphocytes in the central nervous system, and serum IgM, a
pentameric molecule, is too large to pass through an intact
blood-brain barrier. However, because anti-WNV IgM in serum
persists in many patients for 3 to 6 months after symptom onset,
and can persist beyond 15 months (D. A. Martin et al. 2000),
caution should be used when interpreting IgM-positive results from
early-season WNV IgM-positive patients. Solitary serum IgM positive
results must be considered in conjunction with other WNV
surveillance data, e.g., evidence of WNV in mosquitoes or in
reservoir bird populations. Follow-up testing of paired specimens
is recommended. For the same reason, because the duration of the
anti-WNV IgM in chickens or rabbits has been not well defined, IgM
testing should not be performed as the sole testing modality when
these animals are used as sentinels for monitoring WNV activities.
In one study, the IgM persistence was reported to vary in chickens
from 19 days to greater than 61 days post WNV infection (A. J.
Johnson et al.). If the bleeding schedule is less frequent than
once every 3 weeks, a recent infection of the sentinel animals may
be missed through sole reliance on MAC-ELISA (A. J. Johnson et
al.).
[0176] Several limitations were previously reported for the
MAC-ELISA. First, although human IgM response to WNV and other
flaviviruses is more virus-type specific than is the IgG response,
cross-reactivities with other JE serogroup viruses, DEN and YF, do
occur in the IgM tests (D. A. Martin et al. 2002, T. P. Monath et
al., M. Lhullier et al.). Additional information about the patient,
such as travel history to regions where WNV or other flaviviruses
are active, should be helpful in interpreting the diagnostic
results. Second, the sensitivity of the MAC-ELISA may be decreased
when patient serum contains IgM molecules in response to infections
other than WNV. These non-WNV-IgM molecules can nonspecifically
compete against WNV-IgM molecules for binding to the anti-human IgM
antibodies coated on the ELISA plate, resulting in reduced
sensitivity of the assay. Third, false-positive results of
MAC-ELISA may occur due to nonspecific binding of rheumatoid
factors, which often exist in sera from healthy individuals (P. P.
Mortimer et al.). Rheumatoid factors are well known to confound
serological diagnosis through their cross-linking of the capture
antibody to the detector antibody in the absence of any WNV antigen
(P. P. Mortimer et al.).
[0177] Indirect IgG ELISA
[0178] Indirect IgG ELISA is a 2-day assay that is often performed
in tandem with MAC-ELISA. The protocol for diagnosis of anti-WNV
IgG was described by Johnson and coworkers, 2000). A flavivirus E
protein cross-reactive monoclonal antibody (Mab) 4G2 (ATCC,
Manassas, Va.) (A. J. Johnson et al., 2000) is coated onto 96-well
microtiter plates. After blocking of the plates with 3% goat serum
in PBS and multiple washes, WNV antigens are reacted with the Mab
4G2. After several washes, diluted human sera are reacted with the
immobilized viral antigens. Goat anti-human IgG Fc-alkaline
phosphatase conjugate is then reacted with serum-derived IgG. Upon
addition of p-nitrophenyl phosphate (Sigma Aldrich, St. Louis, Mo.)
to the wells, colorimetric absorbance at 405 nm is measured, and a
ratio of 2.0 or greater for the test serum over the negative
control serum is considered positive (A. J. Johnson et al.,
2003).
[0179] The same antigens as used for the MAC-ELISA are used for the
IgG ELISA. Because of the cross reactivities of the structural
proteins during various flavivirus infections, the identity of the
infecting virus can not be determined with certainty. Other assays
such as HI and PRNT are routinely performed to verify the identity
of the virus. Because the level of IgG remains elevated for many
years after an infection, a 4-fold increase in IgG antibody titer
between paired sera are considered essential for estimation of a
recent, acute infection (D. Gubler et al., 2000). Using inactivated
WNV as antigen, Ebel and co-workers recently reported that the IgG
ELISA could also be used to detect anti-WNV antibodies in
birds.
[0180] Indirect Fluorescent Antibody Tests
[0181] IFAT is used to detect anti-WNV IgG, IgM, or total
antibodies (IgG+IgA+IgM) from suspected WNV-infected sera. The
assay can be completed within 2-3 hr. IFAT slides and test kits are
commercially available from PanBio (Baltimore, Md.). WNV-infected
Vero cells are grown until the appearance of cytopathic effects,
mixed with uninfected tissue culture cells, and spotted onto a
microscope slide. The slides are then acetone-fixed and stored
frozen. Patient sera, starting at a 1:8 dilution, are reacted with
the antigens on the slide. After incubations with anti-human
immunoglobulins conjugated with fluorescein isothiocyanate (FITC)
and washings, the cells are examined under a fluorescent
microscope. Positive IgG antibody is indicated by specific
apple-green fluorescence in the cytoplasm cells. Since only 30% to
50% of the cells on the slides are infected with WNV, observation
of 100% cells of positive fluorescence indicates a non-specific
reaction, rather than a serum infected with WNV. Autoantibodies in
patient serum can react with cellular antigens, resulting in
non-specific fluorescence. This possibility can be excluded by
using undiluted serum to react with uninfected tissue culture
cells. If positive, the patient serum is likely to have
autoantibodies to cellular antigens. The sensitivity of the IFAT is
low, with an estimated detection limit of 0.05-1 .mu.g of
virus-specific neutralizing antibody (J. Pillot, 1996). The
detection limit of the IFAT is about a 1,000-fold lower than that
of ELISA. However, IFAT measurement of IgG is slightly more
specific than ELISA (P. Koraka et al., 2002).
[0182] For detection of virus-specific IgM or IgG, serum specimens
are pretreated with rabbit anti-human IgG or IgM, respectively.
Complete depletion of IgG in serum is essential for an accurate
detection of IgM, because residual IgG can compete with IgM to bind
to the antigens on the slide, resulting in inaccurate results. To
increase the sensitivity of the IgM assay, overnight incubation of
IgG depleted serum with antigen slides is recommended. After
binding of the IgM to antigen, anti-human IgM conjugated with FITC
is applied to bind to the antigen-bound IgM. Even through the
IgM-IFAT is less sensitive than MAC-ELISA, it has been applied to
rapid diagnosis of acute serum samples. Because the procedure of
IgM-IFAT requires manual pipetting and reviewing of individual
wells of the IFAT slides under a fluorescent microscope, this assay
does not have the capability to diagnose a large volume of patient
specimens. Further, since the concentration of IgM in spinal fluid
is nearly a 1,000-fold lower than that in serum, neither can the
assay be reliably used to detect IgM in spinal fluid (W. R. Chen et
al., 1992). However, the specificity of the IgM IFAT against DEN,
JE, YF, and WNV was reported to exceed that of the standard EIA (P.
Koraka et al.), with a cross-reactivity that ranged from 4% to 10%,
compared to 30% to 44% for the standard EIA. False IgM-IFAT
positives can be caused by rheumatoid factor; false IgM-IFAT
negatives can be caused by competition from residual IgG molecules
remaining after the IgG depletion.
[0183] Hemagglutination Inhibition Tests
[0184] The HI test is performed essentially as described nearly 60
years ago by Casals and Brown (J. Casals, et al., 1954). Serum is
first treated by acetone extraction, followed by adsorption with
goose erythrocytes to remove nonspecific inhibitors associated with
false-positive results, and to remove hemagglutinins associated
with false-negative results. Treated sera are serially diluted and
mixed with a known amount of suckling mouse brain WNV antigen for
an overnight incubation at 4.degree. C. Goose erythrocytes,
preferably from an adult gander, are added to the serum/virus
mixture in microtiter plates. The HI titer is read after a 1-hr
incubation at room temperature. A thin mat of cells across the well
indicates agglutination. A pellet of cells at the bottom of the
well indicates inhibition of agglutination. The highest dilution of
serum that completely inhibits agglutination of the goose
erythrocytes is taken as the HI titer of the serum. HI tests
provide higher titers than do standard neutralization tests, but
lower titers and lower numbers of positive samples than do micro
PRN tests (H. M. Weingartl et al.). HI tests measure both IgM and
IgG antibody classes, and are considerably less sensitive than
ELISAs. Although HI antibodies appear rapidly, they disappear more
quickly than do neutralizing antibodies, which are detected by the
IgG ELISAs (B. J. Beaty et al., 1995). Reagents for HI tests are
somewhat less stable to long-term storage than are the reagents
used in other methods. Agglutination occurs over a narrow pH range.
In addition, patient sera must be tested by a panel of viruses
known to occur and to cause disease in humans in a given geographic
region.
[0185] Plaque Reduction Neutralization Tests (PRNT)
[0186] PRNT is a 3- to 5-day assay (H. S. Lindsey, 1976). Sera are
first heat-inactivated at 56.degree. C. for 30 min. A set of
serially diluted sera are added to known amounts of virus. After
incubation for 1 hr, the mixture is added to Vero cells, followed
by another 1-hr incubation. Nutrient agar is applied, and the
plates are incubated for 2-3 days in a CO.sub.2 incubator. A second
overlay with a neutral red stain is applied. Plates are checked for
plaque formation over the next 1-2 days. The titer is the
reciprocal of the serum dilution causing a plaque reduction of 90%.
PRNT detects antibodies at an earlier time post-infection, with
higher mean serum antibody titers, than did HI and ELISA tests, in
experimental infections of chickens. PRNT also detects the highest
number of positive serum samples at various times post-infection
(H. M. Weingartl, et al., 2003).
[0187] It will be appreciated that the instant invention provides
methods for assaying biological samples for the presence of
antibodies against flaviviruses that are distinguished from and
advantageous over the previous methods. Several examples of this
invention's advantages over prior art methods for detecting
anti-flavivirus antibodies include, inter alia, reduced time
requirements, greater consistency in results, and enhanced ease of
use. More in particular, the traditional serologic assays such as
HI or particle agglutination are performed with either antigens or
antibodies passively adsorbed onto the surfaces of tanned
erythrocytes or latex microspheres. The recent technology of
lateral flow immunoassays utilizes antibodies or antigens bound to
microspheres and lateral flow immunochromatography to capture zones
on nitrocellulose membranes. Recently, antigens have been
covalently attached to fluorescent polystyrene microspheres for
immunoassays performed in microfilter plates, and the assays are
quantified through a flow cytometer such as the Luminex 100
(Luminex, Austin, Tex.).
[0188] One embodiment of the instant invention relates to the use
of microsphere immunoassays. Although the antigens of the
invention, including WNV E glycoprotein and the NS5 protein, can be
used essentially in any assay format known to one of ordinary skill
in the art, such as the above-mentioned methods, including ELISA
formats, certain embodiments of the instant invention are
advantageous over others. More in particular, the antigens of the
invention, including WNV E glycoprotein and the NS5 protein can be
used in connecting with microsphere immunoassays (MIAs). MIAs are
more quantitative than prior serological testing methods,
including, for example, ELISAs. The microsphere assays have broad
dynamic ranges, often exceeding what can be obtained with ELISAs.
Reaction times are short, since kinetics are enhanced by shaking of
the microspheres in fluid suspension during the incubations. Small
specimen volumes can be used in the microsphere assays, and
replicate testing is not required because of the high precision of
the analyses. Therefore, large specimen volumes of precious
specimens, such as spinal fluid, are not required. MIA results can
be obtained with small amounts of biological sample, such as, for
example, 30 .mu.l of spinal fluid, compared to the 450 .mu.l
required for the MAC-ELISA (Wong et al, submitted). In addition,
microbeads have more surface area and thus more epitopes available
for antigen or antibody binding than do macrobeads, microtiter
plates, or nitrocellulose papers, resulting in an increased
sensitivity. The preferred reporter fluorochrome,
red-phycoerythrin, has an extremely high extinction coefficient,
which also enhances the analytical sensitivity. Finally,
microsphere immunoassays allow a more cost-effective use of
antigen: 1 .mu.g of antigen usually suffices for approximately 50
tests.
[0189] In accordance with one embodiment of the present invention,
an E-based microsphere immunoassay is provided which consistently,
accurately, strongly, and reliably detects a WNV-infection at
around day 2-6 post-symptom onset. Retrospective testing as carried
out by the present inventors on over 800 sera from patients with
suspected viral encephalitis by the polyvalent (anti-IgG+IgA+IgM)
microsphere immunoassay exhibited 95% concordance with results
obtained with the IgG ELISA . The E-based microsphere immunoassay
of the present invention could also be used to detect anti-E IgM
antibodies, and to indicate current or recent WNV infection. In
addition, the inventors have discovered that a substantially
purified WNV E glycoprotein antigen having a substantially
authentic conformation is reliably, consistently, predictably, and
strongly cross-reactive to antibodies against any of WNV, JEV,
SLEV, and DENV, and is therefore useful to broadly assay or test
for flavivirus infection, non-specifically, e.g., in subjects,
donors, blood, organs, etc. In contrast, antigens currently
available in the art for the detection of DENV, SLEV, JEV, and WNV
infections are often concentrated by polyethylene glycol and/or
extracted with acetone, treatments which are likely to alter the
structural domains of a given antigen.
[0190] In another embodiment of the present invention, a NS5-based
microsphere immunoassay is provided. The NS5-based microsphere
immunoassay reliably detects WNV-infection (IgG+IgA+IgM total
antibodies) at around day 6 post-symptom onset. The overall
reactive pattern derived from the NS5-based assay was shown by the
inventors to correlate well with that from the E-based assay.
However, the NS5-based assay has two major diagnostic differences
over the E-based assay. First, the NS5-based assay can be used to
differentiate between WNV infection and vaccinations with either an
inactivated JEV or a live attenuated YFV vaccine. In support, sera
was collected from the JEV vaccine recipients and reacted with the
WNV NS5 antigen. The result was that only 5% of the sera collected
from the YFV-vaccine recipients reacted with the WNV NS5 antigen.
By contrast, 100% of the JEV-vaccinated sera and 53% of the
YFV-vaccinated sera reacted with the E antigen. Second, the
NS5-based assay substantially improves discrimination between
DENV/SLEV and WNV infections. The inventors show herein through
experimentation that only 9% of the DENV sera were marginally
positive in the WNV NS5-based assay, whereas 71% of the same sera
were reactive in the WNV E-based assay (see Examples and FIG. 26).
Further, only 5% of the SLEV sera were positive in the WNV
NS5-based assay, whereas 27.5% of the same panel of sera were
positive in the WNV E-based assay (see Examples and FIG. 27). The
results of the NS5-based immunoassay clearly suggest that NS5 could
be used as an antigen for virus type-specific diagnosis of
flavivirus infections.
[0191] In accordance with one embodiment of the invention, the
NS5-based microsphere immunoassay can be used to distinguish
between WNV infection and vaccination by inactivated JE. Without
being bound by theory, this distinction is possible since only
replicative viruses produce NS proteins, while inactivated JE
vaccines cannot replicate and thus cannot produce NS proteins.
Another reason is that no or very few NS proteins, including NS5,
exist in the inactivated JEV vaccines since the vaccines are
prepared through an extensive purification procedure.
[0192] In another embodiment of the invention, the antigen-based
immunoassays of the present application can be useful for
determining whether animals, such as horses, previously vaccinated
with inactivated WNV have sustained a new exposure to WNV
infections. The first documented case of equine WNV infection was
in Minnesota in 2003 in a horse that had received a vaccine in
2002, but had not had a booster. Since protective immunity wanes
quickly, and there is a chance for reinfection, veterinarians are
increasingly challenged to diagnose neurological illness possibly
owing to WNV infection in previously WNV-vaccinated horses. Such
diagnosis will be problematic for structural protein-based assays,
such as assays based on WNV E glycoprotein, due to the presence of
preexisting antibodies to the immunodominant E protein as a result
of the vaccination. However, WNV infection in previously vaccinated
horses could be assessed using the NS5-based immunoassay of the
present invention. The NS5-based assay will detect only current or
recent WNV infections. It will not show a positive result for an
animal that was solely vaccinated with a WNV or flavivirus vaccine
since there needs to be viral replication in order to produce NS5
in sufficient quantity to provide an immune response and the
production of anti-NS5 antibodies.
[0193] As used herein, the term "polypeptide" is taken to encompass
all the polypeptides, peptides, and fusion proteins described in
this invention and refers to any polymer consisting essentially of
amino acids regardless of its size which maintains a comparable
level of cross-reactivity to the cross-reactivity of the unmodified
polypeptide from which it is derived. Although "protein" is often
used in reference to relatively large polypeptides, and "peptide"
is often used in reference to small polypeptides, usage of these
terms in the art overlaps and varies. The term "polypeptide" as
used herein thus refers interchangeably to peptides, polypeptides,
or fusion proteins unless otherwise noted. The term "amino acid"
refers to a monomeric unit of a peptide, polypeptide or
protein.
[0194] Further, the term "polypeptide" is meant to encompass any
"derivative" thereof. A derivative refers to a modified or altered
form of the native or original polypeptide. As used in the present
application, a derivative will have a comparable level of
cross-reactivity to the cross-reactivity of the unmodified
polypeptide from which it is derived. Such modifications include,
but are not limited to: amino acid substitutions, modifications,
additions or deletions; alterations in the pattern of lipidation,
glycosylation or phosphorylation; reactions of free amino,
carboxyl, or hydroxyl side groups of the amino acid residues
present in the polypeptide with other organic and non-organic
molecules; and other modifications, any of which may result in
changes in primary, secondary or tertiary structure.
[0195] A "substantially pure" polypeptide is a polypeptide that is
free from other WNV components with which it is normally
associated. Further, a substantially pure polypeptide is one which
is free of other undesired protein contamination, such as bovine
serum albumin, which can be carried over from culture medium during
antigen preparation.
[0196] As used herein, an "authentic conformation" of a polypeptide
(e.g. antigen) refers to the native conformation of the polypeptide
(e.g. antigen). The native conformation of the polypeptide refers
specifically to the three-dimensional form of the molecule as it
exists in vivo. Many processes currently used in the art to prepare
various polypeptides (e.g. antigens) involve harsh preparatory
treatments, such as acetone extraction and/or polyethylene glycol
precipitation, both of which are known to deform and/or denature
polypeptides (e.g. antigens). A fully or partially denatured
polypeptide (e.g. antigen) is not as cross-reactive as the same
polypeptide (e.g. antigen) having an authentic conformation since
the epitopes of the polypeptide (e.g. antigen) involved in
cross-reacting interactions become damaged such that they are no
longer or less efficiently recognized by antibodies.
[0197] As it is used herein, the terms "NS5", "NS5 protein", or
"NS5 antigen" are meant to be synonymous with one another. Further
"NS5", "NS5 protein", or "NS5 antigen" are meant to encompass any
immunogenic fragment thereof or any specific portion encompassing
any unique epitope that is immunogenically distinct from NS5
proteins from other flaviviruses. The instant invention
contemplates the use of nonstructural proteins or fragment thereof
obtained from any flavivirus, including but not limited to WNV,
SLEV, JEV, and DENV, especially WNV and DENV. It was the inventors'
discovery that the nonstructural protein NS5 antigen from a first
flavivirus, such as WNV or DENV, can be used to specifically detect
antibodies against said first flavivirus from a biological sample
(e.g. biological fluid, tears, semen, blood, plasma, feces, spinal
fluid, saliva, or mucous) wherein the NS5 antigen from the first
flavivirus is not detectably cross-reactive with antibodies to
other flaviviruses. Moreover, in the case of DENV, the NS5 antigens
from a first strain, such as DENV-1, is not cross-reactive with
antibodies to the remaining DENV strains. Thus, the DENV NS5
antigens are useful for discriminating DENV strains.
[0198] According to various embodiments of the instant invention,
the WNV E glycoprotein utilized by the instant invention is
prepared by a process that results in a substantially purified WNV
E glycoprotein having an authentic conformation. In a further
preferred embodiment, the purification method of the instant
invention utilizes column chromatography in a manner that does not
harshly treat or denature the desired polypeptide to be purified.
Specifically, column chromatography as used by the instant
invention does not require polyethylene glycol precipitation or
acetone extraction.
[0199] As used herein, a "protective epitope" is (1) an epitope
that is recognized by a protective antibody, and/or (2) an epitope
that, when used to immunize a human or animal, elicits an immune
response sufficient to confer WNV immunity or to prevent or reduce
the severity for some period of time, of the resulting symptoms. A
protective epitope may comprise a T cell epitope, a B cell epitope,
or combinations thereof.
[0200] As used herein, "enhanced reaction kinetics" refers to an
antibody-antigen binding reaction that occurs at a rate that
exceeds the expected reaction rate when carried out under
conditions used in prior art methods. "Conditions" that are
suitable for enhanced reaction kinetics according to the present
invention are a discovery of the inventor. Such conditions may
comprise parameters related to incubation time, temperature,
buffers, and pH levels. The conditions further may comprise
physical parameters, such as, shaking or moving the components of
any given reaction sample. In one embodiment, enhanced reaction
kinetics are achieved by incubating together a biological sample
and a WNV antigen, such as, WNV E glycoprotein, and at 37.degree.
C. for about 30 minutes while keeping the reaction mixture in
motion, such as on platform shaker at low speed.
[0201] Various compositions and methods of the aforementioned
embodiments are characterized by immunogenic polypeptides. As used
herein, an "immunogenic polypeptide" is a polypeptide that, when
administered to a human or animal, is capable of eliciting a
corresponding antibody.
[0202] This invention also provides two novel immunogenic fragments
of the WNV E glycoprotein and compositions and methods comprising
these peptides. More specifically, this invention provides the
WNE-121-139 (peptide 3) peptide and WNE-288-301 peptide (peptide
1). It will be appreciated by those of ordinary skill in the art
that similar immunogenic fragments of the flavivirus antigens
contemplated by the present invention, especially immunogenic
fragments of NS5 and E glycoprotein antigens from the flaviviruses
of the invention, especially WNV and DENV, can be obtained and used
in accordance with the methods of the invention.
[0203] Also within the scope of this invention are polypeptides
that are at least 75% identical in amino acid sequence to the
aforementioned polypeptides. Specifically, the invention includes
polypeptides that are at least 80%, 85%, 90% or 95% identical in
amino acid sequence to an amino acid sequence set forth herein. The
term "percent identity" in the context of amino acid sequence
refers to the residues in the two sequences which are the same when
aligned for maximum correspondence. There are a number of different
algorithms known in the art which can be used to measure sequence
similarity or identity. For instance, polypeptide sequences can be
compared using NCBI BLASTp. Alternatively, FASTA, a program in GCG
version 6.1. FASTA provides alignments and percent sequence
identity of the regions of the best overlap between the query and
search sequences (Peterson, 1990).
[0204] Alternatively, nucleotide sequence similarity or homology or
identity can be determined using the "Align" program of Myers and
Miller, ("Optimal Alignments in Linear Space", CABIOS 4, 11-17,
1988) and available at NCBI. The terms "similarity" or "identity"
or "homology", for instance, with respect to a nucleotide sequence,
is intended to indicate a quantitative measure of homology between
two sequences. The percent sequence similarity can be calculated as
(N.sub.ref-N.sub.dif)*100/N.sub.ref, wherein N.sub.dif is the total
number of non-identical residues in the two sequences when aligned
and wherein N.sub.ref is the number of residues in one of the
sequences. Hence, the DNA sequence AGTCAGTC will have a sequence
similarity of 75% with the sequence AATCAATC(N.sub.ref=8;
N.sub.dif=2). Alternatively or additionally, "similarity" with
respect to sequences refers to the number of positions with
identical nucleotides divided by the number of nucleotides in the
shorter of the two sequences wherein alignment of the two sequences
can be determined in accordance with the Wilbur and Lipman
algorithm (Wilbur and Lipman, 1983 PNAS USA 80:726), for instance,
using a window size of 20 nucleotides, a word length of 4
nucleotides, and a gap penalty of 4, and computer-assisted analysis
and interpretation of the sequence data including alignment can be
conveniently performed using commercially available programs (e.g.,
Intelligenetics.TM. Suite, Intelligenetics Inc. CA). When RNA
sequences are said to be similar, or have a degree of sequence
identity with DNA sequences, thymidine (T) in the DNA sequence is
considered equal to uracil (U) in the RNA sequence.
[0205] Various compositions and methods of the aforementioned
embodiments are characterized by WNV polypeptides, such as, for
example, WNV E glycoprotein, that elicit in treated humans or
animals the formation of an immune response. As used herein, an
"immune response" is manifested by the production of antibodies
that recognize the corresponding polypeptide. In an especially
preferred embodiment, the compositions and methods of the invention
are characterized by WNV polypeptides or antibodies that confer
protection against WNV infection or disease.
[0206] In yet another embodiment, this invention relates to
diagnostic means and methods characterized by a WNV polypeptide,
such as, for example, WNV E glycoprotein. The inventor has
discovered that a substantially pure WNV E glycoprotein having an
authentic conformation as described in this application is not only
reactive with antibodies against WNV, but is also strongly,
reliably, predictably and consistently cross-reactive against other
flaviviruses, especially, JEV, SLEV, and DENV.
[0207] As used herein, an antigen, such as, WNV E glycoprotein of
WNV, is "reactive" with an antibody raised against the antigen when
there is a specific binding event/reaction between the antigen and
the antibody.
[0208] As used herein, a first antigen, such as, WNV E glycoprotein
of WNV, is "cross-reactive" with an antibody raised against a
second antigen of a second virus, such as, DENV, when there is a
specific binding event/reaction between the first antigen and the
antibody raised against the second antigen. One of ordinary skill
in the art will understand that similar or related viruses may
comprise similar proteins, e.g., proteins with similar amino acid
sequences and three-dimensional structural features that may
provide similar recognition epitopes such that an antibody raised
against a first antigen may recognize and bind to the second
antigen.
[0209] The WNV polypeptides or derivatives thereof described herein
are immunologically reactive with antisera produced in response to
an infection with WNV. Accordingly, they are useful in methods and
compositions to detect both immunity to WNV or prior infection with
WNV.
[0210] As will be apparent from the disclosure to follow, the
polypeptides in the pharmaceutical compositions of this invention
may also be prepared with the objective of increasing stability or
rendering the molecules more amenable to purification and
preparation. One such technique is to express the polypeptides as
fusion proteins comprising other WNV sequences.
[0211] In accordance with this invention, a derivative of a
polypeptide of the invention may be prepared by a variety of
methods, including by in vitro manipulation of the DNA encoding the
native polypeptides and subsequent expression of the modified DNA,
by chemical synthesis of derivatized DNA sequences, or by chemical
or biological manipulation of expressed amino acid sequences.
[0212] For example, derivatives may be produced by substitution of
one or more amino acids with a different natural amino acid, an
amino acid derivative or non-native amino acid. Those of skill in
the art will understand that conservative substitution is
preferred, e.g., 3-methyl-histidine may be substituted for
histidine, 4-hydroxy-proline may be substituted for proline,
5-hydroxylysine may be substituted for lysine, and the like.
[0213] Furthermore, one of skill in the art will recognize that
individual substitutions, deletions or additions which alter, add
or delete a single amino acid or a small percentage of amino acids
(typically less than 5%, more typically less than 1%) in an encoded
sequence are "conservatively modified variations" where the
alterations result in the substitution of an amino acid with a
chemically similar amino acid. Conservative substitution tables
providing functionally similar amino acids are well known in the
art. The following six groups each contain amino acids that are
conservative substitutions for one another:
[0214] 1) Alanine (A), Serine (S), Threonine (T);
[0215] 2) Aspartic acid (D), Glutamic acid (E);
[0216] 3) Asparagine (N), Glutamine (Q);
[0217] 4) Arginine (R), Lysine (K);
[0218] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);
and
[0219] 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
[0220] See also, Creighton (1984) Proteins W.H. Freeman and Co.
[0221] Conservative substitutions typically include the
substitution of one amino acid for another with similar
characteristics such as substitutions within the following groups:
valine, glycine; glycine, alanine; valine, isoleucine; aspartic
acid, glutamic acid; asparagine, glutamine; serine, threonine;
lysine, arginine; and phenylalanine, tyrosine. The non-polar
(hydrophobic) amino acids include alanine, leucine, isoleucine,
valine, proline, phenylalanine, tryptophan and methionine. The
polar neutral amino acids include glycine, serine, threonine,
cysteine, tyrosine, asparagine and glutamine. The positively
charged (basic) amino acids include arginine, lysine and histidine.
The negatively charged (acidic) amino acids include aspartic acid
and glutamic acid.
[0222] Other conservative substitutions are described by Dayhoff in
the Atlas of Protein Sequence and Structure (1988).
[0223] Causing amino acid substitutions which are less conservative
may also result in desired derivatives, e.g., by causing changes in
charge, conformation or other biological properties. Such
substitutions would include for example, substitution of a
hydrophilic residue for a hydrophobic residue, substitution of a
cysteine or proline for another residue, substitution of a residue
having a small side chain for a residue having a bulky side chain
or substitution of a residue having a net positive charge for a
residue having a net negative charge.
[0224] In another embodiment of this invention, the WNV
polypeptides described herein are prepared as part of a larger
fusion protein. For example, a WNV polypeptide used in a
composition of this invention may be fused at its N-terminus or
C-terminus to a different immunogenic WNV polypeptide, to a non-WNV
polypeptide or to combinations thereof, to produce fusion proteins
comprising the WNV polypeptide.
[0225] In a further embodiment of this invention, fusion proteins
comprising a WNV polypeptide used in a composition are constructed
comprising B cell and/or T cell epitopes from multiple strains of
WNV, each variant differing from another with respect to the
locations or sequences of the epitopes within the polypeptide. Such
fusion proteins are in particular effective in the induction of
immunity against a wide spectrum of WNV strains and can be utilized
to modulate the specificity of detection of antibodies against
flaviviruses.
[0226] In an embodiment of this invention, the WNV polypeptides
used in pharmaceutical compositions are fused to moieties, such as
immunoglobulin domains, which may increase the stability and
prolong the in vivo plasma half-life of the polypeptide. Such
fusions may be prepared without undue experimentation according to
methods well known to those of skill in the art, for example, in
accordance with the teachings of U.S. Pat. No. 4,946,778, or U.S.
Pat. No. 5,116,964. The exact site of the fusion is not critical as
long as the polypeptide retains the desired biological activity.
Such determinations may be made according to the teachings herein
or by other methods known to those of skill in the art.
[0227] The fusion proteins comprising the WNV polypeptides,
according to previous embodiments, may be produced at the DNA
level, e.g., by constructing a nucleic acid molecule encoding the
fusion protein, transforming host cells with the molecule, inducing
the cells to express the fusion protein, and recovering the fusion
protein from the cell culture. Alternatively, the fusion proteins
may be produced after gene expression according to known
methods.
[0228] The polypeptides of the invention may also be part of larger
multimeric molecules which may be produced recombinantly or may be
synthesized chemically. Such multimers may also include the
polypeptides fused or coupled to moieties other than amino acids,
including lipids and carbohydrates.
[0229] It will be readily appreciated by one of ordinary skill in
the art that the polypeptides in the pharmaceutical compositions of
this invention, as well as fusion proteins and multimeric proteins
containing them, may be prepared by recombinant means, chemical
means, or combinations thereof.
[0230] For example, the polypeptides may be generated by
recombinant means using the DNA sequence as set forth in the
sequence listing contained herein. DNA encoding variants of the
polypeptides in other WNV strains may likewise be cloned, e.g.,
using PCR and oligonucleotide primers derived from the sequence
herein disclosed.
[0231] For example, it may be particularly desirable to isolate the
genes encoding WNV polypeptides from any isolates that may differ
antigenically in order to obtain a broad spectrum of different
epitopes which would be useful in the methods and compositions of
this invention.
[0232] Oligonucleotide primers and other nucleic acid probes
derived from the genes encoding the polypeptides in the
compositions of this invention may also be used to isolate and
clone related proteins from other WNV isolates which may contain
regions of DNA sequence homologous to the DNA sequences of the
polypeptides described in this invention.
[0233] In another embodiment, the polypeptides used in the
compositions of this invention are produced recombinantly and may
be expressed in unicellular hosts. As is well known to one of skill
in the art, in order to obtain high expression levels of foreign
DNA sequences in a host, the sequences are generally operably
linked to transcriptional and translational expression control
sequences that are functional in the chosen host. Preferably, the
expression control sequences, and the gene of interest, will be
contained in an expression vector that further comprises a
selection marker.
[0234] The DNA sequences encoding the polypeptides used in the
compositions of this invention may or may not encode a signal
sequence. If the expression host is eukaryotic, it generally is
preferred that a signal sequence be encoded so that the mature
glycoprotein is secreted from the eukaryotic host.
[0235] An amino terminal methionine may or may not be present on
the expressed polypeptides in the compositions of this invention.
If the terminal methionine is not cleaved by the expression host,
it may, if desired, be chemically removed by standard
techniques.
[0236] A wide variety of expression host/vector combinations may be
employed in expressing the DNA sequences encoding the WNV
polypeptides used in the pharmaceutical compositions and vaccines
of this invention. Useful expression vectors for eukaryotic hosts,
include, for example, vectors comprising expression control
sequences from SV40, bovine papilloma virus, adenovirus,
adeno-associated virus, cytomegalovirus and retroviruses including
lentiviruses. Useful expression vectors for bacterial hosts include
bacterial plasmids, such as those from E. coli, including
pBluescript.RTM., pGEX-2T, pUC vectors, col E1, pCR1, pBR322, pMB9
and their derivatives, pET-15, wider host range plasmids, such as
RP4, phage DNAs, e.g., the numerous derivatives of phage lambda,
e.g. .lamda.GT10 and .lamda.GT11, and other phages. Useful
expression vectors for yeast cells include the 2.mu. plasmid and
derivatives thereof. Useful vectors for insect cells include pVL
941.
[0237] In addition, any of a wide variety of expression control
sequences-sequences that control the expression of a DNA sequence
when operably linked to it-may be used in these vectors to express
the polypeptides used in the compositions of this invention. Such
useful expression control sequences include the expression control
sequences associated with structural genes of the foregoing
expression vectors. Examples of useful expression control sequences
include, for example, the early and late promoters of SV40 or
adenovirus, the lac system, the system, the TAC or TRC system, the
T3 and T7 promoters, the major operator and promoter regions of
phage lambda, the control regions of fd coat protein, the promoter
for 3-phosphoglycerate kinase or other glycolytic enzymes, the
promoters of acid phosphatase, e.g., Pho5, the promoters of the
yeast-mating system and other constitutive and inducible promoter
sequences known to control the expression of genes of prokaryotic
or eukaryotic cells or their viruses, and various combinations
thereof.
[0238] In another embodiment, a DNA sequence encoding a WNV
polypeptide used in a pharmaceutical composition of this invention
is cloned in the expression vector lambda ZAP.RTM. II (Stratagene,
La Jolla, Calif.), in which expression from the lac promoter may be
induced by IPTG.
[0239] In yet another embodiment, a DNA sequence encoding a WNV
polypeptide, preferably the WNV E glycoprotein, that is used in a
composition of this invention is cloned in the pBAD/Thiofusion.TM.
expression vector, in which expression of the resulting thioredoxin
fusion protein from the araBAD promoter may be induced by
arabinose.
[0240] In another preferred embodiment, DNA encoding the WNV
polypeptides used in a composition of this invention is inserted in
frame into an expression vector that allows high level expression
of the polypeptide as a glutathione S-transferase fusion protein.
Such a fusion protein thus contains amino acids encoded by the
vector sequences as well as amino acids of the WNV polypeptide.
[0241] The term "host cell" refers to one or more cells into which
a recombinant DNA molecule is introduced. Host cells of the
invention include, but need not be limited to, bacterial, yeast,
animal, insect and plant cells. Host cells can be unicellular, or
can be grown in tissue culture as liquid cultures, monolayers or
the like. Host cells may also be derived directly or indirectly
from tissues.
[0242] In an embodiment of the instant invention, an insect cell
line, such as a mosquito cell line, is used in conjunction with an
appropriate expression vector to express and produce the WNV E
glycoprotein antigen. One of ordinary skill in the art will
appreciate that a eukaryotic host line, such as yeast, plant,
insect and mammalian cells can be necessary to achieve
glycosylation of the WNV polypeptide. Further, since a mosquito is
the natural host of WNV, it will be recognized that an insect host
for the expression and production of a WNV antigen may be optimal.
Although prokaryotic cells provide certain advantages with respect
to ease of genetic manipulation, cell growth, and product yield,
there is no capacity for glycosylation (at least in
naturally-occurring prokaryotic cells). Glycosylation of eukaryotic
or viral proteins raised in eukaryotic cells, such as the WNV E
glycoprotein, can affect protein folding, sorting, stability,
protease resistance, secretion and immunogenicity. Therefore, one
of ordinary skill in the art will recognize that glycosylation of
the viral antigen of the instant invention can be necessary to
achieve an authentic three-dimensional structure, thereby promoting
optimal cross-reactivity of the antigen. A discussion of use of
various types of host cell lines and corresponding expression
vectors for the expression of antigens may be found in J. Schmitt
and W. Papisch, Autoimmunity Reviews, 1: 79-88 (2002).
[0243] A wide variety of unicellular host cells are useful in
expressing the DNA sequences encoding the polypeptides used in the
pharmaceutical compositions of this invention. These hosts may
include well known eukaryotic and prokaryotic hosts, such as
strains of E. coli, Pseudomonas, Bacillus, Streptomyces, fungi,
yeast, insect cells such as Spodoptera frugiperda (SF9), animal
cells such as CHO and mouse cells, African green monkey cells such
as COS 1, COS 7, BSC 1, BSC 40, and BMT 10, and human cells, as
well as plant cells.
[0244] A host cell is "transformed" by a nucleic acid when the
nucleic acid is translocated into the cell from the extracellular
environment. Any method of transferring a nucleic acid into the
cell may be used; the term, unless otherwise indicated herein, does
not imply any particular method of delivering a nucleic acid into a
cell, nor that any particular cell type is the subject of
transfer.
[0245] An "expression control sequence" is a nucleic acid sequence
which regulates gene expression (i.e., transcription, RNA formation
and/or translation). Expression control sequences may vary
depending, for example, on the chosen host cell or organism (e.g.,
between prokaryotic and eukaryotic hosts), the type of
transcription unit (e.g., which RNA polymerase must recognize the
sequences), the cell type in which the gene is normally expressed
(and, in turn, the biological factors normally present in that cell
type).
[0246] A "promoter" is one such expression control sequence, and,
as used herein, refers to an array of nucleic acid sequences which
control, regulate and/or direct transcription of downstream (3')
nucleic acid sequences. As used herein, a promoter includes
necessary nucleic acid sequences near the start site of
transcription, such as, in the case of a polymerase II type
promoter, a TATA element.
[0247] A "constitutive" promoter is a promoter which is active
under most environmental and developmental conditions. An
"inducible" promoter is a promoter which is inactive under at least
one environmental or developmental condition and which can be
switched "on" by altering that condition. A "tissue specific"
promoter is active in certain tissue types of an organism, but not
in other tissue types from the same organism. Similarly, a
developmentally-regulated promoter is active during some but not
all developmental stages of a host organism.
[0248] Expression control sequences also include distal enhancer or
repressor elements which can be located as much as several thousand
base pairs from the start site of transcription. They also include
sequences required for RNA formation (e.g., capping, splicing, 3'
end formation and poly-adenylation, where appropriate); translation
(e.g., ribosome binding site); and post-translational modifications
(e.g., glycosylation, phosphorylation, methylation, prenylation,
and the like).
[0249] The term "operably linked" refers to functional linkage
between a nucleic acid expression control sequence (such as a
promoter, or array of transcription factor binding sites) and a
second nucleic acid sequence, wherein the expression control
sequence directs transcription of the nucleic acid corresponding to
the second sequence.
[0250] It should of course be understood that not all vectors and
expression control sequences will function equally well to express
the WNV polypeptides mentioned herein. Neither will all hosts
function equally well with the same expression system. However, one
of skill in the art may make a selection among these vectors,
expression control sequences and hosts without undue
experimentation and without departing from the scope of this
invention. For example, in selecting a vector, the host must be
considered because the vector must be replicated in it. The
vector's copy number, the ability to control that copy number, the
ability to control integration, if any, and the expression of any
other proteins encoded by the vector, such as antibiotic or other
selection markers, should also be considered.
[0251] In selecting an expression control sequence, a variety of
factors should also be considered. These include, for example, the
relative strength of the promoter sequence, its controllability,
and its compatibility with the DNA sequence of the peptides
described in this invention, in particular with regard to potential
secondary structures. Unicellular hosts should be selected by
consideration of their compatibility with the chosen vector, the
toxicity of the product coded for by the DNA sequences encoding the
glycoproteins used in a pharmaceutical composition of this
invention, their secretion characteristics, their ability to fold
the polypeptide correctly, their fermentation or culture
requirements, and the ease of purification from them of the
products coded for by the DNA sequences.
[0252] Within these parameters, one of skill in the art may select
various vector/expression control sequence/host combinations that
will express the DNA sequences encoding the products used in the
pharmaceutical compositions of this invention on fermentation or in
other large scale cultures.
[0253] The polypeptides described in this invention may be isolated
from the fermentation or cell culture and purified using any of a
variety of conventional methods including: liquid chromatography
such as normal or reversed phase, using HPLC, FPLC and the like;
affinity chromatography (such as with inorganic ligands or
monoclonal antibodies); size exclusion chromatography; immobilized
metal chelate chromatography; gel electrophoresis; and the like.
One of ordinary skill in the art may select the most appropriate
isolation and purification techniques without departing from the
scope of this invention. If the polypeptide is membrane bound or
suspected of being a lipoprotein, it may be isolated using methods
known in the art for such proteins, e.g., using any of a variety of
suitable detergents.
[0254] In a preferred embodiment, the WNV E glycoprotein of the
instant invention is expressed in an insect cell line, such as a
mosquito cell line, using an appropriate vector capable of
replicating and expressing cloned genes therefrom. The purification
of the WNV E glycoprotein will not utilize harsh techniques that
denature or deform the antigen such as polyethylene glycol
precipitation or acetone extraction. Instead, the present
embodiment relates to the use of column chromatography methods,
such as size-exclusion or affinity chromatography, to produce a
substantially purified antigen that has an authentic and native
conformation and/or three-dimensional structure.
[0255] In addition, the polypeptides of the invention may be
generated by any of several chemical techniques. For example, they
may be prepared using the solid-phase synthetic technique
originally described by R. B. Merrifield, J Am Chem Soc, 83, pp.
2149-54 (1963), or they may be prepared by synthesis in solution. A
summary of peptide synthesis techniques may be found in E. Gross
& H. J. Meinhofer, 4 The Peptides: Analysis, Synthesis,
Biology; Modern Techniques Of Peptide And Amino Acid Analysis, John
Wiley & Sons, (1981) and M. Bodanszky, Principles Of Peptide
Synthesis, Springer-Verlag (1984).
[0256] Typically, these synthetic methods comprise the sequential
addition of one or more amino acid residues to a growing peptide
chain. Often peptide coupling agents are used to facilitate this
reaction. For a recitation of peptide coupling agents suitable for
the uses described herein see M. Bodansky, supra. Normally, either
the amino or carboxyl group of the first amino acid residue is
protected by a suitable, selectively removable protecting group. A
different protecting group is utilized for amino acids containing a
reactive side group, e.g., lysine. A variety of protecting groups
known in the field of peptide synthesis and recognized by
conventional abbreviations therein, may be found in T. Greene,
Protective Groups In Organic Synthesis, Academic Press (1981).
[0257] To screen the polypeptides or fragments thereof according to
this invention for their ability to confer protection against WNV
infection or their ability to reduce the severity or duration of
the attendant symptoms, mice are preferred as an animal model. Of
course, while any animal that is susceptible to WNV infection may
be useful, mice are a well-known and particularly convenient model.
Thus, by administering a particular WNV polypeptide or anti-WNV
polypeptide antibody to mice, one of skill in the art may determine
without undue experimentation whether that polypeptide or antibody
would be useful in the methods and compositions claimed herein.
[0258] The administration of the WNV polypeptide or antibody of
this invention to the animal may be accomplished by any of the
methods disclosed herein or by a variety of other standard
procedures. For a detailed discussion of such techniques, see
Antibodies, A Laboratory Manual, supra. Preferably, if a
polypeptide is used, it will be administered with a
pharmaceutically acceptable adjuvant, such as complete or
incomplete Freund's adjuvant, RIBI (muramyl dipeptides) or ISCOM
(immunostimulating complexes). Such adjuvants may protect the
polypeptide from rapid dispersal by sequestering it in a local
deposit, or they may contain substances that stimulate the host to
secrete factors that are chemotactic for macrophages and other
components of the immune system. Preferably, if a polypeptide is
being administered, the immunization schedule will involve two or
more administrations of the polypeptide, spread out over several
weeks.
[0259] According to yet another embodiment, the WNV polypeptides
used in the compositions of this invention, preferably, are useful
as diagnostic agents for detecting immunity to WNV, and recent,
current, or prior infection by a flavivirus, especially WNV, JEV,
SLEV or DENV. The polypeptides are capable of binding to antibody
molecules produced in animals, including humans, that have been
exposed to a flavivirus, especially WNV, JEV, SLEV or DENV, as a
result of infection with said flavivirus or from vaccination. The
detection of WNV or flavivirus antigens is evidence of prior
exposure to a flavivirus infection or vaccine. Such information is
an important aid in the diagnosis of WNV infection.
[0260] Such diagnostic agents may be included in a kit which may
also comprise instructions for use and other appropriate reagents,
preferably a means for detecting when the polypeptide or antibody
is bound. For example, the polypeptide may be labeled with a
detection means that allows for the detection of the polypeptide
when it is bound to an antibody, or for the detection of the
antibody when it is bound to WNV or an antigen thereof.
[0261] The detection means may be a fluorescent labeling agent such
as fluorescein isocyanate (FIC), fluorescein isothiocyanate (FITC),
and the like, an enzyme, such as horseradish peroxidase (HRP),
glucose oxidase or the like, a radioactive element such as
.sup.125I or .sup.51Cr that produces gamma ray emissions, or a
radioactive element that emits positrons which produce gamma rays
upon encounters with electrons present in the test solution, such
as .sup.11C, .sup.15O, or .sup.13N. Binding may also be detected by
other methods, for example via avidin-biotin complexes. Further,
the labeling agent may be any enzyme included in the groups
oxidases (such as horse radish peroxidase), luciferases, peptidases
(such as caspase-3), glycosidases (such as beta-galactosidase) and
phosphatases (such as alkaline phosphatase).
[0262] The linking of the detection means is well known in the art.
For instance, monoclonal antibody molecules produced by a hybridoma
can be metabolically labeled by incorporation of
radioisotope-containing amino acids in the culture medium, or
polypeptides may be conjugated or coupled to a detection means
through activated functional groups.
[0263] The diagnostic kits of the present invention may be used to
detect the presence of antibodies against flaviviruses, especially
WNV, JEV, SLEV or DENV, in a body fluid sample such as serum,
plasma, urine, or spinal fluid. In various embodiments of the
instant invention, a substantially pure WNV polypeptide having an
authentic conformation is bound to a solid support typically by
adsorption from an aqueous medium. Useful solid matrices are well
known in the art, and include crosslinked dextran; agarose;
polystyrene; polyvinylchloride; cross-linked polyacrylamide;
nitrocellulose or nylon-based materials; tubes, plates or the wells
of microtiter plates. The polypeptides of the present invention may
be used as diagnostic agents in solution form or as a substantially
dry powder, e.g., in lyophilized form. In another preferred
embodiment, the instant invention provides an antigen, such as WNV
E glycoprotein, coupled to a solid matrix in the form of a bead or
microsphere, such as those available from Luminex Corporation
(Austin, Tex.). Coupling may be to the surface of the microsphere
or to an internal surface that is accessible from the outside
surface.
[0264] The method of attachment of antigens to microsphere beads
are known in the art. Antigens can be coupled to beads such as
those provided by Luminex Corporation by a two-step carbodiimide
process according to the manufacturer's recommendations. According
to the instant invention, 50 micrograms of purified WNV E
glycoprotein antigen (WNV-E) is coupled to the surface of
6.25.times.10.sup.6 microspheres. Activation is initiated with 50
microliters of 50 mg/ml Sulfo-NHS followed by 50 microliters of 50
mg/ml EDC and a 20 minute incubation at room temperature. Coupling
of the recombinant antigen takes place for 2 hours, in the dark, on
a rotator at room temperature. Microspheres were washed by
centrifugation, twice, in 1.0 ml PBS Azide blocking buffer, (PBN)
composed of PBS, 1 BSA, 0.02% NaN.sub.3.
[0265] In a preferred embodiment of the instant invention, a
plurality of antigens can be used, each coupled to separate or the
same microsphere beads. It is within the scope of the present
invention for additional antigens of WNV or another flavivirus,
such as, the membrane (M) protein or a non-structural (NS) protein,
to be coupled to the microspheres. It will be recognized that
incorporating additional antigens to the microspheres can enable
the further ability to distinguish between related flaviviruses,
such as WNV, JEV, SLEV, DENV strains, or tick-borne encephalitis
virus. Different beads or different regions of beads can be tagged
with fluorescent identifier tags, which allows for the coupling of
specific antigens to specific fluorescent tag identifiers. This
enables the methods of the instant invention to be carried out in a
"multiplexing" approach, wherein more than one type of antigen is
bound to the microspheres, which enables a multi-antigen assay to
be carried out simultaneously. Use of the microsphere immunoassay
approach also allows the method of the instant invention to be
carried out in a high-throughput manner. High-throughput screening
according to the method of the instant invention can be useful for
large-scale screening, such as screening large population sizes for
epidemiological studies or screening blood banks or organs for
samples contaminated with flaviviruses or WNV.
[0266] The present invention also encompasses fragments or portions
of WNV polypeptides, which may provide more specific diagnostic
reagents than full-length WNV polypeptides and thus may alleviate
such pitfalls as false positive and false negative results.
According to the inventor's own discoveries, a substantially pure
WNV E glycoprotein having an authentic conformation is not only
reactive against antibodies against WNV E glycoprotein, but
strongly, reliably, predictably and consistently cross-reactive
with antibodies against JEV, SLEV and DENV. Thus, in one
embodiment, the WNV E glycoprotein is used in a diagnostic method
for detecting a current or prior infection of a flavivirus, such
as, WNV, DENV, JEV, or SLEV. Prior to the inventor's own research,
WNV E glycoprotein was believed mainly to react specifically with
antibodies to WNV. It was not reliably known to cross-react with
antibodies against other flaviviruses, such as JEV, SLEV, or DENV,
with greater sensitivity than JEV, SLEV, or DENV antigens prepared
by polyethylene glycol precipitation and/or acetone extraction,
which can cause denaturation.
[0267] One skilled in the art will realize that it may also be
advantageous in the preparation of detection reagents to utilize
epitopes from more than one WNV protein or more than one WNV
isolate.
[0268] One of ordinary skill in the art will recognize that
serodiagnosis of a WNV infection currently requires a series of
enzyme-linked immunosorbent assays (ELISA) and viral plaque
reduction neutralization (PRN) tests. It will be further recognized
by one of ordinary skill in the art that currently used diagnostic
methods available in the art require between 3 days and 3 weeks to
obtain a reliable result. In a preferred embodiment, the instant
invention provides a method for presumptive serodiagnosis of a WNV
infection using a novel microsphere immunoassay that requires less
than about 3 hours to obtain a reliable preliminary result.
Further, the method of the instant invention requires as little as
10 microliters of biological sample and thus is not a wasteful
method nor does the method require plentiful reaction reagents
since the reaction volumes can be kept small.
[0269] According to the instant invention, antibodies elicited by
WNV and certain other flaviviruses, such as, JEV, SLEV, and DENV,
are detected in a recombinant WNV E glycoprotein microsphere
immunoassay. "Immunoassay" refers to a method of detection of a
specific antigen or a group of related or similar antigens through
their ability to be recognized and bound by a specific antibody
directed against them. It will be understood that antibody-antigen
interactions are very specific and involves the recognition of and
binding to specific epitopes of the antigen. One of ordinary skill
in the art will appreciate that the bound antibody can be detected
in a variety of different ways. In one example, the bound antibody,
for example an IgM antibody, that is bound to the antigen being
assayed can itself be detected by a second antibody that is capable
of binding the first antibody, such as, for example, an anti-IgM
antibody. The second antibody can be coupled to a detectable label,
such as a fluorescent marker, or an enzyme, such as horse radish
peroxidase.
[0270] According to one embodiment of the instant invention, the
microsphere immunoassay can identify an infection by a flavivirus,
such as, WNV, JEV, SLEV, or DENV, from a biological sample from a
patient having no evidence of said infection in less than about 3
hours. Further, a recent or current infection can be determined
following IgG depletion of and subsequent detection of IgM
antibodies to said flaviviruses. Thus, it will be understood by one
of ordinary skill in the art that the microsphere assay of the
instant invention can used to identify suspect cases of WNV or
flavivirus infection within 5 working hours. Accordingly, the
microsphere immunoassay according to the instant invention would
enable the replacement of eight separate assays, namely, MAC ELISA
and IgG ELISA for WNV, JEV, SLEV, or DENV.
[0271] Further, results from testing for WNV and certain flavivirus
infections would be available within less than one testing day,
instead of 3 days as currently taught by the methods available in
the art. A cost analysis for a test result on the microsphere
immunoassay according to the instant invention, calculated on the
basis of supplies and reagents while excluding the cost of the
recombinant polypeptide of the instant invention and staff time was
$0.24. Conversely, the cost per test result for the MAC ELISA is
$4.84 and the cost per test result for the IgG ELISA is $5.25
(excluding antigen and monoclonal antibodies provided by the CDC
and labor). Thus, the method of the instant invention provides a
much less expensive alternative to current art methods of
detection.
[0272] The microsphere immunoassay of the instant invention
requires less labor and less time to generate 100 test results than
the MAC ELISA or the IgG ELISA. The microsphere immunoassay of the
instant invention could be combined with a subsequent
virus-specific plaque reduction neutralization test used to provide
information on the specific flavivirus of the infection.
[0273] In another embodiment of the subject invention, a
microsphere-based suspension flow cytometric immunoassay is used to
detect antibodies to a WNV envelope glycoprotein and antibodies to
other certain flaviviruses, such SLEV, JEV, and DENV. The
immunoassay uses a low serum volume (about 10 microliters) and
exhibits a broad dynamic range of detection over two logarithms of
antibody concentration with a high signal to noise ratio. Reaction
kinetics are enhanced by incubations with continual shaking at
37.degree. C., which enables the entire assay to be completed
within 2.5 hours, depending upon the number of serum samples
processed. One of ordinary skill in the art will understand that an
optimal dilution of biological sample is about 1:25 to 1:250,
preferably a dilution of 1:100.
[0274] In preferred embodiments of the present invention, an
immunodepletion step is performed prior to testing a biological
sample in order to remove a specific antibody population, such as
an IgM or IgG antibody population. Immunodepletion can be carried
out by contacting the biological sample with an antibody against
the specific antibody subpopulation to be removed to form an
insoluble complex which can be removed by a separation process,
such as centrifugation. Accordingly, the instant invention can be
used to determine recent or ongoing infections, for example,
following IgG removal, or to detect a protective immune response,
for example, following IgM removal.
[0275] The subject invention also provides for diagnostic kits,
such as ELISAs, capable of detecting a WNV infection and infections
by other certain flaviviruses, such as SLEV, JEV and DENV that
include a purified and/or isolated polypeptide or fragment thereof
from WNV, in particular, WNV E glycoprotein. As determined by the
inventor's own research, a substantially purified WNV E
glycoprotein having intact conformational epitopes is reactive to
antibodies against WNV and also strongly, reliably, predictably and
consistently cross-reactive to antibodies against other certain
flaviviruses, such as, DENV, SLEV and JEV. In contrast to the
methods currently available in the art, ELISA antigens are
partially denatured by acetone and contaminated with other proteins
from the host cells from which the antigen was expressed or
produced. Impure antigen provides more non-specific binding and
lower detection signals than the pure antigen used in the assay of
the present invention. One of ordinary skill in the art will
appreciate the mechanics of an ELISA and further details thereof
can be found in numerous scientific literature and protocol books,
such as, for example, The ELISA: Enzyme-Linked Immunosorbant Assay
in Veterinary Research and Diagnosis (Current Topics in Veterinary
Medicine and Animal Science, V. 22), R. C. Wardley (Editor), J. R.
Crowther (Editor).
[0276] The diagnostic kits and methods for detecting antibodies
against WNV and other flaviviruses are also useful for detecting a
protective immune response to WNV or flavivirus infection. Further,
the methods of the instant invention are also useful in monitoring
the course of immunization against WNV and other flaviviruses. In
patients previously inoculated with the vaccines against WNV or
other flaviviruses, the detection means and methods disclosed
herein are also useful for determining if booster inoculations are
appropriate.
[0277] The diagnostic kit, such as an ELISA, can be self-contained,
no laboratory equipment is needed. The advantages of such a kit are
apparent, as it facilitates screening for antibodies to WNV or
other certain flaviviruses at any time and virtually at any place,
including remote geographic areas and those locations lacking a 24
hour testing facility.
[0278] The invention also contemplates that the diagnostic kits,
such as ELISAs, can include a nonstructural protein of WNV,
especially NS5, for the specific detection of WNV without
cross-reactivity to other flaviviruses, including for example SLEV,
JEV, and DENV. In addition, a nonstructural protein of DENV,
especially NS5, is contemplated for the diagnostic kits of the
present invention to be used to specifically detect an infection of
DENV. DENV NS polypeptides of a first particular strain show
specificity for antibodies raised against the same first DENV
strain and are not cross-reactive with antibodies against other
DENV strains. For example, NS of DENV-1 will show specificity to
anti-DENV-1 sera, but will not be reactive with sera raised against
DENV-2, -3, or -4. In addition, like WNV NS proteins, the DENV NS
polypeptides are not substantially cross-reactive with antibodies
against one or more members of the genus Flavivirus, such as, for
example, JEV, SLEV, or WNV. Thus, the DENV NS can be used to
discriminate between a general flavivirus infection and a DENV
infection. In addition, since the antibodies to DENV NS proteins
are not persistent, the DENV NS proteins can be used to detect
recently acquired infections or current infections.
[0279] The diagnostic kits and methods for detecting antibodies
against WNV, DENV and other flaviviruses are also useful for
detecting a protective immune response to WNV or flavivirus
infection. Further, the methods of the instant invention are also
useful in monitoring the course of immunization against WNV and
other flaviviruses. In patients previously inoculated with the
vaccines against WNV or other flaviviruses, the detection means and
methods disclosed herein are also useful for determining if booster
inoculations are appropriate.
[0280] The diagnostic kit can be self-contained, no laboratory
equipment is needed, such as with ELISAs. The advantages of such a
kit are apparent, as it facilitates screening for antibodies to WNV
or other certain flaviviruses at any time and virtually at any
place, including remote geographic areas and those locations
lacking a 24 hour testing facility.
[0281] In a further embodiment, the diagnostic methods of the
instant invention can be carried out using a lateral flow
immunoassay. A lateral flow immunoassay (immunochromatographic
test) comprising a simple lateral flow device can be used to
rapidly detect antibodies present in a biological sample against a
flavivirus antigen, such as WNV E glycoprotein, WNV NS5, or DENV
NS5. Such a device consists of a membrane strip, with the membrane
typically of nitrocellulose, cellulose acetate or nylon, through
which the serum (i.e., biological) sample, buffer, and detection
reagent (antigen-coated microparticles) flow by capillary action.
The membrane strip further comprises a reagent application pad onto
which a biological sample and an antigen-coupled microparticle can
be applied. The microparticles can be of known form, size or
constitution deemed useful to one of ordinary skill in the art,
such as polystyrene, fluorescently-labeled polystryrene, magnetic,
latex, or any such polymer.
[0282] The membrane strip can be further divided into "zones,"
which are specific regions of the membrane strip wherein an
immunological reaction takes place between an antigen-coated
microparticle and an antibody. In a preferred embodiment, the test
zones are coated with an anti-immunoglobulin antibody population,
such as, anti-human IgG or anti-human IgM antibodies, which can be
located at different positions along the test membrane. According
to the present embodiment, the membrane strip also comprises a
positive control zone containing an antibody against the antigen of
interest, such as a monoclonal/polyclonal antibody reactive against
WNV E glycoprotein antigen, WNV NS5 antigen, or DENV NS5 antigen.
The invention, however, is not meant to be limited to the detection
of antibodies against WNV E and NS5 or DENV NS5, but rather
antibodies to any flavivirus antigen, especially a flavivirus E
glycoprotein or NS5 antigen, could be detected using the membrane
strip method of the invention, such as antibodies against JEV and
SLEV antigens.
[0283] In another embodiment, an antigen of interest, such as a
flavivirus antigen, especially WNV E glycoprotein, WNV NS5, or DENV
NS5, are adsorbed or alternately dried to the surface of the
membrane strip. The membrane strip can be of any suitable material
known in the art, such as, for example nitrocellulose, cellulose
acetate or nylon. Preferably, the antigens are adsorbed or dried to
the surface of the membrane strip in separate zones to enable
separate detection of antibody types, such as IgG or IgM
antibodies, that will bind to the antigen during the course of the
membrane strip assay. In this embodiment, a biological sample, such
as a bodily fluid, blood, serum, plasma, saliva, tears, feces,
semen, mucous, tissue, tissue homogenate, cellular extract, or
spinal fluid, containing anti-flavivirus antigen antibodies, such
as IgG anti-NS5, IgM anti-NS5, IgG anti-WNV E, or IgM anti-WNV E,
would be applied to the membrane strip at one end to allow the
sample to move through the membrane. Antibodies contained in the
biological sample against the flavivirus antigens of the membrane
strip, such as IgG or IgM antibodies, will recognize, interact
with, and bind to said antigens. One of skill in the art will
appreciate that certain biological samples, such as a bodily fluid,
blood, serum, plasma, saliva, tears, feces, semen, mucous, tissue,
tissue homogenate, cellular extract, or spinal fluid, may require
certain preparatory steps prior to applying the membrane strip to
enable the sample to flow through the membrane strip. Such
pretreatment includes, but is not limited to, dilution or removal
of particulate matter. Once the anti-flavivirus antibodies present
in the sample have bound to the flavivirus antigens of the membrane
strip, a detection reagent comprising secondary antibody-coupled
microparticles, such as anti-human IgG or IgM antibody-coupled
microparticles, are applied to the membrane strip to detect the
anti-flavivirus antibodies already bound to the flavivirus
antigens.
[0284] One of ordinary skill in the art will recognize that the key
components of one embodiment of a lateral flow device consist of:
[0285] 1) a membrane strip consisting of modified nitrocellulose,
cellulose acetate or nylon to which the detection reagent,
consisting of antigen-coupled microparticles and a biological
sample containing antibodies that recognize the antigen is applied;
[0286] 2) a test zone of anti-immunoglobulin capture antibodies,
immobilized at a specific zone, or location on the membrane,
wherein capture of the detection reagent at this zone gives a
colored pattern and indicates the presence of antibodies of
interest; and [0287] 3) a control zone of antibodies specific for
the antigen under study, immobilized in a second zone on the
membrane, wherein capture of the detection reagent at this zone
gives a visual pattern and shows that the test was successfully
completed.
[0288] The detection reagent, which consists of antigen-coupled
microparticles, such as colored latex or metal beads, can be
detected visually. The detection reagent is applied with special
releasing agents and dried near the bottom of the membrane strip.
The microparticles can be applied directly to the membrane, or they
can be applied to an absorbent pad that is in contact with the
membrane. When a biological sample is introduced to the
antigen-coupled microparticles, anti-antigen antibodies, such as,
for example anti-antigen IgG or IgM antibodies, present in the
biological sample bind to the antigen-coupled microparticles. The
microparticles are then carried through the membrane strip by
capillary action and come into contact with the secondary
antibodies coupled at each of the zones along the strip, wherein
the secondary antibodies recognize the specific types of
anti-antigen antibodies bound to the antigen-coupled microparticle,
such as anti-human IgG or IgM antibodies. It will be appreciated
that the membrane strip can be provided with absorbent pads located
at the top of the membrane to act as a reservoirs of buffer or
fluid so that the biological sample/antigen-coupled microparticles
flow continuously through the membrane coming into contact with
each zone of the membrane strip.
[0289] One of ordinary skill in the art will understand that all of
the components and reagents that go into a lateral flow device must
be chosen with care and matched during research and development of
the test to ensure adequate sensitivity, stability and reliability
of the finished test device. When properly constructed, these tests
are sturdy and reliable, but they are delicately balanced, and even
minor changes in materials, reagent processing or raw material
specifications can cause significant loss in test performance. A
discussion of lateral flow methodology may be found in L. B. Bangs,
Manual for The Latex Course, Bangs Laboratories, Inc., Carmel, Ind.
(1996).
[0290] In a preferred embodiment of the instant invention and
referring to FIG. 17, the positive control zone (1702) of the
membrane strip (1701) comprises anti-WNV E antigen antibodies,
which can be monoclonal or polyclonal. A detection reagent,
comprising a substantially pure antigen, such as WNV E
glycoprotein, WNV NS5, or DENV NS5, each having an authentic
conformation, are coupled to microparticles and applied to the
reagent application pad (1705), along with a biological specimen,
and a buffer. The microparticles can be colored polystyrene beads,
fluorescently-labeled polystyrene beads, or metal particles, or any
appropriate type known to one of skill in the art. In the case
where the coupled antigen is WNV E glycoprotein, the coupled
antigen is reactive with IgG and/or IgM antibodies against WNV and
strongly cross-reactive with IgG and/or IgM antibodies against a
flavivirus, especially, JEV, SLEV, or DENV, that may be present in
the biological sample. In the case where the coupled antigen is WNV
NS5 or DENV NS5, the coupled antigen is specifically reactive with
IgG and/or IgM antibodies against WNV or DENV, respectively, but
not cross-reactive with IgG and/or IgM antibodies against another
flavivirus that may be present in the biological sample. Further,
DENV NS5 is specific for antibodies against the same DENV strain
from which it is isolated and not cross-reactive with antibodies to
other DENV strains. For example, DENV-1 NS5 is specific for
antibodies against DENV-1, but not cross-reactive with antibodies
against DENV-2, -3, or -4. It will be appreciated that the DENV NS5
antigens thus can be used to discriminate the four different known
DENV strains.
[0291] The detection reagent (further bound to IgG and/or IgM
antibodies, if present in the biological sample) migrate up through
the membrane strip by capillary action and successively come into
contact with different antibody-containing zones. For example, the
detection reagent first comes into contact with zone 1704, which
can be coated with anti-IgM antibodies (such as, goat anti-human
IgM antibodies). The detection reagent will bind to zone 1704
through the binding interaction between the zone 1704 anti-IgM
antibodies and IgM antibodies of the detection reagent, if present.
The detection reagent will also come into contact with zone 1703,
which can be coated with anti-IgG antibodies (such as goat
anti-human IgG antibodies). The detection reagent will bind to zone
1703 through the binding interaction between the zone 1703 anti-IgG
antibodies and IgG antibodies of the detection reagent, if present.
Further, the detection reagent will come into contact with and bind
to zone 1702, a control zone coated with antibodies specific for
the antigen of the detection reagent. The results of the flow
immunoassay can be determined visually since the microparticles are
held at zones 1702, 1703 and 1704 through antibody-antibody or
antibody-antigen interactions.
[0292] One of ordinary skill in the art will appreciate that the
instant invention encompasses any suitable configuration of the
membrane strip test (immunochromatographic test). For example, the
antigen of interest, such as a flavivirus antigen (e.g. WNV NS5,
WNV E, or DENV NS5), can be coupled either to the microparticle or
directly to the membrane strip. If the antigen of interest is
coupled to the microparticle, detection of any anti-antigen
antibodies present in a biological sample can be conducted by
coupling a secondary antibody, such as anti-human IgG or IgM
antibodies, to a specific location or zone on the membrane strip.
In this case, as the antigen-coated microparticles are allowed
first to interact with a biological sample containing anti-antigen
antibodies such that the anti-antigen antibodies bind to the
antigens of the coated microparticles. Next, the microparticles
migrate through the membrane strip. The microparticles will be
captured at the zones of the strip containing the secondary
antibodies vis-a-vis binding interactions between the secondary
antibody (e.g., anti-human IgG or IgM antibody) and the
anti-antigen antibody bound from the sample bound to the
antigen-coupled microparticle. The captured microparticles can be
directly visualized by inspection thereby confirming either the
presence or absence of anti-antigen antibodies in the biological
sample. One of ordinary skill in the art will also appreciate that
the antigens of interest can also be adsorbed or dried onto the
surface of the membrane strip. In this case, the secondary
antibodies would be coupled to the microparticles.
[0293] In various embodiments described herein, the flavivirus
antigens of the instant invention, especially WNV E glycoprotein,
WNV NS5, and DENV NS5, are covalently coupled to a microparticle.
Microparticles can include, but are not limited to, polystyrene
microparticles, colored or fluorescently labeled polystyrene
microparticles, latex and colored latex microparticles,
paramagnetic microparticles, metal particles, such as gold, glass
microparticles, and plastic microparticles. One of ordinary skill
in the art will understand that "microparticles" one in the same as
"microspheres" or "uniform latex particles." The inventor has
further discovered that the antigens of the instant invention,
especially WNV E glycoprotein, WNV NS5 and DENV NS5, are highly
stable when coupled to microparticles, especially polystyrene
microparticles. The data of FIG. 9 and a plot of 1/T90 (time to 90%
potency of reagent) against 1/T (Kelvin) gives an estimated T90 of
three months. Further, the inventor has discovered that in
practice, the stability of the antigen-coupled microparticles is
greater than three months.
[0294] In various embodiments of the instant invention, WNV E
glycoprotein-coupled or NS5-coupled microspheres are used in a
microsphere immunoassay to detect antibodies against a flavivirus,
especially WNV, JEV, SLEV, or DENV, in a biological sample. The WNV
E glycoprotein is substantially pure and of native conformation,
which allows for strong cross-reactivity of the WNV E glycoprotein
among flaviviruses, especially WNV, JEV, SLEV, and DENV. Any kind
of microsphere immunoassay known in the art is within the scope of
the present invention, such as, but not limited to, agglutination
assays, slide tests, lateral flow tests (previously described), or
fluorescence-based assays, such as flow cytometric analyses and
Luminex-based immunoassays (Austin, Tex.). A discussion of
different immunoassays known in the art may be found in L. B.
Bangs, Manual for The Latex Course, Bangs Laboratories, Inc.,
Carmel, Ind. (1996).
[0295] One of ordinary skill in the art will understand that
microsphere-based immunoassays can be both qualitative and
quantitative and are usually based upon a very specific interaction
of antigen (Ag) with antibody (Ab). Sub-micron sized polystyrene
microspheres are used as a solid support. The microspheres act to
magnify or amplify the Ag-Ab reaction which takes place when they
are mixed with a sample containing the opposite reactant.
[0296] The Luminex microsphere immunoassay allows the performance
of multiplex analysis to detect antibodies against multiple
antigens in a single tube. The antigens of the instant invention,
especially the WNV E glycoprotein, WNV NS5, and DENV NS5 described
above, can be covalently linked to microsphere beads containing
different fluorochromes. During the assay readout, the first laser
excites the intrinsic fluorochrome in the antigen-bearing
microspheres, allowing identification of each bead in the assay
mixture. The second laser excites the fluorochrome tag of the
reporter molecule, measuring the level of antibodies that bind to
the specific antigen. The multiplex assay should allow simultaneous
primary and confirmatory diagnosis of a flavivirus infection,
especially WNV, DENV, and other flaviviruses such as JEV and SLEV.
About 100 different types of fluorescent polystyrene microspheres
are commercially available (Luminex, Austin, Tex.). In principle,
one can perform a multiplex analysis of up to 100 analytes. In
practice, most multiplex immunoassays have included up to 20
analytes measured at one time. This technology should be useful for
simultaneous detection of multiple pathogens in clinical
laboratories.
[0297] Upon virus infection, the immune system first develops
conformational epitopes. Antibodies against linear epitopes are
produced later, as virus particles are broken down and presented in
the context of the T cell receptors and major histocompatibility
complex molecules on the surfaces of infected cells. Therefore,
epitope mapping of various parts of structural and NS proteins is a
good strategy by which to identify virus-type specific peptides.
Synthetic peptides representative of linear, virus-type specific
epitopes may be used as antigens for specific diagnosis of the
particular virus. It should be borne in mind that the use of
synthetic peptides as antigens may result in high background in
immunoassays, depending upon the length of the peptide and the
ionic strength of assay buffers. However, in combination with
antigens that have native conformation (e.g., recombinant NS5),
such virus-type specific peptide could add another layer of
specificity to the current serological diagnosis.
[0298] Agglutination tests are portable, rapid, efficient, and
useful under the most primitive conditions, e.g., when no
laboratory equipment is available, such as a flow cytometer or a
Luminex machine (Austin, Tex.). Diagnosis can occur quickly and
simply (2 minutes from sample preparation). Diagnosis and treatment
can commence promptly, before the patient is transferred or
discharged. Agglutination tests can include liquid reagents made
with plain, white microspheres. Tests can be run on either reusable
glass slides or on disposable plastic or coated paper cards. These
tests often require to operator to constantly mix the sample for
several minutes to achieve agglutination, which is visually
detectable following the formation of particulate clumps.
[0299] Slide tests, such as Roche's OnTrak.TM. (F. Hoffmann-La
Roche Ltd, Basel, Switzerland) device, are more recent refinements
of agglutination tests. In the slide test, the sample and reagent
with coated microspheres are mixed and guided into a "track" or
capillary. As the reactants move down the track by capillary
action, they mix. Agglutination is detected with transmitted light
one the sample travels towards the end of the slide. The test is
mainly operator-independent, and therefore is more amendable to
automation. The microspheres used can also be dyed or fluorescent
to provide different contrasting colors to improve detection.
[0300] One of ordinary skill in the art will understand that slide
tests and/or lateral flow immunoassays are synonymous with
immunochromatographic tests. More discussion on
immunochromatographic tests may be found in: L. Kittigul and K.
Suankeow. Eur. J. Clin. Microbiol. Infect. Dis. 21:224-226 (2002);
Tsuda, S., et al. Plant Disease 76, 466-469 (1992); Brown, W. E.
I., Safford, S. E. & Clemens, J. M. Solid-Phase Analytical
Device and Method for Using Same, U.S. Pat. No. 5,160,701, Nov. 3,
1992; Cole, F. X., MacDonnell, P. C. & Cicia, N. J., Porous
Strip Form Assay Device Method, U.S. Pat. No. 5,141,850, Aug. 25,
1992; Fan, E., et al. Immunochromatographic Assay and Method of
Using Same, WO 91/12336, Aug. 22, 1991; Imrich, M. R., Zeis, J. K.,
Miller, S. P. & Pronovost, A. D. Lateral flow medical
diagnostic assay device U.S. Pat. No. 5,415,994, May 16, 1995; and
May, K., Prior, M. E. & Richards, I. Immunoassays and Devices
Therefore, International Patent Number: WO 88/08534, Nov. 3,
1988.
[0301] Agglutination can be quantitated using instruments such as
spectrophotometers and nephelometers to measure transmitted,
absorbed, or scattered light, as a result of protein precipitation
of the agglutination process.
[0302] With Luminex-based immunoassay technology, molecular
reactions take place on the surface of microscopic beads called
microspheres. For each reaction, thousands of molecules are
attached to the surface of internally color-coded microspheres. The
assigned color-code identifies the reaction throughout the
test.
[0303] The magnitude of the biomolecular reaction is measured using
a second molecule called a reporter. The reporter molecule signals
the extent of the reaction by attaching to the molecules on the
microspheres. Because the reporter's signal is also a color, there
are two sources of color, the color-code inside the microsphere and
the reporter color on the surface of the microsphere.
[0304] To perform a test, the color-coded microspheres, reporter
molecules, and sample are combined. This mixture is then injected
into an instrument that uses microfluidics to align the
microspheres in single file where lasers illuminate the colors
inside and on the surface of each microsphere. Next, advanced
optics capture the color signals. Finally, digital signal
processing translates the signals into real-time, quantitative data
for each reaction. Further descriptions of Luminex-based
immunoassays may be found in U.S. Pat. Nos. 6,449,562, 6,411,904,
6,268,222, 6,139,800, 5,981,180 and 5,736,330.
[0305] In another embodiment, the present invention relates to
monoclonal antibodies specific for flavivirus antigens, especially
WNV NS5 and E glycoprotein and DENV NS5, methods for preparing the
monoclonal antibodies, hybridoma cells lines producing the
monoclonal antibodies, diagnostic methods utilizing the monoclonal
antibodies for detecting a flavivirus infection and antigens
thereof in biological samples suspected of having a flavivirus
infection, methods and compositions for administering the
monoclonal antibodies for imparting passive immunity in a patient
or animal, methods and compositions for administering to patients
or animals the monoclonal antibodies as a means for treating
infections (e.g. monoclonal antibodies as neutralizing antibodies),
kits comprising the monoclonal antibodies and optionally a purified
flavivirus antigen for diagnostic purposes, and methods for
detecting a flavivirus in a tissue in vivo to facilitate studies of
the pathology of WNV.
[0306] In one embodiment, the invention provides for monoclonal
antibodies specific for WNV NS5, which can be referred to as
"anti-WNV NS5 monoclonal antibodies." It has been the discovery of
the present inventors that such monoclonal antibodies are specific
for WNV NS5 and are not detectably or substantially cross-reactive
with NS5 antigens from other non-WNV flaviviruses, especially,
DENV, JEV, and SLEV. (We have not shown this in any experiment to
date. All we have done is select antibodies from clones that react
with the West Nile NS5.) Thus, in accordance with the present
invention, one can consistently, reliably, and accurately determine
using diagnostic assays along with the anti-WNV NS5 monoclonal
antibodies whether there is a recent of ongoing WNV infection in a
biological sample.
[0307] In another embodiment, the invention provides for monoclonal
antibodies specific for DENV NS5, which can be referred to as
"anti-DENV NS5 monoclonal antibodies." It has been a discovery of
the present inventors that such monoclonal antibodies are specific
for DENV NS5 and are not detectably or substantially cross-reactive
with NS5 antigens from other non-DENV flaviviruses, especially,
WNV, JEV, and SLEV. Moreover, the anti-DENV monoclonal antibodies
of the present invention can be "type-specific." Such type-specific
monoclonal antibodies can distinguish between the four-different
serotypes of DENV, namely, DENV-1, -2, -3, and -4. There is no
detectable or substantial cross-reactivity of a particular
anti-DENV NS5 monoclonal antibodies with a first type of DENV NS5
as against a second and different type of DENV NS5. Thus, for
example, an anti-DENV-1 NS5 monoclonal antibody of the invention is
specific for DENV-1 NS5, but not detectably or substantially
cross-reactive with DENV-2 NS5, DENV-3 NS5, or DENV-4 NS5.
Likewise, an anti-DENV-2 NS5 monoclonal antibody of the invention
is specific for DENV-2 NS5, but not detectably or substantially
cross-reactive with DENV-1 NS5, DENV-3 NS5, or DENV-4 NS5.
Similarly, an anti-DENV-3 NS5 monoclonal antibody of the invention
is specific for DENV-3 NS5, but not detectably or substantially
cross-reactive with DENV-1 NS5, DENV-2 NS5, or DENV-4 NS5. As such,
an anti-DENV-4 NS5 monoclonal antibody of the invention is specific
for DENV-4 NS5, but not detectably or substantially cross-reactive
with DENV-1 NS5, DENV-2 NS5, or DENV-3 NS5.
[0308] Monoclonal antibodies may be made by any convention method.
For example, monoclonal antibodies of the invention can be prepared
using the hybridoma method first described by Kohler et al.,
Nature, 256:495 (1975). Any other well-known,
subsequently-developed methods are contemplated.
[0309] For the purposes of the present invention the terms
"monoclonal antibody" (or "MAb") and "hybridoma" (alternatively,
"hybridoma cell line" or "hybridoma clone") are intended to have
their conventional meanings as would be understood by one of
ordinary skill in the art. A monoclonal antibody is known as a
single species of immunoglobulin that recognize only one chemical
structure. In other words, a monoclonal antibody is directed
against (or "specific for" or any other similar conventional
language) a single epitope of the antigenic substance used to raise
the antibody. A hybridoma is a cell line resulting from the fusion
of a specific antibody-producing spleen cell (lymphocyte) with a
myeloma cell, which has the growth characteristics of the myeloma
component and the antibody-secreting characteristics of the
lymphocyte and thus will proliferate and express its monoclonal
antibody product.
[0310] Generally, to prepare a monoclonal antibody, a mouse or
other appropriate host animal is immunized with an antigen of
interest to elicit lymphocytes that produce or are capable of
producing antibodies against the antigen of interest, such as, for
example WNV or DENV NS5. Alternatively, lymphocytes may be
introduced to the antigen of interest in vitro. Lymphocytes then
are fused with myeloma cells using a suitable fusing agent, such as
polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal
Antibodies: Principles and Practice, pp. 59-103 (Academic Press,
1986)).
[0311] The hybridoma cells thus prepared are seeded and grown in a
suitable culture medium that preferably contains one or more
substances that inhibit the growth or survival of the unfused,
parental myeloma cells. For example, if the parental myeloma cells
lack the enzyme hypoxanthine guanine phosphoribosyl transferase
(HGPRT or HPRT), the culture medium for the hybridomas typically
will include hypoxanthine, aminopterin, and thymidine (HAT medium),
which substances prevent the growth of HGPRT-deficient cells.
[0312] It is preferable to use myeloma cells that fuse efficiently,
support stable high-level production of antibody by the selected
antibody-producing cells, and are sensitive to a medium such as HAT
medium. Among these myeloma cell lines are murine myeloma lines,
such as those derived from MOPC-21 and MPC-11 mouse tumors
available from the Salk Institute Cell Distribution Center, San
Diego, Calif. USA, and SP2/0 or X63-Ag8-653 cells available from
the American Type Culture Collection, Rockville, Md. USA. Human
myeloma and mouse-human heteromyeloma cell lines also have been
described for the production of human monoclonal antibodies
(Kozbor, J. Immunol. 133:3001 (1984); Brodeur et al., Monoclonal
Antibody Production Techniques and Applications, pp. 51-63 (Marcel
Dekker, Inc., New York, 1987)). The mouse myeloma cell line NSO may
also be used (European Collection of Cell Cultures, Salisbury,
Wiltshire UK). Any of the above myeloma lines are contemplated, as
well as any additional suitable myeloma line available to one of
ordinary skill in the art.
[0313] The culture medium in which hybridoma cells are grown can be
assayed for production of monoclonal antibodies directed against
the antigen. The binding specificity, with respect to the antigen
of interest, of monoclonal antibodies produced by hybridoma cells
may be determined by any suitable method such as, for example,
immunoprecipitation assays, in vitro binding assays,
radioimmunoassays (RIA) or enzyme-linked immunoabsorbent assay
(ELISA), standard competitive assays, sandwich assays,
immunofluorescence assays, chemiluminescence assays, and lateral
flow tests, agglutination tests, microsphere immunoassays (as
herein discussed in further detail). The assays above can be
carried out in an automated or semi-automated fashion using any
conventional means, such as those further described in U.S. Pat.
No. 6,649,743, which is hereby incorporated in its entirety by
reference. There, available types of automated assay apparatuses
are described which can undertake rapid format assays on a number
of samples contemporaneously. Other automated devices useful for
the present invention include those described by U.S. Pat. Nos.
4,632,901, 4,366,241, 4,906,349, 4,918,025, 5,051,237, 5,138,868,
5,141,871, and 5,147,609.
[0314] The invention further contemplates the use of biosensors or
optical immunosensor systems for the detection of the monoclonal
antibodies of the present invention and their complexes formed with
the flavivirus antigens of the invention, especially the WNV and
DENV NS5 antigens. In general an optical biosensor is a device
which uses optical principles quantitatively to convert chemical or
biochemical concentrations or activities of interest into
electrical signals. These systems can be grouped into four major
categories: reflection techniques; surface plasmon resonance (such
as BIACORE's SPR Biosensor Technology, BIACORE AB, Uppsala,
Sweden), fibre optic techniques and integrated optic devices.
Reflection techniques include ellipsometry, multiple integral
reflection spectroscopy, and fluorescent capillary fill devices.
Fibre-optic techniques include evanescent field fluorescence,
optical fibre capillary tube, and fibre optic fluorescence sensors.
Integrated optic devices include planer evanescent field
fluorescence, input grading coupler immunosensor, Mach-Zehnder
interferometer, Hartman interferometer and difference
interferometer sensors. These examples of optical immunosensors are
described in general in a review article by G. A. Robins (Advances
in Biosensors), Vol. 1, pp. 229-256, 1991. More specific
description of these devices are found for example in U.S. Pat.
Nos. 4,810,658; 4,978,503; 5,186,897; R. A. Brady et al. (Phil.
Trans. R. Soc. Land. B 316, 143-160, 1987) and G. A. Robinson et
al. (in Sensors and Actuators, Elsevier, 1992).
[0315] The BIACORE SPR Biosensor Technology ("the BIACORE system"),
more particularly, does not require a particular molecular
property, such as fluorescence or enzymatic activity, to analyze
interacting molecules, such as, in the present invention, the
interaction between the WNV or DENV NS5 antigens and the their
respective monoclonal antibodies of the invention. Instead, it will
be appreciated that the BIACORE system monitors the binding of
macromolecules in real time using plasmon resonance detection by
distinguishing between complexes and non-complexes vis-a-vis the
accumulation of mass that is associated with forming a binding
complex, such as that between WNV NS5 and its cognate monoclonal
antibody. Further information can be found at BIACORE AB's web site
at www.BIACORE.com or in Nieba et al. "Competitive BIACORE for
measuring true affinities: Large difference from values determined
from binding kinetics," Analytical Biochemistry 234, 155-165
(1996). One of skill in the art will appreciate the steps required
to carry out BIACORE system assays through the available literature
and company technical brochures, all of which are incorporated
herein by reference.
[0316] After hybridoma cells are identified that produce antibodies
of the desired specificity, affinity, and/or activity, the clones
can be subcloned by limiting dilution procedures and grown by
standard methods (Goding, Monoclonal Antibodies: Principles and
Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture
media for this purpose include, for example, D-MEM or RPMI-1640
medium. In addition, the hybridoma cells may be grown in vivo as
ascites tumors in an animal. Procedures for producing ascites
fluids are well-known in the art.
[0317] The monoclonal antibodies secreted by the hybridomas clones
are suitably separated from the culture medium, ascites fluid, or
serum by conventional immunoglobulin purification procedures such
as, for example, protein A-Sepharose, hydroxylapatite
chromatography, gel electrophoresis, dialysis, or affinity
chromatography.
[0318] DNA encoding the monoclonal antibodies can be readily
isolated and sequenced using conventional procedures (Innis M. et
al. In PCR Protocols. A Guide to Methods and Applications,
Academic, San Diego, Calif. (1990), Sanger, F. S, et al. Proc. Nat.
Acad. Sci. 74:5463-5467 (1977)). The hybridoma cells serve as a
source of such DNA. Once isolated, the DNA may be placed into
expression vectors, which are then transfected into host cells such
as, for example, E. coli cells, simian COS cells, Chinese hamster
ovary (CHO) cells, or myeloma cells that do not otherwise produce
immunoglobulin protein, to obtain the synthesis of monoclonal
antibodies in the recombinant host cells.
[0319] Antibodies or antibody fragments can be isolated from
antibody phage libraries generated using the techniques described
in McCafferty, et al., Nature 348:552-554 (1990). Clackson, et al.,
Nature 352:624-628 (1991) and Marks, et al., J. Mol. Biol.
222:581-597 (1991) describe the isolation of murine and human
antibodies, respectively, using phage libraries. Subsequent
publications describe the production of high affinity (nM range)
human antibodies by chain shuffling (Marks, et al., Bio/Technology
10:779-783 (1992)), as well as combinatorial infection and in vivo
recombination as a strategy for constructing very large phage
libraries (Waterhouse, et al., Nuc. Acids. Res. 21:2265-2266
(1993)). Thus, these techniques can be used in accordance with the
present invention as viable alternatives to traditional monoclonal
antibody hybridoma techniques for isolation of monoclonal
antibodies.
[0320] In another aspect of the invention, hybridomas can be
alternatively using well-established electrical fusion methods
rather than chemical fusion methods, as outlined above. Instead of
fusion, one can also transform a B-cell to make it immortal using,
for example, an Epstein Barr Virus, or a transforming gene. (See,
e.g., "Continuously Proliferating Human Cell Lines Synthesizing
Antibody of Predetermined Specificity," Zurawaki, V. R. et al, in
Monoclonal Antibodies, ed. by Kennett R. H. et al, Plenum Press,
N.Y. 1980, pp 19-33.) Anti-NS5 MAbs can be raised by immunizing
rodents (e.g. mice, rats, hamsters and guinea pigs) with either
native WNV or DENV NS5 or recombinant WNV or DENV NS5 or its
fragments expressed by either eukaryotic or prokaryotic systems.
Other animals can be used for immunization, e.g. non-human
primates, transgenic mice expressing human immunoglobulins and
severe combined immunodeficient (SCID) mice transplanted with human
B lymphocytes. Hybridomas can be generated by conventional
procedures by fusing B lymphocytes from the immunized animals with
myeloma cells (e.g. Sp2/0 and NSO), as described earlier (Kohler G
et al., Nature 256: 495-7 (1975)). In addition, anti-NS5 antibodies
can be generated by screening of recombinant single-chain Fv or Fab
libraries from human B lymphocytes in phage-display systems. The
specificity of the MAbs to WNV or DENV NS5 can be tested by enzyme
linked immunosorbent assay (ELISA), as shown in FIG. 1, Western
immunoblotting, or other immunochemical techniques mentioned in
more detail previously. The hybridomas in the positive wells are
cloned by limiting dilution, as described above. The antibodies are
purified for characterization for specificity to WNV or DENV NS5 by
the assays described above.
[0321] In one aspect of the invention, the BIACORE system can be
used as an initial screen of the supernatants of individual
hybridoma clones to determine which hybridomas express monoclonal
antibodies with the highest specificity for the antigen of
interest, such as, to the WNV or DENV NS5 antigen. Once positive
hybridoma clones are identified in this manner, further assays can
be used to verify the identification of the positive hybridoma
clones, such as, by using a microsphere immunoassay. Aspects of the
microsphere immunoassay have been described previously herein.
Here, the microsphere immunoassay can include the steps of
contacting the supernatant of a suspected positive hybridoma clone
expressing a monoclonal antibody candidate that has an apparent (as
determined by BIACORE system) high affinity for the antigen of
interest, such as, for example, WNV or DENV NS5 antigen with a
suspension of microspheres coupled to the antigen of interest. For
example, the supernatant of a candidate hybridoma expressing
monoclonal antibodies against WNV NS5 can be validated by carrying
out the microsphere immunoassay of the invention with a suspension
of microspheres that are coupled to purified WNV NS5 antigen. FIGS.
51a and 51b show the results of such a microsphere immunoassay.
[0322] In FIGS. 51a and b, a series of microtiter plates were
screened wherein each well of the microtiter plates contained a
distinct hybridoma clone. The BIACORE system was used to initially
screen the supernatants of each of the hybridomas to identify
candidate clones expressing monoclonal antibodies with an apparent
high affinity for the WNV NS5 antigen. The top 37 hybridomas were
selected for further validation by the microsphere immunoassay. The
microsphere immunoassay identified 11 hybridomas whose monoclonal
antibodies had the greatest affinities for the tested antigen
(indicated in bold).
[0323] In another aspect of the invention, a method is provided for
detecting the presence of a specific flavivirus infection, such as,
for example, a WNV or DENV infection, in a biological sample that
is suspected of having an infection. In this method, an isolated
monoclonal antibody raised against a specific flavivirus antigen,
especially WNV or DENV NS5, is contacted with the biological
sample.
[0324] It will be appreciated that the present method can be
carried out by the operator on one biological sample at a time or
the method can be carried out on a plurality of biological samples
contemporaneously (e.g. in a "high-throughput manner"). One of
skill in the art will appreciate the variety of known technologies
and methods in the art for carrying out assays such as the present
method in a high-throughput manner. Such technologies include
multi-well reaction plates ("microtiter plates") and any of the
automated systems described herein above. It will also be
appreciated that the biological sample can be any known to the
skilled person and include, for example and as mentioned above, any
bodily fluid, including blood, serum, plasma, saliva, tears, feces,
semen, mucous, tissue, tissue homogenate, cellular extract, and
spinal fluid. The samples can be treated in any necessary manner
prior to carrying out the method of the invention, such as lysing
flavivirus-infected cells. The biological sample can also be from
any suitable source, especially those biological specimens that can
either be infected with a flavivirus, especially WNV, or can be a
carrier of the virus. This includes, for example, but not limited
thereto, humans, horses, and birds.
[0325] As previously mentioned, the present inventors have
discovered, it is believed for the first time that monoclonal
antibodies against WNV NS5 are specific for WNV NS5 and not
detectably cross-reactive with NS5 antigens from flaviviruses other
than WNV. In other words, the anti-WNV NS5 monoclonal antibodies do
not substantially form complexes or interact with the NS5 antigens
of other flaviviruses, and as such, are useful for specifically
detecting WNV infections. Likewise, the monoclonal antibodies
against DENV NS5 are specific for DENV NS5 and not detectably
cross-reactive with NS5 antigens from flaviviruses other than DENV.
In addition and as pointed to above, the anti-DENV NS5 monoclonal
antibodies are "type-specific" and thus, can distinguish between
the four types of DENV, namely DENV-1, -2, -3, and -4.
[0326] The contacting of the monoclonal antibody with the
biological system is done under conditions that are sufficient to
form a complex between the monoclonal antibody and the target
antigen present in the biological system. For example, should an
anti-WNV NS5 monoclonal antibody be contacted with a biological
sample suspected of containing a WNV infection, the WNV NS5 antigen
present in the sample will form a complex with the monoclonal
antibody under appropriate conditions. The skilled artisan will
certainly appreciate the proper and suitable conditions necessary
or preferable to form such complexes, including time, buffer
strength and components, and temperature. For example, these
incubations can be carried out at a pH of 7.00 to 7.50, in a buffer
system such as Phosphate Buffered Saline or TRIS-CL at 0.15M salt,
with 0.05% Tween 20 detergent. The incubations may take place at
temperatures between 4.degree. C. and 37.degree. C., with the
incubation time in inverse proportion to the temperature. For
example, if 4.degree. C. is used, overnight incubations are ideal.
If 37.degree. C. is used, a 30 minute incubation is appropriate, as
longer incubations at elevated temperature may result in
denaturation of the antigen. These assays can also be performed
with an additional incubation in the presence of 7M urea for 10
minutes. When the amount of antibody measured in the presence of 7M
urea is divided by the amount of antibody present without a urea
exposure, and multiplied by 100, you have the avidity percentage
index. Antibodies generated early in acute infection are of low
avidity, whereas antibodies in the convalescent time frame, or
formed during a secondary flavivirus infection, are of higher
avidity. For further information as to avidity, see Fick de Souza,
V A, Fernandes S, Araujo E S, Tateno A F, Oliviera O M, Oliviera R
R and Pannuti C S. Use of an Immunoglobulin G Avidity Test to
Discriminate between Primary and Secondary Dengue Virus Infections.
2004. J. Clin Microbiol. 42:1782-1784.
[0327] Once the complexes are formed between the monoclonal
antibody and the target antigen, such as the WNV NS5 antigen, an
assay or detection scheme can be utilized to test for the presence
of the complex. The positive detection of an antibody-antigen
complex indicates that the tested biological sample was infected
with the specific flavivirus screened, such as WNV or DENV. In the
case of a DENV infection, the specific type can be identified since
the anti-DENV NS5 monoclonal antibodies are type-specific.
[0328] It will be appreciated that the monoclonal antibody can be
labeled to enable the detection of the antibody-antigen complex.
Antibody labeling and procedures for coupling labels to antibodies
is well-known in the art. Any such known technique is contemplated
including labeling and detection by way of a fluorophore (detection
of light emissions, e.g. fluorescence), radioactivity (detection of
a radioactive isotope emitting, e.g., gamma or beta waves), a
biotin-labeled antibody using streptavidin conjugated to a
colorometric marker, or an enzyme, such as horseradish peroxidase,
phosphatase, oxidase, luciferase, peptidase, protease, or
glycosidase along with a colormetric or other detectable substrate.
Thus, it will be appreciated that the label coupled to the
monoclonal antibody can be a colormetric material, a fluorescent
material, a luminescent material, a bioluminescent material, or a
radioactive material. Such materials and examples thereof are
well-known in the art. A secondary antibody that is raised to the
monoclonal antibody that is itself coupled to a label can also be
used to detect the monoclonal antibody-antigen complex.
[0329] In another aspect of the present invention, a microsphere
immunoassay is provided that detects a WNV or DENV infection in a
biological sample using purified WNV or DENV NS5 antigen and
monoclonal antibodies raised thereto. In this assay, a biological
sample that is suspected of having a WNV or DENV infection is
contacted or mixed together with a suspension of microspheres
coupled to anti-IgM antibodies. The microspheres coupled to their
anti-IgM antibodies "capture" the IgM antibodies that are contained
in the biological sample. Anti-WNV IgM and anti-DENV IgM
antibodies, in their respective assays, where present in the
biological samples, will bind to the microspheres, specifically
they will form complexes with the anti-IgM antibodies coupled to
the microspheres. Conditions sufficient for forming such complexes
will be know by the ordinary skilled person and can include
temperature, time, and buffer strength and composition. For
example, these incubations can be carried out at a pH of 7.00 to
7.50, in a buffer system such as Phosphate Buffered Saline or
TRIS-CL at 0.15M salt, with 0.05% Tween 20 detergent. The
incubations may take place at temperatures between 4.degree. C. and
37.degree. C., with the incubation time in inverse proportion to
the temperature. If 4.degree. C. is used, overnight incubations are
ideal. If 37.degree. C. is used, a 30 minute incubation is
appropriate, as longer incubations at elevated temperature may
result in denaturation of the antigen.
[0330] Next, purified and/or isolated WNV NS5 or the DENV NS5
antigen is added to the reaction system and allowed to come into
contact with the microspheres and form a binding complex with the
corresponding anti-WNV NS5 or anti-DENV NS5 IgM antibodies coupled
via their complex with the anti-IgM antibodies bound to the
microspheres. As above, conditions sufficient for forming such
complexes will be know by the ordinary skilled person and can
include temperature, time, and buffer strength and composition.
Subsequently, the corresponding anti-WNV NS5 or anti-DENV NS5
monoclonal antibodies are added to the reaction system and allowed
to contact the microspheres under conditions sufficient so that a
binding complex may form between any present WNV NS5 or DENV NS5
antigen complexed to the microspheres. The presence of such
complexes is then assayed such that their positive detection
indicates the presence of a WNV or DENV infection in the biological
sample. It will be appreciated, that as before, the monoclonal
antibodies can be labeled to enable the detection of the
complexes.
[0331] It will be appreciated that since IgM antibodies are
involved in carrying out the above method, it is possible to detect
recent and/or ongoing flavivirus infections in a biological sample.
It is well-established that IgM antibodies are the generated early
in response to a foreign invader, such as a virus, and are not
persistent once the invader is removed or attenuated. The presence
of IgM antibodies can signify an ongoing or recent infection.
However, in neutrotropic flaviviruses (JE, SLE, TBE, WNF) it is
relatively common to find IgM persistence in blood for more than 6
months. (Reference. Goro Kuno. Serodiagnosis of Vlaviviral
Infections and Vaccinations in Humans. Advances in Virus Research,
Vol. 61. pp. 3-65, 2003.)
[0332] The present invention further contemplates a kit that
comprises the monoclonal antibodies of the invention, especially
the anti-WNV NS5 and anti-DENV NS5 monoclonal antibodies and
instructions for their use in the detection of WNV or DENV
infections, respectively, in any biological sample. The kits of the
invention also can be used in basic medical and scientific research
as a research diagnostic and analytical tool for studying the
pathogenesis of flaviviruses, especially WNV and DENV, in tissues
capable of being infected thereby. For example, the kits can be
used to study the presence of a WNV and/or DENV in tissues, the
relative quantities of the viruses, microscopic analysis, tissue
localization analysis to identify where in the tissue virus is
replicating or persisting, Western blot analysis and other similar
analytical tools of infected tissues and cells, and in situ
analysis of infected tissues. The kits of the invention may also
include ancillary agents such as buffering agents and protein
stabilizing agents, e.g., polysaccharides and the like. The
diagnostic kit may further include, where necessary, other
components of a signal-producing system including agents for
reducing background interference, control reagents, and an
apparatus for conducting a test. The kit can also include reagents
and components to label the monoclonal antibodies and/or the
antigens of the invention. Further, the kits can comprise the
purified and isolated WNV NS5 or DENV NS5 antigens.
[0333] The present invention also contemplates the administration
of a pharmaceutical composition comprising the monoclonal
antibodies of the invention, especially the anti-WNV NS5 and
anti-DENV NS5 monoclonal antibodies, for treating a human patient
or animal, such as a horse, or a bird or any patient or animal in
need thereof. For example, the antibodies described herein can be
administered to a subject, e.g., an animal or a human, to attempt
to alleviate infection by a flavivirus, especially WNV or DENV. In
addition, the pharmaceutical compositions of the invention
comprising the monoclonal antibodies herein described can also be
used to provide a patient or animal passive immunity to a
flavivirus, especially WNV or DENV.
[0334] Passive immunity is defined, for the purposes of this
application, as the transfer to an organism of an immune response
effector that was generated in another organism. The classic
example of establishing passive immunity is to transfer antibodies
produced in one organism into a second, immunologically compatible
animal. The present invention contemplates both of these
approaches. Antibodies and antisera prepared as set forth above,
are injected into hosts to provide passive immunity against the
disease to be treated. For example, an antibody composition is
prepared by mixing, preferably homogeneously mixing, at least one
antibody with at least one pharmaceutically or veterinarally
acceptable carrier, diluent, or excipient using standard methods of
pharmaceutical or veterinary preparation. The amount of antibody
required to produce a single dosage form will vary depending upon
the individual to be treated and the particular mode of
administration. The specific dose level for any particular
individual will depend upon a variety of factors including the age,
body weight, general health, sex, and diet of the individual, time
of administration, route of administration, rate of excretion, drug
combination and the severity of the disease.
[0335] The phrase "pharmaceutically or pharmacologically
acceptable" refer to molecular entities and compositions that do
not produce adverse, allergic, or other untoward reactions when
administered to an animal or a human. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents and the like. The
use of such media and agents for pharmaceutically active substances
is well known in the art. Except insofar as any conventional media
or agent is incompatible with the compositions of the present
invention, its use in therapeutic compositions is contemplated.
Supplementary active ingredients also can be incorporated into the
compositions. Suitable pharmaceutical carriers are described in
Remington's Pharmaceutical Sciences (E. W. Martin), a well-known
reference text in this field, and in the USP/NF (United States
Pharmacopeia and the National Formulary).
[0336] The most effective mode of administration and dosage regimen
for the compositions of this invention can also depend upon the
severity and course of the infection, the patient's health and
response to treatment, and the judgment of the treating physician.
Accordingly, the dosages of the compositions can be titrated to the
individual patient or animal being treated. In general, it is
desirable to provide the recipient with a dosage of antibody which
is in the range of from about 1 pg/kg to 10 mg/kg (body weight of
patient), although a lower or higher dosage may be
administered.
[0337] The treatment may be given in a single dose schedule, or
preferably a multiple dose schedule in which a primary course of
treatment may be with 1-10 separate doses, followed by other doses
given at subsequent time intervals required to maintain and or
reinforce the response, for example, at 1-4 months for a second
dose, and if needed, a subsequent dose(s) after several months.
Examples of suitable treatment schedules include: (i) 0, 1 months
and 6 months, (ii) 0, 7 days and 1 month, (iii) 0 and 1 month, (iv)
0 and 6 months, or other schedules sufficient to elicit the desired
responses expected to reduce disease symptoms, or reduce severity
of disease.
[0338] A pharmaceutical composition is formulated to be compatible
with its intended route of administration. Examples of routes of
administration include parenteral, e.g., intravenous, intradermal,
subcutaneous, oral (e.g., inhalation), transdermal (topical),
transmucosal, and rectal administration. Solutions or suspensions
used for parenteral, intradermal, or subcutaneous application can
include the following components: a sterile diluent such as water
for injection, saline solution, fixed oils, polyethylene glycols,
glycerine, propylene glycol or other synthetic solvents;
antibacterial agents such as benzyl alcohol or methyl parabens;
antioxidants such as ascorbic acid or sodium bisulfite; chelating
agents such as ethylenediaminetetraacetic acid; buffers such as
acetates, citrates or phosphates and agents for the adjustment of
tonicity such as sodium chloride or dextrose. pH can be adjusted
with acids or bases, such as hydrochloric acid or sodium hydroxide.
The parenteral preparation can be enclosed in ampoules, disposable
syringes, or multiple dose vials made of glass or plastic.
[0339] More particularly, the pharmaceutical forms suitable for
injectable use include sterile aqueous solutions or dispersions and
sterile powders for the extemporaneous preparation of sterile
injectable solutions or dispersions. In all cases, the form can be
sterile and can be fluid to the extent that easy syringability
exists. It preferably is stable under the conditions of manufacture
and storage and must be preserved against the contaminating action
of microorganisms, such as bacteria and fungi. The carrier can be a
solvent or dispersion medium containing, for example, water,
ethanol, polyol (for example, glycerol, propylene glycol, and
liquid polyethylene glycol, and the like), suitable mixtures
thereof, and vegetable oils. The proper fluidity can be maintained,
for example, by the use of a coating, such as lecithin, by the
maintenance of the required particle size in the case of dispersion
and by the use of surfactants. The prevention of the action of
microorganisms can be brought about by various antibacterial an
antifungal agents, for example, parabens, chlorobutanol, phenol,
sorbic acid, thimerosal, and the like. In many cases, it will be
preferable to include isotonic agents, for example, sugars or
sodium chloride.
[0340] Additional pharmaceutical methods may be employed to control
the duration of action. Control release preparations may be
achieved through the use of polymers to complex or absorb the
compounds. The controlled delivery may be exercised by selecting
appropriate macromolecules (for example polyesters, polyamino
acids, polyvinyl, pyrrolidone, ethylenevinylacetate,
methylcellulose, carboxymethylcellulose, or protamine sulfate) and
the concentration of macromolecules as well as the method of
incorporation in order to control release. Another possible method
to control the duration of action by controlled release
preparations is to incorporate the compounds of the present
invention into particles of a polymeric material such as
polyesters, polyamino acids, hydrogels, poly(lactic acid) or
ethylene vinylacetate copolymers. Alternatively, instead of
incorporating these agents into polymeric particles, it is possible
to entrap these materials in microcapsules prepared, for example,
interfacial polymerization, for example, hydroxymethylcellulose or
gelatin-microcapsules and poly(methylmethacylate)-microcapsules,
respectively, or in colloidal drug delivery systems, for example,
liposomes, albumin microspheres, microemulsions, nanoparticles, and
nanocapsules or in macroemulsions. Such techniques are disclosed in
Remington's Pharmaceutical Sciences (1980).
[0341] Sterile injectable solutions are prepared by incorporating
the active compounds in the required amount in the appropriate
solvent with various of the other ingredients enumerated above, as
required, followed by filtered sterilization. Generally,
dispersions are prepared by incorporating the various sterilized
active ingredients into a sterile vehicle which contains the basic
dispersion medium and the required other ingredients from those
enumerated above. In the case of sterile powders for the
preparation of sterile injectable solutions, the preferred methods
of preparation are vacuum-drying and freeze-drying techniques which
yield a powder of the active ingredient plus any additional desired
ingredient from a previously sterile-filtered solution thereof.
[0342] For oral administration the antibodies of the present
invention may be incorporated with excipients and used in the form
of non-ingestible mouthwashes and dentifrices. A mouthwash may be
prepared incorporating the active. Alternatively, the active
ingredient may be incorporated into an antiseptic wash containing
sodium borate, glycerin and potassium bicarbonate. The active
ingredient may also be dispersed in dentifrices, including: gels,
pastes, powders and slurries. The active ingredient may be added in
a therapeutically effective amount to a paste dentifrice that may
include water, binders, abrasives, flavoring agents, foaming
agents, and humectants.
[0343] For parenteral administration in an aqueous solution, for
example, the solution should be suitably buffered if necessary and
the liquid diluent first rendered isotonic with sufficient saline
or glucose. These particular aqueous solutions are especially
suitable for intravenous, intramuscular, subcutaneous and
intraperitoneal administration. In this connection, sterile aqueous
media which can be employed will be known to those of skill in the
art in light of the present disclosure. For example, one dosage
could be dissolved in 1 ml of isotonic NaCl solution and either
added to 1000 ml of hypodermoclysis fluid or injected at the
proposed site of infusion, (see Remington's Pharmaceutical Sciences
15th Edition, pages 1035-1038 and 1570-1580). Some variation in
dosage will necessarily occur depending on the condition of the
subject being treated. The person responsible for administration
will, in any event, determine the appropriate dose for the
individual subject. Moreover, for human administration,
preparations should meet sterility, pyrogenicity, general safety
and purity standards as required by FDA Office of Biologics
standards.
[0344] Upon formulation, solutions will be administered in a manner
compatible with the dosage formulation and in such amount as is
therapeutically effective. The formulations are easily administered
in a variety of dosage forms such as injectable solutions, drug
release capsules and the like.
[0345] The present invention contemplates that "humanized"
antibodies can be used to provide for a greater compatibility and
effectiveness in human subjects. It will be appreciated that the
antibody modification process involved in humanizing antibodies for
human subjects is not limited thereto. Analogously, the monoclonal
antibodies can be designed for administration in animal subjects
which are capable of being infected by flaviviruses, especially WNV
and DENV. For example, the antibodies can be "equinized" for
treatment of horses.
[0346] Humanized antibodies are designed to have greater homology
to a human immunoglobulin than animal-derived monoclonal
antibodies. Likewise, equinized antibodies are designed with
greater homology to a horse immunoglobulin than to antibodies
derived from another organism.
[0347] In the particular example of humanized antibodies, non-human
amino acid residues from an "import" (animal) variable domain are
transfected to a human "backbone." Humanization can be essentially
performed following the method of Winter and co-workers (Jones et
al., Nature 321:522-525 (1986); Riechmann et al., Nature
332:323-327 (1988); Verhoeyen, et al., Science, 239:1534-1536
(1988)), by substituting rodent complementarity determining regions
("CDRs") or CDR sequences for the corresponding sequences of a
human antibody. Accordingly, in such "humanized" antibodies, the
CDR portions of the human variable domain have been substituted by
the corresponding sequence from a non-human species. Thus,
humanized antibodies are typically human antibodies in which some
CDR residues and possibly some framework residues are substituted
by residues from analogous sites in rodent antibodies.
[0348] The choice of human variable domains, both light and heavy,
to be used in making the humanized antibodies is important to
reduce antigenicity. According to the so-called "best-fit" method,
the sequence of the variable domain of a rodent antibody is
screened against the entire library of known human variable-domain
sequences. The human sequence which is closest to that of the
rodent is then accepted as the human framework (FR) for the
humanized antibody (Sims et al., J. Immunol., 151:2296 (1993);
Chothia et al., J. Mol. Biol., 196:901 (1987)). Another method uses
a particular framework derived from the consensus sequence of all
human antibodies of a particular subgroup of light or heavy chains.
The same framework may be used for several different humanized
antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285
(1992); Presta et al., J. Immunol., 151:2623 (1993)).
[0349] It is further important that humanized antibodies retain
high affinity for the antigen and other favorable biological
properties. To achieve this goal, according to a preferred method,
humanized antibodies are prepared by a process of analysis of the
parental sequences and various conceptual humanized products using
three-dimensional models of the parental and humanized sequences.
Three-dimensional immunoglobulin models are commonly available and
are familiar to those skilled in the art. Computer programs are
available which illustrate and display probable three-dimensional
conformational structures of selected candidate immunoglobulin
sequences. Inspection of these displays permits analysis of the
likely role of certain residues in the functioning of the candidate
immunoglobulin sequence, i.e., the analysis of residues that
influence the ability of the candidate immunoglobulin to bind its
antigen. In this way, FR residues can be selected and combined from
the recipient and import sequences so that the desired antibody
characteristic, such as increased affinity for the target
antigen(s), is maximized, although it is the CDR residues that
directly and most substantially influence antigen binding.
[0350] One can also produce transgenic animals (e.g., mice) that
are capable, upon immunization, of producing a full repertoire of
human antibodies in the absence of endogenous immunoglobulin
production. Such transgenic mice are available from Abgenix, Inc.,
Fremont, Calif., and Medarex, Inc., Annandale, N.J. It has been
described that the homozygous deletion of the antibody heavy-chain
joining region (1H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al.,
Nature 362:255-258 (1993); Bruggermann et al., Year in Immunol.
7:33 (1993); and Duchosal et al. Nature 355:258 (1992). Human
antibodies can also be derived from phage-display libraries
(Hoogenboom et al., J. Mol. Biol. 227: 381 (1991); Marks et al., J.
Mol. Biol. 222:581-597 (1991); Vaughan, et al., Nature Biotech
14:309 (1996)).
[0351] Chimeric antibodies are produced by recombinant processes
well known in the art, and have an animal variable region and a
human constant region. Humanized antibodies have a greater degree
of human peptide sequences than do chimeric antibodies.
[0352] One can also create single peptide chain binding molecules
in which the heavy and light chain Fv regions are connected. Single
chain antibodies ("scFv") and the method of their construction are
described in U.S. Pat. No. 4,946,778. Alternatively, Fab can be
constructed and expressed by similar means (Evans M J et al. Rapid
expression of an anti-human C5 chimeric Fab utilizing a vector that
replicates in COS and 293 cells. J. Immunol. Meth. 184: 123-38
(1995)). All of the wholly and partially human antibodies are less
immunogenic than wholly murine MAbs, and the fragments and single
chain antibodies are also less immunogenic.
[0353] The present invention further encompasses a method of
transmitting data, for example disclosing the result or results of
any of the methods and assays herein described and contemplated,
such as, for example, the use of ELISAs, microsphere immunoassays,
immunochromatographic assays, and agglutination assays to detect
flavivirus antigens, especially WNV E glycoprotein and NS5 and DENV
NS5, as well as antibodies thereto, present in biological samples.
Such information can be transmitted by digital means, such as by
facsimile, electronic mail, telephone, or a global communications
network, such as the World Wide Web. For example, data can be
transmitted via website posting, such as by subscription or
select/secure access thereto and/or via electronic mail and/or via
telephone, IR, radio, television or other frequency signal, and/or
via electronic signals over cable and/or satellite transmission
and/or via transmission of disks, compact discs (CDs), computers,
hard drives, or other apparatus containing the information in
electronic form, and/or transmission of written forms of the
information, e.g., via facsimile transmission and the like. Thus,
the invention comprehends a user performing according to the
invention and transmitting information therefrom; for instance, to
one or more parties who then further utilize some or all of the
data or information, e.g., in the manufacture of products, such as
therapeutics, assays and diagnostic tests and etc. This invention
comprehends disks, CDs, computers, or other apparatus or means for
storing or receiving or transmitting data or information containing
information from methods and/or use of methods of the invention.
Thus, the invention comprehends a method for transmitting
information comprising performing a method as discussed herein and
transmitting a result thereof.
[0354] Further still, the invention comprehends methods of doing
business comprising performing or using some or all of the herein
methods, monoclonal antibodies, hybridoma cell lines, or flavivirus
antigens, especially WNV E glycoprotein and NS5 and DENV NS5, and
communicating or transmitting or divulging a result or results
thereof, advantageously in exchange for compensation, e.g., a fee.
Advantageously, the communicating, transmitting or divulging of
information is via electronic means, e.g., via internet or email,
or by any other transmission means herein discussed. Thus, the
invention comprehends methods of doing business.
[0355] A first party, a "client," can request information, e.g.,
via any of the herein mentioned transmission means--either
previously prepared information or information specially ordered as
to the results of the methods and assays of the invention--of a
second party, "vendor", e.g., requesting information via electronic
means such as via internet (for instance request typed into
website) or via email. The vendor can transmit that information,
e.g., via any of the transmission means herein mentioned,
advantageously via electronic means, such as internet (for instance
secure or subscription or select access website) or email. The
information can come from performing some or all of a herein method
or use of a herein method in response to the request, or from
performing some or all of a herein method, and generating a library
of information from performing some or all of a herein method or
use of a herein algorithm. Meeting the request can then be by
allowing the client access to the library or selecting data from
the library that is responsive to the request.
[0356] Accordingly, the invention even further comprehends
collections of information, e.g., in electronic form (such as forms
of transmission discussed above), from performing or using a herein
invention.
[0357] For example, a client hospital may find itself in need of
assaying a large blood supply for the presence of WNV. A vendor
proficient at the methods of the present invention can be contacted
by the hospital to quickly test or screen the blood supply using
any of the herein described methods or any other method
contemplated by the invention. The results of the blood supply
screen can be transmitted back to the client hospital for a fee. It
will be recognized by one of ordinary skill in the art that the
methods set forth in the present application are not limited to the
use of WNV E glycoprotein, WNV NS5, or DENV NS5, to detect
antibodies to flaviviruses specific to WNV or DENV. In contrast,
the development of the microsphere immunoassay according to the
present invention can be expanded to achieve other efficiencies in
serologic testing for infectious diseases and/or autoimmune
diseases where symptoms and geographic location of vectors and
reservoirs are held in common, or when a need exists to test sera
for exposure to multiples agents in a time-effective manner.
[0358] It will also be appreciated to one of ordinary skill in the
art that the detection methods of the instant invention can be
carried out using any known assay format readily available, such
as, for example, an ELISA. ELISA methods are well known in the art.
Example 26 sets forth further description on the application of
ELISA with the antigens and methods of the present invention.
[0359] A better understanding of the present invention and of its
many advantages will be had from the following examples which
further describe the present invention and given by way of
illustration. The examples that follow are not to be construed as
limiting the scope of the invention in any manner. In light of the
present disclosure, numerous embodiments within the scope of the
claims will be apparent to those of ordinary skill in the art.
EXAMPLES
[0360] The following Materials and Methods were used in the
examples that follow.
[0361] Reagents
[0362] Recombinant West Nile envelope glycoprotein antigen,
provided by L.sup.2 Diagnostics, New Haven, Conn., was expressed in
a eukaryotic cell expression system and purified by column
chromatography. N-Hydroxysuccinimide (Sulfo-NHS) and
1-ethyl-3-(3-dimethylamino-propyl) carbodiimide-HCL (EDC) were
obtained from Pierce, Rockford, Ill. PBS with Tween 20, pH 7.4
(PBS-T); PBS with bovine serum albumin (BSA) (PBS-BSA), pH 7.4; PBS
and Ultra Sodium Azide were from Sigma/Aldrich, St. Louis, Mo.
[0363] Goat F(ab').sub.2 anti-human immunoglobulins, IgG+IgA+IgM
conjugated to red-phycoerythrin (R-PE); goat F(ab').sub.2
anti-human IgG R-PE conjugate; and goat F(ab').sub.2 anti-human IgM
R-PE conjugate were from Bio-Source International, Camarillo,
Calif.
[0364] Supplies and Equipment
[0365] A Luminex 100 flow analyzer was from Luminex Corporation,
Austin, Tex. CL1/CL2 calibration microspheres, RP1 calibration
microspheres, and multi-analyte microspheres with carboxylated
surface were also obtained from Luminex Corporation. Multiscreen
filter plates with 1.2 micron Durapore filters and a multiscreen
vacuum manifold were from Millipore, Bedford, Mass. Slide-A-Lyzer
mini-dialysis unit floats were from Pierce, Rockford, Ill. A
Labquake Thermolyne tube rotator was from VWR, Bridgeport, N.J.
Costar opaque black EIA/RIA plates with breakaway strips/wells,
were from Corning Inc., Corning, N.Y. An ultrasonic cleaner
(sonicator) was from Cole-Palmer, Vernon Hills, Ill.
[0366] Human Sera
[0367] Patient sera previously tested for WNV antibodies by the MAC
ELISA and the IgG ELISA were coded, with all patient identifiers
removed, and were provided from the serum bank at the Wadsworth
Center, of the New York State Department of Health, or by the
Arbovirus Laboratory of the Centers for Disease Control and
Prevention of the United States Public Health Service (CDC), Ft.
Collins, Colo. All sera were tested and evaluated under conditions
approved by the Institutional Review Board of the New York State
Department of Health.
[0368] Sera from Wadsworth Center archives were chosen to establish
normal MIA ranges for positive and negative samples. Ten sera were
selected on the basis of positive results in standard WN virus
ELISA assays. WNV and St. Louis encephalitis virus (SLEV) PRN test
results for paired acute and convalescent sera confirmed WNV as the
infecting agent. Ten sera that were negative for WNV antibodies in
IgM-capture and IgG ELISAs were selected as negative control sera.
For assay covariance studies, the 10 WNV patient sera were combined
into a positive control serum pool, and the 10 negative sera were
combined into a negative control serum pool.
[0369] A coded panel of 19 sera provided by the CDC Arbovirus
Diseases Branch included: three sera from confirmed WNV
encephalitis patients; six sera from SLEV patients; and 3 sera from
dengue fever virus (DENV) patients. For 10 of 12 sera from infected
patients, the infectious agent was confirmed by virus PRN tests
using WNV, SLEV, or DENV. Cross-neutralization data classified the
sera as specific for WNV, DENV or SLEV infections. Seven negative
control sera were from presumed healthy subjects lacking evidence
of previous flavivirus infection. The CDC provided ELISA data for
these sera when the samples were decoded.
[0370] A third serum panel was from eight individuals vaccinated
with three doses of JE-VAX (Connaught Laboratories, Missisauga, ON,
Canada). These sera were from the Wadsworth Center Arbovirus
Laboratory. JE-VAX is a licensed, formalin-inactivated Japanese
encephalitis virus (JEV) vaccine. The vaccinated individuals had a
history of occupational exposure to flaviviruses and, in some
cases, prior vaccination against a flavivirus. The sera, which
included pre- and post-vaccination sera, were tested for
neutralizing antibodies in JEV PRN assays.
[0371] Another serum panel represented serial specimens from a
patient with WNV infection confirmed by PRN tests. Blood specimens
were collected 2, 18, 72, 260, and 430 days post-disease onset,
and, by coincidence, 3 days prior to virus exposure.
[0372] A fifth serum panel included human sera that previously
tested positive in standard serological assays for antibodies to
Epstein Barr virus (EBV), cytomegalovirus (CMV), herpes simplex
virus (HSV), human immunodeficiency virus (HIV), Treponema pallidum
(the syphilis spirochete), Borrelia burgdorferi, Anaplasma
phagocytophilum, autoimmune nuclear antigens (antinuclear
antibodies), or rheumatoid factor. These sera were from frozen sera
archived at the Wadsworth Center. The syphilis patient sera were
negative in WNV or SLEV PRN tests performed at the Arbovirus
Laboratory of the Wadsworth Center. Sixteen normal human sera were
purchased from United States Biological (Swampscott, Mass.). Twelve
additional sera from healthy individuals were from the Wadsworth
Center or L.sup.2 Diagnostics (New Haven, Conn.).
[0373] The Wadsworth Center provided sera from 833 patients with
suspected viral encephalitis. These sera were submitted to the New
York State Department of Health between June and November of 2002.
These sera had previously been tested for antibodies to WNV using
the IgM-capture and IgG ELISAs.
[0374] IgG or IgM were selectively depleted from serum specimens
with goat anti-human IgG or goat anti-human IgM, respectively. For
IgG depletions, 5 .mu.l of serum was mixed with 45 .mu.l of goat
anti-human IgG (GullSORB from Meridian Diagnostics, Cincinnati,
Ohio). The mixtures were centrifuged at 14,000.times.g to remove
antibody-bound IgG. According to the manufacturer, this is
sufficient to deplete IgG at concentrations up to 15 mg/ml, the
upper limit of normal human IgG concentration. Removal of
detectable IgG antibodies to WN virus was confirmed by negative
results in WNV IgG ELISAs and indirect immunofluorescence assays
with SLEV antigen on arbovirus slides (Focus Technologies, Cypress,
Calif.).
[0375] A similar pretreatment with anti-IgM antibody depleted serum
samples of IgM. Ten .mu.l of serum was mixed with 10 .mu.l 2.5
mg/ml goat anti-human Mu chain (Jackson ImmunoResearch
Laboratories, Inc., West Grove, Pa.) prior to addition of 20 .mu.l
PBS and centrifugation for 4 min at 14,000.times.g to remove
antibody-bound IgM.
[0376] Human Sera for WNV NS5 Studies
[0377] Five panels of human sera were used in this study. (i) WNV
patient sera were from serum archives at the Wadsworth Center, New
York State Department of Health. These sera had previously been
tested WNV-positive by the IgM capture and indirect IgG ELISA for
antibodies reactive to noninfectious recombinant antigen (Davis et
al., Martin et al., Johnson A. J., et al.). (ii) Acute and
convalescent paired sera from DEN patients were provided by the
National Microbiology Laboratory, Health Canada. The patients are
Canadian residents who got infected with DEN during recent travels
to various geographical regions. These sera had been tested by HI
assays and PRNT against DEN, Powassan (POW), or SLE virus. (iii)
Forty SLE patient sera were generously provided by the Centers for
Disease Control and Prevention. These samples had been previously
confirmed by PRNT against SLE and WNV. (iv) JE-vaccinated human
sera were from laboratory employees who had received three doses of
the formalin-inactivated JE vaccine. (v) A panel of human sera from
the Diagnostic Immunology Laboratory of the Wadsworth Center were
used to examine the specificity of the WNV assays, including human
specimens that were reactive in serologic assays for Lyme disease
(Borrelia burgdorferi infection), ehrlichiosis (Anaplasma
cytophilum infection), syphilis (Treponema pallidum infection),
human immunodeficiency virus (HIV), Epstein Barr Virus (EBV),
cytomegalovirus (CMV), antinuclear antibodies (ANA), and rheumatoid
factor. All samples were blind tested with patient identifiers
removed, according to guidelines of the NIH and the Institutional
Review Board of the New York State Department of Health.
[0378] Cross-Species Plaque Reduction Neutralization Test (PRNT)
and Hemagglutination Inhibition (HI) Assays.
[0379] Neutralizing antibodies were evaluated in PRNT with WNV,
SLEV, or JEV virus as previously described by Lindsey H. S. et.
al., which is incorporated herein in its entirety by reference.
Standard HI tests for DENV, POWV, SLEV, and WNV were performed
according to the method of Casals J. et. al., which is incorporated
herein in its entirety by reference.
[0380] Microsphere Immunoassay (MIA)
[0381] Approximately 50 .mu.g of recombinant NS3, NS5, or E protein
was covalently linked to the carboxylated surface of
6.25.times.10.sup.6 microspheres through a two-step carbodiimide
linkage protocol as described by the manufacturer (Luminex
Corporation, Austin, Tex.). A two-step suspension MIA was
performed. A 96-well 1.2 .mu.m-filter plate (Millipore, Bedford,
Mass.) was blocked for 2 min with 100 .mu.l of PBN buffer
[phosphate buffered saline (pH 7.4) with 1% bovine serum albumin
and 0.05% sodium azide], washed once with 150 .mu.l of PBS-T buffer
[phosphate buffered saline (pH 7.4) with 0.05% Tween-20], and then
wetted with 20 .mu.l of PBN buffer. Serum samples (50 .mu.l,
diluted 1:100 in PBN unless otherwise specified) and
antigen-conjugated microspheres (2,500 in 50 .mu.l PBN) were added
to each well. The plate was incubated in the dark on a shaker at
37.degree. C. for 30 min, and then washed three times with PBS-T
using a vacuum manifold. Polyvalent goat anti-human immunoglobulins
(IgG+IgA+IgM, 50 .mu.l of 1:250 dilution in PBN) conjugated with
red-phycoerythrin (Bio-Source International, Camarillo, Calif.)
were added. After incubation at 37.degree. C. for 30 min, the plate
was washed twice with PBS-T. Microspheres were resuspended in 125
.mu.l of PBN per well, and 75 .mu.l of suspension was transferred
to an opaque black EIA/RIA 96-well plate (Costar, Corning, N.Y.).
The microsphere fluorescence intensity was quantified using a
Luminex 100 flow analyzer (Luminex Corporation). The MFI of 100
microspheres was recorded for each well. The mean of 20 normal sera
plus 3 times SD was used as the cutoff value for each assay.
[0382] Human Cerebrospinal Fluid Specimens
[0383] Small volumes of spinal fluid (100-200 ul) were obtained
from the frozen archives of the Encephalitis PCR laboratory and the
Diagnostic Immunology Laboratory of the Wadsworth Center. These
specimens had previously been tested for WNV by PCR and or the MAC
ELISA. Patients with either a positive PCR result for WNV, or with
a detectable IgM antibody to WNV also had follow-up plaque
reduction neutralization testing against the likely flavivirus
infections (WNV, SLE, DEN) on serum specimens. These specimens were
tested with approval of the Institutional Review Board of the New
York State Department of Health. All patient identifiers were
removed from specimens prior to testing.
[0384] The microsphere immunoassay was performed on the spinal
fluids under conditions previously described except that the fluids
were tested at a 1:2 dilution by adding 25 microliters of spinal
fluid to 25 microliters of PBS for the total polyvalent antibody
result, or were tested by adding 25 microliters spinal fluid to 25
microliters of a 1/100 dilution of anti-IgG (Gull SORB) for the IgG
depleted "IgM" result. This concentration of anti-human IgG is
calculated to provide an optimal molar ratio to deplete IgG in the
spinal fluid, based on the assumption that the IgG concentration in
serum is 1000 fold greater than in spinal fluid (Burke et al, JCM
1982).
[0385] Configuration of the rE-MI to Detect IgM in Spinal
Fluids.
[0386] For ease of technical performance and for quality control.
We maintained, as much as possible, the similar assay configuration
for the spinal fluids as used for the analysis of serum. The number
of r-WNV-E coated beads added to spinal fluid in the wells was
maintained at 2500 beads in a volume of 50 ul. Our chosen conjugate
dilution was maintained at 1/250 of R-PE anti human
immunoglobulins. A panel of 11 spinal fluids from patients
confirmed to have flaviviral encephalitis was tested with the
rE-MI. Data from the polyvalent assay and from the "IgM" (IgG
depleted) assay are given in FIG. 15. Note that the P/N values for
IgM assay were higher than the P/N values for the polyvalent assay
in specimens from patients deemed to be "WNV Current or Recent" The
patients determined to be WNV at undetermined time" had the lowest
"IgM" P/N values. For the spinal fluids from "Dengue at
Undetermined Time" patients, the IgM P/N values were less than the
polyvalent P/N values.
[0387] IgM-Capture and Indirect IgG ELISAs.
[0388] Sera provided by the CDC Arbovirus Diseases Branch were
tested by the CDC for antibodies to WNV, SLEV, and/or DENV in
IgM-capture and indirect IgG ELISAs in accordance with A. J.
Johnson et al. (2000) and R. Mariella (2002), which are both
incorporated herein in their entirety by reference. The ELISA
antigens included: a WNV noninfectious recombinant antigen (NRA)
preparation of recombinant E, prM and M proteins (B. S. Davis et
al.); a sucrose acetone extract of SLE virus-infected suckling
mouse brain; or acetone-extracted DENV from supernatants of
infected C6/36 mosquito cell cultures. Control wells were coated
with mock antigen prepared in a similar manner from uninfected
cells or tissue.
[0389] The New York State Department of Health tested sera and CSF
for antibodies to WNV using the WNV NRA and control mock antigen
provided by CDC in the IgM-capture and indirect IgG ELISAs.
[0390] A specimen was considered positive if, at a P/N
ratio.gtoreq.3.0, a two-fold greater immunoreactivity was observed
for viral antigen relative to control antigen. ELISA results were
considered uninterpretable due to nonspecific binding if the latter
criterion was not met.
[0391] Statistical Analysis.
[0392] Microsoft Excel software was used for statistical analysis.
Data from different groups were compared with two-tailed Student's
t tests. Relationships between paired variables were evaluated with
Pearson r correlation. Two way contingency table analysis using
distributed JavaStat software provided the kappa statistic,
sensitivity, specificity and predictive values.
Example 1
Isolation of WNV in Connecticut
[0393] Several WNV isolates were obtained from mosquitoes and birds
in Connecticut. Mosquitoes were captured in dry ice-baited Centers
for Disease Control miniature light traps. One mosquito trap was
placed at each location per night; the numbers of traps per site
ranged from 1 to 6. Mosquitoes were transported alive to the
laboratory where they were identified and grouped (pooled)
according to species, collecting site, and date. The number of
mosquitoes per pool ranged from 1 to 50. The total number of
mosquitoes by species that were collected in 14 towns in Fairfield
County, Conn., and tested for virus from 6 Sep. through 14 Oct.
1999: Aedes vexans, 1688; Ae. cinereus, 172; Ae. trivittatus, 131;
Ae. taeniorhynchus, 123; Ae. sollicitans, 109; Ae. cantator, 63;
Ae. triseriatus, 28; Ae. japonicus, 19; Ae. canadensis, 1;
Anopheles punctipennis, 82; An. quadrimaculatus, 4; An. walkeri, 2;
Coquillettidia perturbans, 15; Culex pipiens, 744; Cx. restuans,
27; Cx. erraticus, 4; Cx. territans, 1; Culiseta melanura, 76; Cs.
morsitans, 1; Psorophora ferox, 4; and Uranotaenia sapphirina, 104.
Mosquitoes were stored at -80.degree. C. until tested for virus.
Additionally, we obtained isolated WNV from mosquitoes collected in
New York City.
[0394] Most dead birds were collected by state or town personnel in
Connecticut and sent to the Pathobiology Department at the
University of Connecticut, Storrs, where they were examined for
postmortem and nutritional condition, gross lesions, and
microscopic evidence indicative of encephalitis. Brain tissue from
birds with presumed encephalitis was frozen at -70.degree. C. and
then sent to the Connecticut Agricultural Experiment Station, New
Haven, for virus testing. Connecticut towns from which dead crows
were collected and virus isolated from brain tissues (number of
isolates in parentheses): Bridgeport (1), Darien (1), Fairfield
(4), Greenwich (3), Hamden (1), Madison (1), Milford (1), New
Canaan (1), New Haven (3), North Haven (1), Norwalk (1), Redding
(1), Stamford (5), Stratford (1), Weston (1), Westport (1), and
Woodbridge (1).
[0395] For viral isolation from mosquitoes, frozen pools were
thawed, triturated in tissue grinders or mortars with pestles in 1
to 1.5 ml of phosphate-buffered saline ("PBS") containing 0.5%
gelatin, 30% rabbit serum, antibiotic, and antimycotic. After
centrifugation for 10 min at 520.times.g, 100 .mu.l samples of each
pool of mosquitoes were inoculated onto a monolayer of Vero cells
grown in a 25-cm.sup.2 flask at 37.degree. C. in 5% CO.sub.2. Cells
were examined microscopically for cytopathologic effect for up to 7
days after inoculation.
[0396] For viral isolation from bird brain tissue samples, a 10%
suspension of each sampled brain tissue was prepared in 1.5 ml of
PBS by triturating with a mortar and pestle as described above for
mosquito samples except that Alundum.RTM. was added to facilitate
homogenization of tissue. Two to seven tissue samples from each
brain were tested for virus as follows. Suspensions were
centrifuged at 520.times.g for 10 min. The supernatant of each
sample was then passed through a 0.22-.mu.m filter before
inoculation of a 100-.mu.l sample onto a monolayer of Vero cells.
Cells were grown in a 25-cm.sup.2 flask at 37.degree. C. in 5%
CO.sub.2 and examined for cytopathologic effect for up to 7 days
after inoculation.
[0397] Viral isolates were tested in an ELISA against reference
antibodies to six viruses, in three families, isolated from
mosquitoes in North America. The antibodies were prepared in mice
and provided by the World Health Organization Center for Arbovirus
Research and Reference, Yale Arbovirus Research Unit, Department of
Epidemiology and Public Health, Yale University School of Medicine.
The antibodies were to Eastern Equine Encephalomyelitis and
Highlands J, Cache Valley, LaCrosse, Jamestown Canyon, and St.
Louis Encephalitis viruses.
Example 2
PCR Amplification of DNA Encoding the WNV Envelope Glycoprotein
[0398] The Connecticut WNV isolate 2741 (GenBank.TM. Accession No.
AF206518), as described Example 1, was grown in Vero cells which
were subsequently scraped from the bottom of the flask and
centrifuged at 4500.times.g for 10 min. The supernatants were
discarded and RNA was extracted from the pellet using the
RNeasy.RTM. mini protocol (Qiagen), eluting the column twice with
40 .mu.l of ribonuclease-free water. Two microliters of each eluate
was combined in a 50-.mu.l reverse transcription-polymerase chain
reaction (RT-PCR) with the SuperScript.RTM. one step RT-PCR system
(Life Technologies), following the manufacturer's protocol.
[0399] PCR primers, WN-233F (5'-GACTGAAGAGGGCAATGTTGAGC-3'; SEQ ID:
1) and WN-1189R (5'-GCAATAACTGCGGACYTCTGC-3'; SEQ ID: 2) were
designed to specifically amplify envelope glycoprotein sequences
from WNV based on an alignment of six flavivirus isolates listed in
GenBank.TM. (accession numbers: M16614 (St. Louis encephalitis
virus); M73710 (Japanese encephalitis virus); D00246 (Kunjin
virus); M12294 (West Nile virus); AF130362 (West Nile virus strain
RO97-50); AF130363 (West Nile virus strain 96-1030)).
[0400] The resultant PCR products were purified with the QIAquick
PCR Purification Kit.RTM. (Qiagen) following the manufacturer's
protocol. The amplified DNA and sequenced by the Sanger method at
the Keck Biotechnology Center at Yale University, New Haven, Conn.
The sequence was confirmed to corresponded to the envelope
glycoprotein encoding sequence by alignment with the envelope
glycoprotein encoding sequence from other flavivirus isolates using
the ClustalX 1.64B program (J. D. Thompson, et al., Nucleic Acids
Res, 22, 4673 (1994)). We further purified the resulting DNA
fragments by electrophoresis on a 1% agarose gel, excised the DNA
band, and isolated the DNA using the QIAquick Gel Extraction
Kit.RTM. (Qiagen) following the manufacturer's protocol.
Example 3
Expression and Purification of Recombinant WNV Envelope
Glycoprotein
[0401] The DNA of Example 2 was expressed in E. coli using the
pBAD/TOPO.TM. ThioFusion Expression System.RTM. (Invitrogen). This
system is designed for highly efficient, five minute, one step
cloning of PCR amplified DNA into the pBAD/TOPO.TM. ThioFusion
expression vector. Fusion protein expression is inducible with
arabinose. Fusion proteins were expressed with thioredoxin (12 kDa)
fused to the N-terminus, and a C-terminal polyhistidine tag. The
polyhistidine tag enables the fusion proteins to be rapidly
purified by nickel affinity column chromatography. An enterokinase
cleavage site in the fusion proteins can be used to remove the
N-terminal thioredoxin leader.
[0402] The pBAD/TOPO ThioFusion Expression System.RTM. expression
system was used to express and purify WNV envelope glycoprotein
encoded by the DNA of Example 2 following the manufacturer's
protocol. Specifically, the PCR product obtained as described above
was added to a reaction containing the pBAD/Thio-TOPO.TM. vector (1
.mu.l) and sterile water to a final volume of 5 .mu.l. The reaction
mix was incubated for five minutes at room temperature.
[0403] One Shot.TM. E. coli cells (Invitrogen) were transformed
with the TOPO.TM. cloning reaction products by mixing the TOPO.TM.
cloning reaction with competent cells, incubating the mixture on
ice for 30 minutes and then heat shocking the cells for 30 seconds
at 42.degree. C. 250 .mu.l of room temperature SOC medium was added
to the cells followed by incubation at 37.degree. C. for 30
minutes. 50 .mu.l of the transformation mixture was spread on a
pre-warmed LB plate containing 50 .mu.g/ml ampicillin and incubated
overnight at 37.degree. C. A clone was identified and the DNA was
isolated by standard methods. DNA sequence analysis of cloned DNA
was used to confirm that the thioredoxin-envelope glycoprotein
fusion protein (TR-env; FIG. 4) coding sequence was correct.
[0404] To analyze expression of the recombinant TR-env protein, E.
coli containing the pBAD-TR-env expression vector was grown in
cultures at 37.degree. C. with vigorous shaking to an OD.sub.600
.about.0.5. Prior to protein expression, an aliquot was removed at
the zero point and centrifuged at maximum speed. The supernatant
was removed and the pellet was stored on ice. Protein expression
was induced with arabinose at a final concentration of 0.02%
followed by growth for an additional 4 hours. An aliquot of the
arabinose-induced sample was centrifuged at maximum speed and the
sample was placed on ice following removal of the supernatant. The
uninduced and arabinose-induced cell pellets were resuspended in
sample buffer, the samples were boiled for 5 minutes, analyzed by
denaturing polyacrylamide (SDS-PAGE) gel and stained with Coomassie
blue. The 71 kDa TR-env protein was the major protein found in the
E. coli cells after arabinose induction.
[0405] The induced E. coli cells were lysed by sonication,
centrifuged, and the TR-env protein was purified from the soluble
supernatant with ThioBond.TM. phenylarsinine oxide resin
(Invitrogen) following the manufacturer's protocol. The TR-env
protein was bound to this affinity resin in a batch mode and then
eluted with increasing concentrations of beta-mercaptoethanol. The
fractions were run on a denaturing polyacrylamide (SDS-PAGE) gel
and stained with Coomassie blue. The procedure yielded highly
purified recombinant TR-env fusion protein (FIG. 5).
[0406] In immunoblots, the TR-env protein was recognized by both
anti-thioredoxin antibody (Invitrogen) and human sera from two
individuals seropositive for antibodies to WNV. The purified TR-env
fusion protein, thus, contained an epitope recognized by antibodies
induced by a natural WNV infection.
[0407] Thioredoxin expressed from the pBAD/TOPO.TM. ThioFusion.RTM.
expression vector was used as a negative control protein. The 16
kDa thioredoxin protein was expressed in E. coli and purified using
ProBond.TM. metal-chelating affinity resin as described for the
TR-env protein. Purified thioredoxin was recognized in immunoblots
only by anti-thioredoxin antibody (Invitrogen) and not by human
sera from two individuals seropositive for antibodies to WNV.
[0408] As an alternative method to express and purify the WNV
envelope glycoprotein, a PCR product encoding the WNV E
glycoprotein was engineered as a fusion protein with maltose
binding protein (MBP). Nucleotides 1-1218 of the WNV E glycoprotein
were amplified by PCR using the following primers which contain
EcoRI and PstI restriction sites to facilitate subcloning:
5'GAATTCTTCAACTGCCTTG GAATGAGC-3' (SEQ ID NO: 6) and
5'CTGCAGTTATTTGCCAATGCTGCTT CC-3' (SEQ ID NO: 7). The resulting PCR
product was digested with EcoRI and PstI and the resulting fragment
was cloned into the pMAL.TM.-c2X vector (New England Biolabs,
Beverly, Mass.), creating a recombination fusion to the E. coli
malE gene which encodes the maltose-binding protein (MBP).
[0409] E. coli DH5.alpha. transformed with the resulting plasmid
was grown to a concentration of 2.times.10.sup.8 cells/ml followed
by the addition of isopropyl-D-thiogalactopyranoside (IPTG) to a
final concentration of 0.3 mM. Following incubation of the culture
for 2 hours at 37.degree. C., the cells were harvested by
centrifugation at 4,000.times.g for 20 minutes. The cells were
lysed by freezing overnight at -20.degree. C. and sonicating the
cells for 10 minutes. The expression of a soluble 82 kDa MBP-env
fusion protein in E. coli was confirmed by SDS-PAGE analysis and
Coomassie blue staining. The MBP-env fusion protein was purified
using a maltose-affinity column according to the manufacturer's
instructions. 3 mg of MBP-env protein was obtained from 250 ml of
cell culture. MBP was purified as a control according to the same
protocol.
[0410] The MBP-env fusion protein was used to analyze sera for the
presence of antibodies to the WNV E glycoprotein. 2 .mu.g of
MBP-env fusion protein or MBP (control) protein was boiled in
SDS-PAGE sample buffer (BioRad) containing 2%
.beta.-mercaptoethanol, and run on a 10% SDS-PAGE gel. The
glycoproteins were transferred to nitrocellulose membrane using a
semi-dry electrotransfer apparatus (Fisher Scientific).
[0411] The nitrocellulose membrane was probed with sera from 5
patients with confirmed WNV infection and sera from uninfected
individuals. The membrane was incubated with the sera (1:100
dilution) for 1 hour, then washed 3 times with Tris-buffered saline
with Tween 20 (TBST) and alkaline phosphatase-conjugated goat
anti-human IgG (1:1,000 dilution; Sigma). The blots were developed
with nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate
(Kirkegaard & Perry Laboratories).
[0412] The MBP-env fusion protein detected IgG antibodies to the E
glycoprotein in western blots with sera from 5 humans with
confirmed WNV infection, but not in the control human sera. In
essentially identical experiments, the MBP-env fusion protein also
detects IgM antibodies to the E glycoprotein in western blots with
sera from 5 humans with confirmed WNV infection, and IgG and IgM
antibodies with sera from 10 horses with confirmed WNV infection,
but not in control human or horse sera.
Example 4
Coupling of Recombinant WNV-E Antigen to Polystyrene
Microspheres
[0413] A two-step carbodiimide process, recommended by Luminex
Corporation, Austin Tex., was used to link 50 micrograms of
purified recombinant WNV envelope glycoprotein antigen (WNV-E) to
the surface of 6.25.times.106 microspheres. Activation was
initiated with 50 microliters of 50 mg/ml Sulfo-NHS followed by 50
microliters of 50 mg/ml EDC and a 20 minute incubation at room
temperature. Coupling of the recombinant antigen took place for 2
hours, in the dark, on a rotator at room temperature. Microspheres
were washed by centrifugation, twice, in 1.0 ml PBS Azide blocking
buffer, (PBN) composed of PBS, 1 BSA, 0.02% NaN.sub.3.
Example 5
Stability of WNV-E-Coated Microspheres
[0414] Microspheres coupled to recombinant WN E glycoprotein were
held at 4.degree. C., 25.degree. C., 37.degree. C. and 50.degree.
C. and tested at 1, 3, 5, and 7 days during the first week, then
weekly thereafter for 3 weeks. A plot of the MFI versus time at
25.degree., 37.degree., and 50.degree. C. is shown in FIG. 9.
Thermal stability, as expressed by T90 (time to 90% potency of
reagents), of this key reagent was 0.1 days at 50.degree. C., 0.1
days at 37 C, and 1.5 days at 25.degree. C. A straight line is
obtained when the T90 is plotted (as ordinate) on a semi log scale
against the 1/T Kelvin (abscissa). This is a recommended
calculation for accelerated thermal stability or shelf-life
studies. Interpolation to the desired storage temperature,
4.degree. C., gives an estimated T90 of three months. Performance
of this key reagent, the WNV-E coated microspheres, to within 10%
of maximal reactivity is a realistic expectation for a robust
clinical laboratory assay.
Example 6
WNV-E Microsphere Immunoassay to Detect Antibodies to West Nile
Virus
[0415] A two-step suspension microsphere immunofluorescence assay
was performed. Multiscreen 96-well filter plates with 1.2 .mu.m
Durapore filters (Millipore, Bedford, Mass.) and a Multiscreen
vacuum manifold (Millipore) facilitated microsphere washing.
Briefly, filter plate wells were blocked for 2 min with 100 .mu.l
PBN buffer, washed once with 150 .mu.l PBS-T buffer
(phosphate-buffered saline, pH 7.4, with 0.05% Tween 20 from Sigma
Aldrich), and then wetted with 20 .mu.l PBN. Diluted serum samples
(50 .mu.l, diluted 1:100 in PBN unless otherwise noted) were added
to test wells. IgG-depleted sera were diluted 10-fold during
depletion, and were diluted an additional 10-fold in PBN for
analysis in the rWNV-E MIA with polyvalent secondary antibody
conjugate. IgM-depleted sera were similarly diluted in PBN to a
final serum dilution of 1:100. Antigen-conjugated microspheres
(2,500 in 50 .mu.l PBN) were added to each well. The filter plates
were incubated in darkness on a plate shaker for 30 min at
37.degree. C., and then washed three times with PBS-T using the
vacuum manifold. Diluted fluorochrome-labeled secondary antibody
(50 .mu.l of a 1:250 dilution in PBN) was added to each well.
Unless otherwise noted, the secondary antibody was polyvalent goat
F (ab').sub.2 anti-human immunoglobulins (IgG+IgA+IgM) conjugated
to red-phycoerythrin (R-PE) from Bio-Source International
(Camarillo, Calif.). Alternative secondary antibodies were goat
F(ab').sub.2 anti-human IgG R-PE conjugate and goat F(ab').sub.2
anti-human IgM R-PE conjugate (Bio-Source International). After
incubation for 30 min at 37.degree. C. in darkness with shaking,
filter plates were washed twice with PBS-T using the vacuum
manifold. Microspheres were then resuspended in 125 .mu.l PBN per
well. Seventy five microliter aliquots were transferred to opaque
black EIA/RIA 96-well plates with breakaway strips (Costar,
Corning, N.Y.), and evaluated for microsphere fluorescence
intensity using a Luminex 100 instrument (Luminex Corp.). This
instrument is a dual laser flow analyzer. The first laser excites
the fluorochrome mixture intrinsic to the microspheres, enabling
the bead identity to be determined as the bead passes single file
through the laser path in the flow cell. The second laser excites
the extrinsic fluorochrome (red-phycoerythrin) that is covalently
attached to the reporter antibodies (goat-anti human
immunoglobulins). The dual lasers allow the operator to mix beads
with different antigens together in a well of a filter plate, thus
enabling multiplex analysis of different antibody specificities at
one time.
[0416] The instrument was calibrated with CL1/CL2 and RP1
calibration microspheres from Luminex Corp. according to the
manufacturer's directions. The median fluorescence intensity (MFI)
of fluorochrome-conjugated secondary antibody bound to individual
microspheres was derived from flow analysis of 100 microspheres per
well. Results for each assay were expressed both as MFI and as a
patient/negative (P/N) MFI ratio, i.e., the MFI for the patient's
specimen divided by the MFI obtained from a pool of 10 negative
control sera. The negative control sera contained no detectable
antibodies to WN virus in IgM-capture and IgG ELISAs. Serum MIA P/N
values .gtoreq.4.0 were considered positive for antibodies to WN
virus E protein.
[0417] The rWNV-E MIA was performed on CSF as described for serum
specimens, except that the CSF was tested at a 1:2 dilution,
prepared by addition of 30 ml CSF to 30 .mu.l PBS. IgG-depleted
CSFs, diluted 1:2 during the IgG removal procedure, were assayed
without further dilution. CSF results were reported as MFI values.
CSF with MFI values >426 were considered positive for antibodies
to WN virus E protein.
Example 7
Determination of the Normal Range of Detection of the WNV-E
Microsphere Immunoassay
[0418] Ten sera from cases of West Nile viral encephalitis,
confirmed by plaque reduction neutralization tests with paired
serum sets, and ten sera that were negative in both MAC ELISA and
IgG ELISA WNV antibody tests were examined to establish normal
ranges of detection of the WNV-E microsphere assay using both
positive and negative WNV samples.
[0419] For each serum specimen, a Median Fluorescence Intensity
(MFI) value was established by measuring immunoreactivity with the
WNV-E antigen on 100 individual microspheres. The mean MFI value of
microspheres for the ten negative sera was 247, with a standard
deviation of 74. The mean MFI of ten positive sera was 7625 with a
range from 2763 to 17188, corresponding to range of P/N ratios of
11.1 (2763/247) to 69.6 (17188/247).
Example 8
Covariance Analysis
[0420] Ten sera from West Nile encephalitis patients, in which
infection with WNV was positively confirmed using MAC ELISA, IgG
ELISA and plaque reduction nuetralization (PRN), were pooled ("ten
positive sera"). A similar pool was made using ten human sera
tested negative for WNV human subjects ("ten negative sera").
[0421] To determine the imprecision of the WNV-E microsphere
immunoassay, ten aliquots of each pool were prepared and tested
separately. Imprecision between measurements in the same assay
(intra-assay measurements) of the aliquots of the positive pool
yielded a covariance of 7%. Imprecision between measurements of the
negative serum pool provided a covariance of 11%.
[0422] Further, the pools of ten positive and negative sera were
analyzed over the course of several days to determine the
imprecision between sets of measurements (inter-assay imprecision).
The pool of positive sera provided an inter-assay covariance of
17%, whereas the pool of negative sera provided an inter-assay
covariance of 32%. Results for 70 sera independently analyzed by
two individuals showed that inter-operator correlation of
measurements of 0.995 with a slope of 1.125. Overall, the data
demonstrating that there is relatively low imprecision in carrying
out the WNV-E microsphere immunoassay.
Example 9
Determination of the Specificity of the WNV-E Microsphere
Immunoassay
[0423] Sera from patients with various viral infections, bacterial
infections, or autoimmune diseases were tested in the rWNV-E MIA.
Twenty-four sera from presumed healthy subjects were also
tested--four (17%) were borderline positive with P/N values
<4.9. Only sera from patients with syphilis had a high frequency
of false-positive results (FIG. 35). Sera in the first syphilis
panel were all treponemal antibody positive. In the second syphilis
serum panel, sera were rapid plasma reagin (RPR) negative and less
cross-reactive. Cross-reactive antibodies in syphilis patient sera
were also detected by the WN virus IgG ELISA (data not shown).
However, the syphilitic sera did not contain virus-specific
neutralizing antibodies detectable in WN virus or SLE PRN tests
(data not shown). A BLAST (1) computer search of the T pallidum
genomic database (C. M. Fraser et al.) failed to reveal possible
cross-reactive epitopes.
Example 10
Separate Detection of Anti-WNV-E Glycoprotein IgM or IgG Antibodies
Following Immunodepletion of IgM or IgG from a Serum Specimen
[0424] Separate detection of IgM or IgG antibodies against the WNV
E glycoprotein in selected sera was carried out using an
immunodepletion step of the sera prior to the microsphere
immunoassay. First, selected sera were tested for anti-WNV-E
glycoprotein antibodies using the WNV-E microsphere immunoassay
along with the polyvalent reporter antibody, which detects IgG, IgA
and IgM. Second, the same sera were retested for either anti-WNV-E
glycoprotein IgG or IgM antibodies using an immunodepletion step
prior to the WNV-E microsphere immunoassay. Treatment with goat
anti-human IgG or goat anti-human IgM resulted in the depletion of
either IgG or IgM, respectively, from the human sera.
[0425] To carry out the immunodepletion step for the removal of
IgG, 5 microliters of human serum was treated with 45 microliters
of goat anti-human IgG, (Gull SORB, from Meridian Diagnostics,
Cincinnati, Ohio). According to the manufacturer, this protocol is
sufficient to deplete IgG at concentrations up to 15 mg/ml, the
upper limit of normal human IgG concentration. This 1:10 serum
dilution was mixed and centrifuged at 13,000 rpm in an Eppendorf
centrifuge with a rotor radius of 7.5 cm. The supernatant is used
for making the further 1:100 sample solution for reanalysis in the
microsphere-based immunoassay. Negative IgG ELISA, and negative IFA
tests using slides from Focus Technologies demonstrated that the
Gull SORB pretreatment efficiently removes detectable IgG
antibodies to WNV. Any remaining fluorescence activity after IgG
depletion would represent the relative amount of IgM antibodies
that recognize WNV E glycoprotein. Further, given that IgM
antibodies typically do not persist for long periods of time, but
are the first antibodies to respond to an antigen, any fluorescence
following IgG depletion would indicate a current or recent
infection.
[0426] To carry out IgM immunodepletion, 10 microliters of serum
was treated with 10 microliters of goat anti-human Mu chain
(anti-human IgM), at a concentration of 2.5 mg/ml. Twenty
microliters of PBS was added to the treated serum prior to
centrifugation at 13,000 rpm in an Eppendorf centrifuge as above.
The supernatant was used to make a further dilution to 1:100, which
was then tested in the WNV-E microsphere immunoassay with the
polyvalent conjugate. The remaining fluorescence activity after IgM
depletion represented the relative amount of IgG antibodies that
recognize WNV E glycoprotein.
[0427] IgA antibodies may also be present, but their role in
detection of flavivirus infection is not well documented in the
literature, nor frequently considered.
Example 11
Comparison Between WNV-E Microsphere Immunoassay And ELISA Methods
Following an Immunodepletion Step to Detect Acute Cases of WNV
[0428] It was desirable to detect the presence of IgM antibody to
detect acute cases of WNV infection. The WNV-E microsphere
immunoassay provided strong positive MFI values for many patients'
first serum specimens, indicating that the polyvalent assay
detected IgM as well as IgG antibodies to WNV.
[0429] Five sequential sera from a WNV encephalitis patient were
treated with anti-human IgG (Gull SORB) at a concentration designed
to deplete all IgG reactivity. These treated sera were then tested
again in the polyvalent WNV-E immunoassay.
[0430] The five sequential sera were also treated with anti human
Mu chain (anti IgM) at a concentration calculated to deplete all
IgM reactivity and then reanalyzed with the polyvalent WNV-E
immunoassay.
[0431] The anti-IgG and anti-IgM treated sera were also analyzed in
a WNV-E immunoassay using an anti-IgM R-PE fluorescent conjugate to
detect IgM antibodies. Results of this experiment are presented in
FIG. 12, along with P/N values from the MAC ELISA and the IgG
ELISA.
[0432] The results showed that the Gull SORB treatment (removal of
IgG) increased the P/N correlation coefficient with IgM ELISA assay
from 0.75 to 0.93. Anti-IgM treatment increased the P/N correlation
coefficient with the IgG ELISA assay from 0.92 to 0.99, with
approximately five-fold higher P/N ratios observed with the
microsphere immunoassay. Note that by the traditional assays, the
third serum at 18 days post onset had the maximal IgM reactivity
and the fourth sample at 72 days post onset had the maximal IgG
reactivity.
[0433] Conclusions from WNV-E microsphere immunoassays on the
different sera are comparable. The fourth serum specimen had the
peak antibody reactivity as measured by MFI. Removal of IgG allowed
once again the identification of the third serum as having peak IgM
reactivity both with the polyvalent conjugate and with the anti-IgM
conjugate. Removal of IgM allowed identification of the fourth
serum as having the most IgG.
[0434] Overall, the results showed that the WNV-E microsphere
immunoassay, unlike the MAC and IgG ELISAs, provided an IgM (IgG
depleted) P/N ratio that was greater than the P/N ratio of the IgG
(IgM depleted) sample for the early bleeds. This relationship may
be an indicator of an active or recent infection. The use of the
anti-IgM R-PE conjugate on the anti-IgM treated sera demonstrated
that the IgM was effectively depleted to a level in the negative
range (P/N<4.0) according to our established assay
conditions.
Example 12
Comparison of the WNV-E Microsphere Immunoassay to Standard ELISAs
by Retrospective Parallel Testing
[0435] Archived sera at the New York State Department of Health
provided an opportunity to parallel test a larger panel of sera
submitted for suspected viral encephalitis. The objective of this
study was to determine whether a cut-off P/N value of 4.0 would
provide test results concordant with the MAC ELISA and IgG ELISA
previously used to screen the sera for antibodies to WNV.
[0436] FIGS. 11A and 11B provide scatter plots with polyvalent
WNV-E microsphere immunoassay P/N vs. IgG ELISA P/N and/or
MAC-ELISA P/N with trendline.
[0437] Out of 107 total sera tested, 20 West Nile reactive sera,
identified previously by the MAC ELISA and or the IgG ELISA, were
also correctly identified by the WNV-E microsphere immunoassay.
Seven sera of 107 tested were just above the cut-off on the WNV-E
microsphere immunoassay, whereas they were non-reactive in both of
the traditional ELISA assays. Since these seven sera were
non-reactive in the ELISAs, no follow up sera were provided to
allow us to definitively rule out infection. The mean of the MFI
for the 20 positive sera by traditional assays was 7804 (range
1084-21038). The mean of the borderline/equivocal samples was 1333
(range 1084-2118). The mean of the 53 sera that tested negative was
349 (range 49-607).
Example 13
Detection of Antibodies to Japanese Encephalitis Vaccine Using the
WNV-E Microsphere Immunoassay
[0438] Retrospective testing of the serum bank of the Wadsworth
Center for flavivirus-reactive antibodies, demonstrated that the
WNV-E microsphere immunoassay could detect antibodies to three
flaviviruses in the Japanese encephalitis serogroup.
[0439] Twenty four human sera were received from the Arbovirus
Research Laboratory of the Wadsworth Center, with all identifiers
as to identity of the recipients of the Japanese Encephalitis (JEV)
vaccine status or time of vaccination. This blinded serum panel
consisted of twelve post-vaccine specimens (collected in June,
2002) and eight pre-vaccine sera (collected in April, 2001) from
eight of the twelve vaccine recipients (the pre-bleed sera of these
employees was not found in the freezer archives.) A further four
serum samples were from new employees who had not received the
vaccine, and who lacked an exposure history to WNV from dead birds
or mosquitoes. These sera were tested by the microsphere
immunoassay employing the polyvalent R-PE anti-human
immunoglobulins conjugate. After testing the blinded specimens, the
pre-vaccine specimens were matched with the post-vaccine specimens,
and plaque reduction neutralization titers for the post-vaccine
sera were obtained.
[0440] Results on the polyvalent WNV-E microsphere immunoassay are
given in FIGS. 13A and 13B. Note that where pre and post samples
were available, 6 of 8 employees made a large increase in
detectable antibodies.
[0441] Since neutralizing protective antibodies are primarily IgG
class, we treated all 24 sera with anti-IgM at a concentration
calculated to provide an optimal proportion to deplete all IgM. We
repeated the assay with the polyvalent red-phycoerythrin anti human
immunoglobulin conjugate on the IgM-depleted sera. As expected,
FIG. 14B demonstrates that the MFI were lower than in the untreated
samples, yet clearly were positive in all but two vaccine
recipients. The two vaccine recipients with negative post-vaccine
MFI levels were the two employees who lacked a detectable
neutralizing antibody response by plaque reduction neutralization
testing.
[0442] The results demonstrated that the WNV-E microsphere
immunoassay could detect antibodies to three flaviviruses in the
Japanese encephalitis serogroup.
Example 14
Detection of WNV Antibody from Serum and Spinal Fluid Samples from
Patients with Acute Viral Encephalitis Using WNV-E Microsphere
Immunoassay as Compared to Results from MAC ELISA
[0443] Seven pairs of serum along with same-day collected spinal
fluid specimens from seven patients were tested using the
recombinant WNV-E microsphere immunoassay using both the polyvalent
antibody reagent and the "IgM" serum (anti-IgG treated serum). The
seven patients were chosen on the basis of having been tested
positive for WNV by either an IgM and/or an IgG ELISA using the
reagents and protocol recommended by the CDC. The data are
presented in FIG. 17.
[0444] The results showed that both patients with confirmed WNV
infection by PRN testing, had high levels of detectable antibody in
spinal fluid, as detected by the WNV-E microsphere immunoassay.
Further, 5 patients who were shown to test negative for a WNV
infection by MAC ELISA were shown to be strongly positive by the
WNV-E assay.
[0445] The data from the paired serum and spinal fluid testing
demonstrated the high sensitivity of the WNV-E assay since the P/N
values of the WNV-E microsphere immunoassay are significantly
greater than the P/N values of the MAC ELISA. The data further
showed that WNV-E microsphere immunoassay is superior to the MAC
ELISA since the WNV-E assay was able to detect a WNV infection in 5
patients who were shown not to have an infection by the MAC
ELISA.
Example 15
Expression and Purification of NTPase/Helicase domain of NS3 and
NS5
[0446] WNV nonstructural proteins NS3 and NS5 were tested as
targets to develop a novel serologic assay for WNV diagnosis. NS3
and NS5 are key enzymes in flavivirus RNA replication. NS3
functions as a serine protease (in the presence of cofactor NS2b),
5'-RNA triphosphatase, NTPase, and helicase; NS5 functions as a
methyltransferase and RNA-dependent RNA polymerase (RdRp). Since
the NS proteins are primarily involved in flavivirus replication,
the immunogenic features of the NS proteins during WNV infection
would be different from those of viral structural proteins. These
unique features could be exploited to improve the current
structural protein-based serologic assay.
[0447] The NTPase/helicase domain (amino acids 182 to 619) of NS3
(see FIG. 23B) and full-length NS5 (see FIG. 23C) of WNV were
expressed and purified using an E. coli expression system as
follows. The NTPase/helicase domain of NS3 (amino acids 182 to 619)
and full-length NS5 were cloned into the pET-21a and pET-28a
vectors, respectively, and expressed in E. coli BL21 cells upon
induction with isopropyl-.beta.-D-thiogalactopyranoside (IPTG) at
30.degree. C. for 3 to 4 h. The recombinant NS5 and NS3
NTPase/helicase domain contained a His.sub.6 tag at the N-terminus
and C-terminus, respectively, and were purified through a nickel
column (Novagen, Madison, Wis.). The NTPase assay was performed as
previously described (Cui, T. et al.). The RdRp activity of NS5 was
assayed using a WNV subgenomic RNA transcript containing a large
deletion from nucleotide 269 to 10408. The reactions were labeled
with [.alpha.-.sup.32P]UTP and analyzed on a 4% denaturing
polyacrylamide gel followed by autoradiography (Ackermann, M. et
al.).
[0448] The recombinant proteins were enzymatically active: the NS3
protein exhibited an NTPase activity in hydrolyzing ATP to ADP and
phosphate (FIG. 23D); and the NS5 protein retained the RdRp
activity, using WNV RNA as a temple to synthesize both
double-stranded RNA (a replicative 2.times. form) and
single-stranded RNA (1.times. form) (FIG. 23E). The enzymatic
activities of WNV NS3 and NS5 are comparable to those of DNEV NS3
and NS5. The enzymatic activities indicate retention of native
conformation by the recombinant NS3 and NS5.
Example 16
Microsphere Immunoassay (MIA) to Test NS5 in Serologic Assay
[0449] A microsphere immunoassay (MIA) was selected to establish
the NS3- and NS5-based serologic assays to detect antibodies
induced by WNV infection. Recombinant NS3 or NS5 was covalently
linked to microsphere beads, and then reacted with patient serum
followed by anti-human immunoglobulins with a fluorescent
conjugate. The levels of reactive antibodies from the sera were
quantified by a flow analyzer. Initially, 20 human sera from
healthy individuals were used to establish cutoff levels for the
assay. The mean median fluorescence intensity (MFI) for NS3 was 909
[standard deviation ("SD") 351], with an assay cut-off (X+3SD) of
1962; the mean MFI for NS5 was 1810 (SD 852), with an assay cut-off
(X+3SD) of 4366. Analyses of five positive WNV sera, which had been
previously confirmed by a subviral particle-based immunoassay
(Davis, B. et al.) and PRNT, revealed that the NS5 MIA had an assay
dynamic range of 32, from 100- to 3200-fold serum dilutions. The
NS3-based MIA did not exhibit consistent signals above the
background level with these sera (see below).
Example 17
NS5-Based MIA Reliably Detects WNV Infection and May Indicate
Recent Infection
[0450] A total of 61 sera from WNV patients with clinical symptoms
and confirmation by PRNT were tested using to NS5- and NS3-based
MIA, along with the recombinant E protein-based MIA for comparison
(S. J. Wong et al., J Clin Microbiol., 2003, 41:2127-2223). The
plot of MFI versus days post symptom onset (FIG. 24A) shows that
the NS5-reactive signals appeared on day 6; the MFI of 35 of 38
(92%) sera collected from day 7 to day 77 were positive; and the
MFI dropped to a negative level for two sera collected on day 259
and day 431. The reactive pattern derived from the NS5-based assay
correlated well with that from the E-based assay, except that, in
the latter assay, reactive signals appeared around day 2 to day 6,
and the MFI remained positive throughout the later time points,
including day 259 and day 431 (compare FIG. 24A with 24C). On the
other hand, the NS3 MIA did not exhibit consistent signals above
the background level, with less than half of the sera showing
positive MFI (FIG. 24B); it therefore was not further analyzed.
These results demonstrate that the NS5-based MIA is a sensitive
assay for detection of human WNV infection.
Example 18
Persistence of Anti-E and Anti-NS5 Antibodies
[0451] To examine the persistence of antibody against WNV E
glycoprotein and NS5 antigen upon WNV infection, we examined a
series of sera collected from a single patient at various time
points post-infection (FIG. 24D). Positive MFI signals were
detected on day 17 post symptom onset in both E and NS5 MIA.
Signals from the E-based MIA remained positive for sera collected
on days 71, 259, and 431 post-symptom onset (indicated by dashed
lines in FIG. 24D). In contrast, signals from the NS5-based MIA
were positive for sera collected on day 17 and 71 post-symptom
onset, however, the MFI declined to a negative range on day 259 and
431 post-symptom onset (indicated by solid lines in FIG. 24D).
These results suggest that a positive NS5-based MIF indicates
current or recent infection.
[0452] The NS5 MIA can likely be used to indicate the timing of WNV
infection. Time-course analysis of WNV patient sera showed that,
after serum conversion at approximately day 6 post symptom onset,
the anti-E antibody signal remained highly positive up to 431 days
post symptom onset (FIGS. 24C and 24D), while antibodies against
NS5 diminished to a negative level between 71 and 259 days post
symptom onset (FIGS. 24A and 24D). More clinical samples at late
time points post-infection are required to confirm this conclusion.
Although not wishing to be bound by theory, since NS5 protein is
only present during viral replication and associates with the
replication complex located at the cytoplasmic side of the
endoplasmic reticulum, NS5 may be more accessible to protein
degradation, resulting in a shorter half-life in cells than the
membrane-spanning E protein. It is also possible that antibodies
generated in response to NS5 are of shorter duration than the
anti-E antibodies.
Example 19
Specificity of NS5-Based MIA
Differentiation of WNV from Other Nonflavivirus Infections or
Diseases and from Flavivirus Vaccination
[0453] The specificity of the NS5-based MIA was demonstrated by
challenging 120 sera from patients with various infections,
autoimmune conditions, JEV vaccination, YFV vaccination, or good
health (FIG. 25). Only one patient with HIV infection showed an MFI
(7,517) above the cut-off level of the NS5 MIA (4,366). It should
be noted in particular that none of the sera from the JEV vaccine
recipients reacted with the WNV NS5 antigen; only 1 of 19 (5%) YFV
vaccine recipients exhibited a positive MFI signal. By contrast,
all 10 (100%) JEV-vaccinated sera and 10 of the 19 (53%)
YFV-vaccinated sera showed positive MFIs in the E-protein-based
MIA. These results demonstrate that the NS5-based assay can be used
to differentiate between WNV infection and vaccinations with either
an inactivated (JEV) or a live attenuated (YFV) flavivirus.
flavivirus
Example 20
Cross-Reactivity of WNV NS5 and E with DENV or SLEV Infections
[0454] The cross-reactivity of WNV NS5 and E with DENV infection
was tested with 17 pairs of acute and convalescent sera from
DENV-infected individuals (FIG. 26). The DENV patient sera reacted
with WNV E protein. The MFI signal and the titer of the E MIA
correlated well with the hemagglutination inhibition (HI) titer of
the sera. Twenty-four of the 34 (71%) DENV sera tested positive in
the E-based MIA; 8 samples with negative E MIA results were either
HI negative or showed low HI titer. For the NS5-based MIA, only 3
of the 34 (9%) DENV sera were marginally positive (samples 3A, 4B,
and 11A), with MFI values very close to the cut-off value. Next, we
examined the potential cross-reactivity of WNV NS5 and E with SLEV
patient sera. Among the 20 pairs of SLEV sera that had been
previously confirmed by plaque reduction neutralization tests, only
2 (5%) sera were MFI positive (samples 3A and 3B) in the WNV
NS5-based assay, while 11 of the 40 (27.5%) SLEV sera were positive
in the E-based assay (FIG. 27). These results suggest that,
compared with the E protein-based MIA, the NS5-based MIA exhibits
substantially improved discrimination between DENV/SLEV and WNV
infections.
Example 21
Three Advantages for NS5 Immunoassays as Compared to WNV E
Immunoassays Flavivirus
[0455] flavivirus To improve the specificity of the diagnosis of a
flavivirus infection using the WNV E glycoprotein, the RNA
replication NS proteins were tested as an alternative to the WNV E
glycoprotein for serologic diagnosis of WNV infection. The active
NTPase/helicase domain of NS3 and full-length NS5 were expressed
and purified. The NS5 protein-although not the NS3 NTPase/helicase
domain-reacted consistently with WNV patient sera. Contrary to the
WNV E glycoprotein, the NS5 when used in the immunoassays (MIA) of
the present invention can provide the following three
advantages.
[0456] First, unlike the WNV E-based MIA, the NS5-based MIA
reliably discriminates between WNV infection and DENV (FIG. 27) or
SLEV infections (FIG. 26) only 3 of the 34 DENV sera and 2 of the
40 SLEV sera showed weak NS5 MFI signals. On the other hand, WNV E
protein cross-reacts with both DENV (26 out of the 34) and SLEV (11
out of 40) patient sera. These results appear to be consistent with
a previous report suggesting that NS antigens can be viral type
specific, whereas structural antigens can be cross-reactive among
flaviviruses (Qureshi, A. A. et al.). However, the ordinary skilled
person in the art would certainly appreciate that one could not
reasonably know or predict the specificity of the WNV NS5 to
anti-WNV sera, indeed as shown by the present inventors, without
providing proof thereof by way of appropriate and necessary
experimentation.flavivirus
[0457] There are likely at least two reasons why the NS5-based
immunoassay shows greater specificity for WNV detection than WNV
E-based immunoassays. First, notwithstanding that the amino acid
sequence homology of NS5 between WNV and DENV (75%) or SLEV (47% %)
could be as high as that of E protein between WNV and DENV (62%) or
SLEV (78% %), epitopes (either structure or sequence) presented by
WNV E could be more conserved among the flaviviruses than those in
the NS5, resulting in greater cross-reactivity in the WNV E-based
assay. Alternatively, the specificity of the WNV NS5-based assay
could have been a consequence of a failure an NS5 immune response
during DENV and SLEV infections. This is unlikely because partially
purified NS proteins of DENV, SLEV, or WNV were demonstrated to be
reactive with only homologous sera, but not with heterologous sera,
indicating the production of antibodies against the NS proteins
during infections (Qureshi, A. A. et al.). Nevertheless, the
specificity of the NS5-based assay may eliminate the need for
plaque reduction neutralization tests, and therefore the
requirement of Level 3 Biocontainment, to discriminate among
infecting flaviviruses. Quick and accurate differentiation between
WNV and DENV/SLEV infections will be important in diagnosing
specimens where WNV co-circulates with DEN and/or SLEV viruses.
[0458] Second, the NS5 MIA differentiates between vaccination with
inactivated flavivirus and natural WNV infection. None of the
JE-vaccinated sera reacted with the WNV NS5. This feature was
expected, because only replicative viruses produce NS proteins,
while inactivated JE vaccines could not replicate and produce NS
proteins. Distinguishing between vaccination and natural viral
infection is important for WNV diagnosis in geographic regions
where inactivated JE vaccination is performed, or in vaccinated
military personnel or travelers. For the same reason, the NS5 MIA
will be useful for testing whether horses previously vaccinated
with inactivated WNV (Davis, B. et. al., Monath, T.) have
encountered a new round of WNV infection.
[0459] Third, the NS5 MIA could potentially be used to indicate the
timing of WNV infection. Time-course analysis of WNV patient sera
showed that, after serum conversion at approximately day 6 post
symptom onset, the anti-E antibody signal remained highly positive
up to 431 days post symptom onset (FIGS. 24C and 24D), while
antibodies against NS5 diminished to a negative level between 71
and 259 days post symptom onset (FIGS. 24A and 24D).
[0460] Overall, the unique features of the NS5-based immunoassay
will be very useful for both clinical and veterinary diagnosis of
WNV infection. The MIA assay format used in this study is highly
sensitive (flow-cytometry based), has a rapid turnaround time (3 to
4 h for testing 96 specimens), and is cost-effective (approximately
50 tests per microgram of recombinant protein). More importantly,
the MIA format allows the performance of multiplex assays to detect
antibodies against E and NS5 proteins in a single tube, allowing
simultaneous primary and confirmatory diagnosis.
Example 22
Animal Studies Show that Antibody Levels to Ns Protein Decline Over
Time while Antibodies to Structural Proteins Increase Over Time
[0461] Animal model studies of WNV infection have added proof of
the concept that antibody levels to nonstructural proteins decline
while antibodies to structural proteins are increasing. In an
experimental mouse model of infection where sequentially timed
serum samples were drawn at 5, 10 and 28 days post infection, total
antibodies to the WNV E, a structural protein, were still
increasing at day 28 whereas the total of antibodies to the NS5
protein was decreasing. In a similar manner, IgM antibodies to WNV
E were still increasing in the day 28 sample, whereas the IgM
antibodies to NS5 were lower at 28 days than in the day 10 sample.
Thus, NS5 appears to be a useful antigen to screen for WNV
infections at an early and/or acute stage.
Example 23
Avian Response to Flavivirus Infection is Strain Specific
[0462] An evaluation of West Nile antibodies in wild birds of
various orders and species, has demonstrated that some birds made
much higher antibody responses to NS5 than to WNV E protein. For
surveillance activities, a bird that has antibodies to NS5 must
have been infected in the recent past, where as a bird that only
has antibodies to WNV E only has evidence of infection in the
remote past. Wild birds (house finches and morning doves) with SLEV
infection made low to moderate antibody responses to WNV E, whereas
they made no response to WNV NS5. This indicates that the avian
response to flavivirus infections is strain specific, as we have
demonstrated in humans. Antibodies to WNV NS5 indicate recent
infection with West Nile virus, whereas antibodies to WNV envelope
protein indicate infection at some time with one of many
flaviviruses.
Example 24
High Antibody Response to NS5 in Naturally Infected Horses
[0463] An evaluation of West Nile antibodies in naturally infected
horses demonstrated high antibody responses to NS5, often greater
than to Envelope protein. Antibodies generated by the Ft. Dodge
inactivated West Nile vaccines were only to the Envelope protein.
Horses with no infection and no vaccination were negative to
Envelope and to NS5. Therefore, a high antibody level to NS5 in a
vaccinated horse means active, recent infection. The duration of
protective antibodies from the vaccine is short, and antibodies
drop off within two months of the last dose of vaccine.
Example 25
High Levels of NS5 Antibodies Even in the Absence of High IgM to
WNV May Indicate Recent WNV Infection
[0464] A serosurvey of 871 solid organ transplant recipients has
identified about 85 persons with antibodies to Envelope protein.
Only 5 of these persons have IgM to WNV E protein, indicating
current WNV infection. Ten of the 85 persons have high levels of
antibodies to NS5. Studies are currently underway to demonstrate
that these patients have evidence of recent WNV infection despite
the lack of IgM to WNV.
Example 26
Enzyme-Linked Immunosorbant Assay (ELISA) Using WNV NS50or E
Antigens
[0465] The NS5 protein can be used to detect human antibodies
specific for NS5 from blood. More in particular, the NS5 protein
can be used in connection with an ELISA to detect NS5-specific
antibodies. Positive detection of NS5-specific antibodies would
indicate a more recent WNV infection since the antibodies to NS5
show limited persistence in the blood; thus anti-NS5 antibodies are
more likely to be present at higher titers early during infection
(see Example 18). The WNV E glycoprotein, owing to the fact that it
shows consistent, reliable, and predictable cross-reactivity
against antibodies to other flavivirus, in particular JEV, SLEV,
and DENV, can be used in an ELISA to detect a flavivirus
infection.
[0466] Although any known ELISA format is contemplated by the
present invention, one standard ELISA assay format assay, the
"three layer sandwich", can be used. In this format, NS5 or WNV
antigen are passively adsorbed to polycarbonate microtiter plates.
The remaining reactive sites on the plate are then blocked with a
solution of serum protein, serum albumin, non-fat dry milk,
gelatin, or detergent (e.g. Tween, Triton X-100, SDS). The antigen
coated plates are then incubated with a dilution of a patient's
serum. If the patient was infected with WNV, anti-WNV E or anti-WNV
NS5 antibodies bind to the WNV E or NS5, respectively, on the
plate. Following several washes, the plate is incubated with
goat-anti-human antibody conjugated to an enzyme such as alkaline
phosphatase or horse radish peroxidase. Following several washes,
the plate is developed with a chromogen (substrate for the enzyme).
The color development is read on a microtiter plate
spectrophotometer. The deepness of the color is proportional to the
amount of human antibody to NS5 in the sample applied to the
plate.
[0467] Another ELISA format contemplated by the instant invention
is the IgM antibody capture ELISA. In this assay, Goat anti-human
IgM is used to coat the microtiter plates. After blocking reagents
are added, such as solutions of serum protein, serum albumin,
non-fat dry milk, gelatin, or detergent (e.g. Tween, Triton X-100,
SDS), a dilution of human serum is added to the wells in the plate.
The goat-anti human IgM captures the patient's IgM. Subsequently,
WNV E or NS5 antigen would be added to the wells. After an
incubation period, the unbound antigen is washed away. Next,
biotinylated monoclonal antibody to WNV NS5 or E is added to wells.
Following an incubation period, the unbound monoclonal antibody is
washed away. Streptavidin linked to a detector enzyme is then added
to the wells of the plates. Following washing steps, the unbound
Streptavidin conjugate is washed away. The assay is developed with
a chromogen and read on a microtiter plate spectrophotometer. The
amount of color developed is proportional to the amount of IgM the
patient had to West Nile NS5.
[0468] The WNV NS5-based and E-based ELISAs can be run separately
or in parallel. It will be appreciated that a positive result in
the E-based assay is indicative of a flavivirus infection,
including for example detection of WNV, SLEV, DENV, or JEV. It will
be further appreciated by one of ordinary skill in the art that the
teachings of the present invention demonstrate by experiment that
substantially purified WNV E glycoprotein antigen having a
substantially authentic conformation is reliably, consistently,
predictably, and strongly cross-reactive to antibodies against any
of WNV, JEV, SLEV, and DENV, and is therefore useful to broadly
assay or test for flavivirus infection. A positive result based on
the NS5 ELISA indicates with specificity a WNV infection, in
particular a recent infection.
[0469] It is again to be appreciated by one of ordinary skill in
the art that any ELISA format currently used to detect WNV
antibodies in serum or spinal fluid or other biological samples,
can easily be adapted to detect antibodies to the NS5 or E antigens
therein. The information obtained with the NS5 assays is more
useful for identifying recent WNV, since the antibody response to
NS5 is of shorter duration that the antibody response to the E
glycoprotein. Further, information obtained with the E glycoprotein
is more useful for identifying a general flavivirus infection, in
particular, a recent or non-recent infection or flavivirus
vaccination with WNV, JEV, SLEV, or DENV.
Example 27
Microsphere Immunofluorescence Assay Parameters
[0470] Recombinant WN virus envelope protein (rWNV-E) conjugated to
fluorescent microspheres provided the basis for a novel immunoassay
to detect antibodies induced by flavivirus infection. The MIA
quantitatively measures anti-E protein antibodies binding over a
broad range of antibody concentrations (FIG. 31). A standardized,
2.5 h MIA procedure was developed to detect antibodies to WN virus
E protein in .ltoreq.30 ml of human serum or CSF, diluted 1:100 and
1:2 respectively. Performance of the suspension assay at 37.degree.
C. with continual shaking enhanced assay kinetics.
Antigen-conjugated microspheres exhibited long-term stability when
stored at 4.degree. C. Conjugated microspheres were held at
4.degree. C., 25.degree. C., 37.degree. C., or 50.degree. C.
Reactivities of the rWNV-E microspheres with a positive control
serum were tested at several intervals during a 35-day storage
period. Thermal stability of this key assay component (expressed as
time to 90% potency), was observed to be <one day at 37.degree.
C. and 50.degree. C., 3.1 days at 25.degree. C., and >35 days at
4.degree. C. The immunoreactivity of antigen-conjugated
microspheres is stable for >4 months when used in serial MIAs
(data not shown).
[0471] MIA ranges for positive and negative control sera were
established by evaluation of 20 human sera. Ten negative control
sera had no detectable virus-specific antibodies in WN virus
IgM-capture and IgG ELISAs. The mean microsphere MFI for these sera
was 247.+-.74, establishing MFI.gtoreq.988 (P/N.gtoreq.4.0) as a
cutoff for a positive result. The 10 sera from WN viral
encephalitis patients all tested positive for antibodies to WN
virus E protein. The mean MFI for the patient sera was
7,626.+-.4,312 (P<0.001; range 2,763 to 17,188) corresponding to
a mean P/N ratio of 30.8.+-.17.4 (range 11.2 to 69.4). MIA results
from repeated experiments were compared to determine inter-operator
reproducibility. For intra-assay imprecision studies, 10 aliquots
of a WN virus encephalitis patient serum pool and 10 aliquots of a
negative control serum pool were tested separately in the rWNV-E
MIA. Intra-assay imprecision of the positive pool provided a
coefficient of variation (CV) of 7%. Intra-assay imprecision of the
negative serum pool provided a CV of 11%. These same negative and
positive serum pools were analyzed on several days for estimates of
inter-assay imprecision. The inter-assay CVs for the positive serum
and negative serum pools were 17% and 32%, respectively. MIA
results for 91 positive and negative sera independently analyzed by
two individuals demonstrated inter-operator assay reproducibility
(kappa=0.85; P/N r.sup.2=0.99; slope=1.12).
Example 28
Anti-E Protein-Based Microsphere Immunoassay (MIA) Detects
Antibodies to Related Flaviviruses
[0472] Testing of a coded serum panel revealed that the rWNV-E MIA
detects human antibodies elicited by SLEV and DENV (FIG. 34). The
serum panel, provided by CDC Arbovirus Diseases Branch, included
sera from patients infected with WNV, SLEV or DENV. Ten of 19 sera
in the panel were positive in the rWNV-E MIA (mean P/N
25.75.+-.20.26; range, 4.28 to 55.23) using P/N>4.0 as a MIA
positive cut-off value. Decoding of the serum panel revealed that
the rWNV-E MIA detected 10 of 12 sera from flavivirus-infected
individuals (kappa=0.79). All six sera from WNV encephalitis or
DENV fever patients (FIG. 34) were positive. The MIA also
identified four sera from patients infected with SLEV (FIG. 34).
Two sera from patients infected with SLEV were negative in the
rWNV-E MIA. These two sera were obtained within one day after
disease onset, when significant anti-SLEV antibody titers may not
be present. The MIA produced no false positive results with seven
sera negative for neutralizing flavivirus antibodies in PRN assays
(FIG. 34). One negative control specimen consistently tested false
positive in IgM-capture ELISAs. Comparison of WNV ELISA results for
negative control sera and sera containing anti-WNV antibodies (FIG.
34) indicated inter-laboratory agreement for IgG ELISA (kappa=1.00;
P/N r.sup.2=0.98) and IgM-capture ELISA (kappa=0.80; P/N
r.sup.2=0.94) results.
[0473] The rWNV-E MIA detects antibodies elicited by JE-VAX.TM.,
the licensed JE virus vaccine. Sera from eight individuals with
occupational exposure to flaviviruses were collected before and
after vaccination with JE-VAX.TM.. Mean polyvalent rWNV-E MIA
values were 4.2.+-.4.5 for pre-vaccination sera, and 13.3.+-.12.7
for post-vaccination sera (P<0.05). The vaccination induced JEV
neutralizing antibodies in six vaccinees that were detectable in
JEV PRN tests.
Example 29
Microsphere Immunoassay (MIA) Assessment of the Humoral Response to
WNV Infection
[0474] Several WN virus antibody detection methods were compared
using serial serum samples from a patient with WN virus infection
(FIG. 32). The polyvalent MIA, which detects both IgM and IgG
(Panel A) and the ELISAs (Panel B) were first used to evaluate the
specimens. The MIA procedure was then modified to measure IgM
antibodies to WN virus E protein. Sera were depleted of IgG with
goat anti-human IgG, and analyzed with polyvalent R-PE-conjugated
detector antibody or with IgM-specific conjugate. These two
detection systems yielded equivalent P/N results (FIG. 32, PanelA;
r.sup.2=0.998; slope=1.14) that correlated with P/N values obtained
with the IgM-capture ELISA (r.sup.2=0.85). Each method detected
maximal IgM reactivity in the serum specimen obtained 18 days post
disease-onset.
[0475] IgG antibodies to WN virus E protein in the serial serum
samples were also evaluated by MIA (FIG. 32, Panel A). Removal of
IgM with anti-human IgM enhanced the correlation between the MIA
and IgG ELISA P/N values (r.sup.2=0.45 and 0.98 before after IgM
depletion, respectively). The MIA detected maximal IgG reactivity
72 days after disease onset whereas the IgG ELISA P/N value was
highest at 430 days post onset. In convalescent specimens obtained
at days 72 and 260 post-disease onset, anti-IgG treatment depleted
>80% of rWNV-E MIA reactivity. Anti-IgG treatment of the day 18
specimen depleted only 11% of the MIA reactivity. These data
indicate that this patient's IgM response to WN virus infection
predominated only during acute infection.
Example 30
rWNV-E MIA analysis of SERA from Patients with Suspected Viral
Encephalitis
[0476] New York State Department of Health serum archives provided
an opportunity to evaluate 833 sera from patients with suspected
viral encephalitis in the rWNV-E MIA. With P/N>4.0 used as a
positive cutoff, 188 (23%) sera were positive in the MIA (mean P/N
18.3.+-.15.8; range 4.07 to 122). A polyvalent (IgM+IgG) MIA result
was obtained for each of the 833 sera. In contrast, IgG ELISA
results for 131 (16%) sera were reported as "uninterpretable" due
to high non-specific background in negative control ELISA assays.
(A. J. Johnson et al., D. A. Martin et al.). One hundred fifty-one
(18%) of the 833 sera were positive in the IgG ELISA (mean P/N
11.51.+-.5.96; range 3.08 to 27.4). The MIA had high sensitivity
(0.94) and specificity (0.95) for sera with anti-WN virus IgG
antibodies detected by the
[0477] IgG ELISA (positive predictive value=0.829; negative
predictive value=0.98, Positive and negative test results for the
two assays were concordant (kappa=0.85). FIG. 33 compares MIA and
IgG ELISA P/N results for sera with interpretable IgG ELISA
results.
[0478] IgM capture ELISA data were available for 806 of the 833
sera from patients with suspected viral encephalitis. Ninety-six
(12%) sera were positive in this assay (mean P/N 12.49.+-.4.13;
range 7.18 to 25.9). Seven hundred (87%) sera were negative. Ten
sera (1%) provided nonspecific results. The polyvalent MIA detected
80 (83%) of the 96 sera that were positive in the IgM-capture
ELISA. Sera positive in the standard polyvalent MIA (n=172) were
re-assayed after depletion of IgG to measure IgM antibodies to WN
virus E protein. 80 (46%) sera were positive after removing IgG
(mean P/N 19.5.+-.29.9; range 4.00 to 178). The IgG-depleted MIA
sensitivity (0.61) and specificity (0.64) for sera positive in the
IgM-capture ELISA (kappa=0.25) suggested that capturing IgM
antibodies may enhance anti-WN virus IgM assay sensitivity.
However, twenty-three (36%) of the 64 discordant samples were
positive in the IgG-depleted MIA, positive in the IgG ELISA, but
negative in the IgM-capture ELISA. These samples apparently have
IgM antibodies to WN virus E protein not detected by the
IgM-capture ELISA. Forty-three (5%) of the 833 sera from patients
with suspected flavivirus infection tested positive (mean P/N
7.28.+-.5.74; range 4.07 to 39.5) in the polyvalent rWNV-E MIA, but
were negative or uninterpretable in IgM-capture and IgG ELISAs.
These sera were initially identified as non-reactive or
non-specific in the ELISAs, and no diagnostic or clinical follow up
was performed that could rule out WN virus infection. The combined
data indicate that the rWNV-E MIA has >95% specificity in
detecting flavivirus antibodies in sera from patients with
suspected viral encephalitis.
Example 31
WNV E-Based Microsphere Immunoassay (MIA) Detects Anti-Flavivirus
Antibodies in Cerebral Spinal Fluid (CFS)
[0479] Thirty-five CSF were evaluated in the rWNV-E MIA after
depletion of IgG from the specimens. Twenty negative control CSF
(mean MFI 69.+-.119; range 5 to 540) were evaluated, establishing
MFI >426 (3 standard deviations above the mean) as a cutoff for
a positive CSF result. The MIA was then used to evaluate CSF from
15 encephalitis patients with flavivirus infection confirmed by
serum PRN tests (FIG. 36). Twelve specimens (80%) were positive in
the MIA before and after depletion of IgG, including eight CSF from
patients infected with WNV, two CSF from patients infected with
dengue virus, and two CSF from patients infected with an
unidentified flavivirus. For most of these CSFs, depletion of IgG
minimally reduced MFI values (FIG. 36), indicating that the anti-E
protein antibodies were predominately IgM. MIA-negative CSF 9 and
10 were from patients with WN virus-specific serum antibodies. PRN
tests of acute and convalescent sera indicated that these two
patients did not have active flavivirus infection.
[0480] Paired CSF and serum obtained on the same day were available
for Patients 1-6. These patients had WN virus infection confirmed
by serum ELISA and PRN tests. All six CSF were positive in MIAs
(FIG. 36). In contrast, only one of these CSF, from Patient 2, was
positive in IgM-capture ELISAs (data not shown).
Example 32
WNV NS5-Based Immunoassay Determines Whether Previously-Vaccinated
Horse has Sustained New Exposure to WNV
[0481] Using the WNV NS5-based immunoassay, in particular the
microsphere immunoassay (MIA), a determination can be made as to
whether the horse has been exposed and infected with WNV. Compared
with live attenuated virus vaccine, the duration of protective
antibody in response to "killed" WNV vaccination is relatively
short. Thus, there exists an ongoing risk that the horse could be
infected with WNV upon exposure or reexposure thereto. In other
words, since protective immunity wanes quickly, veterinarians may
be increasingly challenged to diagnose neurological illness that
could be due to WNV infection in previously WNV-vaccinated horses.
Such diagnosis will be problematic for structural protein-based
assays, such as the WNV E-glycoprotein assay, due to the presence
of preexisting antibodies to the structural protein as a result of
the immune response to the vaccination.
[0482] A two-step suspension microsphere immunofluorescence assay
will be performed. Multiscreen 96-well filter plates with 1.2 .mu.m
Durapore filters (Millipore, Bedford, Mass.) and a Multiscreen
vacuum manifold (Millipore) facilitated microsphere washing.
Briefly, filter plate wells will be blocked for 2 min with PBN
buffer. Diluted serum samples (for example, 50 .mu.l, diluted 1:100
in PBN) will be added to test wells. IgG-depleted sera will be
diluted 10-fold during depletion, and will diluted an additional
10-fold in PBN for analysis in the rWNV-NS5 MIA with polyvalent
secondary antibody conjugate. NS5-antigen-conjugated microspheres
(2,500 in 50 .mu.l PBN) will be added to each well and incubated.
Diluted fluorochrome-labeled secondary antibody (50 .mu.l of a
1:250 dilution in PBN) will be added to each well. As an example,
the secondary antibody can be polyvalent goat F (ab').sub.2
anti-horse immunoglobulins (IgG+IgA+IgM) conjugated to
red-phycoerythrin (R-PE) obtained from a commercial or veterinary
source. After incubation, microspheres will be resuspended in 125
.mu.l PBN per well. Seventy-five microliter aliquots will be
transferred to opaque black EIA/RIA 96-well plates with breakaway
strips (Costar, Corning, N.Y.), and will be evaluated for
microsphere fluorescence intensity using a Luminex 100 instrument
(Luminex Corp.). This instrument is a dual laser flow analyzer. The
first laser excites the fluorochrome mixture intrinsic to the
microspheres, enabling the bead identity to be determined as the
bead passes single file through the laser path in the flow cell.
The second laser excites the extrinsic fluorochrome
(red-phycoerythrin) that is covalently attached to the reporter
antibodies (goat-anti horse immunoglobulins). The dual lasers
allows the operator to mix beads with different antigens together
in a well of a filter plate, thus enabling multiplex analysis of
different antibody specificities at one time.
[0483] The instrument will be calibrated with CL1/CL2 and RP1
calibration microspheres from Luminex Corp. according to the
manufacturer's directions. The median fluorescence intensity (MFI)
of fluorochrome-conjugated secondary antibody bound to individual
microspheres will be derived from flow analysis of 100 microspheres
per well. Results for each assay will be expressed both as MFI and
as a horse/negative (P/N) MFI ratio, i.e., the MFI for the horse's
specimen divided by the MFI obtained from a pool of 10 negative
control sera. The negative control sera will contain no detectable
antibodies to WN virus in IgM-capture and IgG ELISAs.
[0484] Positive detection of anti-NS5 antibodies will indicate
recent exposure of the horse to WNV or an ongoing WNV infection.
Killed WNV vaccine is not expected to generate any immune response
to NS5 proteins since the WNV vaccine is not expected to replicate.
Non-replicating viruses do not produce new NS5 protein. Thus, the
NS5-based microsphere immunoassay will be useful for discriminating
between horses that have been vaccinated previously with killed WNV
vaccine and those that have been previously been vaccinated and
were exposed and infected with WNV.
Example 33
Preparation of Monoclonal Antibodies to WNV NS5 Antigen
[0485] WNV NS5 was expressed in E. coli in accordance with the
teachings of the invention. Affinity column chromatography was used
to purify the WNV NS5 to a final concentration of 7.2 mg/ml and a
purity of 99% as estimated by Coomassie stained PAGE, and by
crystallization. To generate antibodies against WNV NS5, seven week
old female BALB/c ByJ mice were inoculated subcutaneously at
inguinal, axillary, and nuchal sites with a water in oil emulsion
containing 100 micrograms of the purified WNV NS5 in 0.25 ml PBS
buffer and 0.25 ml Complete Freunds Adjuvant. A boost by i.p.
inoculation of 80 micrograms of WNV NS5 was given at day 60. Seven
days after the booster shot, 0.2 ml of blood was collected from the
mice from the retroorbital sinus. The presence of an antibody
against WNV NS5 was confirmed from the sample of blood using the
BIACORE system (described in detail above). After an additional 30
days, and 3 days prior to carrying out the somatic cell
hybridization procedure by polyethylene glycol induced fusion of
myeloma cells with isolated spleen cells, a second boost of 100
micrograms was administered by i.p. inoculation.
[0486] On the third day following the administration of the second
boost of WNV NS5, the mouse was sacrificed and the spleen cells
were harvested. The mouse spleen is dissected and the splenic
capsule removed. Spleen tissue is minced with a scalpel, and added
to a tube containing PBS. The particulate connective tissue is
allowed to settle to the bottom of the tube. The single cells in
suspension are used to fuse with the myeloma cells in the presence
of polyethylene glycol. [The harvested spleen cells were used in
somatic cell hybridization to form a population of hybridomas using
standard techniques. The hybridomas were cultured in a plurality of
culture wells, e.g. 96 well-plates, under standard conditions and
at a concentration such that each culture well was predicted to
contain only one hybridomas cell.
[0487] Next, the clones were screened to identify which wells
contained hybridomas expressing the monoclonal anti-NS5 antibody of
interest. To do this, the culture supernatant from each of the
wells was tested for the presence of the antibody of interest
utilizing a BIACORE system. A total of only three 96-well plates
were tested as to their expression of the monoclonal of interest as
the NS5 protein was a potent immunogen. The 12 hybridoma clones
expressing the highest levels of monoclonal anti-NS5 antibody
identified by the BIACORE system were further screened by a
microsphere immunoassay (e.g. Luminex), in accordance with the
invention. In addition, the 12 hybridomas were measured by IFA
(indirect fluorescence assay) studies of WNV-infected Vero
cells.
[0488] The microsphere immunoassay demonstrated that 11 out of the
12 clones identified by the BIACORE system actually expressed the
anti-NS5 monoclonal antibody of interest. The two highest antibody
producing clones, #215 and #270, were injected into SCID mice on a
BALB/c background (These are an immunodeficient strain of the
BALB/c mice in which the immunizations took place.) to produce
ascites fluids containing antibody. Clones #215 and #270 made very
high-titered monoclonal antibodies to NS5. The titers for clones
#215 and #270 were about 1,000,000 and 100,000, respectively. The
titer is the reciprocal of the serum dilution.
Example 34
Preparation of Monoclonal Antibodies to DENV NS5 Antigen
[0489] A similar preparation scheme to Example 33 will be carried
out to prepare monoclonal antibodies to DENV NS5 antigen. Namely,
the DENV NS5 antigen (e.g. DENV-1, -2, -3, or -4 NS5) will be
expressed by recombinant DNA techniques in a host organism, such as
E. coli, and purified therefrom using standard biochemical
techniques, such as, affinity chromatography. The purified DENV NS5
antigen will be injected subcutaneously into a appropriate mouse
host. Over a period of time, and after booster shots of the antigen
are administered, the spleen cells of the mouse will be harvested
for the purposes of carrying out somatic cell hybridization to
produce hybridomas. The resulting hybridomas will be cultured in
multi-well plates, such as 96-well plates, under conditions
sufficient such that only 1 clone is cultured per well. The clones
in each of the wells of the plates will then be screened to
determine which clone or clones produce the highest titers of
anti-NS5 antibody. This is carried out by detecting whether the
supernatant in the culture wells of each of the clones contains the
anti-NS5 antibody. The screening will be carried out using a
BIACORE instrument, which will detect whether there is an
interaction between the NS5 antigen and the antibody contained in
the supernatant of each culture well. Positive clones are
identified as those clones having a detectable reaction between the
NS5 antigen and the supernatant of the clone culture well. Next,
the positive clones will be further screened using a microsphere
immunoassay to confirm whether the antibody in the clone culture
supernatant is an anti-NS5 monoclonal antibody.
[0490] Once positively identified, the hybridoma clone which
expresses a high titer of anti-NS5 monoclonal antibody will be used
to produce a greater quantity of antibody. This will be carried out
by introducing into another mouse to hybridoma clone and collecting
ascites fluid that form, which will contain high titers of the
anti-NS5 monoclonal antibodies.
[0491] Alternatively, large amounts of the anti-NS5 monoclonal
antibodies can be produced either in vitro using cell culturing
techniques, e.g. growing the hybridoma clone in a large tissue
culture flask at 37.degree. C. in an incubator.
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Sequence CWU 1
1
22110975DNAWest Nile virus 1gctgacaaac ttagtagtgt ttgtgaggat
taacaacaat taacacagtg cgagctgttt 60cttagcacga agatctcgat gtctaagaaa
ccaggagggc ccggcaagag ccgggctgtc 120aatatgctaa aacgcggaat
gccccgcgtg ttgtccttga ttggactgaa gagggctatg 180ttgagcctga
tcgacggcaa ggggccaata cgatttgtgt tggctctctt ggcgttcttc
240aggttcacag caattgctcc gacccgagca gtgctggatc gatggagagg
tgtgaacaaa 300caaacagcga tgaaacacct tctgagtttt aagaaggaac
tagggacctt gaccagtgct 360atcaatcggc ggagctcaaa acaaaagaaa
agaggaggaa agaccggaat tgcagtcatg 420attggcctga tcgccagcgt
aggagcagtt accctctcta acttccaagg gaaggtgatg 480atgacggtaa
atgctactga cgtcacagat gtcatcacga ttccaacagc tgctggaaag
540aacctatgca ttgtcagagc aatggatgtg ggatacatgt gcgatgatac
tatcacttat 600gaatgcccag tgctgtcggc tggtaatgat ccagaagaca
tcgactgttg gtgcacaaag 660tcagcagttt acgtcaggta tggaagatgc
accaagacac gccactcaag acgcagtcgg 720aggtcactga cagtgcagac
acacggagaa agcactctag cgaacaagaa gggggcttgg 780atggacagca
ccaaggccac aaggtacttg gtaaaaacag aatcatggat cttgaggaac
840cctggatatg ccctggtggc agccgtcatt ggttggatgc ttgggagcaa
caccatgcag 900agagttgtgt ttgtcgtgct attgcttttg gtggccccag
cttacagctt caactgcctt 960ggaatgagca acagagactt cttggaagga
gtgtctggag caacatgggt ggatttggtt 1020ctcgaaggcg acagctgcgt
gactatcatg tctaaggaca agcctaccat cgatgtgaag 1080atgatgaata
tggaggcggc caacctggca gaggtccgca gttattgcta tttggctacc
1140gtcagcgatc tctccaccaa agctgcgtgc ccgaccatgg gagaagctca
caatgacaaa 1200cgtgctgacc cagcttttgt gtgcagacaa ggagtggtgg
acaggggctg gggcaacggc 1260tgcggactat ttggcaaagg aagcattgac
acatgcgcca aatttgcctg ctctaccaag 1320gcaataggaa gaaccatctt
gaaagagaat atcaagtacg aagtggccat ttttgtccat 1380ggaccaacta
ctgtggagtc gcacggaaac tactccacac aggttggagc cactcaggca
1440gggagattca gcatcactcc tgcagcgcct tcatacacac taaagcttgg
agaatatgga 1500gaggtgacag tggactgtga accacggtca gggattgaca
ccaatgcata ctacgtgatg 1560actgttggaa caaagacgtt cttggtccat
cgtgagtggt tcatggacct caacctccct 1620tggagcagtg ctggaagtac
tgtgtggagg aacagagaga cgttaatgga gtttgaggaa 1680ccacacgcca
cgaagcagtc tgtgatagca ttgggctcac aagagggagc tctgcatcaa
1740gctttggctg gagccattcc tgtggaattt tcaagcaaca ctgtcaagtt
gacgtcgggt 1800catttgaagt gtagagtgaa gatggaaaaa ttgcagttga
agggaacaac ctatggcgtc 1860tgttcaaagg ctttcaagtt tcttgggact
cccgcagaca caggtcacgg cactgtggtg 1920ttggaattgc agtacactgg
cacggatgga ccttgcaaag ttcctatctc gtcagtggct 1980tcattgaacg
acctaacgcc agtgggcaga ttggtcactg tcaacccttt tgtttcaatg
2040gccacggcca acgctaaggt cctgattgaa ttggaaccac cctttggaga
ctcatacata 2100gtggtgggca gaggagaaca acagatcaat caccattggc
acaagtctgg aagcagcatt 2160ggcaaagcct ttacaaccac cctcaaagga
gcgcagagac tagccgctct aggagacaca 2220gcttgggact ttggatcagt
tggaggggtg ttcacctcag ttgggaaggc tgtccatcaa 2280gtgttcggag
gagcattccg ctcactgttc ggaggcatgt cctggataac gcaaggattg
2340ctgggggctc tcctgttgtg gatgggcatc aatgctcgtg ataggtccat
agctctcacg 2400tttctcgcag ttggaggagt tctgctcttc ctctccgtga
acgtgcacgc tgacactggg 2460tgtgccatag acatcagccg gcaagagctg
agatgtggaa gtggagtgtt catacacaat 2520gatgtggagg cttggatgga
ccggtacaag tattaccctg aaacgccaca aggcctagcc 2580aagatcattc
agaaagctca taaggaagga gtgtgcggtc tacgatcagt ttccagactg
2640gagcatcaaa tgtgggaagc agtgaaggac gagctgaaca ctcttttgaa
ggagaatggt 2700gtggacctta gtgtcgtggt tgagaaacag gagggaatgt
acaagtcagc acctaaacgc 2760ctcaccgcca ccacggaaaa attggaaatt
ggctggaagg cctggggaaa gagtatttta 2820tttgcaccag aactcgccaa
caacaccttt gtggttgatg gtccggagac caaggaatgt 2880ccgactcaga
atcgcgcttg gaatagctta gaagtggagg attttggatt tggtctcacc
2940agcactcgga tgttcctgaa ggtcagagag agcaacacaa ctgaatgtga
ctcgaagatc 3000attggaacgg ctgtcaagaa caacttggcg atccacagtg
acctgtccta ttggattgaa 3060agcaggctca atgatacgtg gaagcttgaa
agggcagttc tgggtgaagt caaatcatgt 3120acgtggcctg agacgcatac
cttgtggggc gatggaatcc ttgagagtga cttgataata 3180ccagtcacac
tggcgggacc acgaagcaat cacaatcgga gacctgggta caagacacaa
3240aaccagggcc catgggacga aggccgggta gagattgact tcgattactg
cccaggaact 3300acggtcaccc tgagtgagag ctgcggacac cgtggacctg
ccactcgcac caccacagag 3360agcggaaagt tgataacaga ttggtgctgc
aggagctgca ccttaccacc actgcgctac 3420caaactgaca gcggctgttg
gtatggtatg gagatcagac cacagagaca tgatgaaaag 3480accctcgtgc
agtcacaagt gaatgcttat aatgctgata tgattgaccc ttttcagttg
3540ggccttctgg tcgtgttctt ggccacccag gaggtccttc gcaagaggtg
gacagccaag 3600atcagcatgc cagctatact gattgctctg ctagtcctgg
tgtttggggg cattacttac 3660actgatgtgt tacgctatgt catcttggtg
ggggcagctt tcgcagaatc taattcggga 3720ggagacgtgg tacacttggc
gctcatggcg accttcaaga tacaaccagt gtttatggtg 3780gcatcgtttc
ttaaagcgag atggaccaac caggagaaca ttttgttgat gttggcggct
3840gttttctttc aaatggctta tcacgatgcc cgccaaattc tgctctggga
gatccctgat 3900gtgttgaatt cactggcggt agcttggatg atactgagag
ccataacatt cacaacgaca 3960tcaaacgtgg ttgttccgct gctagccctg
ctaacacccg ggctgagatg cttgaatctg 4020gatgtgtaca ggatactgct
gttgatggtc ggaataggca gcttgatcag ggagaagagg 4080agtgcagctg
caaaaaagaa aggagcaagt ctgctatgct tggctctagc ctcaacagga
4140cttttcaacc ccatgatcct tgctgctgga ctgattgcat gtgatcccaa
ccgtaaacgc 4200ggatggcccg caactgaagt gatgacagct gtcggcctaa
tgtttgccat cgtcggaggg 4260ctggcagagc ttgacattga ctccatggcc
attccaatga ctatcgcggg gctcatgttt 4320gctgctttcg tgatttctgg
gaaatcaaca gatatgtgga ttgagagaac ggcggacatt 4380tcctgggaaa
gtgatgcaga aattacaggc tcgagcgaaa gagttgatgt gcggcttgat
4440gatgatggaa acttccagct catgaatgat ccaggagcac cttggaagat
atggatgctc 4500agaatggtct gtctcgcgat tagtgcgtac accccctggg
caatcttgcc ctcagtagtt 4560ggattttgga taactctcca atacacaaag
agaggaggcg tgttgtggga cactccctca 4620ccaaaggagt acaaaaaggg
ggacacgacc accggcgtct acaggatcat gactcgtggg 4680ctgctcggca
gttatcaagc aggagcgggc gtgatggttg aaggtgtttt ccacaccctt
4740tggcatacaa caaaaggagc cgctttgatg agcggagagg gccgcctgga
cccatactgg 4800ggcagtgtca aggaggatcg actttgttac ggaggaccct
ggaaattgca gcacaagtgg 4860aacgggcagg atgaggtgca gatgattgtg
gtggaacctg gcaagaacgt taagaacgtc 4920cagacgaaac caggggtgtt
caaaacacct gaaggagaaa tcggggccgt gactttggac 4980ttccccactg
gaacatcagg ctcaccaata gtggacaaaa acggtgatgt gattgggctt
5040tatggcaatg gagtcataat gcccaacggc tcatacataa gcgcgatagt
gcagggtgaa 5100aggatggatg agccaatccc agccggattc gaacctgaga
tgctgaggaa aaaacagatc 5160actgtactgg atctccatcc cggcgccggt
aaaacaagga ggattctgcc acagatcatc 5220aaagaggcca taaacagaag
actgagaaca gccgtgctag caccaaccag ggttgtggct 5280gctgagatgg
ctgaagcact gagaggactg cccatccggt accagacatc cgcagtgccc
5340agagaacata atggaaatga gattgttgat gtcatgtgtc atgctaccct
cacccacagg 5400ctgatgtctc ctcacagggt gccgaactac aacctgttcg
tgatggatga ggctcatttc 5460accgacccag ctagcattgc agcaagaggt
tacatttcca caaaggtcga gctaggggag 5520gcggcggcaa tattcatgac
agccacccca ccaggcactt cagatccatt cccagagtcc 5580aattcaccaa
tttccgactt acagactgag atcccggatc gagcttggaa ctctggatac
5640gaatggatca cagaatacac cgggaagacg gtttggtttg tgcctagtgt
caagatgggg 5700aatgagattg ccctttgcct acaacgtgct ggaaagaaag
tagtccaatt gaacagaaag 5760tcgtacgaga cggagtaccc aaaatgtaag
aacgatgatt gggactttgt tatcacaaca 5820gacatatctg aaatgggggc
taacttcaag gcgagcaggg tgattgacag ccggaagagt 5880gtgaaaccaa
ccatcataac agaaggagaa gcgagagtga tcctgggaga accatctgca
5940gtgacagcag ctagtgccgc ccagagacgt ggacgtatcg gtagaaatcc
gtcgcaagtt 6000ggtgatgagt actgttatgg ggggcacacg aatgaagacg
actcgaactt cgcccattgg 6060actgaggcac gaatcatgct ggacaacatc
aacatgccaa acggactgat cgctcaattc 6120taccaaccag agcgtgagaa
ggtatatacc atggatgggg aataccggct cagaggagaa 6180gagagaaaaa
actttctgga actgttgagg actgcagatc tgccagtttg gctggcttac
6240aaggttgcag cggctggagt gtcataccac gaccggaggt ggtgctttga
tggtcctagg 6300acaaacacaa ttttagaaga caacaacgaa gtggaagtca
tcacgaagct tggtgaaagg 6360aagattctga ggccgcgctg gattgacgcc
agggtgtact cggatcacca ggcactaaag 6420gcgttcaagg acttcgcctc
gggaaaacgt tctcagatag ggctcattga ggttctggga 6480aagatgcctg
agcacttcat ggggaagaca tgggaagcac ttgacaccat gtacgttgtg
6540gccactgcag agaaaggagg aagagctcac agaatggccc tggaggaact
gccagatgct 6600cttcagacaa ttgccttgat tgccttattg agtgtgatga
ccatgggagt attcttcctc 6660ctcatgcagc ggaagggcat tggaaagata
ggtttgggag gcgctgtctt gggagtcgcg 6720acctttttct gttggatggc
tgaagttcca ggaacgaaga tcgccggaat gttgctgctc 6780tcccttctct
tgatgattgt gctaattcct gagccagaga agcaacgttc gcagacagac
6840aaccagctag ccgtgttcct gatttgtgtc atgacccttg tgagcgcagt
ggcagccaac 6900gagatgggtt ggctagataa gaccaagagt gacataagca
gtttgtttgg gcaaagaatt 6960gaggtcaagg agaatttcag catgggagag
tttcttctgg acttgaggcc ggcaacagcc 7020tggtcactgt acgctgtgac
aacagcggtc ctcactccac tgctaaagca tttgatcacg 7080tcagattaca
tcaacacctc attgacctca ataaacgttc aggcaagtgc actattcaca
7140ctcgcgcgag gcttcccctt cgtcgatgtt ggagtgtcgg ctctcctgct
agcagccgga 7200tgctggggac aagtcaccct caccgttacg gtaacagcgg
caacactcct tttttgccac 7260tatgcctaca tggttcccgg ttggcaagct
gaggcaatgc gctcagccca gcggcggaca 7320gcggccggaa tcatgaagaa
cgctgtagtg gatggcatcg tggccacgga cgtcccagaa 7380ttagagcgca
ccacacccat catgcagaag aaagttggac agatcatgct gatcttggtg
7440tctctagctg cagtagtagt gaacccgtct gtgaagacag tacgagaagc
cggaattttg 7500atcacggccg cagcggtgac gctttgggag aatggagcaa
gctctgtttg gaacgcaaca 7560actgccatcg gactctgcca catcatgcgt
gggggttggt tgtcatgtct atccataaca 7620tggacactca taaagaacat
ggaaaaacca ggactaaaaa gaggtggggc aaaaggacgc 7680accttgggag
aggtttggaa agaaagactc aaccagatga caaaagaaga gttcactagg
7740taccgcaaag aggccatcat cgaagtcgat cgctcagcgg caaaacacgc
caggaaagaa 7800ggcaatgtca ctggagggca tccagtctct aggggcacag
caaaactgag atggctggtc 7860gaacggaggt ttctcgaacc ggtcggaaaa
gtgattgacc ttggatgtgg aagaggcggt 7920tggtgttact atatggcaac
ccaaaaaaga gtccaagaag tcagagggta cacaaagggc 7980ggtcccggac
atgaagagcc ccaactagtg caaagttatg gatggaacat tgtcaccatg
8040aagagtggag tggatgtgtt ctacagacct tctgagtgtt gtgacaccct
cctttgtgac 8100atcggagagt cctcgtcaag tgctgaggtt gaagagcata
ggacgattcg ggtccttgaa 8160atggttgagg actggctgca ccgagggcca
agggaatttt gcgtgaaggt gctctgcccc 8220tacatgccga aagtcataga
gaagatggag ctgctccaac gccggtatgg ggggggactg 8280gtcagaaacc
cactctcacg gaattccacg cacgagatgt attgggtgag tcgagcttca
8340ggcaatgtgg tacattcagt gaatatgacc agccaggtgc tcctaggaag
aatggaaaaa 8400aggacctgga agggacccca atacgaggaa gacgtaaact
tgggaagtgg aaccagggcg 8460gtgggaaaac ccctgctcaa ctcagacacc
agtaaaatca agaacaggat tgaacgactc 8520aggcgtgagt acagttcgac
gtggcaccac gatgagaacc acccatatag aacctggaac 8580tatcacggca
gttatgatgt gaagcccaca ggctccgcca gttcgctggt caatggagtg
8640gtcaggctcc tctcaaaacc atgggacacc atcacgaatg ttaccaccat
ggccatgact 8700gacactactc ccttcgggca gcagcgagtg ttcaaagaga
aggtggacac gaaagctcct 8760gaaccgccag aaggagtgaa gtacgtgctc
aacgagacca ccaactggtt gtgggcgttt 8820ttggccagag aaaaacgtcc
cagaatgtgc tctcgagagg aattcataag aaaggtcaac 8880agcaatgcag
ctttgggtgc catgtttgaa gagcagaatc aatggaggag cgccagagaa
8940gcagttgaag atccaaaatt ttgggagatg gtggatgagg agcgcgaggc
acatctgcgg 9000ggggaatgtc acacttgcat ttacaacatg atgggaaaga
gagagaaaaa acccggagag 9060ttcggaaagg ccaagggaag cagagccatt
tggttcatgt ggctcggagc tcgctttctg 9120gagttcgagg ctctgggttt
tctcaatgaa gaccactggc ttggaagaaa gaactcagga 9180ggaggtgtcg
agggcttggg cctccaaaaa ctgggttaca tcctgcgtga agttggcacc
9240cggcctgggg gcaagatcta tgctgatgac acagctggct gggacacccg
catcacgaga 9300gctgacttgg aaaatgaagc taaggtgctt gagctgcttg
atggggaaca tcggcgtctt 9360gccagggcca tcattgagct cacctatcgt
cacaaagttg tgaaagtgat gcgcccggct 9420gctgatggaa gaaccgtcat
ggatgttatc tccagagaag atcagagggg gagtggacaa 9480gttgtcacct
acgccctaaa cactttcacc aacctggccg tccagctggt gaggatgatg
9540gaaggggaag gagtgattgg cccagatgat gtggagaaac tcacaaaagg
gaaaggaccc 9600aaagtcagga cctggctgtt tgagaatggg gaagaaagac
tcagccgcat ggctgtcagt 9660ggagatgact gtgtggtaaa gcccctggac
gatcgctttg ccacctcgct ccacttcctc 9720aatgctatgt caaaggttcg
caaagacatc caagagtgga aaccgtcaac tggatggtat 9780gattggcagc
aggttccatt ttgctcaaac catttcactg aattgatcat gaaagatgga
9840agaacactgg tggttccatg ccgaggacag gatgaattgg taggcagagc
tcgcatatct 9900ccaggggccg gatggaacgt ccgcgacact gcttgtctgg
ctaagtctta tgcccagatg 9960tggctgcttc tgtacttcca cagaagagac
ctgcggctca tggccaacgc catttgctcc 10020gctgtccctg tgaattgggt
ccctaccgga agaaccacgt ggtccatcca tgcaggagga 10080gagtggatga
caacagagga catgttggag gtctggaacc gtgtttggat agaggagaat
10140gaatggatgg aagacaaaac cccagtggag aaatggagtg acgtcccata
ttcaggaaaa 10200cgagaggaca tctggtgtgg cagcctgatt ggcacaagag
cccgagccac gtgggcagaa 10260aacatccagg tggctatcaa ccaagtcaga
gcaatcatcg gagatgagaa gtatgtggat 10320tacatgagtt cactaaagag
atatgaagac acaactttgg ttgaggacac agtactgtag 10380atatttaatt
aattgtaaat agacaatata agtatgcata aaagtgtagt tttatagtag
10440tatttagtgg tgttagtgta aatagttaag aaaattttga ggagaaagtc
aggccgggaa 10500gttcccgcca ccggaagttg agtagacggt gctgcctgcg
actcaacccc aggaggactg 10560ggtgaacaaa gccgcgaagt gatccatgta
agccctcaga accgtctcgg aaggaggacc 10620ccacatgttg taacttcaaa
gcccaatgtc agaccacgct acggcgtgct actctgcgga 10680gagtgcagtc
tgcgatagtg ccccaggagg actgggttaa caaaggcaaa ccaacgcccc
10740acgcggccct agccccggta atggtgttaa ccagggcgaa aggactagag
gttagaggag 10800accccgcggt ttaaagtgca cggcccagcc tggctgaagc
tgtaggtcag gggaaggact 10860agaggttagt ggagaccccg tgccacaaaa
caccacaaca aaacagcata ttgacacctg 10920ggatagacta ggagatcttc
tgctctgcac aaccagccac acggcacagt gcgcc 10975211029DNAWest Nile
virus 2agtagttcgc ctgtgtgagc tgacaaactt agtagtgttt gtgaggatta
acaacaatta 60acacagtgcg agctgtttct tagcacgaag atctcgatgt ctaagaaacc
aggagggccc 120ggcaagagcc gggctgtcaa tatgctaaaa cgcggaatgc
cccgcgtgtt gtccttgatt 180ggactgaaga gggctatgtt gagcctgatc
gacggcaagg ggccaatacg atttgtgttg 240gctctcttgg cgttcttcag
gttcacagca attgctccga cccgagcagt gctggatcga 300tggagaggtg
tgaacaaaca aacagcgatg aaacaccttc tgagttttaa gaaggaacta
360gggaccttga ccagtgctat caatcggcgg agctcaaaac aaaagaaaag
aggaggaaag 420accggaattg cagtcatgat tggcctgatc gccagcgtag
gagcagttac cctctctaac 480ttccaaggga aggtgatgat gacggtaaat
gctactgacg tcacagatgt catcacgatt 540ccaacagctg ctggaaagaa
cctatgcatt gtcagagcaa tggatgtggg atacatgtgc 600gatgatacta
tcacttatga atgcccagtg ctgtcggctg gtaatgatcc agaagacatc
660gactgttggt gcacaaagtc agcagtctac gtcaggtatg gaagatgcac
caagacacgc 720cactcaagac gcagtcggag gtcactgaca gtgcagacac
acggagaaag cactctagcg 780aacaagaagg gggcttggat ggacagcacc
aaggccacaa ggtatttggt aaaaacagaa 840tcatggatct tgaggaaccc
tggatatgcc ctggtggcag ccgtcattgg ttggatgctt 900gggagcaaca
ccatgcagag agttgtgttt gtcgtgctat tgcttttggt ggccccagct
960tacagcttca actgccttgg aatgagcaac agagacttct tggaaggagt
gtctggagca 1020acatgggtgg atttggttct cgaaggcgac agctgcgtga
ctatcatgtc taaggacaag 1080cctaccatcg atgtgaagat gatgaatatg
gaggcggcca acctggcaga ggtccgcagt 1140tattgctatt tggctaccgt
cagcgatctc tccaccaaag ctgcgtgccc gaccatggga 1200gaagctcaca
atgacaaacg tgctgaccca gcttttgtgt gcagacaagg agtggtggac
1260aggggctggg gcaacggctg cggattattt ggcaaaggaa gcattgacac
atgcgccaaa 1320tttgcctgct ctaccaaggc aataggaaga accatcttga
aagagaatat caagtacgaa 1380gtggccattt ttgtccatgg accaactact
gtggagtcgc acggaaacta ctccacacag 1440gttggagcca ctcaggcagg
gagattcagc atcactcctg cggcgccttc atacacacta 1500aagcttggag
aatatggaga ggtgacagtg gactgtgaac cacggtcagg gattgacacc
1560aatgcatact acgtgatgac tgttggaaca aagacgttct tggtccatcg
tgagtggttc 1620atggacctca acctcccttg gagcagtgct ggaagtactg
tgtggaggaa cagagagacg 1680ttaatggagt ttgaggaacc acacgccacg
aagcagtctg tgatagcatt gggctcacaa 1740gagggagctc tgcatcaagc
tttggctgga gccattcctg tggaattttc aagcaacact 1800gtcaagttga
cgtcgggtca tttgaagtgt agagtgaaga tggaaaaatt gcagttgaag
1860ggaacaacct atggcgtctg ttcaaaggct ttcaagtttc ttgggactcc
cgcagacaca 1920ggtcacggca ctgtggtgtt ggaattgcag tacactggca
cggatggacc ttgtaaagtt 1980cctatctcgt cagtggcttc attgaacgac
ctaacgccag tgggcagatt ggtcactgtc 2040aacccttttg tttcagtggc
cacggccaac gctaaggtcc tgattgaatt ggaaccaccc 2100tttggagact
catacatagt ggtgggcaga ggagaacaac agatcaatca ccattggcac
2160aagtctggaa gcagcattgg caaagccttt acaaccaccc tcaaaggagc
gcagagacta 2220gccgctctag gagacacagc ttgggacttt ggatcagttg
gaggggtgtt cacctcagtt 2280gggaaggctg tccatcaagt gttcggagga
gcattccgct tactgttcgg aggcatgtcc 2340tggataacgc aaggattgct
gggggctctc ctgttgtgga tgggcatcaa tgctcgtgat 2400aggtccatag
ctctcacgtt tctcgcagtt ggaggagttc tgctcttcct ctccgtgaac
2460gtgcacgctg acactgggtg tgccatagac atcagccggc aagagctgag
atgtggaagt 2520ggagtgttca tacacaatga tgtggaggct tggatggacc
gatacaagta ttaccctgaa 2580acgccacaag gcctagccaa gatcattcag
aaagctcata aggaaggagt gtgcggtcta 2640cgatcagttt ccagactgga
gcatcaaatg tgggaagcag tgaaggacga gctgaacact 2700cttttgaagg
agaatggtgt ggaccttagt gtcgtggttg agaaacagga gggaatgtac
2760aagtcagcac ctaaacgcct caccgccacc acggaaaaat tggaaattgg
ctggaaggcc 2820tggggaaaga gtattttatt tgcaccagaa ctcgccaaca
acacctttgt ggttgatggt 2880ccggagacca aggaatgtcc gactcagaat
cgcgcttgga atagcttaga agtggaggat 2940tttggatttg gtctcaccag
cactcggatg ttcctgaagg tcagagagag caacacaact 3000gaatgtgact
cgaagatcat tggaacggct gtcaagaaca acttggcgat ccacagtgac
3060ctgtcctatt ggattgaaag caggctcaat gatacgtgga agcttgaaag
ggcagttctg 3120ggtgaagtca aatcatgtac gtggcctgag acgcatacct
tgtggggcga tggaatcctt 3180gagagtgact tgataatacc agtcacactg
gcgggaccac gaagcaatca caatcggaga 3240cctgggtaca agacacaaaa
ccagggccca tgggacgaag gccgggtaga gattgacttc 3300gattactgcc
caggaactac ggtcaccctg agtgagagct gcggacaccg tggacctgcc
3360actcgcacca ccacagagag cggaaagttg ataacagatt ggtgctgcag
gagctgcacc 3420ttaccaccac tgcgctacca aactgacagc ggctgttggt
atggtatgga gatcagacca 3480cagagacatg atgaaaagac cctcgtgcag
tcacaagtga atgcttataa tgctgatatg 3540attgaccctt ttcagttggg
ccttctggtc gtgttcttgg ccacccagga ggtccttcgc 3600aagaggtgga
cagccaagat cagcatgcca gctatactga ttgctctgct agtcctggtg
3660tttgggggca ttacttacac tgatgtgtta cgctatgtca tcttggtggg
ggcagctttc 3720gcagaatcta attcgggagg agacgtggta cacttggcgc
tcatggcgac cttcaagata 3780caaccagtgt ttatggtggc atcgtttctc
aaagcgagat ggaccaacca ggagaacatt 3840ttgttgatgt tggcggctgt
tttctttcaa atggcttatc acgatgcccg ccaaattctg 3900ctctgggaga
tccctgatgt gttgaattca ctggcggtag cttggatgat actgagagcc
3960ataacattca caacgacatc aaacgtggtt gttccgctgc tagccctgct
aacacccggg 4020ctgagatgct
tgaatctgga tgtgtacagg atactgctgt tgatggtcgg aataggcagc
4080ttgatcaggg agaagaggag tgcagctgca aaaaagaaag gagcaagtct
gctatgcttg 4140gctctagcct caacaggact tttcaacccc atgatccttg
ctgctggact gattacatgt 4200gatcccaacc gtaaacgcgg atggcccgca
actgaagtga tgacagctgt cggcctgatg 4260tttgccatcg tcggagggct
ggcagagctt gacattgact ccatggccat tccaatgact 4320atcgcggggc
tcatgtttgc tgctttcgtg atttctggga aatcaacaga tatgtggatt
4380gagagaacgg cggacatttc ctgggaaagt gatgcagaaa ttacaggctc
gagcgaaaga 4440gttgatgtgc ggcttgatga tgatggaaac ttccagctca
tgaatgatcc aggagcacct 4500tggaagatat ggatgctcag aatggtctgt
ctcgcgatta gtgcgtacac cccctgggca 4560atcttgccct cagtagttgg
attttggata actctccaat acacaaagag aggaggcgtg 4620ttgtgggaca
ctccctcacc aaaggagtac aaaaaggggg acacgaccac cggcgtctac
4680aggatcatga ctcgtgggct gctcggcagt tatcaagcag gagcgggcgt
gatggttgaa 4740ggtgttttcc acaccctttg gcatacaaca aaaggagccg
ctttgatgag cggagagggc 4800cgcctggacc catactgggg cagtgtcaag
gaggatcgac tttgttacgg aggaccctgg 4860aaattgcagc acaagtggaa
cgggcaggat gaggtgcaga tgattgtggt ggaacctggc 4920aagaacgtta
agaacgtcca gacgaaacca ggggtgttca aaacacctga aggagaaatc
4980ggggccgtga ctttggactt ccccactgga acatcaggct caccaatagt
ggacaaaaac 5040ggtgatgtga ttgggcttta tggcaatgga gtcataatgc
ccaacggctc atacataagc 5100gcgatagtgc agggtgaaag gatggatgag
ccaatcccag ccggattcga acctgagatg 5160ctgaggaaaa aacagatcac
tgtactggat ctccatcccg gcgccggtaa aacaaggagg 5220attctgccac
agatcatcaa agaggccata aacagaagac tgagaacagc cgtgctagca
5280ccaaccaggg ttgtggctgc tgagatggct gaagcactga gaggactgcc
catccggtac 5340cagacatccg cagtgcccag agaacataat ggaaatgaga
ttgttgatgt catgtgtcat 5400gctaccctca cccacaggct gatgtctcct
cacagggtgc cgaactacaa cctgttcgtg 5460atggatgagg ctcatttcac
cgacccagct agcattgcag caagaggtta catttccaca 5520aaggtcgagc
taggggaggc ggcggcaata ttcatgacag ccaccccacc aggcacttca
5580gatccattcc cagagtccaa ttcaccaatt tccgacttac agactgagat
cccggatcga 5640gcttggaact ctggatacga atggatcaca gaatacaccg
ggaagacggt ttggtttgtg 5700cctagtgtca agatggggaa tgagattgcc
ctttgcctac aacgtgctgg aaagaaagta 5760gtccaattga acagaaagtc
gtacgagacg gagtacccaa aatgtaagaa cgatgattgg 5820gactttgtta
tcacaacaga catatctgaa atgggggcta actttaaggc gagcagggtg
5880attgacagcc ggaagagtgt gaaaccaacc atcataacag aaggagaagg
gagagtgatc 5940ctgggagaac catctgcagt gacagcagct agtgccgccc
agagacgtgg acgtatcggt 6000agaaatccgt cgcaagttgg tgatgagtac
tgttatgggg ggcacacgaa tgaagacgac 6060tcgaacttcg cccattggac
tgaggcacga atcatgctgg acaacatcaa catgccaaac 6120ggactgatcg
ctcaattcta ccaaccagag cgtgagaagg tatataccat ggatggggaa
6180taccggctca gaggagaaga gagaaaaaac tttctggaac tgttgaggac
tgcagatctg 6240ccagtttggc tggcttacaa ggttgcagcg gctggagtgt
cataccacga ccggaggtgg 6300tgctttgatg gtcctaggac aaacacaatt
ttagaagaca acaacgaagt ggaagtcatc 6360acgaagcttg gtgaaaggaa
gattctgagg ccgcgctgga ttgacgccag ggtgtactcg 6420gatcaccagg
cactaaaggc gttcaaggac ttcgcctcgg gaaaacgttc tcagataggg
6480ctcattgagg ttctgggaaa gatgcctgag cacttcatgg ggaagacatg
ggaagcactt 6540gacaccatgt acgttgtggc cactgcagag aaaggaggaa
gagctcacag aatggccctg 6600gaggaactgc cagatgctct tcagacaatt
gccttgattg ccttattgag tgtgatgacc 6660atgggagtat tcttcctcct
catgcagcgg aagggcattg gaaagatagg tttgggaggc 6720gctgtcttgg
gagtcgcgac ctttttctgt tggatggctg aagttccagg aacgaagatc
6780gccggaatgt tgctgctctc ccttctcttg atgattgtgc taattcctga
gccagagaag 6840caacgttcgc agacagacaa ccagctagcc gtgttcctga
tttgtgtcat gacccttgtg 6900agcgcagtgg cagccaacga gatgggttgg
ctagataaga ccaagagtga cataagcagt 6960ttgtttgggc aaagaattga
ggtcaaggag aatttcagca tgggagagtt tcttctggac 7020ttgaggccgg
caacagcctg gtcactgtac gctgtgacaa cagcggtcct cactccactg
7080ctaaagcatt tgatcacgtc agattacatc aacacctcat tgacctcaat
aaacgttcag 7140gcaagtgcac tattcacact cgcgcgaggc ttccccttcg
tcgatgttgg agtgtcggct 7200ctcctgctag cagccggatg ctggggacaa
gtcaccctca ccgttacggt aacagcggca 7260acactccttt tttgccacta
tgcctacatg gttcccggtt ggcaagctga ggcaatgcgc 7320tcagcccagc
ggcggacagc ggccggaatc atgaagaacg ctgtagtgga tggcatcgtg
7380gccacggacg tcccagaatt agagcgcacc acacccatca tgcagaagaa
agttggacag 7440atcatgctga tcttggtgtc tctagctgca gtagtagtga
acccgtctgt gaagacagta 7500cgagaagccg gaattttgat cacggccgca
gcggtgacgc tttgggagaa tggagcaagc 7560tctgtttgga acgcaacaac
tgccatcgga ctctgccaca tcatgcgtgg gggttggttg 7620tcatgtctat
ccataacatg gacactcata aagaacatgg aaaaaccagg actaaaaaga
7680ggtggggcaa aaggacgcac cttgggagag gtttggaaag aaagactcaa
ccagatgaca 7740aaagaagagt tcactaggta ccgcaaagag gccatcatcg
aagtcgatcg ctcagcagca 7800aaacacgcca ggaaagaagg caatgtcact
ggagggcatc cagtctctag gggcacagca 7860aaactgagat ggctggtcga
acggaggttt ctcgaaccgg tcggaaaagt gattgacctt 7920ggatgtggaa
gaggcggttg gtgttactat atggcaaccc aaaaaagagt ccaagaagtc
7980agagggtaca caaagggcgg tcccggacat gaagagcccc aactagtgca
aagttatgga 8040tggaacattg tcaccatgaa gagtggggtg gatgtgttct
acagaccttc tgagtgttgt 8100gacaccctcc tttgtgacat cggagagtcc
tcgtcaagtg ctgaggttga agagcatagg 8160acgattcggg tccttgaaat
ggttgaggac tggctgcacc gagggccaag ggaattttgc 8220gtgaaggtgc
tctgccccta catgccgaaa gtcatagaga agatggagct gctccaacgc
8280cggtatgggg ggggactggt cagaaaccca ctctcacgga attccacgca
cgagatgtat 8340tgggtgagtc gagcttcagg caatgtggta cattcagtga
atatgaccag ccaggtgctc 8400ctaggaagaa tggaaaaaag gacctggaag
ggaccccaat acgaggaaga tgtaaacttg 8460ggaagtggaa ccagggcggt
gggaaaaccc ctgctcaact cagacaccag taaaatcaag 8520aacaggattg
aacgactcag gcgtgagtac agttcgacgt ggcaccacga tgagaaccac
8580ccatatagaa cctggaacta tcacggcagt tatgatgtga agcccacagg
ctccgccagt 8640tcgctggtca atggagtggt caggctcctc tcaaaaccat
gggacaccat cacgaatgtt 8700accaccatgg ccatgactga cactactccc
ttcgggcagc agcgagtgtt caaagagaag 8760gtggacacga aagctcctga
accgccagaa ggagtgaagt acgtgctcaa cgagaccacc 8820aactggttgt
gggcgttttt ggccagagaa aaacgtccca gaatgtgctc tcgagaggaa
8880ttcataagaa aggtcaacag caatgcagct ttgggtgcca tgtttgaaga
gcagaatcaa 8940tggaggagcg ccagagaggc agttgaagat ccaaaatttt
gggagatggt ggatgaggag 9000cgcgaggcac atctgcgggg ggaatgtcac
acttgcattt acaacatgat gggaaagaga 9060gagaaaaaac ccggagagtt
cggaaaggcc aagggaagca gagccatttg gttcatgtgg 9120ctcggagctc
gctttctgga gttcgaggct ctgggttttc tcaatgaaga ccactggctt
9180ggaagaaaga actcaggagg aggtgtcgag ggcttgggcc tccaaaaact
gggttacatc 9240ctgcgtgaag ttggcacccg gcctgggggc aagatctatg
ctgatgacac agctggctgg 9300gacacccgca tcacgagagc tgacttggaa
aatgaagcta aggtgcttga gctgcttgat 9360ggggaacatc ggcgtcttgc
cagggccatc attgagctca cctatcgtca caaagttgtg 9420aaagtgatgc
gcccggctgc tgatggaaga accgtcatgg atgttatctc cagagaagat
9480cagaggggga gtggacaagt tgtcacctac gccctaaaca ctttcaccaa
cctggccgtc 9540cagctggtga ggatgatgga aggggaagga gtgattggcc
cagatgatgt ggagaaactc 9600acaaaaggga aaggacccaa agtcaggacc
tggctgtttg agaatgggga agaaagactc 9660agccgcatgg ctgtcagtgg
agatgactgt gtggtaaagc ccctggacga tcgctttgcc 9720acctcgctcc
acttcctcaa tgctatgtca aaggttcgca aagacatcca agagtggaaa
9780ccgtcaactg gatggtatga ttggcagcag gttccatttt gctcaaacca
tttcactgaa 9840ttgatcatga aagatggaag aacactggtg gttccatgcc
gaggacagga tgaattggta 9900ggcagagctc gcatatctcc aggggccgga
tggaacgtcc gcgacactgc ttgtctggct 9960aagtcttatg cccagatgtg
gctgcttctg tacttccaca gaagagacct gcggctcatg 10020gccaacgcca
tttgctccgc tgtccctgtg aattgggtcc ctaccggaag aaccacgtgg
10080tccatccatg caggaggaga gtggatgaca acagaggaca tgttggaggt
ctggaaccgt 10140gtttggatag aggagaatga atggatggaa gacaaaaccc
cagtggagaa atggagtgac 10200gtcccatatt caggaaaacg agaggacatc
tggtgtggca gcctgattgg cacaagagcc 10260cgagccacgt gggcagaaaa
catccaggtg gctatcaacc aagtcagagc aatcatcgga 10320gatgagaagt
atgtggatta catgagttca ctaaagagat atgaagacac aactttggtt
10380gaggacacag tactgtagat atttaatcaa ttgtaaatag acaatataag
tatgcataaa 10440agtgtagttt tatagtagta tttagtggtg ttagtgtaaa
tagttaagaa aattttgagg 10500agaaagtcag gccgggaagt tcccgccacc
ggaagttgag tagacggtgc tgcctgcgac 10560tcaaccccag gaggactggg
tgaacaaagc cgcgaagtga tccatgtaag ccctcagaac 10620cgtctcggaa
ggaggacccc acatgttgta acttcaaagc ccaatgtcag accacgctac
10680ggcgtgctac tctgcggaga gtgcagtctg cgatagtgcc ccaggaggac
tgggttaaca 10740aaggcaaacc aacgccccac gcggccctag ccccggtaat
ggcgttaacc agggcgaaag 10800gactagaggt tagaggagac cccgcggttt
aaagtgcacg gcccagcctg gctgaagctg 10860taggtcaggg gaaggactag
aggttagtgg agaccccgtg ccacaaaaca ccacaacaaa 10920acagcatatt
gacacctggg atagactagg agatcttctg ctctgcacaa ccagccacac
10980ggcacagtgc gccgacaatg gtggctggtg gtgcgagaac acaggatct
11029310735DNADengue virus type 1 3agttgttagt ctacgtggac cgacaagaac
agtttcgaat cggaagcttg cttaacgtag 60ttctaacagt tttttattag agagcagatc
tctgatgaac aaccaacgga aaaagacggg 120tcgaccgtct ttcaatatgc
tgaaacgcgc gagaaaccgc gtgtcaactg tttcacagtt 180ggcgaagaga
ttctcaaaag gattgctttc aggccaagga cccatgaaat tggtgatggc
240ttttatagca ttcctaagat ttctagccat acctccaaca gcaggaattt
tggctagatg 300gggctcattc aagaagaatg gagcgatcaa agtgttacgg
ggtttcaaga aagaaatctc 360aaacatgttg aacataatga acaggaggaa
aagatctgtg accatgctcc tcatgctgct 420gcccacagcc ctggcgttcc
atctgaccac ccgaggggga gagccgcaca tgatagttag 480caagcaggaa
agaggaaaat cacttttgtt taagacctct gcaggtgtca acatgtgcac
540ccttattgca atggatttgg gagagttatg tgaggacaca atgacctaca
aatgcccccg 600gatcactgag acggaaccag atgacgttga ctgttggtgc
aatgccacgg agacatgggt 660gacctatgga acatgttctc aaactggtga
acaccgacga gacaaacgtt ccgtcgcact 720ggcaccacac gtagggcttg
gtctagaaac aagaaccgaa acgtggatgt cctctgaagg 780cgcttggaaa
caaatacaaa aagtggagac ctgggctctg agacacccag gattcacggt
840gatagccctt tttctagcac atgccatagg aacatccatc acccagaaag
ggatcatttt 900tattttgctg atgctggtaa ctccatccat ggccatgcgg
tgcgtgggaa taggcaacag 960agacttcgtg gaaggactgt caggagctac
gtgggtggat gtggtactgg agcatggaag 1020ttgcgtcact accatggcaa
aagacaaacc aacactggac attgaactct tgaagacgga 1080ggtcacaaac
cctgccgtcc tgcgcaaact gtgcattgaa gctaaaatat caaacaccac
1140caccgattcg agatgtccaa cacaaggaga agccacgctg gtggaagaac
aggacacgaa 1200ctttgtgtgt cgacgaacgt tcgtggacag aggctggggc
aatggttgtg ggctattcgg 1260aaaaggtagc ttaataacgt gtgctaagtt
taagtgtgtg acaaaactgg aaggaaagat 1320agtccaatat gaaaacttaa
aatattcagt gatagtcacc gtacacactg gagaccagca 1380ccaagttgga
aatgagacca cagaacatgg aacaactgca accataacac ctcaagctcc
1440cacgtcggaa atacagctga cagactacgg agctctaaca ttggattgtt
cacctagaac 1500agggctagac tttaatgaga tggtgttgtt gacaatggaa
aaaaaatcat ggctcgtcca 1560caaacaatgg tttctagact taccactgcc
ttggacctcg ggggcttcaa catcccaaga 1620gacttggaat agacaagact
tgctggtcac atttaagaca gctcatgcaa aaaagcagga 1680agtagtcgta
ctaggatcac aagaaggagc aatgcacact gcgttgactg gagcgacaga
1740aatccaaacg tctggaacga caacaatttt tgcaggacac ctgaaatgca
gactaaaaat 1800ggataaactg actttaaaag ggatgtcata tgtaatgtgc
acagggtcat tcaagttaga 1860gaaggaagtg gctgagaccc agcatggaac
tgttctagtg caggttaaat acgaaggaac 1920agatgcacca tgcaagatcc
ccttctcgtc ccaagatgag aagggagtaa cccagaatgg 1980gagattgata
acagccaacc ccatagtcac tgacaaagaa aaaccagtca acattgaagc
2040ggagccacct tttggtgaga gctacattgt ggtaggagca ggtgaaaaag
ctttgaaact 2100aagctggttc aagaagggaa gcagtatagg gaaaatgttt
gaagcaactg cccgtggagc 2160acgaaggatg gccatcctgg gagacactgc
atgggacttc ggttctatag gaggggtgtt 2220cacgtctgtg ggaaaactga
tacaccagat ttttgggact gcgtatggag ttttgttcag 2280cggtgtttct
tggaccatga agataggaat agggattctg ctgacatggc taggattaaa
2340ctcaaggagc acgtcccttt caatgacgtg tatcgcagtt ggcatggtca
cgctgtacct 2400aggagtcatg gttcaggcgg actcgggatg tgtaatcaac
tggaaaggca gagaactcaa 2460atgtggaagc ggcatttttg tcaccaatga
agtccacacc tggacagagc aatataaatt 2520ccaggccgac tcccctaaga
gactatcagc ggccattggg aaggcatggg aggagggtgt 2580gtgtggaatt
cgatcagcca ctcgtctcga gaacatcatg tggaagcaaa tatcaaatga
2640attaaaccac atcttacttg aaaatgacat gaaatttaca gtggtcgtag
gagacgttag 2700tggaatcttg gcccaaggaa agaaaatgat taggccacaa
cccatggaac acaaatactc 2760gtggaaaagc tggggaaaag ccaaaatcat
aggagcagat gtacagaata ccaccttcat 2820catcgacggc ccaaacaccc
cagaatgccc tgataaccaa agagcatgga acatttggga 2880agttgaagac
tatggatttg gaattttcac gacaaacata tggttgaaat tgcgtgactc
2940ctacactcaa gtgtgtgacc accggctaat gtcagctgcc atcaaggata
gcaaagcagt 3000ccatgctgac atggggtact ggatagaaag tgaaaagaac
gagacttgga agttggcaag 3060agcctccttc atagaagtta agacatgcat
ctggccaaaa tcccacactc tatggagcaa 3120tggagtcctg gaaagtgaga
tgataatccc aaagatatat ggaggaccaa tatctcagca 3180caactacaga
ccaggatatt tcacacaaac agcagggccg tggcacttgg gcaagttaga
3240actagatttt gatttatgtg aaggtaccac tgttgttgtg gatgaacatt
gtggaaatcg 3300aggaccatct cttagaacca caacagtcac aggaaagaca
atccatgaat ggtgctgtag 3360atcttgcacg ttaccccccc tacgtttcaa
aggagaagac gggtgctggt acggcatgga 3420aatcagacca gtcaaggaga
aggaagagaa cctagttaag tcaatggtct ctgcagggtc 3480aggagaagtg
gacagttttt cactaggact gctatgcata tcaataatga tcgaagaggt
3540aatgagatcc agatggagca gaaaaatgct gatgactgga acattggctg
tgttcctcct 3600tctcacaatg ggacaattga catggaatga tctgatcagg
ctatgtatca tggttggagc 3660caacgcttca gacaagatgg ggatgggaac
aacgtaccta gctttgatgg ccactttcag 3720aatgagacca atgttcgcag
tcgggctact gtttcgcaga ttaacatcta gagaagttct 3780tcttcttaca
gttggattga gtctggtggc atctgtagaa ctaccaaatt ccttagagga
3840gctaggggat ggacttgcaa tgggcatcat gatgttgaaa ttactgactg
attttcagtc 3900acatcagcta tgggctacct tgctgtcttt aacatttgtc
aaaacaactt tttcattgca 3960ctatgcatgg aagacaatgg ctatgatact
gtcaattgta tctctcttcc ctttatgcct 4020gtccacgact tctcaaaaaa
caacatggct tccggtgttg ctgggatctc ttggatgcaa 4080accactaacc
atgtttctta taacagaaaa caaaatctgg ggaaggaaaa gctggcctct
4140caatgaagga attatggctg ttggaatagt tagcattctt ctaagttcac
ttctcaagaa 4200tgatgtgcca ctagctggcc cactaatagc tggaggcatg
ctaatagcat gttatgtcat 4260atctggaagc tcggccgatt tatcactgga
gaaagcggct gaggtctcct gggaagaaga 4320agcagaacac tctggtgcct
cacacaacat actagtggag gtccaagatg atggaaccat 4380gaagataaag
gatgaagaga gagatgacac actcaccatt ctcctcaaag caactctgct
4440agcaatctca ggggtatacc caatgtcaat accggcgacc ctctttgtgt
ggtatttttg 4500gcagaaaaag aaacagagat caggagtgct atgggacaca
cccagccctc cagaagtgga 4560aagagcagtc cttgatgatg gcatttatag
aattctccaa agaggattgt tgggcaggtc 4620tcaagtagga gtaggagttt
ttcaagaagg cgtgttccac acaatgtggc acgtcaccag 4680gggagctgtc
ctcatgtacc aagggaagag actggaacca agttgggcca gtgtcaaaaa
4740agacttgatc tcatatggag gaggttggag gtttcaagga tcctggaacg
cgggagaaga 4800agtgcaggtg attgctgttg aaccggggaa gaaccccaaa
aatgtacaga cagcgccggg 4860taccttcaag acccctgaag gcgaagttgg
agccatagct ctagacttta aacccggcac 4920atctggatct cctatcgtga
acagagaggg aaaaatagta ggtctttatg gaaatggagt 4980ggtgacaaca
agtggtacct acgtcagcgc catagctcaa gctaaagcat cacaagaagg
5040gcctctacca gagattgagg acgaggtgtt taggaaaaga aacttaacaa
taatggacct 5100acatccagga tcggggaaaa caagaagata tcttccagcc
atagtccgtg aggccataag 5160aaggaacgtg cgcacgctag tcttagctcc
cacaagagtt gtcgcttctg aaatggcaga 5220ggcgctcaag ggaatgccaa
taaggtatca gacaacagca gtgaagagtg aacacacagg 5280aaaagagata
gttgacctta tgtgtcacgc cactttcact atgcgtctcc tgtctcctgt
5340gagagttccc aattataata tgattatcat ggatgaagca cattttaccg
atccagccag 5400catagcagcc agagggtata tctcaacccg agtgggtatg
ggtgaagcag ctgcgatttt 5460catgacagcc actccccccg gatcggtgga
ggcctttcca cagagcaatg cagttatcca 5520agatgaggaa agagacattc
ctgaaagatc atggaactca ggctatgact ggatcactga 5580tttcccaggt
aaaacagtct ggtttgttcc aagcatcaaa tcaggaaatg acattgccaa
5640ctgtttaaga aagaatggga aacgggtggt ccaattgagc agaaaaactt
ttgacactga 5700gtaccagaaa acaaaaaata acgactggga ctatgttgtc
acaacagaca tatccgaaat 5760gggagcaaac ttccgagccg acagggtaat
agacccgagg cggtgcctga aaccggtaat 5820actaaaagat ggcccagagc
gtgtcattct agccggaccg atgccagtga ctgtggctag 5880cgccgcccag
aggagaggaa gaattggaag gaaccaaaat aaggaaggcg atcagtatat
5940ttacatggga cagcctctaa acaatgatga ggaccacgcc cattggacag
aagcaaaaat 6000gctccttgac aacataaaca caccagaagg gattatccca
gccctctttg agccggagag 6060agaaaagagt gcagcaatag acggggaata
cagactacgg ggtgaagcga ggaaaacgtt 6120cgtggagctc atgagaagag
gagatctacc tgtctggcta tcctacaaag ttgcctcaga 6180aggcttccag
tactccgaca gaaggtggtg ctttgatggg gaaaggaaca accaggtgtt
6240ggaggagaac atggacgtgg agatctggac aaaagaagga gaaagaaaga
aactacgacc 6300ccgctggctg gatgccagaa catactctga cccactggct
ctgcgcgaat tcaaagagtt 6360cgcagcagga agaagaagcg tctcaggtga
cctaatatta gaaataggga aacttccaca 6420acatttaacg caaagggccc
agaacgcctt ggacaatctg gttatgttgc acaactctga 6480acaaggagga
aaagcctata gacacgccat ggaagaacta ccagacacca tagaaacgtt
6540aatgctccta gctttgatag ctgtgctgac tggtggagtg acgttgttct
tcctatcagg 6600aaggggtcta ggaaaaacat ccattggcct actctgcgtg
attgcctcaa gcgcactgct 6660atggatggcc agtgtggaac cccattggat
agcggcctct atcatactgg agttctttct 6720gatggtgttg cttattccag
agccggacag acagcgcact ccacaagaca accagctagc 6780atacgtggtg
ataggtctgt tattcatgat attgacagcg gcagccaatg agatgggatt
6840actggaaacc acaaagaagg acctggggat tggtcatgca gctgctgaaa
accaccatca 6900tgctgcaatg ctggacgtag acctacatcc agcttcagcc
tggactctct atgcagtggc 6960cacaacaatt atcactccca tgatgagaca
cacaattgaa aacacaacgg caaatatttc 7020cctgacagct attgcaaacc
aggcagctat attgatggga cttgacaagg gatggccaat 7080atcaaagatg
gacataggag ttccacttct cgccttgggg tgctattctc aggtgaaccc
7140gctgacgctg acagcggcgg tatttatgct agtggctcat tatgccataa
ttggacccgg 7200actgcaagca aaagctacta gagaagctca aaaaaggaca
gcagccggaa taatgaaaaa 7260cccaactgtc gacgggatcg ttgcaataga
tttggaccct gtggtttacg atgcaaaatt 7320tgaaaaacag ctaggccaaa
taatgttgtt gatactttgc acatcacaga tcctcctgat 7380gcggaccaca
tgggccttgt gtgaatccat cacactagcc actggacctc tgactacgct
7440ttgggaggga tctccaggaa aattctggaa caccacgata gcggtgtcca
tggcaaacat 7500ttttagggga agttatctag caggagcagg tctggccttt
tcattaatga aatctctagg 7560aggaggtagg agaggcacgg gagcccaagg
ggaaacactg ggagaaaaat ggaaaagaca 7620gctaaaccaa ttgagcaagt
cagaattcaa cacttacaaa aggagtggga ttatagaggt 7680ggatagatct
gaagccaaag aggggttaaa aagaggagaa ccgactaaac acgcagtgtc
7740gagaggaacg gccaaactga ggtggtttgt ggagaggaac cttgtgaaac
cagaagggaa 7800agtcatagac ctcggttgtg gaagaggtgg ctggtcatat
tattgcgctg ggctgaagaa 7860agtcacagaa gtgaaaggat acacgaaagg
aggacctgga catgaggaac caatcccaat 7920ggcaacctat ggatggaacc
tagtaaagct atactccggg aaagatgtat tctttacacc 7980acctgagaaa
tgtgacaccc tcttgtgtga
tattggtgag tcctctccga acccaactat 8040agaagaagga agaacgttac
gtgttctaaa gatggtggaa ccatggctca gaggaaacca 8100attttgcata
aaaattctaa atccctatat gccgagtgtg gtagaaactt tggagcaaat
8160gcaaagaaaa catggaggaa tgctagtgcg aaatccactc tcaagaaact
ccactcatga 8220aatgtactgg gtttcatgtg gaacaggaaa cattgtgtca
gcagtaaaca tgacatctag 8280aatgttgcta aatcgattca caatggctca
caggaagcca acatatgaaa gagacgtgga 8340cttaggcgct ggaacaagac
atgtggcagt agaaccagag gtggccaacc tagatatcat 8400tggccagagg
atagagaata taaaaaatgg acacaaatca acatggcact atgatgagga
8460caatccatac aaaacatggg cctatcatgg atcatatgag gtcaagccat
caggatcagc 8520ctcatccatg gtcaatggtg tggtgagact gctaaccaaa
ccatgggatg tcattcccat 8580ggtcacacaa atagccatga ctgacaccac
accctttgga caacagaggg tgtttaaaga 8640gaaagttgac acgcgtacac
caaaagcgaa acgaggcaca gcacaaatta tggaggtgac 8700agccaggtgg
ttatggggtt ttctctctag aaacaaaaaa cccagaatct gcacaagaga
8760ggagttcaca agaaaagtca ggtcaaacgc agctattgga gcagtgttcg
ttgatgaaaa 8820tcaatggaac tcagcaaaag aggcagtgga agatgaacgg
ttctgggacc ttgtgcacag 8880agagagggag cttcataaac aaggaaaatg
tgccacgtgt gtctacaaca tgatgggaaa 8940gagagagaaa aaattaggag
agttcggaaa ggcaaaagga agtcgcgcaa tatggtacat 9000gtggttggga
gcgcgctttt tagagtttga agcccttggt ttcatgaatg aagatcactg
9060gttcagcaga gagaattcac tcagtggagt ggaaggagaa ggactccaca
aacttggata 9120catactcaga gacatatcaa agattccagg gggaaatatg
tatgcagatg acacagccgg 9180atgggacaca agaataacag aggatgatct
tcagaatgag gccaaaatca ctgacatcat 9240ggaacctgaa catgccctat
tggccacgtc aatctttaag ctaacctacc aaaacaaggt 9300agtaagggtg
cagagaccag cgaaaaatgg aaccgtgatg gatgtcatat ccagacgtga
9360ccagagagga agtggacagg ttggaaccta tggcttaaac accttcacca
acatggaggc 9420ccaactaata agacaaatgg agtctgaggg aatcttttca
cccagcgaat tggaaacccc 9480aaatctagcc gaaagagtcc tcgactggtt
gaaaaaacat ggcaccgaga ggctgaaaag 9540aatggcaatc agtggagatg
actgtgtggt gaaaccaatc gatgacagat ttgcaacagc 9600cttaacagct
ttgaatgaca tgggaaaggt aagaaaagac ataccgcaat gggaaccttc
9660aaaaggatgg aatgattggc aacaagtgcc tttctgttca caccatttcc
accagctgat 9720tatgaaggat gggagggaga tagtggtgcc atgccgcaac
caagatgaac ttgtaggtag 9780ggccagagta tcacaaggcg ccggatggag
cttgagagaa actgcatgcc taggcaagtc 9840atatgcacaa atgtggcagc
tgatgtactt ccacaggaga gacttgagat tagcggctaa 9900tgctatctgt
tcagccgttc cagttgattg ggtcccaacc agccgtacca cctggtcgat
9960ccatgcccac catcaatgga tgacaacaga agacatgttg tcagtgtgga
atagggtttg 10020gatagaggaa aacccatgga tggaggacaa gactcatgtg
tccagttggg aagacgttcc 10080atacctagga aaaagggaag atcgatggtg
tggatcccta ataggcttaa cagcacgagc 10140cacctgggcc accaacatac
aagtggccat aaaccaagtg agaaggctca ttgggaatga 10200gaattatcta
gacttcatga catcaatgaa gagattcaaa aacgagagtg atcccgaagg
10260ggcactctgg taagccaact cattcacaaa ataaaggaaa ataaaaaatc
aaacaaggca 10320agaagtcagg ccggattaag ccatagcacg gtaagagcta
tgctgcctgt gagccccgtc 10380caaggacgta aaatgaagtc aggccgaaag
ccacggttcg agcaagccgt gctgcctgta 10440gctccatcgt ggggatgtaa
aaacccggga ggctgcaaac catggaagct gtacgcatgg 10500ggtagcagac
tagtggttag aggagacccc tcccaagaca caacgcagca gcggggccca
10560acaccagggg aagctgtacc ctggtggtaa ggactagagg ttagaggaga
ccccccgcac 10620aacaacaaac agcatattga cgctgggaga gaccagagat
cctgctgtct ctacagcatc 10680attccaggca cagaacgcca aaaaatggaa
tggtgctgtt gaatcaacag gttct 10735410724DNADengue virus type 2
4agttgttagt ctacgtggac cgacaaagac agattctttg agggagctaa gctcaacgta
60gttctaacag ttttttaatt agagagcaga tctctgatga ataaccaacg aaaaaaggcg
120agaaataccc ctttcaatat gctgaaacgc gagagaaacc gcgtgtcgac
tgtacaacag 180ctgacaaaga gattctcact tggaatgctg cagggacgag
gaccattaaa actgttcatg 240gccctggtgg cgttccttcg tttcctaaca
atcccaccaa cagcagggat actgaagaga 300tggggaacaa ttaaaaaatc
aaaagccatt aatgttttga gagggttcag gaaagagatt 360ggaaggatgc
tgaacatctt gaacaggaga cgcagaactg caggcatgat cattatgctg
420attccaacag tgatggcgtt ccatttaacc acacgtaacg gagaaccaca
catgatcgtc 480agtagacaag agaaagggaa aagtcttctg tttaaaacag
aggatggtgt gaacatgtgt 540accctcatgg ccatggacct tggtgaattg
tgtgaagata caatcacgta caagtgtcct 600tttctcaggc agaatgaacc
agaagacata gattgttggt gcaactctac gtccacatgg 660gtaacttatg
ggacgtgtac caccacagga gaacacagaa gagaaaaaag atcagtggca
720ctcgttccac atgtgggaat gggactggag acacgaactg aaacatggat
gtcatcagaa 780ggggcctgga aacatgccca gagaattgaa acttggatct
tgagacatcc aggctttacc 840ataatggcag caatcctggc atacaccata
ggaacgacac atttccaaag agccctgatt 900ttcatcttac tgacagctgt
cgctccttca atgacaatgc gttgcatagg aatatcaaat 960agagactttg
tagaaggggt ttcaggagga agctgggttg acatagtctt agaacatgga
1020agctgtgtga cgacgatggc aaaaaacaaa ccaacattgg attttgaact
gataaaaaca 1080gaagccaaac aacctgccac tctaaggaag tactgtatag
aggcaaagct gaccaacaca 1140acaacagatt ctcgctgccc aacacaagga
gaacccagcc taaatgaaga gcaggacaaa 1200aggttcgtct gcaaacactc
catggtggac agaggatggg gaaatggatg tggattattt 1260ggaaaaggag
gcattgtgac ctgtgctatg ttcacatgca aaaagaacat gaaaggaaaa
1320gtcgtgcaac cagaaaactt ggaatacacc attgtgataa cacctcactc
aggggaagag 1380catgcagtcg gaaatgacac aggaaaacat ggcaaggaaa
tcaaaataac accacagagt 1440tccatcacag aagcagagtt gacaggctat
ggcactgtca cgatggagtg ctctccgaga 1500acgggcctcg acttcaatga
gatggtgttg ctgcaaatgg aaaataaagc ttggctggtg 1560cacaggcaat
ggttcctaga cctgccgttg ccatggctgc ccggagcgga cacacaagga
1620tcaaattgga tacagaaaga gacattggtc actttcaaaa atccccatgc
gaagaaacag 1680gatgttgttg ttttgggatc ccaagaaggg gccatgcaca
cagcactcac aggggccaca 1740gaaatccaga tgtcatcagg aaacttactg
ttcacaggac atctcaagtg caggctgagg 1800atggacaaac tacagctcaa
aggaatgtca tactctatgt gcacaggaaa gtttaaagtt 1860gtgaaggaaa
tagcagaaac acaacatgga acaatagtta tcagagtaca atatgaaggg
1920gacggttctc catgtaagat cccttttgag ataatggatt tggaaaaaag
acatgtttta 1980ggtcgcctga ttacagtcaa cccaatcgta acagaaaaag
atagcccagt caacatagaa 2040gcagaacctc cattcggaga cagctacatc
atcataggag tagagccggg acaattgaag 2100ctcaactggt ttaagaaagg
aagttctatc ggccaaatga ttgagacaac aatgagggga 2160gcgaagagaa
tggccatttt aggtgacaca gcttgggatt ttggatccct gggaggagtg
2220tttacatcta taggaaaggc tctccaccaa gttttcggag caatctatgg
ggctgccttc 2280agtggggtct catggactat gaaaatactc ataggagtca
ttatcacatg gataggaatg 2340aattcacgca gcacctcact gtctgtgtca
ctagtattgg tgggagtcgt gacgctgtat 2400ttgggagtta tggtgcaggc
cgatagtggt tgcgttgtga gctggaaaaa caaagaactg 2460aagtgtggca
gtgggatttt catcacagac aacgtgcaca catggacaga acaatacaag
2520ttccaaccag aatccccttc aaagctagct tcagctatcc agaaagctca
tgaagagggc 2580atttgtggaa tccgctcagt aacaagactg gaaaatctga
tgtggaaaca aataacacca 2640gaattgaatc acattctatc agaaaatgag
gtgaagttga ctattatgac aggagacatc 2700aaaggaatca tgcaggcagg
aaaacgatct ctgcagcccc agcccactga gctgaagtat 2760tcatggaaaa
catggggcaa agcgaaaatg ctctctacag agtctcataa ccagaccttt
2820ctcattgatg gccccgaaac agcagaatgc cccaacacaa acagagcttg
gaattcgctg 2880gaagttgaag actatggctt tggagtattc accaccaata
tatggctaaa gttgagagaa 2940aagcaggatg tattctgcga ctcaaaactc
atgtcagcgg ccataaaaga caacagagcc 3000gtccatgccg atatgggtta
ttggatagaa agtgcactca atgacacatg gaagatagag 3060aaagcctctt
tcatcgaagt taaaagctgc cactggccaa agtcacacac cctctggagt
3120aatggagtgt tagaaagtga gatgataatt ccaaagaatt tcgctggacc
agtgtcacaa 3180cacaactaca gaccaggcta ccatacacaa acagcaggac
catggcatct aggtaagctt 3240gagatggact ttgatttctg cgaaggaacc
acagtggtgg tgactgagga ctgtggaaat 3300agaggaccct ctttaagaac
aactactgcc tctggaaaac tcataacaga atggtgctgc 3360cgatcttgca
cattaccacc gctaagatac agaggtgagg acggatgctg gtacgggatg
3420gaaatcagac cattgaaaga gaaagaagag aatttggtca actccttggt
cacagccgga 3480catgggcaga ttgacaactt ttcactagga gtcttgggaa
tggcattgtt cctggaagaa 3540atgctcagga cccgagtagg aacgaaacat
gcaatactac tagttgcagt ttcttttgtg 3600acattgatca cagggaacat
gtcctttaga gacctgggaa gagtgatggt tatggtgggc 3660gctactatga
cggatgacat aggtatgggc gtgacttatc ttgccctact agcagccttc
3720aaagtcagac caacttttgc agctggacta ctcttgagaa agttgacctc
caaggaattg 3780atgatgacta ccataggaat cgtactcctc tcccagagca
ccataccaga gaccattctt 3840gaactgactg atgcgttagc cttgggcatg
atggtcctta aaatggtgag aaaaatggaa 3900aagtatcaat tggcagtgac
tatcatggct atcttgtgcg tcccaaatgc agtgatatta 3960caaaacgcat
ggaaagtgag ttgcacaata ttggcagtgg tgtccgtttc cccactgttc
4020ttaacatcct cacagcagaa agcggattgg ataccattag cattgacgat
caagggtctc 4080aatccaacag ctatttttct aacaaccctt tcaagaacca
acaagaaaag gagctggcca 4140ctaaatgagg ctatcatggc agtcgggatg
gtgagcattt tggccagttc actcctaaag 4200aatgacattc ccatgacagg
accattagtg gctggagggc tcctcactgt gtgctacgtg 4260ctcactggac
gatcggccga tttggaactg gagagagccg ccgatgtcaa atgggaagat
4320caggcagaga tatcaggaag cagtccaatc ctgtcaataa caatatcaga
agatggtagc 4380atgtcgataa aaaacgaaga ggaagaacaa acactgacca
tactcattag aacaggattg 4440ctggtgatct caggactttt tcctgtatca
ataccaatca cggcagcagc atggtacctg 4500tgggaagtga agaaacaacg
ggctggagta ttgtgggatg tcccttcacc cccacccgtg 4560ggaaaggctg
aactggaaga tggagcctat agaatcaagc aaaaagggat tcttggatat
4620tcccagatcg gagccggagt ttacaaagaa ggaacattcc atacaatgtg
gcatgtcaca 4680cgcggcgctg ttctaatgca taaaggaaag aggattgaac
catcatgggc ggacgttaag 4740aaagacctaa tatcatatgg aggaggctgg
aagctagaag gagaatggaa ggaaggagaa 4800gaagtccagg tcttggcatt
ggagcctgga aaaaatccaa gagccgtcca aacaaaacct 4860ggtcttttca
aaaccaacgc cggaaccata ggtgccgtat ctctggactt ttctcctgga
4920acctcaggat ctccaatcat cgacaaaaaa ggaaaagttg tgggtcttta
tggtaatggt 4980gttgttacaa ggagtggagc atatgtgagt gctatagccc
agactgaaaa aagtattgaa 5040gacaatccag agatcgaaga tgatattttt
cgaaagagaa aattgaccat catggacctc 5100cacccaggag cgggaaagac
gaagagatac cttccggcca tagtcagaga ggctataaaa 5160cggggcctga
ggacattaat cctggccccc actagagtcg tggcagctga aatggaggaa
5220gccctaagag gacttccaat aagataccaa accccagcca tcagagctga
gcacaccggg 5280cgggagattg tggacctaat gtgtcatgcc acattcacta
tgaggctgct atcaccagtt 5340agagtgccaa attacaacct gatcatcatg
gacgaagccc atttcacaga cccagcaagt 5400atagcggcta gaggatacat
ctcaactcga gtagagatgg gtgaggcagc tgggattttc 5460atgacagcca
ctcctccggg aagcagagac ccattccctc agagcaatgc accaatcatg
5520gatgaagaaa gagaaatccc tgaacgttcg tggagttctg gacatgagtg
ggtcacggat 5580tttaaaggga agactgtttg gttcgttcca agtataaaag
caggaaatga tatagcagct 5640tgcctgagaa aaaatggaaa gaaagtgata
caactcagta ggaagacctt tgattctgag 5700tatgtcaaga ctagaaccaa
tgattgggac ttcgtggtca caactgacat ttcagaaatg 5760ggtgccaact
tcaaggctga gagggttata gaccccagac gctgcatgaa accagttata
5820ctaacagatg gtgaagagcg ggtgatcctg gcaggaccta tgccagtgac
ccactctagt 5880gcagcacaaa gaagagggag aataggaaga aatccaaaaa
atgaaaatga ccagtacata 5940tacatggggg aacctctgga aaatgatgaa
gactgtgcac actggaaaga agctaaaatg 6000ctcctagata acatcaacac
acctgaagga atcattccta gcatgttcga accagagcgt 6060gaaaaggtgg
atgccattga tggtgaatac cgcttgagag gagaagcaag gaaaaccttt
6120gtggacctaa tgagaagagg agacctacca gtctggttgg cctacagagt
ggcagctgaa 6180ggcatcaact acgcagacag aaggtggtgt tttgatggaa
ttaagaacaa ccaaatcttg 6240gaagaaaatg tggaggtgga aatctggaca
aaagaagggg aaaggaagaa attaaaaccc 6300agatggttgg atgccaggat
ctactctgac ccactgacgc taaaggaatt caaggagttt 6360gcagctggaa
gaaagtccct gaccctgaac ctaatcacag aaatgggtag gcttccaact
6420ttcatgactc agaaggcaag agacgcactg gacaacttag cagtgctgca
cacggctgaa 6480gcaggtggaa gggcgtacaa tcatgctctc agtgaactgc
cggagaccct ggagacattg 6540cttttactga cacttctggc tacagtcaca
ggaggaatct ttttattctt gatgagcgga 6600aggggtatag ggaagatgac
cctgggaatg tgctgcataa tcacggctag tattctccta 6660tggtacgcac
aaatacagcc acactggata gcagcttcaa taatactgga gttttttctc
6720atagttttgc ttattccaga accagaaaag cagagaacac cccaagataa
ccaattgacc 6780tacgttgtca tagccatcct cacagtggtg gccgcaacca
tggcaaacga gatgggtttc 6840ctggaaaaaa cgaagaaaga tctcggattg
ggaagcatta caacccagca acccgagagc 6900aacatcctgg acatagatct
acgtcccgca tcagcatgga cgctgtatgc tgtggccaca 6960acatttgtca
caccaatgtt gagacacagc attgaaaatt cctcagtgaa cgtgtcccta
7020acagctattg ccaaccaagc cacagtgtta atgggtcttg ggaaaggatg
gccattgtca 7080aagatggaca tcggagttcc ccttctcgcc attggatgct
actcacaagt caaccccata 7140actctcacag cagctctttt cttactggta
gcacattatg ccatcatagg gccaggactc 7200caagcaaaag caaccaggga
agctcagaaa agagcagcag cgggcatcat gaaaaaccca 7260actgtcgatg
gaataacagt gattgaccta gatccaatac cctatgatcc aaagtttgaa
7320aagcagttgg gacaagtaat gctcctagtc ctctgcgtga ctcaagtgtt
gatgatgagg 7380actacatggg ctctgtgtga ggctttaacc ttagcgaccg
ggcctatctc cacattgtgg 7440gaaggaaatc cagggaggtt ttggaacact
accattgcag tgtcaatggc taacattttt 7500agagggagtt acttggccgg
agctggactt ctcttttcca tcatgaagaa cacaaccaac 7560acgagaaggg
gaactggcaa cataggagag acgcttggag agaaatggaa aagccgattg
7620aacgcattgg ggaaaagtga attccagatc tacaagaaaa gtggaatcca
ggaagtggat 7680agaaccttag caaaagaagg cattaaaaga ggagaaacgg
accatcacgc tgtgtcgcga 7740ggctcagcaa aactgagatg gttcgtcgag
agaaatatgg tcacaccaga agggaaagta 7800gtggacctcg gttgcggcag
aggaggctgg tcatactatt gtgggggact aaagaatgta 7860agagaagtca
aaggcctgac aaaaggagga ccaggacatg aagaacccat ccccatgtca
7920acatatgggt ggaatctagt acgtcttcaa agtggagttg acgttttctt
cactccgcca 7980gaaaagtgtg acacattgtt gtgtgacata ggggagtcgt
caccaaatcc cacggtagaa 8040gcaggacgaa cactcagagt ccttaactta
gtggaaaatt ggttgaacaa caacacccaa 8100ttttgcataa aggttctcaa
cccatacatg ccctcagtca tagaaaaaat ggaagcacta 8160caaaggaaat
atggaggagc cttagtgagg aatccactct cacgaaactc cacacatgag
8220atgtactggg tatccaatgc ctccgggaac atagtgtcat cagtgaacat
gatttcaagg 8280atgttgatca acagattcac aatgagacac aagaaagcca
cttacgagcc agatgtagac 8340ctcggaagcg gaacccgcaa catcggaatt
gaaagtgaga taccaaacct agacataatc 8400gggaaaagaa tagaaaaaat
aaaacaagag catgaaacat catggcacta tgaccaagac 8460cacccataca
aaacgtgggc ttaccatggc agctatgaaa caaaacaaac tggatcagca
8520tcatccatgg tgaacggagt ggtcagactg ctgacaaaac cttgggacgt
cgtccccatg 8580gtgacacaga tggcaatgac agacacgact ccatttggac
aacagcgcgt ttttaaagaa 8640aaagtggaca cgagaaccca agaaccgaaa
gaaggcacaa agaaactaat gaaaatcacg 8700gcagagtggc tttggaaaga
actagggaag aaaaagacac ctaggatgtg cactagagaa 8760gaattcacaa
gaaaggtgag aagcaatgca gccttggggg ccatattcac tgatgagaac
8820aagtggaagt cggcacgtga ggctgttgaa gatagtaggt tttgggagct
ggttgacaag 8880gaaaggaatc tccatcttga aggaaagtgt gaaacatgtg
tgtataacat gatgggaaaa 8940agagagaaga agctagggga gttcggcaag
gcaaaaggca gcagagccat atggtacatg 9000tggcttggag cacgcttctt
agagtttgaa gccctaggat tcttgaatga agatcactgg 9060ttctccagag
agaactcctt gagtggagtg gaaggagaag ggctgcacaa gctaggttac
9120attttaagag acgtgagcaa gaaagaggga ggagcaatgt atgccgatga
caccgcagga 9180tgggacacaa gaatcacact agaagaccta aaaaatgaag
aaatggtaac aaaccacatg 9240gaaggagaac acaagaaact agccgaggcc
attttcaaat taacgtacca aaacaaggtg 9300gtgcgtgtgc aaagaccaac
accaagaggc acagtaatgg atatcatatc gagaagagac 9360caaagaggta
gtggacaagt tggtacctat ggactcaata ctttcaccaa tatggaagcc
9420caactaatca gacagatgga gggagaagga gtcttcaaaa gcattcagca
cctgacagtc 9480acagaagaaa tcgccgtgca aaactggtta gcaagagtag
ggcgcgaaag gttatcaaga 9540atggccatca gtggagatga ttgtgttgtg
aaacctttag atgacaggtt cgcaagcgct 9600ttaacagctc taaatgacat
gggaaaggtt aggaaagaca tacaacaatg ggaaccttca 9660agaggatgga
acgattggac acaagtgccc ttctgttcac accatttcca tgagttaatc
9720atgaaagacg gccgcgtact tgtagttcca tgcagaaacc aagatgaact
gattggtaga 9780gcccgaattt cccaaggagc tgggtggtct ttgcgagaga
cggcctgttt ggggaagtcc 9840tacgcccaaa tgtggagctt gatgtacttc
cacagacgtg acctcaggct ggcggctaat 9900gctatttgct cggcagtccc
atcacattgg gttccaacaa gtagaacaac ctggtccata 9960cacgccaaac
atgaatggat gacaacggaa gacatgctga cagtctggaa cagggtgtgg
10020attcaagaaa acccatggat ggaagacaaa actccagtgg aatcatggga
ggaaatccca 10080tacttgggga aaagagaaga ccaatggtgc ggctcattga
ttgggctaac aagcagggcc 10140acctgggcaa agaacatcca aacagcaata
aatcaagtta gatcccttat aggcaatgag 10200gaatacacag attacatgcc
atccatgaaa agattcagaa gagaagagga agaggcagga 10260gtcctgtggt
agaaggcaaa actaacatga aacaaggcta gaagtcaggt cggattaagc
10320tatagtacgg aaaaaactat gctacctgtg agccccgtcc aaggacgtta
aaagaagtca 10380ggccattaca aatgccatag cttgagtaaa ctgtggcagc
ctgtagctcc acctgagaag 10440gtgtaaaaaa tctgggaggc cacaaaccat
ggaagctgta cgcatggcgt agtggactag 10500cggttagagg agacccctcc
cttacaaatc gcagcaacaa tgggggccca aggtgagatg 10560aagctgtagt
ctcactggaa ggactagagg ttagaggaga cccccccaaa acaaaaaaca
10620gcatattgac gctgggaaag accagagatc ctgctgtctc ctcagcatca
ttccaggcac 10680agaacgccag aaaatggaat ggtgctgttg aatcaacagg ttct
107245519DNAWest Nile virus 5ttggaaggag tgtctggagc aacatgggtg
gatttggttc tcgaaggcga cagctgcgtg 60actatcatgt ctaaggacaa gcctaccatc
gatgtgaaga tgatgaatat ggaggcggcc 120aacctggcag aggtccgcag
ttattgctat ttggctaccg tcagcgatct ctccaccaaa 180gctgcgtgcc
cgaccatggg agaagctcac aatgacaaac gtgctgaccc agcttttgtg
240tgcagacaag gagtggtgga caggggctgg ggcaacggct gcggactatt
tggcaaagga 300agcattgaca catgcgccaa atttgcctgc tctaccaagg
caataggaag aaccatcttg 360aaagagaata tcaagtacga agtggccatt
tttgtccatg gaccaactac tgtggagtcg 420cacggaaact actccacaca
ggttggagcc actcaggcag ggagattcag catcactcct 480gcagcgcctt
catacacact aaagcttgga gaatatgga 5196171PRTWest Nile virus 6Leu Glu
Gly Val Ser Gly Ala Thr Trp Val Asp Leu Val Leu Glu Gly1 5 10 15Asp
Ser Cys Val Thr Ile Met Ser Lys Asp Lys Pro Thr Ile Asp Val 20 25
30Lys Met Met Asn Met Glu Ala Ala Asn Leu Ala Glu Val Arg Ser Tyr
35 40 45Cys Tyr Leu Ala Thr Val Ser Asp Leu Ser Thr Lys Ala Ala Cys
Pro 50 55 60Thr Met Gly Glu Ala His Asn Asp Lys Arg Ala Asp Pro Ala
Phe Val65 70 75 80Cys Arg Gln Gly Val Val Asp Arg Gly Trp Gly Asn
Gly Cys Gly Leu 85 90 95Phe Gly Lys Gly Ser Ile Asp Thr Cys Ala Lys
Phe Ala Cys Ser Thr 100 105 110Lys Ala Ile Gly Arg Thr Ile Leu Lys
Glu Asn Ile Lys Tyr Glu Val 115 120 125Ala Ile Phe Val His Gly Pro
Thr Thr Val Glu Ser His Gly Asn Tyr 130 135 140Ser Thr Gln Val Gly
Ala Thr Gln Ala Gly Arg Phe Ser Ile Thr Pro145 150 155 160Ala
Ala
Pro Ser Tyr Thr Leu Lys Leu Gly Glu 165 17072715DNAWest Nile virus
7ggtggggcaa aaggacgcac cttgggagag gtttggaaag aaagactcaa ccagatgaca
60aaagaagagt tcactaggta ccgcaaagag gccatcatcg aagtcgatcg ctcagcagca
120aaacacgcca ggaaagaagg caatgtcact ggagggcatc cagtctctag
gggcacagca 180aaactgagat ggctggtcga acggaggttt ctcgaaccgg
tcggaaaagt gattgacctt 240ggatgtggaa gaggcggttg gtgttactat
atggcaaccc aaaaaagagt ccaagaagtc 300agagggtaca caaagggcgg
tcccggacat gaagagcccc aactagtgca aagttatgga 360tggaacattg
tcaccatgaa gagtggggtg gatgtgttct acagaccttc tgagtgttgt
420gacaccctcc tttgtgacat cggagagtcc tcgtcaagtg ctgaggttga
agagcatagg 480acgattcggg tccttgaaat ggttgaggac tggctgcacc
gagggccaag ggaattttgc 540gtgaaggtgc tctgccccta catgccgaaa
gtcatagaga agatggagct gctccaacgc 600cggtatgggg ggggactggt
cagaaaccca ctctcacgga attccacgca cgagatgtat 660tgggtgagtc
gagcttcagg caatgtggta cattcagtga atatgaccag ccaggtgctc
720ctaggaagaa tggaaaaaag gacctggaag ggaccccaat acgaggaaga
tgtaaacttg 780ggaagtggaa ccagggcggt gggaaaaccc ctgctcaact
cagacaccag taaaatcaag 840aacaggattg aacgactcag gcgtgagtac
agttcgacgt ggcaccacga tgagaaccac 900ccatatagaa cctggaacta
tcacggcagt tatgatgtga agcccacagg ctccgccagt 960tcgctggtca
atggagtggt caggctcctc tcaaaaccat gggacaccat cacgaatgtt
1020accaccatgg ccatgactga cactactccc ttcgggcagc agcgagtgtt
caaagagaag 1080gtggacacga aagctcctga accgccagaa ggagtgaagt
acgtgctcaa cgagaccacc 1140aactggttgt gggcgttttt ggccagagaa
aaacgtccca gaatgtgctc tcgagaggaa 1200ttcataagaa aggtcaacag
caatgcagct ttgggtgcca tgtttgaaga gcagaatcaa 1260tggaggagcg
ccagagaggc agttgaagat ccaaaatttt gggagatggt ggatgaggag
1320cgcgaggcac atctgcgggg ggaatgtcac acttgcattt acaacatgat
gggaaagaga 1380gagaaaaaac ccggagagtt cggaaaggcc aagggaagca
gagccatttg gttcatgtgg 1440ctcggagctc gctttctgga gttcgaggct
ctgggttttc tcaatgaaga ccactggctt 1500ggaagaaaga actcaggagg
aggtgtcgag ggcttgggcc tccaaaaact gggttacatc 1560ctgcgtgaag
ttggcacccg gcctgggggc aagatctatg ctgatgacac agctggctgg
1620gacacccgca tcacgagagc tgacttggaa aatgaagcta aggtgcttga
gctgcttgat 1680ggggaacatc ggcgtcttgc cagggccatc attgagctca
cctatcgtca caaagttgtg 1740aaagtgatgc gcccggctgc tgatggaaga
accgtcatgg atgttatctc cagagaagat 1800cagaggggga gtggacaagt
tgtcacctac gccctaaaca ctttcaccaa cctggccgtc 1860cagctggtga
ggatgatgga aggggaagga gtgattggcc cagatgatgt ggagaaactc
1920acaaaaggga aaggacccaa agtcaggacc tggctgtttg agaatgggga
agaaagactc 1980agccgcatgg ctgtcagtgg agatgactgt gtggtaaagc
ccctggacga tcgctttgcc 2040acctcgctcc acttcctcaa tgctatgtca
aaggttcgca aagacatcca agagtggaaa 2100ccgtcaactg gatggtatga
ttggcagcag gttccatttt gctcaaacca tttcactgaa 2160ttgatcatga
aagatggaag aacactggtg gttccatgcc gaggacagga tgaattggta
2220ggcagagctc gcatatctcc aggggccgga tggaacgtcc gcgacactgc
ttgtctggct 2280aagtcttatg cccagatgtg gctgcttctg tacttccaca
gaagagacct gcggctcatg 2340gccaacgcca tttgctccgc tgtccctgtg
aattgggtcc ctaccggaag aaccacgtgg 2400tccatccatg caggaggaga
gtggatgaca acagaggaca tgttggaggt ctggaaccgt 2460gtttggatag
aggagaatga atggatggaa gacaaaaccc cagtggagaa atggagtgac
2520gtcccatatt caggaaaacg agaggacatc tggtgtggca gcctgattgg
cacaagagcc 2580cgagccacgt gggcagaaaa catccaggtg gctatcaacc
aagtcagagc aatcatcgga 2640gatgagaagt atgtggatta catgagttca
ctaaagagat atgaagacac aactttggtt 2700gaggacacag tactg
27158905PRTWest Nile virus 8Gly Gly Ala Lys Gly Arg Thr Leu Gly Glu
Val Trp Lys Glu Arg Leu1 5 10 15Asn Gln Met Thr Lys Glu Glu Phe Thr
Arg Tyr Arg Lys Glu Ala Ile 20 25 30Ile Glu Val Asp Arg Ser Ala Ala
Lys His Ala Arg Lys Glu Gly Asn 35 40 45Val Thr Gly Gly His Pro Val
Ser Arg Gly Thr Ala Lys Leu Arg Trp 50 55 60Leu Val Glu Arg Arg Phe
Leu Glu Pro Val Gly Lys Val Ile Asp Leu65 70 75 80Gly Cys Gly Arg
Gly Gly Trp Cys Tyr Tyr Met Ala Thr Gln Lys Arg 85 90 95Val Gln Glu
Val Arg Gly Tyr Thr Lys Gly Gly Pro Gly His Glu Glu 100 105 110Pro
Gln Leu Val Gln Ser Tyr Gly Trp Asn Ile Val Thr Met Lys Ser 115 120
125Gly Val Asp Val Phe Tyr Arg Pro Ser Glu Cys Cys Asp Thr Leu Leu
130 135 140Cys Asp Ile Gly Glu Ser Ser Ser Ser Ala Glu Val Glu Glu
His Arg145 150 155 160Thr Ile Arg Val Leu Glu Met Val Glu Asp Trp
Leu His Arg Gly Pro 165 170 175Arg Glu Phe Cys Val Lys Val Leu Cys
Pro Tyr Met Pro Lys Val Ile 180 185 190Glu Lys Met Glu Leu Leu Gln
Arg Arg Tyr Gly Gly Gly Leu Val Arg 195 200 205Asn Pro Leu Ser Arg
Asn Ser Thr His Glu Met Tyr Trp Val Ser Arg 210 215 220Ala Ser Gly
Asn Val Val His Ser Val Asn Met Thr Ser Gln Val Leu225 230 235
240Leu Gly Arg Met Glu Lys Arg Thr Trp Lys Gly Pro Gln Tyr Glu Glu
245 250 255Asp Val Asn Leu Gly Ser Gly Thr Arg Ala Val Gly Lys Pro
Leu Leu 260 265 270Asn Ser Asp Thr Ser Lys Ile Lys Asn Arg Ile Glu
Arg Leu Arg Arg 275 280 285Glu Tyr Ser Ser Thr Trp His His Asp Glu
Asn His Pro Tyr Arg Thr 290 295 300Trp Asn Tyr His Gly Ser Tyr Asp
Val Lys Pro Thr Gly Ser Ala Ser305 310 315 320Ser Leu Val Asn Gly
Val Val Arg Leu Leu Ser Lys Pro Trp Asp Thr 325 330 335Ile Thr Asn
Val Thr Thr Met Ala Met Thr Asp Thr Thr Pro Phe Gly 340 345 350Gln
Gln Arg Val Phe Lys Glu Lys Val Asp Thr Lys Ala Pro Glu Pro 355 360
365Pro Glu Gly Val Lys Tyr Val Leu Asn Glu Thr Thr Asn Trp Leu Trp
370 375 380Ala Phe Leu Ala Arg Glu Lys Arg Pro Arg Met Cys Ser Arg
Glu Glu385 390 395 400Phe Ile Arg Lys Val Asn Ser Asn Ala Ala Leu
Gly Ala Met Phe Glu 405 410 415Glu Gln Asn Gln Trp Arg Ser Ala Arg
Glu Ala Val Glu Asp Pro Lys 420 425 430Phe Trp Glu Met Val Asp Glu
Glu Arg Glu Ala His Leu Arg Gly Glu 435 440 445Cys His Thr Cys Ile
Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Pro 450 455 460Gly Glu Phe
Gly Lys Ala Lys Gly Ser Arg Ala Ile Trp Phe Met Trp465 470 475
480Leu Gly Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu
485 490 495Asp His Trp Leu Gly Arg Lys Asn Ser Gly Gly Gly Val Glu
Gly Leu 500 505 510Gly Leu Gln Lys Leu Gly Tyr Ile Leu Arg Glu Val
Gly Thr Arg Pro 515 520 525Gly Gly Lys Ile Tyr Ala Asp Asp Thr Ala
Gly Trp Asp Thr Arg Ile 530 535 540Thr Arg Ala Asp Leu Glu Asn Glu
Ala Lys Val Leu Glu Leu Leu Asp545 550 555 560Gly Glu His Arg Arg
Leu Ala Arg Ala Ile Ile Glu Leu Thr Tyr Arg 565 570 575His Lys Val
Val Lys Val Met Arg Pro Ala Ala Asp Gly Arg Thr Val 580 585 590Met
Asp Val Ile Ser Arg Glu Asp Gln Arg Gly Ser Gly Gln Val Val 595 600
605Thr Tyr Ala Leu Asn Thr Phe Thr Asn Leu Ala Val Gln Leu Val Arg
610 615 620Met Met Glu Gly Glu Gly Val Ile Gly Pro Asp Asp Val Glu
Lys Leu625 630 635 640Thr Lys Gly Lys Gly Pro Lys Val Arg Thr Trp
Leu Phe Glu Asn Gly 645 650 655Glu Glu Arg Leu Ser Arg Met Ala Val
Ser Gly Asp Asp Cys Val Val 660 665 670Lys Pro Leu Asp Asp Arg Phe
Ala Thr Ser Leu His Phe Leu Asn Ala 675 680 685Met Ser Lys Val Arg
Lys Asp Ile Gln Glu Trp Lys Pro Ser Thr Gly 690 695 700Trp Tyr Asp
Trp Gln Gln Val Pro Phe Cys Ser Asn His Phe Thr Glu705 710 715
720Leu Ile Met Lys Asp Gly Arg Thr Leu Val Val Pro Cys Arg Gly Gln
725 730 735Asp Glu Leu Val Gly Arg Ala Arg Ile Ser Pro Gly Ala Gly
Trp Asn 740 745 750Val Arg Asp Thr Ala Cys Leu Ala Lys Ser Tyr Ala
Gln Met Trp Leu 755 760 765Leu Leu Tyr Phe His Arg Arg Asp Leu Arg
Leu Met Ala Asn Ala Ile 770 775 780Cys Ser Ala Val Pro Val Asn Trp
Val Pro Thr Gly Arg Thr Thr Trp785 790 795 800Ser Ile His Ala Gly
Gly Glu Trp Met Thr Thr Glu Asp Met Leu Glu 805 810 815Val Trp Asn
Arg Val Trp Ile Glu Glu Asn Glu Trp Met Glu Asp Lys 820 825 830Thr
Pro Val Glu Lys Trp Ser Asp Val Pro Tyr Ser Gly Lys Arg Glu 835 840
845Asp Ile Trp Cys Gly Ser Leu Ile Gly Thr Arg Ala Arg Ala Thr Trp
850 855 860Ala Glu Asn Ile Gln Val Ala Ile Asn Gln Val Arg Ala Ile
Ile Gly865 870 875 880Asp Glu Lys Tyr Val Asp Tyr Met Ser Ser Leu
Lys Arg Tyr Glu Asp 885 890 895Thr Thr Leu Val Glu Asp Thr Val Leu
900 90592697DNADengue virus type 1 9ggcacgggag cccaagggga
aacactggga gaaaaatgga aaagacagct aaaccaattg 60agcaagtcag aattcaacac
ttacaaaagg agtgggatta tagaggtgga tagatctgaa 120gccaaagagg
ggttaaaaag aggagaaccg actaaacacg cagtgtcgag aggaacggcc
180aaactgaggt ggtttgtgga gaggaacctt gtgaaaccag aagggaaagt
catagacctc 240ggttgtggaa gaggtggctg gtcatattat tgcgctgggc
tgaagaaagt cacagaagtg 300aaaggataca cgaaaggagg acctggacat
gaggaaccaa tcccaatggc aacctatgga 360tggaacctag taaagctata
ctccgggaaa gatgtattct ttacaccacc tgagaaatgt 420gacaccctct
tgtgtgatat tggtgagtcc tctccgaacc caactataga agaaggaaga
480acgttacgtg ttctaaagat ggtggaacca tggctcagag gaaaccaatt
ttgcataaaa 540attctaaatc cctatatgcc gagtgtggta gaaactttgg
agcaaatgca aagaaaacat 600ggaggaatgc tagtgcgaaa tccactctca
agaaactcca ctcatgaaat gtactgggtt 660tcatgtggaa caggaaacat
tgtgtcagca gtaaacatga catctagaat gttgctaaat 720cgattcacaa
tggctcacag gaagccaaca tatgaaagag acgtggactt aggcgctgga
780acaagacatg tggcagtaga accagaggtg gccaacctag atatcattgg
ccagaggata 840gagaatataa aaaatggaca caaatcaaca tggcactatg
atgaggacaa tccatacaaa 900acatgggcct atcatggatc atatgaggtc
aagccatcag gatcagcctc atccatggtc 960aatggtgtgg tgagactgct
aaccaaacca tgggatgtca ttcccatggt cacacaaata 1020gccatgactg
acaccacacc ctttggacaa cagagggtgt ttaaagagaa agttgacacg
1080cgtacaccaa aagcgaaacg aggcacagca caaattatgg aggtgacagc
caggtggtta 1140tggggttttc tctctagaaa caaaaaaccc agaatctgca
caagagagga gttcacaaga 1200aaagtcaggt caaacgcagc tattggagca
gtgttcgttg atgaaaatca atggaactca 1260gcaaaagagg cagtggaaga
tgaacggttc tgggaccttg tgcacagaga gagggagctt 1320cataaacaag
gaaaatgtgc cacgtgtgtc tacaacatga tgggaaagag agagaaaaaa
1380ttaggagagt tcggaaaggc aaaaggaagt cgcgcaatat ggtacatgtg
gttgggagcg 1440cgctttttag agtttgaagc ccttggtttc atgaatgaag
atcactggtt cagcagagag 1500aattcactca gtggagtgga aggagaagga
ctccacaaac ttggatacat actcagagac 1560atatcaaaga ttccaggggg
aaatatgtat gcagatgaca cagccggatg ggacacaaga 1620ataacagagg
atgatcttca gaatgaggcc aaaatcactg acatcatgga acctgaacat
1680gccctattgg ccacgtcaat ctttaagcta acctaccaaa acaaggtagt
aagggtgcag 1740agaccagcga aaaatggaac cgtgatggat gtcatatcca
gacgtgacca gagaggaagt 1800ggacaggttg gaacctatgg cttaaacacc
ttcaccaaca tggaggccca actaataaga 1860caaatggagt ctgagggaat
cttttcaccc agcgaattgg aaaccccaaa tctagccgaa 1920agagtcctcg
actggttgaa aaaacatggc accgagaggc tgaaaagaat ggcaatcagt
1980ggagatgact gtgtggtgaa accaatcgat gacagatttg caacagcctt
aacagctttg 2040aatgacatgg gaaaggtaag aaaagacata ccgcaatggg
aaccttcaaa aggatggaat 2100gattggcaac aagtgccttt ctgttcacac
catttccacc agctgattat gaaggatggg 2160agggagatag tggtgccatg
ccgcaaccaa gatgaacttg taggtagggc cagagtatca 2220caaggcgccg
gatggagctt gagagaaact gcatgcctag gcaagtcata tgcacaaatg
2280tggcagctga tgtacttcca caggagagac ttgagattag cggctaatgc
tatctgttca 2340gccgttccag ttgattgggt cccaaccagc cgtaccacct
ggtcgatcca tgcccaccat 2400caatggatga caacagaaga catgttgtca
gtgtggaata gggtttggat agaggaaaac 2460ccatggatgg aggacaagac
tcatgtgtcc agttgggaag acgttccata cctaggaaaa 2520agggaagatc
gatggtgtgg atccctaata ggcttaacag cacgagccac ctgggccacc
2580aacatacaag tggccataaa ccaagtgaga aggctcattg ggaatgagaa
ttatctagac 2640ttcatgacat caatgaagag attcaaaaac gagagtgatc
ccgaaggggc actctgg 269710899PRTDengue virus type 1 10Gly Thr Gly
Ala Gln Gly Glu Thr Leu Gly Glu Lys Trp Lys Arg Gln1 5 10 15Leu Asn
Gln Leu Ser Lys Ser Glu Phe Asn Thr Tyr Lys Arg Ser Gly 20 25 30Ile
Ile Glu Val Asp Arg Ser Glu Ala Lys Glu Gly Leu Lys Arg Gly 35 40
45Glu Pro Thr Lys His Ala Val Ser Arg Gly Thr Ala Lys Leu Arg Trp
50 55 60Phe Val Glu Arg Asn Leu Val Lys Pro Glu Gly Lys Val Ile Asp
Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Ala Gly
Leu Lys Lys 85 90 95Val Thr Glu Val Lys Gly Tyr Thr Lys Gly Gly Pro
Gly His Glu Glu 100 105 110Pro Ile Pro Met Ala Thr Tyr Gly Trp Asn
Leu Val Lys Leu Tyr Ser 115 120 125Gly Lys Asp Val Phe Phe Thr Pro
Pro Glu Lys Cys Asp Thr Leu Leu 130 135 140Cys Asp Ile Gly Glu Ser
Ser Pro Asn Pro Thr Ile Glu Glu Gly Arg145 150 155 160Thr Leu Arg
Val Leu Lys Met Val Glu Pro Trp Leu Arg Gly Asn Gln 165 170 175Phe
Cys Ile Lys Ile Leu Asn Pro Tyr Met Pro Ser Val Val Glu Thr 180 185
190Leu Glu Gln Met Gln Arg Lys His Gly Gly Met Leu Val Arg Asn Pro
195 200 205Leu Ser Arg Asn Ser Thr His Glu Met Tyr Trp Val Ser Cys
Gly Thr 210 215 220Gly Asn Ile Val Ser Ala Val Asn Met Thr Ser Arg
Met Leu Leu Asn225 230 235 240Arg Phe Thr Met Ala His Arg Lys Pro
Thr Tyr Glu Arg Asp Val Asp 245 250 255Leu Gly Ala Gly Thr Arg His
Val Ala Val Glu Pro Glu Val Ala Asn 260 265 270Leu Asp Ile Ile Gly
Gln Arg Ile Glu Asn Ile Lys Asn Gly His Lys 275 280 285Ser Thr Trp
His Tyr Asp Glu Asp Asn Pro Tyr Lys Thr Trp Ala Tyr 290 295 300His
Gly Ser Tyr Glu Val Lys Pro Ser Gly Ser Ala Ser Ser Met Val305 310
315 320Asn Gly Val Val Arg Leu Leu Thr Lys Pro Trp Asp Val Ile Pro
Met 325 330 335Val Thr Gln Ile Ala Met Thr Asp Thr Thr Pro Phe Gly
Gln Gln Arg 340 345 350Val Phe Lys Glu Lys Val Asp Thr Arg Thr Pro
Lys Ala Lys Arg Gly 355 360 365Thr Ala Gln Ile Met Glu Val Thr Ala
Arg Trp Leu Trp Gly Phe Leu 370 375 380Ser Arg Asn Lys Lys Pro Arg
Ile Cys Thr Arg Glu Glu Phe Thr Arg385 390 395 400Lys Val Arg Ser
Asn Ala Ala Ile Gly Ala Val Phe Val Asp Glu Asn 405 410 415Gln Trp
Asn Ser Ala Lys Glu Ala Val Glu Asp Glu Arg Phe Trp Asp 420 425
430Leu Val His Arg Glu Arg Glu Leu His Lys Gln Gly Lys Cys Ala Thr
435 440 445Cys Val Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly
Glu Phe 450 455 460Gly Lys Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met
Trp Leu Gly Ala465 470 475 480Arg Phe Leu Glu Phe Glu Ala Leu Gly
Phe Met Asn Glu Asp His Trp 485 490 495Phe Ser Arg Glu Asn Ser Leu
Ser Gly Val Glu Gly Glu Gly Leu His 500 505 510Lys Leu Gly Tyr Ile
Leu Arg Asp Ile Ser Lys Ile Pro Gly Gly Asn 515 520 525Met Tyr Ala
Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Asp 530 535 540Asp
Leu Gln Asn Glu Ala Lys Ile Thr Asp Ile Met Glu Pro Glu His545 550
555 560Ala Leu Leu Ala Thr Ser Ile Phe Lys Leu Thr Tyr Gln Asn Lys
Val 565 570 575Val Arg Val Gln Arg Pro Ala Lys Asn Gly Thr Val Met
Asp Val Ile 580 585 590Ser Arg Arg Asp Gln Arg Gly Ser Gly Gln Val
Gly Thr Tyr Gly Leu 595 600 605Asn Thr Phe Thr Asn Met Glu Ala Gln
Leu Ile Arg Gln Met Glu Ser 610 615 620Glu Gly Ile Phe Ser Pro Ser
Glu Leu Glu Thr Pro Asn Leu Ala Glu625 630 635 640Arg Val Leu Asp
Trp Leu Lys Lys His Gly Thr Glu
Arg Leu Lys Arg 645 650 655Met Ala Ile Ser Gly Asp Asp Cys Val Val
Lys Pro Ile Asp Asp Arg 660 665 670Phe Ala Thr Ala Leu Thr Ala Leu
Asn Asp Met Gly Lys Val Arg Lys 675 680 685Asp Ile Pro Gln Trp Glu
Pro Ser Lys Gly Trp Asn Asp Trp Gln Gln 690 695 700Val Pro Phe Cys
Ser His His Phe His Gln Leu Ile Met Lys Asp Gly705 710 715 720Arg
Glu Ile Val Val Pro Cys Arg Asn Gln Asp Glu Leu Val Gly Arg 725 730
735Ala Arg Val Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala Cys
740 745 750Leu Gly Lys Ser Tyr Ala Gln Met Trp Gln Leu Met Tyr Phe
His Arg 755 760 765Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser
Ala Val Pro Val 770 775 780Asp Trp Val Pro Thr Ser Arg Thr Thr Trp
Ser Ile His Ala His His785 790 795 800Gln Trp Met Thr Thr Glu Asp
Met Leu Ser Val Trp Asn Arg Val Trp 805 810 815Ile Glu Glu Asn Pro
Trp Met Glu Asp Lys Thr His Val Ser Ser Trp 820 825 830Glu Asp Val
Pro Tyr Leu Gly Lys Arg Glu Asp Arg Trp Cys Gly Ser 835 840 845Leu
Ile Gly Leu Thr Ala Arg Ala Thr Trp Ala Thr Asn Ile Gln Val 850 855
860Ala Ile Asn Gln Val Arg Arg Leu Ile Gly Asn Glu Asn Tyr Leu
Asp865 870 875 880Phe Met Thr Ser Met Lys Arg Phe Lys Asn Glu Ser
Asp Pro Glu Gly 885 890 895Ala Leu Trp 112701DNADengue virus type 2
11ggaactggca acataggaga gacgcttgga gagaaatgga aaagccgatt gaacgcattg
60gggaaaagtg aattccagat ctacaagaaa agtggaatcc aggaagtgga tagaacctta
120gcaaaagaag gcattaaaag aggagaaacg gaccatcacg ctgtgtcgcg
aggctcagca 180aaactgagat ggttcgtcga gagaaatatg gtcacaccag
aagggaaagt agtggacctc 240ggttgcggca gaggaggctg gtcatactat
tgtgggggac taaagaatgt aagagaagtc 300aaaggcctga caaaaggagg
accaggacat gaagaaccca tccccatgtc aacatatggg 360tggaatctag
tacgtcttca aagtggagtt gacgttttct tcactccgcc agaaaagtgt
420gacacattgt tgtgtgacat aggggagtcg tcaccaaatc ccacggtaga
agcaggacga 480acactcagag tccttaactt agtggaaaat tggttgaaca
acaacaccca attttgcata 540aaggttctca acccatacat gccctcagtc
atagaaaaaa tggaagcact acaaaggaaa 600tatggaggag ccttagtgag
gaatccactc tcacgaaact ccacacatga gatgtactgg 660gtatccaatg
cctccgggaa catagtgtca tcagtgaaca tgatttcaag gatgttgatc
720aacagattca caatgagaca caagaaagcc acttacgagc cagatgtaga
cctcggaagc 780ggaacccgca acatcggaat tgaaagtgag ataccaaacc
tagacataat cgggaaaaga 840atagaaaaaa taaaacaaga gcatgaaaca
tcatggcact atgaccaaga ccacccatac 900aaaacgtggg cttaccatgg
cagctatgaa acaaaacaaa ctggatcagc atcatccatg 960gtgaacggag
tggtcagact gctgacaaaa ccttgggacg tcgtccccat ggtgacacag
1020atggcaatga cagacacgac tccatttgga caacagcgcg tttttaaaga
aaaagtggac 1080acgagaaccc aagaaccgaa agaaggcaca aagaaactaa
tgaaaatcac ggcagagtgg 1140ctttggaaag aactagggaa gaaaaagaca
cctaggatgt gcactagaga agaattcaca 1200agaaaggtga gaagcaatgc
agccttgggg gccatattca ctgatgagaa caagtggaag 1260tcggcacgtg
aggctgttga agatagtagg ttttgggagc tggttgacaa ggaaaggaat
1320ctccatcttg aaggaaagtg tgaaacatgt gtgtataaca tgatgggaaa
aagagagaag 1380aagctagggg agttcggcaa ggcaaaaggc agcagagcca
tatggtacat gtggcttgga 1440gcacgcttct tagagtttga agccctagga
ttcttgaatg aagatcactg gttctccaga 1500gagaactcct tgagtggagt
ggaaggagaa gggctgcaca agctaggtta cattttaaga 1560gacgtgagca
agaaagaggg aggagcaatg tatgccgatg acaccgcagg atgggacaca
1620agaatcacac tagaagacct aaaaaatgaa gaaatggtaa caaaccacat
ggaaggagaa 1680cacaagaaac tagccgaggc cattttcaaa ttaacgtacc
aaaacaaggt ggtgcgtgtg 1740caaagaccaa caccaagagg cacagtaatg
gatatcatat cgagaagaga ccaaagaggt 1800agtggacaag ttggtaccta
tggactcaat actttcacca atatggaagc ccaactaatc 1860agacagatgg
agggagaagg agtcttcaaa agcattcagc acctgacagt cacagaagaa
1920atcgccgtgc aaaactggtt agcaagagta gggcgcgaaa ggttatcaag
aatggccatc 1980agtggagatg attgtgttgt gaaaccttta gatgacaggt
tcgcaagcgc tttaacagct 2040ctaaatgaca tgggaaaggt taggaaagac
atacaacaat gggaaccttc aagaggatgg 2100aacgattgga cacaagtgcc
cttctgttca caccatttcc atgagttaat catgaaagac 2160ggccgcgtac
ttgtagttcc atgcagaaac caagatgaac tgattggtag agcccgaatt
2220tcccaaggag ctgggtggtc tttgcgagag acggcctgtt tggggaagtc
ctacgcccaa 2280atgtggagct tgatgtactt ccacagacgt gacctcaggc
tggcggctaa tgctatttgc 2340tcggcagtcc catcacattg ggttccaaca
agtagaacaa cctggtccat acacgccaaa 2400catgaatgga tgacaacgga
agacatgctg acagtctgga acagggtgtg gattcaagaa 2460aacccatgga
tggaagacaa aactccagtg gaatcatggg aggaaatccc atacttgggg
2520aaaagagaag accaatggtg cggctcattg attgggctaa caagcagggc
cacctgggca 2580aagaacatcc aaacagcaat aaatcaagtt agatccctta
taggcaatga ggaatacaca 2640gattacatgc catccatgaa aagattcaga
agagaagagg aagaggcagg agtcctgtgg 2700t 270112900PRTDengue virus
type 2 12Gly Thr Gly Asn Ile Gly Glu Thr Leu Gly Glu Lys Trp Lys
Ser Arg1 5 10 15Leu Asn Ala Leu Gly Lys Ser Glu Phe Gln Ile Tyr Lys
Lys Ser Gly 20 25 30Ile Gln Glu Val Asp Arg Thr Leu Ala Lys Glu Gly
Ile Lys Arg Gly 35 40 45Glu Thr Asp His His Ala Val Ser Arg Gly Ser
Ala Lys Leu Arg Trp 50 55 60Phe Val Glu Arg Asn Met Val Thr Pro Glu
Gly Lys Val Val Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Ser
Tyr Tyr Cys Gly Gly Leu Lys Asn 85 90 95Val Arg Glu Val Lys Gly Leu
Thr Lys Gly Gly Pro Gly His Glu Glu 100 105 110Pro Ile Pro Met Ser
Thr Tyr Gly Trp Asn Leu Val Arg Leu Gln Ser 115 120 125Gly Val Asp
Val Phe Phe Thr Pro Pro Glu Lys Cys Asp Thr Leu Leu 130 135 140Cys
Asp Ile Gly Glu Ser Ser Pro Asn Pro Thr Val Glu Ala Gly Arg145 150
155 160Thr Leu Arg Val Leu Asn Leu Val Glu Asn Trp Leu Asn Asn Asn
Thr 165 170 175Gln Phe Cys Ile Lys Val Leu Asn Pro Tyr Met Pro Ser
Val Ile Glu 180 185 190Lys Met Glu Ala Leu Gln Arg Lys Tyr Gly Gly
Ala Leu Val Arg Asn 195 200 205Pro Leu Ser Arg Asn Ser Thr His Glu
Met Tyr Trp Val Ser Asn Ala 210 215 220Ser Gly Asn Ile Val Ser Ser
Val Asn Met Ile Ser Arg Met Leu Ile225 230 235 240Asn Arg Phe Thr
Met Arg His Lys Lys Ala Thr Tyr Glu Pro Asp Val 245 250 255Asp Leu
Gly Ser Gly Thr Arg Asn Ile Gly Ile Glu Ser Glu Ile Pro 260 265
270Asn Leu Asp Ile Ile Gly Lys Arg Ile Glu Lys Ile Lys Gln Glu His
275 280 285Glu Thr Ser Trp His Tyr Asp Gln Asp His Pro Tyr Lys Thr
Trp Ala 290 295 300Tyr His Gly Ser Tyr Glu Thr Lys Gln Thr Gly Ser
Ala Ser Ser Met305 310 315 320Val Asn Gly Val Val Arg Leu Leu Thr
Lys Pro Trp Asp Val Val Pro 325 330 335Met Val Thr Gln Met Ala Met
Thr Asp Thr Thr Pro Phe Gly Gln Gln 340 345 350Arg Val Phe Lys Glu
Lys Val Asp Thr Arg Thr Gln Glu Pro Lys Glu 355 360 365Gly Thr Lys
Lys Leu Met Lys Ile Thr Ala Glu Trp Leu Trp Lys Glu 370 375 380Leu
Gly Lys Lys Lys Thr Pro Arg Met Cys Thr Arg Glu Glu Phe Thr385 390
395 400Arg Lys Val Arg Ser Asn Ala Ala Leu Gly Ala Ile Phe Thr Asp
Glu 405 410 415Asn Lys Trp Lys Ser Ala Arg Glu Ala Val Glu Asp Ser
Arg Phe Trp 420 425 430Glu Leu Val Asp Lys Glu Arg Asn Leu His Leu
Glu Gly Lys Cys Glu 435 440 445Thr Cys Val Tyr Asn Met Met Gly Lys
Arg Glu Lys Lys Leu Gly Glu 450 455 460Phe Gly Lys Ala Lys Gly Ser
Arg Ala Ile Trp Tyr Met Trp Leu Gly465 470 475 480Ala Arg Phe Leu
Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp His 485 490 495Trp Phe
Ser Arg Glu Asn Ser Leu Ser Gly Val Glu Gly Glu Gly Leu 500 505
510His Lys Leu Gly Tyr Ile Leu Arg Asp Val Ser Lys Lys Glu Gly Gly
515 520 525Ala Met Tyr Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile
Thr Leu 530 535 540Glu Asp Leu Lys Asn Glu Glu Met Val Thr Asn His
Met Glu Gly Glu545 550 555 560His Lys Lys Leu Ala Glu Ala Ile Phe
Lys Leu Thr Tyr Gln Asn Lys 565 570 575Val Val Arg Val Gln Arg Pro
Thr Pro Arg Gly Thr Val Met Asp Ile 580 585 590Ile Ser Arg Arg Asp
Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr Gly 595 600 605Leu Asn Thr
Phe Thr Asn Met Glu Ala Gln Leu Ile Arg Gln Met Glu 610 615 620Gly
Glu Gly Val Phe Lys Ser Ile Gln His Leu Thr Val Thr Glu Glu625 630
635 640Ile Ala Val Gln Asn Trp Leu Ala Arg Val Gly Arg Glu Arg Leu
Ser 645 650 655Arg Met Ala Ile Ser Gly Asp Asp Cys Val Val Lys Pro
Leu Asp Asp 660 665 670Arg Phe Ala Ser Ala Leu Thr Ala Leu Asn Asp
Met Gly Lys Val Arg 675 680 685Lys Asp Ile Gln Gln Trp Glu Pro Ser
Arg Gly Trp Asn Asp Trp Thr 690 695 700Gln Val Pro Phe Cys Ser His
His Phe His Glu Leu Ile Met Lys Asp705 710 715 720Gly Arg Val Leu
Val Val Pro Cys Arg Asn Gln Asp Glu Leu Ile Gly 725 730 735Arg Ala
Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu Arg Glu Thr Ala 740 745
750Cys Leu Gly Lys Ser Tyr Ala Gln Met Trp Ser Leu Met Tyr Phe His
755 760 765Arg Arg Asp Leu Arg Leu Ala Ala Asn Ala Ile Cys Ser Ala
Val Pro 770 775 780Ser His Trp Val Pro Thr Ser Arg Thr Thr Trp Ser
Ile His Ala Lys785 790 795 800His Glu Trp Met Thr Thr Glu Asp Met
Leu Thr Val Trp Asn Arg Val 805 810 815Trp Ile Gln Glu Asn Pro Trp
Met Glu Asp Lys Thr Pro Val Glu Ser 820 825 830Trp Glu Glu Ile Pro
Tyr Leu Gly Lys Arg Glu Asp Gln Trp Cys Gly 835 840 845Ser Leu Ile
Gly Leu Thr Ser Arg Ala Thr Trp Ala Lys Asn Ile Gln 850 855 860Thr
Ala Ile Asn Gln Val Arg Ser Leu Ile Gly Asn Glu Glu Tyr Thr865 870
875 880Asp Tyr Met Pro Ser Met Lys Arg Phe Arg Arg Glu Glu Glu Glu
Ala 885 890 895Gly Val Leu Trp 9001314PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 13Cys
Arg Val Lys Met Glu Lys Leu Gln Leu Lys Gly Thr Thr1 5
101414PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 14Cys Gln Leu Leu Met Arg Glu Val Lys Thr Gly Thr
Lys Lys1 5 101519PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 15Cys Ser Thr Lys Ala Ile Gly Arg Thr
Ile Leu Lys Glu Asn Ile Lys1 5 10 15Tyr Glu Val1623DNAArtificial
SequenceDescription of Artificial Sequence Synthetic primer
16gactgaagag ggcaatgttg agc 231721DNAArtificial SequenceDescription
of Artificial Sequence Synthetic primer 17gcaataactg cggacytctg c
211827DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 18gaattcttca actgccttgg aatgagc
271927DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 19ctgcagttat ttgccaatgc tgcttcc 27206PRTArtificial
SequenceDescription of Artificial Sequence Synthetic 6xHis tag
20His His His His His His1 521899PRTDengue virus type 3 21Gly Thr
Gly Ser Gln Gly Glu Thr Leu Gly Glu Lys Trp Lys Lys Lys1 5 10 15Leu
Asn Gln Leu Ser Arg Lys Glu Phe Asp Leu Tyr Lys Lys Ser Gly 20 25
30Ile Thr Glu Val Asp Arg Thr Glu Ala Lys Glu Gly Leu Lys Arg Gly
35 40 45Glu Ile Thr His His Ala Val Ser Arg Gly Ser Ala Lys Leu Gln
Trp 50 55 60Phe Val Glu Arg Asn Met Val Ile Pro Glu Gly Arg Val Ile
Asp Leu65 70 75 80Gly Cys Gly Arg Gly Gly Trp Ser Tyr Tyr Cys Ala
Gly Leu Lys Lys 85 90 95Val Thr Glu Val Arg Gly Tyr Thr Lys Gly Gly
Pro Gly His Glu Glu 100 105 110Pro Val Pro Met Ser Thr Tyr Gly Trp
Asn Ile Val Lys Leu Met Ser 115 120 125Gly Lys Asp Val Phe Tyr Leu
Pro Pro Glu Lys Cys Asp Thr Leu Leu 130 135 140Cys Asp Ile Gly Glu
Ser Ser Pro Ser Pro Thr Val Glu Glu Ser Arg145 150 155 160Thr Ile
Arg Val Leu Lys Met Val Glu Pro Trp Leu Lys Asn Asn Gln 165 170
175Phe Cys Ile Lys Val Leu Asn Pro Tyr Met Pro Thr Val Ile Glu His
180 185 190Leu Glu Arg Leu Gln Arg Lys His Gly Gly Met Leu Val Arg
Asn Pro 195 200 205Leu Ser Arg Asn Ser Thr His Glu Met Tyr Trp Ile
Ser Asn Gly Thr 210 215 220Gly Asn Ile Val Ala Ser Val Asn Met Val
Ser Arg Leu Leu Leu Asn225 230 235 240Arg Phe Thr Met Thr His Arg
Arg Pro Thr Ile Glu Lys Asp Val Asp 245 250 255Leu Gly Ala Gly Thr
Arg His Val Asn Ala Glu Pro Glu Thr Pro Asn 260 265 270Met Asp Val
Ile Gly Glu Arg Ile Lys Arg Ile Lys Glu Glu His Asn 275 280 285Ser
Thr Trp His Tyr Asp Asp Glu Asn Pro Tyr Lys Thr Trp Ala Tyr 290 295
300His Gly Ser Tyr Glu Val Lys Ala Thr Gly Ser Ala Ser Ser Met
Ile305 310 315 320Asn Gly Val Val Lys Leu Leu Thr Lys Pro Trp Asp
Val Val Pro Met 325 330 335Val Thr Gln Met Ala Met Thr Asp Thr Thr
Pro Phe Gly Gln Gln Arg 340 345 350Val Phe Lys Glu Lys Val Asp Thr
Arg Thr Pro Arg Ser Met Pro Gly 355 360 365Thr Arg Arg Val Met Gly
Ile Thr Ala Glu Trp Leu Trp Arg Thr Leu 370 375 380Gly Arg Asn Lys
Lys Pro Arg Leu Cys Thr Arg Glu Glu Phe Thr Lys385 390 395 400Lys
Val Arg Thr Asn Ala Ala Met Gly Ala Val Phe Thr Glu Glu Asn 405 410
415Gln Trp Asp Ser Ala Lys Ala Ala Val Glu Asp Glu Asp Phe Trp Lys
420 425 430Leu Val Asp Arg Glu Arg Glu Leu His Lys Leu Gly Lys Cys
Gly Ser 435 440 445Cys Val Tyr Asn Met Met Lys Arg Glu Lys Lys Leu
Gly Glu Phe Gly 450 455 460Lys Ala Lys Gly Ser Arg Ala Ile Trp Tyr
Met Trp Leu Gly Ala Arg465 470 475 480Tyr Leu Glu Phe Glu Ala Leu
Gly Phe Leu Asn Glu Asp His Trp Phe 485 490 495Ser Arg Glu Asn Ser
Tyr Ser Gly Val Glu Gly Glu Gly Leu His Lys 500 505 510Leu Gly Tyr
Ile Leu Arg Asp Ile Ser Lys Ile Pro Gly Gly Ala Met 515 520 525Tyr
Ala Asp Asp Thr Ala Gly Trp Asp Thr Arg Ile Thr Glu Asp Asp 530 535
540Leu His Asn Glu Glu Lys Ile Thr Gln Gln Met Asp Pro Glu His
Arg545 550 555 560Gln Leu Ala Asn Ala Ile Phe Lys Leu Thr Tyr Gln
Asn Lys Val Val 565 570 575Lys Val Gln Arg Pro Thr Pro Thr Gly Thr
Val Met Asp Ile Ile Ser 580 585 590Arg Lys Asp Gln Arg Gly Ser Gly
Gln Val Gly Thr Tyr Gly Leu Asn 595 600 605Thr Phe Thr Asn Met Glu
Ala Gln Leu Ile Arg Gln Met Glu Gly Glu 610 615 620Gly Val Leu Ser
Lys Ala Asp Leu Glu Asn Pro His Leu Pro Glu Lys625 630 635 640Lys
Ile Thr Gln Trp Leu Glu Thr Lys Gly Val Glu Arg Leu Lys Arg 645 650
655Met Ala Ile Ser Gly Asp Asp Cys Val Val Lys Pro Ile Asp Asp Arg
660 665
670Phe Ala Asn Ala Leu Leu Ala Leu Asn Asp Met Gly Lys Val Arg Lys
675 680 685Asp Ile Pro Gln Trp Gln Pro Ser Lys Gly Trp His Asp Trp
Gln Gln 690 695 700Val Pro Phe Cys Ser His His Phe His Glu Leu Ile
Met Lys Asp Gly705 710 715 720Arg Lys Leu Val Val Pro Cys Arg Pro
Gln Asp Glu Leu Ile Gly Arg 725 730 735Ala Arg Ile Ser Gln Gly Ala
Gly Trp Ser Leu Arg Glu Thr Ala Cys 740 745 750Leu Gly Lys Ala Tyr
Ala Gln Met Trp Ser Leu Met Tyr Phe His Arg 755 760 765Arg Asp Leu
Arg Leu Ala Ser Asn Ala Ile Cys Ser Ala Val Pro Val 770 775 780His
Trp Val Pro Thr Ser Arg Thr Thr Trp Ser Ile His Ala His His785 790
795 800Gln Trp Met Thr Thr Glu Asp Met Leu Thr Val Trp Asn Arg Val
Trp 805 810 815Ile Glu Asp Asn Pro Trp Met Glu Asp Lys Thr Pro Val
Thr Thr Trp 820 825 830Glu Asn Val Pro Tyr Leu Gly Lys Arg Glu Asp
Gln Trp Cys Gly Ser 835 840 845Leu Ile Gly Leu Thr Ser Arg Ala Thr
Trp Ala Gln Asn Ile Pro Thr 850 855 860Ala Ile Gln Gln Val Arg Ser
Leu Ile Gly Asn Glu Glu Phe Leu Asp865 870 875 880Tyr Met Pro Ser
Met Lys Arg Phe Arg Lys Glu Glu Glu Ser Glu Gly 885 890 895Ala Ile
Trp 22900PRTDengue virus type 4 22Gly Thr Gly Thr Thr Gly Glu Thr
Leu Gly Glu Lys Trp Lys Arg Gln1 5 10 15Leu Asn Ser Leu Asp Arg Lys
Glu Phe Glu Glu Tyr Lys Arg Ser Gly 20 25 30Ile Leu Glu Val Asp Arg
Thr Glu Ala Lys Ser Ala Leu Lys Asp Gly 35 40 45Ser Lys Ile Lys His
Ala Val Ser Arg Gly Ser Ser Lys Ile Arg Trp 50 55 60Ile Val Glu Arg
Gly Met Val Lys Pro Lys Gly Lys Val Val Asp Leu65 70 75 80Gly Cys
Gly Arg Gly Gly Trp Ser Tyr Tyr Met Ala Thr Leu Lys Asn 85 90 95Val
Thr Glu Val Lys Gly Tyr Thr Lys Gly Gly Pro Gly His Glu Glu 100 105
110Pro Ile Pro Met Ala Thr Tyr Gly Trp Asn Leu Val Lys Leu His Ser
115 120 125Gly Val Asp Val Phe Tyr Lys Pro Thr Glu Gln Val Asp Thr
Leu Leu 130 135 140Cys Asp Ile Gly Glu Ser Ser Ser Asn Pro Thr Ile
Glu Glu Gly Arg145 150 155 160Thr Leu Arg Val Leu Lys Met Val Glu
Pro Trp Leu Ser Ser Lys Pro 165 170 175Glu Phe Cys Ile Lys Val Leu
Asn Pro Tyr Met Pro Thr Val Ile Glu 180 185 190Glu Leu Glu Lys Leu
Gln Arg Lys His Gly Gly Asn Leu Val Arg Cys 195 200 205Pro Leu Ser
Arg Asn Ser Thr His Glu Met Tyr Trp Val Ser Gly Ala 210 215 220Ser
Gly Asn Ile Val Ser Ser Val Asn Thr Thr Ser Lys Met Leu Leu225 230
235 240Asn Arg Phe Thr Thr Arg His Arg Lys Pro Thr Tyr Glu Lys Asp
Val 245 250 255Asp Leu Gly Ala Gly Thr Arg Ser Val Ser Thr Glu Thr
Glu Lys Pro 260 265 270Asp Met Thr Ile Ile Gly Arg Arg Leu Gln Arg
Leu Gln Glu Glu His 275 280 285Lys Glu Thr Trp His Tyr Asp Gln Glu
Asn Pro Tyr Arg Thr Trp Ala 290 295 300Tyr His Gly Ser Tyr Glu Ala
Pro Ser Thr Gly Ser Ala Ser Ser Met305 310 315 320Val Asn Gly Val
Val Lys Leu Leu Thr Lys Pro Trp Asp Val Ile Pro 325 330 335Met Val
Thr Gln Leu Ala Met Thr Asp Thr Thr Pro Phe Gly Gln Gln 340 345
350Arg Val Phe Lys Glu Lys Val Asp Thr Arg Thr Pro Gln Pro Lys Pro
355 360 365Gly Thr Arg Met Val Met Thr Thr Thr Ala Asn Trp Leu Trp
Ala Leu 370 375 380Leu Gly Lys Lys Lys Asn Pro Arg Leu Cys Thr Arg
Glu Glu Phe Ile385 390 395 400Ser Lys Val Arg Ser Asn Ala Ala Ile
Gly Ala Val Phe Gln Glu Glu 405 410 415Gln Gly Trp Thr Ser Ala Ser
Glu Ala Val Asn Asp Ser Arg Phe Trp 420 425 430Glu Leu Val Asp Lys
Glu Arg Ala Leu His Gln Glu Gly Lys Cys Glu 435 440 445Ser Cys Val
Tyr Asn Met Met Gly Lys Arg Glu Lys Lys Leu Gly Glu 450 455 460Phe
Gly Arg Ala Lys Gly Ser Arg Ala Ile Trp Tyr Met Trp Leu Gly465 470
475 480Ala Arg Phe Leu Glu Phe Glu Ala Leu Gly Phe Leu Asn Glu Asp
His 485 490 495Trp Phe Gly Arg Glu Asn Ser Trp Ser Gly Val Glu Gly
Glu Gly Leu 500 505 510His Arg Leu Gly Tyr Ile Leu Glu Glu Ile Asp
Lys Lys Asp Gly Asp 515 520 525Leu Met Tyr Ala Asp Asp Thr Ala Gly
Trp Asp Thr Arg Ile Thr Glu 530 535 540Asp Asp Leu Gln Asn Glu Glu
Leu Ile Thr Glu Gln Met Ala Pro His545 550 555 560His Lys Ile Leu
Ala Lys Ala Ile Phe Lys Leu Thr Tyr Gln Asn Lys 565 570 575Val Val
Lys Val Leu Arg Pro Thr Pro Arg Gly Ala Val Met Asp Ile 580 585
590Ile Ser Arg Lys Asp Gln Arg Gly Ser Gly Gln Val Gly Thr Tyr Gly
595 600 605Leu Asn Thr Phe Thr Asn Met Glu Val Gln Leu Ile Arg Gln
Met Glu 610 615 620Ala Glu Gly Val Ile Thr Gln Asp Asp Met Gln Asn
Pro Lys Gly Leu625 630 635 640Lys Glu Arg Val Glu Lys Trp Leu Lys
Glu Cys Gly Val Asp Arg Leu 645 650 655Lys Arg Met Ala Ile Ser Gly
Asp Asp Cys Val Val Lys Pro Leu Asp 660 665 670Glu Arg Phe Gly Thr
Ser Leu Leu Phe Leu Asn Asp Met Gly Lys Val 675 680 685Arg Lys Asp
Ile Pro Gln Trp Glu Pro Ser Lys Gly Trp Lys Asn Trp 690 695 700Gln
Glu Val Pro Phe Cys Ser His His Phe His Lys Ile Phe Met Lys705 710
715 720Asp Gly Arg Ser Leu Val Val Pro Cys Arg Asn Gln Asp Glu Leu
Ile 725 730 735Gly Arg Ala Arg Ile Ser Gln Gly Ala Gly Trp Ser Leu
Arg Glu Thr 740 745 750Ala Cys Leu Gly Lys Ala Tyr Ala Gln Met Trp
Ser Leu Met Tyr Phe 755 760 765His Arg Arg Asp Leu Arg Leu Ala Ser
Met Ala Ile Cys Ser Ala Val 770 775 780Pro Thr Glu Trp Phe Pro Thr
Ser Arg Thr Thr Trp Ser Ile His Ala785 790 795 800His His Gln Trp
Met Thr Thr Glu Asp Met Leu Lys Val Trp Asn Arg 805 810 815Val Trp
Ile Glu Asp Asn Pro Asn Met Thr Asp Lys Thr Pro Val His 820 825
830Ser Trp Glu Asp Ile Pro Tyr Leu Gly Lys Arg Glu Asp Leu Trp Cys
835 840 845Gly Ser Leu Ile Gly Leu Ser Ser Arg Ala Thr Trp Ala Lys
Asn Ile 850 855 860His Thr Ala Ile Thr Gln Val Arg Asn Leu Ile Gly
Lys Glu Glu Tyr865 870 875 880Val Asp Tyr Met Pro Val Met Lys Arg
Tyr Ser Ala Pro Ser Glu Ser 885 890 895Glu Gly Val Leu 900
* * * * *
References