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.
VECTOR-BORNE AND ZOONOTIC DISEASES Volume XX, Number XX, 2020 ª Mary Ann Liebert, Inc DOI: 10.1089/vbz.2019.2523 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 Li,1 Yande Dong,1 Dan Xing,1 Chengfeng Qin,2 and Tongyan Zhao1 Downloaded by Lund University from www.liebertpub.com at 04/08/20 For personal use only 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 between 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 Keywords: BYDV-1, vector competence, Culex 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 TMUVrelated 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 isolated in Culex mosquitoes collected from Shandong Province Introduction S ince April 2010, 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 outbreak 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 Flaviviridae It has an *11 kb single-stranded positive-sense RNA genome, which contains a single ORF that encodes three Department of Vector Biology and Control, State Key Laboratory of Pathogen and Biosecurity, Beijing Key Laboratory, Institute of Microbiology and Epidemiology, Beijing, China Department of Virology, State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology, Beijing, China Downloaded by Lund University from www.liebertpub.com at 04/08/20 For personal use only (Tang et al 2013b) Two strains of TMUV from Culex tritaeniorhynchus 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 ornithophilic and anthropophilic in their host-feeding preference (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 correlation with vector–host associations support the interpretation 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 mosquitoes in China Materials and Methods Ethics statement All animal experiments were performed strictly in accordance 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 Standardization 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 pallens 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 emergence, oviposited Ae albopictus eggs on filter paper were collected and air dried for days to allow complete embryonation 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 dechlorinated water and newly hatched first instar larvae were transferred to enamel trays measuring 50 cm in length and 36 cm in width and 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 GUO ET AL 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 Epidemiology 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 streptomycin Viruses were harvested and stored as individual mL aliquots in freezing tubes at -80C The BYDV-1 titer was 107 plaque-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 days to minimize the effect of shipping stress Then ducks were prescreened and found to be negative for IgG antibodies 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 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 · 106 pfu/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 immediately followed by the titration Then, fully engorged females were selected, transferred to 300 mL plastic cups and were maintained with 8% sucrose in a climatic chamber 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 mL of DMEM (10% FBS, 10% tryptose phosphate broth, and 100 lg/mL Downloaded by Lund University from www.liebertpub.com at 04/08/20 For personal use only VECTOR COMPETENCE OF MOSQUITOES TO TRANSMIT BYDV 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 days and the number of characteristic plaques counted The viral titers of the mosquitoes were expressed as pfu per sample 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 BYDN-1positive glands out of the number of specimens with BYDN1-positive bodies SYBR Green-I-based real-time RT-PCR 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 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-Ibased real-time RT-PCR in Cx p quinquefasciatus, Cx tritaeniorhynchus, 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 mm stainless steel grinding balls (Next Advance) in a Bullet Blender 24 mixer mill (Next Advance) set at frequency of 12/s for 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 CO2 and blood-engorged females removed for virus detection 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 postfeeding, the 10 ducks were killed, and the brains excised Approximately 100 mg brain tissue was individually transferred to 1.5 mL microtubes containing 100 mL DMEM (GIBCO; Invitrogen) supplemented with 2% FBS Viral titer was expressed in pfu Data analysis Results Susceptibility and replication potential of four mosquito species to BYDV-1 Engorged Cx p quinquefasciatus were collected immediately 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 to dpi For the Ae albopictus over time, with small fluctuations, the average virus copies of BYDV-1 exhibited downward trends at 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 log10RNA 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) Body and salivary gland infection rate The vector competence of mosquito populations was assessed by calculating the body (thorax and abdomen) infection rate and salivary gland infection rate Body infection rate was calculated by dividing the number of body infective for FIG 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 4 GUO ET AL Table 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 120 152 155 108 63 100 65 39 Cx tritaeniorhynchus Cx p pallens Cx p quinquefasciatus Ae Albopictus Infection rate (%) 52.5 65.8 41.9 36.1 (63/120) (100/152) (65/155) (39/108) BYDV-1, BYD-1 virus Downloaded by Lund University from www.liebertpub.com at 04/08/20 For personal use only 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) Infection 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 dpi By day 10 and 13 dpi, 22.2% and 33.3% of salivary glands became infected, respectively At dpi, among 19 dissected mosquitoes, 10 (47.3%) were positive for BYDV-1 in the homogenized body segment, and 40.0% (4/10) developed salivary gland infection The average BYDV-1 titers in salivary gland were 1.5 · 102 pfu/mL and 1.6 · 103 pfu/mL at and 13 dpi, respectively Experimental transmission of BYDV-1 to healthy ducks Healthy ducks bitten by infectious mosquitoes can become infected with the virus, which can break through the blood–brain barrier to replicate in the mouse brain, providing direct evidence that this mosquito species can transmit BYDV-1 and is a potential vector Among 29 blood-engorged Cx p quinquefasciatus mosquitoes, 11 Table Infection and Dissemination Rates for Culex pipiens pallens Orally Fed with BYDV-1 at Various Days Postinfection Body (head, thorax, and abdomen) Salivary gland Days No of No of Infection No of Infection postinfection tested positive rate (%) positive rate (%) 10 13 20 16 20 16 19 16 16 12 20 10 10 100.0 50.0 30.0 56.3 47.3 62.5 56.3 50.0 0 0 4 2 0 0 40.0 40.0 22.2 33.3 (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 days after being bitten by infected Cx p quinquefasciatus mosquitoes, two ducks were infected and BYDV-1 titer of duck brains was 1.9 · 103 pfu/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 · 103 pfu/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 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 In recent research, phylogenetic analyses of the whole polyproteins of flaviviruses showed that the DTMUV and DTMUV-related viruses cluster within the clade of mosquito-borne flaviviruses (Liu et al 2012b, Yun et al 2012) This was supported 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 Downloaded by Lund University from www.liebertpub.com at 04/08/20 For personal use only VECTOR COMPETENCE OF MOSQUITOES TO TRANSMIT BYDV 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 tropical and subtropical 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 encephalitis 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 transmit BYDV in China With regard to increased and extensive transport activities and global warming, BYDV can spread more quickly and broadly and continuously evolve Therefore, the possibility of epidemics of reemerging diseases caused by BYDV cannot be ignored (Liu et al 2013) Although 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 surveillance 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 flaviviruses 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 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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 Downloaded by Lund University from www.liebertpub.com at 04/08/20 For personal use only 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 Sparrows (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 surveillance 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, isolated from ducks and geese in china J Virol 2012; 86:3406– 3407 GUO ET AL 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 ... 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... 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. .. 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 Downloaded