Rutvisuttinunt Wiriya, Klungthong Chonticha, Thaisomboonsuk Butsaya, Chinnawirotpisan Piyawan, Ajariyakhajorn Chuanpis, Manasatienkij Wudtichai, Phonpakobsin Thipwipha, Lon Chanthap, Saunders David, Wangchuk Sonam, Shrestha Sanjaya K, Velasco John Mark S, Alera Maria Theresa P, Simasathien Sriluck, Buddhari Darunee, Jarman Richard G, Macareo Louis R, Yoon In-Kyu, Fernandez Stefan
Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6, Rajavithi Road, Rajathewi, Bangkok, Thailand; Walter Reed/AFRIMS Research Unit Nepal, Kathmandu, Nepal.
Department of Virology, Armed Forces Research Institute of Medical Sciences, 315/6, Rajavithi Road, Rajathewi, Bangkok, Thailand.
J Clin Virol. 2017 Sep;94:91-99. doi: 10.1016/j.jcv.2017.07.004. Epub 2017 Jul 14.
Emerging and re-emerging respiratory pathogens represent an increasing threat to public health. Etiological determination during outbreaks generally relies on clinical information, occasionally accompanied by traditional laboratory molecular or serological testing. Often, this limited testing leads to inconclusive findings. The Armed Forces Research Institute of Medical Sciences (AFRIMS) collected 12,865 nasopharyngeal specimens from acute influenza-like illness (ILI) patients in five countries in South/South East Asia during 2010-2013. Three hundred and twenty-four samples which were found to be negative for influenza virus after screening with real-time RT-PCR and cell-based culture techniques demonstrated the potential for viral infection with evident cytopathic effect (CPE) in several cell lines.
To assess whether whole genome next-generation sequencing (WG-NGS) together with conventional molecular assays can be used to reveal the etiology of influenza negative, but CPE positive specimens.
The supernatant of these CPE positive cell cultures were grouped in 32 pools containing 2-26 supernatants per pool. Three WG-NGS runs were performed on these supernatant pools. Sequence reads were used to identify positive pools containing viral pathogens. Individual samples in the positive pools were confirmed by qRT-PCR, RT-PCR, PCR and Sanger sequencing from the CPE culture and original clinical specimens.
WG-NGS was an effective way to expand pathogen identification in surveillance studies. This enabled the identification of a viral agent in 71.3% (231/324) of unidentified surveillance samples, including common respiratory pathogens (100/324; 30.9%): enterovirus (16/100; 16.0%), coxsackievirus (31/100; 31.0%), echovirus (22/100; 22.0%), human rhinovirus (3/100; 3%), enterovirus genus (2/100; 2.0%), influenza A (9/100; 9.0%), influenza B, (5/100; 5.0%), human parainfluenza (4/100; 4.0%), human adenovirus (3/100; 3.0%), human coronavirus (1/100; 1.0%), human metapneumovirus (2/100; 2.0%), and mumps virus (2/100; 2.0%), in addition to the non-respiratory pathogen herpes simplex virus type 1 (HSV-1) (172/324; 53.1%) and HSV-1 co-infection with respiratory viruses (41/324; 12.7%).
新出现和再次出现的呼吸道病原体对公众健康构成的威胁日益增加。疫情期间的病因诊断通常依赖临床信息，偶尔辅以传统实验室分子或血清学检测。通常，这种有限的检测会导致结果不明确。武装部队医学科学研究院（AFRIMS）在2010年至2013年期间从南亚/东南亚五个国家的急性流感样疾病（ILI）患者中收集了12,865份鼻咽标本。在用实时RT-PCR和基于细胞的培养技术筛查后，发现324份样本流感病毒呈阴性，但在几种细胞系中表现出明显细胞病变效应（CPE），显示出病毒感染的可能性。
评估全基因组下一代测序（WG-NGS）与传统分子检测方法结合是否可用于揭示流感阴性但CPE阳性标本的病因。
将这些CPE阳性细胞培养物的上清液分成32个池，每个池包含2 - 26份上清液。对这些上清液池进行了三次WG-NGS检测。序列读数用于识别含有病毒病原体的阳性池。通过qRT-PCR、RT-PCR、PCR和Sanger测序对CPE培养物和原始临床标本中的阳性池中的单个样本进行确认。
WG-NGS是在监测研究中扩大病原体鉴定的有效方法。这使得能够在71.3%（231/324）的未鉴定监测样本中鉴定出一种病毒病原体，包括常见呼吸道病原体（100/324；30.9%）：肠道病毒（16/100；16.0%）、柯萨奇病毒（31/100；31.0%）、埃可病毒（22/100；22.0%）、人鼻病毒（3/100；3%）、肠道病毒属（2/100；2.0%）、甲型流感病毒（9/100；9.0%）、乙型流感病毒（5/100；5.0%）、人副流感病毒（4/100；4.0%）、人腺病毒（3/100；3.0%）、人冠状病毒（1/100；1.0%）、人偏肺病毒（2/100；2.0%）和腮腺炎病毒（2/100；2.0%），此外还有非呼吸道病原体单纯疱疹病毒1型（HSV-1）（172/324；53.1%）以及HSV-1与呼吸道病毒的共感染（41/324；12.7%）。
