Characterization of H5N1 Influenza Virus Quasispecies with Adaptive Hemagglutinin Mutations from Single-Virus Infections of Human Airway Cells

Abstract

Transmission of avian influenza (AI) viruses to mammals involves phylogenetic bottlenecks that select small numbers of variants for transmission to new host species. However, little is known about the AI virus quasispecies diversity that produces variants for virus adaptation to humans. Here, we analyzed the hemagglutinin (HA) genetic diversity produced during AI H5N1 single-virus infection of primary human airway cells and characterized the phenotypes of these variants. During single-virus infection, HA variants emerged with increased fitness to infect human cells. These variants generally had decreased HA thermostability, an indicator of decreased transmissibility, that appeared to compensate for their increase in α2,6-linked sialic acid (α2,6 Sia) binding specificity and/or in the membrane fusion pH threshold, each of which is an advantageous mutational change for viral infection of human airway epithelia. An HA variant with increased HA thermostability also emerged but could not outcompete variants with less HA thermostability. These results provided data on HA quasispecies diversity in human airway cells. The diversity of the influenza virus quasispecies that emerges from a single infection is the starting point for viral adaptation to new hosts. A few studies have investigated AI virus quasispecies diversity during human adaptation using clinical samples. However, those studies could be appreciably affected by individual variability and multifactorial respiratory factors, which complicate identification of quasispecies diversity produced by selective pressure for increased adaptation to infect human airway cells. Here, we found that detectable HA genetic diversity was produced by H5N1 single-virus infection of human airway cells. Most of the HA variants had increased fitness to infect human airway cells but incurred a fitness cost of less HA stability. To our knowledge, this is the first report to characterize the adaptive changes of AI virus quasispecies produced by infection of human airway cells. These results provide a better perspective on AI virus adaptation to infect humans.

H9N2 Influenza Virus Infections in Human Cells Require a Balance between Neuraminidase Sialidase Activity and Hemagglutinin Receptor Affinity

“…Primary human bronchial epithelial cells were obtained from PromoCell and maintained according to the manufacturer’s recommendations. None of the cell cultures in this study were grown or infected under air-liquid interface conditions as described previously ( 51 – 53 ). Influenza virus A/chicken/Egypt/CL42/2013 (CL42) (H9N2) is a representative strain of the G1-A/B lineage that has been prevalent in Egypt and that has been described previously ( 18 ).…”

“…HA receptor binding specificity was analyzed by solid-phase binding assays with the Sia analogues Neu5Acα2,3Galβ1-4GlcNAc (α2,3 SLN) and Neu5Acα2,6Galβ1-4GlcNAc (α2,6 SLN), as previously described ( 23 , 51 , 52 ), and α2,3 SLN sulfated on its antepenultimate sugar [Neu5Acα2,3Galβ1-4(6-HSO 3 )GlcNAc (Su-α2,3 SLN); GlycoNZ]. Anti-influenza virus NP antibody was used as the primary antibody, and peroxidase-conjugated goat anti-immunoglobulin (Histofine simple stain MAX-PO; Nichirei) was used as the secondary antibody.…”

“…Influenza virus A/chicken/Egypt/CL42/2013 (CL42) (H9N2) is a representative strain of the G1-A/B lineage that has been prevalent in Egypt and that has been described previously (18). All recombinant H9N2 viruses were propagated once in 10-day-old embryonated eggs and purified by ultracentrifugation as described previously (8,52).…”

“…Lung tissue samples were collected from sick and dead chickens from different poultry farms during AI outbreaks in the Delta region of Egypt in 2013. Viral RNAs were extracted directly from lung homogenates and analyzed by reverse transcription-PCR using the Uni-12 RT primer and primer sets specific for HA or NA genes, as described previously (52,54). The PCR products were analyzed by agarose gel electrophoresis.…”

Genetic Compatibility of Reassortants between Avian H5N1 and H9N2 Influenza Viruses with Higher Pathogenicity in Mammals

“…Furthermore, in this study, a mouse infection study was performed after observing no increase in viral growth by the reassortants used in cell cultures compared to previously published studies (2, 16, 41–45).…”