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The Use of Polyacrylamide Gel Electrophoresis of Virus RNA in the Study of Rotavirus Infections
Abstract
Polyacrylamide gel electrophoresis of rotavirus RNA has been used to provide diagnostically and epidemiologically useful information about avian and mammalian rotavirus infections. (1) Rotaviruses possessing four different electropherotypes were detected in a longitudinal survey of a 34,000 bird broiler flock. Two of these had not been described previously in chickens. When virus recognisates were grouped according to electropherotype, infection with different electropherotypes was seen to occur in waves, with each wave lasting about one week. Cross-immunofluorescence indicated that rotaviruses with different electropherotypes were antigenically distinct, with only one sharing an antigen with conventional mammalian rotaviruses. (2) Mich intra-species variation was observed when bovine and porcine rotavirus RNAs obtained from different farms were analysed on high resolution 12.5% polyacrylamide gels. Consistent differences were observed between bovine and porcine rotavirus RNA profiles. The detection of additional bands in some RNA profiles indicated that more than one population of rotavirus was present in some faeces specimens. The finding that only one electropherotype was observed in samples taken from heifer calves from a “closed” dairy farm over a 16 month period suggested that only one rotavirus was present and that its genome was electrophoretically stable. (3) One electrophoretically “atypical” recognisate was observed out of 70 samples from bovine or porcine clinical cases.
Western blotting is an important analytical technique used in cell and molecular biology for last four decades. It involves separation of proteins in SDS-PAGE and then transfer of proteins to a membrane followed by detection. By using a western blot, one can identify specific protein from a complex mixture of proteins. Along with its use as a diagnostic aid, it can also be used to verify proteins of interest in exploratory proteomic studies to identify different disease mechanisms. The ease of performing the technique, low cost, and accessibility further support the use of western blot in proteomic research. However, a good understanding, initial training, and optimization are of utmost importance because being a multi-step technique, it is prone to false results and incorrect interpretation. This chapter attempts to explain the technique and theory behind western blot along with some ways to troubleshoot.Key wordsSDS-PAGEWestern blotProtein blottingImmunoblot
A total of 341 fecal samples obtained from children under five years of age with acute diarrhea disease, attending two nursing homes in Loja city, Ecuador were studied for rotavirus diagnosis. Out of them, 56 (16%) were rotavirus positive and 33 samples were genotyped for the determination of genotypes G and P. The most frequent genotypes were G4 (42%), P [6] (36%) and +9 mixed combination G4 P [6] (21%). This is the first molecular study on rotavirus carried out in Southern Ecuador and the obtained data confirms the variability of the rotavirus circulating strains in Ecuador.
Five different sorts of RNA profiles (electropherogroups 1 to 5) were recognised when avian rotavirus RNAs were electrophoresed in polyacrylamide gels. These could be distinguished from the RNAs of mammalian group A, B and C rotaviruses. Viruses belonging to electropherogroups 1, 2/3 and 4 were detected in chickens and viruses belonging to electropherogroups 1, 2, 3 and 5 were detected in turkeys. A minimum of three electropherogroups was recognised in each of four longitudinal surveys of broiler chicken flocks. On examination of rotaviruses from different clinical outbreaks of diarrhoea and from different longitudinal surveys minor differences involving almost all the RNA segments were observed when rotaviruses from the same electropherogroup were compared. Detailed RNA analysis in two of the surveys detected up to three electropherotypes within each electropherogroup.
Polyacrylamide gel electrophoresis (PAGE) and enzyme-linked immunosorbent assay (ELISA) were employed to investigate the epidemiology of typical and atypical rotavirus infections in five piggeries. Of 152 faecal samples examined, 46 (30 per cent) were positive by ELISA for group A rotavirus. Rotaviruses with electrophoretic patterns resembling groups A, B and C were detected. At least two and up to five different rotavirus electrophoretypes (typical and/or atypical) were detected in each of the five piggeries. Out of 152 faecal samples examined, 28 (18 per cent) contained rotaviruses with group A electrophoretypes, 9 (6 per cent) with group C but only 1 with Group B. Six samples contained both group A and group C rotaviruses. No common electrophoretypes of group A or C rotaviruses were detected in these five piggeries. The PAGE technique was also used to analyze group A rotavirus isolated sequentially from another piggery over a three year period. A single electrophoretype was found during the first two years, but in the third year a different electrophoretype was detected.
A porcine virus with rotavirus morphology, which was antigenically unrelated to previously described rotaviruses, is described. Particles with an outer capsid layer measured 75 nm and those lacking the outer layer were 63 nm in diameter. Particles which resembled cores were also identified. The virus was shown to be antigenically distinct from other rotaviruses as judged by immunofluorescence and immune electron microscopy, and it failed to protect piglets from challenge with porcine rotavirus. Analysis of the genome indicated that it was composed of 11 segments of double-stranded RNA with the same overall size range as other rotaviruses but with an unusual segment pattern.
Rotaviruses contain a double-stranded ribonucleic acid genome consisting of 11 segments. Gel electrophoresis separates genome segments and allows identification of strain differences. This electrophoretic typing technique was applied to rotavirus specimens from 116 children and 72 newborn babies. Between 1973 and 1979, 17 different electropherotypes of rotavirus were observed in children with acute gastroenteritis. These electropherotypes showed a sequential pattern of appearance, with a limited number of electropherotypes present at any given time. By contrast, only two electropherotypes were identified from isolates from newborn babies in seven hospitals during 1975 to 1979. These two electropherotypes were very similar and were never identified in children with acute gastroenteritis. One of the neonatal electropherotypes was found in the nurseries of five different hospitals and persisted in one hospital for 4 years. Electrophoretic typing techniques can be applied routinely and reproducibly to small samples of feces and could prove to be of value in epidemiological studies of rotavirus infection
The changes in human rotavirus electropherotypes, occurring during a period including two rotavirus gastroenteritis epidemics in 1976 and 1979 in relatively remote Central Australia, were determined by polyacrylamide gel electrophoretic analysis of the rotavirus genome ribonucleic acid. A number of different electropherotypes were present during each of the epidemics, although a single type was predominant in each one. The predominant electropherotype of the first epidemic persisted in the area for approximately 2 years afterwards. Apart from this electropherotype, only three others were recognized in the 3 years between the two epidemics. One of these, first seen 1 year before the second epidemic, bore a very close similarity to the predominant type of the second epidemic. Altogether, 12 different electropherotypes were recognized during the period of the survey. No type common to both areas was found when rotavirus electropherotypes recognized in Central Australia were compared with those detected in a 1973-to-1979 survey in Melbourne, Australia.
Some characteristics of a newly recognized porcine enteric virus are described. Tentatively, the virus was referred to as porcine pararotavirus (PaRV) because it resembled rotaviruses in respect to size, morphology, and tropism for villous enterocytes of the small intestine. However, it was antigenically distinct from porcine, human, and bovine rotaviruses and reoviruses 1, 2, and 3, and the electrophoretic migration pattern of PaRV double-stranded RNA was distinct from the electrophoretic migration patterns of the rotaviral and reoviral genomes. By passage in gnotobiotic pigs, PaRV was isolated from two suckling diarrheic pigs originating from two herds. After oral exposure of gnotobiotic pigs, villous enterocytes of the small intestines became infected as judged by immunofluorescence, resulting in villous atrophy and diarrhea. Mortality was high when gnotobiotic pigs less than 5 days old were infected. The C strain of this virus was serially passed 10 times in gnotobiotic pigs, and electron microscopy, immunofluorescence, and serological tests indicated no extraneous agents. The virus was serially passed five times in cell cultures which contained pancreatin in the medium, but replication was negligible or absent, as the number of immunofluorescent cells decreased with each passage. Since rotaviral infections are frequently diagnosed by direct electron microscopy of fecal specimens, the presence of other morphologically similar viruses, such as PaRV, should be considered. The use of immune electron microscopy is suggested as a means of helping recognize this situation.
The genomic RNA patterns of six different bovine rotavirus isolates were analyzed on high-percentage polyacrylamide gels (12.5, 13.6, and 17.5%). In contrast to the RNA patterns exhibited by conventional gel systems, those on the high-percentage gels showed an improvement in segment resolution which consequently aided in the detection of extensive band splitting in these patterns. The ability to clone out various electrophoretically distinct virus subpopulations from each of the six isolates provided an explanation for the band splitting detected by the high-resolution gels. The significance of the coexistence of genetically distinct rotavirus populations within a single host is discussed.
A rotavirus-like agent was detected in the feces of a child with diarrhea. Although morphologically indistinguishable from rotavirus, the agent was serologically distinct, and electrophoresis of its nucleic acid also showed that it was dissimilar.
Stools from 183 babies under 2 years of age admitted to Ruchill Hospital with diarrhoea were examined by electron microscopy, virus culture, bacterial culture and light microscopy. As far as possible, several stools were examined from each patient and the results showed rotaviruses, astroviruses and other viruses in association with symptoms, as well as the expected bacterial pathogens. Examination of several stools from the same patient also showed that in this age group the viral flora of the gut changes rapidly and that the viruses seen by electron microscopy were only rarely grown in cell culture and vice versa. This phenomenon was particularly noted with adenoviruses. In 30% of cases no microbial pathogen was identified and in the remainder the presence of the infecting organism did not always coincide with the symptoms. It is concluded that, with viruses at least, presence of the organism does not constitute proof of causation.
A total of 151 specimens of turkey and chicken faeces and intestinal contents were examined for the presence of viruses by electron microscopy. Viruses were detected in 48 of these specimens (32%). The most frequently observed viruses were rotaviruses and enterovirus-like particles. Rotavirus infection was associated with outbreaks of diarrhoea in turkeys, but symptomless rotavirus infection was seen in broiler chickens. Adeno-viruses and infectious bursal disease virus were also observed in turkey faeces. The best method for preparing faecal material for examination employed initial purification by extraction with a fluorocarbon, followed by concentration in the ultracentrifuge. Examination of the pooled contents of the caeca and large intestine gave better results than examination of small intestinal contents. It is concluded that direct electron microscopic examination of faeces has considerable potential as a diagnostic technique in avian virology.
Six isolates of rotavirus were made from turkeys and two from chickens. Three of these required trypsin treatment for isolation and serial passage in cell cultures. The remainder were isolated without trypsin treatment. Most of these viruses were isolated in chick embryo liver cell cultures from the faeces of birds aged under 1 week. In six of the eight instances, rotavirus isolation was associated with enteric disturbance, characterised by signs such as diarrhoea, poor or abnormal appetite, abnormally fluid or gaseous intestinal contents or increased mortality. Cross immunofluorescence tests showed that while avian and mammalian rotaviruses shared a common group antigen, the avian viruses were more closely related to each other than to the Nebraska calf rotavirus isolate. On the basis of serum neutralisation tests seven of the eight avian rotaviruses were grouped into three serotypes, with two turkey isolates (Ty1 and Ty3) and a chicken (Ch1) virus being the prototype strains. The remaining virus, Ty2, was intermediate in type between Ty1 and Ch1. Analysis of the RNA of these viruses by polyacrylamide gel electrophoresis showed that they could also be grouped into a number of electropherotypes. The isolates which were serologically distinct were also electrophoretically distinct. Similarly the five isolates which belonged to the Ty3 sero-type were electrophoretically identical. Analysis of the serological and electrophoretic differences suggested that RNA segment 5 may code for a type-specific antigen.
Rotaviruses resemble reoviruses and orbiviruses in certain aspects of their morphology and morphogenesis and in possessing a double-stranded segmented RNA genome. However, they are antigenically distinct from reoviruses and orbiviruses, and occupy a position intermediate between reoviruses and orbiviruses in their stability to various inactivating agents. Rotavirus infections are very common in young animals and humans and in many instances are associated with enteritis and diarrhoea. However, as discussed above, symptomless rotavirus infections have also been recorded. While rotaviruses are capable of producing pathological lesions and diarrhoea in colostrum-deprived animals, there is insufficient quantitative information on their ability to cause disease in colostrum-fed animals to allow proper evaluation of their importance as pathogens under field conditions. Other areas for future work include developing a means to grow rotaviruses serially in cell cultures, investigating the biochemical aspects of their replication, establishing the number of serotypes which exist and finding out more about the epidemiology and immunology of rotavirus infections.
Analysis of the polypeptides induced by 29 herpes simplex virus type 1/type 2 intertypic recombinants and correlation of the data with the crossover points in the recombinant DNAs have enabled the map positions of many polypeptides to be deduced. These include 25 polypeptides which label with [35S]methionine, 11 which label with [32P]orthophosphate, and 4 which label with [14C]glucosamine. Together with the data of Preston et al. (J. Virol., in press) on the mapping of five immediate-early polypeptides, the results show that representatives of four groups of proteins--immediate-early, late, phosphorylated, and glycosylated--map in both long and short regions. The functional organization of the herpes simplex virus genome does not therefore restrict any of these four groups to either the long or the short region.
A rapid simple technique for the diagnosis of rotavirus has been developed based on the sensitive detection of rotavirus double-stranded RNA genome segments separated in polyacrylamide gels. The method utilizes a recently described ultrasensitive silver stain for polypeptides, which can also detect subnanogram amounts of nucleic acid. The sensitivity of the technique is comparable with that of electron microscopy or enzyme-linked immunosorbent assay.
The RNAs of 4 avian rotavirus isolates were analysed by polyacrylamide gel electrophoresis. One of these isolates was made from chickens, the other three were from turkeys. The RNAs were separated into 8 or 9 bands consisting of 11 segments, ranging in molecular weight from 0.24 × 106 to 2.09 × 106. In contrast to calf rotavirus, segments 10 and 11 of avian rotavirus RNA had very similar or identical electrophoretic mobilities. This may be a unique feature of avian rotavirus RNA.
Co-electrophoresis experiments showed that the RNAs of 2 of the turkey isolates were electrophoretically identical. However, differences existed between these 2 isolates, the chicken isolate and the remaining turkey isolate with respect to segments 5, 6, the 7, 8, 9 complex and 10. Segment 5 of the chicken isolate was sometimes resolved into 2 components, and was further anomalous in that it was absent in some (analyses).
Detection of human and bovine rotavirus in stools is described using a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) with poly-styrene microtest plates as solid phase, immunoglobulin fraction of rabbit antiserum to rotavirus (human) as catching antibody, and the same reagent labelled with horseradish peroxidase as conjugate. The ELISA has been optimized with regard to simplicity, rapidity, sensitivity, and specificity. In a comparative study, stool specimens from 81 infants and children and 92 neonatal calves with diarrhoea were tested for rotavirus by ELISA, electron microscopy (EM), immunoelectro-osmophoresis (IEOP), and fluorescent antibody technique (FA). The relative sensitivity of the different assays for human and bovine rotavirus was: EM 68%, 76%; IEOP 80%, 76%; FA not determined, 85%; and ELISA 86%, 98%, respectively. Less than 1 ng of purified human rotavirus could be detected in ELISA, whereas 100 ng was the minimal amount detected by IEOP. It is concluded that the developed ELISA is a simple, rapid, reliable, and sensitive method for the diagnosis of human and bovine rotavirus infections.
A virus, designated 132 virus, was isolated from the faeces of chickens in chick embryo liver cell cultures. The morphology and morphogenesis of 132 virus were indistinguishable from that of rotaviruses. The nucleic acid of 132 virus had the nuclease resistance of double-stranded RNA, and was separated by polyacrylamide gel electrophoresis into 11 segments with mol. wt. ranging from 2.07 x 10(6) to 0.20 x 10(6). SPF chickens were susceptible to oral infection with 132 virus, which replicated in the villous epithelial cells of the small intestine. 132 virus was therefore a rotavirus by morphological, biochemical and biological criteria. However, by immunofluorescence it was not possible to demonstrate an antigenic relationship between 132 virus and known avian and mammalian rotaviruses, indicating that 132 virus does not possess the group antigen shared by all previously characterized rotaviruses. This finding has implications for the diagnosis of rotavirus infections by serological tests.
During a survey of 350 different isolates of human rotaviruses, a single isolate with unique properties was obtained. Unlike all rotaviruses tested this unique isolate failed to react in an ELISA test with antibody directed against the group-specific rotavirus antigen. The morphology of this unusual virus isolate was identical in electron micrographs with other rotaviruses and the virus also contained 11 double-stranded RNA segments characteristic of this virus group. The electrophoretic migration of these RNAs (electrophoretype) in polyacrylamide gels did not coincide with the typical pattern of distinct size classes observed with all human rotaviruses. The unique isolate thus had two distinct properties that distinguish it from human rotaviruses, lack of a group-specific antigen and an atypical electrophoretype of the RNA segments. Based upon this, the new human isolate is given the name pararotavirus to distinguish this new type of virus group. Porcine and chicken pararotaviruses have recently been described with similar properties.
Rotaviruses isolated from 22 patients during a local outbreak of infantile gastroenteritis in October/November 1981 were genotyped by establishing their RNA migration patterns on polyacrylamide gels. A highly sensitive silver staining procedure was used to visualize the RNAs. It was found that strains with at least four different RNA patterns cocirculated and also showed serological differences.
Comparative serological and nucleic acid studies were carried out on rotavirus isolates, with and without the original group antigen. Indirect immunofluorescence allowed the atypical isolates to be divided into two groups each with its own distinct group antigen hence giving a total of at least three distinct rotavirus groups. Genome profile analysis of the atypical isolates again divided them into two groups which corresponded with those obtained by immunofluorescence and within which the overall pattern of genome segments was similar. A more rigorous examination of the degree of difference between the three virus groups was carried out using single-dimension terminal fingerprint analysis. This indicated that the viruses in the three groups did not carry any genome segments in common with each other. Therefore, experimental criteria have been established which allow the division of rotaviruses into three groups. Analysis of further atypical isolates, as they become available, will establish whether the division of rotaviruses into a number of separate groups, of the type seen in influenza virus, is justified.
A longitudinal survey of rotavirus infection in heifer calves was carried out on a closed Friesian dairy herd over two successive calving seasons. Rotavirus was detected by electron microscopy in the faeces of 45 of 57 (79 per cent) calves examined. On average the virus was first detected at 6.1 days of age. Clinically the disease associated with rotavirus infection was of mild to moderate severity. Only one infected calf required intravenous fluid therapy. Diarrhoea or excretion of abnormal faeces was associated with rotavirus infection in 58 per cent of infected calves, while in the remaining 42 per cent infection was subclinical. The cycle of rotavirus infection was broken by thorough cleansing and disinfection of the calf house.
In a longitudinal survey of 11 broiler flocks, rotavirus excretion was detected by direct electron microscopic examination of faeces in 10. In most of these flocks, rotavirus excretion was first detected during the third week of life. In some flocks, infection with 2 antigenically distinct serogroups of rotavirus was demonstrated. In a more detailed survey of a 34,000 bird broiler crop, rotavirus excretion was detected intermittently from 9-50 days. Infection with 4 different RNA electropherotypes of rotavirus occurred in waves with each wave of infection lasting about 1 week. Analysis of representatives of the 4 different electropherotypes by cross-immunofluorescence indicated that each electropherotype represented an antigenically distinct serogroup. Two of these serogroups were represented by the previously characterised Ch 1 and 132 chicken rotavirus isolates. The other 2 serogroups have not been previously recognised.