Evolution of rabbit haemorrhagic disease virus (RHDV) in the European rabbit (Oryctolagus cuniculus) from the Iberian Peninsula

To date information on rabbit haemorrhagic disease virus (RHDV) in Spain and Portugal has been scarce, although the disease is endemic and continues to have a considerable impact on species conservation and hunting industry. We analysed RHDVs obtained between 1994 and 2007 at different geographic locations in Portugal (40 samples), Spain (3 samples) and France (4 samples) from wild European rabbits (Oryctolagus cuniculus) that succumbed to the disease. Phylogenetic analyses based on partial VP60 gene sequences allowed a grouping of these RHDVs into three groups, termed "Iberian" Groups IB1, IB2 and IB3. Interestingly, these three Iberian groups clustered separately, though not far from earlier RHDVs of Genogroup 1 (containing e.g., strain "AST89"), but clearly distinct from globally described RHDV strains of Genogroups 2-6. This result, supported by a bootstrap value of 76%, gives rise to the hypothesis that the virus evolved independently since its introduction to wild rabbit populations on the Iberian Peninsula, with the Pyrenees acting as a natural barrier to rabbit and hence to virus dispersal. No differences were observed in RHDV sequences obtained from geographic regions where the rabbit subspecies O. c. algirus prevails compared with those obtained from O. c. cuniculus.     

Immunohistological diagnosis of rabbit haemorrhagic disease (RHD).

In the present study the diagnostic use of a biotinylated serum from an immune rabbit was investigated by means of an Avidin-Biotin-Complex (ABC)-Peroxidase method on paraffin sections. 15 cases of RHD which had been verified histologically and/or by haemagglutination test (HA), 4 suspected cases and 3 cases without history of RHD were included (cases 1 to 22). From 5 prospective cases a wider tissue range was examined (cases 23 to 25 and 29 to 30). Furthermore lungs, liver and placenta of 3 fetuses from a RHD affected dam were investigated (cases 26 to 28). The 20 typical cases had intense intranuclear and diffuse intracytoplasmic immunostaining of hepatocytes, predominantly in the periportal areas. In some cases there was also positive staining of macrophages in the lungs (4 cases), spleen (4 cases) and in lymph nodes (1 case). Positive granular staining in the renal mesangial cells of the glomeruli was observed in 1 case. No positive staining was observed in the 3 negative cases. In contrast to other reports (4), crossreactivity of these antigens to Porcine Parvovirus (PPV) could not be confirmed. Furthermore the RHD virus (RHDV) seems not to crossreact with Feline and Bovine Parvoviruses.     

浅谈兔出血症病毒

兔出血症（Rabbit hemorrhagic disease，RHD），又称兔瘟，由兔出血症病毒（Rabbit hemorrhagic disease virus，RHDV）引起的一种急性、高度传染性、高致死性的疾病。以呼吸系统出血，实质器官水肿、淤血及出血变化为特征。1984年，该病首先在我国爆发，随后朝鲜、韩国、意大利、德国、印度、法国、前苏联、西班牙、日本、美国和非洲等国家和地区相继有该病流行的报道。国内外学者从形态学、生物化学和分子生物学对RHDV进行了系统研究。     

Phagocytosis of neutrophils in rabbits infected with antigenic variants of RHD (rabbit haemorrhagic disease) virus

The present study was aimed at determining changes in chosen elements of phagocytosis in rabbits infected with 3 antigenic variants of RHD - Hartmannsdorf, Pv97 and 9905, which differed in haemagglutination ability. The animals were tested for phagocytosis parameters, and the results revealed that the examined strains showed the differences. These variations regarded mainly Pv97 strain, as the intensity of the changes were 5 times stronger in comparison to strain Hartmannsdorf and 9905. As all of the strains examined are signified as antigenic variants, we have stated that this feature does not determine their immunological picture. The results suggest the existence of immunological dissimilarities among strains of the RHD virus, which was revealed for the first time in antigenic variants.     

Apoptosis of peripheral blood leukocytes from rabbits infected with non-haemagglutinating strains of rabbit haemorrhagic disease virus (RHDV)

The report demonstrates that the induction of apoptosis in peripheral blood granulocytes and lymphocytes of rabbits infected with three non-haemagglutinating RHDV strains (English Rainham, German Frankfurt, and Spanish Asturias) is a crucial determinant of the pathogenesis of rabbit haemorrhagic disease. Apoptosis was measured by flow cytometric detection of caspase activity. These studies demonstrated that the investigated RHDV (rabbit haemorrhagic disease virus) viral strains affected leukocyte apoptosis to varying degrees. Enhanced leukocyte apoptosis was detected between 4 and 36h after infection and was more pronounced in lymphocytes than in granulocytes. The data presented here thus provide a preliminary understanding of the kinetics of apoptosis in leukocytes of rabbits infected with RHDV.     

Comparative analysis of rabbit hemorrhagic disease virus (RHDV) and new RHDV2 virus antigenicity,using specific virus-like particles

In 2010 a new Lagovirus related to rabbit haemorrhagic disease virus (RHDV) emerged in France and has since rapidly spread throughout domestic and wild rabbit populations of several European countries. The new virus, termed RHDV2, exhibits distinctive genetic, antigenic and pathogenic features. Notably, RHDV2 kills rabbits previously vaccinated with RHDV vaccines. Here we report for the first time the generation and characterization of RHDV2-specific virus-like particles (VLPs). Our results further confirmed the differential antigenic properties exhibited by RHDV and RHDV2, highlighting the need of using RHDV2-specific diagnostic assays to monitor the spread of this new virus.     

An outbreak of rabbit hemorrhagic disease (RHD) caused by Lagovirus europaeus GI.2/rabbit hemorrhagic disease virus 2 (RHDV2) in Ehime, Japan

A total of ten 1–2-year-old rabbits died within 2 weeks at a facility in Ehime prefecture in May 2019. Necropsy revealed liver discoloration and fragility, hemorrhage of some organs and blood coagulation failure. On histopathologic examination, necrotizing hepatitis was a common finding, together with fibrin thrombi in the small vessels and hemorrhage in some organs. Rabbit hemorrhagic disease (RHD) virus gene was detected in liver samples, and viral particles of approximately 32 nm in diameter were found in the cytoplasm of degenerated hepatocytes by electron microscopy. Phylogenetic analysis based on the partial VP60 gene sequence classified it as Lagovirus europaeus GI.2/RHDV2. This is the first confirmed outbreak of RHD caused by globally emerging GI.2/RHDV2 in Japan.     

Leukocyte-hepatocyte interaction in calicivirus infection: differences between rabbits that are resistant or susceptible to rabbit haemorrhagic disease (RHD)

Calicivirus infection is lethal for adult rabbits, whereas young rabbits (less than 8-weeks-old) are resistant to the same infectious agent. The virus replicates in the liver and causes a fulminant hepatitis in adult rabbits leading to rabbit haemorrhagic disease (RHD); this is in contrast with the mild and transient hepatitis observed in infected young rabbits. We have used electron microscopy to compare liver leukocyte infiltrates between young (resistant) and adult (susceptible) rabbits, 36-48 h after inoculation of the animals with caliciviruses. In adult rabbits, liver infiltrates were made up mostly of heterophils, and they were located near hepatocytes showing severe cellular damage. In contrast, liver leukocyte infiltrates of RHD-resistant young rabbits were dominated by lymphocytes that depicted membrane contacts with the cell surface of undamaged hepatocytes. We conclude that: (i) the cellular inflammatory response of the liver to calicivirus infection is different in rabbits that are susceptible (adult) or resistant (young) to RHD; (ii) leukocyte infiltration of the adult liver by heterophils is probably directed at the removal of dead hepatocytes, whereas the liver lymphocytic infiltration of young rabbits suggests the expression of viral antigens on the surface of liver cells of the RHD-resistant animals.     

Cytometric analysis of lymphocytes T and B in rabbits infected with non-haemagglutinogenic strains of RHD virus (rabbit haemorrhagic disease)--Rainham, Frankfurt and Asturias

The present paper refers to the cytometric analysis of lymphocytes T (with receptor CD5+), Th (with receptor CD4+), Tc/Ts (with receptor CD8+), lymphocytes CD25+ and lymphocytes B with receptor CD19+ in rabbits experimentally infected with strains of RHD virus--Rainham, Frankfurt and Asturias, not having haemagglutinogenic capacities, which makes them unique, as haemagglutinogenic capacity is a classic and typical property of most strains of this virus. The study was performed in the dynamic system, drawing blood samples from animals at hour 0, namely before the administration of the viral antigen, and then at 4, 8, 12, 24 and 36 h after the infection. The study indicated that Rainham and Asturias strains of RHD virus cause a similar amount of changes as the most immunogenic haemagglutinogenic strains CAMP V-561 and CAMP V-562 of the RHD virus do. In contrast, the Frankfurt strain of the RHD virus is characterised with 5-6-fold lower reactivity in this respect and is most similar to the least immunogenic haemagglutinogenic strain CAMP V-558 of the RHD virus.     

Disseminated intravascular coagulation (DIC) in rabbit haemorrhagic disease.

Seven rabbits experimentally infected with rabbit haemorrhagic disease virus were examined haematologically and histologically. Haematologically, activated partial thromboplastin time and prothrombin time were markedly prolonged in the terminal phase of the disease, just prior to death (all the animals died between 27 and 40 hr after inoculation with rabbit haemorrhagic disease virus). There was an increase in the titre of fibrin degradation products and a decrease in antithrombin III activity during the same interval. Acute necrotic hepatitis and disseminated intravascular coagulation (DIC) in many organs, including the lung, kidney, spleen and heart were the characteristic histopathological changes. Thus, the haematological and histological changes suggested that DIC was induced by rabbit haemorrhagic disease virus infection. Severe liver necrosis was considered to be a factor causing DIC by inducing a hypercoagulable condition in the systemic blood circulation.     

Serological assays to discriminate rabbit haemorrhagic disease virus from Australian non-pathogenic rabbit calicivirus

Serological cross reactivity between the virulent rabbit haemorrhagic disease virus (RHDV) and the closely related but non-pathogenic rabbit calicivirus (RCV) makes it difficult to study the epidemiology of each virus and the interaction between them when both viruses co-circulate in wild rabbit populations. ELISA methods for the diagnosis of RHDV infection are well characterized, but no specific serological tests for RCV have been developed. Following the characterization of Australian non-pathogenic RCV-A1 strains, we used virus-like-particles (VLPs) and anti-RCV-A1 specific antibodies to establish a set of isotype ELISAs for detection of IgG, IgA and IgM in rabbit sera and secretory mucosal IgA in rectal swabs, and two competition ELISAs. These assays were used to discriminate between anti-RCV-A1 and anti-RHDV antibodies in rabbits. The isotype ELISAs were highly sensitive for detection of anti-RCV-A1 antibodies, but varying levels of cross reactivity from anti-RHDV antibodies occurred in the isotype ELISAs and one competition ELISA. However, the second competition ELISA specifically detected antibodies to RCV-A1 and showed no cross reactivity to anti-RHDV sera. These ELISAs provide important tools to monitor RCV-A1 infection when it occurs alone, and to discriminate between RHDV and RCV-A1 infection when they occur in the same rabbit population. When used in parallel with RHDV serology, they could be used to monitor the dynamics of these two closely related but pathogenically distinct viruses in wild and domestic rabbit populations.     

兔病毒性出血症病毒的提纯及部分特性鉴定

应用建立的SDS—蔗糖密度梯度超速离心法,纯化了从兔肝组织中粗提的兔病毒性出血症病毒。通过血凝、电镜观察、SDS-PAGE、琼脂双向免疫扩散、免疫印迹等试验,证明SDS—蔗糖梯度柱分离的病毒纯度高,具有4种结构多肽。     

Liver Enzymes and Ultrastructure in Rabbit Haemorrhagic Disease (RHD)

Rabbit haemorrhagic disease (RHD) is caused by a calicivirus infection that kills most adult rabbits 24–72 h after viral inoculation. Two liver enzymes (AST, aspartate aminotransferase, and ALT, alanine aminotransferase) were monitored in blood samples of calicivirus-infected rabbits during the short course of RHD. Values of AST were used to differentiate three stages of hepatocellular degeneration in RHD: mild (up to 20-fold increase in AST), moderate (150–200-fold elevation of AST) and severe (more than 1000-fold elevation in AST). Liver samples of rabbits from these three biochemical stages of hepatocellular degeneration of RHD were studied by transmission electron microscopy to define the fine structure of the hepatocytes. In the mild hepatocellular degeneration there was proliferation (microvesiculation) of the smooth endoplasmic reticulum and swelling of mitochondria into spheroid bodies with loss of cristae. In moderate hepatocellular degeneration, vacuolization of cytoplasm and mitochondrial damage continued to be present, and there was also formation of autophagic vesicles. In the severe hepatocellular degeneration of RHD, the altered mitochondria also showed loss of density of their matrix; rupture of cytoplasmic vacuoles led to the formation of large vesicles. Marked depletion of liver glycogen was also found in this late stage of RHD. These data offer a correlation between biochemical and cytological features of the liver during the hepatocellular degeneration of RHD.