Whole-genome characterization and genotyping of global WU polyomavirus strains.

Abstract

Exploration of the genetic diversity of WU polyomavirus (WUV) has been limited in terms of the specimen numbers and particularly the sizes of the genomic fragments analyzed. Using whole-genome sequencing of 48 WUV strains collected in four continents over a 5-year period and 16 publicly available whole-genome sequences, we identified three main WUV clades and five subtypes, provisionally termed Ia, Ib, Ic, II, IIIa, and IIIb. Overall nucleotide variation was low (0 to 1.2%). The discriminatory power of the previous VP2 fragment typing method was found to be limited, and a new, larger genotyping region within the VP2/1 interface was proposed.

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JOURNAL OF VIROLOGY, June 2010, p. 6229–6234
0022-538X/10/$12.00doi:10.1128/JVI.02658-09
Copyright © 2010, American Society for Microbiology. All Rights Reserved.
Vol. 84, No. 12
NOTES
Whole-Genome Characterization and Genotyping of
Global WU Polyomavirus Strains?†
Seweryn Bialasiewicz,1,2* Rebecca Rockett,1,2David W. Whiley,1,2Yacine Abed,3Tobias Allander,4
Michael Binks,5Guy Boivin,3Allen C. Cheng,5,6Ju-Young Chung,7Patricia E. Ferguson,8,9
Nicole M. Gilroy,8,9Amanda J. Leach,5Cecilia Lindau,4John W. Rossen,10
Tania C. Sorrell,8,9Michael D. Nissen,1,2,11and Theo P. Sloots1,2,11
Queensland Paediatric Infectious Diseases Laboratory, Sir Albert Sakzewski Virus Research Centre, Queensland Children’s Medical
Research Institute, Children’s Health Service District, Brisbane, Queensland, Australia1; Clinical Medical Virology Centre,
University of Queensland, Brisbane, Queensland, Australia2; Le Centre Hospitalier Universitaire de Que ´bec and Laval University,
Que ´bec City, Quebec, Canada3; Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet and
Department of Clinical Microbiology, Karolinska University Hospital, SE-17176 Stockholm, Sweden4; Menzies School of
Health Research, Darwin, Northern Territory, Australia5; Department of Epidemiology and Preventive Medicine,
Monash University, Melbourne, Victoria, Australia6; Department of Pediatrics, Sanggyepaik Hospital,
Inje University College of Medicine, Obang-dong, Kyungnam, Republic of Korea7; Centre for
Infectious Diseases and Microbiology, Westmead Hospital, Westmead, New South Wales, Australia8;
NHMRC Centre of Clinical Research Excellence—Infection & Bioethics in Haematological Malignancy,
Westmead Hospital, Westmead, New South Wales, Australia9; Laboratory of Medical Microbiology and
Immunology, St. Elisabeth Hospital, Tilburg, Netherlands10; and Pathology Queensland Central,
Brisbane, Queensland, Australia11
Received 18 December 2009/Accepted 22 March 2010
Exploration of the genetic diversity of WU polyomavirus (WUV) has been limited in terms of the specimen
numbers and particularly the sizes of the genomic fragments analyzed. Using whole-genome sequencing of 48
WUV strains collected in four continents over a 5-year period and 16 publicly available whole-genome
sequences, we identified three main WUV clades and five subtypes, provisionally termed Ia, Ib, Ic, II, IIIa, and
IIIb. Overall nucleotide variation was low (0 to 1.2%). The discriminatory power of the previous VP2 fragment
typing method was found to be limited, and a new, larger genotyping region within the VP2/1 interface was
proposed.
In 2007, two new human polyomaviruses isolated from res-
piratory samples of pediatric patients suffering from respira-
tory disease were discovered, with one being KI polyomavirus
(KIV) (2) and the other being WU polyomavirus (WUV) (8).
WU polyomavirus shares most of the genomic characteris-
tics of other polyomaviruses, with a noncoding control region
(NCCR) separating the early and late coding regions on op-
posite strands. However, unlike for JCV and BKV, but similar
to what was observed for KIV, a late-region-residing agnopro-
tein gene has not been identified in WUV (8).
Despite being frequently detected in respiratory samples of
ill patients, no distinct disease associations have so far been
conclusively identified for WUV (1, 2, 4, 8, 10, 27). There have
been some suggestions that sequence variation plays a role in
disease severity and pathogenesis in other polyomaviruses (6,
24). Unfortunately, due to the early nature of research into
WUV, there has been a dearth of available complete genomic
sequences.
In this study, we set out to investigate a large sample set of
whole WUV genomes from diverse geographical, temporal,
and clinical origins. Incorporating existing WUV genomes with
this data set allowed us to investigate global WUV genomic
diversity, to characterize the WUV genome, and to propose a
new robust typing scheme.
Sample selection and sequencing. The study sample set was
obtained from both published and undocumented sources (see
Table S1 in the supplemental material). All candidate samples
were either detected with or confirmed by the WU-B and
WU-C real-time PCR assays (3). Of the 48 samples chosen,
only 4 (B38 to -41) had previously been subjected to sequenc-
ing, and these were limited to the NCCR and VP1 regions (5).
Published sources. A total of 33 samples in which WUV had
been detected by PCR in previously published study popula-
tions in Australia (n ? 19) (4, 5), Canada (n ? 4) (1), Neth-
erlands (n ? 4) (27), South Korea (n ? 3) (10), and Sweden
(n ? 3) (13) were selected (see Table S1 in the supplemental
material).
* Corresponding author. Mailing address: Sir Albert Sakzewski Vi-
rus Research Centre, Building C28, Back Rd., Herston, Queensland
4029, Australia. Phone: 61-7-3636 8719. Fax: 61-7-3636 1401. E-mail:
seweryn@uq.edu.au.
† Supplemental material for this article may be found at http://jvi
.asm.org/.
?Published ahead of print on 31 March 2010.
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Undocumented sources. WUV-positive samples from ongo-
ing studies were also used (n ? 15) and included respiratory
samples collected during 2008 from South-East Queensland
(Australia) hospital patients suffering from respiratory disease
(n ? 5), nose and throat swabs from hematology patients
(Westmead Hospital, NSW, Australia) (including hematopoi-
etic stem cell transplant recipients [n ? 4]), and nasopharyn-
geal swabs from pediatric otitis media (OM) patients in an
isolated Northern Territory (Australia) indigenous community
(n ? 6) (see Table S1 in the supplemental material).
Overlapping regions spanning the entire WUV genome
were amplified utilizing 10 primer pairs (see Table S2 in the
supplemental material). cDNA fragments were sequenced bi-
directionally with BigDye 3.1 chemistry (Applied Biosystems
Pty. Ltd., Australia), with anomalous sequencing results be-
tween overlapping regions reamplified and resequenced.
Alignments, entropy plots, and contig assembly were
achieved using BioEdit version 7.0.5.3 (9). The GenBank
accession numbers for all generated genomes are shown in
Table S1 in the supplemental material. All numbering con-
ventions follow the prototype B0 (GenBank accession num-
ber NC_009539) WUV sequence.
Sequence diversity. The overall genomic variability of the 64
WUV strains investigated in this study was low (0 to 1.2%),
with several islands of dense diversity in the VP2, VP1, and
LTAg N-terminal-end regions (Fig. 1). Variation was greatest
FIG. 1. Entropy plot showing the levels of nucleotide variation in the 64 aligned WUV genomes along each genomic position as well as the
locations of each gene, the NCCR, and the two evaluated typing regions. Numbering is based on prototype strain B0. Open stars along entropy
bars indicate type-specific SNPs, which correlate with the SNP table positioned below each graph. A type-specific SNP was defined as a position
which consistently holds a different nucleotide in one or more subtypes in comparison to the consensus sequence. G/a or G/c indicates a conserved
change where a minority of type-specific SNPs contain an alternate base, such as adenine or cystine. Shaded positions in the SNP table indicate
one or more non-type-specific variations at that position from other publicly available sequences. Dashes indicate a point deletion. The consensus
sequence (a) was defined as the majority nucleotide at that position between all genotypes.
6230NOTESJ. VIROL.

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in the VP1 region on both the nucleic and the amino acid levels
and, to a lesser extent, in the VP2 and STAg genes (see Table
S3 in the supplemental material).
Recurring variant positions were noted in the core NCCR,
including several type-specific conserved changes (Fig. 1). No
variation was observed in the NCCR past position 286, regard-
less of genotype, patient origin, or patient immune status,
making the late-NCCR-to-early-VP2 stretch comprising nucle-
otides (nt) 287 to 722 the largest fully conserved region within
the WUV genome. This conservation was also found in the
variation-rich NCCR sequences described by Sharp et al. (21),
implying that a strong negative selective pressure is being ex-
erted on that region. It is tempting to speculate that the lack of
variation in the region which typically codes for the agnopro-
tein in other polyomaviruses could be an indication of a yet-
to-be-discovered regulatory or coding function critical to
WUV viability or fitness.
Phylogeny of whole-genome ex-NCCR. After removal of the
LTAg and VP2 start codon-framed NCCR, genomic sequences
from this study, along with the 16 WUV genomes available in
GenBank (under accession numbers EU711058, EU711057,
EU711056, EU711055,EU711054,
NC_009539, EU358769, EU358768, EU296475, EF444554,
EF444553, EF444552, EF444551, and EF444550 as of 17 Sep-
tember 2009), were analyzed using neighbor-joining (NJ) ana-
lyses with 1,000 bootstraps and the Tamura-Nei substitution
method (Mega4.1) (26). The best available nucleotide substi-
tution model was chosen with the help of the FindModel ap-
plication (http://www.hiv.lanl.gov/content/sequence/findmodel
/findmodel.html).
Three clear clades supported by high bootstrap values (?99)
were evident and, in accordance with BKV typing terminology,
were putatively named genotypes I, II, and III (Fig. 2). Parallel
likelihood heuristic (PAUP* 4.0b10) (25) and maximum par-
simony (MEGA 4.1) (26) analyses confirmed the distinct na-
ture of the 3 clades, producing equally high bootstrap values
for all 3 genotypes (data not shown). On the basis of the
genomic NJ tree, further subdivisions were evident within ge-
notypes I and III. Genotype I could be split into what could be
considered the main body of the group, or subtype Ia, followed
by the major branch Ib and the divergent subtype Ic (Fig. 2).
Genotype III could be further divided into two groups, subtype
IIIa and subtype IIIb (Fig. 2). Network phylogeny generated in
the SplitsTree4 software package (http://www.splitstree.org/)
(11) by the Neighbor-Net method confirmed the distinct sep-
aration of the three main genotypes as well as the five subtypes
(see Fig. S1 in the supplemental material). Additionally, sub-
type Ic retained an association with the main body of genotype
I while at the same time illustrating its divergent nature (see
Fig. S1 in the supplemental material). All individual gene NJ
tree analyses retained the distinction between genotypes; how-
ever, further subtype and topographic resolutions showed
marked variability (Table 1; see also Fig. S2 in the supplemen-
tal material).
Of particular note was the reorientation of genotype II,
which brought the orientation of type II closer to that of type
III in both the VP2 and the VP1 trees (see Fig. S2 in the
supplemental material), which would suggest a potential re-
combination event. To explore this further, two methods were
used to investigate possible recombination; however, no such
FJ890982,FJ890981,
evidence was detected with the use of the RDP3beta applica-
tion (http://darwin.uvigo.es/rdp/rdp.html) (16, 17), and no sig-
nificant (P ? 0.90) recombination events were identified by
SplitsTree4’s PHI test (11).
FIG. 2. Unrooted consensus neighbor-joining tree using 1,000
bootstraps of the 64 NCCR-excised WUV genomes.
VOL. 84, 2010NOTES6231

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Genotyping methods. Since its description in the original
WUV article written by Gaynor et al., the 207-bp-long VP2
typing region has been the typing target of choice in the ma-
jority of subsequent WUV studies (1, 8, 10, 28). This region
was originally chosen based on sequence data from six whole
WUV genomes; however, no subsequent evaluation of the
accuracy of such a typing scheme has been performed, partially
due to the limited availability of full WUV genomes. Of addi-
tional concern to us was the short length of the typing region,
as similar-sized regions for BKV have been found to lack
discriminatory power to adequately separate all known geno-
types (14). When applied to the full genomic alignment, the
WUV VP2 typing region was sufficient to discriminate between
types I and III but lacked resolution for most subtypes and type
II, as well as having substantially lower bootstrap values for
many of the clades (Table 1 and Fig. 3). Several discrepancies
were noted with the genotyping scheme of Venter et al. (28),
which utilizes the short VP2 typing region, although a thor-
ough comparison was unable to be preformed, due to unavail-
able full genomes from the appropriate isolates. Briefly, geno-
types 1, 3, and 4 as described by Venter et al. generally
correlated with our genomic types I, IIIa, and IIIb, respec-
tively. No equivalents could be found for genotype 2 described
by Venter et al. and our genome type II and subtype Ic se-
quences in each other’s classification schemes. Subtype Ia was
in overall agreement (excluding isolate v367) with our subtype
Ib, although the remaining subtypes, Ib to -e, did not provide
sufficient differentiation or bootstrap values under the VP2
typing region to allow for further comparison with this study’s
proposed classification scheme.
Thus, we set out to design a more robust typing scheme
which aimed to accurately reflect the genomic NJ tree. In brief,
regions of genetic variability which also contained a high pro-
portion of informative single nucleotide polymorphisms
(SNPs) (Fig. 1) were used to generate potential candidate
WUV typing regions. Consensus NJ trees were generated for
each candidate typing region, and candidate typing region
trees were judged for clade and outer taxonomic unit fidelity as
well as node strength.
Several promising sites across the four main genes and of
lengths ranging from 300 to 800 bp were assessed; however,
most were found to be unable to discriminate between all of
the genotypes and subtypes (data not shown). A 679-bp region
spanning the VP2/VP1 genes at positions 1620 to 2299 pro-
vided better overall clade separation and bootstrap values than
the other analyzed genomic regions as well as the original VP2
typing region (Table 1 and Fig. 3).
On the basis of the VP2/1 typing region comprising nt 1620
to 2299, fully conserved flanking typing primers WUT-F (5?-G
GTACTCCCCATTATGCAGCC-3?) and WUT-R (5?-GGTT
GGAGGGGCTGCAA-3?), which were completely conserved
between all genotypes and flanked the typing region producing
an 806-bp-sized amplicon, were designed. The suggested typ-
ing target is much larger than the original VP2 typing region;
however, larger typing fragment sizes have been recognized to
confer a greater ability to hold discriminatory information and
to achieve greater fidelity with the true tree (14, 18).
Genotype geography and clinical features. From available
clinical records, no apparent correlation with genotype and
immune status or clinical features was noted to occur outside
the OM originating sequences (see Table S1 in the supplemen-
tal material). The inability to identify distinct clinical associa-
tions with genotype may be due to the disproportionate num-
ber of respiratory sample-based WUV sequences used,
although the one sequence obtained from feces clustered with
the general respiratory-oriented population. WUV sequences
from all chronic OM samples clustered and were the sole
representatives of groups II and IIIb (Fig. 2). Due to the small
sample size, it is not known if types II and IIIb are associated
with chronic OM or if they merely reflect geographic diversi-
fication. Further studies are under way to clarify this issue.
Our results suggest a trend between geographical origin and
genotype (see Table S1 in the supplemental material), similar
to that found with JCV and BKV (23, 29), although more
sequence data are needed to confirm such a correlation. In
particular, genotype II and subtype IIIb samples originated
from a small indigenous island community and may represent
genotypes unique to that specific geographical region or to
indigenous Australians more generally.
Sequential and spatially related samples. Of the three iden-
tical genotype II WUV sequences originating from children
with OM, two (O61 and O63) were collected from the same
patient, 3 months apart. The third, O3, was collected from a
second patient, but within the same isolated island community
approximately 3 years prior, which suggests that genetically
stable WUV populations can circulate within communities.
Several WUV isolates were obtained sequentially from im-
munosuppressed patients 1, 7, 15, or 112 days apart (see Table
S1, boxed sequences, in the supplemental material). Sequences
from samples collected over a short time span (1, 7, and 15
days) did not change, while the WUV sequences from the
sample collected 112 days apart contained one nonsynonymous
change (830C 3 T) within the VP2 gene. The collection timing
of these two samples correlated with the progression from
flu-induced symptoms to community-acquired pneumonia of
unknown origin; however, we were not able to determine if the
residue change happened in vivo in response to the patient’s
disease progression or if the acquisition of pneumonia facili-
tated reinfection with a new WUV strain.
Genotype impact on predicted function. Overall, there was
little to no variation seen in predicted early and late protein
TABLE 1. Comparison of bootstrap values for various WUV
regions using NJ phylogenetic treesa
Subtype
comparisonb
Value for:
Genome
NCCR
VP1VP2LTAg STAg
VP2 nt
1354–1560c
VP2/1 nt
1620–2299d
I vs II/III
II vs I/III
III vs I/II
Ib vs Ia/c
Ia vs Ic
IIIa vs IIIb
IIIb vs IIIa
99
99
99
63
73
79
99
99
98
99
64
32
45
98
83
99
97
63*
69
67
85
89
99
99
50*
NA
65
76
66
62
96
NA
NA
55
41
41*
74
93
64*
NA
47*
64
99
95
98
63
73
48
91
aAsterisks indicate strains assigned to incorrect clades. NA, not applicable
(due to lack of clade separation).
bThe genome ex-NCCR tree was used as the reference to determine clade
fidelity.
cDetermined by Gaynor et al. (8).
dDetermined in this study.
6232NOTESJ. VIROL.

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FIG. 3. Unrooted consensus neighbor-joining trees using 1,000 bootstraps of the widely used typing region comprising VP2 nt 1354 to 1560 and
the proposed typing region comprising VP2/1 nt 1620 to 2299.
VOL. 84, 2010 NOTES6233

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features throughout the genotypes (see Table S4 in the sup-
plemental material) (2, 7, 8, 12, 15, 19, 22). Within VP2, all
genotype II and subtype IIIa sequences contained a corrupted
stop codon (TAA 3 TCA), allowing for the C-terminal-end
extension of two additional serines. Two conserved point de-
letions were observed outside the LTAg coding region, a T
deletion in all genotype III and Chinese FZTF and FZ18
sequences within the LTAg intron and a T deletion immedi-
ately downstream of the LTAg stop codon within genotypes II
and III (Fig. 1). The conservation of the deletions within ge-
notypes and across continents suggests regulatory or process-
ing functions within those areas. Indeed, one of the deletions
resides in an analogous area which has been shown to produce
regulatory microRNAs (miRNAs) in other polyomaviruses
(20) and thus may betray likely sites of miRNA production in
the WUV genome. These sites are currently being investigated
for their potential miRNA production using independent ex-
perimental infectious system methods.
Of particular interest were the observed type-specific
changes within VP1’s predicted antigenic loops (see Table S4
in the supplemental material) (12), which could indicate that
genotypes II and III may also be serologically distinct, in a
fashion similar to that observed for BKV. The antigenic qual-
ities of the type-specific VP1 sequences therefore need further
investigation to determine if such antigenic diversity exists.
Accurate classification and typing methods will be critical in
the future for cataloguing WUV isolates and investigating what
role these genetic groups play in human biology and disease.
This study has used full genomic sequencing of samples col-
lected from four continents and various patient groups to char-
acterize the diversity of WU polyomavirus and to propose a
new classification scheme. Finally, a typing method was ratio-
nally designed based on genome-wide informative variables,
which accurately represented the genomic phylogeny.
This study was supported by Royal Children’s Hospital Foundation
grant I 922-034, sponsored by the Woolworths Fresh Futures Appeal.
We thank Cheryl Blechly and the Molecular Diagnostics Unit staff at
the Royal Brisbane Hospital for their assistance in obtaining clinical
samples, Tae-Hee Han for providing WUV-positive material, and
Marieke van der Zalm for sharing samples from the WHISTLER
study.
This study was conducted predominantly at the Sir Albert Sakzewski
Virus Research Centre, Queensland Children’s Medical Research In-
stitute, Children’s Health Service District, Brisbane, Queensland, Aus-
tralia. Additional screening was performed in: Le Centre Hospitalier
Universitaire de Que ´bec and Laval University, Que ´bec City, Quebec,
Canada, the Department of Microbiology, Tumor and Cell Biology,
Karolinska Institutet and Department of Clinical Microbiology,
Karolinksa University Hospital, SE-17176 Stockholm, Sweden, the De-
partment of Pediatrics, Sanggyepaik Hospital, Inje University College
of Medicine, Obang-dong, Kyungnam, Republic of Korea, and the
Laboratory of Medical Microbiology and Immunology, St Elisabeth
Hospital, Tilburg, The Netherlands.
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