(en)Viral proteins derived from an enterically transmitted non-A/non-B viral hepatitis agent (HEV) are disclosed. In one embodiment, the protein is immunologically reactive with antibodies present in individuals infected with the viral hepatitis agent. This protein is useful in a diagnostic method for detecting infection by the enterically transmitted agent. Specific epitopes have been identified that are reactive with sera of individual infected with different strains of HEV. Also disclosed are DNA probes derived from a cloned sequence of the viral agent. These probes are useful for identifying and sequencing the entire viral agent and for assaying the presence of the viral agent in an infected sample, by using probe-specific amplification of virus-derived DNA fragments.

1.ApplicationNumber: US-55342700-A
1.PublishNumber: US-6379891-B1
2.Date Publish: 20020430
3.Inventor: REYES GREGORY R.
YARBOUGH PATRICE O.
BRADLEY DANIEL W.
KRAWCZYNSKI KRZYSZTOF Z.
TAM ALBERT
FRY KIRK E.

4.Inventor Harmonized: REYES GREGORY R(US)
YARBOUGH PATRICE O(US)
BRADLEY DANIEL W(US)
KRAWCZYNSKI KRZYSZTOF Z(US)
TAM ALBERT(US)
FRY KIRK E(US)

5.Country: US
6.Claims:
(en)Viral proteins derived from an enterically transmitted non-A/non-B viral hepatitis agent (HEV) are disclosed. In one embodiment, the protein is immunologically reactive with antibodies present in individuals infected with the viral hepatitis agent. This protein is useful in a diagnostic method for detecting infection by the enterically transmitted agent. Specific epitopes have been identified that are reactive with sera of individual infected with different strains of HEV. Also disclosed are DNA probes derived from a cloned sequence of the viral agent. These probes are useful for identifying and sequencing the entire viral agent and for assaying the presence of the viral agent in an infected sample, by using probe-specific amplification of virus-derived DNA fragments.

7.Description:
(en)CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a division of U.S. application Ser. No. 08/478,507, filed Jun. 7, 1995, which is a division of Ser. No. 08/279,823 filed Jul. 25, 1994 now U.S. Pat. No. 5,789,559 which is a continuation of Ser. No. 07/681,078 filed Apr. 5, 1991, abandoned, which is a continuation-in-part of U.S. application Ser. No. 07/505,888, filed Apr. 5, 1990, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 09/420,921, filed Oct. 13, 1989, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 09/367,486, filed Jun. 16, 1989, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 09/336,672, filed Apr. 11, 1989, now abandoned, which is a continuation-in-part of U.S. application Ser. No. 07/208,997, filed Jun. 17, 1988, now abandoned, all of which are herein incorporated by reference.


INTRODUCTION
1. Field of Invention
This invention relates to recombinant proteins, genes, and gene probes and more specifically to such proteins and probes derived from an enterically transmitted nonA/nonB hepatitis viral agent, to diagnostic methods and vaccine applications which employ the proteins and probes, and to gene segments that encode specific epitopes (and proteins artificially produced to contain those epitopes) that are particularly useful in diagnosis and prophylaxis.
2. Background
Enterically transmitted non-A/non-B hepatitis viral agent (ET-NANB; also referred to herein as HEV) is the reported cause of hepatitis in several epidemics and sporadic cases in Asia, Africa, Europe, Mexico, and the Indian subcontinent. Infection is usually by water contaminated with feces, although the virus may also spread by close physical contact. The virus does not seem to cause chronic infection. The viral etiology in ET-NANB has been demonstrated by infection of volunteers with pooled fecal isolates; immune electron microscopy (IEM) studies have shown virus particles with 27-34 nm diameters in stools from infected individuals. The virus particles reacted with antibodies in serum from infected individuals from geographically distinct regions, suggesting that a single viral agent or class is responsible for the majority of ET-NANB hepatitis seen worldwide. No antibody reaction was seen in serum from individuals infected with parenterally transmitted NANB virus (also known as hepatitis C virus or HCV), indicating a different specificity between the two NANB types.
In addition to serological differences, the two types of NANB infection show distinct clinical differences. ET-NANB is characteristically an acute infection, often associated with fever and arthralgia, and with portal inflammation and associated bile stasis in liver biopsy specimens (Arankalle). Symptoms are usually resolved within six weeks. Parenterally transmitted NANB, by contrast, produces a chronic infection in about 50% of the cases. Fever and arthralgia are rarely seen, and inflammation has a predominantly parenchymal distribution (Khuroo, 1980). The course of ET-NANBH is generally uneventful in healthy individuals, and the vast majority of those infected recover without the chronic sequelae seen with HCV. One peculiar epidemiologic feature of this disease, however, is the markedly high mortality observed in pregnant women; this is reported in numerous studies to be on the order of 10-20%. This finding has been seen in a number of epidemiologic studies but at present remains unexplained. Whether this reflects viral pathogenicity, the lethal consequence of the interaction of virus and immune suppressed (pregnant) host, or a reflection of the debilitated prenatal health of a susceptible malnourished population remains to be clarified.
The two viral agents can also be distinguished on the basis of primate host susceptibility. ET-NANB, but not the parenterally transmitted agent, can be transmitted to cynomolgus monkeys. The parenterally transmitted agent is more readily transmitted to chimpanzees than is ET-NANB (Bradley, 1987).
There have been major efforts worldwide to identify and clone viral genomic sequences associated with ET-NANB hepatitis. One goal of this effort, requiring virus-specific genomic sequences, is to identify and characterize the nature of the virus and its protein products. Another goal is to produce recombinant viral proteins which can be used in antibody-based diagnostic procedures and for a vaccine. Despite these efforts, viral sequences associated with ET-NANB hepatitis have not been successfully identified or cloned heretofore, nor have any virus-specific proteins been identified or produced.
RELEVANT LITERATURE
Arankalle, V. A., et al., The Lancet, 550 (Mar. 12, 1988).
Bradley, D. W., et al., J Gen. Virol., 69:1 (1988).
Bradley, D. W. et al., Proc. Nat. Acad. Sci., USA, 84:6277 (1987).
Gravelle, C. R. et al., J. Infect. Diseases, 131:167 (1975).
Kane, M. A., et al., JAMA, 252:3140 (1984).
Khuroo, M. S., Am. J. Med., 48:818 (1980).
Khuroo, M. S., et al., Am. J. Med., 68:818 (1983).
Maniatis, T., et al. Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory (1982).
Seto, B., et al., Lancet, 11:941 (1984).
Sreenivasan, M. A., et al., J. Gen. Virol., 65:1005 (1984).
Tabor, E., et al., J. Infect. Dis., 140:789 (1979).
SUMMARY OF THE INVENTION
Novel compositions, as well as methods of preparation and use of the compositions are provided, where the compositions comprise viral proteins and fragments thereof derived from the viral agent for ET-NANB. A number of specific fragments of viral proteins (and the corresponding genetic sequences) that are particularly useful in diagnosis and vaccine production are also disclosed. Methods for preparation of ET-NANB viral proteins include isolating ET-NANB genomic sequences which are then cloned and expressed in a host cell. The resultant recombinant viral proteins find use as diagnostic agents and as vaccines. The genomic sequences and fragments thereof find use in preparing ET-NANB viral proteins and as probes for virus detection.


BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows vector constructions and manipulations used in obtaining and sequencing cloned ET-NANB fragment; and
FIGS. 2A-2B are representations of Southern blots in which a radiolabeled ET-NANB probe was hybridized with amplified cDNA fragments prepared from RNA isolated from infected (I) and non-infected (N) bile sources ( 2 A), and from infected (I) and non-infected (N) stool-sample sources ( 2 B).


DESCRIPTION OF SPECIFIC EMBODIMENTS
Novel compositions comprising generic sequences and fragments thereof derived from the viral agent for ET-NANB are provided, together with recombinant viral proteins produced using the genomic sequences and methods of using these compositions. Epitopes on the viral protein have been identified that are particularly useful in diagnosis and vaccine production. Small peptides containing the epitopes are recognized by multiple sera of patients infected with ET-NANB.
The molecular cloning of HEV was accomplished by two very different approaches. The first successful identification of a molecular clone was based on the differential hybridization of putative HEV cDNA clones to heterogeneous cDNA from infected and uninfected cyno bile. cDNAs from both sources were labeled to high specific activity with 32 P to identify a clone that hybridized specifically to the infected source probe. A cyno monkey infected with the Burma isolate of HEV was used in these first experiments. The sensitivity of this procedure is directly related to the relative abundance of the specific sequence against the overall background. In control experiments, it was found that specific identification of a target sequence may be obtained with as little as 1 specific part per 1000 background sequences. A number of clones were identified by this procedure using libraries and probes made from infected (Burma isolate) and control uninfected cyno bile. The first extensively characterized clone of the 16 plaques purified by this protocol was given the designation ET1.1.
ET1.1 was first characterized as both derived from and unique to the infected source cDNA. Heterogeneous cDNA was amplified from both infected and uninfected sources using a sequence independent single premier amplification technique (SISPA). This technique is described in copending application Ser. No. 208,512, filed Jun. 17, 1988. The limited pool of cDNA made from Burma infected cyno bile could then be amplified enzymatically prior to cloning or hybridization using putative HEV clones as probes. ET1.1 hybridized specifically to the original bile cDNA from the infected source. Further validation of this clone as derived from the genome of HEV was demonstrated by the similarity of the ET1.1 sequence and those present in SISPA cDNA prepared from five different human stool samples collected from different ET-NANBH epidemics including Somalia, Tashkent, Borneo, Mexico and Pakistan. These molecular epidemiologic studies established the isolated sequence as derived from the virus that represented the major cause of ET-NANBH worldwide.
The viral specificity of ET1.1 was further established by the finding that the clone hybridized specifically to RNA extracted from infected cyno liver. Hybridization analysis of polyadenylated RNA demonstrated a unique 7.5 Kb polyadenylated transcript not present in uninfected liver. The size of this transcript suggested that it represented the full length viral genome. Strand specific oligonucleotides were also used to probe viral genomic RNA extracted directly from semi-purified virions prepared from human stool. The strand specificity was based on the RNA-directed RNA polymerase (RDRP) open reading frame (ORF) identified in ET1.1 (see below). Only the probe detecting the sense strand hybridized to the nucleic acid. These studies characterized HEV as a plus sense, single stranded genome. Strand specific hybridization to RNA extracted from the liver also established that the vast majority of intracellular transcript was positive sense. Barring any novel mechanism for virus expression, the negative strand, although not detectable, would be present at a ratio of less than 1:100 when compared with the sense strand.
ET1.1 was documented as exogenous when tested by both Southern blot hybridization and PCR using genomic DNAs derived from uninfected humans, infected and uninfected cynos and also the genomic DNAs from E. coli and various bacteriophage sources. The latter were tested in order to rule out trivial contamination with an exogenous sequence introduced during the numerous enzymatic manipulations performed during cDNA construction and amplification. It was also found that the nucleotide sequence of the ET1.1 clone was not homologous to any entries in the Genebank database. The translated open reading frame of the ET1.1 clone did, however, demonstrate limited homology with consensus amino acid residues consistent with an RNA-directed RNA polymerase. This consensus amino acid motif is shared among all positive strand RNA viruses and, as noted above, is present at the 3′ end of the HCV genome. The 1.3 Kb clone was therefore presumed to be derived, at least in part, from the nonstructural portion of the viral genome.
Because of the relationship of different strains of ET-NANB to each other that has been demonstrated by the present invention, the genome of the ET-NANB viral agent is defined in this specification as containing a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZKF1 (ET1.1) carried in E. coli strain BB4 and having ATCC deposit no. 67717. The entire sequence, in both directions, has now been identified as set forth below. The sequences of both strands are provided, since both strands can encode proteins. However, the sequence in one direction has been designated as the “forward” sequence because of statistical similarities to known proteins and because the forward sequence is known to be predominately protein-encoding. This sequence is set forth below along with the three possible transportation sequences. There is one long open reading frame that starts at nucleotide 145 with an isoleucine and extends to the end of the sequence. The two other reading frames have many termination codons. Standard abbreviations for nucleotides and amino acids are used here and elsewhere in this specification.
The gene sequence given below is substantially identical to one given in the parent application. The present sequence differs in the omission of the first 37 nucleotides at the 5′ end and last 13 nucleotides at the 3′ end, which are derived from the linker used for cloning rather than from the virus. In addition, a G was omitted at position 227 of the sequence given in the parent application.
The following gene sequence has SEQ ID NO.1; the first amino acid sequence in reading frame beginning with nucleotide 1 has SEQ ID NO.2; the second amino acid sequence in reading frame beginning with nuclectide 2 has SEQ ID NO.3; and the third amino acid sequence in reading frame beginning with nucleotide 3 has SEQ ID NO.4.
Forward Sequence






SEQ ID NO. 1:






AGACCTGTCC CTGTTGCAGC TGTTCTACCA CCCTGCCCCG AGCTCGAACA GGGCCTTCTC
60




TACCTGCCCC AGGAGCTCAC CACCTGTGAT AGTGTCGTAA CATTTGAATT AACAGACATT
120



GTGCACTGCC GCATGGCCGC CCCGAGCCAG CGCAAGGCCG TGCTGTCCAC ACTCGTGGGC
180



CGCTACGGCG GTCGCACAAA GCTCTACAAT GCTTCCCACT CTGATGTTCG CGACTCTCTC
240



GCCCGTTTTA TCCCGGCCAT TGGCCCCGTA CAGGTTACAA CTTGTGAATT GTACGAGCTA
300



GTGGAGGCCA TGGTCGAGAA GGGCCAGGAT GGCTCCGCCG TCCTTGAGCT TGATCTTTGC
360



AACCGTGACG TGTCCAGGAT CACCTTCTTC CAGAAAGATT GTAACAAGTT CACCACAGGT
420



GAGACCATTG CCCATGGTAA AGTGGGCCAG GGCATCTCGG CCTGGAGCAA GACCTTCTGC
480



GCCCTCTTTG GCCCTTGGTT CCGCGCTATT GAGAAGGCTA TTCTGGCCCT GCTCCCTCAG
540



GGTGTGTTTT ACGGTGATGC CTTTGATGAC ACCGTCTTCT CGGCGGCTGT GGCCGCAGCA
600



AAGGCATCCA TGGTGTTTGA GAATGACTTT TCTGAGTTTG ACTCCACCCA GAATAACTTT
660



TCTCTGGGTC TAGAGTGTGC TATTATGGAG GAGTGTGGGA TGCCGCAGTG GCTCATCCGC
720



CTGTATCACC TTATAAGGTC TGCGTGGATC TTGCAGGCCC CGAAGGAGTC TCTGCGAGGG
780



TTTTGGAAGA AACACTCCGG TGAGCCCGGC ACTCTTCTAT GGAATACTGT CTGGAATATG
840



GCCGTTATTA CCCACTGTTA TGACTTCCGC GATTTTCAGG TGGCTGCCTT TAAAGGTGAT
900



GATTCGATAG TGCTTTGCAG TGAGTATCGT CAGAGTCCAG GAGCTGCTGT CCTGATCGCC
960



GGCTGTGGCT TGAAGTTGAA GGTAGATTTC CGCCCGATCG GTTTGTATGC AGGTGTTGTG
1020



GTGGCCCCCG GCCTTGGCGC GCTCCCTGAT GTTGTGCGCT TCGCCGGCCG GCTTACCGAG
1080



AAGAATTGGG GCCCTGGCCC TGAGCGGGCG GAGCAGCTCC GCCTCGCTGT TAGTGATTTC
1140



CTCCGCAAGC TCACGAATGT AGCTCAGATG TGTGTGGATG TTGTTTCCCG TGTTTATGGG
1200



GTTTCCCCTG GACTCGTTCA TAACCTGATT GGCATGCTAC AGGCTGTTGC TGATGGCAAG
1260



GCACATTTCA CTGAGTCAGT AAAACCAGTG CTCGA
1295






SEQ ID NO. 2




Arg Pro Val Pro Val Ala Ala Val Leu Pro Pro Cys Pro Glu Leu Glu

1 5 10 15



Gln Gly Leu Leu Tyr Leu Pro Gln Glu Leu Thr Thr Cys Asp Ser Val

20 25 30



Val Thr Phe Glu Leu Thr Asp Ile Val His Cys Arg Met Ala Ala Pro

35 40 45



Ser Gln Arg Lys Ala Val Leu Ser Thr Leu Val Gly Arg Tyr Gly Gly

50 55 60



Arg Thr Lys Leu Tyr Asn Ala Ser His Ser Asp Val Arg Asp Ser Leu

65 70 75 80



Ala Arg Phe Ile Pro Ala Ile Gly Pro Val Gln Val Thr Thr Cys Glu

85 90 95



Leu Tyr Glu Leu Val Glu Ala Met Val Glu Lys Gly Gln Asp Gly Ser

100 105 110



Ala Val Leu Glu Leu Asp Leu Cys Asn Arg Asp Val Ser Arg Ile Thr

115 120 125



Phe Phe Gln Lys Asp Cys Asn Lys Phe Thr Thr Gly Glu Thr Ile Ala

130 135 140



His Gly Lys Val Gly Gln Gly Ile Ser Ala Trp Ser Lys Thr Phe Cys

145 150 155 160



Ala Leu Phe Gly Pro Trp Phe Arg Ala Ile Glu Lys Ala Ile Leu Ala

165 170 175



Leu Leu Pro Gln Gly Val Phe Tyr Gly Asp Ala Phe Asp Asp Thr Val

180 185 190



Phe Ser Ala Ala Val Ala Ala Ala Lys Ala Ser Met Val Phe Glu Asn

195 200 205



Asp Phe Ser Glu Phe Asp Ser Thr Gln Asn Asn Phe Ser Leu Gly Leu

210 215 220



Glu Cys Ala Ile Met Glu Glu Cys Gly Met Pro Gln Trp Leu Ile Arg

225 230 235 240



Leu Tyr His Leu Ile Arg Ser Ala Trp Ile Leu Gln Ala Pro Lys Glu

245 250 255



Ser Leu Arg Gly Phe Trp Lys Lys His Ser Gly Glu Pro Gly Thr Leu

260 265 270



Leu Trp Asn Thr Val Trp Asn Met Ala Val Ile Thr His Cys Tyr Asp

275 280 285



Phe Arg Asp Phe Gln Val Ala Ala Phe Lys Gly Asp Asp Ser Ile Val

290 295 300



Leu Cys Ser Glu Tyr Arg Gln Ser Pro Gly Ala Ala Val Leu Ile Ala

305 310 315 320



Gly Cys Gly Leu Lys Leu Lys Val Aso Phe Arg Pro Ile Gly Leu Tyr

325 330 335



Ala Gly Val Val Val Ala Pro Gly Leu Gly Ala Leu Pro Asp Val Val

340 345 350



Arg Phe Ala Gly Arg Leu Thr Glu Lys Asn Trp Gly Pro Gly Pro Glu

355 360 365



Arg Ala Glu Gln Leu Arg Leu Ala Val Ser Asp Phe Leu Arg Lys Leu

370 375 380



Thr Asn Val Ala Gln Met Cys Val Asp Val Val Ser Arg Val Tyr Gly

385 390 395 400



Val Ser Pro Gly Leu Val His Asn Leu Ile Gly Met Leu Gln Ala Val

405 410 415



Ala Asp Gly Lys Ala His Phe Thr Glu Ser Val Lys Pro Val Leu

420 425 430






SEQ ID NO. 3




Asp Leu Ser Leu Leu Gln Leu Phe Tyr His Pro Ala Pro Ser Ser Asn

1 5 10 15



Arg Ala Phe Ser Thr Cys Pro Arg Ser Ser Pro Pro Val Ile Val Ser

20 25 30



. His Leu Asn . Gln Thr Leu Cys Thr Ala Ala Trp Pro Pro Arg

35 40 45



Ala Ser Ala Arg Pro Cys Cys Pro His Ser Trp Ala Ala Thr Ala Val

50 55 60



Ala Gln Ser Ser Thr Met Leu Pro Thr Leu Met Phe Ala Thr Leu Ser

65 70 75 80



Pro Val Leu Ser Arg Pro Leu Ala Pro Tyr Arg Leu Gln Leu Val Asn

85 90 95



Cys Thr Ser . Trp Arg Pro Trp Ser Arg Arg Ala Arg Met Ala Pro

100 105 110



Pro Ser Leu Ser Leu Ile Phe Ala Thr Val Thr Cys Pro Gly Ser Pro

115 120 125



Ser Ser Arg Lys Ile Val Thr Ser Ser Pro Gln Val Arg Pro Leu Pro

130 135 140



Met Val Lys Trp Ala Arg Ala Ser Arg Pro Gly Ala Arg Pro Ser Ala

145 150 155 160



Pro Ser Leu Ala Leu Gly Ser Ala Leu Leu Arg Arg Leu Phe Trp Pro

165 170 175



Cys Ser Leu Arg Val Cys Phe Thr Val Met Pro Leu Met Thr Pro Ser

180 185 190



Ser Arg Arg Leu Trp Pro Gln Gln Arg His Pro Trp Cys Leu Arg Met

195 200 205



Thr Phe Leu Ser Leu Thr Pro Pro Arg Ile Thr Phe Leu Trp Val .

210 215 220



Ser Val Leu Leu Trp Arg Ser Val Gly Cys Arg Ser Gly Ser Ser Ala

225 230 235 240



Cys Ile Thr Leu . Gly Leu Arg Gly Ser Cys Arg Pro Arg Arg Ser

245 250 255



Leu Cys Glu Gly Phe Gly Arg Asn Thr Pro Val Ser Pro Ala Leu Phe

260 265 270



Tyr Gly Ile Leu Ser Gly Ile Trp Pro Leu Leu Pro Thr Val Met Thr

275 280 285



Ser Ala Ile Phe Arg Trp Leu Pro Leu Lys Val Met Ile Arg . Cys

290 295 300



Phe Ala Val Ser Ile Val Arg Val Gln Glu Leu Leu Ser . Ser Pro

305 310 315 320



Ala Val Ala . Ser . Arg . Ile Ser Ala Arg Ser Val Cys Met

325 330 335



Gln Val Leu Trp Trp Pro Pro Ala Leu Ala Arg Ser Leu Met Leu Cys

340 345 350



Ala Ser Pro Ala Gly Leu Pro Arg Arg Ile Gly Ala Leu Ala Leu Ser

355 360 365



Gly Arg Ser Ser Ser Ala Ser Leu Leu Val Ile Ser Ser Ala Ser Ser

370 375 380



Arg Met . Leu Arg Cys Val Trp Met Leu Phe Pro Val Phe Met Gly

385 390 395 400



Phe Pro Leu Asp Ser Phe Ile Thr . Leu Ala Cys Tyr Arg Leu Leu

405 41O 415



Leu Met Ala Arg His Ile Ser Leu Ser Gln . Asn Gln Cys Ser

420 425 430






SEQ ID NO. 4




Thr Cys Pro Cys Cys Ser Cys Ser Thr Thr Leu Pro Arg Ala Arg Thr

1 5 10 15



Gly Pro Ser Leu Pro Ala Pro Gly Ala His His Leu . . Cys Arg

20 25 30



Asn Ile . Ile Asn Arg His Cys Ala Leu Pro His Gly Arg Pro Glu

35 40 45



Pro Ala Gln Gly Arg Ala Val His Thr Arg Gly Pro Leu Arg Arg Ser

50 55 60



His Lys Ala Leu Gln Cys Phe Pro Leu . Cys Ser Arg Leu Ser Arg

65 70 75 80



Pro Phe Tyr Pro Gly His Trp Pro Arg Thr Gly Tyr Asn Leu . Ile

85 90 95



Val Arg Ala Ser Gly Gly His Gly Arg Glu Gly Pro Gly Trp Leu Arg

100 105 110



Arg Pro . Ala . Ser Leu Gln Pro . Arg Val Gln Asp His Leu

115 120 125



Leu Pro Glu Arg Leu . Gln Val His His Arg . Asp His Cys Pro

130 135 140



Trp . Ser Gly Pro Gly His Leu Gly Leu Glu Gln Asp Leu Leu Arg

145 150 155 150



Pro Leu Trp Pro Leu Val Pro Arg Tyr . Glu Gly Tyr Ser Gly Pro

165 170 175



Ala Pro Ser Gly Cys Val Leu Arg . Cys Leu . . His Arg Leu

180 185 190



Leu Gly Gly Cys Gly Arg Ser Lys Gly Ile His Gly Val . Glu .

195 200 205



Leu Phe . Val . Leu His Pro Glu . Leu Phe Ser Gly Ser Arg

210 215 220



Val Cys Tyr Tyr Gly Gly Val Trp Asp Ala Ala Val Ala His Pro Pro

225 230 235 240



Val Ser Pro Tyr Lys Val Cys Val Asp Leu Ala Gly Pro Glu Gly Val

245 250 255



Ser Ala Arg Val Leu Glu Glu Thr Leu Arg . Ala Arg His Ser Ser

250 265 270



Met Glu Tyr Cys Leu Glu Tyr Gly Arg Tyr Tyr Pro Leu Leu . Leu

275 280 285



Pro Arg Phe Ser Gly Gly Cys Leu . Arg . . Phe Asp Ser Ala

290 295 300



Leu Gln . Val Ser Ser Glu Ser Arg Ser Cys Cys Pro Asp Arg Arg

305 310 315 320



Leu Trp Leu Glu Val Glu Gly Arg Phe Pro Pro Asp Arg Phe Val Cys

325 330 335



Arg Cys Cys Gly Gly Pro Arg Pro Trp Arg Ala Pro . Cys Cys Ala

340 345 350



Leu Arg Arg Pro Ala Tyr Arg Glu Glu Leu Gly Pro Trp Pro . Ala

355 360 365



Gly Gly Ala Ala Pro Pro Arg Cys . . Phe Pro Pro Gln Ala His

370 375 380



Glu Cys Ser Ser Asp Val Cys Gly Cys Cys Phe Pro Cys Leu Trp Gly

385 390 395 400



Phe Pro Trp Thr Arg Ser . Pro Asp Trp His Ala Thr Gly Cys Cys

405 41O 415



. Trp Gln Gly Thr Phe His . Val Ser Lys Thr Ser Ala Arg

420 425 430






























































































































































































































































































































The complementary strand, referred to here as the “reverse sequence,” is set forth below in the same manner as the forward sequence set forth above. Several open reading frames, shorter than the long open reading frame found in the forward sequence, can be seen this reverse sequence. Because of the relative brevity of the open reading frames in the reverse direction, they are probably not expressed.
The following gene sequence has SEQ ID NO.5.
Reverse Sequence








TCGAGCACTG GTTTTACTGA CTCAGTGAAA TGTGCCTTGC CATCAGCAAC AGCCTGTAGC
60
SEQ ID NO. 5:




ATGCCAATCA GGTTATGAAC GAGTCCAGGG GAAACCCCAT AAACACGGGA AACAACATCC
120



ACACACATCT GAGCTACATT CGTGAGCTTG CGGAGGAAAT CACTAACAGC GAGGCGGAGC
180



TGCTCCGCCC GCTCAGGGCC AGGGCCCCAA TTCTTCTCGG TAAGCCGGCC GGCGAAGCGC
240



ACAACATCAG GGAGCGCGCC AAGGCCGGGG GCCACCACAA CACCTGCATA CAAACCGATC
300



GGGCGGAAAT CTACCTTCAA CTTCAAGCCA CAGCCGGCGA TCAGGACAGC AGCTCCTGGA
360



CTCTGACGAT ACTCACTGCA AAGCACTATC GAATCATCAC CTTTAAAGGC AGCCACCTGA
420



AAATCGCGGA AGTCATAACA GTGGGTAATA ACGGCCATAT TCCAGACAGT ATTCCATAGA
480



AGAGTGCCGG GCTCACCGGA GTGTTTCTTC CAAAACCCTC GCAGAGACTC CTTCGGGGCC
540



TGCAAGATCC ACGCAGACCT TATAAGGTGA TACAGGCGGA TGAGCCACTG CGGCATCCCA
600



CACTCCTCCA TAATAGCACA CTCTAGACCC AGAGAAAAGT TATTCTGGGT GGAGTCAAAC
660



TCAGAAAAGT CATTCTCAAA CACCATGGAT GCCTTTGCTG CGGCCACAGC CGCCGAGAAG
720



ACGGTGTCAT CAAAGGCATC ACCGTAAAAC ACACCCTGAG GGAGCAGGGC CAGAATAGCC
780



TTCTCAATAG CGCGGAACCA AGGGCCAAAG AGGGCGCAGA AGGTCTTGCT CCAGGCCGAG
840



ATGCCCTGGC CCACTTTACC ATGGGCAATG GTCTCACCTG TGGTGAACTT GTTACAATCT
900



TTCTGGAAGA AGGTGATCCT GGACACGTCA CGGTTGCAAA GATCAAGCTC AAGGACGGCG
960



GAGCCATCCT GGCCCTTCTC GACCATGGCC TCCACTAGCT CGTACAATTC ACAAGTTGTA
1020



ACCTGTACGG GGCCAATGGC CGGGATAAAA CGGGCGAGAG AGTCGCGAAC ATCAGAGTGG
1080



GAAGCATTGT AGAGCTTTGT GCGACCGCCG TAGCGGCCCA CGAGTGTGGA CAGCACGGCC
1140



TTGCGCTGGC TCGGGGCGGC CATGCGGCAG TGCACAATGT CTGTTAATTC AAATGTTACG
1200



ACACTATCAC AGGTGGTGAG CTCCTGGGGC AGGTAGAGAA GGCCCTGTTC GAGCTCGGGG
1260



CAGGGTGGTA GAACAGCTGC AACAGGGACA GGTCT
1295


















































Identity of this sequence with sequences in etiologic agents has been confirmed by locating a corresponding sequence in a viral strain isolated in Burma. The Burmese isolate contains the following sequence of nucleotides (one strand and open reading frames shown). The following gene sequence has SEQ ID NO.6; the protein sequence corresponding to ORF1 has SEQ ID NO.7; ORF2 has SEQ ID NO.8; and ORF3 has SEQ ID NO.9.







SEQUENCE OF HEV (BURMA STRAIN)








|-ORF1-->


M E A H Q F I K A P G

AGGCAGACCACATATGTGGTCGATGCCATGGAGGCCCATCAGTTTATTAAGGCTCCTGGC



I T T A I E Q A A L A A A N S A L A N A

ATCACTACTGCTATTGAGCAGGCTGCTCTAGCAGCGGCCAACTCTGCCCTGGCGAATGCT
120



V V V R P F L S H Q Q I E I L I N L M Q

GTGGTAGTTAGGCCTTTTCTCTCTCACCAGCAGATTGAGATCCTCATTAACCTAATGCAA



P R Q L V F R P E V F W N H P I Q R V I

CCTCGCCAGCTTGTTTTCCGCCCCGAGGTTTTCTGGAATCATCCCATCCAGCGTGTCATC
240



H N E L E L Y C R A R S G R C L E I G A

CATAACGAGCTGGAGCTTTACTGCCGCGCCCGCTCCGGCCGCTGTCTTGAAATTGGCGCC



H P R S I N D N P N V V H R C F L R P V

CATCCCCGCTCAATAAATGATAATCCTAATGTGGTCCACCGCTGCTTCCTCCGCCCTGTT
360



G R D V Q R W Y T A P T R G P A A N C R

GGGCGTGATGTTCAGCGCTGGTATACTGCTCCCACTCGCGGGCCGGCTGCTAATTGCCGG



R S A L R G L P A A D R T Y C L D G F S

CGTTCCGCGCTGCGCGGGCTTCCCGCTGCTGACCGCACTTACTGCCTCGACGGGTTTTCT
480



G C N F P A E T G I A L Y S L H D M S P

GGCTGTAACTTTCCCGCCGAGACTGGCATCGCCCTCTACTCCCTTCATGATATGTCACCA



S D V A E A M F R H G M T R L Y A A L H

TCTGATGTCGCCGAGGCCATGTTCCGCCATGGTATGACGCGGCTCTATGCCGCCCTCCAT
600



L P P E V L L P P G T Y R T A S Y L L I

CTTCCGCCTGAGGTCCTGCTGCCCCCTGGCACATATCGCACCGCATCGTATTTGCTAATT



H D G R R V V V T Y E G D T S A G Y N H

CATGACGGTAGGCGCGTTGTGGTGACGTATGAGGGTGATACTAGTGCTGGTTACAACCAC
720



D V S N L R S W I R T T K V T G D H P L

GATGTCTCCAACTTGCGCTCCTGGATTAGAACCACCAAGGTTACCGGAGACCATCCCCTC



V I E R V R A I G C H F V L L L T A A P

GTTATCGAGCGGGTTAGGGCCATTGGCTGCCACTTTGTTCTCTTGCTCACGGCAGCCCCG
840



E P S P M P Y V P Y P R S T E V Y V R S

GAGCCATCACCTATGCCTTATGTTCCTTACCCCCGGTCTACCGAGGTCTATGTCCGATCG



I F G P G G T P S L F P T S C S T K S T

ATCTTCGGCCCGGGTGGCACCCCTTCCTTATTCCCAACCTCATGCTCCACTAAGTCGACC
960



F H A V P A H I W D R L M L F G A T L D

TTCCATGCTGTCCCTGCCCATATTTGGGACCGTCTTATGCTGTTCGGGGCCACCTTGGAT



D Q A F C C S R L M T Y L R G I S Y K V

GACCAAGCCTTTTGCTGCTCCCGTTTAATGACCTACCTTCGCGGCATTAGCTACAAGGTC
1080



T V G T L V A N E G W N A S E D A L T A

ACTGTTGGTACCCTTGTGGCTAATGAAGGCTGGAATGCCTCTGAGGACGCCCTCACAGCT



V I T A A Y L T I C H Q R Y L R T Q A I

GTTATCACTGCCGCCTACCTTACCATTTGCCACCAGCGGTATCTCCGCACCCAGGCTATA
1200



S K G M R R L E R E H A Q K F I T R L Y

TCCAAGGGGATGCGTCGTCTGGAACGGGAGCATGCCCAGAAGTTTATAACACGCCTCTAC



S W L F E K S G R D Y I P G R Q L E F Y

AGCTGGCTCTTCGAGAAGTCCGGCCGTGATTACATCCCTGGCCGTCAGTTGGAGTTCTAC
1320



A Q C R R W L S A G F H L D P R V L V F

GCCCAGTGCAGGCGCTGGCTCTCCGCCGGCTTTCATCTTGATCCACGGGTGTTGGTTTTT



D E S A P C H C R T A I R K A L S K F C

GACGAGTCGGCCCCCTGCCATTGTAGGACCGCGATCCGTAAGGCGCTCTCAAAGTTTTGC
1440



C F M K W L G Q E C T C F L Q P A E G A

TGCTTCATGAAGTGGCTTGGTCAGGAGTGCACCTGCTTCCTTCAGCCTGCAGAAGGCGCC



V G D Q G H D N E A Y E G S D V D P A E

GTCGGCGACCAGGGTCATGATAATGAAGCCTATGAGGGGTCCGATGTTGACCCTGCTGAG
1560



S A I S D I S G S Y V V P G T A L Q P L

TCCGCCATTAGTGACATATCTGGGTCCTATGTCGTCCCTGGCACTGCCCTCCAACCGCTC



Y Q A L D L P A E I V A R A G R L T A T

TACCAGGCCCTCGATCTCCCCGCTGAGATTGTGGCTCGCGCGGGCCGGCTGACCGCCACA
1680



V K V S Q V D G R I D C E T L L G N K T

GTAAAGGTCTCCCAGGTCGATGGGCGGATCGATTGCGAGACCCTTCTTGGTAACAAAACC



F R T S F V D G A V L E T N G P E R H N

TTTCGCACGTCGTTCGTTGACGGGGCGGTCTTAGAGACCAATGGCCCAGAGCGCCACAAT
1800



L S F D A S Q S T M A A G P F S L T Y A

CTCTCCTTCGATGCCAGTCAGAGCACTATGGCCGCTGGCCCTTTCAGTCTCACCTATGCC



A S A A G L E V R Y V A A G L D H R A V

GCCTCTGCAGCTGGGCTGGAGGTGCGCTATGTTGCTGCCGGGCTTGACCATCGGGCGGTT
1920



F A P G V S P R S A P G E V T A F C S A

TTTGCCCCCGGTGTTTCACCCCGGTCAGCCCCCGGCGAGGTTACCGCCTTCTGCTCTGCC



L Y R F N R E A Q R H S L I G N L W F H

CTATACAGGTTTAACCGTGAGGCCCAGCGCCATTCGCTGATCGGTAACTTATGGTTCCAT
2040



P E G L I G L F A D F S P G H V W E S A

CCTGAGGGACTCATTGGCCTCTTCGCCCCGTTTTCGCCCGGGCATGTTTGGGAGTCGGCT



N P F C G E S T L Y T R T W S E V D A V

AATCCATTCTGTGGCGAGAGCACACTTTACACCCGTACTTGGTCGGAGGTTGATGCCGTC
2160



S S P A R P D L G F M S E P S I P S R A

TCTAGTCCAGCCCGGCCTGACTTAGGTTTTATGTCTGAGCCTTCTATACCTAGTAGGGCC



A T P T L A A P L P P P A P D P S P P P

GCCACGCCTACCCTGGCGGCCCCTCTACCCCCCCCTGCACCGGACCCTTCCCCCCCTCCC
2280



S A P A L A E P A S G A T A G A P A I T

TCTGCCCCGGCGCTTGCTGAGCCGGCTTCTGGCGCTACCGCCGGGGCCCCGGCCATAACT



H Q T A R H R R L L F T Y P D G S K V F

CACCAGACGGCCCGGCACCGCCGCCTGCTCTTCACCTACCCGGATGGCTCTAAGGTATTC
2400



A G S L F E S T C T W L V N A S N V D H

GCCGGCTCGCTGTTCGAGTCGACATGCACGTGGCTCGTTAACGCGTCTAATGTTGACCAC



R P G G G L C H A F Y Q R Y P A S F D A

CGCCCTGGCGGCGGGCTTTGCCATGCATTTTACCAAAGGTACCCCGCCTCCTTTGATGCT
2520



A S F V M R D G A A A Y T L T P R P I I

GCCTCTTTTGTGATGCGCGACGGCGCGGCCGCGTACACACTAACCCCCCGGCCAATAATT



H A V A P D Y R L E H N P K R L E A A Y

CACGCTGTCGCCCCTGATTATAGGTTGGAACATAACCCAAAGAGGCTTGAGGCTGCTTAT
2640



R E T C S R L G T A A Y P L L G T G I Y

CGGGAAACTTGCTCCCGCCTCGGCACCGCTGCATACCCGCTCCTCGGGACCGGCATATAC



Q V P I G P S F D A W E R N H R P G D E

CAGGTGCCGATCGGCCCCAGTTTTGACGCCTGGGAGCGGAACCACCGCCCCGGGGATGAG
2760



L Y L P E L A A R W F E A N R P T R P T

TTGTACCTTCCTGAGCTTGCTGCCAGATGGTTTGAGGCCAATAGGCCGACCCGCCCGACT



L T I T E D V A R T A N L A I E L D S A

CTCACTATAACTGAGGATGTTGCACGGACAGCGAATCTGGCCATCGAGCTTGACTCAGCC
2880



T D V G R A C A G C R V T P G V V Q Y Q

ACAGATGTCGGCCGGGCCTGTGCCGGCTGTCGGGTCACCCCCGGCGTTGTTCAGTACCAG



F T A G V P G S G K S R S I T Q A D V D

TTTACTGCAGGTGTGCCTGGATCCGGCAAGTCCCGCTCTATCACCCAAGCCGATGTGGAC
3000



V V V V P T R E L R N A W R R R G F A A

GTTGTCGTGGTCCCGACGCGTGAGTTGCGTAATGCCTGGCGCCGTCGCGGCTTTGCTGCT



F T P H T A A R V T Q G R R V V I D E A

TTTACCCCGCATACTGCCGCCAGAGTCACCCAGGGGCGCCGGGTTGTCATTGATGAGGCT
3120



P S L P P H L L L L H M Q R A A T V H L

CCATCCCTCCCCCCTCACCTGCTGCTGCTCCACATGCAGCGGGCCGCCACCGTCCACCTT



L G D P N Q I P A I D F E H A G L V P A

CTTGGCGACCCGAACCAGATCCCAGCCATCGACTTTGAGCACGCTGGGCTCGTCCCCGCC
3240



I R P D L G P T S W W H V T H R W P A D

ATCAGGCCCGACTTAGGCCCCACCTCCTGGTGGCATGTTACCCATCGCTGGCCTGCGGAT



V C E L I R G A Y P M I Q T T S R V L R

GTATGCGAGCTCATCCGTGGTGCATACCCCATGATCCAGACCACTAGCCGGGTTCTCCGT
3360



S L F W G E P A V G Q K L V F T Q A A K

TCGTTGTTCTGGGGTGAGCCTGCCGTCGGGCAGAAACTAGTGTTCACCCAGGCGGCCAAG



P A N P G S V T V H E A Q G A T Y T E T

CCCGCCAACCCCGGCTCAGTGACGGTCCACGAGGCGCAGGGCGCTACCTACACGGAGACC
3480



T I I A T A D A R G L I Q S S R A H A I

ACTATTATTGCCACAGCAGATGCCCGGGGCCTTATTCAGTCGTCTCGGGCTCATGCCATT



V A L T R H T E K C V I I D A P G L L R

GTTGCTCTGACGCGCCACACTGAGAAGTGCGTCATCATTGACGCACCAGGCCTGCTTCGC
3600



E V G I S D A I V N N F F L A G G E I G

GAGGTGGGCATCTCCGATGCAATCGTTAATAACTTTTTCCTCGCTGGTGGCGAAATTGGT



H Q R P S V I P R G N P D A N V D T L A

CACCAGCGCCCATCAGTTATTCCCCGTGGCAACCCTGACGCCAATGTTGACACCCTGGCT
3720



A F P P S C Q I S A F H Q L A E E L G H

GCCTTCCCGCCGTCTTGCCAGATTAGTGCCTTCCATCAGTTGGCTGAGGAGCTTGGCCAC



R P V P V A A V L P P C P E L E Q G L L

AGACCTGTCCCTGTTGCAGCTGTTCTACCACCCTGCCCCGAGCTCGAACAGGGCCTTCTC
3840



Y L P Q E L T T C D S V V T F E L T D I

TACCTGCCCCAGGAGCTCACCACCTGTGATAGTGTCGTAACATTTGAATTAACAGACATT



V H C R M A A P S Q R K A V L S T L V G

GTGCACTGCCGCATGGCCGCCCCGAGCCAGCGCAAGGCCGTGCTGTCCACACTCGTGGGC
3960



R Y G G R T K L Y N A S H S D V R D S L

CGCTACGGCGGTCGCACAAAGCTCTACAATGCTTCCCACTCTGATGTTCGCGACTCTCTC



A R F I P A I G P V Q V T T C E L Y E L

GCCCGTTTTATCCCGGCCATTGGCCCCGTACAGGTTACAACTTGTGAATTGTACGAGCTA
4080



V E A M V E K G Q D G S A V L E L D L C

GTGGAGGCCATGGTCGAGAAGGGCCAGGATGGCTCCGCCGTCCTTGAGCTTGATCTTTGC



N R D V S R I T F F Q K D C N K F T T G

AACCGTGACGTGTCCAGGATCACCTTCTTCCAGAAAGATTGTAACAAGTTCACCACAGGT
4200



E T I A H G K V G Q G I S A W S K T F C

GAGACCATTGCCCATGGTAAAGTGGGCCAGGGCATCTCGGCCTGGAGCAAGACCTTCTGC



A L F G P W F R A I E K A I L A L L P Q

GCCCTCTTTGGCCCTTGGTTCCGCGCTATTGAGAAGGCTATTCTGGCCCTGCTCCCTCAG
4320



G V F Y G D A F D D T V F S A A V A A A

GGTGTGTTTTACGGTGATGCCTTTGATGACACCGTCTTCTCGGCGGCTGTGGCCGCAGCA



K A S M V F E N D F S E F D S T Q N N F

AAGGCATCCATGGTGTTTGAGAATGACTTTTCTGAGTTTGACTCCACCCAGAATAACTTT
4440



S L G L E C A I M E E C G M P Q W L I R

TCTCTGGGTCTAGAGTGTGCTATTATGGAGGAGTGTGGGATGCCGCAGTGGCTCATCCGC



L Y H L I R S A W I L Q A P K E S L R G

CTGTATCACCTTATAAGGTCTGCGTGGATCTTGCAGGCCCCGAAGGAGTCTCTGCGAGGG
4560



F W K K H S G E P G T L L W N T V W N M

TTTTGGAAGAAACACTCCGGTGAGCCCGGCACTCTTCTATGGAATACTGTCTGGAATATG



A V I T H C Y D F R D F Q V A A F K G D

GCCGTTATTACCCACTGTTATGACTTCCGCGATTTTCAGGTGGCTGCCTTTAAAGGTGAT
4680



D S I V L C S E Y R Q S P G A A V L I A

GATTCGATAGTGCTTTGCAGTGAGTATCGTCAGAGTCCAGGAGCTGCTGTCCTGATCGCC



G C G L K L K V D F R P I G L Y A G V V

GGCTGTGGCTTGAAGTTGAAGGTAGATTTCCGCCCGATCGGTTTGTATGCAGGTGTTGTG
4800



V A P G L G A L P D V V R F A G R L T E

GTGGCCCCCGGCCTTGGCGCGCTCCCTGATGTTGTGCGCTTCGCCGGCCGGCTTACCGAG



K N W G P G P E R A E Q L R L A V S D F

AAGAATTGGGGCCCTGGCCCTGAGCGGGCGGAGCAGCTCCGCCTCGCTGTTAGTGATTTC
4920



L R K L T N V A Q M C V D V V S R V Y G

CTCCGCAAGCTCACGAATGTAGCTCAGATGTGTGTGGATGTTGTTTCCCGTGTTTATGGG



V S P G L V H N L I G M L Q A V A D G K

GTTTCCCCTGGACTCGTTCATAACCTGATTGGCATGCTACAGGCTGTTGCTGATGGCAAG
5040



A H F T E S V K P V L D L T N S I L C R

GCACATTTCACTGAGTCAGTAAAACCAGTGCTCGACTTGACAAATTCAATCTTGTGTCGG



|-ORF3--->

M N N M S F A A P M G S R P C A L G

M R P R P

V E Z |-ORF2-->

GTGGAATGAATAACATGTCTTTTGCTGCGCCCATGGGTTCGCGACCATGCGCCCTCGGCC
5160



L F C C C S S C F C L C C P R H R P V S

I L L L L L M F L P M L P A P P P G Q P



TATTTTGTTGCTGCTCCTCATGTTTTTGCCTATGCTGCCCGCGCCACCGCCCGGTCAGCC

R L A A V V G G A A A V P A V V S G V T

S G R R R G R R S G G S G G G F W G D R



GTCTGGCCGCCGTCGTGGGCGGCGCAGCGGCGGTTCCGGCGGTGGTTTCTGGGGTGACCG
5280



G L I L S P S Q S P I F I Q P T P S P P

V D S Q P F A I P Y I H P T N P F A P D



GGTTGATTCTCAGCCCTTCGCAATCCCCTATATTCATCCAACCAACCCCTTCGCCCCCGA



M S P L R P G L D L V F A N P P D H S A

V T A A A G A G P R V R Q P A R P L G S



TGTCACCGCTGCGGCCGGGGCTGGACCTCGTGTTCGCCAACCCGCCCGACCACTCGGCTC
5400



P L G V T R P S A P P L P H V V D L P Q

A W R D Q A Q R P A V A S R R R P T T A



CGCTTGGCGTGACCAGGCCCAGCGCCCCGCCGTTGCCTCACGTCGTAGACCTACCACAGC



L G P R R Z

G A A P L T A V A P A H D T P P V P D V



TGGGGCCGCGCCGCTAACCGCGGTCGCTCCGGCCCATGACACCCCGCCAGTGCCTGATGT
5520

D S R G A I L R R Q Y N L S T S P L T S



CGACTCCCGCGGCGCCATCTTGCGCCGGCAGTATAACCTATCAACATCTCCCCTTACCTC



S V A T G T N L V L Y A A P L S P L L P



TTCCGTGGCCACCGGCACTAACCTGGTTCTTTATGCCGCCCCTCTTAGTCCGCTTTTACC
5640



L Q D G T N T H I M A T E A S N Y A Q Y



CCTTCAGGACGGCACCAATACCCATATAATGGCCACGGAAGCTTCTAATTATGCCCAGTA



R V A R A T I R Y R P L V P N A V G G Y



CCGGGTTGCCCGTGCCACAATCCGTTACCGCCCGCTGGTCCCCAATGCTGTCGGCGGTTA
5760



A I S I S F W P Q T T T T P T S V D M N



CGCCATCTCCATCTCATTCTGGCCACAGACCACCACCACCCCGACGTCCGTTGATATGAA



S I T S T D V R I L V Q P G I A S E L V



TTCAATAACCTCGACGGATGTTCGTATTTTAGTCCAGCCCGGCATAGCCTCTGAGCTTGT
5880



I P S E R L H Y R N Q G W R S V E T S G



GATCCCAAGTGAGCGCCTACACTATCGTAACCAAGGCTGGCGCTCCGTCGAGACCTCTGG



V A E E E A T S G L V M L C I H G S L V



GGTGGCTGAGGAGGAGGCTACCTCTGGTCTTGTTATGCTTTGCATACATGGCTCACTCGT
6000



N S Y T N T P Y T G A L G L L D F A L E



AAATTCCTATACTAATACACCCTATACCGGTGCCCTCGGGCTGTTGGACTTTGCCCTTGA



L E F R N L T P G N T N T R V S R Y S S



GCTTGAGTTTCGCAACCTTACCCCCGGTAACACCAATACGCGGGTCTCCCGTTATTCCAG
6120



T A R H R L R R G A D G T A E L T T T A



CACTGCTCGCCACCGCCTTCGTCGCGGTGCGGACGGGACTGCCGAGCTCACCACCACGGC



A T R F M K D L Y F T S T N G V G E I G



TGCTACCCGCTTTATGAAGGACCTCTATTTTACTAGTACTAATGGTGTCGGTGAGATCGG
6240



R G I A L T L F N L A D T L L G G L P T



CCGCGGGATAGCCCTCACCCTGTTCAACCTTGCTGACACTCTGCTTGGCGGCCTGCCGAC



E L I S S A G G Q L F Y S R P V V S A N



AGAATTGATTTCGTCGGCTGGTGGCCAGCTGTTCTACTCCCGTCCCGTTGTCTCAGCCAA
6360



G E P T V K L Y T S V E N A Q Q D K G I



TGGCGAGCCGACTGTTAAGTTGTATACATCTGTAGAGAATGCTCAGCAGGATAAGGGTAT



A I P H D I D L G E S R V V I Q D Y D N



TGCAATCCCGCATGACATTGACCTCGGAGAATCTCGTGTGGTTATTCAGGATTATGATAA
6480



Q H E Q D R P T P S P A P S R P F S V L



CCAACATGAACAAGATCGGCCGACGCCTTCTCCAGCCCCATCGCGCCCTTTCTCTGTCCT



R A N D V L W L S L T A A E Y D Q S T Y



TCGAGCTAATGATGTGCTTTGGCTCTCTCTCACCGCTGCCGAGTATGACCAGTCCACTTA
6600



G S S T G P V Y V S D S V T L V N V A T



TGGCTCTTCGACTGGCCCAGTTTATGTTTCTGACTCTGTGACCTTGGTTAATGTTGCGAC



G A Q A V A R S L D W T K V T L D G R P



CGGCGCGCAGGCCGTTGCCCGGTCGCTCGATTGGACCAAGGTCACACTTGACGGTCGCCC
6720



L S T I Q Q Y S K T F F V L P L R G K L



CCTCTCCACCATCCAGCAGTACTCGAAGACCTTCTTTGTCCTGCCGCTCCGCGGTAAGCT



S F W E A G T T K A G Y P Y N Y N T T A



CTCTTTCTGGGAGGCAGGCACAACTAAAGCCGGGTACCCTTATAATTATAACACCACTGC
6840



S D Q L L V E N A A G H R V A I S T Y T



TAGCGACCAACTGCTTGTCGAGAATGCCGCCGGGCACCGGGTCGCTATTTCCACTTACAC



T S L G A G P V S I S A V A V L A P H S



CACTAGCCTGGGTGCTGGTCCCGTCTCCATTTCTGCGGTTGCCGTTTTAGCCCCCCACTC
6960



A L A L L E D T L D Y P A R A H T F D D



TGCGCTAGCATTGCTTGAGGATACCTTGGACTACCCTGCCCGCGCCCATACTTTTGATGA



F C P E C R P L G L Q G C A F Q S T V A



TTTCTGCCCAGAGTGCCGCCCCCTTGGCCTTCAGGGCTGCGCTTTCCAGTCTACTGTCGC
7080



E L Q R L K M K V G K T R E L Z



TGAGCTTCAGCGCCTTAAGATGAAGGTGGGTAAAACTCGGGAGTTGTAGTTTATTTGCTT



GTGCCCCCCTTCTTTCTGTTGCTTATTTCTCATTTCTGCGTTCCGCGCTCCCTGA
7195


































































































































































































































































































































































































































Total number of bases in this sequence as presented is 7195. The poly-A tail present in the cloned sequence has been omitted.
The ability of the methods described herein to isolate and identify genetic material from other NANB hepatitis strains has been confirmed by identifying genetic material from an isolate obtained in Mexico. The sequence of this isolate was about 75% identical to the ET1.1 sequence set forth in SEQ ID NO.1 above. The sequence was identified by hybridization using the conditions set forth in Section II.B below.
In this different approach to isolation of the virus, cDNA libraries were made directly from a semi-purified human stool specimen collected from an outbreak of ET-NANB in Telixtac. The recovery of cDNA and the construction of representative libraries was assured by the application of sequence independent single premier amplification (SISPA). A cDNA library constructed in lambda gt11 from such an amplified cDNA population was screened with a serum considered to have “high” titer anti-HEV antibodies as assayed by direct immunofluorescence on liver sections from infected cynos. Two cDNA clones, denoted 406.3-2 and 406.4-2, were identified by this approach from a total of 60,000 screened. The sequence of these clones was subsequently localized to the 3′ half of the viral genome by homology comparison to the HEV (Burma) sequence obtained from clones isolated by hybridization screening of libraries with the original ET1.1 clone.
These isolated cDNA epitopes when used as hybridization probes on Northern blots of RNA extracted from infected cyno liver gave a somewhat different result when compared to the Northern blots obtained with the ET1.1 probe. In addition to the single 7.5 Kb transcript seen using ET1.1, two additional transcripts of 3.7 and 2.0 Kb were identified using either of these epitopes as hybridization probes. These polyadenylated transcripts were identified using the extreme 3′ end epitope clone (406.3-2) as probe and therefore established these transcripts as co-terminal with the 3′ end of the genome (see below). One of the epitope clones (406.4-2) was subsequently shown to react in a specific fashion with antisera collected from 5 different geographic epidemics (Somalia, Burma, Mexico, Tashkent and Pakistan). The 406.3-2 clone reacted with sera from 4 out of these same 5 epidemics (Yarbough et al., 1990). Both clones reacted with only post inoculation antisera from infected cynos. The latter experiment confirmed that seroconversion in experimentally infected cynos was related to the isolated exogenous cloned sequence.
A composite cDNA sequence (obtained from several clones of the Mexican strain) is set forth below. Composite Mexico strain sequence (SEQ ID NO.10):








GCCATGGAGG CCCACCAGTT CATTAAGGCT CCTGGCATCA CTACTGCTAT TGAGCAAGCA
60
SEQ ID NO. 10:




GCTCTAGCAG CGGCCAACTC CGCCCTTGCG AATGCTGTGG TGGTCCGGCC TTTCCTTTCC
120



CATCAGCAGG TTGAGATCCT TATAAATCTC ATGCAACCTC GGCAGCTGGT GTTTCGTCCT
180



GAGGTTTTTT GGAATCACCC GATTCAACGT GTTATACATA ATGAGCTTGA GCAGTATTGC
240



CGTGCTCGCT CGGGTCGCTG CCTTGAGATT GGAGCCCACC CACGCTCCAT TAATGATAAT
300



CCTAATGTCC TCCATCGCTG CTTTCTCCAC CCCGTCGGCC GGGATGTTCA GCGCTGGTAC
360



ACAGCCCCGA CTAGGGGACC TGCGGCGAAC TGTCGCCGCT CGGCACTTCG TGGTCTGCCA
420



CCAGCCGACC GCACTTACTG TTTTGATGGC TTTGCCGGCT GCCGTTTTGC CGCCGAGACT
480



GGTGTGGCTC TCTATTCTCT CCATGACTTG CAGCCGGCTG ATGTTGCCGA GGCGATGGCT
540



CGCCACGGCA TGACCCGCCT TTATGCAGCT TTCCACTTGC CTCCAGAGGT GCTCCTGCCT
600



CCTGGCACCT ACCGGACATC ATCCTACTTG CTGATCCACG ATGGTAAGCG CGCGGTTGTC
660



ACTTATGAGG GTGACACTAG CGCCGGTTAC AATCATGATG TTGCCACCCT CCGCACATGG
720



ATCAGGTCAA CTAAGGTTGT GGGTGAACAC CCTTTGGTGA TCGAGCGGGT GCGGGGTATT
780



GGCTGTCACT TTGTGTTGTT GATCACTGCG GCCCCTGAGC CCTCCCCGAT GCCCTACGTT
840



CCTTACCCGC GTTCGACGGA GGTCTATGTC CGGTCTATCT TTGGGCCCGG CGGGTCCCCG
900



TCGCTGTTCC CGACCGCTTG TGCTGTCAAG TCCACTTTTC ACGCCGTCCC CACGCACATC
960



TGGGACCGTC TCATGCTCTT TGGGGCCACC CTCGACGACC AGGCCTTTTG CTGCTCCAGG
1020



CTTATGACGT ACCTTCGTGG CATTAGCTAT AAGGTAACTG TGGGTGCCCT GGTCGCTAAT
1080



GAAGGCTGGA ATGCCACCGA GGATGCGCTC ACTGCAGTTA TTACGGCGGC TTACCTCACA
1140



ATATGTCATC AGCGTTATTT GCGGACCCAG GCGATTTCTA AGGGCATGCG CCGGCTTGAG
1200



CTTGAACATG CTCAGAAATT TATTTCACGC CTCTACAGCT GGCTATTTGA GAAGTCAGGT
1260



CGTGATTACA TCCCAGGCCG CCAGCTGCAG TTCTACGCTC AGTGCCGCCG CTGGTTATCT
1320



GCCGGGTTCC ATCTCGACCC CCGCACCTTA GTTTTTGATG AGTCAGTGCC TTGTAGCTGC
1380



CGAACCACCA TCCGGCGGAT CGCTGGAAAA TTTTGCTGTT TTATGAAGTG GCTCGGTCAG
1440



GAGTGTTCTT GTTTCCTCCA GCCCGCCGAG GGGCTGGCGG GCGACCAAGG TCATGACAAT
1500



GAGGCCTATG AAGGCTCTGA TGTTGATACT GCTGAGCCTG CCACCCTAGA CATTACAGGC
1560



TCATACATCG TGGATGGTCG GTCTCTGCAA ACTGTCTATC AAGCTCTCGA CCTGCCAGCT
1620



GACCTGGTAG CTCGCGCAGC CCGACTGTCT GCTACAGTTA CTGTTACTGA AACCTCTGGC
1680



CGTCTGGATT GCCAAACAAT GATCGGCAAT AAGACTTTTC TCACTACCTT TGTTGATGGG
1740



GCACGCCTTG AGGTTAACGG GCCTGAGCAG CTTAACCTCT CTTTTGACAG CCAGCAGTGT
1800



AGTATGGCAG CCGGCCCGTT TTGCCTCACC TATGCTGCCG TAGATGGCGG GCTGGAAGTT
1860



CATTTTTCCA CCGCTGGCCT CGAGAGCCGT GTTGTTTTCC CCCCTGGTAA TGCCCCGACT
1920



GCCCCGCCGA GTGAGGTCAC CGCCTTCTGC TCAGCTCTTT ATAGGCACAA CCGGCAGAGC
1980



CAGCGCCAGT CGGTTATTGG TAGTTTGTGG CTGCACCCTG AAGGTTTGCT CGGCCTGTTC
2040



CCGCCCTTTT CACCCGGGCA TGAGTGGCGG TCTGCTAACC CATTTTGCGG CGAGAGCACG
2100



CTCTACACCC GCACTTGGTC CACAATTACA GACACACCCT TAACTGTCGG GCTAATTTCC
2160



GGTCATTTGG ATGCTGCTCC CCACTCGGGG GGGCCACCTG CTACTGCCAC AGGCCCTGCT
2220



GTAGGCTCGT CTGACTCTCC AGACCCTGAC CCGCTACCTG ATGTTACAGA TGGCTCACGC
2280



CCCTCTGGGG CCCGTCCGGC TGGCCCCAAC CCGAATGGCG TTCCGCAGCG CCGCTTACTA
2340



CACACCTACC CTGACGGCGC TAAGATCTAT GTCGGCTCCA TTTTCGAGTC TGAGTGCACC
2400



TGGCTTGTCA ACGCATCTAA CGCCGGCCAC CGCCCTGGTG GCGGGCTTTG TCATGCTTTT
2460



TTTCAGCGTT ACCCTGATTC GTTTGACGCC ACCAAGTTTG TGATGCGTGA TGGTCTTGCC
2520



GCGTATACCC TTACACCCCG GCCGATCATT CATGCGGTGG CCCCGGACTA TCGATTGGAA
2580



CATAACCCCA AGAGGCTCGA GGCTGCCTAC CGCGAGACTT GCGCCCGCCG AGGCACTGCT
2640



GCCTATCCAC TCTTAGGCGC TGGCATTTAC CAGGTGCCTG TTAGTTTGAG TTTTGATGCC
2700



TGGGAGCGGA ACCACCGCCC GTTTGACGAG CTTTACCTAA CAGAGCTGGC GGCTCGGTGG
2760



TTTGAATCCA ACCGCCCCGG TCAGCCCACG TTGAACATAA CTGAGGATAC CGCCCGTGCG
2820



GCCAACCTGG CCCTGGAGCT TGACTCCGGG AGTGAAGTAG GCCGCGCATG TGCCGGGTGT
2880



AAAGTCGAGC CTGGCGTTGT GCGGTATCAG TTTACAGCCG GTGTCCCCGG CTCTGGCAAG
2940



TCAAAGTCCG TGCAACAGGC GGATGTGGAT GTTGTTGTTG TGCCCACTCG CGAGCTTCGG
3000



AACGCTTGGC GGCGCCGGGG CTTTGCGGCA TTCACTCCGC ACACTGCGGC CCGTGTCACT
3060



AGCGGCCGTA GGGTTGTCAT TGATGAGGCC CCTTCGCTCC CCCCACACTT GCTGCTTTTA
3120



CATATGCAGC GTGCTGCATC TGTGCACCTC CTTGGGGACC CGAATCAGAT CCCCGCCATA
3180



GATTTTGAGC ACACCGGTCT GATTCCAGCA ATACGGCCGG AGTTGGTCCC GACTTCATGG
3240



TGGCATGTCA CCCACCGTTG CCCTGCAGAT GTCTGTGAGT TAGTCCGTGG TGCTTACCCT
3300



AAAATCCAGA CTACAAGTAA GGTGCTCCGT TCCCTTTTCT GGGGAGAGCC AGCTGTCGGC
3360



CAGAAGCTAG TGTTCACACA GGCTGCTAAG GCCGCGCACC CCGGATCTAT AACGGTCCAT
3420



GAGGCCCAGG GTGCCACTTT TACCACTACA ACTATAATTG CAACTGCAGA TGCCCGTGGC
3480



CTCATACAGT CCTCCCGGGC TCACGCTATA GTTGCTCTCA CTAGGCATAC TGAAAAATGT
3540



GTTATACTTG ACTCTCCCGG CCTGTTGCGT GAGGTGGGTA TCTCAGATGC CATTGTTAAT
3600



AATTTCTTCC TTTCGGGTGG CGAGGTTGGT CACCAGAGAC CATCGGTCAT TCCGCGAGGC
3660



AACCCTGACC GCAATGTTGA CGTGCTTGCG GCGTTTCCAC CTTCATGCCA AATAAGCGCC
3720



TTCCATCAGC TTGCTGAGGA GCTGGGCCAC CGGCCGGCGC CGGTGGCGGC TGTGCTACCT
3780



CCCTGCCCTG AGCTTGAGCA GGGCCTTCTC TATCTGCCAC AGGAGCTAGC CTCCTGTGAC
3840



AGTGTTGTGA CATTTGAGCT AACTGACATT GTGCACTGCC GCATGGCGGC CCCTAGCCAA
3900



AGGAAAGCTG TTTTGTCCAC GCTGGTAGGC CGGTATGGCA GACGCACAAG GCTTTATGAT
3960



GCGGGTCACA CCGATGTCCG CGCCTCCCTT GCGCGCTTTA TTCCCACTCT CGGGCGGGTT
4020



ACTGCCACCA CCTGTGAACT CTTTGAGCTT GTAGAGGCGA TGGTGGAGAA GGGCCAAGAC
4080



GGTTCAGCCG TCCTCGAGTT GGATTTGTGC AGCCGAGATG TCTCCCGCAT AACCTTTTTC
4140



CAGAAGGATT GTAACAAGTT CACGACCGGC GAGACAATTG CGCATGGCAA AGTCGGTCAG
4200



GGTATCTTCC GCTGGAGTAA GACGTTTTGT GCCCTGTTTG GCCCCTGGTT CCGTGCGATT
4260



GAGAAGGCTA TTCTATCCCT TTTACCACAA GCTGTGTTCT ACGGGGATGC TTATGACGAC
4320



TCAGTATTCT CTGCTGCCGT GGCTGGCGCC AGCCATGCCA TGGTGTTTGA AAATGATTTT
4380



TCTGAGTTTG ACTCGACTCA GAATAACTTT TCCCTAGGTC TTGAGTGCGC CATTATGGAA
4440



GAGTGTGGTA TGCCCCAGTG GCTTGTCAGG TTGTACCATG CCGTCCGGTC GGCGTGGATC
4500



CTGCAGGCCC CAAAAGAGTC TTTGAGAGGG TTCTGGAAGA AGCATTCTGG TGAGCCGGGC
4560



AGCTTGCTCT GGAATACGGT GTGGAACATG GCAATCATTG CCCATTGCTA TGAGTTCCGG
4620



GACCTCCAGG TTGCCGCCTT CAAGGGCGAC GACTCGGTCG TCCTCTGTAG TGAATACCGC
4680



CAGAGCCCAG GCGCCGGTTC GCTTATAGCA GGCTGTGGTT TGAAGTTGAA GGCTGACTTC
4740



CGGCCGATTG GGCTGTATGC CGGGGTTGTC GTCGCCCCGG GGCTCGGGGC CCTACCCGAT
4800



GTCGTTCGAT TCGCCGGACG GCTTTCGGAG AAGAACTGGG GGCCTGATCC GGAGCGGGCA
4860



GAGCAGCTCC GCCTCGCCGT GCAGGATTTC CTCCGTAGGT TAACGAATGT GGCCCAGATT
4920



TGTGTTGAGG TGGTGTCTAG AGTTTACGGG GTTTCCCCGG GTCTGGTTCA TAACCTGATA
4980



GGCATGCTCC AGACTATTGG TGATGGTAAG GCGCATTTTA CAGAGTCTGT TAAGCCTATA
5040



CTTGACCTTA CACACTCAAT TATGCACCGG TCTGAATGAA TAACATGTGG TTTGCTGCGC
5100



CCATGGGTTC GCCACCATGC GCCCTAGGCC TCTTTTGCTG TTGTTCCTCT TGTTTCTGCC
5160



TATGTTGCCC GCGCCACCGA CCGGTCAGCC GTCTGGCCGC CGTCGTGGGC GGCGCAGCGG
5220



CGGTACCGGC GGTGGTTTCT GGGGTGACCG GGTTGATTCT CAGCCCTTCG CAATCCCCTA
5280



TATTCATCCA ACCAACCCCT TTGCCCCAGA CGTTGCCGCT GCGTCCGGGT CTGGACCTCG
5340



CCTTCGCCAA CCAGCCCGGC CACTTGGCTC CACTTGGCGA GATCAGGCCC AGCGCCCCTC
5400



CGCTGCCTCC CGTCGCCGAC CTGCCACAGC CGGGGCTGCG GCGCTGACGG CTGTGGCGCC
5460



TGCCCATGAC ACCTCACCCG TCCCGGACGT TGATTCTCGC GGTGCAATTC TACGCCGCCA
5520



GTATAATTTG TCTACTTCAC CCCTGACATC CTCTGTGGCC TCTGGCACTA ATTTAGTCCT
5580



GTATGCAGCC CCCCTTAATC CGCCTCTGCC GCTGCAGGAC GGTACTAATA CTCACATTAT
5640



GGCCACAGAG GCCTCCAATT ATGCACAGTA CCGGGTTGCC CGCGCTACTA TCCGTTACCG
5700



GCCCCTAGTG CCTAATGCAG TTGGAGGCTA TGCTATATCC ATTTCTTTCT GGCCTCAAAC
5760



AACCACAACC CCTACATCTG TTGACATGAA TTCCATTACT TCCACTGATG TCAGGATTCT
5820



TGTTCAACCT GGCATAGCAT CTGAATTGGT CATCCCAAGC GAGCGCCTTC ACTACCGCAA
5880



TCAAGGTTGG CGCTCGGTTG AGACATCTGG TGTTGCTGAG GAGGAAGCCA CCTCCGGTCT
5940



TGTCATGTTA TGCATACATG GCTCTCCAGT TAACTCCTAT ACCAATACCC CTTATACCGG
6000



TGCCCTTGGC TTACTGGACT TTGCCTTAGA GCTTGAGTTT CGCAATCTCA CCACCTGTAA
6060



CACCAATACA CGTGTGTCCC GTTACTCCAG CACTGCTCGT CACTCCGCCC GAGGGGCCGA
6120



CGGGACTGCG GAGCTGACCA CAACTGCAGC CACCAGGTTC ATGAAAGATC TCCACTTTAC
6180



CGGCCTTAAT GGGGTAGGTG AAGTCGGCCG CGGGATAGCT CTAACATTAC TTAACCTTGC
6240



TGACACGCTC CTCGGCGGGC TCCCGACAGA ATTAATTTCG TCGGCTGGCG GGCAACTGTT
6300



TTATTCCCGC CCGGTTGTCT CAGCCAATGG CGAGCCAACC GTGAAGCTCT ATACATCAGT
6360



GGAGAATGCT CAGCAGGATA AGGGTGTTGC TATCCCCCAC GATATCGATC TTGGTGATTC
6420



GCGTGTGGTC ATTCAGGATT ATGACAACCA GCATGAGCAG GATCGGCCCA CCCCGTCGCC
6480



TGCGCCATCT CGGCCTTTTT CTGTTCTCCG AGCAAATGAT GTACTTTGGC TGTCCCTCAC
6540



TGCAGCCGAG TATGACCAGT CCACTTACGG GTCGTCAACT GGCCCGGTTT ATATCTCGGA
6600



CAGCGTGACT TTGGTGAATG TTGCGACTGG CGCGCAGGCC GTAGCCCGAT CGCTTGACTG
6660



GTCCAAAGTC ACCCTCGACG GGCGGCCCCT CCCGACTGTT GAGCAATATT CCAAGACATT
6720



CTTTGTGCTC CCCCTTCGTG GCAAGCTCTC CTTTTGGGAG GCCGGCACAA CAAAAGCAGG
6780



TTATCCTTAT AATTATAATA CTACTGCTAG TGACCAGATT CTGATTGAAA ATGCTGCCGG
6840



CCATCGGGTC GCCATTTCAA CCTATACCAC CAGGCTTGGG GCCGGTCCGG TCGCCATTTC
6900



TGCGGCCGCG GTTTTGGCTC CACGCTCCGC CCTGGCTCTG CTGGAGGATA CTTTTGATTA
6960



TCCGGGGCGG GCGCACACAT TTGATGACTT CTGCCCTGAA TGCCGCGCTT TAGGCCTCCA
7020



GGGTTGTGCT TTCCAGTCAA CTGTCGCTGA GCTCCAGCGC CTTAAAGTTA AGGTGGGTAA
7080



AACTCGGGAG TTGTAGTTTA TTTGGCTGTG CCCACCTACT TATATCTGCT GATTTCCTTT
7140



ATTTCCTTTT TCTCGGTCCC GCGCTCCCTG A
7171






















































































































































































































































The above sequence was obtained from polyadenylated clones. For clarity the 3′ polyA “tail” has been omitted.
The sequence above includes a partial cDNA sequence consisting of 1661 nucleotides that was identified in a previous application in this series. The previously identified partial sequence is set forth below, with certain corrections (SEQ ID NO.11). The corrections include deletion of the first 80 bases of the prior reported sequence, which are cloning artifacts; insertion of G after former position 174, of C after 270, and of GGCG after 279; change of C to T at former position 709, of GC to CG at 722-723, of CC to TT at 1238-39, and of C to G at 1606; deletion of T at former position 765; and deletion of the last 11 bases of the former sequence, which are part of a linker sequence and are not of viral origin.
Non-A Non-B T: M′ Mexican Strain; SEQ ID NO.11








GTTGCGTGAG GTGGGTATCT CAGATGCCAT TGTTAATAAT TTCTTCCTTT CGGGTGGCGA
60
SEQ ID NO. 11:




GGTTGGTCAC CAGAGACCAT CGGTCATTCC GCGAGGCAAC CCTGACCGCA ATGTTGACGT
120



GCTTGCGGCG TTTCCACCTT CATGCCAAAT AAGCGCCTTC CATCAGCTTG CTGAGGAGCT
180



GGGCCACCGG CCGGCGCCGG TGGCGGCTGT GCTACCTCCC TGCCCTGAGC TTGAGCAGGG
240



CCTTCTCTAT CTGCCACAGG AGCTAGCCTC CTGTGACAGT GTTGTGACAT TTGAGCTAAC
300



TGACATTGTG CACTGCCGCA TGGCGGCCCC TAGCCAAAGG AAAGCTGTTT TGTCCACGCT
360



GGTAGGCCGG TATGGCAGAC GCACAAGGCT TTATGATGCG GGTCACACCG ATGTCCGCGC
420



CTCCCTTGCG CGCTTTATTC CCACTCTCGG GCGGGTTACT GCCACCACCT GTGAACTCTT
480



TGAGCTTGTA GAGGCGATGG TGGAGAAGGG CCAAGACGGT TCAGCCGTCC TCGAGTTGGA
540



TTTGTGCAGC CGAGATGTCT CCCGCATAAC CTTTTTCCAG AAGGATTGTA ACAAGTTCAC
600



GACCGGCGAG ACAATTGCGC ATGGCAAAGT CGGTCAGGGT ATCTTCCGCT GGAGTAAGAC
660



CTTTTGTGCC CTGTTTGGCC CCTGGTTCCG TGCGATTGAG AAGGCTATTC TATCCCTTTT
720



ACCACAAGCT GTGTTCTACG GGGATGCTTA TGACGACTCA GTATTCTCTG CTGCCGTGGC
780



TGGCGCCAGC CATGCCATGG TGTTTGAAAA TGATTTTTCT GAGTTTGACT CGACTCAGAA
840



TAACTTTTCC CTAGGTCTTG AGTGCGCCAT TATGGAAGAG TGTGGTATGC CCCAGTGGCT
900



TGTCAGGTTG TACCATGCCG TCCGGTCGGC GTGGATCCTG CAGGCCCCAA AAGAGTCTTT
960



GAGAGGGTTC TGGAAGAAGC ATTCTGGTGA GCCGGGCACG TTGCTCTGGA ATACGGTGTG
1020



GAACATGGCA ATCATTGCCC ATTGCTATGA GTTCCGGGAC CTCCAGGTTG CCGCCTTCAA
1080



GGGCGACGAC TCGGTCGTCC TCTGTAGTGA ATACCGCCAG AGCCCAGGCG CCGGTTCGCT
1140



TATAGCAGGC TGTGGTTTGA AGTTGAAGGC TGACTTCCGG CCGATTGGGC TGTATGCCGG
1200



GGTTGTCGTC GCCCCGGGGC TCGGGGCCCT ACCCGATGTC GTTCGATTCG CCGGACGGCT
1260



TTCGGAGAAG AACTGGGGGC CTGATCCGGA GCGGGCAGAG CAGCTCCGCC TCGCCGTGCA
1320



GGATTTCCTC CGTAGGTTAA CGAATGTGGC CCAGATTTGT GTTGAGGTGG TGTCTAGAGT
1380



TTACGGGGTT TCCCCGGGTC TGGTTCATAA CCTGATAGGC ATGCTCCAGA CTATTGGTGA
1440



TGGTAAGGCG CATTTTACAG AGTCTGTTAA GCCTATACTT GACCTTACAC ACTCAATTAT
1500



GCACCGGTCT GAATGAATAA CATGTGGTTT GCTGCGCCCA TGGGTTCGCC ACCATGCGCC
1560



CTAGGCCTCT TTTGC
1575




























































When comparing the Burmese and Mexican strains, 75.7% identity is seen in a 7189 nucleotide overlap beginning at nucleotide 1 of the Mexican strain and nucleotide 25 of the Burmese strain.
In the same manner, a different strain of HEV was identified in an isolate obtained in Tashkent, U.S.S.R. The Tashkent sequence is given below (SEQ ID NO.12):








CGGGCCCCGT ACAGGTCACA ACCTGTGAGT TGTACGAGCT AGTGGAGGCC ATGGTCGAGA
60
SEQ ID. NO. 12:




AAGGCCAGGA TGGCTCCGCC GTCCTTGAGC TCGATCTCTG CAACCGTGAC GTGTCCAGGA
120



TCACCTTTTT CCAGAAAGAT TGCAATAAGT TCACCACGGG AGAGACCATC GCCCATGGTA
180



AAGTGGGCCA GGGCATTTCG GCCTGGAGTA AGACCTTCTG TGCCCTTTTC GGCCCCTGGT
240



TCCGTGCTAT TGAGAAGGCT ATTCTGGCCC TGCTCCCTCA GGGTGTGTTT TATGGGGATG
300



CCTTTGATGA CACCGTCTTC TCGGCGCGTG TGGCCGCAGC AAAGGCGTCC ATGGTGTTTG
360



AGAATGACTT TTCTGAGTTT GACTCCACCC AGAATAATTT TTCCCTGGGC CTAGAGTGTG
420



CTATTATGGA GAAGTGTGGG ATGCCGAAGT GGCTCATCCG CTTGTACCAC CTTATAAGGT
480



CTGCGTGGAT CCTGCAGGCC CCGAAGGAGT CCCTGCGAGG GTGTTGGAAG AAACACTCCG
540



GTGAGCCCGG CACTCTTCTA TGGAATACTG TCTGGAACAT GGCCGTTATC ACCCATTGTT
600



ACGATTTCCG CGATTTGCAG GTGGCTGCCT TTAAAGGTGA TGATTCGATA GTGCTTTGCA
660



GTGAGTACCG TCAGAGTCCA GGGGCTGCTG TCCTGATTGC TGGCTGTGGC TTAAAGCTGA
720



AGGTGGGTTT CCGTCCGATT GGTTTGTATG CAGGTGTTGT GGTGACCCCC GGCCTTGGCG
780



CGCTTCCCGA CGTCGTGCGC TTGTCCGGCC GGCTTACTGA GAAGAATTGG GGCCCTGGCC
840



CTGAGCGGGC GGAGCAGCTC CGCCTTGCTG TGCG
874




































A number of open reading frames, which are potential coding regions, have been found within the DNA sequences set forth above. As has already been noted, consensus residues for the RNA-directed RNA polymerase (RDRP) were identified in the HEV (Burma) strain clone ET1.1. Once a contiguous overlapping set of clones was accumulated, it became clear that the nonstructural elements containing the RDRP as well as what were identified as consensus residues for the helicase domain were located in the first large open reading frame (ORFI). ORFI covers the 5′ half of the genome and begins at the first encoded met, after the 27th bp of the apparent non-coding sequence, and then extends 5079 bp before reaching a termination codon. Beginning 37 bp downstream from the ORF1 stop codon in the plus 1 frame is the second major opening reading frame (ORF2) extending 1980 bp and terminating 68 bp upstream from the point of poly A addition. The third forward ORF (in the plus 2 frame) is also utilized by HEV. ORF3 is only 370 bp in length and would not have been predicted to be utilized by the virus were it not for the identification of the immunoreactive cDNA clone 406.4-2 from the Mexico SISPA cDNA library (see below for detailed discussion). This epitope confirmed the utilization of ORF3 by the virus, although the means by which this ORF is expressed has not yet been fully elucidated. If we assume that the first met is utilized, ORF3 overlaps ORF1 by 1 bp at its 5′ end and ORF2 by 328 bp at its 3′ end. ORF2 contains the broadly reactive 406.3-2 epitope and also a signal sequence at its extreme 5′ end. The first half of this ORF2 also has a high pI value (>10) similar to that seen with other virus capsid proteins. These data suggest that the ORF2 might be the predominant structural gene of HEV.
The existence of subgenomic transcripts prompted a set of experiments to determine whether these RNAs were produced by splicing from the 5′ end of the genome. An analysis using subgenomic probes from throughout the genome, including the extreme 5′ end, did not provide evidence for a spliced transcript. However, it was discovered that a region of the genome displayed a high degree of homology with a 21 bp segment identified in Sindbis as a probably internal initiation site for RNA transcription used in the production of its subgenomic messages. Sixteen of 21 (76%) of the nucleotides are identical.
Two cDNA clones which encode an epitope of HEV that is recognized by sera collected from different ET-NANB outbreaks (i.e., a universally recognized epitope) have been isolated and characterized. One of the clones immunoreacted with 8 human sera from different infected individuals and the other clone immunoreacted with 7 of the human sera tested. Both clones immunoreacted specifically with cyno sera from infected animals and exhibited no immunologic response to sera from uninfected animals. The sequences of the cDNAs in these recombinant phages, designated 406.3-2 and 406.4-2 have been determined. The HEV open reading frames are shown to encode epitopes specifically recognized by sera from patients with HEV infections. The cDNA sequences and the polypeptides that they encode are set forth below.
Epitopes Derived From Mexican Strain of HEV
406.4-2 sequence (nucleotide sequence has SEQ ID NO.13; amino acid sequence has SEQ ID NO.14):








C GCC AAC CAG CCC GGC CAC TTG GCT CCA CTT GGC GAG ATC AGG CCC
46
SEQ ID NO. 13:


Ala Asn Gln Pro Gly His Leu Ala Pro Leu Gly Glu Ile Arg Pro

1 5 10 15



AGC GCC CCT CCG CTG CCT CCC GTC GCC GAC CTG CCA CAG CCG GGG CTG
94

Ser Ala Pro Pro Leu Pro Pro Val Ala Asp Leu Pro Gln Pro Gly Leu

20 25 30



CGG CGC TGA CGGCTGTGGC GCCTGCCCAT GACACCTCAC CCGTCCCGGA
143

Arg Arg .



CGTTGATTCT CGCGGTGCAA TTCTACGCCG CCAGTATAAT TTGTCTACTT CACCCCTGAC
203



ATCCTCTGTG GCCTCTGGCA CTAATTTAGT CCTGTATGCA GCCCCCCTTA ATCCGCCTCT
253



GCCGCTGCAG GACGGTACTA ATACTCACAT TATGGCCACA GAGGCCTCCA ATTATGCACA
323



GTACCGGGTT GCCCGCGCTA CTATCCGTTA CCGGCCCCTA GTGCCTAATG CAGTTGGAGG
383



CTATGCTATA TCCATTTCTT TCTGGCCTCA AACAACCACA ACCCCTACAT CTGTTGACAT
443



GAATTC
449



Ala Asn Gln Pro Gly His Leu Ala Pro Leu Gly Glu Ile Arg Pro Ser

SEQ ID NO. 14:

1 5 10 15



Ala Pro Pro Leu Pro Pro Val Ala Asp Leu Pro Gln Pro Gly Leu Arg

20 25 30

Arg .




































0451 !
406.3-2 sequence (nucleotide sequence has SEQ ID NO.15; amino acid sequence has SEQ ID NO.16):








GGAT ACT TTT GAT TAT CCG GGG CGG GCG CAC ACA TTT GAT GAC TTC TGC
49
SEQ ID NO. 15:


Thr Phe Asp Tyr Pro Gly Arg Ala His Thr Phe Asp Asp Phe Cys

1 5 10 15



CCT GAA TGC CGC GCT TTA GGC CTC CAG GGT TGT GCT TTC CAG TCA ACT
97

Pro Glu Cys Arg Ala Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr

20 25 30



GTC GCT GAG CTC CAG CGC CTT AAA GTT AAG GTT
130

Val Ala Glu Leu Gln Arg Leu Lys Val Lys Val

35 40



Thr Phe Asp Tyr Pro Gly Arg Ala His Thr Phe Asp Asp Phe Cys Pro

SEQ ID NO. 16:

1 5 10 15



Glu Cys Arg Ala Leu Gly Leu Gln Gly Cys Ala Phe Gln Ser Thr Val

20 25 30



Ala Glu Leu Gln Arg Leu Lys Val Lys Val

35 40



























The universal nature of these epitopes is evident from the homology exhibited by the DNA that encodes them. If the epitope coding sequences from the Mexican strains shown above are compared to DNA sequences from other strains, such as the Burmese Strain also set forth above, similarities are evident, as shown in the following comparisons.
Comparison of 406.4-2 Epitopes, HEV Mexico and Burma Strains








10 20 30


MEXICAN (SEQ ID NO. 17)
ANQPGHLAPLGEIRPSAPPLPPVADLPQPGLRR




::.:.: :::: .::::::::.:.:::: : ::



BURMA (SEQ ID NO. 18)
ANPPDHSAPLGVTRPSAPPLPHVVDLPQLGPRR


10 20 30














There is 73.5% identity in a 33-amino acid overlap
Comparison of 406.3-2 Epitopes, HEV Mexico and Burma Strains








10 20 30 40


MEXICAN (SEQ ID No. 19)
TFDYPGRAHTFDDFCPECRALGLQGCAFQSTVAELQRLKVKV




:.:::.:::::::::::::.:::::::::::::::::::.::



BURMA (SEQ ID No. 20)
TLDYPARAHTFDDFCPECRPLGLQGCAFQSTVAELQRLKMKV


10 20 30 40














There is 90.5% identity in the 42-amino acid overlap
It will be recognized by one skilled in the art of molecular genetics that each of the specific DNA sequences given above shows a corresponding complementary DNA sequence as well as RNA sequences corresponding to both the principal sequence shown and the complementary DNA sequence. Additionally, open reading frames encoding peptides are present, and expressible peptides are disclosed by the nucleotide sequences without setting forth the amino acid sequences explicitly, in the same manner as if the amino acid sequences were explicitly set forth as in the ET1.1 sequence or other sequences above.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
The terms defined below have the following meaning herein:
1. “Enterically transmitted non-A/non-B hepatitis viral agent, ET-NANB, or HEV” means a virus, virus type, or virus class which (1) causes water-borne, infectious hepatitis, (ii) is transmissible in cynomolgus monkeys, (iii) is serologically distinct from hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), and hepatitis D virus, and (iv) includes a genomic region which is homologous to the 1.33 kb cDNA insert in plasmid pTZKF1(ET1.1) carried in E. coli strain BB4 identified by ATCC deposit number 67717.
2. Two nucleic acid fragments are “homologous” if they are capable of hybridizing to one another under hybridization conditions described in Maniatis et al., op. cit., pp. 320-323. However, using the following wash conditions: 2×SCC, 0.1% SDS, room temperature twice, 30 minutes each; then 2×SCC, 0.1% SDS, 50° C. once, 30 minutes; then 2×SCC, room temperature twice, 10 minutes each, homologous sequences can be identified that contain at most about 25-30% basepair mismatches. More preferably, homologous nucleic acid strands contain 15-25% basepair mismatches, even more preferably 5-15% basepair mismatches. These degrees of homology can be selected by using more stringent wash conditions for identification of clones from gene libraries (or other sources of genetic material), as is well known in the art.
3. Two amino acid sequences or two nucleotide sequences (in an alternative definition for homology between two nucleotide sequences) are considered homologous (as this term is preferably used in this specification) if they have an alignment score of >5 (in standard deviation units) using the program ALIGN with the mutation gap matrix and a gap penalty of 6 or greater. See Dayhoff, M. O., in Atlas of Protein Sequence and Structure (1972) Vol. 5, National Biomedical Research Foundation, pp. 101-110, and Supplement 2 to this volume, pp. 1-10. The two sequences (or parts thereof, preferably at least 30 amino acids in length) are more preferably homologous if their amino acids are greater than or equal to 50% identical when optimally aligned using the ALIGN program mentioned above.
4. A DNA fragment is “derived from” an ET-NANB viral agent if it has the same or substantially the same basepair sequence as a region of the viral agent genome.
5. A protein is “derived from” an ET-NANB viral agent if it is encoded by an open reading frame of a DNA or RNA fragment derived from an ET-NANB viral agent.
II. Obtaining Cloned ET-NANB Fragments
According to one aspect of the invention, it has been found that a virus-specific DNA clone can be produced by (a) isolating RNA from the bile of a cynomolgus monkey having a known ET-NANB infection, (b) cloning the cDNA fragments to form a fragment library, and (c) screening the library by differential hybridization to radiolabeled cDNAs from infected and non-infected bile sources.
A. cDNA Fragment Mixture
ET-NANB infection in cynomolgus monkeys is initiated by inoculating the animals intravenously with a 10% w/v suspension from human case stools positive for 27-34 nm ET-NANB particles (mean diameter 32 nm). An infected animal is monitored for elevated levels of alanine aminotransferase, indicating hepatitis infection. ET-NANB infection is confirmed by immunospecific binding of seropositive antibodies to virus-like particles (VLPs), according to published methods (Gravelle). Briefly, a stool (or bile) specimen taken from the infected animal 3-4 weeks after infection is diluted 1:10 with phosphate-buffered saline, and the lot suspension is clarified by low-speed centrifugation and filtration successively through 1.2 and 0.45 micron filters. The material may be further purified by pelleting through a 30% sucrose cushion (Bradley). The resulting preparation of VLPs is mixed with diluted serum from human patients with known ET-NANB infection. After incubation overnight, the mixture is centrifuged overnight to pellet immune aggregates, and these are stained and examined by electron microscopy for antibody binding to the VLPs.
ET-NANB infection can also be confirmed by seroconversion to VLP-positive serum. Here the serum of the infected animal is mixed as above with 27-34 nm VLPs isolated from the stool specimens of infected human cases and examined by immune electron microscopy for antibody binding to the VLPs.
Bile can be collected from ET-NANB positive animals by either cannulating the bile duct and collecting the bile fluid or by draining the bile duct during necropsy. Total RNA is extracted from the bile by hot phenol extraction, as outlined in Example 1A. The RNA fragments are used to synthesize corresponding duplex cDNA fragments by random priming, also as referenced in Example 1A. The cDNA fragments may be fractionated by gel electrophoresis density gradient centrifugation to obtain a desired size class of fragments, e.g., 500-4,000 basepair fragments.
Although alternative sources of viral material, such as VLPs obtained from stool samples (as described in Example 4), may be used for producing a CDNA fraction, the bile source is preferred. According to one aspect of the invention, it has been found that bile from ET-NANB-infected monkeys shows a greater number of intact viral particles than material obtained from stool samples, as evidenced by immune electron microscopy. Bile obtained from an ET-NANB infected human or cynomolgus macaque, for use as a source of ET-NANB viral protein or genomic material, or intact virus, forms part of the present invention.
B. CDNA Library and Screening
The CDNA fragments from above are cloned into a suitable cloning vector to form a cDNA library. This may be done by equipping blunt-ended fragments with a suitable end linker, such as an EcoRI sequence, and inserting the fragments into a suitable insertion site of a cloning vector, such as at a unique EcoRI site. After initial cloning, the library may be re-cloned, if desired, to increase the percentage of vectors containing a fragment insert. The library construction described in Example 1B is illustrative. Here cDNA fragments were blunt-ended, equipped with EcoRI ends, and inserted into the EcoRI site of the lambda phage vector gt10. The library phage, which showed less than 5% fragment inserts, was isolated, and the fragment inserts re-cloned into the lambda gt10 vector, yielding more than 95% insert-containing phage.
The cDNA library is screened for sequences specific for ET-NANB by differential hybridization to cDNA probes derived from infected and non-infected sources. cDNA fragments from infected and non-infected source bile or stool viral isolates can be prepared as above. Radiolabeling the fragments is by random labeling nick translation, or ending, according to conventional methods (Maniatis, p. 109). The cDNA library from above is screened by transfer to duplicate nitrocellulose filters, and hybridization with both infected-source and non-infected-source (control) radiolabeled probes, as detailed in Example 2. In order to recover sequences that hybridize at the preferred outer limit of 25-30% basepair mismatches, clones can be selected if they hybridize under the conditions described in Maniatis et al., op. cit., pp. 320-323, but using the following wash conditions: 2×SCC, 0.1% SDS, room temperature—twice, 30 minutes each; then 2×SCC, 0.1% SDS, 50° C.—once, 30 minutes; then 2×SCC, room temperature—twice, 10 minutes each. These conditions allowed identification of the Mexican isolate discussed above using the ET1.1 sequence as a probe. Plaques which show selective hybridization to the infected-source probes are preferably re-plated at low plating density and re-screened as above, to isolate single clones which are specific for ET-NANB sequences. As indicated in Example 2, sixteen clones which hybridized specifically with infected-source probes were identified by these procedures. One of the clones, designated lambda gt101.1, contained a 1.33 kilobase fragment insert.
C. ET-NANB Sequences
The basepair sequence of cloned regions of the ET-NANB fragments from Part B are determined by standard sequencing methods. In one illustrative method, described in Example 3, the fragment insert from the selected cloning vector is excised, isolated by gel electrophoresis, and inserted into a cloning vector whose basepair sequence on either side of the insertion site is known. The particular vector employed in Example 3 is a pTZKF1 vector shown at the left in FIG. 1 . The ET-NANB fragment from the gt10-1.1 phage was inserted at the unique EcoRI site of the pTZKF1 plasmid. Recombinants carrying the desired insert were identified by hybridization with the isolated 1.33 kilobase fragment, as described in Example 3. One selected plasmid, identified as pTZKF1 (ET1.1), gave the expected 1.33 kb fragment after vector digestion with EcoRI. E. coli strain BB4 infected with the pTZKF1 (ET1.1) plasmid has been deposited with the American Type Culture Collection, Rockville, Md., and is identified by ATCC deposit number 67717.
The pTZKF1(ET1.1) plasmid is illustrated at the bottom in FIG. 1 . The fragment insert has 5′ and 3′ end regions denoted at A and C, respectively, and an intermediate region, denoted at B. The sequences in these regions were determined by standard dideoxy sequencing and were set forth in an earlier application in this series. The three short sequences (A, B, and C) are from the same insert strand. As will be seen in Example 3, the B-region sequence was actually determined from the opposite strand, so that the B region sequence shown above represents the complement of the sequence in the sequenced strand. The base numbers of the partial sequences are approximate.
Later work in the laboratory of the inventors identified the full sequence, set forth above. Fragments of this total sequence can readily be prepared using restriction endonucleases. Computer analysis of both the forward and reverse sequence has identified a number of cleavage sites.
III. ET-NANB Fragments
According to another aspect, the invention includes ET-NANB-specific fragments or probes which hybridize with ET-NANB genomic sequences or cDNA fragments derived therefrom. The fragments may include full-length cDNA fragments such as described in Section II, or may be derived from shorter sequence regions within cloned CDNA fragments. Shorter fragments can be prepared by enzymatic digestion of full-length fragments under conditions which yield desired-sized fragments, as will be described in Section IV. Alternatively, the fragments can be produced by oligonucleotide synthetic methods, using sequences derived from the cDNA fragments. Methods or commercial services for Producing selected-sequence oligonucleotide fragments are available. Fragments are usually at least 12 nucleotides in length, preferably at least 14, 20, 30 or 50 nucleotides, when used as probes. Probes can be full length or less than 500, preferably less than 300 or 200, nucleotides in length.
To confirm that a given ET-NANB fragment is in fact derived from the ET-NANB viral agent, the fragment can be shown to hybridize selectively with cDNA from infected sources. By way of illustration, to confirm that the 1.33 kb fragment in the pTZKF1(ET1.1) plasmid is ET-NANB in origin, the fragment was excised from the pTZKF1(ET1.1) plasmid, purified, and radiolabeled by random labeling. The radiolabeled fragment was hybridized with fractionated cDNAs from infected and non-infected sources to confirm that the probe reacts only with infected-source cDNAs. This method is illustrated in Example 4, where the above radiolabeled 1.33 kb fragment from pTZKF1(ET1.1) plasmid was examined for binding to cDNAs prepared from infected and non-infected sources. The infected sources are (1) bile from a cynomolgus macaque infected with a strain of virus derived from stool samples from human patients from Burma with known ET-NANB infections and (2) a viral agent derived from the stool sample of a human ET-NANB patient from Mexico. The cDNAs in each fragment mixture were first amplified by a linker/primer amplification method described in Example 4. Fragment separation was on agarose gel, followed by Southern blotting and then hybridization to bind the radiolabeled 1.33 kb fragment to the fractionated cDNAs. The lane containing cDNAs from the infected sources showed a smeared band of bound probe, as expected (cDNAs amplified by the linker/primer amplification method would be expected to have a broad range of sizes). No probe binding to the amplified cDNAs from the non-infected sources was observed. The results indicate that the 1.33 kb probe is specific for CDNA fragments associated with ET-NANB infection. This same type of study, using ET 1.1 as the probe, has demonstrated hybridization to ET-NANB samples collected from Tashkent, Somalia, Borneo and Pakistan. Secondly, the fact that the probe is specific for ET-NANB related sequences derived from different continents (Asia, Africa and North America) indicates the cloned ET-NANB Burma sequence (ET1.1) is derived from a common ET-NANB virus or virus class responsible for ET-NANB hepatitis infection worldwide.
In a related confirmatory study, probe binding to fractionated genomic fragments prepared from human or cynomolgus macaque genomic DNA (both infected and uninfected) was examined. No probe binding was observed to either genomic fraction, demonstrating that the ET-NANB fragment is not an endogenous human or cynomolgus genomic fragment and additionally demonstrating that HEV is an RNA virus.
Another confirmation of ET-NANB specific sequences in the fragments is the ability to express ET-NANB proteins from coding regions in the fragments and to demonstrated specific sero-reactivity of these proteins with sera collected during documented outbreaks of ET-NANB. Section IV below discusses methods of protein expression using the fragments.
One important use of the ET-NANB-specific fragments is for identifying ET-NANB-derived cDNAs which contain additional sequence information. The newly identified cDNAs, in turn, yield new fragment probes, allowing further iterations until the entire viral genome is identified and sequenced. Procedures for identifying additional ET-NANB library clones and generating new probes therefrom generally follow the cloning and selection procedures described in Section II.
The fragments (and oligonucleotides prepared based on the sequences given above) are also useful as primers for a polymerase chain reaction method of detecting ET-NANB viral genomic material in a patient sample. This diagnostic method will be described in Section V below.
Two specific genetic sequences derived from the Mexican strain, identified herein as 406.3-2 and 406.4-2, have been identified that encode immunogenic epitopes. This was done by isolating clones which encode epitopes that immunologically react specifically with sera from individuals and experimental animals infected with HEV. Comparison of the isolated sequences with those in the Genebank collection of genetic sequences indicate that these viral sequences are novel. Since these sequences are unique, they can be used to identify the presence of HEV and to distinguish this strain of hepatitis from HAV, HBV, and HCV strains. The sequences are also useful for the design of oligonucleotide probes to diagnose the presence of virus in samples. They can be used for the synthesis of polypeptides that themselves are used in immunoassays. The specific 406.3-2 and 406.4-2 sequences can be incorporated into other genetic material, such as vectors, for ease of expression or replication. They can also be used (as demonstrated above) for identifying similar antigenic regions encoded by related viral strains, such as the Burmese strain.
IV. ET-NANB Proteins
As indicated above, ET-NANB proteins can be prepared by expressing open reading-frame coding regions in ET-NANB fragments. In one preferred approach, the ET-NANB fragments used for protein expression are derived from cloned cDNAs which have been treated to produce desired-size fragments, and preferably random fragments with sizes predominantly between about 100 to about 300 base pairs. Example 5 describes the preparation of such fragments by DNAs digestion. Because it is desired to obtain peptide antigens of between about 30 to about 100 amino acids, the digest fragments are preferably size fractionated, for example by gel electrophoresis, to select those in the approximately 100-300 basepair size range. Alternatively, cDNA libraries constructed directly from HEV-containing sources (e.g., bile or stool) can be screened directly if cloned into an appropriate expression vector (see below).
For example, the ET-NANB proteins expressed by the 406.3-2 and 406.4-2 sequences (and peptide fragments thereof) are particularly preferred since these proteins have been demonstrated to be immunoreactive with a variety of different human sera, thereby indicating the presence of one or more epitopes specific for HEV on their surfaces. These clones were identified by direct screening of a gt11 library.
A. Expression Vector
The ET-NANB fragments are inserted into a suitable expression vector. One exemplary expression vector is lambda gt11, which contains a unique EcoRI insertion site 53 base pairs upstream of the translation termination codon of the beta-galactosidase gene. Thus, the inserted sequence will be expressed as a beta-galactosidase fusion protein which contains the N-terminal portion of the beta-galactosidase gene, the heterologous peptide, and optionally the C-terminal region of the beta-galactosidase peptide (the C-terminal portion being expressed when the heterologous peptide coding sequence does not contain a translation termination codon). This vector also produces a temperature-sensitive repressor (c1857) which causes viral lysogeny at permissive temperatures, e.g., 32° C., and leads to viral lysis at elevated temperatures, e.g., 37° C. Advantages of this vector include: (1) highly efficient recombinant generation, (2) ability to select lysogenized host cells on the basis of host-cell growth at permissive, but not non-permissive, temperatures, and (3) high levels of recombinant fusion protein production. Further, since phage containing a heterologous insert produces an inactive beta-galactosidase enzyme, phage with inserts can be readily identified by a beta-galactosidase colored-substrate reaction.
For insertion into the expression vector, the viral digest fragments may be modified, if needed, to contain selected restriction-site linkers, such as EcoRI linkers, according to conventional procedures. Example 1 illustrates methods for cloning the digest fragments into lambda gt11, which includes the steps of blunt-ending the fragments, ligating with EcoRI linkers, and introducing the fragments into EcoRI-cut lambda gt11. The resulting viral genomic library may be checked to confirm that a relatively large (representative) library has been produced. This can be done, in the case of the lambda gt11 vector, by infecting a suitable bacterial host, plating the bacteria, and examining the plaques for loss of beta-galactosidase activity. Using the procedures described in Example 1, about 50% of the plaques showed loss of enzyme activity.
B. Peptide Antigen Expression
The viral genomic library armed above is screened for production of peptide antigen (expressed as a fusion protein) which is immunoreactive with antiserum from ET-NANB seropositve individuals. In a preferred screening method, host cells infected with phage library vectors are plated, as above, and the plate is blotted with a nitrocellulose filter to transfer recombinant protein antigens produced by the cells onto the filter. The filter is then reacted with the ET-NANB antiserum, washed to remove unbound antibody, and reacted with reporter-labeled, anti-human antibody, which becomes bound to the filter, in sandwich fashion, through the anti-ET-NANB antibody.
Typically phage plaques which are identified by virtue of their production of recombinant antigen of interest are re-examined at a relatively low density for production of antibody-reactive fusion protein. Several recombinant phage clones which produced immunoreactive recombinant antigen were identified in the procedure.
The selected expression vectors may be used for scale-up production, for purposes of recombinant protein purification. Scale-up production is carried out using one of a variety of reported methods for (a) lysogenizing a suitable host, such as E. coli , with a selected lambda gt11 recombinant (b) culturing the transduced cells under conditions that yield high levels of the heterologous peptide, and (c) purifying the recombinant antigen from the lysed cells.
In one preferred method involving the above lambda gt11 cloning vector, a high-producer E. coli host, BNN103, is infected with the selected library phage and replica plated on two plates. One of the plates is grown at 32° C., at which viral lysogeny can occur, and the other at 42° C., at which the infecting phage is in a lytic stage and therefore prevents cell growth. Cells which grow at the lower but not the higher temperature are therefore assumed to be successfully lysogenized.
The lysogenized host cells are then grown under liquid culture conditions which favor high production of the fused protein containing the viral insert, and lysed by rapid freezing to release the desired fusion protein.
C. Peptide Purification
The recombinant peptide can be purified by standard protein purification procedures which may include differential precipitation, molecular sieve chromatography, ion-exchange chromatography, isoelectric focusing, gel electrophoresis and affinity chromatography. In the case of a fused protein, such as the beta-galactosidase fused protein prepared as above, the protein isolation techniques which are used can be adapted from those used in isolation of the native protein. Thus, for isolation of a soluble betagalactosidase fusion protein, the protein can be isolated readily by simple affinity chromatography, by passing the cell lysis material over a solid support having surface-bound anti-beta-galactosidase antibody.
D. Viral Proteins
The ET-NANB protein of the invention may also be derived directly from the ET-NANB viral agent. VLPs or protein isolated from stool or liver samples from an infected individual, as above, are one suitable source of viral protein material. The VLPs isolated from the stool sample may be further purified by affinity chromatography prior to protein isolation (see below). The viral agent may also be raised in cell culture, which provides a convenient and potentially concentrated source of viral protein. Co-owned U.S. patent application Ser. No. 846,757, filed Apr. 1, 1986, describes an immortalized trioma liver cell which supports NANB infection in cell culture. The trioma cell line is prepared by fusing human liver cells with a mouse/human fusion partner selected for human chromosome stability. Cells containing the desired NANB viral agent can be identified by immunofluorescence methods, employing anti-ET-NANB human antibodies.
The viral agent is disrupted, prior to protein isolation, by conventional methods, which can include sonication, high- or low-salt conditions, or use of detergents.
Purification of ET-NANB viral protein can be carried out by affinity chromatography, using a purified anti-ET-NANB antibody attached according to standard methods to a suitable solid support. The antibody itself may be purified by affinity chromatography, where an immunoreactive recombinant ETNANB protein, such as described above, is attached to a solid support, for isolation of anti-ET-NANB antibodies from an immune serum source. The bound antibody is released from the support by standard methods.
Alternatively, the anti-ET-NANB antibody may be an antiserum or a monoclonal antibody (Mab) prepared by immunizing a mouse or other animal with recombinant ETNANB protein. For Mab production, lymphocytes are isolated from the animal and immortalized with a suitable fusion partner, and successful fusion products which react with the recombinant protein immunogen are selected. These in turn may be used in affinity purification procedures, described above, to obtain native ET-NANB antigen.
V. Utility
Although ET-NANB is primarily of interest because of its effects on humans, recent data has shown that this virus is also capable of infecting other animals, especially mammals. Accordingly, any discussion herein of utility applies to both human and veterinary uses, especially commercial veterinary uses, such as the diagnosis and treatment of pigs, cattle, sheep, horses, and other domesticated animals.
A. Diagnostic Methods
The particles and antigens of the invention, as well as the genetic material, can be used in diagnostic assays. Methods for detecting the presence of ET-NANB hepatitis comprise analyzing a biological sample such as a blood sample, stool sample or liver biopsy specimen for the presence of an analyte associated with ET-NANB hepatitis virus.
The analyte can be a nucleotide sequence which hybridizes with a probe comprising a sequence of at least about 16 consecutive nucleotides, usually 30 to 200 nucleotides, up to substantially the full sequence of the sequences shown above (cDNA sequences). The analyte can be RNA or cDNA. The analyte is typically a virus particle suspected of being ET-NANB or a particle for which this classification is being ruled out. The virus particle can be further characterized as having an RNA viral genome comprising a sequence at least about 70% homologous to a sequence of at least 12 consecutive nucleotides of the “forward” and “reverse” sequences given above, usually at least about 80% homologous to at least about 60 consecutive nucleotides within the sequences, and may comprise a sequence substantially homologous to the full-length sequences. In order to detect an analyte, where the analyte hybridizes to a probe, the probe may contain a detectable label. Particularly preferred for use as a probe are sequences of consecutive nucleotides derived from the 406.3-2 and 406.4-2 clones described herein, since these clones appear to be particularly diagnostic for HEV.
The analyte can also comprise an antibody which recognizes an antigen, such as a cell surface antigen, in a ET-NANB virus particle. The analyte can also be a ET-NANB viral antigen. Where the analyte is an antibody or an antigen, either a labelled antigen or antibody, respectively, can be used to bind to the analyte to form an immunological complex, which can then be detected by means of the label.
Typically, methods for detecting analytes such as surface antigens and/or whole particles are based on immunoassays. Immunoassays can be conducted either to determine the presence of antibodies in the host that have arisen from infection by ET-NANB hepatitis virus or by assays that directly determine the presence of virus particles or antigens. Such techniques are well known and need not be described here in detail. Examples include both heterogeneous and homogeneous immunoassay techniques. Both techniques are based on the formation of an immunological complex between the virus particle or its antigen and a corresponding specific antibody. Heterogeneous assays for viral antigens typically use a specific monoclonal or polyclonal antibody bound to a solid surface. Sandwich assays are becoming increasingly popular. Homogeneous assays, which are carried out in solution without the presence of a solid phase, can also be used, for example by determining the difference in enzyme activity brought on by binding of free antibody to an enzyme-antigen conjugate. A number of suitable assays are disclosed in U.S. Pat. Nos. 3,817,837, 4,006,360, 3,996,345.
When assaying for the presence of antibodies induced by ET-NANB viruses, the viruses and antigens of the invention can be used as specific binding agents to detect either IgG or IgM antibodies. Since IgM antibodies are typically the first antibodies that appear during the course of an infection, when IgG synthesis may not yet have been initiated, specifically distinguishing between IgM and IgG antibodies present in the blood stream of a host will enable a physician or other investigator to determine whether the infection is recent or convalescent. Proteins expressed by the 406.3-2 and 406.4-2 clones described herein and peptide fragments thereof are particularly preferred for use as specific binding agents to detect antibodies since they have been demonstrated to be reactive with a number of different human HEV sera. Further, they are reactive with both acute and convalescent sera.
In one diagnostic configuration, test serum is reacted with a solid phase reagent having surface-bound ET-NANB protein antigen. After binding anti-ET-NANB antibody to the reagent and removing unbound serum components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-ET-NANB antibody on the solid support. The reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined. Typically, the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric or colorimetric substrate.
The solid surface reagent in the above assay prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, or filter material. These attachment methods generally include non-specific adsorption of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activate carboxyl, hydroxyl, or aldehyde group.
In a second diagnostic configuration, known as a homogeneous assay, antibody binding to a solid support produces some change in the reaction medium which can be directly detected in the medium. Known general types of homogeneous assays proposed heretofore include (a) spin-labeled reporters, where antibody binding to the antigen is detected by a change in reported mobility (broadening of the spin splitting peaks), (b) fluorescent reporters, where binding is detected by a change in fluorescence efficiency, (c) enzyme reporters, where antibody binding effects enzyme/substrate interactions, and (d) liposome-bound reporters, where binding leads to liposome lysis and release of encapsulated reporter. The adaptation of these methods to the protein antigen of the present invention follows conventional methods for preparing homogeneous assay reagents.
In each of the assays described above, the assay method involves reacting the serum from a test individual with the protein antigen and examining the antigen for the presence of bound antibody. The examining may involve attaching a labeled anti-human antibody to the antibody being examined, either IgM (acute phase) or IgG (convalescent phase), and measuring the amount of reporter bound to the solid support, as in the first method, or may involve observing the effect of antibody binding on a homogeneous assay reagent, as in the second method.
Also forming part of the invention is an assay system or kit for carrying out the assay method just described. The kit generally includes a support with surface-bound recombinant protein antigen which is (a) immunoreactive with antibodies present in individuals infected with enterically transmitted nonA/nonB viral agent and (b) derived from a viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZKF1(ET1.1) carried in E. Coli strain BB4, and having ATCC deposit no. 67717. A reporter-labeled anti-human antibody in the kit is used for detecting surface-bound anti-ET-NANB antibody.
B. Viral Genome Diagnostic Applications
The genetic material of the invention can itself be used in numerous assays as probes for genetic material present in naturally occurring infections. One method for amplification of target nucleic acids, for later analysis by hybridization assays, is known as the polymerase chain reaction or PCR technique. The PCR technique can be applied to detecting virus particles of the invention in suspected pathological samples using oligonucleotide primers spaced apart from each other and based on the genetic sequence set forth above. The primers are complementary to opposite strands of a double stranded DNA molecule and are typically separated by from about 50 to 450 nt or more (usually not more than 2000 nt). This method entails preparing the specific oligonucleotide primers and then repeated cycles of target DNA denaturation, primer binding, and extension with a DNA polymerase to obtain DNA fragments of the expected length based on the primer spacing. Extension products generated from one primer serve as additional target sequences for the other primer. The degree of amplification of a target sequence is controlled by the number of cycles that are performed and is theoretically calculated by the simple formula 2 n where n is the number of cycles. Given that the average efficiency per cycle ranges from about 65% to 85%, 25 cycles produce from 0.3 to 4.8 million copies of the target sequence. The PCR method is described in a number of publications, including Saiki et al., Science (1985) 230:1350-1354; Saiki et al., Nature (1986) 324:163-166; and Scharf et al., Science (1986) 233:1076-1078. Also see U.S. Pat. Nos. 4,683,194; 4,683,195; and 4,683,202.
The invention includes a specific diagnostic method for determination of ET-NANB viral agent, based on selective amplification of ET-NANB fragments. This method employs a pair of single-strand primers derived from non-homologous regions of opposite a strands of a DNA duplex fragment, which in turn is derived from an enterically transmitted viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZKF1(ET1.1) carried in E. coli strain BB4, and having ATCC deposit no. 67717. These “primer fragments,” which form one aspect of the invention, are prepared from ET-NANB fragments such as described in Section III above. The method follows the process for amplifying selected nucleic acid sequences as disclosed in U.S. Pat. No. 4,683,202, as discussed above.
C. Peptide Vaccine
Any of the antigens of the invention can be used in preparation of a vaccine. A preferred starting material for preparation of a vaccine is the particle antigen isolated from bile. The antigens are preferably initially recovered as intact particles as described above. However, it is also possible to prepare a suitable vaccine from particles isolated from other sources or non-particle recombinant antigens. When non-particle antigens are used (typically soluble antigens), proteins derived from the viral envelope or viral capsid are preferred for use in preparing vaccines. These proteins can be purified by affinity chromatography, also described above.
If the purified protein is not immunogenic per se, it can be bound to a carrier to make the protein immunogenic. Carriers include bovine serum albumin, keyhole limpet hemocyanin and the like. It is desirable, but not necessary, to purify antigens to be substantially free of human protein. However, it is more important that the antigens be free of proteins, viruses, and other substances not of human origin that may have been introduced by way of, or contamination of, the nutrient medium, cell lines, tissues, or pathological fluids from which the virus is cultured or obtained.
Vaccination can be conducted in conventional fashion. For example, the antigen, whether a viral particle or a protein, can be used in a suitable diluent such as water, saline, buffered salines, complete or incomplete adjuvants, and the like. The immunogen is administered using standard techniques for antibody induction, such as by subcutaneous administration of physiologically compatible, sterile solutions containing inactivated or attenuated virus particles or antigens. An immune response producing amount of virus particles is typically administered per vaccinizing injection, typically in a volume of one milliliter or less.
A specific example of a vaccine composition includes, in a pharmacologically acceptable adjuvant, a recombinant protein or protein mixture derived from an enterically transmitted nonA/nonB viral hepatitis agent whose genome contains a region which is homologous to the 1.33 kb DNA EcoRI insert present in plasmid pTZKF1(ET1.1) carried in E. coli strain BB4, and having ATCC deposit no. 67717. The vaccine is administered at periodic intervals until a significant titer of anti-ET-NANB antibody is detected in the serum. The vaccine is intended to protect against ET-NANB infection.
Particularly preferred are vaccines prepared using proteins expressed by the 406.3-2 and 406.4-2 clones described herein and equivalents thereof, including fragments of the expressed proteins. Since these clones have already been demonstrated to be reactive with a variety of human HEV-positive sera, their utility in protecting against a variety of HEV strains is indicated.
D. Prophylactic and Therapeutic Antibodies and Antisera
An addition to use as a vaccine, the compositions can be used to prepare antibodies to ET-NANB virus particles. The antibodies can be used directly as antiviral agents. To prepare antibodies, a host animal is immunized using the virus particles or, as appropriate, non-particle antigens native to the virus particle are bound to a carrier as described above for vaccines. The host serum or plasma is collected following an appropriate time interval to provide a composition comprising antibodies reactive with the virus particle. The gamma globulin fraction or the IgG antibodies can De obtained, for example, by use of saturated ammonium sulfate or DEAE Sephadex, or other techniques known to those skilled in the art. The antibodies are substantially free of many of the adverse side effects which may be associated with other anti-viral agents such as drugs.
The antibody compositions can be made even more compatible with the host system by minimizing potential adverse immune system responses. This is accomplished by removing all or a portion of the FC portion of a foreign species antibody or using an antibody of the same species as the host animal, for example, the use of antibodies from human/human hybridomas.
The antibodies can also be used as a means of enhancing the immune response since antibody-virus complexes are recognized by macrophages. The antibodies can be administered in amounts similar to those used for other therapeutic administrations of antibody. For example, pooled gamma globulin is administered at 0.02-0.1 ml/lb body weight during the early incubation of other viral diseases such as rabies, measles and hepatitis B to interfere with viral entry into cells. Thus, antibodies reactive with the ET-NANB virus particle can be passively administered alone or in conjunction with another anti-viral agent to a host infected with an ET-NANB virus to enhance the immune response and/or the effectiveness of an antiviral drug.
Alternatively, anti-ET-NANB-virus antibodies can be induced by administering anti-idiotype antibodies as immunogens. Conveniently, a purified anti-ET-NANB-virus antibody preparation prepared as described above is used to induce anti-idiotype antibody in a host animal. The composition is administered to the host animal in a suitable diluent. Following administration, usually repeated administration, the host produces anti-idiotype antibody. To eliminate an immunogenic response to the Fc region, antibodies produced by the same species as the host animal can be used or the Fc region of the administered antibodies can be removed. Following induction of anti-idiotype antibody in the host animal, serum or plasma is removed to provide an antibody composition. The composition can be purified as described above for anti-ET-NANB virus antibodies, or by affinity chromatography using anti-ET-NANB-virus antibodies bound to the affinity matrix. The anti-idiotype antibodies produced are similar in conformation to the authentic ET-NANB antigen and may be used to prepare an ET-NANB vaccine rather than using a ET-NANB particle antigen.
When used as a means of inducing anti-ET-NANB virus antibodies in a patient, the manner of injecting the antibody is the same as for vaccination purposes, namely intramuscularly, intraperitoneally, subcutaneously or the like in an effective concentration in a physiologically suitable diluent with or without adjuvant. One or more booster injections may be desirable. The anti-idiotype method of induction of anti-ET-NANB virus antibodies can alleviate problems which may be caused by passive administration of anti-ET-NANB-virus antibodies, such as an adverse immune response, and those associated with administration of purified blood components, such as infection with as yet undiscovered viruses.
The ET-NANB derived proteins of the invention are also intended for use in producing antiserum designed for pre- or post-exposure prophylaxis. Here an ET-NANB protein, or mixture of proteins is formulated with a suitable adjuvant and administered by injection to human volunteers, according to known methods for producing human antisera. Antibody response to the injected proteins is monitored, during a several-week period following immunization, by periodic serum sampling to detect the presence an anti-ET-NANB serum antibodies, as described in Section IIA above.
The antiserum from immunized individuals may be administered as a pre-exposure prophylactic measure for individuals who are at risk of contracting infection. The antiserum is also useful in treating an individual post-exposure, analogous to the use of high titer antiserum against hepatitis B virus for post-exposure prophylaxis.
E. Monoclonal Antibodies
For both in vivo use of antibodies to ET-NANB virus particles and proteins and anti-idiotype antibodies and diagnostic use, it may be preferable to use monoclonal antibodies. Monoclonal anti-virus particle antibodies or anti-idiotype antibodies can be produced as follows. The spleen or lymphocytes from an immunized animal are removed and immortalized or used to prepare hybridomas by methods known to those skilled in the art. To produce a human-human hybridoma, a human lymphocyte donor is selected. A donor known to be infected with a ET-NANB virus (where infection has been shown for example by the presence of anti-virus antibodies in the blood or by virus culture) may serve as a suitable lymphocyte donor. Lymphocytes can be isolated from a peripheral blood sample or spleen cells may be used if the donor is subject to splenectomy. Epstein-Barr virus (EBV) can be used to immortalize human lymphocytes or a human fusion partner can be used to produce human-human hybridomas. Primary in vitro immunization with peptides can also be used in the generation of human monoclonal antibodies.
Antibodies secreted by the immortalized cells are screened to determine the clones that secrete antibodies of the desired specificity. For monoclonal anti-virus particle antibodies, the antibodies must bind to ET-NANB virus particles. For monoclonal anti-idiotype antibodies, the antibodies must bind to anti-virus particle antibodies. Cells producing antibodies of the desired specificity are selected.
The following examples illustrate various aspects of the invention, but are in no way intended to limit the scope thereof.
MATERIAL
The materials used in the following Examples were as follows:
Enzymes: DNAse I and alkaline phosphatase were obtained from Boehringer Mannheim Biochemicals (BMB, Indianapolis, Ind.); EcoRI, EcoRI methylase, DNA ligase, and DNA Polymerase I, from New England Biolabs (NEB, Beverly Ma.); and RNase A was obtained from Sigma (St. Louis, Mont.)
Other reagents: EcoRI linkers were obtained from NEB; and nitro blue tetrazolium (NBT), S-bromo-4-chloro-3-indolyl phosphate (BCIP) S-bromo-4-chloro-3-indolyl-B-D-galactopyranoside (Xgal) and isopropyl B-D-thiogalactopyranoside (IPTG) were obtained from Sigma.
cDNA synthesis kit and random priming labeling kits are available from Boehringer-Mannheim Biochemical (BMB, Indianapolis, Ind.).
EXAMPLE 1
Preparing CDNA Library
A. Source of ET-NANB Virus
Two cynomolgus monkeys (cynos) were intravenously injected with a 10% suspension of a stool pool obtained from a second-passage cyno (cyno #37) infected with a strain of ET-NANB virus isolated from Burma cases whose stools were positive for ET-NANB, as evidenced by binding of 27-34 nm virus-like particles (VLPs) in the stool to immune serum from a known ETNANB patient. The animals developed elevated levels of alanine aminotransferase (ALT) between 24-36 days after inoculation, and one excreted 27-34 nm VLPS in its bile in the pre-acute phase of infection.
The bile duct of each infected animal was cannulated and about 1-3 cc of bile was collected daily. RNA was extracted from one bile specimen (cyno #121) by hot phenol extraction, using a standard RNA isolation procedure. Double-strand cDNA was formed from the isolated RNA by a random primer for first-strand generation, using a cDNA synthesis kit obtained from Boehringer-Mannheim (Indianapolis, Ind.).
B. Cloning the Duplex Fragments
The duplex CDNA fragments were blunt-ended with T4 DNA polymerase under standard conditions (Maniatis, p. 118), then extracted with phenol/chloroform and precipitated with ethanol. The blunt-ended material was ligated with EcoRI linkers under standard conditions (Maniatis, pp. 396-397) and digested with EcoRI to remove redundant linker ends. Non-ligated linkers were removed by sequential isopropanol precipitation.
Lambda gt10 phage vector (Huynh) was obtained-from Promega Biotec (Madison, Wis.). This cloning vector has a unique EcoRI cloning site in the phage CI repressor gene. The cDNA fragments from above were introduced into the EcoRI site by mixing 0.5-1.0 μg EcoRI-cleaved gt10, 0.5-3 μl of the above duplex fragments, 0.5 μl 10×ligation buffer, 0.5 μl ligase (200 units), and distilled water to 5 μl. The mixture was incubated overnight at 14° C., followed by in vitro packaging, according to standard methods (Maniatis, pp. 256-268).
The packaged phage were used to infect an E. coli hf1 strain, such as strain HG415. Alternatively, E. coli , strain C600 hf1 available from Promega Biotec, Madison, Wis., could be used. The percentage of recombinant plaques obtained with insertion of the EcoRI-ended fragments was less than 5% by analysis of 20 random plaques.
The resultant cDNA library was plated and phage were eluted from the selection plates by addition of elution buffer. After DNA extraction from the phage, the DNA was digested with EcoRI to release the heterogeneous insert population, and the DNA fragments were fractionated on agarose to remove phage fragments. The 500-4,000 basepair inserts were isolated and recloned into lambda gt10 as above, and the packaged phage was used to infect E. coli strain HG415. The percentage of successful recombinants was greater than 95%. The phage library was plated on E. coli strain HG415, at about 5,000 plaques/plate, on a total of 8 plates.
EXAMPLE 2
Selecting ET-NANB Cloned Fragments
A. CDNA Probes
Duplex cDNA fragments from noninfected and ETNANB-infected cynomolgus monkeys were prepared as in Example 1. The cDNA fragments were radiolabeled by random priming, using a random-priming labeling kit obtained from Boehringer-Mannheim (Indianapolis, Ind.).
B. Clone Selection
The plated CDNA library from Example 1 was transferred to each of two nitrocellulose filters, and the phage DNA was fixed on the filters by baking, according to standard methods (Maniatis, pp. 320323). The duplicate filters were hybridized with either infected-source or control CDNA probes from above. Autoradiographs of the filters were examined to identify library clones which hybridized with radiolabeled CDNA probes from infected source only, i.e., did not hybridize with cDNA probes from the non-infected source. Sixteen such clones, out of a total of about 40,000 clones examined, were identified by this subtraction selection method.
Each of the sixteen clones was picked and replated at low concentration on an agar plate. The clones on each plate were transferred to two nitro-cellulose ag duplicate lifts, and examined for hybridization to radiolabeled cDNA probes from infected and noninfected sources, as above. Clones were selected which showed selective binding for infected-source probes (i.e., binding with infected-source probes and substantially no binding with non-infected-source probes). One of the clones which bound selectively to probe from infected source was isolated for further study. The selected vector was identified as lambda gt10-1.1, indicated in FIG. 1 .
EXAMPLE 3
ET-NANB Sequence
Clone lambda gt10-1.1 from Example 2 was digested with EcoRI to release the heterologous insert, which was separated from the vector fragments by gel electrophoresis. The electrophoretic mobility of the fragment was consistent with a 1.33 kb fragment. This fragment, which contained EcoRI ends, was inserted into the EcoRI site of a pTZKF1 vector, whose construction and properties are described in co-owned U.S. patent application for “Cloning Vector System and Method for Rare Clone Identification”, Ser. No. 125,650, filed Nov. 25, 1987. Briefly, and as illustrated in FIG. 1, this plasmid contains a unique EcoRI site adjacent a T7 polymerase promoter site, and plasmid and phage origins of replication. The sequence immediately adjacent each side of the EcoRI site is known. E. coli BB4 bacteria, obtained from Stratagene (La Jolla, Calif., were transformed with the plasmid.
Radiolableled ET-NANB probe was prepared by excising the 1.33 kb insert from the lambda gt10-1.1 phage in Example 2, separating the fragment by gel electrophoresis, and randomly labeling as above. Bacteria transfected with the above pTZKF1 and containing the desired ET-NANB insert were selected by ET-NANB probe, according to methods outlined in Example 2.
One bacterial colony containing a successful recombinant was used for sequencing a portion of the 1.33 kb insert. This isolate, designated pTZKF1(ET1.1), has been deposited with the American Type Culture Collection, and is identified by ATCC deposit no. 67717. Using a standard dideoxy sequencing procedure, and primers for the sequences flanking the EcoRI site, about 200-250 basepairs of sequence from the 5′-end region and 3′-end region of the insert were obtained. The sequences are given above in Section II. Later sequencing by the same techniques gave the full sequence in both directions, also given above.
EXAMPLE 4
Detecting ET-NANB Sequences
cDNA fragment mixtures from the bile of noninfected and ET-NANB-infected cynomolgus monkeys were prepared as above. The cDNA fragements obtained from human stool samples were prepared as follows. Thirty mg of a 10% stool suspension gained from an individual from Mexico diagnosed as infected with ET-NANB as a result of an ET-NANB outbreak, and a similar volume of stool from a healthy, non-infected individual, were layered over a 30% sucrose density gradient cushion, and centrifuged at 25,000×g for 6 hr in an SW27 rotor, at 15° C. The pelleted material from the infected-source stool contained 27-34 nm VLP particles characteristic of ET-NANB infection in the infected-stool sample. RNA was isolated from the sucrose-gradient pellets in both the infected and non-infected samples, and the isolated RNA was used to produce cDNA fragments as described in Example 1.
The CDNA fragment mixtures from infected and non-infected bile source, and from infected and non-infected human-stool source were each amplified by a novel linker/primer replication method described in co-owned patent application Ser. No. 07/208,512 for “DNA Amplification and Subtraction Technique,” filed Jun. 17, 1988. Briefly, the fragments in each sample were blunt-ended with KNA Pol I then extracted with phenol/chloroform and precipitated with ethanol. The blunt-ended material was ligated with linkers having the following sequence (top or 5′ sequence has SEQ ID NO.21; bottom or 3′sequence has SEQ ID NO:22):







5′-GGAATTCGCGGCCGCTCG-3′


3′-TTCCTTAAGCGCCGGCGAGC-5′









The duplex fragments were digested with NruI to remove linker timers, mixed with a primer having the sequence 5′-GGAATTCGCGGCCGCTCG-3′, and then heat denatured and cooled to room temperature to form single-strand DNA/primer complexes. The complexes were replicated to form duplex fragments by addition of Thermus aquaticus (Taq) polymerase and all four deoxynucleotides. The replication procedures, involving successive strand denaturation, formation of strand/primer complexes, and replication, was repeated 25 times.
The amplified CDNA sequences were fractionated by agarose gel electrophoresis, using a 2% agarose matrix. After transfer of the DNA fragments from the agarose gels to nitrocellulose paper, the filters were hybridized to a random-labeled 32p probe prepared by (i) treating the pTZKF1(ET1.1) plasmid from above with EcoRI, (ii) isolating the released 1.33 kb ET-NANB fragment, and (iii) randomly labeling the isolated fragment. The probe hybridization wag performed by conventional Southern blotting methods (Maniatis, pp. 382-389). FIG. 2 shows the hybridization pattern obtained with cDNAs from infected (I) and non-infected (N) bile sources ( 2 A) and from infected (I) and noninfected (N) human stool sources ( 2 B). As seen, the ET-NANB probe hybridized with fragments obtained from both of the infected sources, but was non-homologous to sequences obtained from either of the non-infected sources, thus confirming the specificity of derived sequence.
Southern blots of the radiolabeled 1.33 kb fragment with genomic DNA fragments from both human and cynomolgus-monkey DNA were also prepared. No probe hybridization to either of the genomic fragment mixtures was observed, confirming that the ET-NANB sequence is exogenous to either human or cynomolgus genome.
EXAMPLE 5
Expressing ET-NANB Proteins
A. Preparing ET-NANB Coding Sequences
The pTZKF1(ET1.1) plasmid from Example 2 was digested with EcoRI to release the 1.33 kb ET-NANB insert which was purified from the linearized plasmid by gel electrophoresis. The purified fragment was suspended in a standard digest buffer (0.5 M Tris HCl, pH 7.5; 1 mg/ml BSA; 10 mM MnC12) to a concentration of about 1 mg/ml and digested with DNAse I at room temperature for about 5 minutes. These reaction conditions were determined from a prior calibration study, in which the incubation time required to produce predominantly 100-300 basepair fragments was determined. The material was extracted with phenol/chloroform before ethanol precipitation.
The fragments in the digest mixture were blunt-ended and ligated with EcoRI linkers as in Example 1. The resultant fragments were analyzed by electrophoresis (5-10 V/cm) on 1.2% agarose gel, using PhiX174/HaeIII and lambda/HindIII size markers. The 100-300 bp fraction was eluted onto NA45 strips (Schleicher and Schuell), which were then placed into 1.5 ml microtubes with eluting solution (1 M NaCl, 50 mM arginine, pH 9.0), and incubated at 67° C. for 30-60 minutes. The eluted DNA was phenol/chloroform extracted and then precipitated with two volumes of ethanol. The pellet was resuspended in 20 μl TE (0.01 M Tris HCl, pH 7.5, 0.001 M EDTA).
B. Cloning in an Expression Vector
Lambda gt11 phage vector (Huynh) was obtained from Promega Biotec (Madison, Wis.). This cloning vector has a unique EcoRI cloning site 53 base pairs upstream from the beta-galactosidase translation termination codon. The genomic fragments from above, provided either directly from coding sequences (Example 5) or after amplification of cDNA (Example 4), were introduced into the EcoRI site by mixing 0.5-1.0 μg EcoRI-cleaved gt11, 0.3-3 μl of the above sized fragments, 0.5 μl 10×ligation buffer (above), 0.5 μl ligase (200 units), and distilled water to 5 μl. The mixture was incubated overnight at 14° C., followed by in vitro packaging, according to standard methods (Maniatis, pp. 256-268).
The packaged phage were used to infect E. coli strain KM392, obtained from Dr. Kevin Moore, DNAX (Palo Alto, Calif.). Alternatively, E. Coli strain Y1090, available from the American Type Culture Collection (ATCC #37197), could be used. The infected bacteria were plated and the resultant colonies were checked for loss of beta-galactosidase activity-(clear plaques) in the presence of X-gal using a standard X-gal substrate plaque assay method (Maniatis). About 50% of the phage plaques showed loss of beta-galactosidase enzyme activity (recombinants).
C. Screening for ET-NANB Recombinant Proteins
ET-NANB convalescent antiserum was obtained from patients infected during documented ET-NANB outbreaks in Mexico, Borneo, Pakistan, Somalia, and Burma. The sera were immunoreactive with VLPs in stool specimens from each of several other patients with ET-NANB hepatitis.
A lawn of E. coli KM392 cells infected with about 104 pfu of the phage stock from above was prepared on a 150 mm plate and incubated, inverted, for 5-8 hours at 37° C. The lawn was overlaid with a nitrocellulose sheet, causing transfer of expressed ETNANB recombinant protein from the plaques to the paper. The plate and filter were indexed for matching corresponding plate and filter positions.
The filter was washed twice in TBST buffer (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20), blocked with AIB (TBST buffer with 1% gelatin), washed again in TBST, and incubated overnight after addition of antiserum (diluted to 1:50 in AIB, 12-15 ml/plate). The sheet was washed twice in TBST and then contacted with enzyme-labeled anti-human antibody to attach the labeled antibody at filter sites containing antigen recognized by the antiserum. After a final washing, the filter was developed in a substrate medium containing 33 μl NBT (50 mg/ml stock solution maintained at 4° C.) mixed with 16 μl BCIP (50 mg/ml stock solution maintained at 4° C.) in 5 ml of alkaline phosphatase buffer (100 mM Tris, 9.5, 100 mM NaCl, 5 mM MgC12). Purple color appeared at points of antigen production, as recognized by the antiserum.
D. Screening Plating
The areas of antigen production determined in the previous step were replated at about 100-200 pfu on an 82 mm plate. The above steps, beginning with a 5-8 hour incubation, through NBT-BCIP development, were repeated in order to plaque purify phage secreting an antigen capable of reacting with the ET-NANB antibody. The identified plaques were picked and eluted in phage buffer (Maniatis, p. 443).
E. Epitope Identification
A series of subclones derived from the original pTZKF1(ET1.1) plasmid from Example 2 were isolated using the same techniques described above. Each of these five subclones were immunoreactive with a pool of anti-ET antisera noted in C. The subclones contained short sequences from the “reverse” sequence set forth previously. The beginning and ending points of the sequences in the subclones (relative to the full “reverse” sequence), are identified in the table below.








TABLE 1





Position in “Reverse” Sequence






Subclone
5′-end
3′-end



Y1
522
643

Y2
594
667

Y3
508
665

Y4
558
752

Y5
545
665

























Since all of the gene sequences identified in the table must contain the coding sequence for the epitope, it is apparent that the coding sequence for the epitope falls in the region between nucleotide 594 (5′-end) and 643 (3′-end). Genetic sequences equivalent to and complementary to this relatively short sequence are therefore particularly preferred aspects of the present invention, as are peptides produced using this coding region.
A second series of clones identifying an altogether different epitope was isolated with only Mexican serum.








TABLE 2





Position in “Forward” Sequence






Subclone
5′ end
3′ end



ET 2-2
2
193

ET 8-3
2
135

ET 9-1
2
109

ET 13-1
2
101
























The coding system for this epitope falls between nucleotide 2 (S-end) and 101 (3-end). Genetic sequences related to this short sequence are therefore also preferred, as are peptides produced using this coding region.
Two particularly preferred subclones for use in preparing polypeptides containing epitopes specific for HEV are the 406.3-2 and 406.4-2 clones whose sequences are set forth above. These sequences were isolated from an amplified cDNA library derived from a Mexican stool. Using the techniques described in this section, polypeptides expressed by these clones have been tested for immunoreactivity against a number of different human HEV-positive sera obtained from sources around the world. As shown in Table 3 below, 8 sera immunoreactive with the polypeptide expressed by the 406.4-2, and 6 sera immunoreacted with polypeptide expressed by the 406.3-2 clone.
For comparison, the Table also shows reactivity of the various human sera with the Y2 clone identified in Table 1 above. Only one of the sera reacted with the polypeptide expressed by this clone. No immunoreactivity was seen for normal expression products of the gt11 vector.





TABLE 3



Immunoreactivity of HEV Recombinant

Proteins: Human Sera









Sera
Source
Stage 1
406.3-2
406.4-2
Y2
λgtll



FVH-21
Burma
A
−
−
−
−

FVH-8
Burma
A
−
+
+
−

SOM-19
Somalia
A
+
+
−
−

SOM-20
Somalia
A
+
+
−
−

IM-35
Borneo
A
+
+
−
−

IM-36
Borneo
A
−
−
−
−

PAK-1
Pakistan
A
+
+
−
−

FFI-4
Mexico
A
+
+
−
−

FFI-−125
Mexico
A
−
+
−
−

F 387 IC
Mexico
C
+
+
ND
−

Normal
U.S.A.
−
−
−
−
−



1 A = acute; C = convalescent































While the invention has been described with reference to particular embodiments, methods, construction and use, it will be apparent to those skilled in the art that various changes and modifications can be made without departing from the invention.