Co-infection of Culex tarsalis with Rift Valley fever phlebo-virus strains produces efficient segmental reassortment across midgut and salivary gland tissues

Authors: HARRIS, EMMA - Colorado State University; BALARAMAN, VELMURUGAN - Kansas State University; KEATING, CASSIDY - Colorado State University; MCDOWELL, CHESTER - Kansas State University; Kimble, James; De La Mota-Peynad, Alina; BORLAND, ERIN - Colorado State University; GRAHAM, BARBARA - Colorado State University; WILSON, WILLIAM - Retired ARS Employee; RICHT, JUERGEN - Kansas State University; KADING, REBEKAH - Colorado State University; GAUDREAULT, NATASHA - Kansas State University

Submitted to: Viruses
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/7/2025
Publication Date: 1/11/2025
Citation: Harris, E.K., Balaraman, V., Keating, C., Mcdowell, C., Kimble, J.B., De La Mota-Peynad, A.M., Borland, E., Graham, B., Wilson, W., Richt, J., Kading, R., Gaudreault, N. 2025. Co-infection of Culex tarsalis with Rift Valley fever phlebo-virus strains produces efficient segmental reassortment across midgut and salivary gland tissues. Viruses. 17(1):88. https://doi.org/10.3390/v17010088.
DOI: https://doi.org/10.3390/v17010088

Interpretive Summary: The mosquito-borne Rift Valley fever phlebovirus (RVFV) is endemic to sub-Saharan Africa and the Arabian Peninsula causes disease in ruminant livestock and humans. Co-infection of insect and mammalian cell types with divergent viral strains can produce reassorted genotypes. These novel genotypes may have altered virulence, transmission dynamics, and/or mosquito host range. This is especially of concern in RVFV endemic countries where multiple strains circulate. Previously, we evaluated the frequency of RVFV segmental exchange in a susceptible sheep host and observed low rates of reassortment (0-1.7%). Thus, we hypothesized that reassortment observed in the field instead occurs in the mosquito vector. Here, the reassortment frequency of RVFV was evaluated in a highly permissive mosquito vector. At three days post-infection approximately 2% of virus genotypes isolated from co-infected mosquito cell cultures represented reassorted genotypes. We further examined reassortment among these co-infecting strains in blood-fed mosquitoes. At 14 days post-exposure, 2-60% of plaques isolated from midgut and salivary tissue represented reassortment genotypes. The results suggest that mosquitoes play a crucial role in the reassortment of RVFV and potentially contribute to driving the evolution of the virus.

Technical Abstract: Rift Valley fever phlebovirus (RVFV) is a zoonotic mosquito-borne pathogen endemic to sub-Saharan Africa and the Arabian Peninsula which causes Rift Valley fever in ruminant livestock and humans. Co-infection of insect and mammalian cell types with divergent viral strains can produce segmental reassortment of the tri-segmented (L, M and S segment) RVFV genome. Reassortment events can produce novel genotypes with altered virulence, transmission dynamics, and/or mosquito host range. This is especially of concern in RVFV endemic countries where multiple strains circulate. Previously, we evaluated the frequency of RVFV segmental exchange in a susceptible sheep host and observed low rates of reassortment (0-1.7%). Thus, we hypothesized that reassortment observed in the field instead occurs in the mosquito vector. Here, the reassortment frequency of RVFV was evaluated in a highly permissive vector, Culex tarsalis. Cells derived from Cx. tarsalis were co-infected with either two virulent (Kenya-128B-15 and SA01-1322)or a virulent and attenuated (Kenya-128B-15 and MP-12) strain of RVFV. At three days post-infection approximately 2% of virus genotypes isolated represented reassortants. We further examined reassortment among these co-infecting strains in blood-fed Cx. tarsalis. At 14 days post-exposure, 2-60% of plaques isolated from midgut and salivary tissue represented reassortment genotypes. Reassortant genotypes isolated from the midguts of mosquitoes co-infected with both Kenya-128B-15 and SA01-1322 were similar to that of mosquitoes co-infected with Kenya-128B-15 and MP-12- strains (51.0 vs. 45.5%). However, only approximately 2% of virus isolated from salivary glands of Kenya-128B-15 and SA01-1322 co-infected mosquitoes represented reassortment geno-types. This was contrasted by 54% reassortment in the salivary glands of mosquitoes co-infected with Kenya-128B-15 and MP-12 strains. We observed a preferential inclusion of specific genomic segments from the three parental strains among the reassorted viruses. Replication curves of select reassorted genotypes were significantly higher in Vero cells but not in Culex—derived cells. These data imply that mosquitoes play a crucial role in the reassortment of RVFV and potentially con-tribute to driving the evolution of the virus.