Phylogenomic analysis uncovers a 9-year variation of Uganda influenza type-A strains from the WHO-recommended vaccines and other Africa strains

Abstract

Genetic characterisation of circulating influenza viruses directs annual vaccine strain selection and mitigation of infection spread. We used next-generation sequencing to locally generate whole genomes from 116 A(H1N1)pdm09 and 118 A(H3N2) positive patient swabs collected across Uganda between 2010 and 2018. We recovered sequences from 92% (215/234) of the swabs, 90% (193/215) of which were whole genomes. The newly-generated sequences were genetically and phylogenetically compared to the WHO-recommended vaccines and other Africa strains sampled since 1994. Uganda strain hemagglutinin (n = 206), neuraminidase (n = 207), and matrix protein (MP, n = 213) sequences had 95.23-99.65%, 95.31-99.79%, and 95.46-100% amino acid similarity to the 2010-2020 season vaccines, respectively, with several mutated hemagglutinin antigenic, receptor binding, and N-linked glycosylation sites. Uganda influenza type-A virus strains sequenced before 2016 clustered uniquely while later strains mixed with other Africa and global strains. We are the first to report novel A(H1N1)pdm09 subclades 6B.1A.3, 6B.1A.5(a,b), and 6B.1A.6 (± T120A) that circulated in Eastern, Western, and Southern Africa in 2017-2019. Africa forms part of the global influenza ecology with high viral genetic diversity, progressive antigenic drift, and local transmissions. For a continent with inadequate health resources and where social distancing is unsustainable, vaccination is the best option. Hence, African stakeholders should prioritise routine genome sequencing and analysis to direct vaccine selection and virus control.

Figure 1

Workflow of swab selection and whole genome recovery. (A) Shows how swabs were selected for influenza whole-genome sequencing (WGS). Patients diagnosed with either influenza subtypes A(H1N1)pdm09 or A(H3N2) and whose swab had a PCR CT ≤ 35 had their laboratory codes randomised based on the subtype and year of collection using the R software v3.6.3 (https://www.r-project.org). All available swabs were retrieved for years with less than fifteen swabs. The 697 swabs missing include some shipped to the Centers for Disease Control and Prevention (CDC) for routine surveillance and some lost due to an accidental failure of a freezer. The numbers are based on the UVRI-NIC laboratory dataset only, as of 9th May 2018. (B) Shows how viral samples were excluded before and after sequencing and the rate of whole genome recovery. Eight viruses [2 A(H1N1)pdm09 and 6 A(H3N2)] failed quality control (QC) before sequencing.

Figure 2

Phylogenies showing the temporal divergence of the HA, NA and MP genes of Uganda A(H1N1)pdm09 (A) and A(H3N2) (B) influenza viruses sampled from 2010 to 2018. Trees were rooted using the oldest sequence in the dataset. Shaded clusters are the two and three major co-circulating lineages observed since 2013 and 2012 for A(H1N1)pdm09 and A(H3N2) viruses, respectively. The third A(H3N2) lineage (with one 2011 and 2016–2017 viruses) disappeared in the MP phylogeny.

Figure 3

Genetic clades of influenza A viruses that previously circulated in Uganda during the 2010–2018 seasons. All labelled clades (indicated by black bars) were inferred based on the signature amino acid substitutions in the HA1 protein indicated on the tree trunk in bold. (A) Shows clades for 2010–2018 A(H1N1)pdm09 viruses. Novel clades H1-UG1 and H1-UG2 are indicated. Genetic clade 6 diverged into 6A, 6B, and 6C. All clade 3, 5, and 7 viruses were collected from Entebbe and Kampala (Central Uganda) and circulated in 2010–2011. (B) Shows clades for 2010–2017 A(H3N2) viruses. A novel clade H3-UG1 is indicated. Clade 3 persisted in all 9 years. A similar figure with the full sequence names is provided in Supplementary Fig. 5.