Potent protease inhibitors of deadly lagoviruses: rabbit hemorrhagic disease virus and European brown hare syndrome virus

Rabbit hemorrhagic disease (RHD) and European brown hare syndrome (EBHS) are highly contagious diseases caused by lagoviruses in the Caliciviridae family and mainly affect rabbits and hares, respectively. These infectious diseases are associated with high mortality and a serious threat to domesticated (farmed and pet) and wild rabbits and hares, including endangered species such as Riparian brush rabbits. In the US, only isolated cases of RHD had been reported until Spring 2020. However, RHD caused by RHD type 2 virus (RHDV2) was unexpectedly reported in April 2020 in New Mexico and has subsequently spread to several US states infecting wild rabbits and hares. Since it is almost impossible to control and eradicate the virus from wild animals, it is highly likely RHD will become endemic in the US. Vaccines are available for RHD, however, there is no specific treatment for these deadly diseases. RHDV and EBHSV encode a 3C-like protease (3CLpro), which is essential for virus replication and a promising target for antiviral drug development. We have previously generated focused small molecule libraries of 3CLpro inhibitors and demonstrated the in vitro potency and in vivo efficacy of some protease inhibitors against viruses that encode 3CLpro including caliciviruses and coronaviruses. Here we established the enzyme assay and cell-based assays for these uncultivable viruses to determine the in vitro activity of 3CLpro inhibitors, including GC376, a protease inhibitor being developed for feline infectious peritonitis, and identified potent inhibitors of RHDV1 and 2 and EBHSV. In addition, structure-activity relationship study and homology modelling of the 3CLpros and inhibitors revealed that lagoviruses share similar structural requirements for 3CLpro inhibition with other caliciviruses.

Preliminary Outbreak Investigation and Spatial Epidemiology of Rabbit Hemorrhagic Disease Outbreak in Nigeria

The outbreak of highly contagious transboundary rabbit hemorrhagic disease (RHD) in Nigeria has a severe socio-economic impact on the rabbit industry. We present the outbreak investigation and spatial epidemiology of the first confirmed RHD outbreak in Nigeria from a field survey of 28 stochastic outbreaks in Kwara State, north-central Nigeria. A total of 1,639 rabbits died from 2,053 susceptible rabbits. The serotype “RHDV-2” was detected in tissue samples from some of the outbreaks. The case fatality rate of the RHDV-2 outbreak was 79.8%. The source of the outbreak is still unknown. Most (71.4%) of the farmers had introduced new rabbits into their farms 1–2 weeks before the outbreak. Most of the farmers practiced biosecurity measures such as farm fencing (83.1%) and routine disinfection of the farm materials (53.6%). However, only 17.8% of the farmers enforced movement restrictions into their farms. Some of the farmers (42.8%) had restocked their farms after being affected by the RHD outbreak and 75% of all those farmers that have restocked had used the RHD vaccine. There was no statistically significant association between adherence to biosecurity measures and the RHD outbreak in affected farms (p = 0.408). However, the introduction of new rabbits into rabbit farms significantly pre-disposed farms to the RHD outbreak (p < 0.001). There is a need for active surveillance of RHD across the country to ensure efficient and effective tracking, monitoring, and control of the disease. Equally, understanding the genetic diversity of the Lagoviruses in Nigeria that cause RHD to aid vaccine development is of utmost importance to prevent future RHD outbreaks.

Preventieve diergeneeskunde bij het konijn

In dit artikel wordt een overzicht gegeven van belangrijke maatregelen die kunnen worden genomen in de preventie van ziekten bij konijnen. Op kop staat de jaarlijkse vaccinatie tegen myxomatose en “rabbit hemorrhagic disease” (RHD), een handeling die elke dierenarts zou moeten aanraden aan konijneneigenaars. Daarnaast is het verstrekken van correcte voeding ter preventie van onder andere tand- en darmproblemen welbekend. Verder worden een aantal huisvestingstips gegeven die vaak voorkomende gezondheidsproblemen bij konijnen kunnen helpen voorkomen. Obesitas, pododermatitis en urinewegproblemen zijn er enkele van. Minder aangeraden door dierenartsen, doch zeker niet onbelangrijk, is de preventie van uteriene tumoren bij voedsters door middel van electieve chirurgische wegname van ovaria en uterus, bij voorkeur vóór de leeftijd van twee jaar. Een gedegen kennis van de konijnendiergeneeskunde bij de dierenarts en een goede communicatie met konijneneigenaars over hoe ziekte bij hun dieren te voorkomen, kunnen bijdragen tot een lang en gezond leven van gezelschapskonijnen.

Detection of rabbit hemorrhagic disease virus 2 in formalin-fixed, paraffin-embedded tissues via in situ hybridization

Formalin-fixed, paraffin-embedded tissues from European rabbits ( Oryctolagus cuniculus) that succumbed to rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) during the 2019 outbreak in Washington, USA, were utilized for in situ hybridization via RNAscope (ACDBio). This detection method was both sensitive and specific, with no staining in tissues from RHDV- ( Lagovirus GI.1) and RHDV2-negative rabbits, and only slight background staining of RHDV-positive rabbits; RHDV2-positive tissues had bright-red cytoplasmic staining. Although much of the viral mRNA detection was consistent with previously described antigen detection via immunohistochemistry of the liver, lungs, and spleen, there was also significant glomerular staining in the kidneys, and endothelial staining within blood vessels of almost all organs. We validated the RNAscope technique for detection of RHDV2 mRNA in formalin-fixed, paraffin-embedded tissues, with increased sensitivity from previous techniques, and identified additional affected cell types that may contribute to the understanding of pathogenesis.

Multiple Introductions of Rabbit Hemorrhagic Disease Virus Lagovirus europaeus/GI.2 in Africa

Rabbit hemorrhagic disease (RHD) causes high mortality and morbidity in European rabbits (Oryctolagus cuniculus). In Africa, the presence of the causative agent, the rabbit hemorrhagic disease virus (RHDV), was first confirmed in 1992 (genotype Lagovirus europaeus/GI.1). In 2015, the new genotype Lagovirus europaeus/GI.2 (RHDV2/b) was detected in Tunisia. Currently, GI.2 strains are present in several North and Sub-Saharan African countries. Considerable economic losses have been observed in industrial and traditional African rabbitries due to RHDV. Like other RNA viruses, this virus presents high recombination rates, with the emergence of GI.2 being associated with a recombinant strain. Recombination events have been detected with both pathogenic (GI.1b and GII.1) and benign (GI.3 and GI.4) strains. We obtained complete genome sequences of Tunisian GI.2 strains collected between 2018 and 2020 and carried out phylogenetic analyses. The results revealed that Tunisian strains are GI.3P-GI.2 strains that were most likely introduced from Europe. In addition, the results support the occurrence of multiple introductions of GI.2 into Africa, stressing the need for characterizing complete genome sequences of the circulating lagoviruses to uncover their origin. Continued monitoring and control of rabbit trade will grant a better containment of the disease and reduce the disease-associated economic losses.

Experimental Study of the Mechanical Transmission of Rabbit Hemorrhagic Disease Virus (RHDV2/b) by Aedes albopictus (Diptera: Culicidae) and Phlebotomus papatasi (Diptera: Psychodidae)

Rabbit hemorrhagic disease (RHD) is caused by a lagovirus mainly affecting European rabbits (Oryctolagus cuniculus), although other European and North American lagomorph species are also susceptible to fatal infection by the new viral variant RHDV2/b. In the present work, direct mechanical transmission of the rabbit hemorrhagic disease virus (RHDV2/b variant) by the hematophagous Diptera Aedes albopictus (Skuse) (Diptera: Culicidae) and the sand fly Phlebotomus papatasi (Scopoli) (Diptera: Psychodidae) was tested. For each species, six and three laboratory rabbits were exposed to bites of dipterous females partially fed on RHDV2/b viral suspension 2 h and 24 h prior to exposure, respectively. The rabbits were then monitored for clinical changes and mortality for 35 d, and seroconversion was assessed by indirect ELISA. No rabbit died or showed clinical signs of disease, and seroconversion was recorded in two rabbits challenged with P. papatasi females fed the viral suspension 2 h prior to exposure. The number of RHDV2/b RNA copies/female was higher in Ae. albopictus than in P. papatasi but the decrease over time of RNA load in Ae. albopictus was greater than that in P. papatasi. The results of this study suggest the inability of Ae. albopictus to serve as a direct mechanical vector of RHDV2/b, but sand flies could play a role in the local transmission of RHD.