Molecular and phylogenetic analysis of HIV-1 variants circulating in Italy

Abstract

Objective: 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.

Methods: 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.

Results: 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.

Conclusion: 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.

Figure 1

Analysis of DNA fragments obtained by nested PCR on the C2-V5 env (A) and the p17 gag (B) genes of HIV-1. Examples of specific env fragments of 666 bp (panel A) and gag fragments of 474 bp (panel B), obtained by nested PCR have been separated on a 1% agarose gel. M = λ/Hind III, C1 = I round negative control; C2 = II round negative control; + = positive control.

Figure 2

Average nucleotide divergence of C2-V5 env gene versus standard sequences of different clades. The shown average divergence values have been obtained aligning the Italian sequences with standard sequences of individual clades, Groups and CRF02_AG. (A) The lowest average divergence value of the whole group of sequences is versus B standard sequences (arrow); (B) the sample PR06.07 shows the lowest average divergence value versus CRF02_AG standards (arrow); (C) the sample PR06.03 shows the lowest average divergence versus clade C standard sequences (arrow) (p < 0.01).

Figure 3

Average nucleotide divergence of p17 gag gene versus standard sequences of different clades. The shown average divergence values have been obtained aligning the Italian sequences with standard sequences of individual clades. (A) The lowest average divergence value of the whole group of sequences is versus B standard sequences (arrow); (B) the sample PR06.07 and (C) the sample NA05.05 show the lowest average divergence value versus CRF02_AG standards (arrow); (D) the sample PR06.03 shows the lowest average divergence versus clade C standard sequences (arrow) (p < 0.01).

Figure 4

Phylogenetic classification of the C2-V3 env region. The C2-V3 env region of Italian samples has been aligned with standard sequences of HIV-1 group M including some known CRF. Sequences from groups N and O have been used as outgroup. Italian sequences are indicated as underlined. Reliability has been estimated by boot-strap analysis. The bar shows a 10% divergence.

Figure 5

Phylogenetic classification of the p17 region of gag gene. The p17 region of gag gene from Italian samples has been aligned with standard sequences of HIV-1 group M including some known CRF. Sequences from groups N and O have been used as outgroup. Italian sequences are indicated as underlined. Reliability has been estimated by boot-strap analysis. The bar shows a 10% divergence.

Figure 6

Alignment of the amino acid sequences. (A) V3 env region and (B) p17 gag region have been aligned, and a consensus sequence (top) has been generated. Question marks (?) have been introduced in the consensus when, in the specific position, no residue is found in more than 50% of sequences. Dots (.) indicate agreement with the consensus and dashes (-) indicate a gap inserted to maintain the alignment. Gray areas indicate perfectly conserved residues. Boxed residues indicate mutations possibly conferring resistance to class of drugs.