Unravelling the factors behind differences in Sindbis virus load in Aedes aegypti after oral infection

Abstract

Arbovirus transmission to their vertebrate hosts by mosquito vectors continues to linger on, causing major public health concerns. One of the reasons why mosquitoes that transmit arboviruses persist is due to failure of robust vector control strategies to limit or eradicate mosquitoes of public health importance. The shortfall of effective vector control strategies is due to insufficient understanding of what factors contribute to mosquito vector competence. The arbovirus load, or the amount of infectious virus in a mosquito, has been reported to affect vector competence. Related to this, after oral infection of inbred mosquitoes there is a high level of variation in virus load observed among individual mosquitoes. Unfortunately, there have been few studies conducted to understand what factors contribute to this titer variation. For this reason, Sindbis virus (SINV) was used as a model virus to better understand the factors contributing to variation in virus load among individual Aedes aegypti mosquitoes after oral infection. To achieve this goal, two specific aims were established. In Aim 1, the biological factors associated with variability in virus load in Ae. aegypti after SINV oral infection were investigated, while in Aim 2, the virus factors that contribute to variation in virus load after oral infection were examined. To address the first aim, highly inbred mosquitoes were reared and infected with SINV. The amount of virus load in the mosquitoes was determined and the results were analyzed to ascertain the biological factors contributing to titer variation. Results from this study showed that higher titer variation is observed after oral infection compared to intrathoracic infection, suggesting that the midgut is an important tissue barrier introducing variation in virus load. Additionally, decreased titer variation was observed with increased incubation period. This suggests that Ae. aegypti increasingly resist SINV infection over time following virus exposure. Biological factors such as mosquito age, mosquito strain, virus genotype, mosquito weight, amount of blood mosquito ingested, time after adult mosquito emergence, and the batch of eggs used to produce adult mosquitoes did not affect titer variation. Furthermore, virus collected from high titer mosquitoes replicated better in cultured cells compared to virus from low titer mosquitoes, suggesting there may be genetic differences between these virus populations. To address the second aim, the virus populations from individual low and high titer infected mosquitoes were sequenced, and the amount of sequence variation among the different populations was analyzed. Results from this study showed that there was a significant reduction in sequence variation when SINV moved from midguts to carcasses of low titer mosquitoes but not in high titer mosquitoes, suggesting that the midgut escape barrier is stronger in low titer mosquitoes compared to high titer mosquitoes. Also, carcasses of high titer mosquitoes had significantly higher population diversity and complexity than carcasses of low titer mosquitoes. Stronger purifying selection was also observed in the midguts and carcasses of low titer mosquitoes compared to high titer mosquitoes. Together these findings suggest that the virus populations present in low titer mosquitoes are less fit than those found in high titer mosquitoes, possibly due to genetic founder effect. Surprisingly, when the amount of SINV genomes was determined in the low and high titer mosquitoes it was found that the genome levels did not vary significantly, despite the large difference in infectious virus load. Additional analyses showed that high titer mosquitoes had significantly greater specific infectivity, or the ratio of infectious particles to viral genomes, than low titer mosquitoes. Low titer mosquitoes also had less viral protein translation and virion assembly than viruses from high titer mosquitoes. This could possibly contribute to the lower specific infectivity in low titer mosquitoes than high titer mosquitoes. Finally, immune suppression by antibiotic treatment caused an increase in specific infectivity, indicating that these differences were immune-related. Altogether, this dissertation has contributed fundamental knowledge towards understanding the factors contributing to arbovirus titer in mosquitoes, which is driven by tissue barriers to infection and dissemination and immune-related differences in specific infectivity of SINV in the carcasses of mosquitoes.