Rabbit Hemorrhagic Disease Virus Infection Research Articles

RHDV 3C protein antagonizes type I interferon signaling by cleaving interferon promoter stimulated 1 protein

The host innate immune response to viral infection often involves the activation of type I interferons. Not surprisingly, many viruses have evolved various mechanisms to disable the interferon pathway and evade the antiviral response involving innate immunity. Rabbit hemorrhagic disease (RHD) is caused by RHD virus (RHDV), but whether it can antagonize the production of host interferon to establish infection has not been investigated. In this study, we found that during RHDV infection, the expressions of interferon and the interferon-stimulated gene were not activated. We constructed eukaryotic expression plasmids of all RHDV proteins, and found that RHDV 3C protein inhibited poly(I:C)-induced interferon expressions. Using siRNA to interfere with the expressions of TLR3 and MDA5, we found that the MDA5 signal pathway was used by the 3C protein to inhibit poly(I:C)-induced interferon expression. This effect was mediated by cleaving the interferon promoter stimulated 1 (IPS-1) protein. Finally, our study showed that interferon was effective against RHDV infection. In summary, our findings showed that the RHDV 3C protein was a new interferon antagonist. These results increase our understanding of the escape mechanism from innate immunity mediated by the RHDV 3C protein.

Rabbit hemorrhagic disease virus VP60 protein expressed in recombinant swinepox virus self-assembles into virus-like particles with strong immunogenicity in rabbits.

Rabbit Hemorrhagic Disease (RHD) is an economically significant infectious disease of rabbits, and its infection causes severe losses in the meat and fur industry. RHD Virus (RHDV) is difficult to proliferate in cell lines in vitro, which has greatly impeded the progress of investigating its replication mechanism and production of inactivated virus vaccines. RHDV VP60 protein is a major antigen for developing RHD subunit vaccines. Herein, we constructed a TK-deactivated recombinant Swinepox virus (rSWPV) expressing VP60 protein and VP60 protein coupled with His-tag respectively, and the expression of foreign proteins was confirmed using immunofluorescence assay and western blotting. Transmission electron microscopy showed that the recombinant VP60, with or without His-tag, self-assembled into virus-like particles (VLPs). Its efficacy was evaluated by comparison with available commercial vaccines in rabbits. ELISA and HI titer assays showed that high levels of neutralizing antibodies were induced at the first week after immunization with the recombinant strain and were maintained during the ongoing monitoring for the following 13 weeks. Challenge experiments showed that a single immunization with 106 PFU of the recombinant strain protected rabbits from lethal RHDV infection, and no histopathological changes or antigenic staining was found in the vaccine and rSWPV groups. These results suggest that rSWPV expressing RHDV VP60 could be an efficient candidate vaccine against RHDV in rabbits.

Inconspicuous post-mortem findings in rabbits from Switzerland naturally -infected with Rabbit Haemorrhagic -Disease Virus 2.

Rabbit Haemorrhagic Disease Virus 2 (RHDV-2) emerged in France in 2010. In Switzerland, RHDV-2 was first identified in 2015 and apparently has almost replaced the classical Rabbit Haemorrhagic Disease Virus (RHDV) by now. Like RHDV, RHDV-2 causes a viral hepatitis with a peracute course and an increased mortality rate within the rabbitry. RHDV infection causes consistent gross pathological findings, especially in the liver and respiratory tract. Reports about gross pathology for animals naturally infected with RHDV-2 is scarce. The present study analysed the anamnesis and necropsy reports of 35 rabbits examined during routine diagnostics between March 2015 and May 2017. A reverse transcriptase real-time polymerase chain reaction (RT-qPCR) specific for RHDV-2 and RHDV proved a total of 25 animals to be positive for RHDV-2, while none was positive for RHDV. Additionally, histological examinations were performed on liver, lung, and kidney of 18 rabbits that had tested positive by RHDV-2 RT-qPCR. The anamnestic report more often stated an increased mortality rate in RHDV-2 positive (16/18, 89 %) compared to RHDV-2 negative rabbits (3/9, 33 %). Gross pathology did not reveal any pathognomonic changes in RHDV-2 positive animals. Histologically, the liver showed the most severe lesions followed by lung and kidney. Animals positive for RHDV-2 frequently showed signs of gastro-intestinal disease (n = 5) and/or septicaemia (n = 6) masking possible indicators of an RHDV-2 infection, such as the rather unspecific findings of an enlarged spleen or an enlarged, friable, tan-coloured liver. The authors want to raise awareness among clinicians and pathologists that in case of sudden death in commercial or pet rabbits, RHDV-2 needs to be considered as differential diagnosis and should be confirmed by laboratory diagnosis.

Vimentin as a Cap of Invisibility: Proposed Role of Vimentin in Rabbit Hemorrhagic Disease Virus (RHDV) Infection.

Vimentin is an intermediate filament, a cytoskeleton protein expressed mainly in cells of mesenchymal origin. Increasing evidence indicates that vimentin could play a key role in viral infections. Therefore, changes in tissue and extracellular vimentin expression and associated signal trails may determine/protect the fate of cells and the progression of disease caused by viral infection. Rabbit hemorrhagic disease virus (RHDV), genotype GI.1, is an etiological agent that causes a severe and highly lethal disease—RHD (rabbit hemorrhagic disease). This article evaluates the gene and protein expression of vimentin in the tissues (liver, lungs, spleen, and kidneys) and serum of rabbits experimentally infected with two RHDV variants (GI.1a). The VIM mRNA expression levels in the tissues were determined using reverse transcription quantitative real-time PCR (RT-qPCR). In addition, the amount of vimentin protein in the serum was analyzed by an ELISA test. We observed significantly elevated expression levels of VIM mRNA and protein in the liver and kidney tissues of infected rather than healthy rabbits. In addition, VIM mRNA expression was increased in the lung tissues; meanwhile, we observed only protein-enhanced vimentin in the spleen. The obtained results are significant and promising, as they indicate the role of vimentin in RHDV infection and the course of RHD. The role of vimentin in RHDV infection could potentially rely on the one hand, on creating a cap of invisibility against the intracellular viral spread, or, on the other hand, after the damage of cells, vimentin could act as a signal of tissue damage.

Hemoglobin subunit beta interacts with the capsid, RdRp and VPg proteins, and antagonizes the replication of rabbit hemorrhagic disease virus

Rabbit hemorrhagic disease virus (RHDV) causes a highly contagious disease in rabbits that is associated with high mortality. Because of the lack of a suitable cell culture system for RHDV, its pathogenic mechanism and replication remain unclear. This study found that the expression level of host protein rabbit hemoglobin subunit beta (HBB) was significantly downregulated in RHDV-infected cells. To investigate the role of HBB in RHDV replication, small interfering RNAs for HBB and HBB eukaryotic expression plasmids were used to change the expression level of HBB in RK-13 cells and the results showed that the RHDV replication level was negatively correlated with the expression level of HBB. It was also verified that HBB inhibited RHDV replication using constructed HBB stable overexpression cell lines and HBB knockout cell lines. The interaction of HBB with viral capsid protein VP60, replicase RdRp, and VPg protein was confirmed, as was the activation of the expression of interferon γ by HBB. The results of this study indicated that HBB may be an important host protein in host resistance to RHDV infection.

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 48hr 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.

Genomic insights into a population of introduced European rabbits Oryctolagus cuniculus in Australia and the development of genetic resistance to rabbit hemorrhagic disease virus.

The European rabbit (Oryctolagus cuniculus) is one of the most devastating invasive species in Australia. Since the 1950s, myxoma virus (MYXV) and rabbit haemorrhagic disease virus (RHDV) have been used to manage overabundant rabbit populations. Resistance to MYXV was observed within a few years of the release. More recently, resistance to lethal RHDV infection has also been reported, undermining the efficiency of landscape-scale rabbit control. Previous studies suggest that genetic resistance to lethal RHDV infection may differ locally between populations, yet the mechanisms of genetic resistance remain poorly understood. Here, we used genotyping by sequencing (GBS) data representing a reduced representation of the genome, to investigate Australian rabbit populations. Our aims were to understand the relationship between populations and identify possible genomic signatures of selection for RHDV resistance. One population we investigated had previously been reported to show levels of resistance to lethal RHDV infection. This population was compared to three other populations with lower or no previously reported RHDV resistance. We identified a set of novel candidate genes that could be involved in host-pathogen interactions such as virus binding and infection processes. These genes did not overlap with previous studies on RHDV resistance carried out in different rabbit populations, suggesting that multiple mechanisms are feasible. These findings provide useful insights into the different potential mechanisms of genetic resistance to RHDV virus which will inform future functional studies in this area.

A Review on Rabbit Hemorrhagic Disease with a Special Reference to Egyptian Situation

Rabbit hemorrhagic disease (RHD) is considered as one of the most important viral diseases that affects and threatens rabbit’s flocks. This disease has affected rabbits since mid-1980. Two epidemics of RHD had been discovered; the first was in mid-1980 and known as classical RHD virus (RHDV), while the second was in 2011 and described as variant virus (RHDVb/RHDV2). Domestic and wild rabbits are susceptible to RHD. All ages can be affected, but adults are more susceptible to young kitten. RHD is presented in three forms; per-acute, acute and subacute or chronic form. Mortality rate is usually high especially in per-acute and acute stages and it is associated with disseminated intravascular coagulopathy and necrotic hepatitis. The main lesions have been observed in the liver, lungs and spleen. Diagnosis of RHD is based on the clinical picture and detection of RHDV or specific antibodies. The prevention and control strategies depend mainly on using of preventive inactivated vaccine together with adoptionof hygienic measures. However, there is no specific treatment of RHDV infection. So, this review article puts a spot light on RHD regarding the epidemiology, the clinical and laboratory diagnosis as well as the prevention and control strategies with a special reference to Egyptian situation.

Changes in MicroRNA Expression during Rabbit Hemorrhagic Disease Virus (RHDV) Infection.

Current knowledge on the role of microRNAs (miRNAs) in rabbit hemorrhagic disease virus (RHDV) infection and the pathogenesis of rabbit hemorrhagic disease (RHD) is still limited. RHDV replicates in the liver, causing hepatic necrosis and liver failure. MiRNAs are a class of short RNA molecules, and their expression profiles vary over the course of diseases, both in the tissue environment and in the bloodstream. This paper evaluates the expression of miRNAs in the liver tissue (ocu-miR-122-5p, ocu-miR-155-5p, and ocu-miR-16b-5p) and serum (ocu-miR-122-5p) of rabbits experimentally infected with RHDV. The expression levels of ocu-miR-122-5p, ocu-miR-155-5p, and ocu-miR-16b-5p in liver tissue were determined using reverse transcription quantitative real-time PCR (RT-qPCR), and the expression level of circulating ocu-miR-122-5p was established using droplet digital PCR (ddPCR). The expression levels of ocu-miR-155-5p and ocu-miR-16b-5p were significantly higher in the infected rabbits compared to the healthy rabbits (a fold-change of 5.8 and 2.5, respectively). The expression of ocu-miR-122-5p was not significantly different in the liver tissue from the infected rabbits compared to the healthy rabbits (p = 0.990), while the absolute expression level of the circulating ocu-miR-122-5p was significantly higher in the infected rabbits than in the healthy rabbits (p < 0.0001). Furthermore, a functional analysis showed that ocu-miR-155-5p, ocu-miR-16b-5p, and ocu-miR-122-5p can regulate the expression of genes involved in processes correlated with acute liver failure (ALF) in rabbits. Search tool for the retrieval of interacting genes/proteins (STRING) analysis showed that the potential target genes of the three selected miRNAs may interact with each other in different pathways. The results indicate the roles of these miRNAs in RHDV infection and over the course of RHD and may reflect hepatic inflammation and impairment/dysfunction in RHD.

NF-κB and Keap1 Interaction Represses Nrf2-Mediated Antioxidant Response in Rabbit Hemorrhagic Disease Virus Infection.

The rabbit hemorrhagic disease virus (RHDV), which belongs to the family Caliciviridae and the genus Lagovirus, causes lethal fulminant hepatitis in rabbits. RHDV decreases the activity of antioxidant enzymes regulated by Nrf2 in the liver. Antioxidants are important for the maintenance of cellular integrity and cytoprotection. However, the mechanism underlying the regulation of the Nrf2-antioxidant response element (ARE) signaling pathway by RHDV remains unclear. Using isobaric tags for relative and absolute quantification (iTRAQ) technology, the current study demonstrated that RHDV inhibits the induction of ARE-regulated genes and increases the expression of the p50 subunit of the NF-κB transcription factor. We showed that RHDV replication causes a remarkable increase in reactive oxygen species (ROS), which is simultaneously accompanied by a significant decrease in Nrf2. It was found that nuclear translocation of Keap1 plays a key role in the nuclear export of Nrf2, leading to the inhibition of Nrf2 transcriptional activity. The p50 protein partners with Keap1 to form the Keap1-p50/p65 complex, which is involved in the nuclear translocation of Keap1. Moreover, upregulation of Nrf2 protein levels in liver cell nuclei by tert-butylhydroquinone (tBHQ) delayed rabbit deaths due to RHDV infection. Considered together, our findings suggest that RHDV inhibits the Nrf2-dependent antioxidant response via nuclear translocation of Keap1-NF-κB complex and nuclear export of Nrf2 and provide new insight into the importance of oxidative stress during RHDV infection.IMPORTANCE Recent studies have reported that rabbit hemorrhagic disease virus (RHDV) infection reduced Nrf2-related antioxidant function. However, the regulatory mechanisms underlying this process remain unclear. The current study showed that the NF-κB p50 subunit partners with Keap1 to form the Keap1-NF-κB complex, which plays a key role in the inhibition of Nrf2 transcriptional activity. More importantly, upregulated Nrf2 activity delayed the death of RHDV-infected rabbits, strongly indicating the importance of oxidative damage during RHDV infection. These findings may provide novel insights into the pathogenesis of RHDV.

Construction and immunogenicity of novel bivalent virus-like particles bearing VP60 genes of classic RHDV(GI.1) and RHDV2(GI.2)

Rabbit hemorrhagic disease (RHD) is an acute, inflammatory, septic, and devastating infectious disease caused by Rabbit hemorrhagic disease virus (RHDV), which poses a serious threat to the rabbit industry. RHDV2 (GI.2/RHDVb), a recently reported new variant could cause RHD in wild populations, but also RHDV-vaccinated rabbits. For now, both RHDV and RHDV2 are the main causes of RHD. To develop a new subunit vaccine that could protect rabbits against both classic RHDV and RHDV2 infections, we constructed a recombinant baculovirus (Bac-classic RHDV VP60-RHDV2 VP60) containing the VP60 genes of classic RHDV and RHDV2. Both VP60 genes were well expressed simultaneously in Spodoptera frugiperda cells (Sf9) after infection with the recombinant baculovirus. Transmission electron microscopy showed that the recombinant VP60 self-assembled into virus-like particles (VLPs). The antigenicity and immunogenicity of the bivalent VLPs vaccine were examined with animal experiments. Our results demonstrated that both the humoral and cellular immune responses were efficiently induced in rabbits by a subunit vaccine based on the recombinant baculovirus. In addition, all rabbits immunized with the bivalent VLPs vaccine survived after challenged with classic RHDV, and showed no clinical signs of RHD, whereas all the rabbits in the negative control group died from classic RHDV infection and showed typical clinical signs of RHD. In summary, our results indicated that the recombinant baculovirus carrying two VP60 genes is a candidate construct from which to develop a bivalent VLPs vaccine against both classic RHDV and RHDV2 infections.

Nucleolin mediates the internalization of rabbit hemorrhagic disease virus through clathrin-dependent endocytosis.

Rabbit hemorrhagic disease virus (RHDV) is an important member of the Caliciviridae family and a highly lethal pathogen in rabbits. Although the cell receptor of RHDV has been identified, the mechanism underlying RHDV internalization remains unknown. In this study, the entry and post-internalization of RHDV into host cells were investigated using several biochemical inhibitors and RNA interference. Our data demonstrate that rabbit nucleolin (NCL) plays a key role in RHDV internalization. Further study revealed that NCL specifically interacts with the RHDV capsid protein (VP60) through its N-terminal residues (aa 285–318), and the exact position of the VP60 protein for the interaction with NCL is located in a highly conserved region (472Asp-Val-Asn474; DVN motif). Following competitive blocking of the interaction between NCL and VP60 with an artificial DVN peptide (RRTGDVNAAAGSTNGTQ), the internalization efficiency of the virus was markedly reduced. Moreover, NCL also interacts with the C-terminal residues of clathrin light chain A, which is an important component in clathrin-dependent endocytosis. In addition, the results of animal experiments also demonstrated that artificial DVN peptides protected most rabbits from RHDV infection. These findings demonstrate that NCL is involved in RHDV internalization through clathrin-dependent endocytosis.

Disentangling synergistic disease dynamics: Implications for the viral biocontrol of rabbits.

European rabbits (Oryctolagus cuniculus) have been exposed to rabbit haemorrhagic disease virus (RHDV) and myxoma virus (MYXV) in their native and invasive ranges for decades. Yet, the long-term effects of these viruses on rabbit population dynamics remain poorly understood. In this context, we analysed 17years of detailed capture-mark-recapture data (2000-2016) from Turretfield, South Australia, using a probabilistic state-space hierarchical modelling framework to estimate rabbit survival and epidemiological dynamics. While RHDV infection and disease-induced death were most prominent during annual epidemics in winter and spring, we found evidence for continuous infection of susceptible individuals with RHDV throughout the year. RHDV-susceptible rabbits had, on average, 25% lower monthly survival rates compared to immune individuals, while the average monthly force of infection in winter and spring was ~38%. These combined to result in an average infection-induced mortality rate of 69% in winter and spring. Individuals susceptible to MYXV and immune to RHDV had similar survival probabilities to those having survived infections from both viruses, whereas individuals susceptible to both RHDV and MYXV had higher survival probabilities than those susceptible to RHDV and immune to MYXV. This suggests that MYXV may reduce the future survival rates of individuals that endure initial MYXV infection. There was no evidence for long-term changes in disease-induced mortality and infection rates for either RHDV or MYXV. We conclude that continuous, year-round virus perpetuation (and perhaps heterogeneity in modes of transmission and infectious doses during and after epidemics) acts to reduce the efficiency of RHDV and MYXV as biocontrol agents of rabbits in their invasive range. However, if virulence can be maintained as relatively constant through time, RHDV and MYXV will likely continue realizing strong benefits as biocontrol agents.

Prior exposure to non-pathogenic calicivirus RCV-A1 reduces both infection rate and mortality from rabbit haemorrhagic disease in a population of wild rabbits in Australia.

Mortality caused by rabbit haemorrhagic disease virus (RHDV) in wild rabbits is reduced in parts of Australia where the related, non-pathogenic calicivirus RCV-A1 is endemic. Laboratory experiments previously showed that prior infection with RCV-A1 enabled rabbits to better withstand subsequent infection with highly virulent RHDV, and this was assumed to explain higher survival. Here, we analyse serological data from the field suggesting that reduced mortality rates among wild rabbits may also result from rabbits previously infected with RCV-A1 having a reduced likelihood of RHDV infection. We discuss the possible mechanisms underlying this finding and its implications. The methods we describe for analysing field data gave far greater insights into epidemiological processes and virus interactions than gained from reporting basic seroprevalence rates alone.

The Taishan Robinia pseudoacacia polysaccharides enhance immune effects of rabbit haemorrhagic disease virus inactivated vaccines

Robinia pseudoacacia flower, a common component in traditional Chinese medicine, has long been well-known for its high pharmaceutical value. This study aimed to assess the immunopotentiating effects of Taishan Robinia Pseudoacacia polysaccharides (TRPPS) in rabbits inoculated with a rabbit haemorrhagic disease virus (RHDV) inactivated vaccine. The rabbits were administered with the RHDV vaccine in conjunction with varying concentrations of TRPPS, and their blood samples were collected at different time points to analyze the ratio and number of blood lymphocytes. In addition, sera were prepared and analyzed to determine the overall antibody titer and the level of IL-2, a cytokine commonly used as an indicator of immune activity. The various TRPPS-supplemented vaccines were shown to be more effective in enhancing the immune functions of the inoculated rabbits compared to their polysaccharide-free counterpart, with 200 mg/mL of TRPPS exhibiting the most pronounced benefits that were comparable to those of propolis. In addition, the TRPPS-supplemented RHDV inactivated vaccines could significantly improve the survival rates of the immunized rabbits against RHDV infection. Our studies offered convincing experimental evidence for the development of TRPPS as a new type of plant-derived immunopotentiator.

Expression of IL-1Ra, IL-6, IL-8, IL-18, TNF-α and IFN-γ genes in peripheral blood leukocytes of rabbits infected with RHDV (Rabbit Haemorrhagic Disease Virus)

Rabbit haemorrhagic disease virus (RHDV) induces a highly contagious and extremely lethal disease that fulfils many requirements of an animal model of fulminant hepatic failure (FHF); however, the pathogenesis of RHD has still not been fully elucidated. Cytokines play an important role in regulation of the immune response and pathogenesis of many diseases, including those caused by viral infections. Furthermore, recent studies indicate a role of the immune response, especially peripheral blood leukocytes (PBL), in the pathogenesis of RHD. Thus, in the present study we investigated the expression of IL-1Ra, IL-6, IL-8, IL-18, TNF-α and IFN-γ genes in PBL of RHDV-infected rabbits. We also compared the expression of genes encoding these cytokines in rabbits with different course of RHDV infection (in animals that died 36 h post infection or survived even over 60 h after infection). The study revealed increased expression of genes encoding pro-inflammatory cytokines: IL-6, IL-8, TNF-α, IFN-γ in PBL of RHDV-infected rabbits. Moreover, the level of cytokine gene expression depended on the course of RHD. Hence, the results obtained indicate the potential role of these cytokines in RHDV infection and their influence on the survival time of infected rabbits.

Inclusion of an Arg-Gly-Asp receptor-recognition motif into the capsid protein of rabbit hemorrhagic disease virus enables culture of the virus in vitro

The fact that rabbit hemorrhagic disease virus (RHDV), an important member of the Caliciviridae family, cannot be propagated in vitro has greatly impeded the progress of investigations into the mechanisms of pathogenesis, translation, and replication of this and related viruses. In this study, we have successfully bypassed this obstacle by constructing a mutant RHDV (mRHDV) by using a reverse genetics technique. By changing two amino acids (S305R,N307D), we produced a specific receptor-recognition motif (Arg-Gly-Asp; called RGD) on the surface of the RHDV capsid protein. mRHDV was recognized by the intrinsic membrane receptor (integrin) of the RK-13 cells, which then gained entry and proliferated as well as imparted apparent cytopathic effects. After 20 passages, the titers of RHDV reached 1 × 104.3 50% tissue culture infectious dose (TCID50)/ml at 72 h. Furthermore, mRHDV-infected rabbits showed typical rabbit plague symptoms and died within 48-72 h. After immunization with inactivated mRHDV, the rabbits survived wild-type RHDV infection, indicating that mRHDV could be a candidate virus strain for producing a vaccine against RHDV infection. In summary, this study offers a novel strategy for overcoming the challenges of proliferating RHDV in vitro Because virus uptake via specific membrane receptors, several of which specifically bind to the RGD peptide motif, is a common feature of host cells, we believe that this the strategy could also be applied to other RNA viruses that currently lack suitable cell lines for propagation such as hepatitis E virus and norovirus.

DIFFERENT SEROLOGICAL PROFILES TO CO-OCCURRING PATHOGENIC AND NONPATHOGENIC CALICIVIRUSES IN WILD EUROPEAN RABBITS (ORYCTOLAGUS CUNICULUS) ACROSS AUSTRALIA.

Rabbit hemorrhagic disease virus (RHDV) was released in Australia as a biocontrol agent for wild European rabbits ( Oryctolagus cuniculus ) in 1995-96; however, its effects were variable across Australia with the greatest population reductions seen in lower annual rainfall areas (<400 mm). There is speculation that the reduced effectiveness observed at higher annual rainfall sites is at least partially due to the presence of a nonpathogenic calicivirus (RCV-A1). The RCV-A1 is related to RHDV and confers partial and transient protection against lethal RHDV infection in laboratory tests. What is not well understood is where, how, and to what degree RCV-A1 impedes the effect of RHDV-mediated rabbit control under field conditions. We investigated seven wild rabbit populations across six states and territories representing different seasonal rainfall zones across Australia, four times during 2011-12, to investigate if the presence and prevalence of RCV-A1 coincided with a change in RHDV immunity status within these populations. Besides serology, tissue samples from both trapped and shot rabbits were collected for virus detection by reverse transcription PCR. Overall, 52% (n=258) of the total samples (n=496) tested positive for RHDV antibodies and 42% (n=208) positive for RCV-A1 antibodies; 30% (n=150) of the sera contained antibodies to both viruses. The proportion of rabbits with RHDV antibodies increased significantly at sites where RCV-A1 antibodies were present (χ21, α=0.1, P<0.001). Evidence that preinfection of RCV-A1 may lead to a higher proportion of sampled rabbits with antibodies to both viruses was found at only one site.

Molecular cloning of the rabbit interleukin 6 promoter: Functional characterization of rabbit hemorrhagic disease virus response elements in RK-13 cells

Infection with rabbit hemorrhagic disease virus (RHDV) can cause acute liver failure (ALF), leading to severe mortality in rabbits. Inflammatory response, especially the expression of inflammatory cytokines such as interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-6, may play major roles in mediating and amplifying the ALF. Among these cytokines, IL-6 is a multifunctional cytokine with a central role in various physiological inflammatory and immunological processes. In this study, we found that RHDV infection significantly upregulated IL-6 gene expression in vivo. Next, the rabbit IL-6 promoter was cloned and analyzed. Transfection of full-length RHDV cDNA in RK-13 cells upregulated the activity of the IL-6 promoter. A series of 5′ deletion constructs demonstrated that AP-1 (activator protein 1), NF-IL6 (nuclear factor interleukin-6), and NF-κB (nuclear factor kappa B) elements were critical for RHDV-induced IL-6 transcription. Besides, the CREB (cAMP-response element binding protein) element may also play an accessory effect on RHDV-induced IL-6 transcription. Collectively, the results elucidate the mechanism of IL-6 induction, and enrich the RHDV pathogenesis in rabbit.

Melatonin inhibits the sphingosine kinase 1/sphingosine-1-phosphate signaling pathway in rabbits with fulminant hepatitis of viral origin.

The sphingosine kinase (SphK)1/sphingosine-1-phosphate (S1P) pathway is involved in multiple biological processes, including liver diseases. This study investigate whether modulation of the SphK1/S1P system associates to the beneficial effects of melatonin in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2×10(4) hemagglutination units of a RHDV isolate and received 20mg/kg of melatonin at 0, 12, and 24hr postinfection. Liver mRNA levels, protein concentration, and immunohistochemical labeling for SphK1 increased in RHDV-infected rabbits. S1P production and protein expression of the S1PR1 receptor were significantly elevated following RHDV infection. These effects were significantly reduced by melatonin. Rabbits also exhibited increased expression of toll-like receptor (TLR)4, tumor necrosis factor alpha (TNF-α), interleukin (IL)-6, nuclear factor-kappa B (NF-κB) p50 and p65 subunits, and phosphorylated inhibitor of kappa B (IκB)α. Melatonin administration significantly inhibited those changes and induced a decreased immunoreactivity for RHDV viral VP60 antigen in the liver. Results obtained indicate that the SphK1/S1P system activates in parallel to viral replication and the inflammatory process induced by the virus. Inhibition of the lipid signaling pathway by the indole reveals novel molecular pathways that may account for the protective effect of melatonin in this animal model of ALF, and supports the potential of melatonin as an antiviral agent.