Generation of a variety of stable Influenza A reporter viruses by genetic engineering of the NS gene segment

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.

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Alternative base pairs attenuate influenza A virus when introduced into the duplex region of the conserved viral RNA promoter of either the NS or the PA gene

Journal of General Virology ◽

10.1099/vir.0.18795-0 ◽

2003 ◽

Vol 84 (3) ◽

pp. 507-515 ◽

Cited By ~ 12

Author(s):

A. P. Catchpole ◽

L. J. Mingay ◽

E. Fodor ◽

G. G. Brownlee

Keyword(s):

Influenza Virus ◽

Nuclear Export ◽

Influenza A ◽

Gene Segment ◽

Mrna Levels ◽

Base Pairs ◽

Virus Growth ◽

Ns Gene ◽

Mdbk Cells ◽

Attenuated Viruses

The development of plasmid-based rescue systems for influenza virus has allowed previous studies of the neuraminidase (NA) virion RNA (vRNA) promoter to be extended, in order to test the hypothesis that alternative base pairs in the conserved influenza virus vRNA promoter cause attenuation when introduced into other gene segments. Influenza A/WSN/33 viruses with alternative base pairs in the duplex region of the vRNA promoter of either the polymerase acidic (PA) or the NS (non-structural 1, NS1, and nuclear export, NEP, -encoding) gene have been rescued. Virus growth in MDBK cells demonstrated that one of the mutations, the D2 mutation (U–A replacing G–C at nucleotide positions 12′–11), caused significant virus attenuation when introduced into either the PA or the NS gene. The D2 mutation resulted in the reduction of PA- or NS-specific vRNA and mRNA levels in PA- or NS-recombinant viruses, respectively. Since the D2 mutation attenuates influenza virus when introduced into either the PA or the NS gene segments, or the NA gene segment, as demonstrated previously, this suggests that this mutation will lead to virus attenuation when introduced into any of the eight gene segments. Such a mutation may be useful in the production of live-attenuated viruses.

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Transfectant Influenza A Viruses with Long Deletions in the NS1 Protein Grow Efficiently in Vero Cells

Journal of Virology ◽

10.1128/jvi.72.8.6437-6441.1998 ◽

1998 ◽

Vol 72 (8) ◽

pp. 6437-6441 ◽

Cited By ~ 144

Author(s):

Andrej Egorov ◽

Sabine Brandt ◽

Sabine Sereinig ◽

Julia Romanova ◽

Boris Ferko ◽

...

Keyword(s):

Influenza A ◽

Reverse Genetics ◽

High Efficiency ◽

Mdck Cells ◽

Selection Process ◽

Gene Segment ◽

Vero Cells ◽

Deletion Mutants ◽

Ns1 Protein ◽

Ns Gene

ABSTRACT We established a reverse genetics system for the nonstructural (NS) gene segment of influenza A virus. This system is based on the use of the temperature-sensitive (ts) reassortant virus 25A-1. The 25A-1 virus contains the NS gene from influenza A/Leningrad/134/57 virus and the remaining gene segments from A/Puerto Rico (PR)/8/34 virus. This particular gene constellation was found to be responsible for the ts phenotype. For reverse genetics of the NS gene, a plasmid-derived NS gene from influenza A/PR/8/34 virus was ribonucleoprotein transfected into cells that were previously infected with the 25A-1 virus. Two subsequent passages of the transfection supernatant at 40°C selected viruses containing the transfected NS gene derived from A/PR/8/34 virus. The high efficiency of the selection process permitted the rescue of transfectant viruses with large deletions of the C-terminal part of the NS1 protein. Viable transfectant viruses containing the N-terminal 124, 80, or 38 amino acids of the NS1 protein were obtained. Whereas all deletion mutants grew to high titers in Vero cells, growth on Madin-Darby canine kidney (MDCK) cells and replication in mice decreased with increasing length of the deletions. In Vero cells expression levels of viral proteins of the deletion mutants were similar to those of the wild type. In contrast, in MDCK cells the level of the M1 protein was significantly reduced for the deletion mutants.

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Human HLA-A0201-restricted cytotoxic T lymphocyte recognition of influenza A is dominated by T cells bearing the V beta 17 gene segment.

Journal of Experimental Medicine ◽

10.1084/jem.181.1.79 ◽

1995 ◽

Vol 181 (1) ◽

pp. 79-91 ◽

Cited By ~ 193

Author(s):

P J Lehner ◽

E C Wang ◽

P A Moss ◽

S Williams ◽

K Platt ◽

...

Keyword(s):

T Cell ◽

T Lymphocyte ◽

Influenza A ◽

Cytotoxic T Lymphocyte ◽

Gene Segment ◽

Cell Subpopulation ◽

Specific Lysis ◽

Ctl Response ◽

The Matrix ◽

Limiting Dilution

The major histocompatibility complex class I-restricted cytotoxic T lymphocyte (CTL) response is important in the clearance of viral infections in humans. After influenza A infection, a peptide from the matrix protein, M58-66, is presented in the context of the MHC allele HLA-A0201 and the resulting CTL response is detectable in most HLA-A0201 subjects. An initial study suggested that M58-66-specific CTL clones show conserved T cell receptor (TCR) alpha and beta gene segments. We have addressed the significance of this observation by determining the expression of V beta 17 during the development of M58-66-specific CTL lines in 21 unrelated HLA-A0201 subjects, and analyzing TCR usage by M58-66-specific CTL clones. TCR V beta 17 was the dominant V beta segment used and CD8 V beta 17 expansion correlated with M58-66-specific lysis. Limiting dilution analysis from five subjects showed the M58-66 CTL precursor frequency to vary between 1/54,000 and less than 1/250,000, and that up to 85% of the matrix peptide (M58-66)-specific CTL used the V beta 17 gene segment. The M58-66 specific CTL response was dependent on previous viral exposure and specific V beta 17 expansion, as it was not found in cord blood, despite a readily expandable V beta 17+ CD8+ T cell subpopulation. Sequence analysis of 38 M58-66-specific V beta 17 transcripts from 13 subjects revealed extensive conservation in the CDR3 region including conservation of an arginine-serine motif. To test the dependence of this CTL response on the V beta 17 gene segment, peripheral blood lymphocytes were depleted of CD8+ TCR V beta 17+ cells, before the generation of M58-66-specific CTL. In most cases such depletion blocked or severely reduced the generation of the M58-66-specific response, and under limiting dilution conditions could abolish M58-66-specific CTL precursors. These studies reveal the dependence of this natural human immune response on a particular TCR gene segment.

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The Dynamics of Influenza A H3N2 Defective Viral Genomes from a Human Challenge Study

10.1101/814673 ◽

2019 ◽

Cited By ~ 1

Author(s):

Michael A. Martin ◽

Drishti Kaul ◽

Gene S. Tan ◽

Christopher W. Woods ◽

Katia Koelle

Keyword(s):

Genetic Drift ◽

Influenza A ◽

Evolutionary Dynamics ◽

De Novo ◽

Gene Segment ◽

Influenza Viruses ◽

Genomic Variation ◽

Wild Type Virus ◽

Single Nucleotide Variants ◽

Viral Genomes

AbstractThe rapid evolution of influenza is an important contributing factor to its high worldwide incidence. The emergence and spread of genetic point mutations has been thoroughly studied both within populations and within individual hosts. In addition, influenza viruses are also known to generate genomic variation during their replication in the form of defective viral genomes (DVGs). These DVGs are formed by internal deletions in at least one gene segment that render them incapable of replication without the presence of wild-type virus. DVGs have previously been identified in natural human infections and may be associated with less severe clinical outcomes. These studies have not been able to address how DVG populations evolve in vivo in individual infections due to their cross-sectional design. Here we present an analysis of DVGs present in samples from two longitudinal influenza A H3N2 human challenge studies. We observe the generation of DVGs in almost all subjects. Although the genetic composition of DVG populations was highly variable, identical DVGs were observed both between multiple samples within single hosts as well as between hosts. Most likely due to stochastic effects, we did not observe clear instances of selection for specific DVGs or for shorter DVGs over the course of infection. Furthermore, DVG presence was not found to be associated with peak viral titer or peak symptom scores. Our analyses highlight the diversity of DVG populations within a host over the course of infection and the apparent role that genetic drift plays in their population dynamics.ImportanceThe evolution of influenza virus, in terms of single nucleotide variants and the reassortment of gene segments, has been studied in detail. However, influenza is known to generate defective viral genomes (DVGs) during replication, and little is known about how these genomes evolve both within hosts and at the population level. Studies in animal models have indicated that prophylactically or therapeutically administered DVGs can impact patterns of disease progression. However, the formation of naturally-occurring DVGs, their evolutionary dynamics, and their contribution to disease severity in human hosts is not well understood. Here, we identify the formation of de novo DVGs in samples from human challenge studies throughout the course of infection. We analyze their evolutionary trajectories, revealing the important role of genetic drift in shaping DVG populations during acute infections with well-adapted viral strains.

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Rewiring the RNAs of influenza virus to prevent reassortment

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0908897106 ◽

2009 ◽

Vol 106 (37) ◽

pp. 15891-15896 ◽

Cited By ~ 52

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.

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An R195K Mutation in the PA-X Protein Increases the Virulence and Transmission of Influenza A Virus in Mammalian Hosts

Journal of Virology ◽

10.1128/jvi.01817-19 ◽

2020 ◽

Vol 94 (11) ◽

Cited By ~ 6

Author(s):

Yipeng Sun ◽

Zhe Hu ◽

Xuxiao Zhang ◽

Mingyue Chen ◽

Zhen Wang ◽

...

Keyword(s):

Avian Influenza ◽

Influenza A ◽

Reverse Genetics ◽

Inflammatory Responses ◽

Gene Segment ◽

Genetic Alterations ◽

Influenza Viruses ◽

Interspecies Transmission ◽

Adaptive Mutation ◽

Human Origin

ABSTRACT In the 21st century, the emergence of H7N9 and H1N1/2009 influenza viruses, originating from animals and causing severe human infections, has prompted investigations into the genetic alterations required for cross-species transmission. We previously found that replacement of the human-origin PA gene segment in avian influenza virus (AIV) could overcome barriers to cross-species transmission. Recently, it was reported that the PA gene segment encodes both the PA protein and a second protein, PA-X. Here, we investigated the role of PA-X. We found that an H9N2 avian influenza reassortant virus bearing a human-origin H1N1/2009 PA gene was attenuated in mice after the loss of PA-X. Reverse genetics analyses of PA-X substitutions conserved in human influenza viruses indicated that R195K, K206R, and P210L substitutions conferred significantly increased replication and pathogenicity on H9N2 virus in mice and ferrets. PA-X R195K was present in all human H7N9 and H1N1/2009 viruses and predominated in human H5N6 viruses. Compared with PA-X 195R, H7N9 influenza viruses bearing PA-X 195K showed increased replication and transmission in ferrets. We further showed that PA-X 195K enhanced lung inflammatory responses, potentially due to decreased host shutoff function. A competitive transmission study in ferrets indicated that 195K provides a replicative advantage over 195R in H1N1/2009 viruses. In contrast, PA-X 195K did not influence the virulence of H9N2 AIV in chickens, suggesting that the effects of the substitution were mammal specific. Therefore, future surveillance efforts should scrutinize this region of PA-X because of its potential impact on cross-species transmission of influenza viruses. IMPORTANCE Four influenza pandemics in humans (the Spanish flu of 1918 [H1N1], the Asian flu of 1957 [H2N2], the Hong Kong flu of 1968 [H3N2], and the swine origin flu of 2009 [H1N1]) are all proposed to have been caused by avian or swine influenza viruses that acquired virulence factors through adaptive mutation or reassortment with circulating human viruses. Currently, influenza viruses circulating in animals are repeatedly transmitted to humans, posing a significant threat to public health. However, the molecular properties accounting for interspecies transmission of influenza viruses remain unclear. In the present study, we demonstrated that PA-X plays an important role in cross-species transmission of influenza viruses. At least three human-specific amino acid substitutions in PA-X dramatically enhanced the adaptation of animal influenza viruses in mammals. In particular, PA-X 195K might have contributed to cross-species transmission of H7N9, H5N6, and H1N1/2009 viruses from animal reservoirs to humans.

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Non-Uniform and Non-Random Binding of Nucleoprotein to Influenza A and B Viral RNA

Viruses ◽

10.3390/v10100522 ◽

2018 ◽

Vol 10 (10) ◽

pp. 522 ◽

Cited By ~ 10

Author(s):

Valerie Le Sage ◽

Adalena Nanni ◽

Amar Bhagwat ◽

Dan Snyder ◽

Vaughn Cooper ◽

...

Keyword(s):

Influenza A ◽

Gene Segment ◽

Influenza Viruses ◽

Viral Rna ◽

Influenza B Virus ◽

Viral Gene ◽

Influenza B ◽

Influenza A Viruses ◽

Binding Profile ◽

Random Manner

The genomes of influenza A and B viruses have eight, single-stranded RNA segments that exist in the form of a viral ribonucleoprotein complex in association with nucleoprotein (NP) and an RNA-dependent RNA polymerase complex. We previously used high-throughput RNA sequencing coupled with crosslinking immunoprecipitation (HITS-CLIP) to examine where NP binds to the viral RNA (vRNA) and demonstrated for two H1N1 strains that NP binds vRNA in a non-uniform, non-random manner. In this study, we expand on those initial observations and describe the NP-vRNA binding profile for a seasonal H3N2 and influenza B virus. We show that, similar to H1N1 strains, NP binds vRNA in a non-uniform and non-random manner. Each viral gene segment has a unique NP binding profile with areas that are enriched for NP association as well as free of NP-binding. Interestingly, NP-vRNA binding profiles have some conservation between influenza A viruses, H1N1 and H3N2, but no correlation was observed between influenza A and B viruses. Our study demonstrates the conserved nature of non-uniform NP binding within influenza viruses. Mapping of the NP-bound vRNA segments provides information on the flexible NP regions that may be involved in facilitating assembly.

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Characterization of recombinant influenza A virus as a vector expressing respiratory syncytial virus fusion protein epitopes

Journal of General Virology ◽

10.1099/vir.0.064105-0 ◽

2014 ◽

Vol 95 (9) ◽

pp. 1886-1891 ◽

Cited By ~ 6

Author(s):

Peirui Zhang ◽

Hongjing Gu ◽

Chengrong Bian ◽

Na Liu ◽

Zhiwei Li ◽

...

Keyword(s):

Influenza Virus ◽

Respiratory Syncytial Virus ◽

Cell Line ◽

Nuclear Export ◽

Influenza A ◽

Haemagglutination Inhibition ◽

The Elderly ◽

Ns Gene ◽

Syncytial Virus

Respiratory syncytial virus (RSV) is the most common cause of respiratory infection in infants and the elderly, and no vaccine against this virus has yet been licensed. Here, we report a recombinant PR8 influenza virus with the RSV fusion (F) protein epitopes of the subgroup A gene inserted into the influenza virus non-structural (NS) gene (rFlu/RSV/F) that was generated as an RSV vaccine candidate. The rescued viruses were assessed by microscopy and Western blotting. The proper expression of NS1, the NS gene product, and the nuclear export protein (NEP) of rFlu/RSV/F was also investigated using an immunofluorescent assay. The rescued virus replicated well in the MDCK kidney cell line, A549 lung adenocarcinoma cell line and CNE-2Z nasopharyngeal carcinoma cell line. BALB/c mice immunized intranasally with rFlu/RSV/F had specific haemagglutination inhibition antibody responses against the PR8 influenza virus and RSV neutralization test proteins. Furthermore, intranasal immunization with rFlu/RSV/F elicited T helper type 1-dominant cytokine profiles against the RSV strain A2 virus. Taken together, our findings suggested that rFlu/RSV/F was immunogenic in vivo and warrants further development as a promising candidate vaccine.

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