Serial Passaging of Seasonal H3N2 Influenza A/Singapore/G2-31.1/2014 Virus in MDCK-SIAT1 Cells and Primary Chick Embryo Cells Generates HA D457G Mutation and Other Variants in HA, NA, PB1, PB1-F2, and NS1

Abstract

Influenza remains one of the most prevalent viruses circulating amongst humans and has resulted in several pandemics. The prevention and control of H3N2 influenza is complicated by its propensity for evolution, which leads to vaccine mismatch and reduced vaccine efficacies. This study employed the strategy of serial passaging to compare the evolution of the human seasonal influenza strain A/Singapore/G2-31.1/2014(H3N2) in MDCK-SIAT1 versus primary chick embryo fibroblast (CEF) cells. Genetic analysis of the HA, NS1, NA, and PB1 gene segments by Sanger sequencing revealed the presence of specific mutations and a repertoire of viral quasispecies following serial passaging. Most quasispecies were also found in PB1, which exhibited consistently high transversion-to-transition ratios in all five MDCK-SIAT1 passages. Most notably, passage 5 virus harbored the D457G substitution in the HA2 subunit, while passage 3 virus acquired K53Q and Q69H mutations in PB1-F2. An A971 variant leading to a non-synonymous R316Q substitution in PB1 was also identified in MDCK-SIAT1 passages 2 and 4. With an increasing number of passages, the proportion of D457G mutations progressively increased and was associated with larger virus plaque sizes. However, microneutralization assays revealed no significant differences in the neutralizing antibody profiles of human-influenza-immune serum samples against pre-passaged virus and passage 5 virus. In contrast, viable virus was only detected in passage 1 of CEF cells, which gave rise to multiple viral RNA quasispecies. Our findings highlight that serial passaging is able to drive differential adaptation of H3N2 influenza in different host species and may alter viral virulence. More studies are warranted to elucidate the complex relationships between H3N2 virus evolution, viral virulence changes, and low vaccine efficacy.

Genome characterization of influenza A and B viruses in New South Wales, Australia, in 2019: A retrospective study using high‐throughput whole genome sequencing

“…Multiple amino acid substitutions were identified in the internal proteins of IAVs and IBVs, but their functional consequences are currently unclear. No known virulence markers were detected in any of the examined IAVs (A/H1N1pdm09: D222G/E in HA, T588I, E627K, and D701N in PB2, and R108K and I123V in NS1; A/H3N2: K615E in PA) and IBVs (K338R in PA) 59–62 . Some amino acid substitutions found in A/H1N1pdm09 (e.g., G225S and V667I in PB2, V200I and K386R in PB1, and T80A and A155T in NS1), A/H3N2 (e.g., S107N in PB2 and K158R in PA), and B/Victoria lineage viruses (e.g., V453A in NP) were similar to those found in hospitalized patients in Thailand during the 2018–2019 influenza season 47 .…”

“…No known virulence markers were detected in any of the examined IAVs (A/H1N1pdm09: D222G/E in HA, T588I, E627K, and D701N in PB2, and R108K and I123V in NS1; A/H3N2: K615E in PA) and IBVs (K338R in PA). 59 , 60 , 61 , 62 Some amino acid substitutions found in A/H1N1pdm09 (e.g., G225S and V667I in PB2, V200I and K386R in PB1, and T80A and A155T in NS1), A/H3N2 (e.g., S107N in PB2 and K158R in PA), and B/Victoria lineage viruses (e.g., V453A in NP) were similar to those found in hospitalized patients in Thailand during the 2018–2019 influenza season. 47 However, further investigation is needed to determine if these mutations have an impact on the virulence of the influenza viruses.…”

Genomics: Infectious Disease and Host–Pathogen Interaction

“…On the other hand, the mapping of small RNA reads from Huh7 cells infected with Ebola virus to the Ebola genome revealed the presence of two novel Ebola-virus-derived microRNAs which may target host genes [ 13 ]. Serial passaging of seasonal H3N2 influenza A virus in host cells was able to induce mutations across different ORFs, driving the viral genome to evolve and perhaps altering viral virulence [ 12 ]. In porcine, although the newly identified porcine circovirus (PCV) PCV4 is prevalent in pigs, the genomes of PCV4 are relatively stable among the circulating strains.…”

“…This Special Issue of the International Journal of Molecular Sciences entitled “Genomics: Infectious Disease and Host –Pathogen Interaction” contains twelve original articles and three reviews that provide new insights into infectious diseases with regard to biofilm formation [ 1 , 2 , 3 ], diagnostics [ 4 ], vaccine development [ 5 ], and host–pathogen interaction [ 6 , 7 ]; these insights are based on genomic [ 8 , 9 , 10 , 11 , 12 ], transcriptomic [ 13 , 14 ], and microbiomic [ 15 ] studies.…”