This assay detects adventitious viruses by in vivo culture techniques. Disrupted cells, culture supernatants or other types of test article (i.e., virus stocks) are inoculated into embryonated hens’ eggs, suckling mice and adult mice. Each of these test systems favors the detection of specific viruses which replicate most efficiently in that system. After 14 days, the organs from the suckling mice are homogenized and passaged into a second set of suckling mice. These mice are observed for 14 days. Inoculated adult mice are observed for 28 days. Embryonated eggs are inoculated by two routes (allantoic and yolk sac.) Fluids harvested from the inoculated eggs are passaged into fresh eggs to amplify viruses, thereby increasing test sensitivity. After incubation, a hemagglutination assay is performed on the harvested allantoic fluids.

This test is used in conjunction with Protocol 30027 ["In Vivo Assay for Viral Contaminants (FDA Requirements)"] in order to meet the current EC regulatory specifications. Standing alone, this protocol does not satisfy the EC requirements.
See also Protocol 30027 above.

Notes
· Whenever possible, submit cells harvested in the conditioned medium at a minimum concentration of 1 x 10⁷ cells/ml. Cells do not need to be cryopreserved. Unprocessed bulk material is also acceptable for this test.
· If culture contains selective reagents (e.g., methotrexate), passage twice in medium free of these additives prior to shipment.

Notes
· If the test article is a cell suspension, a minimum of 1 x 10⁷ cells/ml of conditioned growth medium is recommended.
· If the test article is ascites fluid, protein concentration is left to the client’s discretion. Specify concentration on the Sample Submission Form.
· The infectivity of certain viruses decreases rapidly at temperatures higher than -20°C. Therefore, immediately after collection, samples should be frozen on dry ice in sealed, leakproof plastic vial(s).

Notes
· If the test article is a cell suspension, a minimum of 1 x 10⁷ cells/ml of conditioned growth medium is recommended.
· If the test article is ascites fluid, protein concentration is left to the client’s discretion. Specify concentration on the Sample Submission Form.
· The infectivity of certain viruses decreases rapidly at temperatures higher than -20°C. Therefore, immediately after collection, samples should be frozen on dry ice in sealed, leakproof plastic vial(s).

Notes
· If the test article is a cell suspension, a minimum of 1 x 10⁷ cells/ml of conditioned growth medium is recommended.
· If the test article is ascites fluid, protein concentration is left to the client’s discretion. Specify concentration on the Sample Submission Form.
· The infectivity of certain viruses decreases rapidly at temperatures higher than -20°C. Therefore, immediately after collection, samples should be frozen on dry ice in sealed, leakproof plastic vial(s).

Notes
· Contact the laboratory regarding the submission of material for testing. The patient dose as well as the titer of the material must be provided.
· Approximately three (3) patient doses are required to perform this assay with interference controls.
· This assay can also be modified to assay several different levels of test sample to determine the amount of RCA present.

Notes
· If suspension cells, the appropriate number of viable cells should be sent in T75 or T150 flasks which are filled almost to the top with medium, capped tightly and sealed with Parafilm®. Some air space is required to allow for expansion and contraction of liquid during shipping.
· If anchorage-dependent cells, the appropriate number of subconfluent (50% to 80%) cultures should be sent in T75 or T150 flasks as described above.
· Provide pertinent passage instructions on the Sample Submission Form, as well as sufficient growth medium and supplements for two weeks in culture.
· Provide S.O.P. or protocols for growth conditions (+/- trypsin) and media components. Also list any drugs or special additives that are present in media.

Notes
· If test article is submitted as cryopreserved cells, Cell Growth for Assays (Protocol 30040) should be performed.

This protocol utilizes five passages of the inoculated cultures to enhance the ability of any retroviruses. A sample of the test article (10% of volume being tested) is spiked with GaLV (or supernatant) at a low level FFU/ml to determine whether the test article will inhibit the appearance of a positive result. This sample will be run in parallel with the negative control, test article and positive samples. The original test article (if available) and the cell culture supernatants collected after passage 2 and passage 5 from all samples are tested for the presence of RCRs by the PG4 S⁺L^- assay (as described in Protocol 30165).

The test article cells are co-cultivated with amplification cells (the HEK 293 cells) for 5 passages in culture to increase the ability of any potential retroviruses to replicate. The original test article (cell free supernatant) and the cell culture supernatants collected from all samples after passage 2 and 5 are tested for the presence of RCRs by the PG4 S⁺L^- assay (as described in Protocol 30165).

Notes
· Submission of material for testing:
1% of the MCB or 1 x 10⁸ viable cells (whichever is less). If cells are submitted as cryopreserved, Cell Growth for Assays (Protocol 30040) should be performed.

The test article supernatants are amplified on detector cells for 5 passages in culture to increase the ability of any potential retroviruses to replicate. A sample of the TA
(5-10% of volume being tested) will be spiked with AmuLV stock virus at a low level FFU/ml to determine whether the test article will inhibit the appearance of a positive result. This sample will be run in parallel with the negative, test article and positive samples. The original test article (if available) and the control cell culture supernatants collected post passage 5 (pp5) from all samples are tested for the presence of RCRs by the PG4 S⁺L^- assay (Protocol 30165).

Notes
· This protocol should be used when 5% is less than 100 ml. If 5% is greater than 100 ml, see large scale assay (Protocol 30633).

This protocol is for small scale co-cultivation. It is designed to test cell banks where one percent of the bank is no more than approximately 2 x 10⁶ cells total. For larger banks where one percent of the bank is over 2 x 10⁶ cells total, use protocol 30628. For supernatant testing refer to protocol 30629 for small scale (5% is less than 100 ml) or 30633 for large scale (5% is greater than 100 ml.)

Notes
· Submission of material for testing:
1% or 2x10⁶ of total pooled cells. If cells are submitted as cryopreserved, Cell Growth for Assays (Protocol 30040) should be performed.

The test article supernatants are amplified on detector cells for 5 passages in culture to increase the ability of any potential retroviruses to replicate. A sample of the TA (5-10% of volume being tested) will be spiked with AmuLV stock virus at a low level FFU/ml to determine whether the test article will inhibit the appearance of a positive result. This sample will be run in parallel with the negative control, test article and positive samples. The original test article (if available) and the cell culture supernatants from all samples are collected post passage 5 (pp5) and are tested for the presence of RCRs by the PG4 S⁺L^- assay (as described in Protocol 30165).

Notes
· This protocol should be used when 5% is greater than 100 ml.
· Please inquire as to large volumes.

This protocol is for large scale co-cultivation. It is designed to test cell banks where one percent of the bank is greater than 2 x 10⁶ cells total. For smaller scale testing refer to Protocol 30222. For supernatant testing refer to Protocol 30629 for small scale (5% is less than 100 ml.) Current regulations require that 1% of the cell bank or 1 x 10⁸ cells, whichever is less, be tested.

Notes
· Submission of material for testing:
1% or 10⁸ of total pooled cells. If cells are submitted as cryopreserved, Cell Growth for Assays (Protocol 30040) should be performed.

Notes
· Please note that blood bags are prone to cracking when in direct contact with dry ice. If your sample is stored in blood bags, please ensure that appropriate insulation is used to protect the sample.

Notes
· If cells are submitted as cryopreserved, Cell Growth for Assays (Protocol 30040) should be performed.

Notes
· Provide cell-free supernatant from culture in exponential growth or ascites fluid.
· Sample should be shipped in sealed, leakproof plastic vials.
· For many samples, amplification on Mus dunni cells is recommended prior to testing on PG4 S⁺L^- cells.

Notes
· Provide cell-free supernatant from culture in exponential growth or ascites fluid.
· Sample should be shipped in sealed, leakproof plastic vials.

Notes
· Provide cell-free supernatant from culture in exponential growth or ascites fluid.
· Sample should be shipped in sealed, leakproof plastic vials.

Notes
· Supply information on the preparation of all products to be tested.
· If sterility and mycoplasma assays are also requested, samples may be taken from the 500 ml bottle.

Notes
· Whenever possible, submit cells harvested in conditioned medium at a minimum concentration of 1 x 10⁷ cells/ml. Cells do not need to be cryopreserved.
· If cells are submitted as cryopreserved, Cell Growth for Assays (Protocol 30040) should be performed.

The test will follow a general outline as described in the 9 CFR and use three concentrations of the sample to provide a semi-quantitative answer. The detector cells are observed for 21 days for the development of characteristic changes in morphology attributable to replication of viral agents. A blind passage is made on day 21 and the cells are isolated, lysates prepared and fresh BT cells are inoculated and observed for an additional 14 days. Upon completion of the cultivation period, the cells are stained with a cytological stain and are again observed microscopically for changes in morphology.

A final test is run to detect extraneous viral agents by the fluorescent antibody technique as described in the 9 CFR Section 113.47. In this assay, the test article inoculated BT cultures are tested with an anti-BVD antibody. Positive controls consist of negative control monolayers inoculated with 10-30, and 100-300 infection units (IU) of the specific virus. Interference controls inoculated with 10-30, and 100-300 infection units (IU) are also run with each assay.

Notes
· Whenever possible, submit cells harvested in conditioned medium at a minimum concentration of 1 x 10⁷ cells/ml. Cells do not need to be cryopreserved.
· If cells are submitted as cryopreserved, Cell Growth for Assays (Protocol 30040) should be performed.

Notes
· Whenever possible, submit cells harvested in conditioned medium at a minimum concentration of 1 x 10⁷ cells/ml. Cells do not need to be cryopreserved.
· If cells are submitted as cryopreserved, Cell Growth for Assays (Protocol 30040) should be performed.

Notes
· Container/Volume
Trypsin in powder form: 2 x 6 g 
Trypsin in liquid form: 2 x 50 ml
· Shipment Temperature
Powder: Ambient, or as suggested by manufacturer
Liquid: Frozen on dry ice

As described in the 9 CFR the introduction of porcine derived tissues or products, test article cells, culture media supplements or trypsin to a simian kidney cell line (Vero), a porcine cell line (swine testis [ST] cells) and a bovine cell line (bovine turbinate [BT] cells allows the detection of a wide range of viruses. The detector cells are observed for at least 21 days with subculture performed at 7 and 14 days for the development of characteristic changes in morphology attributable to replication of viral agents. Upon completion of the cultivation period, the cells are stained with a cytologic stain and are again observed microscopically for changes in morphology. Certain viruses may replicate in cells with the development of little or no cytopathic effects. The presence of some of these viruses may be detected by their ability to absorb erythrocytes (chicken and guinea pig) to the surface of infected cells. Accordingly, a hemadsorption assay is run on viable cells at the conclusion of the 21 day (or longer) observation period.

A final test is run to detect extraneous viral agents by the fluorescent antibody technique as described in the 9 CFR Section 113.47. Inoculated cultures are tested for the presence of bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), porcine adenovirus (PAV), transmissible gastroenteritis (TGE) [also know as procine coronavirus type 1], hemagglutinating encephomeyelitis virus (HEV), reovirus (RV), and rabies virus.

Notes
· Whenever possible, submit cells harvested in conditioned medium at a minimum concentration of 1 x 10⁷ cells/ml. Cells do not need to be cryopreserved.
· If cells are submitted as cryopreserved, Cell Growth for Assays (Protocol 30040) should be performed.

It has been proven that pig cell lines in culture release retrovirus particles and these particles are capable of infection, replication and serial transfer in a range of human cell lines. To test for these porcine endogenous retroviruses, an eight week co-cultivation assay will be performed with the test article cells and HEK-293 cells, a human cell line, in an effort to infect the HEK-293 cells with any retrovirus being released from the test article. The supernatant from these cultures will be sampled at various time points in the assay and tested for retrovirus by RT assay at the end of the co-cultivation period.

Notes
· If test article cells are submitted as cryopreserved cells, Cell Growth for Assays (Protocol 30040) should be performed.

Notes
· Whenever possible, submit cells harvested in conditioned medium at a minimum concentration of 1 x 10⁷ cells/ml. Cells do not need to be cryopreserved. If cells are submitted as cryopreserved, Cell Growth for Assays (Protocol 30040) should be performed.
· Supply information on components and preparation of your media supplements.
· For serum samples, if sterility and mycoplasma assays are also requested, samples may be taken from the 500 ml bottle.

Notes
· Whenever possible, submit cells harvested in conditioned medium at a minimum concentration of 1 x 10⁷ cells/ml. Cells do not need to be cryopreserved. If cells are submitted as cryopreserved, Cell Growth for Assays (Protocol 30040) should be performed.

This procedure is used to detect and quantitate virus particles. The test article is mixed with a known concentration of latex beads and the mixture is centrifuged onto a copper grid. Samples are stained and visualized by transmission electron microscopy. Concentration of virus particles is determined by comparison to the reference number of latex beads.

Notes
· Provide cell-free supernatant from culture in exponential growth, ascites fluid, or production lot material.
· Sample should be shipped in sealed, leakproof plastic vials.

Notes
· If suspension cells, the appropriate number of viable cells should be sent in T25 or T75 flasks that are filled almost to the top with medium, capped tightly and sealed with Parafilm^®. Some air space is required to allow for expansion and contraction of liquid during shipping.
· If anchorage-dependent cells, the appropriate number of subconfluent (50% to 80%) cultures should be sent in T25 or T75 flasks as described above.
· Provide pertinent passage instructions on Sample Submission Form, as well as sufficient growth medium and supplements to maintain cells for four to five weeks in culture.
· Provide S.O.P. or protocols for growth conditions (+/- trypsin), media components and freezing conditions. Also list any drugs or special additives present in the media.
· For cryopreserved cells, Cell Growth for Assays (Protocol 30040) should be performed.

Notes
· If suspension cells, the appropriate number of viable cells should be sent in T25 or T75 flasks that are filled almost to the top with medium, capped tightly and sealed with Parafilm^®. Some air space is required to allow for expansion and contraction of liquid during shipping.
· If anchorage-dependent cells, the appropriate number of subconfluent (50% to 80%) cultures should be sent in T25 or T75 flasks as described above.
· Provide pertinent passage instructions on Sample Submission Form, as well as sufficient growth medium and supplements to maintain cells for four to five weeks in culture.
· Provide S.O.P. or protocols for growth conditions (+/- trypsin), media components and freezing conditions. Also list any drugs or special additives present in media.
· For cryopreserved cells, Cell Growth for Assays (Protocol 30040) should be performed.