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In June and July 2018, in total, 22, 985 mosquitoes of four genera and four species (Cx. tritaeniorhynchus, An. sinensis, Cx. pipiens quinquefasciatus, and Armigeres subalbatus) and 57, 500 midges (types to be identified) were collected in ten counties/towns of Jiangxi Province (Fig. 1). There were 16, 592 Cx. tritaeniorhynchus and 5822 An. sinensis specimens (72.2% [16, 592/22, 985] and 25.3% [5822/22, 985] of the total mosquitoes, respectively) (Table 1). Cx. tritaeniorhynchus and An. sinensis are local dominant mosquitoes.
Period of sample collection Sampling site (county) Mosquito species Total Midges Culex tritaeniorhynchus Culex quinquefasciatus Armigeres subalbatus Anopheles sinensis June Xiajiang 3130
(1530/1600)a0 80 830
(730/100)3960
(2260/1700)3000 Yongfeng 5100
(2100b/3000)0 0 2600
(1250b/1350)7700
(3350/4350)1100 Anfu 530b 0 0 500b 1030 550 Jing 55 39 0 33 127 4550 Gangshan (1900/2650) Subtotal 8815
(4215/4600)39 80 3963
(2513/1450)12897
(6847/6050)9200
(6550/2650)July Lichuan 270 0 0 230 500 12400 Zixi 15 0 6 91 112 7800 Long-Hushan town 520 0 44 197b 761 8900 Yanshan 5150 0 370 1100 6620 10600 Yushan 1660 0 12 200 1872 5300 Fuliang 162 0 20 41 223 3300 Subtotal 7777 0 452 1859 10088 48300 Total 16592
(11992/4600)39 532 5822
(4372/1450)22985
(16935/6050)57500
(54850/2650)a(1530/1600): (number of ground mosquito specimens/number of unground mosquito specimens).
bZIKV isolated from this mosquito specimen.Table 1. Specimens of blood-sucking insects collected in Jiangxi Province in 2018.
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Eleven virus isolates were stably passaged in tissue culture; these included several JEV and DENV strains (results not shown). Six ZIKV isolates, (JXJA1835-1, JXJA1835-2, JXJA1839-1, JXJA1866, JXJA1874-1, and JXLHS 1807) (Table 2), showed CPE and were stably passaged in tissue culture. Of these, five were isolated from specimens collected in June, and one from a specimen collected in July. Two of the six ZIKV strains were isolated from Cx. tritaeniorhynchus and four from An. sinensis (Table 2). The ZIKV isolate, JXLHS1807, from An. sinensis showed CPE in BHK-21 cells and formed plaques (Fig. 2).
Sampling date Sampling site (county) Strain Mosquito species Breeding ground GenBank Accession No. June 2018.06.12 Yongfeng JXJA1835-1 Anopheles sinensis Cowshed MK696546 2018.06.12 Yongfeng JXJA1835-2 Anopheles sinensis Cowshed MK696547 2018.06.12 Yongfeng JXJA1839-1 Culex tritaeniorhynchus Cowshed MK696548 2018.06.13 Anfu JXJA1866 Anopheles sinensis Sheepfold MK696549 2018.06.13 Anfu JXJA1874-1 Culex tritaeniorhynchus Cowshed MK696550 July 2018.07.10 Long-Hushan town JXLHS1807 Anopheles sinensis Pigsty MK696551 Table 2. ZIKV strains isolated from mosquitoes in Jiangxi Province in 2018.
Figure 2. CPE and plaques caused by ZIKV (JXLHS 1807). Normal BHK-21 cells cultured for 8 days grew densely and were neatly arranged (A), whereas inoculation with ZIKV strain JXLHS1807 reduced the number of adherent cells and induced rounding and exfoliation (B). Manification, 200×. C Plaques in monolayers of BHK-21 cells caused by JXLHS1807 with different dilutions.
Four mosquito species (Cx. tritaeniorhynchus, An. sinensis, Cx. pipiens quinquefasciatus, and Ar. subbalbatus) were collected in Jiangxi Province. Among these, six ZIKV strains were isolated from Cx. tritaeniorhynchus and An. sinensis collected in Yongfeng County, Anfu County, and Long-Hushan Town. The minimum ZIKV infection rate of Cx. tritaeniorhynchus and An. sinensis was 0.76 and 2.05, respectively (Table 3).
Collection date Collection site (county) Mosquito species No. Individuals/no.pools No. Positive Pools/no.pools MIR(/1000) June Yongfeng Culex tritaeniorhynchus 2100/14 1/14 0.48 Yongfeng Anopheles sinensis 1250/8 2/8 1.60 Anfu Culex tritaeniorhynchus 530/5 1/5 1.89 Anfu Anopheles sinensis 500/5 1/5 2.00 July Long-Hushan town Anopheles sinensis 197/4 1/4 5.08 Subtotal Culex tritaeniorhynchus 2630/19 2/19 0.76 Anopheles sinensis 1947/17 4/17 2.05 Table 3. ZIKV infection rate of Cx. tritaeniorhynchus and An. sinensis.
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The number of nucleotide and amino acid sequences of the E gene (Li et al. 2006; Lin et al. 2008) of the six ZIKV strains (JXJA1835-1, JXJA1835-2, JXJA1839-1, JXJA1866, JXJA1874-1, and JXLHS 1807) were 1510 nt and 504 aa, and similarity among the six strains was 99.7%-100.0% and 99.2%-100.0%, respectively.
The nucleotide and amino acid sequences (Li et al. 2006; Lin et al. 2008) of the JXLHS1807 ORF were 10, 296 nt and 3423 aa in length, respectively, and encoded a single reading frame. The details of the six ZIKV strains are listed in Table 2.
The nucleotide and amino acid sequence homologies of the ORF of JXLHS1807 and other ZIKV isolates were 88.4% (African type 1 MR766, Uganda), 88.4% (African type 2 ArD7117, Senegal), 96.2% (Africa type 1 MR766, Uganda), and 96.3% (African type 2 ArD7117, Senegal). The corresponding levels of homology with Asian ZIKV strains were 95.3% (Asian type 1 P6-740, Malaysia), 99.7% (Asian type 2 SZ-WIV01, American Samoa), 98.4% (Asian type 1, P6-740, Malaysia), and 99.5% (Asian type 2 SZ-WIV01, American Samoa) (Table 4).
Genotype Strain Source Homology of nucleotide (amino acid) of virus Divergences of amino acids Country Year Host JXLHS1807(ORF) JXLHS 1807(E) D683E V763M T777M S139N M/t2634V Africa Ⅰ MR766 Uganda 1947 Rhesus money 88.4% (96.2%) D V T s M Africa Ⅱ ArD7117 Senegal 1968 Aedes luteocephalus 88.4% (96.3%) D V T s M Asia Ⅰ P6-740 Malaysia 1966 Aedes aegypti 95.3% (98.4%) D V T s T Asia Ⅰ GZDJ1666-2 China 2016 Armigeres subalbatus 99.9% (99.7%) E M M N M Asia Ⅱ GZDJ1685 China 2016 Culex quinquefasciatus 99.9% (99.7%) E M M N M Asia Ⅱ SZ-WIV01 Americ an-S amon 2016 Human 99.7% (99.5%) E M M N M Asia Ⅱ ECMN2007 Micronesia 2007 Human 97.8% (98.8%) E V T S M Asia Ⅱ H/PF/2013 French-Polynesia 2013 Human 99.5% (99.4%) E M M N M Asia Ⅱ 1_0035_PF French-Polynesia 2014 Human 99.4% (99.4%) E M M N M Asia Ⅱ ZikaSPH2015 Brazil 2015 Human 99.3% (99.2%) E M M N V Asia Ⅱ JXLHS1807a China 2018 Anopheles sinensis E M M N M Asia Ⅱ JXJA1835-1a China 2018 Anopheles sinensis 99.7% (99.2%) E M M Asia Ⅱ JXJA1835-2a China 2018 Anopheles sinensis 99.8% (99.4%) E M M Asia Ⅱ JXJA1839-1a China 2018 Culex tritaeniorhynchus 99.8% (99.4%) E M M Asia Ⅱ JXJA1866a China 2018 Anopheles sinensis 99.8% (99.4%) E M M Asia Ⅱ JXJA1874-1a China 2018 Culex tritaeniorhynchus 99.8% (99.4%) E M M aZIKV strains isolated in this study. Table 4. Variation in the amino acid sequences of ZIKVs isolated from Cx. tritaeniorhynchus and An. sinensis.
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The amino acid substitutions S139N in the PrM protein; D683E, V763M, and T777M in the E protein; and M/t2634V in the NS5 protein of JXLHS1807 were identical to those of the ZIKV strains prevalent in South America since 2015. The E proteins of the other five ZIKV strains isolated from Cx. tritaeniorhynchus and An. sinensis (JXJA1835-1, JXJA1835-2, JXJA1839-1, JXJA1866, and JXJA1874-1) had the D683E, V763M, and T777M amino acid substitutions (Table 4).
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A molecular phylogenetic analysis based on the viral open reading frame nucleotide sequences showed that JXLHS1807 was an Asian type 2 ZIKV, as those isolated from Cx. pipiens quinquefasciatus (GZDJ 1685) and Ar. subbalbatus (GZDJ1666-2) collected in Guizhou Province in 2016. And Asian type 2 ZIKVs were also isolated in Yap Island (ECMN2007, Micronesia), French Polynesia (1_0199_PF, French Polynesia), Brazil (SSABR1, Brazil), Puerto Rico (PRVABC59, Puerto Rico), and Haiti (Haiti 1225/2014, Haiti) since 2007. However, JXLHS1807 was not in the same evolutionary branch as the ZIKV strains prevalent in South America since 2015 (Fig. 3).