Giorgi Metreveli

A Size‐Exclusion Nanocellulose Filter Paper for Virus Removal

This is the first time a 100% natural, unmodified nanofibrous polymer-based membrane is demonstrated capable of removing viruses solely based on the size-exclusion principle, with a log10 reduction value (LRV) ≥ 6.3 as limited by the assay lower detection limit and the feed virus titre, thereby matching the performance of industrial synthetic polymer virus removal filters.

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

BackgroundThe 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.ResultsThe 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.ConclusionThe 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.

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

BackgroundThe 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.ResultsPhylogenetic 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.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.ConclusionsDifferences 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.

Alleles A and B of non-structural protein 1 of avian influenza A viruses differentially inhibit beta interferon production in human and mink lung cells.

Non-structural protein 1 (NS1) counteracts the production of host type I interferons (IFN-α/β) for the efficient replication and pathogenicity of influenza A viruses. Here, we reveal another dimension of the NS1 protein of avian influenza A viruses in suppressing IFN-β production in cultured cell lines. We found that allele A NS1 proteins of H6N8 and H4N6 have a strong capacity to inhibit the activation of IFN-β production, compared with allele B from corresponding subtypes, as measured by IFN stimulatory response element (ISRE) promoter activation, IFN-β mRNA transcription and IFN-β protein expression. Furthermore, the ability to suppress IFN-β promoter activation was mapped to the C-terminal effector domain (ED), while the RNA-binding domain (RBD) alone was unable to suppress IFN-β promoter activation. Chimeric studies indicated that when the RBD of allele A was fused to the ED of allele B, it was a strong inhibitor of IFN-β promoter activity. This shows that well-matched ED and RBD are crucial for the function of the NS1 protein and that the RBD could be one possible cause for this differential IFN-β inhibition. Notably, mutagenesis studies indicated that the F103Y and Y103F substitutions in alleles A and B, respectively, do not influence the ISRE promoter activation. Apart from dsRNA signalling, differences were observed in the expression pattern of NS1 in transfected human and mink lung cells. This study therefore expands the versatile nature of the NS1 protein in inhibiting IFN responses at multiple levels, by demonstrating for the first time that it occurs in a manner dependent on allele type.

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

BackgroundIn 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.ResultsUsing 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.ConclusionsThese 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.

Phylogenetic Analysis of the Hemagglutinin Gene of Low Pathogenic Avian Influenza Virus H7N7 Strains in Mallards in Northern Europe

Abstract The H7 subtype of avian influenza (AI) has the capability to evolve into a highly pathogenic AI (HPAI) virus. In this study, we have characterized the hemagglutinin (HA) genes of three avian H7N7 influenza A viruses isolated from healthy migratory mallards in Northern Europe in three different years to study the natural variation of these viruses in the natural reservoir. Phylogenetic analysis demonstrated that the H7 HA genes were all closely related to recent H7 isolates responsible for influenza outbreaks in poultry in Europe. The A/mallard/Sweden/S90735/2003 isolate clustered together with the HA gene of A/mallard/Netherlands/12/00/H7N3 (AY338460), which has been shown to be closely related to the H7N7 responsible for HPAI outbreaks in the Netherlands and Germany in 2003. In contrast, the HA gene of the two mallard strains A/mallard/Sweden/S90597/2005 and A/mallard/Sweden/100993/2008 were more related to the chicken strain isolated in domestic poultry in England in 2006, A/Ch/England/4054/2006/H7N3 (EF467826), and 2008, A/Ch/England/2008/H7N7 (214011964). Analysis of the deduced HA amino acid sequence shows two different HA cleavage sites in these isolates. Although these HA cleavage sites are consistent with a low pathogenicity AI, the cleavage sites appear to posses an increasing numbers of basic amino acids over time (PEIPKGRGLF in 2003 and 2005 and PEIPKKRGLF in 2008). The conclusion from this study is that H7 subtypes isolated from healthy mallards are closely related to the H7 subtypes that have caused recent influenza outbreaks in poultry in Europe.

Comparison of two H1N2 swine influenza A viruses from disease outbreaks in pigs in Sweden during 2009 and 2010.

The influenza A virus subtypes H1N1, H1N2 and H3N2 are prevalent in pig populations worldwide. In the present study, two relatively uncommon swine influenza virus (SIV) H1N2 subtypes, isolated in Sweden in 2009 and 2010, were compared regarding their molecular composition and biological characteristics. The differences regarding markers purportedly related to pathogenicity, host adaptation or replication efficiency. They included a truncated PB1-F2 protein in the earlier isolate but a full length version in the more recent one; differences in the number of haemagglutinin glycosylation sites, including a characteristic human one; and a nuclear export protein with altered export signal. Of particular interest, the NS1 amino acid sequence of swine H1N2-2009 and 2010 has a ‘unique or very unusual’ PDZ binding domain (RPKV) at the C-terminal of the protein, a motif that has been implicated as a virulence marker. Concerning biological properties, these viruses reached lower titre and showed reduced cytopathogenicity in MDCK cells compared with an avian-like H1N1 isolate A/swine/Lidkoping/1193/2002 belonging to the same lineage as the 2009 and 2010 isolates. The findings should contribute to better understanding of factors related to the survival/extinction of this uncommon reassortant variant.

Characterization of two low pathogenic avian influenza viruses isolated in Hungary in 2007.

Two low pathogenic (LP) avian influenza virus strains, A/mallard/Hungary/19616/07 (H3N8) and A/mute swan/Hungary/5973/07 (H7N7), isolated as part of the National Surveillance Program in Hungary, were fully sequenced and characterized. The two viruses showed the closest phylogenetic relationship regarding their acidic polymerase genes. The H7N7 Hungarian virus and some H5N2 influenza viruses isolated from Korean pigs appeared to have their basic polymerase gene 1 from a relatively recent common ancestor. The matrix gene nucleotide sequence of each Hungarian virus showed close relationship with contemporaneous Czech H3N8 mallard isolates, which belonged to distinct phylogenetic branches. The non-structural protein genes belonged to different alleles, rendering a peculiar characteristic to the H7N7 isolate compared to the so far analyzed Eurasian H7 viruses. The surface glycoprotein genes of the H3N8 isolate showed a close phylogenetic relationship and high nucleotide identities to H3N8 subtype isolates from Northern Europe collected in 2003-2006, and to an H3N2 isolate in Italy in 2006, extending the perceptions of this HA subtype across Northern and Southern Europe close to this period. These findings provide further data to the diversity of influenza viruses found in wild migratory birds and present useful information for large scale studies on influenza virus evolution.

Molecular analysis and characterization of swine and human influenza viruses isolated in Hungary in 2006–2007

In order to provide additional information to the epidemiological situation in Middle Europe and open further possibilities to investigate the transmission of influenza viruses between species, the viral genomes of three influenza A virus isolates (one human and two swine) collected from North-East Hungary in 2006–2007 have been fully sequenced and characterized. The sequence analysis reveals strong geographical relationships between the internal genes of the two swine viruses; the human isolate shows strict conservation to recent H1N1 strains, while the swine strains demonstrate and reflect a mixed avian–human origin, a characteristic of European swine influenza viruses. No evidence of interspecies interaction has been found among the studied isolates.

Evaluation of Biological Indicator Spores as Tools for Assessment of Fumigation Decontamination Effectiveness

In high-containment laboratories and animal facilities common practice is to decontaminate the facilities prior to maintenance or in an emergency situation. Many laboratories use commercially available biological indicators (BIs) to validate the decontamination procedure. In this study the focus was to evaluate the reliability of four different commercial BIs in comparison to control microorganisms that are commonly used in laboratories. Two different fumigation decontamination procedures were chosen: formaldehyde (FA) and vaporized hydrogen peroxide (VHP). The control microorganisms were Salmonella typhimurium, Brucella melitensis, Bacillus thuringiensis spores, porcine parvovirus, equine rhinitis A virus, bovine viral diarrhea virus, and low pathogenic avian influenza virus. Exposure to formaldehyde caused a sufficient reduction of all the control microorganisms, including B. thuringiensis spores, whereas only one of the four commercial BIs was completely negative for growth. The VHP decontamination procedure was not able to reduce any of the control microorganisms more than 4 log10, except for the enveloped viruses, whereas the BIs with the lowest concentration (105 microorganisms per spore strip) indicated a satisfactory decontamination procedure. These results indicate that commercial BIs could be unreliable as general indicators of decontamination effectiveness. To ensure a reliable decontamination process, BIs have to be evaluated for each protocol in parallel with the microorganisms used in the laboratory.