Molecular insights into evolution, mutations and receptor-binding specificity of influenza A and B viruses from outpatients and hospitalized patients in Singapore

Ivan, Fransiskus Xaverius; Zhou, Xinrui; Lau, Suk Hiang; Rashid, Shamima; Teo, J.S.; Lee, Hong Kai; Koay, Evelyn Siew-Chuan; Chan, Kwai Peng; Leo, Yee Sin; Chen, Mark; Kwoh, Chee Keong; Chow, Vincent T. K.

doi:10.1016/j.ijid.2019.10.024

Cited by 5 publications

(5 citation statements)

References 53 publications

(51 reference statements)

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“…Human Influenza B/Victoria lineage (B/Vic) was of the strain B/Singapore/G2-14.1/2014, MOI 0.1. Human Rhinovirus (HRV) used in this study is HRV A16, MOI 2.5 (Qiao et al, 2018;Tan et al, 2018;Tian et al, 2018;Chen et al, 2019;Ivan et al, 2020).…”

Section: Inoculation Of Influenza (H3n2 H1n1 B/victoria) and Rhinovmentioning

confidence: 99%

Host Antiviral Response Suppresses Ciliogenesis and Motile Ciliary Functions in the Nasal Epithelium

Chen

Tan

Liu

et al. 2020

Front. Cell Dev. Biol.

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BackgroundRespiratory viral infections are one of the main drivers of development and exacerbation for chronic airway inflammatory diseases. Increased viral susceptibility and impaired mucociliary clearance are often associated with chronic airway inflammatory diseases and served as risk factors of exacerbations. However, the links between viral susceptibility, viral clearance, and impaired mucociliary functions are unclear. Therefore, the objective of this study is to provide the insights into the effects of improper clearance of respiratory viruses from the epithelium following infection, and their resulting persistent activation of antiviral response, on mucociliary functions.MethodsIn order to investigate the effects of persistent antiviral responses triggered by viral components from improper clearance on cilia formation and function, we established an in vitro air–liquid interface (ALI) culture of human nasal epithelial cells (hNECs) and used Poly(I:C) as a surrogate of viral components to simulate their effects toward re-epithelization and mucociliary functions of the nasal epithelium following damages from a viral infection.ResultsThrough previous and current viral infection expression data, we found that respiratory viral infection of hNECs downregulated motile cilia gene expression. We then further tested the effects of antiviral response activation on the differentiation of hNECs using Poly(I:C) stimulation on differentiating human nasal epithelial stem/progenitor cells (hNESPCs). Using this model, we observed reduced ciliated cell differentiation compared to goblet cells, reduced protein and mRNA in ciliogenesis-associated markers, and increased mis-assembly and mis-localization of ciliary protein DNAH5 following treatment with 25 μg/ml Poly(I:C) in differentiating hNECs. Additionally, the cilia length and ciliary beat frequency (CBF) were also decreased, which suggest impairment of ciliary function as well.ConclusionOur results suggest that the impairments of ciliogenesis and ciliary function in hNECs may be triggered by specific expression of host antiviral response genes during re-epithelization of the nasal epithelium following viral infection. This event may in turn drive the development and exacerbation of chronic airway inflammatory diseases.

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Section: Inoculation Of Influenza (H3n2 H1n1 B/victoria) and Rhinovmentioning

confidence: 99%

Host Antiviral Response Suppresses Ciliogenesis and Motile Ciliary Functions in the Nasal Epithelium

Chen

Tan

Liu

et al. 2020

Front. Cell Dev. Biol.

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“…Most of the influenza-positive samples were subjected to next-generation sequencing of the complete viral genomes to determine their subtypes and phylogenetic relationships [ 20 ]. Among the influenza A subtypes were 11 H3N2, 6 H1N1, and 1 H1N2, while the influenza B samples included 3 Victoria and 4 Yamagata lineages, and their sequences are available in the GenBank database [ 20 ].…”

Section: Resultsmentioning

confidence: 99%

“…After this, 1 mL of virus culture medium was added to each well. The cells were then incubated at 35 °C with 5% CO 2 and examined daily for cytopathic effect or CPE [ 20 ]. Samples without CPE were passaged in MDCK cells on day 7.…”

Section: Methodsmentioning

confidence: 99%

“… *: Vaccinated in last 10 years from date of recruitment; NP: nasopharyngeal; F: fine aerosol fraction; -: influenza not detected using RT-qPCR or viral culture; and (R): assays were repeated with consistent results. a : influenza subtyping and genome sequencing with next-generation sequencing, as previously reported [ 20 ]. b : viral RNA copies in original nasopharyngeal swab sample.…”

Section: Figurementioning

confidence: 99%

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Influenza A and B Viruses in Fine Aerosols of Exhaled Breath Samples from Patients in Tropical Singapore

Chow,

Tay,

Chen

et al. 2023

Viruses

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Influenza is a highly contagious respiratory illness that commonly causes outbreaks among human communities. Details about the exact nature of the droplets produced by human respiratory activities such as breathing, and their potential to carry and transmit influenza A and B viruses is still not fully understood. The objective of our study was to characterize and quantify influenza viral shedding in exhaled aerosols from natural patient breath, and to determine their viral infectivity among participants in a university cohort in tropical Singapore. Using the Gesundheit-II exhaled breath sampling apparatus, samples of exhaled breath of two aerosol size fractions (“coarse” > 5 µm and “fine” ≤ 5 µm) were collected and analyzed from 31 study participants, i.e., 24 with influenza A (including H1N1 and H3N2 subtypes) and 7 with influenza B (including Victoria and Yamagata lineages). Influenza viral copy number was quantified using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Infectivity of influenza virus in the fine particle fraction was determined by culturing in Madin–Darby canine kidney cells. Exhaled influenza virus RNA generation rates ranged from 9 to 1.67 × 105 and 10 to 1.24 × 104 influenza virus RNA copies per minute for the fine and coarse aerosol fractions, respectively. Compared to the coarse aerosol fractions, influenza A and B viruses were detected more frequently in the fine aerosol fractions that harbored 12-fold higher viral loads. Culturable virus was recovered from the fine aerosol fractions from 9 of the 31 subjects (29%). These findings constitute additional evidence to reiterate the important role of fine aerosols in influenza transmission and provide a baseline range of influenza virus RNA generation rates.

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“…In this study, the passage adaptations of the seasonal influenza strain A/Singapore/G2-31.1/2014(H3N2) [ 33 ] in both MDCK-SIAT1 and primary CEF cells were explored and compared. The HA, NA, PB1, and non-structural protein 1 (NS1) genes of the passages were sequenced and analyzed for amino acid substitutions.…”

Section: Introductionmentioning

confidence: 99%

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

Heng²,

Heok³

et al. 2022

IJMS

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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.

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Molecular insights into evolution, mutations and receptor-binding specificity of influenza A and B viruses from outpatients and hospitalized patients in Singapore

Cited by 5 publications

References 53 publications

Host Antiviral Response Suppresses Ciliogenesis and Motile Ciliary Functions in the Nasal Epithelium

Host Antiviral Response Suppresses Ciliogenesis and Motile Ciliary Functions in the Nasal Epithelium

Influenza A and B Viruses in Fine Aerosols of Exhaled Breath Samples from Patients in Tropical Singapore

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

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