Virus assay

Whitcomb JM, Huang W, Fransen S, Limoli K, Toma J, Wiin T, Chappey C, Kiss LD, Paxinos EE, Petropoulos CJ (2007) Development and characterization of a novel single-cycle recombinant-virus assay to determine human immunodeficiency virus type 1 coreceptor tropism, Antimicrob Agents Chemother 51 566-575... 

Penetration. After fusion of viral and host membranes, or uptake into a phagosome, the virus particle is carried into the cytoplasm across the plasma membrane. This penetration process is an active one that requires expenditure of energy by the cell. At this stage the envelope and the capsid are shed, and the viral nucleic acids are released. The uncoating of virus accounts for the drop in infectious virus assayed, because the uncoated virus cannot withstand the assay conditions. 

In a Moloney murine leukemia virus assay that served as a model for HIV, it could be demonstrated that glucosidase inhibitors 1-deoxynojirimycin 2 and castanospermine 4 were active at concentrations of 1-2 p.g/ml [185]. 

Although this is the most crucial step in the recombinant virus assay, the procedure is easy and simple. In general, 10 pg of proviral plasmid and 2 pg of PCR are used for recombination. In most cases, less PCR product will also perform well, but it may take somewhat longer before the virus breaks through. In difficult cases, more PCR product can be used, but one should be aware that, in these cases, the recombinant virus might not be representative for the virus population in the patient. The virus obtained can be directly used for stock titration as described in the protocol for the MT-4/MTT assay in Subheading 4.I.3.I. 

Kellam, P. and Larder, B. A. (1994) Recombinant virus assay a rapid, phenotypic assay for assessment of drug susceptibility of human immunodeficiency virus type 1 isolates. Antimicrob. Agents Chemother. 38, 23-30. 

Abstract The assembly of the N-terminus heptad repeats of the respiratory syncytial virus (RSV) F protein into a trimeric complex that associates with the C-terminus heptad repeats to form a six-helix bundle is a critical step in the process of vims-host fusion and represents an intramolecular protein-protein interaction. Screening campaigns using replicating virus assays have identified several structurally distinct but mechanistically similar chemotypes that interfere with RSV fusion by disrupting the function of the F protein six-helix bundle. This chapter summarizes structure-activity relationships and mechanistic insights associated with the most prominent RSV fusion inhibitors and the key issues in the development of potential clinical candidates. 

CCL 81 Vero African green monkey 199-5 From adult kidney hypodiploid (mode 58) used extensively in virus assay and production... 

Fahr, C., and Schauer, R. Detection of gangliosides with 9-0-acetylated sialic acids in basaliomas and normal human skin by influenza C virus assay. J. Dermatol, submitted. 

Guo, W.-R, Ma, X.-M., Zeng, Y., 2005. Clinical laboratories on a chip for human immunodeficiency virus assay. In Conference proceedings . .. Annual International Conference of the IEEE Engineering in Medicine and Biology Society. IEEE Engineering in Medicine and Biology Society. Conference, vol. 2, pp. 1274-1277. Available at http //www.ncbi.nlm. nih.gov/pubmed/17282427. 

Independent Assays for Provings Virus Removal. Retrovimses and vimses can also be present in culture fluids of mammalian cell lines (15,24). Certainly the absence of vims can be difficult to prove. Model vimses, eg, NIH Rausher leukemia vims and NZB Xenotropic vims, were spiked into fluids being purified, and their removal subsequently vaUdated when subjected to the same purification sequence as used for the product. 

At this stage the interferon preparations were assayed and submitted to safety tests for the absence of contaminating viruses. 

Each interferon preparation was ultracentrifuged at 20,000 revolutions per minute for one hour to remove tissue debris and inactivated virus. The supernatant was dialyzed against distilled water (1 400) for 24 hours at4°C. The material was then freeze-dried. The dried product was reconstituted in one-tenth of the original volume in distilled water and dispensed into ampoules. Reconstituted solutions were assayed for interferon activity, examined for toxicity, and tested for sterility. 

In our screening program for virus inhibitors and stimulators we used cucumber as an assay host, in which starch lesions are formed in proportion to the concentration of virus in the inoculum. 

Ludmerer SW, Graham DJ, Boots E, Murray EM, Simcoe A, Markel EJ, Grobler JA, Flores OA, Olsen DB, Hazuda DJ, LaFemina RL (2005) Replication fitness and NS5B drug sensitivity of diverse hepatitis C virus isolates characterized by using a transient replication assay. Antimicrob Agents Chemother 49 2059-2069... 

A major limitation in the development of anti-HCV compounds was the lack of a virus replication system. This was finally overcome with the development of a novel replicon system that directed persistent replication in a cell culture format (Lohmann et al. 1999). Using such a system, it was possible to demonstrate antiviral activity of an NS3/4A inhibitor in a cell culture assay, and demonstrate potency on par with treatment with interferon-a (Pause et al. 2003). 

Neu5Ac2en 4, a micromolar inhibitor of influenza virus sialidase 4 x 10 M (A/N2)] (Holzer et al. 1993), was first identified as a very good inhibitor in the late 1960s (Meindl and Tuppy 1969). A series of C-5 modified Neu5Ac2en derivatives provided the first improved in vitro inhibitors compared with the parent compound 4. The replacement of the C-5 A-acetyl moiety with a A-trifluoroacetyl group resulted in the most potent inhibitor of this series, 2-deoxy-2,3-didehydro-A-trifluoroacetylneuraminic acid 10 [A] 8 x 10 M (A/Nl)] (Meindl et al. 1974). While these C-5 modified compounds were also very effective in cell culture assays (Palese et al. 1974a Palese and Compans 1976), none, including the parent... 

Table 2 Sensitivity of influenza virus sialidase variants isolated in the clinical setting to sialidase inhibitors in sialidase assays ...

Since the pioneering work of Kleymann et al. (2002), Betz et al. (2002), Baumeister et al. (2007), and Crute et al. (2002), who showed that compounds identified as inhibitors of the helicase-primase enzyme complex could alleviate herpesvirus-induced disease in animal models, the attention of researchers developing antiviral compounds has been drawn more and more towards the virus-encoded helicases, particularly those of Herpes viruses and of RNA viruses such as Hepatitis C Virus (HCV) and SAKS coronavirus (SARS-CoV). Enzyme activity is usually assayed by measuring NTPase activity in the presence of an appropriate nucleic acid co-substrate although, more recently, novel fiuorimetric and luminescence principles have been applied to the measurement of strand unwinding and/or translocation of the protein along the nucleic acid (Frick 2003, 2006). 

The situation is somewhat more complex for virus sample amphfied or isolated directly from patient plasma, as these samples comprise a heterologous mix of related yet genetically distinct quasi-species. Often these samples contain mixtures of CCR5-tropic (R5), CXCR4-tropic (X4), and/or dual-tropic viruses these samples are classified in phenotypic ttopism assays as dual/mixed tropic (D/M) (Whitcomb etal. 2007). 

Delwart EL, Sheppard HW, Walker BD, Goudsmit J, Mullins J1 (1994) Human immunodeficiency virus type 1 evolution in vivo tracked by DNA heteroduplex mobUity assays, J Vhol 68 6672-6683... 

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