Merkel cell polyomavirus and two previously unknown polyomaviruses are chronically shed from human skin

Abstract

Mounting evidence indicates that Merkel cell polyomavirus (MCV), a circular double-stranded DNA virus, is a causal factor underlying a highly lethal form of skin cancer known as Merkel cell carcinoma. To explore the possibility that MCV and other polyomaviruses commonly inhabit healthy human skin, we developed an improved rolling circle amplification (RCA) technique to isolate circular DNA viral genomes from human skin swabs. Complete MCV genomes were recovered from 40% of healthy adult volunteers tested, providing full-length, apparently wild-type cloned MCV genomes. RCA analysis also identified two previously unknown polyomavirus species that we name human polyomavirus-6 (HPyV6) and HPyV7. Biochemical experiments show that polyomavirus DNA is shed from the skin in the form of assembled virions. A pilot serological study indicates that infection or coinfection with these three skin-tropic polyomaviruses is very common. Thus, at least three polyomavirus species are constituents of the human skin microbiome.

Figure 1

Phylogenetic analysis of cloned MCV genomes. Previously reported MCV clones derived from tumor specimens are shown in bold. MCV isolates new in this work were named according to subject identification number followed by an “a” or “b” indicating isolation during the initial or repeat sampling, respectively. Isolates beginning with the letter “R” were cloned directly from restriction digested RCA products. Underlined isolates were derived from individuals born in Asia. Italicized isolates were derived from individuals born in Europe. Other isolates were derived from individuals born in the US. See also Figure S1.

Figure 2

The complete genomic nucleotide sequences of various polyomavirus species were used to construct a phylogenetic tree. Human polyomaviruses are underlined. See also Figure S2.

Figure 3

Detection of virions in skin swab extracts. Polyomavirus virions were extracted from nuclease-treated skin swab specimens and fractionated by ultracentrifugation through Optiprep (panels on right). DNA was extracted from each fraction (x-axes) and subjected to qPCR analysis with primers targeting MCV, HPyV6 or HPyV7. Purified plasmid DNA carrying a cloned MCV genome was ultracentrifuged without nuclease treatment and subjected to qPCR analysis using MCV-specific primers (top left panel). A parallel gradient in which nuclease was added to MCV plasmid DNA showed no MCV qPCR signal in any fraction (data not shown). The middle left panel shows MCV-specific qPCR analysis of a gradient loaded with MCV virions produced in 293TT cells transfected with recombinant MCV genomic DNA. The bottom left panel shows an experiment in which MCV VP1-specific monoclonal antibodies were used to strip MCV DNA out of a pool of fractions 3 and 4 of the swab extract gradient or the MCV-transfected 293TT cell extract gradient. MCV-specific qPCR measurements in the bottom left panel were standardized to an isotype control antibody condition. Error bars represent the standard deviation between three independent experiments.

Figure 4

Serological analysis of HPyV6 and 7 exposure. Serum samples from 95 donors were diluted 1:100 and subjected to ELISA using purified VP1 particles of indicated polyomavirus species. The optical density (OD) values for a representative set of 12 samples are shown graphically. A seropositivity cutoff value of 0.105 (dotted line) was chosen to exclude the maximum signal observed in the MPyV (negative control) ELISA (data not shown). The table at the bottom of the figure shows observed frequency of single, double and triple seropositivity for the three human polyomavirus species, as well as expected frequencies derived by multiplication of individual frequencies. See also Figure S3.