Vector competence of Culex tritaeniorhynchus for duck Tembusu virus

Abstract

Duck Tembusu virus (DTMUV), an emerging infectious disease in ducks, belongs to the Ntaya virus group of the Flavivirus genus in the Flaviviridae family. The emergence of DTMUV has been observed in layer and breeder duck farms in China since 2010 and in layer and broiler duck farms in Thailand since 2013. Infected ducks show neurologic signs, including an incapability to stand, ataxia, and paralysis. A significant drop in egg production is usually observed among layer ducks. The transmission of DTUMV involves mosquito vectors, however, the exact role of mosquitoes in the ecology of DTMUV in Thailand remains unclear. The first objective of this dissertation was to examine the mosquito distribution and their DTMUV detection status in four duck farms in central Thailand. Mosquitoes were collected from two duck farms in Sing Buri province and two duck farms in Ang Thong province from September 2015 to July 2016 using four CDC-light traps. A total of 30,841 mosquitoes were collected and identified to seven species (Anopheles (An.) barbirostris, An. stephensi, Culex (Cx.) gelidus, Cx. quinquefasciatus, Cx. tritaeniorhynchus, Mansonia (Ma.) annulifera and Ma. uniformis). The most common collected species from each duck farm and each collection time was Cx. tritaeniorhynchus. A total of 273 mosquito pools were examined, with only one pool of Cx. tritaeniorhynchus collected from Sing Buri province in November 2015 testing positive for DTMUV by reverse transcription polymerase chain reaction (RT-PCR). Phylogenetic analysis of the polyprotein gene sequence demonstrated that a mosquito-derived Thai DTMUV (Culex/TH/CU_2015) was grouped into subcluster 2.1. Therefore, the second objective of this dissertation was to examine the vector competence of Cx. tritaeniorhynchus and Cx. quinquafasciatus for DTMUV. Four groups of Cx. tritaeniorhynchus were allowed to feed on four levels of DTMUV which were 102, 103, 104, and 105 TCID50/mL and two groups of Cx. quinquefasciatus were allowed to feed on two levels of DTMUV which were 104 and 105 TCID50/mL. The results showed that DTMUV infection in Cx. tritaeniorhynchus was found from all groups. While DTMUV dissemination and transmission in Cx. tritaeniorhynchus were only found from the mosquitoes that fed on the blood meal with 105 TCID50/mL of DTMUV. DTMUV infection in Cx. quinquefasciatus were found from the mosquitoes that fed on the blood meal with 104 and 105 TCID50/mL of DTMUV; however, there was no virus dissemination and transmission found from all tested mosquitoes. The third objective of this dissertation was to study the transovarial transmission of DTMUV in Cx. tritaeniorhynchus. Cx. tritaeniorhynchus were allowed to feed on infected blood meal with 105 TCID50/mL of DTMUV. Each blood-fed mosquito was individually kept in a plastic cup with water to allow the mosquito to lay eggs. After egg-laying, the mosquitoes were tested for DTMUV infection by using RT-PCR. A total of 43 DTMUV infected and 37 non-infected female mosquitoes with eggs were included in this study. A total of 182 (75 male and 107 female) F1 mosquitoes from DTMUV infected mosquitoes and 145 (51 male and 94 female) F1 mosquitoes from non-infected mosquitoes were tested for DTMUV infection; however, all of them were negative for DTMUV. The findings from this dissertation indicated the vector competence of Cx. tritaeniorhynchus for DTMUV and possible role as major vectors in the transmission cycle of DTMUV in Thailand. However, there was no transovarial transmission of DTMUV in Cx. tritaeniorhynchus.