CA2306451A1

CA2306451A1 - Methods and compositions for detecting hepatitis e virus

Info

Publication number: CA2306451A1
Application number: CA002306451A
Authority: CA
Inventors: George G. Schlauder; James C. Erker; Suresh M. Desai; George J. Dawson; Isa K. Mushahwar
Original assignee: Individual
Current assignee: Abbott Laboratories
Priority date: 1997-10-15
Filing date: 1998-10-15
Publication date: 1999-04-22
Also published as: EP1023605A1; JP4475803B2; WO1999019732A1; JP2001520384A

Abstract

Disclosed herein are methods and compositions for detecting the presence in a sample of a US-type or a US-subtype hepatitis E virus, including naturally occurring variants thereof. In particular, the invention provides nucleic acid sequences corresponding to the genome of the US-type or US-subtype hepatitis E virus, amino acid sequences, including epitope sequences, encoded by the genomes of such viruses, and antibodies that bind specifically to such amino acid sequences. The invention further provides methods and compositions for immunizing individuals against infection by, or for treating individuals already infected with such a virus.

Description

METHODS AND COMPOSITIONS
FOR DETECTING HEPATITIS E VIRUS
Related Annlications This application claims priority under 35 U.S.C. ~119(e) to provisional application U.S.S.N. 60/061,199, filed October 15, 1997, the disclosure of which is incorporated by reference herein.
Field of the Invention This invention relates generally to methods and compositions for detecting hepatitis E
virus, and more particularly to methods and compositions for detecting in, or treating individuals infected with US-type and US-subtype strains of hepatitis E virus.
Background of the Invention There are at least five major classes of hepatotropic viruses that cause inflammation of the liver {hepatitis). These viruses include hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus {HCV), hepatitis D virus (HDV) and hepatitis E virus (HEV).
Although only HBV, HCV and HDV cause chronic hepatitis, all five types cause acute disease either directly or as a result of superinfection/co-infection by, for example, HBV and HDV. HEV
causes symptoms of hepatitis that are similar to those of other viral agents including abdominal pain, jaundice, malaise, anorexia, dark urine, fever, nausea and vomiting (see, for example, Reyes et al., "Molecular biology of non-A, non-B hepatitis agents: hepatitis C and hepatitis E viruses" in Advances in Virus Research (1991) 40: 57-102; Bradley, "Hepatitis non-A, non-B
viruses become identified as hepatitis C and E viruses" in Progr. Med. Virol. (1990) 37: 101-135;
Hollinger "Non-A, non-B hepatitis viruses" in Virology, Second Edition (1990), Second Edition, Raven Press, New York pp. 2239-2271; Gust et al., "Report of a workshop:
waterborne non-A, non-B hepatitis" J. Infect. Dis. (1987) 156: 630-635; and Krawcyznski "Hepatitis E" Hepatology ( 1993 ) 17: 932-941 ). Unlike the other hepatoviruses, however, HEV generally has not been perceived as being a significant cause of hepatitis in the US.
Geographic regions where HEV is endemic include eastern and northern Africa, India, Pakistan, Burma and China (Reyes et al. ( 1991 ) supra). The case fatality rate of HEV infection is estimated to be between about 0.1 % to about 1.0% in the general population, where HEV is endemic, and as high as about 20% among pregnant women in developing countries. Most fatalities result from fulminant hepatitis (Reyes et al. ( 1991 ) supra). The occasional reports of infection with HEV in the US, western Europe and Japan, usually are observed in travelers returning home from visits to areas where HEV in endemic. However, there is little information pertaining to the morbidity and/or mortality of infection with HEV in the US
since HEV
infections are not reported to a central agency. Extensive, systematic studies have not been performed to determine the importance of HEV in US. Further, if such studies were performed, the relative importance of HEV in US (and possibly Japan and Western Europe) may continue to be underestimated unless the proper reagents are developed to conduct such a study.
The basic features of HEV is that it is a non-enveloped virus, approximately-2?-30 nm in diameter possessing a positive sense, single stranded RNA genome which comprises three discontinuous open-reading frames (ORFs), referred to in the art as open reading frame 1 (ORF
1), open reading frame 2 (ORF 2), and open reading frame 3 (ORF 3). Based on the overall morphology of the virus and the size and organization of the genome, the virus is tentatively classified as a member of the Caliciviridae. The first two isolates of HEV to be identified and sequenced were obtained from Burma and from Mexico. The overall nucleic acid identity across the genome of both isolates is 76% (Reyes et al. (1990) Science, 247: 1335-1339; Tam et al.
(1991) Virology 185: 120-131; Huang et al. (1992) Virology 191: 550-558). Many of the nucleotide differences were noted at the third codon position, such that the deduced similarities in amino acid sequences between the Burmese and Mexican strains of HEV were 83%, 93% and 87%, for open reading frames ORF 1, ORF 2, and ORF 3, respectively.
In the Burmese strain, there is a short non-translated region of about 27 nucleotides at the S' end of the genome which has not been identified in the Mexican strain. ORF
1 comprises approximately 5,100 nucleotides, which encode several conserved motifs including a putative methyltransferase domain, an RNA helicase domain, a putative RNA-dependent RNA
polymerase (RDRP) domain, and a putative papain-like protease. A tripeptide sequence of Gly-Asp-Asp (GDD), found in all positive-sense RNA plant and animal viruses, is located within ORF 1 and usually signifies RDRP function. Conserved motifs suggestive of purine NTPases activity that is usually associated with cellular and viral helicases also are present in the ORF 1 sequence. There is no consistent immune response to gene products encoded in ORF 1.
The second open reading frame (ORF 2) occupies the carboxyl one-third of the viral genome. ORF 2 comprises approximately 2,000 nucleotides which encode a consensus signal peptide sequence at the amino terminus of ORF 2, and a putative capsid protein, translated in a 1+ reading frame in relation to ORF 1. Frequently, HEV infected individuals produce antibodies that react with peptides or recombinant proteins derived from ORF
2.
The third open reading frame (ORF 3) partly overlaps both ORF l and ORF 2, and comprises 369 nucleotides translated in the +2 reading frame in relation to ORF 1. Although the function of the protein encoded by ORF 3 is unknown, the protein is antigenic, with most HEV
infected individuals producing antibodies to this protein. Accordingly, peptides or recombinant proteins derived from ORF 2 and ORF 3 may serve as serologic markers useful in diagnosing exposure to HEV.
Recently, several additional HEV isolates have been identified and compared to the Burmese and Mexican strains of HEV. Most of the recent isolates are more closely related to the Burmese strain than to the Mexican strain of HEV. Except for a brief appearance in 1986-1987, there have been no additional isolates of the Mexican strain of HEV
(Velasquez et al.
(1992) JAMA, 263: 3281-3286).
One isolate, referred to as SAR-55, recently was isolated from an HEV-infected individual from Pakistan. The SAR-55 isolate is highly related to the Burmese strain with nucleotide and amino acid identities of 94% and 99%, respectively, across the entire genome.

Several other recent isolates have been made from the Chinese province of Xuar, bordering on Pakistan. These Chinese isolates were more closely related to the Pakistani strain (approximately 98% nucleotide identity) than to the Burmese strain (approximately 93%
nucleotide identity).
Prior to the sequencing of the viral genome and the availability of viral-encoded recombinant proteins and synthetic peptides, HEV infection was monitored by electron microscopy and immunofluoresence. Soon after the identification of the HEV
genome, specific laboratory techniques for detecting HEV infection became available including (i) specific immunoassays, for example, western blot assays and ELISA's based on recombinant proteins and/or synthetic peptides, and {ii) polymerase chain reactions (PCR), for example, reverse transcriptase PCR (RT-PCR). RT-PCR has been used successfully to detect HEV
RNA in samples of stool or serum in cases of acute hepatitis infections, and in epidemics of ET-NANBH.
Furthermore, by using recombinant antigens derived from the Mexican and Burmese strains of HEV, specific IgG, IgM and, in some cases, IgA antibodies to HEV have been detected in specimens obtained from ET-NANBH outbreaks in Somalia, Burma, Borneo, Tashkent, Kenya, Pakistan and Mexico. Specific IgG, and sometimes IgM antibodies to HEV have been detected in cases of acute, sporadic hepatitis in geographic regions such as Egypt, India, Tajikistan and Uzbekistan as well as in acute hepatitis cases among patients in industrialized nations (for example, US, UK, Netherlands and Japan) who traveled to areas endemic for HEV.
To date, PCR and immunoassay-based tests based on the Burmese and Mexican isolates of HEV have established that various cases of "waterborne hepatitis" were caused by HEV. The antibody tests also were important in establishing HEV as a cause of acute, sporadic hepatitis in developing nations and among travelers to regions where HEV is endemic.
However, it is unclear as to how many cases of acute HEV currently go undiagnosed due to the inability of current reagents to detect exposure to all strains of HEV. Accordingly, as new isolates of HEV
are identified, it is desirable to develop new compositions and methods for detecting and/or treating hepatitis caused by the new HEV strains, which heretofore remain undetectable by the currently available test kits.

Summar~o~f tl:e Invention The invention is based, in part, upon the discovery of a new family of human hepatitis E
viruses. The newly discovered family of hepatitis E viruses fall within a class referred to hereinafter as a US-type hepatitis E virus. Furthermore, two members of the family were discovered in individuals living in the United States and exhibit considerable similarities when compared at the nucleotide and amino acid levels. The latter two members together belong to a subclass of the US-type hepatitis E virus, referred to hereinafter as US-subtype hepatitis E virus.
Accordingly, in one aspect, the invention provides a method for detecting the presence of a US-type or US-subtype hepatitis E virus in a test sample of interest. The method comprises the steps of (a) contacting the test sample with a binding partner that binds specifically to a marker (or target) for the virus, which if present in the sample binds to the binding partner to produce a marker-binding partner complex, and (b) detecting the presence or absence of the complex. The presence of the complex is indicative of the presence of the virus in the test sample.
In one embodiment, the marker is an anti-US-type or anti-US-subtype antibody, for example, an immunoglobulin G (IgG) or an immunoglobulin M (IgM) molecule, present in the sample of interest, and the binding partner is an isolated polypeptide chain defining an epitope that binds specifically to the marker. In such a case, it is contemplated that the test sample is a body fluid sample, for example, blood, serum or plasma, harvested from an individual under investigation. In a preferred embodiment, the polypeptide chain defining a US-type or US-subtype specific epitope is immobilized on a solid support. Thereafter, the immobilized polypeptide chain is combined with the sample under conditions that permit the marker antibody, for example, an anti-US-type or anti-US-subtype hepatitis E virus specific antibody, present in the sample to bind to the immobilized polypeptide. Thereafter, the presence or absence of bound antibody can be detected using, for example, a second antibody or an antigen binding fragment thereof, for example, an anti-human antibody or an antigen binding fragment thereof, labeled with a detectable moiety.

It is contemplated that many different US-type and US-subtype specific polypeptides may be useful as a binding partner in the practice of this embodiment of the invention. For example, in one preferred embodiment of the invention, it is contemplated that the binding partner may be at least a portion, for example, at least S, preferably at least 8, more preferably at least 15 and even more preferably at least about 25 amino acid residues, of a polypeptide chain selected from the group consisting of SEQ ID NOS:91, 92 and 93, including naturally occurring variants thereof, and which represent a unique amino acid sequence when compared to the corresponding amino acid sequences of members of the Burmese and Mexican families.
Similarly. it is contemplated that the binding partner may be a polypeptide chain comprising the amino acid sequence set forth in SEQ ID NOS:173, 174, or 175. In another preferred embodiment of the invention, it is contemplated that the binding partner may be at least a portion, for example, at least 5, preferably at least 8, more preferably at least 15 and even more preferably at least about 25 amino acid residues, of a polypeptide chain selected from the group consisting of SEQ ID
NOS:166, 167 and 168, including naturally occurring variants thereof, and which represent a unique amino acid sequence when compared to the corresponding amino acid sequences of members of the Burmese and Mexican families. Similarly, it is contemplated that the binding partner may be a polypeptide chain comprising the amino acid sequence set forth in SEQ ID
NOS:176, 223 or 224.
In another embodiment of the invention, the marker is a polypeptide chain unique for a member of the US-type or US-subtype families of HEV, and the binding partner preferably is an isolated antibody, for example, a polyclonal or monoclonal antibody, that binds to an epitope on the marker polypeptide chain. The binding partner may be either labeled with a detectable moiety or immobilized on a solid support. For example, it is contemplated that practice of this embodiment of the invention may be facilitated by immobilizing on a solid support, a first antibody that binds a first epitope on the marker polypeptide of interest. A
test sample to be analyzed then is combined with the solid support under conditions that permit the immobilized antibody to bind the marker polypeptide. Thereafter, the presence or absence of bound marker polypeptide chain may be determined using, for example, a second antibody conjugated with a detectable moiety which binds to a second, different epitope on the marker polypeptide chain.
An antibody useful in the practice of this embodiment of the invention preferably is capable of binding specifically to a polypeptide chain selected from the group consisting of SEQ
ID NOS:91, 92, and 93, including naturally occurring variants thereof, and has a higher binding affinity for such a polypeptide chain relative to the corresponding sequences of members of the Burmese and Mexican families. It is contemplated that an antibody useful in the practice of the invention preferably is capable of binding specifically to a polypeptide chain comprising the amino acid sequence set forth in SEQ ID NOS:173 or 175. This antibody being further characterized as, under similar conditions, preferably having a lower affinity for, and most preferably failing to bind the amino acid sequence set forth in SEQ. ID
NOS:169 or 171 or to the regions in the Burmese and Mexican strains that correspond to SEQ ID N0:175.
Similarly, it is contemplated that an antibody useful in the practice of the invention preferably is capable of binding specifically to a polypeptide chain comprising the amino acid sequence set forth in SEQ
ID NOS:174 or 176. This antibody being further characterized as, under similar conditions, preferably having a lower affinity for, and most preferably failing to bind the amino acid sequence set forth in SEQ. ID NOS:170 or 172 or to the regions in the Burmese and Mexican strains that correspond to SEQ ID N0:176.
Similarly, it is contemplated that an antibody useful in the practice of this embodiment of the invention preferably is capable of binding specifically to a polypeptide chain selected from the group consisting of SEQ ID NOS:166, 167, and 168, including naturally occurring variants thereof, and has a higher binding affinity for such a polypeptide chain relative to the corresponding sequences of members of the Burmese and Mexican families. It is contemplated that an antibody useful in the practice of the invention preferably is capable of binding specifically to a polypeptide chain comprising the amino acid sequence set forth in SEQ ID
N0:223. This antibody being further characterized as, under similar conditions, preferably having a lower affinity for, and most preferably failing to bind the amino acid sequences set forth in SEQ. ID NOS:170 or 172. Similarly, it is contemplated that an antibody useful in the practice of the invention preferably is capable of binding specifically to a polypeptide chain comprising the amino acid sequence set forth in SEQ ID N0:224. This antibody being further characterized as, under similar conditions, preferably having a lower affinity for, and most preferably failing to bind the amino acid sequence set forth in SEQ ID NOS:169 or 171.
In another embodiment of the invention, the marker is a nucleic acid sequence defining at least a portion of a genome of a US-type or US-subtype E virus, or a sequence complementary thereto. Similarly, it is contemplated that the binding partner is an isolated nucleic acid sequence, for example, a deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or peptidyl nucleic acid (PNA) sequence, preferably comprising 8-100 nucleotides, more preferably comprising 10 to 75 nucleotides and mostly preferably comprising 15-50 nucleotides, which is capable of hybridizing specifically, for example, under specific hybridization conditions or under specific PCR annealing conditions, to the nucleotide sequence set forth in SEQ
ID NOS:89 or 164.
Practice of this embodiment of the invention may be facilitated, for example, by isolating nucleic acids from the sample of interest. Thereafter, the resulting nucleic acids, may be fractionated by, for example, gel electrophoresis, transferred to, and immobilized onto a solid support, for example, nitrocellulose or nylon membrane, or alternatively may be immobilized directly onto the solid support via conventional dot blot or slot blot methodologies. The immobilized nucleic acid then may be probed with a preselected nucleic acid sequence labeled with a detectable moiety, that hybridizes specifically to the marker sequence.
Alternatively, the presence of marker nucleic acid in a sample may be determined by standard amplification based methodologies, for example, polymerase chain reaction (PCR) wherein the production of a specific amplification product is indicative of the presence of marker nucleic acid in the sample.
In another aspect, the invention provides isolated US-type and US-subtype specific polypeptides sequences. These polypeptides include those described hereinabove in the section pertaining to US-type and US-subtype hepatitis E specific polypeptides chains useful as binding partners. In a preferred embodiment, the isolated polypeptide chain comprises an amino acid sequence set forth in SEQ ID N0:93, SEQ ID N0:168, SEQ ID N0:173, SEQ ID
N0:174, SEQ
ID N0:175, SEQ ID N0:176, SEQ ID N0:223 or SEQ ID N0:224. It is contemplated that these and other US-type and US-subtype specific poIypeptide chains may be employed in an assay format for detecting the presence of anti-US-type of US-subtype hepatitis E
specific antibodies in a sample. In addition, it is contemplated that these polypeptides may be used either alone or in combination with adjuvants for the production of antibodies in laboratory animals, or similarly, used in combination with pharmaceutically acceptable Garners as vaccines for either the prophylactic or therapeutic immunization of mammals.
In another aspect, the invention provides isolated anti-US-type or anti-US-subtype hepatitis E specific antibodies, which include those discussed hereinabove in the section pertaining to antibodies useful as binding partners. In a preferred embodiment, the isolated antibody is capable of binding specifically to a polypeptide chain selected from the group consisting of a polypeptide encoded by an ORF 1 sequence of a US-type or a US-subtype hepatitis E virus, a polypeptide encoded by an ORF 2 sequence of a US-type or a US-subtype hepatitis E virus, or a polypeptide encoded by an ORF 3 sequence of a US-type or a US-subtype hepatitis E virus. In particular, it is contemplated that useful antibodies are characterized in that they are capable of binding specifically to a polypeptide chain comprising the amino acid sequence set forth in SEQ ID N0:93, SEQ ID N0:168, SEQ ID N0:173, SEQ ID
N0:174, SEQ
ID N0:175, SEQ ID N0:176, SEQ ID N0:223 or SEQ ID N0:224. It is contemplated that these antibodies and other antibodies may be used to advantage in immunoassays for detecting the presence in a sample of members of the US-type or US-subtype hepatitis E
families. The antibody may be used either in a direct immunoassay wherein the antibody itself preferably is labeled with a detectable moiety or in an indirect immunoassay wherein the antibody itself provides a target for a second binding partner, e.g., a second antibody labeled with a detectable moiety. Furthermore, it is contemplated that these antibodies may be used in combination with, for example, a pharmaceutically acceptable carrier for use in the passive, therapeutic or prophylactic immunization of a mammal.

In another aspect, the invention provides isolated nucleic acid sequences such as those discussed in the previous section pertaining to the use of nucleic acids as a marker or a binding partner for detecting the presence of a US-type or US-subtype hepatitis E
virus in a sample. In a preferred embodiment, the invention provides isolated nucleic acid sequences defining at least a portion of an ORF l, ORF 2 or ORF 3 sequence of a US-type or US-subtype hepatitis E virus, or a sequence complementary thereto. It is contemplated that these and other nucleic acid sequences may be used, for example, as nucleotide probes and/or amplification primers for detecting the presence of a US-type or US-subtype hepatitis E virus in a sample of interest. In addition, it is contemplated the nucleic acid sequences or sequences complementary thereto may be combined with a pharmaceutically acceptable carrier for use in anti-sense therapy.
Furthermore, it is contemplated the nucleic acid sequences may be integrated in vectors which may then be transformed or transfected into a host cell of interest. The host cells may then be combined with a pharmaceutically acceptable carrier and used as a vaccine, for example, a recombinant vaccine, for immunizing a mammal, either prophylactically or therapeutically, against a preselected US-type or US-subtype hepatitis E virus.
The foregoing and other objects, features and advantages of the present invention will be made more apparent from the following detailed description of preferred embodiments of the invention.
BriefDescriavtion of tl:e Drawings The objects and features of the invention may be better understood by reference to the drawings described below in which, Figure 1 is a schematic representation of a HEV genome showing the relative positions of the ORF 1, ORF 2, and ORF 3 regions.
Figure 2 is a graph showing levels of serum aspartate aminotransferase (boxes) and serum total bilirubin (diamonds) in patient USP-1 from day 1 of a hospital admission through day 37 post admission.
Figure 3 is a schematic representation of the HEV US-1 genome showing the relative positions of clones isolated during the course of this work.
Figure 4 is a schematic representation of the HEV US-2 genome showing the relative positions of clones isolated during the course of this work.
Figure 5 shows an uprooted phylogenetic tree depicting the relationship of nucleotide sequences from full length HEV US-I, HEV US-2, and 10 other HEV isolates.
Branch lengths are proportional to the evolutionary distances between sequences. The scale representing nucleotide substitutions per position is shown. The internal node numbers indicate the bootstrap values (expressed as a percentage of all trees) obtained from 100 replicates.
Isolates represented are Burmese, B1, B2; Chinese, C1, C2, C3, C4; Pakistan, P1; Indian, I1, I2;
Mexican, M1; and United States. US-1, US-2.
Figure 6 shows an uprooted phylogenetic tree depicting the relationship of nucleotide sequences from the ORF 2/3 regions (i.e., sequences corresponding to nucleotide residue numbers 5094-7114 of SEQ ID N0:89). Branch lengths are proportional to the evolutionary distances between sequences. The scale representing nucleotide substitutions per position is shown. The internal node numbers indicate the bootstrap values (expressed as a percentage of all trees) obtained from I00 replicates. Isolates represented are Burmese, B1, B2;
Chinese, C1, C2, C3, C4; Pakistan, P 1; Indian, I 1, I2; Mexican, M 1; Swine, S I ; and United States, US-1, US-2.
Figure 7 is a graph showing levels of alanine aminotransferase (boxes), serum aspartate transferase (circles), and gamma-glutamyltransferase (triangles) in a macaque before and after inoculation with sera harvested from patient USP-2. Also shown are times when RNA were present in serum and fecal samples, as well as times when anti-HEV US-2 IgM and IgG were detectable.

Figure 8 is a schematic representation of the Itl genome showing the relative positions of clones isolated during the course of this work.
Figures 9 (a-c) are aligments of Burmese (B 1 ), Mexican (M 1 ), Chinese (C 1 ), Pakistan (P 1 ) and US-1 showing the design of HEV consensus primers for a) ORF 1, b) ORF 2/3 and c) ORF 2.
Preferred consensus primers are denoted by the highlighted boxes.
Figure 10 shows an uprooted phylogenetic tree depicting the relationship of nucleotide sequences 371 nucleotides in length and corresponding to residues 26-396 of SEQ ID
N0:89. The scale representing nucleotide substitutions per position is shown.
The internal node numbers indicate the bootstrap values (expressed as a percentage of all trees) obtained from 1000 replicates. Isolates represented are Burmese, B1, B2; Chinese, C1, C2, C3, C4;
Pakistan, P1;
Indian, I1, I2; Mexican, M1; Italian, Itl; Greek, G1, G2; and United States, US-1, US-2.
Figure 11 shows an uprooted phylogenetic tree depicting the relationship of nucleotide sequences 148 nucleotides in length and corresponding to residues 6307-6454 of SEQ
ID N0:89. The scale representing nucleotide substitutions per position is shown. The internal node numbers indicate the bootstrap values (expressed as a percentage of all trees) obtained from 1000 replicates. Isolates represented are Burmese, B1, B2; Chinese, C1, C2, C3, C4; Pakistan, P 1; Indian, I 1. I2; Mexican, M 1; Italian, It 1; Greek, G 1, G2; Swine, S 1;
and United States, US-1 and US-2.
Figure 12 shows a schematic representation of preferred HEV-US recombinant protein constructs. In 12A, the ORF 2 and ORF 3 structural proteins of HEV are shown with the first and last amino acid positions designated. The presence of immunodominant epitopes are indicated by lines within the ORFs. Figure 12B shows an ORF 3 region that was cloned into an expression vector, with the first and last amino acid positions designated (SEQ ID N0:203 or SEQ ID N0:204). Figure 12C shows an ORF 2 region that was cloned into an expression vector, with the first and last amino acid positions designated (SEQ ID N0:199 or 200). Figure 12D
shows an ORF 3/2 chimeric construct cloned into an expression vector with the first and last amino acid positions of each component of the chimeric construct designated (SEQ ID N0:206 or 207). The sequence omitted from the ORF 3/2 construct is indicated with a dashed line. In Figures 12B-12D, the presence of a FLAG~ peptide at the carboxyl terminus of each construct is indicated by a solid box.
Figure 13 is a graph showing levels of alanine aminotransferase (square), IgG
(circle) and IgM (star) in a macaque before and after inoculation with sera harvested from patient USP-2.
Detailed Descri~ tion of the Invention As mentioned above, this invention is based, in part, upon the discovery of a new family of human hepatitis E viruses. The newly discovered family of hepatitis E
viruses fall within a class referred to hereinafter as a US-type hepatitis E virus. Furthermore, as mentioned above, two members of the US-type family were identified in sera obtained from two individuals living in the United States of America. These two members together belong to a subclass of the US-type hepatitis E virus, referred to hereinafter as a US-subtype hepatitis E
virus. The discovery of the US-type and US-subtype hepatitis E viruses enables the development of methods and compositions for detecting the presence of a US-type of US-subtype hepatitis E
virus in individuals who heretofore have not been diagnosed as suffering from hepatitis based on commercially available hepatitis detection kits, as well as methods and compositions for immunizing an individual against such a virus.
In one aspect, the invention pertains to a method of detecting the presence of a US-type or US-subtype hepatitis E virus in a test sample. The method comprises the steps of (a) contacting the sample with a binding partner that binds specifically to a marker for such a virus, which if present in the sample binds to the binding partner to produce a marker-binding protein complex, and (b) detecting the presence or absence of the complex. The presence of the complex is indicative of the presence of the virus in the sample. Based on the discovery of the US-type and US-subtype hepatitis E virus disclosed herein, it will be apparent that a variety of assays, for example, protein- or nucleic acid-based assays, may be produced for detecting the presence of the virus in a sample. Protein-based assays may include, for example, conventional immunoassays, and nucleic acid-based assays may include, for example, conventional probe hybridization or nucleic acid sequence amplification assays, all of which are well known and thoroughly discussed in the art.
In another aspect, the invention provides reagents, for example, antibodies, epitope containing polypeptide chains, and nucleotide sequences that may be used to develop vaccines for immunizing. either prophylactically or therapeutically, an individual against a US-type or US-subtype hepatitis E virus.
1. De~~nitions So that the invention may be more readily understood, certain terms as used herein are defined hereinbelow.
As used herein, the term "US-type" hepatitis E virus is understood to mean any human virus (i. e., capable of infecting a human) that is serologically distinct from hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis G virus (HGV) and comprising a single stranded RNA genome defining at least one open reading frame and having a nucleotide sequence greater than 79.7% identity to the nucleotide sequence defined by residues 6307-6454 of SEQ ID N0:89. It is understood, however, that a virus having a genome, a portion of which is identical to the entire nucleotide sequence identified under the Genbank Accession number AF011921, is excluded from the family of US-type hepatitis E
viruses.
As used herein. the term "US-subtype" hepatitis E is understood to mean any human virus (i.e., capable of infecting a human) that is serologically distinct from hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV) and hepatitis G virus (HGV) and comprising a single stranded RNA genome defining at least one open read frame and having a nucleotide sequence greater than 90.5% identity to the nucleotide sequence defined by residues 6307-b454 of SEQ ID N0:89.

As used herein, the term, "test sample" is understood to mean any sample. for example, a biological sample, which contains the marker (for example, an antibody, antigenic protein or peptide, or nucleotide sequence) to be tested. Preferred test samples include tissue or body fluid samples isolatable from an individual under investigation. Preferred body fluid samples include, for example, blood, serum, plasma, saliva, sputum, semen, urine, feces, bile, spinal fluid, breast exude, ascities, and peritoneal fluid. Another preferred test sample is a cell line and more preferably, a mammalian cell line. A most preferred cell line is a human fetal kidney cell line.
As used herein, the term "open reading frame" or "ORF" is understood to mean a region of a polynucleotide sequence capable of encoding one or more polypeptide chains. The region may represent an entire coding sequence, i.e., beginning with an initiation codon (e.g., ATG
(AUG)) and ending at a termination codon (e.g., TAA (UAA), TAG (UAG), or TGA
(UGA)), or a portion thereof.
As used herein, the term "polypeptide chain" is understood to mean any molecular chain of amino acids and does not refer to a specific length of the product. Thus, peptides, oligopeptides, and proteins are included within the definition of polypeptide chain.
As used herein, the term "epitope", as used synonymously with "antigenic determinant", is understood to mean at least a portion of an antigen capable of being specifically bound (i.e., bound with an affinity greater than about 105 M-', and more preferably with an affinity greater than about 10' M'') by an antibody variable region. Conceivably, an epitope may comprise three amino acids in a spatial conformation unique to the epitope. Generally, an epitope comprises at least five amino acids, and more usually, at least eight to ten amino acids.
Methods of examining spatial conformation are known in the art and include, for example, x-ray crystallography and two-dimensional nuclear magnetic resonance.
A polypeptide is "immunologically reactive" with an antibody when it binds to an antibody due to antibody recognition of a specific epitope defined by the polypeptide chain.
Immunological reactivity may be determined by antibody binding, more particularly by the kinetics of antibody binding, and/or by a competitive binding study. If a preselected antibody is immunologically reactive with a first antigen but is not immunologically reactive or is less immunologically reactive with a second, different antigen, then the two antigens are considered to be serologically distinct. As used herein, the team "affinity" is understood to mean a measure of reversible interaction between two molecules (for example, between an antibody and an antigen). The higher the affinity, the stronger the interaction between the two molecules.
As used herein, the term "detectable moiety" is understood to mean any signal generating compound, for example, chromogen, a catalyst such as an enzyme, a luminescent compound such as dioxetane, acridinium, phenanthridinium and luminol, a radioactive element, and a visually detectable label. Examples of enzymes include alkaline phosphatase, horseradish peroxidase, beta-galactosidase, and the like. Although the selection of a particular detectable moiety is not critical, the detectable moiety will be capable of producing a signal either by itself or in conjunction with one or more additional substances.
As used herein, the term "solid support" is understood to mean any plastic, derivatized plastic, magnetic or non-magnetic metal, glass or silicon surface. Useful surfaces include, for example, the surface of a test tube, microtiter well, sheet, bead, microparticle, chip, sheep (or other suitable animal's) red blood cell, or duracyte. Suitable solid supports are not critical to the practice of the invention and can be selected by one skilled in the art.
Suitable methods for immobilizing peptides on solid phases include ionic, hydrophobic, covalent interactions and the like. The solid support can be chosen for its intrinsic ability to attract and immobilize the capture reagent. Alternatively, the solid support can retain an additional receptor which has the ability to attract and immobilize the capture reagent.
It is contemplated that the solid support also may comprise any suitable porous material with sufficient porosity to allow access by detection antibodies and a suitable surface affinity to bind antigens. Microporous structures generally are preferred, but materials with gel structure in the hydrated state may be used as well. All of these materials may be used in suitable shapes, such as films, sheets, or plates, or they may be coated onto or bonded or laminated to appropriate inert carriers, such as paper, glass, plastic films, or fabrics.
Other embodiments which utilize various other solid supports also are contemplated and are within the scope of this invention. For example, ion capture procedures for immobilizing an immobilizable reaction complex with a negatively charged polymer, described in EP Publication No. 0 326 100 and EP Publication No. 0 406 473, can be employed according to the present invention to effect a fast solution-phase immunochemical reaction. An immobilizable immune complex is separated from the rest of the reaction mixture by ionic interactions between the negatively charged poly-anion/immune complex and the previously treated, positively charged porous matrix and detected by using various signal generating systems previously described, including those described in chemiluminescent signal measurements as described in EP
Publication No. 0 273 115.
Also, the methods of the present invention can be adapted for use in systems which utilize microparticle technology including automated and semi-automated systems wherein the solid phase comprises a microparticle (magnetic or non-magnetic). Such systems include those described in U.S. Patent Nos. 5,089,424 and 5244,630, issued February 18, 1992 and September 14, 1993, respectively.
The use of scanning probe microscopy (SPM) for immunoassays also is a technology to which the monoclonal antibodies of the present invention are easily adaptable.
In scanning probe microscopy, in particular in atomic force microscopy, the capture phase, for example, at least one of the monoclonal antibodies of the invention, is adhered to a solid phase and a scanning probe microscope is utilized to detect antigen/antibody complexes which may be present on the surface of the solid phase. The use of scanning tunneling microscopy eliminates the need for labels which normally must be utilized in many immunoassay systems to detect antigen/antibody complexes. The use of SPM to monitor specific binding reactions can occur in many ways. In one embodiment, one member of a specific binding partner (anaiyte specific substance which is the monoclonal antibody of the invention) is attached to a surface suitable for scanning. The attachment of the analyte specific substance may be by adsorption to a test piece which comprises a solid phase of a plastic or metal surface, following methods known to those of ordinary skill in the art. Or, covalent attachment of a specific binding partner (analyte specific substance) to a test piece which test piece comprises a solid phase of derivatized plastic, metal, silicon, or glass may be utilized. Covalent attachment methods are known to those skilled in the art and include a variety of means to irreversibly link specific binding partners to the test piece.
If the test piece is silicon or glass, the surface must be activated prior to attaching the specific binding partner. Also, polyelectrolyte interactions may be used to immobilize a specific binding partner on a surface of a test piece by using techniques and chemistries described in EP
Publication No. 0 322 100 and EP Publication No. 0 406 473. The preferred method of attachment is by covalent attachment. Following attachment of a specific binding member, the surface may be further treated with materials such as serum, proteins, or other blocking agents to minimize non-specific binding. The surface also may be scanned either at the site of manufacture or point of use to verify its suitability for assay purposes. The scanning process is not anticipated to alter the specific binding properties of the test piece.
As used herein, the terms "nucleotide sequence" or "nucleic acid sequence" is understood to mean any polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. The term refers to the primary structure of the molecule. Thus, the term includes double- and single-stranded DNA, as well as double- and single-stranded RNA. It also includes modifications, for example, by methylation and/or by capping, and unmodified forms of the polynucleotide.
As used herein, the term "primer" is understood to mean a specific oligonucleotide sequence complementary to a target nucleotide sequence which is capable of hybridizing to the target nucleotide sequence and serving as an initiation point for nucleotide polymerization catalyzed by DNA polymerase, RNA polymerase or reverse transcriptase.
When refernng to a nucleic acid fragment, such a fragment is considered to "specifically hybridize" or to "specifically bind" to an HEV US-type or US-subtype polynucleotide or variants thereof, if, within the linear range of detection, the hybridization results in a stronger signal relative to the signal that would result from hybridization to an equal amount of a polynucleotide from other than an HEV US-type, US- subtype or variant thereof. A signal which is "stronger"
than another is one which is measurable over the other by the particular method of detection.
Also, when referring to a nucleic acid fragment, such a fragment is considered to hybridize under specific hybridization conditions if it specifically hybridizes under (i) typical hybridization and wash conditions, such as those described, for example, in Maniatis, ( 1 st Edition, pages 387-389, 1982) where preferred hybridization conditions are those of lesser stringency and more preferred, higher stringency; or (ii) standard PCR
conditions (Saiki, R.K. et al. ) or ''touch-down" PCR conditions (Roux, K.H., ( 1994), Biotechiques, 16:812-814).
As used herein, the term "probe" is understood to mean any nucleotide or nucleotide analog (e.g., PNA) containing a sequence which can be used to identify specific DNA or RNA
present in samples bearing the complementary sequence.
As used herein, the term "PNA" is used to mean peptide nucleic acid analog which may be utilized in a procedure such as an assay described herein to determine the presence of a target.
"MA" denotes a "morpholino analog" which may be utilized in a procedure such as an assay described herein to determine the presence of a target. See, for example, U.S.
Patent No.
5,378,841, which is incorporated herein by reference. PNAs typically are neutrally charged moieties which can be directed against RNA targets or DNA. PNA probes used in assays in place of, for example, the DNA probes of the present invention, offer advantages not achievable when DNA probes are used. These advantages include manufacturability, large scale labeling, reproducibility. stability, insensitivity to changes in ionic strength and resistance to enzymatic degradation which is present in methods utilizing DNA or RNA. These PNAs can be labeled with such signal generating compounds as fluorescein, radionucleotides, chemiluminescent compounds, and the like. PNAs or other nucleic acid analogs such as MAs thus can be used in assay methods in place of DNA or RNA. Although assays are described herein utilizing DNA
probes, it is within the scope of the routine that PNAs or MAs can be substituted for RNA or DNA with appropriate changes if and as needed in assay reagents.

When referring to a nucleic acid fragment, such a fragment is considered to "specifically hybridize" or to "specifically bind" to an HEV US-type or US-subtype polynucleotide or variants thereof if, within the linear range of detection, the hybridization results in a stronger signal relative to the signal that would result from hybridization to an equal amount of a polynucleotide from other than an HEV US-type, US- subtype or variant thereof. A signal which is "stronger"
than another is one which is measurable over the other by the particular method of detection.
Also, when referring to a nucleic acid fragment, such a fragment is considered to hybridize under specific hybridization conditions if it specifically hybridizes under (i) typical hybridization and wash conditions, such as those described, for example, in Maniatis, ( 1 st Edition, pages 387-389, 1982) where preferred hybridization conditions are those of lesser stringency and more preferred, higher stringency; or (ii) standard PCR
conditions (Saiki, R.K. et al.) or ''touch-down" PCR conditions (Roux, K.H., (1994), Biotechiques, 16:812-814).
Il. Detection Methods and Reagents It is contemplated that the detection methods of the invention may employ a variety of protein-based or nucleic acid-based assays which are described in detail below.
It is contemplated that a reagent for the detection of virus or markers thereof may be either an anti-US-type and/or US-subtype hepatitis E virus antibody, a US-type and/or US-subtype specific polypeptide, or a nucleic acid defining at least a portion of the genome of a US-type and/or US-subtype hepatitis E virus or a nucleic acid sequence complementary thereto.
ll. ~(i) Protein-based Assays A. Marker Antibodies: It is contemplated that if the viral marker is an anti-US-type or anti-US-subtype specific antibody, for example, an IgG or an IgM, molecule circulating in the blood stream of an individual of interest, the binding partner preferably is a polypeptide defining an epitope that binds specifically to the marker.

In a preferred protocol for detecting the presence of anti-US-type or anti-US-subtype hepatitis E virus antibodies in a test sample, the protocol preferably comprises the following steps which include: (a) providing an antigen comprising an immunologically reactive US-type or US-subtype specific polypeptide chain comprising at least 5, more preferably at least 8, even more preferably at least 15, and most preferably at least 25 contiguous amino acid residues and bindable by the antibody; {b) incubating the antigen with the test sample under conditions that permit formation of an antibody-antigen complex; and (c) detecting the presence of the complex.
It is contemplated that many, different US-type or US-subtype specific polypeptides may be useful as a binding partner for the detection of anti-US-type or anti-US-subtype antibodies.
For example, it is contemplated that the polypeptide chain may be an amino acid sequence defined by SEQ ID NOS:91, 92 or 93 or an immunologically reactive fragment thereof containing, preferably at least 5, more preferably at least 8, even more preferably at least 15, and most preferably at least about 25 contiguous amino acid residues, of the polypeptide chain set forth in SEQ ID NOS:91, 92, or 93, and which represent a unique amino acid sequence when compared to the corresponding amino acid sequences of members of the Burmese and Mexican families. The Burmese family i. e., "Burmese-like" strains, as used herein, presently comprises strains referred to herein as B 1, B2, I1, I2, C 1, C2, C3, C4 and P 1 and the Mexican family presently comprises strain M 1.
It is contemplated that the binding partner may be a polypeptide selected from the group consisting of polypeptides defined by SEQ ID NOS:91, 92, and 93, including naturally occurring variants thereof. As used herein the term "naturally occurring variants thereof' with respect to the polypeptide defined by SEQ ID N0:91 is understood to mean any amino acid sequence that is at least 84%, preferably at least 86%, more preferably at least 89% and even more preferably at least 95% identical to residues I through 1698 of SEQ ID N0:91. As used herein the term "naturally occurring variants thereof' with respect to the polypeptide defined by SEQ ID N0:92 is understood to mean any amino acid sequence that is at least 93%, preferably at least 95%, and even more preferably at least 98% identical to residues 1 through 660 of SEQ
ID N0:92. As used herein the term ''naturally occurring variants thereoF' with respect to the polypeptide defined by SEQ ID N0:93 is understood to mean any amino acid sequence that is at least 85.4%, preferably at least 87.4%, more preferably at least 90.4% and even more preferably at least 95%
identical to residues 1 through 122 of SEQ ID N0:93.
Furthermore, it is contemplated that the binding partner may be a polypeptide encoded by a portion of an ORF 1 sequence. Proteins encoded by the ORF 1 sequence include, for example, a methyltransferase protein, a protease, a Y domain protein, an X domain protein, a helicase protein, a hypervariable region protein, and an RNA-dependent RNA polymerase protein.
Accordingly, it is contemplated that a useful methyltransferase protein preferably has at least 92.3%, more preferably has at least 94.3%, and most preferably has at least 97.3% identity to residues 1-231 of SEQ ID N0:91. Also, it is contemplated that a useful protease protein preferably has at least 70.3%, more preferably has at least 72.3%, and most preferably has at least 75.3% identity to residues 424-697 of SEQ ID N0:91. Also, it is contemplated that a useful Y domain protein preferably has at least 94.6%, more preferably has at least 96.6% and most preferably has at least 99.6% identity to residues 207-424 of SEQ ID N0:91.
Also it is contemplated that a useful X domain protein preferably has at least 83.4%, more preferably has at least 85.4% and most preferably has at least 88.4% identity to residues 789-947 of SEQ ID
N0:91. Also, it is contemplated that a useful helicase protein has at least 92%, more preferably has at least 94% and most preferably at least 93% identity to residues 965-1197 of SEQ ID
N0:91. Also, it is contemplated that a useful hypervariable region protein has at least 28.7%, more preferably has at least 30.7%, and most preferably has at least 33.7%
identity to the residues 698-788 of SEQ ID N0:91. Also, it is contemplated that a useful RNA-dependent RNA
polymerase has at least 88.8%, more preferably has at least 90.8%, and most preferably has at least about 93.8% identity to residues 1212-1698 of SEQ ID N0:91.
Furthermore. it is contemplated that the binding partner may be a polypeptide chain having an amino acid sequence defined by SEQ ID NOS:166, 167 or 168, or an immunologically reactive fragment thereof containing 5, preferably at least 8, more preferably at least 15 and most preferably at least 25 contiguous amino acid residues of the polypeptide chain set forth in SEQ
ID NOS:166, 167 or 168, and which represent a unique amino acid sequence when compared to the corresponding amino acid sequences of members of the Burmese and Mexican families.
Similarly, it is contemplated that the binding partner may be a polypeptide selected from the group consisting of SEQ ID NOS:166, 167 and 168, including naturally occurring variants thereof. As used herein, the term "naturally occurring variants thereof ' with respect to the polypeptide defined by SEQ ID N0:166 is understood to mean any amino acid sequence that is at least 83.9%, preferably at least 85.9%, more preferably at least 88.9%, and most preferably at least 95% identical to residues 1 through 1708 of SEQ ID N0:166. As used herein. the term "naturally occurring variants thereof ' with respect to the polypeptide defined by SEQ ID N0:167 is understood to mean any amino acid sequence that is at least 93%, preferably at least 95%, and most preferably at least 98% identical to residues 1 through 660 of SEQ ID
N0:167. As used herein, the term "naturally occurring variants thereof ' with respect to the polypeptide defined by SEQ ID N0:168 is understood to mean any amino acid sequence that is at least 85.4%, preferably at least 87.4%, more preferably at least 90.4%, and even more preferably at least 95%
identical to residues 1 through 122 of SEQ ID N0:168.
Furthermore, it is contemplated that the binding partner may be a polypeptide encoded by a portion of the HEV US-2 ORF 1, including, for example, a methyltransferase protein, a protease, a Y domain protein, an X domain protein, a helicase protein, a hypervariable region protein and an RNA-dependent RNA polymerase protein, or a variant thereof.
Accordingly, it is contemplated that a useful methyltransferase protein preferably has at least 92.7%, more preferably has at least 94.7%, and most preferably has at least 97.7% identity to residues 1-240 of SEQ ID N0:166. Also, it is contemplated that a useful protease protein preferably has at least 69.6%, more preferably has at least 71.6%, and most preferably has at least 74.6% identity to residues 433-706 of SEQ ID N0:166. Also, it is contemplated that a useful Y
domain protein preferably has at least 94.6%, more preferably has at least 96.6%, and most preferably has at least 99.6% identity to residues 216-433 of SEQ ID N0:166. Also it is contemplated that a useful X domain protein preferably has at least 82.8%, more preferably has at least 84.8%, and most preferably has at least 87.8% identity to residues 799-957 of SEQ ID
N0:166. Also, it is contemplated that a useful helicase protein has at least 92.8%, more preferably has at least 94.8%, and most preferably has at least 97.8% identity to residues 975-1207 of SEQ ID N0:166.
Also, it is contemplated that a useful hypervariable region protein has at least 27%, more preferably has at least 29%, and most preferably has at least 31 % identity to the residues 707-798 of SEQ ID N0:166. Also, it is contemplated that a useful RNA-dependent RNA
polymerase has at least 88.7%, more preferably has at least 90.7%, and most preferably has at least 93.7%
identity to residues 1222-1708 of SEQ ID N0:166.
With regard to the identification of US-type or US-subtype specific epitopes, it is contemplated that one skilled in the art in possession of nucleic acid sequences defining and/or amino acid sequences encoded by at least a portion of the genome of a US-type or US-subtype hepatitis E virus can map potential epitope sites using conventional technologies well known and thoroughly discussed in the art. In addition to the use of commercially available software packages which identify potential epitope sites in a given sequence, it is possible to identify potential epitopes by comparison of amino acid sequences encoded by such a genome with sequences encoded by the genomes of other strains of HEV whose antigenic sites have already been elucidated. See, for example, U.S. Patent Nos: 5,686,239, 5,741,490 and 5,770,689.
Epitopes currently identified are shown in Figure l, and include epitopes referred to in the art as 8-5 (SEQ ID NOS:93 AND 168), 4-2 (position 90-122 of SEQ ID NOS:93 and 168), SG3 (SEQ
ID NOS:175 AND 176), 3-2 (position 613-654 of SEQ ID NOS:92 and 167) and 3-2e (position 613-660 of SEQ ID NOS:92 and 167). A method for calculating antigenic index is described by Jameson and Wolf (CABIOS, 4(1), 181-186 [1988]).
For example, two epitopes of interest are discussed in detail below and are referred to as 3-2e and 4-2 which are encoded by portions of ORF 2 and ORF 3 of the hepatitis E genome, respectively. These epitopes were identified in the Burmese strains of HEV
(referred to below as B 3-2e (SEQ ID N0:172) and B 4-2 (SEQ IS N0:171 )), and in the Mexican strain of HEV
(referred to below as M 3-2e (SEQ ID N0:170) and M 4-2 (SEQ ID N0:169)).
Similar epitopes were identified in HEV US-1 based on amino acid sequence comparisons, and are referred to below as U3-2e (SEQ ID N0:174) and U4-2 (SEQ ID N0:173). Similar epitopes were identified in HEV US-2, also based on amino acid sequence comparisons, and are referred to below as US-2 3-2e (SEQ ID N0:223) and US-2 4-2 (SEQ ID N0:224).
In addition, potential epitopes may be identified using screening procedures well known and thoroughly documented in the art. For example, based on the nucleic acid sequences defining either the entire or portions of the HEV US-1 or the HEV US-2 genome, it is possible to generate an expression library, which, after expression can be screened to identify epitopes. For example, nucleic acid fragments representative of the HEV US-1 or the HEV US-2 genome can be cloned into the lambda-gtl 1 expression vector to produce a lambda-gtl 1 library, for example, a cDNA library. The library then is screened for encoded epitopes that can bind specifically with sera derived from individuals identified as being infected with HEV US-1 or HEV US-2. See, for example, Glover (1985) in "DNA Cloning Techniques, A Practical Approach", IRL Press, pp.
49-78. Typically, about 106 - 10' phage are screened, from which positive phage are identified, purified, and then tested for specificity of binding to sera from different individuals previously infected with HEV US-1 or HEV US-2. Phage which bind selectively to antibodies present in sera or plasma from the individual are selected for additional characterization. Once identified, an amino acid sequence of interest may be produced in large scale either by use of conventional recombinant DNA methodologies or by conventional peptide synthesis methodologies, well known and thoroughly documented in the art.
b. Marker PolYpeptides: It is contemplated that if the marker is a US-type or US-subtype virus or a specific polypeptide thereof, the binding partner useful in the practice of the invention preferably is an antibody, for example, a polyclonal or monoclonal antibody, that binds to an epitope on the virus or marker polypeptide. The binding partner may be either labeled with a detectable moiety or immobilized on a solid support. In particular, the antibodies useful in the practice of this embodiment preferably are capable of binding specifically to a US-type or US-subtype specific polypeptide chain preferably at least 5, more preferably at least 8, even more preferably at least 15, and most preferably at least 25 contiguous amino acid residues in length which is unique with respect to the corresponding amino acid sequence found in members of the Burmese and Mexican families.
An antibody useful in the practice of this embodiment of the invention preferably is capable of binding specifically to a polypeptide chain selected from the group consisting of SEQ
ID NOS:91, 92, and 93, including naturally occurring variants thereof, and has a higher binding affinity for such a polypeptide chain relative to the corresponding sequences of members of the Burmese and Mexican families. It is contemplated that an antibody useful in the practice of the invention preferably is capable of binding specifically to a polypeptide chain comprising the amino acid sequence set forth in SEQ ID N0:173 or 175. This antibody being further characterized as, under similar conditions, preferably having a lower affinity for, and most preferably failing to bind the amino acid sequence set forth in SEQ. ID
NOS:169 or 171 or regions in the Burmese and Mexican strains that correspond to SEQ ID N0:175.
Similarly, it is contemplated that an antibody useful in the practice of the invention preferably is capable of binding specifically to a polypeptide chain comprising the amino acid sequence set forth in SEQ
ID NOS:174 or 176. This antibody being further characterized as, under similar conditions, preferably having a lower affinity for, and most preferably failing to bind the amino acid sequence set forth in SEQ ID NOS:170 or 172 or regions in the Burmese and Mexican strains that con espond to SEQ ID N0:176.
Similarly, it is contemplated that an antibody useful in the practice of this embodiment of the invention preferably is capable of binding specifically to a polypeptide chain selected from the group consisting of SEQ ID NOS:166, 177, and 168, including naturally occurring variants thereof, and has a higher binding affinity for such a polypeptide chain relative to the corresponding sequences of members of the Burmese and Mexican families. It is contemplated that an antibody useful in the practice of the invention preferably is capable of binding specifically to a polypeptide chain comprising the amino acid sequence set forth in SEQ ID
N0:223. This antibody being further characterized as, under similar conditions, preferably having a lower affinity for, and most preferably failing to bind the amino acid sequences set forth in SEQ. ID NOS:170 or 172. Similarly. it is contemplated that an antibody useful in the practice of the invention preferably is capable of binding specifically to a polypeptide chain comprising the amino acid sequence set forth in SEQ ID N0:224. This antibody being further characterized as, under similar conditions, preferably having a lower affinity for, and most preferably failing to bind the amino acid sequence set forth in SEQ ID NOS:169 or 171.
The antibodies or antigen binding fragments thereof as described herein can be provided individually to detect US-type or US-subtype specific antigens. Combinations of the antibodies (and antigen binding fragments thereof) provided herein also may be used together as components in a mixture or "cocktail" of at least two antibodies, both having different binding specificities to separate US-type or US-subtype specific antigens.
c. Antibody Production: It is contemplated that one skilled in the art, in possession of the nucleic acid sequences defining, or amino acid sequences encoded by at least a portion of the ORF 1, ORF 2 and/or ORF 3 sequences of a US-type or a US-subtype hepatitis E
virus may be able to produce specific antibodies using techniques well known and thoroughly documented in the art. See, for example, Practical Immunology, Butt, N.R., ed., Marcel Dekker, NY, 1984.
Briefly, an isolated target protein is used to raise antibodies in a xenogenic host, such as a mouse, pig, goat or other suitable mammal. Preferred antibodies are antibodies that bind specifically to an epitope on the target protein, preferably having a binding affinity greater than l OSM-', and most preferably having a binding affinity greater than 10'M-' for that epitope. Typically, the target protein is combined with a suitable adjuvant capable of enhancing antibody production in the host, and injected into the host, for example, by intraperitoneal administration. Any adjuvant suitable for stimulating the host's immune response may be used to advantage.
A commonly used adjuvant is Freund's complete adjuvant (an emulsion comprising killed and dried microbial cells, e.g., from Calbiochem Corp., San Diego, CA or Gibco, Grand Island, NY).
Where multiple antigen injections are desired, the subsequent injections comprise the antigen in combination with an incomplete adjuvant (e.g., cell-free emulsion).

Polyclonal antibodies may be isolated from the antibody-producing host by extracting serum containing antibodies to the protein of interest. Monoclonal antibodies may be produced by isolating host cells that produce the desired antibody, fusing these cells with myeloma cells using standard procedures known in the immunology art (See for example, Kohler and Milstein, Nature ( 1975) 256:495), and screening for hybrid cells (hybridomas) that react specifically with the target protein and have the desired binding affinity.
In addition, it is contemplated that when small peptides are used their immunogenicity may be enhanced by coupling to solid supports. For example, an epitope or antigenic region or fragment of a polypeptide generally is relatively small, and rnay comprise about 8 to 10 amino acids or less in length. Fragments of as few as 3 amino acids may characterize an antigenic region. These polypeptides may be linked to a suitable carrier molecule when the polypeptide of interest provided folds to provide the correct epitope but yet is too small to be antigenic.
Preferred linking reagents and methodologies for their use are well known in the art and may include, without limitation, N-succinimidyl-3-(2-pyrdylthio)propionate (SPDP) and succinimidyl 4-(N-maleimidomethyl)cyclohexane-I-carboxylate (SMCC).
Furthermore, polypeptides lacking sulfhydryl groups can be modified by adding a cysteine residue. These reagents create a disulfide linkage between themselves and peptide cysteine residues on one protein and an amide linkage through the epsilonamino on a lysine, or other free amino group in the other. A variety of such disulfide/amide-forming agents are known. Other bifunctional coupling agents form a thioester rather than a disulfide linkage. Many of these thioether-forming agents are commercially available and are known to those of ordinary skill in the art. The carboxyl groups can be activated by combining them with succinimide or 1-hydroxyl-2-nitro-4-sulfonic acid, sodium salt. Any carrier which does not itself induce the production of antibodies harmful to the host can be used. Suitable carriers include proteins, polysaccharides such as latex functionalized sepharose, agarose, cellulose, cellulose beads, polymeric amino acids such as polyglutamic acid, polylysine, and no acid copolymers and inactive virus particles, among others. Examples of protein substrates include serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and yet other proteins known to those skilled in the art.
In addition, it is contemplated that biosynthetically produced antibody binding domains wherein the amino acid sequence of the binding domain is manipulated to enhance binding affinity to a preferred epitope also may be useful in the practice of the invention. A detailed description of their preparation can be found, for example, in Practical Immunology, Butt, W.R., ed., Marcel Dekker, New York, 1984. Optionally, a monovalent antibody fragment such as an Fab or an Fab' fragment may be utilized. Additionally, genetically engineered biosynthetic antibody binding sites may be utilized which comprise either 1 ) non-covalently associated or disulfide bonded synthetic VH and VL dimers, 2) covalently linked VH-VL single chain binding sites; 3) individual VH or V~ domains, or 4) single chain antibody binding sites, as disclosed, for example, in U.S. Patent Nos. 5,091,513 and 5,132,405.
It is contemplated that intact antibodies (for example, monoclonal or polyclonal antibodies), antibody fragments or biosynthetic antibody binding sites that bind a US-type or US-subtype hepatitis E virus specific epitope, will be useful in diagnostic and prognostic applications, and also, will be useful in passive immunotherapy.
d. Assay Formats: It is contemplated that both polypeptides which react immunologically with serum containing anti-US-type or anti-US-subtype hepatitis E virus specific antibodies, or antibodies raised against US-type or US-subtype hepatitis E specific epitopes will be useful in immunoassays to detect the presence of such a virus in a test sample of interest. Furthermore, it is contemplated that the presence of US-type or US-subtype hepatitis E
virus in a sample may be detected using any of a wide range of immunoassay techniques, for example, direct assays, sandwich assays, and/or competition assays, currently known and thoroughly documented in the art. A variety of preferred assay formats are described in more detail below.
In one preferred format, the assay employs a sandwich format. Sandwich immunoassays typically are highly specific and very sensitive, provided that labels with good limits of detection WO 99/19732 PCTlUS98121941 are used. A detailed review of immunological assay design, theory and protocols can be found in numerous texts in the art, including Practical Immunology, Butt, W.R., ed., MarcelI Dekker, New York, 1984.
In one type of sandwich format, a polypeptide {binding partner) which has been immobilized onto a solid support and is immunologically reactive with an anti-US-type or anti-US-subtype hepatitis E virus antibody (marker), is contacted with a test sample from an individual suspected of having been infected with the US-type or US-subtype hepatitis E virus, to form a mixture. The mixture then is incubated for a time and under conditions sufficient to form polypeptide/antibody complexes. Then, an indicator reagent comprising a monoclonal or a polyclonal antibody or a fragment thereof, which specifically binds to the test sample antibody, and labeled with a detectable moiety, is contacted with the antigen/antibody complexes to form a second mixture. The second mixture then is incubated for a time and under conditions sufficient to form antigen/antibody/antibody complexes. The presence of anti-US-type or anti-US-subtype hepatitis E antibody, if any, in the test sample is determined by detecting the presence of detectable moiety immobilized to the solid support. The amount of antibody present in the test sample is proportional to the signal generated. The use of biotin and antibiotin, biotin and avidin, biotin and streptavidin, and the like, may be used to enhance the generated signal in the assay systems described herein.
In an alternative format of the above-described assay, the immunologically reactive polypeptide may be immobilized "indirectly" to the solid support, i.e. through a monoclonal or polyclonal antibody or fragment thereof which specifically binds that polypeptide. Alternatively, in another format, the assay components may be used in the reverse configuration, such that an antibody or antigen binding fragment thereof, which specifically binds the test sample antibody, i.e., marker antibody (for example, IgG or IgM) and immobilized on the solid support is contacted with the test sample, for a time and under conditions sufficient to permit formation of antibody/antibody complexes. Then, an indicator reagent, for example, a US-type or US-subtype hepatitis E polypeptide immunologically reactive with captured test sample antibody and labeled with a detectable moiety, is incubated with the antibody/antibody complexes to form a second mixture for a time and under conditions sufficient to permit formation of antibody/antibody/antigen complexes. As above, the presence of antibody in the test sample, if any, that is captured by the capture antibody or antigen binding fragment thereof immobilized on the solid support is determined by detecting the measurable signal generated by the detectable moiety.
It is contemplated that the aforementioned sandwich assays also may be used to test for the presence of a US-type or US-subtype hepatitis E virus, or immunologically reactive polypeptides thereof in a test sample by routine modification of the above-described assay configurations. It is contemplated that such modifications would be well known to one skilled in the art.
In addition to the aforementioned sandwich assays, it is contemplated that competitive assays may also be employed in the practice of the invention. In this format, one or a combination of at least two antibodies, preferably monoclonal antibodies, which specifically bind to a US-type or US-subtype hepatitis E specific polypeptide chain can be employed as a competitive probe for the detection of antibodies to the US-type or the US-subtype specific protein. For example, a first HEV US-1 specific polypeptide chain such as one of the polypeptides disclosed herein, acting as a binding partner for the marker, is immobilized on a solid support. A test sample suspected of containing antibody to HEV US-1 antigen then is incubated with the solid support together with an indicator reagent comprising, for example, an isolated anti-US-type or anti-US-subtype antibody that binds the immobilized specific polypeptide chain and labeled with a detectable moiety, for a time and under conditions sufficient to form antigen/antibody complexes immobilized to the solid support. If the marker antibody is present in the test sample, then the marker antibody competes with the labeled indicator reagent for binding the immobilized polypeptide. As the amount of marker antibody present in the test sample increases, the amount of labeled indicator reagent that binds the immobilized polypeptide decreases. A reduction in the amount of indicator reagent bound to the solid phase can be quantitated. A measurable reduction in signal compared to the signal generated from a confirmed negative non-A, non-B, non-C, non-D, non-E
hepatitis test sample also is indicative of the presence of anti-HEV US-1 antibody in the test sample. It is contemplated that similar protocols may be used to identify the presence in a test sample of other hepatitis E viruses falling within the US-type or US-subtype classes.
In yet another detection method. the antibodies of the present invention may be employed to detect the presence of US-type or US-subtype hepatitis E specific antigens in fixed tissue sections. as well as fixed cells by immunohistochemical analysis. Cytochemical analysis wherein these antibodies are labeled directly with a detectable moiety (e.g., fluorescein, colloidal gold, horseradish peroxidase, alkaline phosphatase, etc.) or are labeled indirectly, for example, by means of a secondary antibody labeled with a detectable moiety also may be used in the practice of the invention.
In another assay format, the presence of antibody and/or antigen can be detected by means of a simultaneous assay, for example, as described in EP Publication No.
0 473 065. For example, a test sample is contacted simultaneously with (i) a capture reagent of a first analyte, wherein the capture reagent comprises a first binding member specific for a first analyte immobilized on a solid support and (ii) a capture reagent for a second analyte, wherein the capture reagent comprises a first binding member for a second analyte immobilized on a second different solid support, to produce a mixture. The mixture then is incubated for a time and under conditions sufficient to form capture reagent/first analyte and capture reagent/second analyte complexes. The complexes so-formed then are contacted with a first indicator reagent comprising a member of a binding pair specific for the first analyte labeled with a detectable moiety and a second indicator reagent comprising a member of a binding pair specific for the second analyte labeled with a detectable moiety, to produce a second mixture.
The second mixture then is incubated for a time and under conditions sufficient to produce both capture reagent/first analyte/first indicator reagent and capture reagent/second analyte/second indicator reagent complexes. The presence of one or more analytes is determined by detecting a signal generated by the complexes formed on either or both solid phases as an indication of the presence of one or more analytes in the test sample.
Other assay systems may employ an antibody which specifically binds US-type or US-subtype hepatitis E viral particles or sub-viral particles encapsulating the viral genome (or fragments thereof] by virtue of a contact between the specific antibody and the viral protein (peptide, etc.). The captured particles then can be analyzed by methods such as LCR or PCR to determine whether the viral genome is present in the test sample. The advantage of utilizing such an antigen capture amplification method is that it can separate the viral genome from other molecules in the test specimen by use of a specific antibody. Such a method has been described in EP 0 672 176, published September 20, 1995.
In general, immunoassay design considerations include preparation of antibodies {e.g., monoclonal or polyclonal antibodies or antigen binding fragments thereof) having sufficiently high binding specificity for the target protein to form a complex that can be distinguished reliably from products of nonspecific interactions. Typically, the higher the antibody binding specificity, the lower the concentration of target that can be detected.
Both the polypeptide and antibody reagents of the invention may be used to develop assays as described herein to detect either the presence of an antigen from or an antibody that binds to a US-type or US-subtype hepatitis E virus. In addition to their use in immunoassays, it is contemplated that the aforementioned polypeptides may be used either alone or in combination with adjuvants for use in the production of antibodies in laboratory animals, or similarly, used in combination with pharmaceutically acceptable Garners as vaccines for either the prophylactic or therapeutic immunization of individuals. Also, it is contemplated that, in addition to their use in immunoassays, the antibodies of the invention may be used in combination with, for example, a pharmaceutically acceptable Garner for use in passive, therapeutic or prophylactic immunization of an individual. These latter uses are described in more detail in section (III) below. The antibodies of the invention can also be used for the generation of chimeric antibodies for therapeutic use, or other similar applications.

Kits suitable for immunodiagnosis and containing the appropriate reagents may be constructed by packaging the appropriate materials, including, for example, a polypeptide defining a specific epitope of interest or antibodies that bind such epitopes in suitable containers.
In addition, the kit optionally may include additional reagents, for example, suitable detection systems and buffers.
In addition, these antibodies, preferably monoclonal, can be bound to matrices similar to CNBr-activated Sepharose and used for the affinity purification of US-type or US-subtype hepatitis E specific proteins from cell cultures, or biological tissues such as blood and liver such as to purify recombinant and native viral antigens and proteins.
II. (iil Nucleic Acid-based Assays It is contemplated that if the marker is a US-type or US-subtype specific nucleotide sequence, the binding partner preferably also is a nucleotide sequence or an analog thereof that hybridizes specifically to the marker sequence or to regions adjacent thereto.
Based on the unique polynucleotide sequences disclosed herein, it is contemplated that a binding partner may be a nucleotide sequence complementary to a US-type or US-subtype specific nucleotide sequence, for example, a nucleotide sequence or analog thereof complementary to at least a portion of an ORF I sequence, an ORF 2 sequence, or an ORF 3 sequence of a US-type or US-subtype hepatitis E virus, which is unique when compared to the corresponding nucleotide sequences of the Burmese and Mexican families. Furthermore, it is contemplated that noncoding portions of the genome of US-type and US-subtype hepatitis E viruses which are unique relative to the genomes of the Burmese and Mexican families of hepatitis E also may provide useful markers in the practice of the invention. Such nucleotide sequences (either primers or probes) are of a length which allow detection of US-type or US-subtype specific sequences by hybridization and/or amplification and may be prepared using routine, standard methods, including automated oligonucleotide synthesis methodologies, well known and thoroughly discussed in the art. A complement of any unique portion of the HEV US-1 genome will be satisfactory. Complete complementarity is desirable for use as probes, although it may be unnecessary as the length of the fragment is increased.
Similarly, it is contemplated that the binding partner may be a polynucleotide sequence, for example, a DNA, RNA or PNA sequence, preferably comprising 8-100 nucleotides more preferably comprising 10-75 nucleotides and most preferably comprising 15-50 nucleotides, which is capable of hybridizing specifically to the target sequence. It is understood that the target sequence may be a nucleotide sequence defining at least a portion of a genome of a US-type or US-subtype hepatitis E virus. or a sequence complementary thereto. It is known in the art that the particular stringency conditions selected for a hybridization reaction depend largely upon the degree of complementarity of the binding partner nucleic acid sequence with the target sequence, the composition of the binding sequence and the length of the binding sequence. The parameters for determining stringency conditions are well known to those of ordinary skill in the art or are deemed to be readily ascertained from standard textbooks (see for example, Maniatis et al., Molecular Cloning: A Laboratory Manual, (Cold Spring Harbor Press. N.Y., 1989)).
The sequences provided herein may be used to produce probes which can be used in assays for the detection of nucleic acids in test samples. The probes may be designed from conserved nucleotide regions of the polynucleotides of interest or from non-conserved nucleotide regions of the polynucleotide of interest. The design of such probes for optimization in assays is within the skill of the routineer. Generally, nucleic acid probes are developed from non-conserved or unique regions when maximum specificity is desired, and nucleic acid probes are developed from conserved regions when assaying for nucleotide regions that are closely related to, for example. different members of a multigene family or in related species like mouse and man.
One preferred protocol provides a method of detecting the presence or absence of a US-type or US-subtype hepatitis E virus in a test sample. The method comprises the steps of (a) providing a probe comprising a polynucleotide sequence containing at least 15 contiguous nucleotides from a US-type or US-subtype isolate, wherein the sequence is not present in other members of the hepatitis E Burmese and Mexican families; (b) contacting the test sample and the probe under conditions that permit formation of a polynucleotide duplex bet<veen the probe and its complement, in the absence of substantial polynucleotide duplex formation between the probe and non US-type and non US-subtype hepatitis polynucleotide sequences present in the test sample; and (c) detecting the presence of any polynucleotide duplexes containing the probe.
Preferred nucleotide sequences may comprise nucleotide residue numbers 1 through 5097 of SEQ ID N0:89, or a naturally occurring sequence variant thereof. With regard to this sequence, the term "a naturally occurring sequence variant" includes any nucleic acid sequence that is at least 73.3%, preferably at least 75.3%, more preferably at least 78.3%, and most preferably at least 95% identical to residues I through 5097 of SEQ ID N0:89.
Other preferred marker or binding partner sequences may comprise nucleotide residue numbers 5132 through 7114 of SEQ ID N0:89, or a naturally occurring sequence variant thereof. With regard to this sequence, the term "naturally occurring sequence variant" includes any nucleic acid sequence that is at least 87.4%, preferably at least 89.4%, more preferably at least 92.4%, and most preferably at least 95% identical to residues S 132 through 7114 of SEQ ID
N0:89. Other preferred marker or binding partner sequences may comprise nucleotide residue numbers 5094 through 5462 of SEQ ID N0:89, or a naturally occurring sequence variant thereof. With regard to this sequence, the term "naturally occurring sequence variant" includes any nucleic acid sequence that is at least 88.3% identical, preferably at least 90.3%
identical, more preferably at least 93.3% identical, and most preferably at least 95% identical to residues 5094 through 5462 of SEQ ID N0:89.
Furthermore, it is contemplated that useful nucleotide sequences may include, for example, portions of the ORF 1 sequence encoding, for example, a protein selected from the group consisting of the methyltransferase protein, the protease protein, the Y
domain protein, the X domain protein, the helicase protein, the hypervariable region protein and the RNA-dependent RNA polymerase protein, or a variant thereof. Accordingly, it is contemplated that a useful methyltransferase encoding region of ORF 1 preferably has at least 78%, more preferably has at least 80%, and most preferably has at least 83% identity to residues I-693 of SEQ ID N0:89.
Also, it is contemplated that a useful protease encoding region of ORF 1 preferably has at least 66. I %, more preferably has at least 68.1 %, and most preferably has at least 71.1 % identity to residues 1270-2091 of SEQ ID N0:89. Also, it is contemplated that a useful Y
domain encoding region of ORF 1 has at least 80%, more preferably has at least 82%, and most preferably has at least 85% identity to residues 619-1272 of SEQ ID N0:89. Also, it is contemplated that a useful X domain encoding region of ORF 1 has at least 73.5%, more preferably has at least 75.5%, and most preferably has at least 78.5% identity to residues 2365-2841 of SEQ ID
N0:89. Also, it is contemplated that a useful helicase encoding region of ORF I has at least 77.5%, and most preferably has at least 79.5%, and most preferably has at least 81.5% identity to residues 2893-3591 of SEQ ID N0:89. Also, it is contemplated that a useful hypervariable region encoding region of ORF 1 has at least 51.2%, more preferably has at least 53.2%, and most preferably has at least 56.2% identity to residues 2092-2364 of SEQ ID N0:89. Also, it is contemplated that a useful RNA-dependent RNA polymerase encoding region of ORF I has at least 76.3%, more preferably has at least 78.3 %, and most preferably has at least 81.3 %
identity to residues 3634-5094 of SEQ ID N0:89.
Preferred nucleotide sequences may comprise nucleotide residue numbers 36 through 5162 of SEQ ID N0:164, or a naturally occurring sequence variant thereof. With regard to this sequence, the term "a naturally occurring sequence variant" includes any nucleic acid sequence that is at least 73.6%, preferably at least 75.6%, more preferably at least 78.6% and more preferably at least 95% identical to residues 36 through 5162 of SEQ ID
N0:164. Other preferred marker or binding partner sequences may comprise nucleotide residue numbers 5197 through 7179 of SEQ ID NO:I64, or a naturally occurring sequence variant thereof. With regard to this sequence, the term "naturally occurring sequence variant" includes any nucleic acid sequence that is at least 80.7%, preferably at least 82.7%, more preferably at least 85.7% and most preferably at least95% identical to residues 5197 through 7179 of SEQ ID
N0:164. Other preferred marker or binding partner sequences may comprise nucleotide residue numbers 5159 through 5527 of SEQ ID N0:164, or a naturally occurring sequence variant thereof. With regard to this sequence, the term "naturally occurring sequence variant" includes any nucleic acid sequence that is at least 87.9% identical, preferably at least 89.9%
identical, more preferably at least 92.9% identical and even more preferably at least 9S% identical to residues S 159 through SS27 of SEQ ID N0:164.
Furthermore, it is contemplated that useful HEV US-2 nucleotide sequences may include, for example, portions of the ORF 1 sequence encoding, for example, at least a portion of a protein selected from the group consisting of the methyltransferase protein, the protease protein, the Y domain protein, the X domain protein, the helicase protein, the hypervariable region protein and the RNA-dependent RNA polymerase protein. or a variant thereof.
Accordingly, it is contemplated that a useful methyltransferase encoding region of ORF 1 preferably has at least 79.5%, more preferably has at least 81.5%, and most preferably has at least 84.5% identity to residues 36-7SS of SEQ ID N0:164. Also, it is contemplated that a useful protease encoding region of ORF 1 preferably has at least 66.1 %, more preferably has at least 68.1 %, and most preferably has at least 71.1 % identity to residues 1332-21 S3 of SEQ ID
N0:164. Also, it is contemplated that a useful Y domain encoding region of ORF 1 has at least 80.7%, more preferably has at least 82.7%, and most preferably has at least 85.7% identity to residues 680-1334 of SEQ ID N0:164. Also, it is contemplated that a useful X domain encoding region of ORF 1 has at least 73.7%, more preferably has at least 75.7%, and most preferably has at least 78.7% identity to residues 2430-2906 of SEQ ID N0:164. Also, it is contemplated that a useful helicase encoding region of ORF 1 has at least 76.4%, and most preferably has at least 78.4%, and most preferably has at least 81.4% identity to residues 2958-3656 of SEQ
ID N0:164. Also, it is contemplated that a useful hypervariable region encoding region of ORF 1 has at least 50.4%, more preferably has at least 52.8%, and most preferably has at least SS.8% identity to residues 21 S4-2429 of SEQ ID N0:164. Also, it is contemplated that a useful RNA-dependent RNA polymerase encoding region of ORF 1 has at least 76.8%, more preferably has at least 78.8%, and most preferably has at least 81.8% identity to residues 3699-S 1 S9 of SEQ ID
N0:164.

Other useful nucleotide sequences comprise the nucleotide sequences that encode the amino acid sequences selected from the group consisting of SEQ ID NOS:93, 168, 173, 174, 175, 176, 223, and 224 and nucleotide sequences complementary thereto.
It is contemplated that the nucleic acid sequences provided herein may be used to determine the presence of US-type or US-subtype hepatitis E virus in a lest sample by conventional nucleic acid based assays, for example, by polymerase chain reaction (PCR) and/or by blot hybridization studies (described in detail below). In addition to their use in nucleic acid based assays, it is contemplated the aforementioned nucleic acid sequences may be integrated in vectors which may then be transformed or transfected into a host cell of interest, for example, vaccinia or mycobacteria. The resulting host cells may then be combined with a pharmaceutically acceptable carrier and used, for example, as a recombinant vaccine for immunizing a mammal, either prophylactically or therapeutically, against a preselected US-type or US-subtype hepatitis E virus.
The polymerase chain reaction (PCR) is a technique for amplifying a desired nucleic acid sequence (target) contained in a nucleic acid or mixture thereof. In PCR, a pair of primers typically are employed in excess to hybridize at the outside ends of complementary strands of the target nucleic acid. The primers are each extended by a polymerase, for example, a thermostable polymerase, using the target nucleic acid as a template. The extension products become target sequences themselves, following dissociation from the original target strand.
New primers then are hybridized and extended by a polymerase, and the cycle is repeated to geometrically increase the number of target sequence molecules. PCR is disclosed in U.S. patents 4,683,195 and 4,683,202.
The Ligase Chain Reaction (LCR) is an alternate method for nucleic acid amplification.
In LCR, probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess of the target nucleic acid sequence. The first probe hybridizes to a first segment of the target strand and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphate-3'hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion. Once the ligated strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary ligated product. The ligated products are functionally equivalent to either the target or its complement.
By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved.
This technique is described more completely in EP-A- 320 308 to K. Backman published June 16, 1989 and EP-A-439 182 to K. Backman et al, published July 31, 1991.
For amplification of mRNAs, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Patent No. 5,322,770; or to reverse transcribe mRNA into cDNA followed by asymmetric gap ligase chain reaction (RT-AGLCR) as described by R. L. Marshall, et al. , PCR Methods and Applications 4: 80-84 ( 1994).
Other known amplification methods which can be utilized herein include but are not limited to the so-called "NASBA" or "3SR" technique described in Proc. Natl.
Acad. Sci. USA
87: 1874-1878 ( 1990) and also described in Nature 350- (No. 6313): 91-92 ( 1991 ); Q-beta amplification as described in published EP 4544610; strand displacement amplification (as described in G. T. Walker et al., Clin. Chem. 42: 9-13 (1996]) and EP 684315;
and target mediated amplification, as described by PCT Publication WO 9322461.
In one embodiment, the present invention generally comprises the steps of contacting a test sample suspected of containing a target polynucleotide sequence with amplification reaction reagents comprising an amplification primer, and a detection probe that can hybridize with an internal region of the amplicon sequences. Probes and primers employed according to the method herein provided are labeled with capture and detection labels wherein probes are labeled with one type of label and primers are labeled with the other type of label.
Additionally, the primers and probes are selected such that the probe sequence has a lower melt temperature than the primer sequences. The amplification reagents, detection reagents and test sample are placed under amplification conditions whereby, in the presence of target sequence, copies of the target sequence (an amplicon) are produced. The double stranded amplicon then is thermally denatured to produce single stranded amplicon members. Upon formation of the single stranded amplicon members, the mixture is cooled to allow the formation of complexes between the probes and single stranded amplicon members.
After the probe/single stranded amplicon member hybrids are formed, they are detected.
Standard heterogeneous assay formats are suitable for detecting the hybrids using the detection labels and capture labels present on the primers and probes. The hybrids can be bound to a solid phase reagent by virtue of the capture label and detected by virtue of the detection label. In cases where the detection label is directly detectable, the presence of the hybrids on the solid phase can be detected by causing the label to produce a detectable signal, if necessary, and detecting the signal. In cases where the label is not directly detectable, the captured hybrids can be contacted with a conjugate, which generally comprises a binding member attached to a directly detectable label. The conjugate becomes bound to the complexes and the conjugates presence on the complexes can be detected with the directly detectable label. Thus, the presence of the hybrids on the solid phase reagent can be determined. Those skilled in the art will recognize that wash steps may be employed to wash away unhybridized amplicon or probe as well as unbound conjugate.
Test samples for detecting target sequences can be prepared using methodologies well known in the art such as by obtaining a sample and, if necessary, disrupting any cells contained therein to release target nucleic acids. In the case where PCR is employed in this method, the ends of the target sequences are usually known. In cases where LCR or a modification thereof is employed in the preferred method, the entire target sequence is usually known.
Typically, the target sequence is a nucleic acid sequence such as, for example, RNA or DNA.

While the length of the primers and probes can vary, the probe sequences are selected such that they have a lower melt temperature than the primer sequences. Hence, the primer sequences are generally longer than the probe sequences. Typically, the primer sequences are in the range of between 20 and 50 nucleotides long, more typically in the range of between 20 and 30 nucleotides long. Preferred primer sequences typically are greater than 20 nucleotides long.
The typical probe is in the range of between 10 and 25 nucleotides long more typically in the range of between 15 and 20 nucleotides long. Preferred probe sequences typically are greater than 15 nucleotides long.
Alternatively, a probe may be involved in the amplifying a target sequence, via a process known as "nested PCR". In nested PCR, the probe has characteristics which are similar to those of the first and second primers normally used for amplification (such as length, melting temperature etc.) and as such, may itself serve as a primer in an amplification reaction.
Generally in nested PCR, a first pair of primers (P 1 and P2) are employed to fonm primary extension products. One of the primary primers (for example, P 1 ) may optionally be a capture primer (i.e. linked to a member of a first reactive pair), whereas the other primary primer (P2) is not. A secondary extension product is then formed using a probe (P1') and a probe (P2') which may also have a capture type label (such as a member of a second reactive pair) or a detection label at its 5' end. The probes are complementary to and hybridize at a site on the template near or adjacent the site where the 3' termini of P1 and P2 would hybridize if still in solution.
Alternatively, a secondary extension product can be formed using the P1 primer with the probe (P2') or the P2 primer with the probe (P 1 ~) sometimes referred to as "hemi-nested PCR". Thus, a labeled primer/probe set generates a secondary product which is shorter than the primary extension product. Furthermore, the secondary product may be detected either on the basis of its size or via its labeled ends (by detection methodologies well known to those of ordinary skill in the art). In this process, probe and primers are generally employed in equivalent concentrations.
Various methods for synthesizing primers and probes are well known in the art.
Similarly, methods for attaching labels to primers or probes are also well known in the art. For example, it is a matter of routine experimentation to synthesize desired nucleic acid primers or probes using conventional nucleotide phosphoramidite chemistry and instruments available from Applied Biosystems, Inc., (Foster City, CA), Dupont (Wilmington, DE), or Milligen (Bedford MA). Many methods have been described for labeling oligonucleotides such as the primers or probes of the present invention. Enzo Biochemical (New York, NY) and Clontech (Palo Alto, CA) both have described and commercialized probe labeling techniques. For example, a primary amine can be attached to a 3' oligo terminus using 3'-Amine-ON CPGTM
(Clontech, Palo Alto, CA). Similarly. a primary amine can be attached to a 5' oligo terminus using Aminomodifier II~ (Clontech). The amines can be reacted to various haptens using conventional activation and linking chemistries. In addition, WO 92/10506, published 25 June 1992 and U.
S. Patent 5,290,925, issued March l, 1994, teach methods for labeling probes at their 5' and 3' termini, respectively. In addition, WO 92/11388 published 9 July 1992 teaches methods for labeling probes at their ends. According to one known method for labeling an oligonucleotide, a label-phosphoramidite reagent is prepared and used to add the label to the oligonucleotide during its synthesis. See. for example, N.T. Thuong et al., Tet. Letters 246): 5905-5908 (1988); or J. S.
Cohen et al., published U.S. Patent Application 07/246,688 (NTIS ORDER No. PAT-APPL-7-246,688) (1989). Preferably, probes are labeled at their 3' and 5' ends.
Capture labels are carried by the primers or probes and can be a specific binding member which forms a binding pair with the solid phase reagent's specific binding member. It will be understood, of course that the primer or probe itself may serve as the capture label. For example, in the case where a solid phase reagent's binding member is a nucleic acid sequence, it may be selected such that it binds a complementary portion of the primer or probe to thereby immobilize the primer or probe to the solid phase. In cases where the probe itself serves as the binding member, those skilled in the art will recognize that the probe will contain a sequence or "tail"
that is not complementary to the single stranded amplicon members. In the case where the primer itself serves as the capture label, at least a portion of the primer will be free to hybridize with a nucleic acid on a solid phase because the probe is selected such that it is not fully complementary to the primer sequence.

Generally, probe/single stranded amplicon member complexes can be detected using techniques commonly employed to perform heterogeneous immunoassays.
Preferably, in this embodiment, detection is performed according to the protocols used by the commercially available Abbott LCx~ instrumentation (Abbott Laboratories, Abbott Park, IL).
Other useful procedures known in the art include solution hybridization, and dot and slot blot hybridization protocols. The amount of the target nucleic acid present in a sample optionally may be quantitated by measuring the radioactivity of hybridized fragments, using standard procedures known in the art.
III. Vaccines It is contemplated that vaccines may be prepared from one or more immunogenic polypeptides based on US-type and/or US-subtype specific protein sequences or antibodies that bind to such protein sequences. In addition, it is contemplated that vaccines also may comprise dead, live but attenuated US-type or US-subtype hepatitis E virus, or a live, recombinant vaccine comprising a heterologous host cell, for example, a vaccinia virus, expressing a US-type or US-subtype hepatitis E virus specific antigen.
With regard to the polypeptide based vaccines, the polypeptide must define at least one epitope. It is contemplated, however, that the vaccine may comprise a plurality of different epitopes which are defined by one or more polypeptide chains. Furthermore, it is contemplated that nonstructural proteins as well as structural proteins may provide protection against viral pathogenicity, even if they do not cause the production of neutralizing antibodies. Considering the above, multivalent vaccines against the US-type or US-subtype virus may comprise one or more structural proteins, and/or one or more nonstructural proteins. These immunogenic epitopes can be used in combinations, i.e., as a mixture of recombinant proteins, synthetic peptides and/or polypeptides isolated from the virion; which may be co-administered at the same or administered at different time.

Methodologies for the preparation of protein or peptide based vaccines which contain at least one immunogenic peptide as an active ingredient are well known in the art. Typically, such vaccines are prepared as injectables, either as liquid solutions or suspensions. The preparation may be emulsified or the protein may be encapsulated in liposomes. The active immunogenic ingredients may be mixed with pharmacologically acceptable excipients which are compatible with the active ingredient. Suitable excipients include, without limitation, water, saline, dextrose, glycerol, ethanol or a combination thereof. The vaccine also may contain small amounts of auxiliary substances such as wetting or emulsifying reagents, pH
buffering agents, andlor adjuvants which enhance the effectiveness of the vaccine. For example, such adjuvants can include aluminum hydroxide, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-DMP), N-acetyl-nomuramyl-L-alanyl-D-isoglutamine (CGP 11687, also referred to as nor-MDP), N-acetyl-muramyul-L-alanyl-D-isoglutaminyl-L-alanine-2-(1'2'-dipalmitoyl sn-glycero-3-hydroxphosphoryloxy)-ethylamine (CGP 19835A, also referred to as MTP-PE), and RIBI
(MPL + TDM + CWS) in a 2% squalene/Tween-80~ emulsion. The effectiveness of an adjuvant may be determined by measuring the amount of antibodies directed against an immunogenic polypeptide containing a US-type or US-subtype specific antigenic sequence resulting from administration of this polypeptide in vaccines which also comprise various adjuvants under investigation.
The vaccines usually are administered by intravenous or intramuscular injection.
Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and Garners may include but are not limited to polyalkylene glycols or triglycerides. Such suppositories may be formed from-mixtures containing the active ingredient in the range of from about 0.5% to about 10%, preferably, from about 1 % to about 2% (w/w). Oral formulation may include excipients including, for example, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate and the like. These compositions may take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain about 10% to about 95% of active ingredient, preferably about 25% to about 70% (w/w).

The polypeptide chains used in the vaccine may be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include, for example, acid addition salts formed by the addition of inorganic acids such as hydrochloric or phosphoric acids, or such organic acids such as acetic, oxalic, tartaric, malefic, or other acids known to those skilled in the art. Salts formed with the free carboxyl groups also may be derived from inorganic bases such as sodium, potassium, ammonium, calcium or ferric hydroxides and the like, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine procaine, or other bases known to those skilled in the art.
Vaccines typically are administered in a way compatible with the dosage formulation, and in such amounts that will be effective prophylactically and/or therapeutically. The quantity to be administered generally ranges from about 5 pg to about 250 ~cg of antigen per dose, however the actual dose will depend upon the health and size of the subject, the capacity of the subject's immune system to synthesize antibodies, and the degree of protection sought. The vaccine may be given in a single or multiple dose schedule. A multiple dose is one in which a primary course of vaccination may be with one to ten separate doses, followed by other doses given at subsequent time intervals required to maintain and/or to reinforce the immune response, for example, at one to four months for a second dose, and if required by the individual, a subsequent doses) several months later. In addition, the dosage regimen may be determined, at least in part, by the need of the individual, and may be dependent upon the practitioner's judgment.
With regard to dead or otherwise inactivated US-type or US-subtype hepatitis E
virus containing vaccines, inactivation may be facilitated using conventional methodologies well known and thoroughly documented in the art. Preferred inactivation methods include, for example, exposure to one or more of (i) organic solvents, (ii) detergents, (iii) formalin, and (iv) ionizing radiation. It is contemplated that some of the proteins in attenuated vaccines may cross-react with other known viruses, and thus shared epitopes may exist between a US-type or US-subtype hepatitis E virus and other members of the HEV family (for example, members of the Burmese or Mexican families) and thus give rise to protective antibodies against one or more of the disorders caused by these pathogenic agents. Preferred formulations and modes of administration are thoroughly documented in the art and so are not discussed in detail herein.
The various factors to be considered may include one or more features discussed hereinabove for the peptide based vaccines.
With regard to the live, but attenuated vaccines, it may be possible to produce attenuated virus using any of the attenuation methods known and used in the art. Briefly, attenuation may be accomplished by passage of the virus at low temperatures or by introducing missense mutations or deletions into the viral genome. Preferred formulations and modes of administration are thoroughly documented in the art and so are not discussed in detail herein.
The various factors to be considered may include one or more features discussed hereinabove for the peptide based vaccines.
With regard to live, recombinant vaccines (vector vaccines), these may be developed by incorporating into the genome of a living but harmless virus or bacterium, a gene or nucleic acid sequence encoding a US-type or US-subtype hepatitis E specific polypeptide chain defining an antigenic determinant. The resulting vector organism may then be administered to the intended host. Typically, for such a vaccine to be successful, the vector organism must be viable, and either naturally non-virulent or have an attenuated phenotype. Preferred host organisms include, vaccinia virus, adenovirus, adeno-associated virus, salmonella and mycobacteria. Live strains of vaccinia virus and mycobacteria have been administered safely to humans in the forms of the smallpox and tuberculosis (BCG) vaccines, respectively. In addition, they have been shown to express foreign proteins and exhibit little or no conversion into virulent phenotypes. Vector vaccines are capable of carrying a plurality of foreign genes or nucleic acid sequences thereby permitting simultaneous vaccination against a variety of preselected antigenic determinants.
Preferred formulations and modes of administration are thoroughly documented in the art and so are not discussed in detail herein.

IV. Identification of molecules with anti-US-type or anti-US-subtype hepatitis E
virus activity.
In view of the discovery of specific HEV US-type sequences, it is contemplated that one skilled in the art may be able to identify molecules which either inactivate or reduce the activity of HEV US-type specific proteins, e.g., the helicase, methyltransferase, or protease proteins encoded by the ORF 1 portions of the HEV genome. An exemplary protocol for identifying molecules that inhibit the HCV protease is described in U.S. Patent No.
5,597,691, the disclosure of which is incorporated herein by reference. Although, the method pertains to the identification of HCV protease inhibitors, it is contemplated that the same or similar protocols maybe used to identify HEV protease inhibitors, or any other protein encoded by a HEV US-type sequence.
Briefly, a method for identifying HEV protease inhibitors is as follows.
Typically, a substrate is employed which mimics the proteases natural substrate, but which provides a quantifiable signal when cleaved. The signal preferably is detectable by colorimetric or fluorometric means; however, other methods such as HPLC or silica gel chromatography, nuclear magnetic resonance, and the like may also be useful. After optimum substrate and protease concentrations have been determined, candidate protease inhibitors are added one at a time to the reaction mixture at a range of concentrations. The assay conditions preferably resemble the conditions under which the protease is to be inhibited in vivo, i.e., under physiologic pH, temperature, ionic strength, etc. Suitable inhibitors exhibit strong protease inhibition at concentrations which do not raise toxic side effects in the subject. Inhibitors which compete for binding to the protease active site may require concentrations equal to or greater than the substrate concentration, while inhibitors capable of binding irreversibly to the protease active site may be added in concentrations on the order of the enzyme concentration.
It is contemplated that the inhibitors may be organic compounds, which, for example, mimic the cleavage site recognized by the HEV protease, or alternatively, may be proteins, for example, antibodies or antibody fragments capable of binding specifically to and inactivating or reducing the activity of the HEV protease. Once identified, the protease inhibitors may be administered by a variety of methods, such as intravenously, orally, intramuscularly, intraperitoneally, bronchially, intranasally, and so forth. The preferred route of administration will depend upon the nature of inhibitor. Inhibitors prepared as organic compounds may be administered orally (which is generally preferred) if well absorbed. Protein-based inhibitors (such as most antibodies or antibody derivatives) generally are administered by parenteral routes.
Exam~nles Practice of the invention will be more fully understood from the following examples, which are presented herein for illustrative purposes only, and should not be construed as limiting the invention in any way. All citations to the literature, both supra and infra, including patents, patent applications and scientific publications are incorporated by reference herein, in their entirety.
Example 1- Case study HEV strain US-1 was identified in the serum of a patient (USP-1) suffering from acute hepatitis. The patient was a 62 year old, white male who was hospitalized in Rochester, MN
after a three-week history of fever, abdominal pain, jaundice, and pruritis.
Onset of signs and symptoms began two weeks after returning home following a ten day trip to San Jose, California.
His past medical history included a nephrectomy for autosomal dominant polycystic kidney disease accompanied by mild renal insufficiency, and a Iaparoscopic cholecystectomy for symptomatic cholelithiasis. The patient had osteoanthritis and was hypertensive. Lisinopnil therapy had been initiated three months prior to admission. Physical examination revealed an ill appearing icteric white male with an enlarged tender liver, and no asterixis.
Serum aspartate aminotransferase.(AST), alanine aminotransferase (ALT), and bilirubin levels were markedly elevated at the time of hospital admission and peaked 8 days and 16 days after hospitalization, respectively (Figure 2). Lisinopril was discontinued on admission. Serologies for hepatitis A
(IgM and IgG anti-HAV), hepatitis B (HBsAg, IgM and IgG anti-HBc), hepatitis C
(anti-HCV), and HCV RNA were negative. Ceruloplasmin, iron, transferrin, anti-nuclear and anti-smooth muscle antibodies, toxin and drug screen were all normal. Careful questioning of the patient revealed no history of ethanol use. Abdominal ultrasound and computed tomography scan, and endoscopic retrograde cholangiopancreatogram were also normal. A liver biopsy showed a severe, acute lobular hepatitis with striking pyknotic and ballooning degeneration of hepatocytes consistent with autoimmune, drug, or viral hepatitis.
The patient made a complete clinical recovery within 2 months, with normalization of AST, ALT, and bilirubin noted about 5 months after hospital admission. No risk factors for acquiring HEV were identified. He had not traveled outside the US for over 10 years. In the 6 weeks prior to illness onset, the only meals he reported eating that were not prepared at home were at a Mexican restaurant and a large fast food restaurant chain. He had no exposure to untreated drinking water, did not report eating raw shellfish, and had no known exposure to farm animals. None of the food handlers at the Mexican restaurant or the fast food restaurant reported foreign travel since less than 5 months from admission date and none reported signs and/or symptoms of hepatitis. No other cases of non- ABC hepatitis were reported in the county health department where the patient stayed in California, and where the patient lived in Minnesota during the period of admission. No family members had signs and/or symptoms of hepatitis either during the patient's trip to California or in the subsequent 10 weeks.
Serum obtained from 6 family members in California, and from his spouse who lived with him in Minnesota over the period of interest were negative for anti-HEV by EIA.
Example 2 - Identircation o unigue isolate o NEV US-1 The presence of HEV was determined by RT-PCR using HEV primer sequences.
Briefly, nucleic acids were isolated from 25 pL of serum from patient USP-1 as previously described (Schlauder et al. (1995) J. Virological Methods 46: 81-89). Ethanol precipitated nucleic acids were resuspended in 3 ~L of diethyl pyrocarbonate (DEPC) treated water.

cDNA synthesis and PCR were performed using the GeneAmp RNA PCR kit from Perkin-Elmer (Norwalk, CT) in accordance with the manufacturer's instructions.
RNA (1 pL) was used as a template for each 10 p,L cDNA reaction. cDNA synthesis was primed with specific primers added to a final concentration of 4 pM. The subsequent amplification of cDNA
was primed with oligonucleotides added to a final concentration of 0.8 to 1.0 pM. PCR was performed for 40 cycles (94°C, 20 sec; 55°C, 30 sec;
72°C, 30 sec; followed by an extension cycle of 72°C for 3 min). The initial PCR reaction (2 ~tL) then was used as a template for a second round of amplification using a nested set of PCR primers. PCR was performed using the GeneAmp PCR kit from Perkin-Elmer in accordance with the manufacturer's instructions.
Briefly, primers were added to a final concentration of 1 pM. The initial set of experiments used three sets of primers. Two from the S'-end of ORF 1 based on sequences from the Burmese and Mexican strains. One set from the 3'-end of ORF 1 based on the Mexican strain sequence. The three sets of primers used were as follows:
Primer Set 1 Primer Seauence SEO ID NO:
5'-ORF 1-Mexican primer C375M CTGAACATCCCGGCCGAC SEQ ID NO:1 PCR primer A 1-350M AGAAAGCAGCGATGGAGGA SEQ iD N0:2 PCR primer S1-34M GCCCACCAGTTCATTAAGGCT SEQ ID N0:3 nested PCR primer A2-320M TCATTAATGGAGCGTGGGTG SEQ ID N0:4 nested PCR primer S2-SSM CCTGGCATCACTACTGCTAT SEQ ID NO:S
Primer Set 2 Primer Se uence SEO ID NO:
5'-ORF I- Bunmese cDNA primer C375 CTGAACATCACGCCCAAC SEQ ID N0:6 PCR primer A1-350 AGGAAGCAGCGGTGGACCA SEQ ID N0:7 PCR primer S1-34 GCCCATCAGTTTATTAAGGC SEQ ID N0:8 nested PCR primer A2-320 TCATTTATTGAGCGGGGATG SEQ ID N0:9 nested PCR primer S2-55 CCTGGCATCACTACTGCTAT SEQ ID NO:10 Primer Set 3 Primer Seauence SEO ID NO:
3'-ORF I- Mexican cDNA primer M1PR6 CCATGTTCCACACCGTATTCCAGAG SEQ ID NO:11 PCR primer S4294M GTGTTCTACGGGGATGCTTATGACG SEQ ID N0:12 nested PCR primer M 1 PF6 GACTCAGTATTCTCTGCTGCCGTGG SEQ ID N0:13 nested PCR primer A4556 GGCTCACCAGAATGCTTCTTCCAGA SEQ ID N0:14 The S'-ORF 1-Burmese primers are described in Schlauder et al. (1993) Lancet 341: 378.
Primers M 1 PR6 and M 1 PF6 are described in McCaustland et al. ( 1991 ) J.
Virological Methods 35: 331-342. The PCR products were separated by agarose gel electrophoresis and visualized by UV irradiation after ethidium bromide staining. The resulting PCR products were hybridized to a radiolabelled probe after Southern blot transfer to a nitrocellulose filter.
Radiolabelled probes were generated from PCR products purified with the QIAEX
gel extraction purification kit by Qiagen (Chatsworth, CA). Radiolabel was incorporated using the Stratgene~ (La Jolla, CA) Prime-It II kit according to the manufacturer's instructions. Filters were prehybridized in Rapid-hyb buffer from Amersham (Arlington Heights, IL) for 3-5 hours, and then hybridized in Fast-Pair Hybridization Solution with 100-200 cpm/cm2 at 42°C for 15-25 hours. Filters then were washed as described in Schlauder et al. (1992) J.
Virol. Methods 37:
189-200. Phosphorimages of the probed filters were obtained with a Molecular Dynamics Phosphorimager 425E (Sunnyvale, CA).
Ethidium bromide stained bands were detected with the primers from the 5'-end of ORF
1. However, only the primers based on the Mexican strain resulted in a nested product of the expected size of 266 base pairs. Hybridization to a probe derived from a Burmese-like strain (identity > 90%) infected patient resulted in a very weak hybridization signal to the patient USP-1 derived products relative to the signal from the Burmese positive control.
These results gave the first indication that this isolate was not closely related to the Burmese isolate. No probe was available from the Mexican strain.

To confirm these results, RNA was extracted from additional serum aliquots of patient USP-1. RT-PCR was performed using the 5'-ORF 1-Mexican primers, SEQ ID NOS:1-S, as described above. Following agarose gel electrophoresis and staining with ethidium bromide, a 342 by product was visualized in each sample. The PCR products were extracted from the agarose gel using the QIAEXII Agarose Gel Extraction Kit by Qiagen (Chatsworth, CA) and cloned into pT7 Blue T-vector plasmid by Novagen (Madison, WI). The cloned products were sequenced using the SEQUENASE VERSION 2.0 sequencing kit (USB, Cleveland, OH) in accordance with the manufacturers instructions.
The nucleotide sequences obtained from the product of the latter two samples were identical and are shown in SEQ ID NO:15. These results indicate that only the cDNA primer and primer S 1 from both the Burmese and Mexican strains resulted in an ethidium bromide stainable product from the patient USP-1 samples. Only the Mexican strain based nested primers, S2 and A2 generated an ethidium bromide stainable product of the expected size.
In order to determine the degree of relatedness between the HEV US-1 isolate and other known isolates of HEV, alignments of the nucleotide and amino acid sequences were performed using the program GAP of the Wisconsin Sequence Analysis Package (Version 9), available from the Genetics Computer Group, Inc., 575 Science Drive, Madison, Wisconsin, 53711. The program employs the algorithm of Needleman and Wunsch (J. Mol. Biol. (1970) 48:443-453) to calculate the degree of similarity and identity, which are expressed as percentages between the two sequences being aligned. The gap creation and gap extension penalties were 50 and 3.0, respectively, for nucleic acid sequence alignments, and 12 and 4, respectively, for amino acid sequence comparisons.
The complete nucleotide and amino acid sequences of the two 'prototype' HEV
isolates from Burma and Mexico, as well as other sequences used for analyses were obtained from GenBank, with their respective accession numbers are indicated in Table 1 below. Each of the these sequences are incorporated herein by reference.

Isolate Genbank Accession Number Mexican (M1)M7450~~~

Burmese (B M73218 1 ) Pakistan M80581 (P1) Chinese (C4)D11093 A 303 base pair sequence of HEV US-1 (homologous to residues 1-303 of SEQ ID
N0:89) was compared against the homologous regions identified in the Mexican, Burmese, Pakistani, and Chinese strains. The resulting percent identities are summarized in Table 2 below.
TABLE 2. Identity over 303 nucleic acids from the 5'-end ORF 1 product US-1 Mexican Burmese Pakistan Mexican 77.2 Burmese 74.9 83.2 Pakistan75.9 83.2 95.7 Chinese 75.9 83.5 95.7 97.4 The results in Table 2 indicate that the fragment from the 5'-end of ORF 1 from the USP-1 isolate showed a nucleic acid identity from about 74.9 to about 77.2 %
relative to other known isolates of HEV. This was less than the identity between the prototype Mexican and Burmese isolates (83.2%). These results indicate that the product likely was derived from a unique isolate of HEV not previously identified.
Example 3 - Genome Extension and Seauencing ofHEV US 1 The clone obtained and sequenced as described in Example 2 (SEQ ID NO:15) hereinabove was derived from a unique HEV genome, HEV US-1. To obtain sequences from additional regions of the HEV US-1 genome, several reverse transcriptase-polymerise chain reaction (RT-PCR) walking experiments were performed.

Total nucleic acids were extracted by the procedure described in Example 2 (for SEQ ID
N0:19 only) or by one of the following procedures. Aliquots (25 pL) of patient USP-1 serum were extracted using the Total Nucleic Acid Extraction procedure in accordance with the manufacturers instructions (United States Biochemical) in the presence of 10 mg yeast tRNA as earner. Nucleic acids were precipitated and resuspended in 3.75 pL RNase/DNase free water.
Alternatively, total RNA was isolated from 100 ~L of serum using the TOTALLY
RNA isolation kit as recommended by the manufacturer (Ambion, Inc.). The resulting RNAs were treated with DNase and column purified with reagents from S.N.A.P. Total RNA isolation kit (Invitrogen, San Diego, CA). Thereafter, RNA was precipitated with 0.1 volumes of 3M sodium acetate, 2 pL pellet paint (Novagen) as carrier, and 2 volumes ethanol. RNA pellets were dissolved in SO
pL DEPC treated water.
RT-PCR was performed using the GeneAmp RNA PCR kit in accordance with the manufacturers instructions (Perkin-Elmer). Random hexamers were used to prime cDNA
synthesis in a total volume of 25 pL except for the isolation of SEQ ID N0:19 which utilized cDNA specifically primed with primer PA2-5560 (SEQ ID N0:16), as described in Example 2 above. US 1-gap was generated with specifically primed cDNA generated using RNA extracted from 12.5 pL serum equivalents, primer US 1 gap-a0.5 (SEQ ID N0:46), and Superscript II (3' RACE Kit: GIBCO BRL). PCR was performed with the cDNA encompassing one-fifth of the total reaction volume (2 pL for 10 pL reaction or 5 pL for 25 pL reaction, etc.). Standard PCR
was performed in the presence of 2 mM MgCl2 and 0.5 to 1.0 j~M of each primer.
Modified reactions contained 1 x PCR Buffer and 20% Q Solution (Qiagen) in accordance with the manufacturer's instructions for the isolation of SEQ ID NOS:33 and 41.
Reactions used two HEV consensus primers (Table 3), one HEV consensus primer and one HEV-US-1 specific primer (Table 4), two HEV US-1 specific primers (Table 5), one HEV US-1 specific primer and one HEV US-2 (see Example 5) specific primer (Table 6), or two HEV US-2 specific primers (Table 7). Reactions were subjected to thermal cycling as follows:

SEQ ID NOS:19, 24, 27, 30, 33, 41, 44, 60, 64, 68, 73, 78, and 83 were obtained by touchdown PCR. Amplification involved 43 cycles of 94°C for 30 seconds, 55°C for 30 seconds (-0.3°C/cycle), and 72°C for 1 minute. This was followed by 10 cycles of 94°C for 30 seconds, 40°C for 30 seconds, and 72°C for 1 minute. For SEQ ID NOS:38, 49, 52, and SS, cycling involved 35 rounds of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute. All amplifications were preceded by 1-2 minutes at 94°C and followed by 72°C for 5 to 10 minutes.
The reactions were held at 4°C prior to agarose gel analysis.
The isolation of SEQ ID N0:19 required a second round of touch down amplification to isolate the desired product. Here, I pL of first round was placed into a second round 25 pL
reaction. The second round amplification utilized hemi-nested primers as indicated in Table 3 by reactions 1.1.1 and 1.1.2. The isolation of SEQ ID N0:24 required a second round of nested touch down amplification as described above and indicated in Table 4 as reactions 2.1. l and 2.1.2. The isolation of SEQ ID NOS:38 and 49 required a second round of nested PCR (Table 5}
utilizing 1 pL of first round into a 25 uL reaction as described above. The isolation of SEQ ID
NOS:60, 64, 68, and 73 required nested PCR in which 1 p.l of the first round was amplified in a 25 p.L second round reaction (Table 6). Products SEQ ID NOS:78 and 83 were generated from two rounds of amplification (Table 7).
Agarose gel electrophoresis was performed on a fraction or all of the PCR
reaction in a 0.8% to 2% agarose TAE gel in the presence of 0.2 mg/mL ethidium bromide.
Products were visualized by UV irradiation and products of the desired molecular weight were excised, purified using GeneClean, in accordance with the manufacturers' instructions (BIO 101, Inc.), and cloned into pT7-Blue T-Vector plasmid (Novagen) II or pGEM-T Easy Vector (Promega) in accordance with the manufacturers' instructions. Cloned products were sequenced as described in Example 2 or on a ABI Model 373 DNA Sequencer using ABI Sequencing Ready Reaction Kit as specified by the manufacturer. Results of these experiments are presented hereinbelow in Tables 3, 4, 5, 6, and 7.

Reaction Primer 1 Primer 2 A rox. Prod. Size /SEQ ID

1.1.1 SEQ ID N0:17 SEQ ID N0:16_ none 1.1.2 SE ID N0:18 SEQ ID N0:16251 b /SEQ ID N0:19 1.2 SE ID N0:28 SEQ ID N0:29168 b /SEQ ID N0:30 Reaction Primer 1 Primer 2 A ro_x_. Product Size/SEQ
ID NO

2.1.1 SEQ 1D N0:20SEQ ID N0:22 none 2.1.2 SEQ ID N0:21SEQ ID N0:23 899 b /SEQ ID N0:24 2.2 SEQ ID N0:25SEQ ID N0:26 846 b /SEQ ID N0:27 2.3 SEQ ID N0:31SEQ ID N0:32 424 b /SEQ ID N0:33 2.4 SEQ ID N0:39SEQ ID N0:40 460 b /SEQ ID N0:41 2.5 SEQ ID N0:42SEQ ID N0:43 235 b /SEQ ID N0:44 ReactionPrimer Set PCR 1 Primer Set PCR 2 Approx. Product Size/SEQ ID NO:

3.1 SEQ ID N0:34/SEQ SEQ ID N0:36/SEQ. 1186 bp/SEQ ID
1D N0:35 ID N0:37 N0:38 3.2 SEQ ID N0:45/SEQ SEQ ID N0:47/SEQ ID 545 bp/SEQ ID
ID N0:46 N0:48 N0:49 3.3 SEQ ID N0:50/SEQ 344 bp/SEQ ID
ID N0:51 N0:52 3.4 SEQ ID N0:53/SEQ 194 bp/SEQ ID
ID N0:54 N0:55 ReactionPrimer Set PCR I Primer Set PCR 2 Approx. Product Size/SEQ ID NO:

4.1 SEQ ID N0:56/SEQ SEQ ID NO:58/SEQ ID 464 bp/SEQ ID
ID N0:57 N0:59 N0:60 4.2 SEQ ID N0:61/SEQ SEQ ID N0:63/SEQ ID 433 bp/SEQ ID
ID N0:62 N0:62 N0:64 4.3 SEQ ID N0:65/SEQ SEQ ID N0:65/SEQ ID 382 bp/SEQ ID
ID N0:66 N0:67 N0:68 4.4 SEQ ID N0:69/SEQ SEQ ID N0:71/SEQ ID 451 bp/SEQ ID
ID N0:70 N0:72 N0:73 ReactionPrimer Set PCR 1 Primer Set PCR 2 Approx. Product Size/SEQ ID NO:

S.1 SEQ ID N0:74/SEQ SEQ ID N0:76/SEQ ID 334 bp/SEQ ID
ID N0:75 N0:77 N0:78 5.2 SEQ ID N0:79/SEQ SEQ ID N0:81/SEQ ID 413 bp/SEQ ID
ID N0:80 N0:82 N0:83 To obtain the sequence at the 3' end of the genome, ampIif cation utilized the 3' RACE
System of GIBCO BRL in accordance with the manufacturer's instructions. It was assumed that, as an HEV strain, the 3' end of the HEV-US-1 genome would contain a poly-adenosine tail similar to the Mexican, Burmese, and Pakistani strains. RNA extracted as described above from the equivalent of 50 ItL of serum was reverse transcribed utilizing the oligo dT adapter primer S'-GGCCACGCGTCGACTAGTACTTTTTTTTTZ'TTTTTTT -3' of (SEQ ID N0:84) supplied by the manufacturer. First round PCR utilized the AUAP primer supplied 5' GGCCACGCGTCGACTAGTAC -3' (SEQ ID N0:85) and a HEV US- specific primer (Table 8) at 0.2 mM final concentration with PCR Buffer, MgCl2, and cDNA
concentrations as recommended. Amplification involved 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, and 72°C for 1 minute. Amplification was preceded by a 1 minute incubation at 94°C and followed by a 72°C, 10 minute extension. A second round of amplification used 1 ~tL of first round in a 50 ~tL reaction. PCR buffer was 1 X final concentration with 2 mM
MgClz, and 0.5 mM of each of the primers. Primers were hemi-nested with the AUAP primer and a specific primer (Table 8). Amplification conditions were the same as first round. The products were analyzed by agarose gel electrophoresis, cloned, and sequenced as above.

Reaction Primer Set PCR 1 Primer Set PCR 2 Approx. Product Size/SEQ ID
NO:

8.1 SEQ ID N0:86/SEQ SEQ ID N0:87/SEQ 960 bp/SEQ ID
ID N0:85 ID N0:85 N0:88 The sequences obtained from the products described in Tables 3, 4, 5, 6, 7, and 8 hereinabove, and the initial PCR product near the 5' end of the genome, SEQ ID
NO:15, were assembled into contigs using the programs of the GCG package (Genetics Computer Group, Madison, WI, version 9) and a consensus sequence determined. A schematic of the assembled contig is presented in Figure 3. The HEV US-1 genome is 7202 by in length, all of which has been sequenced (SEQ ID N0:89). This sequence was translated into three open reading frames, two of which are shown in SEQ ID N0:90 {the third ORF is positioned at nucleotide positions 5094-5462 but cannot be shown in SEQ ID N0:90 due to overlap with the other two ORFs). The resulting translations (ORF 1, ORF 2, and ORF 3) are set forth in SEQ ID
N0:91, SEQ ID
N0:92, and SEQ ID N0:93, respectively.
Example 4 - Identification of unique isolate of HEV US 2 A patient from the US suffering from acute hepatitis, who tested for IgG class antibodies in the HEV EIA test, also tested positive by means of a US-1 strain-specific ELISA. This patient (USP-2) diagnosed with acute hepatitis, was a 62 year old male who was admitted to the hospital with jaundice and fatigue. Initial laboratory studies indicated an ALT of 1270 U/L (normal 0-40 U/L). Since there was a recent outbreak of hepatitis A virus (HAV) in the area, it was suspected that this individual was infected with HAV. However, the anti-HAV IgM test, HAVAB-M EIA
(Abbott Laboratories) was negative as were tests for serologic markers for hepatitis B virus and hepatitis C virus. This patient's history included a visit to Cancun, Mexico, several weeks prior to the onset of his illness.
The sample from the patient then was analyzed for the presence of HEV specific sequences via PCR amplification using HEV US-1 specific PCR primers. RNA was extracted using Ultraspec as described in Example 2. Random primed cDNA synthesis was performed as described in Example 3 and PCR was performed using standard conditions as described in Example 2 with HEV US-1 specific primers SEQ ID N0:94 and SEQ ID N0:96. Nested PCR
was performed with primers SEQ ID N0:95 and SEQ ID N0:97. Sequencing of the PCR
product was performed as described in Example 3. The sequence of the resulting PCR product is set forth in SEQ ID N0:98. GAP analysis as described in Example 2 showed that the nucleotide sequence, SEQ ID N0:98 was 95% identical to the corresponding or homologous homologous region from HEV US-1.

Example ~ - Genome Extension and Se~ruencing ofHEV US 2 The clone obtained and sequenced in Example 4 (SEQ ID N0:98) was derived from a HEV isolate most closely related to HEV US-1. To obtain additional regions of the HEV US-2 genome, several RT-PCR walking experiments were performed as described in Example 3.
RNA was extracted using the Total Nucleic Acid Extraction procedure (United States Biochemical). Reverse transcription was random primed using the GeneAmp RNA
PCR kit (Perkin-Elmer). Standard PCR was performed in the presence of 2 mM MgClz and 0.5 to 1.0 pM
of each primer. Modified reactions contained 1 x PCR Buffer and 20% Q Solution (Qiagen) for the isolation of SEQ ID NOS:129, 141 and 146. Reactions used two HEV US-1 specific primers (Table 9). one HEV US-1 specific primer and one HEV consensus primer (Table 10), one HEV
US-2 specific primer and one HEV consensus primer (Table 11 ), two HEV US-2 specific primers (Table 12), or two Burmese, Mexican, and US derived Consensus primers (described hereinbelow, Table 13).
The products shown in SEQ ID NOS:101, 102, 105, 108, 1 I0, 113. 117, 120, 124, and I51 were obtained by touchdown PCR. Amplification involved 43 cycles of 94°C for 30 seconds, 55°C for 30 seconds (-0.3°C/cycle), and 72°C for I minute. This was followed by 10 cycles of 94°C for 30 seconds, 40°C for 30 seconds, and 72°C for 1 minute. Cycling involving 35 cycles of 94°C for 30 seconds, SS°C for 30 seconds, and 72°C for 1 minute was used to amplify SEQ ID NOS:129, 132, 136, 141 and 146. All amplifications were preceded by 1-2 minutes at 94°C and followed by 72°C for 5-10 minutes. The reactions were held at 4°C prior to agarose gel analysis. Isolation of many products required a second round of nested or hemi-nested PCR as shown in Tables 9-13. In these reactions 1 pL of the PCR1 product was added to 25-50 pL of the PCR2 reaction mixture and the resulting mixture cycled as in PCRl.
Reactions were analyzed and products cloned and sequenced as described in Example 3 above. The results of these experiments are presented below in Tables 9-13.

ReactionPrimer set PCR1 Primer set PCR2 Approx. Product Size/SEQ ID NO:

7.1 SEQ ID N0:99/SEQ ID 331 bp/SEQ ID
NO:100 NO:101 7.2 SEQ 1D N0:34/SEQ ID SEQ ID N0:36/SEQ ID 1186 bp/SEQ ID
N0.:35 N0.:37 N0:102 7.3 SEQ ID N0:103/SEQ 130bp/SEQ ID
ID N0:104 NO:105 7.4 SEQ ID N0:106/SEQ SEQ ID N0:39/SEQ ID 564 bp/SEQ ID
ID N0:107 N0:107 N0:108 7.5 SEQ ID NO.: 86/SEQ SEQ ID N0:87/SEQ ID 678 bp/SEQ ID
ID N0:109 N0:109 NO:110 ReactionPrimer set PCR1 Primer set PCR2 Approx. Product Size/SEQ ID NO:

8.1 SEQ ID NO:111/SEQ 580 bp/SEQ ID
ID N0:112 N0:113 8.2 SEQ ID NO:I 14/SEQ SEQ ID NO:1 16/SEQ 734 bp/SEQ ID
ID N0:116 ID NO:1 IS NO: I 17 ReactionPrimer set PCR1 Primer set PCR2 Approx. Product Size/
SEQ ID NO:

9.1 SEQ 1D N0:118/SEQ 483 bp/SEQ ID
ID N0:119 N0:120 9.2 SEQ ID N0:121/SEQ SEQ ID N0:121/SEQ ID 431 bp/SEQ ID
ID NO:122 N0:123 N0:124 9.3 SEQ ID N0:125/SEQ SEQ ID N0:127/SEQ ID 1020 bp/SEQ ID
ID N0:126 N0:128 N0:129 ReactionPrimer set PCR1 Primer set PCR2 Approx. Product Size/SEQ ID NO.:

10.1 SEQ ID N0:130/SEQ 407 bp/SEQ ID
ID N0:13 t N0:132 10.2 SEQ ID N0:133/SEQ SEQ ID N0:135/SEQ ID 547 bp/SEQ ID
ID N0:134 N0:134 N0:136 10.3 SEQ ID N0:137/SEQ SEQ ID N0:139/SEQ ID 903 bp/SEQ ID
ID N0:138 N0:140 N0:141 10.4 SEQ ID N0:142/SEQ SEQ ID NO: l44/SEQ 503 bp/SEQ ID
ID N0:143 ID N0:145 N0:146 ReactionPrimer set Approx. Product Size/SEQ
ID

NO.:

11.1 SEQ ID N0:147/SEQ ID N0:148 418 bp/SEQ ID N0:149 I 1.2 SEQ ID NO:150/SEQ ID N0:126 197 bp/SEQ ID NO:I51 To obtain the sequence at the 3' end of the genome, amplification utilized the 3' RACE
System of GIBCO BRL in accordance with the manufacturer's instructions as described Example 3. cDNA was generated using SEQ ID N0:84. PCR1 utilized primers SEQ ID NO:150 and SEQ ID N0:85. PCR2 primers were SEQ ID N0:152 and SEQ ID N0:85 (reaction 12.1 ). The resulting product was 901 by (SEQ ID N0:153).
The isolation of new sequences located at the 5'-terminus of the HEV US-2 viral genome was achieved by inverse PCR (M: Zeiner and U. Gehring, Biotechniques 17: 1051-1053, 1994).
Due to limited availability of sera from USP-1 and USP-2, fecal material from a HEV US-2 infected macaque (described in Example 9 below) was chosen as the source material. A product of 462 nucleotides was amplified from macaque fecal material from within the hypervariable/
proline rich hinge region using RNA extracted, reverse transcribed, and PCR
amplified as described in Example 3 using primers SEQ ID NOS:154, 155, 156 and 157. This product (SEQ
ID N0:158) was 100% identical to HEV US-2 sequences. Therefore, it is contemplated that, any sequences identified at the 5' end of the HEV genome from macaque feces should accurately represent the 5' end of the HEV US-2 genome. Total nucleic acids were extracted from 200 p.L
of a 10% fecal suspension as described above. Reverse transcription reactions, which utilized HEV US specific primers (SEQ ID N0:159), were performed using a kit obtained from BMB (as described in M. Zeiner and U. Gehring, Biotechniques, supra), except that nucleic acids were denatured at 70°C for 5 min and then placed on ice prior to initiation of the RT reaction.
Generation of double-stranded, circular cDNAs was performed as described in M.
Zeiner and U.
Gehring, Biotechniques, supra. The resulting circular cDNA molecules served as template,for subsequent PCR reactions. The primers used in the first PCR reaction (PCR1) are shown in SEQ
ID NOS:160 and 161. The nested primers used in the second PCR reaction (PCR 2) were as shown in SEQ ID NOS:162 and 163.
Products from PCR2 (reaction 13.1 ) were cloned into pGEM-EasyT Vector (Promega) and sequenced using an Applied Biosystems 373 Automated sequencer. One product of 221 nucleotides was identified as having the appropriate primers and HEV US-2 sequences, identifying 63 nucleotides upstream of known HEV US-2 sequences. Additional clones were identified with the appropriate primers and portions of this new sequence.
Primer extension experiments performed on RNA from I00 ~L of USP-2 serum or 100 pL of a 10%
fecal suspension using the sequences shown in SEQ ID NOS:163 and 161 as primers were unsuccessful in confirming the length of this sequence. Pair-wise comparisons of the 63 nucleotides to 5' NTR sequences of Burmese-like isolates revealed identities greater than 94%
suggesting that this is the true sequence of HEV US-2.
The sequences obtained from the products described in this Example and those described in Example 4 were assembled into contigs using programs in the GCG package (Genetics Computer Group, Madison, WI, version 9) and a consensus sequence determined. A
schematic of the assembled contigs is presented in Figure 4. The genome of the HEV US-2 strain is 7277 by in length, all of which has been sequenced and is set forth in SEQ ID
N0:164. This sequence was translated into three open reading frames as indicated in SEQ ID N0:165, with the WO 99/19732 PC'T/US98/21941 translation products of the ORF 1 and ORF 2 sequences only being shown (the third ORF is positioned at nucleotide positions 5159-5527 but cannot be shown within SEQ ID
N0:165 due to overlap with the other two ORFs). The resulting translations of the OItF 1, OIRF 2, and ORF 3 sequences are shown in SEQ ID NOS:166, 167 and 168, respectively.
Example 6 - Seguence Comwarisons Information about the degree of relatedness of viruses typically can be obtained by performing comparisons such as alignments of nucleotide and deduced amino acid sequences.
Alignments of the sequences of the US isolates of HEV (e.g., HEV US-1 and HEV
US-2) with corresponding sequences of other isolates of HEV provide a quantitative assessment of the degree of similarity and identity between the sequences. In general, the calculation of the similarity between two amino acid sequences is based upon the degree of likeness exhibited between the side chains of an amino acid pair in an alignment. The degree of likeness is based upon the physical-chemical characteristics of the amino acid side chains, i.e.
size, shape, charge, hydrogen-bonding capacity, and chemical reactivity. Thus similar amino acids possess side chains that have similar physical-chemical characteristics. The calculation of identity between two aligned amino acid or nucleotide sequences is, in general, an arithmetic calculation that counts the number of identical pairs of amino acids or nucleotides in an alignment and divides this number by the length of the sequences) in the alignment. The calculation of similarity between two aligned nucleotide sequences sometimes uses different values for transitions and transversions between paired (i.e. matched) nucleotides at various positions in the alignment.
However, the magnitude of the similarity and identity scores between pairs of nucleotide sequences, are usually very close, i.e. within one to two percent.
The degree of similarity and identity was determined using the program GAP of the Wisconsin Sequence Analysis Package (Version 9). The gap creation and gap extension penalties were SO and 3.0, respectively, for nucleic acid sequence alignments, and 12 and 4, respectively, for amino acid sequence comparisons.

As indicated previously, a partial identity exists between the initial S'-end ORF 1 clone and other isolates of HEV, which supports the proposition that the HEV
infection associated with patient USP-1 is due to a unique isolate of HEV. In order to more extensively determine the degree of relatedness between this isolate and other known isolates of HEV, alignments of the extended nucleotide and deduced amino acid sequences were performed.
Pair-wise nucleotide and amino acid comparisons of HEV US-1, HEV US-2, and 10 other full length HEV genomes (obtained from a publicly-available database, see Table 14) were performed, as described above, to determine the relationship of the US
isolates to each other and to the known variants of HEV.

Isolate Genbank Accession Number Mexican (M1) M74560 Burmese (B M73218 1 ) Burmese (B2) D10330 Pakistan (PI)M80581 Chinese (C D 1 I 092 1 ) Chinese (C2) L25547 Chinese (C3) M94177 Chinese (C4) D 11093 Indian (I X98292 1 ) Indian (I2) X99441 Nucleotide identity across the entire genomes ofUS-1, US-2, B1, B2, I2, C1, C2, C3, P1, C4 and I1 strains is presented in Table I5. The nucleotide identities of ORF
1, ORF 2, and OItF
3 are shown in Tables 16, 17 and 18, respectively. Tables 17 and 18 also contain comparisons against a recently isolated swine (S1) sequence, available under GenBank accession number AF011921.

TABLE 15 - Nucleotide Identity Across Genome US-l US-2 B1 B2 I2 C1 C2 C3 P1 C4 I1 US-2 92.0 B 73.9 74.0 B2 73.8 74.0 98.5 I2 73.5 73.8 96.1 95.4 C 74.2 74.3 93.9 93.4 92.3 C2 74.2 74.3 93.5 93.0 92.0 98.7 C3 74.1 74.3 93.7 93.0 92.0 98.2 98.7 P 74.1 74.1 93.6 92.8 92.0 98.2 98.8 98.3 C4 73.7 73.9 94.5 94.1 92.7 97.1 97.2 96.8 96.7 I1 74.4 74.4 93.5 93.0 92.2 93.8 94.0 93.8 93.9 93.5 M 73.7 74.5 75.9 75.7 75.0 75.9 75.9 75.9 76.1 75.7 75.7 TABLE 16 - Nucleotide Identity Across ORF 1 US-2 92.0 B1 71.7 71.6 B2 71.7 71.8 98.6 I2 71.2 71.5 95.7 95.1 C 72.1 72.1 93.5 93.1 91.8 C2 72.2 72.3 93.1 92.7 91.598.6 C3 71.9 72.2 93.3 92.8 91.498.1 98.7 P 72.2 72.1 93.1 92.6 91.498.2 99.0 98.4 C4 71.5 71.7 94.6 94.4 92.396.7 98.8 96.3 96.4 I1 72.3 72.3 93.2 92.8 91.593.6 94.0 93.7 93.9 93.3 M 72.0 72.6 73.6 73 72.573.7 73.8 73.8 73.9 73.4 73 1 .5 .5 TABLE 17 - Nucleotide Identity Across ORF 2 US-2 92.2 B 1 79.2 79.6 B2 86.4 79.4 98.5 I2 79.0 79.5 99.2 98.4 C 1 79.3 79.5 94.4 98.4 98.4 C2 79.2 79.4 94.3 97.8 97.8 98.9 C3 79.3 79.4 94.4 97.8 97.8 98.9 98.4 P 1 79.0 79.3 93.8 98.1 98.7 99.7 99.2 99.2 C4 78.8 79.3 94.0 97.8 97.8 98.9 98.4 98.4 97.4 I1 79.4 79.7 94.1 97.6 97.3 97.9 97.0 94.0 93.7 93.9 M 1 78.0 79.3 81.1 90.1 98.5 90.6 90.1 81.0 81.4 90.3 90.3 S 1 92.0 98.9 79.8 84.6 85.4 85.4 85.1 80.2 80.1 84.8 85.1 84.6 TABLE 18 - Nucleotide Identity Across ORF 3 US-1 US-2 B1 B2 I2 C1 C2 C3 P1 C4 I1 Ml US-2 96.2 B 1 87.0 86.6 B2 86.4 86.3 99.2 I2 86.4 86.9 97.8 99.2 C 1 87.3 86.3 99.2 98.4 98.4 C2 86.4 86.1 98.1 97.3 97.8 98.9 C3 86.7 85.6 98.1 97.3 97.8 98.9 98.4 P1 87.0 86.6 98.9 98.1 98.7 99.7 99.2 99.2 C4 86.2 85.8 98.1 97.6 97.8 98.9 98.4 98.4 99.2 I1 86.4 86.6 97.8 97.6 97.6 97.9 97.0 97.0 97.8 97.8 M1 84.6 85.2 87.8 90.1 89.5 90.6 90.1 90.1 90.9 90.3 90.3 S1 94.9 96.7 85.1 84.6 85.4 85.4 85.1 84.8 85.6 84.8 85.1 84.6 In addition, the ORF 1 nucleotide sequences encoding the methyltransferase proteins were compared between each of the US-l, US-2, M1 and P1 isolates. The methyltransferase encoding region of the HEV US-1 genome is represented by residues 1-693 of SEQ
ID N0:89, whereas the methyltransferase encoding region of the HEV US-2 genome is represented by residues 36-755 of SEQ ID N0:164. The comparison results are set forth in Table 19.
TABLE 19 - Methyltransferase Region IDENTITY

US-1 - 93.4 77.0 75.2 US-2 _ - 78.5 76.0 M - - _ 78.8 The ORF 1 nucleotide sequences encoding the Y domain proteins were compared between each ofthe US-1, US-2, M1 and P1 isolates. The Y domain protein encoding region of the HEV US-1 genome is represented by residues 619-1272 of SEQ ID N0:89, whereas the Y
domain protein encoding region of the HEV US-2 genome is represented by residues 680-1334 of SEQ ID N0:164. The comparison results are set forth in Table 20.
TABLE 20 - Y Domain IDENTITY

US-1 US-2 ~~~ M1 P1 US-1 - 94.0 79.0 ~ 77.2 US-2 - _ 79.7 76.8 M 1 - - - 78.3 The ORF 1 nucleotide sequences encoding the protease proteins were compared between each of the US-1. US-2, M 1 and P 1 isolates. The protease protein encoding region of the HEV
US-1 genome is represented by residues 1270-2091 of SEQ ID N0:89, whereas the protease protein encoding region of the HEV US-2 genome is represented by residues 1332-2153 of SEQ
ID N0:164. The comparison results are set forth in Table 21.

TABLE 21 - Protease Region IDENTITY

US-1 US-2 M1 pl US-1 - _ 65.1 64.0 91.8 US-2 - - 65.1 63.1 M1 - _ - 68.1 The ORF 1 nucleotide sequences encoding the hypervariable region were compared between each of the US-l, US-2, M1 and P1 isolates. The hypervariable region encoding region of the HEV US-1 genome is represented by residues 2092-2364 of SEQ IS N0:89, whereas the hypervariable region encoding region of the HEV US-2 genome is represented by residues 2194-2429 of SEQ ID N0:164. The comparison results are set forth in Table 22.
TABLE 22 - Hypervariable Region IDENTITY

US-1 - 83.9 40.3 50.2 US-2 - - 45.8 49.8 M1 - _ - 40.4 The ORF 1 nucleotide sequences encoding the X domain proteins were compared between each of the US-1, US-2, M l and P 1 isolates. The X domain protein encoding region of the HEV US-1 genomes represented by residues 2365-2841 of SEQ ID N0:89, whereas the X
domain probe encoding region of the HEV US-2 genome is represented by residues of SEQ ID N0:164. The comparison results are set forth in Table 23.
TABLE 23 - X Domain IDENTITY

US-1 - 91.6 72.5 71.3 US-2 - - 72.7 70.9 M1 - _ - 72.9 The ORF 1 nucleotide sequences encoding the helicase proteins were compared between each of the US-1, US-2, MI and P1 isolates. The helicase encoding region of the HEV US-1 genomes represented by residues 2893-3591 of SEQ ID N0:89, whereas the helicase encoding region of the HEV US-2 genome is represented by residues 3656 of SEQ ID N0:164. The comparison results are set forth in Table 24.
TABLE 24 - Helicase Region IDENTITY

US-1 US-2 M 1 pl US-1 - 92.8 ~ 76.5 75.2 US-2 - - 75.4 74.1 M 1 - - - 76.2 The ORF 1 nucleotide sequences encoding the RNA-dependent RNA polymerase proteins were compared between each of the US-1, US-2, MI and P1 isolates. The polymerase encoding region of the HEV US-1 genome is represented by residues 3634-5094 of SEQ ID
N0:89, whereas the polymerase encoding region of the HEV US-2 genome is represented by residues 3699-5159 of SEQ ID N0:164. The comparison results are set forth in Table 25.
TABLE 25 - RNA-dependent RNA Polymerase Region IDENTITY

US-1 US-2 M l p 1 US-1 - 93.1 72.9 75.3 US-2 - - 73.6 75.8 M1 - _ -77.1 In addition, the amino acid identities/similarities of the proteins encoded by the ORF 1, ORF 2, and ORF 3 sequences of US-1, US-2, B1, B2, I2, CI, C2, C3, P1, C4 and I1 strains are shown in Tables 26, 27 and 28 respectively. In addition, Tables 27 and 28 also contain comparisons against the swine sequence (S 1 ). In Tables 26, 27 and 28, the similarities are presented in the upper right hand halves of the tables and the identities are presented in the lower left hand halves of the tables.
TABLE 26 - Amino Acid Similarity/Identity Across ORF 1 SIMILARITY

US-1 US-2 BI B2 12 Cl C2 C3 P1 C4 I1 M1 US-I 97.8 86.085.784.485.986.284.986.4 85.786.3 85.4 I US-297.5 86.285.884.585.886.085.086.3 85.786.3 85.5 D B 82.4 82.6 98.796.898.498.597.198.5 98.198.2 87.0 E B2 82.3 82.3 98.6 96.297.897.996.397.8 97.697.6 86.6 N 12 80.7 80.7 96.395.7 96.396.495.096.3 95.995.9 85.2 T Cl 82.5 82.3 98.297.595.7 99.597.999.4 99.098.2 86.9 I C2 82.8 82.6 98.497.895.999.4 98.299.6 99.298.4 87.0 T C3 81.6 81.6 96.996.194.497.798.1 98.1 97.697.0 85.9 Y P1 83.0 82.9 98.497.795.999.299.698.0 99.098.4 87.1 C4 82.5 82.3 98.097.695.498.899.197.498.9 97.8 86.5 I1 82.9 82.9 98.197.595.598.198.496.998.4 97.8 87.3 M1 82.0 82.0 83.883.481.883.783.982.884.0 83.484.2 I , TABLE 27 - Amino Acid Similarity/Identity Across ORF 2 _ %
SIMILARITY

US-1 98.393.3 93.093.093.593.2 92.993.292.4 92.691.597.1 I US-298.0 93.3 93.093.393.393.3 93.093.392.6 92.791.?99.1 D B 91.891.8 98.999.199.899.2 99.299.598.8 98.994.893.0 E B2 91.591.598.9 98.399.198.5 98.598.898.2 98.294.192.7 N 12 91.591.899.1 98.3 99.298.9 98.699.298.5 98.694.591.5 T C 92.092.099.7 98.999.1 99.4 99.199.798.9 99.195.093.2 I C2 91.792.099.1 98.398.899.4 98.899.498.6 98.894.793.0 T C3 91.491.799.1 98.398.599.198.8 99.198.3 98.594.492.7 Y P 91.792.099.4 98.699.199.799.4 99.1 98.9 99.195.093.0 C4 90.991.298.6 98.098.498.998.6 98.398.9 98.394.292.3 II 91.191.498.~ 97.798.298.898.5 98.298.898.0 94.792.4 I M 90.190.693.2 92.492.993.393.0 92.993.392.6 93.0 91.2 I

S 97.798.99 91.491.991.891.7 91.491.790.9 91.190.2 1 i .7 SLIBSTIME SHEET (RULE 26) TABLE 28 - Amino Acid Similarity/Identity Across ORF 3 SIMILARITY

US-1 96.7 85.284.485.285.2 83.6 85.285.2 83.685.279.5 93.5 US-296.7 85.284.485.285.2 83.6 83.685.2 83.685.281.1 96.7 I B 84.484.4 98.4100.0100.098.4 98.4100.098.498.487.0 83.7 D B2 83.683.6 98.4 98.498.4 96.7 96.798.4 96.796.787.0 82.9 E I2 84.484.4 100.098.4 100.098.4 98.4100.098.498.487.0 83.7 N Cl 84.484.4 100.098.4100.0 98.4 98.4100.098.498.487.0 83.7 T C2 82.882.8 98.496.798.498.4 96.798.4 97.696.785.4 82.1 I C3 84.482.8 98.496.798.498.4 96.7 98.4 96.796.785.4 82.1 T P1 84.484.4 100.098.4100.0100.098.4 98.4 98.498.487.0 83.7 Y C4 82.882.8 98.496.798.498.4 97.6 96.798.4 96.785.4 82.1 I1 84.484.4 98.496.79$.498.4 96.7 96.798.4 96.7 88.6 83.7 M 78.780.3 87.087.087.087.0 85.4 85.487.0 85.488.6 79.7 S 93.596.7 82.982.182.982.9 81.3 81.382.9 81.382.978.9 In addition, the ORF 1 amino acid sequences defining the methyltransferase proteins were compared between each of the US-1, US-2, M1 and P1 isolates. The methyltransferase protein encoded by the HEV US-1 genome is represented by residues 1-231 of SEQ
ID
N0:91, whereas the methyltransferase protein encoded by the HEV US-2 genome is represented by residues 1-240 of SEQ ID N0:166. The comparison results are set forth in Table 29.
TABLE 29 - Methyltransferase Region IDENTITY

US-1 US-2 Ml P1 S

1 US-1 - 98.7 91.3 88.7 M

1 US-2 98.7 - 91.7 89.1 L

A M 1 91. 8 92.0 - 92.9 R

1 P 1 90.0 90.4 91.2 -T

Y

SUBSTIT'U'TE SHEET (RULE 26~

The ORF 1 amino acid sequences defining the protease proteins were compared between each of the US-1, US-2, M1 and P1 isolates. The protease protein encoded by the HEV US-1 genome is represented by residues 424-697 of SEQ ID N0:91, whereas the protease protein encoded by the HEV US-2 genome is represented by residues 433-706 of SEQ ID
N0:166. The comparison results are set forth in Table 30.
TABLE 30 - Protease Region IDENTITY

S

I US-1 - 98.5 67.5 69.3 M

I US-2 97.8 - 67.1 68.6 L

A M 1 73.3 73.3 - 76.6 R

I P 1 74.4 74.0 72.2 -~T

',Y

The ORF 1 amino acid sequences defining Y domain proteins were compared between each of the US-1, US-2, M1 and P1 isolates. The Y domain protein encoded by the HEV US-1 genome is represented by residues 207-424 of SEQ ID N0:91, whereas the Y
domain protein encoded by the HEV US-2 genome is represented by residues 216-433 of SEQ ID
N0:166. The comparison results are set forth in Table 31.

TABLE 31 - Y Domain IDENTITY

US-1 US-2 M l P 1 S

I US-1 - 98.2 92.7 93.6 M

I US-2 98.2 - 92.7 93.6 L

A M 1 94.0 94.0 - 93.1 R

I P 1 94.5 94. 5 91.7 T

Y

The ORF 1 amino acid sequences defining the X domain proteins were compared between each of the US-1, US-2, MI and P1 isolates. The X domain encoded by the HEV US-1 genome is represented by residues 789-947 of SEQ ID N0:91, whereas the X
domain protein encoded by the HEV US-2 genome is represented by residues 799-957 of SEQ ID
N0:166. The comparison results are set forth in Table 32.
TABLE 32 - X Domain IDENTITY

S

I US-1 - 97.5 82.4 80.5 M

1 US-2 97.5 - 81.8 79.9 L

A M 1 88.0 87.4 - 86.1 R

I P 1 84.3 83.6 83.0 -T

Y

The ORF 1 amino acid sequences defining helicase proteins were compared between each of the US-1, US-2, M1 and P1 isolates. The helicase encoded by the HEV US-1, US-2, Ml and P1 isolates. The helicase encoded by the HEV US-1 genome is represented by residues 965-1197 of SEQ ID N0:91, whereas the helicase encoded by the HEV US-2 genome is represented by residues 975-1207 of SEQ ID N0:166. The comparison results are set forth in Table 33.
TABLE 33 - HeIicase Region IDENTITY

S

I US-1 - 99.1 89.7 91.0 M

I US-2 99.1 - 90.6 91.8 L

A M 1 93.1 94.0 - 95.2 R

I P 1 94.0 94. 8 91.0 -T

Y

The ORF 1 amino acid sequence defining the hypervariable regions were compared between each end of the US-1, US-2, M1 and PI isolates. The hypervariable region encoded by the HEV US-1 genome is represented by residues 698-788 of SEQ ID N0:91, whereas the hypervariable region encoded by the HEV US-2 genome is represented by residues 707-798 of SEQ ID N0:166. The comparison results are set forth in Table 34.
TABLE 34 - Hypervariable Region IDENTITY

S

I US-1 - 82.4 25.0 27.7 M

I US-2 79.1 - 25.0 21.0 L

-A M1 25.0 25.0 - 20.8 R

I P1 31.9 21.0 18.0 -T

Y

The ORF 1 amino acid sequence defining the RNA-dependent RNA polymerase proteins were compared between each of the US-1, US-2, M1 and P1 isolates. The polymerase encoded by the HEV US-1 genome is represented by residues 1212-1698 of SEQ ID N0:91, whereas the polymerase encoded by the HEV US-2 genome is represented by residues 1222-1708 of SEQ ID
N0:166. The comparison results are set forth in Table 35.
TABLE 35 - RNA-dependent RNA Polymerase Domain IDENTITY

US-1 US-2 /VI I pl S

I US-1 - 99.0 86.0 87.8 M

I US-2 99.0 - 86.2 87.7 L

A M 1 89.7 89.9 - 92.6 R

I P1 91.6 91.6 89.5 -I

'T

Y

In addition to the foregoing, several additional HEV isolates belonging to the HEV US-type family were identified during the course of this work (see, Example 13 below). The additional isolates are denoted as Itl (Italian strain), G1 (first Greek strain) and G2 (second Greek strain). Additional sequence comparisons were performed and include the Itl, G1 and G2 sequences, the results of which are presented below in Tables 36 and 37. Table 36 shows the nucleotide and deduced amino acid identities between isolates of HEV over a 371 base (123 amino acids) ORF 1 fragment. The ORF 1 fragment corresponds to residues 2b-396 of SEQ ID
N0:89. Table 37 shows the nucleotide and deduced amino acid identities between isolates of HEV over a 148 base (49 amino acid) ORF 2 fragment. The ORF 2 fragment corresponds to residues 6307-6454 of SEQ ID N0:89. In both Tables 36 and 37, the isolates represented are Burmese (Bl, B2), Chinese (C1, C2, C3, C4), Indian (I1, I2), Pakistan (P1), Mexican {M1), Swine (SI), United States (US-1, US-2), Greek (G1, G2) and Italian (Itl).

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Pairwise comparisons of the full length nucleotide sequences were.preferred using the nucleotide sequences of the respective genomes of HEV US-l and HEV US-2 together with the other genomes of the other HEV isolates identified in Table 14. The results of the comparison are shown in Table 15. At the nucleotide level, HEV US-l and HEV US-2 were most closely related to each other, with 92.0% identity across the entire genome. The full length Burmese-like isolates demonstrated similar identities ranging from 92.0 to 98.8%. The US
isolates were 73.5 to 74.5% identical to the Burmese-like and Mexican isolates. This is similar to the identity seen between any one Burmese-like isolate and the Mexican isolate, 75.0 to 76.1 %
nucleotide identity. These data indicate that the US isolates are members of a new strain variant of HEV, distinct from the Burmese and Mexican strains.
Similar degrees of identity are found when smaller portions of each genome are analyzed, such as the individual ORFs. These values are presented in Tables 16, I7 and 18 for ORF I, ORF 2, and ORF 3, respectively. Across each region, the Burmese and Pakistani isolates demonstrate the highest degree of identity ranging from 93. I to 98.9%
identity. The Mexican isolate is distinct, with identities of 73.6 to 90.1% to the Burmese-like isolates. HEV US-1 nucleotide sequence analysis reveals a significant degree of divergence with ORF 1 sequences being less than 72% identical to the Burmese-like and Mexican isolates.
Similarly, ORF 2 and ORF 3 sequences were less than 79.1 % and 86.9% identical to the Burmese-like and Mexican isolates, respectively.
The variability seen at the nucleotide level is reflected in the amino acid similarity and identity of the translated open reading frames. ORF 1 is the most divergent product, potentially due to the presence of a hypervariable region. The US isolates possess 97.5%
amino acid identity across this region (Table 26). This is similar to the 94.4 to 99.6%
identity seen between Burmese-like ORF I proteins. The US ORF 1 products are 80.7 to 83.0% identical to Burmese-like and Mexican proteins (Table 26). These values are similar to those observed between any one Burmese-like isolates and the Mexican isolate, ranging from 81.8 to 84.2%
identity. Amino acid similarity values are generally up to 3.5% higher than the identity value, reflecting a large number of conservative amino acid substitutions. The ORF 2 product is the most conserved, potentially due to its role as the viral capsid protein. The US ORF 2 products are 98.0% identical to each other, while being 90.1 to 92% identical to Burmese and Mexican ORF 2 proteins (Table 27). Again, these ranges mirror those observed between Burmese isolates (97.7 to 99.7%
identity). Identity between Burmese and Mexican isolates is slightly greater than that between the US variant and other variants, being 92.4 to 93.3%. Amino acid similarity across ORF 2 adds approximately 1.5% to the identity value. The ORF 3 product of HEV US-I
and HEV US-2 shared 96.7% amino acid identity. The Burmese isolates showed 96.7 to 100%
amino acid identity. ORF 3 amino acid identities of the US isolates to the Burmese and Mexican isolates were 78.7 to 84.4%, slightly less than that observed between Burmese and Mexican isolates, 85.4 to 88.6% identity (Table 28). Amino acid similarity across ORF 3 was generally the same as the identity values, however, some comparisons demonstrated similarity values less than I .0%
greater than the identity value. These amino acid similarity and identity values indicate that the analysis of short amino acid sequences produce similar results to full length and partial nucleotide analyses, indicating that the US isolates are closely related and genetically distinct from previously characterized isolates of HEV.
Tables 27 and 28 also include pairwise amino acid sequence comparisons with a HEV-like isolate recently identified in swine (Meng et al. ( 1997) Proc. Natl.
Acad. Sci. USA 94: 9860-9865. Only 2021 by across the ORF 2/3 region have been characterized (GenBank Accession Number: AF011921). The US swine sequence is 92% identical to the corresponding region of HEV US-1 at the nucleotide level. It is noted that HEV US-1 is very similar at the amino acid level to the recently identified swine virus. For example, the HEV US-1 and swine strains exhibit 97.1% and 93.5% identity over the respective ORF 2 and ORF 3 sequences (Tables 27 and 28, respectively).
Partial sequences of 210 nucleotides from two HEV isolates from China referred to as G9 and G20 (Genbank Accession numbers X87306 and X87307, respectively) recently have been described in the literature by (Huang et al. (1995) J. Med Virology 47: 303-308). These fragments represent nucleotide sequences homologous to residue numbers 4533 to 4742 of SEQ
ID N0:89. Their encoded amino acid sequences (69 amino acid residues in length) are homologous to residue numbers 1 S 12-1580 of SEQ ID N0:91. The results from the pairwise comparisons of the nucleotide sequences and the predicted amino acid sequences of these sequences are shown in Tables 38 and 39. Results indicate that the G9 and G20 isolates are 89%
identical to one another at the nucleotide level across this region. The closely related Burmese and Pakistan isolates are 92.9% identical over this range. The US-1 isolate exhibits a 77.1 and 81.0 across this region suggesting that the US-1 isolate also is unique from these isolates.
Although the G9 and G20 sequences are most closely related at the nucleotide level, the deduced amino acid translation of G20 is most similar/identical to the US sequence from the US-1 isolate (Table 38). This is most likely due to the short length of amino acids utilized in the analysis.
TABLE 38. Identity across 210 nucleotides of ORF 1 Pak Mex US-1 G20 G9 ~

Bur 92.9 74.8 75.7 78.1 76.7 Pak 75.2 _ 76.7 78.1 76.7 Mex 77.1 75.2 71.9 US-1 g 1.0 77.1 G20 89.0 TABLE 39. Similarity/identity across 69 amino acids of ORF 1 Pak Mex US-I G20 G9 Bur 98.6 / 92.8 / 92.8 / 92.8 / 88.482.6 / 79.7 98.6 88.4 85.5 Pak 94.2/89.9 91.3/84.1 91.3/87.0 84.1/81.2 Mex 89.9 / 89.9 / 87.081.2 / 78.3 87.0 US-1 100 / 95.7 88.4 / 88.1 G20 88.4 / 87.0 Example 7 - Phylogenetic Anal ses.
Alignments of nucleotide and amino acid sequences were performed in order to determine the phylogenetic relationships between the novel US-type isolates and other isolates of HEV. The alignments were made using the program PILEUP of the Wisconsin Sequence Analysis Package, version 9 (Genetics Computer Group, Madison, WI).
Evolutionary distances between sequences were determined using the DNADIST program (Kimura 2-parameter method) with a transition-transversion ratio of 2.0 and PROTDIST (Dayhoff PAM matrix) program of the PHYLIP package, version 3.5c (Felsenstein 1993. Department of Genetics, University of Washington, Seattle). The computed distances were used for the construction of phylogenetic trees using the program FITCH (Fitch-Margoliash method). The robustness of the trees was determined by bootstrap resampling of the multiple-sequence alignments (100 sets or 1,000 sets) with the programs SEQBOOT, DNADIST, the neighbor joining method of the program NEIGHBOR, and CONSENSE (PHYLIP package). Bootstrap values of less than 70% are regarded as not providing evidence for a phylogenetic grouping {Muerhoff et al., (1997) Journal of Virology, 71: 6501-6508). The final trees were produced using RETREE
(PHYLIP) with the midpoint rooting option and the graphical output was created with TREEVIEW
(Page, (1996) Computer Applied Biosciences 12: 357-358), the results of which are presented in Figures 5, 6, 10, and 11.
Phvlogenetic analysis with complete ~enomes. To more extensively determine the degree of relatedness between HEV US-1, HEV US-2, and other known isolates of HEV, nucleotide alignments were performed. The full length HEV US-1 and HEV US-2 genomes were aligned with 10 other isolates of HEV from which complete genomes are available (Table 14).
Examination of the phylogenetic distances based upon alignments of the HEV-US
isolates and other isolates of HEV demonstrate that there is considerable evolutionary distance between those from the US and those from other geographical areas as determined using the DNADIST
program (Kimura 2-parameter method) with a transition-transversion ratio of 2.0 (Table 40). The distances calculated also show the close relationship between the isolates originating from Asia.

Within this Burmese-like group the maximum distance calculated from the full length alignment is 0.0850 nucleotide substitutions per base. The minimum distance between a member of this group and a US isolate is 0.3322 substitutions. The Mexican strain shows similar distances to the Burmese-like group of 0.3055 to 0.3132 substitutions and 0.3322 to 0.3462 substitutions to the US isolate. The genetic distance between HEV US-1 and HEV US-2 of 0.0812 substitutions is similar to that seen between Burmese-like isolates. The relative evolutionary distances between the viral sequences analyzed are readily apparent upon inspection of the unrooted phylogenetic tree presented in Figure 5, where the branch lengths are proportional to the evolutionary distances. In the phylogenetic tree, the Burmese=like isolates, the Mexican isolate and the US isolates each represent a major branch. In addition, the branching of the prototype viruses are supported with bootstrap values of 100%. Analysis of smaller segments of the genome (e.g. ORF 1, ORF 2, or ORF 3) were individually analyzed resulting in trees analogous to those obtained with the full length sequence and shown in Figure 5. These analyses demonstrate that the HEV US isolates represent a distinct strain or variant of HEV and that HEV
US-1 and HEV US-2 are as similar to each other as are the most divergent Burmese-like isolates.

TABLE 40 - Phylogenetic distances over the full length sequence B2 0.0149 C 0.06430.0697 C2 0.06800.07330.0136 C3 0.06630.07340.01780.0132 C4 0.05740.06110.03040.02900.0329 I1 0.06770.07280.06450.06250.06470.0681 I2 0.04030.04770.08200.08490.08460.07760.0832 Pl 0.06930.07510.01780.01200.01720.03350.06330.0850 M1 0.30960.31200.30860.30890.30910.31320.31200.32590.3055 US-10.34060.34180.33600.33450.33670.34450.33220.34640.33630.3462 US-20.34130.34080.33700.33610.33740.34450.33330.34610.33770.33670.0812 Comparison to ORF 2/ORF 3 from Swine HEV In order to determine the relationship between a recently described swine-HEV and the human HEV US-1 and HEV
US-2 isolates, comparisons of the nucleotide sequences across the complete ORF
2 and ORF
3 were performed using analogous regions from the 10 full length sequences utilized above 5 (Table 14). Phylogenetic analysis produces genetic distances of 0.0799 to 0.0810 nucleotide substitutions per position between the US and swine HEV isolates (Table 41).
These values are similar to those observed between the most distant Burmese-like isolates.
The US and swine isolates group closely on an unrooted phylogenetic tree when the ORF 2/3 nucleotide sequences are analyzed (See, Figure 6). These isolates form a phylogenetic group distinct 10 from the Mexican isolate and the Burmese-like isolates. These grouping are supported by bootstrap values of 100%.
SUBSTIME SHEET (RULE 26) TABLE 41 - Phylogenetic distances between USswine and human HEV isolates US-2 USswine Burmese Mexican US-1 0.0799 0.0810 0.2441-0.24950.2671 US-2 0.0795 0.2409-0.24790.2486 USswine 0.2348-0.24850:2615 Burmese ~ 0.0119-0.07160.2183-0.2248 Example 8 - HEV Serologic Studies A. Background Early studies indicate that epitopes useful for diagnosis of HEV infections are located near the carboxyl terminus of ORF 2 and ORF 3 of both the Burmese and Mexican strains of HEV. The two antigens from the Mexican strain, referred to hereinafter as M 3-2 and M 4-2, comprise 42 and 32 amino acids near the carboxyl terminus of ORF 2 and ORF 3, respectively (Yarbough et al. (1991) Journal of Virology, 65: 5790-5797). The two antigens from the Burmese strain of HEV, referred to hereinafter as B 3-2 and B 4-2 proteins, comprise 42 and 33 amino acids near the carboxyl terminus of ORF 2 and ORF 3, respectively (Yarbough et al.
(1991 ) supra). Diagnostic tests designed to detect IgG, IgA and IgM class antibodies to HEV
have been developed based on these antigenic regions. Additional HEV
recombinant proteins have been generated that encompass full-length ORF 3 (Dawson et al. (1992) Journal of Virology Methods, 38: 175-186) or additional amino acid sequences from the ORF
2 protein (Dawson et al. (1993) supra), to potentially enhance the detection of antibodies to HEV.
Comparative studies indicate that the original recombinant proteins and synthetic peptides (B4-2, B3-2, M3-2, M4-2) were as effective as the larger recombinant proteins in detecting antibodies to HEV in known cases of acute HEV infection. A licensed test to detect antibodies to HEV is manufactured by Abbott Laboratories and consists of the full length Burmese strain ORF 3 protein and the carboxyl 327 amino acids of the Burmese strain ORF 2 protein.

After initial serological studies demonstrating the utility of B 3-2, B 4-2, M
3-2 and M 4-2, it was established that six additional amino acids reside at the carboxyl terminus of ORF 2 of both the Burmese and Mexican strains of HEV which do not form part of the M 3-2 and B 3-2 antigenic peptides. Since the carboxyl ends of ORF 2 and ORF 3 have been shown to be of value for the Burmese and Mexican strains of HEV, synthetic peptides corresponding to the these regions of the genome were generated for the US-1 strain of HEV. The synthetic peptides corresponding to the 48 amino acids at the carboxyl end of the ORF 2 were generated for the Burmese and Mexican strains of HEV (SEQ ID NOS:172 and 170, respectively), and are referred to as B 3-2e and M 3-2e (where "e" designates extended amino acid sequence).
In addition;
synthetic peptides representing the 33 amino acids at the carboxyl end of the were generated for the Burmese and Mexican strains of HEV (SEQ ID NOS:171 and 169, respectively), and are referred to as B4-2 and M4-2. The synthetic peptide based on the epitope from within ORF 2 for the HEV US-1 strain (SEQ ID N0:174) is referred to as the US 3-2e.
The synthetic peptide based on the epitope at the carboxyl end of the HEV US-1 ORF 3 (SEQ ID
N0:173) is referred to as US 4-2. Each of these peptides derived from the Mexican, Burmese and US strains of HEV were synthesized, coated on a solid phase and utilized in ELISA tests to determine the relative usefulness of these synthetic peptides.
As noted in Table 42, the amino acid identity between HEV US-1 and the Burmese, Mexican, and Pakistani strains of HEV range from about 87.5% to about 91.7%
for the amino acids comprising the 3-2e epitopes within ORF 2, and from about 63.6 to about 72.7% for the amino acids comprising the 4-2 epitopes within ORF 3. Without wishing to be bound by theory, given the degree of variability in the regions encoding for epitopes, it is likely that there may be strain specific antibody responses to theses viruses.

TABLE 42 - (Similarity/Identify) _- 3-2e 4-2 Peptide Peptide Pak Mex US-1 ~ Pak Mex US-1 ur 100/97.9 91.7/91.7 93.7/91.7 100/ 100 72.7/72.7 72.7/72.7 ak 91.7/91.7 93.7/91.7 72.7/72.7 72.7/72.7 ex I I 89.6/87.5 ~ 63.6/63.6 ~ II

B. Use of ELISA's in dia nosin acute HEV infection ~''? It has been reported that most cases of acute HEV infection in man are accompanied by IgM class antibodies v~rhich bind to one or more HEV recombinant proteins or synthetic peptides.
If a person does not have IgM class antibodies to HEV, the basis for diagnosis of acute HEV
infection cannot be made on serology alone but may require, RT-PCR and/or other tests to verify HEV as the etiologic agent.
C. Generation of Synthetic Peptides Peptides were prepared on a Rainin Symphony Multiple Peptide Synthesizer using standard FMOC solid phase peptide synthesis on a 0.025 pmole scale with (HBTU) coupling chemistry by in situ activation provided by N-methyl-morpholine, with 45 minute coupling times at each residue, and double coupling at predetermined residues. Standard cleavage of the resin provided the unprotected peptide, followed by ether precipitation and washing.
The peptides synthesized are shown in Table 43.

PeptideSequence ~ SEQ ID NO:

B 3-2eLDYPARAHTFDDFCPECRPLGLQGCAFQSTVAELQRLKMKVGKTRELSEQ ID N0:172 B 4-2 NPPDHSAPLGVTRPSAPPLPHVVDLPQLGPRR SEQ ID N0:171 M 3-2eFDYPGRAHTFDDFCPECRALGLQGCAFQSTVAELQRLKVKVGKTRELSEQ ID N0:170 M 4-2 NQPGHLAPLGEIRPSAPPLPPVADLPQPGLRR SEQ ID N0:169 US VDYPARAHTFDDFCPECRTLGVQGCAFQSTIAEVQRLKMKVGKTREVSEQ ID N0:174 3-2e US SRPAPSVPLGVTSPSAPPLPPVVDLPQLGLRC SEQ ID N0:173 D. Analysis of Synthesized Peptides The synthesized peptides were analyzed for their amino acid composition as follows.
The crude peptides from the small scale syntheses (0.025 .mole) were analyzed for their quality by C 18 reverse phase high pressure liquid chromatography using an acetonitrile/water gradient with 0.1 % (v/v) 2 trifluoracetic acid (TFA) in each solvent. From the analytical chromatogram, the major peak from each synthesis was collected and the effluent analyzed by mass spectrometry (electrospray and/or laser desorption mass spectrometry.
Purification of the peptides (small and/or large scale) was achieved using C 18 reverse phase HPLC
with an acetonitrile/water gradient with 0.1 % TFA in each solvent. The major peak was collected, and lyophilized until use.
E. ELISA Test The utility of the HEV US-1 epitopes was determined by coating 1/4 inch polystyrene beads with each peptide. Specifically, the peptides were solubilized in water or water plus glacial acetic acid and diluted to contain 10 p.g/mL in phosphate buffer (pH
7.4). A total of 60 polystyrene beads were added to a scintillation vial along with 14 mL of peptide solution ( 10 ~,g/mL) and incubated at 56°C for two hours phosphate buffered saline (PBS). After incubation, the liquid was aspirated and replaced with a buffer containing 0.1% Triton-X100~. The beads were exposed to this solution for 60 minutes, the fluid aspirated and the beads washed twice with SUBSTITUTE SHEET (RULE 26) PBS buffer. The beads then were incubated with S% bovine serum albumin solution for 60 minutes at 40°C. After incubation, the fluid was aspirated and the beads rinsed with PBS. The resulting beads were soaked in PBS containing 5% sucrose for 30 minutes. The fluids then were aspirated and the beads air-dried.
In one study, one-quarter inch polystyrene beads were coated with various concentrations of the synthetic peptide (approximately 50 beads per lot) and evaluated in an ELISA test (described below) using serum from an anti-HEV seronegative human as a negative control and convalescent sera from an HEV-infected person as a positive control. The bead coating conditions providing the highest ratio of positive control signal to negative control signal were selected for scaling up the bead coating process. Two 1,000 bead lots were produced for both HEV US-1 ORF 2 and ORF 3 epitopes and then used as follows.
A sample of sera or plasma was diluted in specimen diluent and mixed with antigen-coated solid phase under conditions that permit an antibody in the sample to bind to the immobilized antigen. After washing, the resulting beads were mixed with horseradish peroxidase (HRPO)-labeled anti-human antibodies that bind to either tamarin or human antibodies bound to the solid phase. Specimens which produced signals above a cutoff value were considered reactive.
More specifically, the preferred ELISA format requires contacting the antigen-coated solid phase with serum pre-diluted with specimen diluent (buffered solution containing animal sera and non-ionic detergents). Specifically, 10 pL of serum was diluted in 1 SO pL of specimen diluent and vortexed. Then 10 pl of this pre-diluted specimen was added to each well of an ELISA plate, followed by the addition of 200 pL of specimen diluent and an antigen coated polystyrene beads. The ELISA plate then was incubated in a Dynamic Incubator (Abbott Laboratories) with constant agitation at room temperature for 1 hour. After the incubation, the fluids were aspirated, and the wells washed three times in distilled water (5 mL per wash). Next, 200 pL of HRPO-labeled goat anti-human immunoglobulin diluted in a conjugate diluent (buffered solution containing animal sera and non-ionic detergents) was added to each well and the ELISA plate incubated for 1 hour, as indicated above. The wells then were washed three times in distilled water, the beads containing antigen and bound immunoglobulins removed from each well, and then placed in a test tube with 300 uL of a solution of 0.1 M
citrate buffer (pH
5.5), 0.3% o-phenylenediamine-2 HCl and 0.02% hydrogen peroxide. After 30 minutes at room temperature, the reaction was terminated by the addition of 1 N sulphuric acid. The resulting absorbance at 492 nm was the recorded. The intensity of the color produced was directly proportional to the amount of antibody present in the test sample. For each group of specimens, a preliminary cutoff value was set to separate specimens which presumably contained antibodies to the HEV epitope from those specimens which did not.
Panel 1: Testing of pre-screened panels In order to demonstrate the utility of epitopes derived from the HEV US-1 strain, a panel of specimens was tested by an ELISA based on the HEV US-1 amino acid sequences (Table 44 These samples had been pre-screened for antibodies to HEV, using a combination of existing peptides and a licensed anti-HEV (Abbott Laboratories) as described above and in published reports (Dawson et al. (1993) supra; Paul et al. (1993) supra).
The first l0 members of the panel consisted of specimens obtained from US
volunteer blood donors whose sera was negative for antibodies to HEV following analysis using a combination of peptides and recombinant proteins derived from Burmese and Mexican strains of HEV. All the specimens were non-reactive with ELISA's derived from HEV US-1.
Five additional specimens were obtained from individuals suffering from acute hepatitis, and who were diagnosed with acute HEV infection because their sera was reactive for both IgG and IgM
class antibodies to HEV recombinant antigens and synthetic peptides based on the Burmese and Mexican strains of HEV. Three of the five samples were from Egypt, one from India and one from Norway (a traveler). HEV RNA was detected by RT-PCR in all five of these individuals.
These five members were tested for antibodies to the HEV US-1 isolate and both IgG and IgM

class antibodies were detected in each of the cases (Table 44). Thus, these data support the use of synthetic peptides from the US-1 strain of HEV as having utility in diagnosing exposure to HEV and for diagnosing acute HEV infections.

Test Licensed US
anti Isolate HEV ~
~
~
~
~~

Specimens IgG IgM

Tested IgG IgM 4-2 3-2e 4-2 3-2e Neg. Control0.061 0.084 0.031 0.041 0.071 0.109 Pos. Control0.567 1.051 1.606 1.619 1.376 1.798 US
Volunteer Donors TG 827 - _ _ _ _ _ EG 549 _ - _- _ _ _ EC 760 - - _ _ _ -RF 762 _ - _ _ _ _ RF 762 - - _ _ _ _ RG 730 - _ _ _ _ _ NH 770 _ _ _ _ _ _ AS 705 - _ _ _ - _ BW494 - _ _ _ _ _ CD 648 - _ _ _ _ _ Egypt 7 + + + + + +

9 + + + + + +

12 + + + _ + +

India + + + + + +

Norway M1 + + + + + +

Panel 2: Detection of antibodies to HEV in biological source of HEV US-I
isolate Serial bleeds were obtained form the patient described in Example I, whose serum served as the biological source for the HEV US-1 strain. Based on serological data obtained for the Burmese and Mexican strains of HEV, this patient would have been misdiagnosed as HEV
negative because of the lack of detectable IgM class antibodies to HEV.
However, both IgM
class (Table 45) and IgG class (Table 46) antibodies to the HEV US-I strain were detected on all four bleed dates (Tables 45 and 46. Had this patient's sera been analyzed for the presence of IgG
and IgM class antibodies to the HEV US 3-2e and US 4-2 peptides, a positive diagnosis of acute HEV infection would have been made. This diagnosis is further supported by the observation that the individual had acute hepatitis and most importantly, had detectable HEV US-1 strain RNA in serum samples. These data indicate that synthetic peptides derived form the HEV US-1 strain may be useful in more accurately diagnosing acute infection due to HEV.

IgM: ORF IgM:
Specimens 3 synthetic ORF
peptide 2 synthetic 4-2 peptide ISOLATES 3-2e ISOLATES

Tested Burmese Mexican US-1 Burmese MexicanUS-1 Negative Control0.059 0.081 0.0310.142 0.065 0.109 Positive Control0.854 0.985 1.3631.309 0.579 1.798 8 days post - - + _ _ +
admission 9 days post - - + _ _ +
admission days post - - + - _ +
admission 37 days post - - + _ _ +
admission IgG: IgG: ORF
Specimens ORF 2 synthetic 3 synthetic peptide peptide 3-2e ISOLATES

Tested BurmeseMexican US-1 Burmese Mexican US-I

Negative Control 0.039 0.055 0.031 0.034 0.057 0.041 Positive Control 1.296 0.666 0.941 1.322 0.893 1.041 USP-~ - - + - _ +

8 days post admission- - + _ - +

9 days post admission- - + _ - +

days post admission- - + _ - +

37 days post admission- - + - - +

Panel 3 - Other cases of potential acute HEV infection A panel of sera from 50 patients diagnosed with acute hepatitis who were negative for IgM class antibodies to the Burmese and Mexican strains was assembled. Ten of 50 sera samples were positive for antibodies to the US strain of HEV (Tables 47 and 48). RT-PCR was performed on these samples, but none of the 10 were positive for HEV RNA.
Thus, as demonstrated in this example, when patient sera is analyzed for the presence of antibodies to HEV US-1, occult viral hepatitis may be diagnosed as acute HEV infection.

IgM: ORF IgM: ORF
Specimens 3 synthetic 2 synthetic peptide peptide 4-2 3-2e ISOLATES ISOLATES

Tested Burmese Mexican US-1 Burmese Mexican US-1 Negative Control0.059 0.081 0.031 0.142 0.065 0.109 Positive Control0.854 0.985 1.363 1.309 0.579 1.798 US - _ - _ _ +

Acute non - - _ _ _ +
A-E

SH 755 - - - _ _ _ +

DT 314 - - _ _ - +

EH 673 _ - _ - - _ +

SG560 - - _ _ _ +

SR681 - - _ _ _ -N11C10 - - + _ - +

35 - - + - _ +

52 - - _ _ _ +

161 - - ~ _ _ +

IgG: ORF IgG: ORF
Specimens 3 synthetic 2 synthetic peptide peptide 4-2 3-2e ISOLATES ISOLATES

Tested Burmese Mexican US-1 Burmese Mexican US-1 Negative Control0.039 0.055 0.031 0.034 0.057 0.041 Positive Control1.296 0.666 0.941 1.322 0.893 1.041 US - - - _ _ _ Acute non - - - _ - -A-E

SH 755 - - _ _ _ -DT 314 - _ _ _ - -EH 673 - - _ - - _ SG560 - - _ - - -SR681 _ - - _ - +

N11C10 - - _ _ - -35 - - - - - +

52 - - _ _ _ -161 - - _ _ - -Example 9 - Animal Transmission Studies Cynomolgus macaques (Macaca fascicularis) were obtained through the Southwest Foundation for Biomedical Research (SFBR) in San Antonio, Texas. The animals were maintained and monitored in accordance with guidelines established by SFBR to ensure humane care and the ethical use of primates. Sera were obtained twice weekly for at least four weeks prior to inoculation in order to establish the baseline levels for serum ALT.
Cut-off (CO) values were determined based on the mean of the baseline plus 3.75 times the standard deviation. Two macaques were inoculated intravenously with 0.4-0.625 mL of HEV positive USP-1 serum and one macaque was inoculated with 2.0 mL of HEV positive USP-2 serum. Serum and fecal samples were collected twice weekly for up to 16 weeks post-inoculation (PI).
Sera were tested for changes in ALT and values greater than the CO were considered positive and suggestive of liver damage. Sera samples were tested for antibodies to HEV as described hereinabove in Example 8 (Table 49, Figure 7). Sera and fecal samples were tested for HEV RNA
by RT-PCR.
25-100 ~L of macaque sera was extracted using the QIAamp Viral RNA Kit (Qiagen). 10%
fecal suspension were extracted as described in Example 1. RT PCR was performed as described below in Example 12 (Figure 7).
Although intravenous inoculation of 0.4-0.625 mL of USP-1 sera into two cynomolgus macaques failed to produce infection (data not shown), inoculation of 2.0 mL
of sera from patient US-2 resulted in viremia and elevations of liver enzyme levels in the serum (Figure 7).
HEV RNA was first detected in fecal material on day 15 PI and remained positive through 64 days PI. Serum specimens collected between days 28-56 PI were HEV RNA
positive. Elevated ALT values were noted on days 15, 44-58, 72 and 93 PI, with the peak ALT value (116 IU/L) on day S 1 PI.
Six ELSIAs based on the Burmese, Mexican and US sequences for the 4-2 and 302e peptides were utilized to assess antibody response. Measurable response was found only to the US 3-2e peptide assay (Table 49) with no noted crossreactivity to the Burmese or Mexican peptides. IgM class antibody directed against HEV was detectable between 28 and 58 days PI.
This was followed by a strong anti-HEV-IgG response at day 44 PI.

Date DPI ALT AST GGT IgG S%IV

06/04/97-82 35 37 102 1.4 09/09/9715 58 42 108 0.8 09/22/9728 39 33 86 1.2 10/01/9737 48 58 90 1.1 10/08/9744 58 38 94 6.2 10/15/9751 116 b2 89 11.9 10/20/9756 87 38 83 33.6 10/22/9758 76 43 85 29.9 10/28/9764 45 42 88 17,2 11 /05/9772 54 47 88 13.3 11/12/9779 50 38 93 12.4 11/17/9784 46 31 91 10.4 11/26/9793 67 104 109 7.2 12/10/97107 ~ 36 ~ - 29 103 2.1 ~

Example 10: Recombinant Protein ELISAs A. Recombinant Constructs E. coli derived recombinant proteins encoded by HEV-US sequence from the ORF 2 and ORF 3 regions of the HEV-US genome were expressed as fusion proteins with CMP-KDO
synthetase (CKS), designated as pJOorf3-29 (SEQ ID N0:191 ); cksorf2m-2 (SEQ
ID N0:192);
and CKSORF32M-3 (SEQ ID N0:193), or as non-fusion proteins, designated as plorf3-12 ( SEQ
ID N0:194); plorf2-2.6 (SEQ ID N0:195); and PLORF-32M-14-5 (SEQ ID N0:196).
The cloning vector pJ0201, as described in U.S. Patent No. 5,124,255, was used in the construction of the recombinant fusion proteins. This vector was digested with the restriction endonucleases Eco RI and Bam HI to allow cloning of HEV-US sequences in frame with CKS. The lambda pL
expression vector pKRR826 was utilized in the construction of recombinant non-fusion proteins.
This vector was digested with the restriction endonucleases Eco RI and Bam HI
to allow for cloning of HEV-US sequences immediately down stream of the ribosome binding site. Since the vector system contains strong lambda promoter, induction of heterologous protein synthesis is accomplished by shift in the temperature from 30°C to 42°C which inactivates the temperature sensitive repressor protein. The constructs were cloned and transformed into E. coli K12 strain HS36 cells for the expression of these HEV proteins.
HEV-US sequences were amplified from nucleic acids extracted from HEV US-2 human serum or macaque 13906 fecal material and reverse transcribed as described above in Example 5.
The ORF 2 sequence, encompassing the carboxyl half of ORF 2 (i. e., encoding amino acid residue numbers 334-660 of SEQ ID N0:167), was generated using a sense primer, SEQ ID
N0:208, which contained an Eco RI restriction site as well as an ATG start codon and an antisense primer, SEQ ID N0:198, which contained a unique peptide sequence termed FLAG
(Eastman Kodak), two consecutive TAA termination codons, and a Bam HI
restriction site. A 50 pl PCR reaction was set up using LA TAQ (Takara) reagents as recommended by the manufacturer. Cycling conditions involved 40 cycles of 94°C for 20 seconds, 55°C for 30 seconds, 72°C for 2 minute. Amplifications were preceded by 1 minute at 94°C and followed by minutes at 72°C. Products were digested with Eco RI and Bam HI and ligated into the desired vector. The nucleotide sequence of the CKS fusion clone, between the restriction sites, is set forth in SEQ ID N0:192, the translation of which is set forth in SEQ ID
N0:199. The nucleotide sequence of the non-fusion clone, between restriction sites, is set forth in SEQ ID N0:195, the translation of which is set forth in SEQ ID N0:200. The ORF 3 sequences, encompassing the entire ORF 3 (amino acids 1-122), was generated using a sense primer, SEQ ID
N0:201, which contained an Eco RI restriction site as well as an ATG start codon and an antisense primer, SEQ
ID N0:202, which contained a unique peptide sequence termed FLAG, two consecutive TAA
termination codons, and a Bam HI restriction site. A 50 ~L PCR reaction was set up using Qiagen reagents as described in Example S. Cycling conditions comprised 35 cycles of 94°C for 30 seconds, 55°C for 30 seconds, 72°C for 1 minute.
Amplifications were preceded by incubation for 1 minute at 94°C, followed by 10 minutes at 72°C.
The resulting products were digested with Eco RI and Bam HI and ligated into the desired vector. The nucleotide sequence of the CKS fusion clone, between the restriction sites, is set forth in SEQ ID
N0:191, the translation of which is set forth in SEQ ID N0:203. The nucleotide sequence of the clone representing the non-fusion construct, between the restriction sites, is set forth in SEQ ID
N0:195, the translation of which is set forth in SEQ ID N0:204.
Additionally, a chimeric construct encompassing the full length ORF 3 (amino acids 1-123) and the carboxyl half of ORF 2 (amino acids 334-660) was generated.
Approximately 100 ng of the plasmids containing SEQ ID N0:191 and SEQ ID N0:192 were utilized as template in 100 ~L PCR reactions. PCR buffers and enzymes were from the LA TAQ kit (Takara), and used in accordance with the manufacturer's instructions. ORF 3 was amplified with primers set forth in SEQ ID NOS:201 and 205. The antisense primer of SEQ ID N0:205 eliminates the FLAG
sequences and stop codons from the carboxyl end of SEQ ID N0:191 and contains the sequence identical to SEQ ID N0:192 which will eliminate the ATG start codon. ORF 2 was amplified with primers of SEQ ID NOS:208 and 198. Cycling conditions were as described above using LA TAQ. The resulting products were fractionated on a 1.2% agarose gel and excised. DNA
was isolated from the gel slices using GeneClean II as described by the manufacturer (Bio 1 O 1 ).

Products were eluted off the glass beads into 15 pL HBO. Approximately equal molar ratios of each product (10 ~L of ORF 3 product and 1 ~L of ORF 2 product) were mixed in a 25 pL end fill reaction using i x PCR buffer, 0.5 pl dNTPs, and 0.25 pL LA TAQ (Takara).
This reaction was cycled as follows: 94°C for 1 minute, 10 cycles of 94°C for 20 seconds, 55°C for 30 seconds, and 72°C for 1.5 minutes, followed by 72°C for 10 minutes. S p,L of this reaction was placed into a 100 p.L amplification reaction utilizing LA TAQ kit (Takara) and primers of SEQ
ID NOS:201 and 198. Cycling conditions were 94°C for 1 minute followed by 35 cycles of 90°C
for 20 seconds, 55°C for 30 seconds, and 72°C for 1.5 minutes.
This was followed by 10 minutes at 72°C and a 4°C soak. Products of the appropriate size were digested with restriction enzymes Eco RI and Bam HI. This product was ligated into pJ0201 and clones with the appropriate sequence identified (SEQ ID N0:193, the translation of which is set forth in SEQ ID N0:206).
The resulting product was ligated into pKRR826 and clones with the appropriate sequence (SEQ
ID N0:196, the translation of which is set forth in SEQ ID N0:207) identified.
B. Protein expression and purification The CKS constructs were expressed in two S00 mL cultures (4 hour induction), as described in U. S. Patent No. 5,312,737. P~ constructs were expressed as described above.
Frozen cell pellets of the induced Ecoli cultures were used as the starting material for the purification of protein. Cells were lysed in buffer containing lysozyme, DNase and proteinase inhibitors. Soluble protein was separated from insoluble (inclusion body) protein by centrifugation at 11,000 x g. The solubility of the recombinant protein was estimated via sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and Western blotting using a FLAGm M2 antibody.
Soluble recombinant protein was purified by affinity chromatography using FLAG~ M2 antibody affinity gel after exchange into suitable buffer (Surowy et al.
(1997) 3ournal of General Virology, 78:18 1-1859). If necessary, additional purification was performed via Sephacryl~ 5-200 gel filtration chromatography, in which the sample and chromatography buffers contained 10 mM ~3-mercaptoethanol. Purified protein was quantitated by measurement of absorbance at 280 nm. An assumed extinction coefficient of 1 was used to convert absorbance to mg of protein.
Protein purity was determined by scanning densitometry (Molecular Dynamics) of protein fractioned by SDS PAGE, using standards of pre-determined purity.
C. ELISA
In order to determine potential utility of the recombinant HEV US constructs, solid phase ELISA's were developed and evaluated. All recombinant HEV US proteins were coated onto solid phase as described below. Briefly, 1/4" polystyrene beads were coated with varying amounts of (PJOORF3-29) which ranged in concentration from 0.5 to 10 pg/mL
diluted in 100 mM sodium phosphate buffer, pH 7.6. Sixty beads per concentration condition were coated in approximately 14 mL of buffer and rotated end-over-end at 40° C for 2 hours. The coating solution was aspirated and the remainder of the coating procedure was performed as described above in Example 8, section E, paragraph 1.
An ELISA was developed using the pJOorf3-29 coated beads. Briefly, sera or plasma was diluted 1:16 in Specimen Diluent (SpD) as described above. A 10 p.L
aliquot of this pre-dilution then was added into the well of a reaction tray, followed by the addition of 200 p,L of SpD. One coated bead was added per well and incubated for 1 hour at 37°C in dynamic mode using a Dynamic Incubator (Abbott Laboratories). After incubation, the fluid was aspirated and each bead washed 3 times with deionized water (5 mL per wash). The beads then were incubated with 200 p,L HRPO-labeled goat anti-human IgG or IgM conjugate, diluted in conjugate diluent {described above) and incubated for 30 minutes at 37°C. The conjugate then was aspirated and the beads washed as above. Color development and absorbance readings were performed as described in Example 8, section E.
To validate the immunoreactivity of this construct, serial bleed specimens from Macaque #13903 experimentally infected with HEV US-2 (described in Example 9) were tested for IgM
and IgG antibody to pJOorf3-29. As shown in Figure 1, IgM antibody was detected at day 51 post-infection (PI) and continued to be elevated through day 72 and corresponded to the peak elevations in ALT values. IgG antibody to pJOorf3-29 was first detected on day Sb PI and remained positive through day 107 (Table 50).
A second construct, plorf3-12, representing HEV US ORF 3 but lacking the CKS
fusion partner was also evaluated in an ELISA format identical to that described above. IgG antibody to plorf3-l2was evaluated on several serial bleeds from the same experimentally infected macaque. IgG antibody to plorf3-l2was detected on day 58 PI and remained positive through day 107 (Table 50).

pJOorf3-29 plorf3-12 Sample Mean OD S/N Mean OD S/N

SpD 0.01 "pre-bleed"0.02 0.01 Post-inoculation bleeds - Days Post-inoculation (DPI) DPI

44 0.02 0.96 0.02 l.0 51 0.05 2.35 0.03 2.2 56 0.24 10.35 0.05 3.4 58 0.44 19 0.16 11.

63 1.14 49.57 0.32 22.

65 NT 0.53 37.

70 NT 1.19 85.

72 2.22 96.52 0.92 65.

98 0.89 38.87 0.39 27.

107 0.49 21.43 0.27 19.

NT:
not tested Due to the high percent homology between Swine HEV and the US-2 isolate, the pJOorf3-29 ELISA also was used to measure the prevalence of both immunoreactive IgG and IgM in sera isolated from U.S. swine herds (Table 51). The assay was performed as described above with the exception of substituting HRPO-conjugated labeled anti-swine immunoglobulin (either IgG or IgM) for the anti-human conjugate.

Prevalence of Antibody to HEV
orf3 in U. S. Swine (pJOorf3-29) No. IgM

IgG No. IgG IgM Only Only Total Swine Reactive Confirmed by Reactive Confirmed Exposure Source No./TotalBlocking or No./Total by Blot Confirmed Blot State (%) (%) (%) (%) Only New Jersey 9114 9 0/ 14 64%

(64) ( 100) Texas 2S/SO 20 0/SO 40%

{S0) (80) Iowa 7/64 1 0/64 2%

(11) (14) Oregon 7/36 S 1/36 1/1 14%

(19) (71) (3) {100) Total 48/164 3S 1 /164 1 /1 36/164 (29} (73) (0.6) (100) (22%) NOTE: A of ~4 gG.
total pigs (all Texas herd) had IgM
in addition to I

In order to confirm reactive specimens, a blocking assay was developed.
Briefly, a 10 pL
aliquot of the 1:16 specimen pre-dilution was added to duplicate wells of a reaction tray; one well to be used for the standard assay and one well to be used for the blocking assay. The ELISA for the standard assay was performed as described above with the exception that there was a 30 minute room temperature pre-incubation step prior to addition of the pJOorf3-29 antigen coated bead. For the blocking assay, pJOorf3-29 was added to the SpD
(blocking reagent) at a 10-fold molar excess to that on the solid phase. 200 p,L of blocking reagent was added per reaction and a 30 minutes room temperature pre-incubation was performed prior to addition of the pJOorf3-29 antigen coated bead. The rest of the assay was performed as described above for the swine assay, except that the HRPO-conjugated anti-swine conjugate (IgG) was used in place of the anti-human conjugate.

The % blocking was determined using the equation:
UA492 nm s~dard assay - A,9z nm blocking assay)/A49z "", standard assay] x 100 Specimens that showed blocking rates of 50% or greater were considered to be reactive for IgG
antibody to HEV pJOorf3-29. Representative IgG positive and IgG negative swine samples and their blocking results are shown in Table 52.

Table 52 - Blocking Assay With pJOorf3-29 and PL-12 at 10-fold molar excess S~tau~ ~ loch y ~I;xTOs~~ B~;~UC'ICIN
,essa at .. ,~"'.,~

. 3 ..#-t~fc~lCT:~: ~. : , REST
..
::
:;.
;
~
, SAMPLE OD MEANOD MEAN %

OD OD BLOCKING

0.02 0.02 NC 0.020.020.03 0.02 1.09 0.56 PC 1.011.050.48 0.52 50.4% +

pregon~S~rrine:~anel :Positives 1 NJS 0.65 0.15 76.5% +

2 N112 1.78 0.46 74.0% +

3 NJ21 0.48 0.16 66.7% +

4 NJ23 0.52 0.09 81.9% +

5 T$ 2 0.81 59.5% +

6 T9 0.52 0.18 64.3% +

7 T32 2 0.9 54.9% +

8 T33 0.3 0.13 57.8% +

9 T48 0.53 0.14 73.7% +

10T49 0.33 0.09 73.3% +

Oregon egatia~c~
a~vin~Panel :.
N

IIT43 0.08 0.07 13.3%

12T46 0.12 0.08 29.1 % -131-23 0.12 0.08 32.2%

14I-24 0.07 0.06 13.2%

15I-27 0.1 0.08 12.6%

16I-28 0.15 O.12 20.4%

17i-33 0.15 0.12 19,9% -181-39 0.23 0.14 37.4%

19I-61 0.19 0.14 25.9%

20O-4 0.15 0.12 22.7%

In addition to the blocking assay, western blots were run on a subset of swine specimens.
Briefly, 50 pg of HEV pJOorf3-29 and SO p.g of "CKS only" proteins were fractionated by SDS-PAGE and the fractionated proteins transferred to nitrocellulose. 3mm strips of the nitrocellulose were cut and incubated overnight at room temperature on an orbital rotator with primary antibody at a 1:100 dilution in protein based buffer containing 10% E. coli lysate. On the following day, strips were washed three times with 0.3% Tween/TBS (TBST), followed by the addition of HRPO-conjugated anti-swine IgG conjugate diluted to 0.5 pg/mL in TBST. Strips were incubated with rotation for 4 hours at room temperature. Blots then were washed three times in TBST, followed by 2 washes in TBS. Blots were developed using 4-chloro-1-naphthol as a substrate. The reaction was stopped by the addition of water and band intensities recorded.
Specimens were determined to have specific reactivity to HEV if they showed a band at the correct molecular weight for pJOorf3-29 (approx. 40 kD) and had no reactivity in the region where "CKS only" bands (approx. 29 kD). Results for 20 swine sera run on the pJOorf3-29 western blot are shown in Table 53. No swine sera showed non-specific reactivity with the "CKS-only" band.

BAND INTENSITY

Swine 1D NumberpJOorf3-29CKS only NJ4 +

NJ7 + _ NJ 14 +++ _ NJ 18 +

NJ25 ++++ _ T6 ++++ _ T10 ++++ _ T14 - _ T15 + _ T18 ++ _ T28 +++ _ T29 - _ T30 +

T34 - _ T36 ++++ _ T37 - _ T43 - _ T44 ++++ _ T45 ++++ _ T46 - _ These data suggest that HEV US recombinant proteins are useful in diagnosing exposure to HEV.

Exam~nle ll - Consensus Primers Consensus oligonucleotide primers for HEV~ORF 1 ORF 2 and ORF 3 were designed based on conserved regions between the full length sequences of isolates from Asia, Mexico, and the US (Figure 9). The ORF 1 primers are positioned within the methyltransferase region at nucleotides 56-79 and 473-451 of the Burmese isolate (GenBank accession number M73218), and amplify a product 418 nucleotides in length. The ORF 1 primers include:
HEVConsORF 1-sl; CTGGCATYACTACTGCYATTGAGC (SEQ ID N0:147); and HEVConsORF 1-al; CCATCRARRCAGTAAGTGCGGTC (SEQ ID N0:148).
The ORF 2 primers, at positions 6298-6321 and 6494-6470 of the Burmese isolate, produce a product 197 nucleotides in length. The ORF 2 primers include:
HEVConsORF 2-sl; GACAGAATTRATTTCGTCGGCTGG (SEQ ID NO:150); and HEVConsORF 2-al; CTTGTTCRTGYTGGTTRTCATAATC (SEQ ID N0:126).
For a second round of amplification, internal primers can be used to produce products 287 and 145 nucleotides in length for ORF 1 and ORF 2, respectively. The ORF 1 primers include:
HEVConsORF 1-s2; CTGCCYTKGCGAATGCTGTGG (SEQ ID N0:177); and HEVConsORF 1-a2; GGCAGWRTACCARCGCTGAACATC (SEQ ID N0:178).
The ORF 2 primers include:
HEVConsORF 2-s2; GTYGTCTCRGCCAATGGCGAGC (SEQ ID N0:152); and HEVConsORF 2-a2; GTTCRTGYTGGTTRTCATAATCCTG (SEQ ID N0:128).
PCR reactions contained 2 mM MgCl2 and 0.5 ~,M of each oligonucleotide primer as per the manufacturer's instructions (Perkin-Elmer) and amplified using Touch-down PCR as described in Example 5. Amplified products were separated on a 1.5% agarose gel and analyzed for the presence of PCR products of the appropriate size. The primers were used to detect the presence of virus in serum and feces containing HEV US-2 as described above in Example 8 and Figure 7. In addition, these primers were found to be reactive with a number of different variants of HEV that included Burmese-like strains 6A, 7A, 9A and 12 A as well as two distinct isolates from Greece (see Example I 3 below) as well as a unique isolate from Italy and the two isolates from the US (see Example 13 below). In addition, these primers have been used to identify an isolate from a patient with a clinical diagnosis of acute sporadic hepatitis from the Liaoning province of China (S 15). The results are presented in Table 54 below.

Sample ORF 1 -PCR1ORF 1 -PCR O ORF 2 -PCR2 6A peg pos _ Pos pos 7A peg pos peg Pos 9A peg peg peg Pos 12A pos pos peg Neg Gl pos pos pos Pos G2 pos pos pos Pos Itl pos pos pos Pos S15 nd pos nd Pos US-2 pos pos pos Pos Example 12 - Detection ofHEV RNA in Primary Human Fetal Kidney Cells Frozen cell pellets containing 1 Ox 1 O6 cells were thawed and resuspended in 1.0 mL
Dulbecco's phosphate buffered saline. RNA was extracted from 20 pL (2x105 cells) of the cell pellet using the Ultraspec Isolation System as described in Example 1. cDNA
synthesis was performed on the above extracted nucleic acid (RNA) and primed with random hexamers. PCR
then was performed on the above cDNA using degenerate primers from the ORF-1 and ORF-2 regions of the viral genome at a final concentration of O.S~M as described in Example 11.

To monitor the performance of the above assay, a positive control utilizing primary human kidney cells and HEV US-2 positive serum was included in the experimental design.
Two positive control sets were prepared by spiking 2x105 HEV negative primary human kidney cells with 2.5 pL and 25 pL of a documented HEV US-2 positive serum specimen.
The positive control serum also was tested without the addition of the human kidney cells.
Nineteen primary human kidney cell pellet lots were tested using the above assay method utilizing the 2 degenerate primer sets from ORF 1 and ORF 2. The results are summarized in Table 55 below. None of the cell pellet lots tested gave positive results as seen in the positive controls.

CELL LINES PCR RESULTS

1757 _ 1851 _ 1690 _ 1853 _ 1955 _ 1893 _ 1895 _ 1921 _ -1946 _ 1846 _ cells + 25 +
PL serum cells + 2.5 +
uL serum 25 uL serum +

Example 13: Identification and Extension ofAdditional US type Isolates A. Identification of isolate from Italy referred to as Itl RNA was extracted from 25 to 50 pL of serum using the QIAamp Viral RNA kit (Qiagen) as described by the manufacturer except that 25 to SOpL of serum was diluted to 100pL
with PBS and the final elution was performed with 100 pL of RNase-free water.
RT reactions were random primed. PCR utilized the HEV US-1 primer as described hereinabove in Example S. A 294 by product was generated after amplification with primers SEQ ID
N0:94 and SEQ ID
N0:96. The product was cloned and sequenced as described in Example 3 and is shown in SEQ
ID N0:179.
Extension of the Itl isolate genome was performed as follows. RNA was extracted from 2S to SO p.L of serum as described hereinabove in Example S. RT reactions were random primed. PCR utilized the HEV CONSENSUS primers described above in Example 11 using touchdown PCR, as described hereinabove in Example 3. Primers shown in SEQ ID
NOS:147 and 148 were used to generate a product having the sequence set forth in SEQ
ID N0:180 (reaction z2, 418 bp). Primers as shown in SEQ ID NOS:1 SO and 126 were used to generate a product having the sequence set forth in SEQ ID N0:181 (reaction z3, 197 bp).
In the presence of 1 x PCR Buffer and 20% Q Solution (Qiagen), primers as shown in SEQ ID
NOS:182 and 183 were used to generate a product having a sequence set forth in SEQ ID N0:184 (reaction z4, 234 bp). The 3' end of the genome was isolated by 3' RACE as described above in Example 3 using primers shown in SEQ ID NOS:1S0 and 8S in PCR1, and primers shown in SEQ ID
NOS:1S2 and 8S in PCR2, to produce a product having the sequence shown in SEQ ID
N0:18S (reaction zS, 890 bp). Products were cloned and sequenced as described in Example 3 and consensus sequences generated. These regions are shown in Figure 8 and are set forth in SEQ ID NOS:180, 184 and 186. The amino acid translations of these regions are represented by the amino acid sequences set forth in SEQ ID NOS:187, 188; 189; 190; and 197.
B. Identification of two isolates from Greece referred to as G 1 and G2 Two patients with acute hepatitis who had no history of travel to endemic areas had been analyzed with primers based on the Burmese isolate (Psichogiou M.A., et al., (1995) "Hepatitis E
virus (HEV) infection in a cohort of patients with acute non-A, non-B
hepatitis," Journal of Hepatology, 23, 668-673). Only patient G2 was found to be PCR positive. RNA
was isolated as described hereinabove in Example 12 and PCR performed with the consensus primers described above in Example 11. The ORF 1 and ORF 2 primer sets generated products of the expected size from both patients. The products were cloned and sequenced as described above in Example 3.
The products generated using the ORF 1 and ORF 2 consensus primers from patient G1 are shown in SEQ ID NOS:209 and 211, respectively. The products generated using the ORF 1 and ORF 2 consensus primers from patient G2 are shown in SEQ ID NOS:213 and 215, respectively.
The identification of G 1 as being PCR positive demonstrates the utility of the consensus primers over Burmese base strain specific primers.
Additional sequence from G1 and G2 was also obtained using primers SEQ ID
N0:16, SEQ ID No: l7. and SEQ ID N0:18 as for the generation of SEQ ID N0:19 as described above in Example 3 except that random primed cDNA was used for PCR and amplification involved 10 cycles of 94°C for 20 seconds, 60°C for 30 seconds, and 72°C for I minute, followed by 10 cycles of 94°C for 20 seconds, 55°C for 30 seconds, and 72°C for 1 minute followed by 30 cycles of 94°C for 20 seconds, 50°C for 30 seconds (-0.3°C/cycle), and 72°C for 1 minute. This was followed by an extension cycle of 72°C for 7 minutes. The product generated from patient G I is shown in SEQ ID N0:217. The product generated from patient G2 is shown in SEQ ID
N0:220.
Alignments of the nucleotide sequences of the US, Chinese, Greek, Italian, Mexican and Burmese-like isolates, were performed to determine the relationship of these isolates to each other. The divergence of the Italian isolate is supported by the comparisons of the product from the ORF 1 region of the genome which has a percent nucleic acid identity of 77.6 %, 78.4 %, and 84.6 % with the prototype isolates from Burma (B 1 ), Mexico (M I ) and the US
(US-1 ), respectively (Table 36). The divergence of the Italian isolate also is supported by the comparisons of the product from the ORF 2 region of the genome which had a percent nucleic acid identity of 83.3 %, 79.7 %, and 87.8 % with the prototype isolates from Burma, Mexico and the US, respectively (Table 37). The nucleotide identities between the prototype isolates from Burma, Mexico and the US, range between 75.5 % to 82.4 % over these two regions. Over these same regions, the isolates that comprise the Burmese-like group have much higher identities of .;

91.2% or greater.
Comparisons of the ORF 1 and ORF 2 amplified sequences indicate that the isolates from the two patients from Greece are quite distinct from each other, exhibiting 84.4 %
and 87.2 nucleotide sequence identity over these regions of ORF l and ORF 2, respectively. At the nucleotide level, the percent identities between the Greek, Italian and US
isolates range from 81.9% to 86.8% for the ORF 1 product (Table 36) and 82.4% to 87.8% for the ORF
2 product (Table 37). These values are lower than the lowest percent nucleotide identities between any Burmese-like isolates, which are greater than 91.2% for both ORF 1 and ORF 2.
Comparisons of the amino acid identities derived from the ORF 1 fragment between the US, Italian or Greek isolates and the Burmese or Mexican isolates range from 87.8% to 93.5 % (Table 36). These values are equal to or less than the differences between the Burmese and Mexican isolates (93.5% to 95.1 %) (Table 36), indicating that the isolates from non-endemic regions are distinct from the isolates originating from endemic regions.
The relative evolutionary distances between the viral sequences analyzed are readily apparent upon inspection of the uprooted phylogenetic trees generated from the pairwise distances, where the branch lengths are proportional to the relative genetic relationships between the isolates. The phylogenetic trees based on alignments of either ORF 1 (Fig. 10) or ORF 2 (Fig. 11 ) sequences are quite similar in overall topology. The Burmese-like isolates and the Mexican isolate represent major branches at one end of the tree. The human US isolates form a distinct group distal to the Mexican and Burmese isolates. The swine HEV-like sequence from ORF 2 is closely related to the US human isolates. The three European isolates form three additional distinct branches with the Italian isolate being most closely related to the US isolates.
Equivalents The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein.
Scope of the invention WO 99/19732 PCTlUS98/21941 is thus indicated by the appended claims rather than by the foregoing description, and alI changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

SEQUENCE LISTING
c110> Schlauder, George G
Erker, James C
Desai, Suresh M
Dawson, George J
Mushawar, Isa K
<120> METHODS AND COMPOSITIONS FOR DETECTING HEPATITIS E
VIRUS
<130> 132/18 <140>
<141>
<160> 242 <170> PatentIri Ver. 2.0 c210> 1 <211> 18 <212> DNA
c213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer C375M
<400> 1 ctgaacatcc cggccgac 18 <210> 2 <211> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer A1-350M
c400> 2 agaaagcagc gatggagga 19 <210> 3 <211> 21 c212> DNA
c213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer Sl-34M
<400> 3 gcccaccagt tcattaaggc t 21 <210> 4 <211> 20 <212> DNA
<213> Artificial Sequence <220>
c223> Description of Artificial Sequence: Primer A2-320M
<400> 4 tcattaatgg agcgtgggtg <210> 5 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer S2-55M
<400> 5 cctggcatca ctactgctat 20 <210> 6 <211> 18 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer C375 <400> 6 ctgaacatca cgcccaac 18 <210> 7 <2I1> 19 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer Al-350 <400> 7 aggaagcagc ggtggacca <210> 8 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer S1-34 <400> 8 gcccatcagt ttattaaggc 20 <210> 9 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer A2-320 <400> 9 tcatttattg agcggggatg 20 <210> ZO
<211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer S2-55 <400> 10 cctggcatca ctactgctat 20 <210> 11 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer M1PR6 <400> 11 ccatgttcca caccgtattc cagag 25 <210> 12 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer 54294M
<400> 12 gtgttctacg gggatgctta tgacg 25 <210> 13 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer M1PF6 <400> 13 gactcagtat tctctgctgc cgtgg 25 <210> 14 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer A4556 <400> 14 ggctcaccag aatgcttctt ccaga 25 <210> 15 <211> 342 <212> DNA
<213> Hepatitis E virus <220>
<223> Clone: USP-15 <400> 15 gcccatcagt ttattaaggc tcctggcatt actactgcca ttgagcaggc tgctctggct 60 gcggccaatt ctgccttggc gaatgctgtg gtggttcggc cgtttttatc tcgcgtgcaa 120 accgagattc ttattaattt gatgcaaccc cggcagttgg ttttccgccc tgaggtactt 180 tggaatcacc ctatccagcg ggttatacat aatgaattag aacagtactg ccgggctcgg 240 gctggtcgtt gcttggaggt tggagctcac ccaagatcca ttaatgacaa ccccaacgtt 300 ctgcatcggt gtttccttag accggtcggg cgtgatgttc ag 342 <210> 16 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 16 taggttatac tgccggcgca 20 <210> 17 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer S1-5287 <400> 17 ttctcagccc ttcgcaatcc 20 <210> 18 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer S2-5310 <400> 18 atattcatcc aaccaacccc 20 <210> 19 <211> 251 <212> DNA
<213> Hepatitis E virus <220>
<223> Clone b421 <400> 19 atattcatcc aaccaacccc ttcgccgccg atgtcgtttc acaacccggg gctggaactc 60 gccctcgaca gccgccccgc cccctcggtt ccgcttggcg tgaccagtcc cagcgcccct 120 ccgttgcccc ccgtcgtcga tctaccccag ctggggctgc gccgctaact gccatatcac 180 cagcccctga tacagctcct gtacctgatg ttgactcacg tggtgctatt ttgcgccggc 240 agtataacct a 251 <210> 20 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer US4.2-69S/20 <400> 20 ttccgcttgg cgtgaccagt <210> 21 <211> 21 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer US4.4/1445 <400> 21 gctaactgcc atatcaccag c 21 <210> 22 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer M6417a <400> 22 cccttatcct gctgagcatt 20 <210> 23 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer M6371a <400> 23 ttggctcgcc attggctgag acaa 24 <210> 24 <211> 899 <2i2> DNA
<213> Hepatitis E virus <220>
<223> Clone df-orf2/3 <400> 24 gctaactgcc atatcaccag cccctgatac agctcctgta cctgatgttg actcacgtgg 60 tgctattttg cgccggcagt acaatttgtc tacgtccccg cttacatcat ctgttgcttc 120 tggtactaat ctggttctct atgctgcccc gctgaaccct ctcttgcctc ttcaggatgg 180 caccaacact catattatgg ctactgaggc atctaattac gcccagtatc gggttgttcg 240 ggctacgatt cgttatcgcc cgttggtgcc aaatgctgtt ggtggttatg ctatctctat 300 ttctttctgg cctcaaacta caactacccc tacttctgtt gacatgaatt ctatcacttc 360 tactgatgtc aggatcttgg tccagcccgg tatagcctcc gagttagtca tccctagtga 420 acgccttcac taccgcaacc aaggctggcg ctctgttgag accacgggtg tggccgaaga 480 ggaggctacc tccggtctgg taatgctttg tattcatggc tcccctgtta actcctacac 540 taatacacct tacaccggtg cattggggct tcttgatttt gcattagaac ttgaatttag 600 aaatttgaca cccgggaaca ctaacacccg tgtttcccgg tatactagca cagcccgcca 660 ccggctgcgc cgcggtgctg atgggaccgc tgagctcacc accacagcag ccacacgctt 720 catgaaggat ttgcatttta ctggtacgaa cggcgttggt gaggtgggtc gtggtattgc 780 cctgactctg tttaatcttg ctgatacgct tcttggtggt ttaccgacag aattgatttc 840 gtcggctggg ggtcaactgt tttactcccg ccctgttgtc tcagccaatg gcgagccaa 899 <210> 25 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer USP
3s/20 <400> 25 tggcattact actgccattg 20 <210> 26 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer M902A
<400> 26 atcgatcgga catagacctc 20 <210> 27 <211> 846 <212> DNA
<213> Hepatitis E virus <220>
<223> Clone df-orfl <400> 27 tggcattact actgccattg agcaggctgc tctggctgcg gccaattctg ccttggcgaa 60 tgctgtggtg gttcggccgt ttttatctcg cgtgcaaacc gagattctta ttaatttgat 120 gcaaccccgg cagttggttt tccgccctga ggtactttgg aatcacccta tccagcgggt 180 tatacataat gaattagaac agtactgccg ggctcgggct ggtcgttgct tggaggttgg 240 agctcaccca agatccatta atgacaaccc caacgttctg catcggtgtt tccttagacc 300 ggttggccga gatgttcagc gctggtactc tgcccccacc cgcggccctg cggctaattg 360 ccgccgctcc gcgttgcgtg gtctcccccc cgctgaccgc acttactgct ttgatggatt 420 ctcccgttgt gcttttgctg cagagaccgg tgtggctctt tactctctgc atgacctttg 480 gccagctgat gttgcagagg ctatggcccg ccacgggatr acacgcttgt atgccgcact 540 gcaccttccc cctgaggtgc tgctaccacc cggcacctac cacacaacct cgtatctcct 600 gattcacgac ggcgaccgcg ctgttgtaac ttacgagggc gatactagtg cgggctataa 660 tcatgatgtc tccatacttc gtgcgtggat ccgtactaca aaaatagttg gtgatcatcc 720 gttggtcata gagcgtgtgc gggccattgg atgtcatttt gtgttgctgc tcaccgcagc 780 ccctgagccg tcacccatgc cttatgttcc ttaccctcgt tcaacggagg tctatgtccg 840 atcgat <210> 28 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer 3750s <400> 28 cttccatcag ttggctgagg agc <210> 29 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer 3900a <400> 29 gccatgcggc agtgcacaat gtc 23 <210.> 30 <211> 168 <212> DNA
<213> Hepatitis E virus <220>
<223> Clone HEV 167 <400> 30 cttccatcag ttggctgagg agctgggcca tcgcccggcc cctgtcgccg ccgtcttgcc 60 cccttgccct gagcttgagc agggcctgct ctacatgcca caggagctca ctgtgtccga 120 tagtgtgttg gtttttgagc ttacggacat tgtgcactgc cgcatggc 168 <210> 31 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer 5000s <400> 31 ctcgttcata acctgattgg catgc 25 <210> 32 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer uf-orf2/3 a3 <400> 32 ggactggtca cgccaagcgg aac 23 <210> 33 <211> 424 <212> DNA
<213> Hepatitis E virus <220>
<223> Clone HEV 426 <400> 33 ctcgttcata acctgattgg catgctgcag accatcgccg atggcaaggc ccactttaca 60 gagactatta aacctgtact tgatctcaca aattccatca tacagcgggt ggaatgaata 120 acatgtcttt tgcatcgccc atgggatcac catgcgccct agggctgttc tgttgttgtt 180 cctcatgttt ctgcctatgc tgcccgcgcc accggccggt cagccgtctg gccgtcgccg 240 tgggcggcgc agcggcggtg ccggcggtgg tttctggagt gacagggttg attctcagcc 300 cttcgccctc ccctatattc atccaaccaa ccccttcgcc gccgatgtcg tttcacaacc 360 cggggctgga actcgccctc gacagccgcc ccgccccctc ggttccgctt ggcgtgacca 420 gtcc 424 <210> 34 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer 167-sl <400> 34 tctacatgcc acaggagctc actg <210> 35 <211> 27 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer 426-a3 <400> 35 gatggaattt gtgagatcaa gtacagg 27 <210> 36 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer 167-s2 <400> 36 ctcactgtgt ccgatagtgt gttgg 25 <210> 37 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer 426-a4 <400> 37 ccttgccatc ggcgatggtc tgc 23 <210> 38 <211> 1186 <212> DNA
<213> Hepatitis E virus <220>
<223> Clone HEV 1186 <400> 38 ctcactgtgt ccgatagtgt gttggttttt gagcttacgg atatagttca ttgccgcatg 60 gccgctccaa gccagcgaaa ggctgttctc tcaacacttg tggggaggta tggccgtagg 120 acgaaactat atgaggcggc gcattcagat gttcgtgagt ccctagctag gttcatccct 180 actatcgggc ctgttcaggc taccacatgt gagttgtatg agttggttga ggctatggtg 240 gagaaaggtc aggacggctc tgcagtctta gagcttgatc tttgtaatcg tgatgtctcg 300 cgcatcacat ttttccaaaa agwctgcaac aagtttacaa ctggtgagac catcgcccac 360 ggcaaggttg gccagggtat atcggcctgg agtaagacct tctgcgctct gttcggcccg 420 tggttccgcg ccattgaaaa agaaatattg gccctgctcc cgcctaatat cttttatggc 480 gacgcttatg aggagtcagt ttttgccgcc gctgtgtccg gggcggggtc atgtatggta 540 tttgaaaatg acttttcaga gtttgacagt acccagaata atttctctct tggccttgag 600 tgtgtggtta tggaggagtg cggcatgcct caatggctaa ttaggttgta ccatctggtt 660 cggtctgcct ggattctgca ggcgccgaag gagtctctta agggtttctg gaagaagcat 720 tctggtgagc ctggtaccct tctttggaat accgtctgga atatggcgat tatagcacat 780 tgctatgagt tccgtgactt tcgtgttgct gcctttaagg gtgatgattc ggtggtcctc 840 tgtagtgact accgacagag ccgcaatgca gctgccttaa ttgctggctg tgggctcaaa 900 ttgaaggttg attaccgccc tatcgggctg tatgctgggg tggtggtggc ccccggtttg 960 gggacactgc ccgatgtggt gcgttttgct ggtcggttgt ctgaaaagaa ttggggcccc 1020 ggcccggaac gtgctgagca gctgcgtctt gctgtctgcg acttccttcg agggttgacg 1080 aatgttgcgc aggtctgtgt tgatgttgtg tcccgtgtct atggagtcag ccccgggctc 1140 gtacataacc ttattggcat gctgcagacc atcgccgatg gcaagg 1186 <210> 39 <211> 25 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer orfl-s2 <400> 39 tcacccatgc cttatgttcc ttacc 25 <210> 40 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer 1300a <400> 40 ggcggcctgg gatgtaatca cg 22 <210> 41 <211> 460 <212> DNA
<213> Hepatitis E virus <220>
<223> Clone HEV 459 <400> 41 tcacccatgc cttatgttcc ttaccctcgt tcaacggagg tgtatgtccg gtccatattt 60 ggccctggcg gctccccatc cttgtttccg tcagcctgct ctactaaatc tactttccat 120 gctgtcccgg tgcatatctg ggatcggctc atgctctttg gtgccaccct ggacgatcag 180 gcgttttgct gttcacggct catgacttac ctccgtggta ttagttacaa ggtcactgtc 240 ggcgcgcttg tcgctaatga ggggtggaac gcctctgaag acgctcttac tgcartgatc 300 actgcagctt atttgactat ttgccatcag cgttatctcc gcacccaggc gatatccaag 360 ggcatgcgcc ggttgggggt tgagcacgcc cagaaattta tcacaagact ctacagttgg 420 ctatttgaga agtctggccg tgattacatc ccaggccgcc 460 <210> 42 <211> 26 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer 459-s2 <400> 42 cagaaattta tcacaagact ctacag 26 <210> 43 <211> 23 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer 1450a <400> 43 aacactcctg accgagccac ttc 23 <210> 44 <211> 235 <212> DNA
<213> Hepatitis E virus <220>
<223> Clone HEV 216 <400> 44 cagaaattta tcacaagact ctacagttgg ctatttgaga agtctggccg tgattatatc 60 cccggccgcc agcttcagtt ctatgcacag tgccgacggt ggctatctgc aggcttccac 120 ctagacccca gggtacttgt ttttgatgag tcagtaccat gccgctgtag gacgtttttg 180 aagaaagttg cgggtaaatt ctgctgtttt atgaagtggc tcggtcagga gtgtt 235 <210> 45 <211> 26 <212> DNA
<213> Hepatitis E virus <220>
<223> usl gap-sl <400> 45 tatagatata acaggttcac ccagcg 26 <210> 46 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> usl gap-a0.5 <400> 46 gctgcaagac cctcacgcat gatg 24 <210> 47 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> us1 gap-s2 <400> 47 cggattatgg ttacaccctg agg 23 <210> 48 <211> 25 <212> DNA
<213> Hepatitis E virus <220>
<223> usl gap-al <400> 48 attcagttgg gtaaaacgct tctgg 25 <210> 49 <211> 545 <212> DNA
<213> Hepatitis E virus <220>
<223> usl-gap <400> 49 cggattatgg ttacaccctg aggggttgct gggtattttc ccccctttct cccctgggca 60 tatctgggag tctgcgaacc ccttttgcgg ggaggggact ttgtataccc gaacttggtc 120 aacatctggc ttttctagtg atttctcccc ccctgaagcg gccgctcctg ctatggctgc 180 taccccgggg ctgccccatt ctaccccacc tgttagcgat atttgggtgc taccaccgcc 240 ctcagaggag tttcaggttg atgcagcacc tgtgccccct gcccctgacc ctgctggatt 300 gcccggtccc gttgtgctta cccccccccc ccctccccct gtgcataagc catcaatacc 360 cccgccttcc cgtaaccgtc gtctcctcta tacctatcct gacggcgcta aggtgtatgc 420 agggtcactg tttgaatcag actgtgactg gctggttaat gcctcaaacc cgggccatcg 480 tcccggaggt ggcctctgcc atgcctttta ccaacgtttt ccagaagcgt tttacccaac 540 tgaat <210> 50 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us1-2600s <400> 50 gtgctcacca taactgagga cacg 24 <210> sl <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us1-2600a <400> 51 cgctgcatat gtaacagcaa cagg 24 <210> 52 <211> 344 <212> DNA
<213> Hepatitis E virus <220>
<223> usl-344 <400> 52 gtgctcacca taactgagga cacggcccgt acggccaacc tggcattgga gattgatgcc 60 gctacagagg tcggccgtgc ttgtgccggt tgcaccatca gccctggcat tgtgcactat 120 cagtttaccg ccggggtccc gggctcgggc aagtcaaggt ccatacaaca gggagatgtc 180 gatgtggtgg ttgtgcccac ccgggagctt cgtaatagtt ggcgccgccg gggttttgcg 240 gccttcacac cccacacagc ggcccgtgtt actatcggcc gccgcgttgt gattgatgag 300 gctccatctc tcccgccaca cctgttgctg ttacatatgc agcg 344 <210> 53 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> usl 3200s <400> 53 gccgatgtgt gcgagctcat acg 23 <210> 54 <211> 25 <212> DNA
<213> Hepatitis E virus <220>
<223> usl 3200a <400> 54 atgattgtgg tctctgtgaa ggtgg 25 <210> 55 <211> 194 <212> DNA
<213> Hepatitis E virus <220>
<223> usl-194 <400> ss gccgatgtgt gcgagctcat acgcggagcc taccctaaaa tccagaccac gagccgtgtg 60 ctacggtccc tgttttggaa tgaaccggcc attggccaga agttggttyt cacgcaggcg 120 gcaaaggctg ctaaccctgg tgcgattacg gtccacgaag ctcagggtgc caccttcaca 180 gagaccacaa tcat 194 <210> 56 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> HEV216-sl <400> 56 cagtaccatg ccgctgtagg acg 23 <210> 57 <211> 26 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-733a1 <400> 57 ccattagatg aaatctttac ctgcag 26 <210> 58 <211> 26 <212> DNA
<213> Hepatitis E virus <220>
<223> HEV216-s2 <400> 58 gtaggacgtt tttgaagaaa gttgcg 26 <210> 59 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-733a2 <400> 59 ggtgagctca taagtgaggc tgtg <210> 60 <211> 464 <212> DNA
<213> Hepatitis E virus <220>
<223> usl-733wb <400> 60 gtaggacgtt tttgaagaaa gttgcgggta aattctgctg ttttatgcgg tggctcgggc 60 aggagtgtac ctgcttcttg gagccggccg agggtttagt cggcgatcat ggccatgaca 120 acgaggccta tgagggttct gaggtcgacc cggctgaacc tgcacatctt gatgtttctg 180 ggacttacgc cgtccacggg caccagcttg aggccctcta tagggcactt aatgtcccac 240 aagatattgc cgctcgagct tcccgactaa cggcaactgt tgagctcgtt gcaagtccag 300 accgcttaga gtgccgcacc gtgctcggta ataagacctt ccggacgacg gtggtcgacg 360 gcgcccatct agaggcgaat ggccctgagc agtatgtctt atcatttgac gcctcccgtc 420 agtctatggg ggccgggtcg cacagcctca cttatgagct cacc 464 <210> 61 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us1 733s1 <400> 61 ttgagctcgt tgcaagtcca gacc 24 <210> 62 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> us2851-r2 <400> 62 ccagaggttg accaggttcg gg 22 <210> 63 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> usl 733s2 <400> 63 ccgtgctcgg taataagacc ttcc 24 <210> 64 <211> 433 <212> DNA
<213> Hepatitis E virus <220>
<223> usl-432 <400> 64 ccgtgctcgg taataagacc ttccggacga cggtggtcga cggcgcccat ctagaggcga 60 atggccctga gcagtatgtc ttatcatttg acgcctcccg tcagtctatg ggggccgggt 120 cgcatagcct cacttatgag ctcacccctg ctggtttgca ggttaggatt tcatctaatg 180 gtctggattg cactgctaca ttcccccccg gtggagcccc tagcgctgcg cccggggagg 240 tggcagcctt ttgcagtgcc ctttatagat ataacaggtt cacccagcgg cactcgctga 300 ctggcggatt atggttacac cctgaggggt tgctgggtat tttcccccct ttctcccctg 360 ggcatatctg ggagtctgcg aacccctttt gcggggaggg gactttgtat acccgaacct 420 ggtcaacctc tgg 433 <210> 65 <211> 26 <212> DNA
<213> Hepatitis E virus <220>
<223> us2851-fl <400> 65 gactgtgatt ggttagtcaa tgcctc 26 <210> 66 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> usl 430-al <400> 66 cgtgtcctca gttatggtga gcac 24 <210> 67 <211> 26 <212> DNA
<213> Hepatitis E virus <220>
<223> usl 430-a2 <400> 67 tattagcctc aaaccaattt gcagcg 26 <210> 68 <211> 382 <212> DNA
<213> Hepatitis E virus <220>
<223> usl-382 <400> 68 gactgtgatt ggttagtcaa tgcctcaaac ccgggccatc gtcccggagg tggcctctgc 60 catgcctttt accaacgttt tccagaagcg ttttacccaa ctgaattcat catgcgtgag 120 ggtcttgcag catacacctt gaccccgcgc cctatcattc atgcagtcgc tcccgattat 180 agggttgagc agaacccgaa gaggcttgag gcagcgtacc gtgaaacttg ttcccgtcgt 240 ggcaccgctg cctacccgct tttgggttcg ggtatatacc aggtccctgt tagcctcagt 300 tttgatgcct gggaacgtaa tcaccgcccc ggcgatgagc tttacttgac cgagcccgct 360 gcaaattggt ttgaggctaa to <210> 69 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-579-sl <400> 69 cagaccacga gccgtgtgct ac 22 <210> 70 <211> 25 <212> DNA
<213> Hepatitis E virus <220>
<223> JE hev167-al <400> 70 ccaacacact atcggacaca gtgag 25 <210> 71 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-579-s2 <400> 71 gctgctaagg ctgccaaccc tg <210> 72 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> JE hev167-a2 <400> 72 cagtgagctc ctgtggcatg taga 24 <220> 73 <211> 451 <212> DNA
<213> Hepatitis E virus <220>
<223> usl-579wb <400> 73 gctgctaagg ctgccaaccc tggtgcgatt acggtccacg aagctcaggg tgccaccttc 60 WO 99/19732 PC1'/US98/21941 acagagacca caatcatagc cacggccgac gccaggggcc ttatccagtc atcccgggct 120 catgctatag ttgcacttac tcgccacact gagaagtgtg ttatcctgga tgcccccggc 180 ctgcttcgtg aggtcggcat ttcggatgtg attgtcaaca actttttcct tgctggtggc 240 gaggtcggcc rccaccgccc ttctgtgata cctcgcggta accctgatca aaacctcggg 300 actttacagg ccttcccgcc gtcctgtcaa attagtgctt accatcagtt ggctgaggaa 360 ctgggccatc gcccggcccc tgtcgccgcc gtcttgcccc cttgccctga gcttgagcag 420 ggcctgctct acatgccaca ggagctcact g 451 <210> 74 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-430s1 <400> 74 ggtatatacc aggtccctgt tagc 24 <210> 75 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-482-al <400> 75 ccgctgtgtg aggtgtgaag gc 22 <210> 76 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-430s2 <400> 76 gttagcctca gttttgatgc ctgg 24 <210> 77 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-482-a2 <400> 77 gacgccagct gttacggagc tcc 23 <210> 78 <211> 334 <212> DNA
<213> Hepatitis E virus <220>
<223> usl-430wb <400> 78 gttagcctca gttttgatgc ctgggaacgt aatcaccgcc ccggcgatga gctttacttg 60 accgagcccg ctgcaaattg gtttgaggct aataagccgg cgcagccggt gctcaccata 120 actgaggaca cggcccgtac ggccaacctg gcattggaga ttgatgccgc tacagaggtc 180 ggccgtgctt gtgccggttg caccatcagc cctggcattg tgcactatca gtttaccgcc 240 ggggtcccgg gctcgggcaa gtcaaggtcc atacaacagg gagatgtcga tgtggtggtt 300 gtgcccaccc gggagctccg taacagctgg cgtc <210> 79 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-482-sl <400> 79 gatgtcgatg tggtggttgt gcc 23 <210> 80 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> JE us2-579-al <400> 80 gtaatcgcac cagggttggc agc <210> 81 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-482-s2 <400> 81 ggagctccgt aacagctggc gtc 23 <210> 82 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> JE us2-579-a2 <400> 82 cagggttggc agccttagca gc 22 <210> 83 <211> 413 <212> DNA
<213> Hepatitis E virus <220>
<223> usl-482wb <400> 83 ggagctccgt aacagctggc gtcgccgggg ttttgcggcc ttcacacccc acacagcggc 60 ccgtgttact atcggccgcc gcgttgtgat tgatgaggct ccatctctcc cgccacacct 120 gttgctgtta catatgcagc gggcctcctc ggtccatctc ctcggtgacc caaatcagat 180 ccctgctatt gattttgagc acgccggcct ggtccctgcg atccgtcccg agcttgcgcc 240 aacgagctgg tggcrcgtta cacaccgttg cccggccgat gtgtgcgagc tcatacgcgg 300 agcctaccct aaaatccaga ccacgagccg tgtgctacgg tccctgtttt ggaatgaacc 360 ggccattggc cagaagttgg ttytcacgca ggctgctaag gctgccaacc ctg 413 <210> 84 <211> 37 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: oligo dT
adapter primer <400> 84 ggccacgcgt cgactagtac tttttttttt ttttttt 37 <210> 85 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: AUAP primer <400> 85 ggccacgcgt cgactagtac 20 <210> 86 <211> 22 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer df-orf3-sl <400> 86 gcgttggtga ggtgggtcgt gg 22 <210> 87 <211> 24 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer df -orf 3-s2 <400> 87 cgcttcttgg tggtttaccg acag 24 <210> 88 <211> 960 <212> DNA
<213> Hepatitis E virus <220>
<223> Clone HEV 3p RACE
<400> 88 cgcttcttgg tggtttaccg acagaattga tttcgtcggc tgggggtcaa ctgttttact 60 cccgccctgt tgtctcggcc aatggcgagc caacagtaaa gttatacaca tctgttgaga 120 atgcgcagca agacaagggc atcaccattc cacacgacat agatttaggt gactcccgtg 180 tggttatcca ggattatgat aaccagcacg aacaagatcg acctaccccg tcacctgccc 240 cctcccgccc tttctcagtt cttcgtgcca atgatgtttt gtggctctct ctcactgccg 300 ctgagtacgr ccagaccacg tatgggtcgt ccaccaaccc tatgtatgtc tctgatacag 360 tcacgcttgt taatgtagcc actggtgctc aggctgttgc ccgctctctt gactggtcta 420 aagttactct ggatggtcgc cctcttacta ccattcagca gtattctaag aaattttatg 480 ttctcccgct tcgsgggaag ctgtcctttt gggaggctgg tacgaccaag gccggctacc 540 cgtataatta taataccact gctagtgacc aaattttgat tgagaacgcg gccggtcacc 600 gtgtcgccat ttctacttat accactagtt tgggtgccgg ccctacctcg atytctgcgg 660 tcggtgtact agctccacat tcggcccttg ctgttctcga ggatactgtt gattatcctg 720 ctcgtgccca tacttttgat gatttctgcc cggagtgtcg cacccttggt ctgcagggtt 780 gtgcattcca atctactatt gctgaacttc agcgtcttaa aatgaaggta ggtaaaaccc 840 gggagtctta attaattcct tttgtgcccc cttcgcagtt ctctttggct ttatttctca 900 tttctgcttt ccgcgctncc ctggaaaaaa aaaaaaaaaa gtactagtcg acgcgtggcc 960 <210> 89 <211> 7202 <212> DNA
<213> Hepatitis E virus <220>
<223> uslfull <400> 89 cctggcatta ctactgccat tgagcaggct gctctggctg cggccaattc tgccttggcg 60 aatgctgtgg tggttcggcc gtttttatct cgcgtgcaaa ccgagattct tattaatttg 120 atgcaacccc ggcagttggt tttccgccct gaggtacttt ggaatcaccc tatccagcgg 180 gttatacata atgaattaga acagtactgc cgggctcggg ctggtcgttg cttggaggtt 240 ggagctcacc caagatccat taatgacaac cccaacgttc tgcatcggtg tttccttaga 300 ccggttggcc gagatgttca gcgctggtac tctgccccca cccgcggccc tgcggctaat 360 tgccgccgct ccgcgttgcg tggtctcccc cccgctgacc gcacttactg ctttgatgga 420 ttctcccgtt gtgcttttgc tgcagagacc ggtgtggctc tttactctct gcatgacctt 480 tggccagctg atgttgcaga ggctatggcc cgccacggga tracacgctt gtatgccgca 540 ctgcaccttc cccctgaggt gctgctacca cccggcacct accacacaac ctcgtatctc 600 ctgattcacg acggcgaccg cgctgttgta acttacgagg gcgatactag tgcgggctat 660 aatcatgatg tctccatact tcgtgcgtgg atccgtacta caaaaatagt tggtgatcat 720 ccgttggtca tagagcgtgt gcgggccatt ggatgtcatt ttgtgttgct gctcaccgca 780 gcccctgagc cgtcacccat gccttatgtt ccttaccctc gttcaacgga ggtgtatgtc 840 cggtccatat ttggccctgg cggctcccca tccttgtttc cgtcagcctg ctctactaaa 900 tctactttcc atgctgtccc ggtgcatatc tgggatcggc tcatgctctt tggtgccacc 960 ctggacgatc aggcgttttg ctgttcacgg ctcatgactt acctccgtgg tattagttac 1020 aaggtcactg tcggcgcgct tgtcgctaat gaggggtgga acgcctctga agacgctctt 1080 actgcartga tcactgcagc ttatttgact atttgccatc agcgttatct ccgcacccag 1140 gcgatatcca agggcatgcg ccggttgggg gttgagcacg cccagaaatt tatcacaaga 1200 ctctacagtt ggctatttga gaagtctggc cgtgattata tccccggccg ccagcttcag 1260 ttctatgcac agtgccgacg gtggctatct gcaggcttcc acctagaccc cagggtactt 1320 gtttttgatg agtcagtacc atgccgctgt aggacgtttt tgaagaaagt tgcgggtaaa 1380 ttctgctgtt ttatgcggtg gctcgggcag gagtgtacct gcttcttgga gccggccgag 1440 ggtttagtcg gcgatcatgg ccatgacaac gaggcctatg agggttctga ggtcgacccg 1500 gctgaacctg cacatcttga tgtttctggg acttacgccg tccacgggca ccagcttgag 1560 gccctctata gggcacttaa tgtcccacaa gatattgccg ctcgagcttc ccgactaacg 1620 gcaactgttg agctcgttgc aagtccagac cgcttagagt gccgcaccgt gctcggtaat 1680 aagaccttcc ggacgacggt ggtcgacggc gcccatctag aggcgaatgg ccctgagcag 1740 tatgtcttat catttgacgc ctcccgtcag tctatggggg ccgggtcgca tagcctcact 1800 tatgagctca cccctgctgg tttgcaggtt aggatttcat ctaatggtct ggattgcact 1860 gctacattcc cccccggtgg agcccctagc gctgcgcccg gggaggtggc agccttttgc 1920 agtgcccttt atagatataa caggttcacc cagcggcact cgctgactgg cggattatgg 1980 ttacaccctg aggggttgct gggtattttc ccccctttct cccctgggca tatctgggag 2040 tctgcgaacc ccttttgcgg ggaggggact ttgtataccc gaacttggtc aacatctggc 2100 ttttctagtg atttctcccc ccctgaagcg gccgctcctg ctatggctgc taccccgggg 2160 ctgccccatt ctaccccacc tgttagcgat atttgggtgc taccaccgcc ctcagaggag 2220 tttcaggttg atgcagcacc tgtgccccct gcccctgacc ctgctggatt gcccggtccc 2280 gttgtgctta cccccccccc ccctccccct gtgcataagc catcaatacc cccgccttcc 2340 cgtaaccgtc gtctcctcta tacctatcct gacggcgcta aggtgtatgc agggtcactg 2400 tttgaatcag actgtgactg gctggttaat gcctcaaacc cgggccatcg tcccggaggt 2460 ggcctctgcc atgcctttta ccaacgtttt ccagaagcgt tttacccaac tgaattcatc 2520 atgcgtgagg gtcttgcagc atacaccttg accccgcgcc ctatcattca tgcagtcgct 2580 cccgattata gggttgagca gaacccgaag aggcttgagg cagcgtaccg tgaaacttgt 2640 tcccgtcgtg gcaccgctgc ctacccgctt ttgggttcgg gtatatacca ggtccctgtt 2700 agcctcagtt ttgatgcctg ggaacgtaat caccgccccg gcgatgagct ttacttgacc 2760 gagcccgctg caaattggtt tgaggctaat aagccggcgc agccggtgct caccataact 2820 gaggacacgg cccgtacggc caacctggca ttggagattg atgccgctac agaggtcggc 2880 cgtgcttgtg ccggttgcac catcagccct ggcattgtgc actatcagtt taccgccggg 2940 gtcccgggct cgggcaagtc aaggtccata caacagggag atgtcgatgt ggtggttgtg 3000 cccacccggg agcttcgtaa tagttggcgc cgccggggtt ttgcggcctt cacaccccac 3060 acagcggccc gtgttactat cggccgccgc gttgtgattg atgaggctcc atctctcccg 3120 ccacacctgt tgctgttaca tatgcagcgg gcctcctcgg tccatctcct cggtgaccca 3180 aatcagatcc ctgctattga ttttgagcac gccggcctgg tccctgcgat ccgtcccgag 3240 cttgcgccaa cgagctggtg gcrcgttaca caccgttgcc cggccgatgt gtgcgagctc 3300 atacgcggag cctaccctaa aatccagacc acgagccgtg tgctacggtc cctgttttgg 3360 aatgaaccgg ccattggcca gaagttggtt ytcacgcagg cggcaaaggc tgctaaccct 3420 ggtgcgatta cggtccacga agctcagggt gccaccttca cagagaccac aatcatagcc 3480 acggccgacg ccaggggcct tatccagtca tcccgggctc atgctatagt tgcacttact 3540 cgccacactg agaagtgtgt tatcctggat gcccccggcc tgcttcgtga ggtcggcatt 3600 tcggatgtga ttgtcaacaa ctttttcctt gctggtggcg aggtcggccr ccaccgccct 3660 tctgtgatac ctcgcggtaa ccctgatcaa aacctcggga ctttacaggc cttcccgccg 3720 tcctgtcaaa ttagtgctta ccatcagttg gctgaggaac tgggccatcg cccggcccct 3780 gtcgccgccg tcttgccccc ttgccctgag cttgagcagg gcctgctcta catgccacag 3840 gagctcactg tgtccgatag tgtgttggtt tttgagctta cggatatagt tcattgccgc 3900 atggccgctc caagccagcg aaaggctgtt ctctcaacac ttgtggggag gtatggccgt 3960 aggacgaaac tatatgaggc ggcgcattca gatgttcgtg agtccctagc taggttcatc 4020 cctactatcg ggcctgttca ggctaccaca tgtgagttgt atgagttggt tgaggctatg 4080 gtggagaaag gtcaggacgg ctctgcagtc ttagagcttg atctttgtaa tcgtgatgtc 4140 tcgcgcatca catttttcca aaaagwctgc aacaagttta caactggtga gaccatcgcc 4200 cacggcaagg ttggccaggg tatatcggcc tggagtaaga ccttctgcgc tctgttcggc 4260 ccgtggttcc gcgccattga aaaagaaata ttggccctgc tcccgcctaa tatcttttat 4320 ggcgacgctt atgaggagtc agtttttgcc gccgctgtgt ccggggcggg gtcatgtatg 4380 gtatttgaaa atgacttttc agagtttgac agtacccaga ataatttctc tcttggcctt 4440 gagtgtgtgg ttatggagga gtgcggcatg cctcaatggc taattaggtt gtaccatctg 4500 gttcggtctg cctggattct gcaggcgccg aaggagtctc ttaagggttt ctggaagaag 4560 cattctggtg agcctggtac ccttctttgg aataccgtct ggaatatggc gattatagca 4620 cattgctatg agttccgtga ctttcgtgtt gctgccttta agggtgatga ttcggtggtc 4680 ctctgtagtg actaccgaca gagccgcaat gcagctgcct taattgctgg ctgtgggctc 4740 aaattgaagg ttgattaccg ccctatcggg ctgtatgctg gggtggtggt ggcccccggt 4800 ttggggacac tgcccgatgt ggtgcgtttt gctggtcggt tgtctgaaaa gaattggggc 4860 cccggcccgg aacgtgctga gcagctgcgt cttgctgtct gcgacttcct tcgagggttg 4920 acgaatgttg cgcaggtctg tgttgatgtt gtgtcccgtg tctatggagt cagccccggg 4980 ctcgtacata accttattgg catgctgcag accatcgccg atggcaaggc ccactttaca 5040 gagactatta aacctgtact tgatctcaca aattccatca tacagcgggt ggaatgaata 5100 acatgtcttt tgcatcgccc atgggatcac catgcgccct agggctgttc tgttgttgtt 5160 cctcatgttt ctgcctatgc tgcccgcgcc accggccggt cagccgtctg gccgtcgccg 5220 tgggcggcgc agcggcggtg ccggcggtgg tttctggagt gacagggttg attctcagcc 5280 cttcgccctc ccctatattc atccaaccaa ccccttcgcc gccgatgtcg tttcacaacc 5340 cggggctgga actcgccctc gacagccgcc ccgccccctc ggttccgctt ggcgtgacca 5400 gtccaagcgc ccctccgttg ccccccgtcg tcgatctacc ccagctgggg ctgcgccgct 5460 aactgccata tcaccagccc ctgatacagc tcctgtacct gatgttgact cacgtggtgc 5520 tattttgcgc cggcagtaca atttgtctac gtccccgctt acatcatctg ttgcttctgg 5580 tactaatctg gttctctatg ctgccccgct gaaccctctc ttgcctcttc aggatggcac 5640 caacactcat attatggcta ctgaggcatc taattacgcc cagtatcggg ttgttcgggc 5700 tacgattcgt tatcgcccgt tggtgccaaa tgctgttggt ggttatgcta tctctatttc 5760 tttctggcct caaactacaa ctacccctac ttctgttgac atgaattcta tcacttctac 5820 tgatgtcagg atcttggtcc agcccggtat agcctccgag ttagtcatcc ctagtgaacg 5880 ccttcactac cgcaaccaag gctggcgctc tgttgagacc acgggtgtgg ccgaagagga 5940 ggctacctcc ggtctggtaa tgctttgtat tcatggctcc cctgttaact cctacactaa 6000 tacaccttac accggtgcat tggggcttct tgattttgca ttagaacttg aatttagaaa 6060 tttgacaccc gggaacacta acacccgtgt ttcccggtat actagcacag cccgccaccg 6120 gctgcgccgc ggtgctgatg ggaccgctga gctcaccacc acagcagcca cacgcttcat 6180 gaaggatttg cattttactg gtacgaacgg cgttggtgag gtgggtcgtg gtattgccct 6240 gactctgttt aatcttgctg atacgcttct tggtggttta ccgacagaat tgatttcgtc 6300 ggctgggggt caactgtttt actcccgccc tgttgtctcg gccaatggcg agccaacagt 6360 aaagttatac acatctgttg agaatgcgca gcaagacaag ggcatcacca ttccacacga 6420 catagattta ggtgactccc gtgtggttat ccaggattat gataaccagc acgaacaaga 6480 tcgacctacc ccgtcacctg ccccctcccg ccctttctca gttcttcgtg ccaatgatgt 6540 tttgtggctc tctctcactg ccgctgagta cgrccagacc acgtatgggt cgtccaccaa 6600 ccctatgtat gtctctgata cagtcacgct tgttaatgta gccactggtg ctcaggctgt 6660 tgcccgctct cttgactggt ctaaagttac tctggatggt cgccctctta ctaccattca 6720 gcagtattct aagaaatttt atgttctccc gcttcgsggg aagctgtcct tttgggaggc 6780 tggtacgacc aaggccggct acccgtataa ttataatacc actgctagtg accaaatttt 6840 gattgagaac gcggccggtc accgtgtcgc catttctact tataccacta gtttgggtgc 6900 cggccctacc tcgatytctg cggtcggtgt actagctcca cattcggccc ttgctgttct 6960 cgaggatact gttgattatc ctgctcgtgc ccatactttt gatgatttct gcccggagtg 7020 tcgcaccctt ggtctgcagg gttgtgcatt ccaatctact attgctgaac ttcagcgtct 7080 taaaatgaag gtaggtaaaa cccgggagtc ttaattaatt ccttttgtgc ccccttcgca 7140 gttctctttg gctttatttc tcatttctgc tttccgcgct ccctggaaaa aaaaaaaaaa 7200 as <210> 90 <211> 7202 <212> DNA
<213> Hepatitis E virus <220>
<221> CDS
<222> (1)..(5097) <223> orfl <220>
<221> CDS
<222> (5132)..(7114) <223> orf2 <220>
<223> orf3 at positions 5094-5462 <220>
<223> uslfull <400> 90 cct ggc att act act gcc att gag cag get get ctg get gcg gcc aat 48 Pro Gly Ile Thr Thr Ala Ile Glu Gln Ala Ala Leu Ala Ala Ala Asn tct gcc ttg gcg aat get gtg gtg gtt cgg ccg ttt tta tct cgc gtg 96 Ser Ala Leu Ala Asn Ala Val Val Val Arg Pro Phe Leu Ser Arg Val caa acc gag att ctt att aat ttg atg caa ccc cgg cag ttg gtt ttc 144 Gln Thr Glu Ile Leu Ile Asn Leu Met Gln Pro Arg Gln Leu Val Phe cgc cct gag gta ctt tgg aat cac cct atc cag cgg gtt ata cat aat 192 Arg Pro Glu Val Leu Trp Asn His Pro Ile Gln Arg Val Ile His Asn gaa tta gaa cag tac tgc cgg get cgg get ggt cgt tgc ttg gag gtt 240 Glu Leu Glu Gln Tyr Cys Arg Ala Arg Ala Gly Arg Cys Leu Glu Val gga get cac cca aga tcc att aat gac aac ccc aac gtt ctg cat cgg 288 Gly Ala His Pro Arg Ser Ile Asn Asp Asn Pro Asn Val Leu His Arg tgt ttc ctt aga ccg gtt ggc cga gat gtt cag cgc tgg tac tct gcc 336 Cys Phe Leu Arg Pro Val Gly Arg Asp Val Gln Arg Trp Tyr Ser Ala ccc acc cgc ggc cct gcg get aat tgc cgc cgc tcc gcg ttg cgt ggt 384 Pro Thr Arg Gly Pro Ala Ala Asn Cys Arg Arg Ser Ala Leu Arg Gly ctc ccc ccc get gac cgc act tac tgc ttt gat gga ttc tcc cgt tgt 432 Leu Pro Pro Ala Asp Arg Thr Tyr Cys Phe Asp Gly Phe Ser Arg Cys get ttt get gca gag acc ggt gtg get ctt tac tct ctg cat gac ctt 480 Ala Phe Ala Ala Glu Thr Gly Val Ala Leu Tyr Ser Leu His Asp Leu tgg cca get gat gtt gca gag get atg gcc cgc cac ggg atr aca cgc 528 Trp Pro Ala Asp Val Ala Glu Ala Met Ala Arg His Gly Xaa Thr Arg ttg tat gcc gca ctg cac ctt ccc cct gag gtg ctg cta cca ccc ggc 576 Leu Tyr Ala Ala Leu His Leu Pro Pro Glu Val Leu Leu Pro Pro Gly acc tac cac aca acc tcg tat ctc ctg att cac gac ggc gac cgc get 624 Thr Tyr His Thr Thr Ser Tyr Leu Leu Ile His Asp Gly Asp Arg Ala gtt gta act tac gag ggc gat act agt gcg ggc tat aat cat gat gtc 672 Val Val Thr Tyr Glu Gly Asp Thr Ser Ala Gly Tyr Asn His Asp Val WO 99/19732 PCT/US9$/Z1941 tcc ata ctt cgt gcg tgg atc cgt act aca aaa ata gtt ggt gat cat 720 Ser Ile Leu Arg Ala Trp Ile Arg Thr Thr Lys Ile Val Gly Asp His ccg ttg gtc ata gag cgt gtg cgg gcc att gga tgt cat ttt gtg ttg 768 Pro Leu Val Ile Glu Arg Val Arg Ala Ile Gly Cys His Phe Val Leu ctg ctc acc gca gcc cct gag ccg tca ccc atg cct tat gtt cct tac 816 Leu Leu Thr Ala Ala Pro Glu Pro Ser Pro Met Pro Tyr Val Pro Tyr cct cgt tca acg gag gtg tat gtc cgg tcc ata ttt ggc cct ggc ggc 864 Pro Arg Ser Thr Glu Val Tyr Val Arg Ser Ile Phe Gly Pro Gly Gly tcc cca tcc ttg ttt ccg tca gcc tgc tct act aaa tct act ttc cat 912 Ser Pro Ser Leu Phe Pro Ser Ala Cys Ser Thr Lys Ser Thr Phe His get gtc ccg gtg cat atc tgg gat cgg ctc atg ctc ttt ggt gcc acc 960 Ala Val Pro Val His Ile Trp Asp Arg Leu Met Leu Phe Gly Ala Thr ctg gac gat cag gcg ttt tgc tgt tca cgg ctc atg act tac ctc cgt 1008 Leu Asp Asp Gln Ala Phe Cys Cys Ser Arg Leu Met Thr Tyr Leu Arg ggt att agt tac aag gtc act gtc ggc gcg ctt gtc get aat gag ggg 1056 Gly Ile Ser Tyr Lys Val Thr Val Gly Ala Leu Val Ala Asn Glu Gly tgg aac gcc tct gaa gac get ctt act gca rtg atc act gca get tat 1104 Trp Asn Ala Ser Glu Asp Ala Leu Thr Ala Xaa Ile Thr Ala Ala Tyr ttg act att tgc cat cag cgt tat ctc cgc acc cag gcg ata tcc aag 1152 Leu Thr Ile Cys His Gln Arg Tyr Leu Arg Thr Gln Ala Ile Ser Lys ggc atg cgc cgg ttg ggg gtt gag cac gcc cag aaa ttt atc aca aga 1200 Gly Met Arg Arg Leu Gly Val Glu His Ala Gln Lys Phe Ile Thr Arg ctc tac agt tgg cta ttt gag aag tct ggc cgt gat tat atc ccc ggc 1248 Leu Tyr Ser Trp Leu Phe Glu Lys Ser Gly Arg Asp Tyr Ile Pro Gly cgc cag ctt cag ttc tat gca cag tgc cga cgg tgg cta tct gca ggc 1296 Arg Gln Leu Gln Phe Tyr Ala Gln Cys Arg Arg Trp Leu Ser Ala Gly ttc cac cta gac ccc agg gta ctt gtt ttt gat gag tca gta cca tgc 1344 Phe His Leu Asp Pro Arg Val Leu Val Phe Asp Glu Ser Val Pro Cys _. .._. _..~.~ _.__. .__.._..__._-_ _ cgc tgt agg acg ttt ttg aag aaa gtt gcg ggt aaa ttc tgc tgt ttt 1392 Arg Cys Arg Thr Phe Leu Lys Lys Val Ala Gly Lys Phe Cys Cys Phe atg cgg tgg ctc ggg cag gag tgt acc tgc ttc ttg gag ccg gcc gag 1440 Met Arg Trp Leu Gly Gln Glu Cys Thr Cys Phe Leu Glu Pro Ala Glu ggt tta gtc ggc gat cat ggc cat gac aac gag gcc tat gag ggt tct 1488 Gly Leu Val Gly Asp His Gly His Asp Asn Glu Ala Tyr Glu Gly Ser gag gtc gac ccg get gaa cct gca cat ctt gat gtt tct ggg act tac 1536 Glu Val Asp Pro Ala Glu Pro Ala His Leu Asp Val Ser Gly Thr Tyr gcc gtc cac ggg cac cag ctt gag gcc ctc tat agg gca ctt aat gtc 1584 Ala Val His Gly His Gln Leu Glu Ala Leu Tyr Arg Ala Leu Asn Val cca caa gat att gcc get cga get tcc cga cta acg gca act gtt gag 1632 Pro Gln Asp Ile Ala Ala Arg Ala Ser Arg Leu Thr Ala Thr Val Glu ctc gtt gca agt cca gac cgc tta gag tgc cgc acc gtg ctc ggt aat 1680 Leu Val Ala Ser Pro Asp Arg Leu Glu Cys Arg Thr Val Leu Gly Asn aag acc ttc cgg acg acg gtg gtc gac ggc gcc cat cta gag gcg aat 1728 Lys Thr Phe Arg Thr Thr Val Val Asp Gly Ala His Leu Glu Ala Asn ggc cct gag cag tat gtc tta tca ttt gac gcc tcc cgt cag tct atg 1776 Gly Pro Glu Gln Tyr Val Leu Ser Phe Asp Ala Ser Arg Gln Ser Met ggg gcc ggg tcg cat agc ctc act tat gag ctc acc cct get ggt ttg 1824 Gly Ala Gly Ser His Ser Leu Thr Tyr Glu Leu Thr Pro Ala Gly Leu cag gtt agg att tca tct aat ggt ctg gat tgc act get aca ttc ccc 1872 Gln Val Arg Ile Ser Ser Asn Gly Leu Asp Cys Thr Ala Thr Phe Pro ccc ggt gga gcc cct agc get gcg ccc ggg gag gtg gca gcc ttt tgc 1920 Pro Gly Gly Ala Pro Ser Ala Ala Pro Gly Glu Val Ala Ala Phe Cys agt gcc ctt tat aga tat aac agg ttc acc cag cgg cac tcg ctg act 1968 Ser Ala Leu Tyr Arg Tyr Asn Arg Phe Thr Gln Arg His Ser Leu Thr ggc gga tta tgg tta cac cct gag ggg ttg etg ggt att ttc ccc cct 2016 Gly Gly Leu Trp Leu His Pro Glu Gly Leu Leu Gly Ile Phe Pro Pro ttc tcc cct ggg cat atc tgg gag tct gcg aac ccc ttt tgc ggg gag 2064 Phe Ser Pro Gly His Ile Trp Glu Ser Ala Asn Pro Phe Cys Gly Glu ggg act ttg tat acc cga act tgg tca aca tct ggc ttt tct agt gat 2112 Gly Thr Leu Tyr Thr Arg Thr Trp Ser Thr Ser Gly Phe Ser Ser Asp ttc tcc ccc cct gaa gcg gcc get cct get atg get get acc ccg ggg 2160 Phe Ser Pro Pro Glu Ala Ala Ala Pro Ala Met Ala Ala Thr Pro Gly ctg ccc cat tct acc cca cct gtt agc gat att tgg gtg cta cca ccg 2208 Leu Pro His Ser Thr Pro Pro Val Ser Asp Ile Trp Val Leu Pro Pro ccc tca gag gag ttt cag gtt gat gca gca cct gtg ccc cct gcc cct 2256 Pro Ser Glu Glu Phe Gln Val Asp Ala Ala Pro Val Pro Pro Ala Pro gac cct get gga ttg ccc ggt ccc gtt gtg ctt acc ccc ccc ccc cct 2304 Asp Pro Ala Gly Leu Pro Gly Pro Val Val Leu Thr Pro Pro Pro Pro ccc cct gtg cat aag cca tca ata ccc ccg cct tcc cgt aac cgt cgt 2352 Pro Pro Val His Lys Pro Ser Ile Pro Pro Pro Ser Arg Asn Arg Arg ctc ctc tat acc tat cct gac ggc get aag gtg tat gca ggg tca ctg 2400 Leu Leu Tyr Thr Tyr Pro Asp Gly Ala Lys Val Tyr Ala Gly Ser Leu ttt gaa tca gac tgt gac tgg ctg gtt aat gcc tca aac ccg ggc cat 2448 Phe Glu Ser Asp Cys Asp Trp Leu Val Asn Ala Ser Asn Pro Gly His cgt ccc gga ggt ggc ctc tgc cat gcc ttt tac caa cgt ttt cca gaa 2496 Arg Pro Gly Gly Gly Leu Cys His Ala Phe Tyr Gln Arg Phe Pro Glu gcg ttt tac cca act gaa ttc atc atg cgt gag ggt ctt gca gca tac 2544 Ala Phe Tyr Pro Thr Glu Phe Ile Met Arg Glu Gly Leu Ala Ala Tyr acc ttg acc ccg cgc cct atc att cat gca gtc get ccc gat tat agg 2592 Thr Leu Thr Pro Arg Pro Ile Ile His Ala Val Ala Pro Asp Tyr Arg gtt gag cag aac ccg aag agg ctt gag gca gcg tac cgt gaa act tgt 2640 Val Glu Gln Asn Pro Lys Arg Leu Glu Ala Ala Tyr Arg Glu Thr Cys tcc cgt cgt ggc acc get gcc tac ccg ctt ttg ggt tcg ggt ata tac 2688 Ser Arg Arg Gly Thr Ala Ala Tyr Pro Leu Leu Gly Ser Gly Ile Tyr cag gtc cct gtt agc ctc agt ttt gat gcc tgg gaa cgt aat cac cgc 2736 Gln Val Pro Val Ser Leu Ser Phe Asp Ala Trp Glu Arg Asn His Arg ccc ggc gat gag ctt tac ttg acc gag ccc get gca aat tgg ttt gag 2784 Pro Gly Asp Glu Leu Tyr Leu Thr Glu Pro Ala Ala Asn Trp Phe Glu get aat aag ccg gcg cag ccg gtg ctc acc ata act gag gac acg gcc 2832 Ala Asn Lys Pro Ala Gln Pro Val Leu Thr Ile Thr Glu Asp Thr Ala cgtacggcc aacctggca ttggagatt gatgccget acagaggtc ggc 2880 ArgThrAla AsnLeuAla LeuGluIle AspAlaAla ThrGluVal Gly cgtgettgt gccggttgc accatcagc cctggcatt gtgcactat cag 2928 ArgAlaCys AlaGlyCys ThrIleSer ProGlyIle ValHisTyr Gln tttaccgcc ggggtcccg ggctcgggc aagtcaagg tccatacaa cag 2976 PheThrAla GlyValPro GlySerGly LysSerArg SerIleGln Gln ggagatgtc gatgtggtg gttgtgccc acccgggag cttcgtaat agt 3024 .

GlyAspVal AspValVal ValValPro ThrArgGlu LeuArgAsn Ser tggcgccgc cggggtttt gcggccttc acaccccac acagcggcc cgt 3072 TrpArgArg ArgGlyPhe AlaAlaPhe ThrProHis ThrAlaAla Arg gttactatcggc cgccgc gttgtgattgat gagget ccatctctcccg 3120 ValThrIleGly ArgArg ValValIleAsp GluAla ProSerLeuPro ccacacctgttg ctgtta catatgcagcgg gcctcc tcggtccatctc 3168 ProHisLeuLeu LeuLeu HisMetGlnArg AlaSer SerValHisLeu ctcggtgaccca aatcag atccctgetatt gatttt gagcacgccggc 3216 LeuGlyAspPro AsnGln IleProAlaIle AspPhe GluHisAlaGly ctggtccctgcg atccgt cccgagcttgcg ccaacg agctggtggcrc 3264 LeuValProAla IleArg ProGluLeuAla ProThr SerTrpTrpXaa gttacacaccgt tgcccg gccgatgtgtgc gagctc atacgcggagcc 3312 ValThrHisArg CysPro AlaAspValCys GluLeu IleArgGlyAla taccctaaaatc cagacc acgagccgtgtg ctacgg tccctgttttgg 3360 TyrProLysIle GlnThr ThrSerArgVal LeuArg SerLeuPheTrp aat gaa ccg gcc att ggc cag aag ttg gtt ytc acg cag gcg gca aag 3408 Asn G1u Pro Ala Ile Gly Gln Lys Leu Val Xaa Thr Gln Ala Ala Lys get get aac cct ggt gcg att acg gtc cac gaa get cag ggt gcc acc 3456 Ala Ala Asn Pro Gly Ala Ile Thr Val His Glu Ala Gln Gly Ala Thr ttc aca gag acc aca atc ata gcc acg gcc gac gcc agg ggc ctt atc 3504 Phe Thr Glu Thr Thr Ile Ile Ala Thr Ala Asp Ala Arg Gly Leu Ile cag tca tcc cgg get cat get ata gtt gca ctt act cgc cac act gag 3552 Gln Ser Ser Arg Ala His Ala Ile Val Ala Leu Thr Arg His Thr Glu aag tgt gtt atc ctg gat gcc ccc ggc ctg ctt cgt gag gtc ggc att 3600 Lys Cys Val Ile Leu Asp Ala Pro Gly Leu Leu Arg Glu Val Gly Ile tcg gat gtg att gtc aac aac ttt ttc ctt get ggt ggc gag gtc ggc 3648 Ser Asp Val Ile Val Asn Asn Phe Phe Leu Ala Gly Gly Glu Val Gly crc cac cgc cct tct gtg ata cct cgc ggt aac cct gat caa aac ctc 3696 Xaa His Arg Pro Ser Val Ile Pro Arg Gly Asn Pro Asp Gln Asn Leu ggg act tta cag gcc ttc ccg ccg tcc tgt caa att agt get tac cat 3744 Gly Thr Leu Gln Ala Phe Pro Pro Ser Cys Gln Ile Ser Ala Tyr His cag ttg get gag gaa ctg ggc cat cgc ccg gcc cct gtc gcc gcc gtc 3792 Gln Leu Ala Glu Glu Leu Gly His Arg Pro Ala Pro Val Ala Ala Val ttg ccc cct tgc cct gag ctt gag cag ggc ctg ctc tac atg cca cag 3840 Leu Pro Pro Cys Pro Glu Leu Glu Gln Gly Leu Leu Tyr Met Pro Gln gag ctc act gtg tcc gat agt gtg ttg gtt ttt gag ctt acg gat ata 3888 Glu Leu Thr Val Ser Asp Ser Val Leu Val Phe Glu Leu Thr Asp Ile gtt cat tgc cgc atg gcc get eca agc cag cga aag get gtt ctc tca 3936 Val His Cys Arg Met Ala Ala Pro Ser Gln Arg Lys Ala Val Leu Ser aca ctt gtg ggg agg tat ggc cgt agg acg aaa cta tat gag gcg gcg 3984 Thr Leu Val Gly Arg Tyr Gly Arg Arg Thr Lys Leu Tyr Glu Ala Ala cat tca gat gtt cgt gag tcc cta get agg ttc atc cct act atc ggg 4032 His Ser Asp Val Arg Glu Ser Leu Ala Arg Phe Ile Pro Thr Ile Gly cct gtt cag get acc aca tgt gag ttg tat gag ttg gtt gag get atg 4080 Pro Val Gln Ala Thr Thr Cys Glu Leu Tyr Glu Leu Val Glu Ala Met gtg gag aaa ggt cag gac ggc tct gca gtc tta gag ctt gat ctt tgt 4128 Val Glu Lys Gly Gln Asp Gly Ser Ala Val Leu Glu Leu Asp Leu Cys aat cgt gat gtc tcg cgc atc aca ttt ttc caa aaa gwc tgc aac aag 4176 Asn Arg Asp Val Ser Arg Ile Thr Phe Phe Gln Lys Xaa Cys Asn Lys ttt aca act ggt gag acc atc gcc cac ggc aag gtt ggc cag ggt ata 4224 Phe Thr Thr Gly Glu Thr Ile Ala His Gly Lys Val Gly Gln Gly Ile tcg gcc tgg agt aag acc ttc tgc get ctg ttc ggc ccg tgg ttr cgc 4272 Ser Ala Trp Ser Lys Thr Phe Cys Ala Leu Phe Gly Pro Trp Phe Arg gcc att gaa aaa gaa ata ttg gcc ctg ctc ccg cct aat atc ttt tat 4320 Ala Ile Glu Lys Glu Ile Leu Ala Leu Leu Pro Pro Asn Ile Phe Tyr ggc gac get tat gag gag tca gtt ttt gcc gcc get gtg tcc ggg gcg 4368 Gly Asp Ala Tyr Glu Glu Ser Val Phe Ala Ala Ala Val Ser Gly Ala ggg tca tgt atg gta ttt gaa aat gac ttt tca gag ttt gac agt acc 4416 Gly Ser Cys Met Val Phe Glu Asn Asp Phe Ser Glu Phe Asp Ser Thr cag aat aat ttc tct ctt ggc ctt gag tgt gtg gtt atg gag gag tgc 4464 Gln Asn Asn Phe Ser Leu Gly Leu Glu Cys Val Val Met Glu Glu Cys ggc atg cct caa tgg cta att agg ttg tac cat ctg gtt cgg tct gcc 4512 Gly Met Pro Gln Trp Leu Ile Arg Leu Tyr His Leu Val Arg Ser Ala tgg att ctg cag gcg ccg aag gag tct ctt aag ggt ttc tgg aag aag 4560 Trp Ile Leu Gln Ala Pro Lys Glu Ser Leu Lys Gly Phe Trp Lys Lys cat tct ggt gag cct ggt acc ctt ctt tgg aat acc gtc tgg aat atg 4608 His Ser Gly Glu Pro Gly Thr Leu Leu Trp Asn Thr Val Trp Asn Met 1525. 1530 1535 gcg att ata gca cat tgc tat gag ttc cgt gac ttt cgt gtt get gcc 4656 Ala Ile Ile Ala His Cys Tyr Glu Phe Arg Asp Phe Arg Val Ala Ala ttt aag ggt gat gat tcg gtg gtc ctc tgt agt gac tac cga cag agc 4704 Phe Lys Gly Asp Asp Ser Val Val Leu Cys Ser Asp Tyr Arg Gln Ser cgc aat gca get gcc tta att get ggc tgt ggg ctc aaa ttg aag gtt 4752 Arg Asn Ala Ala Ala Leu Ile Ala Gly Cys Gly Leu Lys Leu Lys Val gat tac cgc cct atc ggg ctg tat get ggg gtg gtg gtg gcc ccc ggt 4800 Asp Tyr Arg Pro Ile Gly Leu Tyr Ala Gly Val Val Val Ala Pro Gly ttg ggg aca ctg ccc gat gtg gtg cgt ttt get ggt cgg ttg tct gaa 4848 Leu Gly Thr Leu Pro Asp Val Val Arg Phe Ala Gly Arg Leu Ser Glu aag aat tgg ggc ccc ggc ccg gaa cgt get gag cag ctg cgt ctt get 4896 Lys Asn Trp Gly Pro Gly Pro Glu Arg Ala Glu Gln Leu Arg Leu Ala gtc tgc gac ttc ctt cga ggg ttg acg aat gtt gcg cag gtc tgt gtt 4944 Val Cys Asp Phe Leu Arg Gly Leu Thr Asn Val Ala Gln Val Cys Val gat gtt gtg tcc cgt gtc tat gga gtc agc ccc ggg ctc gta cat aac 4992 Asp Val Val Ser Arg Val Tyr Gly Val Ser Pro Gly Leu Val His Asn ctt att ggc atg ctg cag acc atc gcc gat ggc aag gcc cac ttt aca 5040 Leu Ile Gly Met Leu Gln Thr Ile Ala Asp Gly Lys Ala His Phe Thr gag act att aaa cct gta ctt gat ctc aca aat tcc atc ata cag cgg 5088 Glu Thr Ile Lys Pro Val Leu Asp Leu Thr Asn Ser Ile Ile Gln Arg gtg gaa tga ataacatgtc ttttgcatcg cccatgggat cacc atg cgc cct agg 5143 Val Glu Met Arg Pro Arg get gtt ctg ttg ttg ttc ctc atg ttt ctg cct atg ctg ccc gcg cca 5191 Ala Val Leu Leu Leu Phe Leu Met Phe Leu Pro Met Leu Pro Ala Pro ccg gcc ggt cag ccg tct ggc cgt cgc cgt ggg cgg cgc agc ggc ggt 5239 Pro Ala Gly Gln Pro Ser G1y Arg Arg Arg Gly Arg Arg Ser Gly Gly gcc ggc ggt ggt ttc tgg agt gac agg gtt gat tct cag ccc ttc gcc 5287 Ala Gly Gly Gly Phe Trp Ser Asp Arg Val Asp Ser Gln Pro Phe Ala ctc ccc tat att cat cca acc aac ccc ttc gcc gcc gat gtc gtt tca 5335 Leu Pro Tyr Ile His Pro Thr Asn Pro Phe Ala Ala Asp Val Val Ser caa ccc ggg get gga act cgc cct cga cag ccg ccc cgc ccc ctc ggt 5383 Gln Pro Gly Ala Gly Thr Arg Pro Arg Gln Pro Pro Arg Pro Leu Gly tcc get tgg cgt gac cag tcc aag cgc ccc tcc gtt gcc ccc cgt cgt 5431 Ser Ala Trp Arg Asp Gln Ser Lys Arg Pro Ser Val Ala Pro Arg Arg cga tct acc cca get ggg get gcg ccg cta act gcc ata tca cca gcc 5479 Arg Ser Thr Pro Ala Gly Ala Ala Pro Leu Thr Ala Ile Ser Pro Ala cct gat aca get cct gta cct gat gtt gac tca cgt ggt get att ttg 5527 Pro Asp Thr Ala Pro Val Pro Asp Val Asp Ser Arg Gly Ala Ile Leu cgc cgg cag tac aat ttg tct acg tcc ccg ctt aca tca tct gtt get 5575 Arg Arg Gln Tyr Asn Leu Ser Thr Ser Pro Leu Thr Ser Ser Val Ala tct ggt act aat ctg gtt ctc tat get gcc ccg ctg aac cct ctc ttg 5623 Ser Gly Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu Asn Pro Leu Leu cct ctt cag gat ggc acc aac act cat att atg get act gag gca tct 5671 Pro Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala Thr Glu Ala Ser aat tac gcc cag tat cgg gtt gtt cgg get acg att cgt tat cgc ccg 5719 Asn Tyr Ala Gln Tyr Arg Val Val Arg Ala Thr Ile Arg Tyr Arg Pro ttg gtg cca aat get gtt ggt ggt tat get atc tct att tct ttc tgg 5767 Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser Ile Ser Phe Trp cct caa act aca act acc cct act tct gtt gac atg aat tct atc act 5815 Pro Gln Thr Thr Thr Thr Pro Thr Ser Val Asp Met Asn Ser Ile Thr tct act gat gtc agg atc ttg gtc cag ccc ggt ata gcc tcc gag tta 5863 Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile Ala Ser Glu Leu gtc atc cct agt gaa cgc ctt cac tac cgc aac caa ggc tgg cgc tct 5911 Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln Gly Trp Arg Ser gtt gag acc acg ggt gtg gcc gaa gag gag get acc tcc ggt ctg gta 5959 Val Glu Thr Thr Gly Val Ala Glu Glu Glu Ala Thr Ser Gly Leu Val atg ctt tgt att cat ggc tcc cct gtt aac tcc tac act aat aca cct 6007 Met Leu Cys Ile His Gly Ser Pro Val Asn Ser Tyr Thr Asn Thr Pro tac acc ggt gca ttg ggg ctt ctt gat ttt gca tta gaa ctt gaa ttt 6055 Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu Glu Leu Glu Phe WO 99/19732 PC'T/US98/21941 agaaatttgaca cccgggaac actaac acccgtgtttcc cggtat act 6103 ArgAsnLeuThr ProGlyAsn ThrAsn ThrArgValSer ArgTyr Thr agcacagcccgc caccggctg cgccgc ggtgetgatggg accget gag 6151 SerThrAlaArg HisArgLeu ArgArg GlyAlaAspGly ThrAla Glu ctcaccaccaca gcagccaca cgcttc atgaaggatttg catttt act 6199 LeuThrThrThr AlaAlaThr ArgPhe MetLysAspLeu HisPhe Thr ggtacgaacggc gttggtgag gtgggt cgtggtattgcc ctgact ctg 6247 GlyThrAsnGly ValGlyGlu ValGly ArgGlyIleAla LeuThr Leu tttaatcttget gatacgctt cttggt ggtttaccgaca gaattg att 6295 PheAsnLeuAla AspThrLeu LeuGly GlyLeuProThr GluLeu Ile tcgtcggetggg ggtcaactg ttttac tcccgccctgtt gtctcg gcc 6343 SerSerAlaGly GlyGlnLeu PheTyr SerArgProVal ValSer Ala aatggcgagcca acagtaaag ttatac acatctgttgag aatgcg cag 6391 AsnGlyGluPro ThrValLys LeuTyr ThrSerValGlu AsnAla Gln caagacaagggc atcaccatt ccacacgac atagattta ggtgactcc 6439 GlnAspLysGly IleThrIle ProHisAsp IleAspLeu GlyAspSer cgtgtggttatc caggattat gataaccag cacgaacaa gatcgacct 6487 ArgValValIle GlnAspTyr AspAsnGln HisGluGln AspArgPro accccgtcacct gccccctcc cgccctttc tcagttctt cgtgccaat 6535 ThrProSerPro AlaProSer ArgProPhe SerValLeu ArgAlaAsn gatgttttgtgg ctctctctc actgccget gagtacgrc cagaccacg 6583 AspValLeuTrp LeuSerLeu ThrAlaAla GluTyrXaa GlnThrThr tatgggtcgtcc accaaccct atgtatgtc tctgataca gtcacgctt 6631 TyrGlySerSer ThrAsnPro MetTyrVal SerAspThr ValThrLeu gttaatgtagcc actggtget caggetgtt gcccgctct cttgactgg 6679 ValAsnValAla ThrGlyAla GlnAlaVal AlaArgSer LeuAspTrp tctaaagttact ctggatggt cgccctctt actaccatt cagcagtat 6727 SerLysValThr LeuAspGly ArgProLeu ThrThrIle GlnGlnTyr tct aag aaa ttt tat gtt ctc ccg ctt cgs ggg aag ctg tcc ttt tgg 6775 Ser Lys Lys Phe Tyr Val Leu Pro Leu Xaa Gly Lys Leu Ser Phe Trp gag get ggt acg acc aag gcc ggc tac ccg tat aat tat aat acc act 6823 Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr get agt gac caa att ttg att gag aac gcg gcc ggt cac cgt gtc gcc 6871 Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His Arg Val Ala att tct act tat acc act agt ttg ggt gcc ggc cct acc tcg aty tct 6919 Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Thr Ser Xaa Ser gcg gtc ggt gta cta get cca cat tcg gcc ctt get gtt ctc gag gat 6967 Ala Val Gly Val Leu Ala Pro His Ser Ala Leu Ala Val Leu Glu Asp act gtt gat tat cct get cgt gcc cat act ttt gat gat ttc tgc ccg 7015 Thr Val Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro gag tgt cgc acc ctt ggt ctg cag ggt tgt gca ttc caa tct act att 7063 Glu Cys Arg Thr Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Ile get gaa ctt cag cgt ctt aaa atg aag gta ggt aaa acc cgg gag tct 7111 Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Ser taa ttaattcctt ttgtgccccc ttcgcagttc tctttggctt tatttctcat 7164 ttctgctttc cgcgctccct ggaaaaaaaa aaaaaaaa 7202 <210> 91 <211> 1698 <212> PRT
<213> Hepatitis E virus <400> 91 Pro Gly Ile Thr Thr Ala Ile Glu Gln Ala Ala Leu Ala Ala Ala Asn Ser Ala Leu Ala Asn.Ala Val Val Val Arg Pro Phe Leu Ser Arg Val Gln Thr Glu Ile Leu Ile Asn Leu Met Gln Pro Arg Gln Leu Val Phe Arg Pro Glu Val Leu Trp Asn His Pro Ile Gln Arg Val Ile His Asn Glu Leu Glu Gln Tyr Cys Arg Ala Arg Ala Gly Arg Cys Leu Glu Val Gly Ala His Pro Arg Ser Ile Asn Asp Asn Pro Asn Val Leu His Arg Cys Phe Leu Arg Pro Val Gly Arg Asp Val Gln Arg Trp Tyr Ser Ala Pro Thr Arg Gly Pro Ala Ala Asn Cys Arg Arg Ser Ala Leu Arg Gly Leu Pro Pro Ala Asp Arg Thr Tyr Cys Phe Asp Gly Phe Ser Arg Cys Ala Phe Ala Ala Glu Thr Gly Val Ala Leu Tyr Ser Leu His Asp Leu Trp Pro Ala Asp Val Ala Glu Ala Met Ala Arg His Gly Xaa Thr Arg Leu Tyr Ala Ala Leu His Leu Pro Pro Glu Val Leu Leu Pro Pro Gly Thr Tyr His Thr Thr Ser Tyr Leu Leu Ile His Asp Gly Asp Arg Ala Val Val Thr Tyr Glu Gly Asp Thr Ser Ala Gly Tyr Asn His Asp Val Ser Ile Leu Arg Ala Trp Ile Arg Thr Thr Lys Ile Val Gly Asp His Pro Leu Val Ile Glu Arg Val Arg Ala Ile Gly Cys His Phe Val Leu Leu Leu Thr Ala Ala Pro Glu Pro Ser Pro Met Pro Tyr Val Pro Tyr Pro Arg Ser Thr Glu Val Tyr Val Arg Ser Ile Phe Gly Pro Gly Gly Ser Pro Ser Leu Phe Pro Ser Ala Cys Ser Thr Lys Ser Thr Phe His Ala Val Pro Val His Ile Trp Asp Arg Leu Met Leu Phe Gly Ala Thr Leu Asp Asp Gln Ala Phe Cys Cys Ser Arg Leu Met Thr Tyr Leu Arg Gly Ile Ser Tyr Lys Val Thr Val Gly Ala Leu Val Ala Asn Glu Gly Trp Asn Ala Ser Glu Asp Ala Leu Thr Ala Xaa Ile Thr Ala Ala Tyr Leu Thr Ile Cys His Gln Arg Tyr Leu Arg Thr Gln Ala Ile Ser Lys Gly Met Arg Arg Leu Gly Val Glu His Ala Gln Lys Phe Ile Thr Arg Leu Tyr Ser Trp Leu Phe Glu Lys Ser Gly Arg Asp Tyr Ile Pro Gly Arg Gln Leu Gln Phe Tyr Ala Gln Cys Arg Arg Trp Leu Ser Ala Gly Phe His Leu Asp Pro Arg Val Leu Val Phe Asp Glu Ser Val Pro Cys Arg Cys Arg Thr Phe Leu Lys Lys Val Ala Gly Lys Phe Cys Cys Phe Met Arg Trp Leu Gly Gln Glu Cys Thr Cys Phe Leu Glu Pro Ala Glu Gly Leu Val Gly Asp His Gly His Asp Asn Glu Ala Tyr Glu Gly Ser Glu Val Asp Pro Ala Glu Pro Ala His Leu Asp Val Ser Gly Thr Tyr Ala Val His Gly His Gln Leu Glu Ala Leu Tyr Arg Ala Leu Asn Val Pro Gln Asp Ile Ala Ala Arg Ala Ser Arg Leu Thr Ala Thr Val Glu Leu Val Ala Ser Pro Asp Arg Leu Glu Cys Arg Thr Val Leu Gly Asn Lys Thr Phe Arg Thr Thr Val Val Asp Gly Ala His Leu Glu Ala Asn Gly Pro Glu Gln Tyr Val Leu Ser Phe Asp Ala Ser Arg Gln Ser Met Gly Ala Gly Ser His Ser Leu Thr Tyr Glu Leu Thr Pro Ala Gly Leu Gln Val Arg Ile Ser Ser Asn Gly Leu Asp Cys Thr Ala Thr Phe Pro Pro Gly Gly Ala Pro Ser Ala Ala Pro Gly Glu Val Ala Ala Phe Cys Ser Ala Leu Tyr Arg Tyr Asn Arg Phe Thr Gln Arg His Ser Leu Thr Gly Gly Leu Trp Leu His Pro Glu Gly Leu Leu Gly Ile Phe Pro Pro Phe Ser Pro Gly His Ile Trp Glu Ser Ala Asn Pro Phe Cys Gly Glu Gly Thr Leu Tyr Thr Arg Thr Trp Ser Thr Ser Gly Phe Ser Ser Asp Phe Ser Pro Pro Glu Ala Ala Ala Pro Ala Met Ala Ala Thr Pro Gly Leu Pro His Ser Thr Pro Pro Val Ser Asp Ile Trp Val Leu Pro Pro Pro Ser Glu Glu Phe Gln Val Asp Ala Ala Pro Val Pro Pro Ala Pro Asp Pro Ala Gly Leu Pro Gly Pro Val Val Leu Thr Pro Pro Pro Pro Pro Pro Val His Lys Pro Ser Ile Pro Pro Pro Ser Arg Asn Arg Arg Leu Leu Tyr Thr Tyr Pro Asp Gly Ala Lys Val Tyr Ala Gly Ser Leu Phe Glu Ser Asp Cys Asp Trp Leu Val Asn Ala Ser Asn Pro Gly His Arg Pro Gly Gly Gly Leu Cys His Ala Phe Tyr Gln Arg Phe Pro Glu Ala Phe Tyr Pro Thr Glu Phe Ile Met Arg Glu Gly Leu Ala Ala Tyr Thr Leu Thr Pro Arg Pro Ile Ile His Ala Val Ala Pro Asp Tyr Arg Val Glu Gln Asn Pro Lys Arg Leu Glu Ala Ala Tyr Arg Glu Thr Cys Ser Arg Arg Gly Thr Ala Ala Tyr Pro Leu Leu Gly Ser Gly Ile Tyr Gln Val Pro Val Ser Leu Ser Phe Asp Ala Trp Glu Arg Asn His Arg Pro Gly Asp Glu Leu Tyr Leu Thr Glu Pro Ala Ala Asn Trp Phe Glu Ala Asn Lys Pro Ala Gln Pro Val Leu Thr Ile Thr Glu Asp Thr Ala Arg Thr Ala Asn Leu Ala Leu Glu Ile Asp Ala Ala Thr Glu Val Gly Arg Ala Cys Ala Gly Cys Thr Ile Ser Pro Gly Ile Val His Tyr Gln Phe Thr Ala Gly Val Pro Gly Ser Gly Lys Ser Arg Ser Ile Gln Gln Gly Asp Val Asp Val Val Val Val Pro Thr Arg Glu Leu Arg Asn Ser Trp Arg Arg Arg Gly Phe Ala Ala Phe Thr Pro His Thr Ala Ala Arg Val Thr Ile Gly Arg Arg Val Val Ile Asp Glu Ala Pro Ser Leu Pro Pro His Leu Leu Leu Leu His Met Gln Arg Ala Ser Ser Val His Leu Leu Gly Asp Pro Asn Gln Ile Pro Ala Ile Asp Phe Glu His Ala Gly Leu Val Pro Ala Ile Arg Pro Glu Leu Ala Pro Thr Ser Trp Trp Xaa Val Thr His Arg Cys Pro Ala Asp Val Cys Glu Leu Ile Arg Gly Ala Tyr Pro Lys Ile Gln Thr Thr Ser Arg Val Leu Arg Ser Leu Phe Trp Asn Glu Pro Ala Ile Gly Gln Lys Leu Val Xaa Thr Gln Ala Ala Lys 40 .
Ala Ala Asn Pro Gly Ala Ile Thr Val His Glu Ala Gln Gly Ala Thr Phe Thr Glu Thr Thr Ile Ile Ala Thr Ala Asp Ala Arg Gly Leu Ile Gln Ser Ser Arg Ala His Ala Ile Val Ala Leu Thr Arg His Thr Glu Lys Cys Val Ile Leu Asp Ala Pro Gly Leu Leu Arg Glu Val Gly Ile Ser Asp Val Ile Val Asn Asn Phe Phe Leu Ala Gly Gly Glu Val Gly Xaa His Arg Pro Ser Val Ile Pro Arg Gly Asn Pro Asp Gln Asn Leu Gly Thr Leu Gln Ala Phe Pro Pro Ser Cys Gln Ile Ser Ala Tyr His Gln Leu Ala Glu Glu Leu Gly His Arg Pro Ala Pro Val Ala Ala Val Leu Pro Pro Cys Pro Glu Leu Glu Gln Gly Leu Leu Tyr Met Pro Gln Glu Leu Thr Val Ser Asp Ser Val Leu Val Phe Glu Leu Thr Asp Ile Val His Cys Arg Met Ala Ala Pro Ser Gln Arg Lys Ala Val Leu Ser Thr Leu Val Gly Arg Tyr Gly Arg Arg Thr Lys Leu Tyr Glu Ala Ala His Ser Asp Val Arg Glu Ser Leu Ala Arg Phe Ile Pro Thr Ile Gly Pro Val Gln Ala Thr Thr Cys Glu Leu Tyr Glu Leu Val Glu Ala Met Val Glu Lys Gly Gln Asp Gly Ser Ala Val Leu Glu Leu Asp Leu Cys Asn Arg Asp Val Ser Arg Ile Thr Phe Phe Gln Lys Xaa Cys Asn Lys Phe Thr Thr Gly Glu Thr Ile Ala His Gly Lys Val Gly Gln Gly Ile Ser Ala Trp Ser Lys Thr Phe Cys Ala Leu Phe Gly Pro Trp Phe Arg Ala Ile Glu Lys Glu Ile Leu Ala Leu Leu Pro Pro Asn Ile Phe Tyr Gly Asp Ala Tyr Glu Glu Ser Val Phe Ala Ala Ala Val Ser Gly Ala Gly Ser Cys Met Val Phe Glu Asn Asp Phe Ser Glu Phe Asp Ser Thr Gln Asn Asn Phe Ser Leu Gly Leu Glu Cys Val Val Met Glu Glu Cys Gly Met Pro Gln Trp Leu Ile Arg Leu Tyr His Leu Val Arg Ser Ala Trp Ile Leu Gln Ala Pro Lys Glu Ser Leu Lys Gly Phe Trp Lys Lys His Ser Gly Glu Pro Gly Thr Leu Leu Trp Asn Thr Val Trp Asn Met Ala Ile Ile Ala His Cys Tyr Glu Phe Arg Asp Phe Arg Val Ala Ala 1540 _ 1545 1550 Phe Lys Gly Asp Asp Ser Val Val Leu Cys Ser Asp Tyr Arg Gln Ser Arg Asn Ala Ala Ala Leu Ile Ala Gly Cys Gly Leu Lys Leu Lys Val Asp Tyr Arg Pro Ile Gly Leu Tyr Ala Gly Val Val Val Ala Pro Gly Leu Gly Thr Leu Pro Asp Val Val Arg Phe Ala Gly Arg Leu Ser Glu Lys Asn Trp Gly Pro Gly Pro Glu Arg Ala Glu Gln Leu Arg Leu Ala Val Cys Asp Phe Leu Arg Gly Leu Thr Asn Val Ala Gln Val Cys Val Asp.Val Val Ser Arg Val Tyr Gly Val Ser Pro Gly Leu Val His Asn Leu Ile Gly Met Leu Gln Thr Ile Ala Asp Gly Lys Ala His Phe Thr Glu Thr Ile Lys Pro Val Leu Asp Leu Thr Asn Ser Ile Ile Gln Arg Val Glu <210> 92 <211> 660 <212> PRT
<213> Hepatitis E virus <400> 92 Met Arg Pro Arg Ala Val Leu Leu Leu Phe Leu Met Phe Leu Pro Met Leu Pro Ala Pro Pro Ala Gly Gln Pro Ser Gly Arg Arg Arg Gly Arg Arg Ser Gly Gly Ala Gly Gly Gly Phe Trp Ser Asp Arg Val Asp Ser Gln Pro Phe Ala Leu Pro Tyr Ile His Pro Thr Asn Pro Phe Ala Ala Asp Val Val Ser Gln Pro Gly Ala Gly Thr Arg Pro Arg Gln Pro Pro Arg Pro Leu Gly Ser Ala Trp Arg Asp Gln Ser Lys Arg Pro Ser Val Ala Pro Arg Arg Arg Ser Thr Pro Ala Gly Ala Ala Pro Leu Thr Ala Ile Ser Pro Ala Pro Asp Thr Ala Pro Val Pro Asp Val Asp Ser Arg Gly Ala Ile Leu Arg Arg Gln Tyr Asn Leu Ser Thr Ser Pro Leu Thr Ser Ser Val Ala Ser Gly Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu Asn Pro Leu Leu Pro Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala Thr Glu Ala Ser Asn Tyr Ala Gln Tyr Arg Val Val Arg Ala Thr Ile Arg Tyr Arg Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser Ile Ser Phe Trp Pro Gln Thr Thr Thr Thr Pro Thr Ser Val Asp Met Asn Ser Ile Thr Ser Thr Asp Val Arg Ile Leu Val Gln Pro Gly Ile Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln Gly Trp Arg Ser Val Glu Thr Thr Gly Val Ala Glu Glu Glu Ala Thr Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Pro Val Asn Ser Tyr Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn Thr Asn Thr Arg Val Ser Arg Tyr Thr Ser Thr Ala Arg His Arg Leu Arg Arg Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp Leu His Phe Thr Gly Thr Asn Gly Val Gly Glu Val Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Thr Ile Pro His Asp Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Xaa Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val Ser Asp Thr Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Thr Thr Ile Gln Gln Tyr Ser Lys Lys Phe Tyr Val Leu Pro Leu Xaa Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Thr Ser Xaa Ser Ala Val Gly Val Leu Ala Pro His Ser Ala Leu Ala Val Leu Glu Asp Thr Val Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Thr Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Ile Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Ser <210> 93 <211> 122 <212> PRT
<213> Hepatitis E virus <220>
<223> ORF3 HEV US-1 <400> 93 Met Asn Asn Met Ser Phe Ala Ser Pro Met Gly Ser Pro Cys Ala Leu Gly Leu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys Cys Pro Arg His Arg Pro Val Ser Arg Leu Ala Val Ala Val Gly Gly Ala Ala Ala Val Pro Ala Val Val Ser Gly Val Thr Gly Leu Ile Leu Ser Pro Ser Pro Ser Pro Ile Phe Ile Gln Pro Thr Pro Ser Pro Pro Met Ser Phe His Asn Pro Gly Leu Glu Leu Ala Leu Asp Ser Arg Pro Ala Pro Ser Val Pro Leu Gly Val Thr Ser Pro Ser Ala Pro Pro Leu Pro Pro Val Val Asp Leu Pro Gln Leu Gly Leu Arg Arg <210> 94 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 94 tggcattact actgccattg 20 <210> 95 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 95 caattctgcc ttggcgaatg 20 <210> 96 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 96 aggaaacacc gatgcagaac 20 <210> 97 <211> 20 <212> DNA
<213> Artificial Sequence <220>
<223> Description of Artificial Sequence: Primer <400> 97 tccaacctcc aagcaacgac 20 <210> 98 <211> 199 <212> DNA

<213> Hepatitis E virus <220>
<223> Clone 199con <400> 98 caattctgcc ttggcgaatg ctgtggtggt tcggccgttt ctttctcgtg tgcaaactga 60 gattcttatt aatttgatgc aaccccggca gttggtcttc cgccctgagg tgctttggaa 120 tcatcctatc cagcgggtta tacataatga attagagcag tactgccggg cccgggctgg 180 tcgttgcttg gaggttgga <210> 99 <211> 25 <212> DNA
<213> Hepatitis E virus <220>
<223> JE orfl-s <400> 99 gttctgcatc ggtgtttcct tagac 25 <210> 100 <211> 26 <212> DNA
<213> Hepatitis E virus <220>
<223> JE orfl-a <400> 100 gaatcaggag atacgaggtt gtgtgg <210> 101 <211> 331 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-320 <400> 101 gttctgcatc ggtgtttcct tagaccggtc ggccgagatg ttcagcgctg gtattctgcc 60 cctacccgtg gtcctgcggc caattgccgc cgctccgcgt tgcgtggtct cccccctgtc 120 gaccgcacct attgttttga tggattttcc cgttgtgctt ttgctgcaga gaccggtgtg 180 gccctttact ctttgcatga cctttggcca gctgatgttg cagaggctat ggcccgccat 240 gggatgacac gcttatacgc cgcactgcac cttccccccg aggtgctgct accacccggc 300 acctaccaca caacctcgta tctcctgatt c 331 <210> 102 <211> 1186 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-1168 <400> 102 ctcactgtgt ccgatagtgt gttggttttt gagcttacgg atatagtcca ctgccgtatg 60 gccgccccaa gccagcgaaa ggctgttctc tcaacgcttg tggggaggta cggccgtagg 120 actaaattat atgaggcggc gcattcagat gtccgtgagt ccctagcgag gtttatcccc 180 accatcgggc ctgttcgggc taccacatgt gagctgtacg agctggttga agccatggta 240 gagaagggtc aggacggatc tgccgtccta gagctcgacc tttgcaatcg tgacgtctcg 300 cgcatcacat ttttccaaaa ggattgcaat aagtttacaa ctggtgagac tatcgcccat 360 ggcaaggttg gccagggcat atcggcctgg agcaagacct tctgtgctct gtttggcccg 420 tggttccgcg ccattgaaaa ggaaatattg gccctactcc cgcctaatat cttttatggc 480 gacgcctatg aggagtcagt gtttgctgcc gctgtgtccg gggcagggtc atgtatggta 540 tttgaaaatg acttctcaga gtttgacagt acccagaata atttctctct cggccttgag 600 tgtgtggtta tggaggagtg cggcatgccc caatggttaa ttaggttgta ccatctggtc 660 cggtcagcct ggattttgca ggcgccgaag gagtctctta aggggttttg gaagaagcac 720 tctggtgagc ctggtaccct tctctggaac actgtctgga acatggcgat tatagcacat 780 tgctaygagt tccgtgactt tcgtgttgcc gccttcaagg gtgatgattc agtggtcctc 840 tgtagtgact accgacagrg ccgtaacgcg gctgccttaa ttgcaggctg tgggctcaaa 900 ttgaaggttg attaccgccc tatcgggcta tatgctggag tggtggtggc ccccggtttg 960 gggacactgc ccgatgtggt gcgttttgcc ggtcggttat ctgagaagaa ttggggccct 1020 ggcccggagc gtgctgagca gctgcgtctt gctgtttgtg atttccttcg agggttgacg 1080 aatgttgcgc aggtctgtgt tgatgttgtg tcccgtgtct atggagttag ccccgggctg 1140 gtacataacc ttattggcat gctgcagacc atcgccgatg gcaagg 1186 <210> 103 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> JE hevdf2/3 sl <400> 103 gttccgcttg gcgtgaccag tcc 23 <210> 104 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> JE hevdf2/3 al <400> 104 gagtcaacat caggtacagg agc 23 <210> 105 <211> 130 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-135 <400> 105 gttccgcttg gcgtgaccag tcccagcgcc cctccgctgc cccccgtcgt cgatctgccc 60 cagctggggc tgcgccgctg actgccgtgt caccggctcc tgacacagct cctgtacctg 120 atgttgactc 130 <210> 106 <211> 26 <212> DNA
<213> Hepatitis E virus <220>
<223> JE hevdfl-sl <400> 106 gatgtcattt tgtgttgctg ctcacc 26 <210> 107 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> hev216 al <400> 107 cgtcctacag cggcatggta ctg 23 <210> 108 <211> 564 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-563 <400> 108 tcacccatgc cttatgttcc ttaccctcgt tcaacggagg tgtatgtccg gtctatattt 60 ggccctggcg gctccccatc cttgtttcca tcagcctgct ctactaaatc tacctttcat 120 gctgtcccgg ttcacatctg ggatcrgctc atgctctttg gtgccaccct gracgatcag 180 gcgttctgct gttcacggct tatgacttac ctccgtggta ttagttataa ggtcactgtc 240 ggtgcgcttg tcgctaatga ggggtggaac gcctctgagg atgctcttac tgcagtgatc 300 actgcggcct atctgaccat ctgccatcag cgttaccttc gcacccaggc gatttccaag 360 ggcatgcgcc ggttggaggt tgagcatgct cagaaattta tcacaagact ctacagctgg 420 ctatttgaga agtctggccg tgactacatc cccggccgcc agcttcaatt ttatgcacaa 480 tgccgacggt ggctttctgc aggcttccac ctaracccca ggrtgcttgt ctttgatgaa S40 tcagtaccat gccgctgtag gacg <210> 109 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> USorf2.l~
<400> 109 gtggagctag tacaccgacc gcag 24 <210> 110 <211> 678 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-667 <400> 110 cgcttcttgg tggtttaccg acagaattga tttcgtcggc tgggggccaa ctgttttact 60 cccgcccggt tgtctcagcc aatggcgagc caacagtaaa gttatataca tctgttgaga 120 atgcgcagca agacaagggc atcaccattc cacatgatat agacctgggt gactcccgtg 180 tggttatcca ggattatgat aaccagcayg agcaagaccg acctactccg tcacctgccc 240 cctctcgccc cttctcagtt cttcgtgcca atgatgtttt gtggctttcc ctcactgccg 300 ctgagtatga ccagactacg tatgggtcgt ccaccaaccc tatgtatgtc tctgacacag 360 ttacgcttgt taatgtggct actggtgctc aggctgttgc ccgctccctt gattggtcta 420 aagttactct ggacggccgc ccccttacta ccattcagca gtattctaag acattttatg 480 ttctcccgct ccgcgggaag ctgtcctttt gggaggctgg cacgactaag gccggctacc 540 cttacaatta taatactacc gctagtgacc aaattttgat tgagaatgcg gccggccacc 600 gtgtcgctat ttccacctat accactagct taggtgccgg tcctacctcg atctctgcgg 660 tcggtgtact agctccac 678 <210> 111 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> hev3301s <400> 111 gtatgcgagc tcatccgtgg tgc 23 <210> 112 <211> 25 <212> DNA
<213> Hepatitis E virus <220>
<223> JE hev167-al <400> 112 ccaacacact atcggacaca gtgag <210> 113 <211> 580 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-579 <400> 113 gtatgcgagc tcatccgtgg tgcctacccc aaaattcaga ccacgagccg tgtgctacgg 60 tccctgtttt ggaacgaacc ggccatcggc caaaagttgg tttttacgca ggctgctaag 120 gctgccaacc ctggtgcgat tacggttcac gaagctcagg gtgctacttt cacggagacc 180 acaattatag ccacggccga cgctaggggc ctcattcagt catcccgggc ccatgctata 240 gtcgcactca cccgccatac tgagaagtgt gttattttgg atgcccccgg cttgttgcgc 300 gaggtcggca tttcggatgt tattgtcaat aactttttcc ttgccggtgg agaggtcggc 360 catcaccgcc cttctgtgat acctcgcggc aatcctgatc agaacctcgg gactctacag 420 gcctttccgc cgtcatgtca gatcagtgct taccatcagt tggctgagga actaggtcat 480 cgcccggccc ctgtcgccgc cgtcttgccc ccttgccctg agcttgagca gggcctgctc 540 tatatgccac aagaactcac tgtgtccgat agtgtgttgg 5gp <210> 114 <211> 26 <212> DNA
<213> Hepatitis E virus <220>
<223> HEV459 sl <400> 114 cagaaattta tcacaagact ctacag 26 <210> 115 <211> 26 <212> DNA
<213> Hepatitis E virus <220>
<223> HEV459 s3 <400> 115 ctctacagtt ggctatttga gaagtc 26 <210> 116 <211> 25 <212> DNA
<213> Hepatitis E virus <220>
<223> JE1955a <400> 116 ctataaagag ctgagcagaa ggcgg 25 <210> 117 <211> 734 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-733 <400> 117 ctctacagtt ggctatttga gaagtctggc cgtgactaca tccccggccg ccagcttcaa 60 ttttatgcac aatgccgacg gtggctttct gcaggcttcc acctaraccc caggrtgctt 120 gtctttgatg aatcagtgcc atgccgttgc aggacgtttt tgaagaaggt cgcgggtaaa 180 ttctgctgtt ttatgcggtg gctggggcag gagtgtacct gcttcttgga gccagccgag 240 ggtttagttg gtgatcaagg tcatgacaac gaggcctatg aaggttctga ggtcgaccca 300 gctgagcctg cacatcttga tgtctcgggg acttatgccg tccatgggca ccagcttgag 360 gccctctata gggcacttaa tgtcccacat gatattgccg ctcgagcctc ccgactaacg 420 gctactgttg agctcgttgc tagtccggac cgcttagagt gccgcactgt acttggtaat 480 aagaccttcc ggacgacggt ggttgatggc gcccatcttg aagcgaatgg ccctgaggag 540 tatgttctgt catttgacgc ctctcgccag tctatggggg ccgggtcgca cagcctcact 600 tatgagctca cccctgccgg tctgcaggta aagatttcat ctaatggtct ggattgcact .660 gccacattcc ccccyggtgg cgcccctagc gccgcgccgg gggaggtggc cgccttctgc 720 tcagctcttt atag 734 <210> 118 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> JE 2950mex s <400> 118 gtgtccccgg ctctggcaag tc 22 <210> 119 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> JE us2-579-a2 <400> 119 cagggttggc agccttagca gc <210> 120 <211> 483 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-482 <400> 120 gtgtccccgg ctctggcaag tcaaggtcca tacaacaggg agatgtcgat gtggtggttg 60 tgcccacccg ggagctccgt aacagctggc gtcgccgggg ttttgcggcc ttcacacctc 120 acacagcggc ccgtgttact atcggccgcc gcgttgtgat tgatgaggct ccatctctcc 180 caccgcacct gctgctgtta cacatgcagc gggcctcctc ggtccatctc cttggtgatc 240 caaaccagat tcctgctatt gattttgagc atgccggcct ggtccccgcg atccgccccg 300 agcttgcgcc aacgagctgg tggcacgtta cacaccgttg cccggccgat gtgtgcgagc 360 tcatacgtgg ggcctacccc aaaattcaga ccacgagccg tgtgctacgg tccctgtttt 420 ggaacgaacc ggccatcggc caaaagttgg tttttacgca ggctgctaag gctgccaacc 480 ctg 483 <210> 121 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> JE 2600s <400> 121 taacccaaag aggcttgagg ctgc 24 <210> 122 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-482-al <400> 122 ccgctgtgtg aggtgtgaag gc 22 <210> 123 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-482-a2 <400> 123 w 52 gacgccagct gttacggagc tcc 23 <210> 124 <211> 431 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-430 <400> 124 taacccaaag aggcttgagg ctgcgtaccg ggaaacttgc tcccgtcgtg gcaccgctgc 60 ctacccgctt ttgggctcgg gtatatacca ggtccctgtt agcctcagtt ttgatgcctg 120 ggaacgcaat caccgccccg gcgatgagct ttacttgaca gagcccgccg cagcctggtt 180 tgaggctaat aagccggcgc agccggcgct tactataact gaggacacgg cccgtacggc 240 caacctggca ttagagattg atgccgccac agaggttggc cgtgcttgtg ccggctgcac 300 catcagcccc gggattgtgc actatcagtt taccgccggg gtcccgggct caggcaagtc 360 aaggtccata caacagggag atgtcgatgt ggtggttgtg cccacccggg agctccgtaa 420 cagctggcgt c <210> 125 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-orf2/3 sl <400> 125 cgtcgtcgat ctgccccagc tg 22 <210> 126 <211> 25 <212> DNA
<213> Hepatitis E virus <220>
<223> HEVConsORF2-al <400> 126 cttgttcrtg ytggttrtca taatc <210> 127 <211> 21 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-orf2/3 s2 <400> 127 cgctgactgc cgtgtcaccg g 21 <210> 128 <211> 25 <212> DNA
<213> Hepatitis E virus <220>
<223> HEVConsORF2-a2 <400> 128 gttcrtgytg gttrtcataa tcctg 25 <210> 129 <211> 1020 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-1019 <400> 129 cgctgactgc cgtgtcaccg gctcctgaca cagcccctgt acctgatgtt gactcacgtg 60 gtgctattct gcgccggcag tacaatttgt ccacgtcccc gctcacgtca tctgtcgctt 120 cgggtactaa tttggtcctc tatgctgccc cgctgaatcc cctcttgcct ctccaggatg 180 gtaccaacac tcatattatg gctactgagg catccaatta tgcccagtat cgggttgttc 240 gagctacaat ccgttatcgc ccgctggtgc cgaatgccgt tggtggctat gccatttcca 300 tttctttctg gccccaaact acaactaccc ctacttctgt cgatatgaat tctattactt 360 ccacygatgt taggattttg gttcagcccg gtattgcctc cgagctagtc atccccagtg 420 agcgccttca ttaccgtaat caaggctggc gctctgttga gaccacgggt gtggctgagg 480 aggaggctac ttccggtctg gtaatgcttt gcattcatgg ctctcctgtt aattcctaca 540 ctaatacacc ttacactggt gcgctggggc ttcttgattt tgcactagag cttgaattta 600 ggaatttgac acccgggaac accaacaccc gtgtttcccg gtataccagc acagcccgcc 660 accggctgcg ccgtggtgct gatgggactg ctgagcttac taccacagca gccacacgtt 720 tcatgaagga cctgcacttc gctggcacga atggcgttgg tgaggtgggt cgtggtatcg 780 cccbgacact gttcaatctc gctgatacgc ttctcggcgg tttaccgaca gaattgattt 840 cgtcggctgg gggccaactg ttttactccc gcccggttgt ctcagccaat ggcgagccaa 900 cagtaaagtt atatacatct gttgagaatg cgcagcaaga caagggcatc accattccac 960 atgatataga cctgggtgac tcccgtgtgg ttatccagga ttatgataac cagcaygaac 1020 <210> 130 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us2 330s1 <400> 130 cagctgatgt tgcagaggct atgg 24 <210> 131 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us2 563a1 <400> 131 gcaggctgat ggaaacaagg atgg 24 <210> 132 <211> 407 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-406 <400> 132 cagctgatgt tgcagaggct atggcccgcc atgggatgac acgcttatac gccgcactgc 60 accttccccc cgaggtgctg ctaccacccg gcacctacca cacaacctcg tacctcttga 120 ttcacgatgg caaccgcgct gttgtaactt acgagggcga tactagtgcg ggctataatc 180 atgatgtctc catacttcgt gcatggatcc gtactactaa aatagttggt gaccatccat 240 tggtcataga gcgagtgcgg gccattgggt gtcattttgt gctgctgctc accgcagccc 300 ctgaaccgtc acctatgcct tatgttccct accctcgttc aacggaggtg tatgtccggt 360 ctatatttgg ccctggcggc tccccatcct tgtttccatc agcctgc 407 <210> 133 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-579 sl <400> 133 cagaccacga gccgtgtgct ac 22 <210> 134 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-1168 al <400> 134 ccacaagcgt tgagagaaca gcc 23 <210> 135 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-579 s2 <400> 135 gctgctaagg ctgccaaccc tg 22 <210> 136 <211> 547 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-579wb <400> 136 gctgctaagg ctgccaaccc tggtgcgatt acggttcacg aagctcaggg tgctactttc 60 acggagacca caattatagc cacggccgac gctaggggcc tcattcagtc atcccgggcc 120 catgctatag tcgcactcac ccgccatact gagaagtgtg ttattttgga tgcccccggc 180 ttgttgcgcg aggtcggcat ttcggatgtt attgtcaata actttttcct tgccggtgga 240 gaggtcggcc atcaccgccc ttctgtgata cctcgcggca atcctgatca gaacctcggg 300 actctacagg cctttccgcc gtcatgtcag atcagtgctt accatcagtt ggctgaggaa 360 ctaggtcatc gcccggcccc tgtcgccgcc gtcttgcccc cttgccctga gcttgagcag 420 ggcctgctct atatgccaca agaacttact gtgtccgata gcgtgctggt ttttgagctt 480 acggatatag tccactgccg tatggccgcc ccaagccagc gaaaggctgt tctctcaacg 540 cttgtgg <210> 137 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-733s1 <400> 137 cacagcctca cttatgagct cacc 24 <210> 138 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-430a1 <400> 138 cggtgattgc gttcccaggc atc <210> 139 <211> 26 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-733s2 <400> 139 ctgcaggtaa agatttcatc taatgg 26 <210> 140 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-430a2 <400> 140 ccaggcatca aaactgaggc taac <210> 141 <211> 903 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-851 <400> 141 ctgcaggtaa agatttcatc taatggtctg gattgcactg ccacattccc cccyggtggc 60 gcccctagcg ccgcgccggg ggaggtggcs gccttctgca gtgctcttta tagatacaat 120 aggttcaccc agcggcattc gctgacaggc ggactatggc tacatcctga ggggctgctg 180 ggtatcttcc ccccattctc ccctgggcat atttgggagt ctgctaaccc cttttgcggt 240 gaggggactt tgtatacccg aacctggtca acctctggtt tttctagtga tttctccccc 300 cctgaggcgg ccgctcctgc ttcggctgcc gccccggggt tgccctaccc tactccacct 360 gttagtgata tctgggtgtt accaccgccc tcagaggaat ctcatgttga tgcggcatct 420 gtaccctctg ttcctgagcc tgctggattg accagcccta ttgtgcttac cccccccccc 480 ccccctcctc ccgtgcgtaa gccggcaaca tccccgcctc cccgcactcg ccgtetcctt 540 tacacctacc ccgacggcgc caaggtgtat gcggggtcat tgtktgagtc agactgtgat 600 tggttagtca atgcctcaaa ccctggccat cgccccgggg gtggcctctg ccatgctttt 660 tatcaacgtt tcccagaagc gttctactcg actgaattca tcatgcgcga gggccttgca 720 gcatacactt taaccccgcg ccctattatc catgcagtgg ctcccgacta tagggttgag 780 caaaacccga agaggcttga ggcagcgtac cgggaaactt gctcccgtcg tggcaccgct 840 gcctacccgc ttttgggctc gggtatatac caggtccctg ttagcctcag ttttgatgcc 900 tgg 903 <210> 142 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-1168s1 <400> 142 gcaggtctgt gttgatgttg tgtc 24 <210> 143 <211> 21 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-dforf2/3 a2 <400> 143 ccggtgacac ggcagtcagc g 21 <210> 144 <211> 25 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-1168s2 <400> 144 gatgttgtgt cccgtgtcta tggag 25 <210> 145 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> us2 dforf2/3 a3 <400> 145 cagctggggc agatcgacga cg 22 <210> 146 <211> 503 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-502 <400> 146 gatgttgtgt cccgtgtcta tggagttagc cccgggctgg tacataacct tattggcatg 60 ctgcagacca ttgctgatgg caaggcccac tttacagara atattaaacc tgtgcttgac 120 cttacaaatt ccatcataca acgggtggaa tgaataacat gtcttttgca tcgcccatgg 180 gatcaccatg cgccctaggg ctgttctgtt gttgctcttc gtgcttttgc ctatgctgcc 240 cgcgccaccg gccggccagc cgtctggccg ccgtcgtggg cggcgcagcg gcggtgccgg 300 cggtggtttc tggggtgaca gggttgattc tcagcccttc gccctcccct atattcatcc 360 aaccaacccc ttcgccgccg atgtcgtttc acaacccggg gctggaactc gccctcgaca 420 gccgccccgc ccccttggyt ccgcttggcg tgaccagtcc cagcgcccct ccgctgcccc 480 ccgtcgtcga tctgccccag ctg 503 <210> 147 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> HEVConsORFl-sl <400> 147 ctggcatyac tactgcyatt gagc 24 <210> 148 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> HEVConsORF1-a1 <400> 148 ccatcrarrc agtaagtgcg gtc 23 <210> 149 <211> 418 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-orfl <400> 149 ctggcattac tactgctatt gagcaggctg ctctggctgc ggctaattcc gccttggcga 60 atgctgtggt ggttcggccg tttctttctc gtgtgcaaac tgagattctt attaatttga 120 tgcaaccccg gcagttggtc ttccgccctg aggtgctttg gaatcatcct atccagcggg 180 ttatacataa tgaattagag cagtactgcc gggcccgggc tggtcgttgt ttggaggttg 240 gagcccaccc gaggtccatt aatgacaacc ctaatgtctt gcataggtgt tttcttagac 300 cggtcggccg agatgttcag cgctggtatt ctgcccctac ccgtggtcct gcggccaatt 360 gccgccgctc cgcgttgcgt ggtctccccc ctgtcgaccg cacttactgt tttgatgg 418 <210> 150 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> HEVConsORF2-s1 <400> 150 gacagaattr atttcgtcgg ctgg 24 <210> 151 <211> 197 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-orf2 <400> 151 gacagaattg atttcgtcgg ctgggggcca actgttttac tcccgcccgg ttgtctcagc 60 caatggcgag ccaacagtaa agttatatac atctgttgag aatgcgcagc aagacaaggg 120 catcaccatt ccacatgata tagacctggg tgactcccgt gtggttatcc aggattatga 180 taaccagcay gagcaag <210> 152 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> HEVConsORF2-s2 <400> 152 gtygtctcrg ccaatggcga gc 22 <210> 153 <211> 901 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-3p <400> 153 gttgtctcag ccaatggcga gccaacagta aagttatata catctgttga gaatgcgcag 60 caagacaagg gcatcaccat tccacatgat atagacctgg gtgactcccg tgtggttatc 120 caggattatg ataaccagca ygagcaagac cgacctactc cgtcacctgc cccctctcgc 180 cccttctcag ttcttcgtgc caatgatgtt ttgtggcttt ccctcactgc cgctgagtat 240 gaccagacta cgtatgggtc gtccaccaac cctatgtatg tctctgacac agttacgctt 300 gttaatgtgg ctactggtgc tcaggctgtt gcccgctccc ttgattggtc taaagttact 360 ctggacggcc gcccccttac taccattcag cagtattcta agacatttta tgttctcccg 420 ctccgcggga agctgtcctt ttgggaggct ggcacgacta aggccggcta cccttacaat 480 tataatacta ccgctagtga ccaaattttg attgagaatg cggccggcca ccgtgtcgct 540 atttccacct ataccactag cttaggtgcc ggtcctacct cgatctctgc ggtcggcgta 600 ctggctccac actctgccct tgccgttctt gaggatacta ttgattaccc cgcccgtgcc 660 catacttttg atgatttttg cccggagtgc cgtaccctag gtttgcaggg ttgtgcattc 720 cagtctacta ttgctgagct ccagcgttta aaaatgaagg taggtaaaac ccgggagtct 780 taattaattc cttctgtgcc cccttcgtag tttctttcgc ttttatttct tatttctgct 840 ttccgcgctc cctggaaaaa aaaaaaaaaa aaaaaaaaaa agtactagtc gacgcgtggc 900 c 901 <210> 154 <211> 27 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-gap sl <400> 154 tatagataac aataggttca cccagcg 27 <210> 155 <211> 25 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-gap al <400> 155 attcagtcga gtagaacgct tctgg 25 <210> 156 <211> 23 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-gap s2 <400> 156 cggactatgg ctacatcctg agg <210> 157 <211> 26 <212> DNA
<213> Hepatitis E virus <220>
<223> us2-gap a2 <400> 157 ttgactaacc aatcacagtc tgactc 26 <210> 158 <211> 462 <212> DNA
<213> Hepatitis E virus <220>
<223> 13906-gap <400> 158 cggactatgg ctacatcctg aggggctgct gggtatcttc cccccattct cccctgggca 60 tatttgggag tctgctaacc ccttttgcgg tgaggggact ttgtataccc gaacctggtc 120 aacctctggt ttttctagtg atttctcccc ccctgaggcg gccgctcctg cttcggctgc 180 cgccccgggg ttgccctacc ctactccacc tgttagtgat atctgggtgt taccaccgcc 240 ctcagaggaa tctcatgttg atgcggcatc tgtaccctct gttcctgagc ctgctggatt 300 gaccagccct attgtgctta cccccccccc cccccctcct cccgtgcgta agccggcaac 360 atccccgcct ccccgcactc gccgtctcct ttacacctac cccgacggcg ccaaggtgta 420 tgcggggtca ttgtttgagt cagactgtga ttggttagtc as 462 <210> 159 <211> 21 <212> DNA
<213> Hepatitis E virus <220>
<223> us-575a <400> 159 gccgggtggt agcagcacct c 21 <210> 160 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us-426s <400> 160 cgttgtgctt ttgctgcaga gacc <210> 161 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> us-84a <400> 161 gaaacggccg aaccaccaca gc 22 <210> 162 <211> 24 <212> DNA
<213> Hepatitis E virus <220>
<223> us-484s <400> 162 cagctgatgt tgcagaggct atgg 24 <210> 163 <211> 22 <212> DNA
<213> Hepatitis E virus <220>
<223> us-78a <400> 163 gccgaaccac cacagcattc gc 22 <210> 164 <211> 7277 <212> DNA
<213> Hepatitis E virus <220>
<223> us2fu11 <400> 164 tcgacagggg gcagaccacg tatgtggtcg atgccatgga ggcccatcag ttcattaagg 60 ctcctggcat tactactgct attgagcagg ctgctctggc tgcggctaat tccgccttgg 120 cgaatgctgt ggtggttcgg ccgtttcttt ctcgtgtgca aactgagatt cttattaatt 180 tgatgcaacc ccggcagttg gtcttccgcc ctgaggtgct ttggaatcat cctatccagc 240 gggttataca taatgaatta gagcagtact gccgggcccg ggctggtcgt tgtttggagg 300 ttggagccca cccgaggtcc attaatgaca accctaatgt cttgcatagg tgttttctta 360 gaccggtcgg ccgagatgtt cagcgctggt attctgcccc tacccgtggt cctgcggcca 420 attgccgccg ctccgcgttg cgtggtctcc cccctgtcga ccgcacctat tgttttgatg 480 gattttcccg ttgtgctttt gctgcagaga ccggtgtggc cctttactct ttgcatgacc 540 tttggccagc tgatgttgca gaggctatgg cccgccatgg gatgacacgc ttatacgccg 600 cactgcacct tccccccgag gtgctgctac cacccggcac ctaccacaca acctcgtacc 660 tcttgattca cgatggcaac cgcgctgttg taacttacga gggcgatact agtgcgggct 720 ataatcatga tgtctccata cttcgtgcat ggatccgtac tactaaaata gttggtgacc 780 atccattggt catagagcga gtgcgggcca ttgggtgtca ttttgtgctg ctgctcaccg 840 cagcccctga accgtcacct atgccttatg ttccctaccc tcgttcaacg gaggtgtatg 900 tccggtctat atttggccct ggcggctccc catccttgtt tccatcagcc tgctctacta 960 aatctacctt tcatgctgtc ccggttcaca tctgggatcr gctcatgctc tttggtgcca 1020 ccctgracga tcaggcgttc tgctgttcac ggcttatgac ttacctccgt ggtattagtt 1080 ataaggtcac tgtcggtgcg cttgtcgcta atgaggggtg gaacgcctct gaggatgctc 1140 ttactgcagt gatcactgcg gcctatctga ccatctgcca tcagcgttac cttcgcaccc 1200 aggcgatttc caagggcatg cgccggttgg aggttgagca tgctcagaaa tttatcacaa 1260 gactctacag ctggctattt gagaagtctg gccgtgacta catccccggc cgccagcttc 1320 aattttatgc acaatgccga cggtggcttt ctgcaggctt ccacctarac cccaggrtgc 1380 ttgtctttga tgaatcagtg ccatgccgtt gcaggacgtt tttgaagaag gtcgcgggta 1440 aattctgctg ttttatgcgg tggctggggc aggagtgtac ctgcttcttg gagccagccg 1500 agggtttagt tggtgatcaa ggtcatgaca acgaggccta tgaaggttct gaggtcgacc 1560 cagctgagcc tgcacatctt gatgtctcgg ggacttatgc cgtccatggg caccagcttg 1620 aggccctcta tagggcactt aatgtcccac atgatattgc cgctcgagcc tcccgactaa 1680 cggctactgt tgagctcgtt gctagtccgg accgcttaga gtgccgcact gtacttggta 1740 ataagacctt ccggacgacg gtggttgatg gcgcccatct tgaagcgaat ggccctgagg 1800 agtatgttct gtcatttgac gcctctcgcc agtctatggg ggccgggtcg cacagcctca 1860 cttatgagct cacccctgcc ggtctgcagg taaagatttc atctaatggt ctggattgca 1920 ctgccacatt ccccccyggt ggcgccccta gcgccgcgcc gggggaggtg gcsgccttct 1980 gcagtgctct ttatagatac aataggttca cccagcggca ttcgctgaca ggcggactat 2040 ggctacatcc tgaggggctg ctgggtatct tccccccatt ctcccctggg catatttggg 2100 agtctgctaa ccccttttgc ggtgagggga ctttgtatac ccgaacctgg tcaacctctg 2160 gtttttctag tgatttctcc ccccctgagg cggccgctcc tgcttcggct gccgccccgg 2220 ggttgcccta ccctactcca cctgttagtg atatctgggt gttaccaccg ccctcagagg 2280 aatctcatgt tgatgcggca tctgtaccct ctgttcctga gcctgctgga ttgaccagcc 2340 ctattgtgct tacccccccc cccccccctc ctcccgtgcg taagccggca acatccccgc 2400 ctccccgcac tcgccgtctc ctttacacct accccgacgg cgccaaggtg tatgcggggt 2460 cattgtktga gtcagactgt gattggttag tcaatgcctc aaaccctggc catcgccccg 2520 ggggtggcct ctgccatgct ttttatcaac gtttcccaga agcgttctac tcgactgaat 2580 tcatcatgcg cgagggcctt gcagcataca ctttaacccc gcgccctatt atccatgcag 2640 tggctcccga ctatagggtt gagcaaaacc cgaagaggct tgaggcagcg taccgggaaa 2700 cttgctcccg tcgtggcacc gctgcctacc cgcttttggg ctcgggtata taccaggtcc 2760 ctgttagcct cagttttgat gcctgggaac gcaatcaccg ccccggcgat gagctttact 2820 tgacagagcc cgccgcagcc tggtttgagg ctaataagcc ggcgcagccg gcgcttacta 2880 taactgagga cacggcccgt acggccaacc tggcattaga gattgatgcc gccacagagg 2940 ttggccgtgc ttgtgccggc tgcaccatca gccccgggat tgtgcactat cagtttaccg 3000 ccggggtccc gggctcaggc aagtcaaggt ccatacaaca gggagatgtc gatgtggtgg 3060 ttgtgcccac ccgggagctc cgtaacagct ggcgtcgccg gggttttgcg gccttcacac 3120 ctcacacagc ggcccgtgtt actatcggcc gccgcgttgt gattgatgag gctccatctc 3180 tcccaccgca cctgctgctg ttacacatgc agcgggcctc ctcggtccat ctccttggtg 3240 atccaaacca gattcctgct attgattttg agcatgccgg cctggtcccc gcgatccgcc 3300 ccgagcttgc gccaacgagc tggtggcacg ttacacaccg ttgcccggcc gatgtgtgcg 3360 agctcatacg tggggcctac cccaaaattc agaccacgag ccgtgtgcta cggtccctgt 3420 tttggaacga accggccatc ggccaaaagt tggtttttac gcaggctgct aaggctgcca 3480 accctggtgc gattacggtt cacgaagctc agggtgctac tttcacggag accacaatta 3540 tagccacggc cgacgctagg ggcctcattc agtcatcccg ggcccatgct atagtcgcac 3600 tcacccgcca tactgagaag tgtgttattt tggatgcccc cggcttgttg cgcgaggtcg 3660 gcatttcgga tgttattgtc aataactttt tccttgccgg tggagaggtc ggccatcacc 3720 gcccttctgt gatacctcgc ggcaatcctg atcagaacct cgggactcta caggcctttc 3780 cgccgtcatg tcagatcagt gcttaccatc agttggctga ggaactaggt catcgcccgg 3840 cccctgtcgc cgccgtcttg cccccttgcc ctgagcttga gcagggcctg ctctatatgc 3900 cacaagaact tactgtgtcc gatagcgtgc tggtttttga gcttacggat atagtccact 3960 gccgtatggc cgccccaagc cagcgaaagg ctgttctctc aacgcttgtg gggaggtacg 4020 gccgtaggac taaattatat gaggcggcgc attcagatgt ccgtgagtcc ctagcgaggt 4080 ttatccccac catcgggcct gttcgggcta ccacatgtga gctgtacgag ctggttgaag 4140 ccatggtaga gaagggtcag gacggatctg ccgtcctaga gctcgacctt tgcaatcgtg 4200 acgtctcgcg catcacattt ttccaaaagg attgcaataa gtttacaact ggtgagacta 4260 tcgcccatgg caaggttggc cagggcatat cggcctggag caagaccttc tgtgctctgt 4320 ttggcccgtg gttccgcgcc attgaaaagg aaatattggc cctactcccg cctaatatct 4380 tttatggcga cgcctatgag gagtcagtgt ttgctgccgc tgtgtccggg gcagggtcat 4440 gtatggtatt tgaaaatgac ttctcagagt ttgacagtac ccagaataat ttctctctcg 4500 gccttgagtg tgtggttatg gaggagtgcg gcatgcccca atggttaatt aggttgtacc 4560 atctggtccg gtcagcctgg attttgcagg cgccgaagga gtctcttaag gggttttgga 4620 agaagcactc tggtgagcct ggtacccttc tctggaacac tgtctggaac atggcgatta 4680 tagcacattg ctaygagttc cgtgactttc gtgttgccgc cttcaagggt gatgattcag 4740 tggtcctctg tagtgactac cgacagrgcc gtaacgcggc tgccttaatt gcaggctgtg 4800 ggctcaaatt gaaggttgat taccgcccta tcgggctata tgctggagtg gtggtggccc 4860 ccggtttggg gacactgccc gatgtggtgc gttttgccgg tcggttatct gagaagaatt 4920 ggggccctgg cccggagcgt gctgagcagc tgcgtcttgc tgtttgtgat ttccttcgag 4980 ggttgacgaa tgttgcgcag gtctgtgttg atgttgtgtc ccgtgtctat ggagttagcc 5040 ccgggctggt acataacctt attggcatgc tgcagaccat tgctgatggc aaggcccact 5100 ttacagaraa tattaaacct gtgcttgacc ttacaaattc catcatacaa cgggtggaat 5160 gaataacatg tcttttgcat cgcccatggg atcaccatgc gccctagggc tgttctgttg 5220 ttgctcttcg tgcttttgcc tatgctgccc gcgccaccgg ccggccagcc gtctggccgc 5280 cgtcgtgggc ggcgcagcgg cggtgccggc ggtggtttct ggggtgacag ggttgattct 5340 cagcccttcg ccctccccta tattcatcca accaacccct tcgccgccga tgtcgtttca 5400 caacccgggg ctggaactcg ccctcgacag ccgccccgcc cccttggytc cgcttggcgt 5460 gaccagtccc agcgcccctc cgctgccccc cgtcgtcgat ctgccccagc tggggctgcg 5520 ccgctgactg ccgtgtcacc ggctcctgac acagcccctg tacctgatgt tgactcacgt 5580 ggtgctattc tgcgccggca gtacaatttg tccacgtccc cgctcacgtc atctgtcgct 5640 tcgggtacta atttggtcct ctatgctgcc ccgctgaatc ccctcttgcc tctccaggat 5700 ggtaccaaca ctcatattat ggctactgag gcatccaatt atgcccagta tcgggttgtt 5760 cgagctacaa tccgttatcg cccgctggtg ccgaatgccg ttggtggcta tgccatttcc 5820 atttctttct ggccccaaac tacaactacc cctacttctg tcgatatgaa ttctattact 5880 tccacygatg ttaggatttt ggttcagccc ggtattgcct ccgagctagt catccccagt 5940 gagcgccttc attaccgtaa tcaaggctgg cgctctgttg agaccacggg tgtggctgag 6000 gaggaggcta cttccggtct ggtaatgctt tgcattcatg gctctcctgt taattcctac 6060 actaatacac cttacactgg tgcgctgggg cttcttgatt ttgcactaga gcttgaattt 6120 aggaatttga cacccgggaa caccaacacc cgtgtttccc ggtataccag cacagcccgc 6180 caccggctgc gccgtggtgc tgatgggact gctgagctta ctaccacagc agccacacgt 6240 ttcatgaagg acctgcactt cgctggcacg aatggcgttg gtgaggtggg tcgtggtatc 6300 gccctgacac tgttcaatct cgctgatacg cttctcggcg gtttaccgac agaattgatt 6360 tcgtcggctg ggggccaact gttttactcc cgcccggttg tctcagccaa tggcgagcca 6420 acagtaaagt tatatacatc tgttgagaat gcgcagcaag acaagggcat caccattcca 6480 catgatatag acctgggtga ctcccgtgtg gttatccagg attatgataa ccagcaygag 6540 caagaccgac ctactccgtc acctgccccc tctcgcccct tctcagttct tcgtgccaat 6600 gatgttttgt ggctttccct cactgccgct gagtatgacc agactacgta tgggtcgtcc 6660 accaacccta tgtatgtctc tgacacagtt acgcttgtta atgtggctac tggtgctcag 6720 gctgttgccc gctcccttga ttggtctaaa gttactctgg acggccgccc ccttactacc 6780 attcagcagt attctaagac attttatgtt ctcccgctcc gcgggaagct gtccttttgg 6840 gaggctggca cgactaaggc cggctaccct tacaattata atactaccgc tagtgaccaa 6900 attttgattg agaatgcggc cggccaccgt gtcgctattt ccacctatac cactagctta 6960 ggtgccggtc ctacctcgat ctctgcggtc ggcgtactgg ctccacactc tgcccttgcc 7020 gttcttgagg atactattga ttaccccgcc cgtgcccata cttttgatga tttttgcccg 7080 gagtgccgta ccctaggttt gcagggttgt gcattccagt ctactattgc tgagctccag 7140 cgtttaaaaa tgaaggtagg taaaacccgg gagtcttaat taattccttc tgtgccccct 7200 tcgtagtttc tttcgctttt atttcttatt tctgctttcc gcgctccctg gaaaaaaaaa 7260 aaaaaaaaaa aaaaaaa 7277 <210> 165 <211> 7277 <212> DNA

<213> Hepatitis E virus <220>
<221> CDS
<222> (36)..{5162) <223> orfl <220>
<221> CDS
<222> (5197)..(7179) <223> orf2 <220>
<223> orf3 at positions 5159-5527 <220>
<223> us2fu11 <400>

tcgacagggg atg gaggcc catcagttc 53 gcagaccacg tatgtggtcg atgcc Met GluAla HisGlnPhe attaaggetcct ggcatt actactgetattgag cagget getctgget 101 IleLysAlaPro GlyIle ThrThrAlaIleGlu GlnAla AlaLeuAla gcggetaattcc gccttg gcgaatgetgtggtg gttcgg ccgtttctt 149 AlaAlaAsnSer AlaLeu AlaAsnAlaValVal ValArg ProPheLeu tctcgtgtgcaa actgag attcttattaatttg atgcaa ccccggcag 197 SerArgValGln ThrGlu IleLeuIleAsnLeu MetGln ProArgGln ttggtcttccgc cctgaggtg ctttggaat catcctatc cagcgggtt 245 LeuValPheArg ProGluVal LeuTrpAsn HisProIle GlnArgVal atacataatgaa ttagagcag tactgccgg gcccggget ggtcgttgt 293 IleHisAsnGlu LeuGluGln TyrCysArg AlaArgAla GlyArgCys ttggaggttgga gcccacccg aggtccatt aatgacaac cctaatgtc 341 LeuGluValGly AlaHisPro ArgSerIle AsnAspAsn ProAsnVal ttgcataggtgt tttcttaga ccggtcggc cgagatgtt cagcgctgg 389 LeuHisArgCys PheLeuArg ProValGly ArgAspVal GlnArgTrp tattctgcccct acccgtggt cctgcggcc aattgccgc cgctccgcg 437 TyrSerAlaPro ThrArgGly ProAlaAla AsnCysArg ArgSerAla ttg cgt ggt ctc ccc cct gtc gac cgc acc tat tgt ttt gat gga ttt 485 6~
Leu Arg Gly Leu Pro Pro Val Asp Arg Thr Tyr Cys Phe Asp Gly Phe tcc cgt tgt get ttt get gca gag acc ggt gtg gcc ctt tac tct ttg 533 Ser Arg Cys Ala Phe Ala Ala Glu Thr Gly Val Ala Leu Tyr Ser Leu cat gac ctt tgg cca get gat gtt gca gag get atg gcc cgc cat ggg 581 His Asp Leu Trp Pro Ala Asp Val Ala Glu Ala Met Ala Arg His Gly atg aca cgc tta tac gcc gca ctg cac ctt ccc ccc gag gtg ctg cta 629 Met Thr Arg Leu Tyr Ala Ala Leu His Leu Pro Pro Glu Val Leu Leu cca ccc ggc acc tac cac aca acc tcg tac ctc ttg att cac gat ggc 677 Pro Pro Gly Thr Tyr His Thr Thr Ser Tyr Leu Leu Ile His Asp Gly aac cgc get gtt gta act tac gag ggc gat act agt gcg ggc tat aat 725 Asn Arg Ala Val Val Thr Tyr Glu Gly Asp Thr Ser Ala Gly Tyr Asn cat gat gtc tcc ata ctt cgt gca tgg atc cgt act act aaa ata gtt 773 His Asp Val Ser Ile Leu Arg Ala Trp Ile Arg Thr Thr Lys Ile Val ggt gac cat cca ttg gtc ata gag cga gtg cgg gcc att ggg tgt cat 821 Gly Asp His Pro Leu Val Ile Glu Arg Val Arg Ala Ile Gly Cys His ttt gtg ctg ctg ctc acc gca gcc cct gaa ccg tca cct atg cct tat 869 Phe Val Leu Leu Leu Thr Ala Ala Pro Glu Pro Ser Pro Met Pro Tyr gtt ccc tac cct cgt tca acg gag gtg tat gtc cgg tct ata ttt ggc 917 Val Pro Tyr Pro Arg Ser Thr Glu Val Tyr Val Arg Ser Ile Phe Gly cct ggc ggc tcc cca tcc ttg ttt cca tca gcc tgc tct act aaa tct 965 Pro Gly Gly Ser Pro Ser Leu Phe Pro Ser Ala Cys Ser Thr Lys Ser acc ttt cat get gtc ccg gtt cac atc tgg gat crg ctc atg ctc ttt 1013 Thr Phe His Ala Val Pro Val His Ile Trp Asp Xaa Leu Met Leu Phe ggt gcc acc ctg rac gat cag gcg ttc tgc tgt tca cgg ctt atg act 1061 Gly Ala Thr Leu Xaa Asp Gln Ala Phe Cys Cys Ser Arg Leu Met Thr tac ctc cgt ggt att agt tat aag gtc act gtc ggt gcg ctt gtc get 1109 Tyr Leu Arg Gly Ile Ser Tyr Lys Val Thr Val Gly Ala Leu Val Ala aat gag ggg tgg aac gcc tct gag gat get ctt act gca gtg atc act 1157 6$
Asn Glu Gly Trp Asn Ala Ser Glu Asp Ala Leu Thr Ala Val Ile Thr gcg gcc tat ctg acc atc tgc cat cag cgt tac ctt cgc acc cag gcg 1205 Ala Ala Tyr Leu Thr Ile Cys His Gln Arg Tyr Leu Arg Thr Gln Ala att tcc aag ggc atg cgc cgg ttg gag gtt gag cat get cag aaa ttt 1253 Ile Ser Lys Gly Met Arg Arg Leu Glu Val Glu His Ala Gln Lys Phe atc aca aga ctc tac agc tgg cta ttt gag aag tct ggc cgt gac tac 1301 Ile Thr Arg Leu Tyr Ser Trp Leu Phe Glu Lys Ser Gly Arg Asp Tyr atc ccc ggc cgc cag ctt caa ttt tat gca caa tgc cga cgg tgg ctt 1349 Ile Pro Gly Arg Gln Leu Gln Phe Tyr Ala Gln Cys Arg Arg Trp Leu tct gca ggc ttc cac cta rac ccc agg rtg ctt gtc ttt gat gaa tca 1397 Ser Ala Gly Phe His Leu Xaa Pro Arg Xaa Leu Val Phe Asp Glu Ser gtg cca tgc cgt tgc agg acg ttt ttg aag aag gtc gcg ggt aaa ttc 1445 Val Pro Cys Arg Cys Arg Thr Phe Leu Lys Lys Val Ala Gly Lys Phe tgc tgt ttt atg cgg tgg ctg ggg cag gag tgt acc tgc ttc ttg gag 1493 Cys Cys Phe Met Arg Trp Leu Gly Gln Glu Cys Thr Cys Phe Leu Glu cca gcc gag ggt tta gtt ggt gat caa ggt cat gac aac gag gcc tat 1541 Pro Ala Glu Gly Leu Val Gly Asp Gln Gly His Asp Asn Glu Ala Tyr gaa ggt tct gag gtc gac cca get gag cct gca cat ctt gat gtc tcg 1589 Glu Gly Ser Glu Val Asp Pro Ala Glu Pro Ala His Leu Asp Val Ser ggg act tat gcc gtc cat ggg cac cag ctt gag gcc ctc tat agg gca 1637 Gly Thr Tyr Ala Val His Gly His Gln Leu Glu Ala Leu Tyr Arg Ala ctt aat gtc cca cat gat att gcc get cga gcc tcc cga cta acg get 1685 Leu Asn Val Pro His Asp Ile Ala Ala Arg Ala Ser Arg Leu Thr Ala act gtt gag ctc gtt get agt ccg gac cgc tta gag tgc cgc act gta 1733 Thr Val Glu Leu Val Ala Ser Pro Asp Arg Leu Glu Cys Arg Thr Val ctt ggt aat aag acc ttc cgg acg acg gtg gtt gat ggc gcc cat ctt 1781 Leu Gly Asn Lys Thr Phe Arg Thr Thr Val Val Asp Gly Ala His Leu gaa gcg aat ggc cct gag gag tat gtt ctg tca ttt gac gcc tct cgc 1829 Glu Ala Asn Gly Pro Glu Glu Tyr Val Leu Ser Phe Asp Ala Ser Arg cag tct atg ggg gcc ggg tcg cac agc ctc act tat gag ctc acc cct 1877 Gln Ser Met Gly Ala Gly Ser His Ser Leu Thr Tyr Glu Leu Thr Pro gcc ggt ctg cag gta aag att tca tct aat ggt ctg gat tgc act gcc 1925 Ala Gly Leu Gln Val Lys Ile Ser Ser Asn Gly Leu Asp Cys Thr Ala aca ttc ccc ccy ggt ggc gcc cct agc gcc gcg ccg ggg gag gtg gcs 1973 Thr Phe Pro Xaa Gly Gly Ala Pro 8er Ala Ala Pro Gly Glu Val Xaa gcc ttc tgc agt get ctt tat aga tac aat agg ttc acc cag cgg cat 2021 Ala Phe Cys Ser Ala Leu Tyr Arg Tyr Asn Arg Phe Thr Gln Arg His tcg ctg aca ggc gga cta tgg cta cat cct gag ggg ctg ctg ggt atc 2069 Ser Leu Thr Gly Gly Leu Trp Leu His Pro Glu Gly Leu Leu Gly Ile ttc ccc cca ttc tcc cct ggg cat att tgg gag tct get aac ccc ttt 2117 Phe Pro Pro Phe Ser Pro Gly His Ile Trp Glu Ser Ala Asn Pro Phe tgc ggt gag ggg act ttg tat acc cga acc tgg tca acc tct ggt ttt 2165 Cys Gly Glu Gly Thr Leu Tyr Thr Arg Thr Trp Ser Thr Ser Gly Phe tct agt gat ttc tcc ccc cct gag gcg gcc get cct get tcg get gcc 2213 Ser Ser Asp Phe Ser Pro Pro Glu Ala Ala Ala Pro Ala Ser Ala Ala gcc ccg ggg ttg ccc tac cct act cca cct gtt agt gat atc tgg gtg 2261 Ala Pro Gly Leu Pro Tyr Pro Thr Pro Pro Val Ser Asp Ile Trp Val tta cca ccg ccc tca gag gaa tct cat gtt gat gcg gca tct gta ccc 2309 Leu Pro Pro Pro Ser Glu Glu Ser His Val Asp Ala Ala Ser Val Pro tct gtt cct gag cct get gga ttg acc agc cct att gtg ctt acc ccc 2357 Ser Val Pro Glu Pro Ala Gly Leu Thr Ser Pro Ile Val Leu Thr Pro ccc ccc ccc cct cct ccc gtg cgt aag ccg gca aca tcc ccg cct ccc 2405 Pro Pro Pro Pro Pro Pro Val Arg Lys Pro Ala Thr Ser Pro Pro Pro cgc act cgc cgt ctc ctt tac acc tac ccc gac ggc gcc aag gtg tat 2453 Arg Thr Arg Arg Leu Leu Tyr Thr Tyr Pro Asp Gly Ala Lys Val Tyr gcg ggg tca ttg tkt gag tca gac tgt gat tgg tta gtc aat gcc tca 2501 Ala Gly Ser Leu Xaa Glu Ser Asp Cys Asp Trp Leu Val Asn Ala Ser aac cct ggc cat cgc ccc ggg ggt ggc ctc tgc cat get ttt tat caa 2549 Asn Pro Gly His Arg Pro Gly Gly Gly Leu Cys His Ala Phe Tyr Gln cgt ttc cca gaa gcg ttc tac tcg act gaa ttc atc atg cgc gag ggc 2597 Arg Phe Pro Glu Ala Phe Tyr Ser Thr Glu Phe Ile Met Arg Glu Gly ctt gca gca tac act tta acc ccg cgc cct att atc cat gca gtg get 2645 Leu Ala Ala Tyr Thr Leu Thr Pro Arg Pro Ile Ile His Ala Val Ala ccc gac tat agg gtt gag caa aac ccg aag agg ctt gag gca gcg tac 2693 Pro Asp Tyr Arg Val Glu Gln Asn Pro Lys Arg Leu Glu Ala Ala Tyr cgg gaa act tgc tcc cgt cgt ggc acc get gcc tac ccg ctt ttg ggc 2741 Arg Glu Thr Cys Ser Arg Arg Gly Thr Ala Ala Tyr Pro Leu Leu Gly tcg ggt ata tac cag gtc cct gtt agc ctc agt ttt gat gcc tgg gaa 2789 Ser Gly Ile Tyr Gln Val Pro Val Ser Leu Ser Phe Asp Ala Trp Glu cgc aat cac cgc ccc ggc gat gag ctt tac ttg aca gag ccc gcc gca 2837 Arg Asn His Arg Pro Gly Asp Glu Leu Tyr Leu Thr Glu Pro Ala Ala gcc tgg ttt gag get aat aag ccg gcg cag ccg gcg ctt act ata act 2885 Ala Trp Phe Glu Ala Asn Lys Pro Ala Gln Pro Ala Leu Thr Ile Thr gag gac acg gcc cgt acg gcc aac ctg gca tta gag att gat gcc gcc 2933 Glu Asp Thr Ala Arg Thr Ala Asn Leu Ala Leu Glu Ile Asp Ala Ala aca gag gtt ggc cgt get tgt gcc ggc tgc acc atc agc ccc ggg att 2981 Thr Glu Val Gly Arg Ala Cys Ala Gly Cys Thr Ile Ser Pro Gly Ile gtg cac tat cag ttt acc gcc ggg gtc ccg ggc tca ggc aag tca agg 3029 Val His Tyr Gln Phe Thr Ala Gly Val Pro Gly Ser Gly Lys Ser Arg tcc ata caa cag gga gat gtc gat gtg gtg gtt gtg ccc acc cgg gag 3077 Ser Ile Gln Gln Gly Asp Val Asp Val Val Val Val Pro Thr Arg Glu ctc cgt aac agc tgg cgt cgc cgg ggt ttt gcg gcc ttc aca cct cac 3125 Leu Arg Asn Ser Trp Arg Arg Arg Gly Phe Ala Ala Phe Thr Pro His aca gcg gcc cgt gtt act atc ggc cgc cgc gtt gtg att gat gag get 3173 ~l Thr Ala Ala Arg Val Thr Ile Gly Arg Arg Val Val Ile Asp Glu Ala cca tct ctc cca ccg cac ctg ctg ctg tta cac atg cag cgg gcc tcc 3221 Pro Ser Leu Pro Pro His Leu Leu Leu Leu His Met Gln Arg Ala Ser tcg gtc cat ctc ctt ggt gat cca aac cag att cct get att gat ttt 3269 Ser Val His Leu Leu Gly Asp Pro Asn Gln Ile Pro Ala Ile Asp Phe gag cat gcc ggc ctg gtc ccc gcg atc cgc ccc gag ctt gcg cca acg 3317 Glu His Ala Gly Leu Val Pro Ala Ile Arg Pro Glu Leu Ala Pro Thr agc tgg tgg cac gtt aca cac cgt tgc ccg gcc gat gtg tgc gag ctc 3365 Ser Trp Trp His Val Thr His Arg Cys Pro Ala Asp Val Cys Glu Leu ata cgt ggg gcc tac ccc aaa att cag acc acg agc cgt gtg cta cgg 3413 Ile Arg Gly Ala Tyr Pro Lys Ile Gln Thr Thr Ser Arg Val Leu Arg tcc ctg ttt tgg aac gaa ccg gcc atc ggc caa aag ttg gtt ttt acg 3461 Ser Leu Phe Trp Asn Glu Pro Ala Ile Gly Gln Lys Leu Val Phe Thr cag get get aag get gcc aac cct ggt gcg att acg gtt cac gaa get 3509 Gln Ala Ala Lys Ala Ala Asn Pro Gly Ala Ile Thr Val His Glu Ala cag ggt get act ttc acg gag acc aca att ata gcc acg gcc gac get 3557 Gln Gly Ala Thr Phe Thr Glu Thr Thr Ile Ile Ala Thr Ala Asp Ala aggggc ctcattcagtca tcccgggcc catgetata gtcgcactc acc 3605 ArgGly LeuIleGlnSer SerArgAla HisAlaIle ValAlaLeu Thr cgccat actgagaagtgt gttattttg gatgccccc ggcttgttg cgc 3653 ArgHis ThrGluLysCys ValIleLeu AspAlaPro GlyLeuLeu Arg gaggtc ggcatttcggat gttattgtc aataacttt ttccttgcc ggt 3701 GluVal GlyIleSerAsp ValIleVal AsnAsnPhe PheLeuAla Gly ggagag gtcggccatcac cgcccttct gtgatacct cgcggcaat cct 3749 GlyGlu ValGlyHisHis ArgProSer ValIlePro ArgGlyAsn Pro gatcag aacctcgggact ctacaggcc tttccgccg tcatgtcag atc 3797 AspGln AsnLeuGlyThr LeuGlnAla PheProPro SerCysGln Ile agt get tac cat cag ttg get gag gaa cta ggt cat cgc ccg gcc cct 3845 SerAla TyrHisGln LeuAlaGlu GluLeuGly HisArgPro AlaPro gtcgcc gccgtcttg cccccttgc cctgagctt gagcagggc ctgctc 3893 ValAla AlaValLeu ProProCys ProGluLeu GluGlnGly LeuLeu tatatg ccacaagaa cttactgtg tccgatagc gtgctggtt tttgag 3941 TyrMet ProGlnGlu LeuThrVal SerAspSer ValLeuVal PheGlu cttacg gatatagtc cactgccgt atggccgcc ccaagccag cgaaag 3989 LeuThr AspIleVal HisCysArg MetAlaAla ProSerGln ArgLys getgtt ctctcaacg cttgtgggg aggtacggc cgtaggact aaatta 4037 AlaVal LeuSerThr LeuValGly ArgTyrGly ArgArgThr LysLeu tatgag gcggcgcat tcagatgtc cgtgagtcc ctagcgagg tttatc 4085 TyrGlu AlaAlaHis SerAspVal ArgGluSer LeuAlaArg PheIle cccacc atcgggcct gttcggget accacatgt gagctgtac gagctg 4133 ProThr IleGlyPro ValArgAla ThrThrCys GluLeuTyr GluLeu gttgaa gccatggta gagaagggt caggacgga tctgccgtc ctagag 4181 ValGlu AlaMetVal GluLysGly GlnAspGly SerAlaVal LeuGlu ctcgac ctttgcaat cgtgacgtc tcgcgcatc acatttttc caaaag 4229 LeuAsp LeuCysAsn ArgAspVal SerArgIle ThrPhePhe GlnLys gattgc aataagttt acaactggt gagactatc gcccatggc aaggtt 4277 AspCys AsnLysPhe ThrThrGly GluThrIle AlaHisGly LysVal ggc cagggcatatcg gcctggagcaag accttc tgtgetctgttt ggc 4325 Gly GlnGlyIleSer AlaTrpSerLys ThrPhe CysAlaLeuPhe Gly ccg tggttccgcgcc attgaaaaggaa atattg gccctactcccg cct 4373 Pro TrpPheArgAla IleGluLysGlu IleLeu AlaLeuLeuPro Pro aat atcttttatggc gacgcctatgag gagtca gtgtttgetgcc get 4421 Asn IlePheTyrGly AspAlaTyrGlu GluSer ValPheAlaAla Ala gtg tccggggcaggg tcatgtatggta tttgaa aatgacttctca gag 4469 Val SerGlyAlaGly SerCysMetVal PheGlu AsnAspPheSer Glu ttt gac agt acc cag aat aat ttc tct ctc ggc ctt gag tgt gtg gtt 4517 Phe Asp Ser Thr Gln Asn Asn Phe Ser Leu Gly Leu Glu Cys Val Val atg gag gag tgc ggc atg ccc caa tgg tta att agg ttg tac cat ctg 4565 Met Glu Glu Cys Gly Met Pro Gln Trp Leu Ile Arg Leu Tyr His Leu gtc cgg tca gcc tgg att ttg cag gcg ccg aag gag tct ctt aag ggg 4613 Val Arg Ser Ala Trp Ile Leu Gln Ala Pro Lys Glu Ser Leu Lys Gly ttt tgg aag aag cac tct ggt gag cct ggt acc ctt ctc tgg aac act 4661 Phe Trp Lys Lys His Ser Gly Glu Pro Gly Thr Leu Leu Trp Asn Thr gtc tgg aac atg gcg att ata gca cat tgc tay gag ttc cgt gac ttt 4709 Val Trp Asn Met Ala Ile Ile Ala His Cys Xaa Glu Phe Arg Asp Phe cgt gtt gcc gcc ttc aag ggt gat gat tca gtg gtc ctc tgt agt gac 4757 Arg Val Ala Ala Phe Lys Gly Asp Asp Ser Val Val Leu Cys Ser Asp tac cga cag rgc cgt aac gcg get gcc tta att gca ggc tgt ggg ctc 4805 Tyr Arg Gln Xaa Arg Asn Ala Ala Ala Leu Ile.Ala Gly Cys Gly Leu aaa ttg aag gtt gat tac cgc cct atc ggg cta tat get gga gtg gtg 4853 Lys Leu Lys Val Asp Tyr Arg Pro Ile Gly Leu Tyr Ala Gly Val Val gtg gcc ccc ggt ttg ggg aca ctg ccc gat gtg gtg cgt ttt gcc ggt 4901 Val Ala Pro Gly Leu Gly Thr Leu Pro Asp Val Val Arg Phe Ala Gly cgg tta tct gag aag aat tgg ggc cct ggc ccg gag cgt get gag cag 4949 Arg Leu Ser Glu Lys Asn Trp Gly Pro Gly Pro Glu Arg Ala Glu Gln ctg cgt ctt get gtt tgt gat ttc ctt cga ggg ttg acg aat gtt gcg 4997 Leu Arg Leu Ala Val Cys Asp Phe Leu Arg Gly Leu Thr Asn Val Ala cag gtc tgt gtt gat gtt gtg tcc cgt gtc tat gga gtt agc ccc ggg 5045 Gln Val Cys Val Asp Val Val Ser Arg Val Tyr Gly Val Ser Pro Gly ctg gta cat aac ctt att ggc atg ctg cag acc att get gat ggc aag 5093 Leu Val His Asn Leu Ile Gly Met Leu Gln Thr Ile Ala Asp Gly Lys gcc cac ttt aca gar aat att aaa cct gtg ctt gac ctt aca aat tcc 5141 Ala His Phe Thr Xaa Asn Ile Lys Pro Val Leu Asp Leu Thr Asn Ser atc ata caa cgg gtg gaa tga ataacatgtc ttttgcatcg cccatgggat cacc 5196 WO 99/19'732 PCT1US98/21941 Ile Ile Gln Arg Val Glu atg cgc cct agg get gtt ctg ttg ttg ctc ttc gtg ctt ttg cct atg 5244 Met Arg Pro Arg Ala Val Leu Leu Leu Leu Phe Val Leu Leu Pro Met ctg ccc gcg cca ccg gcc ggc cag ccg tct ggc cgc cgt cgt ggg cgg 5292 Leu Pro Ala Pro Pro Ala Gly Gln Pro Ser Gly Arg Arg Arg Gly Arg cgc agc ggc ggt gcc ggc ggt ggt ttc tgg ggt gac agg gtt gat tct 5340 Arg Ser Gly Gly Ala Gly Gly Gly Phe Trp Gly Asp Arg Val Asp Ser cag ccc ttc gcc ctc ccc tat att cat cca acc aac ccc ttc gcc gcc 5388 Gln Pro Phe Ala Leu Pro Tyr Ile His Pro Thr Asn Pro Phe Ala Ala gat gtc gtt tca caa ccc ggg get gga act cgc cct cga cag ccg ccc 5436 Asp Val Val Ser Gln Pro Gly Ala Gly Thr Arg Pro Arg Gln Pro Pro cgc ccc ctt ggy tcc get tgg cgt gac cag tcc cag cgc ccc tcc get 5484 Arg Pro Leu Xaa Ser Ala Trp Arg Asp Gln Ser Gln Arg Pro Ser Ala gcc ccc cgt cgt cga tct gcc cca get ggg get gcg ccg ctg act gcc 5532 Ala Pro Arg Arg Arg Ser Ala Pro Ala Gly Ala Ala Pro Leu Thr Ala gtg tca ccg get cct gac aca gcc cct gta cct gat gtt gac tca cgt 5580 Val Ser Pro Ala Pro Asp Thr Ala Pro Val Pro Asp Val Asp Ser Arg ggt get att ctg cgc cgg cag tac aat ttg tcc acg tcc ccg ctc acg 5628 Gly Ala Ile Leu Arg Arg Gln Tyr Asn Leu Ser Thr Ser Pro Leu Thr tca tct gtc get tcg ggt act aat ttg gtc ctc tat get gcc ccg ctg 5676 Ser Ser Val Ala Ser Gly Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu aat ccc ctc ttg cct ctc cag gat ggt acc aac act cat att atg get 5724 Asn Pro Leu Leu Pro Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala act gag gca tcc aat tat gcc cag tat cgg gtt gtt cga get aca atc 5772 Thr Glu Ala Ser Asn Tyr Ala Gln Tyr Arg Val Val Arg Ala Thr Ile cgt tat cgc ccg ctg gtg ccg aat gcc gtt ggt ggc tat gcc att tcc 5820 Arg Tyr Arg Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser att tct ttc tgg ccc caa act aca act acc cct act tct gtc gat atg 5868 Ile Ser Phe Trp Pro Gln Thr Thr Thr Thr Pro Thr Ser Val Asp Met aat tct att act tcc acy gat gtt agg att ttg gtt cag ccc ggt att 5916 Asn Ser Ile Thr Ser Xaa Asp Val Arg Ile Leu Val Gln Pro Gly Ile gcc tcc gag cta gtc atc ccc agt gag cgc ctt cat tac cgt aat caa 5964 Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln ggc tgg cgc tct gtt gag acc acg ggt gtg get gag gag gag get act 6012 Gly Trp Arg Ser Val Glu Thr Thr Gly Val Ala Glu Glu Glu Ala Thr tcc ggt ctg gta atg ctt tgc att cat ggc tct cct gtt aat tcc tac 6060 Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Pro Val Asn Ser Tyr act aat aca cct tac act ggt gcg ctg ggg ctt ctt gat ttt gca cta 6108 Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu gag ctt gaa ttt agg aat ttg aca ccc ggg aac acc aac acc cgt gtt 6156 Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn Thr Asn Thr Arg Val tcc cgg tat acc agc aca gcc cgc cac cgg ctg cgc cgt ggt get gat 6204 Ser Arg Tyr Thr Ser Thr Ala Arg His Arg Leu Arg Arg Gly Ala Asp ggg act get gag ctt act acc aca gca gcc aca cgt ttc atg aag gac 6252 Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp ctg cac ttc get ggc acg aat ggc gtt ggt gag gtg ggt cgt ggt atc 6300 Leu His Phe Ala Gly Thr Asn Gly Val Gly Glu Val Gly Arg Gly Ile gcc ctg aca ctg ttc aat ctc get gat acg ctt ctc ggc ggt tta ccg 6348 Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro aca gaa ttg att tcg tcg get ggg ggc caa ctg ttt tac tcc cgc ccg 6396 Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro gtt gtc tca gcc aat ggc gag cca aca gta aag tta tat aca tct gtt 6444 Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val gag aat gcg cag caa gac aag ggc atc acc att cca cat gat ata gac 6492 Glu Asn Ala Gln Gln Asp Lys Gly Ile Thr Ile Pro His Asp Ile Asp ctg ggt gac tcc cgt gtg gtt atc cag gat tat gat aac cag cay gag 6540 WO 99!19732 PCT/US98/21941 Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln Xaa Glu caa gac cga cct act ccg tca cct gcc ccc tct cgc ccc ttc tca gtt 6588 Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val ctt cgt gcc aat gat gtt ttg tgg ctt tcc ctc act gcc get gag tat 6636 Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr gaccagact acgtatggg tcgtccaccaac cctatg tatgtctct gac 6684 AspGlnThr ThrTyrGly SerSerThrAsn ProMet TyrValSer Asp acagttacg cttgttaat gtggetactggt getcag getgttgcc cgc 6732 ThrValThr LeuValAsn ValAlaThrGly AlaGln AlaValAla Arg tcccttgat tggtctaaa gttactctggac ggccgc ccccttact acc 6780 SerLeuAsp TrpSerLys ValThrLeuAsp GlyArg ProLeuThr Thr attcagcag tattctaag acattttatgtt ctcccg ctccgcggg aag 6828 IleGlnGln TyrSerLys ThrPheTyrVal LeuPro LeuArgGly Lys ctgtccttt tgggagget ggcacgactaag gccggc tacccttac aat 6876 LeuSerPhe TrpGluAla GlyThrThrLys AlaGly TyrProTyr Asn tataatact accgetagt gaccaaattttg attgag aatgcggcc ggc 6924 TyrAsnThr ThrAlaSer AspGlnIleLeu IleGlu AsnAlaAla Gly caccgtgtc getatttcc acctataccact agctta ggtgccggt cct 6972 HisArgVal AlaIleSer ThrTyrThrThr SerLeu GlyAlaGly Pro acctcgatc tctgcggtc ggcgtactgget ccacac tctgccctt gcc 7020 ThrSerIle SerAlaVal GlyValLeuAla ProHis SerAlaLeu Ala gttcttgag gatactatt gattaccccgcc cgtgcc catactttt gat 7068 ValLeuGlu AspThrIle AspTyrProAla ArgAla HisThrPhe Asp gatttttgc ccggagtgc cgtaccctaggt ttgcag ggttgtgca ttc 7116 AspPheCys ProGluCys ArgThrLeuGly LeuGln GlyCysAla Phe cagtctact attgetgag ctccagcgttta aaaatg aaggtaggt aaa 7164 GlnSerThr IleAlaGlu LeuGlnArgLeu LysMet LysValGly Lys acc cgg gag tct taa ttaattcctt ctgtgccccc ttcgtagttt ctttcgcttt 7219 Thr Arg Glu Ser tatttcttat ttctgctttc cgcgctccct ggaaaaaaaa aaaaaaaaaa aaaaaaaa 7277 <210> 166 <211> 1708 <212> PRT
<213> Hepatitis E virus <400> 166 Met Glu Ala His Gln Phe Ile Lys Ala Pro Gly Ile Thr Thr Ala Ile Glu Gln Ala Ala Leu Ala Ala Ala Asn Ser Ala Leu Ala Asn Ala Val Val Val Arg Pro Phe Leu Ser Arg Val Gln Thr Glu Ile Leu Ile Asn Leu Met Gln Pro Arg Gln Leu Val Phe Arg Pro Glu Val Leu Trp Asn His Pro Ile Gln Arg Val Ile His Asn Glu Leu Glu Gln Tyr Cys Arg Ala Arg Ala Gly Arg Cys Leu Glu Val Gly Ala His Pro Arg Ser Ile Asn Asp Asn Pro Asn Val Leu His Arg Cys Phe Leu Arg Pro Val Gly Arg Asp Val Gln Arg Trp Tyr Ser Ala Pro Thr Arg Gly Pro Ala Ala Asn Cys Arg Arg Ser Ala Leu Arg Gly Leu Pro Pro Val Asp Arg Thr Tyr Cys Phe Asp Gly Phe Ser Arg Cys Ala Phe Ala Ala Glu Thr Gly Val Ala Leu Tyr Ser Leu His Asp Leu Trp Pro Ala Asp Val Ala Glu Ala Met Ala Arg His Gly Met Thr Arg Leu Tyr Ala Ala Leu His Leu Pro Pro Glu Val Leu Leu Pro Pro Gly Thr Tyr His Thr Thr Ser Tyr Leu Leu Ile His Asp Gly Asn Arg Ala Val Val Thr Tyr Glu Gly Asp Thr Ser Ala Gly Tyr.Asn His Asp Val Ser Ile Leu Arg Ala Trp Ile Arg Thr Thr Lys Ile Val Gly Asp His Pro Leu Val Ile Glu Arg Val Arg Ala Ile Gly Cys His Phe VaI Leu Leu Leu Thr Ala Ala Pro Glu Pro Ser Pro Met Pro Tyr Val Pro Tyr Pro Arg Ser Thr Glu Val Tyr Val Arg Ser Ile Phe Gly Pro Gly Gly Ser Pro Ser Leu Phe Pro Ser Ala Cys Ser Thr Lys Ser Thr Phe His Ala Val Pro Val His Ile Trp Asp Xaa Leu Met Leu Phe Gly Ala Thr Leu Xaa Asp Gln Ala Phe Cys Cys Ser Arg Leu Met Thr Tyr Leu Arg Gly Ile Ser Tyr Lys Val Thr Val Gly Ala Leu Val Ala Asn Glu Gly Trp Asn Ala Ser Glu Asp Ala Leu Thr Ala Val Ile Thr Ala Ala Tyr Leu Thr Ile Cys His Gln Arg Tyr Leu Arg Thr Gln Ala Ile Ser Lys Gly Met Arg Arg Leu Glu Val Glu His Ala Gln Lys Phe Ile Thr Arg Leu Tyr Ser Trp Leu Phe Glu Lys Ser Gly Arg Asp Tyr Ile Pro Gly Arg Gln Leu Gln Phe Tyr Ala Gln Cys Arg Arg Trp Leu Ser Ala Gly Phe His Leu Xaa Pro Arg Xaa 435 , 440 445 Leu Val Phe Asp Glu Ser Val Pro Cys Arg Cys Arg Thr Phe Leu Lys Lys Val Ala Gly Lys Phe Cys Cys Phe Met Arg Trp Leu Gly Gln Glu Cys Thr Cys Phe Leu Glu Pro Ala Glu Gly Leu Val Gly Asp Gln Gly His Asp Asn Glu Ala Tyr Glu Gly Ser Glu Val Asp Pro Ala Glu Pro Ala His Leu Asp Val Ser Gly Thr Tyr Ala Val His Gly His Gln Leu Glu Ala Leu Tyr Arg Ala Leu Asn Val Pro His Asp Ile Ala AIa Arg Ala Ser Arg Leu Thr Ala Thr Val Glu Leu Val Ala Ser Pro Asp Arg Leu Glu Cys Arg Thr Val Leu Gly Asn Lys Thr Phe Arg Thr Thr Val Val Asp Gly Ala His Leu Glu Ala Asn Gly Pro Glu Glu Tyr Val Leu Ser Phe Asp Ala Ser Arg Gln Ser Met Gly Ala Gly Ser His Ser Leu Thr Tyr Glu Leu Thr Pro Ala Gly Leu Gln Val Lys Ile Ser Ser Asn Gly Leu Asp Cys Thr Ala Thr Phe Pro Xaa Gly Gly Ala Pro Ser Ala Ala Pro Gly Glu Val Xaa Ala Phe Cys Ser Ala Leu Tyr Arg Tyr Asn Arg Phe Thr Gln Arg His Ser Leu Thr Gly Gly Leu Trp Leu His Pro Glu Gly Leu Leu Gly Ile Phe Pro Pro Phe Ser Pro Gly His Ile Trp Glu Ser Ala Asn Pro Phe Cys Gly Glu Gly Thr Leu Tyr Thr Arg Thr Trp Ser Thr Ser Gly Phe Ser Ser Asp Phe Ser Pro Pro Glu Ala Ala Ala Pro Ala Ser Ala Ala Ala Pro Gly Leu Pro Tyr Pro Thr Pro Pro Val 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Thr Ala Gly Val Pro Gly Ser Gly Lys Ser Arg Ser Ile Gln Gln Gly Asp Val Asp Val Val Val Val Pro Thr Arg Glu Leu Arg Asn Ser Trp Arg Arg Arg Gly Phe Ala Ala Phe Thr Pro His Thr Ala Ala Arg Val Thr Ile Gly Arg Arg Val Val Ile Asp Glu Ala Pro Ser Leu Pro Pro His Leu Leu Leu Leu His Met Gln Arg Ala Ser Ser Val His Leu Leu Gly Asp Pro Asn Gln Ile Pro Ala Ile Asp Phe Glu His Ala Gly Leu Val Pro Ala Ile Arg Pro Glu Leu Ala Pro Thr Ser Trp Trp His Val Thr His Arg Cys Pro Ala Asp Val Cys Glu Leu Ile Arg Gly Ala Tyr Pro Lys Ile Gln Thr Thr Ser Arg Val Leu Arg Ser Leu Phe Trp Asn Glu Pro Ala Ile Gly Gln Lys Leu Val Phe Thr Gln Ala Ala Lys Ala Ala Asn Pro Gly Ala Ile Thr Val His Glu Ala Gln Gly Ala Thr Phe Thr Glu Thr Thr Ile Ile Ala Thr Ala Asp Ala Arg Gly Leu Ile Gln Ser Ser Arg Ala His Ala Ile Val Ala Leu Thr Arg His Thr Glu Lys Cys Val Ile Leu Asp Ala Pro Gly Leu Leu Arg Glu Val Gly Ile Ser Asp Val Ile Val Asn Asn Phe Phe Leu Ala Gly Gly Glu Val Gly His His Arg Pro Ser Val Ile Pro Arg Gly Asn Pro Asp Gln Asn Leu Gly Thr Leu Gln Ala Phe Pro Pro Ser Cys Gln Ile Ser Ala Tyr His Gln Leu Ala Glu Glu Leu Gly His Arg Pro Ala Pro Val Ala Ala Val Leu Pro Pro Cys Pro Glu Leu Glu Gln Gly Leu Leu Tyr Met Pro Gln Glu Leu Thr Val Ser Asp Ser Val Leu Val Phe Glu Leu Thr Asp Ile Val His Cys Arg Met Ala Ala Pro Ser Gln Arg Lys Ala Val Leu Ser Thr Leu Val Gly Arg Tyr Gly Arg Arg Thr Lys Leu Tyr Glu Ala Ala His Ser Asp Val Arg Glu Ser Leu Ala Arg Phe Ile Pro Thr Ile Gly Pro Val Arg Ala Thr Thr Cys Glu Leu Tyr Glu Leu Val Glu Ala Met Val Glu Lys Gly Gln Asp Gly Ser Ala Val Leu Glu Leu Asp Leu Cys Asn Arg Asp Val Ser Arg Ile Thr Phe Phe Gln Lys Asp Cys Asn Lys Phe Thr Thr Gly Glu Thr Ile Ala His Gly Lys Val Gly Gln Gly Ile Ser Ala Trp Ser Lys Thr Phe Cys Ala Leu Phe Gly Pro Trp Phe Arg Ala Ile Glu Lys Glu Ile Leu Ala Leu Leu Pro Pro Asn Ile Phe Tyr Gly Asp Ala Tyr Glu Glu Ser Val Phe Aia Ala Ala Val Ser Gly Ala Gly Ser Cys Met Val Phe Glu Asn Asp Phe Ser Glu Phe Asp Ser Thr Gln Asn Asn Phe Ser Leu Gly Leu Glu Cys Val Val Met Glu Glu Cys Gly Met Pro Gln Trp Leu Ile Arg Leu Tyr His Leu Val Arg Ser Ala Trp Ile Leu Gln Ala Pro Lys Glu Ser Leu Lys Gly Phe Trp Lys Lys His Ser Gly Glu Pro Gly Thr Leu Leu Trp Asn Thr Val Trp Asn Met Ala Ile Ile Ala His Cys Xaa Glu Phe Arg Asp Phe Arg Val Ala Ala Phe Lys Gly Asp Asp Ser Val Val Leu Cys Ser Asp Tyr Arg Gln Xaa Arg Asn Ala Ala Ala Leu Ile Ala Gly Cys Gly Leu Lys Leu Lys Val Asp Tyr Arg Pro Ile Gly Leu Tyr Ala Gly Val Val Val Ala Pro Gly Leu Gly Thr Leu Pro Asp Val Val Arg Phe Ala Gly Arg Leu Ser Glu Lys Asn Trp Gly Pro Gly Pro Glu Arg Ala Glu Gln Leu Arg Leu Ala Val Cys Asp Phe Leu Arg Gly Leu Thr Asn Val Ala Gln Val Cys Val Asp Val Val Ser Arg Val Tyr Gly Val Ser Pro Gly Leu Val His Asn Leu Ile Gly Met Leu Gln Thr Ile Ala Asp Gly Lys Ala His Phe Thr Xaa Asn Ile Lys Pro Val Leu Asp Leu Thr Asn Ser Ile Ile Gln Arg Val Glu <210> 167 <211> 660 <212> PRT
<213> Hepatitis E virus <400> 167 Met Arg Pro Arg Ala Val Leu Leu Leu Leu Phe Val Leu Leu Pro Met Leu Pro Ala Pro Pro Ala Gly Gln Pro Ser Gly Arg Arg Arg Gly Arg Arg Ser Gly Gly Ala Gly Gly Gly Phe Trp Gly Asp Arg Val Asp Ser Gln Pro Phe Ala Leu Pro Tyr Ile His Pro Thr Asn Pro Phe Ala Ala Asp Val Val Ser Gln Pro Gly Ala Gly Thr Arg Pro Arg Gln Pro Pro Arg Pro Leu Xaa Ser Ala Trp Arg Asp Gln Ser Gln Arg Pro Ser Ala Ala Pro Arg Arg Arg Ser Ala Pro Ala Gly Ala Ala Pro Leu Thr Ala Val Ser Pro Ala Pro Asp Thr Ala Pro Val Pro Asp Val Asp Ser Arg Gly Ala Ile Leu Arg Arg Gln Tyr Asn Leu Ser Thr Ser Pro Leu Thr Ser Ser Val Ala Ser Gly Thr Asn Leu Val Leu Tyr Ala Ala Pro Leu Asn Pro Leu Leu Pro Leu Gln Asp Gly Thr Asn Thr His Ile Met Ala Thr Glu Ala Ser Asn Tyr Ala Gln Tyr Arg Val Val Arg Ala Thr Ile Arg Tyr Arg Pro Leu Val Pro Asn Ala Val Gly Gly Tyr Ala Ile Ser Ile Ser Phe Trp Pro Gln Thr Thr Thr Thr Pro Thr Ser Val Asp Met Asn Ser Ile Thr Ser Xaa Asp Val Arg Ile Leu Val Gln Pro Gly Ile Ala Ser Glu Leu Val Ile Pro Ser Glu Arg Leu His Tyr Arg Asn Gln Gly Trp Arg Ser Val Glu Thr Thr Gly Val Ala Glu Glu Glu Ala Thr Ser Gly Leu Val Met Leu Cys Ile His Gly Ser Pro Val Asn Ser Tyr Thr Asn Thr Pro Tyr Thr Gly Ala Leu Gly Leu Leu Asp Phe Ala Leu Glu Leu Glu Phe Arg Asn Leu Thr Pro Gly Asn Thr Asn Thr Arg Val Ser Arg Tyr Thr Ser Thr Ala Arg His Arg Leu Arg Arg Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp Leu His Phe Ala Gly Thr Asn Gly Val Gly Glu Val Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Thr Ile Pro His Asp Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln Xaa Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val Ser Asp Thr Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Thr Thr Ile Gln Gln Tyr Ser Lys Thr Phe Tyr Val Leu Pro Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Thr Ser Ile Ser Ala Val Gly Val Leu Ala Pro His Ser Ala Leu Ala Val Leu Glu Asp Thr Ile Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Thr Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Ile Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Ser <210> 168 <211> 122 <212> PRT
<213> Hepatitis E virus <220>
<223> us2 orf3 <400> 168 Met Asn Asn Met Ser Phe Ala Ser Pro Met Gly Ser Pro Cys Ala Leu Gly Leu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys Cys Pro Arg His Arg Pro Ala Ser Arg Leu Ala Ala Val Val Gly Gly Ala Ala Ala Val Pro Ala Val Val Ser Gly Val Thr Gly Leu Ile Leu Ser Pro Ser Pro Ser Pro IIe Phe Ile Gln Pro Thr Pro Ser Pro Pro Met Ser Phe 65 70 ~5 80 His Asn Pro Gly Leu Glu Leu Ala Leu Asp Ser Arg Pro Ala Pro Leu Xaa Pro Leu Gly Val Thr Ser Pro Ser Ala Pro Pro Leu Pro Pro Val Val Asp Leu Pro Gln Leu Gly Leu Arg Arg <210> 169 <211> 33 <212> PRT
<213> Hepatitis E virus <220>
<223> M 4-2 <400> 169 Ala Asn Gln Pro Gly His Leu Ala Pro Leu Gly Glu Ile Arg Pro Ser Ala Pro Pro Leu Pro Pro Val Ala Asp Leu Pro Gln Pro Gly Leu Arg Arg <210> 170 <211> 48 <212> PRT
<213> Hepatitis E virus <220>
<223> M 3-2e <400> 170 Thr Phe Asp Tyr Pro Gly Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Ala Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Val Ala Glu Leu Gln Arg Leu Lys Val Lys Val Gly Lys Thr Arg Glu Leu <210> 171 <211> 33 <212> PRT
<213> Hepatitis E virus <220>
<223> B 4-2 <400> 171 Ala Asn Pro Pro Asp His Ser Ala Pro Leu Gly Val Thr Arg Pro Ser Ala Pro Pro Leu Pro His Val Val Asp Leu Pro Gln Leu Gly Pro Arg Arg <210> 172 <211> 48 <212> PRT
<213> Hepatitis E virus <220>
<223> B 3-2e <400> 172 Thr Leu TyrProAla Arg His ThrPhe Asp PheCys Asp Ala Asp Pro Glu Cys ProLeuGly Leu Gly CysAla Phe SerThr Arg Gln Gln Val Ala Glu GlnArgLeu Lys Lys ValGly Lys ArgGlu Leu Met Thr Leu <210> 173 <211> 33 <212> PRT
<213> Hepatitis E virus <220>
<223> ORF3 (u4.2) <400> 173 Asp Ser Arg Pro Ala Pro Ser Val Pro Leu Gly Val Thr Ser Pro Ser Ala Pro Pro Leu Pro Pro Val Val Asp Leu Pro Gln Leu Gly Leu Arg Arg <210> 174 <211> 48 <212> PRT
<213> Hepatitis E virus <220>
<223> ORF2 (u3.2e) <400> 174 Thr Val Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Thr Leu Gly Leu G1n Gly Cys Ala Phe Gln Ser Thr Ile Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Ser <210> 175 <211> 327 <212> PRT
<213> Hepatitis E virus <220>
<223> US-1 SG3 <400> 175 Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp Leu His Phe Thr Gly Thr Asn Gly Val Gly Glu Val Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr 65 70 75 gp Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Thr Ile Pro His Asp Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Xaa Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val Ser Asp Thr Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Thr Thr Ile Gln Gln Tyr Ser Lys Lys Phe Tyr Val Leu Pro Leu Xaa Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile GIu Asn Ala Ala Gly His Arg VaI Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Thr Ser Xaa Ser Ala Val Gly Val Leu Ala Pro His Ser Ala Leu Ala Val Leu Glu Asp Thr Val Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Thr Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Ile Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Ser <210> 176 <211> 327 <212> PRT
<213> Hepatitis E virus <220>
<223> US-2 SG3 <400> 176 Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp Leu His Phe Ala Gly Thr Asn Gly Val Gly Glu Val Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Thr Ile Pro His Asp Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln Xaa Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val Ser Asp Thr Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Thr Thr Ile Gln Gln Tyr Ser Lys Thr Phe Tyr Val Leu Pro Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Thr Ser Ile Ser Ala Val Gly Val Leu Ala Pro His Ser Ala Leu Ala Val Leu Glu Asp Thr Ile Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Thr Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Ile Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Ser <210> 177 <211> 21 <212> DNA
<213> Hepatitis E virus <220>
<223> HEVConsORFl-s2 <400> 177 ctgccytkgc gaatgctgtg g 21 <210> 178 <211> 24 <212> DNA

<213> Hepatitis E virus <220>
<223> HEVConsORFl-a2 <400> 178 ggcagwrtac carcgctgaa catc 24 <210> 179 <211> 294 <212> DNA
<213> Hepatitis E virus <220>
<223> zl2-orfl (G. S.) <400> 179 tggcattact actgccattg agcaagctgc tctggctgcg gccaattctg ccttggcgaa 60 tgctgtggtg gttcggccgt ttttatctcg tttacagact gagattctta ttaatttgat 120 gcaaccccga cagttggtct ttcgacctga ggtgttctgg aaccatccca tccaacgtgt 180 tatacataat gaattggagc agtactgccg ggcccgggcc ggtcgctgtc tggaaattgg 240 agcccatcca aggtcaatca atgataatcc taatgttctg catcggtgtt tcct 294 <220> 180 <211> 418 <212> DNA
<213> Hepatitis E virus <220>
<223> z12-orfl.con <400> 180 ctggcattac tactgctatt gagcaagctg ctctgggtgc ggccaattct gccttggcga 60 atgctgtggt ggttcggccg tttttatctc gtttacagac tgagattctt attaatttga 120 tgcaaccccg acagttggtc tttcgacctg aggtgttctg gaaccatccc atccaacgtg 180 ttatacataa tgaattggag cagtactgcc gggcccgggc cggtcgctgt ctggaaattg 240 gagcccatcc aaggtcaatc aatgataatc ctaatgttct gcatcggtgc tttttacgac 300 cggtcgggag ggacgttcag cgctggtact ccgcccccac ccgtggcccc gcggccaact 360 gccgccggtc tgcgctgcgt ggtctccccc ctgtcgaccg cacttactgc ctcgatgg 418 <210> 181 <211> 197 <212> DNA
<213> Hepatitis E virus <220>
<223> z12-orf2.con <400> 181 gacagaatta atttcgtcgg ctgggggtca actgttctac tcccgccctg tcgtctcagc 60 caatggcgag ccgactgtca agttatacac atctgttgag aatgcacagc aggataaggg 120 WO 99/19732 PCT/US98l21941 gatagctatt ccacatgaca tagatttggg cgactctcgt ttggtaatcc aggattatga 180 taaccaacac gaacaag 197 <210> 182 <211> 25 <212> DNA
<213> Hepatitis E virus <220>
<223> HEVConsORF2/3-s1 <400> 182 gtatcggkyk gaatgaataa catgt 25 <210> 183 <211> 25 <212> DNA
<213> Hepatitis E virus <220>
<223> HEVConsORF2/3-ai <400> 183 aggggttggt tggatgaata taggg <210> 184 <211> 234 <212> DNA
<213> Hepatitis E virus <220>
<223> z12-orf23.con <400> 184 gtatcggktt gaatgaataa catgttttgt gcatcgccca tgggatcacc atgcgcccta 60 gggttgttct gttgttgttc ctcgtgtttc tgcctatgct gcccgcgcca ccggccggcc 120 agycgactgg ccgccgtcgt gggcggcgca gcggcggtgc cggcggtggt ttctggggtg 180 acagggttga ttctcagccc ttcgccctcc cctatattca tccaaccaac ccct 234 <210> 185 <211> 890 <212> DNA
<213> Hepatitis E virus <220>
<223> z12-3p. race <400> 185 gtcgtctcgg ccaatggcga gccgactgtc aagttataca catctgttga gaatgcacag 60 caggataagg ggatagctat tccacatgac atagatttgg gcgactctcg tttggtaatc 120 caggattacg ataatcagca cgagcaggac cggcccaccc cttcgcccgc cccgtctcgt 180 cctttctcgg tcctccgcgc taatgatgct ttgtggcttt ctcttaccgc tgctgagtat 240 gaccagacta catatgggtc gtccaccaac ccgatgtatg tctcagacac tgttacattt 300 gtcaatgtgg ccacaggggc tcaggctgtc gcccgttctc ttgattggtc taaagttacc 360 ctggacggcc gccctcttac taccatccag cagtactcta agacatttta tgttctccca 420 cttcgcggga agttatcttt ttgggaggct ggcacaacta aagccggtta cccttataat 480 tataacacaa ctgctagtga ccagattctg attgaaaacg cggctggcca tcgtgtcgct 540 atatctactt atactactag cctgggcgcc ggccctgtgt cagtttctgc ggttggtgtg 600 ttagccccac actcgagcct tgctattctt gaagacactg ttgactatcc ggcccgtgct 660 cacacttttg atgacttctg tccggaatgc cgtgccctgg gtctgcaggg gtgtgctttt 720 caatctacta tcgctgagct ccagcgtctt aaaatgaagg taggcaaaac ccgggagttt 780 taattaattc ttcttgtgcc cccttcacgg ttctcgcttt atttctttct tctgcctccc 840 gcgctccctg gaaaaaaaaa aaaaaaaaaa gtactagtcg acgcgtggcc 89p <210> 186 <211> 919 <212> DNA
<213> Hepatitis E virus <220>
<223> z12-3p. con <400> 186 gacagaatta atttcgtcgg ctgggggtca actgttctac tcccgccctg tcgtctcagc 60 caatggcgag ccgactgtca agttatacac atctgttgag aatgcacagc aggataaggg 120 gatagctatt ccacatgaca tagatttggg cgactctcgt ttggtaatcc aggattacga 180 taatcagcac gagcaggacc ggcccacccc ttcgcccgcc ccgtctcgtc ctttctcggt 240 cctccgcgct aatgatgctt tgtggctttc tcttaccgct gctgagtatg accagactac 300 atatgggtcg tccaccaacc cgatgtatgt ctcagacact gttacatttg tcaatgtggc 360 cacaggggct caggctgtcg cccgttctct tgattggtct aaagttaccc tggacggccg 420 ccctcttact accatccagc agtactctaa gacattttat gttctcccac ttcgcgggaa 480 gttatctttt tgggaggctg gcacaactaa agccggttac ccttataatt ataacacaac 540 tgctagtgac cagattctga ttgaaaacgc ggctggccat cgtgtcgcta tatctactta 600 tactactagc ctgggcgccg gccctgtgtc agtttctgcg gttggtgtgt tagccccaca 660 ctcgagcctt gctattcttg aagacactgt tgactatccg gcccgtgctc acacttttga 720 tgacttctgt ccggaatgcc gtgccctggg tctgcagggg tgtgcttttc aatctactat 780 cgctgagctc cagcgtctta aaatgaaggt aggcaaaacc cgggagtttt aattaattct 840 tcttgtgccc ccttcacggt tctcgcttta tttctttctt ctgcctcccg cgctccctgg 900 aaaaaaaaaa aaaaaaaaa 919 <210> 187 <211> 138 <212> PRT

<213> Hepatitis E virus <220>
<223> z12-orfi.pep <400> 187 Gly Ile Thr Thr Ala Ile Glu Gln Ala Ala Leu Gly Ala Ala Asn Ser Ala Leu Ala Asn Ala Val Val Val Arg Pro Phe Leu Ser Arg Leu Gln Thr Glu Ile Leu Ile Asn Leu Met Gln Pro Arg Gln Leu Val Phe Arg Pro Glu Val Phe Trp Asn His Pro ile Gln Arg Val Ile His Asn Glu Leu Glu Gln Tyr Cys Arg Ala Arg Ala Gly Arg Cys Leu Glu Ile Gly Ala His Pro Arg Ser Ile Asn Asp Asn Pro Asn Val Leu His Arg Cys Phe Leu Arg Pro Val Gly Arg Asp Val Gln Arg Trp Tyr Ser Ala Pro Thr Arg Gly Pro Ala Ala Asn Cys Arg Arg Ser Ala Leu Arg Gly Leu Pro Pro Val Asp Arg Thr Tyr Cys Leu Asp <210> 188 <211> 61 <212> PRT
<213> Hepatitis E virus <220>
<223> z12-orf2-5'. pep <400> 188 Met Arg Pro Arg Val Val Leu Leu Leu Phe Leu Val Phe Leu Pro Met Leu Pro Ala Pro Pro Ala Gly Gln Xaa Thr Gly Arg Arg Arg Gly Arg Arg Ser Gly Gly Ala Gly Gly Gly Phe Trp Gly Asp Arg Val Asp Ser Gln Pro Phe Ala Leu Pro Tyr Ile His Pro Thr Asn Pro <210> 189 <211> 276 <212> PRT
<213> Hepatitis E virus <220>
<223> zl2-orf2-3'. pep <400> 189 Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu_Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp Leu Gly Asp Ser Arg Leu Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Ala Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val Ser Asp Thr Val Thr Phe Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Thr Thr Ile Gln Gln Tyr Ser Lys Thr Phe Tyr Val Leu Pro Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Val Ser Val Ser Ala Val Gly Val Leu Ala Pro His Ser Ser Leu Ala Ile Leu Glu Asp Thr Val Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Ala Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Ile Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Phe <210> 190 <211> 74 <212> PRT
<213> Hepatitis E virus <220>
<223> z12-orf3.pep <400> 190 Met Asn Asn Met Phe Cys Ala Ser Pro Met Gly Ser Pro Cys Ala Leu Gly Leu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys Cys Pro Arg His Arg Pro Ala Ser Arg Leu Ala Ala Val Val Gly Gly Ala Ala Ala Val Pro Ala Val Val Ser Gly Val Thr Gly Leu Ile Leu Ser Pro Ser Pro Ser Pro Ile Phe Ile Gln Pro Thr Pro <210> 191 <211> 408 <212> DNA
<213> Hepatitis E virus <220>
<223> pJOorf3-29.seq <400> 191 gaattcatga ataacatgtc ttttgcatcg cccatgggat caccatgcgc cctagggctg 60 ttctgttgtt gctcttcgtg cttttgccta tgctgcccgc gccaccggcc agccagccgt 120 ctggccgccg tcgtgggcgg cgcagcggcg gtgccggcgg tggtttctgg ggtgacaggg 180 ttgattctca gcccttcgcc ctcccctata ttcatccaac caaccccttc gccgccgatg 240 tcgtttcaca acccggggct ggaactcgcc ctcgacagcc gccccgcccc cttggctccg 300 cttggcgtga ccagtcccag cgcccctccg ctgccccccg tcgtcgatct gccccagctt 360 ggtctgcgcc gcgactacaa ggacgacgat gacaagtaat aaggatcc 408 <210> 192 <211> 1026 <212> DNA
<213> Hepatitis E virus <220>
<223> cksorf2m-2.seq <400> 192 gaattcatgg gtgctgatgg gactgctgag cttactacca cagcagccac acgtttcatg 60 aaggacctgc acttcgctgg cacgaatggc gttggtgagg tgggtcgtgg tatcgccctg 120 acactgttca atctcgctga tacgcttctc ggcggtttac cgacagaatt gatttcgtcg 180 gctgggggcc aactgtttta ctcccgcccg gttgtctcag. ccaatggcga gccaacagta 240 aagttatata catctgttga gaatgcgcag caagacaagg gcatcaccat tccacatgat 300 atagacctgg gtgactcccg tgtggttatc caggattatg ataaccagca tgagcaagac 360 cgacctactc cgtcacctgc cccctctcgc cccttctcag ttcttcgtgc caatgatgtt 420 ttgtggcttt ccctcactgc cgctgagtat gaccagacta cgtatgggtc gtccaccaac 480 cctatgtatg tctctgacac agttacgctt gttaatgtgg ctactggtgc tcaggctgtt 540 gcccgctccc ttgattggtc taaagttact ctggacggcc gcccccttac taccattcag 600 cagtattcta agacatttta tgttctcccg ctccgcggga agctgtcctt ttgggaggct 660 ggcacgacta aggccggcta cccttacaat tataatacta ccgctagtga ccaaattttg 720 attgagaatg cggccggcca ccgtgtcgct atttccacct ataccactag cttaggtgcc 780 ggtcctacct cgatctctgc ggtcggcgta ctggctccac actctgccct tgccgttctt 840 gaggatacta ttgattaccc cgcccgtgcc catacttttg atgatttttg cccggagtgc 900 cgtaccctag gtttgcaggg ttgtgcattc cagtctacta ttgctgagct ccagcgttta 960 aaaatgaagg taggtaaaac ccgggagtct gactacaagg acgacgatga caagtaataa 1020 ggatcc 1026 <210> 193 <211> 1389 <212> DNA
<213> Hepatitis E virus <220>
<223> CKSORF32M-3.seq <400> 193 gaattcatga ataacatgtc ttttgcatcg cccatgggat caccatgcgc cctagggctg 60 ttctgttgtt gctcttcgtg cttttgccta tgctgcccgc gccaccggcc agccagccgt 120 ctggccgccg tcgtgggcgg cgtagcggcg gtgccggcgg tggtttctgg ggtgacaggg 180 ttgattctca gcccttcgcc ctcccctata ttcatccaac caaccccttc gccgccgatg 240 tcgtttcaca acccggggct ggaactcgcc ctcgacagcc gccccgcccc cttggctccg 300 cttggcgtga ccagtcccag cgcccctccg ctgccccccg tcgtcgatct gccccagctt 360 ggtctgcgcc gcggtgctga tgggactgct gagcttacta ccacagcagc cacacgtttc 420 atgaaggacc tgcacttcgc tggcacgaat ggcgttggtg aggtgggtcg tggtatcgcc 480 ctgacactgt tcaatctcgc tgatacgctt ctcggcggtt taccgacaga attgatttcg 540 tcggctgggg gccaactgtt ttactcccgc ccggttgtct cagccaatgg cgagccaaca 600 gtaaagttat atacatctgt tgagaatgcg cagcaagaca agggcatcac cattccacat 660 gatatagacc tgggtgactc ccgtgtggtt atccaggatt atgataacca gcatgagcaa 720 gaccgaccta ctccgtcacc tgccccctct cgccccttct cagttcttcg tgccaatgat 780 gttttgtggc tttccctcac tgccgctgag tatgaccaga ctacgtatgg gtcgtccacc 840 aaccctatgt atgtctctga cacagttacg cttgttaatg tggctactgg tgctcaggct 900 gttgcccgct cccttgattg gtctaaagtt actctggacg gccgccccct tactaccatt 960 cagcagtatt ctaagacatt ttatgttctc ccgctccgcg ggaagctgtc cttttgggag 1020 gctggcacga ctaaggccgg ctacccttac aattataata ctaccgctag tgaccaaatt 1080 ttgattgaga atgcggccgg ccaccgtgtc gctatttcca cctataccac tagcttaggt 1140 gccggtccta cctcgatctc tgcggtcggc gtactggctc cacactctgc ccttgccgtt 1200 cttgaggata ctattgatta cccCgcccgt gcccatactt ttgatgattt ttgcccggag 1260 tgccgtaccc taggtttgca gggttgtgca ttccagtcta ctattgctga gctccagcgt 1320 ttaaaaatga aggtaggtaa aacccgggag tctgactaca aggacgacga tgacaagtaa 1380 taaggatcc 1389 <210> 194 <211> 408 <212> DNA
<213> Hepatitis E virus <220>
<223> plorf3-l2. con <400> 194 gaattcatga ataacatgtc ttttgcatcg cccatgggat caccatgcgc cctagggctg 60 ttctgttgtt gctcttcgtg cttttgccta tgctgcccgc gccaccggcc ggccagccgt 120 ctggccgccg tcgtgggcgg cgcagcggcg gtgccggcgg tggtttctgg ggtgacaggg 180 ttgattctca gcccttcgcc ctcccctata ttcatccaac caaccccttc gccgccgatg 240 tcgtttcaca acccggggct ggaactcgcc ctcgacagcc gccccgcccc cttggctccg 300 cttggcgtga ccagtcccag cgcccctccg ctgccccccg tcgtcgatct gccccagctt 360 ggtctgcgcc gcgactacaa ggacgacgat gacaagtaat aaggatcc 408 <210> 195 <211> 1026 <212> DNA
<213> Hepatitis E virus <220>
<223> plorf2.2-6.seq <400> 195 gaattcatgg gtgctgatgg gactgctgag cttactacca cagcagccac acgtttcatg 60 aaggacctgc acttcgctgg cacgaatggc gttggtgagg tgggtcgtgg tatcgccctg 120 acactgttca atctcgctga tacgcttctc ggcggtttac cgacagaatt gatttcgtcg 180 gctgggggcc aactgtttta ctcccgcccg gttgtctcag ccaatggcga gccaacagta 240 aagttatata catctgttga gaatgcgcag caagacaagg gcatcaccat tccacatgat 300 atagacctgg gtgactcccg tgtggttatc caggattatg ataaccagca tgagcaagac 360 cgacctactc cgtcacctgc cccctctcgc cccttctcag ttcttcgtgc caatgatgtt 420 ttgtggcttt ccctcactgc cgctgagtat gaccagacta cgtatgggtc gtccaccaac 480 cctatgtatg tctctgacac agttacgctt gttaatgtgg ctactggtgc tcaggctgtt 540 gcccgctccc ttgattggtc taaagttact ctggacggcc gcccccttac taccattcag 600 cagtattcta agacatttta tgttctcccg ctccgcggga agctgtcctt ttgggaggct 660 ggcacgacta aggccggcta cccttacaat tataatacta ccgctagtga ccaaattttg 720 attgagaatg cggccggcca ccgtgtcgct atttccacct ataccactag cttaggtgcc 780 ggtcctacct cgatctctgc ggtcggcgta ctggctccac actctgccct tgccgttctt 840 gaggatacta ttgattaccc cgcccgtgcc catacttttg atgatttttg cccggagtgc 900 cgtaccctag gtttgcaggg ttgtgcattc cagtctacta ttgctgagct ccagcgttta 960 aaaatgaagg taggtaaaac ccgggagtct gactacaagg acgacgatga caagtaataa 1020 ggatcc 1026 <210> 196 <211> 1389 <212> DNA
<213> Hepatitis E virus <220>
<223> PLORF32M-14-5.seq <400> 196 gaattcatga ataacatgtc ttttgcatcg cccatgggat caccatgcgc cctagggctg 60 ttctgttgtt gctcttcgtg cttttgccta tgctgcccgc gccaccggcc agccagccgt 120 ctggccgccg tcgtgggcgg cgtagcggcg gtgccggcgg tggtttctgg ggtgacaggg 180 ttgattctca gcccttcgcc ctcccctata ttcatccaac caaccccttc gccgccgatg 240 tcgtttcaca acccggggct ggaactcgcc ctcgacagcc gccccgcccc cttggctccg 300 cttggcgtga ccagtcccag cgcccctccg ctgccccccg tcgtcgatct gccccagctt 360 ggtctgcgcc gcggtgctga tgggactgct gagcttacta ccacagcagc cacacgtttc 420 atgaaggacc tgcacttcgc tggcacgaat ggcgttggtg aggtgggtcg tggtatcgcc 480 ctgacactgt tcaatctcgc tgatacgctt ctcggcggtt taccgacaga attgatttcg 540 tcggctgggg gccaactgtt ttactcccgc ccggttgtct cagccaatgg cgagccaaca 600 gtaaagttat atacatctgt tgagaatgcg cagcaagaca agggcatcac cattccacat 660 gatatagacc tgggtgactc ccgtgtggtt atccaggatt atgataacca gcatgagcaa 720 gaccgaccta ctccgtcacc tgccccctct cgccccttct cagttcttcg tgccaatgat 780 gttttgtggc tttccctcac tgccgctgag tatgaccaga ctacgtatgg gtcgtccacc 840 aaccctatgt atgtctctga cacagttacg cttgttaatg tggctactgg tgctcaggct 900 gttgcccgct cccttgattg gtctaaagtt actctggacg gccgccccct tactaccatt 960 cagcagtatt ctaagacatt ttatgttctc ccgctccgcg ggaagctgtc cttttgggag 1020 gctggcacga ctaaggccgg ctacccttac aattataata ctaccgctag tgaccaaatt 1080 ttgattgaga atgcggccgg ccaccgtgtc gctatttcca cctataccac tagcttaggt 1140 gccggtccta cctcgatctc tgcggtcggc gtactggctc cacactctgc ccttgccgtt 1200 cttgaggata ctattgatta ccccgcccgt gcccatactt ttgatgattt ttgcccggag 1260 tgccgtaccc taggtttgca gggttgtgca ttccagtcta ctattgctga gctccagcgt 1320 ttaaaaatga aggtaggtaa aacccgggag tctgactaca aggacgacga tgacaagtaa 1380 taaggatcc 1389 <210> 197 <211> 74 <212> PRT
<213> Hepatitis E virus <220>
<223> zl2-orf3-5'. pep <400> 197 Met Asn Asn Met Phe Cys Ala Ser Pro Met Gly Ser Pro Cys Ala Leu Gly Leu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys Cys Pro Arg His Arg Pro Ala Xaa Arg Leu Ala.Ala Val Val Gly Gly Ala Ala Ala Val Pro Ala Val Val Ser Gly Val Thr Gly Leu Ile Leu Ser Pro Ser Pro Ser Pro Ile Phe Ile Gln Pro Thr Pro <210> 198 <211> 63 <212> DNA
<213> Hepatitis E virus <220>
<223> Description of Artificial Sequence: Primer orf23p <400> 198 tatatggatc cttattactt gtcatcgtcg tccttgtagt cagactcccg ggttttacct 60 acc 63 <210> 199 <211> 338 <212> PRT
<213> Hepatitis E virus <220>
<223> cksorf2m-2. pep <400> 199 Glu Phe Met Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp Leu His Phe Ala Gly Thr Asn Gly Val Gly Glu Val Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Thr Ile Pro His Asp Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val Ser Asp Thr Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Thr Thr Ile Gln Gln Tyr Ser Lys Thr Phe Tyr Val Leu Pro Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Thr Ser Ile Ser Ala Val Gly Val Leu Ala Pro His Ser Ala Leu Ala Val Leu Glu Asp Thr Ile Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Thr Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Ile Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Ser Asp Tyr Lys Asp Asp Asp Asp Lys <210> 200 <211> 338 <212> PRT
<213> Hepatitis E virus <220>
<223> plorf2.2-6. pep <400> 200 Glu Phe Met Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala 1 5 . 10 15 Thr Arg Phe Met Lys Asp Leu His Phe Ala Gly Thr Asn Gly Val Gly Glu Val Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Thr Ile Pro His Asp Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val Ser Asp Thr Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Thr Thr Ile Gln Gln Tyr Ser Lys Thr Phe Tyr Val Leu Pro Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Thr Ser Ile Ser Ala Val Gly Val Leu Ala Pro His Ser Ala Leu Ala Val Leu Glu Asp Thr Ile Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Thr Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Ile Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Ser Asp Tyr Lys Asp Asp Asp Asp Lys <210> 201 <211> 37 WO 99!19732 PCTNS98/21941 <212> DNA
<213> Hepatitis E virus <220>
<223> Description of Artificial Sequence: Primer orf35p <400> 201 tatatgaatt catgaataac atgtcttttg catcgcc 37 <210> 202 <211> 68 <212> DNA
<213> Hepatitis E virus <220>
<223> Description of Artificial Sequence: Primer orf33p <400> 202 tatatggatc cttattactt gtcatcgtcg tccttgtagt cgcggcgcag accaagctgg 60 ggcagatc 68 <210> 203 <211> 132 <212> PRT
<213> Hepatitis E virus <220>
<223> pJOorf3-29. pep <400> 203 Glu Phe Met Asn Asn Met Ser Phe Ala Ser Pro Met Gly Ser Pro Cys Ala Leu Gly Leu Phe Cys Cys Cys Ser Sex Cys Phe Cys Leu Cys Cys Pro Arg His Arg Pro Ala Ser Arg Leu Ala Ala Val Val Gly Gly Ala Ala Ala Val Pro Ala Val Val Ser Gly Val Thr Gly Leu Ile Leu Ser Pro Ser Pro Ser Pro Ile Phe Ile Gln Pro Thr Pro Ser Pro Pro Met Ser Phe His Asn Pro Gly Leu Glu Leu Ala Leu Asp Ser Arg Pro Ala Pro Leu Ala Pro Leu Gly Val Thr Ser Pro Ser Ala Pro Pro Leu Pro Pro Val Val Asp Leu Pro Gln Leu Gly Leu Arg Arg Asp Tyr Lys Asp Asp Asp Asp Lys <210> 204 <211> 132 <212> PRT
<213> Hepatitis E virus <220>
<223> plorf3-l2. pep <400> 204 Glu Phe Met Asn Asn Met Ser Phe Ala Ser Pro Met Gly Ser Pro Cys Ala Leu Gly Leu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys Cys Pro Arg His Arg Pro Ala Ser Arg Leu Ala Ala Val Val Gly Gly Ala Ala Ala Val Pro Ala Val Val Ser Gly Val Thr Gly Leu Ile Leu Ser Pro Ser Pro Ser Pro Ile Phe Ile Gln Pro Thr Pro Ser Pro Pro Met 65 70 75 gp Ser Phe His Asn Pro Gly Leu Glu Leu Ala Leu Asp Ser Arg Pro Ala Pro Leu Ala Pro Leu Gly Val Thr Ser Pro Ser Ala Pro Pro Leu Pro Pro Val Val Asp Leu Pro Gln Leu Gly Leu Arg Arg Asp Tyr Lys Asp Asp Asp Asp Lys <210> 205 <211> 48 <212> DNA
<2i3> Hepatitis E virus <220>
<223> Description of Artificial Sequence: Primer orf23 <400> 205 ctcagcagtc ccatcagcac cgcggcgcag accaagctgg ggcagatc 48 <210> 206 <211> 459 <212> PRT
<213> Hepatitis E virus <220>
<223> CKSORF32M-3. pep <400> 206 Glu Phe Met Asn Asn Met Ser Phe Ala Ser Pro Met Gly Ser Pro Cys Ala Leu Gly Leu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys Cys Pro Arg His Arg Pro Ala Ser Arg Leu Ala Ala Val Val Gly Gly Val Ala Ala Val Pro Ala Val Val Ser Gly Val Thr Gly Leu Ile Leu Ser Pro Ser Pro Ser Pro Ile Phe Ile Gln Pro Thr Pro Ser Pro Pro Met Ser Phe His Asn Pro Gly Leu Glu Leu Ala Leu Asp Ser Arg Pro Ala Pro Leu Ala Pro Leu Gly Val Thr Ser Pro Ser Ala Pro Pro Leu Pro Pro Val VaI Asp Leu Pro Gln Leu Gly Leu Arg Arg Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp Leu His Phe Ala Gly Thr Asn Gly Val Gly Glu Val Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Thr Ile Pro His Asp Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val Ser Asp Thr Val Thr Leu Val Asn Val Ala Thr Gly Ala Gl.n Ala Val Ala Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Thr Thr Ile Gln Gln Tyr Ser Lys Thr Phe Tyr Val Leu Pro Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Thr Ser Ile Ser Ala Val Gly Val Leu Ala Pro His Ser Ala Leu Ala Val Leu Glu Asp Thr Ile Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Thr Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Ile Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Ser Asp Tyr Lys Asp Asp Asp Asp Lys <210> 207 <211> 459 <212> PRT
<213> Hepatitis E virus <220>
<223> PLORF32M-14-5. pep <400> 207 Glu Phe Met Asn Asn Met Ser Phe Ala Ser Pro Met Gly Ser Pro Cys Ala Leu Gly Leu Phe Cys Cys Cys Ser Ser Cys Phe Cys Leu Cys Cys Pro Arg His Arg Pro Ala Ser Arg Leu Ala Ala Val Val Gly Gly Val Ala Ala Val Pro Ala Val Val Ser Gly Val Thr Gly Leu Ile Leu Ser Pro Ser Pro Ser Pro Ile Phe Ile Gln Pro Thr Pro Ser Pro Pro Met Ser Phe His Asn Pro Gly Leu Glu Leu Ala Leu Asp Ser Arg Pro Ala Pro Leu Ala Pro Leu Gly Val Thr Ser Pro Ser Ala Pro Pro Leu Pro Pro Val Val Asp Leu Pro Gln Leu Gly Leu Arg Arg Gly Ala Asp Gly Thr Ala Glu Leu Thr Thr Thr Ala Ala Thr Arg Phe Met Lys Asp Leu His Phe Ala Gly Thr Asn Gly Val Gly Glu Val Gly Arg Gly Ile Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Thr Ile Pro His Asp Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr Asp Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val Sex Asp Thr Val Thr Leu Val Asn Val Ala Thr Gly Ala Gln Ala Val Ala Arg Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Thr Thr Ile Gln Gln Tyr Ser Lys Thr Phe Tyr Val Leu Pro Leu Arg Gly Lys Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly His Arg Val Ala Ile Ser Thr Tyr Thr Thr Ser Leu Gly Ala Gly Pro Thr Ser Ile Ser Ala Val Gly Val Leu Ala Pro His Ser Ala Leu Ala Val Leu Glu Asp Thr Ile Asp Tyr Pro Ala Arg Ala His Thr Phe Asp Asp Phe Cys Pro Glu Cys Arg Thr Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Ile Ala Glu Leu Gln Arg Leu Lys Met Lys Val Gly Lys Thr Arg Glu Ser Asp Tyr Lys Asp Asp Asp Asp Lys <210> 208 <211> 36 <212> DNA
<213> Hepatitis E virus <220>
<223> Description of Artificial Sequence: Primer orf2mid5p <400> 208 tatatgaatt catgggtgct gatgggactg ctgagc 36 <210> 209 <211> 418 <212> DNA
<213> Hepatitis E virus <220>
<223> 1440o1.seq <220>
<221> CDS
<222> (3) .. (416) <400> 209 ct atyact act att gagcagget getctgget gcggccaat 47 ggc gcy Gly XaaThr Thr Ile GluGlnAla AlaLeuAla AlaAlaAsn Xaa tccgccttggcg aatgetgtg gtggttcgg ccgttttta tcccgtgtt 95 SerAlaLeuAla AsnAlaVal ValValArg ProPheLeu SerArgVal caaactgatatc cttattaac ctgatgcaa ccccgtcag cttgtgttc 143 GlnThrAspIle LeuIleAsn LeuMetGln ProArgGln LeuValPhe cggcctgaagtt ctctggaac catccgatc cagcgagtt atacataat 191 ArgProGluVal LeuTrpAsn HisProIle GlnArgVal IleHisAsn gagctggaacaa tactgtcga gcccgcget ggccgctgt cttgaggtg 239 GluLeuGluGln TyrCysArg AlaArgAla GlyArgCys LeuGluVal ggcgetcaccca aggtctatt aatgataac cccaatgtt ctgcaccgg 287 GlyAlaHisPro ArgSerIle AsnAspAsn ProAsnVal LeuHisArg tgctttctccgc ccggttggg agagacgtc cagcgctgg tattccgcc 335 CysPheLeuArg ProValGly ArgAspVal GlnArgTrp TyrSerAla cccactcgtggt ccagcgget aactgccgc cgttctgcg ctacgcggt 383 ProThrArgGly ProAlaAla AsnCysArg ArgSerAla LeuArgGly ttgccccctgtc gaccgcact tactgtyty gatgg 418 LeuProProVai AspArgThr TyrCysXaa Asp <210> 210 <211> 138 <212> PRT
<213> Hepatitis E virus <400> 210 Gly Xaa Thr Thr Xaa Ile Glu Gln Ala Ala Leu Ala Ala Ala Asn Ser Ala Leu Ala Asn Ala Val Val Val Arg Pro Phe Leu Ser Arg Val Gln Thr Asp Ile Leu Ile Asn Leu Met Gln Pro Arg Gln Leu Val Phe Arg Pro Glu Val Leu Trp Asn His Pro Ile Gln Arg Val Ile His Asn Glu Leu Glu Gln Tyr Cys Arg Ala Arg Ala Gly Arg Cys Leu Glu Val Gly Ala His Pro Arg Ser Ile Asn Asp Asn Pro Asn Val Leu His Arg Cys Phe Leu Arg Pro Val Gly Arg Asp Val Gln Arg Trp Tyr Ser Ala Pro Thr Arg Gly Pro Ala Ala Asn Cys Arg Arg Ser Ala Leu Arg Gly Leu Pro Pro Val Asp Arg Thr Tyr Cys Xaa Asp <210> 211 <211> 197 <212> DNA
<213> Hepatitis E virus <220>
<223> 1440o2.seq <220>
<221> CDS
<222> (2)..(196) <400> 211 g aca gaa ttr att tcg tcg get gga ggt caa ctg ttc tac tcc cgc ccg 49 Thr Glu Xaa Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro gtt gtc tca gcc aat ggc gag ccg act gtt aag tta tac acc tct gtc 97 Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val gag aat gca cag cag gat aag ggc att get ata cca cat gat ata gac 145 Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp tta ggg gat tcc cgt gtg gtt ata caa gat tat gay aac car cay gaa 193 Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Xaa Asn Xaa Xaa Glu caa g 197 Gln <210> 212 <211> 65 <212> PRT
<213> Hepatitis E virus <400> 212 Thr Glu Xaa Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Giy Ile Ala Ile Pro His Asp Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Xaa Asn Xaa Xaa Glu Gln <210> 213 <211> 418 <212> DNA
<213> Hepatitis E virus <220>

<223> 015-l.seq <220>

<221>
CDS

<222> 3) (416) ( .
.

<400> 13 ct atyactact gcyattgag caggetget ctgget gcggetaac 47 ggc Gly XaaThrThr IleGlu GlnAlaAla LeuAla AlaAla Xaa Asn tct ttggcgaat getgtggtg gtccggccg ttcctg tcccgcact 95 gcc Ser LeuAlaAsn AlaValVal ValArgPro PheLeu SerArgThr Ala cag gatattctt attaatttg atgcaaccc cggcaa cttgtattc 143 act Gln AspIleLeu IleAsnLeu MetGlnPro ArgGln LeuValPhe Thr cgc gaggttttg tggaaccat ccgatccag cgagtc atacataat 191 cct Arg GluValLeu TrpAsnHis ProIleGln ArgVal IleHisAsn Pro gag gagcagtat tgccgtget cgtgetggt cgctgc ctggaggtt 239 ctg Glu GluGlnTyr CysArgAla ArgAlaGly ArgCys LeuGluVal Leu ggg catccaaga tctatcaat gacaaccct aatgtt ctgcaccgg 287 get Gly HisProArg SerIleAsn AspAsnPro AsnVal LeuHisArg Ala tgt ctccgtccg gttgggcga gacgtacag cgttgg tattctgcc 335 ttc Cys LeuArgPro ValGlyArg AspValGln ArgTrp TyrSerAla Phe cct cgcggcccg gcggetaat tgccgccgt tccgcg ttacgtggc 383 act Pro ArgGlyPro AlaAlaAsn CysArgArg SerAla LeuArgGly Thr cta cctgtcgac cgcacttac tgtytygat gg 418' cct Leu ProValAsp ArgThrTyr CysXaaAsp Pro <210> 214 <211> 138 <212> PRT
<213> Hepatitis E virus <400> 214 Gly Xaa Thr Thr Xaa Ile Glu Gln Ala Ala Leu Ala Ala Ala Asn Ser Ala Leu Ala Asn Ala Val Val Val Arg Pro Phe Leu Ser Arg Thr Gln Thr Asp Ile Leu Ile Asn Leu Met Gln Pro Arg Gln Leu Val Phe Arg Pro Glu Val Leu Trp Asn His Pro Ile Gln Arg Val Ile His Asn Glu Leu Glu Gln Tyr Cys Arg Ala Arg Ala Gly Arg Cys Leu Glu Val Gly Ala His Pro Arg Ser Ile Asn Asp Asn Pro Asn Val Leu His Arg Cys Phe Leu Arg Pro Val Gly Arg Asp Val Gln Arg Trp Tyr Ser Ala Pro Thr Arg Gly Pro Ala Ala Asn Cys Arg Arg Ser Ala Leu Arg Gly Leu Pro Pro Val Asp Arg Thr Tyr Cys Xaa Asp <210> 215 <211> 197 <212> DNA
<213> Hepatitis E virus <220>
<223> 2015o2.seq <220>
<221> CDS
<222> (2) . . (196) <400> 215 g aca gaa ttr att tcg tcg get gga ggc cag ctc ttc tac tcc cgc cca 49 Thr Glu Xaa Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro gtc gtc tca gcc aat ggc gag ccg act gtt aaa ttg tat aca tcc gtc 97 Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val gag aat gcg cag cag gac aag ggc att gcc ata cca cat gat ata gat 145 Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Iie Pro His Asp Ile Asp cta gga gat tcc cgc gtg gtt atc cag gat tat gay aac car cay gaa 193 Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Xaa Asn Xaa Xaa Glu caa g 197 Gln <210> 216 <211> 65 <212> PRT
<213> Hepatitis E virus <400> 216 Thr Glu Xaa Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp Leu Gly Asp Ser Arg Val Val Ile Gln Asp Tyr Xaa Asn Xaa Xaa Glu Gln <210> 217 <211> 251 <212> DNA

<213> HepatitisE
virus <220>

<223> 14404-2.seq <220>

<221> CDS

<222> (3)..(251) <223> orf2 <220>

<223> orf3 fromposition to osition165 1 p <400> 217 at att cat cca cccttt gcctccgac tcg caatcc 47 acc gtc aac gta Ile His Pro ProPhe AlaSerAsp Ser GlnSer Thr Val Asn Val ggg get get cgccctcgacag ccggcccgc cccctcggc tcctct 95 gga Gly Ala Ala ArgProArgGln ProAlaArg ProLeuGly SerSer Gly tgg cgt cag tcccagcgcccc cccgetgtc ccccgtcgt cgatct 143 gac Trp Arg Gln SerGlnArgPro ProAlaVal ProArgArg ArgSer Asp acc cca ggg getgcgccgcta actgetgtt tcaccagcg cctgat 191 act Thr Pro Gly AlaAlaProLeu ThrAlaVal SerProAla ProAsp Thr acg gcc gtc cctgatgttgac tctcgtggc getatcttg cgccgg 239 cca Thr Ala Val ProAspValAsp SerArgGly AlaIleLeu ArgArg Pro cag tat cta 251 aac Gln Tyr Leu Asn <210> 218 <211> 83 <212> PRT
<213> Hepatitis E virus <400> 218 Ile His Pro Thr Asn Pro Phe Ala Ser Asp Val Val Ser Gln Ser Gly Ala Gly Ala Arg Pro Arg Gln Pro Ala Arg Pro Leu Gly Ser Ser Trp Arg Asp Gln Ser Gln Arg Pro Pro Ala Val Pro Arg Arg Arg Ser Thr Pro Thr Gly Ala Ala Pro Leu Thr Ala Val Ser Pro Ala Pro Asp Thr Ala Pro Val Pro Asp Val Asp Ser Arg Gly Ala Ile Leu Arg Arg Gln Tyr Asn Leu <210> 219 <211> 55 <212> PRT
<213> Hepatitis E virus <220>
<223> 14404-2.seq orf3 <400> 219 Ile Phe Ile Gln Pro Thr Pro Leu Pro Pro Thr Ser Tyr Arg Asn Pro Gly Leu Glu Leu Ala Leu Asp Ser Arg Pro Ala Pro Ser Ala Pro Leu Gly Val Thr Ser Pro Ser Ala Pro Pro Leu Ser Pro Val Val Asp Leu Pro Gln Leu Gly Leu Arg Arg <210> 220 <211> 251 <212> DNA
<213> Hepatitis E virus <220>
<223> 20154-2.seq <220>
<221> CDS
<222> (3)..(251) <223> orf2 <220>
<223> orf3 from position 1 to position 165 <400> 220 at att cat cca acc aac ccc ttt gcc gcc gac gtc gta tca caa ccc 47 Ile His Pro Thr Asn Pro Phe Ala Ala Asp Val Val Ser Gln Pro ggg get gga get cgc cct cga cag ccg ccc cgc ccc ctc ggc tcc tct 95 Gly Ala Gly Ala Arg Pro Arg Gln Pro Pro Arg Pro Leu Gly Ser Ser tgg cgt gat cag tcc cag cgc ccc tcc get gcc ccc cgt cgt cga tct 143 Trp Arg Asp Gln Ser Gln Arg Pro Ser Ala Ala Pro Arg Arg Arg Ser acc cca get ggg get gcg ccg tta act get gtt tcc cct gcg ccc gat 191 Thr Pro Ala Gly Ala Ala Pro Leu Thr Ala Val Ser Pro Ala Pro Asp acg gcc cca gtc ccc gac gtt gat tcc cgt ggt gcc atc ctg cgc cgg 239 Thr Ala Pro Val Pro Asp Val Asp Ser Arg Gly Ala Ile Leu Arg Arg cag tat aac cta 251 Gln Tyr Asn Leu BO

w 112 <210> 221 <211> 83 <212> PRT
<213> Hepatitis E virus <400> 221 Ile His Pro Thr Asn Pro Phe Ala Ala Asp Val Val Ser Gln Pro Giy Ala Gly Ala Arg Pro Arg Gln Pro Pro Arg Pro Leu Gly Ser Ser Trp Arg Asp Gln Ser Gln Arg Pro Ser Ala Ala Pro Arg Arg Arg Ser Thr Pro Ala Gly Ala Ala Pro Leu Thr Ala Val Ser Pro Ala Pro Asp Thr Ala Pro Val Pro Asp Val Asp Ser Arg Gly Ala Ile Leu Arg Arg Gln ' Tyr Asn Leu <210> 222 <211> 55 <212> PRT
<213> Hepatitis E virus <220>
<223> 20154-2.seq orf3 <400> 222 Ile Phe Ile Gln Pro Thr Pro Leu Pro Pro Thr Ser Tyr His Asn Pro Gly Leu Glu Leu Ala Leu Asp Ser Arg Pro Ala Pro Ser Ala Pro Leu Gly Val Ile Ser Pro Ser Ala Pro Pro Leu Pro Pro Val Val Asp Leu Pro Gln Leu Gly Leu Arg Arg <210> 223 <211> 48 <212> PRT

<213> Hepatitis E virus <220>

<223> US-2 3-2e <400> 223 Thr Ile Asp Tyr Pro Ala HisThr Asp PheCys Arg Ala Phe Asp Pro Glu Cys Arg Thr Leu Gly GlyCys Phe SerThr Leu Gln Ala Gln Ile Ala Glu Leu Gln Arg Leu LysVal Lys ArgGlu Lys Met Gly Thr Ser <210> 224 <211> 33 <212> PRT
<213> Hepatitis E virus <220>
<223> US-2 4-2 <400> 224 Asp Ser Arg Pro Ala Pro Leu Val Pro Leu Gly Val Thr Ser Pro Ser Ala Pro Pro Leu Pro Pro Val Val Asp Leu Pro Gln Leu Gly Leu Arg Arg <210> 225 <211> 450 <212> DNA
<213> Hepatitis E virus <220>
<223> 5p. pile {hpesvp}
<400> 225 ggctcctggc atcactactg ctattgagca ggctgctcta gcagcggcca actctgccct 60 ggcgaatgct gtggtagtta ggccttttct ctctcaccag cagattgaga tcctcattaa 120 cctaatgcaa cctcgccagc ttgttttccg ccccgaggtt ttctggaatc atcccatcca 180 gcgtgtcatc cataacgagc tggagcttta ctgccgcgcc cgctccggcc gctgtcttga 240 aattggcgcc catccccgct caataaatga taatcctaat gtggtccacc gctgcttcct 300 ccgccctgtt gggcgtgatg ttcagcgctg gtatactgct cccactcgcg ggccggctgc 360 taattgccgg cgttccgcgc tgcgcgggct tcccgctgct gaccgcactt actgcctcga 420 cgggttttct ggctgtaact ttcccgccga 450 <210> 226 <211> 450 <212> DNA
<213> Hepatitis E virus <220>
<223> 5p. pile {hpeuigh) <400> 226 ggctcctggc atcactactg ctattgagca ggctgctcta gcagcggcca attctgccct 60 tgcgaatgct gtggtagtta ggccttttct ctctcaccag cagattgaga tccttattaa 120 cctaatgcaa cctcgccagc ttgttttccg ccccgaggtt ttctggaacc accccatcca 180 gcgtgtcatc cataatgagc tggagcttta ctgtcgcgcc cgctccggcc gctgccttga 240 aattggtgcc caccctcgct caataaacga caatcctaat gtggtccacc gctgcttcct 300 ccgccctgcc gggcgtgatg ttcagcgttg gtatactgct cctacccgcg ggccggctgc 360 taattgccgg ggttccgcac tgcgcgggct ccccgctgct gaccgcactt actgcttcga 420 cgggttttct ggctgtaact ttcccgccga 450 <210> 227 <211> 450 <212> DNA
<213> Hepatitis E virus <220>
<223> 5p. pile {hpea}
<400> 227 ggctcctggc atcactactg ctattgagca ggctgctcta gcagcggcca actctgccct 60 tgcgaatgct gtggtagtta ggccttttct ctctcaccag cagattgaga tccttattaa 120 cctaatgcaa cctcgccagc ttgttttccg ccccgaggtt ttctggaacc atcccatcca 180 gcgtgttatc cataatgagc tggagcttta ctgtcgcgcc cgctccggcc gctgcctcga 240 aattggtgcc cacccccgct caataaatga caatcctaat gtggtccacc gttgcttcct 300 ccgtcctgcc gggcgtgatg ttcagcgttg gtatactgcc cctacccgcg ggccggctgc 360 taattgccgg cgttccgcgc tgcgcgggct ccccgctgct gaccgcactt actgcttcga 420 cgggttttct ggctgtaact ttcccgccga 450 <210> 228 <211> 446 <212> DNA
<213> Hepatitis E virus <220>
<223> 5p. pile {840455p}
<400> 228 cctggcatta ctactgccat tgagcaggct gctctggctg cggccaattc tgccttggcg 60 aatgctgtgg tggttcggcc gtttttatct cgcgtgcaaa ccgagattct tattaatttg 120 atgcaacccc ggcagttggt tttccgccct gaggtacttt ggaatcaccc tatccagcgg 180 gttatacata atgaattaga acagtactgc cgggctcggg ctggtcgttg cttggaggtt 240 ggagctcacc caagatccat taatgacaac cccaacgttc tgcatcggtg tttccttaga 300 ccggttggcc gagatgttca gcgctggtac tctgccccca cccgcggccc tgcggctaat 360 tgccgccgct ccgcgttgcg tggtctcccc cccgctgacc gcacttactg ctttgatgga 420 ttctcccgtt gtgcttttgc tgcaga 446 <210> 229 <211> 450 <212> DNA
<213> Hepatitis E virus <220>
<223> 5p. pile {hpenssp}
<400> 229 ggctcctggc atcactactg ctattgagca agcagctcta gcagcggcca actccgccct 60 tgcgaatgct gtggtggtcc ggcctttcct ttcccatcag caggttgaga tccttataaa 120 tctcatgcaa cctcggcagc tggtgtttcg tcctgaggtt ttttggaatc acccgattca 180 acgtgttata cataatgagc ttgagcagta ttgccgtgct cgctcgggtc gctgccttga 240 gattggagcc cacccacgct ccattaatga taatcctaat gtcctccatc gctgctttct 300 ccaccccgtc ggccgggatg ttcagcgctg gtacacagcc ccgactaggg gacctgcggc 360 gaactgtcgc cgctcggcac ttcgtggtct gccaccagcc gaccgcactt actgttttga 420 tggctttgcc ggctgccgtt ttgccgccga 450 <210> 230 <211> 450 <212> DNA
<213> Hepatitis E virus <220>
<223> 5p Consensus <220>
<221> variation <222> () . . (450) <223> The nucleotide identity of each n is indicated in Figure 9.
<400> 230 nnnncctggc atnactactg cnattgagca ngcngctctn gcngcggcca antcngccnt 60 ngcgaatgct gtggtngtnn ggccnttnnt ntcncnnnng cannnngaga tnctnatnaa 120 nntnatgcaa ccncgncagn tngtnttncg nccngaggtn ntntggaanc anccnatnca 180 ncgngtnatn cataangann tngancnnta ntgncgngcn cgnncnggnc gntgnntnga 240 nnttggngcn canccnngnt cnatnaanga naanccnaan gtnntncanc gntgnttnct 300 nnnnccngnn ggncgngatg ttcagcgntg gtanncngcn ccnacnngng gnccngcngc 360 naantgncgn ngntcngcnn tncgnggnct nccnncngcn gaccgcactt actgnntnga 420 nggnttnncn ngntgnnnnt ttncngcnga 450 <210> 231 <211> 300 <212> DNA
<213> Hepatitis E virus <220>
<223> 3p.pile {hpea} shown in Figure 9B
<400> 231 actgagtcag tgaagccagt gcttgacctg acaaattcaa ttctgtgtcg ggtggaatga 60 ataacatgtc ttttgctgcg cccatgggtt cgcgaccatg cgccctcggc ctattttgct 120 WO 99/19732 PCT/(TS98/21941 gttgctcctc atgtttctgc ctatgctgcc cgcgccaccg cccggtcagc cgtctggccg 180 ccgtcgtggg cggcgcagcg gcggttccgg cggtggtttc tggggtgacc gggttgattc 240 tcagcccttc gcaatcccct atattcatcc aaccaacccc ttcgcccccg atgtcaccgc 300 <210> 232 <211> 300 <212> DNA
<213> Hepatitis E virus <220>
<223> 3p.pile {hpeuigh} shown in Figure 9B
<400> 232 actgagtcgg tgaagccagt gctcgacttg acaaattcaa tcctgtgtcg ggtggaatga 60 ataacatgtc ttttgctgcg cccatgggtt ggcgaccatg cgccctcggc ctattttgct 120 gttgctcctc atgtttctgc ctatcgtgcc cgcgccaccg cccggtcagc cgtctggccg 180 ccgtcgtggg cggcgcagcg gcggttccgg cggtggtttc tggggtgacc gggttgattc 240 tcagcccttc gcaatcccct atattcatcc aaccaacccc ttcgcccccg atgtcaccgc 300 <210> 233 <211> 300 <212> DNA
<213> Hepatitis E virus <220>
<223> 3p.pile {hpesvp} shown in Figure 9B
<400> 233 actgagtcag taaaaccagt gctcgacttg acaaattcaa tcttgtgtcg ggtggaatga 60 ataacatgtc ttttgctgcg cccatgggtt cgcgaccatg cgccctcggc ctattttgtt 120 gctgctcctc atgtttttgc ctatgctgcc cgcgccaccg cccggtcagc cgtctggccg 180 ccgtcgtggg cggcgcagcg gcggttccgg cggtggtttc tggggtgacc gggttgattc 240 tcagcccttc gcaatcccct atattcatcc aaccaacccc ttcgcccccg atgtcaccgc 300 <210> 234 <211> 300 <212> DNA
<213> Hepatitis E virus <220>
<223> 3p.pile {hpenssp} shown in Figure 9B
<400> 234 acagagtctg ttaagcctat acttgacctt acacactcaa ttatgcaccg gtctgaatga 60 ataacatgtg gtttgctgcg cccatgggtt cgccaccatg cgccctaggc ctcttttgct 120 gttgttcctc ttgtttctgc ctatgttgcc cgcgccaccg accggtcagc cgtctggccg 180 ccgtcgtggg cggcgcagcg gcggtaccgg cggtggtttc tggggtgacc gggttgattc 240 tcagcccttc gcaatcccct atattcatcc aaccaacccc tttgccccag acgttgccgc 300 <210> 235 <211> 297 <212> DNA
<213> Hepatitis E virus <220>
<223> 3p.pile {840453p} shown in Figure 9B
<400> 235 acagagacta ttaaacctgt acttgatctc acaaattcca tcatacagcg ggtggaatga 60 ataacatgtc ttttgcatcg cccatgggat caccatgcgc cctagggctg ttctgttgtt 120 gttcctcatg tttctgccta tgctgcccgc gccaccggcc ggtcagccgt ctggccgtcg 180 ccgtgggcgg cgcagcggcg gtgccggcgg tggtttctgg agtgacaggg ttgattctca 240 gcccttcgcc ctcccctata ttcatccaac caaccccttc gccgccgatg tcgtttc 297 <210> 236 <211> 300 <212> DNA
<213> Hepatitis E virus <220>
<223> 3p Consensus shown in Figure 9B
<220>
<221> variation <222> (1) . . (300) <223> The nucleotide identity of each n is indicated in Figure 9B
<400> 236 acngagncnn tnaanccnnt nctnganntn acanantcna tnntnnnncg gnnngaatga 60 ataacatgtn ntttgcnncg cccatgggnt nnnnaccatg cgccctnggn ctnttntgnt 120 gntgntcctc ntgtttntgc ctatnntgcc cgcgccaccg nccggtcagc cgtctggccg 180 ncgncgtggg cggcgcagcg gcggtnccgg cggtggtttc tggngtgacn gggttgattc 240 tcagcccttc gcnntcccct atattcatcc aaccaacccc ttngccncng angtnnnnnc 300 <210> 237 <211> 250 <212> DNA
<213> Hepatitis E virus <220>
<223> 3p.pile {hpea} shown in Figure 9C
<400> 237 agcgcttacc ctgtttaacc ttgctgacac cctgcttggc ggtctaccga cagaattgat 60 ttcgtcggct ggtggccagc tgttctactc tcgccccgtc gtctcagcca atggcgagcc 120 gactgttaag ctgtatacat ctgtggagaa tgctcagcag gataagggta ttgcaatccc 180 gcatgacatc gacctcgggg aatcccgtgt agttattcag gattatgaca accaacatga 240 gcaggaccga 250 <210> 238 <211> 250 <212> DNA
<213> Hepatitis E virus <220>
<223> 3p.pile {hpeuigh} shown in Figure 9C
<400> 238 agcgcttacc ctgtttaacc ttgctgacac cctgcttggc ggtctaccga cagaattgat 60 ttcgtcggct ggtggccagc tgttctactc tcgccccgtc gtctcagcca atggcgagcc 120 gactgttaag ctgtatacat ctgtagagaa tgctcagcag gataagggta ttgcaatccc 180 gcatgacatc gacctcgggg aatctcgagt tgttattcag gattatgaca accaacatga 240 gcaggaccgg <210> 239 <211> 250 <212> DNA
<213> Hepatitis E virus <220>
<223> 3p.pile {hpesvp} shown in Figure 9C
<400> 239 agccctcacc ctgttcaacc ttgctgacac tctgcttggc ggcctgccga cagaattgat 60 ttcgtcggct ggtggccagc tgttctactc ccgtcccgtt gtctcagcca atggcgagcc 120 gactgttaag ttgtatacat ctgtagagaa tgctcagcag gataagggta ttgcaatccc 180 gcatgacatt gacctcggag aatctcgtgt ggttattcag gattatgata accaacatga 240 acaagatcgg 250 <210> 240 <211> 250 <212> DNA
<213> Hepatitis E virus <220>
<223> 3p.pile {hpenssp} shown in Figure 9C
<400> 240 agctctaaca ttacttaacc ttgctgacac gctcctcggc gggctcccga cagaattaat 60 ttcgtcggct ggcgggcaac tgttttattc ccgcccggtt gtctcagcca atggcgagcc 120 aaccgtgaag ctctatacat cagtggagaa tgctcagcag gataagggtg ttgctatccc 180 ccacgatatc gatcttggtg attcgcgtgt ggtcattcag gattatgaca accagcatga 240 gcaggatcgg 250 <210> 241 <211> 250 <212> DNA
<213> Hepatitis E virus <220>
<223> 3p.pile (840453p} shown in Figure 9C
<400> 241 tgccctgact ctgtttaatc ttgctgatac gcttcttggt ggtttaccga cagaattgat 60 ttcgtcggct gggggtcaac tgttttactc ccgccctgtt cagaattgat ttcgtcggct 120 gggggtcaac tgttttactc ccgccctgtt tgcgcagcaa gacaagggca tcaccattcc 180 acacgacata gatttaggtg actcccgtgt ggttatccag gattatgata accagcacga 240 acaagatcga 250 <210> 242 <211> 250 <212> DNA
<213> Hepatitis E virus <220>
<223> 3p Consensus shown in Figure 9C
<220>
<221> variation <222> ()..(250) <223> The nucleotide identity of each n is indicated in Figure 9C
<400> 242 ngcnctnacn ntnntnaanc ttgctganac nctnctnggn ggnntnccga cagaattnat 60 ttcgtcggct ggnggncanc tgttntantc ncgnccngtn gtctcngcca atggcgagcc 120 nacngtnaag ntntanacat cngtngagaa tgcncagcan ganaagggnn tnncnatncc 180 ncanganatn ganntnggng antcncgngt ngtnatncag gattatgana accancanga 240 ncangancgn 250

Claims

WHAT IS CLAIMED IS:

1. A method of detecting the presence of a US-type or US-subtype hepatitis E
virus (HEV) or a naturally occurring variant thereof in a test sample, the method comprising the steps of:
(a) contacting the sample with a binding partner that binds specifically to a marker for said virus, which if present in the sample binds to the binding partner to produce a marker-binding partner complex, and (b) detecting the presence of said complex, the presence of said complex being indicative of the presence of said virus in the sample.

2. The method of claim 1, wherein said marker is an antibody capable of binding said virus.

3. The method of claim 2, wherein said antibody is an immunoglobulin G or an immunoglobulin M.

4. The method of claim 2, wherein said binding partner is an isolated polypeptide chain.

5. The method of claim 4, wherein said polypeptide chain is immobilized on a solid support.

6. The method of claim 4, wherein said binding partner is a polypeptide chain selected from the group consisting of SEQ ID NOS:91, 92, and 93, including naturally occurring variants thereof.

7. The method of claim 4, wherein said binding partner is a polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:173 or SEQ ID NO:175.

8. The method of claim 4, where said binding partner is a polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:174 or SEQ ID NO:176.

9. The method of claim 4, wherein said binding partner is a polypeptide chain selected from the group consisting of SEQ ID NOS:166, 167 and 168, including naturally occurring variants thereof.

10. The method of claim 4, wherein said binding partner is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:223.

11. The method of claim 4, wherein said binding partner is a polypeptide comprising the amino acid sequence set forth in SEQ ID NO:224.

12. The method of claim 1, wherein said binding partner is an isolated antibody capable of binding specifically to a polypeptide chain selected from the group consisting of SEQ ID
NOS:91, 92, 93, 166, 167, and 168, including naturally occurring variants thereof.

13. The method of claim 12, wherein said antibody is a monoclonal antibody.

14. The method of claim 1, wherein said marker is a polypeptide chain.

15. The method of claim 14, wherein said polypeptide chain is selected from the group consisting of SEQ ID NOS:91, 92, and 93, including naturally occurring variants thereof.

16. The method of claim 14, wherein said polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:173 or SEQ ID NO:175.

17. The method of claim 14, wherein said polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:174 or SEQ ID NO:176.

18. The method of claim 14, wherein said polypeptide chain is selected from the group consisting of SEQ ID NOS:166, 167, and 168, including naturally occurring variants thereof.

19. The method of claim 14, wherein said polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:223.

20. The method of claim 14, wherein said polypeptide chain comprises the amino acid sequence set forth in SEQ ID NO:224.

21. The method of claim 1, wherein said marker is a nucleic acid sequence defining at least a portion of a genome of said virus, or a complementary strand thereof.

22. The method of claim 1 wherein said binding partner is an isolated nucleic acid sequence that is capable of hybridizing under specific hybridization conditions to the nucleic acid sequences set forth in SEQ ID NOS:89 and 164.

23. The method of claim 1 wherein said binding partner is selected from the group consisting of SEQ ID NOS:126, 128, 147, 148, 150, 152, 177 and 178.

24. The method of claim 1 wherein said binding partner is an isolated polypeptide chain.

25. The method of claim 1 wherein said test sample is a mammalian cell line.

26. The method of claim 41 wherein said mammalian cell tine is a human fetal kidney cell line.

27. A method of detecting the presence of a hepatitis E virus (HEV) in a test sample, the method comprising the steps of:
(a) contacting the sample with a binding partner selected from the group consisting of SEQ ID NOS:126, 128, 147, 148, 150, 152, 177 and 178 that binds specifically to a marker for said virus, which if present in the sample binds to the binding partner to produce a marker-binding partner complex, and (b) detecting the presence of said complex, the presence of said complex being indicative of the presence of said virus in the sample.

28. An isolated polypeptide chain comprising the amino acid sequence set forth in SEQ ID
NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:223 and SEQ ID
NO:224.

29. An isolated antibody capable of binding specifically to a polypeptide chain selected from the group consisting of a polypeptide encoded by an ORF 1 sequence of a US-type or a US-subtype HEV, a polypeptide encoded by an ORF 2 sequence of a US-type or a US-subtype HEV, and a polypeptide encoded by an ORF 3 sequence of a US-type or a US-subtype HEV.

30. An isolated antibody capable of binding specifically to a polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:173, SEQ ID NO:175 or SEQ ID
NO:224.

31. An isolated antibody capable of binding specifically to a polypeptide chain comprising the amino acid sequence set forth in SEQ ID NO:174, SEQ ID NO:176 or SEQ ID
NO:223.

32. The isolated antibody of claim 30, wherein said antibody, under similar conditions, has a lower affinity for a polypeptide chain comprising the amino acid sequence set forth in SEQ ID
NO:169 or 171.

33. The isolated antibody of claim 31, wherein said antibody, under similar conditions, has a lower affinity for a polypeptide chain comprising the amino acid sequence set forth SEQ ID
NO:170 or 172.

34. The isolated antibody of claim 29 further comprising a detectable moiety.

35. An isolated nucleic acid sequence defining at least a portion of an ORF 1, ORF 2 or ORF
3 sequence of a US-type or US-subtype hepatitis E virus, or a sequence complementary thereto.

36. An isolated nucleic acid sequence capable of hybridizing under specific hybridization conditions to the nucleotide sequence set forth in SEQ ID NOS:89 and 164.

37. A vector comprising the isolated nucleic acid sequence of claim 35.

38. A host cell containing the vector of claim 37.

39. A method of immunizing a mammal against a US-type or US-subtype HEV, the method comprising administering to the mammal the polypeptide of claim 28 in an amount sufficient to stimulate the production of an antibody capable of binding specifically to the US-type or US-subtype hepatitis E virus.

40. A method of immunizing a mammal against a US-type or US-subtype HEV 1, the method comprising administering to said mammal the antibody of claim 29 in an amount sufficient to immunize said mammal against the US-type or US-subtype hepatitis E virus.

41. A method of immunizing a mammal against a US-type or US-subtype HEV 1, the method comprising administering to said mammal the antibody of claim 30 in an amount sufficient to immunize said mammal against the US-type or US-subtype hepatitis E virus.

42. A method of immunizing a mammal against a US-type or US-subtype HEV 1, the method comprising administering to said mammal the antibody of claim 31 in an amount sufficient to immunize said mammal against the US-type or US-subtype hepatitis E virus.

43. A method of immunizing a mammal against a US-type or US-subtype HEV, the method comprising administering to said mammal the host cell of claim 38 in an amount sufficient to immunize said mammal against the US-type or US-subtype hepatitis E virus.