Inhibition of Cellular DNA Synthesis in Cells Infected with Infectious Pancreatic Necrosis Virus

In asynchronous RTG-2 cell cultures infected with infectious pancreatic necrosis (IPN) virus, inhibition of cellular DNA synthesis, but not protein synthesis, was detected 5 to 6 h postinfection and was 80 to 90% complete by 7 to 8 h. Inhibition of DNA synthesis was largely abolished by UV irradiation of the virus. Sedimentation analyses of phenol-extracted DNA indicated that native cellular DNA was not degraded during infection. Sedimentation on alkaline sucrose gradients of DNA from cells pulsed with radioactive thymidine for varying periods indicated that elongation of nascent DNA chains proceeded normally in infected cells. These and previous results suggest that IPN virus infection results in a reduction of the number of chromosomal sites active in DNA synthesis but does not affect the rate of polymerization at active sites. Cells synchronized with excess thymidine and hydroxyurea and infected with virus at the time of release from the block demonstrated an inhibition of DNA synthesis 3 h postinfection. Cells infected 4 h prior to release continued to synthesize normal amounts of DNA for 1 to 2 h after release. These results indicated that DNA synthesis in early synthetic phase is relatively insensitive to inhibition by IPN virus.     

Immunofluorescent Cell-Counting Assay for Lymphocytic Choriomeningitis Virus

A quantitative assay for lymphocytic choriomeningitis virus was developed and standardized. The assay is based on direct immunofluorescent staining of infected L-929 cell monolayers and enumeration of cells containing fluorescent viral antigens. Maximal adsorption of virus to cells occurred within 1 h. Observations on the sequential development of viral antigens within cells showed that specific cytoplasmic fluorescence appeared within 10 h. The optimal time for enumerating fluorescent cells was from 18 to 20 h after addition of virus. A linear relationship was demonstrated between the number of infected cells and the relative virus concentration. Fluorescent cells were distributed randomly in infected cover slip cell monolayers. The immunofluorescent cell-counting assay for lymphocytic choriomeningitis virus was highly precise and reproducible.     

Induction and decay of thermotolerance in rainbow trout fibroblasts

Thermotolerance was studied in the rainbow trout fibroblast cell line RTG-2. RTG-2 cultures that had been incubated at 28 degrees C for 24 h were better able to withstand ultimately lethal temperatures above 28 degrees C than RTG-2 cultures that had been maintained at the routine growth temperature of 22 degrees C. This thermotolerance developed rapidly between 3 and 6 h and was fully developed by 24 h at 28 degrees C. After development for 24 hr at 28 degrees C, thermotolerance showed little change over 72 h at 0 and 5 degrees C but approximately a 40 and 60% reduction at 10 and 22 degrees C, respectively. This is the first demonstration of heat-induced thermal resistance in the cells of a poikilothermic vertebrate.     

Fluorescent Cell Counting as an Assay Method for Respiratory Syncytial Virus

The fluorescent cell-counting technique was applied to the enumeration of cell-infecting units of respiratory syncytial (RS) virus in human fetal diploid (HFD) cover-slip cell cultures; it was a sensitive, precise, and rapid assay method. Approximately 2 hr was required for maximal adsorption of RS virus to HFD cell monolayers. However, about 15% of the infectious virus in the inoculum remained unadsorbed; this percentage was not significantly reduced even when the adsorption period was extended to 5 hr. A linear relationship between virus concentration and the number of fluorescent cells existed over a range of 1.2 log(10) units. Variation of the mean of replicate determinations in a single experiment was approximately 7.5%. The distribution of single infected HFD cells on cover-slip cell cultures corresponded with the calculated frequencies of the Poisson distribution. The Chi square test for the extent of fit was calculated for several experiments, and the value of P was never less than 0.5. The addition of immune serum after virus adsorption effectively inhibited the development of detectable levels of viral antigen in secondarily infected cells.     

Polyunsaturated fatty acid metabolism in fish cells: differential metabolism of (n-3) and (n-6) series acids by cultured cells originating from a freshwater teleost fish and from a marine teleost fish

1. The incorporation and metabolism of (n-3) and (n-6) polyunsaturated fatty acids (PUFA) supplemented to growing cultures were studied in rainbow trout (RTG-2) and turbot (TF) cell lines. 2. A fatty acid concentration of 20 microM considerably altered the fatty acid composition of the cells without affecting lipid class composition or the appearance of cytoplasmic lipid droplets. 3. Both cell lines exhibited considerable delta 6 desaturase activities. 4. Whereas delta 5 desaturase activity was expressed in RTG-2 cells, delta 4 desaturase activity was absent and, conversely, delta 4 desaturase activity was expressed in TF cells, but there was an apparent deficiency in the C18 to C20 elongase multi-enzyme complex. 5. The delta 6 desaturase activity in both cell lines showed little preference between 18:2(n-6) and 18:3(n-3) but the delta 5 desaturase activity of RTG-2 cells and the delta 4 desaturase activity of TF cells showed a preference for (n-3)PUFA. 6. Two fish oil concentrates were assessed for their ability to generate fatty acid compositions in the cell lines more closely resembling those of intact fish tissues.     

Growth of Fish Cell Lines on Microcarriers

Microcarrier beads were evaluated as substrates for the propagation of five anchorage-dependent fish cell lines. Growth of rainbow trout gonad (RTG-2) and Atlantic salmon cells was limited on microcarriers maintained in suspension. However, stationary microcarriers were suitable substrates for the growth of RTG-2, AS, Chinook salmon embryo (CHSE-214), and fathead minnow cells. Cell yields ranged from 2 × 10⁶ to 2.9 × 10⁶ cells per ml, representing 7- to 10-fold increases over the initial cell concentrations. The yield of new RTG-2 cells per unit volume of growth medium was 2.8 times greater in microcarrier cultures than in standard monolayer cultures. Northern pike cells failed to grow on microcarriers. Yields of infectious pancreatic necrosis virus propagated in microcarrier cultures of RTG-2 cells were more than twice the yields in standard monolayer cultures. The greater economy of microcarrier cultures in terms of growth vessel and medium requirements holds great promise for the large-scale production of anchorage-dependent fish cell cultures and fish viruses.     

Assay of Variola Virus by the Fluorescent Cell-Counting Technique

A quantitative assay for infective variola virus particles was developed which is based on the enumeration of cells containing fluorescent viral antigen after infection of McCoy cell monolayers. The direct fluorescent-antibody technique was employed to stain cells. The efficiency of virus adsorption was markedly enhanced by centrifugation of virus inoculum onto McCoy cell monolayers at 500 x g for 15 min. By this procedure, a proportionality was obtained between the number of fluorescent cells and volume of inoculum. Observations on the sequential development of viral antigen within cells and counts of fluorescent cells showed that the optimal time for enumerating fluorescent cells was after an incubation period of 16 to 20 hr. A linear function existed between virus concentration and cell-infecting units. Fluorescent cells were distributed randomly in infected cover slip cell monolayers. The assay was demonstrated to be highly sensitive, precise, and reproducible.     

Fluorescent brightener inhibits apoptosis in baculovirus-infected gypsy moth larval midgut cells in vitro

Fluorescent brighteners significantly lower the LC₅₀ and LT₅₀ in a variety of nucleopolyhedrovirus-insect host systems. In larvae of the gypsy moth, Lymantria dispar (L.), a European NPV strain of virus (LdMNPV) does not normally replicate in the midgut, but addition of a fluorescent brightener (Calcofluor M2R) to the virus suspension results in productive infections. In the current study, we show that LdMNPV also does not replicate in a larval midgut primary cell culture system unless a fluorescent brightener (Blankophor P167) is added. Morphological and cellular changes characteristic of apoptotic cell death were noted in infected midgut cells in vitro. We used the TUNEL assay to measure apoptosis in virus-challenged midgut cell cultures at 24-48 h post-inoculation. A significant decrease in apoptotic midgut cells was noted in the presence of 0.01 M brightener. The inhibition of apoptosis and presumptive inhibition of shedding of infected midgut cells in the presence of fluorescent brightener in the insect midgut appeared to promote virus replication and are likely to be partly responsible for enhancement of LdMNPV activity that is observed in gypsy moth larvae.     

Primary Virus-Cell Interactions in the Immuno-fluorescence Assay of Venezuelan Equine Encephalomyelitis Virus

The conditions under which Venezuelan equine encephalomyelitis (VEE) virus attached to host cells markedly influenced the assay of virus by the fluorescent cell-counting technique. When virus inoculum was centrifuged onto McCoy cell monolayers, approximately 97% of virus was attached to cells within 10 min, in contrast to 34% after stationary incubation at 35 C for 2 hr. Maximal binding of virus occurred only in the presence of 0.1 to 0.15 m NaCl. This salt requirement, added to evidence of (p)H dependence and temperature independence of VEE virus attachment to cells, indicated that the initial union involved electrostatic forces. Virus penetration, measured by the insensitivity of virus-cell complexes to viral antiserum, was complete in 30 min at 35 C. The process was temperature-dependent and un-affected by the ionic content of medium. For assay of VEE virus by the fluorescent cell-counting technique, infected cells may be enumerated as early as 12 hr after infection of cell monolayers. The relationship between virus concentration and cell-infecting units was linear; the distribution of fluorescent cells was random. The virus assay was equivalent in sensitivity but more precise and rapid than that of intracerebral inoculation of mice.     

Simian virus 40 gene A regulation of cellular DNA synthesis. II. In nonpermissive cells.

The stimulation of host macromolecular synthesis and induction into the cell cycle of serum-deprived G0-G1-arrested mouse embryo fibroblasts were examined after infection of resting cells with wild-type simian virus 40 or with viral mutants affecting T antigen (tsA58) or small t antigen (dl884). At various times after virus infection, cell cultures were analyzed for DNA synthesis by autoradiography and flow microfluorimetry. Whereas mock-infected cultured remained quiescent and displayed either a 2N DNA content (80%) or a 4N DNA content (15%), mouse cells infected with wild-type simian virus 40, tsA58 at 33 degrees C, or dl884 were induced into active cell cycling at approximately 18 h postinfection. Although dl884-infected mouse cells were induced to cycle initially at the same rate as wild type-infected cells, they became arrested earlier after infection and also failed to reach the saturation densities of wild-type simian virus 40-infected cells. Infection with dl884 also failed to induce loss of cytoplasmic actin cables in the majority of the infected cell population. Mouse cells infected with tsA58 and maintained at 39.5 degrees C showed a transient burst of DNA synthesis as reflected by changes in cell DNA content and an increase in the number of labeled nuclei during the first 24 h postinfection; however, after the abortive stimulation of DNA synthesis at 39.5 degrees C shift experiments demonstrated that host DNA replication was regulated by a functional A gene product. It is concluded that both products of the early region of simian virus 40 DNA play a complementary role in recruiting and maintaining simian virus 40-infected cells in the cell cycle.     

A nuclear localization of the infectious haematopoietic necrosis virus NV protein is necessary for optimal viral growth

The nonvirion (NV) protein of infectious hematopoietic necrosis virus (IHNV) has been previously reported to be essential for efficient growth and pathogenicity of IHNV. However, little is known about the mechanism by which the NV supports the viral growth. In this study, cellular localization of NV and its role in IHNV growth in host cells was investigated. Through transient transfection in RTG-2 cells of NV fused to green fluorescent protein (GFP), a nuclear localization of NV was demonstrated. Deletion analyses showed that the (32)EGDL(35) residues were essential for nuclear localization of NV protein, and fusion of these 4 amino acids to GFP directed its transport to the nucleus. We generated a recombinant IHNV, rIHNV-NV-ΔEGDL in which the (32)EGDL(35) was deleted from the NV. rIHNVs with wild-type NV (rIHNV-NV) or with the NV gene replaced with GFP (rIHNV-ΔNV-GFP) were used as controls. RTG-2 cells infected with rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL yielded 12- and 5-fold less infectious virion, respectively, than wild type rIHNV-infected cells at 48 h post-infection (p.i.). While treatment with poly I∶C at 24 h p.i. did not inhibit replication of wild-type rIHNVs, replication rates of rIHNV-ΔNV-GFP and rIHNV-NV-ΔEGDL were inhibited by poly I∶C. In addition, both rIHNV-ΔNV and rIHNV-NV-ΔEGDL induced higher levels of expressions of both IFN1 and Mx1 than wild-type rIHNV. These data suggest that the IHNV NV may support the growth of IHNV through inhibition of the INF system and the amino acid residues of (32)EGDL(35) responsible for nuclear localization are important for the inhibitory activity of NV.     

Inactivated Rabies Vaccine Produced from the Flury LEP Strain of Virus Grown in BHK-21 Suspension Cells

Suspension cultures of BHK-21 cells maintained at 32 to 33 C were infected with the Flury LEP strain of rabies virus. By using a cell concentration of 2.0 x 10(6) to 2.5 x 10(6) cells per ml infected at a multiplicity of 0.05, high titers of extracellular virus were reached in 96 to 120 h, and potent inactivated vaccines were prepared from culture fluids harvested between 96 to 168 h. The addition of 1% bovine serum to the maintenance medium resulted in an increase in virus yields and vaccine potency. Estimation of the number of infected cells by immunofluorescent procedures proved a rapid and reliable guide to the virus content of suspension cultures.     

Culture conditions for arresting and stimulating the proliferation of a rainbow trout fibroblast cell line,RTG-2

Summary Conditions for arresting and stimulating the proliferation of the rainbow trout fibroblast cell line RTG-2 have been examined and the time course of events after stimulation determined. Quiescent populations were achieved in two ways. Cultures grown to confluency without a medium change for at least 7 d had fewer than 5% of the cells in S phase and few mitotic figures. Cultures deprived of serum, which could be done for up to 3 d without a loss in cell number, also achieved quiescence. After 3 d without serum, less than 1% of cells were in S phase and mitotic figures were infrequent. Addition to these cultures of fresh serum-containing medium brought about the synchronous entry of cells into S phase and mitosis. For cultures in which either the medium had been changed after 7 d without a change or serum-containing medium had been added after 3 d of serum deprivation, DNA synthesis increased after a lag period of 20 to 24 h, was pronounced between 30 and 45 h, and then declined. This was followed by a peak in the mitotic index. These protocols for arresting and subsequently stimulating RTG-2 proliferation should allow the G₁-S transition to be studied in a representative of teleosts. This research was supported by Natural Sciences and Engineering Research Council of Canada grant to N. C. B.     

Development of an antibody-based assay for determination of baculovirus titers in 10 hours

The baculovirus expression system has been used to produce large amounts of biologically active proteins by infecting insect cells with a recombinant baculovirus expressing the target protein. For an efficient expression of the target protein, it is necessary to infect insect cells with an adequate amount of virus. However, current methods are time-consuming and either have technical difficulties or are limited as a result of virus expression mechanism using a reporter gene. A novel method is developed to yield virus titers in 10 h that is easy to perform using 96-well plates and applicable to both any Autographa californica nucleopolyhyderovirus (AcNPV) and Bombyx mori nucleopolyhedrovirus (BmNPV)-based recombinant baculovirus. This assay uses an antibody to a DNA-binding protein to detect the infected cells via immunostaining. The titer is determined by counting foci produced as a result of infection of the virus under a fluorescent microscope. The required incubation period was shortened considerably because infected cells expressed viral antigens at the post-infection time of 4 h. Therefore, 10 h was enough to estimate the virus titer including virus infection time, insect cell culture, and estimation of virus titer. Titers determined using this immunological assay are comparable, both in value and validity, to those obtained using a traditional method, provided that the stocks have titers above 10(3) pfu/mL.     

Characterization of a virus obtained from snakeheads Ophicephalus striatus with epizootic ulcerative syndrome (EUS) in the Philippines

This is the first report of the isolation and characterization of a fish virus from the Philippines. The virus was isolated using snakehead spleen cells (SHS) from severely lesioned epizootic ulcerative syndrome (EUS)-affected snakehead Ophicephalus striatus from Laguna de Bay, in January 1991. The virus induced cytopathic effects (CPE) in SHS cells yielding a titer of 3.02 x 10(6) TCID50 ml(-1) at 25 degrees C within 2 to 3 d. Other susceptible cell lines included bluegill fry (BF-2), catfish spleen (CFS) and channel catfish ovary (CCO) cells. Replication in chinook salmon embryo cells (CHSE-214) was minimal while Epithelioma papulosum cyprini cells (EPC) and rainbow trout gonad cells (RTG-2) were refractory. Temperatures of 15 to 25 degrees C were optimum for virus replication but the virus did not replicate at 37 degrees C. The virus can be stored at -10 and 8 degrees C for 30 and 10 d, respectively, without significant loss of infectivity. Viral replication was logarithmic with a 2 h lag phase; viral assembly in the host cells occurred in 4 h and release of virus occurred 8 h after viral infection. A 1-log difference in TCID50 titer between the cell-free virus and the total virus was noted. Freezing and thawing the virus caused a half-log drop in titer. Viral exposure to chloroform or heating to 56 degrees C for 30 min inactivated the virus. Exposure to pH 3 medium for 30 min resulted in a more than 100-fold loss of viral infectivity. The 5-iododeoxyuridine (IUdR) did not affect virus replication, indicating a RNA genome. Neutralization tests using the Philippine virus, the ulcerative disease rhabdovirus (UDRV) and the infectious hematopoietic necrosis virus (IHNV) polyvalent antisera showed slight cross-reaction between the Philippine virus antiserum and UDRV but established no serological relationship with SHRV and IHN virus. Transmission electron microscopy (TEM) of SHS cells infected with the virus showed virus particles with typical bullet morphology and an estimated size of 65 x 175 nm. The Philippine virus was therefore a rhabdovirus, but the present study did not establish its role in the epizootiology of EUS.     

An efficient method for introducing defined lipids into the plasma membrane of mammalian cells

An efficient method has been devised to introduce lipid molecules into the plasma membrane of mammalian cells. This method has been applied to fuse lipid vesicles with the apical plasma membrane of Madin-Darby canine kidney cells. The cells were infected with fowl plague or influenza N virus. 4 h after infection, the hemagglutinin (HA) spike glycoprotein of the virus was present in the apical plasma membrane of the cells. Lipid vesicles containing egg phosphatidylcholine, cholesterol, and an HA receptor (ganglioside) were then bound to the cells at 0 degrees C. More than 85% of the vesicles were released by external neuraminidase at 0 degrees C or by simply warming the cells to 37 degrees C for 10 s, probably because of the action of the viral neuraminidase at the cell surface. However, when the cells were warmed to 37 degrees C in a pH 5.3 medium for 30 s, 50% of the bound vesicles could no longer be released by external neuraminidase. This only occurred when the HA protein had been cleaved into its HA1 and HA2 subunits. When we used influenza N virus, whose HA is not cleaved in Madin-Darby canine kidney cells, cleavage with external trypsin was required. The fact that the HA protein has fusogenic properties at low pH only in its cleaved form suggests that fusion of the vesicles with the plasma membrane had taken place. Further confirmation for fusion was obtained using an assay based on the decrease of energy transfer between two fluorescent phospholipids in a vesicle upon fusion of the vesicle with the plasma membrane (Struck, D. K., D. Hoekstra, and R. E. Pagano. 1981. Biochemistry, 20:4093-4099).     

Association of Virus with Cases of Rubella Studied in Toronto: Propagation of the Agent and Transmission to Monkeys

Using an interference test with indicator virus Echo 11, a virus has been isolated in nine of 18 specimens from cases of typical rubella. The virus will interfere with the development of cytopathology in green monkey kidney cells with viruses Echo 11, Coxsackie B1 and B4, Poliovirus I and III (Sabin strains) and simian virus SV4. In four of five paired sera this virus was neutralized by convalescent but not by the acute phase serum, tested by interference inhibition. No cytopathology was observed in unstained cultures or in sequential cultures stained with acridine orange or fluorescent antibody. The virus was destroyed by exposure to 56° C. for 30 minutes and 15% ether at 4° C. for 24 hours, but survived with some reduction in titre at 4° C. for 24 hours. Green monkeys infected by this virus developed a macular rash, lymphadenopathy and modest rise in white blood cell count.     

Titration and Extensive Serial Passages of Yaba Virus In Vitro

A sensitive assay system of Yaba virus (YV) was established in a cynomolgus monkey kidney cell line, JINET, in which the virus caused multilayered cellular foci countable even with the unaided eye. The specificity of the foci induced by YV in these cells was demonstrated by (1) the focus-forming ability was destroyed by heating at 60 C for 12 min; (2) the focus formation was inhibited by specific antiserum; (3) specific fluorescence was detected only in cells composing the foci when tested by fluorescent antibody technique; (4) a linear relationship was observed between the virus concentration and the number of foci formed; (5) YV preparation passed 20 times in JINET cells still possessed “tumorigenicity” in cynomolgus monkeys. The sensitivity of JINET cells to YV was comparable to that of cynomolgus monkeys, and YV was successively propagated in JINET cells with 2 log increase in infectivity titer during over 40 serial passages. Application of this assay system to growth kinetic studies of YV and quantitation of neutralizing antibody to YV is also discussed.