Multiple gene segment reassortment between Eurasian and American lineages of influenza A virus (H6N2) in Guillemot (Uria aalge)

ABSTRACT The influenza A virus PB1-F2 protein has been implicated as a virulence factor, but the mechanism by which it enhances pathogenicity is not understood. The PB1 gene segment of the H1N1 swine-origin influenza virus pandemic strain codes for a truncated PB1-F2 protein which terminates after 11 amino acids but could acquire the full-length form by mutation or reassortment. It is therefore important to understand the function and impact of this protein. We systematically assessed the effect that PB1-F2 expression has on viral polymerase activity, accumulation and localization of PB1, and replication in vitro and in mice. We used both the laboratory strain PR8 and a set of viruses engineered to study clinically relevant PB1-F2 proteins. PB1-F2 expression had modest effects on polymerase activity, PB1 accumulation, and replication that were cell type and virus strain dependent. Disruption of the PB1-F2 reading frame in a recent, seasonal H3N2 influenza virus strain did not affect these parameters, suggesting that this is not a universal function of the protein. Disruption of PB1-F2 expression in several backgrounds or expression of PB1-F2 from the 1918 pandemic strain or a 1956 H1N1 strain had no effect on viral lung loads in mice. Alternate mechanisms besides alterations to replication are likely responsible for the enhanced virulence in mammalian hosts attributed to PB1-F2 in previous studies.

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Identification of the 5′-Terminal Packaging Signal of the H1N1 Influenza A Virus Neuraminidase Segment at Single-Nucleotide Resolution

Frontiers in Microbiology ◽

10.3389/fmicb.2021.709010 ◽

2021 ◽

Vol 12 ◽

Author(s):

Erika Seshimo ◽

Fumitaka Momose ◽

Yuko Morikawa

Keyword(s):

Influenza A Virus ◽

Influenza A ◽

Synonymous Codon ◽

Gene Segment ◽

H1n1 Influenza ◽

Terminal Region ◽

Selective Assembly ◽

Wild Type ◽

Packaging Signal ◽

Nucleotide Resolution

The genome of the influenza A virus is an eight-segmented negative-strand RNA (vRNA). Progeny vRNAs replicated in the nucleus selectively assemble into a single set of eight different segments, probably in the cytoplasm, and are packaged into progeny virions at the cell membrane. In these processes, a region of approximately 100 nucleotides at both ends of each segment is thought to function as a selective assembly/packaging signal; however, the details of the mechanism, such as the required sequences, are still unknown. In this study, we focused on the 5′-terminus of the sixth neuraminidase gene segment vRNA (Seg.6) to identify the essential sequence for selective packaging. The 5′-terminal region of the A/Puerto Rico/8/34 strain Seg.6 was divided into seven regions of 15 nucleotides each from A to G, and mutations were introduced into each region by complementary base substitutions or synonymous codon substitutions. Mutant viruses were generated and compared for infectious titers, and the relative ratios of the eight segments packaged into virions were measured. We also ascertained whether mutant vRNA was eliminated by competitive packaging with wild-type vRNA. Mutations in the A–C regions reduced infectious titers and eliminated mutant vRNAs by competition with wild-type vRNA. Even under non-competitive conditions, the packaging efficiency of the A or B region mutant Seg.6 was reduced. Next, we designed an artificial vRNA with a 50-nucleotide duplication at the 5′-terminal region. Using this, a virus library was created by randomly replacing each region, which became an untranslated region (UTR), with complementary bases. After selecting proliferative viruses from the library, nine wild-type nucleotides in the A and B regions were identified as essential bases, and we found that these bases were highly conserved in Seg.6 vRNAs encoding the N1 subtype neuraminidase. From these results, we conclude that the identified bases function as the 5′-terminal packaging signal for the N1 subtype Seg.6 vRNA.

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THE NONSTRUCTURAL GENE SEGMENT OF INFLUENZA A VIRUS: EXPRESSION OF NS1 PROTEIN IN MAMMALIAN CELLS; ANALYSIS OF A DELETION MUTANT

Segmented Negative Strand Viruses ◽

10.1016/b978-0-12-183501-9.50026-1 ◽

1984 ◽

pp. 147-157

Author(s):

Mark Krystal ◽

Susumu Nakada ◽

Deborah A. Buonagurio ◽

Dan C. DeBorde ◽

H.F. Maassab ◽

...

Keyword(s):

Influenza A Virus ◽

Mammalian Cells ◽

Influenza A ◽

Deletion Mutant ◽

Gene Segment ◽

Ns1 Protein ◽

Virus Expression

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A compensatory mutagenesis study of a conserved hairpin in the M gene segment of influenza A virus shows its role in virus replication

RNA Biology ◽

10.1080/15476286.2017.1338243 ◽

2017 ◽

Vol 14 (11) ◽

pp. 1606-1616 ◽

Cited By ~ 7

Author(s):

M. I. Spronken ◽

C. E. van de Sandt ◽

E. P. de Jongh ◽

O. Vuong ◽

S. van der Vliet ◽

...

Keyword(s):

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Gene Segment ◽

M Gene ◽

Mutagenesis Study

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The substantia nigra is a major target for neurovirulent influenza A virus.

Journal of Experimental Medicine ◽

10.1084/jem.181.6.2161 ◽

1995 ◽

Vol 181 (6) ◽

pp. 2161-2169 ◽

Cited By ~ 75

Author(s):

M Takahashi ◽

T Yamada ◽

S Nakajima ◽

K Nakajima ◽

T Yamamoto ◽

...

Keyword(s):

Substantia Nigra ◽

Influenza A Virus ◽

Influenza A ◽

Matrix Protein ◽

Gene Segment ◽

Positive Staining ◽

Influenza A Viruses ◽

Postencephalitic Parkinsonism ◽

Causative Agents ◽

Immunohistochemical Studies

Clinical and immunohistochemical studies were done for 3-39 d on mice after intracerebral inoculation with the neurovirulent A/WSN/33 (H1N1; WSN) strain of influenza A virus, the nonneurovirulent A/Aichi/2/68 (H3N2; Aichi) strain, and two reassortant viruses between them. The virus strains with the WSN gene segment coding for neuraminidase induced meningoencephalitis in mice. The mice inoculated with the R96 strain, which has only the neuraminidase gene from the WSN strain, had mild symptoms and weak positive immunostaining to the anti-WSN antibody in meningeal regions. Both the WSN and R404BP strains, which contain the WSN gene segments coding for neuraminidase and matrix protein, were clearly neurovirulent both clinically and pathologically. On day 3 after inoculation with either of these two strains, WSN antigen was detected in meningeal and ependymal areas, neurons of circumventricular regions, the cerebral and cerebellar cortices, the substantia nigra zona compacta, and the ventral tegmental area. On day 7, meningeal reactions and neuronal staining were still seen, and advanced accumulation of the viral antigen was evident in the substantia nigra zona compacta and hippocampus. Double immunostaining demonstrated that the WSN antigen was only seen in neurons and not in microglia or reactive astrocytes. Immunostaining for the lectin maackia amurensis agglutinin, which recognizes the Neu5Ac alpha 2,3 Gal sequence, which serves as a binding site for influenza A virus on target cell membranes, showed that positive staining was localized in the ventral substantia nigra and hippocampus. These results suggest that neurovirulent influenza A viruses could be one of the causative agents for postencephalitic parkinsonism.

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

Journal of Virology ◽

10.1128/jvi.01725-12 ◽

2012 ◽

Vol 86 (22) ◽

pp. 12341-12350 ◽

Cited By ~ 10

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

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Australia as a global sink for the genetic diversity of avian influenza A virus

10.1101/2021.11.30.470533 ◽

2021 ◽

Author(s):

Michelle Wille ◽

Victoria Grillo ◽

Silvia Ban de Gouvea Pedroso ◽

Graham W. Burgess ◽

Allison Crawley ◽

...

Keyword(s):

Avian Influenza ◽

Southern Hemisphere ◽

Influenza A Virus ◽

Influenza A ◽

Gene Segment ◽

Wild Birds ◽

Long Distance ◽

Migratory Shorebirds ◽

Australian Continent ◽

Avian Influenza A Virus

Most of our understanding of the ecology and evolution of avian influenza A virus (AIV) in wild birds is derived from studies conducted in the northern hemisphere on waterfowl, with a substantial bias towards dabbling ducks. However, relevant environmental conditions and patterns of avian migration and reproduction are substantially different in the southern hemisphere. Through the sequencing and analysis of 333 unique AIV genomes collected from wild birds collected over 15 years we show that Australia is a global sink for AIV diversity and not integrally linked with the Eurasian gene pool. Rather, AIV are infrequently introduced to Australia, followed by decades of isolated circulation and eventual extinction. The number of co-circulating viral lineages varies per subtype. AIV haemagglutinin (HA) subtypes that are rarely identified at duck-centric study sites (H8-12) had more detected introductions and contemporary co-circulating lineages in Australia. Combined with a lack of duck migration beyond the Australian-Papuan region, these findings suggest introductions by long-distance migratory shorebirds. In addition, we found no evidence of directional or consistent patterns in virus movement across the Australian continent. This feature corresponds to patterns of bird movement, whereby waterfowl have nomadic and erratic rainfall-dependant distributions rather than consistent intra-continental migratory routes. Finally, we detected high levels of virus gene segment reassortment, with a high diversity of AIV genome constellations across years and locations. These data, in addition to those from other studies in Africa and South America, clearly show that patterns of AIV dynamics in the Southern Hemisphere are distinct from those in the temperate north.

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

Journal of General Virology ◽

10.1099/vir.0.039701-0 ◽

2012 ◽

Vol 93 (5) ◽

pp. 998-1006 ◽

Cited By ~ 14

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.

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