Phylogenomic analysis of Uganda influenza type-A viruses to assess their relatedness to the vaccine strains and other Africa viruses: a molecular epidemiology study

Abstract

Background: Genetic characterisation of circulating influenza viruses is essential for vaccine selection and mitigation of viral transmission. The current scantiness of viral genomic data and underutilisation of advanced molecular analysis methods on influenza viruses circulating in Africa has limited their extensive study and representation in the global influenza ecology. We aimed to sequence influenza type-A viruses (IAVs) that previously circulated in Uganda and characterised their genetic relatedness to the vaccine viruses and publicly available Africa IAVs. Methods: This was an observational study nested to the Uganda national influenza surveillance programme. We used Next-generation sequencing to locally generate genomes from 116 A(H1N1)pdm09 and 118 A(H3N2) viruses collected between 2010 and 2018 from 7 districts across Uganda. A total of 206 hemagglutinin (HA), 207 neuraminidase (NA), and 213 matrix protein (MP) sequences were genetically compared to the WHO-recommended vaccines and other viruses isolated from Africa since 1994. Viral temporal and spatial divergence and circulating genetic clades were characterised using phylogenetic methods. Findings: We successfully generated gene sequences for 91.9% (215/234) viruses. Uganda A(H1N1)pdm09 and A(H3N2) virus HA, NA, and MP proteins had 96.36-99.09%, 96.49-99.39%, and 97.48-99.95% amino acid similarity, respectively, to vaccines recommended from 2010 through 2020. The local viruses incorporated amino acid substitutions (AAS) in their antigenic, receptor binding, and glycosylation sites each year causing them to antigenically drift away from vaccines. For seasons when vaccine formulations differed, Uganda IAV antigenic sites had 1-2 extra AAS relative to the Southern than Northern hemisphere vaccine viruses. All Uganda IAVs carried the adamantine-resistance marker S31N but not the neuraminidase inhibitor (NAI) resistance markers H274Y and H275Y. However, some A(H1N1)pdm09 viruses had permissive substitutions V234I, N369K, and V241I typical of NAI-resistant viruses. The 2017-2018 A(H1N1)pdm09 viruses belonged to global genetic clade 6B.1, while the A(H3N2) viruses isolated in 2017 belonged to clades 3C.2a and 3C.3a. Uganda IAVs obtained before 2016 clustered distinctly from other Africa viruses while later viruses mixed with other Africa, especially Kenya and Congo, and global viruses. Several unique viral lineages (bootstrap >90) persisted in Uganda and other countries for 1-3 years. Interpretation: The study reveals Uganda as part of the global influenza ecology with continuous importation, antigenic drift, and extensive local transmission of IAVs, presenting a potential risk of future outbreaks. For a country with limited health resources and where social distancing is not sustainable, viral prevention by vaccination should be prioritized. The notable viral diversity in Africa is a warning to countries to broaden and incorporate genome analysis in routine surveillance to monitor circulating and detect new viruses. This knowledge can inform virus selection for vaccine production and assist in developing cost-effective virus control strategies.

Whole-genome analysis to determine the rate and patterns of intra-subtype reassortment among influenza type-A viruses in Africa

“…Fifty-six percent (106/189) and 43 · 9 per cent (83/189) of the swabs were sampled from male and female patients, respectively. Thirty-two percent (61/189) of the recovered WGs were from patients aged 1 month to < 2 years old, 38 · 6 per cent (73/189) from 2 to < 5 years old, 18 · 5 per cent (35/189) from 5 to < 15 years old, 9 · 5 per cent (18/189) from 15 to < 50 years old, 1 · 1 per cent (2/189) from 50 to < 65 years old, and none from above 65 year olds ( Nabakooza et al. 2021 ).…”

“…We successfully sequenced and assembled 82 · 5 per cent (193/234) WGs for H1N1pdm09 ( n = 100) and H3N2 ( n = 93) viruses sampled from Uganda between 2010 and 2018. The generated WGs consisted of the complete coding sequences for all eight genes ( Nabakooza et al. 2021 ).…”

“…At the continental level, we analysed 785 H1N1pdm09 and 1,224 H3N2 WGs sampled from 38 · 9 per cent (21/54) countries for 12 (2009–20) and 27 (1994–2020) years, respectively, including the newly generated Uganda WGs. The sampled countries spanned Western, Central, Northern, Eastern, Southeast, and Southern Africa ( Nabakooza et al. 2021 ).…”

“…Following M-RTPCR, amplified viral RNA libraries were loaded and sequenced using the Illumina MiSeq platform (Illumina Inc., San Diego, California, USA) at a KEMRI-Wellcome Trust Programme collaborating laboratory in Kilifi (Kenya), as described previously ( Nabakooza et al. 2021 ).…”

Sequence-matching adapter trimmers generate consistent quality and assembly metrics for Illumina sequencing of RNA viruses

“…Next-generation sequencing (NGS) has revolutionized infectious disease research and public health, enabling faster pathogen discovery, surveillance, and response (1)(2)(3)(4), at a lower cost and higher throughput than traditional Sanger sequencing (5). NGS sample preparation involves attaching adapters and unique barcodes to target genomic DNA or cDNA.…”