Fatal Gastroenteritis AssociatedWith Coronaviruslike ParticlesPhilip J. Rettig, MD, Geoffrey P. Altshuler, MBBS

\s=b\The role of human enteric coro-naviruses in infantile gastroenteritis iscontroversial. We detected coronavirus\x=req-\like particles in the intestinal contentsand within the epithelial cells of the il-eum in a 15-month-old infant who hadpostmortem evidence of severe enter-itis. Ultrastructural findings consistentwith in vivo coronavirus replication inthe human small intestine support a

causative role for this agent in gastroen-teritis.

(AJDC 1985;139:245-248)

Touring the past decade, substantialadvances in electron microscopic

and immunologie techniques havegreatly increased our knowledge ofviral agents of acute gastroenteritis.1Rotaviruses are now known to be thesingle most common cause of diarrheain infants and young children, and theNorwalk-agent and related viruses are

recognized as important causes of epi¬demic gastroenteritis in school-agedchildren and young adults. Other non-

cultivable, putative causes of enteritis,such as caliciviruses and enteric ade-noviruses, have been seen in feces of illsubjects by use of the electron micro¬scope. We recently found coronavirus-like particles (CVLPs) in the fecalfluid and small intestine of an infantwho died of complications of gastroen¬teritis. Ultrastructural findings dem¬onstrated evidence of in vivo replica¬tion of this agent within human intes¬tinal epithelium.

From the Departments of Pediatrics (Dr Ret-tig) and Pathology (Dr Altshuler), College ofMedicine, University of Oklahoma Health Sci-ences Center; and Oklahoma Children's MemorialHospital (Drs Rettig and Altshuler), OklahomaCity.

Reprint requests to Division of Infectious Dis-eases, Department of Pediatrics, Oklahoma Chil-dren's Memorial Hospital, PO Box 26307, Okla-homa City, OK 73126 (Dr Rettig).

REPORT OF A CASE

A 15-month-old male infant was admittedto Oklahoma Children's Memorial Hospital,Oklahoma City, in March 1982 in a postictalcomatose state. He had been in good healthuntil one week earlier when rhinorrhea haddeveloped. Two days prior to admission, hemanifested a nonproductive cough, in¬creased rhinorrhea, and a temperature of40.6 °C. The family physician diagnosed an

upper respiratory tract infection and pre¬scribed ampicillin sodium, diphenhy-dramine hydrochloride, theophylline, andaspirin. There were no siblings, and neitherparent had been ill. The child had no knownexposure to other ill persons or animals.

On the evening of admission, the patientappeared well when put to bed. Severalhours later, he was found to be cyanotic andgasping, with vomitus on the bedcovers. A40-minute generalized grand mal seizurewas successfully treated in the emergencyroom with intravenous diazepam and phé¬nobarbital sodium. The patient had a

temperature of 41.1 °C, systolic BP of92 mm Hg, and pulse rate of 120 beats perminute. He was unresponsive and hadfixed, dilated pupils. He did not appearsubstantially dehydrated. There was no

evidence of upper or lower respiratorytract infection. The abdomen was flat, withactive bowel sounds. There were no focalneurologic signs. Initial laboratory dataincluded a normal complete blood cellcount, a serum urea nitrogen level of27 mg/dL, and a serum creatinine level of1.8 mg/dL. Serum electrolyte levels were as

follows: sodium, 138 mEq/L; potassium,4.2 mEq/L; chloride, 107 mEq/L; and Co2,10 mEq/L. Initial arterial blood gas mea¬

surements showed a pH of 7.13 with a Pco2of 27 mm Hg and a normal Po2. Chestroentgenograms, computed tomographicbrain scan, lumbar puncture, and tox¬icologie studies disclosed no abnormalities.

The patient was admitted to the intensivecare unit with a diagnosis of postictalanoxic brain damage, possibly secondary toaspiration of gastric contents. He was

treated with ventilatory support, with phé¬nobarbital for seizure control, and with

chloramphenicol and ampicillin for sus¬

pected bacterial sepsis. Several hours afteradmission, the patient passed two large,soft, semiformed stools. There was no re¬turn of neurologic responsiveness, and ap¬proximately eight hours after admissionthe patient died after an episode of hypo¬tension and bradycardia. Shortly before hisdeath, the serum urea nitrogen level hadrisen to 58 mg/dL and the creatinine level to2.2 mg/dL.

RESULTSPostmortem Examination

An autopsy was performed withinthree hours of death. Very dry sub¬cutaneous tissue indicated severe de¬hydration. Green gastric fluid was

present in the trachea and major bron¬chi. The spleen showed multiple areasof hemorrhagic infarction under thecapsule and throughout the paren¬chyma. There were large volumes ofintraluminal fluid in both small andlarge bowel but no other noteworthygross gastrointestinal (GI) tractchanges. Examination of the CNS dis¬closed brain-stem herniation associ¬ated with severely swollen gyri andnarrow sulci.

Abnormal microscopic findings were

confined to the lungs, the spleen, andthe small intestine and related lymph-oid tissue. Proteinaceous material was

seen within the bronchi and alveolarducts. Both lungs showed diffuse in¬terstitial infiltrates of lympho-histiocytic cells and lymphocytes. Mul-tinucleated giant cells or viralinclusions were not observed.Sinusoidal fibrin thrombi and patchesof hemorrhagic necrosis were presentin the spleen. There was blunting andloss of microvilli in the small intestine,with severe infiltration of lymphocytesand lymphohistiocytic cells through¬out the lamina propria (Fig 1). Nocolitis was microscopically evident.

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Fig 1.—Small intestine. Note blunted architecture of mucosal surface and severe lympho¬cytic infiltration throughout lamina propria (hematoxylin-eosin, original magnification 200).

Comprehensive light microscopic ex¬amination of the CNS and other organsshowed no lesions.

Microbiologie FindingsPremortem bacterial cultures of

blood, CSF, and stool showed no patho¬gens. Stool cultures were processed byroutine methods for bacterial entericpathogens, including Campylobacterjejuni, Yersinia enterocolitica, and

Clostridium difficile. Postmortemcultures of blood, lung, and intestinalcontents yielded no bacterial patho¬gens. Stool contents were negative forrotavirus antigen by enzyme immu¬noassay. Autopsy samples of stool, in¬testine, liver, and myocardium werecultured for viruses in primary mon¬

key kidney and in a human fibroblastline (MRC-5 cells). No viruses wereisolated.

Electron MicroscopyFecal fluid and sections of small in¬

testine and lung were examined byconventional electron microscopy. Thefecal fluid contained numerous pleo-morphic, enveloped structures withdiameters ranging from 66 to 140 nm.These particles had a peripheralfringelike rim composed of thin projec¬tions with bulbous tips. These projec¬tions measured 15 to 20 nm in length(Fig 2, left). Ultrastructural examina¬tion of the small intestine showedmany double-enveloped particles 50 to60 nm in diameter at the apex of epi¬thelial cells. Some of these particleswere within cytoplasmic vesicles, lo¬cated adjacent to the microvilli of epi¬thelial cells. New daughter particlesappeared to be budding off withinthese vesicles (Fig 2, right). Thesevirions had an envelope 7 nm thick.Electron microscopic examination oflung sections disclosed no viruslikeparticles or inclusions.

COMMENT

The particles in our patient's in¬testinal fluid are typical of CVLPs24previously described in the stools ofhuman beings with gastroenteritis.The size, pleomorphism, presence ofan envelope, and demonstration of a"crownlike" fringe surrounding thevirion all support this morphologicdiagnosis. Of greater interest is ourdemonstration of smaller, nonfringedviruslike particles within epithelialcells of the distal part of the smallbowel. The location, size, shape, andarrangement of these particles withina cytoplasmic vacuole are identical tothe published ultrastructural featuresof canine coronavirus enteritis,5 of en¬

teritis in infant mice caused by mouse

hepatitis virus (also a coronavirus),6and of enteritis in newborn calvescaused by neonatal calf diarrhea coro¬

navirus.7 Similar intracellular vi¬ruslike particles have been seen on

ultrastructural examination of twoother coronaviruses cultivated invitro.8'9 Both a human respiratorycoronavirus, cultivated in human fetaldiploid lung cells,8 and avian infectiousbronchitis virus, cultivated in chorio-allantoic membrane,9 produce double-enveloped particles, 80 to 100 nm in

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Fig 2.—Left, Fecal coronavirus. Labels indicate size range of round and pleomorphic forms. Severalvirions display typical solar-corona projections (phosphotungstic acid, original magnification 150,000). Right, Intestinal mucosa. Budding virions are present within this cytoplasmic vesicle(uranyl acetate-lead citrate, original magnification 60,000).

diameter, that appear to be buddingwithin cytoplasmic vacuoles. Finally,Rousset et al10 published electron mi¬crographs of coronaviruslike particlesin the small intestines of human neo¬

nates with necrotizing enterocolitis.These virions had an average diameterof 70 nm (range, 60 to 125 nm) and adouble envelope and were found withinapical cytoplasmic vesicles. These par¬ticles appear identical in size and loca¬tion to those found in the small bowel ofour patient.

Coronaviruses are enveloped, pleo¬morphic RNA viruses with character¬istic surface projections; they are a

common cause of upper respiratorytract infections, particularly the com¬

mon cold, in human beings. Severalhuman respiratory tract strains havebeen successfully cultivated in tra¬chéal organ culture and adapted togrowth in cell culture. Animal strainscausing enteritis in infant dogs, calves,mice, pigs, and foals have been de¬scribed,4 with ultrastructural findingsas noted earlier.5"7

Definition of the role of human en¬

teric coronaviruses in human diseasehas been problematic. Although out¬breaks of enteritis associated withCVLPs detected by electron micro¬scopic examination of feces have beenreported,1·4·1112 such particles have alsofrequently been observed in the stoolsof subjects without acute diarrhea. In

support of a pathogenetic role forCVLPs in enteritis of human infants,several investigators have recently re¬

ported microscopic agglutination ofthese particles by convalescent serum

samples from previously infected in¬fants.1112 Lack of reproducible and pro¬ductive methods for cultivation ofhuman enteric coronaviruses has ham¬pered the study of their role in infan¬tile gastroenteritis. Caul and Clarke13accomplished limited cultivation of a

single isolate in human embryo in¬testinal organ culture and demon¬strated intracytoplasmic antigen byimmunofluorescence in this systemand in primary human embryonickidney. A putative isolate from theParis outbreak of necrotizing entero¬colitis was cultured in HRT 18 cells (awell-differentiated human rectal ade-nocarcinoma cell line).14 All other at¬tempts to grow CVLPs have been un¬

successful.4The ultrastructural findings in our

patient, particularly those suggestingin vivo replication of CVLPs in in¬testinal epithelium, support an entericcoronavirus being the cause of our

patient's intestinal disease. The in-tracellular location of the CVLPs sug¬gests that the virions in the intestinallumen were produced by local entericmultiplication, rather than merelyrepresenting ingested viruses fromthe respiratory tract. Comprehensive

histopathologic and microbiologie ex¬aminations failed to demonstrate anyother viral or bacterial pathogens inthis patient.

An important lesson from this caseis the occurrence of pathologically se¬

vere enteritis and fluid loss in a patientwho had not experienced remarkableGI tract symptoms. Fatal infection at¬tributed to human enteric corona¬viruses has been reported previouslyin young infants, including cases offatal necrotizing enterocolitis12 and fa¬tal enteritis in neonates.11 In the latteroutbreak, several infants with fataloutcomes appeared to have sepsisrather than a primary GI tract proc¬ess. Fulminant fatal viral gastroen¬teritis, complicated in some cases byaspiration of vomitus or seizures, hasbeen well described in young, previ¬ously healthy children infected withrotavirus.15 The possibility of severe

intraluminal fluid loss or pulmonaryaspiration in the patient without ante¬cedent GI tract symptoms must beconsidered in the young child whoseclinical appearance resembles that ofour patient. Investigation of the causeof such an illness should include theapplication of electron microscopy tosearch for novel noncultivable entericviruses. Our findings in this case sup¬port the role of enteric coronavirusesas one potential cause of such fulmi¬nant illness.

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References1. Blacklow NR, Cukor G: Viral gastroen-

teritis. N Engl J Med 1981;304:397-406.2. Caul EO, Paver WK, Clarke SKR: Coro-

navirus particles in feces from patients withgastroenteritis. Lancet 1975;1:1192.

3. Caul EO, Ashley CR, Egglestone SI: Recog-nition of human enteric coronaviruses by electronmicroscopy. Med Lab Sci 1977;34:259-263.

4. Macnaughton MR, Davies HA: Human en-teric coronaviruses. Arch Virol 1981;70:301-313.

5. Takeuchi A, Binn LN, Jervis HR, et al:Electron microscope study of experimental en-teric infection in neonatal dogs with a caninecoronavirus. Lab Invest 1976;34:539-549.

6. Hierholzer JC, Broderson JR, Murphy FA:New strain of mouse hepatitis virus as the causeof lethal enteritis in infant mice. Infect Immun1979;24:508-522.

7. Doughri AM, Storz J, Hazer I, et al: Mor-phology and morphogenesis of a coronavirus in-fecting intestinal epithelial cells of newborncalves. Exp Mol Pathol 1976;25:355-370.

8. Oshiro LS, Schieble JH, Lennette EH:Electron microscopic studies of coronavirus. JGen Virol 1971;12:161-168.

9. Becker WB, McIntosh K, Dees JH, et al:Morphogenesis of avian infectious bronchitisvirus and a related human virus (strain 229E).J Virol 1967;1:1019-1027.

10. Rousset S, Moscovici 0, Lebon P, et al:Intestinal lesions containing coronavirus-likeparticles in neonatal necrotizing enterocolitis: Anultrastructural analysis. Pediatrics 1984;73:218-224.

11. Vaucher YE, Ray CG, Minnich LL, et al:Pleomorphic, enveloped, virus-like particles as-

sociated with gastrointestinal illness in neonates.J Infect Dis 1982;145:27-36.

12. Chany C, Moscovici 0, Lebon P, et al:Association of coronavirus infection with neo-natal necrotizing enterocolitis. Pediatrics 1982;69:209-214.

13. Caul EO, Clarke SKR: Coronavirus propa-gated from patient with nonbacterial gastroen-teritis. Lancet 1975;2:953-954.

14. Laporte J, Bobulesco P: Growth of humanand canine enteritic coronaviruses in a highlysusceptible cell line: HRT 18. Prospect Virol1981;11:189-193.

15. Carlson JAK, Middleton PJ, SzymanskiMT, et al: Fatal rotavirus gastroenteritis: Ananalysis of 21 cases. AJDC 1978;132:477-479.

International Review of Child Neurology Series, vol 2: Brain Tumorsin Children: Principles of Diagnosis and Treatment, edited by Michael E.Cohen and Patricia K. Duffner; vol 3: Progressive Spinal MuscularAtrophies, edited by Ingrid Gamstorp and Harvey B. Sarnat, New York,Raven Press, 1984.

Raven Press, a leader in publishing good neurology texts,has done it again. These two monographs, the second andthird volumes of the International Review of Child Neu-rology Series, are excellent and timely reviews of twoneurological disorders not recently reviewed elsewhere.The volumes are sponsored by the International ChildNeurological Association, which is devoted to providing anoutlet for the exchange of professional ideas and to advanc-ing neurological sciences in infancy and childhood. Theseofficial publications of the association have definitelyachieved that goal. The authorship is broad, representing a

superb field of child neurology in the United States andthroughout the world.

Brain Tumors in Children includes sections on principlesof diagnosis and treatment, management, and complica-tions. In this single volume new concepts of epidemiology,neuroradiologic diagnosis, surgical excision, radiation ther-apy, chemotherapy, and the long-term effects of radiationand chemotherapy are discussed. Case examples and algo-rithms are used appropriately to illustrate current ap-proaches in diagnosis and management. Controversies,such as total removal of craniopharyngiomas and therapy ofoptic nerve gliomas, are discussed freely. The editors,leaders in efforts to establish a Childhood Brain TumorRegistry in the United States, note sadly that only 50% ofchildren with brain tumors reach university or cancertreatment centers, making trials of new therapy difficult.For this reason, they urge that children with brain tumorsbe referred to university cancer centers affiliated with largecooperative study groups. The results of current brain

tumor therapy certainly justify their recommendation.Drs Gamstorp and Sarnat note in the preface to Progres¬

sive Spinal Muscular Atrophies that they hope their bookwill be regarded as "of historical interest only in the future,because the etiology of this terrible affliction of children andits treatment remain obscure." Despite this caveat, theyhave edited an excellent "state of the art" monograph on thehistorical aspects, genetics, clinical features, pathology,electrophysiologic abnormalities, rehabilitation, and futureresearch strategies for "Lou Gehrig's disease" in children.Dr Gamstorp's scholarly chapters reflect her wealth ofclinical experience, combined with a sensitive and scholarlyapproach to diagnosis and management. The use of pulseecho ultrasound imaging and computed tomography scan¬

ning are reviewed as potentially convenient, noninvasivetools for diagnosing these disorders. Chapters on rehabili¬tation and psychological counseling will be of interest topracticing pediatricians who will be involved in followingup these children. The value of parent groups, rarely dis¬cussed in neurological texts, is also reviewed. Dr Sarnat'sconcluding chapter on research strategies highlights thestudies of fetal motor neuroblasts, the "dying-back" phe¬nomena of retrograde axonal degeneration, and the trans¬plantation of healthy motor neurons.

For excellent state of the art reviews on these grimneurological problems in children, the pediatrician is ad¬vised to consult these two monographs. Pediatrie neu¬rologists should add them to their libraries.

PEGGY C. FERRY, MDDepartment of PediatricsArizona Health Sciences Center1501 N Campbell AveTucson, AZ 85724

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