Whole-genome sequencing reveals origin and evolution of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018

Xiao-Nan Zhao; Han-Ju Zhang; Duo Li; Jie-Nan Zhou; Yao-Yao Chen; Yan-Hong Sun; Adeniyi C Adeola; Xiao-Qing Fu; Yong Shao; Mei-Ling Zhang

doi:10.1371/journal.pone.0234869

. 2020 Jun 24;15(6):e0234869. doi: 10.1371/journal.pone.0234869

Whole-genome sequencing reveals origin and evolution of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018

Xiao-Nan Zhao ^1,^#, Han-Ju Zhang ^2,^#, Duo Li ¹, Jie-Nan Zhou ¹, Yao-Yao Chen ¹, Yan-Hong Sun ¹, Adeniyi C Adeola ³, Xiao-Qing Fu ¹, Yong Shao ^3,^*, Mei-Ling Zhang ^1,^*

Editor: Mrinmoy Sanyal⁴

PMCID: PMC7314029 PMID: 32579578

Abstract

The continuous variation of the seasonal influenza viruses, particularly A(H1N1)pdm09, persistently threatens human life and health around the world. In local areas of southwest china, the large time-scale genomic research on A(H1N1)pdm09 is still insufficient. Here, we sequenced 45 whole-genome sequences of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018, by next-generation sequencing technology to characterize molecular mechanisms of their origin and evolution. Our phylogenetic analyses suggest that the A(H1N1)pdm09 strains circulating in Lincang belong to clade 6B and the subclade 6B.1A predominates in 2018. Further, the strains in 2018 possess elevated evolutionary rate as compared to strains in other years. Several newly emerged mutations for HA (hemagglutinin) in 2018 are revealed (i.e., S183P and R221K). Intriguingly, the substitution R221K falls into the RBS (receptor binding site) of HA protein, which could affect antigenic properties of influenza A(H1N1)pdm09 viruses, and another substitution S183P near to RBS with a high covering frequency (11/14 strains) in 2018 is exactly located at the epitope B. Notably, the NA (neuraminidase) protein harbors a new mutation I23T, potentially involved in N-glycosylation. Based on the background with a higher evolutionary rate in 2018 strains, we deeply evaluate the potential vaccine efficacy against Lincang strains and discover a substantive decline of the vaccine efficacy in 2018. Our analyses reaffirm that the real-time molecular surveillance and timely updated vaccine strains for prevention and control of influenza A(H1N1)pdm09 are crucial in the future.

Introduction

The outbreak of influenza A(H1N1)pdm09 pandemic in 2009–2010 has resulted in a large amount of illness or death globally [1–4]. It has become a serious worldwide public health problem. Since the influenza A(H1N1)pdm09 pandemic during 2009–2010, the activity of seasonal influenza viruses has relatively inhibited and further sustained a low level in China until the national epidemic during Sep 2017 and Feb 2018 with elevated influenza-related hospitalization, illness and death [5, 6]. Meanwhile, of in-hospital mortality during 2017–2018, the vast majority of infections were caused by influenza B and A/H1N1 [5, 6].

It is well-known that the influenza virus genome consists of eight gene segments and their high variability originates from sequence changes located in eight gene segments (HA, NA, M, NP, NS, PA, PB1 and PB2) [7]. The HA gene encodes the viral surface glycoprotein HA, which is responsible for binding to sialic acids (SAs), serving as the viral receptors on host cells and for fusion of the viral and host cell membranes on endocytosis. Another gene NA encodes a neuraminidase helping viruses to cleave SAs from host cells and virus particles [8, 9]. Afterwards, the HA is cleaved by host proteases into HA1 and HA2 subunits. The HA1 subunit harbors the globular head domain containing five distinct antigenic epitopes (Sa, Sb, Ca1, Ca2, and Cb) and the sialic acid receptor-binding sites (RBSs) [10, 11]; The HA2 subunit harbors the stalk domain which induces fusion between the viral envelope and host endosomal membrane [12]. Consequently, mutations within these domains might have potential effectiveness on the attachment of the virus to host cells and the recognition of the virus by the neutralizing antibodies aroused in human [13, 14]. Influenza viruses can utilize the efficient antigenic drift to escape from the elimination of the host’s immune system. Thus, WHO needs to update components of the vaccine strains annually to ensure maximum protection [14]. More importantly, the evaluation of the vaccine efficacy is essential for the selection of high-quality vaccine strains each year [15, 16].

The rapid evolution of genome sequences of seasonal influenza viruses poses an underlying challenge for vaccine development and selection. Therefore, in the present study, we aim at elucidating molecular mechanisms of origin and genetic variability of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018 and further providing real-time monitoring resources for the development of effective vaccine, which is particularly vital for prevention and control of seasonal influenza.

Materials and methods

Ethics consideration

The project entitled “Whole-genome sequencing reveals origin and evolution of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018”, submitted by investigator, Mei-Ling Zhang, Department of acute infectious disease control and prevention, has been approved by the meeting of ethics committee of Yunnan Provincial Centers for Disease Control and Prevention, according to Chinese ethics laws and regulations, with approval number YNCDC2014001. The ethics committee has approved the oral consent procedure to protect patients’ privacy including minor patients, whose oral consent originated from their parents. It is recognized that the right and the welfare of the subject are adequately protected.

Specimen collection and isolation and identification of virus

The surveillance was performed in patients with an influenza-like illness (ILI) who were admitted to Lincang people's hospital in Lincang, China, from January 1, 2014 to December 31, 2018. The ILI was defined by unexpected high fever ≥38°C with respiratory symptoms (i.e., cough and sore throat). The nasopharyngeal swabs were placed using the viral transport media and sent to the Lincang Center for Disease Control and Prevention within 24 hours for further detection of the influenza viruses. The collected samples were screened for influenza virus by RNA extraction and the real-time reverse transcription-polymerase chain reaction (RT-PCR). The positive samples were cultured in the Madin–Darby canine kidney (MDCK) cells for 4–7 days to isolate influenza viruses, which were later confirmed by haemagglutination (HA) assay. The influenza subtypes were identified by hemagglutination inhibition (HI) assay with post-infection ferret antisera raised against vaccine strains (A/California/7/2009(H1N1)pdm09 (2009–2016) and A/Michigan/45/2015 (2017–2018)) and circulating strains from the Chinese National Influenza Center. And specific experimental procedures comply with Technical Guidelines for National Influenza Surveillance (2017 Edition) (http://www.chinaivdc.cn/cnic/zyzx/jcfa/201709/t20170930_153976.htm). The identified results were reviewed by Yunnan Center for Disease Control and Prevention and then sent to the Chinese National Influenza Center for confirmation.

Whole genome sequencing of influenza A(H1N1)pdm09

Viral nucleic acids of isolated strains were extracted using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany). They were subsequently applied for cDNA synthesis and PCR amplification by SuperScript^TM III One-Step RT-PCR System with Platinum^TM Taq High Fidelity. The amplification primers were Uni-12/Inf1 (primer A): 5’-GGGGGGAGCAAAAGCAGG-3’, Uni-12/Inf3 (primer B): 5’-GGGGGGAGCGAAAGCAGG-3’ and Uni-13/Inf1 (primer C): 5’-CGGGTTATTAGTAGAAACAAGG-3’. Then, the amplification products were purified and quantified by QIAquick 96 PCR Purification Kit and Qubit^TM dsDNA HS Assay Kit. Next, we performed a series of procedures such as tagment genomic DNA, amplify libraries, clean up libraries, normalize libraries and dilute libraries to final loading concentration by Nextera XT DNA Library Prep kit referring to the kit instructions. In the end, we thawed reagent cartridge (MiSeq v2 Reagent Tray 300 cycles-PE), loaded the pooled libraries onto the reagent cartridge in the designated reservoir and operated MiSeq sequencer applied to sequencing according to the illumine MiSeq system guide. The raw sequencing paired-end reads were imported to CLC Genomics Workbench 9.5.2 (Qiagen, Denmark) and further were trimmed and performed for de novo assembly into contigs. The contigs were imported to National Center for Biotechnology Information (NCBI) for blast (https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database), and the best-hit reference strains were selected for mapping to produce consensuses. The 45 genome sequencing data of A(H1N1)pdm09 strains have been deposited at NCBI Gene Bank Database with accession codes: MT540604-MT540963.

Construction of phylogenetic trees and prediction of glycosylation sites

The 14 global reference strains including three northern hemisphere vaccine strains (2014–2020) recommended by WHO were retrieved from the Global Initiative on Sharing All Influenza Data (GISAID) database (http://www.gisaid.org) (S1 Table). Multiple sequence alignment was guided by ClustalW 1.82 (http://www.clustal.org/). Phylogenetic analyses for the eight gene segments were executed by the MrBayes v3.2.1 software under HKY+I+G nucleotide substitution model [17], and further we presented and annotated our phylogenies by using FigTree v1.4.3 (https://mac.softpedia.com/get/Graphics/FigTree.shtml). The chain length was set to 10,000,000, with the first 1,000 samples burned in and other parameters were regarded as defaults.

Meanwhile, we utilized the MCMC algorithm of BEAST v1.10.4 (http://tree.bio.ed.ac.uk/software/beast/) to evaluate the most recent common ancestor (TMRCA) from our isolated strains. In detail, HKY substitution model and uncorrelated relaxed clock with lognormal relaxed distribution were used in our analysis, and the 10,000,000 MCMC chain length was performed. The Tracer v1.5 (http://tree.bio.ed.ac.uk/software/tracer/) was used to detect the convergence. Furthermore we used the TreeAnnotator v1.10.4 software of the BEAST v1.10.4 to annotate the target Maximum clade credibility tree (MCCTREE) with 10% Burnin as the number of trees. Additionally, we also used FigTree v1.4.3 to visualize our MCCtree. Divergence time of ancestral nodes with 95% highest density probability (HPD) and mutation rates were estimated by BEAST v1.10.4, respectively. The numbering system of HA amino acid was applied after removing the signal peptide. Potential N-linked glycosylation sites were predicted using the NetNGlyc 2.0 web server (http://www.cbs.dtu.dk/services/NetNGlyc) with a threshold value of >0.5.

3D model visualization of HA and NA protein

The 3D structures of HA and NA proteins were predicted using the SWISS-MODEL web server (https://www.swissmodel.expasy.org/), and the identified mutations within antigenic sites and other important sites were marked with PyMOL (https://www.pymol.org/). The relative amino acid frequency in the epitope of HA1 was obtained using WebLogo [18].

Potential mutants contributing to functional changes

In order to further elucidate functional mutations, we used PROVEAN (http://provean.jcvi.org) to predict potentially functional impact of amino acid substitutions for HA and NA protein. The software provides the conservation degree of amino acids relative to other published sequences and further employs a score to define the potential effect of the substitution on protein function. A default cutoff score of less than −2.5 indicates a high probability of functional mutation [19].

Estimation of vaccine efficacy of influenza A(H1N1)pdm09 strains

The vaccine efficacy of A(H1N1)pdm09 was estimated using the P_epitope method, which is expressed as the proportion of amino acid substitutions in the dominant HA epitope [15]. To determine antigenic distance between the vaccine strain and the circulating strains, we took into account five epitopes (A-E) of the A(H1N1)pdm09 strains in analogy to the A(H3N2) virus [20]. The P_epitope is calculated by dividing the number of amino acid substitutions in HA1 epitope by the total number of amino acids in the epitope. This association between vaccine efficacy and P_epitope is given by E = (0.53–1.19 x P_epitope) x 100 for influenza A(H1N1)pdm09 virus [16, 21, 22].

Results

Isolation rate, demographic characteristic and sampling distribution of influenza

Of the 6,767 influenza-like illness samples collected in Lincang people's hospital, during the study period (January 1, 2014 to December 31, 2018), 392 strains were isolated from the samples of influenza-like cases. Among that, the isolation rates of influenza A and B strains were 3.04% and 2.75%, respectively (S2 Table). Among the 392 cases infected with influenza viruses, male accounted for the majority (61.73%), mainly in the age group under 5 years old, and scattered children were the main infected population (S3 Table). 45 strains were randomly selected from total 112 isolated influenza A(H1N1)pdm09 strains in Lincang, China to perform whole-genome sequencing. The sampling distribution of selected strains was plotted in Fig 1. The number of sequencing strains covered ~44.3% of the total A(H1N1)pdm09 strains per year on average. Because none of A(H1N1)pdm09 strains was isolated in 2015, this year was deleted in our downstream analyses.

Phylogeny and mutation analysis of HA of influenza A(H1N1)pdm09 strains

Here, we integrated phylogenetic and antigenic epitope analyses to understand potential evolutionary mechanisms of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018. Mutations occurred in antigenic epitopes of A(H1N1)pdm09 viruses can potentially influence their pathogenicity [23]. Based on the sequence homology, mutations observed in five epitopes (Sa, Sb, Ca1, Ca2, and Cb) were mapped to the H3 epitopes (B+D, B, C+D, A+D and E), respectively (S4 Table). This was an alternative way to identify antigenic sites in A(H1N1)pdm09 viruses as the previous study [20, 24].

According to the Mrbayes phylogenetic tree of HA genes (Fig 2 and S1 Fig), all the 45 strains characterized by cluster 6B (A/South Africa/3626/2013-like) showed representative amino acid substitutions, including P83S (epitope E), D97N, K163Q (epitope D), S185T (epitope B), S203T, K283E (epitope C), I321V, E374K, S451N and E499K (S4 Table).

Furthermore, the 6B clade grouped into two distinct cluster lineages, represented by A/Michigan/45/2015 (6B.1) and A/Iowa/53/2015 (6B.2). The 2014 strains and most (4/7) of 2016 strains located in 6B.2 lineage with differentiable amino acid substitutions, such as V152T (epitope B), V173I (epitope D), A261S (epitope E), E491G and D501E. All 2017–2018 strains and residual three 2016 strains were clustered into 6B.1 lineage, which are widespread throughout the world since 2016, harboring substitutions S84N (epitope E), I216T (epitope D) and S162N (epitope B), representing a potential N-glycosylation site (S4 Table).

Moreover, two 2016 and four 2017 strains clustered with A/Victoria/55/2017 shared an additional substitution A215G (epitope D), and most (15/20) strains from 2017 shared extra substitutions I324V and K454R. And most (13/14) 2018 strains clustered into the 6B.1A subgroup (A/Brisbane/02/2018-like) with mutations S74R (12/13)/S74K (1/13) (epitope E), I96V (9/13) (epitope D), S164T (13/13) (epitope D), S183P (11/13) (epitope B), I295V (13/13) (epitope C) and A256I (10/13) (S4 Table). Additionally, one 2018 strain (A/Yunnan-Linxiang/SWL1765/2018) has a substitution R221K occurred at the HA RBSs (positions 184–191, 218–225, 131–135, Y91, W150, H180 and Y192) [25]. The substitution S185T also overlapped with RBS. Furthermore, other two substitutions (S183P and I216T) were also observed near RBSs that can increase receptor binding affinity [26–28]. These mutations found at antigenic sites and RBSs were further visualized to illustrate their potential functional effectiveness for the HA protein (Fig 3A and 3B).

Fig 3 — (A) Observed mutations at the epitopes and RBSs of HA protein of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018. The amino acid differences at the epitopes and RBSs of HA, between Lincang 2014–2018 strains and vaccine strain (A/California/07/2009) were compared. We searched and obtained the model template (PDB ID: 6n41.1.A) of HA protein of A/California/07/2009. We conducted a structure prediction of the trimeric HA protein by SWISS-MODEL, then the changes at the epitopes and RBSs were visualized in PyMol. Amino acid substitutions located in different epitopes and RBSs were shown in different colors, respectively. The amino acid substitutions in RBSs were shown in italics, as well. Amino acid substitutions that were associated with the acquisition of a potential N-glycosylation site were framed. (B) Mutational frequencies of amino acid residues at the antigenic epitopes and RBSs in the HA1 protein of A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018. The vaccine strain A/California/07/2009 was regarded as a reference. Relative frequency of the amino acid residues was proportional to the residue height. Mutational frequencies of amino acid changes at non-epitopes were shown in black color.

Additionally, a MCCTREE under a MCMC uncorrelated relaxed molecular clock model using HA gene segments further supported the exact topology of Mrbayes phylogenetic tree and also provided a deep insight on the divergence time for ancestral nodes of the different clades (Fig 4). We discovered that the most recent common ancestors (TMRCAs) of clade 6B.1A, 6B.2 and all Lincang strains aroused in 2016.5683 year with 95% HPD [2016.1408, 2017.1150], 2013.5371 year with 95% HPD [2013.2442, 2013.9384] and 2012.4240 year with 95% HPD [2011.7507, 2013.2182], respectively. Meanwhile, based on BEAST v1.10.4, we estimated the mutation rates of all strain branches in Lincang with a median of 2.6x10^-3 substitutions/site/year, and concluded that 2018 strains possessed a higher evolutionary rate (median: 2.95x10^-3 substitutions/site/year) than that of other years (median: 2.40x10^-3 substitutions/site/year) (S5 Table). Interestingly, we found that 2018 strains also accumulated the mutational amino acids more quickly than other years’ strains (S2A Fig, Wilcoxon rank sum test, W = 123.5, P = 0.01285). And mutational amino acid statistics of each epitope of 2018 strains revealed that the ratio of amino acid substitutions in B epitope was much higher than those of other epitopes (C-E) (S2B Fig, Wilcoxon rank sum test, P<2.855e-06). These accumulated genomic variations in 2018 strains might potentially become dominant in the future.

Fig 4 — The branch length represented time scale. The blue bars showed 95% HPD of nodal divergence time. TMRCAs of several nodes (clade 6B.1A, 6B.1, 6B.2 and all of isolated strains) were highlighted by the red font with 95% highest density probability (HPD). The two strains(A/Lviv/N6/2009 and A/Osaka/1/2009) without the sample collection date were removed in this analysis.

Phylogeny and mutation analysis of NA of influenza A(H1N1)pdm09 strains

The topology of the Mrbayes phylogenetic tree of NA genes from 45 strains was similar to that of HA (Fig 5 and S3 Fig). All the 45 strains clustered into 6B clade including representative substitutions (e.g. L40I, N44S, N200S, V241I, N248D, V264I, N270K, I321V, Y351F, N369K, N386K and K432E) (S6 Table). The strains of the 6B.2 lineage contained the unique substitutions V67I and T381I and the clade 6B.1 strains were defined by V13I, I34V and I314M. Moreover, most (15/20) strains from 2017 harbored one unique mutation E47G and the sub-clade 6B.1A shared the additional substitutions G77R (12/13)/G77K (1/13), V81A and N449D. And seven strains from clade 6B.1A possessed the extra substitutions T289I and D416G (S6 Table).

All the 45 strains carried eight conserved potential N-glycosylation sites in NA (positions 42, 50, 58, 63, 68, 88, 146 and 235), except that A/Yunnan-Linxiang/SWL1766/2018 harbored an additional substitution I23T, resulting in the acquisition of an N-glycosylation motif (NLT) (S6 Table). Like the Guangdong-A(H1N1)pdm09 viruses, the mutations V241I, N369K, N386K and K432E were also observed in Lincang strains which were reported to have significant impacts on the binding affinity between oseltamivir and neuraminidase, and further alter the susceptibility of A(H1N1)pdm09 strains to oseltamivir [29, 30]. Furthermore, the Lincang strains harbored the V106I (2/45) in 2017 and N248D (45/45) substitutions in NA, which could lead to low-PH stability of A(H1N1)pdm09 viruses and may contribute to its adaptation to human’s immune system [9]. These mutations which were likely to bring about structural modification of NA protein of A(H1N1)pdm09 strains were plotted using PyMol (Fig 6).

Fig 6 — The amino acid differences in these important amino acid sites of NA between Lincang 2014–2018 strains and the vaccine strain (A/California/07/2009) were compared. The model template (PDB ID: 5nwe.1.A) of NA protein of A/California/07/2009 was searched and the tetramer structure of NA protein was predicted by SWISS-MODEL. Amino acid substitutions located in different amino acid sites were shown in different colors, respectively. Probable binding position of oseltamivir in NA was shown in light blue color. The common and conserved mutations were indicated in purple and the variations responsible for oseltamivir binding were shown in red, and the substitutions that were associated with low-PH stability of NA were displayed in blue color.

Phylogeny and mutation analysis of six internal segments of influenza A(H1N1)pdm09 strains

Here, we also focused on genetic changes in the internal gene segments (i.e., M, NP, NS, PA, PB1 and PB2). Extremely similar to the Mrbayes phylogenetic trees of HA and NA, their tree topologies exhibited the similar evolutionary relationship (S4–S9 Figs).

The amino acid variations in internal proteins (M, NP, NS, PA, PB1 and PB2) also were analyzed (S7 Table). All the 45 strains were resistant to M2 inhibitors, owing to carrying the mutation S31N in M2 [31] (S7 Table). Since 2009 pandemic, several mutations have been fixed in influenza A(H1N1)pdm09 viruses including M1-V80I, M1-M192V, M1-K230R, NP-A22T, NP-V100I, NP-M105T, NP-S498N, NS1-E55K, NS1-L90I, NS1-I123V, NS1-K131E, NS1-N205S, PA-V100I, PA-P224S, PA-N321K, PA-I330V, PA-R362K, PB1-G154D, PB1-I397M, PB1-I435T, PB2-R54K, PB2-M66I, PB2-D195N, PB2-R293K, PB2-V344M, PB2-I354L and PB2-V731I (S7 Table). In recent years (i.e., 2016–2018), newly accumulated mutations were observed in M1-Q208K, NS1-D2E, NS1-E125D, PB2-R299K and PB2-S453T, which were also the representative mutation sites of 6B.1 clades of their own phylogenetic trees. Additionally, two unique variations (PB1-R211K and PB1-K486R) were only identified in 2018 strains (9/13) (S7 Table).

Estimation of vaccine efficacy of influenza A(H1N1)pdm09 strains

Amino acid residues in five epitope regions A to E, which respectively possess 24, 22, 33, 48, and 34 amino acids, were defined by a previous report [20]. Table 1 provided a basic assessment of the predicted vaccine efficacy of vaccine strain against Lincang 2014–2018 strains.

Table 1. Predicted vaccine efficacy of vaccine strains against influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Year(N)	Vaccine Strain	No. of strains	Dominant Epitope	No. of mutations	Residue differences	P_epitope	Vaccine Efficacy(%)
2014(N = 4)	A/California/07/2009	4	B	2	V152T,S185T	0.091	42.17
		4	C	1	K283E	0.030	49.43
		4	D	2	K163Q,V173I	0.042	48.00
		4	E	2	P83S,A261S	0.059	45.98
2016(N = 7)	A/California/07/2009	7	B	1	S185T	0.045	47.65
		4	B	1	V152T	0.045	47.65
		3	B	1	S162N	0.045	47.65
		7	C	1	K283E	0.030	49.43
		7	D	1	K163Q	0.021	50.50
		4	D	1	V173I	0.021	50.50
		3	D	1	I216T	0.021	50.50
		2	D	1	A215G	0.021	50.50
		7	E	1	P83S	0.029	49.55
		4	E	1	A261S	0.029	49.55
		3	E	1	S84N	0.029	49.55
2017(N = 20)	A/California/07/2009	20	B	2	S162N,S185T	0.091	42.17
		20	C	1	K283E	0.030	49.43
		20	D	2	K163Q,I216T	0.042	48.00
		4	D	1	A215G	0.021	50.50
		20	E	2	P83S,S84N	0.059	45.98
		1	E	1	A73S	0.029	49.55
	A/Michigan/45/2015	4	D	1	A215G	0.021	50.50
	A/Michigan/45/2015	1	E	1	A73S	0.029	49.55
2018(N = 14)	A/California/07/2009	14	B	2	S162N,S185T	0.091	42.17
		11	B	1	S183P	0.045	47.65
		14	C	1	K283E	0.030	49.43
		13	C	1	I295V	0.030	49.43
		1	C	1	V272A	0.030	49.43
		14	D	2	K163Q,I216T	0.042	48.00
		13	D	1	S164T	0.021	50.50
		9	D	1	I96V	0.021	50.50
		14	E	2	P83S,S84N	0.059	45.98
		13	E	1	S74R/K	0.029	49.55
		1	E	1	L70F	0.029	49.55
	A/Michigan/45/2015	11	B	1	S183P	0.045	47.65
		13	C	1	I295V	0.030	49.43
		1	C	1	V272A	0.030	49.43
		13	D	1	S164T	0.021	50.50
		9	D	1	I96V	0.021	50.50
		13	E	1	S74R/K	0.029	49.55
		1	E	1	L70F	0.029	49.55
	A/Brisbane/02/2018	3	B	1	P183S	0.045	47.65
		14	C	2	G45R,V298I	0.061	45.74
		1	C	1	V272A	0.030	49.43
		1	C	1	V295I	0.030	49.43
		14	D	1	R223Q	0.021	50.50
		9	D	1	I96V	0.021	50.50
		1	D	1	T164S	0.021	50.50
		1	E	1	L70F	0.029	49.55
		2	E	1	R74K/S	0.029	49.55

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Dominant epitopes are shown in Bold.

With regards to vaccine strain A/California/07/2009, for 2014 and 2016, the P_epitope between Lincang strains and vaccine strain A/California/07/2009 were 0.042–0.091 (dominant epitopes B, D and E) and 0.021–0.045 (dominant epitopes B-E), suggesting a predicated vaccine efficacy of 42.17%-48.00% and 47.65%-50.50%, respectively. For 2017 and 2018, the amino acid substitutions were observed both on epitopes B, D and E, resulting in the decreased vaccine efficacy of 42.17%-48.00%. However, when compared to the new vaccine strain A/Michigan/45/2015 recommended by WHO for 2017–2018, the P_epitope declined to 0.021 and 0.021–0.03 (dominant epitopes D and C-E), yielding the increased vaccine efficacy ranging between 50.50% and 49.43%-50.50%. Notably, compared with the 2019 vaccine strain A/Brisbane/02/2018, the predicated vaccine efficacy against Lincang 2018 strains ranged of 45.74%-50.50%, with a slight descent. This suggests that the 2019 vaccine strain A/Brisbane/02/2018 may not fully provide modified protection against the circulating strains of 2018, in Lincang. It was mainly attributed to the additional substitutions (G45R, V298I and R223Q) on dominant epitopes C and D.

Discussion

Consistent with global trends [7, 24, 32], our analyses from phylogenies using diverse gene segments indicate that all A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018, originated from the clade 6B, and deeply time-scale analyses reveal that the most recent common ancestor (TMRCA) of all Lincang strains in this study aroused in ~2012.4240 year. And recently the subclade 6B.1A has been predominated with a TMRCA in ~2016.5683 year for the local areas of southwest china.

At the molecular level, based on MCMC uncorrelated relaxed molecular clock model, for the first time, we produce an exact evaluation of mutation rate (median: 2.6x10^-3 substitutions/site/year) for all A(H1N1)pdm09 strains in Lincang, from 2014 to 2018. This evolutionary rate is obviously higher than that (1.5 or 1.6x10^-3) of pandemic (H1N1) 2009 influenza viruses in Sendai, Japan, during 2009–2011 [8]. However, it is significantly lower than that (5.046x10^-3) of Saudi Arabia during the 2009–2011 pandemic seasons [33]. These imply that the mutation rate may potentially be influenced by regional diversification. Interestingly, we discover that Lincang strains in 2018 have evolved more rapidly (mutation rate: ~2.95x10^-3 substitutions/site/year) than those strains in other years (mutation rate: ~2.40x10^-3 substitutions/site/year). After integrating phylogenetic and antigenic epitope analyses, we indeed discover several important recently produced mutations especially in 2018 for diverse gene segments. For example, the mutation R221K (in 2018) is located at the RBS of HA, and it’s well-known that previous studies have shown that mutations in or near the RBS domain of HA have important effects on the antigenic properties of influenza A(H1N1)pdm09 viruses [34, 35]. And another newly produced mutation S183P in HA covering 11/14 strains in 2018 and located at the epitope B is also near RBS, and proves to elevate receptor binding affinity [26–28]. For NA, A/Yunnan-Linxiang/SWL1766/2018 carries a new substitution I23T, leading to a potential gain of N-linked glycosylation site, and further functional experiments could highlight its importance in virus pathogenicity. Additionally, The NA segments of two Lincang strains in 2017 harbor a V106I mutation, and a previous study has indicated that this mutation has contributions to enhance the virulence of A(H1N1)pdm09 viruses in mice [36]. For other segments, two unique newly produced variations (i.e., PB1-R211K and PB1-K486R) were also harbored by 2018 strains with a high coverage (9/13), suggesting potential functional candidates in the future.

The rapid mutation rate of influenza A(H1N1)pdm09 in Lincang especially in recent years poses an enormous challenge for the timely renewal of vaccines. Our analyses in vaccine efficacy consolidate the WHO decision to update northern hemisphere A(H1N1)pdm09 vaccine strain in 2017 [37–39]. As expected, the estimated vaccine efficacy between A/Michigan/45/2015 vaccine strain and the 2017 circulating strains in Lincang increased to 50.50%. Subsequently, the estimated vaccine efficacy against Lincang 2018 strains dropped to a range of 49.43%~50.50%, which further suggests a rapid evolution of strains in 2018. Although the estimated vaccine efficacy of A/Brisbane/02/2018 vaccine strain has not improved as expected, our analyses do not include the isolated strains in 2019 and cannot directly reflect a true vaccine efficacy against the circulating strains in 2019.

Conclusions

In conclusion, consistent phylogenetic trees and those functional mutations elucidate the molecular mechanisms of origin and rapid evolution of the influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018. And we also verifies the need for constant molecular monitoring of the circulating strains to provide prompt supporting data for the better selection of the vaccine strain of influenza A(H1N1)pdm09.

Supporting information

S1 Table. Accession numbers in GISAID databases of applied gene segments of 14 A(H1N1)pdm09 reference strains included in the analysis.

N/A: not applied.

(DOC)

Click here for additional data file.^{(39KB, doc)}

S2 Table. The number (%) of influenza strains isolated from samples of influenza-like cases in Lincang, China, from 2014 to 2018.

(DOC)

Click here for additional data file.^{(31KB, doc)}

S3 Table. Demographic characteristics of the cases infected with influenza viruses in Lincang, China, from 2014 to 2018.

(DOC)

Click here for additional data file.^{(47KB, doc)}

S4 Table. Amino acid substitutions in the epitopes and non-epitopes of HA protein of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Amino acid mutations were referred to A/California/07/2009. “.” showed that the amino acid site was the same as that of A/California/07/2009. “a” represented the receptor binding sites. “+” indicated the gain of a potential glycosylation site. “-” indicated the predicated deleterious mutations.

(XLSX)

Click here for additional data file.^{(27.2KB, xlsx)}

S5 Table. Estimated evolutionary rates of HA genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

The evolutionary rates were estimated by BEAST v1.10.4 software under the uncorrelated relaxed clock model.

(DOC)

Click here for additional data file.^{(60.5KB, doc)}

S6 Table. Amino acid substitutions of NA protein of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Amino acid mutations were referred to A/California/07/2009. “.” showed that the amino acid site was the same as that of A/California/07/2009. “+” indicated the gain of a potential glycosylation site. “-”indicated the predicated deleterious mutations.

(XLSX)

Click here for additional data file.^{(24.8KB, xlsx)}

S7 Table. Important amino acid substitutions of six internal proteins of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Amino acid mutations were referred to A/California/07/2009. “.” showed that the amino acid site was the same as that of A/California/07/2009.

(XLSX)

Click here for additional data file.^{(22KB, xlsx)}

S1 Fig. Mrbayes phylogeny of the HA genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Each nodal number in square brackets represented a Bayesian posterior probability (BPP) range. The ruler value (0.005) represented genetic distance.

(TIF)

Click here for additional data file.^{(760.5KB, tif)}

S2 Fig. Comparative analyses of mutations in HA protein between 2018 and other years.

(A) Comparative analyses of accumulated variations in HA between 2018 and other year strains. (B) Comparative analyses of mutations under different epitopes for HA in 2018 strains. The significant level was decided by Wilcoxon rank sum test (P<0.05).

(TIF)

Click here for additional data file.^{(503.5KB, tif)}

S3 Fig. Mrbayes phylogeny of the NA genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Each nodal number in square brackets represented a Bayesian posterior probability (BPP) range. The ruler value (0.005) represented genetic distance.

(TIF)

Click here for additional data file.^{(790.9KB, tif)}

S4 Fig. Mrbayes phylogeny of the M genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

6B~6B.1A indicated branch Numbers of clades. Each nodal number in phylogeny exhibited a Bayesian posterior probability (BPP). The ruler value (0.2) represented genetic distance.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S5 Fig. Mrbayes phylogeny of the NP genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

6B~6B.1A indicated branch Numbers of clades. Each nodal number in phylogeny exhibited a Bayesian posterior probability (BPP). The ruler value (0.002) represented genetic distance.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S6 Fig. Mrbayes phylogeny of the NS genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

6B~6B.1A indicated branch Numbers of clades. Each nodal number in phylogeny exhibited a Bayesian posterior probability (BPP). The ruler value (0.2) represented genetic distance.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S7 Fig. Mrbayes phylogeny of the PA genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

6B~6B.1A indicated branch Numbers of clades. Each nodal number in phylogeny exhibited a Bayesian posterior probability (BPP). The ruler value (0.002) represented genetic distance.

(TIF)

Click here for additional data file.^{(1.6MB, tif)}

S8 Fig. Mrbayes phylogeny of the PB1 genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

6B~6B.1A indicated branch Numbers of clades. Each nodal number in phylogeny exhibited a Bayesian posterior probability (BPP). The ruler value (0.002) represented genetic distance.

(TIF)

Click here for additional data file.^{(1.6MB, tif)}

S9 Fig. Mrbayes phylogeny of the PB2 genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

6B~6B.1A indicated branch Numbers of clades. Each nodal number in phylogeny exhibited a Bayesian posterior probability (BPP). The ruler value (0.002) represented genetic distance.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

Acknowledgments

The authors thank the staff of the hospital and CDC in Lincang for their assistance with the collection of specimens of influenza-like illness and virus isolation and identification.

Data Availability

The 45 genome sequencing data of A(H1N1)pdm09 strains have been deposited at NCBI Gene Bank Database with accession codes: MT540604-MT540963.

Funding Statement

The authors received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0234869.r001

Decision Letter 0

Mrinmoy Sanyal

18 Feb 2020

PONE-D-20-01217

Whole-Genome Sequencing Reveals Origin and Evolution of Influenza A(H1N1)pdm09 Viruses in Lincang, China, from 2014 to 2018

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Reviewer #1: This is an important paper, about evolution of H1N1 sequences from regions often thought to be the source of new influenza strains. I have a few comments

1) It would be helpful when pEpitope is introduced to mention the relation between Sa, Sb, Ca1, Ca2, and Cb and epitopes A-E. The mapping is "Sa, Sb, Ca1 , Ca2 and Cb" -> "B+D, B, C+D, A+D and E". Plus the A-E epitopes contain an additional 31 amino acids.

2) Since pEpitope is used to compute vaccine efficacy, it would be more helpful to identify epitope membership rather than the Sa, Sb, Ca1, Ca2, and Cb membership in Table 1 and Figure 4. Or, this could be done in SI figures.

3) Please verify the numbering from Table 1 of ref 21 was used to compute pEpitope (or that the .xls or matlab script was used to compute pEpitope).

3) How are years defined? Usually, we group influenza strains per season (e.g. from July 1 year n to June 30 year n+1). I am not sure whether seasonal grouping is correct for China, however, as new strains seem to arise in China in the summer.

4) Regarding this sentence: "Our results suggested that a timely replacement of the vaccine was absolutely necessary because that the mean vaccine efficacy against the circulating strains in 2018 showed a decreased trend, compared to 2017" it would be helpful to compare the 2017 and 2018 strains against the old vaccine (California) to show the new vaccine (Michigan) was expected to have higher efficacy. It would also be interesting to compare the 2018 strains against the newer vaccine (Brisbane) to show Brisbane would be expected to have higher efficacy.

5) It would be interesting to describe in which epitope most of the evolution in 2018 (e.g. figure S1) ocurred.

Reviewer #2: The manuscript by Zhang et al, has described the genomic sequencing of influenza A(pH1N1) viruses from Lincang in China from 2014 to 2018. Samples for this study are sourced from ILI surveillance and WGS was conducted from cell culture isolated virus. While this work and findings are important and relevant, the key messages is lost in the verboseness of the manuscript.

Methods:

1. The methods used appear to be standard methods for China, The authros must refence literature to provide a description of methods – realtime RT-PCR, virus isolation, HA and HI. What was the source of the strain identification kit?

2. Authors mention genome amplification using “Invitrogen SuperScript III One-Step RT-PCR Platinum kit”. IT is not clear what is amplified. The primers used must be stated,

3. What was the protocol for library prep? Should be mentioned briefly.

4. While the authros have states use of a MiSeq for generation of whole genome sequence. A description of sequencing kit used and the run conditions and protocol for data handling is not mentioned.

5. Any reason why contigs were generated de novo? The usual protocol for influenza is to map to reference strains

6. Details on the “Global Reference Strains” are missing.

7. For the phylogenetic analysis, what was the nucleotide substitution model used? This has a bearing on the results

8. What is the tree being presented? MCC tree? Software used for annotation of trees?

9. Vaccine efficacy (16,22,23)

Results

1. This study is a detailed sequencing analysis of influenza A /pH1N1 strains obtained from routine ILI surveillance. However, figure 1 only has details fo the number of strains and seuqneing strains. How many samples were collected and what was the detection rate of influenza ( A and B) in this study? Demographic profile of influenza is not mentioned.

2. The source of these strains as mentioned by the authors is not clear.

3. Authors infer “faster evolutionary rate than other years (Supplementary Fig S1, Wilcoxon rank sum test, W=123.5, P=0.01285)”. However, the parameters being compared are ratio of mutational sites/total sites.

4. The HA and NA phylogeny with listed mutation sites is verbose and difficult to read. It will benefit from a table summarizing the mutations.

5. Has the tMRCA been calculated?

6. HPD of nodes must be presented.

7. Genetic variation presented in the phylogeny has again been presented in the context of the HA antigenic sites and NA sites conferring neuraminidase resistance.

8. The relevance of mutations belongs in the discussion but is presented in the results.

9. THe prediction of “vaccine efficacy” is confusing to read. This section needs to be reworked to allow the reader to understand what terms like “worst-case” efficacy and “perfect match”mean. The table is confusing and must either be removed or presented in a more intelligible fashion.

Discussion: The discussion does not highlight the relevance of the work. The discussion needs to be reworked to summarize key findings of the study and their agreement in a functional or epidemiological or evolutionary framework. Some of the content in the results belongs in the discussion e.g the paragraph on pg 19 “Several substitutions listed above…” etc.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2020 Jun 24;15(6):e0234869. doi: 10.1371/journal.pone.0234869.r002

Author response to Decision Letter 0

10 Apr 2020

April 1, 2020

The Editorial Office,

PloS One

Dear editors and reviewers:

Thank you for your kind reply. We are very grateful for the reviewer’s comments on our manuscript entitled “Whole-genome sequencing reveals origin and evolution of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018” (PONE-D-20-01217). Indeed, your comments are very helpful in improving our manuscript. We have carefully studied your comments point by point and tried our best to revise the manuscript to meet your approvals. We believe that we have settled all of the concerns from the reviewers, and the revisions are documented in the revised manuscript and supplementary information. The responses to the reviewers’ comments are listed as follows (Our replies are in blue).

Kind regards,

Corresponding author: Mei-Ling Zhang

E-mail address: meilingz2011@163.com

Responses to reviewers' comments

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Yes

Reviewer #2: Yes

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: N/A

Reply:

For statistical analysis, we have re-normlized by scientific algorithms and softwares. Please check our improvements.

3. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

Reviewer #2: Yes

4. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: No

Reviewer #2: Yes

Reply:

We have done our best to reduce the specific errors and discuss with local English professors. Please check our revised manuscript.

5. Review Comments to the Author

Reply:

Thank you very much. According to your requests, we have provided consistent answers.

Reviewer #1: This is an important paper, about evolution of H1N1 sequences from regions often thought to be the source of new influenza strains. I have a few comments

Reply:

Thank you very much for the professional suggestions. And we fully agree with your points. Now, according to your suggestions, we have adjusted our revisions as follows:

(1) To make our manuscript more clearer, when we discussed the antigenic epitopes, we explained the theory of the mapping relationships from Sa, Sb, Ca1, Ca2 and Cb to B+D, B, C+D, A+D and E. According to the sequence homology, mutations observed in five epitopes (Sa, Sb, Ca1, Ca2, and Cb) were mapped to the H3 epitopes (B+D, B, C+D, A+D and E), respectively. So, we changed memberships (Sa, Sb, Ca1, Ca2 and Cb) into epitope memberships (B+D, B, C+D, A+D and E).

(2) And further we cited the previous published references to illustrate that these mapping relationships are reliable (Deem and Pan 2009; Opanda, et al. 2020).

(3) These mapping relationships provided a preliminary basis to understand the downstream evaluation of Pepitope.

Please check our revised Result section << Phylogeny and mutation analysis of HA of influenza A(H1N1)pdm09 strains>>. We hope that our revisions can meet your criterion.

Reply:

We thank the reviewer for pointing out this issue. Consistent with above answers, we have identified the epitope relationship in our revised Result section << Phylogeny and mutation analysis of HA of influenza A(H1N1)pdm09 strains>> to improve our manuscript. In detail, the mutation sites and 3D structure analyses of HA protein have been adjusted by the mapped five epitopes (A-E), and please see our revised S4 Table and Fig 3.

3) Please verify the numbering from Table 1 of ref 21 was used to compute pEpitope (or that the .xls or matlab script was used to compute pEpitope).

Reply:

Thanks for your good comments. According to your points, we repeatly validated the numbering and our numbering (Table 1) in this study was accordance with your listed research, which point out that the five epitopes (A to E) respectively possess 24, 22, 33, 48, and 34 amino acids (Deem and Pan 2009). The residue differences on the epitopes were counted and reanalyzed in our Table 1.

Further, the vaccine efficacy was estimated by the formula (E = 0.47–2.47 x Pepitope). Also please check our revised method, especially for this section << Estimation of vaccine efficacy of influenza A(H1N1)pdm09 strains>>. And three related references were cited in this section accordingly (Deem and Pan 2009; Gupta, et al. 2006; Monamele, et al. 2019).

Reply:

We thank the reviewer to point out this issue. We agree with your views that generally you group influenza strains from July 1 year n to June 30 year n+1. Herein, in this study we defined one year from January 1 to December 31. Thus this manuscript has covered the period from January 1, 2014 to December 31, 2018.

Please see our revised sampling strategy in Results. We added according description in this revised manuscript.

Reply:

Thanks for your good suggestions to improve this article. We have compared the 2017 and 2018 strains against the old vaccine strain (A/California/07/2009). Judging from the results, there was an expected increase (from 24.52%-36.63% to 39.59%-41.81%) in vaccine efficacy when the vaccine strain changed to A/Michigan/45/2015 in 2017-2018.

Further, we also compared the 2018 strains against the newer vaccine strain (A/Brisbane/02/2018), but the predicated vaccine efficacy declined to the ranging of 31.93%-41.81%. It was mainly attributed to the additional three substitutions (G45R, V298I and R223Q) on dominant epitopes C and D. We also observe that the vaccine efficacy against 2018 strains was lower than that of 2017. Therefore, it’s essential for the WHO to update the vaccine strain in 2019. However, due to the time lag of research, our study did not include the strains isolated in 2019, so it could not directly reflect the true vaccine efficacy against the circulating strains of 2019, in lincang, China. On the other hand, WHO determined the vaccine strain compositions based on the situation of global circulating strains, so the vaccine strains recommended by WHO could not always provide better vaccine efficacy in local city, which was also supported by previous studies (Opanda, et al. 2020).

Please check our revised Results section <<Estimation of vaccine efficacy of influenza A(H1N1)pdm09 strains>> in detail.

5) It would be interesting to describe in which epitope most of the evolution in 2018 (e.g. figure S1) ocurred.

Reply:

Thanks for your valuable advices. According to your good suggestions, we have analyzed the amino acid replacement rate for each epitope of 2018 strains. Our results revealed that the epitope B accumulated mutational amino acids more quickly than that of other epitopes (C-E) (Wilcoxon rank sum test, P<2.855e-06). The data have been described in S2B Fig.

Reply:

We thank the reviewer for pointing out these issues. According to you suggestions, we have provided careful improvements in this revised manuscript. We hope that our revisions can meet your criteria well.

Methods:

Reply:

Thanks for your valuable comments. According to your suggestions, we have cited Technical Guidelines for National Influenza Surveillance (2017 Edition) (http://www.chinaivdc.cn/cnic/zyzx/jcfa/201709/t20170930_153976.htm) to provide the detailed experimental protocols. The strain identification kit was furnished by Chinese National Influenza Center, and the post-infection ferret antisera was raised by corresponding vaccine strains (A/California/7/2009(H1N1)pdm09 for 2009-2016 and A/Michigan/45/2015 for 2017-2018) and selected representative strains in China.

The related information has been supplemented in Materials and methods section << Specimen collection and isolation and identification of virus>>.

2. Authors mention genome amplification using “Invitrogen SuperScript III One-Step RT-PCR Platinum kit”. IT is not clear what is amplified. The primers used must be stated,

Reply:

Thanks for your suggestions. According to your points, we have provided the information of genome amplification. For example, the SuperScriptTM III One-Step RT-PCR System with PlatinumTM Taq High Fidelity was used to perform cDNA synthesis and PCR amplification of viral nucleic acids.

Next, the amplification primers included Uni-12/Inf1 (primer A): 5’-GGGGGGAGCAAAAGCAGG-3’, Uni-12/Inf3 (primer B): 5’-GGGGGGAGCGAAAGCAGG-3’ and Uni-13/Inf1 (primer C): 5’-CGGGTTATTAGTAGAAACAAGG-3’. They have been added in the revised Materials and methods section <<Whole genome sequencing of influenza A(H1N1)pdm09>>.

3. What was the protocol for library prep? Should be mentioned briefly.

Reply:

Thanks very much for your kind points. According to your suggestions, we have added a clear description to our revised Materials and methods section <<Whole genome sequencing of influenza A(H1N1)pdm09> to briefly describe the protocols for library prep. Please check our revised manuscript.

4. While the authros have states use of a MiSeq for generation of whole genome sequence. A description of sequencing kit used and the run conditions and protocol for data handling is not mentioned.

Reply:

Thanks a lot. We have provided improvements for the Materials and methods section <<Whole genome sequencing of influenza A(H1N1)pdm09> in this revised manuscript. To make our description more clearer, we described as follows in our revised manuscript:

“In the end, we thawed reagent cartridge (MiSeq v2 Reagent Tray 300 cycles – PE) , loaded the pooled libraries onto the reagent cartridge in the designated reservoir and operated MiSeq sequencer applied to sequencing according to the illumine MiSeq system guide. The raw sequencing paired-end reads were imported to CLC Genomics Workbench 9.5.2 (Qiagen, Denmark) and further were trimmed and performed for de novo assembly into contigs. ”

5. Any reason why contigs were generated de novo? The usual protocol for influenza is to map to reference strains

Reply:

Thank you so much. We are in agreement with your comments. As a matter of fact, this has been done according to the usual protocol. We apologize for our unclear descriptions. Now, in this revised manuscript, we have added this part to our updated version. In detail, the raw sequencing paired-end reads were imported to CLC Genomics Workbench 9.5.2 (Qiagen, Denmark) and further were trimmed and performed for de novo assembly into contigs. Then, the contigs were imported to National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/genomes/FLU/Database/nph-select.cgi?go=database) for blast, and the best-hit reference strains were selected for mapping to produce consensuses.

6. Details on the “Global Reference Strains” are missing.

Reply:

Thanks very much. We have revised it in this manuscript. The 14 global reference strains included three northern hemisphere vaccine strains (2014-2020) recommended by WHO. And the detail information (accession numbers) about global reference strains was replenished in S1 Table.

7. For the phylogenetic analysis, what was the nucleotide substitution model used? This has a bearing on the results

Reply:

Thanks. The phylogenetic trees were constructed using Mrbayes method under the HKY+I+G nucleotide substitution model. This has been added to the methods section in our revised manuscript.

Additionally, we also performed the MCCTREE analyses by BEAST v1.10.4 software to estimate divergence time and obtained an according topology (please see below answers for MCCTREE). This suggests our analyses including our model parameters are reliable.

8. What is the tree being presented? MCC tree? Software used for annotation of trees?

Reply:

Many thanks. We apologize for our unclear statements although we have described our Mrbayes algorithm applied to construct phylogenies in Materials and methods. Here, in the revised figure legends (Figs 2 and 5), we also added the ‘Mrbayes phylogeny’ to make our descriptions more clearly. Please check the according revision in main text.

The Mrbayes phylogenetic trees were presented and annotated in Fig Tree v1.4.3 software. This has also been appended to the method section in our revised manuscript.

9. Vaccine efficacy (16,22,23)

Reply:

Thank you for your professional advice. We have re-calculated the vaccine efficacy based on a published reference (Deem and Pan 2009). The results were in good agreement with the trends obtained from your listed references (Falchi, et al. 2011; Pan, et al. 2011; Tewawong, et al. 2015).

Results

Reply:

Thanks greatly for the useful comment. We calculated and supplemented this part in Results section <<Isolation rate, demographic characteristic and sampling distribution of influenza>> in this revised manuscript. 6767 influenza-like illness samples were collected in Lincang people's hospital, during the study period (January 1, 2014 to December 31, 2018). The isolation rates of influenza A and B strains were 3.04% and 2.75%, respectively. Among the 392 cases infected with influenza viruses, male accounted for the majority (61.73%), mainly in the age group under 5 years old, and scattered children were the main infected population. The detailed data were shown in S2 Table.

2. The source of these strains as mentioned by the authors is not clear.

Reply:

Thanks a lot. First, all the 6767 influenza-like illness samples were collected in Lincang people's hospital, which is the only sentinel surveillance hospitals in Lincang. Second, total 392 strains were isolated from the 6767 influenza-like illness samples. Among that, 112 strains were influenza A(H1N1)pdm09 strains. In the end, we randomly selected 45 strains for subsequent sequencing experiments. Please further check our revisions in Result section <<Isolation rate, demographic characteristic and sampling distribution of influenza>> in this revised manuscript.

Reply:

We are grateful to the reviewer for normalizing our vague statements for this section. I’m sorry for unclear descriptions. Actually, our analyses in this section only illustrated that the strains in 2018 have accumulated amino acid variations more quickly than those from all of other years. Therefore, we rephrased our descriptions in this revised manuscript. Additionally, we further provided insights into the statistics for different antigenic epitopes about amino acid substitutions. Please check our revisions in main text accordingly.

Importantly, according to your suggestions, we also provided accurate estimates of virus evolutionary rates (substitutions/site/year) for each branch in our phylogeny using BEAST v1.10.4 under an uncorrelated relaxed molecular clock model. We supplied this information to S5 Table including the evolutionary rates from all of isolated strains. Please check our revisions.

4. The HA and NA phylogeny with listed mutation sites is verbose and difficult to read. It will benefit from a table summarizing the mutations.

Reply:

Thanks. According to your suggestions, we have deleted the listed mutation sites in HA and NA phylogeny, as can be seen in Figs 2 and 5. The mutations of HA and NA were also summarized in S4 and S6 Tables.

5. Has the tMRCA been calculated?

Reply:

We very thank this reviewer for pointing out this issue. We did not calculate the tMRCA from our analyzed strains. But, according to your suggestions, we added this analysis in our revised manuscript.

In this revision, we utilized the MCMC algorithm of BEAST v1.10.4 to evaluate the tMRCA from our isolated strains. In detail, HKY substitution model and uncorrelated relaxed clock with lognormal relaxed distribution were used in our analysis, and the 10,000,000 MCMC chain length was performed. Furthermore, we used the TreeAnnotator v1.10.4 software of the BEAST v1.10.4 toolkit to annotate the target Maximum clade credibility tree (MCCTREE) with 10% Burnin as the number of trees. Please see our method section in the revised manuscript.

Additionally, we used FigTree v1.4.3 to visualize our MCCTREE. Divergence time of ancestral nodes was estimated with 95% highest density probability (HPD). Please see our revised manuscript.

6. HPD of nodes must be presented.

Reply:

Many thanks for this reviewer to help us point out this issue. We fully agree with your suggestions. According to your points, we calculated HPD for each node in our constructed MCCTREE using BEAST v1.10.4 and also demonstrated them in phylogeny using blue bars. And several focused ancestral nodes were highlighted for clade 6B.1A, 6B.1, 6B.2 and clade from all isolated strains by using BEAST v1.10.4 in this study. Please check our added Fig 4 in our revised manuscript.

For Mrbayes phylogeny for HA and NA, we further provided a Bayesian posterior probability range because of the algorithm without 95% HPD compared to MCCTREE. Please check our S1 and S3 Figs.

Hope our revisions to meet your criterions.

7. Genetic variation presented in the phylogeny has again been presented in the context of the HA antigenic sites and NA sites conferring neuraminidase resistance.

Reply:

We thank the reviewer to point out this issue. We approved of your points. According to your suggestions, we have integrated mutation and phylogeny analysis into one section in our revised Results for this manuscript version. Please check our improvements accordingly.

8. The relevance of mutations belongs in the discussion but is presented in the results.

Reply:

Thanks a lot. We have adjusted the relevance of mutations in Results to our Discussion.

Reply:

Thanks for your important suggestions. The section of estimation of vaccine efficacy has been rewritten, and the unclear terms like “worst-case” efficacy and “perfect match” have been suppressed in this revision. The table about vaccine efficacy has been revised in a more intelligible mode. Please check our revisions in Results section <<Estimation of vaccine efficacy of influenza A(H1N1)pdm09 strains>>.

Reply:

We thank the reviewer for pointing out this issue. We agree with your points. According to your suggestions, we have reworked to summarize our key findings in this revised manuscript. Please check our revisions in the Discussion section.

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

References

Deem MW, Pan K 2009. The epitope regions of H1-subtype influenza A, with application to vaccine efficacy. Protein Eng Des Sel 22: 543-546.

Falchi A, Amoros JP, Arena C, Arrighi J, Casabianca F, Andreoletti L, Turbelin C, Flahault A, Blanchon T, Hanslik T, Varesi L 2011. Genetic structure of human A/H1N1 and A/H3N2 influenza virus on Corsica Island: phylogenetic analysis and vaccine strain match, 2006-2010. PLoS One 6: e24471.

Gupta V, Earl DJ, Deem MW 2006. Quantifying influenza vaccine efficacy and antigenic distance. Vaccine 24: 3881-3888.

Monamele CG, Munshili Njifon HL, Vernet MA, Njankouo MR, Kenmoe S, Yahaya AA, Deweerdt L, Nono R, Mbacham W, Anong DN, Akoachere JF, Njouom R 2019. Molecular characterization of influenza A(H1N1)pdm09 in Cameroon during the 2014-2016 influenza seasons. PLoS One 14: e0210119.

Opanda S, Bulimo W, Gachara G, Ekuttan C, Amukoye E 2020. Assessing antigenic drift and phylogeny of influenza A (H1N1) pdm09 virus in Kenya using HA1 sub-unit of the hemagglutinin gene. PLoS One 15: e0228029.

Pan K, Subieta KC, Deem MW 2011. A novel sequence-based antigenic distance measure for H1N1, with application to vaccine effectiveness and the selection of vaccine strains. Protein Eng Des Sel 24: 291-299.

Tewawong N, Prachayangprecha S, Vichiwattana P, Korkong S, Klinfueng S, Vongpunsawad S, Thongmee T, Theamboonlers A, Poovorawan Y 2015. Assessing Antigenic Drift of Seasonal Influenza A(H3N2) and A(H1N1)pdm09 Viruses. PLoS One 10: e0139958.

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PLoS One. doi: 10.1371/journal.pone.0234869.r003

Decision Letter 1

Mrinmoy Sanyal

28 Apr 2020

PONE-D-20-01217R1

Whole-genome sequencing reveals origin and evolution of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018

PLOS ONE

Dear Mrs Zhang,

The revised manuscript is significantly improved. However, one of the experts raised significant concern (see below) which need to be corrected before the manuscript is suitable for publication. Please update the manuscript as advised by the Reviewer # 1 as soon as possible.

We would appreciate receiving your revised manuscript by Jun 12 2020 11:59PM. When you are ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). This letter should be uploaded as separate file and labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. This file should be uploaded as separate file and labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. This file should be uploaded as separate file and labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Mrinmoy Sanyal, PhD

Academic Editor

PLOS ONE

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

**********

6. Review Comments to the Author

Reviewer #1: The authors have largely addressed my comments. There is one significant issue, though. The formula used for efficacy is the one for H3N2. But the authors are studying H1N1. So, the author should use the efficacy formula from from Ref. 16 Figure 2, which is

efficacy = 0.53 - 1.19 p_epitope

All results should be updated with this formula.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

PLoS One. 2020 Jun 24;15(6):e0234869. doi: 10.1371/journal.pone.0234869.r004

Author response to Decision Letter 1

1 Jun 2020

June 1st, 2020

The Editorial Office,

PloS One

Dear editors and reviewers:

Many thanks for receiving your kind reply. We are very grateful for the reviewer’s comments on our manuscript entitled “Whole-genome sequencing reveals origin and evolution of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018” (PONE-D-20-01217R1). We have carefully studied your valuable comments especially for the formula calculating efficacy and tried our best to revise our manuscript. We believe that we have settled all of the concerns from the reviewer. We hope that our revisions can meet your approvals.

Kind regards,

Corresponding author: Mei-Ling Zhang and Yong Shao

E-mail address: meilingz2011@163.com

E-mail address: Yong_Shao_dws@126.com

Response to reviewers

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: (No Response)

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reply: According to suggestions from reviewer#1, we have fully corrected this issue in our revised manuscript.

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #1: Yes

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #1: Yes

6. Review Comments to the Author

Reviewer #1: The authors have largely addressed my comments. There is one significant issue, though. The formula used for efficacy is the one for H3N2. But the authors are studying H1N1. So, the author should use the efficacy formula from from Ref. 16 Figure 2, which is

efficacy = 0.53 - 1.19 pepitope

All results should be updated with this formula.

Reply: We thank this reviewer to point out this issue for improving our manuscript. We really appreciate for your great professional knowledge in virology.

In this revision, according to your kind suggestions and your recommended reference and formula, we have updated all of our results throughout our manuscript including methods, results, and discussions. And we obtained a very similar efficacy trends with the previous formula. Therefore, all of revisions do not influence or change our conclusions.

Please check our revised manuscript in this version. We hope that our revisions can meet our requests sincerely.

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

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PLoS One. doi: 10.1371/journal.pone.0234869.r005

Decision Letter 2

Mrinmoy Sanyal

4 Jun 2020

Whole-genome sequencing reveals origin and evolution of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018

PONE-D-20-01217R2

Dear Dr. Zhang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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PLoS One. doi: 10.1371/journal.pone.0234869.r006

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Whole-genome sequencing reveals origin and evolution of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Accession numbers in GISAID databases of applied gene segments of 14 A(H1N1)pdm09 reference strains included in the analysis.

N/A: not applied.

(DOC)

Click here for additional data file.^{(39KB, doc)}

S2 Table. The number (%) of influenza strains isolated from samples of influenza-like cases in Lincang, China, from 2014 to 2018.

(DOC)

Click here for additional data file.^{(31KB, doc)}

S3 Table. Demographic characteristics of the cases infected with influenza viruses in Lincang, China, from 2014 to 2018.

(DOC)

Click here for additional data file.^{(47KB, doc)}

S4 Table. Amino acid substitutions in the epitopes and non-epitopes of HA protein of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

(XLSX)

Click here for additional data file.^{(27.2KB, xlsx)}

S5 Table. Estimated evolutionary rates of HA genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

The evolutionary rates were estimated by BEAST v1.10.4 software under the uncorrelated relaxed clock model.

(DOC)

Click here for additional data file.^{(60.5KB, doc)}

S6 Table. Amino acid substitutions of NA protein of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

(XLSX)

Click here for additional data file.^{(24.8KB, xlsx)}

S7 Table. Important amino acid substitutions of six internal proteins of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Amino acid mutations were referred to A/California/07/2009. “.” showed that the amino acid site was the same as that of A/California/07/2009.

(XLSX)

Click here for additional data file.^{(22KB, xlsx)}

S1 Fig. Mrbayes phylogeny of the HA genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Each nodal number in square brackets represented a Bayesian posterior probability (BPP) range. The ruler value (0.005) represented genetic distance.

(TIF)

Click here for additional data file.^{(760.5KB, tif)}

S2 Fig. Comparative analyses of mutations in HA protein between 2018 and other years.

(TIF)

Click here for additional data file.^{(503.5KB, tif)}

S3 Fig. Mrbayes phylogeny of the NA genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Each nodal number in square brackets represented a Bayesian posterior probability (BPP) range. The ruler value (0.005) represented genetic distance.

(TIF)

Click here for additional data file.^{(790.9KB, tif)}

S4 Fig. Mrbayes phylogeny of the M genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

6B~6B.1A indicated branch Numbers of clades. Each nodal number in phylogeny exhibited a Bayesian posterior probability (BPP). The ruler value (0.2) represented genetic distance.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S5 Fig. Mrbayes phylogeny of the NP genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

6B~6B.1A indicated branch Numbers of clades. Each nodal number in phylogeny exhibited a Bayesian posterior probability (BPP). The ruler value (0.002) represented genetic distance.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S6 Fig. Mrbayes phylogeny of the NS genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

6B~6B.1A indicated branch Numbers of clades. Each nodal number in phylogeny exhibited a Bayesian posterior probability (BPP). The ruler value (0.2) represented genetic distance.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

S7 Fig. Mrbayes phylogeny of the PA genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

6B~6B.1A indicated branch Numbers of clades. Each nodal number in phylogeny exhibited a Bayesian posterior probability (BPP). The ruler value (0.002) represented genetic distance.

(TIF)

Click here for additional data file.^{(1.6MB, tif)}

S8 Fig. Mrbayes phylogeny of the PB1 genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

6B~6B.1A indicated branch Numbers of clades. Each nodal number in phylogeny exhibited a Bayesian posterior probability (BPP). The ruler value (0.002) represented genetic distance.

(TIF)

Click here for additional data file.^{(1.6MB, tif)}

S9 Fig. Mrbayes phylogeny of the PB2 genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

6B~6B.1A indicated branch Numbers of clades. Each nodal number in phylogeny exhibited a Bayesian posterior probability (BPP). The ruler value (0.002) represented genetic distance.

(TIF)

Click here for additional data file.^{(1.5MB, tif)}

Attachment

Submitted filename: Response_to_Reviewers.doc

Click here for additional data file.^{(116.5KB, doc)}

Attachment

Submitted filename: Response_to_reviewers_ZML_20200601.doc

Click here for additional data file.^{(33KB, doc)}

Data Availability Statement

The 45 genome sequencing data of A(H1N1)pdm09 strains have been deposited at NCBI Gene Bank Database with accession codes: MT540604-MT540963.

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PERMALINK

Whole-genome sequencing reveals origin and evolution of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018

Xiao-Nan Zhao

Han-Ju Zhang

Duo Li

Jie-Nan Zhou

Yao-Yao Chen

Yan-Hong Sun

Adeniyi C Adeola

Xiao-Qing Fu

Yong Shao

Mei-Ling Zhang

Roles

Abstract

Introduction

Materials and methods

Ethics consideration

Specimen collection and isolation and identification of virus

Whole genome sequencing of influenza A(H1N1)pdm09

Construction of phylogenetic trees and prediction of glycosylation sites

3D model visualization of HA and NA protein

Potential mutants contributing to functional changes

Estimation of vaccine efficacy of influenza A(H1N1)pdm09 strains

Results

Isolation rate, demographic characteristic and sampling distribution of influenza

Fig 1. Sample distribution of total and sequencing strains of influenza A(H1N1)pdm09 viruses in Lincang, China, from 2014 to 2018.

Phylogeny and mutation analysis of HA of influenza A(H1N1)pdm09 strains

Fig 2. Mrbayes phylogeny of the HA genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Fig 3. Observed mutations and mutational frequencies of amino acid residues at the antigenic epitopes and RBSs of HA protein of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Fig 4. Maximum clade credibility tree from HA alignment segments of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Phylogeny and mutation analysis of NA of influenza A(H1N1)pdm09 strains

Fig 5. Mrbayes phylogeny of the NA genes of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Fig 6. Observed mutations in the important amino acid sites of NA protein of influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Phylogeny and mutation analysis of six internal segments of influenza A(H1N1)pdm09 strains

Estimation of vaccine efficacy of influenza A(H1N1)pdm09 strains

Table 1. Predicted vaccine efficacy of vaccine strains against influenza A(H1N1)pdm09 strains in Lincang, China, from 2014 to 2018.

Discussion

Conclusions

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Mrinmoy Sanyal

Roles

Author response to Decision Letter 0

Decision Letter 1

Mrinmoy Sanyal

Roles

Author response to Decision Letter 1

Decision Letter 2

Mrinmoy Sanyal

Roles

Acceptance letter

Mrinmoy Sanyal

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Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

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