nucleotide sequence of uk and australian isolates of feline calicivirus (fcv) and phylogenetic...
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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: alanrad@liv.ac.uk
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.
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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).
M. Glenn et al. / Veterinary Microbiology 67 (1999) 175±193 179
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.
180 M. Glenn et al. / Veterinary Microbiology 67 (1999) 175±193
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.
M. Glenn et al. / Veterinary Microbiology 67 (1999) 175±193 181
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).
182 M. Glenn et al. / Veterinary Microbiology 67 (1999) 175±193
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
M. Glenn et al. / Veterinary Microbiology 67 (1999) 175±193 183
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.
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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.
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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.
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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
M. Glenn et al. / Veterinary Microbiology 67 (1999) 175±193 187
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
188 M. Glenn et al. / Veterinary Microbiology 67 (1999) 175±193
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.
M. Glenn et al. / Veterinary Microbiology 67 (1999) 175±193 189
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