Recombination in feline immunodeficiency virus from feral and companion domestic cats

Abstract

Background: Recombination is a relatively common phenomenon in retroviruses. We investigated recombination in Feline Immunodeficiency Virus from naturally-infected New Zealand domestic cats (Felis catus) by sequencing regions of the gag, pol and env genes.

Results: The occurrence of intragenic recombination was highest in env, with evidence of recombination in 6.4% (n = 156) of all cats. A further recombinant was identified in each of the gag (n = 48) and pol (n = 91) genes. Comparisons of phylogenetic trees across genes identified cases of incongruence, indicating intergenic recombination. Three (7.7%, n = 39) of these incongruencies were found to be significantly different using the Shimodaira-Hasegawa test.Surprisingly, our phylogenies from the gag and pol genes showed that no New Zealand sequences group with reference subtype C sequences within intrasubtype pairwise distances. Indeed, we find one and two distinct unknown subtype groups in gag and pol, respectively. These observations cause us to speculate that these New Zealand FIV strains have undergone several recombination events between subtype A parent strains and undefined unknown subtype strains, similar to the evolutionary history hypothesised for HIV-1 "subtype E".Endpoint dilution sequencing was used to confirm the consensus sequences of the putative recombinants and unknown subtype groups, providing evidence for the authenticity of these sequences. Endpoint dilution sequencing also resulted in the identification of a dual infection event in the env gene. In addition, an intrahost recombination event between variants of the same subtype in the pol gene was established. This is the first known example of naturally-occurring recombination in a cat with infection of the parent strains.

Conclusion: Evidence of intragenic recombination in the gag, pol and env regions, and complex intergenic recombination, of FIV from naturally-infected domestic cats in New Zealand was found. Strains of unknown subtype were identified in all three gene regions. These results have implications for the use of the current FIV vaccine in New Zealand.

Figure 1

NJ phylogenetic tree of env sequences from NZ domestic cats. Tree is rooted by subtype B. Subtypes are shown along the right side of the tree. Bootstrap values based on 1000 replicates are shown for the major groups. Sequences with ⇐ are used as reference sequences in the RIP analyses. Outlier sequences are 258, PN22, 259, 168, PN17, PN23, PN27, PN21, PN18, 197, 260, MF14, 214. U-NZenv is a group of NZ sequences that does not group with a known subtype. Reference sequences from GENBANK are; subtype A: Sendai1 (D37813, Japan), Petaluma (M25381, USA), DEBAb91 (AF531043, Germany), UK2 (X69494, UK), SwissZ2 (X57001, Switzerland), Wo (L06135, France) and Ca2 (DQ873714, South Africa); subtype B: TM2 (M59418, Japan), ItalyM2 (X69501, Italy), ATVIa33 (AF531045, Austria), LP9 (D84497, Argentina) and 14-02PalP (DQ072558, Portugal); subtype C: CABCpbar01C (U02393, Canada), TI-2 (AB016026, Taiwan), DEBAfred (U57020, Germany), BM3070 (AF474246, Canada), FIV-C36 (AY600517, USA) and VND-8 (AB083509, Vietnam); subtype D: MC8 (D67062, Japan), Shizuoka (D37811, Japan), Fukuoka (D37815, Japan) and VND-1 (AB083502, Vietnam); subtype E: LP3 (D84496, Argentina) and LP20 (D84498, Argentina).

Figure 2

NJ phylogenetic tree of gag sequences from NZ domestic cats. Tree is rooted by subtype B. Subtypes are shown along the right side of the tree. Bootstrap values based on 1000 replicates are shown for the major groups. Sequences with ⇐ are used as reference sequences in the RIP analyses. Outlier sequence is PN6. U-NZgag is a group of NZ sequences that does not group with a known subtype. Reference sequences from GENBANK are; subtype A: CaONA22 (AY369383, Canada), Petaluma (M25381, USA); subtype B: CaONB06 (AY369381, Canada), TM2 (M59418, Japan), RP1 (AJ304962, Portugal); subtype C: BM3070 (AF474246, Canada), FIVC36 (AY600517, USA), CaONC02 (AY369384, Canada).

Figure 3

NJ phylogenetic tree of pol sequences from NZ domestic cats. Tree is rooted by subtype B. Subtypes are shown along the right side of the tree. Bootstrap values based on 1000 replicates are shown for the major groups. Sequences with ⇐ are used as reference sequences in the RIP analyses. Outlier sequences are Ni and PN9. U-NZpol1 and U-NZpol2 are groups of NZ sequences that do not group with a known subtype. Reference sequences from GENBANK are; subtype A: T90 (S67753, Australia), Petaluma (M25381, USA), FIV-Fca155-1 (U53760, Argentina), FIV-FcaTK1-11 (U53762, England); subtype B: TM2 (E03581, Japan), USIL2489 (U11820, USA); subtype C: BM3070 (AF474246, Canada), FIV-C36 (AY600517, USA).

Figure 4

RIP outputs. Reference sequences used are highlighted in the respective NJ trees in Figs. 1, 2, 3. (A) Two new outlier env sequences. (a) PN18; (b) PN27. Red is similarity to subtype A, green is similarity to subtype C. (B) gag outlier sequence, PN6. Red is similarity to subtype A, green is similarity to U-NZgag. (C) pol outlier sequences. (a) Ni: red is similarity to subtype A, green is similarity to U-NZpol1, blue is similarity to U-NZpol2. (b) PN9: red is similarity to subtype B, green is similarity to U-NZpol1.

Figure 5

RIP outputs for env sequences from Duarte & Tavares (2006). (A) 150_02LisP; (B) 164_02UZP. Red is similarity to reference subtype A (Petaluma); green is similarity to reference subtype B (TM2).

Figure 6

Gag, pol and env ML trees of sequences for which all three genes were amplified. Lines between the trees link sequences from the same sample, to highlight incongruencies. The three thickened lines are the statistically significant intergenic recombinant sequences. Reference sequences used are: subtype A (Petaluma, M25381); subtype B (TM2, M59418); subtype C (FIV-C36, AY600517).

Figure 7

Intrahost recombination evidence from endpoint dilution sequencing of pol gene of TKP94. (a) NJ tree showing the eleven endpoint dilution sequences for the pol gene of TKP94. * represents sequences used as reference sequences in the RIP analysis. ** represents the outlier sequence. (b) RIP output for the outlier sequence. Red is similarity to reference sequence from group one; green is similarity to reference sequence from group two.

Figure 8

Dual subtype infection identified from endpoint dilution sequencing of env gene of TKP95. NJ tree showing the thirteen endpoint dilution sequences (●) and the consensus (*) from the env gene of TKP95. Reference sequences (○) are included to show subtypes.

Figure 9

NZ map showing locations of cats infected with different putative intragenic recombinant sequence crossover patterns. Sample numbers for cats in the upper half of the North Island, the lower half of the North Island, and the South Island refer to companion cats only.