Gangnonngiw Warachin, Anantasomboon Gun, Sang-oum Wiwat, Sriurairatana Siriporn, Sritunyalucksana Kallaya, Flegel Timothy W
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Thailand Science Park, Klong Luang Pratumthani, Thailand.
Virology. 2009 Mar 1;385(1):161-8. doi: 10.1016/j.virol.2008.10.044. Epub 2008 Dec 13.
RT-PCR using a commercial kit for yellow head virus (YHV) detection in growth-retarded shrimp yielded an unusual 777 bp amplicon instead of expected amplicons of 277 bp for YHV type-1 (YHV-1) or 406 bp for YHV type-2 (YHV-2). Cloning and sequencing (GenBank EU170438) revealed approximately 80% identity to non-structural (NS) ORF1b sequences of both YHV-1 (GenBank AA083987) and YHV-2 (GenBank AF227196), indicating an atypical YHV type (A-YHV) phylogenetically equidistant from both types. An RT-PCR test specifically designed for A-YHV revealed that it was uncommon and that its occurrence in shrimp culture ponds did not correlate with growth retardation or mortality. By immunohistochemistry with YHV-specific monoclonal antibodies, the A-YHV gave positive reactions for envelope protein gp64 and capsid protein p20, but not for envelope protein gp116, even though gp116 and gp64 originate from a polyprotein of ORF3. Lack of gp116 immunoreactivity correlated with a large ORF3 deletion (GenBank EU123854) in the region of the protein targeted by an MAb against gp116. Transmission electron microscopy of A-YHV-infected shrimp revealed only unenveloped pre-virions. During manuscript revision, information received revealed that typing of YHV isolates based on sequences of ORF1b and ORF3 had yielded several geographical types, including one virulent type (YHV-1b) with an ORF3 deletion sequence that matched the sequence of A-YHV. Using these sequences and an additional A-YHV sequence (EU853170) from the ORF1b typing region, A-YHV potentially represents a recombinant between type 1b and type 5. SDS-PAGE and Western blot analysis revealed that type 1b produced a gp116 deletion protein that did not bind with the MAb or polyclonal Ab to normal gp116. Overall, the information suggested that lack of A-YHV virulence was associated with the NS gene sequence linked to ORF1b rather than the deletion in ORF3.
使用商业试剂盒对生长迟缓的虾进行黄头病毒（YHV）检测的逆转录聚合酶链反应（RT-PCR）产生了一个异常的777 bp扩增子，而不是YHV-1型预期的277 bp扩增子或YHV-2型预期的406 bp扩增子。克隆和测序（GenBank EU170438）显示，与YHV-1（GenBank AA083987）和YHV-2（GenBank AF227196）的非结构（NS）ORF1b序列具有约80%的同一性，表明这是一种在系统发育上与两种类型等距的非典型YHV类型（A-YHV）。专门为A-YHV设计的RT-PCR检测表明，它并不常见，并且其在对虾养殖池塘中的出现与生长迟缓或死亡率无关。通过使用YHV特异性单克隆抗体进行免疫组织化学分析，A-YHV对包膜蛋白gp64和衣壳蛋白p20呈阳性反应，但对包膜蛋白gp116呈阴性反应，尽管gp116和gp64源自ORF3的多蛋白。缺乏gp116免疫反应性与针对gp116的单克隆抗体靶向的蛋白区域中的大ORF3缺失（GenBank EU123854）相关。对感染A-YHV的虾进行透射电子显微镜检查仅发现无包膜的前病毒粒子。在稿件修订期间收到的信息显示，基于ORF1b和ORF3序列对YHV分离株进行分型产生了几种地理类型，包括一种具有与A-YHV序列匹配的ORF3缺失序列的强毒株（YHV-1b）。使用这些序列以及来自ORF1b分型区域的另一个A-YHV序列（EU853170），A-YHV可能代表1b型和5型之间的重组体。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）和蛋白质免疫印迹分析显示，1b型产生了一种gp116缺失蛋白，该蛋白不与针对正常gp116的单克隆抗体或多克隆抗体结合。总体而言，这些信息表明A-YHV缺乏毒力与与ORF1b相关的NS基因序列有关，而不是与ORF3中的缺失有关。
