Department of Veterinary Pathology, University of Glasgow, Bearsden Road, Glasgow G61 1QH, UK
Correspondence
D. D. Addie
D.D.Addie{at}vet.gla.ac.uk
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ABSTRACT |
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Present address: Physics of Complex Systems, Division of Physics and Astronomy, Vrije Universiteit, De Boelelaan 1081, HV Amsterdam, The Netherlands
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INTRODUCTION |
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An unfortunate, inadvertent result of FCoV infection in a small proportion of cats is the development of the lethal, immune-mediated condition known as feline infectious peritonitis (FIP) (Poland et al., 1996; Vennema et al., 1998
). In many parts of the world, FIP is considered to be the major, infectious cause of death in cats (Vennema et al., 1998
) and is of sufficient veterinary importance to create considerable demand for measures to control the infection, mainly through management practices (Addie & Jarrett, 1992
; Kass & Dent, 1995
). A more complete understanding of the epidemiology of the virus would permit more rational methods of control and may provide a model for the epidemiology of the severe acute respiratory syndrome virus. In particular, more information is required on the way in which the virus persists in the population.
Studies of other coronaviruses, particularly mouse hepatitis virus and infectious bronchitis virus of domestic poultry, have shown clearly that coronaviruses are prone to genetic variation (Cavanagh et al., 1998), and one might imagine that variation would promote persistence through the generation of quasispecies from which escape mutants might emerge. The existence of FCoV quasispecies and variation has been shown already (Gunn-Moore et al., 1998
; Kiss et al., 1999
), which poses the question of whether variants arise in persistently infected cats that contribute both to the maintenance of persistence in these animals and to the re-infection of seronegative cats.
There are two types of FCoV: type I FCoV is wholly feline, whereas type II FCoVs have arisen by recombination events between type I FCoVs and canine coronavirus (CCoV), resulting in a FCoV genome consisting of the spike (S) gene and part of the adjacent genes from CCoV (Herrewegh et al., 1998). The S protein is key: it is the S protein that attaches to the cellular receptor and it is antibodies to the S protein that mediate virus clearance (Gonon et al., 1999
). Type II FCoVs are easier to grow in cell culture and form most laboratory isolates but it is difficult to know how accurately laboratory infections with these strains reflect the real-life situation with type I viruses. The majority (90 %) of field isolates in Japan and the USA are type I (Vennema, 1999
). Amongst cats with FIP in Japan, 30 % of FCoVs were type II (Hohdatsu et al., 1992
). Apart from the possibly greater chance of developing FIP with a type II infection, whether or not there is any correlation between FCoV type and outcome of infection in terms of becoming a carrier or being transiently infected has never been investigated previously. Until now, types I and II FCoV have been differentiated by monoclonal antibodies; in this paper, we describe an RT-PCR developed specifically to differentiate between type I and type II FCoVs.
To begin to answer these questions, we developed a method to identify FCoV isolates through amplification of a region of the S gene by RT-PCR and restriction endonuclease digestions of the product. This reaction distinguished FCoV of types I and II and indicated that the former was by far the more prevalent type in the UK. Phylogenetic analysis of viruses from a larger number of cats was performed to establish the range of variation in nucleotide sequence in our population. This study confirmed previous reports of considerable sequence variation between FCoV from different sources (Vennema et al., 1998). A longitudinal study of cats in households in which the virus was endemic revealed that the amino acid sequence at the C terminus of the S gene of the virus in individual persistently infected cats appeared to be conserved over periods of up to 7 years. Finally, we investigated whether transiently infected cats that had recovered from infection could become re-infected with the same virus strain, excreted either from a persistently infected cat or from a transiently infected cat, or whether they could only be re-infected with a different virus strain. In this way, we expected to learn the relative importance of persistent and transient infections in FCoV survival.
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METHODS |
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In addition, we examined FCoV isolates from pleural and peritoneal effusions from eight naturally infected cats with FIP that had been submitted to our diagnostic laboratory (these were not from survey households).
RT-PCR.
To monitor virus shedding, an RT-PCR that amplified the FCoV 7b gene (Herrewegh et al., 1995) was performed on faecal samples or rectal swabs. In addition, a novel RT-PCR amplifying a partial S gene sequence (S RT-PCR) was developed to differentiate between FCoV types I and II (Fig. 1
). Where there was sufficient material available, samples were also analysed using S RT-PCR. The target for the differentiating RT-PCR is located within the 3' region from the S gene, which encodes the more conserved C-terminal part of the S protein and is believed to be responsible for the incorporation of the S protein into the FCoV particle (Godeke et al., 2000
). The region that was amplified is N-terminal to the proposed transmembrane domain and differences in the S genes of FCoV types I and II are apparent in this region (Motokawa et al., 1995
). A reverse sequence primer (Iubs) targeting a region that is conserved among FCoV and CCoV S genes was used to make cDNA, while the forward primers were directed specifically against the type I or II FCoV sequence, resulting in fragments of different sizes depending on the identity of the FCoV. To increase both the sensitivity and the specificity of the reaction, a nested PCR step was performed. The primers and temperature cycling programmes are described in Table 1
.
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A 10 µl sample of cDNA was added to the primary PCR mixture, which contained 1x PCR buffer II (Perkin Elmer), 15 pmol Iubs, 20 pmol each specific forward primer (Iffs and Icfs), 2 mM MgCl2, 0·8 mM dNTPs and 2·5 U Taq polymerase (Perkin Elmer) in a final volume of 100 µl. From this reaction, 10 µl was added to 90 µl of the secondary PCR mixture, which contained 1x PCR buffer II, 20 pmol each primer (nIubs, nIffles and nIcfs), 1·5 mM MgCl2, 0·8 mM dNTPs and 2·5 U Taq polymerase in a final volume of 100 µl. Samples of 10 µl of the secondary PCR were analysed on a 2 % agarose gel containing 0·5 µg ethidium bromide ml-1.
Restriction enzyme analysis.
The products of the differentiating RT-PCR were analysed by separate digestions with AluI, Sau3AI (both from Gibco-BRL) and RsaI (Quantum-Appligene). Digestions were performed with 10 µl PCR product, 12 µl H2O, 1x appropriate buffer and 5 units enzyme. Samples were analysed on 2 % agarose gels containing 0·5 µg ethidium bromide ml-1.
Nucleotide sequencing and analysis.
Direct sequencing of both strands of the RT-PCR products was performed by MWG-Biotech. At positions where more than one base was called in both forward and reverse reads, a variant position was recorded. This was presumed to be a consequence of a mixed target virus population, as no cloning step that might fix Taq-induced errors was performed. In several cats sampled sequentially, the emergence of a dominant nucleotide could be detected at positions that were variant in earlier samplings.
Sequence data were derived for 50 samples from cats in 14 households and from Primucell (Pfizer) vaccine strain FIPV-DF2 (Christianson et al., 1989). Sequences were assigned the format AN/NNNN, where A is the household, N designates an individual cat and the numbers after the solidus refer to month and year of sampling (for example, J8/0196 was a sample collected from cat 8 in household J in January 1996). Sequences recovered from non-survey cats with FIP are prefaced by the letter F. Sequence alignment and unrooted phylogenetic trees were derived using the CLUSTAL_W program provided in MEGALIGN, version 5, using IUB weight matrix. Unrooted trees were derived through TREEVIEW, version 1.6.6 (Page, 1996
). Pairwise sequence comparisons were performed using the MartinezNeedleman-Wunsch alignment facility within MEGALIGN. Where types I and II FCoVs were compared, phylogenetic trees were derived through analysis of the C-terminal 174 bp of a subset of type I amplicons and the full-length type II amplicons. Where only type I FCoV sequences were analysed, the entire 320 bp was compared. Type I sequences with nucleotide identities of over 97 % (Fig. 3
) or over 99 % (Fig. 4
) to specific samples were excluded from phylogenetic trees for purposes of clarity. Relatedness of these sequences to sequences incorporated in the phylogenetic trees is summarized as follows: G5/1095, (G1/0296 and G1/1100)+[G2/0500, G5/0600, G1/1095 and G5/0100], where the underlined sequence, G5/1095, is included in the tree, sequences in round brackets are >97 % identical to G5/1095 across the C-terminal 174 bp of amplicon and those in square brackets are >99 % identical to G5/1095 across the entire 320 nt amplicon. Other related sequences include: J8/0196, (J10/0897)+[J9/0301 and J10/0895]; J8/0197, [J7/1196]; J8/0201, [J4/0201, J5/0201, J6/0201 and J6/0300]; J10/0897, [J10/0998]; C10/0301, (C11/0301)+[C9/0301]; X1/0201, (H2/1100); P4/0900, (P2/0900)+[P4_0396]; and FQ1/1099, (Q8/1197). Published database sequences were also included as follows: type I strains were UCD1 (AB088222), Black (AB088223) and KU-2 (D32044); and type II strains were 79-1146 (X06170) and 79-1683 (X80799).
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RESULTS |
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Using this method, types I and II could be distinguished by the size of the PCR product (Fig. 1). The first round of the S RT-PCR produced fragments of the predicted 376 bp when used to amplify the UCD-1 and UCD-3 type I strains (Hoskins, 1997
) of FCoV (kindly provided by D. Harbour, University of Bristol, UK). The nested step produced a product of 360 bp. From the type II Wellcome strain of FCoV, bands of 283 bp were produced by first-round PCR and 218 bp by second-round PCR (Fig. 2
).
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Comparative analysis of partial S sequences
To establish the origin of new strains within households, the range of sequence variation from cats across the UK was examined. Two households in which the virus was endemic over periods of up to 6 years were studied in detail. In these households, cats had been identified that were infected either persistently or transiently, as defined previously (Addie & Jarrett, 2001). Initial observations of whether a virus was type I or type II FCoV and the typing of isolates were made by restriction endonuclease digestion and verified by sequence analysis of a selection of samples. The 51 sequences were deposited in the EMBL database (accession nos AY159735AY159785). A phylogenetic tree derived from comparison of a selection of these sequences and five reference type I (KU-2, Black and UCD1) and II (Wellcome and FIPV-DF2) S sequences is shown in Fig. 3
. The range of nucleotide identities for all type I isolates (excluding reference strains) was 79100 %. Sequence identity between 13 isolates each recovered from a distinct household was in the range of 8295 %. Phylogenetic analysis of samples recovered from cats in the same household indicated a common origin of infection in some instances (sequence identities in the range of 95100 %), but in other households, partial S sequences were as related to S genes of other households as they were to isolates circulating within their own household (sequence identities <95 %), implying that novel isolates had been introduced rather than emerging from within the household. Sequence identity between type I and II sequences was in the range of 6067 %.
FCoV in persistently infected cats is highly conserved
Persistently infected carrier cats that shed virus continuously in the faeces (Addie & Jarrett, 2001) were of particular interest since they might be expected to be reservoirs of the virus, as well as sources of virus variants. The 320 bp partial S sequences (exclusive of primer sequences) were obtained from five of these cats over periods of up to 6 years (Table 2
). Sequence variation ranged from 0 changes in 17 months (100 % identity) (cat J3) to 9 nt in 5 years (97 % identity) (cat G1). For comparison, the sequences of three transiently infected cats that had recovered and become re-infected, on serological and molecular evidence, are included. In one of these, cat G5, there was very little change in sequence (>99 % sequence identity) and this cat may have been re-infected by cat G2 (100 % identity), a possible carrier of the type I strain detected initially in the household. However, in cats C5 and J7, which were infected at intervals of 12 and 13 months, respectively, there was over 8 % sequence change, which is suggestive of re-infection by distinct FCoV strains.
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Recovered transiently infected cats can be re-infected with the same or a different strain
We then investigated cats that had been transiently infected and subsequently re-infected with FCoV. We investigated whether the indigenous virus from the persistently infected cats in the same household or from another source had re-infected these cats. PCR products recovered from several of the transiently infected cats throughout the study were sequenced. From the results, presented in Tables 3 and 4, it appeared that both possibilities could occur.
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In household G, at the start of the survey, all but one cat were positive by ORF 7b RT-PCR (Table 4). By 14 months later, several cats had apparently cleared the infection, as their antibody titres had fallen to <10 and viral sequences could not be amplified from their faeces. After a further 9 months, all of the cats, except G5, were virus positive by PCR and rising antibody titres confirmed that cats G3 and G6 had been re-infected. Analysis of the S amplicon of four cats at the 1995 sampling indicated that a type I FCoV was circulating in the majority of cats and cat G6 was infected with a type II FCoV strain. Sequence analysis of samples from cats G5 and G2 collected in 2000 indicated that the region of the S gene that was sequenced varied by fewer than 2 nt from the 1995 isolate. In contrast, the S gene of the confirmed carrier G1 had over nine differences. To summarize, in this household there was evidence for infection of cat(s) with types I and II FCoV. The majority of cats were re-infected with type I FCoV, which, on the basis of the S sequence, was likely to have originated from the same source.
In household J, cat J10 was a known carrier and cat J9 became a carrier. Sequences in this household grouped into three sets, designated a, b and c, in which same-set sequences were related by 97100 % and sequences from different sets were related by 8692 % (Table 3). Sequences were associated with infections in multiple cats in the household at different periods of the 7 year study: a sequences were recovered at the start of the study period, b sequences in 1996/1997 and c sequences at the end of the study period in 2001, consistent with circulation of new strains of FCoV introduced into the household. Most a and b infections were transient infections from which the cats recovered, although cat J10 was a carrier of sequence a. Cats infected with sequence c in 1999 were still shedding virus at the end of the study. Cats J6, J7 and J8 had several periods of virus shedding over this period. As can be seen from Table 3
, cat J8 experienced three different FCoV infections in 1996 (sequence a), 1997 (sequence b) and 1999 (sequence c). That she was truly re-infected in 1999, rather than that her infecting strain had mutated, is confirmed by her antibody titre decreasing to 20 in May 1999 and rising again to 640 in July 1999.
Origin of two novel FCoV strains in household J
Sequence analysis indicated divergence between three sequences, a, b and c, associated with isolates recovered from household J: isolates b and c were as distant from isolates a as they were to sequences derived from isolates outside of this household (Fig. 4). This is suggestive of infection of cats in the household by three distinct strains of FCoV, although it does not preclude that recombination might have occurred between these coronaviruses in genome regions other than the S region sequenced herein.
To attempt to trace the origins of sequences b and c, the S amplicon of the FCoV from an in-contact cat, C9, from household C, who had mated cat J7 was sequenced. The sequence was found to be quite different to strains b (87 % identity) and c (90 % identity), so is unlikely to represent their origin (Fig. 4). Cats in this household had been vaccinated with Primucell but this is a type II strain and identity with sequences b and c is under 65 %. Cat J3 had been to a cat show in June 1999, which may have been the source of the third strain found in this household.
Most cats are not superinfected with several strains of FCoV
Although evidence was obtained for the concurrent presence of multiple strains of FCoV in three households, most cats were infected with only a single FCoV strain. However, three samples (C12, P2 and FZ1) had multiple variant positions within the sequences to an extent that this may reflect co-infection of these cats with two distinct strains. This is the first indication that co-infection or superinfection may occur with FCoV.
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DISCUSSION |
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We have shown for the first time that FCoV of types I and II can be distinguished using molecular biological methods. In addition, the PCR products can be digested using a combination of enzymes to produce a characteristic fingerprint for each FCoV strain. This method provides a useful tool for examining transmission patterns in natural infections in the field. Only 1 of 43 (2 %) cats tested was infected with type II FCoV, which is in agreement with figures from other countries in which type I is the endemic strain (Hohdatsu et al., 1992; Vennema, 1999
). Thus, in a Japanese study, 10 % of healthy FCoV-infected cats and 31 % of infected cats with FIP were infected with a type II FCoV (Hohdatsu et al., 1992
). Since type II FCoV arises by recombination of type I FCoV and CCoV (Herrewegh et al., 1998
), it might have been expected that cats in our households that contained dogs would have had a greater prevalence of type II. However, only five households had the seven dogs that were studied, which may have been too few to expect concurrent CCoV infection and in which to find recombinant virus. The one household that contained a cat with a type II virus did not have a dog. Whether type II viruses are transmitted naturally among cats is not known.
Carrier cats were defined as cats that shed virus continually, as opposed to intermittently, probably for life (Addie & Jarrett, 2001). Although the region of the S gene chosen showed up to 20 % variability across isolates, the amount of variation within lifelong carrier cats over periods of up to 5·5 years was minimal, suggesting that carrier status may not be maintained by quasispecies variation within the individual animal. Virus clearance has been correlated with humoral (Gonon et al., 1999
) and cell-mediated immune responses to the S glycoprotein (de Groot-Mijnes et al., 2002
). However, it is likely that the region of the S gene that we chose to examine was not the region involved in immune clearance of the virus and, therefore, might not be subject to selection pressure. Further sequencing of the whole S gene of these isolates might provide additional information in this regard. Our data demonstrated clearly that virus persistence in the carrier cats was due to the maintenance of the same virus over time and was not due to re-infection by different FCoV strains.
Most cats that become infected with FCoV mount an immune response, eliminate the virus and may then become re-infected (Addie & Jarrett, 2001; Foley et al., 1997
). We have shown that cats can become re-infected not only with different strains but also with the same strain of FCoV. In household G, re-infection of cat G5 was by a strain only 2 nt different from the strain with which he had been infected previously. In household J, over a 3 year period, only one mutation occurred in the sequenced region of the virus that was excreted by the carrier cat, J10. In contrast, there were 31 changes between this sequence (isolate a) and isolates circulating in the household, suggesting that a new virus had been introduced into the household. A possible candidate for introducing the virus was from another survey household, in which matings had occurred between two pairs of cats. However, the new virus in household J did not match any of the sequences recovered from the in-contact household (C).
Interestingly, no cat that became re-infected with FCoV developed FIP, in contrast to laboratory infections, where second infections commonly lead to more rapid and fulminant development of FIP (Vennema et al., 1990), a phenomenon known as antibody-dependent enhancement.
The factors that cause a cat to become a carrier or to be transiently infected are unknown. It has been proposed that a deletion or mutation must occur in FCoV for it to cause FIP in the host (Poland et al., 1996; Vennema et al., 1998
), so it seemed reasonable to expect that carrier status of the host be a consequence of genetic change in the virus. In household J, cats became re-infected with a type of FCoV that appeared to be more persistent than the previous indigenous virus, since it continued to be shed by a high proportion of cats for at least 18 months, including cats that had eliminated previous strains within a year. However, it was notable that the novel strain did not superinfect the existing persistently infected cat. With the exception of the cats mentioned above, most transiently infected cats did not become re-infected while they were shedding virus or were seropositive. However, after recovery from shedding and the loss of antibodies, these cats could become re-infected with the same or a different virus. More research is required to define the roles of both virus variation and host immune response that allow persistence of this virus. One possible explanation for the persistence of FCoV in healthy carrier cats is that they maintain a cell-mediated response indefinitely, with a focus of infection acting as a perpetual antigenic stimulus.
It has been proposed that cats infected with FCoV do not become superinfected, that is, infected with more than one strain of FCoV (Herrewegh et al., 1997). We have conflicting evidence on that subject. Thus, in one household, cat J9 did not become infected with the more infectious strain c virus, while in two other households, two other cats, C12 and P2, appeared to be infected with two virus strains.
In three cats (H2, H9 and Hub1; data not shown) from two households there was a deletion of 6 nt (resulting in the loss of 2 aa). These cats were from different breeds and had never had contact but were from neighbouring counties. This finding supports the conclusion of Vennema et al. (1998): there are many geographical differences in FCoV strains. Had these cats had FIP or been carriers, it might have been tempting to conclude that this deletion was responsible for the phenotypic change, whereas it was merely a geographical difference. This finding illustrates the dangers of drawing conclusions about virus virulence from changes in limited regions of the FCoV genome of only a few isolates.
In conclusion, we present a simple molecular biological method for examination of natural strains of FCoV. This method was used to show that most FCoVs infecting cats in the UK are type I. Carrier cats shed the same strain of FCoV continually. Most cats eliminate FCoV infection but are susceptible to re-infection with the same or a different FCoV strain. Whether a cat becomes a carrier or simply transiently infected appears to be a property of the cat rather than a property of the virus strain.
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ACKNOWLEDGEMENTS |
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Received 29 January 2003;
accepted 8 June 2003.