Human immunodeficiency virus seroconversion and evolution of the hepatitis C virus quasispecies.

When chronic hepatitis C virus (HCV) infections are complicated by acquisition of human immunodeficiency virus (HIV), liver disease appears to accelerate and serum levels of HCV RNA may rise. We hypothesized that HIV might affect the HCV quasispecies by decreasing both complexity (if HIV-induced immunosuppression lessens pressure for selecting HCV substitutions) and the ratio of nonsynonymous (d(N)) to synonymous (d(S)) substitutions, because d(N) may be lower (if there is less selective pressure). To test this hypothesis, we studied the evolution of HCV sequences in 10 persons with chronic HCV infection who seroconverted to HIV and, over the next 3 years, had slow or rapid progression of HIV-associated disease. From each subject, four serum specimens were selected with reference to HIV seroconversion: (i) more than 2 years prior, (ii) less than 2 years prior, (iii) less than 2 years after, and (iv) more than 2 years after. The HCV quasispecies in these specimens was characterized by generating clones containing 1 kb of cDNA that spanned the E1 gene and the E2 hypervariable region 1 (HVR1), followed by analysis of clonal frequencies (via electrophoretic migration) and nucleotide sequences. We examined 1,320 cDNA clones (33 per time point) and 287 sequences (median of 7 per time point). We observed a trend toward lower d(N)/d(S) after HIV seroconversion in 7 of 10 subjects and lower d(N)/d(S) in those with rapid HIV disease progression. However, the magnitude of these differences was small. These results are consistent with the hypothesis that HIV infection alters the HCV quasispecies, but the number of subjects and observation time may be too low to characterize the full effect.

Acute hepatitis C virus structural gene sequences as predictors of persistent viremia: hypervariable region 1 as a decoy.

We hypothesized that hepatitis C virus (HCV) persistence is related to the sequence variability of putative envelope genes. This hypothesis was tested by characterizing quasispecies in specimens collected every six months from a cohort of acutely HCV-infected subjects (mean duration of specimen collection, 72 months after seroconversion). We evaluated 5 individuals who spontaneously cleared viremia and 10 individuals with persistent viremia by cloning 33 1-kb amplicons that spanned E1 and the 5' half of E2, including hypervariable region 1 (HVR1). To assess the quasispecies complexity and to detect variants for sequencing, the first PCR-positive sample was examined by using a previously described method that combines heteroduplex analysis and analysis of single-stranded conformational polymorphisms. The ratio of nonsynonymous to synonymous substitutions (dN/dS) within each sample was evaluated as an indicator of relative selective pressure. Amino acid sequences were analyzed for signature patterns, glycosylation signals, and charge. Quasispecies complexity was higher and E1 dN/dS ratios (selective pressure) were lower in those with persistent viremia; the association with persistence was strengthened by the presence of a combination of both characteristics. In contrast, a trend toward higher HVR1 dN/dS ratios was detected among those with persistent viremia. We did not detect any such association for factors that may affect complexity such as serum HCV RNA concentration. HVR1 had a lower positive charge in subjects with persistent viremia, although no consistent motifs were detected. Our data suggest that HCV persistence is associated with a complex quasispecies and immune response to HVR1.

Assessment of hepatitis C virus sequence complexity by electrophoretic mobilities of both single-and double-stranded DNAs.

To assess genetic variation in hepatitis C virus (HCV) sequences accurately, we optimized a method for identifying distinct viral clones without determining the nucleotide sequence of each clone. Twelve serum samples were obtained from seven individuals soon after they acquired HCV during a prospective study, and a 452-bp fragment from the E2 region was amplified by reverse transcriptase PCR and cloned. Thirty-three cloned cDNAs representing each specimen were assessed by a method that combined heteroduplex analysis (HDA) and a single-stranded conformational polymorphism (SSCP) method to determine the number of clonotypes (electrophoretically indistinguishable cloned cDNAs) as a measure of genetic complexity (this combined method is referred to herein as the HDA+SSCP method). We calculated Shannon entropy, incorporating the number and distribution of clonotypes into a single quantifier of complexity. These measures were evaluated for their correlation with nucleotide sequence diversity. Blinded analysis revealed that the sensitivity (ability to detect variants) and specificity (avoidance of false detection) of the HDA+SSCP method were very high. The genetic distance (mean +/- standard deviation) between indistinguishable cloned cDNAs (intraclonotype diversity) was 0.6% +/- 0.9%, and 98.7% of cDNAs differed by <2%, while the mean distance between cloned cDNAs with different patterns was 4.0% +/- 3.2%. The sensitivity of the HDA+SSCP method compared favorably with either HDA or the SSCP method alone, which resulted in intraclonotype diversities of 1.6% +/- 1.8% and 3.5% +/- 3.4%, respectively. The number of clonotypes correlated strongly with genetic diversity (R2, 0.93), but this correlation fell off sharply when fewer clones were assessed. This HDA+SSCP method accurately reflected nucleotide sequence diversity among a large number of viral cDNA clones, which should enhance analyses to determine the effects of viral diversity on HCV-associated disease. If sequence diversity becomes recognized as an important parameter for staging or monitoring of HCV infection, this method should be practical enough for use in laboratories that perform nucleic acid testing.

Initial report of a hepatitis investigation in rural Belize.

In spring 1991, Belizian health officials expressed concern about a possible hepatitis outbreak in a banana farming district. A study was designed to identify cases and to address the serological prevalence of hepatitis virus markers. Three populations were studied: (i) persons meeting a clinical case definition for hepatitis; (ii) designated banana workers; and (iii) people in a random sample of households in the community. Information was collected using questionnaires and sera were collected for laboratory testing. This report presents the preliminary results of a study conducted in June 1991. Among people who met the clinical case definition, 24% of 42 tested had immunoglobulin M antibody to hepatitis B virus (HBV) core antigen (anti-HBc IgM). In the worker and household survey populations, 284 and 280 people, respectively, were tested for anti-HBc IgM. In each group, 4% were positive. HBV surface antigen was found in 37% of 43 clinical cases, 18% of workers, and 13% of people in the household survey. Among the 3 study populations, the prevalence of HBV core antibody (anti-HBc) ranged from 73% to 81%. Almost all tested persons had evidence of prior hepatitis A virus infection. Evidence of prior infection with hepatitis viruses A and B was widespread, but an aetiology could not be established for most of the clinical cases. However, the prevalence of hepatitis B markers in this population was very high compared to other reports from the Caribbean.

Detection of hepatitis B virus DNA in asymptomatic hepatitis B surface antigen carriers: relation to sexual transmission.

The authors tested, by molecular hybridization, for hepatitis B virus DNA in serum specimens of 182 asymptomatic hepatitis B surface antigen (HBsAg) Greek carriers who were heterosexual partners of patients with acute hepatitis B (group A: 96 cases) or healthy subjects who were susceptible to hepatitis B (group B: 86 cases). The mean age (34.1 +/- 10.4 vs. 33.9 +/- 8.4 years) and the mean duration of sexual contact (6.9 +/- 8.9 vs. 7.2 +/- 6.3 years) were similar in the two groups of carriers. Hepatitis B virus DNA was detected significantly more frequently in group A than in group B (59.4% vs. 11.6%, p less than 0.001). In particular, in group A, hepatitis B virus DNA was detected in 96.9% of hepatitis B e antigen (HBeAg)-positive and 41% of antibody to HBeAg (anti-HBe)-positive carriers. In contrast, in group B, hepatitis B virus DNA was identified in only 10.8% of anti-HBe-positive carriers (p less than 0.001). These differences were especially significant in the young and middle-aged carriers (16-49 years old) and during the first four years of sexual contact. These data suggest that 1) there is a positive correlation between the presence of hepatitis B virus DNA in serum and the epidemiologic evidence of sexual transmission of hepatitis B virus, 2) hepatitis B virus DNA is a better indicator of infectivity than HBeAg/anti-HBe, and 3) the detection of hepatitis B virus DNA in serum probably identified carriers with high infectivity and potentially higher risk of transmitting hepatitis B virus to their sexual partners.

Detection of hepatitis A virus by extraction of viral RNA and molecular hybridization.

Hepatitis A virus (HAV) RNA was extracted from cell culture, serum, liver, and feces and then detected by molecular hybridization with cloned HAV cDNA. Hybridization was approximately 10-fold more sensitive than immune electron microscopy or radioimmunoassay was and less sensitive than was assays of HAV infectivity in primates or in cell culture. As little as 10(3) 50% infective doses of HAV, or approximately 0.1 pg of viral RNA, was detected by this method. Analysis of fecal specimens from an experimentally infected marmoset and an epidemic of hepatitis A showed that HAV excretion could often be detected later in the illness by hybridization than by radioimmunoassay. This technique should be widely applicable for detection and analysis of HAV RNA.

Hepatitis A virus cDNA and its RNA transcripts are infectious in cell culture.

A full-length cDNA copy of an attenuated, cell culture-adapted hepatitis A virus (HAV HM-175/7 MK-5) genome was constructed in the PstI site of plasmid vector pBR322. Transfection of monkey kidney cells with this plasmid failed to induce the production of hepatitis A virus (HAV). The HAV cDNA was excised from pBR322 and inserted, without the oligo(dG) X oligo(dC) tails, into an RNA transcription vector to yield plasmid pHAV/7. Transfection of monkey kidney cells with pHAV/7 DNA induced HAV infection. Transfection with RNA transcripts produced in vitro from pHAV/7 yielded about 10-fold more HAV than did transfection with pHAV/7 DNA. Marmosets inoculated with transfection-derived virus developed anti-HAV antibodies and had liver enzyme patterns that closely resembled the liver enzyme patterns seen in animals inoculated with virus from a comparable level of cell culture passage. Infectious RNA transcripts from HAV cDNA should be useful for studying the molecular basis of cell culture adaptation and attenuation as well as for studying specific viral functions.

Complete nucleotide sequence of an attenuated hepatitis A virus: comparison with wild-type virus.

The complete nucleotide sequence of an attenuated hepatitis A virus, HAV HM-175/7 MK-5, was determined from cloned cDNA. This virus was derived from wild-type HAV HM-175 after 32 passages in African green monkey kidney cells. The resultant cell culture-adapted virus is attenuated for chimpanzees. This virus was passaged an additional three times in monkey kidney cells to obtain sufficient virus for molecular cloning and was designated HM-175/7 MK-5. Three overlapping cDNA clones were obtained that together spanned the entire genome. Comparison of the nucleotide sequence of cDNA from wild-type virus (propagated in marmoset liver in vivo) with attenuated virus (grown in cell culture) showed 24 nucleotide changes distributed throughout the genome. Five base deletions occurred in the 5' noncoding region, and 12 of the 16 base substitutions in the coding region resulted in amino acid changes. Amino acid changes occurred in viral capsid proteins VP1 and VP2 and several of the nonstructural proteins. Thus, a small number of nucleotide changes are responsible for adaptation to cell culture and attenuation of HAV strain HM-175.

Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses.

The complete nucleotide sequence of wild-type hepatitis A virus (HAV) HM-175 was determined. The sequence was compared with that of a cell culture-adapted HAV strain (R. Najarian, D. Caput, W. Gee, S.J. Potter, A. Renard, J. Merryweather, G.V. Nest, and D. Dina, Proc. Natl. Acad. Sci. USA 82:2627-2631, 1985). Both strains have a genome length of 7,478 nucleotides followed by a poly(A) tail, and both encode a polyprotein of 2,227 amino acids. Sequence comparison showed 624 nucleotide differences (91.7% identity) but only 34 amino acid differences (98.5% identity). All of the dipeptide cleavage sites mapped in this study were conserved between the two strains. The sequences of these two HAV strains were compared with the partial sequences of three other HAV strains. Most amino acid differences were located in the capsid region, especially in VP1. Whereas changes in amino acids were localized to certain portions of the genome, nucleotide differences occurred randomly throughout the genome. The most extensive nucleotide homology between the strains was in the 5' noncoding region (96% identity for cell culture-adapted strains versus wild type; greater than 99% identity among cell culture-adapted strains). HAV proteins are less homologous with those of any other picornavirus than the latter proteins are when compared with each other. When the sequences of wild-type and cell culture-adapted HAV strains are compared, the nucleotide differences in the 5' noncoding region and the amino acid differences in the capsid region suggest areas that may contain markers for cell culture adaptation and for attenuation.

Fulminant hepatitis in asymptomatic hepatitis B surface antigen carriers in Greece.

Eleven male fulminant hepatitis (FH) patients (mean age: 47.7 +/- 16 years) positive for hepatitis B surface antigen (HBsAg) but negative for IgM antibody to hepatitis B core antigen (IgM anti-HBc) were admitted consecutively to the Athens Hospital for Infectious Diseases between May 1981 and November 1983. Because of the absence of IgM anti-HBc, determined by an enzyme immunoassay, these patients were considered to be HBsAg carriers with a superimposed acute hepatitis. Three of the 11 patients received immunosuppressive chemotherapy during the six months before the onset of the acute hepatitis. None of the patients was homosexual or a drug addict. Infection with hepatitis A virus (HAV), hepatitis B virus (HBV), or hepatitis delta virus (HDV) was detected with serologic markers and/or molecular hybridization techniques. Fulminant hepatitis was attributed to spontaneous reactivation of chronic hepatitis B in four patients, chemotherapy-induced reactivation of chronic hepatitis B in three patients, HDV superinfection in one patient and possible superinfection by non-A, non-B agent(s), HDV, or HDV-like agents in three patients. Reactivation of chronic hepatitis B was an important cause of apparent acute hepatitis in heterosexual male HBsAg carriers from an area with a high prevalence of HBV infection.

Detection of a genome-linked protein (VPg) of hepatitis A virus and its comparison with other picornaviral VPgs.

The nucleotide sequence corresponding to the P3 region of the hepatitis A virus (HAV) polyprotein genome was determined from cloned cDNA and translated into an amino acid sequence. Comparison of the amino acid sequences of the genome-linked proteins (VPgs) of other picornaviruses with the predicted amino acid sequence of HAV was used to locate the primary structure of a putative VPg within the genome of HAV. The sequence of HAV VPg, like those of other picornaviral VPg molecules, contains a tyrosine residue as a potential binding site for HAV RNA in position 3 from its N terminus. The potential cleavage sites to generate VPg from a putative HAV polyprotein are between glutamic acid and glycine at the N terminus and glutamic acid and serine or glutamine and serine at the C terminus. A synthetic peptide corresponding to 10 amino acids of the predicted C terminus of HAV VPg induced anti-peptide antibodies in rabbits when it was conjugated to thyroglobulin as a carrier. These antibodies were specific for the peptide and precipitated VPg, linked to HAV RNA, from purified HAV and from lysates of HAV-infected cells. The precipitation reaction was blocked by the synthetic peptide (free in solution or coupled to carrier proteins) and prevented by pretreatment of VPg RNA with protease. Thus, our predicted amino acid sequence is colinear with the nucleotide sequence of the VPg gene in the HAV genome. From our results we concluded that HAV has the typical organization of picornavirus genes in this part of its genome. Similarity among hydrophobicity patterns of amino acid sequences of different picornaviral VPgs was revealed in hydropathy plots. Thus, the VPg of HAV appears to be closely related to VPg1 and VPg2 of foot-and-mouth disease virus. In contrast, HAV VPg has a unique isoelectric point (pI = 7.15) among the picornavirus VPgs.

Significance of IgM antibody to hepatitis B core antigen in a Greek population with chronic hepatitis B virus infection.

IgM antibody to hepatitis B core antigen (IgM anti-HBc) may indicate an active immune response to persistent infection with hepatitis B virus (HBV). We studied 186 Greek HBsAg carriers for IgM anti-HBc and attempted to correlate it with other HBV and hepatitis delta virus (HDV) markers. Overall, IgM anti-HBc was detected more frequently than HBV DNA in this population (50% vs 34, p less than 0.001); this was also true for the 149 of the 186 HBsAg carriers with antibody to hepatitis B e antigen (anti-HBe) (48% vs 22%, p less than 0.001). The opposite was found in the carriers positive for hepatitis B e antigen (HBeAg): HBV DNA was observed in 93% and IgM anti-HBc in 64% of the cases (p less than 0.05). The detection of these markers was independent of sex. Serum alanine aminotransferase (ALT) levels were significantly more elevated in patients with positive tests for IgM anti-HBc and HBV DNA than in patients positive only for HBV DNA (p less than 0.001) irrespective of their HBeAg or anti-HBe status. Moreover, the detection of elevated ALT was independent of the intensity of the HBV DNA hybridization signal. Antibodies to hepatitis delta antigen (HDAg) were only found in 4 (2.4%) of 167 patients tested.

Fecal excretion of Greek strains of hepatitis A virus in patients with hepatitis A and in experimentally infected chimpanzees.

The presence of hepatitis A virus (HAV) in stool samples was determined in 36 children (mean age, 8.9 years) and 38 adults (mean age, 19.9 years) with acute type A hepatitis. Three stool samples, taken on admission and thereafter at three-to-five-day intervals, were collected from each patient. The first day of dark urine was considered to be the onset of illness. Molecular hybridization of cloned HAV cDNA to fecal extracts was used to detect HAV RNA; radioimmunoassay was used to detect HAV antigen. In all of the samples tested, HAV RNA was detected significantly more frequently than HAV antigen (28.4% vs. 8.1%, P less than .001). HAV RNA was detected with equal frequency in both children and adults during the first week of illness. However, HAV RNA was detected more frequently in children than in adults during the second week of illness (45.7% vs. 18.9%, P less than .05). Among patients with HAV RNA, detection in multiple samples was more frequent in children than in adults (38.9% vs. 7.9%, P less than .01), especially among males.

Search for hepatitis B virus DNA in sera from patients with acute type B or non-A, non-B hepatitis.

One hundred and three single sera from adults hospitalized with acute type B (78) or non-A, non-B (25) hepatitis were tested for the presence of hepatitis B virus DNA (HBV DNA). All sera from patients with type B hepatitis were IgM anti-HBc-positive. These patients were classified as benign (47) or fulminant (31) hepatitis. The 25 acute non-A, non-B patients were also classified as benign (21) or fulminant (4) hepatitis and were negative for serologic markers of past HBV infection. Serum HBV DNA was detected with similar frequency in benign (38.5%) and fulminant (FH, 34.6%) HBsAg-positive cases. HBV DNA was not detected in either the 26 acute HBsAg-negative hepatitis B cases who were positive for anti-HBc and anti-HBs or the 25 acute non-A, non-B hepatitis cases. The absence of HBV DNA in 43.8% of benign hepatitis B patients who were positive for HBsAg and HBeAg could possibly be attributed to either low level replication of HBV that was not detectable by the [32P]HBV DNA probe or to a period of delayed clearance of free HBeAg following cessation of HBV replication. Emergence of anti-HBs in the presence of HBsAg did not always correspond to clearance of HBV in fulminant type B cases. However, in acute type B hepatitis, irrespectively of severity, disappearance of HBsAg and appearance of anti-HBs was accompanied by reduction of HBV replication to undetectable levels.

Sequence analysis of hepatitis A virus cDNA coding for capsid proteins and RNA polymerase.

We report here the nucleotide sequence corresponding to two large regions of the hepatitis A virus (HAV) genome. These comprise a sequence of 3274 bases corresponding to the 5' end of the genome, which includes the putative capsid protein region of this picornavirus, and 1590 bases corresponding to the 3' end of the genome, terminating in a 15-base poly(A) tract. These sequences revealed that HAV had the characteristic genomic organization of picornaviruses: an open reading frame beginning approximately 750 bases from the 5' end of the RNA and a termination codon 60 bases from the 3' poly(A) tract. The predicted amino acid sequences of both regions have been compared to analogous regions previously determined for other picornaviruses. There was sufficient homology to conclude that the 5' region of HAV codes for capsid proteins and that the 3' region codes for an RNA polymerase. However, these regions of HAV were not found to be closely related to analogous regions of poliovirus, encephalomyocarditis virus, and foot and mouth disease virus.

Molecular cloning and characterization of hepatitis A virus cDNA.

Double-stranded cDNA was synthesized from hepatitis A virus (HAV) RNA and inserted into the Pst I site of pBR322. Restriction endonuclease digestion and cross-hybridization of fragments yielded a map of overlapping cloned cDNAs that included at least 99% of the viral genome. Molecular clones containing HAV cDNA were identified by hybridizing cloned cDNA to electrophoretically resolved RNA from uninfected and HAV-infected tissue culture cells. Cloned cDNA probes specifically hybridized to RNA from infected cells, and the predominant species identified had the characteristic genomic length of picornaviral RNA (approximately equal to 7,500 nucleotides). A partial sequence from the 3' end of the genome revealed 414 bases in an open reading frame followed by two closely spaced stop codons, a 60-base noncoding region, and a tract of poly(A).