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. 2011 Jan 21:8:31.
doi: 10.1186/1743-422X-8-31.

Improved hatchability and efficient protection after in ovo vaccination with live-attenuated H7N2 and H9N2 avian influenza viruses

Affiliations

Improved hatchability and efficient protection after in ovo vaccination with live-attenuated H7N2 and H9N2 avian influenza viruses

Yibin Cai et al. Virol J. .

Abstract

Mass in ovo vaccination with live attenuated viruses is widely used in the poultry industry to protect against various infectious diseases. The worldwide outbreaks of low pathogenic and highly pathogenic avian influenza highlight the pressing need for the development of similar mass vaccination strategies against avian influenza viruses. We have previously shown that a genetically modified live attenuated avian influenza virus (LAIV) was amenable for in ovo vaccination and provided optimal protection against H5 HPAI viruses. However, in ovo vaccination against other subtypes resulted in poor hatchability and, therefore, seemed impractical. In this study, we modified the H7 and H9 hemagglutinin (HA) proteins by substituting the amino acids at the cleavage site for those found in the H6 HA subtype. We found that with this modification, a single dose in ovo vaccination of 18-day old eggs provided complete protection against homologous challenge with low pathogenic virus in ≥ 70% of chickens at 2 or 6 weeks post-hatching. Further, inoculation of 19-day old egg embryos with 10⁶ EID₅₀ of LAIVs improved hatchability to ≥ 90% (equivalent to unvaccinated controls) with similar levels of protection. Our findings indicate that the strategy of modifying the HA cleavage site combined with the LAIV backbone could be used for in ovo vaccination against avian influenza. Importantly, with protection conferred as early as 2 weeks post-hatching, with this strategy birds would be protected prior to or at the time of delivery to a farm or commercial operation.

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Figures

Figure 1
Figure 1
Strategy of modifying the HA cleavage site. (A). The substitution of H7N2 (VIVA/04) HA amino acid cleavage site with alternative cleavage site sequences of H6's. (B). The substitution of H9N2 (WF10) HA amino acid cleavage site with alternative cleavage site sequences of H6's.
Figure 2
Figure 2
Viral replication kinetics of the live-attenuated viruses in MDCK cells at (A) 35°C and (B) 39°C using MOI of 0.001. Viral titers at different time points were determined by TCID50.
Figure 3
Figure 3
Plaque morphologies of the live-attenuated viruses in CEK cell at different temperatures. Confluent CEK cells in six-well plates were infected with 2mH7N2:6WF10att or 2H7N2:6WF10att. The numbers 10-6, 10-5, and 10-3 on the plaque pictures indicate the virus dilution used to infect cells at the indicated temperature. The cells incubated at 37°C, 39°C, or 41°C, respectively, for 4 days post infection and then fixed and the viral antigen was visualized by immunostaining as described in Materials and Methods. The plaques sizes were observed and the plaque numbers were counted and calculated as the log10 PFU/ml, as indicated below the individual plaque picture. A titer of <3.0 log10 PFU/ml indicates that no virus was detected at 10-3 dilution.

References

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