Development of A MERS-CoV Replicon Cell Line for Antiviral Screening

Middle East respiratory syndrome coronavirus(MERS-CoV)is the causative agent of a severe respiratory disease with a high mortality of~35%.The lack of approved treatments for MERS-CoV infection underscores the need for a user-friendly system for rapid drug screening.In this study,we constructed a MERS-CoV replicon containing the Renilla luciferase(Rluc)reporter gene and a stable luciferase replicon-carrying cell line.Using this cell line,we showed that MERS-CoV replication was inhibited by combined application of lopinavir and ritonavir,indicating that this cell line can be used to screen inhibitors of MERS-CoV replication.Importantly,the MERS-replicon cell line can be used for high-throughput screening of antiviral drugs without the need for live virus handling,providing an effective and safe tool for the discovery of antiviral drugs against MERS-CoV.

DUCK HEPATITIS B VIRUS MODEL FOR SCREENING OF ANTIVIRAL AGENTS FROM MEDICINAL HERBS

The effects of the extracts of 20 Chinese medicinal herbs and an antiviral drug foscarnet on duck hepatitis B virus (DHBV) endogenous DNA polymerase (DNAp) activity were compared. The extracts of P. urinaria showed a dose-dependent inhibition on DHBV DNAp. And those of other herbs showed little inhibition effect. Primary duck hepatocyte (PDH) cultures were used for evaluating effects of the extract of P. urinaria, foscarnet and acyclovir (ACV) on DHBV, and all the drugs or the extracts showed inhibition of DHBV DNA replication. Furthermore, in vivo trials were carried out. Peking ducks infected with LJ-76 strain of DHBV were treated with the extract of P. urinaria or ACV and compared with placebo treated control ducks. The treatment results in the loss or reduction of circulating viral DHBV DNA and DHBsAg.

Development of a Mengla virus minigenome and comparison of its polymerase complexes with those of other filoviruses

Ebola virus(EBOV)and Marburg virus(MARV),members of the Filoviridae family,are highly pathogenic and can cause hemorrhagic fevers,significantly impacting human society.Bats are considered reservoirs of these viruses because related filoviruses have been discovered in bats.However,due to the requirement for maximum containment laboratories when studying infectious viruses,the characterization of bat filoviruses often relies on pseudoviruses and minigenome systems.In this study,we used RACE technology to sequence the 30-leader and 50-trailer of Mengla virus(MLAV)and constructed a minigenome.Similar to MARV,the transcription activities of the MLAV minigenome are independent of VP30.We further assessed the effects of polymorphisms at the 50 end on MLAV minigenome activity and identified certain mutations that decrease minigenome reporter efficiency,probably due to alterations in the RNA secondary structure.The reporter activity upon recombination of the 30-leaders and 50-trailers of MLAV,MARV,and EBOV with those of the homologous or heterologous minigenomes was compared and it was found that the polymerase complex and leader and trailer sequences exhibit intrinsic specificities.Additionally,we investigated whether the polymerase complex proteins from EBOV and MARV support MLAV minigenome RNA synthesis and found that the homologous system is more efficient than the heterologous system.Remdesivir efficiently inhibited MLAV as well as EBOV replication.In summary,this study provides new information on bat filoviruses and the minigenome will be a useful tool for high-throughput antiviral drug screening.

A Convenient and Biosafe Replicon with Accessory Genes of SARS-CoV-2 and Its Potential Application in Antiviral Drug Discovery

SARS-CoV-2 causes the pandemic of COVID-19 and no effective drugs for this disease are available thus far.Due to the high infectivity and pathogenicity of this virus,all studies on the live virus are strictly confined in the biosafety level 3(BSL3)laboratory but this would hinder the basic research and antiviral drug development of SARS-CoV-2 because the BSL3 facility is not commonly available and the work in the containment is costly and laborious.In this study,we constructed a reverse genetics system of SARS-CoV-2 by assembling the viral cDNA in a bacterial artificial chromosome(BAC)vector with deletion of the spike(S)gene.Transfection of the cDNA into cells results in the production of an RNA replicon that keeps the capability of genome or subgenome replication but is deficient in virion assembly and infection due to the absence of S protein.Therefore,such a replicon system is not infectious and can be used in ordinary biological laboratories.We confirmed the efficient replication of the replicon by demonstrating the expression of the subgenomic RNAs which have similar profiles to the wild-type virus.By mutational analysis of nsp12 and nsp14,we showed that the RNA polymerase,exonuclease,and cap N7 methyltransferase play essential roles in genome replication and sgRNA production.We also created a SARS-CoV-2 replicon carrying a luciferase reporter gene and this system was validated by the inhibition assays with known anti-SARS-CoV-2 inhibitors.Thus,such a one-plasmid system is biosafe and convenient to use,which will benefit both fundamental research and development of antiviral drugs.