In vivo growth of porcine reproductive and respiratory syndrome virus engineered nsp2 deletion mutants.
In vivo growth of porcine reproductive and respiratory syndrome virus engineered nsp2 deletion mutants.
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DOI：
10.1016/j.virusres.2010.07.024
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发表时间：
2010-12
期刊：
影响因子：
5
通讯作者：
Han J
中科院分区：
文献类型：
--
作者：
Faaberg KS;Kehrli ME Jr;Lager KM;Guo B;Han J
2.具有2型毒株VR-2332 nsp 2氨基酸缺失的病毒的复制动力学受损。在2型毒株VR-2332的nsp 2中含有多达403个氨基酸缺失的工程化病毒在猪中是遗传稳定的。2型毒株VR-2332感染后，观察到双相血清干扰素γ应答。先前对PRRSV毒株VR-2332非结构蛋白2（nsp 2）的研究表明，可以从高变区去除多达403个氨基酸而不丧失体外病毒活力。我们利用选择的nsp 2缺失突变体来检查体内生长。用4种nsp 2缺失突变体（rΔ727-813、rΔ543-726、rΔ324-523、r Δ 324-726）或全长重组病毒（rVR-2332）之一肌内接种幼龄猪（4头猪/组; 5头对照猪）。在接种后的不同日期收集血清样品，并通过HerdChek* ELISA、PRRSV真实的时间RT-PCR、γ干扰素（IFN-γ）ELISA和整个nsp 2编码区的核苷酸序列分析进行分析。记录每只动物的气管支气管淋巴结重量与体重的比较，并用作病毒发病机制的临床测量。结果表明，所有缺失突变体的生长不如全长重组病毒稳健，但在研究结束时，除大缺失病毒（rΔ324-726）外，所有突变体均恢复至亲本病毒RNA水平。与rVR-2332相比，接受rΔ727-813突变体的猪的淋巴结肿大显著降低。虽然猪感染rVR-2332导致血清IFN-γ水平快速升高，但感染4种缺失突变病毒中的3种产生的IFN-γ蛋白显著降低，可能是由于病毒生长动力学的差异。rΔ543-726 nsp 2突变病毒虽然生长受损，但在诱导宿主血清IFN-γ应答方面模拟rVR-2332，但表现出2周延迟。靶向测序显示，在一次猪传代后，所有缺失在编码nsp 2的区域中是稳定的。数据表明，所选nsp 2缺失突变体在猪中生长减弱，改变了血清IFN-γ（一种在PRRSV清除中功能未知的先天性细胞因子）的诱导，并指出了可能影响气管支气管淋巴结大小的结构域。
▶ Replication kinetics of viruses with Type 2 strain VR-2332 nsp2 amino acid deletions were impaired. ▶ Engineered viruses that contain up to a 403 amino acid deletion in nsp2 of Type 2 strain VR-2332 were genetically stable in swine ▶ A small deletion virus was shown to reduce lymph node hyperplasia. ▶ A biphasic serum interferon γ response was seen after infection with Type 2 strain VR-2332. Prior studies on PRRSV strain VR-2332 non-structural protein 2 (nsp2) had shown that as much as 403 amino acids could be removed from the hypervariable region without losing virus viability in vitro. We utilized selected nsp2 deletion mutants to examine in vivo growth. Young swine (4 pigs/group; 5 control swine) were inoculated intramuscularly with one of 4 nsp2 deletion mutants (rΔ727–813, rΔ543–726, rΔ324–523, rΔ324–726) or full-length recombinant virus (rVR-2332). Serum samples were collected on various days post-inoculation and analyzed by HerdChek* ELISA, PRRSV real time RT-PCR, gamma interferon (IFN-γ) ELISA, and nucleotide sequence analysis of the entire nsp2 coding region. Tracheobronchial lymph node weight compared to body weight was recorded for each animal and used as a clinical measurement of viral pathogenesis. Results showed that all deletion mutants grew less robustly than full-length recombinant virus, yet all but the large deletion virus (rΔ324–726) recovered to parental viral RNA levels by study end. Swine receiving the rΔ727–813 mutants had a significant decrease in lymph node enlargement compared to rVR-2332. While swine infection with rVR-2332 caused a rapid rise in serum IFN-γ levels, the IFN-γ protein produced by infection with 3 of the 4 deletion mutant viruses was significantly reduced, perhaps due to differences in viral growth kinetics. The rΔ543–726 nsp2 mutant virus, although growth impaired, mimicked rVR-2332 in inducing a host serum IFN-γ response but exhibited a 2-week delay. Targeted sequencing showed that all deletions were stable in the region coding for nsp2 after one swine passage. The data suggested that the selected nsp2 deletion mutants were growth attenuated in swine, altered the induction of serum IFN-γ, an innate cytokine of unknown function in PRRSV clearance, and pointed to a domain that may influence tracheobronchial lymph node size.
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DOI：
10.1073/pnas.89.22.10915
发表时间：
1992-11-15
影响因子：
11.1
作者：
HENIKOFF, S;HENIKOFF, JG
通讯作者：
HENIKOFF, JG
影响因子：
5.4
作者：
Han, Jun;Liu, Gongping;Faaberg, Kay S.
通讯作者：
Faaberg, Kay S.
影响因子：
1.8
作者：
Plagemann, PGW
通讯作者：
Plagemann, PGW
影响因子：
2.7
作者：
Gao, ZQ;Guo, X;Yang, HC
通讯作者：
Yang, HC
DOI：
10.2460/javma.2005.227.385
发表时间：
2005-08-01
影响因子：
1.9
作者：
Neumann, EJ;Kliebenstein, JB;Zimmerman, JJ
通讯作者：
Zimmerman, JJ