U.S. patent application number 10/029840 was filed with the patent office on 2003-03-27 for avian hepatitis e virus, vaccines and methods of protecting against avian hepatitis-splenomegaly syndrome and mammalian hepatitis e.
This patent application is currently assigned to Virginia Tech Intellectual Properties, Inc.. Invention is credited to Haqshenas, Gholamreza, Huang, Fang-Fang, Meng, Xiang-Jin.
Application Number | 20030059873 10/029840 |
Document ID | / |
Family ID | 26705396 |
Filed Date | 2003-03-27 |
United States Patent
Application |
20030059873 |
Kind Code |
A1 |
Meng, Xiang-Jin ; et
al. |
March 27, 2003 |
Avian hepatitis E virus, vaccines and methods of protecting against
avian hepatitis-splenomegaly syndrome and mammalian hepatitis E
Abstract
The present invention relates to a novel isolated avian
hepatitis E virus having a nucleotide sequence set forth in SEQ ID
NO: 1 or its complementary strand. The invention further concerns
immunogenic compositions comprising this new virus or a recombinant
products such as the nucleic acid and vaccines that protect an
avian or mammalian species from viral infection or
hepatitis-splenomegaly syndrome caused by the hepatitis E virus.
Also included in the scope of the invention is a method for
propagating, inactivating or attenuating a hepatitis E virus
comprising inoculating an embryonated chicken egg with a live,
pathogenic hepatitis E virus and recovering the virus or serially
passing the pathogenic virus through additional embryonated chicken
eggs until the virus is rendered inactivated or attenuated.
Further, this invention concerns diagnostic reagents for detecting
an avian hepatitis E viral infection or diagnosing
hepatitis-splenomegaly syndrome in an avian or mammalian species
comprising an antibody raised or produced against the immunogenic
compositions and antigens such as ORF2 proteins expressed in a
baculovirus vector, E. coli, etc. The invention additionally
encompasses methods for detecting avian HEV nucleic acid sequences
using nucleic acid hybridization probes or oligonucleotide primers
for polymerase chain reaction (PCR).
Inventors: |
Meng, Xiang-Jin;
(Blacksburg, VA) ; Haqshenas, Gholamreza; (Tehran,
IR) ; Huang, Fang-Fang; (Blacksburg, VA) |
Correspondence
Address: |
Anne M. Rosenblum, Esq.
Suite 212
163 Delaware Avenue
Delmar
NY
12054
US
|
Assignee: |
Virginia Tech Intellectual
Properties, Inc.
Blacksburg
VA
|
Family ID: |
26705396 |
Appl. No.: |
10/029840 |
Filed: |
December 31, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60259846 |
Jan 5, 2001 |
|
|
|
Current U.S.
Class: |
435/69.1 ;
424/204.1; 424/225.1; 435/91.33; 514/44R |
Current CPC
Class: |
C07K 14/005 20130101;
C12Q 1/707 20130101; C12N 2770/28034 20130101; C12N 2770/28022
20130101; C12N 7/00 20130101; G01N 33/576 20130101; A61K 2039/5254
20130101; A61K 2039/54 20130101 |
Class at
Publication: |
435/69.1 ;
514/44; 435/91.33; 424/225.1; 424/204.1 |
International
Class: |
A61K 031/70; A01N
043/04; C12P 021/06; C12P 019/34; A61K 039/12; A61K 039/29 |
Goverment Interests
[0002] The project resulting in the present invention has been
supported in part by grants from the National Institutes of Health
(AI01653-01, AI46505-01).
Claims
What is claimed is:
1. An isolated avian hepatitis E virus having a nucleotide sequence
set forth in SEQ ID NO: 1 or its complementary strand.
2. The avian hepatitis E virus according to claim 1, wherein said
virus has no more than about 80% nucleotide sequence identity to an
Australian big liver and spleen disease virus.
3. An isolated polynucleotide comprising a member selected from the
group consisting of (a) a nucleotide sequence set forth in SEQ ID
NO: 1 or its complementary strand; (b) a polynucleotide which
hybridizes to and which is at least 95% complementary to the
nucleotide sequence set forth in SEQ ID NO: 1; and (c) an
immunogenic fragment selected from the group consisting of a
nucleotide sequence in the partial helicase gene of ORF1 set forth
in SEQ ID NO: 3, a nucleotide sequence in the RdRp gene set forth
in SEQ ID NO: 5, a nucleotide sequence in the ORF2 gene set forth
in SEQ ID NO: 7, a nucleotide sequence in the ORF3 gene set forth
in SEQ ID NO: 9 and their complementary strands.
4. A purified, immunogenic protein encoded by the isolated
polynucleotide according to claim 3.
5. The protein according to claim 4, wherein the protein comprises
an ORF2 capsid protein or an ORF3 protein.
6. An immunogenic composition comprising a nontoxic,
physiologically acceptable carrier and an isolated avian hepatitis
E virus having a nucleotide sequence set forth in SEQ ID NO: 1, its
complementary strand, an isolated polynucleotide according to claim
3 or an antigenic protein encoded by the isolated
polynucleotide.
7. A diagnostic reagent for detecting an avian hepatitis E viral
infection or diagnosing hepatitis-splenomegaly syndrome in an avian
or mammalian species comprising an antibody raised or produced
against the immunogenic composition according to claim 6.
8. A vaccine that protects an avian or mammalian species from viral
infection or hepatitis-splenomegaly syndrome caused by an avian or
mammalian hepatitis E virus comprising a nontoxic, physiologically
acceptable carrier and a member selected from the group consisting
of: (a) a modified live avian hepatitis E virus, having a
nucleotide sequence set forth in SEQ ID NO: 1 or its complementary
strand; (b) an inactivated avian hepatitis E virus, having a
nucleotide sequence set forth in SEQ ID NO: 1 or its complementary
strand; (c) an attenuated avian hepatitis E virus, having a
nucleotide sequence set forth in SEQ ID NO: 1 or its complementary
strand; (d) an antigenic subunit of avian hepatitis E virus, having
a nucleotide sequence set forth in SEQ ID NO: 1 or its
complementary strand; and (e) a purified, immunogenic protein
encoded by an isolated polynucleotide comprising a nucleotide
sequence set forth in SEQ ID NO: 1 or its complementary strand; a
polynucleotide which hybridizes to and which is at least 95%
complementary to the nucleotide sequence set forth in SEQ ID NO: 1;
or an immunogenic fragment selected from the group consisting of a
nucleotide sequence in the partial helicase gene of ORF1 set forth
in SEQ ID NO: 3, a nucleotide sequence in the RdRp gene set forth
in SEQ ID NO: 5, a nucleotide sequence in the ORF2 gene set forth
in SEQ ID NO: 7, a nucleotide sequence in the ORF3 gene set forth
in SEQ ID NO: 9 and their complementary strands.
9. The vaccine according to claim 8, wherein said virus is
inactivated or attenuated by serial passage of the virus through
embryonated chicken eggs.
10. The vaccine according to claim 8, wherein said vaccine further
contains an adjuvant.
11. A method of protecting an avian or mammalian species from viral
infection or hepatitis-splenomegaly syndrome caused by the avian or
mammalian hepatitis E virus comprising administering an
immunologically effective amount of the vaccine according to claim
8 to an avian or mammalian species in need of protection against
said infection or syndrome.
12. The method according to claim 11, wherein the vaccine is
administered to a chicken, a pig or a human.
13. The method according to claim 11, wherein the vaccine is
administered orally, intrabuccally, intranasally, transdermally or
parenterally.
14. A method for propagating, inactivating or attenuating a
hepatitis E virus comprising inoculating an embryonated chicken egg
with a live, pathogenic hepatitis E virus and recovering a live,
pathogenic hepatitis E virus or serially passing the pathogenic
virus through additional embryonated chicken eggs until said virus
is rendered inactivated or attenuated.
15. The method according to claim 14, wherein the live, pathogenic
hepatitis E virus is injected intravenously into the embryonated
chicken egg.
16. The method according to claim 14, wherein said hepatitis E
virus is an avian hepatitis E virus having a nucleotide sequence
set forth in SEQ ID NO: 1 or its complementary strand.
17. A method for detecting an avian hepatitis E viral infection or
diagnosing hepatitis-splenomegaly syndrome in an avian or mammalian
species comprising contacting a biological sample of the avian or
mammalian species with the diagnostic reagent according to claim 7
and detecting or observing the presence of an antigen-antibody
complex.
18. The method according to claim 17, wherein the biological sample
is taken from a chicken, a pig or a human.
19. A method for detecting an avian hepatitis E viral nucleic acid
sequence in an avian or mammalian species comprising isolating
nucleic acid from the avian or mammalian species, hybridizing the
nucleic acid and determining the presence or absence of a
hybridized probe complex.
20. The method according to claim 19, wherein the nucleic acid is
hybridized with a radio-labeled or a non-radiolabeled nucleic acid
probe derived from the nucleotide sequence set forth in SEQ ID NO:
1 or hybridized with a pair of oligonucleotide primers derived from
the nucleotide sequence set forth in SEQ ID NO: 1 and further
amplified in a polymerase chain reaction.
Description
CROSS-REFERENCE TO RELATED U.S. APPLICATIONS
[0001] This application claims the benefit under 35 U.S.C. .sctn.
119 (e) of U.S. Provisional Application No. 60/259,846, filed Jan.
5, 2001.
REFERENCE TO A "Sequence Listing"
[0003] The material on a single compact disc containing a Sequence
Listing file provided in this application is incorporated by
reference. The date of creation is ______,2002 and the size is
______ bytes.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] The present invention concerns a novel avian hepatitis E
virus, immunogenic compositions, diagnostic reagents, vaccines and
methods of detecting or protecting against avian
hepatitis-splenomegaly syndrome and mammalian hepatitis E.
[0006] 2. Description of the Related Art
[0007] Human hepatitis E is an important public health disease in
many developing countries, and is also endemic in some
industrialized countries. Hepatitis E virus (hereinafter referred
to as "HEV"), the causative agent of human hepatitis E, is a single
positive-stranded RNA virus without an envelope (R. H. Purcell,
"Hepatitis E virus," FIELDS VIROLOGY, Vol. 2, pp. 2831-2843, B. N.
Fields et al. eds, Lippincott-Raven Publishers, Philadelphia (3d
ed. 1996)). The main route of transmission is fecal-oral, and the
disease reportedly has a high mortality rate, up to 20%, in
infected pregnant women. The existence of a population of
individuals who are positive for HEV antibodies (anti-HEV) in
industrialized countries and the recent identification of numerous
genetically distinct strains of HEV have led to a hypothesis that
an animal reservoir for HEV exists (X. J. Meng, "Zoonotic and
xenozoonotic risks of hepatitis E virus," Infect. Dis. Rev. 2:35-41
(2000); X. J. Meng, "Novel strains of hepatitis E virus identified
from humans and other animal species: Is hepatitis E a zoonosis?"
J. Hepatol. 33:842-845 (2000)). In 1997, the first animal strain of
HEV, swine hepatitis E virus (hereinafter referred to as "swine
HEV"), was identified and characterized from a pig in the U.S. (X.
J. Meng et al., "A novel virus in swine is closely related to the
human hepatitis E virus," Proc. Natl. Acad. Sci. USA 94:9860-9865
(1997)). Swine HEV was shown to be very closely related genetically
to human HEV. Interspecies transmission of HEV has been documented:
swine HEV infects non-human primates and a U.S. strain of human HEV
infects pigs. These data lend further credence to the hypothesis of
an animal reservoir for HEV.
[0008] Numerous genetically distinct strains of HEV have been
identified from patients with acute hepatitis in both developing
and industrialized countries. The two U.S. strains of human HEV
recently identified from hepatitis E patients (US-1 and US-2) are
genetically distinct from other known HEV strains worldwide but are
closely related to each other and to the U.S. strain of swine HEV
(J. C. Erker et al., "A hepatitis E virus variant from the United
States: molecular characterization and transmission in cynomolgus
macaques," J. Gen. Virol. 80:681-690 (1999); X. J. Meng et al.,
"Genetic and experimental evidence for cross-species infection by
the swine hepatitis E virus," J. Virol. 72:9714-9721 (1998); G. G.
Schlauder et al., "The sequence and phylogenetic analysis of a
novel hepatitis E virus isolated from a patient with acute
hepatitis reported in the United States," J. Gen. Virol. 79:447-456
(1998)). Similarly, several isolates of HEV have been identified
from patients in Taiwan with no history of travel to endemic
region. An Italian strain of human HEV was found to share only
about 79.5 to 85.8% nucleotide sequence identity with other known
strains of HEV. Schlauder et al. recently identified another
Italian and two Greek strains of HEV (G. G. Schlauder et al.,
"Novel hepatitis E virus (HEV) isolates from Europe: evidence for
additional genotypes of HEV," J. Med. Virol. 57:243-51 (1999)). The
sequences of the Greek and Italian strains of HEV differed
significantly from other known strains of HEV. In endemic regions,
strains of HEV, which are distinct from the previously known
epidemic strains, have also been identified in Pakistan (H. Van
Cuyck-Gandre et al., "Short report: phylogenetically distinct
hepatitis E viruses in Pakistan," Am. J. Trop. Med. Hyg. 62:187-189
(2000)), Nigeria (Y. Buisson et al., "Identification of a novel
hepatitis E virus in Nigeria," J. Gen. Virol. 81:903-909 (2000))
and China (Y. Wang et al., "A divergent genotype of hepatitis E
virus in Chinese patients with acute hepatitis," J. Gen. Virol.
80:169-77 (1999); Y. Wang et al., "The complete sequence of
hepatitis E virus genotype 4 reveals an alternative strategy for
translation of open reading frames 2 and 3," J. Gen. Virol.
81:1675-1686 (2000)). Six isolates of HEV were identified from
Chinese hepatitis E patients that were negative for anti-HEV
assayed by the serological test used (Y. Wang et al., 1999, supra).
The intriguing fact is that these recently identified strains of
HEV are genetically distinct from each other and from other known
strains of HEV. Although the source of these human HEV strains is
not clear, it is plausible that they may be of animal origins.
[0009] Recently, several U.S. patents have issued which concern the
human hepatitis E virus. U.S. Pat. No. 6,022,685 describes methods
and compositions for detecting anti-hepatitis E virus activity via
antigenic peptides and polypeptides. U.S. Pat. No. 5,885,768
discloses immunogenic peptides which are derived from the ORF1,
ORF2 and ORF3 regions of hepatitis E virus, diagnostic reagents
containing the peptide antigens, vaccines and immunoreactive
antibodies. U.S. Pat. No. 5,770,689 relates to certain ORF Z
peptides of the human HEV genome. U.S. Pat. No. 5,741,490 deals
with a vaccine and vaccination method for preventing hepatitis E
viral infections. U.S. Pat. No. 5,686,239 provides a method of
detecting HEV antibodies in an individual using a peptide antigen
obtained from the human HEV sequence.
[0010] Evidence of HEV infection of domestic and farm animals has
been well documented (X. J. Meng, "Zoonotic and xenozoonotic risks
of hepatitis E virus," Infect. Dis. Rev. 2:35-41 (2000); X. J.
Meng, "Novel strains of hepatitis E virus identified from humans
and other animal species: Is hepatitis E a zoonosis?" J. Hepatol.,
33:842-845 (2000); R. H. Purcell, "Hepatitis E virus," FIELDS
VIROLOGY, Vol. 2, pp. 2831-2843, B. N. Fields et al. eds,
Lippincott-Raven Publishers, Philadelphia (3d ed. 1996)). Anti-HEV
was detected in pigs from developing countries such as Nepal (E. T.
Clayson et al., "Detection of hepatitis E virus infections among
domestic swine in the Kathmandu Valley of Nepal," Am. J. Trop. Med.
Hyg. 53:228-232 (1995)), China (X. J. Meng et al., "Prevalence of
antibodies to the hepatitis E virus in pigs from countries where
hepatitis E is common or is rare in the human population," J. Med.
Virol. 58:297-302 (1999)) and Thailand (id), and from
industrialized countries such as U.S. (X. J. Meng et al., "A novel
virus in swine is closely related to the human hepatitis E virus,"
Proc. Natl. Acad. Sci. USA 94:9860-9865 (1997)), Canada (X. J. Meng
et al., 1999, supra), Korea (X. J. Meng et al., 1999, id), Taiwan
(S. Y. Hsieh et al., "Identity of a novel swine hepatitis E virus
in Taiwan forming a monophyletic group with Taiwan isolates of
human hepatitis E virus," J. Clin. Microbiol. 37:3828-3834 (1999)),
Spain (S. Pina et al., "HEV identified in serum from humans with
acute hepatitis and in sewage of animal origin in Spain," J.
Hepatol. 33:826-833 (2000)) and Australia (J. D. Chandler et al.,
"Serological evidence for swine hepatitis E virus infection in
Australian pig herds," Vet. Microbiol. 68:95-105 (1999)). In
addition to pigs, Kabrane-Lazizi et al. reported that about 77% of
the rats from Maryland, 90% from Hawaii and 44% from Louisiana are
positive for anti-HEV (Y. Kabrane-Lazizi et al., "Evidence for
wide-spread infection of wild rats with hepatitis E virus in the
United States," Am. J. Trop. Med. Hyg. 61:331-335 (1999)). Favorov
et al. also reported the detection of IgG anti-HEV among rodents in
the U.S. (M. O. Favorov et al., "Prevalence of antibody to
hepatitis E virus among rodents in the United States," J. Infect.
Dis. 181:449-455 (2000)). In Vietnam where HEV is endemic, anti-HEV
was reportedly detected in 44% of chickens, 36% of pigs, 27% of
dogs and 9% of rats (N. T. Tien et al., "Detection of
immunoglobulin G to the hepatitis E virus among several animal
species in Vietnam," Am. J. Trop. Med. Hyg. 57:211 (1997)). About
29 to 62% of cows from Somali, Tajikistan and Turkmenistan (HEV
endemic regions), and about 42 to 67% of the sheep and goats from
Turkmenistan and 12% of cows from Ukraine (a non-endemic region)
are positive for anti-HEV (M. O. Favorov et al., "Is hepatitis E an
emerging zoonotic disease?" Am. J. Trop. Med. Hyg. 59:242 (1998)).
Naturally acquired anti-HEV has also been reported in rhesus
monkeys (S. A. Tsarev et al, "Experimental hepatitis E in pregnant
rhesus monkeys: failure to transmit hepatitis E virus (HEV) to
offspring and evidence of naturally acquired antibodies to HEV," J.
Infect. Dis. 172:31 -37 (1995)). These serological data strongly
suggest that these animal species are infected with HEV or a
related agent. Until recently, the source of seropositivity in
these animals could not be definitively demonstrated since the
virus was either not recovered from these animal species or the
recovered virus was not genetically characterized to confirm its
identity. The first and only animal strain of HEV that has been
identified and extensively characterized thus far is swine HEV (X.
J. Meng et al., "A novel virus in swine is closely related to the
human hepatitis E virus," Proc. Natl. Acad. Sci. USA 94:9860-9865
(1997); X. J. Meng et al., "Experimental infection of pigs with the
newly identified swine hepatitis E virus (swine HEV), but not with
human strains of HEV," Arch. Virol. 143:1405-1415 (1998); X. J.
Meng et al., "Genetic and experimental evidence for cross-species
infection by the swine hepatitis E virus," J. Virol. 72:9714-9721
(1998); X. J. Meng et al., "Prevalence of antibodies to the
hepatitis E virus in pigs from countries where hepatitis E is
common or is rare in the human population," J. Med. Virol.
58:297-302 (1999)). However, because swine HEV causes only
subclinical infection and mild microscopic liver lesions in pigs,
it does not provide a good, adaptable animal model to study human
HEV replication and pathogenesis.
[0011] Since the identification and characterization of the first
animal strain of HEV (swine HEV) in the U.S. in 1997, several other
HEV strains of animal origins were genetically identified. Hsieh et
al. identified a second strain of swine HEV from a pig in Taiwan
(S. Y. Hsieh et al., "Identity of a novel swine hepatitis E virus
in Taiwan forming a monophyletic group with Taiwan isolates of
human hepatitis E virus," J. Clin. Microbiol. 37:3828-3834 (1999)).
This Taiwanese strain of swine HEV shared 97.3% nucleotide sequence
identity with a human strain of HEV identified from a retired
Taiwanese farmer but is genetically distinct from other known
strains of HEV including the U.S. strain of swine HEV. Recently,
Pina et al. identified a strain of HEV (E11 strain) from sewage
samples of animal origin from a slaughterhouse that primarily
processed pigs in Spain (S. Pina et al., "HEV identified in serum
from humans with acute hepatitis and in sewage of animal origin in
Spain," J. Hepatol. 33:826-833 (2000)). The E11 strain of possible
animal origin is most closely related to two Spanish strains of
human HEV, and is more closely related to the U.S. swine and human
strains compared to other HEV strains worldwide (id.). In addition
to pigs, a strain of HEV was reportedly identified from tissue and
fecal samples of wild-trapped rodents from Kathmandu Valley, Nepal
(S. A. Tsarev et al., "Naturally acquired hepatitis E virus (HEV)
infection in Nepalese rodents," Am. J. Trop. Med. Hyg. 59:242
(1998)). Sequence analyses revealed that the HEV sequence recovered
from Nepalese rodents is most closely related to the HEV isolates
from patients in Nepal (id.).
[0012] Hepatitis-splenomegaly syndrome (hereinafter referred to as
"HS syndrome") is an emerging disease in chickens in North America.
HS syndrome in chickens was first described in 1991 in western
Canada, and the disease has since been recognized in eastern Canada
and the U.S. HS syndrome is characterized by increased mortality in
broiler breeder hens and laying hens of 30-72 weeks of age. The
highest incidence usually occurs in birds between 40 to 50 weeks of
age, and the weekly mortality rate can exceed 1%. Prior to sudden
death, diseased chickens usually are clinically normal, with pale
combs and wattles although some birds are in poor condition. In
some outbreaks, up to 20% drop in egg production was observed.
Affected chickens usually show regressive ovaries, red fluid in the
abdomen, and enlarged liver and spleen. The enlarged livers are
mottled and stippled with red, yellow and tan foci. Similar to the
microscopic lesions found in the livers of humans infected with
HEV, microscopic lesions in the livers of chickens with HS syndrome
vary from multifocal to extensive hepatic necrosis and hemorrhage,
with infiltration of mononuclear cells around portal triads.
Microscopic lesions in the spleen include lymphoid depletion and
accumulation of eosinophilic materials. Numerous other names have
been used to describe the disease such as necrotic hemorrhage
hepatitis-splenomegaly syndrome, chronic fulminating
cholangiohepatitis, necrotic hemorrhagic hepatomegalic hepatitis
and hepatitis-liver hemorrhage syndrome.
[0013] The cause of HS syndrome is not known. A viral etiology for
HS syndrome has been suspected but attempts to propagate the virus
in cell culture or embryonated eggs were unsuccessful (J. S.
Jeffrey et al., "Investigation of hemorrhagic hepatosplenomegaly
syndrome in broiler breeder hens," Proc. Western Poult. Dis. Conf.,
p. 46-48, Sacramento, Calif. (1998); H. L. Shivaprasad et al.,
"Necrohemorrhagic hepatitis in broiler breeders," Proc. Western
Poult. Dis. Conf, p. 6, Sacramento, Calif. (1995)). The
pathological lesions of HS in chickens, characterized by hepatic
necrosis and hemorrhage, are somewhat similar to those observed in
humans infected with HEV (R. H. Purcell, "Hepatitis E virus,"
FIELDS VIROLOGY, Vol. 2, pp. 2831-2843, B. N. Fields et al. eds,
Lippincott-Raven Publishers, Philadelphia (3d ed. 1996); C.
Riddell, "Hepatitis-splenomegaly syndrome," DISEASE OF POULTRY, p.
1041 (1997)). Since anti-HEV was detected in 44% of chickens in
Vietnam (N. T. Tien et al., "Detection of immunoglobulin G to the
hepatitis E virus among several animal species in Vietnam," Am. J.
Trop. Med. Hyg. 57:211 (1997)), suggesting that chickens have been
infected by HEV (or a related agent), it would be advantageous to
find a link between HEV infection and HS syndrome in chickens. The
link would permit the development of diagnostic assays and vaccines
to protect against both human and chicken HEV infections thereby
providing substantial public health and veterinary benefits. These
goals and other desirable objectives are met by the isolation,
genetic identification and characterization of the novel avian
hepatitis E virus as described herein.
BRIEF SUMMARY OF THE INVENTION
[0014] The present invention concerns a novel avian hepatitis E
virus, immunogenic compositions, vaccines which protect avian and
mammalian species from viral infection or hepatitis-splenomegaly
syndrome and methods of administering the vaccines to the avian and
mammalian species to protect against viral infection or
hepatitis-splenomegaly syndrome. The invention encompasses vaccines
which are based on avian hepatitis E virus to protect against human
hepatitis E. This invention includes methods for propagating,
inactivating or attenuating hepatitis E viruses which uniquely
utilize the inoculation of the live, pathogenic virus in
embryonated chicken eggs. Other aspects of the present invention
involve diagnostic reagents and methods for detecting the viral
causative agent and diagnosing hepatitis E in a mammal or
hepatitis-splenomegaly syndrome in an avian species which employ
the nucleotide sequence described herein, antibodies raised or
produced against the immunogenic compositions or antigens (such as
ORF2, ORF3, etc.) expressed in a baculovirus vector, E. coli and
the like. The invention further embraces methods for detecting
avian HEV nucleic acid sequences in an avian or mammalian species
using nucleic acid hybridization probes or oligonucleotide primers
for polymerase chain reaction (PCR).
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] The background of the invention and its departure from the
art will be further described hereinbelow with reference to the
accompanying drawings, wherein:
[0016] FIG. 1 shows the amplification of the 3' half of the avian
HEV genome by RT-PCR: Lane M, 1 kb ladder; Lanes 1 and 2, PCR with
ampliTaq gold polymerase; Lanes 3 and 4, PCR with ampliTaq gold
polymerase in the presence of 5% v/v dimethyl sulfoxide
(hereinafter referred to as "DMSO"); Lane 5 and 6, PCR
amplification with a mixture of Taq polymerase and pfu containing
in an eLONGase.RTM. Kit (GIBCO-BRL, Gaithersburg, Md.).
[0017] FIGS. 2A and 2B represent the amino acid sequence alignment
of the putative RNA-dependent RNA polymerase (RdRp) gene of avian
HEV with that of known HEV strains. The conserved GDD motif is
underlined. The sequence of the prototype Burmese strain is shown
on top, and only differences are indicated. Deletions are indicated
by hyphens (-).
[0018] FIGS. 3A-3C represent the sequence alignment of the ORFs 1,
2 and 3 overlapping region. The sequence of the prototype Burmese
strain is shown on top, and only differences are indicated in other
HEV strains. The sequence of avian HEV is shown at the bottom. The
start codons are indicated by arrows, and the stop codons are
indicated by three asterisks (***). The two PCR primers (FdelAIHEV
and RdelAHEV) used to amplify the region flanking the deletions are
indicated. Deletions are indicated by hyphens (-).
[0019] FIG. 4 shows the hydropathy plot of the putative ORF2
protein of avian HEV. A highly hydrophobic domain is identified at
the N-terminus of the protein followed by a hydrophilic region. The
hydrophobic domain is the putative signal peptide of ORF2. The
horizontal scale indicates the relative position of amino acid
residues of the ORF2.
[0020] FIGS. 5A-5C represent the amino acid sequence alignment of
the putative capsid gene (ORF2) of avian HEV with that of known HEV
strains. The putative signal peptide sequence is highlighted, and
the predicted cleavage site is indicated by arrowheads. The
N-linked glycosylation sites are underlined in boldface. The
sequence of the prototype Burmese strain is shown on top, and only
differences are indicated in other HEV strains. The conserved
tetrapeptide APLT is indicated (asterisks). Deletions are indicated
by hyphens (-).
[0021] FIG. 6 illustrates the sequence alignments of the 3'
noncoding region (NCR) of avian HEV with that of known HEV strains.
The 3' NCR of avian HEV is shown on top, and only differences are
indicated in other HEV strains. Deletions are indicated by hyphens
(-).
[0022] FIG. 7 represents the RT-PCR amplification of the avian HEV
genomic region with a major deletion: Lane M, 1 kb ladder; Lanes 1
and 2, PCR amplification without DMSO; Lane 3, PCR amplification in
the presence of 5% v/v DMSO; Lane 4, PCR amplification in the
presence of 5% v/v formamide.
[0023] FIGS. 8A-8C provide phylogenetic trees based on the
sequences of different genomic regions of HEV wherein FIG. 8A is a
439 bp sequence of the helicase gene, FIG. 8B is a 196 bp sequence
of the RNA-dependent RNA polymerase gene and FIG. 8C is a 148 bp
sequence of the ORF2 gene. The sequences in the three selected
regions are available for most HEV strains.
[0024] FIGS. 9A-9C represent the entire 4 kb nucleotide sequence
(3931 bp plus poly(a) tract at 3' end and 5' sense primer for
amplification) of the avian hepatitis E virus (which corresponds to
SEQ ID NO: 1).
[0025] FIG. 10 represents the predicted amino acid sequence of the
protein encoded by the helicase gene (which corresponds to SEQ ID
NO: 2).
[0026] FIG. 11 represents the nucleotide sequence (439 bp) of the
helicase gene (which corresponds to SEQ ID NO: 3).
[0027] FIG. 12 represents the predicted amino acid sequence of the
protein encoded by the RdRp gene (which corresponds to SEQ ID NO:
4).
[0028] FIG. 13 represents the nucleotide sequence (1450 bp) of the
RdRp gene (which corresponds to SEQ ID NO: 5)
[0029] FIG. 14 represents the predicted amino acid sequence of the
protein encoded by the ORF2 gene (which corresponds to SEQ ID NO:
6).
[0030] FIG. 15 represents the nucleotide sequence (1821 bp) of the
ORF2 gene (which corresponds to SEQ ID NO: 7).
[0031] FIG. 16 represents the predicted amino acid sequence of the
protein encoded by the ORF3 gene (which corresponds to SEQ ID NO:
8).
[0032] FIG. 17 represents the nucleotide sequence (264 bp) of the
ORF3 gene (which corresponds to SEQ ID NO: 9).
[0033] FIG. 18A (left panel) is a photograph of a normal liver from
a uninoculated control SPF layer chicken. FIG. 18B (right panel) is
a photograph showing hepatomegaly and subcapsular hemorrhage of a
liver from a SPF layer chicken experimentally infected with avian
HEV. Note subcapsular hemorrhage and pronounced enlargement of
right liver lobe. Liver margins are blunted indicating
swelling.
[0034] FIG. 19A (upper panel) shows a liver section from an
uninoculated control SPF layer chicken. Note the lack of
inflammatory cells anywhere in the section. FIG. 19B (lower panel)
shows a liver section from a SPF layer chicken experimentally
infected with avian HEV (hematoxylin-eosin (HE) staining). Note the
infiltration of lymphocytes in the periportal and perivascular
regions.
[0035] FIG. 20 illustrates a phylogenetic tree based on the
helicase gene region of 9 avian HEV isolates and other selected
strains of human and swine HEVs. The avian HEV isolates (shown in
boldface) are all clustered with the prototype avian HEV isolate
(avian HEV USA).
[0036] FIG. 21A represents the expression of the C-terminal 268
amino acid sequence of truncated ORF2 capsid protein of avian HEV:
Lanes 1-6, SDS-PAGE analysis of bacterial lysates at time points 0,
1, 2, 3, 4 and 6 hours after induction with IPTG; Lane 7, soluble
proteins in the supernatant part of cell lysate; Lane 8, insoluble
proteins after solubilization in SDS; Lane 9, SDS-PAGE analysis of
5 .mu.g of the purified fusion protein. FIG. 21B (lower panel)
represents the Western blot analyses of the bacterial cell lysates
at time points 0 and 3 hours after IPTG induction (Lanes 1 and 2,
respectively) and of the purified protein (Lane 3) using monoclonal
antibody (MAb) against Xpress.TM. epitope (Invitrogen Corporation,
Carlsbad, Calif.) located at the N-terminal of the expressed fusion
protein. The product of about 32 kD is indicated by arrows.
[0037] FIG. 22A illustrates Western blot analyses of antigenic
cross-reactivity of avian HEV, swine HEV, human HEV and BLSV.
Purified recombinant proteins of truncated avian HEV ORF2 (Lanes 1,
6, 9, 12-15), swine HEV ORF2 (Lanes 2, 5, 8, 11, 16) and Sar-55
human HEV ORF2 (Lanes 3, 4, 7, 10, 17) were separated by SDS PAGE,
transferred onto a nitrocellulose membrane and incubated with
antibodies against swine HEV (Lanes 1-3), US2 human HEV (Lanes
4-6), Sar-55 human HEV (Lanes 7-9), avian HEV (Lanes 13, 16-17),
and BLSV (Lane 14). Each lane contains about 250 ng of recombinant
proteins. The sera were diluted 1:100 in blocking solution before
added to the membranes. The development step was stopped as soon as
the signal related to the preinoculation ("preimmune") sera started
to appear. Preinoculation pig (Lanes 10-12) and chicken sera (Lane
15) were used as negative controls. FIGS. 22B and 22C present the
same data in a comparative format.
[0038] FIG. 23 illustrates the ELISA results generated from
cross-reactivity of different antigens with different antisera and
measured by optical density ("OD").
[0039] FIG. 24 represents the alignment of the C-terminal 268 amino
acid sequence of avian HEV with the corresponding regions of swine
HEV, US2 and Sar-55 strains of human HEV. The sequence of avian HEV
is shown on top. The deletions are indicated by minus (-)
signs.
[0040] FIGS. 25A-25D show hydropathy and antigenicity plots of the
truncated ORF2 proteins of avian HEV (FIG. 25A), swine HEV (FIG.
25B), Sar-55 strain of human HEV (FIG. 25C) and US2 strain of human
HEV (FIG. 25D).
DETAILED DESCRIPTION OF THE INVENTION
[0041] In accordance with the present invention, there is provided
a novel avian hepatitis E virus (hereinafter referred to as "avian
HEV"). The new animal strain of HEV, avian HEV, has been identified
and genetically characterized from chickens with HS syndrome in the
United States. Like swine HEV, the avian HEV identified in this
invention is genetically related to human HEV strains. Unlike swine
HEV that causes only subclinical infection and mild microscopic
liver lesions in pigs, avian HEV is associated with a disease (HS
syndrome) in chickens. Advantageously, therefore, avian HEV
infection in chickens provides a superior, viable animal model to
study human HEV replication and pathogenesis.
[0042] Electron microscopy examination of bile samples of chickens
with HS syndrome revealed virus-like particles. The virus was
biologically amplified in embryonated chicken eggs, and a novel
virus genetically related to human HEV was identified from bile
samples. The 3' half of the viral genome of approximately 4 kb was
amplified by reverse-transcription polymerase chain reaction
(RT-PCR) and sequenced. Sequence analyses of this genomic region
revealed that it contains the complete 3' noncoding region, the
complete ORFs 2 and 3 genes, the complete RNA-dependent RNA
polymerase (RdRp) gene and a partial helicase gene of the ORF1. The
helicase gene is most conserved between avian HEV and other HEV
strains, displaying 58 to 60% amino acid sequence identities.
[0043] By comparing the ORF2 sequence of avian HEV with that of
known HEV strains, a major deletion of 54 amino acid residues
between the putative signal peptide sequence and the conserved
tetrapeptide APLT of ORF2 was identified in the avian HEV. As
described herein, phylogenetic analysis indicated that avian HEV is
related to known HEV strains such as the well-characterized human
and swine HEV. Conserved regions of amino acid sequences exist
among the ORF2 capsid proteins of avian HEV, swine HEV and human
HEV. The close genetic-relatedness of avian HEV with human and
swine strains of HEV suggests avian, swine and human HEV all belong
to the same virus family. The avian HEV of the present invention is
the most divergent strain of HEV identified thus far. This
discovery has important implications for HEV animal model,
nomenclature and epidemiology, and for vaccine development against
chicken HS, swine hepatitis E and human hepatitis E.
[0044] Schlauder et al. recently reported that at least 8 different
genotypes of HEV exist worldwide (G. G. Schlauder et al.,
"Identification of 2 novel isolates of hepatitis E virus in
Argentina," J. Infect. Dis. 182:294-297 (2000)). They found that
the European strains (Greek 1, Greek 2, and Italy) and two
Argentine isolates represent distinct genotypes. However, it is now
found that the European strains (Greek 1, Greek 2 and Italy) appear
to be more related to HEV genotype 3 which consists of swine and
human HEV strains from the U.S. and a swine HEV strain from New
Zealand. The phylogenetic tree was based on only 148 bp sequence
that is available for these strains. Additional sequence
information from these strains of human HEV is required for a
definitive phylogenetic analysis. HEV was classified in the family
Caliciviridae (R. H. Purcell, "Hepatitis E virus," FIELDS VIROLOGY,
Vol. 2, pp. 2831-2843, B. N. Fields et al. eds, Lippincott-Raven
Publishers, Philadelphia (3d ed. 1996)). The lack of common
features between HEV and caliciviruses has led to the recent
removal of HEV from the Caliciviridae family, and HEV remains
unclassified.
[0045] Avian HEV represents a new genotype 5. Sequence analyses
revealed that the new avian HEV is genetically related to swine and
human HEV, displaying 47% to 50% amino acid sequence identity in
the RdRp gene, 58% to 60% identity in the helicase gene, and 42% to
44% identity in the putative capsid gene (ORF2) with the
corresponding regions of known HEV strains. The genomic
organization of avian HEV is very similar to that of human HEV:
non-structural genes such as RdRp and helicase are located at the
5' end and structural genes (ORF2 and ORF3) are located at the 3'
end of the genome. The putative capsid gene (ORF2) of avian HEV is
relatively conserved at its N-terminal region (excluding the signal
peptide) but is less conserved at its C-terminal region. The ORF3
gene of avian HEV is very divergent compared to that of known HEV
strains. However, the C-terminus of the ORF3 of avian HEV is
relatively conserved, and this region is believed to be the
immuno-dominant portion of the ORF3 protein (M. Zafrullah et al.,
"Mutational analysis of glycosylation, membrane translocation, and
cell surface expression of the hepatitis E virus ORF2 protein," J.
Virol. 73:4074-4082 (1999)). Unlike most known HEV strains, the
ORF3 of avian HEV does not overlap with the ORF1. The ORF3 start
codon of avian HEV is located 41 nucleotides downstream that of
known HEV strains. Similar to avian HEV, the ORF3 of a strain of
human HEV (HEV-T1 strain) recently identified from a patient in
China does not overlap with ORF1, and its ORF3 start codon is
located 28 nucleotides downstream the ORF1 stop codon (Y. Wang et
al., "The complete sequence of hepatitis E virus genotype 4 reveals
an alternative strategy for translation of open reading frames 2
and 3," J. Gen. Virol. 81:1675-1686 (2000)).
[0046] A major deletion was identified in the ORFs 2 and 3
overlapping region of the avian HEV genome, located between the
ORF2 signal peptide and the conserved tetrapeptide APLT. It has
been shown that, for certain HEV strains, this genomic region is
difficult to amplify by conventional PCR methods (S. Yin et al., "A
new Chinese isolate of hepatitis E virus: comparison with strains
recovered from different geographical regions," Virus Genes 9:23-32
(1994)), and that an addition of 5% v/v of formamide or DMSO in the
PCR reaction results in the successful amplification of this
genomic region. The region flanking the deletion in avian HEV
genome is relatively easy to amplify by a conventional PCR modified
by the method of the present invention. To rule out potential
RT-PCR artifacts, the region flanking the deletion was amplified
with a set of avian HEV-specific primers flanking the deletion.
RT-PCR was performed with various different parameters and
conditions including cDNA synthesis at 60.degree. C., PCR
amplification with higher denaturation temperature and shorter
annealing time, and PCR with the addition of 5% v/v of formamide or
DMSO. No additional sequence was identified, and the deletion was
further verified by direct sequencing of the amplified PCR product
flanking the deletion region. It is thus concluded that the
observed deletion in avian HEV genome is not due to RT-PCR
artifacts.
[0047] Ray et al. also reported a major deletion in the ORF2/ORF3
overlapping region of an Indian strain of human HEV (R. Ray et al.,
"Indian hepatitis E virus shows a major deletion in the small open
reading frame," Virology 189:359-362 (1992)). Unlike the avian HEV
deletion, the deletion in the Indian strain of human HEV eliminated
the ORF2 signal peptide sequence that overlaps with the ORF3. The
sequence of other genomic regions of this Indian HEV strain is not
available for further analysis. The biological significance of this
deletion is not known. It has been shown that, when the ORF2 of a
human HEV is expressed in the baculovirus system, a truncated
version of ORF2 protein lacking the N-terminal 111 amino acid
residues is produced. The truncated ORF2 protein was cleaved at
amino acid position 111-112 (Y. Zhang et al., "Expression,
characterization, and immunoreactivities of a soluble hepatitis E
virus putative capsid protein species expressed in insect cells,"
Clin. Diag. Lab. Immunol. 4:423-428 (1997)), but was still able to
form virus-like particles (T. C. Li et al., "Expression and
self-assembly of empty virus-like particles of hepatitis E virus,"
J. Virol. 71:7207-7213 (1997)). Avian HEV lacks most of the
N-terminal 100 amino acid residues of the ORF2, however, the
conserved tetrapeptide APLT (pos. 108-111 in ORF2) and a distinct
but typical signal peptide sequence are present in the ORF2 of
avian HEV. Taken together, these data suggest that the genomic
region between the cleavage site of the ORF2 signal peptide and the
conserved tetrapeptide APLT is dispensable, and is not required for
virus replication or maturation.
[0048] It has been shown that the ORF2 protein of human HEV pORF2
is the main immunogenic protein that is able to induce immune
response against HEV. Recently, the C-terminal 267 amino acids of
truncated ORF2 of a human HEV was expressed in a bacterial
expression system showing that the sequences spanning amino acids
394 to 457 of the ORF2 capsid protein participated in the formation
of strongly immunodominant epitopes on the surface of HEV particles
(M. A. Riddell et al., "Identification of immunodominant and
conformational epitopes in the capsid protein of hepatitis E virus
by using monoclonal antibodies," J. Virology 74:8011-17 (2000)). It
was reported that this truncated protein was used in an ELISA to
detect HEV infection in humans (D. A. Anderson et al., "ELISA for
IgG-class antibody to hepatitis E virus based on a highly
conserved, conformational epitope expressed in Escherichia coli,"
J. Virol. Methods 81:131-42 (1999)). It has also been shown that
C-terminus of the protein is masked when expression of the entire
pORF2 is carried out in a bacterial expression system, and that the
112 amino acids located at N-terminus of ORF2 and the 50 amino
acids located at the C-terminus are not involved in the formation
of virus-like particles (T. C. Li et al., 1997, supra). The
expression and characterization of the C-terminal 268 amino acid
residues of avian HEV ORF2 in the context of the present invention
corresponds to the C-terminal 267 amino acid residues of human
HEV.
[0049] The present invention demonstrates that avian HEV is
antigenically related to human and swine HEVs as well as chicken
BLSV. The antigenic relatedness of avian HEV ORF2 capsid protein
with human HEV, swine HEV and chicken BLSV establishes that
immunization with an avian HEV vaccine (either an attenuated or a
recombinant vaccine) will protect not only against avian HEV
infection, HS syndrome and BLSV infection in chickens but also
against human and swine HEV infections in humans and swine. Thus, a
vaccine based on avian HEV, its nucleic acid and the proteins
encoded by the nucleic acid will possess beneficial, broad
spectrum, immunogenic activity against avian, swine and human HEVs,
and BLSV.
[0050] Western blot analyses revealed that antiserum to each virus
strongly reacted with homologous antigen. It was also demonstrated
that the antiserum against BLSV reacted with the recombinant ORF2
protein of avian HEV, indicating that BLSV is antigenically related
to avian HEV. The reaction between Sar-55 human HEV and swine HEV
antigens with convalescent antiserum against avian HEV generated
strong signals while the cross-reactivity of antisera with
heterologous antigens was relatively weak. In ELISA, the optical
densities ("ODs") obtained from the reaction of avian HEV antigen
with Sar-55 HEV and swine HEV antisera were lower than the ODs
obtained from the reaction of avian HEV antiserum with the
HPLC-purified Sar-55 HEV and swine HEV antigens. This result may
have occurred because the Sar-55 HEV and swine HEV antigens of the
examples were the complete ORF2 proteins instead of the truncated
avian ORF2 protein lacking the N-terminal amino acid residues.
[0051] Schofield et al. generated neutralizing MAbs against the
capsid protein of a human HEV (D. J. Schofield et al.,
"Identification by phage display and characterization of two
neutralizing chimpanzee monoclonal antibodies to the hepatitis E
virus capsid protein," J. Virol. 74:5548-55 (2000)). The
neutralizing MAbs recognized the linear epitope(s) located between
amino acids 578 and 607. The region in avian HEV corresponding to
this neutralizing epitope is located within the truncated ORF2 of
avian HEV that reacted with human HEV and swine HEV anti-sera.
[0052] So far, HS syndrome has only been reported in several
Provinces of Canada and a few States in the U.S. In Australia,
chicken farms have been experiencing outbreaks of big liver and
spleen disease (BLS) for many years. BLS was recognized in
Australia in 1988 (J. H. Handlinger et al., "An egg drop associated
with splenomegaly in broiler breeders," Avian Dis. 32:773-778
(1988)), however, there has been no report regarding a possible
link between HS in North America and BLS in Australia. A virus
(designated BLSV) was isolated from chickens with BLS in Australia.
BLSV was shown to be genetically related to HEV based on a short
stretch of sequence available (C. J. Payne et al., "Sequence data
suggests big liver and spleen disease virus (BLSV) is genetically
related to hepatitis E virus," Vet. Microbiol. 68:119-25 (1999)).
The avian HEV identified in this invention is closely related to
BLSV identified from chickens in Australia, displaying about 80%
nucleotide sequence identity in this short genomic region (439 bp).
It appears that a similar virus related to HEV may have caused the
HS syndrome in North American chickens and BLS in Australian
chickens, but the avian HEV nevertheless remains a unique strain or
isolate, a totally distinct entity from the BLS virus. Further
genetic characterization of avian HEV shows that it has about 60%A
nucleotide sequence identities with human and swine HEVs.
[0053] In the past, the pathogenesis and replication of HEV have
been poorly understood due to the absence of an efficient in vitro
cell culture system for HEV. In this invention, it is now
demonstrated that embryonated SPF chicken eggs can unexpectedly be
infected with avian HEV through intravenous route (I.V.) of
inoculation. Earlier studies showed that bile samples positive by
EM for virus particles failed to infect embryonated chicken eggs
(J. S. Jeffrey et al., 1998, supra; H. L. Shivaprasad et al., 1995,
supra). The I.V. route of inoculation has been almost exclusively
used in studies with human and swine HEV. Other inoculation routes
such as the oral route have failed to infect pigs with swine HEV,
even when a relatively high infectious dose (10.sup.4.5 50% pig
infectious dose) of swine HEV was used. Based on the surprising
success of the present egg inoculation experiments, it illustrates
that embryonated eggs are susceptible to infection with human and
avian strains of HEV making embryonated eggs a useful in vitro
method to study HEV replication and a useful tool to manufacture
vaccines that benefit public health.
[0054] The identification of avian HEV from chickens with HS in the
context of this invention further strengthens the hypothesis that
hepatitis E is a zoonosis. The genetic close-relatedness of avian
HEV to human and swine HEV strains raises a potential public health
concern for zoonosis. Recent studies showed that pig handlers are
at increased risk of zoonotic HEV infection (X. J. Meng et al.,
1999, supra). Karetnyi et al. reported that human populations with
occupational exposure to wild animals have increased risks of HEV
infection (Y. V. Karetnyi et al., "Hepatitis E virus infection
prevalence among selected populations in Iowa," J. Clin. Virol.
14:51-55 (1999)). Since individuals such as poultry farmers or
avian veterinarians may be at potential risk of zoonotic infection
by avian HEV, the present invention finds broad application to
prevent viral infections in humans as well as chickens and other
carrier animals.
[0055] The present invention provides an isolated avian hepatitis E
virus that is associated with serious viral infections and
hepatitis-splenomegaly syndrome in chickens. This invention
includes, but is not limited to, the virus which has a nucleotide
sequence set forth in SEQ ID NO: 1, its functional equivalent or
complementary strand. It will be understood that the specific
nucleotide sequence derived from any avian HEV will have slight
variations that exist naturally between individual viruses. These
variations in sequences may be seen in deletions, substitutions,
insertions and the like. Thus, to distinguish the virus embraced by
this invention from the Australian big liver and spleen disease
virus, the avian HEV virus is characterized by having no more than
about 80% nucleotide sequence homology to the BLSV.
[0056] The source of the isolated virus strain is bile, feces,
serum, plasma or liver cells from chickens or human carriers
suspected to have the avian hepatitis E viral infection. However,
it is contemplated that recombinant DNA technology can be used to
duplicate and chemically synthesize the nucleotide sequence.
Therefore, the scope of the present invention encompasses the
isolated polynucleotide which comprises, but is not limited to, a
nucleotide sequence set forth in SEQ ID NO: 1 or its complementary
strand; a polynucleotide which hybridizes to and which is at least
95% complementary to the nucleotide sequence set forth in SEQ ID
NO: 1; or an immunogenic fragment selected from the group
consisting of a nucleotide sequence in the partial helicase gene of
ORF1 set forth in SEQ ID NO: 3, a nucleotide sequence in the RdRp
gene set forth in SEQ ID NO: 5, a nucleotide sequence in the ORF2
gene set forth in SEQ ID NO: 7, a nucleotide sequence in the ORF3
gene set forth in SEQ ID NO: 9 or their complementary strands. The
immunogenic or antigenic coding regions or fragments can be
determined by techniques known in the art and then used to make
monoclonal or polyclonal antibodies for immunoreactivity screening
or other diagnostic purposes. The invention further encompasses the
purified, immunogenic protein encoded by the isolated
polynucleotides. Desirably, the protein may be an isolated or
recombinant ORF2 capsid protein or an ORF3 protein.
[0057] Another important aspect of the present invention is the
unique immunogenic composition comprising the isolated avian HEV or
an antigenic protein encoded by an isolated polynucleotide
described hereinabove and its use for raising or producing
antibodies. The composition contains a nontoxic, physiologically
acceptable carrier and, optionally, one or more adjuvants. Suitable
carriers, such as, for example, water, saline, ethanol, ethylene
glycol, glycerol, etc., are easily selected from conventional
excipients and co-formulants may be added. Routine tests can be
performed to ensure physical compatibility and stability of the
final composition.
[0058] Vaccines and methods of using them are also included within
the scope of the present invention. Inoculated avian or mammalian
species are protected from serious viral infection,
hepatitis-splenomegaly syndrome, hepatitis E and other related
illness. The vaccines comprise, for example, an inactivated or
attenuated avian hepatitis E virus, a nontoxic, physiologically
acceptable carrier and, optionally, one or more adjuvants.
[0059] The adjuvant, which may be administered in conjunction with
the immunogenic composition or vaccine of the present invention, is
a substance that increases the immunological response when combined
with the composition or vaccine. The adjuvant may be administered
at the same time and at the same site as the composition or
vaccine, or at a different time, for example, as a booster.
Adjuvants also may advantageously be administered to the mammal in
a manner or at a site different from the manner or site in which
the composition or vaccine is administered. Suitable adjuvants
include, but are not limited to, aluminum hydroxide (alum),
immunostimulating complexes (ISCOMS), non-ionic block polymers or
copolymers, cytokines (like IL-1, IL-2, IL-7, IFN-.alpha.,
IFN-.beta., IFN-.gamma., etc.), saponins, monophosphoryl lipid A
(MLA), muramyl dipeptides (MDP) and the like. Other suitable
adjuvants include, for example, aluminum potassium sulfate,
heat-labile or heat-stable enterotoxin isolated from Escherichia
coli, cholera toxin or the B subunit thereof, diphtheria toxin,
tetanus toxin, pertussis toxin, Freund's incomplete or complete
adjuvant, etc. Toxin-based adjuvants, such as diphtheria toxin,
tetanus toxin and pertussis toxin may be inactivated prior to use,
for example, by treatment with formaldehyde.
[0060] The new vaccines of this invention are not restricted to any
particular type or method of preparation. The vaccines include, but
are not limited to, modified live vaccines, inactivated vaccines,
subunit vaccines, attenuated vaccines, genetically engineered
vaccines, etc. These vaccines are prepared by general methods known
in the art modified by the new use of embryonated eggs. For
instance, a modified live vaccine may be prepared by optimizing
avian HEV propagation in embryonated eggs as described herein and
further virus production by methods known in the art. Since avian
HEV cannot grow in the standard cell culture, the avian HEV of the
present invention can uniquely be attenuated by serial passage in
embryonated chicken eggs. The virus propagated in eggs may be
lyophilized (freeze-dried) by methods known in the art to enhance
preservability for storage. After subsequent rehydration, the
material is then used as a live vaccine.
[0061] The advantages of live vaccines is that all possible immune
responses are activated in the recipient of the vaccine, including
systemic, local, humoral and cell-mediated immune responses. The
disadvantages of live virus vaccines, which may outweigh the
advantages, lie in the potential for contamination with live
adventitious viral agents or the risk that the virus may revert to
virulence in the field.
[0062] To prepare inactivated virus vaccines, for instance, the
virus propagation and virus production in embryonated eggs are
again first optimized by methods described herein. Serial virus
inactivation is then optimized by protocols generally known to
those of ordinary skill in the art or, preferably, by the methods
described herein.
[0063] Inactivated virus vaccines may be prepared by treating the
avian HEV with inactivating agents such as formalin or hydrophobic
solvents, acids, etc., by irradiation with ultraviolet light or
X-rays, by heating, etc. Inactivation is conducted in a manner
understood in the art. For example, in chemical inactivation, a
suitable virus sample or serum sample containing the virus is
treated for a sufficient length of time with a sufficient amount or
concentration of inactivating agent at a sufficiently high (or low,
depending on the inactivating agent) temperature or pH to
inactivate the virus. Inactivation by heating is conducted at a
temperature and for a length of time sufficient to inactivate the
virus. Inactivation by irradiation is conducted using a wavelength
of light or other energy source for a length of time sufficient to
inactivate the virus. The virus is considered inactivated if it is
unable to infect a cell susceptible to infection.
[0064] The preparation of subunit vaccines typically differs from
the preparation of a modified live vaccine or an inactivated
vaccine. Prior to preparation of a subunit vaccine, the protective
or antigenic components of the vaccine must be identified. Such
protective or antigenic components include certain amino acid
segments or fragments of the viral capsid proteins which raise a
particularly strong protective or immunological response in
chickens; single or multiple viral capsid proteins themselves,
oligomers thereof, and higher-order associations of the viral
capsid proteins which form virus substructures or identifiable
parts or units of such substructures; oligoglycosides, glycolipids
or glycoproteins present on or near the surface of the virus or in
viral substructures such as the lipoproteins or lipid groups
associated with the virus, etc. Preferably, the capsid protein
(ORF2) is employed as the antigenic component of the subunit
vaccine. Other proteins may also be used such as those encoded by
the nucleotide sequence in the ORF3 gene. These immunogenic
components are readily identified by methods known in the art. Once
identified, the protective or antigenic portions of the virus
(i.e., the "subunit") are subsequently purified and/or cloned by
procedures known in the art. The subunit vaccine provides an
advantage over other vaccines based on the live virus since the
subunit, such as highly purified subunits of the virus, is less
toxic than the whole virus.
[0065] If the subunit vaccine is produced through recombinant
genetic techniques, expression of the cloned subunit such as the
ORF2 (capsid) and ORF3 genes, for example, may be optimized by
methods known to those in the art (see, for example, Maniatis et
al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor
Laboratory, Cold Spring Harbor, Mass. (1989)). On the other hand,
if the subunit being employed represents an intact structural
feature of the virus, such as an entire capsid protein, the
procedure for its isolation from the virus must then be optimized.
In either case, after optimization of the inactivation protocol,
the subunit purification protocol may be optimized prior to
manufacture.
[0066] To prepare attenuated vaccines, the live, pathogenic virus
is first attenuated (rendered nonpathogenic or harmless) by methods
known in the art or, preferably, as described herein. For instance,
attenuated viruses may be prepared by the technique of the present
invention which involves the novel serial passage through
embryonated chicken eggs. Attenuated viruses can be found in nature
and may have naturally-occurring gene deletions or, alternatively,
the pathogenic viruses can be attenuated by making gene deletions
or producing gene mutations. The attenuated and inactivated virus
vaccines comprise the preferred vaccines of the present
invention.
[0067] Genetically engineered vaccines, which are also desirable in
the present invention, are produced by techniques known in the art.
Such techniques involve, but are not limited to, the use of RNA,
recombinant DNA, recombinant proteins, live viruses and the
like.
[0068] For instance, after purification, the wild-type virus may be
isolated from suitable clinical, biological samples such as feces
or bile by methods known in the art, preferably by the method
taught herein using embryonated chicken eggs as hosts. The RNA is
extracted from the biologically pure virus or infectious agent by
methods known in the art, preferably by the guanidine
isothiocyanate method using a commercially available RNA isolation
kit (for example, the kit available from Statagene, La Jolla,
Calif.) and purified by methods known in the art, preferably by
ultracentrifugation in a CsCl gradient. RNA may be further purified
or enriched by oligo(dT)-cellulose column chromatography. The cDNA
of viral genome is cloned into a suitable host by methods known in
the art (see Maniatis et al., id.), and the virus genome is then
analyzed to determine essential regions of the genome for producing
antigenic portions of the virus. Thereafter, the procedure is
generally the same as that for the modified live vaccine, an
inactivated vaccine or a subunit vaccine.
[0069] Genetically engineered vaccines based on recombinant DNA
technology are made, for instance, by identifying the portion of
the viral gene which encodes for proteins responsible for inducing
a stronger immune or protective response in chickens (e.g.,
proteins derived from ORF1, ORF2, ORF3, etc.). Such identified
genes or immunodominant fragments can be cloned into standard
protein expression vectors, such as the baculovirus vector, and
used to infect appropriate host cells (see, for example, O'Reilly
et al., "Baculovirus Expression Vectors: A Lab Manual," Freeman
& Co. (1992)). The host cells are cultured, thus expressing the
desired vaccine proteins, which can be purified to the desired
extent and formulated into a suitable vaccine product.
[0070] Genetically engineered proteins, useful in vaccines, for
instance, may be expressed in insect cells, yeast cells or
mammalian cells. The genetically engineered proteins, which may be
purified or isolated by conventional methods, can be directly
inoculated into an avian or mammalian species to confer protection
against avian or human hepatitis E.
[0071] An insect cell line (like HI-FIVE) can be transformed with a
transfer vector containing polynucleic acids obtained from the
virus or copied from the viral genome which encodes one or more of
the immuno-dominant proteins of the virus. The transfer vector
includes, for example, linearized baculovirus DNA and a plasmid
containing the desired polynucleotides. The host cell line may be
co-transfected with the linearized baculovirus DNA and a plasmid in
order to make a recombinant baculovirus.
[0072] Alternatively, RNA or DNA from the HS infected carrier or
the isolated avian HEV which encode one or more capsid proteins can
be inserted into live vectors, such as a poxvirus or an adenovirus
and used as a vaccine.
[0073] An immunologically effective amount of the vaccine of the
present invention is administered to an avian or mammalian species
in need of protection against said infection or syndrome. The
"immunologically effective amount" can be easily determined or
readily titrated by routine testing. An effective amount is one in
which a sufficient immunological response to the vaccine is
attained to protect the bird or mammal exposed to the virus which
causes chicken HS, human hepatitis E, swine hepatitis E or related
illness. Preferably, the avian or mammalian species is protected to
an extent in which one to all of the adverse physiological symptoms
or effects of the viral disease are found to be significantly
reduced, ameliorated or totally prevented.
[0074] The vaccine can be administered in a single dose or in
repeated doses. Dosages may contain, for example, from 1 to 1,000
micrograms of virus-based antigen (dependent upon the concentration
of the immuno-active component of the vaccine), but should not
contain an amount of virus-based antigen sufficient to result in an
adverse reaction or physiological symptoms of viral infection.
Methods are known in the art for determining or titrating suitable
dosages of active antigenic agent based on the weight of the bird
or mammal, concentration of the antigen and other typical
factors.
[0075] The vaccine can be administered to chickens, turkeys or
other farm animals in close contact with chickens, for example,
pigs. Also, the vaccine can be given to humans such as chicken or
poultry farmers who are at high risk of being infected by the viral
agent. It is contemplated that a vaccine based on the avian HEV can
be designed to provide broad protection against both avian and
human hepatitis E. In other words, the vaccine based on the avian
HEV can be preferentially designed to protect against human
hepatitis E through the so-called "Jennerian approach" (i.e.,
cowpox virus vaccine can be used against human smallpox by Edward
Jenner). Desirably, the vaccine is administered directly to an
avian or mammalian species not yet exposed to the virus which
causes HS, hepatitis E or related illness. The vaccine can
conveniently be administered orally, intrabuccally, intranasally,
transdermally, parenterally, etc. The parenteral route of
administration includes, but is not limited to, intramuscular,
intravenous, intraperitoneal and subcutaneous routes.
[0076] When administered as a liquid, the present vaccine may be
prepared in the form of an aqueous solution, a syrup, an elixir, a
tincture and the like. Such formulations are known in the art and
are typically prepared by dissolution of the antigen and other
typical additives in the appropriate carrier or solvent systems.
Suitable carriers or solvents include, but are not limited to,
water, saline, ethanol, ethylene glycol, glycerol, etc. Typical
additives are, for example, certified dyes, flavors, sweeteners and
antimicrobial preservatives such as thimerosal (sodium
ethylmercurithiosalicylate). Such solutions may be stabilized, for
example, by addition of partially hydrolyzed gelatin, sorbitol or
cell culture medium, and may be buffered by conventional methods
using reagents known in the art, such as sodium hydrogen phosphate,
sodium dihydrogen phosphate, potassium hydrogen phosphate,
potassium dihydrogen phosphate, a mixture thereof, and the
like.
[0077] Liquid formulations also may include suspensions and
emulsions which contain suspending or emulsifying agents in
combination with other standard co-formulants. These types of
liquid formulations may be prepared by conventional methods.
Suspensions, for example, may be prepared using a colloid mill.
Emulsions, for example, may be prepared using a homogenizer.
[0078] Parenteral formulations, designed for injection into body
fluid systems, require proper isotonicity and pH buffering to the
corresponding levels of mammalian body fluids. Isotonicity can be
appropriately adjusted with sodium chloride and other salts as
needed. Suitable solvents, such as ethanol or propylene glycol, can
be used to increase the solubility of the ingredients in the
formulation and the stability of the liquid preparation. Further
additives which can be employed in the present vaccine include, but
are not limited to, dextrose, conventional antioxidants and
conventional chelating agents such as ethylenediamine tetraacetic
acid (EDTA). Parenteral dosage forms must also be sterilized prior
to use.
[0079] Also included within the scope of the present invention is a
novel method for propagating, inactivating or attenuating the
pathogenic hepatitis E virus (avian, swine, human, etc.) which
comprises inoculating an embryonated chicken egg with a live,
pathogenic hepatitis E virus contained in a biological sample from
bile, feces, serum, plasma, liver cell, etc., preferably by
intravenous injection, and either recovering a live, pathogenic
virus for further research and vaccine development or continuing to
pass the pathogenic virus serially through additional embryonated
chicken eggs until the pathogenic virus is rendered inactivated or
attenuated. Propagating live viruses through embryonated chicken
eggs according to the present invention is a unique method which
others have failed to attain. Vaccines are typically made by serial
passage through cell cultures but avian HEV, for example, cannot be
propagated in conventional cell cultures. Using embryonated chicken
eggs provides a novel, viable means for inactivating or attenuating
the pathogenic virus in order to be able to make a vaccine product.
The inactivated or attenuated strain, which was previously
unobtainable, can now be incorporated into conventional vehicles
for delivering vaccines.
[0080] Additionally, the present invention provides a useful
diagnostic reagent for detecting the avian or mammalian HEV
infection or diagnosing hepatitis-splenomegaly syndrome in an avian
or mammalian species which comprise a monoclonal or polyclonal
antibody purified from a natural host such as, for example, by
inoculating a chicken with the avian HEV or the immunogenic
composition of the invention in an effective immunogenic quantity
to produce a viral infection and recovering the antibody from the
serum of the infected chicken. Alternatively, the antibodies can be
raised in experimental animals against the natural or synthetic
polypeptides derived or expressed from the amino acid sequences or
immunogenic fragments encoded by the nucleotide sequence of the
isolated avian HEV. For example, monoclonal antibodies can be
produced from hybridoma cells which are obtained from mice such as,
for example, Balb/c, immunized with a polypeptide antigen derived
from the nucleotide sequence of the isolated avian HEV. Selection
of the hybridoma cells is made by growth in hyproxanthine,
thymidine and aminopterin in a standard cell culture medium like
Dulbecco's modified Eagle's medium (DMEM) or minimal essential
medium. The hybridoma cells which produce antibodies can be cloned
according to procedures known in the art. Then, the discrete
colonies which are formed can be transferred into separate wells of
culture plates for cultivation in a suitable culture medium.
Identification of antibody secreting cells is done by conventional
screening methods with the appropriate antigen or immunogen.
Cultivating the hybridoma cells in vitro or in vivo by obtaining
ascites fluid in mice after injecting the hybridoma produces the
desired monoclonal antibody via well-known techniques.
[0081] For another alternative method, avian HEV capsid protein can
be expressed in a baculovirus expression system or E coli according
to procedures known in the art. The expressed recombinant avian HEV
capsid protein can be used as the antigen for diagnosis of HS or
human hepatitis E in an enzyme-linked immunoabsorbent Assay
(ELISA). The ELISA assay based on the avian recombinant capsid
antigen, for example, can be used to detect antibodies to avian HEV
in avian and mammalian species. Although the ELISA assay is
preferred, other known diagnostic tests can be employed such as
immunofluorescence assay (IFA), immunoperoxidase assay (IPA),
etc.
[0082] Desirably, a commercial ELISA diagnostic assay in accordance
with the present invention can be used to diagnose avian HEV
infection and HS syndrome in chickens. The examples illustrate
using purified ORF2 protein of avian HEV to develop an ELISA assay
to detect anti-HEV in chickens. Weekly sera collected from SPF
chickens experimentally infected with avian HEV, and negative sera
from control chickens are used to validate the assay. This ELISA
assay has been successfully used in the chicken studies to monitor
the course of seroconversion to anti-HEV in chickens experimentally
infected with avian HEV. Further standardization of the test by
techniques known to those skilled in the art may optimize the
commercialization of a diagnostic assay for avian HEV. Other
diagnostic assays can also be developed as a result of the findings
of the present invention such as a nucleic acid-based diagnostic
assay, for example, an RT-PCR assay and the like. Based on the
description of the sequences of the partial genomes of the nine new
strains of avian HEV, the RT-PCR assay and other nucleic acid-based
assays can be standardized to detect avian HEV in clinical
samples.
[0083] The antigenic cross-reactivity of the truncated ORF2 capsid
protein (pORF2) of avian HEV with swine HEV, human HEV and the
chicken big liver and spleen disease virus (BLSV) is shown in the
below examples. The sequence of C-terminal 268 amino acid residuals
of avian HEV ORF2 was cloned into expression vector pRSET-C and
expressed in Escherichia coil (E. coli) strain BL21(DE3)pLysS. The
truncated ORF2 protein was expressed as a fusion protein and
purified by affinity chromatography. Western blot analysis revealed
that the purified avian HEV ORF2 protein reacted with the antisera
raised against the capsid protein of Sar-55 human HEV and with
convalescent antisera against swine HEV and US2 human HEV as well
as antiserum against BLSV. The antiserum against avian HEV also
reacted with the HPLC-purified recombinant capsid proteins of swine
HEV and Sar-55 human HEV. The antiserum against US2 strain of human
HEV also reacted with recombinant ORF2 proteins of both swine HEV
and Sar-55 human HEV. Using ELISA further confirmed the cross
reactivity of avian HEV putative capsid protein with the
corresponding genes of swine HEV and human HEVs. The results show
that avian HEV shares some antigenic epitopes in its capsid protein
with swine and human HEVs as well as BLSV, and establish the
usefulness of the diagnostic reagents for HEV diagnosis as
described herein.
[0084] The diagnostic reagent is employed in a method of the
invention for detecting the avian or mammalian hepatitis E viral
infection or diagnosing hepatitis-splenomegaly syndrome in an avian
or mammalian species which comprises contacting a biological sample
of the bird or mammal with the aforesaid diagnostic reagent and
detecting the presence of an antigen-antibody complex by
conventional means known to those of ordinary skill in the art. The
biological sample includes, but is not limited to, blood, plasma,
bile, feces, serum, liver cell, etc. To detect the antigen-antibody
complex, a form of labeling is often used. Suitable radioactive or
non-radioactive labeling substances include, but are not limited
to, radioactive isotopes, fluorescent compounds, dyes, etc. The
detection or diagnosis method of this invention includes
immunoassays, immunometric assays and the like. The method
employing the diagnostic reagent may also be accomplished in an in
vitro assay in which the antigen-antibody complex is detected by
observing a resulting precipitation. The biological sample can be
utilized from any avian species such as chickens, turkeys, etc. or
mammals such as pigs and other farm animals or humans, in
particular, chicken farmers who have close contact with chickens,
If the bird or the mammal is suspected of harboring a hepatitis E
viral infection and exhibiting symptoms typical of
hepatitis-splenomegaly syndrome or other related illness, the
diagnostic assay will be helpful to determine the appropriate
course of treatment once the viral causative agent has been
identified.
[0085] Another preferred embodiment of the present invention
involves methods for detecting avian HEV nucleic acid sequences in
an avian or mammalian species using nucleic acid hybridization
probes or oligonucleotide primers for polymerase chain reaction
(PCR) to further aid in the diagnosis of viral infection or
disease. The diagnostic tests, which are useful in detecting the
presence or absence of the avian hepatitis E viral nucleic acid
sequence in the avian or mammalian species, comprise, but are not
limited to, isolating nucleic acid from the bird or mammal and then
hybridizing the isolated nucleic acid with a suitable nucleic acid
probe or probes, which can be radio-labeled, or a pair of
oligonucleotide primers derived from the nucleotide sequence set
forth in SEQ ID NO: 1 and determining the presence or absence of a
hybridized probe complex. Conventional nucleic acid hybridization
assays can be employed by those of ordinary skill in this art. For
example, the sample nucleic acid can be immobilized on paper, beads
or plastic surfaces, with or without employing capture probes; an
excess amount of radio-labeled probes that are complementary to the
sequence of the sample nucleic acid is added; the mixture is
hybridized under suitable standard or stringent conditions; the
unhybridized probe or probes are removed; and then an analysis is
made to detect the presence of the hybridized probe complex, that
is, the probes which are bound to the immobilized sample. When the
oligonucleotide primers are used, the isolated nucleic acid may be
further amplified in a polymerase chain reaction or other
comparable manner before analysis for the presence or absence of
the hybridized probe complex. Preferably, the polymerase chain
reaction is performed with the addition of 5% v/v of formamide or
dimethyl sulfoxide.
[0086] The following examples demonstrate certain aspects of the
present invention. However, it is to be understood that these
examples are for illustration only and do not purport to be wholly
definitive as to conditions and scope of this invention. It should
be appreciated that when typical reaction conditions (e.g.,
temperature, reaction times, etc.) have been given, the conditions
both above and below the specified ranges can also be used, though
generally less conveniently. The examples are conducted at room
temperature (about 23.degree. C. to about 28.degree. C.) and at
atmospheric pressure. All parts and percents referred to herein are
on a weight basis and all temperatures are expressed in degrees
centigrade unless otherwise specified.
[0087] A further understanding of the invention may be obtained
from the non-limiting examples that follow below.
EXAMPLE 1
Biological Amplification of the Virus in Embryonated Chicken
Eggs
[0088] A sample of bile collected from a chicken with HS in
California was used in this study. Electron microscopy (EM)
examination showed that this bile sample was positive for virus
particles of 30 to 40 nm in diameter. The limited bile materials
containing the virus prevented the performance of extensive genetic
identification and characterization of the virus. A preliminary
study was conducted to determine if the virus could be biologically
amplified in embryonated chicken eggs. SPF eggs were purchased at
one day of age (Charles River SPAFAS, Inc., North Franklin, Conn.)
and incubated for 9 days in a 37.degree. C. egg incubator. At 9
days of embryonated age, 6 eggs were inoculated intravenously with
100 .mu.l of a 10.sup.-3 dilution and 6 eggs with a 10.sup.-4
dilution in phosphate buffered saline (PBS) of the positive bile
sample. Six eggs were uninoculated as controls. The inoculated eggs
were incubated at 37.degree. C. until 21 days of age (before
natural hatching), at which time the embryos were sacrificed. Bile
and liver samples were collected and tested by RT-PCR for evidence
of virus replication. The virus recovered from infected eggs was
used as the virus source for further characterization.
EXAMPLE 2
Amplification of the 3' Half of the Viral Genome
[0089] Based on the assumption that the putative virus associated
with HS in chickens shared nucleotide sequence similarity with
human and swine HEV, a modified 3' RACE (Rapid Amplification of
cDNA Ends) system was employed to amplify the 3'-half of the viral
genome. Briefly, the sense primer, F4AHEV (Table 1 below), was
chosen from a conserved region in ORF1 among known swine and human
HEV strains including the big liver and spleen disease virus (BLSV)
identified from chickens in Australia (C. J. Payne et al.,
"Sequence data suggests big liver and spleen disease virus (BLSV)
is genetically related to hepatitis E virus," Vet. Microbiol.
68:119-25 (1999)). The antisence primers included two anchored
commercial primers of nonviral origin (GIBCO-BRL, Gaithersburg,
Md.): AUAP (Abridged Universal Amplification Primer) and AP
(Adapter Primer) with a poly (T) stretch (Table 1, below). Total
RNA was extracted from 100 .mu.l of the bile by TriZol reagent
(GIBCO-BRL), and resuspended in 11.5 .mu.l of DNase-, RNase- and
proteinase-free water (Eppendorf Scientific, Inc., now Brinkmann
Instruments, Inc., Westbury, N.Y.). Total RNA was
reverse-transcribed at 42.degree. C. for 90 minutes in the presence
of reverse transcription reaction mixtures consisting of 11.5 .mu.l
of the total RNA, 1 .mu.l of Superscript II reverse transcriptase
(GIBCO-BRL), 1 .mu.l of 10 .mu.M antisense primer, 0.5 .mu.l of
RNase inhibitor (GIBCO-BRL), 0.5 .mu.l of dithioteritol, and 4
.mu.l of 5.times.RT buffer.
[0090] PCR was performed with a mixture of a Taq DNA polymerase and
a proofreading pfu polymerase contained in an eLONGase.RTM. Kit
(GIBCO-BRL, Gaithersburg, Md.). The PCR reaction was carried out
according to the instructions supplied with the kit and consisted
of 10 .mu.l of cDNA, 1.7 mM MgCL.sub.2 and 1 .mu.l of each 10 .mu.M
sense and antisense primers. Alternatively, AmpliTaq gold
polymerase (Perkin-Elmer, Wellesley, Mass.) with and without 5% v/v
dimethyl sulfoxide (DMSO) was used. The PCR reaction consisted of a
denaturation at 94.degree. C. for 1 minute, followed by 5 cycles of
denaturation at 94.degree. C. for 40 seconds, annealing at
42.degree. C. for 40 seconds, extension at 68.degree. C. for 5
minutes, 16 cycles of a touch down PCR with the starting annealing
temperature at 59.degree. C. which was reduced by 1 degree every 2
cycles, and then 11 cycles of amplification with an annealing
temperature at 51.degree. C., followed by a final extension at
74.degree. C. for 10 minutes. The resulting PCR product was
analyzed on a 0.8% w/v agarose gel. When AmpliTaq gold polymerase
was used, the thermal cycle profile and parameters remained the
same except that the enzyme was first activated by incubation at
95.degree. C. for 9 minutes.
1TABLE 1 Synthetic oligonucleotide primers used for PCR
amplification and DNA sequencing of the avian HEV genome 1
.sup.aThe positions are relative to the 3931 bp sequence of avian
HEV determined in the present invention.
EXAMPLE 3
Cloning of the Amplified PCR Product
[0091] A PCR product of approximately 4 kb was amplified by the
modified 3' RACE system. The PCR product was excised and eluted
from the agarose gel with the CoNcERT.TM. Rapid Gel Extraction
System (GIBCO-BRL). The purified PCR product was subsequently
cloned into a TA vector. The recombinant plasmid was used to
transform competent cells supplied in the AdvanTAge.TM. PCR Cloning
Kit (Clontech Laboratories, Inc., Palo Alta, Calif.) according to
the manufacturer's instruction. White colonies were selected and
grown in LB broth containing 100 .mu.g/ml of ampicillin. The
recombinant plasmids containing the insert were isolated with a
Plasmid DNA Isolation kit (Qiagen Inc., Valencia, Calif.).
EXAMPLE 4
DNA Sequencing
[0092] Three independent cDNA clones containing the approximately 4
kb insert were selected and sequenced at Virginia Tech DNA
Sequencing Facility with an Automated DNA Sequencer (Applied
Biosystem, Inc., Foster City, CA). Primer walking strategy was
employed to determine the nucleotide sequence of both DNA strands
of the three independent cDNA clones. The M13 forward and reverse
primers as well as sixteen avian HEV specific primers (Table 1,
above) were used to determine the nucleotide sequence of the
approximately 4 kb viral genome. To facilitate DNA sequencing, a
unique EcoR I restriction site that is present in this 4 kb viral
genomic fragment was utilized. The recombinant plasmid with the 4
kb insert was digested by the EcoR I restriction enzyme, and the
resulting two EcoR I fragments were subdloned into pGEM-9zf (-)
(Promega, Madison, Wis.). The cDNA subdlones were also used to
determine the sequence by primer walking strategy. The sequence at
the 5' end of the fragment was further confirmed by direct
sequencing of the PCR product amplified with avian HEV-specific
primers.
EXAMPLE 5
Sequence and Phylogenetic Analyses
[0093] The complete sequence of the approximately 4 kb viral
genomic fragment was assembled and analyzed with the MacVector.RTM.
(Oxford Molecular, Inc., Madison, Wis.) and DNAstar (DNASTAR, Inc.,
Madison, Wis.) computer programs. For any given region, the
consensus sequence was derived from at least three independent cDNA
clones. The putative signal peptide of the ORF2 protein was
predicted with the SignalP V1.1 program
(http://www.cbs.dtu.dk/services/SignalP). The hydrophobicity
analysis of the putative ORF2 protein was performed with the
MacVector program using Sweet/Eisenberg method (R. M. Sweet et al.,
"Correlation of sequence hydrophobicities measures similarity in
three-dimensional protein structure," J. Mol. Biol. 171:479-488
(1983)). Phylogenetic analyses were conducted with the aid of the
PAUP program (David L. Swofford, Smithsonian Institution,
Washington, D.C., and distributed by Sinauer Associates, Inc.,
Sunderland, Mass).
[0094] For most HEV strains, the sequences are available only in
certain genomic regions. Therefore, to better understand the
phylogenetic relationship of known HEV strains, phylogenetic
analyses were based on three different genomic regions: a 148 bp
fragment of the ORF2 gene in which the sequences of most HEV
strains are available, a 196 bp fragment of the RdRp gene, and a
439 bp fragment of the helicase gene in which the sequence of BLSV
is known. Phylogenetic analyses were also performed with the
complete RdRp and ORF2 genes from known HEV strains. The
branch-and-bound and midpoint rooting options were used to produce
the phylogenetic trees. The sequences of known HEV strains used in
the sequence and phylogenetic analyses were either published or
available in the Genbank database: Nepal (V. Gouvea et al.,
"Hepatitis E virus in Nepal: similarities with the Burmese and
Indian variants," Virus Res. 52:87-96 (1997)), Egypt 93 (S. A.
Tsarev et al., "Phylogenetic analysis of hepatitis E virus isolates
from Egypt," J. Med. Virol . 57:68-74 (1999)), Egypt 94 (id),
Morroco (J. Meng et al., "Primary structure of open reading frame 2
and 3 of the hepatitis E virus isolated from Morocco," J. Med.
Virol. 57:126-133 (1999)), Pakistan (strain Sar55) (S. A. Tsarev et
al., "Characterization of a prototype strain of hepatitis E virus,"
Proc. Natl. Acad. Sci. U S A. 89:559-63 (1992)), Burma (G. R. Reyes
et al., "Isolation of a cDNA from the virus responsible for
enterically transmitted non-A, non-B hepatitis," Science
247:1335-1339 (1990)), Myanmar (A. W. Tam et al., "Hepatitis E
virus (HEV): molecular cloning and sequencing of the full-length
viral genome," Virology 185:120-131 (1991)), Vietnam (accession no.
AF 170450), Greek 1 (G. G. Schlauder et al., "Novel hepatitis E
virus (HEV) isolates from Europe: evidence for additional genotypes
of HEV," J. Med. Virol. 57:243-51 (1999)), Greek 2 (id), Italy
(id.), Mexico (C. C. Huang et al., "Molecular cloning and
sequencing of the Mexico isolate of hepatitis E virus (HEV),"
Virology 191:550-558 (1992)), USI (G.G. Schlauder et al., "The
sequence and phylogenetic analysis of a novel hepatitis E virus
isolated from a patient with acute hepatitis reported in the United
States," J. Gen. Virol. 79:447-456 (1998)), US2 (J. C. Erker et
al., "A hepatitis E virus variant from the United States: molecular
characterization and transmission in cynomolgus macaques," J. Gen.
Virol. 80:681-690 (1999)), the U.S. strain of swine HEV (X. J. Meng
et al., "A novel virus in swine is closely related to the human
hepatitis E virus," Proc. Natl. Acad. Sci. USA 94:9860-9865 (1997);
X. J. Meng et al., "Genetic and experimental evidence for
cross-species infection by the swine hepatitis E virus," J. Virol.
72:9714-9721 (1998)), the New Zealand strain of swine HEV
(accession no. AF200704), Indian strains including Hyderabad (S. K.
Panda et al., "The in vitro-synthesized RNA from a cDNA clone of
hepatitis E virus is infectious," J. Virol. 74:2430-2437 (2000)),
Madras (accession no. X99441), X98292 (strain HEV037) (M. C. Donati
et al., "Sequence analysis of full-length HEV clones derived
directly from human liver in fulminant hepatitis E," VIRAL
HEPATITIS AND LIVER DISEASE, pp. 313-316 (M. Rizzetto et al., eds.,
Edizioni Minerva Medica, Torino, 1997)), AKL 90 (V. A. Arankalle et
al., "Phylogenetic analysis of hepatitis E virus isolates from
India (1976-1993)," J. Gen. Virol. 80:1691-1700 (1999)), and U22532
(S. K. Panda et al., "An Indian strain of hepatitis E virus (HEV):
cloning, sequence, and expression of structural region and antibody
responses in sera from individuals from an area of high-level HEV
endemicity," J. Clin. Microbiol. 33:2653-2659 (1995)), Taiwanese
strains including TW4E, TW7E and TW8E, and Chinese strains
including 93G (accession no. AF145208), L25547, Hetian, KS2, D11093
(strain Uigh 179), D11092, HEV-T1, Ch-T11 (accession no. AF151962)
and Ch-T21 (accession no. AF151963).
EXAMPLE 6
Propagation of Avian HEV in Embryonated Chicken Eggs
[0095] The aim of this preliminary experiment was to generate, by
biological amplification of the virus in embryonated eggs,
sufficient amounts of virus for further studies, and to determine
if avian HEV replicates in eggs. The undiluted positive bile sample
contained about 10.sup.7 genomic equivalents (GE) of avian HEV per
ml of bile. Embryonated SPF chicken eggs were intravenously
inoculated with a diluted bile sample containing avian HEV. Five
out of six embryos inoculated with 10.sup.-3 dilution and three out
of six embryos inoculated with 10.sup.-4 dilution died before 21
days of embryonated age. At 12 days postinoculation (21 days of
embryonated age), the remaining 4 inoculated embryos were
sacrificed. The inoculated embryos showed congestion of yolk sac
and hemorrhage in the liver. There are no apparent gross lesions in
uninoculated embryos. Samples of bile and liver collected from
inoculated eggs at the day of natural hatching (12 days
postinoculation) were tested by RT-PCR. Avian HEV RNA was detected
in both bile and liver samples. The titer of virus in the bile
recovered from embryos was about 10.sup.7 genomic equivalent per ml
(GE/ml), indicating that avian HEV replicates in embryonated
chicken eggs. The virus recovered from inoculated eggs was used as
the source for subsequent genetic characterization.
EXAMPLE 7
Amplification and Sequence Determination of the 3' Half of the
Avian HEV Genome
[0096] An attempt to amplify an approximately 4 kb fragment at the
3' half of the avian HEV genome was pursued. The attempt initially
failed to amplify the fragment with AmpliTaq Gold polymerase.
However, in the presence of 5% v/v DMSO, a weak signal of a PCR
product of approximately 4 kb was generated with AmpliTaq Gold
polymerase (FIG. 1). To increase the amplification efficiency, the
PCR with a mixture of pfu polymerase and Taq DNA polymerase was
performed in the presence of 10 .mu.l of cDNA and 1.7 mM MgCL.sub.2
by using an eLONGase.RTM. kit. After 32 cycles of amplification, an
abundant amount of PCR product of approximately 4 kb was generated
(FIG. 1). The resulting PCR product was subsequently cloned into a
TA vector. Three cDNA clones were selected and sequenced for both
DNA strands. The number of poly (A) residues at the 3' end of each
of the three cDNA clones was different (19, 23, and 26 residues,
respectively), indicating that these 3 clones sequenced represent
independent cDNA clones. This 4 kb genomic fragment contains the
complete ORFs 2 and 3 (set forth in SEQ ID NO: 7 and SEQ ID NO: 9,
respectively), the complete RNA-dependent RNA polymerase (RdRp)
gene (set forth in SEQ ID NO: 5), a partial helicase gene of the
ORF1 (set forth in SEQ ID NO: 3), and the complete 3' noncoding
region (NCR).
EXAMPLE 8
Sequence Analysis of the ORF1 Region
[0097] The sequences of the three independent cDNA clones have the
same size but differ in 16 nucleotide positions. However, at any
given position, two of the three cDNA clones have the same
nucleotide. Therefore, a consensus sequence was produced. The
resulting consensus sequence of the 3' half genomic fragment of
avian HEV is 3,931 nucleotides in length, excluding the poly (A)
tract at the 3' end and the sequence of the 5' sense primer used
for amplification. Sequence analysis revealed that the novel virus
associated with HS in chickens is genetically related to human and
swine HEV. Two complete ORFs (ORFs 2 and 3), and one incomplete
ORF1 were identified in this genomic region.
[0098] The incomplete ORF1 sequence of avian HEV was aligned with
the corresponding regions of human and swine HEV strains.
Significant nucleotide and amino acid sequence identities were
found in the ORF1 region between avian HEV and known HEV strains
(Table 2, below). The avian HEV ORF1 region sequenced thus far
contained the complete RdRp gene and a partial helicase gene. The
RdRp gene of avian HEV encodes 483 amino acid residues and
terminates at the stop codon of ORF1. A GDD motif (positions
343-345 in RdRp gene) that is believed to be critical for viral
replication was identified (FIGS. 2A-2B), and this motif was found
in all RdRps (G. Kamer et al., "Primary structural comparison of
RNA-dependent polymerases from plant, animal and bacterial
viruses," Nucleic Acids Res. 12:7269-7282 (1984)). The RdRp gene of
avian HEV is 4 amino acid residues shorter than that of known HEV
strains (FIGS. 2A-2B), and shared 47% to 50% amino acid and 52% to
53% nucleotide sequence identity with that of known HEV strains
(Table 2, below). The C-terminal 146 amino acid residues of the
incomplete helicase gene of avian HEV shared approximately 57-60%
nucleotide sequence and 58-60% amino acid sequence identities with
the corresponding region of other HEV strains. The helicase gene of
avian HEV is the most conserved region compared to known HEV
strains. There is no deletion or insertion in this partial helicase
gene region between avian HEV and other HEV strains. A 439 bp
sequence of BLSV is available in the helicase gene region (C. J.
Payne et al., 1999, supra), and avian HEV shared 80% nucleotide
sequence identity with BLSV in this region.
2TABLE 2 Pairwise comparison of the RNA-dependent RNA polymerase
(RdRp) gene of the avian HEV with that of known HEV strains Avian
HEV Burma D11092 China D11093 China HEV-T1 China Hetian China
Hydarabad India K52-87 China Avian HEV 53.sup.a 53 53 53 53 52 53
Burma 49 93 93 76 93 96 93 D11092 China 47 94 97 75 98 92 98 D11093
China 49 98 94 74 97 92 98 HEV-T1 China 50 86 82 86 75 75 75 Hetian
China 49 98 93 98 86 92 98 Hydarabad India 49 97 93 97 85 97 92
K52-87 China 49 99 94 98 87 98 98 Madras India 47 95 90 94 82 94 94
95 Mexico 48 88 84 88 85 88 87 89 Myanmar 49 99 93 98 86 97 97 98
Nepal 49 98 93 98 86 98 97 98 Sar-55 Pakistan 49 99 94 98 87 98 98
99 Swine HEV USA 49 87 83 87 89 87 86 88 US1 USA 49 87 82 87 89 87
86 87 US2 USA 49 87 82 87 88 87 86 87 X98292 India 49 98 94 98 87
98 97 99 Madras Mexico Myanmar Nepal Sar-55 Pakistan Swine HEV USA
US1 USA US2 USA X98292 India Avian HEV 52 52 53 53 53 52 52 52 53
Burma 95 74 98 96 93 75 74 75 93 D11092 China 91 76 93 92 98 75 75
75 94 D11093 China 91 76 93 91 97 75 74 75 94 HEV-T1 China 74 73 75
76 75 76 75 75 75 Hetian China 90 74 93 92 98 75 74 75 94 Hydarabad
India 94 76 96 95 92 75 74 74 92 K52-87 China 91 76 93 92 98 75 75
75 94 Madras India 75 94 95 91 74 73 74 91 Mexico 85 76 76 77 74 62
73 76 Myanmar 95 88 95 93 75 74 75 92 Nepal 94 88 98 92 75 75 75 92
Sar-55 Pakistan 95 89 98 98 75 75 75 94 Swine HEV USA 84 86 87 87
88 92 92 76 US1 USA 83 86 87 87 87 99 92 75 US2 USA 83 86 87 87 87
99 98 75 X98292 India 94 89 98 98 99 88 88 88 .sup.aThe values in
the table are percentage identity of amino acids (lower left half)
or nucleotides (upper right half).
EXAMPLE 9
Sequence Analysis of the ORFs 2 and 3
[0099] The ORF2 gene of avian HEV consists of 1,821 nucleotides
with a coding capacity of 606 amino acids, about 60 amino acids
shorter than that of other HEV strains. The ORF2 gene of avian HEV
overlaps with ORF3 (FIGS. 3A-3C), and terminates at stop codon UAA
located 130 bases upstream the poly (A) tract. The predicted amino
acid sequence of ORF2 contains a typical signal peptide at its
N-terminus followed by a hydrophilic domain (FIG. 4). The sequence
of the avian HEV signal peptide is distinct from that of known HEV
strains (FIGS. 5A-5C). However, it contains common signal peptide
features that are necessary for the translocation of the peptide
into endoplasmic reticulum: a positively charged amino acid
(Arginine) at its N-terminus, a core of highly hydrophobic region
(rich in Leucine residues) and a cleavage site (SRG-SQ) between
position 19 and 20 (FIGS. 5A-5C). Sequence analysis of the ORF2
revealed that the region between the signal peptide and the
conserved tetrapeptide APLT (positions 108-111) is hypervariable,
and 54 amino acid residues of avian HEV are deleted in this region
(FIGS. 5A-5C). Three putative N-linked glycosylation sites were
identified in the ORF2 of avian HEV: NLS (pos. 255-257), NST (pos.
510-512) and NGS (pos. 522-524). Three N-linked glycosylation sites
were also identified in known HEV strains but the locations are
different from those of avian HEV. The first glycosylation site in
known HEV strains is absent in avian HEV (FIGS. 5A-5C), and the
third glycosylation site in avian HEV is absent in the known HEV
strains.
[0100] The ORF2 gene of known HEV strains varies slightly in size,
ranging from 655 to 672 amino acid residues, but most strains have
a ORF2 gene of 660 amino acid residues. The ORF2 of avian HEV has
606 amino acid residues, which is 54 amino acids shorter than that
of most known HEV strains. The deletions are largely due to the
shift of the ORF2 start codon of avian HHEV to 80 nucleotides
downstream from that of known HEV strains (FIGS. 3A-3C). The
putative capsid gene (ORF2) of avian HEV shared only 42% to 44%
amino acid sequence identity with that of known HEV strains (Table
3, below), when the major deletion at the N-terminus is taken into
consideration. However, when the N-terminal deletion is not
included in the comparison, avian HEV shared 48% to 49% amino acid
sequence identity with the corresponding region of other HEV
strains.
[0101] Multiple sequence alignment revealed that the normal start
codon of the ORF3 gene in known HEV strains does not exist in avian
HEV due to base substitutions (FIGS. 3A-3C). Avian HEV utilizes the
ORF2 start codon of other HEV strains for its ORF3, and
consequently the ORF3 of avian HEV starts 41 nucleotides downstream
from the start codon of known HEV strains (FIGS. 3A-3C). Unlike
known HEV strains, the ORF3 gene of avian HEV does not overlap with
the ORF1 and locates 33 bases downstream from the ORF1 stop codon
(FIGS. 3A-3C). The ORF3 of avian HEV consists of 264 nucleotides
with a coding capacity of 87 amino acid residues, which is 24 to 37
amino acid residues shorter than that of known HEV strains.
Sequence analysis indicated that the ORF3 of avian HEV is very
divergent compared to that of known HEV strains.
3TABLE 3 Pairwise comparison of the putative capsid gene (ORF2) of
avian HEV with that of known HEV strains D11092 D11093 Avian
HEV.sup.a Avian HEV.sup.b Burma China China HEV-T1 China Hetian
China Hydarabad India KS2-87 China Avian HEV.sup.a 47 47 47 44 47
47 47 Avian HEV.sup.b 51 51 51 48 51 51 51 Burma 44 49 94 93 77 94
96 94 D11092 China 44 49 99 97 77 98 93 98 D11093 China 44 49 98 98
77 97 93 98 HEV-T1 China 42 48 88 88 87 77 76 77 Hetian China 44 49
99 99 98 88 93 98 Hydarabad India 44 49 97 97 96 86 96 93 KS2-87
China 44 49 99 99 98 88 98 97 Madras India 44 49 99 99 98 88 98 96
98 Mexico 43 48 93 93 92 86 92 91 92 Myanmar 43 48 98 98 98 87 98
96 98 Nepal 44 49 98 98 98 87 98 96 98 Sar-55 Pakistan 44 49 99 99
98 88 99 97 99 Swine HEV USA 43 49 91 91 90 90 91 89 91 US1 USA 43
49 91 92 91 88 91 90 91 US2 USA 44 49 91 91 91 90 91 90 91 Egypt93
44 49 98 98 97 88 98 96 98 Egypt94 44 49 99 99 98 88 98 96 98
Morroco 44 49 99 99 98 88 98 97 98 AKL90 44 49 99 99 98 88 98 97 98
Sar-55 Swine HEV Madras Mexico Myanmar Nepal Pakistan USA US1 USA
US2 USA Egypt93 Egypt94 Morroco AKL90 Avian HEV.sup.a 47 45 47 47
47 46 45 46 47 47 48 47 Avian HEV.sup.b 51 49 51 51 51 50 49 50 51
51 51 51 Burma 96 80 97 98 93 79 79 79 91 90 89 97 D11092 China 93
81 93 93 98 80 79 79 91 91 90 93 D11093 China 93 80 93 93 97 79 78
79 91 91 90 93 HEV-T1 China 77 77 78 77 78 78 78 79 77 77 78 77
Hetian China 93 80 93 93 98 80 79 79 91 91 90 93 Hydarabad India 95
80 95 97 92 79 78 79 90 90 89 97 KS2-87 China 93 81 93 93 98 80 79
79 91 91 90 93 Madras India 80 96 96 92 97 97 97 90 90 90 96 Mexico
92 80 80 81 78 77 79 80 80 81 80 Myanmar 98 92 96 93 79 79 79 91 90
89 96 Nepal 98 92 98 93 79 79 79 90 90 90 97 Sar-55 Pakistan 99 93
98 98 80 79 79 91 91 91 93 Swine HEV USA 91 90 91 90 91 92 92 79 79
80 79 US1 USA 91 90 91 91 91 97 91 78 79 79 79 US2 USA 91 90 92 91
91 98 98 79 79 79 79 Egypt93 98 92 98 97 98 91 92 92 96 91 91
Egypt94 98 93 98 98 98 91 92 91 99 91 90 Morroco 98 93 98 98 99 91
91 91 98 99 90 AKL90 98 93 98 98 99 91 91 91 98 98 99 The values in
the table are percentage identity of amino acids (lower left half)
or nucleotides (upper right half). .sup.aPercentage identity when
the major deletion at the N-terminal region of ORF2 is taken into
consideration. .sup.bPercentage identity when the major deletion is
not included in the comparison.
EXAMPLE 10
Sequence Analysis of the 3' NCRs
[0102] The region between the stop codon of the ORF2 and the poly
(A) tail of avian HEV, the 3' NCR, is 130 nucleotides. Sequence
analysis revealed that the 3' NCR of avian HEV is the longest among
all known HEV strains. The 3 NCRs of known HEV strains range from
65 to 74 nucleotides (FIG. 6). Multiple sequence alignment
indicated that the 3' NCRs of HEV is highly variable, although a
stretch of sequence immediately proceeding the poly (A) tract is
relatively conserved (FIG. 6).
EXAMPLE 11
Identification of a Major Deletion in the ORFs 2 and 3 Overlapping
Region of Avian HEV
[0103] Sequence analyses revealed a major deletion of 54 amino acid
residues in avian HEV between the putative signal peptide and the
conserved tetrapeptide APLT of the ORF2 (FIGS. 5A-5C). To rule out
the possibility of RT-PCR artifacts, a pair of avian HEV-specific
primers flanking the deleted region was designed (Table 1, FIGS.
3A-3C). The 3' antisense primer (RdelAHEV) located before the ORF3
stop codon of avian HEV, and the 5' sense primer (FdelAHEV) located
within the C-terminal region of the ORF1. To minimize potential
secondary structure problems, reverse transcription was performed
at 60.degree. C. with a One Step RT-PCR Kit (Qiagen Inc., Valencia,
Calif.). PCR was performed with 35 cycles of denaturation at
95.degree. C. for 40 seconds, annealing at 55.degree. C. for 30
seconds and extension at 72.degree. C. for 1 minute. In addition,
PCR was also performed with shorter annealing time and higher
denaturation temperature to avoid potential problems due to
secondary structures. The PCR reaction consisted of an initial
enzyme activation step at 95.degree. C. for 13 minutes, followed by
35 cycles of denaturation at 98.degree. C. for 20 seconds,
annealing at 55.degree. C. for 5 seconds and extension at
73.degree. C. for 1 minute. It has been reported that formamide or
DMSO could enhance the capability of PCR to amplify certain genomic
regions of HEV (S. Yin et al., "A new Chinese isolate of hepatitis
E virus: comparison with strains recovered from different
geographical regions," Virus Genes 9:23-32 (1994)). Therefore, a
sufficient amount to make 5% (v/v) of formamide or DMSO was added
in the PCR reactions. A PCR product of the same size (502 bp) as
observed in a conventional PCR is produced with various different
RT-PCR parameters and conditions including the addition of 5% (v/v)
of formamide or DMSO, the use of higher denaturation temperature
and short annealing time, and the synthesis of cDNA at 60.degree.
C. (FIG. 7). The deletion was further confirmed by directly
sequencing the 502 bp PCR product.
EXAMPLE 12
Phylogenetic Evidence of Avian HEV as a New Genotype
[0104] Phylogenetic analyses based on three different genomic
regions of HEV (a 439 bp of the helicase gene, a 196 bp of the RdRp
gene, and a 148 bp of the ORF2 gene) identified at least 5 distinct
genotypes of HEV (FIG. 8). The topology of the three trees based on
different genomic regions is very similar. Similar phylogenetic
trees were also produced with the complete RdRp and ORF2 genes of
HEV strains in which their sequences are known. Most Asian strains
of HEV are related to the prototype Burmese strain and clustered
together, and these Burmese-like Asian strains of HEV represent
genotype 1. The African strains of HEV (Egypt 93, Egypt 94 and
Morroco) were related to, but distinct from, Burmese-like strains
in the genotype 1. The limited sequences available for these
African strains do not allow for a determination of whether they
represent a distinct genotype or a subgenotype within the genotype
1. The single Mexican strain of HEV represents genotype 2. The
genotype 3 of HEV consists of two U.S. strains of human HEV (US1,
US2), a U.S. strain of swine HEV, a New Zealand strain of swine
HEV, and several European strains of human HEV (Greek 1, Greek 2,
Italy). The genotype 4 includes several strains of HEV identified
from patients in China (HEV-T1, Ch-T11, Ch-T21, 93G) and Taiwan
(TW7E, TW4E, TW8E). Avian HEV is the most divergent and represents
the new genotype 5. Based on the limited sequence available for
BLSV, it appears that the BLSV identified from chickens in
Australia clustered with the genotype 5 of avian HEV, but the avian
HEV retained significant differences in nucleotide sequence
indicating that the avian HEV represents a new and distinct viral
strain. Phylogenetic evidence that avian HEV is the most divergent
strain of HEV identified thus far and represents a new
genotype.
EXAMPLE 13
Isolation of Avian HEV in Embryonated Chicken Eggs
[0105] Others have failed to isolate the agent associated with HS
syndrome in chicken embryos with conventional routes of egg
inoculation (H. L. Shivaprasad et al., "Necrohemorrhagic hepatitis
in broiler breeders," Proc. Western Poult. Dis. Conf., p. 6,
Sacramento, Calif. (1995)). Previous studies in pigs and primates
showed that the I.V. route of inoculation is the most sensitive
method to infect animals with the hepatitis E virus (HEV) (P. G.
Kasorndorkbua et al., "Use of a swine bioassay and a RT-PCR assay
to assess the risk of transmission of swine hepatitis E virus in
pigs," J. Virol. Methods, In Press (2001); P. G. Halbur et al.,
"Comparative pathogenesis of infection of pigs with hepatitis E
viruses recovered from a pig and a human," J. Clin. Microbiol.
39:918-923 (2001); X. J. Meng et al., "Experimental infection of
pigs with the newly identified swine hepatitis E virus (swine HEV),
but not with human strains of HEV," Arch. Virol. 143:1405-1415
(1998); X. J. Meng et al., "Genetic and experimental evidence for
cross-species infection by the swine hepatitis E virus," J. Virol.
72:9714-9721 (1998)).
[0106] Surprisingly, the present attempt to isolate the agent
associated with HS syndrome by I.V. inoculation of embryonated eggs
was successful. A sample of bile collected from a 42-week-old
Leghorn chicken with HS syndrome in California was used as the
virus source (G. Haqshenas et al., "Genetic identification and
characterization of a novel virus related to the human hepatitis E
virus from chickens with Hepatitis-Splenomegaly Syndrome in the
United States," J. Gen. Virol. 82:2449-2462 (2001)). The undiluted
positive bile contained about 10.sup.7 genomic equivalents (GE) of
avian HEV per ml measured by an avian HEV-specific
semi-quantitative PCR (id.). Specific-pathogen-free (SPF) eggs were
purchased at 1 day of embryonated age (Charles River SPAFAS, Inc.
North Franklin, Conn.). At 9 days of embryonated age, 40 eggs were
I.V.-inoculated with 100 .mu.l of a 10.sup.-4 dilution of the
original positive bile, and 20 eggs remain uninoculated as
controls. On the day of natural hatching (21 days of embryonated
age), half of the inoculated embryos were sacrificed, and bile and
samples of liver and spleen were harvested. The other half of the
inoculated embryos were allowed to hatch, and most of the hatched
chickens were necropsied at 2 to 3 days of age. Bile and liver
collected from the necropsied embryos and chickens were tested
positive for avian HEV RNA. The titer of virus in the bile
recovered from inoculated embryos was about 10.sup.6 GE/ml,
indicating that avian HEV replicates in embryonated chicken eggs.
Four hatched chickens were monitored continuously. The hatched
chickens seroconverted to anti-HEV, and avian HEV shed in feces.
The feces collected from a hatched chicken at 8 days of age contain
about 10.sup.5 to 10.sup.6 GE/ml of 10% fecal suspension, and this
was the source of avian HEV for the subsequent animal studies.
EXAMPLE 14
Experimental Infection of Young SPF Chickens with Avian HEV
[0107] As a first step to determine if chickens can be infected
experimentally with avian HEV, 12 SPF chickens of 3-to-6 days of
age were I.V.-inoculated, each with about 2.times.10.sup.4 GE/ml of
avian HEV. Two uninoculated chickens were kept in the same cage
with the inoculated ones as contact controls. Eight uninoculated
chickens housed in a separate room served as negative controls.
Fecal swabs were collected from all chickens every 3 days and
tested for avian HEV RNA. Weekly sera from all chickens were tested
for anti-HEV antibodies. Avian HEV RNA was detected in the feces of
all inoculated chickens but not of the controls. Fecal shedding of
avian HEV lasted about 2 to 3 weeks from 9 to 28 days
post-inoculation (DPI). As expected with a fecal-orally transmitted
virus, the two uninoculated contact control chickens (housed in the
same cage with the inoculated ones) also became infected, and fecal
virus shedding in the two contact control chickens started late
from 18 to 35 DPI. Seroconversion to anti-HEV antibodies in
inoculated chickens (but not in controls) occurred at about 32 to
38 DPI. Two infected and two control chickens were necropsied each
at 25 and 35 DPI. The biles and feces of the necropsied chickens
were positive for avian HEV RNA. There were no significant gross
lesions in the infected young chickens. Microscopic liver lesions
in infected chickens (but not in controls) were characterized by
lymphoplasmacytic hepatitis with moderate to severe periportal,
perivascular/vascular and occasional random foci of infiltration of
lymphocytes mixed with a few plasma cells. The results demonstrate
the successful reproduction of avian HEV infection in young
chickens of 3-to-6 days of age but not the full-spectrum of HS
syndrome.
EXAMPLE 15
Experimental Reproduction of Avian HEV Infection and HS Syndrome in
Leghorn SPF Layer Chickens and Broiler Breeder Chickens
[0108] The failure to reproduce the full-spectrum of HS syndrome in
young chickens is not surprising since, under field conditions,
only broiler breeder and laying hens of 30-72 weeks of age
developed HS syndrome (H. L. Shivaprasad et al., "Necrohemorrhagic
hepatitis in broiler breeders," Proc. Western Poult. Dis. Conf., p.
6, Sacramento, Calif. (1995); C. Riddell, "Hepatitis-splenomegaly
syndrome," DISEASE OF POULTRY, p. 1041 (1997)); S. J. Ritchie et
al., "Hepatitis-splenomegaly" syndrome in commercial egg laying
hens, Can. Vet. J. 32:500-501(1991)). Thus, two additional studies
were performed to determine if avian HEV infection and HS syndrome
could be experimentally reproduced in SPF layer chickens and
broiler breeder chickens.
[0109] Layer chickens: Twenty (20) Leghorn SPF layer chickens of 60
weeks of age were purchased from Charles River SPAFAS, Inc. North
Franklin, Conn. Ten chickens were I.V.-inoculated each with
10.sup.4 GE/ml of avian HEV, and housed in 5 isolators of 2
chickens each. Another 10 chickens, kept in 5 isolators in a
separate room, were uninoculated as negative controls. Fecal swabs
were collected from all chickens every 4 days. Avian HEV RNA was
detected by RT-PCR from 8 to 27 DPIs in feces of infected chickens
but not of controls. Sera were collected every 10 days, and
seroconversion to anti-HEV antibodies occurred as early as 20 DPI.
Two infected and two control chickens were necropsied each at 13,
17 and 21 DPIs. Avian HEV RNA was detected in the biles and feces
of necropsied inoculated chickens but not of controls. Gross
lesions characteristic of HS syndrome were observed in infected
chickens, including hepatomegaly, subcapsular hemorrhages in livers
(FIG. 18B) and pale foci on splenic capsular. Ovarian regression
was also noticed in some infected chickens.
[0110] Significant microscopic lesions of liver and spleen
consistent with HS syndrome were observed in infected SPF layer
chickens. Livers from infected chickens had lymphoplasmacytic
hepatitis with mild to moderate infiltration of lymphocytes in the
periportal and perivascular regions (FIG. 19B). There were also
foci of lymphocytes randomly scattered throughout the liver. A few
focal hepatocellular necrosis with lymphocyte infiltration was also
observed. Spleens from infected chickens had a mild increase in
mononuclear phagocytic system (MPS) cells. No significant gross or
microscopic lesions were seen in control chickens.
[0111] Broiler breeder chickens: Six broiler breeder chickens of 64
weeks of age were I.V.-inoculated each with 10.sup.4 GE/ml of avian
HEV. Another 6 chickens were uninoculated as controls. Fecal swabs
were collected every 4 days, and avian HEV RNA was detected in
feces of all inoculated chickens from 12 to 27 DPI but not from
controls. Sera were collected every 10 days and, like SPF layer
chickens, seroconversion to anti-HEV antibodies also occurred in
broiler breeder chickens as early as 20 DPI. Two infected and two
control chickens were each necropsied at 14 and 21 DPI. Like layer
chickens, the infected broiler breeders also had gross lesions
consistent with HS syndrome including swollen liver and hemorrhages
in the live and spleen. Microscopic liver lesions were
characterized by lymphoplasmacytic hepatitis with infiltration of
lymphocytes in the periportal and perivascular regions, and mild to
severe vacuolation of most hepatocytes. Sections of spleens had a
mild increase in MPS cells. No significant gross or microscopic
lesions were observed in controls.
[0112] These two studies demonstrate the successful reproduction of
avian HEV infection and HS syndrome with characteristic gross and
microscopic lesions in SPF layers and broiler breeder chickens.
Avian HEV with a sequence identical to the virus in the inoculum
was re-isolated from experimentally infected chickens. Thus, avian
HEV as a causative agent of HS syndrome in chickens is confirmed in
accordance with Koch's germ theory of disease (Koch, R., 1876,
Untersuchungen ueber Bakterien V. Die Aetiologie der
Milzbrand-Krankheit, begruendent auf die Entwicklungsgeschichte des
Bacillus Anthracis. Beitr. z. Biol. D. Pflanzen 2: 277-310, In
Milestones in Microbiology: 1556 to 1940, translated and edited by
Thomas D. Brock, ASM Press. 1998, p. 89).
EXAMPLE 16
Evaluation of Field Isolates of Avian HEV from Chickens with HS
Syndrome
[0113] Strains of human and swine HEVs are genetically heterogenic.
To determine the extent of heterogeneity among avian HEV isolates,
the helicase gene region of 8 additional avian HEV isolates from
chickens with HS syndrome from different geographic regions of the
U.S. was amplified by RT-PCR and sequenced (Table 4, below),
showing that field isolates of avian HEV from chickens with HS
syndrome are heterogeneic. Sequence and phylogenetic analyses
revealed that, like swine and human HEVs, avian HEV isolates
identified from different geographic regions of the United States
are also heterogeneic (FIG. 20). Avian HEV isolates shared 79 to
96% nucleotide sequence identities with each other, 76-80%
nucleotide sequence identities with BLSV and about 60% identities
with swine and human HEVs (Table 4, below). The data also suggested
that the BLS disease in Australian chickens and the HS syndrome in
North American chickens are caused by a similar virus with about
76-80/% sequence identities.
4TABLE 4 Pairwise comparison of the nucleotide sequences of the
helicase gene region of 8 field isolates of avian HEV (shown in
boldface) identified from chickens with HS syndrome in the U.S.
with that of other selected HEV strains Flock Year 2966G 0242 4449
4090 3690 3158.5 3077 9318B aHEV BLSV T1 Mexico US2 Swine Sar-55
location Isol. 2966G 83 81 79 81 98 79 96 86 77 56 60 59 59 61 WI
2000 0242 83 88 86 83 83 86 83 80 79 56 59 59 60 59 CA 1994 4449 81
88 96 84 83 96 82 80 77 56 60 60 60 59 NY 2000 4090 79 86 96 83 80
94 81 79 76 56 60 59 59 59 East 2000 coast 3690 81 83 84 83 81 84
80 80 80 57 60 59 60 59 CT 2000 3158.5 98 83 83 80 81 80 96 86 78
57 61 59 60 61 CA 1997 3077 79 86 96 94 84 80 81 79 77 56 60 60 60
59 CA 1993 9318B 96 83 82 81 80 96 81 88 78 57 60 59 59 60 Mid 2000
west aHEV* 86 80 80 79 80 86 79 88 77 57 61 57 58 60 CA 1993
BLSV.dagger. 77 79 77 76 80 78 77 78 77 56 59 60 60 59 T1 56 56 56
56 57 57 56 57 57 56 73 75 75 76 Mexico 60 59 60 60 60 61 60 60 61
59 73 72 75 78 US2 59 59 60 59 59 59 60 59 57 60 75 72 91 75
Swine.dagger-dbl. 59 60 60 59 60 60 60 59 58 60 75 75 91 75
Sar-55.paragraph. 61 59 59 59 59 61 59 60 60 59 76 78 75 75 *aHEV,
the prototype avian HEV. .dagger.BLSV, the causative agent of BLS
disease in Australian chickens. .dagger-dbl.Swine, the prototype
U.S. swine HEV. .paragraph.Sar-55, the Pakistani strain of human
HEV.
EXAMPLE 17
Expression and Purification of the Truncated ORF2 Capsid Protein of
Avian HEV in a Bacterial Expression System
[0114] The truncated ORF2 protein of avian HEV containing the
C-terminal 268 amino acid residues of ORF2 was expressed and
characterized. The 804 bp sequence of the C-terminus of the avian
HEV ORF2 was amplified with a set of avian HEV-specific primers: a
sense primer (5'-GGGGGATCCAGTACATGTA- CGGCCGGCCTG-3', which
corresponds to SEQ ID NO: 10) with an introduced BamHI site
(underlined), and an antisense primer (5'-GGGGAATTCTTAGGGTGGTG-
AGGGGAATG-3', which corresponds to SEQ ID NO: 11) with an
introduced EcoRI site (underlined). The BamHI and EcoRI sites were
introduced at the 5' ends of the sense and antisense primers,
respectively, to facilitate subsequent cloning steps. Proofreading
Pfu DNA polymerase (Stratagene, La Jolla, Calif.) was used for PCR
amplification of the fragment. The obtained PCR amplified fragment
was purified and digested with BamHI and EcoRI restriction enzymes
and cloned into the pRSET-C expression vector (Clontech
Laboratories, Inc., Palo Alta, Calif.). The truncated ORF2 gene was
in-frame with the coding sequence of the Xpress.TM. epitope
(Invitrogen Corporation, Carlsbad, Calif.) located upstream of the
multiple-cloning site of the expression vector. E. coli DH5.alpha.
cells were transformed with the recombinant plasmids. The
recombinant expression vector was isolated with a Qiagen Plasmid
Mini Kit (Qiagen Inc., Valencia, Calif.), and confirmed by
restriction enzyme digestions and DNA sequencing.
[0115] The recombinant plasmids were transformed into
BL21(DE3)pLysS competent cells that have been engineered to produce
T7 RNA polymerase. Expression of the fusion protein was driven by a
T7 promoter sequence upstream of the Xpress.TM. epitope sequence
(Invitrogen Corporation, Carlsbad, Calif.). By using pRSET-C
vector, the recombinant fusion protein is tagged by six tandem
histidine residues at the amino terminus (N-terminus) that have a
high affinity for ProBond.TM. resin (Invitrogen Corporation,
Carlsbad, Calif.). The bacterial cells were grown in SOB broth
containing 50 .mu.g/ml of ampicillin and 25 .mu.g/ml of
chloramphenicol. Expression of the fusion protein was induced by
the addition of 1 mM isopropyl-beta-D-thiogalactopyranoside (IPTG)
for 4-5 hrs at 37.degree. C. The fusion protein was expressed in E.
coli strain BL21(DE3)pLysS as inclusion bodies. To confirm that the
over-expressed protein contains Xpress.TM. epitope (Invitrogen
Corporation, Carlsbad, Calif.), the crude bacterial lysates
separated on a 12% polyacrylamide gel containing 0.1% SDS and
transferred onto a nitrocellulose membrane (Osmonics, Inc.,
Minnetonka, Minn.). The immobolized protein on the membrane was
incubated with a with horseradish peroxidase (HRP)-conjugated
monoclonal antibody, known to be against Xpress.TM. epitope
(Invitrogen Corporation, Carlsbad, Calif.) at 1:5,000 dilution. The
immunocomplexes were detected using 4-chloro-1-naphthol (Sigma, St.
Louis, Mo.).
[0116] From 50 ml of bacterial cultures, the fusion protein was
purified by the use of ProBond.TM. Purification System (Invitrogen
Corporation, Carlsbad, Calif.) based on the affinity of ProBond.TM.
resin for His-tagged recombinant fusion protein. Bacterial cells
were lysed with guanidinium lysis buffer (6 M guanidine
hydrochloride, 20 mM sodium phosphate, 500 mM sodium chloride, pH
7.8) and insoluble debris was clarified by centrifugation at 3,000
g for 10 minutes at 40 C. The supernatant was added to the resin
pre-equilibrated with the binding buffer and gently agitated for 10
minutes at room temperature to allow the fusion protein to bind the
resin. The protein-bound resin was serially washed six times with
denaturing binding buffer (8 M urea, 20 mM sodium phosphate, 500 mM
sodium chloride) twice at each pH of 7.8, 6.0 and 5.3. The fusion
protein was eluted in the elution buffer containing 8 M urea, 20 mM
sodium phosphate and 500 mM sodium chloride (pH 4.0). The fractions
containing the highest concentrations of protein were determined by
the use of Bio-Rad protein assay reagent (BioRad, Carlsbad,
Calif.). Five micrograms of the purified protein was analyzed by
SDS-PAGE. The purified fusion protein hybridized with the MAb
against Xpress.TM. epitope (Invitrogen Corporation, Carlsbad,
Calif.).
[0117] The nucleotide sequence of the insert was confirmed by
automated cycle sequencing. The recombinant plasmid containing the
truncated ORF2 gene of avian HEV was transformed into E. coli
strain BL21(DE3)pLysS. Upon induction with IPTG, the truncated ORF2
capsid protein of avian HEV was expressed in this bacterial strain
with a very high yield. The expressed protein was observed on the
gel at the size of about 32 kD (FIG. 21A). Samples taken at
different time points revealed that the maximum expression occurred
at about 4 to 5 hrs after induction with IPTG (FIG. 21A). Western
blot analysis using a monoclonal antibody against Xpress.TM.
epitope (Invitrogen Corporation, Carlsbad, Calif.) of the fusion
protein confirmed the expression of the avian HEV ORF2 protein
(FIG. 21B). Although the bacterial cells used in this study contain
pLysS plasmid to minimize the background protein expressions,
background expression was still observed. The fusion protein was
expressed as inclusion bodies in the bacterial cells and was shown
to be insoluble. The protein purification method was very efficient
and about 6 mg of protein were obtained from 50 ml of the bacterial
culture.
EXAMPLE 18
Evaluation of Antigenic Epitopes of Capsid Protein of Avian HEV,
Human HEV, Swine HEV and Australian Chicken BLSV
[0118] In Western blot analysis, the purified truncated ORF2
protein of avian HEV reacted with the antiserum obtained from
chickens experimentally infected with avian HEV but not with sera
from normal control chickens. To prepare antiserum against avian
HEV, specific-pathogen-free (SPF) chickens (SPAFAS Inc.) were
inoculated intravenously with a diluted bile sample containing
10.sup.3 GE/ml of avian HEV. The inoculated chickens excreted avian
HEV in the feces and seroconverted to avian HEV antibodies. The
convalescent sera collected at 30 days post inoculation were used
as the avian HEV antiseum in this experiment. The antiserum against
Sar-55 strain of human HEV was prepared by immunizing SPF pigs with
baculovirus expressed and HPLC-purified capsid protein of the
Sar-55 HEV. The antisera against swine HEV and US2 strain of human
HEV were convalescent sera from pigs experimentally co-infected
with these two HEV strains. The antiserum against Australia chicken
BLSV was also kindly provided by Dr. Christine Payne (Murdoch
University, Australia). The putative capsid protein of human HEV
Sar-55 and swine HEV were expressed in baculovirus systems as
described herein. The recombinant proteins were a gift from Drs
Robert Purcell and Suzanne Emerson (NIH, Bethesda, Md.). The
HPLC-purified recombinant ORF2 capsid proteins of human HEV Sar-55
and swine HEV were used in this study.
[0119] Western blot analyses were used to determine if the
truncated ORF2 protein of avian HEV shares antigenic epitopes with
that of human HEV, swine HEV and BLSV. The purified recombinant
truncated ORF2 protein (250 ng/lane) of avian HEV was separated by
SDS-PAGE and transferred onto a nitrocellulose membrane. The blots
were cut into separate strips and then blocked in blocking solution
(20 mM Tris-Cl, 500 mM NaCl, pH 7.5) containing 2% bovine serum
albumin (BSA) for 1 hour. The strips were then incubated overnight
at room temperature with 1:100 dilutions of antisera against avian
HEV, swine HEV, human HEV and BLSV in Tris-buffered saline (20 mM
Tris-Cl, 500 mM NaCl, pH 7.5) (TBS) containing 0.05% Tween.RTM. 20
(polysorbate 20, commercially available from Mallinckrodt Baker,
Inc., Phillipsburg, N.J.) (TBST) and 2% BSA. The original purified
antibody against BLSV was diluted 1:1000 in TBST. Dilutions 1:100
of preinoculation swine sera were used as the negative controls.
The strips were washed 2 times with TBST and once with TBS.
Following 3 hrs incubation with HRP-conjugated goat anti-swine IgG
(1:2000, Research Diagnostics Inc., Flanders, N.J.) and
HRP-conjugated rabbit anti-chicken IgY (1:2000, Sigma, St. Louis,
Mo.), the strips were washed as described above and the
immunocomplexes were detected using 4-chloro-1-naphthol.
[0120] To further confirm the cross-reactivity between avian, swine
and human HEVs, approximately 250 ng of HPLC purified recombinant
capsid proteins of swine HEV and Sar-55 human HEV were separated by
SDS-PAGE and blotted onto a nitrocellulose membrane. The blot was
incubated with antisera against avian HEV, swine HEV and human HEV.
Serum dilutions, incubation and washing steps were carried out as
described above. Anti-chicken IgY conjugated with HRP was used as
the secondary antibody as described above.
[0121] The purified truncated ORF2 protein of avian HEV reacted
strongly in Western blot analyses with convalescent sera from SPF
chickens experimentally infected with avian HEV, HEV antibodies
(antisera) raised against the capsid protein of Sar-55 human HEV
and convalescent sera against the US2 strain of human HEV and swine
HHEV, and the antiserum against the Australian chicken BLSV (FIGS.
22A and 22B). The purified truncated avian HEV ORF2 protein did not
react with the preinoculation control chicken sera. Convalescent
antisera from chickens experimentally infected with avian HEV
reacted with the HPLC-purified recombinant ORF2 protein of Sar-55
human HEV. Swine HEV antiserum reacted strongly with the
recombinant swine HEV ORF2 antigen. The US2 and Sar-55 human HEV
antisera reacted with the recombinant swine HEV ORF2 capsid
protein. The Sar-55 human HEV antiserum reacted strongly with
Sar-55 ORF2 capsid antigen, but to a lesser extent with
heterologous antisera against swine and avian HEVs (FIGS. 22A and
22B). The reaction signals between avian HEV antiserum, Sar-55
human HEV and swine HEV ORF2 proteins were also strong. These
results showed that avian HEV shares antigenic epitopes in its ORF2
capsid protein with swine and human HEVs as well as BLSV.
EXAMPLE 19
Cross-Reactivity of Avian HEV, Swine HEV and Human HEV Using
ELISA
[0122] To assess the cross-reactivity of avian HEV, swine HEV and
human HEV under a different condition than above study, this
experiment was conducted. The ELISA plates (commercially available
from Viral Antigens, Inc., Memphis, Tenn.; BD Biosciences, Bedford,
Mass. and others) were coated for 2 hrs with recombinant avian HEV,
swine HEV and human HEV capsid antigens at 37.degree. C. Each
antigen was used at a concentration of 2 .mu.g/ml of sodium
carbonate buffer, pH 9.6. The potential non-specific binding sites
were blocked with blocking solution (10% fetal bovine serum and
0.5% gelatin in washing buffer). The antisera, used in Western blot
analyses, were diluted 1/200 in blocking solution. The
preinoculation sera from a pig and a chicken were used as the
negative controls. Following 30 minutes incubation at 37.degree.
C., the plates were washed 4 times with washing solution (PBS
containing 0.05% Tween.RTM. 20 (polysorbate 20, commercially
available from Mallinckrodt Baker, Inc., Phillipsburg, N.J.), pH
7.4). The HRP-conjugated secondary antibodies were used as
described for Western blot analysis. Following 30 minutes
incubation at 37.degree. C., the plates were washed as described
above and the antigen-antibody complexes were detected using 2,
2'-Azino-bis (3-ethylbenthiazoline-6-sulfonic acid). After 10
minutes incubation at room temperature, the optical density (OD)
was measured at 405 nm.
[0123] The cross-reactivity of avian HEV, swine HEV and human HEV
were further confirmed using ELISA. As can be seen from FIG. 23,
each antiserum strongly reacted with the corresponding antigen. The
OD generated by interaction of avian HEV antiserum against
recombinant antigens of Sar-55 human HEV strain and swine HEV was
as high as 0.722 and 0.655, respectively, while the OD indicating
non-specific binding of preinoculation ("preimmune") chicken serum
remained as low as 0.142 and 0.103, respectively. The OD values
obtained from cross-reacting of avian HEV antigen to antiserum
against Sar-55 human HEV was almost twice the OD recorded when the
preinoculation pig serum was used. The OD obtained from reaction of
US2 human HEV almost did not differ from the OD obtained for the
preinoculation serum.
EXAMPLE 20
Computer Analysis of Amino Acid Sequences
[0124] The predicted amino acid sequences of the truncated ORF2
protein of avian, swine and human HEV strains were compared with
MacVector.RTM. program (Oxford Molecular, Inc., Madison, Wis.).
Hydropathy and antigenic plots of the amino acid sequences were
determined according to Kyte-Doolittle (J. Kyte & R. F.
Doolittle, "A simple method for displaying the hydropathic
character of a protein," J. Mol. Biol. 157:105-32 (1982)) and
Welling (Welling et al., "Prediction of sequential antigenic
regions in proteins," FEBS Letters 188:215-18 (1985)) methods using
the MacVector.RTM. computer program (Oxford Molecular, Inc.,
Madison, Wis.).
[0125] Analyses of the predicted amino acid sequences of the entire
ORF2 revealed that avian HEV shares only about 38% amino acid
sequence identities with swine, US2 and Sar-55 HEV strains. Swine
HEV ORF2 shared about 98% and 91% amino acid identities with US2
and Sar-55 HEV strains, respectively. The ORF2 of Sar-55 human HEV
shared 91% amino acid sequence identity with the US2 strain of
human HEV. Amino acid sequence alignment of the truncated ORF2
protein of avian HEV with the corresponding regions of swine HEV,
Sar-55 human HEV and US2 human HEV also revealed that the most
conserved region of the truncated 267 amino acid sequence is
located at its N-terminus (FIG. 24) which contains hydrophilic
amino acid residues (FIGS. 25A-25D). By using the Welling method
(Welling et al., 1985, supra) to predict antigenic domains of the
protein, three antigenic regions located at amino acids 460-490,
556-566 and 590-600 were also hydrophilic (FIGS. 25A-25D).
[0126] In the foregoing, there has been provided a detailed
description of particular embodiments of the present invention for
the purpose of illustration and not limitation. It is to be
understood that all other modifications, ramifications and
equivalents obvious to those having skill in the art based on this
disclosure are intended to be included within the scope of the
invention as claimed.
* * * * *
References