Alternative reassortment events leading to transmissible H9N1 influenza viruses in the ferret model.
Kimble JB, Angel M, Wan H, Sutton TC, Finch C, Perez DR.
J Virol. 2014 Jan;88(1):66-71. doi: 10.1128/JVI.02677-13. Epub 2013 Oct 16.
Kimble et al., describe inoculation of ferrets with MDCK (dog) cells transfected by 15 plasmids for avian H9N2 flu strain and human pandemic 2009 H1N1 (minus the H1 hemagglutinin). Theoretically 128 reassortant viruses can be made. Two of three animals so inoculated became infected giving rise to what were called lineage A and B viruses. Within 4 days of transfection, the dye was cast and one combination quickly dominated in each infected animal. This contrasts to the findings for tissue culture that once again highlights the need for animal studies.
At the end of 4 manual passages (P1-P4 virus) a fifth passage by aerosol transmission with P4 virus was attempted for three infected animals and it worked (RCP5 virus, RC=respiratory contact). P1 and RCP5 viruses were sequenced. There were 4 non-synonymous nucleotide changes (i.e. resulting in amino acid changes) for lineage A virus and 6 (5 non-synonymous, 1 synonymous, or silent change) for lineage B virus. None of the mutations overlapped. None were in the HA receptor binding pocket. This shows that there are many constellations that compatible with aerosol transmission using 6 common genes (PB2, PA, HA, NP, NA, and NS, Table 1). 2/4 and 5/5 mutations were of unknown phenotype indicating once again the wide gap in correlating genotype and phenotype.
The authors did not sequence the P4 virus used to initiate the aerosol transmission experiment hence we cannot work out which mutations arose during serial mechanical passage and which resulted from selection via transmission. It is unlikely that all 4 and 6 mutations arose during the single aerosol transmission passage, hence the numbers so acquired must be <4 and <5 for lineage A and B. Sequencing the P4 virus would have established the precise number involved.
Their interest in GOF is shown by the last sentence: “A similar approach can be implemented to ascertain the gene constellation that would most likely lead to more effective respiratory-droplet transmission of other avian influenza viruses in mammals.” Sequencing the P4 virus would have helped.
