2014-2019年青岛市A型流感病毒进化分析

赵丹; 张晓月; 董礼艳; 孙睿; 汪照国; 高汝钦

doi:10.16462/j.cnki.zhjbkz.2020.05.016

2014-2019年青岛市A型流感病毒进化分析

doi: 10.16462/j.cnki.zhjbkz.2020.05.016

1.
266033 青岛, 青岛市疾病预防控制中心/青岛市预防医学研究院
2.
250014 济南, 山东省第一医科大学第一附属医院/山东省千佛山医院

基金项目: 青岛市民生科技计划项目(17-3-3-2-nsh)

详细信息

通讯作者:
高汝钦, E-mail:gaoruqin@sohu.com

中图分类号: R181
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出版历程
- 收稿日期: 2019-10-30
- 修回日期: 2020-01-31
- 刊出日期: 2020-05-10

Evolutionary analysis of influenza A viruses in Qingdao from 2014 to 2019

1.
Qingdao Municipal Center for Disease Prevention and Control/Qingdao Institute of Preventive Medicine, Qingdao 266033, China
2.
The First Affiliated Hospital of Shandong First Medical University/Shandong Provincial Qianfoshan Hospital, Jinan 250014, China

Funds: Livelihood and Science Program of Qingdao(17-3-3-2-nsh)

More Information

Corresponding author: GAO Ru-qin, E-mail:gaoruqin@sohu.com

摘要

摘要: 目的了解2014-2019年青岛市人群A型流感病毒(influenza A virus, IAV)流行病学和遗传特征。方法提取9 807份流感样病例咽拭子标本的病毒RNA, 采用多重实时反转录PCR方法鉴定分型; 采用一步法反转录PCR扩增IAV血凝素(hemagglutinin, HA)和神经氨酸酶(neuraminidase, NA)基因, 并进行序列测定和基因分析。结果 2014-2019年青岛市流感病毒流行具有北半球典型季节性流感特征。夏季小量流行主要是H3N2。青岛市IAV中H1N1pdm09和H3N2分别占57.6%和42.4%, 并呈现交替流行。基因群分属于6B和3C。两者HA和NA基因进化都处在净化选择下, 但抗原进化可能具有不同的进化模式。与2009年毒株相比, 2019年青岛市H1N1pdm09和H3N2的HA分别已发生21个和30个氨基酸替换, 均主要发生在头部, 分别包含5个和18个抗原位点。H1N1pdm09检测到两株神经氨酸酶抑制剂抗性突变, 1株为H275Y突变, 另1株为S247N突变; H3N2中未发现NAI抗性突变。结论 H1N1pdm09和H3N2是2014-2019年青岛市季节性IAV中的主要组分, 进化迅速。加强流感监测和研究十分必要, 流感疫苗株需要及时更新。
- A型流感病毒 /
- 血凝素 /
- 神经氨酸酶 /
- 基因进化分析
Abstract: Objective To analyze the epidemiology and molecular characteristics of influenza A virus(IAV) circulated in Qingdao from 2014 to 2019. Methods Throat swab samples from 9 807 influenza-like cases were collected and viral RNA was extracted. All samples were screened for influenza virusby multiplex real-time reverse transcription PCR. Hemagglutinin(HA) and neuraminidase(NA) genes were amplifyed by one-step reverse transcription PCR and sequenced, and then molecular characteristics of them were analyzed. Results The characteristics of influenza virus in Qingdao from 2014 to 2019 were similar to those of the typical seasonal influenza in the northern hemisphere. A small peak of summer influenza was mainly attributed to H3 N2. Of these IAV positive cases, H1 N1 pdm09 and H3 N2 accounted for 57.6% and 42.4% respectively, both alternately popular in different years. Phylogenetic tree indicated that they belonged to the 6 B and 3 C subgroup respectively. The genetic evolution of H1 N1 pdm09 and H3 N2 was under purifying selection, but antigenic evolution might have different evolution patterns. Compared with strains in 2009, twenty-one and thirty amino acid substitutions in HA occurred respectively in H1 N1 pdm09 and H3 N2 viruses in 2019, and they were mainly located in the head domain, including 5 and 18 antigen sites respectively. Only one isolate with H275 Y mutation and one with S247 N known to reduce susceptibility to NA inhibitors(NAI) were detected in H1 N1 pdm09, and no NAI-resistant mutation was found in H3 N2. Conclusions H1 N1 pdm09 and H3 N2 are the most prevalent strains of seasonal IAV with rapid evolution in Qingdao. Strengthening influenza surveillance and research is necessary and influenza vaccine strains need to be updated in time.
- Influenza A virus /
- Hemagglutinin /
- Neuraminidase /
- Molecular evolution

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图 1 2014-2019年青岛市流感病毒流行的时间分布

Figure 1. Time distribution of influenza viruses in Qingdao from 2014 to 2019

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图 2 2014-2019年青岛市pH1N1的HA和NA基因系统进化分析

注:(A)为HA, (B)为NA。○标注为参考株, ■标注为2015-2016年毒株, ▲标注为2016-2017年毒株; ▼标注为2017-2018年毒株; ◆标注为2018-2019年毒株; Bootstrap进行1 000次运算, > 70数值列于各节点。

Figure 2. Phylogenetic trees of HA and NA nucleotide sequences of pH1N1 viruses in Qingdao from 2014 to 2019

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图 3 2014-2019年青岛市H3N2的HA和NA基因系统进化分析

注:(A)为HA, (B)为NA。○标注为参考株, ●标注为2014-2015年毒株, ■标注为2015-2016年毒株, ▲标注为2016-2017年毒株; ▼标注为2017-2018年毒株; ◆标注为2018-2019年毒株; Bootstrap进行1 000次运算, > 70数值列于各节点。

Figure 3. Phylogenetic trees of HA and NA nucleotide sequences of H3N2 viruses in Qingdao from 2014 to 2019

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图 4 2019年青岛pH1N1和H3N2毒株HA蛋白的三维结构

注:黑色圆球标注为氨基酸变异位点。

Figure 4. The 3D crystal structures of HAs of pH1N1 and H3N2 viruses circulated in 2019

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表 1 2014-2019年青岛市流感病毒检测阳性率

Table 1. Positive rate of influenza virus in Qingdao from 2014 to 2019

年度	阳性数(例)	样本总数(例)	阳性率[%(95%CI)]	基因型(例)
年度	阳性数(例)	样本总数(例)	阳性率[%(95%CI)]	IBV	pH1N1	H3N2
2014-	190	2 004	9.5(8.2~10.8)	106	0	84
2015-	249	2 026	12.3(10.9~13.7)	140	95	14
2016-	184	1 967	9.4(8.1~10.6)	14	15	155
2017-	372	1 890	19.7(17.9~21.5)	207	142	23
2018-2019	239	1 920	12.4(11.0~13.9)	22	177	40
合计	1 234	9 807	12.6(11.9~13.2)	489	429	316

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表 2 2014-2019年青岛市pH1N1和H3N2基因进化速率和阳性选择分析

Table 2. Evolution rate and positive selection for HA and NA genes of pH1N1 and H3N2 viruses in Qingdao from 2014 to 2019

	序列数	核苷酸替代速率(×10^-3)		阳性选择位点
		平均值	95%HPD	SLAC			FEL
		平均值	95%HPD	ω平均值	数量	位点	ω平均值	数量	位点
H1	60	3.56	2.61~4.46	0.240	0	-	0.240	0	-
H3	54	2.12	1.55~2.69	0.242	0	-	0.242	0	-
N1	60	2.80	2.07~3.56	0.368	0	-	0.368	0	-
N2	54	2.51	1.82~3.24	0.175	0	-	0.175	0	-

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表 3 pH1N1和H3N2基因群主要分支的HA特征位点变异

Table 3. Feature of amino acid substitutions identified in HAs of pH1N1 and H3N2 viruses

亚型	分支	特征变异位点
pH1N1	6B	D97N、K163Q、S185T、S203T、A256T、K283E、E374K、S451N、E499K
	6B.1	S84N、S162N、I216T
	6B.2	V152T、V173I、E491G、D501E
	6B.1A	S74R、S164T、I295V
H3N2	3C.3a	T128A、A138S、R142G、N145S、F159S、N225D
	3C.2a	L3I、N145S、F159Y、K160T、N225D、Q311H、D489N
	3C.2a1	N171K、I406V、G484E
	3C.2a2	T131K、R142K、R261Q
	3C.2a1b	K92R、H311Q
注:D代表天冬氨酸, N代表天冬酰胺, K代表赖氨酸, Q代表谷氨酰胺, S代表丝氨酸, T代表苏氨酸, A代表丙氨酸, E代表谷氨酸, I代表异亮氨酸, V代表缬氨酸, G代表甘氨酸, R代表精氨酸, F代表苯丙氨酸, L代表亮氨酸, Y代表酪氨酸, Q代表谷氨酰胺, H代表组氨酸。

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表 4 2019年青岛市pH1N1和H3N2毒株HA氨基酸位点变异

Table 4. Summary of amino acid substitutions identified in the HA of pH1N1 and H3N2 viruses in Qingdao in 2019

基因	毒株	氨基酸位点
H1		74	83	84	97	129	162	163	164	183	185	203	216
	Cal09	S	P	S	D	N	S	K	S	S	S	S	I
	1QD2019	R	S	N	N	D/N	N	Q	T	P/S	I/T	T	T
		256	260	283	295	321	374	451	499	504
	Cal09	A	N	K	I	I	E	S	E	K
	1QD2019	T	D/N	E	V	V	K	N	K	R/K
H3		3	33	45	48	62	92	121	128	131	142	144	145
	Perth09	L	Q	S	T	K	K	N	T	T	R	K	N
	3QD2019	I	R	N	I	G	R	K	T/A	K/T	G	S	S
		159	160	171	183	194	197	198	212	214	219	223	225
	Perth09	F	K	N	L	L	Q	A	T	S	S	V	N
	3QD2019	Y	T	K	H	L	Q/R	S	A	I	S/F	I	D
		278	311	312	347	406	484	489	529
	Perth09	N	Q	N	V	I	G	D	V
	3QD2019	K	Q	S	M/V	V	G/E	N	I/V
注:Cal09代表A/California/07/2009, 1QD2019代表2019年青岛pH1N1;Perth09代表A/Perth/16/2009, 3QD2019代表2019年青岛H3N2。D是天冬氨酸, N是天冬酰胺, K是赖氨酸, Q是谷氨酰胺, S是丝氨酸, T是苏氨酸, A是丙氨酸, E是谷氨酸, I是异亮氨酸, V是缬氨酸, G是甘氨酸, R是精氨酸, F是苯丙氨酸, L是亮氨酸, Y是酪氨酸, H是组氨酸, P是脯氨酸, M是甲硫氨酸。

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