Infectious Diseases Pathology Branch, Division of High-Consequence Pathogens and Pathology, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
J Clin Virol. 2013 Mar;56(3):232-7. doi: 10.1016/j.jcv.2012.11.014. Epub 2012 Dec 14.
Pandemic 2009 H1N1 influenza A (pH1N1) virus has caused substantial morbidity and mortality globally and continues to circulate. Although pH1N1 viral antigens have been demonstrated in various human tissues by immunohistochemistry (IHC), cellular localization of pH1N1 RNA in these tissues has largely remained uninvestigated.
To examine the distribution of pH1N1 RNA in tissues of fatal cases in order to understand the virus tissue tropism, replication and disease pathogenesis.
Formalin-fixed, paraffin embedded autopsy tissues from 21 patients with confirmed pH1N1 infection were analyzed by influenza A IHC and by in situ hybridization (ISH) using DIG-labeled sense (detects viral RNA) and antisense probes (detects positive-stranded mRNA and cRNA) targeting the nucleoprotein gene of pH1N1 virus.
pH1N1 RNA was localized by ISH in 57% of cases while viral antigens were detected by IHC in 76%. However, in cases with a short duration of illness (1-3 days), more cases (69%) were positive by ISH than IHC (62%). Strong ISH staining was detected by antisense probes in the alveolar pneumocytes of the lungs, mucous glands and in lymph nodes. IHC staining of viral antigens was demonstrated in the lung pneumocytes and mucous glands, but no immunostaining was detected in any of the lymph nodes examined.
This study demonstrates cellular localization of positive-stranded pH1N1 RNA in the lungs, mucous glands and lymph nodes that suggests viral replication in these tissues. The novel ISH assay can be a useful adjunct for the detection of pH1N1 virus in tissues and for pathogenesis studies.
大流行性 2009 年 H1N1 流感 A（pH1N1）病毒在全球范围内造成了大量发病率和死亡率，并仍在继续传播。虽然免疫组织化学（IHC）已在各种人体组织中检测到 pH1N1 病毒抗原，但 pH1N1 RNA 在这些组织中的细胞定位在很大程度上仍未得到研究。
研究致命病例组织中 pH1N1 RNA 的分布，以了解病毒的组织嗜性、复制和疾病发病机制。
使用流感 A IHC 和针对 pH1N1 病毒核蛋白基因的 DIG 标记的 sense（检测病毒 RNA）和 antisense 探针（检测正链 mRNA 和 cRNA）对 21 例经证实 pH1N1 感染的福尔马林固定、石蜡包埋的尸检组织进行分析。
ISH 检测到 pH1N1 RNA 在 57%的病例中定位，而 IHC 检测到病毒抗原在 76%的病例中定位。然而，在病程较短（1-3 天）的病例中，ISH 阳性病例（69%）多于 IHC（62%）。在肺部的肺泡上皮细胞、粘液腺和淋巴结中检测到强烈的 antisense 探针 ISH 染色。在肺上皮细胞和粘液腺中检测到病毒抗原的 IHC 染色，但在检查的任何淋巴结中均未检测到免疫染色。
本研究证明了正链 pH1N1 RNA 在肺部、粘液腺和淋巴结中的细胞定位，提示这些组织中的病毒复制。新型 ISH 检测方法可作为检测组织中 pH1N1 病毒和发病机制研究的有用辅助手段。
