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Co-infection status of porcine circoviruses (PCV2 and PCV3) and porcine epidemic diarrhea virus (PEDV) in pigs with watery diarrhea in Henan province, central China
Journal Pre-proof Co-infection status of porcine circoviruses (PCV2 and PCV3) and porcine epidemic diarrhea virus (PEDV) in pigs with watery diarrhea in Henan province, central China Zhenhua Guo, Haiyu Ruan, Songlin Qiao, Ruiguang Deng, Gaiping Zhang PII:
S0882-4010(19)31796-6
DOI:
https://doi.org/10.1016/j.micpath.2020.104047
Reference:
YMPAT 104047
To appear in:
Microbial Pathogenesis
Received Date: 12 October 2019 Revised Date:
4 February 2020
Accepted Date: 5 February 2020
Please cite this article as: Guo Z, Ruan H, Qiao S, Deng R, Zhang G, Co-infection status of porcine circoviruses (PCV2 and PCV3) and porcine epidemic diarrhea virus (PEDV) in pigs with watery diarrhea in Henan province, central China, Microbial Pathogenesis (2020), doi: https://doi.org/10.1016/ j.micpath.2020.104047. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Author Statement Zhenhua Guo: Conceptualization, Investigation, Project administration, Writing – original draft. Haiyu Ruan: Methodology, Formal analysis, Data curation. Songlin Qiao: Resources, Writing – review & editing. Ruiguang Deng: Writing – review & editing. Gaiping Zhang: Supervision, Writing–review & editing.
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Co-infection status of porcine circoviruses (PCV2 and PCV3) and porcine epidemic
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diarrhea virus (PEDV) in pigs with watery diarrhea in Henan province,
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Central China
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Short Title：Co-infection of PCV2, PCV3 and PEDV
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Zhenhua Guo1#, Haiyu Ruan2#, Songlin Qiao1, Ruiguang Deng1, Gaiping Zhang1,2,3*
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1
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Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences,
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Zhengzhou, PR China.
Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Provincial
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2
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Zhengzhou, PR China.
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3
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Diseases and Zoonoses, Yangzhou, PR China.
College of Animal Science and Veterinary Medicine, Henan Agricultural University,
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious
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*
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Prof. Gaiping Zhang. College of Animal Science and Veterinary Medicine, Henan
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Agricultural University, Zhengzhou 450002, PR China. Fax: +86-371-63550369;
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E-mail address: [email protected]
Corresponding author:
19 20
#
These authors contributed equally to this work.
21 22 1
23
Abstract
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Porcine circoviruses (PCV2 and PCV3) and porcine epidemic diarrhea virus (PEDV) are
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important swine viruses that threaten the swine industry worldwide. Here, we evaluated the
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co-infection status of PCV2, PCV3 and PEDV in 76 enteric samples from piglets with severe
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diarrhea disease in Henan, China. All samples were tested by PCR/RT-PCR. Our results
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showed that the infection rate of PCV2, PCV3 and PEDV was 82.89%, 76.32% and 68.42%,
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respectively. Interestingly, most of these samples exhibited mixed infections. The co-infection
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rates of PCV2 and PCV3, PCV2 and PEDV, PCV3 and PEDV were 69.74%, 57.89% and
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53.95%, respectively. And the triple infection rate was 48.68%. Furthermore, the genetic
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characteristics of PCV2 and PCV3 were analyzed based on the cap genes. Two PCV2
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genotypes, PCV2b and PCV2d, were circulating in the fields. The cap gene of PCV2b and
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PCV2d isolates only shared 94.6%-95.0% nucleotide identities. The PCV3 isolates together
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with the reference strains could be divided into four clades (clade1-4), and the cap genes of
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these isolates have 98.6%-100% nucleotide identities to each other. Distinctive amino acid
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subsititutions were also characterized on the cap protein of PCV2 and PCV3 isolates. Our
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studies provide the new knowledge on the co-infectious status of PCV2, PCV3 and PEDV in
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China. The results also provide insight into the genetic diversity and molecular epidemiology
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of PCV2 and PCV3.
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Keywords: PCV2; PCV3; PEDV; co-infection; phylogeny; genetic diversity.
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1. Introduction Porcine circoviruses (PCVs), members of the genus Circovirus in the Circoviridae family, 2
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are the smallest non-enveloped virus with a circular, single-stranded DNA genome [1]. Three
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species of PCVs are currently identified, porcine circovirus type 1 (PCV1), porcine circovirus
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type 2 (PCV2) and porcine circovirus type 3 (PCV3) [1, 2]. PCV1 was originally found as a
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contaminant of continuous porcine kidney cell culture PK-15 and is considered to be
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nonpathogenic to pigs [3]. On the other hand, PCV2 infection can cause numerous types of
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syndromes and is a major pathogen for porcine circovirus-associated disease (PCVAD),
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including post-weaning multi-systemic wasting syndrome (PMWS), porcine dermatitis and
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nephropathy syndrome (PDNS), interstitial pneumonia, enteric disease and reproductive
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failure [4]. PCV2 is recognized as one of the most economically important causative agents to
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the swine industry worldwide. PCV3, a novel circovirus species, was first identified in pigs
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with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure in USA
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in 2015 [2]. Subsequently, it has been reported in swine-farming countries in North and South
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America, Europe and Asia, exhibiting an increasing geographic distribution [5-8].
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The genome size is about ~1.76 kb for PCV2 and ~2.0 kb for PCV3. It contains two major
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open reading frames, ORF1 and ORF2, encoding replicase (Rep) and capsid (Cap) protein,
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respectively [9]. The major structural capsid protein is associated with viral entry and triggers
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the production of virus neutralization antibodies. Compared with the ORF1 gene, the ORF2
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gene shows more genetic variations and is always used as a phylogenetic and epidemiological
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marker. Currently, based on the evolutionary analysis of cap genes, PCV2 could be divided
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into PCV2a-PCV2e genotypes and at least three clades of PCV3 have been reported [10-14].
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Co-infections of viruses play a critical role in pathogenicity and complicate the disease
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control and prevention [15]. For example, porcine reproductive and respiratory syndrome 3
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virus (PRRSV) and swine influenza virus (SIV) infection can exacerbate PCV2 pathogenesis
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[4]. The situation of suckling piglets infected by porcine epidemic diarrhea virus (PEDV)
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would be much worse if being co-infected with transmissible gastroenteritis virus (TGEV) or
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PCV2 [16, 17]. However, the co-infection status of PCV2, PCV3, and PEDV is rarely studied
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in Chinese swine herds. Here, we evaluated the co-infection of PCV2, PCV3, and PEDV in
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the piglets with severe diarrhea disease. A mixed infection of 2 or 3 viral pathogens was
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observed in most of the infected samples. In addition, we also characterized the genetic
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variations of PCV2 and PCV3 based on the cap gene analysis.
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2. Materials & methods
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2.1 Clinical samples
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During 2017, a total of 76 enteric tissues were collected from 22 pig farms distributed in 10
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areas of Henan province, which markets about 60 million pigs each year and is a major
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pig-producing province in China (Fig 1). The pig farms were commercial farrow-to-finisher
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farms with 300 to 2000 sows and carried out batch production system. These farms suffered
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from a severe watery diarrhea disease in suckling piglets with high morbidity (>60%) and
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mortality (>30%), which could not be cured with any antibiotics. Then, 2 to 5 enteric samples
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from each farm were collected. Differential diagnosis of porcine epidemic diarrhea virus
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(PEDV), porcine transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus
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(PDcoV) were performed with the specific primers by a RT-PCR test. After that, these
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samples were stored at -80℃ until further studies.
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2.2 Nucleic acid extraction 4
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Viral DNA and RNA were extracted from homogenized tissues using the MiniBEST Viral
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RNA/DNA Extraction Kit Ver.5.0 (TaKaRa) and the transcription was carried out with the
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PrimeScriptTMRT Master Mix reagents (TaKaRa). All the operations were finished according
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to the manufacture instructions. The DNA and cDNA were stored at -40℃ for further tests.
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2.3 PCR and RT-PCR
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All the primers used in this study were listed in Table 1. Each sample went through a PCR
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or RT-PCR detection with the PCV2, PCV3 and PEDV specific primers, respectively. The cap
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gene was further amplified from the positive samples of PCV2 or PCV3. The PCR reaction
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volume was 25μl containing 1.5μl of templates (DNA or cDNA), 1µl of primer pairs
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(2.5µM), 12.5µl of 2 x Premix TaqTM or 2x PrimeSTAR MAX Premix (TaKaRa) and 10µl
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deionized water. The reaction condition was as follows: one cycle at 98°C for 2 min; 30
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cycles at 98°C for 10 s, 55°C for 15 s and 72°C for 30 s or 45 s according to the length of the
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products, followed by elongation at 72°C for 5 min and hold at 16°C. The PCR products were
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visualized by 1.0% agarose gel electrophoresis and ultraviolet light.
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2.4 Cloning and sequencing
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The PCR products were purified by E.Z.N.A.® Gel Extraction Kit (Omega Bio-tek, Inc.)
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and then cloned into pEASY-Blunt Cloning vector (TransGen Biotech) following the
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manufacture’s instruction. At least three positive clones were submitted to Sangon Biotech
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(Shanghai, China) for sequencing. The target sequences were further uploaded to GenBank
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database, and the sequence information was listed in Table 2.
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2.5 Phylogeny, homology and sequence alignment
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The sequence homology was analyzed with the MegAlign program of DNASTAR package 5
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(DNASTAR, Inc., Madison, WI, USA) using the nucleotide and deduced amino acid (AA)
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sequences. To evaluate the evolution of PCV2 and PCV3 in central China, phylogenetic tree
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was constructed based on the cap genes with the neighbour-joining method in MEGA6.0
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software. The reliability of the generated trees was determined with 1000 bootstrap replicates.
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Multiple sequence alignments were also performed by clustal W method to analyze the
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genetic variations of Cap proteins [18]. All the reference sequence information was listed in
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Table S2 (PCV2 reference strains) and Table S3 (PCV3 reference strains).
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3. Results
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3.1 Detection results of PCV2, PCV3, and PEDV
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Among the 22 pig farms, 19 out of 22 (86.36%) pig farms were diagnosed as PEDV
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infection. Of the 76 enteric tissues, the positive rate of PEDV was 68.42% (52/76). Both
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TGEV and PDcoV were negative in all samples based on RT-PCR tests (data not shown).
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Then, we evaluated the PCV2 and PCV3 infection status in all samples. As shown in Table S1
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and Fig 2, the infection rate was 82.89% (63/76) for PCV2 and 76.32% (58/76) for PCV3 at
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the sample level. At the farm level, both of PCV2 and PCV3 had an infection rate of 90.91%
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(19/22). For the mixed infection assay, the co-infection by PCV2 and PCV3 was 86.36%
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(19/22) at farm level and 69.74% (53/76) at sample level, the combined infection rate of
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PCV2 and PEDV was 81.82% (18/22) and 57.89% (44/76), the positive rate of PCV3 and
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PEDV co-infection was 86.36% (19/22) and 53.95% (41/76), the triple infection by PCV2,
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PCV3 and PEDV was 77.27% (17/22) and 48.68% (37/76), respectively at farm level and
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sample level. 6
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3.2 Phylogenetic and homological analysis
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To evaluate the evolutionary relationships of the PCV2 and PCV3 isolates in this study, the
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samples with a relatively strong positive signal from each farm were chose to amplify the
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complete cap genes. Finally, 16 PCV2 and 15 PCV3 cap genes were successfully obtained.
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Then, the phylogenetic trees were constructed by the MEGA 6.0 software with a p-distance
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model based on the PCV2 or PCV3 cap genes. PCV2 could be divided into five genotypes
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(PCV2a-PCV2e). 7 out of 16 (43.75%) isolates were clustered with PCV2b genotype, and the
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other 9 isolates (56.25%) were grouped into PCV2d genotype (Fig 3). The ORF2 sequences in
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this study were 702 nt (PCV2b) and 705nt (PCV2d) in length, encoding capsid proteins of
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233 and 234 amino acids, respectively. The homology of cap nucleotides and amino acid
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sequences was 94.6%-100% and 88.9%-100% among the 16 isolates, respectively. Further on,
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within a genotype, the cap gene of PCV2b isolates shared 99.4%-100% nucleotide identities
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and PCV2d isolates shared 99.7%-100% nucleotide identities, while the isolates between
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PCV2b and PCV2d only have 94.6%-95% nucleotide homology.
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ORF2 sequences of all the PCV3 strains in this study were 645 nt encoding a 214 aa capsid
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protein. PCV3 could be divided into 4 evolutionary branches: clades 1-4. 11 out of 15 isolates
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belonged to clade 1 and the other 4 isolates (HeNZK1, HeNZK2, HeNLY1, and HeNLY2) in
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this study formed an independent branch-clade 3 (Fig 4). The cap gene homological analysis
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showed that the isolates are 98.6%-100% identical to each other, and shared 98.1%-99.8%
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nucleotide identities and 98.6%-100% amino acid similarities with the reference strains
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(GenBank no. KX778720, KX966193) , respectively.
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3.3 Sequence alignment of cap protein 7
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To evaluate the amino acid mutations of Cap protein, the multiple alignments of PCV2 and
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PCV3 Cap proteins was executed by a Clustal W method. The PCV2 Cap alignment showed
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similar results with previous reports. Briefly, typical motifs 86SNPRSV91 and
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were observed in PCV2b strains, while
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PCV2d strains. Besides, several amino acid substitutions were also found at Y8thF, F53thI,
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I57thV, A68thN, I/L80thL, S/121thT, T134thN, S169thR/G and V215thI sites. Most of these aa
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changes were distributed in the reported antibody epitope regions, immunodominant decoy
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epitope and the virulence related sites 133-135, except for those of Y8thF and V215thI sites
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(Fig 5).
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SNPRSV91 and
190 191
T
190 191
T G206I210E
G206I210D were conserved in
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Compared to PCV2, low genetic diversity was observed among these PCV3 capsid proteins.
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Only six sites, at V24thA, K27thR, N56thD, S77thT, F104thY and I150thL, display genetic
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variation of amino acids. The relationships between these amino acid substitutions and genetic
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evolution and virulence of PCV3 need to be further investigated.
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4. Discussion
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PCV2 has been emerging as a major pathogen to pigs worldwide since the 1990s [19, 20].
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Now it was present in nearly all swine-farming countries with a high prevalence. Although
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PCV3 was reported in USA in 2015, the retrospective studies showed that PCV3 existed in
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pigs as early as 1993 in Sweden and 1996 in China [21, 22]. Currently, PCV3 commonly
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circulated in the North and South America, Europe and Asia [5-8, 11]. Similar to PCV2,
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existing studies also suggest that PCV3 is likely to be associated with a variety of clinical
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diseases, including reproductive failure, PDNS, diarrhea, respiratory disease, multi-systemic 8
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inflammations etc [2, 9, 23-25]. In this study, we evaluated the co-infectious status of PCV2,
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PCV3, and PEDV in enteric samples of piglets with severe watery diarrhea disease. The
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results showed that all the pig farms showed a mixed infection by 2 or 3 viral pathogens and
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the singular infection of the samples was very low, only 2.63% (2/76) for PCV2, 3.95% (3/76)
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for PCV3 and 6.58% (5/76) for PEDV. Surprisingly, a high mixed infection rate of PCV2,
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PCV3, and PEDV was observed. PCV2 is related to enteric clinical disorders in
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PCV2-systemic disease (PCV2-SD). The transplacental infection of PCV2 can markedly
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affect the clinical course of PEDV and produce a more severe disease [17]. However, the
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pathogenicity of PCV3 to pigs is still not clarified because of the difficulty of PCV3 isolation.
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Recently studies displayed that the PCV3 positive rate was 10.4% (50/480) in the
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digestive-disease-affected samples and 17.14% (6/35) in the 35 diarrheal weaned pig samples
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[9, 25]. Further studies need to be done to clarify the effect of PCV3 on porcine diarrhea
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diseases.
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Currently, PCV2 can be divided into five genotypes based on the phylogenetic analysis of
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cap genes, defined as PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e [10, 26, 27]. PCV2e is a
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new genotype identified recently which contains an additional five amino acid at the
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C-terminal end resulting in 238 amino acids of ORF2. All other PCV2 ORF2 proteins are 233
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or 234 aa in length [10, 26]. In China, all the genotypes are circulating in the fields, except
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PCV2c which was only reported in Denmark [28-30]. A genotype shifting from PCV2a to
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PCV2b has happened since 2007. Recently, researchers discovered a new genotype switching
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from PCV2b to PCV2d in many countries since 2012 [4, 13]. Here, we found two genotypes,
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PCV2b and PCV2d, were prevalent in the fields in Henan, central China. The cap genes of 9
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PCV2b and PCV2d isolates in this study only share 94.6%-95.0% nucleotide identities,
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meaning that the genetic divergence is more than 5.0%. Furthermore, we also identified the
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specific motifs,
202
190 191
203
most of the amino acid mutations, such as F53thI, I57thV, A68thN, I/L80thL, S/121thT, T134thN
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and S169thR/G, are related to antibody recognitions and virulence of the PCV2, which may be
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associated with the immune escape, since all the vaccine strains are PCV2a and PCV2b
206
genotypes [4, 13, 30].
86
SNPRSV91 and
190 191
T G206I210E in PCV2b strains,
86
SNPRSV91 and
T G206I210D in PCV2d strains, which are consistent with previous studies [4]. Besides,
207
Compared with PCV2, PCV3 ORF2 sequence in this study exhibits a low mutation rate of
208
less than 2% at the nucleotide and amino acid level. Only 6 amino acid sites, V24A, K27R,
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N56D, S77T, F104Y and I150L, display genetic variations. These amino acids have been used
210
as a molecular marker to define the evolutionary clades of PCV3 and at least three clusters of
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PCV3 have been reported [11, 12, 31]. Considering the Cap is the main structural protein of
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PCVs, the amino acid changes may be linked to the antigenic variations. However, the
213
biological significance of these amino acid changes is still not clear and needs to be further
214
clarified.
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Overall, we provide evidence of a high co-infection rate of PCV2, PCV3, and PEDV in
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enteric samples from piglets with severe watery diarrheal disease. It suggests that PCV3, may
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be similar to PCV2, plays a role in the enteric disorder disease, which would complicate the
218
piglet diarrheal disease control and prevention. Furthermore, the genetic variations of cap
219
protein of PCV2 and PCV3 were characterized. Our studies insight into the molecular
220
epidemiology and evolution of porcine circovirus in China. 10
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Ethics approval and consent to participate
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This study was approved by the Institutional Animal Care and Use Committee of Henan
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Academy of Agricultural Sciences. All sampling and publication of the data were approved by
224
the farm owners.
225
Acknowledgments
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We are grateful to Dr. Leyi Wang (College of Veterinary Medicine, University of Illinois) for
227
careful revision of the manuscript.
228
Conflict of Interest
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The authors declare that they have no conflict of interest.
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Funding
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This work was funded by grants from the National Key Research and Development Program
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of China (2018YFD0501102, 2016YFD0500709), China Agriculture Research System
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(CARS-35) and the Special Fund for Henan Agriculture Research System (S2012-06).
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Data availability
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All data generated or analysed during this study are included in this published article and its
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supplementary information files.
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References
239
[1] Rosario K, Breitbart M, Harrach B, Segales J, Delwart E, Biagini P, et al. Revisiting the
240
taxonomy of the family Circoviridae: establishment of the genus Cyclovirus and removal of
241
the genus Gyrovirus. Arch Virol. 2017;162:1447-63.
242
[2] Palinski R, Piñeyro P, Shang P, Yuan F, Guo R, Fang Y, et al. A Novel Porcine Circovirus 11
243
Distantly Related to Known Circoviruses Is Associated with Porcine Dermatitis and
244
Nephropathy Syndrome and Reproductive Failure. Journal of Virology. 2017;91.
245
[3] Tischer I, Rasch R, Tochtermann G. Characterization of papovavirus-and picornavirus-like
246
particles in permanent pig kidney cell lines. Zentralbl Bakteriol Orig A. 1974;226:153-67.
247
[4] Ssemadaali MA, Ilha M, Ramamoorthy S. Genetic diversity of porcine circovirus type 2
248
and implications for detection and control. Research in veterinary science. 2015;103:179-86.
249
[5] Tochetto C, Lima DA, Varela APM, Loiko MR, Paim WP, Scheffer CM, et al.
250
Full-Genome Sequence of Porcine Circovirus type 3 recovered from serum of sows with
251
stillbirths in Brazil. Transboundary and emerging diseases. 2018;65:5-9.
252
[6] Franzo G, Legnardi M, Hjulsager CK, Klaumann F, Larsen LE, Segales J, et al.
253
Full-genome sequencing of porcine circovirus 3 field strains from Denmark, Italy and Spain
254
demonstrates a high within-Europe genetic heterogeneity. Transboundary and emerging
255
diseases. 2018;65:602-6.
256
[7] Ku X, Chen F, Li P, Wang Y, Yu X, Fan S, et al. Identification and genetic characterization
257
of porcine circovirus type 3 in China. Transboundary and emerging diseases. 2017;64:703-8.
258
[8] Kwon T, Yoo SJ, Park CK, Lyoo YS. Prevalence of novel porcine circovirus 3 in Korean
259
pig populations. Vet Microbiol. 2017;207:178-80.
260
[9] Zhai S-L, Zhou X, Zhang H, Hause BM, Lin T, Liu R, et al. Comparative epidemiology of
261
porcine circovirus type 3 in pigs with different clinical presentations. Virology Journal.
262
2017;14.
263
[10] Davies B, Wang X, Dvorak CM, Marthaler D, Murtaugh MP. Diagnostic phylogenetics
264
reveals a new Porcine circovirus 2 cluster. Virus research. 2016;217:32-7. 12
265
[11] Fux R, Sockler C, Link EK, Renken C, Krejci R, Sutter G, et al. Full genome
266
characterization of porcine circovirus type 3 isolates reveals the existence of two distinct
267
groups of virus strains. Virol J. 2018;15:25.
268
[12] Fu X, Fang B, Ma J, Liu Y, Bu D, Zhou P, et al. Insights into the epidemic characteristics
269
and evolutionary history of the novel porcine circovirus type 3 in southern China.
270
Transboundary and emerging diseases. 2018;65:e296-e303.
271
[13] Karuppannan A, Opriessnig T. Porcine Circovirus Type 2 (PCV2) Vaccines in the
272
Context of Current Molecular Epidemiology. Viruses. 2017;9:99.
273
[14] Franzo G, Cortey M, Olvera A, Novosel D, De Castro AMMG, Biagini P, et al. Revisiting
274
the taxonomical classification of Porcine Circovirus type 2 (PCV2): still a real challenge.
275
Virology Journal. 2015;12.
276
[15] Chen N, Huang Y, Ye M, Li S, Xiao Y, Cui B, et al. Co-infection status of classical swine
277
fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and
278
porcine circoviruses (PCV2 and PCV3) in eight regions of China from 2016 to 2018. Infect
279
Genet Evol. 2019;68:127-35.
280
[16] Zhao J, Shi B-j, Huang X-g, Peng M-y, Zhang X-m, He D-n, et al. A multiplex RT-PCR
281
assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field
282
samples from pig farms in East China from 2010 to 2012. Journal of Virological Methods.
283
2013;194:107-12.
284
[17] Jung K, Kim J, Ha Y, Choi C, Chae C. The effects of transplacental porcine circovirus
285
type 2 infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets.
286
Vet J. 2006;171:445-50. 13
287
[18] Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis
288
Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33:1870-4.
289
[19] Nayar GP, Hamel A, Lin L. Detection and characterization of porcine circovirus
290
associated with postweaning multisystemic wasting syndrome in pigs. Can Vet J.
291
1997;38:385-6.
292
[20] Allan GM, McNeilly F, Kennedy S, Daft B, Clarke EG, Ellis JA, et al. Isolation of
293
porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J Vet
294
Diagn Invest. 1998;10:3-10.
295
[21] Ye X, Berg M, Fossum C, Wallgren P, Blomstrom AL. Detection and genetic
296
characterisation of porcine circovirus 3 from pigs in Sweden. Virus genes. 2018;54:466-9.
297
[22] Sun J, Wei L, Lu Z, Mi S, Bao F, Guo H, et al. Retrospective study of porcine circovirus
298
3 infection in China. Transboundary and emerging diseases. 2018;65:607-13.
299
[23] Phan TG, Giannitti F, Rossow S, Marthaler D, Knutson TP, Li L, et al. Detection of a
300
novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation. Virology
301
Journal. 2016;13.
302
[24] Jiang H, Wang D, Wang J, Zhu S, She R, Ren X, et al. Induction of Porcine Dermatitis
303
and Nephropathy Syndrome in Piglets by Infection with Porcine Circovirus Type 3. J Virol.
304
2019;93.
305
[25] Qi S, Su M, Guo D, Li C, Wei S, Feng L, et al. Molecular detection and phylogenetic
306
analysis of porcine circovirus type 3 in 21 Provinces of China during 2015-2017.
307
Transboundary and emerging diseases. 2019;66:1004-15.
308
[26] Liu J, Wei C, Dai A, Lin Z, Fan K, Fan J, et al. Detection of PCV2e strains in Southeast 14
309
China. PeerJ. 2018;6:e4476.
310
[27] Xiao C-T, Halbur PG, Opriessnig T. Global molecular genetic analysis of porcine
311
circovirus type 2 (PCV2) sequences confirms the presence of four main PCV2 genotypes and
312
reveals a rapid increase of PCV2d. Journal of General Virology. 2015;96:1830-41.
313
[28] Liu C, Liu Y, Chen H, Feng H, Chen Y, Wang Y, et al. Genetic and immunogenicity
314
analysis of porcine circovirus type 2 strains isolated in central China. Arch Virol.
315
2018;163:937-46.
316
[29] Yang S, Yin S, Shang Y, Liu B, Yuan L, Zafar Khan MU, et al. Phylogenetic and genetic
317
variation analyses of porcine circovirus type 2 isolated from China. Transboundary and
318
emerging diseases. 2018;65:e383-e92.
319
[30] Zhai SL, Chen SN, Xu ZH, Tang MH, Wang FG, Li XJ, et al. Porcine circovirus type 2 in
320
China: an update on and insights to its prevalence and control. Virol J. 2014;11:88.
321
[31] Saraiva GL, Vidigal PMP, Assao VS, Fajardo MLM, Loreto ANS, Fietto JLR, et al.
322
Retrospective Detection and Genetic Characterization of Porcine circovirus 3 (PCV3) Strains
323
Identified between 2006 and 2007 in Brazil. Viruses. 2019;11.
324 325
Table 1. Primers used in this study.
326
Table 2. The PCV2 and PCV3 isolates collected in this study.
327
Supplementary Material
328
Table S1. The sample information and detection results.
329
Table S2. The PCV2 reference sequences in this study.
330
Table S3. The PCV3 reference sequences in this study.
331
Figure Legends： ： 15
332
Fig 1. Geographical distribution of pig farms where the enteric samples were collected in
333
Henan province, China. The stars (★) show the distribution of pig farms in Henan province,
334
China.
335
Fig 2. The analysis of detection results in this study.
336
Fig 3. The phylogenetic analysis based on the cap genes of PCV2. The phylogenetic tree
337
was constructed using the neighbour-joining method in MEGA 6.0 software with a bootstrap
338
test of 1,000 replicates. Scale bar indicates nucleotide substitutions per site. The black solid
339
circles (●) indicate the PCV2 strains isolated in this study. The black triangles (▲) show the
340
representative strains of different PCV2 genotypes.
341
Fig 4. The phylogenetic analysis based on the cap gene of PCV3. The phylogenetic tree
342
was constructed using the neighbour-joining method in MEGA 6.0 software with a bootstrap
343
test of 1,000 replicates. Scale bar indicates nucleotide substitutions per site. The black solid
344
circles (●) indicate the PCV3 strains isolated in this study.
345
Fig 5. Amino acid sequence alignment of cap protein of 16 PCV2 isolates. The 16 cap
346
protein sequences in this study were aligned by clustal W method in MEGA6.0 software. The
347
grey regions display four antibody recognition domains. The specific motifs of PCV2b and
348
PCV2d are shown in the solid line boxes. The dashed line indicates the location of decoy
349
epitope.
350
Fig 6. Amino acid sequence alignment of cap protein of PCV3 isolates. The 15 cap protein
351
sequences in this study and 10 reference strains were aligned by clustal W method in
352
MEGA6.0 software. The genetic variation sites are shown in the solid line boxes.
16
Table 1 Primers used in this study. Primer name
Sequence
PCV2-D-F
AAGGGCTGGGTTATGGTATG
PCV2-D-R
CGCTGGAGAAGGAAAAATGG
PCV2-cap-F
ATGACGTATCCAAGGAGGCGT
PCV2-cap-R
TCACTTAGGGTTAAGTGG
PCV3-D-F
ACAAARAAATACTCCACCAT
PCV3-D-R
ACTTTCCGCATAAGGGTC
PCV3-N-F
TTGCACTTGTGTACAATTATTGCG
PCV3-N-R
ATCTTCAGGACACTCGTAGCACCAC
PCV3-cap-F
ATGAGACACAGAGCTATATT
PCV3-cap-R
TTAGAGAACGGACTTGTAAC
PEDV-F
TTCTGAGTCATGAACAGCCA
PEDV-R
CATATGCAGCCTGCTCTGAA
Purpose
Product size（bp）
Detection
353
Sequence for PCV2 cap
705
Detection
270
Nested PCR for 1075 PCV3 cap Sequence for PCV3 cap
645
Detection
650
Table 2 The PCV2 and PCV3 isolates collected in this study cap gene Strain name
Regions Accession No.
HeNZMD-1
PCV2/ Length(bp/aa) PCV3
Zhumadian
MK641810
705/234
PCV2d
HeNHB-1
Hebi
MK641811
705/234
PCV2d
HeNKF-1
Kaifeng
MK641812
702/233
PCV2b
HeNKF-2
Kaifeng
MK641813
702/233
PCV2b
HeNLH-1
Luohe
MK641814
702/233
PCV2b
HeNLY-1
Luoyang
MK641815
705/234
PCV2d
HeNLY-2
Luoyang
MK641816
705/234
PCV2d
HeNLY-3
Luoyang
MK641817
705/234
PCV2d
HeNNY-1
Nanyang
MK641818
705/234
PCV2d
HeNNY-2
Nanyang
MK641819
705/234
PCV2d
HeNNY-3
Nanyang
MK641820
705/234
PCV2d
HeNPDS-1
Pingdingshan
MK641821
705/234
PCV2d
HeNXX-1
Xinxinag
MK641822
702/233
PCV2b
HeNZK-1
Zhoukou
MK641823
702/233
PCV2b
HeNZM-1
Kaifeng
MK641824
702/233
PCV2b
HeNZM-2
Kaifeng
MK641825
702/233
PCV2b
HeNPDS
Pingdingshan
MK641826
645/214
PCV3
HeNLH
Luohe
MK641836
645/214
PCV3
HeNZM1
Kaifeng
MK641837
645/214
PCV3
HeNZM2
Kaifeng
MK641838
645/214
PCV3
HeNKF
Kaifeng
MK641839
645/214
PCV3
HeNZK1
Zhoukou
MK641840
645/214
PCV3
HeNZK2
Zhoukou
MK641841
645/214
PCV3
HeNXX1
Xinxinag
MK641842
645/214
PCV3
HeNXX2
Xinxiang
MK641843
645/214
PCV3
HeNLY1
Luoyang
MK777996
645/214
PCV3
HeNLY2
Luoyang
MK777997
645/214
PCV3
HeNNY1
Nanyang
MK777998
645/214
PCV3
HeNNY2
Nanyang
MK777999
645/214
PCV3
HeNHB1
Hebi
MK778000
645/214
PCV3
Zhamadian
MK778001
645/214
PCV3
HeNZMD1
1
Highlights
2
High co-infection rate of PCV2, PCV3, and PEDV was determined in pigs with
3
watery diarrhea in Henan province, Central China.
4
Two PCV2 genotypes, PCV2b and PCV2d, were circulating in the fields in
5
central China.
6
PCV3 also exhibits a degree of genetic divergence.
7
Distinctive amino acid substitutions were characterized on the cap protein of
8
PCV2 and PCV3 isolates, which may be linked to the antigenic variations and
9
immune escape.
1
S0882-4010(19)31796-6
DOI:
https://doi.org/10.1016/j.micpath.2020.104047
Reference:
YMPAT 104047
To appear in:
Microbial Pathogenesis
Received Date: 12 October 2019 Revised Date:
4 February 2020
Accepted Date: 5 February 2020
Please cite this article as: Guo Z, Ruan H, Qiao S, Deng R, Zhang G, Co-infection status of porcine circoviruses (PCV2 and PCV3) and porcine epidemic diarrhea virus (PEDV) in pigs with watery diarrhea in Henan province, central China, Microbial Pathogenesis (2020), doi: https://doi.org/10.1016/ j.micpath.2020.104047. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier Ltd.
Author Statement Zhenhua Guo: Conceptualization, Investigation, Project administration, Writing – original draft. Haiyu Ruan: Methodology, Formal analysis, Data curation. Songlin Qiao: Resources, Writing – review & editing. Ruiguang Deng: Writing – review & editing. Gaiping Zhang: Supervision, Writing–review & editing.
1 2
Co-infection status of porcine circoviruses (PCV2 and PCV3) and porcine epidemic
3
diarrhea virus (PEDV) in pigs with watery diarrhea in Henan province,
4
Central China
5
Short Title：Co-infection of PCV2, PCV3 and PEDV
6
Zhenhua Guo1#, Haiyu Ruan2#, Songlin Qiao1, Ruiguang Deng1, Gaiping Zhang1,2,3*
7
1
8
Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences,
9
Zhengzhou, PR China.
Key Laboratory of Animal Immunology of the Ministry of Agriculture, Henan Provincial
10
2
11
Zhengzhou, PR China.
12
3
13
Diseases and Zoonoses, Yangzhou, PR China.
College of Animal Science and Veterinary Medicine, Henan Agricultural University,
Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious
14 15
*
16
Prof. Gaiping Zhang. College of Animal Science and Veterinary Medicine, Henan
17
Agricultural University, Zhengzhou 450002, PR China. Fax: +86-371-63550369;
18
E-mail address: [email protected]
Corresponding author:
19 20
#
These authors contributed equally to this work.
21 22 1
23
Abstract
24
Porcine circoviruses (PCV2 and PCV3) and porcine epidemic diarrhea virus (PEDV) are
25
important swine viruses that threaten the swine industry worldwide. Here, we evaluated the
26
co-infection status of PCV2, PCV3 and PEDV in 76 enteric samples from piglets with severe
27
diarrhea disease in Henan, China. All samples were tested by PCR/RT-PCR. Our results
28
showed that the infection rate of PCV2, PCV3 and PEDV was 82.89%, 76.32% and 68.42%,
29
respectively. Interestingly, most of these samples exhibited mixed infections. The co-infection
30
rates of PCV2 and PCV3, PCV2 and PEDV, PCV3 and PEDV were 69.74%, 57.89% and
31
53.95%, respectively. And the triple infection rate was 48.68%. Furthermore, the genetic
32
characteristics of PCV2 and PCV3 were analyzed based on the cap genes. Two PCV2
33
genotypes, PCV2b and PCV2d, were circulating in the fields. The cap gene of PCV2b and
34
PCV2d isolates only shared 94.6%-95.0% nucleotide identities. The PCV3 isolates together
35
with the reference strains could be divided into four clades (clade1-4), and the cap genes of
36
these isolates have 98.6%-100% nucleotide identities to each other. Distinctive amino acid
37
subsititutions were also characterized on the cap protein of PCV2 and PCV3 isolates. Our
38
studies provide the new knowledge on the co-infectious status of PCV2, PCV3 and PEDV in
39
China. The results also provide insight into the genetic diversity and molecular epidemiology
40
of PCV2 and PCV3.
41
Keywords: PCV2; PCV3; PEDV; co-infection; phylogeny; genetic diversity.
42 43 44
1. Introduction Porcine circoviruses (PCVs), members of the genus Circovirus in the Circoviridae family, 2
45
are the smallest non-enveloped virus with a circular, single-stranded DNA genome [1]. Three
46
species of PCVs are currently identified, porcine circovirus type 1 (PCV1), porcine circovirus
47
type 2 (PCV2) and porcine circovirus type 3 (PCV3) [1, 2]. PCV1 was originally found as a
48
contaminant of continuous porcine kidney cell culture PK-15 and is considered to be
49
nonpathogenic to pigs [3]. On the other hand, PCV2 infection can cause numerous types of
50
syndromes and is a major pathogen for porcine circovirus-associated disease (PCVAD),
51
including post-weaning multi-systemic wasting syndrome (PMWS), porcine dermatitis and
52
nephropathy syndrome (PDNS), interstitial pneumonia, enteric disease and reproductive
53
failure [4]. PCV2 is recognized as one of the most economically important causative agents to
54
the swine industry worldwide. PCV3, a novel circovirus species, was first identified in pigs
55
with porcine dermatitis and nephropathy syndrome (PDNS) and reproductive failure in USA
56
in 2015 [2]. Subsequently, it has been reported in swine-farming countries in North and South
57
America, Europe and Asia, exhibiting an increasing geographic distribution [5-8].
58
The genome size is about ~1.76 kb for PCV2 and ~2.0 kb for PCV3. It contains two major
59
open reading frames, ORF1 and ORF2, encoding replicase (Rep) and capsid (Cap) protein,
60
respectively [9]. The major structural capsid protein is associated with viral entry and triggers
61
the production of virus neutralization antibodies. Compared with the ORF1 gene, the ORF2
62
gene shows more genetic variations and is always used as a phylogenetic and epidemiological
63
marker. Currently, based on the evolutionary analysis of cap genes, PCV2 could be divided
64
into PCV2a-PCV2e genotypes and at least three clades of PCV3 have been reported [10-14].
65
Co-infections of viruses play a critical role in pathogenicity and complicate the disease
66
control and prevention [15]. For example, porcine reproductive and respiratory syndrome 3
67
virus (PRRSV) and swine influenza virus (SIV) infection can exacerbate PCV2 pathogenesis
68
[4]. The situation of suckling piglets infected by porcine epidemic diarrhea virus (PEDV)
69
would be much worse if being co-infected with transmissible gastroenteritis virus (TGEV) or
70
PCV2 [16, 17]. However, the co-infection status of PCV2, PCV3, and PEDV is rarely studied
71
in Chinese swine herds. Here, we evaluated the co-infection of PCV2, PCV3, and PEDV in
72
the piglets with severe diarrhea disease. A mixed infection of 2 or 3 viral pathogens was
73
observed in most of the infected samples. In addition, we also characterized the genetic
74
variations of PCV2 and PCV3 based on the cap gene analysis.
75 76
2. Materials & methods
77
2.1 Clinical samples
78
During 2017, a total of 76 enteric tissues were collected from 22 pig farms distributed in 10
79
areas of Henan province, which markets about 60 million pigs each year and is a major
80
pig-producing province in China (Fig 1). The pig farms were commercial farrow-to-finisher
81
farms with 300 to 2000 sows and carried out batch production system. These farms suffered
82
from a severe watery diarrhea disease in suckling piglets with high morbidity (>60%) and
83
mortality (>30%), which could not be cured with any antibiotics. Then, 2 to 5 enteric samples
84
from each farm were collected. Differential diagnosis of porcine epidemic diarrhea virus
85
(PEDV), porcine transmissible gastroenteritis virus (TGEV) and porcine deltacoronavirus
86
(PDcoV) were performed with the specific primers by a RT-PCR test. After that, these
87
samples were stored at -80℃ until further studies.
88
2.2 Nucleic acid extraction 4
89
Viral DNA and RNA were extracted from homogenized tissues using the MiniBEST Viral
90
RNA/DNA Extraction Kit Ver.5.0 (TaKaRa) and the transcription was carried out with the
91
PrimeScriptTMRT Master Mix reagents (TaKaRa). All the operations were finished according
92
to the manufacture instructions. The DNA and cDNA were stored at -40℃ for further tests.
93
2.3 PCR and RT-PCR
94
All the primers used in this study were listed in Table 1. Each sample went through a PCR
95
or RT-PCR detection with the PCV2, PCV3 and PEDV specific primers, respectively. The cap
96
gene was further amplified from the positive samples of PCV2 or PCV3. The PCR reaction
97
volume was 25μl containing 1.5μl of templates (DNA or cDNA), 1µl of primer pairs
98
(2.5µM), 12.5µl of 2 x Premix TaqTM or 2x PrimeSTAR MAX Premix (TaKaRa) and 10µl
99
deionized water. The reaction condition was as follows: one cycle at 98°C for 2 min; 30
100
cycles at 98°C for 10 s, 55°C for 15 s and 72°C for 30 s or 45 s according to the length of the
101
products, followed by elongation at 72°C for 5 min and hold at 16°C. The PCR products were
102
visualized by 1.0% agarose gel electrophoresis and ultraviolet light.
103
2.4 Cloning and sequencing
104
The PCR products were purified by E.Z.N.A.® Gel Extraction Kit (Omega Bio-tek, Inc.)
105
and then cloned into pEASY-Blunt Cloning vector (TransGen Biotech) following the
106
manufacture’s instruction. At least three positive clones were submitted to Sangon Biotech
107
(Shanghai, China) for sequencing. The target sequences were further uploaded to GenBank
108
database, and the sequence information was listed in Table 2.
109
2.5 Phylogeny, homology and sequence alignment
110
The sequence homology was analyzed with the MegAlign program of DNASTAR package 5
111
(DNASTAR, Inc., Madison, WI, USA) using the nucleotide and deduced amino acid (AA)
112
sequences. To evaluate the evolution of PCV2 and PCV3 in central China, phylogenetic tree
113
was constructed based on the cap genes with the neighbour-joining method in MEGA6.0
114
software. The reliability of the generated trees was determined with 1000 bootstrap replicates.
115
Multiple sequence alignments were also performed by clustal W method to analyze the
116
genetic variations of Cap proteins [18]. All the reference sequence information was listed in
117
Table S2 (PCV2 reference strains) and Table S3 (PCV3 reference strains).
118 119
3. Results
120
3.1 Detection results of PCV2, PCV3, and PEDV
121
Among the 22 pig farms, 19 out of 22 (86.36%) pig farms were diagnosed as PEDV
122
infection. Of the 76 enteric tissues, the positive rate of PEDV was 68.42% (52/76). Both
123
TGEV and PDcoV were negative in all samples based on RT-PCR tests (data not shown).
124
Then, we evaluated the PCV2 and PCV3 infection status in all samples. As shown in Table S1
125
and Fig 2, the infection rate was 82.89% (63/76) for PCV2 and 76.32% (58/76) for PCV3 at
126
the sample level. At the farm level, both of PCV2 and PCV3 had an infection rate of 90.91%
127
(19/22). For the mixed infection assay, the co-infection by PCV2 and PCV3 was 86.36%
128
(19/22) at farm level and 69.74% (53/76) at sample level, the combined infection rate of
129
PCV2 and PEDV was 81.82% (18/22) and 57.89% (44/76), the positive rate of PCV3 and
130
PEDV co-infection was 86.36% (19/22) and 53.95% (41/76), the triple infection by PCV2,
131
PCV3 and PEDV was 77.27% (17/22) and 48.68% (37/76), respectively at farm level and
132
sample level. 6
133
3.2 Phylogenetic and homological analysis
134
To evaluate the evolutionary relationships of the PCV2 and PCV3 isolates in this study, the
135
samples with a relatively strong positive signal from each farm were chose to amplify the
136
complete cap genes. Finally, 16 PCV2 and 15 PCV3 cap genes were successfully obtained.
137
Then, the phylogenetic trees were constructed by the MEGA 6.0 software with a p-distance
138
model based on the PCV2 or PCV3 cap genes. PCV2 could be divided into five genotypes
139
(PCV2a-PCV2e). 7 out of 16 (43.75%) isolates were clustered with PCV2b genotype, and the
140
other 9 isolates (56.25%) were grouped into PCV2d genotype (Fig 3). The ORF2 sequences in
141
this study were 702 nt (PCV2b) and 705nt (PCV2d) in length, encoding capsid proteins of
142
233 and 234 amino acids, respectively. The homology of cap nucleotides and amino acid
143
sequences was 94.6%-100% and 88.9%-100% among the 16 isolates, respectively. Further on,
144
within a genotype, the cap gene of PCV2b isolates shared 99.4%-100% nucleotide identities
145
and PCV2d isolates shared 99.7%-100% nucleotide identities, while the isolates between
146
PCV2b and PCV2d only have 94.6%-95% nucleotide homology.
147
ORF2 sequences of all the PCV3 strains in this study were 645 nt encoding a 214 aa capsid
148
protein. PCV3 could be divided into 4 evolutionary branches: clades 1-4. 11 out of 15 isolates
149
belonged to clade 1 and the other 4 isolates (HeNZK1, HeNZK2, HeNLY1, and HeNLY2) in
150
this study formed an independent branch-clade 3 (Fig 4). The cap gene homological analysis
151
showed that the isolates are 98.6%-100% identical to each other, and shared 98.1%-99.8%
152
nucleotide identities and 98.6%-100% amino acid similarities with the reference strains
153
(GenBank no. KX778720, KX966193) , respectively.
154
3.3 Sequence alignment of cap protein 7
155
To evaluate the amino acid mutations of Cap protein, the multiple alignments of PCV2 and
156
PCV3 Cap proteins was executed by a Clustal W method. The PCV2 Cap alignment showed
157
similar results with previous reports. Briefly, typical motifs 86SNPRSV91 and
158
were observed in PCV2b strains, while
159
PCV2d strains. Besides, several amino acid substitutions were also found at Y8thF, F53thI,
160
I57thV, A68thN, I/L80thL, S/121thT, T134thN, S169thR/G and V215thI sites. Most of these aa
161
changes were distributed in the reported antibody epitope regions, immunodominant decoy
162
epitope and the virulence related sites 133-135, except for those of Y8thF and V215thI sites
163
(Fig 5).
86
SNPRSV91 and
190 191
T
190 191
T G206I210E
G206I210D were conserved in
164
Compared to PCV2, low genetic diversity was observed among these PCV3 capsid proteins.
165
Only six sites, at V24thA, K27thR, N56thD, S77thT, F104thY and I150thL, display genetic
166
variation of amino acids. The relationships between these amino acid substitutions and genetic
167
evolution and virulence of PCV3 need to be further investigated.
168 169
4. Discussion
170
PCV2 has been emerging as a major pathogen to pigs worldwide since the 1990s [19, 20].
171
Now it was present in nearly all swine-farming countries with a high prevalence. Although
172
PCV3 was reported in USA in 2015, the retrospective studies showed that PCV3 existed in
173
pigs as early as 1993 in Sweden and 1996 in China [21, 22]. Currently, PCV3 commonly
174
circulated in the North and South America, Europe and Asia [5-8, 11]. Similar to PCV2,
175
existing studies also suggest that PCV3 is likely to be associated with a variety of clinical
176
diseases, including reproductive failure, PDNS, diarrhea, respiratory disease, multi-systemic 8
177
inflammations etc [2, 9, 23-25]. In this study, we evaluated the co-infectious status of PCV2,
178
PCV3, and PEDV in enteric samples of piglets with severe watery diarrhea disease. The
179
results showed that all the pig farms showed a mixed infection by 2 or 3 viral pathogens and
180
the singular infection of the samples was very low, only 2.63% (2/76) for PCV2, 3.95% (3/76)
181
for PCV3 and 6.58% (5/76) for PEDV. Surprisingly, a high mixed infection rate of PCV2,
182
PCV3, and PEDV was observed. PCV2 is related to enteric clinical disorders in
183
PCV2-systemic disease (PCV2-SD). The transplacental infection of PCV2 can markedly
184
affect the clinical course of PEDV and produce a more severe disease [17]. However, the
185
pathogenicity of PCV3 to pigs is still not clarified because of the difficulty of PCV3 isolation.
186
Recently studies displayed that the PCV3 positive rate was 10.4% (50/480) in the
187
digestive-disease-affected samples and 17.14% (6/35) in the 35 diarrheal weaned pig samples
188
[9, 25]. Further studies need to be done to clarify the effect of PCV3 on porcine diarrhea
189
diseases.
190
Currently, PCV2 can be divided into five genotypes based on the phylogenetic analysis of
191
cap genes, defined as PCV2a, PCV2b, PCV2c, PCV2d, and PCV2e [10, 26, 27]. PCV2e is a
192
new genotype identified recently which contains an additional five amino acid at the
193
C-terminal end resulting in 238 amino acids of ORF2. All other PCV2 ORF2 proteins are 233
194
or 234 aa in length [10, 26]. In China, all the genotypes are circulating in the fields, except
195
PCV2c which was only reported in Denmark [28-30]. A genotype shifting from PCV2a to
196
PCV2b has happened since 2007. Recently, researchers discovered a new genotype switching
197
from PCV2b to PCV2d in many countries since 2012 [4, 13]. Here, we found two genotypes,
198
PCV2b and PCV2d, were prevalent in the fields in Henan, central China. The cap genes of 9
199
PCV2b and PCV2d isolates in this study only share 94.6%-95.0% nucleotide identities,
200
meaning that the genetic divergence is more than 5.0%. Furthermore, we also identified the
201
specific motifs,
202
190 191
203
most of the amino acid mutations, such as F53thI, I57thV, A68thN, I/L80thL, S/121thT, T134thN
204
and S169thR/G, are related to antibody recognitions and virulence of the PCV2, which may be
205
associated with the immune escape, since all the vaccine strains are PCV2a and PCV2b
206
genotypes [4, 13, 30].
86
SNPRSV91 and
190 191
T G206I210E in PCV2b strains,
86
SNPRSV91 and
T G206I210D in PCV2d strains, which are consistent with previous studies [4]. Besides,
207
Compared with PCV2, PCV3 ORF2 sequence in this study exhibits a low mutation rate of
208
less than 2% at the nucleotide and amino acid level. Only 6 amino acid sites, V24A, K27R,
209
N56D, S77T, F104Y and I150L, display genetic variations. These amino acids have been used
210
as a molecular marker to define the evolutionary clades of PCV3 and at least three clusters of
211
PCV3 have been reported [11, 12, 31]. Considering the Cap is the main structural protein of
212
PCVs, the amino acid changes may be linked to the antigenic variations. However, the
213
biological significance of these amino acid changes is still not clear and needs to be further
214
clarified.
215
Overall, we provide evidence of a high co-infection rate of PCV2, PCV3, and PEDV in
216
enteric samples from piglets with severe watery diarrheal disease. It suggests that PCV3, may
217
be similar to PCV2, plays a role in the enteric disorder disease, which would complicate the
218
piglet diarrheal disease control and prevention. Furthermore, the genetic variations of cap
219
protein of PCV2 and PCV3 were characterized. Our studies insight into the molecular
220
epidemiology and evolution of porcine circovirus in China. 10
221
Ethics approval and consent to participate
222
This study was approved by the Institutional Animal Care and Use Committee of Henan
223
Academy of Agricultural Sciences. All sampling and publication of the data were approved by
224
the farm owners.
225
Acknowledgments
226
We are grateful to Dr. Leyi Wang (College of Veterinary Medicine, University of Illinois) for
227
careful revision of the manuscript.
228
Conflict of Interest
229
The authors declare that they have no conflict of interest.
230
Funding
231
This work was funded by grants from the National Key Research and Development Program
232
of China (2018YFD0501102, 2016YFD0500709), China Agriculture Research System
233
(CARS-35) and the Special Fund for Henan Agriculture Research System (S2012-06).
234
Data availability
235
All data generated or analysed during this study are included in this published article and its
236
supplementary information files.
237 238
References
239
[1] Rosario K, Breitbart M, Harrach B, Segales J, Delwart E, Biagini P, et al. Revisiting the
240
taxonomy of the family Circoviridae: establishment of the genus Cyclovirus and removal of
241
the genus Gyrovirus. Arch Virol. 2017;162:1447-63.
242
[2] Palinski R, Piñeyro P, Shang P, Yuan F, Guo R, Fang Y, et al. A Novel Porcine Circovirus 11
243
Distantly Related to Known Circoviruses Is Associated with Porcine Dermatitis and
244
Nephropathy Syndrome and Reproductive Failure. Journal of Virology. 2017;91.
245
[3] Tischer I, Rasch R, Tochtermann G. Characterization of papovavirus-and picornavirus-like
246
particles in permanent pig kidney cell lines. Zentralbl Bakteriol Orig A. 1974;226:153-67.
247
[4] Ssemadaali MA, Ilha M, Ramamoorthy S. Genetic diversity of porcine circovirus type 2
248
and implications for detection and control. Research in veterinary science. 2015;103:179-86.
249
[5] Tochetto C, Lima DA, Varela APM, Loiko MR, Paim WP, Scheffer CM, et al.
250
Full-Genome Sequence of Porcine Circovirus type 3 recovered from serum of sows with
251
stillbirths in Brazil. Transboundary and emerging diseases. 2018;65:5-9.
252
[6] Franzo G, Legnardi M, Hjulsager CK, Klaumann F, Larsen LE, Segales J, et al.
253
Full-genome sequencing of porcine circovirus 3 field strains from Denmark, Italy and Spain
254
demonstrates a high within-Europe genetic heterogeneity. Transboundary and emerging
255
diseases. 2018;65:602-6.
256
[7] Ku X, Chen F, Li P, Wang Y, Yu X, Fan S, et al. Identification and genetic characterization
257
of porcine circovirus type 3 in China. Transboundary and emerging diseases. 2017;64:703-8.
258
[8] Kwon T, Yoo SJ, Park CK, Lyoo YS. Prevalence of novel porcine circovirus 3 in Korean
259
pig populations. Vet Microbiol. 2017;207:178-80.
260
[9] Zhai S-L, Zhou X, Zhang H, Hause BM, Lin T, Liu R, et al. Comparative epidemiology of
261
porcine circovirus type 3 in pigs with different clinical presentations. Virology Journal.
262
2017;14.
263
[10] Davies B, Wang X, Dvorak CM, Marthaler D, Murtaugh MP. Diagnostic phylogenetics
264
reveals a new Porcine circovirus 2 cluster. Virus research. 2016;217:32-7. 12
265
[11] Fux R, Sockler C, Link EK, Renken C, Krejci R, Sutter G, et al. Full genome
266
characterization of porcine circovirus type 3 isolates reveals the existence of two distinct
267
groups of virus strains. Virol J. 2018;15:25.
268
[12] Fu X, Fang B, Ma J, Liu Y, Bu D, Zhou P, et al. Insights into the epidemic characteristics
269
and evolutionary history of the novel porcine circovirus type 3 in southern China.
270
Transboundary and emerging diseases. 2018;65:e296-e303.
271
[13] Karuppannan A, Opriessnig T. Porcine Circovirus Type 2 (PCV2) Vaccines in the
272
Context of Current Molecular Epidemiology. Viruses. 2017;9:99.
273
[14] Franzo G, Cortey M, Olvera A, Novosel D, De Castro AMMG, Biagini P, et al. Revisiting
274
the taxonomical classification of Porcine Circovirus type 2 (PCV2): still a real challenge.
275
Virology Journal. 2015;12.
276
[15] Chen N, Huang Y, Ye M, Li S, Xiao Y, Cui B, et al. Co-infection status of classical swine
277
fever virus (CSFV), porcine reproductive and respiratory syndrome virus (PRRSV) and
278
porcine circoviruses (PCV2 and PCV3) in eight regions of China from 2016 to 2018. Infect
279
Genet Evol. 2019;68:127-35.
280
[16] Zhao J, Shi B-j, Huang X-g, Peng M-y, Zhang X-m, He D-n, et al. A multiplex RT-PCR
281
assay for rapid and differential diagnosis of four porcine diarrhea associated viruses in field
282
samples from pig farms in East China from 2010 to 2012. Journal of Virological Methods.
283
2013;194:107-12.
284
[17] Jung K, Kim J, Ha Y, Choi C, Chae C. The effects of transplacental porcine circovirus
285
type 2 infection on porcine epidemic diarrhoea virus-induced enteritis in preweaning piglets.
286
Vet J. 2006;171:445-50. 13
287
[18] Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis
288
Version 7.0 for Bigger Datasets. Mol Biol Evol. 2016;33:1870-4.
289
[19] Nayar GP, Hamel A, Lin L. Detection and characterization of porcine circovirus
290
associated with postweaning multisystemic wasting syndrome in pigs. Can Vet J.
291
1997;38:385-6.
292
[20] Allan GM, McNeilly F, Kennedy S, Daft B, Clarke EG, Ellis JA, et al. Isolation of
293
porcine circovirus-like viruses from pigs with a wasting disease in the USA and Europe. J Vet
294
Diagn Invest. 1998;10:3-10.
295
[21] Ye X, Berg M, Fossum C, Wallgren P, Blomstrom AL. Detection and genetic
296
characterisation of porcine circovirus 3 from pigs in Sweden. Virus genes. 2018;54:466-9.
297
[22] Sun J, Wei L, Lu Z, Mi S, Bao F, Guo H, et al. Retrospective study of porcine circovirus
298
3 infection in China. Transboundary and emerging diseases. 2018;65:607-13.
299
[23] Phan TG, Giannitti F, Rossow S, Marthaler D, Knutson TP, Li L, et al. Detection of a
300
novel circovirus PCV3 in pigs with cardiac and multi-systemic inflammation. Virology
301
Journal. 2016;13.
302
[24] Jiang H, Wang D, Wang J, Zhu S, She R, Ren X, et al. Induction of Porcine Dermatitis
303
and Nephropathy Syndrome in Piglets by Infection with Porcine Circovirus Type 3. J Virol.
304
2019;93.
305
[25] Qi S, Su M, Guo D, Li C, Wei S, Feng L, et al. Molecular detection and phylogenetic
306
analysis of porcine circovirus type 3 in 21 Provinces of China during 2015-2017.
307
Transboundary and emerging diseases. 2019;66:1004-15.
308
[26] Liu J, Wei C, Dai A, Lin Z, Fan K, Fan J, et al. Detection of PCV2e strains in Southeast 14
309
China. PeerJ. 2018;6:e4476.
310
[27] Xiao C-T, Halbur PG, Opriessnig T. Global molecular genetic analysis of porcine
311
circovirus type 2 (PCV2) sequences confirms the presence of four main PCV2 genotypes and
312
reveals a rapid increase of PCV2d. Journal of General Virology. 2015;96:1830-41.
313
[28] Liu C, Liu Y, Chen H, Feng H, Chen Y, Wang Y, et al. Genetic and immunogenicity
314
analysis of porcine circovirus type 2 strains isolated in central China. Arch Virol.
315
2018;163:937-46.
316
[29] Yang S, Yin S, Shang Y, Liu B, Yuan L, Zafar Khan MU, et al. Phylogenetic and genetic
317
variation analyses of porcine circovirus type 2 isolated from China. Transboundary and
318
emerging diseases. 2018;65:e383-e92.
319
[30] Zhai SL, Chen SN, Xu ZH, Tang MH, Wang FG, Li XJ, et al. Porcine circovirus type 2 in
320
China: an update on and insights to its prevalence and control. Virol J. 2014;11:88.
321
[31] Saraiva GL, Vidigal PMP, Assao VS, Fajardo MLM, Loreto ANS, Fietto JLR, et al.
322
Retrospective Detection and Genetic Characterization of Porcine circovirus 3 (PCV3) Strains
323
Identified between 2006 and 2007 in Brazil. Viruses. 2019;11.
324 325
Table 1. Primers used in this study.
326
Table 2. The PCV2 and PCV3 isolates collected in this study.
327
Supplementary Material
328
Table S1. The sample information and detection results.
329
Table S2. The PCV2 reference sequences in this study.
330
Table S3. The PCV3 reference sequences in this study.
331
Figure Legends： ： 15
332
Fig 1. Geographical distribution of pig farms where the enteric samples were collected in
333
Henan province, China. The stars (★) show the distribution of pig farms in Henan province,
334
China.
335
Fig 2. The analysis of detection results in this study.
336
Fig 3. The phylogenetic analysis based on the cap genes of PCV2. The phylogenetic tree
337
was constructed using the neighbour-joining method in MEGA 6.0 software with a bootstrap
338
test of 1,000 replicates. Scale bar indicates nucleotide substitutions per site. The black solid
339
circles (●) indicate the PCV2 strains isolated in this study. The black triangles (▲) show the
340
representative strains of different PCV2 genotypes.
341
Fig 4. The phylogenetic analysis based on the cap gene of PCV3. The phylogenetic tree
342
was constructed using the neighbour-joining method in MEGA 6.0 software with a bootstrap
343
test of 1,000 replicates. Scale bar indicates nucleotide substitutions per site. The black solid
344
circles (●) indicate the PCV3 strains isolated in this study.
345
Fig 5. Amino acid sequence alignment of cap protein of 16 PCV2 isolates. The 16 cap
346
protein sequences in this study were aligned by clustal W method in MEGA6.0 software. The
347
grey regions display four antibody recognition domains. The specific motifs of PCV2b and
348
PCV2d are shown in the solid line boxes. The dashed line indicates the location of decoy
349
epitope.
350
Fig 6. Amino acid sequence alignment of cap protein of PCV3 isolates. The 15 cap protein
351
sequences in this study and 10 reference strains were aligned by clustal W method in
352
MEGA6.0 software. The genetic variation sites are shown in the solid line boxes.
16
Table 1 Primers used in this study. Primer name
Sequence
PCV2-D-F
AAGGGCTGGGTTATGGTATG
PCV2-D-R
CGCTGGAGAAGGAAAAATGG
PCV2-cap-F
ATGACGTATCCAAGGAGGCGT
PCV2-cap-R
TCACTTAGGGTTAAGTGG
PCV3-D-F
ACAAARAAATACTCCACCAT
PCV3-D-R
ACTTTCCGCATAAGGGTC
PCV3-N-F
TTGCACTTGTGTACAATTATTGCG
PCV3-N-R
ATCTTCAGGACACTCGTAGCACCAC
PCV3-cap-F
ATGAGACACAGAGCTATATT
PCV3-cap-R
TTAGAGAACGGACTTGTAAC
PEDV-F
TTCTGAGTCATGAACAGCCA
PEDV-R
CATATGCAGCCTGCTCTGAA
Purpose
Product size（bp）
Detection
353
Sequence for PCV2 cap
705
Detection
270
Nested PCR for 1075 PCV3 cap Sequence for PCV3 cap
645
Detection
650
Table 2 The PCV2 and PCV3 isolates collected in this study cap gene Strain name
Regions Accession No.
HeNZMD-1
PCV2/ Length(bp/aa) PCV3
Zhumadian
MK641810
705/234
PCV2d
HeNHB-1
Hebi
MK641811
705/234
PCV2d
HeNKF-1
Kaifeng
MK641812
702/233
PCV2b
HeNKF-2
Kaifeng
MK641813
702/233
PCV2b
HeNLH-1
Luohe
MK641814
702/233
PCV2b
HeNLY-1
Luoyang
MK641815
705/234
PCV2d
HeNLY-2
Luoyang
MK641816
705/234
PCV2d
HeNLY-3
Luoyang
MK641817
705/234
PCV2d
HeNNY-1
Nanyang
MK641818
705/234
PCV2d
HeNNY-2
Nanyang
MK641819
705/234
PCV2d
HeNNY-3
Nanyang
MK641820
705/234
PCV2d
HeNPDS-1
Pingdingshan
MK641821
705/234
PCV2d
HeNXX-1
Xinxinag
MK641822
702/233
PCV2b
HeNZK-1
Zhoukou
MK641823
702/233
PCV2b
HeNZM-1
Kaifeng
MK641824
702/233
PCV2b
HeNZM-2
Kaifeng
MK641825
702/233
PCV2b
HeNPDS
Pingdingshan
MK641826
645/214
PCV3
HeNLH
Luohe
MK641836
645/214
PCV3
HeNZM1
Kaifeng
MK641837
645/214
PCV3
HeNZM2
Kaifeng
MK641838
645/214
PCV3
HeNKF
Kaifeng
MK641839
645/214
PCV3
HeNZK1
Zhoukou
MK641840
645/214
PCV3
HeNZK2
Zhoukou
MK641841
645/214
PCV3
HeNXX1
Xinxinag
MK641842
645/214
PCV3
HeNXX2
Xinxiang
MK641843
645/214
PCV3
HeNLY1
Luoyang
MK777996
645/214
PCV3
HeNLY2
Luoyang
MK777997
645/214
PCV3
HeNNY1
Nanyang
MK777998
645/214
PCV3
HeNNY2
Nanyang
MK777999
645/214
PCV3
HeNHB1
Hebi
MK778000
645/214
PCV3
Zhamadian
MK778001
645/214
PCV3
HeNZMD1
1
Highlights
2
High co-infection rate of PCV2, PCV3, and PEDV was determined in pigs with
3
watery diarrhea in Henan province, Central China.
4
Two PCV2 genotypes, PCV2b and PCV2d, were circulating in the fields in
5
central China.
6
PCV3 also exhibits a degree of genetic divergence.
7
Distinctive amino acid substitutions were characterized on the cap protein of
8
PCV2 and PCV3 isolates, which may be linked to the antigenic variations and
9
immune escape.
1