Horizontal Transmissible Protection against Myxomatosis and Rabbit Hemorrhagic Disease by Using a Recombinant Myxoma Virus

ABSTRACT We have developed a new strategy for immunization of wild rabbit populations against myxomatosis and rabbit hemorrhagic disease (RHD) that uses recombinant viruses based on a naturally attenuated field strain of myxoma virus (MV). The recombinant viruses expressed the RHDV major capsid protein (VP60) including a linear epitope tag from the transmissible gastroenteritis virus (TGEV) nucleoprotein. Following inoculation, the recombinant viruses induced specific antibody responses against MV, RHDV, and the TGEV tag. Immunization of wild rabbits by the subcutaneous and oral routes conferred protection against virulent RHDV and MV challenges. The recombinant viruses showed a limited horizontal transmission capacity, either by direct contact or in a flea-mediated process, promoting immunization of contact uninoculated animals.

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Towards a unique and transmissible vaccine against myxomatosis and rabbit haemorrhagic disease for rabbit populations

Wildlife Research ◽

10.1071/wr06160 ◽

2007 ◽

Vol 34 (7) ◽

pp. 567 ◽

Cited By ~ 31

Author(s):

Elena Angulo ◽

Juan Bárcena

Keyword(s):

Recombinant Virus ◽

Horizontal Transmission ◽

Myxoma Virus ◽

Rabbit Hemorrhagic Disease Virus ◽

Hemorrhagic Disease ◽

Wild Rabbit ◽

The European Union ◽

Vaccine Strategy ◽

Rabbit Hemorrhagic Disease ◽

Rabbit Haemorrhagic Disease

Currently available vaccines against myxomatosis and rabbit hemorrhagic disease virus (RHDV) are not suited to immunise wild rabbit populations, as vaccines need to be delivered individually by conventional veterinary practices. As an alternative approach, research in Spain has focused on the development of a transmissible vaccine. A recombinant virus has been constructed based on a naturally attenuated myxoma virus (MV) field strain, expressing the RHDV capsid protein (VP60). Following inoculation of rabbits, the recombinant virus (MV-VP60) induced specific antibody responses against MV and RHDV, conferring protection against lethal challenges with both viruses. Furthermore, the recombinant MV-VP60 virus showed a limited horizontal transmission capacity, either by direct contact or in a flea-mediated process, promoting immunisation of contact uninoculated animals. Efficacy and safety of the vaccine have been extensively evaluated under laboratory conditions and in a limited field trial. The development of the transmissible vaccine strategy and the steps being taken to obtain the marketing authorisation for the vaccine in the European Union are presented in this review.

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Punctuated evolution of myxoma virus: rapid and disjunct evolution of a recent viral lineage in Australia

10.1101/465583 ◽

2018 ◽

Cited By ~ 1

Author(s):

Peter J. Kerr ◽

John-Sebastian Eden ◽

Francesca Di Giallonardo ◽

David Peacock ◽

June Liu ◽

...

Keyword(s):

Myxoma Virus ◽

Rabbit Hemorrhagic Disease Virus ◽

Hemorrhagic Disease ◽

Partial Recovery ◽

Wild Rabbit ◽

Genome Sequences ◽

Selection Pressures ◽

Rabbit Hemorrhagic Disease ◽

Evolutionary Event ◽

European Rabbits

ABSTRACTMyxoma virus (MYXV) has been evolving in a novel host species – European rabbits – in Australia since 1950. Previous studies of viruses sampled from 1950 to 1999 revealed a remarkably clock-like evolutionary process across all Australian lineages of MYXV. Through an analysis of 49 newly generated MYXV genome sequences isolated in Australia between 2008 and 2017 we show that MYXV evolution in Australia can be characterized by three lineages, one of which exhibited a greatly elevated rate of evolutionary change and a dramatic break-down of temporal structure. Phylogenetic analysis revealed that this apparently punctuated evolutionary event occurred between 1996 and 2012. The branch leading to the rapidly evolving lineage contained a relatively high number of non-synonymous substitutions, and viruses in this lineage reversed a mutation found in the progenitor standard laboratory strain (SLS) and all previous sequences that disrupts the reading frame of theM005L/Rgene. Analysis of genes encoding proteins involved in DNA synthesis or RNA transcription did not reveal any mutations likely to cause rapid evolution. Although there was some evidence for recombination across the MYXV phylogeny, this was not associated with the increase in evolutionary rate. The period from 1996 to 2012 saw significant declines in wild rabbit numbers, due to the introduction of rabbit hemorrhagic disease and prolonged drought in south-eastern Australia, followed by the partial recovery of populations. We therefore suggest that a rapidly changing environment for virus transmission changed the selection pressures faced by MYXV and altered the course of virus evolution.IMPORTANCEThe co-evolution of myxoma virus (MYXV) and European rabbits in Australia is one of the most important natural ‘experiments’ in evolutionary biology, providing insights into virus adaptation to new hosts and the evolution of virulence. Previous studies of MYXV evolution have also shown that the virus evolves both relatively rapidly and in a strongly clock-like manner. Using newly acquired MYXV genome sequences from Australia we show that the virus has experienced a dramatic change in evolutionary behavior over the last 20 years, with a break-down in clock-like structure, the appearance of a rapidly evolving virus lineage, and the accumulation of multiple non-synonymous and indel mutations. We suggest that this punctuated evolutionary event likely reflects a change in selection pressures as rabbit numbers declined following the introduction of rabbit hemorrhagic disease virus and drought in the geographic regions inhabited by rabbits.

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Punctuated Evolution of Myxoma Virus: Rapid and Disjunct Evolution of a Recent Viral Lineage in Australia

Journal of Virology ◽

10.1128/jvi.01994-18 ◽

2019 ◽

Vol 93 (8) ◽

Cited By ~ 2

Author(s):

Peter J. Kerr ◽

John-Sebastian Eden ◽

Francesca Di Giallonardo ◽

David Peacock ◽

June Liu ◽

...

Keyword(s):

Myxoma Virus ◽

Rabbit Hemorrhagic Disease Virus ◽

Hemorrhagic Disease ◽

Partial Recovery ◽

Wild Rabbit ◽

Genome Sequences ◽

Selection Pressures ◽

Rabbit Hemorrhagic Disease ◽

Evolutionary Event ◽

European Rabbits

ABSTRACTMyxoma virus (MYXV) has been evolving in a novel host species—European rabbits—in Australia since 1950. Previous studies of viruses sampled from 1950 to 1999 revealed a remarkably clock-like evolutionary process across all Australian lineages of MYXV. Through an analysis of 49 newly generated MYXV genome sequences isolated in Australia between 2008 and 2017, we show that MYXV evolution in Australia can be characterized by three lineages, one of which exhibited a greatly elevated rate of evolutionary change and a dramatic breakdown of temporal structure. Phylogenetic analysis revealed that this apparently punctuated evolutionary event occurred between 1996 and 2012. The branch leading to the rapidly evolving lineage contained a relatively high number of nonsynonymous substitutions, and viruses in this lineage reversed a mutation found in the progenitor standard laboratory strain (SLS) and all previous sequences that disrupts the reading frame of theM005L/Rgene. Analysis of genes encoding proteins involved in DNA synthesis or RNA transcription did not reveal any mutations likely to cause rapid evolution. Although there was some evidence for recombination across the MYXV phylogeny, this was not associated with the increase in the evolutionary rate. The period from 1996 to 2012 saw significant declines in wild rabbit numbers, due to the introduction of rabbit hemorrhagic disease and prolonged drought in southeastern Australia, followed by the partial recovery of populations. It is therefore possible that a rapidly changing environment for virus transmission changed the selection pressures faced by MYXV, altering the course and pace of virus evolution.IMPORTANCEThe coevolution of myxoma virus (MYXV) and European rabbits in Australia is one of the most important natural experiments in evolutionary biology, providing insights into virus adaptation to new hosts and the evolution of virulence. Previous studies of MYXV evolution have also shown that the virus evolves both relatively rapidly and in a strongly clock-like manner. Using newly acquired MYXV genome sequences from Australia, we show that the virus has experienced a dramatic change in evolutionary behavior over the last 20 years, with a breakdown in clock-like structure, the appearance of a rapidly evolving virus lineage, and the accumulation of multiple nonsynonymous and indel mutations. We suggest that this punctuated evolutionary event may reflect a change in selection pressures as rabbit numbers declined following the introduction of rabbit hemorrhagic disease virus and drought in the geographic regions inhabited by rabbits.

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Minor structural protein VP2 in rabbit hemorrhagic disease virus downregulates the expression of the viral capsid protein VP60

Journal of General Virology ◽

10.1099/vir.0.015081-0 ◽

2009 ◽

Vol 90 (12) ◽

pp. 2952-2955 ◽

Cited By ~ 5

Author(s):

Liu Chen ◽

Guangqing Liu ◽

Zheng Ni ◽

Bin Yu ◽

Tao Yun ◽

...

Keyword(s):

Capsid Protein ◽

Disease Virus ◽

Luciferase Assay ◽

Rabbit Hemorrhagic Disease Virus ◽

Hemorrhagic Disease ◽

Mrna Levels ◽

Structural Protein ◽

Rabbit Hemorrhagic Disease ◽

Minor Structural Protein ◽

Capsid Protein Vp60

Rabbit hemorrhagic disease virus (RHDV) has two structural proteins: the major capsid protein VP60 and the minor capsid protein VP2. VP2 is speculated to play an important role in the virus life cycle. To investigate the effect of VP2 on VP60 expression, three types of experiment (baculovirus–insect cell system, mammalian–luciferase assay system and in vitro coupled transcription/translation system) were used to express VP60 alone or co-expressed with VP2. Both forms of VP60 were able to form virus-like particles in insect cells. Western blot analysis and dual-luciferase assays demonstrated that the presence of VP2 results in downregulation of the expression of VP60 in vivo. Real-time RT-PCR of mRNA levels showed that downregulation of VP60 occurs at the transcriptional level. The ability of the viral minor structural protein VP2 to regulate capsid protein levels may contribute to effective virus infection.

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Progress towards using recombinant myxoma virus as a vector for fertility control in rabbits

Reproduction Fertility and Development ◽

10.1071/r96067 ◽

1997 ◽

Vol 9 (1) ◽

pp. 77 ◽

Cited By ~ 22

Author(s):

A. J. Robinson ◽

R. Jackson ◽

P. Kerr ◽

J. Merchant ◽

I. Parer ◽

...

Keyword(s):

Recombinant Virus ◽

Fertility Control ◽

Myxoma Virus ◽

Wild Rabbit ◽

Recombinant Viruses ◽

History Of ◽

Insertion Sites ◽

Influenza Virus Haemagglutinin ◽

Double Recombinant ◽

Virus Haemagglutinin

The history of myxoma virus, its use in Australia as a mortality agent and the development of the virus as a vector for controlling fertility in wild rabbit populations in Australia is reviewed. Myxoma virus recombinants have been constructed to express model antigens. Four potential insertion sites in the genome have been identified and two have been used to construct single and double recombinant viruses expressing Escherichia colienzymes β-galactosidase and β-glucuronidase. Another recombinant expressing an influenza virus haemagglutinin gene (A/PR8/34) induced high and sustained antibody responses following intradermal inoculation in rabbits. To demonstrate the potential of introducing a recombinant virus into wild rabbit populations, a virus containing a natural deletion was released at four field locations. Preliminary analysis of the data has shown that the introduced virus spread well on 3 of the 4 locations. The steps being taken to address the ethical and safety implications of the introduction of a recombinant virus into the field are discussed.

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Recombinant Lactobacillus casei Expressing Capsid Protein VP60 can Serve as Vaccine Against Rabbit Hemorrhagic Disease Virus in Rabbits

Vaccines ◽

10.3390/vaccines7040172 ◽

2019 ◽

Vol 7 (4) ◽

pp. 172 ◽

Cited By ~ 1

Author(s):

Li Wang ◽

Tian Xia ◽

Tiantian Guo ◽

Yi Ru ◽

Yanping Jiang ◽

...

Keyword(s):

Capsid Protein ◽

Disease Virus ◽

Lactobacillus Casei ◽

Rabbit Hemorrhagic Disease Virus ◽

Economic Losses ◽

High Morbidity ◽

Hemorrhagic Disease ◽

Rabbit Hemorrhagic Disease ◽

Inactivated Vaccines ◽

Capsid Protein Vp60

Rabbit hemorrhagic disease virus (RHDV) is the causative agent of rabbit hemorrhagic disease (RHD). RHD, characterized by hemorrhaging, liver necrosis, and high morbidity and mortality in rabbits and hares, causes severe economic losses in the rabbit industry worldwide. Due to the lack of an efficient in-vitro propagation system for RHDV, the current vaccine is produced via chemical inactivation of crude RHDV preparation derived from the livers of infected rabbits. Inactivated vaccines are effective for controlling RHD, but the potential problems of biosafety and animal welfare have negative effects on the application of inactivated vaccines. In this study, an oral Lactobacillus casei (L. casei) vaccine was used as an antigen delivery system to express RHDV capsid protein VP60(VP1)-eGFP fusion protein. The expression of the recombinant protein was confirmed via western blotting and immunofluorescence (IFA). Our results indicate that oral administration of this probiotic vaccine can stimulate secretory immunoglobulin A (SIgA)-based mucosal and IgG-based humoral immune responses in rabbits. The immunized rabbits were completely protected against challenge with RHDV. Our findings indicate that the L. casei expression system is a new strategy for the development of a safe and efficient vaccine against RHDV.

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Construction and characterization of recombinant porcine adenovirus serotype 5 expressing the transmissible gastroenteritis virus spike gene

Journal of General Virology ◽

10.1099/0022-1317-82-1-183 ◽

2001 ◽

Vol 82 (1) ◽

pp. 183-190 ◽

Cited By ~ 45

Author(s):

Tamás Tuboly ◽

Éva Nagy

Keyword(s):

Clinical Signs ◽

Secretory Iga ◽

Transmissible Gastroenteritis Virus ◽

Recombinant Viruses ◽

S Gene ◽

Gastroenteritis Virus ◽

A Genome ◽

Serotype 5 ◽

Transmissible Gastroenteritis ◽

E3 Region

Five recombinant porcine adenoviruses of serotype 5 (PAdV-5) carrying the full-length or the 5′ 2·2 kb half of the transmissible gastroenteritis virus (TGEV) spike (S) gene were generated by homologous recombination in E. coli strain BJ5183 cells and subsequent transfection of swine testicle cells. The foreign genes were inserted into the E3 region of PAdV-5. One recombinant virus had no deletion in the E3 region, whereas a 1·2 kb fragment was removed from the E3 region in the remainder of the recombinant viruses. One stable construct with a 4·4 kb insertion had a genome size of 109·6% of the wild-type genome, the largest reported for any recombinant adenovirus. Only those viruses that carried the S gene in the left to right orientation expressed the S gene. Three recombinant viruses were tested by oral immunization of pigs and both antibody response and virus shedding were monitored. None of the pigs showed clinical signs and the virus was recovered from rectal swabs until 6–7 days post-infection. Viruses expressing the S gene induced TGEV- and PAdV-5-specific virus-neutralizing antibodies. Moreover, TGEV-specific secretory IgA was detected in the small intestine and in the lungs of the immunized animals.

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Long-term decline of the European wild rabbit (Oryctolagus cuniculus) in south-western Spain

Wildlife Research ◽

10.1071/wr06142 ◽

2007 ◽

Vol 34 (8) ◽

pp. 652 ◽

Cited By ~ 59

Author(s):

Sacramento Moreno ◽

Juan F. Beltrán ◽

Irene Cotilla ◽

Beatriz Kuffner ◽

Rafael Laffite ◽

...

Keyword(s):

Oryctolagus Cuniculus ◽

Population Trends ◽

Hemorrhagic Disease ◽

Lynx Lynx ◽

Wild Rabbit ◽

Doñana National Park ◽

Lynx Pardinus ◽

Rabbit Hemorrhagic Disease ◽

Rabbit Population ◽

Important Prey

The European wild rabbit (Oryctolagus cuniculus) is a species native to the Iberian Peninsula, where it was once extremely abundant. It is considered the most important prey item for the peninsula’s assemblage of Mediterranean vertebrate predators, which includes two endangered specialist rabbit feeders, the Spanish imperial eagle (Aquila adalberti) and the Iberian lynx (Lynx pardinus). However, rabbit population trends in Spain have not been accurately documented. In the present study, we analysed trends in a population of European rabbits monitored over 23 years in the Doñana National Park, home to one of the most diverse and densest predator communities in Europe. Rabbit abundance and population trends were estimated using roadside counts. Results show that the rabbit population declined sharply by ~60% during the first wave of epizootic rabbit hemorrhagic disease (RHD) in 1990. Since then, rabbit numbers have declined at a relatively constant rate and the species has become progressively scarcer in the area. The current population is less than 10% of that before the arrival of RHD. However, after the RHD epizootic we observed increasing intra-annual population recruitment. We hypothesise that density-dependent factors caused by enzootic viral diseases (myxomatosis, RHD) and higher predation of rabbits are the main factors preventing recovery of rabbit numbers. The effects of a decline in the prey species on the ecology of sympatric rabbit predators are discussed, and measures to improve ongoing rabbit conservation efforts are suggested.

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Rabbit Hemorrhagic Disease Virus 2 (RHDV2; GI.2) Is Replacing Endemic Strains of RHDV in the Australian Landscape within 18 Months of Its Arrival

Journal of Virology ◽

10.1128/jvi.01374-17 ◽

2017 ◽

Vol 92 (2) ◽

Cited By ~ 26

Author(s):

Jackie E. Mahar ◽

Robyn N. Hall ◽

David Peacock ◽

John Kovaliski ◽

Melissa Piper ◽

...

Keyword(s):

Disease Virus ◽

Rabbit Hemorrhagic Disease Virus ◽

Effective Vaccine ◽

Hemorrhagic Disease ◽

Wild Rabbit ◽

New Host ◽

Content Type ◽

Rabbit Hemorrhagic Disease ◽

Initial Description ◽

Ongoing Surveillance

ABSTRACTRabbit hemorrhagic disease virus 2(RHDV2;LagovirusGI.2) is a pathogenic calicivirus that affects European rabbits (Oryctolagus cuniculus) and various hare (Lepus) species. GI.2 was first detected in France in 2010 and subsequently caused epidemics in wild and domestic lagomorph populations throughout Europe. In May 2015, GI.2 was detected in Australia. Within 18 months of its initial detection, GI.2 had spread to all Australian states and territories and rapidly became the dominant circulating strain, replacingRabbit hemorrhagic disease virus(RHDV/GI.1) in mainland Australia. Reconstruction of the evolutionary history of 127 Australian GI.2 isolates revealed that the virus arrived in Australia at least several months before its initial description and likely circulated unnoticed in wild rabbit populations in the east of the continent prior to its detection. GI.2 sequences isolated from five hares clustered with sequences from sympatric rabbit populations sampled contemporaneously, indicating multiple spillover events into hares rather than an adaptation of the Australian GI.2 to a new host. Since the presence of GI.2 in Australia may have wide-ranging consequences for rabbit biocontrol, particularly with the release of the novel biocontrol agent GI.1a/RHDVa-K5 in March 2017, ongoing surveillance is critical to understanding the interactions of the various lagoviruses in Australia and their impact on host populations.IMPORTANCEThis study describes the spread and distribution ofRabbit hemorrhagic disease virus 2(GI.2) in Australia since its first detection in May 2015. Within the first 18 months following its detection, RHDV2 spread from east to west across the continent and became the dominant strain in all mainland states of Australia. This has important implications for pest animal management and for owners of pet and farmed rabbits, as there currently is no effective vaccine available in Australia for GI.2. The closely related RHDV (GI.1) is used to control overabundant wild rabbits, a serious environmental and agricultural pest in this country, and it is currently unclear how the widespread circulation of GI.2 will impact ongoing targeted wild rabbit management operations.

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