Neglected Spleen Transcriptional Profile Reveals Inflammatory Disorder Conferred by Rabbit Hemorrhagic Disease Virus 2 Infection.

Rabbit hemorrhagic disease (RHD) is an acute fatal disease caused by the rabbit hemorrhagic disease virus (RHDV). Since the first outbreaks of type 2 RHDV (RHDV2) in April 2020 in China, the persistence of this virus in the rabbit population has caused substantial economic losses in rabbit husbandry. Previous failures in preventing RHDV2 prompted us to further investigate the immune mechanisms underlying the virus's pathogenicity, particularly concerning the spleen, a vital component of the mononuclear phagocyte system (MPS). For this, a previous RHDV2 isolate, CHN/SC2020, was utilized to challenge naive adult rabbits. Then, the splenic transcriptome was determined by RNA-Seq. This study showed that the infected adult rabbits had 3148 differentially expressed genes (DEGs), which were associated with disease, signal transduction, cellular processes, and cytokine signaling categories. Of these, 100 upregulated DEGs were involved in inflammatory factors such as IL1α, IL-6, and IL-8. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these DEGs were significantly enriched in the cytokine-cytokine receptor interaction signaling pathway, which may play a vital role in CHN/SC2020 infection. At the same time, proinflammatory cytokines and chemokines were significantly increased in the spleen at the late stages of infection. These findings suggested that RHDV2 (CHN/SC2020) might induce dysregulation of the cytokine network and compromise splenic immunity against viral infection, which expanded our understanding of RHDV2 pathogenicity.

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.

Vaccination against Rabbit Hemorrhagic Disease Virus 2 (RHDV2) Using a Baculovirus Recombinant Vaccine Provides Durable Immunity in Rabbits.

Rabbit hemorrhagic disease virus 2 (RHDV2) emerged in the United States in 2018 and has spread in both domestic and wild rabbits nationwide. The virus has a high mortality rate and can spread rapidly once introduced in a rabbit population. Vaccination against RHDV2 provides the best protection against disease and should be considered by all rabbit owners. Here, we investigate the duration of immunity provided by vaccination with the Medgene Platform conditionally licensed commercial vaccine 6 months following the initial series. Rabbits received either the vaccination or a placebo and were challenged with RHDV2 6 months later. All vaccinated rabbits survived challenge whereas 18/19 non-vaccinated controls succumbed to infection within 10 or fewer days post-challenge. These results demonstrate lasting immunity following vaccination with the Medgene RHDV2 vaccine.

Adipose-derived stem cells can alleviate RHDV2 induced acute liver injury in rabbits.

Rabbit hemorrhagic disease virus (RHDV) can cause fatal fulminant hepatitis, which is very similar to human acute liver failure. The aim of this study was to investigate whether adipose-derived stem cells (ADSCs) could alleviate RHDV2-induced liver injury in rabbits. Twenty 50-day-old rabbits were divided randomly into two groups (RHDV2 group, ADSCs + RHDV2 group). Starting from the 1st day, two groups of rabbits were given 0.5 ml of viral suspensions by subcutaneous injection in the neck. Meanwhile, the ADSCs + RHDV2 group was injected with ADSCs cell suspension (1.5 × 107 cells/ml) via a marginal ear vein, and the RHDV2 group was injected with an equal amount of saline via a marginal ear vein. At the end of the 48 h experiment, the animals were euthanized and gross hepatic changes were observed before liver specimens were collected. Histopathological analysis was performed using hematoxylin-eosin (HE), periodic acid schiff (PAS) and Masson's trichrome staining. For RHDV2 affected rabbits, HE staining demonstrated disorganized hepatic cords, loss of cellular detail, and severe cytoplasmic vacuolation within hepatocytes. Glycogen was not observed with PAS staining, and Masson's Trichrome staining showed increased hepatic collagen deposition. For rabbits treated with ADSCs at the time of inoculation, hepatic pathological changes were significantly less severe, liver glycogen synthesis was increased, and collagen fiber deposition was decreased. For RHDV2 affected rabbits, Tunel and immunofluorescence staining showed that the number of apoptotic cells, TGF-ß, and MMP-9 protein expression increased. And that in the ADSC treated group there was less hepatocyte apoptosis. In addition, RHDV2 induces liver inflammation and promotes the expression of IL-1ß, IL-6, and TNF-α. In rabbits administered ADSCs at time of inoculation, the expression of inflammatory factors in liver tissue decreased significantly. Our experiments show that ADSCs can protect rabbits from liver injury by RHDV2 and reduce the pathological and inflammatory response of liver. However, the specific protective mechanism needs further study.

Epidemiological characterization and risk assessment of rabbit haemorrhagic disease virus 2 (RHDV2/b/GI.2) in the world.

A novel variant of rabbit haemorrhagic disease virus, designated RHDV2/b/GI.2, was first discovered in France in 2010. Subsequently, RHDV2 rapidly spread to Africa, North America, Australia, and Asia. RHDV2 outbreaks have resulted in significant economic losses in the global rabbit industry and disrupted the balance of natural ecosystems. Our study investigated the seasonal characteristics of RHDV2 outbreaks using seasonal indices. RHDV2 is prone to causing significant outbreaks within domestic and wild rabbit populations during the spring season and is more likely to induce outbreaks within wild rabbit populations during late autumn in the Southern Hemisphere. Furthermore, based on outbreak data for domestic and wild rabbits and environmental variables, our study established two MaxEnt models to explore the relationship between RHDV2 outbreaks and the environmental factors and conducted outbreak risk predictions for RHDV2 in global domestic and wild rabbit populations. Both models demonstrated good predictive performance, with AUC values of 0.960 and 0.974, respectively. Road density, isothermality, and population density were identified as important variables in the outbreak of RHDV2 in domestic rabbits, while road density, normalized difference vegetation index, and mean annual solar radiation were considered key variables in the outbreak of RHDV2 in wild rabbits. The environmental factors associated with RHDV2 outbreaks identified in our study and the outbreak risk prediction maps generated in our study will aid in the formulation of appropriate RHDV2 control measures to reduce the risk of morbidity in domestic and wild rabbits.

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.

Recombination between non-structural and structural genes as a mechanism of selection in lagoviruses: The evolutionary dead-end of an RHDV2 isolated from European hare.

The genus Lagovirus, belonging to the family Caliciviridae, emerged around the 1980s. It includes highly pathogenic species, rabbit hemorrhagic disease virus (RHDV/GI.1) and European brown hare syndrome virus (EBHSV/GII.1), which cause fatal hepatitis, and nonpathogenic viruses with enteric tropism, rabbit calicivirus (RCV/GI.3,4) and hare calicivirus (HaCV/GII.2). Lagoviruses have evolved along two independent genetic lineages: GI (RHDV and RCV) in rabbits and GII (EBHSV and HaCV) in hares. To be emphasized is that genomes of lagoviruses, like other caliciviruses, are highly conserved at RdRp-VP60 junctions, favoring intergenotypic recombination events at this point. The recombination between an RCV (genotype GI.3), donor of non-structural (NS) genes, and an unknown virus, donor of structural (S) genes, likely led to the emergence of a new lagovirus in the European rabbit, called RHDV type 2 (GI.2), identified in Europe in 2010. New RHDV2 intergenotypic recombinants isolated in rabbits in Europe and Australia originated from similar events between RHDV2 (GI.2) and RHDV (GI.1) or RCV (GI.3,4). RHDV2 (GI.2) rapidly spread worldwide, replacing RHDV and showing several lagomorph species as secondary hosts. The recombination events in RHDV2 viruses have led to a number of viruses with very different combinations of NS and S genes. Recombinant RHDV2 with NS genes from hare lineage (GII) was recently identified in the European hare. This study investigated the first RHDV2 (GI.2) identified in Italy in European hare (RHDV2_Bg12), demonstrating that it was a new virus that originated from the recombination between RHDV2, as an S-gene donor and a hare lagovirus, not yet identified but presumably nonpathogenic, as an NS gene donor. When rabbits were inoculated with RHDV2_Bg12, neither deaths nor seroconversions were recorded, demonstrating that RHDV2_Bg12 cannot infect the rabbit. Furthermore, despite intensive and continuous field surveillance, RHDV2_Bg12 has never again been identified in either hares or rabbits in Italy or elsewhere. This result showed that the host specificity of lagoviruses can depend not only on S genes, as expected until today, but potentially also on some species-specific NS gene sequences. Therefore, because RHDV2 (GI.2) infects several lagomorphs, which in turn probably harbor several specific nonpathogenic lagoviruses, the possibility of new speciation, especially in those other than rabbits, is real. RHDV2 Bg_12 demonstrated this, although the attempt apparently failed.

Bayesian evaluation of temporal changes in sensitivity and specificity of three serological tests for multiple circulating strains of rabbit haemorrhagic disease virus.

Competition and indirect ELISAs are currently being used to monitor rabbit haemorrhagic disease viruses (RHDV1 and RHDV2) in rabbits worldwide. Temporal changes in the sensitivity (Se) and specificity (Sp) of RHDV1 competition-ELISA (cELISA1), RHDV2 competition-ELISA (cELISA2), and RHDV1 Immunoglobulin G (IgG1) ELISA, were investigated using Bayesian Latent Class models (BCLM) in the Australian wild rabbit population where both viruses circulate simultaneously and a long-term serological dataset exists. When cELISA1 was compared to IgG1 ELISA, the Se of cELISA1 improved while the Sp of IgG1 ELISA declined over the 2011-21. This corresponded with a decline in the true RHDV1 prevalence in 2018-21, suggesting that a large proportion of RHDV1 exposed rabbits survived the introduction and dominance of RHDV2 up to approximately 2017/2018, after which they died and were not replaced. The Se and Sp estimates for 2014-15 for both cELISA1 and IgG1 ELISA, and the true prevalence when analysing all three tests together were similar to those obtained from the analysis of cELISA1/IgG1 ELISA. The same was also true for the Se and Sp of cELISA2 and IgG1 ELISA estimates from 2018 onwards. This suggests that RHDV1 was the dominant infection type in 2014-15, but RHDV2 was the dominant infection type in 2018-21. Further, the increase in Se of cELISA2 and the low Sp of IgG1 ELISA in the cELISA2/IgG1 ELISA analysis, compared to the Se of cELISA2 and Sp of IgG1 ELISA when analysing all three tests together suggests that the underlying infection status was more influenced by RHDV2 and that the higher Se of IgG1 ELISA is due to cross-reaction of RHDV2 antibodies on IgG1 ELISA. The true prevalence data suggest that RHDV2 exposure peaked in 2017. Our findings show that test characteristics changed in response to the changing virus prevalences over time. IgG1 ELISA, currently having a high Se, should be used to monitor both viruses and will perform better than both cELISAs.

Utilizing Molecular Epidemiology and Citizen Science for the Surveillance of Lagoviruses in Australia.

Australia has multiple lagoviruses with differing pathogenicity. The circulation of these viruses was traditionally determined through opportunistic sampling events. In the lead up to the nationwide release of RHDVa-K5 (GI.1aP-GI.1a) in 2017, an existing citizen science program, RabbitScan, was augmented to allow members of the public to submit samples collected from dead leporids for lagovirus testing. This study describes the information obtained from the increased number of leporid samples received between 2015 and 2022 and focuses on the recent epidemiological interactions and evolutionary trajectory of circulating lagoviruses in Australia between October 2020 and December 2022. A total of 2771 samples were tested from January 2015 to December 2022, of which 1643 were lagovirus-positive. Notable changes in the distribution of lagovirus variants were observed, predominantly in Western Australia, where RHDV2-4c (GI.4cP-GI.2) was detected again in 2021 after initially being reported to be present in 2018. Interestingly, we found evidence that the deliberately released RHDVa-K5 was able to establish and circulate in wild rabbit populations in WA. Overall, the incorporation of citizen science approaches proved to be a cost-efficient method to increase the sampling area and enable an in-depth analysis of lagovirus distribution, genetic diversity, and interactions. The maintenance of such programs is essential to enable continued investigations of the critical parameters affecting the biocontrol of feral rabbit populations in Australia, as well as to enable the detection of any potential future incursions.

Seromolecular surveillance of rabbit haemorrhagic disease virus in Nigeria.

Following the first 2020 rabbit haemorrhagic disease virus (RHDV) outbreak in Nigeria which caused massive mortalities in several rabbitries, there was a need to know the spread and strains circulating in the affected states. Over 100 rabbitries still existing post-RHDV outbreak in Ogun and Kwara States were investigated. A commercial enzyme-linked immunosorbent assay kit was used to screen for RHDV immunoglobulin G in 192 rabbit sera, while RHDV VP60 gene was amplified in RNA extracted from these sera and tissues (liver and/or spleen harvested from 37 carcasses necrotized) by reverse transcription-polymerase chain reaction (RT-PCR). Sequences obtained from the amplicons were subjected to phylogenetic analysis. The results revealed a seroprevalence of 82.3% (158/192). RHDV VP60 gene was detected in 15/17 (88.2%) and 2/20 (10.0%) carcasses from Ogun and Kwara States, respectively, while none of the sera was positive. Sequences of the two positive amplicons selected (one from each states) shared 98.95% nucleotide identity and belonged to RHDV 2/GI.2 strain. Also, nBLAST of these sequences revealed 98.43-99.55% homology with the prototype Nigerian RHDV strain RHDV/NGR/ILN/001 (MT996357.1). Furthermore, these strains clustered with this prototype and a German RHDV strain (LR899166.1). Pathologic lesions affecting the respiratory, cardiovascular, renal, lymphatic, and digestive systems were observed in necropsied carcasses. This study indicated that RHDV 2/GI.2 strain was the cause of 2020 RHD outbreak in Nigeria. Thus, while continuous public sensitization about RHD especially among rabbit farmers in Nigeria is important, efforts aimed at design and implementation of RHD vaccination policy, preferably using indigenous seed, should be expedited.

Optimising the Delivery of RHDV to Rabbits for Biocontrol: An Experimental Evaluation of Two Novel Methods of Virus Delivery.

Rabbit haemorrhagic disease virus (RHDV) is established as a landscape-scale biocontrol that assists the management of invasive European rabbits and their impacts in both Australia and New Zealand. In addition to this, it is also available to land managers to augment rabbit control efforts at a local scale. However, current methods of deploying RHDV to rabbits that rely on the consumption of virus-treated baits can be problematic as rabbits are reluctant to consume bait when there is abundant, green, protein-rich feed available. We ran a suite of interrupted time-series experiments to compare the duration of infectivity of two conventional (carrot and oat baits) and two novel (meat bait and soil burrow spray) methods of deploying RHDV to rabbits. All methods effectively killed exposed rabbits. Soil burrow spray and carrot baits resulted in infection and mortality out to 5 days post their deployment in the field, and meat baits caused infection out to 10 days post their deployment. In contrast, oat baits continued to infect and kill exposed rabbits out to 20 days post deployment. Molecular assays demonstrated high viral loads in deployed baits beyond the duration for which they were infectious or lethal to rabbits. Based on our results, we suggest that the drying of meat baits may create a barrier to effective transmission of RHDV by adult flies within 10 days. We therefore hypothesise that fly larvae production and development on infected tissues is critical to prolonged viral transmission from meat baits, and similarly from carcasses of RHDV mortalities, via mechanical fly vectors. Our study demonstrates that meat baits and soil spray could provide additional virus deployment options that remove the need for rabbits to consume baits at times when they are reluctant to do so.

Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses.

The genus Lagovirus of the family Caliciviridae contains some of the most virulent vertebrate viruses known. Lagoviruses infect leporids, such as rabbits, hares and cottontails. Highly pathogenic viruses such as Rabbit haemorrhagic disease virus 1 (RHDV1) cause a fulminant hepatitis that typically leads to disseminated intravascular coagulation within 24-72 h of infection, killing over 95 % of susceptible animals. Research into the pathophysiological mechanisms that are responsible for this extreme phenotype has been hampered by the lack of a reliable culture system. Here, we report on a new ex vivo model for the cultivation of lagoviruses in cells derived from the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus). We show that three different lagoviruses, RHDV1, RHDV2 and RHDVa-K5, replicate in monolayer cultures derived from rabbit hepatobiliary organoids, but not in monolayer cultures derived from cat (Felis catus) or mouse (Mus musculus) organoids. Virus multiplication was demonstrated by (i) an increase in viral RNA levels, (ii) the accumulation of dsRNA viral replication intermediates and (iii) the expression of viral structural and non-structural proteins. The establishment of an organoid culture system for lagoviruses will facilitate studies with considerable implications for the conservation of endangered leporid species in Europe and North America, and the biocontrol of overabundant rabbit populations in Australia and New Zealand.

Digital PCR (dPCR) Quantification of miR-155-5p as a Potential Candidate for a Tissue Biomarker of Inflammation in Rabbits Infected with Lagovirus europaeus/Rabbit Hemorrhagic Disease Virus (RHDV).

MicroRNAs (miRNAs, miRs) are a group of small, 17-25 nucleotide, non-coding RNA sequences that, in their mature form, regulate gene expression at the post-transcriptional level. They participate in many physiological and pathological processes in both humans and animals. One such process is viral infection, in which miR-155 participates in innate and adaptive immune responses to a broad range of inflammatory mediators. Recently, the study of microRNA has become an interesting field of research as a potential candidate for biomarkers for various processes and disease. To use miRNAs as potential biomarkers of inflammation in viral diseases of animals and humans, it is necessary to improve their detection and quantification. In a previous study, using reverse transcription real-time quantitative PCR (RT-qPCR), we showed that the expression of ocu-miR-155-5p in liver tissue was significantly higher in rabbits infected with Lagovirus europaeus/Rabbit Hemorrhagic Disease Virus (RHDV) compared to healthy rabbits. The results indicated a role for ocu-miR-155-5p in Lagovirus europaeus/RHDV infection and reflected hepatitis and the impairment/dysfunction of this organ during RHD. MiR-155-5p was, therefore, hypothesized as a potential candidate for a tissue biomarker of inflammation and examined in tissues in Lagovirus europaeus/RHDV infection by dPCR. The objective of the study is the absolute quantification of ocu-miR-155-5p in four tissues (liver, lung, kidney, and spleen) of rabbits infected with Lagovirus europaeus/RHDV by digital PCR, a robust technique for the precise and direct quantification of small amounts of nucleic acids, including miRNAs, without standard curves and external references. The average copy number/µL (copies/µL) of ocu-miRNA-155-5p in rabbits infected with Lagovirus europaeus GI.1a/Rossi in the liver tissue was 12.26 ± 0.14, that in the lung tissue was 48.90 ± 9.23, that in the kidney tissue was 16.92 ± 2.89, and that in the spleen was 25.10 ± 0.90. In contrast, in the tissues of healthy control rabbits, the average number of copies/µL of ocu-miRNA-155-5p was 5.07 ± 1.10 for the liver, 23.52 ± 2.77 for lungs, 8.10 ± 0.86 for kidneys, and 42.12 ± 3.68 for the spleen. The increased expression of ocu-miRNA-155-5p in infected rabbits was demonstrated in the liver (a fold-change of 2.4, p-value = 0.0003), lung (a fold-change of 2.1, p-value = 0.03), and kidneys (a fold-change of 2.1, p-value = 0.01), with a decrease in the spleen (a fold-change of 0.6, p-value = 0.002). In the study of Lagovirus europaeus/RHDV infection and in the context of viral infections, this is the first report that shows the potential use of dPCR for the sensitive and absolute quantification of microRNA-155-5p in tissues during viral infection. We think miR-155-5p may be a potential candidate for a tissue biomarker of inflammation with Lagovirus europaeus/RHDV infection. Our report presents a new path in discovering potential candidates for the tissue biomarkers of inflammation.

[Antigenic and immunogenic activity of virus-like particles based on rabbit hemorrhagic disease virus (Caliciviridae: Lagovirus) genotypes GI1 and GI2 recombinant major capsid proteins].

INTRODUCTION: Rabbit hemorrhagic disease is an acute highly contagious infection associated with two genotypes of pathogenic Lagovirus. Antibodies to major capsid protein (Vp60) are protective. The aim of the work ‒ is an evaluation of antigenic and immunogenic activity of virus-like particles (VLPs) based on recombinant major capsid proteins of both genotypes of rabbit hemorrhagic disease virus (RHDV) (recVP60-GI1 and recVP60-GI2). MATERIALS AND METHODS: Baculovirus-expressed VLPs were evaluated using electron microscopy and administered to clinically healthy 1.53 month old rabbits in a dose of 50 g. Rabbits were challenged with 103 LD50 of virulent strains Voronezhsky-87 and Tula 21 days post immunization. Serum samples were tested for the presence of RHDV-specific antibodies. RESULTS: VLPs with hemagglutination activity forming VLP 3040 nm in size were obtained in Hi-5 cell culture. Specific antibody titers in rabbits measured by ELISA were 1 : 200 to 1 : 800 on 21th day post immunization with VLPs. Immunogenic activity of recVP60-GI1 VLPs was 90 and 40%, while it was 30 and 100% for recVP60-GI2 VLPs after the challenge with RHDV genotypes 1 and 2 respectively. The immunogenicity of two VLPs in mixture reached 100%. DISCUSSION: VLPs possess hemagglutinating, antigenic and immunogenic activity, suggesting their use as components in substances designed for RHDV specific prophylaxis in rabbits. Results of the control challenge experiment demonstrated the need to include the antigens from both RHDV genotypes in the vaccine. CONCLUSION: Recombinant proteins recVP60-GI1 and recVP60-GI2 form VLPs that possess hemagglutinating an antigenic activity, and provide 90100% level of protection for animals challenged with RHDV GI1 and GI2 virulent strains.

Dynamics of Humoral Immunity to Myxoma and Rabbit Hemorrhagic Disease Viruses in Wild European Rabbits Assessed by Longitudinal Semiquantitative Serology.

Myxoma virus (MYXV) and rabbit hemorrhagic disease virus (RHDV) are important drivers of the population decline of the European rabbit, an endangered keystone species. Both viruses elicit strong immune responses, but the long-term dynamics of humoral immunity are imperfectly known. This study aimed to assess the determinants of the long-term dynamics of antibodies to each virus based on a longitudinal capture-mark-recapture of wild European rabbits and semiquantitative serological data of MYXV and RHDV GI.2-specific IgG. The study included 611 indirect enzyme-linked immunosorbent assay (iELISA) normalized absorbance ratios for each MYXV and RHDV GI.2 from 505 rabbits from 2018 to 2022. Normalized absorbance ratios were analyzed using log-linear mixed models, showing a significant positive relationship with the time since the first capture of individual rabbits, with monthly increases of 4.1% for antibodies against MYXV and 2.0% against RHDV GI.2. Individual serological histories showed fluctuations over time, suggesting that reinfections boosted the immune response and likely resulted in lifelong immunity. Normalized absorbance ratios significantly increased with the seroprevalence in the population, probably because of recent outbreaks, and with body weight, highlighting the role of MYXV and RHDV GI.2 in determining survival to adulthood. Juvenile rabbits seropositive for both viruses were found, and the dynamics of RHDV GI.2 normalized absorbance ratios suggest the presence of maternal immunity up to 2 months of age. Semiquantitative longitudinal serological data provide epidemiological information, otherwise lost when considering only qualitative data, and support a lifelong acquired humoral immunity to RHDV GI.2 and MYXV upon natural infection. IMPORTANCE This study addresses the long-term dynamics of humoral immunity to two major viral pathogens of the European rabbit, an endangered keystone species of major ecological relevance. Such studies are particularly challenging in free-ranging species, and a combination of longitudinal capture-mark-recapture and semiquantitative serology was used to address this question. Over 600 normalized absorbance ratios of iELISA, obtained from 505 individual rabbits in 7 populations over 5 years, were analyzed using linear mixed models. The results support a lifelong acquired humoral immunity to myxoma virus and rabbit hemorrhagic disease virus upon natural infection and suggest the presence of maternal immunity to the latter in wild juvenile rabbits. These results contribute to understanding the epidemiology of two viral diseases threatening this keystone species and assist in developing conservation programs.

Etiology of viral induced acute liver failure and defensins as potential therapeutic agents in ALF treatment.

Acute liver failure (ALF) is a rare and severe disease, which, despite continuous advances in medicine, is still characterized by high mortality (65-85%). Very often, a liver transplant is the only effective treatment for ALF. Despite the implementation of prophylactic vaccinations in the world, the viral background of ALF is still a problem and leads to many deaths. Depending on the cause of ALF, it is sometimes possible to reverse this condition with appropriate therapies, which is why the search for effective antiviral agents seems to be a very desirable direction of research. Defensins, which are our natural antimicrobial peptides, have a very high potential to be used as therapeutic agents for infectious liver diseases. Previous studies on the expression of human defensins have shown that increased expression of human α and ß-defensins in HCV and HBV infections is associated with a better response to treatment. Unfortunately, conducting clinical trials for ALF is very difficult due to the severity of the disease and the low incidence, therefore animal models are important for the development of new therapeutic strategies. One of the best animal models that has real reference to research on acute liver failure (ALF) is rabbit hemorrhagic disease in rabbits caused by the Lagovirus europaeus virus. So far, there have been no studies on the potential of defensins in rabbits infected with Lagovirus europaeus virus.

Genetics of the rabbit haemorrhagic disease virus strains responsible for rabbit haemorrhagic disease outbreaks in Japan between 2000 and 2020.

Rabbit haemorrhagic disease (RHD) is a highly contagious and fatal disease in rabbits caused by the rabbit haemorrhagic disease virus (RHDV), which includes two genotypes, RHDV-GI.1 and RHDV2-GI.2. RHDVs tend to recombine among different strains, resulting in significant genetic evolution. This study evaluated the genetics of Japanese RHDV strains associated with six outbreaks between 2000 and 2020 using whole-genome sequencing, genomic recombination and phylogenetic analyses. Genomic recombination analysis using near-complete genomic sequences revealed that two Japanese strains detected in 2000 and 2002 were non-recombinant GI.1 (variant RHDVa-GI.1a) strains of different origins, most closely related to strains identified in PR China in 1997 and the USA in 2001, respectively. In contrast, four recent Japanese GI.2 strains detected between 2019 and 2020 were recombinant viruses harbouring structural protein (SP) genes from GI.2 strains and non-SP (NSP) genes from a benign rabbit calicivirus (RCV) strain of genotype RCV-E1-GI.3 (GI.3P-GI.2) or an RHDV G1-GI.1b variant (GI.1bP-GI.2). Phylogenetic analysis based on SP and NSP regions revealed that the GI.1bP-GI.2 recombinant virus detected in Ehime prefecture and the GI.3P-GI.2 recombinant viruses detected in Ibaraki, Tochigi and Chiba prefectures were most closely related to recombinant viruses identified in Australia in 2017 and Germany in 2017, respectively. These results suggested that past RHD outbreaks in Japan did not result from the evolution of domestic RHDVs but rather represented incursions of foreign RHDV strains, implying that Japan is constantly at risk of RHDV incursion from other countries.

Comparison of magnetic bead and rapid swab RNA extraction methods for detecting rabbit hemorrhagic disease virus 2 in rabbit liver samples.

We compared a bead RNA extraction method with a one-tube method that required only a heat block and ice. RNA was first extracted from liver samples from nine rabbits dying from rabbit hemorrhagic disease virus 2 (RHDV2) using magnetic beads, and RT-PCR was used to detect RHDV2 sequence. Following freezing, RNA was extracted a second time using the SwiftX™ Swabs Viral RNA Extraction Reagent. RHDV2 was detected in all nine samples. Cycle threshold values were higher in the RT-PCR following SwiftX extraction (mean: 3.79), indicating that the second extraction method resulted in approximately a 1 log10 reduction in sensitivity. A second freeze-thaw for the samples and less tissue extracted using SwiftX may have contributed additionally to the loss in sensitivity.

Sustained Impact of RHDV2 on Wild Rabbit Populations across Australia Eight Years after Its Initial Detection.

Following the arrival of rabbit haemorrhagic disease virus 2 (RHDV2) in Australia, average rabbit population abundances were reduced by 60% between 2014 and 2018 based on monitoring data acquired from 18 sites across Australia. During this period, as the seropositivity to RHDV2 increased, concurrent decreases were observed in the seroprevalence of both the previously circulating RHDV1 and RCVA, a benign endemic rabbit calicivirus. However, the detection of substantial RHDV1 seropositivity in juvenile rabbits suggested that infections were continuing to occur, ruling out the rapid extinction of this variant. Here we investigate whether the co-circulation of two pathogenic RHDV variants was sustained after 2018 and whether the initially observed impact on rabbit abundance was still maintained. We monitored rabbit abundance and seropositivity to RHDV2, RHDV1 and RCVA at six of the initial eighteen sites until the summer of 2022. We observed sustained suppression of rabbit abundance at five of the six sites, with the average population reduction across all six sites being 64%. Across all sites, average RHDV2 seroprevalence remained high, reaching 60-70% in adult rabbits and 30-40% in juvenile rabbits. In contrast, average RHDV1 seroprevalence declined to <3% in adult rabbits and 5-6% in juvenile rabbits. Although seropositivity continued to be detected in a low number of juvenile rabbits, it is unlikely that RHDV1 strains now play a major role in the regulation of rabbit abundance. In contrast, RCVA seropositivity appears to be reaching an equilibrium with that of RHDV2, with RCVA seroprevalence in the preceding quarter having a strong negative effect on RHDV2 seroprevalence and vice versa, suggesting ongoing co-circulation of these variants. These findings highlight the complex interactions between different calicivirus variants in free-living rabbit populations and demonstrate the changes in interactions over the course of the RHDV2 epizootic as it has moved towards endemicity. While it is encouraging from an Australian perspective to see sustained suppression of rabbit populations in the eight years following the arrival of RHDV2, it is likely that rabbit populations will eventually recover, as has been observed with previous rabbit pathogens.

The potential role of scavenging flies as mechanical vectors of Lagovirus europaeus/GI.2.

The European rabbit (Oryctolagus cuniculus) populations of the Iberian Peninsula have been severely affected by the emergence of the rabbit haemorrhagic disease virus (RHDV) Lagovirus europaeus/GI.2 (RHDV2/b). Bushflies and blowflies (Muscidae and Calliphoridae families, respectively) are important RHDV vectors in Oceania, but their epidemiological role is unknown in the native range of the European rabbit. In this study, scavenging flies were collected between June 2018 and February 2019 in baited traps at one site in southern Portugal, alongside a longitudinal capture-mark-recapture study of a wild European rabbit population, aiming to provide evidence of mechanical transmission of GI.2 by flies. Fly abundance, particularly from Calliphoridae and Muscidae families, peaked in October 2018 and in February 2019. By employing molecular tools, we were able to detect the presence of GI.2 in flies belonging to the families Calliphoridae, Muscidae, Fanniidae and Drosophilidae. The positive samples were detected during an RHD outbreak and absent in samples collected when no evidence of viral circulation in the local rabbit population was found. We were able to sequence a short viral genomic fragment, confirming its identity as RHDV GI.2. The results suggest that scavenging flies may act as mechanical vectors of GI.2 in the native range of the southwestern Iberian subspecies O. cuniculus algirus. Future studies should better assess their potential in the epidemiology of RHD and as a tool for monitoring viral circulation in the field.