The isolation of viruses from this patient containing different HA receptor binding site polymorphisms at position 222 demonstrates that mutations in the HA can occur rapidly in a human host increasing quasispecies diversity with different predominance in the upper and lower respiratory tract. Rapid increase in quasispecies diversity was also seen in the ferret model when clinical isolates were used to inoculate the animals, and a great increase in quasispecies was observed especially after transmission in both the upper and lower airways. In contrast, much less diversity developed after infection with the plaque-purified virus. This was likely due to a lack of significant quasispecies present and/or selective pressures minimizing the ability of the pure G222 population to change. The clinical NP wash isolate that was identified as only containing D222 did develop diversity at this site rapidly, but it is possible that there were pre-existing quasispecies present in this clinical isolate below the limits of detection in this study. This highlights the complex nature of development of these polymorphisms and that multiple host and virological factors are likely involved in their development.
The presence of the H275Y mutation in the neuraminidase (NA) gene of the BAL and plaque purified sample did not seem to have any effect on viral fitness as the BAL isolate, NP isolate, and plaque-purified virus all grew well in culture and grew to similar titers from the lungs of the ferrets (). In a previously published study we demonstrated that this NP isolate, containing H at position 275, and another NP isolate from later in this same patient’s infection that contained the H275Y mutation maintained fitness and transmissibility in the ferret model (Memoli et al., 2010a
). These data in conjunction with the increased presence of H275Y in seasonal H1N1 and increasing evidence that the H275Y mutation in A(H1N1)pdm09 does not effect viral fitness, transmissibility, or pathogenesis (Hamelin et al.; Seibert et al.; van der Vries et al.); it seems likely that the presence of the H275Y mutation did not play a role in the differences observed here.
Viral Titers in Ferret Lung Tissues
Although in vitro
studies have demonstrated that the D222G mutation may alter receptor specificity and cell tropism allowing binding of a broader range of α2-3-linked sialyl receptor sequences on ciliated bronchial epithelial cells and on lung epithelia (Liu et al., 2010
; Watanabe et al., 2011
), we did not observe any differences in viral antigen distribution, viral titer, or range of histopathology in the upper and lower airways in the ferret model. Additionally, regardless of HA residue 222 sequence, all viruses grew well and were fully transmissible. These data are consistent with a recent study demonstrating similar disease in ferrets infected with a reverse genetics derived mutant virus and a high passage vaccine strain derived from A/CA/04/2009 (H1N1) (Belser et al., 2011
Although the results of the above studies were similar to what we observed, the current study has some significant differences. By using two very low passage clinical isolates (< 3 passages) and comparing them to a plaque-purified virus, we were able to explore not only the role of the D222G mutation itself, but also that of the described diversity of quasispecies at this position. While we observed differences in quasispecies proportions depending on location in the ferret respiratory tree, it is unclear if this difference in proportion of a certain amino acid at position 222 in the quasispecies present in the upper or lower airways was related to differences in viral binding or cell tropism, or rather reflected stochastic processes related to which viral variant predominated in early infection.
The other significant finding was that the plaque-purified virus containing the G222 caused the least severe illness in ferrets, again suggesting that it is likely that the 222G mutation is not a sole virulence determinant of severe disease as supported by the nonhuman primate infections described by Watanabe et al (Watanabe et al., 2011
). Interestingly, in the current study, those viral isolates with more diversity at HA position 222 were the most pathogenic, causing the most clinical disease, weight loss, and histopathology as demonstrated by those animals infected by transmission with the BAL isolate. Those animals developed the most severe clinical course, and the viruses isolated from those animals were the most diverse at position 222, particularly in virus isolated from the lungs. Although those animals infected by exposure to the NP isolate also developed considerable diversity at position 222, they developed slightly less overall disease. This difference may have been due to the fact that the virus causing the infection did not contain any detectable G222 variants; suggesting that the G222 mutation may play a role in enhancing pathogenesis as part of a diverse population.
The least diverse viruses were isolated from the animals infected with the plaque-purified G222 isogenic clone, and these animals demonstrated the least clinical disease and pathology compared to the other 2 diverse infections. This difference in pathogenicity may signify that HA receptor-binding region diversity may allow virus to bind to more diverse cell types in the initial infection, thus spreading more easily throughout the upper and lower airways. This is similar to quasispecies diversity determined neurotrophism and pathogenesis of polioviruses as described by Vignuzzi et al
., suggesting that diverse quasispecies populations are the unit of selection, rather than individual variants (Vignuzzi et al., 2006
). Thus, diversity at this HA receptor-binding site, rather than specific polymorphisms themselves, may be a better determinant of disease progression than a specific amino acid change.
Although diverse 222 variants are generated at the intrahost level and show a specificity for codon usage bias, phylogenetic analysis of 2009–2011 gene sequence data revealed that G222 variants are a result of sporadic mutations that occur with no forward persistence and very little transmission over time. In contrast, E222 forms a pronounced, monophyletic clade over time. These data suggest that variation at the amino acid site 222 is likely driven by intrahost adaptation, and that the population snap-shot of these variants in the context of phylogenetics is likely not indicative of pathogenic or circulating variants.