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Atypical human rotaviruses in Hungary
©
Ann. Inst. Pasteur/Viral.
ELSEVIER
Paris
1987
1987, 138, 391-395
ATYPICAL HUMAN ROTAVIRUSES IN HUNGARY by G. Szucs, M. Kende and M. Uj Laboratory of Virology, Public Health Station, Szabadsdg u. 7., H-7623 Pees (Hungary)
SUMMARY
Of 105 human rotaviruses, two atypical, antigenically distinct rotaviruses (pararotaviruses) were isolated in Hungary. A 4-year old boy and a 3~-year old girl, both with gastroenteritis, vomiting and moderate fever, shed these viruses. Electrophoretic patterns of viral RNA extracted from the two faecal specimens appeared to be related to patterns of group C rotaviruses. KEY-WORDS: Rotavirus, Pararotavirus, Infantile gastroenteritis; RNA, Electrophoretypes, Hungary.
INTRODUCTION
Human rotaviruses with unusual RNA electrophoretic patterns have been reported in Australia [13], France [10], Bulgaria [5], Mexico [6], Netherlands [4], China [8], England [7], Italy [1], Argentina [14] and recently in South Africa [2]. Different terms have been suggested, such as pararotavirus [3], group B rotavirus [5] and atypical rotavirus [8]. All these rotavirus isolates were morphologically identical to the prototype virus, and only 6 strains from Johannesburg, South Africa contained the common group A antigen and were detectable by the current ELISA test. When viral RNA was analysed by PAGE and electrophoretypes were characterized, three different patterns could be observed. Isolates reported in China and in Sou th Africa shared apparently different electrophoretypes which were easily distinguishable from all others. Electrophoretyping is currently a useful tool for revealing these viruses. Here we report the first two atypical, antigenically distinct rotaviruses detected by RNA gel electrophoresis in Hungary.
Submitted November 14, 1986, accepted January 24, 1987.
392
G. SZUCS. M. KENDE AND M. VI
MATERIALS AND METHODS
Patients. One of the samples containing rotaviruses with an unusual pattern was obtained from a male child, aged 4, admitted on March 11, 1982 to the «UiilZ16)} Hospital for Infectious Disease in Budapest, Hungary. The child had diarrhoea with moderate fever and vomiting. A faecal sample was taken on the first day of admission. Rotavirus was detected by EM, but rotavirus antigens were undetectable by the ELISA test using the double sandwich technique in the Department of Virology, National Institute of Public Health, Budapest. Approximately 20 OJo of the stool suspension was stored at - 70°C and received for electrophoretyping in October, 1983. The second child, a 3~-year old girl, was admitted on May 25, 1984 with a diagnosis of gastroenteritis, vomiting and bronchitis to the Department of Pediatrics, University Medical School, Pees. A stool sample was taken on the second day. The ELISA test for rotaviruses (<
RESULTS
The two virus strains recovered from children in 1982 and 1984, when analysed by polyacrylamide gel electrophoresis, exhibited completely different migration patterns from those of human rotavirus strains which had been isolated up to then in Hungary (fig. 1); 9 and 10 bands of RNA were identified clearly for the two samples, respectively, but from the intensity of the bands after staining, it was suggested that 3-4 segments and 8-9 segments were co-migrating in the first case and 8-9 segments were also not resolved in the sample taken from the girl. In a comparison of distribution of segments in four size groups of «typical» rotaviruses, these two patterns exhibited different positions of the segments. Migration of all segments except for segments 2, 7, 8 and 9 were faster, showing an «extra-long» profile. Nevertheless, segment 2 and segment 7 ran very close to segments 1 and 6, respectively, under our electrophoretic conditions.
ELISA EM
enzyme-linked imrnunosorbent assay. electron microscopy.
PAGE
polyacrylamide gel electrophoresis.
ATYPICAL HUMAN ROTA VIR USES IN HUNGAR Y
A
FIG.
1. -
c
o
B
393
D
RNA electrophoretic migration patterns of two Hungarian atypical human rotaviruses (A and B) and of «typical» human rotavirus (C).
Simian rotavirus SAll genomic RNA were included as controls CD).
In both cases, rotavirus particles were observed by EM in stool samples, and results of ELISA tests (« Rotazyme kit», Abbott and «Rotavirus ELISA kit», Dakopatts) applying guinea-pig and rabbit anti-rotavirus sera, respectively, against conventional rotavirus group-specific antigen(s), were negative.
DISCUSSION
To date, three types of unconventional rotaviruses based on their electrophoretic pattern have been reported in different geographic area of the world [2, 5, 10, 13]. Isolates from neonates in South Africa share the common group A antigen [2], unlike the other atypical, antigenically distinct human rotaviruses. Ofthem, strains described in adults in China [8] also differ from
394
G. szUCS, M. KENDE AND M. UJ
all others in migration of their segments. According to Flewett, the majority of atypical rotaviruses represent a distinct group classified as group C [7], in which one of segments 7, 8 and 9 is heavier and migrates close to segment 6. Recently described atypical human rotaviral strains in China and South Africa are not included in this group. These two independent isolates of human rotaviruses are the first demonstration of the presence of antigenically distinct atypical rotaviruses (pararotaviruses) in Hungary. A prevalence of this electrophoretype appears to be rare. From October, 1983 to December, 1984, i05 rotaviruses with many distinct electrophoretic patterns were isolated, but only these two viruses shared a characteristic atypical electrophoretype. RNA profiles of our two viruses are very similiar to the others reported thus far; however, some minor differences can be observed in migration of segments 3 and 4, and 8 and 9. Unfortunately, no additional samples were available for further analysis. Applying less samples and using silver staining, we assume that all 11 RNA segments would be resolved. It must also be noted that clinical symptoms and duration of disease were no different from those of cases caused by «typical» rotaviruses. Nevertheless, the real prevalence and medical significance of these agents cannot be assessed at this time. The low frequency of detection may be due partly to the fact that many laboratories routinely use only the ELISA test for screening rotaviruses. Without electrophoretyping, the prevalence and real role of these viruses in the aetiology of gastroenteritis remain unknown. Therefore, further investigations are important to evaluate the clinical, serological and epidemiological significance of these distinct human rotaviruses. RESUME
MISE EN EVIDENCE DE ROTAVIRUS ATYPIQUES EN HONORIE Lors d'une enquete qui a permis d'isoler en Hongrie 105 rotavirus humains nous avons mis en evidence deux rotavirus atypiques, ne possedant pas l'antigene du groupe A (pararotavirus). Ce virus a ete excrete par un garcon de quatre ans et par une fille de trois ans et demi, tous les deux etant atteints de gastroenterite avec fievre et vomissements. Le profil electrophoretique de l' ARN viral extrait de ces deux echantillons de selles semble indiquer que ces «isolats » peuvent etre rattaches au rotavirus du groupe C. MOTS-CLl3.S: Rotavirus, Pararotavirus, Gastroenterite infantile; ARN, Electrophoretypes, Hongrie. ACKNOWLEDGEMENT We wishto express our thanks to Drs M. Koller, E. Szarka and M. Csik, Department of Virology, National Institute of Public Health, Budapest for supplying us with faecal samples, and Mrs M. Nemeth for excellent technical assistance.
ATYPICAL HUMAN ROTA VIRUSES IN HUNGARY
395
REFERENCES
[1] ARISTA, S., GIOVANNELLI, L. & TITONE, L., Detection of an antigenicallydistinct
[2] [3] [4] [5] [6]
[7] [8]
[9]
[10] [11]
[12]
[13] [14]
human rotavirus in Palermo, Italy. Ann. Inst. Pasteur/Virol., 1985,136 E, 229-235. BESSELAAR, T.G., ROSENBLATT, A. & KIDD, A.H., Atypical rotavirus from South African neonates. Arch. Viral., 1986, 87, 327-330. BOHL, B.H., SAIF, L.J., THEIL, K.W., AGNES, A.G. & CROSS, R.F., Porcine pararotavirus: detection, differentiation from rotavirus and pathogenesis in gnotobiotic pigs. J. din. Microbiol., 1982, 15, 312-319. BUITENWERF, J., MUILWIJK-VAN ALPHEN, M. & SCHAAP, G.J.P., Characterization of rotaviral RNA isolated from children with gastroenteritis in two hospitals in Rotterdam. J. med. Virol., 1983, 12, 71-78. DIMITROV, D.H., ESTES, M.K., RANGELOVA, S.M., SHINDAROV, L.M., MELNICK, J.L. & GRAHAM, D.Y., Detection of antigenically distinct rotaviruses from infants. Inject. Immun., 1983, 41, 523-526. ESPEJO, R.T., PUERTO, F., SOLER, C. & GONZALEZ, N., Characterization of a human pararotavirus. Inject. Immun., 1984, 44, 112-116. FLEWETT, T.R., BEARDS, G.M., SANDERS, R.C. & HALL, C.J., New diarrhoea viruses of man, in « Abstracts Sixth Intern. Congr. Virology», Sendai, Japan, Sept. 1-7, 1984, p. 212. HUNG, T., CHEN, G., WANG, C., YAO, R., FANG, Z., CHAO, T., CHOU, Z., YE, W., CHANG, X., DEN, S., LIONG, X. & CHANG, W., Waterborne outbreak of rotavirus diarrhoea in adults in China caused by a novel rotavirus. Lancet, 1984, I, 1139-1142. LAEMMLI, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 1970, 227, 680-685. NICOLAS, J.C., COHEN, J., FORTIER, B., LOURENCO, M.H. & BRICOUT, F., Isolation of a human pararotavirus. Virology, 1983, 124, 181-184. NICOLAS, J.C., LOURENCO, M.H., MARCHAL, S., COHEN, J., SCHERRER, R. & BRICOUT, F., Description of a very simplified method for rotavirus dsRNA extraction. Ann. Viral. (Inst. Pasteur), 1983, 134 E, 135-139. RODGER, S.M., BISHOP, R.F., BIRCH, C., McLEAN, B. & HOLMES, I.H., Molecular epidemiology of human rotaviruses in Melbourne, Australia, from 1973 to 1979, as determined by electrophoresis of genome ribonucleic acid. J. din. Microbiol., 1981, 13, 272-278. RODGER, S.M., BISHOP, R.F. & HOLMES, LH., Detection of a rotavirus-like agent associated with diarrhoea in an infant. J. din. Microbiol., 1982, 16, 724-726. SORRENTINO, A., SCODELLER, B.A.,. BELLINZONI, R., MUCHINIK, 'G.R. & LA TORRE, J.L., Detection of an atypical rotavirus associated with diarrhoea in Chaco, Argentina. Trans. roy. Soc. trop. Med. Hyg., 1986,80, 120-122.
Ann. Inst. Pasteur/Viral.
ELSEVIER
Paris
1987
1987, 138, 391-395
ATYPICAL HUMAN ROTAVIRUSES IN HUNGARY by G. Szucs, M. Kende and M. Uj Laboratory of Virology, Public Health Station, Szabadsdg u. 7., H-7623 Pees (Hungary)
SUMMARY
Of 105 human rotaviruses, two atypical, antigenically distinct rotaviruses (pararotaviruses) were isolated in Hungary. A 4-year old boy and a 3~-year old girl, both with gastroenteritis, vomiting and moderate fever, shed these viruses. Electrophoretic patterns of viral RNA extracted from the two faecal specimens appeared to be related to patterns of group C rotaviruses. KEY-WORDS: Rotavirus, Pararotavirus, Infantile gastroenteritis; RNA, Electrophoretypes, Hungary.
INTRODUCTION
Human rotaviruses with unusual RNA electrophoretic patterns have been reported in Australia [13], France [10], Bulgaria [5], Mexico [6], Netherlands [4], China [8], England [7], Italy [1], Argentina [14] and recently in South Africa [2]. Different terms have been suggested, such as pararotavirus [3], group B rotavirus [5] and atypical rotavirus [8]. All these rotavirus isolates were morphologically identical to the prototype virus, and only 6 strains from Johannesburg, South Africa contained the common group A antigen and were detectable by the current ELISA test. When viral RNA was analysed by PAGE and electrophoretypes were characterized, three different patterns could be observed. Isolates reported in China and in Sou th Africa shared apparently different electrophoretypes which were easily distinguishable from all others. Electrophoretyping is currently a useful tool for revealing these viruses. Here we report the first two atypical, antigenically distinct rotaviruses detected by RNA gel electrophoresis in Hungary.
Submitted November 14, 1986, accepted January 24, 1987.
392
G. SZUCS. M. KENDE AND M. VI
MATERIALS AND METHODS
Patients. One of the samples containing rotaviruses with an unusual pattern was obtained from a male child, aged 4, admitted on March 11, 1982 to the «UiilZ16)} Hospital for Infectious Disease in Budapest, Hungary. The child had diarrhoea with moderate fever and vomiting. A faecal sample was taken on the first day of admission. Rotavirus was detected by EM, but rotavirus antigens were undetectable by the ELISA test using the double sandwich technique in the Department of Virology, National Institute of Public Health, Budapest. Approximately 20 OJo of the stool suspension was stored at - 70°C and received for electrophoretyping in October, 1983. The second child, a 3~-year old girl, was admitted on May 25, 1984 with a diagnosis of gastroenteritis, vomiting and bronchitis to the Department of Pediatrics, University Medical School, Pees. A stool sample was taken on the second day. The ELISA test for rotaviruses (<
RESULTS
The two virus strains recovered from children in 1982 and 1984, when analysed by polyacrylamide gel electrophoresis, exhibited completely different migration patterns from those of human rotavirus strains which had been isolated up to then in Hungary (fig. 1); 9 and 10 bands of RNA were identified clearly for the two samples, respectively, but from the intensity of the bands after staining, it was suggested that 3-4 segments and 8-9 segments were co-migrating in the first case and 8-9 segments were also not resolved in the sample taken from the girl. In a comparison of distribution of segments in four size groups of «typical» rotaviruses, these two patterns exhibited different positions of the segments. Migration of all segments except for segments 2, 7, 8 and 9 were faster, showing an «extra-long» profile. Nevertheless, segment 2 and segment 7 ran very close to segments 1 and 6, respectively, under our electrophoretic conditions.
ELISA EM
enzyme-linked imrnunosorbent assay. electron microscopy.
PAGE
polyacrylamide gel electrophoresis.
ATYPICAL HUMAN ROTA VIR USES IN HUNGAR Y
A
FIG.
1. -
c
o
B
393
D
RNA electrophoretic migration patterns of two Hungarian atypical human rotaviruses (A and B) and of «typical» human rotavirus (C).
Simian rotavirus SAll genomic RNA were included as controls CD).
In both cases, rotavirus particles were observed by EM in stool samples, and results of ELISA tests (« Rotazyme kit», Abbott and «Rotavirus ELISA kit», Dakopatts) applying guinea-pig and rabbit anti-rotavirus sera, respectively, against conventional rotavirus group-specific antigen(s), were negative.
DISCUSSION
To date, three types of unconventional rotaviruses based on their electrophoretic pattern have been reported in different geographic area of the world [2, 5, 10, 13]. Isolates from neonates in South Africa share the common group A antigen [2], unlike the other atypical, antigenically distinct human rotaviruses. Ofthem, strains described in adults in China [8] also differ from
394
G. szUCS, M. KENDE AND M. UJ
all others in migration of their segments. According to Flewett, the majority of atypical rotaviruses represent a distinct group classified as group C [7], in which one of segments 7, 8 and 9 is heavier and migrates close to segment 6. Recently described atypical human rotaviral strains in China and South Africa are not included in this group. These two independent isolates of human rotaviruses are the first demonstration of the presence of antigenically distinct atypical rotaviruses (pararotaviruses) in Hungary. A prevalence of this electrophoretype appears to be rare. From October, 1983 to December, 1984, i05 rotaviruses with many distinct electrophoretic patterns were isolated, but only these two viruses shared a characteristic atypical electrophoretype. RNA profiles of our two viruses are very similiar to the others reported thus far; however, some minor differences can be observed in migration of segments 3 and 4, and 8 and 9. Unfortunately, no additional samples were available for further analysis. Applying less samples and using silver staining, we assume that all 11 RNA segments would be resolved. It must also be noted that clinical symptoms and duration of disease were no different from those of cases caused by «typical» rotaviruses. Nevertheless, the real prevalence and medical significance of these agents cannot be assessed at this time. The low frequency of detection may be due partly to the fact that many laboratories routinely use only the ELISA test for screening rotaviruses. Without electrophoretyping, the prevalence and real role of these viruses in the aetiology of gastroenteritis remain unknown. Therefore, further investigations are important to evaluate the clinical, serological and epidemiological significance of these distinct human rotaviruses. RESUME
MISE EN EVIDENCE DE ROTAVIRUS ATYPIQUES EN HONORIE Lors d'une enquete qui a permis d'isoler en Hongrie 105 rotavirus humains nous avons mis en evidence deux rotavirus atypiques, ne possedant pas l'antigene du groupe A (pararotavirus). Ce virus a ete excrete par un garcon de quatre ans et par une fille de trois ans et demi, tous les deux etant atteints de gastroenterite avec fievre et vomissements. Le profil electrophoretique de l' ARN viral extrait de ces deux echantillons de selles semble indiquer que ces «isolats » peuvent etre rattaches au rotavirus du groupe C. MOTS-CLl3.S: Rotavirus, Pararotavirus, Gastroenterite infantile; ARN, Electrophoretypes, Hongrie. ACKNOWLEDGEMENT We wishto express our thanks to Drs M. Koller, E. Szarka and M. Csik, Department of Virology, National Institute of Public Health, Budapest for supplying us with faecal samples, and Mrs M. Nemeth for excellent technical assistance.
ATYPICAL HUMAN ROTA VIRUSES IN HUNGARY
395
REFERENCES
[1] ARISTA, S., GIOVANNELLI, L. & TITONE, L., Detection of an antigenicallydistinct
[2] [3] [4] [5] [6]
[7] [8]
[9]
[10] [11]
[12]
[13] [14]
human rotavirus in Palermo, Italy. Ann. Inst. Pasteur/Virol., 1985,136 E, 229-235. BESSELAAR, T.G., ROSENBLATT, A. & KIDD, A.H., Atypical rotavirus from South African neonates. Arch. Viral., 1986, 87, 327-330. BOHL, B.H., SAIF, L.J., THEIL, K.W., AGNES, A.G. & CROSS, R.F., Porcine pararotavirus: detection, differentiation from rotavirus and pathogenesis in gnotobiotic pigs. J. din. Microbiol., 1982, 15, 312-319. BUITENWERF, J., MUILWIJK-VAN ALPHEN, M. & SCHAAP, G.J.P., Characterization of rotaviral RNA isolated from children with gastroenteritis in two hospitals in Rotterdam. J. med. Virol., 1983, 12, 71-78. DIMITROV, D.H., ESTES, M.K., RANGELOVA, S.M., SHINDAROV, L.M., MELNICK, J.L. & GRAHAM, D.Y., Detection of antigenically distinct rotaviruses from infants. Inject. Immun., 1983, 41, 523-526. ESPEJO, R.T., PUERTO, F., SOLER, C. & GONZALEZ, N., Characterization of a human pararotavirus. Inject. Immun., 1984, 44, 112-116. FLEWETT, T.R., BEARDS, G.M., SANDERS, R.C. & HALL, C.J., New diarrhoea viruses of man, in « Abstracts Sixth Intern. Congr. Virology», Sendai, Japan, Sept. 1-7, 1984, p. 212. HUNG, T., CHEN, G., WANG, C., YAO, R., FANG, Z., CHAO, T., CHOU, Z., YE, W., CHANG, X., DEN, S., LIONG, X. & CHANG, W., Waterborne outbreak of rotavirus diarrhoea in adults in China caused by a novel rotavirus. Lancet, 1984, I, 1139-1142. LAEMMLI, U.K., Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (Lond.), 1970, 227, 680-685. NICOLAS, J.C., COHEN, J., FORTIER, B., LOURENCO, M.H. & BRICOUT, F., Isolation of a human pararotavirus. Virology, 1983, 124, 181-184. NICOLAS, J.C., LOURENCO, M.H., MARCHAL, S., COHEN, J., SCHERRER, R. & BRICOUT, F., Description of a very simplified method for rotavirus dsRNA extraction. Ann. Viral. (Inst. Pasteur), 1983, 134 E, 135-139. RODGER, S.M., BISHOP, R.F., BIRCH, C., McLEAN, B. & HOLMES, I.H., Molecular epidemiology of human rotaviruses in Melbourne, Australia, from 1973 to 1979, as determined by electrophoresis of genome ribonucleic acid. J. din. Microbiol., 1981, 13, 272-278. RODGER, S.M., BISHOP, R.F. & HOLMES, LH., Detection of a rotavirus-like agent associated with diarrhoea in an infant. J. din. Microbiol., 1982, 16, 724-726. SORRENTINO, A., SCODELLER, B.A.,. BELLINZONI, R., MUCHINIK, 'G.R. & LA TORRE, J.L., Detection of an atypical rotavirus associated with diarrhoea in Chaco, Argentina. Trans. roy. Soc. trop. Med. Hyg., 1986,80, 120-122.