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First isolation and genetic characterization of porcine circovirus type 3 using primary porcine kidney cells
Journal Pre-proof First isolation and genetic characterization of porcine circovirus type 3 using primary porcine kidney cells Taehwan Oh, Chanhee Chae
PII:
S0378-1135(19)31281-7
DOI:
https://doi.org/10.1016/j.vetmic.2020.108576
Reference:
VETMIC 108576
To appear in:
Veterinary Microbiology
Received Date:
31 October 2019
Revised Date:
26 December 2019
Accepted Date:
1 January 2020
Please cite this article as: Oh T, Chae C, First isolation and genetic characterization of porcine circovirus type 3 using primary porcine kidney cells, Veterinary Microbiology (2020), doi: https://doi.org/10.1016/j.vetmic.2020.108576
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. © 2019 Published by Elsevier.
Veterinary Microbiology (Short Communication 1st revised VETMIC_2019_1192)
First isolation and genetic characterization of porcine circovirus type 3 using primary porcine kidney cells
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Running title: Isolation of porcine circovirus type 3
Taehwan Oh, Chanhee Chae
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Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National
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University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea
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Correspondence
Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National
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University, Gwanak-ro 1, Gwanak-gu, Seoul, 08826, Republic of Korea Tel: +82 2880 1277;
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Fax: +82 2873 1213;
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E-mail address: [email protected] (Chanhee Chae)
Highlights
The primary porcine kidney cells were used for isolation and propagation of PCV3.
The intracytoplasmic inclusion bodies contained virus-like particles arranged in 1
paracrystallline arrays on PCV3-infected primary porcine kidney cell.
ABSTRACT Porcine circovirus type 3 (PCV3) was first detected in aborted fetuses in 2015 when sows displaying clinical signs that looked like porcine dermatitis and nephropathy syndrome died suddenly. Primary porcine kidney cells were selected for both the isolation and propagation
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of PCV3 strain SNUVR181115 (GenBank accession number MK503331) as these cells were permissive to PCV3 infection. PCV3 did not produce cytopathic effect on infected monolayers, therefore PCV3 infection was confirmed by in situ hybridization with a PCV3
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specific DNA probe. Electron microscopy was used to analyze cell culture for the presence of
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virus. The intracytoplasmic inclusion bodies contained virus-like particles arranged in paracrystallline arrays on PCV3-infected primary porcine kidney cell. Virus replication
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peaked at 6th passage yielding titers close to 106 genomic copies of PCV3 per mL. PCV3 strain SNUVR181115 isolated from primary porcine kidney cells was highly conservative
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and was clustered with the Korean and Chinese strains. These results demonstrated that
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primary porcine kidney cells are useful for PCV3 isolation and replication.
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Keywords: isolation, genetic characteristics, porcine circovirus type 3
1. Introduction
The porcine circoviruses (PCVs) are members of the genus Circovirus and the family
Circoviridae. PCVs are known as the smallest non-enveloped, single-stranded, circular DNA viruses (Mankertz et al., 1997). Previously, two types of porcine circoviruses have been 2
identified and are known as PCV1 and PCV2 (Meehan et al., 1998). PCV1 is considered nonpathogenic for pigs (Allan et al., 2012) and is traditionally propagated in porcine kidney cells (PK-15) (Tischer et al., 1982). By contrast, PCV2 is the primary etiological agent of porcine circovirus-associated diseases (PCVAD) which have resulted in severe economic losses to the global pig industry (Chae, 2005). A new study recently analyzed aborted fetuses from sows which displayed symptoms similar to porcine dermatitis and nephropathy
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syndrome (PDNS) and were found deceased shortly afterward, identified a new species of PCV which was designated as PCV3 (Palinski et al., 2017). A second study reported the detection of PCV3 from a case of multisystemic disease with the absence of any other significant pathogens (Phan et al., 2016). Subsequently, multiple studies from a number of
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pig producing countries have reported detection of PCV3 (Faccini et al., 2017; Franzo et al.,
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2018; Kwon et al., 2017; Stadejek et al., 2017; Tochetto et al., 2018; Ye et al., 2018; Zhai et al., 2017). Despite the apparent wide distribution of PCV3 and the reported association with
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clinical disease similar to that of PCV2, the debate regarding the pathogenicity of PCV3 is still ongoing. To answer this question, it was necessary to isolate PCV3 and conduct clinical
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studies in an attempt to mimic the clinical signs and lesions observed in the field. Although the PK-15 cell line is widely used for the isolation and production of PCV1 and PCV2 current
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research exists proving the cell line is not permissive to PCV3 infection (Palinski et al., 2017). This study describes the isolation and propagation of PCV3 using primary porcine kidney cell
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lines.
2. Materials and methods 2.1. Preparation of primary porcine kidney cell culture 3
Primary cells were harvested from the kidneys of 21 day-old crossbred piglets. Commercial farms from which the swine neonates were obtained were selected based on four factors: availability of long-term (12-month period) of clinical and slaughter history, and breeding herd serological testing that produced negative results for porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae. The neonatal piglets were transferred to an incubator after farrowing to avoid to contact with their sows and raised
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within the incubator for 21days. Sera from piglets produced negative results for PCV1, PCV2, PCV3, porcine parvovirus (PPV) and PRRSV during screening via polymerase chain reaction (PCR) (Do et al., 2016; Kim and Chae, 2003; Palinski et al., 2017).
The tissue collection process was performed by anesthetizing the animals prior to
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euthanasia. Post-euthanasia, the piglets were removed to an aseptic laboratory environment
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for kidney collection during necropsy.
The method of preparing primary porcine kidney cells from the kidneys was performed as
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previously described (Takenouchi et al., 2014). Harvested primary porcine kidney cells were confirmed as free of PCV1, PCV2, PCV3, PPV, and PRRSV by PCR ((Do et al., 2016; Kim
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and Chae, 2003; Palinski et al., 2017) prior to freeze-down. Upon addition of DMSO (Nacalai tesque, Kyoto, Japan), cells were immediately frozen in liquid nitrogen where they were
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stored for 1–5 months.
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2.2. PCV3 isolation using primary porcine kidney cells After a stock of primary porcine kidney cells was established, PCV3 was isolated from
lymph node using the previously referenced method (Allan et al., 1998) as guidance for the virus isolation conditions. It should be noted that the referenced study differs from the current 4
study as it isolated PCV2 on PK-15 cells. A clinically healthy 91-day old pig was selected and purchased from a commercial farm which was confirmed to be positive exclusively for PCV3. The growing pig was confirmed sera-negative for PCV1, PCV2, PPV, and PRRSV by PCR methodology. Inguinal lymph nodes were collected from this pig post- euthanasia. Lymph node tissue samples were prepared for PCV3 isolation as follows: Approximately 15% of the tissues were suspended into RPMI-1640 medium containing 100 IU/mL of
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penicillin and 100 mg/ mL of streptomycin. The suspensions were then homogenized by a laboratory blender for 2 minutes. The homogenate was sonicated for 4 seconds at 20kHz, clarified by centrifugation at 2,000 g, and sterilized through a 0.2 m filter. Homogenate prepared in this manner was used as the inoculum for virus isolation studies. Prepared lymph
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nodes homogenate was tested with PCR and confirmed positive for PCV3 and negative for
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PCV1, PCV2, PPV, and PRRSV. The complete genome of PCV3 from the homogenated lymph node was amplified and sequenced as previously described (Kwon et al., 2017).
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Homogenated lymph nodes were evaluated with real-time PCR for PCV3 isolation, and resulted in 2.33 104 genomic copies of PCV3 per mL.
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Once primary porcine kidney cells reached confluency, cells were dissociated from the T75 flasks with trypsin–versene. Trypsin was then neutralized and cells were resuspended in
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RPMI-1640 medium containing 10% pestivirus-free fetal bovine serum (FBS) at a concentration of 1×106 cells/ mL. The cell suspension was divided into 20 mL aliquots which
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were then mixed with 4-mL aliquots of the inoculum described above. Each 24 mL aliquot was used to seed two new T-75 tissue culture flasks (12 mL per flask, or a 1:2 split). These cultures were placed inside of a 37°C, 5% CO2 incubator for 18 hours. The resulting semiconfluent monolayers were treated with 300 mM D-glucosamine (Allan et al., 1994) and then 5
incubated an additional 48–72 hours at 37°C. After 48-72 hours of incubation, three freezethaw cycles were performed on one representative flasks per each set of inoculum. The second flask for each set of inoculum was fresh-passaged with trypsin–versene. Trypsin was then neutralized while the detached cells were resuspended in 12 mL of RPMI-1640 medium containing 10% FBS. Fresh T-75 tissue culture flasks were seeded at a concentration of 1×106 cells/mL. The freshly seeded flasks were then ‘‘superinfected’’ by adding 4 mL of the cell
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lysate obtained from the partnered flask that had gone through 3 freeze-thaw cycles.
2.3. In situ hybridization
A 349 base pair (bp) DNA fragment from open reading frame (ORF) 1 was used as a PCV1
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probe, with the forward primer as 5’-TTG CTG AGC CTA GCG ACA CC-3’ and the reverse
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primers as 5'-TCC ACT GCT TCA AAT CGG CC-3'. The PCR method was carried out as described (Larochelle et al., 1999). A 481 bp DNA fragment from ORF 2 was used for the
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PCV2 probe with the forward primer as 5'-CGG ATA TTG TAG TCC TGG TCG-3' and the reverse primer as 5'-ACT GTC AAG GCT ACC ACA GTC A-3'. A 330 bp DNA fragment
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from cap gene was used as the PCV3 probe with the forward primer as 5'-CCA CAG AAG GCG CTA TGT C-3' and the reverse primer as 5'-CCG CAT AAG GGT CGT CTT G-3'. The
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PCR was carried out as described (Ellis et al., 1999; Palinski et al., 2017). Prior to use, Wizard PCR Preps (Promega Biotech, Madison, WI, USA) were used to purify the PCR
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products. After purification, random primed labeling with digoxigenin (DIG)-dUTP was performed on the PCR products by use of a commercial kit (Boehringer Mannheim, Indianapolis, IN, USA). 2-well chamber slides were used to propagate infected primary porcine kidney cell cultures for each of PCV1, PCV2, and PCV3 (respectively). The same 6
type of chamber slides were also used to propagate PCV1-infected, PCV2-infected, and PCV3-infected PK-15 cell cultures. In situ hybridization for the detection of PCV1, PCV2, and PCV3 was performed as previously described (Kim and Chae, 2001).
2.4. Electron microscopy Primary porcine kidney cells cultures were infected with approximately 4.11 × 105
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genomic copies/mL of PCV3, and mock-infected cell cultures (primary porcine kidney cell lysate) were treated in a similar fashion. Both the PCV3 and mock-infected cell cultures were incubated for 72 hr. at 37℃ in a 5% (v/v) CO2 atmosphere. The cells were dissociated from their vessels using trypsin–versene. Trypsin-versene was neutralized and cells were
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resuspended in RPMI-1640 medium. Cells were pelleted by centrifugation at 2,000xg for 10
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min. After processing, one set of samples was observed with conventional transmission electron microscopy (TEM) for the detection of PCV3. Subsequently, the culture of infected
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cells was checked by in situ hybridization at 48 hpi.
Preparation of the electron microscopy was done in accordance as previously described
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(Rodríguez-Cariño et al., 2011). Ultra-thin sections (70 nm in thickness) were cut from sections deemed visually sufficient in cell concentration with a diamond knife (45°, Diatome,
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Biel, Switzerland). These were placed on non-coated, 200-mesh copper grids and contrasted with conventional uranyl acetate solution for 10 minutes followed by Reynolds lead citrate
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for 3 minutes. Sections were then observed with a Talos L120C transmission electron microscope (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
2.5. Replication kinetics of PCV3 in primary porcine kidney cells 7
Replication kinetics were determined for the PCV3 isolates. Confluent monolayers in 75 cm2 cells culture flasks were inoculated with 2ml of a viral suspension containing a titer of 2.15 × 104 genomic copies/mL. The subcultures were passaged eight times, reaching confluency in 23 weeks. One flask was frozen at 70℃ at each passage until the end of the experiment. Finally, all collected samples were thawed, clarified by low-speed centrifugation
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and the resulting supernatants were titrated by real-time PCR (Kwon et al., 2017).
2.6. Viral genome sequencing analysis
The complete genome of the isolated virus strain was amplified and sequenced as previously described (Kwon et al., 2017). These sequences were analyzed using BioEdit
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7.0.5.3 and designated as SNUVR181115 with the accession number MK503331 in GenBank
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(www.ncbi.nlm.nih.gov/genbank/). Complete genome sequences of other PCV3 strains were obtained from the GenBank for study comparison. These included four Korean strains, seven
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Chinese strains, one Thai strain, five US strains, two Russian strains, two Italian strains and two Brazilian strains. The phylogenetic analysis was conducted by Neighbor-joining method
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3. Results
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with 1,000 bootstrap values in MEGA 6.06 software.
3.1. Isolation of PCV3 using primary porcine kidney cells
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Uninfected primary porcine kidney cells consisted of 2 major cell types: a large polygonal
cell arranged in islands and a predominantly spindle shaped cell-type which was organized in a network of strands surrounding the islands of polygonal cells (Fig. 1A). No cytopathic effects (CPE) were observed on infected monolayers, therefore in situ hybridization with a 8
PCV3 specific probe to confirm the infection on primary porcine kidney cell monolayers was utilized, which detected a PCV3-positive signal predominately found in the cytoplasm (Fig. 1B). Some occurrences also appeared in the nuclei of the infected cells. Primary porcine kidney cells were detached during the process of in situ hybridization. In contrast, PCV3 DNA was not detected in the cytoplasm or the nuclei of PCV3-infected PK-15 cell (Fig. 1C). As expected, no signal was detected when PCV1 and PCV2 specific probes were used as
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controls. PCV1 or PCV2 specific probes also detected positive signals in PK-15 cells infected with either PCV1 or PCV2, respectively to confirm specificity. In addition, the PCV3 specific
3.2. Electron microscopic observation of PCV3
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probe used in this study did not hybridize with PCV1- and PCV2-infected PK-15 cell lines.
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Electron microscopy was used to analyze cell culture for the presence of virus. Small aggregates of PCV-like particles that were circular and had a diameter of approximately 17
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nm were present. Non-membrane bound intracytoplasmic inclusion bodies were also observed in infected cultures at 72 hour post-inoculation. The intracytoplasmic inclusion
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bodies contained virus-like particles arranged in paracrystallline arrays on PCV3-infected primary porcine kidney cell (Fig. 2). No other virus-like particles were observed in these
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preparations nor were any virus particle observed in uninoculated control cell cultures.
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3.3. Replication kinetics of PCV3 in primary kidney cells Growth curves were established for the PCV3 isolate by monitoring infectious progency
production through synthesis of viral DNA using RT-PCR. The observed increase of the infectious virus after a primary porcine kidney cells passage correlated with an increase in the 9
amount of viral DNA detected (Fig. 3). Viral genomic copies continued to increase as late as the 6th passage at which point they plateaued until the end of the experiment at passage 8.
3.4. Phylogenetic analysis of PCV3 isolate The comparative genomic analyses of the PCV3 strain isolated from the lymph node and the cell-adapted isolate were compared and found to be identical, further confirming that the
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PCV3 isolation was successful. The complete genome of the PCV3 cell culture isolate was 2,000 nucleotides in length, similar to other PCV3 strains. A phylogenetic tree based on the whole genome showed that PCV3 strain SNUVR181115 (GenBank accession number MK50333) was closely related to multiple other strains evaluated in this study. The Chinese
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PCV3 strains reported from 7 Provinces were compared with SNUVR181115 to identify the
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genetic differences between each other. The genome nucleotide similarity of Chinese strains with SNUVR181115 ranged from 98.9% to 99.6%. Also, the strain isolated in this study
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shares 99.0%, 99.2%, 99.4%, 99.3% and 99.3% identity with US strains (PCV3-US/MO2015, PCV3-US/MN2016, PCV3-US/SD2016, 2164, 29160) respectively. PCV3 (SNUVR181115)
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showed the highest genomic nucleotide identity (99.8%) with PCV3/KU-1605 from among the Korean strains. SNUVR181115 share 98.8% and 99.4% identity with Russian strains
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(PCV3-RU/SM17, PCV3-RU/TY17), 99.0% and 98.9% identity with Italian strains (PCV3IT/CO2017, PCV3-IT/MN2017), 99.2% and 99.6% identity with Brazilian strains (PCV3-
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BR/RS/6 and PCV3-BR/RS/8) and 99.1 % with Thai strain (PCV3/Thailand/PB01/17), respectively (Fig. 4).
4. DISCUSSION 10
The successful isolation and in vitro culture of PCV3 is the first major step that will enable us to study this novel virus, its pathogenesis, its interaction with the host immune system and its adverse effect on the global pork industry. In this study we were able to successfully culture a PCV3 isolate on primary porcine kidney cells. Examination of cell cultures with electron microscopy revealed the presence of virus particles of a size and morphology identical to porcine circovirus (Tischer et al., 1982). In additions, in situ hybridization using a
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PCV3 specific probe on formalin-fixed cells inoculated with PCV3 showed a positive signal in the cytoplasm of infected cells. These cells were negative when in situ hybridization was performed using a PCV1 or PCV2 specific probe confirming the infection was specific to PCV3. As in situ hybridization is not capable of differentiating replicating virus from
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nonreplicating virus, growth kinetics of PCV3 in primary porcine kidney cells were used to
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demonstrate the PCV3 replication in primary porcine kidney cells. These results indicate that PCV3 is likely replicated in the cytoplasm of infected cells and did not accumulate by uptake
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from the culture medium. An increase of infectious virus and viral DNA synthesis was seen at several passages. We saw a successful serial propagation of PCV3 in primary porcine kidney
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cells over the course of 8 passages. Therefore, primary porcine kidney cells are considered a useful tool for the isolation and propagation of virus, despite the setback of not using a
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continuous cell line where a larger number of passages are allowed. The fact that they can support replication of PCV3 underscores the potential of these cells for diagnostic and
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research applications. Growth kinetics are not an efficient method for measuring the amount of virus in primary porcine kidney cells. This is because during PCV2 replication, there are some PK-15 cell subpopulations that are highly permissive and others that are lowly permissive to PCV2 infection from the heterogeneous PK-15 parent cells (Zhu et al., 2007). 11
Similar to PK-15 cells, the primary porcine kidney cells are heterogeneous in the sense that they contain these selectively permissive populations. Further studies are needed to clone and produce a homologous, highly permissive primary porcine kidney cell subpopulation for PCV3 infection. The complete genome of PCV3 strain SNUVR181115 (GenBank accession number MK503331) shares 99.3% of its nucleotide identity with both PCV3 29160 and PCV3 2164.
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Both are associated with PDNS and reproductive failure (Palinski et al., 2017). The genome nucleotide homology of PCV3 strain SNUVR181115 with other Korean isolates (PCV3/KU1605, PCV3/KU-1607, PCV3/KU-1608, and PCV3/KU-1609) range from 99.2% to 99.8%. The genome nucleotide homology of PCV3 strain SNUVR181115 with 7 Chinese isolates
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(PCV3/CN/Fujian-12/2016, PCV3-CN-HeBei1-2018, PCV3 CN Beijing-3 2017SJYH,
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PCV3/CN/Xinjiang-12/2018, PCV3/Guangxi-CZ/05, PCV3/CN/Chongqing-147/2016, and PCV3/CN/Guangdong-HY1/2016) range from 98.9% to 99.6%. The phylogenetic analysis
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performed in this study demonstrates that the first PCV3 cell culture isolate (SNUVR181115) is highly conservative and within the same cluster as the Korean and Chinese strains.
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A homogeneous PCV3 infectious virus stock, which was derived from an infectious PCV3 molecular DNA clone, is capable of reproducing PDNS-like disease when administered to 4-
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and 8-week-old piglets via the intranasal route (Jiang et al., 2019). This is consistent with a previous study which reported that PCV3 was detected in aborted fetuses which were
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collected from sows that died while exhibiting similar to PDNS (Palinski et al., 2017). PCV3 may also be associated with porcine respiratory disease complex (PRDC). The PRDCaffected pigs exhibited higher prevalence of PCV3 infection and higher PCV3 titers compare to clinically healthy pigs (Kedkovid et al., 2018). However, so far, no one has been able to 12
reproduce these various clinical manifestations with experimental infection using PCV3 isolates and further studies are necessary to demonstrate a correlation.
Conflict of interest statement The authors declare that there is no conflict of interest.
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Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2019R1F1A1044419). The authors’ research was also supported by the Research Institute for Veterinary Science (RIVS) from the College
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of Veterinary Medicine and by the BK 21 Plus Program (Grant no. 5260-20150100) for
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Creative Veterinary Science Research.
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Virology 369, 423-430.
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Legend of Figures FIG. 1. Uninfected normal primary porcine kidney cells with islands of polygonal and strands of spindle cells (A). In situ hybridization on primary porcine kidney cell culture preparations inoculated with lymph node materials using PCV3 specific probe. Note strong hybridization signal in the cytoplasm of cells (B). In situ hybridization on PK-15 cell culture preparations inoculated with lymph node materials using PCV3 specific probe. No
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hybridization signal in the cytoplasm and nuclei of cells (C).
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FIG. 2. Electron micrographys of PCV3-infected primary porcine kidney cells at 72 hours post inoculation. Large irregular intracytoplasmic inclusion (ICI) body with virus-like
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particles arranged in paracrystalline arrays (inset at higher manificantion).
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FIG. 3. Serial passage of PCV3 in primary porcine kidney cells. Real-time polymerase chain reaction (PCR) results are displayed as log10 genomic copies/mL. Viral DNA was quantified
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by three independent real-time PCR. Error bars represent the standard deviation.
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FIG. 4. Maximum likelihood phylogenetic tress of Korean and other PCV3 strains based on
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full-genome nucleotide sequences. GenBank accession numbers and the country of origin and
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the collection data are in parenthesis.
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PII:
S0378-1135(19)31281-7
DOI:
https://doi.org/10.1016/j.vetmic.2020.108576
Reference:
VETMIC 108576
To appear in:
Veterinary Microbiology
Received Date:
31 October 2019
Revised Date:
26 December 2019
Accepted Date:
1 January 2020
Please cite this article as: Oh T, Chae C, First isolation and genetic characterization of porcine circovirus type 3 using primary porcine kidney cells, Veterinary Microbiology (2020), doi: https://doi.org/10.1016/j.vetmic.2020.108576
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. © 2019 Published by Elsevier.
Veterinary Microbiology (Short Communication 1st revised VETMIC_2019_1192)
First isolation and genetic characterization of porcine circovirus type 3 using primary porcine kidney cells
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Running title: Isolation of porcine circovirus type 3
Taehwan Oh, Chanhee Chae
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Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National
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University, 1 Gwanak-ro, Gwanak-gu, Seoul, 08826, Republic of Korea
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Correspondence
Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National
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University, Gwanak-ro 1, Gwanak-gu, Seoul, 08826, Republic of Korea Tel: +82 2880 1277;
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Fax: +82 2873 1213;
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E-mail address: [email protected] (Chanhee Chae)
Highlights
The primary porcine kidney cells were used for isolation and propagation of PCV3.
The intracytoplasmic inclusion bodies contained virus-like particles arranged in 1
paracrystallline arrays on PCV3-infected primary porcine kidney cell.
ABSTRACT Porcine circovirus type 3 (PCV3) was first detected in aborted fetuses in 2015 when sows displaying clinical signs that looked like porcine dermatitis and nephropathy syndrome died suddenly. Primary porcine kidney cells were selected for both the isolation and propagation
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of PCV3 strain SNUVR181115 (GenBank accession number MK503331) as these cells were permissive to PCV3 infection. PCV3 did not produce cytopathic effect on infected monolayers, therefore PCV3 infection was confirmed by in situ hybridization with a PCV3
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specific DNA probe. Electron microscopy was used to analyze cell culture for the presence of
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virus. The intracytoplasmic inclusion bodies contained virus-like particles arranged in paracrystallline arrays on PCV3-infected primary porcine kidney cell. Virus replication
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peaked at 6th passage yielding titers close to 106 genomic copies of PCV3 per mL. PCV3 strain SNUVR181115 isolated from primary porcine kidney cells was highly conservative
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and was clustered with the Korean and Chinese strains. These results demonstrated that
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primary porcine kidney cells are useful for PCV3 isolation and replication.
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Keywords: isolation, genetic characteristics, porcine circovirus type 3
1. Introduction
The porcine circoviruses (PCVs) are members of the genus Circovirus and the family
Circoviridae. PCVs are known as the smallest non-enveloped, single-stranded, circular DNA viruses (Mankertz et al., 1997). Previously, two types of porcine circoviruses have been 2
identified and are known as PCV1 and PCV2 (Meehan et al., 1998). PCV1 is considered nonpathogenic for pigs (Allan et al., 2012) and is traditionally propagated in porcine kidney cells (PK-15) (Tischer et al., 1982). By contrast, PCV2 is the primary etiological agent of porcine circovirus-associated diseases (PCVAD) which have resulted in severe economic losses to the global pig industry (Chae, 2005). A new study recently analyzed aborted fetuses from sows which displayed symptoms similar to porcine dermatitis and nephropathy
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syndrome (PDNS) and were found deceased shortly afterward, identified a new species of PCV which was designated as PCV3 (Palinski et al., 2017). A second study reported the detection of PCV3 from a case of multisystemic disease with the absence of any other significant pathogens (Phan et al., 2016). Subsequently, multiple studies from a number of
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pig producing countries have reported detection of PCV3 (Faccini et al., 2017; Franzo et al.,
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2018; Kwon et al., 2017; Stadejek et al., 2017; Tochetto et al., 2018; Ye et al., 2018; Zhai et al., 2017). Despite the apparent wide distribution of PCV3 and the reported association with
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clinical disease similar to that of PCV2, the debate regarding the pathogenicity of PCV3 is still ongoing. To answer this question, it was necessary to isolate PCV3 and conduct clinical
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studies in an attempt to mimic the clinical signs and lesions observed in the field. Although the PK-15 cell line is widely used for the isolation and production of PCV1 and PCV2 current
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research exists proving the cell line is not permissive to PCV3 infection (Palinski et al., 2017). This study describes the isolation and propagation of PCV3 using primary porcine kidney cell
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lines.
2. Materials and methods 2.1. Preparation of primary porcine kidney cell culture 3
Primary cells were harvested from the kidneys of 21 day-old crossbred piglets. Commercial farms from which the swine neonates were obtained were selected based on four factors: availability of long-term (12-month period) of clinical and slaughter history, and breeding herd serological testing that produced negative results for porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae. The neonatal piglets were transferred to an incubator after farrowing to avoid to contact with their sows and raised
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within the incubator for 21days. Sera from piglets produced negative results for PCV1, PCV2, PCV3, porcine parvovirus (PPV) and PRRSV during screening via polymerase chain reaction (PCR) (Do et al., 2016; Kim and Chae, 2003; Palinski et al., 2017).
The tissue collection process was performed by anesthetizing the animals prior to
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euthanasia. Post-euthanasia, the piglets were removed to an aseptic laboratory environment
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for kidney collection during necropsy.
The method of preparing primary porcine kidney cells from the kidneys was performed as
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previously described (Takenouchi et al., 2014). Harvested primary porcine kidney cells were confirmed as free of PCV1, PCV2, PCV3, PPV, and PRRSV by PCR ((Do et al., 2016; Kim
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and Chae, 2003; Palinski et al., 2017) prior to freeze-down. Upon addition of DMSO (Nacalai tesque, Kyoto, Japan), cells were immediately frozen in liquid nitrogen where they were
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stored for 1–5 months.
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2.2. PCV3 isolation using primary porcine kidney cells After a stock of primary porcine kidney cells was established, PCV3 was isolated from
lymph node using the previously referenced method (Allan et al., 1998) as guidance for the virus isolation conditions. It should be noted that the referenced study differs from the current 4
study as it isolated PCV2 on PK-15 cells. A clinically healthy 91-day old pig was selected and purchased from a commercial farm which was confirmed to be positive exclusively for PCV3. The growing pig was confirmed sera-negative for PCV1, PCV2, PPV, and PRRSV by PCR methodology. Inguinal lymph nodes were collected from this pig post- euthanasia. Lymph node tissue samples were prepared for PCV3 isolation as follows: Approximately 15% of the tissues were suspended into RPMI-1640 medium containing 100 IU/mL of
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penicillin and 100 mg/ mL of streptomycin. The suspensions were then homogenized by a laboratory blender for 2 minutes. The homogenate was sonicated for 4 seconds at 20kHz, clarified by centrifugation at 2,000 g, and sterilized through a 0.2 m filter. Homogenate prepared in this manner was used as the inoculum for virus isolation studies. Prepared lymph
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nodes homogenate was tested with PCR and confirmed positive for PCV3 and negative for
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PCV1, PCV2, PPV, and PRRSV. The complete genome of PCV3 from the homogenated lymph node was amplified and sequenced as previously described (Kwon et al., 2017).
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Homogenated lymph nodes were evaluated with real-time PCR for PCV3 isolation, and resulted in 2.33 104 genomic copies of PCV3 per mL.
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Once primary porcine kidney cells reached confluency, cells were dissociated from the T75 flasks with trypsin–versene. Trypsin was then neutralized and cells were resuspended in
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RPMI-1640 medium containing 10% pestivirus-free fetal bovine serum (FBS) at a concentration of 1×106 cells/ mL. The cell suspension was divided into 20 mL aliquots which
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were then mixed with 4-mL aliquots of the inoculum described above. Each 24 mL aliquot was used to seed two new T-75 tissue culture flasks (12 mL per flask, or a 1:2 split). These cultures were placed inside of a 37°C, 5% CO2 incubator for 18 hours. The resulting semiconfluent monolayers were treated with 300 mM D-glucosamine (Allan et al., 1994) and then 5
incubated an additional 48–72 hours at 37°C. After 48-72 hours of incubation, three freezethaw cycles were performed on one representative flasks per each set of inoculum. The second flask for each set of inoculum was fresh-passaged with trypsin–versene. Trypsin was then neutralized while the detached cells were resuspended in 12 mL of RPMI-1640 medium containing 10% FBS. Fresh T-75 tissue culture flasks were seeded at a concentration of 1×106 cells/mL. The freshly seeded flasks were then ‘‘superinfected’’ by adding 4 mL of the cell
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lysate obtained from the partnered flask that had gone through 3 freeze-thaw cycles.
2.3. In situ hybridization
A 349 base pair (bp) DNA fragment from open reading frame (ORF) 1 was used as a PCV1
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probe, with the forward primer as 5’-TTG CTG AGC CTA GCG ACA CC-3’ and the reverse
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primers as 5'-TCC ACT GCT TCA AAT CGG CC-3'. The PCR method was carried out as described (Larochelle et al., 1999). A 481 bp DNA fragment from ORF 2 was used for the
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PCV2 probe with the forward primer as 5'-CGG ATA TTG TAG TCC TGG TCG-3' and the reverse primer as 5'-ACT GTC AAG GCT ACC ACA GTC A-3'. A 330 bp DNA fragment
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from cap gene was used as the PCV3 probe with the forward primer as 5'-CCA CAG AAG GCG CTA TGT C-3' and the reverse primer as 5'-CCG CAT AAG GGT CGT CTT G-3'. The
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PCR was carried out as described (Ellis et al., 1999; Palinski et al., 2017). Prior to use, Wizard PCR Preps (Promega Biotech, Madison, WI, USA) were used to purify the PCR
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products. After purification, random primed labeling with digoxigenin (DIG)-dUTP was performed on the PCR products by use of a commercial kit (Boehringer Mannheim, Indianapolis, IN, USA). 2-well chamber slides were used to propagate infected primary porcine kidney cell cultures for each of PCV1, PCV2, and PCV3 (respectively). The same 6
type of chamber slides were also used to propagate PCV1-infected, PCV2-infected, and PCV3-infected PK-15 cell cultures. In situ hybridization for the detection of PCV1, PCV2, and PCV3 was performed as previously described (Kim and Chae, 2001).
2.4. Electron microscopy Primary porcine kidney cells cultures were infected with approximately 4.11 × 105
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genomic copies/mL of PCV3, and mock-infected cell cultures (primary porcine kidney cell lysate) were treated in a similar fashion. Both the PCV3 and mock-infected cell cultures were incubated for 72 hr. at 37℃ in a 5% (v/v) CO2 atmosphere. The cells were dissociated from their vessels using trypsin–versene. Trypsin-versene was neutralized and cells were
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resuspended in RPMI-1640 medium. Cells were pelleted by centrifugation at 2,000xg for 10
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min. After processing, one set of samples was observed with conventional transmission electron microscopy (TEM) for the detection of PCV3. Subsequently, the culture of infected
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cells was checked by in situ hybridization at 48 hpi.
Preparation of the electron microscopy was done in accordance as previously described
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(Rodríguez-Cariño et al., 2011). Ultra-thin sections (70 nm in thickness) were cut from sections deemed visually sufficient in cell concentration with a diamond knife (45°, Diatome,
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Biel, Switzerland). These were placed on non-coated, 200-mesh copper grids and contrasted with conventional uranyl acetate solution for 10 minutes followed by Reynolds lead citrate
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for 3 minutes. Sections were then observed with a Talos L120C transmission electron microscope (Thermo Fisher Scientific, Inc., Waltham, MA, USA).
2.5. Replication kinetics of PCV3 in primary porcine kidney cells 7
Replication kinetics were determined for the PCV3 isolates. Confluent monolayers in 75 cm2 cells culture flasks were inoculated with 2ml of a viral suspension containing a titer of 2.15 × 104 genomic copies/mL. The subcultures were passaged eight times, reaching confluency in 23 weeks. One flask was frozen at 70℃ at each passage until the end of the experiment. Finally, all collected samples were thawed, clarified by low-speed centrifugation
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and the resulting supernatants were titrated by real-time PCR (Kwon et al., 2017).
2.6. Viral genome sequencing analysis
The complete genome of the isolated virus strain was amplified and sequenced as previously described (Kwon et al., 2017). These sequences were analyzed using BioEdit
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7.0.5.3 and designated as SNUVR181115 with the accession number MK503331 in GenBank
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(www.ncbi.nlm.nih.gov/genbank/). Complete genome sequences of other PCV3 strains were obtained from the GenBank for study comparison. These included four Korean strains, seven
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Chinese strains, one Thai strain, five US strains, two Russian strains, two Italian strains and two Brazilian strains. The phylogenetic analysis was conducted by Neighbor-joining method
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3. Results
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with 1,000 bootstrap values in MEGA 6.06 software.
3.1. Isolation of PCV3 using primary porcine kidney cells
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Uninfected primary porcine kidney cells consisted of 2 major cell types: a large polygonal
cell arranged in islands and a predominantly spindle shaped cell-type which was organized in a network of strands surrounding the islands of polygonal cells (Fig. 1A). No cytopathic effects (CPE) were observed on infected monolayers, therefore in situ hybridization with a 8
PCV3 specific probe to confirm the infection on primary porcine kidney cell monolayers was utilized, which detected a PCV3-positive signal predominately found in the cytoplasm (Fig. 1B). Some occurrences also appeared in the nuclei of the infected cells. Primary porcine kidney cells were detached during the process of in situ hybridization. In contrast, PCV3 DNA was not detected in the cytoplasm or the nuclei of PCV3-infected PK-15 cell (Fig. 1C). As expected, no signal was detected when PCV1 and PCV2 specific probes were used as
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controls. PCV1 or PCV2 specific probes also detected positive signals in PK-15 cells infected with either PCV1 or PCV2, respectively to confirm specificity. In addition, the PCV3 specific
3.2. Electron microscopic observation of PCV3
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probe used in this study did not hybridize with PCV1- and PCV2-infected PK-15 cell lines.
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Electron microscopy was used to analyze cell culture for the presence of virus. Small aggregates of PCV-like particles that were circular and had a diameter of approximately 17
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nm were present. Non-membrane bound intracytoplasmic inclusion bodies were also observed in infected cultures at 72 hour post-inoculation. The intracytoplasmic inclusion
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bodies contained virus-like particles arranged in paracrystallline arrays on PCV3-infected primary porcine kidney cell (Fig. 2). No other virus-like particles were observed in these
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preparations nor were any virus particle observed in uninoculated control cell cultures.
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3.3. Replication kinetics of PCV3 in primary kidney cells Growth curves were established for the PCV3 isolate by monitoring infectious progency
production through synthesis of viral DNA using RT-PCR. The observed increase of the infectious virus after a primary porcine kidney cells passage correlated with an increase in the 9
amount of viral DNA detected (Fig. 3). Viral genomic copies continued to increase as late as the 6th passage at which point they plateaued until the end of the experiment at passage 8.
3.4. Phylogenetic analysis of PCV3 isolate The comparative genomic analyses of the PCV3 strain isolated from the lymph node and the cell-adapted isolate were compared and found to be identical, further confirming that the
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PCV3 isolation was successful. The complete genome of the PCV3 cell culture isolate was 2,000 nucleotides in length, similar to other PCV3 strains. A phylogenetic tree based on the whole genome showed that PCV3 strain SNUVR181115 (GenBank accession number MK50333) was closely related to multiple other strains evaluated in this study. The Chinese
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PCV3 strains reported from 7 Provinces were compared with SNUVR181115 to identify the
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genetic differences between each other. The genome nucleotide similarity of Chinese strains with SNUVR181115 ranged from 98.9% to 99.6%. Also, the strain isolated in this study
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shares 99.0%, 99.2%, 99.4%, 99.3% and 99.3% identity with US strains (PCV3-US/MO2015, PCV3-US/MN2016, PCV3-US/SD2016, 2164, 29160) respectively. PCV3 (SNUVR181115)
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showed the highest genomic nucleotide identity (99.8%) with PCV3/KU-1605 from among the Korean strains. SNUVR181115 share 98.8% and 99.4% identity with Russian strains
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(PCV3-RU/SM17, PCV3-RU/TY17), 99.0% and 98.9% identity with Italian strains (PCV3IT/CO2017, PCV3-IT/MN2017), 99.2% and 99.6% identity with Brazilian strains (PCV3-
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BR/RS/6 and PCV3-BR/RS/8) and 99.1 % with Thai strain (PCV3/Thailand/PB01/17), respectively (Fig. 4).
4. DISCUSSION 10
The successful isolation and in vitro culture of PCV3 is the first major step that will enable us to study this novel virus, its pathogenesis, its interaction with the host immune system and its adverse effect on the global pork industry. In this study we were able to successfully culture a PCV3 isolate on primary porcine kidney cells. Examination of cell cultures with electron microscopy revealed the presence of virus particles of a size and morphology identical to porcine circovirus (Tischer et al., 1982). In additions, in situ hybridization using a
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PCV3 specific probe on formalin-fixed cells inoculated with PCV3 showed a positive signal in the cytoplasm of infected cells. These cells were negative when in situ hybridization was performed using a PCV1 or PCV2 specific probe confirming the infection was specific to PCV3. As in situ hybridization is not capable of differentiating replicating virus from
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nonreplicating virus, growth kinetics of PCV3 in primary porcine kidney cells were used to
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demonstrate the PCV3 replication in primary porcine kidney cells. These results indicate that PCV3 is likely replicated in the cytoplasm of infected cells and did not accumulate by uptake
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from the culture medium. An increase of infectious virus and viral DNA synthesis was seen at several passages. We saw a successful serial propagation of PCV3 in primary porcine kidney
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cells over the course of 8 passages. Therefore, primary porcine kidney cells are considered a useful tool for the isolation and propagation of virus, despite the setback of not using a
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continuous cell line where a larger number of passages are allowed. The fact that they can support replication of PCV3 underscores the potential of these cells for diagnostic and
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research applications. Growth kinetics are not an efficient method for measuring the amount of virus in primary porcine kidney cells. This is because during PCV2 replication, there are some PK-15 cell subpopulations that are highly permissive and others that are lowly permissive to PCV2 infection from the heterogeneous PK-15 parent cells (Zhu et al., 2007). 11
Similar to PK-15 cells, the primary porcine kidney cells are heterogeneous in the sense that they contain these selectively permissive populations. Further studies are needed to clone and produce a homologous, highly permissive primary porcine kidney cell subpopulation for PCV3 infection. The complete genome of PCV3 strain SNUVR181115 (GenBank accession number MK503331) shares 99.3% of its nucleotide identity with both PCV3 29160 and PCV3 2164.
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Both are associated with PDNS and reproductive failure (Palinski et al., 2017). The genome nucleotide homology of PCV3 strain SNUVR181115 with other Korean isolates (PCV3/KU1605, PCV3/KU-1607, PCV3/KU-1608, and PCV3/KU-1609) range from 99.2% to 99.8%. The genome nucleotide homology of PCV3 strain SNUVR181115 with 7 Chinese isolates
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(PCV3/CN/Fujian-12/2016, PCV3-CN-HeBei1-2018, PCV3 CN Beijing-3 2017SJYH,
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PCV3/CN/Xinjiang-12/2018, PCV3/Guangxi-CZ/05, PCV3/CN/Chongqing-147/2016, and PCV3/CN/Guangdong-HY1/2016) range from 98.9% to 99.6%. The phylogenetic analysis
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performed in this study demonstrates that the first PCV3 cell culture isolate (SNUVR181115) is highly conservative and within the same cluster as the Korean and Chinese strains.
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A homogeneous PCV3 infectious virus stock, which was derived from an infectious PCV3 molecular DNA clone, is capable of reproducing PDNS-like disease when administered to 4-
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and 8-week-old piglets via the intranasal route (Jiang et al., 2019). This is consistent with a previous study which reported that PCV3 was detected in aborted fetuses which were
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collected from sows that died while exhibiting similar to PDNS (Palinski et al., 2017). PCV3 may also be associated with porcine respiratory disease complex (PRDC). The PRDCaffected pigs exhibited higher prevalence of PCV3 infection and higher PCV3 titers compare to clinically healthy pigs (Kedkovid et al., 2018). However, so far, no one has been able to 12
reproduce these various clinical manifestations with experimental infection using PCV3 isolates and further studies are necessary to demonstrate a correlation.
Conflict of interest statement The authors declare that there is no conflict of interest.
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Acknowledgements This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. 2019R1F1A1044419). The authors’ research was also supported by the Research Institute for Veterinary Science (RIVS) from the College
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of Veterinary Medicine and by the BK 21 Plus Program (Grant no. 5260-20150100) for
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Creative Veterinary Science Research.
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Legend of Figures FIG. 1. Uninfected normal primary porcine kidney cells with islands of polygonal and strands of spindle cells (A). In situ hybridization on primary porcine kidney cell culture preparations inoculated with lymph node materials using PCV3 specific probe. Note strong hybridization signal in the cytoplasm of cells (B). In situ hybridization on PK-15 cell culture preparations inoculated with lymph node materials using PCV3 specific probe. No
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hybridization signal in the cytoplasm and nuclei of cells (C).
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FIG. 2. Electron micrographys of PCV3-infected primary porcine kidney cells at 72 hours post inoculation. Large irregular intracytoplasmic inclusion (ICI) body with virus-like
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particles arranged in paracrystalline arrays (inset at higher manificantion).
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FIG. 3. Serial passage of PCV3 in primary porcine kidney cells. Real-time polymerase chain reaction (PCR) results are displayed as log10 genomic copies/mL. Viral DNA was quantified
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by three independent real-time PCR. Error bars represent the standard deviation.
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FIG. 4. Maximum likelihood phylogenetic tress of Korean and other PCV3 strains based on
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full-genome nucleotide sequences. GenBank accession numbers and the country of origin and
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the collection data are in parenthesis.
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