Development of an in vitro model for animal species susceptibility to SARS-CoV-2 replication based on expression of ACE2 and TMPRSS2 in avian cells

ABSTRACTThe SARS-CoV-2 (SC2) virus has caused a worldwide pandemic because of the virus’s ability to transmit efficiently human-to-human. A key determinant of infection is the attachment of the viral spike protein to the host receptor angiotensin-converting enzyme 2 (ACE2). Because of the presumed zoonotic origin of SC2, there is no practical way to assess every species susceptibility to SC2 by direct challenge studies. In an effort to have a better predictive model of animal host susceptibility to SC2, we expressed the ACE2 and/or transmembrane serine protease 2 (TMPRSS2) genes from humans and other animal species in the avian fibroblast cell line, DF1, that is not permissive to infection. We demonstrated that expression of both human ACE2 and TMPRSS2 genes is necessary to support SC2 infection and replication in DF1 and a non-permissive sub-lineage of MDCK cells. Titers of SC2 in these cell lines were comparable to those observed in control Vero cells. To further test the model, we developed seven additional transgenic cell lines expressing the ACE2 and TMPRSS2 derived from Felis (cat), Equus (horse), Sus (pig), Capra (goat), Mesocricetus (Golden hamster), Myotis lucifugus (Little Brown bat) and Hipposideros armiger (Great Roundleaf bat) in DF1 cells. Results demonstrate permissive replication of SC2 in cat, Golden hamster, and goat species, but not pig or horse, which correlated with the results of reported challenge studies. The development of this cell culture model allows for more efficient testing of the potential susceptibility of many different animal species for SC2 and emerging variant viruses.IMPORTANCESARS-CoV-2 (SC2) is believed to have originated in animal species and jumped into humans where it has produced the greatest viral pandemic of our time. Identification of animal species susceptible to SC2 infection would provide information on potential zoonotic reservoirs, and transmission potential at the human-animal interface. Our work provides a model system to test the ability of the virus to replicate in an otherwise non-permissive cell line by transgenic insertion of the ACE2 and TMPRSS2 genes from human and other animal species. The results from our in vitro model positively correlate with animal infection studies enhancing the predicative capability of the model. Importantly, we demonstrate that both proteins are required for successful virus replication. These findings establish a framework to test other animal species for susceptibility to infection that may be critical zoonotic reservoirs for transmission, as well as to test variant viruses that arise over time.

Environmental transmission of Pseudogymnoascus destructans to hibernating little brown bats

Pathogens with persistent environmental stages can have devastating effects on wildlife communities. White-nose syndrome (WNS), caused by the fungus Pseudogymnoascus destructans, has caused widespread declines in bat populations of North America. In 2009, during the early stages of the WNS investigation and before molecular techniques had been developed to readily detect P. destructans in environmental samples, we initiated this study to assess whether P. destructans can persist in the hibernaculum environment in the absence of its conclusive bat host and cause infections in naive bats. We transferred little brown bats (Myotis lucifugus) from an unaffected winter colony in northwest Wisconsin to two P. destructans contaminated hibernacula in Vermont where native bats had been excluded. Infection with P. destructans was apparent on some bats within 8 weeks following the introduction of unexposed bats to these environments, and mortality from WNS was confirmed by histopathology at both sites 14 weeks following introduction. These results indicate that environmental exposure to P. destructans is sufficient to cause the infection and mortality associated with WNS in naive bats, which increases the probability of winter colony extirpation and complicates conservation efforts.

Integrating Multiple Survey Techniques to Document a Shifting Bat Community in the Wake of White-Nose Syndrome

The long-term study of bat communities often depends on a diverse set of sampling methodologies that are chosen based on the species or habitat management priorities of the research project. Integrating the data from a diverse set of methodologies (such as acoustic monitoring and mist net sampling) would improve our ability to characterize changes in community structure or composition over time, such as one would expect following an emergent infectious disease such as white-nose syndrome. We developed a Bayesian state-space model to integrate these disparate data into a common currency (relative abundance). We collected both acoustic monitoring and mist net capture data over an 8-y period (2006–2014) to document shifts in the bat community in central New England, USA, in response to the onset of white-nose syndrome in 2009. The integrated data model shows a significant decline in the abundance of little brown bat Myotis lucifugus, northern long-eared bat Myotis septentrionalis, and hoary bat Lasiurus cinereus, and an increase in abundance of the eastern small-footed bat Myotis leibii and the eastern red bat Lasiurus borealis. There was no evidence for a change in abundance in the big brown bat Eptesicus fuscus since the onset of white-nose syndrome. The consistency of this model with regional estimates of decline over the same time period support the validity of our relative abundance estimate. This model provides the opportunity to quantify shifts in other communities where multiple sampling methodologies were employed, and therefore provides natural resource managers with a robust tool to integrate existing sampling data to quantify changes in community composition that can inform conservation and management recommendations.

Hepatic lipid signatures of little brown bats (Myotis lucifugus) and big brown bats (Eptesicus fuscus) at early stages of white-nose syndrome

White-nose syndrome (WNS) is an emergent wildlife fungal disease of cave-dwelling, hibernating bats that has led to unprecedented mortalities throughout North America. A primary factor in WNS-associated bat mortality includes increased arousals from torpor and premature fat depletion during winter months. Details of species and sex-specific changes in lipid metabolism during WNS are poorly understood and may play an important role in the pathophysiology of the disease. Given the likely role of fat metabolism in WNS and the fact that the liver plays a crucial role in fatty acid distribution and lipid storage, we assessed hepatic lipid signatures of little brown bats (Myotis lucifugus) and big brown bats (Eptesicus fuscus) at an early stage of infection with the etiological agent, Pseudogymnoascus destructans (Pd). Differences in lipid profiles were detected at the species and sex level in the sham-inoculated treatment, most strikingly in higher hepatic triacylglyceride (TG) levels in E. fuscus females compared to males. Interestingly, several dominant TGs (storage lipids) decreased dramatically after Pd infection in both female M. lucifugus and E. fuscus. Increases in hepatic glycerophospholipid (structural lipid) levels were only observed in M. lucifugus, including two phosphatidylcholines (PC [32:1], PC [42:6]) and one phosphatidylglycerol (PG [34:1]). These results suggest that even at early stages of WNS, changes in hepatic lipid mobilization may occur and be species and sex specific. As pre-hibernation lipid reserves may aid in bat persistence and survival during WNS, these early perturbations to lipid metabolism could have important implications for management responses that aid in pre-hibernation fat storage.

Evaluating the risk of SARS-CoV-2 transmission to bats using a decision analytical framework

Preventing wildlife disease outbreaks is a priority issue for natural resource agencies, and management decisions can be urgent, especially in epidemic circumstances. With the emergence of SARS-CoV-2, wildlife agencies were concerned whether the activities they authorize might increase the risk of viral transmission from humans to North American bats but had a limited amount of time in which to make decisions. We provide a description of how decision analysis provides a powerful framework to analyze and re-analyze complex natural resource management problems as knowledge evolves. Coupled with expert judgment and avenues for the rapid release of information, risk assessment can provide timely scientific information for evolving decisions. In April 2020, the first rapid risk assessment was conducted to evaluate the risk of transmission of SARS-CoV-2 from humans to North American bats. Based on the best available information, and relying heavily on formal expert judgment, the risk assessment found a small possibility of transmission during summer work activities. Following that assessment, additional knowledge and data emerged, such as bat viral challenge studies, that further elucidated the risks of human-to-bat transmission and culminated in a second risk assessment in the fall of 2020. We update the first SARS-CoV-2 risk assessment with new estimates of little brown bat (Myotis lucifugus) susceptibility and new management alternatives, using findings from the prior two risk assessments and other empirical studies. We highlight the strengths of decision analysis and expert judgment not only to frame decisions and produce useful science in a timely manner, but also to serve as a framework to reassess risk as understanding improves. For SARS-CoV-2 risk, new knowledge led to an 88% decrease in the median number of bats estimated to be infected per 1000 encountered when compared to earlier results. The use of facemasks during, or a negative COVID-19 test prior to, bat encounters further reduced those risks. Using a combination of decision analysis, expert judgment, rapid risk assessment, and efficient modes of information distribution, we provide timely science support to decision makers for summer bat work in North America.

White-nose syndrome-related changes to Mid-Atlantic bat communities across an urban-to-rural gradient

Background White-nose Syndrome (WNS) has reduced the abundance of many bat species within the United States’ Mid-Atlantic region. To determine changes within the National Park Service National Capital Region (NCR) bat communities, we surveyed the area with mist netting and active acoustic sampling (2016–2018) and compared findings to pre-WNS (2003–2004) data. Results The results indicated the continued presence of the threatened Myotis septentrionalis (Northern Long-eared bat) and species of conservation concern, including Perimyotis subflavus (Tri-colored bat), Myotis leibii (Eastern Small-footed bat) and Myotis lucifugus (Little Brown bat). However, we documented a significant reduction in the abundance and distribution of M. lucifugus and P. subflavus, a decrease in the distribution of M. septentrionalis, and an increase in the abundance of Eptesicus fuscus (Big Brown bat). Conclusions Documented post-WNS M. septentrionalis recruitment suggests that portions of the NCR may be important bat conservation areas. Decreases in distribution and abundance of P. subflavus and M. lucifugus indicate probable extirpation from many previously occupied portions of the region.