key: cord-0009321-f6rj21wy authors: Buesa, F.J.; Duato, M.; Gimeno, C.; de Lomas, J. García title: Sequential variation in genomic RNA patterns of human rotaviruses isolated from infantile gastroenteritis date: 2009-09-23 journal: Ann Inst Pasteur Virol DOI: 10.1016/s0769-2617(87)80017-5 sha: bf1c99af172ac20a11305a8d191792500724e6f7 doc_id: 9321 cord_uid: f6rj21wy The incidence and RNA electrophoretypes of rotaviral isolates obtained from infants and young children with acute gastroenteritis were studied from October, 1985 through April, 1986. Analysis of the viral RNA was carried out by Polyacrylamide gel electrophoresis followed by silver staining. Fourteen electrophoretypes were identified. A single dominant electrophoretype was found during the first months of the rotavirus seasonal outbreak. In contrast, a large variety of RNA patterns were observed during the latter portion of the study period. Subgrouping of rotavirus isolates by a double-sandwich enzyme-linked immunosorbent assay using monoclonal-detecting antibodies showed that all strains belonged to subgroup II. Mixed rotavirus electrophoretypes appeared in 4 cases (8.16 %). Rotaviruses have been recognized as being one of the main causes of childhood diarrhoea [7] . The viral genome consists of 11 segments of linear double-stranded RNA (dsRNA), with molecular weights ranging from 2.5 x 10 6 to 0.4 x 10 6 daltons [14] . Polyacrylamide gel electrophoresis (PAGE) of rotavirus dsRNA, in conjunction with a sensitive silver staining technique Submitted November 5, 1986 , accepted February 9, 1987. [6] , has proven to be a very useful method for the identification and distinction of rotavirus isolates. Human rotaviruses show a large diversity in their segmented genomic patterns, and electrophoretic analysis has been established as a valuable means of studying the epidemiology of rotavirus infections [10, 12, 15, 17] . In the present report, we analysed faecal samples collected from infants and young children with gastroenteritis between October, 1985 and April, 1986, a period which included an outbreak of rotavirus infection, in order to conduct an epidemiological survey of the propagation of the virus. We describe the sequential variation in genomic RNA patterns detected. Two-hundred and sixty faecal specimens were obtained from October, 1985 through April, 1986 from infants and young children under 5 years of age who had been admitted to the Paediatric Department of the Hospital Clinico Universitario of Valencia, Spain, with symptoms of gastroenteritis or diarrhoea with dehydration. Preparation of specimens. Ten to twenty percent suspensions of faeces in phosphate-buffered saline (PBS), pH 7.4, were made up, homogenized and clarified by centrifugation at 1500 g for 10 min at 4°C. Liquid faecal specimens were diluted to 1/2 with PBS and centrifuged. The supernatants were used to perform electron microscopic examination of samples, nucleic acid analysis and enzyme-linked immunosorbent assay (ELISA) for sub grouping of rotavirus strains. Previously prepared 10-20 070 faecal suspensions were centrifuged at 50,000 rpm for 90 min (L8-70M Ultracentrifuge, Beckman Instruments), absorbed onto carbon-Formvar-coated 400-mesh grids and negatively stained with 3 % phosphotungstic acid, pH 7.0. The preparations were examined under an electron microscope (Zeiss EM lOC/CR, voltage 80 KV) at a magnification of 31,500X. Samples wereconsidered negative when no virus particles were found during 15 min of observation. The PAGE technique for rotavirus RNA detection has been previously described [3, 4, 8, 10] . Electrophoresis was performed on 10 070 polyacrylamide gelslabs without SDS by using the discontinuous buffer system of Laemmli 16 h at room temperature at 15 rnA constant current. The gels were stained with silver nitrate as described by Herring et al. [6] . Comparisons of different rotavirus strains were made by mixing and then co-electrophoresing them. ELISA for sub grouping of rotaviruses. Double-sandwich ELISA subgroup assays were performed as previously described by Beards et al. [2] . Capture antibody was a hyperimmune rabbit anti-rotavirus serum raised against complete and incomplete rotavirus particles of subgroups I and II. As «detecting» antibody, three monoclonal antibodies were used: (1) a rotavirus-group-specific monoclonal antibody, (2) a rotavirus subgroup-l-specific monoclonal antibody and (3) Fourteen different electrophoretypes were found. Figure 1 shows typical RNA migration patterns displayed by rotavirus isolates. Whenever appropriate, differences between isolates with similar RNA patterns were confirmed by co-electrophoresis (results not shown). All rotavirus isolates analysed during this period had the so-called «long» electrophoresis pattern, with fast-moving segments 10 and 11, but contained RNA segments with differences in their electrophoretic mobility. Mixed rotavirus electrophoretypes showing extra RNA fragments with respect to the 11 regular genome segments appeared in four cases (8.16 0/0). The classification scheme proposed by Lourenco et al. [10] to characterize and compare rotavirus electrophoretypes was used in this study as follows: the II RNA bands were divided into 4 groups including, respectively, bands 1, 2, 3 and 4 (group I); bands 5 and 6 (group II); bands 7, 8 and 9 (group III); and bands 10 and 11 (group IV). Differences in the relative migration of RNA bands within a group are indicated by a small letter , with each pattern referred to as a, b, c, etc. The monthly incidence and distribution of the different rotaviral electrophoretypes detected are summarized in table II. One of the electrophoretic migration patterns, Ib,IIb,IIIb,IVa, accounted for the largest proportion (36.7 070) of all rotaviruses identified. Five isolates obtained during the first two months of the study period had this electrophoretic pattern. In contrast, strains collected during the following months had a large variety of RNA patterns, although the electrophoretype that had appeared first occurred more frequently than the others. During the final months, it was difficult to find any predominant electrophoretype. In an attempt to more precisely characterize the rotavirus isolates, we determined their serological subgroups using two subgrouping monoclonal antibodies in an ELISA test. The specificity of both monoclonal antibodies was assessed by titering them against dilutions of two known subgroup I and subgroup II strains. The reactivity appeared to be highly specific. As expected, all rotavirus isolates studied were identified as subgroup II, with OD values higher than 1.200 when a monoclonal antibody specific for this subgroup was used as the detecting antibody. With the rotavirus subgroup-I-specific monoclonal antibody, OD values between 0.165 and 0.380 were obtained ( fig. 2) . Capture antibody was a hyperimmune rabbit anti-rotavirus serum . As detecting antibodies, three monoclonal antibodies were used: (1) a rotavirus group-specific monoclonal antibody, Our results have further demonstrated the extensive genomic heterogeneity of rotaviruses and the occurrence of different RNA patterns within an outbreak, as published by others [3, 10, 12] . However, some remarkable findings of our survey should be noted: no «short» electrophoresis patterns were detected, in contrast to many other reports. The predominance ofthe «long» electrophoretype has also been universally observed in recent years [17] . This fact could have immunological consequences, as sequential illnesses associated with different rotavirus serotypes have been described [13] . The observation of a predominant electrophoretype in a.given rotavirus season is in agreement with results of other studies-reported from many parts of the world [4, 11, 12] . We have detected 8.16 070 of RNA patterns with more than 11 bands, suggesting the possibility of simultaneous or sequential infection by more than, one rotavirus strain. The frequency of such a finding is approximately 101076 according to different reports [10, 16] . Mixed rotavirus infection could be the first step in genetic reassortment in vivo, with an intermediate step being necessary in the establishment of stable reassorted virus [5] . Electrophoresis of rotaviral RNA unfortunately cannot be used to determine antigenic serotypes of field isolates [1] . Whentype-specific monoclonal antibodies become available, serotyping of rotavirus strains will be transferable to many laboratories. On a etudie l'incidence et les profils electrophoretiques de l'ARN genomique des souches de rotavirus obtenues d'octobre 1985 aavril 1986, de gastroenterites algues de l'enfant. L'analyse de l'ARN par electrophorese sur gel de polyacrylamide a permis l'identification de 14 electrophoretypes differents, Pendant les premiers mois de la periode d'etude, on a detecte un electrophoretype dominant; au contraire, danslesmoissuivants, on a observe une grande variete de profils electrophoretiques. La determination des sous-groupes des rotavirus par analyse irnrnunoenzymatique avec des anticorps rnonoc1onaux detecteurs, a permis de constater que toutes les souches sont du sous-groupe II. DansA. . echantillons (8,16 %) on a detecte des electrophoretypes mixtes. MOTs-cLES: Rotavirus, Gastroenterite, ARN; Enfant, Epiderniologie moleculaire, Electrophoretypes, Profils de l' ARN genomique, Variation sequentielle. Polymorphism of genomic RNAs within rotavirus serotypes and subgroups Enzymelinked immunosorbent assays based on polyclonal and monoclonal antibodies for rotavirus detection electrophoretic migration of their double-stranded ribonucleic acid genome segments Molecular epidemiology of human rotaviruses. Analysis of outbreaks of acute gastroenteritis in Glasgow and the West of Scotland Study of genetic reassortment between two human rotaviruses Rapid diagnosis of rotavirus infection by direct detection of viral nucleic acid in silver-stained polyacrylamide gels Human reovirus-like agent as the major pathogen associated with «winter» gastroenteritis in hospitalized infants and young children Changing RNA patterns in rotaviruses of human origin: demonstration of a single dominant pattern at the start of an epidemic and various patterns thereafter Cleavage of structural proteins during the assembly of the head Stbdy.of human rotavirus genome by electrophoresis: attempt of classification among strains isolated in France Survey of human rotavirus propagation as studied by electrophoresis of genomic RNA Molecular epidemiology of human rotaviruses in Melbourne, Australia from 1973 to 1979, as determined by electrophoresis of genome ribonucleic acid Sequential enteric illness associated with different rotavirus serotypes Rotavirus RNA segments sized by electron microscopy Molecular epidemiology of human rotavirus infections Analysis of human rotavirus mixed electrophoretypes Molecular epidemiology of human rotavirus infection in children in Hong Kong We thank Dr GvM, Beards and Dr R.C. Sanders, Regional-Virus Laboratory and World Health Organization Collaborating Centre for Reference and Research on Rotaviruses, East Birmingham Hospital, Birmingham, United.Kingdom, for supplying polyclonal sera and human rotavirus-specific monoclonal antibodies.