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

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 (χ² = 10.385, p = 0.016), there was no significant differences between infection rates of four mosquito species (χ² = 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 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 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 isolated in Culex mosquitoes collected from Shandong Province (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 (N27°58′, E109°98′), Guangdong Province. Cx. tritaeniorhynchus was collected as larvae in Xi'an (N34°16′, E108°54′), Shananxi province. Culex pipiens pallens was collected as larvae in Beijing (N39°28′, E116°28′). 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 2 days to allow complete embryonation under laboratory conditions and were maintained at a temperature of 26°C ± 1°C 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 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 26°C ± 1°C 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 37°C 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 1 mL aliquots in freezing tubes at −80°C. 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 5 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 1 mL aliquots in freezing tubes at −80°C 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 37°C 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 28°C ± 1°C 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 μg/mL streptomycin) and sterilized by syringe filtration (0.22 μm). 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′-GGAATGACCTACCCGATGTG-3′ and reverse: 5′-TTATCTTGGCACCCTTGGAG-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. 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 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 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 8 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.

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 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-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 immediately after bloodmeals containing BYDV-1. The average titer was10^5.3 ± 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 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 (χ² = 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). 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 (χ² = 3.98, p = 0.137).

Table 1.

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 (%)
Cx. tritaeniorhynchus	120	63	52.5 (63/120)
Cx. p. pallens	152	100	65.8 (100/152)
Cx. p. quinquefasciatus	155	65	41.9 (65/155)
Ae. Albopictus	108	39	36.1 (39/108)

BYDV-1, BYD-1 virus.

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) 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 7 and 13 dpi, respectively.

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
Days postinfection	No. of tested	No. of positive	Infection rate (%)	No. of positive	Infection rate (%)
0	20	20	100.0	0	0
1	16	8	50.0	0	0
3	20	6	30.0	0	0
5	16	9	56.3	0	0
7	19	10	47.3	4	40.0
9	16	10	62.5	4	40.0
10	16	9	56.3	2	22.2
13	12	6	50.0	2	33.3

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 (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 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 (χ² = 10.385, p = 0.016), there was no significant difference between infection rates of four mosquito species (χ² = 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 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.

Footnotes

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).

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