Isolation of cytopathic porcine rotavirus in cell roller culture in the presence of trypsin

Summary Cytopathic porcine rotavirus was isolated in roller tube cultures of MA-104 cells. Faeces of a piglet suffering from diarrhoea, were treated with trypsin which was also added to the maintenance medium. Using stationary cultures, virus was not isolated from the same materials. The cytopathic effect was clearly observed after 8 serial passages and the virus titer at the 14th passage level was 10⁷ median tissue culture infective doses per ml, or higher. At the 27th passage, rotavirus particles were seen by negative contrast electron microscopy at a density of 1.36 to 1.38 g/cm³ in CsCl centrifugation gradients. There are partial cross-neutralization between the Lincoln strain of bovine rotavirus and porcine rotavirus from pigs or tissue cultures. Gnotobiotic piglets were inoculated with pig-passaged virus and viral antigen was detected in epithelial cells of small intestinal villi by immunofluorescence. The highest titer of virus was detected in faeces 72 hours after inoculation. The cell culture adapted virus which produce a cytopathic effect was designated the strain S80 of porcine rotavirus.With 6 Figures     

Replication of human rotavirus in cell culture

Nine strains of human rotavirus were adapted to growth in CV-1 and/or MA-104 cells following pretreatment of virus with trypsin, incorporation of trypsin into culture medium, and use of roller cultures. Immunofluorescence was the most reliable method for the detection of virus replication, although characteristic cytopathic effects were produced sporadically by most isolates. Virus could be readily detected in supernatant fluids of cell cultures and in cell sections by electron microscopy.     

Radioimmunofocus assay for detection and quantitation of human rotavirus.

A radioimmunofocus assay suitable for quantitation of cell culture-adapted human rotavirus was developed. The method was reproducible, more sensitive than plaque assay, and useful to detect and quantify strains of rotavirus which do not produce plaques. Preliminary results also suggested that the technique will be a useful means of serotyping cell culture-adapted strains of the virus.     

Isolation from faecal specimens of new strains of human rotavirus primarily cytopathic for stationary cell cultures without trypsin

Summary Eight cytopathic human rotavirus strains were isolated in LLC-MK2 cells and in human embryo fibroblasts. The strains were isolated from faeces collected from pediatric and adult patients. Pretreatment of specimens with trypsin and trypsin incorporation in maintenance medium were not performed. Inoculated monolayers were not subjected to centrifugation and were incubated stationary at 36° C. Viruses were identified by electron microscopy and by fluorescent antibody techniques.It is suggested that these rotaviruses are different from any previously recovered from man.With 5 Figures     

Serotyping of cell culture-adapted subgroup 2 human rotavirus strains by neutralization

Nine human rotavirus strains from stools of infants with gastroenteritis were serially propagated in MA-104 cell cultures. All strains were identified as subgroup 2 rotaviruses by RNA gel electrophoresis, complement fixation, and enzyme-linked immunosorbent assay. The human rotavirus strains were propagated for 15 to 20 passages and then used for immunization of guinea pigs and rabbits. Animal antisera were also raised against a subgroup 1 human strain purified from stools and against the cell culture-adapted Wa strain, a reference subgroup 2 rotavirus of human origin. Cross-neutralization studies revealed the existence of two distinct serotypes within the cell culture-adapted subgroup 2 human rotaviruses: strains related and unrelated to strain Wa were classified as serotypes 1 and 3, respectively. Results with convalescent-phase sera from infants with primary rotavirus infections confirmed the existence of two serotypes within subgroup 2, and the serotypes responsible for primary subgroup 2 infections could be determined on the basis of the neutralizing reactivity of convalescent sera.     

Single gene substitution rotavirus reassortants containing the major neutralization protein (VP7) of human rotavirus serotype 4.

A series of reassortants was isolated from coinfection of cell cultures with wild-type bovine rotavirus (UK strain [serotype 6]) or rhesus rotavirus (strain MMU18006 [serotype 3]) and a tissue culture-adapted human rotavirus strain, ST3 (serotype 4). Monospecific antiserum or a set of monoclonal antibodies to the major outer capsid neutralization glycoprotein, VP7, of the animal rotavirus parent was used to select for reassortants with human rotavirus serotype 4 neutralization specificity. The majority of reassortants contained only gene 9 of the human rotavirus parent, ST3, whereas the remaining genes were derived from the animal rotavirus parent. These single human rotavirus gene substitution reassortants were neutralized to high titer by hyperimmune serum directed at ST3, thus demonstrating that gene 9 of ST3 codes for the major neutralization protein of this strain. Moreover, these single gene substitution, reassortants were also neutralized to low titer by antiserum directed at their animal rotavirus parent, probably because they derived gene 4, which codes for another outer capsid protein, VP3, from their animal rotavirus parent. None of the reassortants derived gene 4, which had previously been shown to be responsible for host range restriction of human rotaviruses in tissue culture, from ST3, despite the fact that the ST3 strain used for gene reassortment had been tissue culture adapted.     

Rapid serotyping of human rotavirus strains by solid-phase immune electron microscopy.

Nine cell culture-adapted, as well as 30 clinical, human rotavirus (HRV) strains from fecal extracts of children with primary HRV infection were typed by rapid solid-phase immune electron microscopy with protein A and absorbed DS-1 (HRV serotype 2), Wa (serotype 1), and VA70 (assumed serotype 3) rabbit immune sera. As a reference typing test for cell culture-adapted strains, the neutralization assay was used, whereas for noncultivatable strains typing was done for comparison, indirectly, based upon the differential neutralization reactivity of convalescent-phase serum samples from patients with primary HRV infection versus the three reference HRV serotypes. Typing results by solid-phase immune electron microscopy for all strains examined were in complete agreement with those obtained by the neutralization assay, both on cell culture-adapted strains with the three reference rabbit antisera and on three reference HRV strains with human convalescent-phase serum samples. Since adaptation to growth in cell cultures of clinical HRV strains from stool specimens is a time-consuming procedure and is often unsuccessful, solid-phase immune electron microscopy is preferred over the neutralization assay, giving results in about 16 h and also allowing typing of HRV strains from stool specimens low in virus particles. In addition, HRV strains reacting differently from the three reference serotypes may be easily selected by solid-phase immune electron microscopy for further characterization, as was the case for one strain in this study.     

Isolation, propagation, and characterization of a second equine rotavirus serotype.

A rotavirus designated strain H-2 was isolated in primary African green monkey kidney cells from a foal with diarrhea. This cell culture-adapted strain was found to be similar, if not identical, to simian rotavirus (strains MMU18006 and SA-11) and canine rotavirus (strain CU-1) and, in addition, demonstrated a one-way antigenic relationship with five human rotavirus strains (P, B, no. 14, no. 15, and YO) of the third human rotavirus serotype by the plaque reduction neutralization test. This is the fifth example of an animal rotavirus which shares serotypic specificity with a human rotavirus. The H-2 strain is distinct from the H-1 strain (Y. Hoshino et al., J. Clin. Microbiol., in press) of equine rotavirus not only in serotypic specificity by neutralization but also in subgroup specificity, hemagglutinating activity, and RNA electrophoretic migration pattern, thus establishing the existence of a second equine rotavirus serotype. This H-2 isolate is also distinct by neutralization from three other human rotavirus serotypes, 1 (Wa), 2 (DS-1), and 4 (St. Thomas no. 4), as well as bovine (NCDV), and porcine (OSU) rotaviruses.     

The VP8 fragment of VP4 is the rhesus rotavirus hemagglutinin.

The amino-terminal trypsin cleavage fragment of VP4, called VP8, was expressed from a recombinant baculovirus in Sf-9 cells. The baculovirus-expressed VP8 protein is antigenically conserved as demonstrated by its recognition by a library of neutralizing monoclonal antibodies. In Sf-9 cell sonicates, the expressed VP8 protein is capable of agglutinating human type O erythrocytes, indicating that the functionally intact rhesus rotavirus viral hemagglutinin is contained in the 247-amino acid VP8 trypsin cleavage fragment. Amino acid similarities between VP8 and the amino-terminal 282 amino acids of the reovirus sigma 1 protein suggests that the sigma 1 hemagglutination function resides within these amino-terminal amino acids as well. When the expressed VP8 protein was used to immunize mice, a broadly cross-reactive neutralizing antibody response was obtained. Antibodies elicited to the expressed VP8 protein neutralized viruses of serotypes 1-4 and 6 but not porcine strains OSU (st5) or Gottfried (st4). The neutralizing antibody response to VP8 appeared to be more cross-reactive than the immune response to expressed VP4 or to whole RRV virion. This suggests that subunit protein immunizations may broaden the neutralizing antibody immune responses to rotaviruses and enhance protective immunity to serotypically distinct strains.     

Two modes of human rotavirus entry into MA 104 cells

Summary Entry of the KUN strain of human rotavirus into MA 104 cells was studied by electron microscopy. Virus particles attached to the cell membrane appeared to be almost exclusively double-shelled virions. These attached virions followed two distinct pathways into the cytoplasm depending on pretreatment with trypsin. Using infectious rotavirus which had been pretreated with trypsin, the viral nucleoids passed directly into the cytoplasm within 5 minutes after inoculation, through dissolution of the viral capsid and cell membrane. Using non-infectious rotavirus that had not been pretreated with trypsin, phagocytosis or pinocytosis occurred in which virions were sequestered into lysosomes 20 minutes after virus attachment to the cell membrane. After being sequestered, uncoating of the rotavirus virions within lysosomes was seen, but it did not result in release of the genome.On the basis of these observations it was concluded that when virions were pretreated with trypsin, virus replication occurred following the direct passage of viral nucleoids into the cell cytoplasm. However, mere phagocytosis of virus particles into cell lysosomes, which occurred when trypsinuntreated virus was used, does not appear to be related to rotavirus replication.With 7 Figures     

Isolation of human rotavirus in cell cultures

Summary Three cytopathic rotavirus strains were isolated in MA104 cells from faecal specimens of pediatric patients with acute gastroenteritis. Pre-treatment of virus with trypsin and incorporation of a small amount of trypsin in maintenance medium were important for establishment of the strains in these cells. The isolates were antigenically closely related with strain Wa of human rotavirus and had some antigenic relationship with strain Lincoln of bovine rotavirus.With 1 Figure     

An improved enzyme-linked immunosorbent assay for the detection of rotavirus in faeces of neonates

A direct enzyme-linked immunosorbent assay (EIA) for the detection of rotavirus in neonatal stools was developed. Rabbit antiserum against SA 11 rotavirus was incorporated as both coating and detector antibody, and rotavirus-negative rabbit serum was applied as a coating antibody control to eliminate false positive results. Pretreatment of stools with EDTA was found to increase both the sensitivity and specificity of the assay. This effect was greatest when 0.25 M EDTA (tetrasodium salt) was included in homogenized stool suspensions before the removal of solid debris by centrifugation. By electron microscopy, this EDTA pretreatment appeared to partly uncoat human rotavirus particles in faeces. Potentially suitable solid phase supports and horseradish peroxidase substrates were evaluated in the development of the assay. Screening of stool samples revealed that repeated freezing and thawing of stools eliminated positive EIA reactions. The SA 11 coating antibody compared favourably with a reference coating antiserum prepared against human faecal rotavirus strains. This EIA showed greater sensitivity for rotavirus detection than electron microscopy of stool concentrates prepared by ultracentrifugation, on testing 143 stools from 99 neonates and children. The assay has been applied successfully to detection of rotavirus in stools of neonates containing meconium, smaller amounts of viral antigen than in older children, and lacteal antirotaviral antibody. It is likely to be particularly useful for cross-infection studies in hospital wards and neonatal nurseries.     

Investigation of immunoperoxidase-labelled rotavirus in tissue culture by light and electron microscopy

A tissue culture-adapted strain of bovine rotavirus, grown in calf kidney monolayers, has been examined by light and electron microscopy after immunoperoxidase labelling. Some of the characteristic problems associated with pre-embedding methods have been demonstrated. Preparative techniques involving pretreatment of infected cells with a detergent followed by a detergent/fixative combination have enabled virus antigen to be labelled while maintaining satisfactory ultrastructural preservation.     

Isolation, characterization, and serial propagation of a bovine group C rotavirus in a monkey kidney cell line (MA104).

A virus (designated the Shintoku strain) which was morphologically indistinguishable from group A rotaviruses was detected in the feces of adult cows with diarrhea in Japan. The virus contained 11 segments of double-stranded RNA and had an electrophoretic migration pattern in polyacrylamide gels similar to that of other group C rotaviruses (4-3-2-2). Feces containing the bovine virus reacted with antiserum to porcine group C rotavirus (Cowden strain) but not group A or B rotaviruses in immunoelectron microscopy. The virus was adapted to serial propagation in roller tube cultures of a rhesus monkey kidney cell line (MA104) by using high concentrations of trypsin. Evidence for viral replication in MA104 cell cultures was demonstrated by immunoelectron microscopy and indirect immunofluorescence by using antiserum to porcine group C rotavirus and by electrophoretic analysis of extracted viral double-stranded RNA. A significant antibody response against the isolate was detected in convalescent-phase sera of cows which excreted the virus: no increased antibody response to bovine group A rotavirus was observed. To our knowledge, this is the first isolation of a group C rotavirus from cattle.     

Morphological and antigenic relationships between viruses (rotaviruses) from acute gastroenteritis of children, calves, piglets, mice, and foals.

The reovirus-like particles present in the feces of young pigs and foals with acute enteritis and the virus causing epizootic diarrhea of infant mice were found to be indistinguishable morphologically from each other, from the South African SA. 11 and "O" viruses, and from the rotaviruses of children and calves. The inner capsid layer of each of these viruses reacted seriologically with sera of children, calves, mice, piglets, and foals convalescent from infection with their respective rotaviruses. These sera reacted by immunofluorescence with human, bovine, porcine, and murine rotaviruses, SA.11, and "O" viruses in tissue cultures and with human bovine, procine, nad murine viral antigens by complement fixation and gel diffusion. However, the antisera differed in their ability to react serologically with the outer capsid layer of the viruses investigated and in their ability to neutralize tissue culture-adapted calf virus. These two tests may demonstrate strain or host specificity among rotaviruses. Since the porcine, murine, and equine viruses are closely related serologically to and are morphologically identical to the human and bovine viruses, they should be included in the group of viruses for which the term "rotavirus" has been suggested. All known members of this proposed group of viruses share a common antigen, probably situated within the inner capsid layer; thus, any one of the viruses may be used for the preparation of antigen or antibody for diagnostic tests, and this will aid in the diagnosis of virus infection in those species from which a rotavirus has not been cultured.     

Identification of two subtypes of serotype 4 human rotavirus by using VP7-specific neutralizing monoclonal antibodies

Two distinct subtypes of human rotavirus serotype 4 were identified by using neutralizing monoclonal antibodies directed to the major outer capsid glycoprotein, VP7, of strains ST3 (subtype 4A) and VA70 (subtype 4B). Specimens containing serotype 4 rotavirus, obtained from different countries, were examined for subtyping by using solid-phase immune electron microscopy, enzyme-linked immunosorbent assay, and, for cell culture-adapted strains, neutralization assay. All 59 human rotavirus strains identified as serotype 4 by using animal antisera were classified into either subtype by monoclonal antibodies. This suggests that the antigenic difference between the two subtypes is a consequence of critical variations within the immunodominant serotype 4-specific neutralization site of rotavirus VP7. Subtype 4A (ST3-like) strains were predominant and were detected in stools from patients with gastroenteritis, as well as from healthy infants and young children.     

Isolation and characterization of an equine rotavirus.

A rotavirus, designated as the H-1 strain, was isolated from a diarrheic foal in primary African green monkey kidney cells and MA104 cells. This cell culture-adapted strain hemagglutinated erythrocytes of human group O, rhesus monkeys, guinea pigs, and sheep. It was found to be similar, if not identical, to porcine rotaviruses (strains OSU, EE, and A-580) by plaque reduction neutralization and hemagglutination inhibition tests, and, in addition, it was found to belong to subgroup 1. This equine rotavirus has an RNA electrophoretic migration pattern which was distinct from those of the three strains of porcine rotavirus. The serological relationship established by plaque reduction neutralization and hemagglutination inhibition tests between the equine (H-1) and porcine (OSU, EE, and A-580) rotaviruses is an example of a rotavirus of the same serotype being isolated from different species. The H-1 strain was distinct from four human rotavirus serotypes (Wa, DS-1, P, and St. Thomas 4) as well as from bovine rotavirus NCDV, simian rotavirus MMU18006, and canine rotavirus CU-1 by plaque reduction neutralization tests. This equine isolate (H-1) was found to be related antigenically to canine CU-1 and bovine NCDV rotaviruses in a one-way fashion by hemagglutination inhibition tests.     

人轮状病毒G2和G3型主要中和抗原VP7基因在重组腺病毒中的表达

目的 研究我国G2和G3型轮状病毒主要中和抗原VP7基因在重组腺病毒中的表达。方法 在前期成功表达Gl型VP7的基础上，选用我国G2和G3型主要流行株97S43和97S48 VP7基因，用非复制型腺病毒载体对上述基因进行表达。结果 获得了表达我国G2型和G3型人轮状病毒流行株VP7基因的非复制型重组腺病毒rvAdG2VP7和rvAdG3VP7，应用PCR及Southem blot技术证实在重组腺病毒中整合有轮状病毒G2型VP7和G3型VP7基因，RT-PCR证明重组腺病毒在感染的293细胞内均能有效地转录插入基因，Western blot检测到轮状病毒VP7基因的表达。结论 这一工作为进一步进行动物实验，发展多价轮状病毒疫苗打下了基础。     

Isolation of rotaviruses from turkeys and chickens: demonstration of distinct serotypes and RNA electropherotypes

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.     

Isolation and serial propagation of human group C rotaviruses in a cell line (CaCo-2)

Rotaviruses were detected via electron microscopy in fecal specimens collected from school children during an outbreak of diarrhea and from a sporadic case in 1993 in Japan. All of the viruses were found to belong to human group C rotavirus by reverse passive hemagglutination assay (RPHA). These viruses replicated well in a human colon carcinoma (CaCo-2) cell line cultured in the presence of trypsin (4 μg/ml). This report demonstrates that human group C rotaviruses can be propagated efficiently in a cell line cultured in the presence of trypsin. The infected cells did not show any apparent cytopathic changes. However, virus was detected in the cell cytoplasm by immunofluorescence (IF) staining and in the culture supernatant by RPHA. On the basis of immune electron microscopy (IEM), virus particles collected from infected CaCo-2 cell cultures were confirmed to aggregate specifically with anti-human group C rotavirus anti-body. The electrophoretic patterns of RNA segments extracted from viral particles found in the fecal specimens or infected cells were identical to those of human group C rotavirus. These results indicated that human group C rotaviruses were the causal agent of the diarrhea outbreak. © 1996 Wiley-Liss, Inc.