Impact of temperature on vector competence of Culex pipiens molestus: implications for Usutu virus transmission in temperate regions

Abstract

IntroductionUsutu virus (USUV) has been detected annually in the southeast of England since 2020. USUV RNA has been identified in wild birds and mosquito populations, and exposure of captive birds to USUV at Zoological Society of London (ZSL) has also been confirmed. Since its first detection in London, USUVs distribution has expanded across the South East, highlighting a need to understand the transmission dynamics of this virus in the UK. The primary vectors of USUV in the UK are likely Culex pipiens mosquitoes. Two biotypes have been identified, the bird-feeding Cx. pipiens pipiens and Cx. pipiens molestus which shows no restriction in host preference. The latter may play an important role in transmitting USUV from birds to humans. MethodsA laboratory colony of Cx. pipiens molestus mosquitoes were orally infected with the London strain of USUV and, incubated at 22 {degrees}C, 20 {degrees}C and 18{degrees} C for up to 28 days. Body samples and mosquito saliva samples were collected and analysed using a quantitative real-time reverse transcription PCR to determine infection and transmission potential, respectively. ResultsUSUV RNA was detected in all sample times at all temperatures assessed; with the highest temperature (22 {degrees}C) showing the greatest proportion of saliva and body positive samples. At this temperature, there was also an eight-fold increase in the relative viral copy number in the mosquito bodies, which was unobserved at other experimental temperatures. When a more sensitive PCR assay was used at the lowest experimental temperature used (18 {degrees}C) USUV RNA was present in the mosquito saliva and body samples for longer and showed a greater proportion of positive samples when compared to 20 {degrees}C. ConclusionThis study has demonstrated that Cx. pipiens molestus may be able to transmit USUV at 22 {degrees}C. Active replication of USUV was identified in the mosquito bodies at 22 {degrees}C but could not be demonstrated at lower temperatures, suggesting that 20 {degrees}C to 22 {degrees}C may be an important temperature threshold in USUV replication and transmission. Utilisation of a more sensitive assay for the lower experimental temperatures revealed that USUV was detectable at 18 {degrees}C. Therefore, when conducting infection studies on temperate mosquito-borne viruses, it is important to consider assay sensitivity.