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Isolation in cell cultures and initial characterisation of two novel bocavirus species from swine in Northern
Ireland
John Mckillen, Francis Mcneilly, Catherine Duffy, Michael Mcmenamy, Irene Mcnair, Bernt Hjertner, Andrena Millar, Karen Mckay, Paula Lagan, Brian
Adair, et al.
To cite this version:
John Mckillen, Francis Mcneilly, Catherine Duffy, Michael Mcmenamy, Irene Mcnair, et al.. Isolation in cell cultures and initial characterisation of two novel bocavirus species from swine in Northern Ireland. Veterinary Microbiology, Elsevier, 2011, 152 (1-2), pp.39. �10.1016/j.vetmic.2011.04.013�.
�hal-00719078�
Accepted Manuscript
Title: Isolation in cell cultures and initial characterisation of two novel bocavirus species from swine in Northern Ireland Authors: John McKillen, Francis McNeilly, Catherine Duffy, Michael McMenamy, Irene McNair, Bernt Hjertner, Andrena Millar, Karen McKay, Paula Lagan, Brian Adair, Gordon Allan
PII: S0378-1135(11)00227-6
DOI: doi:10.1016/j.vetmic.2011.04.013
Reference: VETMIC 5276
To appear in: VETMIC
Received date: 3-8-2010 Revised date: 29-3-2011 Accepted date: 14-4-2011
Please cite this article as: McKillen, J., McNeilly, F., Duffy, C., McMenamy, M., McNair, I., Hjertner, B., Millar, A., McKay, K., Lagan, P., Adair, B., Allan, G., Isolation in cell cultures and initial characterisation of two novel bocavirus species from swine in Northern Ireland, Veterinary Microbiology (2010), doi:10.1016/j.vetmic.2011.04.013 This is a PDF file of an unedited manuscript that has been accepted for publication.
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Accepted Manuscript
Isolation in cell cultures and initial characterisation of two novel bocavirus species 1
from swine in Northern Ireland 2
John McKillen
a*, Francis McNeilly
a, Catherine Duffy
b, Michael McMenamy
b, Irene 3
McNair
a, Bernt Hjertner
b, Andrena Millar
b, Karen McKay
a, Paula Lagan
a, Brian 4
Adair
a, Gordon Allan
a. 5
*Corresponding author.
6 7
a
Veterinary Sciences Division, Agri-Food and Biosciences Institute, Stormont, Belfast 8
BT4 3SD, United Kingdom 9
b
School of Biological Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast, 10
BT9 7BL, United Kingdom 11
12
*Corresponding author. Tel.: +44 (0)28 90525752: +44 (0)28 90525823 13
E-mail address: john.mckillen@afbini.gov.uk (John McKillen).
14
Correspondence address: Veterinary Sciences Division, Agri-Food and Biosciences 15
Institute, Stoney Road, Stormont, Belfast BT4 3SD, United Kingdom 16
17
*Manuscript
Accepted Manuscript
Abstract 18
We report the isolation in cell cultures of two novel bocavirus species in pigs 19
from farms in Northern Ireland with clinical postweaning multisystemic wasting 20
syndrome (PMWS). We have designated the isolates as porcine bocavirus-3 (PBoV3) 21
and porcine bocavirus-4 (PBoV4). To date 5082 and 4125 bps of PBoV3 and PBoV4 22
have been sequenced, respectively. PBoV3 and PBoV4 show nucleotide homology to 23
other known bocaviruses in swine and other organisms. Open reading frame (ORF) 24
analysis has shown that these viruses have a third small ORF, equivalent to the NP1 ORF 25
that distinguishes the bocaviruses from other parvoviruses.
26
A panel of porcine field sera was screened by indirect immunofluorescence against both 27
viruses. Of the 369 samples analysed, 32 (8.7%) and 35 (9.5%) sera were seropositive 28
for PBoV3 and PBoV4 respectively, thus providing serological evidence of the exposure 29
of swine in the field to bocavirus-like viruses. To date, the clinico-pathological 30
significance of these novel swine bocaviruses, as primary pathogens or as 31
immunosuppresive triggers for other infectious agents, is undetermined.
32 33
Key Words 34
Swine, bocavirus, virus isolation 35
36
1. Introduction 37
38
Parvoviruses are widespread pathogens causing a variety of diseases in animals. The 39
family parvoviridae is sub-divided into two sub-families; Parvovirinae which infect
40
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vertebrates and Densovirinae which infect arthropods. Further subdivision of 41
Parvovirinae into genera has not been well classified but currently at least five genera 42
have been described; Parvovirus, Erythrovirus, Dependovirus, Amdovirus and Bocavirus.
43
Two other genera have recently been proposed; Hokovirus (Lau et al., 2008) and Cnvirus 44
(Hijikata et al., 2001; Wang et al., 2010). Parvoviruses are small non-enveloped viruses 45
and have a single stranded DNA genome of approximately 5 kb.
46
The genus Bocavirus, currently consists of bovine parvovirus-1 (BVP-1) (Abinanti and 47
Warfield, 1961), canine minute virus (CMV) (Carmichael, 2004), gorilla bocavirus 48
(Kapoor et al., 2010a), four genotypes of human bocavirus (HBoV) (Kapoor et al., 49
2010b) and several species of porcine bocavirus.
50
Recently 1879 base pairs (bps) of a novel bocavirus referred to as porcine boca-like virus 51
(PBo-likeV) has been sequenced from porcine lymph nodes (Genbank accession no.
52
FJ872544) (Blomström et al., 2009 and 2010) using next-generation sequencing.
53
Another virus called porcine parvovirus 4 (PPV4) has been detected in a pig in the USA.
54
This virus is related to bovine parvovirus 2, but is similar to the genus bocavirus in terms 55
of genomic arrangement (Genbank accession no. GQ387499) (Cheung et al., 2010). In 56
China a further two putative bocavirus species have been detected and named porcine 57
bocavirus 1 (PBoV1), (Genbank accession no. HM053693) and porcine bocavirus 2 58
(PBoV2) (Genbank accession no. HM053694). The same researchers have also identified 59
two other partial bocavirus-like sequences, referred to as porcine bocavirus isolates 6V 60
and 7V (Genbank accession nos. HM053672 and HM053673).
61
This manuscript describes the isolation and initial characterization of two novel porcine 62
bocavirus isolates (Genbank accession nos. JF512472 and JF512473) that are distinct
63
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from each other and from other porcine boca-like viruses previously reported. The viral 64
genomes are compared to the other swine bocaviruses, the other known parvoviruses and 65
to representative members of the sub-family Parvovirinae.
66 67
2. Materials and Methods 68
69
2.1 Origin of bocavirus-like virus isolates.
70
Two studies were carried out in 2004.
71
Farm A: A longitudinal study was conducted on a pig farm in Northern Ireland with an 72
ongoing problem with post-weaning multisystemic wasting syndrome (PMWS).
73
Nasopharyngeal swabs, faecal swabs and clotted blood samples were taken from 51 74
piglets derived from five sows when the youngest litter was one week old and thereafter 75
at days 19, 29, 40, 49, 57, 68 and 93 post farrowing.
76
Farm B: In a separate study, tissue samples were taken from three 3-week old, two 6- 77
week old and two 9-week old pigs submitted from a farm with an ongoing problem with 78
PMWS.
79 80
2.2 Virus isolation 81
Tonsil, or faecal swab suspensions and tissue homogenates were inoculated into roller 82
tube cultures of semi-confluent primary pig kidney cell lines using standard methods.
83
Cultures were examined for signs of cytopathic effect (CPE) after 72 hours and thereafter 84
at daily intervals. After six days the cultures were subjected to three freeze/thaw cycles 85
and the cell lysates (200µl) were inoculated into fresh primary pig kidney cells.
86
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Cultures that exhibited an evident CPE after two passages were expanded into cell culture 87
flasks of primary pig kidney, frozen and thawed as before and the lysates stored at -80
oC.
88
Unidentified cultures of particular interest were purified by either sucrose gradient 89
purification alone or sucrose coupled with CsCl gradient purification using standard 90
methods. Viral-containing aliquots were identified by electron microscopy and by 91
immunofluorescent assay using pooled sera from the five maternal sows from farm A.
92 93
2.3 Testing of isolates for common porcine viruses 94
Cell lysates (200 l) of cultures that exhibited a CPE or were positive by IFA with 95
the sow sera were screened by PCR or reverse-trancriptase PCR (RT-PCR) for common 96
porcine viruses. DNA and RNA was extracted from 200 μl of culture material using the 97
Qiagen DNA Blood Mini Kit (Qiagen, Crawley, U.K.) and QIAamp RNA Blood Mini 98
Kit (Qiagen), respectively, according to the manufacturer’s instructions.
99
PCV1, PCV2 (Ouardani et al., 1999), PPV (Wilhelm et al., 2006) and adenovirus 100
PCR assays were carried out using HotStarTaq Master Mix (Qiagen) according to the 101
manufacturer’s instructions. Enterovirus CPE groups 1, 2 and 3 (Palmquist et al., 2002, 102
Krumbholz et al., 2003) and reovirus (Leary et al., 2002) RT-PCR assays were carried 103
out using Superscript III SuperScript™ One-Step RT-PCR System with Platinum Taq 104
DNA Polymerase (Invitrogen, Paisley, U.K.) according to the manufacturer’s 105
instructions.
106 107
2.4 Production of swine antisera 108
Six 26-day old snatch farrowed colostrum deprived piglets were inoculated oro-nasally
109
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with PCV2. Five days later each animal was infected oro-nasally with a 4 ml tissue 110
culture pool of unidentified isolates from both farm studies. The piglets were euthanized 111
32 days later. Blood samples were taken immediately prior to euthanasia. PCV2 112
antibodies were detected by IPMA as previously described (McNair et al., 2004). The 113
sera were pooled and screened by IIF (1/100 dilution) against coverslip cultures of 114
primary kidney cells infected with the unidentified viruses, using an anti-swine FITC 115
conjugate (1/30). The pooled serum was subsequently used as an immunoreagent to 116
detect the unidentified isolates in cell culture.
117 118
2.5 Cloning and sequencing of PBoV3 and PBoV4 119
An aliquot of nucleic acid (100 µl), extracted from a CsCl purified fraction (1.2 120
ml) of one of the isolates of interest (PBoV3) using a standard phenol/chloroform method 121
(Russell and Sambrook, 2001), was analysed on a 1% TAE agarose gel. The single 122
stranded or double stranded status of the resulting bands was confirmed by acridine 123
orange staining using a variation of the method described by McMaster et al., 1977.
124
A double stranded DNA band of approximately 5 kb was excised from the gel, 125
digested using a selection of common restriction enzymes, cloned into pUC19 vector, 126
transformed into One Shot
®TOP10 cells (Invitrogen) and incubated on agar containing 127
X-gal. Seventy five white colonies were selected for further analysis. Half of each 128
colony was scrapped of using a pipette tip, placed in 10 µl of dH
2O and boiled for 10 129
minutes. Two µl of this was used for PCR using M13 primers. Colonies with PCR 130
products of greater than 500bps were selected and plasmid minipreps were prepared 131
using QIAprep Spin Miniprep Kit (Qiagen). The plasmid preparations were used as
132
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template for sequencing reactions using BigDye Terminator Kit version 3.1 (Applied 133
Biosystems, Warrington, U.K.), primed with M13 forward and reverse primers. The 134
resulting sequences were assembled, trimmed and analysed using Vector NTI software 135
(Invitrogen).
136
These sequences or consensus sequences from fragments that aligned with each other 137
were submitted for nucleotide (blastn) query and translated nucleotide query (blastx) 138
(http://blast.ncbi.nlm.nih.gov/Blast.cgi).
139
PCR primers were designed for each sequence or alignment that showed homology to the 140
known bocaviruses by blastx (Oligo 5.0 software, Molecular Biology Insights).
141
Combinations of these primers were then used to amplify the areas between the 142
clones using Expand Long Template PCR System (Roche Diagnostics L.T.D., Burgess 143
Hill, U.K.). The PCR amplicons were sequenced by primer walking.
144
An alignment was made of complete genome sequences of HBoV, CMV and 145
BPV1. A number of short degenerate reverse primers were designed as near as possible 146
to the 3’ of the three genomes based on regions of homology. These primers were then 147
used to amplify extracts of PBoV using already existing forward primers also located 148
near the 3’ end of the known sequence PBoV3.
149
Amplicons produced by both these sequencing strategies were sequenced and analysed in 150
Vector NTI and blastn as described.
151
All the existing primers designed during the sequencing of PBoV3 were used for 152
preliminary sequencing and analysis of a second isolate of interest (PBoV4).
153 154
2.6 Genomic and phylogenetic analyses of PBoV3 and PBoV4 nucleotide sequences
155
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The assembled sequences of PBoV3 and PBoV4 were compared to those of 156
HBoV, BVP1 and CMV and the other swine bocaviruses (Table 1). The sequences were 157
converted to Fasta format and anlysed using
ClustalW1.81(http://align.genome.jp/) for 158
nucleotide similarity (Table 1). Included in the analysis is a range of other parvoviruses.
159
A phylogenetic tree was created using MEGA version 4 (Tamura et al., 2007) 160
(bootstrapped neighbour joining analysis) with parvovirus complete genome or almost 161
complete genome sequences from GenBank, along with the PBoV3 and PBoV4 partial 162
sequences.
163
The assembled sequences of PBoV3 and PBoV4 were analysed for potential open 164
reading frames (ORFs) using ORF finder software on the National Centre for 165
Biotechnology Information (NCBI) website, http://www.ncbi.nlm.nih.gov/gorf/gorf.html.
166 167
2.7 Swine sera survey 168
A panel of 369 miscellaneous swine sera (1/50 and 1/250 dilutions) were screened by IIF 169
using an anti-swine FITC conjugate (1/30) against coverslip cultures of primary pig 170
kidney inoculated with the unidentified isolates. These sera dated from 1985-2007 but the 171
majority of samples were collected in 2004 and 2007.
172 173
3. Results 174
3.1 Virus isolation 175
As expected, a number of common porcine viruses including porcine adenovirus, 176
enteroviruses and reoviruses, circovirus and porcine parvovirus were isolated from 177
animals in both farm studies. Two unidentified isolates that were recovered were subject
178
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to further investigation.
179
In the longitudinal study, the faecal suspension from piglet 41 at the fourth sampling 180
exhibited an evident CPE in the form of rounded cells after four passages in primary pig 181
kidney cell culture. The isolate was subsequently referred to as PBoV4.
182
In the second study, a primary pig kidney cell culture inoculated with a homogenate of 183
small intestine (SI
3) from a six-week old piglet also exhibited a CPE in the form of 184
rounded cells. Post-mortem analysis reported that this animal was in poor condition and 185
had pneumonia and severe diarrhoea. This virus is designated as PBoV3.
186 187
3.2 Testing of isolates for common porcine viruses 188
PCR and RT-PCR analyses established that the CPE or IFA staining with sow sera from 189
PBoV3 and PBoV4 isolates were not due to the presence of PCV1, PCV2, PPV, porcine 190
enterovirus types 1, 2 and 3, porcine adenovirus or porcine reovirus.
191
Indirect immunofluorescence analyses using specific sera against porcine 192
adenovirus, PCV1, PCV2 and PPV also proved negative. The isolates therefore remained 193
unidentified.
194 195
3.3 Swine antiserum production 196
Following experimental infection with PCV2 and a selection of the unidentified 197
isolates of interest, a pool of terminal sera was produced. This pool was used to detect 198
the unidentified isolates in cell culture by indirect immunofluorescence. Strong nuclear 199
and faint cytoplasmic staining was observed in PBoV3 infected cell cultures (Fig. 1a). A 200
similar pattern of staining was detected against isolate PBoV4 (Fig. 1b).
201
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202
3.4 Electron microscopy 203
Viral-containing fractions from sucrose gradient and CsCl gradient purification 204
were identified by electron microscopy (Fig. 2a and Fig. 2b). Virus particles were clearly 205
evident in the samples. Virus particles of both isolates were estimated to be 25-30nm in 206
diameter and similar in size to other known members of the sub-family Parvovirinae.
207 208
3.5 Cloning and sequencing of PBoV3 and PBoV4 209
By sequencing cloned restriction fragments as well as the amplicons from PCR generated 210
by amplifying between the clones and by consensus PCR, 5082 bps of PBoV3 and 4125 211
bps of PBoV4 were sequenced.
212
Initial partial sequencing of PBoV3 lower band produced an identical sequence to that of 213
the upper band.
214
Partial PBoV3 and PBoV4 sequence was submitted to GenBank (Acc. No. JF512472- 215
JF512473).
216 217
3.6 Phylogenetic comparisons of PBoV3 and PBoV4 nucleotide sequences to existing 218
bocaviruses 219
Table 1 is a table of similarities (ClustalW 1.81) of the sequences of PBoV3 and 220
PBoV4 compared to each other and to the sequences of the known bocavirus sequences 221
and representatives of the sub-family parvovirinae. PBoV3 and PBoV4 are 84%
222
homologous. PBoV3 is 41-49% homologous to non-porcine bocaviruses (HBoV1-4, 223
CMV and BVP1) PBoV4 is 36-46% homologous to these viruses.
224
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Among the swine bocaviruses PBoV3 and PBoV4 partial sequences show least similarity 225
to PBoV-like virus from Sweden, with 42% and 11% homology respectively. The two 226
viruses are more similar to the Chinese viruses (PBoV1 and 2), showing homologies of 227
50% and 51% (PBoV3) and 41% and 51% (PBoV4). PBoV3 is 73% and 78%
228
homologous to the partial Chinese sequences 6V and 7V respectively, whereas PBoV4 is 229
52% homologous to both.
230
Figure 3 shows the Mega version 4 generated phylogenetic tree of PBoV partial 231
sequences and complete or nearly complete genome sequences of other parvoviruses.
232
With both viruses there were three putative ORFs on the positive strand over 200 amino 233
acids in size. These corresponded closely in size and position to the NS1, NP1 and 234
(partial) VP1/2 ORFs of the other known bocaviruses according to ORF analysis of their 235
NCBI Genbank files. The putative ORFs corresponding to NS1 in PBoV3 and PBoV4 236
were considerably different in size at 800 and 667 amino acids respectively.
237 238
3.7 Swine sera survey 239
Of the 369 sera analysed, 32 (8.7%) and 35 (9.5%) samples were seropositive for PBoV3 240
and PBoV4, respectively.
241 242
4. Discussion 243
The application of molecular methods to the discovery of new viruses has led to the 244
identification of several new members of the sub-family Parvovirinae. Two new 245
parvoviruses named as hokoviruses have been isolated recently from pigs and cattle and 246
phylogenetic analysis of these has determined them to be related to the human parvovirus
247
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Parv4 (Lau et al., 2008). These viruses may eventually be assigned to a new genus, 248
Hokovirus. A virus referred to as porcine parvovirus 2 (PPV2) has been isolated from 249
swine in Myanmar (Hijikata et al., 2001) and has been subsequently reported in China 250
(Wang et al., 2010). It has been suggested that this virus could be designated as another 251
genus called Cnvirus. Nucleic acids from another virus, referred to as porcine parvovirus 252
4 (PPV4) has been sequenced from a lung lavage taken from a diseased pig co-infected 253
with PCV2. PPV4 is most closely related to bovine parvovirus 2 in terms of genomic 254
homology, yet it has the three ORF genomic arrangement associated with bocaviruses.
255
As well as the known primate (Kapoor et al., 2010a+b), bovine (Abinanti and Warfield, 256
1961) and canine bocaviruses (Carmichael, 2004), a number of bocavirus-like sequences 257
have recently been detected in pig samples. One of these was isolated from the lymph 258
nodes of a PMWS-affected pig in Sweden using next generation sequencing (Blomström 259
et al., 2009). Investigation of the prevalence of this virus found that it was almost twice 260
as prevalent in PMWS affected than non-PMWS affected pigs sampled in Sweden from 261
2003-2007 (Blomström et al., 2010). This virus has subsequently been found to be 262
widespread in pigs in China where it has been reported to have a statistically significant 263
association with respiratory disease (Zhai et al., 2010), and the full coding sequence has 264
been described (Zeng et al., 2011). Two further putative bocavirus-like species detected 265
in China (PBoV1 and PBoV2), (Genbank accession nos. HM053693 and HM053694) 266
also have the characteristic three ORF genomic arrangement associated with bocaviruses.
267
Two partial sequences identified by the same researchers (isolates 6V and 7v, Genbank 268
accession nos. HM053672 and HM053673) will likely represent another two novel swine 269
bocavirus species. The two bocavirus isolates described in this manuscript (PBov3 and
270
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PBoV4) also represent two distinct bocavirus species and to date are the only swine 271
bocaviruses isolated that have been adapted to grow in tissue culture.
272
These viruses were isolated as part of two swine farm studies conducted in Northern 273
Ireland, were grown in cell cultures and using a variety of molecular strategies 5082 274
nucleotides of PBoV3 and 4125 nucleotides of PBoV4 were sequenced.
275
Clustal W analysis of the nucleic acid sequences, phylogeny and the ORF analysis 276
confirmed the designation of the two isolates as bocaviruses. At a nucleic acid level the 277
similarities of PBoV3 and PBoV4 with the other known bocaviruses are consistent with 278
the similarities among the known bocaviruses. According to the International Committee 279
for the Taxonomy of Viruses the criteria for defining a new species in the bocavirus 280
genus is less than 95% homology in the non-structural genes (http://www.ictvdb.org/). It 281
is likely therefore that PBoV1-4, PBo-likeV and viruses 6V and 7V represent seven 282
distinct species of swine bocavirus, although more sequencing of 6V and 7V will be 283
required as well as more specific analysis of the non-structural genes of the other viruses.
284
Mega 4 analysis produced a phylogenetic tree confirming that the PBoV3 and 4 isolates 285
are related to the other bocavirus. Phylogentic analyses of the protein sequences of the 286
three genes in the bocaviruses will be carried out after full sequencing of PBoV3 and 287
PBoV4.
288
Agarose gel electrophoresis of nucleic acid isolated from purified viral particles showed 289
one double and one single stranded DNA band. Parvoviruses vary regarding the amount 290
and sense of DNA that is encapsidated. For example, bovine parvovirus 1 encapsidates 291
approximately 10 to 15% of the plus sense strand (Chen et al., 1988). Therefore the gel 292
electrophoresis pattern observed could be consistent with a parvovirus that encapsulated
293
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both positive and negative sense strands in different amounts / proportions.
294
The sequences were subjected to ORF analysis using NCBI ORF finder. Bocaviruses are 295
known to have three major open reading frames encoding two non-structural proteins and 296
a nested set of structural proteins. The 5' ORF codes for a large non-structural protein 297
NS-1, the smaller middle ORF encodes another non-structural protein and the right hand 298
ORF encodes a nested set of structural proteins. ORF analysis found that both PBoV3 299
and PBoV4 have three potential ORFs over 200 amino acids in size that correspond with 300
those of the known bocaviruses. Analysis of the NS1 ORF shows for PBoV4 that two 301
nucleotide changes coding for putative amino acid 668 result in termination of the NS-1 302
ORF at this point, significantly shorter than the 800 aa size of the PBoV3 counterpart.
303
However, both the NS1 and NP1 ORFs from both PBoV3 and PBoV4 fit into the range 304
of sizes of the ORFs of the other bocaviruses. No stop codon was found for the PBoV 305
VP1/2 sequence using NCBI ORF Finder, but comparison with the other bocaviruses 306
would suggest that this is very close to being the complete VP1/2 coding sequence.
307
To date, the clinico-pathological significance of these novel swine bocaviruses has not 308
been determined. The initial association of PBo-likeV with respiratory infections and 309
increased prevalence in PMWS-affected pigs is of significance. Indeed in this study, 310
PBoV3 was recovered from a piglet that presented with pneumonia and severe diarrhoea.
311
Even if PBoV3 and PBoV4 are not directly associated with any disease they may have 312
the potential to function as immunosuppresive triggers for other infectious agents. An 313
initial serological survey detected that 32 out of 369 sera tested were seropositive for 314
PBoV3 and 35 were seropositive for PBoV4. With the exception of two serum samples 315
dating from 1990, all of the seropositive sera were sampled in either 2004 or 2007.
316
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However, given the relatively low levels of antibody detected in positive samples, it 317
cannot be discounted that negative results were a reflection of antibody degradation over 318
time. Nevertheless, these results confirmed that swine in the field had been exposed to 319
bocavirus-like viruses. Further serological investigation will be carried out on archival 320
samples. Optimised real-time PCR based assays will be developed to detect the viral 321
genome. It is likely that these viruses are widespread in pig populations. It is intended 322
that the pathogenicity of the virus will be determined by experimental infection in 323
colostrum deprived pigs and monoclonal antibodies produced for use as a serological 324
tool.
325 326
5. Acknowledgments:
327
This work was supported in part by the U.K. Biotechnology and Biological Sciences 328
Research Council, grant no. BB/F020171/1 and by Merial L.T.D.
329 330
6. References:
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Figure Captions
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Fig. 1 Indirect immunofluorescence detection of (a) PBoV3 and (b) PBoV4 in primary pig
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kidney cells with polyclonal swine antiserum (1/100 dilution). Strong nuclear staining is
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evident and faint cytoplasmic staining can also be seen.
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Fig. 2a Electron microscopy of virus (a) PBoV3 and (b) PBoV4.
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Fig. 3 Mega version 4 bootstraped neighbour joining phylogenetic tree of PBoV3 and
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PBoV4 partial sequences with full or nearly full genome sequences of representatives of
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the Parvovirinae. Bootstrapping was carried out with 1000 replicates.
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% NA homology Porcine bocavirus-3
% NA homology Porcine bocavirus-4
Porcine bocavirus-3 - 84
Porcine bocavirus-4 84 -
Porcine Bocavirus-SX-CHN 42 11
Porcine bocavirus-1 51 51
Porcine bocavirus-2 50 41
Porcine bocavirus 6V 78 52
Porcine bocavirus 7V 73 52
Human bocavirus-1 45 46
Human bocavirus-2 49 43
Human bocavirus-3 46 45
Human bocavirus-4 46 42
Bovine parvovirus-1 42 39
Canine minute virus 41 36
Porcine parvovirus-4 4 5
Porcine hokovirus 4 4
Porcine parvovirus 1 2
Porcine parvovirus-2 3 5
Bovine parvovirus-2 4 5
Table 1. Clustal W analysis showing % nucleic acid homologies of the Northern Ireland PBoV isolates compared to representatives of the other bocaviruses, other porcine parvovirus and bovine parvovirus 2. Sequence lengths and accession nos. for known bocaviruses are: Porcine Bocavirus-SX-CHN (4786 bps, HQ223038), human bocavirus-1 ST1 (5217 bps, DQ000495), human bocavirus-2 PK-2255 (5134 bps, FJ170279), human bocavirus-3 W855 (5164 bps, FJ948861), human bocavirus-4 NI-385 (5104bps, NC_012729), bovine parvovirus-1 Abinanti (5515bps, DQ335247), canine minute virus GA3 (5402 bps, FJ214110), porcine parvovirus-4 (5905 bps,GQ387499), bovine parvovirus-2 (5610bps, NC_006259), porcine hokovirus HK1 (5043 bps, EU200671), porcine parvovirus-2 (Myanmar erythrovirus, 5118 bps, AB076669), porcine parvovirus NADL-2 (5075 bps, NC_001718), porcine bocavirus-1_Chn (PBoV1, 5173bps, HM053693), porcine bocavirus-2_Chn (PBoV2, 5186bps, HM053694), porcine bocavirus 6V Chn (2407bps, HM053672), porcine bocavirus 7V Chn (2434bps, HM053673).
PBoV3 and PBoV4 sequences are 5082 and 4125 bps respectively.
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Fig. 1a
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Fig. 2a
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Fig. 3
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