Rapid evolution of the env gene leader sequence in cats naturally infected with feline immunodeficiency virus

Abstract

Analysing the evolution of feline immunodeficiency virus (FIV) at the intra-host level is important in order to address whether the diversity and composition of viral quasispecies affect disease progression. We examined the intra-host diversity and the evolutionary rates of the entire env and structural fragments of the env sequences obtained from sequential blood samples in 43 naturally infected domestic cats that displayed different clinical outcomes. We observed in the majority of cats that FIV env showed very low levels of intra-host diversity. We estimated that env evolved at a rate of 1.16×10(-3) substitutions per site per year and demonstrated that recombinant sequences evolved faster than non-recombinant sequences. It was evident that the V3-V5 fragment of FIV env displayed higher evolutionary rates in healthy cats than in those with terminal illness. Our study provided the first evidence that the leader sequence of env, rather than the V3-V5 sequence, had the highest intra-host diversity and the highest evolutionary rate of all env fragments, consistent with this region being under a strong selective pressure for genetic variation. Overall, FIV env displayed relatively low intra-host diversity and evolved slowly in naturally infected cats. The maximum evolutionary rate was observed in the leader sequence of env. Although genetic stability is not necessarily a prerequisite for clinical stability, the higher genetic stability of FIV compared with human immunodeficiency virus might explain why many naturally infected cats do not progress rapidly to AIDS.

Fig. 1.

ML tree, based on the HKY model, rooted on a clade C reference FIV env. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The ML phylogeny includes 47 entire env nucleotide sequences (representative of a total of 355 sequences from Chicago and Memphis), 15 entire env sequences derived from GenBank: Aomori 1 (GenBank accession number D37816), Aomori 2 (GenBank accession number D37817.1), FIV C (GenBank accession number AF474246.1), Dixon (GenBank accession number L00608.1), Dutch (GenBank accession number X60725), Fukuoka (GenBank accession number D37815.1), Sendai 1 (GenBank accession number D37813.1), Shizuoka (GenBank accession number D37811.1), UK2 (GenBank accession number X69494.1), UK8 (GenBank accession number X69496.1), USIL2489 (GenBank accession number U11820.1), Yokohama (GenBank accession number D37812.1), Petaluma (GenBank accession number M25381.1), PPR (GenBank accession number M36968.1), Leviano (GenBank accession number FJ374696.1), three V3–V5 region sequences representing clade E: LP3 (GenBank accession number D84496), LP20 (GenBank accession number D84498) and LP24 (GenBank accession number D84500), and one shorter 504 bp in length RUS14 (GenBank accession number EF447297) sequence. Taxa with inconsistent clade assignment are represented with a star (P8, P21). Non-monophyletic taxa from cat M5 are marked with a triangle. Only bootstrap values >80 are shown.

Fig. 2.

Comparison of mean pairwise distances between env fragments and entire env sequences amplified from three samplings (A, B and C on the x-axis). Median values for the leader, V3–V5, NV and env, respectively, for time points A (0.20, 0.11, 0.16 and 0.13 %), B (0.20, 0.12, 0.12 and 0.11 %) and C (0.27, 0.19, 0.14 and 0.17 %).

Fig. 3.

Frequency and location of predicted PNGSs (a) and sites under positive selection (b) with corresponding dN/dS (ω) values. PNGSs at positions 303, 335, 347, 536, 553 and 738 represent fixed sequons among all clades of FIV from different geographical origins (n = 329). Sequons at positions 263, 279, 423, 427, 504, 734 and 341, 523 and 746 were also fixed, and were present in >99 % of sequences. Positively selected sites (n = 10) were consistently identified by three different detection methods (for details, see Table S2). Leader sequence and B-cell epitope regions identified in previous studies are highlighted in grey. The putative leader sequence cleavage site is located at the position 528 bp from the start codon of the env ORF of reference FIV Petaluma (GenBank accession number M25381.1). Location of V1–V8 regions is highlighted below the graphs.

Fig. 4.

Distribution of the evolutionary rates calculated for the leader, V3–V5 fragment, NV fragment and entire env gene. Evolutionary rates are compared between sequences amplified from Memphis (M), Chicago (C) and all cats (SG). Rate estimates were either based on strict or relaxed clock models (for details, see Table S3).

Fig. 5.

Comparison of evolutionary rates of the leader, V3–V5, NV fragments and the entire env genes from six alive (healthy, A) and 11 deceased (unhealthy, D) cats from the Memphis cohort during the 12 month observation period. Rate estimates were either based on strict or relaxed clock models (for details, see Table S3).