key: cord-022349-z8w1wkm8 authors: Beeler, Judy A. title: Human and Animal Viruses date: 2007-09-02 journal: Maintaining Cultures for Biotechnology and Industry DOI: 10.1016/b978-012361946-4/50010-7 sha: doc_id: 22349 cord_uid: z8w1wkm8 This chapter provides an overview of the human and animal viruses. Viruses held to a low number of passages in animals or cell cultures represent a viral population that is similar to that found in nature, and freezing these pools guards against genetic mutations that occur during subsequent passage. Aliquots of viral stocks frozen at a designated passage level can then be used for multiple and repeatable experiments with the same viral population. Furthermore, it is important that consistency should be maintained during the production of viral vaccines; new lots of final product are prepared with frozen viral seed stocks that consistently reproduce the desired immunogenic and attenuation characteristics. To better appreciate the requirements for freezing and freeze drying of human and animal viruses, some consideration is given to understanding the structural and functional organization of this diverse group of microorganisms. The classification of viruses is based on morphological and physiochemical properties. Thus, viruses are divided into those with DNA or RNA genomes and subdivided into families based on size and structural properties. Several methods for preservation of viruses are included in the chapter. Shackell's successful treatment of rabies virus in 1909 represents the first published account of preservation of an animal virus using a process that was an early version of freeze-drying (Shackell, 1909) . Prior to this, viruses were maintained by passage in animals. However, freeze-drying provided a way to maintain virally infected material over long periods of time with relative ease when compared to serial passage in a susceptible animal host. The first experiment to demonstrate that this method could provide long-term stability was reported for a bovine virus that had been freeze-dried in 1916 and was shown to be viable after being maintained at room temperature for 30 years (Fasquelle and Barbier, 1950) . Later, as mechanical freezers became more available, freezing viruses at -25~ or less was adopted as a practical and reliable method of virus preservation. Influenza virus-infected mouse lung tissue and yellow fever virus were successfully frozen at -76~ by Turner (1938) , and Horsfall (1940) , using Turner's methods, demonstrated successful storage of influenza, mouse pneumonitis, and canine distemper viruses. Following these early successes, viral repositories, diagnostic and research laboratories, and vaccine manufacturing facilities have relied on freezing and freeze-drying for preservation of viruses and viral vaccines. The reasons for preserving viruses are similar to those for the preservation of other microorganisms and cells: successful preservation allows for long-term maintenance of consistent stocks and for reproducibility in the testing of viral samples and manufacture of vaccines. For example, field and clinical isolates need to be preserved for transport to testing laboratories; harvested virus from animals or cell cultures may not be processed immediately and require preservation until used. Viruses held to a low number of passages in animals or cell cultures represent a viral population that is similar to that found in nature, and freezing these pools guards against genetic mutations that may occur during subsequent passage. Aliquots of viral stocks frozen at a designated passage level can then be used for multiple and repeatable experiments with the same viral population. Furthermore, it is extremely important that consistency be maintained during the production of viral vaccines; new lots of final product can be prepared with frozen viral seed stocks that consistently reproduce the desired immunogenic and attenuation characteristics. For example, the World Health Organization (WHO) maintains aliquots of the original attenuated poliovirus strains developed by Dr. Albert Sabin in the 1950s. These aliquots are frozen and, when amplified properly and kept to the prescribed low passage level, should produce a vaccine today with the same properties that characterized the original Sabin vaccine. Similarly, viruses that are currently under development for use as vectors for gene therapy must be highly characterized and homogeneous for safe and effective use and need to be maintained as frozen stocks. In order to better appreciate the requirements for freezing and freezedrying of human and animal viruses, some consideration must first be given to understanding the structural and functional organization of this diverse group of microorganisms. The classification of viruses is based on morphological and physiochemical properties. Thus viruses are divided into those with DNA or RNA genomes and further subdivided into families based on size and structural properties. There are 6 families of DNA viruses and 13 families of RNA viruses (see Table 1 ). The genomes of viruses within a given family are similar in terms of their polarity, genome organization, and method of replication and have morphologic features in common, including size, shape, nucleocapsid symmetry, and presence or absence of an envelope. Other characteristics that help to classify viruses include sensitivity to low pH, heat, and lipid solvents. Sensitivity to pH is determined by subjecting a virus to a pH of 3.0. Loss of titer greater than one log10 indicates that the virus is pH sensitive and acid labile. Heat treatment of a virus for 30 min at 50~ is a test of thermal stability. If the virus survives this treatment, with no more than a one log10 loss of infectivity, it is considered to be thermostable. Treatment with lipid solvents, commonly ether and Despite the similarities among the physical properties of these families of viruses, relatively few general rules for freezing or freeze-drying viruses can be made. For example, it is common to use membrane-stabilizing agents to preserve viruses with a lipid envelope. Picornaviruses, which lack a lipid membrane, maintain infectivity in the presence of MgC12. However, the optimal conditions for freezing specific viruses need to be determined empirically. Significant reduction of morbidity and mortality for many viral infections has been accomplished by the successful application of viral vaccines. In the case of smallpox, eradication of this important human pathogen has been realized. Polio has now been targeted as the next viral disease that can be eradicated by immunization. Vaccine production requires the use of the "seed lot system" for consistent reproduction and quality control of vaccine lots. Vaccine virus seeds are usually frozen at two passage levels prior to vaccine lot production and these stocks of virus are designated the master seed and the production or working seed, respectively. Multiple aliquots of these seeds are prepared for reference as well as for characterization and standardized testing for possible contaminants. Finally, it is also important to distribute the storage of the master virus seed and working seed among multiple freezers in order to be assured of preserving part of the stock in the event of a mechanical failure. The seed lot system establishes a viral stock that can be standardized and provides a source of viruses so that vaccine lots manufactured over decades will be closely related to the original seed lot. This is especially important for live-attenuated vaccines that in most cases have been derived by multiple serial passage of a clinical isolate of a virus. Deviation from the attenuated passage number may result in undesirable phenotypic changes of the attenuated vaccine virus. This was the case with yellow fever vaccine when uncontrolled passage of this virus resulted in a vaccine with a higher than normal incidence of side reactions (Fox et al., 1942) . Presently, researchers are using the tools of molecular biology to produce a new class of recombinant viral agents that will be used for immunization or genetic therapy. For example, mammalian retroviruses that integrate into the host cell genome are being developed as vectors for delivery of human DNA sequences. The newly acquired DNA is expressed in the form of protein(s) that may be deficient in the host cell or may require de novo synthesis. This form of genetic therapy has been demonstrated routinely in vitro and in vivo in animal experiments and is being proposed as therapy for some human genetic diseases (for review, see Roemer and Friedmann, 1992) . In addition, recombinant viral vectors are also paving the way for a novel type of therapy in which cancer cells can be specifically targeted for infection by a recombinant virus; following infection the inserted gene is expressed and the gene product selectively makes the infected cancer cells more susceptible to subsequent chemotherapy or to natural immune surveillance in the host (Collins et al., 1992; Culver et al., 1992) . Alternatively, recombinant vectors may express antisense RNA that can bind to and block the activity of the host gene sequences. Successful experiments of this kind require viral vectors that are thoroughly characterized and safe. Seed stocks of the recombinant viral vectors constructed to express the desired nonviral gene sequences must be frozen or freeze-dried in order to be preserved for future experiments and clinical trials. A somewhat similar approach is being developed for vaccination using viral vectors as delivery vehicles for genes important for immunization. For example, one approach uses a highly attenuated vaccinia virus that carries, as part of its genome, various immunogenic proteins that would evoke protection against infection (Moss and Flexner, 1987) . Other viral vectors used similarly include adenovirus and canarypox. Antigens used for viral diagnostic purposes must also be quality controlled and, in general, would also be produced from a virus seed lot system. Variation of quality or quantity of viral antigens in diagnostic kits and reagents may directly influence results in the diagnostic laboratory. Regulation of the manufacture of diagnostics is similar to those for vaccine manufacture. Regulations concerning the manufacture of biological products are published by the United States Public Health Service (21 CFR 600) and outline the requirements for control of reagents and raw materials in the manufacture of diagnostics to screen blood and blood products. For these purposes, it is essential to use viral seed stocks that are appropriately characterized and preserved by freezing. Viability of viruses is usually determined by the use of infectivity assays. These include assays whereby viral cytopathology is measured in cell cultures or, when this is not possible, potency may be determined by inoculation of animals or eggs. By far the most accepted and accurate quantitative measure of viral infectivity is the plaque assay. Most viruses form plaques in susceptible host cells. Generally, after virus inoculation on suitably sensitive cell monolayers, an agar overlay is applied so that viral cytopathology is localized. After an incubation period, viable cells are stained with a vital dye, revealing areas of cell death, the plaque-forming unit. Most viral freezing and recovery experiments use the plaque assay to titer virus before and after treatment as a measure of viral activity. In this way, one can determine the efficiency of the freezing or freeze-drying process. Electron microscopy (EM) has also been used on occasion to visualize the effects of freezing and freeze-drying. These EM experiments are limited by the purity and titer of the viral preparation as well as the subjective interpretation of observations. The success of freezing or freeze-drying of viruses is dependent on the proper preparation of the harvested virus. The source of virus may be animal tissues or organs, animal sera, virus-infected cell cultures, and, on occasion, egg allantoic or amniotic fluids. The harvested tissue will depend on the particular host-range of the virus being grown. Harvesting methods are also dependent on the properties of viral replication. Some viruses, e.g., herpes viruses, stay cell associated whereas other are readily released from the cell into the culture fluids. In general, viruses grown after passage in vivo require disruption of the infected animal tissue or organ by homogenizers or aerosol-controlled blenders. Diluent consisting of buffer or cell culture media is added for release of the virus into the aqueous phase during cell disruption. Depending on the virus, serum may be required as an additive to the diluent for optimal freezing ( Table 2 ). Viruses that are found in blood may be harvested and frozen directly in the serum after separation from blood cells or by cocultivating infected peripheral blood lymphocytes (PBLs) with susceptible cell lines. These preparations may be frozen without further treatment, although it is generally recommended that serum or PBLs be frozen and thawed only once to achieve optimal virus recovery. Similarly, cell culture harvests may require disruption of the cell monolayer as a first step. Cells may be scraped from the culture vessel surface into the culture fluids or these fluids may be removed and the cells treated separately. In either case, cells are disrupted and virus is released. In certain cases, virus-infected cells are harvested for a seed preparation. Gentle scraping and harvesting of these cells into the culture fluids or into flesh buffer or cell culture medium is required. The specific details for optimal harvesting procedures for individual viruses may be found in the literature. Usually animal serum is used as a component of cell culture medium. Fetal bovine serum (FBS) is the most frequently used additive by cell (1979) culturists and virologists because it is nontoxic to most cells and the protective effect during freezing is consistent. Therefore, FBS doubles for cell nutrition as well as a cryoprotectant for freezing and freeze-drying. Concentrations used for cell culturing and growing viruses range from 0.5 to 5% in cell culture medium. In many cases the cell culture medium containing serum constitutes the cyroprotective diluent that will be used for freezing. However, further addition of serum is required for many viruses (see Table 2 ). Infectious materials should be handled according to the biosafety guidelines that apply for each virus. The rate of freezing and the final temperature must be considered for virus preparations being frozen for long-term storage. To maintain viral infectivity throughout this process, the integrity of the viral capsid, the viral envelope (if there is one), and the viral nucleic acid must be preserved. Dimmock (1967) demonstrated that viral infectivity may be reduced by heat damage to both viral proteins and nucleic acid. He showed that the stability of these components varies with elevated temperature so that inactivation at a particular temperature takes place through whichever component is least stable at that temperature. In general, most viruses may be "snap frozen" in a dry ice/ethanol bath prior to storage at temperatures _<-70~ It may be preferable to freeze some viruses, such as cytomegalovirus or varicella-zoster virus, as viable infected cells. In this case, a slow controlled rate of freezing is recommended: cooling should be done at l~ to-40~ and then at 10~ to -90~ (Simione and Brown, 1991) . It has also been reported that influenza and measles virus may benefit from this two-step freezing process (Rowe and Snowman, 1976) . Storage temperature is also critical for some viruses with envelopes, such as influenza virus, respiratory syncytial virus, and measles virus. These all require storage at -65~ or lower for optimal recovery after long-term storage (Rightsel and Greiff, 1967; Greiff et al., 1964; Law and Hull, 1968) . It is common practice for today's virologist to store frozen viruses in ultralow-temperature freezers at temperatures of -65 to -85~ Freezing in liquid nitrogen or its vapor phase, a common practice for preservation of mammalian cells, is not necessary for most viruses. Liquid nitrogen storage may be used as a "backup" system for viral seed repositories. No known conventional virus requires -196~ (liquid) or -150~ (vapor) nitrogen for short-or long-term storage. CRYOPROTECTANTS In one of the first comprehensive studies of freezing and freeze-drying with representative members of most of the RNA and DNA virus families, Rightsel and Greiff (1967) demonstrated the importance of the suspending medium used for freezing and freeze-drying. Medium containing skim milk, calcium lactobionate, normal serum albumin, dimethyl sulfoxide (DMSO), glycerol, or magnesium chloride promoted better freezing and freeze-drying recovery with a range of viruses, especially enveloped RNA viruses. Wallis and Melnick (1968) further demonstrated that DMSO or serum acted as a cryoprotectant for four different enveloped viruses, herpes virus, measles virus, Sindbis virus, and vesicular stomatitis virus. Under the conditions described DMSO performed somewhat better than serum as a cryoprotectant. The nonenveloped viruses, vaccinia, adenovirus, and poliovirus, did not require DMSO or serum for retention of viability during freezing. The authors concluded that enveloped viruses were similar to mammalian cells and that DMSO stabilizes the viral envelope much as it stabilizes the mammalian plasma membrane. However, another member of the herpes virus family, varicella-zoster (VZ), is especially sensitive to freezing and freeze-drying. Grose et al., (1981) used sucrose, potassium phosphate, sodium glutamate, and albumin in a cryoprotective "cocktail" for VZ virus. For both cell-associated and cell-free virus, the cocktail was fully protective, with 100% recovery of infectivity after freezing to -70~ and freeze-drying. Additionally, these authors found that glutamate and albumin could be removed from the cocktail without loss of protection. In contrast, Scott and Woodside (1976) showed that sodium glutamate was essential for the stability of freeze-dried herpes virus. Certain viruses are stabilized by the addition of divalent cations. For example, respiratory syncytial virus was found to be stable for up to 1 month at 4~ following the addition of magnesium sulfate, and the titer of virus was maintained through several cycles of freezing and thawing when MgSO4 was added (Fernie and Gerin, 1980) . Likewise, poliovirus was stabilized in the presence of MgC12 (Melnick et al., 1963) . Alternatively, 300 mM sodium phosphate at pH 6.7 stabilized poliovirus at 0-6~ for up to 12 months (Mauler and Gruschkau, 1978) . Cryoprotectants and the rate of freezing minimize the formation of ice crystals that can damage the viruses; similarly, it is common practice to rapidly thaw frozen viral stocks at a previously determined optimal temperature to minimize ice crystal formation. Freeze-drying, or lyophilization, is a method for preserving viruses and other biological materials by removing water from frozen samples via sublimation. The result is a dried preparation that is usually more stable than wet-frozen preparations at various temperatures and for longer periods. The process is usually divided into three stages: prefreezing, sublimation drying under vacuum (primary drying), and desorption or secondary drying. Prefreezing can be accomplished in special freezers or in the freeze-dryer itself. A freeze-dryer has a vaccuum pump that removes air from a chamber that holds vials of the material to be freeze-dried. Water vapor generated through this process is condensed into a refrigerated trap and is not allowed to enter the vacuum pump. The rate of sublimation can be controlled by regulating the heat transfer to the frozen material. After final drying, inert gas is used to fill the vials and protect the freeze-dried material from the deleterious effects of oxygen. The freeze-drying cycle requires optimization for each virus. A typical cycle that has been used successfully for dengue liveattenuated virus vaccines (all four serotypes) consisted of freezing to -40~ for 2 hours and allowing the condenser to reach a temperature of -60~ (K. Eckels, unpublished data). Vacuum was drawn to 100/zm of Hg, at which time heat was applied at a controlled rate overnight (approximately 18 hours). Final drying occurred at 20-23~ for 2 hours followed by backfilling the vials with dry, sterile nitrogen and capping. Vaccine vials were finally stored at -30~ Another cycle that preserves viral infectivity has been used for dengue candidate vaccines; this consisted of freezing to -60~ overnight followed by raising the temperature to -20, 0, and finally to 10~ over a 2-day period while vaccuum was applied (K. Eckels, unpublished data). In both cases, care was taken to avoid subjecting this thermolabile virus to prolonged periods of high temperatures that could result in inactivation of viral infectivity. Greiff and Rightsel (1967) have successfully freeze-dried influenza and measles virus with cycles extending over a 48-hour period with shelf and product temperatures not exceeding 0~ Greiff (1991) has outlined an approach for the development of a successful freeze-drying cycle. Similarly, by varying drying time, shelf temperature, and vacuum pressure an optimal lyophilization cycle for varicella-zoster vaccine was determined (Bennett et al., 1991) . Freeze-drying methods and cycles used by the American Type Culture Collection (ATCC) for their virus stocks can be found in Simione and Brown (1991) . Stabilizers added to the viral suspension medium for freeze-drying are many of the same used for freezing. These additives promote preservation of viral infectivity by acting as antioxidants and by providing "bulk" to the virus preparation to allow easier reconstitution. Also, they should be nonhygroscopic to avoid moisture contamination of the freeze-dried mate-rial. For vaccines, the stabilizers must also be nonimmunogenic and nonreactive in the vaccine recipient. One of the first stabilizers used for viral freeze-drying was 2.5% gum acacia to preserve the infectivity of vaccinia virus (Rivers and Ward, 1935) . Peptone and normal horse serum were also successfully used for freeze-drying of vaccinia virus (Collier, 1951; Sparkes and Fenje, 1972) . Calcium lactobionate and human serum albumin were cryoprotective for measles virus during freeze-drying (Greiff and Rightsel, 1967) , whereas cell culture medium alone was less protective. A combination of sucrose, phosphate, sodium glutamate, and albumin was used for freeze-drying pseudorabies and herpes viruses (Scott and Woodside, 1976; Calnek et al., 1970) . Varicella-zoster virus was also freeze-dried successfully in this combination of stabilizers, but Grose (1981) found that he could eliminate glutamate and albumin from the mixture and still retain 100% recovery of this very labile virus. DMSO cannot be used for stabilization in freeze-dried preparations because it becomes concentrated to toxic levels (Greiff and Rightsel, 1967) . A group of viruses that requires special attention to stabilizing media is the enteroviruses, for example, poliovirus and hepatitis A virus. Early work with poliovirus demonstrated that it had a high degree of lability when freeze-dried, even with stabilizers. The lability is due to the presence of inorganic salts that may be present in the culture medium. When these are removed by dialysis prior to freeze-drying, losses do not occur (Berge et al., 1971) . The ATCC uses ultrafiltration, a more rapid type of dialysis to remove salts prior to freeze-drying of enteroviruses (Simione and Brown, 1991) . Vaccines that require freeze-drying have special stabilizer requirements that allow them to be used safely in humans. Animal serum cannot be used as a stabilizer and must be completely removed from the vaccine prior to bottling. When a protein substitute is required, human serum albumin has been used successfully for many vaccines. More recently, hydrolyzed gelatin has been used as a replacement for albumin (Table 3 ). Both of these stabilizers are acceptable for human vaccines because they usually do not stimulate deleterious immune responses in the vaccinee. Sugars such as sucrose, lactose, and sorbitol are also used either alone or in combination with a protein stabilizer. Cell culture medium is often used as a buffer because many live vaccines are harvested in their culture medium without further processing. Optimal moisture content of the freeze-dried product needs to be empirically determined for each viral preparation. A good freeze-drying cycle can attain a moisture content of 1% or less in the final product. However, depending on the virus, a higher moisture content may be desirable. A varicella virus vaccine with 6-8% moisture following freeze-drying was found to be more stable than vaccines containing less moisture (Bennett et aL, 1991) . Various viruses have been freeze-dried to approximately 1% moisture and shown to be stable over periods of time at 4~ but better long-term stability can be achieved by storage at -20~ STABILITY Assays for retained infectivity following freeze-drying are done on rehydrated vials of freeze-dried virus. A volume of sterile, distilled water is used to reconstitute the vial, with titration of the virus done immediately in the appropriate plaque or infectivity assay. The best control for baseline infectivity would be the virus harvest that was assayed prior to freezedrying. Often, frozen and thawed specimens are used for baseline titers. Freeze-dried viruses are normally stored at 4~ or -20~ Accelerated stability studies can be done to test thermostability for shorter periods at elevated temperatures. Often an ambient temperature of 25~ or temperatures of 35-37~ are used for accelerated studies. These temperatures are chosen to study thermostability for viruses, mainly vaccines, that may not have continuous refrigeration available up to the time of use. This is a problem for many live viral vaccines that are being used in developing countries (Widdus et al., 1989) . The largest application of freeze-drying of viruses occur for liveattenuated viral vaccines that are thermolabile. The first viral vaccine to be freeze-dried was yellow fever vaccine (Penna, 1956) . Recent advances have resulted in more stable, freeze-dried yellow fever vaccines (Robin et al., 1971; Burfoot et al., 1977) . The WHO standard for stability of this vaccine is no more than one log10 loss of potency held at 37~ for 2 weeks. A recent collaborative study of yellow fever vaccines from 12 manufacturers demonstrated that 7 of 12 vaccines met this requirement. Similar WHO stability standards are in place for live, freeze-dried,measles vaccine. A method to freeze-dry and stabilize trivalent, live, attenuated, oral polio vaccine is being sought so that this vaccine can be delivered and used in developing countries without significant loss in potency. It has been proposed that thermoresistant mutants of polioviruses might serve as the prototype for the development of heat-stable vaccine strains (Kew, 1989) . Alternatively, organic compounds such as WIN 51711 and R 78206, which bind to polivirus VP1, have been found to increase the half-life of poliovirus antigen 50-to 250-fold at temperatures up to 48~ (Rombaut et al., 1991) . These drugs stabilize the conformation of the viral capsid and exhibit a potent antiviral effect that would not make them suitable as stabilizers for a live virus vaccine. In the future, similar compounds could be designed to stabilize but not inhibit the replication of live poliovirus strains. Other licensed, freeze-dried live-viral vaccines are those for mumps and rubella, whereas vaccines for varicella (chicken pox) and cytomegaloviruses are being developed that will require stabilization by freeze-drying. Table 3 lists licensed vaccines as well as those still being tested and the available published data on freeze-drying of these vaccines. American Type Culture Collection 12301 Parklawn Drive Rockville, Maryland 20852 Telephone: 1-800-638-6597 (United States and Canada, only) or 1-301-881-2600 FAX: 1-301-231-5826 Request for international reference materials should be made with a statement of intent from the investigator. The Catalogue of Animal Viruses and Antisera, Chlamydiae, and Rickittsiae ATCC Preservation Methods: Freezing and Freeze-drying Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections Freezing and Drying Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections Biological Product Freeze Drying and Formulation Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections Temperature-Stable Vaccines for Developing Countries World Health Organization Edwards Freeze-Drying Handbook Diagnostic Procedures for Viral, Rickettsial, and Chlamydial Infections ATCC Preservation Methods: Freezing and Freeze-drying Temperature-Stable Vaccines for Developing Countries: Significance and Development Strategies The author would like to thank Dr. Kenneth Eckels for guidance and sharing data on freeze-drying dengue viruses and Drs. Karen Goldenthal, Ron Lundquist, and Hana Golding for reviewing the manuscript.