Serological survey of neutralizing antibodies to eight major enteroviruses among healthy population

Figures & data

Demographic characteristics of subjects in serological survey

Age (years)	Age (mean ± SD)	Male n (%)	Female n (%)	Total n (%)
<1	0.7 ± 0.2	20 (44.4)	25 (55.6)	45 (8.74)
1–3	1.7 ± 0.7	35 (52.2)	32 (47.8)	67 (13.01)
4–6	4.7 ± 0.9	49 (51.0)	47 (49.0)	96 (18.64)
7–19	13.1 ± 4.3	45 (52.3)	41 (47.7)	86 (16.7)
20–39	29.9 ± 5.7	30 (44.8)	37 (55.2)	67 (13.01)
40–59	49.3 ± 5.7	43 (50)	43 (50)	86 (16.7)
≥60	66.3 ± 5.8	33 (48.5)	35 (51.5)	68 (13.2)
Total		255 (49.5)	260 (50.5)	515

All healthy subjects were recruited from an status surveillance of infection diseases at the Xiamen City Center for Disease Control and Prevention in the Fujian Province in China in 2016.

The information on enterovirus strains used in the CPE assay

Virus strain^a	Origin	Year	Genebank No.	Ref.
EV-A71/ C4	Jiangsu CDC	2008	FJ600325	Xu et al.^Citation30
CV-A16/190	National Taiwan University	2007	JF420555	Hou et al.^Citation33
CV-A6/141	Infectious cDNA clone	2007	KR706309	Yang et al.^Citation27
CV-A10	Xiamen CDC	2014	KY012321	Li et al.^Citation29
CV-B3/2035	Xiamen CDC	2008	JQ042700	Yang et al.^Citation34
CV-B5	Xiamen University	2009	JQ042699	/
ECHO25	Infectious cDNA clone	2013	KP099941	Hou et al.^Citation14
ECHO30	Xiamen University	2016	MF177223	/

^aThe EV-A71/C4 strain was kindly provided by Jiangsu CDC; the CV-A16/190 strain was kindly provided by National Taiwan University; the CV-A10 and CV-B3 strains were kindly provided by Xiamen CDC; the CV-A6/141 and ECHO25 strains were infectious cDNA clones constructed by our laboratory; the CV-B5 and ECHO30 strains were isolated by our laboratory

Fig. 1 Overview of micro-neutralization assay (a) and the age distributions of seroprevalence (b) and GMT (c) against eight types of EVs.

The main steps of the micro-neutralization assay are shown in a. After step 3, the diluted heat inactivated sera, enterovirus and RD cells were incubated at 37 °C or 33°C (EV-D68) for 5 to 7 days. The detection of CPE was observed by microscope. The neutralizing titer was defined as the highest dilution that exhibited > 50% neutralization of the well. Heat maps illustrating the age distributions of seroprevalence (b) and GMT (c) against eight types of EVs (EV-A71, CV-A16, CV-A6, and CV-A10 are belong to EV-A species; CV-B3, CV-B5, ECHO25, and ECHO30 are belong to EV-B species). A higher seroprevalence or GMT is shown as a darker red or blue color. The values of seroprevalence and GMT are given in the map

Seroprevalence and GMT of nAb against different types of enteroviruses by gender and district

	Pathogen, seroprevalence % (GMT)
	EV-A71	CV-A16	CV-A6	CV-A10	CV-B3	CV-B5	ECHO25	ECHO30
Gender
Male (n = 255)	34.5 (52.6)	20.8 (47.4)	36.1 (48.1)	38.4 (43.1)	31.4 (100.4)	30.6 (58.0)	13.7 (32.0)	51.0 (78.0)
Female (n = 260)	40.7 (37.2)	28.8 (44.6)	42.7 (47.4)	46.9 (47.4)	37.7 (97.1)	33.8 (58.7)	15.0 (33.2)	25.7 (73.2)
District
Siming (n = 83)	26.5 (51.3)	18.0 (33.5)	20.1 (28.3)	34.9 (43.7)	25.3 (66.1)	20.5 (48.1)	7.2 (22.6)	16.9 (45.3)
Huli (n = 95)	47.4 (39.1)	28.4 (45.8)	34.7 (50.8)	41.1 (41.0)	30.5 (65.5)	33.7 (55.0)	7.4 (32.0)	30.5 (105.7)
Haicang (n = 84)	33.3 (34.5)	29.8 (64.0)	54.8 (54.2)	42.7 (41.1)	31.0 (112.0)	35.7 (53.2)	19.1 (51.4)	29.8 (73.5)
Jimei (n = 82)	41.5 (44.3)	25.6 (49.1)	47.6 (47.3)	39.0 (52.7)	30.5 (147.0)	36.6 (51.2)	18.3 (29.2)	20.7 (104.4)
Xiang’an (n = 82)	40.2 (45.7)	29.3 (39.2)	42.7 (55.7)	57.3 (43.0)	42.7 (135.8)	32.9 (80.6)	22.0 (34.6)	24.4 (43.7)
Tong’an (n = 89)	36.0 (51.5)	18.1 (41.5)	37.1 (42.0)	41.6 (54.0)	47.2 (89.0)	33.7 (64.0)	13.5 (22.6)	28.1 (86.8)

nAb neutralizing antibody; A neutralizing titer of ≥ 1:16 was used as a threshold for seropositive; n the number of seropositive samples, GMT geometric mean titer

Fig. 2 Co-existence of nAbs against different types of enterovirus in healthy subjects

Fig. 3 Seroprevalence and GMT of nAb against different types of enterovirus by age groups.

Clustering analysis in the existence of nAb against eight types of EVs in 0–6 years age group (a). Co-existence of nAbs in < 1 year age group (b), 1–3 years age group (c) and 4–6 years age group (d). “All + ”, nAbs against all eight EVs are positive; “7 + ” means that nAb against any seven of the EVs are positive and so on; “All-”, nAbs against eight EVs are negative

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