| Peste des petits ruminants (PPR) and Bluetong (BT) are severe and highly contagious diseases of wild and domestic ruminants (goats and sheep), which caused by Peste des petits ruminants virus (PPRV) and Bluetong virus (BTV) respectively. Until now, there are no effective medical therapy have been established for Peste des petits ruminants and Bluetongue.Reverse genetics is new viral technology and adopt a research route from gene to characteristics. It became a reliable laboratory method that can be used to study lots of economically important viruses and provided a powerful tool for studying structure and function of viral genome, the role of viral proteins in pathogenicity, virus assembly and interaction of viral proteins with compomemts of the host cell immune reponse, especially for application of RNA virus as a novel vaccine vector. Although minigenome rescue of PPRV has been described in 2007, several groups have attempted to establish the reverse genetics system for PPRV. However, the attempts have been unsuccessful to date. For serologically distinguishing vaccinated animals from those that have recovered from natural infection, we have used this reverse genetics technique to construct viruses expressing two different types of a tracer protein (green fluorescent protein, GFP) and improved the virus neutralization test. At the same time, recombinant viruses expressing bluetongue proteins provides a useful platform for development of novel polyvalent vaccine for BT. Part ?Development of PPRV reverse genetics according to full-length sequence by GeneBank and constructed a cDNA clone which was inserted GFP gene between P and M gene then named pN75/l-GFP. Transfected with the helper plasmids pCA-N (2 ?g), pCA-P (1?g) and pCA-L (1?g) together with 4 ?g of pN75/1-GFP using Lipofatamine2000. The growth curves of rPPRV/GFP strain was similar to widetype PPRV Nigeria75/1 in Vero cells as high as 107.4 TCID50/mL. Analysis of IFA and western-blot showed that recombinant virus expressed antigens of itself and GFP; meantime, during 10 successive passages in Vero cells, the recombinant virus stably kept the expression of GFP and virus titer. Three groups of four goats each were vaccined intramuscularly with 103 TCID50 of rPPRV/GFP, wtPPRV/N75/1 and PBS. The sera were collected at 0 (pretest),2,4 and 6 weeks after vaccination and viral neutralization antibody titers against PPRV were tested. VNA titers were not detected from any animals pretest, and VNA was detected at all other time points from the animals inoculated with the rPPRV/GFP, wtPPRV/N75/1 viruses. The titers among the three time points and the two groups were not significantly different (P>0.05). The PBS-inoculated goats showed negative responses to PPRV (VNA titer<10). The results indicated that the insertion of a foreign gene between the P and M genes dose not affect the immunogenicity of the parent PPRV.Part ?It is clear that the exisiting vaccine is highly effective in protecting animals from Peste Des Petits infection, it has a drawback in that vaccinated animals are serologically indistinguishable from those that have recovered from natural infection, it would be desirable to use a marker vaccine that enables this distinction to be made.In this study, we modified GFP gene which was introduced signal sequence of tPA and anchor sequence of influenza virus HA protein at 5'and 3'end respectively. Then introduced the modified GFP gene into full-length cDNA clones of PPRV/N75/1 to rescue a marked recombinant PPRV. The recombinant virus named rPPRV/GFP-SA has a similar biological and immunological characteristics with wide type PPRV, the virus titer can reached a peak of at 5th day in Vero cells, and also expresses for exogenous protein via western-blotting. The modified GFP was efficiently expressed by Vero cells and was localized to the surface of infected cells. When five sheep were inoculated with 105 TCID50 dose of rPPRV/GFP-SA and rPPRV/GFP by subcutaneous respectively, the PPRV neutralization antibody titers among the two time points and the two groups were not significantly different (P>0.05). There is a linear correlation between PPRV antibodies and GFP antibodies in some extent, possibly the needle of syringes through the skin resulting in vaccine failure. In contast, no anti-GFP antibodies were produced in the five sheep vaccinated with rPPRV/GFP, only one sheep showed a detectable level antibody after priming immunity but decreased quickly.Part ?Antigen of Bluetongue virus (BTV) on VP2 are responsible for virus neutralization, although VP5 may affect indirectly neutralization through its conformational influence on VP2. VP3 together with VP7 form inner capsid, VP7 may induce a protective and serotype cross reactive immune response to BTV (possibly via a cell mediated mechanism), meanwhile, VP2, VP5, VP7 contain cytotoxic T-lymphocyte epitopes.Peste des petits virus (PPRV) is the promising and suitable live vaccine vector for the control and prevention of infectious diseases in dometic and wild small ruminants. In this study, the gene encoding the VP2, VP3, VP5 and VP7 protein of the bluetongue virus of serotype 12 was inserted into the site between P and M of in full-length genomic cDNA of PPRV N75/1 for expression then named pN75/1-BTV12-VP2/VP3/VP5/VP7. The full-length cDNA plasmid was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large polymerase protein into Vero cells. Recused rPPRV/BTV12-VP5 and rPPRV/BTV12-VP7 replicated to a titer similar to that of parental PPRV strain Nigeria75/1 in Vero cells. Analysis of IFA and Western-blot showed that recombinant virus expressed antigens of VP5, VP7 and itself. Administration of 107TCID50 dose of rPPRV/BTV12-VP5 into goats twice, the VNA levels were similar to paretal virus introduced (P>0.05), but antibodies to VP5 protein wasn't significant high. For time-limitting, recombinant virus expressing VP7 protein didn't carry out animal test, at the same time, the recombinant viruses expressing VP2 or VP3 proteins haven't rescued for some reasons. Our research on recombinant viruses provides a useful platform for development of novel polyvalent vaccine for BTV.Part ?The measurement of virus-netralizing antibody (VNA) against PPRV is an indispensable and important technology to determine the levels of neutralizing antibodies. Without such a test, it is virtually impossible to evaluate new vaccines or to carry out serosurvey. Vitrus neutralization test which is considered the gold standard for the detection of antibodies to PPRV, was recommended by OIE. The currently used VNT which is sensitivity, specificity and reliable however, is time-consuming to perform. Usually the inhibition of virus replication is assessed by monitoring cytopathologic changes in cell culture, especially need skilled investigator to observe CPE.In this study, a recombinant PPRV vaccine strain carrying a green fluorescent protein (GFP) gene, rPPRV/GFP, was used as a challenge virus in the virus neutralization test. Expression of the GFP could be readily detected in the cells under a fluorescent microcope. The CPE can be observed with the help of GFP fluorescence as early as 4th day, and VNT results could be available as early as 6th day. Thus,8 days could be saved compared with traditional methods using wtPPRV/N75/1. Meantime, viral CPEs could be distinguished easily from CPE-like cell death caused by serum with the help of GFP fluorescence, even though limited cells are infected by rPPRV/GFP. All sera from vaccinated 20 goats and 10 sheep were assayed for PPR VNA titer using wtPPRV/N75/1 and rPPRV/GFP, respectively, there was no statistical significant difference (P>0.05) between the VNA titer results using the two viral stains. These results show that rPPRV/GFP can be used as a rapid, specific, and reliable test for detecting PPRV-netralizing antibodied as an effective substitute for the use of native PPRV/N75/1. |
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