Virus Infectivity Assays Research Articles

An international collaborative study on foot and mouth disease virus assay methods. 1. Virus infectivity and neutralizing antibody assays

In a collaborative study sponsored by the European Commission for the Control of Foot and Mouth Disease, workers in 19 laboratories participated in the early phases (1, 2 and 4). All three phases were devoted to assessments of virus infectivity and the neutralizing activity of sera. Virus preparations and antisera distributed from one laboratory were tested either by 'in house' or suggested methods. Analyses of the results clearly showed that whilst 'within' laboratory variation in the results of replicate tests was low, there were significant differences in the results from various laboratories. By attempting to standardize test conditions with distributed materials, e.g. media and cells, etc., the 'between' laboratory variation was reduced.

Determinants of Susceptibility and Resistance to Feline Leukemia Virus Infection. II. Susceptibility of Feline Lymphocytes to Productive Feline Leukemia Virus Infection<xref ref-type="fn" rid="FN2">2</xref><xref ref-type="fn" rid="FN3">3</xref>

The interplay between feline leukemia virus (FeLV) and feline lymphocytes (lc) infected in vitro or in vivo was investigated. Surface marker analysis and viral infectivity (VI) assays of lc populations were used to determine susceptibility of lc subsets to FeLV. The principal FeLV-replicating cell in the mesenteric lymph node of persistently infected, preleukemic cats was a nonadherent, complement receptor (CR)-bearing lc (B-cell). The lymph nodes of preleukemic cats also had increased numbers of uninfected T-cells [cells forming rosettes with guinea pig erythrocytes (GPE)] and cells with receptors for the Fc portion of 7S IgG (Fc gamma R cells) as compared with lymph nodes of age-matched specific-pathogen-free (SPF) cats. The induction of productive infection of feline peripheral blood mononuclear leukocytes (PBL) in vitro depended on a 48-hour in vitro preincubation period before virus exposure. The equivalent susceptibility of whole and adherent cell-depleted PBL to productive infection and the failure of hydrocortisone to enhance viral infection were compatible with identification of the FeLV-replicating cell as an lc. Furthermore, lc from susceptible SPF kittens replicated 50 times as much FeLV as did lc from resistant adult SPF cats. The Ic productively infected with FeLV after in vitro exposure were more precisely identified with the use of Ficoll-Isopaque density gradient separations of rosetted and nonrosetted lc. Whole PBL, GPE rosette-positive PBL (T-cells), and CR-positive PBL (B-cells) were permissive to FeLV infection, and maximal VI was evident at 14 days after exposure. The substantial (1,325-fold) increment in VI found in the Fc gamma R-depleted PBL suggested a role for Fc gamma R cells in the containment of FeLV infection. Unstimulated mononuclear leukocytes from blood, spleen, lymph node, thymus, and marrow were susceptible to productive FeLV infection after in vitro exposure. The degree of spontaneous DNA synthesis in marrow, thymus, and spleen but not lymph node or PBL was inversely related to permissiveness to viral replication. Mitogen activation of lc was associated with decreased viral replication when either T-cell mitogens (concanavalin A, phytohemagglutinin, or pokeweed mitogen) or a B-cell mitogen (dextran sulfate) was used. Virus production by spleen cells and PBL was enhanced twofold to tenfold by prior lc stimulation by the B-cell mitogen, lipopolysaccharide, or protein A-bearing Staphylococcus aureus, a mitogen for feline T-cells with Fc gamma R. Both productively infected (preincubated) and nonproductively infected (freshly isolated) PBL transferred infectious FeLV to autochthonous peritoneal macrophages (M theta); most of the virus in PBL-peritoneal M theta cocultures was produced by adherent cells, irrespective of whether the adherent or nonadherent cell population was inoculated originally.

Human lymphoblastoid cells as hosts for parvoviruses H-1 and rat virus

A human T-cell line (Molt-4) was shown by viral hemagglutination and infectivity assays to support the replication of rat virus (RV) and H-1 virus. In addition, H-1 virus, but not RV, multiplied in two human B-cell lines, AV-1 and NC-37. The ability to bind radioactively labeled RV was demonstrated for each of the cell lines, but viral adsorption occurred to a greater degree with Molt-4 cells than with either AV-1 or NC-37 cells. After challenge with RV, virus-specific antigens were detected in cells of the B-cell lines by the indirect immunofluorescence technique. Infection of AV-1 or NC-37 cells by RV apparently results in an abortive cycle of virus replication. Differences among the three cell lines that might influence with H-1 virus or RV are discussed.

Antibody-dependent cellular protection against herpes simplex virus dissemination as revealed by viral plauqe and infectivity assays

Mouse nonimmune peripheral blood lymphocytes (PBL) plus antibody to herpes simplex virus inhibited virus dissemination in herpes simplex virus-infected 3T3 cell culture as revealed by development of viral plaque, size of immunofluorescent foci, appearance of polycaryocytes, and viral infectivity appearing in the culture. These nonimmune PBL did not act alone in inibiting virus dissemination, but did act synergistically in combination with antibody. The ratio of PBL to target monolayer cells needed to produce this effect was 20. Splenic lymphocytes had weak activity, whereas thymocytes were without effect, even in the presence of antibody. Neither interferon nor lymphotoxin was detected in this lymphocyte-mediated response. These findings support previous observations, based on cytotoxicity assays, that antibody-dependent cellular immune processes could be important in control of and recovery from herpesvirus infection.

RD-114 virus infectivity assay by measurements of DNA polymerase activity and virus group specific antigen.

RD-114 virus infectivity assay by measurements of DNA polymerase activity and virus group specific antigen.

Improved Techniques for Staining and Enumerating Focus Formation in Viral Infectivity Assays

Improved techniques consisting of glutaraldehyde fixation, Giemsa staining, and colony counter enumeration of foci in viral infectivity assays were compared to microscope scanning and lantern slide projection methods. These techniques broadened the scope of application to potentially hazardous viruses and increased the efficiency of assaying.

Haemadsorption-negative assay of rubella virus in baby hamster kidney cells.

A method is described for the assay of rubella virus infectivity in BHK-21 cells, by counting haemadsorption-negative plaques, and using Sendai virus as challenge agent instead of NDV.

Assay of the infectivity of Epstein-Barr virus by transformation of human leucocytes in vitro.

One of the important biological activities of EB virus (Epstein, Achong & Barr, 1964) is the transformation of human leucocytes in vitro (Henle et al. 1967; Pope, Horne & Scott, 1968). Transformation in this context is defined as morphological change in a proportion of the cells, associated with continued cell proliferation leading to establishment of a lymphoid cell line. The transformation factor was identified as EB virus by showing that it had some of the general properties of herpes viruses and was neutralized specifically by human sera containing antibody to EB virus (Pope, Horne & Scott, 1969). The all-or-none nature of the transformation response to EB virus makes it a suitable basis for assay of virus infectivity. Some experiments have been reported of titration of EB virus by transformation assay in 1 ml leucocyte cultures (Pope et al. 1969, 1971) and of the distribution of infectivity in a sucrose density gradient (Walters & Pope, 1971).

Assay of C-Type virus infectivity by measurement of RNA-dependent DNA polymerase activity

Assay of C-Type virus infectivity by measurement of RNA-dependent DNA polymerase activity

Fluorescent plague assay of rubella virus infectivity.

Plaques of rubella virus infected cells demonstrating granular, cytopathic fluorescence were counted in RK13 cell cultures fixed 72 hr after infection. There was a linear relationship between virus concentration and fluorescent plaque count. Counts on replicate cultures have demonstrated that the technique is very precise. The sensitivity of the fluorescent plaque assay was equal to that of the conventional GMK-interference titration method.

Plaque formation by myxovirus parainfluenza type 2 in grivet monkey kidney cells.

SummaryA method has been described for plaque formation of parainfluenza type 2 virus in grivet kidney monolayer cell culture. The method has shown utility in the assay of virus infectivity. The plaquing technique should also be useful for cloning of this virus for genetic studies.