Rabbit Hemorrhagic Disease Virus in Mexico in 2020-2021: Risk Areas and Climatic Distribution.

Mexico is home to 14 species of lagomorphs, 6 of which are endemic. Studies on diseases affecting native lagomorphs are scarce, and in most cases, the impact on their populations remains largely unknown. Rabbit hemorrhagic disease virus (RHDV), especially the RHDV2 variant, causes a serious and extremely contagious disease, resulting in high mortality rates and major declines in wild lagomorph populations. The objectives of this study were to identify disease hotspots and critical biodiversity regions in Mexico through the combined use of disease information and lagomorph distribution maps and to determine the areas of greatest concern. In total, 19 states of Mexico recorded RHDV2 from April 2020 to August 2021, and 12 of them reported the wild species Sylvilagus audubonii, Lepus californicus, and unidentified Leporidae species. The distribution of RHDV2 in Mexico can be closely predicted from climatic variables. RHDV2 hotspots are located in the central-southern area of the Mexican Highlands and the Trans-Mexican Volcanic Belt, where the virus affects multiple species. This knowledge is essential for proposing specific actions to manage and preserve lagomorph populations at risk and address these issues as soon as possible.

First Detection of Benign Rabbit Caliciviruses in Chile.

Pathogenic lagoviruses (Rabbit hemorrhagic disease virus, RHDV) are widely spread across the world and are used in Australia and New Zealand to control populations of feral European rabbits. The spread of the non-pathogenic lagoviruses, e.g., rabbit calicivirus (RCV), is less well studied as the infection results in no clinical signs. Nonetheless, RCV has important implications for the spread of RHDV and rabbit biocontrol as it can provide varying levels of cross-protection against fatal infection with pathogenic lagoviruses. In Chile, where European rabbits are also an introduced species, myxoma virus was used for localised biocontrol of rabbits in the 1950s. To date, there have been no studies investigating the presence of lagoviruses in the Chilean feral rabbit population. In this study, liver and duodenum rabbit samples from central Chile were tested for the presence of lagoviruses and positive samples were subject to whole RNA sequencing and subsequent data analysis. Phylogenetic analysis revealed a novel RCV variant in duodenal samples that likely originated from European RCVs. Sequencing analysis also detected the presence of a rabbit astrovirus in one of the lagovirus-positive samples.

A novel vaccine candidate against rabbit hemorrhagic disease virus 2 (RHDV2) confers protection in domestic rabbits.

OBJECTIVE: To evaluate efficacy of a novel vaccine against rabbit hemorrhagic disease virus 2 (RHDV2) in domestic rabbits. ANIMALS: 40 New Zealand White rabbits obtained from a commercial breeder. PROCEDURES: Rabbits were vaccinated and held at the production facility for the duration of the vaccination phase and transferred to Colorado State University for challenge with RHDV2. Rabbits were challenged with oral suspensions containing infectious virus and monitored for clinical disease for up to 10 days. Rabbits that died or were euthanized following infection were necropsied, and livers were evaluated for viral RNA via RT-PCR. RESULTS: None of the vaccinated animals (0/9) exhibited clinical disease or mortality following infection with RHDV2 while 9/13 (69%) of the control animals succumbed to lethal disease following infection. CLINICAL RELEVANCE: The novel vaccine described herein provided complete protection against lethal infection following RHDV2 challenge. Outside of emergency use, there are currently no licensed vaccines against RHDV2 on the market in the United States; as such, this vaccine candidate would provide an option for control of this disease now that RHDV2 has become established in North America.

Secretion and assembly of calicivirus-like particles in high-cell-density yeast fermentations: strategies based on a recombinant non-specific BPTI-Kunitz-type protease inhibitor.

The yeast Pichia pastoris is one of the most robust cell factories in use for the large-scale production of biopharmaceuticals with applications in the fields of human and animal health. Recently, intracellular high-level expression of rabbit hemorrhagic disease virus (RHDV) capsid protein (VP1) as a self-assembled multipurpose antigen/carrier was established as a production process from P. pastoris. Since recovery of VP1 from the culture media implies technological and economic advantages, the secretion of VP1 variants was undertaken in this work. Conversely, extensive degradation of VP1 was detected. Variations to culture parameters and supplementation with different classes of additives were unable to diminish degradation. Strategies were then conducted during fermentations using a recombinant variant of a non-specific BPTI-Kunitz-type protease inhibitor (rShPI-1A) isolated from the sea anemone Stichodactyla helianthus. The presence of the inhibitor in the culture medium at the recombinant protein induction phase, as well as co-culture of the yeast strains expressing VP1 and rShPI-1A, led to VP1 protection from proteolysis and to production of ordered virus-like particles. A yeast strain was also engineered to co-express the rShPI-1A inhibitor and intact VP1. Expression levels up to 116 mg L(-1) of VP1 were reached under these approaches. The antigen was characterized and purified in a single chromatography step, its immunogenic capacity was evaluated, and a detection test for specific antibodies was developed. This work provides feasible strategies for improvements in P. pastoris heterologous protein secretion and is the first report on co-expression of the ShPI-1A with a recombinant product otherwise subjected to proteolytic degradation.

Single dose adenovirus vectored vaccine induces a potent and long-lasting immune response against rabbit hemorrhagic disease virus after parenteral or mucosal administration.

Rabbit hemorrhagic disease virus (RHDV) is the etiological agent of a lethal and contagious disease of rabbits that remains as a serious problem worldwide. As this virus does not replicate in cell culture systems, the capsid protein gene has been expressed in heterologous hosts or inserted in replication-competent viruses in order to obtain non-conventional RHDV vaccines. However, due to technological or safety issues, current RHDV vaccines are still prepared from organs of infected rabbits. In this work, two human type 5 derived replication-defective adenoviruses encoding the rabbit hemorrhagic disease virus VP60 capsid protein were constructed. The recombinant protein was expressed as a multimer in mouse and rabbit cell lines at levels that ranged from approximately 120 to 160 mg/L of culture. Mice intravenously or subcutaneously inoculated with a single 10(8) gene transfer units (GTU) dose of the AdVP60 vector (designed for VP60 intracellular expression) seroconverted at days 7 and 14 post-immunization, respectively. This vector generated a stronger response than that obtained with a second vector (AdVP60sec) designed for VP60 secretion. Rabbits were then immunized by parenteral or mucosal routes with a single 10(9)GTU dose of the AdVP60 and the antibody response was evaluated using a competition ELISA specific for RHDV or RHDVa. Protective hemagglutination inhibition (HI) titers were also promptly detected and IgG antibodies corresponding with inhibition percentages over 85% persisted up to one year in all rabbits, independently of the immunization route employed. These levels were similar to those elicited with inactivated RHDV or with VP60 obtained from yeast or insect cells. IgA specific antibodies were only found in saliva of rabbits immunized by intranasal instillation. The feasibility of VP60 production and vaccination of rabbits with replication-defective adenoviral vectors was demonstrated.

Biochemical and structural characterization of RHDV capsid protein variants produced in Pichia pastoris: advantages for immunization strategies and vaccine implementation.

Rabbit hemorrhagic disease virus (RHDV) VP60 capsid protein was recently expressed at approximately 1.5 gL(-1) associated with the disruption pellet of Pichia pastoris, thus requiring an additional process of extraction by solubilization. Consequently, the expression of a soluble variant of VP60 was undertaken in order to attain an easier approach for vaccine production. The VP60 gene was cloned without secretion signal under the transcriptional control of the AOX1 yeast promoter. The antigen obtained was intracellular and soluble at approximately 480 mg L(-1). Its characterization by size-exclusion HPLC, ultracentrifugation, and electron microscopy, showed the presence of high molecular weight structures similar in mass, size and buoyant density to native RHDV. The antigenic profile was similar to that from authentic virions as determined with monoclonal antibodies directed against RHDV conformational epitopes. These analyses, conducted on VP60 obtained insoluble in P. pastoris revealed the formation of protein aggregates rather than the presence of ordered multimeric structures. An immunization trial was conducted in which the soluble VP60 was employed by subcutaneous (s.c.) injection either purified by a single chromatographic step or contained within raw disruption supernatant, emulsified in Montanide 888. The insoluble variant was administered as a yeast extract powder by oral and s.c. routes. The earliest IgG response, titers and persistence of antibodies were studied by competition ELISA and hemagglutination inhibition (HI) assays. All rabbits immunized with the yeast-derived antigens developed a strong RHDV-specific response (including the "RHDVa" subtype) that lasted over one year after the primary immunization. Early HI titers up to 1/40 960 were generated. The immune response was similar to that induced by VP60 from Sf9 cells and superior to the response elicited with inactivated RHDV. Overall it was found that the soluble VP60 multimers, safely and easily produced in P. pastoris, are a valuable candidate for the rational implementation of a low-cost, scalable subunit vaccine against RHDV.

Molecular and antigenic characterization of rabbit hemorrhagic disease virus isolated in Cuba indicates a distinct antigenic subtype.

Phylogenetic analyses conducted on isolates of rabbit hemorrhagic disease virus (RHDV) from throughout the world have shown well-defined genogroups comprising representative strains of the virus and antigenic variants. In this work, we have isolated and characterized RHDV from the major epizootic that occurred in Cuba in 2004-2005. Sequence analysis of the capsid protein gene and antigenic characterization of this strain has allowed its inclusion as a member of the distinct RHDVa subtype. We also found that specific antibodies directed against RHDV reference strains bound to the Cuban isolate in a competition ELISA and inhibited virus hemagglutination in vitro. This is the second report on the molecular characterization of RHDVa circulating in the American region.