In vitro characterization of naturally occurring influenza H3NA− viruses lacking the NA gene segment: Toward a new mechanism of viral resistance?

Moulés, Vincent; Ferraris, Olivier; Terrier, B.; Giudice, Emmanuel; Yver, Matthieu; Rolland, Jean-Paul; Bouscambert‐Duchamp, Maude; Bergeron, Corinne; Ottmann, Michèle; Fournier, E; Traversier, Aurélien; Boule, C.; Rivoire, Annie; Lin, Yi Pu; Hay, Alan J.; Valette, Martine; Marquet, Roland; Rosa‐Calatrava, Manuel; Naffakh, Nadia; Schoehn, Guy; Thomas, Daniel; Lina, Bruno

doi:10.1016/j.virol.2010.04.030

Cited by 31 publications

(36 citation statements)

References 43 publications

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“…However, in the latter case and in contrast to our variants with NA deletions, attempts to isolate a neuraminidase-negative virus strain were not successful, and the authors therefore suggested that full-length segment 6 remained in the virus population at a lower frequency. A complete loss of a neuraminidaseencoding segment or neuraminidase function is deemed impossible (41), with the only example of this being in a human H3 isolate which was claimed to lack the complete segment 6 and addressed as a seven-segmented influenza A virus (42). Alternatively, the growth of a neuraminidase-negative influenza A virus without supplementation of exogenous sialidase was demonstrated in vitro and in mice only if the loss of neuraminidase activity was accompanied by mutations around the HA receptor-binding pocket, which lowered the avidity for receptors (43,44).…”

Section: Discussionmentioning

confidence: 99%

Truncation and Sequence Shuffling of Segment 6 Generate Replication-Competent Neuraminidase-Negative Influenza H5N1 Viruses

Kalthoff

Röhrs

Höper

et al. 2013

J Virol

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Influenza viruses are highly genetically variable and escape from immunogenic pressure by antigenic changes in their surface proteins, referred to as "antigenic drift" and "antigenic shift." To assess the potential genetic plasticity under strong selection pressure, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was passaged 50 times in embryonated chicken eggs in the presence of a neutralizing, polyclonal chicken serum. The resulting mutant acquired major alterations in the neuraminidase (NA)-encoding segment. Extensive deletions and rearrangements were detected, in contrast to only 12 amino acid substitutions within all other segments. Interestingly, this new neuraminidase segment resulted from complex sequence shuffling and insertion of a short fragment originating from the PA segment. Characterization of that novel variant revealed a loss of the neuraminidase protein and enzymatic activity, but its replication efficiency remained comparable to that of the wild type. Using reverse genetics, a recombinant virus consisting of the wild-type backbone and the shortened NA segment could be generated; however, generation of this recombinant virus required the polybasic hemagglutinin cleavage site. Two independent repetitions starting with egg passage 30 in the presence of alternative chicken-derived immune sera selected mutants with similar but different large deletions within the NA segment without any neuraminidase activity, indicating a general mechanism. In chicken, these virus variants were avirulent, even though the HPAIV polybasic hemagglutinin cleavage site was still present. Overall, the variants reported here are the first HPAIV H5N1 strains without a functional neuraminidase shown to grow efficiently without any helper factor. These novel HPAIV variants may facilitate future studies shedding light on the role of neuraminidase in virus replication and pathogenicity.

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Section: Discussionmentioning

confidence: 99%

Truncation and Sequence Shuffling of Segment 6 Generate Replication-Competent Neuraminidase-Negative Influenza H5N1 Viruses

Kalthoff

Röhrs

Höper

et al. 2013

J Virol

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“…To do so, viral RNA was extracted from the infected MDCK cell-culture supernatant by using the QIAamp viral RNA minikit (Qiagen) according to the manufacturer's instructions. A twostep RT-PCR was carried out for full-length amplification of each viral RNA gene segment, by using the influenza A universal RT primer 5′-AGC AAA AGC AGG-3′ (Eurogentec) (46). The cDNAs obtained from the different genes were cloned into the pHW2000 vector (47) allowing expression of both negative-and positive-strand RNAs and viral proteins from two different promoters.…”

Section: Methodsmentioning

confidence: 99%

Critical role of segment-specific packaging signals in genetic reassortment of influenza A viruses

Essere

Yver

Gavazzi

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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The fragmented nature of the influenza A genome allows the exchange of gene segments when two or more influenza viruses infect the same cell, but little is known about the rules underlying this process. Here, we studied genetic reassortment between the A/Moscow/10/99 (H3N2, MO) virus originally isolated from human and the avian A/Finch/England/2051/91 (H5N2, EN) virus and found that this process is strongly biased. Importantly, the avian HA segment never entered the MO genetic background alone but always was accompanied by the avian PA and M fragments. Introduction of the 5′ and 3′ packaging sequences of HA MO into an otherwise HA EN backbone allowed efficient incorporation of the chimerical viral RNA (vRNA) into the MO genetic background. Furthermore, forcing the incorporation of the avian M segment or introducing five silent mutations into the human M segment was sufficient to drive coincorporation of the avian HA segment into the MO genetic background. These silent mutations also strongly affected the genotype of reassortant viruses. Taken together, our results indicate that packaging signals are crucial for genetic reassortment and that suboptimal compatibility between the vRNA packaging signals, which are detected only when vRNAs compete for packaging, limit this process.hemagglutinin | RNA packaging

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“…We used an eight-plasmid system to generate viruses in the A/Moscow/10/99 H3N2 background (28). cDNAs were cloned in the pHW2000 plasmid as already described (42). Mutations were introduced in the NA plasmid using the QuikChange site-directed mutagenesis kit (Stratagene).…”

Section: Methodsmentioning

confidence: 99%

“…Infectious titers (doses infecting 50% of the cell culture [TCID 50 ]/50 l) were performed in confluent MDCK and MDCK-SIAT1 cells as previously described (42). The cytopathic effect was visible after incubation at 34°C for 96 h, and the test was revealed by hemagglutination tests performed with 0.5% chicken red blood.…”

Section: Methodsmentioning

confidence: 99%

Combinatorial Effect of Two Framework Mutations (E119V and I222L) in the Neuraminidase Active Site of H3N2 Influenza Virus on Resistance to Oseltamivir

Richard

Ferraris

Erny

et al. 2011

Antimicrob Agents Chemother

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Neuraminidase (NA) inhibitors (NIs) are the first line of defense against influenza virus. Reverse genetics experiments allow the study of resistance mechanisms by anticipating the impacts of mutations to the virus. To look at the possibility of an increased effect on the resistance phenotype of a combination of framework mutations, known to confer resistance to oseltamivir or zanamivir, with limited effect on virus fitness, we constructed 4 viruses by reverse genetics in the A/Moscow/10/99 H3N2 background containing double mutations in their neuraminidase genes: E119D؉I222L, E119V؉I222L, D198N؉I222L, and H274Y؉I222L (N2 numbering). Among the viruses produced, the E119D؉I222L mutant virus was not able to grow without bacterial NA complementation and the D198N؉I222L mutant and H274Y؉I222L mutant were not stable after passages in MDCK cells. The E119V؉I222L mutant was stable after five passages in MDCK cells. This E119V-and-I222L combination had a combinatorial effect on oseltamivir resistance. The total NA activity of the E119V؉I222L mutant was low (5% compared to that of the wild-type virus). This drop in NA activity resulted from a decreased NA quantity in the virion in comparison to that of the wild-type virus (1.4% of that of the wild type). In MDCK-SIAT1 cells, the E119V؉I222L mutant virus did not present a replicative advantage over the wild-type virus, even in the presence of oseltamivir. Double mutations combining two framework mutations in the NA gene still have to be monitored, as they could induce a high level of resistance to NIs, without impairing the NA affinity. Our study allows a better understanding of the diversity of the mechanisms of resistance to NIs.The influenza A virus presents two major surface glycoproteins: hemagglutinin (HA) and neuraminidase (NA). HA mediates virus entry into the cell by binding to terminal sialic acid (N-acetyl neuraminic acid) of cellular glycoconjugates (52). NA, through its sialidase activity, facilitates the elution of progeny virions from infected cells through the cleavage of terminal sialic acid from viral and cellular glycoconjugates (36,44,45).Among the nine NA subtypes described, the crystal structures of the N9 (5, 57), N2 (58, 59), N6, and N1/N4/N8 subtypes (51) have been resolved. The NA head is a tetramer composed of four identical subunits arranged with a circular 4-fold symmetry. Each monomer contains six beta sheets composed of 4 antiparallel strands. The NA active site is located centrally on each unit and forms a pocket composed of conserved residues found in all influenza A virus NA (16). All these residues form the wall of the catalytic pocket. It includes catalytic sites (R118, D151, R152, R224, E276, R292, R371, and Y406) that interact directly with the substrate, sialic acid, and framework sites (E119, R156, W178, S179, D/N198, I222, E227, H274, E277, N294, and E425) that interact with catalytic residues to stabilize the active site (15).Due to its conserved structure and its essential role in viral replication, NA is an attractive antiviral t...

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In vitro characterization of naturally occurring influenza H3NA− viruses lacking the NA gene segment: Toward a new mechanism of viral resistance?

Cited by 31 publications

References 43 publications

Truncation and Sequence Shuffling of Segment 6 Generate Replication-Competent Neuraminidase-Negative Influenza H5N1 Viruses

Truncation and Sequence Shuffling of Segment 6 Generate Replication-Competent Neuraminidase-Negative Influenza H5N1 Viruses

Critical role of segment-specific packaging signals in genetic reassortment of influenza A viruses

Combinatorial Effect of Two Framework Mutations (E119V and I222L) in the Neuraminidase Active Site of H3N2 Influenza Virus on Resistance to Oseltamivir

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