The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets

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The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets

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Yanhui Wang b,1 , Xiaojing Gao b,1 , Yali Yao b,1 , Yunjing Zhang b , Chaochao Lv b , Zhe Sun b , Yuzhou Wang b , Xiangrui Jia b , Jinshan Zhuang b , Yan Xiao b , Xiangdong Li b,∗ , Kegong Tian a,b,∗ a

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College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, Henan 450002, PR China National Research Center for Veterinary Medicine, Road Cuiwei, High-Tech District, Luoyang 471003, PR China

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a r t i c l e

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a b s t r a c t

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Article history: Received 13 April 2015 Received in revised form 5 June 2015 Accepted 5 June 2015 Available online xxx

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Keywords: Porcine epidemic diarrhea virus Serial propagation in cell culture Immunoperoxidase monolayer assay Dynamics of virus production

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1. Introduction

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A severe porcine epidemic diarrhea (PED) epizootic has been affecting pigs of all ages that are characterized by high mortality among suckling piglets in China since late 2010, causing significant economic losses. Obtaining a current-circulating PEDV variant isolate that can grow efficiently in cell culture is prerequisite for the development of efficient vaccines. In this study, PEDV strain HN1303 was isolated successfully on Vero cells with supplemental trypsin, and the isolate has been serially propagated in cell culture for over 95 passages. The infectious titers of the virus during the first 10 passages ranged from 102.6 to 105.8 50% tissue culture infective doses (TCID50 )/ml, and the titers of 20–95 passages ranged from 106.2 to 108.0 TCID50 /ml. The growth curve of Vero cell-adapted HN1303 in cell culture was determined, and dynamics of virus production was confirmed by immunoperoxidase monolayer assay (IPMA). Sequence and phylogenetic analysis based on spike gene indicate that the HN1303 strain belongs to genotype IIa. In addition, the fourth passage cell-culture HN1303 was subjected to 2-day old piglets. All piglets orally inoculated developed severe watery diarrhea and vomiting within 24 hours post-inoculation (hpi) and died within 72 hpi. The results of animal experiments reveal that this strain is highly pathogenic to 2-day old piglets. © 2015 Published by Elsevier B.V.

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Porcine epidemic diarrhea virus (PEDV), an agent causing a serious and highly contagious swine enteric disease, is an enveloped, single-stranded, positive-sense RNA virus belonging to the order Nidovirale, the family Coronaviridae, subfamily Coronavirinae, and genus Alphacoronavirus (International Committee on Taxonomy of Viruses, 2012). This disease was first observed in England in 1971, and subsequently reported in many swine-producing countries in Europe and Asia (Pensaert and de Bouck, 1978; Song and Park, 2012). Severe PED epizootics have been affecting pigs of all ages characterized by high morbidity and mortality rates among suckling piglets in China since late 2010, resulting in huge economic

∗ Corresponding authors at: National Research Center for Veterinary Medicine, Road Cuiwei, High-Tech District, Luoyang 471003, PR China. Tel.: +86 10 59198895; fax: +86 10 59198899. E-mail addresses: [email protected] (X. Li), [email protected] (K. Tian). 1 These authors contributed equally to this work.

losses, although most herds previously had received CV777-based inactivated or attenuated vaccines (Sun et al., 2012; Wang et al., 2013). Developing efficient live attenuated or inactivated vaccines against PEDV remains the best solution for prevention and control of the disease. Obtaining an epidemic PEDV isolate that can grow efficiently in cell culture is critical for the development of vaccines. There are previous reports of successful propagation of several classical PEDV strains using Vero cells and trypsin in the medium, such as CV777, KPEDV-9, and 83P-5 (Hofmann and Wyler, 1988; Kusanagi et al., 1992; Kweon et al., 1999). Recently, Oka et al. (2014) and Chen et al. (2014) reported successful serially propagation of the highly virulent US PEDV strains in cell cultures for over 30 passages. In this study, we attempted to isolate and propagate PEDV from four PEDV-positive intestinal samples using Vero cells supplemented with trypsin, and successfully isolated one PEDV strain designated as HN1303. The PEDV HN1303 was successfully serially passaged for over 95 passages, and the growth and titers of which in cell culture were characterized. Furthermore, the 4th passage

http://dx.doi.org/10.1016/j.virusres.2015.06.009 0168-1702/© 2015 Published by Elsevier B.V.

Please cite this article in press as: Wang, Y., et al., The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.06.009

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of PEDV HN1303 was used for experimental infection of 2-day old piglets to determine its virulence. Immunochemistry staining was applied to different parts of small intestines at different time points after infection to understand the progression of infection.

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2. Materials and methods

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2.1. Clinical samples

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chamber at 37 ◦ C for 40 min. After three washes with PBS, the cells were incubated for 30 min at 37 ◦ C with HRP-labeled goat antimouse IgG (Biomedical Technologies Inc., USA) diluted 1:500 in PBS. The dishes were washed 3 times with PBS, followed by incubation for 4–5 min at room temperature in diaminobenzidine solution (ZSGB-BIO, Beijing, China). Cell staining was examined by using a light microscope. 2.5. S gene sequencing and phylogenetic analysis

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In January 2013, four intestinal samples were obtained from 3-day old piglets with diarrhea from a farm in Henan Province, China. These samples were tested positive by a PEDV M gene-based RT-PCR at National Research Center for Veterinary Medicine, and were selected for virus isolation attempts. Each small-intestine tissue was homogenated in sterile phosphate-buffered saline (PBS), and was vortexed briefly followed by centrifugation at 8000 × g for 10 min at 4 ◦ C. The supernatant went through 0.22 ␮m Millipore filters, and was used as an inoculum for virus isolation.

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2.2. Virus isolation, serial propagation, and titration

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Virus isolation of PEDV was attempted on Vero cells (ATCC CCL81) as previously described (Hofmann and Wyler, 1988) with some modifications. Briefly, Vero cells were grown in Dulbecco Modified Eagle Medium (DMEM, Gibco, USA) supplemented with 5% fetal bovine serum (Hyclone, USA) and antibiotics (100 units/ml of penicillin and 100 ␮g/ml of streptomycin). Confluent Vero cell monolayers were used for virus inoculation. Prior to inoculation, growth medium was removed and the monolayers of cells were washed with PBS twice in T25 flask. Then 0.5 ml of inoculum and 0.5 ml maintenance medium was added. Maintenance medium was consisted of DMEM supplemented with antibiotics (100 units/ml of penicillin and 100 ␮g/ml of streptomycin), 0.3% tryptose phosphate broth (Sigma, USA), 0.02% yeast extract (Sigma, USA), and 10 ␮g/ml trypsin 1:250 (BD, USA). After adsorption at 37 ◦ C for 60 min, 4 ml maintenance medium were added to each flask. The cultures were incubated at 37 ◦ C with 5% CO2 , and examined for cytopathic effect (CPE) twice daily. Eighty percent of maintenance medium was changed daily. When CPE appeared in more than 80% of cells (∼5 days after inoculation), the flasks were subjected to freeze-thaw twice. The cells and supernatants were mixed and stored at −70 ◦ C. These samples were used as seed stocks for the next passage. If no CPE was observed within 5 days, 4 blind passages were performed using inoculated culture fluid as inoculum. Virus titration was performed in 96-well plates with 10-fold serial dilutions performed in eight replicates per dilution. After 5 days of inoculation, the virus titers were determined according to the Reed and Muench method (Reed and Muench, 1938) and expressed as the 50% tissue culture infective dose (TCID50 )/ml.

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2.3. Determination of virus growth curve

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To determine virus growth curve, 750 TCID50 3rd and 79th passages of PEDV HN1303 were inoculated onto cell monolayers in 35 mm dishes. After adsorption at 37 ◦ C for 60 min, the cells were fed with maintenance medium, and incubated at 37 ◦ C with 5% CO2 . At 6, 9, 12, 15, 18, 21, 24, and 27 hours post-inoculation (hpi), cells were collected for virus titration. Virus titration at different time points was performed in triplicates.

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2.4. IPMA

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PEDV-infected Vero cells in 35 mm dishes were fixed with 80% acetone for 20 min, and then washed by PBS, air dried, and incubated with a 1:500 dilution mouse monoclonal antibody 3F12 (MEDIAN, Korea) specifically against PEDV spike in a humidity

The full-length spike (S) gene sequence of PEDV strain HN1303 was determined by the traditional Sanger method. Viral RNA extraction was performed with 200 ␮l HN1303 isolation at the passage 4 by using a viral nucleic acid extraction kit (Geneaid Biotech Ltd., Taiwan, China) following the instructions of the manufacturers. cDNA for the S gene was generated by EasyScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Beijing, China) using a reverse primer PEDV-N (5 -GGCATAGAGAGATAATGGCA-3 ). The S gene was amplified in three fragments using 2× TransStart FastPfu PCR SuperMix (TransGen Biotech, Beijing, China). Primers used were previously described by Park et al. (2007). The three fragments were amplified under same program of 3 min at 94 ◦ C, 35 cycles of 20 s at 94 ◦ C, 15 s at 58 ◦ C and 1 min at 72 ◦ C, and a final extension at 72 ◦ C for 10 min. Purified PCR products were cloned using pEASYBlunt Cloning Kit (TransGen Biotech, Beijing, China) according to the manufacturer’s instructions. Recombinant DNA clones were sequenced by Invitrogen of Life Technologies (Shanghai, China). Sequence obtained was aligned with reference sequences in the GenBank using ClustalX version 1.81, and the default setting with manual adjustment. Neighbor-joining tree based on the S gene was generated using the software PHYLIP version 3.67 (Retief, 2000). The reliability of branches in the tree was assessed by bootstrap analysis using 1000 replicates. The degree of similarity among sequences was determined using MegAlign version 7.0 (a tool in the software DNAStar). The full-length S gene nucleotide sequence of PEDV strain HN1303 in this study was deposited in the GenBank database under accession number KR080551. 2.6. Experimental infection of 2-day old piglets Two experiments were performed and approved by the Animal Care and Ethics Committee of China National Research Center for Veterinary Medicine. Colostrum-deprived piglets used in both experiments were obtained from sows free of PEDV, porcine transmissible gastroenteritis virus, group A rotavirus, porcine circovirus 2, porcine reproductive and respiratory syndrome virus, and pseudorabies virus. In the first experiment, to determine the virulence of the PEDV strain HN1303, seven 2-day old piglets were randomly allocated into a PEDV-infected group (n = 5) or a negative control group (n = 2). Piglets in infected group were inoculated orally 2 ml (105.2 TCID50 /ml) virus of HN1303 at passage 4. Piglets in negative control group were similarly treated with 2 ml virus-free cell culture media. All 5 infected piglets exhibited severe diarrhea and vomiting, and died within 70 hpi. To understand the progression of PEDV infection, we carried out the second experiment. Nine 2-day old piglets were randomly allocated into an infected group (pig number 1–7, n = 7) or a negative control group (pig number 8–9, n = 2), and were administered by the same dose and route as in the first experiment. Clinical signs were monitored continuously. In infected groups, pig7 and pig6 were euthanized at 8 and 12 hpi individually before the appearance of clinical symptoms. All the other piglets in infected group showed the clinical symptoms between 12 and 21 hpi and were euthanized at 8 (pig3), 17 (pig1), 27 (pig5), 35 (pig2), and 50 (pig4) hours postappearance of clinical symptoms (hpacs) of each individual piglets. Pig8 and pig9 in negative control group were euthanized at 24 h and

Please cite this article in press as: Wang, Y., et al., The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.06.009

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Table 1 Summary of virus titers of PEDV HN1303 during serially passages on Vero cells. Passage no.

Infectious titer (log10 TCID50 /ml)

Passage no.

Infectious titer (log10 TCID50 /ml)

Passage no.

Infectious titer (log10 TCID50 /ml)

P1 P2 P3 P4 P5 P6 P7 P8 P9

2.8 3.6 4.4 5.2 4.6 4.8 5.5 5.2 5.6

P10 P15 P20 P25 P30 P35 P40 P45 P50

5.8 6.3 6.2 6.5 6.6 6.2 6.3 6.6 7.2

P55 P60 P65 P70 P75 P80 P85 P90 P95

6.3 7.5 7.3 7.6 7.2 7.5 8.0 7.7 7.4

Note: Virus was titrated every passage for the first 10 passages and then every 5 passages thereafter.

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72 h after mock inoculation. Rectal swabs were collected daily and were used for detection of virus shedding by RT-PCR as described below. Formalin-fixed five sections of small intestine including duodenum, proximal jejunum, mid-jejunum, distal jejunum, and ileum were collected, and were subjected to antigen detection by immunohistochemistry (IHC).

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2.7. RT-PCR and quantification of virus fecal shedding

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Each rectal swab was prepared at 10-fold serial dilutions with sterilized PBS, from 100 to 10−4 . The highest dilution that yielded a positive band of 663 bp PCR amplicons was considered to be the endpoint, and the PCR titers of fecal shedding were calculated according to the endpoint dilution. 2.8. IHC

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Tissues were de-waxed with xylene, rehydrated through graded alcohols, and air dried. Endogenous peroxidase was quenched with 3% hydrogen peroxide at room temperature for 20 min, and then the slides were washed with PBS (pH 7.2) for 3 min, twice. Antigen retrieval was accomplished with 0.01 mol/L citrate buffer (pH 6.0) boiling for 15 min followed by three PBS rinses. To saturate nonspecific protein-binding sites, all slides were treated for 20 min at room temperature with 1:20 dilution normal horse serum (ZSGB-BIO, Beijing, China). The slides were next treated overnight at 4 ◦ C in a humidity chamber with PEDV monoclonal antibody 3F12, diluted 1:500 with PBS. After three washes with PBS, sections were flooded and incubated for 1 h at 37 ◦ C with HRP-goat anti-mouse IgG (Biomedical Technologies Inc., USA) diluted 1 in 100 in PBS. The slides were washed 3 times in PBS, followed by incubation for 4–5 min at room temperature in Diaminobenzidine solution (ZSGB-BIO, Beijing, China). The sections were lightly counterstained with Mayer’s hematoxylin, dehydrated through graded concentrations of ethanol and xylene, and mounted. Antigen detection was semi-quantitatively scored based on the following criteria: − = no signal, + = 1–25% of villous enterocytes within the section showing a positive signal, ++ = 26–50% of villous enterocytes showing a positive signal, +++ = 51–75% of villous enterocytes showing a positive signal, ++++ = greater than 75% of villous enterocytes showing a positive signal.

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3. Results

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3.1. Virus isolation and serial propagation Virus isolation was performed on 4 individual PEDV PCRpositive intestine homogenates on Vero cells. PEDV HN1303 was successfully isolated from one of these four samples. At 3 days postinoculation (dpi), typical cytopathic effect (CPE) characterized by cell fusion and syncytium formation was observed. Thereafter, the

appearance and progression of CPE were expedited as the passage in Vero cells advanced. In the passages 2–10, CPE was visible within 24 hpi. CPE was observed as early as 10–12 hpi after passage 11. These distinctive CPE was not observed in uninfected control cultures and differed distinctly from cellular injuries caused by trypsin. During the first 10 serial passages, the virus titers of HN1303 ranged from 102.6 to 105.8 TCID50 /ml, and increased gradually in an irregular zigzag pattern in the ensuing passages (maximum 108.0 TCID50 /ml). This virus has been serially propagated in Vero cell culture over 95 passages. The infectious titers of PEDV strain HN1303 during serially passages in Vero cell culture are summarized in Table 1.

3.2. Virus growth characterization The cell-culture PEDV HN1303 was adapted to serial propagation on Vero cells, and at passage 4 and 80, the growth curve of the viruses were determined (Fig. 1). CPE caused by passage 4 virus infection was observed at 16 dpi and virus titer reached to the maximum (5.2log10 TCID50 /ml) at 24 hpi. It is hardly to make the conclusion that passage 4 was cell-adapted since the virus titer at this passage was still low. As compared with passage 4, CPE caused by passage 80 virus infection was observed at 12 hpi and spread rapidly all over the cell monolayer; at 27 hpi, syncytial cells detached from the flask, leaving gaps in the monolayer. Virus titer reached to the maximum (7.5log10 TCID50 /ml) at 24 hpi. Virus growth of passage 80 in infected Vero cells was also confirmed by IPMA by using anti-PEDV S protein monoclonal antibody 3F12. As shown by Fig. 2, consistent with above virus titration results, the positively signal of staining could be detected as early as 6 hpi and reached to the maximum at 24 hpi before infected cells detached from the well.

3.3. S gene sequencing and phylogenetic analysis The S gene of HN1303 has 4161 nucleotides, encoding a protein of 1386 amino acids. N-terminal domain of the S protein possesses 2 discontinuous amino-acid insertions (59 QGVN62 and 140 N) and an amino acid deletion (161 GK162 ) as compared to CV777 strain (Fig. 3). According to the genotyping method performed by Chen et al. (2014), HN1303 and other 29 PEDV viruses could be divided into group I and II based on the S gene. Two groups can be further divided into subgroups Ia, Ib, IIa, and IIb (Fig. 4). The HN1303 strain belongs to genotype IIa which consists of partial recent Chinese and US variant strains. HN1303 exhibited highest nucleotide sequence similarity (99.6%) to strains JS-HZ2012 and HB-2012-1 from China, but most closely related to strain CH/XCYL/11 that was not supported by bootstrap analysis (Fig. 4). HN1303 shared 94.0% and 92.8% nucleotide and amino acid identities with CV777 vaccine strain, respectively.

Please cite this article in press as: Wang, Y., et al., The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.06.009

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hpi Fig. 1. Growth curve of PEDV strain HN1303 at passage 4 and 80 in Vero cell culture. Data was presented as mean ± SD by triplicates.

Fig. 2. Detection of PEDV antigen from Vero cell cultures by IPMA at different time points. Panels A–H represent 6, 9, 12, 15, 18, 21, 24, and 27 hpi, respectively, and panel I represents Vero cells without infection at 27 dpi. Magnification 200×.

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3.4. Animal experiments In the first animal experiment, all five piglets developed suddenonset, severe watery diarrhea and vomiting at 12–21 hpi, and died at 39–70 hpi (data not shown). Vomiting was observed in all infected pigs within 24 h after onset of clinical signs, but not persistent after 24 h. At the onset of diarrhea, appetite was maintained in piglets until they became too weak. Dilated gas- and fluid-filled

small intestines with thin, translucent walls were observed, and small intestines contained coagulated liquid milk replacement diet. The pigs in uninfected control group had normal feces and no clinical signs of illness. In the 2nd animal experiment, similar onset of clinical signs was observed. The infected piglets exhibited severe diarrhea and vomiting at 12–21 hpi. Detection of fecal viral shedding coincided with onset of clinical signs. High level PCR titer (10−4 dilution)

Fig. 3. Amino acid comparison of N-terminal domain of S protein between PRV HN1303 and CV777 strain.

Please cite this article in press as: Wang, Y., et al., The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.06.009

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Fig. 4. Phylogenetic relationship among PEDV HN1303 in this study and reference sequences in the GenBank, as inferred by a neighbor-joining analysis of the full-length S gene nucleotide sequences based on distance calculated using the Kimura 2-parameter model. Bootstrap values (in percentage) above 50 from 1000 pseudoreplicates are shown. The isolate in this study is marked by closed circle.

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of viral RNAs was detected from rectal swab samples within 12 h after onset of clinical signs, and decreased subsequently (PCR titers ranged 10−3 –100 ) (Table 2). PEDV antigen was detected initially by IHC in the cytoplasm of villous enterocytes of challenged pig6 at 12 hpi with absent of clinical signs; apparent immunostaining was visible in the upper small intestine (duodenum, proximal jejunum and mid-jejunum) (Fig. 5). A largest number of antigens were detected in the jejunum and ileum in infected piglets within 24 h after onset of clinical signs, and decreased at latter time points. Trends that globally gradual decrease in duodenum of viral antigens were observed, following by extend time after onset of clinical signs (Fig. 5).

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4. Discussion

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In the present study, we successfully isolated PEDV HN1303 from one of four intestine homogenates of PEDV-infected piglets. Low successful rates of virus isolation have been also recently

reported by Oka et al. (2014) and Chen et al. (2014) with 14.3% (9/63) and 11.8% success (2/17) from PEDV-positive intestine samples, respectively. Multiple factors might affect virus isolation, such as the type of sample, virus titer, substances in intestinal contents, strain-specific factors, Vero cell lines and culture conditions. Furthermore, the isolation procedures need to be improved to increase the success rate of PEDV isolation. The PEDV growth in cell culture was reported difficult and the virus may gradually lose infectivity upon further passages in cell culture (Chen et al., 2014). Recently, several US PEDV strains were serially propagated in Vero cell cultures for over 30 passages (Chen et al., 2014; Oka et al., 2014). In this study, strain HN1303 was adapted to Vero cell culture after several passaging and continuously propagated to high passages with high infectious titers. The complete S gene of PEDV HN1303 was sequenced and analyzed, and the results showed that HN1303 shares 94.0% and 92.8% nucleotide and amino acid identities with CV777 vaccine strain, respectively. Phylogenetic tree based on S gene indicates that the

Table 2 Fecal shedding of virus, clinical signs and immunohistochemistry findings after inoculation of pigs with PEDV strain HN1303. Pig status, pig no

Fecal shedding, PCR titer*, by hpi

Onset of clinical signs, hpi

0

12

24

36

48

60

72

PEDV-inoculated 7 6 3 1 5 2 4

− − − − − − −

ND 0 −2 0 0 −1 −2

ND ND −4 −4 −4 −4 −3

ND ND ND −4 −3 −3 −2

ND ND ND ND ND −1 −1

ND ND ND ND ND ND 0

ND ND ND ND ND ND ND

None None 16 21 17 13 12

Negative control 8 9

− −

− −

− −

ND −

ND −

ND −

ND −

None None

hpi at euthanasia (after onset)

Antigen detection by IHC

Duodenum

Proximal jejunum

Mid-jejunum

Distal jejunum

Ileum

8 12 24 (8) 38 (17) 44 (27) 48 (35) 62 (50)

− ++ + ++ − − −

− ++ +++ +++ ++ ++ +

− +++ ++++ ++ ++ ++ +

− + ++++ +++ +++ +++ ++

− + ++++ +++ +++ +++ ++

NA NA

− −

− −

− −

− −

− −

−, negative; *, dilution 10n ; NA, not applicable; hpi, hour post-inoculation.

Please cite this article in press as: Wang, Y., et al., The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.06.009

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Fig. 5. Immunohistochemistry analysis of small-intestine sections from infected and control pigs. PEDV antigen (brown reaction product) was detected in the villous enterocytes. Magnification 200×. Note: hpi and hpacs are the abbreviations for “hour post-inoculation” and “hour post-appearance of clinical symptoms”.

HN1303 strain belongs to genotype IIa, a recently prevalent genotype in China (Huang et al., 2013; Song et al., 2015) and USA (Chen et al., 2014; Huang et al., 2013). In the future study, we will exam315 ine the genetic changes of strain HN1303 complete genome during 316 serial passages in cell culture. 317 In this study, all PEDV-infected piglets developed sudden-onset, 318 severe watery diarrhea and vomiting within 1 dpi, and died within 319 3 dpi. The result reveals high virulence of HN1303 strain to 2-day 320 old piglets which was consistent with the devastating diarrhea 321 observed in the field outbreaks. Similar results were also observed 322 in the previous studies. Shibata et al. (2000) described that all 2323 and 7-day old piglets inoculated with PED virus developed severe 324 watery diarrhea at 1 dpi and died at 3–4 dpi. Kim and Chae, 2003 325 Q3 reported that 1-day old colostrum-deprived piglets inoculated with 326 PEDV strain SNUVR971496 showed diarrhea at 12–36 hpi. In the 327 present study, vomiting was an inconsistent early clinical sign in 328 disease progression which coincide with that described by Madson 329 et al. (2014). 330 313 314

Saif et al. (2012) described that porcine epidemic diarrhea virus replicates in the cytoplasm of mature enterocytes within 12–18 h post-infection and typically reaches maximum replication around 24–36 h. In this study, we confirmed rapid replication of PEDV since antigen was detected as early as 12 hpi from a pig absent of clinical signs. The maximum virus replication occurred within 24 h after onset of clinical signs based on antigen detection by IHC, coincided with results of severe watery diarrhea and dehydration. Apparent antigen immunostaining was firstly detected in the upper small intestine at early time post-inoculation, and spread in entire jejunum and ileum. From 24 h after onset of clinical signs, viral antigens in duodenum gradually decreased. In conclusion, PEDV variant strain HN1303 was successfully isolated, serially propagated in cell culture and characterized. The results of animal experiments reveal that this strain is highly pathogenic to 2-day old piglets. Phylogenetic analysis based on S gene indicates that HN1303 strain belongs to a currently prevalent genotype. The information presented in this study is important

Please cite this article in press as: Wang, Y., et al., The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.06.009

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Please cite this article in press as: Wang, Y., et al., The dynamics of Chinese variant porcine epidemic diarrhea virus production in Vero cells and intestines of 2-day old piglets. Virus Res. (2015), http://dx.doi.org/10.1016/j.virusres.2015.06.009

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