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Myxoma virus (MYXV) is a member of the Poxviridae family and prototype for the genus Leporipoxvirus. It is pathogenic only for European rabbits (Oryctolagus cuniculus), in which it causes a lethal disease called myxomatosis, and for two North American species, Sylvilagus audubonni and Sylvilagus nuttalli, in which it causes a less severe disease. MYXV replicates exclusively in the cytoplasm of the host cell, and its genome encodes 171 open reading frames. A number of these genes encode proteins that can interfere with or modulate host defense mechanisms, and several show promise in a clinical setting. Serp-1, for example, is in clinical trials for treatment of acute unstable coronary syndromes, and M-T7 has been shown to inhibit inflammatory responses in rabbit models of balloon angioplasty arterial injury. Additionally, although MYXV is not infectious in humans, it is able to productively infect a number of human cancer cell lines, but not normal human cells, and has also been shown to increase survival time in mouse models of human glioma. These characteristics suggest that MYXV could prove to be a viable therapeutic agent in a variety of clinical settings, including as an anti-inflammatory or anti-immune therapy, or as an oncolytic agent. MYXV is also an excellent model for poxvirus biology, pathogenesis, and host tropism studies. It is easily propagated in a number of cell lines, including adherent cells and suspension cultures, and minimal purification is required to provide a stock useful for in vivo and in vitro studies.
Myxoma virus (MYXV) is a poxvirus that is the prototype of the genus Leporipoxvirus, and a member of the Poxviridae family. It is pathogenic only for European rabbits and two North American species of rabbits, and is harmless to humans. However, MYXV has been shown to productively infect a variety of human cancer cell lines originated from a diverse group of tissues (Sypula et al., 2004), and therefore has the potential for development as an oncolytic virus useful in treatment against a variety of cancers. Furthermore, MYXV is adept at evading and interfering with the host immune response and might serve as a source of immunomodulatory proteins that can be used as therapeutic agents in a variety of clinical settings (Lucas and McFadden, 2004). MYXV has also been established as a model to study poxvirus biology, pathogenesis, and host tropism.
Producing pure, high titer stocks of virus thus becomes critical for studying MYXV in the laboratory and developing it as a useful clinical agent. The virus is easily propagated on adherent cells or in suspension cultures, and grows in several cell lines, including RK13 (rabbit kidney epithelial), BHK-21 (baby hamster kidney), BGMK (Buffalo green monkey kidney), Vero (African green monkey kidney epithelial), BSC-40 (African green monkey kidney), and CV-1 (African green monkey kidney fibroblast) cells. Minimal purification is needed to provide a stock that is appropriate for both in vitro and in vivo work. The following protocols, which include propagation on both adherent and suspension cells, describe these methods for propagating, purifying, and quantifying stocks of MYXV that are untagged or that express fluorescent proteins such as green fluorescent protein (GFP), red fluorescent protein (RFP), tomato Red (tdRed), or other fluorescent proteins.
CAUTION: Although MYXV is not pathogenic for humans, it is considered a BSL-2 agent, and precautions for working with BSL-2 agents, including the proper use of personal protective equipment and handling in a Class II biosafety cabinet, are indicated. For more information regarding the safe handling of BSL-2 agents, refer to the U.S. Department of Health and Human Services’ “Biosafety in Microbiological and Biomedical Laboratories” (http://www.cdc.gov/od/OHS/biosfty/bmbl5/BMBL_5th_Edition.pdf).
Furthermore, additional precautions may be required by individual Institutional Biosafety Committees when working with other poxviruses in concert with MYXV because of concerns for the possibility of recombination between poxviruses. Potential institutional requirements may include, for instance, propagating two different poxviruses in separate incubators. Contact the local institutional biosafety office for policies and advice.
This protocol describes propagation of MYXV on adherent CV-1, BSC-40, or BGMK cells.
In a 37°C water bath, preheat the MEM medium, PBS, and 1X trypsin-EDTA-PBS.
MYXV can also be grown in suspension cultures of BHK-21 cells. Adherent BHK-21 cells used to seed spinner flasks are grown in dishes in DMEM; when transferred to the spinner flasks, the cells are cultured in IMDM medium, an enriched medium that is ideal for culturing cells at high density. Bellco makes a spinner flask that has an impeller, a built-in suspended rotating magnet, and side arms with removable caps that allow for easy transfer of media and virus in and out of the flask. These caps are left loose but secure to allow gas exchange in a CO2 incubator. This alternate protocol is adapted for following the growth of MYXV that is tagged with a fluorescent protein and is not intended for growing untagged virus.
MYXV stock can also be amplified on BGMK cells using 4–6 confluent 150cm2 flasks. This method is useful for preparing a smaller crudely purified stock of virus after making, for example, a new recombinant virus. The virus preparation will contain cellular debris that cannot be removed by centrifugation and may inhibit certain applications.
Virus is first homogenized and then pelleted through a 36% w/v sucrose cushion. This level of purification is sufficient for many protocols, including in vivo work. However, for some procedures, a more purified preparation is necessary. An assay that measures cytokine induction, for example, would require the use of highly purified virus that is free from any cellular components; this assures that any activity measured is solely a result of viral factors. To obtain a level of purification sufficient for such assays, virus that is first pelleted through 36% sucrose is then banded over a 24–40% sucrose step gradient, collected, and concentrated.
MYXV forms foci, areas of clumped infected cells, rather than plaques, and titer is expressed in foci forming units/ml of culture (FFU/ml). Determining the titer can be done in a variety of ways, depending on whether the virus is untagged, has a fluorescent tag, or other reporter gene. For untagged virus, the infected cell monolayer and foci are stained with crystal violet (Kotwal and Abrahams, 2004). For a fluorescent protein-tagged MYXV, titer is determined by counting foci under a fluorescent microscope, and for titration of MYXV having the reporter gene lac Z (vMyxlac), X-gal staining can be used.
Titrating MYXV that is tagged with a fluorescent protein such as GFP, RFP, or tdRed is readily accomplished by counting foci under a fluorescent microscope. View each well of the plate under the microscope and count the foci in wells that have <100 foci. It should be possible to get counts for at least two wells. Calculate the titer (FFU/ml), which = number of foci × dilution × 2. Average the FFU/ml for each countable well.
If the virus is not tagged with a fluorescent protein, the cells are stained with crystal violet and foci counted under an inverted microscope.
For titration of MYXV having the reporter gene lac Z (vMyxlac), X-gal staining can be used for β-galactosidase activity. After staining, blue foci can be counted with the naked eye (Stanford et al., 2007)
Use ultra-pure pyrogen free water in all recipes and protocols steps. Information on common stock solutions can be found in APPENDIX 2A, and for suppliers, refer to SUPPLIERS APPENDIX.
Per 500 ml:
Per 500 ml:
MYXV is a rabbit specific poxvirus that causes myxomatosis, a devastating and lethal disease in European rabbits (Oryctolagus cuniculus). It has also been shown to cause disease in two North American species, Sylvilagus audubonni and Sylvilagus nuttalli, but in these rabbits pathogenesis differs from that seen with classical myxomatosis (Regnery, 1971; Silvers et al., 2009). Myxomatosis is characterized by severe systemic cellular immunosuppression which leads to respiratory complications and death. However, in its evolutionary hosts, which belong to the genus Sylvilagus, and include the South American tapeti (Sylvilagus brasilensis) and the North American brush rabbit (Sylvilagus bachmani), MYXV is slowly cleared and causes only mild disease and a benign tumor at the infection site (Stanford, Werden, and McFadden, 2007; McFadden, 2005). To date, neither MYXV infection nor seroconversion in humans has been observed, even where the virus is widely distributed in rabbits and in carrier mosquitoes (Stanford and McFadden, 2007). Because of its high species specificity, MYXV was used in Australia in the 1950’s as a biological control for rabbits that were introduced in 1859, and which quickly multiplied and caused devastating damage to agriculture and the environment (Fenner and Ratcliffe, 1965).
Like other members of the poxvirus family, MYXV replicates exclusively in the cytoplasm of infected cells, and its genome consists of a single double stranded DNA (dsDNA) which encodes 171 open reading frames (Cameron et al., 1999). The central part of the genome encodes approximately 100 essential genes that are conserved among the members of poxvirus genera. The rest of the genes, including two copies each of the 12 genes that map within the terminal inverted repeats, encode proteins that interfere with and modulate host defense mechanisms. A number of these proteins share a sequence similarity with host cellular genes, suggesting a coevolutionary path (Johnston and McFadden, 2003). Some, called viroceptors, are secreted and able to bind specific ligands such as TNF, for example. Others, known as virokines, are also secreted, and imitate host immune inhibitors, while viromitigators function as host range factors that inhibit apoptosis (Johnston and McFadden, 2003; Kerr and McFadden, 2002). These characteristics give MYXV possible utility in a number of therapeutic settings. One of the MYXV-encoded immunomodulatory proteins, Serp-1, is now in clinical trials (conducted by VIRON Therapeutics, Inc.) for acute unstable coronary syndromes (unstable angina and small heart attacks). The M-T7 protein of MYXV, a secreted glycoprotein that inhibits rabbit gamma interferon, has also been shown to inhibit inflammatory responses in rabbit models of balloon angioplasty injury to arteries (Liu et al., 2000), and it is likely that a variety of other immunomodulatory proteins can be developed as anti-inflammatory or anti-immune therapeutics.
MYXV is also able to productively infect a variety of human cancer cell lines, but not normal human cells, and in in vivo studies, MYXV has also been shown to provide long-term survival in mouse models of human glioma (Lun et al., 2005). Although it was previously unknown whether MYXV could reliably target human primary cancer cells while sparing normal cells, in recent studies, normal and leukemic hematopoietic stem and progenitor cells (HSPCs) were treated with MYXV, resulting in infection of a significant number of the leukemic cells, and in only a small number of the normal cells. Additionally, normal HSPCs from healthy human bone marrow treated ex vivo with MYXV and xenotransplanted into sublethally irradiated NOG mice readily engrafted, and no evidence for MYXV replication was found in tissues at necropsy (Kim et al., 2009). These results open the door for MYXV to potentially join vaccinia virus, another poxvirus, as a new oncolytic virotherapy candidate.
Although contamination with bacteria, fungi, and yeast is common in cell culture, there are preventive measures that can be taken. Reduce contamination by using careful aseptic technique and performing all cell culture activities in a biosafety cabinet; regularly wipe down cabinet and equipment surfaces with 70% ethanol. Change gloves often, keep the time flasks and media bottles are open to a minimum, and if possible, use individually wrapped sterile disposable pipettes. Antibiotics such as penicillin and streptomycin may be used routinely in media; alternatively, an antibiotic-antimycotic agent (Invitrogen cat. no. 15240-062) containing a 100X concentration of penicillin and streptomycin, as well as 25 µg amphotericin B/ml, may be used. Commercially prepared media is recommended, but if media is prepared in the lab from a powdered mix, it is important to use very clean glassware and ultra-pure, pyrogen-free water when preparing it.
Mycoplasma is a more difficult contaminant to detect and treat, because unlike other types of contamination, it is not visible under the microscope, nor is it sensitive to antibiotics commonly used in cell culture. It can affect both cell growth and the propagation of virus in those cells, so it is important to test cultures regularly and treat if necessary.
A PCR-based test kit for mycoplasma detection is available from Sigma (cat. no. MP0025-1KT), and should cultures test positive, Plasmocin (InvivoGen), an antibiotic effective against mycoplasma, is available in concentrations for both prophylaxis and treatment. For treatment of contaminated cultures, cells should be split into media containing Plasmocin (cat. no. ant-mpt) at a concentration of 25µg/ml, maintained at that concentration for two weeks, and retested. If mycoplasma is still present, an additional one week treatment and/or increase of Plasmocin concentration to 37.5µg/ml may be necessary. For prophylactic maintenance of cell cultures, Plasmocin (cat. no. ant-mpp) is used in media at a concentration of 5µg/ml, but is not typically used unless mycoplasma is a recurring problem.
Mycoplasma is a leading cause of low virus yield when propagating MYXV; cells should be routinely tested and, if necessary, treated. It is also important to determine an optimum multiplicity of infection (MOI) when infecting cells, which for MYXV is MOI = 0.02 – 0.05. A CO2 level of 5% and temperature of 37°C should be carefully maintained in incubators, as well.
Virus yields of 1 × 1010 ffu/ml are typical with Basic Protocol 1 and Alternate Protocol 1 after purification. Yields from Alternate Protocol 2 range from 1 × 108 to1 × 109 ffu/ml.
For Basic Protocol 1 and Alternate Protocols 1 and 2, most time will be spent waiting for cells to reach confluency after seeding. It may be convenient to seed cells in the middle of the week to be ready to inoculate with virus on a Friday; the infection can be incubated over the weekend and virus harvested on a Monday in most instances. Harvesting virus involves only a 5 min spin to collect cells from Basic Protocol 1, and a 15 min spin for Alternate Protocol 1. If following Alternate Protocol 2, virus is collected by scraping cells from flasks, followed by a 10–15 minute centrifugation step, and 20 minute cell-swelling step. Harvested virus from all three protocols can then be frozen and purified at a later time. For Basic Protocol 1 and Alternate Protocol 1 this involves homogenizing virus, pelleting through a sucrose cushion, and purifying on sucrose step gradients, all of which can be easily accomplished in a single day. However, the sucrose step gradients must be prepared the day before needed and stored at 4°C overnight prior to use. For Alternate Protocol 2, the crude purification can be completed in a morning and includes Dounce homogenizing, two 5 minute spins, and a 1 hr centrifugation step to pellet virus.
Titrating virus is done over several days. On day one, cells are seeded into a 6-well dish, and on day two, virus is diluted and applied to the plate for a 1 hr incubation, followed by removal of the inoculum and addition of media. This process takes about 2 hrs, and is followed by a 2–5 day incubation. Staining and/or counting foci can be easily accomplished in about 2 hrs unless the virus requires X-gal staining. The wash and staining process takes less than an hour, but it can take from 4–8 hr for color to develop so that foci can be counted.
MYXV is stable for a number of years when stored at −80°C, but a reduction in titer by one log is likely after 10 years or more. It should be stored in 0.2–1.0 ml aliquots in cryotubes, as repeated freezing/thawing will reduce the titer by one or two logs. Aliquot in an appropriate volume according to specific needs.