title: Influenza A virus polymerase is a site for adaptive changes during experimental evolution in bat cells.
abstract: The recent identification of highly divergent influenza A viruses in bats revealed a new, geographically dispersed viral reservoir. To investigate the molecular mechanisms of host-restricted viral tropism and the potential for transmission of viruses between humans and bats, we exposed a panel of cell lines from bats of diverse species to a prototypical human-origin influenza A virus. All of the tested bat cell lines were susceptible to influenza A virus infection. Experimental evolution of human and avian-like viruses in bat cells resulted in efficient replication and created highly cytopathic variants. Deep sequencing of adapted human influenza A virus revealed a mutation in the PA polymerase subunit not previously described, M285K. Recombinant virus with the PA M285K mutation completely phenocopied the adapted virus. Adaptation of an avian virus-like virus resulted in the canonical PB2 E627K mutation that is required for efficient replication in other mammals. None of the adaptive mutations occurred in the gene for viral hemagglutinin, a gene that frequently acquires changes to recognize host-specific variations in sialic acid receptors. We showed that human influenza A virus uses canonical sialic acid receptors to infect bat cells, even though bat influenza A viruses do not appear to use these receptors for virus entry. Our results demonstrate that bats are unique hosts that select for both a novel mutation and a well-known adaptive mutation in the viral polymerase to support replication.
  
  
  OBJECTIVE
  Bats constitute well-known reservoirs for viruses that may be transferred into human populations, sometimes with fatal consequences. Influenza A viruses have recently been identified in bats, dramatically expanding the known host range of this virus. Here we investigated the replication of human influenza A virus in bat cell lines and the barriers that the virus faces in this new host. Human influenza A and B viruses infected cells from geographically and evolutionarily diverse New and Old World bats. Viruses mutated during infections in bat cells, resulting in increased replication and cytopathic effects. These mutations were mapped to the viral polymerase and shown to be solely responsible for adaptation to bat cells. Our data suggest that replication of human influenza A viruses in a nonnative host drives the evolution of new variants and may be an important source of genetic diversity.
authors.affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Poole
authors.affiliation: NIH/NIAID Integrated Research Facility at Fort Detrick, Frederick, Maryland, USA.
authors.forename: Shuǐqìng
authors.initials: S
authors.lastname: Yú
authors.affiliation: NIH/NIAID Integrated Research Facility at Fort Detrick, Frederick, Maryland, USA.
authors.forename: Yíngyún
authors.initials: Y
authors.lastname: Caì
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA.
authors.forename: Jorge M
authors.initials: JM
authors.lastname: Dinis
authors.affiliation: Institute of Virology, University of Bonn Medical Centre, Bonn, Germany.
authors.forename: Marcel A
authors.initials: MA
authors.lastname: Müller
authors.affiliation: ProBioGen AG, Berlin, Germany.
authors.forename: Ingo
authors.initials: I
authors.lastname: Jordan
authors.affiliation: Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin, USA Wisconsin National Primate Research Center, Madison, Wisconsin, USA.
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Friedrich
authors.affiliation: NIH/NIAID Integrated Research Facility at Fort Detrick, Frederick, Maryland, USA.
authors.forename: Jens H
authors.identifier: http://orcid.org/0000-0002-7800-6045
authors.initials: JH
authors.lastname: Kuhn
authors.affiliation: Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, Wisconsin, USA amehle@wisc.edu.
authors.forename: Andrew
authors.initials: A
authors.lastname: Mehle
mesh_terms: D000220:Adaptation, Biological; D000818:Animals; D002460:Cell Line; D002685:Chiroptera; D003588:Cytopathogenic Effect, Viral; D004252:DNA Mutational Analysis; D059014:High-Throughput Nucleotide Sequencing; D009980:Influenza A virus / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D020125:Mutation, Missense; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
chemical_list: C061290:PA protein, influenza viruses; C057027:PB2 protein, Influenzavirus A; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase