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Goblet cell depletion in small intestinal villous and crypt epithelium of conventional nursing and weaned pigs infected with porcine epidemic diarrhea virus
Research in Veterinary Science 110 (2017) 12–15
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Goblet cell depletion in small intestinal villous and crypt epithelium of conventional nursing and weaned pigs infected with porcine epidemic diarrhea virus Kwonil Jung ⁎, Linda J. Saif ⁎ Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA
a r t i c l e
i n f o
Article history: Received 21 April 2016 Received in revised form 26 August 2016 Accepted 22 October 2016 Available online xxxx Keywords: PEDV Pathogenesis Goblet cell Pig Virus
a b s t r a c t Intestinal goblet cells secret mucins to form mucus layers critical for maintaining the integrity of the intestinal epithelium. Porcine epidemic diarrhea virus (PEDV) causes watery diarrhea and high mortality of suckling pigs. PEDV mainly infects villous epithelial cells of the small intestine, and infected cells undergo acute, massive necrosis, followed by severe villous atrophy. Conventional 9-day-old nursing pigs [PEDV-inoculated (n = 9); Mock (n = 11)] and 26-day-old weaned [PEDV-inoculated (n = 11); Mock (n = 9)] were inoculated orally [8.9 log10 genomic equivalents/pig] with PEDV strain PC21A or mock. We used alcian blue or Periodic-Acid-Schiff staining for the detection of acidic or neutral mucin-secreting goblet cells in the small intestine. We demonstrated that PEDV infection of the nursing pigs at post-inoculation days (PIDs) 1–5 and weaned pigs at PIDs 3–5 led to depletion or significant reduction in the number of goblet cells (and also the number of villous goblet cells normalized by jejunal villous crypt height to crypt depth ratios) in the villi or crypts. These findings coincided with the development of intestinal villous atrophy. By immunohistochemistry, a few PEDV antigen-positive goblet cells were identified in the jejunal or ileal villous epithelium of the infected nursing or weaned pigs. During the early stages of PEDV infection, goblet cell mucins in the small intestine may be decreased, possibly leading to an impaired mucus layer and increased susceptibility to secondary enteric bacterial infection. © 2016 Elsevier Ltd. All rights reserved.
Intestinal villous epithelium lining the gastrointestinal tract mainly consists of the absorptive enterocytes and specialized secretory cells such as goblet cells (McCauley and Guasch, 2015). For both lubrication and barrier function of the intestinal epithelium against enteric pathogens, the intestinal mucosal surface is covered by the mucus layer(s) that mainly consist of mucins secreted by goblet cells (McCauley and Guasch, 2015). Goblet cells are critical to maintain intestinal homeostasis and integrity of intestinal epithelium. Loss or dysfunction of intestinal goblet cells is implicated in enteric disease (McCauley and Guasch, 2015). Porcine epidemic diarrhea virus (PEDV) (family Coronaviridae, genus Alphacoronavirus) causes acute watery diarrhea, dehydration and high mortality of suckling pigs and substantial economic losses (Saif et al., 2012; Jung and Saif, 2015). PEDV is highly enteropathogenic and acutely infects villous epithelial cells of the entire small and large intestines, but the jejunum and ileum are the primary sites of infection (Jung and Saif, 2015; Jung et al., 2014). During the early stages of PEDV infection, villous epithelial cells are infected, followed by acute, massive necrosis and exfoliation of infected cells, resulting in severe
⁎ Corresponding authors. E-mail addresses: [email protected] (K. Jung), [email protected] (L.J. Saif).
http://dx.doi.org/10.1016/j.rvsc.2016.10.009 0034-5288/© 2016 Elsevier Ltd. All rights reserved.
villous atrophy (Madson et al., 2016). The severity of clinical disease in suckling pigs may be exacerbated by secondary co-infections of other enteropathogens, such as Esherichia coli or other viruses (Jung and Saif, 2015). Another enteropathogenic Alphacoronavirus, transmissible gastroenteritis virus (TGEV), binds to goblet cells via sialic acid receptors expressed on the surface (Schwegmann-Wessels et al., 2003; Schwegmann-Wessels et al., 2011). Whether or how, after attaching to the surface sialic acids, TGEV enters them via a cellular receptor to replicate in goblet cells is unclear. Further studies are also needed to investigate whether the sialic acid binding activity of TGEV contributes to stabilizing and anchoring the virus in the intestine or mainly to providing the virus access to the cellular receptor, porcine aminopeptidase N (APN), expressed on the surface of enterocytes (SchwegmannWessels et al., 2011). However, similar to TGEV, whether PEDV also binds to goblet cells is unknown. A previous study showed that the number of goblet cells per intestinal villi of gnotobiotic pigs infected with an original US PEDV strain PC21A was reduced at post-inoculation hours 30–72, compared to the corresponding negative controls (Jung and Saif, 2015), suggesting a potential effect of PEDV infection on intestinal goblet cells. Therefore, our aim was to determine whether PEDV infection causes a reduced number of goblet cells in the villous and crypt epithelium of the small intestine of infected conventional nursing and weaned pigs. We used alcian blue (pH 2.5) (AB) and Periodic-Acid-
K. Jung, L.J. Saif / Research in Veterinary Science 110 (2017) 12–15
Schiff (PAS) staining for the detection of the acidic and neutral sialylated mucin-secreting goblet cells, respectively. All tissue samples tested were archival formalin-fixed, paraffin-embedded tissues acquired from twenty 9-day-old [PEDV infected (n = 9) and Mock (n = 11)] and twenty 26-day-old [PEDV infected (n = 11) and Mock (n = 9)] conventional pigs, inoculated orally with 8.9 log10 genomic equivalents of PEDV strain PC21A, or mock (modified Eagle's medium) (Jung et al., 2015). The clinical disease, fecal virus shedding, and gross and histopathology, including measurement of mean jejunal villous height: crypt depth (VH:CD) ratios to evaluate the severity of intestinal villous atrophy, were reported in a previous paper (Jung et al., 2015). Pigs (n = 3–4/time-point) were euthanized for pathologic examination at post-inoculation days (PIDs) 1, 3, and 5. The mid-jejunal and ileal tissues were stained with AB or PAS. Only well-orientated, intestinal tissue sections were evaluated to count the number of goblet cells per intestinal villus or crypt. Mean numbers of AB or PAS-stained goblet cells per villus or crypt were estimated by measuring at least 10 villi and crypts for each PID and from infected or control pigs. All values were expressed as the means ± standard deviation of the means (SDM). Mean numbers of AB or PAS-stained goblet cells per villus or crypt between PEDV-infected and uninfected nursing or weaned pigs at the same time-points were analyzed and compared by a Student's t-test using GraphPad Prism software (GraphPad Prism Inc.). A value of P b 0.05 was considered statistically significant. The intestinal tissues were also tested by immunohistochemistry (IHC), as described previously (Jung et al., 2007; Jung et al., 2014), for the detection of PEDV antigen, using monoclonal antibody 6C8-1 against the spike protein of PEDV strain DR13 that was shown previously to cross-react with the US PEDV strain PC21A (provided by Daesub Song, Korean Research Institute of Bioscience and Biotechnology, Daejeon, Korea) (Jung et al., 2014). Briefly, endogenous alkaline phosphatase in rehydrated tissues was quenched with 3% glacial acetic acid 20% for 20 min at room temperature. Antigen retrieval was performed using 100 μg/ml of proteinase K (Invitrogen, Carlsbad, CA). The tissue slides were then washed in PBTS [phosphate-buffered saline (PBS) containing Tween 20, 0.1%] three times and blocked with 1× buffered solution of casein (Universal Blocking Reagent; Biogenex, Fremont, CA) in
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distilled water for 30 min at room temperature. Sections were coated with monoclonal antibody 6C8-1 diluted 1 in 200 in PBTS and incubated overnight at 4 °C in a humid chamber. After three washes with PBTS, the sections were incubated for 1 h at 36 °C with goat anti-mouse IgG labeled with alkaline phosphatase (Dako, Glostrup, Denmark) diluted 1 in 200 in PBTS. After three washes with PBTS, the final reaction was generated by immersing the tissue sections in a staining solution [1 tablet of Fast Red in 2 ml of 0.1 M Tris-buffer (pH 8.2); Roche Applied Science, Mannheim, Germany] for 10 min at room temperature. Sections were lightly counterstained with Gill's hematoxylin. In infected nursing pigs, mean numbers of AB (acidic mucin)-stained goblet cells per villus and crypt were significantly (P b 0.05) reduced in mid-jejunum and ileum at PIDs 1, 3, and 5 (except for crypt in ileum at PID 5), compared to the corresponding negative controls (Table 1A; Fig. 1A and B). Similarly, mean numbers of PAS (neutral mucin)-stained goblet cells per villus and crypt were also significantly (P b 0.05) reduced in the mid-jejunum and ileum of the infected nursing pigs at PIDs 1, 3, and 5, but not in crypt in ileum at PIDs 1, 3, and 5 and in jejunum at PID 5 (Table 1A; Fig. 1C and D). The reduction rate, ranging from 1.8 to 16.5%, in the number of AB or PAS-stained goblet cells per villus in the jejunum of infected nursing pigs at PIDs 1–5 was also less than that (15.2 to 48.0%) in the ileum during the same period. Relative to the jejunal crypts of infected nursing pigs, goblet cells in the ileal crypts were less depleted at PIDs 1–5 (Table 1A), possibly contributing to more efficient compensation for loss of villous goblet cells. Additionally to investigate if the reduced numbers of goblet cells observed per villus could be an expected outcome of the shortened villus following PEDV infection, mean numbers of goblet cells per villus were normalized by dividing by the mean intestinal VH:CD ratios acquired from infection and non-infection groups at each PID. All infected nursing pigs at PIDs 1–5 exhibited severe watery diarrhea and jejunal villous atrophy (mean VH:CD ratios of 1.1–1.4), whereas none of the uninfected control nursing pigs showed clinical signs. Their mean jejunal VH:CD ratios were 7.1–8.7 during the same period, as reported previously (Jung et al., 2015). In the jejunal villous epithelium of uninfected nursing pigs, the numbers of goblet cells normalized by mean jejunal VH:CD ratios were 2.3 (AB) or 2.5 (PAS), 2.6 (AB) or 3.0 (PAS), and 3.1
Table 1 Mean numbers (±SDM) of alcian blue (acidic mucin) or Periodic Acid Schiff (neutral mucin)-stained goblet cells per villi or crypt in the mid-jejunum and ileum of conventional 9-day-old nursing (A) and 26-day-old weaned (B) pigs infected with the original US PEDV strain PC21A at post-inoculation days (PIDs) 1, 3, and 5. (A) Conventional 9-day-old nursing pigs PID 1
PID 3
Jejunum Villus
Ileum Crypt
PID 5
Jejunum
Ileum
Jejunum
Ileum
Villus
Crypt
Villus
Crypt
Villus
Crypt
Villus
Crypt
Villus
Crypt
Alcian blue-stained goblet cells 19.9 (6.8) 3.4 (1.8) Uninfecteda 0.5 (0.8)b 1.4 (1.3) Infecteda
30.5 (6.7) 10.8 (8.8)
8.9 (3.8) 6.3 (3.4)
19.9 (6.0) 1.1 (1.3)
4.7 (1.9) 2.9 (1.4)
33.6 (5.6) 5.1 (4.3)
10.0 (3.3) 5.8 (1.7)
21.8 (7.4) 1.6 (1.1)
4.7 (1.9) 2.5 (1.9)
31.9 (11.6) 15.3 (5.0)
12.3 (6.0) 9.5 (1.4)
Periodic Acid Schiff-stained goblet cells Uninfecteda 21.7 (5.0) 4.9 (1.6) 0.4 (0.7)b 1.9 (1.8) Infecteda
34.1 (6.8) 12.5 (14.1)
8.7 (3.6) 6.7 (3.2)
23.0 (5.7) 2.0 (1.5)
4.6 (2.0) 2.9 (1.9)
34.3 (6.3) 9.6 (2.6)
10.8 (3.6) 11.4 (5.3)
25.5 (9.0) 4.2 (3.8)
4.6 (1.9) 6.2 (3.5)
35.6 (6.6) 15.2 (4.3)
12.4 (4.2) 13.8 (5.3)
(B) Conventional 26-day-old weaned pigs PID 1
PID 3
Jejunum Villus
Ileum
Ileum
Jejunum
Ileum
Villus
Crypt
Villus
Crypt
Villus
Crypt
Villus
Crypt
Villus
Crypt
Alcian blue-stained goblet cells Uninfectedc 10.2 (3.2) 5.6 (2.2) 8.1 (3.4) 7.7 (4.3) Infectedc
18.9 (8.1) 20.0 (5.8)
10.3 (3.6) 8.5 (2.8)
7.2 (3.4) 4.0 (3.9)b
9.6 (4.7) 5.2 (3.5)
13.9 (6.3) 21.9 (4.5)
9.7 (3.2) 10.7 (5.7)
11.4 (2.9) 1.8 (1.6)
7.6 (3.7) 7.1 (2.6)
23.1 (7.7) 15.9 (6.1)
10.1 (3.3) 11.8 (5.0)
Periodic Acid Schiff-stained goblet cells 11.9 (4.2) 7.5 (3.4) Uninfectedc 11.7 (4.9) 9.4 (4.6) Infectedc
24.6 (11.0) 18.7 (6.7)
10.2 (4.6) 12.5 (4.9)
12.4 (4.1) 4.3 (4.1)b
6.9 (5.0) 6.8 (2.5)
24. 2 (9.2) 21.0 (9.5)
11.0 (5.0) 13.1 (3.5)
19.8 (5.8) 3.2 (2.6)
9.1 (2.7) 4.5 (3.0)
27.3 (4.8) 13.6 (8.2)
10.8 (3.1) 9.3 (5.0)
a b c
Crypt
PID 5
Jejunum
Uninfected, n = 3 at PID 1 and n = 4 at each PID 3 and 5; PEDV infected, n = 3 at each PID. Bold numbers, P b 0.05 (statistically significant differences between the PEDV-infected and uninfected pigs by Student's t-test). Uninfected, n = 3 at each PID; PEDV infected, n = 3 at PID 1 and n = 4 at each PID 3 and 5.
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K. Jung, L.J. Saif / Research in Veterinary Science 110 (2017) 12–15
Fig. 1. Goblet cells in the jejunum of conventional 9-day-old nursing or 26-day-old weaned pigs inoculated with the original US porcine epidemic diarrhea virus (PEDV) strain PC21A, as stained by alcian blue (A,B), Periodic Acid Schiff (C,D), or immunohistochemistry for the detection of PEDV antigen (E,F). (A) Jejunum of a PEDV-inoculated nursing pig at post-inoculation day (PID) 3, showing few (acidic mucin-secreting) goblet cells (blue color, arrowheads) per villus or crypt. (B) Jejunum of a corresponding negative control pig, showing higher numbers of (acidic mucin-secreting) goblet cells (blue color, arrowheads) per villus or crypt. (C) Jejunum of a PEDV-inoculated nursing pig at PID 3, showing few (neutral mucin-secreting) goblet cells (magenta color, arrowheads) per villus or crypt. (D) Jejunum of a corresponding negative control pig, showing higher numbers of (neutral mucin-secreting) goblet cells (magenta color, arrowheads) per villus or crypt. (E) Jejunum of a PEDV-inoculated weaned pig at PID 3, showing a few PEDV antigen-positive goblet cells (red color, arrowheads) (highlighted in the inset). (F) Jejunum of a corresponding negative control pig, showing no PEDV antigen-positive enterocytes or goblet cells. Fast Red, Gill's hematoxylin counterstaining. Original magnification, all ×200. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(AB) or 3.6 (PAS) at PIDs 1, 3, and 5, respectively. Comparatively, in the jejunal villous epithelium of PEDV-infected nursing pigs, similar to the reduction in the number of goblet cells per villus, the numbers of goblet cells normalized by mean jejunal VH:CD ratios were 0.3 (AB) or 0.4 (PAS) and 0.9 (AB) or 1.7 (PAS) at PIDs 1 and 3, respectively, confirming the depleted or reduced number of goblet cells in the villi of jejunum during the early stages of PEDV infection (summarized in Table 1A) and a significant effect of PEDV infection on the number of villous goblet cells. Thereafter, at PID 5, the numbers were increased slightly to 1.5 (AB) or 3.8 (PAS), implying a potential recovery of the number of villous goblet cells during the later stage of infection. Considering PEDV antigens were mostly found in jejunal villous epithelial cells of infected nursing or weaned pigs tested at PIDs 1–5 (Jung et al., 2015), a significant decrease in the number of crypt goblet cells in jejunum was remarkable, which might reflect increased movement of crypt goblet cells onto the villous epithelium to compensate for loss of villous goblet cells. By IHC, a few PEDV antigen-positive goblet cells
were identified in the jejunal or, to a greater extent, ileal villous epithelium of the infected nursing pigs at PIDs 1 (1/3 pigs tested), 3 (3/3 pigs tested), and 5 (2/3 pigs tested) or infected weaned pigs at PIDs 3 (3/4 pigs tested) (Fig. 1E) and 5 (4/4 pigs tested). No PEDV antigen-positive cells were detected in intestinal tissues of negative control pigs (Fig. 1F). In infected pigs, positive IHC staining of goblet cells mostly appeared to be a circular, thin red stain around the mucin droplet occupying the intracellular space. However, IHC failed to fully differentiate between the intracytoplasmic and cell surface stains of PEDV antigen-positive goblet cells due to the characteristic cell morphology, although some PEDV antigen-positive goblet cells evidently had only intracellular staining with no cell surface staining (Fig. 1E). The dramatic loss of goblet cells in the villous epithelium of PEDV-infected nursing or weaned pigs may indicate a significant effect of PEDV infection on the number of goblet cells, possibly mediated via PEDV binding to the sialic acids expressed on the surface (Deng et al., 2016), although further studies are needed to confirm whether or how, after attaching to the surface sialic acids,
K. Jung, L.J. Saif / Research in Veterinary Science 110 (2017) 12–15
PEDV may enter them via a cellular receptor and replicate in goblet cells. Additional studies are also needed to investigate if, similar to that of TGEV (Schwegmann-Wessels et al., 2011), the sialic acid binding activity of PEDV (Deng et al., 2016) may enable virus particles to penetrate the mucus layer and reach a major cellular receptor of the virus on the surface of other cell types, such as enterocytes. It is noteworthy that although porcine APN which is abundantly expressed on enterocytes was previously indicated as the cellular receptor of PEDV, a recent paper reported that porcine APN is not likely the cellular receptor of the virus, but its role is in enhancing viral infectivity via its enzymatic activity (Shirato et al., 2016). In infected weaned pigs that were resistant to PEDV infection at PID 1 but then showed mild to moderate histological lesions at PIDs 3 and 5 (Jung et al., 2015), similar to the onset and severity of villous atrophy (Jung et al., 2015), mean numbers of AB (acidic mucin)-stained goblet cells per villus or crypt were significantly (P b 0.05) reduced in mid-jejunum at PID 3 and 5 and ileum at PID 5 (Table 1B), compared to the corresponding negative controls. Similarly, mean numbers of PAS (neutral mucin)-stained goblet cells per villus or crypt were significantly (P b 0.05) reduced in the mid-jejunum at PID 3 and 5 and ileum at PID 5 (Table 1B). The reduction in the number of goblet cells per villus in the jejunum of infected weaned pigs peaked at PID 5 (Table 1B), which was delayed by 4 days compared to the infected nursing pigs (Table 1A), confirming age-dependent resistance of weaned pigs to PEDV infection and the disease, such as pathological outcomes (Jung et al., 2015). Mean jejunal VH:CD ratios of uninfected weaned pigs were 2.9–3.1 at PIDs 1–5, whereas mean jejunal VH:CD ratios of the infected weaned pigs were 3.0 at PID 1, but were then reduced to 2.3 and 1.2 at PIDs 3 and 5, respectively (Jung et al., 2015). In the jejunal villous epithelium of uninfected weaned pigs, the numbers of goblet cells normalized by mean jejunal VH:CD ratios were 3.5 (AB) or 4.1 (PAS), 2.3 (AB) or 4.0 (PAS), and 3.9 (AB) or 6.8 (PAS) at PIDs 1, 3, and 5, respectively. However, in the jejunal villous epithelium of infected weaned pigs, similar to the reduction in the number of goblet cells per villus, the numbers of goblet cells normalized by mean jejunal VH:CD ratios were 2.7 (AB) or 3.9 (PAS) at PID 1, but they were decreased to 1.7 (AB) or 1.9 (PAS) at PID 3 and 1.5 (AB) or 2.7 (PAS) at PID 5, confirming the reduced number of goblet cells in the villi of jejunum during the period (summarized in Table 1B). Collectively, PEDV infection resulted in depleted or significantly reduced numbers of goblet cells in the villi or crypts of infected nursing and weaned pigs at the time-points when intestinal villous atrophy was evident (Jung et al., 2015). As a result of reduced numbers of goblet cells for the period, the amount of mucins stained by AB or PAS in the small intestine of infected nursing and weaned pigs also appeared to be less compared to the corresponding negative controls (Fig. 1A–D). Based on these findings, we speculate that during early stages of PEDV infection, goblet cell mucins in the small intestine may be reduced, possibly leading to an impaired mucus layer and increased susceptibility to secondary enteric bacterial or viral infection (Jung and Saif, 2015). Preventive or therapeutic antibiotic therapy is indicated, if concurrent infection with enteric bacterial pathogens, such as Esherichia coli, occurs
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on farms (Jung and Saif, 2015). In addition, the number of goblet cells might be used as an additional parameter for histopathological evaluation of PEDV-infected nursing and weaned pigs, because decreased goblet cell numbers were positively related to severe villous atrophy in the infected jejunum. Further studies are also needed during the later recovery stages of infection of pigs. Conflict of interest statement Neither of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgements We thank Dr. Juliette Hanson, Andrew Wright, Megan Strother, and Ronna Wood for assistance with animal care; and Xiaohong Wang, Zhongyan Lu, John Blakenship, and Thavamathi Annamalai for technical assistance. Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. This work was supported by a grant from the OARDC SEEDS, Grant no. OAOH1536 (Jung K, PI). References Deng, F., Ye, G., Liu, Q., Navid, M.T., Zhong, X., Li, Y., Wan, C., Xiao, S., He, Q., Fu, Z.F., Peng, G., 2016. Identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains. Virus 8, 55. Jung, K., Saif, L.J., 2015. Porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis. Vet. J. 204, 134–143. Jung, K., Alekseev, K.P., Zhang, X., Cheon, D.S., Vlasova, A.N., Saif, L.J., 2007. Altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus. J. Virol. 81, 13681–13693. Jung, K., Wang, Q., Scheuer, K.A., Lu, Z., Zhang, Y., Saif, L.J., 2014. Pathology of US porcine epidemic diarrhea virus strain PC21A in gnotobiotic pigs. Emerg. Infect. Dis. 20, 662–665. Jung, K., Annamalai, T., Lu, Z., Saif, L.J., 2015. Comparative pathogenesis of US porcine epidemic diarrhea virus (PEDV) strain PC21A in conventional 9-day-old nursing piglets vs. 26-day-old weaned pigs. Vet. Microbiol. 178, 31–40. Madson, D.M., Arruda, P.H., Magstadt, D.R., Burrough, E.R., Hoang, H., Sun, D., Bower, L.P., Bhandari, M., Gauger, P.C., Stevenson, G.W., Wilberts, B.L., Wang, C., Zhang, J., Yoon, K.J., 2016. Characterization of porcine epidemic diarrhea virus isolate US/Iowa/ 18984/2013 infection in 1-day-old cesarean-derived colostrum-deprived piglets. Vet. Pathol. 53, 44–52. McCauley, H.A., Guasch, G., 2015. Three cheers for the goblet cell: maintaining homeostasis in mucosal epithelia. Trends Mol. Med. 21, 492–503. Saif, L.J., Pensaert, M.P., Sestak, K., Yeo, S.G., Jung, K., 2012. Coronaviruses. In: Zimmerman, J.J., Karriker, L.A., Ramirez, A., Schwartz, K.J., Stevenson, G.W. (Eds.), Diseases of Swine. Wiley-Blackwell, Iowa State University, pp. 501–524. Schwegmann-Wessels, C., Zimmer, G., Schroder, B., Breves, G., Herrler, G., 2003. Binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins. J. Virol. 77, 11846–11848. Schwegmann-Wessels, C., Bauer, S., Winter, C., Enjuanes, L., Laude, H., Herrler, G., 2011. The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus. Virol. J. 8, 435. Shirato, K., Maejima, M., Islam, M.T., Miyazaki, A., Kawase, M., Matsuyama, S., Taguchi, F., 2016. Porcine aminopeptidase N is not a cellular receptor of porcine epidemic diarrhoea virus, but promotes its infectivity via aminopeptidase activity. J. Gen. Virol. http://dx.doi.org/10.1099/jgv.0.000563.
Contents lists available at ScienceDirect
Research in Veterinary Science journal homepage: www.elsevier.com/locate/rvsc
Goblet cell depletion in small intestinal villous and crypt epithelium of conventional nursing and weaned pigs infected with porcine epidemic diarrhea virus Kwonil Jung ⁎, Linda J. Saif ⁎ Food Animal Health Research Program, Ohio Agricultural Research and Development Center, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, Ohio, USA
a r t i c l e
i n f o
Article history: Received 21 April 2016 Received in revised form 26 August 2016 Accepted 22 October 2016 Available online xxxx Keywords: PEDV Pathogenesis Goblet cell Pig Virus
a b s t r a c t Intestinal goblet cells secret mucins to form mucus layers critical for maintaining the integrity of the intestinal epithelium. Porcine epidemic diarrhea virus (PEDV) causes watery diarrhea and high mortality of suckling pigs. PEDV mainly infects villous epithelial cells of the small intestine, and infected cells undergo acute, massive necrosis, followed by severe villous atrophy. Conventional 9-day-old nursing pigs [PEDV-inoculated (n = 9); Mock (n = 11)] and 26-day-old weaned [PEDV-inoculated (n = 11); Mock (n = 9)] were inoculated orally [8.9 log10 genomic equivalents/pig] with PEDV strain PC21A or mock. We used alcian blue or Periodic-Acid-Schiff staining for the detection of acidic or neutral mucin-secreting goblet cells in the small intestine. We demonstrated that PEDV infection of the nursing pigs at post-inoculation days (PIDs) 1–5 and weaned pigs at PIDs 3–5 led to depletion or significant reduction in the number of goblet cells (and also the number of villous goblet cells normalized by jejunal villous crypt height to crypt depth ratios) in the villi or crypts. These findings coincided with the development of intestinal villous atrophy. By immunohistochemistry, a few PEDV antigen-positive goblet cells were identified in the jejunal or ileal villous epithelium of the infected nursing or weaned pigs. During the early stages of PEDV infection, goblet cell mucins in the small intestine may be decreased, possibly leading to an impaired mucus layer and increased susceptibility to secondary enteric bacterial infection. © 2016 Elsevier Ltd. All rights reserved.
Intestinal villous epithelium lining the gastrointestinal tract mainly consists of the absorptive enterocytes and specialized secretory cells such as goblet cells (McCauley and Guasch, 2015). For both lubrication and barrier function of the intestinal epithelium against enteric pathogens, the intestinal mucosal surface is covered by the mucus layer(s) that mainly consist of mucins secreted by goblet cells (McCauley and Guasch, 2015). Goblet cells are critical to maintain intestinal homeostasis and integrity of intestinal epithelium. Loss or dysfunction of intestinal goblet cells is implicated in enteric disease (McCauley and Guasch, 2015). Porcine epidemic diarrhea virus (PEDV) (family Coronaviridae, genus Alphacoronavirus) causes acute watery diarrhea, dehydration and high mortality of suckling pigs and substantial economic losses (Saif et al., 2012; Jung and Saif, 2015). PEDV is highly enteropathogenic and acutely infects villous epithelial cells of the entire small and large intestines, but the jejunum and ileum are the primary sites of infection (Jung and Saif, 2015; Jung et al., 2014). During the early stages of PEDV infection, villous epithelial cells are infected, followed by acute, massive necrosis and exfoliation of infected cells, resulting in severe
⁎ Corresponding authors. E-mail addresses: [email protected] (K. Jung), [email protected] (L.J. Saif).
http://dx.doi.org/10.1016/j.rvsc.2016.10.009 0034-5288/© 2016 Elsevier Ltd. All rights reserved.
villous atrophy (Madson et al., 2016). The severity of clinical disease in suckling pigs may be exacerbated by secondary co-infections of other enteropathogens, such as Esherichia coli or other viruses (Jung and Saif, 2015). Another enteropathogenic Alphacoronavirus, transmissible gastroenteritis virus (TGEV), binds to goblet cells via sialic acid receptors expressed on the surface (Schwegmann-Wessels et al., 2003; Schwegmann-Wessels et al., 2011). Whether or how, after attaching to the surface sialic acids, TGEV enters them via a cellular receptor to replicate in goblet cells is unclear. Further studies are also needed to investigate whether the sialic acid binding activity of TGEV contributes to stabilizing and anchoring the virus in the intestine or mainly to providing the virus access to the cellular receptor, porcine aminopeptidase N (APN), expressed on the surface of enterocytes (SchwegmannWessels et al., 2011). However, similar to TGEV, whether PEDV also binds to goblet cells is unknown. A previous study showed that the number of goblet cells per intestinal villi of gnotobiotic pigs infected with an original US PEDV strain PC21A was reduced at post-inoculation hours 30–72, compared to the corresponding negative controls (Jung and Saif, 2015), suggesting a potential effect of PEDV infection on intestinal goblet cells. Therefore, our aim was to determine whether PEDV infection causes a reduced number of goblet cells in the villous and crypt epithelium of the small intestine of infected conventional nursing and weaned pigs. We used alcian blue (pH 2.5) (AB) and Periodic-Acid-
K. Jung, L.J. Saif / Research in Veterinary Science 110 (2017) 12–15
Schiff (PAS) staining for the detection of the acidic and neutral sialylated mucin-secreting goblet cells, respectively. All tissue samples tested were archival formalin-fixed, paraffin-embedded tissues acquired from twenty 9-day-old [PEDV infected (n = 9) and Mock (n = 11)] and twenty 26-day-old [PEDV infected (n = 11) and Mock (n = 9)] conventional pigs, inoculated orally with 8.9 log10 genomic equivalents of PEDV strain PC21A, or mock (modified Eagle's medium) (Jung et al., 2015). The clinical disease, fecal virus shedding, and gross and histopathology, including measurement of mean jejunal villous height: crypt depth (VH:CD) ratios to evaluate the severity of intestinal villous atrophy, were reported in a previous paper (Jung et al., 2015). Pigs (n = 3–4/time-point) were euthanized for pathologic examination at post-inoculation days (PIDs) 1, 3, and 5. The mid-jejunal and ileal tissues were stained with AB or PAS. Only well-orientated, intestinal tissue sections were evaluated to count the number of goblet cells per intestinal villus or crypt. Mean numbers of AB or PAS-stained goblet cells per villus or crypt were estimated by measuring at least 10 villi and crypts for each PID and from infected or control pigs. All values were expressed as the means ± standard deviation of the means (SDM). Mean numbers of AB or PAS-stained goblet cells per villus or crypt between PEDV-infected and uninfected nursing or weaned pigs at the same time-points were analyzed and compared by a Student's t-test using GraphPad Prism software (GraphPad Prism Inc.). A value of P b 0.05 was considered statistically significant. The intestinal tissues were also tested by immunohistochemistry (IHC), as described previously (Jung et al., 2007; Jung et al., 2014), for the detection of PEDV antigen, using monoclonal antibody 6C8-1 against the spike protein of PEDV strain DR13 that was shown previously to cross-react with the US PEDV strain PC21A (provided by Daesub Song, Korean Research Institute of Bioscience and Biotechnology, Daejeon, Korea) (Jung et al., 2014). Briefly, endogenous alkaline phosphatase in rehydrated tissues was quenched with 3% glacial acetic acid 20% for 20 min at room temperature. Antigen retrieval was performed using 100 μg/ml of proteinase K (Invitrogen, Carlsbad, CA). The tissue slides were then washed in PBTS [phosphate-buffered saline (PBS) containing Tween 20, 0.1%] three times and blocked with 1× buffered solution of casein (Universal Blocking Reagent; Biogenex, Fremont, CA) in
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distilled water for 30 min at room temperature. Sections were coated with monoclonal antibody 6C8-1 diluted 1 in 200 in PBTS and incubated overnight at 4 °C in a humid chamber. After three washes with PBTS, the sections were incubated for 1 h at 36 °C with goat anti-mouse IgG labeled with alkaline phosphatase (Dako, Glostrup, Denmark) diluted 1 in 200 in PBTS. After three washes with PBTS, the final reaction was generated by immersing the tissue sections in a staining solution [1 tablet of Fast Red in 2 ml of 0.1 M Tris-buffer (pH 8.2); Roche Applied Science, Mannheim, Germany] for 10 min at room temperature. Sections were lightly counterstained with Gill's hematoxylin. In infected nursing pigs, mean numbers of AB (acidic mucin)-stained goblet cells per villus and crypt were significantly (P b 0.05) reduced in mid-jejunum and ileum at PIDs 1, 3, and 5 (except for crypt in ileum at PID 5), compared to the corresponding negative controls (Table 1A; Fig. 1A and B). Similarly, mean numbers of PAS (neutral mucin)-stained goblet cells per villus and crypt were also significantly (P b 0.05) reduced in the mid-jejunum and ileum of the infected nursing pigs at PIDs 1, 3, and 5, but not in crypt in ileum at PIDs 1, 3, and 5 and in jejunum at PID 5 (Table 1A; Fig. 1C and D). The reduction rate, ranging from 1.8 to 16.5%, in the number of AB or PAS-stained goblet cells per villus in the jejunum of infected nursing pigs at PIDs 1–5 was also less than that (15.2 to 48.0%) in the ileum during the same period. Relative to the jejunal crypts of infected nursing pigs, goblet cells in the ileal crypts were less depleted at PIDs 1–5 (Table 1A), possibly contributing to more efficient compensation for loss of villous goblet cells. Additionally to investigate if the reduced numbers of goblet cells observed per villus could be an expected outcome of the shortened villus following PEDV infection, mean numbers of goblet cells per villus were normalized by dividing by the mean intestinal VH:CD ratios acquired from infection and non-infection groups at each PID. All infected nursing pigs at PIDs 1–5 exhibited severe watery diarrhea and jejunal villous atrophy (mean VH:CD ratios of 1.1–1.4), whereas none of the uninfected control nursing pigs showed clinical signs. Their mean jejunal VH:CD ratios were 7.1–8.7 during the same period, as reported previously (Jung et al., 2015). In the jejunal villous epithelium of uninfected nursing pigs, the numbers of goblet cells normalized by mean jejunal VH:CD ratios were 2.3 (AB) or 2.5 (PAS), 2.6 (AB) or 3.0 (PAS), and 3.1
Table 1 Mean numbers (±SDM) of alcian blue (acidic mucin) or Periodic Acid Schiff (neutral mucin)-stained goblet cells per villi or crypt in the mid-jejunum and ileum of conventional 9-day-old nursing (A) and 26-day-old weaned (B) pigs infected with the original US PEDV strain PC21A at post-inoculation days (PIDs) 1, 3, and 5. (A) Conventional 9-day-old nursing pigs PID 1
PID 3
Jejunum Villus
Ileum Crypt
PID 5
Jejunum
Ileum
Jejunum
Ileum
Villus
Crypt
Villus
Crypt
Villus
Crypt
Villus
Crypt
Villus
Crypt
Alcian blue-stained goblet cells 19.9 (6.8) 3.4 (1.8) Uninfecteda 0.5 (0.8)b 1.4 (1.3) Infecteda
30.5 (6.7) 10.8 (8.8)
8.9 (3.8) 6.3 (3.4)
19.9 (6.0) 1.1 (1.3)
4.7 (1.9) 2.9 (1.4)
33.6 (5.6) 5.1 (4.3)
10.0 (3.3) 5.8 (1.7)
21.8 (7.4) 1.6 (1.1)
4.7 (1.9) 2.5 (1.9)
31.9 (11.6) 15.3 (5.0)
12.3 (6.0) 9.5 (1.4)
Periodic Acid Schiff-stained goblet cells Uninfecteda 21.7 (5.0) 4.9 (1.6) 0.4 (0.7)b 1.9 (1.8) Infecteda
34.1 (6.8) 12.5 (14.1)
8.7 (3.6) 6.7 (3.2)
23.0 (5.7) 2.0 (1.5)
4.6 (2.0) 2.9 (1.9)
34.3 (6.3) 9.6 (2.6)
10.8 (3.6) 11.4 (5.3)
25.5 (9.0) 4.2 (3.8)
4.6 (1.9) 6.2 (3.5)
35.6 (6.6) 15.2 (4.3)
12.4 (4.2) 13.8 (5.3)
(B) Conventional 26-day-old weaned pigs PID 1
PID 3
Jejunum Villus
Ileum
Ileum
Jejunum
Ileum
Villus
Crypt
Villus
Crypt
Villus
Crypt
Villus
Crypt
Villus
Crypt
Alcian blue-stained goblet cells Uninfectedc 10.2 (3.2) 5.6 (2.2) 8.1 (3.4) 7.7 (4.3) Infectedc
18.9 (8.1) 20.0 (5.8)
10.3 (3.6) 8.5 (2.8)
7.2 (3.4) 4.0 (3.9)b
9.6 (4.7) 5.2 (3.5)
13.9 (6.3) 21.9 (4.5)
9.7 (3.2) 10.7 (5.7)
11.4 (2.9) 1.8 (1.6)
7.6 (3.7) 7.1 (2.6)
23.1 (7.7) 15.9 (6.1)
10.1 (3.3) 11.8 (5.0)
Periodic Acid Schiff-stained goblet cells 11.9 (4.2) 7.5 (3.4) Uninfectedc 11.7 (4.9) 9.4 (4.6) Infectedc
24.6 (11.0) 18.7 (6.7)
10.2 (4.6) 12.5 (4.9)
12.4 (4.1) 4.3 (4.1)b
6.9 (5.0) 6.8 (2.5)
24. 2 (9.2) 21.0 (9.5)
11.0 (5.0) 13.1 (3.5)
19.8 (5.8) 3.2 (2.6)
9.1 (2.7) 4.5 (3.0)
27.3 (4.8) 13.6 (8.2)
10.8 (3.1) 9.3 (5.0)
a b c
Crypt
PID 5
Jejunum
Uninfected, n = 3 at PID 1 and n = 4 at each PID 3 and 5; PEDV infected, n = 3 at each PID. Bold numbers, P b 0.05 (statistically significant differences between the PEDV-infected and uninfected pigs by Student's t-test). Uninfected, n = 3 at each PID; PEDV infected, n = 3 at PID 1 and n = 4 at each PID 3 and 5.
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K. Jung, L.J. Saif / Research in Veterinary Science 110 (2017) 12–15
Fig. 1. Goblet cells in the jejunum of conventional 9-day-old nursing or 26-day-old weaned pigs inoculated with the original US porcine epidemic diarrhea virus (PEDV) strain PC21A, as stained by alcian blue (A,B), Periodic Acid Schiff (C,D), or immunohistochemistry for the detection of PEDV antigen (E,F). (A) Jejunum of a PEDV-inoculated nursing pig at post-inoculation day (PID) 3, showing few (acidic mucin-secreting) goblet cells (blue color, arrowheads) per villus or crypt. (B) Jejunum of a corresponding negative control pig, showing higher numbers of (acidic mucin-secreting) goblet cells (blue color, arrowheads) per villus or crypt. (C) Jejunum of a PEDV-inoculated nursing pig at PID 3, showing few (neutral mucin-secreting) goblet cells (magenta color, arrowheads) per villus or crypt. (D) Jejunum of a corresponding negative control pig, showing higher numbers of (neutral mucin-secreting) goblet cells (magenta color, arrowheads) per villus or crypt. (E) Jejunum of a PEDV-inoculated weaned pig at PID 3, showing a few PEDV antigen-positive goblet cells (red color, arrowheads) (highlighted in the inset). (F) Jejunum of a corresponding negative control pig, showing no PEDV antigen-positive enterocytes or goblet cells. Fast Red, Gill's hematoxylin counterstaining. Original magnification, all ×200. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
(AB) or 3.6 (PAS) at PIDs 1, 3, and 5, respectively. Comparatively, in the jejunal villous epithelium of PEDV-infected nursing pigs, similar to the reduction in the number of goblet cells per villus, the numbers of goblet cells normalized by mean jejunal VH:CD ratios were 0.3 (AB) or 0.4 (PAS) and 0.9 (AB) or 1.7 (PAS) at PIDs 1 and 3, respectively, confirming the depleted or reduced number of goblet cells in the villi of jejunum during the early stages of PEDV infection (summarized in Table 1A) and a significant effect of PEDV infection on the number of villous goblet cells. Thereafter, at PID 5, the numbers were increased slightly to 1.5 (AB) or 3.8 (PAS), implying a potential recovery of the number of villous goblet cells during the later stage of infection. Considering PEDV antigens were mostly found in jejunal villous epithelial cells of infected nursing or weaned pigs tested at PIDs 1–5 (Jung et al., 2015), a significant decrease in the number of crypt goblet cells in jejunum was remarkable, which might reflect increased movement of crypt goblet cells onto the villous epithelium to compensate for loss of villous goblet cells. By IHC, a few PEDV antigen-positive goblet cells
were identified in the jejunal or, to a greater extent, ileal villous epithelium of the infected nursing pigs at PIDs 1 (1/3 pigs tested), 3 (3/3 pigs tested), and 5 (2/3 pigs tested) or infected weaned pigs at PIDs 3 (3/4 pigs tested) (Fig. 1E) and 5 (4/4 pigs tested). No PEDV antigen-positive cells were detected in intestinal tissues of negative control pigs (Fig. 1F). In infected pigs, positive IHC staining of goblet cells mostly appeared to be a circular, thin red stain around the mucin droplet occupying the intracellular space. However, IHC failed to fully differentiate between the intracytoplasmic and cell surface stains of PEDV antigen-positive goblet cells due to the characteristic cell morphology, although some PEDV antigen-positive goblet cells evidently had only intracellular staining with no cell surface staining (Fig. 1E). The dramatic loss of goblet cells in the villous epithelium of PEDV-infected nursing or weaned pigs may indicate a significant effect of PEDV infection on the number of goblet cells, possibly mediated via PEDV binding to the sialic acids expressed on the surface (Deng et al., 2016), although further studies are needed to confirm whether or how, after attaching to the surface sialic acids,
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PEDV may enter them via a cellular receptor and replicate in goblet cells. Additional studies are also needed to investigate if, similar to that of TGEV (Schwegmann-Wessels et al., 2011), the sialic acid binding activity of PEDV (Deng et al., 2016) may enable virus particles to penetrate the mucus layer and reach a major cellular receptor of the virus on the surface of other cell types, such as enterocytes. It is noteworthy that although porcine APN which is abundantly expressed on enterocytes was previously indicated as the cellular receptor of PEDV, a recent paper reported that porcine APN is not likely the cellular receptor of the virus, but its role is in enhancing viral infectivity via its enzymatic activity (Shirato et al., 2016). In infected weaned pigs that were resistant to PEDV infection at PID 1 but then showed mild to moderate histological lesions at PIDs 3 and 5 (Jung et al., 2015), similar to the onset and severity of villous atrophy (Jung et al., 2015), mean numbers of AB (acidic mucin)-stained goblet cells per villus or crypt were significantly (P b 0.05) reduced in mid-jejunum at PID 3 and 5 and ileum at PID 5 (Table 1B), compared to the corresponding negative controls. Similarly, mean numbers of PAS (neutral mucin)-stained goblet cells per villus or crypt were significantly (P b 0.05) reduced in the mid-jejunum at PID 3 and 5 and ileum at PID 5 (Table 1B). The reduction in the number of goblet cells per villus in the jejunum of infected weaned pigs peaked at PID 5 (Table 1B), which was delayed by 4 days compared to the infected nursing pigs (Table 1A), confirming age-dependent resistance of weaned pigs to PEDV infection and the disease, such as pathological outcomes (Jung et al., 2015). Mean jejunal VH:CD ratios of uninfected weaned pigs were 2.9–3.1 at PIDs 1–5, whereas mean jejunal VH:CD ratios of the infected weaned pigs were 3.0 at PID 1, but were then reduced to 2.3 and 1.2 at PIDs 3 and 5, respectively (Jung et al., 2015). In the jejunal villous epithelium of uninfected weaned pigs, the numbers of goblet cells normalized by mean jejunal VH:CD ratios were 3.5 (AB) or 4.1 (PAS), 2.3 (AB) or 4.0 (PAS), and 3.9 (AB) or 6.8 (PAS) at PIDs 1, 3, and 5, respectively. However, in the jejunal villous epithelium of infected weaned pigs, similar to the reduction in the number of goblet cells per villus, the numbers of goblet cells normalized by mean jejunal VH:CD ratios were 2.7 (AB) or 3.9 (PAS) at PID 1, but they were decreased to 1.7 (AB) or 1.9 (PAS) at PID 3 and 1.5 (AB) or 2.7 (PAS) at PID 5, confirming the reduced number of goblet cells in the villi of jejunum during the period (summarized in Table 1B). Collectively, PEDV infection resulted in depleted or significantly reduced numbers of goblet cells in the villi or crypts of infected nursing and weaned pigs at the time-points when intestinal villous atrophy was evident (Jung et al., 2015). As a result of reduced numbers of goblet cells for the period, the amount of mucins stained by AB or PAS in the small intestine of infected nursing and weaned pigs also appeared to be less compared to the corresponding negative controls (Fig. 1A–D). Based on these findings, we speculate that during early stages of PEDV infection, goblet cell mucins in the small intestine may be reduced, possibly leading to an impaired mucus layer and increased susceptibility to secondary enteric bacterial or viral infection (Jung and Saif, 2015). Preventive or therapeutic antibiotic therapy is indicated, if concurrent infection with enteric bacterial pathogens, such as Esherichia coli, occurs
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on farms (Jung and Saif, 2015). In addition, the number of goblet cells might be used as an additional parameter for histopathological evaluation of PEDV-infected nursing and weaned pigs, because decreased goblet cell numbers were positively related to severe villous atrophy in the infected jejunum. Further studies are also needed during the later recovery stages of infection of pigs. Conflict of interest statement Neither of the authors of this paper has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Acknowledgements We thank Dr. Juliette Hanson, Andrew Wright, Megan Strother, and Ronna Wood for assistance with animal care; and Xiaohong Wang, Zhongyan Lu, John Blakenship, and Thavamathi Annamalai for technical assistance. Salaries and research support were provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. This work was supported by a grant from the OARDC SEEDS, Grant no. OAOH1536 (Jung K, PI). References Deng, F., Ye, G., Liu, Q., Navid, M.T., Zhong, X., Li, Y., Wan, C., Xiao, S., He, Q., Fu, Z.F., Peng, G., 2016. Identification and comparison of receptor binding characteristics of the spike protein of two porcine epidemic diarrhea virus strains. Virus 8, 55. Jung, K., Saif, L.J., 2015. Porcine epidemic diarrhea virus infection: etiology, epidemiology, pathogenesis and immunoprophylaxis. Vet. J. 204, 134–143. Jung, K., Alekseev, K.P., Zhang, X., Cheon, D.S., Vlasova, A.N., Saif, L.J., 2007. Altered pathogenesis of porcine respiratory coronavirus in pigs due to immunosuppressive effects of dexamethasone: implications for corticosteroid use in treatment of severe acute respiratory syndrome coronavirus. J. Virol. 81, 13681–13693. Jung, K., Wang, Q., Scheuer, K.A., Lu, Z., Zhang, Y., Saif, L.J., 2014. Pathology of US porcine epidemic diarrhea virus strain PC21A in gnotobiotic pigs. Emerg. Infect. Dis. 20, 662–665. Jung, K., Annamalai, T., Lu, Z., Saif, L.J., 2015. Comparative pathogenesis of US porcine epidemic diarrhea virus (PEDV) strain PC21A in conventional 9-day-old nursing piglets vs. 26-day-old weaned pigs. Vet. Microbiol. 178, 31–40. Madson, D.M., Arruda, P.H., Magstadt, D.R., Burrough, E.R., Hoang, H., Sun, D., Bower, L.P., Bhandari, M., Gauger, P.C., Stevenson, G.W., Wilberts, B.L., Wang, C., Zhang, J., Yoon, K.J., 2016. Characterization of porcine epidemic diarrhea virus isolate US/Iowa/ 18984/2013 infection in 1-day-old cesarean-derived colostrum-deprived piglets. Vet. Pathol. 53, 44–52. McCauley, H.A., Guasch, G., 2015. Three cheers for the goblet cell: maintaining homeostasis in mucosal epithelia. Trends Mol. Med. 21, 492–503. Saif, L.J., Pensaert, M.P., Sestak, K., Yeo, S.G., Jung, K., 2012. Coronaviruses. In: Zimmerman, J.J., Karriker, L.A., Ramirez, A., Schwartz, K.J., Stevenson, G.W. (Eds.), Diseases of Swine. Wiley-Blackwell, Iowa State University, pp. 501–524. Schwegmann-Wessels, C., Zimmer, G., Schroder, B., Breves, G., Herrler, G., 2003. Binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins. J. Virol. 77, 11846–11848. Schwegmann-Wessels, C., Bauer, S., Winter, C., Enjuanes, L., Laude, H., Herrler, G., 2011. The sialic acid binding activity of the S protein facilitates infection by porcine transmissible gastroenteritis coronavirus. Virol. J. 8, 435. Shirato, K., Maejima, M., Islam, M.T., Miyazaki, A., Kawase, M., Matsuyama, S., Taguchi, F., 2016. Porcine aminopeptidase N is not a cellular receptor of porcine epidemic diarrhoea virus, but promotes its infectivity via aminopeptidase activity. J. Gen. Virol. http://dx.doi.org/10.1099/jgv.0.000563.