Attenuation And Infectious Clone Construction Of Feline Calicivirus

Feline calicivirus（FCV）is one of the most common pathogens in domestic cats worldwide.It can infect cats with oral ulcers,upper respiratory disease and lameness,which is called feline infectious rhinoconjunctivitis.Felines is generally susceptible to FCV.which may cause a long-term release of quantities of virus.At present,as for the prevention and treatment of FCV,the most effective solution is vaccination,which can reduce the incidence of severe clinical diseases.However,the constant emergence of highly infectious and transmissible FCV variants was associated with considerable reductions in the protective efficacy of the current vaccines.In recent years,the emergence of virulent systemic strains of FCV（VS-FCV）has caused multiple outbreaks with a fatality rate of40%,creating a serious health concern worldwide.Therefore,it is urgent to study the variation mechanism of FCV and develop new vaccines.In this paper,we studied the attenuation of FCV-S7 via cell culture passage and construct the infectious clone of FCV strains,which laid a foundation for the research and development of FCV vaccine and the mechanism of attenuation.The specific research content and results are as follows.1.Attenuation of FCV-S7 via cell culture passageFirstly,FCV-S7,which was independently isolated and identified in the laboratory,was passed for 150 successive generations in F81 cells.Then,we selected F10,F40,F60,F80,F100,F120,F130,F140 and F150 viruses for whole genome sequencing,compared those strains with parent virus（F3）in the aspects of the nucleotide and amino acid.At the same time,we measured and compared the plaque diameter of each generation of virus,evaluating the infectious titer of each generation of virus by multistep growth curve.The results showed that F150 had 16 amino acid mutations in the whole genome sequence,which were mainly located in the coding region of non-structural protein P30,Pro-Pol and structural protein VP1.With the increase of generation,the diameter of the plaque of FCV on F81 cells was larger,the average diameter of the plaque of F150 strain was 1.71 times of the parent virus,and the infectious titer was also significantly increased.When0.001MOI F150 virus infected F81,the titer was up to 8.5 Lg TCID₅₀/m L.Based on above results,we carried out the pathogenicity study of passaged virus FCV-S7-F150.Groups of3 cats were intranasally with 1ml of 10^8.75TCID₅₀/m L per cat for each virus.And then assess the virulence phenotype of the passaged virus through observing the manifestations and detoxification of the cats.The results showed that that the parent virus could cause clinical symptoms such as fever,mental depression,eye purulent secretions,tongue and face swelling and ulceration and even death in cats,the detoxification was continuous.On the contrary,cats inoculated with F150 virus did not show any clinical signs,with body temperature increasing in the normal range.The form of virus-shedding was intermittent.These results indicated that the virulence of F150 to be fully attenuated,which will be developed as an attenuated live vaccine.2.The construction of infectious full-length c DNA clone of the FCV-S7 genomeFirst,getting segment c DNA of FCV-S7 by extracting RNA and RT-PCR.According to the characteristics of the cleavage site of the FCV-S7 genome,the whole FCV-S7 genome was divided into two segments（G1 and G2）for amplification.G1 and G2 are cloned and connected to the modified infectious clone vector p BR322 to construct infectious clone.Then,introducing genetic markers to distinguish the rescue virus from the parent virus The virus was rescued by two methods:in vitro transcription and in vivo transcription.The rescued virus was identified through cytopathic observation,indirect immunofluorescence trial,sequencing and find the differences between the rescued virus and the parent virus in growth curve.The results showed that rescue FCV-S7（r FCV-S7）with genetic markers was successfully rescued and r FCV-S7 had similar growth curve and the diameter of plaque as the parent virus,which proving the feasibility of this reverse genetic system.It provides a powerful tool for the subsequent research on FCV.In conclusion,we got an attenuated FCV-S7 virus via cell culture passage,which will be expected to be developed as an attenuation vaccine.And we constructed infectious full-length c DNA clone of the FCV-S7 genome.