Surveillance of Wildlife Viruses: Insights from South Australia's Monitoring of Rabbit Haemorrhagic Disease Virus (RHDV GI.1 and GI.2).

Surveillance of wildlife virus impacts can be passive or active. Both approaches have their strengths and weaknesses, especially regarding cost and knowledge that can be gained. Monitoring of rabbit haemorrhagic disease virus (GI.1 and GI.2) in South Australia has utilised both strategies and their methods and gained insights are discussed. Active strategies to monitor the continuing impact of rabbit haemorrhagic disease virus 2 (GI.2) on susceptible lagomorphs in countries such as the USA, Mexico, South Africa, Spain, France and Portugal are encouraged to gain critical insights into the evolution, spread and impact of this virus. Furthermore, there are lessons here for the international monitoring of diseases in wildlife, particularly where there is a risk of them becoming zoonotic.

MicroRNAs Regulate the Expression of Genes Related to the Innate Immune and Inflammatory Response in Rabbits Infected with Lagovirus europaeus GI.1 and GI.2 Genotypes.

MicroRNAs (miR) are a group of small, non-coding RNAs of 17-25 nucleotides that regulate gene expression at the post-transcriptional level. Dysregulation of miRNA expression or function may contribute to abnormal gene expression and signaling pathways, leading to disease pathology. Lagovirus europaeus (L. europaeus) causes severe disease in rabbits called rabbit hemorrhagic disease (RHD). The symptoms of liver, lung, kidney, and spleen degeneration observed during RHD are similar to those of acute liver failure (ALF) and multi-organ failure (MOF) in humans. In this study, we assessed the expression of miRs and their target genes involved in the innate immune and inflammatory response. Also, we assessed their potential impact on pathways in L. europaeus infection-two genotypes (GI.1 and GI.2)-in the liver, lungs, kidneys, and spleen. The expression of miRs and target genes was determined using quantitative real-time PCR (qPCR). We assessed the expression of miR-155 (MyD88, TAB2, p65, NLRP3), miR-146a (IRAK1, TRAF6), miR-223 (TLR4, IKKα, NLRP3), and miR-125b (MyD88). We also examined biomarkers of inflammation: IL-1ß, IL-6, TNF-α, and IL-18 in four tissues at the mRNA level. Our study shows that the main regulators of the innate immune and inflammatory response in L. europaeus/GI.1 and GI.2 infection, as well as RHD, are miR-155, miR-223, and miR-146a. During infection with L. europaeus/RHD, miR-155 has both pro- and anti-inflammatory effects in the liver and anti-inflammatory effects in the kidneys and spleen; miR-146a has anti-inflammatory effects in the liver, lungs and kidneys; miR-223 has anti-inflammatory effects in all tissues; however, miR-125b has anti-inflammatory effects only in the liver. In each case, such an effect may be a determinant of the pathogenesis of RHD. Our research shows that miRs may regulate three innate immune and inflammatory response pathways in L. europaeus infection. However, the result of this regulation may be influenced by the tissue microenvironment. Our research shows that infection of rabbits with L. europaeus/GI.1 and GI.2 genotypes causes an overexpression of two critical acute phase cytokines: IL-6 in all examined tissues and TNF-α (in the liver, lungs, and spleen). IL-1ß was highly expressed only in the lungs after L. europaeus infection. These facts indicate a strong and rapid involvement of the local innate immune and inflammatory response in L. europaeus infection-two genotypes (GI.1 and GI.2)-and in the pathogenesis of RHD. Profile of biomarkers of inflammation in rabbits infected with L. europaeus/GI.1 and GI.2 genotypes are similar regarding the nature of changes but are different for individual tissues. Therefore, we propose three inflammation profiles for L. europaeus infection for both GI.1 and GI.2 genotypes (pulmonary, renal, liver, and spleen).

Rabbit hemorrhagic disease virus 2, 2010-2023: a review of global detections and affected species.

Rabbit hemorrhagic disease virus 2/genotype GI.2 (RHDV2/GI.2; Caliciviridae, Lagovirus) causes a highly contagious disease with hepatic necrosis and disseminated intravascular coagulation in several Leporidae species. RHDV2 was first detected in European rabbits (Oryctolagus cuniculus) in France in 2010 and has since spread widely. We gather here data on viral detections reported in various countries and affected species, and discuss pathology, genetic differences, and novel diagnostic aspects. RHDV2 has been detected almost globally, with cases reported in Europe, Africa, Oceania, Asia, and North America as of 2023. Since 2020, large scale outbreaks have occurred in the United States and Mexico and, at the same time, cases have been reported for the first time in previously unaffected countries, such as China, Japan, Singapore, and South Africa, among others. Detections have been notified in domestic and wild European rabbits, hares and jackrabbits (Lepus spp.), several species of cottontail and brush rabbits (Sylvilagus spp.), pygmy rabbits (Brachylagus idahoensis), and red rock rabbits (Pronolagus spp.). RHDV2 has also been detected in a few non-lagomorph species. Detection of RHDV2 causing RHD in Sylvilagus spp. and Leporidae species other than those in the genera Oryctolagus and Lepus is very novel. The global spread of this fast-evolving RNA virus into previously unexploited geographic areas increases the likelihood of host range expansion as new species are exposed; animals may also be infected by nonpathogenic caliciviruses that are disseminated by almost all species, and with which genetic recombination may occur.

Evaluation of comparative effect between aluminum hydroxide gel and montanide (ISA 70) in potency and protection of locally prepared rabbit hemorrhagic disease virus 2 (RHDV2) vaccines in rabbits.

BACKGROUND: Rabbit hemorrhagic disease (RHD) is an acute infectious disease that damages the rabbit industry by producing significant mortality rates in young and adult rabbits. RHD is better controlled by vaccination. OBJECTIVE: The current study's goal was to prepare and evaluate the immuno-enhancing effect of montanide ISA70 and aluminum hydroxide (Al(OH)3) gel incorporated within the inactivated RHDV2 vaccine and assess the vaccine's protective efficacy against the homologous and heterologous local RHDV2 strains in rabbits. METHODS: Inactivated RHDV vaccines were prepared using Montanide ISA70 oil or Al(OH)3 gel adjuvants and submitted to sterility, safety, and potency tests. 200 rabbits were equally divided into 4 groups: G1 (control), G2 (vaccinated with gel-incorporated vaccine), G3 (vaccinated with montanide-incorporated vaccine), and G4 (vaccinated with gel- and montanide-incorporated vaccines). Individual blood samples were collected from one week to six months from all groups. The vaccine's potency was measured by the HI test and protection percentage post challenge. RESULTS: Data revealed slightly increasing HI titer means reaching the 1st peak at 4 weeks post-vaccination (7.33, 7.67, and 7.33 log2 in the 2nd, 3rd, and 4th groups, respectively), then slightly decreasing and peaked again, giving 9.33 log2 for the2nd group at 3 months post-vaccination (MPV), 10.67 log2 for 3rd the group, and 10.33 log2 for the 4th group at 5 months post-vaccination. Titer gradually decreased but remained protective. The protection rate ranged from 80-100% and 80-90% for homologous and heterologous local RHDV2 vaccines, respectively, within 3 weeks and 6 months post-challenge. The montanide oil RHDV2 vaccine induced better protection than the aluminum gel RHDV2 vaccine. CONCLUSION: The results demonstrated evidence of cross-protection between RHDV2 strains. The oil emulsion vaccine induced higher and longer-lasting antibody titers than those obtained with the RHDV2 aluminum gel vaccine.

Antibody response of endangered riparian brush rabbits to vaccination against rabbit hemorrhagic disease virus 2.

Rabbit hemorrhagic disease virus 2 (RHDV2; Caliciviridae, Lagovirus europaeus), the cause of a highly transmissible and fatal lagomorph disease, has spread rapidly through the western United States and Mexico, resulting in substantial mortality in domestic and wild rabbits. The disease was first detected in California in May 2020, prompting an interagency/zoo/academia/nonprofit team to implement emergency conservation actions to protect endangered riparian brush rabbits (Sylvilagus bachmani riparius) from RHDV2. Prior to vaccinating wild rabbits, we conducted a vaccine safety trial by giving a single SC dose of Filavac VHD K C+V (Filavie) vaccine to 19 adult wild riparian brush rabbits captured and temporarily held in captivity. Rabbits were monitored for adverse effects, and serum was collected before vaccination, and at 7-10, 14-20, and 60 d post-vaccination. Sera were tested using an ELISA to determine antibody response and timing of seroconversion. Reverse-transcription quantitative real-time PCR (RT-qPCR) was performed on rectal swabs to evaluate infection status. No adverse effects from the vaccine were observed. Before vaccination, 18 of 19 rabbits were seronegative, and RHDV2 was not detected by RT-qPCR on any rectal swabs. After vaccination, all rabbits developed an antibody response, with titers of 1:10-1:160. Seroconversion generally occurred at 7-10 d. The duration of antibody response was ≥60 d in 12 of 13 rabbits. Sixteen animals were released and 4 were recaptured several months later, offering a glimpse into longer duration immune response. Our study has informed vaccination strategies for this species and serves as a model for protecting other vulnerable lagomorphs against RHDV2.

Practical Suggestions for Assessing Rabbit Haemorrhagic Disease Virus 2 Risk to Endangered Native Lagomorphs in North America and Southern Africa.

A new form of the rabbit haemorrhagic disease virus, RHDV2, first observed in European rabbits, has spread widely among different species of hares in Europe, jackrabbits and cottontails in North America, and hares in southern Africa. However, only limited surveillance studies have been undertaken so far. It is suggested that methods developed for controlling the disease in farmed rabbits in Europe and studying the efficacy of RHDV as a biological control agent in Australia could facilitate epidemiological research on those recently affected lagomorph species. This would enable the assessment of the risk of RHDV2 to native lagomorphs, including endangered species, and the determination of the main host species of RHDV2. Because RHDV2 has not spread equally through all lagomorph species, epidemiological studies could give insights into factors important for determining host susceptibility.

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.

Development of a peptide-generated antibody to rabbit hemorrhagic disease virus 2 VP60 and its immunohistochemical application in natural cases.

Rabbit hemorrhagic disease virus 2 (RHDV2) has spread across the United States infecting and causing death in domestic and wild rabbits. Immunohistochemistry (IHC) would be a useful tool for the detection of RHDV2 antigen in tissues as it is inexpensive and readily achievable in most diagnostic laboratories. However, there is no readily available antibody for this purpose. To fill this void, we generated an RHDV2 capsid protein VP60-specific antibody in chicken eggs and validated the antibody using formalin-fixed tissues from 5 domestic rabbits naturally infected with RHDV2. Viral antigen was detected immunohistochemically in various tissues, most prominently in hepatocytes and macrophages in liver, and in macrophages in spleen and cecal lymphoid tissue. Intravascular mononuclear cells in lung and renal tubular and biliary epithelium also were immunolabeled. Both nuclear and cytoplasmic immunolabeling were observed. This peptide-generated antibody is a potentially useful tool as an adjunct to reverse-transcription PCR or in situ hybridization for detection of RHDV2 in tissues.

Detections of Rabbit Hemorrhagic Disease Virus 2 (RHDV2) Following the 2020 Outbreak in Wild Lagomorphs across the Western United States.

Rabbit hemorrhagic disease virus 2 (RHDV2) is a highly infectious, often fatal viral disease that affects both domestic and wild lagomorph species. In the United States (U.S.), the virus first was detected in wild lagomorph populations in the southwest in March 2020 and has continued to be detected in native North American lagomorph species over several years. The susceptibility of host species and exact mechanisms of environmental transmission across the U.S. landscape remain poorly understood. Our study aims to increase the understanding of RHDV2 in wild lagomorph populations by providing a history of detection. We present and summarize results from all RHDV2-suspect wild lagomorph morbidity and mortality samples submitted for diagnostic testing in the U.S. from March 2020 to March 2024. Samples were submitted from 916 wild lagomorphs across eight native North American species in 14 western states, of which 313 (34.2%) tested positive by RHDV2 RT-qPCR. Detections of RHDV2 in pygmy rabbits (Brachylagus idahoensis) and riparian brush rabbits (Sylvilagus bachmani riparius) suggest that the risk to threatened and endangered species warrants more attention. Continuing to investigate wild lagomorph morbidity and mortality events and tracking RHDV2 detections over time can help inform on disease epidemiology and wild lagomorph population trends.

Causes of morbidity and mortality in wild cottontail rabbits in the eastern United States, 2013-2022.

Interest in causes of mortality of free-ranging, native North American lagomorphs has grown with the emergence of rabbit hemorrhagic disease virus 2 (RHDV2). Over the years 2013-2022, the Southeastern Cooperative Wildlife Disease Study received 119 Sylvilagus spp. case submissions from the central and eastern United States, comprising 147 rabbits. Most (86%) of these submissions occurred after detecting RHDV2 in the United States in 2020. Laboratory data from these rabbits were retrospectively evaluated for major causes, contributors to mortality, and pathogen detections. Gross and histologic examination was performed for 112 rabbits. Common primary causes of death included trauma (n = 49), bacterial disease (n = 31), emaciation (n = 6), and parasitism (n = 6). Among the 32 rabbits with bacterial disease, 12 were diagnosed with tularemia and 7 with pasteurellosis. Rabbits with pasteurellosis had disseminated abscessation, septicemia, and/or polyserositis. Less commonly, cutaneous fibroma (n = 2), notoedric mange (n = 2), encephalitozoonosis (n = 2), neoplasia (round-cell sarcoma; n = 1), and congenital abnormalities (n = 1) were diagnosed. RHDV2 was not detected in 123 rabbits tested. Although RHDV2 has not been detected in wild lagomorphs in the eastern United States, detections in domestic rabbits from the region emphasize the need for continued surveillance. Furthermore, continued surveillance for Francisella tularensis informs public health risk. Overall, increased knowledge of Sylvilagus spp. health furthers our understanding of diseases affecting these important prey and game species.

Screening optimal DC-targeting peptide to enhance the immune efficacy of recombinant Lactobacillus expressing RHDV VP60.

Dendritic cells (DCs) present an ideal target for delivering immunogenic cargo due to their potent antigen-presenting capabilities. This targeting approach holds promise in vaccine development by enhancing the efficiency of antigen recognition and capture by DCs. To identify a high-affinity targeting peptide binding to rabbit DCs, rabbit monocyte-derived DCs (raMoDCs) were isolated and cultured, and a novel peptide, HS (HSLRHDYGYPGH), was identified using a phage-displayed peptide library. Alongside HS, two other DC-targeting peptides, KC1 and MY, previously validated in our laboratory, were employed to construct recombinant Lactgobacillus reuteri fusion-expressed rabbit hemorrhagic disease virus (RHDV) capsid protein VP60. These recombinant Lactobacillus strains were named HS-VP60/L. reuteri, KC1-VP60/L. reuteri, and MY-VP60/L. reuteri. The ability of these recombinant Lactobacillus to bind rabbit DCs was evaluated both in vivo and in vitro. Results demonstrated that the DC-targeting peptide KC1 significantly enhanced the capture efficiency of recombinant Lactobacillus by raMoDCs, promoted DC maturation, and increased cytokine secretion. Furthermore, oral administration of KC1-VP60/L. reuteri effectively induced SIgA and IgG production in rabbits, prolonged rabbit survival post-challenge, and reduced RHDV copies in organs. In summary, the DC-targeting peptide KC1 exhibited robust binding to raMoDCs, and recombinant Lactobacillus expressing KC1-VP60 protein antigens efficiently induced systemic and mucosal immune responses in rabbits, conferring protective efficacy against RHDV. This study offers valuable insights for the development of novel RHDV vaccines.

Characterisation of Lagovirus europaeus GI-RHDVs (Rabbit Haemorrhagic Disease Viruses) in Terms of Their Pathogenicity and Immunogenicity.

Rabbit haemorrhagic disease viruses (RHDV) belong to the family Caliciviridae, genus Lagovirus europaeus, genogroup GI, comprising four genotypes GI.1-GI.4, of which the genotypes GI.1 and GI.2 are pathogenic RHD viruses, while the genotypes GI.3 and GI.4 are non-pathogenic RCV (Rabbit calicivirus) viruses. Among the pathogenic genotypes GI.1 and GI.2 of RHD viruses, an antigenic variant of RHDV, named RHDVa-now GI.1a-RHDVa, was distinguished in 1996; and in 2010, a variant of RHDV-named RHDVb, later RHDV2 and now GI.2-RHDV2/b-was described; and recombinants of these viruses were registered. Pathogenic viruses of the genotype GI.1 were the cause of a disease described in 1984 in China in domestic (Oryctolagus (O.) cuniculus domesticus) and wild (O. cuniculus) rabbits, characterised by a very rapid course and a mortality rate of 90-100%, which spread in countries all over the world and which has been defined since 1989 as rabbit haemorrhagic disease. It is now accepted that GI.1-RHDV, including GI.1a-RHDVa, cause the predetermined primary haemorrhagic disease in domestic and wild rabbits, while GI.2-RHDV2/b cause it not only in rabbits, including domestic rabbits' young up to 4 weeks and rabbits immunised with rabbit haemorrhagic disease vaccine, but also in five various species of wild rabbits and seven different species of hares, as well as wild ruminants: mountain muskoxen and European badger. Among these viruses, haemagglutination-positive, doubtful and harmful viruses have been recorded and described and have been shown to form phylogenogroups, immunotypes, haematotypes and pathotypes, which, together with traits that alter and expand their infectious spectrum (rabbit, hare, wild ruminant, badger and various rabbit and hare species), are the determinants of their pathogenicity (infectivity) and immunogenicity and thus shape their virulence. These relationships are the aim of our consideration in this article.

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.

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.

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.

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.