The Special Neuraminidase Stalk-Motif Responsible for Increased Virulence and Pathogenesis of H5N1 Influenza A Virus

Zhou, Hongbo; Yu, Zhengjun; Hu, Yong; Tu, Jiagang; Zou, Wei; Peng, Yaping; Jun, Zhu; Li, Yongtao; Zhang, Anding; Yu, Ziniu; Ye, Zhiping; Chen, Huanchun; Jin, Meilin

doi:10.1371/journal.pone.0006277

Cited by 125 publications

(140 citation statements)

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“…1a). The longer stalk region of the H5N1 NA has been shown to contribute to pathogenicity (Zhou et al, 2009). Here, we confirmed that removing this region reduced pathogenicity, with or without the PB1 M5 allele, by determining mean death time (MDT).…”

supporting

confidence: 73%

“…We have also described the improved growth and protein yield of a reassortant containing the H5N1 A/Vietnam/1203/2004 (A/VN/1203) HA segment and a chimeric NA segment comprising the C terminus (non-coding region and first 72 aa) from the A/ VN/1203 strain with the N terminus from PR/8 (Adamo et al, 2009). The stalk region of the NA protein differs between different influenza virus strains in both length and amino acid identity, and has been shown to affect growth, protein yield and pathogenesis (Adamo et al, 2009;Baigent & McCauley, 2001;Zhou et al, 2009). Here, we continued these studies by creating reassortants containing PB1 wt or PB1 m5 and the A/VN/1203 HA with either the WT (NA wt ) or chimeric (NA Ch ) NA from A/VN/1203.…”

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confidence: 99%

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Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus

Plant

2015

Journal of General Virology

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We previously showed that a mutated PB1 gene improved the growth kinetics of a H3N2 influenza reassortant. Here, we showed that the same mutations improved the growth kinetics of a virus containing the A/Vietnam/1203/2004 (H5N1) haemagglutinin and neuraminidase (NA). Total protein yield and NA activity were increased when a chimeric NA was included. These increases indicated that the synergistic effect was due to the gene constellation containing both the altered PB1 gene and the chimeric NA gene.The object of this work was to improve the genomic backbone segments used in vaccine candidate viruses whilst avoiding detrimental gene constellation effects. It is not known what attributes from each segment affect the gene constellation or reassortment, but it is desirable that vaccine seed strains containing contemporary haemagglutinin (HA) and neuraminidase (NA) segments in the context of known backbone segments can be recovered. It is also desirable that those strains replicate well and that satisfactory protein yields can be harvested. We have previously shown that five mutations to the PB1 gene of the high-growth backbone virus PR/8 improved the growth kinetics of a reassortant containing the HA and NA segments from an H3N2 virus (Plant et al., 2012). These mutations (G180E, S216G, S361R, Q621R and N654S) were not selected based on any structural or functional information. The five mutations made to the PB1 gene (PB1 m5 ) were based on sequence variations found in vaccine strain reassortants. We have also described the improved growth and protein yield of a reassortant (Table 1) and all the viruses produced large plaques on MDCK cells (Fig. 1a). The longer stalk region of the H5N1 NA has been shown to contribute to pathogenicity (Zhou et al., 2009). Here, we confirmed that removing this region reduced pathogenicity, with or without the PB1 M5 allele, by determining mean death time (MDT). Virus particles (10 6 ) were inoculated into the allantoic fluid of eight eggs, which were then incubated at 35 u C and candled at 12 h intervals. Eggs were scored as alive or dead and the time of death recorded. MDT was obtained by multiplying the number of embryos dead at each interval post-inoculation by the hours post-inoculation and dividing by the total number of embryos inoculated. The MDT range was similar for all viruses (Table 1).Optimal polymerase activity is required for efficient replication (Giesendorf et al., 1986;Li et al., 2009;Ngai et al., 2013). PB1 from a seasonal strain or with targeted mutations has been shown to improve growth or HA yield of H3 reassortants (Cobbin et al., 2013;Plant et al., 2012); however, mixed results have been observed for H5 reassortants (Abt et al., 2011;Rudneva et al., 2007;Wanitchang et al., 2010). All three polymerase segments reassort together more frequently in experimental settings than natural reassortment, suggesting gene constellation effects (Macken et al., 2006;Varich et al., 2008;Wille et al., 2013). Here, we examined the growth of H5 reassortants with the PB1 m5 allele w...

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confidence: 73%

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confidence: 99%

Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus

Plant

2015

Journal of General Virology

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“…Castrucci and Kawaoka demonstrated in 1993 that mutated A/WSN/33 (H1N1) strains with a shortened stalk replicated poorly in eggs and did not cause systemic disease (18). In 2009 Matsuoka et al (19) and Zhou et al (20) both showed higher virulence of H5N1 viruses with longer NA stalks in chickens, but their studies reported varying effects in mice (19,20). Although isolates with the NA stalk deletion did grow, we did not proceed with them because of the importance of egg growth for current manufacturing of influenza vaccines.…”

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confidence: 92%

Feasibility of reconstructed ancestral H5N1 influenza viruses for cross-clade protective vaccine development

Ducatez

Bahl

Griffin

et al. 2010

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

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Since the reemergence of highly pathogenic H5N1 influenza viruses in humans in 2003, these viruses have spread throughout avian species in Asia, Europe, and Africa. Their sustained circulation has resulted in the evolution of phylogenetically diverse lineages. Viruses from these lineages show considerable antigenic variation, which has confounded vaccine planning efforts. We reconstructed ancestral protein sequences at several nodes of the hemagglutinin (HA) and neuraminidase (NA) gene phylogenies that represent ancestors to diverse H5N1 virus clades. By using the same methods that have been used to generate currently licensed inactivated H5N1 vaccines, we were able to produce a panel of replication competent influenza viruses containing synthesized HA and NA genes representing the reconstructed ancestral proteins. We identified two of these viruses that showed promising in vitro cross-reactivity with clade 1, 2.1, 2.2, 2.3.4, and 4 viruses. To confirm that vaccine antigens derived from these viruses were able to elicit functional antibodies following immunization, we created whole-virus vaccines and compared their protective efficacy versus that of antigens from positive control, naturally occurring, and broadly reactive H5N1 viruses. The ancestral viruses’ vaccines provided robust protection against morbidity and mortality in ferrets challenged with H5N1 strains from clades 1, 2.1, and 2.2 in a manner similar to those based on the control strains. These findings provide proof of principle that viable, computationally derived vaccine seed viruses can be constructed within the context of currently licensed vaccine platforms. Such technologies should be explored to enhance the cross reactivity and availability of H5N1 influenza vaccines.

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“…The NA stalk of SC-H5N1 viruses had a 20 aa deletion (positions 49-68), which might enhance virus adaptation to domestic fowl and increase virulence in mammals (Cauldwell et al, 2014;Matsuoka et al, 2009;Zhou et al, 2009). This 20 aa deletion was not found in the NA stalk of SC-H5N6 and the human-H5N6 strain.…”

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Two novel reassortants of avian influenza A (H5N6) virus in China

Shi

et al. 2015

Journal of General Virology

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The Special Neuraminidase Stalk-Motif Responsible for Increased Virulence and Pathogenesis of H5N1 Influenza A Virus

Cited by 125 publications

References 33 publications

Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus

Chimeric neuraminidase and mutant PB1 gene constellation improves growth and yield of H5N1 vaccine candidate virus

Feasibility of reconstructed ancestral H5N1 influenza viruses for cross-clade protective vaccine development

Two novel reassortants of avian influenza A (H5N6) virus in China

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