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.

Nucleolin interacts with the rabbit hemorrhagic disease virus replicase RdRp, nonstructural proteins p16 and p23, playing a role in virus replication.

Rabbit hemorrhagic disease virus (RHDV) is a member of the Caliciviridae family and cannot be propagated in vitro, which has impeded the progress of investigating its replication mechanism. Construction of an RHDV replicon system has recently provided a platform for exploring RHDV replication in host cells. Here, aided by this replicon system and using two-step affinity purification, we purified the RHDV replicase and identified its associated host factors. We identified rabbit nucleolin (NCL) as a physical link, which mediating the interaction between other RNA-dependent RNA polymerase (RdRp)-related host proteins and the viral replicase RdRp. We found that the overexpression or knockdown of NCL significantly increased or severely impaired RHDV replication in RK-13 â€‹cells, respectively. NCL was identified to directly interact with RHDV RdRp, p16, and p23. Furthermore, NCL knockdown severely impaired the binding of RdRp to RdRp-related host factors. Collectively, these results indicate that the host protein NCL is essential for RHDV replication and acts as a physical link between viral replicase and host proteins.

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.

RPS5 interacts with the rabbit hemorrhagic disease virus 3' extremities region and plays a role in virus replication.

Rabbit hemorrhagic disease virus (RHDV), a member of Caliciviridae family, causes a highly contagious disease in rabbits. The RHDV replication mechanism is poorly understood due to the lack of a suitable culture system in vitro. This study identified RHDV 5' and 3' extremities (Ex) RNA binding proteins from the rabbit kidney cell line RK-13 based on a pull-down assay by applying a tRNA scaffold streptavidin aptamer. Using mass spectrometry (MS), several host proteins were discovered which interact with RHDV 5' and 3' Ex RNA. The ribosomal protein S5 (RPS5) was shown to interact with RHDV 3' Ex RNA directly by RNA-pulldown and confocal microscopy. To further investigate the role of RPS5 in RHDV replication, small interfering RNAs for RPS5 and RPS5 eukaryotic expression plasmids were used to change the expression level of RPS5 in RK-13 cells and the results showed that the RHDV replication and translation levels were positively correlated with the expression level of RPS5. It was also verified that RPS5 promoted RHDV replication by constructing RPS5 stable overexpression cell lines and RPS5 knockdown cell lines. In summary, it has been identified that RPS5 interacted with the RHDV 3' Ex RNA region and played a role in virus replication. These results will help to understand the mechanism of RHDV replication.

Strict Assembly Restriction of Peptides from Rabbit Hemorrhagic Disease Virus Presented by Rabbit Major Histocompatibility Complex Class I Molecule RLA-A1.

Rabbits are pivotal domestic animals for both the economy and as an animal model for human diseases. A large number of rabbits have been infected by rabbit hemorrhagic disease virus (RHDV) in natural and artificial pandemics in the past. Differences in presentation of antigenic peptides by polymorphic major histocompatibility complex (MHC) molecules to T-cell receptors (TCR) on T lymphocytes are associated with viral clearance in mammals. Here, we screened and identified a series of peptides derived from RHDV binding to the rabbit MHC class I molecule, RLA-A1. The small, hydrophobic B and F pockets of RLA-A1 capture a peptide motif analogous to that recognized by human class I molecule HLA-A*0201, with more restricted aliphatic anchors at P2 and PΩ positions. Moreover, the rabbit molecule is characterized by an uncommon residue combination of Gly53, Val55, and Glu56, making the 310 helix and the loop between the 310 and α1 helices closer to the α2 helix. A wider A pocket in RLA-A1 can induce a special conformation of the P1 anchor and may play a pivotal role in peptide assembly and TCR recognition. Our study broadens the knowledge of T-cell immunity in domestic animals and also provides useful insights for vaccine development to prevent infectious diseases in rabbits.IMPORTANCE We screened rabbit MHC class I RLA-A1-restricted peptides from the capsid protein VP60 of rabbit hemorrhagic disease virus (RHDV) and determined the structures of RLA-A1 complexed with three peptides, VP60-1, VP60-2, and VP60-10. From the structures, we found that the peptide binding motifs of RLA-A1 are extremely constraining. Thus, there is a generally restricted peptide selection for RLA-A1 compared to that for human HLA-A*0201. In addition, uncommon residues Gly53, Val55, and Glu56 of RLA-A1 are located between the 310 helix and α1 helix, which makes the steric position of the 310 helix in RLA-A1 much closer to the α2 helix than that found in other mammalian MHC class I molecules. This special conformation between the 310 helix and α1 helix plays a pivotal role in rabbit MHC class I assembly. Our results provide new insights into MHC class I molecule assembly and peptide presentation of domestic mammals. Furthermore, these data also broaden our knowledge on T-cell immunity in rabbits and may also provide useful information for vaccine development to prevent infectious diseases in rabbits.

Multi-target chimaeric VLP as a therapeutic vaccine in a model of colorectal cancer.

BACKGROUND: Colorectal cancer is responsible for almost 700,000 deaths annually worldwide. Therapeutic vaccination is a promising alternative to conventional treatment for colorectal cancer, using vaccines to induce targeted immune responses against tumour-associated antigens. In this study, we have developed chimaeric virus-like particles (VLP), a form of non-infectious non-replicative subunit vaccine consisting of rabbit haemorrhagic disease virus (RHDV) VP60 capsid proteins containing recombinantly inserted epitopes from murine topoisomerase IIα and survivin. These vaccines were developed in mono- (T.VP60, S.VP60) and multi-target (TS.VP60) forms, aiming to elucidate the potential benefits from multi-target vaccination. METHODS: Chimaeric RHDV VLP were developed by recombinantly inserting immune epitopes at the N-terminus of VP60. Vaccines were tested against a murine model of colorectal cancer by establishing MC38-OVA tumours subcutaneously. Unmethylated CpG DNA oligonucleotides (CpGs) were used as a vaccine adjuvant. Statistical tests employed included the Mantel-Cox log-rank test, ANOVA and unpaired t-tests depending on the data analysed, with a post hoc Bonferroni adjustment for multiple measures. RESULTS: Chimaeric RHDV VLP were found to form a composite particle in the presence of CpGs. Overall survival was significantly improved amongst mice bearing MC38-OVA tumours following vaccination with T.VP60 (60%, 9/15), S.VP60 (60%, 9/15) or TS.VP60 (73%, 11/15). TS.VP60 significantly prolonged the vaccine-induced remission period in comparison to each mono-therapy. CONCLUSIONS: Chimaeric VLP containing multiple epitopes were found to confer an advantage for therapeutic vaccination in a model of colorectal cancer based on the prolongation of remission prior to tumour escape.

Multiple mechanisms of reinitiation on bicistronic calicivirus mRNAs.

Reinitiation is a strategy used by viruses to express several cistrons from one mRNA. Although extremely weak after translation of long open reading frames (ORFs) on cellular mRNAs, reinitiation occurs efficiently on subgenomic bicistronic calicivirus mRNAs, enabling synthesis of minor capsid proteins. The process is governed by a short element upstream of the restart AUG, designated "termination upstream ribosomal binding site" (TURBS). It contains the conserved Motif 1 complementary to h26 of 18S rRNA, displayed in the loop of a hairpin formed by species-specific Motifs 2/2(∗). To determine the advantages conferred on reinitiation by TURBS, we reconstituted this process in vitro on two model bicistronic calicivirus mRNAs. We found that post-termination ribosomal tethering of mRNA by TURBS allows reinitiation by post-termination 80S ribosomes and diminishes dependence on eukaryotic initiation factor 3 (eIF3) of reinitiation by recycled 40S subunits, which can be mediated either by eIFs 2/1/1A or by Ligatin following ABCE1-dependent or -independent splitting of post-termination complexes.

Development of CpG-oligodeoxynucleotides for effective activation of rabbit TLR9 mediated immune responses.

CpG-oligodeoxynucleotides (CpG-ODN) are potent immune stimuli being developed for use as adjuvants in different species. Toll-like receptor 9 (TLR9) is the cellular receptor for CpG-ODN in mammalian cells. The CpG-ODN with 18-24 deoxynucleotides that are in current use for human and mouse cells, however, have low activity with rabbit TLR9. Using a cell-based activation assay, we developed a type of CpG-ODN containing a GACGTT or AACGTT motif in 12 phosphorothioate-modified deoxynucleotides with potent stimulatory activity for rabbit TLR9. The developed CpG-ODN have higher activities than other developed CpG-ODN in eliciting antigen-nonspecific immune responses in rabbit splenocytes. When mixed with an NJ85 peptide derived from rabbit hemorrhagic disease virus, they had potent activities to boost an antigen-specific T cell activation and antibody production in rabbits. Compared to Freund's adjuvant, the developed CpG-ODN are capable of boosting a potent and less toxic antibody response. The results of this study suggest that both the choice of CpG-motif and its length are important factors for CpG-ODN to effectively activate rabbit TLR9 mediated immune responses.

Melatonin modulates the autophagic response in acute liver failure induced by the rabbit hemorrhagic disease virus.

Autophagy is an important survival pathway and participates in the host response to infection. Beneficial effects of melatonin have been previously reported in an animal model of acute liver failure (ALF) induced by the rabbit hemorrhagic disease virus (RHDV). This study was aimed to investigate whether melatonin protection against liver injury induced by the RHDV associates to modulation of autophagy. Rabbits were infected with 2 × 10(4) hemagglutination units of a RHDV isolate and received 20 mg/kg melatonin at 0, 12, and 24 hr postinfection. RHDV induced autophagy, with increased expression of beclin-1, ubiquitin-like autophagy-related (Atg)5, Atg12, Atg16L1 and sequestrosome 1 (p62/SQSTM1), protein 1 light chain 3 (LC3) staining, and conversion of LC3-I to autophagosome-associated LC3-II. These effects reached a maximum at 24 hr postinfection, in parallel to extensive colocalization of LC3 and lysosome-associated membrane protein (LAMP)-1. The autophagic response induced by RHDV infection was significantly inhibited by melatonin administration. Melatonin treatment also resulted in decreased immunoreactivity for RHDV viral VP60 antigen and a significantly reduction in RHDV VP60 mRNA levels, oxidized to reduced glutathione ratio (GSSG/GSH), caspase-3 activity, and immunoglobulin-heavy-chain-binding protein (BiP) and CCAAT/enhancer-binding protein homologous protein (CHOP) expression. Results indicate that, in addition to its antioxidant and antiapoptotic effects, and the suppression of ER stress, melatonin induces a decrease in autophagy associated with RHDV infection and inhibits RHDV RNA replication. Results obtained reveal novel molecular pathways accounting for the protective effect of melatonin in this animal model of ALF.

The new French 2010 Rabbit Hemorrhagic Disease Virus causes an RHD-like disease in the Sardinian Cape hare (Lepus capensis mediterraneus).

Lagovirus is an emerging genus of Caliciviridae, which includes the Rabbit Hemorrhagic Disease Virus (RHDV) of rabbits and the European brown hare syndrome virus (EBHSV) of hares that cause lethal hepatitis. In 2010, a new RHDV related virus (RHDV2) with a unique genetic and antigenic profile and lower virulence was identified in France in rabbits. Here we report the identification of RHDV2 as the cause in Sardinia of several outbreaks of acute hepatitis in rabbits and Cape hare (Lepus capensis mediterraneus). This is the first account of a lagovirus that causes fatal hepatitis in both rabbits and hares.

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.

Melatonin treatment reduces endoplasmic reticulum stress and modulates the unfolded protein response in rabbits with lethal fulminant hepatitis of viral origin.

Hepatocyte apoptosis plays an important role in the development of fulminant hepatic failure (FHF). The objective of this study was to investigate whether endoplasmic reticulum (ER) stress and unfolded protein response (UPR) inhibition is an underlying mechanism of melatonin anti-apoptotic effects in an animal model of FHF of viral origin induced by the rabbit hemorrhagic disease virus (RHDV). Rabbits were experimentally infected with 2 × 10(4) hemagglutination units of a RHDV isolate and received melatonin at two concentrations of 10 mg/kg and 20 mg/kg at 0 hr, 12 hr and 24 hr postinfection. RHDV infection induced increased expression of CCAAT/enhancer-binding protein homologous protein (CHOP), immunoglobulin heavy chain binding protein (BiP/GRP78), glucose-regulated protein 94 (GRP94), phospho-c-Jun N-terminal kinase (JNK) and caspase-12. These effects were attenuated by melatonin. Double immunofluorescence staining showed colocalization of CHOP and cleaved caspase-3 in liver sections of RHDV-infected rabbits, while immunostaining decreased markedly with melatonin treatment. RHDV infection resulted in significant increases in the mRNA levels of activating transcription factor 6 (ATF6), ATF4, inositol-requiring enzyme 1 (IRE1), spliced X-box binding protein-1 (XBP1s) and tumor necrosis factor receptor-associated factor 2 (TRAF2). Melatonin attenuated the extent of the changes. Data obtained provide evidence that in rabbits with experimental infection by RHDV, reduction in apoptotic liver damage by melatonin is associated with attenuation of ER stress through a modulation of the three arms of UPR signaling and further support a potential hepatoprotective role of melatonin in FHF.

Analysis of two VP60 capsid protein genes of rabbit hemorrhagic disease virus from viruses obtained from the same farm.

Two distinct clones of the VP60 capsid protein gene of rabbit hemorrhagic disease virus were amplified from mixed liver tissue of rabbits collected from the same farm in the Xinjiang Uygur Autonomous Region of China in 2002. The results of DNA sequence analysis showed that the length of the VP60 gene in the first clone was 1,740 bp, similar to other VP60 genes. The length of the VP60 gene in the second clone was only 1,536 bp. The two clones were predicted to encode 579 and 511 amino acids, respectively.

Minor structural protein VP2 in rabbit hemorrhagic disease virus downregulates the expression of the viral capsid protein VP60.

Rabbit hemorrhagic disease virus (RHDV) has two structural proteins: the major capsid protein VP60 and the minor capsid protein VP2. VP2 is speculated to play an important role in the virus life cycle. To investigate the effect of VP2 on VP60 expression, three types of experiment (baculovirus-insect cell system, mammalian-luciferase assay system and in vitro coupled transcription/translation system) were used to express VP60 alone or co-expressed with VP2. Both forms of VP60 were able to form virus-like particles in insect cells. Western blot analysis and dual-luciferase assays demonstrated that the presence of VP2 results in downregulation of the expression of VP60 in vivo. Real-time RT-PCR of mRNA levels showed that downregulation of VP60 occurs at the transcriptional level. The ability of the viral minor structural protein VP2 to regulate capsid protein levels may contribute to effective virus infection.

Detection of positive selection in the major capsid protein VP60 of the rabbit haemorrhagic disease virus (RHDV).

Mutations were analysed in the major capsid protein VP60 of the rabbit haemorrhagic disease virus (RHDV), a calicivirus responsible for high mortality rates in both wild and domestic European rabbits (Oryctolagus cuniculus). Likelihood of positive selection was estimated using the PAML software applied to 43 non-identical complete sequences of the major capsid protein. Three codons showed signs of positive selection (with posterior probabilities over 95%), one of them is located in the region containing the major antigenic determinants (region E). The presence of positively selected codons (PSCs) in other regions may suggest the existence of other antigenic regions on the major capsid protein that stimulate protective immune responses. At all the 3 PSCs, variation contributes to putative N-glycosylation sites of the protein. An N-glycosylation site is deleted in the non-pathogenic strain RCV. Some of the substitutions at PSCs may alter the polarity and the charge of the protein with possible implications in the protein structure and host interaction. The detection of PSCs should allow a better understanding of the interaction between RHDV and the rabbit immune system.

Rabbit hemorrhagic disease virus: identification of a cleavage site in the viral polyprotein that is not processed by the known calicivirus protease.

The positive-strand RNA genome of the calicivirus rabbit hemorrhagic disease virus (RHDV) contains one long open reading frame (ORF) covering almost 95% of the genomic RNA. Translation of this ORF leads to a polyprotein that is proteolytically processed at eight sites. Site nos. 4 and 5 from the amino-terminus are located within the protein p41 and obviously belong to alternative cleavage pathways leading to p23/2 and p18 or p29 and p13. Seven of the eight cleavage sites were identified before and the flanking sequences fulfill the requirements of the known RHDV protease, so that it is very likely that all theses sites are cleaved by this enzyme. The last unknown cleavage site was no. 4, one of the two alternative sites within p41 that separates the nonstructural proteins p23/2 and the VPg precursor p18. Mutagenesis studies identified aspartic acid at position 936 and arginine at position 937 as the residues that flank the cleavage site. The sequence at the processing site is unusual for the RHDV 3C-like protease since other sites display glutamic acid or glutamine at the P1 site and glycine, aspartic acid, or threonine at the P1' site. Expression of a polyprotein fragment lacking the viral protease revealed that the newly identified site is not cleaved by the RHDV protease but by an unknown proteolytic activity.

Crystal structures of active and inactive conformations of a caliciviral RNA-dependent RNA polymerase.

The structure of the RNA-dependent RNA polymerase (RdRP) from the rabbit hemorrhagic disease virus has been determined by x-ray crystallography to a 2.5-A resolution. The overall structure resembles a "right hand," as seen before in other polymerases, including the RdRPs of polio virus and hepatitis C virus. Two copies of the polymerase are present in the asymmetric unit of the crystal, revealing active and inactive conformations within the same crystal form. The fingers and palm domains form a relatively rigid unit, but the thumb domain can adopt either "closed" or "open" conformations differing by a rigid body rotation of approximately 8 degrees. Metal ions bind at different positions in the two conformations and suggest how structural changes may be important to enzymatic function in RdRPs. Comparisons between the structures of the alternate conformational states of rabbit hemorrhagic disease virus RdRP and the structures of RdRPs from hepatitis C virus and polio virus suggest novel structure-function relationships in this medically important class of enzymes.

Binding of rabbit hemorrhagic disease virus to antigens of the ABH histo-blood group family.

The ability of rabbit hemorrhagic disease virus to agglutinate human erythrocytes and to attach to rabbit epithelial cells of the upper respiratory and digestive tracts was shown to depend on the presence of ABH blood group antigens. Indeed, agglutination was inhibited by saliva from secretor individuals but not from nonsecretors, the latter being devoid of H antigen. In addition, erythrocytes of the rare Bombay phenotype, which completely lack ABH antigens, were not agglutinated. Native viral particles from extracts of infected rabbit liver as well as virus-like particles from the recombinant virus capsid protein specifically bound to synthetic A and H type 2 blood group oligosaccharides. Both types of particles could attach to adult rabbit epithelial cells of the upper respiratory and digestive tracts. This binding paralleled that of anti-H type 2 blood group reagents and was inhibited by the H type 2-specific lectin UEA-I and polyacrylamide-conjugated H type 2 trisaccharide. Young rabbit tissues were almost devoid of A and H type 2 antigens, and only very weak binding of virus particles could be obtained on these tissues.