ANSES, Ploufragan-Plouzané Laboratory, Swine Virology Immunology Unit, National Reference Laboratory for Swine Influenza, Ploufragan, France.
Bretagne Loire University, Rennes, France.
Virol J. 2018 Jan 10;15(1):7. doi: 10.1186/s12985-018-0920-z.
Swine influenza is a respiratory infection of pigs that may have a significant economic impact in affected herds and pose a threat to the human population since swine influenza A viruses (swIAVs) are zoonotic pathogens. Due to the increasing genetic diversity of swIAVs and because novel reassortants or variants may become enzootic or have zoonotic implications, surveillance is strongly encouraged. Therefore, diagnostic tests and advanced technologies able to identify the circulating strains rapidly are critically important.
Several reverse transcription real-time PCR assays (RT-qPCRs) were developed to subtype European swIAVs in clinical samples previously identified as containing IAV genome. The RT-qPCRs aimed to discriminate HA genes of four H1 genetic lineages (H1, H1, H1, H1pdm) and one H3 lineage, and NA genes of two N1 lineages (N1, N1pdm) and one N2 lineage. After individual validation, each RT-qPCR was adapted to high-throughput analyses in parallel to the amplification of the IAV M gene (target for IAV detection) and the β-actin gene (as an internal control), in order to test the ten target genes simultaneously on a large number of clinical samples, using low volumes of reagents and RNA extracts.
The RT-qPCRs dedicated to IAV molecular subtyping enabled the identification of swIAVs from the four viral subtypes that are known to be enzootic in European pigs, i.e. H1N1, H1N2, H3N2 and H1N1pdm. They also made it possible to discriminate a new antigenic variant (H1N2) among H1N2 viruses, as well as reassortant viruses, such as H1N1 or H1N2 for example, and virus mixtures. These PCR techniques exhibited a gain in sensitivity as compared to end-point RT-PCRs, enabling the characterization of biological samples with low genetic loads, with considerable time saving. Adaptation to high-throughput analyses appeared effective, both in terms of specificity and sensitivity. This new development opens novel perspectives in diagnostic capacities that could be very useful for swIAV surveillance and large-scale epidemiological studies.
猪流感是一种猪的呼吸道感染病，在受感染的畜群中可能会造成重大的经济影响，并且由于甲型流感病毒（swIAVs）是人畜共患病病原体，因此对人类构成威胁。由于 swIAVs 的遗传多样性不断增加，并且新型重配体或变体可能成为地方性或具有人畜共患意义，因此强烈鼓励进行监测。因此，能够快速识别循环毒株的诊断测试和先进技术至关重要。
开发了几种逆转录实时 PCR 检测（RT-qPCR）方法，以在先前鉴定为含有 IAV 基因组的临床样本中对欧洲 swIAVs 进行亚型分型。RT-qPCR 的目的是区分四个 H1 遗传谱系（H1、H1、H1、H1pdm）和一个 H3 谱系的 HA 基因，以及两个 N1 谱系（N1、N1pdm）和一个 N2 谱系的 NA 基因。在单独验证后，每个 RT-qPCR 都经过了适应性调整，以便与 IAV M 基因（IAV 检测的靶标）和 β-肌动蛋白基因（作为内部对照）的扩增同时进行高通量分析，以便在大量临床样本上同时测试十个目标基因，同时使用小体积的试剂和 RNA 提取物。
专门用于 IAV 分子分型的 RT-qPCR 使我们能够从已知在欧洲猪中地方性流行的四种病毒亚型中鉴定出 swIAVs，即 H1N1、H1N2、H3N2 和 H1N1pdm。它们还能够区分 H1N2 病毒中的新型抗原变体（H1N2），以及重配病毒，例如 H1N1 或 H1N2 等，以及病毒混合物。与终点 RT-PCR 相比，这些 PCR 技术的灵敏度有所提高，能够对具有低遗传负荷的生物样本进行特征描述，大大节省了时间。适应高通量分析在特异性和敏感性方面似乎都很有效。这一新发展为诊断能力开辟了新的前景，这对于 swIAV 监测和大规模流行病学研究可能非常有用。
