SARS-CoV-2 and mosquitoes: an extreme challenge

Yan-Jang S. Huang Biosecurity Research Institute and Department of Diagnostic Medicine and Pathobiology, Kansas State University https://orcid.org/0000-0002-9687-9341 Dana L. Vanlandingham Biosecurity Research Institute and Department of Diagnostic Medicine and Pathobiology, Kansas State University Ashley N. Bilyeu Biosecurity Research Institute and Department of Diagnostic Medicine and Pathobiology, Kansas State University Haelea M. Sharp Biosecurity Research Institute and Department of Diagnostic Medicine and Pathobiology, Kansas State University Susan M. Hettenbach Biosecurity Research Institute, Kansas State University Stephen Higgs (  shiggs@bri.ksu.edu ) Biosecurity Research Institute and Department of Diagnostic Medicine and Pathobiology, Kansas State University


Introduction
The question has been asked as to whether or not SARS-CoV-2, the causative agent of COVID-19, can infect and be transmitted by mosquitoes. The WHO has de nitively stated that mosquitoes cannot transmit the virus 3 , and in interviews, various experts have unanimously and de nitively also dispelled the suggestion that SARS-CoV-2 could be transmitted by mosquitoes. The presumption may be based on various observations and facts extrapolated from other coronaviruses. For example, neither the closely related SARS-CoV nor MERS produce the level of virus in the blood that for typical arthropod-borne viruses such as dengue and yellow fever viruses, would be regarded as high enough to infect mosquitoes.
Recent studies with infected humans and non-human primates infected with SARS-CoV-2, found no detectable virus in peripheral blood 4,5 . Lack of viremia is also suggested by the fact that neither SARS-COV nor MERS infections have resulted from blood transfusions or organ transplantations. Despite the lack of detectable viremia, experiments to determine the potential role of mosquitoes in SARS-CoV-2 transmission, are necessary because previous experiments have demonstrated that mosquitoes may become infected with viruses even when exposed to levels of infectious virus that are below the level of detection [6][7][8] .
To be a biological vector of viruses, mosquitoes must take up su cient virus to infect midgut epithelial cells, and the virus must then disseminate to infect other organs in the hemocoel, notably the salivary glands. Overcoming the midgut infection and escape barriers is essential for a virus to be transmissible by mosquitoes. If these barriers are bypassed by direct inoculation of virus into the hemocoel, then even non-susceptible mosquitoes can be infected. Inoculation can accomplish short-term infection of insects that could never be naturally infected because they do not feed on blood. These include not only nonhematophagous mosquitoes such as Toxorhynchites spp, but also male mosquitoes and even beetles and butter ies 9,10 .
Like the over 500 viruses that are transmitted by arthropods 11 , with the exception of African swine fever virus, coronaviruses have an RNA genome. In spite of the recovery of coronavirus or coronavirus-like agents from various arthropods 12,13 , no virus in the family has been isolated from mosquitoes. To date, only one report related to epidemic coronaviruses and mosquitoes has been published 14 . This study evaluated the potential use of mosquitoes for surveillance, included feeding of MERS virus to Anopheles gambiae mosquitoes. Residual viral RNA, probably in the remains of the bloodmeal in the midgut, was detected up to 1 day post-feeding. Similarly, positive PCR detection was observed for Bacillus anthracis, Trypanosoma brucei gambiensis, and Zika virus, none of which infect or are transmitted by An. gambiae. Levels of detected RNA were equal to or below the input level, indicating a lack of replication.

Results And Discussion
In this study, the susceptibility of three mosquito species, Ae. aegypti, Ae. albopictus and Cx. quinquefasciatus, were determined through the intrathoracic inoculation with SARS-CoV-2. Infectious viruses could only be recovered from mosquitoes collected within two hours of inoculation. Quantities of infectious viruses corresponded to the amount of inocula, producing infectious titers at approximately 1.5 logTCID 50 /ml and suggesting the lack of replication after injection. Waning of infectious titers was rapid and led to the lack of infectious viruses isolated from the majority of mosquitoes obtained between one and fourteen days post challenge suggests that mosquitoes in the Aedes and Culex genera are refractory to SARS-CoV-2 and unlikely to contribute to viral maintenance and transmission in nature (Table 1.).   16,17 . All mosquitoes were reared as previously described and have proven to be susceptible to several arboviruses. Viral challenge of mosquitoes: For intrathoracic inoculation, mosquitoes were cold-anaesthetized on ice, transferred to a secure glove box, and then inoculated with approximately 0.5µl of viral stock. It was anticipated that each mosquito received approximately 2.0 logTCID 50 /ml of infectious viruses. L-15 medium was inoculated as a negative control. The results were compiled from two experiments using Ae. aegypti and Ae. albopictus and one single experiment using for Cx. quinquefasciatus. Experimentally challenged mosquitoes were maintained and sampled under conditions as previously described 18 .
Declarations Data Availability Statement: The data that support the nding of this study are available in the table of this manuscript.