Increased genetic variation of A(H3N2) virus from influenza surveillance at the end of the 2016/2017 season for Shanghai port, China | Scientific…

Virological influenza surveillance during flu season 2016/2017

Virological influenza surveillance data in the Shanghai port were collected weekly. From February 2016 to September 2017, a total of 64 swab samples were collected from passengers of different countries, including 41 passed through Asia (25 from Hong Kong and 12 from Southeast Asia, especially), 16 passed through Europe, 7 passed through the America, and 7 passed through Oceania.

A(H3N2) virus activity increased from the 44th week of 2016, peaked in the 1st week of 2017 and decreased afterwards. The highest proportion of A(H3N2) was observed in summer (28/64, 43.7%), followed by winter (22/64, 34.3%) which outnumbered by that in spring and fall (11/64, 21.8%).

Of the 610 genetically characterized viruses, 546 were provided from GISAID EpiFlu databases. All 64 HA genes sequenced by the Shanghai Port belonged to the H3N2 3C.2a clade. This clade also included the vaccine strain A/HongKong/4801/2014, supporting the vaccine recommendation in the 20162018 northern hemisphere influenza season by WHO. Among the 64 viruses, the majority (n=20, 31.2%) belonged to the subclade 3C.2a.1 represented by A/Singapore/INFIMH-16-0019/2016. The proportions for other subclades were 26.5% (3C.2a.2, n=17), 25% (3C.2a.3, n=16) and 6.2% (3C.2a.4, n=4) (Fig.1).

Phylogenetic analysis of the HA segments circulating between 2016/2017.

Individual clades of A(H3N2) are typically defined by amino acid substitutions that occur as they diversify from parental strains. Analysis of HA sequences indicated co-circulation of multiple variants in clade 3C.2a. All variants within subclade 3C.2a.1 shared four substitutions N121K, N171K, I406V and G484E. Three additional substitutions were observed in the 3C.2a.1 subcluster: S92R and H311Q in cluster I, G479E in cluster II. Variants 3C.2a.3 shared N121K/E and S144K (I58V and S219R in cluster I and T135K and R150K in cluster II), Variants 3C.2a.2 were characterized by T131K and R142K substitutions and variants 3C.2a.4 were characterized by D53N, R142G, S144R, I182T and Q197H (Fig.2).

Schematic diagram demonstrating the shared amino acid changes between clades 3C.2a, 3C.2a.1, 3C.2a.2, 3C.2a.3 and 3C.2a.4 on HA gene.

There were more 3C.2a.1 variants identified from samples collected in the 2017 summer (n=11) than in the 2016/2017 winter (n=7). This subclade was further divided into two homogenous sub-clusters (cluster I and II; Fig.1). The strains from cluster I were concentrated in winter, and the cluster II strains were persisted more common in the summer months. Most viruses in the subgroup 3C.2a.3 happened in summer. And we also found that there was no prominent summer or winter trend of viruses clustered in 3C.2a.2.

To analyze the geographical distribution of A(H3N2) in China, 31 provinces were classified into six regions based on geographic proximity: North (Beijing), East-coastal (Shanghai), East-inland (Anhui), South-coastal (Guangdong), South-inland (Guizhou), Northeast (Jilin), Northwest (Shanxi) and West (Sichuan). According to our phylogenetic analysis, the A(H3N2) number of the above six regions be counted (Fig.3A). The Proportions for A(H3N2) in these regions were 5%, 43%, 7.2%, 19%, 4%, 7% ,5% and 7%, respectively. Interestingly, higher epidemic waves of A(H3N2) were observed in Eastern and Southern in China coastal areas, and we presumed that convenient transportation and dense population contributed to it19.

The Proportions of Influenza A(H3N2) in China six regions (A) and H3N2 clade patterns in China eight provinces (B).

The genetic diversity results (Fig.3B) indicated that the diversity increased in the East and South, especially coastal cities, Shanghai and Guangzhou. All clades and subclades of the current A(H3N2) were detected in both cities. 3C.2a.3 (60%) was dominant in Guangzhou, with a small proportion of 3C.2a.4 (10%), 3C.2a (3%), 3C.1 (2%), 3C.3 (5%), 3C.2a.1-I (10%) and 3C.2a.1 (5%). In contrast, 3C.2a.1, 3C.2a.1-I and 3C.2a.2 were the major subcluster in Shanghai, with proportions of 19.21%, 32.36% and 30.34%, respectively. 3C.2a.3-II (4%), 3C.1 (1%), 3C.3 (2%) and 3C.2a (1%) were also detected in this region. The diversity of the clade pattern and the dominant clade in these two coastal cities matched well with the trends of the current global A (H3N2), likely because of the higher density of migration and subtropical monsoon climate20.

There were two models of predicted glycosylation sites in the HA proteins of the A(H3N2) clade 3C.2a: 12 potential glycosylation sites (N8ST, N22GT, N38AT, N45SS, N63CT, N126WT, N133GT, N158YT, N165VT, N246ST, N285GS and N483GT) and 11 potential glycosylation sites (N8ST, N22GT, N38AT, N45SS, N63CT, N126WT, N133GT, N158YT, N165VT, N246ST and N285GS). All of virus strains detected at the Shanghai Port in the clade 3C.2a.1 had 11 potential glycosylation sites, and the rest in the other clades had 12 sites. Comparing to the vaccine strains 2016/2017 A/HongKong/4801/2014 (N8ST, N22GT, N38AT, N45SS, N63CT, N126WT, N133GT, N165VT, N246ST, N285GS and N483GT), the clade 3C.2a.1 virus did not have potential glycosylation site 483(N483GT), the viruses in the clade 3C.2a.2, the clade 3C.2a.3 and the clade 3C.2a.4 had the potential glycosylation site 158(N158YT).

To assess the effect of the accumulated mutations in the HA1 domain on predicted vaccine efficacy in a given year, the p epitope method was used to evaluate how closely the vaccine strain resembles the imported strain (Table 1). Theoretically, when p epitope in the dominant epitope is higher than 0.19, the vaccine efficacy becomes negative21,22. For the 2016/2017 season, the average p epitope for all A(H3N2) strains was 0.090, which indicated the vaccine efficacy (VE) against those strains was 52.96% (E=24.89% of 47%, p epitope=0) of that of a perfect match with the vaccine strain. However, from the 2016/2017 winter to the 2017 autumn, the VE value fluctuated first and then decreased, with the highest value in spring (VE=58.51%), the lowest value in autumn (VE=58.51%), and the inflection point in summer (VE=49.29%). These results suggest that the A(H3N2) strains circulating in 2017 were separated from the vaccine strain and effectively reduced the VE starting in the summer.

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Increased genetic variation of A(H3N2) virus from influenza surveillance at the end of the 2016/2017 season for Shanghai port, China | Scientific...