Virus preparation

Since the introduction of the mumps vaccine in 1967 there has been a 99% reduction in reported cases. The mumps vaccine is a live virus preparation of the leryl-Lynn strain. It produces a sub-clinical, non-communicable infection following vaccination. Single doses of mumps vaccine will elicit immunity in 75% to 95% of individuals. Vaccine-induced immunity lasts for more than 30 years. 

Calberg-Bacq, C.-M. Francois, C., Gosselin, L., Osterrieth, P. M. and Rentier-Delrue, F. 1976. Comparative study of the milk fat globule membrane and the mouse mammary tumour virus prepared from the milk of an infected strain of Swiss albino mice. Biochim. Biophys. Acta 419, 458-478. 

Milne, R. G. and Luisoni, E. (1977) Rapid immune electron microscopy of virus preparations, in Methods in Virology (Maramorosch, K. and Koprowski, H. eds.), Academic Press, New York, Vol. 6, pp. 265-281. 

Remove the medium from a cell monolayer and wash the mono-layer with BSS or PBS (Appendix 1) to remove inhibitors (antibodies) which may be present in the medium. Apply the virus preparation suspended in a small volume of BSS or PBS and allow 30-60 min for adsorption. Replace or supplement the salt solution with fresh medium. 

Following growth of the virus in batch cell culture the culture fluid containing virus particles and cell debris is clarified by low-speed centrifugation or filtration to remove the latter. The virus preparation (inactivated when required) must then be tested for... 

Preparative RP-HPLC separation of viral proteins was performed on a 4.6-X 250-mm Jupiter C4 column at 50°C. Approximately 6mL of the column-purihed virus preparation ( 1 x 10 particles/mL) was loaded onto the column. Each chromatographic peak was collected manually for further MS characterization. 

Cumulative experience from 106 monkey-years and 23 human-years of retroviral gene therapy had revealed no side-effects or malignancies. Recently, however, three monkeys used in clinical trials developed T cell lymphomas which were traced to a helper virus-contaminated retroviral vector preparation (Anderson, 1992a). Contamination of defective viral vector stocks by infectious viruses produced in helper cells was a serious problem with the early version of the helper cells. New helper cell lines which greatly reduce the probability of virus production have been developed (Miller, 1990). Eurther, it is also imperative to test the lots of vector virus preparations used in therapy for the presence of other infectious viruses. The probability... 

The data also demonstrated that the subtle differences in viruses should not be underestimated. The different HAV strains/clones and the method of virus preparation had a significant impact on virus clearance. Virus stocks used in virus validation studies are produced in cell culture and the behavior of tissue culture derived viruses may be different from that of native viruses. Laboratory adapted strains of viruses may also have impre-dicted properties, such as association with lipids, which... 

Resuspend virus pellets in 8 mL RPMI-1640 medium with 1.5% FCS. This virus preparation represents a 250-fold concentration. 

Determine the transforming titers of the virus preparation by end-point titration on human umbilical cord blood lymphocytes (see Subheading 3.3.2.), and calculate by the method of Reed and Muench (8). 

Three years after smallpox eradication, in 1980, the World Health Organization (WHO) recommended that aU countries cease smallpox vaccination. In addition, WHO recommended that all laboratories destroy stocks of the virus or transfer them to either of two WHO reference laboratories, the Institute of Virus Preparations in Moscow, Russia, or the Centers for Disease Control and Prevention (CDC) in the United States. However, there may have been stocks of virus elsewhere (26,27). Although the WHO Advisory Committee on Variola Virus research recommend eradication of aU smallpox stocks by June 30,2002, the WHO Health Assembly has delayed this each year because of concerns that stocks of virus are needed for continued study (28). 

By early 2004, 15 million doses of smallpox vaccine, Dryvax, were available in the United States. Dryvax, a Wyeth product, is a live virus preparation of vaccinia. The vaccine comes as a lyophilized (freeze-dried) powder in 100 dose vials containing the antibiotics polymyxin B, streptomycin, tetracycline, and neomycin. Fifty percent glycerin, containing a small amount of phenol as a preservative, serves as the diluent for reconstitution (25). Studies have shown that diluting the vaccine in a 1 5 ratio could expand the supply without reducing vaccine efficacy (25). In addition, 200 million antibiotic-free doses are in production. The CDC Web site has detailed directions on how to reconstitute and administer the vaccine at http //www.bt.cdc.gov/agent/smallpox/vaccination/ administration.asp. 

Fig. 2.4 Flowchart of the steps for rAAV production as described in Protocol 1. HEK293 cells are expanded, transfected, and harvested at 60h posttransfection. The cells are lysed, and loaded onto either a cesium chloride gradient or an iodixanol gradient to separate infectious virions from empty capsids. Virus is purified using either heparin affinity chromatography or ion-exchange chromatography. Virus preparations are formulated and concentrated, and characterized...

Dialyze the virus preparation into storage buffer or suitable formulation and freeze at -20°C. 

Dialyze the virus preparation into storage buffer (50 mM Tris, lOOmM NaCl, pH 8.0) or suitable formulation aliquot, and freeze at -20°C. 

As the aggregated particles become larger than 300 nm, other techniques become necessary for their detection. One useful option is that of dynamic hght scattering (DLS), which also provides size information based on the diffusivity (and can be calibrated with polystyrene beads). The DLS analysis of a non-aggregated and an aggregated virus preparation is shown in Fig. 6.6. 

Dryvax powder for injection dried, calf lymph type live-virus preparation of vaccinia virus (Polymyxin B sulfate, dihydrostreptomycin sulfate, chlortetracycline hydrochloride, and neomycin sulfate are added in trace amounts). The reconstituted vaccine contains approximately 100 milUon infectious vaccinia viruses/mL)... 

The remaining vaccinia vaccine licensed in the United States (Dryvax, manufactured by Wyeth, Philadelphia, Pa.) is a live, infectious virus prepared from calf lymph. Like all smallpox vaccines that were marketed in the United States, it derived from the NYCBOH strain and contains 108 plaque-forming units per milliliter. Current vaccinia vaccine stocks (> 12 million doses) are held by the CDC. It must be noted that the potency of several lots of this lyophilized vaccine has fallen. Pharmaceutical companies in the United States lack interest in manufacturing new lots of vaccine, owing to the absence of a profitable retail market, antiquation of calf-lymph production techniques and facilities, and the manufacturer s legal liability for vaccination complications. 

Fig. 17.9. Spectra of virus preparations and normal host components processed by 3 cycles of differential centrifugation (,4) influenza A. A, B, mumps, and normal host components (B) Newcastle disease (C) psittacosis, lymphogranuloma venereum, meningopneumonitis, feline pneumonitis, and mouse pneumonitis (f>) vaccinia and fowl-pox. (Benedict, 1955.)...

In MCMV infected macrophages and fibroblast, M78 is mainly localized inside the cells in vacuole like structures which colocalizes with markers for medial and peripheral Golgi bodies. Furthermore, M78 is present in virus preparations, and is as a possible virion envelope protein, presumably delivered to newly infected target cells. M78 has been proposed to be involved in modulating the intracellular milieu and regulating the transcription of selected immediate early viral mRNAs. ... 

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