Characterization of novel bat influenza viruses and mammalian orthoreoviruses
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Emerging and reemerging RNA viruses including both segmented and nonsegmented viruses cause significant threats to public and animal health. Segmented RNA viruses such as influenza virus and reovirus are featured with the capacity to exchange viral RNA segments during co-infection through a process called reassortment which results in creating novel viral progenies that contain genes derived from more than one parent. The reassortant virus strains may be conferred with critical fitness advantages or disadvantages that are closely related to viral evolution in emergence of novel genotypes or subtypes, host expansion or jumps, drug resistance, immune escape, and viral virulence and transmissibility. The discovery of novel bat H17N10 and H18N11 Influenza A viruses (IAVs) has resulted in numerous research to understand the biology of these novel viruses. In contrast to classical IAVs, these novel bat viruses are highly divergent and have unique features such as using MHC-II, not sialic acid as the receptor. Although no reassortment has been detected in co-infected cells with bat and classical IAVs, the underlying mechanisms of why they cannot reassort effectively remains poorly understood. Herein, we used bat influenza M and NS genes in the PR8 H1N1 genetic background to determine potential mechanisms that hinder reassortment between the novel bat and conventional IAVs. We hypothesized that bat influenza NEP or M1 cannot efficiently interact with PR8 M1 or NEP, resulting in that PR8 vRNPs cannot be transported to cytoplasm from nucleus for packaging, thereby failing to generate infectious viruses. To test our hypothesis, we performed Co-IP, virus-like particle and polymerase activity assays using combinations of NEP and M1 from bat and PR8 IAVs. Results showed that bat NEP or M1 can interact with PR8 M1 or NEP in the presence of PR8 vRNP, and their combinations can medicate PR8 vRNPs nucleus export and form virus-like particles, indicating a compatibility of both NEP and M1 from bat and PR8 IAVs at the protein level. Furthermore, we demonstrated that the mismatched packaging signals NS and M segments are the major reasons to hinder the reassortment between bat and classical IAVs by using revere genetics. Additionally, we revealed that attenuation of recombinant PR8 viruses having bat M gene ORFs correlates with a low efficiency of virus uncoating likely due to impaired functions of bat M2 in contrast to PR8 M2. Our studies uncover molecular mechanisms of both NS and M genes that hinder reassortment between bat and conventional IAVs, which help understand the biology and zoonotic potential of bat IAVs. Mammalian orthoreovirus (MRV) is another segmented RNA virus that can infect multiple mammalian species including humans and bats. A novel reassortant MRV, which contains viral genomic segments from three MRV serotypes that infect human, bovine and swine, respectively, was isolated from the diseased pigs from a United States Midwest swine farm in which more than 300 pigs showed neurological signs, like "down and peddling", with approximately 40 percent mortality. Further characterizations show that this novel reassortant MRV is able to replicate in human, swine, monkey and canine cells without prior adaptation and is pathogenic and transmissible in experimentally infected pigs. Considering that reassortment occurs among different genotypes and serotypes of MRVs in nature, we hypothesize that bats are the mixing vessels of MRVs and potentially spread wild type and reassortant MRVs to different species including humans. To test our hypothesis, we screened more than 900 bats of different species collected in the United States during 2015-2019 and successfully isolated 13 MRVs belonging to four different strains/genotypes of viruses in serotypes 1 or 2, which contain genes from MRVs detected in humans, bats, bovine, and deer. Further characterization showed that these four MRV strains replicated efficiently in human, canine, monkey, ferret, and swine cell lines. The 40/Bat/USA/2018 strain belonging to the serotype 1 demonstrated the ability to infect and transmit in pigs without prior adaptation. Our studies provide evidence for different genotypes and serotypes of MRVs circulating in US bats, which can be a mixing vessel of MRVs that may spread to other species, including humans, resulting in cross-species infections. Taken together, above studies unveil the underlying mechanisms of how NS and M segments hamper the reassortment of bat and classical IAVs and characterize virus replication and pathogenicity of novel reassortant MRV isolates from pigs and bats, and provide new perspectives on the evolution of the segmented RNA viruses.
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Ph. D.