VIROLOGY
189, 359-362
(1992)
Indian Hepatitis
E Virus Shows a Major Deletion RATNA RAY,’ SHAHID JAMEEL,
international
VENKATASAMY
in the Small Open Reading MANIVEL,
AND RANJIT RAY*
Centre for Genetic Engineering and Biotechnology, and *National Shahid Jeet Singh Marg, New Delhi- 110067, India Received January
13, 1992; accepted
March
Frame
Institute of Immunology,
17, 1992
Enterically transmitted non-A, non-B hepatitis virus (HEV), the causative agent for sporadic and large epidemic outbreaks in developing countries, contains a positive-sense single-stranded RNA genome. The genome of the virus encodes three open reading frames (ORFl, ORF2, and ORF3). The gene segment corresponding to the small open reading frame (ORF3), overlapping between ORFl and ORF2, was synthesized by reverse transcription-polymerase chain reaction (RT-PCR) from a number of previously identified HEV-positive clinical specimens. A DNA fragment of 166 bp was consistently obtained from all the clinical specimens. This small fragment was cloned, sequenced, and found to contain an open reading frame encoding only 41 amino acid residues. Comparison of our results with that of geographically related Burma HEV suggests a major inframe deletion of 246 bp in the ORF3 of Indian strain. The protein encoded by ORF3 does not appear to be useful for early serodiagnosis as a synthetic peptide deduced from the truncated ORF3 failed to show any demonstrable immunoreactivity against HEV-infected acute phase sera in an o 1992 Academic PWS. I~C. enzyme-linked immunosorbent assay.
Enterically transmitted hepatitis virus (HEV) is associated with sporadic and large epidemics of viral hepatitis particularly in the developing countries (1-3). The virus has been shown to be serologically unrelated to infection with the more commonly described hepatitis A and B viruses (4, 53. The disease is transmitted by the fecal-oral route through contamination of food or water sources (5, 6). A high mortality rate, approaching 20%, has been noted in HEV-infected pregnant women (7, 8). A majority of the work reported so far on HEV is based on clinical and/or epidemiological studies (911). Experimental models of HEV infection were established in tamarins and cynomolgus macaques (8). Immunoelectron microscopy with acute phase sera showed aggregation of 27-34-nm diameter virus-like particles (VLPs) from stools of infected human and experimental primates (8, 9). Recently, the full-length genome of HEV has been identified and sequenced (12, 13). The viral genome contains a positive-stranded, polyadenylated RNA of approximately 7.5 kb. The nucleotide sequence of the HEV genome did not show homology with wild-type hepatitis A virus or polio virus (14). On the other hand, the HEV particle size compares quite closely to that of feline calicivirus, an etiological agent of orthopharyngitis (2, 12). Nucleotide sequence analysis of the HEV genome revealed three positive-polarity open reading frames (ORF). A portion of ORFl appears to encode the RNA-dependent RNA polymerase gene of the virus.
The highly conserved amino acid residues in ORFl, including the invariant Gly-Asp-Asp (GDD) tripeptide found in all positive-strand animal and plant RNA viruses, can be located in the deduced amino acid sequence (12, 13). The deduced amino acid sequence of ORF2 suggests that it encodes a capsid-like protein following the canonical signal sequence at the 5’ end (13). ORF2 appeared to be the major open reading frame encoding the viral structural protein(s). The third open reading frame, ORF3, was found to overlap between ORFl and ORF2. An immunoreactive epitope was identified from ORF3 using infected human and monkey sera (15). Similar to the feline calicivirus (FCV), nonstructural genes are located in the 5’ direction relative to the structural genes in HEV (16). However, there is no sequence homology between the small ORF of FCV and HEV. The small ORF of calicivlrus encodes a polypeptide of approximately 10 kDa which is covalently attached to the genomic RNA. This polypeptide was thought to be important for viral infectivity (17). We initiated studies on the small ORF (ORF3) of HEV to determine its structure and functional activity. A synthetic peptide corresponding to ORF3 was also tested to determine its immunoreactivity and to explore the possibility for use in early serodiagnosis of HEV infection. Human stool samples were collected from two epidemics of water-borne hepatitis during September to October, 1990, in Hyderabad, and January to April, 199 1, in Kanpur. Authencity of the virus infection was diagnosed from the presence of hepatitis E virus putative polymerase gene sequence (12) in clinical specimens by reverse transcription-polymerase chain reac-
’ To whom correspondence and reprint requests should be addressed at Department of Hematology/Oncology, University of Alabama at Birmingham, 1918 Unlverslty Blvd., Birmingham, AL 35294. 359
0042.6822/92
$5.00
Copyright : 1992 by Academic Press. Inc All rights of reproductun in any form resewed
360
SHORT COMMUNICATIONS
tion (RT-PCR) (3). HEV-positive human stool samples were separately transmitted in rhesus monkeys (n/lacaca mulatta). Monkeys showed rise in serum transaminase level and presence of HEV putative polymerase gene sequence in the bile and stool. Six HEV-positive human stool samples from the Kanpur epidemic, two stool samples from monkeys transmitted with the virus from the Kanpur epidemic, one stool sample from a monkey transmitted with the virus from the Hyderabad epidemic, and two bile samples from monkeys transmitted with the virus from the Kanpur and Hyderabad epidemics were used in this study. Five human stool samples thought to be uninfected, stool from three uninfected monkeys, and bile from two uninfected monkeys were also included as negative control in this study. RNA from stool sample was isolated using the acid guanidinium thiocyanate-phenol-chloroform extraction method (18). For RNA extraction from bile, 25 ~1 of bile was mixed with 225 ~1 solution containing 10 mMTris, pH 8.0, 5 mNI EDTA, 0.5% SDS, 10 pg tRNA, and 100 pg proteinase K and incubated at 55” for 45 min. An equal volume of phenol-chloroform (1 :l) was added to the mixture and incubated at 65” for 15 min. RNA was precipitated with ethanol. The pelleted RNA was rinsed with 70% ethanol, dried, and used for amplification by RT-PCR. First-strand cDNA was synthesized using a random hexanucleotide primer and murine leukemia virus reverse transcriptase following the supplier’s procedure (Bethesda Research Laboratory, MD). cDNA was precipitated along with 1 pg of carrier tRNA. The pelleted DNA was dried and suspended in 20 ~1of water. PCR was performed using a gene amplification kit (Perkin-Elmer Cetus, CT) and a pair of sense Pl (5’ AGGCTGTTGCTGATGGCAAGGCACA 3’) and antisense P2 (5’ACATCAGGCACTGGCGGGGTGTCAT 3’) oligonucleotide primers at nucleotide positions 5021 and 5495, respectively (13). A 5-p.1 cDNA was used for PCR amplification in a total reaction volume of 50 ~1 containing 10 mMTris-HCI (pH 8) 50 PAI! KCI; 1.5 mn/l MgCI,, 200 mlL/I of each of the four dNTPs; 30 pmol of sense (Pl) and antisense (P2) primers; and 1.5 unit of Taq DNA polymerase. The amplification reaction was performed by using an automatic thermocycler (Perkin-Elmer Cetus) for 30 cycles of denaturation at 94” for 1 min, primer annealing at 45” for 1.5 min, and primer extension at 72” for 2 min. In the second round of PCR, 5 ~1 of the first PCR product was amplified as above except the primers were internal sense RR2 (5’ ATTCAAGCTTGTGTCGGGTGG 3’) and antisense RR3 (5’ GGCCGGAGGGATCCGCGGTTA 3’) at nucleotide positions 5087 and 5476, respectively (13). Underlined sequences represent restriction enzyme sites created in the primerfor unidirec-
x45
6
7 8910.
FIG. 1. Amplification of ORF3 DNA from experimental samples by RT-PCR. Amplified products from HEV-positive bile (lanes 1 and 2) and stool (lanes 5 and 6) of transmitted monkeys, and five human stool samples (lanes 8, 9, 10, 1 1, and 12) of hepatitis patients were analyzed by 1.5% agarose gel electrophoresis followed by ethidium bromide staining. Amplified products from bile (lanes 3 and 4) and stool (lane 7) of uninfected monkeys, and human stool samples thought to be HEV negative (lanes 13 and 14) are also shown as negative control In RT-PCR amplification. Molecular weight marker pBR322/Mspl digest (M) IS shown on the left. The arrow indicates the position of 166 bp-amplified DNA fragment.
tional cloning. The PCR products were analyzed by 1.5% agarose or 5% polyacrylamide gel electrophoresis and the results are illustrated in Fig. 1. The amplified 166-bp DNA band was electroeluted and doubly digested with HindIll and BarnHI restriction enzymes for cloning into pGEM4 plasmid vector. The resulting clones were analyzed for nucleotide and predicted amino acid sequences. Sequence analyses were performed with the clones generated from two HEV-positive human stool samples from the Kanpur epidemic, two stool samples from monkeys transmitted with the virus from the Kanpur epidemic, and from bile of two monkeys transmitted with the virus from the Hyderabad and Kanpur epidemics. Results suggested that all the six clones contain an ORF of 123 nucleotide encoding 41 amino acid residues (Fig. 2). Comparison of our results with the known Burmese HEV sequence (13) shows that the nucleotide sequence at 5’ and 3’ ends of the clones match exactly, although a major deletion of 246 bp (nucleotide position 5187 to 5433) was observed in the Indian HEV genome. Secondary structure analysis using the DNASIS program (LKB/ Pharmacia, Sweden) of Burmese HEV sequence does not show any unusual stem or loop structure. An internal oligonucleotide RR4 (5’ mGCCTATGCTGCCCGCGC 3’) from the Burma HEV sequence (13) was also synthesized and PCR amplification performed using RR4 and RR3 primers. However, amplification was
361
SHORT COMMUNICATIONS
1 1
AG
CTT GTG TCG GGT GGG ATG AAT AAC ATG TCT TTT M N N M S F
30 10
ATG GGT TCG CGA CCA TGC GCC CTC GGC CTA TTT M G S R P C A L G L F
75 25
TCA TGT TTC TTG CCT CAC GTC GTA GAC CTA CCG CAG CTG GGG CCG S C F L P H V V D L P Q L G P
130 40
CGC CGC TAA CCG CGG ATC R R *
Bl 11
MNNMSFAAPMGSRPCALGLFCCCSSCFCLC MNNMSFAAPMGSRPCALGLFCCCSSCF---
B31
CPRHRPVSRLAAVVGGAAAVPAVVSGVPGL ------------------------------
B61
ILSPSQSPIFIQPTPSPPMSPLRPGLDLVF ------__----------------------
B91
128
ANPPDHSAPLGVPRPSAPPLPHVVDLPQLG -----------v---v--
B121 139
PRR* PRR*
GCT GCG CCC A A P
TGT TGC TGT TCC C C C S
-LPHVVDLPQLG
FIG. 2. Sequence analysis of amplified DNA fragments. (A) Nucleotide sequence and predicted amino acid sequence of ORF3 from Indian HEV. The amplified DNA fragment was dlgested with HindIll and BarnHI and cloned Into pGEM4 vector, The resulted clone was sequenced by dideoxy chain termination method using SP6 and T7 primers. Six clones generated from different HEV-posltlve samples showed identical nucleotide sequence. (B) Amino acid sequence alignment for ORF3 from Burmese (6) and Indian (I) HEV strains
not observed under various PCR conditions. This further suggested the absence of the RR4 nucleotide sequence in the viral genome of Indian HEV strains. A major deletion consistently observed in the ORF3 of Indian HEV by RT-PCR under careful experimental conditions does not appear to be an artifact of PCR amplification. Further indirect evidences of inframe deletion from sequence analysis and failure to amplify by PCR using synthetic oligonucleotide primers from the deleted region (RR4) and RR3 support our experimental observation. A 41 -mer peptide corresponding to the ORF3 of Indian HEV was synthesized by stepwise solid phase procedure using a Model 430A peptide synthesizer (Applied Biosystems). Purified peptide was analyzed by 15% SDS-polyacrylamide gel electrophoresis. A Coomassie blue-stained single band protein similar to that of the calculated molecular weight of the peptide was obtained under both reducing and nonreducing conditions. Polyvinyl plates (Greiner, Germany) were coated with 2 pg of synthetic peptide per well in 100 ~1 of phosphate-buffered saline (PBS), pH 7.2, by incubation
overnight at 4”. After three washes with PBS containing 1% Tween 20, the plates were filled with 1% bovine serum albumin (Sigma Chemical Co., St. Louis, MO) in PBS for 3 hr at 37” to block the remaining unadsorbed sites. Next, 100 ~1 of twofold dilutions of sera was added to the peptide-coated well and incubated overnight at 4’. The plates were washed three times and an appropriate dilution (determined by checkerboard titration) of goat antibody to human IgM (u chain specific) or IgG conjugated to horseradish peroxidase (Sigma) was added to each well and incubated for 2 hr at 37”. The plates were washed and 100 ~1 of ortho-phenylene-diamine dihydrochloride (Sigma) in 0.15 M citratephosphate buffer, pH 5.0, containing 0.0006% hydrogen peroxide was added. After incubation for 30 min the reaction was stopped by the addition of 2 Nsulfuric acid and the color reaction read at wavelength 490 nm with a spectrophotometer (Fisher Scientific, Springfield, NJ). Absorbance values of negative control wells (reagent blank) were substracted, and the end point was determined by the highest dilution of the test material showing an OD of at least 0.5. A number of HEV-in-
362
SHORT COMMUNICATIONS TABLE 1
PEPTIDE-SPECIFICANTIBODY RESPONSEIN HEV-INFECTED SEFIABY ELISA ELISA Titer Sera Infected human sera (Acute phase) 117 201 207 208 601 801 811 Infected monkey sera (Acute phase) 100 141 Infected monkey sera (Convalescent phase) 100 141 Preimmune monkey sera 100 141
W
IgM
189, 359-362
(1992)
Indian Hepatitis
E Virus Shows a Major Deletion RATNA RAY,’ SHAHID JAMEEL,
international
VENKATASAMY
in the Small Open Reading MANIVEL,
AND RANJIT RAY*
Centre for Genetic Engineering and Biotechnology, and *National Shahid Jeet Singh Marg, New Delhi- 110067, India Received January
13, 1992; accepted
March
Frame
Institute of Immunology,
17, 1992
Enterically transmitted non-A, non-B hepatitis virus (HEV), the causative agent for sporadic and large epidemic outbreaks in developing countries, contains a positive-sense single-stranded RNA genome. The genome of the virus encodes three open reading frames (ORFl, ORF2, and ORF3). The gene segment corresponding to the small open reading frame (ORF3), overlapping between ORFl and ORF2, was synthesized by reverse transcription-polymerase chain reaction (RT-PCR) from a number of previously identified HEV-positive clinical specimens. A DNA fragment of 166 bp was consistently obtained from all the clinical specimens. This small fragment was cloned, sequenced, and found to contain an open reading frame encoding only 41 amino acid residues. Comparison of our results with that of geographically related Burma HEV suggests a major inframe deletion of 246 bp in the ORF3 of Indian strain. The protein encoded by ORF3 does not appear to be useful for early serodiagnosis as a synthetic peptide deduced from the truncated ORF3 failed to show any demonstrable immunoreactivity against HEV-infected acute phase sera in an o 1992 Academic PWS. I~C. enzyme-linked immunosorbent assay.
Enterically transmitted hepatitis virus (HEV) is associated with sporadic and large epidemics of viral hepatitis particularly in the developing countries (1-3). The virus has been shown to be serologically unrelated to infection with the more commonly described hepatitis A and B viruses (4, 53. The disease is transmitted by the fecal-oral route through contamination of food or water sources (5, 6). A high mortality rate, approaching 20%, has been noted in HEV-infected pregnant women (7, 8). A majority of the work reported so far on HEV is based on clinical and/or epidemiological studies (911). Experimental models of HEV infection were established in tamarins and cynomolgus macaques (8). Immunoelectron microscopy with acute phase sera showed aggregation of 27-34-nm diameter virus-like particles (VLPs) from stools of infected human and experimental primates (8, 9). Recently, the full-length genome of HEV has been identified and sequenced (12, 13). The viral genome contains a positive-stranded, polyadenylated RNA of approximately 7.5 kb. The nucleotide sequence of the HEV genome did not show homology with wild-type hepatitis A virus or polio virus (14). On the other hand, the HEV particle size compares quite closely to that of feline calicivirus, an etiological agent of orthopharyngitis (2, 12). Nucleotide sequence analysis of the HEV genome revealed three positive-polarity open reading frames (ORF). A portion of ORFl appears to encode the RNA-dependent RNA polymerase gene of the virus.
The highly conserved amino acid residues in ORFl, including the invariant Gly-Asp-Asp (GDD) tripeptide found in all positive-strand animal and plant RNA viruses, can be located in the deduced amino acid sequence (12, 13). The deduced amino acid sequence of ORF2 suggests that it encodes a capsid-like protein following the canonical signal sequence at the 5’ end (13). ORF2 appeared to be the major open reading frame encoding the viral structural protein(s). The third open reading frame, ORF3, was found to overlap between ORFl and ORF2. An immunoreactive epitope was identified from ORF3 using infected human and monkey sera (15). Similar to the feline calicivirus (FCV), nonstructural genes are located in the 5’ direction relative to the structural genes in HEV (16). However, there is no sequence homology between the small ORF of FCV and HEV. The small ORF of calicivlrus encodes a polypeptide of approximately 10 kDa which is covalently attached to the genomic RNA. This polypeptide was thought to be important for viral infectivity (17). We initiated studies on the small ORF (ORF3) of HEV to determine its structure and functional activity. A synthetic peptide corresponding to ORF3 was also tested to determine its immunoreactivity and to explore the possibility for use in early serodiagnosis of HEV infection. Human stool samples were collected from two epidemics of water-borne hepatitis during September to October, 1990, in Hyderabad, and January to April, 199 1, in Kanpur. Authencity of the virus infection was diagnosed from the presence of hepatitis E virus putative polymerase gene sequence (12) in clinical specimens by reverse transcription-polymerase chain reac-
’ To whom correspondence and reprint requests should be addressed at Department of Hematology/Oncology, University of Alabama at Birmingham, 1918 Unlverslty Blvd., Birmingham, AL 35294. 359
0042.6822/92
$5.00
Copyright : 1992 by Academic Press. Inc All rights of reproductun in any form resewed
360
SHORT COMMUNICATIONS
tion (RT-PCR) (3). HEV-positive human stool samples were separately transmitted in rhesus monkeys (n/lacaca mulatta). Monkeys showed rise in serum transaminase level and presence of HEV putative polymerase gene sequence in the bile and stool. Six HEV-positive human stool samples from the Kanpur epidemic, two stool samples from monkeys transmitted with the virus from the Kanpur epidemic, one stool sample from a monkey transmitted with the virus from the Hyderabad epidemic, and two bile samples from monkeys transmitted with the virus from the Kanpur and Hyderabad epidemics were used in this study. Five human stool samples thought to be uninfected, stool from three uninfected monkeys, and bile from two uninfected monkeys were also included as negative control in this study. RNA from stool sample was isolated using the acid guanidinium thiocyanate-phenol-chloroform extraction method (18). For RNA extraction from bile, 25 ~1 of bile was mixed with 225 ~1 solution containing 10 mMTris, pH 8.0, 5 mNI EDTA, 0.5% SDS, 10 pg tRNA, and 100 pg proteinase K and incubated at 55” for 45 min. An equal volume of phenol-chloroform (1 :l) was added to the mixture and incubated at 65” for 15 min. RNA was precipitated with ethanol. The pelleted RNA was rinsed with 70% ethanol, dried, and used for amplification by RT-PCR. First-strand cDNA was synthesized using a random hexanucleotide primer and murine leukemia virus reverse transcriptase following the supplier’s procedure (Bethesda Research Laboratory, MD). cDNA was precipitated along with 1 pg of carrier tRNA. The pelleted DNA was dried and suspended in 20 ~1of water. PCR was performed using a gene amplification kit (Perkin-Elmer Cetus, CT) and a pair of sense Pl (5’ AGGCTGTTGCTGATGGCAAGGCACA 3’) and antisense P2 (5’ACATCAGGCACTGGCGGGGTGTCAT 3’) oligonucleotide primers at nucleotide positions 5021 and 5495, respectively (13). A 5-p.1 cDNA was used for PCR amplification in a total reaction volume of 50 ~1 containing 10 mMTris-HCI (pH 8) 50 PAI! KCI; 1.5 mn/l MgCI,, 200 mlL/I of each of the four dNTPs; 30 pmol of sense (Pl) and antisense (P2) primers; and 1.5 unit of Taq DNA polymerase. The amplification reaction was performed by using an automatic thermocycler (Perkin-Elmer Cetus) for 30 cycles of denaturation at 94” for 1 min, primer annealing at 45” for 1.5 min, and primer extension at 72” for 2 min. In the second round of PCR, 5 ~1 of the first PCR product was amplified as above except the primers were internal sense RR2 (5’ ATTCAAGCTTGTGTCGGGTGG 3’) and antisense RR3 (5’ GGCCGGAGGGATCCGCGGTTA 3’) at nucleotide positions 5087 and 5476, respectively (13). Underlined sequences represent restriction enzyme sites created in the primerfor unidirec-
x45
6
7 8910.
FIG. 1. Amplification of ORF3 DNA from experimental samples by RT-PCR. Amplified products from HEV-positive bile (lanes 1 and 2) and stool (lanes 5 and 6) of transmitted monkeys, and five human stool samples (lanes 8, 9, 10, 1 1, and 12) of hepatitis patients were analyzed by 1.5% agarose gel electrophoresis followed by ethidium bromide staining. Amplified products from bile (lanes 3 and 4) and stool (lane 7) of uninfected monkeys, and human stool samples thought to be HEV negative (lanes 13 and 14) are also shown as negative control In RT-PCR amplification. Molecular weight marker pBR322/Mspl digest (M) IS shown on the left. The arrow indicates the position of 166 bp-amplified DNA fragment.
tional cloning. The PCR products were analyzed by 1.5% agarose or 5% polyacrylamide gel electrophoresis and the results are illustrated in Fig. 1. The amplified 166-bp DNA band was electroeluted and doubly digested with HindIll and BarnHI restriction enzymes for cloning into pGEM4 plasmid vector. The resulting clones were analyzed for nucleotide and predicted amino acid sequences. Sequence analyses were performed with the clones generated from two HEV-positive human stool samples from the Kanpur epidemic, two stool samples from monkeys transmitted with the virus from the Kanpur epidemic, and from bile of two monkeys transmitted with the virus from the Hyderabad and Kanpur epidemics. Results suggested that all the six clones contain an ORF of 123 nucleotide encoding 41 amino acid residues (Fig. 2). Comparison of our results with the known Burmese HEV sequence (13) shows that the nucleotide sequence at 5’ and 3’ ends of the clones match exactly, although a major deletion of 246 bp (nucleotide position 5187 to 5433) was observed in the Indian HEV genome. Secondary structure analysis using the DNASIS program (LKB/ Pharmacia, Sweden) of Burmese HEV sequence does not show any unusual stem or loop structure. An internal oligonucleotide RR4 (5’ mGCCTATGCTGCCCGCGC 3’) from the Burma HEV sequence (13) was also synthesized and PCR amplification performed using RR4 and RR3 primers. However, amplification was
361
SHORT COMMUNICATIONS
1 1
AG
CTT GTG TCG GGT GGG ATG AAT AAC ATG TCT TTT M N N M S F
30 10
ATG GGT TCG CGA CCA TGC GCC CTC GGC CTA TTT M G S R P C A L G L F
75 25
TCA TGT TTC TTG CCT CAC GTC GTA GAC CTA CCG CAG CTG GGG CCG S C F L P H V V D L P Q L G P
130 40
CGC CGC TAA CCG CGG ATC R R *
Bl 11
MNNMSFAAPMGSRPCALGLFCCCSSCFCLC MNNMSFAAPMGSRPCALGLFCCCSSCF---
B31
CPRHRPVSRLAAVVGGAAAVPAVVSGVPGL ------------------------------
B61
ILSPSQSPIFIQPTPSPPMSPLRPGLDLVF ------__----------------------
B91
128
ANPPDHSAPLGVPRPSAPPLPHVVDLPQLG -----------v---v--
B121 139
PRR* PRR*
GCT GCG CCC A A P
TGT TGC TGT TCC C C C S
-LPHVVDLPQLG
FIG. 2. Sequence analysis of amplified DNA fragments. (A) Nucleotide sequence and predicted amino acid sequence of ORF3 from Indian HEV. The amplified DNA fragment was dlgested with HindIll and BarnHI and cloned Into pGEM4 vector, The resulted clone was sequenced by dideoxy chain termination method using SP6 and T7 primers. Six clones generated from different HEV-posltlve samples showed identical nucleotide sequence. (B) Amino acid sequence alignment for ORF3 from Burmese (6) and Indian (I) HEV strains
not observed under various PCR conditions. This further suggested the absence of the RR4 nucleotide sequence in the viral genome of Indian HEV strains. A major deletion consistently observed in the ORF3 of Indian HEV by RT-PCR under careful experimental conditions does not appear to be an artifact of PCR amplification. Further indirect evidences of inframe deletion from sequence analysis and failure to amplify by PCR using synthetic oligonucleotide primers from the deleted region (RR4) and RR3 support our experimental observation. A 41 -mer peptide corresponding to the ORF3 of Indian HEV was synthesized by stepwise solid phase procedure using a Model 430A peptide synthesizer (Applied Biosystems). Purified peptide was analyzed by 15% SDS-polyacrylamide gel electrophoresis. A Coomassie blue-stained single band protein similar to that of the calculated molecular weight of the peptide was obtained under both reducing and nonreducing conditions. Polyvinyl plates (Greiner, Germany) were coated with 2 pg of synthetic peptide per well in 100 ~1 of phosphate-buffered saline (PBS), pH 7.2, by incubation
overnight at 4”. After three washes with PBS containing 1% Tween 20, the plates were filled with 1% bovine serum albumin (Sigma Chemical Co., St. Louis, MO) in PBS for 3 hr at 37” to block the remaining unadsorbed sites. Next, 100 ~1 of twofold dilutions of sera was added to the peptide-coated well and incubated overnight at 4’. The plates were washed three times and an appropriate dilution (determined by checkerboard titration) of goat antibody to human IgM (u chain specific) or IgG conjugated to horseradish peroxidase (Sigma) was added to each well and incubated for 2 hr at 37”. The plates were washed and 100 ~1 of ortho-phenylene-diamine dihydrochloride (Sigma) in 0.15 M citratephosphate buffer, pH 5.0, containing 0.0006% hydrogen peroxide was added. After incubation for 30 min the reaction was stopped by the addition of 2 Nsulfuric acid and the color reaction read at wavelength 490 nm with a spectrophotometer (Fisher Scientific, Springfield, NJ). Absorbance values of negative control wells (reagent blank) were substracted, and the end point was determined by the highest dilution of the test material showing an OD of at least 0.5. A number of HEV-in-
362
SHORT COMMUNICATIONS TABLE 1
PEPTIDE-SPECIFICANTIBODY RESPONSEIN HEV-INFECTED SEFIABY ELISA ELISA Titer Sera Infected human sera (Acute phase) 117 201 207 208 601 801 811 Infected monkey sera (Acute phase) 100 141 Infected monkey sera (Convalescent phase) 100 141 Preimmune monkey sera 100 141
W
IgM
