Experimental pathogenicity and comparative genome analysis of high- and low-virulence strains of rabbit-origin Pasteurella multocida.

Pasteurella multocida, the causative pathogen of rabbit pasteurellosis, causes significant economic losses in the commercial rabbit industry. However, the associated pathogenic mechanism of P. multocida remains unclear. The aim of this study is to compare the genomes and pathogenicity of high- and low-virulence strains of P. multocida to advance the current understanding of rabbit pasteurellosis. The high-virulence strain rapidly proliferates in the lung and spleen of infected mice within approximately 9 h, maintaining a high bacterial load until host death. Meanwhile, the low-virulence strain only proliferates in mouse organs for a short time, with the bacterial load beginning to decrease 13 h post-infection. Moreover, the expressions of inflammatory cytokines MCP-1, TNF-α, and IL-1ß are upregulated in all infected mouse lung and spleen tissue, however, the high-virulence strain induced significantly higher expression than the low-virulence strain. Histopathological analysis revealed greater inflammation and tissue lesions in the lung and spleen of mice infected with the high-virulence strain. Two pathogenicity-associated regions unique to the genome of the high-virulence strain harbor approximately 199 genes, including functional genes related to virulence factors, such as lipopolysaccharide biosynthesis, iron acquisition, biosynthesis of outer membrane proteins, and adhesion. These two genomic regions are shared by three previously sequenced, highly virulent P. multocida strains in rabbits. In conclusion, the increased pathogenicity of high-virulence P. multocida may be due to the presence of virulence-associated genes in two unique genomic regions, resulting in strong proliferative activity, significant inflammation, and pathological lesions in the mouse model. These findings provide important insights regarding the pathogenic mechanism underlying rabbit pasteurellosis.

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.

Emergence of rabbit haemorrhagic disease virus 2 in China in 2020.

Rabbit haemorrhagic disease (RHD) is an acute fatal disease caused by the Lagovirus rabbit haemorrhagic disease virus (RHDV), which was first reported in 1984 in China. Strains of two different genotypes (GI.1a and GI.1c) have been detected in China to date. In 2010, a new RHDV variant with a unique genetic and antigenic profile was identified in France, designated RHDV2, which rapidly spread throughout continental Europe and nearby islands. Here, we report the first outbreak of RHD induced by RHDV2 (GI.2) in rabbit farms in the Sichuan province of China. We conducted haemagglutination tests and phylogenetic analysis of the new RHDV isolate SC2020/04, which was identified as a non-haemagglutinating strain belonging to the RHDV2 (GI.2) genogroup. Considering the serious risk of RHDV2 to the Chinese rabbit industry, the circulation of RHDV2 in the population should be carefully monitored in China.

Characterization of Pasteurella multocida isolates from rabbits in China.

Pasteurella multocida is the causative agent of a wide range of diseases (pasteurellosis) and is a zoonotic pathogen in humans. The molecular epidemiology of P. multocida from rabbits in some southern European countries has been characterized, and the associations of some populations with the respiratory niche or virulence factors have been suggested. However, the population structure of P. multocida from rabbits in China has not been well characterized. In this study, 30 P. multocida isolates from rabbits without epidemiological relations in China were clustered using mutilocus sequence typing (MLST). Then, the genotypes of virulence factors (capsule, lipopolysaccharides, HgbB, and PfhA) of these isolates were determined via multiplex PCR methods. Next, the virulence of the isolates in a mice model was established by determining the 50 % lethal dose. Finally, the associations between MLST types and the prevalence of genotypes, virulent strains, or clinical origins were characterized. The P. multocida isolates identified in this work included 3 major clonal complexes: CC9, CC74, and ST129. CC9 exhibited cpsA(F)L3, and was associated with a higher prevalence of rhinitis; CC74 exhibited cpsAL6, and was associated with higher prevalences of hgbB+pfhA- and pneumonia; ST129 exhibited cpsAL1, and was associated with higher prevalences of high-virulence strains and septicemia. The results provided insights into P. multocida from rabbits in China and suggested the use of strains from different populations in future P. multocida pathogenesis and vaccine studies.

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.

Characterization of host plasminogen exploitation of Pasteurella multocida.

Pasteurella multocida is the causative agent of a wide range of disease (pasteurellosis) and a zoonotic pathogen in humans. Some pathogenic bacteria are able to exploit host plasminogen for migration across tissue barriers or evade from host innate immunity. However, there is no study on host plasminogen exploitation of P. multocida. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) has been reported to be a plasminogen receptor in many pathogenic bacteria, but its role in P. multocida exploiting plasminogen has not yet been characterized. The aim of this study was to detect the activity of P. multocida to exploit host plasminogen and evaluate the ability of GAPDH to act as a receptor in the recruitment process. P. multocida could recruit host plasminogen and exhibited plasmin activity when stimulated by urokinase. GAPDH exhibited binding activity to plasminogen. GAPDH Antiserum significantly decrease the plasminogen recruitment activity of P. multocida. In conclusion, P. multocida is able to exploit host plasminogen via GAPDH. To our knowledge, this is the first report on host plasminogen exploitation of P. multocida.

Detection of mucoid enteropathy syndrome disease in rabbit farms in East China.

During the past years epizootic outbreaks of mucoid enteropathy syndrome (MES) disease have emerged and spread rapidly in rabbit farms in East China causing great economic losses. To investigate the nature and evolution of the disease, two trials were conducted in an attempt to reproduce the disease and to assess the microbiota cecal profile changes associated with the disease. In the first trial, twenty 6-week-old New Zealand White healthy rabbits were assigned randomly to one of two treatments: 1) Inoculation with 2.0 mL of cecal content (inoculum) from dead rabbits affected with MES; 2) No inoculation or control group. In the second trial, V3 - V4 regions of 16S ribosomal genes from three virulent (MES) and two non-virulent (healthy) cecal samples were amplified and sequenced for microbiota genomic characterization. The mucoid enteropathy syndrome was reproduced using a virulent cecal material (inoculum). The mortality rates for treatments 1 and 2 were 70% and 0%, respectively. Clinical signs and gross lesions of affected rabbits consisted of bloated abdomen, cecal impaction, presence of variable amounts of gelatinous mucus particularly in the colon, mucus excretion and diarrhea of low intensity. The disease that emerged in China is similar to the epizootic rabbit enteropathy (ERE) described in Europe. However, microbiota cecal changes associated with the disease differed from those in ERE. Here, a comprehensive analysis of mucoid enteropathy syndrome disease is presented, which should be carefully monitored.

Rabbit Hemorrhagic Disease Virus Non-structural Protein 6 Induces Apoptosis in Rabbit Kidney Cells.

Rabbit hemorrhagic disease (RHD) is a highly contagious disease caused by rabbit hemorrhagic disease virus (RHDV). Previous research has shown that RHDV induces apoptosis in numerous cell types, although the molecular mechanisms underlying the apoptosis induced by RHDV are not well understood. One possible factor is non-structural protein 6 (NSP6), a 3C-like protease that plays an important role in processing viral polyprotein precursors into mature non-structural proteins. To fully establish a role for NSP6, the present study examined the effects of ectopic expression of the protein in rabbit (RK13) and human (HeLa and HepG2) cells. We found that NSP6 suppressed cell viability and promoted apoptosis in all three cell types in a dose-dependent manner. We also identified increased caspase-3, -8, and -9 activities in RK13 cell, and an increased Bax to Bcl2 mRNA ratio. Mechanistically, the ability of NSP6 to induce apoptosis was impaired by mutation of the catalytic His27 residue. Our study has shown that RHDV NSP6 can induce apoptosis in host cells and is likely an important contributor to RHDV-induced apoptosis and pathogenesis.

Binding of rabbit hemorrhagic disease virus-like particles to host histo-blood group antigens is blocked by antisera from experimentally vaccinated rabbits.

During infection host histo-blood group antigens (HBGAs) act as attachment factors that interact with rabbit hemorrhagic disease virus (RHDV) and participate in the infectious process. In the present study, baculovirus expressing recombinant RHDV capsid protein (VP60r) as a vaccine immunogen was used to test its antigenicity and immunogenicity via immunization experiments. Each group of rabbits immunized with VP60r was found to be fully protected against RHDV challenge. The duration of immunity of the vaccine following the inoculation of a single dose was determined to be at least 240 days. RHDV-specific humoral responses in antisera from inoculated rabbits were analyzed using VP60r virus-like particle (VLP)-based ELISA. Anti-VP60-specific antibody was produced by 7 days post-primary immunization. Following this stage, the levels of this antibody increased steadily, peaking at 90 days and maintaining a high level until 240 days. We developed a synthetic carbohydrate assay to detect blockage in attachment of RHDV VLPs to HBGAs by the rabbit antisera. On day 7 post-immunization, serum samples were demonstrated to block the binding of H type 2 to RHDV VLPs, with a blocking rate of almost 60%, a value that then increased steadily over time. From day 60 to day 240 post-immunization, serum samples completely blocked the binding of H type 2 to RHDV VLPs, with a blocking rate of almost 100%. This indicated that VP60-induced antibodies neutralize the interaction of RHDV with HBGAs.

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)-1ß, 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.

A new variant of rabbit hemorrhagic disease virus G2-like strain isolated in China.

To investigate the genetic variability and evolution of rabbit hemorrhagic disease virus (RHDV) strains in China, VP60 gene sequences of eight new isolates collected from farms with RHD occurrences in China between 2009 and 2014 were analyzed, and compared with the reference sequence of the vaccine strain WF/China/2007. We conducted a comprehensive analysis of the Chinese RHDV strains, including hemagglutination tests, western blot and immunosassays of capsid proteins, and phylogenetic analysis, and identified a new distinct antigenic variant. Specifically, strain HB/2014 collected in North China was identified as a non-hemagglutinating strain, and belongs to the original RHDV (G1-G5) group. The other seven isolates were classified in genogroup G6 (RHDVa), which was widely distributed across China before 2014, and was thought to replace the earlier groups. Antigenic characterization of the VP60 genes revealed a large degree of nucleotide sequence divergence between HB/2014 and the other Chinese strains. However, the current vaccine showed complete cross-protection against HB/2014 challenge in inoculated rabbits. Collectively, these data provide new tools and insight for further understanding the molecular evolution of RHDV in China.

Identification of novel rabbit hemorrhagic disease virus B-cell epitopes and their interaction with host histo-blood group antigens.

Rabbit haemorrhagic disease, caused by rabbit hemorrhagic disease virus (RHDV), results in the death of millions of adult rabbits worldwide, with a mortality rate that exceeds 90%. The sole capsid protein, VP60, is divided into shell (S) and protruding (P) domains, and the more exposed P domain likely contains determinants for cell attachment and antigenic diversity. Nine mAbs against VP60 were screened and identified. To map antigenic epitopes, a set of partially overlapping and consecutive truncated proteins spanning VP60 were expressed. The minimal determinants of the linear B-cell epitopes of VP60 in the P domain, N(326)PISQV(331), D(338)MSFV(342) and K(562)STLVFNL(569), were recognized by one (5H3), four (1B8, 3D11, 4C2 and 4G2) and four mAbs (1D4, 3F7, 5G2 and 6B2), respectively. Sequence alignment showed epitope D(338)MSFV(342) was conserved among all RHDV isolates. Epitopes N(326)PISQV(331) and K(562)STLVFNL(569) were highly conserved among RHDV G1-G6 and variable in RHDV2 strains. Previous studies demonstrated that native viral particles and virus-like particles (VLPs) of RHDV specifically bound to synthetic blood group H type 2 oligosaccharides. We established an oligosaccharide-based assay to analyse the binding of VP60 and epitopes to histo-blood group antigens (HBGAs). Results showed VP60 and its epitopes (aa 326-331 and 338-342) in the P2 subdomain could significantly bind to blood group H type 2. Furthermore, mAbs 1B8 and 5H3 could block RHDV VLP binding to synthetic H type 2. Collectively, these two epitopes might play a key role in the antigenic structure of VP60 and interaction of RHDV and HBGA.

Immunogenicity of different recombinant rabbit hemorrhagic disease virus-like particles carrying CD8+ T cell epitope from chicken ovalbumin (OVA).

To explore the capacity and immunogenicity of virus-like particles (VLPs) of rabbit hemorrhagic disease virus (RHDV) accommodating foreign amino acid sequences, integrations were performed at the following four locations of the structural protein VP60 of RHDV using the OVA257-264 CD8+ T cell epitope (SIINFEKL): (1) inserting at the N-terminus of the VP60 protein (N1); (2) replacing amino acid positions 2-14 of the VP60 protein (N2); (3) replacing amino acid positions 196-207 of the VP60 protein (I1); and (4) replacing amino acid positions 217-228 of the VP60 protein (I2). The recombinant proteins were expressed by baculovirus expression system. The ability to form RHDV-like particles was confirmed by electron microscopy. The immunogenicity of the four recombinant proteins (N1, N2, I1 and I2) was evaluated in mice without any adjuvants. The results indicated that the four recombinant proteins (N1, N2, I1 and I2) could assemble into VLPs. All of the recombinant proteins could induce a specific immune response. Recombinant proteins I1 and I2 were able to elicit both high levels of IFN-γ secretion and anti-VP60 specific immune responses in the murine model. The levels of the VP60-specific IgG antibody in groups I1 and I2 displayed higher optical density (OD) values than those of groups N1 and N2 (P<0.001, P<0.001). The number of IFN-γ-producing splenocytes in mice that were immunized with recombinant proteins I1 and I2 was also significantly greater compared with mice that were immunized with recombinant proteins N1 and N2 (P<0.01). All of these above mentioned results might be beneficial to the establishment of the RHDV-VLPs display system.