Isolation and Characterization of Wuxiang Virus from Sandflies Collected in Yangquan County, Shanxi Province, China

Abstract

Keywords: sandfly, Phlebovirus In this study, we isolated a group of viruses that can cause a cytopathic effect in BHK-21 cells from sandfly specimens collected in Yangquan County, Shanxi Province, central China. The representative virus strain is SXYQ1860 and, with increased viral injection, the suckling mice became ill and died, with viral titers continually increasing. The results of molecular genetic evolution analysis of the three gene segments of the virus, L, M, and S, indicate that the newly isolated viruses from Yangquan County are the same as the Wuxiang virus (WUXV) previously isolated from sandflies collected in Wuxiang county, China. This is the first time that the WUXV was also isolated outside Wuxiang County. Therefore, strengthening the surveillance of neglected sandflies and the viruses they transmit to help prevent and control arboviruses and the associated diseases is essential for public health.

Full text

Isolation and Characterization of Wuxiang Virus
from Sandflies Collected in Yangquan County,
Shanxi Province, China
Jing Wang,
1,2,
*Na Fan,
1,2,
*Shihong Fu,
1,
*Jingxia Cheng,
3
Bin Wu,
4
Ziqian Xu,
1
Jingdong Song,
1
Xiaodong Tian,
3
Yan Li,
4
Ying He,
1
Fan Li,
1
Songtao Xu,
1
Xiaoqing Lu,
2
Huanyu Wang,
1
Bin Wang,
5
and Guodong Liang
1
Abstract
Keywords: sandfly, Phlebovirus
In this study, we isolated a group of viruses that can cause a cytopathic effect in BHK-21 cells from sandfly
specimens collected in Yangquan County, Shanxi Province, central China. The representative virus strain is
SXYQ1860 and, with increased viral injection, the suckling mice became ill and died, with viral titers con-
tinually increasing. The results of molecular genetic evolution analysis of the three gene segments of the virus,
L, M, and S, indicate that the newly isolated viruses from Yangquan County are the same as the Wuxiang virus
(WUXV) previously isolated from sandflies collected in Wuxiang county, China. This is the first time that the
WUXV was also isolated outside Wuxiang County. Therefore, strengthening the surveillance of neglected
sandflies and the viruses they transmit to help prevent and control arboviruses and the associated diseases is
essential for public health.
, Wuxiang virus, evolution
Introduction
Sandflies belong to the family Phlebotominae within
the Psychodidae and Diptera and are distributed world-
Sandfly-borne phleboviruses are distributed worldwide
and its infection in humans can cause fever, and even severe
wide. Sandflies have a blood-sucking habit and are fond of
human blood in addition to a variety of animal sources, in-
cluding dog, chicken, and sheep (Killick-Kendrick 1999).
Sandflies can spread various viruses through blood sucking
(Depaquit et al. 2010). Due to the broad geographic distri-
bution of sandflies and the variety of pathogens that they can
transmit, sandflies are important public health harmful in-
sects. They can transmit diseases that cause fever, bleeding,
and even encephalitis, thereby threatening public health and
imposing a burden of disease (Depaquit et al. 2010, Ayhan
et al. 2017).
clinical symptoms such as meningitis and encephalitis,
making sandfly-borne phleboviruses a significant public
health issue (Tesh et al. 1976, Charrel et al. 2005, Izri et al.
2008). According to the report of the International Commit-
tee on Taxonomy of Viruses (ICTV), the genus Phlebovirus
contains numerous viruses, including Rift Valley fever virus
and sandfly fever Naples virus (Kuhn et al. 2020). In recent
years, new viral species with genomic characteristics of
phleboviruses have been isolated from sandfly specimens
collected around the world (Elliott and Brennan 2014).
Our research group previously reported that a virus was
isolated from sandfly specimens collected in Wuxiang
County, China. Molecular genetic analysis of the virus
showed that it was a new member of the genus Phlebovirus,
and it was named Wuxiang virus (WUXV). This is the first
time that a phlebovirus has been isolated from sandfly
specimens collected from the wild in China (Wang et al.
2020). In the present study, we report that WUXV was iso-
lated again from wild sandfly specimens collected in
1
State Key Laboratory of Infectious Disease Prevention and Control, National Institute for Viral Disease Control and Prevention, Chinese
Center for Disease Control and Prevention, Beijing, China.
2
School of Public Health, Qingdao University, Qingdao, China.
3
Shanxi Provincial Center for Disease Control and Prevention, Taiyuan, China.
4
Yangquan Center for Disease Control and Prevention, Yangquan, China.
5
School of Basic Medicine, Qingdao University, Qingdao, China.
*These authors contributed equally to this work.
VECTOR-BORNE AND ZOONOTIC DISEASES
Volume 21, Number 6, 2021
ªMary Ann Liebert, Inc.
DOI: 10.1089/vbz.2020.2699
446
Downloaded by 221.218.171.207 from www.liebertpub.com at 06/08/21. For personal use only.

Yangquan, Shanxi Province, China (Fig. 1). This is the first
time that WUXV has been isolated from outside Wuxiang
County, suggesting that WUXV has a relatively broad geo-
graphic distribution.
Materials and Methods
Collection of blood-sucking insect specimens
The specimen collection tools used for this study are Wen
Taitai, a collection tool of sucking insects (Battery 12 V 15
Ah, bulb 0.1 A, fan 0.14 A, MM200BL;Guangzhou Chang-
sheng Chemical Technology Service Co., Ltd.,) and a carbon
dioxide mosquito trap lamp (MT-1; Beijing Detelong Tech-
nology Development Co., Ltd.). The collection tool was hung
in a livestock pen from 18:00 pm to 7:00 am the next morning
to collect specimens. All specimens were morphologically
identified, packaged, and numbered under ice bath condi-
tions. Specimens were stored in liquid nitrogen until labo-
ratory testing (Huemer et al. 2017, Song et al. 2017, Wang
et al. 2020). To identify the sandfly species of the positive
pools, PCR was used to amplify the cytochrome c oxidase I
(COI) gene (Wang et al. 2020).
Cell culture and virus isolation
BHK-21 cells (Golden hamster kidney cells) and C6/36
cells (Aedes albopictus eggcells)werestoredinthe
laboratory. BHK-21 cell culture: 90% Eagle’s medium
(laboratory preparation), 7% fetal bovine serum (FBS;
Invitrogen), 1% penicillin and streptomycin (100 U/mL),
1% glutamine (30 g/L), and 1% NaHCO
3
; and C6/36 cell
culture: 89% RMPI 1640 (Invitrogen), 10% FBS (Invitro-
gen), and 1% penicillin and streptomycin (100 U/mL).
BHK-21 and C6/36 cells were cultured in an incubator
containing 5% CO
2
at 37C and 28C, respectively (Song
et al. 2017, Wang et al. 2020).
The sandflies were pooled in groups of 50–100, and
mosquitoes were pooled in groups of 50. After washing,
abrasive fluid was added and specimens were ground re-
peatedly in an ice bath with 1.5 mL of grinding fluid. After
grinding and centrifugation (4C, 12, 000 rpm, 30 min),
100 lL of the supernatant was removed to inoculate BHK-21
cells or C6/36 cells in 24-well plates (Corning, Incorporated),
then the cells were continuously cultured to observe the cy-
topathic effect (CPE) (Huemer et al. 2017, Song et al. 2017,
Wang et al. 2020). SXWX1813-2 isolate had been isolated
previously in the Wuxiang county in 2018 (Wang et al. 2020).
Viral plaque assay and viral titer determination
Viral plaque assay. BHK-21 cells were introduced into
a six-well culture plate (Corning, Incorporated) and grown
into a monolayer with coverage of 80%. A series of 10-fold
viral dilutions (10
-1
–10
-6
) was successively added to the
six-well culture plate (0.1 mL/well). After incubation for
1h at 37C under 5% CO
2
, 1% agarose-MEM (mineral
FIG. 1. Collection of blood-sucking insect specimens in Yangquan County, Shanxi Province, 2018. The shaded area in
the figure is Shanxi Province in central China. Yangquan County (YQ), where specimens were collected for this study, is
represented by a circle, and the triangle represents Wuxiang County (WX), where WUXV was first isolated in China. The
distance between the two sites is about 200 km. WUXV, Wuxiang virus.
WUXIANG VIRUS IN CHINA 447
Downloaded by 221.218.171.207 from www.liebertpub.com at 06/08/21. For personal use only.

essential medium) containing 2% FBS was added to cover
the cells (3 mL/well). After culturing the cells for 3 days,
we added a second layer of 1% agarose-MEM containing
7% neutral red and 2% FBS to cover the cells (3 mL/well).
The appearance time and number of plaques were ob-
served and the diameter of plaques was measured. The data
are statistically processed by SPSS software, and inde-
pendent sample ttest is used (Song et al. 2017, Wang et al.
2020).
Viral titer. A series of 10-fold dilutions of the virus were
added to the 6-well culture plate. After incubation, 1.3%
methylcellulose-MEM semisolid medium containing 2%
FBS was added to each well (4 mL/well). After culturing for
several days, plaques were obvious, and the cells were stained
with crystal violet solution. Then, the plaque-forming units
(pfu) and viral titers were calculated (Song et al. 2017, Wang
et al. 2020).
Electron microscope observation
Brain tissues of suckling mice that had died of the virus
after inoculation were collected, and the samples were fixed
with 2% formaldehyde and 2.5% glutaraldehyde solution.
The samples were sliced on an ultramicrotome (80 nm), dried
at room temperature, stained with uranium acetate and lead
citrate, and then observed using a transmission electron mi-
croscope (TF20; FEI Company) (Feng et al. 2017).
Animal experiment
Suckling mice (Kunming mice) were purchased from
Beijing Vital River Laboratory Animal Technology Co., Ltd.
(quality code SCXK Beijing 2016–0006). All mice were
maintained under sterile conditions in the animal facility of
the Chinese Center for Disease Control and Prevention. All
animal experiments were conducted in strict accordance with
the animal experiment regulations established by the Ethics
Committee of the Chinese Center for Disease Control and
Prevention.
Each suckling mouse that was aged 3 days was inocu-
lated with 20 lL of viral infection supernatant and allowed
to continue feeding. Seven to ten suckling mice were in-
oculated in each group, and the mice were sacrificed and
dissected 14 days after viral inoculation. Then brain tissue
of each passage of suckling mice was ground separately.
The supernatant was obtained after centrifugation at
12,000 rpm, 30 min, without filtration. The supernatant was
collected and inoculated into the next group of suckling mice.
Viral isolates were passed continuously four times in suck-
ling mice. The virus inoculated in the brain of each passage
was the grinding supernatant of the brain tissue of suckling
mice have already become ill and died after the last inocu-
lation. The morbidity and mortality of suckling mice were
observed and recorded every day, and the plaque assay was
used for the determination of virus titer (Cao et al. 2016).
Viral RNA extraction and complementary DNA
synthesis preparation
We used the Viral RNA Mini Kit (QIAamp; Qiagen,
Valencia, CA), following the instructions to extract total
RNA from each sample, and used the Ready-To-Go kit
(GE Healthcare, Little Chalfont, Buckinghamshire, United
Kingdom) to prepare a complementary DNA (cDNA) syn-
thesis of the extracted viral RNA for subsequent viral gene
amplification (Song et al. 2017, Wang et al. 2020).
Viral gene amplification and nucleotide
sequence determination
The PCR system was 25 lL in volume, including cDNA
template, GoTaq

Green Master Mix 2 ·(Promega, Madi-
son, WI), and 10 pmol/lL each of the upstream and down-
stream primers. WUXV L, M, and S gene amplification
primers were used for viral gene amplification from viral
isolates (Wang et al. 2020). The success of gene amplification
was detected using 1% agarose gel electrophoresis before
nucleotide sequence determination. The sequencing method
is Sanger dideoxy sequencing.
Nucleotide sequence analysis
BLAST (NCBI) alignment of nucleotide sequences. Seq-
Man software (DNAStar, Madison, WI) was used for nu-
cleotide sequence splicing and quality analysis. BioEdit
(Version 7.0, Thomas) software was used for nucleotide
multiple sequence alignment (Bootstrap NJ tree). MEGA6.0
software was used for phylogenetic analysis based on the
neighbor-joining method with a bootstrap value of 1,000.
MegAlign was used for homology analysis of nucleotide and
amino acid sequences (Song et al. 2017, Feng et al. 2019,
Wang et al. 2020).
Results
Blood-sucking insect collection
In June 2018, blood-sucking insects were collected from
chicken pens, sheep pens, and abandoned caves in villages in
Yangquan County (1125¢–1144¢east longitude, 3740¢–
3831¢north latitude), Shanxi Province. A total of 4070
blood-sucking insect specimens were collected, including
3996 sandflies. In addition, 74 mosquito specimens were
collected (including Culex [52], Armigeres [13], Aedes [8],
and Anopheles [1]). In addition, the sandflies species for each
of the 10 phlebovirus isolates positive pools were identified
as Phlebotomus chinensis.
Virus isolation
The blood-sucking insect specimens were divided into 54
pools (51 pools of sandfly specimens and three pools of
mosquito specimens). After grinding and centrifugation, the
supernatant was inoculated with BHK-21 cells and C6/36
cells in parallel. Three days after inoculation of BHK-21 cells
with SXYQ1860 (representative strain), CPE was apparent,
showing that the cells contracted and detached (Fig. 2A).
CPE could be stably serially passaged in BHK-21 cells. After
51 pools of sandfly grinding supernatant were inoculated onto
BHK-21 cells, a total of 10 isolates similar to the SXYQ1860
virus strain, which could cause CPE in BHK-21 cells, were
obtained. The grinding supernatant of three pools of mos-
quitoes did not cause CPE in BHK-21 cells. All 51 pools of
sandfly specimens and three pools of mosquito specimens
were also inoculated into C6/36 cells after grinding, and no
CPE was observed after three consecutive passages.
448 WANG ET AL.
Downloaded by 221.218.171.207 from www.liebertpub.com at 06/08/21. For personal use only.

In the supernatants of 10 strains of virus isolates that
caused CPE in BHK-21 cells, WUXV S gene amplification
primers showed a positive result (Table 1) and WUXV was
identified through nucleotide sequence determination and
analysis. The corresponding 10 pools of C6/36 supernatants
(3 consecutive passages) did not show positive amplification
of WUXV genes. The three pools of mosquito specimens did
not show positive WUXV gene amplification in either BHK-
21 or C6/36 cell supernatants.
SXYQ1860 virus can form plaques in BHK-21 cells
(Fig. 2B1), and the average diameter of those plaques is about
2.6 –0.4 mm (n=10) (Table 2). The SXYQ1860 virus was
subjected to two rounds ofplaque purification in BHK-21 cells
to obtain a single viral plaque. The purified plaques were in-
oculated into BHK-21 cells to amplify the virus, then aliquoted
and frozen in an ultra-low temperature refrigerator. The virus-
induced CPE, electron microscopy, animal experiments, and
viral genome sequencing conducted in this study used the
FIG. 2. SXYQ1860 virus-caused CPE of BHK-21 cells, viral titer and electron micrograph. (A1, A2) Show normal
BHK-21 cells and BHK-21 cells inoculated with SXYQ1860 virus, respectively. After inoculation with SXYQ1860, BHK-
21 cells contracted and shed viral particles. (B1, B2) Shows the plaques formed on BHK-21 cells with dilutions of
SXYQ1860 virus and SXWX1813-2 virus. (C) Shows the morphology of viral particles in tissue sections of suckling mice
inoculated with SXYQ1860 virus and the arrows indicate viral particles. CPE, cytopathic effect. Color images are available
online.
Table 1. Isolation and Identification of Wuxiang Virus in Yangquan County, Shanxi Province
Strain No. No. of sandflies Breeding place
BHK-21 C6/36
Genbank acc. no.CPE WUXV(S)
a
CPE WUXV(S)
SXYQ1849-1 50 Sheep pen ++— — MT786481
SXYQ1860 39 Chicken pen ++— — MT786480
SXYQ1861-4 100 .++— — MT786482
SXYQ1864-2 100 .++— — MT786483
SXYQ1867-1 100 .++— — MT786484
SXYQ1869 40 .++— — MT786485
SXYQ1870-1 100 .++— — MT786486
SXYQ1872-1 120 .++— — MT786487
SXYQ1872-2 130 .++— — MT786488
SXYQ1875-2 120 Cave ++— — MT786489
a
S gene of WUXV amplification primers were used for viral gene amplification from viral isolates and 10 isolates of S gene amplification
results are all positive. SXYQ1849-1 virus isolate came from sheep pens, SXYQ1875-2 virus isolate came from an abandoned cave, and the
remaining 8 virus isolates came from chicken pens.
CPE, cytopathic effect; WUXV, Wuxiang virus.
WUXIANG VIRUS IN CHINA 449
Downloaded by 221.218.171.207 from www.liebertpub.com at 06/08/21. For personal use only.

SXYQ1860 virus after plaque purification. Spherical viral
particles were visible on ultrathin sections of infected suckling
mouse brain tissue, which had a diameter of 80–100 nm
(Fig. 2C).
Animal challenge with SXYQ1860 virus
We studied the pathogenicity of SXYQ1860 virus in
suckling mice. After inoculating 20 lL(1·10
5.6
pfu/mL) of
SXYQ1860 virus (BHK-21 7th passage) supernatant into
the suckling mouse brain, we observed and recorded the
morbidity and mortality of suckling mice. No morbidity or
mortality occurred in the first or second passage of suckling
mice after inoculation and incubation for 14 days. On the
10th day after the third passage (n=10), onset of symptoms
began, such as failing to consume milk, convulsion and rigor
of the limbs, lateral lying position, and negative turning
reflex. The suckling mice died on the 10th (n=1), 11th
(n=1), 12th (n=1), 13th (n=4), and 14th days (n=1). The
remaining two suckling mice showed no morbidity or death
on the 14th day after viral inoculation. All animals (n=7) at
thefourthpassagebecameillonthe6thdayanddiedsuc-
cessivelyonthe7th(n=1), 8th (n=2), 9th (n=2), and 10th
days (n=2) (Fig. 3).
Titers of SXYQ1860 virus in the brain tissues of four
consecutive passages of suckling mouse brains were detected
through plaque formation experiments. The results showed
that the viral titers of suckling mouse brains in the first to
fourth passages were 1 ·10
5.86
,1·10
6.05
,1·10
6.32
, and
1·10
6.60
pfu/mL (Fig. 2B1), respectively.
Characteristics of molecular biology
Use the virus cDNA prepared in this study and primers to
use gene amplification methods to obtain the whole virus
genome. The genome of the SXYQ1860 virus contains three
segments. The nucleotide sequences of the coding regions of
these three segments showed that the nucleotide and amino
acid counts of the L segment (GenBank acc. no. MT767755)
were 6273 nt and 2090 aa, encoding RNA-dependent RNA
polymerase (RdRp); the M segment (GenBank acc. no.
MT767756) contained 4089 nt and 1362 aa, encoding gly-
coprotein (GP); and the S segment (GenBank acc. no.
MT786480) includes 1611 nt of genes and contains two open
reading frames (ORFs) encoding nonstructural protein (NS;
260 aa) and nucleocapsid protein (N; 246 aa). The length of
nucleotide and amino acid sequences of the S segments of the
other nine isolates were the same as those of SXYQ1860.
FIG. 3. Animal challenge with
SXYQ1860. The two polynomial curves
shown are the survival rates of suckling
mice in the third and fourth passages after
inoculation with SXYQ1860, respectively.
Color images are available online.
Table 2. Phenotypic Characteristics of SXWX1813-2Virus and SXYQ1860 Virus
SXWX1813-2 SXYQ1860
Time of CPE (h) BHK-21 cells 72 72
C6/36 cells — —
Plaque diameter (mm, n=10, 3d) 2.2 2.6
Titer (pfu/mL) BHK-21 cells (Passage 7th) 1 ·10
6.08
1·10
5.6
Suckling mice brain
(Passage 4th)
1·10
8.09
1·10
6.6
Time from morbidity to death
after inoculation of virus (days)
Passage 3th 9–13 10–14
Passage 4
th
6–9 6–10
Virus particle diameter under electron
microscope (nm)
Negative stain 80–100 —
Ultra-thin section
electron microscope
80–100 80–100
‘‘—’’ Indicates that the test result was negative.
pfu, Plaque-forming units.
450 WANG ET AL.
Downloaded by 221.218.171.207 from www.liebertpub.com at 06/08/21. For personal use only.

Table 3. Genome Sequence and Homology of SXYQ1860
Virus strains
L segment M segment S segment
RdRp GP SNS N
nt (%) aa (%) nt (%) aa (%) nt (%) nt (%) aa (%) nt (%) aa (%)
SXYQ1860 6273 2090 4089 1362 1611 783 260 741 246
SXWX1813-2 6273 (97.7) 2090 (99.3) 4089 (96.6) 1362 (97.3) 1611 (97.5) 783 (97.2) 260 (98.1) 741 (98) 246 (99.6)
TORV (213/Turkey/2012) 6273 (76.9) 2090 (88) 4080 (71.9) 1359 (75.1) 1621 (78) 783 (75.4) 260 (85.1) 741 (82.2) 246 (96.4)
TORV (292/Turkey/2012) 6273 (77) 2090 (88.1) 4081 (71.9) 1359 (75) 1621 (77.8) 783 (75.2) 260 (85.1) 741 (81.9) 246 (96)
CFUV (Pa Ar 814/1981/Greece) 6273 (76.5) 2090 (88.2) 4080 (71.2) 1359 (75.7) 1622 (77.5) 783 (74.3) 260 (84.3) 741 (81.9) 246 (96.4)
SFSV (Ethiopia-2011/Ethiopia/2011) 6273 (71.9) 2090 (78.4) 4026 (62.7) 1341 (57.1) / / / 741 (74.4) 246 (84.2)
SFSV (U30500) / / 4026 (61.7) 1341 (56.5) / ////
SFTV (Izmir 19/Turkey/2008) 6273 (71.8) 2090 (78.6) 4026 (61.6) 1341 (56.9) 1700 (1.6) 804 (53.3) 267 (63.6) 741 (74.5) 246 (83.8)
DASHV (131/Iran/2011) 6273 (72.3) 2090 (79.2) 4029 (65.2) 1342 (58.6) 1741 (70.5) 786 (57.7) 261 (60.9) 741 (76.5) 246 (85)
SFSV ( J04418) / / / / 1686 (2.9) 804 (53.1) 267 (62.8) 741 (75.4) 246 (74.5)
SFSV (I-701735/India/1970) / / / / 1693 (69.8) 789 (55.9) 262 (63.2) 741 (76) 246 (85.8)
SFSV (R-18/Cyprus/1985) / / / / 1558 (69.9) 786 (53.3) 261 (60.9) 741 (74.1) 246 (83.8)
SFSV (RM-09/Cyprus/1985) / / / / 1682 (69.6) 789 (53.3) 262 (63.2) 741 (73.8) 246 (83.4)
SFSV (91045I/Iran/1975) / / / / 1586 (70.2) 786 (57.2) 261 (61.3) 741 (76.8) 246 (84.6)
SFSV (91025B/Iran/1975) / / / / 1700 (70.5) 786 (62.8) 261 (60.9) 741 (76.8) 246 (85)
SFSV (Sabin/Italy/1943) / / / / 1673 (69.7) 789 (53.1) 262 (62.8) 741 (75.3) 246 (83.8)
SFSV (Cyprus/Cyprus/2002) / / / / 1685 (69.3) 787 (52.7) 261 (63.2) 741 (73.8) 246 (84.6)
SFSV (AJ811547) / / / / 1688 (2.9) 804 (53) 267 (62.1) 741 (75.2) 246 (83)
RVF (35/74/South Africa/1974) 6279 (59.5) 2092 (56) 3594 (33.3) 1197 (5.3) 1619 (26.2) 798 (15.1) 265 (24.9) 738 (56.5) 245 (52.8)
SFNV (HM566170) 6288 (56.5) 2095 (50.6) 3972 (31.4) 1323 (6.6) 1753 (28.7) 930 (7.4) 309 (14.9) 762 (44.5) 253 (41.7)
‘‘/’’ Indicates that no sequences were available in the GenBank database.
CFUV, Corfou virus; GP, glycoprotein; N, nucleocapsid protein; NS, nonstructural protein; RdRp, RNA polymerase; TORV, Toros virus.
451
Downloaded by 221.218.171.207 from www.liebertpub.com at 06/08/21. For personal use only.

The nucleotide and amino acid homology results for the
SXYQ1860 virus isolated in Yangquan sandflies in this study
and the WUXV isolated previously show that the SXYQ1860
virus has a homologous nucleotide (amino acid) sequence
of the L gene with WUXV (SXWX1813-2), with similarity of
97.7% (99.3%); the nucleotide (amino acid) homology
between SXYQ1860 and WUXV (17 isolates, including
SXWX1813-2) M gene, NS gene, and N gene are 96.5–
96.9% (97.1–97.8%), 96.6–98% (97.3–98.1%), and 98–
99.2% (99.2–99.6%), respectively, and their homology with
other viruses isolated from sandflies outside China is signif-
icantly lower than that with WUXV.
The viruses isolated from Wuxiang and Yangquan
showed the highest levels of homology with Toros virus
(TORV) and Corfou virus (CFUV), which had L segment
nucleotide (amino acid) homology of 76.5–77.0% (88–
88.2%), M segment nucleotide (amino acid) homology of
71.2–71.9% (75.0–75.7%), S segment NS gene nucleotide
(amino acid) homology of 74.3–75.4% (84.3–85.1%), and
S segment N gene nucleotide (amino acid) homology of
81.9–82.2% (96–96.4%) (Table 3). These results indicate
that the viruses isolated from Yangquan sandflies can be
attributed to WUXV, representing a newly isolated strain of
this virus.
The results of molecular genetic evolution analysis (Sup-
plementary Table S1) of the nucleotide sequence of the viral
genome show that regardless of the L, M, and S (N) genes,
Drin virus (Bino et al. 2019), CFUV, TORV, and WUXV,
including the 10 isolates isolated in Yangquan in the present
study, belong to the mosquito- and sandfly-borne virus group,
FIG. 4. Molecular genetic evolution of the virus isolated from sandflies in Yangquan County. (A) Molecular genetic
evolution of the virus isolated from sandflies in Yangquan County (L gene). (B) Molecular genetic evolution of the virus
isolated from sandflies in Yangquan County (M gene). (C) Molecular genetic evolution of the virus isolated from sandflies
in Yangquan County (N gene). (A–C) Show phylogenetic analyses of the nucleotide sequences of the L, M, and N genes of
viruses isolated from sandfly in Yangquan County, respectively. (A, B) Show phylogenetic analysis of the L and M genes of
SXYQ1860. MEGA 6.0 and the neighbor-joining method were used for genetic evolution analysis with 1000 bootstrap
replicates. As can be seen in the figure, regardless of the L gene or M gene, the SXYQ1860 formed a common evolutionary
cluster with the original strain of WUXV (SXWX1813-2), and had the closest genetic relationship with Drin virus, Corfou
virus, and Toros virus. (C) Shows that the 27 WUXV isolates [17 from Wuxiang (Wang et al. 2020) and the 10 isolates in
this study] can be divided into 2 subgroups. The first subgroup contains 16 isolates from Wuxiang and Yangquan, and the
isolates of the 2 places intersect in the evolution analysis. The second subgroup of viruses did not contain any Yangquan
isolates, and all the 11 isolates were isolated from Wuxiang County.
(continued)
452 WANG ET AL.
Downloaded by 221.218.171.207 from www.liebertpub.com at 06/08/21. For personal use only.

which shows the closest relationships with TORV, CFUV,
and Drin virus (subbranch II) (Fig. 4A–C).
Phylogenetic analysis of the S gene showed that, inde-
pendently of the phylogenetic analysis results for the NS
and N protein gene nucleotide sequences, all 10 isolates
obtained from sandflies in Yangquan County, including
SXYQ1860, and the 17 strains of WUXV isolated from
sandflies in Wuxiang County form a separate evolutionary
branch (subbranch I) that is closely related to the Drin virus,
TORV, and CFUV (subbranch II). This result suggests that
the sandfly-borne virus isolated from Yangquan County and
that isolated from Wuxiang County are the same virus, but
that the 10 isolates isolated from Yangquan represent new
strains of that virus.
Discussion
Previously, our research group reported that a new member
of Phlebovirus, WUXV, was isolated from sandfly specimens
collected in Wuxiang County, China (Wang et al. 2020). The
present study reports that 10 strains of the same virus were
isolated from sandflies collected in Yangquan County, Shanxi
Province, China. Molecular genetic evolution analysis of the
virus showed that these viruses belong to the WUXV group
within the genus Phlebovirus and represent different isolates of
WUXV from different regions (Wang et al. 2020).
Why can WUXV be isolated from sandflies
in Yangquan County?
Yangquan County (1125¢–1144¢east longitude, 3740¢–
3831¢north latitude) and Wuxiang County (11226¢–
11322¢east longitude, 3639¢–378¢north latitude) are
located in the central and eastern parts of Shanxi Province,
China, and the distance between the two areas is about
200 km (Fig. 1). Yangquan and Wuxiang counties are located
in the central and western portions of the Taihang Mountains,
and the geographical climate and natural environment of the
two places are similar. The average altitude of the two sites is
similar (between about 700 and 1700 meters), and both have a
warm temperate continental climate with arid conditions and
low rainfall (Wuxiang 2019, Yangquan 2019).
In addition, villagers in both places commonly raise sheep
and chickens. Our research team used the same type and number
of insect collectors in chicken and sheep pen environments in
Wuxiang and Yangquan counties as collection sites.
On June 11–12, 3819 sandflies and 250 mosquitoes were
collected in Wuxiang (Wang et al. 2020). In this study, we
collected sandflies in Yangquan on June 13–14, obtaining 3996
sandflies and 74 mosquitoes. In both Wuxiang and Yangquan,
natural populations of sandflies and mosquitoes are breeding in
mid-June, and sandflies are denser than mosquitoes. In addition,
the number of sandflies breeding in chicken and sheep pens in
the two places is very similar. The numbers of breeding sandflies
FIG. 4. (Continued).
(continued)
WUXIANG VIRUS IN CHINA 453
Downloaded by 221.218.171.207 from www.liebertpub.com at 06/08/21. For personal use only.

captured in chicken pens were 2665 (Yangquan) and 1604
(Wuxiang), and the breeding populations in sheep pens were
1029 (Yangquan) and 2032 (Wuxiang) (Wang et al. 2020).
Thus, the climate and natural environment of the Yang-
quan and Wuxiang areas are suitable for the growth and re-
production of sandflies. Therefore, the isolation of
Phlebovirus (WUXV) from sandfly specimens collected in
Yangquan is not surprising.
Molecular genetic characteristics of phleboviruses
isolated in China
The results of molecular genetic evolution analysis of
the viral genomes show that the genus Phlebovirus can be
divided into five evolutionary groups, namely the mosquito- and
sandfly-borne group, severe fever with thrombocytopenia syn-
drome (SFTS)/Heartland group, Bhanja group, Kaisodi group,
and Uukuniemi group (Shen et al. 2018, Kuhn et al. 2020).
The mosquito- and sandfly-borne group can be further
divided into two subgroups, of which the second subgroup
contains sandfly fever Naples virus, transmitted by sandflies
(Verani et al. 1980), and Rift valley fever virus, transmitted
by mosquitoes (El Mamy et al. 2014). The first subgroup
contains three evolutionary branches.
The first branch includes the SXYQ1860 virus isolated in
this study and the WUXV isolated for the first time in China;
the second branch contains the Toros (Alkan et al. 2016) virus
and Corfou (Rodhain et al. 1985) virus. The third branch
comprised sandfly fever Sicilian virus (Carhan et al. 2010)
and Dashli virus (Alkan et al. 2017) (Fig. 4A). The
SXYQ1860 virus isolated in Yangquan in this study is a
member of Phlebovirus and forms a common evolutionary
cluster with the WUXV (SXWX1813-2 virus) isolated pre-
viously (Fig. 4A: Viral L gene phylogenetic analysis and
Fig. 4B: viral M gene phylogenetic analysis). Figure 4B and
C show the molecular genetic evolution analysis results for
FIG. 4. (Continued).
(continued)
454 WANG ET AL.
Downloaded by 221.218.171.207 from www.liebertpub.com at 06/08/21. For personal use only.

17 WUXVs (Wang et al. 2020) and 10 viruses isolated from
Yangquan sandflies, indicating that the 27 isolates obtained
from Chinese sandflies formed an independent evolutionary
cluster. These 27 isolates are completely independent of
epidemic phleboviruses of the Mediterranean, such as the
TORV (Turkey) and CFUV (Greece) in the second cluster
and sandfly fever Sicilian virus and Dashi virus in the third
cluster. The virus isolated from sandflies in Yangquan and the
virus previously isolated in Wuxiang are the same virus.
Although the currently WUXV isolates [17 from Wuxiang
(Wang et al. 2020) and the 10 isolated in this study] are all
closely related to Drin virus, CFUV, and TORV, and the
length of gene sequence of WUXV is the same. However, it is
obvious from the evolution analysis of the virus N gene
(Fig. 4C) and NS gene (Supplementary Fig. S1) that WUXVs
are divided into two subgroups. The number of isolates and
the distribution of isolates in the two subgroups were com-
pletely different. The second subgroup of N gene analysis
contained 11 isolates from Wuxiang County (Fig. 4C), while
the second subgroup of NS gene evolution analysis only
contained 2 isolates from Wuxiang County (SXWX1816-1
and SXWX1841-3), which were in the first subgroup of N
gene evolution analysis (Fig. 4C).
In addition, the branch length of different WUXV isolates
in the evolutionary tree was also very different, suggesting
that there were evolutionary differences among different vi-
rus isolates. In conclusion, WUXV is a group of sandfly-
borne virus that is constantly mutating.
The Mediterranean coast is hot and dry in summer, and
warm and humid in winter, with an average temperature in
the coldest month of 4–10C. Winter is rainy, whereas
summer is dry with bright sun, and annual precipitation is
300–1000 mm, defining a unique geographic climate, namely
the Mediterranean climate (Giorgi and Lionello 2008).
FIG. 4. (Continued).
WUXIANG VIRUS IN CHINA 455
Downloaded by 221.218.171.207 from www.liebertpub.com at 06/08/21. For personal use only.

Wuxiang and Yangquan are located in central China. The
winter is cold and dry, with temperatures below 0C, and
summer is hot and rainy. The annual rainfall is about
1000 mm, with two thirds of precipitation occurring in
summer. Thus, the Mediterranean area and central Shanxi
Province in China have common characteristics of cold
winters, hot summers, drought and low rainfall, which are
suitable geographic and climatic conditions for the breeding
of sandflies. The Mediterranean region and Shanxi Province
in central China are nearly 10,000 km apart.
However, WUXV isolated from a sandfly sample from
Shanxi Province in China had a very close genetic evolu-
tionary relationship with the TORV (Turkey) and CFUV
(Greece), which are epidemic to Mediterranean countries.
Biological characteristics between SXYQ1860
and SXWX1813-2
We analyzed the virus isolated from Yangquan
(SXYQ1860) and that virus isolated from Wuxiang
(SXWX1813-2) in parallel to examine their viral biologi-
cal characteristics. These phenotypic characteristics are as
follows: they showed the same CPE timing and charac-
teristics in BHK-21 cells; they had similar viral plaque
characteristics and the same particle size; and the time
between the onset and death after viral infection of suck-
ling mice is not much different (Table 2). Thus, it indicates
that the two virus isolates are the same virus and should
both be considered isolates of WUXV from different
geographic regions.
In this study, WUXV was isolated from sandflies collected
in the wild in Yangquan County. Our results suggest that
WUXV may have a wide geographical distribution except
Wuxiang county. Therefore, it is of great public health sig-
nificance to advance the detection and monitoring of local
sandfly-borne pathogens and determine the potential infec-
tion of humans and animals.
Authors’ Contributions
J.W.,N.F.,andS.F.involvedindraftingthearticle.
J.W., N.F., S.F., and G.L. involved in revising the article.
J.W., N.F., S.F., J.C., B. Wu, Z.X., J.S., X.T., Y.L., Y.H.,
F.L., and S.X. participated in the collection of sandfly
specimens and acquisition of data. J.W., N.F., S.F., and
G.L. participated in the analysis of data. J.W., N.F., S.F.,
and G.L. contributed to the interpretation of data. H.W., B.
Wang, and G.L. contributed to conception and design of the
article. H.W., B. Wang, J.C., and G.L. supported funding
for the study.
Author Disclosure Statement
No conflicting financial interests exist.
Funding Information
This work was supported by the Ministry of Science
and Technology of the People’s Republic of China
(2018ZX10711001, 2018ZX10734-404-003); National Key
Research and Development Program (No. 2018YFA0900800);
Key Research and Development (R&D) Projects of Shanxi
Province, China (201803D31205); Development Grant of the
State Key Laboratory of Infectious Disease Prevention and
Control (2014SKLID103, 2015SKLID505).
Supplementary Material
Supplementary Figure S1
Supplementary Table S1
References
Alkan C, Erisoz Kasap O, Alten B, de Lamballerie X,et al.
Sandfly-borne phlebovirus isolations from Turkey: new in-
sight into the sandfly fever Sicilian and sandfly fever Naples
species. PLoS Negl Trop Dis 2016; 10:e0004519.
Alkan C, Moin Vaziri V, Ayhan N, Badakhshan M,et al. Iso-
lation and sequencing of Dashli virus, a novel Sicilian-like
virus in sandflies from Iran; genetic and phylogenetic evi-
dence for the creation of one novel species within the Phle-
bovirus genus in the Phenuiviridae family. PLoS Negl Trop
Dis 2017; 11:e0005978.
Ayhan N, Baklouti A, Prudhomme J, Walder G,et al. Practical
guidelines for studies on sandfly-borne phleboviruses: part I:
important points to consider ante field work. Vector Borne
Zoonotic Dis 2017; 17:73–80.
Bino S, Velo E, Kadriaj P, Kota M,et al. Detection of a novel
phlebovirus (Drin virus) from sand flies in Albania. Viruses
2019; 11:469.
Cao L, Fu S, Gao X, Li M,et al. Low protective efficacy of the
current Japanese encephalitis vaccine against the emerging
genotype 5 Japanese encephalitis virus. PLoS Negl Trop Dis
2016; 10:e0004686.
Carhan A, Uyar Y, Ozkaya E, Ertek M,et al. Characterization
of a sandfly fever Sicilian virus isolated during a sandfly fever
epidemic in Turkey. J Clin Virol 2010; 48:264–269.
Charrel RN, Gallian P, Navarro-Mari JM, Nicoletti L,et al.
Emergence of Toscana virus in Europe. Emerg Infect Dis
2005; 11:1657–1663.
Depaquit J, Grandadam M, Fouque F, Andry PE,et al.
Arthropod-borne viruses transmitted by Phlebotomine sand-
flies in Europe: a review. Euro Surveill 2010; 15:19507.
El Mamy AB, Lo MM, Thiongane Y, Diop M,et al. Compre-
hensive phylogenetic reconstructions of Rift Valley fever virus:
the 2010 northern Mauritania outbreak in the Camelus drome-
darius species. Vector Borne Zoonotic Dis 2014; 14:856–861.
Elliott RM, Brennan B. Emerging phleboviruses. Curr Opin
Virol 2014; 5:50–57.
Feng Y, Li Y, Fu S, Li X,et al. Isolation of Kaeng Khoi virus
(KKV) from Eucampsipoda sundaica bat flies in China. Virus
Res 2017; 238:94–100.
Feng Y, Ren X, Xu Z, Fu S,et al. Genetic diversity of the
Yokose virus, XYBX1332, isolated from bats (Myotis dau-
bentonii) in China. Virol J 2019; 16:8.
Giorgi F, Lionello P. Climate change projections for the Med-
iterranean region. Glob Planet Change 2008; 63:90–104.
Huemer H, Prudhomme J, Amaro F, Baklouti A,et al. Practical
guidelines for studies on sandfly-borne phleboviruses: part II:
important points to consider for fieldwork and subsequent
virological screening. Vector Borne Zoonotic Dis 2017; 17:
81–90.
Izri A, Temmam S, Moureau G, Hamrioui B,et al. Sandfly fever
Sicilian virus, Algeria. Emerg Infect Dis 2008; 14:795–797.
Killick-Kendrick R. The biology and control of phlebotomine
sand flies. Clin Dermatol 1999; 17:279–289.
456 WANG ET AL.
Downloaded by 221.218.171.207 from www.liebertpub.com at 06/08/21. For personal use only.

Kuhn JH, Adkins S, Alioto D, Alkhovsky SV,et al. 2020
Taxonomic update for phylum Negarnaviricota (Riboviria:
Orthornavirae), including the large orders Bunyavirales and
Mononegavirales. Arch Virol 2020; 165:3023–3072.
Rodhain F, Madulo-Leblond G, Hannoun C, Tesh R. Le virus
Corfou. The virus Corfou, a new Phlebovirus isolated from
Phlebotomus in Greece. Ann Insl Pasteur/Virol 1985; 126E:
161–166.
Shen S, Duan X, Wang B, Zhu L,et al. A novel tick-borne
phlebovirus, closely related to severe fever with thrombocy-
topenia syndrome virus and Heartland virus, is a potential
pathogen. Emerg Microbes Infect 2018; 7:95.
Song S, Li Y, Fu S, Liu H,et al. Could Zika virus emerge in
Mainland China? Virus isolation from nature in Culex quin-
quefasciatus, 2016. Emerg Microbes Infect 2017; 6:e93.
Tesh RB, Saidi S, Gajdamovic SJ, Rodhain F,et al. Serological
studies on the epidemiology of sandfly fever in the Old
World. Bull World Health Organ 1976; 54:663–674.
Verani P, Lopes M, Nicoletti L, Balducci M. Studies on
Phlebotomus-transmitted viruses in Italy: I. Isolation and
characterization of a Sandfly fever Naples-like virus. In:
Arboviruses in the Mediterranean Countries,Zbl.Bakt.
Suppl. 9. Stuttgart, New York: Gustav Fischer Verlag, 1980:
195–201.
Wang J, Fu S, Xu Z, Cheng J, et al. Emerging Sand Fly–Borne
Phlebovirus in China. Emerg Infect Dis 2020;26:2435–2438.
Wuxiang. PsGo. WuXiang overview. 2019 (cited June 2019).
Available at www.wuxiang.gov.cn/hswx/wxgk/rwdl
Yangquan. PsGo. Yangquan overview. 2019 (cited October
2019). Available at www.yq.gov.cn/scfq/yqgl/zhgl_1411/
201509/t20150902_44194.shtml
Address correspondence to:
Huanyu Wang
State Key Laboratory of Infectious Disease
Prevention and Control
National Institute for Viral Disease
Control and Prevention
Chinese Center for Disease Control and Prevention
Beijing 102206
China
E-mail: wanghy@ivdc.chinacdc.cn
Bin Wang
School of Basic Medicine
Qingdao University
Qingdao 266071
China
E-mail: wangbin532@126.com
Guodong Liang
State Key Laboratory of Infectious Disease
Prevention and Control
National Institute for Viral Disease
Control and Prevention
Chinese Center for Disease Control and Prevention
Beijing 102206
China
E-mail: gdliang@hotmail.com
WUXIANG VIRUS IN CHINA 457
Downloaded by 221.218.171.207 from www.liebertpub.com at 06/08/21. For personal use only.