Eilat Virus Isolated from Culex Univittatus Mosquitoes from the Namibian Zambezi Region Influences in Vitro Superinfection with Alpha- and Flaviviruses in a Virus-species-dependent Manner

The genus Alphavirus harbors arboviruses of great concern, such as the Chikungunya virus and the equine encephalitis viruses. Transmission of pathogenic alphaviruses by mosquitoes could be influenced by insect-specific alphaviruses such as Eilat virus (EILV). However, insect-specific alphaviruses are rarely found in wild mosquitoes and only a few have been described in the literature. Here, we report the detection of EILV in a Culex univittatus mosquito from the north-eastern Namibian Zambezi region. Full genome analysis of MP458-NA-2018 showed 94.5% nucleotide identity to an EILV isolate from Israel. MP458-NA-2018 grouped with EILV in phylogenetic analysis and was placed within the clade of insect-specific alphaviruses. The virus was isolated in mosquito cells and shown to be restricted to insects as hosts by the inability to infect different vertebrate cell lines and a complete block of virus replication at 34°C. We further showed that infection of cells with EILV MP458-NA-2018 reduced production of infectious particles of Sindbis virus by 2000-fold over the entire course of infection, whereas reduction rates of Bagaza and Middleburg virus were approximately 3-10-fold and dependent on time after infection. While production of infectious particles of cells superinfected with the Chikungunya virus were approximately 30-fold reduced and more pronounced at a lower multiplicity of infection of 0.01, EILV seemed to enhance production of West Nile virus infectious particles by >5-fold when superinfected at a multiplicitiy of infection of 0.1. In summary, EILV from the Namibian Zambezi region influences in vitro replication of endemic flavi- and alphaviruses.