Molecular Insights into Innate Immune Response in Captive Koala Peripheral Blood Mononuclear Cells Co-Infected with Multiple Koala Retrovirus Subtypes.

Koala retrovirus (KoRV) exists in both endogenous and exogenous forms and has appeared as a major threat to koala health and conservation. Currently, there are twelve identified KoRV subtypes: an endogenous subtype (KoRV-A) and eleven exogenous subtypes (KoRV-B to -I, KoRV-K, -L, and -M). However, information about subtype-related immune responses in koalas against multiple KoRV infections is limited. In this study, we investigated KoRV-subtype (A, B, C, D, and F)-related immunophenotypic changes, including CD4, CD8b, IFN-γ, IL-6, and IL-10 mRNA expression, in peripheral blood mononuclear cells (PBMCs) obtained from captive koalas (n = 37) infected with multiple KoRV subtypes (KoRV-A to F) reared in seven Japanese zoos. Based on KoRV subtype infection profiles, no significant difference in CD4 and CD8b mRNA expression was observed in the study populations. Based on the different KoRV subtype infections, we found that the IFN-γ mRNA expression in koala PMBCs differs insignificantly (p = 0.0534). In addition, IL-6 and IL-10 mRNA expression also did not vary significantly in koala PBMCs based on KoRV subtype differences. We also investigated the Toll-like receptors (TLRs) response, including TLR2-10, and TLR13 mRNA in koala PBMCs infected with multiple KoRV subtypes. Significant differential expression of TLR5, 7, 9, 10, and 13 mRNA was observed in the PBMCs from koalas infected with different KoRV subtypes. Therefore, based on the findings of this study, it is assumed that co-infection of multiple KoRV subtypes might modify the host innate immune response, including IFN-γ and TLRs responses. However, to have a more clear understanding regarding the effect of multiple KoRV subtypes on host cytokines and TLR response and pathogenesis, further large-scale studies including the koalas negative for KoRV and koalas infected with other KoRV subtypes (KoRV-A to -I, KoRV-K, -L and -M) are required.

Subtype distribution and expression of the koala retrovirus in the Japanese zoo koala population.

We investigated the proviral copies and RNA expression in koala retrovirus (KoRV)-infected koalas. To ascertain any variation in viral load by institution, age, sex, or body condition score, we quantified KoRV proviral DNA and RNA loads in captive koalas (n = 37) reared in Japanese zoos. All koalas were positive for KoRV genes (pol, LTRs, and env of KoRV-A) in genomic DNA (gDNA), and 91.89% were positive for the pol gene in RNA. In contrast, the distribution rates of KoRV-B, KoRV-C, KoRV-D, and KoRV-F env genes in gDNA were 94.59%, 27.03%, 67.57%, and 54.05%, respectively. A wide inter-individual variation and/or a significant inter-institutional difference in proviral DNA (p < 0.0001) and RNA (p < 0.001) amounts (copies/103 koala ß-actin copies) were observed in Awaji Farm England Hill Zoo koalas, which were obtained from southern koala populations, suggesting exogenous incorporation of KoRV in these koalas. Significant (p < 0.05) age differences were noted in KoRV RNA load (p < 0.05) and median total RNA load (p < 0.001), with loads higher in younger koalas (joeys and juveniles). Thus, the current study provides the distribution of KoRV subtypes in Japanese zoo koala populations and identifies several additional risk factors (sex, age, and body condition) associated with KoRV expression.