key: cord-0838170-s2vdazq0 authors: Huang, Yan-Jang S.; Vanlandingham, Dana L.; Bilyeu, Ashley N.; Sharp, Haelea M.; Hettenbach, Susan M.; Higgs, Stephen title: SARS-CoV-2 failure to infect or replicate in mosquitoes: an extreme challenge date: 2020-07-17 journal: Sci Rep DOI: 10.1038/s41598-020-68882-7 sha: f2f2a8c1b50c0557d96621e4215a3117d6e2f2fc doc_id: 838170 cord_uid: s2vdazq0 This research addresses public speculation that SARS-CoV-2 might be transmitted by mosquitoes. The World Health Organization has stated “To date there has been no information nor evidence to suggest that the new coronavirus could be transmitted by mosquitoes”. Here we provide the first experimental data to investigate the capacity of SARS-CoV-2 to infect and be transmitted by mosquitoes. Three widely distributed species of mosquito; Aedes aegypti, Ae. albopictus and Culex quinquefasciatus, representing the two most significant genera of arbovirus vectors that infect people, were tested. We demonstrate that even under extreme conditions, SARS-CoV-2 virus is unable to replicate in these mosquitoes and therefore cannot be transmitted to people even in the unlikely event that a mosquito fed upon a viremic host. www.nature.com/scientificreports/ from various arthropods 14, 15 , no virus in the family has been isolated from mosquitoes. To date, only one report related to epidemic coronaviruses and mosquitoes has been published 16 . This study that 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. By analyzing samples using in vitro cultivation, rather than using molecular approaches, we focused specifically on detection of infectious virus rather than on RNA. As illustrated by, for example, the use of inactivation techniques specifically developed to enable safe handling and shipping of viral material, the mere presence of RNA does not mean that any infectious virus is actually present. It is well known that viral RNA can be detected in mosquitoes simply because they have fed on a viremic host, and so RNA detection should never be interpreted as proof of mosquito susceptibility to infection and competence to transmit the virus. 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 were recovered from 13/15 mosquitoes collected within two hours of inoculation. It is possible, that in the two negative mosquitoes, the inoculated virus lost infectivity during the holding period. No virus was detected in the 277 inoculated mosquitoes collected and titrated at time points beyond 24 h, suggesting a rapid loss of infectivity and the lack of replication after injection. From a total of 48 mosquitoes analyzed, infectious viruses were only recovered from one Ae. albopictus collected at 24 h post-inoculation. The quantity of infectious virus in this mosquito corresponded to the amount of inocula, producing infectious titers at approximately 1.5 logTCID 50 /ml. No virus was detected in control L-15 medium inoculated mosquitoes. Collectively, our findings suggest 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. ). The most extreme approach for viral challenge of mosquitoes, namely intrathoracic inoculation, was used as an ultimate test of the capacity of SARS-CoV-2 to infect and replicate in mosquitoes. The hypothesis was that if the virus did not replicate in mosquitoes after intrathoracic inoculation, then even if mosquitoes did feed on viremic people, and the virus disseminated from the midgut, the lack of replication would preclude the possibility of biological transmission. Three widely distributed species of mosquito, representing the two most significant genera of arbovirus vectors that infect people, were tested. All three of the species: Aedes aegypti, Ae. albopictus, and Culex quinquefasciatus are present in China, the country of origin of SARS-CoV-2. Samples collected within two hours of inoculation confirmed efficient delivery of infectious viruses to mosquitoes. Based upon the lack of detectable infectious virus in any of the 277 samples collected at all time points beyond 24 h post-inoculation, we conclude that SARS-CoV-2 is unable to replicate in mosquitoes and that even if a mosquito fed on a person with virus in the blood, that the mosquito would not be a vector if feeding on a naïve host. Virus: SARS-CoV-2 virus WA1/2020 strain was obtained from BEI Resources (Catalog # NR-52281). Virus was propagated in Vero76 cells at the approximate multiplicity of infection of 0.01. Using serial tenfold dilutions in 96-well plates 17 , infectious titers of viral stocks used for intrathoracic injection were approximately 5.5 logTCID 50 /ml. Mosquitoes: The colonized Aedes aegypti strain Rex D, Higgs white eye was originally obtained from Puerto Rico 18 , Ae. albopictus generation F11 originated from New Jersey, and Culex quinquefasciatus F43 were from Florida 19, 20 . All mosquitoes were reared at 28 °C, relative humidity of 80% and a 12 h light:12 h dark photoperiod. These colonized mosquitoes have proven to be susceptible to several arboviruses 19, [21] [22] [23] [24] [25] [26] . Viral challenge of mosquitoes: For intrathoracic inoculation 9 , mosquitoes were cold-anaesthetized on ice, transferred to a secure glove box, and then inoculated with approximately 0.5 µl of viral stock. It was anticipated that each mosquito received approximately 2.0 logTCID 50 /ml of infectious viruses. L-15 medium was inoculated Table 1 . Recovery rates of SARS-CoV-2 in mosquitoes receiving intrathoracic injection. * Mosquitoes obtained at day 0 post infection were collected within 2 h from the time of intrathoracic injection. ** All mock control groups of mosquitoes received Leibovitz's L-15 media. www.nature.com/scientificreports/ as a negative control. 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Vector Borne Zoo Differential outcomes of Zika virus infection in Aedes aegypti orally challenged with infectious blood meals and infectious protein meals Culex tarsalis is a competent vector species for Cache Valley virus Infection and transmission of Cache Valley virus by Aedes albopictus and Aedes aegypti mosquitoes A single mutation in chikungunya virus affects vector specificity and epidemic potential Evaluation of simultaneous transmission of chikungunya virus and dengue virus type 2 in infected Aedes aegypti and Aedes albopictus (Diptera: Culicidae) North American Culex pipiens and Culex quinquefasciatus are competent vectors for Usutu virus /scientificreports/ Reprints and permissions information is available at www.nature.com/reprints This work was performed in the ACL-3 insectary at Kansas State University's Biosecurity Research Institute. The research was in part supported by the State of Kansas National Bio and Agro-defense Facility (NBAF) Transition Fund. 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