Viruses reverse transcriptase assays

Berberine chloride was evaluated in the human immunodeficiency virus reverse transcriptase assay and found to be moderately active (50 pg/ml < IC50 < 150 pg/ml)[229]. 

A Rapid Microtiter Assay for Duck Hepatitis Virus Reverse Transcriptase... 

Assays based on sandwich-hybridization are available in several platforms, such as sequential injection analysis (55), microtiter plate assays (61), and microfluidic devices (62). The LFA biosensor assays described in this chapter rely on the sandwich-hybridization of a nucleic acid sequence based amplified (NASBA) RNA target between a membrane immobilized capture probe and SRB-encapsulating liposome conjugated reporter probe. NASBA uses the enzymes avian myeloblastosis virus reverse transcriptase (AMV-RT), RNaseH, and T7 DNA dependent RNA polymerase in the presence of deoxyribonucleoside triphosphates and appropriate primers to amplify relatively few copies of target RNA into... 

Detailed overviews on test methods and vimses detected are provided. For retro-vimses and other endogenous vimses, electron microscopy, infectivity assays or reverse transcriptase assays are proposed. Non-endogenous or adventitious vimses may be detected by suitable in vitro or in vivo assays or (in case of rodent cell fines) by a corresponding antibody production test in the respective species. Other virus-specific tests might be useful alternatives. 

Studies have demonstrated that one such method is to examine the effects of disinfectants on endogenous RNA-dependent DNA polymerase (i.e. reverse transcriptase) activity. In essence, HIV is an RNA virus after it enters a cell the RNA is converted to DNA under the influence of reverse transcriptase. The virus induces a cytopathic effect on T lymphocytes, and in the assay reverse transcriptase activity is determined after exposure to different concentrations of various disinfectants. However, it has been suggested that monitoring residual viral reverse transcriptase activity is not a satisfactory alternative to tests whereby infectious HIV can be detected in systems employing fresh human peripheral blood mononuclear cells. 

Some viruses have secondary structure, which can prevent the production of cDNA detectable in a PCR assay by early termination of the synthesis reaction. To overcome this problem one can raise the temperature of incubation used in first-strand synthesis to 42°C or higher. This will reduce some secondary structures, but will also reduce the half-life of the reverse transcriptase. AMV reverse transcriptase may be used instead, because it has an optimal temperature of 42°C. Unfortunately, AMV RT has more endogenous RNaseH activity than M-MLV RT, thus on average AMV RT produces shorter cDNA fragments. RNaseH deficient RT enzymes are also available (e.g., the Superscript enzymes from Invitrogen), and there is some evidence that these may be the most sensitive type of RT enzymes for PCR assays. The RT conditions required for the efficient detection of individual viruses can only be determined empirically. 

Cys202 [208]. Human immunodeficiency virus (HIV) is the primary cause of acquired immunodeficiency syndrome (AIDS). In an effort to find new drugs preventing the growth of HIV, Masao et al developed an in vitro assay method of RNase H activity associated with reverse transcriptase (RT) from HIV-1. Some 1,4-naphthoquinones moderately inhibited RNase H activity [209]. Several natural occurring naphtoquinones have showed antiretroviral activity [210-211],... 

Palmer S, Wiegand AP, Maldarelli F, Bazmi H, Mican JM, PoUs M, Dewar RL, Planta A, Liu S, Metcalf JA, Mellors JW, Coffin JM. New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J. Clin. Microbiol. 2003 41 4531-4536. 

Stuyver, L., Wyseur, A., Rombout, A., Louwagie, J., Scarcez, T Verhofstede C., Rimland, D., Schinazi, R. F., and Rossau, R. (1997) Line Probe Assay for rapid detection of drug-selected mutations in the human immunodeficiency virus type 1 reverse transcriptase gene. Antimicrobiol. Agents. Chem. 41, 284-291. 

Heneine, W., Yamamoto, S., Switzer, W. M., Spira, T. J., and Folks, T. M. (1995) Detection of reverse transcriptase by a highly sensitive assay in sera from persons infected with human immunodeficiency virus type l.J. Inf. Dis. 171,1210-1216. 

Enzyme-linked immunoabsorbent assays (ELISAs) for VHP infections can be performed on samples inactivated by treatment with P-propiolactone. Reverse transcriptase polymerase chain reaction (RT-PCR) tests on samples following RNA extraction using chloroform and methanol can detect most of the VHP agents rapidly. RT-PCR is particularly useful when isolation of the virus is difficult or impractical, and was effective in detecting the agent causing HPS months before it was isolated in culture (48). 

It is now almost exactly 50 years since I went as a postdoctoral fellow learn about cell culture in Harry Rubin s group in the Virus Laboratory at the University of California at Berkeley. The use of cell cultures had created the breakthroughs in quantitative animal virology, which led, inter alia, to the production of polio vaccines (albeit at the cost of the lives of hundreds of thousands of rhesus monkeys, whose kidney cells were used to produce the viruses for the vaccines). We worked on Rous sarcoma virus (RSV) and chicken leucosis viruses in chick embryo fibroblast cell cultures. Rubin and Temin had developed an assay for RSV, based on the production of foci of virus-transformed fibroblasts, and Temin did the crucial experiments which showed that RSV, an RNA virus, made a DNA copy of itself, which was used to produce new virus particles. The enzyme involved was reverse transcriptase, and its discovery was one of the most important leaps forward in cell and molecular biology. 

Some RNA aptamers that were isolated in vitro have also been expressed in vivo to study their biological function within the context of a living pro- or eucaryotic cell. Among them is an aptamer which binds to the reverse transcriptase (Rev) protein of the human immunodeficiency virus type 1 (HIV-1) [42,43,70],This anti-HIV-l-Rev aptamer was cloned into an expression cassette based on the U6 snRNA promoter, in which aptamer transcripts are protected against nuclease degradation to some extent. Transient expression in the nucleus of cultured cells led to 107-109 full-length aptamer transcripts per cell. When anti-HIV-l-Rev aptamer-expressing cells were co-transfected with the HIV-1 provirus, viral replication was efficiently inhibited in these cells, as shown by an assay in which the production of HIV-1 reverse transcriptase was measured [71],... 

Lanciotti RS, Kerst AJ, Nasci RS, et al. Rapid detection of West Nile virus from human clinical specimens, field-collected mosquitoes, and avian samples by a TaqMan reverse transcriptase PCR assay. J Clin Microbiol. 2000 38 4066-4071. 

This table is intended to hold results of assays testing compounds in reg-istry.structure for activity as human immunodeficiency virus (HIV) protease inhibitors. As new assays are added, the test results can be added to newly created tables with similar definitions. For example, there might be tables for HIV reverse transcriptase inhibitors stored in a table named hiv.rt. Other assay results might be stored in new schemas, for example, fpr.htfc for high-throughput flow cytometry results for the formyl peptide receptor (FPR), or f pr.ca for FPR cell adhesion assay results. Each of these tables would have columns of data named and typed appropriately for each assay. Each table would have a column containing a compound id that references compounds in the registry, structure table. 

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