An improvement in the plaque assay of rabies virus in chick embryo cells

Summary The faint plaques formed with HEP Flury strain of rabies virus in chick embryo cells by the original method were thought ascribable to acid production from virus-infected cells coupled by the weak buffering action of the agar overlay medium. When the overlay medium contained appropriate concentrations of alkalies, clear plaques could be observed. The optimal conditions were (i) incorporation of 0.02% NaOH, 0.0025m Tris-HCl buffer of pH 8.2 and 2% calf serum in the base overlay medium, and (ii) neutral red staining after 7 days' incubation at 35 C. Under these conditions the plaque size was approximately 2 mm in diameter and autointerference caused by higher multiplicity of infection showed a diminishing trend. Mouse passaged NOPM strain also formed fairly clear plaques when agarose was substituted for agar, the plaque titer being almost equal to its mouse LD titer. That these plaques were formed by infection of the virus was testified by a neutralization test using a standard antiserum prepared with rabbit passaged CVS strain. Adsorption of rabies virus to chick embryo cells was found to proceed slowly, the maximal adsorption requiring 4 hours or longer. The adsorption efficiency was equal between 35 and 37C.     

Studies with rinderpest virus in tissue culture

Summary The stability of cultured rinderpest virus, in maintenance medium containing 5% normal ox serum, was studied at 4°, 37°, and 56° C. The half-life at these temperatures was calculated and the results compared with figures available for other strains of rinderpest virus in cattle tissues and for measles virus in tissue culture fluids.Data were also provided on the freezing of the same virus at –25° C and –70° C, with storage for periods of up to four months. The accuracy of replicate virus titrations, in primary or serially-cultivated calf-kidney cells, was determined.Details were given of tissue culture techniques for the detection and titration of neutralizing antibody to rinderpest virus in the sera of animals, especially cattle.Box titrations of a standard ox immune serum showed that a 1 log increase in virus dose lowered the SN₅₀ titre of the serum by a mean 0.56 log units. The error in replicate titrations of two standard immune sera, using different batches of calf-kidney cells as substrate, was determined.The effect of normal ox serum on rinderpest virus was investigated and the sera of over 3.000 experimental cattle were examined by a screening test for immunity. There were no false positives and only 0.25% of the serologically-negative cattle gave later evidence of resistance to challenge.Tissue culture techniques for the detection and titration of rinderpest neutralizing antibody are briefly compared with the methods hitherto available.     

Some properties of the virus of duck plague

Summary Experiments were conducted to determine some of the physical and chemical characteristics of duck plague virus. As measured by filtration through membranes of graded porosity, the infectious particles were more than 100 m in diameter but less than 220 m The virus is sensitive to ether and chloroform and has the acridine orange-staining characteristics of DNA. Its infectivity is destroyed by heating at 50° C for 2 hours or at 56°C or 60°C for 10 minutes. At 22°C infectivity declines slowly and is no longer detectable after 30 days. Drying over calcium chloride at 22° C results in complete inactivation in 9 days. The virus is fairly stable over the pH range from 5 to 10 but is very rapidly inactivated at pH 11 or higher and pH 3 or lower. Infectivity is destroyed by trypsin, chymotrypsin and pancreatic lipase.The Long Island duck disease was designated duck virus enteritis by New York State and ARS regulatory officials (9-code of Federal Regulations-Part 83-duck virus enteritis (duck plague).     

The Tween-ether treated hemagglutinin of parainfluenza 2 virus

Summary Several properties of the Tween-ether treated hemagglutinin of parainfluenza 2 virus have been, studied and compared with those of the untreated virus. When the untreated preparation contains a large proportion of infective or complete particles, Tween-ether treatment results in a 64- to 128-fold enhancement of the hemagglutinin titer. With less complete particles, the enhancement is only 16- to 32-fold. By ultrafiltration it is shown that Tween-ether treated hemagglutinin consists of particles much smaller than untreated virus particles, including some which pass through 10 m pores. This hemagglutinin can be stored at 4°C for long periods without loss of titer. At 37°C, 41°C, 56°C and –70°C it is less stable than untreated virus. It was found to be efficient in eliciting antibodies in rabbits but more sensitive in detecting hemagglutination inhibition antibodies in human sera.This investigation was supported by the National Institutes of Allergy and Infectious Diseases, N.I.H., Bethesda, Md., contract No. 43-62-477.     

Removal of contaminating protein from high titre virus suspensions to be used in the production of antisera for immimofluorescence

Summary A method is described for the removal of contaminating (endogenous) protein from high titre virus suspension. The method depends upon adsorption of the virus to barium sulphate suspension at 37° C and its subsequent elution at 4° C into sodium citrate solution.The efficiency of protein removal varied from 90 to 98.9%, corresponding to a fall from 1400 to 150 g/ml, and from 9500 to 110 g/ml with Coxsackie B 4 and mumps viruses, respectively. Other viruses, for which intermediate figures were obtained included several myxoviruses, vaccinia, herpes simplex and respiratory syncytial virus.Subsequent immunization of guinea-pigs with protein-reduced virus suspensions confirmed the retention of viral immunogenicity after treatment in terms of complement-fixing, neutralizing and immunofluorescent antibody titres induced. All the treated viruses except respiratory syncytial virus produced an antiserum in guinea-pigs completely free from non-specific fluorescent when used without dilution in a homologous immunofluorescent test system.     

Physicochemical properties of transmissible gastroenteritis virus hemagglutinin

Summary Transmissible gastroenteritis virus was readily adsorbed onto chicken erythrocytes at 4°C. The hemagglutinin thus adsorbed could be eluted from the erythrocytes by incubating in phosphate buffered saline at 37°C. The on chicken erythrocytes for the hemagglutinin was inactivated by neuraminidase and potassium periodate, but not by trypsin, 2-mercaptoethanol and formalin. The hemagglutinin was inactivated by trypsin, papain, pepsin, -amylase, phospholipase C, neuraminidase, formalin, 2-mercaptoethanol, potassium periodate, ethylether, chloroform, Tween-80 and -propiolactone, but not by sodium deoxycholate and trichlorotrifluoroethane, suggesting that the active component of the hemagglutinin involved glycoproteins. The hemagglutinin was stable at 37°C or lower temperatures but not at 60°C or higher temperatures. The hemagglutinin activity was resistant to ultraviolet irradiation, while the infectivity was very susceptible. The hemagglutinin and the infectivity were readily sedimented by ultracentrifugation at 45,000 × g for 60 minutes. In rate zonal centrifugation of the hemagglutinin preparation on a sucrose density gradient, the hemagglutinin activity showed a sharp peak at 1.19 g/ml coinciding with the peak of infectivity. The activity in the peak fraction seemed to be structually associated with virus particles.     

Acid- and cryoactivation of renin in human plasma

Summary Inactive renin in normal human plasma was activated in vitro either by cryoactivation (incubation at 0° C/–5° C up to 3 months) or acid-activation (dialysis to pH 3.0 for 48 h followed by dialysis to pH 7.5). Plasma-renin-concentration was similar after either activation procedure (96±50µU/ml vs 100±43µU/ml). There was no further significant acid-activable renin after cryoactivation. These data suggest that conversion of inactive to active renin by optimized procedures is complete. The term total renin for activation results under these conditions seems to be valid.     

Studies of laryngotracheitis virus in avian tissue cultures II. Deficient adsorption of a strain of laryngotracheitis virus

Summary In the plaque assay of the N71851 strain of laryngotracheitis virus (LTV) in chicken kidney (CK) monolayer cultures, the number of plaques which developed depended upon the treatment between adsorption and addition of agar overlay medium. Cultures in which the inoculum was aspirated after the adsorption period had a greater number of plaques than cultures in which the inoculum was aspirated and the cultures were washed. This effect was found to be due to a deficiency of virus to adsorb to cells. Under the conditions of the assay, the efficiency with which virus adsorbed to monolayers was too low to be measured. Therefore, the efficiency of the assay was also low. The initial association between virus and cells was easily disrupted by washing when adsorption was carried out at 5°C to prevent penetration. Treatment of cultures with diethylaminoethyl-dextran for one hour prior to inoculation increased the number of plaques in treated compared with untreated cultures by a factor of five.     

Studies on a measles virus variant inducing persistent infections in cultured cells

Summary Attempts were made to characterize by a plaque assay two variants of the Edmonston strain of measles virus and to obtain plaque purified virus populations.The UP non-cytocidal variant, in all the examined cell systems, mainly produced small but also large plaques; the DP cytocidal variant always large plaques.Three clones, UP-SP₄, UP-LP₄ and DP-LP₄, were derived by plaque purification respectively of the UP small plaque, UP large plaque and DP large plaque forming particles. The virus populations of the clones could be distinguished by some other biological and physical characters: cytopathic effect in roller tube cultures, growth potential in HeLa cells, thermal stability at 45° C, stability of the properties during serial passages at different input multiplicity.The hypothesis was supported that the typical properties of the UP and DP variants are host-independent and genetically controlled viral markers.With 3 FiguresThis investigation was supported by the Consiglio Nazionale delle Ricerche, Grant No. 73.01400.44.     

Latent measles virus infection in Vero cells depending on a temperature-sensitive phenomenon

Summary A latent infection by measles virus in a line of Vero cells could be maintained only at 37° C. The conditions of temperature nonpermissiveness were associated with some block in virus production and/or release and with the establishment of an autointerference phenomenon. Reduction of the incubation temperature to 33.5° C induced a rather rapid transition from the latent to a lytical infection with a rescue of virus. The rescued virus exhibited a restricted capacity to grow at 37° C.With 1 FigureThis investigation was supported by the Consiglio Nazionale delle Ricerche—Progetto Finalizzato Virus, Grant No. 76.00655.84.     

Bovine ephemeral fever

Summary Thermal and pH stability of ephemeral fever virus (EFV) were studied. Infectivity of EFV was inactivated within 10 minutes at 56°C, by 18 hours at 37°C, and within 120 hours at 25°C. Infected mouse-brain virus had little titer loss after 30 days' storage at 4°C. Infected culture fluids stored at –35°C lost about 0.04 log units of virus per day. EFV was inactivated within 10 minutes at pH 2.5 and 12.0. Infectivity was lost in 60 minutes at pH 5.1 and in 90 minutes at pH 9.1. EFV was adapted to monkey kidney cell lines (Vero and MS); a plaque assay in Vero cells is described. The virus sized between 100 and 220 m by filtration. Growth studies of EFV in MS cells are described.     

Growth of Junin virus,the etiological agent of argentinian hemorrhagic fever,in cell cultures

Summary Propagation of Junin virus, the etiological agent of Argentinian hemorrhagic fever, has been studied in a variety of cell cultures. One line of African green monkey kidney cells (Vero) at the 107th to 165th passage levels supported the multiplication of the virus and developed a marked cytopathic effect (CPE), while no CPE appeared in another line (BS-C-1) at the 34th passage level. CPE was markedly enhanced by rolling the cultures, and the cytocidal nature of virus replication was markedly more pronounced under these conditions. Junin virus also produced well-defined plaques in monolayer cultures of Vero cells under agar overlay. Virus assays obtained by titration endpoint of CPE or by plaque counts in Vero cells were in almost all instances higher than those obtained by titration in suckling mice. A linear relationship was observed between plaque counts and virus concentration. Maximum adsorption was obtained by 120 minutes incubation at 37°C. When the cultures were infected with <0.001 PFU per cell, the maximum virus titer was obtained on the 5th day after inoculation.Neutralization tests employing the 80% plaque reduction endpoint give reproducible results and indicate the absence of antibody for Junin virus in the Tacaribe and Machupo antisera that were tested.     

Characterization of a small Porcine DNA virus

Summary Characteristics of a small DNA virus isolated from kidney cell cultures of healthy 3 week-old pigs are described.The virus isolate multiplies in kidney cell cultures of pig origin, produces intranuclear inclusion bodies, and hemagglutinates guinea pig, human group 0, chicken, cat, rat and mouse red blood cells. It multiplies in pigs resulting in antibody production, but is not pathogenic for newborn hamsters and mice.The virus particle is 20–22 m in size, hexagonal in shape and without a lipid containing envelope. Buoyant density is between 1.37 and 1.38g/ml. This virus is stable within a wide range of pH, resistant to heat (56°C), against treatment with trypsin, and lipid solvents.The porcine virus was proposed as a member of the picodna virus group, and named Porcine Picodna Virus (PPV).Supported by a grant of the Common Market Research Program for control of European and African Swine Fever.     

Interaction of Newcastle disease virus strains differing in virulence with chicken red blood cell receptors

Summary Nine NDV strains belonging to lentogenic, mesogenic and velogenic groups were studied. Virus adsorption to chicken red blood cell (RBC) surface was performed at 4° C, and after a temperature shift from 4° to 37° C elution of pre-adsorbed virus and accumulation of free N-acetyl-neuraminic acid (NANA) split from RBC receptors as a result of neuramindase (Nase) activity was detected. In the case of high multiplicity of adsorption the elution was very fast (complete elution within 5 minutes) for all the strains irrespective of their virulence. Although physical saturation of RBC surface with the adsorbed virus was not achieved, a certain minimal (strain-specific) amount of the pre-adsorbed virus which splits a maximally possible (for a given strain) quantity of the NANA was found (a state of enzymatic saturation). Below a certain low multiplicity of adsorption elution was delayed for about 20–30 minutes while the accumulation of the split NANA began immediately after the temperature shift. This phenomenon was interpreted as a result of crawling of the adsorbed virions upon the RBC surface followed by browsing of RBC recptors and liberation of NANA. Thus, the Nase activity of the attached virus (in situ Nase activity) is a factor providing both elution and crawling of the virus (depending on the multiplicity of adsorption).Thein situ Nase activity of all the strains used was determined quantitatively by (1) parameters of enzymatic kinetics (V_max, K_m and K_m/V_max) and (2) parameters of enzymatic efficiency related to a certain quantity of the adsorbed virus, namely, per amount of: a) crawling virus, b) that providing enyzmatic saturation, and c) that equal to K_m. Computation of these parameters revealed inverse correlation between thein situ Nase activity and the strain virulence. Thus, these indications can bein vitro markers of thein vivo virulence.With 8 Figures     

Parvovirus (feline panleucopaenia virus) plaque formation

Summary A plaque assay was developed for feline parvovirus (FPV; feline panleucopaenia virus) in a feline embryo (FEmb) cell line. Higher numbers and larger diameter plaques were obtained with a) seeding rates of 0.7 × 10⁵ and 1.5×10⁵ cells cf. 3×10⁵ and 6×10⁵ cells/well of 35 mm diameter, b) synchronised cells infected at the G₁—S interface cf. nonsynchronised cells and c) 5 to 6 days incubation post inoculation.The plaque assay was standardised by using serum deprivation for 24 hours to synchronize cells, a seeding rate of 1.5×10⁵ cells/35 mm diameter well, inoculation of virus 16 hours post seeding followed by 5 days incubation. The standardised assay gave consistent, reproducible results. A dose-response curve using the assay showed a linear, 45° slope, relationship between plaque forming units and virus dilution which further verified the sensitivity and reliability of the assay. Plaques produced by wild type and plaque purified virus were inavriably non uniform in diameter; diameter of plaques in fact followed a normal frequency distribution under standard assay conditions.With 3 Figures     

Biophysical studies of teschen disease virus (Talfan strain)

Summary Studies have been made of the heterogeneity of infectivity and CFA in Teschen virus (Talfan strain) suspensions. Most of the infectivity was contained in two components of densities 1.46 gm./ml. and 1.35 gm./ml. The physical, chemical and immunological properties of these components have been compared. It was possible, however, to convert a large proportion of 1.46 component to 1.35 component by treating the 1.46 component with sodium dodecyl sulphate. This would indicate that the 1.46 component was a complex formed between the infective particles and cellular debris.Further studies on the growth characteristics and electron microscopy of the virus have been made.     

Formaldehyde inactivation of foot-and-mouth disease virus. Conditions for the preparation of safe vaccine

Summary The inactivation of foot-and-mouth disease virus by formaldehyde was studied under different conditions, both as free virus and (as in routine vaccine production) after adsorption of the virus to aluminium hydroxide gel (alhydrogel). In the latter case infectivity was monitored after elution of the virus from the gel by isopycnic ultracentrifugation of the virusalhydrogel mixture in CsCl. By this method good virus recoveries were obtained. Adsorption of the virus to alhydrogel (without formaldehyde) did not reduce infectivity significantly. Both adsorbed and non-absorbed virus lost infectivity at a rate of about one log₁₀ per day (at pH 8.5, 25° C—no formaldehyde).Kinetics of formaldehyde inactivation of adsorbed and non-adsorbed virus were also identical, with a fast reduction in the initial phase (in case of O₁ and A₁₀-virus approximately one log₁₀/hour). After this initial phase inactivation became linear and rather slow (for O₁ and A₁₀-virus 0.2 log₁₀/hour). No tailing-off was observed. Under standard conditions (0.04 per cent formaldehyde, pH 8.5, 25° C) C_D-virus was inactivated approximately 1.5 times faster than O₁ and A₁₀-virus. At 4° C the inactivation of the three strains continued at about one log₁₀/day.Increased lactalbumin hydrolysate concentrations reduced the inactivation rate, especially at the formaldehyde concentration of 0.02 per cent, which was originally applied. Quaternary amines like Tris strongly inhibited formaldehyde activity. These findings might explain some data of others who observed tailing off.Analysis of formaldehyde inactivated antigen by SDS-PAGE and electrofocusing showed that extensive cross-linking occurs especially of VP₁, probably with other virus proteins but also with non-virus proteins from the medium. VP₂ and VP₃ are less affected. Cross-linking was enhanced when the virus had been adsorbed to alhydrogel during inactivation.Progressive cross-linking was observed during storage of the vaccine at 4° C, which also indicated that inactivation continued at this temperature.These data show that formaldehyde inactivated adsorbate vaccines can be safe.With 7 Figures     

Temperature-and UV-light resistance of rubella virus infectivity

Summary The thermal and UV-light sensitivity of a laboratory-adapted rubella virus (strain Wright) was assayed quantitatively and by electron microscopic observation. As judged by the appearence of cytopathic effects in cultures of African green monkey kidney cells (Vero) and/or by the presence of rubella virus particles in electron microscopic preparations, infectivity was found to withstand inactivation temperatures up to 70° C for 30 minutes. Infectious tissue culture fluid treated by UV-irradiation for 60 minutes also produced cytopathic effects in Vero cells. Ultrastructurally, however, only a few rubella virus-like particles could be detected. Virus suspensions filtered through 0.22 membrane filters prior to heat-or UV-treatment failed to reveal residual infectivity. Some of the possible mechanisms responsible for this marked thermal and UV-resistance of the rubella virus strain used are discussed.This work was supported by Grant Nr. 4804 from the Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung.     

Ultrastructural examination of phytohemagglutinin stimulated lymphocytes infected with vesicular stomatitis virus

Summary Human peripheral blood lymphocytes treated with PHA and subsequently infected with VSV supported extensive viral replication. Maximum titers of virus were obtained 16–24 hours after infection and represented several hundred fold increase over the input virus concentration. Untreated control cultures infected with VSV showed no significant viral increase. In electron micrographs of stimulated cultures infected with VSV, a large number of virions were produced by a few transforming cells of intermediate size. Viral penetration into the lymphocytes at the cell membrane, accompanied by cellular invagination, maturation at the marginal membrane surface, and release of virus to the outside by budding were consistent findings. The appearance of the nucleus in virus producing cells was similar to that in stimulated, uninfected cells. However, stimulated lymphocytes supporting extensive viral replication demonstrated some structural changes in the cytoplasm characterized by fewer mitochondria and polyribosomal aggregates and less active Golgi zones.     

Wesselsbron virus haemagglutination: studies with erythrocytes of various animal species at different pH and temperatures

Summary The haemagglutinating (HA) properties of the Nigerian strain of Wesselsbron virus have been investigated using erythrocytes from a wide range of animals. The results showed that Wesselsbron virus possesses HA activity when extracted using the sucrose and acetone method. The erythrocytes of goose, horse, donkey, pig, cattle, sheep, goat, monkey, man, rabbit, rat, guinea pig and chicken were agglutinated by Wesselsbron virus at different pH values (5.75–7.0) and temperatures of 4°C, room (25±2°C) and 37°C.The ability to haemagglutinate fell as pH increased, but the effect of incubation at different temperature was not marked. However, under the conditions of the experiment HA pattern was clearest at 37°C. High HA titres (1:16) were consistently obtained using goose, horse, donkey and human erythrocytes at different temperatures.