Characterization of Neuraminidases from the Highly Pathogenic Avian H5N1 and 2009 Pandemic H1N1 Influenza A Viruses

Abstract

To study the precise role of the neuraminidase (NA), and its stalk region in particular, in the assembly, release, and entry of influenza virus, we deleted the 20-aa stalk segment from 2009 pandemic H1N1 NA (09N1) and inserted this segment, now designated 09s60, into the stalk region of a highly pathogenic avian influenza (HPAI) virus H5N1 NA (AH N1). The biological characterization of these wild-type and mutant NAs was analyzed by pseudotyped particles (pseudoparticles) system. Compared with the wild-type AH N1, the wild-type 09N1 exhibited higher NA activity and released more pseudoparticles. Deletion/insertion of the 09s60 segment did not alter this relationship. The infectivity of pseudoparticles harboring NA in combination with the hemagglutinin from HPAI H5N1 (AH H5) was decreased by insertion of 09s60 into AH N1 and was increased by deletion of 09s60 from 09N1. When isolated from the wild-type 2009H1N1 virus, 09N1 existed in the forms (in order of abundance) dimer>>tetramer>monomer, but when isolated from pseudoparticles, 09N1 existed in the forms dimer>monomer>>>tetramer. After deletion of 09s60, 09N1 existed in the forms monomer>>>dimer. AH N1 from pseudoparticles existed in the forms monomer>>dimer, but after insertion of 09s60, it existed in the forms dimer>>monomer. Deletion/insertion of 09s60 did not alter the NA glycosylation pattern of 09N1 or AH N1. The 09N1 was more sensitive than the AH N1 to the NA inhibitor oseltamivir, suggesting that the infectivity-enhancing effect of oseltamivir correlates with robust NA activity.

Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice

“…The NA VLPs were spheroidal and approximately 50–200 µm in diameter, similar in morphology to influenza virions (Figure 1) and similar to a previous published full characterization of VLPs expressing only NA and M (Lai et al, 2010). When standardized by the amount of protein, the activity of CA/09 NA VLPs was about 2-fold higher than VN/1203 NA VLPs for both the small NA-XTD substrate (1704 and 879 mU/mg protein, respectively), as well as for the larger fetuin substrate (576 and 341 mU/mg protein, respectively), consistent with previously reported higher NA enzymatic activities of H1N1pdm09 viruses as compared with highly pathogenic H5N1 viruses (Lin et al, 2009; Wu et al, 2010). NA proteins are inherently unstable, and may be even less stable when originating from H5N1 viruses because several potential glycosylation sites and a cysteine that are likely to contribute to tetrameric stability are deleted in the short-stalk NA of HPAI H5N1 viruses (Air, 2011; Blok and Air, 1982).…”

Cross-Reactive Neuraminidase-Inhibiting Antibodies Elicited by Immunization with Recombinant Neuraminidase Proteins of H5N1 and Pandemic H1N1 Influenza A Viruses

“…Each NA monomer consists of six identical ␤ sheets (␤) plus several strands (S) and loops (L). Influenza NA enzyme catalytic sites are located at the upper corners of each monomer (tetrameric structure); amino acids encircling active catalytic sites are highly conserved among various influenza A and B virus strains (11,33) NI antibodies are important for NA-based vaccine development. Other researchers have reported that NI antibodies induced by pH1N1 immunizations are cross-reactive with H5N1 viruses in mice (34) and ferrets (21,22).…”

Construction high-yield candidate influenza vaccine viruses in Vero cells by reassortment

“…Among these new technologies, the cells culture‐based manufacturing vaccine offers some significant advantages. In several recent publications, it has described selection of highly productive vaccine strain in Vero cells [Wu et al, ; Zhang et al, ].…”