Multinucleate Enterocytes Associated with Experimental Group A Porcine Rotavirus Infection

Abstract

It has been recently demonstrated that atypical (non-group A) porcine rotaviruses are capable of producing villous enterocyte syncytia both in experimental and spontaneous cases of porcine Epithelial syncytia may also occur in atypical rotavirus infections of lambs,3 calves,8 and rats.I0 Since group A rotavirus infection is not known to be associated with small intestinal syncytia, it has been suggested that in the pig, this phenomenon is specific enough to allow its use as a histologic diagnostic ~r i ter ion.~ We present evidence indicating that enterocyte syncytia occasionally occur in group A rotavirus infection of piglets; therefore, the presence of these cells does not denote a non-group A infection. For this study, cesarian-derived piglets were maintained in gnotobiotic isolators. One ml of group A rotavirus (type OSU)6 was further passaged in seven newborn gnotobiotic piglets. Feces and intestinal contents from these pigs were collected 24 hours after infection, pooled, clarified, and filtered (0.45 pm). Electropherotyping6 performed on RNA extracted from this material yielded a banding pattern identical to reference group A OSU porcine rotavirus. No adventitial agents were detected. Two ml of filtered feces ( lo7 plaqueforming units titrated in MA-104 cells) were used to orally infect six gnotobiotic pigs varying in age from 2 to 4 days. One pig, infected at 2 days of age and euthanatized 24 hours after infection, forms the basis for this report. Tissue sections from seven equidistant regions of small intestine7 were fixed in 10% neutral buffered formalin, processed in low-melt paraffin (Peel-A-Way, American Scientific Products, McGaw Park, IL), and sectioned 5 pm thick. They were then processed for immunohistochemical localization of group A specific rotavirus antigen. The glass slides containing the paraffin sections were incubated for 30 minutes at 56 C and then deparaffinized in xylene. After hydration through a series of graded ethanols, endogenous peroxidase activity was blocked by 0.3% hydrogen peroxide for 30 minutes. After washing the slides in 0.0 1 M phosphate buffered saline (pH 7.6, wash buffer and diluent), diluted normal goat serum was added to the sections and the slides were incubated at room temperature for 30 minutes. Excess serum was shaken from the slides, and a 1 : 200 dilution of rabbit antihuman group A rotavirus antibody (DAKO Corp., Santa Barbara, CA) was added to the tissue sections followed by overnight incubation in a humid chamber at 4 C. After washing, a secondary antibody, biotinylated goat antirabbit IgG (Vector Labs, Burlingame, CA), was incubated on the tissue sections for 30 minutes at room temperature. Following washing, horseradish peroxidase-conjugated avidin biotin complex (Vector Labs, Burlingame, CA) was incubated on the sections for 60 minutes at room temperature, after which the slides were washed. A peroxidase substrate solution (0.05% 3,3’diaminobenzidine in 0.05 M Tris buffer [pH 7.21 with 0.0 1% hydrogen peroxide) was added to the tissue sections. After incubation for 5 minutes at room temperature to allow for color development, the slides were rinsed in tap water, counterstained in Mayer’s hematoxylin, dehydrated through a series of graded alcohols, cleared in xylene, and mounted with a coverslip. Paired control sections of porcine intestine, known to be immunohistochemically positive or negative for group A rotavirus antigen, were stained as described above and also with substitution of the primary antibody with normal rabbit serum diluted to the same protein concentration as the pnmary antibody. In addition, a positive control section was reacted with the primary rabbit antibody that had been incubated overnight at 4 C with OSU rotavirus. Enterocytes containing group A rotavirus antigen had variable numbers of cytoplasmic brown granules as opposed to negative tissues and negative controls that lacked such granules. Preincubation of virus with antibody completely blocked specific staining of rotavirus antigen in tissue sections. Examination of the paraffin-embedded, avidin-biotin stained tissues from the experimental pigs demonstrated the presence of brown granules in the cytoplasm of large numbers of villous enterocytes from the mid-jejunum through the ileum. In addition, many cells were vacuolated, many were exfoliating from the villous tips, and there were areas where the villi were denuded of epithelium. These results were similar to previous reports of the pathogenesis of rotavirus infection in Several multinucleated enterocytes were present along the villous sides and tips of one pig. For the most part, these cells lacked the specific cytoplasmic staining indicative of group A rotavirus infection. An occasional sloughed multinucleate cell was seen, however, that had vacuolated cytoplasm and virus-specific immunoperoxidase positive cytoplasmic granules (Fig. 1) indicating replication of group A rotavirus within the cell. We were unable to ascertain from this study whether multinucleate enterocytes are occasionally found in the intestines