Construction And Application Of An Infectious CDNA Clone For Rabies Virus CVS-11Strain

Rabies virus (RABV), strict neurotropism virus, induces fatal encephalomyelitisin all warm-blooded animals and humans. Globally, an estimated55,000humandeaths are attributed to RABV annually, including3,000cases in China. Dogs arethe principal reservoir in Africa and Asia. The majority of human rabies victims areinfected by the bites of domestic dogs. A study from the World Health Organization(WHO) showed that70%immunization of the dog population could efficiently blockrabies virus transmission. Elimination of canine rabies is necessary for the control ofhuman rabies in developing countries. Virus-neutralizing antibody (VNA) plays animportant role in protecting against RABV. In order to eradicate rabies, on the onehand, we have to strengthen the management of dogs, immune dogs routinely, andimprove the quality of our animal rabies vaccine. On the other hand, it is necessaryto monitor timely immunization coverage of rabies neutralizing antibody in dogs andeliminate stray dogs. Fluorescent antibody virus neutralization (FAVN) and the rapidfluorescent focus inhibition test (RFFIT) approved by the Office International DesEpizooties (OIE) and WHO, respectively, have been widely used to measure levelsof VNAs. Both methods require high-quality, expensive fluorescent isothiocyanate(FITC) labelled monoclonal antibody, therefore can not be spreaded in thedeveloping world. Due to poor quality but expensive animal rabies vaccine indomestic, it is important for prevention and control of rabies to develop aconservation and accurate VNA test and high efficiency and affordable animal rabiesinactived vaccine.With the development of transcribed RNA technology in vitro, many reversegenetics of animal RNA viruses were also frequently created, which allow to operatetheir relatively stable cDNA and do further studies from the molecular level in recenttwo decades. The first infectious clone for RABV SAD B19strain is made by Schnell in1994. Scientists have pursued constantly to improve the reverse geneticsystem, and increase the efficiency of virus rescue. Several RABV strains, includingthe attenuated and vaccine strains, have been successfully rescued during20years.The recovery of virulent RABV strainis more difficult for the viruses are poorlyadapted to the cells in vitro. Therefore, construction of infectious cDNA clone forRABV virulent strains is still relatively a few at home and abroad. RABV CVS-11was isolated from a mad cow in1882in Paris, France, and then passaged in rabbitbrain, murine brain and cells. Nowadays, CVS-11strain is a laboratory pathogenicfixed strain. The CVS-11strain is widely used for antibody detection, immunizationevaluation of RABV vaccines, and challenge strain approved by WHO and OIE.Furthermore, the CVS-11strain is approved as an inactivated vaccine strain by theMinistry of Agriculture in China. Given the CVS-11strain has been widely practicalapplication, it is important to build reverse genetics platform for CVS-11strain.Therefore, an infectious cDNA clone for CVS-11strain was explored to develop forits applicant in RABV detection and vaccines. The main contents are as follows:First, development of an infectious cDNA clone for RABV CVS-11strain.To obtain the complete genome of CVS-11strain and establish a reverse geneticsystem of CVS-11for in-depth studying RABV detections and vaccines,12fragments repeated each other was amplified by RT-PCR to cover the completegenome of CVS-11, and then cloned to pEASY-Blunt vector for sequencing thecomplete genome of CVS-11. Four pairs of specific primers were designed and wereused for amplification of the full-length cDNA of CVS-11by RT-PCR. Fourfragments were cloned into pcDNA3.1step by step and the full-length plasmid wasnamed pcDNA3.1-CVS-11. We also cloned help plasmids pcDNA3.1-N, P, L and Gexpressing N, P, L and G protein of CVS-11strain. Full-length and four helpplasmids were co-transfected NA cell and then rescued virus. We confirmed theresuced CVS-11by fluorescence antibody test (FAT), RT-PCR. The rCVS-11has thesame growth characteristics with parental strain wtCVS-11in NA cells. Wesuccessfully established an infectious cDNA clone for RABV CVS-11strain. Second, generation of recombinant RABV CVS-11expressing eGFP appliedtothe rapid virus neutralization test.A recombinant rabies virus rCVS-11-eGFP stably expressing eGFP is generatedbased on an infectious clone of wtCVS-11strain.Compared to the rCVS-11strain,the rCVS-11-eGFP strain showed a similar growth property with passaging stabilityin vitro and pathogenicity in vivo. The eGFP gene doesn’t affect growth property andpathogenicity of rCVS-11.The rCVS-11-eGFP strain was utilized as a detectionantigen to determine the levels of rabies VNAs in23human and29canine sera; thistechnique was termed the FAVN-eGFP method. The good reproducibility ofFAVN-eGFP was tested with partial serum samples and showed96.6%agreement.The FAVN-eGFP method did not need a FITC-conjugated monoclonal antibody toidentify positive or negative cells. However, good-quality FITC-conjugated anti-Nprotein monoclonal antibodies are scarce in developing countries. Additionally, theFITC-conjugated anti-N protein monoclonal antibody requires a good transportenvironment or is likely to be degraded during long distance transport. Therefore, theFAVN-eGFP allows rapid economical, specific, and high-throughput assessment forthe titration of rabies VNAs.Third, an inactivated recombinant rabies CVS-11virus expressing twocopies ofthe glycoprotein elicits a higher level of neutralizing antibodies and provides betterprotection in mice.We generated a recombinant rCVS-11-G strain containing two copies of the Gprotein derived from the pathogenic wild-type (wt) CVS-11strain and based on itsinfectious clone. Compared with the wtCVS-11strain, the rCVS-11-G strainpossessed a larger virion and1.4-fold more G protein, but it exhibited asimilargrowth property to the rCVS-11strain, including passaging stability in vitro. qPCRresults showed that the two G genes were over-expressed in BHK-21cells infectedwith the rCVS-11-G strain. However, the rCVS-11-G strain presented an80%lowerLD50than the wtCVS-11strain when intracranially (i.c.) inoculated in adult mice.Adult mice that were either intracranially (i.c.) or intramuscularly(i.m.) inoculated with rCVS-11-G strain developed moreacute neurological symptoms and greatermortality thanthose inoculated with the wtCVS-11strain. Furthermore, therCVS-11-G strain was more easily and rapidly taken up by NA cells. These dataindicated that the rCVS-11-G strain might have increased neurotropism because ofthe over-expression of the pathogenic G protein.The inactivated rCVS-11-G straininduced significantly higher levels of virus neutralization antibodies and providedbetter protection from street rabies virus challenge in mice. Therefore, therCVS-11-G strain may be a promising inactivated candidate vaccine strain due to itsbetter immunogenicity.