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

Abstract

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.

Induction of humoral and cell-mediated immunity to the NS1 protein of TBEV with recombinant Influenza virus and MVA affords partial protection against lethal TBEV infection in mice

“…Insertion of the NS1 gene did not affect the attenuated phenotype of both recombinant vectors. In the absence of exogenous NA, NA-deficient IAV-RBD and IAV-NS1 replicated only to low titers, which is in concordance with previous studies (56,57). Attenuation is achieved by the replacement of a large part of the IAV NA, which as receptor destroying enzyme plays an important role in the IAV replication cycle and release of newly budded IAV virions.…”

RNA Elements in Open Reading Frames of the Bluetongue Virus Genome Are Essential for Virus Replication

“…This explains the high recombination incidence examined. Recently, similar events have also been shown for influenza virus [53] .…”

Bat-Borne Influenza A Viruses: An Awakening

“…Although an explanation for the selection of truncated N11 NA is still lacking, it is tempting to speculate that the negatively charged N11 NA head domain might disturb proper H18 HA-mediated entry caused by repulsions with the also negatively charged cellular membrane. Importantly, even though NA-negative mutants have been described for classical IAVs before, the ability of H18N11 to compensate for the lack of a functional NA without impaired viral growth in vitro and in vivo is unprecedented among all other known classical subtypes of influenza (Hughes et al 2000;Kalthoff et al 2013;Samson et al 2014;Ann et al 2016). Whether H17N10 possesses a comparable N10 NA-independent replication capacity remains to be determined.…”