Single Assay for Simultaneous Detection and Differential Identification of Human and Avian Influenza Virus Types, Subtypes, and Emergent Variants

For more than four decades the cause of most type A influenza virus infections of humans has been attributed to only two viral subtypes, A/H1N1 or A/H3N2. In contrast, avian and other vertebrate species are a reservoir of type A influenza virus genome diversity, hosting strains representing at least 120 of 144 combinations of 16 viral hemagglutinin and 9 viral neuraminidase subtypes. Viral genome segment reassortments and mutations emerging within this reservoir may spawn new influenza virus strains as imminent epidemic or pandemic threats to human health and poultry production. Traditional methods to detect and differentiate influenza virus subtypes are either time-consuming and labor-intensive (culture-based) or remarkably insensitive (antibody-based). Molecular diagnostic assays based upon reverse transcriptase-polymerase chain reaction (RT-PCR) have short assay cycle time, and high analytical sensitivity and specificity. However, none of these diagnostic tests determine viral gene nucleotide sequences to distinguish strains and variants of a detected pathogen from one specimen to the next. Decision-quality, strain- and variant-specific pathogen gene sequence information may be critical for public health, infection control, surveillance, epidemiology, or medical/veterinary treatment planning. The Resequencing Pathogen Microarray (RPM-Flu) is a robust, highly multiplexed and target gene sequencing-based alternative to both traditional culture- or biomarker-based diagnostic tests. RPM-Flu is a single, simultaneous differential diagnostic assay for all subtype combinations of type A influenza viruses and for 30 other viral and bacterial pathogens that may cause influenza-like illness. These other pathogen targets of RPM-Flu may co-infect and compound the morbidity and/or mortality of patients with influenza. The informative specificity of a single RPM-Flu test represents specimen-specific viral gene sequences as determinants of virus type, A/HN subtype, virulence, host-range, and resistance to antiviral agents.

四十余年来，绝大多数人类甲型流感病毒感染的致病原因仅被归因于两种病毒亚型：A/H1N1或A/H3N2。与之形成鲜明对比的是，禽类与其他脊椎动物是甲型流感病毒基因组多样性的储存宿主，可容纳16种病毒血凝素（hemagglutinin）与9种病毒神经氨酸酶（neuraminidase）的144种组合中至少120种的毒株。该储存宿主中出现的病毒基因组片段重配与突变，可能催生新的流感病毒毒株，对人类健康与家禽生产构成迫在眉睫的流行病或大流行病威胁。传统的流感病毒亚型检测与区分方法要么耗时耗力（基于培养法），要么灵敏度极低（基于抗体法）。基于逆转录酶-聚合酶链反应（reverse transcriptase-polymerase chain reaction, RT-PCR）的分子诊断检测方法具有检测周期短、分析灵敏度与特异性高的优势。然而，此类诊断检测均无法测定病毒基因核苷酸序列，以区分不同样本中检测到的病原体毒株与变异株。具备决策价值的、针对毒株与变异株的病原体基因序列信息，对于公共卫生、感染控制、监测、流行病学，或是医疗/兽医治疗方案制定均至关重要。重测序病原体微阵列（Resequencing Pathogen Microarray, RPM-Flu）是一种性能可靠、高度多重化且基于靶基因测序的替代方案，可替代传统的基于培养或生物标志物的诊断检测方法。RPM-Flu是一种单一的同步鉴别诊断检测方法，可覆盖甲型流感病毒的所有亚型组合，以及30余种可能引发类流感疾病的其他病毒与细菌病原体。RPM-Flu的这些其他病原体靶标可能会引发合并感染，加重流感患者的发病率和/或死亡率。单次RPM-Flu检测所提供的信息特异性，可将样本特异性的病毒基因序列作为判定病毒型别、A/HN亚型、毒力、宿主范围以及抗病毒药物耐药性的依据。