A sensitive and specific blocking ELISA for the detection of rabbit calicivirus RCV-A1 antibodies

BACKGROUND: Antibodies to non-pathogenic rabbit caliciviruses (RCVs) cross-react in serological tests for rabbit hemorrhagic disease virus (RHDV) and vice versa, making epidemiological studies very difficult where both viruses occur. It is important to understand the distribution and interaction of the two viruses because the highly pathogenic RHDV has been used as a biocontrol agent for wild rabbits in Australia and New Zealand for the past 17 years. The presence of the benign RCV Australia 1 (RCV-A1) is considered a key factor for the failure of RHDV mediated rabbit control in cooler, wetter areas of Australia. RESULTS: A highly sensitive and specific blocking ELISA was developed for the detection of RCV-A1 antibodies. When sera from rabbits with a known infection history for either RCV-A1 or RHDV were tested, this assay showed 100% sensitivity and no cross-reactivity with RHDV sera (100% specificity). CONCLUSIONS: This new ELISA not only allows the detection of RCV-A1 at a population level, but also permits the serological status of individual rabbits to be determined more reliably than previously described methods. This robust and simple to perform assay is therefore the tool of choice for studying RCV-A1 epidemiology in Australian wild rabbit populations

Calicivirus Non-structural Proteins:Potential Functions in Replication and Host Cell Manipulation

The Caliciviridae are a family of viruses with a single-stranded, non-segmented RNA genome of positive polarity. The ongoing discovery of caliciviruses has increased the number of genera in this family to 11 (Norovirus, Nebovirus, Sapovirus, Lagovirus, Vesivirus, Nacovirus, Bavovirus, Recovirus, Salovirus, Minovirus, and Valovirus). Caliciviruses infect a wide range of hosts that include fishes, amphibians, reptiles, birds, and marine and land mammals. All caliciviruses have a genome that encodes a major and a minor capsid protein, a genome-linked viral protein, and several non-structural proteins. Of these non-structural proteins, only the helicase, protease, and RNA-dependent RNA polymerase share clear sequence and structural similarities with proteins from other virus families. In addition, all caliciviruses express two or three non-structural proteins for which functions have not been clearly defined. The sequence diversity of these non-structural proteins and a multitude of processing strategies suggest that at least some have evolved independently, possibly to counteract innate and adaptive immune responses in a host-specific manner. Studying these proteins is often difficult as many caliciviruses cannot be grown in cell culture. Nevertheless, the study of recombinant proteins has revealed many of their properties, such as intracellular localization, capacity to oligomerize, and ability to interact with viral and/or cellular proteins; the release of non-structural proteins from transfected cells has also been investigated. Here, we will summarize these findings and discuss recent in silico studies that identified previously overlooked putative functional domains and structural features, including transmembrane domains that suggest the presence of viroporins

The non-pathogenic Australian rabbit calicivirus RCV-A1 provides temporal and partial cross protection to lethal Rabbit Haemorrhagic Disease Virus infection which is not dependent on antibody titres

The endemic non-pathogenic Australian rabbit calicivirus RCV-A1 is known to provide some cross protection to lethal infection with the closely related Rabbit Haemorrhagic Disease Virus (RHDV). Despite its obvious negative impacts on viral biocontrol of introduced European rabbits in Australia, little is known about the extent and mechanisms of this cross protection. In this study 46 rabbits from a colony naturally infected with RCV-A1 were exposed to RHDV. Survival rates and survival times did not correlate with titres of serum antibodies specific to RCV-A1 or cross reacting to RHDV, but were instead influenced by the time between infection with the two viruses, demonstrating for the first time that the cross protection to lethal RHDV infection is transient. These findings are an important step towards a better understanding of the complex interactions of co-occurring pathogenic and non-pathogenic lagoviruses

Comparative quantitative monitoring of rabbit haemorrhagic disease viruses in rabbit kittens

BACKGROUND: Only one strain (the Czech CAPM-v351) of rabbit haemorrhagic disease virus (RHDV) has been released in Australia and New Zealand to control pest populations of the European rabbit O. cuniculus. Antigenic variants of RHDV known as RHDVa strains are reportedly replacing RHDV strains in other parts of the world, and Australia is currently investigating the usefulness of RHDVa to complement rabbit biocontrol efforts in Australia and New Zealand. RHDV efficiently kills adult rabbits but not rabbit kittens, which are more resistant to RHD the younger they are and which may carry the virus without signs of disease for prolonged periods. These different infection patterns in young rabbits may significantly influence RHDV epidemiology in the field and hence attempts to control rabbit numbers. METHODS: We quantified RHDV replication and shedding in 4–5 week old rabbits using quantitative real time PCR to assess their potential to shape RHDV epidemiology by shedding and transmitting virus. We further compared RHDV-v351 with an antigenic variant strain of RHDVa in kittens that is currently being considered as a potential RHDV strain for future release to improve rabbit biocontrol in Australia. RESULTS: Kittens were susceptible to infection with virus doses as low as 10 ID(50). Virus growth, shedding and transmission after RHDVa infection was found to be comparable or non-significantly lower compared to RHDV. Virus replication and shedding was observed in all kittens infected, but was low in comparison to adult rabbits. Both viruses were shed and transmitted to bystander rabbits. While blood titres indicated that 4–5 week old kittens mostly clear the infection even in the absence of maternal antibodies, virus titres in liver, spleen and mesenteric lymph node were still high on day 5 post infection. CONCLUSIONS: Rabbit kittens are susceptible to infection with very low doses of RHDV, and can transmit virus before they seroconvert. They may therefore play an important role in RHDV field epidemiology, in particular for virus transmission within social groups during virus outbreaks

Purification and biochemical characterisation of rabbit calicivirus RNA-dependent RNA polymerases and identification of non-nucleoside inhibitors

Rabbit haemorrhagic disease virus (RHDV) is a calicivirus that causes acute infections in both domestic and wild European rabbits (Oryctolagus cuniculus). The virus causes significant economic losses in rabbit farming and reduces wild rabbit populations. The recent emergence of RHDV variants capable of overcoming immunity to other strains emphasises the need to develop universally effective antivirals to enable quick responses during outbreaks until new vaccines become available. The RNA-dependent RNA polymerase (RdRp) is a primary target for the development of such antiviral drugs. In this study, we used cell-free in vitro assays to examine the biochemical characteristics of two rabbit calicivirus RdRps and the effects of several antivirals that were previously identified as human norovirus RdRp inhibitors. The non-nucleoside inhibitor NIC02 was identified as a potential scaffold for further drug development against rabbit caliciviruses. Our experiments revealed an unusually high temperature optimum (between 40 and 45 °C) for RdRps derived from both a pathogenic and a non-pathogenic rabbit calicivirus, possibly demonstrating an adaptation to a host with a physiological body temperature of more than 38 °C. Interestingly, the in vitro polymerase activity of the non-pathogenic calicivirus RdRp was at least two times higher than that of the RdRp of the highly virulent RHDV

Uncovering the microbiome of invasive sympatric European brown hares and European rabbits in Australia

Background European brown hares (Lepus europaeus) and European rabbits (Oryctolagus cuniculus) are invasive pest species in Australia, with rabbits having a substantially larger environmental impact than hares. As their spatial distribution in Australia partially overlaps, we conducted a comparative microbiome study to determine how the composition of gastrointestinal microbiota varies between these species, since this may indicate species differences in diet, physiology, and other internal and external factors. Methods We analysed the faecal microbiome of nine wild hares and twelve wild rabbits from a sympatric periurban reserve in Canberra, Australia, using a 16S rRNA amplicon-based sequencing approach. Additionally, we compared the concordance between results from Illumina and Nanopore sequencing platforms. Results We identified significantly more variation in faecal microbiome composition between individual rabbits compared to hares, despite both species occupying a similar habitat. The faecal microbiome in both species was dominated by the phyla Firmicutes and Bacteroidetes, typical of many vertebrates. Many phyla, including Actinobacteria, Proteobacteria and Patescibacteria, were shared between rabbits and hares. In contrast, bacteria from phylum Verrucomicrobia were present only in rabbits, while phyla Lentisphaerae and Synergistetes were represented only in hares. We did not identify phylum Spirochaetes in Australian hares; this phylum was previously shown to be present at high relative abundance in European hare faecal samples. These differences in the composition of faecal microbiota may be indicative of less discriminate foraging behaviour in rabbits, which in turn may enable them to adapt quicker to new environments, and may reflect the severe environmental impacts that this species has in Australia.Funding was provided by a Centre for Biodiversity Analysis ‘Ignition’ Grant, a collaborative initiative of the Australian National University, CSIRO and the University of Canberra