Genetic subtyping and V3 serotyping of HIV type 1 isolates in Congo.

Abstract

309 THE HUMAN IMMUNODEFICIENCY VIRUS type 1 (HIV-1) displays a high genetic variability that is rapidly evolving. Phylogenetic analyses have resulted in the classification of HIV-1 isolates into a major group (M) with corresponding subtypes A±J, and an outlier group (O).1 In addition, some isolates have been reported as a uncertaino with respect to their phylogenetic classification or as the result of recombination between different subtypes of group M. So far, little is known about the biological significance of these genetic subtypes. Generally, genetic subtypes are determined using molecular biological methods such as gag and env sequencing or the heteroduplex mobility assay.3 In addition, large epidemiological studies regarding HIV-1 subtypes have been performed using V3 serotyping. These serotyping assays provide an HIV-1 classification based on antibody binding to V3 peptides derived from env genetic subtypes.4,5 However, although HIV-1 serotyping has proved to be appropriate for studying the frequency distribution of V3 serotypes, its ability to predict genetic subtypes has been questioned. HIV-1 subtypes may have a predominant geographical distribution although geographic intermixing of genetic subtypes has been reported, too.7 All the HIV-1 subtypes, except subtype I, have been detected in sub-Saharan Africa, where HIV1 is endemic.8 Previous epidemiologic studies have shown that the dominant HIV-1 subtypes were subtypes A in West Africa (Ivory Coast, Benin, Gambia, Ghana, Guinea-Bisseau, Nigeria, Togo), A and D in East Africa (Uganda, Rwanda, Kenya, Tanzania, Burundi), and C in southern Africa (Malawi, Zambia, Zimbabwe, South Africa) and northern Africa (Ethiopia, Djibouti).8 A more complex subtype distribution was observed in Central Africa (Cameroon, Gabon, Central African Republic, and former Zaire). In this area, subtype A predominated but all the other subtypes and even group O isolates were also detected. To our knowledge, there are no data on HIV-1 subtypes in Congo, which is close to this area of major HIV-1 diversity. We investigated the distribution of HIV-1 subtypes in a population of 32 HIV-1-infected patients living in Congo. After obtaining informed consent, whole blood samples were collected from 9 patients (BAY, EKE, ITO, MAH, MAY, MOU, POA, SON, and TAN) in 1988 and from 23 patients (C10 to C32) in 1992 in Hopital General Brazzaville, Congo. All patients included in this study (21 females and 11 males) met the clinical criteria of AIDS according to the Bangui definition for AIDS in sub-Saharan Africa.10 The dates of seroconversion and geographic sites where infection occurred were not known. All patients were living in Brazzaville except for BAY, EKE, and MOU, who were living in Pointe Noire. Plasma and peripheral blood mononuclear cells (PBMCs) isolated by Ficoll-Hypaque gradient centrifugation were frozen at 2 80°C and shipped to the Laboratory of Virology (Hopital Piti...-Salp‘tri†re, Paris, France). No plasma was available for the 9 patient samples collected in 1988 and PBMC samples were missing for 11 patients (C10, C18, C19, C23, C24, C26, C27, C28, C29, C30, and C32) owing to problems during cold storage. DNA was obtained from the PBMCs by detergent lysis and proteinase K digestion.9 A nested polymerase chain reaction (PCR) protocol was used to generate a fragment of approximately 350 nucleotides from the C2±V3 region of the HIV-1 env gene as previously described. Primer combinations used were ED5/ED12 (outer primers) and ED31/ED33 (inner primers).11 These primer combinations have been reported to amplify HIV-1 subtypes A to H and are commonly used for HIV-1 subtyping.11 The PCR products were purified on a 1.8% TBE agarose gel and subsequently sequenced directly using a Thermo Sequenase sequencing kit and Vistra DNA automatic sequencer 725 (Amersham, Little Chalfont, Buckinghamshire, England). Nucleotide sequences were aligned by the CLUSTAL W software program, together with reference sequences from the Los Alamos HIV sequence database. Phylogenetic analyses of the alignments including distance calculation, neighbor-join-