Selection Of Susceptible Cell System For Infection With Hepatits B Virus

Hepatitis B virus(HBV)infection is a small partially double-stranded circular DNA virus with highly exclusive liver tropism that favors replication in hepatocytes.Because of high species and tissue specificity,HBV only infect human and troglodyte.There are several hepatitis B virus(HBV)replication cell models,as follow:(1)Integrated HBV genomes into hepatocarcinoma cells,which could express hepatitis B virus replicative intermediates and secrete HBV particles,among which HepG2.2.15 cell line is most widely used.(2)Transfection with whole HBV genes into hepatocarcinoma cells.(3)Adenovirus vectors in which HBV genomes were inserted.(4)Recombinant baculoviruses to deliver HBV genomes.All of the above cell models cannot represent the life period of HBV,especially role of specific viral cell receptor and the process of infection.Human or tree shrew cells can be directly infected by HBV particles,but it is difficult to prepare the cells,and the infection efficiency is neither high enough nor sustainable.The HepaRG cell line was established by French researcher Gripon,which can be directly infected by serum containing HBV after the HepaRG cells were differentiated.The level of viral replication in infected HepaRG cells is low,and the infection efficiency is uncertain.Up to now,there is no efficient susceptible HBV infection cell model available,it is urgent to establish a cell line susceptible to natural HBV infection.Objective: To provide a cell line that is susceptible to HBV natural infection and replication.Methods: Hepatocellular carcinoma cells were obtained by separation culture and successive passage from the cancer tissues in patients with hepatocellular carcinoma.Among the cells,15 strains were confirmed with the better growth rate.The primary cells along with carcinoma cell lines stored in our lab,including HepG2,Huh7,QSG-7701 and SMMC-7721 cells,were infected with recombinant HBV-BsdR viral particles expressing Bsd resistance gene and then screened by blasticidin.A cell line(S0)was selected and subjected to another 8 rounds of screening(S1-S8).The multigeneration cells from QSG-7701-S0 to QSG-7701-S8 were infected with HBV serum.To demonstrate that the cells infectivity increased after several rounds of screening,HBsAg and HBV DNA in cell culture supernatant were measured by ELISA and RT-PCR separately whereas HBV DNA in cell lysate was measured by Southern blot.To further study the characteristics of HBV susceptible cells,HBV infectivity was observed in a dose-dependent manner by Southern blot;GFP expression was observed after the cells were infected with recombinant HBV-hrGFP viral particles.The virus replicative intermediate,HBV RNA and HBs Ag in different infected times were measured by Southern blot,Northern blot and ELISA separately.The antivirus effect of Lamivudine was observed by Southern blot..Results: Eight days after using the high titer serum of HBV to infect the cell lines,S1 and S8,the quantity of HBsAg in culture supernatant was detected by chemiluminescence.The result showed that the basical susceptibility of QSG-7701 is the highest than the other lines.QSQ-7701 cell line were chosen for final 8 rounds of selection,named S1-S8.The cells(S0?S1?S3?S5?S7?S8)were infected by HBV serum,and the amount of HBs Ag in the cell culture supernatant were quantified by chemiluminescence.The results showed that the quantity of HBsAg increased obviously,and reached 15.8ng/ml in S8.HBV DNA in supernatant was detected by the real-time PCR,and the HBV DNA levels also increased obviously.HBV DNA was detected in cell lysates by Southern Blot.HBV repliation enhanced gradually and HBV replicative intermediates were detected in high level.QSG-7701 cells were infected respectively by HBV serum of different doses and HBsAg in the medium were detected by chemiluminescence.We observed that the quantity of HBsAg were increased dose-dependently by infective virus particles.QSG-7701-S1?QSG-77-1-S8 were infected by the recombinant virus particles i.e.,HBV-hrGFP.The susceptibility enhanced obviously in S8.After infected by HBV serum,cell lysates in different time points were collected and intracellular HBV DNA was detected by Southern blot.HBV DNA replicative intermediates were observed at 6 days,and increased with time.The quantity of HBsAg in supernatants increased from 5 day after infection,peaked in 8-10 days(16ng/ml),and then gradually decreased.Southern blot demonstrated that HBV replication were enhanced by DMSO.We tested the inhibition of lamivudine on HBV replication.HBV replication was repressed dose-dependently by lamivudine.Conclusions: It was demonstrated that QSG-7701 cell line is naturally susceptible to HBV infection,and the viral replication was stable.Selected by blasticidine,the infection efficiency and replication ability increased obviously.We provided here a cell line that is naturally susceptible to HBV infection and replication-competent,which can facilitate the selection of anti-viral drugs and the investigation of HBV molecular biology.