nucleotide sequence of uk and australian isolates of feline calicivirus (fcv) and phylogenetic...

Nucleotide sequence of UK and Australian

isolates of feline calicivirus (FCV) and

phylogenetic analysis of FCVs

Mark Glenna, Alan D. Radfordb,*, Philip C. Turnera, Mike Carterc,David Loweryd, Dwynwen A. DeSilverd, Jayesh Meangere,

Cindy Baulch-Browne, Malcolm Bennettb, Rosalind M. Gaskellb

aSchool of Biological Sciences, University of Liverpool, Liverpool, UKbDepartment of Veterinary Pathology, Leahurst Field Station, Neston, Cheshire, CH64 7TE, UK

cDepartment of Biological Sciences, University of Surrey, Guildford, Surrey, UKdPharmacia and Upjohn Animal Health, Kalamazoo, MI, USA

eChildren's Virology Research Unit, Macfarlane Burnet Centre for Medical Research,

P.O. Box 254, Fairfield 3078, Australia

Received 4 December 1998; accepted 16 April 1999

Abstract

We have determined the first complete genome sequence and capsid gene sequences of feline

calicivirus (FCV) isolates from the UK and Australia. These were compared with other previously

published sequences. The viruses used in the comparisons were isolated between 1957 and 1995

from various geographical locations and obtained from cats showing a range of clinical signs.

Despite these diverse origins, comparisons between all strains showed a similar degree of sequence

variation within both ORF1 (non-structural polyprotein) and ORF2 (major capsid protein) (amino

acid distances of 7.7±13.0% and 8.8±18.6%, respectively). In contrast, ORF3 (putative minor

structural protein) sequences indicated a more heterogenous distribution of FCV relatedness (amino

acid distances of 1.9±17.9%). Phylogenetic analysis suggested that, unlike some other caliciviruses,

FCV isolates within the current data set fall into one diverse genogroup. Within this group, there

was an overall lack of geographic or temporal clustering which may be related to the epidemiology

of FCV infection in cats. Analysis of regions of variability in the genome has shown that, as well as

the previously identified variable regions in ORF2, similar domains exist within ORFs 1 and 3 also,

although to a lesser extent. In ORF1, these variable domains largely fall between the putative non-

structural protein functional domains. # 1999 Elsevier Science B.V. All rights reserved.

Keywords: Cat; Feline calicivirus; Phylogenetic analysis; Sequence

Veterinary Microbiology 67 (1999) 175±193

* Corresponding author. Tel.: +44-151-794-6012; fax: +44-151-794-6005; e-mail: [email protected]

0378-1135/99/$ ± see front matter # 1999 Elsevier Science B.V. All rights reserved.

PII: S 0 3 7 8 - 1 1 3 5 ( 9 9 ) 0 0 0 4 3 - 7

1. Introduction

The Caliciviridae contains a number of viruses of veterinary importance, including

feline calicivirus (FCV), vesicular exanthema of swine virus, San Miguel sea lion virus

(SMSV) and the recently emerged rabbit haemorrhagic disease virus (RHDV) (Cubitt

et al., 1995). The family also includes important human pathogens, such as classic

morphology human caliciviruses and small round structured viruses (SRSVs), both of

which are major causes of gastroenteritis in man (Caul, 1996).

FCV is an important pathogen of domestic cats, predominantly inducing acute oral and

upper respiratory tract disease (Gaskell and Dawson, 1998). Some isolates may also

induce a febrile lameness syndrome (Pedersen et al., 1983; Dawson et al., 1994), while

other strains appear to cause an unapparent infection (Fastier, 1957). FCV has also been

associated with chronic stomatitis and gingivitis (Thompson et al., 1984; Knowles et al.,

1989), and on occasion, may be isolated from cases of abortion (Ellis, 1981). On recovery

from clinical disease, a proportion of cats develops an asymptomatic carrier state in

which virus is shed from the oropharynx (Povey et al., 1973; Wardley and Povey, 1977).

Such carrier cats appear to be widespread in the cat population with up to 20±25% of

apparently healthy cats shedding virus (Wardley et al., 1974; Tenorio et al., 1991; Coutts

et al., 1994). In some cases, FCV carriers have been shown to shed virus for at least 2

years (Povey et al., 1973; Wardley, 1976). As well as in domestic cats, FCV has been

isolated from cheetahs with mouth ulcers (Baulch-Brown et al., 1998).

Caliciviruses possess a single-stranded, positive sense RNA genome approximately

7.2±7.7 kb in length (Meyers et al., 1991b; Carter et al., 1992a; Jiang et al., 1993; Liu

et al., 1995). The non-structural proteins, which, based on sequence similarity with

picornaviruses (Neill, 1990; Meyers et al., 1991b; Oshikamo et al., 1994) and functional

studies (Boniotti et al., 1994; VaÂzquez et al., 1998), include a putative 2C-helicase, a

3C-protease and a 3D RNA-dependent RNA polymerase, are translated as a polyprotein

from a single open reading frame (ORF), ORF1 at the 50 end of the genome. In FCV,

SMSV and the SRSVs, the major capsid protein is encoded by a second ORF (ORF2),

located towards the 30 end of the viral genome. In the case of RHDV and classic

morphology human caliciviruses, ORFs 1 and 2 are fused in frame such that the non-

structural and major capsid proteins are expressed as a single polyprotein (Meyers et al.,

1991a; Liu et al., 1995). As well as from genomic RNA, the capsid protein is translated

from a 30 co-terminal sub-genomic RNA (Clarke and Lambden, 1997). Similar conserved

sequences identified at the 50 end of the both genomic and sub-genomic RNAs are

thought to be involved in RNA replication and/or packaging (Clarke and Lambden,

1997). A short ORF (ORF3 in FCV, ORF2 in RHDV) is located at the extreme 30 end of

the virus, which in RHDV has been shown to encode a minor structural protein (Wirblich

et al., 1996).

Based on comparisons of limited sequence data from FCV, SMSV and RHDV, the

major capsid gene has been divided into conserved (B, D and F) and more variable (A, C

and E) regions (Neill, 1992; Seal et al., 1993). Region E in FCV (amino acid 426±523 of

the F9 capsid (Carter et al., 1992a)) contains a central conserved domain separating the 50

and 30 hypervariable regions (Seal et al., 1993), and is believed to contain the major

antigenic determinants (Guiver et al., 1992; Milton et al., 1992; Shin et al., 1993; Neill

176 M. Glenn et al. / Veterinary Microbiology 67 (1999) 175±193

et al., 1997; Tohya et al., 1997). Sequence analysis of part of this variable region has been

used in the epidemiological investigation of FCV infection (Radford et al., 1997).

Previous attempts to define a viral basis for the observed spectrum of FCV related

disease based on serological (Dawson et al., 1993b; McArdle et al., 1996) and capsid

gene sequence analysis (Geissler et al., 1997) have, in general, been unsuccessful.

Antigenic differences between FCVs based on virus neutralisation assays have allowed

some grouping of isolates (Dawson et al., 1993a; Lauritzen et al., 1997). However, such

antigenic differences are relatively small and FCV strains are considered to belong to a

single serotype (Povey, 1974; Kalunda et al., 1975; Burki et al., 1976). In contrast, the

human caliciviruses show distinct serological (Carter and Cubitt, 1995) and genotypic

groupings (Carter and Cubitt, 1995; Green et al., 1997), the latter based on sequence

analysis of both RNA dependent RNA polymerase (Green et al., 1997) and major capsid

(Green et al., 1995) genes. Isolates of SMSV fall into multiple serotypes (Smith et al.,

1981), and more recently, isolates of RHDV have been shown to fall into three distinct

genogroups (Le Gall et al., 1998).

Failure to identify genogroups within FCV may reflect the limited amount of sequence

data that is currently available. To date, four strains of FCV have been sequenced

completely, and capsid gene sequences are available for a further 10 strains (for

references, see Table 1). However, no data is available for isolates from the UK or

Australia. In this study, we have determined the complete genome sequence of UK isolate

F65, which has previously been shown experimentally to induce a febrile lameness

syndrome (Dawson et al., 1994), and which is less related antigenically than other isolates

to the commonly used vaccine strain F9 (Dawson, 1991; Dawson et al., 1993b). We have

also determined the capsid sequence from a further eight isolates, four from the UK and

four from Australia, and ORF3 sequences of two of these UK isolates. These sequences

are compared with currently available FCV sequence data. The analysis includes viruses

isolated between 1957 and 1995 from cats with different clinical conditions, and obtained

from different parts of the world. Despite this broad range of sources, and unlike some

other caliciviruses, all FCV isolates examined so far appear to comprise a single

genotype. It is suggested that this may relate to the epidemiology of the disease in the cat.

2. Materials and methods

2.1. Viruses

Where known, the origins of isolates used in this study are shown in Table 1. Complete

sequence was obtained for UK isolate F65; the capsid gene sequence for UK isolates

LS015, A4, JOK 63 and LS012 and Australian isolates V77, V83, V274 and 182cvs5A;

and ORF3 sequence for LS015, JOK 63 and LS012. All other sequence data were

obtained from GenBank (Table 1).

2.2. Cloning and sequencing

The precise methods used to generate sequences from FCV varied according to the

laboratory where the virus was sequenced. Briefly, ORF2 and ORF3 of F65 was reverse

M. Glenn et al. / Veterinary Microbiology 67 (1999) 175±193 177

Table 1Origins of FCV isolates

Virus Year ofisolationa

Country ofisolation

Clinical origin Gen Bank accession numbers Reference (isolation/sequence)

ORF1 ORF2 ORF3

LS015 1985±1988b UK chronic stomatitis ND AF109464 ND (Knowles, 1988)/this paperF65 1990b UK lameness and OD AF109465 AF109465 AF109465 (Dawson, 1991)/this paperA4 1973b UK URTD ND AF109468 ND (Wardley, 1974)/this paperJOK63 1985±1988b UK URTD and OD ND AF109466 AF109466 (Knowles, 1988)/this paperLS012 1985±1988b UK chronic stomatitis ND AF109467 AF109467 (Knowles, 1988)/this paperCFI/68 1960 USA URTD and stiffness U13992 U13992 U13992 (Crandell et al., 1960)/(Neill, 1990; Neill et al., 1991)Urbana late 1960sc USA URTD and OD L40021 L40021 L40021 (Sosnovtsev and Green, 1995)/(Sosnovtsev and Green, 1995)255 1970 USA pneumonia and OD ND U07130 U07130 (Kahn and Gillespie, 1970)/(Seal, 1994)F9 1958c USA URTD and OD M86379 M86379 M86379 (Bittle et al., 1960; Bittle and Rubic, 1976)/

(Carter et al., 1992a)NADC 1983 USA apparently healthy ND L09718 L09718 (Seal, 1994)/(Seal et al., 1993)KCD 1957 New Zealand avirulent ND L09719 L09719 (Fastier, 1957)/(Seal et al., 1993)2280 1982d Canada lameness ND X99445 ND (Pedersen et al., 1983)/(Geissler et al., 1997)LLK 1982d Canada lameness ND U07131 U07131 (Pedersen et al., 1983)/(Seal and Neill, 1994)F4 1971 Japan URTD D31836 D31836 D31836 (Takahashi et al., 1971)/(Tohya et al., 1991;

Oshikamo et al., 1994)KS8 1994e Germany acute stomatitis ND X99449 ND (Geissler et al., 1997)/(Geissler et al., 1997)KS20 1994e Germany chronic stomatitis ND X99447 ND (Geissler et al., 1997)/(Geissler et al., 1997)KS40 1995e Germany acute respiratory disease ND X99448 ND (Geissler et al., 1997)/(Geissler et al., 1997)KS109 1995e Germany chronic stomatitis ND X99446 ND (Geissler et al., 1997)/(Geissler et al., 1997)V77 1979 Australia abortion, throat ulcers ND AF038126 ND (Huxtable et al., 1979)/(Baulch-Brown et al., 1998)V83 1972 Australia pneumonia, OD, death ND AF031876 ND (Love and Baker, 1972)/(Baulch-Brown et al., 1998)V274f 1989 Australia URTD, OD ND AF031877 ND (Baulch-Brown et al., 1998)/(Baulch-Brown et al., 1998)182cvs5A 1980 Australia conjunctivitis, OD ND AF031875 ND (Baulch-Brown et al., 1998)/(Baulch-Brown et al., 1998)

a Unless otherwise indicated, year of isolation is given as the year the isolate was first mentioned in publications.b R.M. Gaskell (unpublished).c Date of isolation given as quoted in isolation paper.d Personal communication, N. Pedersen.e Personal communication, U. Truyen.f Isolated from a cheetah.URTD: Upper respiratory tract disease.OD: Oral disease.ND: Not determined.

17

8M

.G

lenn

eta

l./Veterin

ary

Micro

bio

logy

67

(1999)

175±193

transcribed with the primer dT-Sma (50-GCACCCGGGATGAAAATGCATA-

TAGCCCGCG(T)18-30), followed by PCR amplification using the primer 30-Sma

(identical to dT-Sma but lacking the oligo-d(T) region, and primer 50-Sma (50-GCACCCGGGAGATCGAYWCGAGTGCATGACGCG-30). Products were digested with

Xma I and ligated into pBluescript (Stratagene). The ORF1 of F65 was reverse

transcribed with primer 30-NSR (50-GCGCCCGGGAAGCACGTTAGCGGCAGGTT-30)and amplified as two fragments using the primer pair 30-NSR and ORF 1b (50-TACGGGCCCCGACAAGTATCCCTGCAATGTTGCG-30) and primer pair 50-NSR (50-GCGCCCGGGTAAAAGAAATTTGAGACAATG-30) and ORF 1a (50-AGACTGCAG-

GATGACTATGCCACCAGGTGTTAC-30). Amplicons were cloned into pBluescript

digested with either Sma I and Pst I (for the primer pair 50-NSR and ORF 1b) or Sma I

and Apa I (for the primer pair 30-NSR and ORF 1a). A 50 RACE was used to identify the

sequence at the 50 end of F65 using primer F65pe (50-GTGCACAAAGTCCTTGCGGA-

CA CTGTGAGT-30).A cDNA clone for strain A4 was produced in pBR322 by homopolymeric tailing. A Pst

I digest released the FCV capsid which was subsequently digested with Sau 3A. The

resulting fragments were ligated into Bam HI restricted M13mp18.

Complementary DNA libraries of FCVs LS015, JOK 63 and LS012 were prepared

using both random and oligo-d(T) primers, as described by Gubler and Hoffman (1983).

Resulting cDNA was ligated to Eco RI or Bst XI linkers, and cloned into plasmid pSP72

(Promega Corp.) or pcDNAII (Invitrogen). Recombinant clones were identified by

hybridisation with probes prepared from clones of the CFI/68 or F9 capsid.

For isolates V77, V83, V274 and 182cvs5a, total mRNA was extracted and RT-PCR

carried out using primers 50-TACACTGTGATGTGTTCGAAGTTTGAGC-30 and 50-GTGTATGAGTAAGG GTCAACCC-30. Resulting amplicons were cloned into pBlue-

script.

Clones derived from ORF1 of F65 and ORF2 of the Australian isolates were sequenced

using an ABI automated sequencer using conditions specified by the manufacturer (ABI

prism dye terminator cycle sequencing ready reaction kit; Perkin Elmer). All other

sequencing was performed essentially according to Sanger et al. (1977).

2.3. Sequence analysis

The University of Wisconsin GCG package (Deveraux et al., 1984) was used to align

sequence data using the PILEUP and LINEUP programs. Uncorrected nucleotide and amino

acid distances (average number of substitutions per 100 compared) were calculated using

the DISTANCES program. A comparison of amino acid sequence variability within regions

of the genome was performed using PLOTSIMILARITY. Phylogenetic analysis was performed

on sequence from ORFs 2 and 3 using programs available in the PHYLIP package

(Felsenstein, 1989). Trees based on the conserved regions of ORF2 were rooted using

sequence derived from SMSV-1 (GenBank accession number M87481). SMSV-1 has

been shown to be more closely related to FCV than other members of the Caliciviridae

(Berke et al., 1997). The precise regions used for comparison between SMSV-1 and FCV

were chosen using the DOTPLOT program (Ramin Nakisa, Biophysics Section, Imperial

College of Science, Technology and Medicine, London).


3. Results

3.1. Sequence analysis

The FCV F65 genome is 7681 nucleotides (nt) in length and contains three potential

ORFs, similar to other published sequences (CFI/68 ± 7677 nt, F4 ± 7681 nt, Urbana ±

7683 nt and F9 ± 7690 nt) (for references, see Table 1). ORF1 of F65 is 1763 amino acids

(aa) in length, as in F9, F4 and Urbana and one aa longer than CFI/68. Conserved motifs

described by Neill (1990), and suggestive of a 2C protease, a 3C helicase and an RNA-

dependent RNA polymerase are also present. ORF2 of F65 encodes 668 aa. Other capsids

range from 668 to 671 aa. As for other FCVs, the stop codon of ORF1 is separated from

the start codon of ORF2 by two nt creating a ÿ1 frameshift. The third ORF of F65

encodes 106 aa, as in all other published ORF3 sequences. The start codon of ORF3

overlaps the stop codon of ORF2 by two nt (ATGA) as in other FCVs, creating a ÿ1

frameshift between these two ORFs. ORF3 is followed by a short untranslated region of

44 nt, which precedes the poly (A) tail.

Comparison of sequences within ORFs 1 and 2 showed a similar degree of variability

with nt distances of 20.2±22.1% and 17.8±25.3%, respectively, and aa distances of 7.7±

13.0% and 8.8±18.6%, respectively (Table 2; Fig. 1(a) and (b)). In contrast, ORF3 shows

a broader range of distance values (10.0±20.6% and 1.9± 17.9% for nt and aa,

respectively), with the majority of isolates being more closely related than in ORFs 1 and

2 (Table 2; Fig. 1(c)).

The first 56 nucleotides of ORF1 are completely conserved amongst the five

isolates analysed. However, ORF1 was shown to contain discrete regions of variability,

as shown in Fig. 2(a) and numbered 1±6. Interestingly, peaks 4 and 5 appear to

be due largely to variation in a single isolate (F4). Similarity plots based upon the

remaining four ORF1 sequences result in the loss of these peaks but in the persistence

of peaks 1, 2, 3 and 6. Whether peaks 4 and 5 represent genuine regions of variability

or a peculiarity of isolate F4 will only become apparent as more ORF1 sequences

become available. These regions of variability largely identify the extremities of putative

ORF1 proteins that have been defined on the basis of sequence similarity to non-

structural protein motifs in the picornaviruses (Fig. 2(a)) (Neill, 1990). In particular,

Table 2Uncorrected nucleotide and amino acid distance distributions (Deveraux et al., 1984) for sequences from ORFs1, 2 and 3. The range of values and the mean are expressed as % differences (average number of substitutions per100 nucleotides or amino acids compared)

Nucleotide distances (%) Amino acid distances (%)

Range Mean SDa Range Mean SD

ORF1 (N � 5) 20.2±22.1 20.9 0.6 7.7±13.0 10.6 1.7

ORF2 (N � 22) 17.8±25.3 22.1 1.1 8.8±18.6 12.5 1.85

ORF3 (N � 12) 10.0±20.6 15.5 2.4 1.9±17.9 8.5 3.9

a Standard deviation.


Fig. 1. Graph representing uncorrected distance distributions (Deveraux et al., 1984) within (a) ORF1, (b) ORF2

and (c) ORF3. Within each ORF, sequences for all available isolates were compared to each other. Distance

values were treated as whole numbers and the frequency with which each distance value occurred was expressed

as a percentage of the total number of comparisons. Black bars represent amino acid distances. White bars

represent nucleotide sequences. n is the number of sequences used in the comparison.


variable regions 2 and 3 border the putative 2C-like helicase, regions 4 and 5 border the

3C-like protease and regions 5 and 6 border the 3D-like RNA-dependent RNA

polymerase.

Fig. 2. Amino acid sequence variability within each ORF of FCV using PLOTSIMILARITY (Deveraux et al., 1984).

Amino acid positions are numbered according to the beginning of each ORF. Window sizes were 20, 15 and 10

for (a) ORF1, (b) ORF2 and (c) ORF3, respectively. The approximate regions believed to encode the 2C, 3C and

3D proteins are indicated in (a) (Neill, 1990). The regions of the capsid as defined by Neill (1992) and Seal et al.

(1993) are indicated in (b). The arrow in (b) indicates the position of the capsid precursor cleavage site (Carter

et al., 1992b; Sosnovtsev et al., 1998).


The first 32 nucleotides of ORF2 are completely conserved in 21 of the 22 isolates

examined. Conserved sequences identified previously and partially shared by the 50 end

of both genomic and sub-genomic RNAs are maintained (Clarke and Lambden, 1997).

These conserved regions are predicted to form similar stem and loop secondary structures

in both regions (FOLDRNA program (Deveraux et al., 1984); data not presented). Such

regions of secondary structure may prove to be important for the replication of the viral

RNA. Conservation of nucleotide sequence may also suggest a role for these regions in

sequence-specific protein binding.

Analysis of the variation within the capsid gene confirmed that variation was greatest

within regions C and E (amino acids 397±401 and 426±523 inclusive, respectively) (all

amino acid numbers relate to the FCV F9 capsid (Carter et al., 1992a)) (Fig. 2(b)).

Variation in region E ranges from 50% to 65% at the amino acid level, with 66% of

residues subject to change (data not presented). This analysis also confirmed that region E

contains a highly conserved central region (amino acids 461±488) (Fig. 2(b)). Region A

is moderately variable, particularly around the A/B junction, although the capsid

precursor cleavage site (FRLE/AD; (Carter et al., 1992b; Sosnovtsev et al., 1998))

between regions A and B is highly conserved.

ORF3 appears to have at least two regions of variability, although the extent of these

variable regions is not as marked as in ORFs 1 and 2 (Fig. 2(c)).

3.2. Phylogenetic analysis

Phylogenetic trees were constructed for ORFs 2 (Figs. 3 and 4) and 3 (Fig. 5).

Rooted phylogeny construction was performed on FCV ORF2 sequences between

aa 169 and 428 and nt 457 and 1163 (Fig. 3), where there was sufficient similarity

between the outgroup (SMSV-1) and FCV sequence, as defined by DOTPLOT (data not

presented). Whilst the outgroup was always supported by a bootstrap value of 100,

its position relative to the FCV isolates was not consistent (Fig. 3). Variability

between the proposed outgroup and other regions of the FCV capsid and ORF3

precluded the inclusion of an outgroup in other analyses. Unrooted phylogeny

reconstructions were, therefore, performed on sequence from whole ORF2, conserved

ORF2 (made up of regions B, D, central E and F; Fig. 4), variable ORF2 (region E) and

ORF3 (Fig. 5).

For all trees, distance and parsimony-based algorithms were used on both nucleotide

and amino acid sequences. In general, the trees generated showed a radial distribution of

FCV isolates around a centrally placed node, suggesting a `star-like' evolution (e.g.

Fig. 4). Not surprisingly, attempts to identify significant phylogenetic clustering of FCVs

within this star-like evolution were largely unsuccessful. In the majority of cases,

individual nodes were inconsistent between methods, irrespective of whether nt or aa

sequences were used. This lack of consistency of individual nodes was supported by low

bootstrap values, indicating a lack of confidence in the majority of nodes. The only

possible exceptions to this were the KS20/KS40 node (as reported by Geissler et al.,

1997), the F65/LS015 node and the cluster containing isolates V77, V83 and V274 in

trees based on capsid sequence (Figs. 3 and 4). These groupings were observed

frequently in other trees, and were often supported by significant bootstrap values (data


Fig. 3. Rooted phylogenetic construction for FCV using SMSV-1 as an outgroup. Dendrograms were derived for conserved capsid sequences using both nucleotides (a)

and amino acids (b), as described in the methods. Each neighbor-joining tree was produced using evolutionary distances corrected by the Kimura-2-parameter model and

neighbor joining (PHYLIP; (Felsenstein, 1989)). The origins of each isolate are also indicated by a country letter and year, as in Table 1 (A: Australia, C: Canada, G:

Germany, J: Japan). Bootstrap values greater than 60 are included at the relevant nodes. Nodes supported by significant bootstraps are in bold. Evolutionary distances are

to scale except within the outgroup SMSV.

18

4M

.G

lenn

eta

l./Veterin

ary

Micro

bio

logy

67

(1999)

175±193

Fig. 4. Unrooted phylogenetic construction of FCV using conserved capsid nucleotide (a) and amino acid (b) sequences. Each tree was produced using the Kimura-2-

parameter model and neighbor-joining (PHYLIP; (Felsenstein, 1989)). The origins of each isolate are also indicated by a country letter and year, as in Table 1 (A:

Australia, C: Canada, G: Germany, J: Japan). For clarity, significant bootstrap values (>70) are included at the relevant node and detailed below each tree; the first figure

represents bootstraps based on the above analysis, and the second figure is based on parsimony. Nodes supported by significant bootstraps are in bold. Evolutionary

distances are to scale.

M.

Glen

net

al./V

eterina

ryM

icrob

iolo

gy

67

(1999)

175±193

185

Fig. 5. Unrooted phylogenetic construction of FCV using ORF3 nucleotide (a) and amino acid (b) sequences. Each tree was produced using the Kimura-2-parameter

model and neighbor-joining (PHYLIP; (Felsenstein, 1989)). The origins of each isolate are also indicated by a country letter and year as in Table 1 (A: Australia, C:

Canada, G: Germany, J: Japan). Where bootstrap values exceeded 70, they are included; the first figure represents bootstraps based on the above analysis, and the second

figure is based on parsimony. Nodes supported by significant bootstraps are in bold. Evolutionary distances are to scale.

18

6M

.G

lenn

eta

l./Veterin

ary

Micro

bio

logy

67

(1999)

175±193

not presented). There was also significant bootstrap support for the LLK/255 node in

trees based on ORF3 sequence (Fig. 5), but this was not supported by the trees based on

ORF2.

4. Discussion

FCV is an extremely successful pathogen of cats. It causes clinical signs ranging from

subclinical infection through to oral and respiratory signs, lameness, and pneumonia

(Gaskell and Dawson, 1998). FCV occurs worldwide, and is very common even in

apparently healthy cat populations, in part, due to persistent shedding of virus from

the oropharynx of carrier cats (Wardley et al., 1974; Wardley, 1976; Coutts et al.,

1994). Although host and other factors may play a role in disease outcome,

experimentally, some strains of FCV do appear to have differing disease potential,

suggesting that there may be an underlying viral genetic basis for differences in

pathogenicity (Fastier, 1957; Povey and Hale, 1974; Hoover and Kahn, 1975; Pedersen

et al., 1983; Dawson et al., 1994).

This paper compares new sequence data from five UK and four Australian isolates with

all previously published data. The comparison covers viruses isolated over a period of

approximately 38 years from many parts of the world, and includes viruses obtained from

cats showing different clinical signs. However, despite these diverse origins, all strains

examined demonstrated a similar degree of sequence relatedness to each other over the

majority of the genome, and there was little apparent clustering of isolates observed on

phylogenetic analysis.

The overall lack of significant clustering within FCVs is consistent with the findings of

Geissler et al., 1997 and suggests that FCV exists as a single diverse genotype. This is in

broad agreement with existing serological data, where, despite extensive comparisons,

and the presence of minor antigenic differences between strains, FCV isolates are

considered to belong to a single serotype (Povey, 1974; Kalunda et al., 1975; Burki et al.,

1976). Even in the present study, where strain F65 was chosen for sequence analysis

because it is antigenically more distinct from the broadly cross-reactive vaccine strain F9

than other isolates (Dawson, 1991; Dawson et al., 1993b), no marked differences were

found compared to other FCV isolates. However, minor sequence differences may have a

significant impact on antigenicity (Mateu et al., 1990; Radford et al., 1998).

In terms of pathogenicity, it is interesting to note that strains with apparently different

pathogenic potential (e.g. strains F65, LLK and 2280 which can induce lameness

(Pedersen et al., 1983; Dawson et al., 1994)) do not show marked sequence or

phylogenetic differences from other isolates, confirming the findings of Geissler et al.

(1997). It is likely that, as for the effect of sequence changes on antigenicity, small and

possibly localised differences are responsible for the different clinical signs associated

with some FCV isolates. Further work is required to identify regions of the FCV genome

that may relate to virulence, such as receptor binding sites.

Although there was evidence for the clustering of some isolates (F65/LS015, KS20/

KS40 and V77/V83/V274), most FCVs failed to group on a geographic or temporal basis.

For example, LLK and 2280 (isolated in Canada in 1982) exhibit a greater degree of


sequence divergence when compared to each other than when KCD is compared to the

KS isolates (isolated at opposite ends of the world and at least 37 years apart). There are a

number of possible explanations for the apparent lack of genetic clades in the current data

set including the limitation of sample size and sampling methodology. It is also possible

that FCV has not been present in the cat population long enough for distinct clades to

have evolved. Nevertheless, there is considerable variability between isolates of FCV.

These multiple variants appear to exist within a defined spectrum of relatedness, and are

possibly of equal fitness: they seem to have co-circulated in different geographic areas

over a number of years. Although it is possible that similar pathways of evolution have

occurred independently in different locations over time, it seems more likely that the rate

of geographical movement of FCV isolates may be greater than the ability of the virus to

evolve into and exploit new geographical niches.

This apparent lack of geographic clustering for FCV is in contrast to feline

immunodeficiency virus (FIV), where isolates may be broadly grouped according to

their region of isolation (Rigby et al., 1993; Bachmann et al., 1997). Such a distinction

may reflect fundamental differences in the epidemiology of these two viruses. FIV is

transmitted relatively inefficiently, typically in older male cats, by biting (Hopper et al.,

1994). In contrast, FCV is transmitted very efficiently between cats, with the most acute

infection occurring in young kittens (Gaskell and Dawson, 1998). In addition, a large

proportion of clinically recovered animals (approximately 20±25%) then become carriers,

providing a frequent source of virus for transmission to susceptible cats. Therefore, FCV

is likely to circulate continuously around the globe with its host, for example, through

trading and showing, as pets, and also naturally across land borders. Therefore, the

genetic pool of isolates is constantly being mixed, which may reduce opportunities for

genotypes to form within distinct geographic areas.

Lack of chronological sequence evolution, despite the presence of considerable

variability amongst FCV isolates, suggests that FCV evolution has not been associated

with any gain in fitness. This suggests that selection pressures operating within or on the

FCV population are either absent or are inconsistent. Whilst it is difficult to characterise

the nature of selection pressures acting on the FCV population, one candidate may be

vaccine-induced immunity. Vaccination against FCV has been widespread since the

1970s, and the majority of vaccines have been based on F9 or F9-like isolates. Until

relatively recently, antisera to F9 have been found to neutralise a consistently high

percentage of field isolates (Kalunda et al., 1975; Knowles et al., 1990). However, there is

serological evidence emerging to suggest that recent FCV isolates are becoming less

efficiently neutralised by F9 antisera than older isolates (Geissler et al., 1997; Lauritzen

et al., 1997). Such vaccine-driven selection does not appear to be reflected in the current

study by the clustering of more recent isolates. However, this may reflect the small

number of recent isolates examined or the fact that the small number of changes

necessary to alter antigenicity may be lost easily amongst the high levels of FCV

sequence variation.

These studies, which now include 22 capsid genes, have confirmed the variability of

the FCV capsid gene, in particular, within regions C and E (Neill, 1992; Seal et al., 1993).

Despite the classification of region E as variable, a distinct region of sequence

conservation exists in the middle of region E separating the 50 and 30 hypervariable


regions (Seal et al., 1993). It seems likely that this high degree of conservation reflects

significant function within the capsid, such as that imposed by structural requirements of

the protein or for interaction with host cells. Indeed, the presence of conserved receptors

between variable domains has been demonstrated in other viruses, such as foot-and-

mouth disease virus (Mateu et al., 1990; Leippert et al., 1997).

Although the variability of ORF2 is well recognised, our analysis suggests that there is

also significant variability within ORFs 1 and 3. In ORF1, this variability is clustered in

up to six regions which approximately border the putative functional domains of the

non-structural proteins. The significance of these ORF1 islands of variability is un-

certain but may reflect parts of the FCV genome that are less subject to functional

constraints, in contrast to the conserved regions they border. Some of these conserved

regions are considered to encode a 2C-like helicase, 3C-like protease and 3D-like

RNA polymerase based on sequence similarity with picornaviruses (Neill, 1990; Meyers

et al., 1991b; Oshikamo et al., 1994) and some functional studies (Boniotti et al., 1994;

VaÂzquez et al., 1998). However, the function of the other conserved regions is, as yet,

uncertain. In ORF3, the extent of variability between isolates is more heterogenous than

within ORFs 1 and 2. The function of this ORF in FCV is unknown, although the closely

related ORF2 of RHDV has been shown to encode a minor structural protein (Wirblich

et al., 1996).

In conclusion, we have found that regions of variability in the genome of FCV,

previously identified only in ORF2, exist in other regions also. Overall, the variability in

all three ORFs of FCV is marked. Despite this variability, however, all FCV isolates

examined so far appear to represent a single diverse genetic group consisting of multiple

variants co-circulating with equal fitness. This is in contrast to some other caliciviruses,

such as the human caliciviruses and RHDV, which form distinct genogroups based on

sequence analysis of both capsid and non-structural genes (Carter and Cubitt, 1995;

Green et al., 1997; Le Gall et al., 1998). It is possible that the apparent lack of genotypes

within FCV may be a reflection of the epidemiology of the disease in the cat. In FCV

infection, there is a widespread carrier state which may allow continuous global

movement of feline caliciviruses within the cat population, ensuring that isolates of

maximum fitness will always tend to predominate. However, it is also possible that the

examination of more isolates from different disease outbreaks and locations, including

sequence from ORF1 and ORF3, will disclose distinct FCV genotypes: indeed, such a

situation occurred with influenza C virus (Buonagurio et al., 1985; Muraki et al., 1996;

Tada et al., 1997). Alternatively, further selection pressures, such as vaccination, may

lead to their development.

Acknowledgements

The authors thank Dr. E. Gould, Dr. S. Butcher, Dr. E. Holmes and colleagues for

helpful discussion, Dr. Ian Head and Dr. Vincent Moulton for assistance with

phylogenetic analysis and Chris McCracken and Ruth Ryvar for skilful technical

assistance. This work was supported by grants from The Wellcome Trust, SERC and the

Whitley Animal Protection Trust.


References

Bachmann, M.H., Mathiason-Dubard, C., Learn, G.H., Rodrigo, A.G., Sodora, D.L., Mazzetti, P., Hoover, E.A.,

Mullins, J.I., 1997. Genetic diversity of feline immunodeficiency virus: dual infection, recombination, and

distinct evolutionary rates among envelope sequence clades. J. Virol. 71, 4241±4253.

Baulch-Brown, C., Love, D.N., Meanger, J., 1998. Variation in the capsid protein of Australian feline calicivirus

isolates. In: Proceedings of the 2nd Veterinary Virology in Australia Conference, Melbourne, Australia.

Berke, T., Golding, B., Jiang, X., Cubitt, D.W., Wolfaardt, M., Smith, A.W., Matson, D.O., 1997. Phylogenetic

analysis of the caliciviruses. J. Med. Virol. 52, 419±424.

Bittle, J.L., Rubic, W.J., 1976. Immunization against feline calicivirus infection. Am. J. Vet. Res. 37, 275±278.

Bittle, J.L., York, C.J., Newberne, J.W., Martin, M., 1960. Serological relationship of new feline cytopathogenic

viruses. Am. J. Vet. Res. 21, 547±550.

Boniotti, B., Wirblich, C., Sibillia, M., Meyers, G., Thiel, H.-J., Rossi, C., 1994. Identification and

characterization of a 3C-like protease from rabbit haemorrhagic disease virus, a calicivirus. J. Virol. 68,

6487±6495.

Buonagurio, D.A., Nakada, S., Desselberger, U., Krystal, M., Palese, P., 1985. Noncumulative sequence changes

in the hemagglutinin genes of influenza C virus isolates. Virology 146, 221±232.

Burki, F., Starustka, B., Ruttner, O., 1976. Attempts to serologically classify caliciviruses on a national and an

international basis. Infect. Immun. 14, 876±881.

Carter, M.J., Cubitt, W.D., 1995. Norwalk and related viruses. Curr. Opin. Infect. Dis. 8, 403±409.

Carter, M.J., Milton, I.D., Meanger, J., Bennett, M., Gaskell, R.M., Turner, P.C., 1992a. The complete nucleotide

sequence of feline calicivirus. Virology 190, 443±448.

Carter, M.J., Milton, I.D., Turner, P.C., Meanger, J., Bennett, M., Gaskell, R.M., 1992b. Identification and

sequence determination of the capsid protein gene of feline calicivirus. Arch. Virol. 122, 223±235.

Caul, E.O., 1996. Viral gastroenteritis: small round structured viruses, caliciviruses and astroviruses 1. The

clinical and diagnostic perspective. J. Clin. Pathol. 49, 874±880.

Clarke, I.N., Lambden, P.R., 1997. The molecular biology of caliciviruses. J. Gen. Virol. 78, 291±301.

Coutts, A.J., Dawson, S., Willoughby, K., Gaskell, R.M., 1994. Isolation of feline respiratory viruses from

clinically healthy cats at UK cat shows. Vet. Rec. 135, 555±556.

Crandell, R.A., Niemann, W.H., Ganaway, J.R., Maurer, F.D., 1960. Isolation of cytopathic agents from the

nasopharyngeal region of the domestic cat. Virology 10, 283±285.

Cubitt, D., Bradley, D.W., Carter, M.J., Chiba, S., Estes, M.K., Saif, L.J., Schaffer, F.L., Smith, A.W., Studdert,

M.J., Thiel, H.J., 1995. Virus taxonomy. Classification and nomenclature of viruses. In: Murphy, F.A.,

Fauquet, C.M., Bishop, D.H.L., Ghabrial, S.A., Jarvis, A.W., Martelli, G.P., Mayo, M.A., Summers, M.D.

(Eds.), Sixth report of the international committee on taxonomy of viruses, Springer, New York, pp. 359±

363.

Dawson, S., 1991. Studies of feline calicivirus and its role in feline disease, Ph.D. Thesis, University of

Liverpool, Liverpool.

Dawson, S., Bennett, D., Carter, S.D., Bennett, M., Meanger, J., Turner, P.C., Carter, M.J., Milton, I., Gaskell,

R.M., 1994. Acute arthritis of cats associated with feline calicivirus infection. Res. Vet. Sci. 56, 133±

143.

Dawson, S., McArdle, F., Bennett, D., Carter, S.D., Bennett, M., Ryvar, R., Gaskell, R.M., 1993a. Investigation

of vaccine reactions and breakdowns after feline calicivirus vaccination. Vet. Rec. 132, 346±350.

Dawson, S., McArdle, F., Bennett, M., Carter, M., Milton, I.P., Turner, P., Meanger, J., Gaskell, R.M., 1993b.

Typing of feline calicivirus isolates from different clinical groups by virus neutralisation tests. Vet. Rec.

133, 13±17.

Deveraux, J., Haeberli, P., Smithies, O., 1984. A comprehensive set of sequence analysis programs for the VAX.

Nucl. Acid Res. 12, 387±395.

Ellis, T.M., 1981. Jaundice in a siamese cat with in utero feline calicivirus infection. Aust. Vet. J. 57, 383±385.

Fastier, L.B., 1957. New feline virus isolated in tissue culture. Am. J. Vet. Res. 18, 382±389.

Felsenstein, J., 1989. PHYLIP: phylogenetic inference package (version 3.2). Cladistics 5, 164±166.

Gaskell, R.M., Dawson, S.D., 1998. Feline respiratory disease. In: Greene, C.E. (Ed.), Infectious diseases of the

dog and cat, 2nd ed., W.B. Saunders Co., Philadelphia.


Geissler, K., Schneider, K., Platzer, G., Truyen, B., Kaaden, O.-R., Truyen, U., 1997. Genetic and antigenic

heterogeneity among feline calicivirus isolates from distinct disease manifestations. Virus Res. 48, 193±

206.

Green, J., VinjeÂ, J., Lewis, D.C., Gallimore, C.I., Koopmanns, M., Brown, D.W.G., 1997. Genomic diversity

among human caliciviruses. In: Chasey, D., Gaskell, R.M., Clarke, I.N. (Eds.), First International

Symposium on Caliciviruses, Proceedings of a European Society for Veterinary Virology (ESVV)

Symposium, Reading, UK, 1996, ESVV and Central Veterinary Laboratory, pp. 37±49.

Green, S.M., Lambden, P.R., Caul, E.O., Ashley, C.R., Clarke, I.N., 1995. Capsid diversity in small round-

structured viruses: molecular characterization of an antigenically distinct human enteric calicivirus. Virus

Res. 37, 271±283.

Gubler, U., Hoffman, B.J., 1983. A simple and very efficient method for generating cDNA libraries. Gene 25,

263±269.

Guiver, M., Littler, E., Caul, E.O., Fox, A.J., 1992. The cloning, sequencing and expression of a major antigenic

region from the feline calicivirus capsid protein. J. Gen. Virol. 73, 2429±2433.

Hoover, E.A., Kahn, D.E., 1975. Experimentally induced feline calicivirus infection: clinical signs and lesions.

J. Am. Vet. Med. Assoc. 166, 463±468.

Hopper, C.D., Sparkes, A.H., Harbour, D.A., 1994. Feline immunodeficiency virus. In: Chandler, E.A., Gaskell,

C.J., Gaskell, R.M. (Eds.), Feline Medicine and Therapeutics, 2nd ed., Blackwell, Oxford, pp. 488±505.

Huxtable, C.R., Duff, B.C., Bennett, A.M., Love, D.N., Buthcher, D.R., 1979. Placental lesions in habitually

aborting cats. Vet. Pathol. 16, 283±291.

Jiang, X., Wang, M., Wang, K., Estes, M.K., 1993. Sequence and genomic organisation of Norwalk virus.

Virology 195, 51±61.

Kahn, D.E., Gillespie, J.H., 1970. Feline viruses X. Characterization of a newly-isolated picornavirus causing

interstitial pneumonia and ulcerative stomatitis in the domestic cat. Cornell Vet. 60, 669±683.

Kalunda, M., Lee, K.M., Holmes, D.F., Gillespie, J.H., 1975. Serological classification of feline caliciviruses by

plaque-reduction and immunodiffusion. Am. J. Vet. Res. 36, 353±356.

Knowles, J.O., 1988. Studies on feline calicivirus with particular reference to chronic stomatitis in the cat, Ph.D.

Thesis, University of Liverpool, Liverpool.

Knowles, J.O., Dawson, S., Gaskell, R.M., Gaskell, C.J., Harvey, C.E., 1990. Neutralisation patterns among

recent British and North American feline calicivirus isolates from different clinical origins. Vet. Rec. 127,

125±127.

Knowles, J.O., Gaskell, R.M., Gaskell, C.J., Harvey, C.E., Lutz, H., 1989. Prevalence of feline calicivirus, feline

leukaemia virus and antibodies to FIV in cats with chronic stomatitis. Vet. Rec. 124, 336±338.

Lauritzen, A., Jarrett, O., Sabara, M., 1997. Serological analysis of feline calicivirus isolates from the United

States and United Kingdom. Vet. Microbiol. 56, 55±63.

Le Gall, G., Arnauld, C., Boilletot, E., Morisse, J.P., Rasschaert, D., 1998. Molecular epidemiology of rabbit

haemorrhagic disease virus outbreaks in France during 1988 to 1995. J. Gen. Virol. 79, 11±16.

Leippert, M., Beck, E., Weiland, F., Pfaff, E., 1997. Point mutations within the bG-bH loop of foot-and-mouth

disease virus 01K affect virus attachment to target cells. J. Virol. 71, 1046±1051.

Liu, B.L., Clarke, I.N., Caul, E.O., Lambden, P.R., 1995. Human enteric caliciviruses have a unique genome

structure and are distinct from the Norwalk-like viruses. Arch. Virol. 140, 1345±1356.

Love, D.N., Baker, K.D., 1972. Sudden death in kittens associated with a feline picornavirus. Aust. Vet. J. 48,

643.

Mateu, M.G., MartõÂnez, M.A., Capucci, L., Andreu, D., Giralt, E., Sobrino, F., Brocchi, E., Domingo, E., 1990.

A single amino acid substitution affects multiple overlapping epitopes in the major antigenic site of foot-

and-mouth disease virus of serotype C. J. Gen. Virol. 71, 629±637.

McArdle, F., Dawson, S., Carter, M.J., Milton, I.D., Turner, P.C., Meanger, J., Bennett, M., Gaskell, R.M., 1996.

Feline calicivirus strain differentiation using monoclonal antibody analysis in an enzyme-linked immuno-

flow-assay. Vet. Microbiol. 51, 197±206.

Meyers, G., Wirblich, C., Thiel, H.-J., 1991a. Genomic and subgenomic RNAs of rabbit hemmorrhagic disease

virus are both protein-linked and packaged into particles. Virology 184, 677±686.

Meyers, G., Wirblich, C., Thiel, H.-J., 1991b. Rabbit hemorrhagic disease virus ± molecular cloning and

nucleotide sequencing of a calicivirus genome. Virology 184, 664±676.


Milton, I.D., Turner, J., Teelan, A., Gaskell, R., Turner, P.C., Carter, M.J., 1992. Location of monoclonal

antibody binding sites in the capsid protein of feline calicivirus. J. Gen. Virol. 73, 2435±2439.

Muraki, Y., Hongo, S., Sugawara, K., Kitame, F., Nakamura, K., 1996. Evolution of the haemagglutinin-esterase

gene of influenza C virus. J. Gen. Virol. 77, 673±679.

Neill, J.D., 1990. Nucleotide sequence of a region of the feline calicivirus genome which encodes picornavirus-

like RNA-dependent RNA polymerase, cysteine protease and 2C polypeptides. Virus Res. 17, 145±160.

Neill, J.D., 1992. Nucleotide sequence of the capsid protein gene of two serotypes of San Miguel sea lion virus:

identification of conserved and non-conserved amino acid sequences among calicivirus sequences. Virus

Res. 24, 211±222.

Neill, J.D., Reardon, I.M., Heinrikson, R.L., 1991. Nucleotide sequence and expression of the capsid protein

gene of feline calicivirus. J. Virol. 65, 5440±5447.

Neill, J.D., Sosnovtsev, S., Green, K.Y., 1997. Structure/function studies of the capsid protein of caliciviruses:

domain swaps between different feline calicivirus strains. In: Chasey, D., Gaskell, R.M., Clarke, I.N. (Eds.),

First International Symposium on Caliciviruses, Proceedings of a European Society for Veterinary Virology

(ESVV) Symposium, Reading, UK, 1996, ESVV and Central Veterinary Laboratory, pp. 120±124.

Oshikamo, R., Tohya, Y., Kawaguchi, Y., Tomonaga, K., Maeda, K., Takeda, N., Utagawa, E., Kai, C., Mikami,

T., 1994. The molecular cloning and sequencing of an open reading frame encoding non-structural proteins

of feline calicivirus F4 strain isolated in Japan. J. Vet. Med. Sci. 56, 1093±1099.

Pedersen, N.C., Laliberte, L., Ekman, S., 1983. A transient febrile `limping' syndrome of kittens caused by two

different strains of feline calicivirus. Feline Practice 13, 26±35.

Povey, R.C., 1974. Serological relationships among feline caliciviruses. Infect. Immun. 10, 1307±1314.

Povey, R.C., Hale, C.J., 1974. Experimental infections with feline caliciviruses (picornaviruses) in specific-

pathogen-free cats. J. Comp. Pathol. 84, 245±256.

Povey, R.C., Wardley, R.C., Jessen, H., 1973. Feline picornavirus infection: the in vivo carrier state. Vet. Rec. 92,

224±229.

Radford, A.D., Bennett, M., McArdle, F., Dawson, S., Turner, P.C., Glenn, M.A., Gaskell, R.M., 1997. The use

of sequence analysis of a feline calicivirus (FCV) hypervariable region in the epidemiological investigation

of FCV related disease and vaccine failures. Vaccine 15, 1451±1458.

Radford, A.D., Turner, P.C., Bennett, M., McArdle, F., Dawson, S., Glenn, M.A., Williams, R.A., Gaskell, R.M.,

1998. Quasispecies evolution of a hypervariable region of the feline calicivirus capsid gene in cell culture

and in persistently infected cats. J. Gen. Virol. 79, 1±10.

Rigby, M.A., Holmes, E.C., Pistello, M., Mackay, A., Leigh Brown, A.J., Neil, J.C., 1993. Evolution of

structural proteins of feline immunodeficiency virus: molecular epidemiology and evidence of selection for

change. J. Gen. Virol. 74, 425±436.

Sanger, F., Nicklen, S., Coulson, A.R., 1977. DNA sequencing with chain terminating inhibitors. Proc. Natl.

Acad. Sci. (USA) 74, 5463±5467.

Seal, B.S., 1994. Analysis of capsid protein gene variation among divergent isolates of feline calicivirus. Virus

Res. 33, 39±53.

Seal, B.S., Neill, J.D., 1994. Capsid protein gene sequence of feline calicivirus isolates 255 and LLK: further

evidence for capsid protein configuration among feline caliciviruses. Virus Genes 9, 183±187.

Seal, B.S., Ridpath, J.F., Mengeling, W.L., 1993. Analysis of feline calicivirus capsid protein genes:

identification of variable antigenic determinant regions of the protein. J. Gen. Virol. 74, 2519±2524.

Shin, Y.-S., Tohya, Y., Oshikamo, R., Kawaguchi, Y., Tomonaga, K., Miyazawa, T., Kai, C., Mikami, T., 1993.

Antigenic analysis of feline calicivirus capsid precursor protein and its polypeptides produced in a

mammalian cDNA expression system. Virus Res. 30, 17±26.

Smith, A.W., Skilling, D.E., Latham, A.B., 1981. Isolation and identification of five new serotypes of

caliciviruses from marine mammals. Am. J. Vet. Res. 42, 693±694.

Sosnovtsev, S., Green, K.Y., 1995. RNA transcripts derived from a cloned full-length copy of feline calicivirus

genome do not require VpG for infectivity. Virology 210, 383±390.

Sosnovtsev, S.V., Sosnovtseva, S.A., Green, K.Y., 1998. Cleavage of the feline calicivirus capsid precursor is

mediated by a virus-encoded proteinase. J. Virol. 72, 3051±3059.

Tada, Y., Hongo, S., Muraki, Y., Sugawara, K., Kitame, F., Nakamura, K., 1997. Evolutionary analysis of

influenza C virus M genes. Virus Genes 15, 53±59.


Takahashi, E., Konishi, S., Ogata, M., 1971. Studies on cytopathogenic viruses from cats with respiratory

infections II. Characterization of feline picornaviruses. Jpn. J. Vet. Sci. 33, 81±87.

Tenorio, A.P., Franti, C.E., Madewell, B.R., Pedersen, N.C., 1991. Chronic oral infections of cats and their

relationship to persistent oral carriage of feline calici-, immunodeficiency, or leukemia viruses. Vet.

Immunol. Immunopathol. 29, 1±14.

Thompson, R.R., Wilcox, G.E., Clark, W.T., Jansen, K.L., 1984. Association of calicivirus infection with

chronic gingivitis and pharyngitis in cats. J. Sm. Anim. Pract. 25, 207±210.

Tohya, Y., Taniguchi, Y., Takahashi, E., Utagawa, E., Takeda, N., Miyamura, K., Yamazaki, S., Mikami, T.,

1991. Sequence analysis of the 30-end of the feline calicivirus genome. Virology 183, 810±814.

Tohya, Y., Yokoyama, N., Maeda, K., Kawaguchi, Y., Mikami, T., 1997. Mapping of antigenic sites involved in

neutralization on the capsid protein of feline calicivirus. J. Gen. Virol. 78, 303±305.

VaÂzquez, A.L., MartõÂn Alonso, J.M., Casais, R., Boga, J.A., Parra, F., 1998. Expression of the enzymatically

active rabbit hemorrhagic disease virus RNA-dependent RNA polymerase in Escherichia coli. J. Virol. 72,

2999±3004.

Wardley, R.C., 1974. Studies on feline calicivirus with particular reference to persistent infections, Ph.D. Thesis,

University of Bristol, Bristol.

Wardley, R.C., 1976. Feline calicivirus carrier state: a study of the host/virus relationship. Arch. Virol. 52, 243±

249.

Wardley, R.C., Gaskell, R.M., Povey, R.C., 1974. Feline respiratory viruses ± their prevalence in clinically

healthy cats. J. Sm. Anim. Pract. 15, 579±586.

Wardley, R.C., Povey, R.C., 1977. The clinical disease and patterns of excretion associated with three different

strains of feline calicivirus. Res. Vet. Sci. 23, 7±14.

Wirblich, C., Thiel, H.-J., Meyers, G., 1996. Genetic map of the calicivirus rabbit hemorrhagic disease virus as

deduced from in vitro translation studies. J. Virol. 70, 7974±7983.


nucleotide sequence of uk and australian isolates of feline calicivirus (fcv) and phylogenetic...

Documents