Infectious centers

The infectious center assay can also be used to measure the level of rcAAV contamination in an rAAV preparation. In this case, dilutions of the rAAV preparation are used to infect HEK293 cells in the presence of adenovirus. Since no additional wild-type AAV is added, only particles in the rAAV preparation that have packaged a wild-type or pseudo-wtAAV genome are capable of replication and produce a signal following probing with the AAV rep gene. 

Fig. 2.4. Vector characterization. (A). SDS acrylamide gel electrophoresis of a rAAV-UF-11 vector. Approximately 2.5 x 1011 drp were loaded on a 10% SDS-polyacrylamide gel. The three capsid protein bands in the correct stoichiometry are visualized by silver staining. (B) Infectious center assay of a rAAV-CB-hAAT stock when probed for the transgene each spot represents a vector infected cell. (C) Dot blot analysis of the rAAV stock to determine the titer of DNase-resistant particles of rAAV. Amounts (ng) refer to a serial dilution of the packaged rAAV vector plasmid used to construct the standard curve. The stock has a calculated titer of 4.3 x 1013 drp/ml.

Infectious Infectious Center Assay (ICA) [192, 193] Serial Dilution Replication Assay (dRA) [194] Determine titer of infectious particles produced... 

Fig. 2. Inhibition of spread of infection by immunotoxins. A kinetic analysis of the effect of an anti-gpl20 immunotoxin is shown. PHA blast cells were infected with a primary clinical isolate of HIV in the presence or absence of immunotoxin. Beginning 4 d after infection, an aliquot of cells was taken and plated onto a monolayer of Hl-J cells. The cells were washed off the monolayer 1 d later and 2 d later the cells were stained for HIV foci. The percent of cells secreting infectious HIV was determined by dividing the number of foci by the number of input cells. Note both the small proportion of cells that were infected and the increase in infectious centers with time postinfection.

Cell-free virus or infected cells (infectious centers, ICs) can be measured at frequencies as low as 10 6. The sensitivity of the FIA rivals that of PCR, where examining the input from 106 cells is an upper limit. The FIA detects all strains of HIV, including primary isolates from patients (9,57,61,64,67-69). ICs may be detected in tissue culture cell lines and PHA blast cultures infected in vitro. The assay has also been used to quantify infectious HIV in AIDS patients (69). The assay is performed by plating either infected cells or cell-free virus on a monolayer of HeLa cells expressing CD4, CXCR4, and CCR5. Detection of clinical isolates is highly dependent on the HeLa-CD4 cell line used and is a... 

The importance of the conclusion that bacterial multiplication stops should not be ignored. This fact has since been affirmed by colony counts of bacteria multiply infected with T2r+ (54), T3 (225), and T7 (262), by cytochemical observation, and by the constancy of the infectious centers during the latent period of one-step growth curves of multiply infected bacteria. Indeed, no one has ever demonstrated that phage-infected E. coli can multiply. ... 

The penetration of infectious RF-RNA, can also be measured by following the effect of RNase on infective center formation (Fig. 8). In an isotonic environment, most of the RF-RNA infectivity is lost rapidly when exposed to RNcise (Bishop and Koch, 1967 Mittelstaedt and Koch, 1974). Only those cells which have already engulfed one complete RNA molecule before RNase is added will register as an infectious center in the plaque assay. Both... 

The concentration of tissue culture cells can be varied from 2 XIO to 9 xiO /ml without a significant influence on the titer of a standard RNA preparation. This is apparently due to the fact that the number of cells competent for infection is in excess of the number of RNA molecules capable of initiating infection. Within a certain limit, greater quantities of input RNA would give a higher number of infectious centers (Fig. 11). 

Fig. 9 illustrates another aspect of the dose-response relationship the significance of the amount of polycations present per cell. These experiments, performed by varying the concentrations of both polycation and cells, show that the yield of infectious centers is determined primarily by the polycation ceU ratio rather than the absolute concentration of the polybasic compound. The important practical implication of these data is that an appropriate adjustment in the polycation concentration must be made to obtain an optimal infection by viral RNA at a given ceU concentration. 

Fig. 10. Relationship between time of polycation additions and yield of infectious centers induced by RNA. Polycations were added to suspended cells at various times either before or after the addition of RNA and the mixture incubated at 37 C. The following final concentrations were used poly-L-omithine 20 (xg/ml, methylated albumin 40 xg/ml, and DEAE-dextran 80 [xg/ml and 3X10 cells/ml. The 0 time data were obtained by mixing RNA and polycations and then adding them immediately to the cells. The RNA in the incubation mixtures with methylated albumine was fourfold more concentrated...

Within a certain range of RNA concentrations, the yield of infectious centers is directly proportional to the amount of RNA present in the incubation mixture. This has the same implication as the similar relationship observed with intact virus a single molecule of RNA is sufficient to initiate infection. 

Vaccines are used in either the general population of children or adults or for special groups. Recommendations for vaccine usage are made by the Advisory Committee on Immunization Practices (ACIP) of the Centers for Disease Control. The Committee on Infectious Diseases of the American Academy of Pediatrics (Redbook Committee) also makes recommendations for infants through adolescents, and the American Academy of Family Physicians makes recommendations for adults. An excellent review of vaccine history, development, usage, and related regulatory issues is available (2). 

American Thoracic Society/Centers for Disease Control/Infectious Disease Society of America. Treatment of tuberculosis. Am I Respir Crit Care Med 2003 167 603-662. 

Owing to the morbidity associated with these opportunistic infections in HCT recipients, optimal pharmacotherapy for preventing and treating infections in this patient population is critical. In 2000, the Center for Disease Control and Prevention (CDC) published guidelines for preventing these infections among HCT recipients.8 These guidelines were constructed from available data by an expert panel from the CDC, the Infectious Disease Society of America, and the American... 

Infectious Disease Pharmacokinetics Laboratory National Jewish Medical and Research Center Denver, Colorado Chapter 72 Tuberculosis... 

Infectious Diseases Physician Assistant University of Nebraska Medical Center Omaha, Nebraska... 

Achievements in Public Health 1900 1999 Control of Infectious Diseases. Atlanta, GA Centers for Disease Control. 48, no. 29 (July 30, 1999) 621-629. Source for infant mortality rates. 

Centers for Disease Control (1998) Preventing Emerging Infectious Diseases A Strategyfor the 21st Century. Centers for Disease Control, Atlanta, GA. http // medi-smart.com/infect emerging2.htm... 

Centers for Disease Control and Prevention. "Case Definitions for Infectious Conditions Under Public Health Surveillance." Morbidity and Mortality Weekly Report 46 (RR-10) (1997). 

Center for Infectious Disease Research Policy, University of Minnesota. Agricultural Biosecurity, Animal Diseases, http //www.cidrap.umn.edu/cidrap/content/biosecurity/ag-biosec/anim-disease/index.html. 2006. 

Guidance for Industry Considerations for Developmental Toxicity Studies for Preventive and Therapeutic Vaccines for Infectious Disease Indications. U.S. Department of Health and Human Services, Food and Drug Administration Center for Biologies Evaluation and... 

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