A control for fetal bovine serum.

The FBS Control is designed to assist cell culture scientists who have been frustrated with lack of consistency in FBS lots. Continued use will reduce the time and expense needed in classical lot selection methods.

Influence of malnutrition and alterations in dietary protein on murine rotaviral disease.

The possible correlation between malnutrition and degree of severity of rotavirus-associated infantile diarrhea which appears to occur in human populations was studied using a mouse model. To determine the effects of general malnutrition or altered levels of dietary protein, female mice were fed throughout pregnancy and infection periods with diets diluted with 0, 300, or 600 g glucose/kg, designated as normal nutrient to calorie ratio (N/C) diet, 70% N/C diet, or 40% N/C diet or with diets containing 75, 150, or 300 g casein/kg, as low-, normal-, or high-protein diets. Murine rotavirus was given by gavage to the 2-day-old offspring of these dams, and the extent of infection determined. Marked increases in severity of diarrheal disease were seen in the infants from dams receiving the 40 and 70% N/C diets and the low-protein diet. Severity of infection was seen as increased deaths, reduced weight gain, and increased passage of diarrheic feces. Intestinal viral levels and intestinal diarrhea scores did not vary appreciably. Serum interferon remained below detectable limits throughout the studies, but serum antibody was determined in dams 30 days post-virus exposure. The latter titers were lower in the infected mice from dams fed the 40 and 70% N/C diets, but were essentially the same in all the protein diet groups. Cross-fostering was done using the 40 and 100% N/C diets, wherein mice from dams fed either diet were placed on mothers fed the opposite diet. Increased severity of infection was again seen when the virus was given 2 days after the exchange, although the greatest infection occurred in animals from dams fed 40% N/C diet which were then fostered by other similarly fed dams. The increased host sensitivity to the rotaviral infection appeared to be a result of both pre- and postnatal dietary effects.

Aerosol stability of infectious and potentially infectious reovirus particles.

The aerosol stability of two particle forms, infectious and potentially infectious, of reovirus were examined under static conditions for a range of relative humidities at 21 and 24 degrees C. Virus aerosolization efficiency was determined for two methods of dissemination: Collison nebulizer and Chicago atomizer. Suspensions of Bacillus subtilis var. niger spores were added to reovirus preparations that included both particle forms and disseminated into a dynamic aerosol toroid to estimate the physical decay of the aerosols. At 90 to 100% relative humidity, both reovirus particle forms showed less than 10-fold loss of infectivity after 12 h of aging. At lower relative humidities the aerosol decay curve showed rapid initial decay followed by a markedly lower decay rate. Our findings reveal that reovirus particles are relatively stable in the airborne state.

Protamine precipitation of two reovirus particle types from polluted waters.

Two forms of virus particle are released from reovirus-infected cell cultures, infectious reovirus and potentially infectious reovirus (PIV). PIV particle forms have a complete outer coat and are not infectious until the outer coat is altered or removed. The PIV concentration in polluted waters, however, has not been determined. Protamine sulfate precipitation, using 0.25% fetal bovine serum and 0.005% protamine sulfate for the first precipitation of the sample and 0.0025% for the second, was employed to concentrate infectious reovirus and PIV from water and sewage. Infectious reovirus and PIV particles were concentrated over 500-fold from river water inoculated with virus, and virus recoveries of between 80 and 100% were achieved. Virus precipitates stored at -20 degrees C as a protamine-virus concentrate showed a 5% loss of PIV after 14 days. Virus preparations were assayed, before and after treatment, with 200 micrograms of chymotrypsin per ml, using a fluorescent-antibody procedure. Protamine sulfate precipitation and fluorescent-antibody detection are effective ways to recover and assay reoviruses present in raw sewage.

Evaluation of cell lines and immunofluorescence and plaque assay procedures for quantifying reoviruses in sewage.

Twelve continuous cell lines were tested to determine their sensitivity to reovirus types 1, 2, and 3 isolated from sewage. Madin-Darby bovine kidney (MDBK), rhesus monkey kidney (LLC-MK2), and human embryonic intestinal (intestinal 407) cells were most sensitive, respectively. In a similar study, MDBK cells were more sensitive than LLC-MK2 and Buffalo green monkey kidney (BGM) cells to sewage-isolated, protamine-precipitated reoviruses which had not been serotyped and had no previous cell contact. Sewage-isolated, protamine-precipitated reoviruses were also used in conjunction with MDBK cells in a comparative evaluation of immunofluorescent cell count and plaque assay procedures. The immunofluorescence assay is more sensitive and more rapid than the plaque assay. Reoviruses in excess of 10(4)/liter of raw sewage were detected by the immunofluorescent cell count assay.

Inhibition of rotaviruses by selected antiviral substances: mechanisms of viral inhibition and in vivo activity.

Several RNA virus inhibitors were evaluated against simian (SA11) rotavirus infections in vitro and murine rotavirus gastroenteritis in vivo. Test compounds included 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin), 3-deazaguanine (3-DG), 3-deazauridine, and 9-(S)-(2,3-dihydroxypropyl)adenine [(S)-DHPA]. All drugs inhibited total infectious SA11 virus yields in MA-104 cells. Ribavirin, 3-DG, and (S)-DHPA affected [3H]uridine uptake into uninfected MA-104 cells in both the acid-soluble and -insoluble fractions. All drugs reduced the levels of dense (precursor) and light (complete) SA11 particle yields compared with control but did not alter the relative amounts of dense compared with light particles, suggesting that the agents did not interfere with virus assembly. Ribavirin and 3-DG inhibited SA11 polypeptide synthesis, as determined by polyacrylamide gel electrophoresis studies. None of the agents or mono- and triphosphate derivatives of ribavirin inhibited SA11 RNA polymerase activity. In murine rotavirus studies, oral therapy with ribavirin-2',3',5'-triacetate and (S)-DHPA increased mean survival time, but no increase in survivor rate was observed. 3-DG- and (S)-DHPA-treated mice had a more rapid weight gain than controls, suggesting a probable lessening of the severity of the disease.

Inhibition of bluetongue and Colorado tick fever orbiviruses by selected antiviral substances.

The effects of four ribonucleic acid virus inhibitors were evaluated in cell cultures and in mice to determine inhibitory effects against bluetongue virus and Colorado tick fever virus (CTFV). Test compounds included 1-beta-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin), 3-deazaguanine, 3-deazauridine, and 9-(S)-(2,3-dihydroxypropyl)adenine. Ribavirin-2',3',5'-triacetate (ribavirin triacetate) was evaluated in vivo against CTFV. Inhibition of cytopathic effect and plaque reduction were used to evaluate antiviral activity. In cytopathic effect inhibition studies, bluetongue virus was markedly inhibited by 3-deazaguanine and 3-deazauridine in Vero cells with moderate inhibition by the other agents. Ribavirin and 3-deazaguanine markedly inhibited CTFV in MA-104 cells, 3-deazauridine was slightly less active, and 9-(S)-(2,3-dihydroxypropyl)adenine was negative. Ribavirin was less effective in Vero cells against CTFV. When mice were inoculated intracerebrally with CTFV and treated by a single intracerebral injection with drug, ribavirin triacetate increased the number of survivors, 3-deazaguanine increased mean survival time, and ribavirin was negative. Intraperitoneal treatment of infected mice with ribavirin triacetate for 1 week significantly increased the number of survivors and mean survival time, providing strong evidence that the agent is active across the blood-brain barrier.

Trypsin enhancement of rotavirus infectivity: mechanism of enhancement.

The infectivity of most rotaviruses is enhanced by treatment with trypsin. We studied the mechanism of enhancement of examining the effect of trypsin on rotavirus infectivity, aggregation, early interactions with host cells, and structure. The results indicated that trypsin does not increase levels of infectious virus by dispersion of aggregates or affect the efficiency or rate of attachment of virus to cells. A fraction of virus that was not infections without trypsin treatment was found to attach to cells, but did not initiate antigen synthesis. When cells were infected with labeled, purified virus, increased levels of uncoated particles were found in cells infected with trypsin-treated virus. Infection of cells with trypsin-treated virus also led to greater levels of RNA synthesis early in the infection. The results suggest that trypsin converts a noninfectious fraction of virus into infectious virus by allowing this fraction to uncoat in the infected cell. Trypsin was found to cleave an 88,000-dalton structural polypeptide of bovine rotavirus generating 67,000- and 20,000-dalton cleavage products.

Development of antiviral levels of ribavirin in serum and urine of orally treated rats.

The levels of ribavirin or its antivirally active metabolic products were determined in the serum and urine of rats treated with single oral doses of 1,000 or 100 mg/kg of the compound, using a newly developed micromethod in which measles virus inhibition was assayed in BS-C-1 cells. At the high dosage level, maximum ribavirin serum levels of 24 microgram/ml were observed 2 h postribavirin administration. Approximately 10-fold less active material was seen in the rats receiving the lower ribavirin dosage; this peak effect was seen 1 h after treatment. Urine excretion was maximal between 4 and 20 h after treatment in both dosage groups.

Bioassay system for determining ribavirin levels in human serum and urine.

A simple, sensitive, micromethod was developed to determine levels of ribavirin, or its active metabolic products, in human serum or urine. The procedure utilized the inhibition of measles virus cytopathic effect in BS-C-1 cells. Based upon maximum dilutions of human serum or urine containing ribavirin which inhibited the measles virus, the bioassay detected ribavirin in concentrations as low as 0.006 microgram/ml in serum or 0.03 microgram/ml in urine. Herpesvirus 1, parainfluenza virus 3 and reovirus 1 were also tested for sensitivity to ribavirin. Minimum inhibitory concentrations of ribavirin in serum or urine against these other viruses were no lower 0.32 microgram/ml.

Role of two particle types in bovine rotavirus morphogenesis.

The involvement of light (L) and dense (D) bovine rotavirus particle types during virus replication has been studied. It was found that infectious parental L virions are uncoated in vivo to a particle similar to native D particles. Differences in the rate of synthesis and relative yields of L and D particles in MDBK and MA-104 cells have been detected. Results from pulse-chase labeling experiments indicate that D particles serve as morphogenic precursors to the complete L virion.

Effect of enzymes on rotavirus infectivity.

The infectivity of a bovine rotavirus was enhanced 140-, 8-, and 3-fold, respectively, by trypsin, protease, and lactase. Ficin, carboxypeptidases A and B, lysozyme, and beta-galactosidase had little effect on the infectivity. Chymotrypsin caused a threefold decrease in the infectivity. Trypsin acts directly on the rotavirus and not on the host cell.

Production of high-titer bovine rotavirus with trypsin.

Titers of bovine rotavirus in excess of 10(9) immunofluorescent infectious units per ml of culture fluids have been produced, using trypsin treatment of the virus. Infectivity of preparations of the virus can be increased with as little as 1 ng of trypsin per ml, with maximum increases of 1 to 2 log10 with 1 microgram of trypsin per ml. The virus grows to titers in excess of 10(5) immunofluorescent units per ml in MDBK, LLC-MK2, MA-104, and HeLa cells. When MDBK cells are infected with a multiplicity of infection of 20, maximum yields of cell-associated, trypsin-enhanceable virus are obtained 4 to 8 h postinfection. Maximum yields of cell-free, trypsin-enhanceable virus are produced 16 to 20 h postinfection. The results presented here indicate that trypsin can be used to produce high-titer stocks of bovine rotavirus.

Characteristics of neonatal calf diarrhea virus ribonucleic acid.

The nucleic acids of neonatal calf diarrhea virus were characterized by isopycnic centrifugation in Cs2SO4, electron microscopy, ultraviolet absorbance temperature profiles and polyacrylamide gel electrophoresis. These studies indicated that the neonatal calf diarrhea virus genome consists of 11 segments of double stranded RNA with a total molecular weight of 10.75 million daltons.

Detection of neonatal calf diarrhea virus, infant reovirus-like diarrhea virus, and a coronavirus using the fluorescent virus precipitin test.

Thirty-four calf and five infant fecal specimens were tested for the neonatal calf diarrhea virus (NCDV) and for the reovirus-like infantile diarrhea agent; respectively. The procedures used were the fluorescent virus precipitin test and immune electron microscopy. Fourteen of the calf stools contained detectable NCDV, and four of the five infant stools contained the reovirus-like human agent. Infectious NCDV was detected in four of the 34 calf fecal specimens when Madin-Darby bovine kidney cell cultures that had been inoculated with supernatant fluids from stool suspensions were stained with fluorescent antibody. The 20 calf stools that did not have detectable virus were examined for the bovine corona diarrhea virus. Coronavirus was found in two of these specimens.

Fluorescent virus precipitin test.

A fluorescent virus precipitin test (FVPT) for the serologic identification of small particulate antigens such as viruses has been described. The test has several advantageous characteristics: (a) It is probably as sensitive as any serologic test (i.e., aggregates with dimensions of 0.2 mum are detectable; therefore, complexes containing as few as three large viruses would give a positive test). (b) Cultivation of the virus is not required. (c) Since an indirect test can be used, only a single fluorescent conjugate is needed to permit the detection of a number of viruses. (d) The indirect test can be used to detect antiviral antibody. (e) The FVPT is rapid and reliable. (f) Its simplicity should enhance its general acceptance and application.

Immunofluorescent cell assay of neonatal calf diarrhea virus.

A reliable plaque assay procedure has not yet been described for the neonatal calf diarrhea virus. Therefore, a previously developed immunofluorescent cell counting procedure was adapted to assay this virus. Adsorption of the virus to bovine kidney cells plateaued at 60 minutes. The optimal staining time was between 20 and 24 hours postinfection. Infected cells begun releasing from the coverslips if the cultures were incubated longer than 24 hours. This procedure has proven successful with virus grown in cell culture as well as virus present in fecal samples.

Effect of temperature on survival and growth of infectious pancreatic necrosis virus.

Infectious pancreatic necrosis virus was stable for 10 days at 4 C in stream and well water, after which the virus had a half-life of 7.5 days. At 15 C, the virus was stable for 5 days, and then had a half-life between 5 and 6 days. Viral antigen in infected cells developed much more slowly at 4 C than at 20 C. Infected cells released infectious viral particles at temperatures as low as 4 C. Nutrition had a greater effect on the production of infectious virus at 4 C than at 20 C.

Occurrence of spontaneous hemorrhagic necrosis of the central nervous system in fetal hamsters.

Lesions of hamster fetal neuraxial tissues, characterized by multifocal and coalescent zones of hemorrhage, edema, and necrosis in the cerebral mantle, brainstem, and spinal cord, were observed in experiments designed to test the teratogenicity of potato preparations. Retrospective and prospective data indicated, however, that the potato preparations were not responsible but that the disease occurred spontaneously in the colony and was associated with direct breeding contact of virgin females with certain males. Observations suggest that an infectious agent may be responsible, but no agent was recovered. Immunofluorescence assay of inoculated cultures indicated that reovirus was not present in affected fetal tissues.

Immunofluorescent cell assay of infectious pancreatic necrosis virus.

An immunofluorescent cell (IFC) assay technique was developed for the quantification of infectious pancreatic necrosis (IPN) virus of salmonid fishes. Cover slip cultures of rainbow trout gonad (RTG-2) cells were infected with diluted virus preparations. After incubation to permit antigen development, the cells were stained with antiviral fluorescent antibody, and the number of fluorescing (infected) cells was counted. Optimal conditions for the IFC assay procedure are: (i) the use of RTG-2 cells cultured for at least 3 days at 20 C; (ii) 1-h absorption of IPN virus to RTG-2 cells at 20 C or alternatively, 4 h at 4 C; (iii) staining the infected cell cultures at 10 to 12 h postinfection. A linear relationship between the relative concentration of virus in the inoculum and the number of fluorescent cells in the first cycle of infection was observed. The IFC assay method is more sensitive than the plaque method for the assay of IPN virus.