Comparative Usutu and West Nile virus transmission potential by local Culex pipiens mosquitoes in north-western Europe

Abstract

Originating from Africa, Usutu virus (USUV) first emerged in Europe in 2001. This mosquito-borne flavivirus caused high mortality rates in its bird reservoirs, which strongly resembled the introduction of West Nile virus (WNV) in 1999 in the United States. Mosquitoes infected with USUV incidentally transmit the virus to other vertebrates, including humans, which can result in neuroinvasive disease. USUV and WNV co-circulate in parts of southern Europe, but the distribution of USUV extends into central and northwestern Europe. In the field, both viruses have been detected in the northern house mosquito Culex pipiens, of which the potential for USUV transmission is unknown. To understand the transmission dynamics and assess the potential spread of USUV, we determined the vector competence of C. pipiens for USUV and compared it with the well characterized WNV. We show for the first time that northwestern European mosquitoes are highly effective vectors for USUV, with infection rates of 11% at 18 °C and 53% at 23 °C, which are comparable with values obtained for WNV. Interestingly, at a high temperature of 28 °C, mosquitoes became more effectively infected with USUV (90%) than with WNV (58%), which could be attributed to barriers in the mosquito midgut. Small RNA deep sequencing of infected mosquitoes showed for both viruses a strong bias for 21-nucleotide small interfering (si)RNAs, which map across the entire viral genome both on the sense and antisense strand. No evidence for viral PIWI-associated RNA (piRNA) was found, suggesting that the siRNA pathway is the major small RNA pathway that targets USUV and WNV infection in C. pipiens mosquitoes.

Keywords: Antiviral RNAi; Culex pipiens; Mosquitoes; Transmission; Usutu virus; West Nile virus.

Graphical abstract

Fig. 1

Culex pipiens is a highly competent vector for USUV. Mosquitoes were offered an infectious blood meal (A) or were injected (B) with either WNV or USUV. Fourteen days post infection mosquito saliva was collected and the mosquito body was homogenized in a cell culture medium. The mosquito homogenate and saliva were incubated on Vero E6 cells to detect the presence of either WNV or USUV in the mosquito bodies and saliva. Bars represent the percentage of positive samples. Asterisk indicates a significant difference (Fisher's exact test, P < 0.05).

Fig. 2

USUV and WNV replicate to equally high titers in Culex pipiens after an infectious blood meal but not after injection of the viruses. Homogenates from infected mosquitoes were used in end point dilution assays and the tissue culture infectious dose 50% (TCID₅₀)/ml was determined. Data points represent individual mosquitoes infected with either USUV or WNV via the indicated route. Lines show the mean and whiskers the standard error of the mean. Asterisk indicates a significant difference (t-test, P < 0.05).

Fig. 3

RNAi activity in WNV and USUV infected mosquitoes. (A) Size profile of the small RNAs mapping to the genome of WNV (left panel) or USUV (right panel). Reads mapping to the positive viral RNA strand (black) are shown above and reads mapping to the negative strand (gray) below the x-axes. (B) Genome distribution of 21 nucleotides vsiRNAs across the WNV (left panel) or USUV (right panel) genomes. Reads that map the positive strands are depicted in red, reads that map to the negative strands are depicted in blue. The number of small RNA reads in A and B was normalized against the total size of the library and is displayed as the percentage of the library.

Fig. 4

r. After infectious blood meals, engorged mosquitoes were incubated at three different temperatures for fourteen days, before determining the presence of WNV and USUV. Data points indicate the percentage of infected mosquitoes from the total sample size (n > 25). Asterisks indicate significant differences between the temperatures (*P < 0.001, Fisher's exact test, for both WNV and USUV) and between WNV and USUV at 28 °C (**P < 0.01, Fisher's exact test).