Plaque formation by B cell colonies

Three techniques for separating free antigen from antigen-antibody complexes have been applied to radioimmunoassay of myelin basic protein: cold ethanol precipitation of complexes, dextran-coated charcoal precipitation of free antigen, and second antibody precipitation of complexes. After optimization of the incubation and separation steps, the 3 methods were evaluated for precision and accuracy when applied to both spinal fluid and brain tissue homogenates. For determinations in brain tissue all 3 methods showed the same precision and gave largely the same values, though the ethanol method gave slightly lower levels. For spinal fluid the ethanol and dextran-charcoal methods gave the same values, but the double antibody method gave values only 1/3 as high. With spinal fluid, the precision of the dextran-charcoal method was poor compared with that of the other two. The double antibody method proved to be the method of choice for brain tissue samples, when the results of the incubation and separation steps, and the precision and accuracy of the determinations were taken into account. However, for an unknown reason values for spinal fluid were too low by this method. Therefore the ethanol precipitation method is recommended for spinal fluid samples and the double antibody method for brain tissue samples.     

Giant cell pneumonia associated with parainfluenza virus type 3 infection

Giant cell pneumonia associated with parainfluenza virus type 3 infection and chronic poliovirus type 2 meningoencephalomyelitis are documented in an infant with combined immunologic deficiency (Swiss type). Caution should be exercised in attributing cases of giant cell pneumonia to measles virus without serologic or virologic evidence.     

Growth studies of parainfluenza virus (type 2)

Summary Single cycle and multiple cycle growth of type 2 parainfluenza virus in stable amnion cells was examined. Hemadsorption plaque counts constituted a reliable method for quantitative estimation of infective virus. Virus from cells grown in serum-free medium supplemented with lactalbumin hydrolysate (LH) gave a consistently lower p.f.u./hemagglutinin ratio than virus from cells grown in medium containing calf serum. Infectivity titers, however, were the same in all media at the end of a single cycle of growth. Factors are discussed which might bear on the effect of LH on the production of virus in the presence or absence of calf serum. An unstable variant of type 2 parainfluenza virus is described which is characterized by the formation of hemadsorption plaques larger than those produced by 90% of the particles in stock preparations of virus. A strain of type 2 parainfluenza virus (clone 13) free of the large plaque variant was derived from stock cultures by limiting dilution and plaque purification techniques.Supported by grant No. AI 03168 from the National Institutes of Health, United States Public Health Service.     

Plaque formation by non-cytopathogenic bovine virus diarrhea strains

Summary Five NCP-BVD strains, isolated from clinical cases of BVD and 3 similar strains recovered from non-inoculated BET cultures were found to produce plaques in BET-monolayers under agar overlay when kept at room temperature in the dark for several days after staining.These plaque-forming agents were identified as BVD by neutralization with a BVD antiserum prepared in rabbits.The NCP-BVD strains New York-1 and Sanders-78 also produced plaques in this cell system.     

Viral RNA and protein synthesis in two LLC-MK2 cell lines persistently infected with human parainfluenza virus 3.

Two lines of LLC-MK2 cells persistently infected with human parainfluenza virus 3 (HPIV-3) have been maintained in culture for approximately 3 years. Subgenomic RNAs (putative defective interfering particle genomes) were detected in virions released from both persistently infected cultures. In one of the persistently infected cell lines cyclic variation in the production of virions containing standard virus genomic-size (50S) RNA and subgenomic RNA was observed. The molar ratio of subgenomic RNA to 50S RNA ranged from less than 0.1/1 to 8.7/1. Northern blot analyses revealed that the patterns of viral mRNA synthesis in persistently infected cells from both cultures were similar to those of standard virus infected cells. Furthermore, the intracellular viral-specific proteins had electrophoretic mobilities similar to the corresponding proteins in standard virus-infected cells. Nucleotide sequence analysis of cloned M gene from virus after 29 months of persistence (147 passages) revealed only one variable conservative amino acid change in two clones analyzed from each cell line, indicating that the M protein is not likely to be involved in the maintenance of the persistent infections. The possible mechanisms by which the persistent state is maintained are discussed.     

Syncytium formation induced by baboon endogenous virus in several transformed human cell lines

Several types of cultured human cells derived from malignant tumors and transformedin vitro by DNA tumor viruses. RNA tumor viruses, chemical carcinogens, or⁶⁰Co γ radiation were fused into syncytia when cocultivated with baboon endogenous virus producing human embryonic cells (BaEV-HEC). On the other hand, neither diploid human cell lines nor cells from normal human embryos were fused when cocultivated with BaEV-HEC. Thus, syncytium formation induced by BaEV is dependent upon transformation of human indicator cells rather than upon transforming agents themselves. Concentrated cell-free BaEV suspensions also induced syncytia in human indicator cells within 2 hr after inoculation. The presence of cycloheximide in culture medium had no effect on early syncytium formation. Human indicator cells exposed to low concentrations of BaEV did not form syncytia but produced the virus. These findings strongly suggest that cell fusion induced by BaEV is fusion from without. Specific antiserum against BaEV (M7) blocked this syncytium formation but did not block cell fusion mediated by Mason-Pfizer monkey virus or simian sarcoma virus type I. These observations indicate that the syncytium formation is BaEV specific. The findings in this study suggest that syncytium formation induced by BaEV is a specific characteristic of malignant or transformed human cells.     

Characterization of naturally occurring parainfluenza virus type 2 (hPIV-2) variants

BACKGROUND: Human parainfluenza viruses (hPIV) are respiratory pathogens responsible for upper and lower respiratory tract infections. In most labs, the clinical diagnosis of hPIV is routinely done using techniques based on the detection of viral antigens such as immunofluorescence assay or/and viral isolation. STUDY DESIGN: Five hPIV-2 isolated from respiratory samples exhibited unusual phenotypic and antigenic characteristics. These isolates showed important syncytial cytopathic effect and failed to react with one specific monoclonal antibody. These variant strains were subsequently compared with hPIV-2 prototype strain by cellular and molecular techniques. RESULTS: Both variant and prototype strains showed similar growth kinetics. Observation of plaque formation and syncytia assay indicated a more important fusogenic activity for the variant strains. Sequencing of fusion (F) and hemagglutinin-neuraminidase (HN) genes showed differences between the "atypical" hPIV-2 isolates and the Greer hPIV-2 prototype strain. These differences were analyzed with molecular modelling and structure prediction soft wares. A potential new glycosylation site in HN, in addition to minor changes observed in the predicted structure for the variant strains could explain their antigenic variation. Genetic changes in the fusion peptide and the cleavage site of F could also explain the difference observed in the fusion activity. CONCLUSIONS: Continuous global viral surveillance is essential to monitor antigenic changes that may occur in nature particularly with regards to the implementation of diagnostic assays. The differences observed in F and HN between the prototype strain and clinical hPIV-2 variants could also provide new data for the analysis of Paramyxovirus fusion mechanisms and their pathogenesis.     

Incomplete replication of human parainfluenza virus type 2 in mouse L929 cells

Summary Human parainfluenza virus type 2 (HPIV-2) was tested for its ability to replicate in murine L929 cells. L929 cells were non-permissive for replication of HPIV-2. Interferon produced endogenously played no role in its incomplete replication. The mechanism by which growth of HPIV-2 was suppressed in L929 cells was studied. Synthesis of virus-specific polypeptides, particularly glycoprotein(s), was suppressed in HPIV-2-infected L929 cells. The HN mRNA could scarcely be detected in virus-infected L929 cells.     

Cell-mediated immunity in experimental parainfluenza type 3 virus infection

Local respiratory-tract infection was produced experimentally in guinea-pigs by intranasal instillation of a suspension of parainfluenza virus type 3. Histologically, interstitial pneumonitis developed within 10 days and persisted for at least 70 days. Cell-mediated immunity was measured at intervals for 70 days after infection. Dermal reactivity could not be elicited. Leucocyte-migration inhibition and macrophage-migration inhibition were increased. Macrophage aggregation was present. Increased cell-mediated immunity could be transferred from infected donor animals to normal recipient animals by adoptive spleen-cell transfer even 60 and 70 days after infection.