Cultivation and subgroup determination of human rotaviruses from Egyptian infants and young children.

Primary African green monkey kidney cells were more sensitive than primary cynomolgus monkey kidney and MA104 cells for supporting the growth of human rotaviruses detected in diarrheal stools of Egyptian infants and young children. In attempts to characterize these Egyptian rotaviruses, only 31% of the strains tested in the form of fecal suspensions were identified as subgroup 1 or 2. After one passage in African green monkey kidney cells, 80% of the strains were identified as subgroup 1 or 2. Of these 43 rotaviruses for which the subgroup was determined, 28% were subgroup 1 and 72% were subgroup 2. Thus, cultivation in African green monkey kidney cell cultures facilitated the antigenic characterization of rotaviruses by subgrouping; cultivation also represents an initial step in determining serotype and in developing potential vaccine candidates.     

Isolation and characterization of a canine rotavirus

Summary Canine rotavirus particles were visualized by direct electron microscopy in the feces from a clinically normal dog. The virus was subsequently propagated in cell cultures; it was chracterized and compared with rotaviruses from other species. Replication of the virus in cell culture was found to be less dependent upon trypsin than that of human, bovine and porcine rotaviruses. Reproducible, sharp-edged plaques of various sizes were produced by the canine rotavirus in an established cell line of fetal rhesus monkey kidney, MA104, under overlays of carboxymethyl cellulose or agarose. Intracytoplasmic inclusion bodies of different sizes and shapes were produced in infected MA104 cells. By plaque reduction neutralization assay, a two-way antigenic relationship was found between the canine (CU-1) and simian (rhesus MMU 18006 and SA-11) rotaviruses. The canine rotavirus had a one-way antigenic relationship with feline (Taka), bovine (NCDV), and porcine (OSU) rotaviruses.With 4 Figures     

Efficiency of human rotavirus propagation in cell culture.

This study was designed to find methods to reproducibly propagate human rotaviruses from fecal specimens and to determine the relationship between particle numbers and infectivity. Growth of virus was initially compared in primary and continuous lines of monkey kidney cells. Primary cells (African green and cynomolgus monkey kidney) supported virus growth directly from fecal specimens much more efficiently than did continuous lines of African green (CV-1) or rhesus (MA104) monkey kidney cells. Rotaviruses were grown in primary cells from 14 of 14 fecal specimens of different individuals collected over a 3-year period. Although rotaviruses in fecal samples could not always be grown in the continuous cell lines, two passages in primary cells appeared to fully adapt the viruses for propagation in the continuous cell line tested (MA104). The efficiency of rotavirus growth was quantified with five of the fecal isolates. It was calculated that, on the average, 1 out of every 46,000 particles in fecal specimens infected monkey kidney cells. After three passages in primary cells, an average of 1 out of every 6,600 progeny virus particles appeared to be infectious. Thus, rotaviruses in fecal specimens were consistently grown in primary cells, and passage in these cells both increased virus infectivity and adapted the viruses for growth in continuous cell lines.     

Cell culture propagation of rotaviruses

Summary Rotaviruses are ubiquitous intestinal pathogens that infect the young of many species. Their adaptation to cell culture has met with variable success depending on the strain and serogroup of virus. Successful cultivation of rotaviruses has been achieved through sequential use of animal passage and kidney cell cultures. Roller tubes were beneficial for adaption of several strains as opposed to use of stationary cell cultures. Activation of rotaviruses with proteolytic enzymes has increased their adaptability to culture and is standard practice with many strains. Alternative methods for cultivation of rotavirus, particularly human strains, have involved use of reassortants between animal and human rotaviruses. Continuous cell lines for rotavirus passage include rhesus monkey kidney (MA-104 & LLC-MK₂), African green monkey kidney (BSC-1 & CV-1), and Madin-Darby bovine kidney. Fluorescent foci, cytopathic effects, and plaque assays are used to titrate infectious virus. Enzyme-linked immunosorbent assay, hemagglutination, dsRNA electropherotyping, and electron microscopy are used to detect rotavirus growth. Optimal conditions for the growth of two group A rotaviruses (OSU & NCDV) in MA-104 cells are described in this report.     

Comparison of human, simian, and bovine rotaviruses for requirement of sialic acid in hemagglutination and cell adsorption

Human rotaviruses (Wa, KUN, MO) showed hemagglutination (HA) only with fixed 1-day-old chicken erythrocytes, and their HA activities were completely destroyed by trypsin activation of virions. Simian SA-11 and bovine NCDV had HA activities not only against fixed erythrocytes but also against fresh erythrocytes from various species. Their HA activities against fixed erythrocytes were also inhibited by trypsin activation, but those against fresh erythrocytes were not. Neuraminidase treatment of fixed erythrocytes did not inhibit HA by trypsin-untreated rotaviruses. In contrast, HA of fresh human erythrocytes by SA-11 and NCDV was completely inhibited by neuraminidase treatment of erythrocytes or glycophorin A, the major erythrocyte sialoglycoprotein. Adsorption and infection of SA-11 and NCDV to monkey kidney MA104 cells were also inhibited by neuraminidase treatment of cells. Adsorption and infection of human rotaviruses were not, however, affected by treatment of cells with neuraminidase from Vibrio cholerae or Arthrobacter ureafaciens or with potassium periodate. Therefore, HA of fixed chicken erythrocytes by trypsin-untreated human and animal rotaviruses may be independent of sialic acids, whereas that of fresh erythrocytes by SA-11 and NCDV is sialic acid dependent and probably mediated by glycophorin A. Sialic acids also constitute an essential part of the cellular receptors for SA-11 and NCDV, whereas those for human rotaviruses were quite resistant to treatments known to destroy major types of sialic acids.     

Isolation of murine rotavirus in cell cultures

Summary 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.With 2 Figures     

Direct isolation in cell culture of human rotaviruses and their characterization into four serotypes.

Of 73 rotavirus-positive fecal specimens tested, 39 yielded a human rotavirus that could be cultivated serially in MA104 or primary African green monkey kidney cells or both; 18 were serotyped. Four distinct serotypes were identified by plaque reduction or tube neutralization assay or both, and three of these serotypes were the same as those established previously by plaque reduction, using human rotaviruses cultivated by genetic reassortment with a cultivable bovine rotavirus. Ten human rotavirus strains received from Japan were found to be similar, if not identical, to our candidate prototype strains representing these four human rotavirus serotypes.     

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.     

The use of human foreskin cell cultures for isolation of herpesvirus group in the diagnostic laboratory.

Cell cultures originated from human foreskin (HFS) tissues were used for isolation of viruses from diagnostic specimens. The foreskins were collected in Hank's balanced salt solution and then processed on the same day by dispersion in trypsin. A week after the trypsin treatment of the tissues, the first cell cultures were ready to use. Continuous subcultures in vitro of the cells gave rise to a colony of cells that multiplied freely in vitro and supported the growth of viruses from the herpes group. In three cases tested in our laboratory in the last 6 months, viruses from the herpes group were isolated on the HES. The cytopathic changes of the HFS cells were observed 5 to 8 days after infection. They were not detected on two other human-origin cell cultures (WI-38 and HEp2) or on primary monkey kidney cells. The viruses isolated from these three cases were cytomegalovirus (CMV) from urine of a 2-week-old baby, a second CMV from a cutaneous lesion of a renal-transplant patient and herpes simplex virus from the eye swab of a young girl. After a few subcultures on the HFS cells, the three viruses produced CPE on the other susceptible human cells. The preparation of HFS cells is easy, the availability of the tissue is high, and the diagnostic value is unquestionable. It is suggested that this tissue and its cell cultures be used more often in diagnostic and research laboratories.     

Replication and plaque formation of parainfluenza viruses in an established line of monkey kidney cells

All four types of parainfluenza virus produced distinct plaques in an established line of monkey kidney cells (LLCMK2) under agar overlay containing trypsin and DEAE dextran. Parallel titration of these viruses in LLCMK2 and primary cynomologous monkey kidney (MK) cells showed that LLCMK2 cells were about tenfold more sensitive than MK cells. When trypsin was added to the fluid medium, the virus yield in LLCMK2 cells was significantly higher than in MK cells.     

Plaque formation by human-origin parainfluenza type 2 virus in established cell lines

Summary Two strains of parainfluenza type 2 virus formed well-defined plaques in cultures of Vero cells, an established line of African green monkey kidney cells. In the absence of trypsin, satisfactory plaques were formed by the Toshiba strain of virus. When trypsin was added in the overlay medium of Vero cell monolayers, another strain (62-M786) of virus produced plaques. The Toshiba strain was also able to make plaques in HeLa, BHK, and LLC-MK₂ (an established line of monkey kidney) cells without trypsin, but not in mouse L cells. The sensitivity of plaque assay was about equal to that of the hemadsorption method.With 3 Figures     

Neutralization activity of some Coxsackie B3 antibodies is compromized by protein factor(s) secreted by Vero cells

Summary When various anti-Coxsackie B3 virus antibodies were examined for the neutralizing activity in cultures under liquid medium, some antibodies including monoclonal antibodies gave abnormally low titers in the neutralization test in Vero cells, in comparison with the other cells such as HeLa, FL, HEp-2, or primary monkey kidney cells. The neutralization titer of these antibodies was, however, similar in all these cells by plaque reduction assays under agar overlay, i.e., the above phenomenon was restricted to cultures under liquid medium. The reduced neutralization titer in Vero cells under liquid medium was found to be brought about by protease-sensitive factor(s) released by Vero cells, because (1) the addition of Vero cell culture fluid resulted in a marked reduction of neutralizing titer in primary monkey kidney cells, and (2) the activity of the Vero cell factor was destroyed by trypsin (20 µg/ml for 1 h). As three-day incubation of virus-antibody complex in Vero cell culture fluid resulted in a partial restoration of virus infectivity, the binding of antibody to virions appears to be competed by the Vero cell factor.     

Isolation and cell culture propagation of rotaviruses from turkeys and chickens

Summary Rotaviruses were detected by electron microscopy in the faeces of turkey poults and broiler chickens with diarrhoea. Apparently symptomless infection was also observed in broilers. The avian rotaviruses could not be isolated in cell cultures by conventional techniques, but were adapted to serial growth in chick cell cultures following trypsin treatment of the virus and the cells. Immunofluorescence studies showed that the avian and mammalian rotaviruses are antigenically related. Antibodies to rotavirus were widespread in sera collected from turkeys, chickens and ducks.With 3 Figures     

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.     

Cytolar microcarriers for the cultivation of surface-dependent cells

The results of trials of one of the microcarriers (MC) developed by the authors, Cytolar, synthesized from denaturated collagen are presented. Both primary chick embryo cell and green monkey kidney cell cultures and diploid fibroblast-like human embryo cells were successfully cultivated on these MC. The properties of the developed MC were not inferior to those of the best known analogues.     

Established cell line sensitive to influenza C virus.

Various strains of influenza C virus grew productively in an established line of monkey kidney cells (LLCMK2) without prior adaptation. When trypsin was added to the medium, higher virus yields were obtained than in other cell cultures. All influenza C virus strains tested formed well defined plaques under the agar overlay medium containing trypsin. Infectivity determined by plaque assay in LLCMK2 cells was higher than that determined by amniotic inoculation of fertile hens' eggs.     

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.     

NCI-H292 as an alternative cell line for the isolation and propagation of the human paramyxoviruses

Summary Primary rhesus monkey kidney (MK) cells have long been the cells of choice for isolation and propagation of the human paramyxoviruses (parainfluenza 1, 2, 3, 4A, 4B, and mumps). However, problems with the supply and cost of MK cells and the presence of endogenous viruses, including herpes B virus and SV-5, necessitated a search for an alternative cell line. Continuous cell cultures of human origin (L132, A-549, HuT-292, HEK, G-293, G-401, A-498, A-704, CAKI-1, RD) and simian origin (LLC-MK2, BSC-1, MA-104, Vero) were evaluated for their capacity to support the growth of the human paramyxoviruses, as followed by cytopathic effect, hemadsorption, hemagglutination, and EIA. NCI-H292 (HuT-292) human lung mucoepidermoid carcinoma cells (ATCC # CRL-1848) proved to be the most sensitive line for cultivating all serotypes and strains of the paramyxoviruses. These cells were also shown to be a suitable substitute for MK in primary isolation of paramyxoviruses from clinical specimens. RPMI-1640 with 1.5µg/ml trypsin was the preferred maintenance medium; alternatively, Eagle's MEM supplemented with 1.5µg/ml trypsin and 0.1% ITS was satisfactory. NCI-H292 cells are a continuous line with excellent growth characteristics, although the genetic polyploidy of the cells may limit the number of passages of usable cells.     

Enhanced isolation of respiratory syncytial virus in cell culture.

During two winter seasons, we found that the combination of WI-38 or MRC-5 human lung fibroblasts plus primary rhesus monkey kidney (RhMK) and HEp-2 cell cultures yielded maximal isolation of respiratory syncytial virus. Cytopathic effects (CPE) developed earliest in RhMK cells and slowest in the human fibroblast lines. In RhMK cells, 50% of ultimately positive cultures showed CPE in 5 days, and 90% of positive cultures showed CPE within 7 days during both respiratory syncytial virus seasons.     

Isolation of the human rotavirus in a cell culture of green monkey kidneys

Properties of a virus isolated from a patient with rotavirus gastroenteritis (the diagnosis verified by direct electron microscopy) were studied. The virus was identified as a rotavirus in tissue culture neutralization and complement fixation tests. It was shown to replicate through a significant number of passages in continuous cell cultures (Vero, RH) as well as in primary cell cultures of human and animal origin (human embryo kidney, green monkey kidney); some features associated with rotavirus propagation are described.