Vincent V. Hamparian

India ink staining of proteins on nylon and hydrophobic membranes

India ink was found to be an acceptable stain for proteins blotted or dotted onto positively charged nylon or hydrophobic membranes. The hydrophobic membrane, Immobilon, was an outstanding matrix for binding proteins and displayed low levels of background staining. The least amount of protein detected by india ink staining was between 1.0 and 10 ng. India ink staining of proteins on nylon membranes is an easy, inexpensive, and quick method for the unequivocal detection of both standards and unknowns in the same blot. However, inks, ink concentrations, fixing conditions, staining times, pH, washing conditions, and membrane lots all need to be controlled to achieve maximum sensitivity for protein detection following india ink staining.

Acid Lability of Rhinovirus Type 14: Effect of pH, Time, and Temperature

Summary The kinetics of acid inactivation of human rhinovirus type 14 are affected by pH and temperature of incubation. At pH 3.0, inactivation was complete within 10 sec with total loss of infectivity. At pH 5.0, inactivation reached a maximum by 20 min and yielded a persistent fraction of infectious virus. This surviving fraction was eliminated when the pH was lowered to 3.0. At 0° no loss of infectivity was observed after 5 min of incubation at pH 5.0, while at room temperature 99% of the infectivity was lost. The characteristics of rhinovirus inactivation at low pH appears to be different from all other picornaviruses. This investigation was supported by Research Contract 69-2062 from the Infectious Disease Branch of the National Institutes of Health, and by Public Health Service General Research Support Grant RR-05504 from the Childrens Hospital Research Foundation, Columbus, Ohio 43205. The authors would like to thank Miss Helen Rehn for typing the manuscript.

Inhibition of rotavirus in vitro transcription by optimal concentrations of monoclonal antibodies specific for rotavirus VP6

Three monoclonal antibodies (MAbs) obtained from inoculation of mice with either a serotype 1 human rotavirus or rotavirus SA11 (serotype 3) inhibited the in vitro transcription of rotavirus SA11. Two of the MAbs exhibited a biphasic inhibitory response. Removal of antibody from MAb preparations by adsorption with Sepharose-Protein G reduced the inhibitory activity completely for all three MAb preparations. Analysis by radioimmunoprecipitation and Western blotting indicated that all three MAbs reacted with VP6. All MAbs also reacted with four group A rotavirus serotypes by ELISA, but did not cross-react with reovirus type 1, poliovirus type 2 or MA-104 cell lysates. Transcription of four rotavirus serotypes as well as epizootic diarrhoea of infant mice rotavirus was inhibited when tested with two of the MAbs. Transcription of both purified single-shelled virus and purified heat-activated double-shelled SA11 rotavirus was inhibited by purified MAb. Our results indicate that these MAbs can be used effectively to study the events associated with rotavirus transcription.

Rhinovirus replication in suspension cultures of HeLa cells.

Summary Rhinovirus-infected HeLa suspension cultures did not increase in cell number and lost viability late in the infectious cycle. Maximum adsorption occurred within 30 min with approximately 40% of the virus being adsorbed. Yields of intracellular virus of about 8-10 PFU/cell were obtained at 12 hr after infection. Comparable levels of extracellular virus were not obtained until 30 hr after infection. The suspended cell culture system should have practical value in studies on replication and for obtaining concentrated preparations of virions.

Plaque formation by rhinoviruses.

Summary A reliable plaque assay is described for 60 rhinovirus strains representing 55 distinct serotypes and one subtype. Using HeLa cell bottle cultures, plaques were produced under starch gel and Ionagar overlay media. Employing the methodology described in this report, certain basic aspects of rhinovirus plaque formation were investigated.

Isolation and Genetic Analysis of Temperature-Sensitive Mutants of Rhinovirus Type 2

Temperature-sensitive mutants of human rhinovirus type 2 were isolated by random clonings of mutagenized virus. All mutants were stable. Temperature sensitivity was not affected by different host cell systems. Complementation was observed in 3 of 10 dual viral mixtures, with complementation indices being as high as 4.0. Recombination frequencies fluctuated widely between experiments with different mutants, but positive recombination occurred with mean frequencies ranging from 0.03 to 1.25%. The complementation and recombination results obtained are similar to those reported for other picornaviruses.

Properties of Rhinovirus Plaque Mutants

Summary Under Ionagar overlay medium, the prototype strain of rhinovirus type 34 was found to consist of a mixture of two plaque types designated m (minute) and m + (large). Large plaques were obtained with m virus when agarose was used or when polycations (protamine or DEAE-dextran) were incorporated into Ionagar medium. Starch gel medium completely inhibited the appearance or m but not m + plaques. However, the m virus formed plaques under starch when polycations were incorporated. The nature of the inhibitory principle in starch is unknown. The pH of overlay media did not appear to influence the plaque size of either mutant. Temperature of incubation had little if any effect on replication of m virus. At 37°, the m + mutant required an adaptation period of three to four cell culture passages before maximum yields of infectious virus were obtained. No differences between the mutants were observed in buoyant density, rate of adsorption, host cell tropism, or antibody sensitivity.

The effect of rolling and orbital motion on herpesvirus replication in tube cultures and shell vials

BACKGROUND Isolation of viruses from clinical specimens remains a viable diagnostic manoeuvre but positive isolation rates may be low and time to observe cytopathic effects (CPE) may be longer than preferred for optimal management. Both shell vial (SV) centrifugation and rolling of conventional tube cultures (TC) have been used to enhance the isolation process. OBJECTIVES To determine the effect of rolling and orbital motion on the replication of herpes simplex virus (HSV) and cytomegalovirus (CMV) TC and SV. STUDY DESIGN TC were inoculated with HSV or CMV at varying dilutions and subjected to rolling or incubated stationary. Samples were selected to determine the impact of motion on viral yield, time to CPE according to initial multiplicity of infection (MOI) and stage of the infection process. Similar studies were performed with SV centrifuged with virus followed by stationary incubation or not centrifuged but incubated on an orbital shaker. RESULTS Rolling HSV-infected TC 383 rotations per minute (RPM) (30.2 x g) for 4 days enhanced viral yields by 53-fold over TC without motion. The optimal RPM response for HSV replication occurred at 96 RPM (1.9 x g) where an 89-fold increase in viral yield was detected, P < 0.01. One-step growth studies at 0,2 and 96 RPM demonstrated enhancement of HSV replication at 2 and 96 RPM. TC infected with HSV at low MOI and rolled at 96 RPM had more CPE-positive cultures after 1-3 days than controls. Late in the infection process, no differences in CPE-positive were detected between rolled and non-rolled TC. Studies with CMV and rolling at 96 RPM resulted in more positive TC and greater CPE. SV that were not centrifuged but incubated for 16 h on an orbital shaker had significantly more HSV foci than those handled by a conventional SV method. CONCLUSION Our studies indicate that orbital motion and motion yielding a force near [Formula: see text] enhances the isolation and growth of HSV and CMV in TC and SV.

Detection of serotypic variants in viral preparations: Sensitivity of a procedure for selection

The efficiency of an in vitro method (the breakthrough neutralization procedure) for selecting serotypic variants from preparations of human rhinovirus (HRV) 17 and a temperature sensitive strain (Ts-1) of HRV-2 was examined. Viruses were plaqued in the presence of homologous polyclonal antisera, and plaques which escaped neutralization were isolated. For control purposes, isolates were obtained by plaquing in the absence of antisera. Any clone that consistently (minimum of 2 tests) yielded a 4-fold or greater difference in serum neutralization titer compared to the parent virus when tested with antiserum to the parent, was considered a serotypic variant. The efficiency of the breakthrough neutralization procedure was calculated using varying mixtures of 2 related rhinovirus serotypes. Using this method, the ability to isolate serotypic subpopulations from a mixture was enhanced at least 7000-fold. This procedure allows the rapid isolation of variants which differ from the parent virus by as little as 4-fold in a serum neutralization test. Hence, viral preparations used to prepare reference grade reagents such as seed stocks and polyclonal and monoclonal antisera, can be tested for specificity prior to use.

RNA and Protein Synthesis in Rhinovirus Infected HeLa Cells

Summary Initial inhibition of label accumulation into RNA of rhinovirus infected cells occurs 5 hr postinfection when viral RNA synthesis is at a maximum. Infection lowers the level of label accumulation into protein with maximum inhibition occurring late in the cycle. Actinomycin D does not significantly affect protein synthesis in infected cells. These results support the hypothesis that two separate functions are responsible for inhibition of host RNA and protein synthesis. This investigation was supported by Research Contract NIH 69-2062 from the National Institute for Allergy and Infectious Diseases, and by U.S. Public Health Service General Research Support Grants 22-14-54 and 22-15-74 from the Childrens Hospital Research Foundation. Miss Judy Halstead provided valuable technical assistance.