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.

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.

Social interactions of juvenile rabbits (Oryctolagus cuniculus) and their potential role in lagovirus transmission.

Rabbit Haemorrhagic Disease Virus (RHDV), which is a calicivirus, is used as a biocontrol agent to suppress European wild rabbit populations in Australia. The transmission of RHDV can be influenced by social interactions of rabbits; however, there is a paucity of this knowledge about juvenile rabbits and the roles they may play in the transmission of RHDV. We aimed to quantify the social interactions of juvenile (< 900 g) and adult (> 1200 g) rabbits in a locally abundant population in the Central Tablelands of New South Wales, Australia. Twenty-six juvenile and 16 adult rabbits were fitted with VHF proximity loggers to monitor intra- and inter-group pairings. Use of multiple warrens by these rabbits was investigated using VHF base stations at nine warrens and on foot with a hand-held Yagi antenna. Juvenile rabbits were strongly interconnected with both juveniles and adults within and outside their warren of capture, and almost all juveniles were well-connected to other individuals within their own social group. Inter-group pairings were infrequent and fleeting between adults. Both juvenile and adult rabbits used multiple warrens. However, visits to warrens outside their warren of capture, particularly those within 50 m, were more common and longer in duration in juveniles than in adults. The high connectivity of juveniles within and between warrens in close proximity increases potential pathogen exchange between warrens. Therefore, juvenile rabbits could be of greater importance in lagovirus transmission than adult rabbits. The strength of juvenile rabbit inter- and intra-group pairings, and their tendency to use multiple warrens, highlight their potential to act as 'superspreaders' of both infection and immunity for lagoviruses and other pathogens with similar lifecycles. Confirmation of this potential is required through examination of disease progress and rabbit age-related immune responses during outbreaks.

Potent Protease Inhibitors of Highly Pathogenic Lagoviruses: Rabbit Hemorrhagic Disease Virus and European Brown Hare Syndrome Virus.

Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are highly contagious diseases caused by lagoviruses in the Caliciviridae family. These infectious diseases are associated with high mortality and a serious threat to domesticated and wild rabbits and hares, including endangered species such as riparian brush rabbits (Sylvilagus bachmani riparius). In the United States (U.S.), only isolated cases of RHD had been reported until Spring 2020. However, RHD caused by GI.2/rabbit hemorrhagic disease virus (RHDV)2/b was unexpectedly reported in April 2020 in New Mexico and has subsequently spread to several U.S. states, infecting wild rabbits and hares and making it highly likely that RHD will become endemic in the U.S. Vaccines are available for RHD; however, there is no specific treatment for this disease. Lagoviruses encode a 3C-like protease (3CLpro), which is essential for virus replication and a promising target for antiviral drug development. We have previously generated focused small-molecule libraries of 3CLpro inhibitors and demonstrated the in vitro potency and in vivo efficacy of some protease inhibitors against viruses encoding 3CLpro, including caliciviruses and coronaviruses. Here, we report the development of the enzyme and cell-based assays for the 3CLpro of GI.1c/RHDV, recombinant GI.3P-GI.2 (RHDV2/b), and GII.1/European brown hare syndrome virus (EBHSV) as well as the identification of potent lagovirus 3CLpro inhibitors, including GC376, a protease inhibitor being developed for feline infectious peritonitis. In addition, structure-activity relationship study and homology modeling of the 3CLpro and inhibitors revealed that lagovirus 3CLpro share similar structural requirements for inhibition with other calicivirus 3CLpro. IMPORTANCE Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are viral diseases that affect lagomorphs with significant economic and ecological impacts. RHD vaccines are available, but specific antiviral treatment for these viral infections would be a valuable addition to the current control measures. Lagoviruses encode 3C-like protease (3CLpro), which is essential for virus replication and an attractive target for antiviral drug discovery. We have screened and identified potent small-molecule inhibitors that block lagovirus 3CLpro in the enzyme- and cell-based assays. Our results suggest that these compounds have the potential for further development as antiviral drugs for lagoviruses.

Hemoglobin subunit beta interacts with the capsid, RdRp and VPg proteins, and antagonizes the replication of rabbit hemorrhagic disease virus.

Rabbit hemorrhagic disease virus (RHDV) causes a highly contagious disease in rabbits that is associated with high mortality. Because of the lack of a suitable cell culture system for RHDV, its pathogenic mechanism and replication remain unclear. This study found that the expression level of host protein rabbit hemoglobin subunit beta (HBB) was significantly downregulated in RHDV-infected cells. To investigate the role of HBB in RHDV replication, small interfering RNAs for HBB and HBB eukaryotic expression plasmids were used to change the expression level of HBB in RK-13 cells and the results showed that the RHDV replication level was negatively correlated with the expression level of HBB. It was also verified that HBB inhibited RHDV replication using constructed HBB stable overexpression cell lines and HBB knockout cell lines. The interaction of HBB with viral capsid protein VP60, replicase RdRp, and VPg protein was confirmed, as was the activation of the expression of interferon γ by HBB. The results of this study indicated that HBB may be an important host protein in host resistance to RHDV infection.

Infectivity of rabbit haemorrhagic disease virus excreted in rabbit faecal pellets.

Rabbit haemorrhagic disease (RHD) is caused by a lagovirus affecting European rabbits (Oryctolagus cuniculus). Viral RNA is detected in tissues or faeces of convalescent rabbits, suggesting persistent infections; however, this RNA has not been shown to be related to infective viruses to date. In the present work, seven laboratory rabbits were challenged with the RHDV2/b virus variant. Viral RNA was individually detected by duplex qPCR in faeces collected for four weeks after infection, and the infective capacity of viral RNA excreted in the faeces of surviving rabbits was tested by challenging new rabbits with faecal inocula. As results, viral RNA was detected in faeces until the end of the assay. Viral RNA detected in the fourth week was infective only in the case of one rabbit that did not exhibit clear seroconversion, suggesting persistent infection as a result of an impaired immune response. Since the surviving rabbits were apparently healthy individuals, the importance of detecting carriers and the correct management of faeces to control RHD outbreaks in rabbitries are highlighted.

Melatonin modulates mitophagy, innate immunity and circadian clocks in a model of viral-induced fulminant hepatic failure.

The haemorrhagic disease virus (RHDV) is a non-cultivable virus that promotes in rabbits an acute disease which accomplishes many characteristics of an animal model of fulminant hepatic failure (FHF). Beneficial effects of melatonin have been reported in RHDV-infected rabbits. This study investigated whether protection against viral-derived liver injury by melatonin is associated with modulation of mitophagy, innate immunity and clock signalling. Rabbits were experimentally infected with 2 × 104 haemagglutination units of a RHDV isolate and killed at 18, 24 and 30 hours after infection (hpi). Melatonin (20 mg/kg body weight ip) was administered at 0, 12 and 24 hpi. RHDV infection induced mitophagy, with the presence of a high number of mitophagosomes in hepatocytes and increased expression of mitophagy genes. Greater expression of main innate immune intermediaries and inflammasome components was also found in livers with RHDV-induced FHF. Both mitophagy and innate immunity activation was significantly hindered by melatonin. FHF induction also elicited an early dysregulation in clock signalling, and melatonin was able to prevent such circadian disruption. Our study discloses novel molecular routes contributing to RHDV-induced damage progression and supports the potential of melatonin as a promising therapeutic option in human FHF.

Worldwide rapid spread of the novel rabbit haemorrhagic disease virus (GI.2/RHDV2/b).

We describe the extremely rapid worldwide spread of the Lagovirus europaeus/GI.2/RHDV2/b (henceforth GI.2), the causative infectious agent of the so-called 'novel' rabbit haemorrhagic disease of the European rabbit (Oryctolagus cuniculus). We tracked down all novel confirmed detections of GI.2 between May 2010 and November 2018 by carrying out a two-step in-depth review. We suggest that such spread would not have been possible without anthropogenic involvement. Our results also point out the importance of reviewing and adapting the protocols of virus detection and management in order to control, mitigate and contain properly, not only GI.2, but also new viruses that may emerge in the future.

Nucleolin mediates the internalization of rabbit hemorrhagic disease virus through clathrin-dependent endocytosis.

Rabbit hemorrhagic disease virus (RHDV) is an important member of the Caliciviridae family and a highly lethal pathogen in rabbits. Although the cell receptor of RHDV has been identified, the mechanism underlying RHDV internalization remains unknown. In this study, the entry and post-internalization of RHDV into host cells were investigated using several biochemical inhibitors and RNA interference. Our data demonstrate that rabbit nucleolin (NCL) plays a key role in RHDV internalization. Further study revealed that NCL specifically interacts with the RHDV capsid protein (VP60) through its N-terminal residues (aa 285-318), and the exact position of the VP60 protein for the interaction with NCL is located in a highly conserved region (472Asp-Val-Asn474; DVN motif). Following competitive blocking of the interaction between NCL and VP60 with an artificial DVN peptide (RRTGDVNAAAGSTNGTQ), the internalization efficiency of the virus was markedly reduced. Moreover, NCL also interacts with the C-terminal residues of clathrin light chain A, which is an important component in clathrin-dependent endocytosis. In addition, the results of animal experiments also demonstrated that artificial DVN peptides protected most rabbits from RHDV infection. These findings demonstrate that NCL is involved in RHDV internalization through clathrin-dependent endocytosis.

Robust Innate Immunity of Young Rabbits Mediates Resistance to Rabbit Hemorrhagic Disease Caused by Lagovirus Europaeus GI.1 But Not GI.2.

The rabbit caliciviruses Lagovirus europaeus GI.1 and GI.2 both cause acute necrotizing hepatitis in European rabbits (Oryctolagus cuniculus). Whilst GI.2 is highly virulent in both young and adult rabbits, rabbits younger than eight weeks of age are highly resistant to disease caused by GI.1, although they are still permissive to infection and viral replication. To investigate the underlying mechanism(s) of this age related resistance to GI.1, we compared liver transcriptomes of young rabbits infected with GI.1 to those of adult rabbits infected with GI.1 and young rabbits infected with GI.2. Our data suggest that kittens have constitutively heightened innate immune responses compared to adult rabbits, particularly associated with increased expression of major histocompatibility class II molecules and activity of natural killer cells, macrophages, and cholangiocytes. This enables them to respond more rapidly to GI.1 infection than adult rabbits and thus limit virus-induced pathology. In contrast, these responses were not fully developed during GI.2 infection. We speculate that the observed downregulation of multiple genes associated with innate immunity in kittens during GI.2 infection may be due to virally-mediated immunomodulation, permitting fatal disease to develop. Our study provides insight into the fundamental host⁻pathogen interactions responsible for the differences in age-related susceptibility, which likely plays a critical role in defining the success of GI.2 in outcompeting GI.1 in the field.

Elucidation of the pathology and tissue distribution of Lagovirus europaeus GI.2/RHDV2 (rabbit haemorrhagic disease virus 2) in young and adult rabbits (Oryctolagus cuniculus).

Lagovirus europaeus GI.2, also known as RHDV2 or RHDVb, is an emerging virus that causes rabbit haemorrhagic disease (RHD) in European rabbits (Oryctolagus cuniculus). In contrast to L. europaeus GI.1 (or RHDV/RHDVa) viruses that are only pathogenic for adults, GI.2 causes clinical disease in both adults and kittens. However, detailed descriptions of the pathology of this virus that may provide insight into its pathogenicity and emergence are lacking. Using an Australian GI.2 field strain isolated in 2015, we provide the first detailed description of pathology, viral antigen distribution and tissue load of GI.2 in adult and 5-week old New Zealand white rabbits using histology, immunohistochemistry and RT-qPCR. Liver was the target organ, but in contrast to GI.1 viruses, lesions and inflammatory responses did not differ between adults and kittens. Lymphocytic inflammation, proposed to be protective in kittens infected with GI.1, was notably absent. We also present the first descriptions of bone marrow changes in RHD, including decreased myeloid-to-erythroid ratio. Consistent with other pathogenic lagoviruses, intracellular viral antigen was demonstrated in hepatocytes and cells of the mononuclear phagocytic system. In terminal stages of disease, viral loads were highest in liver, serum and spleen. Despite the small sample size, our data suggest that unlike early European GI.2 strains, the pathogenicity of the Australian GI.2 virus is similar to GI.1 viruses. Additionally, GI.2 was fatal for all (n = 5) inoculated kittens in this study. This may significantly alter RHD epidemiology in the field, and may impact biocontrol programs for invasive rabbits in Australia where GI.1 viruses are intentionally released.

Host-Specific Glycans Are Correlated with Susceptibility to Infection by Lagoviruses, but Not with Their Virulence.

Rabbit hemorrhagic disease virus (RHDV) and European brown hare syndrome virus (EBHSV) are two lagoviruses from the family Caliciviridae that cause fatal diseases in two leporid genera, Oryctolagus and Lepus, respectively. In the last few years, several examples of host jumps of lagoviruses among leporids were recorded. In addition, a new pathogenic genotype of RHDV emerged, and many nonpathogenic strains of lagoviruses have been described. The molecular mechanisms behind host shifts and the emergence of virulence are unknown. Since RHDV uses glycans of the histo-blood group antigen type as attachment factors to initiate infection, we studied if glycan specificities of the new pathogenic RHDV genotype, nonpathogenic lagoviruses, and EBHSV potentially play a role in determining the host range and virulence of lagoviruses. We observed binding to A, B, or H antigens of the histo-blood group family for all strains known to primarily infect European rabbits (Oryctolagus cuniculus), which have recently been classified as GI strains. However, we could not explain the emergence of virulence, since similar glycan specificities were found in several pathogenic and nonpathogenic strains. In contrast, EBHSV, recently classified as GII.1, bound to terminal ß-linked N-acetylglucosamine residues of O-glycans. Expression of these attachment factors in the upper respiratory and digestive tracts in three lagomorph species (Oryctolagus cuniculus, Lepuseuropaeus, and Sylvilagus floridanus) showed species-specific patterns regarding susceptibility to infection by these viruses, indicating that species-specific glycan expression is likely a major contributor to lagovirus host specificity and range.IMPORTANCE Lagoviruses constitute a genus of the family Caliciviridae comprising highly pathogenic viruses, RHDV and EBHSV, that infect rabbits and hares, respectively. Recently, nonpathogenic strains were discovered and new pathogenic strains have emerged. In addition, host jumps between lagomorphs have been observed. The mechanisms responsible for the emergence of pathogenicity and host species range are unknown. Previous studies showed that RHDV strains attach to glycans expressed in the upper respiratory and digestive tracts of rabbits, the likely portals of virus entry. Here, we studied the glycan-binding properties of novel pathogenic and nonpathogenic strains looking for a link between glycan binding and virulence or between glycan specificity and host range. We found that glycan binding did not correlate with virulence. However, expression of glycan motifs in the upper respiratory and digestive tracts of lagomorphs revealed species-specific patterns associated with the host ranges of the virus strains, suggesting that glycan diversity contributes to lagovirus host ranges.

Multi-target chimaeric VLP as a therapeutic vaccine in a model of colorectal cancer.

BACKGROUND: Colorectal cancer is responsible for almost 700,000 deaths annually worldwide. Therapeutic vaccination is a promising alternative to conventional treatment for colorectal cancer, using vaccines to induce targeted immune responses against tumour-associated antigens. In this study, we have developed chimaeric virus-like particles (VLP), a form of non-infectious non-replicative subunit vaccine consisting of rabbit haemorrhagic disease virus (RHDV) VP60 capsid proteins containing recombinantly inserted epitopes from murine topoisomerase IIα and survivin. These vaccines were developed in mono- (T.VP60, S.VP60) and multi-target (TS.VP60) forms, aiming to elucidate the potential benefits from multi-target vaccination. METHODS: Chimaeric RHDV VLP were developed by recombinantly inserting immune epitopes at the N-terminus of VP60. Vaccines were tested against a murine model of colorectal cancer by establishing MC38-OVA tumours subcutaneously. Unmethylated CpG DNA oligonucleotides (CpGs) were used as a vaccine adjuvant. Statistical tests employed included the Mantel-Cox log-rank test, ANOVA and unpaired t-tests depending on the data analysed, with a post hoc Bonferroni adjustment for multiple measures. RESULTS: Chimaeric RHDV VLP were found to form a composite particle in the presence of CpGs. Overall survival was significantly improved amongst mice bearing MC38-OVA tumours following vaccination with T.VP60 (60%, 9/15), S.VP60 (60%, 9/15) or TS.VP60 (73%, 11/15). TS.VP60 significantly prolonged the vaccine-induced remission period in comparison to each mono-therapy. CONCLUSIONS: Chimaeric VLP containing multiple epitopes were found to confer an advantage for therapeutic vaccination in a model of colorectal cancer based on the prolongation of remission prior to tumour escape.

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.

RNA-Dependent RNA Polymerases of Both Virulent and Benign Rabbit Caliciviruses Induce Striking Rearrangement of Golgi Membranes.

The extremely pathogenic Rabbit haemorrhagic disease virus (RHDV) and the completely benign Rabbit calicivirus (RCV) are closely related members of the genus Lagovirus (family Caliciviridae). The molecular mechanisms that determine the dramatic difference in virulence are unknown, but indirect evidence suggests that different properties of their RNA-dependent RNA polymerases (RdRps) may at least partially be responsible for the contrasting phenotypes. Here we report that the unusual ability of the RHDV RdRp to induce a striking rearrangement of the Golgi network is not specific to RHDV, but a common feature of virulent and benign rabbit caliciviruses alike. Expression of rabbit calicivirus RdRps induced a redistribution of both cis/medial and medial/trans Golgi membrane markers, but not that of an endoplasmic reticulum membrane marker. Inactivating mutations in the conserved GDD motif did not abolish the ability of RHDV RdRp to rearrange the Golgi network, suggesting that polymerase activity and metal co-factors are not required for this function. Finally, we discuss possible implications of RdRp-induced membrane rearrangements on virus replication and host immune responses.

Spillover Events of Infection of Brown Hares (Lepus europaeus) with Rabbit Haemorrhagic Disease Type 2 Virus (RHDV2) Caused Sporadic Cases of an European Brown Hare Syndrome-Like Disease in Italy and Spain.

Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that can cause fatal hepatitis (rabbit haemorrhagic disease, RHD) with mortality of 80-90% in farmed and wild rabbits. Since 1986, RHDV has caused outbreaks in rabbits (Oryctolagus cuniculus) in Europe, but never in European brown hares (Lepus europaeus, EBH). In 2010, a new RHDV-related virus, called RHDV2, emerged in Europe, causing extended epidemics because it largely overcame the immunity to RHDV present in most rabbit populations. RHDV2 also was identified in Cape hare (Lepus capensis subsp. mediterraneus) and in Italian hare (Lepus corsicanus). Here, we describe two distinct incidents of RHDV2 infection in EBH that occurred in Italy (2012) and Spain (2014). The two RHDV2 strains caused macroscopic and microscopic lesions similar to European brown hare syndrome (EBHS) in hares, and they were genetically related to other RHDV2 strains in Europe. EBHs are common in Europe, often sharing habitat with rabbits. They likely have been exposed to high levels of RHDV2 during outbreaks in rabbits in recent years, yet only two incidents of RHDV2 in EBHs have been found in Italy and Spain, suggesting that EBHs are not a primary host. Instead, they may act as spillover hosts in situations when infection pressure is high and barriers between rabbits and hares are limited, resulting in occasional infections causing EBHS-like lesions. The serological survey of stocked hare sera taken from Italian and Spanish hare populations provided an understanding of naturally occurring RHDV2 infection in the field confirming its sporadic occurrence in EBH. Our findings increase the knowledge on distribution, host range and epidemiology of RHDV2.

Effects of myxoma virus and rabbit hemorrhagic disease virus on the physiological condition of wild European rabbits: Is blood biochemistry a useful monitoring tool?

Myxomatosis and rabbit hemorrhagic disease (RHD) are the major viral diseases that affect the wild European rabbit (Oryctolagus cuniculus). These diseases arrived in Europe within the last decades and have caused wild rabbit populations to decline dramatically. Both viruses are currently considered to be endemic in the Iberian Peninsula; periodic outbreaks that strongly impact wild populations regularly occur. Myxoma virus (MV) and rabbit hemorrhagic disease virus (RHDV) alter the physiology of infected rabbits, resulting in physical deterioration. Consequently, the persistence and viability of natural populations are affected. The main goal of our study was to determine if blood biochemistry is correlated with serostatus in wild European rabbits. We carried out seven live-trapping sessions in three wild rabbit populations over a two-year period. Blood samples were collected to measure anti-MV and anti-RHDV antibody concentrations and to measure biochemical parameters related to organ function, protein metabolism, and nutritional status. Overall, we found no significant relationships between rabbit serostatus and biochemistry. Our main result was that rabbits that were seropositive for both MV and RHDV had low gamma glutamyltransferase concentrations. Given the robustness of our analyses, the lack of significant relationships may indicate that the biochemical parameters measured are poor proxies for serostatus. Another explanation is that wild rabbits might be producing attenuated physiological responses to these viruses because the latter are now enzootic in the study area.

Extensive characterization of a lentiviral-derived stable cell line expressing rabbit hemorrhagic disease virus VPg protein.

Rabbit hemorrhagic disease virus (RHDV) is an important member of the caliciviridae family. Currently, no suitable tissue culture system is available for proliferating RHDV, which limits the study of its pathogenesis. To bypass this obstacle, we established a cell line, RK13-VPg, stably expressing the VPg gene with a lentivirus packaging system in this study. In addition, the recently constructed RHDV replicon in our laboratory provided an appropriate model for studying the pathogenesis of RHDV without in vitro RHDV propagation and culture. Using this RHDV replicon and RK13-VPg cell line, we further demonstrated that the presence of VPg protein is essential for efficient translation of an RHDV replicon. Therefore, the RK13-VPg cell line is a powerful tool for studying the replication and translation mechanisms of RHDV.

Expression of IL-1ß, IL-2, IL-10, TNF-ß and GM-CSF in peripheral blood leukocytes of rabbits experimentally infected with rabbit haemorrhagic disease virus.

Rabbit haemorrhagic disease (RHD) is a highly morbid and mortal viral infection of European rabbits. This disease is one of the main causes of death in wild rabbits, and results in large economic losses in farms of rabbits worldwide. Although the first outbreak of this disease was noted in 1984, the pathogenesis of RHD and mechanisms of RHDV (rabbit haemorrhagic disease virus) pathogenecity have still not been fully elucidated. Recent studies indicate a role of the immune response, especially peripheral blood leukocytes (PBL), in the pathogenesis of this disease. Thus, in the present study we investigated the expression of IL-1ß, IL-2, IL-10, TNF-ß and GM-CSF genes in PBL of RHDV-infected rabbits. We also compared the expression of genes encoding these cytokines in rabbits with different course of RHDV infection (in animals that died 36h postinfection or survived until 60th h after infection). The study revealed that three (IL-10, TNF-ß and GM-CSF) out of five investigated genes encoding cytokines showed increased expression in PBL of RHDV-infected rabbits, and the level of expression depended on the course of RHD. The results indicate the potential role of these cytokines in RHDV infection and their influence on the survival time of infected rabbits.

Expression and partial characterisation of rabbit haemorrhagic disease virus non-structural proteins.

The intracellular replication and molecular virulence mechanisms of Rabbit haemorrhagic disease virus (RHDV) are poorly understood, mainly due to the lack of an effective cell culture system for this virus. To increase our understanding of RHDV molecular biology, the subcellular localisation of recombinant non-structural RHDV proteins was investigated in transiently transfected rabbit kidney (RK-13) cells. We provide evidence for oligomerisation of p23, and an ability of the viral protease to cleave the p16:p23 junction in trans, outside the context of the nascent polyprotein chain. Notably, expression of the viral polymerase alone and in the context of the entire RHDV polyprotein resulted in a redistribution of the Golgi network. This suggests that, similar to other positive-strand RNA viruses, RHDV may recruit membranes of the secretory pathway during replication, and that the viral polymerase may play a critical role during this process.