Rash Adam, Woodward Alana, Bryant Neil, McCauley John, Elton Debra
Animal Health Trust, Lanwades Park, Kentford, Newmarket CB8 7UU, UK.
Virol J. 2014 Sep 2;11:159. doi: 10.1186/1743-422X-11-159.
H3N8 equine influenza virus (EIV) has caused disease outbreaks in horses across the world since its first isolation in 1963. However, unlike human, swine and avian influenza, there is relatively little sequence data available for this virus. The majority of published sequences are for the segment encoding haemagglutinin (HA), one of the two surface glycoproteins, making it difficult to study the evolution of the other gene segments and determine the level of reassortment occurring between sub-lineages.
To facilitate the generation of full genome sequences for EIV, we developed a simple, cost-effective and efficient method. M13-tagged primers were used to amplify short, overlapping RT-PCR products, which were then sequenced using Sanger dideoxynucleotide sequencing technology. We also modified a previously published method, developed for human H3N2 and avian H5N1 influenza viruses, which was based on the ligation of viral RNA and subsequent amplification by RT-PCR, to sequence the non-coding termini (NCRs). This necessitated the design of novel primers for an N8 neuraminidase segment.
Two field isolates were sequenced successfully, A/equine/Lincolnshire/1/07 and A/equine/Richmond/1/07, representative of the Florida sublineage clades 1 and 2 respectively. A total of 26 PCR products varying in length from 400-600 nucleotides allowed full coverage of the coding sequences of the eight segments, with sufficient overlap to allow sequence assembly with no primer-derived sequences. Sequences were also determined for the non-coding regions and revealed cytosine at nucleotide 4 in the polymerase segments. Analysis of EIV genomes sequenced using these methods revealed a novel polymorphism in the PA-X protein in some isolates.
These methods can be used to determine the genome sequences of EIV, including the NCRs, from both clade 1 and clade 2 of the Florida sublineage. Full genomes were covered efficiently using fewer PCR products than previously reported methods for influenza A viruses, the techniques used are affordable and the equipment required is available in most research laboratories. The adoption of these methods will hopefully allow for an increase in the number of full genomes available for EIV, leading to improved surveillance and a better understanding of EIV evolution.
H3N8马流感病毒（EIV）自1963年首次分离以来，已在全球范围内引发马匹疾病暴发。然而，与人类、猪和禽流感不同，该病毒的序列数据相对较少。大多数已发表的序列是针对编码血凝素（HA）的片段，血凝素是两种表面糖蛋白之一，这使得研究其他基因片段的进化以及确定亚谱系之间发生的重配水平变得困难。
为了便于生成EIV的全基因组序列，我们开发了一种简单、经济高效的方法。使用M13标记的引物扩增短的重叠RT-PCR产物，然后使用桑格双脱氧核苷酸测序技术进行测序。我们还修改了一种先前发表的方法，该方法是针对人类H3N2和禽流感H5N1病毒开发的，基于病毒RNA的连接和随后的RT-PCR扩增，用于对非编码末端（NCRs）进行测序。这需要为N8神经氨酸酶片段设计新的引物。
成功对两个野外分离株进行了测序，A/equine/Lincolnshire/1/07和A/equine/Richmond/1/07，分别代表佛罗里达亚谱系分支1和2。总共26个长度在400 - 600个核苷酸之间的PCR产物能够完全覆盖八个片段的编码序列，具有足够的重叠以允许进行序列组装且无引物衍生序列。还确定了非编码区域的序列，并在聚合酶片段的核苷酸4处发现了胞嘧啶。使用这些方法对EIV基因组进行分析，发现在一些分离株的PA-X蛋白中存在一种新的多态性。
这些方法可用于确定佛罗里达亚谱系分支1和分支2的EIV基因组序列，包括NCRs。与先前报道的甲型流感病毒方法相比，使用更少的PCR产物就能有效地覆盖全基因组，所使用的技术经济实惠，且大多数研究实验室都具备所需设备。采用这些方法有望增加可获得的EIV全基因组数量，从而加强监测并更好地了解EIV的进化。
