SMRT sequencing revealed the diversity and characteristics of defective interfering RNAs in influenza A (H7N9) virus infection

Abstract

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.

Influenza virus DI particles: Defective interfering or delightfully interesting?

“…Analysis of DelVG sequences has provided contradictory information about the importance of complementarity at DelVG deletion junctions. Two recent studies reported the presence of overlapping sequences at the flanking regions of IV DelVG junction sites [17,18]. In contrast, we and others failed to detect a significant enrichment of overlapping sequences at the junction sites of many DelVGs [19,20].…”

Differential alphavirus defective RNA diversity between intracellular and encapsidated compartments is driven by subgenomic recombination events

“…However, we have definitively shown that a pathogenic alphavirus, CHIKV, forms D-RNAs with substantial incapacitating deletions in a mammalian host. Importantly, many of the recombination events we observed in vivo were similar to ones observed in vitro during early passages, suggesting that expression of sgD-RNAs is a programmed phenomenon with potential biological functions, recapitulating observations made with IAV (21).…”

Nanopore Sequencing Reveals Novel Targets for Detection and Surveillance of Human and Avian Influenza A Viruses

“…For patients with A(H1N1)pdm09 virus infection, Saira et al showed that only the 5= or 3= end of the polymerase genes were abundant, with few reads in the internal region (28). Using long-read sequencing, we previously showed that Ͼ90% of the reads mapping to PB2, PB1, or PA were defective interfering (DI) sequences for patients with A(H7N9) virus infection (19). In this study, we designed primers and probes to target the 5= end of the PB2 gene because the number of reads at the 5= end of PB2 was much higher than at those of PA and PB1 for most patients.…”

“…For the evaluation of the detection of A(H7N9), a total of 10 A(H7N9)-positive specimens were included. These were collected from patients in Hong Kong from 2014 and 2017, and six were included in our previous study (19).…”