Development and evaluation of an efficient cell-culture system for Hepatitis E virus
Development and evaluation of an efficient cell-culture system for Hepatitis E virus
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DOI：
10.1099/vir.0.82535-0
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发表时间：
2007-03-01
影响因子：
3.8
通讯作者：
Okamoto, Hiroaki
中科院分区：
文献类型：
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作者：
Tanaka, Toshinori;Takahashi, Masaharu;Okamoto, Hiroaki
利用高负荷戊型肝炎病毒（HEV）（2.0 × 10（7）拷贝ml（-1），基因型3）的粪便悬浮液，我们建立了一个高效的肝癌细胞系（PLC/PRF/5）中HEV的细胞培养系统。在PLC/PRF/5细胞中连续传代5次。接种后培养上清中检出HEV的起始天数和载量取决于接种物中种子病毒的滴度。当将6.4 × 10（4）拷贝的HEV接种到6孔微孔板中的PLC/PRF/5细胞单层上时，接种后第14天在培养基中首次检测到HEV RNA，并在第60天增加至9.1 × 10（5）拷贝ml（-1）。当接种8.6 × 105拷贝的HEV时，在第12天首次检测到HEV RNA，并在第60天达到最高滴度8.6 × 107拷贝ml-1。在高于70 ℃的温度下孵育的HEV在PLC/PRF/5细胞中不生长，而在56 ℃孵育30分钟的HEV具有感染性。从感染基因型1、3或4的HEV的患者获得的具有IgM类HEV抗体的恢复期血清样品中和基因型3病毒，表明HEV抗体具有广泛的交叉反应性。从HEV感染发作后8.7或24.0年的患者中获得的血清样品也阻止了PLC/PRF/5细胞中HEV的传播，这表明在过去HEV感染的个体中存在具有中和活性的持久HEV抗体。
Using a faecal suspension with high load of Hepatitis E virus (HEV) (2.0 x 10(7) copies ml(-1), genotype 3), we developed an efficient cell-culture system for HEV in a hepatocarcinoma cell line (PLC/PRF/5). HEV progeny released in the culture medium were passaged five times successively in PLC/PRF/5 cells. The initial day of appearance and load of HEV detectable in the culture supernatant after inoculation were dependent on the titre of seed virus in the inoculum. When 6.4 x 10(4) copies of HEV were inoculated on monolayers of PLC/PRF/5 cells in six-well microplates, HEV RNA was first detected in the culture medium on day 14 post-inoculation and increased to 9.1 x 10(5) copies ml(-1) on day 60. When 8.6 x 10(5) copies of HEV were inoculated, HEV RNA was initially detected on day 12 and reached the highest titre of 8.6 x 107 copies ml-1 on day 60. HEV incubated at temperatures higher than 70 degrees C did not grow in PLC/PRF/5 cells, while HEV incubated at 56 degrees C for 30 min was infectious. Convalescent serum samples with IgM-class HEV antibodies obtained from patients infected with HEV of genotype 1, 3 or 4 neutralized the genotype 3 virus, indicating that HEV antibodies are broadly cross-reactive. Serum samples obtained from patients 8.7 or 24.0 years after the onset of HEV infection also prevented the propagation of HEV in PLC/PRF/5 cells, suggesting the presence of long-lasting HEV antibodies with neutralizing activity in individuals with past HEV infection.