Bovine lymphosarcoma: development of a radioimmunologic technique for detection of the etiologic agent.

A highly sensitive and specific radioimmunoassay has been developed for the major structural protein of an oncornavirus etiologically associated with bovine lymphosarcoma. This test can be used to identify cattle which have been exposed to the bovine leukemia virus and may thus develop or transmit the disease. Analysis of randomly obtained serums indicates that infection that infection with this virus is widespread among cattle.

In vivo identification of the transforming gene product of simian sarcoma virus.

Simian sarcoma virus (SSV) deletion mutants were constructed from a molecular clone containing the entire infectious provirus. Transfection analysis of these mutants localized the SSV transforming gene to a small region of the viral genome encompassing its cell-derived sequence (v-sis). Antiserum to a peptide synthesized on the basis of the predicted amino acid sequence of the SSV transforming gene detected a 28,000-dalton protein that was specifically expressed in SSV transformed cells and that corresponded in size to that predicted from the v-sis coding sequence. The v-sis gene product designated p28sis was not a phosphoprotein, nor did it possess detectable protein kinase activity. These findings distinguish p28sis from a number of other retroviral onc proteins.

Simian sarcoma virus onc gene, v-sis, is derived from the gene (or genes) encoding a platelet-derived growth factor.

The transforming protein of a primate sarcoma virus and a platelet-derived growth factor are derived from the same or closely related cellular genes. This conclusion is based on the demonstration of extensive sequence similarity between the transforming protein derived from the simian sarcoma virus onc gene, v-sis, and a human platelet-derived growth factor. The mechanism by which v-sis transforms cells could involve the constitutive expression of a protein with functions similar or identical to those of a factor active transiently during normal cell growth.

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.

Simplified radioimmunoassay for viral antigens: use of Staphylococcus aureus as an adsorbent for antigen-antibody complexes.

In a rapid method for the radioimmunoassay (RIA) of viral antigens, Staphylococcus aureus was used as the adsorbent for antigen-antibody complexes ("protein A" molecules on the cell walls of certain strains of staphylococci have a strong affinity toward IgG molecules). The results showed that this method could be used instead of the double-antibody technique, with the same or probably higher sensitivity in precipitation as well as competition RIA's.

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.

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.

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.

Prevalence of antibodies to the core protein P17, a serological marker during HIV-1 infection.

Studies on monitoring the immune response to viral structural proteins during human immunodeficiency virus (HIV-1) infection have established the significance of antibodies to the core protein p24 during the progression of the disease. We have studied the prevalence of antibodies to the core protein p17 in order to study their diagnostic and prognostic significance in the pathogenesis of HIV-1. Full-length HIV-1 p17, molecularly cloned and expressed in Escherichia coli was purified by immunoaffinity chromatography using an HIV-1 p17-specific monoclonal antibody. A highly sensitive enzyme-linked immunoassay was developed using the purified recombinant p17 as the serological target to detect antibodies to p17. The results indicated that antibodies to p17 decline during progression of disease, with the decline being more dramatic as patients moved from asymptomatic to AIDS-related complex (ARC). Patient specimens deficient in p24 antibody, but having detectable levels of antibody to p17 were almost always positive for p24 antigen. Under these conditions, p17 antibody is an important serological marker because it provides a more consistent marker for core antigens during HIV-1 infection.

Mouse monoclonal antibody 5-21-3 recognizes a contiguous, conformation-dependent epitope and maps to a hydrophilic region in HIV-1 gp41.

Mouse monoclonal antibody 5-21-3 is mapped to an epitope within a hydrophilic region of HIV-1 gp41 between amino acids 642 and 665 (numbering by Meyers et al. based on HXB2 isolate). The epitope is formed from amino acids within the sequence IHSLIEESQNQQEKNEQELLELDK; however, antibody 5-21-3 is unable to recognize the epitope-forming sequence when it is presented to the antibody in the form of a short (642-665) synthetic polypeptide. The epitope apparently is partially formed when additional native sequence of varying length is added to the amino and/or carboxy ends of the epitope-forming sequence, and 5-21-3 binds these larger synthetic polypeptides to varying degrees depending on the position and length of the flanking sequences. The 5-21-3 epitope apparently is formed from contiguous amino acids which require a specific, conformation-dependent, secondary structure for proper epitope formation. Binding preferences exhibited by 5-21-3 toward synthetic polypeptides and recombinant proteins may reflect the conformational nature of the epitope in disrupted HIV which elicited formation of the monoclonal.

Diagnostic utility of a mouse monoclonal antibody (5-21-3) employed as a competitive probe in an immunoassay to detect antibody to HIV-1 gp41.

A mouse monoclonal antibody, designated 5-21-3, was raised against HIV-1 gp41 using detergent-disrupted virus as the immunogen. Antibody 5-21-3 was conjugated to horseradish peroxidase (HRP) and employed as a competitive probe against normal and HIV-1 antibody-positive sera in an immunoassay to detect the presence of antibody to HIV-1 gp41. The diagnostic utility of the competitive monoclonal immunoassay was assessed by correlation to a similar assay which employed HRP-labeled polyclonal IgG from a gp41-seropositive donor as the competitive probe. The monoclonal immunoassay was greater than 98% as sensitive and 99% as specific as the polyclonal immunoassay, regardless of the geographic source or disease state of the donor. The monoclonal immunoassay also was nearly as effective as the polyclonal immunoassay in detecting points of seroconversion in individuals enrolled in longitudinal studies. Of particular interest was the finding that the epitope recognized by monoclonal antibody 5-21-3 did not map to the well-characterized gp41 immunodominant region.

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.

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

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.