Dissemination of western equine encephalomyelitis virus in the potential vector, Culex pipiens pallens



Two western equine encephalomyelitis virus (WEEV) strains have been isolated in China. Our previous studies have verified that the mosquito Culex pipiens pallens Coquillett (Diptera: Culicidae) infected with WEEV was capable of transmitting this arbovirus, but it was not clear how the sequential multiplication and spread of virus occurred within the mosquito. In this study, we observed the distribution of WEEV antigen in orally-infected Cx. p. pallens by immunohistochemistry in order to better understand the initial infection, dissemination, and transmission of WEEV in the potential vector. Orally-infected WEEV dissemination varied within the different tissues of Cx. p. pallens, with virus antigen consistently observed in the salivary glands, foregut, midgut epithelial cells, Malpighian tubules, hindgut, and ovarian follicles of some individuals after various days of extrinsic incubation. We suggest that Cx. p. pallens, the potential vector of WEEV, has the ability to harbor the virus through the alimentary system, and the midgut epithelial cell may be the initial site of WEEV replication after ingestion of a viremic blood meal.


Western equine encephalomyelitis virus (WEEV), family Togaviridae, genus Alphavirus, is an historically enzootic virus transmitted by mosquitoes in North America and South America. In the northern hemisphere, Culex tarsalis is an important vector (Hardy et al. 1979). Although no disease in humans has been reported in China, two WEEV strains have been isolated from Anopheles hyrcanus and Ixodes persulcatus in 1990 and 1991, respectively (Lv et al. 2001, He et al. 2001), from Xinjiang Uygur, an autonomous region of China. Western equine encephalomyelitis is a potential health threat to equines and humans in China, and previous studies have verified that Culex pipiens pallens Coquillett infected with WEEV is capable of transmitting this arbovirus in the laboratory (Wang et al. 2007). Cx. pipiens pallens is distributed widely in northern China and, because it feeds on both humans and birds, would be an important potential vector of WEEV.

Our previous study found that the infection and transmission rate of Cx. pipiens pallens in laboratory studies was approximately 45% and 40%, respectively (Wang 2007). It indicated that many barriers exist to the infection, dissemination, and transmission of the virus, and that an understanding of these mechanisms is important for developing strategies to interrupt transmission. Most studies agree that posterior midgut epithelial cells of the mosquito are the primary sites of arbovirus initial replication after ingestion of a viremic blood meal (Chamberlain and Sudia 1961, McLintock 1978, Olson et al. 2000, Romoser et al. 2004), but the sequential multiplication and spread of the virus within an individual mosquito is less clear. It is necessary to elucidate the sequential infection and multiplication of this arbovirus in the different tissues of a vector as the mode of the epidemiology of infectious diseases caused by these agents. In this study we observed the distribution of WEEV antigen in orally-infected Cx. p. pallens by immunohistochemistry (IHC), in order to better understand the initial infection, dissemination, and transmission of WEEV.



Cx. pipiens pallens (Beijing strain), originally collected from Beijing and in culture for more than 100 generations, were colonized at the Beijing Institute of Microbiology and Epidemiology. They were maintained at 26 ± 1° C with a photoperiod of 14:10 (L:D) and 80 ± 5% RH. Adults were maintained on an 8% sucrose solution.

Preparation of the WEEV strain

The WEEV strain had been preserved in the Microbial Culture Collection Center of the Beijing Institute of Microbiology and Epidemiology since the 1950s. A 324 fragment of the E1 region of the WEEV genome was obtained by reverse transcription-polymerase chain reaction (RT-PCR) amplification, using the WEEV-specific forward primer (P1) 5′-GTTCTGCCCGTATTGCAGACACTCA-3′ and reverse primer (P2) 5′-CCTCCTGATCTTTTTCTCCACG-3′. The homology with the 71V-1658 strain (GenBank Accession no. NC-003908) (Netolitzky et al. 2000) was 98.15% compared with the same E1 region (8,478∼8,802bp). The virus stock was made with suckling mouse brains harvested 24 to 48 h after intracerebral inoculation and was prepared as a 10% W/V suspension. The titer of WEEV was approximately 108.0–108.3 Vero cell culture plaque-forming units (PFU) of virus.

Virus infection of mosquitoes for immunohistochemistry

Four- to six-day-old female mosquitoes were ether-anesthetized and individually transferred to transparent plastic cups. They were deprived of sucrose for 24 h before infection. The mosquitoes fed on two-day-old chickens that were inoculated subcutaneously with approximately 105.0–106.0 Vero cell culture plaque-forming units (PFU) of virus. Fully-engorged mosquitoes were separated from those partially or unengorged and incubated within a biosafety level 3 laboratory. All mosquitoes were maintained at 26 ± 1° C and 50 ± 5% RH with a photoperiod of 14:10 (L:D) h. The adult mosquitoes were provided with an 8% sucrose solution.

Immunohistochemical staining

After infection, thirty mosquitoes per cohort were collected on days 1, 4, 7, 10, and 14. The legs and wings were removed and the bodies were placed into 60° C Bouin's fixative for 10 min, and then at room temperature for 24 h. They were then washed three times with 70% ethanol and dehydrated in a graded ethanol series (70%, 95%, 100%I, 100%II, 100%III) for 1 h and paraffinized sequentially in xylene I and II for 30 min. Before the paraffinization procedure, the mosquito tissues were kept in the mixture of xylene and paraplast for 30 min and then pure paraplast solution in an incubator overnight at 56–60° C. Mosquitoes embedded in paraffin were prepared in 5 μm serial sagittal sections and dried on slides at room temperature. These slides were incubated overnight at 56° C before deparaffinization in xylene and rehydration in the graded ethanol series above. When the antigens of the slides were retrieved in citrate buffer solution (0.1 M, pH 7.4) at 97° C, the primary antibody mouse ascetic fluid containing anti-WEEV strain, diluted 1:50 in blocking buffer, was incubated with the section and was followed by goat anti-mouse IgG horseradish peroxidase-conjugated secondary antibody diluted 1:500 in the same blocking buffer. Aminoethylcarbazole (AEC) peroxidase substrate allowed the visualization of antigen as a red precipitate, and the slides were developed according to the manufacturers protocol. Fifteen engorged mosquitoes, which feed on asepsis chicken, were treated the same as the negative control. The slides were observed under a compound light microscope.


On day 1 post-infection, the remnants of the blood meal were observed in the midgut of each individual adult mosquito (Figure 1A), but there were no other parts of the body showed any stained at this time. After four days extrinsic incubation periods, the antigens of WEEV were detected in four individuals out of 25 mosquitoes (Table 1). Viral antigen was concentrated in epithelial cells adjacent to the midgut lumen (Figure 1B) but was not found in any other tissues and negative controls. Most tissues of Cx. p. pallens, including the Malpighian tubules, ovarian follicles, and hindgut, were not infected by day 7 except for the midgut (Figure 2). The maximum infection rate of WEEV in the midgut epithelial cells was 36% at 10 days, which was higher than the infective doses in the other tissues of Cx. p. pallens. This is understandable because the blood was digested and assimilated in the midgut lumen when the posterior midgut was distended with the blood meal (Figures 1 and 2A).

Figure 1.

WEEV antigen detection in the midgut (MG) of Cx. p. pallens after oral infection (100×). (A) blood residue at day 1; (B) four days after blood meal.

Table 1.  Positive WEEV infections in different staining tissues of Cx. p. pallens after a blood meal.
  Positive numbers in the different tissues after post infection
Days since infectionNumber feedingmidguthindgutovarian folliclesMalpighian tubulesforegutsalivary gland
Figure 2.

WEEV antigen-positive results in different tissues of the alimentary tract of Cx. p. pallens on day 7 (100×). (A) in the dissemination of MG, ovarian follicle (OF), and Malpighian tubules (MT), respectively. (B) in the hindgut (HG).

The dissemination of WEEV outside of the midgut was initially detected in the other organs on day 7, including ovarian follicles, the Malpighian tubules (Figure 2A), and hindgut (Figure 2B). The infective rates of all organs were below 24%. There were no obvious differences among mosquitoes at 7, 10, or 14 days after virus infection (Table 1). Only 12% of individuals showed infections of the foregut after 10 day post-infection (Figure 3A). The salivary glands were not found to be infected (8%) until day 14 (Figure 3B). In summary, the posterior midgut epithelial cells of Cx. p. pallens were the primary sites of WEEV initial replication after ingestion of a viremic blood meal.

Figure 3.

Immunocytochemical staining analysis in the foregut (FG) at ten days. (A), 100×) and salivary gland (SG) (B), 400× after 14 days during the WEEV oral infection of Cx. p. pallens.


Understanding the dissemination of arboviruses from the mosquito midgut into other tissues is important for understanding vector-virus interactions. This study contributes to our understanding of the initial infection, dissemination, and transmission of WEEV in Cx. p. pallens. According to our results, the antigen staining first appeared in midgut epithelial cells three days later after the blood meal. This result was similar to several other reports of arbovirus replication in the anterior region of the midgut soon after infection (Doi 1970, Romoser et al. 1987, Brault et al. 2004, McElroy et al. 2008). Other organs, including Malpighian tubules, ovarian follicles, and the hindgut, were observed with stained antigen some samples at 7 days post-infection. Reisen et al. (2002) suggested that female Cx. tarsalis became infected orally and transmitted WEE in vitro. We agree that a WEEV suspension from infected mosquito midgut epithelial cells into the hemocele was digested normally and stimulated ovarian development, but the adverse effect of WEE infection on longevity and spontaneous flight activity may decrease the vectorial capacity of the primary vector of WEE (Lee et al. 2000). Salivary gland infection is necessary for horizontal, biological transmission of arboviruses. Weaver et al. (1984) reported that the salivary glands of Culex (Melanoconion) taeniopus mosquitoes are first infected with VEEV by day 4, consistent with initial transmission on day 5. Our results were not similar, in that salivary glands infected by WEEV were first appeared by day 14. This means that at least two weeks of incubation are necessary for Cx. p. pallens transmission of the virus.

Members of the Cx. pipiens complex have been found to be refractory for dissemination and transmission of American strains of WEEV (Gabriela et al. 1990, Hardy et al. 1979). However, Villa et al. (1962) reported that Cx. pipiens from Argentina were able to transmit an Argentine strain of WEE virus under experimental conditions. Our study shows that Cx. p. pallens could be infected with WEEV by feeding on viremic chickens and the virus could be disseminated within them. It is not known whether the vector competence of the species for WEE virus varies with time or whether the differences observed are related to variations in procedures or to the virus strains used. There were no detectable WEEV antibodies in mosquito tissues initially. The infection rate was very low in the foregut and it was possible that WEEV is directly disseminated from the cardia to the adjacent foregut by cell-to-cell communication without traversing a basal lamina, similar to the spread of Venezuelan equine encephalitis virus (VEEV) in Aedes taeniorhynchus (Smith et al. 2007). The cardial tissues including the foregut provide a ready conduit for virus in orally and parenterally infected mosquitoes (Lerdthusnee et al. 1995, Romoser et al. 2004).

The midgut epithelial cells may be the initial site of viral replication in Cx. p. pallens, although some antigen staining in the midgut did not appear in other tissues. It is possible that the midgut escaped barrier of those individuals to WEEV leads to a significant reduction of viremic infections, so no midgut disseminated infection with WEEV occurred in some individuals of Cx. p. pallens during the whole extrinsic incubation period. Dasgupta et al. (2003) also considered that the midgut functions as the first barrier that a pathogen encounters when ingested with a blood meal. Although the mosquito salivary glands and midgut are key entry and exit points for pathogens, only few individuals had WEEV salivary gland infections by day 14 in our study, possibly because the salivary gland infection barrier may be reason for the differences in transmission rate among individuals, where is necessary place for horizontal, mechanical transmission of arboviruses by mosquito (Trevejo and Reeves 2005, Smith et al. 2007).

Based on our findings, it suggested that Cx. p. pallens, the potential vector of WEEV, have the ability to transmit arboviruses through the alimentary system (Weaver et al. 1993), and the midgut epithelial cells may be the initial site of WEEV replication. The mechanisms associated with barriers in incompetent vectors are not clear and deserve further study.


We thank the staff of the vector biology laboratory for their valuable assistance. Tis work was funded by the Ministry of Science and Technology of the People's Republic of China (2003BA712A09–02).