Molecular and phylogenetic analysis of HIV-1 variants circulating in Italy
© Buonaguro et al; licensee BioMed Central Ltd. 2008
Received: 24 July 2008
Accepted: 10 October 2008
Published: 10 October 2008
The continuous identification of HIV-1 non-B subtypes and recombinant forms in Italy indicates the need of constant molecular epidemiology survey of genetic forms circulating and transmitted in the resident population.
The distribution of HIV-1 subtypes has been evaluated in 25 seropositive individuals residing in Italy, most of whom were infected through a sexual route during the 1995–2005 period. Each sample has been characterized by detailed molecular and phylogenetic analyses.
18 of the 25 samples were positive at HIV-1 PCR amplification. Three samples showed a nucleotide divergence compatible with a non-B subtype classification. The phylogenetic analysis, performed on both HIV-1 env and gag regions, confirms the molecular sub-typing prediction, given that 1 sample falls into the C subtype and 2 into the G subtype. The B subtype isolates show high levels of intra-subtype nucleotide divergence, compatible with a long-lasting epidemic and a progressive HIV-1 molecular diversification.
The Italian HIV-1 epidemic is still mostly attributable to the B subtype, regardless the transmission route, which shows an increasing nucleotide heterogeneity. Heterosexual transmission and the interracial blending, however, are slowly introducing novel HIV-1 subtypes. Therefore, a molecular monitoring is needed to follow the constant evolution of the HIV-1 epidemic.
Human immunodeficiency virus type 1 (HIV-1) shows an extensive genetic variability and can be classified into 9 phylogenetic subtypes (A-K), which are approximately equidistant from one another, and several circulating recombinant forms (CRFs), resulting from recombination events occurring between different HIV-1 subtypes co-circulating in a specific geographic region .
The first phase of the HIV epidemic in Italy has been mainly confined to the injecting drug users (IDUs) risk group, with an absolute predominance of HIV-1 B subtype, in accordance with other Western Countries. In particular, among the total AIDS cases reported in the adult population during the period between 1982 and 2006, 56.0% were IDUs (including also homosexual IDUs) with similar percentages in men and women groups (57.3% and 51.5%, respectively) . The annual percentages of AIDS cases reported in IDUs have gradually decreased from 65.8% in 1987 to 27.6% in 2006 , in part as consequence of prevention programs implemented in Italy to discourage syringe sharing [3, 4]. In parallel, the overall AIDS cases reported in heterosexual individuals account for the 19.5% of total epidemic cases, with a significantly higher percentage in the women category compared to men (41.2% vs 13.6%). However, the annual percentage of AIDS cases related to the heterosexual transmission has dramatically increased over the years, becoming in 2006 the most prevalent risk factor for AIDS (40.4%) .
Although almost 25% of heterosexual individuals diagnosed with AIDS in Italy are partners of long-term HIV-1 infected individuals, carrying a "historical" B-subtype virus, more than 10% of them are either immigrants from endemic regions for HIV-1 (6.87%) or their Italian partners (3.03%), while the risk is unidentified for 64% of them . This epidemiological evidence, based only on the AIDS reported cases and not considering all the HIV-1 infections derived also from travelling abroad, suggests that at least 10% of the viruses transmitted through heterosexual contacts could potentially belong to non-B subtypes and CRFs. In fact, HIV-1 isolates genetically related to subtypes novel to the Italian epidemic have been recently increasingly identified and described [5–17].
This has been recently reported in other European Countries, with a higher prevalence of non-B subtypes and CRFs due to an older tradition of immigration waves and much tighter historical as well as economic links with countries endemic for HIV-1 infection [18–30].
In this framework, the introduction and the possible spread of different HIV-1 subtypes and/or recombinant forms, which could require the future development of adequate diagnostic, treatment, and prevention strategies, needs to be constantly monitored.
For the present study, 25 HIV-seropositive individuals residing in Italy were enrolled at sentinel Centers, with a HIV-infection diagnosed in the period 1995–2005. The molecular study has been performed on the hypervariable C2-V3 region of the env gene as well as the more conserved 5' region of the gag p17 sequence. Three non-B-subtype HIV-1 isolates have been identified and phylogenetically classified as C (1 isolate) and G (2 isolates) subtype.
Blood samples were collected from 25 HIV-positive individuals attending Italian sentinel Centers in Bologna, Parma and Naples. For all of them, HIV infection was diagnosed during the 1995 to 2005 period, and most participants were infected through sexual contact (18 of 25, 72%). HIV-1 infection was diagnosed by immunologic methods (ELISA, Western blot), and the viral load was evaluated by viral RNA quantification. The full designation of samples, according to WHO-proposed nomenclature, is CI05.00XE or CI05.00XG, where CI stands for the city of enrolment, 05 stands for the year of study, 00 for the enrolment number and E (or G) stands for env (or gag). For the sake of simplicity, however, in this paper the samples have been indicated with CI05.01 (i.e. BO05.01).
Polymerase Chain Reaction (PCR)
Peripheral blood mononuclear cells (PBMCs) were purified from fresh HIV-1-positive blood samples by Leucoprep density gradient centrifugation, and cellular lysates (approximately 6 × 106 cells) were prepared by Proteinase K digestion at 56°C.
The quality of target DNA was verified by PCR amplification of the housekeeping p53 cellular gene. The amplification of HIV-1 V3-V5 region of the env gene and p17 region of the gag gene were performed by nested PCR analysis, using 1.5 × 105 cells (corresponding to approximately 1 μg of genomic DNA) as a template. The V3-V5 region of the HIV-1 env gene (666 bp) was amplified, as previously described, using the primer pairs ED5-ED12 and ES7-ES8 for the first and the second round of amplification, respectively [31, 32]. The p17 region (474 bp) was amplified, as previously described, using the primer pairs CL1028-CL1033 and CL1029-CL1032 for the first and the second round of amplification, respectively [14, 33].
Direct sequencing reactions were performed on PCR products purified with a rapid method developed in our laboratory, following the Sequenase protocol (United States Biochemical, Cleveland OH), modified in the labelling step (3 minutes on ice) [31, 34]. The internal annealing oligonucleotides, V3B and GAG B SENSE (annealing to a 19 bp fragment of the C2-V3 region and a 17 bp of the p17 region, respectively), were used to prime sense sequence reactions. Sequences were then analyzed on 6% polyacrylamide wedge sequencing gel.
Analysis of Sequences
The env and gag sequences obtained were aligned. Multiple sequence alignments were performed with the MegAlign application of the Lasergene software (DNASTAR Inc., Madison, WI) using the Clustal method. Phylogenetic trees were generated by using the neighbor-joining method with the PHYLIP software package (version 3.52c; Joseph Felsenstein, University of Washington). Briefly, the SEQBOOT program was carried out to generate 100 data sets that represent randomly re-sampled versions of the input-aligned sequences, to test the reliability of the final tree topology. Evolutionary distances were estimated by the DNADIST program, using either the Kimura 2-parameter method or the maximum likelihood distance method, and the phylogenetic relationships were determined by the NEIGHBOR program. A consensus tree was constructed using the CONSENSE program with the majority rule criterion and was drawn with the NJPLOT application.
Epidemiologic and clinical parameters
Epidemiologic and clinical parameters of enrolled subjects.
Presumed infection date
Amplification of Italian samples by PCR
Molecular analysis of env gene
Molecular analysis of gag gene
Phylogenetic analysis of C2-V3 env and p17 gag sequences
Nucleotide sequences have been pairwise aligned with HIV-1 reference standards of different subtypes and CRFs, discounting gaps due to nucleotide insertions/deletions. The alignments of HIV-1 env and gag nucleotide sequences have been used to generate phylogenetic trees by the neighbor-joining method. Confidence values for individual branches have been determined by a bootstrap analysis. The reference standards of different subtypes as well as CRFs included in this study cluster in the expected distinct phylogenetic branches in both env and gag sub-genomic regions, indicating that the length of sequence fragments used in this analysis is sufficient for the identification of known subtypes.
Moreover, all isolates show a concordant subtype classification in both env and gag sub-genomic regions, suggesting the absence of intra-genomic recombination events.
Peptide analysis and comparison
In regards to the non-B clade isolates identified in this study, and distributed in two different clades (C and CRF02-AG), the too limited number of samples hampers the possibility to derive a consensus sequence and to infer conclusions; nevertheless, it is worthwhile to mention that they all show the "fingerprint" GPGQ tetramer at the tip of their V3 loop sequences (data not shown).
Moreover, mutations in amino acid residues conferring resistance to fusion/binding inhibitors, in association with other mutations along the env sequence not analyzed in the present study, have been observed (i.e. Q296K), suggesting the transmission of isolates resistant to this class of antiretrovirals (Figure 6A) [35–37].
The p17 region of the B clade isolates shows a low amino-acidic variability and the consensus derived from the alignment shows an overall rate of amino acid conservation of 44.44% (36 of 81 residues). None of the observed amino acid changes is found in gag residues conferring drug resistance (Figure 6B). Similar results are observed also for non-B clade isolates.
A molecular and phylogenetic characterization was performed on HIV-1 variants identified in individuals residing in Italy and infected in the 1995–2005 period. The groups at high risk of HIV-1 infection (heterosexuals, homosexuals and IDUs) were equally represented in the present study. The C2-V5 and p17 regions of the env and gag gene have been amplified from uncultured PBMCs of Italian samples by the standardized nested PCR conditions.
Informative regions of the 2 structural gag and env genes have been studied in parallel, to have an immediate picture of the evolution pattern in viral regions under different immune pressure and to identify possible intra-genomic recombinants. In fact, considering the worldwide represented CRFs described until now, the p17 gag and the C2-V3 env regions show a different subtype designation in most of cases and are highly diagnostic for the identification of novel recombinants.
The average nucleotide divergences versus standard sequences of different subtypes suggest the presence of B and non-B subtypes among the Italian sequences identified in the present study. Moreover, within the B subtype sequences, the average divergence in the env C2-V3 region is 18.08%, with values ranging from 7.1 to 29.9%. On the contrary, the average divergence in the gag p17 region is 11.13%, with values ranging from 7.79 to 16.86%. These observed divergence values are compatible with samples identified in a geographical area characterized by a long-lasting HIV epidemic and confirm the more pronounced genetic evolution of env gene compared to gag.
The phylogenetic analysis confirmed the nucleotide divergence subtype prediction for both B and non-B-subtype classification; in particular, of the 3 non-B isolates, 2 cluster with CRF02-AG (NA05.05 and PR06.07), 1 with the C subtype (PR06.03). Moreover, for all the B- and non-B-subtype sequences, the phylogenetic classification matches in the gag and env sub-genomic regions, suggesting the absence of intra-genomic recombination events.
The phenetic analysis of the V3 region shows a significant amino acid stability in 9 residues of the V3 loop, for B-subtype isolates, confirming the strong selection for specific sequences involved in strategic functions regarding the immune response as well as cellular tropism and transmission. The different HIV-1 isolates identified in the current study show the subtype-specific tetrameric sequence at the apex of the V3 loop (GPGR for B clade, GPGQ for non-B clades), which is considered the target for the anti-V3 neutralizing antibodies. However, less-represented sequences have been identified at the tip of the B clade isolates (GPGG, GPGS, GPGQ), suggesting a constant diversification in this clade.
The overall results suggest that the B subtype is still largely predominant in the HIV-1 epidemic in Italy and is circulating among all risk groups. On the contrary, HIV-1 non-B subtypes in Italy are strictly associated with the heterosexual transmission and are identified in infections acquired in the last period (2001–2005). The three non-B-subtype HIV-1 isolates are strictly associated with heterosexual transmission. The samples PR06.03 and PR06.07, corresponding to an Ethiopian and a Nigerian subject respectively, belong to the subtype/CRF predominant in their respective Country of origin, suggesting an infection either prior to the immigration to Italy or subsequent but through heterosexual contacts with HIV-infected subjects from those Countries. The sample NA05.05, on the contrary, corresponds to an Italian-born subject with a possible partner/s from Regions with high endemicity for HIV infections.
These results confirm that in Italy, as in other Western European countries, non-B subtypes or recombinant forms are introduced by immigrants/migrants and transmitted at a low rate to the indigenous population. This would also explain the lower prevalence of non-B subtypes in Italy compared with other European countries with an older tradition of immigration waves and much tighter historic and economic links with African countries.
The presented data are representative of a nationwide molecular survey and, regardless the small sample size, are part of recurrent studies giving a constant updated picture of the genetic evolution of the HIV-1 epidemics in different risk groups in Italy.
This study was supported by grant from the Ministero Italiano della Sanità (Ricerca Corrente; Progetto Finalizzato AIDS 2006, #20G.19 and the SIVIM study group for DNA test standardization). The GenBank accession numbers for the sequences included in this manuscript are FJ447882 to FJ447897, for the env sequences, and FJ447898 to FJ447916, for the gag sequences.
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