Virus inactivation, chemical methods

Photolysis of riboflavin leads to the formation of lumiflavin in edkaline solution emd lumichrome in acidic or neutral solution (see Figure 7.2). Because lumiflavin is chloroform extractable, photolysis in alkedine solution, followed by chloroform extraction emd fluorimetric determination, is the basis of commonly used chemical methods of assaying riboflavin. The photolysis proceeds byway of intermediate formation of cytotoxic riboflavin radicals, auid the addition of riboflavin md exposure to light has been suggested as a means of inactivating viruses and bacteria in blood products (Goodrich, 2000). 

During inactivation steps, viral infectivity is reduced by treatment with chemicals and/or physical methods. Remnants of virus particles (e.g., viral nucleic acids) may remain in the product-containing fraction but are not infectious. Chemical methods of virus inactivation, such as treatment with solvent-detergent or acetone, must be placed upstream, since subsequent steps are needed to remove or reduce the levels of the toxic chemicals. Terminal inactivation is often achieved using physical methods, such as heat and low pH, because these methods leave no chemical residues. After treatment, the final products are delivered to patients, so aseptic processing conditions must be maintained throughout terminal inactivation steps and the parameters for virus inactivation must be balanced with the conditions to preserve product quality and yield. 

Screening and selection of the source plasma will only avoid contamination by known pathogens. The protein purification steps and specific virus reduction methods used in production processes, however, will inactivate and/or remove both known and unknown viruses. Terminal virus inactivation treatments are applied to product in final container and must balance virus inactivation with any modifications to protein immunogenicity, activity, and yield. While many upstream virus inactivation steps rely on chemical methods that involve the addition and subsequent removal of toxic agents (e.g., solvent/detergent), physical methods for virus inactivation, such as pH and heat, are used for terminal steps. 

Manufacturing processes have evolved dramatically over the last few years. In the late 1980s, 76 /o of hemophiliacs were HCV positive, ° and between 1979 and 1985, approximately 50 /o of hemophiliacs had acquired HIV from plasma-derived FVIII. Since then, however, most U.S.-licensed plasma derivatives have not transmitted HBV, HCV, or HIV as a result of improvements in donor screening and test methods, and the inclusion of effective upstream virus-reduction and terminal virus-inactivation steps in manufacturing processes. Residual risks of virus transmission from plasma-derived products are now largely associated with non-enveloped viruses. " Thus, the need for additional terminal or upstream virus inactivation/removal steps still exists, but the current challenge is to develop cost effective methods against physico-chemically resistant non-enveloped viruses, such as human parvovirus B19. 

Among chemical methods, monochloramine and free chlorine are used extensively to inactivate viruses." Inactivation can also occur hy degradation or disruption of any one of the viruses structures or hy all of them, depending on the chemistiy of the disinfectant." ... 

Duck hepatitis B virus (DHBV) has been proposed as a possible model for the inactivation of human HBV by chemical disinfectants. The principle of the test method uses viral DNA polymerase (DNAP) as a target, total inhibition in vitro of DNAP by chemical disinfectants being predictive of inactivation of infectivity in vivo. 

The phenomenon of viral adsorption to various surfaces was extensively studied from an environmental standpoint as reviewed by Daniels (14) and Gerba (15) for prevention of various waterborne viral transmissions. The problem of virus removal from complex protein solutions is very different from that of sewage and drinking water treatment processes because most protein molecules compete for the active sites of the adsorbents. Hence, both the adsorption rate and capacity diminish in the presence of protein molecules (16). It is the intention of this paper to demonstrate and to compare the antiviral activity of a surface-bonded QAC in aqueous solutions against 2 model viruses with and without the presence of proteins. The efficacy of the accepted antiviral thermo-inactivation was compared with the viral inactivation method by the surface-bonded QAC treatment. Beta-lactamase was used as a thermolabile model protein (17), and bacteriophage T2 and herpes simplex virus type 1 (HSV-1, an enveloped animal virus) were used as model hydrophilic and hydrophobic viruses to test these chemical inactivation methods. 

A third type of contamination is unique to PCR and other amplification methods, such as the ligase chain reaction. It involves the inadvertent contamination of a new reaction with the aerosolized products of a previous reaction. As shown in Table 2, as little as 10"7 pi of a tube of amplified DNA can contain 103 molecules of target (C4). Recommended precautions (K13) involve the use of positive-displacement pipets and the physical separation of areas where PCR reactions are analyzed from those where new reactions are setup. In laboratories that use these precautions, contamination is infrequent, and, when it does occur, is usually at the 1- to 100-molecule level. However, in addition to these procedural measures, it would be useful to have chemical or enzymatic methods of selectively inactivating amplified DNA—similar to the sterilization procedures used to inactivate large numbers of cultured viruses or bacteria. 

Methodologies most often used for formulation and application of viral insecticides are those developed for conventional chemical insecticides. Viral insecticides are most effectively formulated as wettable powders by lyophilization or spray dry methods. These formulations are best standardized using both counts of occluded virus particle concentration and bioassay activity. Viral insecticides are typically applied as sprays against larval pests of Lepidoptera and Hymenoptera (sawfly) using both aerial and ground equipment. Spray parameters for viral insecticides are not well understood and available equipment is not suitable for their most efficaceous use. Much of the research on virus application has been on development of adjuvants for tank mixtures to overcome problems with plant coverage and sunlight inactivation. 

Painstaking attempts to identify the size of the infectious unit have in the past been necessitated for plant viruses, and also for animal viruses for which hitherto no method of assay for single virus particles has materialized. These include chemical fractionation, physical fractionation by means of separation cells in the ultracentrifuge and electrophoresis apparatus, and inactivation of the virus (Lauffer, 181,182,184,299). Because of electron microscopy and the excellent linearity of the plaque count, these methods have not proved necessary with the bacteriophages. 

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