Real time RT-PCR assays for detection and typing of African horse sickness virus.
Real time RT-PCR assays for detection and typing of African horse sickness virus.
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DOI：
10.1371/journal.pone.0093758
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Mertens PP
中科院分区：
文献类型：
--
作者：
Bachanek-Bankowska K;Maan S;Castillo-Olivares J;Manning NM;Maan NS;Potgieter AC;Di Nardo A;Sutton G;Batten C;Mertens PP
虽然非洲马瘟（AHS）可导致高达95%的马死亡率，但未感染的动物可通过接种抗同源AHSV血清型的疫苗来保护。基因组片段2（Seg-2）编码外衣壳蛋白VP 2，其是AHSV蛋白中最可变的。VP 2也是AHSV特异性中和抗体的主要靶标，因此决定了9种AHSV血清型的同一性。相反，分别由Seg-1和Seg-3编码的VP 1（病毒聚合酶）和VP 3（亚核壳蛋白）是高度保守的，代表病毒种/环状病毒血清群特异性抗原。我们报告了针对AHSV Seg-1或Seg-3的实时RT-PCR检测方法的开发和评价，该方法可以检测任何AHSV类型（病毒种属/血清群特异性检测），以及针对9种AHSV血清型中Seg-2的类型特异性检测。使用来自The Pirbright Institute的“Orbivirus Reference Collection”（ORC）的不同AHSV血清型和其他密切相关的环状病毒分离株对这些检测进行了评价。该试验显示具有AHSV病毒种属特异性或类型特异性（如设计），可用于快速、灵敏和可靠地检测和鉴定（分型）感染血液、组织样本、均质库蠓或组织培养上清液中的AHSV RNA。没有一种检测方法扩增了来自密切相关的异源环状病毒或未感染宿主动物或细胞培养物的cDNA。
Although African horse sickness (AHS) can cause up to 95% mortality in horses, naïve animals can be protected by vaccination against the homologous AHSV serotype. Genome segment 2 (Seg-2) encodes outer capsid protein VP2, the most variable of the AHSV proteins. VP2 is also a primary target for AHSV specific neutralising antibodies, and consequently determines the identity of the nine AHSV serotypes. In contrast VP1 (the viral polymerase) and VP3 (the sub-core shell protein), encoded by Seg-1 and Seg-3 respectively, are highly conserved, representing virus species/orbivirus-serogroup-specific antigens. We report development and evaluation of real-time RT-PCR assays targeting AHSV Seg-1 or Seg-3, that can detect any AHSV type (virus species/serogroup-specific assays), as well as type-specific assays targeting Seg-2 of the nine AHSV serotypes. These assays were evaluated using isolates of different AHSV serotypes and other closely related orbiviruses, from the ‘Orbivirus Reference Collection’ (ORC) at The Pirbright Institute. The assays were shown to be AHSV virus-species-specific, or type-specific (as designed) and can be used for rapid, sensitive and reliable detection and identification (typing) of AHSV RNA in infected blood, tissue samples, homogenised Culicoides, or tissue culture supernatant. None of the assays amplified cDNAs from closely related heterologous orbiviruses, or from uninfected host animals or cell cultures.
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通讯作者：
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