U.S. patent application number 11/622124 was filed with the patent office on 2007-08-23 for surrogate markers for viral infections and other inflammatory responses.
Invention is credited to Kathleen J. Austin, Thomas R. Hansen, Lea A. Rempel, Alberto van Olphen.
Application Number | 20070196823 11/622124 |
Document ID | / |
Family ID | 38257117 |
Filed Date | 2007-08-23 |
United States Patent
Application |
20070196823 |
Kind Code |
A1 |
Hansen; Thomas R. ; et
al. |
August 23, 2007 |
SURROGATE MARKERS FOR VIRAL INFECTIONS AND OTHER INFLAMMATORY
RESPONSES
Abstract
Compositions and methods for the detection, diagnosis and
treatment of BVDV and other viruses are provided.
Inventors: |
Hansen; Thomas R.; (Fort
Collins, CO) ; Austin; Kathleen J.; (Laramie, WY)
; van Olphen; Alberto; (Tampa, FL) ; Rempel; Lea
A.; (Edgerton, KS) |
Correspondence
Address: |
DANN, DORFMAN, HERRELL & SKILLMAN
1601 MARKET STREET
SUITE 2400
PHILADELPHIA
PA
19103-2307
US
|
Family ID: |
38257117 |
Appl. No.: |
11/622124 |
Filed: |
January 11, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60757965 |
Jan 11, 2006 |
|
|
|
Current U.S.
Class: |
435/5 ;
435/287.2 |
Current CPC
Class: |
C12Q 1/6883 20130101;
C12Q 2600/158 20130101 |
Class at
Publication: |
435/005 ;
435/006; 435/287.2 |
International
Class: |
C12Q 1/70 20060101
C12Q001/70; C12Q 1/68 20060101 C12Q001/68; C12M 3/00 20060101
C12M003/00 |
Goverment Interests
[0002] Pursuant to 35 U.S.C. Section 202(c), it is acknowledged
that the United States Government has certain rights in the
invention described herein, which was made in part with funds from
the USDA/CSREES, grant numbers 2004-35204-14916 and
2004-35204-17005.
Claims
1. A method for diagnosing BVDV in a ruminant test animal
comprising: a) obtaining a biological sample from a test animal and
from a non-BVDV infected animal; b) contacting said sample with an
agent having affinity for at least one differentially expressed
BVDV surrogate marker shown in Tables 1-9; and d) diagnosing the
presence of BVDV via detection of at least one differentially
expressed BVDV surrogate marker, an alteration in the expression
level of said BVDV surrogate marker obtained from said test animal,
relative to that obtained from said non-BVDV infected animal being
indicative of BVDV in said test animal.
2. The method of claim 1, wherein said at least one BVDV surrogate
marker is a nucleic acid and said expression level is up-regulated
in response to BVDV infection.
3. The method of claim 1, wherein said at least one BVDV surrogate
marker is a nucleic acid and said expression level is
down-regulated in response to BVDV infection.
4. The method of claim 1, wherein said at least one BVDV surrogate
marker is a nucleic acid immobilized on a gene chip.
5. The method of claim 1, wherein said biological sample is
selected from the group consisting of blood, urine, skin, serum,
milk, sputumr, saliva, and tears.
6. The method of claim 1 wherein said biological sample comprises
blood cells or skin cells which are lysed to release nucleic acids
therein.
7. The method of claim 6, wherein said released nucleic acids are
amplified.
8. The method of claim 1, wherein said ruminant animal is a
persistently infected steer or heifer and at least one BVDV
surrogate marker is a nucleic acid which is upregulated and is
selected from the group consisting of CK945739,
CK777968,NM.sub.--173941 and CK960499. which is upregulated and is
selected from the group consisting of CK945739, CK777968,
NM.sub.--173941 and CK960499.
9. The method of claim 1, wherein said ruminant animal is a
persistently infected steer and at least one BVDV surrogate marker
is a nucleic acid which is downregulated and is selected from the
group consisting of CK848330, BP 102272, AU278490 and BE723387.
10. The method of claim 1, wherein said ruminant animal is a heifer
carrying a persistently infected fetus and at least one BVDV
surrogate marker is a nucleic acid which is upregulated and is
selected from the group consisting of CK977019, CA923353, CB461169,
and CB445920.
11. The method of claim 1, wherein said ruminant animal is a heifer
carrying a persistently infected fetus and at least one BVDV
surrogate marker is a nucleic acid which is down regulated and is
selected from the group consisting of NM.sub.--175827, CB425639,
NM.sub.--174511 and CB534503.
12. The method of claim 1, wherein said ruminant animal is a heifer
carrying a transiently infected fetus and at least one BVDV
surrogate marker is a nucleic acid which is upregulated and is
selected from the group consisting of CK960499, CB464371,
NM.sub.--174366, and CB433489.
13. The method of claim 1, wherein said ruminant animal is a heifer
carrying a transiently infected fetus and at least one BVDV
surrogate marker is a nucleic acid which is down regulated and is
selected from the group consisting of CB444277, AB008573, CB433789
and AV609250.
14. A gene chip for performing the method of claim 1 comprising a
plurality of BVDV surrogate markers selected from the group
consisting of CK945739, CK777968, NM.sub.--173941, CK960499,
CK848330, BP102272, AU278490, BE1723387, CK977019, CA923353,
CB461169, CB445920, NM.sub.--175827, CB425639, NM.sub.--174511,
CB534503. CK960499, CB464371, NM.sub.--174366, CB433489, CB444277,
AB008573, CB433789 and AV609250.
15. The method of claim 1, wherein said at least one BVDV surrogate
marker is a polypeptide and said expression level is up-regulated
in response to BVDV infection.
16. The method of claim 1, wherein said at least one BVDV surrogate
marker is a polypeptide and said expression level is down-regulated
in response to BVDV infection.
17. The method of claim 1, wherein said at least one BVDV surrogate
marker is a polypeptide or fragment thereof immobilized on a solid
support.
18. The method of claim 1, wherein said ruminant animal is a
persistently infected steer and at least one BVDV surrogate marker
is a polypeptide or fragments thereof which is upregulated and is
selected from the group consisting of polypeptides thereof encoded
by CK945739, CK777968, NM.sub.--173941 and CK960499.
19. The method of claim 1, wherein said ruminant animal is a
persistently infected steer and at least one BVDV surrogate marker
is a polypeptide or fragments thereof which is downregulated and is
selected from the group consisting of polypeptides encoded by
CK848330, BP102272, AU278490 and BE723387.
20. The method of claim 1, wherein said ruminant animal is a heifer
carrying a persistently infected fetus and at least one BVDV
surrogate marker is a polypeptide or fragment thereof which is
upregulated and is selected from the group consisting of
polypeptides encoded by CK977019, CA923353, CB461169, and
CB445920.
21. The method of claim 1, wherein said ruminant animal is a heifer
carrying a persistently infected fetus and at least one BVDV
surrogate marker is a polypeptide or fragment thereof which is down
regulated and is selected from the group consisting of polypeptides
encoded by NM.sub.--175827, CB425639, NM.sub.--174511 and
CB534503.
22. The method of claim 1, wherein said ruminant animal is a heifer
carrying a transiently infected fetus and at least one BVDV
surrogate marker is a polypeptide or fragment thereof which is
upregulated and is selected from the group consisting of
polypeptides encoded by CK960499, CB464371, NM.sub.--174366, and
CB433489.
23. The method of claim 1, wherein said ruminant animal is a heifer
carrying a transiently infected fetus and at least one BVDV
surrogate marker is a polypeptide or fragment thereof which is down
regulated and is selected from the group consisting of polypeptides
encoded by CB444277, AB008573, CB433789 and AV609250.
24. A solid support for performing the method of claim 1 comprising
a plurality of BVDV surrogate polypeptide markers selected from the
group consisting of polypeptides encoded by CK945739, CK777968,
NM.sub.--173941, CK960499, CK848330, BP102272, AU278490, BE723387,
CK977019, CA923353, CB461169, CB445920, NM.sub.--175827, CB425639,
NM.sub.--174511, CB534503. CK960499, CB464371, NM.sub.--174366,
CB433489, CB444277, AB008573, CB433789 and AV609250.
25. The method of claim 1, wherein said ruminant test animal is
selected from the group consisting of a bovine a steer, a pregnant
bovine, a bovine fetus and a bovine calf.
26. The method of claim 1, wherein said agent having affinity for
said BVDV surrogate marker is selected from the group consisting of
at least one nucleic acid which specifically hybridizes with at
least one nucleic acid shown in Tables 1-9, and an antibody
immunologically specific for at least one polypeptide encoded by a
nucleic acid shown in Tables 1-9.
27. The method of claim 26, wherein said agent is detectably
labeled.
28. A kit for differentially diagnosing BVDV infection via the
method of claim 1, comprising at least one BVDV surrogate marker
detector molecule, and reagents for detection of the same said
optionally comprising a gene chip comprising a plurality of BVDV
surrogate markers selected from the group consisting of CK945739,
CK777968, NM_173941, CK960499, CK848330, BP102272, AU278490,
BE723387, CK977019, CA923353, CB461169, CR445920, NM.sub.--175827,
CB425639, NM.sub.--174511, CB534503. CK960499, CB464371,
NM.sub.--174366, CB433489, CB444277, AB008573, CB433789 and
AV609250 or a solid support comprising a plurality of BVDV
surrogate polypeptide markers selected from the group consisting of
polypeptides encoded by CK945739, CK777968, NM.sub.--173941,
CK960499, CK848330, BP102272, AU278490, BE723387, CK977019,
CA923353, CB461169, CB445920, NM.sub.--175827, CB425639,
NM.sub.--174511, CB534503. CK960499, CB464371, NM.sub.--174366,
CB433489, CB444277, AB008573, CB433789 and AV609250.
29. The kit of claim 28, wherein said BVDV surrogate marker
detector molecule is selected from the group consisting of a probe
or primer which specifically hybridizes with a BVDV surrogate
marker nucleic acid, and an antibody which specifically binds to a
BVDV surrogate marker polypeptide.
30. A method as claimed in claim 1 further comprising detection of
markers associated with transient or acute BVDV infection.
31. A method for identifying agents useful for the treatment of
viral infections, comprising: a) providing a host cell expressing
at least BVDV surrogate marker in Tables 1-9 which is
differentially expressed in response to viral infection and
exposing said cell to an RNA virus under conditions suitable for
infection to occur; b) incubating said host cells in the presence
and absence of said agent; and c) identifying those agents which
modulate the expression of at least one BVDV surrogate marker in
treated cells when compared to untreated cells.
32. A method for detecting viral surrogate marker molecules in a
test animal comprising: a) obtaining a plurality of biological
samples from said test animal and from a non-virally infected
animal; b) contacting said biological sample with a composition
comprising one or more viral surrogate marker molecule detection
reagents in an amount effective to permit detection and
quantitation of a viral surrogate molecule, if present, in said
sample; and c) determining from b) the amount of said viral
surrogate marker molecule, wherein an alteration of levels of said
viral surrogate marker molecule, relative to those obtained from
non-virally infected animals, is indicative of viral infection in
said test animal.
33. The method of claim 32, wherein a lack of alteration of levels
of said viral surrogate marker molecule indicates that the test
animal is not virally infected.
34. The method of claim 32, wherein said viral surrogate marker
molecule is increased in a test subject infected with a virus
selected from the group consisting of BVDV, Influenza, HIV, Ebola
virus, FcLv, FIP virus, Bluetongue virus, West Nile Virus,
hepatitis C Virus and Epizootic Hemorrhagic Disease Virus.
Description
[0001] This application claims priority to U.S. provisional
Application No. 60/757,965 filed Jan. 11, 2006, the entire contents
of which are incorporated by reference herein.
FIELD OF THE INVENTION
[0003] This invention relates to the field of molecular biology and
virology. More specifically, the present invention provides
materials and methods for the diagnosis and staging of bovine viral
diarrhea virus (BVDV).
BACKGROUND OF THE INVENTION
[0004] Several publications and patent documents are cited
throughout this application in order to more fully describe the
state of the art to which this invention pertains. The disclosure
of each of these citations is incorporated by reference herein.
[0005] Bovine viral diarrhea virus (BVDV) costs the United States
cattle industry more than 400 million dollars per year. The
pathogenesis of BVDV infection has features that are unique to this
virus and vary with the time of infection, virulence of the viral
strain, and age of the animals at the time of infection.
[0006] When the infection occurs after 150 days of gestation
(post-development of the immune system) or after birth, including
adult animals, the infection is referred to as acute infection. The
clinical manifestation of acute infections with BVDV range from
sub-clinical or unapparent infections to embryonic death,
abortions, stillborn, malformed or slow growing calves.
[0007] Certain strains of BVDV can cause a hemorrhagic syndrome
with high morbidity and moderate mortality in adult animals.
Acutely infected animals usually recover and eliminate the virus
within 10 to 14 days post infection.
[0008] Animals vaccinated with modified live vaccines against BVDV
have an immune response similar to the one induced by natural,
acute infection. In contrast, infection of the fetus during the
first 150 days of gestation, when the immune system has not yet
developed, can lead to the generation of persistently infected (PI)
calves. Some of these PI calves die soon after birth, but others
live for relatively long periods of time without showing any
clinical signs. PI animals cannot eliminate the infecting BVDV from
their system, and continuously release high amounts of virus in
their bodily secretions and excretions, making them a continuous
source of infection within the herd and potentially to other herds
as well. Furthermore, nursing PI calves can acutely infect their
mothers and other normal nursing calves, which in turn infect their
own mothers while they are pregnant, producing a new cycle of
infection and eventually more PI calves.
[0009] Mucosal disease, an uncommon but fatal complication observed
in PI calves, occurs when the virus mutates or the animal is
superinfected with an antigenically related BVDV virus. Current
vaccines are relatively inefficient in preventing fetal infections,
therefore the identification and elimination of PI animals is
essential to any successful program for control or eradication of
BVDV.
[0010] Currently available tests for the detection of PI animals
are based on the identification of the viral antigen in a blood or
tissue sample (most commonly a skin biopsy) using detection methods
that depend on the specific binding of anti-BVDV antibodies.
Although these tests are widely used for the detection of PI
animals they frequently fail to identify all infected animals
(false negatives) resulting in the failure to remove all PI animals
from the infected herd. Moreover, serological tests cannot
differentiate between PIs and uninfected animals, or between
acutely infected and vaccinated animals.
[0011] Identification and elimination of PI animals from an
affected herd is the most cost effective measure to control and
eradicate BVDV, underscoring the criticality of an inexpensive and
convenient diagnostic test. It is an object of the invention to
provide such a test and kit for performing the same.
SUMMARY OF THE INVENTION
[0012] In accordance with the present invention, methods and
compositions for diagnosis of Bovine Viral Diarrhea Virus (BVDV)
are disclosed. Specifically, a simple, convenient test for
accurately diagnosing BVDV is provided. The instant method provides
the means to differentiate cattle persistently infected with BVDV
(PI) from control non-infected steers. Other markers are provided
which enable the skilled person to identify 1) heifers carrying
persistently infected fetuses; 2) heifers carrying transiently
virally infected fetuses and 3) heifers carrying control,
uninfected fetuses. Such differentiation may be accomplished by
detecting altered expression levels of one or more markers shown in
Tables 1-9, or the proteins or peptide fragments encoded thereby.
Most preferably, the test can be easily conducted in the field by
veterinarians or cattle producers.
[0013] In one aspect of the invention, BVDV surrogate markers are
provided. A BVDV surrogate marker may be a nucleic acid or
polypeptide or fragments thereof. Such markers are provided herein
at Tables 1-9. Also provided in accordance with the invention are
oligonucleotides, including probes and primers, that specifically
hybridize with the nucleic acid sequences set forth in Tables 1-9.
Antibodies immunologically specific for the BVDV marker
polypeptides described herein are also within the scope of the
invention.
[0014] In a further aspect of the invention, recombinant DNA
molecules comprising the nucleic acid molecules set forth above,
operably linked to a vector are provided. The invention also
encompasses host cells comprising a vector encoding a BVDV specific
marker of the invention.
[0015] In another aspect of the invention, methods for detecting a
differentially expressed BVDV specific marker molecules in a
biological sample are provided. Such molecules can be BVDV specific
marker nucleic acids, such as mRNA, DNA, cDNA, or BVDV specific
marker polypeptides or fragments thereof. Preferably the BVDV
surrogate marker exhibits expression levels which differ at least 2
fold from normal, uninfected cattle. The BVDV markers of the
invention may be up or down regulated relative to the levels
observed in non-infected control cattle. Exemplary methods comprise
detection of isolated biological molecules which hybridize to BVDV
specific markers which are affixed to a solid support, or mRNA
analysis, for example by RT-PCR. Immunological methods include for
example contacting a sample with a detectably labeled antibody
immunologically specific for a BVDV specific marker polypeptide and
determining the presence of the polypeptide as a function of the
amount of detectably labeled antibody bound by the sample relative
to control cells. In a preferred embodiment, these assays may be
used to detect differentially expressed proteins encoded by the
nucleic acids set forth in Tables 1-9.
[0016] In a further aspect of the invention, kits for detection of
BVDV infection or lack thereof are provided. An exemplary kit
comprises a BVDV specific marker protein, polynucleotide or a gene
chip comprising a plurality of such polynucleotides, or antibody,
which are optionally linked to a detectable label. The kits may
also include solid supports, pharmaceutically acceptable carriers
and/or excipients, a suitable container, and instructions for
use.
[0017] In yet another aspect of the invention, the differentially
regulated BVDV markers described herein may be used in screening
methods to identify new therapeutic agents for the treatment of
viral infections, including BVDV infection.
[0018] Agents which affect the differential expression of nucleic
acids or proteins associated with BVDV infection may prove
efficacious for the treatment of BVDV.
BRIEF DESCRIPTION OF THE FIGURE
[0019] FIG. 1 is a schematic diagram showing some of the features
of persistent and acute or transient BVDV infection.
[0020] FIG. 2 is a graph showing upregulation of interferon
stimulated gene 15 (ISG15) in blood from persistently infected
calves. ISGIS mRNA levels were determined using semi-quantitative
(adjusted for GAPDH) Sybr green Real Time PCR. Blood from three
persistently infected (PI) and three calves that had been
vaccinated against BVDV (TI) are represented in the analysis. ISG15
means differ between PI and TI (P<0.05).
[0021] FIG. 3 is a graph showing select blood cell markers that are
upregulated in bloods from persistently infected, when compared to
non-infected steers using semi-quantitative Real Time PCR (GAPDH
used as a control). The 28 kD (interferon induced 28 kD protein;
CK771386), BST2 (bone marrow stromal cell surface antigen 2;
CK846889), MX2 (myxovirus resistance 2; NM.sub.--173941) and ISG15
(Interferon stimulated 15 kDa;NM.sub.--174366) markers were all
useful blood cell mRNA markers for distinguishing persistent viral
infection (positive) when compared to control non-infected steers
(negative). In this illustration, ISG15 and MX2 are preferred
markers.
[0022] FIG. 4 is a heat plot illustrating three-Way ANOVA analysis
of blood cell gene expression from mothers carrying control vs. TI
vs. PI virally infected fetuses described in Table 9. This analysis
represents a fold change of 1.5 fold or greater with P<0.01,
Table 9 provides the actual P values for each comparison and a more
complete description of each gene.
DETAILED DESCRIPTION OF THE INVENTION
[0023] Bovine viral diarrhea virus (BVDV) provides a challenge to
cattle producers, because BVDV is a contagious and potentially
lethal disease that is currently difficult and expensive to
differentially diagnose. Current tests are performed on samples
collected from young calves after birth. Thus, many of these
infected calves have already shed virus and have infected other
pregnant cows. Therefore reinfection of pregnant cows helps
maintain the infectious cycle.
[0024] The complex host-viral interactions resulting from
persistent infection are minimally understood, particularly in the
bovine host. Thus, one purpose of the present research was to
identify those genes and associated biological pathways which are
activated or down-regulated in response to viral infection to
facilitate a better understanding of the mechanism of virus action.
Another objective of the research was to identify peripheral blood
markers that will help distinguish pregnant cattle that are
carrying persistently infected from those carrying transiently
virally infected fetuses. Depending on the time of infection during
gestation, noncytopathic (ncp) bovine viral diarrhea virus (BVDV)
causes persistent infection (PI, <150 d) or transient infection
(TI, >150 d.) in fetuses. TI fetuses develop immunity to the
viral strain and clear the virus. PI fetuses do not recognize the
virus as a foreign agent and once born continually shed the virus
and infect other cattle. Detection and removal of pregnant cows or
heifers carrying PI fetuses would greatly benefit the successful
implementation of control programs. Provided herein is a simple and
effective test for diagnosing BVDV, and identifying persistently
infected (PI) animals.
[0025] Experimental evidence is provided which indicates that the
pattern of gene expression in vaccinated or acutely infected and PI
animals is different, and therefore the differential expression of
genes can be used as a diagnostic marker for these types of BVDV
infection. Genes that are differentially expressed in the cells of
the blood or the skin of persistently infected animals (surrogate
markers) are identified using gene chip analysis of mRNA of PI when
compared to vaccinated or acutely infected animals. Antibodies
produced against such surrogate markers can be used to develop a
diagnostic test to detect PI animals, by analyzing the presence of
the surrogate marker in an animal's blood or skin.
[0026] Thus, in accordance with the present invention, gene chip
analysis has been performed on nucleic acids obtained from blood
cells collected from bovines that are persistently infected with
BVDV when compared to vaccinated control bovines.
[0027] In yet another aspect, the differentially regulated BVDV
markers described herein may be used in screening methods to
identify new therapeutic agents for the treatment of viral
infections, particularly BVDV infections. For example, agents which
down regulate the expression of genes which are upregulated in
response to infection may have efficacy as antiviral agents.
I. Definitions
[0028] The following definitions are provided to facilitate an
understanding of the present invention: The term "surrogate marker"
or infection marker is a marker which is differentially expressed
in animals infected with a pathological condition, such as a
virus.
[0029] Specifically, a surrogate marker may be any gene expression
product which is differentially expressed in persistently infected
animals when compared to vaccinated or acutely infected animals,
transiently infected and non-infected or non-vaccinated (normal)
animals. A surrogate marker can be a polynucleotide, a protein, a
peptide, or any gene expression product, but is preferably an mRNA
or protein expression product. The surrogate markers described
herein may also be useful for diagnosing invention with other RNA
viruses which include for example, Influenza, HIV, Ebola virus,
FeLv, FIP virus, Bluetongue virus, West Nile Virus, hepatitis C
Virus and Epizootic Hemorrhagic Disease Virus. Thus, the term
"surrogate marker" as used herein refers to those biological
molecules which are differentially expressed in response to
infection with any RNA virus.
[0030] A persistently infected calf is one that is infected in
utero prior to 150 days of gestation, does not clear the virus and
if it survives will continue to shed virus. A transiently infected
calf is one that is infected in utero after 150 days of gestation,
recovers and clears the virus. An acutely infected animal is one
that is infected postnatally and recovers, clearing the virus, A
control animal is one that was never infected with virus.
[0031] A "BVDV surrogate marker" refers to a marker which is
differentially expressed in animals infected with BVDV.
Specifically, a BVDV surrogate marker may be any gene expression
product which is differentially expressed in any or all of acutely
infected BVDV animals, persistently infected BVDV animals,
vaccinated BVDV animals, and normal animals. A surrogate marker can
be a polynucleotide, a protein or peptide, or any gene expression
product, but is preferably an mRNA or protein expression
product.
[0032] A "BVDV surrogate marker profile" is an expression pattern
of surrogate BVDV markers which correlates specifically to acute
BVDV infection, persistent BVDV infection, BVDV vaccinated cattle,
or non-BVDV infected cattle.
[0033] A "sample" or "patient sample" or "biological sample"
generally refers to a sample which may be tested for a particular
molecule, preferably a surrogate BVDV marker, including one or more
surrogate BVDV polynucleotide, polypeptide, or antibody. Samples
may include but are not limited to blood or skin, serum, plasma,
urine, saliva, and the like. Most preferably, the sample is a skin
sample or a blood sample from cattle.
[0034] "Blood" includes but is not limited to whole blood, blood
treated or mixed with anticoagulants, and any component of whole
blood, including but not limited to serum, plasma, buffy coat, and
purified peripheral blood mononuclear cells.
[0035] A "ruminant" is an even-toed, herbivorous, ungulate mammal
(Order Artiodactyla) that chews cud (ruminate) and has a complex,
usually four-chambered stomach containing micro-organisms that
break down cellulose. Ruminants include but are not limited to
cattle, sheep, antelope, deer, giraffes, elk, moose, caribou, yak
and camelids (e.g., camel, llama, alpaca, vicuna and guanaco).
[0036] The term "cattle" as used herein includes any of numerous
types of domestic quadrupeds held as property or raised for use,
such as livestock, cows, bulls, bovine, steer, oxen, bison, and the
like. The term "cattle" generally refers to multiple animals, but
may also describe a single animal.
[0037] The term "ruminant nucleic acid" or "ruminant protein"
refers to a nucleic acid or protein whose sequence is of ruminant
origin. Preferably, a ruminant nucleic acid or ruminant protein is
of bovine origin.
[0038] A "BVDV surrogate marker detector molecule" is a molecule
which facilitates detecting or quantitating a BVDV surrogate
marker. A BVDV surrogate marker detector molecule can be any
molecule which facilitates detection of BVDV surrogate marker,
including but not limited to a probe or primer which specifically
hybridizes with a BVDV surrogate marker nucleic acid, or an
antibody or fragment thereof which specifically binds to a BVDV
surrogate marker polypeptide or peptide fragment.
[0039] The term "differential diagnosis" refers to a diagnosis
which is able to differentiate between two or more different types
of BVDV infection (for example, acute infection, persistent
infection, or not infected.) This test also identifies previously
vaccinated animals.
[0040] The phrase "consisting essentially of" when referring to a
particular nucleotide or amino acid means a sequence having the
properties of a given SEQ ID NO:.
[0041] For example, when used in reference to an amino acid
sequence, the phrase includes the sequence per se and molecular
modifications that would not affect the functional and unique
characteristics of the sequence.
[0042] The term "nucleic acid molecule" describes a polymer of
deoxyribonucleotides (DNA) or ribonucleotides (RNA). The nucleic
acid molecule may be isolated from a natural source by cDNA cloning
or subtractive hybridization or synthesized manually. The nucleic
acid molecule may be synthesized manually by the triester synthetic
method or by using an automated DNA synthesizer.
[0043] With regard to nucleic acids used in the invention, the term
"isolated nucleic acid" is sometimes employed. This term, when
applied to DNA, refers to a DNA molecule that is separated from
sequences with which it is immediately contiguous (in the 5' and 3'
directions) in the naturally occurring genome of the organism or
virus from which it was derived. For example, the "isolated nucleic
acid" may comprise a DNA molecule inserted into a vector, such as a
plasmid or virus vector, or integrated into the genomic DNA of a
prokaryote or eukaryote cells.
[0044] An "isolated nucleic acid molecule" may also comprise a eDNA
molecule. An isolated nucleic acid molecule inserted into a vector
is also sometimes referred to herein as a recombinant nucleic acid
molecule.
[0045] The term "complementary" describes two nucleotides that can
form multiple favorable interactions with one another. For example,
adenine is complementary to thymine as they can form two hydrogen
bonds. Similarly, guanine and cytosine are complementary since they
can form three hydrogen bonds. Thus if a nucleic acid sequence
contains the following sequence of bases, thymine, adenine, guanine
and cytosine, a "complement" of this nucleic acid molecule would be
a molecule containing adenine in the place of thymine, thymine in
the place of adenine, cytosine in the place of guanine, and guanine
in the place of cytosine. Because the complement can contain a
nucleic acid sequence that forms optimal interactions with the
parent nucleic acid molecule, such a complement can bind with high
affinity to its parent molecule.
[0046] With respect to single stranded nucleic acids, particularly
oligonucleotides, the term "specifically hybridizing" refers to the
association between two single-stranded nucleotide molecules of
sufficiently complementary sequence to permit such hybridization
under pre-determined conditions generally used in the art
(sometimes termed "substantially complementary"). In particular,
the term refers to hybridization of an oligonucleotide with a
substantially complementary sequence contained within a
single-stranded DNA or RNA molecule of the invention, to the
substantial exclusion of hybridization of the oligonucleotide with
single- stranded nucleic acids of non-complementary sequence.
Appropriate conditions enabling specific hybridization of single
stranded nucleic acid molecules of varying complementarity are well
known in the art.
[0047] For instance, one common formula for calculating the
stringency conditions required to achieve hybridization between
nucleic acid molecules of a specified sequence homology is set
forth below (Sambrook et al. , Molecular Cloning, Cold Spring
Harbor Laboratory (1989):
[0048] Tm 81.5.degree. C +16.6 Log [Na+]+0.41 (% G+C)-0.63 (%
formamide)-600/#bp in duplex.
[0049] As an illustration of the above formula, using [Na+]=[0.368]
and 50% formamide, with GC content of 42% and an average probe size
of 200 bases, the Tm is 57.degree. C. The Tm of a DNA duplex
decreases by 1-1.5.degree. C. with every 1% decrease in homology.
Thus, targets with greater than about 75% sequence identity would
be observed using a hybridization temperature of 42.degree. C.
[0050] The stringency of the hybridization and wash depend
primarily on the salt concentration and temperature of the
solutions. In general, to maximize the rate of annealing of the
probe with its target, the hybridization is usually carried out at
salt and temperature conditions that are 20-25.degree. C. below the
calculated Tm of the hybrid.
[0051] Wash conditions should be as stringent as possible for the
degree of identity of the probe for the target. In general, wash
conditions are selected to be approximately 12-20.degree. C. below
the Tm of the hybrid. In regards to the nucleic acids of the
current invention, a moderate stringency hybridization is defined
as hybridization in 6.times.SSC, 5.times. Denhardt's solution, 0.5%
SDS and 100 pg/ml denatured salmon sperm DNA at 42.degree. C., and
washed in 2.times.SSC and 0.5% SDS at 55.degree. C. for 15 minutes.
A high stringency hybridization is defined as hybridization in
6.times.SSC, 5.times. Denhardt's solution, 0.5% SDS and 100 pg/ml
denatured salmon sperm DNA at 42.degree. C., and washed in
1.times.SSC and 0.5% SDS at 65.degree. C. for 15 minutes. A very
high stringency hybridization is defined as hybridization in
6.times.SSC, 5.times. Denhardt's solution, 0.5% SDS and 100 pg/ml
denatured salmon sperm DNA at 42.degree. C., and washed in
0.1.times.SSC and 0.5% SDS at 65.degree. C. for 15 minutes.
[0052] The term "oligonucleotide" as used herein refers to primers
and probes of the present invention, and is defined as a nucleic
acid molecule comprised of two or more ribo-or
deoxyribonucleotides, preferably more than three. The exact size of
the oligonucleotide will depend on various factors and on the
particular application and use of the oligonucleotide.
Oligonucleotides, which include probes and primers, can be any
length from 3 nucleotides to the full length of the nucleic acid
molecule, and explicitly include every possible number of
contiguous nucleic acids from 3 through the full length of the
polynucleotide. Preferably, oligonucleotides, which include probes
and/or primers are at least about 10 nucleotides in length, more
preferably at least 15 nucleotides in length, more preferably at
least about 20 nucleotides in length.
[0053] The term "probe" as used herein refers to an
oligonucleotide, polynucleotide or nucleic acid, either RNA or DNA,
whether occurring naturally as in a purified restriction enzyme
digest or produced synthetically, which is capable of annealing
with or specifically hybridizing to a nucleic acid with sequences
complementary to the probe. A probe may be either single-stranded
or double-stranded. The exact length of the probe will depend upon
many factors, including temperature, source of probe and use of the
method. For example, for diagnostic applications, depending on the
complexity of the target sequence, the oligonucleotide probe
typically contains 15-25 or more nucleotides, although it may
contain fewer nucleotides. The probes herein are selected to be
complementary to different strands of a particular target nucleic
acid sequence.
[0054] This means that the probes must be sufficiently
complementary so as to be able to "specifically hybridize" or
anneal with their respective target strands under a set of
pre-determined conditions. Therefore, the probe sequence need not
reflect the exact complementary sequence of the target. For
example, a non-complementary nucleotide fragment may be attached to
the 5' or 3'end of the probe, with the remainder of the probe
sequence being complementary to the target strand. Alternatively,
non-complementary bases or longer sequences can be interspersed
into the probe, provided that the probe sequence has sufficient
complementarity with the sequence of the target nucleic acid to
anneal therewith specifically.
[0055] The term "primer" as used herein refers to an
oligonucleotide, either RNA or DNA, either single- stranded or
double-stranded, either derived from a biological system, generated
by restriction enzyme digestion, or produced synthetically which,
when placed in the proper environment, is able to functionally act
as an initiator of template-dependent nucleic acid synthesis. When
presented with an appropriate nucleic acid template, suitable
nucleoside triphosphate precursors of nucleic acids, a polymerase
enzyme, suitable cofactors and conditions such as a suitable
temperature and pH, the primer may be extended at its 3 terminus by
the addition of nucleotides by the action of a polymerase or
similar activity to yield a primer extension product. The primer
may vary in length depending on the particular conditions and
requirement of the application. For example, in diagnostic
applications, the oligonucleotide primer is typically 15-25 or more
nucleotides in length. The primer must be of sufficient
complementarity to the desired template to prime the synthesis of
the desired extension product, that is, to be able anneal with the
desired template strand in a manner sufficient to provide the 3
'hydroxyl moiety of the primer in appropriate juxtaposition for use
in the initiation of synthesis by a polymerase or similar enzyme.
It is not required that the primer sequence represent an exact
complement of the desired template.
[0056] For example, a non-complementary nucleotide sequence may be
attached to the 5'end of an otherwise complementary primer.
Alternatively, non-complementary bases may be interspersed within
the oligonucleotide primer sequence, provided that the primer
sequence has sufficient complementarity with the sequence of the
desired template strand to functionally provide a template-primer
complex for the synthesis of the extension product. Polymerase
chain reaction (PCR) has been described in U.S. Pat. Nos.
4,683,195, 4,800,195, and 4,965,188, the entire disclosures of
which are incorporated by reference herein.
[0057] The term "vector" relates to a single or double stranded
circular nucleic acid molecule that can be transfected or
transformed into cells and replicate independently or within the
host cell genome. A circular double stranded nucleic acid molecule
can be cut and thereby linearized upon treatment with restriction
enzymes. An assortment of vectors, restriction enzymes, and the
knowledge of the nucleotide sequences that are targeted by
restriction enzymes are readily available to those skilled in the
art. A vector of the invention includes any replicon, such as a
plasmid, cosmid, bacmid, phage or virus, to which another genetic
sequence or element (either DNA or RNA) may be attached so as to
bring about the replication of the attached sequence or element. A
nucleic acid molecule of the invention can be inserted into a
vector by cutting the vector with restriction enzymes and ligating
the two pieces together.
[0058] Many techniques are available to those skilled in the art to
facilitate transformation, transfection, or transduction of the
expression construct into a prokaryotic or eukaryotic organism. The
terms "transformation", "transfection", and "transduction" refer to
methods of inserting a nucleic acid and/or expression construct
into a cell or host organism. These methods involve a variety of
techniques, such as treating the cells with high concentrations of
salt, an electric field, or detergent, to render the host cell
outer membrane or wall permeable to nucleic acid molecules of
interest, microinjection, PEG-fusion, and the like.
[0059] The term "promoter element" describes a nucleotide sequence
that is incorporated into a vector that, once inside an appropriate
cell, can facilitate transcription factor and/or polymerase binding
and subsequent transcription of portions of the vector DNA into
mRNA. In one embodiment, the promoter element of the present
invention precedes the Send of the BVDV surrogate marker nucleic
acid molecule such that the latter is transcribed into mRNA. Host
cell machinery then translates mRNA into a polypeptide.
[0060] Those skilled in the art will recognize that a nucleic acid
vector can contain nucleic acid elements other than the promoter
element and the BVDV surrogate marker gene nucleic acid molecule.
These other nucleic acid elements include, but are not limited to,
origins of replication, ribosomal binding sites, nucleic acid
sequences encoding drug resistance enzymes or amino acid metabolic
enzymes, and nucleic acid sequences encoding secretion signals,
periplasm or peroxisome localization signals, or signals useful for
polypeptide purification.
[0061] An "expression operon" refers to a nucleic acid segment that
may possess transcriptional and translational control sequences,
such as promoters, enhancers, translational start signals (e.g.,
ATG or AUG codons), polyadenylation signals, terminators, and the
like, and which facilitate the expression of a polypeptide coding
sequence in a host cell or organism.
[0062] As used herein, the terms "reporter", "reporter system",
"reporter gene", or "reporter gene product" shall mean an operative
genetic system in which a nucleic acid comprises a gene that
encodes a product that when expressed produces a reporter signal
that is a readily measurable, e.g., by biological assay,
immunoassay, radio immunoassay, or by calorimetric, fluorogenic,
chemiluminescent or other methods. The nucleic acid may be either
RNA or DNA, linear or circular, single or double stranded,
antisense or sense polarity, and is operatively linked to the
necessary control elements for the expression of the reporter gene
product. The required control elements will vary according to the
nature of the reporter system and whether the reporter gene is in
the form of DNA or RNA, but may include, but not be limited to,
such elements as promoters, enhancers, translational control
sequences, poly A addition signals, transcriptional termination
signals and the like.
[0063] The introduced nucleic acid may or may not be integrated
(covalently linked) into nucleic acid of the recipient cell or
organism. In bacterial, yeast, plant and mammalian cells, for
example, the introduced nucleic acid may be maintained as an
episomal element or independent replicon such as a plasmid.
Alternatively, the introduced nucleic acid may become integrated
into the nucleic acid of the recipient cell or organism and be
stably maintained in that cell or organism and further passed on or
inherited to progeny cells or organisms of the recipient cell or
organism. Finally, the introduced nucleic acid may exist in the
recipient cell or host organism only transiently.
[0064] The term "selectable marker gene" refers to a gene that when
expressed confers a selectable phenotype, such as antibiotic
resistance, on a transformed cell.
[0065] The term "operably linked" means that the regulatory
sequences necessary for expression of the coding sequence are
placed in the DNA molecule in the appropriate positions relative to
the coding sequence so as to effect expression of the coding
sequence. This same definition is sometimes applied to the
arrangement of transcription units and other transcription control
elements (e.g., enhancers) in an expression vector.
[0066] The term "tag", "tag sequence", or "protein tag" refers to a
chemical moiety, either a nucleotide, oligonucleotide,
polynucleotide or an amino acid, peptide or protein or other
chemical, that when added to another sequence, provides additional
utility or confers useful properties, particularly in the detection
or isolation, of that sequence. Thus, for example, a homopolymer
nucleic acid sequence or a nucleic acid sequence complementary to a
capture oligonucleotide may be added to a primer or probe sequence
to facilitate the subsequent isolation of an extension product or
hybridized product. In the case of protein tags, histidine residues
(e. g., 4 to 8 consecutive histidine residues) may be added to
either the amino-or carboxy-terminus of a protein to facilitate
protein isolation by chelating metal chromatography. Alternatively,
amino acid sequences, peptides, proteins or fusion partners
representing epitopes or binding determinants reactive with
specific antibody molecules or other molecules (e. g., flag
epitope, c-mye epitope, transmembrane epitope of the influenza A
virus hemaglutinin protein, protein A, cellulose binding domain,
calmodulin binding protein, maltose binding protein, chitin binding
domain, glutathione S-transferase, and the like) may be added to
proteins to facilitate protein isolation by procedures such as
affinity or immunoaffinity chromatography.
[0067] Chemical tag moieties include such molecules as biotin,
which may be added to either nucleic acids or proteins and
facilitates isolation or detection by interaction with avidin
reagents, and the like. Numerous other tag moieties are known to,
and can be envisioned by the trained artisan, and are contemplated
to be within the scope of this definition.
[0068] A "specific binding pair" comprises a specific binding
member (sbm) and a binding partner (bp) which have a particular
specificity for each other and which in normal conditions bind to
each other in preference to other molecules. Examples of specific
binding pairs are antigens and antibodies, ligands and receptors
and complementary nucleotide sequences. The skilled person is aware
of many other examples. Further, the term "specific binding pair"
is also applicable where either or both of the specific binding
member and the binding partner comprise a part of a large molecule.
In embodiments in which the specific binding pair comprises nucleic
acid sequences, they will be of a length to hybridize to each other
under conditions of the assay, preferably greater than 10
nucleotides long, more preferably greater than 15 or 20 nucleotides
long.
[0069] An "antibody" or "antibody molecule" is any immunoglobulin,
including antibodies and fragments thereof that binds to a specific
antigen. The term includes polyclonal, monoclonal, chimeric, and
bispecific antibodies. Exemplary antibody fragments, capable of
binding an antigen or other binding partner, are Fab fragment
consisting of the VL, VH, Cl and CH1 domains; the Fd fragment
consisting of the VH and CH1 domains; the Fv fragment consisting of
the VL and VH domains of a single arm of an antibody; the dAb
fragment which consists of a VH domain; isolated CDR regions and F
(ab') 2 fragments, a bivalent fragment including two Fab fragments
linked by a disulphide bridge at the hinge region. Single chain Fv
fragments are also included.
[0070] With respect to antibodies, the term "immunologically
specific" refers to antibodies that bind to one or more epitopes of
a protein or compound of interest, but which do not substantially
recognize and bind other molecules in a sample containing a mixed
population of antigenic biological molecules. Exemplary antibodies
bind to a protein or peptide fragment encoded by a nucleotide
sequence set forth in Tables 1-9.
[0071] A "detection reagent" or a "marker detection reagent" is any
substance which has binding affinity for a BVDV specific molecule,
and includes but is not limited to nucleic acid molecules with
sufficient affinity to hybridize to the BVDV specific marker,
probes, primers, antibodies, fragments thereof and the like. The
"detection reagent" or "marker detection reagent" may optionally be
detectably labeled.
[0072] The term "detectable label" is used herein to refer to any
substance whose detection or measurement, either directly or
indirectly, by physical or chemical means, is indicative of the
presence of a target bioentity in a test sample. Representative
examples of useful detectable labels, include, but are not limited
to the following: molecules or ions directly or indirectly
detectable based on light absorbance, fluorescence, reflectance,
light scatter, phosphorescence, or luminescence properties;
molecules or ions detectable by their radioactive properties;
molecules or ions detectable by their nuclear magnetic resonance or
paramagnetic properties. Included among the group of molecules
indirectly detectable based on light absorbance or fluorescence,
for example, are various enzymes which cause appropriate substrates
to convert, e. g., from non-light absorbing to light absorbing
molecules, or from non-fluorescent to fluorescent molecules.
II. Surrogate BVDV Nucleic Acid Molecules, Probes, and Primers and
Methods of Preparing the Same
[0073] Encompassed by the invention are surrogate BVDV nucleic acid
molecules, nucleic acid molecules which encode isolated, enriched,
or purified surrogate BVDV proteins or peptides, including allelic
variations, analogues, fragments, derivatives, mutants, and
modifications of the same.
[0074] Surrogate BVDV nucleic acid molecules, and nucleic acid
sequences encoding surrogate BVDV proteins may be isolated from
appropriate biological sources using methods known in the art. In a
preferred embodiment, a cDNA clone is isolated from a cDNA
expression library of bovine origin. Preferably, the sample is
isolated from a bovine which has been vaccinated for, or has acute,
or persistent BVDV infection, Surrogate BVDV marker polynucleotides
can be any one of, or any combination of the markers shown in
Tables 1-9, and further may include variants which are at least
about 75%, or 80% or 85% or 90% or 95%, and often, more than 90%,
or more than 95% homologous to the markers shown in Tables 1-9,
over the full length sequence. Surrogate BVDV marker
polynucleotides also may be 60% or 65% or 70% or 75% or 80% or 85%
or 90% or 95% or 97% or 98% or 99% or greater than 99% homologous
to the markers shown in Tables 1-9, over the full length sequence.
All homology may be computed by algorithms known in the art, such
as BLAST, described in Altschul et al. (1990), J. Mol. Biol. 215:
403-10, or the Smith-Waterman homology search algorithm as
implemented in MPSRCH program (Oxford Molecular). Someone of
ordinary skill in the art would readily be able to determine the
ideal gap open penalty and gap extension penalty for a particular
nucleic acid sequence.
[0075] Exemplary search parameters for use with the MPSRCH program
in order to identify sequences of a desired sequence identity are
as follows: gap open penalty:-16; and gap extension penalty:-4.
[0076] Degenerate variants are also encompassed by the instant
invention. The degeneracy of the genetic code permits substitution
of certain codons by other codons, which specify the same amino
acid and hence would give rise to the same protein. The nucleic
acid sequence can vary substantially since, with the exception of
methionine and tryptophan, the known amino acids can be coded for
by more than one codon. Thus, portions or all of the markers could
be synthesized to give a nucleic acid sequence significantly
different from that shown in Tables 1-9. The encoded amino acid
sequence thereof would, however, be preserved.
[0077] In addition, the nucleic acid sequence may comprise a
nucleotide sequence which results from the addition, deletion or
substitution of at least one nucleotide to the 5'-end and/or the
3'-end of one or more of the markers shown in Tables 1-9, or a
derivative thereof. Any nucleotide or polynucleotide may be used in
this regard, provided that its addition, deletion or substitution
does not alter the amino acid sequence which is encoded by the
nucleotide sequence, or it still shares a region of homology with
one or more of the markers shown in Tables 1-9. For example, the
present invention is intended to include any nucleic acid sequence
resulting from the addition of ATG as an initiation codon at the
5'-end of the surrogate BVDV marker nucleic acid sequence or its
functional derivative, or from the addition of TTA, TAG or TGA as a
termination codon at the 3'-end of the inventive nucleotide
sequence or its derivative. Moreover, the nucleic acid molecule of
the present invention may, as necessary, have restriction
endonuclease recognition sites added to its 5'-end and/or
3'-end.
[0078] Such functional alterations of a given nucleic acid sequence
afford an opportunity to promote secretion and/or processing of
heterologous proteins encoded by foreign nucleic acid sequences
fused thereto. All variations of the nucleotide sequence of the
markers shown in Tables 1-9 and fragments thereof permitted by the
genetic code are, therefore, included in this invention.
[0079] In an alternative embodiment, utilizing the sequence
information provided by the eDNA sequence, genomic clones encoding
a surrogate BVDV marker gene may be isolated.
[0080] Alternatively, eDNA or genomic clones having homology with
the markers shown in Tables 1-9 may be isolated from other species,
such as mouse or human, using oligonucleotide probes corresponding
to predetermined sequences within surrogate BVDV marker gene.
III. Surrogate BVDV Proteins (Antigens) and Methods of Making the
Same
[0081] Encompassed by the invention are isolated, purified, or
enriched surrogate BVDV polypeptides, including allelic variations,
analogues, fragments, derivatives, mutants, and modifications of
the same which are differentially expressed in BVDV animals.
Preferably, surrogate BVDV marker polypeptides include polypeptides
encoded by one or more of the sequences shown in Tables 1-9.
Surrogate BVDV marker function is defined above, and includes
increased or decreased expression in response to BVDV infection,
cross-reactivity with an antibody reactive with the polypeptides
encoded by one or more of the sequences shown in Tables 1-9 or
sharing an epitope with the same (as determined for example by
immunological cross-reactivity between the two polypeptides).
Surrogate BVDV marker polypeptides or proteins can be encoded by
one or more of the sequences shown in Tables 1-9, and further may
include variants which are at least about 75%, or 80% or 85% or 90%
or 95%, and often, more than 90%, or more than 95% homologous to
the same over the full length sequence.
[0082] Surrogate BVDV marker polypeptides also may be 60% or 65% or
70% or 75% or 80% or 85% or 90% or 95% or 97% or 98% or 99% or
greater than 99% homologous to polypeptides encoded by one or more
of the sequences shown in Tables 1-9 over the full length sequence.
All homology may be computed by algorithms known in the art, such
as BLAST, described in Altschul et al. (1990), J. Mol. Biol. 215:
403-10, or the Smith-Waterman homology search algorithm as
implemented in MPSRCH program (Oxford Molecular). Someone of
ordinary skill in the art would readily be able to determine the
ideal gap open penalty and gap extension penalty for a particular
protein sequence. Exemplary search parameters for use with the
MPSRCH program in order to identify sequences of a desired sequence
identity are as follows: gap open penalty:-12 ; and gap extension
penalty:-2.
[0083] A full-length or truncated surrogate BVDV protein of the
present invention may be prepared in a variety of ways, according
to known methods. The protein may be purified from appropriate
sources, e.g., transformed bacterial or animal cultured cells or
tissues, by immunoaffinity purification. Additionally, the
surrogate BVDV protein may be produced using in vitro expression
methods known in the art. For example, a cDNA or gene may be cloned
into an appropriate in vitro transcription vector, such as pSP64 or
pSP65 for in vitro transcription, followed by cell-free translation
in a suitable cell-free translation system, such as wheat germ or
rabbit reticulocyte lysates. In vitro transcription and translation
systems are commercially available, e. g., from Promega Corp.,
Madison, Wis. or Invitrogen Corp., Carlsbad, Calif.
[0084] The surrogate BVDV proteins produced by gene expression in a
recombinant prokaryotic or eukaryotic system may be purified
according to methods known in the art. In a preferred embodiment, a
commercially available expression/secretion system can be used,
whereby the recombinant protein is expressed and thereafter
secreted from the host cell, to be easily purified from the
surrounding medium. If expression/secretion vectors are not used,
an alternative approach involves purifying the recombinant protein
by affinity separation, such as by immunological interaction with
antibodies that bind specifically to the recombinant protein or
nickel columns for isolation of recombinant proteins tagged with
6-8 histidine residues at their N-terminus or C-terminus.
[0085] Alternative tags may comprise the FLAG epitope or the
hemagglutinin epitope. Such methods are commonly used by skilled
practitioners.
IV. Anti-Surrogate BVDV Protein Antibodies and Methods of Making
the Same
[0086] The present invention also provides methods of making and
using antibodies capable of immunospecifically binding to surrogate
BVDV proteins. Polyclonal antibodies directed toward surrogate BVDV
proteins may be prepared according to standard methods. In a
preferred embodiment, monoclonal antibodies are prepared, which
react immunospecifically with the various epitopes on the surface
of the surrogate BVDV protein. Monoclonal antibodies may be
prepared according to general methods of Kohler and Milstein,
following standard protocols.
[0087] Purified BVDV antigens, or fragments thereof, may be used to
produce polyclonal or monoclonal antibodies which also may serve as
sensitive detection reagents for the various types of BVDV
infection (acute, PI, vaccination reaction, and not infected).
Recombinant techniques enable expression of fusion proteins
containing part or all of BVDV. The surrogate BVDV protein itself,
or surface proteins or antigens from the surrogate BVDV protein may
be used to advantage to generate an array of monoclonal antibodies
specific for various epitopes of the surrogate BVDV protein,
thereby providing even greater sensitivity for detection of the
surrogate BVDV protein (and thus BVDV infection) in samples.
[0088] Polyclonal or monoclonal antibodies that immunospecifically
interact with BVDV antigens can be utilized for identifying and
diagnosing BVDV. For example, antibodies may be utilized for
affinity separation of proteins with which they immunospecifically
interact. Antibodies may also be used to immunuoprecipitate
proteins from a sample containing a mixture of proteins and other
biological molecules.
[0089] Other uses of anti-surrogate BVDV protein antibodies are
described below.
V. Methods of Using Surrogate BVDV Polynucleotides, Polypeptides,
and Antibodies for Screening and Diagnostic Assays
[0090] Surrogate BVDV nucleic acids may be used for a variety of
purposes in accordance with the present invention. Surrogate BVDV
nucleic acids (DNA, RNA, fragments thereof, etc.), or
protein-encoding DNA, RNA, or fragments thereof may be used as
probes to detect the presence of surrogate BVDV nucleic acids or
protein in a sample. Methods in which surrogate BVDV nucleic acids
and protein-encoding nucleic acids may be utilized as probes for
such assays include, but are not limited to, (1) in situ
hybridization; (2) Southern hybridization (3) northern
hybridization; (4) gene chip analysis and (5) assorted
amplification reactions such as polymerase chain reactions
(PCR).
[0091] Exemplary surrogate BVDV nucleic acids and nucleic acids
encoding exemplary surrogate BVDV proteins or peptides are
described in Tables 1-9.
[0092] The surrogate BVDV nucleic acids of the invention may also
be utilized as probes to identify related surrogate BVDV variants.
As is well known in the art, hybridization stringencies may be
adjusted to allow hybridization of nucleic acid probes with
complementary sequences of varying degrees of homology. Thus, BVDV
surrogate marker nucleic acids may be used to advantage to identify
and characterize other genes of varying degrees of relation to BVDV
surrogate markers, thereby enabling further characterization of
BVDV surrogate markers. Additionally, they may be used to identify
genes encoding proteins that interact with BVDV surrogate markers
(e. g., by the "interaction trap" technique-see for example Current
Protocols in Molecular Biology, ed. Ausubel, F. M., et al., John
Wiley & Sons, NY, 1997), which should further accelerate
identification of the molecular components involved in BVDV.
Finally, they may be used in assay methods to detect BVDV.
[0093] Polyclonal or monoclonal antibodies immunologically specific
for proteins encoded by BVDV surrogate markers or peptide fragments
thereof may be used in a variety of assays designed to detect and
quantitate the protein, as well as to detect ruminant BVDV by
detecting upregulation of BVDV surrogate markers. Such assays
include, but are not limited to: (1) flow cytometric analysis; (2)
immunochemical localization of BVDV specific markers in a body
cell, tissue, or fluid; and (3) immunoblot analysis (e. g., dot
blot, Western blot) (4) ELISA; (5) radioimmunoassay of extracts
from various cells.
[0094] Additionally, as described above, anti-surrogate BVDV marker
protein can be used for purification of surrogate BVDV markers (e.
g., affinity column purification, immunoprecipitation).
[0095] Further, assays for detecting and quantitating surrogate
BVDV markers, or to detect ruminant BVDV by detecting upregulation
of BVDV specific markers may be conducted on any type of biological
sample where upregulation of these molecules is observed, including
but not limited to body fluids (including blood, serum, plasma,
milk, or saliva), any type of cell (such as skin cells, or blood
cells, or endothelial cells), or body tissue.
[0096] From the foregoing discussion, it can be seen that surrogate
BVDV marker nucleic acids, surrogate BVDV marker expressing
vectors, surrogate BVDV marker proteins and anti-surrogate BVDV
marker antibodies of the invention can be used to detect surrogate
BVDV marker expression in body tissue, cells, or fluid, and alter
BVDV specific marker protein expression for purposes of assessing
the genetic and protein interactions involved in BVDV and
infection.
[0097] In most embodiments for screening for surrogate BVDV mRNA,
surrogate BVDV nucleic acid in the sample will initially be
amplified, e. g. using PCR, to increase the amount of the template
as compared to other sequences present in the sample. This allows
the target sequences to be detected with a high degree of
sensitivity if they are present in the sample.
[0098] Thus any of the aforementioned techniques may be used as a
diagnostic tool for detecting surrogate BVDV markers.
[0099] Further, these techniques could be used to diagnose
infectious diseases in humans, by detection of a surrogate marker
(rather than a viral antigen). For example, differential gene
expression could be measured in HIV, Ebola, Hepatitis, and Herpes
viral infections, etc. These tests are advantageous in that they
are directed to detection of a theoretically harmless surrogate
marker, rather than the infectious agent itself.
[0100] Such techniques could also be used to diagnose infectious
diseases in companion animals by detection of a surrogate marker
(rather than a viral antigen). An example of a potential
application would be the diagnosis of feline infectious peritonitis
of cats and latent viral infections caused by herpes viruses for
which current diagnostic tests (based on isolation and
characterization of the virus) have a marginal reliability. In
addition, this technology could also be used for the diagnosis of
cancer through the identification of surrogate cancer markers.
[0101] The instant inventive method improves upon the accuracy of
current BVDV tests. A combination test, which measures both BVDV
itself, and also one or more BVDV surrogate marker, to
differentially diagnose BVDV infection provides superior diagnostic
results in the field.
VI. Assays for Differentially Diagnosing BVDV Using Specific
Surrogate Markers
[0102] In accordance with the present invention, it has been
discovered that Bovine Viral Diarrhea Virus (BVDV) is correlated
with increased expression levels of certain markers, including but
not limited to mRNAs and proteins.
[0103] Thus, these molecules may be utilized in conventional assays
to differentially diagnose BVDV. The detection of one or more of
these differentially expressed BVDV surrogate molecules in a sample
is indicative of BVDV. Similarly, specific patterns of expression
allow detection of acute versus persistent infection.
Alternatively, the absence of these molecules in a sample indicates
that a ruminant is not infected with BVDV.
[0104] In an exemplary method, a blood sample is obtained from a
bovine suspected of having an acute or persistent BVDV infection.
Optionally, the blood may be centrifuged through a Hypaque gradient
to obtain the buffy coat. The blood or buffy coat preparation is
diluted and subjected to polymerase chain reaction conditions
suitable for amplification of the BVDV surrogate marker encoding
mRNA.
[0105] In certain applications, it may be necessary to include an
agent, which lyses cells prior to performing the PCR.
[0106] Such agents are well known to the skilled artisan. The
reaction products are then analyzed, e.g., via gel electrophoresis.
An increase in BVDV surrogate marker mRNA levels relative to levels
obtained from a non-infected bovine is indicative of BVDV in the
animal being tested. Alternatively, an increase in BVDV surrogate
markers in Al animals relative to Pt animals, or in PI animals,
relative to Al animals, can differentially diagnose acute
infection, or persistent infection.
[0107] In an alternative method, a skin tissue sample is obtained
from the bovine suspected of having acute or persistent BVDV
infection. The cells are then lysed and PCR performed. As above, an
increase in BVDV surrogate marker mRNA expression levels relative
to those observed in a non-BVDV infected animal being indicative of
BVDV in the test animal.
[0108] It is also possible to detect BVDV using immunoassays. In an
exemplary method, blood is obtained from a bovine suspected of
being infected with BVDV. As above, the blood may optionally be
centrifuged through a Hypaque gradient to obtain a buffy coat. The
blood or buffy coat sample is diluted and at least one antibody
immunologically specific for BVDV surrogate markers is added to the
sample. In a preferred embodiment, the antibody is operably linked
to a detectable label. Also as described above, the cells may
optionally be lysed prior to contacting the sample with the
antibodies immunologically specific for BVDV surrogate markers.
[0109] Increased production of BVDV surrogate markers is assessed
as a fuaction of an increase in the detectable label relative to
that obtained in parallel assays using blood from non-BVDV infected
cow. In yet another embodiment, the blood or buffy coat preparation
is serially diluted and aliquots added to a solid support.
[0110] Suitable solid supports include multi-well culture dishes,
blots, filter paper, and cartridges. The solid support is then
contacted with the detectably labeled antibody and the amount of
BVDV surrogate marker protein (e.g., a protein or peptide encoded
by a nucleic acid of Tables 1-9) in the animal suspected of being
infected with BVDV is compared with the amount obtained from a
non-AI or PI animal as a function of detectably labeled antibody
binding. An alteration in the BVDV surrogate marker protein level
in the test animal relative to the non-AI or PI infected control
animal is indicative of acute or persistent BVDV.
[0111] In another embodiment, a first antibody which binds to a
first epitope on a target protein is placed in the well of a
cartridge. Whole blood, blood collected in the presence of
anticoagulants (e.g., sodium citrate, heparin), plasma, or serum is
placed into the well of the cartridge. The target protein, if
present in the sample, is bound by the first antibody, and then
migrates laterally by a wicking action, through a filter which has
been sprayed with second antibody. The second antibody has affinity
for a second epitope on the target protein, or alternatively for
the first antibody. The second antibody is optionally labeled with
a detectable label (e. g. radiolabel, gold, biotin, enzyme, etc.)
The second antibody localizes the antigen, and results in the
appearance of a line on the filter. The first and second antibodies
may be generated against the full length target protein, or against
the N-terminal or C-terminal halves of the target protein, so that
they recognize different epitopes of the target protein.
[0112] The foregoing immunoassay methods may also be applied to any
type of sample, including a urine sample.
VII. Kits and Articles of Manufacture
[0113] Any of the aforementioned products or methods can be
incorporated into a kit which may contain a BVDV specific
polynucleotide, an oligonucleotide, a polypeptide, a peptide, a
solid support (e.g., filters, cartridges, gene chips) an antibody,
a label, marker, or reporter, a pharmaceutically acceptable
carrier, a physiologically acceptable carrier, instructions for
use, negative and positive control samples, a container, a vessel
for administration, an assay substrate, or any combination
thereof.
[0114] The following materials and methods are provided to
facilitate the practice of the invention. The Examples illustrate
certain embodiments of the invention. They are not intended to
limit its scope in any way.
EXAMPLE 1
[0115] Bovine viral diarrhea virus (BVDV) infections are
responsible for important economic losses due to reproductive
wastage, and respiratory and digestive disease in cattle. Infection
of the early developing fetus frequently results in persistent
infection (Pt). PI animals are the main source of new infection in
herd mates. The complex host-viral interactions resulting from
persistent infection are minimally understood, particularly in the
bovine host. We hypothesized that gene expression would differ in
bloods collected from PI when compared to non-infected steers. In
preliminary studies, bloods were collected from three PI or three
control steers and were processed to yield total cellular RNA.
Labeled RNA was used to screen six independent bovine Affymetrix
DNA chips, and analyzed for fold-changes at the University of
Colorado Health Sciences Center Microarray Facility. The top 100
up-regulated genes belonged to MHC class I (45-fold), antiviral
(32-100 fold), transcription factor (8-12 fold), interferon
stimulated genes (5-28 fold), bone remodeling (4-9 fold), and
chemokine (2-4) families. The top 100 down-regulated genes belonged
to adhesion (5-10 fold), T-cell receptor (5-10 fold), extracellular
matrix (3-5 fold), growth factor (2-3 fold), and transcription
factor (2-3 fold) families. We conclude from these findings that
persistent infection with BVDV results in antiviral responses in
blood cells which includes induction of type 1 interferon-induced
genes, chemokine-mediated immune responses and bone remodeling with
a concomitant suppression of extracellular remodeling, adhesion and
T-cell-mediated responses. Thus, in accordance with the present
invention, single and/or multiplexing diagnostics for identifying
cattle that are persistently infected with BVDV are provided.
[0116] BVDV is divided into two biotypes: cytopathic (cp) and
non-cytopathic (nep). There are at least two recognized genotypes
based on the sequence of the 5' untranslated region, type I and
type II, and additional sub-genotypes. Within each genotype, there
are several strains that cause different degrees of clinical signs.
The pathogenesis of BVDV infection has features that are unique to
this virus and that vary with the virulence of the viral strain and
age of the animal at the time of infection.
[0117] Particularly interesting are the outcomes of infection of
the fetus. Infection of the fetus during the first 150 days of
gestation, when the immune system has not yet developed, can lead
to the generation of PI calves (FIG. 1). Some of these PI calves
die soon after birth, but others live for relatively long periods
of time without showing any clinical signs. PI animals cannot
eliminate the infecting BVDV from their system and continuously
release high amounts of virus in their bodily secretions. This
makes them a continuous source of infection within the herd and
potentially to other herds. When the infection occurs after 150
days of gestation (post-development of the immune system) or after
birth, the infection is referred to as acute and these animals,
which clear the virus, are frequently called transiently infected
(TI). The clinical manifestations of acute infections with BVDV
range from sub-clinical, or unapparent infections, to embryonic
death, abortions, stillbirths, and malformed or slow-growing
calves. Although recent studies indicate that transient infection
of the fetus may cause long-term detrimental effects in the
development of the calf, the basis for these effects is not clearly
understood.
[0118] Acute infection with the nep biotype results in inhibition
of double-stranded RNA-induced apoptosis and type 1 interferon,
both indicated as plausible contributors to the establishment of
persistent infection. However, in vitro and in vivo infections with
the cp biotype induce apoptosis and are associated with increased
type I interferon production.
[0119] The role of PI animals in the perpetuation of BVDV
infections cannot be overemphasized. The presence of a single PI
calf in a herd can cause severe losses within that herd and any
herd with which the PI calf makes contact. The large percentage of
seropositive cattle due to vaccination in the USA diminishes the
value of serology as a monitoring test. In addition, PI animals
have absent, extremely low or fluctuating titers against the strain
that causes the persistent infection, potentially leading to
misdiagnosis, particularly when serology is used as the only test.
Current diagnostic tests based on the detection of viral antigen or
viral RNA are effective tools for the identification of postnatal
detection of PI animals. However, since PI animals shed high
amounts of virus, early elimination of PI fetuses should greatly
contribute to disrupting the infectious viral cycle.
[0120] In other preliminary studies, we demonstrated that calves
persistently infected with BVDV present a differential pattern of
gene expression in the blood when compared to vaccinated or acutely
infected age-matched herd mates. We investigated the gene
expression profiles in the whole blood of one year old PI and
non-infected calves that were naturally infected with BVDV using
DNA microarray approaches.
[0121] Blood samples were collected from three BVDV PI and three
vaccinated, non-infected control calves in sodium citrate tubes and
placed on ice. RNA was isolated using the QIAamp RNA Blood Mini Kit
following the manufacturer's instructions (Qiagen). Red blood cells
were selectively lysed, and white cells were collected by
centrifugation. White cells were then lysed using highly denaturing
conditions, which immediately inactivate RNases. After
homogenization using the QIAshredder spin column, the sample was
applied to the QIAamp spin column. Total RNA binds to the QIAamp
membrane and contaminants were washed away, leaving pure RNA which
was eluted in 30-100 .mu.l RNase-free water.
[0122] The total RNA was isolated and used for transcriptional
profiling by screening the Affymetrix bovine DNA chip. Biotinylated
cRNA (15 .mu.g), generated from each RNA sample (n=6 total), was
hybridized to the bovine Affymetrix GeneChip (features of which are
shown below) Array (n=6 total). Data were analyzed using Affymetrix
Microarray Suite Software version 5.0 for absolute and pair-wise
comparison analyses. Normalized expression values for the mean and
standard deviation of three replicate average difference scores
were calculated for each gene. Comparisons were performed using the
Student's t test (P<0.05 was considered significant). The raw
data were interpreted using GeneSpring (version 5.0, Silicon
Genetics, Redwood, Calif.) and GeneSifter software (vizX Labs, LLC,
Seattle, Wash.) TABLE-US-00001 Critical Specifications Bos taurus
(Bovine) probe sets 24,072 Bos taurus (Bovine) transcripts
approximately 23,000 UniGene clusters approximately 19,000 Unique
probe sets to single species: Number of arrays in set one Array
format 100 Feature size 11 .mu.m Oligonucleotide probe length
25-mer Probe pairs/sequence 11 Hybridization controls: bioB, bioC,
bioD, from E. coli and cre from P1 B. subtilis Poly-A controls:
dap, lys, phe, thr, trp from B. subtilis Housekeeping/Control
genes: actin, GAPDH, efl.alpha., 5.8S rRNA, 12S rRNA, 18S rRNA,
cyclophilin B, glutathione S-transferase, lactophorin, translation
initiation factor elF-4E Detection sensitivity 1:100,000.sup.1
.sup.1As measured by detection in comparative analysis between a
complex target containing spiked control transcriptions and a
complex target with no spikes
Results
[0123] Two hundred genes were up-regulated in blood from PI when
compared to vaccinated control calves. Known attributes of the top
100 up-regulated genes and fold changes are listed below.
TABLE-US-00002 45 fold MHC Class I Molecules 10-32 fold Antiviral
genes 8-12 fold Signal Transduction Molecules 5-28 fold Type 1
Interferon-Induced Genes (FIG. 2) 4-9 fold Bone Remodeling Genes
3-6 fold Cytoskeletal Remodeling Genes 2-4 fold Chemokine Ligands
and Receptors
[0124] One hundred genes were down-regulated in blood from PI when
compared to control calves. Known attributes of the top 100
down-regulated genes and fold changes are listed below.
TABLE-US-00003 5-10 fold Adhesion Molecules 5-10 fold T Cell
Receptors 3-5 fold Extracellular Matrix 2-3 fold Growth Factors 2-3
fold Chemokine Ligands 2-3 fold Transcription Factors
See FIG. 2.
[0125] Following more extensive and stringent analysis of
microarray data described above, the following up-regulated (Table
1) and down-regulated (Table 2) genes were identified as preferred
bovine blood markers for BVDV persistent infection (n =2 steers)
when compared with controls (n=3 steers). MX2, 2'-5' oligoadenylate
synthetase (OAS) and Interferon-Stimulated Protein, 15 kDa serve as
examples of good markers for cattle with a BVDV PI infection when
compared to non-infected controls. See FIG. 3.
[0126] As can be seen by the data presented herein, persistent
infection with BVDV causes up-regulation and down-regulation of
genes in blood cells. Persistent infection with BVDV results in
antiviral responses in blood cells. The results show induction of
interferon-induced genes, chemokine-mediated immune responses and
bone remodeling genes. Suppression of extracellular remodeling,
adhesion and T-cell-mediated responses is also observed. One gene,
called interferon stimulated gene 15 or ISG15 was confirmed to be
up-regulated in bloods from PI when compared to control vaccinated
steers using blood cell mRNA and Real Time PCR approaches (FIGS. 2
and 3).
EXAMPLE 2
[0127] We hypothesized that gene expression in white blood cells
would differ in pregnant heifers carrying PI, TI or uninfected
(control) fetuses. Non-vaccinated heifers were purchased, confirmed
to be seronegative for BVDV and were placed on growing rations
until they were old enough to be artificially inseminated and
confirmed to be pregnant. Heifers were infected with noncytopathic
BVDV2 on day 75 to generate PI fetuses, on day 175 to generate TI
fetuses, or were not infected (n=6 heifers per treatment). Bloods
were collected on days 0, 37, 75, 78, 82, 90, 120, 160, 175, 178,
182 and 190 of gestation for RNA, serology and virology. Fetuses
were delivered on d. 190 by C-section and necropsied. Maternal
blood mRNA on day 190 of gestation was screened using the bovine
Affymetrix gene chips.
[0128] BVDV infection in heifers and fetuses was confirmed using
ELISA and qRT-PCR. Infected pregnant heifers were seropositive for
BVDV by days 15-45 post infection. PI fetuses weighed less and were
smaller with maldeveloped bone and muscle tissue (P<0.05) when
compared to TI or UI fetuses. Screening of 24,000 transcripts on
the bovine Affymetrix DNA chip using mRNA from blood cells of
heifers on day 190 of pregnancy revealed 67 differentially
expressed genes (1.5 fold or greater; P<0.05) based on infection
status of the fetus: 32 genes in PI vs. TI, 26 genes in PI vs.
control and 47 genes in TI vs. control. These genes were classified
based on ontology analysis in primary categories of immune
response, antigen presentation, inflammatory response, chemotaxis,
protein folding and modification, transport, and defense response
to bacteria. Specific genes that are differentially expressed are
described in Tables 3-9. TABLE-US-00004 TABLE 9 Analysis of blood
gene expression using a three-way ANOVA in heifers carrying control
vs. TI vs. PI fetuses. Analysis represents the top 24 genes that
were differentially expressed in at least one of the treatment
groups. The value in the ANOVA column represents the P value when
determining if one of the three means differs. Please refer to the
heat plot in FIG. 4 for an illustration of these data. Control TI
PI Control PI TI Gene ANOVA Mean Mean Mean SEM 1 SEM 2 SEM 3
Identifier Gene Title 0.006032 8.00309 10.9193 8.96783 0.188264
0.138741 0.660686 CK960499 2'-5'-oligoadenylate synthetase 1 (OAS1)
0.003154 10.115 11.9521 10.7336 0.256113 0.176554 0.231053
NM_174366 interferon-stimulated protein. 15 kDa 0.003028 6.90223
8.43096 7.26529 0.101244 0.219103 0.222812 CK955157 XP_513514.1
similar to Interferon-induced protein 44 (Antigen p44) 0.003101
8.16371 9.68817 8.57334 0.191589 0.15614 0.212549 CB460780
XP_513514.1 similar to Interferon-induced protein 44 (Antigen p44)
0.003419 6.74951 8.13573 7.04487 0.104431 0.170809 0.23341 CK777675
XP_513514.1 similar to Interferon-induced protein 44 (Antigen p44)
0.001684 7.22418 8.76654 7.80757 0.137263 0.093874 0.232921
CB433489 pir: S48218 (H. sapiens) S48218 microtubular aggregate
protein - hu 0.008299 8.21365 9.31672 8.57504 0.110353 0.233574
0.116391 CB432365 XP_520524.1 similar to DEAD/H
(Asp-Glu-Ala-Asp/His) box polype 0.00315 8.16901 9.22019 9.12652
0.048718 0.109524 0.209197 CB445920 Transcribed sequences 0.002321
9.87333 9.15127 9.88773 0.12978 0.095443 0.033274 CK953227
XP_518477.1 similar to beta-tubulin cofactor C [Pan troglodytes]
0.009597 5.57083 5.66441 6.21629 0.161879 0.077099 0.026107
CK971667 XP_524698.1 similar to TAL1 (SCL) interrupting locus; SCL
interrup 0.000202 8.26679 7.64432 8.09498 0.036558 0.065628
0.028333 BP103941 XP_521146.1 similar to ATRX [Pan troglodytes]
0.005323 6.91205 6.14205 7.06362 0.125015 0.061868 0.179441
CB534327 Component 3 0.003199 5.25886 5.54151 5.89927 0.099747
0.072327 0.051183 CB443429 NP_001804.1 centromere protein E, 312
kDa [Homo sapiens] 0.00661 7.76552 7.11499 8.06201 0.028002
0.114012 0.201076 CB460964 ATP-binding cassette transporter
subfamily B, member 1 (ABCB1) 0.006969 5.88815 6.50156 6.25403
0.108627 0.090517 0.04993 CK770915 XP_218427.2 similar to
poliovirus receptor homolog [Rattus norvegi 0.002739 4.50737
4.44439 5.47889 0.218718 0.048776 0.067073 CB461169 Transcribed
sequences 0.003618 7.02454 6.11085 7.19863 0.035911 0.234515
0.075039 M37974 osteoglycin (osteoinductive factor, mimecan)
0.007258 7.29919 7.80462 7.90756 0.145198 0.007311 0.065739
CK950633 similar to NP_612565.1 kinesin family member 23 [Homo
sapiens] 0.006348 9.11153 10.0777 9.28636 0.01768 0.189202 0.15531
CK848208 similar to XP_524747.1 similar to histocompatibility 28
[Pan troglod 0.000656 4.52089 5.16352 5.09881 0.106526 0.017049
0.016545 CB168658 Transcribed locus 0.0004 5.60571 6.28826 6.3913
0.053773 0.063446 0.087055 CB463330 Succinate semialdehyde
dehydrogenase (NAD(+)-dependent succir 0.002006 4.94446 3.99497
3.79603 0.173649 0.094759 0.123417 CB534503 Transcribed locus,
weakly similar to XP_519213.1 paraoxonase 2 [ 0.009713 10.8281
9.92825 9.33185 0.253205 0.201907 0.221374 NM_175827 chemokine
(C--C motif) ligand 5 0.00064 11.0449 7.57803 10.8921 0.258042
0.509224 0.189688 AB008573 MHC class I heavy chain, partial cds,
clone P5647.6m
[0129] Early detection of persistently infected calves is the best
method of controlling and eradicating BVDV from infected herds.
Current diagnostic tests for BVDV include skin biopsies and
serology which do not distinguish acutely infected animals from
persistently animals. These PI animals go undetected and continue
to propagate virus within the herd.
[0130] Current methods of detection also have a high rate of false
negatives due to the fact that they are based on mutating viral
antigens. Most importantly, current tests cannot detect a pregnant
cow/heifer which is carrying a persistently infected calf. The
present invention describes a method whereby BVDV can be detected
in acutely infected calves, persistently infected calves as well as
cows or heifers carrying an infected fetus through the
identification of differentially expressed surrogate markers
described in Tables 1-9. Surrogate markers can exist as mRNA or
protein antigens and allow differentiation of type of BVDV
infection. By the methods listed previously persistently infected
(PI) calves can be differentiated from acutely infected calves and
acutely infected calves can be distinguished from non-infected
calves. Heifers and cows carrying PI fetuses can also be
distinguished from heifers/cows carrying transiently infected
fetuses and cows/heifers carrying PI or TI fetuses can be
distinguished from cows/heifers carrying non-infected (normal)
calves. The same markers can be used to create therapeutics for
treating secondary effects of viral disease and monitoring the
anti-viral response in infected cattle. The global approach
presented herein will aid the prevention, diagnosis, control and
treatment of cattle infected with BVDV as well as other RNA
viruses.
[0131] While certain of the preferred embodiments of the present
invention have been described and specifically exemplified above,
it is not intended that the invention be limited to such
embodiments. Various modifications may be made thereto without
departing from the scope and spirit of the present invention, as
set forth in the following claims.
* * * * *