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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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Serological tests for the presence of antibodies to European brown hare syndrome virus and rabbit hemorrhagic disease virus type a and type 2 were performed on 275 samples of blood serum from the European brown hare by hemagglutination inhibition test and enzyme-linked immunosorbent assay. The presence of antibodies against European brown hare syndrome virus was 28% in the Czech Republic while in the Slovak Republic between 9–33%. Furthermore, the results showed the possibility of interspecies transmission, both rabbit hemorrhagic disease virus type a and rabbit hemorrhagic disease virus type 2 on the European brown hare. In case of RHDVa it is the first documented interspecies transmission, which has not yet been described. This study improved our knowledge about circulation of RHDV in Central European ecosystems and its possible ability to cross interspecies barriers. However, from an epizootiological point of view, it is likely European brown hare is not a significant source of RHD infection for domesticated rabbits. [ABSTRACT FROM AUTHOR]

Rabbit hemorrhagic disease virus 2 (RHDV2), recently detected in the western United States, has the potential to cause mass mortality events in wild rabbit and hare populations. Currently, few management strategies exist other than vaccination. We developed a spatially explicit model of RHDV2 for a population of riparian brush rabbits (Sylvilagus bachmani riparius), a subspecies of brush rabbit classified as endangered in the United States, on a subsection of the San Joaquin River National Wildlife Refuge. The goal of our model was to provide guidance regarding vaccination strategies for an endangered rabbit species. Our model predicts that increased interactions between rabbits (a proxy for landscape connectivity) and disease transmission rates among susceptible hosts (individual brush rabbits and conspecifics) have the greatest influence on the outcome of a potential vaccination campaign. Our model projects that across a range of parameter estimates (given an RHDV2 incursion), the median estimated population size with a 0%–10% vaccination rate after 1 year is 538 rabbits (95% Confidence Interval [C.I.] 69–1235), approximately 36% of the expected size of the study population of 1470 rabbits without an RHDV2 introduction. With a 10%–20%, 20%–30%, or 30%–40% vaccination rate, the median estimated population size increased to 628 rabbits (95% C.I. 130–1298), 723 rabbits (95% C.I. 198–1317), and 774 rabbits (95% C.I. 228–1410), respectively. These estimates represent 43%, 49%, and 53% of the expected population size without an RHDV2 introduction. Overall, a 1% increase in vaccination rate was associated with a six rabbit (95% C.I. 5–7) increase in total remaining population size. This result is dependent on assumptions regarding environmental transmission, home range size (and contact rates of rabbits). Given the relatively short lifespan of rabbits and the potential need for boosters, vaccination programs are most likely to be successful for small target populations where relatively high vaccination rates can be maintained. [ABSTRACT FROM AUTHOR]

Abstract Rabbit hemorrhagic disease virus 2 (RHDV2), recently detected in the western United States, has the potential to cause mass mortality events in wild rabbit and hare populations. Currently, few management strategies exist other than vaccination. We developed a spatially explicit model of RHDV2 for a population of riparian brush rabbits (Sylvilagus bachmani riparius), a subspecies of brush rabbit classified as endangered in the United States, on a subsection of the San Joaquin River National Wildlife Refuge. The goal of our model was to provide guidance regarding vaccination strategies for an endangered rabbit species. Our model predicts that increased interactions between rabbits (a proxy for landscape connectivity) and disease transmission rates among susceptible hosts (individual brush rabbits and conspecifics) have the greatest influence on the outcome of a potential vaccination campaign. Our model projects that across a range of parameter estimates (given an RHDV2 incursion), the median estimated population size with a 0%–10% vaccination rate after 1 year is 538 rabbits (95% Confidence Interval [C.I.] 69–1235), approximately 36% of the expected size of the study population of 1470 rabbits without an RHDV2 introduction. With a 10%–20%, 20%–30%, or 30%–40% vaccination rate, the median estimated population size increased to 628 rabbits (95% C.I. 130–1298), 723 rabbits (95% C.I. 198–1317), and 774 rabbits (95% C.I. 228–1410), respectively. These estimates represent 43%, 49%, and 53% of the expected population size without an RHDV2 introduction. Overall, a 1% increase in vaccination rate was associated with a six rabbit (95% C.I. 5–7) increase in total remaining population size. This result is dependent on assumptions regarding environmental transmission, home range size (and contact rates of rabbits). Given the relatively short lifespan of rabbits and the potential need for boosters, vaccination programs are most likely to be successful for small target populations where relatively high vaccination rates can be maintained.

Rabbit hemorrhagic disease (RHD) is a highly contagious viral disease that causes fatal acute hepatitis in domestic and wild lagomorphs. It has taken on major economic importance in countries like Morocco. In addition to the classical virus (RHDV), a novel emerged genotype (RHDV2) is circulating, especially in the north shore of the Mediterranean basin since 2010. Many small animal farmers reported clinical cases from several rabbitries in Agadir (Morocco) despite systematic vaccination against the RHDV. The main objective was to characterize the current RHDV strains circulating in the studied area to help to choose an adequate vaccine. For that, we extracted viral RNA from rabbit livers, carried out the PCR analyses, and we sequenced the viral structural capsid protein (VP60) of the RHDV. The phylogenetic analysis results allowed us to state that the novel genotype (RHDV2) is circulating in the studied geographical area, and to characterize the isolated sequences. As a conclusion, we recommend updating RHD epidemiological relating data and reviewing the vaccine protocols by both targeting RHDV (GI.1) and RHDV2 (GI.2) in any future preventive program.

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 β-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. [ABSTRACT FROM AUTHOR]

Rabbit hemorrhagic disease virus 2 (RHDV2 or Lagovirus GI.2) began circulating in wild lagomorph populations in the US in March 2020. To date, RHDV2 has been confirmed in several species of cottontail rabbits (Sylvilagus spp.) and hares (Lepus spp.) throughout the US. In February 2022, RHDV2 was detected in a pygmy rabbit (Brachylagus idahoensis). Pygmy rabbits are sagebrush obligates that only occur in the US Intermountain West and are a species of special concern due to the continual degradation and fragmentation of sagebrush-steppe landscapes. The spread of RHDV2 into occupied pygmy rabbit sites may pose a significant threat to their populations because of already declining numbers associated with habitat loss and high mortality rates. [ABSTRACT FROM AUTHOR]

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 β-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.

Four pet rabbits (Oryctolagus cuniculus cuniculus) diagnosed with a fatal infection by rabbit hemorrhagic disease virus (RHDV GI.2) were identified in the same week and further investigated. All animals lived in an urban environment (Lisbon, Portugal), were between 8 months and 2 years old and none had been vaccinated against RHDV2 (GI.2). Three animals arrived at the clinic and died shortly afterward and it was only possible to collect material for RT-qPCR (RHDV) test. These rabbits tested positive for RHDV2, with high viral loads. In the fourth case, additional clinical and post-mortem gross and histological evaluations were performed. This 8 month old intact female indoor pet rabbit was presented with apathy, tachypnea and tachycardia. Radiographic projections revealed no clinical revealed no clinical abnormalities. Serum biochemistry revealed a significant increase in AST and ALT with a small hypoglycemia. Abdominal ultrasound revealed an acute hepatitis. Despite hospitalization support, after 30 h of admission, the rabbit lost consciousness and developed anorexia and pyrexia in the last minutes before death. Post-mortem analysis and molecular testing by RT-qPCR, confirmed the diagnosis of RHDV2 (GI.2) infection also with high viral load. In conclusion, this paper reports a case series that demonstrates the severe infectious ability and the high mortality associated with RHDV even in rabbits from urban environments. Furthermore, it highlights the importance of always considering rabbit hemorrhagic disease (RHD) as a differential diagnosis in pet rabbits with non-specific clinical signs, and should warn veterinarians that pet rabbits living indoors can also be infected with a fatal outcome.

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Until the early 1980s, it was totally unknown that lagomorphs were the hosts of several caliciviruses, which were included in the genus Lagovirus by the International Committee on Taxonomy of Viruses (ICTV) in 2000. In those years, two new diseases appeared, with very similar clinical and pathological profiles and associated high mortality rates: rabbit haemorrhagic disease (RHD) in rabbits and European Brown Hare Syndrome (EBHS) in European brown hares. It took a few years to ascertain that both diseases, actually acute and fatal forms of hepatitis, were caused by two genetically related caliciviruses, but they were finally classified by ICTV into two distinct viral species on the basis of their molecular characterisation and epidemiological data: RHDV in rabbit and EBHSV in brown hare. RHD has had a devastating effect on rabbit farms, causing great economic damage, especially in China, where RHD was first noticed around 1982, and in Europe. RHD has also severely affected wild rabbit populations, whose drastic decline has caused serious ecological imbalances in territories such as Spain, where rabbits are a central link in the wildlife food chain. Since the early 1990s, with the increased availability on the market of RHDV vaccines effective in protecting rabbits from RHD, the impact of the disease on rabbit farms has been significantly reduced. In the following years, also considering that RHDV is an endemic virus that cannot be eradicated, farmers learned how to manage the continuous use of RHDV vaccine in relation to the epidemiological situation, the type of breeding farm and the costs of vaccination prophylaxis. Although precarious, management of the RHD risk for rabbit farmers reached an acceptable equilibrium, which was, however, completely upset starting from 2010 by the emergence of another lagovirus also causing RHD. The genome of the newly emerged virus shows limited differences from that of RHDV, but the phenotypic traits of the two viruses are distinctive in at least three main respects: 1) The antigenic profile of the virus (the “face” of the virus recognised by the antibodies) is largely different from that of RHDV. 2) Newborn rabbits only a couple of weeks old die of RHD when infected with the new virus, while RHDV infections run asymptomatic until 7-8 wk of age. 3) The new virus, which started in Europe, has spread over the years to several continents, affecting wild and/or domestic rabbit populations. During this worldwide distribution, the new virus infected several lagomorph species and was shown to cause RHD in most of them. Considering these marked differences and the fact that the new virus is not a variant of RHDV, we proposed the name RHDV type 2 (RHDV2). All these main distinctive traits that differentiate RHDV from RHDV2 have the following consequences in practice: 1) The antigenic difference between RHDV and RHDV2 (their ‘faces’) is so great that we need “new” specific vaccines to control RHDV2 (i.e. RHDV2 is a new serotype). 2) In the event of an RHDV2 infection in suckling rabbits, the presence of maternal antibodies to RHDV2 in the blood is the only way to prevent RHD. In contrast, newborns are naturally resistant to RHD if infected with RHDV and therefore, in terms of protection, the presence of maternal antibodies is useless. 3) When RHD outbreaks occur in territories where rabbits live in sympatry with populations of other lagomorphs, viral contamination in the environment reaches sufficiently high levels to facilitate the transmission of RHDV2 to other lagomorphs, including those with a lower susceptibility to infection than the rabbit. Taken together, these phenotypic traits characteristic of RHDV2 are the reason for its rapid spread across the territory and the concomitant disappearance of RHDV. Probably the most striking example of the epidemiological consequences related to the peculiar features of RHDV2 is its rapid spread in the USA and Mexico, where it is now practically endemic. There, despite repeated isolated outbreaks of RHD caused by RHDV from 2000 onwards in small rabbit farms, RHDV has never been able to become endemic.

ABSTRACT Lagovirus europaeus (rabbit hemorrhagic disease virus [RHDV]) is a small, nonenveloped, single-stranded RNA virus that causes a severe, highly infectious, and fatal disease in rabbits (Oryctolagus cuniculus) called rabbit hemorrhagic disease (RHD). Since its discovery in the 1980s, it has posed a very serious threat to the global rabbit industry and the rabbit population in the wild. According to data from 2005 to 2018, the occurrence of RHD has been reported or suspected in 50 countries, with more than one-half of the reports being recorded in European countries. The main aim of the study was to detect Lagovirus europaeus (RHDV) strains found in domestic rabbits that died suddenly in the city of Wrocław in southwest Poland. All animals (n = 14) tested in this study died naturally and showed macroscopic features at necropsy that indicated the possibility of death from RHD. As a result of the research, the presence of L. europaeus virus was confirmed in 8 samples of all 14 samples collected. All strains of Lagovirus europaeus isolated in the present study showed 100% nucleotide identity to L. europaeus GI.1 strain FRG and a strain isolated in New Zealand, as well as the L. europaeus GI.1a Erfurt strain. This suggests that it is likely that L. europaeus GI.2 strains have so far not displaced L. europaeus GI.1 strains from the environment in Poland. IMPORTANCE Lagovirus europaeus (RHDV) causes a severe, highly infectious, and fatal disease in rabbits called RHD. The disease is a very serious threat to the global rabbit industry and the rabbit population in the wild. The aim of the study was to detect Lagovirus europaeus (RHDV) strains in domestic rabbits that died suddenly in Poland. The presence of RHDV was confirmed in 8 samples of all 14 samples collected. This is one of the very few reports on the existence of this virus in pet rabbits in Poland.

In September 2019, high mortality in commercial rabbits was reported in the Greater Accra Region of Ghana. Rabbit hemorrhagic disease virus 2 phylogenetically related to isolates from 2015–2017 outbreaks in the Netherlands was confirmed as the causative agent. The virus has not yet been detected in native rabbits in Ghana.

ABSTRACT 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.

Introduced rabbits are a continuing threat to native Australian flora and fauna. Three interventions using biological control agents, myxomatosis, European rabbit fleas, and rabbit hemorrhagic disease, have reduced rabbit abundance and kept numbers low over the last 70 years. We considered the benefits of biological control for native fauna to put the role of rabbits in influencing vegetation cover, food supply, and predation into better perspective. Numerous examples exist demonstrating increases in native vegetation and the expansion and recovery of native animal populations at landscape scales following intense rabbit suppression. Ongoing research on methods for supplementing the impact of biological control agents and managing introduced predators are needed to restore Australia's arid‐zone ecosystems. However, many biologists and rangeland managers first need to reevaluate the misconception that removing rabbits also introduces other serious and insurmountable problems such as prey‐switching by introduced cats and foxes. [ABSTRACT FROM AUTHOR]

To mitigate the negative impacts of invasive rabbits in Australia, land managers are permitted to release the biocontrol virus, rabbit hemorrhagic disease virus (RHDV), to reduce rabbit numbers. However, it is strongly recommended that RHDV is not released when young rabbits are present in the population as infection in this cohort is sublethal and induces life‐long virus immunity. The recruitment of these rabbits into the breeding population may make the population harder to control in future, potentially leading to increasing rather than decreased population size. To investigate whether the recommended release guidelines are followed, we obtained data on the supply and release of RHDV by land managers. We then used generalized additive models to investigate Australia‐wide and state‐specific annual and long‐term temporal trends in the supply and release of RHDV. Half of all RHDV supply (47%) and three quarters of reported releases (74%) Australia‐wide occurred during the anticipated major rabbit breeding seasons and when the risk of immunizing young rabbits is greatest. We found evidence of both RHDV supply and release during the anticipated major rabbit breeding seasons in almost all states for which data existed. RHDV supply increased with below average annual rainfall. This may indicate a tendency for land managers to notice, and want to control, rabbits and their impacts more following drier years when both rabbits and their impacts are potentially more damaging. Our study raises concerns regarding the inappropriate release of RHDV by land managers and whether its supply should be restricted to ensure ongoing and effective management of invasive rabbits. More broadly, our study serves as a warning to other conservation and pest management activities reliant on land managers or citizens following implementation guidelines. In some cases, good intentions may have adverse outcomes. [ABSTRACT FROM AUTHOR]

European rabbits (Oryctolagus cuniculus) are affected by rabbit hemorrhagic disease (RHD), which is caused by a lagovirus responsible for significant mortality in European wild rabbit populations. Our study aimed to evaluate the potential for detecting viral RNA by duplex real-time PCR in rabbit fecal pellets collected in the field, as a noninvasive method to monitor RHD virus circulation in wild populations. To do this, monthly discoveries of rabbits that died from RHD and detection of viral RNA in fecal pellets were recorded in two enclosed populations of wild rabbits throughout a year. The results suggested a low performance of this procedure to monitor viral infection incidence and a weak concordance with monthly discoveries of rabbits that died from RHD. This poor association was probably due to the low amount of viral RNA in feces, the prolonged time of excretion after infection, and that the number of rabbits found dead from RHD does not necessarily correlate with RHD incidence. Nevertheless, this procedure may be a complementary noninvasive method to assist in determining the presence of RHD viruses in populations. Additional research is needed to determine the suitability of this methodology to perform epidemiologic surveys on wild populations of European rabbits and, especially, other European or North American lagomorph species affected by lagoviruses. [ABSTRACT FROM AUTHOR]

The counter-immunoelectrophoresis (CIE) is a highly specific, simple and rapid method, which does not require expensive equipment, input materials and long-term staff training as compared with other express methods of viral infections diagnostic. Despite the CIE high informativeness, the results in practice can be doubtful at times. Therefore, the purpose of the study was to improve CIE method by strengthening the antigen-antibody interaction with the use of synthesized amphiphilic fluorescein-containing copolymer. It was demonstrated that the use of the fluorescein copolymer -antigen complex enhanced the interaction with serum/plasma antibodies of rabbits immunized against hemorrhagic disease and increased protein content in the area of precipitation. This effect was more pronounced when more hydrophobic copolymer containing 5.87% fluorescein was introduced into the complex with antigen. The ability of the amphiphilic fluorescein copolymer to enhance the antigen-antibody interaction and the ability to visualize the fluorescent precipitation zone may increase CIE effectiveness for express diagnostic of infectious diseases.

Abstract To mitigate the negative impacts of invasive rabbits in Australia, land managers are permitted to release the biocontrol virus, rabbit hemorrhagic disease virus (RHDV), to reduce rabbit numbers. However, it is strongly recommended that RHDV is not released when young rabbits are present in the population as infection in this cohort is sublethal and induces life‐long virus immunity. The recruitment of these rabbits into the breeding population may make the population harder to control in future, potentially leading to increasing rather than decreased population size. To investigate whether the recommended release guidelines are followed, we obtained data on the supply and release of RHDV by land managers. We then used generalized additive models to investigate Australia‐wide and state‐specific annual and long‐term temporal trends in the supply and release of RHDV. Half of all RHDV supply (47%) and three quarters of reported releases (74%) Australia‐wide occurred during the anticipated major rabbit breeding seasons and when the risk of immunizing young rabbits is greatest. We found evidence of both RHDV supply and release during the anticipated major rabbit breeding seasons in almost all states for which data existed. RHDV supply increased with below average annual rainfall. This may indicate a tendency for land managers to notice, and want to control, rabbits and their impacts more following drier years when both rabbits and their impacts are potentially more damaging. Our study raises concerns regarding the inappropriate release of RHDV by land managers and whether its supply should be restricted to ensure ongoing and effective management of invasive rabbits. More broadly, our study serves as a warning to other conservation and pest management activities reliant on land managers or citizens following implementation guidelines. In some cases, good intentions may have adverse outcomes.

In September 2019, high mortality in commercial rabbits was reported in the Greater Accra Region of Ghana. Rabbit hemorrhagic disease virus 2 phylogenetically related to isolates from 2015-2017 outbreaks in the Netherlands was confirmed as the causative agent. The virus has not yet been detected in native rabbits in Ghana. [ABSTRACT FROM AUTHOR]

Twenty-three of 42 European rabbits (Oryctolagus cuniculus), belonging to the same rabbit colony, died in March 2020 (55% mortality) in Chiba prefecture, Japan. The disease course was extremely acute without indicators of death or hemorrhage. Necropsy revealed liver swelling, discoloration, cloudiness and fragility, and pulmonary edema. Histologically, severe hepatocellular necrosis (mainly peripheral) and intra-glomerular capillary hyalin thrombi were observed. On molecular-biological examination, reverse transcription polymerase chain reaction analysis of RNA from tissues detected a rabbit hemorrhagic disease virus, confirmed as a RHDV-2 VP60 fragment, which shared 99.42% nucleotide identity with the homologous fragment of RHDV-2 German isolate by nucleotide sequence analysis. This report shows the outbreak of rabbit hemorrhagic disease caused by RHDV-2, an emerging infectious disease, in Japan. [ABSTRACT FROM AUTHOR]

Autophagy is a process focused on maintaining the homeostasis of organisms; nevertheless, the role of this process has also been widely documented in viral infections. Thus, xenophagy is a selective form of autophagy targeting viruses. However, the relation between autophagy and viruses is ambiguous—this process may be used as a strategy to fight with a virus, but is also in favor of the virus’s replication. In this paper, we have gathered data on autophagy in viral hepatitis and viral hemorrhagic fevers and the relations impacting its viral pathogenesis. Thus, autophagy is a potential therapeutic target, but research is needed to fully understand the mechanisms by which the virus interacts with the autophagic machinery. These studies must be performed in specific research models other than the natural host for many reasons. In this paper, we also indicate Lagovirus europaeus virus as a potentially good research model for acute liver failure and viral hemorrhagic disease.

Featured Application: Our experience with real-time PCR, together with several different studies in this field show that this is a very useful method for rapid, specific and effective virus detection.Lagovirus europaeus GI.1/GI.2 is an etiological agent causing the highly dangerous rabbit hemorrhagic disease (RHD). Molecular research is the basic tool today that can help solve epidemic problems related to the expansion of pathogens in the world. By using the real-time polymerase chain reaction technique (PCR), we detected three different strains of Lagovirus europaeus/GI.1, which is an RNA virus infecting mainly rabbits. The results showed that the method used was fast, very specific, and effective. [ABSTRACT FROM AUTHOR]

Abstract Background Prior to 2010, the lagoviruses that cause rabbit hemorrhagic disease (RHD) in European rabbits (Oryctolagus cuniculus) and European brown hare syndrome (EBHS) in hares (Lepus spp.) were generally genus-specific. However, in 2010, rabbit hemorrhagic disease virus 2 (RHDV2), also known as Lagovirus europaeus GI.2, emerged and had the distinguishing ability to cause disease in both rabbits and certain hare species. The mountain hare (Lepus timidus) is native to Sweden and is susceptible to European brown hare syndrome virus (EBHSV), also called Lagovirus europaeus GII.1. While most mountain hare populations are found on the mainland, isolated populations also exist on islands. Here we investigate a mortality event in mountain hares on the small island of Hallands Väderö where other leporid species, including rabbits, are absent. Results Post-mortem and microscopic examination of three mountain hare carcasses collected from early November 2016 to mid-March 2017 revealed acute hepatic necrosis consistent with pathogenic lagovirus infection. Using immunohistochemistry, lagoviral capsid antigen was visualized within lesions, both in hepatocytes and macrophages. Genotyping and immunotyping of the virus independently confirmed infection with L. europaeus GI.2, not GII.1. Phylogenetic analyses of the vp60 gene grouped mountain hare strains together with a rabbit strain from an outbreak of GI.2 in July 2016, collected approximately 50 km away on the mainland. Conclusions This is the first documented infection of GI.2 in mountain hares and further expands the host range of GI.2. Lesions and tissue distribution mimic those of GII.1 in mountain hares. The virus was most likely initially introduced from a concurrent, large-scale GI.2 outbreak in rabbits on the adjacent mainland, providing another example of how readily this virus can spread. The mortality event in mountain hares lasted for at least 4.5 months in the absence of rabbits, which would have required virus circulation among mountain hares, environmental persistence and/or multiple introductions. This marks the fourth Lepus species that can succumb to GI.2 infection, suggesting that susceptibility to GI.2 may be common in Lepus species. Measures to minimize the spread of GI.2 to vulnerable Lepus populations therefore are prudent.

The highly virulent rabbit hemorrhagic disease virus (RHDV) has been widely used in Australia and New Zealand since the mid-1990s to control wild rabbits, an invasive vertebrate pest in these countries. In January 2014, an exotic RHDV was detected in Australia, and 8 additional outbreaks were reported in both domestic and wild rabbits in the 15 months following its detection. Full-length genomic analysis revealed that this virus is a recombinant containing an RHDVa capsid gene and nonstructural genes most closely related to nonpathogenic rabbit caliciviruses. Nationwide monitoring efforts need to be expanded to assess if the increasing number of different RHDV variants circulating in the Australian environment will affect biological control of rabbits. At the same time, updated vaccines and vaccination protocols are urgently needed to protect pet and farmed rabbits from these novel rabbit caliciviruses.

Rabbit hemorrhagic disease (RHD) is a highly contagious disease caused by rabbit hemorrhagic disease virus (RHDV). Previous research has shown that RHDV induces apoptosis in numerous cell types, although the molecular mechanisms underlying the apoptosis induced by RHDV are not well understood. One possible factor is non-structural protein 6 (NSP6), a 3C-like protease that plays an important role in processing viral polyprotein precursors into mature non-structural proteins. To fully establish a role for NSP6, the present study examined the effects of ectopic expression of the protein in rabbit (RK13) and human (HeLa and HepG2) cells. We found that NSP6 suppressed cell viability and promoted apoptosis in all three cell types in a dose-dependent manner. We also identified increased caspase-3, -8, and -9 activities in RK13 cell, and an increased Bax to Bcl2 mRNA ratio. Mechanistically, the ability of NSP6 to induce apoptosis was impaired by mutation of the catalytic His27 residue. Our study has shown that RHDV NSP6 can induce apoptosis in host cells and is likely an important contributor to RHDV-induced apoptosis and pathogenesis.

The European wild rabbit (Oryctolagus cuniculus) is a key prey species on the Iberian Peninsula, and several predator species that are at risk of extinction are dependent on them as prey. A new rabbit hemorrhagic disease (RHD) virus genotype (GI.2/RHDV2/b) emerged in 2010 and posed a threat to wild rabbit populations. During a survey aimed at investigating RHD epidemiology in wild rabbits, GI.2/RHDV2/b was detected by duplex real-time PCR in carcasses of one Mediterranean pine vole (Microtus duodecimcostatus) and two white-toothed shrews (Crocidura russula). Laboratory New Zealand white rabbits that were challenged with inocula obtained from the liver of the small mammals died showing RHD lesions, confirming the infectiousness of the isolates. Phylogenetic analysis of the VP60 gene nucleotide sequences showed complete homology between the isolates from the two small mammal species and a high degree of similarity, but not complete homology, to GI.2/RHDV2/b sequences from wild rabbits. The GI.2/RHDV2/b genotype has not been reported in species outside the order Lagomorpha. [ABSTRACT FROM AUTHOR]

Lagovirus europaeus GI.1/GI.2 is an etiological agent causing the highly dangerous rabbit hemorrhagic disease (RHD). Molecular research is the basic tool today that can help solve epidemic problems related to the expansion of pathogens in the world. By using the real-time polymerase chain reaction technique (PCR), we detected three different strains of Lagovirus europaeus/GI.1, which is an RNA virus infecting mainly rabbits. The results showed that the method used was fast, very specific, and effective.

Infectious diseases, due to their massive scale, are the greatest pain for all rabbit breeders. Viral infections cause enormous economic losses in farms. Treating sick rabbits is very difficult and expensive, so it is very important to prevent disease by vaccinating. In order to successfully fight viral infections, it is important to know about the immune response of an infected animal. The aim of this study was to analyze the immune response mediated by antimicrobial peptides (myeloperoxidase (MPO) and lysozyme (LZM)) in peripheral blood neutrophils and rabbit serum by non-invasive immunological methods. The study was carried out on mixed breed rabbits that were experimentally infected with two strains (Erfurt and Rossi) of the Lagovirus europaeus/GI.1a virus. It has been observed that virus infection causes changes in the form of statistically significant increases in the activity of MPO and LZM concentration, while in the case of LZM activity only statistically significant decreases were noted. Additionally, clinical symptoms typical for the course of the disease were noted, and the probability of survival of the animals at 60 h p.i. (post infection) was 30% for the Erfurt strain, and −60% for the Rossi strain. The obtained results of MPO and LZMs suggest that these enzymes, especially MPO, may serve as a prognostic marker of the state of the immune system of rabbits.

Rabbit hemorrhagic disease (RHD) was detected in European rabbits (Oryctolagus cuniculus) for the first time ever in Finland in 2016. Reports of dead feral rabbits in Helsinki started to accumulate from April 2016. The Finnish Food Safety Authority Evira received the first animals in late April, and the main necropsy finding was severe, acute necrotizing hepatitis. Genetic material from RHD virus (RHDV) was detected in the liver and was further characterized as RHDV2. The Finnish virus did not group with RHDV strains from a concurrent outbreak in neighboring Sweden, suggesting another origin. The outbreak peaked in May and lasted until August, after which sightings of both live and dead rabbits became rare. No major outbreaks in domestic rabbits were observed, although infection in one pet rabbit was confirmed. [ABSTRACT FROM AUTHOR]

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 μ-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, Lepus europaeus, 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. [ABSTRACT FROM AUTHOR]

The highly virulent rabbit hemorrhagic disease virus (RHDV) has been widely used in Australia and New Zealand since the mid-1990s to control wild rabbits, an invasive vertebrate pest in these countries. In January 2014, an exotic RHDV was detected in Australia, and 8 additional outbreaks were reported in both domestic and wild rabbits in the 15 months following its detection. Full-length genomic analysis revealed that this virus is a recombinant containing an RHDVa capsid gene and nonstructural genes most closely related to nonpathogenic rabbit caliciviruses. Nationwide monitoring efforts need to be expanded to assess if the increasing number of different RHDV variants circulating in the Australian environment will affect biological control of rabbits. At the same time, updated vaccines and vaccination protocols are urgently needed to protect pet and farmed rabbits from these novel rabbit caliciviruses. [ABSTRACT FROM AUTHOR]

We report a disease outbreak in a Michigan rabbitry of a rabbit calicivirus distinct from the foreign animal disease agent, rabbit hemorrhagic disease virus (RHDV). The novel virus has been designated Michigan rabbit calicivirus (MRCV). Caliciviruses of the Lagovirus genus other than RHDV have not been described in US rabbit populations. The case-fatality rate was 32.5% (65/200). Clinical signs included hemorrhage and sudden death, with hepatic necrosis. Analysis of viral RNA sequence from >95% of the viral genome showed an average similarity of 79% with RHDV. Similarity of the predicted MRCV capsid amino acid sequence ranged from 89.8% to 91.3%, much lower than the 98% amino acid similarity between RHDV strains. Experimentally infected rabbits lacked clinical disease, but MRCV was detected in tissues by PCR. We propose that MRCV primarily causes subclinical infection but may induce overt RHD-like disease under certain field conditions.

Four pet rabbits ( Oryctolagus cuniculus cuniculus ) diagnosed with a fatal infection by rabbit hemorrhagic disease virus (RHDV GI.2) were identified in the same week and further investigated. All animals lived in an urban environment (Lisbon, Portugal), were between 8 months and 2 years old and none had been vaccinated against RHDV2 (GI.2). Three animals arrived at the clinic and died shortly afterward and it was only possible to collect material for RT-qPCR (RHDV) test. These rabbits tested positive for RHDV2, with high viral loads. In the fourth case, additional clinical and post-mortem gross and histological evaluations were performed. This 8 month old intact female indoor pet rabbit was presented with apathy, tachypnea and tachycardia. Radiographic projections revealed no clinical revealed no clinical abnormalities. Serum biochemistry revealed a significant increase in AST and ALT with a small hypoglycemia. Abdominal ultrasound revealed an acute hepatitis. Despite hospitalization support, after 30 h of admission, the rabbit lost consciousness and developed anorexia and pyrexia in the last minutes before death. Post-mortem analysis and molecular testing by RT-qPCR, confirmed the diagnosis of RHDV2 (GI.2) infection also with high viral load. In conclusion, this paper reports a case series that demonstrates the severe infectious ability and the high mortality associated with RHDV even in rabbits from urban environments. Furthermore, it highlights the importance of always considering rabbit hemorrhagic disease (RHD) as a differential diagnosis in pet rabbits with non-specific clinical signs, and should warn veterinarians that pet rabbits living indoors can also be infected with a fatal outcome., Competing Interests: FP was employed by HIPRA. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Pinto, Abrantes, Ferreira, Nóbrega and Marcos.)

Lagovirus europaeus (rabbit hemorrhagic disease virus [RHDV]) is a small, nonenveloped, single-stranded RNA virus that causes a severe, highly infectious, and fatal disease in rabbits (Oryctolagus cuniculus) called rabbit hemorrhagic disease (RHD). Since its discovery in the 1980s, it has posed a very serious threat to the global rabbit industry and the rabbit population in the wild. According to data from 2005 to 2018, the occurrence of RHD has been reported or suspected in 50 countries, with more than one-half of the reports being recorded in European countries. The main aim of the study was to detect Lagovirus europaeus (RHDV) strains found in domestic rabbits that died suddenly in the city of Wrocław in southwest Poland. All animals ( n  = 14) tested in this study died naturally and showed macroscopic features at necropsy that indicated the possibility of death from RHD. As a result of the research, the presence of L. europaeus virus was confirmed in 8 samples of all 14 samples collected. All strains of Lagovirus europaeus isolated in the present study showed 100% nucleotide identity to L. europaeus GI.1 strain FRG and a strain isolated in New Zealand, as well as the L. europaeus GI.1a Erfurt strain. This suggests that it is likely that L. europaeus GI.2 strains have so far not displaced L. europaeus GI.1 strains from the environment in Poland. IMPORTANCE Lagovirus europaeus (RHDV) causes a severe, highly infectious, and fatal disease in rabbits called RHD. The disease is a very serious threat to the global rabbit industry and the rabbit population in the wild. The aim of the study was to detect Lagovirus europaeus (RHDV) strains in domestic rabbits that died suddenly in Poland. The presence of RHDV was confirmed in 8 samples of all 14 samples collected. This is one of the very few reports on the existence of this virus in pet rabbits in Poland.

Highlights: [•] There have been three major viral biocontrol programs against invasive vertebrates. [•] Rabbits and cats have been successfully targeted with three different viruses. [•] We have identified the most important lessons from these programs. [•] The lessons are applied to biocontrol of carp in Australia with cyprinid herpesvirus-3. [Copyright &y& Elsevier]

Autophagy is an important survival pathway and participates in the host response to infection. Beneficial effects of melatonin have been previously reported in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). This study was aimed to investigate whether melatonin protection against liver injury induced by the RHDV associates to modulation of autophagy. Rabbits were infected with 2 × 104 hemagglutination units of a RHDV isolate and received 20 mg/kg melatonin at 0, 12, and 24 hr postinfection. RHDV induced autophagy, with increased expression of beclin-1, ubiquitin-like autophagy-related (Atg)5, Atg12, Atg16L1 and sequestrosome 1 (p62/SQSTM1), protein 1 light chain 3 (LC3) staining, and conversion of LC3-I to autophagosome-associated LC3-II. These effects reached a maximum at 24 hr postinfection, in parallel to extensive colocalization of LC3 and lysosome-associated membrane protein (LAMP)-1. The autophagic response induced by RHDV infection was significantly inhibited by melatonin administration. Melatonin treatment also resulted in decreased immunoreactivity for RHDV viral VP60 antigen and a significantly reduction in RHDV VP60 m RNA levels, oxidized to reduced glutathione ratio (GSSG/GSH), caspase-3 activity, and immunoglobulin-heavy-chain-binding protein (BiP) and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. Results indicate that, in addition to its antioxidant and antiapoptotic effects, and the suppression of ER stress, melatonin induces a decrease in autophagy associated with RHDV infection and inhibits RHDV RNA replication. Results obtained reveal novel molecular pathways accounting for the protective effect of melatonin in this animal model of ALF. [ABSTRACT FROM AUTHOR]

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV. [ABSTRACT FROM AUTHOR]