Adult rabbits acquire resistance to lethal calicivirus infection by adoptive transfer of sera from infected young rabbits

Calicivirus infection of adult rabbits induces the so-called rabbit haemorrhagic disease (RHD) that kills 90% or more of the infected animals; in contrast, young rabbits (up to 8-week-old animals) are resistant to the same infectious agent. We report that calicivirus inoculation of young rabbits induced moderate titres of antiviral antibodies. When these rabbits reached adulthood, a second calicivirus inoculation resulted in resistance to RHD and boosting of antibody titres in half of the rabbits. Adoptive transfer of sera from calicivirus-infected young rabbits to na?ve adult rabbits conferred resistance to RHD. We conclude that calicivirus infection of young rabbits induces specific anti-calicivirus antibodies that will protect them from RHD when they reach adulthood.     

西藏兔病毒性出血症的研究--病原鉴定及疫苗免疫效力试验

１９９４年６￣７月,西藏林芝地区家兔大批急性死亡同,经过流行病学调查,临诊断症状观察,病理剖检等结果与兔病毒性出血症特征相符。进一步作病毒学检查表明,病变组织超薄发切片经电子显微镜观察发现有大量直径约３０ｎｍ的球状裸听病毒粒子,自然病死兔肝组织匀交涉 清能凝集入）型红细胞,而且血凝兔出血症病毒阳性抗血清所抑制,致病性试验结果表明,该病毒对兔有很强的致病性;用西藏自然病死兔肝组织匀浆试制的矩形疫苗兔     

Novel bivalent vectored vaccine for control of myxomatosis and rabbit haemorrhagic disease

A novel, recombinant myxoma virus-rabbit haemorrhagic disease virus (RHDV) vaccine has been developed for the prevention of myxomatosis and rabbit haemorrhagic disease (RHD). A number of laboratory studies are described illustrating the safety and efficacy of the vaccine following subcutaneous administration in laboratory rabbits from four weeks of age onwards. In these studies, both vaccinated and unvaccinated control rabbits were challenged using pathogenic strains of RHD and myxoma viruses, and 100 per cent of the vaccinated rabbits were protected against both myxomatosis and RHD.     

Transient decrease in blood heterophils and sustained liver damage caused by calicivirus infection of young rabbits that are naturally resistant to rabbit haemorrhagic disease

Young rabbits are naturally resistant to rabbit haemorrhagic disease (RHD) caused by the same calicivirus that kills, within 3 days, nearly all adult animals. We have investigated changes in blood leukocytes, and in the morphology and biochemistry of the liver (the organ where caliciviruses replicate) of young rabbits undergoing benign infection by the RHD virus. Four-week-old rabbits were infected with a calicivirus inoculum having a titre of 2(12) haemagglutination units either sacrificed 18, 24, 48 and 72 h later, or kept for follow-up studies up to 21 days after inoculation. The infection caused an acute and transient decrease in blood heterophils, and sustained enhancement in hepatic transaminases. Inflammatory infiltrates of the liver were seen in all animals after 24 h of infection; they had a predominant midlobular location. Hepatocytes could present different degrees of cell damage, including cell death; these lesions were limited to the liver cells located around the inflammatory infiltrates. Liver transaminases peaked 24-48 h after calicivirus infection; this was the same timing when liver infiltration and hepatocyte damage were more evident. No alterations of other parameters of liver biochemistry were observed. We conclude that calicivirus infection of young rabbits causes a subclinical disorder characterised by an acute and transient decrease in circulating heterophils, and focal liver damage that is expressed by intralobular infiltration by heterophils, initially, and, later on, by mononuclear cells. Our finding of persistence of increased values of liver transaminases suggests chronicity of the infection in young rabbits. We propose that, although resistant to RHD, young rabbits infected by calicivirus may be long-term carriers of the infectious agent and, thus, become a major source of transmission of the virus.     

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.     

“黄白散”对兔接种RHD疫苗后免疫功能的影响

在注射兔病毒性出血症 (RHD)疫苗后 ,30只兔随机分为 2组 ,试验组兔饲喂含 1 5 %黄白散的饲料。从接种到第 1 80天 ,每组取兔 8只 ,每隔 1 5d采血 1次 ,检测血凝抑制 (HI)抗体 ;在接种前 1天及接种后 5、 1 0、 1 7、 2 4、 31、 41、 5 1d ,每组取兔 1 0只 ,采血检测ANAE+淋巴细胞百分率 ;在 1 80、 2 4 0、 30 0d时 ,每组分别取兔 5只做攻毒保护试验。结果表明 :2组兔HI抗体峰值分别为 7 3log2和 9 8log2 ,差异极显著 (P <0 0 1 ) ;试验组疫苗保护期可延长 60d ;试验组ANAE+率从第 1 0天至 5 1天与对照组比较 ,差异极显著(P<0 0 1 )。所以黄白散可增强兔接种RHD疫苗后的特异性免疫功能 ,增强RHD疫苗的免疫效果。     

Possible interaction between myxomatosis and calicivirosis related to rabbit haemorrhagic disease affecting the European rabbit

Serological data on myxoma virus, rabbit haemorrhagic disease (RHD) virus and RHD-like viruses in juvenile rabbits (Oryctolagus cuniculus) trapped in 1995, 1996 and 1997 in two areas of France were analysed. For each disease, the effects of bodyweight, year, month and seropositivity for the other disease were modelled by using logistic regressions. In one area, a model including RHD seropositivity was selected to explain the myxoma virus seropositivity. Models including myxoma virus seropositivity were selected to explain the RHD seropositivity in both areas, and the odds of a rabbit being seropositive to both viruses were 5.1 and 8.4 times higher than the odds of a rabbit being seronegative to myxoma virus and seropositive to RHD. The year and bodyweight had significant effects for myxomatosis in one area and for RHD in both areas.     

Haemorrhagic disease of lagomorphs: evidence for a calicivirus.

Studies on the aetiological agents of rabbit haemorrhagic disease (RHD) and European brown hare syndrome show that the viruses responsible for these infections can be placed in the family Caliciviridae. Established members of this group are vesicular exanthema virus (prototype), San Miguel sea lion virus and feline calcivirus. The human hepatitis E virus and the Norwalk agent may soon be included. The RHD virus genome consists of a positive stranded RNA molecule composed of 7437 nucleotides. A major subgenomic RNA of 2.2 kb, colinear with the 3' end of the genomic RNA, can also be recovered from infected liver tissue, and both RNAs are enclosed within viral capsids formed by a single major protein of approximately 60 kDa. Electron microscopic examination of organ suspensions from diseased animals shows two types of particle; 35-40 nm complete virions have the regularly arranged cup-shaped depressions typical of calcivirus morphology, and 23-25 nm smooth particles resulting from degradation of the outer surface structures of the complete virions.     

兔病毒性出血症基因工程苗研究概况

由于兔病毒性出血症 (RHD)组织灭活苗涉及的成本、生物安全及动物福利等问题 ,国内外学者正致力于 RHD新型疫苗的研制和开发。以 VP60表达为基础的亚单位疫苗经历了最初的原核、真核表达 ,目前正寻求在转基因植物中进行表达 ,以期获得大量、低成本的口服疫苗 ;而以牛痘、金丝雀痘病毒、黏液瘤病毒等作载体的重组活载体苗 ,无疑使 RHD疫苗正在向更安全、实用及对家兔、野兔均有保护作用方向发展 ,具有很大的发展潜力和前景。国外学者在RHD基因工程苗方面研究较为广泛和深入 ,而国内尚未有此方面报道     

Serological evidence for a non-protective RHDV-like virus

The data were recorded during a Rabbit haemorrhagic disease outbreak that occurred in France in 2001 in a wild population of rabbits that we have been monitoring since 2000. These data suggested the existence of non-protective antibodies due to a putative RHDV-like virus. Twenty-one blood and 22 liver samples were taken from the 26 corpses of recently dead rabbits that were found. RHDV was found in all liver samples. A first screening for RHD antibodies, carried out using an ELISA based on the detection of VP60-RHDV antigen, showed that 20 of the rabbits were seropositive. Moreover, we determined antibody titres for 13 of these 20 seropositive samples. All were > or = 1/400. Such titres normally indicate antibody levels sufficient to confer protection to all known RHDV or RHDV-like strains. For 16 samples, we determined whether these rabbits had died of a chronic or an acute form of the disease, by employing monoclonal antibody (Mabs)--based differential ELISA. All had died of an acute form of RHD. Because the antibodies detected by this VP60-ELISA test are known to appear 5-6 days after infection and since acute RHD generally kills the rabbits 2-3 days after infection, we assumed that the detected antibodies must have been present before the exposure to the virus that killed these rabbits. A second detection of antibodies was made with Mabs that are specific for RHDV. The results were negative, showing that the antibodies detected with the VP60 ELISA test were not specific for RHDV. We sequenced a portion of the VP60 gene of viruses isolated in 17 rabbits. All RHDV isolates were very similar to the RHDV strains commonly isolated in France during this period, suggesting that this viral strain was not a putative variant that is not neutralised by antibodies. Therefore we conclude that the detected antibodies were probably due to a RHDV-like virus that induces the production of detectable but non-protective antibodies.     

An experimental contribution to the epizootiology of viral hemorrhagic septicemia of rabbits (rabbit hemorrhagic disease, RHD)--transmission by flies

In experiments adapted to natural conditions it was established that--a conjunctival infection with about 100 RHD-virus particles was successful--in a milieu without flies transmission over a distance of only 50 cm did not take place--iridescent flies (Phormia spp.) seven hours after contamination with RHD-virus material from died rabbits transmitted RHD to susceptible rabbits in an isolated cage. It ist supposed that transmission in this way will be an epizootiological important one for spreading in narrow distances.     

实验性兔出血症病兔肝脏酶组织化学变化研究

用组织化学染色法,对实验性感染兔出血症(RHD)病毒后不同病期(潜伏期、高热期、濒死期和死亡时)病兔的肝脏碱性磷酸酶(AKP)、酸性磷酸酶(ACP)、三磷酸腺苷酶(ATPase)、琥珀酸脱氢酶(SDH)和乳酸脱氢酶(LDH)的活性变化进行了动态观察。结果表明,肝脏AKP、ACP、和LDH活性在潜伏期至高热期增强,以后各期均减弱;ATPase和SDH在病程各期均明显减弱。从而推论,肝细胞的坏死与ACP活性增强、溶酶体活跃有关;黄疸的发生与ATPase活性减弱及肝细胞线粒体受损有关;病兔的肝脏代谢以无氧酵解为主。     

Regulatory T cells are decreased in acute RHDV lethal infection of adult rabbits

Rabbit hemorrhagic disease virus (RHDV) is the etiologic agent of rabbit hemorrhagic disease (RHD), an acute lethal infection that kills 90% of adult rabbits due to severe acute liver inflammation. Interestingly, young rabbits are naturally resistant to RHDV infection. Here, we have compared naturally occurring CD4(+)Foxp3(+) regulatory T cells (Tregs) between young and adult rabbits after infection by RHDV. The number and frequency of Tregs was decreased in the spleen of adult rabbits 24h after the RHDV infection; this was in contrast with the unchanged number and frequency of splenic Tregs found in young rabbits after the same infection. Also, serum levels of IL-10 and TGF-β were enhanced in the infected adult rabbits whereas no alteration was observed in infected young rabbits. However, this increase is accompanied by a burst of pro-inflammatory cytokines, but seems not able to prevent the death of the animals with severe acute liver inflammation in few days after infection. Since Tregs downregulate inflammation, we conclude that their decrease may contribute to the natural susceptibility of adult rabbits to RHDV infection.     

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.     

Immunohistological diagnosis of rabbit haemorrhagic disease in experimentally infected rabbits in Spain.

The immunoreactivity of routinely processed liver and lung tissue samples obtained from rabbits inoculated with tissue explants from naturally infected animals when antisera directed against parvovirus from different species (canine, feline and porcine) as well as a RHD virus antiserum were employed has been tested by different immunoperoxidase methods. Cross-reactivity between RHD-virus antigens and parvovirus antigens was present. Best results were obtained with RHD and canine parvovirus antisera with the ABC method. The immunoreactivity in the liver was found in hepatocytes, Kupffer and bile duct cells. In the lung, it was exclusively observed in intravascular macrophages.     

A simple and rapid method for isolation of caliciviruses from liver of infected rabbits

Rabbit Haemorrhagic Disease Virus (RHDV), a member of the Caliciviridae family, is the etiologic agent of Rabbit Haemorrhagic Disease (RHD); this viral disease is highly contagious and kills more than 90% of infected adult rabbits. Research on experimental calicivirus infection uses inocula obtained from livers of rabbits dying from calicivirus infection. This implies that caliciviruses have to be purified from liver homogenates. Current methods to isolate caliciviruses from rabbit livers are time consuming. We propose here a new procedure for fast purification of rabbit caliciviruses from liver homogenates that uses centrifugation through an iodixanol gradient. This method offers in approximately 2 h a sample with a high degree of calicivirus purity, as shown by its biochemical and immunocytochemistry analysis, which is also able to kill adult rabbits from RHD within 48 h of inoculation.     

Severe leukopenia and liver biochemistry changes in adult rabbits after calicivirus infection

Calicivirus infection is the major cause of the severe decrease in the stocks of wild and farm rabbits that has occurred worldwide during the last two decades. Adult rabbits (10-weeks-old) were experimentally infected with a calicivirus inoculum that killed all animals by causing rabbit haemorrhagic disease (RHD) within 24-62 h of infection. The rabbits were used to evaluate blood cell numbers and serum biochemistry every 6h, starting 12h after the inoculation of the caliciviruses. No significant changes in blood parameters were observed in most of the rabbits up to 18 h of infection. Severe leukopenia was seen 6h before death of the infected rabbits; both heterophils and lymphocytes contributed to the decrease in circulating white blood cells. Platelets were also severely decreased in number. Marked enhancement in liver enzymes was seen 6-12 h before death of the infected rabbits. There was also evidence both for cholestasis, as expressed by the elevated levels of direct (conjugated) bilirubin, and for hypoglycemia, an alteration that it is likely to contribute for the seizures that rabbits show during the late stages of RHD. Liver ultrastructure of rabbits that died from RHD revealed extensive hepatocyte vacuolization, severe changes in mitochondrial structure, and depletion of glycogen granules. We conclude that: (i) severe leukopenia characterizes the final hours of calicivirus-induced RHD; (ii) hypoglycemia and cholestasis precede death of rabbits from RHD; (iii) the kinetics of liver enzymes allows an accurate prediction of the time of death of rabbits from calicivirus-induced RHD.