Expression of influenza virus nonstructural protein 1 (NS1)

Avian influenza poses a threat to many species including man, as shown by the current scenario in Southeast Asia. It appears that this particular type of influenza virus can spread to many species causing severe disease in these new species. For example, from 258 human cases 154 were fatal (WHO, 2006-11-29). Why some avian influenza virus have the ability to infect other species remains to be understood. One viral gene, coding for the non-structural protein 1 (NS1), appears to be an important factor for successfully transmission into a new host, by counteracting the new host’s immune system. The exact mechanism of action of NS1 is still unclear, but leads to down-regulation of various pathways of the immune defence. The aim of this study was to express NS1 proteins of influenza viruses originating from different hosts, including highly pathogenic avian influenza viruses, for use in investigations on the mechanism used by NS1 to interfere with the immune system of various hosts. In particular, studies on the interaction of NS1 with the RNA dependent protein kinase (PKR) were initiated, to determine differences in this interaction between high and low pathogenic influenza viruses from different hosts. By establishing tools and optimised assays, the work enables further studies on the role NS1 in immune evasion

Swine influenza viruses isolated in 1983, 2002 and 2009 in Sweden exemplify different lineages

Swine influenza virus isolates originating from outbreaks in Sweden from 1983, 2002 and 2009 were subjected to nucleotide sequencing and phylogenetic analysis. The aim of the studies was to obtain an overview on their potential relatedness as well as to provide data for broader scale studies on swine influenza epidemiology. Nonetheless, analyzing archive isolates is justified by the efforts directed to the comprehension of the appearance of pandemic H1N1 influenza virus. Interestingly, this study illustrates the evolution of swine influenza viruses in Europe, because the earliest isolate belonged to 'classical' swine H1N1, the subsequent ones to Eurasian 'avian-like' swine H1N1 and reassortant 'avian-like' swine H1N2 lineages, respectively. The latter two showed close genetic relatedness regarding their PB2, HA, NP, and NS genes, suggesting common ancestry. The study substantiates the importance of molecular surveillance for swine influenza viruses

The annotated checklist of plant species that occur in the wetland habitats of Georgia (the Caucasus)

Abstract The checklist includes 270 species that belong to 80 families and 183 genera. Each species has been annotated with the following information: life form, wetland indicator status, and location. In this checklist, Angiosperms are represented by 252 species (93.3%), Bryophytes – 10 species (3.7%), Pteridophytes – 8 (3%), Gymnosperms – 1 (0.4%). The largest families by the number of species are Cyperaceae – 39 (14.4%), Poaceae – 29 (10.7%), Rosaceae – 19 (7.1%), Asteraceae – 17 (6.3%), Fabaceae – 11 (4.1%) and Juncaceae – 11 (4.1%). The checklist is dominated by 55 Palaearctic species (20.4%), followed by 46 Holarctic (17.1%), 31 Euro-Mediterranean (11.5%), 31 Cosmopolitan (11.5%), and 27 Euro-Siberian (10.03%) species. The endemism rate is 4.8%, and the proportion of invasive and naturalized plants is 8.5%. Obligate wetland plants, mainly belonging to the families Cyperaceae and Juncaceae, make up 34.2% of the floristic composition. This is the first comprehensive published checklist of the flora of Georgian wetlands, annotated with wetland indicator values

Influenza virus differentially activates mTORC1 and mTORC2 signaling to maximize late stage replication

<div><p>Influenza A virus usurps host signaling factors to regulate its replication. One example is mTOR, a cellular regulator of protein synthesis, growth and motility. While the role of mTORC1 in viral infection has been studied, the mechanisms that induce mTORC1 activation and the substrates regulated by mTORC1 during influenza virus infection have not been established. In addition, the role of mTORC2 during influenza virus infection remains unknown. Here we show that mTORC2 and PDPK1 differentially phosphorylate AKT upon influenza virus infection. PDPK1-mediated phoshorylation of AKT at a distinct site is required for mTORC1 activation by influenza virus. On the other hand, the viral NS1 protein promotes phosphorylation of AKT at a different site via mTORC2, which is an activity dispensable for mTORC1 stimulation but known to regulate apoptosis. Influenza virus HA protein and down-regulation of the mTORC1 inhibitor REDD1 by the virus M2 protein promote mTORC1 activity. Systematic phosphoproteomics analysis performed in cells lacking the mTORC2 component Rictor in the absence or presence of Torin, an inhibitor of both mTORC1 and mTORC2, revealed mTORC1-dependent substrates regulated during infection. Members of pathways that regulate mTORC1 or are regulated by mTORC1 were identified, including constituents of the translation machinery that once activated can promote translation. mTORC1 activation supports viral protein expression and replication. As mTORC1 activation is optimal midway through the virus life cycle, the observed effects on viral protein expression likely support the late stages of influenza virus replication when infected cells undergo significant stress.</p></div

The first Swedish H1N2 swine influenza virus isolate represents an uncommon reassortant

The European swine influenza viruses (SIVs) show considerable diversity comprising different types of H1N1, H3N2, and H1N2 strains. The intensifying full genome sequencing efforts reveal further reassortants within these subtypes. Here we report the identification of an uncommon reassortant variant of H1N2 subtype influenza virus isolated from a pig in a multisite herd where H1N2 swine influenza was diagnosed for the first time in Sweden during the winter of 2008-2009. The majority of the European H1N2 swine influenza viruses described so far possess haemagglutinin (HA) of the human-like H1N2 SIV viruses and the neuraminidase (NA) of either the European H1N2 or H3N2 SIV-like viruses. The Swedish isolate has an avian-like SIV HA and a H3N2 SIV-like NA, which is phylogenetically more closely related to H3N2 SIV NAs from isolates collected in the early '80s than to the NA of H3N2 origin of the H1N2 viruses isolated during the last decade, as depicted by some German strains, indicative of independent acquisition of the NA genes for these two types of reassortants. The internal genes proved to be entirely of avian-like SIV H1N1 origin. The prevalence of this SIV variant in pig populations needs to be determined, as well as the suitability of the routinely used laboratory reagents to analyze this strain

Molecular characterization of highly pathogenic H5N1 avian influenza viruses isolated in Sweden in 2006

<p>Abstract</p> <p>Background</p> <p>The analysis of the nonstructural (NS) gene of the highly pathogenic (HP) H5N1 avian influenza viruses (AIV) isolated in Sweden early 2006 indicated the co-circulation of two sub-lineages of these viruses at that time. In order to complete the information on their genetic features and relation to other HP H5N1 AIVs the seven additional genes of twelve Swedish isolates were amplified in full length, sequenced, and characterized.</p> <p>Results</p> <p>The presence of two sub-lineages of HP H5N1 AIVs in Sweden in 2006 was further confirmed by the phylogenetic analysis of approximately the 95% of the genome of twelve isolates that were selected on the base of differences in geographic location, timing and animal species of origin. Ten of the analyzed viruses belonged to sub-clade 2.2.2. and grouped together with German and Danish isolates, while two 2.2.1. sub-clade viruses formed a cluster with isolates of Egyptian, Italian, Slovenian, and Nigerian origin. The revealed amino acid differences between the two sub-groups of Swedish viruses affected the predicted antigenicity of the surface glycoproteins, haemagglutinin and neuraminidase, rather than the nucleoprotein, polymerase basic protein 2, and polymerase acidic protein, the main targets of the cellular immune responses. The distinctive characteristics between members of the two subgroups were identified and described.</p> <p>Conclusion</p> <p>The comprehensive genetic characterization of HP H5N1 AIVs isolated in Sweden during the spring of 2006 is reported. Our data support previous findings on the coincidental spread of multiple sub-lineage H5N1 HPAIVs via migrating aquatic birds to large distance from their origin. The detection of 2.2.1. sub-clade viruses in Sweden adds further data regarding their spread in the North of Europe in 2006. The close genetic relationship of Swedish isolates sub-clade 2.2.2. to the contemporary German and Danish isolates supports the proposition of the introduction and spread of a single variant of 2.2.2. sub-clade H5N1 avian influenza viruses in the Baltic region. The presented findings underline the importance of whole genome analysis.</p

Differences in the ability to suppress interferon β production between allele A and allele B NS1 proteins from H10 influenza A viruses

BACKGROUND: In our previous study concerning the genetic relationship among H10 avian influenza viruses with different pathogenicity in mink (Mustela vison), we found that these differences were related to amino acid variations in the NS1 protein. In this study, we extend our previous work to further investigate the effect of the NS1 from different gene pools on type I IFN promoter activity, the production of IFN-β, as well as the expression of the IFN-β mRNA in response to poly I:C. RESULTS: Using a model system, we first demonstrated that NS1 from A/mink/Sweden/84 (H10N4) (allele A) could suppress an interferon-stimulated response element (ISRE) reporter system to about 85%. The other NS1 (allele B), from A/chicken/Germany/N/49 (H10N7), was also able to suppress the reporter system, but only to about 20%. The differences in the abilities of the two NS1s from different alleles to suppress the ISRE reporter system were clearly reflected by the protein and mRNA expressions of IFN-β as shown by ELISA and RT-PCR assays. CONCLUSIONS: These studies reveal that different non-structural protein 1 (NS1) of influenza viruses, one from allele A and another from allele B, show different abilities to suppress the type I interferon β expression. It has been hypothesised that some of the differences in the different abilities of the alleles to suppress ISRE were because of the interactions and inhibitions at later stages from the IFN receptor, such as the JAK/STAT pathway. This might reflect the additional effects of the immune evasion potential of different NS1s

Full genome comparison and characterization of avian H10 viruses with different pathogenicity in Mink (Mustela vison) reveals genetic and functional differences in the non-structural gene

<p>Abstract</p> <p>Background</p> <p>The unique property of some avian H10 viruses, particularly the ability to cause severe disease in mink without prior adaptation, enabled our study. Coupled with previous experimental data and genetic characterization here we tried to investigate the possible influence of different genes on the virulence of these H10 avian influenza viruses in mink.</p> <p>Results</p> <p>Phylogenetic analysis revealed a close relationship between the viruses studied. Our study also showed that there are no genetic differences in receptor specificity or the cleavability of the haemagglutinin proteins of these viruses regardless of whether they are of low or high pathogenicity in mink.</p> <p>In poly I:C stimulated mink lung cells the NS1 protein of influenza A virus showing high pathogenicity in mink down regulated the type I interferon promoter activity to a greater extent than the NS1 protein of the virus showing low pathogenicity in mink.</p> <p>Conclusions</p> <p>Differences in pathogenicity and virulence in mink between these strains could be related to clear amino acid differences in the non structural 1 (NS1) protein. The NS gene of mink/84 appears to have contributed to the virulence of the virus in mink by helping the virus evade the innate immune responses.</p

Swine influenza A virus subtype H1N2 in Sweden

The influenza A virus subtypes H1N1, H1N2 and H3N2 are prevalent in pig populations worldwide. All scientific data point towards swine as the key host species when new human influenza pandemics arise. All previous pandemics have been suggested to evolve in pigs from viral genes of avian, human and porcine origin. Therefore, it is of major importance to monitor the evolution of swine influenza viruses in pigs, and in particular monitor hallmarks of species adaptation to humans.The scope of this project was to increase the understanding of the genetics of swine influenza virus (SIV), with special emphasis on its zoonotic potential, and to investigate the importance of different viral gene markers for species specificity and adaptation. Since clinical manifestation of swine influenza is rare in Sweden, and SIV strains are of particular concern due to the novel human H1N1 epidemic, viruses were isolated in primary swine kidney or Madin Darby Canine Kidney (MDCK) cells, based on standard protocols and the isolates were subjected to full genome sequencing and comparative sequence analysis of the viral genomes. The results describe the analysis of the whole genome sequences from two swine influenza viruses isolated from Sweden in 2009 and 2010. Moreover, this study demonstrates, for the first time, natural reassortment in H1N2 viruses in the pig populations of Sweden. Biological characterization of the two viruses revealed a weaker growing potential, compared to the Swedish 2002 H1N1 isolate. Sequence comparison revealed significant differences between the two consecutive H1N2 isolates. The most remarkable of these was a truncated coding region for PB1-F2 in the earlier isolates and a full length coding region in the more recent isolates. In order to determine the effect of these viruses on the swine industry and on influenza ecology, further surveillance investigations and detailed analyses are needed

Swine influenza A virus subtype H1N2 in Sweden

The influenza A virus subtypes H1N1, H1N2 and H3N2 are prevalent in pig populations worldwide. All scientific data point towards swine as the key host species for new human influenza pandemics, which have been suggested to evolve in pigs from viral genes of avian, human and porcine origin. Therefore, it is of major importance to record the evolution of swine influenza viruses in pigs, and in particular monitor hallmarks of adaptation to humans. The scope of this thesis was to increase the understanding of the genetics of swine influenza virus (SIV), and to investigate the importance of different viral gene markers in association with differences in pathogenicity of two viruses of H1N2 subtype in pigs. The results from this study demonstrate, for the first time, natural reassortment in H1N2 viruses in the pig populations of Sweden. These H1N2 viruses have an avian-like SIV H1N1 haemagglutinin (HA) and a European H3N2 SIV-like neuraminidase (NA). Nucleotide sequence comparison revealed significant differences between the two consecutive H1N2 isolates. To be able to understand the genotypic differences observed in the genomes of these H1N2s, and to identify the genetic markers responsible for the differences, a reverse genetic system was developed. Four recombinant SIV H1N2 viruses were constructed that displayed differences in virulence in mice, r1021 (more virulent) and r9706 (less virulent), as well as the same viruses with swapped PB1 segments. Interestingly, the current findings showed that the replacement of the PB1 segment of r9706 by that of r1021 increases the virulence of the virus that replicate with higher titer in mice lungs, while the opposite is true when PB1 r9706 is introduced into r1021. This study demonstrates that differences in virulence of swine influenza virus subtype H1N2 are attributed at least in part to the PB1 segment. The findings presented in this thesis support the observations concerning the continuous reassortment processes of SIVs in pigs, resulting in repeated and independent emergence of certain HA/NA combinations. This may lead to emergence of new viral variants of severe pathogenicity of pigs. Continuous and efficient surveillance and further detailed genetic and phenotypic analysis can help to identify such novel viral variants, having more potential to cross species barriers and to pose health risks even to humans and to other host species