Effect of gene constellation and postreassortment amino acid change on the phenotypic features of H5 influenza virus reassortants

Руднева, И. А.; Тимофеева, Т. А.; Шилов, А. А.; Kochergin-Nikitsky, Konstantin S.; Varich, N. L.; Ilyushina, Natalia A.; Gambaryan, Alexandra; Krylov, P. S.; Каверин, Н. В.

doi:10.1007/s00705-006-0931-8

Cited by 30 publications

(23 citation statements)

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“…Thus, the reassortants, which contain the HA and NA genes of a human epidemic virus and the other genes of PR8 virus were shown to be as high-yield as the parent PR8 strain (6). Conversely, the reassortants having HA and NA of avian influenza viruses often produce moderate yields in the embryonated chicken eggs, intermediate between the yield of the avian parent virus and the A/Puerto Rico/8/34 (H1N1) strain (8,14). In the present studies we detected a similar situation with respect to the pandemic "swine-like" virus of 2009.…”

supporting

confidence: 68%

“…In our previous studies with avian-human reassortants we observed an expressed increase of virus yield produced by the introduction of the avian virus PB1 gene in the genetic content of the reassortant (14). An enhancement of virus yield by the introduction of the PB1 gene of the 2009 pandemic virus in the genetic content of a reverse genetics-derived reassortant was also reported (16).…”

mentioning

confidence: 79%

“…The immunization of guinea pigs was performed as described in our earlier publication (14). The sera were heated for 30 min at 56°C.…”

mentioning

confidence: 99%

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Yields of virus reassortants containing the HA gene of pandemic influenza 2009 virus

Ta¹,

Av²,

Ia³

et al. 2012

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The recent pandemic, due to the swine-origin 2009 H1N1 influenza virus (11), raises a concern about the future appearance of drift variants and the new outbreaks caused by the H1N1 subtype. There is a need to develop high-yield strains for the production of inactivated, split, or subunit vaccines. The strains for the production of inactivated or subunit vaccines produced in embryonated chicken eggs usually contain hemagglutinin (HA) and neuraminidase (NA) of an epidemic isolate, and the other genes derived from the high-yield A/ Puerto Rico/8/34 (H1N1) strain. The vaccine strains against the 2009 H1N1 virus have been developed on the basis of several 2009 isolates (10). Some reassortants contained not only the HA and NA genes of the pandemic virus, but other genes as well. The vaccine strains were produced by both reverse genetics technique and by conventional virus crossing.We produced a series of reassortants of different genetic content by crossing of A/Moscow/IIV01/2009 (H1N1) pandemic strain with a high-yield H3N2 strain containing the genes of internal and non-structural proteins of the PR8 virus. The H1N1 and H1N2 reassortants were characterized with respect to the virus yield in the embryonated chicken eggs. The initial goal was to produce a candidate vaccine strain against the 2009 pandemic influenza virus (2). However, the characterization of the H1N1 and H1N2 reassortants of different genetic content with respect to the virus yield in the embryonated chicken eggs produced some data on the effect of gene constellation on the high-yield properties of influenza vaccine strains.The strain A/Moscow/IIV01/2009 (H1N1) was isolated during the 2009 pandemic in parallel in MDCK cell culture and in embryonated chicken eggs (9). The variant isolated in eggs was used in our studies for the crossing with X-31 (H3N2) reassortant. The X-31 strain contains the HA and NA genes of A/Aichi/2/68 (H3N2) virus and 6 genes of internal and non-structural viral proteins derived from PR8 virus (1). The viruses were propagated in 10-day-old embryonated chicken eggs, and the virus-containing allantoic fluid was aliquoted and stored at -80°C. The infectivity titration was determined by limiting dilution method in embryonated chicken eggs. The assessment of the hemagglutination titer was performed using conventional technique. For virus concentration and partial purification the virus-containing allantoic fluid was clarified by low-speed centrifugation and layered on top of 4 ml of 20% sucrose. The virus was pelleted by the centrifugation at 22,000 rpm for 90 min in SW27.1 rotor.Polyclonal antisera were obtained by immunization of guinea pigs (Laboratory Animal Breeding Institution of the Russian Academy of Medical Sciences, Andreevka, Moscow Region, Russia) weighing 250 g. The purified virus to be used for immunization of animals was treated with 0.1% glutaraldehyde for 7 days at 4°C. The immunization of guinea pigs

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Yields of virus reassortants containing the HA gene of pandemic influenza 2009 virus

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Av²,

Ia³

et al. 2012

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“…Recently, Rudneva et al reported that the introduction of the PB1 gene from a low pathogenic avian virus of the H5N2 subtype increased the replication of a corresponding 5 + 2 + 1 PR8 reassortant compared to the conventional 6 + 2 reassortant [37]. Moreover, two reassorted PR8-based vaccine seeds carrying the surface glycoprotein genes of the seasonal Pan99 (H3N2) virus had also received the PB1 gene of the epidemic strain [26].…”

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confidence: 99%

Improvement of H5N1 influenza vaccine viruses: Influence of internal gene segments of avian and human origin on production and hemagglutinin content

Abt

Jonge²,

Laue

et al. 2011

Vaccine

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“…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).…”

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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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Effect of gene constellation and postreassortment amino acid change on the phenotypic features of H5 influenza virus reassortants

Cited by 30 publications

References 16 publications

Yields of virus reassortants containing the HA gene of pandemic influenza 2009 virus

Yields of virus reassortants containing the HA gene of pandemic influenza 2009 virus

Improvement of H5N1 influenza vaccine viruses: Influence of internal gene segments of avian and human origin on production and hemagglutinin content

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

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