tetano
Editor, Senior Moderator
J Gen Virol. 2015 May 21. pii: vir.0.000193. doi: 10.1099/vir.0.000193. [Epub ahead of print]
[h=1]H5N1 Influenza A Virus with K193E and G225E Double Mutations in Hemagglutinin is Attenuated and Immunogenic in Mice.[/h] Han PF[SUP]1[/SUP], Li J[SUP]1[/SUP], Hu Y[SUP]1[/SUP], Sun W[SUP]1[/SUP], Zhang S[SUP]1[/SUP], Yang YH[SUP]1[/SUP], Li YC[SUP]1[/SUP], Kang XP[SUP]1[/SUP], Wu XY[SUP]1[/SUP], Zhu SY[SUP]1[/SUP], Zhang Y[SUP]1[/SUP], Zhu QY[SUP]1[/SUP], Jiang T[SUP]1[/SUP], Qin CF[SUP]1[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] Live attenuated influenza vaccines (LAIV) are now available for the prevention of influenza, with LAIV strains generally derived from serial passaging in cultures or by reverse genetics. The receptor binding domain (RBD) in the hemagglutinin (HA) of influenza virus is responsible for viral binding to avian type, α2,3-linked or human type α2,6-linked sialic acid receptor; however the virulence determinants in the RBD of H5N1 virus remain largely unknown. In the present study, serial passaging of H5N1 virus A/Vietnam/1194/2004 (VN1194) in MDCK cells resulted in the generation of adapted variants with large-plaque morphology, and genomic sequencing of selected variants revealed two specific amino acid substitutions (K193E and G225E) in the RBD. Reverse genetics (RG) was used to generate H5N1 viruses containing either single or double substitutions in the HA. The RG virus containing K193E and G225E mutations (rVN-K193E/G225E) demonstrated large plaque morphology, enhanced replication, and genetic stability after serial passaging, without changing the receptor binding preference. Importantly, in vivo virulence assessment demonstrated that rVN-K193E/G225E was significantly attenuated in mice. Microneutralization and hemagglutination inhibition assays demonstrated that immunization with rVN-K193E/G225E efficiently induced a robust antibody response against wild type H5N1 virus in mice. Taken together, our experiments demonstrate that K193E and G225E mutations synergistically attenuated H5N1 virus without enhancing the receptor binding avidity, and that the RG virus rVN-K193E/G225E represents a potential H5N1 LAIV strategy that deserves further development. These findings identify the RBD as novel attenuation target for live vaccine development and highlight the complexity of RBD interactions.
PMID: 25998916 [PubMed - as supplied by publisher]
[h=1]H5N1 Influenza A Virus with K193E and G225E Double Mutations in Hemagglutinin is Attenuated and Immunogenic in Mice.[/h] Han PF[SUP]1[/SUP], Li J[SUP]1[/SUP], Hu Y[SUP]1[/SUP], Sun W[SUP]1[/SUP], Zhang S[SUP]1[/SUP], Yang YH[SUP]1[/SUP], Li YC[SUP]1[/SUP], Kang XP[SUP]1[/SUP], Wu XY[SUP]1[/SUP], Zhu SY[SUP]1[/SUP], Zhang Y[SUP]1[/SUP], Zhu QY[SUP]1[/SUP], Jiang T[SUP]1[/SUP], Qin CF[SUP]1[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] Live attenuated influenza vaccines (LAIV) are now available for the prevention of influenza, with LAIV strains generally derived from serial passaging in cultures or by reverse genetics. The receptor binding domain (RBD) in the hemagglutinin (HA) of influenza virus is responsible for viral binding to avian type, α2,3-linked or human type α2,6-linked sialic acid receptor; however the virulence determinants in the RBD of H5N1 virus remain largely unknown. In the present study, serial passaging of H5N1 virus A/Vietnam/1194/2004 (VN1194) in MDCK cells resulted in the generation of adapted variants with large-plaque morphology, and genomic sequencing of selected variants revealed two specific amino acid substitutions (K193E and G225E) in the RBD. Reverse genetics (RG) was used to generate H5N1 viruses containing either single or double substitutions in the HA. The RG virus containing K193E and G225E mutations (rVN-K193E/G225E) demonstrated large plaque morphology, enhanced replication, and genetic stability after serial passaging, without changing the receptor binding preference. Importantly, in vivo virulence assessment demonstrated that rVN-K193E/G225E was significantly attenuated in mice. Microneutralization and hemagglutination inhibition assays demonstrated that immunization with rVN-K193E/G225E efficiently induced a robust antibody response against wild type H5N1 virus in mice. Taken together, our experiments demonstrate that K193E and G225E mutations synergistically attenuated H5N1 virus without enhancing the receptor binding avidity, and that the RG virus rVN-K193E/G225E represents a potential H5N1 LAIV strategy that deserves further development. These findings identify the RBD as novel attenuation target for live vaccine development and highlight the complexity of RBD interactions.
PMID: 25998916 [PubMed - as supplied by publisher]