Molecular Epidemiology of HIV Type 1 Isolates from the Czech Republic: Identification of an env E Subtype Case

Abstract

85 GENETIC DIVERSITY is a hallmark of human immunodeficiency virus type 1 (HIV-1) biology and is generally responsible for the failure of both drug and vaccine interventions. Phylogenetic relationships based on this sequence heterogeneity have ben employed to subdivide circulating strains of HIV1 into 2 genetic groups (M and O) and at least 19 subtypes (A to J).1 Although specific subtypes tend to predominate in geographic regions (i.e., subtype B in Europe and America), increases in travel due to social±economic development and migration will lead to the spread of different HIV-1 subtypes in most countries. UNAIDS/WHO now estimates that more than 30 million people are infected with HIV-1 worldwide.2 Only 0.5% of this total were reported in eastern Europe and central Asia even though this region has experienced the most rapid increase in HIV-1 seroprevalence in the last decade. Fewer than 30,000 cases of HIV-1 infection were reported in eastern European countries in the early 1990s but this total has risen dramatically to more than 150,000 in 1997.2 Although the first HIV-1 infection in the Czech Republic was diagnosed in 1985,3 only 2000 HIV-infected individuals and fewer than 200 cumulative deaths have been reported since the beginning of the epidemic. Homosexual and intravenous drug user (IDU) transmission are responsible for most infections in the Czech Republic, but the rate of heterosexual transmission appears to be increasing.4 In this report, we have performed the first molecular characterization of HIV-1 isolates from the Czech Republic, identifying at least two different group M subtypes (B and E) of HIV-1 circulating in this country. Peripheral blood was obtained in 1997 from 22 HIV-1seropositive individuals with diverse clinical status, transmission routes, age, and sex (Fig. 1A). All patients were participating in diagnostic and research program in the National Institute of Public Health (Prague, Czech Republic). Homosexual transmission was responsible for the majority of HIV-1 infections in this sample population (Fig. 1A). Patients CZ5 and CZ19 may have contracted HIV-1 from confirmed sexual partners in Germany whereas patients CZ9 and CZ10 may have seroconverted while living in the Ukraine and South America, respectively. A sequence analysis of the HIV-1 envelope gene in all 22 samples established a possible phylogenetic relationship between HIV-1 strains found in the Czech Republic. In addition, a potential course of HIV-1 evolution in this country has been tracked, using samples from patients with different seroconversion dates scattered over the past 10 years. Proviral DNA was extracted directly from uncultured peripheral blood mononuclear cells (PBMCs) as previously described. A fragment of approximately 1.2 kb, spanning the V1±V5-coding region of gp120 (env gene), was amplified by polymerase chain reaction (PCR), using two sets of primer pairs (ED3±ED14 and ED5±ED126) for nested PCR as previously described.6 Owing to failure to amplify HIV-1 DNA from five different samples (CZ2, CZ5, CZ6, CZ7, and CZ14), we resorted to amplify a smaller segment of HIV-1 env DNA (C2±C3 region), using primer pairs ED5±ED12 and ED31±ED336 for external and nested PCR, respectively. The subtype of the env PCR products of each sample was determined using the heteroduplex mobility assay (HMA)6 and env fragments of 14 subtype-specific HIV-1 strains (belonging to subtypes A to H of group M). Nucleotide sequences between positions 7074 and 7286 (positions determined on the HIV-1-HXB2 genome, GenBank accession number K03455), corresponding to 70 amino acids in gp120env (i.e., the entire V3 loop and portions of the C2 and C3 region), were sequenced in both directions using primers E110 (5 9 -CTGTTAAATGGCAGTCTAGCAGGA-3 9 ) and E125 (5 9 -CAATTTCTGGGTCCCCTCCTGAGG-3 9 ) using the fmol method (Promega, Madison, WI).5 Nucleotide sequences were edited and translated by EDITSEQ software (DNASTAR, Madison, WI), then aligned using the CLUSTAL X version 1.63b program.8 Pairwise DNA matrices and phylogenetic analyses were performed with the MEGA version 1.02 program. As suspected, HMA results showed that 21 of 22 (95%) HIV1 isolates from the Czech Republic were most closely related to group M subtype B (data not shown). However, a specific heteroduplex of the CZ4 env sample and the clade E reference env fragments suggested a subtype E HIV-1 infection of this