Cell culture viruses

Hepatitis B Virus Cell Culture Assays for Antiviral Activity... 

Novel isosteres 545 of the antiviral agent acyclovir were synthesized in three stages (84JHC697) by dehydrative coupling of 3-amino-6-aminomethyl[ 1,2,4]triazin-5-(4//)-one with 2-(benzoyloxy)ethoxyacetic acid to give the respective amide that cyclized and deprotected to give 545. It showed no activity against herpes simplex virus types I and II in cell culture (Scheme 112). 

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... 

Viruses that contain amino acid substitutions in the sialidase that impart resistance to the developed inhibitors have been isolated from serial passage of virus in the presence of drug in cell culture and from the clinical setting (reviewed in McKimm-Breschkin 2000 Zambon and Hayden 2001 Cinatl et al. 2007a Reece 2007). In addition, influenza B virus variants with reduced drug sensitivity have been isolated from previously untreated patients (Hurt et al. 2006 Hatakeyama et al. 2007). The types of mutations that are observed are sub-type specific. The mutations present in variants isolated from clinical samples are shown in Table 1, and their locations within the sialidase active site are shown diagrammatically in Fig. 9. 

Yamamoto M, Hayashi N, Takehara T, Ueda K, Mita E, Tatsumi T, Sasaki Y, Kasahara A, Hori M (1999) Intracellular single-chain antibody against hepatitis B virus core protein inhibits the replication of hepatitis B virus in cultured cells. Hepatology 30 300-307 Yang 00, Tran AC, Kalams SA, Johnson RP, Roberts MR, Walker BD (1997) Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells, Proc Natl Acad Sci USA 94 11478-11483... 

Verini, M.A. and M. Ghione. Activity of distamycin A on vaccinia virus infection of cell cultures. Chemotherapia 1964, 9, 144-157. 

Hoschele D (2006) Cell culture models for the investigation of NRTI-induced mitochondrial toxicity. Relevance for the prediction of clinical toxicity. Toxicol In Vitro 20(5) 535-546 Itescu S, Brancato LJ et al (1989) A sicca syndrome in HIV infection association with HLA-DR5 and CDS lymphocytosis. Lancet 2(8661) 466 68 Itescu S, Brancato LJ et al (1990) A diffuse infiltrative CDS lymphocytosis syndrome in human immunodeficiency virus (HIV) infection a host immune response associated with HLA-DR5. Ann Intern Med 112(1) 3-10... 

Inoculation of cell cultures with virus-containing material produces characteristic changes in the cells. The replication of many types of viruses produces the cytopathic effect (CPE) in which cells degenerate. This effect is seen as the shrinkage or sometimes ballooning of cells and the disruption of the monolayer by death and detachment of the cells (Fig. 3.6). The replicating virus can then be identified by inoculating a series of cell cultures with mixtures of the virus and different known viral antisera. If the virus is the same as one of the types used to prepare the various antisera, then its activity will be neutralized by that particular antiserum and CPE will not be apparent in that tube. Alternatively viral antisera labelled with a fluorescent dye can be used to identify the virus in the cell culture. 

A standardized viral suspension is exposed, in the presence of yeast suspension, to appropriate dilutions of disinfectant in WHO hard water. At appropriate times, dilutions are made in inactivated horse serum and each dilution is inoculated into tissue cell culture or embryonated eggs (as appropriate for the test virus). The drop in infectivity of the treated virus is compared with that of the control (untreated) virus. 

Viruses replicate only in living cells so the first viral vaccines were necessarily made in animals smallpox vaccine in the dermis of calves and sheep and rabies vaccines in the spinal cords of rabbits and the brains of mice. Such methods are no longer used in advanced vaccine production and the only intact animal hosts that are used are embryonated hens eggs. Almost all of the vims that is needed for viral vaccine production is obtained from cell cultures infected with vims of the appropriate strain. 

Hepatitis At Human diploid cells infected with hepatitis A virus 1 Separation of virus from cells 2 Inactivation with HCHO 3 Adsorption toAI(OH)3gel Assay of antigen content by ELISA Inoculation of cell cultures to exclude presence of live virus... 

Measles Chick embryo cell cultures infected with attenuated measles virus 1 Clarification 2 Freeze-drying Infectivity titration in cell cultures Tests to exclude presence of extraneous viruses... 

Poliomyelitis (inactivated)t (Salktype) Human diploid cell cultures infected with each of the three serotypes of poliovirus 1 Clarification 2 Inactivation with formalin 3 Concentration 4 Blending of virus of each serotype Induction of antibodies to polioviruses in chicks or guinea-pigs Inoculation of cell cultures and monkey spinal cords to exclude live virus... 

Poliomyelitis (live or oral) (Sabin type) Cell cultures infected with attenuated poliovirus of each of the three serotypes 1 Clarification 2 Blending of virus of three serotypes in stabilizing medium Infectivity titration of each of three virus serotypes Test for attenuation by Inoculation of spinal cords of monkeys and comparison of lesions with those produced by a reference vaccine... 

Measles Neutralization of the infectivity of measles virus for cell cultures Not less than 50IU ml" ... 

NoV are readily transferred from hands to fomites and vice versa (Bidawid et al., 2004 D Souza et al., 2006). The pronoimced environmental stability of NoV particles also contributes to the spread of outbreaks from point sources of surface contamination. All stability studies have made use of surrogate organisms to model NoV response to conditions, since the human virus is not easily grown in cell culture (Duizer et al., 2004b Straub et al., 2007). The murine norovirus (MNV) and the feline calicivirus (FCV) have both been used, with the mouse virus providing more... 

Once potent ligands for a viral protein are identified, further advancement depends on demonstrating activity in cells. Unfortunately, reproducible in vitro viral replication assays for HCV have not been reported. There are scattered reports that a very low level of genome replication, or even virus production, can be observed under certain circumstances [56]. However, recently specific sequences yielding relatively reproducible replication, at consistently detectable levels have been reported [57]. In the coming years these may allow routine assays suitable for compound evaluation to be developed, but to date drug discovery must rely on other cell culture models. 

HIV phenotype A type of resistance testing for human immunodeficiency virus (HIV) in which a patient s blood sample is obtained, and the patient s HIV genes that encode for reverse transcriptase and protease are removed and placed in an HIV viral vector. This viral vector is replicated in a cell culture system with varying concentrations of antiretrovirals. A drug concentration-viral inhibition curve is developed and the concentration needed to inhibit 50% of the patient s virus is reported. This is used to predict resistance versus susceptibility. 

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