Research For Pathogenicity Of VSV With Its Mutants And Establishment Of Antibody Assay Of Matrix Protein

Vesicular stornatitis (VS) ranks as class A infectious animal diseases by World Organization for Animal Health (OIE), which also belongs to class B animal infectious diseases in China. VS is caused by vesicular stomatitis virus (VSV). VSV genome consists of five structural protein genes. Matrix protein (M) is an important virulence factor for VSV, which can inhibit mRNA transport from nucleus outside to cytoplasm in host cells. As the result, production of type I interferon by host cells can be inhibited, whereas viruses can replicate rapidly in infected cell. Glycoprotein (G) is another important pathogenic factor, which plays a key role in budding and packaging of VSV. Engineered VSV can be a promising oncolytic virus and vaccine vector. It can be toxic specifically with tumor cells whereas safe for human due to function of type I interferon signaling. However, it has been indicated that VSV still could cause pathogenesis in animal model. For example, it could be neurotoxic when mice were injected with high titer of VSV. Therefore, the safety of VSV needs to be improved.To characterize the two recombinant VSV with mutant M or/and G proteins (VSV△M51and VSVAM51-G△28) in vivo and in vitro, we compared their ability of formation of plaque and multi-step growth curve and animal pathogenic experiments. It was proved that attenuation is due to type I interferon in host cell and reduction of viral reproduction together.Moreover, M gene was cloned and expressed in E.coli. Based on the recombinant M protein, indirect ELISA assay was established to detect antibody against M in VSV-inoculated SPF mice. Furthermore, kinetics of antibody change in VSV infected mice has been proved. Expression and purification of M protein provides a convenient way to prepare polyclonal antibody and monoclonal antibody.The research shows as followed:1. In Vero cell which is short of type I interferon signal, the area of plaque does not have significant change between VSV and VSV△M51, however, both of them are larger than VSV△M51-G△28significantly; in A549cell which contain type I interferon signal, the area of plaque of VSV is larger than the VSV△M51significantly and the VSV△M51-G△28is smaller than both of them. The result shows that the attenuation of VSV△M51-G△28with both effects of mutant M and G proteins is more obvious than that of VSV△M51.2. The multi-step growth curve in PC3cell shows that the highest titer of virus is VSV, VSV△M51is the second and VSV△M51-G△28is the last. The peak of VSV titer shows at48h post infected (p.i), however the peak of VSV△M51titer shows at24h p.i and the titer reduces after72h p.i; the tendency of VSV△M51-G△28is similar with VSV△M51. The expression of IFNβ shows the characteristic of VSV<VSV△M51-G△28<VSV△M51. The result shows that the attenuation of VSV△M51-G△28is due to type I interferon and the attenuation of virus replication.3. In the animal experiment, the lost weight of VSV and VSV△M51group is more obvious than that of PBS group. The reduction of VSV group begins with1d p.i and is about30%in6d p.i and meanwhile some mice show neurological sign; the symptom of VSV△M51group is similar with VSV group, but its weight reduce about30%in8or9d p.i, most mice have littery fur. The group of VSV△M51-G△28has nothing obvious symptoms; its weight is increasing steadily and is similar with PBS group. The result shows that the pathogenicity of VSV△M51-G△28is weaker than other viruses.4. According to M gene sequence, a pair of primers is designed to clone a stripe of690bp. The M gene and plasmid pET32a(+)are linked after digestion and then the recombinant M protein is expressed and purified and verified by SDS-PAGE and Western-blotting. The result shows that the recombinant M protein is right and has biological activity.5. ELISA method is established by using M protein. According to square titration, the best coating concentration of the M protein is12μg/ml and the dilution of primary antibody is1:5000.6. The experiment of kinetics of antibody change and body weight change shows that M protein antibody can be aroused after infection of VSV. When the dosage is106PFU, the antibody of M protein has nothing change after1week p.i and increase remarkably after2weeks p.i, the body weight reduces rapidly after1week p.i and restores after2weeks p.i and the antibody of M protein have an effective increase; however when the dosage is103PFU, both the antibody and body weight have nothing change. The result shows that expression of virus antibody is associated with emergence of pathogenicity. Low dosage of VSV can’t cause mice pathogenicity or express effective level antibody, that’s because virus does not have an effective reproduction; however, high dosage of VSV can do so.