通过基因组定量研究猪瘟病毒在细胞中的增殖特性

Replicative kinetics of classical swine fever virus in PK-15 cells

摘要:

应用间接免疫荧光、Real-time PCR和病毒感染滴度(TCID₅₀)测定技术,分别从病毒抗原、病毒基因组RNA复制水平和病毒感染滴度变化3个方面,研究了猪瘟病毒(CSFV)在PK-15细胞中增殖的特点,用猪瘟病毒石门株感染96孔板培养的细胞,1×10²个TCID₅₀/孔,间接免疫荧光检测结果显示感染后8h能检测到被荧光抗体染色的感染细胞,随感染时间的延长,出现荧光的细胞数量逐渐增多,在感染后72h,几乎所有细胞均能出现荧光。Real-time PCR结果显示在细胞感染初期的8~24h,病毒的基因组RNA复制呈加速趋势,其拷贝数在感染后72h达到高峰。此外,在感染后8h能检测到病毒基因组负链RNA转录,不过负链RNA在病毒增殖过程中维持在较低的水平。TCID50测定结果表明CSFV的感染滴度增加趋势与基因组类似,在病毒感染8h后能检测到具有感染性的子代病毒,感染滴度在8~20h之间逐渐增长,24~48h之间增长速度稍减慢,在感染后48~52h达到高峰,能在72h之内维持较高的感染滴度。

Abstract:

In order to understand the replication kinetics of classical swine fever virus(CSFV) in in vitro cells PK-15 cells were seeded in 96-well tissues culture plates. After overnight incubation at 37℃ in 5% CO₂ environment when growing to 80% confluence，the cells were infected with CSFV strain Shimen at 100 TCID₅₀ per well. At various time post infection(p.i.) the replication of the virus in the cells were analyzed repectively by detection of viral antigen using indirect immunofluorescent assay(IFA)，RNA replication using reverse transcription real-time PCR and viral production using titration of TCID₅₀. In the results of the IFA the viral antigen could be detected as early as 8hrs p.i. and at 72h hrs p.i. almost all cells showed positive staining.the real-time PCR showed that the synthesis of viral genomic RNA was gradually increased between 8-24 hrs p.i. and reached its peak at 72 hrs p.i.. However，the synthesis of negative strand RNA was maintained at a low level for a whole period of culture although it could be detected at 8hrs p.i.. Titration of TCID₅₀ demonstrated that the production of live virions increased at 8h and peaked between 48-72 hrs p.i. without significant lose of titer.