Genetic Analysis of Influenza A/H1N1pdm Strains Isolated in Bangladesh in Early 2020

Abstract

Influenza is one of the most common respiratory virus infections. We analyzed hemagglutinin (HA) and neuraminidase (NA) gene segments of viruses isolated from influenza patients who visited Evercare Hospital Dhaka, Bangladesh, in early 2020 immediately before the coronavirus disease 2019 (COVID-19) pandemic. All of them were influenza virus type A (IAV) H1N1pdm. Sequence analysis of the HA segments of the virus strains isolated from the clinical specimens and the subsequent phylogenic analyses of the obtained sequences revealed that all of the H1N1pdm recent subclades 6B.1A5A + 187V/A, 6B.1A5A + 156K, and 6B.1A5A + 156K with K209M were already present in Bangladesh in January 2020. Molecular clock analysis results suggested that the subclade 6B.1A5A + 156K emerged in Denmark, Australia, or the United States in July 2019, while subclades 6B.1A5A + 187V/A and 6B.1A5A + 156K with K209M emerged in East Asia in April and September 2019, respectively. On the other hand, sequence analysis of NA segments showed that the viruses lacked the H275Y mutation that confers oseltamivir resistance. Since the number of influenza cases in Bangladesh is usually small between November and January, these results indicated that the IAV H1N1pdm had spread extremely rapidly without acquiring oseltamivir resistance during a time of active international flow of people before the COVID-19 pandemic.

Keywords: Bangladesh; COVID-19; H1N1pdm09; HA; NA; evolution; influenza virus.

Figure 1

Alignment of the coding amino acid sequences of HA1 antigenic sites in the four strains isolated in Bangladesh in the present study. The coding amino acid sequences of HA1 were aligned to that of the reference strain, A/Michigan/45/2015. The H1N1pdm09 subclades and strain names are indicated on the left side of the figure. The amino acid sequences of each antigenic site are indicated as follows: Sa site (red box), Sb site (light blue box), Ca site (green box), and Cb site (dark blue box).

Figure 2

The H1N1pdm09 subclade in Bangladesh in 2018 to 2020. A maximum-likelihood tree of H1N1pdm09 was generated based on the HA gene with TVN + F + G4 and 1000 ultrafast bootstrap replicates using IQ-TREE. The percentage of trees (>80) in which the associated taxa clustered together is shown on the branches. The tree is drawn to scale, with branch lengths being directly proportional to the number of substitutions per site. Nucleotide sequences of the HA genes retrieved from EpiFlu GISAID are labeled as follows: isolate name|isolate ID|collection date. The Bangladesh 2018, 2019, and 2020 sequences are shown in brown, green, and blue, respectively, and the number of collapsed sequences is shown in parentheses at the end. The four sequences obtained in the present study are shown in red. The H1N1pdm09 subclade reference sequences are shown in black, with the subclade names highlighted in gray at the end. Subclade ranges are shown by vertical bars. Asterisks indicate the vaccine strains.

Figure 3

Evolution of Bangladesh H1N1pdm subclade 6B.1A5A strain 2020. (A) A root-to-tip analysis of the dataset. (B) The maximum clade credibility (MCC) tree based on the HA gene of H1N1pdm was estimated in BEAST package v1.10.4 with HKY + F + I, an exponential population growth model, and the relaxed molecular clock. The nucleotide sequences of the HA genes retrieved from EpiFlu GISAID are labeled as follows: isolate name|isolate ID|collection date (yyyy-mm-dd). The H1N1pdm strains, including the H1N1pdm subclade reference strains, are shown in black, with the clade name highlighted in gray at the end. Ranges of subclades 6B.1A1, 6B.1A2, 6B.1A3, 6B.1A4, 6B.1A5A, 6B.1A5B, 6B.1A6A, and 6B.1A7A are shown by vertical bars. Ranges of 6B.1A5A + 156K, 6B.1A5A + 187V/A, and 6B.1A5A + 156K with K209M are shown by vertical doted bars. Asterisks indicate the vaccine strains. The Bangladesh 2020 strains in EpiFlu and the newly obtained Bangladesh 2020 sequences in the present study are shown in blue and red, respectively. The regions in which the strains circulated are indicated at the branch tips and are color-coded as indicated in the legend on the left. The x-axis of the tree indicates the time (year). The tMRCA and interval, posterior probability (PP), and the occurrence of amino acid mutations of the key nodes are indicated by arrows.