First Detection and Circulation of RHDV2 in New Zealand.

Rabbit haemorrhage disease virus 2 (RHDV2) is a highly pathogenic lagovirus that causes lethal disease in rabbits and hares (lagomorphs). Since its first detection in Europe in 2010, RHDV2 has spread worldwide and has been detected in over 35 countries so far. Here, we provide the first detailed report of the detection and subsequent circulation of RHDV2 in New Zealand. RHDV2 was first detected in New Zealand in 2018, with positive samples retrospectively identified in December 2017. Subsequent time-resolved phylogenetic analysis suggested a single introduction into the North Island between March and November 2016. Genetic analysis identified a GI.3P-GI.2 variant supporting a non-Australian origin for the incursion; however, more accurate identification of the source of the incursion remains challenging due to the wide global distribution of the GI.3P-GI.2 variant. Furthermore, our analysis suggests the spread of the virus between the North and South Islands of New Zealand at least twice, dated to mid-2017 and around 2018. Further phylogenetic analysis also revealed a strong phylogeographic pattern. So far, no recombination events with endemic benign New Zealand rabbit caliciviruses have been identified. This study highlights the need for further research and surveillance to monitor the distribution and diversity of lagoviruses in New Zealand and to detect incursions of novel variants.

A single introduction of wild rabbits triggered the biological invasion of Australia.

Biological invasions are a major cause of environmental and economic disruption. While ecological factors are key determinants of their success, the role of genetics has been more challenging to demonstrate. The colonization of Australia by the European rabbit is one of the most iconic and devastating biological invasions in recorded history. Here, we show that despite numerous introductions over a 70-y period, this invasion was triggered by a single release of a few animals that spread thousands of kilometers across the continent. We found genetic support for historical accounts that these were English rabbits imported in 1859 by a settler named Thomas Austin and traced the origin of the invasive population back to his birthplace in England. We also find evidence of additional introductions that established local populations but have not spread geographically. Combining genomic and historical data we show that, contrary to the earlier introductions, which consisted mostly of domestic animals, the invasive rabbits had wild ancestry. In New Zealand and Tasmania, rabbits also became a pest several decades after being introduced. We argue that the common denominator of these invasions was the arrival of a new genotype that was better adapted to the natural environment. These findings demonstrate how the genetic composition of invasive individuals can determine the success of an introduction and provide a mechanism by which multiple introductions can be required for a biological invasion.

Benign Rabbit Calicivirus in New Zealand.

The Czech v351 strain of rabbit hemorrhagic disease virus (RHDV1) is used in Australia and New Zealand as a biological control agent for rabbits, which are important and damaging introduced vertebrate pests in these countries. However, nonpathogenic rabbit caliciviruses (RCVs) can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with effective rabbit biocontrol. Antibodies that cross-reacted against RHDV antigens were found in wild rabbits before the release of RHDV1 in New Zealand in 1997, suggesting that nonpathogenic RCVs were already present in New Zealand. The aim of this study was to confirm the presence of nonpathogenic RCV in New Zealand and describe its geographical distribution. RCV and RHDV antibody assays were used to screen serum samples from 350 wild rabbits from 14 locations in New Zealand. The serological survey indicated that both RCV and RHDV are widespread in New Zealand wild rabbits, with antibodies detected in 10 out of 14 and 12 out of 14 populations, respectively. Two closely related RCV strains were identified in the duodenal tissue from a New Zealand wild rabbit (RCV Gore-425A and RCV Gore-425B). Both variants are most closely related to Australian RCV strains, but with 88% nucleotide identity, they are genetically distinct. Phylogenetic analysis revealed that the New Zealand RCV strains fall within the genetic diversity of the Australian RCV isolates, indicating a relatively recent movement of RCVs between Australia and New Zealand.IMPORTANCE Wild rabbits are important and damaging introduced vertebrate pests in Australia and New Zealand. Although RHDV1 is used as a biological control agent, some nonpathogenic RCVs can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with its effectiveness for rabbit control. The presence of nonpathogenic RCVs in New Zealand wild rabbits has been long hypothesized, but earlier attempts to isolate a New Zealand RCV strain have been unsuccessful. Therefore, it is important to determine if such nonpathogenic viruses exist in New Zealand rabbits, especially considering the proposed introduction of new RHDV strains into New Zealand as biocontrols.

Resolving the Origin of Rabbit Hemorrhagic Disease Virus: Insights from an Investigation of the Viral Stocks Released in Australia.

To resolve the evolutionary history of rabbit hemorrhagic disease virus (RHDV), we performed a genomic analysis of the viral stocks imported and released as a biocontrol measure in Australia, as well as a global phylogenetic analysis. Importantly, conflicts were identified between the sequences determined here and those previously published that may have affected evolutionary rate estimates. By removing likely erroneous sequences, we show that RHDV emerged only shortly before its initial description in China.

Comparative Phylodynamics of Rabbit Hemorrhagic Disease Virus in Australia and New Zealand.

UNLABELLED: The introduction of rabbit hemorrhagic disease virus (RHDV) into Australia and New Zealand during the 1990s as a means of controlling feral rabbits is an important case study in viral emergence. Both epidemics are exceptional in that the founder viruses share an origin and the timing of their release is known, providing a unique opportunity to compare the evolution of a single virus in distinct naive populations. We examined the evolution and spread of RHDV in Australia and New Zealand through a genome-wide evolutionary analysis, including data from 28 newly sequenced RHDV field isolates. Following the release of the Australian inoculum strain into New Zealand, no subsequent mixing of the populations occurred, with viruses from both countries forming distinct groups. Strikingly, the rate of evolution in the capsid gene was higher in the Australian viruses than in those from New Zealand, most likely due to the presence of transient deleterious mutations in the former. However, estimates of both substitution rates and population dynamics were strongly sample dependent, such that small changes in sample composition had an important impact on evolutionary parameters. Phylogeographic analysis revealed a clear spatial structure in the Australian RHDV strains, with a major division between those viruses from western and eastern states. Importantly, RHDV sequences from the state where the virus was first released, South Australia, had the greatest diversity and were diffuse throughout both geographic lineages, such that this region was likely a source population for the subsequent spread of the virus across the country. IMPORTANCE: Most studies of viral emergence lack detailed knowledge about which strains were founders for the outbreak or when these events occurred. Hence, the human-mediated introduction of rabbit hemorrhagic disease virus (RHDV) into Australia and New Zealand from known starting stocks provides a unique opportunity to understand viral evolution and emergence. Within Australia, we revealed a major phylogenetic division between viruses sampled from the east and west of the country, with both regions likely seeded by viruses from South Australia. Despite their common origins, marked differences in evolutionary rates were observed between the Australian and New Zealand RHDV, which led to conflicting conclusions about population growth rates. An analysis of mutational patterns suggested that evolutionary rates have been elevated in the Australian viruses, at least in part due to the presence of low-fitness (deleterious) variants that have yet to be selectively purged.

Vaccinia virus as a vaccine delivery system for marsupial wildlife.

Vaccines based on recombinant poxviruses have proved successful in controlling diseases such as rabies and plague in wild eutherian mammals. They have also been trialled experimentally as delivery agents for fertility-control vaccines in rodents and foxes. In some countries, marsupial mammals represent a wildlife disease reservoir or a threat to conservation values but, as yet there has been no bespoke study of efficacy or immunogenicity of a poxvirus-based vaccine delivery system in a marsupial. Here, we report a study of the potential for vaccination using vaccinia virus in the Australian brushtail possum Trichosurus vulpecula, an introduced pest species in New Zealand. Parent-strain vaccinia virus (Lister) infected 8/8 possums following delivery of virus to the oral cavity and outer nares surfaces (oronasal immunisation), and persisted in the mucosal epithelium around the palatine tonsils for up to 2 weeks post-exposure. A recombinant vaccinia virus construct (VV399, which expresses the Eg95 antigen of the hydatid disease parasite Echinococcus granulosus) was shown to infect 10/15 possums after a single-dose oronasal delivery and to also persist. Both parent vaccinia virus and the VV399 construct virus induced peripheral blood lymphocyte reactivity against viral antigens in possums, first apparent at 4 weeks post-exposure and still detectable at 4 months post-exposure. Serum antibody reactivity to Eg95 was recorded in 7/8 possums which received a single dose of the VV399 construct and 7/7 animals which received triple-dose delivery, with titre end-points in the latter case exceeding 1/4000 dilution. This study demonstrates that vaccinia virus will readily infect possums via a delivery means used to deploy wildlife vaccines, and in doing is capable of generating immune reactivity against viral and heterologous antigens. This highlights the future potential of recombinant vaccinia virus as a vaccine delivery system in marsupial wildlife.

Humoral immune responses in brushtail possums (Trichosurus vulpecula) induced by bacterial ghosts expressing possum zona pellucida 3 protein.

The introduced common brushtail possum (Trichosurus vulpecula) is a major pest in New Zealand and immunocontraceptive vaccines are being developed for biocontrol of possum populations, with bacterial ghosts (BGs) being evaluated as a means of oral delivery. Recombinant BGs expressing possum zona pellucida 3 protein (ZP3) as an L' membrane-anchored protein (ZP3-L') or as an S-layer SbsA-fusion protein (MBP-SbsA-ZP3) were produced by the expression of the cloned bacteriophage phiX174 lysis gene E in E. coli NM522. The humoral immune responses of possums immunised with BGs expressing possum ZP3 were investigated following oral, intranasal/conjunctival, parenteral, and intraduodenal administration to evaluate the BG-ZP3 system for possum fertility control. Antibodies to possum ZP3 were detected in the serum, oviduct secretions, and follicular fluid of immunised animals. Intranasal/conjunctival immunisation elicited reliable antibody immune response in serum and at a key effector site, the ovarian follicular fluid. Intraduodenal administration of possum ZP3 BG vaccine as a priming immunisation elicited significant systemic immune responses, but oral immunisation did not, indicating that protection of BG vaccines from degradation by gastric acidity would enhance the effectiveness of orally delivered vaccines. The detection of antibodies at elevated levels at target sites in the reproductive tract following mucosal delivery demonstrates, for the first time, the potential of BGs as an effective system for vaccine delivery to wild animals, and intranasal/conjunctival immunisation as a promising means for delivery of immunocontraceptive vaccines to wild animals.

Identification and evaluation of an infertility-associated ZP3 epitope from the marsupial brushtail possum (Trichosurus vulpecula).

Immunologically based fertility control vaccines against zona pellucida (ZP) proteins are being developed in New Zealand for biocontrol of the brushtail possum (Trichosurus vulpecula), an introduced Australian marsupial pest. We have shown that immunization of female possums with recombinant possum ZP3 protein (rZP3) reduced fertility by 79%. To enhance the specificity of possum immunocontraceptive vaccines, B-cell epitopes on possum ZP3 protein were mapped using sera of female possums immunized with possum rZP3 and subjected to a fertility trial. The amino acid sequence of the full-length possum ZP3 protein was used to synthesize a complete set of 83 (12-mer) biotinylated peptides each with an overlap of five amino acids with the neighboring peptides. The peptides were used in a modified enzyme-linked immunosorbent assay (ELISA) to identify continuous epitopes recognized by antibodies in the sera of possums immunized with possum rZP3. Sixteen epitopes were identified on the possum ZP3 protein. Comparison of the ELISA binding patterns of these peptides to antibodies in the individual sera with the fertility status of rZP3-immunized possums identified only one epitope (amino acids 156-172) to be associated with infertility. However, female possums immunized with this epitope showed no significant reduction in fertility. The possible reasons for the failure of this potential infertility epitope are discussed.

MHC haplotypes and response to immunocontraceptive vaccines in the brushtail possum.

The possum is a major invasive pest in New Zealand. One option for its control is the use of immunocontraceptive vaccines. Initial trials of vaccines have shown individual variation in response. The use of vaccines on wild populations could result in the evolution of a resistant population through selection for possums that remain fertile because of low or no response. Understanding the basis of this variation is therefore important. The major histocompatibility complex (MHC) is an important influence on the nature of immune responses. This study has investigated the relationship between MHC alleles and individual immune responses to immunocontraceptive vaccines comprising zona pellucida peptides. We identified MHC alleles and putative haplotypes, and compared these between individuals with measured responses to immunocontraceptive vaccines. Two haplotypes were found to associate significantly with differences in vaccine response. Possums that carried haplotype 6 showed reduced responsiveness to one vaccine, while possums that carried haplotype 9 showed increased responsiveness to a separate vaccine. The identification of MHC haplotypes associated with different responses to immunocontraceptive vaccines offers the opportunity to understand what factors trigger non-response and the persistence of fertility in some individuals, and may allow vaccines to be optimised to minimise non-responsiveness.

Bacterial ghosts as a delivery system for zona pellucida-2 fertility control vaccines for brushtail possums (Trichosurus vulpecula).

The introduced brushtail possum is a serious pest in New Zealand and there is much interest in the development of an immunocontraceptive vaccine for population control. Immunisation of female possums against recombinant possum zona pellucida protein-2 (ZP2) is known to reduce embryo production by 72-75% but successful development of fertility control will depend on a delivery system that is effective for field use. Bacterial ghost vaccine technology is a promising system to formulate a non-living vaccine for bait or aerosol delivery. The N-terminal (amino acid residues 41-316, ZP2N) and C-terminal (amino acid residues 308-636, ZP2C) regions of possum ZP2 were fused to maltose-binding protein and expressed in the periplasmic space of Escherichia coli NM522 bacterial ghosts. Female possums (n=20 per treatment group) were immunised with 20mg of either plain ghosts, ZP2N ghosts, or ZP2C ghosts in phosphate-buffered saline applied to the nostrils and eyes (nasal/conjunctival mucosa) at weeks 0, 2 and 4. Effects of immunisation on fertility were assessed following superovulation and artificial insemination. Both constructs evoked humoral (antibody) and cell-mediated immune responses in possums and significantly fewer eggs were fertilised in females immunised against ZP2C ghosts. Results in this study indicate that bacterial ghosts containing possum ZP antigens can reduce possum fertility when delivered by mucosal immunisation and offer a promising delivery system for fertility control of wild possum populations.

Immunogenicity and contraceptive potential of three infertility-relevant zona pellucida 2 epitopes in the marsupial brushtail possum (Trichosurus vulpecula).

In a previous study, three infertility-relevant epitopes of possum ZP2 (Pep12 (amino acids 111-125), Pep31 (amino acids 301-315), and Pep44 (amino acids 431-445)) were identified using sera from possums (Trichosurus vulpecula) immunized with recombinant possum zona pellucida 2 (ZP2) constructs, and a synthetic peptide library of possum ZP2 protein. In this study, the three peptides were conjugated to keyhole limpet hemocyanin and 300 mug of each conjugated peptide were administered subcutaneously to female possums (n = 20 per peptide) in complete Freund's adjuvant. Immunogen doses were repeated 3 and 6 weeks later using incomplete Freund's adjuvant. Control animals were immunized with either phosphate-buffered saline only (n = 10) or 300 mug keyhole limpet hemocyanin (n = 10), administered with the same adjuvants. Serum antibodies from animals immunized against these three epitopes bound to the corresponding possum ZP2 peptides, recombinant possum ZP2 protein constructs, and native zona. Possum fertility was assessed following superovulation and artificial insemination. Peptides Pep12 and Pep31 had no significant effects on fertility parameters (P > 0.05). However, animals immunized with Pep44 had lower egg fertilization rates (immunized 19.5% versus control 60.5%, P < 0.05) and produced significantly fewer embryos than control animals (immunized 0.5 embryos versus control 2.4 embryos, P < 0.05). The number of Pep44-immunized females that produced embryos was reduced by 64%. Identification and characterization of possum infertility-relevant epitopes on possum ZP2 protein will assist development of safe, humane, and possum-specific immunocontraceptive vaccines for controlling the introduced possums in New Zealand.

Expression in Escherichia coli and immunological characterization of three zona pellucida proteins (ZP1, ZP2, and ZP3) from a marsupial, the brushtail possum (Trichosurus vulpecula).

The brushtail possum (Trichosurus vulpecula) zona pellucida (ZP) is composed of three major glycoproteins, designated ZP1, ZP2, and ZP3 based on their size and homology with eutherian ZP proteins. These proteins are candidate antigens for the development of an immunocontraceptive vaccine to control the fertility of the brushtail possum in New Zealand, where it is an introduced pest. In order to further their immunological and functional characterization, recombinant possum ZP proteins were produced in Escherichia coli (E. coli) strain JM109, M15, SG13009, or BL21 codon plus. Each of the proteins produced possessed a N-terminal six histidine tag (His)(6) to facilitate purification and consisted of amino acid (aa) residues 18-471 of possum ZP1, aa residues 40-311 of ZP2 (ZP2-N), aa residues 305-634 of ZP2 (ZP2-C), and aa residues 23-342 of ZP3. Immunoblot using anti-RGS(His)(4) antibodies and polyclonal rabbit anti-porcine ZP antibodies detected major bands at 54 kDa for ZP1, 32 kDa for ZP2-N, 39 kDa for ZP2-C, and 40 kDa for ZP3. Immunization of male and female rabbits with ZP2-N, ZP2-C, and ZP3 purified on Ni-NTA resin under denaturing conditions generated antibodies reactive with recombinant ZP proteins on Western blot and with native ZP proteins in possum ovarian sections using immunofluorescence. Antibodies generated against ZP1 in the same way were reactive with recombinant ZP proteins on Western blot only. The recombinant possum ZP proteins and specific antibodies produced in this study give an indication of the antigenic relationship of the possum ZP proteins and are vital tools for future studies of sperm-ZP binding in marsupials and for the evaluation of ZP-based contraceptive vaccines in possums and other marsupials.