Naturally occurring sequence polymorphisms within HIV type 1 group O protease.

Mutations within the protease gene associated with reduced susceptibility to protease inhibitors have been well documented for HIV-1 group M subtype B strains. In contrast, limited genotypic and phenotypic information is available for the genetically diverse HIV-1 group O strains. Preexisting resistance-associated polymorphisms have the potential to contribute to a poor virological response to antiviral drug treatment in group O-infected patients. In the present study, the protease genes of 28 protease inhibitor-naive HIV-1 group O-infected patients were analyzed to identify any naturally occurring amino acid polymorphisms associated with drug resistance. Comparison of the consensus group O protease sequence with subtype B of group M indicated that both groups have almost identical sequences in the protease active site, the flap and the substrate-binding site. Analysis of the 28 individual protease sequences revealed polymorphisms at 34% of the positions within the protease gene, but no primary mutations associated with protease inhibitor resistance. In contrast, each of the strains harbored multiple secondary or accessory mutations associated with resistance to protease inhibitors in group M viruses. Residues 10I, 15V, 36I, 41K, 62V, 63T/A/K/I, 64V, 71V, and 93L were identified in most strains. The presence of multiple natural sequence polymorphisms associated with drug resistance in the protease gene of group O viruses may contribute to a more rapid emergence of drug resistance phenotype and treatment failure in group O-infected patients.

Seven human immunodeficiency virus (HIV) antigen-antibody combination assays: evaluation of HIV seroconversion sensitivity and subtype detection.

In this study, we evaluated the performance of two prototype human immunodeficiency virus (HIV) antigen-antibody (Ag-Ab) combination assays, one from Abbott Laboratories (AxSYM HIV Ag-Ab) and the other from bioMerieux (VIDAS HIV Duo Ultra), versus five combination assays commercially available in Europe. The assays were Enzygnost HIV Integral, Genscreen Plus HIV Ag-Ab, Murex HIV Ag-Ab Combination, VIDAS HIV Duo, and Vironostika HIV Uniform II Ag-Ab. All assays were evaluated for the ability to detect p24 antigen from HIV-1 groups M and O, antibody-positive plasma samples from HIV-1 groups M and O, HIV-2, and 19 HIV seroconversion panels. Results indicate that although all combination assays can detect antibodies to HIV-1, group M, subtypes A to G, circulating recombinant form (CRF) A/E, and HIV-1 group O, their sensitivity varied considerably when tested using diluted HIV-1 group O and HIV-2 antibody-positive samples. Among combination assays, the AxSYM, Murex, and VIDAS HIV Duo Ultra assays exhibited the best antigen sensitivity (at approximately 25 pg of HIV Ag/ml) for detection of HIV-1 group M, subtypes A to G and CRF A/E, and HIV-1 group O isolates. However, the VIDAS HIV Duo Ultra assay had a lower sensitivity for HIV-1 group M and subtype C, and was unable to detect subtype C antigen even at 125 pg of HIV Ag/ml. The HIV antigen sensitivity of the VIDAS HIV Duo and Genscreen Plus combination assays was approximately 125 pg of HIV Ag/ml for detection of all HIV-1 group M isolates except HIV-1 group O while the sensitivity of Vironostika HIV Uniform II Ag-Ab and Enzygnost HIV Integral Ag-Ab assays for all the group M subtypes was >125 pg of HIV Ag/ml. Among the combination assays, the AxSYM assay had the best performance for detection of early seroconversion samples, followed by the Murex and VIDAS HIV Duo Ultra assays.

Comparative performance of three viral load assays on human immunodeficiency virus type 1 (HIV-1) isolates representing group M (subtypes A to G) and group O: LCx HIV RNA quantitative, AMPLICOR HIV-1 MONITOR version 1.5, and Quantiplex HIV-1 RNA version 3.0.

The performance of the LCx HIV RNA Quantitative (LCx HIV), AMPLICOR HIV-1 MONITOR version 1.5 (MONITOR v1.5), and Quantiplex HIV-1 RNA version 3.0 (bDNA v3.0) viral load assays was evaluated with 39 viral isolates (3 A, 7 B, 6 C, 4 D, 8 E, 4 F, 1 G, 4 mosaic, and 2 group O). Quantitation across the assay dynamic ranges was assessed using serial fivefold dilutions of the viruses. In addition, sequences of gag-encoded p24 (gag p24), pol-encoded integrase, and env-encoded gp41 were analyzed to assign group and subtype and to assess nucleotide mismatches at primer and probe binding sites. For group M isolates, quantification was highly correlated among all three assays. In contrast, only the LCx HIV assay reliably quantified group O isolates. The bDNA v3.0 assay detected but consistently underquantified group O viruses, whereas the MONITOR v1.5 test failed to detect group O viruses. Analysis of target regions revealed fewer primer or probe mismatches in the LCx HIV assay than in the MONITOR v1.5 test. Consistent with the high level of nucleotide conservation is the ability of the LCx HIV assay to quantify efficiently human immunodeficiency virus type 1 group M and the genetically diverse group O.

Quantification of HIV-1 group M (subtypes A-G) and group O by the LCx HIV RNA quantitative assay.

Human immunodeficiency virus type 1 (HIV-1) genetic diversity presents a challenge to nucleic acid-based assays with regard to sensitivity of detection and accuracy of quantification. The Abbott LCx HIV RNA Quantitative assay (LCx(R) HIV assay), a competitive RT-PCR targeting the pol integrase region, was evaluated using a panel of 297 HIV-1 seropositive plasma samples from Cameroon, Uganda, Brazil, Thailand, Spain, Argentina and South Africa. The panel included group M subtypes A-G, mosaics, and group O based on sequence analysis of gag p24, pol integrase, and env gp41. The LCx HIV assay quantified 290 (97.6%) of the samples, including all the group O samples tested. In comparison, the Roche AMPLICOR HIV-1 MONITOR test versions 1.0 and 1.5 quantified 67.3 and 94.6% of the samples, respectively. No group O specimens were quantified by either version of AMPLICOR HIV-1 MONITOR. Seven specimens were below the detectable limits of all the three assays. The LCx HIV assay had fewer nucleotide mismatches at primer/probe binding sites as compared with both AMPLICOR HIV-1 MONITOR tests. The high degree of nucleotide conservation within the pol target region enables the LCx HIV assay to efficiently quantify the HIV-1 subtypes A-G and the most genetically diverse HIV-1, group O.

Comparative performances of an HTLV-I/II EIA and other serologic and PCR assays on samples from persons at risk for HTLV-II infection.

BACKGROUND: HTLV-I and HTLV-II are related exogenous pathogenic human retroviruses. Until recently, ELISAs based on HTLV-I antigens have been used to screen for the presence of HTLV-I or -II antibodies. The HTLV-I-based assays have not been as sensitive in detecting antibodies to HTLV-II as in detecting antibodies to HTLV-I. The Abbott HTLV-I/HTLV-II ELISA uses a combination of HTLV-I and HTLV-II antigens to detect antibodies to the whole HTLV group. The performance of this ELISA was compared to that of several HTLV-I-based serologic assays and an HTLV-II PCR assay in cohorts of South American Indians and New York City IV drug users (IVDUs) in whom HTLV-II is endemic. STUDY DESIGN AND METHODS: Sera from 429 South American Indians and New York City IVDUs were evaluated for HTLV antibodies by the use of three ELISAs (rgp21-enhanced HTLV-I/II, Cambridge Biotech; Vironostika HTLV-I/II, Organon Teknika; and HTLV-I/HTLV-II, Abbott), and a Western blot (WB) assay. Peripheral blood leukocyte DNA from each person was analyzed for HTLV-I and HTLV-II pol DNA via PCR. The HTLV-II PCR-positive samples were further subtyped via cloning and sequencing and/or oligomer restriction. RESULTS: Two hundred four samples (48%) were positive for HTLV-II by serologic and/or PCR assays. All of the positive samples from the Indians and approximately one-third of the positive samples from the IVDUs were of the HTLV-IIB subtype. Comparative analyses indicate that the sensitivity and specificity of the various assays were: PCR, 98 and 100 percent; Abbott HTLV-I/HTLV-II, 78 and 95 percent; Cambridge Biotech HTLV-I/II, 76 and 96 percent; Vironostika HTLV-I/II, 71 and 98 percent; and WB, 73 and 100 percent, respectively. CONCLUSION: There were no significant differences among the sensitivities and specificities of the HTLV-I/II ELISAs (p values, 0.056-0.438). The WB and PCR assays were much more specific than the other serologic assays (p

Identification of a new HIV-2 subtype based on phylogenetic analysis of full-length genomic sequence.

Human immunodeficiency virus type 2 (HIV-2) and simian immunodeficiency virus from sooty mangabey (SIV(SM) form one of the six primate lentivirus lineages. The close phylogenetic relationship and geographic coincidence indicate that HIV-2 originated from cross-species transmission of SIV(SM) to humans. HIV-2 exhibits considerable genetic diversity, with subtypes A-F identified. Previously, we reported the partial gag and env sequences of an unusual HIV-2 isolate, Abt96. Abt96 was collected in Ivory Coast from an asymptomatic blood donor. Here we describe the near full-length genomic sequence of Abt96. The genome was assembled from overlapping PCR fragments amplified from viral RNA isolated from plasma. Phylogenetic analysis of sequences derived from segments of the Abt96 genome demonstrate that the Abt96 isolate branches independently of all other characterized HIV-2 isolates. On the basis of the phylogenetic data being presented, we propose that Abt96 is a new HIV-2 subtype and designate it subtype G.

Rapid assay for simultaneous detection and differentiation of immunoglobulin G antibodies to human immunodeficiency virus type 1 (HIV-1) group M, HIV-1 group O, and HIV-2.

A rapid immunodiagnostic test that detects and discriminates human immunodeficiency virus (HIV) infections on the basis of viral type, HIV type 1 (HIV-1) group M, HIV-1 group O, or HIV-2, was developed. The rapid assay for the detection of HIV (HIV rapid assay) was designed as an instrument-free chromatographic immunoassay that detects immunoglobulin G (IgG) antibodies to HIV. To assess the performance of the HIV rapid assay, 470 HIV-positive plasma samples were tested by PCR and/or Western blotting to confirm the genotype of the infecting virus. These samples were infected with strains that represented a wide variety of HIV strains including HIV-1 group M (subtypes A through G), HIV-1 group O, and HIV-2 (subtypes A and B). The results showed that the HIV genotype identity established by the rapid assay reliably (469 of 470 samples) correlates with the HIV genotype identity established by PCR or Western blotting. A total of 879 plasma samples were tested for IgG to HIV by a licensed enzyme immunoassay (EIA) (470 HIV-positive samples and 409 HIV-negative samples). When they were tested by the rapid assay, 469 samples were positive and 410 were negative (99.88% agreement). Twelve seroconversion panels were tested by both the rapid assay and a licensed EIA. For nine panels identical results were obtained by the two assays. For the remaining three panels, the rapid assay was positive one bleed later in comparison to the bleed at which the EIA was positive. One hundred three urine samples, including 93 urine samples from HIV-seropositive individuals and 10 urine samples from seronegative individuals, were tested by the rapid assay. Ninety-one of the ninety-three urine samples from HIV-seropositive individuals were found to be positive by the rapid assay. There were no false-positive results (98.05% agreement). Virus in all urine samples tested were typed as HIV-1 group M. These results suggest that a rapid assay based on the detection of IgG specific for selected transmembrane HIV antigens provides a simple and reliable test that is capable of distinguishing HIV infections on the basis of viral type.

Serologic and phylogenetic characterization of HIV-1 subtypes in Uganda.

OBJECTIVES: To determine the HIV genetic subtypes present in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate serologic detection of infection by commercial immunoassays; to evaluate samples for HIV-1 group O infections. METHODS: Sixty-four HIV-seropositive plasma samples were collected from the Nakasero Blood Bank, Kampala, Uganda. The plasma were evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. HIV-1 group M and O infections were identified on the basis of discordant seroreactivity in EIA and reactivity to group M and O antigens on the immunoblot. Regions of gag p24 and env gp41 were amplified using reverse transcriptase polymerase chain reaction, and genetic subtypes were determined by phylogenetic analysis. RESULTS: Serologic testing confirmed that 63 out of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of HIV-1 group O infections. Genetic subtyping determined that 25 samples were subtype A, three subtype C, 22 subtype D, and nine were heterogeneous for subtypes A and D. CONCLUSIONS: Despite the sequence variation observed in Uganda, commercial EIA based on HIV-1 subtype B proteins detected all the infections. In contrast, a peptide-based assay failed to detect three infections by subtype D viruses. This emphasizes the negative impact of HIV genetic variation on assays that rely on peptides to detect HIV infections. The number of infections with heterogeneous subtype (due to mixed infections or recombinant viruses) is high and reflects the growing complexity of the HIV epidemic in endemic regions where multiple subtypes are present in the population.

PIP: Extensive sequence heterogeneity between HIV-1 isolates has led to the classification of HIV-1 into group M (major) subtypes A-J, and group O (outlier). Some isolates have also been found to be the result of recombination between different group M subtypes. Findings are reported from a study conducted to determine the various HIV genetic subtypes in HIV-1-infected asymptomatic blood donors in Uganda and to evaluate the serologic detection of infection by commercial immunoassays. 64 HIV-seropositive plasma samples were collected from the Nakasero Blood Bank in Kampala and evaluated using commercial HIV enzyme immunoassays (EIA) and a research immunoblot. 63 of 64 plasma units were positive for HIV-1 group M infection and showed no evidence of group O infections. According to phylogenetic analysis, 25 samples were subtype A, 3 subtype C, 22 subtype D, and 9 heterogenous for subtypes A and D. Despite the sequence variation observed in this study population, commercial EIA based upon HIV-1 subtype B proteins detected all of the infections. A peptide-based assay failed to detect 3 infections by subtype D viruses.

Envelope sequence variability and serologic characterization of HIV type 1 group O isolates from equatorial guinea.

Four sera from Equatorial Guinea (EG) suspected to contain antibody against HIV-1 group O-related viruses were identified on the basis of unusual and differential serologic reactivity in selected commercial assays and Western blot. Degenerate primers, designed from HIV-1 group O published sequences, were used to PCR amplify envelope (env) gene sequences from the suspect EG sera. A complete envelope gene sequence from each serum was determined from the overlapping env gene fragments. Analysis (PHYLIP package of programs) of Env amino acid sequences (translated from nucleotide sequences) indicated that the amino acid sequences obtained from EG sera clustered more closely with HIV Env sequences of group O compared to group M. The amino acid sequences at the octameric tip of the V3 loop were either RIGPLAWY (one isolate), RIGPMAWY (two isolates), or GLGPLAVY (one isolate). The V3 tip tetrameric sequence GPLA is represented only once in the 1995 HIV (Los Alamos) database, but was present in two of our group O-related EG samples. The gp41 immunodominant regions (IDR) protein sequences were identical for sequences from three of the sera, RLLALETLIQNQQLLNLWGCKGR(K)L(I)VCYTSVK(T)W, whereas sequence from the fourth serum contained three changes as noted in parentheses. IDR sequences derived from EG sera were unique compared to those reported for other HIV-1 group O isolate ANT70, VAU, or MVP5180. Antibody in each EG serum directed against the IDR could be detected using synthetic peptides comprising sequences from the ANT70 or MVP5180 IDRs, but were most reactive against the sequences derived from the samples themselves. Little or no serologic reactivity was detected when EG sera were reacted against peptides comprising the IDR of HIV-1 group M (subtype B consensus) or HIV-2 (consensus).

PIP: The genetic variation and epidemiology of HIV-1 group O isolates are of considerable importance to the design of HIV-1 diagnostic and screening assays, especially since current serologic and genetic methods to detect HIV-1 have been developed mainly on the basis of sequences from isolates belonging to HIV-1 group M. The HIV envelope protein, especially the gp41 immunodominant region, plays a major antigenic role in the detection of HIV infection and for discriminating HIV-1 from HIV-2 antibody. This paper reports upon genetic variation and the serologic characterization of env sequences from 4 people living in Equatorial Guinea (EG) who were infected with HIV-1 group O. Selected commercial assays and Western blot were first used to identify the sera, then degenerate primers, designed from HIV-1 group O published sequences, were used to PCR amplify envelope (env) gene sequences. A complete envelope gene sequence from each serum was determined from the overlapping env gene fragments. The env amino acid sequence analysis found the EG sera sequences to be clustered more closely with the HIV env sequences of group O rather than to group M. The amino acid sequences at the octameric tip of the V3 loop were either RIGPLAWY, RIGPMAWY, or GLGPLAVY. Although the V3 tip tetrameric sequence GPLA is represented only once in the 1995 HIV database, it was present in 2 of the group O-related EG samples. The gp41 immunodominant regions (IDR) protein sequences were identical for sequences from 3 of the sera. IDR sequences derived from the EG sera were unique compared to those reported for other HIV-1 group O isolates ANT70, VAU, or MVP5180. Other findings are discussed in detail.

Sequence of gp41env immunodominant region of HIV type 1 group O from west central Africa.

PIP: HIV-1 group O is endemic in the west central region of Africa, where the frequency of infection is estimated to be 3-10% of all HIV-1-infected individuals. However, international travel and immigration have led to group O cases being identified in France, Germany, Belgium, Spain, and the US. With the exception of an infected French woman, all reported group O-infected individuals originate from or have a connection to west central Africa. Since most immunoassay reagents are based upon HIV-1 group M, many HIV immunoassays have lower sensitivity for the detection of group O infections. Serum samples were collected from patients at hospitals, tuberculosis (TB) clinics, and STD clinics in endemic regions of Cameroon and Equatorial Guinea in a study of the sequence divergence with group O isolate infections. Screening of the 1086 samples using a range of research and commercial immunoassays found 255 to be HIV-1 seropositive. On the basis of differential reactivity in the various immunoassays, 8 individuals were identified as potentially being infected with group O virus, of which 4 were drawn from TB patients. 7 of the group-O samples were then subjected to polymerase chain reaction (PCR) amplification to verify group O infection. The gp41(env) immunodominant region was successfully amplified and sequenced from 4 of the 7 samples, 2 of which were from the TB patients; 4 of 1086 samples were definitely infected with HIV-1 group O.^ieng

Molecular analyses of HIV-1 group O and HIV-2 variants from Africa.

Genetic variation among HIV isolates creates challenges for their detection by serologic and genetic techniques. To characterize the sequence variation and its correlation to serologic diversity of HIV-1 Group O and HIV-2 isolates, samples were identified by differential reactivity in selected commercial and research assays. Analysis of sera from Equatorial Guinea (EG) led to identification of 4 HIV-1 Group O variants. Viral RNA, extracted from these samples was used to PCR amplify overlapping sequences of the entire envelope gene using multiple primer pairs. Sequence analysis indicated that the V3 loop nucleotide and protein sequences aligned more closely with HIVANT70 compared to other Group O sequences. The amino acid sequences at the octameric tip of the V3 loop were RIGPLAWY, RIGPMAWY, or GLGPLAVY. The tetrameric tip GPLA is represented only once in the published 1994 HIV database (Los Alamos) but was present in 2 of 4 of EG samples. The immuno-dominant region (IDR) sequences derived from EG sera were unique in that none of the sequences were completely homologous to other HIV-1 group O variants. Further, the HIV-1 group O sequence variation could be correlated with differential serologic reactivity using IDR peptides. Compared to HIV-1, the sequence information on HIV-2 isolates is relatively limited, though the HIV-2 isolates also show genetic variation similar to HIV-1. To further establish a correlation between the genetic diversity and serologic detection of HIV-2, plasma samples from Western Africa were evaluated. Eight samples were selected based on weak serologic reactivity to env proteins. PCR amplification and sequence analysis of the gag, env V3 loop, and env IDR regions indicated that the samples could be classified as subtypes A (4 samples), B (3 samples) and D (1 sample). Across the subtypes, there was conservation in the IDR region of the sequence WGCAFRQVCHT. This region is absolutely conserved among the majority of currently known HIV-2 and related SIV viruses (1994 HIV database). One subtype B sample had a unique sequence immediately adjacent to the IDR, however, this did not change the serologic detection using a HIV-2 IDR specific monoclonal antibody.

Search for retrovirus in patients with multiple sclerosis.

Multiple sclerosis (MS) patient samples were screened for known or novel retroviruses using an ultrasensitive technique, IMx PERT, that detects the presence of reverse transcriptase (RT). This procedure has 10(5)- to 10(7)-fold greater sensitivity than conventional RT assays and is capable of detecting 10 to 50 virions. Moreover, IMx PERT is at least as sensitive as polymerase chain reaction, and requires no previous knowledge of viral nucleotide sequence. The MS specimens analyzed in this study included 136 sera from 79 patients and 128 cerebrospinal fluid samples from 53 patients with relapsing or chronic progressive disease. In addition, peripheral blood mononuclear cells from 19 MS patients were cultured in an attempt to amplify or induce expression of low-copy number or cell-associated retrovirus. No evidence of retrovirus was found in any of the specimens obtained from MS patients.

Expression of HCV envelope proteins and the serological utility of the anti-E2 immune response.

The 5' end of the hepatitis C virus (HCV) genome encodes structural proteins of the virion. The first gene encodes a highly basic core protein. Immediately downstream of the core gene are regions which encode the envelope proteins (E1 and E2) of the virus. Artificial expression and secretion of immunologically active envelope proteins have proven to be a substantial challenge due to the high degree of glycosylation and the existence of certain hydrophobic domains contained within these sequences. Bacterial cell expression of recombinant HCV envelope proteins results in products that are not glycosylated and are poorly immunogenic. Emphasis has shifted to the use of mammalian cell lines (human embryonic kidney [HEK] and Chinese hamster ovary [CHO] cells) for the expression of glycosylated, immunologically active envelope proteins. Using HEK cells, E1 is expressed intracellularly but is not secreted from the cells. When E1 is cloned in fusion with a C-terminal truncated E2 protein, both proteins are detected intracellularly; however, only E2 is secreted. When the E1/E2 processing site is interrupted by constructing deletion mutants, the unprocessed E1/E2 fusion protein can be secreted from the cells. Quantifiable expression and secretion of a truncated E2 protein is now possible using CHO cells and SV40-based vectors. The HCV E2 glycoprotein expressed from CHO cells is highly antigenic; a strong humoral response to this antigen develops in persons infected with HCV. Antibodies to E2 are found in 95% of patients with detectable HCV RNA in their sera. The presence of antibodies to E2 is not indicative of viral clearance and therefore the role these antibodies play in protective immunity, if any, is unclear.

Hypervariable 5'-terminus of hepatitis C virus E2/NS1 encodes antigenically distinct variants.

Synthetic peptides representing sequences encoded at the 5'-terminus of E2/NS1 in hepatitis C virus (HCV) were constructed. Peptides synthesized based on the sequences of four distinct HCV isolates were used to develop enzyme immunoassays (EIAs) for detection of antibodies in chronic HCV patients and in HCV-infected plasma donors. HCV sequence-specific antibodies were detected among patients with chronic HCV from the United States and Italy at frequencies of 22.2% and 55.8%, respectively. Similarly, sequence-specific antibodies were detected in 54.6% of U.S. and 55.6% of Japanese commercial plasma donors who had previous evidence of HCV exposure. Our data support earlier findings of geographic variability among HCV variants. The region encoded by amino acids (aa) 380-436 was shown to contain at least one variant-specific and one conserved epitope. The data further indicate that a majority of patients chronically infected with HCV (58.1% U.S., 68.8% Italy) have antibodies directed to the 5'-terminus of the E2/NS1 gene product. We conclude that genotypic variability within the E2/NS1 gene of HCV results in antigenically distinct variants.

Immune response to GOR, a marker for non-A, non-B hepatitis and its correlation with hepatitis C virus infection.

Recently, identification and molecular cloning of a host cellular gene designated GOR from chimpanzees experimentally infected with non-A, non-B hepatitis (NANBH) agent was reported. It was further demonstrated that there is a close association between the immune response to an antigenic peptide of GOR (GOR2) and NANBH. In order to define the specificity of the immune response, in the present study we have identified an additional epitope in the GOR gene sequence, upstream from GOR2, and studied its correlation with the immune response to hepatitis C virus (HCV) in NANBH patients. An enzyme-linked immunoassay (EIA) was developed which utilizes synthetic peptides designated spGOR346 and spGOR2 as the serological target for the detection of anti-GOR antibodies in patient serum samples from various hepatic and non-hepatic disease categories. GOR peptides identified 80-90% of the NANBH samples that were positive for HCV C100-3 and about 70% of the NANBH samples that were positive by Abbott prototype second-generation HCV antibody assay. Among a normal donor population(s), only 2-3% of the samples were positive for antibodies to GOR sequences, whereas from the patient categories unrelated to viral hepatitis as well as various nonhepatic diseases, the immune response to both GOR peptides was closely associated with the presence of antibodies to HCV. The data indicate that antibodies to GOR is a marker associated with NANBH.

Evidence for persistent hepatitis C virus (HCV) infection in hemophiliacs.

Hepatitis C virus (HCV) is the major etiologic agent associated with non-A, non-B hepatitis. This study was designed to assess virologic and serologic markers in hemophiliacs exposed to non-heat-treated and/or virus-inactivated plasma derivatives. Serial bleeds from 48 hemophilic patients were analyzed for the presence of HCV viral RNA sequences as detected by polymerase chain reaction (PCR) and antibodies to structural (core) and nonstructural (C-100 and 33C) proteins by specific dot immunoblot assay. All patients exposed to non-heat-treated products, and four of six patients exposed only to virus inactivated products, had evidence of HCV infection. However, over the 5-yr study period, six exposed patients (13%) consistently lacked detectable anti-C-100 and seven (15%) lost this antibody. HCV viremia (PCR positive) was found in 91% of exposed patients, and was significantly more frequent in HIV seropositive hemophiliacs (P less than 0.05). Six patients had high antibody level to HCV and elevated ALT, but appeared to clear viremia. Four hemophiliacs were HCV seropositive but lacked detectable viremia. These data indicate that hemophiliacs remain persistently infected by HCV and that antibody to the core antigen of HCV is a reliable marker of this transfusion transmissible agent.

Detection of antibodies to hepatitis C virus in U.S. blood donors.

An enzyme immunoassay (EIA) which utilizes a solid phase coated with a recombinant antigen (c100-3) derived from the hepatitis C virus (HCV) genome was evaluated for efficacy in the detection of antibodies to HCV (anti-HCV). The sensitivity of the antibody test was demonstrated by the detection of anti-HCV in a well-characterized panel of human specimens known to contain the infectious agent of non-A, non-B hepatitis. The specificity of the anti-HCV test was evaluated by testing 6,118 serum specimens from volunteer blood donors considered to be at low risk for exposure to HCV. The specificity of the anti-HCV EIA was demonstrated to be 99.56%, since 6,069 of 6,096 specimens from this low-risk group were nonreactive. A total of 49 (0.80%) of the 6,118 specimens were repeatedly reactive in the test, and 22 (46.81%) of the 47 specimens available for additional testing were confirmed as positive for antibodies to HCV c100-3. Among commercial plasma donors, 390 (10.49%) of 3,718 specimens were repeatedly reactive in the EIA. A total of 375 (97.40%) of the 385 specimens available for further testing were confirmed as positive. These limited data indicate that the prevalence of antibodies to HCV is 0.36% (22 confirmed positives among 6,118 specimens) among volunteer blood donors and 10.08% (375 confirmed positives among 3,718 specimens) among commercial plasma donors. The importance of confirmatory testing is discussed.