| Bluetongue(BT) is an arthropod-borne viral disease throughout many tropical and subtropical regions of the world. Bluetongue virus(BTV), the etiological agent of BT, is usually transmitted by biting midges(Culicoides), and can infect all species of ruminants including sheep, goats, cattle, deer and camels(particularly the sheep and lamb). Generally, the average mortality of infected animals is 20-30%, however, up to 90% mortality in sheep of some susceptible flocks. BT can have major effects on animal health, restrict international trade in livestock and result in important economic losses. It is therefore listed as a ‘notifiable’ disease by the Office International des Epizooties(OIE).The genome of BTV was composed of ten linear segments of double-stranded RNA(ds RNA), encoding seven structural proteins(VP1 to VP7) and four nonstructural proteins(NS1 to NS4). M5 gene encoding protein NS1 and S10 gene encoding protein NS3/3a are highly conserved of the 10 segments while L2 segment encoding protein VP2 is the most variable gene in BTV. BTV genome evolution is driven by genetic drift and shift as well as intragenic recombination, resulting in the possibility of novel genetically distinct strains of the virus. Twenty-six BTV serotypes have been recognized in the world and seven of theses(BTV-1,-2,-3,-4,-12,-15 and-16) have been isolated in China. Additionally, the antigenic diversity among different BTV serotypes poses great difficulties for the prevention and control of BT. Immune responses elicited by one BTV serotype are generally poorly cross-protective against other BTV serotypes.For prevention and control of any kind of infection, the rapid and accurate pathogen diagnostic methods and effective means of vaccination are the most fundamental strategy. Based on the characteristics of BTV, it is important to establish early detection and differential diagnosis methods for prevention and control, especially for rapid pathogen detection methods for each serotype of BTV. In this study, the genomes of all BTV serotypes were compared, which were available through Gen Ban K or previously sequenced by our laboratory. Then, One-step RT-PCR and nested RT-PCR assays were developed for the detection of BTV serogroup and the specific test, sensitivity test, repeatability test and clinical sample detection were performed. The results showed that the One-step RT-PCR method can be applied to detect BTV serotypes 1-24 with a detection limit of 100 TCID50 but no cross-reaction with related viruses. And this method was capable to detect BTV from the infection blood samples of sheep. In addition, nested RT-PCR strategy can be applied to detect BTV serotypes 1-24 and 26 with an amplified fragment of 93 base pairs, no cross-reaction with related viruses. The analytical sensitivities were evaluated by using 10-fold serial dilutions of BTV-16(107-10-1 TCID50/ mL) and this assay was able to detect ≥10 TCID50/ m L, and could faithfully detect the presence of the M5 gene target sequence in the blood of sheep inoculated with BTV.In this study, it is the first to establish serotype-specific RT-PCR detection methods for seven serotypes of BTV prevalence in China, and also set up serotype-specific real-time RT-PCR detection methods for 22 different serotypes of BTV. L2 gene, the most variable gene in BTV, obtained from network database and sequenced by ourselves was used as a target to develop RTPCR-based and real-time RT-PCR based typing assays for each BTV type. 279 nucleotide sequences of L2 gene were aligned and the phylogenetic tree was analysis. Based on the sequences analysis, multiple primer-pairs and Taq Man-MGB probes were designed. The binding sites for type specific primers and probes were chosen with intra-typic conservation and hetero-typic variation in particular to ensure the binding sites designed for Taq Man-MGB probes was conservative of the same BTV serotype. Specific test, sensitivity test, repeatability test, standard curves and sample detection were performed to evaluate the serotype RT-PCR and real-time RTPCR assays of BTV. The specific test results showed that the serotype-specific primers and probes were no cross amplification with other closely related heterologous BTV types and control viruses, but positive amplification of the target serotype BTV nucleic acid. Sensitivity test results showed that the sensitivity of genotyping RT-PCR assays were 102 to 104 copies respectively and 10 to 102 copies for real-time RT-PCR assays. The real-time RT-PCR assays were more than 10 times sensitivity than tradition RT-PCR assay. Accurate quantitation can be achieved with standard samples containing between 102 and 108 copies. Repeatability test results showed that both traditional RT-PCR assays and real-time RT-PCR assays were good. The coefficient of variation(CV) of inter-assay and intra-assay of real-time RT-PCR assays were less than 1% and 4%, respectively. The nucleic acid detection methods developed in this study provide a rapid and reliable method for the identification and differentiation of the 22 BTV serotypes, and are very important for vaccination programmes of BTV.This study firstly used the replication defective adenovirus serotype 5 as a live vehicle for the delivery of BTV-16 VP2 and VP5 antigens. Because this vector only carries the transgenes of interest devoid of other molecular regulatory elements of the parental virus, it is inherently safe and highly reduced the risk of gene segment re-assortment with field bluetongue strains. However, there is a dearth of experience using recombinant adenoviruses(r Ad) as an antigen delivery system for BTV proteins. One potential obstacle to constructing r Ad-vectored BTV vaccines involves multiple E. coli promoter sequences within the genomes of BTV-16 VP2 and VP5 gene that led to the instability of full-length BTV c DNA in E. coli. Here, we introduced silent mutations in predicted E. coli promoter regions of the BTV-16 VP2 and VP5 genes to stabilize the full-length VP2 and VP5 clones in E. coli. Using these modified sequences, we successfully generated recombinant adenovirus vectors to facilitate VP2 and VP5 protein expression and evaluated the immunization of BALB/c mice and sheep.BALB/c mice were immunized with the r Ad-VP2 and r Ad-VP2-IRES-VP5 respectively. The results showed that VP2/VP5-specific Ig G/Ig A was detected in serum of mice immunized intraperitoneally with rAd-VP2 and r Ad-VP2-IRES-VP5 at 3 weeks following the primary immunization. VP2/VP5-specific Ig G was not detected in serum of mice immunized intranasally while the VP2/VP5-specific Ig A chould be detected. Following in vitro re-stimulation with VP2 or VP5 protein, splenocytes from r Ad-immunized mice displayed significantly higher proliferative responses and secreted significantly higher levels of IFN-γ, IL-2 and IL-12 when compared to control mice. In contrast, very little IL-4 and IL-10 was noted following splenocyte restimulation. These results suggest that vaccination with the the r Ad-VP2 and r Ad-VP2-IRES-VP5 may induce pronounced Th1 cellular immune responses. Serum antibodies elicited by r Ad-VP2 and r Ad-VP2-IRES-VP5 vaccination also showed significantly higher levels of virus neutralizing activity in vivo and in vitro as compared to serum from control mice.Sheep were immunized with the r Ad-VP2 and r Ad-VP2-IRES-VP5. The results showed that significantly higher levels of virus-specific Ig G and Ig A antibodies were detected from one week after the second immunization when compared to control sheep. The results of lymphocyte proliferation showed that r Ad-immunized sheep displayed significantly higher proliferative responses and secreted significantly higher levels of IFN-γ, TNF-α, IL-2 and IL-12 when compared to control sheep. In contrast, very little IL-4 and IL-10 was noted. These results suggest that vaccination with the r Ad-VP2 and r Ad-VP2-IRES-VP5 may induce pronounced Th1 cellular immune responses. Serum antibodies elicited by r Ad-VP2 and rAd-VP2-IRES-VP5 vaccination also showed significantly higher levels of virus neutralizing activity in vivo and in vitro as compared to serum from control sheep.In the present study, detection of BTV serogroup(including BTV-1 to 24) by One-step RTPCR and BTV-1 to 24 and 26 by nested RT-PCR were established successfully. Identification and differentiation of Chinese domestic epidemic BTV strains(BTV-1,-2,-3,-4,-12,-15 and-16) by RT-PCR and typing of BTV-1~19, 22~24 by real-time RT-PCR were also established successfully. These nucleic acid detection methods had the potential for the early diagnosis and epidemic surveys of BTV, but also providing the theoretical to the vaccination programmes of BT. At the same time, this study successfully constructed r Ad-VP2 and r Ad-VP2-IRES-VP5. And both of them can elicited humoral and cellular immune response with significantly higher levels of neutralization antibodies in mice and sheep. These vaccines contributed a foundation for prevent and control of BT. |
PDF Full Text Request