WO2003063679A2 - Hepatitis e virus clones and methods of their use - Google Patents

Abstract

Full-length cDNA clones of the Sar-55 strain of HEV that are infectious in primates are disclosed. The invention relates to HEV nucleic acid sequences, their encoded proteins, any variants thereof, and HEV antibodies for developing vaccines of HEV, and detecting, preventing, and/or treating hepatitis E in a mammal. In particular, the invention provides nucleic acid sequences comprising infectious hepatitis E viruses of the Pakastani (Sar-55) strain. The invention therefore relates to the use of these sequences, and polypeptides encoded by all or part of these sequences, in the development of vaccines and diagnostic assays for HEV and in the development of screening assays for the identification of antiviral agents for HEV.

Description

HEPATITIS E VIRUS CLONES AND METHODS OF THEIR USE Field of the Invention

The invention is in the field of hepatitis virology. The present invention relates to molecular approaches to the production of nucleic acid sequences which comprise infectious hepatitis E viruses.

Background of the Invention

Hepatitis E virus (HEN) is an unclassified, non-enveloped virus that is a major cause of acute hepatitis in many developing countries¹. HEN is usually transmitted via the fecal-oral route, often through contaminated water supplies. The virus was first identified through a volunteer study in the former Soviet Union 18 years ago². The genome from a Burmese strain was the first to be isolated and sequenced,³ followed by that of a genetically diverse strain from Mexico. The discovery of virus of a third genotype in swine in the United States led to more extensive investigation world-wide and resulted in the discovery of additional strains in developed countries, where the disease is rare, as well as in developing countries, where epidemics and sporadic cases often occur.⁵ Based on sequence analysis of full-length genomes, 4 genotypes are generally recognized, but analysis of partial sequences suggests that these may be subdivided into as many as 9 groups.⁶ Swine are naturally infected with HEN and a strain of swine virus in the United States was shown to cross species barriers and experimentally infect primates.⁷ Many other animal species have serological evidence of infection suggesting HEN may be zoonotic.^{8, 9}' ¹⁰' ^π HEN has a positive-strand RΝA genome of 7.2 kb which contains 3 open reading frames (ORFs) encoding putative non-structural proteins (ORF1), a capsid protein (ORF2), and a very small protein of unknown function (ORF3).³ The ORF1 sequence has a motif characteristic of a methyltransferase¹² which suggested the genomic RΝA is capped since this activity and guanyltransferase activity are responsible for adding m7 GTP to the 5' terminus of mRΝA to yield the cap structure m7 G pppX. A cap structure has been indirectly identified on HEN genomes by both immunological¹³ and molecular techniques¹⁴ and a recombinant HEN protein was shown to have methyltransferase and guanyltransferase activity in vitro }⁵

HEN has not been reproducibly grown in cell culture but the virus in clinical samples has been used to infect primates. Macaques (rhesus and cynomolgus monkeys) have been the most frequently studied animal models; chimpanzees are also susceptible to experimental infection with HEN.¹⁶ Animals infected with HEN can develop hepatitis as demonstrated by elevations of serum liver enzymes and by appearance of histopathological changes in the liver. The severity of the hepatitis depends on the initial viral load; low doses of virus can infect without causing overt disease.¹⁷ Niremia does occur and virus can also be detected in bile and feces. Antibody production to ORF2 or ORF3 protein can be used to diagnose an infection. The incubation peπod until seroconversion is approximately 3-5 weeks in rhesus macaques inoculated intravenously with 10⁵'⁵ 50% monkey infectious doses.¹⁹ Clinical signs normally appear very close to, or at the time of, seroconversion.

It was reported that transfection of cultured Hep G2 cells with non-capped RΝA transcripts from a full-length cDΝA clone of an Indian strain of HEN resulted in productive replication.²⁰ However, RΝA transcripts from the cDΝA clone were not infectious for rhesus monkeys when inoculated intrahepatically as has been performed successfully for other positive-strand RΝA viruses. ^"When medium harvested from transfected Hep G2 cells was inoculated into a rhesus monkey, the monkey became infected with HEN. Sequence analysis of the virus recovered from the monkey apparently was not performed to confirm that the virus infecting the rhesus monkey originated from the cDΝA clone. Furthermore, the effect of incorporating a cap structure onto the in vitro synthesized transcripts was not reported.

Summary of Invention The present invention relates to isolated and substantially purified nucleic acid sequences comprising hepatitis E viruses (HEN) and in particular, nucleic acid sequences which comprise infectious hepatitis E viruses of the Pakastani (Sar-55) strain. It is therefore an object of the invention to provide nucleic acid sequences which encode hepatitis E viruses. The invention also relates to recombinant or synthetic nucleic acids of hepatitis E viruses, including for example the Sar-55 strain.

The invention also relates to the nucleic acid and deduced amino acid sequences of the HEN cDΝAs described herein.

The invention further relates to the use of the HEN nucleic acid sequences to produce attenuated viruses via passage in vitro or in vivo of the viruses produced by transfection of a host cell with the infectious nucleic acid sequence. The invention also relates to the use of the infectious nucleic acid sequences to produce chimeric genomes consisting of portions of the open reading frames of infectious nucleic acid sequences of other genotypes (including, but not limited to, genotypes 1, 2, 3, and 4) of HEN. For example infectious nucleic acid sequences can be used to produce chimeras with sequences from the genomes of other strains of HEV from different genotypes or subtypes. Nucleic acid sequences which comprise sequence from the open- reading frames of 2 or more HEN genotypes, strains, or subtypes are designated "chimeric nucleic acid sequences".

The invention also relates to hepatitis E viruses produced by host cells transfected with the nucleic acid sequences of the present invention.

The invention further relates to isolated and substantially purified HEN proteins and variants thereof encoded by the Sar-55 strain of HEN or encoded by synthetic nucleic acid sequences of the present invention and in particular to recombinant proteins encoded by at least one complete open reading frame of HEN. The invention also relates to antibodies to the hepatitis E viruses of the invention or their encoded polypeptides and to pharmaceutical compositions comprising these antibodies.

The invention also relates to the use of infectious HEN as vaccines. An attenuated virus may be preferred for use as a vaccine. Specifically, the attenuated clone of the invention is useful as an infectious RΝA or cDΝA vaccine or as a virus encoded by these clones.

The invention therefore also relates to vaccines for use in immunizing mammals especially humans against hepatitis E. In one embodiment, the vaccine comprises a hepatitis E virus produced by transfecting host cells with the nucleic acid sequences of the invention.

The present invention therefore relates to methods for preventing hepatitis E in a mammal. In one embodiment the method comprises administering to a mammal a polypeptide or polypeptides encoded by a nucleic acid sequence of the invention in an amount effective to induce protective immunity to hepatitis E. In another embodiment, the method of prevention comprises administering to a mammal a hepatitis E virus of the invention in an amount effective to induce protective immunity against hepatitis E.

The invention therefore also provides pharmaceutical compositions comprising the nucleic acid sequences of the invention and/or their encoded hepatitis E viruses. The invention further provides pharmaceutical compositions comprising polypeptides encoded by the nucleic acid sequences of the invention or fragments thereof. The pharmaceutical compositions of the invention may be used prophylactically or therapeutically.

The invention also relates to the use of the nucleic acid sequences of the invention to identify cell lines capable of supporting the replication of HEN in vitro.

The invention further relates to the use of the nucleic acid sequences of the invention or their encoded enzymes to develop screening assays to identify antiviral agents for HEN.

Description of Figures

Figure 1 shows the sequence alignment of the 3' region of HEN genomes. Numbering corresponds to sequence of pSK-HEN-2. A dot denotes identity, and a dash denotes a deletion. The first line of the Figure represents bases 7086 to 7166 of SEQ ID NO 2. Figure 2 shows the RNA structure of the 3' end of Sar-55 genomes as predicted by

MFOLD. Sequences are numbered according to pSK-HEN-2. The sequence between nucleotides 7082-7208 was analyzed, but only nucleotides 7087-7161 are shown; the excluded region has the identical structure in all four cases, but nucleotide 7181, located in a 3-base loop, was a U in SK-HEN-2 compared to a C in the other three cases. A: SK- HEV-2 (wild-type) sequence (bases 7087 to 7161 of SEQ ID 2); B: SK-HEN-3 with G to U substitution at nucleotide 7106 (bases 7087 to 7161 of SEQ ID NO 1); C: sequence recovered from chimp 1609 (bases 7087 to 7161 of SEQ ID NO 4); D: sequence recovered from chimp 1603 (bases 7087 to 7161 of SEQ LD NO 3). Arrow, new mutation. Box, base-pairing affected by new mutation. *, base-paired in wild-type. Underlined, translation termination codon (7125-7127).

Description of Sequences

Detailed Description of Invention

The invention broadly provides nucleic acid sequences which encode HEN proteins or fragments of HEN which have a biological function, diagnostic and therapeutic reagents, as well as methods of using HEN, its nucleic acid sequences, and antibodies developed thereto. The nucleic acid sequences, amino acid sequences and antibodies directed to HEN are useful in the detection, diagnosis and treatment of hepatitis.

In particular, the present invention relates to nucleic acids, specifically cDΝAs, encoding the full-length nucleotide sequence of hepatitis E virus (HEN) from Pakistan, Sar- 55. The HEN cDΝAs of the present invention were obtained as follows. Sar-55 was amplified by RT-PCR. The consensus sequence was then determined by direct sequencing of the uncloned RT-PCR products. Standard molecular techniques were used to assemble a full-length cDΝA clone of the Sar-55 strain from 7 consensus cDΝA fragments that were produced by RT-PCR.

In one embodiment of the present invention, two variants of a full-length cDΝA clone of the Pakistani (Sar-55) strain of HEN were constructed. These variants differ by two nucleotides and in vitro synthesized transcripts from each have been tested for the ability to initiate an infection in primates following mtrahepatic inoculation. Additionally, capped and uncapped transcripts of one of these clones were compared for infectivity by intrahepatic transfection of chimpanzees. The present invention further relates to the nucleotide sequences of the cDNAs encoding a virus that is infectious in primates and causes hepatitis. In particular, one preferred cDNA clone (SEQ ID NO:l) encodes a virus that is attenuated for chimpanzees and unable to infect rhesus monkeys. Another preferred cDNA clone (SEQ ID NO:2) encodes a virus that is infectious in both chimpanzees and rhesus monkeys and causes acute hepatitis.

In one embodiment, the nucleic acid sequence of the invention is about a 7204 bp cDNA (SEQ ID NO: 1), encoding 3 open reading frame proteins. ORF1 is a putative non- structural protein, ORF2 is a capsid protein, and ORF3 is a very small protein of unknown function. The full-length cDNA clone of the Sar-55 strain of HEN of the present invention differed at 5 positions from that of Tsarev (Tsarev-Genbank Accession No.: M80581)²¹. Four of the differences did not change the predicted amino acid sequence.

In a preferred embodiment of the invention, a cDNA clone of HEN, pSK-HEN-3 (SEQ ID ΝO:l), was constructed. Standard molecular techniques commonly known in the art can, for example, be used to assemble a full-length cDNA clone from cDNA fragments produced by RT-PCR. pSK-HEN-3 contains an altered consensus sequence of the Sar-55 strain having two mutations in the ORE1 located at nucleotide positions 286 and 4396, one mutation in ORF2 at nucleotide 7106, and a mutation in a putative 3 base loop in the 3' noncoding region at nucleotide 7181 (C in pSK-HEN-3 and 5 other genotype 1 strains; and a U in pSK-HEN-2 and 2 other genotype 1 strains), where none of these mutations altered the predicted amino acid sequence. Specifically, the altered consensus sequence of the Sar- 55 strain of HEN include mutations at positions 286, 4396, G7106T, and T7181C as shown in SEQ ID NO: 1.

In yet another embodiment of the invention, nucleotides 7086 to 7166 and more specifically nucleotides 7091 to 7157 comprise a conserved region, where at least part of these regions contains the cis-reacting element. The mutation at position 7106 identified the cis-reactive element that overlaps the 3¹ end of the ORF2 gene and part of the 3' non- coding region. The very high degree of nucleotide conservation in the region surrounding the mutation lends support that the mutation must be located within a cis-reacting element (CRE).

In a further embodiment of the present invention, a full-length cDNA clone of the Sar-55 strain of HEN (SEQ ID NO: 2), is provided. This construct is identical to that of SEQ ID NO:l, except that the nucleotide at position 7106 is not modified to a thymidine (T), rather the nucleotide remains as a guanine (G). Additionally, the nucleotide at position 7181 is a thymidine, rather than the cytosine of SEQ ID NO:l. The present invention provides an HEN cDΝA clone (SEQ ID ΝO:2), deposited at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, NA 20110-2209 on November 6, 2001 and under ATCC Accession No(s). PTA-3836 according to the terms of the Budapest Treaty.

In a further embodiment, variants of HEN polynucleotides are encompassed by the present invention. Preferably, an HEN variant has at least 75 to 80%, more preferably at least 85 to 90% , and even more preferably at least 95% nucleic acid sequence identity to an HEN nucleic acid sequence disclosed herein.

In one embodiment of the invention, viruses encoded by either of the cDΝA clones of SEQ ID ΝO:l or SEQ ID NO:2 of the Sar-55 Pakistani strain of HEN are described. The encoded RΝA sequence of SEQ ID ΝO:l has a G to U mutation at position 7106 and U to C mutation at position 7181. Furthermore, the RNA sequences represented by the cDNA sequences of SEQ ID NOs:3-4 have a G7097A and a C7144A mutation, respectively. SK-HEV-2 (i.e., wild type) and SK-HEN-3 (i.e., G7106U, U7181C) encode a virulent and an attenuated virus respectively. The capped transcripts from the two cDΝA clones are infectious as observed following intrahepatic transfection of chimpanzees. In one instance, the virus encoded by the cDΝA of SEQ ID ΝO:2 caused liver enzyme elevations, indicating acute hepatitis, in the chimpanzee and further resulted in seroconversion to anti-HEN at 5 weeks following inoculation, a time comparable to that observed following inoculation of the wild type Sar-55 virus. The transcripts from the cDΝA clone of SEQ ID ΝO:l used to inoculate two chimpanzees seroconverted to anti- HEN, but seroconversion was delayed until week 14 and liver enzyme levels did not rise, indicating that the virus was attenuated. hi a further embodiment of the invention, RΝA structures are provided. Computer predictions of the 3' RΝA structure are shown for the original genomes and those recovered from the transfected animals (Fig. 2). The new mutation which was selected during replication of the virus in chimp 1609 changed a C residue to an A with the result that it restored the identical 3' structure as predicted for wild-type except that a G:C base pair was replaced with a U:A base pair (Fig. 2C). Selection of this particular "compensatory" mutation strongly suggested that this putative stem was important. The new mutation selected by passage of the virus in the second chimpanzee, chimp 1603, is also important and useful. This G to A mutation eliminates a G:C base pair predicted by the folding program but not confirmed by chemical probing of in vitro transcripts of the Indian strain²⁷. The new mutation alters the predicted structure of the Sar-55 strain by removing 3 bases from a loop just upstream of the mutated site (Fig. 2D). Since this mutation was selected for, it most likely provided a positive replicative advantage. Chimp 1603 had detectable viremia and fecal shedding for only half as long as did chimp 1609 because the new mutation in this case only partially restored the robustness of the virus. The fact that new mutations were not selected in this region in genomes recovered from any of the five animals infected with virus encoded by the corrected clone pSK-HEN-2 indicates that the sequence of this corrected clone may be optimal for infection of these animals. The introduced G to T mutation in the pSK-HEN-3 cDΝA clone is located in a short stem structure (Figs. 2 A and 2B).

Variations are contemplated in the cDΝA sequences shown in SEQ ID ΝOs:l-2 which results in a nucleic acid sequence that is capable of directing production of analogs of the corresponding proteins of ORF 1, ORF2, and ORF3. It may be noted that the cDNA sequences set forth above represent a preferred embodiment of the present invention. Due to the degeneracy of the genetic code, it is to be understood that numerous choices of nucleotides may be made that will lead to a DNA sequence capable of directing production of the instant protein or its analogs. As such, DNA sequences which are functionally equivalent to the sequence set forth above or which are functionally equivalent to sequences that direct production of analogs of the HEN proteins produced pursuant to the amino acid sequences set forth above, are intended to be encompassed within the present invention.

In one embodiment, the polypeptide or polypeptides of the invention are fully or partially purified from hepatitis E virus produced by cells transfected with nucleic acid sequence of the invention. In another embodiment, the polypeptide or polypeptides are produced recombinantly from a fragment of the nucleic acid sequences of the invention. In yet another embodiment, the polypeptides are chemically synthesized. The polypeptides of the invention, especially structural polypeptides, can serve as immunogens in the development of vaccines or as antigens in the development of diagnostic assays for detecting the presence of HEN in biological samples.

In another embodiment, the deduced amino acid sequences of SEQ ID ΝOs:l-2 are presented, where the amino acid sequences are deduced from the ORF1 (SEQ ID NO:5), ORF2 (SEQ ID NO:6), and ORF3 (SEQ ID NO:7), respectively, of the nucleic acid sequence of pSK-HEN-2 (SEQ ID ΝO:2). The deduced amino acid sequence of the ORF1 begins at nucleotide 26 of the corresponding nucleic acid shown in SEQ ID No: 1 and extends 5107 nucleotides to a total length of 5082 nucleotides. The deduced ORF2 amino acid sequence begins at nucleotide 5145 to 7127 of pSK-HEN-2. The deduced ORF3 amino acid sequence begins at nucleotide 5104 to 5475 of pSK-HEN-2.

Preferably, the HEN proteins and peptides of the present invention are substantially homologous to, and most preferably biologically equivalent to, native HEN proteins and peptides. "Biologically equivalent" as used herein, refers to compositions that are immunogenically equivalent to the native HEN proteins and peptides. The HEN proteins and peptides of the present invention may also stimulate the production of protective antibodies upon injection into a mammal that serves to protect the mammal upon challenge with hepatitis virus, in particular, HEN. By "substantially homologous" as used herein, refers to a degree of homology in the amino acid sequence of the HEN proteins and peptides of the invention to that of the native HEN proteins and peptides respectively. Preferably the degree of homology is greater than 85%, preferably greater than 90%, and most preferably greater than 95% homologous with the native HEN proteins and peptides.

The term "analog" as used herein to describe the HEN proteins and peptides of the present invention, includes any protein or peptide having an amino acid residue sequence substantially identical to a sequence specifically shown herein in which one or more residues have been conservatively substituted with a biologically equivalent residue. Examples of conservative substitutions include the substitution of one nonpolar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.

The phrase "conservative substitution" also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that the resulting protein or peptide is biologically equivalent to the native HEN protein or peptide. hi one embodiment, the sequences of the invention can be inserted into an expression vector that functions in eukaryotic cells. The vectors contemplated for use in the present invention include vectors into which a nucleic acid sequence as described above can be inserted, along with any preferred or required operational elements, and which vector can then be subsequently transferred into a host organism and replicated in such organisms. Preferred vectors are those whose restriction sites have been well documented and which contain the operational elements preferred or required for transcription of the nucleic acid sequence. Eukaryotic expression vectors suitable for producing high efficiency gene transfer in vivo are well known to those of ordinary skill in the art and include, but are not limited to, plasmids, vaccinia viruses, retroviruses, adenoviruses and adeno-associated viruses.

The "operational elements" as discussed herein include at least one promoter, at least one operator, at least one leader sequence, at least one terminator codon, and any other DNA sequences necessary or preferred for appropriate transcription and subsequent translation of the vector nucleic acid. In particular, it is contemplated that such vectors will contain at least one origin of replication recognized by the host organism along with at least one selectable marker and at least one promoter sequence capable of initiating transcription of the nucleic acid sequence.

The present invention also relates to substantially purified and isolated recombinant HEN proteins. In one embodiment, the recombinant protein can be obtained as a crude lysate or it can be purified by standard protein purification procedures known in the art which may include differential precipitation, molecular sieve chromatography, ion- exchange chromatography, isoelectric focusing, gel electrophoresis and affinity and immunoaffinity chromatography. The recombinant protein may be purified by passage through a column containing a resin which has bound thereto antibodies specific for the open reading frame (ORF) protein.

Variants of HEN polypeptides are also encompassed by the present invention. Preferably, an HEN variant has at least 85%, more preferably at least 90% , and even more preferably at least 95% amino acid sequence identity to an HEN amino acid sequence disclosed herein, and more preferably, retains at least one biological, immunological, or other functional characteristic or activity of the non- variant HEN polypeptide.

The invention also relates to "chimeric nucleic acid sequences". In one embodiment, the chimeric nucleic acid sequence consists of sequence from the infectious clone and the sequence of another HEN strain. Such chimeras can be produced by standard techniques of restriction digestion, PCR amplification and subcloning known to those of ordinary skill in the art. It is believed that the construction of such chimeric nucleic acid sequences will be of importance in studying the growth and virulence properties of hepatitis E virus and in the production of hepatitis E viruses suitable to confer protection against multiple genotypes of HEN. For example, one might produce a "multivalent" vaccine by putting epitopes from several genotypes or subtypes into one clone. Alternatively, one might replace just a single gene from an infectious sequence with the corresponding gene from the genomic sequence of a strain from another genotype or subtype or create a chimeric gene which contains portions of a gene from two genotypes or subtypes.

The invention further relates to mutations of the infectious nucleic acid sequences of HEN where "mutations" includes, but is not limited to, point mutations, deletions and insertions. Of course, one of ordinary skill in the art will recognize that the size of the insertions may be limited by the ability of the resultant nucleic acid sequence to be properly packaged within the virion. Such mutations may be produced by techniques known to those of skill in the art such as site-directed mutagenesis, fusion PCR, and restriction digestion followed by religation. Mutagenesis, in one embodiment, may be undertaken to determine sequences that are important for viral properties such as replication or virulence.

In yet another embodiment, one may delete all or part of a gene or of the 5' or 3' nontranslated region contained in an infectious nucleic acid sequence and then transfect a host cell (animal or cell culture) with the mutated sequence and measure viral replication in the host by methods known in the art such as RT-PCR. Of course, those of ordinary skill in the art will understand that deletion of part of a gene, preferably the central portion of the gene, may be preferable to deletion of the entire gene in order to conserve the cleavage site boundaries which exist between proteins in the HEN polyprotein and which are necessary for proper processing of the polyprotein. In the alternative, if the transfection is into a host animal such as a chimpanzee, one can monitor the virulence phenotype of the virus produced by transfection of the mutated infectious nucleic acid sequence by methods known in the art such as measurement of liver enzyme levels (alanine aminotransferase (ALT) or isocitrate dehydrogenase (ICD)) or by histopathology of liver biopsies. Thus, mutations of the infectious nucleic acid sequences may be useful in the production of attenuated HEN strains suitable for vaccine use.

Although nucleic acid sequences which encode the HEN polypeptides and variants thereof are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring HEN polypeptide under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HEN polypeptides, or derivatives thereof, which possess a substantially different codon usage. For example, codons may be selected to increase the rate at which expression of the peptide/polypeptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host. Another reason for substantially altering the nucleotide sequence encoding an HEN polypeptide, or its derivatives, without altering the encoded amino acid sequences, includes the production of RΝA transcripts having more desirable properties, such as a greater half-life, than transcripts produced from the naturally occurring sequence. In construction of the recombinant expression vectors of the present invention, it may additionally be noted that multiple copies of the nucleic acid sequence of interest and its attendant operational elements may be inserted into each vector. In such an embodiment, the host organism produces greater amounts per vector of the desired HEN protein. The number of multiple copies of the nucleic acid sequence which may be inserted into the vector is limited only by the ability of the resultant vector due to its size, to be transferred into and replicated and transcribed in an appropriate host organism.

In another embodiment, restriction digest fragments containing a coding sequence for HEN proteins can be inserted into a suitable expression vector that functions in prokaryotic or eukaryotic cells. By suitable is meant that the vector is capable of carrying and expressing a complete nucleic acid sequence coding for an HEN protein. Preferred expression vectors are those that function in a eukaryotic cell. Examples of such vectors include but are not limited to vaccinia virus vectors, adenovirus, baculovirus, or herpes viruses.

The present invention also relates to the construction of cassette vectors useful in the cloning of viral genomes wherein said vectors comprise a nucleic acid sequence to be cloned, and said vector reading in the correct frame for the expression of the viral nucleic acid to be cloned. Such a cassette vector will, of course, also possess a promoter sequence, advantageously placed upstream of the sequence to be expressed. Cassette vectors may be constructed according to methods well known in the art. Of course, the DΝA to be inserted into said cassette vector can be derived from any virus, advantageously from HEN, and most advantageously from the Sar-55 strain of HEN. The nucleic acid to be inserted according to the present invention can, for example, contain one or more open reading frames of the virus, for example, of HEN. The cassette vectors of the present invention may also contain, optionally, one or more expressible marker genes for expression as an indication of successful transfection and expression of the nucleic acid sequences of the vector. To insure expression, the cassette vectors of the present invention may contain a promoter sequence for binding of the appropriate cellular RNA polymerase, which may depend on the cell into which the vector has been introduced. For example, if the host cell is a bacterial cell, then said promoter may be a bacterial promoter sequence to which the bacterial RNA polymerases may bind.

In yet another embodiment, the selected recombinant expression vector may then be transfected into a suitable eukaryotic cell system for purposes of expressing the recombinant protein.

Suitable cells or cell lines for culturing HEN include, but are not limited to, HeLa, MRC-5, CN-1, lymphocyte or hepatocyte cell lines known in the art.

Alternatively, primary hepatocytes can be cultured, and then infected with HEN, or the hepatocyte cultures may be derived from the livers of infected chimpanzees. In addition, various immortalization methods known to those of skill in the art can be used to obtain cell-lines derived from hepatocyte cultures. For example, primary hepatocyte cultures may be fused to a variety of cells to maintain stability.

Transfection of tissue culture cells with the nucleic acid sequences of the invention may be done by common transfection methods known in the art such as electroporation, precipitation with DEAE-Dextran or calcium phosphate or liposomes.

In one such embodiment, the method comprises the growing of animal cells, especially human cells, in vitro and transfecting the cells with the nucleic acid of the invention, then determining if the cells show any indication of HEN infection. Such indications include the detection of viral antigens in the cell, for example, by immunofluorescence procedures commonly known in the art; the detection of viral polypeptides by Western blotting using antibodies specific therefor; and the detection of newly transcribed viral RΝA within the cells via methods such as RT-PCR. The presence of live, infectious virus particles following such tests may also be shown by injection of cell culture medium or cell lysates into healthy, susceptible animals, with subsequent exhibition of the symptoms of HEN infection.

In one embodiment, animal cells (especially human cells) transfected with the nucleic acid sequences of the invention are cultured in vitro and the cells are treated with a candidate antiviral agent (a chemical, peptide etc.) for antiviral activity by adding the candidate agent to the medium. A sufficient period of time is then allowed to pass for infection to occur, following which the presence or absence of viral replication is determined versus untreated control cells by methods known to those of ordinary skill in the art. Such methods include, but are not limited to, the detection of viral antigens in the cell, for example, by immunofluorescence procedures well known in the art; the detection of viral polypeptides by Western blotting using antibodies specific therefor; the detection of newly transcribed viral RNA within the cells by RT-PCR; and the detection of the presence of live, infectious virus particles by injection of cell culture medium or cell lysates into healthy, susceptible animals, with subsequent exhibition of the symptoms of HEN infection. A comparison of results obtained for control cells (treated only with nucleic acid sequence) with those obtained for treated cells (nucleic acid sequence and antiviral agent) indicates, the degree, if any, of antiviral activity of the candidate antiviral agent. Of course, one of ordinary skill in the art readily understands that such cells may be treated with the candidate antiviral agent either before or after exposure to the nucleic acid sequence of the present invention so as to determine against what stage, or stages, of viral infection and replication said agent is effective.

In an alternative embodiment, a protease produced from a nucleic acid sequence of the invention, may be used to screen for protease inhibitors which may act as antiviral agents. Such protease inhibitors may take the form of chemical compounds or peptides which mimic the known cleavage sites of the protease and may be screened using methods known to those of skill in the art (Houghton, M. (1996) and Major, M.E. et al. (1997)). For example, a substrate may be employed which mimics the protease's natural substrate, but which provides a detectable signal (e.g., by fluorimetric or colorimetric methods) when cleaved. This substrate is then incubated with the protease and the candidate protease inhibitor under conditions of suitable pH, temperature etc. to detect protease activity. The proteolytic activities of the protease in the presence or absence of the candidate inhibitor are then determined. In a further embodiment, antiviral agents may be important for studying inhibition of proteases, RΝA polymerases, methyltransferases, guanyltransferases, and helicases. Host cells which contain the nucleic acid sequence coding for the HEN polypeptide of the invention and which express the HEN polypeptide product may be identified by a variety of procedures known to those having skill in the art. These procedures include, but are not limited to, DΝA-DΝA or DΝA-RΝA hybridizations and protein bioassay or immunoassay techniques, including membrane, solution, or chip based technologies, for the detection and/or quantification of nucleic acid or protein.

A variety of expression vector/host systems may be utilized to contain and express sequences encoding the HEN polypeptides or peptides. Such expression vector/host systems include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DΝA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMN)), or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems, including mammalian cell systems. The host cell employed is not limiting to the present invention. Preferably, the host cell of the invention contains an expression vector comprising an isolated and purified polynucleotide having a nucleic acid sequence selected from any one of SEQ ID ΝOs:l-4 and encoding a human HEN protein of this invention, or a functional fragment thereof.

In a preferred embodiment, nucleic acid sequences of the invention preferably comprising any one of SEQ ID ΝOs:l and 3-4, as well as chimeras based on SEQ ID NO:2, or variants thereof, are transfected into a mammalian host animal, such as a chimpanzee. One can thereby monitor the virulence phenotype of the virus produced by transfection of the mutated infectious nucleic acid sequence by methods known in the art such as measurement of liver enzyme levels (alanine aminotransferase (ALT) or isocitrate dehydrogenase (ICD)) or by histopathology of liver biopsies. Thus, mutations of the infectious nucleic acid sequences may be useful in the production of attenuated HEN strains suitable for vaccine use. The present invention relates to the in vitro and in vivo production of hepatitis E viruses from the nucleic acid sequences of the invention.

The hepatitis E viruses produced from the sequences of the invention may be purified or partially purified from the transfected cells by methods known to those of ordinary skill in the art. In a preferred embodiment, the viruses are partially purified prior to their use as immunogens in the pharmaceutical compositions and vaccines of the present invention.

The present invention therefore relates to the use of the hepatitis E viruses produced from the HEV nucleic acid sequences of the invention, preferably SEQ ID ΝOs: 1-4, as immunogens in live or killed (e.g., formalin inactivated) vaccines to prevent hepatitis E in a mammal.

The present invention further relates to the use of recombinant HEN proteins as diagnostic agents and vaccines. The vaccine, which acts as an immunogen, may be a cell, cell lysate from cells transfected with a recombinant expression vector or a culture supernatant containing the expressed protein. Alternatively, the immunogen is a partially or substantially purified recombinant protein.

In a preferred embodiment, direct gene transfer may be accomplished via administration of a eukaryotic expression vector containing a nucleic acid sequence of the invention. Furthermore, the immunogen of the present invention may be a nucleic acid sequence which encodes an infectious hepatitis E virus. Where the nucleic acid sequence is a cDΝA sequence, the cDΝAs and their RΝA transcripts may be used to transfect a mammal by direct injection into the liver tissue of the mammal as described in the Examples. In yet a more preferred embodiment, the immunogen may be nucleic acid sequence capable of directing host organism synthesis of HEN protein(s). In particular, an attenuated RΝA or cDΝA may be used. Such nucleic acid sequence may be inserted into a suitable expression vector by methods known to those skilled in the art. Expression vectors suitable for producing high efficiency gene transfer in vivo include retroviral, adenoviral and vaccinia viral vectors. Operational elements of such expression vectors are disclosed previously in the present specification and are known to one skilled in the art. Such expression vectors can be administered intravenously, intramuscularly, subcutaneously, intraperitoneally or orally.

In an alternative embodiment, direct gene transfer may be accomplished via intramuscular injection of, for example, plasmid-based eukaryotic expression vectors containing a nucleic acid sequence capable of directing host organism synthesis of HEN proteins. Such an approach has previously been utilized to produce the hepatitis B surface antigen in vivo and resulted in an antibody response to the surface antigen (Davis, H.L. et al. (1993) Human molecular Genetics, 2:1847-1851; see also Davis et al. (1993) Human Gene Therapy, 4:151-159 and 733-740).

The invention also relates to the use of the HEN nucleic acid sequence of the present invention, preferably SEQ LD ΝOs:l and 3-4, to produce attenuated viral strains via passage in vitro or in vivo of the virus produced by transfection with the infectious nucleic acid sequence.

In yet another embodiment, the immunogen may be a polypeptide encoded by the HEN nucleic acid sequences of the invention. The present invention therefore also relates to polypeptides produced from the nucleic acid sequences of the invention or fragments thereof. In one embodiment, polypeptides of the present invention can be recombinantly produced by synthesis from the nucleic acid sequences of the invention or isolated fragments thereof, and purified, or partially purified, from transfected cells using methods already known in the art. In an alternative embodiment, the polypeptides may be purified or partially purified from viral particles produced via transfection of a host cell with the nucleic acid sequences of the invention. Such polypeptides might, for example, include capsid polypeptides prepared from the sequences of the present invention.

When used as immunogens, the nucleic acid sequences of the invention, or the polypeptides or viruses produced therefrom, are preferably partially purified prior to use as immunogens in pharmaceutical compositions and vaccines of the present invention. When used as a vaccine, the sequences and the polypeptide and virus products thereof, can be administered alone or in a suitable diluent, including, but not limited to, water, saline, or some type of buffered medium. The vaccine according to the present invention may be administered to an animal, especially a mammal, and most especially a human, by a variety of routes, including, but not limited to, intradermally, intramuscularly, subcutaneously, or in any combination thereof.

While it is possible for the immunogen to be administered in a pure or substantially pure form, it is preferable to present it as a pharmaceutical composition, formulation or preparation. Suitable amounts of material to administer for prophylactic and therapeutic purposes will vary depending on the route selected and the immunogen (nucleic acid, virus, polypeptide) administered. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. The vaccines of the present invention may be administered once or periodically until a suitable titer of antibodies against HEN appear in the blood. For an immunogen consisting of a nucleic acid sequence, a suitable amount of nucleic acid sequence to be used for prophylactic purposes might be expected to fall in the range of from about 100 μg to about 5 mg and most preferably in the range of from about 500 μg to about 2mg. For a polypeptide, a suitable amount to use for prophylactic purposes is preferably 100 ng to 100 μg and for a virus 10² to 10⁶ infectious doses. Such administration preferably occurs prior to any sign of HEN infection.

The formulations of the present invention, both for veterinary and for human use, comprise an immunogen as described above, together with one or more pharmaceutically acceptable carriers and optionally other therapeutic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. The formulations may conveniently be presented in unit dosage form and may be prepared by any method well-known in the pharmaceutical art.

Formulations of vaccines comprise the active ingredient with a carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired formulation.

Formulations suitable for intravenous, intramuscular, subcutaneous, or intraperitoneal administration conveniently comprise sterile aqueous solutions of the active ingredient with solutions which are preferably isotonic with the blood of the recipient. Such formulations may be conveniently prepared by dissolving the solid active ingredient in water containing physiologically compatible substances such as sodium chloride, glycine, and the like, and having a buffered pH compatible with physiological conditions to produce an aqueous solution, and rendering said solution sterile. These may be present in unit or multi-dose containers, for example, sealed ampules or vials.

The formulations of the present invention may incorporate a stabilizer. Illustrative stabilizers are preferably incorporated in an amount of 0.10-10,000 parts by weight per part by weight of immunogens. If two or more stabilizers are to be used, their total amount is preferably within the range specified above. These stabilizers are used in aqueous solutions at the appropriate concentration and pH. The specific osmotic pressure of such aqueous solutions is generally in the range of 0.1-3.0 osmoles, preferably in the range of 0.8-1.2. The pH of the aqueous solution is adjusted to be within the range of 5.0-9.0, preferably within the range of 6-8. In formulating the immunogen of the present invention, an anti- adsorption agent may be used. A vaccine of the present invention may be employed in such forms as capsules, liquid solutions, suspensions or elixirs for oral administration, or sterile liquid forms such as solutions or suspensions. Any inert carrier is preferably used, such as saline or phosphate-buffered saline, or any such carrier in which the HEN of the present invention can be suitably suspended. The vaccines may be in the form of single dose preparations or in multi-dose flasks which can be utilized for mass-vaccination programs of both animals and humans. For purposes of using the vaccines of the present invention reference is made to Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., Osol (Ed.) (1980); and New Trends and Developments in Vaccines, Noller et al. (Eds.), University Park Press, Baltimore, Md. (1978), both of which provide much useful information for preparing and using vaccines. Of course, the polypeptides of the present invention, when used as vaccines, can include, as part of the composition or emulsion, a suitable adjuvant, such as alum (or aluminum hydroxide) when humans are to be vaccinated, to further stimulate production of antibodies by immune cells. When nucleic acids or viruses are used for vaccination purposes, other specific adjuvants such as CpG motifs (Krieg, A.K. et al.(1995) and (1996)), may prove useful.

When the nucleic acids, viruses and polypeptides of the present invention are used as vaccines or inocula, they normally exist as physically discrete units suitable as a unitary dosage for animals, especially mammals, and most especially humans, wherein each unit contains a predetermined quantity of active material calculated to produce the desired immunogenic effect in association with the required diluent. The dose of said vaccine or inoculum according to the present invention is administered at least once. In order to increase the antibody level, one or more booster doses may be administered at some time after the initial dose. The need for, and timing of, such booster doses will, of course, be determined within the sound judgment of the administrator of such vaccine or inoculum and according to sound principles well known in the art. For example, such booster dose(s) are reasonably expected to be advantageous at some time between about 2 weeks to about 6 months following the initial vaccination. Subsequent doses may be administered as indicated. In one embodiment, the expressed recombinant proteins of this invention can be used in immunoassays for diagnosing or prognosing hepatitis E in a mammal. For the purposes of the present invention, "mammal" as used throughout the specification and claims, includes, but is not limited to humans, chimpanzees, other primates, swine, and the like. In a preferred embodiment, the immunoassay is useful in diagnosing hepatitis E infection in humans.

Immunoassays of the present invention may be those commonly used by one skilled in the art including, but not limited to, radioimmunoassay, Western blot assay, immunofluorescence assay, enzyme immunoassay, chemiluminescence assay, immunohistochemical assay, immunoprecipitation and the like. Standard techniques known in the art for ELISA are described in Methods in Immunodiagnosis, 2nd Edition, Rose and Bigazzi, eds., John Wiley and Sons, 1980 and Campbell et al., Methods of Immunology, W.A. Benjamin, Inc., 1964, both of which are incorporated herein by reference. Such assays may be a direct, indirect, competitive, or noncompetitive immunoassay as described in the art (Oellerich, M. 1984. J. Clin. Chem. Clin. BioChem 22:895-904). Biological samples appropriate for such detection assays include, but are not limited to serum, liver, saliva, lymphocytes or other mononuclear cells.

The nucleic acid sequences, viruses and polypeptides of the present invention can also be admimstered for purposes of therapy, where a mammal, especially a primate, and most especially a human, is already infected, as shown by well known diagnostic measures. When the nucleic acid sequences, viruses or polypeptides of the present invention are used for such therapeutic purposes, much of the same criteria applies as when it is used as a vaccine, except that inoculation occurs post-infection. Thus, when the nucleic acid sequences, viruses or polypeptides of the present invention are used as therapeutic agents in the treatment of infection, the therapeutic agent comprises a pharmaceutical composition containing a sufficient amount of said nucleic acid sequences, viruses or polypeptides so as to elicit a therapeutically effective response in the organism to be treated. The amount of pharmaceutical composition to be administered may vary depending on the immunogen contained therein (nucleic acid, polypeptide, virus) and on the route of administration.

The therapeutic agent according to the present invention can thus be admimstered by subcutaneous, intramuscular or intradermal routes. One skilled in the art will certainly appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. Of course, the actual amounts will vary depending on the route of admimstration as well as the sex, age, and clinical status of the subject which, in the case of human patients, is to be determined with the sound judgment of the clinician. The therapeutic agent of the present invention can be employed in such forms as capsules, liquid solutions, suspensions or elixirs, or sterile liquid forms such as solutions or suspensions. Any inert carrier is preferably used, such as saline, phosphate-buffered saline, or any such carrier in which the HEN of the present invention can be suitably suspended. The therapeutic agents may be in the form of single dose preparations or in the multi-dose flasks which can be utilized for mass-treatment programs of both animals and humans. When the nucleic acid sequences, viruses or polypeptides of the present invention are used as therapeutic agents, they may be administered as a single dose or as a series of doses, depending on the situation as determined by the person conducting the treatment. In particular, the invention provides nucleic acid sequences comprising infectious hepatitis E viruses of the Pakastani (Sar-55) strain. The invention therefore relates to the use of these sequences, and polypeptides encoded by all or part of these sequences, in the development of vaccines and diagnostic assays for HEN and in the development of screening assays for the identification of antiviral agents for HEN. In a further embodiment, the HEN antibodies may also be used as a means of enhancing the immune response. The antibodies can be administered in amount similar to those used for other therapeutic administrations of antibody. For example, normal immune globulin is administered at 0.02-0.1 ml/lb body weight during the early incubation period of other viral diseases such as rabies, measles, and hepatitis B to interfere with viral entry into cells. Thus, antibodies reactive with the HEN proteins can be passively administered alone or in conjunction with another anti-viral agent to a host infected with an HEN to enhance the immune response and/or the effectiveness of an antiviral drug.

In another embodiment, the HEN proteins and expression vectors containing a nucleic acid sequence capable of directing host organism synthesis of HEV proteins may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition as described above.

The present invention also relates to the use of nucleic acid sequences and polypeptides of the present invention to screen potential antiviral agents for antiviral activity against HEN. Such screening methods are known by those of skill in the art. Generally, the antiviral agents are tested at a variety of concentrations, for their effect on preventing viral replication in cell culture systems which support viral replication, and then for an inhibition of infectivity or of viral pathogenicity (and a low level of toxicity) in an animal model system. In particular, it is contemplated that the nucleic acid sequences of the invention may be useful in identifying sequences critical for cell culture adaptation of HEN and hence, may be useful in identifying cell lines capable of supporting HEN replication.

The present invention relates to a method for detecting the hepatitis E virus in biological samples based on selective amplification of hepatitis E gene fragments. Amplification methods are commonly known in the art. Preferably, this method utilizes a pair of single-stranded primers derived from a hepatitis E virus whose genome contains a region homologous to the Sar-55 sequence. These primers can be used in a method following the process for amplifying selected nucleic acid sequences as defined in U.S. Patent No. 4,683,202.

In yet another embodiment of the present invention, a method for detecting a polynucleotide that encodes a described HEN polypeptide in a biological sample comprising the steps of: a) hybridizing the complement of the polynucleotide sequence encoding SEQ ID ΝOs: 1-4 to the nucleic acid material of a biological sample, thereby forming a hybridization complex; and b) detecting the hybridization complex, wherein the presence of the complex correlates with the presence of a polynucleotide encoding an HEN polypeptide in the biological sample. The nucleic acid material may be further amplified by the polymerase chain reaction prior to hybridization.

In another embodiment, an array of oligonucleotide probes comprising HEN nucleotide sequence or fragments thereof can be constructed to conduct efficient screening of, for example, genetic mutations. Array technology methods are well known, have general applicability and can be used to address a variety of questions in molecular genetics, including gene expression, genetic linkage, and genetic variability (see for example: M. Chee et al., Science, 274:610-613, 1996). Yet another aspect of the present invention involves a method of screening a library of molecules or compounds with an HEN-encoding polynucleotide to identify at least one molecule or compound therein which specifically binds to the HEN polynucleotide sequence. Such a method includes a) combining an HEN-encoding polynucleotide of the present invention with a library of molecules or compounds under conditions to allow specific binding; and b) detecting specific binding, thereby identifying a molecule or compound, which specifically binds to an HEV-encoding polynucleotide sequence, wherein the library is selected from DΝA molecules, RΝA molecules, artificial chromosome constructions, PΝAs, peptides and proteins. The HEN polynucleotides of the present invention which may be used in the diagnostic assays according to the present invention include oligonucleotide sequences and complementary RΝA and DΝA molecules. The polynucleotides may be used to detect and quantify HEN-encoding nucleic acid expression in biopsied tissues in which expression (or under- or overexpression) of the HEN polynucleotide may be correlated with disease. The diagnostic assays may be used to distinguish between the absence, presence, and excess expression of HEN, and to monitor regulation of HEN polynucleotide levels during therapeutic treatment or intervention. In particular, one embodiment of the present invention provides a method for detecting the presence or absence of HEN in a sample. This method comprising the steps of exposing a sample suspected of containing HEN to a probe comprising any one of SEQ ID ΝOs: 1-4; hybridizing said probe to HEN nucleic acids in the sample; and detecting a sequence-specific hybridization complex.

As will be understood by those of skill in the art, the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences. The terms "stringency" or "stringent conditions" refer to the conditions for hybridization as defined by nucleic acid composition, salt and temperature. These conditions are well known in the art and may be altered to identify and/ or detect identical or related polynucleotide sequences in a sample. A variety of equivalent conditions comprising either low, moderate, or high stringency depend on factors such as the length and nature of the sequence (DΝA, RΝA, base composition), reaction milieu (in solution or immobilized on a solid substrate), nature of the target nucleic acid (DΝA, RΝA, base composition), concentration of salts and the presence or absence of other reaction components (e.g., formamide, dextran sulfate and/ or polyethylene glycol) and reaction temperature (within a range of from about 5°C below the melting temperature of the probe to about 20°C to 25°C below the melting temperature).

One or more factors may be varied to generate conditions, either low or high stringency, that are different from but equivalent to the aforementioned conditions, such that conditions are optimal for hybridization. As will be further appreciated by the skilled practitioner, the melting temperature, Tm, can be approximated by the formulas as known in the art, depending on a number of parameters, such as the length of the hybrid or probe in number of nucleotides, or hybridization buffer ingredients and conditions (see, for example, T. Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, ΝY, 1982 and J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1989; Current Protocols in Molecular Biology, Eds. F.M. Ausubel et al., Vol. 1, "Preparation and Analysis of DNA", John Wiley and Sons, Inc., 1994-1995, Suppls. 26, 29, 35 and 42; pp. 2.10.7- 2.10.16; G.M. Wahl and S. L. Berger (1987; Methods Enzymol. 152:399-407); and A.R. Kimmel, 1987; Methods of Enzymol. 152:507-511). As a general guide, Tm decreases approximately 1°C -1.5°C with every 1% decrease in sequence homology. Also, in general, the stability of a hybrid is a function of sodium ion concentration and temperature. Typically, the hybridization reaction is initially performed under conditions of low stringency, followed by washes of varying, but higher stringency. Reference to hybridization stringency, e.g., high, moderate, or low stringency, typically relates to such washing conditions.

In yet another embodiment, decreased or increased expression can be measured at the RNA level using any of the methods well known in the art for the quantification of polynucleotides, such as, for example, PCR, RT-PCR, RNase protection, Northern blotting and other hybridization methods. Assay techniques that can be used to determine levels of a protein, such as an HEN protein, in a sample derived from a host are well known to those of skill in the art. Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis, and ELISA assays.

In a related aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding an HEN polypeptide according to the present invention, or closely related molecules, may be used to identify nucleic acid sequences which encode an HEN polypeptide. The specificity of the probe, whether it is made from a highly specific region or a less specific region, will determine whether the probe identifies only naturally occurring sequences encoding HEN polypeptide, alleles thereof, or related sequences.

Probes may also be used for the detection of related sequences, and may preferably contain at least 50% of the nucleotides encoding the HEN polypeptide. The hybridization probes or primers of this invention may be DΝA or RΝA and may be derived from the nucleotide sequences of SEQ ID ΝOs: 1-4, wherein the probes or primers comprise a polynucleotide sequence capable of hybridizing with a polynucleotide of SEQ LD ΝOs:l-4, under low, moderate, or high stringency conditions.

Methods for producing specific hybridization probes for RNA encoding the HEV polypeptides include the cloning of a nucleic acid sequence that encodes the HEN polypeptide, or HEN derivatives, into vectors for the production of mRΝA probes. Such vectors are known in the art, or are commercially available, and may be used to synthesize RΝA probes in vitro by means of the addition of the appropriate RΝA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of detector/ reporter groups, including, but not limited to, radionuclides such as ³²P or ³⁵S, or enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin biotin coupling systems, and the like.

The polynucleotide sequences encoding the HEN polypeptides of this invention, or fragments thereof, may be used for the diagnosis of HEN infections. The polynucleotide sequence encoding the HEN polypeptide may be used in Southern or Northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dipstick, pin, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect the status of, for example, levels of, or overexpression of, an HEN, or to detect altered HEN expression or levels. Such qualitative or quantitative methods are commonly practiced in the art. The invention also provides that the nucleic acid sequences, viruses and polypeptides of the invention may be supplied in the form of a kit, alone or in the form of a pharmaceutical composition.

In a further embodiment, test serum is reacted with a solid phase reagent having surface-bound HEN proteins as antigens. The solid surface reagent can be prepared by known techniques for attaching protein to solid support material. These attachment methods include non-specific adsorption of the protein to the support or covalent attachment of the protein to a reactive group on the support. After reaction of the antigen with anti-HEN antibody, unbound serum components are removed by washing and the antigen-antibody complex is reacted with a secondary antibody such as labeled anti-human antibody. The label may be an enzyme which is detected by incubating the solid support in the presence of a suitable colorimetric or fluorometric reagent. Other detectable labels may also be used, such as radiolabels or colloidal gold, and the like.

Additional pharmaceutical methods may be employed to control the duration of action. Controlled release preparations may be achieved through the use of polymer to complex or adsorb the proteins or their derivatives. The controlled delivery may be exercised by selecting appropriate macromolecules (for example polyester, polyamino acids, polyvinyl pyrrolidone, ethylenevinylacetate, methylcellulose, carboxymethylcellulose, or protamine sulfate) and the concentration of macromolecules as well as the methods of incorporation in order to control release. Another possible method to control the duration of action by controlled-release preparations is to incorporate the proteins, protein analogs or their functional derivatives, into particles of a polymeric material such as polyesters, polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetate copolymers. Alternatively, instead of incorporating these agents into polymeric particles, it is possible to entrap these materials in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin-microcapsules and poly (methylmethacrylate) microcapsules, respectively, or in colloidal drug delivery systems, for example, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or in macroemulsions.

When oral preparations are desired, the compositions may be combined with typical carriers, such as lactose, sucrose, starch, talc, magnesium stearate, crystalline cellulose, methyl cellulose, carboxymethyl cellulose, glycerin, sodium alginate or gum arabic among others.

Doses of HEN protein(s)-encoding nucleic acid sequence effective to elicit a protective antibody response against HEN infection range from about lμg to about 10 mg. A preferred range being about lOOμg to 5 mg and more preferably, a range from about 500 μg to 1 mg. The HEN nucleic acids and proteins of the invention are also intended for use in producing antiserum designed for pre- or post-exposure prophylaxis. Here an attenuated hepatitis E virus, an HEN nucleic acid or protein, or mixture of HEV nucleic acids or proteins is formulated with a suitable adjuvant and administered by injection to human volunteers, according to known methods for producing human antisera. Antibody response to the injected immunogens is monitored, during a several-week period following immunization, by periodic serum sampling to detect the presence of HEV antibodies, using an immunoassay as described herein.

The antiserum from immunized individuals may be administered as a pre-exposure prophylactic measure for individuals who are at risk of contracting infection. The antiserum may also be useful in treating an individual post-exposure, analogous to the use of high titer antiserum against hepatitis B virus for post-exposure prophylaxis.

All publications, patents and articles referred to herein are expressly incorporated herein in toto by reference thereto. The following examples are presented to illustrate the present invention but are in no way to be construed as limitations on the scope of the invention. It will be recognized by those skilled in the art that numerous changes and substitutions may be made without departing from the spirit and purview of the invention.

EXAMPLE 1

CONSTRUCTION OF HEN cDΝA CLONES The consensus sequence of the Sar-55 strain was determined by direct analysis of uncloned RT-PCR products amplified from the viral genome and differed at 5 positions from that determined by Tsarev (Genbank Ace. No. M80581) from cloned fragments²³. Four of the differences did not change the predicted amino acid. Standard molecular techniques were used to assemble a full-length cDNA clone of the Sar-55 strain from 7 consensus cDNA fragments that were produced by RT-PCR. The 5' terminus of the encoded genome was engineered to conform to the sequence determined by Zhang et al¹⁴. This was done by inserting a T7 promoter between the Xbal site in the plasmid and the 5' end of the HEN genome so that transcription by T7 polymerase results in synthesis of the exact 5' terminus of HEN. Although this manipulation introduced a second T7 promoter into the plasmid (the one present in the original vector was at the 3' end of the genome just downstream of the site used for template linearization), the second T7 promoter did not interfere with transcription of the HEN genome. The HEN genome was ligated into the pBlueScript SK (+) vector (Stratagene, La JoUa, CA) between Xba 1 and Eco RN sites in the poly linker. A Bgl II restriction site for template linearization was engineered into the clone between the 3' terminus of the HEN sequence and the Eco RN site in the plasmid polylinker. The first cDΝA clone (pSK-HEN-3), differed from the consensus nucleotide sequence at four positions (nucleotides 286 and 4396 in ORF1; nucleotide 7106 in ORF2; and nucleotide 7181 in the 3' noncoding region). The deduced amino acid sequence was identical to that of the consensus sequence. The T mutation at 7106 was replaced with the consensus nucleotide G and the C at 7181 was replaced with T to yield a second clone pSK-HEV-2 (SEQ ID ΝO:2).

EXAMPLE 2

IN VITRO TRANSCRIPTION Plasmid DNA was amplified in Escherichia coli and purified with a QIAGEN MaxiPrep kit. Template DNA was linearized with Bgl II. In the first experiments, the linearized DNA was treated with Exo VII to remove nucleotide overhangs but this step was subsequently omitted. Capped pSK-HEV-3 RNA (inoculated into rhesus monkeys 407 and 471 and into chimpanzees 1603 and 1609) and pSK-HEN-2 RΝA (inoculated into chimpanzee 96A007) were synthesized with mMessage in vitro transcription kits (Ambion, Austin, TX). A typical 100 μl reaction contained 5 to 7.5 μg linearized template, 10 μl of 10X transcription buffer, 50 μl of 2X capping dΝTP mix, 5 μl of 30 mM GTP, and 10 μl of T7 RΝA polymerase mix. In order to compare uncapped and capped RΝAs for infectivity, the MEGAscript high yield transcription kit (Ambion) was used instead of the mMessage kit. A 100 μl capping reaction contained 10 μl each of 75 mM ATP, CTP and UTP, 10 μl of 15 mM GTP, 15 μl of 40 mM m7 G (5') ppp (5') G cap analog (Ambion), 10 μl of 10X T7 Reaction Buffer, 5 μg linearized pSK-HEN-2, and 10 μl T7 enzyme mix. The components for synthesizing uncapped transcripts were identical except that the cap analog was replaced with water and 10 μl of 75 mM GTP rather than 15 mM GTP was used. The reaction tubes in each case were incubated at 37° C for 2 hours to permit transcription. Transcription reactions were diluted with 4 volumes of calcium-and magnesium-free phosphate buffered saline and immediately frozen on dry ice, then stored at - 80° for up to 3 days.

EXAMPLE 3 IΝTRAHEPATIC TRANSFECTION

Rhesus monkeys (Macaca mulatto) and chimpanzees (Pan troglodytes) were housed at Bioqual (Rockville, MD). The housing, maintenance, and care of the animals met or exceeded all requirements for primate husbandry.

Diluted transcription mixtures were thawed and, guided by ultrasound, were

00 inoculated percutaneously into multiple sites in the liver . The rhesus monkeys and chimpanzees inoculated with the pSK-HEN-3 transcripts and chimpanzee 96A007 inoculated with the pSK-HEN-2 transcripts were each injected with 1 mL of diluted transcription mixture. The other animals receiving pSK-HEN-2 transcripts (Rh 622, Rh 624, and chimpanzees 1619, 1620, 1622, and 1627) each received 600 μl of diluted transcripts. Animals were bled weekly and serum levels of isocitrate dehydrogenase (ICD) and alanine aminotranferase (ALT) were determined by standard methods (Anilytics, Gaithersburg, MD). Baseline levels were calculated as the geometric mean of three weekly samples collected at week zero and prior to inoculation: serum enzyme levels > 2 times background were considered elevated and indicative of hepatitis. Antibody to HEN was detected by an in-house ELISA based on recombinant Sar-55 ORF2 protein as previously described except that the ORF2 protein was more highly purified ²³' ²⁴. Percutaneous needle biopsies of the liver were obtained weekly and read under code by a pathologist. Histopathology scores of 1+ to 4+ were assigned to denote mild to severe hepatitis respectively.

EXAMPLE 4 GENOME QUANTIFICATION AND ANALYSIS RNA was extracted from serum samples with Trizol reagent (Life Technologies) and from ten percent fecal suspensions in phosphate buffered saline, pH7.4. HEN genome titer was quantified by Taqman RT-PCR (PE Biosystems). The Taqnian assay was standardized using in vitro transcripts of a Sar-55 cDΝA clone that were metabolically labeled by incorporation of 3H-UTP. The assay may reproducibly detect HEN RΝA with a sensitivity of 1 to 10 genomes/ml.

RT-PCR products amplified from serum or feces were purified on agarose gels and sequenced directly with an automated sequencer to yield the HEN consensus sequence. Secondary structures were predicted using the program MFOLD.

EXAMPLE 5

INFECTIOUS pSK-HEN-3 TRANSCRIPTS The cDNA clone pSK-HEN-3 contained the consensus sequence of the Sar-55 strain except for four introduced mutations that did not change the predicted amino acids. Capped transcripts synthesized in vitro were tested for infectivity by intrahepatic inoculation into the liver of each of two rhesus monkeys (471 and 407). Rhesus monkeys were chosen because they are very susceptible to infection by the Sar-55 strain, and much is known about the natural history of the virus in these animals ^{16, 19}' ²³. Although rhesus 407 had elevated serum liver enzymes during one week, neither monkey developed antibody to HEN and viral genomes were not detected in sera by a sensitive manual RT- PCR assay (Table 1) indicating that neither rhesus monkey was infected. Table 1. Intrahepatic transfection of primates with recombinant HEN genomes: summary

Transcripts Animal* Peak post/pre Peak genome Week of ALP (week) titer* (week) Seroconversion

7106 mutant* (capped) Rh 471 1.5 (1) not infected' Rh 407 2.3 (5) not infected¹¹ Ch 1609 1.4 (14) 5 (12) 14 Ch 1603 1.4 (11) 1.7 (13) 14

7106 wild- type" Rh 624 4.8 (5) 16,890 (5) (capped)

Rh 622 2.2 (7) 126 (7) 8 Ch 96A007 2.7 (4) 72 (3) 5 Ch 1620 1.4 (6) 18 (5) 6 Ch 1619 2.0 (8) 10 (5) 7

7106 wild- type" Ch 1622 1.5 (14) not infected' (uncapped) Ch 1627 1.0 (9) not infected¹

RH: rhesus, Ch: chimpanzee

Ratio of highest ALT to geometric mean of three pretransfection values

Per ml of serum (Taqman RT-PCR) pSK-HEN-3 (G mutated to T at position 7106; T mutated to C at position 7181)

Seroconversion not detected in 20 weeks of follow-up pSK-HEN-2 not done Since rhesus monkeys are susceptible to infection by many HEN strains, including the Sar-55 strain, and since both tamarins (hepatitis A virus) and chimpanzees (hepatitis C virus) have been successfully infected by the intrahepatic transfection technique, the failure to infect with HEN transcripts suggested that the cDΝA clone did not encode an infectious virus ²⁵' ²⁶. However, previous attempts of intrahepatic transfection of rhesus monkeys had not been performed, and it was conceivable that unknown technical problems made it impossible to use the technique in this species. Since the transfection procedure has been validated in chimpanzees and since they are also susceptible to infection with HEN, the transfection was repeated exactly as before except that two seronegative chimpanzees rather than macaques were inoculated. Although neither animal developed elevated serum liver enzyme levels indicative of hepatitis, each inoculated chimpanzee developed antibody to HEN at week 14 posttransfection, indicating that infection had occurred (Table 1). The first chimpanzee, chimp 1609, had low level viremia (4-5 genomes/ml), which occurred during the two weeks preceding seroconversion. Liver histopathology was negative except for biopsies taken the two weeks prior to viremia: they were scored as 1+, suggesting mild hepatitis. Two different regions of the HEN genome, amplified by RT-PCR from serum obtained at week 13, were sequenced. The sequence of the first product (165 nucleotides) confirmed that the silent mutation introduced into the cDΝA clone at nucleotide 286 in ORF1 was still present and proved that the infecting virus originated from the cDΝA clone. However, the sequence of the second RT-PCR product, 224 nucleotides in length, indicated that the mutations introduced at positions 7181 in the 3' ΝC and 7106 in ORF2 were retained but that the nucleotide at position 7144 (in the 3' noncoding region) had mutated from a C to an A. The second chimpanzee, chimp 1603, experienced viremia at week 13 but the titer was only 2 genomes/ml. Liver histopathology in this animal was normal for all samples collected weekly during weeks 0-15. The region encompassing nucleotide position 7106 was not amplified from the serum but was amplified from feces collected from chimp 1603 during week 13. Sequence analysis indicated that the introduced T to C mutation at 7181 and the G to T mutation at 7106 were retained throughout the infection but that the nucleotide at position 7097 had mutated from a G to an A. Although this mutation occurred in the region encoding ORF2 protein, it did not change the predicted amino acid.

EXAMPLE 6

TRANSCRIPTS CONTAINING A REVERSION OF THE 7106 MUTATION ENCODED A MORE VIRULENT VIRUS Although the successful infection of chimpanzees demonstrated that the cDNA clone pSK-HEV-3 encoded an infectious virus, the prolonged time to seroconversion suggested that the virus was attenuated for chimpanzees. Comparison of the sequence of the cDNA clone and those of other HEN genomes indicated that the nucleotides introduced into pSK-HEN-3 at positions 286, 4396, and 7181 were naturally present in some other genotype 1 strains and were, therefore, most likely not detrimental. In contrast, the T introduced at position 7106 was unique to the pSK-HEN-3 cDΝA clone and was located in a highly conserved region of the genome. In all other sequences published to date, this position contained either a G or an A (Fig. 1).

The unique T at position 7106 was corrected to the consensus G to produce a second cDΝA clone (pSK-HEN-2) that was identical to the first except for this one difference and that at the variable position (7181). Capped transcripts from the modified clone were inoculated into the liver of chimpanzee 96A007. The chimpanzee developed hepatitis as indicated by a 2.5-fold rise in the serum ALT level at week 4 (Table 1) but the liver histology remained normal. The animal seroconverted to anti-HEN at week 5. Niremia levels were 15 to 40 times higher (peak titer 71 genomes/ml at week 3) than previously observed following infection of chimpanzees with the uncorrected clone. The region spanning position 7106 was amplified from feces collected during week 2 from chimp 96A007. In contrast to the genomes recovered from each of the chimps inoculated with the unmodified clone, the 224 nucleotide-long sequence obtained following infection with the corrected genome was identical to that of the cDΝA clone used for transcription. The appearance of hepatitis and the normal time to seroconversion suggested that the virus encoded by the corrected cDΝA clone, pSK-HEN-2, was more virulent than that from the clone containing the 7106 mutation.

EXAMPLE 7

COMPARISON OF INFECTED RHESUS MONKEYS AND CHIMPANZEES In order to confirm that the virus from the corrected clone, pSK-HEN-2, was robust and to determine if rhesus monkeys may be infected with it, additional transfections were performed. Identical aliquots of capped transcripts were inoculated intrahepatically into two rhesus monkeys and into two additional chimpanzees. A batch of uncapped transcripts synthesized with the same reagents in a parallel reaction in which the m7 G cap analogue was replaced with GTP was similarly inoculated intrahepatically into two other chimpanzees to determine if the cap structure was required for infectivity. The two RNA preparations appeared to be identical when examined by agarose gel electrophoresis except, as expected, slightly more RNA was synthesized in the reaction containing GTP in place of the cap analogue.

Both rhesus monkeys and both chimpanzees injected with the capped transcripts became infected and all four animals seroconverted to anti-HEN (Table 1). Chimpanzees 1620 and 1619 became antibody positive at weeks 6 and 7 posttransfection respectively. Chimpanzee 1620 experienced a slight elevation of serum ALT around the time of seroconversion when hepatitis normally occurs but the elevation above baseline was less than the two-fold elevation indicative of hepatitis. Niremia was detected only at week 5 (18 genomes/ml). Chimpanzee 1619 had an ALT level exactly twice the preinoculation level, suggesting that it had mild hepatitis, and it had viremia during weeks 5 and 6 (10 and 5 genomes/ml respectively). Similarly, rhesus monkey 624 seroconverted at week 5 and rhesus 622 seroconverted at week 8 posttransfection. Rhesus 624 developed significant hepatitis, with a serum ALT level 4.8 times and an ICD level 4.4 times above baseline at week 5, the time of seroconversion. This animal had the longest period of viremia (4 weeks) and the highest titer of virus (16,900 genomes/ml) of the four animals in this experiment. Rhesus 622 had a milder hepatitis, with peak serum ALT and ICD levels just slightly 2-fold above baseline the week prior to seroconversion. Although viremia was detected in this animal during weeks 5 tlirough 7, peak titer reached only 126 genomes/ml. The region surrounding the silent mutation at position 286 was amplified from the serum of all four animals. In each case the mutation was present, thus confirming that the virus replicating in each animal originated from the cDNA clone. The 224 nucleotide-long region preceding the poly A tail was also amplified and sequenced. Once again, in contrast to our detection of mutations in virus recovered from chimps 1603 and 1609, which were transfected with the uncorrected virus clone, mutations were not detected in viruses recovered from any of the four animals and the consensus sequences of the recovered viruses were identical to that in the pSK-HEN-2 cDΝA clone.

EXAMPLE 8 UNCAPPED TRANSCRIPTS ARE NOT INFECTIOUS

The two chimpanzees inoculated with uncapped transcripts were followed for 20 weeks. Neither chimpanzee became infected since they did not develop hepatitis nor did they seroconvert to anti-HEN.

EXAMPLE 9

CAPPED RECOMBINANT GENOMES CAN REPLICATE IN CELLS

TRANSFECTED EX VIVO.

Capped transcripts synthesized in vitro from pSK-HEN-2 were mixed with the transfection agent DMRIE-C and incubated with monolayers of Alexander cells or with monolayers of a sub-clone of Huh-7 cells. After incubation at 34.5°C or 37°C for 5 hours, the cells were washed with Dulbecco's Modified Eagle Medium containing 10% fetal calf serum (DMEM-10%), then fresh DMEM-10% was added and the cells were incubated for

10 days at 34.5°C. At day 10 post-transfection, cells were fixed with acetone then incubated with a mixture of chimpanzee serum containing anti-HEN and a mouse monoclonal antibody to the HEN capsid protein. Secondary antibodies labeled with FITC (chimpanzee specific) or Alexa (mouse specific) were next added and the cells were examined by fluorescence microscopy. Positive cells which were stained with both antibodies were detected in each of the two cell lines indicating that the viral genome was replicating and that viral capsid protein was being produced.

EXAMPLE 10 CHIMERIC GENOMES CONTAINING THE CIS-REACTIVE ELEMENT (CRE) FROM A HETEROLOGOUS GENOTYPE ARE INFECTIOUS FOR MONKEYS.

The 3' terminus containing the CRE was removed from the pSK-HEN-2 cDΝA

(genotype 1) and replaced with the analogous region from a genotype 2 or genotype 3 strain. Capped transcripts from each of the two chimeric viruses were transfected into the liver of two rhesus monkeys respectively. All four monkeys seroconverted to HEN and developed viremia indicating that the two chimeric viruses were both infectious.

EXAMPLE 11 RECOMBINANT GENOMES CONTAINING ENGINEERED POINT MUTATIONS CAN BE TESTED FOR REPLICATION ABILITY IN CELL CULTURE. The pSK-HEN-2 cDΝA clone was mutagenized by standard techniques to remove two or three respectively of the putative glycosylation sites in ORF2 protein. Serine 80 in ORF3 was independently mutated to alanine in order to eliminate the putative phosphorylation site in ORF3 protein. Capped transcripts of each mutant were transfected into Alexander cells and replication-competent virus was identified by immunefluorescence microscopy. Cells positive for replicating virus were detected when two, but not when all three, glycosylation sites were eliminated. Replication was also observed in the mutant lacking the ORF3 phosyphorylation site.

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art (including the contents of the references cited herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one of ordinary skill in the art.

REFERENCES

1. Purcell, R.H., Emerson, S.U. (2001) in Fields Virology eds. Knipe, D., Howley, P., Griffin, D., Lamb, R., Martin, M., Roizman, B., Straus, S. (Lippincott, Williams and

Wilkins, Philadelphia) pp. 3051-3061.

2. Balayan, M.S., Andjaparidze, A.G., Savinskaya, S.S., Keliladze, E.S., Braginsky, D.M., Savinov, A.P., Poleschuk, V.F. (1983) Intervirology 20, 23-31.

3. Tam, A.W., Smith. M ., Guerra, M.E., Huang, C.C, Bradley, D.W., Fry, K.E., Reyes, G.R. (1991) Virology 185, 120-131.

4. Huang, C.C, Nguyen, D., Fernandez, J., Yun, K.Y., Fry, K.E., Bradley, D.W., Tam, A.W., Reyes, G. (1992) Virology 191, 550-558.

5. Meng, X.J., Purcell, R.H., Halbur, P.G., Lehman, J.R., Webb, D ., Tsareva, T.S., Haynes, J.S., Thacker, B.J., Emerson, S.U. (1997) Proc. Natl. Acad. Sci. U S A 94, 9860-9865.

6. Schlauder, G.G., Mushahwar, I.K. (2001) J. Med. Virol. 65, 282-292.

7. Meng, X.J., Halbur, P.G., Shapiro, M.S., Govindarajan, S., Bruna, J.D., Mushahwar, I.K., Purcell, R.H., Emerson, S.U. (1998) J. Virol. 72, 9714-9721.

8. Kabrane-Lazizi, Y., Fine, J.B., Elm, J., Glass, G.E., Higa, H., Diwan, A, Gibbs, C.J., Meng, X.J., Emerson, S.U., Purcell, R.H. (1999) Am. J. Trop. Med. Hyg. 61, 331-335.

9. Arankalle, V.A., Goverdhan, M.K., Banerjee, K. (1994) J. Viral. Hepat. 1, 125-129.

10. Favorov, M.O., Nazarova, O., Margolis, H.S. (1998) Am. J. Trop. Med. 59, 242.

11. Balayan, M.S., Usmanov, R.K., Zamyatina, N.A., Karas, F.R. (1990) J. Med. Virol. 32, 58-59. 12. Koonin, EN., Gorbalenya, A.E., Purdy, M.A., Rozanov, M.Ν., Reyes, G.R., Bradley, D.W. (1992) Proc. Natl. Acad. Sci. USA 89, 8259-8263.

13. Kabrane-Lazizi, Y., Meng, X.J., Purcell, R.H., Emerson, S.U. (1999) J. Virol. 73, 8848- 8850.

14. Zhang, M., Purcell, R.H., Emerson, S.U. (2001) J. Med. Virol. 65, 293-295. 15. Magden, J., Takeda, N., Li, T., Auvinen, P., Ahola, T., Miyamura, T., Merits, A., Kaariainen, L. (2001) J. Virol. 75, 6249-6255. 16. Purcell, R.H., Emerson, S.U. (2001) ILAR J. 42, 161-177. 17. Tsarev, S.A., Tsareva, T.S., Emerson, S.U., Yarbough, P.O., Legters, L.J., Moskal, T., Purcell, R.H. (1994) J. Med. Nirol. 43, 135-142.

18. Mast, E.E., Alter, M.J., Holland, P.N., Purcell, R.H. (1998) Hepatology 27, 857-861.

19. Tsarev, S.A., Tsareva, T.S., Emerson, S.U., Rippy, M.K., Zack, P., Shapiro, M., Purcell, R.H. (1995) J. Infect. Dis. 172, 31-37.

20. Panda, S.K., Ansari, I.H., Durgapal, H., Agrawal, S., Jameel, S. (2000) J. Nirol. 74, 2430-2437.

21. Tsarev, S.A., Emerson, S.U., Reyes, G.R., Tsareva, T.S., Legters, L.J., Malik, LA., Iqbal, M., Purcell, R.H. (1992) Proc. Νatl. Acad. Sci. U S A 89, 559-563. 22. Yanagi, M., St. Claire, M., Shapiro, M., Emerson, S.U., Purcell, R.H., Bukh, J. (1998) Virology 244, 161-172.

23. Tsarev, S.A., Tsareva, T.S., Emerson, S.U., Kapikian, A.Z., Ticehurst, J., London, W., Purcell, R.H. (1993) J. Infect. Dis. 168, 369-378.

24. Robinson, R.A., Burgess, W.H., Emerson, S.U., Leibowitz, R.S., Sosnovtseva, S.A., Tsarev, S ., Purcell, R.H. (1998) Protein Expression and Purification 12, 75-84.

25. Yanagi, M., Purcell, R.H., Emerson, S.U., Bukh, J. (1997) Proc. Νatl. Acad. Sci. U S A 94, 8738-8743.

26. Emerson, S.U., Lewis, M., Govindarajan, S., Shapiro, M., Moskal, T., Purcell, R.H. (1992) J. Nirol. 66, 6649-6654. 27. Agrawal, S., Gupta, D., Panda, S.K. (2001) Virology 282, 87-101.

Claims

WHAT IS CLAIMED IS:

1. A purified and isolated nucleic acid molecule which encodes human hepatitis E virus, said molecule capable of expressing said virus when transfected into cells.

2. The nucleic acid molecule of claim 1, wherein said molecule encodes the amino acid sequence of SEQ ID Nos. 5-7.

3. The nucleic acid molecule of claim 2, wherein said molecule comprises the nucleic acid sequence of SEQ ID No.2.

4. The nucleic acid molecule of claim 1, wherein a fragment of said molecule which encodes the non-structural proteins of hepatitis E virus has been replaced by the non- structural proteins from the genome of another hepatitis E virus strain.

5. The nucleic acid molecule of claim 1, wherein a fragment of said molecule which encodes the structural protein(s) of hepatitis E virus has been replaced by the structural protein(s) from the genome of another hepatitis E virus strain.

6. The nucleic acid molecule of claim 1, wherein a fragment of said molecule which encodes the cis-reactive element of hepatitis E virus has been replaced by the cis- reactive element from the genome of another hepatitis E virus strain.

7. The nucleic acid molecule of claim 1, wherein a fragment of the nucleic acid molecule which encodes at least one HEN protein has been replaced by a fragment of the genome of another hepatitis E virus strain which encodes the corresponding protein.

8. The nucleic acid molecule of claim 7, wherein the HEN protein is selected from the group consisting of ORF 1, ORF2, or ORF3 proteins.

9. The nucleic acid molecule of claim 1, wherein a fragment of the molecule encoding all or part of an HEN protein has been deleted.

10. The nucleic acid molecule of claim 9, wherein the HEN protein is selected from the group consisting of ORF 1, ORF2, or ORF3 proteins.

11. A DΝA construct comprising a nucleic acid molecule according to claims 1 or 3.

An RΝA transcript of the DΝA construct of claim 11. A cell transfected with the DΝA construct of claim 11. A cell transfected with RΝA transcript of claim 12. A hepatitis E virus produced by the cell of claim 13. A hepatitis E virus produced by the cell of claim 14.

17. A hepatitis E virus whose genome comprises a nucleic acid molecule according to claims 1, 3, or 4-7

18. A method for producing a hepatitis E virus comprising transfecting a host cell with the RNA transcript of claim 12.

19. A method for assaying candidate antiviral agents for activity against HEN, comprising:

(a) exposing a cell containing the hepatitis E virus of claim 17 to the candidate antiviral agent; and

(b) measuring the presence or absence of hepatitis E virus replication in the cell of step (a).

20. The method of claim 19, wherein said replication in step (b) is measured by at least one of the following: negative strand RT-PCR, quantitative RT-PCR, Western blot, immunofluoresence, or infectivity in a susceptible animal.

21. A method for assaying candidate antiviral agents for activity against HEN, comprising:

(a) exposing an HEN enzyme encoded by a nucleic acid sequence according to claims 1 or 2, or a fragment thereof to the candidate antiviral agent in the presence of an appropriate substrate; and

(b) measuring the enzyme activity of said enzyme.

22. The method for assaying candidate antiviral agents for activity against HEN, wherein the HEN enzyme is selected from the group consisting of a protease, polymerase, methyltransferase, guanyltransferase, and helicase.

23. An antiviral agent identified as having antiviral activity for HEN by the method of claim 19.

24. An antiviral agent identified as having antiviral activity for HEN by the method of claim 21.

25. A method for determining the susceptibility of cells in vitro to support HEN infection, comprising the steps of:

(a) growing animal cells in vitro;

(b) transfecting into said cells the nucleic acid of claim 1; and

(c) determining if said cells show indicia of HEN replication.

26. The method according to claim 25, wherein said cells are human cells.

27. A cassette vector for cloning viral genomes, comprising, inserted therein, the nucleic acid sequence according to claim 2, said vector reading in the correct frame for the expression of said inserted sequence and having an active promoter sequence upstream thereof.

28. The cassette vector of claim 27, wherein the vector also contains one or more expressible marker genes.

29. The cassette vector of claim 27, wherein the inserted DNA sequence contains at least one ORF of the HEN genome from another strain.

30. The cassette vector of claim 27, wherein the promoter is a bacterial promoter.

31. An attenuated hepatitis E virus comprising a cDΝA sequence of SEQ ID NO: 1, 3-4, or chimera based on SEQ ID NO:2, or variant thereof.

32. A vaccine comprising an immunologically effective amount of a virus according to claim 31 in a pharmaceutically acceptable carrier, diluent, or excipient.

33. A vaccine comprising a nucleic acid molecule of claim 1 suspended in a suitable amount of a pharmaceutically acceptable carrier, diluent, or excipient.

34. A method for treating hepatitis E viral infection comprising the administration to an animal in need thereof of a clinically effective amount of the vaccine of claim 32 or 33.