Viral respiratory infections are usually mild and self-limiting; still they exceptionally result in life-threatening infections in previously healthy children. To investigate a potential genetic cause, we recruited 120 previously healthy children requiring support in intensive care because of a severe illness caused by a respiratory virus. Using exome and transcriptome sequencing, we identified and characterized three rare loss-of-function variants in IFIH1, which encodes an RIG-I-like receptor involved in the sensing of viral RNA. Functional testing of the variants IFIH1 alleles demonstrated that the resulting proteins are unable to induce IFN-β, are intrinsically less stable than wild-type IFIH1, and lack ATPase activity. In vitro assays showed that IFIH1 effectively restricts replication of human respiratory syncytial virus and rhinoviruses. We conclude that IFIH1 deficiency causes a primary immunodeficiency manifested in extreme susceptibility to common respiratory RNA viruses. V iral respiratory tract infections are the most common childhood infections worldwide, with close to 100% of children being infected during the first years of life. Whereas the vast majority of viral respiratory infections are mild and self-limiting, more severe disease leads to the hospitalization of about 3% of individuals in each birth cohort (1). In-hospital mortality rates are limited to <1% with intensive care support; still these infections account for 21% of childhood mortality worldwide (2, 3). The main viral pathogens causing lower respiratory tract infections are human respiratory syncytial virus (RSV), enteroviruses [including human rhinoviruses (HRV)], adenoviruses, human metapneumovirus, coronavirus, influenza, and parainfluenza viruses, with RSV being responsible for the majority of the hospitalized pediatric cases (4, 5).A number of risk factors including socioeconomic and environmental influences, preterm birth, chronic diseases, and immunosuppression are associated with more severe clinical presentation (6). However, ∼1 out of 1,000 children without any known risk factor will require intensive care support due to life-threatening manifestations of common viral respiratory infections. In the absence of established differences in pathogen virulence, we hypothesized that human genetic variation contributes to unusual susceptibility to severe disease due to common viruses. Supporting evidence is provided by a recent study, which showed that rare variants in IRF7 resulted in life-threatening influenza in an otherwise healthy child (7).We combined exome sequencing, transcriptomic analysis, and in vitro functional testing to identify and characterize potentially causal genetic variants in a prospective cohort of previously healthy children requiring intensive care support for common respiratory viral infections. We report the identification of a pathogen-restricted immunodeficiency due to loss-of-function variants in IFIH1, which result in defective innate recognition of RNA viruses, preventing the activation of an efficie...

Many RNA viruses are detected by retinoic acid-inducible gene i (RIG-I), a cytoplasmic sensor that triggers an antiviral response upon binding non-self-RNA that contains a stretch of double-stranded RNA (dsRNA) bearing a base-paired 5′ ppp nucleotide. To gain insight into how RIG-I discriminates between self-RNA and non-self-RNA, we used duplexes whose complementary bottom strand contained both ribo- and deoxynucleotides. These duplexes were examined for their binding to RIG-I and their relative abilities to stimulate ATPase activity, to induce RIG-I dimerization on the duplex, and to induce beta interferon (IFN-β) expression. We show that the chemical nature of the bottom strand is not critical for RIG-I binding. However, two key ribonucleotides, at positions 2 and 5 on the bottom strand, are minimally required for the RIG-I ATPase activity, which is necessary but not sufficient for IFN-β stimulation. We find that duplexes with shorter stretches of dsRNA, as model self-RNAs, bind less stably to RIG-I but nevertheless have an enhanced ability to stimulate the ATPase. Moreover, ATPase activity promotes RIG-I recycling on RIG-I/dsRNA complexes. Since pseudo-self-RNAs bind to RIG-I less stably, they are preferentially recycled by ATP hydrolysis that weakens the helicase domain binding of dsRNA. Our results suggest that one function of the ATPase is to restrict RIG-I signaling to its interaction with non-self-RNA. A model of how this discrimination occurs as a function of dsRNA length is presented.

Influenza virus RNA (vRNA) promoter panhandle structures are believed to be sensed by retinoic acid-inducible gene I (RIG-I). The occurrence of mismatches in this double-stranded RNA structure raises questions about their effect on innate sensing. Our results suggest that mismatches in vRNA promoters decrease binding to RIG-I in vivo, affecting RNA/RIG-I complex formation and preventing RIG-I activation. These results can be inferred to apply to other viruses and suggest that mismatches may represent a general viral strategy to escape RIG-I sensing. Detection of viral infections at the cellular level is crucial for the establishment of an innate immune response. Accordingly, viruses have developed strategies to circumvent this response. They can actively block the pathways involved or prevent the formation of viral molecular patterns sensed by specific cellular receptors, such as retinoic acid-inducible gene I (RIG-I) and melanoma differentiation-associated protein 5, that recognize viral RNA patterns. For RIG-I, this pattern consists of double-stranded RNA (dsRNA) structures of various lengths, with 5= tri-or diphosphate (5= ppp or 5= pp, respectively) base-paired ribonucleotides (1, 2). For segmented negative-strand RNA viruses, dsRNA structures are found in panhandles formed by base pairing of conserved and complementary 5= and 3= genome ends, which activate RIG-I

Frankia alni strain ACN14a's genome was scanned for the presence of determinants involved in interactions with its host plant, Alnus spp. One such determinant type is lectin, proteins that bind specifically to sugar motifs. The genome of F. alni was found to contain 7 such lectin-coding genes, five of which were of the ricinB-type. The proteins coded by these genes contain either only the lectin domain, or also a heat shock protein or a serine-threonine kinase domain upstream. These lectins were found to have several homologs in Streptomyces spp., and a few in other bacterial genomes among which none in Frankia EAN1pec and CcI3 and two in strain EUN1f. One of these F. alni genes, FRAAL0616, was cloned in E. coli, fused with a reporter gene yielding a fusion protein that was found to bind to both root hairs and to bacterial hyphae. This protein was also found to modify the dynamics of nodule formation in A. glutinosa, resulting in a higher number of nodules per root. Its role could thus be to permit binding of microbial cells to root hairs and help symbiosis to occur under conditions of low Frankia cell counts such as in pioneer situations.

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Implementation of reverse genetics for influenza A virus, that is, the DNA-based generation of infectious viral particles in cell culture, opened new avenues to investigate the function of viral proteins and their interplay with host factors on a molecular level. This powerful technique allows the introduction, depletion, or manipulation of any given sequence in the viral genome, as long as it gives rise to replicating virus progeny. Reverse genetics can be used to generate targeted reassortant viruses by mixing segments of different viral strains, thus providing insight into phenotypes of potentially pandemic viruses arising from natural reassortment. It was further instrumental for the development of novel vaccine strategies, allowing rapid and targeted exchange of viral surface antigens on a well-replicating genetic backbone of cell culture-adapted or cold-adapted/attenuated viral strains. Establishment of reverse genetics and rescue of molecular clones of influenza A virus have been extensively described before. Here we give a detailed stand-alone protocol encompassing clinical sampling of influenza A virus specimens and subsequent plasmid-based genetics to rescue, manipulate, and confirm a fully infectious molecular clone. This protocol is based on the combined techniques and experience of a number of influenza laboratories, which are credited and referenced whenever appropriate.

There is an urgent need for next-generation COVID-19 vaccines that are safe, demonstrate high protective efficacy against SARS-CoV-2 variants and can be manufactured at scale. We describe a vaccine candidate (CoVac-II) that is based on stabilized, trimeric spike antigen produced in an optimized, scalable and chemically defined production process.

Global control of COVID-19 will require the deployment of vaccines capable of inducing long-term protective immunity against SARS-CoV-2 variants. In this report, we describe an adjuvanted subunit candidate vaccine that affords elevated, sustained and cross-variant SARS-CoV-2 neutralising antibodies (NAbs) in multiple animal models. Alhydroxiquim-II is a TLR7/8 small-molecule agonist chemisorbed on aluminium hydroxide. Vaccination with Alhydroxiquim-II combined with a stabilized, trimeric form of the SARS-CoV-2 spike protein (termed CoVac-II) resulted in high-titre NAbs in mice, with no decay in responses over an 8-month period. NAbs from sera of CoVac-II-immunized mice, horses and rabbits were broadly neutralising against SARS-CoV-2 variants. Boosting long-term CoVac-II-immunized mice with adjuvanted spike protein from the Beta variant markedly increased levels of NAb titres against multiple SARS-CoV-2 variants; notably high titres against the Delta variant were observed. These data strongly support the clinical assessment of Alhydroxiquim-II-adjuvanted spike proteins to protect against SARS-CoV-2 variants of concern.