Gamma Radiation Inactivation of Coxsackievirus B-2

The radioresistance of coxsackievirus B-2 was studied when the virus was suspended in Eagle minimal essential medium, distilled water, cooked ground beef, and raw ground beef and irradiated at various temperatures in a cobalt-60 gamma radiation source. The number of surviving viruses at given doses of radiation was determined by a plaque assay system. All destruction curves indicated a first-order reaction. When the virus was irradiated in minimal essential medium at temperatures of -30, -60, and -90 C, D values (in Mrad) were 0.69, 0.59, and 0.64, respectively. When the virus was suspended in water and irradiated at -90 C, the D value was 0.53. Cooked ground beef containing the virus was irradiated at temperatures ranging from 16 to -90 C. The D values were 0.70 (16 C), 0.76 (0.5 C), 0.68 (-30 C), 0.78 (-60 C), and 0.81 (-90 C). Raw ground beef containing the virus was irradiated at -30, -60, and -90 C, and the D values were respectively 0.75, 0.71, and 0.68. The D values indicate that the rate of viral inactivation was dependent on the suspending menstrum.     

Chicken kidney cell culture in medium without serum

When chicken kidney cell (CKC) culture in a petri dish was prepared in medium with or without serum and incubated in a humidified incubator at 38 degreesC with no addition of CO2, monolayers of CKCs were formed completely on the 5th day of cultivation. Growth medium used for CKC culture was Eagle's minimum essential medium containing 0.3% of dehydrated tryptose phosphate broth. The number of cells in both cultures prepared in medium with or without serum was the same when measured on the 5th day of cultivation. Monolayers of CKC culture prepared in medium with or without serum were maintained up to 21 days of cultivation, while maintenance medium was changed every 4th day. The time of appearance and degree of cytopathic effect, plaque-forming ability, and propagation of some avian viruses were similar in both cultures prepared in medium with or without serum.     

Growth of Chikungunya Virus in Baby Hamster Kidney Cell (BHK-21-Clone 13) Suspension Cultures

This report describes the methods used to obtain high titers of chikungunya virus with suspension cultures of BHK-21-clone 13 cells. The cells were grown at 37 C to a cell concentration of 10(6) to 2 x 10(6) per ml. After maximum cell growth, the cells were inoculated with chikungunya virus at a multiplicity of 1 to 2 50% suckling mouse intracerebral lethal doses (SMICLD(50)) per cell in the spent Eagle's minimum essential medium for suspension cultures (MEMS), or the cell cultures were centrifuged at 200 x g and resuspended in either fresh MEMS or medium 199 prior to inoculation. The medium used had no effect on virus titer. The inoculated cultures were incubated at 34 C until the cell viability dropped to 30%, which usually occurred 28 to 30 hr postinoculation. After these procedures, chikungunya virus titers of log(10) 10.3 to 11.8 SMICLD(50) per ml were obtained.     

Cultivation of mosquito cell lines in serum-free media and their effects on dengue virus replication

Seven mosquito cell lines from five species (Aedes aegypti, Ae. albopictus, Ae. pseudoscutellaris, Culex tarsalis, and Toxorhynchites amboinensis) were adapted to three kinds of serum-free media (SEM), which were composed of equal volumes of tryptose phosphate broth and of either Leibovitz (L15) medium, Eagle's minimum essential medium, or Medium 199 with Hanks' salts. Population growth rates of the cells cultivated in the SMFs were generally slower than those of original cell cultures maintained in conventional media containing bovine sera. A karyological study showed a significant shift to heteroploidy in two of the four cell lines examined. Four SMF-adapted sublines were compared with parental cultures for replication of dengue viruses. Ae. aegypti RML-12, Ae. albopictus C6/36, Ae. pseudoscutellaris AP-61, and Tx. amboinensis TRA-171 demonstrated different levels of alteration in virus replication ranging from lower titers (as in Ae. albopictus C6/36) to comparable or higher titers (as in Ae. aegypti RML-12) when they were simultaneously inoculated with four dengue serotypes.     

Requirement of carbohydrates for cell fusion induced by vesicular stomatitis virus

The fusion of BHK-21-KB cells by vesicular stomatitis virus was not induced in Eagle's minimal essential medium without glucose. In medium containing glucose, the rate of polykaryocyte formation decreased as the concentration of glucose was reduced below 5 mM. However, no reduction in virus production 24 hr after infection was seen under this condition. Addition of pyruvate or mannose to the culture medium caused a reversal of cell fusing activity. Cell fusion and virus growth were significantly suppressed by sodium azide and 2,4-dinitrophenol.     

Interacting Factors That Influence Long-term Storage of Live Pasteurella tularensis Vaccine and Rift Valley Fever Virus

Studies were conducted on the interaction of various parameters which affect the storage stability and growth potential of liquid cultures of Pasteurella tularensis live vaccine strain (LVS) and Rift Valley fever virus Van Wyk strain (RVFV). Storage variables studied with LVS included four storage temperatures (4, -20, -65, -175 C), single and multiple freeze-thaw cycles, two freezing and two thawing rates (slow and fast), various inoculum levels (1, 3, 5, and 10%) for the determination of growth potential, and the retention of immunizing potential (mice and guinea pig) after storage. Neither the freezing rate nor the number of freeze-thaw cycles seriously affected the growth of LVS after storage at -175C; however, the slow rate of thaw proved deleterious as were all temperatures of storage except -175 C after 1 year of storage, as shown by both criteria of evaluation. RVFV produced in two combinations of cell lines and media (LM cell line-199 peptone medium and LDR cell line-Eagle's minimum essential medium) was stored at three serum levels (10, 20, 40%), three pH values (6.2., 7.0, 7.8), and three temperatures (-20, -65, -175 C). These studies indicated: (i) virus produced in the LDR cell line and Eagle's medium was more stable than that produced in the LM cell line and 199 peptone medium for either short- or long-term storage; (ii) serum levels did not affect stability; and (iii) low pH resulted in losses during long-term storage under all conditions tested. Thus, cryogenic storage is advantageous for stock culture maintenance of bacteria and viruses and for other similar applications.     

Fish cell lines: establishment and characterization of nine cell lines from salmonids

Nine permanent cell lines have been established from five species of salmonids native to America's Pacific Northwest. With the exception of a hepatoma from an adult trout, the lines were derived from normal tissues of embryonic or juvenile fish. Cells were routinely grown in Eagle's minimum essential medium with 10% fetal bovine serum. Optimum growth temperatures for these lines ranged from 21 to 24 degrees C. All survived storage for at least 1 yr at -65 degrees C and at least 5 yr in liquid nitrogen. Six of the lines were demonstrably free of any microbial contamination but mycoplasmas were found in three. Eight of the lines were heteroploid. The morphology of only one was fibroblastic. All the lines effectively replicated one or more of the common salmonid viruses. Isozyme patterns were consistent with those of the species of origin. These cell lines have significant application in fish virology.     

Primary culture of human diploid cells and its long-term transfer in a serum-free medium

Using a serum-free culture medium, primary human embryo fibroblasts can be grown in long-term serial culture. The basal medium consists of the components of modified Eagle's minimum essential medium (MEM) and non-essential amino acids, various growth factors and trace metals. Human fibronection (FN) and bovine serum albumin (BSA) were added. BSA was found to be essential for long-term serial culture in the presence of FN. Incubation in a gaseous environment of low oxygen (7% O₂) and low-temperature trypsinization at the time of transfer were also found to be important for growth in serum-free medium.     

Effect of room fluorescent light on the deterioration of tissue culture medium.

A major cause of tissue culture medium deterioration is exposure to room fluorescent light. Riboflavin and tryptophan present in Dulbecco's modified Eagle's minimum essential medium, when exposed to light, yield toxic photoproducts responsible for loss of the ability of the medium to support clonal growth of human, mouse and Chinese hamster cell lines. Procedures for minimizing medium deterioration are discussed.     

Effect of visible light on canine distemper virus

Nemo, George J. (The Catholic University of America, Washington, D.C.), and Ernest C. Cutchins. Effect of visible light on canine distemper virus. J. Bacteriol. 91:798-802. 1966.-Canine distemper virus (CDV) was inactivated by visible light. The virus was light-sensitive in fluid suspension (in vitro) as well as during intracellular replication (in vivo). The addition of calf serum or glutathione reduced the extent of inactivation. CDV was less sensitive when suspended in distilled water or in the amino acid or Earle's salts components of the minimal essential medium of Eagle than when suspended in the vitamin component of the minimal essential medium of Eagle or in riboflavine (0.1 mg per liter). These findings indicate that, whereas some ingredient of the medium may enhance light sensitivity, its presence is not necessary for light inactivation of CDV. It is proposed that some substance derived from the host cell and intimately associated with the virus particle serves to render CDV light-sensitive.     

Propagation of CAR bacillus in artificial media.

CAR bacillus propagated successfully in an artificial medium, and the number of CAR bacillus was about 30 times the original number after 8 days of cultivation. The medium consisted of Eagle's minimum essential medium supplemented with 10% fetal calf serum and 20% hamster tracheal organ culture soup. By intranasal inoculation to mice, two strains of the CAR bacillus passaged 5 and 6 times in this artificial medium produced the same lung lesions as natural CAR bacillus infection.     

Collection of eggs and hatching and culturing second-stage larvae of Toxocara vitulorum in vitro.

Eggs of Toxocara vitulorum were harvested from the feces of infected buffalo calves and embryonated in vitro. Optimum conditions for hatch and culture of the second-stage larvae were determined. Maximum hatch of larvae occurred from eggs that were decoated by treatment with saturated Ca(OCl)2 for 16-24 min followed by treatment with CO2 and incubation at 37 C. Larvae could be cultured in RPMI-1640 medium for up to 3 mo but survived for only 3 wk in Eagle's minimum essential medium.     

Continuous culture of Reuber hepatoma cells in serum-free arginine-, glutamine- and tyrosine-deprived chemically defined medium

The rat hepatoma cell line, H4-II-E, was grown serially over a 1-year period and about 30 passages in arginine-, glutamine-, and tyrosine-deprived and ornithine-supplemented Eagle's minimum essential medium with no supplements other than biotin. The adapted cell line, R-Y121B, proliferates in the above mentioned medium with a doubling time of about 4 days and maintains hepatic "marker" enzymes such as tyrosine aminotransferase, phenylalanine hydroxylase, and all the enzymes of the urea cycle.     

Host-Specific and Segment-Specific Evolutionary Dynamics of Avian and Human Influenza A Viruses: A Systematic Review

Understanding the evolutionary dynamics of influenza viruses is essential to control both avian and human influenza. Here, we analyze host-specific and segment-specific Tajima’s D trends of influenza A virus through a systematic review using viral sequences registered in the National Center for Biotechnology Information. To avoid bias from viral population subdivision, viral sequences were stratified according to their sampling locations and sampling years. As a result, we obtained a total of 580 datasets each of which consists of nucleotide sequences of influenza A viruses isolated from a single population of hosts at a single sampling site within a single year. By analyzing nucleotide sequences in the datasets, we found that Tajima’s D values of viral sequences were different depending on hosts and gene segments. Tajima’s D values of viruses isolated from chicken and human samples showed negative, suggesting purifying selection or a rapid population growth of the viruses. The negative Tajima’s D values in rapidly growing viral population were also observed in computer simulations. Tajima’s D values of PB2, PB1, PA, NP, and M genes of the viruses circulating in wild mallards were close to zero, suggesting that these genes have undergone neutral selection in constant-sized population. On the other hand, Tajima’s D values of HA and NA genes of these viruses were positive, indicating HA and NA have undergone balancing selection in wild mallards. Taken together, these results indicated the existence of unknown factors that maintain viral subtypes in wild mallards.     

Predicted inactivation of viruses of relevance to biodefense by solar radiation

UV radiation from the sun is the primary germicide in the environment. The goal of this study was to estimate inactivation of viruses by solar exposure. We reviewed published reports on 254-nm UV inactivation and tabulated the sensitivities of a wide variety of viruses, including those with double-stranded DNA, single-stranded DNA, double-stranded RNA, or single-stranded RNA genomes. We calculated D(37) values (fluence producing on average one lethal hit per virion and reducing viable virus to 37%) from all available data. We defined "size-normalized sensitivity" (SnS) by multiplying UV(254) sensitivities (D(37) values) by the genome size, and SnS values were relatively constant for viruses with similar genetic composition. In addition, SnS values were similar for complete virions and their defective particles, even when the corresponding D(37) values were significantly different. We used SnS to estimate the UV(254) sensitivities of viruses for which the genome composition and size were known but no UV inactivation data were available, including smallpox virus, Ebola, Marburg, Crimean-Congo, Junin, and other hemorrhagic viruses, and Venezuelan equine encephalitis and other encephalitis viruses. We compiled available data on virus inactivation as a function of wavelength and calculated a composite action spectrum that allowed extrapolation from the 254-nm data to solar UV. We combined our estimates of virus sensitivity with solar measurements at different geographical locations to predict virus inactivation. Our predictions agreed with the available experimental data. This work should be a useful step to understanding and eventually predicting the survival of viruses after their release in the environment.     

Utilization and stability of vitamins in serum-containing and serum-free media in CHO cell culture

The concentrations of four vitamins, ascorbic acid, nicotinamide, choline and thiamine were evaluated in the culture supernatant of Chinese hamster ovary (CHO) cells. The media used were -modified Eagle's minimum essential medium (MEM-) supplemented with 10% fetal calf serum, and a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (DME/F12), containing neither serum nor protein. The reference experiment without cells revealed instability of ascorbic acid and thiamine. Moreover, a significant amount of each vitamin decreased in the culture supernatant. The possibility of growth limitation by vitamin depletion is strongly suggested.     

Effect of room fluorescent light on the deterioration of tissue culture medium

Summary A major cause of tissue culture medium deterioration is exposure to room fluorescent light. Riboflavin and tryptophan present in Dulbecco's modified Eagle's minimum essential medium, when exposed to light, yield toxic photoproducts responsible for loss of the ability of the medium to support clonal growth of human, mouse and Chinese hamster cell lines. Procedures for minimizing medium deterioration are discussed. This work was supported by American Cancer Society Research Grant No. VC-100B and US PHS Research Career Development Award No. 5 K04 GM70537 from the National Institute of General Medical Sciences.     

Inactivation of viral agents in bovine serum by gamma irradiation

Cell culture origin or suckling mouse brain origin viruses of Akabane disease, Aino, bovine ephemeral fever, swine vesicular disease, hog cholera, bluetongue, and minute virus of mice were each suspended in bovine serum. Aliquots (1 mL) were exposed to various doses of gamma radiation from a 60Co source while at -68 degrees C. Aliquots (100-mL) of serum from a steer experimentally infected with foot-and-mouth disease virus were similarly irradiated. The samples were assayed for infectivity in cell culture systems before and after irradiation, and the data points were analyzed by linear regression. The irradiation doses (in megarads) necessary to inactivate one log10 of viral infectivity (D10) was calculated for each virus. D10 is otherwise known as the slope of the regression line. The r2 value, a measure of association with 1.0 = perfect fit, was also calculated for each regression line. The values (D10, r2) for each virus were as follows: Akabane, 0.25, 0.998; Aino, 0.35, 0.997; bovine ephemeral fever, 0.29, 0.961; swine vesicular disease, 0.50, 0.969; foot-and-mouth disease, 0.53, 0.978; hog cholera, 0.55, 0.974; bluetongue, 0.83, 0.958; and minute virus of mice, 1.07, 0.935.     

Class Specificity of Avian and Mammalian Sera in Regards to Myogenic Cell Growth in vitro

Chick myogenic cells grew in the presence of a small amount of avian serum in a culture medium composed of Eagle's minimum essential medium (MEM) and horse serum. Mammalian sera, except for fetal bovine serum at high concentrations, could not substitute for the avian serum.
Rat myogenic cells grew in the presence of a small amount of mammalian serum in a culture medium composed of MEM and chick serum: avian sera, except for dove serum at high concentrations, could not substitute for the mammalian serum.
Serum from animals of the class from which the myoblasts were obtained was needed for cell growth. It is thus concluded that there is a class specificity among sera in regards to myogenic cell growth. The only exceptions to this hypothesis found so far were fetal bovine and dove sera.     

Comparison of Methods for the Recovery of Virus Inoculated into Ground Beef

Various methods for the recovery of virus inoculated into ground beef were investigated in an attempt to develop a sensitive system that could be used to detect viral contaminants in market foods. A 100-g sample, inoculated with poliovirus 1, was suspended in 150 to 900 ml of Eagle minimum essential medium, pH 8.5, and mixed in either plastic bags or plastic cups on a mechanical shaker. The particulate materials were removed by means of cheese cloth, glass wool, woven fiber glass, or low-speed centrifugation. Large volumes of fluid were concentrated by ultrafiltration. Microbiological contamination was controlled by high antibiotic concentrations or by filtration. Quantitative plaque-forming-unit recovery of the virus was determined by utilizing an agar overlay technique on Vero cell cultures. The data indicated that from 20 to 50% of the seeded virus could be recovered from a 100-g sample of ground beef. The glass wool and woven fiber glass methods were the most effective, with recovery of approximately 50% of the inoculated virus.