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

Usutu 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 has also been confirmed in the UK. Since its first detection in London, USUV's distribution has expanded across the South East, highlighting necessity to understand USUV transmission dynamics in the UK. The primary vectors of USUV in the UK are likely Culex pipiens mosquitoes. Culex pipiens molestus is one biotype which shows no restriction in host preference and may play an important role in transmitting USUV from birds to humans.

A laboratory colony of Cx. pipiens molestus mosquitoes were orally infected with the London strain of USUV and incubated at 22 ℃, 20 ℃ and 18 ℃ 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.

USUV RNA was detected in all sample times at all temperatures assessed, with the 22 ℃ 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 ℃), USUV RNA was present in the mosquito saliva and body samples for longer and showed a greater proportion of positive samples compared to 20 ℃.

This study has demonstrated that Cx. pipiens molestus may be able to transmit USUV at 22 ℃. Active replication of USUV was identified in the mosquito bodies at 22 ℃ but not at lower temperatures, suggesting that 20 ℃ to 22 ℃ may be an important threshold in USUV replication and transmission. Utilisation of a more sensitive assay for the lower experimental temperatures revealed that USUV was detectable at 18 ℃. Therefore, when conducting infection studies on temperate mosquito-borne viruses, it is important to consider assay sensitivity.