May Phylogenetic Analysis Support Epidemiological Investigation in Identifying the Source of HIV Infection?

Abstract

Editor: The human immunodeficiency virus (HIV) is characterized by great genetic heterogeneity driven by several factors, such as the lack of proofreading ability of the reverse transcriptase (RT), the rapid turnover of HIV-1 in vivo, host selective immune pressures, and recombination events during replication. In particular, HIV-1 group M subtypes are responsible for most of the infections worldwide. At the end of the year 2008, in Italy, 60,346 persons were cumulatively diagnosed with AIDS. Overall, it is estimated that 140,000/150,000 HIV-positive people live within the country. At the beginning of the epidemic the most common route of HIV transmission was needle sharing among intravenous drug users (IDUs) whereas in more recent years heterosexual contacts have become the leading cause of infection (44.7%). Phylogenetic analysis is an important way to trace epidemiological relationship in cases with dubious or unknown links. Moreover, phylogeny is also an easy way to describe and to identify groups of viral isolates forming genetically related sister clades, which appear to be more closely related to each other. We describe here a case of HIV-1 infection in a 24-year-old Italian soldier in service in Kabul, Afghanistan, where his military command was placed. The epidemiological investigation was conducted in three different phases. In September 2009, during a laboratory investigation in the Military Barracks based on demand screening, he was found to be positive for anti-HIV antibodies (by an ELISA test), which was confirmed by Western blot. The viral load was 39,882 copies/ml and the CD4 cell count was 568 cells/ml. During the month of October he was hospitalized in Catanzaro Hospital for diagnostic controls. His viral load was of 18,000 copies/ml and his CD4 cell count was 701 cells/ml. The epidemiological investigation did not identify any known risk factors for HIV infection (i.e., drug use, tattoos, homosexual or heterosexual contact, biological fluid exposure). Thus, we tried to reconstruct the epidemiological network using the pol viral sequences (1271 nucleotides) detected by a sample collected in November 2009, before starting antiretroviral therapy. RNA extraction, polymerase chain reaction (PCR) amplification, and sequences were obtained as previously described. The pol gene sequence was aligned and compared with reference sequences of the HIV-1 pol genes downloaded from the HIV Los Alamos database (htpp://hiv-web.lanl .gov) as previously described; the HIV-1 pol sequences were obtained with the Blast similarity search (using the Italian soldier’s sequence as the query sequence). HIV-1 subtype B pol sequences obtained by Blast similarity search analysis are reported in Supplementary Table 1 (Supplementary data are available online at www.liebertonline.com/aid). The tree was generated using the F84 model of substitution using both neighbor-joining (NJ) and maximum-likelihood (ML) tree building methods. The evolutionary model was chosen as the best-fitting nucleotide substitution model according to the hierarchical likelihood ratio test (HLRT) implemented with Model Test v3.0 software. The statistical robustness and reliability of the branching order within each phylogenetic tree were confirmed by bootstrap analysis using 1000 replicates for the NJ tree and with the zero branch length test for the ML tree. All calculations were performed with PAUP*4.0 software. SimPlot software version 3.2 and the split-decomposition analysis were used to investigate the recombinant. Separate phylogenetic analyses of the subalignments generated by the different break-points were also used to better investigate the supposed recombinant sequences. Phylogenetic analysis classified our pol gene sequence as a ‘‘nonpure’’ B subtype and showed that the patient’s isolate was closely related to B subtype sequences from Spain, forming a significant monophyletic cluster (bootstrap value >70%) (Fig. 1). Because our sequence was in a marginal position within the cluster, SimPlot and split-decomposition analyses were performed and our sequence was found to be a possible BFB recombinant form; however, we classified it as ‘‘BUB’’ (U for unclassified) since the F trait and the break-point of recombination were under the threshold value (70%) useful for statistical significance (Supplementary Figs. 1 and 2). The young man was enrolled in the peace military mission of the ISAF (International Security Assistance Force). In this context, soldiers from different nations (Italy, the United States,