Virus quantification

About: Virus quantification is a research topic. Over the lifetime, 1600 publications have been published within this topic receiving 60866 citations.

Isolation of a virus from the pancreas of a child with diabetic ketoacidosis.

Abstract: A healthy 10-year-old boy was admitted to the hospital in diabetic ketoacidosis within three days of onset of symptoms of a flu-like illness. He died seven days later and post-mortem examination showed lymphocytic infiltration of the islets of Langerhans and necrosis of beta cells. Inoculation of mouse, monkey and human cell cultures with homogenates from the patient's pancreas led to isolation of a virus. Serologic studies revealed a rise in the titer of neutralizing antibody to this virus from less than 4 on the second hospital day to 32 on the day of death. Neutralization data showed that the virus was related to a diabetogenic variant derived from Coxsackievirus B4. Inoculation of mice with the human isolate produced hyperglycemia, inflammatory cells in the islets of Langerhans and beta-cell necrosis. Staining of mouse pancreatic sections with fluorescein-labeled antiviral antibody revealed viral antigens in beta cells. Both the clinical picture and animal studies suggested that the patient's diabetes was virus induced.

Infection of HTLV-III/LAV in HTLV-I-carrying cells MT-2 and MT-4 and application in a plaque assay

Abstract: The human T-cell lines MT-2 and MT-4 carry the human T-cell leukemia virus type I (HTLV-I). When MT-2 and MT-4 were infected with HTLV-III, the probable etiologic agent of the acquired immune deficiency syndrome (AIDS), rapid cytopathogenic effects and cytotoxicity were observed that made it possible to titrate the biologically active virus in a plaque-forming assay. The cytopathogenic effects were preceded by the rapid induction and increase of HTLV-III antigens as revealed by immunofluorescence and immunoprecipitation. Activities of HTLV-III were neutralized by the human antibodies against the virus when immunofluorescence and plaque assays were used. Essentially the same results were obtained with the lymphadenopathy-associated virus (LAV1).

Evaluation of the concentration and bioactivity of adenovirus vectors for gene therapy.

Abstract: Development of adenovirus vectors as potential therapeutic agents for multiple applications of in vivo human gene therapy has resulted in numerous preclinical and clinical studies. However, lack of standardization of the methods for quantifying the physical concentration and functionally active fraction of virions in these studies has often made comparison between various studies difficult or impossible. This study was therefore carried out to define the variables for quantification of the concentration of adenovirus vectors. The methods for evaluation of total virion concentration included electron microscopy and optical absorbance. The methods for evaluation of the concentration of functional virions included detection of gene transfer (transgene transfer and expression) and the plaque assay on 293 cells. Enumeration of total virion concentration by optical absorbance was found to be a precise procedure, but accuracy was dependent on physical disruption of the virion to eliminate artifacts from light scattering and also on a correct value for the extinction coefficient. Both biological assays for enumerating functional virions were highly dependent on the assay conditions and in particular the time of virion adsorption and adsorption volume. Under optimal conditions, the bioactivity of the vector, defined as the fraction of total virions which leads to detected target cell infection, was determined to be 0.10 in the plaque assay and 0.29 in the gene transfer assay. This difference is most likely due to the fact that detection by gene transfer requires only measurement of levels of transgene expression in the infected cell whereas plaque formation is dependent on a series of biological events of much greater complexity. These results show that the exact conditions for determination of infectious virion concentration and bioactivity of recombinant adenovirus vectors are critical and must be standardized for comparability. These observations may be very useful in comparison of data from different preclinical and clinical studies and may also have important implications for how adenovirus vectors can optimally be used in human gene therapy.

Vaccinia virus expression vector: coexpression of beta-galactosidase provides visual screening of recombinant virus plaques.

Abstract: We constructed a plasmid coexpression vector that directs the insertion of a foreign gene of interest together with the Escherichia coli beta-galactosidase (beta gal) gene into the thymidine kinase (TK) locus of the vaccinia virus genome. Tissue culture cells that had been infected with vaccinia virus were transfected with a plasmid vector containing a foreign gene. TK- recombinants could be selected by a plaque assay on TK- cells in the presence of 5-bromodeoxyuridine and distinguished from spontaneous TK- mutants by the addition of a beta-gal indicator to the agarose overlay. Plaques that expressed beta-gal stained dark blue within several hours at 37 degrees C. Alternatively, TK- selection could be eliminated, and recombinant plaques could be readily identified solely by their blue color. The reverse procedure, in which the starting virus expresses beta-gal (i.e., forms blue plaques) and the desired recombinant has deleted the entire beta-gal gene (i.e., forms white plaques), is another alternative. Each protocol was tested by constructing vaccinia virus recombinants that express hepatitis B virus surface antigen.