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Virus Research 140 (2009) 199–204

Contents lists available at ScienceDirect

Virus Research

journa l homepage: www.e lsev ier .com/ locate /v i rusres

hort communication

nalysis of Foot-and-mouth disease virus nucleotide sequenceariation within naturally infected epithelium

leanor M. Cottama,b,∗, Donald P. Kinga, Anthony Wilsona,avid J. Patona, Daniel T. Haydonb

Institute for Animal Health, Pirbright Laboratory, Ash Road, Pirbright, Woking, Surrey, England GU24 0NF, United KingdomDivision of Environmental and Evolutionary Biology, Graham Kerr Building, University of Glasgow, Scotland G12 8QQ, United Kingdom

r t i c l e i n f o

rticle history:eceived 12 June 2008eceived in revised form 23 October 2008ccepted 28 October 2008vailable online 12 December 2008

eywords:oot-and-mouth disease virus

a b s t r a c t

An important epidemiological tool in the control of epidemics of Foot-and-mouth disease (FMD) is genetictracing using complete virus genome sequence data. However to interpret these genetic data, it is impor-tant to quantify underlying variation present in FMDV populations from individual tissue samples. Clonedcomplete capsid sequences from two virus populations from epithelium from a cow (n = 26) and from asheep (n = 15) infected during the UK 2001 outbreaks were generated. Genetic diversity of the two viruspopulations differed significantly, with sequences representing virus from the cow having a mutationfrequency of 2.79 × 10−4 mutations per nucleotide sequenced (mpns) and those from the sheep having

−4 2

equenceloningonsensusenetic diversityithin host diversity

icornavirus

3.94 × 10 mpns (� = 8.24, P = 0.004). The dN/dS ratio of sequences from the cow was higher (1.228) thanthat from the sheep (0.187) although not significantly so. The sequences from the cow epithelium exhib-ited significantly higher than expected number of changes within neutralising antigenic sites (P = 0.0007).The performance of two different reverse transcriptase enzymes was found not to differ with respectto the frequency (P = 0.559, �2 = 0.341) or dN/dS ratio (P = 0.863, �2 = 0.03) of the mutations observed.These data provide insight into the population diversity that exists within a single lesion and help toward

nisms

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understanding the mecha

Foot-and-mouth disease virus (FMDV) is a single stranded pos-tive sense RNA virus in the Aphthovirus genus of the familyicornaviridae. Although FMDV isolates are normally geneticallyescribed by a single sequence, this represents the consensus ofhat is thought to be an underlying swarm of closely related genetic

ariants. While conventional sequencing has provided insightsnto the genetic variation of the consensus sequence from dis-inct virus populations from different animals (Carrillo et al., 2007),tudies on the range and distribution of genetic variation withinhese swarms have previously been limited to cell culture pop-lations (Airaksinen et al., 2003; Arias et al., 2001; Pariente etl., 2001; Sierra et al., 2000; Gu et al., 2006). These studies typi-ally report mutation frequencies of the order of 10−4 mutations

er nucleotide sequenced (mpns). However, it is not known howell these studies represent what occurs within an infected animalhere the virus could be subject to different selection pressures and

onstraints.

∗ Corresponding author. Present address: Biomedical Research Centre, Faculty ofealth, School of Medicine, University of East Anglia, Norwich, England NR4 7TJ,nited Kingdom. Tel.: +44 1603591798; fax: +44 1603591698.

E-mail address: E.Cottam@uea.ac.uk (E.M. Cottam).

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168-1702/$ – see front matter © 2008 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2008.10.012

that underpin sequence evolution of FMDV.© 2008 Elsevier B.V. All rights reserved.

It is probable that nucleotide changes become fixed within theMDV consensus sequence due to the transmission of the virushrough ‘bottlenecks’ leading to a founder effect in the new popu-ation (Cottam et al., 2006, 2008a). If this is true, then the geneticiversity that surrounds the consensus will help to determine theate and manner in which the virus consensus sequence evolves.his motivates further study of the extent and characteristics of theequence diversity surrounding the consensus sequence. Under-tanding fine scale molecular epidemiology of FMDV and preciselyow nucleotide changes become fixed is important, as demon-trated by real time tracing of the UK 2007 O1BFS FMDV outbreaksCottam et al., 2008b).

In this study, the genetic variation surrounding the virus con-ensus sequence within the capsid region upon natural infectionf both a cow and a sheep (infected during the 2001 UK FMDpidemic) was investigated, providing an estimate of mutant fre-uency. The samples were selected from the library of infectedpithelium tissue held at the World Reference Laboratory for

oot-and-mouth disease (Institute for Animal Health), Pirbright,ollected during the FMDV PanAsia O serotype outbreaks in the UKn 2001. The virus populations investigated were from epitheliumriginating from a cow with 3-day-old lesions (UKG 14524/2001)rom Infected Premises (IP) 1970, and a sheep with 1-day-old

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00 E.M. Cottam et al. / Virus

esions (UKG 15101/2001) from IP2027. The consensus sequencef the complete genome for both had been determined previ-usly (DQ404160 and DQ404158, respectively (Cottam et al., 2006)).omplete capsids from 26 individual clones from UKG 14524/200113 reverse transcribed by Superscript III and 13 by Accuscript) and5 individual clones from UKG15101/2001 (reverse transcribed byuperscript III) were sequenced.

RNA was extracted from original sample suspensions asescribed previously using TRIzol reagent (Invitrogen) (Reid et al.,998). Reverse transcription reactions were carried out according tohe manufacturer’s protocol using either Superscript III (Invitrogen)r Accuscript High Fidelity (Stratagene). The complete capsid wasmplified by PCR using Platinum Taq Hi-fidelity (Invitrogen) withrimers UKFMD/For3 (5′ cca cgc tgg cat ctt cct gaa ag 3′) and Rev 75′ cca gtg gcc agt tcc tca aat g 3′), and cloned into the pGEM-T Easyit (Promega) according to the manufacturer’s protocol. The clonesere sequenced using the Beckman Coulter sequencing protocol onBeckman Coulter Sequencing (CEQ800) machine. All sequencesere submitted to GenBank and assigned the accession numbers

U552169–EU552209.The mutation frequency was calculated as the number of unique

ubstitutions/total number of nucleotides sequenced (mpns). Thei (�) statistic, defined as the average number of nucleotide dif-erences per site between any two nucleotide sequences chosenandomly from the sample population (Nei and Li, 1979), wasalculated using DNAsp (Rozas and Rozas, 1999). Maximum likeli-ood trees were determined using Phylip version 3.66 (Felsenstein,004), assuming a constant rate among sites. Likelihoods of these

rees were compared using models of nucleotide substitutionhat differed in their assumptions regarding evolutionary ratesnd dN/dS ratios between branches using the codeml programmeithin the PAML3.15 freeware package (Yang, 1997). Codeml was

un assuming the standard genetic code, with the equilibrium

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able 1ariant nucleotides observed in all sequences.

lone Capsid protein Nucleotide posit

KG 14524 S 09 VP1 2017KG 14524 S 10 VP3 1168KG 14524 S 11 VP3 1053KG 14524 S 12 VP2 551KG 14524 S 12 VP4 156KG 14524 S 13 VP3 1075KG 14524 S 13 VP1 1588KG 14524 A 08 VP3 1462KG 14524 A 09 VP2 350KG 14524 A 10 VP2 650KG 14524 A 11 VP2 575KG 14524 A 11 VP3 1080KG 14524 A 12 VP3 1080KG 14524 A 12 VP3 1530KG 14524 A 12 VP3 1567KG 14524 A 13 VP2 308KG 14524 A 13 VP3 1080KG 14524 A 13 VP1 1970

KG 15101 S 05 VP3 1098KG 15101 S 06 VP1 1929KG 15101 S 07 VP2 654KG 15101 S 08 VP3 1491KG 15101 S 09 VP3 1518KG 15101 S 10 VP3 1262KG 15101 S 11 VP2 385KG 15101 S 12 VP2 606KG 15101 S 13 VP1 1668KG 15101 S 14 VP1 2136KG 15101 S 15 VP4 240KG 15101 S 15 VP3 1352KG 15101 S 15 VP1 1741

hown in bold are non-synonymous changes in antigenic sites, and in italics are those cha

rch 140 (2009) 199–204

odon frequencies in the codon substitution model calculated fromhe average nucleotide frequencies at the three codon positions,nd with no variation in dN/dS ratios between sites. Estimatedodel likelihoods were compared by performing likelihood ratio

ests. When comparing two evolutionary models (the simpler [null]odel, having p0 parameters and associated log-likelihood l0, and

he more complicated [alternative] model having p1 parameters,ith associated log-likelihood l1), twice the log-likelihood dif-

erence 2�l = 2(l1 − l0) has a chi-squared (�2) distribution withegrees of freedom (d.f.) = p1 − p0 if the null model is assumed toe true. Thus the test statistic 2�l was compared to the �2 dis-ribution with appropriate d.f. to test whether the more complex

odel is supported over the simpler model. The exact binomialest was used to determine whether or not a significantly highumber of mutations had occurred within sites of known antigenic

mportance.Fifteen of the 26 sequences representing UKG 14524/2001 and

of the 15 sequences representing UKG15101/2001 were identi-al to the consensus for that sample. Twenty-nine unique variantucleotides (Table 1), an insertion of a single nucleotide, and aeletion of 31 nucleotides were detected following comparison ofhe cloned sequences to their respective consensus sequences. Theequences from UKG 14524/2001 have a mutation frequency of.79 × 10−4 mpns, and those from UKG 15101/2001 have a mutationrequency of 3.94 × 10−4 mpns. Properties of the two sets of clonedequences are summarised in Table 2. Maximum likelihood phylo-enies were constructed for the sequences representing the clonalopulations individually and combined (Fig. 1a–c). To determine

hether or not the performances of the two reverse transcrip-

ase enzymes were significantly different (Accuscript is reportedo have threefold higher fidelity than other reverse transcriptases),he model likelihood of a single evolutionary rate for all of theequences from virus population UKG 14524/2001 was compared

ion Nucleotide change Amino acid change

G-A V-IT-C S-PT-C –C-T A-VG-T –C-T H-YT-C S-PT-C –T-C V-AT-C I-TT-C V-AC-T –C-T –A-G E-GC-T STOPG-A R-HC-T –G-A C-Y

C-T –G-T –C-T –T-G –T-C –A-G K-RA-G S-GC-T –G-A –G-A –C-T –A-G D-GG-A A-T

nges that occurred in more than one sequence.

E.M. Cottam et al. / Virus Research 140 (2009) 199–204 201

Table 2Genetic characteristics of two natural populations of FMDV.

No. of clones No. of mutant-freeclones

Mutant frequency(×10−4)

Ts:Tv ratio No. of non-synonymousmutations

No. of synonymousmutations

dN/dS �

14524 26 15 2.79 14.27 12 4 1.2286.2 × 10−4a

8.0 × 10−4b

5.6 × 10−4c

Accuscript 13 7 3.14 49.50 7 2 1.3527.3 × 10−4a

9.8 × 10−4b

6.5 × 10−4c

Superscript III 13 8 2.45 6.03 5 2 1.0864.9 × 10−4a

5.6 × 10−4b

4.7 × 10−4c

15101 (Superscript III) 15 4 3.94 4.21 4 9 0.1878.0 × 10−4a

2.2 × 10−3b

3.3 × 10−4c

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a � calculated for all nucleotide sites.b � calculated for synonymous sites.c � calculated for non-synonymous sites.

o the likelihood of separate rates for each of the sequence setsenerated by the different reverse transcriptases Supercript III andccuscript. No significant difference was found between the tworoups (P = 0.559, �2 = 0.341, d.f. = 1). The model likelihood of sepa-ate dN/dS ratios for the two sequence sets was not supported overhe model likelihood of one dN/dS ratio for all (P = 0.863, �2 = 0.03,.f. = 1). Thus the clones generated by the two enzymes from theame starting material did not differ significantly in number or type

f mutations (Fig. 2).

A significant difference in the extent of genetic variation wasound upon comparing a model where branch lengths in a treeere restricted to a single model for both virus populations to

hat which allowed a different evolutionary model for the two

UtbfU

ig. 1. Maximum likelihood analysis of complete capsid sequences from UKG 14524/20equences representing sample UKG 14524/2001; in dark grey are the sequences obtainedbtained from cDNA transcribed by Superscript III reverse transcriptase; (b) maximum5101/2001 and (c) maximum likelihood analysis of all 41 capsid sequences from UKG 14

P = 0.004, �2 = 8.24, d.f. = 1), consistent with the observed � statis-ics for sequences from each sample (Table 2). The dN/dS ratioer nucleotide site for the cloned population representing UKG4524/2001 was 1.228, for UKG 15101/2001 it was 0.187 (Table 2).aximum likelihood analysis did not significantly support a modelith two separate dN/dS ratios in preference to a single ratio model

or all (P = 0.097, �2 = 1.572, d.f. = 1). The transition to transversionts:tv) ratio for the two sets of sequences UKG 14524/2001 and

KG 15101/2001 was 14.27 and 4.21, respectively. The relative posi-

ions within the capsid coding region of all the mutations found, foroth groups of sequences, are shown in Fig. 3. The only mutationound in more than one sequence was the silent C-T mutation in 3KG 14524/2001 sequences. Overall, taking into consideration cod-

01 and UKG 15101/2001. (a) Maximum likelihood analysis of 26 complete capsidfrom cDNA transcribed by Accuscript reverse transcriptase, in black are sequenceslikelihood analysis of 15 complete capsid sequences representing sample UKG

524/2001 (dark grey and black) and UKG 15101/2001 (light grey).

202 E.M. Cottam et al. / Virus Research 140 (2009) 199–204

Fig. 2. Statistical parsimony analysis of capsid sequences. Shown are the genetic relationships between sequences, with each branch representing a single nucleotidesubstitution: dashed branches represent synonymous changes, complete lines non-synonymous, and in thick lines are those that occurred within antigenic sites. In thecentre of each tree the proportion of sequences that were identical to the consensus is represented by a pie diagram, with the exact number printed in black. Each ovalr s from1 UKG 1r

ittosotTaGVOl2

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tttfirHtt(abotftatdmmmc

epresents a sequence and each dot a putative ancestral sequence. (a) 13 sequence3 sequences from cDNA transcribed by Superscript III reverse transcriptase fromepresenting UKG 15101/2001.

ng region length, proportionally more mutations occurred withinhe VP3 than VP1 region (12 out of the 29 substitutions, comparedo 8 in VP1). Interestingly, three non-synonymous mutations werebserved in known neutralising antigenic sites within the clonedequences of virus population UKG 14524/2001. The probability ofbserving 3 of the 12 amino acid changes seen in virus popula-ion UKG 14524/2001 in antigenic sites was 0.0007 (Exact Binomialest). One of these non-synonymous substitutions resulted in anmino acid change to a critical residue in the VP1 antigenic site 1-H loop, another was found to occur in the antigenic site 4 withinP3 and was an amino-acid change that has been seen before in/PHI/5/95 (N Knowles, personal communication), and the third

ay between two residues that form the �-EF loop of antigenic sitein VP2.

The average mutant frequency for PanAsia O FMDV genomesithin populations analysed from naturally infected animals found

n this study was 3.06 × 10−4 mpns. This is close to a previous initro study, for serotype O FMDV that sequenced cloned capsids,hich found a mutation frequency of 2.1 × 10−4 mpns (Gu et al.,

006). Studies on serotype C virus report mutant frequency rangesrom 0.7 × 10−4 to 7.7 × 10−4 mpns (Airaksinen et al., 2003; Arias etl., 2001; Pariente et al., 2001; Sierra et al., 2000). The distributionf the mutations across the sequences and the presence of identi-

al sequences to the consensus provide support for the dominancef the consensus sequence. The majority of substitutions seen inhis study were in VP3, and this corresponds to the majority ofxed mutations noted during the UK 2001 outbreak (Cottam et al.,006).

vtea

cDNA transcribed by Accuscript reverse transcriptase from UKG 14524/2001; (b)4524/2001; (c) 26 sequences representing UKG 14524/2001 and (d) 15 sequences

It is possible that the mutations observed could be an artefact ofhe amplification process, introduced, for example, by the reverseranscription enzyme, the PCR enzyme, the E. coli replication sys-em, or even during the sequencing reaction. In this study, a highdelity PCR enzyme was used that is reported to give an overalleverse-transcription PCR error rate of 7.7 × 10−6 mpns (Arezi andogrefe, 2007; Malet et al., 2003). It has also been suggested that

he reverse transcriptase can be responsible for mutations seen andhis was investigated by assessing the performance of AccuscriptStratagene), which is reported to have proof reading qualities,nd Superscript III (Invitrogen). No statistical difference was seenetween the sequences generated by the two enzymes in termsf evolutionary rate (P = 0.559) or dN/dS (P = 0.863), suggestinghat their performances did not significantly differ. The mutationrequency estimates reported in this paper are thus susceptibleo bias from mutations arising as artefacts of the amplificationnd sequencing process but it seems reasonable to suggest thathe maximum number of mutants were identified because theata represents the real mutant frequency plus minimal artificialutants. However, it is also important to note that some of the sameutations may have arisen twice independently and that the esti-ates represent the minimum number of mutational events that

ould have occurred.

A significant difference in diversity was found between the

irus population from the cow epithelium UKG 14524/2001 andhe sheep epithelium UKG 15101/2001 (P = 0.004). This can bexplained by the fact that the populations were of different agesnd possibly different sizes. However, it is also possible that

E.M. Cottam et al. / Virus Research 140 (2009) 199–204 203

F to thw s withs orizonb ing.

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ig. 3. The schematic shows all mutant sequences reported in this study comparedhite circles, non-synonymous by filled circles and non-synonymous substitution

ubstitution resulting in a stop codon by a filled rectangle, and the deletion by two hy the dashed lines, with antigenically important residues represented by grey shad

ifferences in environment (i.e. host animal type) contribute tontra-population variation. Although not significant the disparityn dN/dS ratios between the two populations could also be due tohese reasons. During the UK 2001 epidemic non-synonymous andynonymous mutations were fixed into the consensus sequence atratio of 0.09 (Cottam et al., 2006), which could thus be regarded

s the dN/dS ratio expected under normal purifying selection. Itay be that transmission between animals presents stronger puri-

ying selection than replication within an animal, however theseata sets are small and more study is required to determine this.

For the virus population analysed from UKG 14524/2001 thereere three changes that occurred in antigenic sites, which is signif-

cantly higher than what would be expected if mutations arise atandom with respect to site. It is possible that this represents pos-tive selection of variants that can escape the immune response,

s supported by the high dN/dS ratio of 1.228. However, both virusopulations were sampled from relatively early infections and it

s unlikely that the adaptive immune response would be impli-ated so soon following infection. An alternative explanation coulde that these sites represent parts of the capsid that are more

nr

(b

eir respective consensus sequences. Synonymous substitutions are represented byin antigenic sites by black stars. The insertion is indicated by a down arrow, thetal arrows. The sections encoding the separate capsid proteins VP1-4 are indicated

asily mutated than others, either due to some unknown mecha-ism during replication, or due to relative resistance to deleteriousutation.Three identical synonymous mutations were found within the

6 sequences from the virus population UKG 14524-2001 (con-ensus cytosine (C) replaced with thymine (T)). A comparisonf the consensus with the sequences from the UK 2001 out-reak revealed that ‘T’ is found at this position in all the otheriruses sequenced; indeed it may be more accurate to say thathose sequences harbouring a ‘C’ at this position are the deriva-ive sequences. This observation demonstrates an instance of aucleotide change becoming fixed within the consensus. The pres-nce of three sequences containing the original nucleotide (‘T’) athis position suggests that those viruses with a ‘C’ at this residueeplicated more abundantly than those with a ‘T’. This may not

ecessarily be due to increased fitness, but perhaps a result of theelative diversity of the starting population.

If we extrapolate the findings in this study to the entire genome∼8K), it suggests that each genome within a population differsy at least one nucleotide from the consensus virus sequence. As

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as been shown for other RNA viruses it is feasible that the withinopulation variation of FMDV differs depending upon the hostnvironment and stage of disease, and may also be affected byhe rate of transmission events and the bottlenecks that arise asresult. For this reason within host genetic variation and evolu-

ion of FMDV needs to be studied further to assess its potential tomprove our understanding of the fixation of mutations into theonsensus sequence, variation in disease severity between hosts,he progression to persistence (a previous study has shown thatenetic change may be a factor in persistence (Horsington andhang, 2007)), and the probability of immune and vaccine escapey the virus. Although the difference in the genetic make up ofhe virus populations noted in this study between the infected cownd the sheep epithelium is intriguing it cannot be regarded as con-lusive because only one sample from each representative animalas been sampled. However, the differences seen encourage furtheresearch into virus evolution at the single population level in vivo,hich may provide additional insight into the fixation of mutationsuring the evolution of the virus in the field.

cknowledgements

This work was funded by a Biotechnology and Biological Sci-nces Research Council (BBSRC) Ph.D. studentship awarded to EC.he funding of other IAH staff was supported by Department ofnvironment, Food and Rural Affairs and BBSRC. The collectionnd archiving of UKG 2001 samples was undertaken by the Foodnd Agricultural Organization World Reference Laboratory for Foot-nd-mouth disease.

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