Squirrels (family Sciuridae) are globally distributed members of the order Rodentia with wildlife occurrence in indigenous and non-indigenous regions (as invasive species) and frequent presence in zoological gardens and other holdings. Multiple species introductions, strong inter-species competition as well as the recent discovery of a novel zoonotic bornavirus resulted in increased research interest on squirrel pathogens. Therefore we aimed to test a variety of squirrel species for representatives of three virus families. Several species of the squirrel subfamilies Sciurinae, Callosciurinae and Xerinae were tested for the presence of polyomaviruses (PyVs; family Polyomaviridae) and herpesviruses (HVs; family Herpesviridae), using generic nested polymerase chain reaction (PCR) with specificity for the PyV VP1 gene and the HV DNA polymerase (DPOL) gene, respectively. Selected animals were tested for the presence of bornaviruses (family Bornaviridae), using both a broad-range orthobornavirus- and a variegated squirrel bornavirus 1 (VSBV-1)-specific reverse transcription-quantitative PCR (RT-qPCR). In addition to previously detected bornavirus RNA-positive squirrels no more animals tested positive in this study, but four novel PyVs, four novel betaherpesviruses (BHVs) and six novel gammaherpesviruses (GHVs) were identified. For three PyVs, complete genomes could be amplified with long-distance PCR (LD-PCR). Splice sites of the PyV genomes were predicted in silico for large T antigen, small T antigen, and VP2 coding sequences, and experimentally confirmed in Vero and NIH/3T3 cells. Attempts to extend the HV DPOL sequences in upstream direction resulted in contiguous sequences of around 3.3 kilobase pairs for one BHV and two GHVs. Phylogenetic analysis allocated the novel squirrel PyVs to the genera Alpha- and Betapolyomavirus, the BHVs to the genus Muromegalovirus, and the GHVs to the genera Rhadinovirus and Macavirus. This is the first report on molecular identification and sequence characterization of PyVs and HVs and the detection of bornavirus coinfections with PyVs or HVs in two squirrel species. Multiple detection of PyVs and HVs in certain squirrel species exclusively indicate their potential host association to a single squirrel species. The novel PyVs and HVs might serve for a better understanding of virus evolution in invading host species in the future.

中文翻译：

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在Sciuliedae家族的松鼠中搜索多瘤病毒，疱疹病毒和博纳病毒。

松鼠（鼠尾草科）是啮齿类动物的全球分布成员，野生生物在土著和非土著地区（作为入侵物种）发生，并经常在动物园和其他饲养场中存在。多种物种的引进，强大的种间竞争以及新型人畜共患病毒博纳病毒的最新发现导致对松鼠病原体的研究兴趣增加。因此，我们旨在测试代表三个病毒家族的各种松鼠物种。测试了几种松鼠亚纲Sciurinae，Callosciurinae和Xerinae的多瘤病毒（PyVs; Polyomaviridae家族）和疱疹病毒（HVs; Herpesviridae家族）的存在，使用通用巢式聚合酶链反应（PCR）分别对PyV VP1基因和HV DNA聚合酶（DPOL）基因具有特异性。使用广泛的正畸博达病毒和杂种松鼠博达病毒1（VSBV-1）特异性逆转录定量PCR（RT-qPCR），测试了选定的动物是否存在博纳病毒（博纳病毒科）。除了先前检测到的博纳病毒RNA阳性松鼠外，在本研究中没有更多动物被检测为阳性，但是鉴定出了四种新型PyV，四种新型β疱疹病毒（BHV）和六种新型γ疱疹病毒（GHV）。对于三个PyV，可以使用长距离PCR（LD-PCR）扩增完整的基因组。在计算机上预测了大T抗原，小T抗​​原和VP2编码序列的PyV基因组剪接位点，在Vero和NIH / 3T3细胞中得到了实验证实。尝试在上游方向上扩展HV DPOL序列会导致一个BHV和两个GHV的大约3.3千碱基对的连续序列。系统发育分析将新的松鼠PyVs分配给Alpha和Betapolyomavirus属，将BHV分配给Muromegalovirus属，将GHV分配给Rhadinovirus和Macavirus属。这是有关两个松鼠物种中PyV和HV分子鉴定和序列表征以及检测带有PyV或HV的鼻病毒共感染的第一份报告。多次检测某些松鼠物种中的PyV和HV仅表明它们与单个松鼠物种的潜在宿主关联。新型的PyV和HV可能有助于将来更好地了解入侵宿主物种中的病毒进化。