Safety evaluation of a recombinant myxoma-RHDV virus inducing horizontal transmissible protection against myxomatosis and rabbit haemorrhagic disease

We have recently developed a transmissible vaccine to immunize rabbits against myxomatosis and rabbit haemorrhagic disease based on a recombinant myxoma virus (MV) expressing the rabbit haemorrhagic disease virus (RHDV) capsid protein [Bárcena et al. Horizontal transmissible protection against myxomatosis and rabbit haemorragic disease using a recombinant myxoma virus. J. Virol. 2000;74:1114-23]. Administration of the recombinant virus protects rabbits against lethal RHDV and MV challenges. Furthermore, the recombinant virus is capable of horizontal spreading promoting protection of contact animals, thus providing the opportunity to immunize wild rabbit populations. However, potential risks must be extensively evaluated before considering its field use. In this study several safety issues concerning the proposed vaccine have been evaluated under laboratory conditions. Results indicated that vaccine administration is safe even at a 100-fold overdose. No undesirable effects were detected upon administration to immunosuppressed or pregnant rabbits. The recombinant virus maintained its attenuated phenotype after 10 passages in vivo.     

Characterization of protective humoral and cellular immune responses against RHDV2 induced by a new vaccine based on recombinant baculovirus

Rabbit hemorrhagic disease (RHD) is a lethal disease in rabbits caused by RHD virus (RHDV). Protection is only possible through vaccination. A new virus variant (RHDV2) which emerged in 2010 in France differed from the classical RHDV1 variant in certain aspects and vaccines against RHDV1 induced limited cross protection only. In a previous study, we designed a recombinant baculovirus based RHDV2-VP1 vaccine, which provided a protective immunity in rabbits against RHDV2. In the present study this newly created vaccine is characterized with regard to onset and duration of protection, and possible cross protection against classical RHDV1. Furthermore, humoral and cellular immune mechanisms in vaccinated and infected rabbits were analyzed. In all experiments, the recombinant vaccine was compared to a conventional liver-based RHDV2 vaccine.The RHDV2-VP1 vaccine induced a protective immune response already seven days after single vaccination and fully protected for at least 14 months. A booster vaccination 21 days after the first had a negative influence on long-term protection. The cross protection provided by the RHDV2-VP1 vaccine against classical RHDV1 was limited since only 50% of vaccinated rabbits survived the infection. Conclusively, the new, baculovirus-based RHDV2-VP1 vaccine has the potential to protect rabbits against the infection with RHDV2, blocks completely the disease progression and prevents the spread of RHDV2 at the population level.     

Molecular characterization of SG33 and Borghi vaccines used against myxomatosis

Myxoma virus is a poxvirus responsible for myxomatosis in European Rabbits (Oryctolagus cuniculus). The entire genome of the myxoma virus has been sequenced, allowing a systemic survey of the functions of a large number of putative pathogenic factors that this virus expresses to subvert the immune and inflammatory pathways of infected rabbit hosts. In Italy, industrial rabbits are mostly vaccinated against myxomatosis using the attenuated myxoma virus strains Borghi or SG33. We have identified genetic markers specific for Borghi or SG33 vaccine strains and established a PCR-based assay that could be used to: (a) rapidly diagnose the presence of myxoma virus in infected organs; (b) discriminate between field strain-infected and vaccinated rabbits and (c) differentiate between Borghi or SG33 vaccine strain.     

A new recombinant Orf virus (ORFV, Parapoxvirus) protects rabbits against lethal infection with rabbit hemorrhagic disease virus (RHDV)

This report describes the generation of a new recombinant Orf virus (ORFV; Parapoxvirus) expressing the major capsid protein VP1 (VP60) of the calicivirus, rabbit hemorrhagic disease virus (RHDV). Authentic expression of VP1 could be demonstrated in cells infected with the recombinant D1701-V-VP1 without the need for production of infectious ORFV progeny. Notably, infected cells also released empty calicivirus-like particles (VLPs). Challenge experiments showed that even a single immunization with ≥10⁵ PFU of D1701-V-VP1 protected rabbits against lethal RHDV infection. ELISA tests indicated that the protective immunity mediated by D1701-V-VP1 did not strictly depend on the presence of detectable RHDV-specific serum antibodies. The induction of interleukin-2 found only in the sera of rabbits immunized with the D1701-V-VP1, but not in sera of rabbits immunized with the inactivated commercial vaccine RIKA-VACC, might indicate also some involvement of T-cells in protection. Collectively, this work adds another example of the successful use of the ORFV vector system for the generation of a recombinant vaccine, and demonstrates its potential as an alternative vaccine to protect rabbits against RHDV infection.     

Limitations of plasmid vaccines to complex viruses: selected myxoma virus antigens as DNA vaccines were not protective

Myxoma virus, a poxvirus of the genus Leporipoxvirus, is the causative agent of the disease myxomatosis which is highly lethal in European rabbits (Oryctolagus cuniculus). Current vaccines to protect against myxomatosis are either attenuated live strains of the virus or the antigenically related rabbit fibroma virus. We examined the immune response of outbred domestic rabbits to the individual myxoma virus antigens M055R, M073R, M115L and M121R, delivered as DNA vaccines co-expressing rabbit interleukin-2 or interleukin-4. M115L and M121R were also delivered simultaneously. None of the vaccine constructs were able to protect the rabbits from disease or reduce mortality after challenge with virulent myxoma virus, despite induction of antigen-specific cell-mediated and humoral immune responses.     

Exposure of rabbits to ultraviolet light-inactivated rabbit haemorrhagic disease virus (RHDV) and subsequent challenge with virulent virus

This study investigated whether exposure to inactivated rabbit haemorrhagic disease virus (RHDV) can produce an antigenic response in rabbits and protect them from a subsequent challenge with virulent virus. The aim was to determine if the spreading of baits containing RHDV, which is a common management practice in New Zealand to reduce rabbit numbers, could result in protective immunity in wild rabbits. RHDV was inactivated by ultraviolet (UV) light using an electronic UV crosslinker with a UV dose of 168.48 W-s/cm2 and a UV intensity of 0.0078 W/cm2. Two groups of four rabbits were then inoculated with inactivated virus via oral and intramuscular routes. Rabbits were monitored for 30 days post-inoculation and then challenged orally with virulent virus. No rabbit exposed to inactivated RHDV developed clinical signs of RHD or had antibodies at day 30 post-infection and all animals died within 82 h after challenge with virulent virus. No antibodies were detected at the time of death. These findings suggest that exposure to virus completely inactivated by UV light in the field or on baits will not protect rabbits against challenge with virulent virus.     

Limitations of plasmid vaccines to complex viruses: selected myxoma virus antigens as DNA vaccines were not protective

Myxoma virus, a poxvirus of the genus Leporipoxvirus, is the causative agent of the disease myxomatosis which is highly lethal in European rabbits (Oryctolagus cuniculus). Current vaccines to protect against myxomatosis are either attenuated live strains of the virus or the antigenically related rabbit fibroma virus. We examined the immune response of outbred domestic rabbits to the individual myxoma virus antigens M055R, M073R, M115L and M121R, delivered as DNA vaccines co-expressing rabbit interleukin-2 or interleukin-4. M115L and M121R were also delivered simultaneously. None of the vaccine constructs were able to protect the rabbits from disease or reduce mortality after challenge with virulent myxoma virus, despite induction of antigen-specific cell-mediated and humoral immune responses.     

Myxomatosis: population dynamics of rabbits (Oryctolagus cuniculus Linnaeus, 1758) and ecological effects in the United Kingdom.

In 1953-1955, myxomatosis spread among rabbits (Oryctolagus cuniculus) in the United Kingdom, causing 99% mortality. Subsequently, there was a gradual increase in rabbit numbers. By 1955, the Ministry of Agriculture, Fisheries and Food (MAFF) had already found attenuated strains of myxoma virus. By 1970, genetic resistance had appeared. In the 1970s, mortality declined to 47-69% with only approximately 25% of rabbits infected, giving a field mortality of 12-19%. However, myxomatosis is persistent, generally showing a major prevalence peak in autumn and often a minor peak in spring. An eight-year MAFF experiment in which prevalence of the disease was artificially reduced indicates that myxomatosis remains a significant factor in population regulation. After rabbit numbers fell in the 1950s, important ecological changes took place: vegetation altered due to reduced grazing pressure, predators were affected by the reduction of a major prey species and these changes also affected many other animals. Currently, rabbit numbers have returned to approximately one-third of pre-myxomatosis levels and this is causing damage to farm and conservation habitats.     

Field experimental vaccination campaigns against myxomatosis and their effectiveness in the wild

We conducted a field experiment in SW Spain to test the efficacy of a myxomatosis vaccine, a viral disease strongly affecting wild rabbit populations, by assessing individual survival and antibody seroprevalence of monthly live-trapped, vaccinated (N = 466) and unvaccinated (N = 558) juvenile wild rabbits, between April and October 2007. Eight percent of all juveniles caught from April to June showed maternal antibodies against myxomatosis, whereas all animals were seropositive to the disease after the outbreak. Juveniles vaccinated before the outbreak showed 17% higher survival (31% vs. 14%) and an increased mortality probability of 8% after the outbreak. Results suggest that only a costly and systematic vaccination performed before the annual myxomatosis outbreak, would improve the survival of juvenile rabbits, a premise not always accomplished that compromises its efficacy in the field.     

Novel Calicivirus Identified in Rabbits, Michigan, USA

We report a disease outbreak in a Michigan rabbitry of a rabbit calicivirus distinct from the foreign animal disease agent, rabbit hemorrhagic disease virus (RHDV). The novel virus has been designated Michigan rabbit calicivirus (MRCV). Caliciviruses of the Lagovirus genus other than RHDV have not been described in US rabbit populations. The case-fatality rate was 32.5% (65/200). Clinical signs included hemorrhage and sudden death, with hepatic necrosis. Analysis of viral RNA sequence from >95% of the viral genome showed an average similarity of 79% with RHDV. Similarity of the predicted MRCV capsid amino acid sequence ranged from 89.8% to 91.3%, much lower than the 98% amino acid similarity between RHDV strains. Experimentally infected rabbits lacked clinical disease, but MRCV was detected in tissues by PCR. We propose that MRCV primarily causes subclinical infection but may induce overt RHD-like disease under certain field conditions.     

Monitoring the spread of myxoma virus in rabbit Oryctolagus cuniculus populations on the southern tablelands of New South Wales,Australia. III. Release,persistence and rate of spread of an identifiable strain of myxoma virus

An identifiable strain of myxoma virus was introduced into four local populations of wild rabbits Oryctolagus cuniculus on the southern tablelands of New South Wales (NSW) and its spread in the presence of other field strains was monitored for 6 months. The main vector in this region was considered to be the European rabbit flea Spilopsyllis cuniculi. Each population of rabbits was of a high density and living in groups of warrens covering areas from 59 to 87 hectares. Rabbits occupying centrally located warrens were inoculated with the virus in late September or early October (spring) and the subsequent appearance of myxomatosis across the sites monitored by trapping, shooting and visual observations. Samples, taken from rabbits with myxomatosis, were examined by polymerase chain reaction (PCR) that allowed identification of the introduced strain. On all four sites the introduced virus spread from the inoculated rabbits in the centrally located warrens to rabbits in surrounding warrens. On Sites 1 and 3, this spread continued across the entire site persisting for at least 118 and 174 days respectively. On Sites 2 and 4, the virus was detected for 78 and 62 days respectively and the subsequent inability to detect the introduced virus correlated with the appearance of an unrelated field strain. Using three different methods of calculation, rates of spread ranged from 3.7 to 17.8 m d(-1).     

Survival of rabbit haemorrhagic disease virus (RHDV) in the environment

A study was conducted to investigate the persistence of rabbit haemorrhagic disease virus (RHDV) in the environment. Virus was impregnated onto two carrier materials (cotton tape and bovine liver) and exposed to environmental conditions on pasture during autumn in New Zealand. Samples were collected after 1, 10, 44 and 91 days and the viability of the virus was determined by oral inoculation of susceptible 11- to 14-week-old New Zealand White rabbits. Evidence of RHDV infection was based on clinical and pathological signs and/or seroconversion to RHDV. Virus impregnated on cotton tape was viable at 10 days of exposure but not at 44 days, while in bovine liver it was still viable at 91 days. The results of this study suggest that RHDV in animal tissues such as rabbit carcasses can survive for at least 3 months in the field, while virus exposed directly to environmental conditions, such as dried excreted virus, is viable for a period of less than 1 month. Survival of RHDV in the tissues of dead animals could, therefore, provide a persistent reservoir of virus, which could initiate new outbreaks of disease after extended delays.     

Detection of rabbit haemorrhagic disease virus (RHDV) in nonspecific vertebrate hosts sympatric to the European wild rabbit (Oryctolagus cuniculus)

Since its detection in China in 1984, rabbit haemorrhagic disease (RHD) has been the subject of numerous studies. Yet, the evolutionary origin of rabbit haemorrhagic disease virus (RHDV) is still under debate. For example, some aspects related to the epidemiology of the disease are still unknown, such as where the virus is hosted between RHD outbreaks. To detect the presence of RHDV in rabbit-sympatric micromammals, 51 rodents (29 Mus spretus and 22 Apodemus sylvaticus) and 31 rabbits (Oryctolagus cuniculus) from the same location in central Spain were analyzed. In those samples in which the virus was detected, a fragment of the VP60 protein gene from the RHDV capsid was sequenced and the phylogenetic relationships between them and other strains of RHDV in the Iberian Peninsula were analyzed. In total, five viral strains were identified in A. sylvaticus, M. spretus and O. cuniculus. All strains were found to be well supported within the clade of RHDV found in rabbits in the Iberian Peninsula. Moreover, one of the strains was found in all three species under study, which suggests the capability of RHDV to infect other mammals apart from the rabbit which have not yet been investigated. The transmission of the virus is discussed as well as its ecoepidemiological implications.     

Emerging rabbit haemorrhagic disease virus 2 (RHDV2) at the gates of the African continent

Until the beginning of this decade, the genetic characterization of rabbit haemorrhagic disease virus (RHDV) from Iberian Peninsula had revealed the existence of two genogroups, G1 and sporadically G6. In 2010, the new emerging rabbit haemorrhagic disease variant, RHDV2 or RHDVb, was described in France, from where it has rapidly spread throughout Europe, including Iberian Peninsula countries. Nevertheless, although cases of rabbit haemorrhagic disease (RHD) have been reported in the Canary Islands, a Spanish archipelago located 100 km off the coast of Morocco, no genetic characterization of RHDV had been carried out. Consequently, in order to identify the circulating RHDV strains in this archipelago, liver samples of six farm rabbits and fifteen wild rabbits were collected from several areas of the largest island, Tenerife, and analyzed for the presence of RHDV by antigen capture double antibody sandwich ELISA. In case of positive ELISA result, we amplified and sequenced two fragments of the vp60 gene, which were concatenated for phylogenetic purposes. The sequences analysis revealed the presence of RHDV2 in both farm and wild rabbits from several areas of Tenerife. This result constitutes the first finding of RHDV2 in the Canary Islands. These RHDV2 strains found in Tenerife shared two exclusive SNPs that have not been observed in the rest of RHDV2 strains. The identification of RHDV2 and the absence of classic RHDV strains in this study suggest that RHDV2 may be replacing classic strains in Tenerife, as has been also proposed in Iberian Peninsula, France and Azores. Given the proximity of the Canary Islands to the African continent, this result should raise awareness about a possible dispersal of RHDV2 from the Canary Islands to the North of Africa.     

Protection of rabbits against cutaneous papillomavirus infection using recombinant tobacco mosaic virus containing L2 capsid epitopes

Cottontail rabbit papillomavirus (CRPV) and rabbit oral papillomavirus (ROPV) represent distantly related, cutaneous and mucosal tissue tropic papillomaviruses respectively that can infect the same host. These two viruses were used to test the effectiveness of an L2 peptide-based vaccine (aa 94-122) that was delivered on the surface of recombinant tobacco mosaic virus (rTMV) particles. Groups of NZW rabbits received combinations of CRPVL2, ROPVL2 and CRPV+ROPVL2 rTMV vaccines, and were then challenged with infectious CRPV and ROPV. The rabbits developed antibodies that reacted to whole L2 protein and these sera were able to neutralize CRPV pseudovirions at half-maximal titers that were between 50 and 500. Rabbits receiving the CRPV L2 vaccine alone or in combination with ROPV L2 vaccines were completely protected against CRPV infections. Those rabbits vaccinated with the ROPV L2 vaccines showed a weak response in some rabbits against CRPV infection. These studies demonstrate that L2-based vaccines provide strong protection against experimental papillomavirus infection that is most likely based upon the induction of virus-neutralizing antibody. Notably, we observed some limited cross-protection induced by the L2 sequences tested in these vaccines. Finally, the study demonstrated that rTMV were excellent agents for the induction of strong protection in a pre-clinical disease model of papillomavirus infection.     

Myxomatosis in farmland rabbit populations in England and Wales

The overall pattern and consequences of myxomatosis in wild rabbit populations were studied at three farmland sites in lowland southern England and upland central Wales between 1971 and 1978. When results from all years were combined, the disease showed a clear two-peaked annual cycle, with a main autumn peak between August and January, and a subsidiary spring peak during February to April. Rabbit fleas, the main vectors of myxomatosis in Britain, were present on full-grown rabbits in sufficient numbers for transmission to occur throughout the year, but the observed seasonal pattern of the disease appeared to be influenced by seasonal mass movements of these fleas. However other factors were also important including the timing and success of the main rabbit breeding season, the proportion of rabbits which had recovered from the disease and the timing and extent of autumn rabbit mortality from other causes. Significantly more males than females, and more adults and immatures than juveniles, were observed to be infected by myxomatosis. Only 25-27% of the total populations were seen to be infected during outbreaks. Using two independent methods of calculation, it was estimated that between 47 and 69% of infected rabbits died from the disease (much lower than the expected 90-95% for fully susceptible rabbits with the partly attenuated virus strains that predominated). Thus it was estimated that 12-19% of the total rabbit populations were known to have died directly or indirectly from myxomatosis. Although the effects of myxomatosis were much less than during the 1950s and 1960s, it continued to be an important mortality factor. It may still have a regulatory effect on rabbit numbers, with autumn/winter peaks of disease reducing the numbers of rabbits present at the start of the breeding season.     

Monitoring the spread of myxoma virus in rabbit Oryctolagus cuniculus populations on the southern tablelands of New South Wales,Australia. I. Natural occurrence of myxomatosis

A survey of rabbit populations in the southern tablelands of New South Wales, Australia, was carried out to establish the pattern of occurrence of myxomatosis in preparation for a deliberate release of myxoma virus. Myxomatosis was first detected in December and cases were found on most sites through to May. The serological profiles of rabbit populations suggested that their susceptibility to myxoma virus was generally low in winter and highest in spring and summer reflecting the presence of increasing numbers of susceptible young rabbits. This was consistent with the pattern of rabbit breeding, as determined from the distribution of births and reproductive activity in females and males, which occurred maximally in spring and early summer. The serology and age structure of rabbit populations on sites suggested that some rabbit populations can escape an annual myxomatosis epizootic. Although fleas were present on rabbits throughout the year and therefore not considered to be a limiting factor in the spread of myxomatosis, their numbers peaked at times coincident with peak rabbit breeding. It was concluded that mid to late spring was an optimal time for a deliberate release.     

Monitoring the spread of myxoma virus in rabbit Oryctolagus cuniculus populations on the southern tablelands of New South Wales,Australia. II. Selection of a strain of virus for release

To be able to study the dynamics of myxoma virus spread following a release in the field, a strain of virus is required that is both highly transmissible and readily differentiated from other field strains. Eight strains of virus of known virulence for laboratory rabbits and with previously mapped and sequenced restriction fragment length polymorphisms, were used to infect groups of seronegative wild rabbits. Based on these trials, and on the nature of the DNA polymorphism, a virus designated Brooklands/2-93 was chosen as a strain suitable for experimental release. These trials confirmed that resistance to myxomatosis within wild rabbit populations continues to be substantial and that some rabbits are highly resistant. These rabbits probably have little role in transmission of virus. Most of the virus strains tested induced very small or invisible primary lesions at the inoculation site. Thus the secondary skin sites such as eyelids, face and ears may be critical for transmission.     

The development of genetic resistance to myxomatosis in wild rabbits in Britain.

The presence of genetic resistance to myxomatosis in a sample of wild rabbits from one area in England was reported in 1977. Rabbits from three other areas in Great Britain have been tested subsequently, and all cases showed similar resistance to a moderately virulent strain of myxoma virus. Rabbits from one area also showed a significant degree of resistance to a fully virulent strain of virus. It is concluded that genetic resistance to myxomatosis is widespread in wild rabbit populations in Britain. The implications of the results are discussed in relation to the co-evolution of the disease and its host.     

The development of genetic resistance to myxomatosis in wild rabbits in Britain

The presence of genetic resistance to myxomatosis in a sample of wild rabbits from one area in England was reported in 1977. Rabbits from three other areas in Great Britain have been tested subsequently, and all cases showed similar resistance to a moderately virulent strain of myxoma virus. Rabbits from one area also showed a significant degree of resistance to a fully virulent strain of virus. It is concluded that genetic resistance to myxomatosis is widespread in wild rabbit populations in Britain. The implications of the results are discussed in relation to the co-evolution of the disease and its host.