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. IMPORTANCE Bats constitute well-known reservoirs for viruses that may be transferred into human populations

Development and applications of single-cycle infectious influenza A virus (sciIAV)

“…To identify a bat cell line permissive to H1N1 virus infection, Poole et al utilized GFP-expressing sciIAV ΔHA for single-round infections at various MOIs (Poole et al, 2014). Prior to this work, this important potential reservoir for IAV lacked experimental evidence suggesting permissiveness to human IAVs (Mehle, 2014).…”

“…Prior to this work, this important potential reservoir for IAV lacked experimental evidence suggesting permissiveness to human IAVs (Mehle, 2014). The permissive bat cell line, Tb 1 Lu, that was identified to support H1N1 replication and infection did so through binding of sialic acid-linked receptors, as treatment of cells with receptor destroying enzyme prior to sciIAV ΔHA inoculation inhibited subsequent GFP expression (Poole et al, 2014). With reports that continually suggest new host species for IAV, sciIAV can be used to evaluate virus tropism to and permissivity of new species (Yoon et al, 2014).…”

Structure of influenza A polymerase bound to the viral RNA promoter

“…1). Bat polymerase can replicate efficiently in human cells 16 and vice versa 17 , suggesting that the bat structure will be a good model for all FluA polymerases. Here we describe the overall architecture of the polymerase, the structure of each subunit and their interfaces, and how the conserved 39 and 59 sequences of the vRNA promoter are bound.…”

Competitive detection of influenza neutralizing antibodies using a novel bivalent fluorescence-based microneutralization assay (BiFMA)

“…Replication-deficient influenza viruses that express reporter proteins are useful in studying virus reassortment [26, 40, 41] and entry [42], and have been adapted to high throughput methodologies to screen for host factors involved in replication [43] and to detect NAbs [22, 23, 44]. To determine the amenability of sciIAV mRFP to a microneutralization assay incorporating a fluorescence plate reader, sciIAV GFP [22] and sciIAV mRFP were used in individual fluorescent microneutralization assays with the anti-WSN NAb 2G9.…”