Members of the family Polyomaviridae are small viruses characterized by a non-enveloped icosahedral capsid and a circular double-stranded DNA genome of approximately 5000 bp. Early and late genes are transcribed bi-directionally, starting from a short non-coding regulatory region. Early genes encode two or three proteins, designated tumor antigens, which participate in viral genome replication and cellular transformation. The late genes encode the major and minor capsid proteins VP1, VP2, and VP3 [1]. In addition, some of the primate and human polyomaviruses encode an additional non-structural multifunctional protein, the agnoprotein [2]. The International Committee on Taxonomy of Viruses (ICTV) officially lists 73 species as polyomaviruses and divides the family into 4 genera including Alpha-, Beta-, Gamma- and Deltapolyomavirus [3].

Many mammalian polyomaviruses cause subclinical infections with life-long persistence in their immune competent hosts [4]. Only some of the polyomaviruses have been described to cause disease in immune-compromised hosts [5]. Polyomavirus-related diseases, which include nephritis [6], encephalitis [7], Merkel cell carcinoma [8], skin dysplasia [9, 10], and pneumonitis [11, 12], can occur among immune-suppressed individuals, including post-transplantation and AIDS patients. Polyomaviruses are widely distributed among mammalian and avian species and besides humans they have been identified in monkeys, cattle, rabbits, raccoons, rodents, bats, elephants, badgers, and a wide variety of birds [13–24].

In the present study, using viral metagenomics we detected a novel polyomavirus, the giant panda polyomavirus 1 (GPPyV1), in the nasal cavity of a giant panda (Ailuropoda melanoleuca) who died for unknown reasons after released into the wild.

DNA libraries from samples of giant pandas were generated and sequenced using the lllumina MiSeq platform. In the nasal sample 17 sequence reads generating 3 different contigs were found to have strong amino acid homology to polyomaviruses. PCR screening of all the samples from the dead wild giant panda, plus 30 other nasal swab samples and 200 fecal samples collected from healthy captive giant pandas in the the Chengdu Research Base of Giant Panda Breeding in China with a set of nested primers designed on the VP1 sequence. Results indicated that one nasal swab and two fecal samples from healthy captive giant pandas and only the nasal swab from the dead animal were positive, which suggests that polyomavirus may establish latency infection in the gastrointestinal tract and nasal cavity of giant pandas.

Sequence analysis based on the 299 bp fragments generated PCR screening, the 3 sequences from the healthy giant pandas were identical and showed only one nucleotide difference from the sequence from the died giant panda, suggesting these polyomavirus sequences may be from the same ancestor virus which was prevalent in the giant panda group. Using inverse PCR, the complete genome was then acquired and sequenced by Sanger walking from the nasal sample of the dead giant panda. The circular genome of the giant panda polyomavirus (named GPPyV1) was 5144 bases, with the overall GC content of 41.8%, which is similar to those of BK (39%), JC (40%), WU (39%) and other polyomaviruses [29]. The genome organization includes an early region coding on one strand for the small T antigen (STAg) and the large T antigen (LTAg), and a late region coding on the opposite strand for the capsid proteins VP1, VP2, and VP3, with a noncoding regulatory region between the beginning of the early region and the beginning of the late region, homologous to earlier described polyomaviruses (Fig. 1). Two copies of the consensus pentanucleotide LTAg binding site GAGGC, or the reverse complement GCCTG were present in the a noncoding regulatory region, where the sequence is GAGGCAGAAGGCCTC which might represent the possible core of the origin of replication [2]. The noncoding region between the ends of the late and early regions (Fig. 1) showed an AT-rich region. The sizes of the proteins deduced from the genome sequences of GPPyV1, their calculated molecular weights and isoelectric points are shown in Table 1.

ORF	Position	Length		MW (kDa)	pI
		nt	aa
URR*	1–354	n/a	n/a	n/a	n/a
VP2	355–1278	924	307	33.69	4.73
VP3	571–1278	708	235	26.55	6.37
VP1	1247–2575	1329	442	48.07	6.88
NCR**	2576–2616	n/a	n/a	n/a	n/a
LTAg	2617–4548	2172	723	81.57	6.38
	4905–5144
STAg	4566–5144	579	192	22.54	8.43

^*Upstream regulatory region

^**Noncoding region

A more detailed analysis of the amino acid sequences of GPPyV1 revealed conserved sequences in functionally important regions of the encoded proteins. The proteins encoded by the 5 GPPyV1 open-reading frames contain the typical elements that are necessary to accomplish their function in the viral life cycle of polyomaviruses. The unspliced early mRNA of GPPyV1 encodes the STAg, which is 192 aa in length, where the CXCX₂C consensus sequence for protein phosphatase 2A binding is present. The initial 80 amino acids of the N-terminus of the STAg and LTAg of GPPyV1 are identical, which is a general characteristic in all polyomaviruses. The LTAg is generated by partial splicing of the early mRNA transcript. Conserved features like the J-domain, which is important for efficient DNA replication and transformation, is found in the LTAg sequence. This domain contains the highly conserved HPDKGG box and a pRB-binding motif LXCXE, which are crucial for DNA replication [9].

To determine the genetic relationship between GPPyV1 and the other related polyomaviruses. Phylogenetic analysis based on the putative LTAg amino acid sequences of polyomaviruses, including GPPyV1, the representative polyomavirus species of the four genera, and the best BLASTp searching matches of GPPyV1 in GenBank, were performed and maximum-likelihood (ML) tree was generated. Results indicated that GPPyV1 is highly divergent from these other polyomaviruses and that it fell within the clade of genus Deltapolyomavirus together with four species from human, sharing the highest amino acid sequence identity of 53% of with MW polyomavirus (GenBank no. JQ898291) (Fig. 2A). BLASTp searches in GenBank based on the amino acid sequences of VP1 and VP2 indicated GPPyV1 showed the closest relationship with polyomaviruses within genus Alphapolyomavirus which includes bat polyomavirus 5 (BatPyV-5) and Pan troglodytes verus polyomavirus 3 and 4 (PtroPyV3 and PtroPyV4), sharing 67–68% and 56–57% with them, respectively. ML trees were then established based on the amino acid sequence of VP1 and VP2, respectively, from the representative strains in genus Deltapolyomavirus and genus Alphapolyomavirus. Both trees revealed that GPPyV1 clustered within the clade of genus Alphapolyomavirus, discordant with the tree based on LTAg (Fig. 2 B and C), suggesting this ployomavirus strain from giant panda was involved in genomic recombination which frequently occurred during polyomavirus evolution [30].

Recently, the ICTV Polyomaviridae Study Group designed the new criteria for definition of a new polyomavirus species [3], which includes four major points: i) the complete genome sequence is available in public databases and published; ii) the genome displays an organization typical for polyomaviruses; iii) sufficient information on the natural host is available; iv) the observed genetic distance to members of the most closely related species is >15% for the large T antigen (LTAg) coding sequence. Based on these criteria, GPPyV1 qualifies as a novel polyomavirus species.

In the present study, using viral metagenomic method, a new polyomavirus was detected in the nasal cavity of a dead giant panda recently released into the wild who died from unknown causes. Our PCR analysis of 13 nasal swabs and 25 fecal samples from healthy captive giant pandas showed no other positive animals, suggesting GPPyV1 is not highly prevalent in captive giant pandas. In mammals polyomaviruses infections are highly common during childhood and adolescence [31]. These persistent infections are not generally associated with acute disease in their natural non-immunocompromised hosts. On the other hand, mammalian polyomaviruses are known to induce tumors after inoculation into non-permissive laboratory rodents [32] and recently, the human polyomavirus, Merkel cell polyomavirus, has been linked to the development of skin carcinoma [8, 33]. In this study, phylogenetic analysis based on LTAg of GPPyV1 showed it had the closest relationship with polyomaviruses from genus Deltapolyomavirus which currently includes four defined species: HPyV-6, HPyV-7, MW polyomavirus, and STL polyomavirus. HPyV-6 and HPyV-7 were mainly found on human skin, while MW polyomavirus and STL polyomavirus have been isolated out of human feces, some of which are associated with human disease, for example, HPyV-7 was associated pruritic rash and viremia in transplant recipients, while MW and STL polyomavirus were reported to be present in tonsillar tissues from children with chronic tonsillar disease [10, 34]. Using an alternative classification scheme both the LT and the VP1 proteins cluster in the Almi clade [35]. The polyomaviruses (HPyV-6, HPyV-7, MW, and STL) whose LT are most closely related to that of GPPyV1 have VP1s that cluster in a different (Wuki) clade than GPPyV1 (Almi) indicating that these genomes may be recombinant of an original virus whose VP1 gene was more related to the GPPyV1 genome described here. During the evolution of polyomavirus, recombination does not seem to occur randomly across the genome but has rearranged the early and late genomic regions of major polyomavirus lineages. For instance, a lineage comprising KIPyV, WUPyV and two related rodent polyomaviruses belongs to the clade corresponding to the genus Betapolyomavirus in LTAg phylogenetic trees, however it clusters within a lineage of the genus Deltapolyomavirus based on the phylogenetic analysis over the VP1 sequences [35–37].

A novel polyomavirus, GPPyV1, was detected in the nasal cavity of a dead giant panda, whose complete genome was determined. Phylogenetic analysis based on the LTAg suggested GPPyV1 belonged to a putative new species within genus Deltapolyomavirus. Epidemiological results suggested GPPyV1 is also prevalent in captive giant pandas. Whether GPPyV1 cause specific disease in giant pandas remain unknown.