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Human polyomavirus 9 (HPyV9) was discovered recently in immunocompromised patients and shown to be genetically closely related to B-lymphotropic polyomavirus (LPyV). No serological data are available for HPyV9, but human antibodies against LPyV have been reported previously. To investigate the seroepidemiology of HPyV9 and the sero-cross-reactivity between HPyV9 and LPyV, a capsomer-based IgG ELISA was established using the major capsid protein VP1 of HPyV9 and LPyV. VP1 of an avian polyomavirus was used as control. For HPyV9, a seroprevalence of 47 % was determined in healthy adults and adolescents (n5328) and 20 % in a group of children (n5101). In both groups, the seroreactivities for LPyV were less frequent and the ELISA titres of LPyV were lower. Of the HPyV9-reactive sera, 47 % reacted also with LPyV, and the titres for both PyVs correlated. Sera from African green monkeys, the natural hosts of LPyV, reacted also with both HPyV9 and LPyV, but here the HPyV9 titres were lower. This potential sero-cross-reactivity between HPyV9 and LPyV was confirmed by competition assays, and it was hypothesized that the reactivity of human sera against LPyV may generally be due to cross-reactivity between HPyV9 and LPyV. The HPyV9 seroprevalence of liver transplant recipients and patients with neurological dysfunctions did not differ from that of age-matched controls, but a significantly higher seroprevalence was determined in renal and haematopoietic stem-cell transplant recipients, indicating that certain immunocompromised patient groups may be at a higher risk for primary infection with or for reactivation of HPyV9. INTRODUCTIONHuman polyomavirus 9 (HPyV9) is the most recently identified of the nine human polyomaviruses (PyVs) reported to date, and was first detected in a renal transplant patient (Scuda et al., 2011). Later, the same virus was also found in human skin (Sauvage et al., 2011).PyVs are small, non-enveloped, circular dsDNA viruses. Primary infection with BK virus (BKV) and JC virus (JCV) occurs in childhood and is usually asymptomatic (Moens & Johannessen, 2008). Subsequently, these viruses establish a latent infection. Reactivation can occur in immunocompromised patients and cause serious disease, such as BKVassociated nephropathy or haemorrhagic cystitis (Gardner et al., 1971;Jiang et al., 2009), JCV-associated progressive multifocal leukoencephalopathy (Hou & Major, 2000;Jiang et al., 2009;Padgett et al., 1971) and trichodysplasia spinulosa caused by trichodysplasia spinulosa-associated PyV (Matthews et al., 2011;van der Meijden et al., 2010). PyVs have been shown to transform cells in vitro and to be tumorigenic in small laboratory rodents (Chen et al., 1989;Eddy et al., 1962;Gross, 1953;Stewart, 1953). BKV and JCV have been implicated aetiologically in a number of human cancers, but this issue is still controversial Maginnis & Atwood, 2009;zur Hausen, 2008). Merkel cell PyV (MCPyV) plays a causative role in Merkel cell carcinoma, a rare but aggressive skin cancer Feng et al., 2008).PyV serology has bee...

Genome replication of Porcine circovirus type 1 (PCV1) relies upon expression of the full-length protein Rep and a spliced isoform (Rep'), and the presence of a 111-bp genomic fragment comprising the origin of replication. Using an electrophoretic mobility shift assay (EMSA), the capability of both Rep proteins to bind to partial fragments of the origin of replication of PCV1 was investigated in vitro. Both proteins formed complexes with double-stranded DNA origin fragments containing a stem-loop structure with a conserved nonamer and four hexamer repeats (5'-CGGCAG; H1 to H4). Use of truncated EMSA substrates identified minimal binding sites (MBS) for Rep and Rep' protein: The Rep binding site was mapped to the right leg of the stem-loop and the two inner hexamer repeats H1/H2, while binding of Rep' required only the presence of two hexamer repeats. Two differentially retarded complexes were observed with Rep protein, which presumably result from alternative binding to the MBS or to H3/4.

The replication of porcine circovirus type 1 (PCV1) is thought to occur by rolling-circle replication (RCR), whereby the introduction of a single-strand break generates a free 3-hydroxyl group serving as a primer for subsequent DNA synthesis. The covalently closed, single-stranded genome of PCV1 replicates via a doublestranded replicative intermediate, and the two virus-encoded replication-associated proteins Rep and Rep have been demonstrated to be necessary for virus replication. However, although postulated to be involved in RCR-based virus replication, the mechanism of action of Rep and Rep is as yet unknown. In this study, the ability of PCV1 Rep and Rep to "nick" and "join" strand discontinuities within synthetic oligonucleotides corresponding to the origin of replication of PCV1 was investigated in vitro. Both proteins were demonstrated to be able to cleave the viral strand between nucleotides 7 and 8 within the conserved nonanucleotide motif (5-TAGTATTAC-3) located at the apex of a putative stem-loop structure. In addition, the Rep and Rep proteins of PCV1 were demonstrated to be capable of joining viral single-stranded DNA fragments, suggesting that these proteins also play roles in the termination of virus DNA replication. This joining activity was demonstrated to be strictly dependent on preceding substrate cleavage and the close proximity of origin fragments accomplished by base pairing in the stem-loop structure. The dual "nicking/joining" activities associated with PCV1 Rep and Rep are pivotal events underlying the RCR-based replication of porcine circoviruses in mammalian cells.

Although PCV gene expression and replication took place in human cells, the infection is non-productive. Alteration of protein localization suggests that protein targeting may be disturbed in human cells.

Porcine circovirus type 2 (PCV2) is an important pathogen in swine, whereas porcine circovirus type 1 (PCV1) is apathogenic. To analyze the interactions between PCV and its host, we have used a yeast two-hybrid assay to identify cellular proteins interacting with Cap and Rep proteins of both PCV genotypes. Six cellular proteins were found to interact with Cap (MKRN1, gC1qR, Par-4, NAP1, NPM1 and Hsp40) and three with Rep (ZNF265, TDG and VG5Q). These interactions were confirmed by co-immunoprecipitation. Investigation of the localisation of the proteins by immunofluorescence revealed in some cases only limited spatial overlapping with Cap, while in others a clear co-localisation and prominent protein redistribution was observed. The nine cellular proteins are associated with distinct aspects of viral lifecycle and our data is likely to support future research in the field of PCV2 pathogenesis.

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