Department of Pathobiology, University of Guelph, Guelph, ON, Canada, N1G 2W1
Correspondence
D. Bienzle
dbienzle{at}uoguelph.ca
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ABSTRACT |
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The GenBank accession numbers of the sequences reported in this article are AY220046AY220074 and AY369376AY369384 (LTRgag), and AY221627AY221629 (envLTR).
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INTRODUCTION |
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FIV is distributed worldwide (Bachmann et al., 1997; Ishida et al., 1989
; Nishimura et al., 1998
; Pedersen et al., 1989
). The prevalence of infection is highly variable ranging from 1 % in cats at low risk in the USA and Canada (Yamamoto et al., 1989
) to 44 % in symptomatic cats in Japan (Hohdatsu et al., 1998
), depending upon factors such as age, gender and indoor or outdoor housing. Currently, FIV isolates are classified into five different subtypes designated A, B, C, D and E, based on envelope sequence analysis (Nishimura et al., 1998
). Subtype A has been reported from California and Northern Europe while subtype B was prevalent in the central and eastern USA and in southern European countries (Sodora et al., 1994
). Subtype C has been identified from California and British Columbia (Bachmann et al., 1997
; Sodora et al., 1994
), while subtypes D and E have been reported from Japan (Hohdatsu et al., 1996
, 1998
; Nishimura et al., 1998
) and Argentina (Pecoraro et al., 1996
), respectively. Classification into subtypes may be accomplished by different methods including subtype-specific PCR (Nishimura et al., 1998
), heteroduplex mobility assays (Bachmann et al., 1997
), restriction fragment polymorphisms (Hohdatsu et al., 1998
; Kurosawa et al., 1999
) and phylogenetic analysis of sequences (Worobey & Holmes, 1999
). Although most studies are based on env gene variability, comparison of sequences of the gag gene can be useful, since this gene also has marked variability among different FIV isolates (Hohdatsu et al., 1998
; Kakinuma et al., 1995
; Pistello et al., 1997
).
Co-infection with two different lentiviral strains might occur following exposure to a second virus shortly after the initial infection or after the initial infection has been established, a condition termed superinfection (Jost et al., 2002). This phenomenon has been experimentally induced with FIV subtypes A and B, both in vitro and in vivo (Okada et al., 1994
; Pistello et al., 1999
). In another study, consecutive exposure of cats to two different FIV strains resulted in superinfection in one cat and recombination in another (Kyaw-Tanner et al., 1994
). This conclusion was based on sequence analysis of 516 bp of env, showing that 106 bp were highly homologous to one parental strain and the remainder to the other parental strain. It was suggested that a high inoculation dose of virus and the route of infection may have favoured superinfection, and that superinfection and recombination should be studied under natural conditions (Kyaw-Tanner et al., 1994
). Since genetic diversity among lentiviruses is extensive, it has been debated whether a vaccine should include a single virus strain, a broad spectrum of viral variants or only those that are relevant to a particular geographical area (Gao et al., 1998
). Similar to HIV, intersubtype recombination might be an important consideration for development of vaccines effective against multiple FIV subtypes (Siepel et al., 1995
).
Neither the prevalence of FIV infection in Canada nor the prevailing subtypes are known. Identification of circulating subtypes is essential to develop strategies for molecular diagnosis, since the genetic diversity is high. In addition, the coexistence of more than one subtype in a particular location raises the possibility of recombination, creating variants with novel features that might interfere with diagnostic tests. Knowing prevalent FIV subtypes is also an important consideration for designing and testing vaccines under field conditions and would aid in establishing potential associations between specific subtypes and the severity of clinical outcomes. Thus, the aims of this study were to identify primary FIV subtypes present in Canada, to detect associated unique clinical conditions and to investigate the occurrence of intersubtype recombination. For this purpose, we sequenced the LTRgag gene region from 40 FIV primary isolates and the envLTR region from selected samples. Recombination was assessed by phylogenetic analysis of nucleotide and amino acid sequences as well as by analysis of similarities to reference sequences.
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METHODS |
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LTRgag PCR.
Primers were designed to amplify the LTRgag region of proviral DNA. Base pair identification of all sequences including primers and amplicons was in reference to FIV subtype A, strain Petaluma (GenBank accession no. M25381; Talbott et al., 1989). The relative locations of the primers are indicated in Fig. 1
. First-round reactions produced amplicons of 1287 bp using primers LTR1-f and Gag1-r (Table 2
). The second round of PCR yielded products of 1127 bp using a different forward primer (LTR2-f) and primer Gag1-r. This PCR product included the LTR regions R and U5, the gag region encoding the p15 protein and 421 bp of the gag gene encoding the p24 protein. Amplifications were performed in 25 µl total volume with 1·25 units Taq DNA polymerase (Life Technologies), 1·5 mM MgCl2, 0·2 mM each dNTP, 0·5 µM each primer and 2 µl purified DNA. For second-round reactions, 2 µl of the first-round PCR product was used as a template. Reactions were cycled 35 times with denaturation at 94 °C for 45 s, annealing at 60 °C for 20 s and elongation at 72 °C for 90 s. Second-round conditions were identical except that the annealing temperature was 64 °C for 20 s. PCR products were identified by electrophoresis in 1 % agarose gels and ethidium bromide staining.
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PCR-based limiting dilution assay (PLDA).
To minimize the likelihood of resampling the same DNA molecule during PCR amplification, the number of copies of provirus from selected samples was estimated. Serial twofold dilutions of template were PCR-amplified using LTR-1 and Gag1-r primers. Five replicates of four template dilutions that yielded a mixture of positive and negative PCR results were then amplified with the same primers. The template copy number was estimated with the software QUALITY, as described previously (Rodrigo et al., 1997).
Cloning experiments and sample preparation for sequencing.
Amplicons of the appropriate size were cut out of agarose gels with sterile blades and the DNA was extracted (QIAquick Gel Extraction kit; Qiagen). Purified PCR products for the LTRgag region from all recombinant samples were cloned into a plasmid (TOPO TA Cloning kit; Life Technologies) and sequenced. The concentration of purified DNA samples was determined by fluorimetry (PicoGreen; Roche) and adjusted to 30 ng µl1. Due to the size of the envLTR fragments, it was not possible to get full sequences with primers MB-1 and LTR3-r; therefore, sequencing primers Env2-f and Env3-f were designed to anneal at nt 78487872 and nt 83408361 (Table 2), 535 and 1025 bp along the 2 kb fragment, respectively. Results from the four overlapping subfragments were aligned to reassemble contiguous sequences. Sequences were determined by the BigDye Terminator method on an ABI Prism 377 XL DNA Sequencer (DNA Sequencing Facility, Robarts Research Institute, London, ON).
Alignments and phylogenetic analysis.
Nucleotide sequence analysis was performed to account for the non-coding function of the LTR. This analysis included 1040 bp (310 bp of LTR and 730 bp of gag) from four reference sequences and 40 unknown FIV samples. Sequences were edited using VECTOR NTI (Version 7.0; InforMax, Calgary, AB) and multiple alignments were performed with CLUSTAL_X (Thompson et al., 1997). Gaps were inserted to compensate for genetic variation among sequences and penalties for gaps and for gap extensions were assigned to minimize the number of possible insertions without breaking the homology of contiguous sequences. Further adjustments were done manually to maximize similarities. Nucleotide alignments were analysed with MODELTEST (Posada & Crandall, 1998
) to determine the correct substitution model. Settings from the best-fit model (selected by hierarchical likelihood ratio tests) were then used to create trees with PAUP (Swofford, 2002
), based on the criteria of parsimony, maximum-likelihood and minimum evolution. Bootstrap analyses were performed on 1000 iterations for parsimony and minimum evolution and on 100 iterations for maximum-likelihood. Amino acid sequence analysis was performed using PHYLIP (Felsenstein, 1993
) to compare clade assignment of selected sequences based on two different genomic regions. This included 242 and 491 aa sequences deduced from gag and env sequences, respectively. Alignments were analysed with PROTDIST to create distance matrices based on the Dayhoff PAM model for substitutions between each pair of amino acids. Neighbour-joining trees were then constructed. To compare different measures, additional trees were created based on the criteria of parsimony and maximum-likelihood. For statistical support in consistency of the clades, bootstraps were performed on 1000 iterations for parsimony and minimal evolution and on 100 iterations for maximum-likelihood. Final editing of the trees and graphical representations were obtained using TREEVIEW (Page, 1996
).
Analysis of recombinants.
Identification of specific patterns of recombination was by direct examination of multiple alignments and sequence analysis using SIMPLOT. This software was developed to demonstrate recombination among HIV sequences and is similar in design to the Recombination Inference Program (RIP) (Lole et al., 1999; Papa et al., 2002
; Siepel et al., 1995
). Briefly, the similarities of a testing sequence compared with two reference sequences and a third sequence included as an outgroup are analysed. In a window of user-specified size (200 bp in this study), similarity values are calculated and repeated for the entire region with 20 bp overlaps. Similarity values are then plotted to indicate the percentage of similarity along the entire sequence. To predict the location of breakpoints, the software analyses the alignments by maximization of
2 (Smith, 1992
).
Subtype-specific PCR.
To identify co-infection with a recombinant virus plus a subtype A or B virus, subtype-specific PCR assays were designed. A common forward primer (LTR1-f) annealed to a conserved region in the LTR and was combined with reverse primers homologous to subtype A or B. The reverse primers, Gag(A)-r and Gag(B)-r, had two or more mismatches at the 3' end relative to the other subtype (Table 2). Each was designed to anneal upstream or downstream of the consistent A/B breakpoint identified in gag at the junction between the p15 and p24 coding regions (Fig. 2
). Gag(A)-r annealed on the 5' side of the breakpoint, while primer Gag(B)-r annealed to the 3' side, but neither matched the sequence of the A/B recombinants in this region. Therefore, these primers identified subtypes A and B in the presence of A/B recombinant sequences. PCR reagents and cycling conditions were as described above for the first round of the LTRgag PCR.
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RESULTS |
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Phylogenetic analysis
To classify unknown isolates within their respective subtypes, a parsimony tree was constructed based on 1040 bp from the LTRgag region, including reference sequences from subtypes A, B and C (Fig. 3a). To assess the consistency of clade assignment (Hall, 2001
), additional trees were created based on the criteria of maximum-likelihood (Fig. 3b
) and minimum evolution (not shown). Trees from the three different methods showed that similar structure and group assignment of samples was well supported by bootstrapping (Fig. 3a, b
). All four US samples were classified as subtype B. Of the 36 Canadian samples, 23 (64 %) were identified as subgroup A, seven (19 %) as subgroup B and two (6 %) as subgroup C. Four (11 %) samples (CaONAB01, CaONAB02, CaONAB03 and CaONAC01) showed inconsistent subgroup assignment. Three of these sequences (CaONAB01, CaONAB02 and CaONAB03) appeared related to subtype B while the remaining sequence (CaONAC01) was most similar to subtype C. This suggested that these four sequences were either highly divergent or potentially derived from more than one subtype. To investigate these possibilities further, the relationship with prototype FIV subtypes was examined through minimum evolution trees based on translated Gag and Env regions. Additional parsimony and maximum-likelihood trees were also created to compare different methods (not shown). Trees from the three different methods showed similar structure and the group assignment was well supported by bootstrapping (Fig. 4
). Three samples (CaONAB01, CaONAB02 and CaONAB03) clustered with subtype B and the remaining sample (CaONAC01) was a subtype C within the Gag region (Fig. 4a
). In contrast, three sequences (CaONAB02, CaONAB03 and CaONAC01) clustered with subtype A in the Env region (Fig. 4b
) while the other sample (CaONAB01) remained within subtype B. Inconsistent clade assignment in LTRgag and discrepancies between Gag and Env indicated that these samples were A/B (CaONAB01, CaONAB02 and CaONAB03) and A/C (CaONAC01) intersubtype recombinants. To investigate these possibilities, an additional 2 kb of proviral DNA encompassing the envLTR junction was amplified and sequenced.
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Co-infection
For recombination to occur within an infected cat, genomic RNA from two different viruses has to be present simultaneously within the same cell. Cats infected with recombinant viruses might concurrently have been infected with their parental subtypes. To distinguish parental subtypes A and/or B from A/B recombinants, DNA from these samples was subjected to subtype-specific PCR. Isolates CaONAB01 and CaONAB03 yielded neither subtype A- nor subtype B-specific products, indicating absence of co-infection; however, subtype-specific PCR amplification of CaONAB02 yielded a product with both sets of primers. Close visual inspection of the sequence from CaONAB02 revealed high similarity relative to subtype A and B sequences in the primer-annealing region. To characterize this sample further, PLDA was performed. Multiple alignments with at least five replicate sequences indicated identities of 99 %. We therefore concluded that in this case the subtype-specific PCR had insufficient discriminatory ability and that the cat was infected with a single recombinant virus.
FIV subtypes and clinical outcomes
Relevant clinical information was available for the majority of FIV-infected cats (Table 1). Clinical conditions ranged from no abnormalities to stomatitis, abscessation, diarrhoea, lymphoma and neuropathy. Associations between clinical conditions and FIV subtype could not be identified.
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DISCUSSION |
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Recovery of recombinant virus sequences from four (11 %) of 36 Canadian samples is an intriguing finding that may be due to the intrinsic propensity of retroviruses for recombination or perhaps due to transmission of recombinant strains among cats. Sequence and phylogenetic analysis are considered effective tools to identify recombination (Worobey & Holmes, 1999). DNA phylogenetic analysis from LTRgag was performed because 30 % of the sequences corresponded to the LTR, which are non-coding regions, thus providing the advantage of analysing longer sequences. However, this analysis showed that four samples did not group within any particular subtype. Since nucleotide changes have different impacts on the amino acid sequence depending on the relative location of the substitutions, parsimony trees were created with translated Gag and Env sequences. These analyses not only have more biological relevance but they also allowed for comparison between two distant genomic regions of the virus, thus providing further consistency of clade assignment. Phylogenetic trees based on Gag classified the A/C recombinant as subtype C, while all A/B samples were subtype B. In contrast, analysis of Env sequences indicated that the A/C and two of the A/B recombinants were subtypes A, while the remaining sample was consistently classified as subtype B. If subtyping had been based solely on data from Gag or Env amino acid sequences, these viruses would have been erroneously classified as subtypes A, B or C, without recognition of intersubtype recombination in the gag gene. These findings illustrate the advantage of contrasting nucleotide with amino acid sequence analyses. They also show the limitations of phylogenetic assignment based on subgenomic DNA fragments, suggesting that full genome sequence analysis should be the preferred method for accurate characterization of FIV isolates. Thus, additional recombination sites could have been identified through whole-genome sequencing of the four recombinant proviruses.
The extensive genetic diversity of lentiviruses derives from their high mutation, recombination and turnover rates (Blackard et al., 2002; Thomson et al., 2002
). The high recombinogenic capacity of retroviruses is due to their peculiar mode of replication involving alternating jumps of the reverse transcriptase from one RNA template to the other, combined with its limited proofreading activity (Peliska & Benkovic, 1992
; Robertson et al., 1995
; Thomson et al., 2002
). Recombination as studied in vitro occurs at a very high rate. Using single-replication vector systems, recombination occurred at a frequency of between 2 and 3 % per genome per cycle (Zhang & Temin, 1993
). Factors that predispose a viral genomic site to recombination are not clearly understood but strand transfers in retroviruses appear to occur non-randomly, involving specific sites (hot spots) at high frequency (Zhuang et al., 2002
). In addition, it seems that once recombination occurs, chimeric molecules become more susceptible to undergoing additional events (Zhuang et al., 2002
). A recombination-prone site may exist in FIV since all A/B recombinant samples, including the two different strains co-infecting one cat (CaONAB03a and CaONAB03b), had a common breakpoint between the p24 and p15 coding regions. However, it is unlikely that all four A/B recombinants had the same breakpoint as a consequence of independent recombination events around a hot spot. Instead, this might indicate transmission of a related field strain among cats. Similarly, in an HIV study in Argentina, 13 out of 15 samples harbouring a BF intersubtype recombinant shared a common breakpoint at the 5' end of env (gp120). Sequencing of the genomic region including the breakpoint enabled samples to be readily identified (Gomez Carrillo et al., 2002
). HIV-1 isolates are classified as circulating recombinant forms' if they show inconsistent clade assignment when different genome regions are compared and if they are identified from at least three individuals with no direct linkage (Blackard et al., 2002
; Lole et al., 1999
; Robertson et al., 2000
). The A/B recombinant samples described here were classified differently depending on the analysed genomic region; they originated from three different cities in Ontario (Table 1
) and all of them shared a common recombination site in gag. Although full sequencing would be desirable, these findings suggest transmission of a circulating recombinant form of FIV among cats in Ontario.
Intersubtype recombination results from coincident infection of a cell by more than one subtype and subsequent exchange of gene regions producing replication-competent heterozygous virions (Goff, 2001). Superinfection with different FIV strains has been reported and concurrent infection with subtypes A and B has been experimentally induced both in vivo and in vitro (Okada et al., 1994
; Pistello et al., 1999
). In another study, recombination occurred in one out of three cats inoculated with two different FIV strains (Kyaw-Tanner et al., 1994
). Since both FIV subtypes A and B were present in Ontario, theoretically opportunities for dual infection of cats and therefore recombination existed. Using subtype A- or B-specific PCR assays, we investigated the possibility of co-infection in recombinant samples. Results suggested that only recombinant sequences were present. We then further investigated the possibility of co-infection by PLDA. While sequence identities among at least five replicates from each sample were of 99 %, one replicate from one sample was 10 % different. This particular sequence was 99·8 % similar to a recombinant identified in a different cat. Parental strains of subtypes A and/or B were not identified in either cat after limiting dilutions. Thus, one cat was infected with at least two different A/B recombinants, one of which was almost identical to the virus in another cat from a different geographical region and neither cat had parental A or B strains. These findings further support transmission of an A/B recombinant strain of FIV among cats in Ontario.
Recombination greatly accelerates the evolutionary rate of retroviruses by creating new variants much faster than mutations will, thereby rapidly increasing genetic diversity. Large-scale recombination allows selection of traits that increase virus fitness and permits the elimination of disadvantageous gene regions (Worobey & Holmes, 1999). The sudden emergence of new variants may further serve as a mechanism to evade the selective pressure imposed by the immune system, leading to better adaptations between the virus and the host or perhaps to more severe clinical outcomes. The evolution of recombinant viruses with enhanced pathogenicity and immune evasion was recently described in an HIV-infected individual with an exceptionally rapid disease course (Liu et al., 2002
). In another report, superinfection and subsequent recombination were necessary to create replicative-competent viruses, suggesting that superinfection may favour virus synergism (Blackard et al., 2002
). Whether recombination had an impact on the clinical outcomes of the cats in this study was unknown, since the available clinical data resulted from one assessment and the duration of infection was unknown in most cases. Similar difficulties would be expected under field conditions, since the disease affects predominantly feral, sexually intact cats that are relatively inaccessible for study.
At least 13 different HIV-1 circulating recombinant forms have been characterized (Thomson et al., 2002). In particular geographical areas, some of these variants are currently more prevalent than the native subtypes, which might be an indication of increased virus fitness after recombination. For example, the recombinant virus CRF02_AG is one of two predominant HIV-1 variants in Africa (Vergne et al., 2003
) and in the year 2000 was found to be the second most important cause of the global pandemic (Osmanov et al., 2002
). Little is known about FIV recombination under natural conditions. Here, we have shown evidence that three of the four main FIV subtypes are represented in Canada and provide the first indication that a recombinant form of FIV circulates among cats in Ontario. These findings have implications for molecular diagnosis and vaccine development.
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ACKNOWLEDGEMENTS |
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Received 22 October 2003;
accepted 24 February 2004.