Peak Virus Production Research Articles

Human Alpha- and Beta-Interferon But Not Gamma- Suppress theIn VitroReplication of LAV, HTLV-III, and ARV-2

The effect of human interferons (IFNs) (alpha, beta, and gamma) on the in vitro replication of AIDS viruses (LAV, HTLV-III, and ARV-2) in human peripheral blood lymphocytes was investigated. At the time of peak virus production, IFN-alpha preparations (leukocyte, Namalwa, alpha 1, and alpha 2) at 100 U/ml, suppressed LAV, HTLV-III, and ARV-2 replication as measured by reverse transcriptase (RT) activity by greater than 50%. This suppression was dose dependent and high dosages (500 U/ml) of IFN-alpha resulted in almost complete suppression of RT activities (77-99%). A low dose (100 U/ml) of IFN-beta suppressed all three AIDS viruses by 75%. In contrast, human IFN-gamma at a dose range from 100 U/ml to 500 U/ml had no significant effect on the production of infectious viruses. These results indicate that only IFN-alpha and -beta are effective against LAV, HTLV-III, and ARV-2 replication. A continuous supply of IFN appeared to be essential for the constant suppression of RT activity. In fact, upon termination of single IFN treatment, enhanced virus production resulted.

Anti-influenza A activity of combinations of amantadine and ribavirin in ferret tracheal ciliated epithelium.

The anti-influenza A activities of amantadine and ribavirin were investigated separately and in combination. Ferret tracheal ciliated epithelium was continuously exposed to the drugs at concentrations (0.25, 0.5, and 1 mg/l) comparable to those found in human serum after oral administration. Each drug alone produced a modest delay in A/Alaska/6/77 (H3N2) induced cytopathic effect. The combination of drugs synergistically delayed cytopathic effect. At 1 mg/l of each, cytopathic effect was prevented in 75% of rings for the 28-day duration of the experiment. This effect was greater than that of 32 mg/l of amantadine or 64 mg/l of ribavirin as single antiviral drugs. Peak virus production was suppressed 4.4 log-fold by the combination of 1 mg/l of each drug. This is in contrast to amantadine alone which suppressed peak virus production by 1 log-fold and ribavirin along which at 1 mg/l suppressed peak virus production by 1.9 log-fold. At lower concentrations, the drugs were at least additive in suppression of virus production.

In vitro production of Rauscher murine leukemia virus: influence of culture age on biological properties.

Large-scale production and concentration procedures have been standardized to study the biological properties of Rauscher leukemia virus produced from the high-passaged JLS-V9-H mouse bone marrow cell line. Virus produced early (days 4 to 6) in the harvest and refeed cycle contained higher levels of ribonucleic acid-directed deoxyribonucleic acid polymerase activity and was more infectious than Rauscher leukemia virus produced later (days 7 to 10) in the growth period. The peak of virus production as detected by physical assays (virus particle count, protein, and p30 antigen) was highest at day 6, whereas the optimum biological and ribonucleic acid-directed deoxyribonucleic acid polymerase activity occurred 24 h earlier. When product characterization values of each concentrate were adjusted to a specific activity (i.e., per milligram of protein) basis, virus particle counts averaged 4 x 10(11) through days 5 to 9, and the peak infectivity occurred at day 4, whereas ribonucleic acid-directed deoxyribonucleic acid polymerase activity was highest at day 4 (endogenous) and 5 (exogenous). Sodium dodecyl sulfate-polyacrylamide gel analysis revealed only slight differences in the polypeptide pattern of Rauscher leukemia virus harvested from cultures of varying age, although Rauscher leukemia virus produced between days 3 and 5 contained more glycoprotein than either earlier or later harvests.

The synthesis and structure of visna virus DNA

The synthesis of proviral DNA of visna virus was measured at various intervals after inoculation of sheep cell cultures at multiplicities of 0.3, 1, and 10 PFU/cell. The DNA from the infected cells was fractionated by the Hirt procedure into low (Hirt supernatant) and high (Hirt precipitate) molecular weight DNAs, and each fraction was quantitated for infectivity by plaque assay using the calcium phosphate transfection technique of Graham and Van Der Eb (1973). The appearance of infectious DNA in the Hirt supernatant (low molecular weight viral DNA) was biphasic at all multiplicities, apparently reflecting two rounds of synthesis of this DNA. The amount of infectious DNA in the Hirt precipitate fraction increased with time, reaching maximum levels in all cultures at the time of peak virus production. Hirt supernatant DNA consisted predominantly of molecules of molecular weight 6 × 10 6, which appeared to be present as double-stranded linear molecules. Infectious DNA in the Hirt precipitate, in contrast, had a molecular weight of 166 x 106, suggesting its association with cellular sequences. The network test strongly suggested that the viral sequences were covalently linked with or integrated into the cellular DNA.

Purification and biophysical properties of human coronavirus 229E

Coronavirus 229E was grown to high titers in diploid fibroblast cells under medium containing twice the normal concentrations of amino acids and vitamins. Growth curves showed maximum virus production at multiplicities of infection of 0.1 and 1; maximum titers of intracellular virus occurred at 22–24 hr and of extracellular virus at 26 hr postadsorption. Tube infectivity titers ranged from 10 9.0–10 9.5 TCID 50/ml and plaque titers from 10 10.2–10 10.9 y PFU/ml at the time of peak virus production, when no cytopathology was evident. Virus titer dropped rapidly between 26 and 56 hr, coincident with increasing cytopathology. A single precipitin band was observed in immunodiffusion and immunoelectrophoresis between concentrated virus preparations and antiserum to purified 229E. Neuraminidase and hemagglutinin assays were negative. Virus was purified by two procedures: adsorption to and elution from human “0” erythrocytes and CaHPO 4 gel followed by equilibrium sucrose gradient centrifugation, and PEG precipitation followed by equilibrium glycerol/tartrate gradients and rate zonal sucrose or glycerol/tartrate gradients. Final lots of purified virus containing <0.02% of the crude tissue culture proteins had absorption maxima at 256 nm and minima at 241.2 nm and a mean extinction coefficient of E 1cm 1% = 54.3 at 256 nm. The fully corrected sedimentation coefficient for the intact virion was S 20,v 0 = 381 S. PAGE by different techniques revealed seven polypeptides of mean apparent molecular weights between 16,900 and 196,100. Six contained carbohydrate and one contained lipid. Electropherograms of 3H- and 14C-labeled virus were identical to those of stained gels. Two glycoproteins constituting 25% of the virion protein were identified by bromelin digestion as the spike proteins. The density in sucrose and in potassium tartrate was 1.18 g/ml for the virion and 1.15 g/ml for the “despiked” particle.

Studies on the budding process of a temperature-sensitive mutant of murine leukemia virus with a scanning electron microscope.

The scanning electron microscope was used to study the budding process of the wild-type Moloney murine leukemia virus and one of its temperature-sensitive mutants, designated ts 3. A considerably larger number of budding particles was observed on TB cells infected with ts 3 at the nonpermissive temperature (39 C) than at the permissive temperature (34 C). No apparent difference was noted between the number of particles on ts 3-infected cells at (34 C) and wild-type-infected cells at 34 or 39 C. Virions were detected at the cell membrane of ts 3-infected cells at 39 C as early as 8 h postinfection. Virion density increased progressively up to 48 h after which no increase was observed. An average of 1,600 virus particles was observed at the cell surface at the peak of virus production. The distribution of these on the cell membrane appeared to be random. The maximum proportion of the cell surface occupied by the viral particles did not exceed 10%. After temperature shift from 39 to 34 C, approximately 90% of the particles had dissociated from the cell membrane within 1 h.

Induction of type C viruses in cultured guinea pig cells.

Particles morphologically resembling type C viruses were activated by bromodeoxyuridine (BUdR; 10(-4) M) treatment of cultured guinea pig cells. Virus particles were isolated from the cells of normal and leukemic strain 2 and random-bred guinea pigs (adult and embryonic). Immature virus particles with electron-lucent cores were found in the cytoplasmic matrix. The mature particles with electron-dense cores were found outside the cells and some appeared in the process of budding from the plasma membrane. The peak of virus production was observed within 4 days of BUdR treatment. When compared to the amount of virus produced in darkness, visible light enhanced virus production, whereas exposure of BUdR-treated cells to UV light either had no effect or inhibited virus production. Virus particles had a density of 1.16 gm/ml, possessed an oncornavirus-specific reverse transcriptase, and contained a large-molecular-weight RNA (65-70S) which dissociated into 36S subunits after heat denaturation. The BUdR-activated virus particles, therefore, possessed the morphological, biophysical, and biochemical characteristics of type C oncornaviruses.

Inhibition of ribonucleic acid methylation in the foot-and-mouth disease virus-infected host cell

Inhibition of baby hamster kidney (BHK) host cell protein and RNA synthesis following infection with foot-and-mouth disease virus (FMDV) has been described (Brown et al. , 1966; Polatnick et al. , in press). Foot-and-mouth disease virus (type A, strain 119) infection causes a more drastic inhibition of host cell protein synthesis than of RNA synthesis throughout the infectious cycle. For example, early in infection (90 min postinfection [PI]), and at peak virus production (300 min PI), protein synthesis is inhibited approximately 50 and 80%, respectively, while RNA synthesis is inhibited only 20 and 50% (Polatnick et al. , in press). In this report, the effect of FMDV infection on host cell RNA methylation is examined at 300 min PI. Methylation of total host cell RNA, fractionated 1 M NaCl insoluble RNA and sRNA is shown to be inhibited to the same degree as host cell protein synthesis in the FMDV infected cell.