a Department of Microbiology, Li Ka Shing Faculty of Medicine , University of Hong Kong , Hong Kong , People's Republic of China.
b Center for Genome Sciences, Li Ka Shing Faculty of Medicine , University of Hong Kong , Hong Kong , People's Republic of China.
Emerg Microbes Infect. 2019;8(1):662-674. doi: 10.1080/22221751.2019.1611346.
Influenza defective interfering (DI) particles are replication-incompetent viruses carrying large internal deletion in the genome. The loss of essential genetic information causes abortive viral replication, which can be rescued by co-infection with a helper virus that possesses an intact genome. Despite reports of DI particles present in seasonal influenza A H1N1 infections, their existence in human infections by the avian influenza A viruses, such as H7N9, has not been studied. Here we report the ubiquitous presence of DI-RNAs in nasopharyngeal aspirates of H7N9-infected patients. Single Molecule Real Time (SMRT) sequencing was first applied and long-read sequencing analysis showed that a variety of H7N9 DI-RNA species were present in the patient samples and human bronchial epithelial cells. In several abundantly expressed DI-RNA species, long overlapping sequences have been identified around at the breakpoint region and the other side of deleted region. Influenza DI-RNA is known as a defective viral RNA with single large internal deletion. Beneficial to the long-read property of SMRT sequencing, double and triple internal deletions were identified in half of the DI-RNA species. In addition, we examined the expression of DI-RNAs in mice infected with sublethal dose of H7N9 virus at different time points. Interestingly, DI-RNAs were abundantly expressed as early as day 2 post-infection. Taken together, we reveal the diversity and characteristics of DI-RNAs found in H7N9-infected patients, cells and animals. Further investigations on this overwhelming generation of DI-RNA may provide important insights into the understanding of H7N9 viral replication and pathogenesis.
流感缺陷干扰 (DI) 颗粒是复制缺陷型病毒，基因组中带有大片段内部缺失。缺失必需的遗传信息会导致病毒复制失败，但可通过与具有完整基因组的辅助病毒共感染来挽救。尽管有报道称季节性甲型流感 H1N1 感染中存在 DI 颗粒，但尚未研究其在禽流感病毒（如 H7N9）引起的人类感染中的存在情况。在这里，我们报告了 H7N9 感染患者的鼻咽抽吸物中普遍存在 DI-RNAs。我们首次应用单分子实时 (SMRT) 测序并进行长读测序分析，结果显示患者样本和人支气管上皮细胞中存在多种 H7N9 DI-RNA 种类。在几种大量表达的 DI-RNA 中，在断点区域及其缺失区域的另一侧周围已鉴定出长重叠序列。已知流感 DI-RNA 是一种具有单个大片段内部缺失的缺陷性病毒 RNA。SMRT 测序的长读特性对我们有帮助，在一半的 DI-RNA 种类中鉴定出了双和三内部缺失。此外，我们在不同时间点用亚致死剂量的 H7N9 病毒感染小鼠后，检查了 DI-RNAs 的表达情况。有趣的是，DI-RNAs 在感染后第 2 天就大量表达。总之，我们揭示了 H7N9 感染患者、细胞和动物中发现的 DI-RNAs 的多样性和特征。进一步研究这种压倒性的 DI-RNA 产生可能会为理解 H7N9 病毒复制和发病机制提供重要见解。
