Porcine circovirus-like agent P1 is a newly discovered virus containing a single-strand circular genome. The genome of P1 is a DNA molecule of 648 nucleotides which contains eight open reading frames (ORFs) that probably encode potential proteins or polypeptides. Thus it is very important to clarify these proteins' function. Here we provide the methods and analysis of microarray data in detail to characterize the transcriptome profile of P1 with and without the ORF. The relevant microarray data sets have been deposited in Gene Expression Omnibus (GEO) database under accession number GSE71945

Dan Zhang

Fengzhi Wang

Kongwang He

Libin Wen

Genomics Data

PubMed

AbstractPorcine circovirus-like agent P1 is a newly discovered virus containing a single-strand circular genome. The genome of P1 is a DNA molecule of 648 nucleotides which contains eight open reading frames (ORFs) that probably encode potential proteins or polypeptides. Thus it is very important to clarify these proteins' function. Here we provide the methods and analysis of microarray data in detail to characterize the transcriptome profile of P1 with and without the ORF. The relevant microarray data sets have been deposited in Gene Expression Omnibus (GEO) database under accession number GSE71945

Wen, Libin

Wang, Fengzhi

Zhang, Dan

He, Kongwang

Elsevier - Publisher Connector 

Genomics Data 6 (2015) 197–198
Contents lists available at ScienceDirect
Genomics Data
j ou rna l homepage: ht tp : / /www. journa ls .e lsev ie r .com/genomics-data /Data in Brief
Function analysis of proteins encoded by ORFs 1 to 8 of porcine circovirus-like virus P1
by microarray assay
Libin Wen, Fengzhi Wang, Dan Zhang, Kongwang He ⁎
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, Key Laboratory of Veterinary Biological Engineering and Technology, Ministry of Agriculture, National Center for
Engineering Research of Veterinary Bio-products, Nanjing, PR ChinaO
Se
Se
D
E
E
C
⁎ Corresponding author.
http://dx.doi.org/10.1016/j.gdata.2015.09.017
2213-5960/© 2015 Elsevier Inc. This is an open access arta b s t r a c ta r t i c l e i n f oArticle history:
Received 12 September 2015
Accepted 14 September 2015
Available online 18 September 2015
Keywords:
Porcine circovirus-like agent P1
Function of protein
MicroarrayPorcine circovirus-like agent P1 is a newly discovered virus containing a single-strand circular genome. The ge-
nome of P1 is a DNAmolecule of 648 nucleotideswhich contains eight open reading frames (ORFs) that probably
encode potential proteins or polypeptides. Thus it is very important to clarify these proteins' function. Here we
provide the methods and analysis of microarray data in detail to characterize the transcriptome proﬁle of P1
with and without the ORF. The relevant microarray data sets have been deposited in Gene Expression Omnibus
(GEO) database under accession number GSE71945.
© 2015 Elsevier Inc. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).Speciﬁcationsrganism/cell line/tissue Porcine kidney cells (PK15)
x N/A
quencer or array type Affymetris pig gene 1.1 ST Array
ata format Raw
xperimental factors PK15 cells transfected with the ORFs mutant
plasmids and parental clone control
xperimental features Identify gene expression proﬁles in the PK15
cells at 12-hour post-transfection
onsent N/A
mple source location N/ASa1. Direct link to deposited data
The deposited data can be found at: http://www.ncbi.nlm.nih.gov/
geo/query/acc.cgi?acc=GSE71945
2. Experimental design, materials and methods
Porcine circovirus (PCV), a member of the genus Circovirus in the
family Circoviridae, is a small non-enveloped virus with a single-
stranded circular DNA genome of approximately 1.8 kb [1]. To date,
two distinct genotypes of PCV have been identiﬁed: PCV1 and PCV2.
PCV2 has been regarded as amajor pathogen of PCV associated diseases
(PCVAD), of which postweaning multisystemic wasting syndrome
(PMWS) is the major syndrome, having a severe economic impact on
swine production wordwide [2,3].icle under the CC BY-NC-ND license (PCV-like agent P1, discovered in 2007 from the serum of the porcine
with PMWS, contains a circular DNA molecule of 648 nucleotides in
length, which is highly homologous to the genome of PCV2with the ex-
ception of 16 nucleotides [4]. Our previous study conﬁrmed that the P1
virus has been epidemic in China's pig farms and can infect pigs
displaying PMWS-like signs [5,6]. The genome of P1 contains eight
open reading frames (ORFs) [7]. In this work, functions of the viral pro-
teins of P1 were studied by microarray assay.
2.1. Construction of the molecular clone of P1
A tandem dimmer of P1 genome was cloned into the BamHI site of
Bluescript plasmid (Invitrogen), which was regarded as the “parent”
to construct all other P1mutant plasmids. The targeted RNA from a spe-
ciﬁc ORF would no longer be generated by speciﬁc mutation. Mean-
while, it did not cause any amino acid change in other overlappingORFs.
2.2. Transfection, RNA isolation and microarray hybridization
PK15 cells seeded in 6-well tissue culture plates were transfected
with the plasmids using lipofectamine 2000 (Invitrogen) according to
the manufacturer's instruction. We have designed nine experimental
groups, each containing 3 repeats. At 12 post-transfection, total RNA
was extracted from the cells using TRIzol (Invitrogen) following the
manufacturer's recommendations. The quantity and quality of each
RNA sample were evaluated using a NanoDrop spectrophotometer
(Thermo Scientiﬁc, USA).
The total RNA was used to synthesize double-strand cDNA and
in vitro transcribed to cRNA, which was used to synthesize 2nd-cyclehttp://creativecommons.org/licenses/by-nc-nd/4.0/).
198 L. Wen et al. / Genomics Data 6 (2015) 197–198cDNAand then hydrolyzed byRNaseH usingAmbionWTExpression Kit
for Affymetrix GeneChip (Affymetrix, Lot. No. 4011974, USA), and then
2nd-cycle cDNAwas fragmented and the single-stranded cDNA was la-
beled with WT Terminal Labeling Kit (Affymetrix, Lot. No. 900671) and
hybridized on Porcine Gene 1.1 ST Array Strip (Affymetrix, Lot. No.
901798). The hybridized arrays were washed and stained on the
GeneAtlas Fluidics Station (Affymetrix). The ﬂuorescent signals were
detected with a GeneChip Scanner 3000 (Affymetrix).2.3. Microarray data analysis
The scanned imageswereﬁrst assessed by visual inspection then an-
alyzed to generate CEL ﬁles using the default setting of Affymetrix®
GeneAtlas™ Workstation Software. Then the raw data (.CEL ﬁles)
were normalized and summarized with the Affymetrix Microarray
Suite 5.0 (MAS5) and with the Robust Multi-array Average (RMA)
algorithm [8]. In a comparison analysis, a two class unpaired method
in the Signiﬁcant Analysis of Microarray software (SAM, version 3.02)
was applied to identify signiﬁcantly differentially expressed genes
between TEST and CONTROL groups. Q value was set to ﹤0.05 and
transcripts were ﬁltered on the basis of ≥1.2-fold difference.
The microarray CEL ﬁles, normalized data and experimental infor-
mation have been deposited in NCBI's Gene Expression Omnibus data-
base under accession number GSE71945.2.4. Function enrichment analysis
Based on hypergeometric distribution, all differentially expressed
genes were analyzed using two databases, Gene Ontology (GO) and
Kyoto Encyclopedia of Genes andGenomes (KEGG) pathway, and statis-
tical signiﬁcance was calculated when their q-values were b0.05.2.5. Basic analysis
Here, we described that functions of the proteins encoded by ORFs 1
to 8 were characterized bymicroarray. Among these, by KEGG pathway
analysis, the pathways involved in the digestive gland (gastric acid, sal-
ivary and pancreatic) secretion and the neurodegenerative diseases
(Alzheimer's disease and Huntington's disease) induced by ORF1 were
the most interesting.
Acknowledgments
This work was supported by National Natural Science Foundation of
China (31272574, 30972184) and Fund for Independent Innovation of
Agricultural Sciences in Jiangsu Province CX(14)2045.
References
[1] I. Tischer, H. Gelderblom, W. Vettermann, M.A. Koch, A very small porcine virus with
circular single-stranded DNA. Nature 295 (1982) 64–66.
[2] G.M. Allan, J.A. Ellis, Porcine circoviruses: a review. J. Vet. Diagn. Investig. 12 (2000)
3–14.
[3] J. Segales, M. Domingo, Postweaning multisystemic wasting syndrome (PMWS) in
pigs. A review. Vet. Q. 24 (2002) 109–124.
[4] L. Wen, K. He, Z. Yu, A. Mao, Y. Ni, X. Zhang, R. Guo, B. Li, X. Wang, J. Zhou, L. Lv,
Complete genome sequence of a novel porcine circovirus-like agent. J. Virol. 86
(2012) 639.
[5] L. Wen, K. He, H. Yang, Z. Yu, A. Mao, S. Zhong, Y. Ni, X. Zhang, B. Li, X. Wang, J. Zhou,
R. Guo, L. Lv, J. Jiang, Prevalence of porcine circovirus-like agent P1 in Jiangsu, China.
Virol. J. 8 (2011) 543.
[6] L. Wen, K. He, Q. Xiao, Z. Yu, A. Mao, Y. Ni, X. Zhang, B. Li, X. Wang, R. Guo, J. Zhou, L.
Lv, J. Jiang, A novel porcine circovirus-like agent P1 is associated with wasting syn-
dromes in pigs. PLoS One 7 (8) (2012) e41565.
[7] L. Wen, F.Wang, K. He, B. Li, X.Wang, R. Guo, J. Xie, Transcriptional analysis of porcine
circovirus-like virus P1. BMC Vet. Res. 10 (2014) 287–294.
[8] R.A. Irizarry, B. Hobbs, F. Collin, Y.D. Beazer-Barclay, K.J. Antonellis, U. Scherf, T.P.
Speed, Exploration, normalization, and summaries of high density oligonucleotide
array probe level data. Biostatistics 4 (2) (2003) 249–264.


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Function analysis of proteins encoded by ORFs 1 to 8 of porcine circovirus-like virus P1 by microarray assay

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