Competence of mosquitoes native to the United Kingdom to support replication and transmission of Rift Valley fever virus

Abstract

Background: Rift Valley fever phlebovirus (RVFV) is a mosquito-borne arbovirus causing severe disease in humans and livestock. It is endemic in Africa and spread to the Arabian Peninsula in 2000 raising concerns it could emerge in Europe. The ability of temperate mosquitoes from the United Kingdom (UK) to support replication and transmission of RVFV is unknown.

Methods: In this study, two colonised lines of Culex pipiens, wild-caught Aedes detritus and Ae. rusticus from the UK were infected with pathogenic strains of RVFV to assess their vector competence. Mosquitoes were offered artificial blood-meals containing 10⁶ or 10⁷ plaque forming units (PFU)/ml RVFV, simulating natural peak viraemia in young ruminants, and maintained at 20 °C or 25 °C for up to 21 days. Bodies, legs and saliva were collected and tested for the presence of viral RNA and infectious virus to determine the infection, dissemination and transmission potential.

Results: Across temperatures, doses and strains the average infection, dissemination and transmission rates were: 35, 13 and 5% (n = 91) for Cx. pipiens (Caldbeck); 23, 14 and 5% (n = 138) for Cx. pipiens (Brookwood); 36, 28 and 7% (n = 118) for Ae. detritus. However, despite 35% (n = 20) being susceptible to infection, Ae. rusticus did not transmit RVFV. Survival of Aedes species was negatively affected by maintenance at 25 °C compared to the more representative peak average British summer temperature of 20 °C. Increased mortality was also observed with some species infected with 10⁷ PFU/ml compared to 10⁶ PFU/ml.

Conclusions: It can be concluded that temperate mosquito species present in the UK demonstrate a transmission potential for RVFV in the laboratory but, even at high temperatures, this occurred at low efficiency.

Keywords: Arbovirus; Mosquito; Rift Valley fever virus; UK; Vector competence.

Fig. 1

Timeline of events effecting mosquito viral transmission. a Schematic of a typical livestock viraemia profile based on experimental infection of ruminants reviewed by Golnar et al. [28]. Viraemia is affected by both age and species of the host, dose, strain and route of inoculum [1], the schematic is therefore representative not absolute. Dotted lines represent a high threshold set by Golnar et al. [28] and a low dose threshold required to infect mosquitoes based on the findings of Vloet et al. [44], the latter facilitates onward infection of the mosquito across a longer duration than the high infectious dose [1]. b Interplay of the extrinsic incubation period (EIP) and mosquito lifespan on transmission. The EIP is the time from virus ingestion to the first point the mosquito is capable of expectorating virus via its saliva during feeding. The shorter the EIP and longer the lifespan the higher the potential for transmission events to occur, both are affected by multiple factors including environment and species specific traits. Scenario 1: short EIP but long lifespan is optimal for a transmission event; Scenario 2: long EIP with a long lifespan; Scenario 3: short EIP with a short lifespan; Scenario 4: long EIP coupled with short a lifespan does not support transmission. Arrows represent a feeding event, with the arrowhead indicating the direction of virus transmission. Red arrows and red sheep: events support transmission; black arrows and black sheep: events do not support transmission

Fig. 2

Percentage survival of mosquitoes. a Effect of temperature on Ae. detritus survival. Mosquitos ingesting a blood meal containing at 10⁶ PFU/mL RVFV strain ZH501, Lunyo or an uninfected control were maintained at 20 or 25 °C. Survival did not differ statistically between strains or controls at 20 °C (Log-rank (Mantel-Cox) χ² = 1.174, df = 2, P = 0.5560) or at 25 °C (χ² = 0.9600, df = 2, P = 0.9600) so datasets were pooled for analysis, demonstrating reduced survival at 25 °C compared to 20 °C. The effects of viral dose on survival are presented in panels b-d comparing 10⁶ PFU/ml vs 10⁷ PFU/ml vs uninfected controls. b Cx. pipiens f. pipiens (Caldbeck). c Cx. pipiens hybrid (Brookwood). d Ae. detritus. Survival was monitored periodically for up to 21 days. Kaplan-Meier plots were generated in GraphPad Prism, bars represent the 95% confidence interval, the numbers tested are written within parentheses (n). Statistical differences were calculated by Log-Rank test with Bonferroni-corrected thresholds, dotted lines compare strain: Lunyo, solid lines: ZH501. Demonstrating reduced survival for Cx. pipiens Caldbeck and Ae. detritus after ingestion of blood containing 10⁷ PFU/ml compared to controls and their lower dose counterparts but no differences between doses or controls ingested by the Brookwood line mosquitoes

Fig. 3

Percentage of mosquitoes infected with Rift Valley fever virus. Mosquitoes were fed a blood-meal containing 10⁶ or 10⁷ PFU/ml Rift Valley fever virus strain Lunyo or ZH501 and maintained at 20 or 25 °C. Results are based on the proportion of mosquitoes positive for RVFV by qRT-PCR results. Day 0 was excluded from the totals. Numbers below bars represent the total number tested; error bars indicate 95% confidence intervals calculated by a modified Wald method. Fisher’s exact test was used to compare between time points, doses and virus strains using a Bonferroni-corrected threshold for multiple comparisons; comparisons between mosquito species are depicted by dotted bars, and comparison of doses by solid bars. Abbreviations: nt, not tested; CBK, Cx. pipiens (Caldbeck); BKW, Cx. pipiens (Brookwood); Ae.d, Ae. detritus; Ae.r, Ae. rusticus