Nine sets of nested PCR primers from a 2.6-kb region of the hepatitis G virus (HGV) genome at nuc... more Nine sets of nested PCR primers from a 2.6-kb region of the hepatitis G virus (HGV) genome at nucleotide positions 5829 to 8421 were designed and used to analyze serum specimens obtained from patients with community-acquired non-A, non-B hepatitis who were HGV RNA positive. One set of primers was found to be most efficient in detecting HGV and was subsequently used to test 162 HCV-positive and 11 HCV-negative plasma units obtained from individual paid donors. HGV RNA was detected in 30 (17.3%) plasma units, 2 of which were found among the 11 HCV-negative specimens. A complete set of nine PCR fragments was obtained from two patients with community-acquired acute non-A, non-B hepatitis and from four paid donors. All PCR fragments were sequenced and were shown to have a nucleotide similarity of 85.9 to 92.3% and a derived amino acid similarity of 96.0 to 99.0%. The majority of nucleotide changes occurred in the third position of codons. The HGV nucleotide and protein sequences obtained...
A nonradioactive hybridization assay for the detection of hepatitis delta virus RNA in serum is d... more A nonradioactive hybridization assay for the detection of hepatitis delta virus RNA in serum is described. This assay utilizes a digoxigenin-labeled RNA hybridization probe and chemiluminescent immunodetection. The probe can detect as little as 0.4 pg of cDNA with a 60-min exposure. Results obtained are in agreement with serological indicators of hepatitis delta virus infection and are comparable to those obtained by hybridization assays employing radioactively labeled RNA.
Hepatitis delta virus, discovered in 1977, requires the help of hepatitis B virus to replicate in... more Hepatitis delta virus, discovered in 1977, requires the help of hepatitis B virus to replicate in hepatocytes and is an important cause of acute, fulminant, and chronic liver disease in many regions of the world. Because of the helper function of hepatitis delta virus, infection with it occurs either as a coinfection with hepatitis B or as a superinfection of a carrier of hepatitis B surface antigen. Although the mechanisms of transmission are similar to those of hepatitis B virus, the patterns of transmission of delta virus vary widely around the world. In regions of the world in which hepatitis delta virus infection is not endemic, the disease is confined to groups at high risk of acquiring hepatitis B infection and high-risk hepatitis B carriers. Because of the propensity of this viral infection to cause fulminant as well as chronic liver disease, continued incursion of hepatitis delta virus into areas of the world where persistent hepatitis B infection is endemic will have serio...
A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatit... more A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by us...
A modified micro solid-phase radioimmunoassay (RIA) for antibody to hepatitis A virus (anti-HAV) ... more A modified micro solid-phase radioimmunoassay (RIA) for antibody to hepatitis A virus (anti-HAV) was developed. This double antibody procedure was performed by coating the surface of a polyvinyl microtiter plate "well" with 200 microliter of a 1:1,000 dilution of a patient's test serum. Purified HAV and 125I-labeled immunoglobulin G (IgG) anti-HAV were then sequentially added to form an antibody sandwich. The specificity and sensitivity of the RIA procedure for anti-HAV were verified by examination of coded human and chimpanzee serum specimens. Radioimmunoassay of early-acute-phase serum specimens from human cases of hepatitis A revealed the presence of anti-HAV activity. Differential examination by RIA of IgG and IgM fractions of acute-phase sera from experimentally infected chimpanzees demonstrated that IgM contained the bulk of the anti-HAV activity. A modification of the RIA procedure for anti-HAV (RIA-IgM blocking), incorporating an incubation step with anti-IgM (...
A competitive binding radioimmunoassay (CBA) for antibody to hepatitis A virus (HAV) was evaluate... more A competitive binding radioimmunoassay (CBA) for antibody to hepatitis A virus (HAV) was evaluated and compared with a standard solid-phase radioimmunoassay for anti-HAV, CBA was found to be sensitive and specific for the detection of anti-HAV, as demonstrated by the 98% concordance of CBA and solid-phase radioimmunoassay test results. The standard CBA test was modified for the differential detection of acute (immunoglobulin M) and convalescent (immunoglobulin G) anti-HAV by incorporation of a step in which immunoglobulin G anti-HAV was preferentially absorbed with S. aureus cells (protein A). The modified CBA test was shown to be capable of differentiating between acute- and convalescent-phase sera. The modified CBAM test was able to detect immunoglobulin M anti-HAV up to approximately 4 weeks after the onset of illness.
Hepatitis E is recognized as a zoonosis, and swine are known reservoirs, but how broadly enzootic... more Hepatitis E is recognized as a zoonosis, and swine are known reservoirs, but how broadly enzootic its causative agent, hepatitis E virus (HEV), is remains controversial. To determine the prevalence of HEV infection in animals, a serological assay with capability to detect anti-HEV-antibody across a wide variety of animal species was devised. Recombinant antigens comprising truncated capsid proteins generated from HEV-subgenomic constructs that represent all four viral genotypes were used to capture anti-HEV in the test sample and as an analyte reporter. To facilitate development and validation of the assay, serum samples were assembled from blood donors ( n = 372), acute hepatitis E patients ( n = 94), five laboratory animals (rhesus monkey, pig, New Zealand rabbit, Wistar rat, and BALB/c mouse) immunized with HEV antigens, and four pigs experimentally infected with HEV. The assay was then applied to 4,936 sera collected from 35 genera of animals that were wild, feral, domesticated,...
Milligram amounts of highly purified hepatitis A virus (HAV) were obtained from persistently infe... more Milligram amounts of highly purified hepatitis A virus (HAV) were obtained from persistently infected cell cultures. The HAV polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for detection by an enzyme-linked immunotransfer blot procedure. The HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. Four of six anti-HAV peptide sera were strongly reactive. Antipeptide serum generated against amino acids (a.a.) 75 through 82 reacted with the 27,000-molecular-weight (MW) polypeptide; serum against a.a. 279 through 285 reacted with the 29,000-MW HAV polypeptide; and sera against a.a. 591 through 602 and 606 through 618 reacted with the 33,000-M...
The nucleotide sequence and genome organization of a new member of Caliciviridae was determined. ... more The nucleotide sequence and genome organization of a new member of Caliciviridae was determined. Cell culture inoculated with fecal matter from walrus was used to recover fragments of a new virus by Suppression Subtractive Hybridization (SSH). The isolate was identified as a member of the Vesivirus genus of Caliciviridae and designated the name Walrus Calicivirus (WCV). Sets of PCR primers spanning the entire putative genome were designed using known sequences of other vesiviruses. The assembled genome was 8289 nucleotides (nt) long and shared no more than 87% identity with sequences of the other members of the genus Vesivirus. The largest open reading frame (ORF1) between positions 4-5646 encoded a polyprotein. ORF2, found at position 5652-7778, encoded a putative capsid protein. ORF3 overlapped ORF2 and encoded a small basic protein. Comparative analysis of multiple caliciviral capsid proteins was performed to propose a uniform capsid structural organization for this viral family.
Nine sets of nested PCR primers from a 2.6-kb region of the hepatitis G virus (HGV) genome at nuc... more Nine sets of nested PCR primers from a 2.6-kb region of the hepatitis G virus (HGV) genome at nucleotide positions 5829 to 8421 were designed and used to analyze serum specimens obtained from patients with community-acquired non-A, non-B hepatitis who were HGV RNA positive. One set of primers was found to be most efficient in detecting HGV and was subsequently used to test 162 HCV-positive and 11 HCV-negative plasma units obtained from individual paid donors. HGV RNA was detected in 30 (17.3%) plasma units, 2 of which were found among the 11 HCV-negative specimens. A complete set of nine PCR fragments was obtained from two patients with community-acquired acute non-A, non-B hepatitis and from four paid donors. All PCR fragments were sequenced and were shown to have a nucleotide similarity of 85.9 to 92.3% and a derived amino acid similarity of 96.0 to 99.0%. The majority of nucleotide changes occurred in the third position of codons. The HGV nucleotide and protein sequences obtained...
A nonradioactive hybridization assay for the detection of hepatitis delta virus RNA in serum is d... more A nonradioactive hybridization assay for the detection of hepatitis delta virus RNA in serum is described. This assay utilizes a digoxigenin-labeled RNA hybridization probe and chemiluminescent immunodetection. The probe can detect as little as 0.4 pg of cDNA with a 60-min exposure. Results obtained are in agreement with serological indicators of hepatitis delta virus infection and are comparable to those obtained by hybridization assays employing radioactively labeled RNA.
Hepatitis delta virus, discovered in 1977, requires the help of hepatitis B virus to replicate in... more Hepatitis delta virus, discovered in 1977, requires the help of hepatitis B virus to replicate in hepatocytes and is an important cause of acute, fulminant, and chronic liver disease in many regions of the world. Because of the helper function of hepatitis delta virus, infection with it occurs either as a coinfection with hepatitis B or as a superinfection of a carrier of hepatitis B surface antigen. Although the mechanisms of transmission are similar to those of hepatitis B virus, the patterns of transmission of delta virus vary widely around the world. In regions of the world in which hepatitis delta virus infection is not endemic, the disease is confined to groups at high risk of acquiring hepatitis B infection and high-risk hepatitis B carriers. Because of the propensity of this viral infection to cause fulminant as well as chronic liver disease, continued incursion of hepatitis delta virus into areas of the world where persistent hepatitis B infection is endemic will have serio...
A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatit... more A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by us...
A modified micro solid-phase radioimmunoassay (RIA) for antibody to hepatitis A virus (anti-HAV) ... more A modified micro solid-phase radioimmunoassay (RIA) for antibody to hepatitis A virus (anti-HAV) was developed. This double antibody procedure was performed by coating the surface of a polyvinyl microtiter plate "well" with 200 microliter of a 1:1,000 dilution of a patient's test serum. Purified HAV and 125I-labeled immunoglobulin G (IgG) anti-HAV were then sequentially added to form an antibody sandwich. The specificity and sensitivity of the RIA procedure for anti-HAV were verified by examination of coded human and chimpanzee serum specimens. Radioimmunoassay of early-acute-phase serum specimens from human cases of hepatitis A revealed the presence of anti-HAV activity. Differential examination by RIA of IgG and IgM fractions of acute-phase sera from experimentally infected chimpanzees demonstrated that IgM contained the bulk of the anti-HAV activity. A modification of the RIA procedure for anti-HAV (RIA-IgM blocking), incorporating an incubation step with anti-IgM (...
A competitive binding radioimmunoassay (CBA) for antibody to hepatitis A virus (HAV) was evaluate... more A competitive binding radioimmunoassay (CBA) for antibody to hepatitis A virus (HAV) was evaluated and compared with a standard solid-phase radioimmunoassay for anti-HAV, CBA was found to be sensitive and specific for the detection of anti-HAV, as demonstrated by the 98% concordance of CBA and solid-phase radioimmunoassay test results. The standard CBA test was modified for the differential detection of acute (immunoglobulin M) and convalescent (immunoglobulin G) anti-HAV by incorporation of a step in which immunoglobulin G anti-HAV was preferentially absorbed with S. aureus cells (protein A). The modified CBA test was shown to be capable of differentiating between acute- and convalescent-phase sera. The modified CBAM test was able to detect immunoglobulin M anti-HAV up to approximately 4 weeks after the onset of illness.
Hepatitis E is recognized as a zoonosis, and swine are known reservoirs, but how broadly enzootic... more Hepatitis E is recognized as a zoonosis, and swine are known reservoirs, but how broadly enzootic its causative agent, hepatitis E virus (HEV), is remains controversial. To determine the prevalence of HEV infection in animals, a serological assay with capability to detect anti-HEV-antibody across a wide variety of animal species was devised. Recombinant antigens comprising truncated capsid proteins generated from HEV-subgenomic constructs that represent all four viral genotypes were used to capture anti-HEV in the test sample and as an analyte reporter. To facilitate development and validation of the assay, serum samples were assembled from blood donors ( n = 372), acute hepatitis E patients ( n = 94), five laboratory animals (rhesus monkey, pig, New Zealand rabbit, Wistar rat, and BALB/c mouse) immunized with HEV antigens, and four pigs experimentally infected with HEV. The assay was then applied to 4,936 sera collected from 35 genera of animals that were wild, feral, domesticated,...
Milligram amounts of highly purified hepatitis A virus (HAV) were obtained from persistently infe... more Milligram amounts of highly purified hepatitis A virus (HAV) were obtained from persistently infected cell cultures. The HAV polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose for detection by an enzyme-linked immunotransfer blot procedure. The HAV nucleotide-derived amino acid sequence was subjected to computer analysis to identify potential immunogenic regions within the HAV capsid polypeptides. Synthetic peptides corresponding to selected regions of each of the larger putative capsid polypeptides were coupled to keyhole limpet hemocyanin and used to immunize rabbits. Four of six anti-HAV peptide sera were strongly reactive. Antipeptide serum generated against amino acids (a.a.) 75 through 82 reacted with the 27,000-molecular-weight (MW) polypeptide; serum against a.a. 279 through 285 reacted with the 29,000-MW HAV polypeptide; and sera against a.a. 591 through 602 and 606 through 618 reacted with the 33,000-M...
The nucleotide sequence and genome organization of a new member of Caliciviridae was determined. ... more The nucleotide sequence and genome organization of a new member of Caliciviridae was determined. Cell culture inoculated with fecal matter from walrus was used to recover fragments of a new virus by Suppression Subtractive Hybridization (SSH). The isolate was identified as a member of the Vesivirus genus of Caliciviridae and designated the name Walrus Calicivirus (WCV). Sets of PCR primers spanning the entire putative genome were designed using known sequences of other vesiviruses. The assembled genome was 8289 nucleotides (nt) long and shared no more than 87% identity with sequences of the other members of the genus Vesivirus. The largest open reading frame (ORF1) between positions 4-5646 encoded a polyprotein. ORF2, found at position 5652-7778, encoded a putative capsid protein. ORF3 overlapped ORF2 and encoded a small basic protein. Comparative analysis of multiple caliciviral capsid proteins was performed to propose a uniform capsid structural organization for this viral family.
Uploads