Antigenic characterization and ELISA detection of adult diarrhea rotaviruses

Recently, severe epidemics of diarrhea among both adults and children in China have been associated with an agent called adult diarrhea rotavirus (ADRV). We have studied ADRV from two areas of China (Jinzhou and Guangxi) and compared them with prototype group A, B, and C rotaviruses. The viral genomes were compared by electrophoresis of the RNA genome segments in polyacrylamide gels, and the antigenic relatedness of these viruses was examined by immune electron microscopic studies using virus preparations of either double- or single-capsid particles. Hyperimmune antisera (guinea pig and rabbit) to one strain of ADRV reacted with high titers with other strains of ADRV and with single-shelled capsids of a porcine group B rotavirus. The antisera to ADRV did not react with either group A or group C rotaviruses. Antisera to either bovine or porcine group B rotaviruses reacted with ADRV but not with the group A or group C viruses. Antisera to group A or group C virus reacted only with their respective homologous virus. These results and those of immunofluorescence studies place the human ADRV rotaviruses from China among the group B rotaviruses. We also report the development of an ELISA to detect ADRV; this ELISA should be useful for defining the epidemiology of these recently described rotaviruses.     

44株人A组轮状病毒武汉流行株的培养鉴定及其意义

目的对A组轮状病毒武汉流行毒株进行培养增殖并确定其基因型和血清型。材料经RT-PCR确定基因型和ELISA确定VP6亚组血清型后初步判定的57株不常见型、混合型及未确定型别的A组轮状病毒流行毒株。方法MA104细胞分离培养病毒;聚丙烯酰胺凝胶电泳确定电泳型;逆转录巢式聚合酶链反应确定G、P、VP6和NSP4基因型;ELISA确定VP6亚组血清型。结果分离率77.2%(44/57)。分离了一株具备双重亚组血清型特征的人轮状病毒R479。分离后基因混合型、非常见基因型别或非常见G、P组合、不能确定的基因型别及VP6亚组血清混合型的样本数量与分离前相比显著减少。结论轮状病毒流行毒株的分离培养有助于提高其基因分型的敏感性,减少基因分型和血清分型的非特异性反应。     

Epidemiology of rotavirus infection around Tokyo area

Epidemiology of rotavirus infection was studied from 1981 to 1988 mainly in three hospitals around Tokyo area. Major serotypes of rotaviruses in the three places were different from those in two hospitals around Kansai area in Japan (Ref. 6, 13), while, major serotypes were same among three hospitals. Both of serotypes 1 and 4 in group A were mostly found around Tokyo area. Frequencies of type 2, 3, and 9 in group A were low, although the frequencies were various among periods. Detail examinations of rotavirus RNA electropherotypes showed the results as follows; different electropherotypes were found during one winter season and at one hospital, the identical electropherotype was found cross a year and cross a hospital. We could not find the identical electropherotype which belong to two serotypes so far. Seven group C rotaviruses were found since 1987 in three hospitals. It would be important to examine RNA electropherotypes and serotypes for long period not only for epidemiological studies but also for development of vaccine.     

An outbreak of gastroenteritis associated with acute rotaviral infection in schoolchildren

In April 1988 a large outbreak of group C rotavirus infection associated with acute gastroenteritis occurred among schoolchildren and their teachers simultaneously at seven elementary schools in Fukui city, Japan. Of 3,102, 675 (21.8%) became ill. Clinical symptoms were mild, predominantly abdominal pain and vomiting, with diarrhea reported in only 27.6%. The outbreak subsided within 2 d. No pathogenic bacteria were found in fecal specimens; the virus particles detected by electron microscopy were morphologically indistinguishable from conventional infantile rotavirus. Immune electron microscopy showed that these virions formed large aggregates with convalescent serum and with the reference serum specific to group C rotavirus. Polyacrylamide gel electrophoresis showed similar RNA patterns for virus from this outbreak and typical group C rotavirus.     

Serological and genetic characterization of bovine rotaviruses in Thailand by ELISA and RNA-RNA hybridization: detection of numerous non-serotype 6 strains

A total of 62 fecal specimens positive for rotavirus were collected from diarrheic cows in Thailand in 1988 and 1989. The antigenic properties of rotaviruses in stool were examined by enzyme-liked immunosorbent assays using specific monoclonal antibodies directed at VP4, VP6 or VP7: all the bovine rotavirus strains were determined as subgroup I; none of the strains were reactive with serotype 6-specific monoclonal antibody; and different reactivities of the bovine strains with two anti-VP4 monoclonal antibodies were observed. Polyacrylamide gel electrophoresis of viral RNA exhibited three different RNA electropherotypes. In RNA-RNA hybridization experiments using cell culture-adapted three strains as well as a reference bovine strain (NCDV), RNA from the Thai bovine strains showed very low homology to that from NCDV; only three or four RNA segments were hybridized between the RNAs from Thai samples and NCDV. These results suggested that bovine rotaviruses isolated in Thailand are serologically and genetically distinct from a reference serotype 6 bovine strain, NCDV.     

Serotypes and electropherotypes of group A human rotaviruses in patients with acute gastroenteritis in Saitama area, 1988-1991]

Serotypes and RNA electropherotypes of group A human rotaviruses were identified in stool samples from patients with acute gastroenteritis in Kasukabe Kosei Hospital, Saitama, Japan, during three rotavirus seasons from 1988 to 1991. Of the 665 stool samples from patients with acute gastroenteritis, 169 (25.4%) stool samples were positive for group A human rotaviruses. Of these 169 samples, 98 (58.0%) were serotype 1, 7 (4.1%) serotype 2, 14 (8.3%) serotype 3, 3 (1.8%) serotype 4 and 47 (27.8%) were untypable. Serotype 1 was predominant over all three rotavirus seasons. Serotype 2 was most prevalent in the second rotavirus season, serotype 3 in the third season. Serotype 4 was detected only the second rotavirus season. The ratio of subtype I to subtype II was 4.5:95.5. Among the 162 strains examined, 139 specimens were available to be electropherotyped. Fifteen different RNA electropherotypes were detected. Three to four different electropherotypes were more prevalent in each rotavirus season and the detection rate in each year was different within the same electropherotypes. The rotavirus strains of the same electropherotypes were observed with high prevalence for every rotavirus seasons. The same electropherotypes was found in human rotavirus strains of different serotypes that were appeared at different epidemic seasons.     

健康人群及腹泻患儿轮状病毒和腺病毒检测

用PAGE法于1987年10月～1988年1月对衡水市健康幼儿和中小学生及腹泻患儿进行了轮状病毒和腺病毒的调查。307名健康人做3次追踪检查未查出带病毒者;腹泻患儿带病毒率58.6％,其中轮状病毒和腺病毒感染分别占病原检出例数的72.1％、18.1％,双重及三重混合感染约占10.0％。轮状病毒以长型为主,发现了3种过去我省未见的基因组合型。     

Clinical and immunological studies of rotavirus vaccines

The RIT 4237 bovine rotavirus vaccine has served as a useful model for rotavirus vaccination, but the vaccine will not be further developed or tested. The main problem encountered with this vaccine was its poor "take" rate in developing countries. The reasons for this are unclear, and it is not known whether other bovine rotavirus vaccines are more efficacious in this respect. WC-3 bovine rotavirus vaccine will shortly be tested at several sites in developing countries. The rhesus rotavirus vaccine RRV-1 does not appear by itself to be a practical vaccine either. It has induced only moderate protection against human rotavirus serotypes other than the vaccine type. However, the fact that RRV-1 vaccine has induced substantial protection against severe diarrhoea caused by serotype 3 rotavirus, even in young infants, is promising and supports the concept that serotype-specific neutralizing antibodies play a role in protection against human rotavirus disease. Based on this concept, it has been possible to develop reassortant rhesus rotaviruses in which one RNA segment of human rotavirus, which encodes the expression of VP7 antigen, has been incorporated (Kapikian et al., 1986; 1987). Such rhesus-human reassortant rotaviruses, representing serotypes 1, 2 and 4 of human rotavirus and serotype 3 of rhesus rotavirus, can be combined to make a tetravalent vaccine that might induce neutralizing antibodies against each of the main serotypes of human rotavirus. Although its efficacy is unproven, such a combination vaccine is presently regarded as the most promising candidate rotavirus vaccine for the prevention of human rotavirus disease.     

Rescue of noncultivatable human rotavirus by gene reassortment during mixed infection with ts mutants of a cultivatable bovine rotavirus.

Fastidious human rotaviruses that did not undergo productive infection in tissue culture were rescued by genetic reassortment during mixed infection with a temperature-sensitive (ts) mutant of a cultivatable bovine rotavirus. In this manner, the genes of the fastidious rotavirus that restricted growth in vitro were replaced by the corresponding genes from a tissue culture-adapted rotavirus. We recovered genetically reassorted viruses that grew to high titer and were neutralized specifically by hyperimmune guinea pig type 1 or type 2 human rotavirus antiserum. Preliminary RNA analysis of these clones disclosed that they were indeed viruses with reassorted genes.     

Antigenic and genetic analyses of human rotaviruses in Chiang Mai, Thailand: evidence for a close relationship between human and animal rotaviruses.

Serotyping of group A rotaviruses obtained from stools of 158 diarrheic patients in Chiang Mai, Thailand, by ELISA with monoclonal antibodies revealed a yearly change in the prevalence of individual serotypes. Three unusual human rotavirus strains were isolated. Strain Mc35 had subgroup I-serotype 10 antigen and a long RNA electrophoretic type, a property hitherto found only in bovine rotaviruses. RNA-RNA hybridization tests showed that the strain is more closely related genetically to bovine than to human rotaviruses. Strain Mc323, although serologically closely related to serotype 9, had subgroup I specificity and a long RNA electrophoretic type, a characteristic common to nonhuman rotaviruses. Strain Mc345, with an aberrant RNA pattern possibly due to genome rearrangement, had the same antigenic specificity as Mc323. These 2 strains were genetically very closely related to each other and were more related to porcine than to human rotaviruses. These results provide insights into the evolutionary mechanisms of human rotaviruses.     

Epidemiology of rotavirus in Guayaquil, Ecuador

Detection of rotavirus by electron microscopy was conducted with fecal specimens from 1,722 infants and young children with acute diarrhea, during a 41-month survey from April 1978 through December 1981 in Guayaquil, Ecuador; 376 of these specimens (21.8%) were positive. The detection rate was higher during the dry season (May to November; 25.2%) than during the rainy season (December to April; 14.7%). When rotaviruses isolated from 59 patients hospitalized with diarrhea (from April 1979 to July 1981) were subjected to genome RNA analysis by polyacrylamide gel electrophoresis, a single dominant electropherotype was found with other less common electropherotypes. An atypical rotavirus with a unique property was also found.     

Maternal antibody-mediated protection against gastroenteritis due to rotavirus in newborn mice is dependent on both serotype and titer of antibody

In order to evaluate the role of passively acquired, rotavirus-specific antibodies in protection against diarrhea, we inoculated mouse dams with rotaviruses of various serotypes, and their newborns were orally challenged with a primate rotavirus (simian SA-11). Dams were immunized by using a regimen that included repeated inoculations administered either orally or intraperitoneally with adjuvant. The serum antibody response detected in dams by radioimmunoassay and plaque-reduction neutralization after parenteral immunization was approximately 15-fold and 80-fold greater, respectively, than that found after oral "hyperimmunization." Parenteral immunization with rotavirus serotypes either homotypic or heterotypic to the challenge virus protected suckling mice against diarrhea; protection was closely correlated with the in vitro neutralizing activity of maternal serum against the challenge virus. Oral immunization with only rotavirus strains homotypic to the challenge virus afforded protection; the lower immune response after oral immunization with rotaviruses heterotypic to the challenge virus resulted in a titer of neutralizing antibody to the challenge virus below the protective threshold. From our current studies it appears that antibody-mediated passive protection against rotavirus challenge is dependent on both serotype and titer of antibody.     

Atypical rotavirus isolated from acute infectious nonbacterial gastroenteritis in Fukuoka

An outbreak of epidemic diarrhea (nonbacterial acute gastroenteritis) in Fukuoka was studied electron microscopically and electrophoretically. From the result of electron microscopy, rotavirus particles about 70 nm in diameter were observed in fecal samples. However, the serological study did not reveal any antigenic relatedness to the ordinary rotaviruses by RPHA. The outbreak appeared to be caused by group C rotaviruses, based on the pattern of the RNA genome segments in polyacrylamide gels.     

猴轮状病毒的灭活、纯化及抗原性鉴定

目的探讨经β丙内酯灭活的猴轮状病毒SA11的抗原性。方法将猴轮状病毒适应到vero细胞上,用β丙内酯灭活后,接种MA104细胞,鉴定灭活效果,经超滤浓缩、凝胶过滤后,加入弗氏完全佐剂免疫豚鼠,确定豚鼠产生的中和抗体效价。结果利用β丙内酯在4℃作用24、48、72或96h,可以有效的灭活轮状病毒SA11,通过凝胶过滤可得到初步纯化的轮状病毒,以1.33mg/只的剂量与弗氏完全佐剂混合免疫豚鼠后,中和抗体的滴度为1∶1024。结论用β丙内酯可以有效的灭活轮状病毒。     

Detection of rotaviruses by nucleic acid hybridization with cloned DNA of simian rotavirus SA11 genes

We developed a dot-blot hybridization assay to detect rotaviral RNA sequences in tissue culture or in clinical samples. 32P-labeled cloned cDNA probes of the simian rotavirus SA11 specifically detected rotaviral RNA sequences and were more sensitive for detecting SA11 than was the commercial enzyme-linked immunosorbent assay Rotazyme test. A full-length probe of SA11 gene 6 detected 2.5 X 10(5) SA11 particles or approximately 0.27 ng of purified SA11 dsRNA. Combined probes from genes 6 and 9 detected 0.135 ng of purified SA11 dsRNA. The assay detected group A rotaviruses from different subgroups and serotypes, but the sensitivity of RNA detection varied from 0.5 to 31 ng when RNA from heterologous strains of virus was analyzed. An analysis of coded stool samples correctly identified 31 (91%) of 34 samples positive for rotavirus by electron microscopy and 100% of 26 samples negative for rotavirus by electron microscopy. Preliminary experiments also showed the assay has potential to directly characterize (subgroup and serotype) rotaviral isolates.     

福建省首次检出新的成人轮状病毒

本文应用PAGE方法,对福州地区腹泻病人的粪便标本,进行轮状病毒RNA基因分析。首次从1例不明原因成人腹泻病人粪便中检出1株RNA电泳图型基因模式为4:2:5的成人轮状病毒。其图型与资料报道的成人轮状病毒和人副轮状病毒的RNA电泳图型不同。此外,该标本的上清液负染后电镜观察,发现形态学上与轮状病毒一致的病毒颗粒,大小约70～75nm,同时还观察到大小约50～52nm的轮状病毒核心样结构。鉴于以上检测结果,我们认为,该株病毒可能系B组新的成人轮状病毒。它的出现提示该病毒已在我省境内流行,并存在感染者,应引起高度重视。     

Human serogroup C rotavirus in Finland

Among 600 rotavirus specimens collected in Finland during 1985-1987, 6 isolates were found to be related to serogroup C rotavirus by RNA profile analysis and solid phase immune electron microscopy (SPIEM). The group C isolates originated from 4 children (4.5, 4.5, 6.5 and 7 years old) with diarrhoea.     

Conservation of amino acid sequence of VP8 and cleavage region of 84-kDa outer capsid protein among rotaviruses recovered from asymptomatic neonatal infection.

Within the past few years, rotavirus strains were recovered from four discrete prolonged outbreaks of infection in newborn nurseries in which affected infants failed to develop significant symptoms. The virus strains recovered from each outbreak belonged to a different human rotavirus serotype and thus each of the four human rotavirus serotypes was associated with asymptomatic infection of neonates. Marked conservation of sequence was observed among the fourth genes of the nursery rotavirus strains in a previous study using RNA X RNA hybridization, while a different conserved set of fourth gene sequences was identified among virulent human rotaviruses representing the four known serotypes. In the present study, this sequence dimorphism was further evaluated by comparing the sequence of the region of the fourth gene of virulent and asymptomatic human rotaviruses that codes for the VP8 protein, downstream cleavage sites, and the NH2 terminus of VP5. The corresponding sequences of a simian rotavirus were also determined. The fourth segment (+) strand RNA has a 5' conserved nontranslated sequence of nine nucleotides and encodes a VP8 protein of 240 amino acids in human rotavirus strains and 241 amino acids in simian rotavirus strains. Human and simian rotaviruses exhibit many similarities in this region of their genome, including identical NH2-terminal amino acid sequences, conservation of arginine at the two trypsin cleavage sites, and the position of a cysteine residue. Alignment of amino acid sequences of the VP8 protein, the downstream cleavage region, and the NH2 terminus of VP5 of asymptomatic and virulent human rotavirus strains indicates a high degree of homology (96% or more) among the asymptomatic viruses (serotypes 1, 2, 3, and 4), while homology between asymptomatic strains and virulent viruses is considerably less (68-72%). A high degree of conservation of amino acid sequence (92-97%) is also observed among three of the virulent strains (serotypes 1, 3, and 4). At 48 positions in the protein sequence of VP8, the cleavage region, and the NH2 terminus of VP5, an amino acid is conserved among asymptomatic rotaviruses, while a different amino acid is conserved among virulent rotaviruses. Notably, three of these differences are located within the cleavage region between VP8 and VP5. These findings suggest that the fourth genes of virulent and asymptomatic human rotavirus strains represent two lines of divergent evolution from a common ancestor. Also, it is possible that this sequence dimorphism may be responsible in part for the difference in virulence between these two groups of human rotaviruses.     

Rotavirus associated diarrhoea during infancy in the city of S. Luís (MA), Brazil: a two-year longitudinal study.

A total of 479 diarrhoeic children and 337 children without diarrhoea (controls) less than 5 years old were investigated in a two-year study in the city of S. Luís (MA), with the purpose to determine the incidence, the age distribution and the seasonality of rotaviruses, as well as to establish the severity of the disease in this region between the North and the Northeast of Brazil. rotavirus incidence was highest in children of the 1st. year of life, showing an average of 25% per year among the diarrhoeic patients attending the two main hospitals and three health units at the periphery of the city. It was shown that rotaviruses are significant enteropathogens in children less than 18 months old. Frequency of rotaviruses dropped in diarrhoeic patients 18 to 23 months old to only 4%, the same percentage observed in children of the control group. A typical seasonal distribution of rotaviruses was not seen during the two years of study. There was a peak in the incidence of rotaviruses in 1986, during the rainy season, and two peaks in 1987, one in the rainy season and one in the dry season. It was also shown that severity of diarrhoea in rotavirus positive cases was higher than in the negative cases. Rotavirus diarrhoeic patients had more loose stools per day, and higher frequencies of vomiting and fever, resulting more often (> 2 times) in moderate or severe dehydration. Finally, it is concluded that the introduction of immunoprophylaxis may reduce significantly the high mortality rates in early childhood observed in S. Luís.     

Some Structural and Antigenic Properties of Intracisternal A Particles Occurring in Mouse Tumors

Intracisternal A-particles were isolated from three different myeloma lines in BALB/c mice and from cultured neuroblastoma cells of A/J origin. All preparations contained a major structural protein with an apparent molecular weight near 70,000 as estimated by electrophoretic mobility in sodium dodecyl sulfate-containing polyacrylamide gels. Solubilization of this component by sodium dodecyl sulfate was dependent on prior or concomitant treatment with sulfhydryl compounds. The size distribution of A-particle proteins was markedly different from that observed for extracellular murine leukemia and mammary tumor viruses. Rabbit antiserum was developed that reacted with the major A-particle protein in both complement fixation and immunodiffusion assays. The antigen was detected in isolated neuroblastoma A-particles, in cytoplasmic membrane fractions prepared from various mouse tumors known to contain intracisternal particles, but not in preparations from normal mouse cells, in samples of leukemia and mammary tumor virus, or in JLS-V9 cells infected with Rauscher leukemia virus. Conversely, isolated A-particles did not react in complement fixation or immunodiffusion assays with antisera against leukemia virus antigens.