Molecular characterization of a human group A rotavirus isolated from an adult with severe dehydrating diarrhea and its relationship to strains concurrently circulating among children

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ELSEVIER

\ ir.h,gy

Molecular characterization of a human group A rotavirus isolated from an adult with severe dehydrating diarrhea and its relationship to strains concurrently circulating among children Eiji Kaga a Masako Tobita b Takashi Saito c Masahiro Iizuka Osamu Urayama b, Toyoko Nakagomi a, Shiro Uesugi b

a

Osamu Nakagomi a'* " Department qfMicrobiology, Akita Universi O, School q/'Medich~e, Akita 010, Japan Department q/'Lahoratory Medic#w..4kita O)#versi O" School ~?/'Medicbte, Akita OlO. Japan ' The Second Department ofhtternal AIedicine. Akita UniveJwit)' School o/Medic#w, Akim 010, Japall

Received 19 November 1993; revised 22 Marcia 1994:accepted 23 March 1994

Abstract

Background: While group A rotavirus is widely accepted as an important etiology of acute gastroenteritis in children, this agent rarely causes severe diarrhea in adults and, thus, is not considered by physicians to be an etiological agent for such diseases. Objectives: None of the reports describing such rare cases in adults has examined the causative strains genetically in detail. Study design: We determined the G type, the gene 4 genotype, the electropherotype, and the genomic RNA constellation (genogroup) of a group A rotavirus strain isolated l¥om an adult with severe diarrhea. This patient, the first documented case of rotavirus diarrhea in adult in Japan, suffered from severe dehydrating diarrhea with 'rice-water' appearance, vomiting and little abdominal pain, presenting a clinical picture typical of cholera. Results: Rotavirus antigen and genomic RNA were detected in the stool but other enteric pathogens including Vihrio choh, rae responsible for the disease were not isolated. Molecular characterization revealed that the patient was infected with a strain of the DS-1 genogroup with G2 and gene 4 genotype 4 which was circulating among children during the same period. Conclusions: The result that the same group A rotavirus strain was isolated from children and an adult underlines the necessity of examining rotavirus in adults with acute diarrhea. Key words. Rotavirus;

Adult

diarrhea;

Molecular characterization; Electropherotype;

Serotype: Genogroup

* Corresponding author. Fax: (+81) 188-36-2607. 0928-0197,,'94,,'$7,00r© 1994 Elsevier Science B.V. All rights reserved SSD1 0928-0197(94)00014-L

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1. Introduction

Group A human rotavirus is the most important etiological agent of acute gastroenteritis in infants and young children ( Kapikian and Chanock, 1990). However, symptomatic infection in adults is uncommon and the disease, even if it occurs, is usually mild and severe cases are reported mainly in the elderly (Christensen, 1989). Since molecular epidemiologic studies designed to examine antigenicities of strains causing the disease in adults or the relationship to strains concurrently circulating among children had been rare, it was not known whether such strains were distinct from strains circulating among children. The electropherotyping and serotyping analyses have been used in epidemiologic studies (Nakagomi et al., 1988; Tam et al., 1990). The electropherotype is the migration pattern of 11 segments of genomic RNA and is strain-specific, providing an excellent tool to trace the spread of a given strain (Tam et al., 1990). Rotavirus has two different neutralization proteins, called the G and P types (Estes and Cohen, 1989). Recently, Steel et al. (1992) reported that strains causing diarrhea in a geriatric ward had G I or G4, both ot" which are frequently found in strains recovered from children (Nakagomi et al., 1988). The sharing of G type specificities, however, does not necessarily imply the similarity of gene 4 genotype (P genotype) among them. For example, the gene 4 genotype of strains circulating among neonates was distinct from those of strains circulating among children despite they shared G types (Flores et al., 1986: Gorziglia ct al., 1990: Hoshino et al., 1985). Thus it was possible that strains causing diarrhea in adults had a distinct gene 4 genotype. Further, none of the studies documenting rotavirus infection in adults examined the genetic constellation of causative strains, leaving a question whether strains causing the disease in adults were genotypically distinct from strains circulating among children. Since human rotaviruses can be classified on the basis of the genetic homology into three genogroups (each of which is represented by Wa, DS-I, or AU-I. respectively) (Nakagomi et al., 1989), genogroup analysis is of value to address this question. We report the first case of rotavirus gastroenteritis in the adult in Japan, which was clinically very similar to that caused by Vibrio cholerae. The causative strain was examined to determine whether it was antigenically or genotypically distinct from strains circulating among children.

2. Case and methods 2.1. Case report

A 63-year-old male was admitted to the hospital in April 1992 because of acute diarrhea and vomiting starting from the previous day. He passed 'rice water-like" stools more than 10 times a day. He also suffered from vomiting 7 8 times a day but did not complain of abdominal pain. His body temperature was normal. On the basis of his clinical picture, cholera was suspected and the patient was arranged lbr hospitalization in an isolated ward. His laboratory data at the time of admission

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were as follows: white blood cell count 9700/~tl, hematocrit 42.1%, C-reactive protein 2.8 mg/dl, total protein 9.7 g/all, phospholipids 262 mg/dl, BUN 26 mg/dl, creatinine 1.6 mg/dl, [Na +] 142 mEq/1, [ K +] 4. l mEq/1, [C1 -] 104 mEq/1. The patient responded well to intravenous rehydration therapy and oliguria was resolved on the following day. The patient had no contact with children with diarrhea. 2.2. Microbiology The stool specimen was tested for the presence of rotavirus antigen with Rotalex (Orion Diagnostica, Espoo, Finland) and cultured on selective media to isolate Salmonella species, Shigella species, Campylobacter jejuni, Yersinia enterocolitica, Aeromonas hydrophila, Plesiomonas shigelloides, Escherichia eoli, and Vibrio cholerae. 2.3. Viruses and stool specimens The following cell culture-adapted human rotavirus strains were used in this study: AU801 (GI, P genotype 8, subgroup IlL K U N (G2, P genotype 4, subgroup I) (Kutsuzawa et al., 1982), DS-1 (G2, P genotype 4, subgroup I) (Wyatt et al., 1983), and AU-1 (G3, P genotype 9, subgroup I) (Nakagomi et al., 1987). AU801 was isolated from a stool specimen 88A001 in our laboratory. In this report, the assignment of gene 4 genotype (P genotype) was according to the system proposed by Estes and Cohen (1989). Rotavirus-positive stool specimens (n = 12) were collected from children with diarrhea in Akitm Japan, during the period from February to April 1992. 2.4. Molecular characterization of the causative strain Rotaviral genomic RNA was extracted from the stool specimen with phenol and analyzed by polyacrylamide gel electrophoresis (PAGE). The P genotype and G type were determined by polymerase chain reaction (PCR)-based typing assays (Gentsch et al., 1992; Gunasena et al., 1993). The genogroup was determined by RNA-RNA hybridization as previously described (Nakagomi et al., 1989). Briefly, heat-denatured genomic RNA was hybridized with each of [32P]-labeled transcription probes from AU801, KUN and AU-1 strains (representatives of the Wa, DS-I and AU-1 genogroups, respectively) in solution under high stringency conditions. Resultant hybrids were separated by PAGE and detected by autoradiography. 2. 5. Titration of rotavirus-speeific antibodies The titers of serum rotavirus-specific antibodies at the acute (time of admission) and in the convalescent phase (18 days later) were determined by indirect immunofluorescent assay. Briefly, after 16 h of infection with DS-1, cells were fixed with methanol and incubated with two-fold serial dilutions of the patient serum. Bound antibodies were detected by anti-human IgG conjugated with fluorescein isothiocya-

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nate. The titers were described as the reciprocals of the highest dilution of the serum showing the positive fluorescence.

3. Results

3.1. IdenHtication o/lhe causative patlto~en Rotavirus antigen and genomic R N A were detected in the patient stool. A strongly positive agglutination reaction and intensely stained genomic R N A on the gel indicated that the patient excreted a large number of virus particles. Its electrophoretic pattern was typical of that of group A rotavirus with short R N A pattern. A few colonies of enteropathogenic Escherichia con ( EPEC 1 was detected but neither Vibrio cholerae nor other enteric pathogens was detected. From the number of colonies of EPEC and the clinical picture, EPEC was not considered to be an etiology of the disease of the patient.

3.2. Molecuhlr characteri-alion ~?/ llle causative strain To further characterize the stool rotavirus shed by tile patient, we isolated a rotavirus strain and named 92A013. The electropherotype of 92A013 was identical with that of the stool rotavirus. Fig. 1 shows the hybridization patterns obtained between the genomic R N A of 92A013 and the transcription probes from AU801, K U N , and AU-1 strains. While neither the AU801 nor AU-I probe produced any hybrids, the K U N probe produced 11 hybrids with the genomic RNA of 92A013. These results indicated that 92A013 belonged to the DS-1 genogroup. PCR-based G and P typing assays showed that 92A013 had G2 and P genotype 4 (corresponding to serotype P1B), which are usually carried by strains belonging to the DS-1 genogroup. 92A013 was, therefore, not novel in terms of either its serotype or its genotype. Since strains of the DS-1 genogroup circulate among children in most rotavirus seasons, our results led us to speculate that the patient was infected with a strain circulating among children during the same rotavirus season. We thus sought for a strain with the identical electropherotype among strains concurrently circulating among children. We found that a field rotavirus 92A01 l isolated from a 15-month-old child with diarrhea had the identical electropherotype, as shown by co-electrophoretic comparison ( Fig. 2). In addition, this strain was 1\rand in the stool specimen from another child with diarrhea (data not shown). Taken together, this patient was mfected with a strain circulating among the child population.

3.3. Serore,v~onse An 8-[bld increase of rotavirus-specific antibodies was detected (the titers at tile acute and in the convalescent phase were 16 and 128, respectively). This provided serologic evidence of rotavirus infection.

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1 2 3 4

5 6

7 8 9

10 i [

II AUiI01

it, KU N

i AU--1

probe Fig. 1. Hybridization patterns obtained from genomic RNAs of the strains indicated on the top of panel and [32p]-labeled probes from AUS01, KUN, and AU-I strains.

4. Discussion This report describes, to our knowledge, the first documented case in Japan of diarrhea in the adult caused by group A human rotavirus. Symptomatic rotavirus infection in adults occurs only occasionally in adults and rotaviruses rarely cause severe disease in adults (Kapikian and Chanock, 1990). Such unusual cases of rotavirus gastroenteritis have been reported under various epidemiological settings in Brazil (Linhares et al., 1981 ), Canada (Marrie et al., 1982; Wenman et al., 1979), England (Cubitt and Holzel 1980; Jewkes et al., 1981; Steel et al., 1992), Finland (Meurman and Laine 1977; von Bonsdorff et al., 1976; von Bonsdorff et al., 1978), Israel (Galil et al., 1986), New Zealand (Grimwood et al., 1983), Pacific Island (Foster et al., 1980), Sweden (Lycke et al., 1978), Switzerland (Loosli et al., 1985), Thailand (Echeverria et al., 1983), and the United States (Kim et al., 1977: Rodriguez et al., 1979). In these documented cases, the disease was mild and severe disease enough to require hospitalization, like the case in this report, was rare and

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J 2 3 4

5 6

7 8 9

10 11

Fig. 2. Comparison of electropherot,,pes of 92A1tl 1 {recovered fronl tt 15-monlh-old child with diarrhea and 92A013 (recovered from the adult patient 1.

restricted mainly to the elderly. For example, rotavirus infection occurring in geriatric wards could become extensive and severe: some fatal cases were reported (Cubitt and Holzel 1980; Marrie et all., 1982). This was possibly due to increased susceptibility to dehydration and relative malnutrition (Hrdy, 1987). Thus, it is critical to make diagnosis of rotavirus infection in people at risk early in the clinical course. Since rotavirus has not been examined routinely for acute diarrhea in adults, we may have missed such cases. Because only a few colonies of EPEC were isolated, this agent contributed minimally, if at all, to the symptoms of the patient. The case reported in this paper reminds us of the fact that the clinical picture of rotavirus infection can be indistinguishable from that of cholera (rotavirus diarrhea had long been called in Japan Pseu~Ncholera il~limtum). We encourage physicians to include rotavirus infection in the differential diagnosis of diarrheal diseases in adults. The causative strain was similar in both its serotype and its genotype to strains circulating among children, indeed, we showed that the electrophoretically-identical strains were circulating a m o n g children with diarrhea during the same rotavirus season. These results provided for the first time evidence that a strain causing the

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disease in children was capable of causing epidemiologically unrelated diarrheal disease in adults.

References Christensen, M.L. (1989) Human viral gastroenteritis. Clin. Microbiol. Rev. 2, 51 89. Cubitt, W.D. and Holzel, H. (1980) An outbreak of rotavirus infection in a long-stay ward of a geriatric hospital. J. Clin. Pathol. 33, 306 308. Echeverria, P., Blacklow, N.R., Cukor, G,G., Vibulbandhitkit, S., Changchawalit, S. and Boonthai, P. (1983) Rotavirus as a cause of severe gastroenteritis in adults. J. Clin. Microbiol. 18, 663 667. Estes, M.K. and Cohen, J. (1989) Rotavirus gene structure and function. Microbiol. Rev. 53,410 449. Flores, J., Midthun, K., Hoshino, Y., Green, K., Gorziglia, M., Kapikian, A.Z. and Chanock, R.M. (1986) Conservation of the fourth gene among rotaviruses recovered l¥om asymptomatic newborn infants and its possible role in attenuation. J. Virol. 60, 972- 979. Foster, S.O., Palmer, E,L., Gary, G.W.J., Martin, M.L., Herrmann, K.L., Beasley, P. and Sampson. J, (1980) Gastroenteritis due to rotavirus in an isolated pacific island group: an epidemic of 3,439 cases. J. Infect. Dis. 141, 32 39. Galil, A., Antverg. R., Katzir, G., Zentner, G., Margalith, M., Friedman, M.G., Sarov, B. and Saro~, I. (1986) Involvement of infants, children, and adults in a rotavirus gastroenteritis outbreak in a kibbutz in southern Israel. J. Med. Virol. 18, 317 326. Gentsch, J.R., Glass, R.I., Woods, P., Gouvea, V., Gorziglia, M,, Flores, J., Das, B.K. and Bhan, M.K. (1992) Identification of group A rotavirus gene 4 types by polymerase chain reaction. J. Clin. Microbiol. 30, 1365 1373. Gorziglia, M., Larralde, G., Kapikian, A.Z. and Chanock, R.M. (1990) Antigenic relationships among human rotaviruses as determined by outer capsid protein VP4. Proc. Natl. Acad. Sci. USA 87, 7155 7159. Grimwood, K., Abbott, G.D., Fergusson, D.M., Jennings, L.C. and Allan, J.M. (1983) Spread o f rotavirus within families: a community based study. Brit. Med. J. 287, 575 577. Gunasena, S., Nakagomi, O., lsegawa, Y., Kaga, E., Nakagomi, T., Steele, A.D., Flores, J. and Ueda, S. (1993) Relative frequency of VP4 gene alleles among human rotaviruses recovered over a 10-year period ( 1982 1991 ) from Japanese children with diarrhea. J. Clin. Microbiol. 31, 2195 2197. Hrdy, D.B. (1987) Epidemiology of rotaviral infection in adults. Rev. Infect. Dis. 9, 461 469. Hoshino, Y., Wyatt, R.G., FIores, J., Midthun, K. and Kapikian, A,Z. (1985) Serotypic characterization of rotaviruses derived from asymptomatic human neonatal infection. J. Clin. Microbiol, 21,425 43{). Jewkes, J,, Larson, H.E., Price, A.B., Sanderson, P.J. and Davies, H.A. (1981) Aetiology of acute diarrhoea in adults. Gut 22, 388 392. Kapikian, A.Z. and Chanock, R.M. (1990). Rota~,iruses. In: B.N. Fields, D.M. Knipe, R.M. Chanock, M.S. Hirsch, J.L. Melnick. T.P. Monath and B. Roizman (Eds,). Virology, Raven Press, New York. pp. 1353 1404. Kim, H.W., Brandt, C.D., Kapikian, A.Z., Wyatt, R.G., Arrobio, J.O., Rodriguez, W.J,, Chanock, R.M. and Parron, R.H. (1977) Human reovirus-like agent infection. Occurrence in adult contracts of pediatric patients with gastroenteritis. JAMA 238, 404 407. Kutsuzawa, T., Konno, T., Suzuki, H., Kapikian, A.Z., Ebina, T. and Ishida. N. (1982) Isolation of human rotavirus subgroups 1 and 2 in cell culture. J. Clin. Microbiol. 16, 727 730. Linhares, A.C., Pinheiro, F.P., Freitas. R.B., Gabbay, Y.B., Shirley, J.A. and Beards, G.B. ( 1981 ) An outbreak of rotavirus diarrhea among a nonimmune, isolated South American Indian community. Am. J. Epidemiol. 113. 703 710. Loosli. J., Gyr, K., Stalder, H., Stalder, G.A., Vischer, W., Voegtlin, J., Gasser, M. and Reichlin, B. (1985) Etiology of acute infectious diarrhea in a highly industrialized area of Switzerland. Gastroenterology 88, 75 79.

366

K Kaga el etL '('/il;iual & Dia~m~sliu ITro/oKv 2 : 1994:,?59 36:)

Lycke, E., Blomberg, J.. Berg, G., Eriksson, A. and Madsen. L. 11978) Epidemic acute diarrhea in adults associated with infantile gastroenteritis virus. I.ancet 2. 1056 1057. Marrie. TJ., Lee, S.H.S., Faulkner, R.S., Ethier, J. and Young, ('.R. 11982) Rotavirus infection in at geriatric population. Arch. Intern. Med. 142, 313 316. Meurman, O.H. and Laine, M.J. (19771 Rotavirus epidemic in adults. N. Eng. J. Med. 296, 1298 1299. Nakagomi, O.. Nakagomi. I . , Hoshino. Y.. Elorc~,, J. and Kapikian, A.Z. (1987) Genetic anal~,sis of a h u m a n rotavirus that belongs to subgroup 1 but has an R N A pattern typical o1" subgroup II h u m a n rotavirus. J. Clin. Microbiol. 25. 115¢) 11()4. Nakagomi, O.. Nakagomi, T., Akatani, K. and Ikegami. N. (1989) Identification of rotavirus genogroups b y ' R N A - R N A hybridization. MoI. Cell. Probes 3. 251 261. Nakagomi. T.. Akatani. K., lkegami, N.. Katsushima. N. and Nakagomi, O. (1988) Occurrence of changes in h u m a n rotavirus serotypes with concl_lrrellt changes in genomic R N A elec|ropherotypes. J. Clm. Microbiol. 26, 2586 2592. Rodriguez, W.J., Kim. H.W., Brandl, C.D., Yolken, R.H., Richard, M., Arrobio, J,O., Scllwartz, R.I t,, Kapikian. A.Z., Chanock, R.M. and Parrot, R.H. (1979) C o m m o n exposure outbreak of gastroenteritis due to type 2 rotavirus with high secondary attack rate within families. J. Infect. Dis. 14(L 353 357. Steel. H.M.. Garnham. S., Beards. G.M. and Bro~n, D,W.G. (1992) Investigation of an outbreak of rotavirus infection in geriatric patients by serolyping and polyacrylamide gel electrophoresis ( PAGE I. J. Med. Virol. 37. 132 136. Tam, J.S.I_., Zheng, B.J.. Lo, S.K.. "feung. ('.Y., ko. M. and Ng, M.H. (1990) Distinct populations of rotaviruses circulating a m o n g neonates and older infants. J. Clin. microbiol. 28. 1033 1038. yon Bonsdorfl', C.H.. Hovi. T., Makcla, P., lira, i, L. and Tevalvoto-Aai'nio, M. (1976) Rotaxirus as>ociated with acute gastroenteritis in adults. Lancet 2. 423. yon Bonsdorll', ('.H., Hovi, T., MfikcEi. P. and M6rttmen. A. (1978) Rotavirus infections in adults in association with acute gastroenteritis. J. Med. Virol, 2, 21 28. Wenman, W.M., Hmde, D., Feltham, S. and Gurwith. M. (19791 Rolavirus infection in adults. Resuhs of a prospectixe family study. N. Eng. J. Mcd. 301. 303 306. Wyatt, R.G., James, H.D.J., Pittman, A.[,.. Hoshino. Y., Greenberg, H.B.. Kalica. A.R.,Flores, .I. and Kapikian. A.Z. (1983) Direct isolation in cell culttu'c of h u m a n rotaviruses and their characterization into four serotypes. J. Clin. Microbiol 18, 31I) 317