Large-Scale Spatial and Temporal Genetic Diversity of FelineCalicivirus

Karen P. Coyne,a Rob M. Christley,a Oliver G. Pybus,a,b Susan Dawson,a Rosalind M. Gaskell,a and Alan D. Radforda

University of Liverpool, Institute of Infection and Global Health, School of Veterinary Science, Leahurst Campus, Neston, South Wirral,a and Department of Zoology,University of Oxford, Oxford,b United Kingdom

Feline calicivirus (FCV) is an important pathogen of domestic cats and a frequently used model of human caliciviruses. Here weuse an epidemiologically rigorous sampling framework to describe for the first time the phylodynamics of a calicivirus at re-gional and national scales. A large number of FCV strains cocirculated in the United Kingdom at the national and communitylevels, with no strain comprising more than 5% and 14% of these populations, respectively. The majority of strains exhibited arelatively restricted geographical range, with only two strains (one field virus and one vaccine virus) spreading further than 100km. None of the field strains were identified outside the United Kingdom. Temporally, while some strains persisted locally forthe majority of the study, others may have become locally extinct. Evolutionary analysis revealed a radial phylogeny with littlebootstrap support for nodes above the strain level. In most cases, spatially and temporally diverse strains intermingled in thephylogeny. Together, these data suggest that current FCV evolution is not associated with selective competition among strains.Rather, the genetic and antigenic landscape in each geographical location is highly complex, with many strains cocirculating.These variants likely exist at the community level by a combination of de novo evolution and occasional gene flow from thewider national population. This complexity provides a benchmark, for the first time, against which vaccine cross-protection atboth local and national levels can be judged.

One of the major challenges of molecular epidemiology is toprogress from qualitative descriptions of pathogen diversity

to an understanding of the processes that generate the temporaland spatial distributions of pathogen strains and to explain howthese are affected by both epidemiology and interactions with thehost immune response (15). The interpretation of such data will,however, inevitably be affected by the samples chosen for analysis.Although structured sampling techniques are well established inepidemiology, they appear to be used less commonly in molecularepidemiology, and sampling is often carried out on samples ob-tained by convenience. For this study, we obtained a large, strati-fied random sample of feline caliciviruses (FCVs), which are im-portant pathogens of cats that exist at a high prevalence in thegeneral cat population. Evolutionary analysis was used to explorethe complex temporal and spatial dynamics of this virus at thenational and community levels in the United Kingdom and torelate the dynamics to the international spread of the virus.

FCV is a member of the Caliciviridae (14, 33). It has a 7.7-kbpositive-sense RNA genome with three open reading frames(ORFs), encoding the nonstructural proteins, the major capsidprotein, and a minor structural protein. Genetically, FCV strainsbelong to one diverse genogroup, with little evidence for subspe-cies clustering. This genetic diversity is accompanied by antigenicdiversity, although there is sufficient cross-reactivity that all iso-lates are deemed to belong to a single serotype (28). Several vac-cines, both live and inactivated, are licensed for disease control,although they do not prevent infection (30). Infection is generallyassociated with relatively mild oral and respiratory tract disease.However, the virus’s genetic plasticity has led to the repeatedemergence of variants (or pathotypes), some of which can be le-thal (8, 18, 25, 33, 42, 43). In addition to its significance for felinehealth, FCV is frequently used as a model for human norovirus, animportant cause of vomiting and diarrhea in people (36).

Several studies have explored the evolution of FCV in different

populations. In individual cats, positive selection for FCV anti-genic change is believed to contribute to the establishment of per-sistent infections (7, 19, 21, 39). Evolutionary rates of the variableregions of the capsid protein have been estimated to be up to 1.3 !10"2 to 2.6 ! 10"2 substitution per nucleotide per year (7), one ofthe highest evolutionary rates reported for any virus. In largegroups of cats, such as those in breeding households, immunolog-ical selection may operate at the level of the colony, leading toextremely high levels of observed strain diversity and long-termendemic infection (5, 7). As a result of persistent infections, theprevalence of FCV is high, ranging from approximately 10% in thegeneral cat population to as much as 90% in some colonies, mak-ing it relatively easy to obtain FCV isolates from randomly sam-pled cats (27, 33, 54).

Studies on FCV diversity and evolution have led to a generallyaccepted level of genetic distance by which strains can be differen-tiated. Nucleotide sequences of the variable and immunodomi-nant regions C to E of the capsid gene (44) differ at more than 20%of sites when epidemiologically unrelated viruses are compared,and these are considered distinct strains (33). Epidemiologicallyrelated viruses such as those found in acute outbreaks of diseaseare usually less than 1 to 2% divergent (and rarely more than 5%divergent) and are considered variants of the same strain (31, 35).However, during endemic infections, such as those that exist insome household colonies, variants of a single strain have been

Received 22 March 2012 Accepted 26 July 2012

Published ahead of print 1 August 2012

Address correspondence to Alan D. Radford, alanrad@liv.ac.uk.

Supplemental material for this article may be found at http://jvi.asm.org/.

Copyright © 2012, American Society for Microbiology. All Rights Reserved.

doi:10.1128/JVI.00701-12

11356 jvi.asm.org Journal of Virology p. 11356–11367 October 2012 Volume 86 Number 20

shown to vary by up to 18% (7). Using such definitions, we haveshown that individual cats are generally infected with a singlestrain but may be infected with two, leading to the possibility ofinterstrain recombination at the ORF1-ORF2 junction (9).Within household colonies and rescue shelters, many strains oftencocirculate, with the number of strains probably reflecting the sizeof the colony and its degree of isolation and turnover (6, 34, 38).

The evolution of FCV is well studied at the individual and smallcolony scales. The aim of this study was to investigate the evolu-tion and diversity of FCV at the community and national levelsand to test whether the observations from small populations applyover larger temporal and spatial scales. In order to address thesequestions, we have undertaken a comprehensive repeat cross-sec-tional national study of FCV genetic diversity, using cats attendingrandomly selected veterinary practices from across the UnitedKingdom, in tandem with an in-depth longitudinal communitystudy centered on two local veterinary practices. Taken togetherwith questionnaire data, the data in this work represent an un-

precedented molecular epidemiological study of the evolution ofan RNA virus of veterinary importance over previously unex-plored spatial and temporal scales.

MATERIALS AND METHODSCross-sectional national survey. Two individual cross-sectional surveyswere carried out to collect oropharyngeal (OP) samples from cats attend-ing veterinary practices during a 12-month period in 2005 to 2006. Inorder to reduce the risk of bias in the sample collection, three veterinarypractices were chosen by random selection from each of the 23 geograph-ical regions of the United Kingdom (identified in the 2004 Royal Collegeof Veterinary Surgeons practice register) and were asked to participate inthis study (Fig. 1A). If a practice declined to take part in the study, thenanother practice from the same region was randomly selected. Each prac-tice was asked to take oropharyngeal swabs between October and Decem-ber 2005 from cats belonging to 30 owners who gave their informed con-sent (cross-sectional study 1 [CS1]). Each practice was asked to repeat thesampling procedure in April to June 2006 (CS2). The dates chosen forsampling were based on convenience and did not relate to any perceived

FIG 1 (A) Locations of the veterinary practices sampled in the two national cross-sectional surveys (filled circles) and the longitudinal community surveys (opentriangle [L] and open diamond [W]). The colors of the circles correspond to the node tips in Fig. 2. (B) Pairwise geographic distances between all veterinarypractices in the national survey. (C) Comparison of the prevalences of FCV per practice in CS1 and CS2. Only practices submitting 10 or more swabs wereincluded in the analysis. (D) Number of sequences isolated per practice (national and community) compared to the number of strains isolated per practice.

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seasonal pattern of FCV dynamics. The mean geographical distance be-tween practices was 317 km (range, 7 to 1,096 km) (Fig. 1B).

Longitudinal community survey. To obtain a more detailed under-standing of the local diversity and dynamics of FCV, oropharyngeal sam-ples were collected over a 14-month period by sampling cats attendingtwo veterinary practices in northwestern England (L and W) (Fig. 1A).These practices were chosen based on compliance and convenience. Eachpractice was asked to take oropharyngeal swabs from up to 30 cats a weekfor cats belonging to compliant owners who gave their informed consentand who visited their surgery for the 60 weeks between 22 October 2006and 14 December 2007.

For both the national and local community virus collections, the geo-graphical location of each sample was determined by the owner’s post-code, and a short questionnaire which included the cat’s age, sex, breed,vaccination status, and brief medical status relating to mouth ulcers andrespiratory disease was administered. This questionnaire was completedby each cat’s owner or veterinary surgeon. Oropharyngeal swabs werecollected into 2 ml of virus transport medium, batched, and sent to ourlaboratories for virus isolation.

Virus isolation. Feline calicivirus and feline herpesvirus 1 (FeHV-1)were isolated as described previously on feline embryo A (FEA) cells orCrandall Reese feline kidney (CRFK) cells (20, 50) and were stored at"80°C for further analysis.

RNA extraction, reverse transcription-PCR (RT-PCR), and consen-sus sequencing. RNAs were extracted from positive cell cultures (secondpassage or less) (QIAmp viral RNA minikit; Qiagen) and transcribed intocDNAs (Superscript III; Invitrogen) by use of 500 ng of random hexamers(Abgene) as the first-strand primer, according to the manufacturers’ in-structions. A 529-nucleotide region of the capsid gene, equivalent to res-idues 6406 to 6934 of FCV strain F9 (4) and incorporating the immuno-dominant region E (23, 40, 44), was amplified as previously described (7).We have previously shown that sequences generated from cell culture are#99% accurate compared to sequences obtained directly from OPswabs (7).

In order to allow comparisons between polymerase and capsid se-quences, a PCR targeting part of the relatively conserved 3= polymerase-encoding region of the FCV genome was carried out on representativeFCV isolates from CS1 for which the corresponding capsid sequence wasavailable. A single-stage PCR was performed to amplify a 486-nucleotideregion of the 3= polymerase-encoding region of the FCV genome, equiv-alent to residues 4766 to 5251 of FCV strain F9 (4), essentially according topreviously described protocols (9).

All capsid and polymerase amplicons were purified (QIAquick PCRpurification kit; Qiagen Ltd.) and sequenced bidirectionally using corre-sponding PCR primers according to standard protocols (ABI Prism Big-Dye Terminator, version 3.0, cycle sequencing kit; Applied Biosystems).For each amplicon, a consensus sequence was produced using Chro-masPro v1.32 (Technelysium Pty. Ltd.). Ambiguities identified consis-tently on both strands of sequence were included in the consensus se-quence. All primer sites were removed prior to sequence analysis. For thecapsid amplicons, a final useable sequence of 420 nucleotides, corre-sponding to codons 383 to 522 of the FCV capsid (44) and includingvariable regions C and E, was used for analysis.

Nucleotide sequence and phylogenetic analysis. Geographical dis-tances between isolates were calculated using free mapping tools (http://www.freemaptools.com/) and R (The R Project for Statistical Comput-ing, Vienna, Austria [http://www.r-project.org/]).

To assign sequences to strains, we used the previously defined nucle-otide distance threshold of 20% for hypervariable region E of the capsid(31, 35). Pairwise genetic distances between sequences were calculatedusing the Jukes-Cantor approach as implemented in MEGAv4.0 (47).

For phylogenetic analyses, sequence alignments were initially esti-mated using MUSCLE v3.6 (11) and manually corrected usingGENEDOC v2.7. Maximum likelihood trees with 1,000 bootstrap repli-cates were calculated using PHYML 3.0 (16), using the most appropriate

model of evolution, which was identified using the model selection ap-proach implemented in Topali v2.5 (22). Amino acid phylogenies wereestimated using MEGAv4.0. Sequences from the community study wereanalyzed alongside published FCV sequences from historical isolates dat-ing between 1958 and 1995 and retrieved from GenBank (13). Quantifi-cation of phylogenetic structure was undertaken using likelihood map-ping as implemented in TREEPUZZLE (45).

Associations between phylogeny and the sampling locations of se-quences were explored (for the community data sets) using the parsimonymethods implemented in BaTS v2 (24), using published global and ances-tral sequences as controls. Tests for nucleotide sequence saturation werecarried out using plots of the transition/transversion ratio versus diver-gence and using the Xia test for saturation as implemented in DAMBE(51).

Finally, to explore the level of phylogenetic signal across the genome,JC neighbor-joining phylogenies estimated from polymerase and capsidsequences were compared. Selection pressures on codons were estimatedusing the SLAC method as implemented in DATAMONKEY (10).

Epidemiological analysis. Univariable and multivariable multilevellogistic regression analyses were carried out using MLWIN (v2.1; Centrefor Multilevel Modeling, University of Bristol). The samples were clus-tered within region and within veterinary practice, and hence these factorswere included in the models as random effects. Separate analyses wereundertaken to identify risk factors for three outcomes of interest: FCVcarriage, presence (at time of sampling) of mouth ulcers, and presence (attime of sampling) of signs of upper respiratory tract disease (URTD). Foreach analysis, initial univariable screening was undertaken for all potentialrisk factors for which data were available. Potential risk factors consideredincluded the age of the cat (categorized into $48 months and #48months, based on generalized additive models [not shown]), sex, breed,vaccination status, time of study (i.e., CS1 or CS2), and FCV carriage (thelatter for the models for ulcers and URTD only). Those variables with Pvalues of $0.25 in univariable screening were considered in the multivari-able model by backward elimination. In all cases, models were fitted usingMarkov chain Monte Carlo (MCMC) simulation, using a Metropolis-Hastings sampler with diffuse priors (41). The number of iterations usedwas determined by examination of the Raftery-Lewis and Brooks-DraperNhat statistics. In each case, a chain length of 50,000 integrations wassufficient, so this length was used throughout. The burn-in was set at 1%of the chain length (12). Model fit was assessed by examining the posteriordistributions of the fixed and random variables included in the mixed-effects models. In each case, the fits were smooth and regular and approx-imated a normal distribution.

A quadratic assignment procedure correlation test (UCInet v 6.288;Analytic Technologies, Boston, MA) was used to assess the relationshipbetween genetic distance (%) and geographical distance. This methodcomputes standard measures of correlation between the genetic and geo-graphic distance matrices and then computes an estimate of the signifi-cance of the correlation by permuting the elements of one of the matrices5,000 times and counting the number of correlations between the ob-served and permuted matrices that are larger or smaller than the empiricalestimate.

Nucleotide sequence accession numbers. Nucleotide sequences ob-tained in this study have been submitted to GenBank under accessionnumbers JX123959 to JX124213.

RESULTSCharacteristics of study population. For the national survey, ofthe 69 practices recruited for CS1, 57 (83%) returned samples(range, 3 to 34 samples; mean, 26 samples). Of these, 52 practicesalso submitted samples for CS2. For those regions where practicesdid not return samples for CS1, a replacement practice was re-cruited for CS2 (n % 12). Of the 69 practices recruited for CS2, 57(83%) returned samples (range, 4 to 33 samples; mean, 25 sam-ples).

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In total, 2,818 swabs were received in the CS1 and CS2 nationalsurveys, including swabs from 23 cats that were sampled twice,with 21 testing negative on both occasions, 1 testing negative inCS1 and positive in CS2, and 1 testing positive in both. The overallFCV prevalence in this population by virus isolation was 9.3% forCS1 (0 to 33% for individual practices) and 11.0% for CS2 (0 to37% for individual practices) (Table 1). There appeared to be littlecorrelation between the prevalence obtained in CS1 and CS2 foreach practice (r2 % 0.11) (Fig. 1C), nor was there an effect ofseason/date of sampling.

For the community survey, a total of 1,047 samples were sub-mitted over a 60-week period (n % 633 for practice L and n % 414for practice W). The overall FCV prevalence by virus isolationwithin these communities was 13.9% for practice L and 9.2% forpractice W.

Risk factors for FCV carriage and for signs of FCV disease.There was a significant effect of age on FCV carriage, with a reduc-tion in risk each month for the first 4 years, followed by a plateau.

This resulted in an odds ratio of 0.9 per year (0.99 per month) foreach of the first 4 years of life (Table 2). There was also a significanteffect of FCV vaccination (Wald &2 % 22.042; P $ 0.001), withvaccinated cats being approximately half as likely to test positivefor FCV as those that were not vaccinated. The group whose vac-cination status was unknown was not significantly different fromthe unvaccinated cats.

There was a significant association between FCV carriage andvaccination, age, and the presence of oral ulceration and signs ofURTD (Table 2). For oral ulceration, age significantly increasedthe odds (after allowing for FCV carriage and vaccination status).Those cats positive for FCV were around 12 times as likely to haveoral ulceration, whereas vaccinated cats were approximately halfas likely to have oral ulceration. The relationship between URTDand vaccination and FCV status was more complex due to aninteraction between these risk factors. In short, compared to un-vaccinated, FCV-negative cats (the reference group), there was asignificantly higher odds of signs of URTD in unvaccinated, FCV-positive cats and a significantly lower odds in vaccinated, FCV-negative cats. Other categories were not significantly differentfrom the reference group.

Sequence analysis for strain identification. Partial sequencesof the immunodominant region of the FCV capsid were obtainedfrom 55 to 70% of the isolates obtained for each population (Table1). Amplification success rates reached 88% when we includedeither capsid or polymerase PCR results (data not presented). Us-ing a JC pairwise genetic divergence of 20% as the threshold be-tween variants of the same strain, a total of 171 strains were iden-tified within the national and community surveys (Table 1). Intotal, 45 strains contained more than one variant, including 28from the national study and 17 from the community study. Forty-

TABLE 1 Summary of samples, sequences, and strains identified in eachpopulation

Study

No. ofcatssampled

No. (%) ofFCV-positivecats

No. (%)of isolatessequenced

No. ofstrains

National surveysCS1 1,468 137 (9.3) 85 (62) 71CS2 1,350 149 (11.0) 86 (58) 58

Community surveysL 633 88 (13.9) 62 (70) 27W 414 38 (9.2) 21 (55) 15

TABLE 2 Final multivariable models of risk factors associated with FCV carriage, presence of oral ulcers, and presence of signs of URTD

Model and factor Beta value SE OR Lower CI Upper CI Wald chi-square value P value

Multivariable model for FCV carriageAge (per month for up to 48 months) "0.009 0.003 0.991 0.985 0.997Vaccination status

No Reference 22.042 $0.001Yes "0.568 0.121 0.567 0.447 0.718Unknown "0.223 0.185 0.800 0.557 1.150

Multivariable model for oral ulcersAge (per month for up to 48 months) 0.028 0.005 1.028 1.018 1.039 26.5 $0.001Vaccination status

No Reference 10.243 0.001Yes "0.532 0.207 0.587 0.392 0.881Unknown 0.265 0.301 1.303 0.723 2.351

FCV carriageNo Reference 155.301 $0.001Yes 2.508 0.201 12.280 8.282 18.210

Multivariable model for signs of URTDAge (per month for up to 48 months) 0.007 0.004 1.007 0.999 1.015 3.185 0.07FCV carriage- vaccination interaction

FCV negative, not vaccinated Reference 68.403 $0.001FCV negative, vaccinated "0.471 0.174 0.624 0.444 0.878FCV negative, unknown vaccination status "0.258 0.321 0.773 0.412 1.449FCV positive, not vaccinated 1.473 0.249 4.362 2.678 7.107FCV positive, vaccinated "0.862 0.498 0.422 0.159 1.121FCV positive, unknown vaccination status "0.332 0.853 0.717 0.135 3.819

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four of these 45 strains were supported by bootstrap values of#80%; the exception was clade J in the cross-sectional study (Fig.2). No strains were common to both the national and communitystudies (data not presented), except for a vaccine strain (F9) thatappeared in both populations. Apart from FCV F9, no strainsidentified here were also seen outside the United Kingdom (basedon BLAST searches of GenBank [data not presented]). All but fiveof these strains (E, J, L, and W in the national survey [Fig. 2 andTable 3] and LW in the community survey [see Fig. 4]) were re-stricted to a single practice. Only in rare instances (7 cases in thenational study and 1 case [L12] in the community study) weresequences with pairwise nucleotide divergences of #20% foundclustered together with bootstrap scores of #80% (Fig. 2; also seeFig. 4).

Using this definition of a strain, there was a clear linear rela-tionship (r2 % 0.89) between the number of sequences obtainedfrom a practice and the number of strains within it, with the num-ber of strains being equal to approximately half the number ofsequences (r2 % 0.7398 for the national survey alone [data notpresented]). This suggests that despite the extensive nature of oursurvey, the extent of the genetic diversity in any given practiceremains far from fully characterized (Fig. 1D).

Evolutionary analysis. (i) National survey. The results of thephylogenetic analysis of the CS1 and CS2 samples are shown inFig. 2. This diverse phylogeny contained 71 and 58 strains, iden-tified in CS1 and CS2, respectively (Table 1). The phylogeny wasstrongly radial or star-like in nature (short external branches andlong internal branches); correspondingly, there is a consistent lackof bootstrap support for most nodes near the root of the phylog-eny. To quantify the lack of phylogenetic structure, we performeda likelihood mapping analysis (not shown) which concluded thatapproximately 25% of quartets are unresolvable or poorly resolv-able.

Since the sequences are highly diverse, this lack of phylogeneticstructure is unlikely due to a lack of polymorphism. Indeed, visualinspection of the alignments suggests that individual nucleotidesites are either invariant or very highly variable. We therefore per-formed further tests to determine if the lack of phylogenetic struc-ture was caused by sequence saturation. For the national study,plots of the transition/transversion ratio against divergence indi-cated substantial saturation in the capsid sequences for geneticdistances of #0.5 but provided little evidence of saturation in thepol data set (data not presented). However, with the Xia test forsaturation (51), both alignments showed significant levels of nu-cleotide saturation. As a result of this saturation, we also estimateda phylogeny by using amino acid sequences, which showed that 35of the 45 strains were supported by high bootstrap values (over 90)(data not shown). Of the remaining 10 strains, 7 were still mono-phyletic, but with lower bootstrap support, and 3 were no longermonophyletic. In addition to clarifying the role of sequence satu-ration, these results support the decision to classify FCV strains byuse of a pairwise genetic divergence threshold of 20%.

Returning to Fig. 2, the most prevalent strains made up only3% (strain A; 5 of 171 strains) and 5% (strain L [F9 vaccine strain];

FIG 2 Unrooted maximum likelihood tree of 172 FCV capsid consensus se-quences obtained from the national study (including the sequence of FCVvaccine strain F9 [GenBank accession no. M86379]). Circles indicate se-quences from CS1, and triangles indicate sequences from CS2. The colors ofthe node tips correspond to the regions of sampling shown in Fig. 1. Grayboxed areas represent strains A and AB (as defined using a JC genetic distanceof $20%). All other nodes with bootstrap support of #80% are indicated byasterisks. Minimum-maximum genetic diversity (%) and minimum-maxi-mum geographical distances (km) are indicated next to the corresponding

strains. Evolutionary distances were calculated using a GTR model and themaximum likelihood method, with the scale bar indicating the number ofestimated substitutions per site. Numbers at major nodes are % bootstrapvalues (for 1,000 replicates; only values of #80% are shown). iom, Isle of Man.

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8 of 171 strains) of the total sequenced population. Even withinindividual practices, high levels of strain diversity sometimes ex-isted, with up to five strains identified in some practices in a singlesampling period (with averages of 2.0 and 1.7 for CS1 and CS2,respectively) (data not shown).

The sequences from CS1 and CS2 were not phylogeneticallydistinct, with sequences from both surveys intermingled in thetree and low bootstrap support for nodes joining most pairs ofstrains. There were no differences between nucleotide distances incomparing viral sequences obtained within and between the twosurveys (within-CS1 distances, 0 to 53% [average, 38%]; within-CS2 distances, 0 to 55% [average, 38%]; and distances betweenCS1 and CS2, 4 to 55% [average, 38%]) (see Fig. S1 in the supple-mental material). The program BaTS revealed some correlationbetween the date of sampling (CS1 versus CS2) and the phylogeny(P $ 0.005 using the AI and PS statistics), although significancewas lost when multiple variants of individual strains were re-moved from the alignment.

In addition, BaTS uncovered evidence for clustering of strainsaccording to geographic region (Fig. 1A and 2) (P $ 0.005 usingthe AI and PS statistics). With the exception of the Orkney Islandsand Wales, this was evident for all geographic regions (P $ 0.05).With that said, all regions contained multiple strains, even smallerislands such as the Isle of Man, the Channel Islands, and the Isle ofWight.

Given the weak structure and bootstrap support for the es-timated phylogenies, we further explored the spatial clusteringof strains by plotting pairwise genetic distances versus geo-graphic distances. Regardless of the genomic region (pol orcapsid gene) used or whether 3rd codon positions were ex-cluded, we observed a weak association between genetic andspatial distances (Pearson correlation, 0.066 to 0.117) (Fig. 3).The weak signal that was present was driven mainly by variantsof individual strains (divergence of $20%) confined to a singlepractice. In contrast, sequence pairs above the strain threshold(pairwise divergence of #20%) showed no association withdistance.

Of the 28 strains for which more than one variant was iden-tified, 21 were restricted to a single practice and obtained dur-

ing a single cross-sectional sampling period. The maximumgeographical range among these 21 strains was 16 km (strainM) (Fig. 2). For the other seven strains, six were found duringboth CS1 and CS2, suggesting that they had persisted in thepopulation, and four were present in more than one practice(Table 3). Only two strains were found at locations #100 kmdistant (strains L and W), suggesting that they had spread morewidely. The most numerous (n % 8) and widely dispersed (upto 438 km) strain was FCV F9, a strain commonly used in livevaccines.

Previous studies have suggested that FCV and other calicivi-ruses have a predilection for recombination at the ORF1-ORF2junction (3, 9). To assess the significance of recombination in thispopulation, the polymerase sequence was obtained for 48 of thesamples whose capsid sequence was also available in CS1. Phylo-genetic analysis of these two ORFs failed to identify any recentrecombination events (see Fig. S2 in the supplemental material),although the radial phylogeny with associated low bootstrap sup-port for internal nodes observed in both trees likely precludes theidentification of older recombination events in this data set. Fur-thermore, the selection analysis revealed no evidence of positiveselection; rather, 127 of 140 codons were predicted to be underpurifying selection (P $ 0.05) (data not shown).

(ii) Community survey. The results of phylogenetic analysisfor the community samples are shown in Fig. 4. As with the na-tional survey, the genetic diversity within both communities washigh, with 27 and 15 strains in total identified in veterinary prac-tices L and W, respectively (Table 1). All but one of these strains(strain LW) were confined to a single community/practice. Nostrain made up more than 14% of the sequences obtained from itspractice (strains L1, L9, and L11 each comprised 7 of 62 [11%]sequences, and strains W5 and W7 each comprised 3 of 21 [14%]sequences). One cat from practice L tested positive three times(strain L5). A single F9-like virus was found in the samples fromcommunity W.

At the community level, 15 of the 16 strains for which morethan one variant was identified were seen in more than 1 month(range, 2 to #10 months). Longitudinal sampling allowed us toassess the persistence of the most numerous strains (five ormore variants) within community L (Fig. 5A). Strain L1 wasfound at a low prevalence (6 of 74 sequences [8%]) over a10-month period, suggesting its persistence in this community.In contrast, strain L11 was seen only in the first 2 months, at arelatively high prevalence (7 of 25 sequences [28%]), suggest-ing that it may have become extinct in this population, whereasstrain L6 was absent for the first 9 months (0 of 68 sequences) ofthe study and was observed only at a high prevalence in the last3 months (5 of 19 strains; 26%), suggesting that this virus mayhave been newly introduced into this community. Strains L8and L9 appeared only transiently, during the middle of thesampling period.

The geographical footprint of strains in the community surveyis shown in Fig. 5B and C. For the 7 strains for which completepostcode information was available, the maximum geographicrange was 22 km (strain L11). There appeared to be no correlationbetween the genetic diversity and the spatial distribution of vari-ants within strains (Fig. 5C).

Of the three populations of viruses included in the phylogeny(i.e., L, W, and published world sequences), none were monophy-letic (Fig. 4). However, there was a tendency for the sequences

TABLE 3 Summary of strains from the national survey that were eitherpresent in both cross-sectional surveys or present at more than onepracticea

Strain

Presence incross-sectionalsurvey

No. ofpracticeswherestrain waspresent

No. ofsequences

Maximumdistance

CS1 CS2 kmDNA(%)

A ! ! 1 5 3–26 0–13E ! ! 2 2 70 19J ! 2 2 29 18Lc ! ! 5 8 2–438 0–18U ! ! 1 2 2 17W ! ! 2 2 205 20Z ! ! 1 2 IOMb 18a Strain identity is the same as that in Fig. 2.b Isle of Man. No postcode data were available; the size of the island is 52 km by 22 km(http://www.gov.im/isleofman/geography.xml).c Strain L is phylogenetically related to FCV F9.

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obtained in this study to be phylogenetically distinct from thoseobtained from GenBank. Most notably, there is a cluster contain-ing six published global reference sequences and one sequencefrom our survey (from community L).

Strong bootstrap support for nodes containing sequences ob-tained in this study was generally restricted to variants of the samestrain. Bayesian phylogenetic analysis suggested some geographi-cal structure above the “strain” level (P $ 0.01 using BaTS whenonly one sequence per strain is retained). This spatial structure isapparently driven by sequences from community L (mean ob-served clade size, 7.4; P % 0.01) and the global reference sequencesmentioned above (mean observed clade size, 3.5; P % 0.05). Se-quences from community W lacked any geographical correlationwith the phylogeny.

DISCUSSION

Our aim is to understand the genetic diversity of FCV across a rangeof temporal and spatial phylodynamic scales. In previous studies, wehave shown that FCV is subject to positive selection within individualcats (7, 39). Within cat colonies, high FCV prevalence is often associ-ated with high levels of strain diversity, with colony persistenceachieved by evolution within a small number of persistently infectedindividuals, and by reinfection of other members of the population(7, 34). We have suggested that immune-mediated positive selectionthus operates at the colony level to generate viral diversity. In thisstudy, we determined the extent to which these strains persist andmix in the wider population. Specifically, we measured the ge-netic diversity and persistence of FCV in rigorously sampled

FIG 3 Comparison of genetic distances (%) and geographical distances (km) for FCV sequences obtained from the national cross-sectional studies, based oncapsid (A and B) and polymerase (C and D) sequences. Panels A and C are based on codon positions 1, 2, and 3, and panels B and D are based on codon positions1 and 2.

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communities, as well as nationally. The prevalence and riskfactors (effects of age and vaccination status) obtained in thisstudy are in close agreement with those in previous work forboth owned and unowned cats, suggesting that these resultsmay be generalizable to other cat populations (6, 7, 27).

It is clear from the current study that the number of strainscirculating in these populations is high, with 123 strains observedover 9 months and 41 strains observed over 14 months in thenational and community studies, respectively. This level of straincomplexity was surprising and previously unseen. It is associatedwith a radial, star-like phylogeny which exhibits low bootstrapsupport for most nodes above the strain level. Even individualpractices in the cross-sectional survey had up to five strains at anyone time, and the Isle of Man, which is only 21 km by 37 km,contained 12 strains.

This complex pattern of strain cocirculation is unusual andquite distinct from that seen for the related noroviruses, which aredivided into five genogroups and a total of 29 genotypes (52). Thediversity within a norovirus genogroup is '15 to 45%, based onuncorrected amino acid distances over the entire capsid. Aminoacid diversity of $15% represents variants of the same genotype,and a diversity of #45% represents viruses in distinct genogroups.The capsid amino acid diversity between strains of FCV rangesfrom 9 to 19% (13), which is somewhat comparable to that for asingle genogroup of norovirus, with FCV strains (as we definethem) somewhat equivalent to norovirus genotypes (e.g., II.1 andII.12) (52). However, in this study, we identified over 100 strains

in the United Kingdom alone, and our sampling suggests that thetrue number could be very much higher. This indicates a greaterdegree of lineage cocirculation for FCV than for noroviruses.However, norovirus studies are generally performed on clinicalsamples obtained by convenience; it would be informative togather similar structured data for the human viruses to see if thesedata would better define the temporal and spatial circulation ofthese viruses.

Within this overall complexity, the maximum prevalence ofany given field strain was 5% and 14% in the national and com-munity studies, respectively. This diversity is reminiscent of thatfound within feline rescue shelters with good biosecurity, which inthe absence of large-scale within-shelter transmission have beensuggested to act as samplers of community pathogen diversity (6,38). This complex pattern of strain coexistence again contrastswith the case for the noroviruses, for which individual genotypespredominate in any given season. Most notably, genotype II.4 hascomprised 44% of 773 norovirus outbreaks in the United Statesover a 12-year period (1994 to 2006), with 51 to 85% of recentoutbreaks being associated with GII.4 (46, 48, 53). The nationaland international epidemiological dominance of genotype II.4 hasbeen attributed to a higher error rate of its viral RNA polymeraseand an associated higher rate of evolution within the virus capsid(2). In contrast, extensive lineage cocirculation of FCV indicatesthat no strain has been able to outcompete the others. This couldbe explained by geographic subdivision of thehost population pre-venting strains from competing directly. However, this seems un-

FIG 4 Unrooted maximum likelihood phylogeny of 83 FCV consensus sequences obtained from the community study and 21 published FCV sequencesobtained from GenBank (accession numbers are shown in reference 13). Strains (L1 to L11, W1 to W4, and L plus W) are represented by shaded arcs. For eachstrain, the JC genetic diversity (%) and, where available, the geographic dispersal (km) and number of calendar months in which it was present are indicatedwithin the box. Published sequences are presented by name, country, and year of origin. Numbers at major nodes are bootstrap values of #800 of 1,000 replicates(expressed as percentages).

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FIG 5 Temporal and spatial distributions of strains from the community survey. (A) Bar chart showing the number of selected strain types (#3 sequences) incommunity L (same colors as in Fig. 4). White areas represent all strains seen only once; shaded areas (ns) are isolates that were refractory to capsid sequencing.The gray line represents the number of swabs received per month. (B) Spatial distribution of selected sequences obtained in the community study, based onpostcode data. Gray circles and squares represent all samples from communities L and W, respectively. Colored circles represent locations of strains fromcommunity L, and colored squares represent locations of strains from community W. (C) Genetic distance (JC %) compared to geographical distance (km) forstrains in the community study. Colored circles represent individual strains.

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likely, since we have shown that even small islands and individualveterinary practices can have many coexisting strains with appar-ently overlapping geographical footprints. It is more likely thatinsufficient immunological interaction between FCV strainswould allow cats to undergo cycles of reinfection or coinfection,both of which have been observed in the field (7). These observa-tions are consistent with the low or absent cross-protection be-tween some strains in vitro and with a failure of the host immuneresponse to prevent heterologous infection (28, 29). Such weakantigenic cross-protection is predicted to cause chaotic or cyclicalpatterns of strain persistence in populations (17), may explain thelack of positive selection observed in the present community andnational studies, and contrasts with previous studies of individualand colony cats infected with single strains (7, 39).

In this study, for the first time, we have mapped FCV isolates ata high resolution by using the owner’s postcode, allowing us todefine the geographical distribution of individual strains. Themost widely dispersed lineage (strain L) contained variants of FCVF9, a common strain included in several live attenuated vaccines(4, 33). This vaccine virus is occasionally shed by vaccinated indi-viduals (26), and variants of this strain are regularly, albeit occa-sionally, found in the general cat population (1, 6, 7, 37). As such,the geographical range of this strain is unsurprising and likelyrepresents the widespread use of live F9 vaccines rather than nat-ural virus transmission. To date, this level of apparently vaccine-derived viruses is tolerated, although pressure for change maycome from those companies that market inactivated vaccines(30).

For those strains confined to an individual practice, the maxi-mum geographical spread was 26 km in the national survey and 22km in the community survey. This is likely to partly reflect thecatchment area of a practice. A more informative estimate of thefootprint of a given strain may be obtained from the four strainsthat were present in more than one practice. The geographicalranges for these strains were 29 km (strain J), 70 km (strain E), and205 km (strain W) in the national survey and 29 km (strain LW) inthe community survey, proving that FCV strains can be transmit-ted more widely, albeit rarely. The mechanism for these occasionalwidespread dissemination events remains unknown, but the mostlikely possibility is the movement of animals, especially those thatare persistently infected (7, 33, 49), though indirect transmissionmay also occur. These data suggest that FCV has some limitedability for widespread geographical spread, and this is further sup-ported by Bayesian analysis, which shows some limited correla-tion between geography and phylogeny for both the national andcommunity studies. However, we cannot reject the hypothesisthat geographically dispersed variants are more widespread, fortwo reasons. First, high overall strain diversity means that if theprevalence of a strain is sufficiently low in the population, then itis unlikely to be detected in multiple locations by our study design.Second, the majority of practices in the national study were over300 km apart, reflecting the shape of the United Kingdom. Thissuggests that intermediate geographic distances may have beenrepresented relatively poorly in this study design, restricting ourability to detect spread of viruses beyond individual veterinarypractice catchment areas.

Knowing the date that each cat was sampled meant we couldprecisely map the temporal persistence of FCV strains. In the na-tional survey, there was no evidence of phylogenetic clustering bydate of sampling, with only 6 of 28 strains being identified in both

the cross-sectional surveys. In the community survey, sheddingpatterns suggested that some strains persisted at a low prevalenceover many months. In contrast, within the limits of our studydesign, others appeared to exist at a high prevalence for shorterperiods but were undetectable at other times, suggesting their ar-rival in the community or extinction from it. Our data indicatehighly dynamic strain behavior at the local community level,where high prevalence is maintained by the cocirculation of mul-tiple strains, associated with ongoing strain extinction and intro-duction, with some other strains persisting for 10 months ormore. The mechanisms that drive these dynamics are as yet un-clear, but it is likely that population immunity plays an importantrole.

Bayesian analyses suggested that the overall temporal structurein our data was comparatively weak. Although there was evidencethat older published sequences clustered distinctly from our com-munity survey isolates, this was not consistent across the analyses.In addition to a lack of temporal signal, our phylogenies werestar-like (radial) in shape, lacking bootstrap support for internalnodes. We concluded that a combination of high evolutionaryrates coupled with strong evolutionary constraint at other nucle-otide positions has led to sequence saturation at many of thosesites that are able to vary and resulted in a loss of statistical supportfor temporal evolution, phylogenetic structure, and genetic isola-tion by distance. This may be an issue for other studies focusing onhypervariable regions of viral surface proteins.

Throughout this and previous studies, we have used the term“strain” to define closely related viral sequences that share a clearepidemiological link. This remains a useful classification for dis-cussing the dynamics of the virus over short, clinically relevanttime scales. However, at what point does the genetic distance be-tween two sequences become too large to reliably indicate epide-miological linkage? Previously, we suggested that new strains take'5 to 10 years to evolve (7). Our sequence saturation results sug-gest that beyond that time, FCV phylogenies based on subgenomiccapsid (and pol) gene sequences become uninformative abouttransmission history. Thus, the radial FCV phylogenies that weand others have observed represent distinct strains emerging froman opaque cloud of diversity, within which ancestral relationshipscannot be resolved due to sequence saturation. The cloud repre-sents all transmission events older than '10 years; hence, onlyvariants of strains sampled within a decade of each other formstatistically supported clades. Thus, a key conclusion of our studyis that it will be necessary to sequence longer genome regions,possibly full genomes, to overcome saturation and provide greaterphylogenetic resolution. Sequence-nonspecific methods may alsoallow us to address the fraction of FCV isolates that remain refrac-tory to amplification (32).

The structured and detailed sampling methods used in thisstudy improve our understanding of FCV phylodynamics and en-able us to propose a new model for the global evolution and di-versification of FCV strains. Strain diversification occurs in catsand colonies, probably aided by immune-mediated positive selec-tion (7). Over many years, this has generated a large pool of dis-tinct strains which act as a reservoir of genetic and antigenic di-versity. However, sequence saturation among these means that thephylogenetic relationships among strains cannot reliably be dis-cerned until larger genome regions are sequenced. At the commu-nity/practice level, there are two possible explanations for the highstrain diversity observed. Strains could have evolved locally, with

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little widespread dissemination. If this is so, sequence saturationwould have to be strong enough to erase nearly all evidence of acorrelation between genetic and geographic distances. Alterna-tively, strains could be spread rapidly on a larger (perhaps na-tional) level, with communities simply sampling from this diver-sity based on gaps in their local population immunity. Our dataprovide partial support for both of these models, with rare strainstransmitted over relatively large distances and with evidence forsome spatial clustering. Whichever is correct, it seems unlikelythat endemic FCV evolution is driven by strong strain selection atthe national level, which would favor strain turnover and limitedstrain diversity at any one time. Cats are exposed to a wide varietyof antigenically variable FCV strains, including vaccine strains,and the dynamics of herd immunity that result are likely verycomplex. Clearly, a further understanding of the cross-reactivityof responses to different FCV strains is necessary and will alsofacilitate vaccine design. We hope that our study design, in whichepidemiological principles are used to inform sample collection,may provide a framework for others seeking to understand thetemporal and spatial evolution of viruses.

ACKNOWLEDGMENTSKaren Coyne was funded by the PetPlan Charitable Trust.

We are very grateful to all the veterinary practitioners who willinglyhelped in this ambitious project, especially to the two practices partakingin the longitudinal community study. We are grateful to Marco Salemi forassistance with the saturation analyses and to Fiona Physick for assistancewith the polymerase gene sequences.

REFERENCES1. Abd-Eldaim M, Potgieter L, Kennedy M. 2005. Genetic analysis of feline

caliciviruses associated with a hemorrhagic-like disease. J. Vet. Diagn. In-vest. 17:420 – 429.

2. Bull RA, Eden JS, Rawlinson WD, White PA. 2010. Rapid evolution ofpandemic noroviruses of the GII.4 lineage. PLoS Pathog. 6:e1000831. doi:10.1371/journal.ppat.1000831.

3. Bull RA, Tanaka MM, White PA. 2007. Norovirus recombination. J.Gen. Virol. 88:3347–3359.

4. Carter MJ, et al. 1992. The complete nucleotide sequence of a felinecalicivirus. Virology 190:443– 448.

5. Coyne KP, et al. 2006. Long-term analysis of feline calicivirus prevalenceand viral shedding patterns in naturally infected colonies of domestic cats.Vet. Microbiol. 118:12–25.

6. Coyne KP, et al. 2007. Longitudinal molecular epidemiological analysisof feline calicivirus infection in an animal shelter: a model for investigatingcalicivirus transmission within high-density, high-turnover populations.J. Clin. Microbiol. 45:3239 –3244.

7. Coyne KP, Gaskell RM, Dawson S, Porter CJ, Radford AD. 2007.Evolutionary mechanisms of persistence and diversification of a calicivi-rus within endemically infected natural host populations. J. Virol. 81:1961–1971.

8. Coyne KP, et al. 2006. Lethal outbreak of disease associated with felinecalicivirus infection in cats. Vet. Rec. 158:544 –550.

9. Coyne KP, et al. 2006. Recombination of feline calicivirus within anendemically infected cat colony. J. Gen. Virol. 87:921–926.

10. Delport W, Poon AF, Frost SD, Kosakovsky Pond SL. 2010. Datamon-key 2010: a suite of phylogenetic analysis tools for evolutionary biology.Bioinformatics 26:2455–2457.

11. Edgar RC. 2004. MUSCLE: multiple sequence alignment with high accu-racy and high throughput. Nucleic Acids Res. 32:1792–1797.

12. Geyer CJ. 1992. Practical Markov chain Monte Carlo. Stat. Sci. 7:473–511.13. Glenn M, et al. 1999. Nucleotide sequence of UK and Australian isolates

of feline calicivirus (FCV) and phylogenetic analysis of FCVs. Vet. Micro-biol. 67:175–193.

14. Green KY, et al. 2000. Taxonomy of the caliciviruses. J. Infect. Dis.181(Suppl 2):S322–S330.

15. Grenfell BT, et al. 2004. Unifying the epidemiological and evolutionarydynamics of pathogens. Science 303:327–332.

16. Guindon S, Gascuel O. 2003. A simple, fast, and accurate algorithm toestimate large phylogenies by maximum likelihood. Syst. Biol. 52:696 –704.

17. Gupta S, Ferguson N, Anderson R. 1998. Chaos, persistence, and evolu-tion of strain structure in antigenically diverse infectious agents. Science280:912–915.

18. Hurley KE, et al. 2004. An outbreak of virulent systemic feline calicivirusdisease. J. Am. Vet. Med. Assoc. 224:241–249.

19. Johnson RP. 1992. Antigenic change in feline calicivirus during persistentinfection. Can. J. Vet. Res. 56:326 –330.

20. Knowles JO, Dawson S, Gaskell RM, Gaskell CJ, Harvey CE. 1990.Neutralisation patterns among recent British and North American felinecalicivirus isolates from different clinical origins. Vet. Rec. 127:125–127.

21. Kreutz LC, Johnson RP, Seal BS. 1998. Phenotypic and genotypic vari-ation of feline calicivirus during persistent infection of cats. Vet. Micro-biol. 59:229 –236.

22. Milne I, et al. 2004. TOPALi: software for automatic identification ofrecombinant sequences within DNA multiple alignments. Bioinformatics20:1806 –1807.

23. Neill JD. 1992. Nucleotide sequence of the capsid protein gene of twoserotypes of San Miguel sea lion virus: identification of conserved andnon-conserved amino acid sequences among calicivirus capsid proteins.Virus Res. 24:211–222.

24. Parker J, Rambaut A, Pybus OG. 2008. Correlating viral phenotypes withphylogeny: accounting for phylogenetic uncertainty. Infect. Genet. Evol.8:239 –246.

25. Pedersen NC, Elliott JB, Glasgow A, Poland A, Keel K. 2000. An isolatedepizootic of hemorrhagic-like fever in cats caused by a novel and highlyvirulent strain of feline calicivirus. Vet. Microbiol. 73:281–300.

26. Pedersen NC, Hawkins KF. 1995. Mechanisms for persistence of acuteand chronic feline calicivirus infections in the face of vaccination. Vet.Microbiol. 47:141–156.

27. Porter CJ, et al. 2008. Comparison of the ability of feline calicivirus (FCV)vaccines to neutralise a panel of current UK FCV isolates. J. Feline Med.Surg. 10:32– 40.

28. Povey C, Ingersoll J. 1975. Cross-protection among feline caliciviruses.Infect. Immun. 11:877– 885.

29. Povey RC. 1974. Serological relationships among feline caliciviruses. In-fect. Immun. 10:1307–1314.

30. Radford AD, et al. 2009. Feline calicivirus infection. ABCD guidelines onprevention and management. J. Feline Med. Surg. 11:556 –564.

31. Radford AD, et al. 1997. The use of sequence analysis of a feline calicivirus(FCV) hypervariable region in the epidemiological investigation of FCVrelated disease and vaccine failures. Vaccine 15:1451–1458.

32. Radford AD, et al. Application of next-generation sequencing technolo-gies in virology. J. Gen. Virol., in press.

33. Radford AD, Coyne KP, Dawson S, Porter CJ, Gaskell RM. 2007. Felinecalicivirus. Vet. Res. 38:319 –335.

34. Radford AD, et al. 2003. High genetic diversity of the immunodominantregion of the feline calicivirus capsid gene in endemically infected catcolonies. Virus Genes 27:145–155.

35. Radford AD, Dawson S, Wharmby C, Ryvar R, Gaskell RM. 2000.Comparison of serological and sequence-based methods for typing felinecalicivirus isolates from vaccine failures. Vet. Rec. 146:117–123.

36. Radford AD, Gaskell RM, Hart CA. 2004. Human norovirus infectionand the lessons from animal caliciviruses. Curr. Opin. Infect. Dis. 17:471–478.

37. Radford AD, et al. 2001. Endemic infection of a cat colony with a felinecalicivirus closely related to an isolate used in live attenuated vaccines.Vaccine 19:4358 – 4362.

38. Radford AD, et al. 2001. Molecular analysis of isolates of feline calicivirusfrom a population of cats in a rescue shelter. Vet. Rec. 149:477– 481.

39. Radford AD, et al. 1998. Quasispecies evolution of a hypervariable regionof the feline calicivirus capsid gene in cell culture and in persistently in-fected cats. J. Gen. Virol. 79:1–10.

40. Radford AD, Willoughby K, Dawson S, McCracken C, Gaskell RM.1999. The capsid gene of feline calicivirus contains linear B-cell epitopes inboth variable and conserved regions. J. Virol. 73:8496 – 8502.

41. Rasbach J, et al. 2000. A user’s guide to MLwiN. Institute of Education,London, United Kingdom.

42. Reynolds BS, et al. 2009. A nosocomial outbreak of feline calicivirus

Coyne et al.

11366 jvi.asm.org Journal of Virology

associated virulent systemic disease in France. J. Feline Med. Surg. 11:633–644.

43. Schulz BS, et al. 2011. Two outbreaks of virulent systemic feline calicivirus infec-tion in cats in Germany. Berl. Munch. Tierarztl. Wochenschr. 124:186–193.

44. Seal BS, Ridpath JF, Mengeling WL. 1993. Analysis of feline caliciviruscapsid protein genes: identification of variable antigenic determinant re-gions of the protein. J. Gen. Virol. 74:2519 –2524.

45. Strimmer K, von Haeseler A. 1997. Likelihood-mapping: a simplemethod to visualize phylogenetic content of a sequence alignment. Proc.Natl. Acad. Sci. U. S. A. 94:6815– 6819.

46. Sukhrie FH, et al. 2011. Using molecular epidemiology to trace transmissionof nosocomial norovirus infection. J. Clin. Microbiol. 49:602–606.

47. Tamura K, Dudley J, Nei M, Kumar S. 2007. MEGA4: molecular evolutionarygenetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24:1596–1599.

48. Verhoef L, et al. 2011. An integrated approach to identifying interna-tional foodborne norovirus outbreaks. Emerg. Infect. Dis. 17:412– 418.

49. Wardley RC, Povey RC. 1977. The clinical disease and patterns of excre-tion associated with three different strains of feline caliciviruses. Res. Vet.Sci. 23:7–14.

50. Wardley RC, Povey RC. 1977. The pathology and sites of persistenceassociated with three different strains of feline calicivirus. Res. Vet. Sci.23:15–19.

51. Xia X, Xie Z, Salemi M, Chen L, Wang Y. 2003. An index of substitutionsaturation and its application. Mol. Phylogenet. Evol. 26:1–7.

52. Zheng DP, et al. 2006. Norovirus classification and proposed strain no-menclature. Virology 346:312–323.

53. Zheng DP, Widdowson MA, Glass RI, Vinje J. 2010. Molecular epide-miology of genogroup II-genotype 4 noroviruses in the United States be-tween 1994 and 2006. J. Clin. Microbiol. 48:168 –177.

54. Zicola A, Saegerman C, Quatpers D, Viandier J, Thiry E. 2009. Felineherpesvirus 1 and feline calicivirus infections in a heterogeneous cat pop-ulation of a rescue shelter. J. Feline Med. Surg. 11:1023–1027.

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