scholarly journals Inclusion of site-specific mutations in the PB1-gene of a virulent A/WSN/33 (H1N1) strain of influenza A virus changes its phenotypic characteristics

2019 ◽  
pp. 21-29
Author(s):  
V. Yu. Kost ◽  
O. A. Sukhova ◽  
I. I. Akopova ◽  
E. O. Gorbacheva ◽  
K. V. Lisovskaya ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Crucial Importance ◽  
Phenotypic Characteristics ◽  
Site Specific ◽  
Ann Arbor ◽  
H1n1 Strain ◽  
New Generation ◽  
Ts Mutations ◽  
Functional Defects

Aim. Study of changes in the phenotypic characteristics of the virulent A/WSN/33 (H1N1) strain of influenza A virus under the influence of the inclusion of site-specific mutations in the PB1-gene of this strain.Materials and methods. Using a two-step polymerase reaction in the PB1 gene of A/ WSN/33 (H1N1) strain were included ts mutations taken from the genome of attenuated CA donors-strains: A/Ann Arbor/6/60 (H2N2), A/Leningrad 134/17/57 (H2N2) and A/ Krasnodar/101/35/59. Ts-phenotype, att-phenotype, immunogenicity, as well as weight loss in mice infected with these mutants were studied in the obtained site-specific mutants.Results. It was shown that the inclusion of ts mutations from the genome of CA donorsstrains of attenuation in the PB1 gene of the virulent A/WSN/33 (H1N1) strain leads to a change in the phenotypic characteristics of this strain to different degrees.Discussion. Analysis of the genome of CA strains- donors of attenuation of influenza virus indicates the crucial importance of the presence of functional defects in the PB1– protein for the formation of the attenuation phenotype of the virus.Conclusion. The technology of site-specific mutagenesis canbe successfully used to modify the PB1 gene of a virulent influenza A virus strain in order to construct a new generation of live influenza vaccines.

scholarly journals Bacteria meets influenza A virus: A bioluminescence mouse model of Escherichia coli O157:H7 following influenza A virus/Puerto Rico/8/34 (H1N1) strain infection

2018 ◽  
Vol 46 (7) ◽  
pp. 2875-2882
Author(s):  
Zhongyi Wang ◽  
Hang Chi ◽  
Xiwen Wang ◽  
Wenliang Li ◽  
Zhiping Li ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Puerto Rico ◽  
Bacterial Infection ◽  
Influenza A Virus ◽  
Influenza A ◽  
Bacterial Colonization ◽  
Infection Model ◽  
Escherichia Coli O157 ◽  
E Coli ◽  
H1n1 Strain

Objective To develop a bioluminescence-labelled bacterial infection model to monitor the colonization and clearance process of Escherichia coli O157:H7 in the lungs of mice following influenza A virus/Puerto Rico/8/34 (H1N1) strain (IAV/PR8) infection. Methods BALB/c mice were administered IAV/PR8 or 0.01 M phosphate-buffered saline (PBS; pH 7.4) intranasally 4 days prior to intranasal administration of 1 × 107 colony-forming units (CFU) of E. coli O157:H7-lux. Whole-body bioluminescent signals were monitored at 10 min, 4 h, 8 h, 12 h, 16 h and 24 h post-bacterial infection. Lung bioluminescent signals and bacterial load (CFU/g) were monitored at 4 h, 8 h, 12 h, 16 h and 24 h post-bacterial infection. Results Prior IAV/PR8 infection of mice resulted in a higher level of bacterial colonization and a lower rate of bacterial clearance from the lungs compared with mice treated with PBS. There were also consistent findings between the bioluminescence imaging and the CFU measurements in terms of identifying bacterial colonization and monitoring the clearance dynamics of E. coli O157:H7-lux in mouse lungs. Conclusion This novel bioluminescence-labelled bacterial infection model rapidly detected bacterial colonization of the lungs and monitored the clearance dynamics of E. coli O157:H7-lux following IAV/PR8 infection.

scholarly journals Measuring site-specific glycosylation similarity between influenza A virus variants with statistical certainty

2020 ◽  
Author(s):  
Deborah Chang ◽  
William E. Hackett ◽  
Lei Zhong ◽  
Xiu-Feng Wan ◽  
Joseph Zaia
Keyword(s):  
Influenza A Virus ◽  
Protein Sequence ◽  
Influenza A ◽  
Viral Fitness ◽  
Antigenic Drift ◽  
Viral Escape ◽  
Strain Selection ◽  
Expression Vectors ◽  
Site Specific ◽  
Measuring Site

AbstractInfluenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between wild-type IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

scholarly journals Measuring Site-specific Glycosylation Similarity between Influenza a Virus Variants with Statistical Certainty

2020 ◽  
Vol 19 (9) ◽  
pp. 1533-1545
Author(s):  
Deborah Chang ◽  
William E. Hackett ◽  
Lei Zhong ◽  
Xiu-Feng Wan ◽  
Joseph Zaia
Keyword(s):  
Influenza A Virus ◽  
Protein Sequence ◽  
Influenza A ◽  
Viral Fitness ◽  
Antigenic Drift ◽  
Viral Escape ◽  
Strain Selection ◽  
Expression Vectors ◽  
Site Specific ◽  
Measuring Site

Influenza A virus (IAV) mutates rapidly, resulting in antigenic drift and poor year-to-year vaccine effectiveness. One challenge in designing effective vaccines is that genetic mutations frequently cause amino acid variations in IAV envelope protein hemagglutinin (HA) that create new N-glycosylation sequons; resulting N-glycans cause antigenic shielding, allowing viral escape from adaptive immune responses. Vaccine candidate strain selection currently involves correlating antigenicity with HA protein sequence among circulating strains, but quantitative comparison of site-specific glycosylation information may likely improve the ability to design vaccines with broader effectiveness against evolving strains. However, there is poor understanding of the influence of glycosylation on immunodominance, antigenicity, and immunogenicity of HA, and there are no well-tested methods for comparing glycosylation similarity among virus samples. Here, we present a method for statistically rigorous quantification of similarity between two related virus strains that considers the presence and abundance of glycopeptide glycoforms. We demonstrate the strength of our approach by determining that there was a quantifiable difference in glycosylation at the protein level between WT IAV HA from A/Switzerland/9715293/2013 (SWZ13) and a mutant strain of SWZ13, even though no N-glycosylation sequons were changed. We determined site-specifically that WT and mutant HA have varying similarity at the glycosylation sites of the head domain, reflecting competing pressures to evade host immune response while retaining viral fitness. To our knowledge, our results are the first to quantify changes in glycosylation state that occur in related proteins of considerable glycan heterogeneity. Our results provide a method for understanding how changes in glycosylation state are correlated with variations in protein sequence, which is necessary for improving IAV vaccine strain selection. Understanding glycosylation will be especially important as we find new expression vectors for vaccine production, as glycosylation state depends greatly on the host species.

scholarly journals The influenza A virus host shutoff factor PA-X is rapidly turned over in a strain-specific manner

2021 ◽  
Author(s):  
Rachel Emily Levene ◽  
Shailab D. Shrestha ◽  
Marta Maria Gaglia
Keyword(s):  
Immune Responses ◽  
Influenza A Virus ◽  
Half Life ◽  
Influenza A ◽  
Pandemic H1n1 ◽  
Host Gene Expression ◽  
X Protein ◽  
Protein Levels ◽  
H1n1 Strain ◽  
Host Shutoff

The influenza A endoribonuclease PA-X regulates virulence and transmission of the virus by reducing host gene expression and thus regulating immune responses to influenza A virus. Despite this key function in viral biology, the levels of PA-X protein remain markedly low during infection, and previous results suggest that these low levels are not solely the result of regulation of the level of translation and RNA stability. How PA-X is regulated post-translationally remains unknown. We now report that the PA-X protein is rapidly turned over. PA-X from multiple viral strains are short-lived, although the half-life of PA-X ranges from ∼30 minutes to ∼3.5 hours depending on the strain. Moreover, sequences in the variable PA-X C-terminal domain are primarily responsible for regulating PA-X half-life, although the N-terminal domain also accounts for some differences among strains. Interestingly, we find that the PA-X from the 2009 pandemic H1N1 strain has a longer half-life compared to the other variants we tested. This PA-X isoform has been reported to have a higher host shutoff activity, suggesting a role for protein turnover in regulating PA-X activity. Collectively, this study reveals a novel regulatory mechanism of PA-X protein levels that may impact host shutoff activity during influenza A virus infection. IMPORTANCE The PA-X protein from influenza A virus reduces host immune responses to infection through suppressing host gene expression, including genes encoding the antiviral response. Thus, it plays a central role in influenza A virus biology. Despite its key function, PA-X was only discovered in 2012 and much remains to be learned including how PA-X activity is regulated to promote optimal levels of viral infection. In this study, we reveal that PA-X protein levels are very low likely because of rapid turnover. We show that instability is a conserved property among PA-X variants from different strains of influenza A virus, but that the half-lives of PA-X variants differ. Moreover, the longer half-life of PA-X from the 2009 pandemic H1N1 strain correlates with its reported higher activity. Therefore, PA-X stability may be a way to regulate its activity and may contribute to the differential virulence of influenza A virus strains.

scholarly journals Cold-adapted reassortants of influenza A virus: Pathogenicity of A/Ann Arbor/6/60�A/Alaska/6/77 reassortant virusesIn vivo andIn vitro

1986 ◽  
Vol 91 (1-2) ◽  
pp. 53-60 ◽  
Author(s):  
A. W. Heath ◽  
H. F. Maassab ◽  
T. Odagiri ◽  
D. C. DeBorde ◽  
C. W. Potter
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Cold Adapted ◽  
Ann Arbor

scholarly journals The influenza A virus host shutoff factor PA-X is rapidly turned over in a strain-specific manner

2020 ◽  
Author(s):  
Rachel Emily Levene ◽  
Shailab D. Shrestha ◽  
Marta Maria Gaglia
Keyword(s):  
Immune Responses ◽  
Influenza A Virus ◽  
Half Life ◽  
Influenza A ◽  
Pandemic H1n1 ◽  
Host Gene Expression ◽  
X Protein ◽  
Protein Levels ◽  
H1n1 Strain ◽  
Host Shutoff

ABSTRACTThe influenza A endoribonuclease PA-X regulates virulence and transmission of the virus by reducing host gene expression and thus regulating immune responses to influenza A virus. Despite this key function in viral biology, the levels of PA-X protein remain markedly low during infection, and previous results suggest that these low levels are not solely the result of regulation of the level of translation and RNA stability. How PA-X is regulated post-translationally remains unknown. We now report that the PA-X protein is rapidly turned over. PA-X from multiple viral strains are short-lived, although the half-life of PA-X ranges from ∼30 minutes to ∼3.5 hours depending on the strain. Moreover, sequences in the variable PA-X C-terminal domain are primarily responsible for regulating PA-X half-life, although the N-terminal domain also accounts for some differences among strains. Interestingly, we find that the PA-X from the 2009 pandemic H1N1 strain has a longer half-life compared to the other variants we tested. This PA-X isoform has been reported to have a higher host shutoff activity, suggesting a role for protein turnover in regulating PA-X activity. Collectively, this study reveals a novel regulatory mechanism of PA-X protein levels that may impact host shutoff activity during influenza A virus infection.IMPORTANCEThe PA-X protein from influenza A virus reduces host immune responses to infection through suppressing host gene expression, including genes encoding the antiviral response. Thus, it plays a central role in influenza A virus biology. Despite its key function, PA-X was only discovered in 2012 and much remains to be learned including how PA-X activity is regulated to promote optimal levels of viral infection. In this study, we reveal that PA-X protein levels are very low likely because of rapid turnover. We show that instability is a conserved property among PA-X variants from different strains of influenza A virus, but that the half-lives of PA-X variants differ. Moreover, the longer half-life of PA-X from the 2009 pandemic H1N1 strain correlates with its reported higher activity. Therefore, PA-X stability may be a way to regulate its activity and may contribute to the differential virulence of influenza A virus strains.

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