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
HAL Id: hal-00719078 https://hal.archives-ouvertes.fr/hal-00719078 Submitted on 19 Jul 2012
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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
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1 Isolation in cell cultures and initial characterisation of two novel bocavirus species
2 from swine in Northern Ireland
3 John McKillena*, Francis McNeillya, Catherine Duffyb, Michael McMenamyb, Irene
4 McNaira, Bernt Hjertnerb, Andrena Millarb, Karen McKaya, Paula Lagana, Brian
5 Adaira, Gordon Allana.
6 *Corresponding author.
7
8 aVeterinary Sciences Division, Agri-Food and Biosciences Institute, Stormont, Belfast
9 BT4 3SD, United Kingdom
10 bSchool of Biological Sciences, Queen’s University Belfast, 97 Lisburn Road, Belfast,
11 BT9 7BL, United Kingdom
12
13 *Corresponding author. Tel.: +44 (0)28 90525752: +44 (0)28 90525823
14 E-mail address: [email protected] (John McKillen).
15 Correspondence address: Veterinary Sciences Division, Agri-Food and Biosciences
16 Institute, Stoney Road, Stormont, Belfast BT4 3SD, United Kingdom
17 Accepted Manuscript
1 Page 1 of 22 18 Abstract
19 We report the isolation in cell cultures of two novel bocavirus species in pigs
20 from farms in Northern Ireland with clinical postweaning multisystemic wasting
21 syndrome (PMWS). We have designated the isolates as porcine bocavirus-3 (PBoV3)
22 and porcine bocavirus-4 (PBoV4). To date 5082 and 4125 bps of PBoV3 and PBoV4
23 have been sequenced, respectively. PBoV3 and PBoV4 show nucleotide homology to
24 other known bocaviruses in swine and other organisms. Open reading frame (ORF)
25 analysis has shown that these viruses have a third small ORF, equivalent to the NP1 ORF
26 that distinguishes the bocaviruses from other parvoviruses.
27 A panel of porcine field sera was screened by indirect immunofluorescence against both
28 viruses. Of the 369 samples analysed, 32 (8.7%) and 35 (9.5%) sera were seropositive
29 for PBoV3 and PBoV4 respectively, thus providing serological evidence of the exposure
30 of swine in the field to bocavirus-like viruses. To date, the clinico-pathological
31 significance of these novel swine bocaviruses, as primary pathogens or as
32 immunosuppresive triggers for other infectious agents, is undetermined.
33
34 Key Words
35 Swine, bocavirus, virus isolation 36 Accepted Manuscript 37 1. Introduction
38
39 Parvoviruses are widespread pathogens causing a variety of diseases in animals. The
40 family parvoviridae is sub-divided into two sub-families; Parvovirinae which infect
2 Page 2 of 22 41 vertebrates and Densovirinae which infect arthropods. Further subdivision of
42 Parvovirinae into genera has not been well classified but currently at least five genera
43 have been described; Parvovirus, Erythrovirus, Dependovirus, Amdovirus and Bocavirus.
44 Two other genera have recently been proposed; Hokovirus (Lau et al., 2008) and Cnvirus
45 (Hijikata et al., 2001; Wang et al., 2010). Parvoviruses are small non-enveloped viruses
46 and have a single stranded DNA genome of approximately 5 kb.
47 The genus Bocavirus, currently consists of bovine parvovirus-1 (BVP-1) (Abinanti and
48 Warfield, 1961), canine minute virus (CMV) (Carmichael, 2004), gorilla bocavirus
49 (Kapoor et al., 2010a), four genotypes of human bocavirus (HBoV) (Kapoor et al.,
50 2010b) and several species of porcine bocavirus.
51 Recently 1879 base pairs (bps) of a novel bocavirus referred to as porcine boca-like virus
52 (PBo-likeV) has been sequenced from porcine lymph nodes (Genbank accession no.
53 FJ872544) (Blomström et al., 2009 and 2010) using next-generation sequencing.
54 Another virus called porcine parvovirus 4 (PPV4) has been detected in a pig in the USA.
55 This virus is related to bovine parvovirus 2, but is similar to the genus bocavirus in terms
56 of genomic arrangement (Genbank accession no. GQ387499) (Cheung et al., 2010). In
57 China a further two putative bocavirus species have been detected and named porcine
58 bocavirus 1 (PBoV1), (Genbank accession no. HM053693) and porcine bocavirus 2 59 (PBoV2) (GenbankAccepted accession no. HM053694). TheManuscript same researchers have also identified 60 two other partial bocavirus-like sequences, referred to as porcine bocavirus isolates 6V
61 and 7V (Genbank accession nos. HM053672 and HM053673).
62 This manuscript describes the isolation and initial characterization of two novel porcine
63 bocavirus isolates (Genbank accession nos. JF512472 and JF512473) that are distinct
3 Page 3 of 22 64 from each other and from other porcine boca-like viruses previously reported. The viral
65 genomes are compared to the other swine bocaviruses, the other known parvoviruses and
66 to representative members of the sub-family Parvovirinae.
67
68 2. Materials and Methods
69
70 2.1 Origin of bocavirus-like virus isolates.
71 Two studies were carried out in 2004.
72 Farm A: A longitudinal study was conducted on a pig farm in Northern Ireland with an
73 ongoing problem with post-weaning multisystemic wasting syndrome (PMWS).
74 Nasopharyngeal swabs, faecal swabs and clotted blood samples were taken from 51
75 piglets derived from five sows when the youngest litter was one week old and thereafter
76 at days 19, 29, 40, 49, 57, 68 and 93 post farrowing.
77 Farm B: In a separate study, tissue samples were taken from three 3-week old, two 6-
78 week old and two 9-week old pigs submitted from a farm with an ongoing problem with
79 PMWS.
80
81 2.2 Virus isolation 82 Tonsil, or faecalAccepted swab suspensions and tissue homogenates Manuscript were inoculated into roller 83 tube cultures of semi-confluent primary pig kidney cell lines using standard methods.
84 Cultures were examined for signs of cytopathic effect (CPE) after 72 hours and thereafter
85 at daily intervals. After six days the cultures were subjected to three freeze/thaw cycles
86 and the cell lysates (200µl) were inoculated into fresh primary pig kidney cells.
4 Page 4 of 22 87 Cultures that exhibited an evident CPE after two passages were expanded into cell culture
88 flasks of primary pig kidney, frozen and thawed as before and the lysates stored at -80oC.
89 Unidentified cultures of particular interest were purified by either sucrose gradient
90 purification alone or sucrose coupled with CsCl gradient purification using standard
91 methods. Viral-containing aliquots were identified by electron microscopy and by
92 immunofluorescent assay using pooled sera from the five maternal sows from farm A.
93
94 2.3 Testing of isolates for common porcine viruses
95 Cell lysates (200 l) of cultures that exhibited a CPE or were positive by IFA with
96 the sow sera were screened by PCR or reverse-trancriptase PCR (RT-PCR) for common
97 porcine viruses. DNA and RNA was extracted from 200 μl of culture material using the
98 Qiagen DNA Blood Mini Kit (Qiagen, Crawley, U.K.) and QIAamp RNA Blood Mini
99 Kit (Qiagen), respectively, according to the manufacturer’s instructions.
100 PCV1, PCV2 (Ouardani et al., 1999), PPV (Wilhelm et al., 2006) and adenovirus
101 PCR assays were carried out using HotStarTaq Master Mix (Qiagen) according to the
102 manufacturer’s instructions. Enterovirus CPE groups 1, 2 and 3 (Palmquist et al., 2002,
103 Krumbholz et al., 2003) and reovirus (Leary et al., 2002) RT-PCR assays were carried
104 out using Superscript III SuperScript™ One-Step RT-PCR System with Platinum Taq 105 DNA PolymeraseAccepted (Invitrogen, Paisley, U.K.) according Manuscript to the manufacturer’s 106 instructions.
107
108 2.4 Production of swine antisera
109 Six 26-day old snatch farrowed colostrum deprived piglets were inoculated oro-nasally
5 Page 5 of 22 110 with PCV2. Five days later each animal was infected oro-nasally with a 4 ml tissue
111 culture pool of unidentified isolates from both farm studies. The piglets were euthanized
112 32 days later. Blood samples were taken immediately prior to euthanasia. PCV2
113 antibodies were detected by IPMA as previously described (McNair et al., 2004). The
114 sera were pooled and screened by IIF (1/100 dilution) against coverslip cultures of
115 primary kidney cells infected with the unidentified viruses, using an anti-swine FITC
116 conjugate (1/30). The pooled serum was subsequently used as an immunoreagent to
117 detect the unidentified isolates in cell culture.
118
119 2.5 Cloning and sequencing of PBoV3 and PBoV4
120 An aliquot of nucleic acid (100 µl), extracted from a CsCl purified fraction (1.2
121 ml) of one of the isolates of interest (PBoV3) using a standard phenol/chloroform method
122 (Russell and Sambrook, 2001), was analysed on a 1% TAE agarose gel. The single
123 stranded or double stranded status of the resulting bands was confirmed by acridine
124 orange staining using a variation of the method described by McMaster et al., 1977.
125 A double stranded DNA band of approximately 5 kb was excised from the gel,
126 digested using a selection of common restriction enzymes, cloned into pUC19 vector,
127 transformed into One Shot® TOP10 cells (Invitrogen) and incubated on agar containing 128 X-gal. SeventyAccepted five white colonies were selected Manuscript for further analysis. Half of each 129 colony was scrapped of using a pipette tip, placed in 10 µl of dH2O and boiled for 10
130 minutes. Two µl of this was used for PCR using M13 primers. Colonies with PCR
131 products of greater than 500bps were selected and plasmid minipreps were prepared
132 using QIAprep Spin Miniprep Kit (Qiagen). The plasmid preparations were used as
6 Page 6 of 22 133 template for sequencing reactions using BigDye Terminator Kit version 3.1 (Applied
134 Biosystems, Warrington, U.K.), primed with M13 forward and reverse primers. The
135 resulting sequences were assembled, trimmed and analysed using Vector NTI software
136 (Invitrogen).
137 These sequences or consensus sequences from fragments that aligned with each other
138 were submitted for nucleotide (blastn) query and translated nucleotide query (blastx)
139 (http://blast.ncbi.nlm.nih.gov/Blast.cgi).
140 PCR primers were designed for each sequence or alignment that showed homology to the
141 known bocaviruses by blastx (Oligo 5.0 software, Molecular Biology Insights).
142 Combinations of these primers were then used to amplify the areas between the
143 clones using Expand Long Template PCR System (Roche Diagnostics L.T.D., Burgess
144 Hill, U.K.). The PCR amplicons were sequenced by primer walking.
145 An alignment was made of complete genome sequences of HBoV, CMV and
146 BPV1. A number of short degenerate reverse primers were designed as near as possible
147 to the 3’ of the three genomes based on regions of homology. These primers were then
148 used to amplify extracts of PBoV using already existing forward primers also located
149 near the 3’ end of the known sequence PBoV3.
150 Amplicons produced by both these sequencing strategies were sequenced and analysed in 151 Vector NTI andAccepted blastn as described. Manuscript 152 All the existing primers designed during the sequencing of PBoV3 were used for
153 preliminary sequencing and analysis of a second isolate of interest (PBoV4).
154
155 2.6 Genomic and phylogenetic analyses of PBoV3 and PBoV4 nucleotide sequences
7 Page 7 of 22 156 The assembled sequences of PBoV3 and PBoV4 were compared to those of
157 HBoV, BVP1 and CMV and the other swine bocaviruses (Table 1). The sequences were
158 converted to Fasta format and anlysed using ClustalW1.81 (http://align.genome.jp/) for
159 nucleotide similarity (Table 1). Included in the analysis is a range of other parvoviruses.
160 A phylogenetic tree was created using MEGA version 4 (Tamura et al., 2007)
161 (bootstrapped neighbour joining analysis) with parvovirus complete genome or almost
162 complete genome sequences from GenBank, along with the PBoV3 and PBoV4 partial
163 sequences.
164 The assembled sequences of PBoV3 and PBoV4 were analysed for potential open
165 reading frames (ORFs) using ORF finder software on the National Centre for
166 Biotechnology Information (NCBI) website, http://www.ncbi.nlm.nih.gov/gorf/gorf.html.
167
168 2.7 Swine sera survey
169 A panel of 369 miscellaneous swine sera (1/50 and 1/250 dilutions) were screened by IIF
170 using an anti-swine FITC conjugate (1/30) against coverslip cultures of primary pig
171 kidney inoculated with the unidentified isolates. These sera dated from 1985-2007 but the
172 majority of samples were collected in 2004 and 2007.
173 174 3. Results Accepted Manuscript 175 3.1 Virus isolation
176 As expected, a number of common porcine viruses including porcine adenovirus,
177 enteroviruses and reoviruses, circovirus and porcine parvovirus were isolated from
178 animals in both farm studies. Two unidentified isolates that were recovered were subject
8 Page 8 of 22 179 to further investigation.
180 In the longitudinal study, the faecal suspension from piglet 41 at the fourth sampling
181 exhibited an evident CPE in the form of rounded cells after four passages in primary pig
182 kidney cell culture. The isolate was subsequently referred to as PBoV4.
183 In the second study, a primary pig kidney cell culture inoculated with a homogenate of
184 small intestine (SI3) from a six-week old piglet also exhibited a CPE in the form of
185 rounded cells. Post-mortem analysis reported that this animal was in poor condition and
186 had pneumonia and severe diarrhoea. This virus is designated as PBoV3.
187
188 3.2 Testing of isolates for common porcine viruses
189 PCR and RT-PCR analyses established that the CPE or IFA staining with sow sera from
190 PBoV3 and PBoV4 isolates were not due to the presence of PCV1, PCV2, PPV, porcine
191 enterovirus types 1, 2 and 3, porcine adenovirus or porcine reovirus.
192 Indirect immunofluorescence analyses using specific sera against porcine
193 adenovirus, PCV1, PCV2 and PPV also proved negative. The isolates therefore remained
194 unidentified.
195
196 3.3 Swine antiserum production 197 FollowingAccepted experimental infection with PCV2 Manuscript and a selection of the unidentified 198 isolates of interest, a pool of terminal sera was produced. This pool was used to detect
199 the unidentified isolates in cell culture by indirect immunofluorescence. Strong nuclear
200 and faint cytoplasmic staining was observed in PBoV3 infected cell cultures (Fig. 1a). A
201 similar pattern of staining was detected against isolate PBoV4 (Fig. 1b).
9 Page 9 of 22 202
203 3.4 Electron microscopy
204 Viral-containing fractions from sucrose gradient and CsCl gradient purification
205 were identified by electron microscopy (Fig. 2a and Fig. 2b). Virus particles were clearly
206 evident in the samples. Virus particles of both isolates were estimated to be 25-30nm in
207 diameter and similar in size to other known members of the sub-family Parvovirinae.
208
209 3.5 Cloning and sequencing of PBoV3 and PBoV4
210 By sequencing cloned restriction fragments as well as the amplicons from PCR generated
211 by amplifying between the clones and by consensus PCR, 5082 bps of PBoV3 and 4125
212 bps of PBoV4 were sequenced.
213 Initial partial sequencing of PBoV3 lower band produced an identical sequence to that of
214 the upper band.
215 Partial PBoV3 and PBoV4 sequence was submitted to GenBank (Acc. No. JF512472-
216 JF512473).
217
218 3.6 Phylogenetic comparisons of PBoV3 and PBoV4 nucleotide sequences to existing
219 bocaviruses 220 TableAccepted 1 is a table of similarities (ClustalW Manuscript 1.81) of the sequences of PBoV3 and 221 PBoV4 compared to each other and to the sequences of the known bocavirus sequences
222 and representatives of the sub-family parvovirinae. PBoV3 and PBoV4 are 84%
223 homologous. PBoV3 is 41-49% homologous to non-porcine bocaviruses (HBoV1-4,
224 CMV and BVP1) PBoV4 is 36-46% homologous to these viruses.
10 Page 10 of 22 225 Among the swine bocaviruses PBoV3 and PBoV4 partial sequences show least similarity
226 to PBoV-like virus from Sweden, with 42% and 11% homology respectively. The two
227 viruses are more similar to the Chinese viruses (PBoV1 and 2), showing homologies of
228 50% and 51% (PBoV3) and 41% and 51% (PBoV4). PBoV3 is 73% and 78%
229 homologous to the partial Chinese sequences 6V and 7V respectively, whereas PBoV4 is
230 52% homologous to both.
231 Figure 3 shows the Mega version 4 generated phylogenetic tree of PBoV partial
232 sequences and complete or nearly complete genome sequences of other parvoviruses.
233 With both viruses there were three putative ORFs on the positive strand over 200 amino
234 acids in size. These corresponded closely in size and position to the NS1, NP1 and
235 (partial) VP1/2 ORFs of the other known bocaviruses according to ORF analysis of their
236 NCBI Genbank files. The putative ORFs corresponding to NS1 in PBoV3 and PBoV4
237 were considerably different in size at 800 and 667 amino acids respectively.
238
239 3.7 Swine sera survey
240 Of the 369 sera analysed, 32 (8.7%) and 35 (9.5%) samples were seropositive for PBoV3
241 and PBoV4, respectively.
242 243 4. DiscussionAccepted Manuscript 244 The application of molecular methods to the discovery of new viruses has led to the
245 identification of several new members of the sub-family Parvovirinae. Two new
246 parvoviruses named as hokoviruses have been isolated recently from pigs and cattle and
247 phylogenetic analysis of these has determined them to be related to the human parvovirus
11 Page 11 of 22 248 Parv4 (Lau et al., 2008). These viruses may eventually be assigned to a new genus,
249 Hokovirus. A virus referred to as porcine parvovirus 2 (PPV2) has been isolated from
250 swine in Myanmar (Hijikata et al., 2001) and has been subsequently reported in China
251 (Wang et al., 2010). It has been suggested that this virus could be designated as another
252 genus called Cnvirus. Nucleic acids from another virus, referred to as porcine parvovirus
253 4 (PPV4) has been sequenced from a lung lavage taken from a diseased pig co-infected
254 with PCV2. PPV4 is most closely related to bovine parvovirus 2 in terms of genomic
255 homology, yet it has the three ORF genomic arrangement associated with bocaviruses.
256 As well as the known primate (Kapoor et al., 2010a+b), bovine (Abinanti and Warfield,
257 1961) and canine bocaviruses (Carmichael, 2004), a number of bocavirus-like sequences
258 have recently been detected in pig samples. One of these was isolated from the lymph
259 nodes of a PMWS-affected pig in Sweden using next generation sequencing (Blomström
260 et al., 2009). Investigation of the prevalence of this virus found that it was almost twice
261 as prevalent in PMWS affected than non-PMWS affected pigs sampled in Sweden from
262 2003-2007 (Blomström et al., 2010). This virus has subsequently been found to be
263 widespread in pigs in China where it has been reported to have a statistically significant
264 association with respiratory disease (Zhai et al., 2010), and the full coding sequence has
265 been described (Zeng et al., 2011). Two further putative bocavirus-like species detected 266 in China (PBoV1Accepted and PBoV2), (Genbank accession Manuscript nos. HM053693 and HM053694) 267 also have the characteristic three ORF genomic arrangement associated with bocaviruses.
268 Two partial sequences identified by the same researchers (isolates 6V and 7v, Genbank
269 accession nos. HM053672 and HM053673) will likely represent another two novel swine
270 bocavirus species. The two bocavirus isolates described in this manuscript (PBov3 and
12 Page 12 of 22 271 PBoV4) also represent two distinct bocavirus species and to date are the only swine
272 bocaviruses isolated that have been adapted to grow in tissue culture.
273 These viruses were isolated as part of two swine farm studies conducted in Northern
274 Ireland, were grown in cell cultures and using a variety of molecular strategies 5082
275 nucleotides of PBoV3 and 4125 nucleotides of PBoV4 were sequenced.
276 Clustal W analysis of the nucleic acid sequences, phylogeny and the ORF analysis
277 confirmed the designation of the two isolates as bocaviruses. At a nucleic acid level the
278 similarities of PBoV3 and PBoV4 with the other known bocaviruses are consistent with
279 the similarities among the known bocaviruses. According to the International Committee
280 for the Taxonomy of Viruses the criteria for defining a new species in the bocavirus
281 genus is less than 95% homology in the non-structural genes (http://www.ictvdb.org/). It
282 is likely therefore that PBoV1-4, PBo-likeV and viruses 6V and 7V represent seven
283 distinct species of swine bocavirus, although more sequencing of 6V and 7V will be
284 required as well as more specific analysis of the non-structural genes of the other viruses.
285 Mega 4 analysis produced a phylogenetic tree confirming that the PBoV3 and 4 isolates
286 are related to the other bocavirus. Phylogentic analyses of the protein sequences of the
287 three genes in the bocaviruses will be carried out after full sequencing of PBoV3 and
288 PBoV4. 289 Agarose gel electrophoresisAccepted of nucleic acid isolated Manuscript from purified viral particles showed 290 one double and one single stranded DNA band. Parvoviruses vary regarding the amount
291 and sense of DNA that is encapsidated. For example, bovine parvovirus 1 encapsidates
292 approximately 10 to 15% of the plus sense strand (Chen et al., 1988). Therefore the gel
293 electrophoresis pattern observed could be consistent with a parvovirus that encapsulated
13 Page 13 of 22 294 both positive and negative sense strands in different amounts / proportions.
295 The sequences were subjected to ORF analysis using NCBI ORF finder. Bocaviruses are
296 known to have three major open reading frames encoding two non-structural proteins and
297 a nested set of structural proteins. The 5' ORF codes for a large non-structural protein
298 NS-1, the smaller middle ORF encodes another non-structural protein and the right hand
299 ORF encodes a nested set of structural proteins. ORF analysis found that both PBoV3
300 and PBoV4 have three potential ORFs over 200 amino acids in size that correspond with
301 those of the known bocaviruses. Analysis of the NS1 ORF shows for PBoV4 that two
302 nucleotide changes coding for putative amino acid 668 result in termination of the NS-1
303 ORF at this point, significantly shorter than the 800 aa size of the PBoV3 counterpart.
304 However, both the NS1 and NP1 ORFs from both PBoV3 and PBoV4 fit into the range
305 of sizes of the ORFs of the other bocaviruses. No stop codon was found for the PBoV
306 VP1/2 sequence using NCBI ORF Finder, but comparison with the other bocaviruses
307 would suggest that this is very close to being the complete VP1/2 coding sequence.
308 To date, the clinico-pathological significance of these novel swine bocaviruses has not
309 been determined. The initial association of PBo-likeV with respiratory infections and
310 increased prevalence in PMWS-affected pigs is of significance. Indeed in this study,
311 PBoV3 was recovered from a piglet that presented with pneumonia and severe diarrhoea. 312 Even if PBoV3Accepted and PBoV4 are not directly associated Manuscript with any disease they may have 313 the potential to function as immunosuppresive triggers for other infectious agents. An
314 initial serological survey detected that 32 out of 369 sera tested were seropositive for
315 PBoV3 and 35 were seropositive for PBoV4. With the exception of two serum samples
316 dating from 1990, all of the seropositive sera were sampled in either 2004 or 2007.
14 Page 14 of 22 317 However, given the relatively low levels of antibody detected in positive samples, it
318 cannot be discounted that negative results were a reflection of antibody degradation over
319 time. Nevertheless, these results confirmed that swine in the field had been exposed to
320 bocavirus-like viruses. Further serological investigation will be carried out on archival
321 samples. Optimised real-time PCR based assays will be developed to detect the viral
322 genome. It is likely that these viruses are widespread in pig populations. It is intended
323 that the pathogenicity of the virus will be determined by experimental infection in
324 colostrum deprived pigs and monoclonal antibodies produced for use as a serological
325 tool.
326
327 5. Acknowledgments:
328 This work was supported in part by the U.K. Biotechnology and Biological Sciences
329 Research Council, grant no. BB/F020171/1 and by Merial L.T.D.
330
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394
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396 (MEGA) software version 4.0.
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402 parvovirus in field samples. J Virol Methods. 2006 Jun;134(1-2):257-60.
403
404 Zeng S, Wang D, Fang L, Ma J, Song T, Zhang R, Chen H, Xiao S. Complete coding sequences and
405 phylogenetic analysis of porcine bocavirus. J Gen Virol. 2011 Apr;92(Pt 4):784-8.
406
407 Zhai S, Yue C, Wei Z, Long J, Ran D, Lin T, Deng Y, Huang L, Sun L, Zheng H, Gao F, Zheng H, Chen S,
408 Yuan S. High prevalence of a novel porcine bocavirus in weanling piglets with respiratory tract symptoms
409 in China. Arch Virol. 2010 May 22.
410
411 Figure Captions
412
413 Fig. 1 Indirect immunofluorescence detection of (a) PBoV3 and (b) PBoV4 in primary pig
414 kidney cells with polyclonal swine antiserum (1/100 dilution). Strong nuclear staining is
415 evident and faint cytoplasmic staining can also be seen.
416
417 Fig. 2a Electron microscopy of virus (a) PBoV3 and (b) PBoV4.
418
419 Fig. 3 Mega version 4 bootstraped neighbour joining phylogenetic tree of PBoV3 and
420 PBoV4 partialAccepted sequences with full or nearly full genomeManuscript sequences of representatives of
421 the Parvovirinae. Bootstrapping was carried out with 1000 replicates.
422
423
18 Page 18 of 22 Table
% NA homology % NA homology
Porcine bocavirus-3 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),Accepted human bocavirus-4 NI-385 (5104bps, Manuscript 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
Fig. 1b Accepted Manuscript
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Fig. 2a
Fig. 2b
Accepted Manuscript
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Fig. 3 Accepted Manuscript
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