Construction Of Japanese Encephalitis Virus Reverse Genetics System

Japanese encephalitis(Japanese encephalitis,JE)is a mosquito-born virus caused by the Japanese encephalitis virus,which can infect people and many kinds of animals and cause irreversible neurological damage.Swine is the most important amplifying host of Japanese encephalitis virus(JEV)when virus transmit to human.Pregnant sows can be manifested as abortion,fever,stillbirth and boar appears to have orchitis.Therefore,Japanese encephalitis has become a serious public health issue.So far little is known about the life cycle and pathogenic mechanism of Japanese encephalitis virus.Reverse genetics system is a powerful tool in virus research,because reverse genetics system is easy to operate.It can shorten the viurs replication cycle,facilitate engineered virus rapid phenotypic expression.Modified virus can become candidate of vaccine or vector.It can also help us study virus life cycle,pathogenesis and transmission.However,JEV genome is unstable in E.Coli in which JEV genome suffer from deletions,insertions or frameshifts.Instability,which may cause by viral protein expression,has greatly raised the difficulty of construction of japanese encephalitis virus reverse genetics system.Aiming of construction of JEV reverse genetics system,we used several strategy to stabilize JEV genome.Virus genome was respectively cloned into two different vectors.First,we cloned 5' UTR and structural protein gene(1~2590 nucleotide)into pJET1.2 with silent mutation 90A>C?101T>G?104A>G?107A>G?1355T>C,1385A>G which could reduce the cryptic bacterial promoter activity.After constructing of pJET-NS,JEV replicon in pSh-JEV-In-EGFP which included non-structural protein gene and 3' UTR(2591~10977 nucleotide)was cloned into low copy vector pOK12.Two fragment of JEV genome was assembled by fusion PCR.Meanwhile,T7 promotor was introduced into the upstream of JEV genome.Using the full length cDNA as template,JEV genome RNA was synthesized by in vitro transcription.After that,genome RNA was transfected into BHK21BHK-21 and japanese encephalitis virus SA14-14-2 strain was successfully rescued.In summary,our study successfully construct japanese encephalitis virus reverse genetics system,making a foundation for further exploration of virus protein biological function and pathogenic mechanism.