key: cord-0039233-l1j6bmws
authors: Jensen, J.L.; Cliver, D.O.
title: Enterovirus replication in ileal explants
date: 2006-11-07
journal: Ann Inst Pasteur Virol
DOI: 10.1016/s0769-2617(84)80050-7
sha: 1c230465786539b455654c5d9f963f1db8d3ac02
doc_id: 39233
cord_uid: l1j6bmws

Porcine enterovirus 3 replicated in explants of fœtal pig intestine, as was shown using photosensitization with neutral red to distinguish between inoculum and progeny virus. Progeny virus titres reached 10(5)–10(6)/ml with 2–4 days' incubation.

Little has been learned of how viruses initiate infections in cells of the host organism because the target cells are not readily accessible to study in vivo (2) Author to whom reprint requests should be addressed.

.Ann. Virol. (Inst. Pasteur), 135 E, n ~ 2, 1984. 10 and tend to lose their differential properties when separated, for culture, from the tissue milieu. Receptor specificities, for example, are sometimes modified in this way, such that cells cultured in vitro in monolayers will support replication of viruses to which they are not susceptible in vivo. We wanted a system with which to study the early interactions of enteroviruses with cells of the ileum, which appears to be the site at which most enterovirus infections are initiated in vivo [1] . Explant cultures, in which the component cells are retained in their normal relationships with each other in the tissue, were examined for this purpose. The use of explant cultures for the detection and study of viruses has been reported in recent years [2-5, 7-9, 11] . Since a close analogy exists between the porcine and human digestive tracts and their respective enteroviruses, use of porcine intestinal explants with porcine enteroviruses provides an animal model system relevant to enterovirus infection of human intestines. This report describes replication of porcine enterovirus 3 (PE3) in an explant system from foetal pig intestine, using photosensitization with neutral red (NR) to distinguish between inoculum and progeny virus [10] ,

Mini-pig kidney (MPK) cells, a continuous line obtained from the American Type Culture Collection, were maintained in Eagle's minimal essential medium with non-essential amino acids, plus 20% foetal calf serum (FCS) and 100 U/ml of penicillin G sodium and 100 ag/ml of dihydrostreptomycin sulphate, buffered to pH 6.8 with NaHCO~. Monolayers were grown in 25-cm ~ polystyrene flasks (Falcon) at 37 ~ C.

PE3, strain ECPO-6, was obtained from Dr E. H. Bohl, Ohio Agricultural Research and Development Station, Wooster, OH. Virus titre was determined by cytopathic effects (CPE) produced in MPK. Virus was diluted serially in Dulbecco's phosphate-buffered saline containing 2~o FCS and antibiotics as in the cell culture medium; and duplicate cultures were inoculated with 0.5 ml of each dilution, incubated at 37 ~ C for 7 days, and scored for CPE. The titres were calculated as most probable numbers of cytopathic units (MPNCU) by the formula of Thomas [6] , and expressed as the loglo of this number, to two significant digits.

Virus was sensitized with NR by a modification of procedures described for poliovirus [10] in the dark. PE3 stock (5 • 105 plaque-forming units in 0.5 ml) was inoculated into MPK cultures that had been incubated for 55 h at 37 ~ C with medium containing 5 ag/ml NR. The monolayers were rocked for 60 min in the dark, the inoculum was decanted, the monolayers were rinsed twice with medium containing NR, fresh medium containing NR was added, and the monolayers were incubated for 72 h at 37 ~ C in the dark. At 72 h, one of the cultures was examined and showed an advanced stage of CPE. The remaining cultures were frozen at --20 ~ C and thawed at room temperature three times, and stored in the dark at 4 ~ C. The pooled suspension (titre~l • was diluted 100-fold in NR medium and passed a second time in the presence of NR. Virus from the second passage was stored in the dark at 4 ~ C.

Two samples of this PE3 stock were diluted 100-fold into 0.03 M phosphate buffer, pH 7.1. One sample was placed in a test tube 15 cm from two 15-watt fluorescent bulbs and light-shocked for 60 rain at room temperature. The titres of the samples were 1 • 109/ml without and 1 X 104/ml with light-shocking, a 1 • 105fold difference.

Explants were prepared from portions of lower ileum excised aseptically from foetal pigs 22-30 cm in length within 50 min after death. Pieces 2.5-5 cm in length were immediately placed into Petri dishes containing chilled Hanks' balanced salt solution (HBSS) and held on ice. The intestinal segments were opened longitudinally and rinsed gently with HBSS to remove meconium debris. Segments approximately 2-3 mm square were cut with scalpels, then gently placed with forceps, mucosal side up, onto prescored areas in 60 • 15-mm Petri dishes that had been incubated overnight in a moist chamber at 37 ~ C. Five explants per dish were placed at positions roughly equidistant from each other. In each experiment, an equal number of control dishes, which had been incubated overnight in the moist chamber but into which no explants were placed, was also used. Immediately after placement of the explants, all dishes were removed to the dark and further manipulations done under a red safety lamp.

The photosensitized virus stock was diluted 100-or 1,000-fold, and 0.1 ml was dripped onto each explant; 0.5 ml of the Same suspension was inoculated into each control dish. All dishes were incubated in the moist chamber in the dark for 3 h at 370 C. Then 1.3 ml of fresh medium was added to each dish (total volume, 1.8 ml per dish), and the dishes were placed into the moist chamber and il~cubated in the dark at 37 ~ C in 5% CO~ in air. Both explant cultures and control ~ishes received Eagle's medium with 0.2% bovine serum albumin and antibiotics as in the cell culture medium, pH 6.8-7.0. At 2, 4 and 6 days, all of the medium (1.8 ml) was harvested from each dish, and 1.8 ml of fresh medium was added. In a typical experiment, three dishes of explants and three control dishes were prepared. Harvested medium from the three explant dishes was pooled, as was that from the three control dishes, on each collection day. All harvested medium was stored at 4o C in the dark. The titres of light-resistant virus inoculated on day 0 were determined by adding 0.5 ml of inoculum to 1.3 ml of medium and holding this mixture at 4 ~ C in the dark until light-shocking (experiments A and E) or by harvesting the inoculum, with medium, from an extra set of plates with and without explants at the end of the 3-h initial adsorption period (experiments B, C and D). Day 0 samples and medium harvested from experimental and control dishes were diluted 100-fold in phosphate buffer (pH 7.1), light-shocked, and assayed.

Explants were also prepared for microscopic observation. In a typical experiment, six dishes of explants were prepared, only three of which were inoculated with virus. All dishes were incubated as described previously. On days 2, 4 and 6, one inoculated and one uninoculated dish were removed and observed under a dissecting microscope for explant attachment and villous structure.

Replication of PE3 in explants was demonstrated in five experiments ( fig. 1 ). The titres of the light-resistant virus peaked on day 2 or 4 with maximal yields of 1 • 10 s to 1 • 10 ~ MPNCU/ml and average yields of 5 X 104 to 1 • 10 s MPNCU/ml. Attachment of explants to scored areas of the petri dishes remained good (60-100%) for 4 days and then declined; similarly, villous structure appeared typical for 4 days, but by 6 days there was shortening and thinning of villi and some buildup of mucus on the surface of the explants. Differences between virus-inoculated and uninoculated explants were not perceptible. The open portion of each bar shows the titre of the virus incubated with ileal explants, and the filled portion of the bar shows the titre of the same quantity of virus incubated in dishes without tissue (controls). Where only a filled bar appears, only a suspension without tissue was tested (experiments A and E) or the titres of the two suspensions were the same (experiment C). Arrowheads pointing downward indicate that the control suspension gave a negative test result at the lower limit of virus detection. The photosensitized virus suspension had been diluted 100-fold initially in experiments A, B, and C, and 1,000-fold initially in experiments D and E.

It is clear that substantial replication of the virus took place in these explant cultures, for the yields of light-resistant virus were considerable and were slightly higher than expected on the basis of in vivo data. Perorally infected 4-week-old pigs shed PE3 virus at levels of 1-4 x l0 s PFU/g of stool, with an average daily faecal output of 25 g [unpublished data]. The ileal tissue present in one dish of 5 explants is estimated to be 0.13-0.15% of the total quantity of ileal tissue in a 4-week old pig, so we calculated that one dish should yield 1-2 • 10 ~ MPNCU/ml in 2 days.

Replication of PE3 virus proceeded to substantial titres in the ileal explants without perceptible destruction of the epithelium, which is probably to be expected of an enterovirus which, in vivo, replicates in the intestine without causing overt signs of gastroenteritis. The replication of the virus to peak titres within 2-4 days is also expected in view of the short replicative cycle of this virus (12 h at 37 ~ C).

Intestinal organ cultures have been used by other investigators to study virus replication. Coronaviruses have been shown to replicate in organ cultures of pig and bovine intestine [2, 9] . Echovirus 11, a human enterovirus that causes gastroenteritis, has been studied using human foetal intestinal explants [5, 8, 11] . Derbyshire and Collins [3] studied Talfan virus, a porcine enterovirus that does not cause enteritis, and found no substantial virus replication in explants of small intestine until 6-8 days after inoculation; more importantly, virus replication could not be demonstrated consistently and appeared to take place after structural degeneration of the epithelium (which occurred within 48 h), presumably in the lamina propria. Bubenstein and Tyrrell [8] reported replication of poliovirus 1 to 1 •215 10 ~ PFU/ml in explants of human foetal ileum after 6 days' incubation; however, the virus continued to grow to maximal titres in the explants after the original intestinal epithelium disappeared. Wyatt [11] reported replication of poliovirus type 1 in human foetal small intestinal explants, with peak titres of more than 1 • 106 PFU/ml after 6 days; no distinction was made in this study between explants of the ileum and other segments of small intestine, and, surprisingly, virus replication was associated with destruction of the epithelium.

In this study, we have used photosensitized virus to distinguish input virus from progeny virus. Other methods for verifying virus replication, such as net virus output, immunofluorescence [5] and electron microscopy [111, do not allow confirmation of enterovirus replication until 6-8 days after infection, i. e. until substantial virus replication has occurred. The use of photosensitized virus allows detection of even low levels of early virus replication (within 48 h) in explant cultures.

Porcine intestinal explants offer a method of studying cell-enterovirus interactions using an animal system (pig) which is closely analogous to the human gastrointestinal system. They offer an advantage over other animal systems and a source of data relevant to human systems without the problems of obtaining human tissue. The use of photosensitized virus allows detection of low levels of early virus replication (within 48 h) in explants and will be valuable in quantitative studies of the initial events in viral infection and replication. Explant culture systems will also allow identification of enterovirus receptor sites in the ileum and study of the early interactions of enteroviruses with receptors on susceptible target cells.

The present explant culture system would not permit maintenance ot cultures for long enough periods of time to model the entire course of an enteric viral infection, but it does seem to preserve the structure and function of the explants sufficiently during the first 4 days to allow studies of how intestinal infections are established and for substantial viral replication to proceed. In particular, this technique might permit comparison between various segments of the intestine as to their ability to serve as the initial site of infection; this is a question which has been much discussed but not totally resolved. Such cultures may also serve eventually to explain the apparent inefficiency with which enteroviruses initiate infection in vivo.

Une technique de photosensibilisation h l'aide de rouge neutre pour distinguer les virus de l'inoculum des virus n~oform~s, a permis de montrer qu'un ent6rovirus porcin de type 3 se r~plique dans des explants intestinaux de f~etus porcin. Les titres des virus n~oform~s atteignent 105 ~ 106/ml aprbs 2/~ 4 jours d'incubation.

MOTS-CLt~S : Entfirovirus, Ilfion, R~plieation ; Explant, Foetus de pore.

Mechanisms of infection with polioviruses, in (( Cellular biology

Replication of an enteric bovine coronavirus in intestinal organ cultures

The multiplication of Talfan virus in pig intestinal organ cultures

Establishment of human fetal intestinal organ cultures for growth of viruses

Detection and localization of viruses in human fetal intestinal organ cultures by immunofluorescence

Estimation of bacterial density, in (( Standard methods for the examination of water and wastewater )~

Immunofluorescent and electron microscopic studies of feline small intestinal organ cultures infected with feline infectious peritonitis virus

Growth of viruses in organ cultures of intestine

Growth of porcine transmissible gastroenteritis (TGE) virus in organ cultures of pig tissue

The effect of light on poliovirus grown in neutral red

Ultrastructural study of viral replication in human fetal intestinal organ culture. Arch. ges. Virus/orsch