Construction And Verification Of Recombinant Canine Distemper Virus Expressing IL-7

Canine distemper is a widespread infectious disease in dog,raccoon and others carnivores.It was caused by CDV,a morbillivirus of family Paramyxoviridae.CD is highly infectious,which seriously affects the development of dog industry and fur animal husbandry.The extensive use of live attenuated CDV has greatly reduced the outbreak of canine distemper,but some cases of canine distemper infection in immunized dogs are still reported.The immune effect of the live attenuated canine distemper vaccine is interfered by the maternal antibody.In addition,the susceptibility of these susceptible animals to these vaccines is also different,which may lead to the occurrence of encephalitis and other adverse reactions.Therefore,it is essential to develop a safe and efficient vaccine to control the epidemic of canine distemper.CDV,a non-segmented single strand negative strand RNA virus,can package and contain stable and express large fragment of foreign protein gene.Under natural conditions,there is little or no gene exchange phenomenon.The above biological characteristics suggest that CDV can be used as a good vaccine carrier.In this study,a full-length cDNA infectious clone pAC-CDV11 was constructed by using canine distemper attenuated vaccine strain CDV11 as the maternal virus.The recombinant canine distemper virus rCDV11 was successfully rescued,and the reverse genetics operation platform of the strain was established.The infection ability and growth characteristics of rCDV11 on Vero cells were not significantly different from that of the parent virus strain CDV11.In order to find the appropriate insertion site within CDV11 genome for the foreign genes,we inserted GFP into the non-coding region between different genes of CDV11,rescued the recombinant CDV,and evaluated the ability to express foreign proteins at different locations.The results showed that rCDV-P/GFP/M with GFP inserted between the P and M genes has the similar biological characteristics to parental rCDV11 in Vero cells,which can express GFP in a stable and efficient way in Vero cells.These results indicated that the CDV11 has the potential to serve as a live vaccine vector and the non-coding region between the P and M genes can be used as a suitable insertion site for exogenous gene.Interleukin-7?IL-7?plays an important role in regulating the growth and development of lymphocytesand,which plays an important role in a variety of virus-induced immune response.Next,we constructed a recombinant CDV expressing murine IL-7,rCDV-IL7.Its growth characteristics in Vero cells were not significantly different from that of rCDV11,indicating that the expression of IL-7 did not affect the replication and diffusion of recombinant CDV.The rCDV-IL7 was continuously transmitted to the 10 generation in Vero cells.The titer of each generation was between 10^6.0and 10^6.5TCID₅₀/mL,indicating that the rCDV-IL7 could be stable in Vero cells.The results of ELISA showed that rCDV-IL7 could express IL-7 in Vero cells and had a dose-dependent manner.The cell activity test showed that the expression of IL-7 had no toxic and side effects in Vero cells.To assess the pathogenicity of recombinant CDV,mice were immunized through an intramuscular route or an intracerebral route with the recombinant CDV.No clinical signs were observed in any of the infected mice.The body weight changes of mice infected with rCDV-IL7 were similar to those of mice infected with rCDV11.These results demonstrated that the expression of IL-7 gene did not increase the virulence of the CDV vector in mice.To further investigate the immunogenicity of rCDV-IL7,mice were i.m.inoculated with 100?L 10⁵ TCID₅₀0 of rCDV-IL7 and rCDV11.At 3 weeks after initial vaccination,the mice received a second vaccine dose,respectively.The blood from each group was collected every week from 1 to 10 weeks post immunization to detect the level of virus neutralizing antibody.The VNA test results showed that the immune response of these two groups rapidly increased after strengthening the immunization.Compared with rCDV11,rCDV-IL7 induced a higher titer of virus neutralizing antibody in mice.In summary,we successfully established a reverse genetics system of the CDV vaccine strain CDV11,and constructed the rCDV-IL7.The rCDV-IL7 has good biological safety and immunogenicity.It can stimulate mice to produce high level neutralizing antibodies against CDV.It is expected to become an effective vaccine strain of CDV.