The polyomavirus family contains four genera, Alpha-, Beta-, Gamma-, and Deltapolyomavirus. To date, a total of 98 polyomaviruses have been reported around the world, 12 of which are human polyomaviruses (HPyVs), including BKPyV and JCPyV identified in the early 1970s, and 10 types of HPyV detected since 2007, including KIPyV, WUPyV, and MCPyV [2]. HPyVs can cause diseases of the nervous system, hematopoietic system, urogenital tract, and skin [6], with two main characteristics. First, polyomavirus infections are usually persistent but asymptomatic, only leading to illness in immunosuppressed and immunocompromised cases, such as TSPyV causing skin disease trichodysplasia spinulosa (TS) in severely immunocompromised patients [22]. Second, some virus-encoded oncogenic proteins can induce cell transformation and malignancies, such as MCPyV acting as a major causal factor in the development of Merkel cell carcinoma (MCC) [23]. WUPyV has been widely detected from respiratory tract samples around the world since it was first identified in 2007. WUPyV was usually asymptomatic or only caused mild symptomatic infections, and was often co-detected with other common respiratory viruses, with co-infection rates ranging from 26.7–100.0% [4]. However, WUPyV was the only pathogen detected in some patients [3, 24], prompting the study of its infectivity and pathogenicity. Since WUPyV is difficult to isolate in vitro, establishing a suitable culture system is especially important.
To date, there have been no cell lines available for the proliferation of HPyVs from clinical samples, with the exception of JCPyV and BKPyV [25]. Much of the research on polyomaviruses was performed using recombinant virions and pseudoviruses [26, 27]. Previous studies showed that WUPyV-VP1 could be detected in cytokeratin+ cells (epithelial cells), CD68+ cells (macrophage/monocyte lineage), and MUC5AC+ cells (goblet cells) in the lung tissue by immunohistochemistry [11, 28], indicating that WUPyV has obvious tropism for respiratory epithelia cells. Reconstructed HAE cells can develop a pseudostratified structure, and differentiate into basal cells, ciliated cells, and goblet cells, which could realistically simulate the virus infection environment in vivo, and was therefore proposed for use in the isolation of WUPyV derived from respiratory samples.
In this study, ALI-HAE cells cultured on Transwell plates were used for clinical sample inoculation, and a WUPyV strain (BJ0771) was successfully isolated from a 3-year-old child with respiratory infection. The replication curves showed that WUPyV grew rapidly in the early stage, with viral loads reaching a plateau at 6 dpi. Despite the growth rate slowing after day 6, viral loads remained at a constant high level (~ 109 copies/µL), indicating that WUPyV could cause a persistent infection. Viral nucleic acids were detected in the basolateral chamber, suggesting that WUPyV could break through the respiratory epithelial barrier, being released into the basal medium from the surface of pseudostratified cells. The ability to break through this cell barrier is crucial for virus release and disease development, as the progeny viruses then have the opportunity to infect other cells with the same receptors.
HAE cells were re-infected with the harvested viruses, and immunofluorescence revealed that WUPyV could express the late region-encoded protein (VP1) in the HAE cell system. Electron microscopy provided evidence that complete WUPyV virions could be released. These two experimental techniques confirmed that HAE cells could produce infectious WUPyV.
Moens et al. [29] explored the activity of early and late promoters of HPyVs in 10 cell lines and the results showed that WUPyV has both higher early and late promoter activity on A375 cells, suggesting that the A375 cell line may be suitable for WUPyV isolation. We cultured the harvested viruses in A375 cells and four other traditional cell lines (293T, Hep2, LLC-MK2, and MDCK); however, nucleic acid tests did not show significant proliferation (data not shown).
At present, international classification standards for WUPyV have not been published. Previous studies indicated that WUPyV could be divided into three main clusters (Ⅰ–Ⅲ) and several sub-clusters (Ⅰa–Ⅰd and Ⅲ a–Ⅲc) [4, 30], but the topological structure of the phylogenetic tree shows inconsistencies according to different algorithms [2]. In this study, we observed three clusters in the maximum likelihood tree, but no sub-groups were evident in cluster Ⅰ. BJ0771 strain belonged to gene cluster Ⅰ and was most closely related to a strain isolated in Hangzhou, China. Homology analysis showed that the genome of BJ0771 was highly conserved, at both the nucleotide and amino acid level, with other isolated strains.