Influenza A viruses (IAVs) cause more than 2 million annual episodes of seasonal acute respiratory infections (ARI) and approximately 500,000 deaths worldwide. Depending on virus strain and host immune status, acute infections by IAV may reach sites other than the respiratory tract. In the present study, IAV RNA and antigens were searched for in tissues of palatine tonsils and adenoids removed from patients without ARI symptoms. A real-time reverse transcriptase PCR (RT-PCR) screening revealed that 8 tissue samples from 7 patients out of 103 were positive for IAV. Positive samples were subjected to next-generation sequencing (NGS) and 3 of 8 tissues yielded complete IAV pH1N1 genomes, whereas in 5 samples, the PB1 gene was not fully assembled. Phylogenetic analysis placed tonsil-derived IAV in clusters clearly segregated from contemporaneous Brazilian viruses. Flow cytometry of dispersed tissue fragments and serial immunohistochemistry of paraffin-embedded sections of naturally infected biopsies indicated that CD20+ B lymphocytes, CD8+ T lymphocytes, and CD11c+ cells are susceptible to IAV infection. We sought to investigate whether these lymphoid tissues could be sites of viral replication and sources of viable virus particles. MDCK cells were inoculated with tissue lysates, enabling recovery of one IAV isolate confirmed by immunofluorescence, reverse transcriptase quantitative PCR (RT-qPCR), and NGS. The data indicate that lymphoid tissues not only harbor expression of IAV proteins but also contain infectious virus. Asymptomatic long-term infection raises the possibility of IAV shedding from tonsils, which may have an impact on host-to-host transmission.IMPORTANCE Influenza A virus (IAV) infections are important threats to human health worldwide. Although extensively studied, some aspects of virus pathogenesis and tissue tropism remain unclear. Here, by different strategies, we describe the asymptomatic infection of human lymphoid organs by IAV in children. Our results indicate that IAV was not only detected and isolated from human tonsils but displayed unique genetic features in comparison with those of contemporaneous IAVs circulating in Brazil and detected in swabs and nasal washes. Inside the tissue microenvironment, immune cells were shown to be carrying IAV antigens, especially B and T CD8+ lymphocytes. Taken together, these results suggest that human lymphoid tissues can be sites of silent IAV infections with possible impact on virus shedding to the population.

中文翻译：

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扁桃体肥大患儿甲型流感病毒对B和CD8 + T淋巴细胞的无声感染。

甲型流感病毒（IAV）导致每年超过200万例季节性急性呼吸道感染（ARI）发作，全球范围内约有50万人死亡。根据病毒株和宿主的免疫状态，IAV的急性感染可能会到达呼吸道以外的其他地方。在本研究中，从无ARI症状的患者中取出的tons扁桃体和腺样体组织中搜索IAV RNA和抗原。实时逆转录PCR（RT-PCR）筛选显示103例患者中有7例患者的8个组织样本IAV呈阳性。对阳性样品进行下一代测序（NGS），在8个组织中有3个产生了完整的IAV pH1N1基因组，而在5个样品中，PB1基因并未完全组装。系统发育分析将扁桃体来源的IAV置于与同期巴西病毒明显分开的簇中。分散的组织碎片的流式细胞仪和自然感染的活检组织石蜡包埋的切片的连续免疫组织化学表明，CD20 + B淋巴细胞，CD8 + T淋巴细胞和CD11c +细胞易受IAV感染。我们试图调查这些淋巴组织是否可能是病毒复制的场所和活病毒颗粒的来源。用组织裂解物接种MDCK细胞，从而能够通过免疫荧光，逆转录酶定量PCR（RT-qPCR）和NGS确认一种IAV分离株的恢复。数据表明，淋巴样组织不仅包含IAV蛋白的表达，而且还包含传染性病毒。无症状的长期感染增加了IAV从扁桃体脱落的可能性，这可能会影响宿主之间的传播。重要信息甲型流感病毒（IAV）感染是对全世界人类健康的重要威胁。尽管已进行了广泛研究，但病毒发病机制和组织嗜性的某些方面仍不清楚。在这里，通过不同的策略，我们描述了儿童IAV对人淋巴器官的无症状感染。我们的结果表明，与在巴西流行的同时在拭子和鼻洗液中检测到的同期IAV相比，不仅从人扁桃体中检出并分离出IAV，而且显示出独特的遗传特征。在组织微环境内部，免疫细胞被证明携带IAV抗原，尤其是B和T CD8 +淋巴细胞。在一起