INFECTION AND IMMUNITY, JUlY 1977, P. 157-160Copyright © 1977 American Society for Microbiology

Vol. 17, No. 1Printed in U.S.A.

Tubular Structures Associated with Acute NonbacterialGastroenteritis in Young Children

TERUO KIMURA* AND TSUKASA MURAKAMI

Osaka City Institute ofPublic Health and Environmental Sciences, Osaka 543, Japan

Received for publication 8 December 1976

We clearly demonstrated a fine morphology ofthe tubular structures antigeni-cally related to the reovirus-like particles associated with acute nonbacterialgastroenteritis in young children. The tubular structures with a caplike struc-ture on each end seemed to be complete forms, 75 to 80 nm in width andapproximately 1,000 nm in length.

Tubular structures have been observed bothwithin infected cells and in negatively stainedsuspensions of several orbiviruses, such asbluetongue, epizootic hemorrhagic disease ofdeer, Tribec, and African horse sickness viruses(6, 7). In 1974 it was reported (3) that a tubularstructure was found in a clump ofhuman reovi-rus-like particles aggregated by convalescentserum and that this was presumably an aggre-gate of capsid protein. Tubular structures havesince been detected in negatively stained prepa-rations of human, bovine, murine, and equinereovirus-like particles (1, 4, 8), but a fine mor-phology of the tubular structures seems not tohave been described.

In this paper we clearly demonstrate a finemorphology of tubular structures from fecal ex-tracts of young children with acute nonbacte-rial gastroenteritis by negative-contrast elec-tron microscopy. Furthermore, we confirm, byusing immunoelectron microscopy, that the tu-bular structures are antigenically related to thereovirus-like particles.

MATERIALS AND METHODSClinical specimens. Feces were obtained from 102

young children of 2 to 51 months ofage who had beenhospitalized in Taniuchi Hospital for Sick Children,Osaka, Japan, with acute nonbacterial gastroenter-itis during December 1975 and January and Febru-ary 1976. Feces were also obtained from 13 youngchildren who did not have gastroenteritis. The agerange of the control group was similar to the groupwith gastroenteritis. All feces from patients and thecontrol group were stored at -20°C until extraction.

Preparation of fecal extracts for electron micro-scopic studies. Fecal extracts for electron micro-scopic studies were prepared by the method de-scribed previously (2) with some modifications. Sus-pensions of feces (about 20%, vol/vol) were made inphosphate-buffered saline and centrifuged at 1,500rpm for 10 min to remove large debris. The superna-tants were then centrifuged at 7,000 rpm for 30 min

in a Sorvall SS-34 rotor to deposit bacteria and de-bris. The supernatant fluids were then centrifugedat 35,000 rpm for 2 h in a Hitachi RP40 rotor, and thedeposits were suspended in 0.1 to 0.2 ml of distilledwater containing 0.2% (wt/vol) sucrose and usedimmediately for electron microscopic studies.

Negative-contrast electron microscopy and im-munoelectron microscopy. A drop ofthe sample wasplaced for 1 min on a 400-mesh collodion-coated cop-per grid covered with a carbon film, the excess wasdrawn off with a piece of filter paper, a drop of thecontrasting agent (2% phosphotungstate, pH 7.0, or2% uranyl acetate, pH 4.2) was added to the sample,and the excess liquid was quickly removed with apiece of filter paper. After drying, the grid wasexamined in a JEOL JEM-1OOB electron microscopeat a magnification of 40,000, using an operatingvoltage of 80 kV. Immunoelectron microscopy wasperformed by the method described previously (3).

RESULTSReovirus-like particles were observed in fecal

extracts of 60 of 102 young children with acutenonbacterial gastroenteritis collected in theacute phase, but not in fecal extracts from 14infants in the convalescent stage and 13 infantswithout gastroenteritis (unpublished data).They seem to be similar to the reovirus-likeparticles detected by numerous other investiga-tors in many parts of the world (5).Tubular structures were observed in 3 of 60

fecal extracts (positive for reovirus-like parti-cles) of young children with acute nonbacterialgastroenteritis. The appearance of the nega-tively stained tubular structures is shown inboth Fig. 1 and Fig. 2. The diameter of thetubular structures was 75 to 80 nm, similar tothat of the reovirus-like particles, and thelength of the tubular structures varied (Fig. 1and 2). The tubular structures shown in Fig. 1appeared to be similar to those described previ-ously (3, 4). The tubular structures with a cap-

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158 KIMURA AND MURAKAMI

FIG. 1. Electron micrograph of tubular structures. Negative staining was with 2% uranyl acetate at pH4.2. x200,000.

FIG. 2. Electron micrograph of tubular structures with caplike structures. Negative staining was with 2%phosphotungstate at pH 7.0. x100,000.

like structure (Fig. 2, arrow) on each end werealso observed in the same negatively stainedpreparation. The tubular structures with cap-like structures seemed to be complete forms, 75to 80 nm in width and approximately 1,000 nmin length. The surfaces of the tubular struc-tures were composed of hexagonally arrayedsubunits, which appeared to be hexagonal inoutline. Each subunit was about 15 nm in diam-eter with a central hole of approximately 5 nm

(Fig. 1). Furthermore, similar hexagonally ar-rayed subunits were seen on the capsid surfaceof complete reovirus-like particles (Fig. 3, ar-row).On the other hand, the tubular structures

were agglutinated by convalescent serum. Atthe same time the complete reovirus-like parti-cles and coreless particles were also aggluti-nated by convalescent serum, as shown in animmunoelectron micrograph (Fig. 4).

INFECT. IMMUN.

FIG. 3. Electron micrograph of reovirus-like particles. A complete virus particle 80 nm in diameter withhexagonally arrayed subunits on its surface and coreless particles 65 to 70 nm in diameter. Negative stainingwas with 2% phosphotungstate at pH 7.0. x200,000.

k-W-~~~~~~~a~h

-N

*~~~~~.:

FIG. 4. Immunoelectron micrograph of tubular structures, complete reovirus-like particles, and corelessparticles agglutinated by convalescent serum. Negative staining was with 2% phosphotungstate at pH 7.0.x120,000.

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DISCUSSIONIn negatively stained preparations the tubu-

lar structures were observed in 3 out of 60 fecalextracts ofyoung children with acute nonbacte-rial gastroenteritis. The tubular structuresusually observed are shown in Fig. 1. They areconstant in width similar to that of the reovi-rus-like particles, but they vary in length andappear to be similar to tubular structures de-scribed by previous workers (3, 4). Further-more, tubular structures with a caplike struc-ture on each end were also observed (Fig. 2).They are about 1,000 nm in length and seem tobe complete forms. Therefore, both the tubularstructures shown in Fig. 1 and those describedpreviously (3, 4) may be fragments of the com-plete form.Hexagonally arrayed subunits similar to

those found on the surfaces of the tubular struc-tures were observed on the capsid surfaces ofcomplete reovirus-like particles (Fig. 3, arrow).It was further confirmed, by immunoelectronmicroscopy (Fig. 4) as described previously (3,4), that the tubular structures were antigeni-cally related to the reovirus-like particles.These findings suggest that the hexagonallyarrayed subunits composing the surfaces of thetubular structures may represent virus sub-units.

In this study we have clearly demonstrated afine morphology of the tubular structures anti-genically related to reovirus-like particles, butthe role ofthe tubular structures in virus matu-ration does not yet seem to be determined.However, we should like to support the sugges-

tion that the tubular structures are formed byaberrant assembly of viral capsid material, asproposed previously (4).

ACKNOWLEDGMENTS

We are greatly indebted to K. Taniuchi, Taniuchi Hospi-tal for Sick Children, Osaka, and A. Tada and A. Mori,Department of Pediatrics, Osaka City University, School ofMedicine, for supplying the clinical specimens, and wewould also like to thank T. Hotta in this laboratory for hisvarious comments.

LITERATURE CITED

1. Banfield, W. G., G. Kasnic, and J. H. Blackwell. 1968.Further observations on the virus of epizootic diar-rhea of infant mice -an electron microscopic study.Virology 36:411-421.

2. Flewett, T. H., A. S. Bryden, and H. A. Davies. 1973.Virus particles in gastroenteritis. Lancet ii:1497.

3. Flewett, T. H., H. Davies, A. S. Bryden, and M. J.Robertson. 1974. Diagnosic electron microscopy offaeces. II. Acute gastroenteritis associated with reo-virus-like particles. J. Clin. Pathol. 27:608-614.

4. Holmes, I. H., B. J. Ruck, R. F. Bishop, and G. P.Davidson. 1975. Infantile enteritis viruses: morpho-genesis and morphology. J. Virol. 16:937-943.

5. Lancet. 1975. Rotaviruses of man and animals. Lanceti:257-259.

6. Murphy, F. A., E. C. Borden, R. E. Shope, and A.Harrison. 1971. Physicochemical and morphologicalrelationships of some arthropod-borne viruses tobluetongue virus-a new taxonomic group. Electronmicroscopic studies. J. Gen. Virol. 13:273-288.

7. Oellermann, R. A., H. J. Els, and B. J. Erasmus. 1970.Characterization of African horsesickness virus.Arch. Gesamte Virusforsch. 29:163-174.

8. Woode, G. N., J. C. Bridger, J. M. Jones, T. H. Flewett,A. S. Bryden, H. A. Davies, and G. B. B. White. 1976.Morphological and antigenic relationships betweenviruses (rotaviruses) from acute gastroenteritis ofchildren, calves, piglets, mice, and foals. Infect. Im-mun. 14:804-810.

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