Analysis Of Virulence Of Japanese Encephalitis Chimeric Genotype Ⅰ/Ⅴ Virus And Cross-protection With Genotype Ⅰ And Ⅲ Viruses

Japanese encephalitis（JE）is an important zoonotic disease caused by Japanese encephalitis virus（JEV）.JEV can be divided into five genotypes（GⅠ,GⅠI,GⅢ,GⅣ and GⅤ）.The GⅤ Muar strain is first isolated in 1952 and has been the only known example of this genotype for more than 50 years.In recent years,GⅤ has been detected and isolated in Culex mosquitoes in China and South Korea,showing an increase in epidemic areas,there by threatening public health and animal husbandry.Previous studies have shown that GⅤ exhibits higher pathogenicity in mice than the prevalent GⅠ and GⅢ strains,and the current vaccines derived from GⅢ strains cannot provide complete protection for GⅤ.In addition,the cross-protection between GⅠ and GⅤ is known.Therefore,it is necessary to examine the cross-protection between GⅠ,GⅢ and GⅤ strainsIn this study,the reverse genetic operating system was used to generate chimeric GⅠ/GⅤ virus using the backbone of attenuated GⅠ SD12-F120 strain and the structural protein of GⅤ XZ0934 strain.The characteristics and pathogenicity of the generated chimeric GⅠ/GⅤ virus containing the structural protein of GⅤ was analyzed.The results showed that the chimeric GⅠ/GⅤ virus was rescued as identified by an indirect immunofluorescence assay.Compared with the parental GⅠ SD12-F120,the chimeric GⅠ/GⅤ virus showed higher titers and larger plaque size than GⅠ SD12-F120,Intracerebral（i.c.）and intraperitoneal（i.p.）infections of mice with the chimeric GⅠ/GⅤ virus indicated that the LD₅₀ of i.c.and i.p.was 10^-2.2PFU and 10^-1.5PFU,respectively,suggesting a high neurovirulence and neuroinvasiveness.In order to map the virulence determinants of GⅠ/GⅤ type JEV chimeric virus,we constructed the recombinant GⅠ/GⅤ type JEV chimeric virus attenuated strain.Comparison of amino acid sequences of E protein among17 GⅠ,GⅢ and GⅤ strains found that the GⅤ XZ0934 strain shared 89.2-91.6%homology with other strains and showed 42-54 residues different from other strains.Among the different residues,five（E47K,L107F,H123R,L138K,L176R）were selected based on their acid-base property,polarity and structure to analyze their role in pathogenicity.A total of six mutated GⅠ/GⅤ viruses including five single-point mutants and one five-point mutant were generated by site-directed mutation.Analysis of the characteristics of all mutants showed that all mutants replicated more efficiently than the parental GⅠ/GⅤ strain and displayed a different plaque sizes compared with the GⅠ/GⅤ virus on BHK-21 cells.Pathogenetic analysis in 3-week-old C57BL/6 mice indicated that substitution at E138 significantly reduced the neuroinvasiveness and that a combination of five substitutions resulted in a loss of the neuroinvasiveness and a significant reduction of neurovirulence.In order to explore the cross-protection efficacy among GⅠ/GⅤ type JEV chimeric virus and GⅠ,GⅢ strains,we used a mouse model to examine the cross-protection.The outcoming indicated that the attenuated GⅠ and GⅢ strains could not provide the immunized C57BL/6 mice a sufficient protection against virulent GⅠ/GⅤ type JEV chimeric virus challenge.While attenuated GⅠ/GⅤ type JEV chimeric virus provided the immunized mice a complete protection against the homologous GⅠ/GⅤ type JEV chimeric virus challenge,but not against GⅠ and GⅢ challenge,suggesting that the attenuated JEV strain can provide 100%protection against the homologous genotype strain challenge,but not the heterologous genotype strains challenge.Moreover,the neutralizing ability titers to GⅠ,GⅢ and GⅠ/GⅤ type JEV chimeric virus were significantly higher than those to the heterologousgenotypes.In summary,the GⅠ/GⅤ chimeric virus was rescued using the reverse genetic operating system and the biological characteristics and pathogenicity was analyzed.The generated GⅠ/GⅤ chimeric virus showed a high neurovirulence and neuroinvasiveness in mice.Substitution analysis indicated that E138was the virulence determinant responsible for the neuroinvasiveness of GⅠ/GⅤ type JEV chimeric virus strain,while tthere is a synergistic effect of multiple amino acid sites played a synergistical role in determination of the neurovirulence and neuroinvasiveness.The cross-protection data suggested that the attenuated GⅠ,GⅢ and GⅠ/GⅤ type JEV chimeric virus strains could not produce sufficient levels of protection against heterologous genotype virus challenge,showing a need of development of serotype based vaccines.