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<title>High prevalence of porcine Hokovirus in German wild boar populations</title>
<meta name="Subject" content="Virology Journal 2010 7:171. doi:10.1186/1743-422X-7-171"/>
<meta name="Author" content="Cornelia Adlhoch"/>
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Adlhoch et al. Virology Journal 2010, 7:171
http://www.virologyj.com/content/7/1/171
SHORT REPORT
Open Access
High prevalence of porcine Hokovirus in German
wild boar populations
Cornelia Adlhoch1*, Marco Kaiser1,2, Heinz Ellerbrok1, Georg Pauli1
Abstract
Porcine Hokovirus (PHoV) was recently discovered in Hong Kong. This new Parvovirus of pigs is closely related to
the human Parvoviruses 4 and 5 (PARV4/5) and bovine Hokovirus (BHoV). So far, nothing is known about the
presence and prevalence of PHoV in regions of the world other than Hong Kong. A study was initiated to investigate PHoV in German wild boars from five different geographical regions, using a newly established quantitative
real-time PCR assay. Analysis of collected liver and serum samples revealed high overall prevalence (32.7%; 51/156)
of PHoV in wild boars. The prevalence differed between the regions and increased with age. Two near full-length
genomes and a large fragment for three additional isolates from different regions were sequenced and used for
phylogenetic analysis. The German PHoV sequences from wild boars showed a close relationship with sequences
of isolates from Hong Kong.
Findings
A broad spectrum of parvoviruses is circulating worldwide in different species causing diseases in animals and
humans. One of several novel animal parvoviruses is the
porcine Hokovirus (PHoV), a putative member of the
genus Parvovirus within the family of Parvoviridae. This
new parvovirus PHoV has been described in pigs from
Hong Kong [1]. The non-enveloped parvovirus harbours
a single-stranded DNA genome of approximately 5 kb.
The genome has two open reading frames (ORFs) coding for non-structural and capsid proteins. Closely
related human Parvoviruses PARV 4 and PARV5 were
detected in various samples from healthy and diseased
individuals [2-5].
Up to now no information is available about the presence of PHoV in other pig populations. This study was
initiated to analyse PHoV in German wild boars. Wild
boars in Germany are carrier of Hepatitis E virus (HEV)
and it was of interest to analyse whether this species
habours additional viruses with a zoonotic potential [6].
Liver, serum and bile samples from a total of 156 wild
boars were tested for the presence of PHoV genomes.
Samples (n = 127) were collected during the hunting
season 2007/2008 at several sites in Germany. Collection
points in the different federal states were described in a
* Correspondence: adlhochc@rki.de
1
Robert Koch-Institut, Centre for Biological Security ZBS1, Nordufer 20, 13353
Berlin, Germany
previous study on HEV [6]. Additional samples (9 wild
boar livers) were collected at sites in the federal state of
Hesse (HE) near Herleshausen/Werra, Bauhaus, Oberellen, Friedewald and Lengers between January and March
2008 and 20 wild boar serum samples were collected
between November 2005 and January 2006 in the federal state of Baden Württemberg (BW) at different sites
in the nature reserve Schönbuch which were nearly
identical to the later sampling places in the hunting season 2007/2008. In general, sampling, age determination
of animals, storing and handling of samples were carried
out as published previously [6]. Briefly, liver samples (20
to 40 mg) were homogenized in 500 μl PBS using Precellys ceramic beads (diameter of 1.4 mm; Peqlab Biotechnology, Erlangen, Germany) and the FastPrep ®
FP220A homogenizer (Qbiogene, MP Biomedicals,
Solon, OH, USA). A volume of 200 μl of supernatant of
the centrifugated homogenized liver, bile or serum samples was used for DNA extraction using the NucleoSpin ® Blood preparation kit (Macherey-Nagel, Düren,
Germany). A quantitative real-time PCR (qPCR) assay
using the PHoV_TM 5’ nuclease probe (TaqMan® probe)
in combination with 3 primers PHoV_F/PHoV_R/HPV_R
(Table 1; TIB MOLBIOL, Berlin, Germany) was applied
in this study to determine the copy numbers of PHoV
genomes. The assay was established to detect the newly
described parvovirus PHoV and the human PARV4/
PARV5 using primers binding within a conserved region
© 2010 Adlhoch et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
Adlhoch et al. Virology Journal 2010, 7:171
http://www.virologyj.com/content/7/1/171
Page 2 of 4
Table 1 Overview of the analysed samples for PHoV
Region
npositive/ntotal
<1 year
1-2 years
Adult
Unknown
BW_2005
2/20
(10.0%)
0/9
2/8
0/3
0/0
BW_2007
5/27
(18.5%)
0/10
2/7
0/4
3/6
HE
2/9
(22.2%)
1/3
1/4
0/2
0/0
RP
4/53
(7.5%)
1/27
0/14
3/11
0/1
BB
12/19
(63.2%)
4/9
3/3
3/3
2/4
SA
26/28
(92.9%)
9/9
10/10
7/9
0/0
Total
51/156
(32.7%)
15/67
(22.4%)
18/46
(39.1%)
13/32
(40.6%)
5/11
(45.5%)
n CT<30
17
7
9
1
0
%/Pos*
%/Total†
33.3
10.9
46.7
10.5
50.0
19.6
7.7
3.1
0
0
Number of PHoV positive and total tested animals with prevalences and virus
load (CT-values) overall and divided by age groups and regions of sample
collection.
*: estimated percentages per total samples tested positive; †: estimated
percentages per total samples tested; n: number of animals; BW: Baden
Württemberg, HE: Hesse, RP: Rhineland Palatinate, BB: Brandenburg, SA: Saxony
for each virus. DNA samples in a volume of 2.5 μl were
analysed using the following qPCR protocol in a final
volume of 25 μl with 10xbuffer, 4 mM of MgCl2, dNTP
0.2 mM each, 0.3 μM of each primer, 0.1 μM of probe,
ROX 0.1 μM and Platinum® Taq 0.5 U. Platinum® Taq
DNA polymerase, MgCl2 and dNTPs were obtained from
Invitrogen (Carlsbad, CA, USA) and water (Molecular
Biology Grade) from Eppendorf (Hamburg, Germany).
General reaction conditions for the real-time assay were
95°C for 10 min and 45 cycles with 95°C for 15 sec, 60°C
for 35 sec. Reactions were run in an ABI GeneAmp ®
7500 Detection System (Applied Biosystems, Foster City,
CA, USA). Plasmid pHoko containing the 83 nucleotide
(nt) amplification product from the isolate
PHoV_BW2117 [GQ869539] was established. Insert was
verified by sequencing and copy numbers in this preparation were calculated using standard methods. The plasmid was tenfold serially diluted in water containing gDNA (1 ng/μl) from 106 copies to 1 copy as standards for
quantification of viral genomes. For qPCR each sample
was analysed in duplicate. Copy numbers in samples
were determined using a standard curve. The detection
limit was estimated to be 10 copies of DNA per reaction.
The b-Actin-qPCR assay was used as internal control [6].
The near full-length genomes were generated with PCR
and nested PCR using several primer pairs in combination with primers for sequencing (Table 1) with the Platinum® Taq DNA polymerase kit as described previously
for HEV [6]. Sequence of amplicons was determined
either directly using the PCR product or after cloning
into vector pCR II TOPO (Invitrogen) by sequencing
both strands with the Big Dye3.1 protocol using an automated sequencer (Genetic Analyzer 3130 xl, Applied Biosystems). Sequence data were analysed using ABI PRISM
DNA Sequencing Analysis Software (Version 3.7, Applied
Biosystems). Phylogenetic tree analysis was performed
using MEGA 4.01 [7] program http://www.megasoftware.
net and BLAST network program (National Center for
Biotechnology Information, Bethesda, MD, USA).
The prevalence of PHoV in liver or serum samples of
wild boars differed between sampling regions: While a
low prevalence was seen in Rhineland Palatinate (RP),
BW and HE, the samples collected in Saxony (SA) and
Brandenburg (BB) showed high values (Table 2). The
overall prevalence was 32.7% (51/156), 17 of 51 (33.3%)
animals tested positive with C T -values lower than 30
indicating high copy numbers of more than 106 genome
equivalents per mg of liver tissue, 16 of the 17 animals
(94%) showing high copy numbers were below 2 years
of age (7 animals <1 year, 9 animals 1-2 years). The analysis of the age distribution showed an increase in prevalence for animals older than 1 year, but the highest
proportion of animals with high virus loads was seen in
the group below 2 years of age (Table 1). Corresponding
serum and liver tissue samples from six animals were
tested in parallel showing comparable values for both
compartments. Although it was shown previously that
HEV was detected in high virus load in bile samples [6],
quantification for PHoV in samples from three animals
yielded virus loads that were up to 1000 times lower in
bile samples than in the liver (data not shown). This
result implicates, that PHoV has an organ tropism different from HEV. It can be assumed that a high virus
Table 2 Used primers and probe for the quantitative
analysis and generation of genome fragments of PHoV
Primer name
Orientation 5’-3’
PHoV_F
gTT ggT CCT ggT AAT CCT YTg g
PHoV_R
TCg TAC CgT TCA TCg Tgg Tg
HPV_R
TgC gTA CCg TTC ATC ATg ATg TT
PHoV_TM
PHoV_240F
FAM-Agg gAC CAg Tgg ATg ARg CAg C-BBQ
CAC ACC TAC CTC gCC TAT AAg AAT C
PHoV_1273F
ggT AYT TTg CWg CHT ggg C
PHoV_1408R
CAA TTC ACR CAR CCR TAA gAW ggA
PHoV_1847F
CCg ATC TCC CCg TCT gCC
PHoV_2293F
CCg CAC TgA ggg CTA Cg
PHoV_2492F
ggT AAg MAA WCA TgW CWg CYg C
PHoV_2492R
gCR gCW gWC ATg WTT KCT TAC C
PHoV_4115F
PHoV_4395R
ggg ARA ATT ATg TTY TKC CTC ART ATg g
ATC WAC MCC TgT CAT RAT MgC
PHoV_5288R
CAC TgA TCA gAA ggM ACY TCR TAC AC
F: forward; R: reverse orientation; TM: TaqMan probe
Adlhoch et al. Virology Journal 2010, 7:171
http://www.virologyj.com/content/7/1/171
load of PHoV in liver tissue and serum indicates an
acute or persistent infection with a simultaneous
viraemia.
In order to analyse the phylogenetic relationship
between PHoV in Hong Kong and in Germany near
full-length genome sequences with 4942 nt and 4944 nt
were amplified from isolates BW2117 [GQ869539] and
Sa15 [GQ869540], respectively. Additionally, discontinuous genome sequences of isolates from isolate
BW22 [GQ869543], RP1754 [GQ869541] and BB09
[GQ869542] were generated with total sizes of 4564,
3027 and 3928 nt, respectively. All isolates were incomplete at the 3’-end of the VP1 and VP2 ORFs. The phylogenetic analysis showed that the PHoV isolates from
German wild boars were closely related to Hong Kong
isolates but formed a separate branch in the phylogenetic tree of all known porcine, bovine and human isolates from the GeneBank database (Figure 1). All
German sequences were closely related to each other.
The generated full-length sequences BW2117 and Sa15
differed in 44 of 4796 nucleotides (99.1% identity). A
divergence of up to 40% was found to complete PARV4/
5 sequences, and of 37% to bovine isolates. Compared to
the isolates from Hong Kong a difference of 1.8-2.0%
(Sa15) and 2.0-2.3% (BW2117) was seen for the German
full-length sequences on nucleotide level.
Within NS1 7 (1.1%) unique amino acid exchanges
were observed in the German isolates BW2117 and
Sa15 in comparison to the Hong Kong isolates The
Page 3 of 4
phylogenetic analysis showed that within the ORFs coding for VP1 and VP2 proteins only one (VP1; 0.2%) and
three (VP2; 0.3%) unique amino acid exchanges were
found in the German isolates (BW2117 and Sa15) in
comparison to the Hong Kong isolates.
In this study it was shown that the newly discovered
PHoV is present in European wild boar populations.
The virus was detectable in approximately every third
animal tested. PHoV prevalence showed regional variation as determined in samples from animals collected in
5 geographic regions in Germany. The presence of high
copy numbers of viral genomes in younger animals
(≤ two years) points to an infection early in life. The
increase of the prevalence in older animals supports the
hypothesis of PHoV persistence in liver comparable to
the situation observed for PARV4 and PARV5 infections
in humans [8]. Therefore persistence might be a common feature for this new group of parvoviruses. So far,
no clear disease has been linked to the infection or persistence of these new parvoviruses. The phylogenetic
analysis showed a close relationship of the German
PHoV sequences with the isolates from Hong Kong,
although the European isolates clustered together in one
separate branch. It can be speculated that the virus has
been distributed through pigs that have been imported
from Europe to Hong Kong.
Although the qPCR assay was established to detect all
known isolates of the new Parvovirus group (PARV4,
PARV5, PHoV and BHoV), only PHoV was found in the
wild boar samples. The fact that approximately 600.000
wild boars are shot and consumed every year in Germany clearly highlights a potential route for a zoonotic
transmission to humans. While the prevalence of PHoV
in commercial pigs is yet unknown PHoV has been
detected in a variety of porcine tissues with high virus
load [1] indicating yet another potential risk of zoonotic
transmission of PHoV to humans that urgently needs to
be evaluated.
Acc. numbers of generated sequences:
[P._Hokovirus_BW2117: GQ869539; P._Hokovirus_Sa15: GQ869540; P._Hokovirus_1754: GQ869541; P.
_Hokovirus_BB09: GQ869542; P._Hokovirus_BW22:
GQ869543]
Abbreviations
BB: Brandenburg; SA: Saxony; RP: Rhineland Palatinate; BW: Baden
Württemberg; HE: Hesse; qPCR: quantitative real-time PCR; PHoV: porcine
Hokovirus; BHoV: Bovine Hokovirus; HEV: Hepatitis E virus
Figure 1 Phylogenetic analysis. A phylogenetic tree of new
porcine, bovine and human parvoviruses was constructed using the
neighbor joining method. Bootstrap values for the major branch
points are given in percent. The trees were statistically evaluated in
a bootstrap analysis with 1,000 replicates. The new German PHoV
sequences are indicated with a dot.
Acknowledgements
The authors thank Anna Löwa and Markus Ulrich for excellent technical
support and Ursula Erikli for copy-editing. The authors are grateful to
P. Linderoth at the LVVG Aulendorf (BW), who provided samples from the
nature reserve, U. Hohmann and D. Huckschlag from the FAWF Trippstadt
(RP), H.-J. Hormel at the forestry management (BW) and the forest officials J.
P. Huber in Welzow-Proschim (SA), F. Wehnert in Krausnick (BB) and D.
Adlhoch et al. Virology Journal 2010, 7:171
http://www.virologyj.com/content/7/1/171
Page 4 of 4
Preißel-Baranowsky (HE) as well as all collaborating hunters for the
opportunity and the support in collecting wild boar samples.
Author details
1
Robert Koch-Institut, Centre for Biological Security ZBS1, Nordufer 20, 13353
Berlin, Germany. 2GenExpress GmbH, Eresburgstr. 22-23, 12103 Berlin,
Germany.
Authors’ contributions
CA: Study design, sampling, interpretation of the data and manuscript draft.
MK: Sample analysis, phylogenetic analysis, interpretation of the data and
manuscript draft. HE: Critical interpretation of the data and manuscript draft.
GP: Study design, interpretation of the data and approval of the manuscript.
All authors have read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 21 May 2010 Accepted: 25 July 2010 Published: 25 July 2010
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doi:10.1186/1743-422X-7-171
Cite this article as: Adlhoch et al.: High prevalence of porcine Hokovirus
in German wild boar populations. Virology Journal 2010 7:171.
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