The study was conducted in the Animal Biosafety Level 3 (ABSL-3) facility. All of the experimental protocols involving animals were approved by the Laboratory Animal Center of the State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology, and Epidemiology Institutional Animal Care and Use Committee (IACUC, the permit number is BIME 2011-2009). The animals were performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

Four geographical strains of Ae. Albopictus were originally collected in Guangzhou city (GPS location: 23°08′N and 113°14′W) in 1996, Shanghai city (GPS location: 31°15′N and 121°30′W) in 1995, Chengdu city (GPS location: 30°05′N and 102°54′W) in 1996, and Beijing city (GPS location: 39°56′N and 116°20′W) in 1997, respectively. Mosquitoes were maintained at 25 ± 1°C and 75 ± 5% relative humidity (RH) under a 14-h light/10-h dark (LD) photoperiod. F30 generation of Ae. Albopictus mosquitoes was used in this study. In addition, the Guangzhou strain and Beijing strain of Ae. Albopictus were collected in the wild in 2004 and 2005, separately. Mosquitoes were domesticated to F3 generation in the laboratory for transmission efficiency evaluation.

The WNV strain (GenBank AY490240) was provided by the Microbial Culture Collection Center, Beijing Institute of Microbiology and Epidemiology. It had been passaged six times in Vero cells. Viral titer was 10⁷.⁰–10⁷.⁵ PFU/ml. Vero cells were cultivated in Dulbecco’s modified Eagle’s medium (DMEM) (GIBCO™, Invitrogen, Beijing, China) containing 10% fetal bovine serum (FBS) and placed in an incubator of 37°C and 5% CO₂.

Three- to five-day-old female mosquitoes were deprived of glucose solution and water for 24 h prior to infection. The infectious blood meal was composed of 1:1:1 mouse blood and virus and an 8% sugar solution. The viral titer of infectious blood meals was 10⁶.⁷–10⁶.⁹ PFU/ml. Mosquitoes were offered a WNV infectious blood meal for 1 h using a Hemotek membrane feeding system housed in a feeding chamber that was constantly warmed to 37°C. One hundred and fifty engorged female mosquitoes were selected under CO₂ sedation for each geographical strain and transferred to three small cages (50 mosquitoes/cage), respectively. Engorged mosquitoes were reared under CO₂ sedation and incubated under 29 ± 1°C and 80 ± 5% RH with a 14:10 (light:dark) photocycle.

West Nile virus-exposed samples were detected by reverse transcription-polymerase chain reaction (RT-PCR). The PCR primers included a pair of universal primers, Primer 1 (P1): P1:5′-TTG TGT TGG CTC TCT TGG CGT TCTT and Primer 2 (P2): 5′-CAG CCG ACA GCA CTG GAC ATT CATA. The RNA extraction was performed using TRIzol reagent (Invitrogen) according to the recommendations of manufacturers. Viral RNA extract was transcribed into cDNA using RNA PCR Kit (AMV) version 2.1 [Takara Biotechnology (Dalian) Co., Ltd., Shiga, Japan]. The resulting cDNA was used as a template in the subsequent PCR reaction. The PCR amplifications were carried out through 45 cycles at 94°C for 30 s, 58°C for 30 s, 72°C for 1 min, and final extension at 72°C for 7 min. Furtherly, some WNV-exposed samples were selected randomly to identify by C6/36 culture isolation. Virus isolation was processed as described in Jiang et al.’s (2006) study. That is, the supernatants of infected mosquitoes or Leghorn chicken blood were passed through a filter with pores of 0.22 μm in diameter and inoculated into duplicate wells of C6/36 cell microplate cultures. After 1.5 h incubation at 37°C, cells were maintained at 37°C in DMEM supplemented with penicillin (100 U/ml), streptomycin (100 μg/ml), and 5% FBS for 7 days.

Mosquitoes were deprived of glucose solution and water for 24 h after a 14-day incubation period. One mosquito was allowed to feed on one 1- to 3-day-old Leghorn chicken individually for 30 min. After sucking blood, mosquitoes were killed by freezing at 20°C and were ground individually in 1.0 ml of mosquito diluents to test for the presence of the virus by RT-PCR. Then RNA positive samples were inoculated into C6/36 cells to isolate WNV. On day 2 post-sucking, the chicken blood was collected and serum was separated. The serum was then used to detect viral RNA by RT-PCR and to isolate WNV by inoculation into C6/36 cells.

The infection rate describes the number of mosquitoes infected with WNV in their body in relation to the total number of mosquitoes examined. The transmission rate was calculated as the ratio of the number of mosquitoes that can transmit the virus by biting to the total number of infected mosquitoes (Goddard et al., 2002; Tiawsirisup et al., 2005). The transmission efficiency is the percentage of WNV-positive mosquitoes that can transmit the virus by biting in relation to the total number of engorged mosquitoes.

Eggs from the second and third gonotrophic cycles of Ae. Albopictus Beijing strain were used in this study. Filter paper used as an oviposition substrate was placed in a small container of water after blood meal. After 3 days, filter papers were removed and kept in humid environment for 48 h. Then, eggs were held in desiccators at 25 ± 1°C, 90% RH with a daily photoperiod of 14-h light/10-h dark (LD) for 6 days to permit embryonation (Hanson and Craig, 1994). A schematic representation of the experimental design and infection, transmission, and diapause induction is shown in Figure 1.

Following embryonation, eggs were placed into a light cultivation chamber (HPG-280H) (Guangdong Medical Instrument Factory Co., Ltd.) and subjected to a 10-h light/14-h dark (LD) photoperiod and 75% RH was controlled constantly with a supersaturated salt solution (Winston and Bates, 1960). The temperature was decreased 5°C every 2 days from 25 to 10°C. At last, eggs were held at a daily photoperiod of 8-h light/16-h dark (LD) and thermoperiod of 10°C:4°C (LD) and 50% RH for 6 weeks. The diapause induction procedure for uninfected eggs was the same. The control group not in diapause (non-diapause) was maintained at 25 ± 1°C and 14-h light/10-h dark (LD) photoperiod and 75% RH.

Reverse transcription-PCR and virus isolation in C6/36 cells were used to detect WNV in diapausing eggs from the second and third gonotrophic cycles of infected Ae. Albopictus. Pools of diapausing eggs (200 eggs per pool) were detected. Minimum infection rates (MIRs) were calculated using Centers for Disease Control and Prevention (CDC) protocols.¹

Diapausing eggs of Ae. Albopictus Beijing strain were submerged in deoxygenated solution (500 mg larval food was added into 500 ml water for 24 h, the dissolved oxygen concentration was about 0.3–0.5 ppm) to terminate diapause. After 24 h, the number of larvae was counted. All samples then were transferred to non-diapausing conditions [25 ± 1°C and 14-h light/10-h dark (LD)] and hatch was attempted weekly. Unhatched eggs were bleached to clear the chorion to determine embryonation. The bleach solution consisted of 40 g sodium hypochlorite, 10 ml acetic acid, and 1 L of water (Hanson and Craig, 1994). Pools of F1 generation larvae from diapausing and non-diapausing eggs were detected by RT-PCR and virus isolation with C6/36. MIRs were calculated.

The significance of any differences in infection and transmission rates between four geographical strains of Ae. Albopictus were tested with Chi-square and Fisher’s exact tests implemented in the SPSS (GraphPad Software, San Diego, CA, United States) Version 10.0. Values of p < 0.05 were considered significant.

Four geographical strains of Ae. Albopictus were susceptible to infection with WNV after imbibing the virus dose of 10⁶.⁷–10⁶.⁹ PFU/ml. The infection rates of four different geographical strains of Ae. Albopictus are shown in Table 1. The infection rates of different strains tested in the study were ranged from 16.7 to 60.0%. There were significant differences in infection rates between the four strains (χ² = 12.81, p < 0.05). The highest infection rate was found in the Shanghai strain, which was 60.0%. There was no significant difference between the Ae. Albopictus 30 generations and 3 generations mosquito (χ² = 2.73, p > 0.05) of Guangzhou and Beijing strains. Four RNA-positive samples were inoculated into C6/36 cells to isolate WNV. Cytopathic effect (CPE) was observed on the fifth day after inoculation.

Four geographical strains of Ae. Albopictus were able to transmit WNV to 1- to 3-day-old Leghorn chicken by biting. Transmission rates varied from 40.0 to 47.6% (Table 1). There were no significant differences in transmission rates between the different strains (χ² = 4.49, p > 0.05). However, there were significant differences in transmission efficiency between the four geographical strains of Ae. Albopictus (χ² = 103.2, p < 0.05). The transmission efficiency of the Shanghai and Chengdu strains was significantly different (χ² = 5.149, p < 0.05). The Leghorn chickens were bled on the second day for virus assay by RT-PCR. Leghorn chickens had viral RNA in their blood. The serum of three Leghorn chickens was inoculated into C6/36 cells. CPE was observed on the seventh day after inoculation.

Pools of diapausing and non-diapausing eggs (200 eggs per pool) from the second and third gonotrophic cycles were tested by RT-PCR. An amplification product of the predicted size of 408 bp was evident in diapausing eggs. All RNA-positive homogenates of diapausing and non-diapausing eggs were inoculated in C6/36 cells for virus isolation. CPE was observed 4-day post-inoculation in diapausing and non-diapausing eggs. Both the PCR amplification results and virus isolation in C6/36 cells confirmed that WNV infected diapausing eggs (Table 2).

West Nile virus was detected in F1 generation larvae from diapausing eggs. An amplification product of the predicted size of 408 bp was evident in F1 generation larvae. RNA-positive samples were inoculated into C6/36 cells to isolate the virus. CPE was observed 6-day post-inoculation in F1 generation larvae. The results showed that WNV can be detected in the larvae hatched from diapausing eggs. The MIR of F1 generation larvae from diapausing eggs was 1:1180 in the second gonotrophic cycle and 1:1120 in the third gonotrophic cycle (Table 3). There were no significant differences in the MIR of F1 generation larvae between diapausing and non-diapausing eggs (1:960 and 1:1120, respectively; p > 0.05). There was no significant difference in the MIR in larvae between the second and third gonotrophic cycles (p > 0.05).