Infectious KoRV-related retroviruses circulating in Australian bats

Abstract

Bats are reservoirs of emerging viruses that are highly pathogenic to other mammals, including humans. Despite the diversity and abundance of bat viruses, to date they have not been shown to harbor exogenous retroviruses. Here we report the discovery and characterization of a group of koala retrovirus-related (KoRV-related) gammaretroviruses in Australian and Asian bats. These include the Hervey pteropid gammaretrovirus (HPG), identified in the scat of the Australian black flying fox (Pteropus alecto), which is the first reproduction-competent retrovirus found in bats. HPG is a close relative of KoRV and the gibbon ape leukemia virus (GALV), with virion morphology and Mn²⁺-dependent virion-associated reverse transcriptase activity typical of a gammaretrovirus. In vitro, HPG is capable of infecting bat and human cells, but not mouse cells, and displays a similar pattern of cell tropism as KoRV-A and GALV. Population studies reveal the presence of HPG and KoRV-related sequences in several locations across northeast Australia, as well as serologic evidence for HPG in multiple pteropid bat species, while phylogenetic analysis places these bat viruses as the basal group within the KoRV-related retroviruses. Taken together, these results reveal bats to be important reservoirs of exogenous KoRV-related gammaretroviruses.

Keywords: GALV; KoRV; bats; retroviruses; viruses.

Fig. 1.

The genome of the HPG contains conserved functional motifs and is analogous to KoRV-A and GALV. R, terminal repeat sequence; U5/U3, unique 5′/3′ region; PBS(Pro), proline tRNA primer-binding site; gag, group-specific antigen; MHR, major homology region; zf, zinc finger; DxG, protease active site motif; DDD, reverse transcriptase active site motif; pol, polymerase; DDE, integrase active site motif; env, envelope; CET(S/A/T)G, pathogenicity motif; PolyA, polyadenylation signal.

Fig. 2.

Evolutionary relationships among KoRV-related viruses. Maximum likelihood phylogeny of the complete (nucleotide) sequence genome of 19 gammaretroviruses. All branches are scaled according to the number of nucleotide substitutions per site, and branches representing bat retroviruses are shown in red. Support for key nodes on the phylogeny are shown in the form of SH-like branch support/bootstrap support. Silhouettes represent the host species: top left, mice; right (in descending order), pteropid bats, koalas, woolly monkeys, microbats, and gibbons. The tree was rooted using the McERV (Mus caroli endogenous retrovirus) KC460271 sequence.

Fig. 3.

Electron micrographs of M-MLV and HPG. (A) An extracellular, roughly spherical enveloped virus-like particle with a concentric icosahedral core (arrow). The cores have variable electron translucence ranging from lucent to dense, indicating variable stages of particle maturation. (B) An immature extracellular virus-like particle with tooth-like appearance of the viral envelope (red arrow) surrounding the double-layered shell of the core. Distinct banding can also be seen in the envelope of the particle (black arrowheads). (C) Virus-like particle exiting the cell demonstrating a type C budding profile, characteristic of viruses belonging to the genus Gammaretrovirus (59, 60). (D) Evidence of virus assembly and budding from the plasma membrane of the cell, including the presence of a tether-like structure connecting the cell membrane to the newly budded virus (black arrow). (E) A mature virus-like particle with an electron-dense core encapsulated in an envelope. (F) Immature virus-like particle exhibiting a tooth-like appearance of the viral envelope (black arrows) surrounding the double-layered shell of the core (red arrow). (G) A mature virus-like particle with an electron-dense core encapsulated in an envelope. (H–J) Evidence of virus-like particle assembly and budding from the plasma membrane of the cell. Budding begins with the formation of electron-dense material under the membrane (H), which progresses until the nascent virus-like particle pushes out from the membrane and is pinched off to form a free particle. (Scale bars: 50 nm in A, 100 nm in B, 250 nm in C, 200 nm in D, 100 nm in E, 200 nm in F, 200 nm in G, and 250 nm in H, I, and J.) Negative controls were untransfected cells and cells mock transfected with the empty vector pcDNA3.1. These controls were not observed to contain or produce viral particles (SI Appendix, Fig. S5).

Fig. 4.

Replication kinetics of M-MLV and HPG in human, mouse, and bat cells. M-MLV and HPG virions were generated by transfection of human 293T cells with pNCS (61) and pCC1-HPG retroviral expression plasmids, respectively, and used to infect human 293T, mouse 3T3, and bat PaKi cell lines. Culture supernatants were collected daily for 5 d and assessed for the presence of virus by measuring virion-associated reverse transcriptase activity. Error bars represent the SEM (n = 6).

Fig. 5.

Human and mouse cell tropism of pseudotyped gammaretroviral virus-like particles and HPG-induced resistance to cross-infection. (A) Gammaretrovirus Env or VSV-G pseudotyped retroviral particles, containing the lacZ reporter gene, were generated using the Retro-X packaging system. The infectivity of pseudotyped viral particles was determined in human HeLa cells and mouse 3T3 cells. Infected cells were quantified by counting blue cell-forming units (BFU) following incubation with X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside). Uninfected cells served as a control (Mock). (B) Human HeLa cells were persistently infected with HPG and then challenged with infection by a reporter virus pseudotyped with gammaretrovirus Env or VSV-G pseudotyped retroviral particles. Infected cells were quantified as in A, and infection in persistently HPG-infected cells is expressed as a proportion of the amount of uninfected HeLa cells infected by the same reporter virus.