Comparison of complete env gene sequences from individuals with symptomatic primary HIV type 1 infection.

PRIM A RY HIV-1 INFEC TIO N is characterized by a burst of viral replication that occurs 2 to 6 weeks after the transmission of the virus. In 30–90% of newly infected individuals, virus dissemination can cause clinical manifestations of varying severity, sometimes requiring hospitalization. The main symptoms associated with primary HIV-1 infection are fever, skin rash, lymphadenopath y, headache, and fatigue. 1 Viremia levels decrease rapidly within 2 weeks, as a result of both specific and nonspecific cellular and humoral immune responses. Because the diagnosis of primary HIV-1 infection is difficult, little is known concerning the viral population before seroconversion. 2 Most of the sequence data concerning the early phase of infection was obtained at the time of seroconversion or shortly thereafter. 3,4 In France, as in most areas of Western Europe and the United States, the AIDS epidemic is caused mainly by HIV-1 subtype B strains. 5 However, sequence data pertaining to HIV-1 variants circulating in France are limited and existing studies have focused only on the C2V3 region.6–8 To date, except for the prototype strain LAI and its derivatives, and the group O strain VAU, no full-length envelope gene sequence from HIV-1-positive patients in France is available. Here we describe complete envelope sequences obtained during the initial peak of viremia from 11 individuals with primary HIV-1 infection and living in Lyon, France. Individuals with symptomatic primary HIV-1 infection were identified among patients referred to the Department of Infectious Disease of the Croix-Rousse Hospital (Lyon, France) during the period 1992–1996. For each patient presenting clinical manifestations such as fever, skin rash, and adenopathy and claiming to have had risk contacts during the month before the consultation, the earliest blood sample available was used to test the anti-HIV antibody status, using a third-generation enzyme immunoassay (EIA; bioMerieux, Marcy l’Etoile, France), as well as Western blotting (Sanofi Diagnostics Pasteur, Marnes-la-Coq uette, France). In addition, HIV-1 RNA levels in plasma and/or p24 antigen levels were determined using the Amplicor HIV-1 Monitor test (Roche Molecular Systems, Branchburg, NJ) and the Vidas HIV p24 test (bioMerieux), respectively. All patients were confirmed to be HIV-1 positive by Western blots performed on samples collected after the regression of symptoms. Clinical, virological, and serological data concerning patients with symptomatic primary HIV-1 infection are shown in Table 1. For nucleotide sequence determination, genomic DNA from peripheral blood mononuclear cells was extracted directly from the earliest blood sample available. The full-length env gene was amplified by nested polymerase chain reaction (PCR), using outer primer pair EA1 (5 9 CTATGGCAGGAAGAAGCGGA 3 9 ) and EB1 (5 9 TCCTCTTGTGCTTCTAGCCA 3 9 ), and inner primer pair EA3 (5 9 AGAGAAAGAGCAGAAGACAGTGG 3 9 ) and EA4 (5 9 TTTGACCACTTGCCACCCAT 3 9 ). The outer primers are located, respectively, at positions 5968–5987 and 8965–8984 of the HXB2 isolate. The inner primers correspond to nucleotides 6200–6222 and 8797–8816. PCR products were cloned into the pCR2.1 vector (InVitrogen BV, Groningen, The Netherlands), and sequenced using the universal primers of the plasmid and a set of env-specific internal primers, following the dye terminator cycle sequencing protocol (Applied Biosystem s, Foster City, CA). Nucleotide sequences were aligned with other subtype B sequences obtained from the Los Alamos HIV Sequence Database, using the CLUSTAL X program version 1.7 (IGBMC, Strasbourg, France). 9 Modifications to improve the alignment were made by hand. Phylogenetic analysis was realized using the PHYLIP analysis package version 3.57c (University of Washington, Seattle, WA). The neighbor-joining tree constructed using full-length env sequences showed that all patients were infected with subtype B viruses (Fig. 1). The node separating the subtype B sequences from other subtypes was highly significant (bootstrap value 100%), but within this subtype there was no significant subclustering of primary infection sequences, except for patients 160 and 384. Their env nucleotide sequences are homologous at 97.5% and their deduced amino acid sequences are homolo-