Construction And Preliminary Application Of Human Adenovirus 55 Reporter Virus

BackgroundAdenovirus type 55(HAdV-55)is one of the most common respiratory pathogens,posing a major public health threat.Due to the rapid person-to-person transmission,HAdV-B55 has caused severe respiratory disease in individuals in schools,hospitals,and the military training facilities.The biological characteristics and pathogenesis of adenovirus type 55 remained incompletely understood.There are still no specific treatments for adenovirus infection.The only FDA-approved HAdV-4 and HAdV-7 oral vaccines are available for military use.Reporter viruses are an important tool in viral research,which can not only be used for tracking virus infections,but also for rapid assessment of human antibody levels and high-throughput antiviral screening.PurposeThis study is mainly aimed at adenovirus type 55,exploring the construction of a reporter virus expressing green fluorescent protein and firefly luciferase,and conducting preliminary applications in drug screening,especially in the evaluation of human antibody levels.Method and Results1.Amplify the homologous arms HAL and HAR at each end of the adenovirus genome,and insert into the cloning vector pAdBone through EcoRⅠ,HindⅢ,and PmeⅠ restriction endonuclease sites to construct the plasmid pAdBone-LRA.Transform Pme I-linearized pAdBone-LRA and the adenovirus type 55 genomic DNA into BJ5183 competent cells.Under the action of homologous recombinant enzymes,the full-length genome infectious clone plasmid pAd55-FL of HAdV-55 was constructed.Transfect pAd55-FLinto 293 A cells to rescue the virus rAd55.2.The upstream and downstream segments of the E3 region of HAdV-55 were successively cloned into the plasmid pEGFP-N1 to construct pEGFP-N1-E3 LR.pEGFP-N1-E3LR/Mlu Ⅰ and pAd55-FL/Avr Ⅱ was transformed into BJ5183 competent cell to achieve pAd55-d3-EGFP with the knockout of E3 and the introduction of EGFP.3.The EGFP gene in pEGFP-E3 LRA was replaced with FLuc gene to construct p Luc-d E3 LRA.pAd55-d E3-Fluc was constructed with the same method to achieve E3 region knockout and luciferase introduction.4.The rAd55-d E3-Luc reporter virus was used to apply and analyze the assessment of adenovirus neutralization antibody levels.The seropositivity rates for type 55 were 28.70% and 37.04% in Beijing and Gansu province,respectively,while the seropositivity rates for type 4 were 28.50% and 39.81%,and for type 7 were 28.24%and 37.04%,respectively.ConclusionIn this study,we established a reverse genetics system for adenovirus type 55 and successfully constructed recombinant adenovirus expressing green fluorescent protein and firefly luciferase,respectively.Based on the above-mentioned recombinant adenovirus type 55 expressing green fluorescent protein,we established a rapid visualization platform for adenovirus antiviral drug screening and tested the antiviral ability of documented cidofovir and brincidofovir.Based on the above-mentioned recombinant adenovirus type 55 expressing firefly luciferase,a rapid,efficient neutralizing antibody assay was established and applied in the assessment of population serum adenovirus antibody levels.By analyzing 1100 serum samples from healthy adult populations in Beijing and Gansu province,it was initially clear that neutralization antibody levels of HAdV-4,7 and 55 were low,which increased the risk of adenovirus outbreaks.