Abstract The genealogy of the hepatitis C virus (HCV) and the genus Hepacivirus remains elusive despite numerous recently discovered animal hepaciviruses (HVs). Viruses from evolutionarily ancient mammals might elucidate the HV macro-evolutionary patterns. Here, we investigated sixty-seven two-toed and nine three-toed sloths from Costa Rica for HVs using molecular and serological tools. A novel sloth HV was detected by reverse transcription polymerase chain reaction (RT-PCR) in three-toed sloths (2/9, 22.2%; 95% confidence interval (CI), 5.3–55.7). Genomic characterization revealed typical HV features including overall polyprotein gene structure, a type 4 internal ribosomal entry site in the viral 5′-genome terminus, an A–U-rich region and X-tail structure in the viral 3′-genome terminus. Different from other animal HVs, HV seropositivity in two-toed sloths was low at 4.5 per cent (3/67; CI, 1.0–12.9), whereas the RT-PCR-positive three-toed sloths were seronegative. Limited cross-reactivity of the serological assay implied exposure of seropositive two-toed sloths to HVs of unknown origin and recent infections in RT-PCR-positive animals preceding seroconversion. Recent infections were consistent with only 9 nucleotide exchanges between the two sloth HVs, located predominantly within the E1/E2 encoding regions. Translated sequence distances of NS3 and NS5 proteins and host comparisons suggested that the sloth HV represents a novel HV species. Event- and sequence distance-based reconciliations of phylogenies of HVs and of their hosts revealed complex macro-evolutionary patterns, including both long-term evolutionary associations and host switches, most strikingly from rodents into sloths. Ancestral state reconstructions corroborated rodents as predominant sources of HV host switches during the genealogy of extant HVs. Sequence distance comparisons, partial conservation of critical amino acid residues associated with HV entry and selection pressure signatures of host genes encoding entry and antiviral protein orthologs were consistent with HV host switches between genetically divergent mammals, including the projected host switch from rodents into sloths. Structural comparison of HCV and sloth HV E2 proteins suggested conserved modes of hepaciviral entry. Our data corroborate complex macro-evolutionary patterns shaping the genus Hepacivirus, highlight that host switches are possible across highly diverse host taxa, and elucidate a prominent role of rodent hosts during the Hepacivirus genealogy.

中文翻译：

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来自树懒的新型肝炎病毒说明丙型肝炎相关病毒的交叉顺序宿主转换

摘要 尽管最近发现了许多动物肝炎病毒 (HV)，但丙型肝炎病毒 (HCV) 和肝炎病毒属的谱系仍然难以捉摸。来自进化上古老哺乳动物的病毒可能阐明 HV 宏观进化模式。在这里，我们使用分子和血清学工具调查了来自哥斯达黎加的 67 只二趾树懒和九只三趾树懒的 HV。通过逆转录聚合酶链反应 (RT-PCR) 在三趾树懒中检测到一种新型树懒 HV（2/9，22.2%；95% 置信区间 (CI)，5.3-55.7）。基因组表征揭示了典型的 HV 特征，包括整体多蛋白基因结构、病毒 5'-基因组末端的 4 型内部核糖体进入位点、富含 A-U 的区域和病毒 3'-基因组末端的 X 尾结构。与其他动物HV不同，二趾树懒的 HV 血清阳性率较低，为 4.5%（3/67；CI，1.0-12.9），而 RT-PCR 阳性的三趾树懒为血清阴性。血清学检测的有限交叉反应性意味着血清反应阳性的两趾树懒在血清转化之前暴露于来源不明的 HV 和近期感染的 RT-PCR 阳性动物。最近的感染与两个树懒 HV 之间仅 9 个核苷酸交换一致，主要位于 E1/E2 编码区域内。NS3 和 NS5 蛋白的翻译序列距离和宿主比较表明树懒 HV 代表了一种新的 HV 物种。基于事件和序列距离的 HV 及其宿主系统发育的协调揭示了复杂的宏观进化模式，包括长期进化关联和宿主转换，最引人注目的是从啮齿动物到树懒。祖先状态重建证实啮齿动物是现存 HV 谱系中 HV 宿主开关的主要来源。序列距离比较、与 HV 进入相关的关键氨基酸残基的部分保守性以及编码进入和抗病毒蛋白直向同源物的宿主基因的选择压力特征与基因不同的哺乳动物之间的 HV 宿主转换一致，包括预计的宿主从啮齿动物转换为树懒。HCV 和懒惰 HV E2 蛋白的结构比较表明肝病毒进入的保守模式。我们的数据证实了塑造 Hepacivirus 属的复杂宏观进化模式，强调在高度多样化的宿主分类群中宿主切换是可能的，