| Japanese encephalitis(JE) is an acute epidemic disease of the central nervous system caused by infection with Japanese encephalitis virus(JEV). JEV belongs to the family Flaviviridae, genus Flavivirus and mainly affects humans, pigs, horses and ardeid birds. JEV is nuerovirulence, its live attenuated vaccin is not yet widely accepted because of some safety concerns. In order to further study the JEV pathogenicity and develop a safe and effective novel vaccine, a research on molecular mechanism of JEV virulence difference is necessary. In this study, we used JEV virulent HEN0701 strain and its attenuated virus HEN-10S3, on the basis of reverse genetics, to explore the genetic basis and the molecular mechanism of virulence enhancement or attenuation.The development of JEV infectious cDNA clones has been hampered because of the genetic instability of the cloned cDNAs while they are propagated in E. coli. Herein, a cDNA fragment of the nucleotide(nt) 1 to 2,913 of the HEN0701 genome was used to investigate factors that caused the instability of cDNA clones. The results showed that the sequences of 54 to 120 nt of the JEV genome exhibited high prokaryotic promoter activity at 37 °C, and the activity declined markedly at 25 °C. Those findings revealed that the high prokaryotic promoter activity of the 54 to 120 nt sequences of the JEV genome together with expression of JEV structural genes determined the instability of a JEV cDNA clone. Growth at room temperature may reduce the prokaryotic promoter activity of 5’-sequences of the JEV genome and could represent an effective way to improve the stability of flavivirus cDNA clones in host bacteria.By culturing cDNA clones at room temperature and using medium-copy-number pBR322 M as a vector, an infectious full-length cDNA clone pBAHC was constructed from the cloned HEN-10S3 genome. We added T7 promoter sequences to the 5’ end of HEN-10S3 genomic cDNA to launch the RNA transcripts in vitro, and then added HDVr sequences to the 3’ end to generate a natural 3’ terminus of JEV. Infectious JEV was generated after transfection with RNA transcripts derived from linearized pBAHC. The rescued virus vAHEN exhibited similar plaque morphology and growth characteristic in vitro to the parental HEN-10S3 strain, and also had little neurovirulence to mouse. The stable pBAHC together with the infectious cDNA clone of HEN0701 were two key platforms for further research on molecular mechanism of JEV virulence difference.To identify the molecular determinants for attenuation of JEV HEN0701, two chimeric viruses, vJE-H/5’CPrME(S) containing the 5’-C-prM-E region of 10S3 in the HEN0701 background and vJE-S/5’CPrME(H) containing the 5’-C-prM-E region of HEN0701 in the 10S3 background, were constructed respectively. vJE-H/5’CPrME(S) was avirulent to mouse, while vJE-S/5’CPrME(H) showed highly neurovirulence in mouse, which indicated that HEN0701 5’ untranslated region(5’ UTR) and structure protein region determined virus neurovirulence. There was only 1 amino acid in the structure protein region of HEN0701 different from that of 10S3(E-138 Glu/ Arg), indicating that the position 138 in the E protein may exhibit a determined effect on the neurovirulence of JEV.Then the E-138 Glu(E) of HEN0701 virus was mutated into acidic aa Asp(D), basic aa Arg(R) and Lys(K), neutral aa Phe(F), Ala(A) and Gln(Q), respectively. The E-138 R of HEN-10S3 was mutated into E-138 E. The recovered viruses vHE138 D, vHE138 R, vHE138 K, vHE138 F, vHE138 A, vHE138 Q and vSE138 E were examined for virulence in 21 day old Kunming mice. The mutated virues vHE138 D and vSE138 E showed significantly strong virulence; vHE138 R and vHE138 K were avirulent; vHE138 F, vHE138 A and vHE138 Q had a certain virulence. These results suggested that the E-138 was a critical residue and its acidity/ basicity property was a determinant for JEV neurovirulence.Bioinformatics analysis of the structure of HEN0701 E protein showed that E-47 residue was contiguous and linked with E-138 through hydrogen bond in different forms. In order to explore the role of E-47 on virus virulence and its relationship with E-138, next, the E-47 Asn(N) of HEN0701 virus was mutated into acidic aa D, basic aa K, neutral aa A, respectively. The virulence of recovered viruses vHE47 D, vHE47 K and vHE47 A were examined. The results showed that vHE47 D had strong virulence in mice, vHE47 A was certain and vHE47 K was little virulent, which demonstrated that the acidity/ basicity property of E-47 residue also had an effect on JEV virulence. According to the causal relationship between neurovirulence difference and genetic sequence variation among 14 recombinant viruses constructed in this study, we found that the acidity/ basicity property of E-138 and E-47 altogether affect JEV neurovirulence. The stronger the acidity of two amino acids, the stronger the virulence. If there were two acidic amino acids at E-138 and E-47 respectively, the virulence was the strongest.Taken together, in this study we concluded that: culturing JEV cDNA clones at room temperature was an effective way to improve the stability of JEV cDNA clones. In this way, we constructed a stable full-length infectious cDNA clone pBAHC from HEN-10S3 genome. Using pBAHC and the infectious cDNA clone of HEN0701, we constructed various chimeric and mutant viruses. We firstly revealed that the E-47 is a key site influencing neurovirulence, just like E-138. The acidity/ basicity property of E-138 and E-47 altogether affect JEV neurovirulence. Analysis based on crystal structure of E protein indicating that the structure containing E-138 and E-47 may be effective on JEV neurovirulence. |
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