The molecular processes that determine the outcome of influenza virus infection in humans are multifactorial and involve a complex interplay between host, viral and bacterial factors. However, it is generally accepted that a strong innate immune dysregulation known as 'cytokine storm' contributes to the pathology of infections with the 1918 H1N1 pandemic or the highly pathogenic avian influenza viruses of the H5N1 subtype. The RNA sensor retinoic acid-inducible gene I (RIG-I) plays an important role in sensing viral infection and initiating a signalling cascade that leads to interferon expression. Here, we show that short aberrant RNAs (mini viral RNAs (mvRNAs)), produced by the viral RNA polymerase during the replication of the viral RNA genome, bind to and activate RIG-I and lead to the expression of interferon-β. We find that erroneous polymerase activity, dysregulation of viral RNA replication or the presence of avian-specific amino acids underlie mvRNA generation and cytokine expression in mammalian cells. By deep sequencing RNA samples from the lungs of ferrets infected with influenza viruses, we show that mvRNAs are generated during infection in vivo. We propose that mvRNAs act as the main agonists of RIG-I during influenza virus infection.

and propose an intramolecular copy-choice mechanism for mvRNA generation. 43 By deep-sequencing RNA samples from lungs of ferrets infected with influenza 44viruses we show that mvRNAs are generated during infection of animal models. 45 We propose that mvRNAs act as main agonists of RIG-I during influenza virus 46infection and the ability of influenza virus strains to generate mvRNAs should be 47 considered when assessing their virulence potential. 48The negative sense viral RNA (vRNA) genome segments of influenza A viruses, 49 as well as the complementary RNA (cRNA) replicative intermediates, contain 5ʹ 50 triphosphates and partially complementary 5ʹ and 3ʹ termini that serve as the viral 51 promoter for replication and transcription of the viral RNA genome 6 . RIG-I has been 52 shown to bind and be activated by the dsRNA structure formed by the termini of 53 influenza virus RNAs 7,8 . However, it remains unclear how RIG-I gains access to this 54 dsRNA structure. Both vRNA and cRNA are assembled into ribonucleoprotein 55 complexes (vRNP and cRNP, respectively) in which the viral RNA polymerase, a 56 heterotrimeric complex of the viral proteins PB1, PB2 and PA, associates with the 57 partially complementary termini, while the rest of the RNA is bound by oligomeric 58 nucleoprotein (NP) 6 (Fig. 1a). The tight binding of the 5ʹ and 3ʹ termini of vRNA and 59for use under a CC0 license.This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.The copyright holder for this preprint . http://dx.doi.org/10.1101/385716 doi: bioRxiv preprint first posted online Aug. 6, 2018; 3 cRNA by the RNA polymerase 9 is likely to preclude an interaction with RIG-I. 60Moreover, it has been demonstrated that IFN expression is triggered only in a fraction 61 of influenza virus infected cells 10,11 , suggesting that influenza viruses efficiently hide 62 their genome segments during infection by replicating them in the context of RNPs 11 . 63This led to the proposal that an aberrant RNA replication product might be binding to 64 RIG-I and triggering IFN expression 12 . Indeed, the influenza virus polymerase is known 65 to generate defective interfering (DI) RNAs, which are ≥178 nt long subgenomic RNAs 66 generated during high multiplicity infections 13 , and small viral RNAs (svRNAs), which 67 are 22-27 nt long and correspond to the 5ʹ end of vRNA segments. However, svRNAs 68 have been shown not to be involved in the induction of antiviral cellular defences 14 and 69 DI RNAs assemble into RNP structures (Fig. 1a) NP and a luciferase reporter to measure the activation of the IFN-b promoter (Fig. 1b). 82We found that the expression of mvRNAs induced significantly higher IFN expression 83 than full-length vRNA or DI RNA, comparable to the levels induced by transfection of 84 2 µg of poly(I:C), a known activator of IFN expression 18 . Similar results were ob...

Seasonal influenza epidemics and occasional pandemics threaten public health worldwide. New alternative strategies for generating recombinant viruses with vaccine potential are needed. Interestingly, influenza viruses circulating in different hosts have been found to have distinct codon usage patterns, which may reflect host adaptation. We therefore hypothesized that it is possible to make a human seasonal influenza virus that is specifically attenuated in human cells but not in eggs by converting its codon usage so that it is similar to that observed from avian influenza viruses. This approach might help to generate human live attenuated viruses without affecting their yield in eggs. To test this hypothesis, over 300 silent mutations were introduced into the genome of a seasonal H1N1 influenza virus. The resultant mutant was significantly attenuated in mammalian cells and mice, yet it grew well in embryonated eggs. A single dose of intranasal vaccination induced potent innate, humoral, and cellular immune responses, and the mutant could protect mice against homologous and heterologous viral challenges. The attenuated mutant could also be used as a vaccine master donor strain by introducing hemagglutinin and neuraminidase genes derived from other strains. Thus, our approach is a successful strategy to generate attenuated viruses for future application as vaccines. IMPORTANCE Vaccination has been one of the best protective measures in combating influenza virus infection. Current licensed influenza vaccines and their production have various limitations. Our virus attenuation strategy makes use of the codon usage biases of human and avian influenza viruses to generate a human-derived influenza virus that is attenuated in mammalian hosts. This method, however, does not affect virus replication in eggs. This makes the resultant mutants highly compatible with existing egg-based vaccine production pipelines. The viral proteins generated from the codon bias mutants are identical to the wild-type viral proteins. In addition, our massive genome-wide mutational approach further minimizes the concern over reverse mutations. The potential use of this kind of codon bias mutant as a master donor strain to generate other live attenuated viruses is also demonstrated. These findings put forward a promising live attenuated influenza vaccine generation strategy to control influenza. Seasonal influenza strikes every year, and the threat of avian influenza outbreaks and worldwide pandemics together make influenza a significant health risk to the general public, particularly young children, pregnant women, the elderly, and patients with underlying medical conditions (1). Vaccination remains one of the best control measures against influenza. However, currently licensed inactivated and live attenuated influenza vaccines have their limitations. Therefore, new options for vaccine development are needed.Vaccination with an updated virus strain is required every year in response to the frequent occurrence of antigenic drift. Even w...

Significant biases of dinucleotide composition in many RNA viruses including influenza A virus have been reported in recent years. Previous studies have showed that a codon-usage-altered influenza mutant with elevated CpG usage is attenuated in mammalian in vitro and in vivo models. However, the relationship between dinucleotide preference and codon usage bias is not entirely clear and changes in dinucleotide usage of influenza virus during evolution at segment level are yet to be investigated. In this study, a Monte Carlo type method was applied to identify under-represented or over-represented dinucleotide motifs, among different segments and different groups, in influenza viral sequences. After excluding the potential biases caused by codon usage and amino acid sequences, CpG and UpA were found under-represented in all viral segments from all groups, whereas UpG and CpA were found over-represented. We further explored the temporal changes of usage of these dinucleotides. Our analyses revealed significant decrease of CpG frequency in Segments 1, 3, 4, and 5 in seasonal H1 virus after its re-emergence in humans in 1977. Such temporal variations were mainly contributed by the dinucleotide changes at the codon positions 3-1 and 2-3 where silent mutations played a major role. The depletions of CpG and UpA through silent mutations consequently led to over-representations of UpG and CpA. We also found that dinucleotide preference directly results in significant synonymous codon usage bias. Our study helps to provide details on understanding the evolutionary history of influenza virus and selection pressures that shape the virus genome.

No abstract