key: cord-0962217-lseuie52
authors: Pospischil, A.; Hess, R. G.; Bachmann, P. A.
title: Light Microscopy and Ultrahistology of Intestinal Changes in Pigs Infected with Epizootic Diarrhoea Virus (EVD): Comparison with Transmissible Gastroenteritis (TGE) Virus and Porcine Rotavirus Infections
date: 2010-05-13
journal: J Vet Med B Infect Dis Vet Public Health
DOI: 10.1111/j.1439-0450.1981.tb01774.x
sha: cfed8b9145f06610357e36ee8ee58b7261044fd3
doc_id: 962217
cord_uid: lseuie52

SUMMARY: Light microscopic and ultrahistological changes were investigated in the intestines of piglets naturally and experimentally infected with epizootic viral diarrhoea (EVD) virus and compared to those occuring during intestinal TGE and rotavirus infections in piglets. The course of the EVD virus infection is comparable to that of TGE. Villous atrophy develops throughout the jejunum and the ileum as in TGE. In this respect it differs from rotavirus infection. The ultrastructural picture of intestinal EVD virus infection resembles that of TGE and other enteropathogenic coronaviruses. Virus replication, however, was observed mainly in enterocytes located on the lateral walls of the villi extending to the villus/crypt border, and sometimes virus‐infected cells were demonstrated in or near the Lieberkühn's crypts. EVD virus was also demonstrated in macrophages in the intestinal lamina propria and in phagocytes in regional lymph nodes. ZUSAMMENFASSUNG: Lichtmikroskopische und ultrahistologische intestinale Veränderungen bei Ferkeln nach Infektion mit dem Epizootischen Diarrhoe‐Virus (EVD): Vergleich mit Übertragbaren Gastroenteritis (TGE) Virus‐ und Schweinerotavirusinfektionen Lichtmikroskopische und ultrahistologische Veränderungen von Därmen natürlich und experimentell mit dem Virus der Epizootischen Virusdiarrhoe (EVD) infizierten Ferkeln wurden untersucht und mit denen nach Infektion mit TGE‐Virus und Rotavirus verglichen. Der Verlauf der EVD‐Virusinfektion ist vergleichbar mit dem der TGE. Eine Zottenatrophie tritt ähnlich wie bei der TGE im gesamten Jejunum und Ileum auf. In dieser Beziehung unterscheiden sich die Befunde von der Rotavirusinfektion. Die ultrastrukturellen Darmveränderungen bei der EVD‐Virusinfektion entsprechen denen der TGE und anderen enteropathogenen Coronaviren. Virusvermehrung war jedoch hauptsächlich auf die laterale Epithelschicht der Zotten begrenzt. Sie reichte bis in den Zotten/Krypten‐Bereich. In einigen Fällen ließen sich infizierte Zellen in oder nahe den Lieberkühnschen Krypten nachweisen. EVD‐Viruspartikel wurden weiterhin in Makrophagen der Lamina propria und in Phagozyten der regionalen Lymphknoten gefunden. RÉSUMÉ: Lésions intestinales au microscope optique et ultrahistologiques chez des porcelets après une infection avec le virus diarrhéique épizootique (EVD): Comparaison avec le virus de la gastro‐entérite transmissible (TGE) et des infections à Rotavirus chez des porcs On a examiné les lésions intestinales ultrahistologiquement et au microscope optique chez des porcelets infectés naturellement et expérimentalement avec le virus épizootique de la diarrhée (EVD). On a comparé cette infection avec celle du virus TGE et Rotavirus. Le déroulement de l'infection au virus EVD est comparable à celui de TGE. Une atrophie des villosités dans tout le jejunum et l'ileum est constatée comme avec TGE. Les résultats trouvés avec une infection à Rotavirus se différencient sur ce point. Les lésions intestinales ultrastructurelles lors d'une infection avec le virus EVD correspondent à celles rencontrées avec TGE et d'autres virus Corona. La multiplication du virus fut cependant principalement localisée à la couche épithéliale latérale des villosités. Elle atteignit la région des villosités/cryptes. Des cellules infectées ont été mises en évidence dans quelques cas dans ou près des cryptes de Lieberkühn. On a trouvé des particules virales EVD dans des macrophages de Lamina propria et dans des phagocytes des ganglions régionaux. RESUMEN: Fotomicroscopía e ultrahistología de las modificaciones intestinales en lechones infectados con el virus de la diarrea virósica epizoótica (DVE): Comparación con el virus de la gastroenteritis transmisible (GET) e infecciones con el virus rota porcino Se examinaron las modificaciones fotomicroscópicas y ultrahistológicas en los intestinos de lechones infectados de forma natural y experimental con el virus de la diarrea virósica epizoótica (DVE), comparándose con las habidas tras la infección con el virus GET y el virus rota porcino. El curso de la infección con el virus de la DVE se puede comparar con el de la GET. La atrofia de villosidades aparece, tal y como en la GET, en la totalidad del yeyuno y del íleon. A este respecto se diferencian los hallazgos de la infección con el virus rota. Las modificaciones entéricas ultraestructurales en la infección con el virus de la DVE corresponden a los de la GET y otros virus corona enteropatógenos. Sin embargo, la multiplicación de virus se hallaba limitada sobre todo a la capa epitelial lateral de las villosidades. La misma alcanzaba hasta el ámbito de las villosidades/criptas. En algunos casos se pudieron identificar células infectadas en las criptas de Lieberkühn o cerca de las mismas. Partículas virales DVE se hallaron además en los macrófagos de la lámina propia y en los fagocitos de los ganglios regionales.

Specific antigen was demonstrated using immunofluorescence methods with FITC antibody conjugates for epizootic diarrhoea virus, transmissible gastroenteritis virus and rotavirus. There was no cross-reaction between any of the three viruses, indicating that epizootic diarrhoea virus is antigenically not related to TGE virus.

Localization of EVD antigen in the villus epithelium is illustrated in Fig. 2 . In some cases EVD antigen-containing cells were also found in crypt epithelium.

At the onset of diarrhoea (24 hours post infection) histological findings in the jejunum of experimentally infected piglets consisted of marked vacuolization (vacuoles contained fat and PAS-positive material) of the enterocyte ' + We thank Prof. Dr. M. PENSAERT, Veterinary Faculty, University of Ghent, Belgium, for supplying the anti-EVD-virus conjugate. 

Immunof luorescen t g of intestinal sec-?om a piglet experi-.ly infected with epidiarrhoea virus using virus antibody conjugate. X 370 lesions could be detected throughout the jejunum and in the ileum. Morphology of enterocytes varied from cylindrical to cuboidal. With increasing degeneration of the brush border of the enterocytes, cuboidal cell morphology predominated.

Electron microscopy of the intestines from infected piglets demonstrated virus-containing enterocytes on the !lateral wall of the villi. Occasionally, virusinfected cells were seen in or near jejunal crypts (Fig. 3) .

Virus particles were seen in varying numbers in vesicles which were localized mainly in the supranuclear cytoplasm. The vesicles were irregular in morphology and size, measuring between 500 and 1,000 nm. Virus particles were highly pleomorphic. They appeared mainly as round or oval, but also bowl-like or elongated forms measuring between 50 and 105 nm. Open and closed rings could be distinguished. In round particles the diameter averaged about 73 nm. The inner structure consisted of concentrically arranged osmiophilic material, sometimes around a central electron-lucent space measuring about 32 nm in diameter (Fig. 4) . The nucleocapsid was surrounded by a double membrane of about 8 nm width. Surface projections could not be demonstrated.

The mitochondria of infected cells were swollen and cristae were partly absent, partly disrupted or degenerated. Only a reduced number of cytoplasmic organelles, such as RER or Golgi apparatus were left. They tended to be concentrated more towards the microvillus border, leaving the basal parts of the cytoplasm more or less empty. Absorptive vesicles were not found. Nuclei did not show morphological changes.

In cells containing large amounts of virus particles, microvilli appeared broad and short. Their number was considerably reduced (Fig. 5 a) . In less infected cells, microvilli appeared intact; virus particles, however, could be seen between microvilli (Fig. 5 b) . In addition, virus particles were also present in intercellular spaces between adjacent jejunal enterocytes and near the basal membrane of infected cells. However, budding from the cytoplasmic membrane was not observed. Some degenerated, infected enterocytes shed from the lamina propria could be demonstrated in the intestinal lumen. Occasionally, macrophages were found in the lamina propria just under the basal membrane. They contained vesicles filled with virus particles as well as phagosomes with cellular detritus and mucous droplets (Fig. 6 ) . Virus particles which reeched the regional lymph nodes were mainly degraded (Fig. 7 a) . There were, however, also morphologically intact particles within a granular matrix. Some of these structures were completely surrounded by a definite membrane, while others were not (Fig. 7 b) . They were round in shape and electron-dense. Viral structures of different morphology were embedded in these bodies. Budding processes were not detected. Virus infected cells were not found in the colon.

The histological findings of naturally infected piglets revealed a marked age dependence difference between neonatal piglets (up to 2,500 g) and feeder pigs (20-30 kg). Villous enterocytes of neonatal piglets also showed a distinct vacuolization. The vacuoles mainly contained fat and PAS-positive substances. Villous atrophy was absent, whereas in feeder pigs, villous atrophy predominated and cytoplasmic vacuolization was rare. Villous atrophy occurred throughout the jejunum and in the ileum.

Electron microscopy also demonstrated the presence of enterocytes containing virus particles, mainly on the lateral walls of the villi. The virus was found neither in enterocytes located at the tip of the villi nor in the crypts. Virus particles were seen in varying numbers in vesicles which were distributed over the whole cytoplasm. Further alterations in the cytoplasm were swollen mitochondriae and fragmentation of cristae. Remaining cytoplasmic organelles were concentrated near the apical cell membrane just as in experimentally infected piglets. There were no changes in the nucleus. The microvillous border of infected enterocytes containing large numbers of virions seemed to be morphologically intact. Only few virus particles were seen between the microvilli. Virus-containing detached cells, however, were found in the jejunal lumen.

In feeder pigs, ultrastructure and localization of alterations were comparable to those seen in the microvilli of neonatal piglets. However, microvilli were markedly shortened and their number reduced. Free virus particles were found in the microvillous border. Infected enterocytes sporadically protruded into the jejunal lumen. Virus could not be demonstrated in the colon, epithelium or lymph nodes. The intestines of the non-infected control piglets showed well defined villi with cylindrical epithelial cells (Fig. 1 a) . Enterocytes had deep basal nuclei with regular microvillus borders and well defined terminal webs. Cell organelles appeared unaltered when examined by electron microscopy.

In experimentally as well as in naturally infected piglets, histological investigation of the jejunum and ileum revealed the well-known distinct villous atrophy (Fig. 1 c) . In the cytoplasm of infected enterocytes, vacuoles were sometimes present which contained mainly fat. In addition, there was a remarkable deposition of PAS-positive material in the lamina propria near the villus tips. The observed histological changes were similar, independent of age or weight of the piglets. Usually enterocytes were cylindrical to cuboidal in form, tending more towards cuboidal in villi with distinct histological lesions.

The ultrastructural picture of lesions in TGE virus-infected jejunal enterocytes is roughly comparable to that seen in EVD-virus infected cells. Virus particles were found in vesicles of enterocytes from the tip of the villus to the villus/crypt border. Virus-containing cells were not detectable in the crypt area.

The cytoplasm of infected cells revealed swollen mitochondria and partial fragmentation of the cristae mitochondriales. In experimentally infected piglets, the smooth endoplasmic reticulum showed a marked dilatation. Remaining organelles did not display any morphological alterations. The appearance of vacuoles of varying osmiophility in infected enterocytes reaching nuclear size was striking. The brush border of cells containing virus appeared broadened, shortened, and microvilli were greatly reduced in number. Frequently viral particles could be demonstrated between the microvilli. Virus particles were neither detected in lymph nodes nor in epithelial cells of colon mucosa.

Severe histological lesions were found in the middle and distal parts of the jejunum as well as in the ileum. They consisted of focal necrosis and epithelial cell desquamation, mainly at the tip of the villi. As a result, marked villous atrophy was present in the distal jejunum and the ileum (Fig. 1 d) . No pathological lesions were detected in the proximal part of the jejunum or in the colon. Enterocytes appeared cylindrical in form regardless of whether the brush border was degenerated or intact.

Virus particles were mainly found in epithelial cells located at the tip of the villi. From large granular viroplasma areas or regions of convoluted membranes in the cytoplasm, mature virions budded into the dilated smooth (SER) and rough (RER) endoplasmic reticulum. These particles were double-shelled and measured 80-90nm in diameter and contained an inner core 40nm in diameter. Virus parti'cles were also demonstrated in cytoplasmic vesicles derived from the SER or RER. These particles lacked the outer shell and measured approx. 65 nm in diameter. Both morphological types of virions were observed free in the intestinal lumen.

Cytoplasmic changes in infected cells began with a dilatation of SER and RER and an aggregation of ribosomes. Mitochondria were swollen and the Zbl. Vet. Med., Reihe B, Bd. 28, Heft 7

cristae underwent degeneration. Virus particles could not be detected in mitochondria. At this early stage the brush border did not reveal any alteration.

As soon as viroplasmic zones appeared in the cytoplasm, the microvilli formed blebs and then gradually disappeared. Finally, degenerated enterocytes desquamated and appeared free in the intestinal lumen. Intranuclear or intracytoplasmic tubular formations could not be found. The colon did not show any ultrastructural alterations or evidence for rotaviral replication.

Comparison of histological intestinal lesions in EVD and TGE virus infected piglets did not reveal significant differences. Both virus infections lead to severe villous atrophy from proximal to distal jejunum and in the ileum, which is in contrast to the findings for rotavirus infection where villous atrophy was only found in the distal parts of the jejunum and in the ileum. The findings for TGE and rotavirus-induced intestinal lesions confirm earlier results obtained by others hours p. inf. These differences may be dependent on the virulence of the strain used or on the virus dose given. The lack of villous atrophy in naturally EVD virus-infected newborn piglets compared to weanlings which were investigated only a few hours after the onset of diarrhoea may have been due to milk antibodies which could have a delaying influence on the development of morphological lesions in the intestine.

Electron microscopical findings in EVD virus-infected intestinal cells revealing swollen mitochondria, degenerated cristae, reduced numbers of cell organelles such as RER and Golgi apparatus, dilatation of the terminal web and irregular microvillus borders resembled those for TGE virus (16) and other enteropathogenic coronaviruses (2, 3, 6, 19) . They are also consistent with the replication of coronaviruses in tissue culture cells (13) .

Dense filamentous structures as demonstrated in intestinal infections with bovine and canine coronaviruses (6,19) were, however, not found.

There were no apparent differences in the ultrastructural picture between naturally and experimentally infected piglets.

EVD virus-containing cells were mainly found on the lateral walls of the villi, leaving enterocytes on the tip of the villus uninfected. In some cases EVD virus-infected cells could also be demonstrated in or near Lieberkuhn's crypts.

Such observations were not made with any of the other enteric coronaviruses studied in vivo, including TGE virus (2, 6, 16) .

Whether these findings, which were also made using the fluorescent antibody assay (5, 8) are pathognomonic for EVD virus remains to be seen. If these results can be confirmed in future studies, the localization of virus replication could possibly be a property which could be used for differentiation of TGE virus from EVD virus in infected small intestines. In this respect rotavirus infection does not present difficulties because of its different morphology and mode of replication. The results obtained in this study on the infection of the small intestine with rotaviruses are well in accordance with data reported by others (10, 11, 17, 20) . The ultrastructural investigation of the lamina propria and regional lymph nodes revealed virus-containing cells in piglets experimentally infected with EVD virus. This suggests uptake of virus-containing cells by macrophages which are present in the lamina propria, and transport of the macro hages to the lymph nodes where virus degradation takes tact virus particles resemble lysosomes, or are similar to structures described in connection with the replication of a human coronavirus in cell cultures (13), cannot be ascertained. Because replication of enteropathogenic coronaviruses has not been reported in other cells than differentiated small intestine enterocytes, one would be hesitant to compare these structures to the membranebound viral factories reported by DOUGHRI et al. (6) in a study on intestinal bovine coronavirus infection, although their resemblance is striking. place (Fig. 7 a) . W K ether the granular inclusions in phagocytes containing in-

The authors thank ANGELA SIEBERT, ANNELIESE BEER and URSULA GOEP-FERT for their assistance. Summary Light microscopic and ultrahistological changes were investigated in the intestines of piglets naturally and experimentally infected with epizootic viral diarrhoea (EVD) virus and compared to those occuring during intestinal TGE and rotavirus infections in piglets. The course of the EVD virus infection is comparable to that of TGE. Villous atrophy develops throughout the jejunum and the ileum as in TGE. In this respect it differs from rotavirus infection. The ultrastructural picture of intestinal EVD virus infection resembles that of TGE and other enteropathogenic coronaviruses. Virus replication, however, was observed mainly in enterocytes located on the lateral walls of the villi extending to the villus/crypt border, and sometimes virus-infected cells were demonstrated in or near the Lieberkuhn's crypts. EVD virus was also demonstrated in macrophages in the intestinal lamina propria and in phagocytes in regional lymph nodes.

Zusammenf assung Lichtmikroskopische und ultrahistologische intestinale Verhderungen bei Ferkeln nach Infektion mit dem Epizootischen Diarrhoe-Virus (EVD):

Virus-und Schweinerotavirusinfektionen

Lichtmikroskopische und ultrahistologische Veranderungen von Darmen natiirlich und experimentell mit dem Virus der Epizootischen Virusdiarrhoe (EVD) infizierten Ferkeln wurden untersucht und mit denen nach Infektion mit TGE-Virus und Rotavirus verglichen. Der Verlauf der EVD-Virusinfektion ist vergleichbar mit dem der TGE. Eine Zottenatrophie tritt ahnlich wie Lei der TGE im gesamten Jejunum und Ileum auf. In dieser Beziehung unterscheiden sich die Befunde von der Rotavirusinfektion.

Die ultrastrukturellen Darmveranderungen bei der EVD-Virusinfektion entsprechen denen der TGE und anderen entero athogenen Coronaviren.

Virusvermehrung war jedoch hauptsachlich auf die P aterale Epithelschicht der Zotten begrenzt. Sie reichte bis in den Zotten/Krypten-Bereich. In einigen Fallen lieiSen sich infizierte Zellen in oder nahe den Lieberkuhnschen Krypten nachweisen. EVD-Viruspartikel wurden weiterhin in Makrophagen der Lamina propria und in Phagozyten der regionalen Lymphknoten gefunden. Resumen Fotomicroscopia e ultrahistologia de las modificaciones intestinales en lechones infectados con el virus de la diarrea virbica epizo6tica (DVE): Comparacibn con el virus de la gastroenteritis transmisible (GET) e infecciones con el virus rota porcino Se examinaron las modificaciones fotomicroscbpicas y ultrahistolbgicas en 10s intestinos de lechones infectados de forma natural y experimental con el virus de la diarrea vir6sica epizobtica (DVE), compardndose con las habidas tras la infeccibn con el virus GET y el virus rota porcino. El curso de la infeccibn con el virus de la DVE se puede comparar con el de la GET. La atrofia de villosidades aparece, tal y como en la GET, en la totalidad del yeyuno y del ileon. A este respecto se diferencian 10s hallazgos de la infeccibn con el virus rota.

Las modificaciones entkricas ultraestructurales en la infeccibn con el virus de la DVE corresponden a 10s de la GET y otros virus corona enteropatbgenos. Sin embargo, la multiplicacibn de virus se hallaba limitada sobre todo a la capa epitelial lateral de las villosidades. La misma alcanzaba hasta el imbito de las villosidades/criptas. En algunos casos se pudieron identificar cklulas infectadas en las criptas de Lieberkuhn o cerca de las mismas. Particulas virales DVE se hallaron ademds en 10s macrbfagos de la lamina propia y en 10s fagocitos de 10s ganglios regionales.

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