Vector Competence of field-collected and laboratory reared floodwater mosquitoes for West Nile virus Lineages 1 and 2

West Nile virus (WNv) is a zoonotic arbovirus (family Flaviviridae, genus Flavivirus) that has recently established an autochthone transmission cycle in Germany. In its natural enzootic transmission cycle WNv is maintained between birds and ornithophilic mosquitoes. Bridge vector species might transmit WNv to other vertebrate host species, including dead-end hosts such as humans and horses, in which the WNv infection may cause severe central nerve system symptoms. The ability to transmit a pathogen may vary between different mosquito populations of the same species. Several mosquito species, widely distributed in Germany, can act as bridge vectors like members of the Culex pipiens complex and Aedes spp.. However, the vector competence of northern German mosquito populations to transmit WNv is largely unknown until now. Therefore, the aim of this study was to investigate the vector competence of indigenous mosquito populations of Aedes cinereus/Ae. geminus and Cx. pipiens biotype pipiens collected from north-eastern Germany (federal state of Mecklenburg-Western Pomerania) in addition to laboratory reared floodwater mosquito species Ae. vexans Green River (GR) for WNv lineage 1 and 2 under temperatures typical for spring/ autumn respectively summer in northern Germany. Long-term cultivation and immortalization of cells might interfere with the interactions between cell and pathogen. Furthermore, high prerequisites (facilities, expertise) need to be fulfilled for the in vivo investigation of virus-vector interactions. This indicates the need for alternative models to investigate virus-vector interactions. For which reason, a protocol for the generation of primary mosquito cells was also established. For assessment of vector competence under BSL3 conditions, 7-14 day old female mosquitoes were offered a blood meal containing either WNv lineage 1 (strain: Magpie/ Italy/ 203204) or WNv lineage 2 (strain: ‘Österreich’), via artificial membrane feeding Hemotek or cotton-stick feeding. After incubation at 18 °C respectively 24 °C for 14 days fully engorged female mosquitoes were salivated and dissected in order to determine dissemination and transmission rates by reverse transcriptase quantitative real-time PCR (RT-qPCR) and cell culture multiplication. For the establishment of the primary cell cultures, mosquito larvae in L1 stage were collected ~24 h after hatching and washed in 70 % ethanol, 0.115 % sodium hypochlorite, altered Whites solution, Rinaldinis solution and initiation medium supplemented with antibiotics. Cells were mechanically isolated and incubated at 26 °C and 2.5 % CO2. In field collections between April and September 2018 Ae. cinereus/Ae. geminus mosquitoes were found in four and Cx. pipiens biotype pipiens in one of 12 evaluated locations in Mecklenburg-Western Pomerania. During in vivo vector competence assessment, Ae. vexans GR laboratory colony mosquitoes displayed overall feeding rates of 16.0 % (87/545) when fed via artificial membrane feeding Hemotek, while this was the case for 7.8 % (4/51) Ae. cinereus/Ae. geminus mosquitoes. When fed with WNv spiked blood via artificial membrane feeding 3.4 % (17/499) of Cx. pipiens biotype pipiens mosquitoes took a bloodmeal. The change of the feeding method to cotton sticks lead to increased feeding rates (20.1 %; 32/145). The determination of dissemination rates 14 dpi after incubation at 24 °C with WNv lineage 1 (Magpie/ Italy/ 203204) showed 1/13 (7.7 %) in Ae. vexans GR mosquitoes, 1/9 (11.1 %) in Cx. pipiens biotype pipiens and 0/3 (0 %) Ae. cinereus/Ae. geminus mosquitoes. After salivation assay, WNv lineage 1 was detected in saliva of 1/9 (11.1 %) Cx. pipiens biotype pipiens by RT-qPCR. Disseminated infections with WNv lineage 2 ‘Österreich’ were detected in 1/7 (14.3 %) Ae. vexans GR mosquitoes after incubation at 18 °C, while none was detected after incubation at 24 °C (0/9). Based on these observations the Ae. vexans GR laboratory colony probably needs prolonged extrinsic incubation period (more than standard 14 dpi) under the given temperatures of 18 °C and 24 °C. Due to the small number of Ae. cinereus/Ae. geminus mosquitoes available in trials, further experiments are required to allow a statement regarding the vector competence of this northern German mosquito species. Since WNv lineage 1 was detected in saliva of Cx. pipiens biotype pipiens mosquitoes after incubation for 14 days at 24 °C, this northern German mosquito population may be a competent vector for WNv lineage 1 under the given conditions. Nevertheless, a reliable statement regarding the transmission efficiency is not possible at the current time point. Primary cells generated from different mosquito species (Ae. vexans, Cx. pipiens), showed bacterial contamination after the isolation process. Modifications of the isolation protocol and tested solutions reduced the bacterial contamination, which was further depleted by usage of antibiotics and mechanical washing.