Hyper-antigenic variation occurs with human rhinovirus type 17.

Of 90 human rhinovirus (RV) serotypes tested, 50 can be placed into 16 groups according to antigenic relationships. It has been suggested that antigenic variants might arise in nature by immunologic pressure. To investigate this possibility, attempts were made to select variants by cloning 16 different plaque-purified RV serotypes in the presence of homologous, polyclonal antisera. Isolates were examined for evidence of variation in serum cross-neutralization tests using parental antisera and, in some cases, antisera prepared for the isolates. Only RV type 17 (RV-17) yielded major antigenic variants after cloning. With some variants, as much as a 500-fold difference in neutralizing titer was obtained with the parental antiserum. By using antisera for two of the variants, it was determined that they are prime strains of the parental RV-17. Continuing attempts at immunoselection by using antisera for one of these prime strains yielded additional antigenic variants. By using antisera prepared for three of these new variants, it was determined that one of them is a prime strain of the virus from which it was derived. Cross-neutralization tests with the two remaining isolates indicate that, according to conventional practice, they no longer would be classified as RV-17.

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.

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.

Continuous high-speed rolling versus centrifugation for detection of herpes simplex virus.

Specimens submitted for diagnosis of herpes simplex virus infections were inoculated into shell vials and conventional culture tubes. Inoculated culture tubes were incubated with rolling at 96 rpm. Immunoperoxidase (IP) staining and cytopathic effects (CPE) were used to detect positive cultures. At 24 h, 42 (53%) of the rolled cultures were positive for CPE, while only 16 (21%) of the shell vials were CPE positive (P less than 0.01). No difference in sensitivity was seen between rolled and shell vial cultures that were inoculated with high-titered viral preparations and IP stained at 16 h. However, when low-titered preparations were used, 39 of 41 (95%) were IP positive by the high-speed roller method at 64 h postinoculation, while only 24 of 41 (58%) were IP positive with shell vials (P less than 0.01). These results indicate that high-speed roller method at 64 h postinoculation, while only 24 of 41 (58%) were IP positive with shell vials (P less than 0.01). These results indicate that high-speed rolling is better than the shell vial technique for the detection of herpes simplex virus by IP staining.

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.

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.

Detection of respiratory syncytial virus in clinical specimens by viral culture, direct and indirect immunofluorescence, and enzyme immunoassay.

We evaluated prospectively the detection of respiratory syncytial virus (RSV) by culture and by direct antigen detection using an indirect immunofluorescence assay (IFA), a direct monoclonal immunofluorescence assay (DFA), and a monoclonal enzyme immunoassay (EIA). Of 221 specimens, 95 (43%) were culture positive for RSV, 4 (1.8%) contained more than one virus, and 17 (7.6%) contained a virus other than RSV. Overall, HEp-2 and Flow 6000 cells grew significantly more RSV isolates (82 and 72%, respectively) than A549 cells, which grew only 29% of the isolates. The mean time for RSV detection with HEp-2 cells was 2.9 days. This was significantly less than the mean time for RSV detection with either Flow 6000 cells (6.1 days) or A549 cells (6.4 days). Of 221 specimens, 129 were tested simultaneously by culture, IFA, and DFA. Of these 129 specimens, 62 (48%) were positive by culture, 69 (53%) were positive by IFA, and 70 (54%) were positive by DFA. For 92 specimens screened simultaneously by culture, IFA, and EIA, positive results were obtained for 33 (36%) of the specimens by both culture and IFA and for 29 (32%) of the specimens by EIA. Of 126 culture-negative specimens, 21 (17%) were positive for RSV when determined by IFA. Conversely, 14 (15%) of 95 RSV culture-positive specimens were negative by IFA, whereas DFA missed 19% of the culture-positive specimens. Compared with culture, the Kallestad EIA kit had a sensitivity and specificity of 73 and 92% respectively, but missed 9 (27%) of 33 culture-positive specimens. These data demonstrate that isolation by culture continues to be important for viral diagnosis of REV infections and that for valid comparative studies between viral isolations and rapid detection methods, both sensitive host cells and appropriate test conditions are required.

Polyacrylamide gel electrophoresis of RNA compared with polyclonal- and monoclonal-antibody-based enzyme immunoassays for rotavirus.

Polyacrylamide gel electrophoresis (PAGE) of rotaviral RNA, a sensitive and highly specific test for detecting rotavirus in stool, was compared with two commercially available enzyme immunoassays (EIAs), monoclonal (Pathfinder) and polyclonal (Rotazyme II). Stool samples from 204 children with nosocomial diarrhea were tested for rotavirus by both EIAs and by PAGE of RNA extracted from raw stools or 10% stool suspensions. Samples which tested positive by either EIA but were negative by PAGE were subjected to blocking EIA with rabbit or goat anti-SA11. Rotavirus was detected by PAGE and Pathfinder in 62 stools, but only 47 of these were positive by Rotazyme II. Blocking assays of EIA-positive, PAGE-negative samples suggested the presence of rotavirus in four additional stools. Sensitivity and specificity measured against PAGE and blocking assays were: Pathfinder, 0.985 and 0.934; and Rotazyme II, 0.731 and 0.927, respectively. False-positive rates were 0.134 for Pathfinder and 0.149 for Rotazyme II. The specificity and rate of false-positive results of Pathfinder were improved by using an adjusted optical density cutoff 4.36 times greater than that recommended by the manufacturer (specificity, 0.993; sensitivity, 0.985; false-positive rate, 0.015).

Nosocomial rotaviral diarrhea: pattern of spread on wards in a children's hospital.

Nosocomial gastroenteritis in a 315-bed hospital for children was examined prospectively from January 11 through May 31, 1985. There were 85 cases of nosocomial diarrhea during the study period, and these were identified on each of the 13 hospital wards. Rotavirus was identified in 40% of cases. Incidence of nosocomial rotavirus was highest on wards where most children were less than 2 years of age, except for the infectious diseases (isolation) ward (0.24 versus 2.30 cases per 100 admissions, p = 1.70 x 10(-4), Fisher exact test). The lower incidence on the infectious diseases ward occurred despite the greater potential for exposure to rotavirus, since 70% of children admitted with community-acquired rotavirus diarrhea were placed on the infectious diseases ward. Better infection control, especially hand washing, aided by the structure of the infectious diseases ward, may have been responsible for this difference.

A collaborative report: rhinoviruses--extension of the numbering system from 89 to 100.

To define the number of rhinovirus serotypes, cross neutralization tests and characterization studies were completed on 25 candidate prototype rhinoviruses submitted to a third phase of a collaborative program. Based on the results, 11 distinct prototype strains were designated and the numbering system was extended to include 100 rhinoviruses. In addition, recent evidence indicates that over 90% of rhinoviruses isolated in three areas of the country could be typed with antisera for rhinovirus types 1-89.

Multiyear study of sludge application to farmland: prevalence of bacterial enteric pathogens and antibody status of farm families.

We describe our experience with the isolation of salmonellae from sewage sludge from four treatment plants in different geographic areas of Ohio. Over 3 years, we isolated salmonellae 50 times from 311 sludge samples. Most isolations were made after enrichment in Selenite broth (BBL Microbiology Systems, Cockeysville, Md.). The largest proportion of isolations came from the plant serving the population of Columbus, a large metropolitan area. A significantly greater number of isolations from this plant were made during the first quarter of the year. Twenty-one different serotypes were isolated, along with five untypable strains. The most frequently isolated serotype was Salmonella infantis. Five of the strains were multiply resistant to antibiotics. We also describe the prevalence of antibodies to salmonellae in members of the families residing on the farms in the study. It was found that antibodies to group C salmonellae predominated.

Viral isolation versus immune staining of infected cell cultures for the laboratory diagnosis of herpes simplex virus infections.

The Difco Laboratories Cellmatics kit, an immune staining method for the detection of herpes simplex virus after amplification in culture, was found to be 94.6% (138 of 146) sensitive and 100% (302 of 302) specific. However, conventional culturing detected 10 (6.4%) additional viruses that were not herpes simplex. For a full-service diagnostic laboratory, viral isolation is important.

Enteroviruses in sludge: multiyear experience with four wastewater treatment plants.

We describe our experience with the isolation of viruses from four treatment plants located in different geographic areas. Over a period of 3 years, 297 enteroviruses were isolated from 307 sludge samples. The highest frequency of viral isolation (92%), including multiple isolates from single samples, was obtained from a treatment plant serving the smallest population. Excluding the polioviruses, 22 different enterovirus serotypes were isolated. The methods used to isolate the viruses were relatively simple and included an elution procedure in which beef extract was used and a disinfection step. No concentration procedure was used. Of three cell culture systems used, the RD line of human rhabdomyosarcoma cells was by far the most useful for the isolation of echoviruses; BGM and HeLa cells were particularly useful for the isolation of group B coxsackieviruses. A seasonal effect on viral isolation rates from sludge was observed.

Preliminary characterization of a persistent infection of HeLa cells with human rhinovirus type 2.

We were able to initiate a persistent infection (PI) in HeLa cells with a temperature-sensitive (ts-) mutant of rhinovirus type 2 (TS-1), but not with the corresponding wildtype (wt) virus. The ability to initiate a PI may be related to the multiplicity of infection. Persistence was established at 37 degrees C but not at 32 degrees C and the virus isolated from the PI was no longer temperature-sensitive. Infectious virus was continually produced at low levels throughout the course of the PI and cell cultures underwent multiple episodes of partial destruction (crisis) and subsequent recovery. PI virus and the initiating virus were neutralized to the same extent by hyperimmune polyclonal TS-1 antiserum indicating that no significant change had occurred with respect to serological type. The presence of either interferon or virus-related interfering activity could not be demonstrated in the PI cultures. Superinfection experiments in cells that were 'cured' of PI virus indicated the selection of a cell population during persistence that could no longer support the growth of homologous-type virus. This effect became less pronounced upon further passage of the cured cells. When compared with the wt and ts viruses, the PI virus yielded comparable amounts of infectious virus in HeLa cells but with decreased synthesis of RNA.

Latex immunoassay for rapid detection of rotavirus.

A latex agglutination (LA) test was evaluated for the detection of human rotaviruses in stool specimens. Both antiserum and immunoglobulin G (IgG)-sensitized latex particles were used, with IgG-coated beads being more sensitive for human rotavirus antigen detection. Latex beads sensitized with anti-simian-SA-11 IgG were stable for at least 8 months when stored at 4 degrees C. The sensitivity of the test was compared with that of the Rotazyme (Abbott Laboratories, Diagnostics Div., North Chicago, Ill.) test. The least number of particles detected was 9.0 X 10(5) particles by the LA test versus 4.5 X 10(5) particles by the Rotazyme test. When 10 stool specimens were serially diluted for antigen endpoint determinations, the geometric mean titer by the LA test was 592 versus 1,280 by the Rotazyme test. Forty-three stool samples positive by the Rotazyme test were all positive by the LA test, and no false negative results were detected. Unconfirmed false positive reactions ranged between 8 and 24%. The LA test for rotavirus antigen detection is direct, easy to perform, sensitive, quick, and may have application for use in diagnostic laboratories, emergency rooms, and physician's offices.

Commercial simian virus antisera that inhibit virus replication in primary monkey kidney cell cultures.

The incorporation of hyperimmune serum into cell culture medium to control endogenous viral infections of primary cells can have a significant effect on the replication of other viruses. When commercial simian virus 5 or simian virus 40 antiserum was used with primary monkey kidney cell cultures, we found a significant inhibition (greater than 90%) of the replication of parainfluenza virus types 2 and 3 and reovirus type 1. In the viral diagnostic laboratory, the use of hyperimmune serum with primary monkey kidney cells may result in failure to isolate certain viruses if these cells are not first washed free of hyperimmune serum.

Antiviral activity of antilipidemic compounds on herpes simplex virus type 1.

Two antilipidemic compounds, clofibrate and procetofene, inhibited the replication of herpes simplex virus type 1 (HSV-1) in African green monkey kidney cells. Clofibrate, at a concentration of 400 mumol/liter caused a 63% reduction (P < 0.001) in HSV-1 yield and at 100 mumol/liter caused a 62% reduction (P < 0.001) in plaque formation. Two stereoisomeric analogs of clofibric acid, (-)- and (+)-desmethyl clofibric acid, also caused a significant inhibition of HSV-1 replication. Procetofene at 5 mumol/liter caused a 56% reduction (P < 0.001) in HSV-1 plaques and at 10 mumol/liter caused a significant reduction (P < 0.001) in both viral yield (42 to 54%) and plaque formation (65%). Procetofene also inhibited the development of HSV-1 plaques. A concentration of 5 mumol/liter resulted in a 26% reduction (P < 0.001) in plaque diameter. Because of their nonspecific inhibitory effect on the uptake of cellular macromolecular precursors for nucleic acid and protein biosynthesis, these antilipidemic compounds may exert their antiviral activity by affecting one or more key metabolic host cell pathways.

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.