Characterization of Uncultivable Bat Influenza Virus Using a Replicative Synthetic Virus

Abstract

Bats harbor many viruses, which are periodically transmitted to humans resulting in outbreaks of disease (e.g., Ebola, SARS-CoV). Recently, influenza virus-like sequences were identified in bats; however, the viruses could not be cultured. This discovery aroused great interest in understanding the evolutionary history and pandemic potential of bat-influenza. Using synthetic genomics, we were unable to rescue the wild type bat virus, but could rescue a modified bat-influenza virus that had the HA and NA coding regions replaced with those of A/PR/8/1934 (H1N1). This modified bat-influenza virus replicated efficiently in vitro and in mice, resulting in severe disease. Additional studies using a bat-influenza virus that had the HA and NA of A/swine/Texas/4199-2/1998 (H3N2) showed that the PR8 HA and NA contributed to the pathogenicity in mice. Unlike other influenza viruses, engineering truncations hypothesized to reduce interferon antagonism into the NS1 protein didn't attenuate bat-influenza. In contrast, substitution of a putative virulence mutation from the bat-influenza PB2 significantly attenuated the virus in mice and introduction of a putative virulence mutation increased its pathogenicity. Mini-genome replication studies and virus reassortment experiments demonstrated that bat-influenza has very limited genetic and protein compatibility with Type A or Type B influenza viruses, yet it readily reassorts with another divergent bat-influenza virus, suggesting that the bat-influenza lineage may represent a new Genus/Species within the Orthomyxoviridae family. Collectively, our data indicate that the bat-influenza viruses recently identified are authentic viruses that pose little, if any, pandemic threat to humans; however, they provide new insights into the evolution and basic biology of influenza viruses.

The NS1 gene from bat-derived influenza-like virus H17N10 can be rescued in influenza A PR8 backbone

“…None of the reassortants possessing any segment from H17N10 and the other seven segments from the parental virus PR8 were successfully rescued, as monitored by the production of replicative viruses in Madin-Darby canine kidney (MDCK) cells or chicken egg embryos. Considering the interaction between HA and NA and their importance for infecting cells, several attempts to rescue a reassortant with HA and NA from PR8 and the other six gene segments from H17N10 were unsuccessful, which is consistent with the results from previous studies (Juozapaitis et al, 2014;Zhou et al, 2014). We did not perform these experiments with HA and NA from H17N10 and the other six segments from PR8, considering that the receptors for H17N10 HA and NA are still unknown and that HA was unable to bind any known cell line Sun et al, 2013;Zhu et al, 2012Zhu et al, , 2013.…”

“…They incorporated 95 nt from bat-H17 5¢ terminus with ATG mutated and 163 nt from bat-H17 3¢ terminus into the two terminals of the ORF of PR8 H1. A similar strategy was also applied to the ORF of PR8 N1 gene; that is, the PR8 N1 coding region was flanked by the 206 nt packing region sequence (with ATG mutated) from the 5¢ terminal of bat-N10 and 194 nt from the 3¢ terminal of bat-N10 (Zhou et al, 2014). Our work, together with the previous studies, partly demonstrated the importance of packing signals for virus rescue.…”

“…In those experiments, the ORFs of H7 and N7 were flanked with the NCRs and about 100 nt of the 5¢ and 3¢ coding sequences from H17N10. Moreover, Zhou et al (2014) also successfully rescued chimeric H1N1/H17N10 recombinants with PR8 H1 and N1 mutants and six internal genes from bat-H17N10. They incorporated 95 nt from bat-H17 5¢ terminus with ATG mutated and 163 nt from bat-H17 3¢ terminus into the two terminals of the ORF of PR8 H1.…”

“…These segments were subsequently tested in virus rescue experiments as described in the previous paragraph, but again without yielding any viable recombinant viruses, whereas Juozapaitis et al (2014) successfully rescued one chimeric influenza virus harbouring ORF of HA and NA genes from H7N7, but with H17N10 NCRs and part of the 5¢ and 3¢ coding sequences from H17N10 at the two terminals (Juozapaitis et al, 2014). This the case with H1N1/ H17N10 recombinant virus with HA and NA from PR8, in which HA and NA genes carry an extra bat H17N10 NCR sequence and a packing sequence with ATG mutated at the 5¢ terminal (Zhou et al, 2014). This led us to conclude that the difference in NCR alone could not explain the incompatibility of complete gene segments derived from H17N10 with those from PR8.…”

“…These results suggest that H17N10 and H18N11 are influenza-like viruses that are related to categorized influenza A viruses but seem incompatible in function and reassortment capability. In 2014, chimeric replicating hybrid viruses with six internal genes from bat influenza viruses and the remaining HA and NA coding regions that originated from H1N1, H3N2 or H7N7 could be rescued (Juozapaitis et al, 2014;Zhou et al, 2014). Moreover, the crystal structure of the heterotrimeric bat-derived H17N10 polymerase, comprising PB2, PB1 and PA subunits, reveals some canonical RNA polymerase motifs and domains (Pflug et al, 2014).…”