Interaction of Polymerase Subunit PB2 and NP with Importin α1 Is a Determinant of Host Range of Influenza A Virus

We have previously reported that mutations in the polymerase proteins PB1, PB2, PA, and the nucleocapsid protein NP resulting in enhanced transcription and replication activities in mammalian cells are responsible for the conversion of the avian influenza virus SC35 (H7N7) into the mouse-adapted variant SC35M. We show now that adaptive mutations D701N in PB2 and N319K in NP enhance binding of these proteins to importin α1 in mammalian cells. Enhanced binding was paralleled by transient nuclear accumulation and cytoplasmic depletion of importin α1 as well as increased transport of PB2 and NP into the nucleus of mammalian cells. In avian cells, enhancement of importin α1 binding and increased nuclear transport were not observed. These findings demonstrate that adaptation of the viral polymerase to the nuclear import machinery plays an important role in interspecies transmission of influenza virus

Human box C/D snoRNAs with miRNA like functions: expanding the range of regulatory RNAs

Small nucleolar RNAs (snoRNAs) and microRNAs are two classes of non-protein-coding RNAs with distinct functions in RNA modification or post-transcriptional gene silencing. In this study, we introduce novel insights to RNA-induced gene activity adjustments in human cells by identifying numerous snoRNA-derived molecules with miRNA-like function, including H/ACA box snoRNAs and C/D box snoRNAs. In particular, we demonstrate that several C/D box snoRNAs give rise to gene regulatory RNAs, named sno-miRNAs here. Our data are complementing the increasing number of studies in the field of small RNAs with regulatory functions. In massively deep sequencing of small RNA fractions we identified high copy numbers of sub-sequences from >30 snoRNAs with lengths of ≥18 nt. RNA secondary structure prediction indicated for a majority of candidates a location in predicted stem regions. Experimental analysis revealed efficient gene silencing for 11 box C/D sno-miRNAs, indicating cytoplasmic processing and recruitment to the RNA silencing machinery. Assays in four different human cell lines indicated variations in both the snoRNA levels and their processing to active sno-miRNAs. In addition we show that box D elements are predominantly flanking at least one of the sno-miRNA strands, while the box C element locates within the sequence of the sno-miRNA guide strand

Tacaribe Virus but Not Junin Virus Infection Induces Cytokine Release from Primary Human Monocytes and Macrophages

The mechanisms underlying the development of disease during arenavirus infection are poorly understood. However, common to all hemorrhagic fever diseases is the involvement of macrophages as primary target cells, suggesting that the immune response in these cells may be of paramount importance during infection. Thus, in order to identify features of the immune response that contribute to arenavirus pathogenesis, we have examined the growth kinetics and cytokine profiles of two closely related New World arenaviruses, the apathogenic Tacaribe virus (TCRV) and the hemorrhagic fever-causing Junin virus (JUNV), in primary human monocytes and macrophages. Both viruses grew robustly in VeroE6 cells; however, TCRV titres were decreased by approximately 10 fold compared to JUNV in both monocytes and macrophages. Infection of both monocytes and macrophages with TCRV also resulted in the release of high levels of IL-6, IL-10 and TNF-α, while levels of IFN-α, IFN-β and IL-12 were not affected. However, we could show that the presence of these cytokines had no direct effect on growth of either TCRV of JUNV in macrophages. Further analysis also showed that while the production of IL-6 and IL-10 are dependent on viral replication, production of TNF-α also occurs after exposure to UV-inactivated TCRV particles and is thus independent of productive virus infection. Surprisingly, JUNV infection did not have an effect on any of the cytokines examined indicating that, in contrast to other viral hemorrhagic fever viruses, macrophage-derived cytokine production is unlikely to play an active role in contributing to the cytokine dysregulation observed in JUNV infected patients. Rather, these results suggest that an early, controlled immune response by infected macrophages may be critical for the successful control of infection of apathogenic viruses and prevention of subsequent disease, including systemic cytokine dysregulation

Integration of governmental risk perception into a Covid-19 model for the Netherlands

Models are valuable tools to evaluate and select Covid-19 mitigation measures, but rarely model governmental behaviour and the social and economic impact of interventions. We enhanced a spatial Covid-19 disease model with an agent-based component, modelling a government agent's risk perception and coping strategy. We tested the model using an age-specific intervention scenario and a RoadMap scenario. Results show that both scenarios lead to a similar reduction in disease numbers, but the age-specific scenario has less social impact as school closure is avoided

Protease activation mutants elicit protective immunity against highly pathogenic avian influenza viruses of subtype H7 in chickens and mice

Protease activation mutants of the highly pathogenic avian influenza virus A/FPV/Rostock/34 (H7N1) have been generated that are fully dependent on the presence of trypsin for growth in cell culture. Unlike wild-type virus, the mutants do not induce systemic infection in chicken embryos and show low pathogenicity in both chicken embryos and adult chickens. Inactivated vaccines prepared from the mutants protected chickens and mice very efficiently against infection with highly pathogenic wild-type virus in a cross-reactive manner. The potential of these mutants to be used as veterinary and prepandemic vaccines will be discussed

Acylation-Mediated Membrane Anchoring of Avian Influenza Virus Hemagglutinin Is Essential for Fusion Pore Formation and Virus Infectivity

Attachment of palmitic acid to cysteine residues is a common modification of viral glycoproteins. The influenza virus hemagglutinin (HA) has three conserved cysteine residues at its C terminus serving as acylation sites. To analyze the structural and functional roles of acylation, we have generated by reverse genetics a series of mutants (Ac1, Ac2, and Ac3) of fowl plague virus (FPV) containing HA in which the acylation sites at positions 551, 559, and 562, respectively, have been abolished. When virus growth in CV1 and MDCK cells was analyzed, similar amounts of virus particles were observed with the mutants and the wild type. Protein patterns and lipid compositions, characterized by high cholesterol and glycolipid contents, were also indistinguishable. However, compared to wild-type virus, Ac2 and Ac3 virions were 10 and almost 1,000 times less infectious, respectively. Fluorescence transfer experiments revealed that loss of acyl chains impeded formation of fusion pores, whereas hemifusion was not affected. When the affinity to detergent-insoluble glycolipid (DIG) domains was analyzed by Triton X-100 treatment of infected cells and virions, solubilization of Ac2 and Ac3 HAs was markedly facilitated. These observations show that acylation of the cytoplasmic tail, while not necessary for targeting to DIG domains, promotes the firm anchoring and retention of FPV HA in these domains. They also indicate that tight DIG association of FPV HA is essential for formation of fusion pores and thus probably for infectivity

Influenza B Virus With Modified Hemagglutinin Cleavage Site as a Novel Attenuated Live Vaccine

Background: Both pandemic and interpandemic influenza is associated with high morbidity and mortality worldwide. Seasonal epidemics are caused by both influenza A and B virus strains that cocirculate with varying predominance and may give rise to severe illness equally. According to World Health Organization recommendations, current annual vaccines are composed of 2 type A and 1 type B virus-specific component. Methods: As a novel attenuated live vaccine against influenza B virus, we generated a hemagglutinin cleavage site mutant of strain B/Lee/40 by replacing the common monobasic cleavage site recognized by trypsinlike proteases with an elastase-sensitive site, and we investigated the in vitro properties, attenuation, humoral responses, and efficacy in mice. Results. This mutant virus replicated in cell culture equally well as the wild type but in a strictly elastase-dependent manner. In contrast to the mouse-pathogenic parental virus, the cleavage site mutant was fully attenuated in mice and not detectable in their lungs. After 1 intranasal immunization, the animals survived lethal challenge with wild-type virus without weight loss or any other signs of disease. Furthermore, no challenge virus could be reisolated from the lungs of vaccinated mice. Conclusions: These findings demonstrate that proteolytic activation mutants can serve as live vaccine against influenza B virus