Genetic divergence of HIV-1 B subtype in Italy over the years 2003-2016 and impact on CTL escape prevalence

Abstract

HIV-1 is characterized by high genetic variability, with implications for spread, and immune-escape selection. Here, the genetic modification of HIV-1 B subtype over time was evaluated on 3,328 pol and 1,152 V3 sequences belonging to B subtype and collected from individuals diagnosed in Italy between 2003 and 2016. Sequences were analyzed for genetic-distance from consensus-B (Tajima-Nei), non-synonymous and synonymous rates (dN and dS), CTL escapes, and intra-host evolution over four time-spans (2003-2006, 2007-2009, 2010-2012, 2013-2016). Genetic-distance increased over time for both pol and V3 sequences (P < 0.0001 and 0.0003). Similar results were obtained for dN and dS. Entropy-value significantly increased at 16 pol and two V3 amino acid positions. Seven of them were CTL escape positions (protease: 71; reverse-transcriptase: 35, 162, 177, 202, 207, 211). Sequences with ≥3 CTL escapes increased from 36.1% in 2003-2006 to 54.0% in 2013-2016 (P < 0.0001), and showed better intra-host adaptation than those containing ≤2 CTL escapes (intra-host evolution: 3.0 × 10^-3 [2.9 × 10^-3-3.1 × 10^-3] vs. 4.3 × 10^-3 [4.0 × 10^-3-5.0 × 10^-3], P[LRT] < 0.0001[21.09]). These data provide evidence of still ongoing modifications, involving CTL escape mutations, in circulating HIV-1 B subtype in Italy. These modifications might affect the process of HIV-1 adaptation to the host, as suggested by the slow intra-host evolution characterizing viruses with a high number of CTL escapes.

Figure 1

Genetic distance of 3,328 (A) and 210 (B) HIV-1 B subtype pol sequences, belonging to newly diagnosed and recently infected patients, respectively, divided into four time frames in accordance with year of diagnosis. ^aGenetic distance was calculated by comparing sequences with HIV-1 consensus B using Tajima Nei model, MEGA 6. ^bBy Kruskal-Wallis test, corrected for Benjamini-Hochberg method. SE: standard error.

Figure 2

Genetic distance of 1,152 (A) and 66 (B) HIV-1 B subtype V3 sequences, belonging to newly diagnosed and recently infected patients, respectively, divided into three time frames. ^aGenetic distance was calculated by comparing HIV-1 sequences with HIV-1 consensus B using Tajima Nei model, MEGA 6. ^bBy Kruskal-Wallis test, corrected for Benjamini-Hochberg method. SE: standard error.

Figure 3

Amino acid positions characterized by an increased entropy value in the last time frame. Pol sequences obtained in the period 2003–2006 and those obtained in the period 2013–2016 were submitted for entropy analysis at HIV Los Alamos National Laboratory Entropy-Two tool (https://www.hiv.lanl.gov/content/sequence/ENTROPY/entropy.html) in order to obtain delta entropy values. The same analysis was performed by comparing V3 sequences obtained in the period 2007–2009 and in the period 2013–2016. Only delta values ≤0.10 and >0.10 followed by a P ≤ 0.05 after Benjamini-Hockberg correction were considered statistically significant.