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Identification of critical residues in the hemagglutinin and neuraminidase of influenza virus H1N1pdm for vaccine virus replication in embryonated chicken eggs.

ABSTRACT: In 2009, we successfully produced a high-yield live attenuated H1N1pdm A/California/7/2009 vaccine (CA/09 LAIV) by substitution of three residues (K119E, A186D, and D222G) in the hemagglutinin (HA) protein. Since then, we have generated and evaluated additional H1N1pdm vaccine candidates from viruses isolated in 2010 and 2011. The 2010 strains with the new HA substitutions near the HA receptor binding site (N125D and D127E or D127E and K209E) grew well in eggs and formed large plaques in Madin-Darby canine kidney (MDCK) cells. Introduction of these acidic amino acids into the HA of CA/09 also improved vaccine virus growth in eggs to a titer comparable to that of CA/09 LAIV. However, the high growth of A/Gilroy/231/2011 (Gil/11) vaccine virus required modification in both the HA and the NA segments. The residue at position 369 of the NA was found to be critical for virus replication in MDCK cells and eggs. These HA and NA residues had minimal impact on viral entry but greatly improved viral release from infected cells. Our data implied that the HA receptor binding and NA receptor cleaving function of the poor-growth H1N1pdm virus was not well balanced for virus replication in host cells. The high-growth vaccine candidates described in this study maintained vaccine virus antigenicity and induced high levels of neutralizing antibodies in immunized ferrets, making them suitable for vaccine production. The identification of the amino acids and their roles in viral replication should greatly help vaccine manufacturers to produce high-yield reassortant vaccine viruses against the future drifted H1N1pdm viruses.

SUBMITTER: Wang W

PROVIDER: S-EPMC3624345 | biostudies-literature | 2013 Apr

REPOSITORIES: biostudies-literature

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Project description:Avian infectious bronchitis (IB) is an acute, highly infectious and contagious viral disease of chickens caused by avian infectious bronchitis virus (IBV) belonging to the genus Coronavirus and family Coronaviridae. It can affect all age groups of birds. The toll-like receptors (TLRs) are a major class of innate immune pattern recognition receptors that have a key role in immune response and defense against various infections.The TLRs are essential for initiation of innate immune responses and in the development of adaptive immune responses. An in ovo model was employed to study the antiviral activity of TLR ligands (Pam3CSK4, LPS and CpG ODN) on replication of IBV. It was hypothesized that optimum dose and specific timing of TLR ligands may reduce viral load of IBV in specific pathogen free (SPF) embryonated chicken eggs (ECEs). Further, the mechanism involved in the TLR-mediated antiviral response in chorioallantoic membrane (CAM) of ECEs was investigated. The ECEs of 9-11 days old were treated with different doses (high, intermediate and low) of TLR-2 (Pam3CSK4), TLR-4 (LPS) and TLR-21 (CpG ODN) ligands. In addition, to know the timing of TLR ligand treatment, six time intervals were analyzed viz. 36, 24 and 12?h prior to infection, time of infection (co-administration of TLR ligands and avian IBV) and 12 and 24?h post-IBV infection. For studying the relative expression of immuno-stimulatory genes (IFN-?, IFN-?, IFN-?, IL-1?, iNOS and OAS) in CAM, TLR ligands were administered through intra-allantoicroute and CAM were collected at 4, 8 and 16?h post treatment. The results demonstrated that intermediate dose of all the three TLR ligands significantly reduced virus titers and used in the present study. However, the LPS reduced virus titer pre- and post-IBV infection but Pam3CSK4 and CpG ODN reduced only pre-IBV infection. Further analysis showed that TLR ligands induced IFN-?, IL-1? and IFN stimulated genes viz. iNOS and OAS genes in CAM. The present study pointed towards the novel opportunities for rational design of LPS as immuno-stimulatory agent in chickens with reference to IBV. It may be speculated that in ovo administration of these TLR ligands may enhance resistance against viral infection in neonatal chicken and may contribute towards the development of more effective and safer vaccines including in ovo vaccines.

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Identification of critical residues in the hemagglutinin and neuraminidase of influenza virus H1N1pdm for vaccine virus replication in embryonated chicken eggs.

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