Feline Infectious Peritonitis Virus Nsp1/nsp15 Mutant Strain Construction And Evaluation Of Pathogenicity And Immune Protection

Feline Infectious Peritonitis(FIP)is a fatal feline disease caused by Feline Infectious Peritonitis Virus(FIPV).However,there is no effective vaccine for it.In this research,three mutant strains,named QS-79dnsp15,79dnsp1 and 79dnsp1/15,respectively,were constructed through the reverse genetics operating system.Moreover,their pathogenicity and immune protection were evaluated.This work provided reference for FIPV vaccine or drug research.1.Construction of FIPV mutant virusesPrevious studies have shown that nsp1 and nsp15 are FIPV virulence proteins.Our group currently has two FIPV infectious cloning platforms: QS-79 BAC and 79-1146＿CA BAC.In this study,nsp1 and nsp15 of FIPV were mutated by CRISPR/Cas9 system and homologous recombination technology,and three FIPV mutant plasmids were successfully constructed.The corresponding mutant strains(QS-79dnsp15,79dnsp1 and79dnsp1/15)were successfully rescued by transfecting the plasmids into CRFK cells,which were verified by indirect immunofluorescence,western blotting and sequencing.Growth kinetics experiments showed that the growth curves of the three mutant strains were similar to the parental strains,and the proliferation ability was good.2.Pathogenicity evaluation of mutant virusesThe three FIPV mutant viruses were orally infected into adult cats to verify their pathogenicity.Infected with QS-79dnsp15,adult cats developed obvious clinical symptoms and the mortality rate was 100%.The result shows that the pathogenicity of the QS-79 is not weakened.Infected with 79dnsp1 or 79dnsp1/15 respectively,the adult cats had no obvious clinical symptoms and no death,and the result shows that 79dnsp1 and 79dnsp1/15 are not pathogenic.3.Immune protection evaluation of mutant virusesAfter immunized 3 times with 79,79dnsp1 and 79dnsp1/15 respectively,adult cats were infected with virulent strains to evaluate their immune protection.The result shows that after 3 times of immunization,the cats in the three groups could produce high neutralizing antibodies,and then they were challenged with the strong pathogenic strain(QS-79),and the mortality rate was 100%.However,compared with the control group that was not immunized and directly attacked QS-79,the 79 group and the 79dnsp1/15 group had later onset,less symptoms,and longer life cycle,while 79dnsp1 did not have this clinical manifestation.The above result indicates that although the high neutralizing antibodies can’t completely protect adult cats from highly pathogenic FIPV infection,they may delay the onset time,alleviate their clinical symptoms,extend its life and provide valuable treatment time for cats with FIP.