Transboundary Animal Diseases Center, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, 890-0065, Japan.
Laboratory of Animal Hygiene, Joint Faculty of Veterinary Medicine, Kagoshima University, Kagoshima, 890-0065, Japan.
BMC Vet Res. 2024 Oct 30;20(1):496. doi: 10.1186/s12917-024-04357-5.
Koala retrovirus (KoRV), a major pathogen of koalas, exists in both endogenous (KoRV-A) and exogenous forms (KoRV-A to I and K to M) and causes multiple disease phenotypes, including carcinomas and immunosuppression. However, the direct association between the different KoRV subtypes and carcinogenesis remains unknown. Differentially expressed gene (DEG) analysis of peripheral blood mononuclear cells (PBMCs) of koalas carrying both endogenous (KoRV-A) and exogenous (KoRV-A, B, and C) subtypes was performed using a high-throughput RNA-seq approach. PBMCs were obtained from three healthy koalas: one infected with endogenous (KoRV-A; Group I) and two infected with exogenous (KoRV-B and/or KoRV-C; Group II) subtypes. Additionally, spleen samples (n = 6) from six KoRV-infected deceased koalas (K1- K6) and blood samples (n = 1) from a live koala (K7) were collected and examined to validate the findings.
All koalas were positive for the endogenous KoRV-A subtype, and eight koalas were positive for KoRV-B and/or KoRV-C. Transcription of KoRV gag, pol, and env genes was detected in all koalas. Upregulation of cytokine and immunosuppressive genes was observed in koalas infected with KoRV-B or KoRV-B and -C subtypes, compared to koalas infected with only KoRV-A. We found 550 DEG signatures with significant (absolute p < 0.05, and absolute log Fold Change (FC) > 1.5) dysregulation, out of which 77.6% and 22.4% DEGs were upregulated (logFC > 1.5) and downregulated (logFC < - 1.5), and downregulated (logFC < - 1), respectively. We identified 17 unique hub genes (82.3% upregulated and 17.7% down-regulated), with KIF23, CCNB2, POLR3F, and RSL24D1 detected as the potential hub genes modified with KoRV infection. Real-time RT-qPCR was performed on seven koalas to ascertain the expression levels of four potential hub genes, which were subsequently normalized to actin copies. Notably, all seven koalas exhibited distinct expression signatures for the hub genes, especially, KIF23 and CCNB2 show the highest expression in healthy koala PBMC, and POLR3F shows the highest expression in koala with lymphoma (K1).
Thus, it can be concluded that multiple KoRV subtypes affect disease progression in koalas and that the predicted hub genes could be promising prognostic biomarkers for pathogenesis.
考拉逆转录病毒（KoRV）是考拉的主要病原体，存在内源性（KoRV-A）和外源性（KoRV-A 到 I 和 K 到 M）两种形式，可引起多种疾病表型，包括癌和免疫抑制。然而，不同 KoRV 亚型与致癌作用之间的直接关联尚不清楚。使用高通量 RNA-seq 方法对携带内源性（KoRV-A）和外源性（KoRV-A、B 和 C）亚型的考拉外周血单核细胞（PBMC）进行差异表达基因（DEG）分析。从三只健康考拉中获得 PBMC：一只感染内源性（KoRV-A；第 I 组），两只感染外源性（KoRV-B 和/或 KoRV-C；第 II 组）亚型。此外，从六只 KoRV 感染死亡的考拉（K1-K6）中采集了脾脏样本（n=6），并从一只活考拉（K7）中采集了血液样本（n=1），以验证研究结果。
所有考拉均为内源性 KoRV-A 亚型阳性，8 只考拉为 KoRV-B 和/或 KoRV-C 阳性。所有考拉均检测到 KoRV gag、pol 和 env 基因的转录。与仅感染 KoRV-A 的考拉相比，感染 KoRV-B 或 KoRV-B 和 -C 亚型的考拉中细胞因子和免疫抑制基因的表达上调。我们发现了 550 个具有显著差异（绝对 p<0.05，绝对对数倍数变化（logFC）>1.5）的 DEG 特征，其中 77.6%和 22.4%的 DEG 上调（logFC>1.5）和下调（logFC<-1.5），下调（logFC<-1）。我们确定了 17 个独特的枢纽基因（82.3%上调和 17.7%下调），其中 KIF23、CCNB2、POLR3F 和 RSL24D1 被检测为受 KoRV 感染修饰的潜在枢纽基因。对 7 只考拉进行实时 RT-qPCR 以确定四个潜在枢纽基因的表达水平，然后将其标准化为肌动蛋白拷贝数。值得注意的是，七只考拉的枢纽基因表达特征均不同，尤其是健康考拉 PBMC 中 KIF23 和 CCNB2 的表达最高，淋巴瘤考拉（K1）中 POLR3F 的表达最高。
因此，可以得出结论，多种 KoRV 亚型影响考拉的疾病进展，预测的枢纽基因可能是发病机制有前途的预后生物标志物。
