Rotavirus In Cell Cultures Research Articles

Genomic changes detected after serial passages in cell culture of virulent human G1P[8] rotaviruses

Serial passages of a virulent mouse rotavirus in cell cultures caused a loss of virulence in mice. To gain insight into the genomic mutations in human rotavirus during cell culture and its attenuation in humans, we serially passaged three wild type human G1P[8] rotavirus strains (Wa, DC3695, DC5685) derived from diarrheal stool samples up to 60 times in two different cell cultures (human colon adenocarcinoma cell line: HT29, and primary African green monkey kidney cells: primary AGMK). We sequenced the whole genomes of 60 times-passaged strains and compared them with those of the original viruses. Most substitutions were detected in VP4, followed by substitutions in VP7 and NSP4 genes. Substitution at amino acid 385 in the putative VP4 fusion domain and substitution T45M in NSP4 genes were detected in all AGMK-passaged strains, respectively. These genomic changes are likely to correlate with a loss of rotavirus virulence in humans.

Comparative efficacy of fluorescent antibody test, immunoperoxidase test and enzyme linked immuno sorbent assay in detection of rotavirus in cell culture.

Rotavirus is prevalent worldwide and has been established as a leading cause of mortality due to severe diarrhoea in neonates. Isolation of the virus is a gold standard method for confirmation of rotavirus infection in the host. Propagation of rotavirus in cell culture is a challenge as in many instances the virus does not produce detectable cytopathic effect. This study was undertaken to evaluate the efficacy of fluorescent antibody test (FAT), immunoperoxidase test (IPT) and sandwich ELISA (S-ELISA) to detect rotavirus antigen propagated in MA 104 cell line. The intensity of fluorescence and colour development for I-FAT and I-IPT was categorized and the ELISA OD values were analyzed. The overall mean of detection were 5.16±0.47, 5.16±0.54 and 5.66±0.33 for I-FAT, I-IPT and S-ELISA, respectively. Significantly less number of samples were positive in the initial one or two passage, which increased up to 100% from third/fourth passage onwards. The study concluded that I-FAT, I-IPT and S-ELISA were equally effective in detecting propagated rotavirus in cell line, and the former two tests are suitable for in situ demonstration of the virus while the later could be used to assay antigen in cell culture fluid.

Effect of nitazoxanide for treatment of severe rotavirus diarrhoea: randomised double-blind placebo-controlled trial

Rotavirus is a leading cause of morbidity and mortality in children younger than 5 years, but there is no effective treatment. We assessed the activity of nitazoxanide, a broad-spectrum anti-infective drug, against rotavirus in cell culture and in a clinical trial in paediatric patients hospitalised with severe rotavirus diarrhoea. We did a randomised double-blind placebo-controlled trial in 50 children admitted to the Cairo University Children's Hospital between June 15 and Aug 23, 2005, with severe rotavirus diarrhoea. 38 children aged 5 months to 7 years (median age 11 months) with rotavirus as the sole identified cause of gastroenteritis were enrolled in the clinical study. Patients were randomly assigned either 7.5 mg/kg nitazoxanide as an oral suspension or placebo twice a day for 3 days, and all remained in hospital for 7 days after start of treatment. The primary endpoint was time from first dose to resolution of illness, and analysis was by modified intention-to-treat. This study is registered with ClinicalTrials.gov, number NCT00302640. Survival analysis showed that the median time to resolution of illness was 31 h (IQR 22-73) for the nitazoxanide-treated group compared with 75 h (51-124) for the placebo group (p=0.0137). No significant adverse events were reported. A 3-day course of nitazoxanide significantly reduced the duration of rotavirus disease in hospitalised paediatric patients. These results are encouraging, and might lead us to think about new approaches to managing rotavirus disease in children.

Immunochemiluminescent focus assays for the quantitation of hepatitis A virus and rotavirus in cell cultures

Two new immunological methods, the luminescent immunofocus assay (LIFA) and the luminescent immunofocus inhibition assay (LIF-IA), are described for the quantitation of cytopathic and non-cytopathic viruses propagated on cell culture monolayers. These methods use enhanced chemiluminescent detection to identify foci (luminescent immunofoci, LIF) of virus-infected cells. Viruses are propagated in susceptible cells under an agarose overlay, inactivated with ultraviolet irradiation, lifted onto nitrocellulose membranes, and probed with virus-specific monoclonal or polyclonal antibody followed by a second antibody conjugated to horseradish peroxidase. Membranes are then treated with a luminol-based detection reagent and exposed to light sensitive film for up to 10 min. The film is developed and foci appear as dark, discrete spots which are proportional to the dose of each virus. The LIFA detected both cytopathic and non-cytopathic hepatitis A viruses (HAV) and simian rotavirus. For the cytopathic HAV, the LIFA and plaque counts were comparable. The LIF-IA was developed for HAV using virus-specific antiserum which effectively attenuated LIF formation. The LIFA and LIF-IA may be completed 5 days faster than conventional radioimmunofocus assays for HAV and rotavirus and do not require the use of radiolabeled antibodies, offering safety advantages and making these techniques more adaptable for general use. Luminescent immunofocus assays should be useful for the detection and quantitation of virtually any cytopathic or non-cytopathic virus that can be propagated in monolayer cultures when virus-specific antiserum is available.

Isolation of group B porcine rotavirus in cell culture.

While group A and C rotaviruses have been grown in cell culture, group B rotavirus has never been cultured. In this study we successfully isolated porcine group B rotavirus in swine kidney cells. Pancreatin treatment is essential for the propagation of group B rotavirus.

A new immune complex dot assay for detection of rotavirus antigen in faeces

A new immune complex dot assay (ICDA) using immune gold/silver staining is described for the sensitive and rapid detection of rotavirus in cell culture and stool specimens. The method involves spotting preformed antigen-antibody complexes onto nitrocellulose paper, followed by incubation with colloidal goldlabelled secondary antibody and silver enhancement. ICDA was sensitive and specific and detected rotavirus antigens over a wide range of concentrations. It was more sensitive than a conventional immunodot assay (CIDA) and two commercial enzyme immunoassays (EIA) based on testing serial dilutions of a positive stool specimen. Of 26 stool specimens tested ICDA detected rotavirus antigen in 17; 14 were positive by Pathfinder Rotavirus EIA, 16 by Testpack Rotavirus EIA, and direct electron microscopy (DEM) detected only 12. The ICDA offers improved sensitivity over commercial EIAs and DEM.

Sensitive solid-phase immune electron microscopy double-antibody technique with gold-immunoglobulin G complexes for detecting rotavirus in cell culture and feces

A new solid-phase immune electron microscopy double-antibody colloidal-gold technique (SPIEMDAGT) was developed and compared with direct electron microscopy, direct immune electron microscopy, and enzyme immunoassay for detecting rotavirus. Guinea pig and rabbit antirotavirus antisera were used as capture and detector antibodies, respectively, and goat anti-rabbit immunoglobulin G-gold complexes were employed as a label. Animal rotavirus in cell culture media and human virus in stool specimens were detected by this method. On average, SPIEMDAGT detected 800 times more virus particles than direct electron microscopy and 45 times more particles than direct immune electron microscopy and yielded 20% more positives than enzyme immunoassay. SPIEMDAGT could detect not only viral antigen associated with morphologically recognizable particles but also antigen present when whole virus particles were not visible.

Isolation of ovine rotavirus in cell cultures. Brief report.

Three cytopathic rotavirus strains were isolated in MA 104 cells from intestinal contents of lambs with diarrhea. Pretreatment of virus with pancreatin and incorporation of a small amount of pancreatin in maintenance medium were important for establishment of the strains in these cells. The isolates showed marked cross reactions with bovine rotavirus in immunofluorescence, but no cross reaction with bovine, human, simian, equine, porcine and lapine rotaviruses in neutralization.

Isolation of murine rotavirus in cell cultures. Brief report.

Murine rotavirus was isolated in primary monkey kidney cells. Electrophoretic pattern of genome RNA of murine rotavirus was different from that of human rotaviruses. Oral administration of cultured murine rotavirus caused mild diarrhea in newborn BALB/C mice.

A simple immunoperoxidase method for detecting enteric adenovirus and rotavirus in cell culture

A technique which includes the use of indirect immunoperoxidase antibody (IPA) has been developed for detecting enteric adenovirus and rotavirus antigens in cell cultures and has been compared with immunofluorescence antibody assay (IFA). The IPA technique was as sensitive as the IFA. The number of positive cells detected by both techniques in tissue cultures was the same; false positive results were not observed. The applicability of IPA in clinical virology is discussed.

Isolation of equine rotavirus in cell cultures from foals with diarrhea.

Survey of rotavirus was conducted in 40 fecal samples which were divided into 4 groups according to diarrheal conditions to clarify the relationship between rotavirus and foals with diarrhea. Particles characteristic of rotavirus were directly detected by electron microscopy from 5 of 14 fecal samples collected from foals with acute diarrhea. Moreover, rotaviruses were isolated from 4 of 5 fecal samples with MA-104 cells and with trypsin treatment, and were capable of being successively passaged in these cells. On the other hand, no rotavirus was detected in 26 fecal samples collected from foals with various non-acute diarrhea. In the cross serum neutralization test, 4 isolated strains were antigenically recognized as the same serotype, which was slightly differed from BI strain of equine rotavirus and clearly separated from Lincoln strain of bovine rotavirus antigenically. From these results, it was revealed that rotavirus was closely related only to acute diarrhea in foals. Moreover, it was confirmed that equine rotavirus in field cases could be isolated in MA-104 cells with trypsin treatment.

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.

Isolation of lapine rotavirus in cell cultures. Brief report.

Two cytopathic rotavirus strains were isolated in MA 104 cells from diarrheal rabbits. The isolates showed marked cross reactions with strain Lincoln of bovine rotavirus in immunofluorescence, but no cross reaction in neutralization.

Genes of human (strain Wa) and bovine (strain UK) rotaviruses that code for neutralization and subgroup antigens

Sixteen rotaviral reassortants recovered from mixed infection with a ts mutant of bovine rotavirus (UK strain) and noncultivatable or cultivatable human rotavirus (Wa strain) were genotyped by polyacrylamide gel electrophoresis. Analysis of the reassortants showed that the ninth RNA gene segment regularly segregated with the neutralization specificity and the sixth RNA gene segment regularly segregated with the subgroup antigenic specificity detected by immune adherence hemagglutination assay. This indicates that the ninth RNA segment codes for the protein that induces and reacts with neutralizing antibodies while the sixth RNA segment codes for the subgroup antigen. In addition, it appears that the fourth and/or fifth RNA segments may be responsible for restriction of growth of the noncultivatable human rotavirus in cell culture.

Isolation of human rotavirus in cell cultures: brief report.

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.