Correlation between infectivity and physical virus particles in human cytomegalovirus

Benyesh-Melnick, Matilda (Baylor University College of Medicine, Houston, Tex.), Fern Probstmeyer, Robert McCombs, Jean P. Brunschwig, and Vladimir Vonka. Correlation between infectivity and physical virus particles in human cyto-megalovirus. J. Bacteriol. 92:1555-1561. 1966.-Infectivity titers [measured as plaque-forming units (PFU)] and particle counts by the sedimentation pseudo-replication technique were determined for crude, unpurified, intracellular preparations of two different strains of human cytomegalovirus. Unlike the high particle-infectivity ratio of 10(6) to 10(8) previously reported for these viruses, the number of total particles per PFU ranged from 160 to 490 with strain AD-169 and from 176 to 1,050 for strain C-87. Interpretation of particle-PFU ratios of intracellular cytomegalovirus in terms of particle morphology is not conclusive at this time. The number of enveloped forms found varied between 0 and 34% of the total particles counted. However, the true proportion is probably greater, because envelopes were found to be destroyed by the enzyme treatment used in preparing the specimens for examination in the electron microscope. The number of full particles found ranged between 4 and 31% of the total particles counted. The particle per PFU ratio of extracellular virus was found to be three- to fivefold lower than that of intracellular virus.     

Preparation of Poliovirus Labeled with Phosphorus-33

Phosphorus-33 ((33)P), a weak (0.25 Mev) beta-emitting isotope of phosphorus with a half-life of 25 days, has been used to label poliovirus in cell culture. HeLa cell monolayers were depleted of phosphate and then labeled by incubating at 37 C in a medium (LM) containing about 10 muCi of (33)P as orthophosphate per ml. Labeled cells were infected at a high multiplicity with poliovirus type 1 and incubated for 8 hr in LM medium. Virus from infected cells was then concentrated and purified. Virus purity was confirmed by comparison of virus infectivity and radioactivity after CsCl density gradient centrifugation and by observing purified virus preparations with electron microscopy. With the method described, yields of about 10(10) to 5 x 10(10) plaque-forming units (PFU) of highly purified poliovirus with specific activities of about 3 x 10(-4) to 10(-3) disintegrations per min per PFU have been obtained from 1.5 x 10(8) to 3.0 x 10(8) HeLa cells.     

Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells

Gilden, R. V. (Wistar Institute, Philadelphia, Pa.), and R. I. Carp. Effects of cycloheximide and puromycin on synthesis of simian virus 40 T antigen in green monkey kidney cells. J. Bacteriol. 91:1295-1297. 1966.-Synthesis of the simian virus 40 (SV40) T antigen in primary African green monkey kidney cells was abolished when cycloheximide was added up to 10 hr postinfection. In contrast, puromycin, another inhibitor of protein synthesis, did not suppress antigen production. The basis of this differential effect was the inability of puromycin to inhibit protein synthesis in the cells used. This was shown by the failure of the drug to depress the incorporation of labeled amino acid into protein and also failure to inhibit poliovirus synthesis. The puromycin preparation used was very effective in inhibiting poliovirus synthesis in HeLa cells. Thus, appearance of the SV40 T antigen is dependent on protein synthesis in infected cells.     

Mechanism by Which Fiber Antigen Inhibits Multiplication of Type 5 Adenovirus

Purified fiber antigen of type 5 adenovirus inhibited the multiplication of type 5 adenovirus by 50% when 35 mug of fiber antigen protein was added to 10(6) KB cells in suspension culture. Although the fiber antigen reduced the number of virions adsorbed per cell when a multiplicity of infection of 50,000 plaque-forming units (PFU)/cell was employed, the number of cells infected was not diminished under these conditions. If a low multiplicity of infection (1.1 PFU/cell) was used, viral adsorption was not detectably decreased. The fiber antigen did not reduce the capability of virions to liberate their viral deoxyribonucleic acid (DNA). The biosyntheses of DNA, ribonucleic acid (RNA), and protein were blocked about 20 to 25 hr after the addition of fiber antigen to cultures of uninfected or type 5 adenovirus-infected KB cells. Most of the fiber antigen protein became cell-associated between 22 and 36 hr after it was added to cells. The hexon antigen neither inhibited viral multiplication nor blocked the biosynthesis of DNA, RNA, or protein. Moreover, the hexon did not attach to KB cells. The profound effects of the fiber antigen were not due to the induction of an interferon-like substance, for actinomycin D did not reduce the ability of the fiber to inhibit multiplication of type 1 poliovirus.     

Translation and replication of human rhinovirus type 14 and mengovirus in Xenopus oocytes

We have previously shown that Xenopus oocytes require coinjection of both poliovirus RNA and HeLa cell extracts to support a complete cycle of viral replication yielding high levels of infectious viral particles. This novel system provides a tool for identifying host factors and for biochemically dissect individual steps that lead to virus production. Here we demonstrate that Xenopus oocytes are able to support replication of other picornaviruses such as human rhinovirus 14 and mengovirus. Unlike poliovirus, microinjection of mengovirus RNA yields high viral titers (about 10(7) PFU/oocyte) without the need for coinjection of additional cell extracts. In contrast, formation of infectious rhinovirus particles requires coinjection of human cell extracts. We found that one of these human factors is required for efficient rhinovirus translation. Our findings uncover differences in the host factor requirements among members of the picornavirus family and provide the means to identify the human protein(s) involved in rhinovirus production.     

Characterization of a synthetic bacterial self-destruction device for programmed cell death and for recombinant proteins release

Background

In a globalized word, prevention of infectious diseases is a major challenge. Rapid detection of viable virus particles in water and other environmental samples is essential to public health risk assessment, homeland security and environmental protection. Current virus detection methods, especially assessing viral infectivity, are complex and time-consuming, making point-of-care detection a challenge. Faster, more sensitive, highly specific methods are needed to quantify potentially hazardous viral pathogens and to determine if suspected materials contain viable viral particles. Fourier transform infrared (FTIR) spectroscopy combined with cellular-based sensing, may offer a precise way to detect specific viruses. This approach utilizes infrared light to monitor changes in molecular components of cells by tracking changes in absorbance patterns produced following virus infection. In this work poliovirus (PV1) was used to evaluate the utility of FTIR spectroscopy with cell culture for rapid detection of infective virus particles.

Results

Buffalo green monkey kidney (BGMK) cells infected with different virus titers were studied at 1 - 12 hours post-infection (h.p.i.). A partial least squares (PLS) regression method was used to analyze and model cellular responses to different infection titers and times post-infection. The model performs best at 8 h.p.i., resulting in an estimated root mean square error of cross validation (RMSECV) of 17 plaque forming units (PFU)/ml when using low titers of infection of 10 and 100 PFU/ml. Higher titers, from 10³ to 10⁶ PFU/ml, could also be reliably detected.

Conclusions

This approach to poliovirus detection and quantification using FTIR spectroscopy and cell culture could potentially be extended to compare biochemical cell responses to infection with different viruses. This virus detection method could feasibly be adapted to an automated scheme for use in areas such as water safety monitoring and medical diagnostics.     

Survival of Virus in Chilled, Frozen, and Processed Oysters

Samples of whole and shucked Pacific and Olympia oysters, contaminated with 10(4)-plaque-forming units (PFU) of poliovirus Lsc-2ab per ml, were held refrigerated at two temperatures, 5 and - 17.5 C. To study the survival of virus in the oysters under these conditions, samples were assayed for virus content at weekly intervals for as long as 12 weeks. Results indicated that poliovirus would survive in refrigerated oysters for a period varying from 30 to 90 days, depending upon temperature. The survival rate varied from 10 to 13%. To study the extent of the hazard presented by oysters contaminated with virus, samples of whole and shucked Pacific oysters contaminated with 10(4) PFU of poliovirus Lsc-2ab per ml were heat processed in four ways: by stewing, frying, baking, and steaming. Results indicated that virus in oysters withstood these methods of processing. The survival rate varied from 7 to 10% and appeared dependent upon the processing method used. Heat penetration studies showed that the internal temperature in the oyster was not sufficient to inactivate all of the virus present. These results suggest that not only fresh but also refrigerated and cooked oysters can serve as vectors for the dissemination of virus disease if the shellfish are harvested from a polluted area.     

Growth and immunogenicity of foot-and-mouth disease virus in baby hamster kidney cells adapted to and continuously grown in a serum-free chemically defined media

The production of foot-and-mouth disease (FMD) virus in baby hamster kidney (BHK) suspension cells grown in serum-free media for subsequent use in vaccines was attempted because of the limited availability of serum in quantities sufficient for propagation of large amounts of cells, as well as the possible presence of mycoplasma, viral contaminants, and interfering antibodies in sera. Suspension cultures (50 to 600 ml) of BHK-21 cells adapted to and continually passed in a glutamine-free autoclavable, chemically defined medium (BHK-S system) were infected with all seven types of FMD virus. Cells were infected at multiplicities of infection (MOI) ranging from 10^?1 to 10^?7 plaque-forming units per cell (PFU/cell). The time course of infectious virus release and the amount of complement-fixing (CF) antigen produced were then followed. Peak harvest infectivities of approximately 10^8.5 PFU/ml were obtained from 12 to 24 hr after inoculation, depending on input MOI, and were apparently independent of cell concentration over the range 1.5 to 4.0 million cells/ml; the CF endpoint dilutions increased from 1:12 at the lower cell concentrations to 1:48 at the highest cell concentration. Monovalent and trivalent vaccines have been produced using viruses from the BHK-S system, inactivated with acetylethyleneimine and emulsified in oil, and the results of tests in steers and guinea pigs are presented.     

Stable high-producer cell clone expressing virus-like particles of the Japanese encephalitis virus e protein for a second-generation subunit vaccine

We produced and characterized a cell clone (J12#26 cells) that stably expresses Japanese encephalitis virus (JEV) cDNA, J12, which encodes the viral signal peptide, premembrane (prM), and envelope (E) proteins (amino acid positions 105 to 794). Rabbit kidney-derived RK13 cells were transfected with a J12 expression plasmid, selected by resistance to marker antibiotics, and cloned by two cycles of a limiting-dilution method in the presence of antibiotics, a procedure that prevents the successful generation of E-producing cell clones. J12#26 cells secreted virus-like particles containing the authentic E antigen (E-VLP) into the culture medium in a huge enzyme-linked immunosorbent assay-equivalent amount (2.5 micro g per 10(4) cells) to the internationally licensed JE vaccine JE-VAX. E-VLP production was stable after multiple cell passages and persisted over 1 year with 100% expressing cells without detectable cell fusion, apoptosis, or cell death, but was suspended when the cells grew to 100% confluency and contact inhibition occurred. Mice immunized with the purified J12#26 E-antigen without adjuvant developed high titers of neutralizing antibodies for at least 7 months and 100% protection against intraperitoneal challenge with 5 x 10(6) PFU of JEV when examined according to the JE vaccine standardization protocol. These results suggest that the recombinant E-VLP antigen produced by the J12#26 cell clone is an effective, safe, and low-cost second-generation subunit JE vaccine.     

Density of infectious virus and complement-fixing antigens of two rhinovirus strains

Dans, P. E. (National Institute of Allergy and Infectious Diseases, Bethesda, Md.), B. R. Forsyth, and R. M. Chanock. Density of infectious virus and complement-fixing antigens of two rhinovirus strains. J. Bacteriol. 91:1605-1611. 1966.-Two rhinovirus serotypes (echovirus 28 and HGP) and poliovirus type 1 were banded by isopycnic centrifugation in cesium chloride. The rhinovirus virions had a density of 1.41 g/ml, whereas that of poliovirus was 1.34. Since a number of other enteroviruses also have a density of 1.34 g/ml in cesium chloride, a basic difference in density may exist between the rhinovirus and enterovirus subgroups of the picornavirus family. Whether this difference reflects differences in ribonucleic acid content or binding of cesium ions remains to be determined. In tests with echovirus 28 two peaks of CF activity were detected: one in association with the virion (1.41 g/ml), and a larger peak of lower density (1.30 g/ml). With echovirus 28 antiserum, a heterotypically reactive complement-fixing (CF) antigen was detected in the HGP virus suspension at a density less than that of the virion (1.30 g/ml). This antigen corresponded in density to the less dense CF antigen of echovirus 28.     

Structural basis for the anticoagulant activity of heparin. 1. Relationship to the number of charged groups.

This study was undertaken to provide further information concerning the chemical heterogeneity of heparins and the relationships between the anticoagulant activity (USP assay) and the anionic density of the heparin. A sample of commercial heparin was fractionated into 13 fractions by sequential extraction in a two-phase system of 1-butanol-aqueous NaCl containing excess hexadecylpyridinium chloride. The anionic density distribution was characterized by the fractional distribution of uronate among the fractions. The fractions were characterized by several molar ratios of constituents, molecular weight, charge density, and anticoagulant activity in recalcified sheep plasma. The heparin was broadly distributed among the last 10 fractions; the first three contained impurities which were completely separated from the heparin fractions. The heparin fractions differ systematically in anionic density but are of substantially the same molecular weight. Anticoagulant activity increased markedly with anionic density, ranging from 81 units/mg for the heparin fraction with the lowest anionic density up to a high of 243 units/mg. The relationship between anticoagulant activity and either anionic density or its square is nonlinear. However, in the latter case an initial linear relationship was observed for anticoagulant activities of less than 200 units/mg.     

Fluorescent-Antibody Studies with Antisera Against Heated and Unheated Poliovirus Type 1

Fluorescent-antibody (FA) reagents were prepared from sera of guinea pigs immunized with either native infectious poliovirus type 1 or poliovirus type 1 which had been heated at 56 C for 30 min. Conjugates made from sera of animals immunized with heated virus gave higher direct FA staining titers on air-dried, acetone-fixed, infected cells than conjugates made from sera of animals immunized with native infectious virus. Evidence was obtained that complement-fixing antibody reactive with heated antigen was responsible for the FA staining. Two conjugates prepared from sera of guinea pigs immunized with heated poliovirus type 1 were successfully used to identify 21 type 1 viruses isolated from a group of 44 stool suspensions studied as unknowns. These conjugates did not stain any of 23 heterologous enteroviruses present in the remainder of the stools and gave minimal non-specific staining.     

Comparison of talc-Celite and polyelectrolyte 60 in virus recovery from sewage: development of technique and experiments with poliovirus (type 1, Sabin)-contaminated multilitre samples.

For virus recovery from sewage, a mixture of talc and Celite was tested as a possible inexpensive substitute for polyelectrolyte 60 (PE 60). After adjustment of pH to 6 and the addition of 45-60 plaque forming units (PFU)/ml of poliovirus type I (Sabin) to the sewage sample under test, 100 ml of it was passed through either a PE 60 (400 mg) or a talc (300 mg)-Celite (100 mg) layer; the layer-adsorbed virus was eluted with 10 ml of 10% fetal calf serum (FCS) in saline (pH 7.2). In these experiments, PE 60 layers recovered 73-80% (mean 76%) of the input virus. In comparison, virus recoveries with the talc-Celite layers were 65-70% (mean 68%). Passage of 5 litres of raw sewage (containing 50 to 1.26 X 10(5) PFU/100 ml of the poliovirus) through the talc (15 g)-Celite (5 g) layers and virus elution with 50 ml of 10% FCS in saline gave virus recoveries of 33-63% (mean 49%). Except for pH adjustment and prefiltration through two layers of gauze to remove large solids, no other sample pretreatment was found to be necessary. Application of this technique to recovery of indigenous viruses from field samples of raw sewage and effluents has been highly satisfactory.     

Presentation and immunogenicity of the hepatitis B surface antigen and a poliovirus neutralization antigen on mixed empty envelope particles.

Insertion of a synthetic DNA fragment encoding a poliovirus neutralization epitope into the S gene encoding the major envelope protein of hepatitis B virus has yielded hybrid (HBsPolioAg) particles closely resembling authentic 22-nm antigen (HBsAg) particles by expression of the modified gene in mammalian cells. In mice, these hybrid particles induce neutralizing antibodies against poliovirus but only weak immune response to HBsAg (F. Delpeyroux, N. Chenciner, A. Lim, Y. Malpièce, B. Blondel, R. Crainic, S. Van der Werf, and R. E. Streeck, Science 233:472-474, 1986). By cotransfection with different plasmids carrying either modified or unmodified S genes, we have now obtained mixed particles presenting both HBsAg and HBsPolioAg. When such particles were inoculated into rabbits, antibodies to both poliovirus and to HBsAg were induced. Moreover, the titers of neutralizing antibodies to poliovirus induced by HBsPolioAg were much higher than those previously obtained in mice. The design of multivalent particles carrying various peptide sequences or presenting several heterologous epitopes may therefore be possible.     

Buoyant Density Analysis of Staphylococcal Bacteriophage 80 Transducing Particles

Densities of pase, tet, and ol transducing particles were indistinguishable, and about 0.002 g x cm(-3) less dense than the plaque-forming units (PFU). The densities of the PFU and nov transducing particles were indistinguishable, the PFU having a density of 1.507 g x cm(-3).     

Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus

Duc-Nguyen, Huu (National Cancer Institute, Bethesda, Md.), Edith N. Rosenblum, and Robert F. Zeigel. Persistent infection of a rat kidney cell line with Rauscher murine leukemia virus. J. Bacteriol. 92:1133-1140. 1966.-The propagation of a murine leukemia virus (Rauscher) in a kidney cell line, derived from a rat with lymphoid leukemia, was studied. A complement-fixing (CF) antigen reacting with Rauscher immune sera was detected at various passage levels, which correlated with the visualization by use of electron microscopy of viral buds and viral particles in different stages of maturation in all passages. Five-month-old monolayers continued to shed virus and to yield high CF antigen titers. The cell-free supernatant fluid from cultures of the 14th passage was shown to be infectious for a normal rat kidney cell line, as evidenced by the appearance of the CF antigen in this line. Interferon production was not demonstrated in infected cultures. The overall data indicated that rat kidney cells could be used to propagate Rauscher virus in a carrier state.     

Cholesterol removal by methyl-beta-cyclodextrin inhibits poliovirus entry

Upon binding to the poliovirus receptor (PVR), the poliovirus 160S particles undergo a conformational transition to generate 135S particles, which are believed to be intermediates in the virus entry process. The 135S particles interact with host cell membranes through exposure of the N termini of VP1 and the myristylated VP4 protein, and successful cytoplasmic delivery of the genomic RNA requires the interaction of these domains with cellular membranes whose identity is unknown. Because detergent-insoluble microdomains (DIMs) in the plasma membrane have been shown to be important in the entry of other picornaviruses, it was of interest to determine if poliovirus similarly required DIMs during virus entry. We show here that methyl-beta-cyclodextrin (MbetaCD), which disrupts DIMs by depleting cells of cholesterol, inhibits virus infection and that this inhibition was partially reversed by partially restoring cholesterol levels in cells, suggesting that MbetaCD inhibition of virus infection was mediated by removal of cellular cholesterol. However, fractionation of cellular membranes into DIMs and detergent-soluble membrane fractions showed that both PVR and poliovirus capsid proteins localize not to DIMs but to detergent-soluble membrane fractions during entry into the cells, and their localization was unaffected by treatment with MbetaCD. We further demonstrate that treatment with MbetaCD inhibits RNA delivery after formation of the 135S particles. These data indicate that the cholesterol status of the cell is important during the process of genome delivery and that these entry pathways are distinct from those requiring DIM integrity.     

Excretion of live attenuated polioviruses in the faeces of orally vaccinated children. Comparison of two immunization schedules

The excretion of live, attenuated poliovirus vaccine strains was determined in the feces of Prague Infants home children given 10(5) PFU of type 1 and 2 and 2.10(5) PFU of type 3 vaccine in a routine annual mass campaign. The first two faeces specimens examined in each vaccinee prior to immunization were negative for the virus. A total of 476 stool specimens were collected from 37 children at weekly intervals for a period of 18 weeks. The presence of type 1 poliovirus in the faeces of children given monovalent type 1 vaccine was detectable for 9 weeks, with a maximum in first week, and the virus was isolated in 74.2% of vaccinees. The timing of bivalent type 2 and type 3 vaccine was 9 weeks after monovalent type 1 immunization. The excretion of these two types of poliovirus was found to persist for at least 6 weeks. Type 2 poliovirus was isolated in all vaccinees, type 3 in 70.4% of children. The highest percentage of children excreting type 2 poliovirus was recorded in the first week, the excretion of type 3 peaked three weeks after bivaccine administration. The excretion peaks were reached relatively early postvaccination, with type poliovirus reaching the highest titre per 1 g of faeces. After revaccination (one year later) with monovalent type 1 vaccine, the vaccine strain of type 1 poliovirus could be detected for 6 weeks and was present in the highest percentage of positive stool samples.(ABSTRACT TRUNCATED AT 250 WORDS)     

Genetic control of quantitative variation in carbonic anhydrase isozymes of mammals. I. The concentration within single erythrocytes of the mouse

The levels of the carbonic anhydrase isozymes (CA and CA II) in single erythrocytes of DBA/2J mice were assayed by measuring the specific immunofluorescence of CA I and CA II with a microspectrofluorometric technique. Measurements of 100 randomly selected cells showed a range (in relative fluorescence units) of 21-52 (mean 31.3 +/- 7.5) for CA I and 30-80 (mean 45.6 +/- 10.7) for CA II. The CA II/CA I ratio of the means obtained by the single-cell fluorescence assay was similar to the ratios obtained for the two isozymes from hemolysates of DBA/2J mice using an immunodiffusion assay. The influence of cell age on the variation in carbonic anhydrase levels was determined by separating red cells into several fractions by a gravity sedimentation procedure. The younger cells showed higher levels of CA I and CA II than the older cells; however, the extensive overlap in the variability between the cells from the different fractions indicated that although cell age was contributing to the overall heterogeneity, its influence was not pronounced.