Browsing by Author "Grubaugh, Nathan D."
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- Adventitious viruses persistently infect three commonly used mosquito cell linesWeger-Lucarelli, James; Rückert, Claudia; Grubaugh, Nathan D.; Misencik, Michael J.; Armstrong, Philip M.; Stenglein, Mark D.; Ebel, Gregory D.; Brackney, Doug E. (2018-08)Mosquito cell lines have been used extensively in research to isolate and propagate arthropod-borne viruses and understand virus-vector interactions. Despite their utility as an in vitro tool, these cell lines are poorly defined and may harbor insect-specific viruses. Accordingly, we screened four commonly-used mosquito cell lines, C6/36 and U4.4 cells from Aedes albopictus, Aag2 cells from Aedes aegypti, and Hsu cells from Culex quinquefasciatus, for the presence of adventitious (i.e. exogenous) viruses. All four cell lines stained positive for double-stranded RNA, indicative of RNA virus replication. We subsequently identified viruses infecting Aag2, U4.4 and Hsu cell lines using untargeted next-generation sequencing, but not C6/36 cells. PCR confirmation revealed that these sequences stem from active viral replication and/or integration into the cellular genome. Our results show that these commonly-used mosquito cell lines are persistently-infected with several viruses. This finding may be critical to interpreting data generated in these systems.
- Genomic and phenotypic analyses suggest moderate fitness differences among Zika virus lineagesOliveira, Glenn; Vogels, Chantal B. F.; Zolfaghari, Ashley; Saraf, Sharada; Klitting, Raphaelle; Weger-Lucarelli, James; Leon, Karla P.; Ontiveros, Carlos O.; Agarwal, Rimjhim; Tsetsarkin, Konstantin A.; Harris, Eva; Ebel, Gregory D.; Wohl, Shirlee; Grubaugh, Nathan D.; Andersen, Kristian G. (Public Library of Science, 2023-02)Zika virus was introduced to the Western Hemisphere, spread rapidly, and led to the 2015-2016 Zika epidemic and a rise in congenital microcephaly. It remains unclear whether Zika virus evolved to become more transmissible directly before or during the epidemic. To investigate whether Zika evolved to become more transmissible, we engineered a library of recombinant viruses that represent twelve major Zika virus lineages that circulated throughout the Americas. We measured the replicative fitness of each of these lineages by infecting live mosquitoes and human cells that are relevant for disease or transmission. We found that two of the lineages, one that dominated Central America and another that existed mostly in the Caribbean, appear to replicate more efficiently in human cells. While the fitness changes do not appear to have significant effects on the 2015-2016 Zika epidemic, our analysis suggests Zika virus evolved at least twice during this outbreak. Monitoring the phenotypic evolution during the course of an outbreak can help control spread and mitigate disease. We believe this framework can be applied to study phenotypic evolution during future epidemics caused by emerging RNA viruses. RNA viruses have short generation times and high mutation rates, allowing them to undergo rapid molecular evolution during epidemics. However, the extent of RNA virus phenotypic evolution within epidemics and the resulting effects on fitness and virulence remain mostly unknown. Here, we screened the 2015-2016 Zika epidemic in the Americas for lineage-specific fitness differences. We engineered a library of recombinant viruses representing twelve major Zika virus lineages and used them to measure replicative fitness within disease-relevant human primary cells and live mosquitoes. We found that two of these lineages conferred significant in vitro replicative fitness changes among human primary cells, but we did not find fitness changes in Aedes aegypti mosquitoes. Additionally, we found evidence for elevated levels of positive selection among five amino acid sites that define major Zika virus lineages. While our work suggests that Zika virus may have acquired several phenotypic changes during a short time scale, these changes were relatively moderate and do not appear to have enhanced transmission during the epidemic.
- Small RNA responses of Culex mosquitoes and cell lines during acute and persistent virus infectionRückert, Claudia; Prasad, Abhishek N.; Garcia-Luna, Selene M.; Robison, Alexis; Grubaugh, Nathan D.; Weger-Lucarelli, James; Ebel, Gregory D. (2019-06)RNA interference is a crucial antiviral mechanism in arthropods, including in mosquito vectors of arthropod-borne viruses (arboviruses). Although the exogenous small interfering RNA (siRNA) pathway constitutes an efficient antiviral response in mosquitoes, virus-derived P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) have been implicated in the response to alpha-, bunya- and flaviviruses in Aedes spp. mosquitoes. Culex mosquitoes transmit several medically important viruses including West Nile virus (WNV), but are considerably less well studied than Aedes mosquitoes and little is known about antiviral RNA interference in Culex mosquitoes. Therefore, we sequenced small RNA (sRNA) libraries from different Culex cell lines and tissues infected with WNV. The clear majority of virus-derived sRNA reads were 21 nt siRNAs in all cell lines and tissues tested, with no evidence for a role of WNV-derived piRNAs. Additionally, we aligned sRNA reads from Culex quinquefasciatus Hsu cells to the insect-specific rhabdovirus, Merida virus, which persistently replicates in these cells. We found that a significant proportion of the sRNA response to Merida virus consisted of piRNAs. Since viral DNA forms have been implicated in siRNA and piRNA responses of Aedes spp. mosquitoes, we also tested for viral DNA forms in WNV infected Culex cells. We detected viral DNA in Culex tarsalis cells infected with WNV and, to a lesser amount, WNV and Merida virus-derived DNA in Culex quinquefasciatus Hsu cells. In conclusion, Hsu cells generated Merida virus-derived piRNAs, but our data suggests that the major sRNA response of Culex cells and mosquitoes to WNV infection is the exogenous siRNA response. It is also evident that sRNA responses differ significantly between specific virus-mosquito combinations. Future work using additional Culex-borne viruses may further elucidate how virus-derived piRNAs are generated in Culex cells and what role they may play in controlling replication of different viruses.