Isolation and Characterization of a Distinct Influenza A Virus from Egyptian Bats

Abstract

Recently, two genetically distinct influenza viruses were detected in bats in Guatemala and Peru. We conducted influenza A virus surveillance among four bat species in Egypt. Out of 1,202 swab specimens, 105 were positive by real-time PCR. A virus was successfully isolated in eggs and propagated in MDCK cells in the presence of N-tosyl-l-phenylalanine chloromethyl ketone-treated trypsin. Genomic analysis revealed that the virus was phylogenetically distinct from all other influenza A viruses. Analysis of the hemagglutinin gene suggested a common ancestry with other H9 viruses, and the virus showed a low level of cross-reactivity with serum raised against H9N2 viruses. Bats were seropositive for the isolated viruses. The virus replicated in the lungs of experimentally infected mice. While it is genetically distinct, this virus shares several avian influenza virus characteristics suggesting a more recent avian host origin.IMPORTANCE Through surveillance, we isolated and characterized an influenza A virus from Egyptian fruit bats. This virus had an affinity to avian-like receptors but was also able to infect mice. Our findings indicate that bats may harbor a diversity of influenza A viruses. Such viruses may have the potential to cross the species barrier to infect other species, including domestic birds, mammals, and, possibly, humans.

Keywords: influenza; public health; surveillance studies; veterinary epidemiology.

FIG 1

Phylogenetic tree of the nucleotide sequences of the eight genome segments of the characterized bat influenza virus in Egypt. Maximum likelihood trees were generated in RAxML (v8.0.0) for the eight gene segments with the GTR substitution model and the gamma distribution of rate heterogeneity. Bootstrapping analyses were conducted with the extended majority rule consensus tree criterion (the autoMRE option in RAxML). Trees were midpoint rooted and visualized in FigTree (v1.4.2; http://tree.bio.ed.ac.uk/software/figtree/). Bootstrapping values are shown on the tree nodes. The red star represents the Egyptian bat influenza virus. Orange branches represent H17/H18 bat influenza viruses. The scale bars represent 0.2 substitution per site for each associated tree.

FIG 2

Distribution of antibodies against the bat influenza virus (bat flu) as well as avian H9N2 and H4N6 viruses.

FIG 3

Receptor-binding specificity of Egyptian bat influenza virus. The direct binding of Egyptian bat influenza virus to biotinylated sialylglycopolymers containing 3′-sialyllactose (α2,3-SL) or 6′-sialyllactose (α2,6-SL) was measured. Influenza A/chicken/Egypt/7100/2013 (H9N2), A/Hong Kong/1073/99 (H9N2), and A/duck/Hong Kong/365/78 (H4N6) were used as controls for the binding assay. O.D., optical density.

FIG 4

Weight change of mice infected with A/bat/Egypt/381OP/2017 and avian H9N2 virus. Female C57BL/6 and BALB/c mice were intranasally infected. The virus-infected mice were monitored for 10 days, and the weight was determined daily. Results from each group and each time point are expressed as the means and standard deviations (SD) for infected mice. Analysis and visualization were performed using GraphPad Prism (v5) software.