Potential vector competence of mosquitoes to transmit baiyangdian virus, a new tembusu related virus in china

VBZ 2019 2523 ver9 Guo 2P 1 6 Potential Vector Competence of Mosquitoes to Transmit Baiyangdian Virus, a New Tembusu Related Virus in China Xiaoxia Guo,1 Tao Jang,2 Yuting Jiang,1 Teng Zhao,1 Chunxiao.

Potential Vector Competence of Mosquitoes to Transmit Baiyangdian Virus, a New Tembusu-Related Virus in China

Xiaoxia Guo,1Tao Jang,2Yuting Jiang,1Teng Zhao,1Chunxiao Li,1Yande Dong,1

Dan Xing,1 Chengfeng Qin,2and Tongyan Zhao1

Abstract

A new duck Tembusu-related flavivirus, Baiyangdian virus (BYDV), caused duck egg-drop syndrome in China The rapid spread, unknown transmission routes, and zoonotic nature, raise serious concern about BYDV as a potential threat to human health The study provides the first evaluation on the vector competence of Culex and Aedes mosquitoes to transmit BYDV in China The results show that Culex tritaeniorhynchus, Culex pipiens pallens, Culex pipiens quinquefasciatus, and Aedes albopictus can become infected with BYD-1 virus (BYDV-1)

on different days after oral infection Although the viral copies in Ae albopictus was higher than that in Cx.

p quinquefasciatus at 13 days postinfection (v2= 10.385, p = 0.016), there was no significant differences be-tween infection rates of four mosquito species (v2= 3.98, p = 0.137) In transmission experiment, healthy ducks were infected after being bitten by virus-positive mosquitoes and BYDV-1 disseminated to and replicated in the duck brains These findings verified the potential role of Cx p quinquefasciatus and Cx tritaeniorhynchus as vectors of BYDV-1 BYDV-1 was also detected in salivary gland of Cx p pallens, which indicated that this virus could be transmitted by mosquitoes These results provide evidence for the role of Culex mosquitoes in the transmission cycles involving BYDV-1 and avian hosts in China.

Introduction

Since April2010, the sudden outbreak and quick spread of

a duck egg-drop syndrome (DEDS) was throughout in the

major duck-producing regions in China The etiological agent

was a newly emerging pathogenic flavivirus, Baiyangdian

(BYD) virus (BYDV), which was first isolated in Hebei

provinces in 2010 (Su et al 2011) Since the epidemic

out-break of 2010, BYDV has been isolated from a variety of

avian specimens including ducks (Su et al 2011), geese

(Huang et al 2013), chickens (Liu et al 2012a), pigeons (Liu

et al 2012b), and sparrows (Tang et al 2013a), which had

spread to 12 provinces and cities causing huge economic

losses and raising social concern

BYDV belongs to the genus Flavivirus of family

Flavi-viridae It has an *11 kb single-stranded positive-sense RNA

genome, which contains a single ORF that encodes three

structural proteins (C, prM, and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5), flanked by the 5¢ and 3¢ untranslated regions (Su et al 2011) Further study proved that BYDV was a new genotype of Tembusu virus (TMUV) belonging to Ntaya virus group of family Flaviviridae, genus Flavivirus (Cao et al 2011) TMUV was first isolated from mosquitoes of the genus Culex

in 1970s in Malaysia (Platt et al 1975) TMUV and TMUV-related viruses have also been isolated in other regions of Southeast Asia, including Thailand and China (Petz et al 2014) It has been isolated from a variety of Culex spp mosquito pools (Platt et al 1975, Pandey et al 1999) and Culex vishnui was able to transmit this virus in the laboratory, which provided evidence for the involvement of Culex mosquitoes in the transmission of TMUV in the environment (O’Guinn et al 2013) In China, a strain of TMUV was iso-lated in Culex mosquitoes collected from Shandong Province

1

Department of Vector Biology and Control, State Key Laboratory of Pathogen and Biosecurity, Beijing Key Laboratory, Institute of Microbiology and Epidemiology, Beijing, China

2

Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Beijing, China

Volume XX, Number XX, 2020

ª Mary Ann Liebert, Inc.

DOI: 10.1089/vbz.2019.2523

1

(Tang et al 2013b) Two strains of TMUV from Culex

tri-taeniorhynchus were isolated from Yunnan Province near

China–Myanmar–Laos Border in 2012 (Lei et al 2017)

Based on phylogenetic, phylogeographic, and ecological

data, BYDV clusters with a group of viruses diverged from

the Japanese encephalitis virus ( JEV) serocomplex cluster

(Liu et al 2012b) Of importance, these two clades primarily

contain Culex spp -associated viruses, which tend to be

or-nithophilic and anthropophilic in their host-feeding

prefer-ence (Gaunt et al 2001, Gould et al 2003) Furthermore,

BYDV grows well in mosquito cell line C6/36 and duck

embryo fibroblast cell line, and causes significant cytopathic

effects in these cell cultures (Yun et al 2012, Tang et al

2013b) Although the natural arthropod vector of BYDV has

not been identified, the phylogenetic data and their

correla-tion with vector–host associacorrela-tions support the interpretacorrela-tion

that ornithophilic Culex spp mosquitoes most likely transmit

BYDV To date, the transmission of BYDV remains largely

unknown There was no evidence indicating that mosquitoes

are involved in the spread of BYDV in China The rapid

spread, unknown transmission routes, and zoonotic nature

raise serious concern about BYDV as a potential threat to

human health in the future The aim of this study was to

evaluate the vector competence of Culex and Aedes

mos-quitoes in China

Materials and Methods

Ethics statement

All animal experiments were performed strictly in

accor-dance with the guidelines of the Chinese Regulations of

Laboratory Animals (Ministry of Science and Technology of

the People’s Republic of China) and the Laboratory Animal

Requirements of Environment and Housing Facilities

(GB 14925-2010, National Laboratory Animal

Standardiza-tion Technical Committee) The experimental protocols were

approved by Animal Experiment Committee of the Beijing

Institute of Microbiology and Epidemiology, Beijing, China

(IME no 2015012)

Mosquito feeding regimes

Culex pipiens quinquefasciatus was collected as larvae

from Guangzhou (N2758¢, E10998¢), Guangdong Province

Cx tritaeniorhynchus was collected as larvae in Xi’an

(N3416¢, E10854¢), Shananxi province Culex pipiens

pal-lens was collected as larvae in Beijing (N3928¢, E11628¢)

Aedes albopictus mosquitoes were from the F8 generation of a

Guangzhou strain originally collected as larvae in Guangzhou

city, Guangdong Province All species collected as larvae were

then taken back to the laboratory and feeding After

emer-gence, oviposited Ae albopictus eggs on filter paper were

collected and air dried for 2 days to allow complete

embry-onation under laboratory conditions and were maintained at a

temperature of 26C – 1C and a relative humidity (RH) of

75%– 5% Oviposited Culex eggs were hatched in

de-chlorinated water and newly hatched first instar larvae were

transferred to enamel trays measuring 50 cm in length and

36 cm in width and 5 cm high, with a density of 2000 larvae

Larval diet was provided in a total feeding regime of 550 mg at

once for each cohort The food for Culex is evenly sprinkled on

the water surface with 20-mesh sieve The food for Aedes was

made into paste with clear water and dripped on the bottom Pupae were sieved and transferred into plastic cups, which were then placed inside cages for adult emergence Adults were maintained under standard insectary condition at 26C – 1C and 75%– 5% RH, with a photoperiod of 14 h:10 h light:dark (L:D) cycles Before the infectious feed, adult mosquitoes were provided with 8% sugar water In general, more than the five generations were used for infection experiment

Virus and cells

TMUV-related BYD-1 virus (BYDV-1) (GenBankacc no JF312912) obtained from the Microbial Culture Collection Center of the Beijing Institute of Microbiology and Epide-miology It was prepared in BHK-21 cells, followed by the titration of the viruses by a standard plaque assay in BHK-21 cells

C6/36 (Ae albopictus) and BHK-21 (baby hamster kidney) cells were maintained in our laboratory C6/36 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO; Invitrogen, Beijing, China) supplemented with 10% heat inactivated fetal bovine serum (FBS; Invitrogen),

100 U/mL penicillin and 100 mg/mL streptomycin (GIBCO)

at 37C with 5% CO2 Infected cells were incubated for 3–5 days in DMEM with 2% FBS and 1% penicillin and strep-tomycin Viruses were harvested and stored as individual

1 mL aliquots in freezing tubes at-80C The BYDV-1 titer was 107plaque-forming units (pfu)

Animals

Experimental infection and transmission was conducted

in 30-day-old healthy ducks Ducks were transferred to our laboratory and adapted to the new environment for 5 days

to minimize the effect of shipping stress Then ducks were prescreened and found to be negative for IgG anti-bodies against DTMUV by enzyme-linked immunosorbent assay

Oral infection of mosquitoes

Seven-day-old female mosquitoes (n> 500) were starved for 12 h before the infectious bloodmeal The bloodmeal consisted of 1:1 mouse blood and virus suspension Before feeding, 0.5 mL virus–blood mixture was stored as individual

1 mL aliquots in freezing tubes at-80C and followed by the titration of the viruses by a standard plaque assay in BHK-21 cells The titer of virus–blood mixture was 1.2· 106pfu/mL Mosquitoes were fed with an infectious bloodmeal that was constantly warmed to 37C using a Hemotek membrane feeding system housed in a feeding chamber After 30-min blood feeding, mosquitoes were cold anesthetized and three engorged Cx p quinquefasciatus were collected immedi-ately followed by the titration Then, fully engorged fe-males were selected, transferred to 300 mL plastic cups and were maintained with 8% sucrose in a climatic cham-ber at 28C – 1C and 75% – 5% RH with a 14 h/10 h L:D cycle Concurrently, uninfected control mosquitoes were blood fed

Plaque assay

Samples were individually titrated in 1 mL of DMEM (10% FBS, 10% tryptose phosphate broth, and 100 lg/mL

streptomycin) and sterilized by syringe filtration (0.22 lm).

The supernatants were serially diluted in the same diluents

and inoculated at 0.1 mL volumes into BHK-21 cell culture

under agar overlay The cell cultures were examined over

2 days and the number of characteristic plaques counted

The viral titers of the mosquitoes were expressed as pfu

per sample

SYBR Green-I-based real-time RT-PCR

Viral RNA was extracted directly from mosquitoes for

BYDV-1 using the QIAamp Viral RNA Mini kit (Qiagen)

following the manufacturer’s protocol cDNA was synthesized

using the Reverse Transcription System for RT-PCR (Promega)

A quantitative real-time PCR for the detection of BYDV-1

based on SYBR Green I dye The primer pair was designed

according to BYD-1 polyprotein gene (GenBank acc no

JF312912), with the sequences of forward: 5¢-GGAATGA

CCTACCCGATGTG-3¢ and reverse: 5¢-TTATCTTGGCA

CCCTTGGAG-3¢ The targeted amplification is a 220-bp

segment of BYDV-1 genome SYBR Premix ExTaq

(perfect real time) kit was purchased from TaKaRa (Dalian,

China)

Growth kinetics

Viral growth curves were performed by SYBR

Green-I-based real-time RT-PCR in Cx p quinquefasciatus, Cx

tri-taeniorhynchus, Cx p pallens, and Ae albopictus About 30

female mosquitoes of each species were sampled on 0, 1, 3, 5,

7, 9, 11, and 13 days postinfection (dpi) Body (thorax and

abdomen) from each mosquito were rinsed in PBS twice and

transferred to 1.5 mL microtubes containing 100 mL of

DMEM (GIBCO; Invitrogen) supplemented with 2% FBS

individually These organs were then homogenized using

5 mm stainless steel grinding balls (Next Advance) in a Bullet

Blender 24 mixer mill (Next Advance) set at frequency of

12/s for 1 min

Transmission experiments

To determine the ability of Cx p pallens to transmit

BYDV-1, on day 7dpi, ten 30-day-old healthy ducks were

provided as a blood source for mosquitoes that had previously

fed on the virus–blood mixture described previously One

duck was placed in a cage containing 20 mosquitoes After 5-h

exposure to mosquitoes, ducks were removed from the cage

and reared In addition, mosquitoes were anesthetized with

CO2and blood-engorged females removed for virus

detec-tion Salivary glands of each mosquito were removed and

individually transferred to 1.5 mL microtubes containing

100 mL DMEM (GIBCO; Invitrogen) supplemented with 2%

FBS On day 8 postfeeding, the 10 ducks were killed, and the

brains excised Approximately 100 mg brain tissue was

in-dividually transferred to 1.5 mL microtubes containing

100 mL DMEM (GIBCO; Invitrogen) supplemented with 2%

FBS Viral titer was expressed in pfu

Body and salivary gland infection rate

The vector competence of mosquito populations was

as-sessed by calculating the body (thorax and abdomen)

infec-tion rate and salivary gland infecinfec-tion rate Body infecinfec-tion rate

was calculated by dividing the number of body infective for

BYDV-1 by the total number of mosquitoes exposed to virus

at each sampling day Salivary gland infection rate was calculated as the number of the specimens with 1-positive glands out of the number of specimens with BYDN-1-positive bodies

Data analysis

The significance of any differences in body infection rate between four species mosquitoes were tested with chi-square and Fisher’s exact tests implemented in the SPSS (GraphPad Software, San Diego, CA) version 14.0 statistical package Values of p< 0.05 were considered significant

Results Susceptibility and replication potential of four mosquito species to BYDV-1

Engorged Cx p quinquefasciatus were collected imme-diately after bloodmeals containing BYDV-1 The average titer was105.3– 0.46 pfu/mL (n = 3) Growth curves of BYDV-1in Cx p quinquefasciatus, Cx tritaeniorhynchus,

Cx p pallens, and Ae albopictus were compared on 0, 1, 3, 5,

7, 9, 11, and 13 dpi, respectively (Fig 1) BYDV-1 could be detected in Cx p quinquefasciatus, Cx tritaeniorhynchus, and Cx p pallens mosquitoes on each sampling day and increased rapidly from 3 to 9 dpi For the Ae albopictus over time, with small fluctuations, the average virus copies of BYDV-1 exhibited downward trends at 7 dpi with average values of 2.68– 0.35 log10RNA copies/mL in body For the

Cx tritaeniorhynchus and Ae albopictus, the highest RNA copies were detected at 13 dpi in bodies with average values

of 4.86– 0.06 log10RNA copies/mL and 5.20– 0.68 log 10-RNA copies/mL, respectively Although the average virus copies of BYDV-1 exhibited downward trends at 11 dpi in

Cx p quinquefasciatus, Cx p pallens, and Ae albopictus

on the curve, there was no significant differences between three Culex mosquitoes at 13 dpi ( p< 0.05) Thus, the viral copies in Ae albopictus was higher than that in Cx

p quinquefasciatus (v2= 10.385, p = 0.016)

FIG 1 Growth curves of BYDV-1 in Culex pipiens quinquefasciatus, Culex tritaeniorhynchus, Culex pipiens pallens, and Aedes albopictus were compared on 0, 1, 3, 5,

7, 9, 11, and 13 days postinfection, respectively BYDV-1, BYD-1 virus

Body and salivary glands infection rate of three

Culex mosquitoes

A total of 120 Cx tritaeniorhynchus, 152 Cx p pallens,

155 Cx p quinquefasciatus, and 108 Ae albopictus were

tested for BYDV-1 competence All three Culex and one

Aedes mosquitoes were infected after 13 dpi (Table 1)

In-fection rates were 52.5% for Cx tritaeniorhynchus, 65.8%

for Cx p pallens, 41.9% for Cx p quinquefasciatus, and

36.1% for Ae albopictus However, no significant difference

was recorded between the infection rates of four mosquito

species (v2= 3.98, p = 0.137)

Body and salivary glands infection rate of Cx p pallens

were detected on 0, 1, 3, 5, 7, 9, 11, and 13 dpi, respectively

(Table 2) BYDV-1 was first detected in salivary glands on

day 7 dpi By day 10 and 13 dpi, 22.2% and 33.3% of salivary

glands became infected, respectively At 7 dpi, among 19

dissected mosquitoes, 10 (47.3%) were positive for BYDV-1

in the homogenized body segment, and 40.0% (4/10)

devel-oped salivary gland infection The average BYDV-1 titers in

salivary gland were 1.5· 102pfu/mL and 1.6· 103pfu/mL at

7 and 13 dpi, respectively

Experimental transmission of BYDV-1 to healthy ducks

Healthy ducks bitten by infectious mosquitoes can

be-come infected with the virus, which can break through the

blood–brain barrier to replicate in the mouse brain,

pro-viding direct evidence that this mosquito species can

transmit BYDV-1 and is a potential vector Among 29

blood-engorged Cx p quinquefasciatus mosquitoes, 11

(37.9%) had viral RNA in their salivary glands Among 36 blood-engorged Cx tritaeniorhynchus mosquitoes, 12 (33.3%) had viral RNA in their salivary glands At 7 days after being bitten by infected Cx p quinquefasciatus mos-quitoes, two ducks were infected and BYDV-1 titer of duck brains was 1.9· 103pfu/mL The infection rate of duck was 20% At the same time, two ducks were positive after being bitten by Cx tritaeniorhynchus, and BYDV-1 titer of duck brains was 7.3· 103pfu/mL

Discussion

The novel TMUV-related mosquito-borne flavivirus was shown to infect multiple avian species and posed a significant threat to public health The urgent necessity is to determine the unknown routes of virus transmission In this study, we provide the first evaluation of vector competence testing to show that Cx tritaeniorhynchus, Cx p pallens, Cx

p quinquefasciatus, and Ae albopictus can become infected with BYDV-1 on different days after oral infection Although the viral copies in Ae albopictus was higher than that in the

Cx p quinquefasciatus at 13 dpi (v2= 10.385, p = 0.016), there was no significant difference between infection rates of four mosquito species (v2= 3.98, p = 0.137) In the other transmission experiment, healthy ducks were infected after being bitten by virus-positive mosquitoes and BYDV-1 dis-seminated to and replicated in the duck brains These findings verified the potential role of Cx p quinquefasciatus and Cx tritaeniorhynchus as vectors of BYDV-1 In recent research, phylogenetic analyses of the whole polyproteins of flavi-viruses showed that the DTMUV and DTMUV-related viruses cluster within the clade of mosquito-borne flavi-viruses (Liu et al 2012b, Yun et al 2012) This was sup-ported by the findings that Cx vishnui and Cx vishnui subgroups were able to transmit TMUV to naive chickens through feeding on TMUV-infected leghorn chicks in the laboratory (Monica et al 2013) The genome strategy of BYDV is the same as that of all the other mosquito-borne flaviviruses Likewise, the distribution of cysteine residues in

C, prM, and E are identical to the other flaviviruses BYDV has an ImRCS3-CS3-RCS2-CS2-CS1 pattern, which is the same as that of the JEV group viruses (Liu et al 2012b) In this study, we demonstrated that Cx p quinquefasciatus and

Cx tritaeniorhynchus were able to be infected with and transmit BYDV-1 after oral exposure BYDV-1 was also detected in salivary gland of Cx p pallens, which indicated that this virus could be transmitted by mosquitoes Taken together, the data presented here extend the role of Culex in the transmission cycles involving BYDV-1 and domestic

Table1 The Infection Rates of Culex tritaeniorhynchus, Culex pipiens pallens, Culex pipiens quinquefasciatus, and Aedes albopictus Orally Fed with BYDV-1 After 13 Days Postinfection Species No of total mosquitoes No of positive mosquitoes Infection rate (%)

BYDV-1, BYD-1 virus

Table 2 Infection and Dissemination Rates for

Culex pipiens pallens Orally Fed with BYDV-1

at Various Days Postinfection

Days

postinfection

No of tested

Body (head, thorax, and abdomen)

Salivary gland

No of positive

Infection rate (%)

No of positive

Infection rate (%)

avian hosts The results also support the role of mosquitoes in

the spread of BYDV in China in addition to the possible oral

route of spread as described for sparrows in China (Tang et al

2013a)

Culex mosquito species breed in groundwater, such as

puddles, rice paddies, ponds, and ditches They prefer to feed

on birds, poultry (domestic chickens, turkeys, and ducks),

pigs and then on humans (Samuel et al 2004, Guo et al 2014,

Azmi et al 2015) Cx tritaeniorhynchus is known to bite pigs

and birds frequently (Hill et al 1969) Cx p quinquefasciatus

is the predominant species in south China and in other

trop-ical and subtroptrop-ical area of the world, especially in urban

areas (Wang et al 2015, Lu et al 2016) Given the close

phylogenetic relationship with dengue virus, tick-borne

en-cephalitis virus, and JVE group viruses, and the same CS

pattern with JEV group viruses (Liu et al 2012b), BYDV has

high potential to be a zoonotic pathogen Furthermore, our

result confirmed the potential of Culex mosquitoes to

trans-mit BYDV in China With regard to increased and extensive

transport activities and global warming, BYDV can spread

more quickly and broadly and continuously evolve

There-fore, the possibility of epidemics of reemerging diseases

caused by BYDV cannot be ignored (Liu et al 2013)

Al-though BYDV has not emerged as a recognized disease in

humans in China, it has had a significant impact on the duck

industry (first recognized in April of 2010), with reports of

*90% drop in egg production and 5–30% mortality in the

birds (Su et al 2011, Yan et al 2011) Continuous

surveil-lance will be necessary to prevent economic losses caused by

the emergence of a more virulent TMUV strain In addition,

more adult mosquito control methods should be implemented

to control mosquitoes if TMUV-related mosquito-borne

fla-viviruses epidemic occur

Conclusions

This study presents the first evidence suggesting that

Culex and Aedes mosquitoes can become infected with

BYDV-1 after oral infection in China Healthy ducks were

infected after being bitten by virus-positive mosquitoes

and BYDV-1 disseminated to and replicated in the duck

brains These findings verified the potential role of Cx

p quinquefasciatus and Cx tritaeniorhynchus as vectors of

BYDV-1 BYDV-1 was also detected in salivary gland of

Cx p pallens, which indicated that this virus could be

transmitted by mosquitoes

Author Disclosure Statement

No competing financial interests exist

Funding Information

This work was funded by grants from the Infective

Diseases Prevention and Cure Project of China (No

2017ZX10303404)

References

Azmi SA, Das S, Chatterjee S Seasonal prevalence and blood

meal analysis of filarial vector Culex quinquefasciatus in

coastal areas of Digha, West Bengal, India J Vector Borne

Dis 2015; 52:252–256

Cao Z, Zhang C, Liu Y, Liu Y, et al Tembusu virus in ducks, China Emerg Infect Dis 2011; 17:1873–1875

Gaunt MW, Sall AA, de Lamballerie X, Falconar AKI, et al Phylogenetic relationships of flaviviruses correlate with their epidemiology, disease association and biogeography J Gen Virol 2001; 82:1867–1876

Gould EA, De Lamballerie X, Zanotto PMA, Holmes EC, et al Origins, evolution, and vector/host coadaptations within the Genus Flavivirus 2003; 59:277–314

Guo XX, Li CX, Wang G, Zheng Z, et al Host feeding patterns

of mosquitoes in a rural malaria-endemic region in hainan island, china J Am Mosq Control Assoc 2014; 30:309–311 Hill MN, Varma MGR, Mahadevan S, Meers PD Arbovirus infections in Sarawak: Observations on mosquitoes in the pre-monsoon period, September to December 1966 J Med En-tomol 1969; 6:398–406

Huang X, Han K, Zhao D, Liu Y, et al Identification and molecular characterization of a novel flavivirus isolated from geese in China Res Vet Sci 2013; 94:774–780

Lei WW, Guo XF, Fua SH, Feng Y, et al The genetic char-acteristics and evolution of Tembusu virus Vet Microbiol 2017; 201:32–41

Liu M, Chen S, Chen Y, Liu C, et al Adapted Tembusu-like virus in chickens and geese in China J Clin Microbiol 2012a; 50:2807–2809

Liu P, Lu H, Li S, Gregory M, et al Genomic and antigenic characterization of the newly emerging Chinese duck egg-drop syndrome flavivirus: Genomic comparison with Tembusu and Sitiawan viruses J Gen Virol 2012b; 93: 2158–2170

Liu PP, Lu H, L1 SH, Wu Y, George F G, et al Duck egg drop syndrome virus: an emerging Tembusu-related flavivirus in China Science China 2013; 56:701–710

Lu RZ, Liu QY, Wu HX, Guo YH Zoogeographical analysis of Culex pipiens pallens/Cx pipiens quinquefasciatus in China,

2014 Chin J Vector Biol Control 2016; 27:107–111 Monica L, Guinn O, Michael J, Turell MJ, et al Field detection

of Tembusu Virus in Western Thailand by RT-PCR and vector competence determination of select Culex mosquitoes for transmission of the virus Am J Trop Med Hyg 2013; 89: 1023–1028

O’Guinn ML, Turell MJ, Kengluecha A, Jaichapor B, et al Field detection of Tembusu virus in western Thailand by RTPCR and vector competence determination of select Culex mosquitoes for transmission of the virus Am J Trop Med Hyg 2013; 89:1023–1028

Pandey BD, Karabatsos N, Cropp B, Tagaki M, et al Identifi-cation of a flavivirus isolated from mosquitos in Chiang Mai Thailand Southeast Asian J Trop Med Public Health 1999; 30:161–165

Petz LN, Turell, MJ, Padilla S, Long LS, et al Development

of conventional and real-time reverse transcription poly-merase chain reaction assays to detect Tembusu virus in Culex tarsalis mosquitoes Am J Trop Med Hyg 2014; 91: 666–671

Platt GS, Way HJ, Bowen ET, Simpson DI, et al Arbovirus infections in Sarawak, October 1968–February 1970 Tem-busu and Sindbis virus isolations from mosquitoes Ann Trop Med Parasitol 1975; 69:65–71

Samuel PP, Arunachalam N, Hiriyan J, Thenmozhi V, et al Host-feeding pattern of Culex quinquefasciatus Say and Mansonia annulifera (Theobald) (Diptera: Culicidae), the major vectors of filariasis in a rural area of south India J Med Entomol 2004; 41:442–446

Su J, Li S, Hu X, Yu X, et al Duck egg-drop syndrome caused

by BYD virus, a new Tembusu-related flavivirus PLoS One

2011; 6:1–10

Tang Y, Diao Y, Yu C, Gao X, et al Characterization of a

Tembusu virus isolated from naturally infected House

Spar-rows (Passer domesticus) in northern China Transbound

Emerg Dis 2013a; 60:152–158

Tang Y, Gao X, Diao Y, Feng Q, et al Tembusu virus in

human, China Transbound Emerg Dis 2013b; 60:193–196

Wang XH, Yang XY, Zhao W, Lin CY Analysis of

surveil-lance data of mosquito density from 2012 to 2014 in Haikou

city, Hainan, China Chin J Vector Biol Control 2015; 26:

424–426

Yan P, Zhao Y, Zhang X, Xu D, et al An infectious disease of

ducks caused by a newly emerged Tembusu virus strain in

mainland China Virology 2011; 417:1–8

Yun T, Zhang D, Ma X, Cao Z, et al Complete genome

sequence of a novel flavivirus, duck Tembusu virus,

iso-lated from ducks and geese in china J Virol 2012; 86:3406–

3407

Address correspondence to:

Cheng-Feng Qin Department of Virology State Key Laboratory of Pathogen and Biosecurity Institute of Microbiology and Epidemiology

Beijing 100071

China

E-mail: chengfeng_qin@126.com

Tong-Yan Zhao Department of Vector Biology and Control State Key Laboratory of Pathogen and Biosecurity

Beijing Key Laboratory Institute of Microbiology and Epidemiology

Beijing 100071

China

E-mail: tongyanzhao@126.com