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 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).

DETAILED DESCRIPTION OF THE INVENTION

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.

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.

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.

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.

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)).

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° 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.

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.

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.

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.

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.

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.

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% nucleotide sequence identities with human and swine HEVs.

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 (10^4.5 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.

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.

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.

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.

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.

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.

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-α, IFN-β, IFN-γ, 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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.
 

Claim 1 of 7 Claims

1. An isolated avian hepatitis E virus having the nucleotide sequence set forth in SEQ ID NO:1 or its complementary strand.
 

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