Construction And Application Of Human Enterovirus D68 Reverse Genetics System

Human enterovirus D68(EV-D68)is a member of the Picornaviridae family.It was first isolated from the pediatric patients in California,USA,in 1962.EV-D68 can cause severe respiratory diseases in children,and severe patients may have acute flaccid paralysis such as nervous system diseases.In recent years,EV-D68 infections have been reported in many countries around the world.In 2014,a large EV-D68 outbreak caused serious respiratory diseases and neurological complications in US,with 1153 confirmed cases and 14 deaths.Unfortunately,no effective medications or prophylactic vaccines are currently available for controlling EV-D68 infections.It is important to study the molecular biological characteristics,replication mechanism and pathogenic mechanism of EV-D68.The use of reverse genetics technology can solve the problem that RNA virus is difficult to operate.By modifying the viral genomic at the level of cDNA,we can study the structure and function of viral genome,and provide a pathway for the study of virus replication mechanism and pathogenic mechanism.In this study,we describe an EV-D68 reverse genetics system for the EV-D68 original prototype Fermon strain,based on the use of Pol I promoter.Both,a minireplicon and infectious full-length cDNA were cloned.The minireplicon is constructed by replacing the EV-D68 coding region with firefly luciferase reporter gene,and we find RNA polymerase I(pol I)was more efficient than T7 RNA polymerase to rescue minireplicon.Additionally,our result show that the translation of EV-D68 virus protein depends on the function of the viral non-coding region,and a shorter poly(A)tails affect the transcription of the minireplicon.Moreover,we successfully constructed EV-D68 infectious cDNA clone based on the Pol I polymerase.The transfection of recombinant plasmid to RD cells can be used to rescue virus.Using sequencing analysis and restriction analysis to determine that the rescue virus was rescued from the derived plasmids.By comparing the plaque morphology and growth kinetics of rescued virus and parentalvirus,we found that the mutation of G394 C reduce the translation of viral protein and the decrease the proliferation of virus.The establishment of reverse genetic system lays the foundation for finding the virulence sites of EV-D68,and opens up new approaches to the design of genetically modified anti-EV-D68 vaccines.