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Herpesviruses Assays

M. Prichard, Ph.D., University of Alabama Birmingham

E.        Laboratory Procedures for Determining Antiviral Efficacy and Toxicity for Herpesviruses

1.     Preparation of compounds for in vitro testing

        The letter of agreement with the drug sponsor included in the RFP indicates that the sponsor will provide pertinent information regarding structure, molecular weight, solubility, toxicity, and any handling precautions the sponsor is aware of. After receipt of the compound, they are entered into a log book and into the drug screening inventory data base, and are stored according to the location assigned. The compounds are then weighed using an analytical balance and reconstituted in the appropriate vehicle. It is critical at this point that solubility data be provided by the sponsor so drug is not wasted determining the solubility. If the compound is water soluble, it will be dissolved in tissue culture media without serum at 1 mg/ml and diluted for use as indicated below in the description of the assay system. If the compound is not water soluble, then it is automatically dissolved in DMSO at a concentration of 10 mg/ml and diluted for use in each assay. This has worked very well for both the assays in HFF cells as well as the lymphoblastic cells as DMSO is not toxic at the final concentration utilized (<1.0%).

2.     Screening and confirmation assays for HSV, VZV, and CMV

a.     Preparation of Human Foreskin Fibroblast Cells

        Newborn human foreskins are obtained from UAB or Brookwood Hospital as soon as possible after circumcisions are performed and placed in minimal essential medium (MEM) containing vancomycin, fungizone, penicillin, and gentamicin, at the usual concentrations, for four hours at room temperature. The medium is then removed, the foreskin minced into small pieces and washed repeatedly until red cells are no longer present. The tissue is then trypsinized using trypsin at 0.25% with continuous stirring for 15 min at 37C in a CO₂ incubator. At the end of each 15 min period, the tissue is allowed to settle to the bottom of the flask. The supernatant containing cells is poured through sterile cheesecloth into a flask containing MEM and 10% fetal bovine serum (FBS). The flask containing the medium is kept on ice throughout the trypsinizing procedure. After each decanting of cells, the cheese cloth is washed with a small amount of MEM containing serum. Fresh trypsin is added each time to the foreskin pieces and the procedure repeated until no more cells become available. The cell-containing medium is then centrifuged at 1000 RPM at 4C for ten min. The supernatant liquid is discarded and the cells resuspended in a small amount of MEM with 10% FBS. The cells are counted using a Coulter Counter and then placed in an appropriate number of 25 cm² tissue culture flasks. As cells become confluent and need trypsinization, they are gradually expanded into 175 cm² flasks. The cells are maintained on vancomycin and fungizone to passage three. Cell lines will be tested periodically for the presence of mycoplasma contamination using the Hoechst fluorescent stain for mycoplasma DNA.  Cells are utilized only until passage 10.

b.     Cytopathic Effect Inhibition Assay.

        Low passage (3-10) human foreskin fibroblast (HFF) cells are trypsinized, counted, and seeded into 96 well tissue culture plates at a cell concentration of 2.5 x 10⁴ cells in 0.1 ml of MEM supplemented with 10% FBS. The cells are then incubated for 24h at 37^oC in a 5% CO₂ - 95% air, 90% humidified atmosphere. The media is then removed and 100 µl of MEM containing 2% FBS is added to all but the first row. In the first row, 125 µl of media containing the experimental drug is added in triplicate wells. Media alone is added to both cell and virus control wells. The drug is the first row of wells is then diluted serially 1:5 throughout the remaining wells by transferring 25 µl using a Beckman Bio-Mek Liquid Handling Machine. The plates are then incubated for 60 min and 100 µl of an appropriate virus concentration added to each well, excluding cell control wells which received 100 µl of MEM. For HSV-1 and HSV-2 assays, the virus concentration utilized is 1000 Plaque Forming Units (PFU) per well. For CMV and VZV assays, the virus concentration added is 2500 and 1000 PFU per well, respectively. The plates are then incubated at 37^oC in a CO₂ incubator for three days for HSV-1 and HSV-2, 10 days for VZV, or 14 days for CMV. After the incubation period, media is aspirated and the cells stained with a 0.1% crystal violet in formalin solution for 4h. The stain is then removed and the plates rinsed using tap water until all excess stain is removed. The plates are allowed to dry for 24h and the amount of CPE is each row determined using a BioTek Multiplate Autoreader. EC₅₀ and IC₅₀ values are determined by comparing drug treated and untreated cells using a computer program.

c.     Plaque Reduction Assay for HSV-1 and HSV-2 using Semi-Solid Overlay.

        Two days prior to use, HFF cells are trypsinized, counted, and plated into six well plates and incubated at 37^oC with 5% CO₂ and 90% humidity. On the date of assay, the drug is made up at twice the desired concentration in 2x MEM and then serially diluted 1:5 in 2x MEM to give six concentrations of drug. The drug concentrations utilized are usually 200 µg/ml down to 0.06 µg/ml. The virus to be used is diluted in MEM containing 10% FBS to a desired concentration which will give 20-30 plaques per well. The media is then aspirated from the wells and 0.2 ml of virus is added to each well in triplicate with 0.2 ml of media being added to drug toxicity wells. The plates are then incubated for 1h with shaking every fifteen min. After the incubation period, an equal amount of 1% agarose is added to an equal volume of each drug dilution. This will give final drug concentrations beginning with 100 µg/ml and ending with 0.03 µg/ml and a final agarose overlay concentration of 0.5%. The drug agarose mixture is applied to each well in 2 ml volume and the plates are incubated for three days, after which the cells are stained with a 1.5% solution of neutral red. At the end of the 4-6h incubation period, the stain is aspirated, and plaques counted using a stereomicroscope at 10x magnification.

d.     VZV Plaque Reduction Assay - Semi-Solid Overlay.

        The procedure is essentially the same as for the HSV plaque assay described above with two exceptions:

        After addition of the drug, the plates are incubated for ten days. On days 3 and 6, an additional 1 ml overlay with equal amounts of 2x MEM and 1% agarose are added.

e.     CMV Plaque Assay - Semi-Solid Overlay.

        The procedure again is nearly the same as for HSV with a few minor changes. The agarose used for both the initial overlay and the two subsequent overlays is 0.8% rather than 1%. The assay is incubated for 14 days with the additional 1 ml overlays being applied on days 4 and 8.

f.      Plaque Reduction Assays Using Liquid Medium Overlay.

        During a previous contract period, we found that some large or highly charged molecules that are active in the CPE - inhibition assay were inactive in the plaque assay because the drug failed to diffuse through the agarose overlay. Therefore, we have modified the plaque assay for confirmation studies, so that the overlay medium is liquid rather than semi-solid. The procedure for the liquid overlay plaque assay is similar to that using the agarose overlay. The procedure for adding the virus is the same as for the regular plaque assay. The drugs are made up at the desired concentrations in MEM with 2% FBS. For HSV-1 and HSV-2 assays, an antibody preparation obtained from Baxter Health Care Corporation is diluted 1:500 and added to the media that the drug is diluted in to limit extracellular spread of virus through the media. For VZV and CMV, no antibody in the overlay is necessary. For the CMV and VZV assays, additional medium without new drug is added on day five and allowed to incubate for a total of 8 and 10 days, respectively. At the end of the incubation period for all of the assays, 2 ml of a 6.0% neutral red solution is added to each well and incubated for six h. The liquid is then aspirated off and plaques enumerated using a stereomicroscope.

3.     Screening and Confirmation Assays for EBV

a.     Cells.

        The two lymphoid cell lines, Raji and Daudi derived from Burkitt's lymphoma are used. The Raji cell line is a non-producer of viral gene products associated with the productive viral cycle. The Daudi cell line is a low level producer, i.e., fewer than 1% of the cells express EA spontaneously. These cells are equally susceptible to superinfection by the P3HR-1 virus as determined by EBV VCA expression. The cells are maintained at 37^oC in a humidified atmosphere with 5% CO₂, in culture with RPMI-1640 medium containing 10% heat inactivated FBS, 100 u/ml Penicillin, 25 μg/ml gentamicin and 2mM L-glutamine. The cells are passaged twice weekly and the cell concentration adjusted to 2x10⁶/ml for use.

b.     Virus.

        There are two prototypes of infectious EBV. One is exemplified by the virus derived from supernatant fluids of the P3HR-1 cell line. This cell line produces non-transforming virus that induces the production of VCA after primary infection or superinfection of B cell lines. The other prototype is exemplified by the B95-8 virus. This virus immortalizes cord blood lymphocytes and induces tumors in marmosets. It does not, however, induce an abortive productive infection even in cell lines harboring EBV genome copies. We use the P3HR-1 strain in all screening assays. For virus production, P3HR-1 cells are cultured at a concentration of 2x10⁵/ml for two weeks in medium containing 2% FCS at 34^oC in a humidified atmosphere with 5% CO₂. Concentrated virus then is prepared from the supernatant of the culture by centrifugation at 12,000g for 90 min in a Sorvall Centrifuge. The pellets are resuspended in RPMI-1640 medium at 1/100 of the original volume and stored at -70^oC.

c.        Antibodies.

        Murine monoclonal antibody to EBV VCA, (Chemicon International, Inc., Temecula, Calif.) is used in immunofluorescence assays and ELISA. Optimal monoclonal antibody concentration is determined by antibody titration for each assay system. For single fluorochrome analyses FITC-labelled goat anti-mouse total IgG (Southern Biotechnology Associates, Birmingham, Ala.) is used as the second antibody. 

d.     EBV superinfection and drug treatment.

        Superinfection is initiated by the incubation of 0.5 ml of an appropriate concentration of EBV with 10⁶ cells/tube in a total of 1 ml/tube. In most cases this amounts to a multiplicity of infection (MOI) of 0.1-0.2 based on VCA induction in Daudi cells. After adsorption at 37^oC for 1h, 3 ml of RPMI-1640 medium is added. The cells are pelleted by centrifugation and supernatants discarded. Drug concentrations (0.08, 0.4, 2, 10, 50 µg/ml) in 4 ml of RPMI-1640 are added to the appropriate tubes. RPMI-1640 is added to positive and negative control tubes and each drug concentration is added to Daudi cells without virus for toxicity controls. After incubation the cells in each tube are counted using a Coulter Counter and washed three times with phosphate buffered saline solution (PBS) (without Ca and Mg). Each cell suspension is adjusted to a concentration of 4.0 x 10⁶ cells/ml in PBS. For EBV IFA and DNA hybridization assays, two sets of slides are prepared with 4 x 10⁴ cells/spot for each cell suspension, and air-dried overnight.

e.        Immunofluorescence assay.

        The infected and drug treated cells are counted and washed three times with PBS. 4 x 10⁴ cells in PBS are spotted on multiwell slides and air dried. The cells are then fixed for 10 min in acetone, washed in PBS and stained for immunofluorescence with the mouse monoclonal antibodies and FITC-labeled goat anti-mouse IgG. EBV VCA specific antibodies are used in the immunofluorescence assays. FITC-labeled goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, Ala.) is used as the second antibody. The slides are counterstained with 0.1% Evan's blue for 5 min and mounted with 10% glycerin in PBS. The number of FITC-positive cells on each smear is determined using a Nikon fluorescence microscope. 500 cells are counted in each spot. The number of cells expressing EBV VCA is calculated by multiplying the fraction of antigen positive cells by the number of cells/ml in the culture at the time of harvest. The drug concentration is plotted against the number of antigen positive cells/ml using a computer program and EC₅₀ and EC₉₀ values are calculated.

f.      ELISA.

        During the previous contract period we developed an ELISA for screening antiviral drugs for activity against EBV VCA. Daudi cells infected with P3HR-1 virus and treated with drug are harvested by centrifugation and washed three times with PBS. The cells are pelleted and suspended to a concentration of 4 x 10⁶ cells/ml in PBS. 100 µl of each suspension is dispensed in triplicate into a 96-well plate, air-dried and fixed with 95% Ethanol and 5% Acetic Acid. Uninfected cells are prepared in the same manner and used as controls. After washing the plate, primary and secondary antibodies diluted in 1% bovine serum albumin containing 0.05% Tween-20 are added sequentially to each well and incubated at room temperature. Antibody additions are separated by 3 washes with PBS containing 0.005% Tween-20. O-phenyldiamine (OPD) substrate is added and the reaction stopped with 3N H₂SO₄ after ~10 min. The optical density is measured at 492 nm and the EC₅₀ extrapolated using the computer software program described earlier.

g.     Evaluation of antiviral agents against EBV DNA replication.

        The Enzo Simply Sensitive Horseradish Peroxidase-AEC In Situ Detection System for EBV (Enzo Diagnostics, Farmingdale, N.Y.) is used to determine antiviral activity against DNA synthesis. Detection and staining is performed according to the manufacturer's instructions. The following is a brief description: Three days after superinfection and drug treatment, slides are prepared with 4 x 10⁴ cells/spot for each cell suspension, air-dried overnight. The slides are fixed in Acetone for 10 min. A biotin labelled EBV probe is added to each spot of fixed cells and the slide is covered with a glass coverslip. The slide is then heated on a hot plate at 95C for five min. After heating, the slide is placed at 37C on a slide warmer for 30-60 min, for the DNAs to anneal. The coverslips are then removed and the Post Hybridization Reagent is added to each spot. After incubation for 10 min and rinsing with washing buffer, Detection Reagent is applied. This is left on for 30-60 min on a slide warmer and then washed off with washing buffer. Chromogen Substrate Solution is added and incubated for 20 min on a slide warmer. The slides are washed and counter stained with Blue Counterstain. The slides are then rinsed with deionized water and mounted with water. The slides are viewed in a light microscope under a magnification of 400X. Positive cells appear as red spots. All the cells are counted in several fields. The fraction of red spots in the total number of cells counted multiplied by 100 reflects the percent hybridization.

h.     Primary infection assay using B95-8 in human cord blood lymphocytes.

        The primary infection of umbilical cord blood lymphocytes with the transforming strain B95-8 of EBV induces the expression of the virus-associated nuclear antigen (EBNA) in the cell (140). It is also known that B95-8 virus induces cellular DNA synthesis after infection of CBL (141). The availability of EBNA virus-infected cells in culture allows the identification and quantitation of EBV-positive cell antigens by indirect IFA staining and FACS. Cord blood lymphocytes separated by ficoll-hypaque gradient are cultured in complete RPMI-1640 as described below. The EBV-B95-8 is produced by incubating the B95-8 cell line in RPMI -1640 plus 10% Fetal calf serum for 10-14 days. The supernatant is collected and stored at 0-4C. One million CBL are infected by incubation with 1 ml of the B95-8 supernant for 1h. The virus is removed by centrifugation. After one wash with RPMI-1640 the infected cells are treated with antiviral drugs as described earlier for P3HR-1 superinfection. The cell cultures are incubated for 4-6 days. Cell harvesting and immunofluorescent staining is the same as described earlier.

4.     Assays Against HHV-6A, HHV-6B, and HHV-8.

a.     Cells.

        Cord Blood Lymphocytes (CBL) Cells:  Fresh heparinized umbilical cord blood was obtained  from the University of Alabama at Birmingham Hospital and diluted 1:1 with Hank’s balanced salt solution and layered on a Histopaque 1077 (Sigma Chemical Co., St. Louis, Mo.) gradient.  The tubes were centrifuged at 1600 rpm for 30 min at room temperature and serum was carefully aspirated off.  The lymphocytes were removed, washed with Hank’s balanced salt solution and centrifuged at 1200 rpm for 10 min.  The supernatant was aspirated and the cells were resuspended in RPMI 1640 containing 10% heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 0.25 µg/ml fungizone, 25 µg/ml gentamicin, 0.1 U/ml Interleukin-2 (Sigma, St. Louis, Mo.) and 0.5 µg/ml Phaseolus Vulagaris agglutinin protein (PHAP).  CBLs were used in the HHV-6, Z-29 (Variant B) assays.

        Human T Cell Lymphoblastoid Line, HSB-2 Cells:  The HSB-2 cells were obtained through the NIH AIDS Research and Reference Reagent Program (Rockville, Md.), and were propagated in RPMI 1640 containing 10% heat-inactivated FBS, 100 U/ml penicillin, 25 μg/ml gentamicin and 2mM L-glutamine.  They were split 1:5 in a 175 cm² flask every 3-4 days and used in the HHV-6, GS (Variant A) assays.

        BCBL-1 (body cavity-based lymphoma) Cells:  BCBL-1 cells (NIH AIDS Research and Reference Program, Rockville, Md.)  propagated in RPMI 1640 media containing 10% FBS, 2 mM L-Glutamine, 10 µM β-Mercaptoethanol 100 μ/l penicillin, and 25 μg/ml gentamicin were utilized in the HHV-8 assay.

b.     Viruses.

        There are two variants of HHV-6 known as type A variants or type B variants. Our prototype HHV-6 type A variant is the GS strain which is propagated in the HSB-2 cells and has been obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. These cells, referred to as HSB-2/HHV-6_GS, are maintained at 5 x 10⁵ cells/ml under the same conditions and in the same media as the uninfected HSB-2 cells. They are split every 3-4 days by addition of uninfected cells at 9 parts to 1 part infected cells. Stock titers of this virus of 1 X 10⁵ both in cell-associated and cell-free virus can be obtained by growth for 5 days. Our prototype HHV-6 type B variant is Z29 (ATCC, Rockville, Md.) which is grown as a stock in CBL by incubation for 10 days followed by collection, centrifugation and freezing of the supernatant. 

        HHV-8, latently expressed in the primary effusion lymphoma derived BCBL-1 cell line (NIH AIDS Research and Reference Program, Rockville, Md.) was induced into lytic HHV-8 expression by addition of 100 ng/ml phorbol 12-myristate 13-acetate.  BCBL-1 cells were cultured in RPMI 1640 media containing 10% FBS, 2 mM L-glutamine, 10 µM β-mercaptoethanol 100 U/ml penicillin and 25 μg/ml gentamicin.

c.     Primary Antibodies.

        The primary antibodies used for the indirect IFA and FACS were selected for their antigen specificity, low cross-reactivity with other herpesviruses and fluorescence intensity as monitored by FACS.  Monoclonal antibodies selected for use in the HHV-6 assay systems were screened for variant specificity and demonstrated no A or B variant cross-reactivity in the assay systems.  Monoclonal antibody 8532 (Chemicon, Temecula, Calif.) targets HHV-6 induced early nuclear proteins and was used as a primary antibody in the HHV-6_GS assay systems at a 5 µg/ml concentration.  Monoclonal antibody 8535 (Chemicon, Temecula, Calif.) which targets a B variant 101 kDa virion protein was used as a primary antibody in the HHV-6_Z-29 assay system at a 5 µg/ml concentration.  The HHV-8 monoclonal antibody KS8.1 (Bala Chandran, University of Kansas Department of Microbiology, Molecular Genetics and Immunology) targets the HHV-8 viral envelope associated glycoprotein 8.1 expressed in the late lytic phase of HHV-8 replication (Zoeteweij et al., 1999) and was used at approximately 5 µg/ml.  Monoclonal antibody to the EBV VCA glycoprotein 125 (Chemicon, Temecula, Calif.) was used at a concentration of 2.5 µg/ml for ELISA and 5 µg/ml for IFA.

d.        Determination of Antiviral Drug Efficacy Against HHV-6.

        Serial 5-fold dilutions of drug starting at 50 µg/ml were prepared in media.  CDV was used as a positive control.  Samples for determining antiviral efficacy were prepared by incubating 1 x 10⁶ cells for one hour with sufficient virus to infect approximately 35% of the cells.  After infection, the appropriate dilution of drug was added and cells incubated for 4 to 6 days at 37C.  Virus free controls were prepared by incubating 1 x 10⁶ cells in drug-free media for the designated period and virus controls were prepared by incubating 1 x 10⁶ cells for one hour with sufficient virus to infect 35% of the cells followed by incubation in drug-free media for the designated period.  After incubation, cells were rinsed with PBS and permeabilized overnight in methanol at -80C for use in FACS.

e.     FACS Assay for HHV-6 and HHV-8.

        Cells were rinsed thoroughly with PBS and a blocking solution containing 5% FBS, 4% Normal goat serum (NGS) and 0.5% DMSO.  Cells were then incubated with the appropriate monoclonal antibody (HHV-6 early nuclear proteins (Chemicon, Temecula, Calif.) for HHV-6, GS variant A, a 101 kDa virion protein (Chemicon, Temecula, Calif.) for HHV-6, Z-29 variant B, and KS8.1 for HHV-8 (Bala Chandran, University of Kansas, Department of Microbiology, Molecular Genetics and Immunology).

5.     Screening and Confirmation Assays for Toxicity

a.     Neutral red uptake assay – HFF Cells.

        Twenty-four h prior to assay, HFF cells are plated into 96 well plates at a concentration of 2.5 x 10⁴ cells per well. After 24h, the media is aspirated and 125 µl of each drug concentration is added to the first row of wells and then diluted serially 1:5 using the automated Bio-Mek Liquid Handling System in a manner similar to that used in the CPE assay. The plates are then incubated in a CO₂ incubator at 37C for seven days. At this time the media/drug is aspirated and 200 µl/well of 0.01% neutral red in DPBS is added. This is incubated in the CO₂ incubator for 1h. The drug is aspirated and the cells are washed using a Nunc Plate Washer. After removing the DPBS wash, 200 µl/well of 50% ETOH/1% glacial acetic acid (in H₂0) is added. The plates are rotated for 15 min and the optical densities are read at 550 nm on a plate reader. CC₅₀ values are calculated using a computer program.

b.     Cell proliferation assay in HFF cells.

        Twenty-four h prior to assay, HFF cells are seeded in 6-well plates at a concentration of 2.5 x 10⁴ cells per well in MEM containing 10% FBS. On the day of the assay, drugs are diluted serially in MEM containing 10% FBS at increments of 1:5 covering a range from 100 µg/ml to 0.03 µg/ml. For drugs that have to be solubilized in DMSO, control wells receive MEM containing 1.0% DMSO. The media from the wells is then aspirated and 2 ml of each drug concentration is then added to each well. The cells are then incubated in a CO₂ incubator at 37C for 72h. At the end of this time, the media-drug solution is removed and the cells washed. One ml of 0.25% trypsin is added to each well and incubated until the cells start to come off of the plate. The cell-media mixture is then pipetted up and down vigorously to break up the cell suspension and 0.2 ml of the mixture is added to 9.8 ml of Isoton III and counted using a Coulter Counter. Each sample is counted 3 times with 2 replicate wells per sample.

c.        Determination of antiviral drug cytotoxicity and cell proliferation for non-adherent cell lines (HSB-2, CBL, Daudi, BCBL-1).

        MTS tetrazolium cytotoxicity assay:  Serial 5-fold dilutions of drug starting at 50 µg/ml were prepared in media and added to 1 x 10⁶ cells.  Controls were prepared by incubating 1 x 10⁶ cells in drug-free media.  After an incubation period of 3-6 days depending on the assay system, 200 µl was transferred to a 96 well plate in duplicate.  20 µl of MTS was added and the plate was wrapped in foil and incubated at 37C for 4 h.  MTS was bioreduced by dehydrogenase enzymes found in metabolically active cells into an aqueous soluble formazan.  The quantity of formazan product as measured by the amount of 490 nm absorbance was directly proportional to the number of living cells in culture.  Drug concentration was plotted against the optical density of each sample and CC₅₀ values were calculated using MacSynergy II. 

        Cell proliferation assay:  Serial 5-fold dilutions of drug starting at 50 µg/ml were prepared in media and added to 1 x 10⁶ cells.  Controls were prepared by incubating 1 x 10⁶ cells in drug-free media.  After an incubation period of 3-4 days depending on the assay system, a Coulter Counter is used to determine the total number of cells for each sample (for HSB-2 and Daudi cell lines only).  Drug concentration was plotted against the total concentration of cells for each sample and IC₅₀ values were calculated using MacSynergy II. 

        Bone marrow assay:  In vitro toxicity can be determined by inhibition of myeloid [colony-forming units granulocyte/macrophage (CFU-GM)] and erythroid [burst-forming unit-erythroid (BFU-E)] colony formation in soft agar clonal assays.  Using a 21-23 gauge needle attached to a syringe, rodent bone marrow cells are collected from the leg bone of rats or mice by flushing with Isocoves’ Modified Dulbecco’s medium (IMDM).  A single cell suspension is obtained by repeated aspiration through the needle.  Nucleated cells are enumerated with a hemacytometer and adjusted to the desired cell concentration in IMDM. Murine CFU-GM assays are prepared with 2.5 x 10⁵ nucleated cells/ml, 20% FBS, 10 ng/ml rmGM-CSF, and 0.2% agarose.  BFU-E cultures include 30% FBS, 1% deionized BSA, 0.1 mM 2-ME, 4 U/ml rhEpo, 10 ng/ml rmIL-3, 2.5 x 10⁵  nucleated cells/ml and 0.2% agarose (140).  Triplicate wells (in 6 well plates) containing 0.1ml of drug (10X) receive 1 ml of either culture mixture for each concentration group and slowly mixed.  The cultures are allowed to gel at 4 C and then incubated for 7 (CFU-GM) or 9 (BFU-E) days at 37 C in a humidified atmosphere of 5% CO₂ in air.  Colonies are counted using an inverted microscope.  CFU-GM colonies are identified as cell clones containing at least 40 cells.  BFU-E cultures are stained with dianisidine, and aggregates of greater than 60 hemoglobin-containing cells are counted as erythroid colonies (141).  The median inhibitory concentration (IC₅₀) and the 90% inhibitory concentration (IC₉₀) are derived from linear regression analysis of the logarithm of drug concentration versus CFU-GM or BFU-E survival fraction. 

6.     Reporting of results.

a.     Screening and confirmation of efficacy and toxicity.

            A monthly reporting form for screening and evaluation of antiviral efficacy and toxicity is used for transmitting the data on a monthly basis to the project officer. Included on the form are the month and year of report, the profile and dates of testing, EC₅₀ and EC₉₀ data for efficacy of the test and control compounds, the CC₅₀ and IC₅₀ for the toxicity tests, the SI for each test and a space for comments and recommendations.