The primary GP5 neutralization epitope of North American isolates of porcine reproductive and respiratory syndrome virus
The primary GP5 neutralization epitope of North American isolates of porcine reproductive and respiratory syndrome virus
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DOI：
10.1016/j.vetimm.2004.09.011
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发表时间：
2004-12-08
影响因子：
1.8
通讯作者：
Plagemann, PGW
中科院分区：
文献类型：
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作者：
Plagemann, PGW
我使用间接ELISA法与重叠合成肽代表GP5外结构域来研究在子宫内感染猪繁殖与呼吸综合征病毒（PRRSV） VR2332株的猪和用于PRRSV感染诊断的北美野外血清中肽结合抗体的产生和特异性。vr2332感染猪的血清在分娩后约30天内出现肽结合抗体，达到最高滴度100-200 dpf，然后缓慢下降至最高滴度的一半左右，约400 dpf。肽结合抗体和病毒中和抗体的形成是相关的，它们最初的出现与病毒从循环中消失的时间一致。抗体对vr2332特异性肽具有特异性。相比之下，HerdCheck ELISA检测到的抗n蛋白抗体出现在7 dpf内，在100 dpf左右达到最高水平，并在200 dpf左右降至可检测水平以下。27份具有病毒中和活性的现场血清样本均具有高水平的肽结合抗体，但抗体与VR2332和菌株Lelystad病毒（LV）特异性肽的结合水平大致相同。采用多种大肽段的间接ELISA和采用小肽段（8或9个氨基酸长）的竞争ELISA结果证实，抗体识别的表位位于VR2332的GP5外结构域序列（37）SHLQLIYNL。在竞争ELISA中使用突变肽发现，I-42对T和(HL)-H-38对TY的替换阻断了Ab识别，而L-41的删除没有影响。此外，两家农场提交的26份血清样本用于诊断测试，发现具有低水平的与GP5外结构域肽结合的抗体，尽管这些血清在HerdChek ELISA中呈血清阴性，并且缺乏中和活性。ELISA结果显示，抗体能够识别PRRSV中和表位下游的一个或多个表位。与BSA结合的GP5外结构域肽免疫小鼠后产生的抗体能够识别位于同一区域的表位。这些抗体也缺乏中和活性。(C) 2004 Elsevier B.V.版权所有
I have used indirect ELISA with overlapping synthetic peptides representing the GP5 ectodomain to study the generation and specificity of peptide-binding Abs in pigs that were infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV) strain VR2332 and in North American field sera submitted for PRRSV infection diagnosis. Peptide-binding Abs appeared in sera of the VR2332-infected pigs within about 30 days post-farrowing (dpf), reaching maximum titers 100-200 dpf and then decreasing slowly to about half of maximum titer by about 400 dpf. The formation of peptide-binding Abs and of virus neutralizing Abs correlated and their initial appearance coincided with disappearance of virus from the circulation. The Abs were specific for VR2332-specific peptides. In contrast, anti-N-protein Abs as measured by HerdCheck ELISA appeared within 7 dpf, reached maximum levels at about 100 dpf and had decreased below detectable levels by about 200 dpf. Twenty-seven field serum samples with virus neutralizing activity all possessed high levels of peptide binding Abs, but the Abs bound about equally to VR2332 and strain Lelystad virus (LV)-specific peptides. The indirect ELISA results using various large peptides and competition ELISA using small peptides (8 or 9 amino acids long) confirmed that the epitope recognized by the Abs is located in the GP5 ectodomain sequence (37)SHLQLIYNL of VR2332. Use of mutated peptides in the competition ELISA showed that I-42 to T and (HL)-H-38 to TY substitutions blocked Ab recognition, whereas deletion of L-41 had no effect. In addition, 26 serum samples submitted by two farms for diagnostic tests were found to possess low levels of Abs that bound to GP5 ectodomain peptides, even though the sera were sero-negative in the HerdChek ELISA and lacked neutralizing activity. Competition ELISA showed that the Abs recognized one or more epitopes located downstream of the PRRSV neutralization epitope. An epitope(s) located in the same area was recognized by Abs generated in mice by immunization with a GP5 ectodomain peptide conjugated to BSA. These Abs also lacked neutralizing activity. (C) 2004 Elsevier B.V. All rights reserved.