Bachanek-Bankowska Katarzyna, Maan Sushila, Castillo-Olivares Javier, Manning Nicola M, Maan Narender Singh, Potgieter Abraham C, Di Nardo Antonello, Sutton Geoff, Batten Carrie, Mertens Peter P C
Vector-borne Viral Diseases Programme, The Pirbright Institute, Pirbright, Surrey, United Kingdom.
Deltamune (Pty) Ltd, Lyttelton, Centurion, South Africa; Department of Biochemistry, Centre for Human Metabonomics, North-West University, Private Bag X6001, Potchefstroom, South Africa.
PLoS One. 2014 Apr 10;9(4):e93758. doi: 10.1371/journal.pone.0093758. eCollection 2014.
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.
尽管非洲马瘟（AHS）可导致马匹高达95%的死亡率，但未接触过该病毒的动物可通过接种针对同源AHSV血清型的疫苗得到保护。基因组片段2（Seg-2）编码外衣壳蛋白VP2，它是AHSV蛋白中变异最大的。VP2也是AHSV特异性中和抗体的主要靶点，因此决定了九种AHSV血清型的特性。相比之下，分别由Seg-1和Seg-3编码的VP1（病毒聚合酶）和VP3（亚核心壳蛋白）高度保守，代表病毒种属/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估，这些方法可检测任何AHSV类型（病毒种属/血清群特异性检测），以及针对九种AHSV血清型Seg-2的型特异性检测。使用来自皮尔布赖特研究所“环状病毒参考库”（ORC）的不同AHSV血清型分离株和其他密切相关的环状病毒对这些检测方法进行了评估。结果表明，这些检测方法具有AHSV病毒种属特异性或型特异性（如设计），可用于快速、灵敏和可靠地检测和鉴定感染血液、组织样本、匀浆库蠓或组织培养上清液中的AHSV RNA。这些检测方法均未扩增来自密切相关的异源环状病毒、未感染宿主动物或细胞培养物的cDNA。
