Studying Viral Cytopathic Effects

One of the problems encountered in studies of viral cytopathic effects is to quantitate the number of cells undergoing changes or dying. Some indication of the number of cells involved can be obtained by estimating those that fail to exclude dyes because of altered membrane impermeability. A more accurate, but somewhat tedious technique was designed by Marcus and Puck (1958). These authors devised a means to study quantitatively the destruction of mammalian cell reproductive capacity by scoring the fraction of monodisperse HeLa cells infected with Newcastle disease virus to survive and form colonies over a period of 8-10 days incubation. They concluded that one virus particle was sufficient to kill a single cell. This method has been extended to other viruses by Marcus (1959) and can be used to... 

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. 

The virus reduction studies of the three process steps discussed here were performed with HFV-l, Bovine viral diarrhea virus (BVDV), Pseudorabies virus (PRV), Reovirus type 3 (Reo), Hepatitis A virus (HAV), and Porcine parvovirus (PPV). HIV-1 was included as a relevant enveloped virus, while BVDV and PRV were tested as specific model viruses for HCV and HBV, respectively (Table 1). Reo was chosen as a non-specific model non-enveloped virus, HAV was included as a relevant virus and PPV was used as a surrogate for human parvovirus B19. All viruses were propagated using standard cell culture conditions. " The appropriate cell lines were infected, at a low multiplicity of infection, and incubated until 4-1- cytopathic effects were observed. The infected cells were frozen and thawed three times to release virus, centrifuged at low speed to remove cell debris and the clarified supernatants were removed for use as virus spikes. 

A variety of mammalian cellular systems have been used as experimental models for documenting the in vitro effects of cannabinoids on immune responsiveness to viruses, bacteria, and amoebae. Blevins and Dumic (1980) indicated that THC had a protective effect against HSV infection in vitro. It was found that both HSV-1 and HSV-2 failed to replicate and produce extensive cytopathic effect (c.p.e.) in human cell monolayer cultures exposed before infection, at infection, or post infection to various concentrations of THC. In contrast, other studies indicate that THC compromises resistance to virus infection. It has been reported that THC inhibits macrophage extrinsic anti-viral activity (Cabral and Vasquez 1991 Cabral and Vdsquez 1992) whereby macrophages normally suppress virus replication in cells to which they attach (Morahan et al. 1980 Stohlman et al. 1982). Noe et al. (1998) reported that a variety of cannabinoid receptor agonists enhanced syncytia formation in human T cell leukemia virus-I (HTLV-I)-transformed human T (MT-2) cells infected with cell free human immunodeficiency virus (HIV-IMN). It was found that CP 55,940, THC, WIN 55,212-2, and WIN 55,212-3 significantly increased syncytia formation, a phenomenon that has been reported to serve as an indicator of HIV infection and cytopathicity. 

The above studies generally support the concept that VSV can cause two cytopathic effects, early cytotoxicity at high multiplicity and late cytopathic effects at lower multiplicity. However, as will be discussed in later chapters on viral effects on cellular macromolecular synthesis, the concept of two sharply separable effects is too simplistic as indicated by variation in cellular susceptibility reported by Baxt and Bablanian (1976a). Related studies by these authors on VSV effects on cellular macromolecular synthesis (Baxt and Bablanian, 1976 ), as well as studies by other investigators will be discussed in a later chapter. 

A major advance in technique for studying cytopathic effects of viruses, particularly relating to the cytoskeleton, has come from Penman s laboratory, in which the cytoskeletal framework is prepared by detergent lysis these suspended cells retain the major features of cell morphology, such as polyribosomes and major structural filaments. Lenk and Penman (1979) found extensive cytoskeletal changes by this technique in poliovirus-infected cells even when guanidine blocks synthesis of most viral products. The skeleton preparation... 

Infection of cultured cells with many lytic viruses results in a marked decrease in the rate of cellular protein synthesis. Usually, this decrease is accompanied by increasing rates of viral protein synthesis, marked cytopathic effects, and ultimately cell death. In most cases, it is not known whether the shut-off of host cell protein synthesis results from an active process induced by the virus evolved for that (or some other) purpose, or whether it is merely a passive result of another viral function, such as production of large quantities of viral mRNA which compete effectively with their cellular counterparts. In the case of poliovirus, however, three types of studies suggested that the former, active type of mechanism was at work. Kinetic analysis of the rate of protein synthesis in cells synchronously infected with high multiplicities of virus showed that cellular protein synthesis could be virtually completely inhibited prior to the synthesis of significant quantities of viral RNA and protein (Summers et ai, 1965). In addition, infection in the presence of 1-3 mM guanidine, which prevents detectable replication of viral RNA, nevertheless results in viral inhibition of host cell protein synthesis (Holland, 1%4 ... 

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