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1 Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca, NY 14853, USA
2 Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine, University of California, Davis, CA 95616, USA
Correspondence
Patricia A. Pesavento
papesavento{at}ucdavis.edu
John S. L. Parker
jsp7{at}cornell.edu
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers of the sequences reported in this paper are DQ910786–DQ910795.
Present address: Biological Engineering Division, Massachusetts Institute of Technology, Cambridge, MA 02139, USA. 
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Pesavento et al., 2004; Coyne et al., 2006). The prevalence of avirulent or mildly virulent FCV in multiple-cat environments is as high as 36 % (Binns et al., 2000; Bannasch & Foley, 2005). During the last decade, however, epizootics of a severe form of FCV disease with mortality rates as high as 50 % have occurred (Hurley et al., 2004; Coyne et al., 2006). These epizootics, designated virulent systemic (VS-) FCV, have been reported in the USA and recently in the UK (Pedersen et al., 2000; Schorr-Evans et al., 2003; Hurley et al., 2004; Coyne et al., 2006). The clinicopathological features of VS-FCV disease differ substantially from those of ‘classical’ FCV disease; reported signs of VS-FCV disease include high persistent fever, anorexia, depression, facial and limb oedema, sores or alopecia on the face, pinnae and feet, pulmonary oedema, coagulation abnormalities, pancreatitis and hepatic necrosis (Pedersen et al., 2000; Hurley et al., 2004; Pesavento et al., 2004). In all of the reported outbreaks, vaccinated cats have been affected, suggesting that current vaccines may not protect against VS disease (Coyne et al., 2006).
FCV belongs to the genus Vesivirus of the family Caliciviridae and contains a positive-sense RNA genome (approx. 7.6 kb) packaged within a non-enveloped capsid. The icosahedral capsid is assembled from 90 homodimers of the major capsid protein VP1. Two genogroups of FCV have been defined by phylogenetic analysis. Of the isolates characterized, only isolates from Japan are members of genogroup II (Sato et al., 2002), whilst all other VS-FCV isolates studied to date appear to be members of genogroup I (Geissler et al., 1997; Glenn et al., 1999). All FCVs are considered to belong to a single, antigenically diverse serotype, but individual isolates vary in their levels of cross-neutralization (Kalunda et al., 1975). Initial reports indicate that VS-FCV isolates cannot be distinguished genetically from non-VS-FCV isolates (Hurley et al., 2004; Abd-Eldaim et al., 2005; Foley et al., 2006).
Here, we characterized the in vitro growth properties, stabilities, cytopathic effects and sequences of seven VS-FCV isolates from five distinct VS-FCV outbreaks and compared them with those of the F9 vaccine strain and three non-VS-FCV clinical isolates. Based on the results herein, we conclude that the VS-FCV isolates that we analysed share the common properties of rapid growth kinetics and an increased propensity to spread in tissue culture in vitro. These properties appear to differ from those of both the F9 vaccine strain and the three non VS-FCV isolates and may in part explain the increased virulence of these viruses in vivo.
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METHODS |
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. FCV isolate VS-FCV-Ari was isolated from an infected cat during a VS-FCV outbreak in Sacramento, CA, USA, in 1998. FCV isolates Kaos, George Walder and Jengo were isolated from sick cats during a VS-FCV outbreak in Los Angeles, CA, USA, in 2002. Isolates FCV-5, Deuce and Georgie were isolated from VS-FCV-infected cats from Massachusetts in 2001, North Carolina in 2004 and Florida in 2003, respectively. Isolates FCV-127, FCV-131 and FCV-796 were all obtained from Dr Ed Dubovi at the New York State Animal Health Diagnostic Center at Cornell University, NY, USA. FCV-131 originated from a shelter in Harrisburg, PA, USA, that experienced an outbreak of non-VS-FCV in 2003. FCV-127 came from a shelter in North Adams, MA, USA, where numerous cats suffered from a pneumonic form of FCV in 2004. FCV-796 was isolated from a cat in Tampa, FL, USA, that presented with excessive drooling and lingual ulceration in 2004. For the duration of the paper, strains FCV-127, -131 and -796 will be referred to as ‘non-VS’. This designation is made based on the clinical history that accompanied the isolates. The F9 vaccine strain (VR-782) of FCV was obtained from the ATCC. Third-passage virus stocks were prepared from twice-plaque-purified viruses amplified in CRFK cells.
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Recovery of viral RNA, RT-PCR, sequencing and analysis.
Total RNA was extracted from infected-cell lysates using a commercial kit (RNeasy Mini; Qiagen). First-strand cDNA from the capsid region of the genome was synthesized from viral RNA by using a degenerate antisense primer (5'-YTGACCMAGTGCAGYCTTRTCCAATTC-3') [adapted from Poulet et al. (2005)], predicted to anneal to bp 7380–7405 of the FCV-Urbana sequence (GenBank accession no. L40021
[GenBank]
), and Accuscript High-Fidelity reverse transcriptase (Stratagene) as per the manufacturer's directions. The capsid open reading frame (ORF) was amplified by PCR using Pfu UltraDNA polymerase (Stratagene) from the first-strand cDNA template using the antisense primer described above and a sense primer (5'-TACACTGTGATGTGTTCGAAGTTTGAGC-3') (Martella et al., 2002) that anneals to bp 5286–5313 of the FCV-Urbana genome. Thermal-cycling conditions were 95 °C for 2 min, followed by 40 cycles of 95 °C for 30 s, 50 °C for 1 min and 68 °C for 3 min, with a final extension step of 68 °C for 10 min. The resulting approximately 2.1 kb PCR products, encompassing the entirety of the capsid ORF, were purified and sequenced directly.
cDNA from the region encompassing the proteinase–polymerase (pro–pol) region of ORF1 was amplified from viral RNA by using the SuperScript One-Step RT-PCR system (Invitrogen) and sense (5'-ATTGGVAARGGYGGYGTNAARMAY-3') and antisense (5'-AGCACGTTAGCGCAGGTT-3') primers predicted to anneal to bp 3143–3166 and 5322–5339 of the FCV-Urbana genome, respectively. Thermal-cycling conditions consisted of 50 °C for 30 min, 94 °C for 2 min, five cycles of 94 °C for 15 s, 52 °C for 1 min and 72 °C for 2.5 min, followed by 35 cycles of 94 °C for 15 s, 52 °C for 30 s and 72 °C for 2.5 min, with a final extension step of 72 °C for 10 min. The resulting approximately 2.2 kb PCR products were cloned into the pGEM-T Easy vector (Promega) and sequenced by the Cornell University Life Sciences Core Laboratories Center.
Sequences were compared with other FCV capsid or pro–pol sequences contained in GenBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide) (Table 2). Determination of ORFs and amino acid sequence was performed by using Vector NTI Advance (Invitrogen Life Technologies). Sequence alignments were carried out by using CLUSTAL_X (Thompson et al., 1997). Similarity tables were constructed by using Vector NTI Advance. Construction of phylogenetic trees inferred from nucleotide sequence was performed by using three methods: the distance (neighbour-joining) method using the CLUSTAL_X software, the Bayesian method with the software MRBAYES (Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003) and the maximum-likelihood method using the software PAUP* version 4.0 beta (Sinauer Associates). Neighbour joining- and maximum likelihood-generated trees were bootstrapped with the corresponding, aforementioned software. Construction and bootstrapping of phylogenetic trees inferred from amino acid sequence were performed by using CLUSTAL_W, utilizing the BLOSUM series (80, 62, 40 and 30) weight matrices for proteins. Constructed trees were visualized by using NJPLOT (Perriere & Gouy, 1996).
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Generation of single- and multiple-cycle growth curves.
CRFK cell monolayers were infected with different FCV isolates at multiplicities of 5 and 0.01 for single- and multiple-cycle growth curves, respectively. Virus was adsorbed to cells for 1 h at room temperature in DMEM plus 0.1 % BSA; EMEM plus 5 % FBS supplemented as above was then added and the cells were incubated for various times at 37 °C in 5 % CO2. At various times post-infection (p.i.), samples were frozen and stored at –80 °C for later titration. Prior to plaque assay, all samples were frozen and thawed three times. Virus growth at each time point was calculated by subtracting the log10(p.f.u. ml–1) at T=0 from the log10(p.f.u. ml–1) measured at the time point. Results are expressed as the change in plaque titre over time, with the mean and SD of three replicates shown.
Cell-viability assay.
The viability of infected CRFK cells was evaluated by using a commercial assay of ATP levels (CellTitre-Glo reagent; Promega) according to the manufacturer's recommendations. Ninety-six-well plates were read by using a Veritas microplate luminometer (Turner Biosystems). Relative luminescence units of experimental wells were compared with those of control wells to determine percentage change in ATP levels.
Statistical analyses.
Comparisons of titres between sets of three replicates were analysed by ANOVA using the Analyse-it (Analyse-it Software) statistical analysis add-in for Microsoft Excel. Graphs were prepared by using KaleidaGraph (Synergy Software).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). We found that all of the FCV isolates tested grew rapidly during a single round of virus replication that was characterized by a lag phase of about 3.5 h, followed by exponential growth that lasted 3–4.5 h (Fig. 1a). Peak yields of virus were produced for all except the F9 vaccine strain at 8–12 h p.i. We noted that virus yield of all the isolates except for F9 had decreased by 0.5–1 log10 from peak titre (at 8–12 h p.i.) by 24 h p.i. The single-cycle growth kinetics of VS-FCV isolates were not statistically significantly different from those of non-VS isolates; however, cells infected with the VS-FCV isolates Ari and Deuce yielded the most virus (2.5 log10). We conclude that FCV isolates have a short replicative cycle in CRFK cells (10–12 h) and that FCV isolates of differing virulence have similar single-cycle growth kinetics, but may differ in the final yield of virus produced.
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). By 4 h p.i., the titres of VS-FCV isolates were increasing exponentially and had yielded 2 log10 infectious virus. In contrast, the F9 vaccine strain and isolate FCV-796 had not yet entered exponential growth. FCV-127, a non-VS isolate that produced large plaques, was growing exponentially at 4 h p.i., but had produced 10-fold less infectious virus than the three VS-FCV isolates. The differences in log titres at 4 and 8 h p.i. between the VS-FCV and non-VS isolates were statistically significant, as determined by ANOVA (P<0.0001). The final yields of virus varied; however, the two highest yields (approx. 5.5 and 4.8 log10) were attained by VS-FCV isolates Deuce and Kaos, respectively. From these results, we conclude that VS-FCV isolates appear to produce infectious virions earlier when cells are infected at low multiplicity and may attain higher yields than less virulent isolates in CRFK cells.
Cytopathic effect of different FCV isolates
During the multiple-cycle growth-kinetics experiments, we noted that cells infected with VS-FCV isolates displayed morphological changes of rounding and detachment from the vessel surface earlier than other FCV isolates (Fig. 2a). From 14 h p.i. onwards, we frequently observed clusters of VS-FCV-infected cells that were rounded up and detached partially or fully from the plate surface. In contrast, few non-VS isolate-infected cells displayed such morphological changes at 14 h p.i., and larger clusters of cells with such morphological alterations were absent. Non-VS isolate FCV-127, which was isolated from a kitten with severe pneumonia, caused more severe morphological changes than isolate FCV-796, suggesting that the severity of the cytopathic effects observed correlated in part with virulence. These observations suggested that VS-FCV isolates caused increased cytopathogenicity. The mechanisms that underlie FCV cytopathogenicity are not fully understood, although FCV infection is known to induce apoptosis (Roberts et al., 2003; Sosnovtsev et al., 2003; Natoni et al., 2006). To test the possibility that VS-FCV isolates caused increased cytopathic effect in infected cells, we measured the intracellular ATP levels of cells infected with VS-FCV, non-VS isolates and the F9 vaccine strain at different times after infection in cells infected at multiplicities of 0.01 and 5 (Fig. 2b, c). Intracellular ATP levels have been used to quantify the cytotoxic effect of the cytokine tumour necrosis factor alpha (Crouch et al., 1993) and as an indicator of the cytopathic effect of murine leukemia virus on CHO-K1 cells (Bruce et al., 2005). Under multiple-cycle growth conditions (m.o.i.=0.01), at 14 h p.i., cells infected with VS-FCV isolates had ATP levels that were equal to or higher than control levels, despite obvious changes in cell morphology (Fig. 2a). ATP levels in cells infected with all FCV isolates diverged from control levels by 16–20 h p.i. (Fig. 2b). Non-VS isolates FCV-796 and -131 demonstrated the most rapid decrease in ATP, with levels at 50 % of control levels at 20 h p.i. For all of the isolates that we investigated, the cellular ATP levels dropped to 25–50 % of control levels by 24 h p.i. Under single-cycle growth conditions (m.o.i.=5), ATP levels in infected cells fell below control levels at 10–14 h p.i. (Fig. 2c). Cells infected with the F9 vaccine strain displayed the earliest divergence from uninfected-control ATP levels at 10 h p.i. By 14 h p.i., cells infected with non-VS isolates had ATP levels that were only 25–50 % of the levels of the uninfected control, whereas cells infected with VS-FCV isolates maintained ATP levels that were 70–90 % of the control. We conclude that, under multiple-cycle conditions, cells infected with VS-FCV isolates show cytopathic effects as early as 14 h p.i., although the ATP content of these cells is similar to that of uninfected cells. In addition, under single-cycle conditions, cells infected with VS-FCV isolates maintain ATP levels longer than cells infected with the non-VS isolates and the vaccine strain F9.
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; Schorr-Evans et al., 2003; Hurley et al., 2004). These observations suggested to us that VS-FCV isolates might be more environmentally stable than non-VS isolates. To address this possibility, we examined the resistance of selected isolates to thermal inactivation (Table 1; Fig. 3). Aliquots of virus were incubated at 37.0, 41.8, 46.2, 52.2, 56.9 and 62.0 °C for 30 min in a thermal cycler and then assayed for infectivity by plaque assay. We found that all isolates lost infectivity substantially following a 30 min incubation at 46.2 °C (Fig. 3). The F9 vaccine strain was most sensitive to thermal inactivation, losing all infectivity following 30 min incubation at 52.2 °C. The other isolates were inactivated fully at 56.9 °C, except for isolate FCV-5 (inactivated fully at 62 °C). We conclude that all of the isolates that we examined were more resistant to thermal inactivation than F9, but exhibited differing temperature stabilities with respect to each other.
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). Isolates stored at room temperature lost 2–5 log10 infectivity after 3 days, with the two VS-FCV isolates Deuce and FCV-5 and the non-VS isolate FCV-127 decreasing in infectivity by 2–3 log10 and the F9 vaccine strain and non-VS isolate FCV-796 decreasing by 4–5 log10. By day 6, isolates FCV-5, -796 and F9 had lost infectivity completely. FCV-Deuce lost infectivity completely by day 10, whilst isolate FCV-127 retained a low level of infectivity at the conclusion of the 12 day experiment (Fig. 4). We noted that, as virus titres dropped, the variability of the plaque assays increased, with SD values ranging from 0.75 log10 to 2 log10. We found no obvious correlation between the temperature stability of different FCV isolates and their virulence.
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). The VS-FCV isolates maintained full infectivity until day 21. By the third week, all isolates exhibited similar rates of infectivity loss and, by 6–10 weeks, had lost infectivity completely. Although the two VS-FCV strains demonstrated enhanced stability over the vaccine strain at 4 °C during the first 2 weeks of storage, further testing will be necessary to confirm that VS-FCV isolates are more stable at 4 °C than other FCV isolates. Similar to our findings above, as virus titres dropped, the variability of the plaque assays increased. None of the isolates tested lost infectivity when stored at –80 °C for 8 weeks (Fig. 4).
Sequence comparison of VS-FCV isolates and other FCV isolates
Abd-Eldaim et al. (2005) and Foley et al. (2006) have suggested that certain residues within the hypervariable region of the capsid protein sequence are unique to VS-FCV isolates. We therefore sequenced the capsid VP1-encoding second ORF of the FCV genome for all of the isolates and performed nucleotide and primary amino acid sequence alignments and phylogenetic analyses to investigate the relatedness of these sequences and 37 other FCV complete capsid sequences (Table 2). Capsid amino acid sequence alignments (CLUSTAL_W) revealed that no residues were unique to the VS-FCV isolates that we investigated (data not shown). We used Rabbit hemorrhagic disease virus (RHDV), a calicivirus in the genus Lagovirus, as an outgroup to root the phylogenies. By using nucleotide (neighbour-joining method)- and amino acid (BLOSUM)-based phylogenies, we found that the VS-FCV isolates did not form a unique clade (Fig. 5a, b). As expected, isolates collected from the same outbreak of VS-FCV in Los Angeles, CA, USA, in 2002 (George Walder, Jengo and Kaos) clustered together in nucleotide and amino acid analyses, with determined bootstrap values of 100 %. The VS-FCV isolates Ari, Georgie, Deuce and FCV-5, as well as two other previously described VS-FCV isolates (Abd-Eldaim et al., 2005), were not related closely. Trees generated by utilizing the Bayesian method and maximum-likelihood method of phylogenetic inference also showed a clustering of the three VS-FCV isolates from the Los Angeles outbreak, whilst the remainder of the VS-FCV isolates were scattered throughout the tree (data not shown).
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). Similar to results from the capsid region, pro–pol sequence alignments (CLUSTAL_W) revealed no residues unique to the VS-FCV isolates investigated. The overall nucleotide identity was 80.6±3.1 % and the amino acid identity was 92.9±2.1 %. This was not significantly different from the nucleotide (82.6±5.4 %) or primary amino acid (94.4±2.2 %) sequence similarity between the VS-FCV isolates investigated. Phylogenetic trees were rooted as described above, using the sequence of the RHDV pro–pol. Both nucleotide (neighbour-joining method)- and amino acid (BLOSUM)-based phylogenies were similar to those of the capsid sequences, and VS-FCV isolates did not segregate from other FCV isolates (Fig. 6a, b). The three isolates from the 2002 Los Angeles outbreak grouped together (bootstrap values of 100 %). The sequences of the other VS-FCV isolates Deuce, Georgie, FCV-5, Ari and the two Tennessee isolates were not related closely. Similar results were obtained when phylogenies were inferred by using Bayesian and maximum-likelihood methods (data not shown). We conclude that VS-FCV isolates do not represent a unique clade of FCV, but instead appear to have arisen independently multiple times in different geographical locations.
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DISCUSSION |
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; Schorr-Evans et al., 2003; Hurley et al., 2004; Pesavento et al., 2004; Coyne et al., 2006). As vaccinated cats appear to be as susceptible to this form of FCV as unvaccinated cats, disease control is currently problematic. A major difficulty is that viruses associated with VS disease cannot be distinguished from other FCV isolates in vitro. One study reported that sequences from a small set of VS-FCV isolates were no more similar to each other than to other non-VS field isolates of FCV (Abd-Eldaim et al., 2005). Despite this, the disease has been reproduced in experimental animals, indicating that the genetic differences are probably hidden by the high variability of the FCV genome (Pedersen et al., 2000). The initial goal of our study was to identify in vitro phenotypes of VS-FCV isolates that would distinguish them from other, less virulent FCV isolates and that could be used as a correlate to identify the underlying genotype(s) associated with FCV virulence.
Previous reports have described the single-cycle growth kinetics of ‘classical’ isolates of FCV (Studdert et al., 1970; Kreutz & Seal, 1995). This is the first report of single-cycle growth kinetics of VS-FCV isolates, as well the first report of multiple-cycle growth kinetics for any FCV isolate. All of the FCV isolates that we investigated displayed similar single-cycle growth kinetics, and our findings are generally similar to those published previously (Studdert et al., 1970; Kreutz & Seal, 1995). Although VS- and non-VS-FCV isolates displayed similar kinetics, the VS-FCV isolates tended to replicate to higher titres, suggesting higher yields per cell. In contrast, during multiple-cycle growth, we observed that the three VS-FCV isolates produced infectious virions earlier and/or to substantially higher titres than the vaccine/non-VS isolate group. Given that the VS- and non-VS isolates had similar kinetics of growth during a single cycle of replication, these findings indicate that VS-FCV isolates infect CRFK cells more efficiently than do non-VS strains. Such efficiency suggests that differences in virus attachment, entry, establishment of replication sites or release from cells may determine the increased growth of VS-FCV isolates and perhaps play a role in the increased pathogenicity of VS-FCV isolates.
In addition to more efficient infection, we also observed that cells infected with VS-FCV isolates at low multiplicities displayed cytopathic effects earlier than those infected with non-VS field isolates or the vaccine strain. Surprisingly, VS-FCV-infected cells had similar or higher ATP levels than did the uninfected controls at time points when cytopathic effects were first readily seen. Under single-cycle conditions, VS-FCV-infected cells maintained ATP levels for longer than the non-VS isolates. By maintaining cellular ATP levels, VS-FCV isolates may delay the onset of the final stages of apoptosis, thus allowing the production and/or release of more virus particles. It was reported recently that intracellular pools of ATP and other nucleotides are able to block cytochrome c-initiated apoptosome formation and caspase activation directly (Chandra et al., 2006). Alternatively, maintaining intracellular ATP levels may serve to promote energy-dependent apoptotic cell death and prevent cellular necrosis (Chiarugi, 2005). This may prove advantageous to virus replication in vivo by limiting the inflammatory reaction initiated by necrotic cells.
Fomite transmission has been implicated as an important factor in the rapid spread observed during the documented VS-FCV outbreaks (Pedersen et al., 2000; Hurley et al., 2004). We therefore hypothesized that VS-FCV isolates might possess increased environmental stability over non-VS isolates. However, whilst individual isolates displayed minor differences in their sensitivity to temperature inactivation, there was no correlation with virulence. We noted, however, that the F9 vaccine strain was inactivated completely at a lower temperature than the other isolates that we tested, suggesting that some capsid stability was lost during the selection for attenuation of the F9 strain. The F9 strain has been used extensively to evaluate the sensitivity of caliciviruses to environmental inactivation by various methods (Duizer et al., 2004; Tree et al., 2005; Malik et al., 2006). Our results suggest that these findings should be tempered by the knowledge that field strains may be more resistant to inactivation than vaccine strains.
Minor differences in stability between isolates after extended incubation at room temperature or 4 °C did not correlate with virulence. In contrast to a previous report that indicated that virions were more stable when maintained at 4 °C (Komolafe, 1979), we found that all of the isolates that we tested lost infectivity when stored for longer than 2–3 weeks at 4 °C, but retained full infectivity when stored at –80 °C. The reasons for these differences are unclear, but may be due to stabilizing effects of serum proteins within the lysate (Komolafe, 1979). Additionally, we also found that infectivity was maintained after as many as 56 freeze–thaw cycles (data not shown). Therefore, FCV appears to tolerate multiple freeze–thaw cycles without loss of infectivity, indicating that storage of viral lysates at –80 °C is optimal.
Previous analyses of the capsid and pro–pol sequences of VS-FCV isolates found them to be no more similar to each other than to other non-VS isolates (Hurley et al., 2004; Abd-Eldaim et al., 2005; Foley et al., 2006). Our sequence analyses support these conclusions and suggest that the different VS-FCV isolates have arisen independently in distinct geographical locations. These studies also suggested that there were amino acid sequences within the hypervariable region (residues 426–452) of the capsid that were unique to VS-FCV isolates. However, when we analysed all of the available VS-FCV sequences together, we found that there were no sequences or amino acid changes unique to all VS isolates in the capsid or pro–pol regions.
We recognize that many of our conclusions are based on two assumptions. The first is that the VS-FCV samples that we are characterizing still possess the virulent nature of the original isolate. Passage in tissue culture has been limited to prevent selection of variants. Furthermore, three of the isolates that we characterized have been used to reproduce VS-FCV disease in cats [Ari (Pedersen et al., 2000); FCV-5 (Rong et al., 2006); Kaos (P. A. Pesavento, unpublished data)]. Whilst the other isolates have not been tested in vivo, the experience thus far is that VS-FCV isolates can be passaged at least three times in tissue culture without loss of virulence, and perhaps up to 20 times (Rong et al., 2006). The second is that all strains considered non-VS isolates do not cause VS disease. However, it is possible that many cases of VS-FCV go undetected. The investigated isolate FCV-127 originated from a shelter where numerous cats died due to pneumonia. In all experiments, isolate FCV-127 behaved more like a VS-FCV isolate than the other non-VS field isolates. Thus, we speculate that the differences in virus growth patterns that we noted between VS and non-VS viruses may in fact be shared by other ‘virulent’ FCV isolates. Currently, the only means to verify that a particular isolate causes VS disease is by reproducing the disease syndrome in an experimentally infected cat.
In summary, we have investigated several viral characteristics in an attempt to define an in vitro phenotype unique to VS strains. Our findings indicate that there are differences between VS and non-VS isolates that may serve as markers for virulence. We are currently evaluating further the genetic bases of these differences.
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ACKNOWLEDGEMENTS |
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Received 21 August 2006;
accepted 27 October 2006.
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FCV field isolates sequenced in this paper. Vaccine strains are underlined and the strain name is provided in parentheses. Bootstrap values >90 % are indicated.