scholarly journals An influenza reassortant with polymerase of pH1N1 and NS gene of H3N2 influenza A virus is attenuated in vivo

2012 ◽  
Vol 93 (5) ◽  
pp. 998-1006 ◽  
Author(s):  
Holly Shelton ◽  
Matt Smith ◽  
Lorian Hartgroves ◽  
Peter Stilwell ◽  
Kim Roberts ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Gene Segment ◽  
Influenza Viruses ◽  
Interferon Response ◽  
H3n2 Virus ◽  
M Gene ◽  
Ns Gene

Influenza viruses readily mutate by accumulating point mutations and also by reassortment in which they acquire whole gene segments from another virus in a co-infected host. The NS1 gene is a major virulence factor of influenza A virus. The effects of changes in NS1 sequence depend on the influenza polymerase constellation. Here, we investigated the consequences of a virus with the polymerase of pandemic H1N1 2009 acquiring an NS gene segment derived from a seasonal influenza A H3N2 virus, a combination that might arise during natural reassortment of viruses that currently circulate in humans. We generated recombinant influenza viruses with surface HA and NA genes and matrix M gene segment from A/PR/8/34 virus, but different combinations of polymerase and NS genes. Thus, any changes in phenotype were not due to differences in receptor use, entry, uncoating or virus release. In Madin–Darby canine kidney (MDCK) cells, the virus with the NS gene from the H3N2 parent showed enhanced replication, probably a result of increased control of the interferon response. However, in mice the same virus was attenuated in comparison with the virus containing homologous pH1N1 polymerase and NS genes. Levels of viral RNA during single-cycles of replication were lower for the virus with H3N2 NS, and this virus reached lower titres in the lungs of infected mice. Thus, virus with pH1N1 polymerase genes did not increase its virulence by acquiring the H3N2 NS gene segment, and MDCK cells were a poor predictor of the outcome of infection in vivo.

scholarly journals Mutations in the M-Gene Segment Can Substantially Increase Replication Efficiency of NS1 Deletion Influenza A Virus in MDCK Cells

2012 ◽  
Vol 86 (22) ◽  
pp. 12341-12350 ◽  
Author(s):  
R. van Wielink ◽  
M. M. Harmsen ◽  
D. E. Martens ◽  
B. P. H. Peeters ◽  
R. H. Wijffels ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Gene Segment ◽  
M Gene ◽  
Replication Efficiency

scholarly journals Rewiring the RNAs of influenza virus to prevent reassortment

2009 ◽  
Vol 106 (37) ◽  
pp. 15891-15896 ◽  
Author(s):  
Qinshan Gao ◽  
Peter Palese
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Nonstructural Protein ◽  
Influenza Viruses ◽  
Ha Gene ◽  
Ns Gene ◽  
Virus Rna ◽  
Reassortant Viruses ◽  
Packaging Signals

Influenza viruses contain segmented, negative-strand RNA genomes. Genome segmentation facilitates reassortment between different influenza virus strains infecting the same cell. This phenomenon results in the rapid exchange of RNA segments. In this study, we have developed a method to prevent the free reassortment of influenza A virus RNAs by rewiring their packaging signals. Specific packaging signals for individual influenza virus RNA segments are located in the 5′ and 3′ noncoding regions as well as in the terminal regions of the ORF of an RNA segment. By putting the nonstructural protein (NS)-specific packaging sequences onto the ORF of the hemagglutinin (HA) gene and mutating the packaging regions in the ORF of the HA, we created a chimeric HA segment with the packaging identity of an NS gene. By the same strategy, we made an NS gene with the packaging identity of an HA segment. This rewired virus had the packaging signals for all eight influenza virus RNAs, but it lost the ability to independently reassort its HA or NS gene. A similar approach can be applied to the other influenza A virus segments to diminish their ability to form reassortant viruses.

scholarly journals Influenza A Virus Inhibits RSV Infection via a Two-Wave Expression of IFIT Proteins

Viruses ◽  
2020 ◽  
Vol 12 (10) ◽  
pp. 1171
Author(s):  
Yaron Drori ◽  
Jasmine Jacob-Hirsch ◽  
Rakefet Pando ◽  
Aharona Glatman-Freedman ◽  
Nehemya Friedman ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Influenza Viruses ◽  
Mass Spectrometry Analysis ◽  
Influenza A Viruses ◽  
Rsv Infection ◽  
Syncytial Virus ◽  
Mouse Lungs

Influenza viruses and respiratory syncytial virus (RSV) are respiratory viruses that primarily circulate worldwide during the autumn and winter seasons. Seasonal surveillance has shown that RSV infection generally precedes influenza. However, in the last four winter seasons (2016–2020) an overlap of the morbidity peaks of both viruses was observed in Israel, and was paralleled by significantly lower RSV infection rates. To investigate whether the influenza A virus inhibits RSV, human cervical carcinoma (HEp2) cells or mice were co-infected with influenza A and RSV. Influenza A inhibited RSV growth, both in vitro and in vivo. Mass spectrometry analysis of mouse lungs infected with influenza A identified a two-wave pattern of protein expression upregulation, which included members of the interferon-induced protein with the tetratricopeptide (IFITs) family. Interestingly, in the second wave, influenza A viruses were no longer detectable in mouse lungs. In addition, knockdown and overexpression of IFITs in HEp2 cells affected RSV multiplicity. In conclusion, influenza A infection inhibits RSV infectivity via upregulation of IFIT proteins in a two-wave modality. Understanding the immune system involvement in the interaction between influenza A and RSV viruses will contribute to the development of future treatment strategies against these viruses.

scholarly journals Strong interferon-inducing capacity of a highly virulent variant of influenza A virus strain PR8 with deletions in the NS1 gene

2009 ◽  
Vol 90 (12) ◽  
pp. 2990-2994 ◽  
Author(s):  
Georg Kochs ◽  
Luis Martínez-Sobrido ◽  
Stefan Lienenklaus ◽  
Siegfried Weiss ◽  
Adolfo García-Sastre ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Influenza Viruses ◽  
Ns1 Protein ◽  
Efficient Induction ◽  
Ns1 Gene ◽  
Mouse Lungs ◽  
Highly Virulent ◽  
Complete Deletion

Influenza viruses lacking the interferon (IFN)-antagonistic non-structural NS1 protein are strongly attenuated. Here, we show that mutants of a highly virulent variant of A/PR/8/34 (H1N1) carrying either a complete deletion or C-terminal truncations of NS1 were far more potent inducers of IFN in infected mice than NS1 mutants derived from standard A/PR/8/34. Efficient induction of IFN correlated with successful initial virus replication in mouse lungs, indicating that the IFN response is boosted by enhanced viral activity. As the new NS1 mutants can be handled in standard biosafety laboratories, they represent convenient novel tools for studying virus-induced IFN expression in vivo.

scholarly journals Variation at Extra-epitopic Amino Acid Residues Influences Suppression of Influenza Virus Replication by M158-66 Epitope-Specific CD8+ T Lymphocytes

2018 ◽  
Vol 92 (11) ◽  
pp. e00232-18 ◽  
Author(s):  
Carolien E. van de Sandt ◽  
Mark R. Pronk ◽  
Carel A. van Baalen ◽  
Ron A. M. Fouchier ◽  
Guus F. Rimmelzwaan
Keyword(s):  
Influenza Virus ◽  
T Lymphocytes ◽  
Virus Replication ◽  
Influenza A ◽  
Gene Segment ◽  
Influenza Viruses ◽  
Amino Acid Residues ◽  
Human Influenza ◽  
M Gene ◽  
Specific Ctls

ABSTRACT Influenza virus-specific CD8+ T lymphocytes (CTLs) contribute to clearance of influenza virus infections and reduce disease severity. Variation at amino acid residues located in or outside CTL epitopes has been shown to affect viral recognition by virus-specific CTLs. In the present study, we investigated the effect of naturally occurring variation at residues outside the conserved immunodominant and HLA*0201-restricted M158-66 epitope, located in the influenza virus M1 protein, on the extent of virus replication in the presence of CTLs specific for the epitope. To this end, we used isogenic viruses with an M1 gene segment derived from either an avian or a human influenza virus, HLA-transgenic human epithelial cells, human T cell clones specific for the M158-66 epitope or a control epitope, and a novel, purposely developed in vitro system to coculture influenza virus-infected cells with T cells. We found that the M gene segment of a human influenza A/H3N2 virus afforded the virus the capacity to replicate better in the presence of M158-66-specific CTLs than the M gene segment of avian viruses. These findings are in concordance with previously observed differential CTL activation, caused by variation at extra-epitopic residues, and may reflect an immune adaptation strategy of human influenza viruses that allows them to cope with potent CTL immunity to the M158-66 epitope in HLA-A*0201-positive individuals, resulting in increased virus replication and shedding and possibly increasing disease severity. IMPORTANCE Influenza viruses are among the leading causes of acute respiratory tract infections. CD8+ T lymphocytes display a high degree of cross-reactivity with influenza A viruses of various subtypes and are considered an important correlate of protection. Unraveling viral immune evasion strategies and identifying signs of immune adaptation are important for defining the role of CD8+ T lymphocytes in affording protection more accurately. Improving our insight into the interaction between influenza viruses and virus-specific CD8+ T lymphocyte immunity may help to advance our understanding of influenza virus epidemiology, aid in risk assessment of potentially pandemic influenza virus strains, and benefit the design of vaccines that induce more broadly protective immunity.

The influenza virus PB1-F2 protein has interferon antagonistic activity

2011 ◽  
Vol 392 (12) ◽  
pp. 1135-1144 ◽  
Author(s):  
Sabine E. Dudek ◽  
Ludmilla Wixler ◽  
Carolin Nordhoff ◽  
Alexandra Nordmann ◽  
Darisuren Anhlan ◽  
...  
Keyword(s):  
Influenza A ◽  
Molecular Mechanisms ◽  
Nonstructural Protein ◽  
Gene Segment ◽  
Antagonistic Activity ◽  
Influenza Viruses ◽  
Type I ◽  
Influenza A Viruses ◽  
Reading Frame

Abstract PB1-F2 is a nonstructural protein of influenza viruses encoded by the PB1 gene segment from a +1 open reading frame. It has been shown that PB1-F2 contributes to viral pathogenicity, although the underlying mechanisms are still unclear. Induction of type I interferon (IFN) and the innate immune response are the first line of defense against viral infection. Here we show that influenza A viruses (IAVs) lacking the PB1-F2 protein induce an enhanced expression of IFN-β and IFN-stimulated genes in infected epithelial cells. Studying molecular mechanisms underlying the PB1-F2-mediated IFN antagonistic activity showed that PB1-F2 interferes with the RIG-I/MAVS protein complex thereby inhibiting the activation of the downstream transcription factor IFN regulatory factor 3. These findings were also reflected in in vivo studies demonstrating that infection with PR8 wild-type (wt) virus resulted in higher lung titers and a more severe onset of disease compared with infection with its PB1-F2-deficient counterpart. Accordingly, a much more pronounced infiltration of lungs with immune cells was detected in mice infected with the PB1-F2 wt virus. In summary, we demonstrate that the PB1-F2 protein of IAVs exhibits a type I IFN-antagonistic function by interfering with the RIG-I/MAVS complex, which contributes to an enhanced pathogenicity in vivo.

scholarly journals Extending the Cytoplasmic Tail of the Influenza A Virus M2 Protein Leads to Reduced Virus Replication In Vivo but Not In Vitro

2007 ◽  
Vol 82 (2) ◽  
pp. 1059-1063 ◽  
Author(s):  
Wai-Hong Wu ◽  
Andrew Pekosz
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Cytoplasmic Tail ◽  
M2 Protein ◽  
Coding Region ◽  
Epitope Tag ◽  
Carboxy Terminal

ABSTRACT A carboxy-terminal epitope tag introduced into the coding region of the A/WSN/33 M2 protein resulted in a recombinant virus (rWSN M2myc) which replicated to titers similar to those of the parental virus (rWSN) in MDCK cells. The rWSN M2myc virus was attenuated in its ability to induce mortality and weight loss after the intranasal inoculation of BALB/c mice, indicating that the M2 cytoplasmic tail plays a role in virus virulence. Mice infected with rWSN M2myc were completely protected from subsequent challenge with rWSN, suggesting that epitope tagging of the M2 protein may be a useful way of attenuating influenza A virus strains.

scholarly journals Defective interfering influenza A virus protects in vivo against disease caused by a heterologous influenza B virus

2011 ◽  
Vol 92 (9) ◽  
pp. 2122-2132 ◽  
Author(s):  
Paul D. Scott ◽  
Bo Meng ◽  
Anthony C. Marriott ◽  
Andrew J. Easton ◽  
Nigel J. Dimmock
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Influenza Viruses ◽  
Influenza B Virus ◽  
Influenza B ◽  
Type I ◽  
Type I Ifn ◽  
B Virus ◽  
The Family

Influenza A and B viruses are major human respiratory pathogens that contribute to the burden of seasonal influenza. They are both members of the family Orthomyxoviridae but do not interact genetically and are classified in different genera. Defective interfering (DI) influenza viruses have a major deletion of one or more of their eight genome segments, which renders them both non-infectious and able to interfere in cell culture with the production of infectious progeny by a genetically compatible, homologous virus. It has been shown previously that intranasal administration of a cloned DI influenza A virus, 244/PR8, protects mice from various homologous influenza A virus subtypes and that it also protects mice from respiratory disease caused by a heterologous virus belonging to the family Paramyxoviridae. The mechanisms of action in vivo differ, with homologous and heterologous protection being mediated by probable genome competition and type I interferon (IFN), respectively. In the current study, it was shown that 244/PR8 also protects against disease caused by a heterologous influenza B virus (B/Lee/40). Protection from B/Lee/40 challenge was partially eliminated in mice that did not express a functional type I IFN receptor, suggesting that innate immunity, and type I IFN in particular, are important in mediating protection against this virus. It was concluded that 244/PR8 has the ability to protect in vivo against heterologous IFN-sensitive respiratory viruses, in addition to homologous influenza A viruses, and that it acts by fundamentally different mechanisms.

scholarly journals Novel Ranking System for Identifying Efficacious Anti-Influenza Virus PB2 Inhibitors

2015 ◽  
Vol 59 (10) ◽  
pp. 6007-6016 ◽  
Author(s):  
Alice W. Tsai ◽  
Colleen F. McNeil ◽  
Joshua R. Leeman ◽  
Hamilton B. Bennett ◽  
Kwame Nti-Addae ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Influenza A Virus ◽  
Influenza A ◽  
Therapeutic Agents ◽  
Influenza Viruses ◽  
Antigenic Drift ◽  
Whole Body ◽  
Infection Model ◽  
Virus Infections

ABSTRACTThrough antigenic drift and shifts, influenza virus infections continue to be an annual cause of morbidity in healthy populations and of death among elderly and at-risk patients. The emergence of highly pathogenic avian influenza viruses such as H5N1 and H7N9 and the rapid spread of the swine-origin H1N1 influenza virus in 2009 demonstrate the continued need for effective therapeutic agents for influenza. While several neuraminidase inhibitors have been developed for the treatment of influenza virus infections, these have shown a limited window for treatment initiation, and resistant variants have been noted in the population. In addition, an older class of antiviral drugs for influenza, the adamantanes, are no longer recommended for treatment due to widespread resistance. There remains a need for new influenza therapeutic agents with improved efficacy as well as an expanded window for the initiation of treatment. Azaindole compounds targeting the influenza A virus PB2 protein and demonstrating excellentin vitroandin vivoproperties have been identified. To evaluate thein vivoefficacy of these PB2 inhibitors, we utilized a mouse influenza A virus infection model. In addition to traditional endpoints, i.e., death, morbidity, and body weight loss, we measured lung function using whole-body plethysmography, and we used these data to develop a composite efficacy score that takes compound exposure into account. This model allowed the rapid identification and ranking of molecules relative to each other and to oseltamivir. The ability to identify compounds with enhanced preclinical properties provides an opportunity to develop more-effective treatments for influenza in patients.

scholarly journals High antiviral effect of TiO2·PL–DNA nanocomposites targeted to conservative regions of (−)RNA and (+)RNA of influenza A virus in cell culture

2016 ◽  
Vol 7 ◽  
pp. 1166-1173 ◽  
Author(s):  
Asya S Levina ◽  
Marina N Repkova ◽  
Elena V Bessudnova ◽  
Ekaterina I Filippova ◽  
Natalia A Mazurkova ◽  
...  
Keyword(s):  
Influenza A Virus ◽  
Influenza A ◽  
Mdck Cells ◽  
Titanium Dioxide Nanoparticles ◽  
Antiviral Effect ◽  
H3n2 Virus ◽  
Dna Fragments ◽  
Efficient System ◽  
Dna Fragment ◽  
Viral Subtypes

Background: The development of new antiviral drugs based on nucleic acids is under scrutiny. An important problem in this aspect is to find the most vulnerable conservative regions in the viral genome as targets for the action of these agents. Another challenge is the development of an efficient system for their delivery into cells. To solve this problem, we proposed a TiO2·PL–DNA nanocomposite consisting of titanium dioxide nanoparticles and polylysine (PL)-containing oligonucleotides. Results: The TiO2·PL–DNA nanocomposites bearing the DNA fragments targeted to different conservative regions of (−)RNA and (+)RNA of segment 5 of influenza A virus (IAV) were studied for their antiviral activity in MDCK cells infected with the H1N1, H5N1, and H3N2 virus subtypes. Within the negative strand of each of the studied strains, the efficiency of DNA fragments increased in the direction of its 3’-end. Thus, the DNA fragment aimed at the 3’-noncoding region of (−)RNA was the most efficient and inhibited the reproduction of different IAV subtypes by 3–4 orders of magnitude. Although to a lesser extent, the DNA fragments targeted at the AUG region of (+)RNA and the corresponding region of (−)RNA were also active. For all studied viral subtypes, the nanocomposites bearing the DNA fragments targeted to (−)RNA appeared to be more efficient than those containing fragments aimed at the corresponding (+)RNA regions. Conclusion: The proposed TiO2·PL–DNA nanocomposites can be successfully used for highly efficient and site-specific inhibition of influenza A virus of different subtypes. Some patterns of localization of the most vulnerable regions in IAV segment 5 for the action of DNA-based drugs were found. The (−)RNA strand of IAV segment 5 appeared to be more sensitive as compared to (+)RNA.

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