Alston Virus, a Novel Paramyxovirus Isolated from Bats Causes Upper Respiratory Tract Infection in Experimentally Challenged Ferrets

Abstract

Multiple viruses with zoonotic potential have been isolated from bats globally. Here we describe the isolation and characterization of a novel paramyxovirus, Alston virus (AlsPV), isolated from urine collected from an Australian pteropid bat colony in Alstonville, New South Wales. Characterization of AlsPV by whole-genome sequencing and analyzing antigenic relatedness revealed it is a rubulavirus that is closely related to parainfluenza virus 5 (PIV5). Intranasal exposure of mice to AlsPV resulted in no clinical signs of disease, although viral RNA was detected in the olfactory bulbs of two mice at 21 days post exposure. Oronasal challenge of ferrets resulted in subclinical upper respiratory tract infection, viral shedding in respiratory secretions, and detection of viral antigen in the olfactory bulb of the brain. These results imply that AlsPV may be similar to PIV5 in its ability to infect multiple mammalian host species. This isolation of a novel paramyxovirus with the potential to transmit from bats to other mammalian species reinforces the importance of continued surveillance of bats as a source of emerging viruses.

Keywords: bat-borne; paramyxovirus; zoonoses.

Figure 1

Phylogenetic analysis of rubulaviruses. (a) L protein or (b) N gene of rubulaviruses with Hendra virus as an outgroup. (c) L protein or (d) N gene of multiple parainfluenza virus 5 strains, AlsPV and human parainfluenza virus 2. All trees are maximum-likelihood tree reconstructed with MEGA 6.06, bootstrapping to 1000 replicates. AlsPV is highlighted in bold and with an asterisk.

Figure 1

Phylogenetic analysis of rubulaviruses. (a) L protein or (b) N gene of rubulaviruses with Hendra virus as an outgroup. (c) L protein or (d) N gene of multiple parainfluenza virus 5 strains, AlsPV and human parainfluenza virus 2. All trees are maximum-likelihood tree reconstructed with MEGA 6.06, bootstrapping to 1000 replicates. AlsPV is highlighted in bold and with an asterisk.

Figure 1

Phylogenetic analysis of rubulaviruses. (a) L protein or (b) N gene of rubulaviruses with Hendra virus as an outgroup. (c) L protein or (d) N gene of multiple parainfluenza virus 5 strains, AlsPV and human parainfluenza virus 2. All trees are maximum-likelihood tree reconstructed with MEGA 6.06, bootstrapping to 1000 replicates. AlsPV is highlighted in bold and with an asterisk.

Figure 2

Effect of Arthrobacter ureafaciens neuraminidase treatment on AlsPV, PIV5, hPIV2 and TioPV infection of Vero cells. Infected cells were counted and averaged across nine fields of view and compared to infected but untreated cells. TioPV was included as a control because it lacks the NRKSCS motif and was therefore not expected to bind sialic acid. Values represent a percentage of the number of infected cells counted in untreated samples. Error bars represent standard error of the mean. Significance calculated by one-way ANOVA followed by Dunnett’s multiple comparison test (compared to untreated cells). *** represents a p value of <0.001. n = 2 independent experiments.

Figure 3

Antigenic cross-reactivity between PIV5 and AlsPV by immunofluorescence assay. Vero cells were infected AlsPV or PIV5 and stained with either rabbit sera raised against an N protein peptide of AlsPV, ferret sera resulting from infection with AlsPV, or anti-PIV5 guinea pig sera (magnification ×20).

Figure 4

Growth kinetics of AlsPV in multiple mammalian cell lines. Mammalian cell lines were infected with AlsPV at MOI 0.01 for 1 h in triplicate. Cells were washed and aliquots were collected every 24 h for 6 days. The TCID₅₀/mL was determined by virus titration. Error bars represent standard error of the mean.

Figure 5

Shedding of AlsPV in ferret respiratory secretions following oronasal exposure. Graphs present log transformations of the (a) copy number of AlsPV N gene per ml of nasal wash sample, (b) titer of AlsPV isolated from nasal wash, (c) copy number of AlsPV N gene per ml of oral swab sample, and the (d) titer of AlsPV isolated from oral swabs.

Figure 6

Shedding of AlsPV in ferret respiratory secretions following oronasal exposure. Ferrets were sampled prior to euthanasia, either 3, 5, 7 or 10 days post infection. Graphs present log transformations of the (a) copy number of AlsPV N gene per ml of nasal wash sample, (b) titer of AlsPV isolated from nasal wash, (c) copy number of AlsPV N gene per ml of oral swab sample, and the (d) titer of AlsPV isolated from oral swabs.

Figure 7

Detection of viral RNA in ferret tissues at euthanasia. Graphs represent log transformations of the copy number of AlsPV N gene RNA per 10¹⁰ copies of 18S rRNA detected in (a) nasal turbinates (b) tonsils (c) retropharyngeal lymph nodes (d) olfactory bulb of the brain.