Evolution of circoviruses in lorikeets lags behind its hosts
Graphical abstract
Introduction
The presence of endogenous viral elements in host genomes hints towards much older host–virus relationships than predicted by exogenous phylogenies of double-stranded DNA (dsDNA) viruses in particular (Belyi et al., 2010, Cui et al., 2014, Theze et al., 2011), alluding to long-term, attenuating, host associations (Firth et al., 2010). In contrast, highly mutable single-stranded DNA (ssDNA) viruses, such as parvoviruses and circoviruses, behave more like RNA viruses (Duffy et al., 2008, Lauring and Andino, 2010, Sarker et al., 2014b), often occupying entangled multispecies ecological niches (Pagan and Holmes, 2010). Resolving such lineages, and the sometimes great discrepancy amongst viral evolutionary timescales, is highly problematic, especially under the influence of high rates of purifying selection or recombination which can significantly alter the accuracy of phylogenetic reconstruction methods (Wertheim et al., 2014). Amongst the milieu of admixture the challenge is to find meaningful host co-divergences, or other discrete viral speciation events that correlate with paleobiological chronology, or endogenous viral features that retain function (Theze et al., 2011). There have been few attempts to unravel the long-term evolutionary history of vertebrate virus-host relationships and determinants of viral speciation (Gilbert and Feschotte, 2010, Gilbert et al., 2014, Rector et al., 2007). Multi-host scenarios may increase this difficulty but our results show they also provide opportunities to identify allopatric or sympatric signals which can occur with host-switching or when ecological sequestration by a host is sufficient to exert strong selection pressure (Fournier and Giraud, 2008, Pagan and Holmes, 2010). Multi-host pathogens are theoretical candidates for sympatric speciation through ecological sequestration but natural examples of sympatry are rare and difficult to substantiate (Schliewen et al., 2006). Infectious agents with broad host-ranges are prime candidates if individual host species exert strong disruptive selection alongside continued evolution of both in response to each other (Fournier and Giraud, 2008).
Circoviruses are small, nonenveloped icosahedral viruses with circular ssDNA genomes of only 2 kilobases making them the smallest autonomously replicating pathogens. They encode only two major genes, one involved in replication (Rep) and the other encoding a single structural protein (Cap) with multiple functions whereas the intergenic region is relatively small in comparison to the whole genome. Because of their small size and compact genome circoviruses provide a test model for sympatry in the absence of strong internal epistatic or pleiotropic influences, since evolutionary innovation in multi-host niches is likely to be heavily constrained by their simple structure and genomes. We suspected that endogenous circovirus elements (Cui et al., 2014) in Kea (Nestor notabilis), a member of the uniquely New Zealand parrot Superfamily Strigopoidea, could unlock a potentially ancient host–virus interaction amongst circovirus progenitors in the Gondwanan-Australasian landscape corresponding to the Cretaceous origin of parrots (Aves: Psittaciformes) (Joseph et al., 2012, Wright et al., 2008). The presence of endogenous circovirus elements in a Strigopoidea, which are an allopatrically diverged relic of three extant parrot species confined to New Zealand, is intriguing since there is little evidence of beak and feather disease virus (BFDV) or any other related exogenous avian circovirus currently circulating in the Strigopoidea or any other New Zealand parrot species prior to human colonisation, despite extensive surveys for BFDV in native New Zealand parrots (Ha et al., 2007, Jackson et al., 2014, Massaro et al., 2012, Raidal et al., 2015). Circoviruses may have been lost in Kea due to fadeout and stochastic disease extinction in refugia associated with New Zealand’s relatively chaotic geological past (Knapp et al., 2007). If as is presumed for other circoviruses, BFDV has its origins, and has co-evolved with psittacine birds, then the paucity of distinctly non-Australian BFDV genotypes from parts of the world that have endemic psittacine birds such as New Zealand, Southern Africa and South America is very difficult to explain without a post-Gondwanan entry into psittacine birds (Raidal et al., 2015).
Phylogeographic evidence points to Australia as the origin of BFDV (Harkins et al., 2014) with BFDV from lorikeets proposed as the true Australasian variant (Harkins et al., 2014, Heath et al., 2004) due to its basal location, although genomes from budgerigars, in the sibling tribe Melopsitticini, are also contenders (Sarker et al., 2015a). Lorikeets and budgerigars are members of the relatively young (10 Ma) parrot Subfamily Loriinae chiefly confined to Australasia (Joseph et al., 2011, Joseph et al., 2012, Schweizer et al., 2015, Wright et al., 2008). By probing the Kea genome with non-psittacine and mammalian circovirus sequences we identified conserved motifs that aligned with contemporary BFDV in lorikeets indicating a more recent introduction of BFDV into the Psittaciformes than predicted by endogenous viral elements in Kea. Given the high degree of host-switching that occurs with BFDV and a lack of virulence motifs in the BFDV genome, or antigenic diversity, this has been considered to be most likely an effect of innate host resistance in Loriini but highlights a potential ecological niche that might favour BFDV exploration (Raidal et al., 2015). Nevertheless, evolutionary innovation in such niches is likely to be heavily constrained by the small genome size of circoviruses and differing expressions of disease could therefore be entirely host-based.
High rates of recombination in an organism, such as occurs in BFDV (Sarker et al., 2014a), can interfere with assessment of genetic diversity and substitution rates, and alter significantly the accuracy of phylogenetic reconstruction methods (Schierup and Hein, 2000). Genome-wide patterns of sequence variation can efficiently infer fine-scale genetic structures within closely related virus populations (Prasanna et al., 2010). Analyses that consider admixture or gene flow between different strata can help resolve sensible species/subspecies/strain classification criteria, the detection of geographical or biological barriers to gene flow, or the identification of demographic, epidemiological and evolutionary processes responsible for virus differentiation (Pritchard et al., 2000, Rosenberg et al., 2002). We used these methods in the present study to infer the genetic population structure of BFDV alongside Bayesian phylogenetic reconstruction to help resolve the evolutionary process with greater resolution, revealing evidence of niche based sympatric differentiation based on the host tribe Loriini. We show how a virus that mutates rapidly might also retain phenotypic stability and wait millennia to change hosts or become a recognisable new virus species, a process that could lag well behind the evolution of potential hosts.
Section snippets
Virus samples, phylogenetic, genetic population structure and recombination analyses of extant BFDV genomes
A total of 39 new full length BFDV genomes (5 budgerigar and 34 lorikeets) sourced over 13 years from different regions of Australia were sequenced and compiled with publicly available BFDV genomes (n = 303) from NIH GenBank for conducting sequence based bioinformatics analysis. Individual sequences were annotated with accession number, geographic origin, host species and taxonomic tribe and sampling year. Genomes were aligned in Geneious with MAFFT v7.017 using G-INS-i (gap open penalty 1.53;
Phylogeny and population structure of BFDV in global context
Along with 39 new BFDV genomes we sequenced from budgerigars (n = 5) and lorikeets (n = 34) from various geographical locations of Australia (Table S1) we compiled other BFDV genomes from GenBank for sequence based molecular analysis. Individual sequences were annotated according to the accession number, geographic origin, host species, taxonomic tribe name and sampling year. A Bayesian phylogenetic tree representing global BFDV genomes (n = 155) demonstrated 9 admixed genetic subpopulations with
Discussion
BFDV is recognised for its rich genetic diversity and flexible host-switching (Sarker et al., 2014a). The alignment of endogenous BFDV sequences in Kea with BFDV was not surprising. However, it was intriguing that BFDV in lorikeets provided the best match and not viral genomes from psittacine host groups of greater antiquity such as the Subfamilies Platycercinae or Cacatuinae which superficially appeared to harbour BFDV derived from those in lorikeets, contradicting existing theories of
Conclusions
The results show how high mutation signals in contemporary viruses may grossly underestimate evolutionary timescales compared to paleovirological chronology. Our data demonstrates a basally located and sympatrically sequestered BFDV lineage in Australian lorikeets produced by the recombination of viral lineages infecting ancient psittacopasserine hosts. Divergence estimates for circoviruses calibrated with paleobiological events that gave rise to the speciation of lorikeets and lories provided
Author contributions
S.D. and S.S. performed the diagnoses, DNA sequencing and bioinformatic analyses, D.F. provided BFDV samples for DNA sequencing. S.D. A.P., S.G., J.F. and S.R. provided analysis and bioinformatic support. The manuscript was written by S.D., S.S, A.P., J.F. and S.R.
Author information
DNA sequences of circovirus genomes can be accessed through GenBank database at the U.S. National Library of Medicine, National Institutes of Health (NIH) National Center for Biotechnology Information (NCBI), http://www.ncbi.nlm.nih.gov/. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to [email protected].
Acknowledgement
This research was supported by Australian Research Council’s Discovery Projects funding scheme (Grant number: DP1095408).
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