Emergence of a new lagovirus related to Rabbit Haemorrhagic Disease Virus

Since summer 2010, numerous cases of Rabbit Haemorrhagic Disease (RHD) have been reported in north-western France both in rabbitries, affecting RHD-vaccinated rabbits, and in wild populations. We demonstrate that the aetiological agent was a lagovirus phylogenetically distinct from other lagoviruses and which presents a unique antigenic profile. Experimental results show that the disease differs from RHD in terms of disease duration, mortality rates, higher occurrence of subacute/chronic forms and that partial cross-protection occurs between RHDV and the new RHDV variant, designated RHDV2. These data support the hypothesis that RHDV2 is a new member of the Lagovirus genus. A molecular epidemiology study detected RHDV2 in France a few months before the first recorded cases and revealed that one year after its discovery it had spread throughout the country and had almost replaced RHDV strains. RHDV2 was detected in continental Italy in June 2011, then four months later in Sardinia.     

云南省楚雄州部分地区猪瘟血清学调查

为了解楚雄州部分地区的猪瘟免疫情况,利用酶联免疫法(ELISA)对楚雄市、南华县和禄丰县随机采取的393份血清进行猪瘟抗体检测,并对各县(市)的调查数据加以比较,了解猪瘟在楚雄州部分地区的免疫情况。结果显示,楚雄州部分地区均有较高的猪瘟抗体阳性率,各县(市)的猪瘟抗体阳性率都在80%以上,有的县(市)猪瘟抗体甚至达到了100%。说明楚雄州部分地区的猪瘟免疫效果较好,猪瘟免疫成功。     

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 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.     

Antibody responses to rabbit haemorrhagic disease virus in predators, scavengers, and hares in New Zealand during epidemics in sympatric rabbit populations

AIM: To test for antibodies to rabbit haemorrhagic disease (RHD) virus (RHDV) in sera from mammals and birds associated with rabbit populations infected with RHDV. METHODS: Sera from feral and domestic cats, feral ferrets, stoats, hedgehogs, hares, harrier hawks, and black-backed gulls were taken (apart from some of the hares) from areas in New Zealand where RHD was active among rabbit populations. The presence of antibodies to RHD was investigated using a competition enzyme-linked immunosorbent assay (ELISA). RESULTS: Some individual animals of all species were seropositive. Thirty eight of 71 feral cats, but only 1/80 domestic cats were seropositive at a 1:40 dilution. The latter had not been exposed to RHDV. Also reactive in the ELISA were 2/8 stoats; 11/115 ferrets, with significantly more females having antibodies than males; 4/73 hedgehogs; 2/18 hawks, and 1/30 gulls. Three of 66 hares, comprising 3/14 from one population, were seropositive. CONCLUSIONS: Apart from the hares, all these species are known to prey upon rabbits or scavenge their carcasses, a possible means of exposure to RHDV. The possibility that the positive test reactions were due to cross-reactions with other caliciviruses cannot be ruled out, especially for the hares. Nor could the study differentiate whether the positive results were due to an antigenic reaction to ingestion of RHDV, as suggested by overseas work, or to infection of new species by RHDV. These possibilities are being investigated further.     

应用单克隆抗体夹心酶联免疫吸附试验检测兔出血症病毒

应用建立的单克隆抗体夹心酶联兔疫吸附试验（ＭｃＡｂ－ＥＬＩＳＡ）,检测了人工感染兔出血症病毒（ＲＨＤＶ）ＤＪＲＫ细胞毒、肝毒的兔以及自然感染ＲＨＤＶ的兔的组织样品。结果表明,感染死亡兔的肝、脾、肾、骨髓样品病毒抗原的检出率为１００％,淋巴结和肌肉的检出率分别为９７．５％和７９．５％。ＭｃＡｂ－ＥＬＩＳＡ能检出肌肉中血凝试验不能检出的ＲＨＤＶ抗原。此外,还用ＭｃＡｂ－ＥＬＩＳＡ检测了肝毒人工感染兔血中ＲＨＤＶ的动态,并对１０份兔出血症脏器灭活苗的效价作了滴定。     

A serial cross-sectional study of the prevalence of rabbit haemorrhagic disease on three farms in the lower North Island of New Zealand

AIM: To estimate over a 3-year period following the first release of rabbit haemorrhagic disease virus (RHDV) the prevalence of rabbit haemorrhagic disease (RHD) and the abundance of rabbits (Oryctolagus cuniculus) in an area that historically had low rabbit densities.

METHODS: Three farms grazing predominantly sheep and beef cattle, located close together and with low initial rabbit densities, were selected for study. RHDV had been deliberately released on all farms in December 1997. Farms were visited 2–3 times per year between June 1998 and April 2001. At each visit, rabbits were shot with the aid of spotlights at night and blood samples were collected for detection of RHDV antibodies. Rabbit carcasses were necropsied and the age of the animals was determined. Rabbit abundance on each property was measured throughout the study using spotlight night counts. Logistic regression was used to identify factors associated with the risk of carcasses being seropositive for RHDV.

RESULTS: Rabbit density differed initially between farms (8.2, 9.9, 2.3 rabbits per spotlight km in June 1998), and declined on all three properties over time (1.2, 2.4, 1.1 rabbits per spotlight km in November 2000). Highest antibody titres to RHDV were initially evident on the farm on which rabbits were most abundant. The average prevalence of seropositive rabbits overall was 21% (95% CI=15–28%). Female rabbits tended to be less likely to be seropositive for RHDV than males (OR=0.47; 95% CI=0.21–1.02). The odds of becoming seropositive were reduced for rabbits born in the breeding season of 1999–2000 (OR=0.17; 95% CI=0.05–0.64).

CONCLUSIONS: The temporal pattern of outbreaks measured by peaks of seroprevalence differed between closely-spaced farms when they had different rabbit densities, but were similar when rabbit densities were similar. Microclimate and vegetation influencing abundance of insect vectors for RHDV and intrinsic population-related factors like rabbit breeding behaviour are also likely to be involved in local patterns of spread.     

An epidemiological study of the spread of rabbit haemorrhagic disease virus amongst previously non-exposed rabbit populations in the North Island of New Zealand

AIM: To monitor the initial releases of rabbit haemorrhagic disease virus (RHDV) into previously unexposed rabbit populations in the North Island of New Zealand. METHODS: The study programme consisted of pre-release spotlight counts of rabbits on the study farms, pre-release serological samples to check for prior exposure to RHDV, a farmer-completed questionnaire and post-release spotlight counts to measure any change in rabbit numbers following the release of RHDV. In total, 23 sites within the lower North Island where RHDV was released during the period November 1997 to June 1998, were monitored. The most common release method involved the spreading of chopped carrot bait laced with a solution of virus-infected material obtained from dead rabbits. RESULTS: Eighty percent of farmers thought that the disease had spread away from the release sites to areas where virus had not been liberated, although only 27% reported finding dead rabbits more than 300 m away from release locations. Seventy-three percent of farmers were satisfied with the overall effectiveness of rabbit haemorrhagic disease (RHD) as a means of reducing rabbit numbers, but 56% indicated they would modify the way they released the virus in the future. Average pre-release night spotlight counts per property ranged from 2.2 rabbits/km to 36.9 rabbits/km, the median being 12.8 rabbits/km. The time interval from initial release to when the first dead rabbit was seen which the farmer believed to have died from RHD varied from 3 to 21 days, the mean being 7.4 days and the median 7 days. The median change in night spotlight counts per site at 3 weeks after release, expressed as a percentage relative to pre-release counts, was -15.5% (range +18.9% to -76.9%) and at 6 weeks was -49.7% (range 0% to -76.9%). The time of the estimated peak of the disease epidemic ranged from 1 to 7 weeks after release of RHDV, the mean being 3.1 and the median 3 weeks. CONCLUSION: Rabbit haemorrhagic disease reduced rabbit numbers on the majority of farms where the virus was released, and appears to be an effective measure for controlling rabbit populations in New Zealand.     

A serial cross-sectional study of the prevalence of rabbit haemorrhagic disease on three farms in the lower North Island of New Zealand

AIM: To estimate over a 3-year period following the first release of rabbit haemorrhagic disease virus (RHDV) the prevalence of rabbit haemorrhagic disease (RHD) and the abundance of rabbits (Oryctolagus cuniculus) in an area that historically had low rabbit densities. METHODS: Three farms grazing predominantly sheep and beef cattle, located close together and with low initial rabbit densities, were selected for study. RHDV had been deliberately released on all farms in December 1997. Farms were visited 2-3 times per year between June 1998 and April 2001. At each visit, rabbits were shot with the aid of spotlights at night and blood samples were collected for detection of RHDV antibodies. Rabbit carcasses were necropsied and the age of the animals was determined. Rabbit abundance on each property was measured throughout the study using spotlight night counts. Logistic regression was used to identify factors associated with the risk of carcasses being seropositive for RHDV. RESULTS: Rabbit density differed initially between farms (8.2, 9.9, 2.3 rabbits per spotlight km in June 1998), and declined on all three properties over time (1.2, 2.4, 1.1 rabbits per spotlight km in November 2000). Highest antibody titres to RHDV were initially evident on the farm on which rabbits were most abundant. The average prevalence of seropositive rabbits overall was 21% (95% CI=15-28%). Female rabbits tended to be less likely to be seropositive for RHDV than males (OR=0.47; 95% CI=0.21-1.02). The odds of becoming seropositive were reduced for rabbits born in the breeding season of 1999-2000 (OR=0.17; 95% CI=0.05-0.64). CONCLUSIONS: The temporal pattern of outbreaks measured by peaks of seroprevalence differed between closely-spaced farms when they had different rabbit densities, but were similar when rabbit densities were similar. Microclimate and vegetation influencing abundance of insect vectors for RHDV and intrinsic population-related factors like rabbit breeding behaviour are also likely to be involved in local patterns of spread.     

An epidemiological study of the spread of rabbit haemorrhagic disease virus amongst previously non-exposed rabbit populations in the North Island of New Zealand

Aim: To monitor the initial releases of rabbit haemorrhagic disease virus (RHDV) into previously unexposed rabbit populations in the North Island of New Zealand.

Methods: The study programme consisted of pre-release spotlight counts of rabbits on the study farms, pre-release serological samples to check for prior exposure to RHDV, a farmer-completed questionnaire and post-release spotlight counts to measure any change in rabbit numbers following the release of RHDV. In total, 23 sites within the lower North Island where RHDV was released during the period November 1997 to June 1998, were monitored. The most common release method involved the spreading of chopped carrot bait laced with a solution of virus-infected material obtained from dead rabbits.

Results: Eighty percent of farmers thought that the disease had spread away from the release sites to areas where virus had not been liberated, although only 27% reported finding dead rabbits more than 300 m away from release locations. Seventy-three percent of farmers were satisfied with the overall effectiveness of rabbit haemorrhagic disease (RHD) as a means of reducing rabbit numbers, but 56% indicated they would modify the way they released the virus in the future. Average pre-release night spotlight counts per property ranged from 2.2 rabbits/km to 36.9 rabbits/km, the median being 12.8 rabbits/km. The time interval from initial release to when the first dead rabbit was seen which the farmer believed to have died from RHD varied from 3 to 21 days, the mean being 7.4 days and the median 7 days. The median change in night spotlight counts per site at 3 weeks after release, expressed as a percentage relative to pre-release counts, was -15.5% (range + 18.9% to -76.9%) and at 6 weeks was -49.7% (range 0% to -76.9%). The time of the estimated peak of the disease epidemic ranged from 1 to 7 weeks after release of RHDV, the mean being 3.1 and the median 3 weeks.

Conclusion: Rabbit haemorrhagic disease reduced rabbit numbers on the majority of farms where the virus was released, and appears to be an effective measure for controlling rabbit populations in New Zealand.     

应用免疫组化PAP法检测兔出血症病毒抗原的动态分布

应用免疫组化PAP法检测了人工感染成年患兔、30～40日龄患兔以及自然感染患兔体内兔出血症病毒（RHDV）抗原的动态分布。结果表明,在成年患兔的肝、肾、脾、胃、十二指肠、睾丸以及幼兔的肝、肾、脾中检出了RHDV抗原;无论在成年或幼龄患兔,RHDV抗原主要位于受侵害细胞胞浆中,少部分位于核中;在成年患兔,RHDV抗原阳性细胞的数量随病程发展而增加,而在幼龄患兔,这种增加趋势不明显。本文还分析了RHDV抗原在患兔体内的动态分布与病变形成之间的关系。     

Rabbit haemorrhagic disease: advantages of cELISA in assessing immunity in wild rabbits (Oryctolagus cuniculus)

Rabbit haemorrhagic disease (RHD) is an acute fatal disease of domestic and wild European rabbits (Oryctolagus cuniculus) caused by RHD virus (RHDV). Accurate assessment of immunity is of great importance for the conservation and control of wild rabbits. We evaluated a competitive ELISA (cELISA) against isotype ELISAs for assessing the protective immunity against the disease by challenging 50 wild-caught rabbits with a lethal dose of RHDV. Death or survival to the challenge was used as a criterion to determine the performance characteristics of the assay for the assessment of immunity in rabbits. At 1:10 dilution, a serum exhibiting ≥ 25% inhibition (1:10(25)) was regarded as the presence of RHDV-specific antibodies. Eleven of 16 (68.8%) rabbits with antibodies at 1:10(25) (<1:40) died of RHD. When the cut-off was moved from 25% to 50% inhibition (1:10(50)) at 1:10 serum dilution, the assay sensitivity, specificity and accuracy for the protective immunity were improved from 84%, 54.2% and 69.4% to 84%, 100% and 91.8%, respectively. We also demonstrated at the epitope amino acid sequence level why the presence of the RHDV-cross reactive benign rabbit calicivirus, which interfered with isotype ELISAs, had little impact on the specificity of the cELISA for the diagnosis of RHDV infection. The presence of RHDV-specific antibody at 1:10(50) by the cELISA is a reliable indicator for the protective immunity. In contrast to isotype ELISAs, the cELISA is a valuable specific tool for monitoring the herd immunity to RHD for the conservation and management of wild rabbits in the field.     

Immunosuppression abrogates resistance of young rabbits to Rabbit Haemorrhagic Disease (RHD)

Rabbit Haemorrhagic Disease (RHD) is caused by a calicivirus (RHDV) that kills 90% of infected adult European rabbits within 3 days. Remarkably, young rabbits are resistant to RHD. We induced immunosuppression in young rabbits by treatment with methylprednisolone acetate (MPA) and challenged the animals with RHDV by intramuscular injection. All of these young rabbits died within 3 days of infection due to fulminant hepatitis, presenting a large number of RHDV-positive dead or apoptotic hepatocytes, and a significant seric increase in cytokines, features that are similar to those of naïve adult rabbits infected by RHDV. We conclude that MPA-induced immunosuppression abrogates the resistance of young rabbits to RHD, indicating that there are differences in the innate immune system between young and adult rabbits that contribute to their distinct resistance/susceptibility to RHDV infection.     

Identification of Polish RHDVa subtype strains based on the analysis of a highly variable part of VP60 gene

In order to determine the genetic variability of Polish RHD virus strains and to confirm the presence of genetic variant (RHDVa) subtype the partial nucleotide sequences of capsid protein gene, including two highly variable regions C and E, were examined. Phylogenetic analyses of 15 viral strains obtained over 18 years revealed the presence of three genetic groups. The oldest RHDV strains exhibit very close amino acid sequence similarity (98-99%) to the German FRG89 reference strain and most of European strains of the same period, as well as Chinese isolate from 1984. The HA-negative strains and isolates with variable reactivity in the HA test belong to the second subgroup and exhibit an intermediate level of variability (about 3%) in the analysed VP60 gene fragment. The most genetically variable strains (6-7%) clustered to RHDVa subtype. The analysis of nucleotides and amino acid sequences demonstrated three pairs of well conserved RHDV strains, isolated over 3, 6 and 10-year period.     

Crypt abscesses in the caeca of feral rabbits in a rabbit haemorrhagic disease endemic region of New Zealand

AIM: To describe the pathology of crypt abscesses in the caeca of feral rabbits in the Manawatu region of New Zealand, and to examine the possible relationship between their prevalence and rabbit haemorrhagic disease (RHD) virus (RHDV) infection.

METHODS: During the course of a 3-year study of RHD, 173 wild rabbits (Oryctolagus cuniculus) were shot on three pastoral livestock farms in the Manawatu region of New Zealand. All rabbits were necropsied, and tissue samples of the caeca were examined histologically. The age of each animal was determined, and blood samples collected for the detection of RHDV antibodies. Logistic regression was used to model the odds of rabbits having crypt abscesses.

RESULTS: At necropsy, 63/173 (36.4%) rabbits were found to have small circular black nodules on the mucosal surface of their appendix caecum and/or sacculus rotundus. Microscopically, these were identified as small crypt abscesses composed of dilated sacs at the base of the mucosa that were often lined by a thin layer of attenuated epithelial cells. They usually contained large amounts of concentrically-laminated mucopolysaccharide material that was sometimes pigmented, inflammatory debris, and were often the site of a moderate multifocal appendicitis. Although RHDV was active in the study area, no association was found between RHDV antibodies in serum and the presence of lesions. The lesions were more common in older individuals and those born in summer or autumn.

CONCLUSIONS: This is the first report of crypt abscesses with inflammation and necrotic debris in the caeca of rabbits. No association between the occurrence of crypt abscesses and RHDV infection was identified. Wild rabbits born in a particular season were presumably exposed very early in life to conditions that caused the crypt abscesses to develop. Alternatively, association with season of birth may represent rabbits that were of similar age in a later stage of their lives, when they became exposed to the cause of the lesions, which remains unidentified.     

Crypt abscesses in the caeca of feral rabbits in a rabbit haemorrhagic disease endemic region of New Zealand

AIM: To describe the pathology of crypt abscesses in the caeca of feral rabbits in the Manawatu region of New Zealand, and to examine the possible relationship between their prevalence and rabbit haemorrhagic disease (RHD) virus (RHDV) infection. METHODS: During the course of a 3-year study of RHD, 173 wild rabbits (Oryctolagus cuniculus) were shot on three pastoral livestock farms in the Manawatu region of New Zealand. All rabbits were necropsied, and tissue samples of the caeca were examined histologically. The age of each animal was determined, and blood samples collected for the detection of RHDV antibodies. Logistic regression was used to model the odds of rabbits having crypt abscesses. RESULTS: At necropsy, 63/173 (36.4%) rabbits were found to have small circular black nodules on the mucosal surface of their appendix caecum and/or sacculus rotundus. Microscopically, these were identified as small crypt abscesses composed of dilated sacs at the base of the mucosa that were often lined by a thin layer of attenuated epithelial cells. They usually contained large amounts of concentrically-laminated mucopolysaccharide material that was sometimes pigmented, inflammatory debris, and were often the site of a moderate multifocal appendicitis. Although RHDV was active in the study area, no association was found between RHDV antibodies in serum and the presence of lesions. The lesions were more common in older individuals and those born in summer or autumn. CONCLUSIONS: This is the first report of crypt abscesses with inflammation and necrotic debris in the caeca of rabbits. No association between the occurrence of crypt abscesses and RHDV infection was identified. Wild rabbits born in a particular season were presumably exposed very early in life to conditions that caused the crypt abscesses to develop. Alternatively, association with season of birth may represent rabbits that were of similar age in a later stage of their lives, when they became exposed to the cause of the lesions, which remains unidentified.     

基因重组抗原酶联免疫法检测兔出血症病毒抗体及其标准化

以重组兔出血症病毒(RHDV)VP60蛋白为抗原，建立了RHDV抗体间接ELISA检测方法。优化的试验反应务件为：重组VP60的包被质量浓度为1．0mg／L，用10％牛血清封闭，以大肠杆菌提取物稀释被检血清以消除非特异性反应。将所建立的ELISA与现行血凝抑制(HI)试验比较发现，不同免疫状态的兔血清的RHDV ELISA抗体与HI抗体均呈正相关。对11个RHD免疫兔场1130份血清样品的抗体检测表明，各免疫兔群血清RHDV抗体水平不完全一致，D值在1．09～1．76之间，显著高于非免疫兔(0．05)及SPF兔(0．02)，低于高免兔(2．34)。在此基础上，研制了RHDV抗体酶联免疫检测试剂盒，测定了其主要指标，制定了各成分的质量控制标准，为兔群进行免疫学监测及评价疫苗的免疫效果提供了便利。     

Clinical and pathologic findings in an outbreak in rabbits of natural infection by rabbit hemorrhagic disease virus 2 in the northwestern United States

Rabbit hemorrhagic disease virus 2 (RHDV2) causes an often-fatal disease of rabbits that has resulted in outbreaks in rabbitries in Europe, Africa, Australia, and Asia. RHD has historically been characterized as a foreign animal disease in the United States. In July 2019, RHDV2 was detected in rabbits on Orcas Island along the northwestern coast of Washington (WA) State following reports of deaths in multiple feral and domestic rabbits. We document and highlight here the unique clinical presentation and gross and histologic lesions observed in this recent WA outbreak. Affected rabbits died without premonitory signs or displayed hyporexia and/or lethargy for ≤1 d prior to death. The most consistent pathologic finding was random, multifocal hepatocellular necrosis, often with concurrent multifocal-to-diffuse splenic necrosis. The lack of significant clinical signs in conjunction with the random distribution of hepatic necrosis in the WA outbreak contrasts with previous reports of RHDV2 disease progression.     

Codon optimization of the rabbit hemorrhagic disease virus (RHDV) capsid gene leads to increased gene expression in Spodoptera frugiperda 9 (Sf9) cells

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.