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Composition of the Hemagglutinin Polybasic Proteolytic Cleavage Motif Mediates Variable Virulence of H7N7 Avian Influenza Viruses

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      Abstract

      Acquisition of a polybasic cleavage site (pCS) in the hemagglutinin (HA) is a prerequisite for the shift of low pathogenic (LP) avian influenza virus (AIV) to the highly pathogenic (HP) form in chickens. Whereas presence of a pCS is required for high pathogenicity, less is known about the effect of composition of pCS on virulence of AIV particularly H7N7. Here, we investigated the virulence of four avian H7N7 viruses after insertion of different naturally occurring pCS from two HPAIV H7N7 (designated pCSGE and pCSUK) or from H7N1 (pCSIT). In vitro, the different pCS motifs modulated viral replication and the HA cleavability independent on the HA background. However, in vivo, the level of virulence conferred by the different pCS varied significantly. Within the respective viral backgrounds viruses with pCSIT and pCSGE were more virulent than those coding for pCSUK. The latter showed also the most restricted spread in inoculated birds. Besides the pCS, other gene segments modulated virulence of these H7N7 viruses. Together, the specific composition of the pCS significantly influences virulence of H7N7 viruses. Eurasian LPAIV H7N7 may shift to high pathogenicity after acquisition of “specific” pCS motifs and/or other gene segments from HPAIV.

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      MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability

      We report a major update of the MAFFT multiple sequence alignment program. This version has several new features, including options for adding unaligned sequences into an existing alignment, adjustment of direction in nucleotide alignment, constrained alignment and parallel processing, which were implemented after the previous major update. This report shows actual examples to explain how these features work, alone and in combination. Some examples incorrectly aligned by MAFFT are also shown to clarify its limitations. We discuss how to avoid misalignments, and our ongoing efforts to overcome such limitations.
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        Evolution and ecology of influenza A viruses.

        In this review we examine the hypothesis that aquatic birds are the primordial source of all influenza viruses in other species and study the ecological features that permit the perpetuation of influenza viruses in aquatic avian species. Phylogenetic analysis of the nucleotide sequence of influenza A virus RNA segments coding for the spike proteins (HA, NA, and M2) and the internal proteins (PB2, PB1, PA, NP, M, and NS) from a wide range of hosts, geographical regions, and influenza A virus subtypes support the following conclusions. (i) Two partly overlapping reservoirs of influenza A viruses exist in migrating waterfowl and shorebirds throughout the world. These species harbor influenza viruses of all the known HA and NA subtypes. (ii) Influenza viruses have evolved into a number of host-specific lineages that are exemplified by the NP gene and include equine Prague/56, recent equine strains, classical swine and human strains, H13 gull strains, and all other avian strains. Other genes show similar patterns, but with extensive evidence of genetic reassortment. Geographical as well as host-specific lineages are evident. (iii) All of the influenza A viruses of mammalian sources originated from the avian gene pool, and it is possible that influenza B viruses also arose from the same source. (iv) The different virus lineages are predominantly host specific, but there are periodic exchanges of influenza virus genes or whole viruses between species, giving rise to pandemics of disease in humans, lower animals, and birds. (v) The influenza viruses currently circulating in humans and pigs in North America originated by transmission of all genes from the avian reservoir prior to the 1918 Spanish influenza pandemic; some of the genes have subsequently been replaced by others from the influenza gene pool in birds. (vi) The influenza virus gene pool in aquatic birds of the world is probably perpetuated by low-level transmission within that species throughout the year. (vii) There is evidence that most new human pandemic strains and variants have originated in southern China. (viii) There is speculation that pigs may serve as the intermediate host in genetic exchange between influenza viruses in avian and humans, but experimental evidence is lacking. (ix) Once the ecological properties of influenza viruses are understood, it may be possible to interdict the introduction of new influenza viruses into humans.
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          Universal primer set for the full-length amplification of all influenza A viruses.

          To systematically identify and analyze the 15 HA and 9 NA subtypes of influenza A virus, we need reliable, simple methods that not only characterize partial sequences but analyze the entire influenza A genome. We designed primers based on the fact that the 15 and 21 terminal segment specific nucleotides of the genomic viral RNA are conserved between all influenza A viruses and unique for each segment. The primers designed for each segment contain influenza virus specific nucleotides at their 3'-end and non-influenza virus nucleotides at the 5'-end. With this set of primers, we were able to amplify all eight segments of N1, N2, N4, N5, and N8 subtypes. For N3, N6, N7, and N9 subtypes, the segment specific sequences of the neuraminidase genes are different. Therefore, we optimized the primer design to allow the amplification of those neuraminidase genes as well. The resultant primer set is suitable for all influenza A viruses to generate full-length cDNAs, to subtype viruses, to sequence their DNA, and to construct expression plasmids for reverse genetics systems.
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            Author and article information

            Affiliations
            [1 ]Institute of Molecular Virology and Cell Biology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health , Greifswald-Insel Riems, Germany
            [2 ]Department of Experimental Animal Facilities and Biorisk Management, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health , Greifswald-Insel Riems, Germany
            [3 ]Institute of Epidemiology, Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health , Greifswald-Insel Riems, Germany
            Author notes
            Journal
            Sci Rep
            Sci Rep
            Scientific Reports
            Nature Publishing Group
            2045-2322
            22 December 2016
            2016
            : 6
            28004772
            5177941
            srep39505
            10.1038/srep39505
            Copyright © 2016, The Author(s)

            This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

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