Immunomics of the koala (Phascolarctos cinereus)

Abstract

The study of the koala transcriptome has the potential to advance our understanding of its immunome--immunological reaction of a given host to foreign antigens--and to help combat infectious diseases (e.g., chlamydiosis) that impede ongoing conservation efforts. We used Illumina sequencing of cDNA to characterize genes expressed in two different koala tissues of immunological importance, blood and spleen. We generated nearly 600 million raw sequence reads, and about 285 million of these were subsequently assembled and condensed into ~70,000 subcomponents that represent putative transcripts. We annotated ~16% of these subcomponents and identified those related to infection and the immune response, including Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), major histocompatibility complex (MHC) genes, and koala retrovirus (KoRV). Using phylogenetic analyses, we identified 29 koala genes in these target categories and report their concordance with currently accepted gene groups. By mapping multiple sequencing reads to transcripts, we identified 56 putative SNPs in genes of interest. The distribution of these SNPs indicates that MHC genes (34 SNPs) are more diverse than KoRV (12 SNPs), TLRs (8 SNPs), or RLRs (2 SNPs). Our sequence data also indicate that KoRV sequences are highly expressed in the transcriptome. Our efforts have produced full-length sequences for potentially important immune genes in koala, which should serve as targets for future investigations that aim to conserve koala populations.

Phylogenetic Diversity of Koala Retrovirus within a Wild Koala Population

“…In particular, although subtypes C and D have been described previously based upon differences in the hypervariable region (17,19,23), our more detailed analysis revealed that these subtypes in reality belong to a highly diverse paraphyletic group (i.e., subtype D) that contains a number of other subtypes (E, F, G, H, and I) within its component genetic diversity (Fig. 1).…”

“…Specifically, we collected samples from 18 diseased individuals and analyzed KoRV genetic diversity using deep sequencing of an approximately 500-nucleotide region of the env gene that encodes a previously identified hypervariable region within the receptor binding domain. This region is of particular significance as it has been implicated in alternate receptor usage and has been used to define unique KoRV subtypes (14,(16)(17)(18)(19)(20)(21). Our study represents the first analysis of KoRV genetic diversity in a wild koala population and the first application of deep sequencing to profile the viral population within infected animals.…”

“…To determine the robustness of individual nodes on the phylogeny, a bootstrap resampling exercise was undertaken, utilizing 1,000 bootstrap replications under the same substitution model defined above but employing NNI branch swapping. Each representative sequence was assigned to a KoRV subtype (A to G) based on clustering within the phylogenetic tree and analysis of patterns of amino acid sequence variation within the hypervariable region, as in previous studies (14,19,20,23).…”

“…KoRV-B was subsequently identified in captive koalas housed in zoos in the United States and Germany (14,18), as well as in wild Australian koalas (23). KoRV-C has also been identified in a captive koala housed in a zoo in the United States (19), while KoRV-D has been identified in wild Australian koalas (23). Another potentially new subtype was recently reported and tentatively designated KoRV-F (21), although this sequence does not differ substantially from that of KoRV-D.…”

“…Five subtypes of KoRV, denoted KoRV-A, -B/J, -C, -D, and -E, have been reported to date; they differ primarily in sequences encoding the envelope protein, particularly within a hypervariable region of the receptor binding domain (RBD) (amino acids [aa] 91 to 135 [KoRV-A numbering]) (14,(16)(17)(18)(19)(20)(21). KoRV-A was first identified in wild and captive Australian koala populations (16) and is the most commonly identified subtype (6,14,18,20,(22)(23)(24).…”