Phylogenetic examination of HIV type 1 glycoprotein 120-V3 sequences in patients from rural Georgia.

399 THE HIV/AIDS EPIDEMIC in the United States has been dynamic both in its geographic distribution and in the demographic profiles of annual AIDS cases and HIV incidence. During the first decade of the HIV-1 epidemic in the United States the majority of AIDS cases were localized within large urban centers, with most new infections due either to homosexual contact or intravenous drug use.1 This was in marked contrast to the HIV-1 epidemic in Southeast Asia and sub-Saharan Africa in which the virus was spread primarily through heterosexual contact.2 In the United States, male-to-male sexual contact (MSM) and intravenous drug use (IVDU) remain the most frequently reported risk factors for HIV-1 transmission. However, a survey of AIDS cases indicates an increase in the number of persons infected through heterosexual exposure. During the period from 1990 to 1995 the number of AIDS cases attributed to heterosexual contact increased at a rate faster than the numbers of those acquired by other routes.3 During this same period, the AIDS epidemic has expanded into rural areas, especially in the southeastern United States. Since 1988, the Southeast has reported greatest number of new AIDS cases of any region of the United States. Many occurring in small towns and rural communities.4 Evidence suggests that the continuing expansion of the AIDS epidemic in the rural, southeastern United States is due in large part to the acquisition of HIV-1 via heterosexual contact with HIV-1-positive individuals who were infected by MSM and/or IVDU.5 In an attempt to describe the HIV-1 epidemic in rural Georgia more fully, we conducted an epidemiological study of HIV1 in patients attending the Infectious Diseases Clinic at the Medical College of Georgia (MCG, Augusta, GA). These patients are drawn from a large area of Georgia and several adjacent counties of South Carolina. The majority are residents of cities and towns of 10,000±50,000 population. We report proviral DNA sequences for the V3 region of the HIV-1 surface protein, gp120, derived from lymphocyte (mononuclear cell) DNA of 27 patients. Individuals who were at least 18 years of age and HIV-1 positive, with T lymphocyte counts of 400/mm whole blood, were recruited for this study. All patients in the cohort were confirmed for HIV-1 infection by Western blot analysis. Diagnosis of AIDS was made according to the revised 1993 Centers for Disease Control (CDC, Atlanta, GA) AIDS classification system. Blood was collected by venipuncture into acid citratedextrose tubes and fractionated by standard Ficoll gradient centrifugation, producing plasma, peripheral blood mononuclear cell (PBMC), and red cell fractions.8 The cells in the PBMC fraction were lysed and DNA was prepared using the reagents in the HCV blood DNA kit (Qiagen, Chatsworth, CA). Patient DNA was amplified by nested polymerase chain reaction (PCR), using a limiting dilution protocol. For each patient sample a series of fourfold dilutions was prepared. Firstround PCR was done with the HIV-1 envelope primers C207 and CO728 using the following thermocycler profile: 95°C for 1 min, 55°C for 1.5 min, 72°C for 2 min; repeated for a total of 40 cycles. A 5-m l aliquot of the first-round PCR products was template for a second-round PCR, using the same profile, with a nested HIV-1 envelope primer pair: sense primer 207NT7 (59 -ATTAATACGACTCACTATAGGG CTGTTAAATGGCAGTCTA-3 9 , which contains the T7 promoter primer [underlined] and nucleotides 7002±7019 of HIV HXB2) and antisense primer V3S (59 ATGTAAAACGACGGCCAGT CAGTAGAAAAATTCCCCT-3 9 , which contains the M13-21 primer [underlined] and the complement to nucleotides 7359±7377 of HIV-1 HXB2).9 The PCR products were gel purified and sequenced directly using M13-21 and T7 promoter primers. Sequencing reactions were performed with the Amplitaq FS dye terminator kit and electrophoresed on a 373A DNA sequencer (ABI-Perkin Elmer, Foster City, CA). For each DNA sample duplicate dilution sets were made. For each set of serial dilutions the least concentrated dilution yielding a PCR product was used for analysis. Most of the duplicate PCRs generated identical PCR products. When the duplicate PCRs produced slightly differing sequences, a third PCR was done, after which the product sequences were aligned and a