[Improved replication of enteric adenovirus type 41 in Hep2 cell line expressing E1B55K].
Adenovirus type 40 and 41 (Ad40, Ad41), which belong to human adenovirus subgroup F, are called fastidious adenoviruses due to their property of poor growth in cultured cell lines in vitro The effect of expression of exogenous E1B55K in Hep2 on Ad41 replication in this cell line was investigated. E1B55K gene was amplified by PCR with DNA extracted from Ad41-positive feces supernatant as template. Eukaryotic expression plasmid (pcDNA3) carrying E1B55K was constructed, purified, and transferred into Hep2 cell. Expression of E1B55K in G418-resistant clones was assayed by RT-PCR, and one clone named as Hep2-E1B4#4 could produce more Ad41 progenies when compared with other clones by the method of inducing complete cytopathic effect (CPE) in 293 cells. Infection of equivalent Ad41 caused more significant cytopathic effect (CPE) in Hep2-E1B#4 than that in the control cells of Hep2 or Hep2-DNA3, also suggesting enhanced viral replication in Hep2-E1B#4. The titer of Ad41 was further determined by method of immunocytochemical staining, and semi-quantity PCR was employed to compare the copy number of Ad41 genome DNA. The results showed that the yield of Ad41 in Hep2-E1B#4 was more than 9 times of that in control cells when equal amount of seed viruses were incubated, and the copy number of Ad41 genome increased 4 times in the raw extract from the infected Hep2-E1B#4 when compared with that from control cells. In conclusion, E1B55K gene transfer improved the ability of Hep2 in packaging Ad41, and the Hep2-E1B#4 cell line, which expressed E1B55K constitutively, would be helpful in isolation, cultivation and amplification of Ad41.
Han
Bing-juan
BJ
Institute of Virology, Shandong Academy of Medical Sciences, Jinan 250062, China.
Guo
Li
L
Qu
Jian-guo
JG
Wang
Min
M
Wang
Jian-Wei
JW
Lu
Zhuo-zhuang
ZZ
Hong
Tao
T
D017501:Adenovirus E1B Proteins / Q000235:genetics* / Q000378:metabolism; D000260:Adenoviruses, Human / Q000235:genetics*; D045744:Cell Line, Tumor; D015870:Gene Expression; D006801:Humans; D007150:Immunohistochemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014779:Virus Replication / Q000235:genetics*
D017501:Adenovirus E1B Proteins