Coronavirus?

560 PEDIATRICS Vol. 74 No. 4 October1984

Letters

to

the Editor

Statements appearing here are those of the writers and do not represent the official position of the American Academy of Pediatrics, Inc., or its Committees. Comments on any topic, including the contents of PEDIATRICS, are invited from all members of the profession: those accepted for publication will not be subject to major editorialrevision, but generally must be no more than 400 words in length. Shorterletters willbe published earlier. The editors reserve the right to publish replies, and may solicit responses from authors and others.

Letters should be submitted in duplicate in double-spaced typing on plain white paper. Send them

to Jerold F. Lucey, M.D., Editor, Pediatrics Editorial Office, Mary Fletcher Hospital, Colchest#{233}rAvenue, Burlington, VT 05401.

Coronavirus?

To the

Editor.-We read with great interest the paper by Rousset et al’ in which the presence of coronavirus-like particles in intestinal lesions of neonates with necrotizing enterocoli-tis was described. The presence of viral particles in thin-sectioned material is often difficult to demonstrate be-cause of the striking similarity of some viral particles to normal cell organelles and structures. We agree with the authors that coronavirus-like particles may be an etio-logic agent in some cases of neonatal necrotizing entero-colitis of nonbacterial origin2’3; we do not agree, however, that their published electron micrographs unequivocally demonstrate viral particles. In fact, the purported viral particles look strikingly similar to normal structures seen in intestinal epithelial cells. For example, the intracyto-plasmic vesicle shown in their figure 3C is purported to contain virus-like particles. These vesicles are, in our opinion, indistinguishable from an R-body (rod-contain-ing body) which appears to be associated with the secretory activities of both normal and neoplastic intes-tinal epithelial cells. In their figure 4B, there are pur-ported viral particles lining up on the edge of damaged microvilli. In our opinion, these particles cannot be dis-tinguished from the glycocalyceal or coccoid bodies (C-bodies) found in abundance in normal colorectal

epithe-liumfr Glycocalyceal bodies have even been utilized as one of several ultrastructural markers useful in identify-ing adenocarcinomas as arising in colorectal

epithe-lium. The long microvillous core rootlets present in the

cells shown in figure 4B are most consistent with colonic epithelium.

The identification of specific viral particles in conjunc-tion with a clinical syndrome is invaluable to our under-standing of gastrointestinal illnesses. It is our opinion that more sophisticated techniques such as immune

elec-tron microscopy will be needed for the unequivocal iden-tification of pleomorphic viruses such as coronavirus-like particles in human intestinal epithelial cells.

REFERENCES

CLAIRE M. PAYNE, PHD

C. GEORGE RAY, MD

Departments of Pathology and Pediatrics University of Arizona

College of Medicine Tucson, AZ 85724

1. Rousset 5, Moscovici 0, Lebon P, et al: Intestinal lesions

containing coronavirus-like particles in neonatal necrotizing enterocolitis: An ultrastructural analysis. Pediatrics 1984; 73:218

2. Vaucher YE, Ray CG, Minnich LL, et al: Pleomorphic,

enveloped viral-like particles associated with gastrointes-tinal illness in neonates. J Infect Dis 1982;145:27

3. Payne CM, Ray CG, Borduin VF, et al: Electron microscopy

in diagnostic virology: A five year study of the etiologic agents of acute non-bacterial gastroenteritis, abstracted.

Proc EMSA 1982, p 354

4. Biempica L, Sternlieb I, Sohn HB, et al: R-bodies of human rectal epithelial cells. Arch Pat/wI Lab Med 1976;100:78 5. Stone J, Mukherjee TM, Hecker R: C bodies and R bodies

in the epithelial cells of normal and diseased human rectum.

Arch Pat/wI Lab Med 1977;101:437

6. Marcus PB: Glycocalyceal bodies and their role in tumor typing. J Submicrosc Cytol 1981;13:483

7. Hickey WF, Seiler MW: Ultrastructural markers of colonic adenocarcinoma. Cancer 1981;47:140

8. Dvorak AM, Monahan RA: Metastatic adenocarcinoma of unknown primary site: Diagnostic electron microscopy to determine the site of tumor origin. Arch Pathol Lab Med

1982;106:21

To the

Editor.-Rousset et al’ described particles within cytoplasmic vesicles in cells of intestinal mucosa and particles asso-ciated with the microvilli of these cells as coronavirus-like particles. Their conclusion is based on the

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Fig 1. Two membrane-bound cytoplasmic R-bodies

containing 40- to 90-nm membrane-bound particles seen

in epithelial cell of rectal mucosa of patient with bloody stools (x55,500).

LETTERS

TO THE

EDITOR

561

ogy of the particles in section, and the fact that these particles were found in the intestinal mucosa of several patients with necrotizing enterocolitis who were shown

by negative stain techniques to have coronavirus particles in their stools. Other authors2” have also proposed a viral nature for both types of particles.

However, in other previous studies of intestinal mu-cosa, these particles-particles within cytoplasmic vesi-des and particles associated with microvilli-have been

reported in both normal and diseased tissue.4 These

structures have been characterized in the literature as bodies4 and C-bodies,’ respectively, and more recently the latter as glycocalyceal bodies.7 The R-bodies have been associated with goblet cells and a secretory nature

has been proposed for them.4 The C-bodies, first

de-scribed by Shnitka8 in 1964, are felt by some to be the extruded particles of the R-bodies2’5 (viral or otherwise); others feel that they arise by budding from the microvilli.8 We have also seen in rectal mucosa both the “R”-bodies (Fig 1) and “C”-bodies (Fig 2) with evidence of budding from microvilli.

We are puzzled why Rousset and colleagues have not alluded to these reports in their paper, especially since some authors refute the viral nature of the two types of particles, or at least question the etiologic role of these particles. Additionally, we feel the strong inference made

by Rousset et al in their discussion that their coronavirus-like particles are indeed the tissue counterpart of the

Fig 2. Several 35- to 55-nm membrane-bound particles (C-bodies) in glycocalyx of luminal surface of rectal epi-thelial cell of same patient shown in Fig 1. A few particles are seen (arrows) budding from microvilli (xllO,000).

coronavirus particles found in the stools of these patients and thus the causative agent of the necrotizing entero-colitis is not substantiated by the evidence presented. Other proofs are required, such as growth of the virus in tissue or organ culture,’ or demonstration of coronavirus

antigen by immunofluorescence9 or immunoelectron

mi-croscopy.’#{176}

The presence of glycocalyceal particles throughout the gastrointestinal tract, as high as the lower esophagus, and the finding of similar particles in a variety of tumors (intestinal, bronchiolar, ovarian, etc)7 strongly suggest that they are not viral in nature. From our material, we would like to suggest the possibility that the particles in

cytoplasmic bags represent phagocytosed glycocalyceal particles as there is no evidence of their assembly in the cytoplasm and their appearance is similar.

EDUARDO J. YuNIs, MD Rocco M. AGOSTINI, JR, BS Department of Pathology

Children’s Hospital of Pittsburgh and University of Pittsburgh School of Medicine 125 DeSoto St

Pittsburgh, PA 15213

REFERENCES

1. Rousset 5, Moscovici 0, Lebon P, et al: Intestinal lesions containing coronavirus-like particles in neonatal necrotizing

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562

PEDIATRICS

Vol. 74 No. 4 October1984

enterocolitis: An ultrastructural analysis. Pediatrics 1984;

73:218

2. Steer HW: The pseudomembranous colitis associated with clindamycm therapy-A viral colitis. Gut 1975;16:695 3. Caul EO, Clarke SKR: Coronavirus propagated from patient

with nonbacterial gastroenteritis. Lancet 1975;2:953

4. Biempica L, Sternlieb I, Sohn HB, et al: R-Bodies of human rectal epithelial cells. Arch Pathol Lab Med 1976;100:78

5. Stone J, Mukherjee TM, Hecker R: C Bodies and R bodies in the epithelial cells of normal and diseased human rectum.

Arch Pat/wI Lab Med 1977;101:437

6. Gonzalez-Licea A, Yardley JH: A comparative ultrastruc-tural study of the mucosa in idiopathic ulcerative colitis, shigellosis and other human colonic diseases. Bull Hopkins

Hospital 1966;118:444

7. Ghadially FN: Ultrastructural Pathology of the Cell and

Matrix, ed 2. London, Butterworths, 1982, pp 782-785 8. Shnitka TK: Current concepts of the pathogenesis and

pathology of inflammatory lesions in the intestine. Can Med

Assoc J 1964;91:7

9. Keenan KP, Jervis HR, Marchwicki RH, et al: Intestinal infection in neonatal dogs with canine coronavirus 1-71:

Studies by virologic, histologic, histochemical and immu-nofluorescent techniques. Am J Vet Res 1976;37:247 10. Oshiro LS, Schieble JH, Lennette EH: Electron microscopic

studies of coronavirus. J Gen Virol 1971;12:161

In

Reply.-In both of our papers,”2 we referred to coronavirus-associated disease and termed the particles “coronavirus-like.” In order to make our position even less equivocal, we stated in our first paper (pages 213-214)’: “At present, because of the lack of viral propagation in tissue culture, the virions found in the stools are only identified on the basis oftheir morphologic appearance (see Figure). These differences could either represent various aspects of one virus, or different viruses presently classified as corona-viruses. Their final identification must await further developments in genetic structure and RNA sequencing.”

We, therefore, agree with our colleagues that, based on morphology, the definition of a virus is far from complete and we never stated that the etiologic role was demon-strated. The only definition satisfactory for viruses is the structure oftheir RNA (or DNA). Thus, immune electron microscopy without the identification of the antigen in-volved is also unsatisfactory.

However, our epidemiologic studies performed in a previously unexposed population (which was followed up for 4 years by electron microscopy negative staining) enabled us to associate neonatal necrotizing enterocolitis with the presence of coronavirus-like particles in stool specimens. This was possible because of the clustered appearance of the disease in a limited population. If occurring in endemic areas, such an identification would be impossible.

Furthermore, it is important to stress that the hypoth-esis we propose is based not only on the morphology of

the particles, but also on the immune responses they

elicit. Indeed, in repeated stool specimens of the diseased patients, first immunoglobulin (IG) A type antibodies aggregate the particles, then circulating antibodies

ap-pear, as identified by immune electron microscopy. If

these particles were of secretory or glycocalyceal origin (considered as normal tissue constituents), such immune responses in diseased but not in control populations would be rather surprising.

Probably, because of the important enzymatic

activi-ties that occur in the digestive track, these particles are

polymorphic and not easy to identify in situ. We agree that one or another could be artificial, but at the present state of our knowledge, we prefer to show them without

further comment. Anyhow, the particles found in the

lesions are reminiscent of similar particles seen during animal epidemics in which comparable diseases could be identified as being associated with, or even induced by,

coronaviruses. They have also been reproduced in

gno-tobiotic piglets and calves, which brings us closer to the fulfillment of Koch’s postulate.

We are grateful to the authors for their commentaries, drawing our attention to the particles called R-bodies3 and C-bodies.4 The C-bodies are apparently somewhat smaller than those we reported, whereas the R-bodies are more comparable but not further identified.

In our report, the particles are detected in the necrotic

areas and as far as one can judge from the electron

micrographs, those reported by Stone et a14 and shown in their figure 5, could be altered too.

Some authors question or refute the possible viral

origin of the R-bodies and C-bodies because of their

occurrence in apparently normal subjects. We feel,

how-ever, that on this basis such a possibility cannot be

excluded, knowing that the overwhelming majority of

viral infections are luckily clinically latent.

We all must await further identification of some of these particles in order to label them unequivocally. This

does not, however, decrease the attractiveness of the

hypothesis associating coronavirus-like agents with nec-rotizing enterocolitis.

REFERENCES

SOLANGE ROUSSET, PHD

CHARLES CHANY, MD PIERRE LEBON, MD

Orro MoScovlcl Laboratory of Virology

INSERM Unite 43

St Vincent-de-Paul Hospital

74, Avenue Denfert-Rochereau 75674 Paris cedex 14, France

1. Chany C, Moscovici 0, Lebon P, et al: Association of coron-avirus infection with neonatal necrotizing enterocolitis.

Pe-diatrics 1982;69:209

2. Rousset 5, Moscovici 0, Lebon P, et al: Intestinal lesions containing coronavirus-like particles in neonatal necrotizing

enterocolitis: An ultrastructural analysis. Pediatrics

1984;73:218

3. Biempica L, Sternlieb I, Sohn HB, et al: R-Bodies of human rectal epithelial cells. Arch Pat/wI Lab Med 1976;100:78 4. Stone J, Mukherjee TM, Hecker R: C Bodies and R bodies

in the epithelial cells of normal and diseased human rectum.

Arch Pat/wI Lab Med 1977;101:437

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1984;74;560

Pediatrics

EDUARDO J. YUNIS and ROCCO M. AGOSTINI, JR

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