Virus provenance and propagation
BATV (strain 5.3) was isolated in Germany from An. maculipennis s.l. mosquitoes [8]. All following procedures were carried out in a dedicated biosafety level 3 laboratory. BATV was propagated and titrated in Vero cells using a previously described protocol [22]. This resulted in virus stocks maintained in Eagles minimum essential media of suitable concentrations which were kept in a -80oC freezer until required.
Mosquitoes and virus inoculation
Laboratory colonies of Ae. aegypti strain AEAE, West Africa, donated by the London School of Hygiene and Tropical Medicine, and Cx. pipiens strain Brookwood, UK (hybrid of forms pipiens and molestus), supplied by The Pirbright Institute, were maintained at 25oC on sucrose solution. Pupae Ae. detritus were caught from Dee Marsh, Cheshire (53o 16’39.48’’N, 3o 4’5.286’’W) and reared to adult stage similar to protocols described in [22, 23].
Three to five day old, unfed, adult females of each mosquito species were tested for the susceptibility to infection by oral challenge and the competency to vector BATV at 20oC. Prior to feeding, mosquitoes were transferred to an insect cage (22 x 22 x 22 cm, bugzaare.co.uk, Suffolk, UK) and starved of sucrose for 5 hours to stimulate feeding. Groups of mosquitoes were offered a blood meal containing defibrinated horse blood, adenosine 5’-triphosphate (final concentration 0.02 mM) and virus at a final concentration between 1.4 x 104 plaque-forming units (PFU)/mL and 5.5 x 106 PFU/mL (Table 1) through a membrane feeding system (Hemotek Ltd, Accrington, Lancashire, UK) and allowed to feed overnight. Following this, cages of mosquitoes were anaesthetized with trimethylamine (FlyNap®, Blades Biological Limited, Edenbridge, UK) and engorged mosquitoes separated from unfed individuals. Blood-fed mosquitoes were held in cages within an incubator set at 20oC for seven or fourteen days. At the designated time point, mosquitoes were caught using a battery-powered, hand-held aspirator and placed for two minutes, whilst in the aspirator, at -80oC to immobilise the specimens. They were then place on a surface chilled by ice to ensure they remained immobile during removal of legs/wings and saliva collection, bodies were retained, then RNA extracted as previously described [22]. A control group of Ae. detritus was provided with a blood-meal without a virus.
Table 1
Infection, dissemination and transmission rates of Ae. aegypti, Ae. detritus and Cx. pipiens following consumption of a blood meal containing BATV. Groups of mosquitoes were maintained at 20°C for the indicated time periods. Infection rate: number of positive mosquitoes/number of blood fed; Dissemination rate: number of mosquitoes with virus detected in legs/total number infected; Transmission rate: number of mosquitoes with virus detected in saliva/total number with disseminated infection.
Mosquito species | Blood-meal titre (In PFU) | Blood feeding rate (%) | Rate | DPI 7 (%) | DPI 14 (%) |
Aedes aegypti | 5.5 x 106 | 145/320 (45) | Infection | 4/16 (25) | 3/44 (7) |
| | | Dissemination | 0 | 0 |
| | | Transmission | 0 | 0 |
Aedes detritus | 1.4 x 104 | 80/112 (74) | Infection | 12/15 (80) | 13/16 (81.2) |
| | | Dissemination | 5/12 (41.6) | 4/13 (30.7) |
| | | Transmission | 5/5 (100) | 1/4 (24) |
Culex pipiens | 5.5 x 106 | 60/188 (32) | Infection | 1/15 (7) | 1/28 (4) |
| | | Dissemination | 0/1 (0) | 0/1 (0) |
| | | Transmission | 0 | 0 |
Molecular detection of Batai virus in bloodfed female mosquitoes
Batai virus RNA was detected using a semi-quantitative RT-PCR that targets a 99 bp sequence of the S segment using the primers BATV-Forward: 5’-GCTGGAA GGTTACT GTA TTTAAT AC-3’; BATV-Reverse: 5’-CAAGGAATCCACTGAGTCTGTG-3’; and BATV-Probe: 5’-FAM-AACAGTCCAGTTCCAGACG ATGGTC-BHQ-1-3’ [8]. Reactions were performed with iTaqTM Universal Probes One-Step Kit (Bio-Rad, UK) using the following reaction mix per microtube: RNase-free water (7 µl); 2x iTaq universal probes reaction mix (12 µl); 1 µl of each primer and probe at 10 pmol/µl; and 1 µl of iScript reverse transcriptase, and 2 µl of extracted RNA. Amplification was conducted using a MxPro 3005 thermal cycler (Agilent Technologies, US) using the following reaction conditions: reverse transcription 50°C for 10 min; reverse transcriptase inactivation 95°C for 5 min; and PCR amplification and detection 40 cycles consisting of 95°C for 10 sec, 55°C for 30 sec. Amplification files were visualised and analysed in MX3000p v4. Software (Agilent Technologies, US).