RESUMEN
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.
Asunto(s)
Infecciones por Caliciviridae/veterinaria , Brotes de Enfermedades/veterinaria , Virus de la Enfermedad Hemorrágica del Conejo/aislamiento & purificación , Conejos , Animales , Infecciones por Caliciviridae/epidemiología , Infecciones por Caliciviridae/virología , China/epidemiologíaRESUMEN
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 2.0â¯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.
Asunto(s)
Enfermedades Intestinales/veterinaria , Conejos , Animales , Ciego , China , Granjas , Enfermedades Intestinales/diagnóstico , Enfermedades Intestinales/microbiología , Conejos/microbiologíaRESUMEN
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.
Asunto(s)
Antígenos de Grupos Sanguíneos/clasificación , Infecciones por Caliciviridae/veterinaria , Virus de la Enfermedad Hemorrágica del Conejo , Sueros Inmunes/inmunología , Conejos/sangre , Vacunas Virales/inmunología , Animales , Antígenos de Grupos Sanguíneos/química , Antígenos de Grupos Sanguíneos/metabolismo , Infecciones por Caliciviridae/prevención & control , Infecciones por Caliciviridae/virología , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Regulación Viral de la Expresión Génica/fisiología , Unión ProteicaRESUMEN
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.
Asunto(s)
Infecciones por Caliciviridae/veterinaria , Virus de la Enfermedad Hemorrágica del Conejo/clasificación , Virus de la Enfermedad Hemorrágica del Conejo/aislamiento & purificación , Filogenia , Animales , Western Blotting , China , Genotipo , Pruebas de Hemaglutinación , Virus de la Enfermedad Hemorrágica del Conejo/genética , Virus de la Enfermedad Hemorrágica del Conejo/inmunología , Conejos , Análisis de Secuencia de ADN , Proteínas Estructurales Virales/genéticaRESUMEN
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.
Asunto(s)
Antígenos de Grupos Sanguíneos/metabolismo , Epítopos de Linfocito B/inmunología , Epítopos de Linfocito B/metabolismo , Virus de la Enfermedad Hemorrágica del Conejo/inmunología , Proteínas Estructurales Virales/inmunología , Proteínas Estructurales Virales/metabolismo , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/aislamiento & purificación , Mapeo Epitopo , Femenino , Ratones Endogámicos BALB C , Unión Proteica , Mapeo de Interacción de Proteínas , ConejosRESUMEN
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.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Portadores de Fármacos , Epítopos de Linfocito T/inmunología , Vectores Genéticos , Virus de la Enfermedad Hemorrágica del Conejo/genética , Ovalbúmina/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Animales , Anticuerpos Antivirales/sangre , Células Cultivadas , Pollos , Epítopos de Linfocito T/genética , Femenino , Inmunoglobulina G/sangre , Interferón gamma/metabolismo , Ratones Endogámicos C57BL , Microscopía Electrónica de Transmisión , Ovalbúmina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas de Partículas Similares a Virus/administración & dosificación , Vacunas de Partículas Similares a Virus/genética , Virosomas/ultraestructuraRESUMEN
In order to validate the immune-enhancement efficacy of compound Chinese herbal medicinal ingredients (cCHMIs), made with astragalus polysaccharide (APS), epimedium polysaccharide (EPS), propolis flavone (PF) and ginsenosides (GS), as immune potentiator or vaccine adjuvants for rabbits, the effects of two cCHMIs on rabbit lymphocyte proliferation and IFN-gamma and IL-10 mRNA expression of T lymphocyte in vitro were determined. At the same time, two cCHMIs were injected into 35-day-old rabbits after mixed with rabbit hemorrhagic disease (RHD) vaccine taking aluminum adjuvant and phosphate-buffered saline (PBS) as controls. On days 7, 14, 21, 35 and 49 after the vaccination, the dynamic changes of peripheral lymphocyte proliferation and serum antibody titers of the rabbits were analyzed. On day 63, all rabbits were challenged with RHD virus. The results showed that the two cCHMIs could significantly promote rabbit lymphocyte proliferation and IFN-gamma and IL-10 mRNA expression of T lymphocyte in vitro. In vivo, two cCHMIs could significantly enhance serum antibody titers and lymphocyte proliferation. Their adjuvanticity was slightly superior to aluminum adjuvant. All the rabbits vaccinated with the cCHMIs adjuvant vaccine were protected. These findings confirmed that two cCHMIs possessed better immune-enhancement efficacy and would be used as effective immune adjuvant of RHD vaccine.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Infecciones por Caliciviridae/veterinaria , Medicamentos Herbarios Chinos/farmacología , Virus de la Enfermedad Hemorrágica del Conejo/inmunología , Plantas Medicinales , Vacunas Virales/inmunología , Adyuvantes Inmunológicos/aislamiento & purificación , Animales , Anticuerpos Antivirales/sangre , Infecciones por Caliciviridae/prevención & control , Proliferación Celular , Medicamentos Herbarios Chinos/aislamiento & purificación , Ginsenósidos/aislamiento & purificación , Ginsenósidos/farmacología , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Leucocitos Mononucleares/inmunología , Própolis/aislamiento & purificación , Própolis/farmacología , Conejos , Linfocitos T/inmunologíaRESUMEN
Reverse transcriptase polymerase chain reaction (RT-PCR) for the diagnosis of rabbit hemorrhagic disease virus (RHDV) was developed by examining sensitivity and specificity. Samples from rabbits infected with rabbit hemorrhagic disease (RHD) were examined to investigate the distribution of the virus in the body. The results showed that the RT-PCR method had good specificity. The sensitivity of the RT-PCR was 1 x 10(4) times higher than that of the hemagglutination assay (HA). RT-PCR was able to detect RHDV in all viscera, but not in feces. In the second part of the study, in order to investigate the prevalence of RHDV, 400 meat samples from the Entry-exit Inspection and Quarantine Bureau and 512 nasal secretion samples from rabbits in three provinces of China were collected and tested by RT-PCR. The results showed significant differences in the prevalence of RHDV in rabbits of different ages, but no significant differences among different provinces and years. Some random isolates were sequenced and compared. The homology of sequences among three new isolates and other isolates ranged between 93.7% and 99.6%. It is recommended that RHD vaccine should be used in China to protect rabbits against RHDV.