key: cord-1018778-blv75dwx
authors: Hu, Hui; Jung, Kwonil; Kenney, Scott P.; Saif, Linda J.
title: Isolation and Tissue Culture Adaptation of Porcine Deltacoronavirus: A Case Study
date: 2020-05-11
journal: Coronaviruses
DOI: 10.1007/978-1-0716-0900-2_6
sha: 38681206a65320591771a7b2116803c58ea068de
doc_id: 1018778
cord_uid: blv75dwx

Porcine deltacoronavirus (PDCoV) has emerged as a novel, contagious swine enteric coronavirus that causes watery diarrhea and/or vomiting and intestinal villous atrophy in nursing piglets. PDCoV-related diarrhea first occurred in the USA in 2014 and was subsequently reported in South Korea, China, Thailand, Vietnam, and Lao People’s Democratic Republic, leading to massive economic losses and posing a threat to the swine industry worldwide. Currently, no treatments or vaccines for PDCoV are available. The critical step in the development of potential vaccines against PDCoV infection is the isolation and propagation of PDCoV in cell culture. This chapter provides a detailed protocol for isolation and propagation of PDCoV in swine testicular (ST) and LLC porcine kidney (LLC-PK) cell cultures supplemented with pancreatin and trypsin, respectively. Filtered clinical samples (swine intestinal contents or feces) applied to ST or LLC-PK cells produce cytopathic effects characterized by rounding, clumping, and detachment of cells. PDCoV replication in cells can be quantifiably monitored by qRT-PCR, immunofluorescence assays, and immune-electron microscopy. Infectious viral titers can be evaluated by using plaque assays or 50% tissue culture infectious dose (TCID(50)) assays. The ST or LLC-PK cells efficiently supported serial passage and propagation of PDCoV. After serial passage of PDCoV in either ST or LLC-PK cells, the virus can be purified further in ST cells by plaque assays.

Porcine deltacoronavirus (PDCoV), a member of the genus Deltacoronavirus in the family Coronaviridae of the order Nidovirales, is a novel, contagious swine enteropathogenic coronavirus that causes watery diarrhea and/or vomiting, dehydration, and intestinal villous atrophy in nursing pigs [1] . PDCoV was first detected in pig feces during a molecular surveillance of coronaviruses in mammals and birds in Hong Kong in 2012 [2] . However, the first outbreak of PDCoV-related diarrhea in pigs was identified in the USA in 2014 [3] . Since then, PDCoV-related diarrhea has been reported in many countries, including China, Canada, South Korea, Lao People's Democratic Republic, Thailand, and Vietnam [4] [5] [6] [7] [8] .

PDCoV is enteropathogenic in young pigs [1] . Wild-type or cell culture-adapted strains of PDCoV caused severe gastrointestinal disease in gnotobiotic (Gn) and conventional, 5-19-day-old pigs [9] [10] [11] . Apart from pigs, PDCoV was also reported to have a limited ability to infect Gn calves [12] . This was evident by the detection of high titers of fecal PDCoV RNA and serum PDCoVspecific IgG antibody in inoculated calves (but with a lack of clinical signs and histological lesions). Similarly, PDCoV infected and replicated within various cell lines of human and chicken origin in vitro [13] . In in vivo settings, PDCoV could also infect and serially propagate in chicken embryos, and orally inoculated chickens showed mild diarrhea and low viral RNA titers in the feces, providing evidence of the possibility of interspecies transmission of deltacoronavirus between birds and pigs [14] .

Cell culture-adapted and plaque-purified strains of PDCoV are useful for studies of PDCoV pathogenesis, and development of virological and serological assays and vaccines. Many cell culturegrown strains of PDCoV have been reported in the USA, China and Korea. The strains USA/IL/2014 [10] , Michigan/8977/ 2014 [11] , and OH-FD22 [15] were isolated in the USA, and the strain OH-FD22 has been serially passaged more than 100 times in LLC porcine kidney (LLC-PK) cells. The Chinese strains HNZK-02 [14] , NH [16] , CHN-HN-2014 [17, 18] , CHN-GD-2016 [19] , and CHN-HG-2017 [20] and Korean strain KNU16-07 [21] were all isolated in cell culture primarily utilizing methods first reported by our laboratory [15] .

In our earlier studies, various cell lines of swine and monkey origin, including Vero cells, commonly used for isolation and propagation of porcine epidemic diarrhea virus, were tested for primary isolation of PDCoV using cell culture medium supplemented with different additives (small intestinal contents, trypsin, and pancreatin). However, only the LLC-PK or swine testicular (ST) cells of swine origin efficiently supported the isolation and serial propagation of PDCoV in cell cultures supplemented with exogenous trypsin or pancreatin, respectively [15] . Recently, porcine, human, and avian aminopeptidase N (APN) was identified as a major cell entry receptor for PDCoV in vitro [17, 22, 13] . Therefore, possible differences in APN expression levels among the cell lines used could influence their susceptibility to infection with PDCoV. In our studies, PDCoV replicated in LLC-PK cells without trypsin treatment, but it did not induce obvious cytopathic effects (CPE), and the virus titer was lower when compared with the trypsin supplemented cell cultures [15] . We also found that PDCoV could be serially propagated in ST cells supplemented with pancreatin or small intestinal contents from Gn piglets, but not with trypsin [15] . The addition of trypsin and pancreatin in PDCoV-inoculated LLC-PK and ST cells, respectively, might be essential for both growth of PDCoV and induction of CPE [15, 23] . Similarly, a porcine small intestinal epithelial cell line, IPEC-J2, was also susceptible to PDCoV infection, accompanied by CPE, when the cell culture medium was supplemented with 10 μg/ml of trypsin [24] . The cell culture systems currently available for PDCoV are useful for an understanding of the mechanisms related to PDCoV infection and generation of live or inactivated vaccine strains of PDCoV.

This chapter is focused on the description of basic protocols for the isolation and propagation of PDCoV in LLC-PK or ST cells, titration of infectious virus by TCID 50 or plaque assays, and purification of PDCoV by plaque assay, based on our experience in isolating and adapting US PDCoV OH-FD22 or Chinese HNZK-02 strains in these cell lines. Details regarding quantification of viral RNA by TaqMan real-time quantitative RT-PCR (qRT-PCR), immunofluorescence assay for detection of PDCoV antigen in cells, and immune-electron microscopy for identification of viral particles in the culture medium have been published previously [15] .

1. Clinical swine samples, such as feces or intestinal contents, are collected from diarrheic pigs positive for PDCoV RNA by RT-PCR or qRT-PCR and stored at À80 C (see Note 1).

1. LLC-PK cell growth medium: Minimal essential media (MEM) supplemented with 5% heat inactivated fetal bovine serum (FBS), 1% MEM nonessential amino acids (NEAA), 1% antibiotic-antimycotic, and 1% HEPES.

2. LLC-PK cell maintenance medium (LLC-PK cell MM): MEM is supplemented with 1% NEAA, 1% antibiotic-antimycotic, 1% HEPES, and 5 μg/ml of trypsin (see Note 2). 5. Add 300 μl of filtered samples to each well and incubate for 1 h at 37 C in 5% CO 2 (see Note 7).

6. Remove the inoculum, and wash cells three times with MM or DPBS (see Note 6).

7. Add 2 ml of MM supplemented with 5 μg/ml of trypsin (LLC-PK cells) or 1% pancreatin (ST cells) to each well.

8. Incubate cells at 37 C in 5% CO 2 until CPE, such as rounding, clumping, or detachment of cells (Fig. 1) , is observed usually by 5 days postinoculation (see Note 8).

9. Freeze the cell plates at À80 C once CPE is observed in 80% of the cell monolayer.

10. Thaw and freeze the cells twice at À80 C and harvest the cell supernatants for qRT-PCR or titration of infectious virus. 

5. Remove the inoculum, and wash cells three times with MM or DPBS.

6. Add 5 ml of MM supplemented with 5 μg/ml of trypsin (LLC-PK cells) or 1% pancreatin (ST cells) per T25 cell culture flask and incubate at 37 C in 5% CO 2 until CPE, such as rounding, clumping, or detachment of cells, is observed.

7. Freeze the cell flasks at À80 C once CPE is observed in more than 80% of the cell monolayer.

8. Thaw and freeze the cells once at À80 C and harvest the cell supernatants for qRT-PCR or titration of infectious virus by TCID 50 or plaque assays. 7. Heat the agarose gel in a microwave oven until fully melted, and then keep it in a water bath at 42 C. Approximately 7 ml is needed per plate.

8. Mix equal volumes of 2Â MEM (step 6) and agarose (step 7) (see Note 17).

10. Add 2 ml of the agarose-MEM mixture to each well (see Note 18).

11. Cool the plates for 10 min to solidify the agarose.

12. Incubate the plates upside down at 37 C in 5% CO 2 for 2-3 days postinoculation (see Note 19) .

13. Prepare the working solution of neutral red (0.01%). Dilute 1 ml of the stock neutral red (0.33%) in 25 ml of DPBS.

14. Add 2 ml of 0.01% neutral red solution to each agarosecoated well.

15. Incubate the plates at room temperature for 2-4 h, and then remove the neutral red solution. Plaques are visualized as clean spots in the red background (Fig. 2) . 16 . Count plaques under oblique light. Plaques should be confirmed by light microscopy. The plaque titers are expressed as plaque forming units (PFU) per ml. The titer of PFU/ml is calculated by using the following formula: Y (viral titer, PFU/ml) ¼ X (mean numbers of plaques from the duplicate wells)/300 Â 1000 Â viral dilution factor.

17. Pick a uniform and clear plaque by using a sterile pipette tip, and then place the agarose plug into a 1.5 ml microcentrifuge tube containing 0.5 ml of ST cell MM (see Note 20) .

18. Inoculate the selected plaque clones (0.5 ml) onto ST cell monolayers prepared in a 6-well cell culture plate.

19. Incubate the plate for 1 h at 37 C in 5% CO 2 .

20. Add 1.5 ml of ST cell MM supplemented with 1% pancreatin and incubate the plate at 37 C in 5% CO 2 for 4-5 days until CPE is observed.

Harvest CPE-positive clones and store them at À80 C (see Note 21).

1. Detailed procedures, including isolation of viral RNA from clinical samples and qRT-PCR for the detection of the membrane (M) gene of PDCoV were published previously [15, 26] . The qRT-PCR was conducted by using Qiagen One-step RT-PCR kit (Qiagen Inc., Valencia, CA, USA) and a real-time thermocycler (RealPlex; Eppendorf, Germany) [15] . The stock of trypsin (2.5%) is diluted (1:5000) in MEM (e.g., 10 μl of 2.5% trypsin is added to 50 ml of MEM supplemented with 1% NEAA, 1% antibiotic-antimycotic, and 1% HEPES), and the working concentration of trypsin in LLC-PK MM is 5 μg/ml.

A 10Â pancreatin stock is made by dissolving 2.50 g of pancreatin and 0.85 g of NaCl in 100 ml of MilliQ water followed by sterilizing through a 0.22 μm filter. The 10Â pancreatin solution is further diluted (1:10) in sterile PBS or MEM (1Â pancreatin). Aliquot into 1 ml vials, and store at À20 C until use. Store at 4 C after thawing. Finally, 1 ml of the 1Â pancreatin solution is added to 99 ml of the maintenance medium.

4. Aliquot into sterile 15 ml conical centrifuge tubes, and store at À20 C until use. Prior to use, thaw and warm at room temperature.

5. 2 g agarose is added to 100 ml of distilled water, autoclaved, and stored at room temperature. Prepare the agarose overlay by melting the agarose gel in a microwave oven. Keep it in liquid form by incubation at 42 C.

6. Trypsin-containing MM solutions can also be used for washing the LLC-PK cells.

7. Frequently, the filtered clinical samples can be toxic to cells, in which case the early detachment of cells is observed at 4-6 h postinoculation. To reduce their cell cytotoxicity, the samples can be diluted by 1:10, 1:100, or 1:1000 (v/v) in MM, or MM with trypsin (LLC-PK cells).

8. If CPE is not observed by 5 days postinoculation, the cell plates are frozen at À80 C and thawed three times, and the cell culture medium is harvested and further inoculated onto fresh LLC-PK or ST cells according to the steps described.

After three continuous passages of the samples, if there is no CPE and cell culture supernatants are negative for increased PDCoV RNA during the serial passages, the samples are considered as negative for growth in cell culture. 9 . Cells should not be allowed to dry out before virus inoculation. 10 . The MOI can also be estimated by the extent of CPE observed and is associated with the extent of adaptation of PDCoV to the LLC-PK or ST cells. After viral inoculation, flasks are also gently shaken by hand every 15 min.

11. Each plate should include one row of negative control (MM alone).

12. If the samples are already diluted with the MM supplemented with 5 μg/ml of trypsin, step 6 is not necessary. The final concentration of trypsin in the cell culture medium in each well should be 5 μg/ml.

13. The TCID 50 assay using ST cells is also similar to the TCID 50 assay using LLC-PK cells, except for use of ST MM supplemented with 1% pancreatin, instead of LLC-PK MM. The CPE observed can be further confirmed as PDCoV-specific by immunofluorescence staining with PDCoV-specific antibodies.

14. ST cells can be used to titrate PDCoV grown in either ST or LLC-PK cells by plaque assay. 15 . For virus inoculation, 100% confluence cell monolayers are further allowed to attach to the plate for 24-36 h. 16 . We believe that this step where cells are incubated without FBS will contribute to an enhanced adsorption of PDCoV to cells.

17. Prepare agarose only prior to use. Dissolve agarose completely. Be sure that the temperature of the agarose used for overlay is around 37 C.

18. Dispense the agarose mixture thoroughly throughout the well.

19. Duration of the incubation is dependent on the extent of plaque formation.

20. The plaques can be stored in the maintenance medium at À80 C until use or promptly mixed using a vortex mixer for 1 min and then used to inoculate cells prepared in 6-well cell culture plates.

21. Viral titers of the plaque clones can be determined by qRT-PCR, TCID 50 , or plaque assays. Based on the titers, some clones can be selected for further passage.

Porcine deltacoronavirus infection: etiology, cell culture for virus isolation and propagation, molecular epidemiology and pathogenesis

Discovery of seven novel mammalian and avian coronaviruses in the genus deltacoronavirus supports bat coronaviruses as the gene source of alphacoronavirus and betacoronavirus and avian coronaviruses as the gene source of gammacoronavirus and deltacoronavirus

Detection and genetic characterization of deltacoronavirus in pigs

Newly emerged porcine Deltacoronavirus associated with Diarrhoea in swine in China: identification, prevalence and full-length genome sequence analysis

Porcine Deltacoronavirus in mainland China

Prevalence, complete genome sequencing and phylogenetic analysis of porcine deltacoronavirus in South Korea

Different lineage of porcine Deltacoronavirus in Thailand, Vietnam and Lao PDR in 2015

Pathogenicity of 2 porcine deltacoronavirus strains in gnotobiotic pigs

Pathogenicity and pathogenesis of a United States porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets

Origin, evolution, and virulence of porcine deltacoronaviruses in the United States

Calves are susceptible to infection with the newly emerged porcine deltacoronavirus, but not with the swine enteric alphacoronavirus, porcine epidemic diarrhea virus

Broad receptor engagement of an emerging global coronavirus may potentiate its diverse crossspecies transmissibility

Susceptibility of chickens to porcine Deltacoronavirus infection

Isolation and characterization of porcine deltacoronavirus from pigs with diarrhea in the United States

Pathogenicity of porcine deltacoronavirus (PDCoV) strain NH and immunization of pregnant sows with an inactivated PDCoV vaccine protects 5-day-old neonatal piglets from virulent challenge

Contribution of porcine aminopeptidase N to porcine deltacoronavirus infection

Isolation, genomic characterization, and pathogenicity of a Chinese porcine deltacoronavirus strain CHN-HN-2014

A highly pathogenic strain of porcine Deltacoronavirus caused watery diarrhea in newborn piglets

Genomic characterization and pathogenicity of porcine deltacoronavirus strain CHN-HG-2017 from China

Isolation and characterization of Korean porcine deltacoronavirus strain KNU16-07

Porcine deltacoronavirus engages the transmissible gastroenteritis virus functional receptor porcine aminopeptidase N for infectious cellular entry

Porcine deltacoronavirus induces apoptosis in swine testicular and LLC porcine kidney cell lines in vitro but not in infected intestinal enterocytes in vivo

Susceptibility of porcine IPEC-J2 intestinal epithelial cells to infection with porcine deltacoronavirus (PDCoV) and serum cytokine responses of gnotobiotic pigs to acute infection with IPEC-J2 cell culture-passaged PDCoV

A simple method of estimating fifty percent endpoints

Rapid detection, complete genome sequencing, and phylogenetic analysis of porcine deltacoronavirus

Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio, USA.