feline caliciviruses (fcvs) isolated from cats with virulent systemic disease possess in vitro...

7/30/2019 Feline caliciviruses (FCVs) isolated from cats with virulent systemic disease possess in vitro phenotypes distinct fro

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Feline caliciviruses (FCVs) isolated from cats withvirulent systemic disease possess in vitrophenotypes distinct from those of other FCV

isolatesRobert J. Ossiboff,1 Alexander Sheh,13 Justine Shotton,1

Patricia A. Pesavento2 and John S. L. Parker1

Correspondence

Patricia A. Pesavento

[email protected]

John S. L. Parker

[email protected]

1Baker Institute for Animal Health, College of Veterinary Medicine, Cornell University, Ithaca,NY 14853, USA

2Department of Pathology, Microbiology and Immunology, School of Veterinary Medicine,University of California, Davis, CA 95616, USA

Received 21 August 2006

Accepted 27 October 2006

During the past decade, several outbreaks of severe systemic disease associated with Feline

calicivirus (FCV) have occurred in the USA and the UK. This new disease has caused high mortality

in the affected animals and has been termed virulent systemic (VS)-FCV disease. Currently, there

are no genetic or in vitro diagnostic methods to distinguish viruses isolated from cases of VS-FCV

disease from other isolates. Here, five in vitro properties, as well as the capsid and

proteinasepolymerase (propol) sequences, of a set of FCV isolates that included seven isolates

from five distinct VS-FCV outbreaks (VS isolates) were investigated. Although all of the FCV

isolates investigated had similar kinetics of growth under single-cycle conditions, VS isolates

infected tissue-culture cells more efficiently under multiple-cycle growth conditions. Moreover, it

was found that cells infected with VS isolates showed cytopathic effects earlier than cells infected

with non-VS isolates, although no difference in relative ATP levels were noted at times when

morphological changes were first seen. Both VS- and other (non-VS) isolates of FCV demonstrated

similar temperature stabilities. Phylogenetic analyses and alignments of the capsid and propolregions of the genome did not reveal any conserved changes that correlated with virulence, and the

VS isolates did not segregate into a unique clade. These results suggest that VS isolates have

arisen independently several times since first being described and can spread more efficiently in

tissue culture than other isolates when infected at low multiplicity.

INTRODUCTION

Feline calicivirus (FCV) is a common pathogen of cats.Infected cats can be clinically normal or show signs of oralulceration and/or mild upper respiratory disease. Lesscommonly, limping, abortion and severe pneumonia canoccur; however, fatal disease is unusual (Hurleyet al., 2004;Pesavento et al., 2004; Coyne et al., 2006). The prevalence ofavirulent or mildly virulent FCV in multiple-cat environ-ments is as high as 36 % (Binns et al., 2000; Bannasch &Foley, 2005). During the last decade, however, epizootics ofa severe form of FCV disease with mortality rates as high as50 % have occurred (Hurleyet 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; Hurleyet al.,2004; Coyne et al., 2006). The clinicopathological features ofVS-FCV disease differ substantially from those of classicalFCV disease; reported signs of VS-FCV disease include highpersistent fever, anorexia, depression, facial and limb

oedema, sores or alopecia on the face, pinnae and feet,pulmonary oedema, coagulation abnormalities, pancreatitisand hepatic necrosis (Pedersen et al., 2000; Hurley et al.,2004; Pesavento et al., 2004). In all of the reportedoutbreaks, vaccinated cats have been affected, suggestingthat current vaccines may not protect against VS disease(Coyne et al., 2006).

FCV belongs to the genus Vesivirus of the familyCaliciviridae 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 ofthe major capsid protein VP1. Two genogroups of FCV have

3Present address: Biological Engineering Division, MassachusettsInstitute of Technology, Cambridge, MA 02139, USA.

The GenBank/EMBL/DDBJ accession numbers of the sequencesreported in this paper are DQ910786DQ910795.

506 0008-2488 G 2007 SGM Printed in Great Britain

Journal of General Virology (2007), 88, 506517 DOI 10.1099/vir.0.82488-0


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been defined by phylogenetic analysis. Of the isolatescharacterized, only isolates from Japan are members ofgenogroup II (Sato et al., 2002), whilst all other VS-FCVisolates studied to date appear to be members of genogroup I(Geissler et al., 1997; Glenn et al., 1999). All FCVs areconsidered to belong to a single, antigenically diverseserotype, but individual isolates vary in their levels of cross-neutralization (Kalunda et al., 1975). Initial reports indicatethat VS-FCV isolates cannot be distinguished geneticallyfrom non-VS-FCV isolates (Hurleyet al., 2004; Abd-Eldaimet al., 2005; Foley et al., 2006).

Here, we characterized the in vitro growth properties,stabilities, cytopathic effects and sequences of seven VS-FCVisolates from five distinct VS-FCV outbreaks and comparedthem with those of the F9 vaccine strain and three non-VS-FCV clinical isolates. Based on the results herein, weconclude that the VS-FCV isolates that we analysed share thecommon properties of rapid growth kinetics and anincreased propensity to spread in tissue culture in vitro.

These properties appear to differ from those of both the F9vaccine strain and the three non VS-FCV isolates and may inpart explain the increased virulence of these viruses in vivo.

METHODS

Cells and viruses. Crandell feline kidney (CRFK) cells were grownin Eagles minimal essential medium (EMEM) (CellGro) supplemen-ted with 5 % fetal bovine serum (FBS; HyClone), 100 U penicillinml21, 100 mg streptomycin ml21, 0.25 mg amphotericin B ml21,1 mM sodium pyruvate and non-essential amino acids (CellGro).The clinical FCV isolates investigated in this study and the analyses

performed are listed in Table 1. FCV isolate VS-FCV-Ari was iso-lated from an infected cat during a VS-FCV outbreak inSacramento, CA, USA, in 1998. FCV isolates Kaos, George Walderand Jengo were isolated from sick cats during a VS-FCV outbreak inLos Angeles, CA, USA, in 2002. Isolates FCV-5, Deuce and Georgiewere isolated from VS-FCV-infected cats from Massachusetts in2001, 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 Centerat Cornell University, NY, USA. FCV-131 originated from a shelterin 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 FCVin 2004. FCV-796 was isolated from a cat in Tampa, FL, USA, thatpresented with excessive drooling and lingual ulceration in 2004. Forthe duration of the paper, strains FCV-127, -131 and -796 will bereferred to as non-VS. This designation is made based on the clini-cal history that accompanied the isolates. The F9 vaccine strain (VR-782) of FCV was obtained from the ATCC. Third-passage virusstocks were prepared from twice-plaque-purified viruses amplifiedin CRFK cells.

Plaque assay. Viruses were titrated on CRFK cells. In brief, con-fluent CRFK monolayers were inoculated with serial virus dilutionsin Dulbeccos modified Eagless medium (DMEM) (CellGro) with0.1 % BSA (Calbiochem). Virus adsorption was carried out at roomtemperature for 60 min with gentle agitation every 10 min.Monolayers were then overlaid with 3 ml EMEM containing 5 %FBS, 100 U penicillin ml21, 100 mg streptomycin ml21, 0.25 mgamphotericin B ml21, 1 mM sodium pyruvate, non-essential aminoacids and 1 % Bacto-agar (Difco Laboratories). After incubation at37 uC for 48 h in humidified 5 % CO2, the overlay was removed, thecells were fixed with 10 % buffered formalin (Fisher) and stainedwith 1 % (w/v) crystal violet solution. After washing, the plaqueswere counted and titre was expressed as p.f.u. ml21.

Table 1. FCV isolates investigated

Isolate Origin Description Analysis Viability* SequenceD

Kinetics Stability

SCd MC TI|| TM

Ari Sacramento, CA, USA VS-FCV, 1998 6 6

FCV-5 Bellingham, MA, USA VS-FCV, 2001 6 6 6 6 6 6

Kaos Los Angeles, CA, USA VS-FCV, 2002 6 6 6 6 6

Jengo Los Angeles, CA, USA VS-FCV, 2002 6 6

George Walder Los Angeles, CA, USA VS-FCV, 2002 6 6

Georgie FL, USA VS-FCV, 2003 6

Deuce NC, USA VS-FCV, 2004 6 6 6 6 6 6

FCV-131 Harrisburg, PA, USA Non-VS-FCV, 2003 6 6

FCV-127 North Adams, MA, USA Non-VS-FCV, 2004 6 6 6 6 6 6

FCV-796 Tampa, FL, USA Non-VS-FCV, 2004 6 6 6 6 6 6

F9 (VR-782) ATCC Vaccine strain 6 6 6 6 6 6

*ATP cell-viability assay.

DProteinasepolymerase and capsid sequencing.

dSingle-cycle growth curve.

Multiple-cycle growth curve.

||Thermal inactivation.

Temperature maintenance.

http://vir.sgmjournals.org 507

Feline calicivirus growth kinetics


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Recovery of viral RNA, RT-PCR, sequencing and analysis.

Total RNA was extracted from infected-cell lysates using a commer-cial kit (RNeasy Mini; Qiagen). First-strand cDNA from the capsidregion of the genome was synthesized from viral RNA by using adegenerate antisense primer (59-YTGACCMAGTGCAGYCTTRT-CCAATTC-39) [adapted from Poulet et al. (2005)], predicted toanneal to bp 73807405 of the FCV-Urbana sequence (GenBankaccession no. L40021), and Accuscript High-Fidelity reverse tran-

scriptase (Stratagene) as per the manufacturers directions. Thecapsid open reading frame (ORF) was amplified by PCR using PfuUltraDNA polymerase (Stratagene) from the first-strand cDNA tem-plate using the antisense primer described above and a sense primer(59-TACACTGTGATGTGTTCGAAGTTTGAGC-39) (Martella et al.,2002) that anneals to bp 52865313 of the FCV-Urbana genome.Thermal-cycling conditions were 95 uC for 2 min, followed by 40cycles of 95 uC for 30 s, 50 uC for 1 min and 68 uC for 3 min, with afinal extension step of 68 uC for 10 min. The resulting approximately2.1 kb PCR products, encompassing the entirety of the capsid ORF,were purified and sequenced directly.

cDNA from the region encompassing the proteinasepolymerase (propol) region of ORF1 was amplified from viral RNA by using theSuperScript One-Step RT-PCR system (Invitrogen) and sense (59-

ATTGGVAARGGYGGYGTNAARMAY-39) and antisense (59-AGCACGTTAGCGCAGGTT-39) primers predicted to anneal to bp31433166 and 53225339 of the FCV-Urbana genome, respectively.Thermal-cycling conditions consisted of 50 uC for 30 min, 94 uC for2 min, five cycles of 94 uC for 15 s, 52 uC for 1 min and 72 uC for2.5 min, followed by 35 cyclesof 94 uCfor15 s,52 uCfor30 sand72 uCfor 2.5 min, with a final extension step of 72 uC for 10 min. Theresulting approximately 2.2 kb PCR products were cloned into thepGEM-T Easy vector (Promega) and sequenced by the CornellUniversity Life Sciences Core Laboratories Center.

Sequences were compared with other FCV capsid or propol sequencescontained in GenBank (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Nucleotide) (Table 2). Determination of ORFs and aminoacid sequence was performed by using Vector NTI Advance

(Invitrogen Life Technologies). Sequence alignments were carriedout by using CLUSTAL_X(Thompson etal., 1997). Similarity tables wereconstructed by using Vector NTI Advance. Construction of phyloge-netic trees inferred from nucleotide sequence was performed by usingthree methods: the distance (neighbour-joining) method using theCLUSTAL_X software, the Bayesian method with the software MRBAYES(Huelsenbeck & Ronquist, 2001; Ronquist & Huelsenbeck, 2003) andthe maximum-likelihood method using the software PAUP* version 4.0beta (Sinauer Associates). Neighbour joining- and maximum like-lihood-generated trees were bootstrapped with the corresponding,aforementioned software. Construction and bootstrapping of phylo-genetic trees inferred from amino acid sequence were performed byusing CLUSTAL_W, utilizing the BLOSUM series (80, 62, 40 and 30)weight matrices for proteins. Constructed trees were visualized byusing NJPLOT (Perriere & Gouy, 1996).

Temperature-inactivation and -stability experiments. Thermalinactivation of virus isolates was performed by using a thermalcycler (MyCycler; Bio-Rad). Virus samples were incubated at 37.0,41.8, 46.2, 52.2, 56.9 and 62.0 uC in thin-wall PCR tubes (Fisher) for30 min, then plaque-titrated. Virus stability was investigated bymaintaining virus in aliquots at room temperature and at 4 and280 uC for up to 10 weeks. Aliquots in triplicate were plaque-titrated at regular intervals.

Generation of single- and multiple-cycle growth curves.

CRFK cell monolayers were infected with different FCV isolates atmultiplicities of 5 and 0.01 for single- and multiple-cycle growthcurves, respectively. Virus was adsorbed to cells for 1 h atroom temperature in DMEM plus 0.1 % BSA; EMEM plus 5 % FBS

supplemented as above was then added and the cells were incubatedfor various times at 37 uC in 5% CO2. At various times post-infec-tion (p.i.), samples were frozen and stored at 280 uC for later titra-tion. Prior to plaque assay, all samples were frozen and thawed threetimes. Virus growth at each time point was calculated by subtractingthe log10(p.f.u. ml

21) at T=0 from the log10(p.f.u. ml21) measured

at the time point. Results are expressed as the change in plaque titreover time, with the mean and SD of three replicates shown.

Cell-viability assay. The viability of infected CRFK cells was evalu-ated by using a commercial assay of ATP levels (CellTitre-Gloreagent; Promega) according to the manufacturers recommenda-tions. Ninety-six-well plates were read by using a Veritas microplateluminometer (Turner Biosystems). Relative luminescence units ofexperimental wells were compared with those of control wells todetermine percentage change in ATP levels.

Statistical analyses. Comparisons of titres between sets of threereplicates were analysed by ANOVA using the Analyse-it (Analyse-itSoftware) statistical analysis add-in for Microsoft Excel. Graphs wereprepared by using KaleidaGraph (Synergy Software).

RESULTS

Single-cycle kinetics of growth of different FCV

isolates in CRFK cells

We compared the growth kinetics of nine FCV isolates thatdiffered in virulence (Table 1). We found that all of the FCVisolates tested grew rapidly during a single round of virusreplication that was characterized by a lag phase of about3.5 h, followed by exponential growth that lasted 34.5 h(Fig. 1a). Peak yields of virus were produced for all exceptthe F9 vaccine strain at 812 h p.i. We noted that virus yieldof all the isolates except for F9 had decreased by 0.51 log10from peak titre (at 812 h p.i.) by 24 h p.i. The single-cycle

growth kinetics of VS-FCV isolates were not statisticallysignificantly different from those of non-VS isolates;however, cells infected with the VS-FCV isolates Ari andDeuce yielded the most virus (2.5 log10). We conclude thatFCV isolates have a short replicative cycle in CRFK cells(1012 h) and that FCV isolates of differing virulence havesimilar single-cycle growth kinetics, but may differ in thefinal yield of virus produced.

Multiple-cycle kinetics of growth of FCV inCRFK cells

We noted that virulent FCV isolates tended to produce

larger plaques (data not shown) and hypothesized that theseisolates would spread faster than less virulent isolates intissue culture. We therefore examined the growth kinetics ofthree VS-FCV isolates, two non-VS isolates and the F9vaccine strain during multiple replicative cycles to evaluatetheir ability to spread in tissue culture. Consistent with theirlarger plaque size, we found that the growth kinetics of VS-FCV isolates were faster than those of non-VS isolates(Fig. 1b). By 4 h p.i., the titres of VS-FCV isolates wereincreasing exponentially and had yielded 2 log10 infectiousvirus. In contrast, the F9 vaccine strain and isolate FCV-796had not yet entered exponential growth. FCV-127, a non-VS isolate that produced large plaques, was growing

508 Journal of General Virology 88

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Table 2. FCV isolates used in sequence analysis

Isolate GenBank accession no. Reference*

FCV-127 DQ910786Dd This paper



Deuce DQ910789Dd This paper


Georgie DQ910791Dd This paper

George Walder DQ910792Dd This paper

Jengo DQ910793Dd This paper

Ari DQ910794Dd This paper

Kaos DQ910795Dd This paper

UTCVM-NH1 AY560113Dd Abd-Eldaim et al. (2005)



UTCVM-H1 AY560116Dd Abd-Eldaim et al. (2005)

UTCVM-H2 AY560117Dd Abd-Eldaim et al. (2005)

USDA AY560118Dd Abd-Eldaim et al. (2005)

182cvs5A AF031875D Baulch-Brown et al. (1999)

V83A AF031876D Baulch-Brown et al. (1999)

V274 AF031877D Baulch-Brown et al. (1999)



F9|| M86379Dd Carter et al. (1992)

2280 X99445D Geissler et al. (1997)

KS109 X99446D Geissler et al. (1997)




LS015 AF109464 Glenn et al. (1999)

F65 AF109465Dd Glenn et al. (1999)

JOK63 AF109466D Glenn et al. (1999)

LS012 AF109467D Glenn et al. (1999)

A4 AF109468D Glenn et al. (1999)

213/95 AF283778D Martella et al. (2002)

CVXPOLYCYS M32296d Neill (1990)

CFI/68 M32819D; U13992d Neill et al. (1991); unpublished

NADC L09718D Seal et al. (1993)

KCD L09719D Seal et al. (1993)

255 U07130D Seal & Neill (1995)

LLK U07131D Seal & Neill (1995)

Urbana L40021Dd Sosnovtsev & Green (1995)

FCV2024|| AF479590Dd Thumfart & Meyers (2002)

F4|| D90357D; D31836d Tohya et al. (1991); Oshikamo et al. (1994)

FCV-U1 AF357010D Unpublished

AF486286 AF486286D Unpublished

V66/97 AJ009721D Unpublished

FCV-U2 AY053460D Unpublished

Cranleigh AY299541D Unpublished

FCV/DD/2006/GE DQ424892Dd Unpublished

FRG NC_001543d Meyers et al. (2000)

whn/China/03/2005 DQ069282D Unpublished

*The reference cited is the first publication to provide sequence for the isolate. Isolates designated unpublished do not have a publication

associated with the accession.

DCapsid sequence.

dProteinasepolymerase sequence.

VS-FCV isolate.

||Vaccine strain.

Rabbit hemorrhagic disease virus (RHDV) isolate used as outgroup.




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exponentially at 4 h p.i., but had produced 10-fold lessinfectious virus than the three VS-FCV isolates. Thedifferences in log titres at 4 and 8 h p.i. between the VS-FCV and non-VS isolates were statistically significant, asdetermined by ANOVA (P


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third week, all isolates exhibited similar rates of infectivityloss and, by 610 weeks, had lost infectivity completely.Although the two VS-FCV strains demonstrated enhancedstability over the vaccine strain at 4 uC during the first2 weeks of storage, further testing will be necessary toconfirm that VS-FCV isolates are more stable at 4 uC thanother 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 at280 uC for 8 weeks (Fig. 4).

Sequence comparison of VS-FCV isolates andother FCV isolates

Abd-Eldaim et al. (2005) and Foley et al. (2006) havesuggested that certain residues within the hypervariableregion of the capsid protein sequence are unique to VS-FCVisolates. We therefore sequenced the capsid VP1-encodingsecond ORF of the FCV genome for all of the isolates andperformed nucleotide and primary amino acid sequencealignments and phylogenetic analyses to investigate therelatedness of these sequences and 37 other FCV completecapsid sequences (Table 2). Capsid amino acid sequencealignments (CLUSTAL_W) revealed that no residues wereunique to the VS-FCV isolates that we investigated (data notshown). We used Rabbit hemorrhagic disease virus(RHDV),a calicivirus in the genus Lagovirus, as an outgroup to root

the phylogenies. By using nucleotide (neighbour-joiningmethod)- and amino acid (BLOSUM)-based phylogenies, wefound that the VS-FCV isolates did not form a unique clade(Fig. 5a, b). As expected, isolates collected from the sameoutbreak of VS-FCV in Los Angeles, CA, USA, in 2002(George Walder, Jengo and Kaos) clustered together innucleotide and amino acid analyses, with determinedbootstrap values of 100 %. The VS-FCV isolates Ari,Georgie, Deuce and FCV-5, as well as two other previouslydescribed VS-FCV isolates (Abd-Eldaim et al., 2005), werenot related closely. Trees generated by utilizing the Bayesianmethod and maximum-likelihood method of phylogeneticinference also showed a clustering of the three VS-FCV

isolates from the Los Angeles outbreak, whilst the remainderof the VS-FCV isolates were scattered throughout the tree(data not shown).

We also sequenced the portion of the first ORF that encodespropol. Sequences from isolates investigated in this paperwere compared with 14 previously published propolsequences (Table 2). Similar to results from the capsidregion, propol sequence alignments (CLUSTAL_W) revealedno residues unique to the VS-FCV isolates investigated. Theoverall nucleotide identity was 80.63.1 % and the aminoacid identity was 92.92.1%. This was not significantlydifferent from the nucleotide (82.65.4%) or primary

amino acid (94.42.2 %) sequence similarity between theVS-FCV isolates investigated. Phylogenetic trees wererooted as described above, using the sequence of theRHDV propol. Both nucleotide (neighbour-joiningmethod)- and amino acid (BLOSUM)-based phylogenieswere similar to those of the capsid sequences, and VS-FCVisolates did not segregate from other FCV isolates (Fig. 6a,b). The three isolates from the 2002 Los Angeles outbreakgrouped together (bootstrap values of 100 %). Thesequences of the other VS-FCV isolates Deuce, Georgie,FCV-5, Ari and the two Tennessee isolates were not relatedclosely. Similar results were obtained when phylogenies wereinferred by using Bayesian and maximum-likelihood

Fig. 1. Single- and multiple-cycle growth kinetics of selectedFCV isolates. CRFK monolayers were infected with viruses atan m.o.i. of (a) 5 or (b) 0.01 and change in virus titre wasdetermined by plaque assay. The mean log10 titre (log10 titre ateach time point log10 titre at T=0) of three replicates isshown. Isolates from cats diagnosed with VS disease are

shown with solid lines and hollow markers. The vaccine strainand non-VS isolates are displayed with dashed lines and solidmarkers. Significant time points as determined by ANOVA areindicated by an asterisk.




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methods (data not shown). We conclude that VS-FCVisolates do not represent a unique clade of FCV, but insteadappear to have arisen independently multiple times indifferent geographical locations.

DISCUSSIONThe reported outbreaks of VS-FCV have been characterizedby severe clinical disease, rapid spread and high mortalityrates (Pedersen et al., 2000; Schorr-Evans et al., 2003; Hurleyet al., 2004; Pesavento et al., 2004; Coyne et al., 2006). Asvaccinated cats appear to be as susceptible to this form ofFCV as unvaccinated cats, disease control is currentlyproblematic. A major difficulty is that viruses associatedwith VS disease cannot be distinguished from other FCV

Fig. 2. Intracellular ATP levels in FCV-infected cells. (a)Confluent monolayers of CRFK cells were infected with theindicated FCV isolates at an m.o.i. of 0.01. At 14 h p.i., phase-contrast images of cell morphology were collected by using a640 objective with a charge-coupled device (CCD) camera.(b, c) Intracellular ATP levels in CRFK monolayers at differenttimes following infection with viruses under (b) multiple-cycle

conditions (m.o.i. of 0.01) or (c) single-cycle conditions (m.o.i.of 5). ATP levels were expressed as a percentage of the mean

ATP level of mock-infected cells. Each data point representsthe meanSD of three replicates. For clarity, the T=0 h datapoint in graph (b) has been omitted and an axis break is indi-

cated between 0 and 12 h p.i. The omitted data point was notstatistically significantly different from the results determined atT=12 h. Isolates from cats diagnosed with VS disease areshown with solid lines and hollow markers. The vaccine strain

and non-VS isolates are displayed with dashed lines and solidmarkers.

Fig. 3. Thermal inactivation of FCV. Virus aliquots were incubatedfor 30 min at the indicated temperatures and the remaining infec-tivity was then determined by plaque assay. Each data point repre-sents the mean log10(p.f.u ml

1)SD of three replicates. Isolatesfrom cats diagnosed with VS disease are shown with solid linesand hollow markers. The vaccine strain and non-VS isolates aredisplayed with dashed lines and solid markers.




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isolates in vitro. One study reported that sequences from asmall set of VS-FCV isolates were no more similar to eachother 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 thegenetic differences are probably hidden by the highvariability of the FCV genome (Pedersen et al., 2000). Theinitial goal of our study was to identifyin vitrophenotypes ofVS-FCV isolates that would distinguish them from other,less virulent FCV isolates and that could be used as acorrelate to identify the underlying genotype(s) associatedwith FCV virulence.

Previous reports have described the single-cycle growthkinetics of classical isolates of FCV (Studdert et al., 1970;Kreutz & Seal, 1995). This is the first report of single-cyclegrowth kinetics of VS-FCV isolates, as well the first report of

multiple-cycle growth kinetics for any FCV isolate. All of theFCV isolates that we investigated displayed similar single-cycle growth kinetics, and our findings are generally similarto those published previously (Studdert et al., 1970; Kreutz& Seal, 1995). Although VS- and non-VS-FCV isolatesdisplayed similar kinetics, the VS-FCV isolates tended toreplicate to higher titres, suggesting higher yields per cell. Incontrast, during multiple-cycle growth, we observed that thethree VS-FCV isolates produced infectious virions earlierand/or to substantially higher titres than the vaccine/non-VS isolate group. Given that the VS- and non-VS isolateshad similar kinetics of growth during a single cycle ofreplication, 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 cellsmay determine the increased growth of VS-FCV isolates and

perhaps play a role in the increased pathogenicity of VS-FCVisolates.

In addition to more efficient infection, we also observed thatcells infected with VS-FCV isolates at low multiplicitiesdisplayed cytopathic effects earlier than those infected withnon-VS field isolates or the vaccine strain. Surprisingly, VS-FCV-infected cells had similar or higher ATP levels than didthe uninfected controls at time points when cytopathiceffects were first readily seen. Under single-cycle conditions,VS-FCV-infected cells maintained ATP levels for longerthan 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 ofmore virus particles. It was reported recently thatintracellular pools of ATP and other nucleotides are ableto block cytochrome c-initiated apoptosome formation andcaspase activation directly (Chandra et al., 2006).Alternatively, maintaining intracellular ATP levels mayserve to promote energy-dependent apoptotic cell death andprevent cellular necrosis (Chiarugi, 2005). This may proveadvantageous to virus replication in vivo by limiting theinflammatory reaction initiated by necrotic cells.

Fomite transmission has been implicated as an importantfactor in the rapid spread observed during the documented

Fig. 4. Stability of FCV over time when maintained at room temperature (RT) and at 4 and 80 6C. Aliquots of virus were

maintained at room temperature or at 4 or 80 6C for the indicated number of days, then the remaining infectivity wasdetermined by plaque assay. Each data point represents the mean log10(p.f.u ml

1) of three replicates. As virus titres dropped,

the variability of the plaque assays increased. Standard deviations ranged from 0.75 log10 to 2 log10 beginning on the fourthday of the RT experiment, and from 1.5 log10 to 3 log10 beginning on day 35 of the 4 6C experiment. Therefore, the SD bars

have been omitted for clarity. Titres were also observed to oscillate around the zero titre mark for isolates FCV-5, Deuce, F9and FCV-96 by values up to 1.5 log10 (RT assay) and 3 log10 (4 6C assay). These data points have also been omitted forclarity. The first data point indicating loss of infectivity is indicated by an asterisk. Isolates from cats diagnosed with VSdisease are shown with solid lines and hollow markers. The vaccine strain and non-VS isolates are displayed with dashed linesand solid markers.




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Fig. 5. Phylogenetic analysis of FCV capsid nucleotide and protein sequences. Phylogenetic analysis was performed on the(a) nucleotide sequences and (b) protein sequences of ORF2 for all of the FCV isolates used in this study and 37 other FCVsequences obtained from GenBank. Trees were generated by using the nearest-neighbour distance method. Rabbithemorrhagic disease virus (RHDV) was used as an outgroup for each analysis; outgroup branch lengths have been shortenedfor simplicity. *VS-FCV isolates sequenced in this paper; DVS-FCV isolates sequenced previously; dFCV field isolatessequenced in this paper. Vaccine strains are underlined and the strain name is provided in parentheses. Bootstrap values>90 % are indicated.




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VS-FCV outbreaks (Pedersen et al., 2000; Hurley et al.,2004). We therefore hypothesized that VS-FCV isolatesmight possess increased environmental stability over non-VS isolates. However, whilst individual isolates displayedminor differences in their sensitivity to temperatureinactivation, there was no correlation with virulence. We

noted, however, that the F9 vaccine strain was inactivatedcompletely at a lower temperature than the other isolatesthat we tested, suggesting that some capsid stability was lostduring the selection for attenuation of the F9 strain. The F9strain has been used extensively to evaluate the sensitivity ofcaliciviruses to environmental inactivation by variousmethods (Duizer et al., 2004; Tree et al., 2005; Malik et al.,2006). Our results suggest that these findings should betempered by the knowledge that field strains may be moreresistant to inactivation than vaccine strains.

Minor differences in stability between isolates after extendedincubation at room temperature or 4 uC did not correlate

with virulence. In contrast to a previous report thatindicated that virions were more stable when maintainedat 4 uC (Komolafe, 1979), we found that all of the isolatesthat we tested lost infectivity when stored for longer than23 weeks at 4 uC, but retained full infectivity when stored at280 uC. The reasons for these differences are unclear,

but may be due to stabilizing effects of serum proteinswithin the lysate (Komolafe, 1979). Additionally, we alsofound that infectivity was maintained after as many as 56freezethaw cycles (data not shown). Therefore, FCVappears to tolerate multiple freezethaw cycles withoutloss of infectivity, indicating that storage of viral lysates at280 uC is optimal.

Previous analyses of the capsid and propol sequences ofVS-FCV isolates found them to be no more similar to eachother than to other non-VS isolates (Hurley et al., 2004;Abd-Eldaim et al., 2005; Foley et al., 2006). Our sequenceanalyses support these conclusions and suggest that the

Fig. 6. Phylogenetic analysis of FCV proteinasepolymerase (propol) nucleotide and protein sequences. Phylogeneticanalysis was performed on the (a) nucleotide sequence and (b) protein sequence of propol for all FCV isolates used in this

study and 14 other FCV sequences obtained from GenBank. Trees were generated by using the nearest-neighbour distancemethod. Rabbit hemorrhagic disease virus (RHDV) was used as an outgroup for each analysis; outgroup branch lengths havebeen shortened for simplicity. *VS-FCV isolates sequenced in this paper; DVS-FCV isolates sequenced previously; dFCV fieldisolates sequenced in this paper. Vaccine strains are underlined and the strain name is provided in parentheses. Bootstrapvalues >90 % are indicated.




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different VS-FCV isolates have arisen independently indistinct geographical locations. These studies also suggestedthat there were amino acid sequences within the hypervari-able region (residues 426452) of the capsid that wereunique to VS-FCV isolates. However, when we analysed allof the available VS-FCV sequences together, we found thatthere were no sequences or amino acid changes unique to allVS isolates in the capsid or propol regions.

We recognize that many of our conclusions are based on twoassumptions. The first is that the VS-FCV samples that weare characterizing still possess the virulent nature of theoriginal isolate. Passage in tissue culture has been limited toprevent selection of variants. Furthermore, three of theisolates that we characterized have been used to reproduceVS-FCV disease in cats [Ari (Pedersen et al., 2000); FCV-5(Rong et al., 2006); Kaos (P. A. Pesavento, unpublisheddata)]. Whilst the other isolates have not been tested in vivo,the experience thus far is that VS-FCV isolates can bepassaged 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 donot cause VS disease. However, it is possible that many casesof VS-FCV go undetected. The investigated isolate FCV-127originated from a shelter where numerous cats died due topneumonia. In all experiments, isolate FCV-127 behavedmore like a VS-FCV isolate than the other non-VS fieldisolates. Thus, we speculate that the differences in virusgrowth patterns that we noted between VS and non-VSviruses may in fact be shared by other virulent FCV isolates.Currently, the only means to verify that a particular isolatecauses VS disease is by reproducing the disease syndrome inan experimentally infected cat.

In summary, we have investigated several viral character-istics in an attempt to define an in vitrophenotype unique toVS strains. Our findings indicate that there are differencesbetween VS and non-VS isolates that may serve as markersfor virulence. We are currently evaluating further the geneticbases of these differences.

ACKNOWLEDGEMENTS

We thank Dr Neils Pedersen and Dr Kate F. Hurleyfor the generous giftof reagents. Aziza Solomon, Lynne Anguish and Christian Nelsonprovided excellent technical assistance. We thank Dr Ed Dubovi of the

New York State Animal Health Diagnostic Center at CornellUniversity, NY, USA, for providing VS and other FCV isolates. Wethank Karin Hoelzer for advice on phylogenetic analysis. We thank DrHollisErb foradvice on statistical analyses.This work wassupported bygrants from the Cornell Feline Health Center and the WinnFoundation Miller Trust. R. J. O. is the recipient of a scholarshipfrom Cornell University.

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