Isolation and Genome Phylogenetic Analysis of Arthropod-Borne Viruses,Including Akabane Virus,from Mosquitoes Collected in Hunan Province,China

Abstract

This study investigated the abundance of mosquitoes and circulation of mosquito-borne arboviruses from 16 villages in 8 cities of Hunan Province, China, in July–August of 2010 and in August of 2011. In total, 16,076 mosquitoes consisting of seven species from four genera were collected by ultraviolet-light trap. Culex quinquefasciatus was the most common species, accounting for 50.63% (8140/16,076) of the total. Anopheles sinensis (24.26%, 3900/16,076) made up the second most common species, followed by Culex tritaeniorhynchus (9.76%, 1569/16,076). The proportions of Culex pipiens pallens, Armigeres subalbatus, and Culex modestus were 6.7%, 5.2%, and 3.31%, respectively. Fourteen Aedes albopictus were detected. The mosquitoes were identified by morphologic characteristics and frozen in liquid nitrogen. The mosquitoes were pooled, triturated, and centrifuged. The clarified supernatant was used to inoculate monolayers of C6/36 and baby hamster kidney-21 cells. We obtained six virus isolates that caused cytopathic effects. Phylogenetic analysis revealed that two isolates were Akabane virus (AKAV, from A. sinensis and C. quinquefasciatus), two isolates were Japanese encephalitis virus (from C. pipiens pallens and C. quinquefasciatus), and two isolates were Tibet orbivirus (from C. quinquefasciatus and C. tritaeniorhynchus). This is the first report of AKAV isolated from A. sinensis and C. quinquefasciatus in nature in China. The detection of AKAV in these species confirms circulation of AKAV in Hunan province and suggests potential challenges to the prevention and control of arthropod-borne animal viruses in mainland China.

Introduction

Mosquitoes can spread many viruses through hematophagy. More than 300 species of arboviruses were identified that could be transmitted by mosquitoes, including dengue virus, Japanese encephalitis virus (JEV), West Nile virus (WNV), and Zika virus (Karabatsos 1985). Arthropod-borne viruses, such as JEV and WNV, can cause serious infectious disease symptoms, including fever, hemorrhage, and encephalitis, in humans, with worldwide prevalence. JEV was found mainly in Asia and Oceania. JEV causes ∼70,000 Japanese Encephalitis (JE) cases each year worldwide (Campbell et al. 2011). Besides, Akabane virus (AKAV) can cause abortion, stillbirth, and congenital defects in calves. From 1972 to 1975, Akabane disease caused abnormalities in 42,000 calves in Japan, resulting in large economic losses (Kurogi et al. 1977). Therefore, infectious diseases caused by mosquito-borne viruses not only pose a threat to human health but also result in great harm to animal husbandry. Mosquito-borne viruses and their associated diseases have become a worldwide public health issue (Liang et al. 2015, Li et al. 2016).

Hunan Province (108°47′–114°15′E and 24°39′–30°08′N), located in the southern part of China, has a humid subtropical climate with high average temperatures and heavy rainfall (Government of Hunan Province 2017). Hunan Province has numerous rivers and abundant water systems and is located near Dongting Lake, the second largest freshwater lake in China. The prolonged high temperatures and rainy weather in the summer provide an optimal geographical environment for breeding and propagation of various mosquitoes. Mosquito species surveys in Hunan Province show that the mosquito density begins to increase from June, peaks in mid-August, and decreases in mid-September; the mosquito density decreases significantly after October. Culex tritaeniorhynchus, Anopheles sinensis, Culex quinquefasciatus, and Armigeres subalbatus are the dominant mosquitoes in the area (Dai et al. 2015).

Hunan Province is a JE endemic area. From 1951 to 2000, JE cases were reported every year, and a total of 142,190 cases were reported with 18,328 deaths. The average annual incidence rate was 5.82/100,000 with a 12.89% fatality rate and a 0.75/100,000 mortality rate (Guo and Li 2002). In addition, JEV genotype I was isolated from mosquitoes collected in Yueyang, located northeast of Hunan Province (Xiang et al. 2017). To understand the circulation of mosquito-borne viruses in Hunan Province, we focused on the identification of mosquito species and mosquito-borne viruses from 16 collection sites in 8 cities of Hunan Province in 2010 and 2011. AKAV, JEV, and Tibet orbivirus (TIBOV) were isolated in this study. AKAV was isolated from A. sinensis and C. quinquefasciatus for the first time in mainland China.

Materials and Methods

Mosquito collections

Mosquitoes were collected from 16 villages in Huaihua, Zhangjiajie, Yueyang, Chenzhou, Xiangtan, Shaoyang, Zhuzhou, and Changsha cities of Hunan Province (Fig. 1). It was the first time that investigation on arboviruses was carried out in Hunan Province. Collection sites were selected based on the proximity to the breeding sites of mosquitoes and in villages inhabited by 10,000–20,000 people and containing pigpens, cattle farms, and ponds. These environments are suitable for the circulation of arboviruses in nature.

FIG. 1.

Locations of mosquito collection sites in Hunan Province, China (triangles: collection sites in eight counties in 2010; spots: collection sites in eight counties in 2011). The map of China (upper left) shows the location of Hunan Province, China.

Mosquitoes in every collection site were trapped for two nights from 20 July to 6 August of 2010 and in 1–17 August of 2011. Mosquitoes were collected from 18:00 in the evening to 6:00 the following morning using ultraviolet (UV)-light traps (Wuhan Lucky Star Environmental Protection Co., Ltd.). The UV-light traps were placed in the pigpens and under the eaves of the houses on sites, where the traps were hung about 2 m high. Female mosquitoes were identified by species using morphologic keys (Lu 2003) and pooled into groups by species, date, and site of collection. Approximately 50 mosquitoes were placed in each pool and frozen in liquid nitrogen (Wang et al. 2011).

Cell culture

Aedes albopictus C6/36 cells and baby hamster kidney (BHK)-21 cells from the American Type Culture Collection (ATCC) were used in this study, and both cell lines were maintained in our laboratory. C6/36 cells were cultured in 90% RMPI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% inactive fetal bovine serum (FBS; Invitrogen) and 100 U/mL of penicillin and streptomycin. BHK-21 cells were cultured in 93% Dulbecco's modified Eagle's medium supplemented with 7% FBS and 100 U/mL of penicillin and streptomycin. C6/36 cells were cultured at 28°C and BHK-21 cells were propagated at 37°C under 5% CO₂ atmosphere (Wang et al. 2011).

Virus isolation

The mosquito pools (50 mosquitoes/pool) were triturated in minimum essential medium (containing 1% FBS, 100 U/mL of penicillin, 100 U/mL of streptomycin, and 1 μL/mL of fungizone) using the TissueLyser (Qiagen, Valencia, CA) at room temperature for 3 min. The homogenates were clarified by centrifugation at 12,000 rpm and 4°C for 10 min. Clarified homogenates (100 μL) were inoculated onto monolayers of BHK-21 and C6/36 cells for 1 h at 37°C and 28°C, respectively. Then, the cells were washed with minimum essential medium and were maintained at 37°C and 28°C after adding the minimum essential medium containing 5% FBS, 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 1 μL/mL of fungizone. Cells were observed daily for cytopathic effects (CPEs) from days 1–7 postinfection (Li et al. 2010).

Molecular identification

Viral RNA was extracted from the cell culture supernatants using the QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA), and first-strand complementary DNA (cDNA) was synthesized using Ready-to-Go You Prime First Strand Beads (GE Health care, Uppsala, Sweden) according to the manufacturer's protocol. Polymerase chain reaction (PCR) was conducted with primers (Table 1) designed for cDNA amplification of bunyaviruses, flaviviruses, and alphaviruses in addition to JEV, TIBOV, and AKAV (Kuno 1998, Feng et al. 2016). We tested CPE-positive cultures using genus-specific primers first and then proceeding to species-specific amplification. Primers designed to amplify the whole genome of AKAV were used to sequence the virus isolates (Table 2). Amplified DNA fragments were visualized on 1% agarose gels.

Table 1.

Primers Used to Identify and Sequence the Viruses Isolated in this Study

Primer	Orientation	Amplified fragment	Sequence (5′→3′)	Size (bp)
Flavivirus	Sense	NS5	TACCACATGATGGGAAAGAGAGAGAA	310
Flavivirus	Antisense		GTGTCCCAGCCGGCGGTGTCATCAGC
Japanese encephalitis virus	Sense	C/PrM	CGTTCTTCAAGTTTACAGCATTAGC	674
	Antisense		CCYRTgTTYCTgCCAAgCATCCAMCC
	Sense		CGY TTG GAA TGY CTR GTCCG	492
Dengue virus (I–IV)	Sense	C/PrM	GTGACAATGCTCCTTATGCTGCTG	391
	Antisense		TCCAGGCACCTTCAGAGGACATC
	Sense	PrM	TGGACCTTGGTGAATTGTGTGAA	319
	Antisense		CCTATGGTGTATGCCAGGATTGC
	Sense	NS4a	ACCAGGTAAAGGGATTGGAAAGAC	216
	Antisense		GCCAAGGTAAGTATGCCTATCACG
	Sense	PrM-E	ATCACGAATCCAGCGAACTGTCTT	152
	Antisense		CGCATCCTCCATGTTCTAGCACC
Alphavirus	Sense	NSP1	YAGAGCDTTTTCGCAYSTRGCHW	434
	Antisense		ACATRAANKGNGTNGTRTCRAANCCDAYCC
	Sense		TGYCCNVTGMDNWSYVCNGARGAYCC	310
Sindbis virus	Sense	nsP4	GATGAA ATCNGGVATGTT	600
Sindbis virus	Antisense		TTAGGACCACCGTAGAGA
Getah virus	Sense	NS3	CTTTCAGAGAGGACAGAGCGT	810
Getah virus	Antisense		CTCTTCTGCCGTTATCAAAGTTAG
Chikungunya virus	Sense	E1	TATCCTGACCACCCAACGCTCC	290
Chikungunya virus	Antisense		ACATGCACATCCCACCTGCC
Banna virus	Sense	12 segment	AAATTGATAGYGYTTGCGTAAGAC	850
Banna virus	Antisense		GTTCTAAATTGGATACGGCGTGC
Liaoning virus	Sense	12 segment	CACTGGCTCCGGCTGTAGTAACAG	435
Liaoning virus	Antisense		CTGTTCGGATCATCTGGAATTTGA
Kadipiro virus	Sense	12 segment	GACGCTTTGAGATTATCTCGAC	354
Kadipiro virus	Antisense		GCTCAATCGCATTCTCACC
Orthobunyavirus	Sense	S segment	ATGACTGAGTTGGAGTTTGATGTCGC	251
Orthobunyavirus	Antisense		TGTTCCTGTTGCCAGGAAAAT
Tahyna virus	Sense	S segment	TTTGGAGAGTGGCAGGTGGAGGTTG	601
Tahyna virus	Antisense		TGCGACATCCATTTGTCTGCAGTG
Batai virus	Sense	S segment	ATGACTGAGTTGGAGTTTCATGATGTCGC	251
Batai virus	Antisense		TGTTCCTGTTGCCAGGAAAAT
Cypovirus	Sense	10 segment	AGGTCTCGTAGACCGTGCAC	964
Cypovirus	Antisense		-GGTGCACGGTCTACGAGACCT
Totivirus	Sense	RdRp	TTACGTTGCTGACGAGTACAAGG	982
Totivirus	Antisense		CAGCGGTGTTTAACTCAGTAATAC
Tibet orbivirus	Sense	10 segment	ATGCGACCCATACATAAA	929
Tibet orbivirus	Antisense		CTCGTCCTCCGTCACAAC
Akabane virus	Sense	S segment	TAACTACGCATTGCAATGGC	720
Akabane virus	Antisense		TAA GCT TAG ATC TGG ATA CC

M: C/A; W: A/T; Y: C/T; K: G/T; R: G/A; V: G/A/C; D: T/A/G.

PrM, premembrane; E, envelope; NS, nonstructural; RdRp, RNA-dependent RNA polymerase.

Table 2.

Primers Used to Obtain the Entire Coding Sequence of Akabane Virus

Primer	Orientation	Segment	Sequence (5′→3′)	Size (bp)
AKAV-M-71F	Sense	M segment	GCA ATG CCT CCT CGT AAT A	770
AKAV-M-851R	Antisense		TTG TTG GAC ATG GGC TAA A
AKAV-M-766F	Sense	M segment	GCT TTA TGG CTT ATT GTT GCTA	963
AKAV-M-1729R	Antisense		TGT TCT CCA CTG TAT GCT GTC T
AKAV-M-1708F	Sense	M segment	AGA CAG CAT ACA GTG GAG AAC A	867
AKAV-M-2575R	Antisense		ACA ATA CTC ATC AAC CAA ATC A
AKAV-M-2438F	Sense	M segment	AGG TCT ATT CGA CTC CTC AAA	886
AKAV-M-3324R	Antisense		ACA CTG TGG ACT TCA AAC TGC
AKAV-M-3230F	Sense	M segment	TTT TGT GTT TCA GAG CCT AC	1025
AKAV-M-4255R	Antisense		CCT CCC GTT ATG TCT ATT TT
AKAV-L1–1F	Sense	L segment	AGT AGT GTA CCC CTA AAT ACA ACT	1189
AKAV-L1-1190R	Antisense		ATT GCT GTT CCC ACA TTA CAG TTG
AKAV-L2-1045F	Sense	L segment	GAC GCA TTT AAA GCC ATT GG	1308
AKAV-L2-2353R	Antisense		CAT ATA CCA GGT ATA TTG AG
AKAV-L3-2214F	Sense	L segment	AGGGTAAAGTGTCATTGAAAGA	1067
AKAV-L3-3281R	Antisense		TGT CCA TGT AGT TGC ATA AGA A
AKAV-L4-3126F	Sense	L segment	AGCCACACTCAACAAAAATAG	810
AKAV-L4-4262R	Antisense		TTA ACA GAG ATC GAG AAC GCA
AKAV-L4-4130F	Sense	L segment	TGGGATTTAAGTAAGTTGACGC	1123
AKAV-L4-5253R	Antisense		TCTAACGATGTTGCTATCTGGG
AKAV-L5-5122F	Sense	L segment	CGA GCT TAC ACT CCA ATT GAC	290
AKAV-L5-6193R	Antisense		GAA CTC AAA TGC ATC TTG ATC
AKAV-L6-5992F	Sense	L segment	GCT GTT AGC GAT ACT GCA ATG GTT	850
AKAV-L6-6845	Antisense		AGT AGT GTG CCC CTA AAT GCA ATC

AKAV, Akabane Virus.

The positive DNA fragments were extracted (Li et al. 2010) using the TaKaRa DNA Purification Kit (TaKaRa, Japan). The PCR fragments were sequenced using the BigDye Terminator V3.1 cycle sequencing kit (PE Applied Biosystems, Foster City, CA) and analyzed using an ABI 3730 XL DNA Analyzer (PE Applied Biosystems).

Sequences of cDNA amplicons were assembled using the SeqMan program (DNASTAR; www.dnastar.com, Madison, WI). Sequences were compared with the GenBank database using the BLAST program. Alignment and nucleotide sequence identity analyses were carried out using the Clustal X software (version 1.83; www.clustal.org) and the MegAlign program (DNASTAR; www.dnastar.com). MEGA software (version 3.1; www.megasoftware.net) was used for phylogenetic analysis. Phylogenetic trees were constructed based on the neighbor-joining method, in which the bootstrap value corresponded to 1000 replications (Li et al. 2010, Wang et al. 2011).

Results

Distribution of mosquitoes

In July–August 2010 and August 2011, we collected a total of 16,076 mosquitoes consisting of seven species from the Culex, Anopheles, Armigeres, and Aedes genera, which included C. quinquefasciatus, Culex pipiens pallens, C. tritaeniorhynchus, A. sinensis, Armigeres obturbans, and A. albopictus (Table 3). C. quinquefasciatus was the most common species, accounting for 50.63% (8140/16,076) of the total. A. sinensis (24.26%, 3900/16,076) was the second most abundant species, followed by C. tritaeniorhynchus (9.76%, 1569/16,076). The proportions of C. pipiens pallens, A. subalbatus, and Culex modestus were 6.7% (1078/16,076), 5.2% (843/16,076), and 3.31% (532/16,076), respectively. Fourteen A. albopictus mosquitoes were collected.

Table 3.

Mosquitoes Collected in Hunan Province, China in 2010 and 2011

Species	No. of mosquitoes								Total
	2010				2011
	Huaihua	Zhangjiajie	Yueyang	Chenzhou	Changsha	Zhuzhou	Xiangtang	Shaoyang	Number	Percent
Culex quinquefasciatus	553	269	96	747	1409	1237	1596	2233	8140	50.63
Culex tritaeniorhynchus	123	0	704	0	536	0	186	20	1569	9.76
Culex pipiens pallens	0	706	350	22	0	0	0	0	1078	6.71
Culex modestus	150	90	292	0	0	0	0	0	532	3.31
Anopheles sinensis	1735	855	270	858	25	71	20	66	3900	24.26
Armigeres subalbatus	0	0	779	38	3	0	0	23	843	5.24
Aedes albopictus	9	0	1	1	2	1	0	0	14	0.09
Total	2570	1920	2492	1666	1975	1309	1802	2342	16,076	100

Isolation and preliminary identification of viruses

The 16,076 mosquitoes were sorted into 347 pools according to species, location, and date of collection. Pools contained ∼50 mosquitoes each, and all the pools were processed for virus isolation using BHK-21 and C6/36 cells. Six isolates causing CPEs were obtained (Table 4). Isolates HN10169 and HN10174 caused CPE on day 2 in BHK-21 cells, but not in C6/36 cells (Fig. 2). Isolates HN10064 and HN11084 caused CPE on day 4 in BHK-21 cells and on day 3 in C6/36 cells. Isolates HN11066 and HN11121 caused CPEs on day 3 in BHK-21 cells, but not in C6/36 cells. Each virus isolate was detected by PCR using a variety of genus-specific and species-specific primers. The PCR tests showed that the six isolates were AKAV, JEV, and TIBOV.

FIG. 2.

Cytopathic effect of HN10169 and HN10174 isolates on BHK-21 cells (200× magnification). (A, D) Uninfected BHK-21 control cells at 24 and 48 h, respectively. (B). HN10169 strain-infected BHK-21 cells, 24 h postinfection. (E). HN10169 strain-infected BHK-21 cells, 48 h postinfection, showing cell aggregation and detachment. (C) HN10174 strain-infected BHK-21 cells, 24 h postinfection. (F) HN10174 strain-infected BHK-21 cells, 48 h postinfection, showing cell aggregation and detachment. BHK, baby hamster kidney.

Table 4.

Virus Isolates from Mosquitoes Collected in Hunan Province, China

No.	Isolate	Collection date	Collection site	Mosquito species	No. of mosquitoes	Cytopathic effect (h)		Virus
No.	Isolate	Collection date	Collection site	Mosquito species	No. of mosquitoes	BHK-21	C6/36	Virus
1	HN10064	July 30, 2010	Zhangjiajie Yongding	C. pipiens pallens	50	96	72	JEV
2	HN10169	August 5, 2010	Chenzhou Yizhang	A. Sinensis	50	48	—	AKAV
3	HN10174	August 5, 2010	Chenzhou Yizhang	C. quinquefasciatus	25	48	—	AKAV
4	HN11066	August 3, 2011	Shaoyang Shuangqing	C. quinquefasciatus	50	72	—	TIBOV
5	HN11084	August 3, 2011	Shaoyang Shuangqing	C. quinquefasciatus	60	96	72	JEV
6	HN11121	August 8, 2011	Changsha Kaifu	C. Tritaeniorhynchus	55	72	—	TIBOV

JEV, Japanese encephalitis virus; TIBOV, Tibet orbivirus; BHK, baby hamster kidney.

Molecular biological characteristics of AKAV isolates

The HN10169 and HN10174 isolates were initially identified as AKAV, and the nucleotide sequences of the S, M, and L segments of both isolates were obtained using AKAV-specific primers (Table 2). Nucleotide sequence analysis showed that the S and M fragments of HN10169 and HN10174 isolates were 702 and 4206 bp in length, and encoded 234 and 1402 amino acids, respectively. The nucleotide/amino acid sequence homologies of the S segment of the HN10169 and HN10174 isolates were 99.1% and 99.4%, respectively, while the nucleotide/amino acid sequence homologies of the M segment of both isolates were 99.9% and 99.8%. The nucleotide sequence of the L segment of both HN10174 and HN10169 was 6756 bp, which encoded 2252 amino acids.

Comparison of nucleotide and amino acid homologies of the open reading frame sequences of AKAVs isolated from various countries, collection dates, and vectors (hosts) showed that the S segment of AKAV isolated from China was closely related to that in isolates from Japan and South Korea, (95.9–97.2% and 98.3–99.1%, respectively). The AKAV M genes in isolates from China and Japan in recent years (1993 and 2006) showed higher homology (96.7–98.1%) than that found in earlier isolates (93–96.9%) from China and Japan (1959 and 1974). The nucleotide and amino acid sequences of the S and M segments of AKAVs isolated from Australia and Kenya ranged from 70.1% to 92.9% and from 74.2% to 97.9%, respectively (Table 5).

Table 5.

Nucleotide and Amino Acid Sequence Homology of Akabane Virus Isolated from Mosquitoes Collected in Hunan Province, China

Country	Strain	Source	HN10169 (S segment)		HN10174 (S segment)		HN10169 (M segment)		HN10174 (M segment)
Country	Strain	Source	Nucleotide homology, %	Amino acid homology, %	Nucleotide homology, %	Amino acid homology, %	Nucleotide homology, %	Amino acid homology, %	Nucleotide homology, %	Amino acid homology, %
China	DHL10M110	Anopheles vagus	97.9	99.6	97.9	99.6	93.6	96.1	97.9	96.1
China	GXLCH04	Rhizomys pruinosus	98.9	99.1	98.9	99.1	91.7	96.1	98.9	96.2
China	CY-77	Bovine	97.2	99.6	97.2	99.6	94	96.8	97.2	96.9
China	NB/BS/1	—	99.4	99.1	99.4	99.1	99.1	99.2	99.4	99.3
Japan	JaGAr39	Aedes vexans	96.6	98.7	96.6	98.7	88.7	94.6	96.6	94.6
Japan	OBE-1	Bovine	95.9	98.7	95.9	98.7	88.2	93.9	95.9	94
Japan	ON-1/P/93	Bovine	97.2	98.7	97.2	98.7	96.9	98.1	97.2	98.1
Japan	KM-1/Br/06	Bovine	96.2	99.1	96.2	99.1	93	96.7	96.2	96.8
Korea	K9	Bovine	95.9	98.3	95.9	98.3	88.3	93.9	95.9	93.9
Kenya	MP496	Anopheles funestus	92.9	97.9	92.9	97.9	84.9	92.4	92.9	74.3
Australia	R7949	Culicoides brevitarsis	83.2	90.6	83.2%	90.6%	70.1	74.2	83.2	92.5

Comparison of the deduced amino acid sequences of the S and M segments from the two AKAV isolates in this study with those of AKAVs in GenBank showed that there were several unique differences in the M and S segments between the two AKAV isolates in this study and other AKAV isolates (Supplementary Table S1; Supplementary Data are available online at www.liebertpub.com/vbz).

We constructed a phylogenetic tree based on the open reading frame sequences of S, M, and L segments of AKAVs isolated from various countries, host animals, and vectors. The cladogram showed that the sequence of S, M, and L segments of HN10169 and HN10174 isolates generated highly comparable topologies, indicating that these isolates have a high level of sequence homology with AKAV (Fig. 3A–C). Phylogenetic analysis of the S segment of AKAV showed that Chinese AKAV isolates formed a separate clade, while AKAV isolates from Japan, South Korea, and Australia were grouped in a different clade (Fig. 3A). The phylogenetic tree of the M segment revealed that the AKAV isolates in this study were in the same group as the AKAV strains isolated from cattle, midge, and mosquitoes in Japan (Fig. 3B).

FIG. 3.

Phylogenetic analysis of AKAV isolates based on predicted complete protein coding sequences. This analysis applied the neighbor-joining method using MEGA 7.0 software. Bootstrap probabilities for each node were calculated using 1000 replicates. Scale bars indicate the number of nucleotide substitutions per site. HN10169 and HN10174 strains are indicated with black dots. (A) Phylogenetic tree constructed using the S segment coding region nucleotide sequences of HN10169 and HN10174 strains. (B) Phylogenetic tree constructed using the M segment coding region nucleotide sequences of HN10169 and HN10174 strains. (C) Phylogenetic tree constructed using the L segment coding region nucleotide sequences of HN10169 and HN10174 strains. AKAV, Akabane virus.

Phylogenetic classification of TIBOV isolates

We conducted PCR using specific primers to amplify the 10th segment gene of TIBOV from the HN11066 and HN11121 isolates and constructed a phylogenetic tree based on the sequences of the PCR products. The cladogram showed that both HN11066 and HN11121 isolates were grouped into the TIBOV clade and were closely related to TIBOV (XZ0906 strain) (Fig. 4).

FIG. 4.

Phylogenetic analysis of TIBOV isolates based on the 10th segment coding region nucleotide sequence. This analysis applied the neighbor-joining method using MEGA 7.0 software. Bootstrap probabilities for each node were calculated using 1000 replicates. Scale bars indicate the number of nucleotide substitutions per site. HN11066 and HN11121 strains are indicated by black dots. TIBOV, Tibet orbivirus.

Phylogenetic classification of JEV isolates

We also conducted PCR on the HN10064 and HN11084 isolates using JEV E gene-specific primers and constructed a phylogenetic tree based on the PCR product sequences. The cladogram showed that both HN10064 and HN11084 isolates were grouped with JEV genotype I (Fig. 5).

FIG. 5.

Phylogenetic analysis of Japanese encephalitis virus isolates based on nucleotide sequences of the E gene coding region. This analysis applied the neighbor-joining method using MEGA 7.0 software. Bootstrap probabilities for each node were calculated using 1000 replicates. Scale bars indicate the number of nucleotide substitutions per site. HN10064 and HN11084 isolates are indicated by black dots.

Discussion

AKAV belongs to the genus Orthobunyavirus of Bunyaviridae. AKAV was first isolated from Aedes vexans in Akabane of Gumma, Japan, in 1959 (Karabatsos 1985). It was subsequently isolated from vector specimens (biting midges and other species of mosquito) and sick animal specimens in Australia, Kenya, Israel, Saudi Arabia, Korea, and Taiwan Province of China (Metselaar and Robin 1976, Coverdale et al. 1978, Liao et al. 1996, AI-Afaleq et al. 1998, Strm et al. 2004, An et al. 2010). Recent studies have shown that two AKAV strains were isolated from C. tritaeniorhynchus (DHL10M117 virus strain) and Anopheles vagas (DHL10M110 virus strain) in Yunnan, China (Feng et al. 2016). In this study, the AKAVs (HN10169 and HN10174 isolates) were isolated from A. sinensis and C. quinquefasciatus in Hunan Province, further suggesting that AKAV can be carried by various mosquito vectors. C. tritaeniorhynchus, C. quinquefasciatus, and A. sinensis are the dominant mosquitoes in the local areas, and there is a wide range of distribution of these mosquitoes in China, spanning from the southern part of China (south of 30°N) (Liu et al. 2011 and Zheng et al. 2012). Therefore, AKAV is likely to exist in other parts of southern China as well.

Previous studies reported that the seroprevalence of AKAV neutralizing antibody in cattle and sheep was 19.06% (85/446) in Xinjiang, and the antibody seroprevalence rates of cattle and sheep were 20.32% (38/187) and 18.15% (47/259), respectively (Jun et al. 2012). The results suggested that the circulation of AKAV in mosquito vectors in China not only exists but also has caused the infection of domesticated animals such as cattle and sheep. Up to now, no studies have reported an AKAV-caused epidemic of animal diseases in China. Therefore, it is important to strengthen the detection and monitoring of AKAV in mosquitoes, midges, and animals. In particular, it is extremely important to detect and monitor the disease caused by AKAV infection in animals.

TIBOV was first isolated from Anopheles maculatus collected in Tibet, China. TIBOV contains a double-stranded RNA genome, consisting of 10 segments of various lengths and is a new member of the genus Orbivirus (Li et al. 2014). Since the first report, TIBOV has been isolated many times from midges collected in China (Lei et al. 2015, Wang et al. 2017). TIBOV was isolated from C. quinquefasciatus and C. tritaeniorhynchus collected in Hunan Province, suggesting that Culex could also become the transmission vector of TIBOV. In serum samples of domestic animals in southern China, the positive rate of TIBOV neutralizing antibody was 26% (35/135). The infection rate was highest in cattle (44%, 22/50), followed by buffalo (20%, 12/60), and goat (4%, 1/25), suggesting that TIBOV was generally infectious in domestic animals, and the susceptibility levels in livestock are distinct in different animals (Wang et al. 2017). These results suggest that TIBOV can not only be isolated from various species of mosquitoes but also infect domestic animals.

China is a JE epidemic area, and Hunan Province is a highly endemic area for JE infection in China. JE cases have been reported in Hunan Province annually since 1950 (Li et al. 2014). In recent years, the incidence rate of JE in Hunan Province has decreased year by year due to the inclusion of JE vaccine in the China Expanded Program of Immunization. In this study, mosquito collection sites were located near latrines, domestic animal pens, and sewage ditches around rural houses. JEV was isolated from two species of mosquitoes (C. pipiens pallens and C. quinquefasciatus) collected in Zhangjiajie in 2010 and Shaoyang in 2011. The results suggest that JEV was circulating in those mosquitoes in those years in Hunan. In view of the fact that C. pipiens pallens and C. quinquefasciatus can spread many species of mosquito-borne viruses such as JEV, WNV, and St. Louis encephalitis virus, it is of great significance for local public health agencies to strengthen the monitoring of the species and density of mosquitoes to detect timely mosquito-borne viruses and related diseases.

In this study, AKAV was isolated from A. sinensis and C. quinquefasciatus collected in Hunan Province, China. This finding represents not only the first isolation of AKAV from mosquitoes in Hunan Province but also the first AKAV isolation from these two mosquitoes in mainland China. AKAV has been isolated from C. tritaeniorhynchus and Anopheles vagas in China (Feng et al. 2016). These results suggest the existence of AKAV in many mosquito species in China. In Japan and Australia, AKAV has been isolated from blood samples of seemingly healthy young bulls and various tissue samples of dead fetal calves (Karabatsos 1985, Kobayashi et al. 2007). At present, although AKAV has been isolated from a variety of mosquito specimens in China, it has not yet been detected in animal specimens, and no animal infection of AKAV has been reported. Therefore, it is important to strengthen the detection and monitoring of AKAV in animal specimens of healthy cattle and sheep, and to quickly identify AKAV in sick animal specimens to reduce economic losses caused by the virus infection.

In this study, we carried out an investigation on mosquitoes and mosquito-borne viruses in 16 counties of Hunan province located in southern China, and three species of virus (AKAV, JEV, and TIBOV) belonging to 3 families were isolated from mosquito specimens. The mosquito samples were frozen in liquid nitrogen immediately after being collected, so the blood in the midgut of mosquitoes was not digested. Therefore, the viruses isolated from these mosquitoes may come from the recent engorgement of viremic blood or be carried by the mosquitoes themselves. In view of the fact that these virus isolates are all derived from CPE-positive culture cells, which we specially focused on, some viruses that cannot cause CPE in these selected culture cells may be neglected. Therefore, it is necessary to increase the application of more tissue culture cell lines to improve the chance of detecting various species of virus and to broaden the range of virus genes detection using advanced sequencing techniques such as Next-Generation Sequencing in the future study.

Footnotes

Acknowledgments

We thank the staff from the local Center for Disease Control in Hunan Province for helping to collect the mosquitoes on site. This work was supported by the National Key R&D Program of China (2016YFC1201904), the National Natural Science Foundation of China (81501757), the National Key Plan for Scientific Research and Development of China (2016YFD0500300), the Development Grants of State Key Laboratory of Infectious Disease Prevention and Control (2014SKLID103, 2015SKLID505). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the article.

Author Disclosure Statements

No competing financial interests exist.

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