Construction And Optimization Of Lisa Pseudovirus Model

Rabies is a zoogenous infectious disease caused by lyssavirus.Lyssavirus does not enter the blood circulation and there are no clinical symptoms during primary infection.The disease can only be diagnosed after the onset of symptoms,such as fever,hypersalivation,difficulty swallowing,hydrophobia,paralysis etc.Once symptoms appear,the disease results in fatality.Currently there are no drugs or vaccines available to treat symptom onset.In recent years,rabies ranks among the top fatal infectious diseases in China,which poses a serious threat to people’s life and public health.Lyssavirus is a typical viral family of togavirus.The surface glycoprotein(G)is the only participation factor involved in viral entry,yet its cell surface receptors are not clear.Although it is clear that lyssavirus enter the host cell via pH dependent endocytosis,it is different from other pH dependent togavirus(such as influenza viruses,HCV,filamentous virus,etc.).Our previous research focuses on the discovery of virus entry inhibitors and their mechanisms,and has found some entry inhibitors for filovirus,coronavirus and arenaviruses.A pseudovirus model with a lentivirus core packaged by Lyssavirus glycoprotein will help understanding the mechanism of viral entry.In addition,considering the complexity of the Lyssavirus receptors and the different sensitivity to the hydrogen ion concentration,our pseudovirus model will help promote understanding of lyssavirus entry mechanism and provide valuable suggestions on the treatment of rabies.Based on this purpose,this study constructed and optimized pseudovirus infection models of the earliest seven genotypes of Lyssavirus,i.e.Rabies lyssavirus(RABV),Lagos bat lyssavirus(LBV),Mokola lyssavirus(MOKV),Duvenhage lyssavirus(DUVV)、European bat 1 lyssavirus(EBLV-1)、European bat 2 lyssavirus(EBLV-2),and Australian bat lyssavirus(ABLV).First,we constructed glycoprotein expression plasmids of the above viruses and examined their expression in eukaryotic cells.The recombinant viruses were assembled via an HIV-1-luc core packed with lyssavirus glycoproteins and the packing conditions were optimized.Finally the infectivity of recombinant viruses was investigated.The results show that the 7 recombinant virus(RABV,ABLV,DUVV,EBLV-1,LBV,MOKV and EBLV-2)glycoproteins can all achieve high level of expression in 293T cells.However only five of them(DUVV,EBLV-1,EBLV-2,LBV and MOKV)can be packaged into infectious particles.RABV and MOKV recombinant virus infectivity were affected by C-terminal His tags and by removing it,their infectivity get a 100-times increase.In this study,we optimized the recombinant virus packing conditions by changing the amount and radio of the plasmid transfection with the HIV core and glycoprotein plasmids.The results show that the optimal plasmid transfection amount in 6-well plate is 2 μg per well,and the best plasmid transfection ratios are as follows:DUVV-G:HIV-luc=1:8;EBLV-1-G:HIV-luc=1:8;EBLV-2-G:HIV-luc=1:8;MOKV-G:HIV-luc=1:8;RABV-G:HIV-luc =1:4 and LBV-G:HIV-luc =1:16,respectively.The optimized recombinant virus(DUVV-G/HIV-luc,EBLV-1-G/HIV-luc,EBLV-2-G/HIV-luc,LBV-G/HIV-luc,MOKV-G/HIV-luc and RABV-G/HIV-luc)infection signal can reach above 1 ×106 RLUs,and the luciferase activity signal value is in correlation to the amount of recombinant virus infection.Finally,this study examined the infectivity of recombinant lyssavirus on different cell lines.The results showed that human lung(A549),nerve(U87MG)and kidney(293T)cell lines are most susceptible to recombinant lyssaviruses,and their infectivities are above 1 × 106 RLUs.This is in accordance with the neurological and tissue preference of lyssavirus infections.In addition,hamster kidney cells(BHK-21)is also susceptible to most recombinant lyssaviruses,which indicates the possibility of rodents as the host of lyssavirus.In summary,this study established six lyssavirus infection models(DUVV-G/HIV-luc,EBLV-1-G/HIV-luc,EBLV-2-G/HIV-luc,LBV-G/HIV-luc,MOKV-G/HIV-luc and RABV-G/HIV-luc),and explored the infectivity of six recombinant lyssavirus on cells with different species and tissue origins.