In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication.
In vitro assembled, recombinant infectious bronchitis viruses demonstrate that the 5a open reading frame is not essential for replication.
复制标题
DOI：
10.1016/j.virol.2004.10.045
复制
发表时间：
2005-02-05
期刊：
影响因子：
3.7
通讯作者：
Collisson EW
中科院分区：
文献类型：
--
作者：
Youn S;Leibowitz JL;Collisson EW
用体外组装法构建了鸡传染性支气管炎病毒（IBV）Vero细胞Beaudette株的分子克隆。将从包含IBV全基因组的7个cDNA片段构建的cDNA模板体外转录的RNA电穿孔到BHK-21细胞中。将细胞覆盖在易感Vero细胞上，并从分子克隆中回收活病毒。分子克隆的IBV（MIBV）表现出生长动力学，噬斑大小和形态，类似于父母Beaudette株IBV。通过用增强型绿色荧光蛋白（EGFP）ORF替换组特异性基因ORF 5a的开放阅读框（ORF），进一步操作重组病毒以表达增强型EGFP。与亲本病毒和MIBV相比，拯救的表达EGFP的重组病毒（GIBV）复制至较低的病毒滴度并形成较小的噬斑。GIBV传代6次后，观察到少数噬斑恢复为较大的噬斑大小，并且来自这些噬斑的病毒不再表达EGFP。直接测序的RT-PCR产物来自感染的细胞与空斑纯化的病毒，其中失去了表达的EGFP，证实了损失的EGFP ORF。EGFP表达的丧失（Δ5a IBV）还伴随着类似于标准病毒和完整重组病毒的生长动力学的逆转。该研究表明，5a ORF对于病毒在Vero细胞中的增殖不是必需的。
Molecular clones of infectious bronchitis virus (IBV), derived from the Vero cell adapted Beaudette strain, were constructed, using an in vitro assembly method. In vitro transcribed RNA from a cDNA template that had been constructed from seven cDNA fragments, encompassing the entire genome of IBV, was electroporated into BHK-21 cells. The cells were overlaid onto the susceptible Vero cells and viable virus was recovered from the molecular clone. The molecularly cloned IBV (MIBV) demonstrated growth kinetics, and plaque size and morphology that resembled the parental Beaudette strain IBV. The recombinant virus was further manipulated to express enhanced green fluorescent protein (EGFP) by replacing an open reading frame (ORF) of the group-specific gene, ORF 5a, with the EGFP ORF. The rescued recombinant virus, expressing EGFP (GIBV), replicated to lower viral titers and formed smaller plaques compared to the parental virus and the MIBV. After six passages of GIBV, a minority of plaques were observed that had reverted to the larger plaque size and virus from these plaques no longer expressed EGFP. Direct sequencing of RT-PCR products derived from cells infected with the plaque-purified virus, which had lost expression of EGFP, confirmed loss of the EGFP ORF. The loss of EGFP expression (Δ5a IBV) was also accompanied by reversion to growth kinetics resembling the standard virus and intact recombinant virus. This study demonstrates that the 5a ORF is not essential for viral multiplication in Vero cells.
登录
查看更多内容
影响因子：
5.4
作者：
Sola, I;Alonso, S;Enjuanes, L
通讯作者：
Enjuanes, L
影响因子：
5.4
作者：
Thiel, V;Karl, N;Siddell, SG
通讯作者：
Siddell, SG
影响因子：
5.6
作者：
Snijder EJ;Bredenbeek PJ;Dobbe JC;Thiel V;Ziebuhr J;Poon LL;Guan Y;Rozanov M;Spaan WJ;Gorbalenya AE
通讯作者：
Gorbalenya AE
影响因子：
5.4
作者：
COMPTON, SR;ROGERS, DB;MCGOWAN, JJ
通讯作者：
MCGOWAN, JJ
影响因子：
5.4
作者：
Bosch, BJ;van der Zee, R;Rottier, PJM
通讯作者：
Rottier, PJM