Genetic subtyping and V3 serotyping of HIV type 1 isolates in Congo.
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Biomedical subjects
Publications and source records attributed to F Barin.
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At least 10 different genetic human immunodeficiency virus type 1 (HIV-1) subtypes (A-J) are responsible for the AIDS pandemic. Much of the understanding of HIV-1 disease progression derives from studies in the developed world where HIV infection is almost exclusively subtype B. This has led many to question whether the properties and consequences of HIV-1 infection can be generalized across subtypes that afflict the majority of infected persons in the developing world. From 1985 to 1997, a prospective study of registered female sex workers in Senegal tracked the introduction and spread of HIV-1 subtypes A, C, D, and G. In clinical follow-up, the AIDS-free survival curves differed by HIV-1 subtype. Women infected with a non-A subtype were 8 times more likely to develop AIDS than were those infected with subtype A (hazard ratio=8.23; P=. 009), the predominant subtype in the study. These data suggest that HIV-1 subtypes may differ in rates of progression to AIDS.
Reported are the results of a cross-sectional survey in Burkina Faso to identify reliable, practical strategies for the serological diagnosis of HIV-1 and/or HIV-2 infections, using less-expensive commercial test kits in various combinations, as an alternative to the conventional Western blot (WB) test, which costs US$ 60. Serum samples, collected from blood donors, patients with acquired immunodeficiency syndrome (AIDS) and pregnant women, were tested between December 1995 and January 1997. Twelve commercial test kits were available: five Mixt enzyme-linked immunosorbent assays (ELISA), three Mixt rapid tests, and four additional tests including monospecific HIV-1 and HIV-2 ELISA. The reference strategy utilized a combination of one ELISA or one rapid test with WB, and was conducted following WHO criteria. A total of 768 serum samples were tested; 35 were indeterminate and excluded from the analysis. Seroprevalence of HIV in the remaining 733 sera was found to be 37.5% (95% confidence interval: 34.0-41.1). All the ELISA tests showed 100% sensitivity, but their specificities ranged from 81.4% to 100%. GLA (Genelavia Mixt) had the highest positive delta value, while ICE HIV-1.0.2 (ICE) produced the most distinct negative results. Among the rapid tests, COM (CombAIDS-RS) achieved 100% sensitivity and SPO (HIV Spot) 100% specificity. Various combinations of commercial tests, according to recommended WHO strategies I, II, III, gave excellent results when ICE was included in the sequence. The best combination of tests for strategy II, which achieved 100% sensitivity and specificity, was to use ICE and COM, the cost of which was US$ 2.10, compared with US$ 55.60 for the corresponding conventional strategy. For strategy III, the best combination, which achieved 100% sensitivity and specificity, was to use ICE, ZYG (Enzygnost Anti HIV-1/HIV-2 Plus) and COM, the cost of which was US$ 2.90 (19.2 times lower than the corresponding strategy requiring WB). No rapid test combination showed 100% sensitivity and specificity. Our results indicate that the serodiagnosis of HIV in Burkina Faso is possible by using reliable, less-expensive strategies which do not require Western blot testing. Moreover, there is a choice of strategies for laboratories working with or without an ELISA chain.
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The Semliki Forest virus (SFV) system seems to be a useful new approach for generating effective immune responses against HIV-1 in animal models. We evaluated this system by comparing the humoral immune responses raised in mice immunized against the HIV-1 envelope with the SFV system, a DNA vaccine, and a recombinant Env glycoprotein. gp160 ELISA antibody titers (204,800) were highest in the sera from mice immunized with recombinant Semliki Forest virus particles. These sera contained antibodies to the CD4-binding site and recognized linear epitopes on gp120 and gp41 that were also recognized by a pool of sera from HIV1-infected individuals. This demonstrates that the HIV-1 envelope produced in vivo by the SFV system does not fold aberrantly. A low level of neutralizing antibodies against the HIV-1LAI strain was also detected in the serum of one mouse immunized with recombinant SFV particles, suggesting that booster injections should be given to achieve a more effective immune response. SFV recombinant particles induced the strongest humoral responses to the HIV-1 envelope of all the potential HIV env vaccines tested.
OBJECTIVE: To compare the performance of V3-loop peptide enzyme immunoassay (PEIA) methodologies from four different laboratories for subtyping HIV-1, and to determine the causes for the lack of correlation between V3-loop PEIA serotyping and subtyping by sequencing. MATERIALS AND METHODS: Synthetic peptides derived from the amino-acid consensus sequences of the V3-loop of group M strains representing genetic subtypes A-F as well as reference strains were evaluated in PEIA by four different laboratories for their ability to accurately determine the subtype in a panel of 85 sera obtained from persons infected with known HIV-1 subtypes (28 subtype A, 34 subtype B, four subtype C, 10 subtype D, seven subtype F, one each of subtype H and G). Furthermore, the V3 loop of the corresponding virus was compared with the V3 loop of the peptides used in PEIA. RESULTS: The correlation between HIV-1 subtyping by sequencing and V3-loop PEIA from the different laboratories varied considerably for the different HIV-1 subtypes: subtype A (46-68%), B (38-85%), C (75-100%), D (29-50%), and F (17-57%). A 70% agreement between PEIA and sequencing subtypes was observed for samples with the concordant presence of the same octameric sequences in the V3 loop of the virus and the V3 loop of the peptide used in PEIA; however, only 42% of specimens with different V3-loop octameric viral and peptide sequences yielded concordant results in V3-loop serotyping and genetic subtyping. CONCLUSION: Our results indicate that V3-loop PEIA methodologies used in different laboratories correlate poorly with genetic subtyping, and that their accuracy to predict HIV-1 subtypes in sera of Belgian individuals infected with different HIV-1 subtypes (A, B, C, D, F, G and H) vary considerably. The poor correlation between serotyping and genetic subtyping was partly due to the simultaneous occurrence of subtype-specific octameric sequences at the tip of the V3 loop of viruses belonging to different genetic subtypes.
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V3 serotyping refers to a system based on binding of antibody in patient sera to V3-loop peptides derived from HIV-1 env genetic subtypes. The V3x serotype represents reactivity of serum from an HIV-1-infected patient (regardless of viral genetic subtype), which reacts preferentially to a V3 peptide derived from the X subtype sequence. We have classified HIV-1 serotypes, determined the relationship between the HIV-1 V3 serotypes and viral genetic subtypes in a large study (n = 125), and evaluated the performance of three different V3 peptide-binding assays. Seven HIV-1 V3 serotypes were identified: A, B, B-Br, B-Th, C, D, and E. Serotypes B-Br and B-Th represent sera that react specifically to peptides derived from Brazilian B (B-Br, GWGR) and Thai B (B-Th, GPGQ) strains. The HIV-1 V3 B, C, and E serotypes correlated closely with their viral env genetic subtypes; 19-26 of 32 B sera (59-79%), 3-4 of 4 C sera (75-100%), and 19-22 of 23 E sera (83-96%) were identified as serotypes B, C, and E, respectively. In contrast, two major V3 serotypes were classified in A sera: A (14-18 of 36 [40-50%]) and C (12-19 of 36 [33-54%]). Similarly, two major V3 serotypes were classified in D sera: B (6-10 of 20 [30-50%]) and D (9-12 of 20 [45-60%]). Serotyping of subtype E sera showed the best concordance with genetic subtypes by all assays. Overall, HIV-1 V3 serotyping produced consistent results among three laboratories. However, HIV-1 V3 serotypes do not distinguish all HIV-1 genetic subtypes. The relative biological significance of the V3 serotypes remains to be elucidated.
HIV-1 V3 serotyping is used to classify immunodeficiency viruses on the basis of antibody binding to V3 peptides derived from env genetic subtypes. Although it shows a reasonable overlap, it has been reported to be distinct from viral genetic subtypes. The aim of this study is to determine the feasibility of HIV-1 serotyping to predict genetic subtypes in an East African setting, where multiple HIV-1 subtypes have coexisted for many years. HIV-1 genetic subtypes of 86 AIDS patients in Mbeya Town, southwest Tanzania, were determined, using env nucleic acid sequencing as the basis for comparison. Those data were compared with V3 serotyping results obtained by four different methodologies. Four HIV-1 genetic subtypes were identified, including A (25, 29%), C (47, 55%), D (13, 15%), and G (1, 1%). The sensitivity and specificity of those serotyping assays varied considerably: sensitivity for genetic subtype A (40-48%), C (52-96%), and D (9-31%); and specificity for genetic subtype A (77-95%), C (46-63%), and D (97-100%). We further tried to identify reasons for the discrepancies between serotyping results and genetic subtypes. By means of logistic regression analysis three amino acid residues within the V3 loop (positions 12, 13, and 19; V, H, and A for serotype A, I, R, and T for serotype C) were found to be most important for antibody binding; a deviation from the subtype-specific amino acids was highly related to mismatched results. In addition, we have shown that phenetic analysis of V3 amino acid sequence data could be used to predict the majority of V3 serotypes (93-94%). Our data demonstrated that for the majority of specimens HIV-1 V3 serotyping results closely match the subtype of the analyzed sample as revealed by the V3 loop amino acid sequence. However, our data demonstrate that HIV-1 serotyping is not sufficiently accurate to predict genetic subtypes in Tanzania, where subtypes A, C, D, and G are circulating. This was due to highly similar amino acid sequences throughout the prevalent genetic subtypes, which caused the inability of HIV-1 V3 serotyping to differentiate subtype A from C as well as D from C. Instead, the serotyping results reflect the frequency distribution of V3 serotypes. To investigate HIV-1 genetic subtypes in population-based studies in this African setting additional or modified algorithms are needed.
Hepatitis C virus (HCV) morphology and physicochemical properties remain unclear because HCV usually circulates in a complexed form in association with immunoglobulins. In the present work, we were interested in the characterization of HCV particles derived from the serum of an anti-HCV negative/HCV RNA positive agammaglobulinemic patient suffering from chronic type C hepatitis. Physicochemical properties of the virus particles were determined by serum centrifugation on a 10-60% isopycnic sucrose density gradient. HCV RNA quantified by bDNA was found in a major peak at density 1.13 g/ml and in a minor peak at densities 1.05-1.07 g/ml. By electron microscopy, 45 nm large core-like particles were found at the 1.13 g/ml density while 60 nm large virus-like particles similar to other members of the Flaviviridae family were visualized at the 1.06-1.07 g/ml densities. This confirms some studies reporting the low density of HCV as compared to other members of the Flaviviridae family.
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Several studies have reported the spontaneous loss of hepatitis C virus (HCV) antibodies in HCV-exposed persons. However, the relationship between seroreversion and spontaneous virus clearance has yet to be precisely determined in a single homogeneous population of untreated immunocompetent patients. In this study, 32 human immunodeficiency virus-seronegative hemophiliacs who had been exposed to HCV were followed for a mean duration of 141 months; 22 remained chronic carriers (68.8%). All but 1 of the nonviremic patients (90.0%) showed partial (8 cases) or complete (2 cases) seroreversion. In contrast, all but 1 of the viremic patients (95.1%) had a stable serologic profile when analyzed by a recombinant immunoblot assay. The results indicate that any HCV antibody-positive immunocompetent patient with no detectable serum HCV RNA and normal alanine aminotransferase values and whose serial samples show a progressive decrease in the level of HCV antibodies present may be considered as having a resolved infection.
This study reports four cases of transmission of the rare hepatitis C virus genotype 4 which occurred in a hemodialysis unit and originated from a single source of infection. Phylogenetic analyses performed with the NS5b domain showed that all four patients with secondary infections were infected with virus strains very similar to that of the index patient. Cross infections probably resulted from breaches in safety precautions and lack of dialysis machine sterilization.
Human immunodeficiency virus type 1 (HIV-1) may be studied by molecular or immunological approaches. Most analyses have been performed by genetic comparison of isolates and have led to the definition of clades or subtypes within the major (M) group of HIV-1. Five subtypes (A to E) were initially identified by comparison of genomic sequences. Four new subtypes (F to I) were identified more recently. Amino acid differences in the immunogenic V3 loop between isolates have also been studied, leading to a phenetic classification of at least 14 clusters (1 to 14) of sequences (B. T. M. Korber, K. McInnes, R. F. Smith, and G. Myers, J. Virol. 68:6730-6744, 1994). In this study, we compared the antigenicity of the V3 consensus sequences defined by phylogenetic analysis to the antigenicity of those defined by phenetic analysis. We used a recently developed subtype-specific enzyme immunoassay (SSEIA) that uses the principle of blocking with an excess of peptide in the liquid phase. Two SSEIAs were performed, the first with five V3 sequences defined by phylogenetic analysis and the second with 14 V3 sequences defined by phenetic analysis. A total of 168 HIV-1 sera taken from seropositive individuals from seven different countries or regions were studied. Experimental and statistical data, including correlation matrix and cluster analyses, demonstrated associations between the genetic subtypes and phenetically associated groups. Most of these were predicted by Korber et al. (J. Virol. 68:6730-6744, 1994) by theoretical analysis. We also found that V3 sequences can be grouped into between three and five antigenically unrelated categories. Residues that may be responsible for major antigenic differences were identified at the apex of the V3 loop, within the octapeptide xIGPGxxx, where x represents the critical positions. Our study provides evidence that there is a limited number of V3 serotypes which could be easily monitored by serological assays to study the diversity and dynamics of HIV-1 strains.
BACKGROUND AND OBJECTIVES: In order to compare sensitivity, five anti-hepatitis C virus (HCV) immunoblot assays were tested (Deciscan plus, Inno-Lia III, Matrix, Murex Western blot and RIBA-3). MATERIALS AND METHODS: The test panel (50 samples for each assay) included 6 anti-HCV-negative samples and 44 samples from 36 HCV-infected subjects. RESULTS: There were minor differences in core reactivity among the assays. The smallest number of NS3-reactive results occurred with the Murex and Matrix assays, and the smallest number of NS4 reactives with RIBA-3 and Matrix. Among the 20 discrepant results for NS5 there was one clear false-negative with Inno-Lia. Only 28 of the 50 samples of the panel gave the same results in all the assays: 5 negatives and 23 positives. One of the 6 negative samples were indeterminate in 3 assays. Eighteen of the 21 other divergent results were interpreted as either indeterminate or positive, a common reactivity being exhibited by all 5 assays. The most important discrepancies occurred on 3 HCV-RNA-positive samples which came up negative in some assays: 2 samples with isolated NS3 reactivity were negative by Matrix and Murex Western blot, 1 of them being also negative by Inno-Lia III; another sample was negative by RIBA-3 and Matrix due to weak signals (< 1) on core and NS3 proteins, which did not exceed 1+ with the other assays. CONCLUSIONS: With more uniform criteria for interpretation, the results would have been less divergent. Some assays should improve their sensitivity to the NS3 protein.