Apoptotic Cell Death in an Animal Model of Virus-Induced Acute Liver Failure-Observations during Lagovirus europaeus/GI.2 Infection.

Lagovirus europaeus/GI.2 causes severe and highly fatal Rabbit Hemorrhagic Disease (RHD). Because of its characteristics, this infection is used as an animal model for acute liver failure (ALF). Apoptosis is one of the key processes underlying ALF and has been described as one of the mechanisms of RHD pathogenesis. Apoptotic cell death has been quite well characterized in infection with different variants of GI.1 strains, but so far, the GI.2 genotype has not been widely studied. In this study, we performed an evaluation of apoptotic cell death in hepatocytes of rabbits infected with Lagovirus europaeus/GI.2. We analyzed the expression of genes involved in apoptotic cell death by real-time PCR and performed immunohistochemical (IHC) assays. We showed a significant increase in the expression of caspase-3 and the proapoptotic Bax and anti-apoptotic Bcl-2 in infected animals. In addition, we recorded increased Bax/Bcl-2 ratios. IHC analyses showed the presence of morphological signs of apoptosis in the hepatocytes of infected rabbits. Our results indicate that caspase-3 and proteins from the Bcl-2 families play a key role in apoptosis induced by Lagovirus europaeus/GI.2 infection.

Genetics of the rabbit haemorrhagic disease virus strains responsible for rabbit haemorrhagic disease outbreaks in Japan between 2000 and 2020.

Rabbit haemorrhagic disease (RHD) is a highly contagious and fatal disease in rabbits caused by the rabbit haemorrhagic disease virus (RHDV), which includes two genotypes, RHDV-GI.1 and RHDV2-GI.2. RHDVs tend to recombine among different strains, resulting in significant genetic evolution. This study evaluated the genetics of Japanese RHDV strains associated with six outbreaks between 2000 and 2020 using whole-genome sequencing, genomic recombination and phylogenetic analyses. Genomic recombination analysis using near-complete genomic sequences revealed that two Japanese strains detected in 2000 and 2002 were non-recombinant GI.1 (variant RHDVa-GI.1a) strains of different origins, most closely related to strains identified in PR China in 1997 and the USA in 2001, respectively. In contrast, four recent Japanese GI.2 strains detected between 2019 and 2020 were recombinant viruses harbouring structural protein (SP) genes from GI.2 strains and non-SP (NSP) genes from a benign rabbit calicivirus (RCV) strain of genotype RCV-E1-GI.3 (GI.3P-GI.2) or an RHDV G1-GI.1b variant (GI.1bP-GI.2). Phylogenetic analysis based on SP and NSP regions revealed that the GI.1bP-GI.2 recombinant virus detected in Ehime prefecture and the GI.3P-GI.2 recombinant viruses detected in Ibaraki, Tochigi and Chiba prefectures were most closely related to recombinant viruses identified in Australia in 2017 and Germany in 2017, respectively. These results suggested that past RHD outbreaks in Japan did not result from the evolution of domestic RHDVs but rather represented incursions of foreign RHDV strains, implying that Japan is constantly at risk of RHDV incursion from other countries.

The impact of preoperative sarcopenia on postoperative ileus following colorectal cancer surgery.

PURPOSE: Sarcopenia is associated with poor short- and long-term patient outcomes following colorectal surgery. Despite postoperative ileus (POI) being a major complication following colorectal surgery, the predictive value of sarcopenia for POI is unclear. We assessed the association between sarcopenia and POI in patients with colorectal cancer. METHODS: Elective colorectal cancer surgery patients were retrospectively included (2018-2022). The cross-sectional psoas area was calculated using preoperative staging imaging at the level of the 3rd lumbar vertebrae. Sarcopenia was determined using gender-specific cut-offs. The primary outcome POI was defined as not achieving GI-2 by day 4. Demographics, operative characteristics, and complications were compared via univariate and multivariate analyses. RESULTS: Of 297 patients, 67 (22.6%) were sarcopenic. Patients with sarcopenia were older (median 74 (IQR 67-82) vs. 69 (58-76) years, p < 0.001) and had lower body mass index (median 24.4 (IQR 22.2-28.6) vs. 28.8 (24.9-31.9) kg/m2, p < 0.001). POI was significantly more prevalent in patients with sarcopenia (41.8% vs. 26.5%, p = 0.016). Overall rate of complications (85.1% vs. 68.3%, p = 0.007), Calvien-Dindo grade > 3 (13.4% vs. 10.0%, p = 0.026) and length of stay were increased in patients with sarcopenia (median 7 (IQR 5-12) vs. 6 (4-8) days, p = 0.013). Anastomotic leak rate was higher in patients with sarcopenia although the difference was not statistically significant (7.5% vs. 2.6%, p = 0.064). Multivariate analysis demonstrated sarcopenia (OR 2.0, 95% CI 1.1-3.8), male sex (OR 1.9, 95% CI 1.0-3.5), postoperative hypokalemia (OR 3.2, 95% CI 1.6-6.5) and increased opioid use (OR 2.4, 95% CI 1.3-4.3) were predictive of POI. CONCLUSION: Sarcopenia demonstrates an association with POI. Future research towards truly identifying the predictive value of sarcopenia for postoperative complications could improve informed consent and operative planning for surgical patients.

Effect of neuromuscular reversal with neostigmine/glycopyrrolate versus sugammadex on postoperative ileus following colorectal surgery.

BACKGROUND: Postoperative ileus (POI) is a common complication following colorectal surgery and is mediated in part by the cholinergic anti-inflammatory pathway (CAIP). Neostigmine (acetylcholinesterase inhibitor), co-administered with glycopyrrolate, is frequently given for neuromuscular reversal before tracheal extubation and modulates the CAIP. An alternative reversal agent, sugammadex (selective rocuronium or vecuronium binder), acts independently from the CAIP. The aim of our study was to assess the impact of neuromuscular reversal agents used during anaesthesia on gastrointestinal recovery. METHODS: Three hundred thirty-five patients undergoing elective colorectal surgery at the Royal Adelaide Hospital between January 2019 and December 2021 were retrospectively included. The primary outcome was GI-2, a validated composite measure of time to diet tolerance and passage of stool. Demographics, 30-day complications and length of stay were collected. Univariate and multivariate analyses were performed. RESULTS: Two hundred twenty-four (66.9%) patients (129 [57.6%] males and 95 [42.4%] females, median age 64 [19-90] years) received neostigmine/glycopyrrolate and 111 (33.1%) received sugammadex (62 [55.9%] males and 49 [44.1%] females, median age 67 [18-94] years). Sugammadex patients achieved GI-2 sooner after surgery (median 3 (0-10) vs. 3 (0-12) days, p = 0.036), and reduced time to first stool (median 2 (0-10) vs. 3 (0-12) days, p = 0.035). Rates of POI, complications and length of stay were similar. On univariate analysis, POI was associated with smoking history, previous abdominal surgery, colostomy formation, increased opioid use and postoperative hypokalaemia (p < 0.05). POI was associated with increased complications, including anastomotic leak and prolonged hospital stay (p < 0.001). On multivariate analysis, neostigmine, bowel anastomoses and increased postoperative opioid use (p < 0.05) remained predictive of time to GI-2. CONCLUSIONS: Patients who received sugammadex had a reduced time to achieving first stool and GI-2. Neostigmine use, bowel anastomoses and postoperative opioid use were associated with delayed time to achieving GI-2.

PyRICo-Pilot: pyridostigmine to reduce the duration of postoperative ileus after colorectal surgery - a phase II study.

AIM: Postoperative ileus (POI) is a major problem after colorectal surgery. Acetylcholinesterase inhibitors such as pyridostigmine increase gastrointestinal (GI) motility through a cholinergic anti-inflammatory pathway. The purpose of this phase II pilot study is to determine the safety of oral pyridostigmine after elective colorectal surgery. METHOD: This is a Stage 2b safety study (IDEAL framework). All adult patients undergoing elective colorectal resection or formation or reversal of stoma at the Royal Adelaide Hospital between September 2020 and January 2021 were eligible. The primary outcomes were 30-day postoperative complications, reported adverse events and GI-2 - a validated composite outcome measure of recovery of GI function after surgery, defined as the interval from surgery until first passage of stool and tolerance of a solid intake for 24 h (in whole days) in the absence of vomiting. RESULTS: Fifteen patients were included in the study. The median age was 58 (range 50-82) years and seven (47%) were men. Most participants had an American Society of Anesthesiologists grade ≥2 (53%) and the median body mass index was 27 (24-35) kg/m2 . There were 13 postoperative complications [seven were Clavien-Dindo (CD) 1, five CD 2 and one CD 3]. None appeared directly related to pyridostigmine administration, and none of the patients had any overt symptoms of excessive parasympathetic activity. Median GI-2 was 2 (1-4) days. CONCLUSION: Oral pyridostigmine appears to be safe to use after elective colorectal surgery in a select group of patients. However, considering this is a pilot study with a small sample size, larger controlled studies are needed to confirm this finding and establish efficacy for prevention of POI.

Lagovirus europeus GI.2 (rabbit hemorrhagic disease virus 2) infection in captive mountain hares (Lepus timidus) in Germany.

BACKGROUND: Rabbit hemorrhagic disease virus (RHDV, Lagovirus europeus GI.1) induces a contagious and highly lethal hemorrhagic disease in rabbits. In 2010 a new genotype of lagovirus (GI.2), emerged in Europe, infecting wild and domestic population of rabbits and hares. CASE PRESENTATION: We describe the infection with a GI.2 strain, "Bremerhaven-17", in captive mountain hares (Lepus timidus) in a zoo facility in Germany. Postmortem examination revealed RHD-like lesions including necrotizing hepatitis. RT-qPCR and AG-ELISA confirmed presence of GI.2. Recombination and phylogenetic analysis grouped the identified strain with other GI.2 strains, sharing nucleotide identity of 91-99%. CONCLUSION: Our findings confirm that mountain hares are susceptible to GI.2 infection, due to a past recombination event facilitating virus spillover from sympatric rabbits.

Overcoming species barriers: an outbreak of Lagovirus europaeus GI.2/RHDV2 in an isolated population of mountain hares (Lepus timidus).

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.

Role of a heterotrimeric G-protein, Gi2, in the corticogenesis: possible involvement in periventricular nodular heterotopia and intellectual disability.

We analyzed the role of a heterotrimeric G-protein, Gi2, in the development of the cerebral cortex. Acute knockdown of the α-subunit (Gαi2) with in utero electroporation caused delayed radial migration of excitatory neurons during corticogenesis, perhaps because of impaired morphology. The migration phenotype was rescued by an RNAi-resistant version of Gαi2. On the other hand, silencing of Gαi2 did not affect axon elongation, dendritic arbor formation or neurogenesis at ventricular zone in vivo. When behavior analyses were conducted with acute Gαi2-knockdown mice, they showed defects in social interaction, novelty recognition and active avoidance learning as well as increased anxiety. Subsequently, using whole-exome sequencing analysis, we identified a de novo heterozygous missense mutation (c.680C>T; p.Ala227Val) in the GNAI2 gene encoding Gαi2 in an individual with periventricular nodular heterotopia and intellectual disability. Collectively, the phenotypes in the knockdown experiments suggest a role of Gαi2 in the brain development, and impairment of its function might cause defects in neuronal functions which lead to neurodevelopmental disorders.

First detection and molecular characterization of rabbit hemorrhagic disease virus (RHDV) in Algeria.

Since the first detection of rabbit hemorrhagic disease (RHD), the rabbit hemorrhagic disease virus (RHDV) has been responsible for high morbidity and mortality worldwide, both in domestic and in wild rabbits. Despite the apparent control of RHD in rabbitries through vaccination, several studies highlighted the rapid evolution of RHDV by recombination, which may facilitate the emergence of new pathogenic strains. The aim of this study was to confirm the presence and characterize RHDV in Algeria. For this, rabbit samples were collected in the north of Algeria, between 2018 and 2021, from small farms where the virus was suspected after the sudden death of a high number of rabbits, and from healthy hunted wild rabbits. The domestic rabbits revealed clinical signs and lesions that were suggestive of RHD. RT-PCR showed that 79.31% of the domestic rabbit samples were positive for RHDV, while in 20.69%, including the hunted rabbits, the virus was not detected. Phylogenetic analysis of the Algerian strains allowed the confirmation and identification as GI.2 (RHDV2), and showed a close relation to GI.3P-GI.2 recombinant strains, suggesting a potential introduction from other countries, with an older strain potentially originated from neighboring Tunisia, while more recent isolates grouped with strains from North America. Our study reports for the first time the presence of GI.2 (RHDV2) in Algeria with multiple routes of introduction. Consequently, we propose that RHDV control in Algeria should be based on epidemiological surveys in association with an adequate prophylactic program.

Comparison of the Impact between Classical and Novel Strains of Rabbit Haemorrhagic Disease on Wild Rabbit Populations in Spain.

The outbreaks of two strains of rabbit haemorrhagic disease (RHD) (GI.1 and GI.2) in the Iberian Peninsula have caused substantial economic losses in commercial rabbitries and have affected the conservation of rabbit-sensitive predators due to the dramatic decline of their natural populations. However, the assessment of the impact of both RHD strains on wild rabbit populations has been limited to a few small-scale studies. Little is known about the overall impact within its native range. In this study, we described and compared the effects of GI.1 and GI.2 countrywide by using time series of hunting bag data widely available across the country and compared their trend during the first eight years after the first outbreak of GI.1 (i.e., 1998) and GI.2 (i.e., 2011), respectively. We used Gaussian generalised additive models (GAM) with the number of hunted rabbits as the response variable and year as the predictor to evaluate the non-linear temporal dynamics of the population at the national and regional community levels. The first GI.1 caused a population decline of around 53%, affecting most Spanish regional communities where the disease occurred. The positive trend observed after GI.1 in Spain ended with the initial outbreak of GI.2, which did not appear to cause a national population decline. In contrast, we found significant variability in the rabbit population trend among regional communities, where some increased, and others decreased. Such a disparity is unlikely to be explained by a single factor; rather, it appears to result from several factors, such as climatic conditions, host resistance improvement, virulence attenuation, or population density. Our study suggests that a national comprehensive hunting bag series could aid in elucidating the differences in the impact of emerging diseases on a large scale. Future research should focus on national longitudinal serological studies to shed light on the immunological status of rabbit populations in different regions to better understand the evolution of RHD strains and the resistance gained by the wild populations.

Chapter 3.7.2. Detection of Rabbit hemorrhagic disease virus 2 in the Pygmy Rabbit (Brachylagus idahoensis) in Nevada, USA.

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.

Dynamics of Humoral Immunity to Myxoma and Rabbit Hemorrhagic Disease Viruses in Wild European Rabbits Assessed by Longitudinal Semiquantitative Serology.

Myxoma virus (MYXV) and rabbit hemorrhagic disease virus (RHDV) are important drivers of the population decline of the European rabbit, an endangered keystone species. Both viruses elicit strong immune responses, but the long-term dynamics of humoral immunity are imperfectly known. This study aimed to assess the determinants of the long-term dynamics of antibodies to each virus based on a longitudinal capture-mark-recapture of wild European rabbits and semiquantitative serological data of MYXV and RHDV GI.2-specific IgG. The study included 611 indirect enzyme-linked immunosorbent assay (iELISA) normalized absorbance ratios for each MYXV and RHDV GI.2 from 505 rabbits from 2018 to 2022. Normalized absorbance ratios were analyzed using log-linear mixed models, showing a significant positive relationship with the time since the first capture of individual rabbits, with monthly increases of 4.1% for antibodies against MYXV and 2.0% against RHDV GI.2. Individual serological histories showed fluctuations over time, suggesting that reinfections boosted the immune response and likely resulted in lifelong immunity. Normalized absorbance ratios significantly increased with the seroprevalence in the population, probably because of recent outbreaks, and with body weight, highlighting the role of MYXV and RHDV GI.2 in determining survival to adulthood. Juvenile rabbits seropositive for both viruses were found, and the dynamics of RHDV GI.2 normalized absorbance ratios suggest the presence of maternal immunity up to 2 months of age. Semiquantitative longitudinal serological data provide epidemiological information, otherwise lost when considering only qualitative data, and support a lifelong acquired humoral immunity to RHDV GI.2 and MYXV upon natural infection. IMPORTANCE This study addresses the long-term dynamics of humoral immunity to two major viral pathogens of the European rabbit, an endangered keystone species of major ecological relevance. Such studies are particularly challenging in free-ranging species, and a combination of longitudinal capture-mark-recapture and semiquantitative serology was used to address this question. Over 600 normalized absorbance ratios of iELISA, obtained from 505 individual rabbits in 7 populations over 5 years, were analyzed using linear mixed models. The results support a lifelong acquired humoral immunity to myxoma virus and rabbit hemorrhagic disease virus upon natural infection and suggest the presence of maternal immunity to the latter in wild juvenile rabbits. These results contribute to understanding the epidemiology of two viral diseases threatening this keystone species and assist in developing conservation programs.

First report of GI.1aP-GI.2 recombinants of rabbit hemorrhagic disease virus in domestic rabbits in China.

The rabbit hemorrhagic disease virus 2 (RHDV2 or GI.2) is a highly contagious agent leading to lethal disease in rabbits. It frequently recombines with other Lagovirus genus, generating epidemical variants with high pathogenicity. In this study, twenty-two liver samples tested positive for GI.2 VP60 gene, were collected in rabbit farms from several geographical regions in China. All GI.2 positive specimens were submitted for RT-PCR detection, nucleotide sequencing and phylogenetic analysis. In addition, suspected GI.2 recombinants were evaluated for virus virulence. The results showed that nine presumptive recombinants were identified by testing for RdRp-VP60 recombination. In these recombinants, four were selected to fully characterize the genome of novel GI.2 recombinant variants, which were described as GI.1aP-GI.2. The nucleotide sequence of these novel variants showed unique recombination pattern and phylogenetic features compared to currently prevalent GI.2 variants. Furthermore, this distinctive recombination of new variant SCNJ-2021 moderately enhanced the virulence of GI.2, even for rabbits vaccinated against parental GI.2. In conclusion, the novel GI.1aP-GI.2 recombinants were identified in rabbit industry in China for the first time, which expanded the knowledge on the phylodynamics and genomic diversity of GI.2 genotype. The rapid molecular evolution and varied pathogenicity of these virus recombinants highlight the urgent need for epidemiological surveillance and for future prevention of these neglected GI.2 variants.

Exposing the incidence of ileus in pelvic and acetabular fractures: a retrospective case analysis.

BACKGROUND: Paralytic ileus is a temporary inhibition of gastrointestinal mobility in the absence of mechanical obstruction. Ileus has previously been observed in up to 40% of patients undergoing bowel surgery, leading to increased morbidity and length of stay. Pelvic and acetabular fractures are often caused by high energy trauma and are associated with a risk of visceral injury.  Prior to this study, there were no reported figures for the incidence of ileus in patients presenting with pelvic and/or acetabular fractures. METHODS: All patients over the age of 16 presenting to a major trauma centre throughout 2019 were included. Data collected included patient demographics, injury pattern, fracture management and presence of ileus. As in previous studies, patients were identified as having ileus if they failed to tolerate an oral diet and open their bowels for more than three days (GI-2). Analysis assessed risk factors for ileus as well as its effect on length of stay. RESULTS: An incidence of ileus of 40.35% was observed in the 57 included patients. Across all patients, ileus was three times more common in patients with a diagnosis of diabetes mellitus (p= 0.56) and 2.5 times more common in the presence of an open pelvic/ acetabular fracture (p= 0.73). Length of stay was significantly longer in patients under 65 years identified as having ileus (p= 0.046). Gender, age, opiate use, fracture management and surgical approach were not identified as risk factors for ileus. CONCLUSION/ FINDINGS: This is the first study to report the incidence of and risk factors for ileus following admission with pelvic and/or acetabular fractures. Due to the morbidity and cost associated with this condition, further research is required to assess the effect of interventions to reduce its incidence in this patient subgroup.

First detection of rabbit haemorrhagic disease virus (RHDV2) in Singapore.

Rabbit haemorrhagic disease (RHD) is a significant viral disease caused by infection with Rabbit haemorrhagic disease virus (RHDV). The first documented cases of RHDV in Singapore occurred in adult pet European rabbits (Oryctolagus cuniculus) in September 2020. Rabbits presented with acute hyporexia, lethargy, huddled posture, and varying degrees of pyrexia and tachypnoea. Clinical pathology consistently reflected markedly elevated alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALKP). Hepatic lobe torsion was ruled out using ultrasonography and colour Doppler studies in all patients. A total of 11 rabbits owned by 3 families were presented to the clinics; 8/11 rabbits died within 48 hr of presentation, while the remaining two rabbits had recovered after prolonged hospitalization and one rabbit was aclinical. Histopathology revealed acute, marked diffuse hepatocellular necrosis and degeneration, findings which were suggestive for RHDV infection and prompted the undertaking of further molecular diagnostics. Subsequent polymerase chain reaction of the liver samples detected RHDV RNA. Molecular characterization of viral genomes by whole genome sequencing revealed that the outbreak strain was of the genotype GI.2 (RHDV2/RHDVb). Nucleotide sequences of the VP60 gene were compared with various RHDV variants using phylogenetic analysis. The sample genome shared highest sequence identity with a GI.2-genotyped virus from GenBank (RHDV isolate Algarve 1 polyprotein and minor structural protein (VP10) genes, GenBank accession KF442961). The combination of clinical, histopathological, molecular and sequencing technologies enabled rapid detection and detailed genetic characterization of the RHDV virus causing the present outbreak for prompt implementation of disease control measures in Singapore. Further epidemiological investigations of potential virus introduction into Singapore are ongoing.

Pathogenicity of the newly emerged Lagovirus europaeus GI.2 strain in China in experimentally infected rabbits.

In April 2020, rabbit hemorrhagic virus type 2 (Lagovirus europaeus GI.2), which causes highly infectious fatal rabbit hemorrhagic disease, was emerged in China. The phylogenetic analyses of the complete genome sequence of GI.2 showed that it belonged to the non-recombinant GI.3/GI.2 genotype. However, the pathogenicity of this GI.2 strain differed from that of early typical GI.2 strains in Europe. To prevent the spread of the new strain in China, its pathogenicity urgently needs to be studied. Thus, viral shedding and distribution as well as clinical symptoms, histopathological changes, and serum cytokines were studied in experimentally GI.2/SC2020-infected rabbit adults and kits. The kit group showed a shorter survival time after the challenge than the adult group did. The mortality rate was higher in the kits (80 %) than in the adults (30 %). Viral RNA could be detected in both nasal and fecal swabs, and the main dissemination route appeared to be the fecal route. Viral RNA rapidly increased in the blood of the adults and kits at 6 h post-infection, indicating that blood viral load testing can be used for early diagnosis. The most affected organs were the liver and spleen, and the lesions were more severe in the kits than in the adults. The liver contained the highest viral RNA levels. Moreover, serum interleukin (IL)-6, IL-8, IL-10, and tumor necrosis factor-alpha levels were increased in the infected rabbits. In conclusion, our findings will help to understand the evolutionary trends and pathogenic characteristics of GI.2 strains in China.

Resolution of rabbit haemorrhagic disease virus 2 (RHDV2; Lagovirus europeus GI.2) outbreak in Singapore.

Rabbit haemorrhagic disease (RHD) is a highly contagious viral disease affecting lagomorphs. The first documented cases of RHD in Singapore occurred in adult pet European rabbits in September 2020. Singapore subsequently declared the outbreak resolved in December 2020. Epidemiological investigations ruled out introductions via importation of infected rabbits and contaminated feed. The source could not be definitively determined. However, the findings suggested that the incident involved both inter- and intra-household transmission and veterinary clinic-household transmission. This incident demonstrated the importance of sustained application of biosecurity measures, epidemiological investigations including active case finding, control measures such as expedient vaccine dissemination and risk communications. It showed that even without a wild lagomorph population, an urbanized city-state like Singapore could still encounter emerging diseases such as RHD. Given its social impact on rabbit owners, the National Parks Board, Singapore and private veterinarians worked together to communicate with rabbit owners in order to urge them to adopt biosecurity measures and to address their concerns.

Spillover events of rabbit haemorrhagic disease virus 2 (recombinant GI.4P-GI.2) from Lagomorpha to Eurasian badger.

Rabbit haemorrhagic disease (RHD) is a major threat to domestic and wild European rabbits. Presently, in Europe, the disease is caused mainly by Rabbit haemorrhagic disease virus 2 (RHDV2/b or Lagovirus europaeus GI.2), the origin of which is still unclear, as no RHDV2 reservoir hosts were identified. After the RHDV2 emergence in 2010, viral RNA was detected in a few rodent species. Furthermore, RHDV2 was found to cause disease in some hare species resembling the disease in rabbits, evidencing the ability of the virus to cross the species barrier. In this study, through molecular, histopathologic, antigenic and morphological evidences, we demonstrate the presence and replication of RHDV2 in Eurasian badgers (Meles meles) found dead in the district of Santarém, Portugal, between March 2017 and January 2020. In these animals, we further classify the RHDV2 as a Lagovirus europaeus recombinant GI.4P-GI.2. Our results indicate that Meles meles is susceptible to RHDV2, developing systemic infection, and excreting the virus in the faeces. Given the high viral loads seen in several organs and matrices, we believe that transmission to the wild rabbit is likely. Furthermore, transmission electron microscopy data show the presence of calicivirus compatible virions in the nucleus of hepatocytes, which constitutes a paradigm shift for caliciviruses' replication cycle.

Establishment of a duplex TaqMan RT-PCR for the differential detection of RHDV GI.1 and GI.2.

BACKGROUND: Rabbit haemorrhagic disease (RHD) is a highly contagious and acute fatal hepatitis of the European rabbit (Oryctolagus cuniculus), caused by a calicivirus (genus Lagovirus). Up to 2010, all RHD viruses (RHDV) isolated belonged to one genotype. In 2010, a new genotype of RHDV (RHDV2/b, currently designated GI.2 based on phylogenetic analysis) emerged in France. The aim of this study was to develop a rapid, simple, specific and sensitive TaqMan real-time PCR assay for the classic strain of RHDV and RHDV2 detection. Specific primers and probes were designed for the VP60 gene of RHDV and RHDV2 within the conserved region of viral genome. RESULTS: This study was demonstrated to be highly specific for RHDV and RHDV2, without cross-reactions with other non-targeted viruses. The detection limit of this work was 102 copies of RHDV and RHDV2, respectively. The coefficient of variation of the assay was less than 5% for both intra-assay and inter-assay. The reproducibility of method was assessed using plasmids and the coefficient of variation obtained was 0.2-3.70. Of 79 clinical samples, 68 were positive samples (86.08%), of which 60 were classic RHDV variants (75.9%), 4 were co-infected (5.06%) and 8 were RHDV2 (10.12%), those results are more sensitivity compare with conventional RT-PCR RT-PCR. CONCLUSIONS: In conclusion, this duplex TaqMan RT-qPCR based on VP60 gene of RHDV and RHDV2 could be a valuable tool in diagnose and molecular epidemiological study of the RHDV and RHDV2.

Monitoring of Rabbit Hemorrhagic Disease Virus in European Wild Rabbit (Oryctolagus cuniculus) Populations by PCR Analysis of Rabbit Fecal Pellets.

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.