SARS-CoV-2 wastewater monitoring in rural and small metropolitan communities in Central Michigan.

Central Michigan University (CMU) participated in a state-wide SARS-CoV-2 wastewater monitoring program throughout the 2021-2022 academic year. Wastewater samples were collected weekly from ten on-campus sites and nine off-campus wastewater treatment plants servicing small metropolitan and rural communities. SARS-CoV-2 genome copies were quantified using droplet digital PCR. Case data reported by Central Michigan District Health Department and CMU were collected and compared with wastewater data. During the delta wave, wastewater detection and on-campus case reports increased rapidly with the start of the academic semester and peaked quickly, compared with a more gradual and prolonged increase in detection and case reports off-campus. During the omicron wave, transmission dynamics were similar on-campus and off-campus. Normalization of on-campus and off-campus wastewater data with pepper mild mottle virus gene expression suggested lower SARS-CoV-2 shedding per person in on-campus compared to off-campus samples during the delta wave, but no difference in virus shedding during the omicron wave. We discuss the possibility that a higher on-campus vaccination rate may have reduced virus shedding per person during the delta wave, but that this effect was lost with the omicron variant. This study suggests that wastewater monitoring is effective in rural and small metropolitan communities when used in conjunction with case reports to understand regional transmission dynamics and the impact of public health policies at a public university on virus shedding in the community.

Chronic depletion of vertebrate lipids in Aedes aegypti cells dysregulates lipid metabolism and inhibits innate immunity without altering dengue infectivity.

Aedes aegypti is the primary vector of dengue virus (DENV) and other arboviruses. Previous literature suggests that vertebrate and invertebrate lipids and the nutritional status of mosquitoes modify virus infection. Here, we developed a vertebrate lipid-depleted Ae. aegypti cell line to investigate if chronic depletion of vertebrate lipids normally present in a blood meal and insect cell culture medium would impact cell growth and virus infection. Chronic depletion of vertebrate lipids reduced cell size and proliferation, although cells retained equivalent total intracellular lipids per cell by reducing lipolysis and modifying gene expression related to sugar and lipid metabolism. Downregulation of innate immunity genes was also observed. We hypothesized that chronic depletion of vertebrate lipids would impact virus infection; however, the same amount of DENV was produced per cell. This study reveals how Ae. aegypti cells adapt in the absence of vertebrate lipids, and how DENV can replicate equally well in cells that contain predominately vertebrate or invertebrate lipids.

Extracellular vesicles restrict dengue virus fusion in Aedes aegypti cells.

Aedes aegypti is the primary vector of dengue virus (DENV), and acquires this virus from a vertebrate host during blood feeding. Previous literature has shown that vertebrate blood factors such as complement protein C5a and low-density lipoprotein (LDL) influence DENV acquisition in the mosquito. Here, we show that extracellular vesicles in cell culture medium inhibit DENV infection in mosquito cells. Specifically, extracellular vesicles enter into mosquito cells and inhibit an early stage of infection. Extracellular vesicles had no effect on virus cell attachment or entry. Instead, extracellular vesicles restricted virus membrane fusion. Extracellular vesicles only inhibited DENV infection in mosquito cells and not vertebrate cells. These data highlight a novel virus-vector-host interaction that limits virus infection in mosquito cells by restricting virus membrane fusion.

Validamycin A Delays Development and Prevents Flight in Aedes aegypti (Diptera: Culicidae).

Trehalose is a disaccharide that is the major sugar found in insect hemolymph fluid. Trehalose provides energy, and promotes growth, metamorphosis, stress recovery, chitin synthesis, and insect flight. The hydrolysis of trehalose is under the enzymatic control of the enzyme trehalase. Trehalase is critical to the role of trehalose in insect physiology, and is required for the regulation of metabolism and glucose generation. Trehalase inhibitors represent a novel class of insecticides that have not been fully developed. Here, we tested the ability of trehalose analogues to function as larvacides or adulticides in an important disease vector-Aedes aegypti. We show that validamycin A, but not 5-thiotrehalose, delays larval and pupal development and prevents flight of adult mosquitoes. Larval mosquitoes treated with validamycin A were hypoglycemic and pupae had increased levels of trehalose. Treatment also skewed the sex ratio toward male mosquitoes. These data reveal that validamycin A is a mosquito adulticide that can impair normal development of an important disease vector.