1 Centre for Ecology and Hydrology (formerly Institute of Virology), Mansfield Road, Oxford OX1 3SR, UK
2 Birch Brae, Knapp, Perth and Kinross PH14 9SW, UK
3 Rabbit-Wise, Holtside, Batts Corner, Dockenfield, Surrey GU10 4EX, UK
4 Institute of Biological Science, University of Stirling, Stirling FK9 4LA, UK
Correspondence
Naomi Forrester
nlf{at}ceh.ac.uk
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ABSTRACT |
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Present address: Department of Infectious Disease Epidemiology, Imperial College Faculty of Medicine, Norfolk Place, London W2 1PG, UK.
Present address: Biology Department, Penn State University, PA 16870, USA.
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INTRODUCTION |
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The high virulence of RHDV and its rapid and efficient dispersal through the rabbit populations led to it being tested as a bio-control agent in Australia, where the rabbit population was devastating the indigenous flora and fauna (Asgari et al., 1999; Sandell, 2002
). In 1995, a series of field trials was initiated on Wardang Island, 5 km off the coast of Port Victoria on the Yorke Peninsula, south coast of Australia. During the course of these trials, the virus inadvertently escaped from the island to mainland Australia and spread at a phenomenal rate throughout the southern states (Asgari et al., 1998
; Cooke et al., 2000
; Kovaliski, 1998
). Although the local rabbit population was initially severely affected, subsequent analysis has shown that the high lethality of the virus has not been maintained, and whilst sporadic epidemics are still being recorded, they do not occur with the same intensity that was observed initially (Lugton, 1999
).
The New Zealand Government had previously rejected the use of RHDV as a control agent; nevertheless, the virus was subsequently introduced deliberately and illegally into New Zealand in 1997 (Motha & Clark, 1998; O'Keefe et al., 1998
; Thompson & Clark, 1997
). Many farmers introduced the virus onto their farms by baiting oats and carrots with RHDV. This was done quite arbitrarily but, in spite of this, the disease was established and the government then approved the importation, manufacture and sale of a known strain of RHDV. The nature of its introduction may have had a detrimental effect on the subsequent efficacy and dispersal of the virus. It has been suggested that many rabbits may have received sublethal doses and did not succumb to the virus, as would have been expected if the virus had been disseminated under carefully regulated conditions (O'Keefe et al., 1999
).
The virus that was introduced inadvertently into Australia was the Czech strain V351. The complete genome sequence of this strain has been determined (Gould et al., 1997) and subsequently monitored after its escape and dispersal from Wardang Island (Asgari et al., 1999
). Two years after its release into Australia, the virus recovered from dead rabbits had diverged by only 2·7 %. The virus subsequently introduced illegally into New Zealand was confirmed to be the same strain (O'Keefe et al., 1998
). However, 4 years after its introduction into New Zealand, there are many healthy rabbits in areas where the virus had been released, raising questions about the suitability of this virus as a bio-control agent (Parkes et al., 2002
). Recent evidence has shown the presence of RNA in the tissues of healthy rabbits from New Zealand (Zheng et al., 2002
). This study focuses on the phylogenetic relationships of the viruses identified in the livers of healthy rabbits.
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METHODS |
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The VP60 gene sequences were aligned using PILEUP (GCG, Wisconsin Package). Phylogenetic analyses were undertaken using PAUP*, version 4.0.10b (Swofford, 2000). The optimal evolutionary model to use with each data set was estimated using MODELTEST, version 3.06 (Posada & Crandall, 1998
). The optimal maximum-likelihood tree was then estimated using the K80+
model, a heuristic search and TBR branch swapping, estimating variable parameters from the data, where necessary. Neighbour-joining bootstrap support (1000 replicates) was calculated for each tree using the maximum-likelihood settings.
Attempts to isolate infectious virus.
Domestic rabbits supplied by Harlan were inoculated with liver suspension that had been demonstrated to be positive by RT-PCR. Five of the freeze-dried liver samples were prepared as a clarified suspension in sterile PBS using mortar and pestles. Of each suspension, 100 µl was injected by the intramuscular route into individual Swiss White rabbits. Rabbit sera were monitored using ELISA tests to detect the presence of antibodies and livers were examined using nested RT-PCR, as described above.
ELISA tests
IgG detection.
An optimized concentration of recombinant RHDV capsid protein (Marin et al., 1995) was coated onto ELISA plates overnight at 4 °C in coating buffer. After washing the plates in PBS/Tween (0·1 %), twofold dilutions of serum were added for 1 h at 37 °C. The plates were washed and a 1 : 1000 dilution of polyvalent goat anti-rabbit serum conjugated with horseradish peroxidase (Sigma) was added for 1 h at 37 °C. After washing, substrate was added and the absorption of each test was estimated. Absorption readings equivalent to at least twice the equivalent dilution of negative control serum (serum from a commercially supplied rabbit certificated as negative) were considered positive for RHDV.
IgM detection.
A similar procedure was followed as with the polyvalent antibody, but instead of the polyvalent goat anti-rabbit serum, a 1 : 40 000 dilution of IgM goat anti-rabbit serum conjugated with horseradish peroxidase (Bethyl Laboratories) was used.
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RESULTS |
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A phylogenetic tree was constructed using the PAUP program. The deduced maximum-likelihood tree (Fig. 2), which includes representatives of RHDV from New Zealand, Europe and China, shows that most of the positive liver samples from Central Otago produced sequences that were related closely to the Czech strain V351, which was introduced deliberately into New Zealand in 1997. However, the sequences of these samples formed two distinct groups. One group clustered close to the Czech strain introduced deliberately and the New Zealand sample sequenced in the first 2 years after release. The second group was similar to, but distinct from, the Czech strain. Very little variability was detected within each group and both groups appear to have varied equally, although they remain distinct. At five of the eight sites, we found either group 1 or group 2 viruses, but not both. From sites 3 and 10, we found viruses from both groups, and from site 6 there were group 2 viruses and a different European strain of RHDV. Within each sampling site, we cannot rule out the possibility that the rabbit population was subdivided; if this is the case, we do not know from which subpopulation the samples came. As can be seen in Fig. 2
, one sample (NZ51) amplified from rabbit liver (site 6) was positioned within a clade containing European viruses (Fig. 2
) and is related most closely to the Spanish AST/89 strain (Boga et al., 1992
). Although we have worked with the capsid gene of this Spanish strain in the past, we have never introduced DNA representing sequence outside the capsid gene. As a control test, we amplified a region of the NZ51 genome outside the capsid genome (01 kb) to confirm that this Spanish-related sequence was not a laboratory contaminant. It is also important to emphasize that, at the time of collection of these rabbits, there were no signs of fatal epidemic RHDV.
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Attempts to identify infectious virus in RT-PCR-positive liver samples
In view of the fact that we had detected genomic length sequence in two rabbit livers, we attempted to isolate infectious virus from RT-PCR-positive liver suspensions. Since RHDV does not grow in cell culture, five rabbits were injected by the intramuscular route with five liver samples known to be positive. None of the rabbits showed clinical signs of infection. Sera were collected before inoculation and at the termination of the experiment. Sera were tested for the presence of RHDV-specific IgG and IgM antibody by ELISA (Moss et al., 2002). There was no evidence of an immune response.
Liver suspensions were analysed by RT-PCR for the presence of viral RNA using the methods described above. No evidence of viral RNA was found in the livers of any of these animals. While these are only limited tests, the data imply that the RT-PCR-positive livers of healthy rabbits do not contain readily demonstrable quantities of infectious virus.
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DISCUSSION |
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The consequences of the deliberate release of a supposedly highly virulent Czech strain of RHDV into New Zealand in 1997 must, therefore, be considered in the light of the evidence that there may have been a background level of immunity and/or virus-specific resistance to infection in the rabbit population. This might have influenced the variable mortality figures (1050 % mortality in the first epidemics), which resulted following the deliberate release of the virus by the farmers (Cooke, 2002).
We found RNA related closely to two supposedly virulent strains of RHDV in the livers of healthy rabbits in New Zealand, i.e. the Czech V351 strain and the Spanish 89 strain. The presence of similar viral sequences in healthy rabbits implies virus attenuation has occurred either before or, more likely, during the deliberate attempts to control the rabbit population. Alternatively, but less likely, the viruses are only virulent under defined circumstances that did not prevail in New Zealand. It is interesting to note that one liver sample produced a cDNA product from first-round PCR amplification, which indicates that there is more virus present within the liver. In our experience, only recognized lethal viruses, i.e. viruses obtained from dead or dying rabbits, have yielded PCR products on first-round PCR amplification (unpublished data). Therefore, it could be argued that this sample represents a virus related more closely to virulent RHDV. However, the fact remains that it was obtained from an apparently healthy rabbit and its sequence was not distinct from the other sequences in its subgroup. Since the livers were freeze-dried, it was not possible to confirm this by histopathological methods.
Of the 38 rabbit liver samples that contained viral RNA, all but one were related closely to the Czech strain known to have been used by the farmers who spread the virus deliberately. However, phylogenetic analysis distinguished two distinct groups. The sequences of the first group were closest to the imported Czech strain and the New Zealand strain that was sequenced in the first 3 years after the virus was introduced (O'Keefe et al., 1998). The second group was related more distantly and it appeared that the divergence point of the virus occurred before the virus was introduced into New Zealand (Fig. 2
). This could correspond to the virus that was imported from Australia, which would presumably have diverged from the original Czech strain before being introduced into New Zealand. Therefore, the two groups probably correspond to the virus introduced originally by the farmers (group 2) and the second virus that was made available by the New Zealand Government for introduction (group 1). Nevertheless, since both groups of viruses were isolated from at least two sites, it is clear that they have co-circulated in the New Zealand rabbit population. Whether or not this type of interaction has influenced the epidemiology and pathogenesis of RHDV in these rabbit populations remains to be determined.
The clustering of these virus sequences around the introduced Czech strain argues strongly against the idea that they are the original New Zealand virus that is believed to have been present before 1997. However, it is possible that the unique European strain we identified at site 6 represents the pre-1997 virus. In view of the surprising nature of this observation, we considered whether or not this sequence could have been introduced inadvertently as a laboratory contaminant in Oxford when we were analysing the samples. However, although we use a recombinant baculovirus that expresses the Spanish 89 capsid gene for our ELISA test (Marin et al., 1995), we have never knowingly worked with other regions of the genome of this virus in our laboratory. Therefore, as a further control, we amplified Spanish 89 sequence from outside the capsid gene of the New Zealand isolate. These experiments confirmed that the Spanish strain is present and circulating in New Zealand but at the present time we are unable to explain how it was introduced.
Since both the Czech and Spanish viruses have been recognized previously as virulent viruses, the presence of the RNA of either strain in healthy rabbits implies that even the most virulent RHDV may, in addition to causing lethal epidemics, also cause subclinical persistent and/or latent infections. It has been suggested that the unorthodox and unregulated introduction of the virus could have caused a sublethal dose of the virus to be administered to the rabbits (O'Keefe et al., 1999). However, there is good experimental evidence showing that either low or high input concentrations of virus are lethal in immunologically naïve adult hosts (Teifke et al., 2002
). Thus, the mechanism by which the virus enters this persistent state is as yet undetermined.
The detection of RHDV-specific RNA in the liver of healthy New Zealand rabbits known to have been infected by the released Czech virus presents us with the problem of whether or not the RNA represents infectious virus? We failed to demonstrate infectivity or an immune response following inoculation of liver suspensions from known carriers of viral RNA identified by RT-PCR sequencing. However, only five samples have been tested thus far and although the RT-PCR sequencing results have been reproduced, they are not always positive in every experiment, indicating that the levels of viral RNA are extremely low. It is, therefore, possible, that the animals we injected were given insufficient RNA to demonstrate infectivity. The presence of full-genome sequences of viral RNA indicates that the RNA may be maintained in a potentially infectious form. The area from which the rabbits were obtained showed no evidence of any RHDV epidemic at the time of collection (unpublished data). Therefore, the rabbits have either been carrying the viral RNA since the last epidemic or, as was suggested previously, there is a virus circulating that does not cause severe morbidity and mortality (Capucci et al., 1998; Chasey et al., 1997
; Rodak et al., 1990
; Trout et al., 1997
). One possible explanation is that RHDV establishes a persistent infection in rabbits. The nature of persistence of RNA viruses is still not understood. Recent work suggests that the pathology of RHDV is caused by apoptosis (Alonso et al., 1998
; Jung et al., 2000
; Ramiro-Ibanez et al., 1999
). It is likely that persistence is caused by the avoidance of this pathway, so that the cells are not destroyed and the usual pathology of RHDV is prevented. Our failure to demonstrate infectivity may indicate that the virus is replicating very slowly in the rabbits. It has been suggested that apoptosis could be triggered by the accumulation of viral proteins within the cell. However, if the virus is replicating slowly then there would be only small quantities of viral proteins in the infected cells and apoptosis would probably not be triggered, thus modulating the infection by the virus. How the virus enters this persistent state has yet to be determined. However, it appears from our results that the viral RNA is able to persist in cells for a significant period of time. Perhaps in a manner similar to some flaviviruses, the virus can lie dormant in the host cells as double-stranded RNA (i.e. replicative form) associated closely with membranes to protect it from degradative enzymes (Mackenzie et al., 1999
; Westaway et al., 1999
). Whether or not this form of RHDV can be reactivated in a manner reminiscent of that described in sheep by Takamatsu et al. (2003)
, causing a virulent outbreak of Bluetongue virus, remains to be determined. Indeed, this could explain the first reported outbreak of RHDV when apparently healthy rabbits from Germany were introduced into China (Cooke, 2002
).
Until the complete genome sequences of several of these putatively attenuated viral RNA products have been determined and compared with the assumed virulent parent viruses, it is not yet possible to identify changes in virus sequence that might determine the changes in virus phenotype. There are of course, other explanations. For example, the deliberate introduction of a virulent virus into a rapidly breeding rabbit population will inevitably result in many young rabbits being exposed to virus at a time when they are either naturally resistant to RHDV or have residual maternal antibody that could protect them against lethal infection. In either situation, these young rabbits could be exposed to released infectious virus and develop immunity that would subsequently protect them from further exposure to virulent released virus. This is entirely consistent with the detection of healthy immune domestic and wild adult rabbits in the UK dating back to 1955 that carry viral RNA virtually indistinguishable from the RNA of apparently virulent virus. Further studies to investigate RHDV persistence and latency are ongoing.
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ACKNOWLEDGEMENTS |
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Received 10 March 2003;
accepted 24 June 2003.
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