Geographic patterns of koala retrovirus genetic diversity, endogenization, and subtype distributions

Abstract

Koala retrovirus (KoRV) subtype A (KoRV-A) is currently in transition from exogenous virus to endogenous viral element, providing an ideal system to elucidate retroviral-host coevolution. We characterized KoRV geography using fecal DNA from 192 samples across 20 populations throughout the koala's range. We reveal an abrupt change in KoRV genetics and incidence at the Victoria/New South Wales state border. In northern koalas, pol gene copies were ubiquitously present at above five per cell, consistent with endogenous KoRV. In southern koalas, pol copies were detected in only 25.8% of koalas and always at copy numbers below one, while the env gene was detected in all animals and in a majority at copy numbers above one per cell. These results suggest that southern koalas carry partial endogenous KoRV-like sequences. Deep sequencing of the env hypervariable region revealed three putatively endogenous KoRV-A sequences in northern koalas and a single, distinct sequence present in all southern koalas. Among northern populations, env sequence diversity decreased with distance from the equator, suggesting infectious KoRV-A invaded the koala genome in northern Australia and then spread south. The exogenous KoRV subtypes (B to K), two novel subtypes, and intermediate subtypes were detected in all northern koala populations but were strikingly absent from all southern animals tested. Apart from KoRV subtype D, these exogenous subtypes were generally locally prevalent but geographically restricted, producing KoRV genetic differentiation among northern populations. This suggests that sporadic evolution and local transmission of the exogenous subtypes have occurred within northern Australia, but this has not extended into animals within southern Australia.

Keywords: endogenization; evolution; genetic diversity; geography; koala retrovirus.

Fig. 1.

(A) Geographic location, (B) KoRV pol incidence, and (C) average pol proviral copies per koala cell for each population. (D) Boxplots of the number of KoRV pol and env copies per koala cell, values shown for pol-positive koalas and southern koalas for which single env melt curve peaks were obtained, respectively. MI, Magnetic Island; C, Clermont; BE, St. Bee’s Island; MB, Mt. Byron; NS, North Stradbroke Island; SQ, southwest Queensland; G, Gunnedah; N, Nowendoc; PM, Port Macquarie; ML, Mountain Lagoon; MO, Monaro; U, Ulupna; BS, Boho South; SZ, Strzelecki 1; SB, Strzelecki 2; CO, Cape Otway; BB, Bessiebelle; AH, Adelaide Hills; KI, Kangaroo Island; MK, Mikkira; QLD, Queensland; NSW, New South Wales; Vic, Victoria; SA, South Australia.

Fig. 2.

Proportion of intact KoRV-A env reads from each KoRV pol-positive koala belonging to the four dominant sequences. Mutation codes are relative to the original KoRV-A sequence (37) with numbering relative to the beginning of the env gene. Population and state codes are as described in Fig. 1.

Fig. 3.

Geographic distribution of (A) KoRV-B and A/B intermediates, (B) KoRV-D and A/D intermediates, and (C) the other detected subtypes.

Fig. 4.

Env sequence sharing between populations and sequence richness (number of unique sequence clusters) within populations and regions. Network diagrams of (A) all (intact and nonfunctional) KoRV env, (B) intact KoRV-A env (excluding the original KoRV-A cluster), and (C) intact KoRV-B-M env sequence clusters shared between populations. Nodes (circles) represent sites, with the size of the node indicating the number of sequence clusters shared with other populations. Black nodes indicate northern populations, and light green nodes indicate southern populations. A line joining two nodes indicates that those two populations shared sequence clusters, with the thickness of the line indicating the number of sequence clusters shared. Gray lines indicate 5 or fewer sequence clusters shared; gradient of blue to red lines indicates 6 to 20 clusters shared. (D–I) Rarefaction curves depicting KoRV sequence richness. The total number of intact KoRV env sequence clusters (D and G), intact KoRV-A env sequence clusters (E and H), and intact KoRV-B-M env sequence clusters (F and I) detected in a population (G–I) or region (north and south; D–F) as the number of koalas sampled is increased. The 95% confidence intervals are shown for D–F.

Fig. 5.

(A) The MND among all sequence clusters detected within each population by geographic location and (B) for northern populations in relation to latitude. Dotted line indicates fitted regression, blue points indicate populations included in regression analysis, and the orange point indicates North (Nth) Stradbroke Island. (C) The mean number of pairwise nucleotide differences among KoRV-A, KoRV-B-M, and nonfunctional sequences are also shown in relation to latitude for the northern populations.

Fig. 6.

Schematic of proposed KoRV evolution and spread.

Fig. 7.

Schematic of proposed KoRV evolution hypothesis.