Vector Competence of Five Common Mosquito Species in the People's Republic of China for Western Equine Encephalitis Virus

Abstract

Two strains of the Western equine encephalitis virus (WEEV) were first detected and isolated in China in 2001. The maintenance and transmission cycles of WEEV in China are currently not well understood, and the mosquito vectors involved in these cycles are unknown. To understand the ability of the local mosquitoes in China to transmit WEEV, the vector competence of five mosquito species, namely, Culex pipiens pallens Coquillett, Cx. p. quinquefasciatus Say, Aedes (Stegomyia) albopictus Skuse, Ae. (Stegomyia) aegypti Linnaeus, and C. tritaeniorhynchus Giles, for WEEV were evaluated. Infection rates for Cx. p. pallens, Cx. p. quinquefasciatus, Cx. tritaeniorhynchus, Ae. Albopictus, and Ae. aegypti were 46%, 60%, 80%, 37%, and 25%, respectively. Dissemination rates for the same species were 60%, 61%, 75%, 55%, and 50%, respectively. Transmission rates were 41%, 53%, 57%, and 45% for Cx. p. pallens, Cx. p. quinquefasciatus, Ae. Albopictus, and Ae. Aegypti, respectively. Infection rates were significantly different between species, but the difference between dissemination and transmission rates were nonsignificant. These results suggest that several local mosquito species in China are competent laboratory vectors for WEEV.

Introduction

W estern equine encephalomyelitis virus (WEEV) is an Alphavirus within the family Togaviridae that is historically endemic in North and South America Reisen, and Monath (1989). Culex tarsalis Coquillett is considered a primary vector in North America (Reeves and Hammon 1962).

In 2001, WEEV strain XJ-90260 was isolated from a pool of Anopheles hyrcanus species collected in 1990 from Wusu County in the Xinjiang Uygur Autonomous Region of China (Lv et al. 2001). Another WEEV strain was isolated from Ixodes persulcatus in Bole County, Xinjiang, in 1991 (He et al. 2001). Further, Lv et al. (2001) detected WEEV strain XJ-90260 polyclonal antibodies in 886 human sera from nine provinces in China suggesting widespread WEEV transmission in this country. In addition, WEEV-positive sera were detected in Xingjiang, Gansu, and Henan provinces, and the percent prevalence in humans was estimated at 2.71% (Lv et al. 2001). Interestingly, numerous unidentified fever and encephalitis cases have been reported from specific areas in China (Liang Guo-dong 1997) that may have been caused by WEEV especially since the virus is not routinely screened in this country. This assumption suggests the possibility of WEEV as an emerging new pathogen in China. Although WEEV has not caused widespread epidemics, this virus poses a potential public health and veterinary threat in China because the maintenance and transmission cycles of this virus are currently not well understood.

Culex pipiens pallens and Cx. p. quinquefasciatus are two members of Cx. pipiens complex that are distributed widely in North and South China, respectively (Zhao and Lv 1994, 1996). Both species feed readily on avian and human hosts. Cx. tritaeniorhynchus, widely distributed in rice-growing areas, feeds frequently on swine but also occasionally feeds on humans and birds. Aedes albopictus and Ae. aegypti are the main vectors of dengue viruses in China. There are no reports concerning the vector competence of Cx. tritaeniorhynchus for WEEV. Members of the Cx. pipiens complex have gut limited infections and are refractory for dissemination and transmission of American strains of WEEV (Gabriela et al. 1990, Hardy and Reeves 1990). Additionally, Ae. albopictus and Ae. aegypti have been reported to possess the ability to be infected with WEEV (Chamberlain et al. 1954, Kelser 1993, Malcolm et al. 1935, Mitchell 1991). However, susceptibility for the same virus varies in different strains of the same mosquito species (Gubler and Rosen 1976, Hardy et al. 1976). The authors previously verified that Cx. p. pallens can be infected with and has the ability to transmit WEEV (Wang et al. 2007, 2010). In the present study, five important indigenous mosquito species, including Cx. p. pallens, were selected to determine their susceptibility to infection and their capability to orally transmit WEEV.

Materials and Methods

Mosquitoes

Mosquitoes tested were acquired from established laboratory colonies (Table 1). All mosquito strains, except Cx. tritaeniorhynchus, were reared at 26°C±1°C with a light:dark cycle of 14 h: 10 h and 80%±5% RH. Cx. tritaeniorhynchus was reared at 29°C±1°C with the same light and RH conditions. Adult mosquitoes were provided with 8% sucrose solution.

Table 1.

Mosquito Species Tested for Susceptibility to Western Equine Encephalitis Virus

Species	Strain	Source	Generation
Culex pipiens Pallens	Beijing	Beijing (1996)	F > 100
Cx. p. quinquefasciatus	Guangzhou	Guangzhou (1996)	F > 100
Cx. tritaeniorhynchus	Dali	Dali (2003)	F30–45
Aedes albopictus	Chengdu	Chengdu	F > 50
Ae. aegypti	Haikou	Haikou	F > 50

WEEV strain

The virus stock (McMillan strain) was made with mouse brains harvested 24–48 h after intracerebral inoculation and prepared as a 10% (W/V) suspension. The WEEV stock was titered by plaque assay in six-well tissue culture plates.

Infection of mosquito

Mosquitoes anesthetized by diethyl ether were transferred to transparent plastic cups with a sealed gauze top 24 h prior to infection. Two-day-old chickens were inoculated subcutaneously with approximately 105.0–106.0 Vero cell culture plaque-forming units (PFU) of virus held for 24 h before serum samples were collected. Then, the chickens were restrained on individual cups to allow mosquitoes to blood feed. Blood was diluted 1:10 in a heparinized diluent. Supernates were frozen at −70°C until virus testing. Fully engorged mosquitoes were incubated in a secure room inside a laboratory with level 3 biosafety (State Key Laboratory of Pathogen and Biosecurity, Institute of Microbiology and Epidemiology). All mosquitoes were maintained at 26°C±1°C and 50%±5% RH, with a light:dark cycle of 14 h: 10 h and provided with 8% sucrose solution. After 14 days postfeeding, the mosquitoes were frozen at −70°C for subsequent virus detection. The mosquitoes and blood samples from chickens were tested for virus by reverse transcription–polymerase chain reaction (RT-PCR) (Wang et al. 2007). The specific primers were P₁: 5′-GTTCTGCCCGTATTGCAGACACTCA-3′ and P₂ : 5′-CCTCCTGATCTTTTT CTCCACG-3′ (Linssen et al. 2000). The infection rates of mosquitoes were established by considering the numbers infected and the total number tested 14 days postinfection.

WEEV dissemination in the body of the mosquito

To detect virus dissemination from the midgut, the wings, and the legs, were removed from the body and frozen separately at −70°C. If the body tested positive for the virus, the wings and the legs were triturated in 400 μL trizol and tested by RT-PCR. The dissemination rate (%) was the number of mosquitoes with positive wings and legs divided by the number of infected mosquitoes tested.

Experimental transmission

On 14 days postinfection, mosquitoes were allowed to feed individually on one- to three-day-old Leghorn chickens. The brain tissues of chicken were removed and tested two to three days after blood feeding to determine infection. Transmission was confirmed by the detection of WEEV from the chickens and the transmission rate was defined as (the number of mosquitoes that can transmit virus by biting/total number of engorged mosquitoes)×100%.

Statistical analysis

Infection, dissemination, and transmission rates were compared among species by χ ² test using SPSS10.0. Differences were considered statistically significant at P<0.05.

Results

Given that mosquitoes were bred singly in a cup in absl-3 lab with 50%±5% RH, which was not fit for the survival of mosquitoes, over a thousand from each mosquito species were included in the infection experiment. Only approximately 10% of the mosquitoes survived the transmission experiment. Only 10 Cx. Tritaeniorhynchus mosquitoes remained, and none of them fed on susceptible chickens after 14 days incubation. In the transmission experiment, the remaining mosquitoes were allowed to feed on chickens. Thirty-five mosquitoes that successfully transmitted and tested for the virus were selected randomly from each species, except Cx. tritaeniorhynchus.

All mosquito species tested were susceptible to infection with WEEV after feeding on the viremic chickens. The virus titers of donor chicken serum sample ranged from 10^3.4 to 10^7.6 PFU/mL by plaque assay in six-well tissue culture plates. The infection rates of mosquitoes ranged from 25% to 80% (Table 2). The infection rates were significantly higher for Cx. tritaeniorhynchus than Cx. p. pallens, Ae. Albopictus, and Ae. aegypti (χ ²=13.7, P=0.008). The infection rate of Cx. p. quinquefasciatus was significantly higher than those of Ae. albopictus and Ae. aegypti (χ ²=7.8, P=0.05).

Table 2.

Infection, Dissemination, and Transmission Rates for Mosquitoes After Oral Exposure to Western Equine Encephalitis Virus

Species	Infection rate (%)	Dissemination rates (%)	Transmission rate (%)
Cx. p. pallens	46	60	41
Cx. p. quinquefasciatu	60	61	53
Cx. tritaeniorhynchus	80	75
Ae. albopictus	37	55	57
Ae. aegypti	25	50	45

At 14 days post feeding, >50% of infected mosquitoes transmitted infections (Table 2). There were no significant differences in the dissemination rate among the five species of mosquitoes tested (χ²=1.2, P=0.9).

Given that Cx. tritaeniorhynchus failed to feed, the transmission experiment of this species had to be suspended. The other four species orally transmitted WEEV to one- to three-day-old Leghorn chickens. Transmission rates of Cx. p. pallens, Cx. p. quinquefasciatus, Ae. Albopictus, and Ae. aegypti were 40.74%, 53.13%, 57.14%, and 45.16%, respectively. No significant differences among the four species was recorded (χ ²=1.9, p=0.6) (Table 2).

Discussion

The current study is the first evaluation of the capability of Chinese mosquito species to be infected with and to transmit WEEV.

Cx. p. pallens and Cx. p. quinquefasciatus are two members of the Cx. pipiens complex (Zhao and Lv 1994, 1996), both of which had been shown to be refractory to oral infection by WEEV and probably do not play roles in the WEEV cycle in North and South America (Reeves et al. 1962, Hardy et al. 1979, Houk et al. 1986, Gabriela et al. 1990). By contrast, the present study showed that both species could be infected with and could transmit WEEV. Whether the vector competence of these two species for WEEV varied with time or whether the differences observed are related to variations in procedures or to the virus strains used is unknown. Both species are widely distributed in North and South China and are considered vectors of Japanese encephalitis virus, a flavivirus, in China (Li et al. 1998). The infection, dissemination, and transmission rates were similar, and both species are known to feed readily on avian and human hosts (Xiong et al. 1997, Zhu et al. 2006). Therefore, Cx. p. quinquefasciatus and Cx. p. pallens might serve as both epizootic and enzootic vectors for WEEV in South and North China, respectively.

The highest rates of infection and dissemination were obtained with Cx. tritaeniorhynchus under the present laboratory conditions. Cx. tritaeniorhynchus is distributed widely in the rice culture region of Asia and recognized as the primary vector of the Japanese Encephalitis Virus (JEV) in China (Li et al. 1998). This species primarily feeds on swine, the amplifying host of Japanese Encephalitis Virus (JEV), but it also feeds on humans and birds. Therefore, Cx. tritaeniorhynchus may also play an important role in the possible outbreak of WEEV in the future based on extremely extensive distribution, susceptibility to infection, and dissemination rate of this virus. Further incrimination of this species as a vector of WEEV must await more definitive experiments on vector competence that include determination of transmission efficiency.

Aedes mosquitoes, such as Ae. albifasciatus in Argentina, are considered potential vectors of WEEV (Gabriela et al. 1990). Hardy and Bruen (1974) have shown that Ae. melanimon is an important vector of WEEV in California. Ae. albopictus is widely distributed, whereas Ae. aegypti is prevalent in South China. In the present study, these two Aedes species were also infected with and transmitted WEEV under the laboratory conditions. The infection and dissemination rates of these species were relatively low but the transmission rates were not significantly different from the two Culex mosquitoes. The multiple blood feeding habit of the two Aedes mosquitoes enhances the chances of these species to acquire or transmit a pathogen in each gonotropic cycle. Considering that Ae. Albopictus overwinters as diapaused eggs, vertical transmission experiments need to be conducted to determine whether this species plays a role in WEEV preservation.

The vector competence of mosquitoes for aboviruses partially depends on intrinsic barriers that influence the ability of a mosquito to be infected with and to transmit virus. Nondisseminated infections indicate the existence of a mesenteronal escape barrier to virus. Kramer et al. (1981)found this barrier to be highly dose-dependent in Cx. tarsalis infected with WEEV. In the current study, the dissemination rates of five select mosquito species were all >50%. Most Cx. p. pallens and Cx. p. quinquefasciatus with disseminated infections were able to transmit virus per os to chickens. Results suggest that virus dissemination is correlated with the infection of the salivary glands in the two Culex species in China.

The demonstrated susceptibility of all five mosquito species to WEEV infection induced orally is further evidence for considering these species as potential WEEV vectors in China.

Footnotes

Acknowledgments

The authors thank the staff of the Vector Biology Laboratory for their valuable assistance. This work was funded by the Ministry of Science and Technology of the People's Republic of China (2003BA712A09-02).

Disclosure Statement

No competing financial interests exist.

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