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Screening Agreement
Compound Submission Form

Screening Assays
-Biodefense Virus Panel
-BK Virus
-Hepatitis B Virus
-Hepatitis C Virus
-Herpesviruses
-Human Papilloma Virus
-Orthopoxviruses
-Respiratory Virus Panel
-SARS CoV

Data Interpretation
Medicinal Chemistry

Contact Info

D. Smee, Ph.D., Utah State University

1.      Viruses used in primary screen

A.    Adenovirus type 1

Virus strain: 65089/Chicago (Source: M.F. Smaron 1/95 isolate)

Cell line: Human lung carcinoma (A549) cells.

B.     Dengue type 2 virus

Virus strain:  New Guinea C.  Clinical isolate (Source:  N. Karabatsos of the Division of Vector-Borne Diseases, CDC)

Cell line:  African green monkey kidney (Vero) cells.

C.    Influenza types A and B viruses

Virus strains:  A/New Caledonia/20/99 (H1N1).   Recent clinical isolate used in latest vaccine (Source: Center for Disease Control and Prevention [CDC]); A/Panama/2007/99 (H3N2).  Recent clinical isolate used in latest vaccine (Source:  CDC); B/Hong Kong/330/02.  Recent clinical isolate used in latest vaccine (Source:  CDC); A/NWS/33 (H1N1).  A well-recognized laboratory strain (Source:  K. W. Cochran, Univ. Michigan).  (All are tested in the presence of trypsin, although in certain studies the A/NWS/33 virus is used without trypsin when requested)

 Cell line: Madin Darby canine kidney (MDCK) cells.

D.    Measles virus

Virus strain: Chicago.  A 1989 clinical isolate (Source: J. Rota, CDC).

Cell line: African green monkey kidney (CV-1) cells.

E.    Parainfluenza type 3 virus

Virus strain: 14702.  Clinical isolate (Source: J. Boivin, Hosp. St. Justine, Montreal Canada, 5/95 isolate).

Cell line: African green monkey kidney (MA-104) cells.

F.   Pichinde virus

Virus strain:  An 4763.  Clinical isolate (Source: Dr. J. D. Gangemi, Univ. of South Carolina School of Medicine, Columbia, SC).

Cell line:  African green monkey kidney (BS-C-1) cells

G. Punta Toro virus

Virus strain:  Adames.  An isolate taken from a patient in Panama in 1972.  (Source:  U. S. Army Medical Research Institute for Infectious Diseases, Fort Detrick, Frederick, MD).

Cell line:  Rhesus monkey kidney (LLC-MK2) cells

H.    Respiratory syncytial virus

Virus strain: A2.  A recognized laboratory strain which also infects cotton rats (Source: ATCC).

Cell line: African green monkey kidney (MA-104) cells.

I.    Rhinovirus type 2

Virus strain: HGP.  Isolated from throat washings of a patient with respiratory illness.  (Source:  ATCC).

Cell line: Human epidermoid carcinoma of the nasopharynx (KB) cells.

J.     Severe acute respiratory syndrome (SARS) virus

Virus strain:  200300592.  Clinical isolate.  (Source:  CDC)

Cell line:  Vero 76 cells.

K.     Venezuelan equine encephalitis virus

Virus strain:  TC-83.  Attenuated strain (Source:  ATCC).

Cell line: Vero cells.

L.    West Nile virus

Virus strain:  New York.  Isolated from a crow brain in New York (Source:  R. Lanciotti, CDC)

Cell line:  Vero cells.

M.   Yellow fever virus

Virus strain:  17D.   Attenuated human isolate (Source:  ATCC).

Cell line:  Vero cells.

2.      Methods for assay of antiviral activity

A.    Rapid Screening Assay

When relatively large numbers (10 or more) of test compounds are submitted at the same time from a single sponsor, the compounds are evaluated in a 2-concentration test.  In this procedure, 2 concentrations (200, 20 µg/ml unless otherwise directed) will be tested.  These are diluted 1:2 when virus is added, making final concentrations 100 and 10 µg/ml.  The standard CPE test uses an 18 h monolayer (80-100% confluent) of the appropriate cells, medium is drained and each of the concentrations of test compound or placebo are added, followed within 15 min by virus or virus diluent.  Two wells are used for each concentration of compound for both antiviral and cytotoxicity testing.  The plate is sealed and incubated the standard time period required to induce near-maximal viral CPE.  The plate is then stained with neutral red by the method described below and the percentage of uptake indicating viable cells read on a microplate autoreader at dual wavelengths of 405 and 540 nm, with the difference taken to eliminate background.  An approximated virus-inhibitory concentration, 50% endpoint (EC50) and cell-inhibitory concentration, 50% endpoint (IC50) will be determined from which a general selectivity index is calculated:  SI = (IC50) / (EC50).  An SI of 3 or greater indicates confirmatory testing is needed.

B.  Standard Assay:  Inhibition of Viral Cytopathic Effect (CPE)

This test, run in 96 well flat-bottomed microplates, will be used for the initial antiviral evaluation of all new test compounds.  In this CPE inhibition test, four log₁₀ dilutions of each test compound (e.g. 1000, 100, 10, 1 µg/ml) will be added to 3 cups containing the cell monolayer; within 5 min, the virus is then added and the plate sealed, incubated at 37°C and CPE read microscopically when untreated infected controls develop a 3 to 4+ CPE (approximately 72  to 120 hr).  A known positive control drug is evaluated in parallel with test drugs in each test.  This drug is ribavirin for dengue, influenza, measles, respiratory syncytial, parainfluenza, Pichinde, Punta Toro and Venezuelan equine encephalitis viruses, cidofovir for adenovirus, pirodovir for rhinovirus, 6-azauridine for West Nile and yellow fever viruses, and alferon (interferon alfa-n3) for SARS virus.  Follow-up testing with compounds found active in initial screening tests are run in the same manner except 8 one-half log₁₀ dilutions of each compound are used in 4 cups containing the cell monolayer per dilution.

The data are expressed as 50% effective concentrations (EC50).

C.  Standard Assay:  Increase in Neutral Red (NR) Dye Uptake

This test is run to validate the CPE inhibition seen in the initial test, and utilizes the same 96-well micro plates after the CPE has been read. Neutral red is added to the medium; cells not damaged by virus take up a greater amount of dye, which is read on a computerized micro plate autoreader. The method as described by McManus (Appl. Environment. Microbiol. 31:35-38, 1976) is used.  An EC50 is determined from this dye uptake.

D.    Decrease in Virus Yield Assay

Compounds considered active by CPE inhibition and by NR dye uptake will be re-tested if additional, fresh material is available, using both CPE inhibition and, using the same plate, effect on reduction of virus yield by assaying frozen and thawed eluates from each cup for virus titer by serial dilution onto monolayers of susceptible cells. Development of CPE in these cells is the indication of presence of infectious virus. As in the initial tests, a known active drug is run in parallel as a positive control. The 90% effective concentration (EC90), which is that test drug concentration that inhibits virus yield by 1 log10, is determined from these data.

3.      Methods for assay of cytotoxicity

A.  Visual Observation

In the CPE inhibition tests, two wells of uninfected cells treated with each concentration of test compound will be run in parallel with the infected, treated wells. At the time CPE is determined microscopically, the toxicity control cells will also be examined microscopically for any changes in cell appearance compared to normal control cells run in the same plate. These changes may be enlargement, granularity, cells with ragged edges, a filmy appearance, rounding, detachment from the surface of the well, or other changes. These changes are given a designation of T (100% toxic), PVH (partially toxic–very heavy–80%), PH (partially toxic–heavy–60%), P (partially toxic–40%), Ps (partially toxic–slight–20%), or 0 (no toxicity–0%), conforming to the degree of cytotoxicity seen.

A 50% cell inhibitory (cytotoxic) concentration (IC50) is determined by regression analysis of these data.

B.  Neutral Red Uptake

In the neutral red dye uptake phase of the antiviral test described above, the two toxicity control wells also receive neutral red and the degree of color intensity is determined spectrophotometrically. A neutral red IC50 (NR IC50) is subsequently determined.

C.  Viable Cell Count

Compounds considered to have significant antiviral activity in the initial CPE and NR tests are re-tested for their effects on cell growth. In this test, 96-well tissue culture plates are seeded with cells (sufficient to be approximately 20% confluent in the well) and exposed to varying concentrations of the test drug while the cells are dividing rapidly. The plates are then incubated in a CO2 incubator at 37°C for 72 hr, at which time neutral red is added and the degree of color intensity indicating viable cell number is determined spectrophotometrically; an IC50 is determined by regression analysis.

4.      Data analysis

Each test compound’s antiviral activity is expressed as a selectivity index (SI), which is the IC50 or IC90 divided by the EC50. Generally, an SI of 10 or greater is indicative of positive antiviral activity, although other factors, such as a low SI for the positive control, are also taken into consideration.

               5.            Secondary Testing

Compounds having confirmed SI values of 10 or greater may, at the discretion of the Project Officer, be evaluated against additional strains of the original virus inhibited in order to more fully determined the spectrum of antiviral activity of the compound.

References

1.      Sidwell, R. W. and J. H. Huffman.  1971.  Use of disposable micro tissue culture plates for antiviral and interferon induction studies.  Appl. Microbiol. 22:797–801.

2.      Sidwell, R. W., J. H. Huffman, G.P. Khare, L. B. Allen, J. T. Witkowski, and R. K. Robins.  1972.  Broad-spectrum antiviral activity of Virazole:  1-b-d-Ribofuranosyl-1,2,4-triazole-3-carboxamide.  Science 177:705–706.

3.      Sidwell, R. W., J. H. Huffman, B. B. Barnett, and D. Y. Pifat.  1988.  In vitro and in vivo Phlebovirus inhibition by ribavirin.  Antimicrob. Ag. Chemother. 32:331–336.

4.      Barnard, D.L., J.H. Huffman, L.R. Meyerson, and R.W. Sidwell.  1993.  Mode of inhibition of respiratory syncytial virus by a plant flavonoid, SP-303.  Chemotherapy 39:212–217.

5.      Barnard, D.L., C.L. Hill, T. Gage, J.L. Matheson, J.H. Huffman, R.W. Sidwell, M.I. Otto and R.F. Schinazi.  1997.  Potent inhibition of respiratory syncytial virus by polyoxometalates of several structural classes.  Antiviral Res.  34:27-37.

6.      Huffman, J.H., R.W.Sidwell, D.L. Barnard, A. Morrison, M.J. Otto, C.L. Hill and R.F. Schinazi.  1997.  Influenza virus-inhibitory effects of a series of germanium and silicon-centered polyoxometalates.  Antiviral Chem. and Chemother.  8:75-83.

7.      Barnard, D.L., N. Bischofberger, C.U. Kim, J.H. Huffman, R.W. Sidwell, J.P. Dougherty, W. Lew, M.A. Williams, and W.Yang.  1997.  Acyclic phosphonomethylether nucleoside inhibitors and respiratory viruses.  Antiviral Chem. and Chemother.  8:223-233.

8.      Lin, Y-M., M. T. Flavin, R. Schure, F-C. Chen, R. W. Sidwell, D. L. Barnard, J. H. Huffman, and E. R. Kern.  1999.  Antiviral activities of biflavonoids.  Planta Med. 65:120-125.

9.      Barnard, D. L., R. W. Sidwell, W. Xiao, M. R. Player, S. Adah, and P. F. Torrence.  1999.  2-5A-Antisense inhibition of respiratory syncytial virus replication:  Effects of oligonucleotide structure modifications and RNA target site selection.  Antiviral Res. 41:119-134.

10.  Sidwell, R. W., and D. F. Smee. 2000. In vitro and  in vivo assay systems for study of influenza virus inhibitors. Antiviral Res. 48:1-16.

11.  Yasuda, S., J. H. Huffman, D. F. Smee, R. W. Sidwell, and K. Miyata. 2000. Spectrum of virus inhibition by consensus interferon YM643. Antiviral Chem. and Chemother. 11:261-271.

12.  Smee, D. F., Huffman, J. H., Morrison, A. C., Barnard, D. L., and Sidwell, R. W. 2000. Cyclopentane neuraminidase inhibitors with potent in vitro anti-influenza virus activity. Antimicrob. Agents Chemother. 45:743-748.

13.  Barnard, D.L, Stowell, V., Seley, K.L., Hegde, V.R., Subha, R., Rajappan, P., Schneller, S.W. Smee, D.F. and Sidwell, R.W.  2001.  Inhibition of measles virus replication by 5'-nor carbocyclic nucleoside analogs.  Antiviral Chem. & Chemother.  12: 241-250.

14.  Song, C.Y., V. Paul, H. Choo, J. Morrey, R.W. Sidwell, R.F. Schinazi, and C.K. Chu. 2001. Enantiomeric synthesis of D- and L- cyclopentenyl nucleosides and their antiviral activity against HIV and West Nile viruses. J. Med. Chem. 44:3985-3993.

15.  Morrey, J.D., D.F. Smee, R.W. Sidwell, and C. Tseng. 2002. Identification of active antiviral compounds against a New York isolate of West Nile virus. Antiviral Res. 55:107-116

16.  Smee, D.F., K.W. Bailey, A.C. Morrison, and R.W. Sidwell. 2002. Combination treatment of influenza A (H1N1) virus infections in cell culture and in mice with the cyclopentane neuraminidase inhibitor RWJ-270201 and ribavirin. Chemotherapy. 48: 88-93.

17.  Sidwell, R.W., and D.F. Smee. 2002. Viruses of the Bunya- and Togaviridae Families: Potential as bioterrorism agents and means of control. Antiviral Res. 57: 101-111.

18.  Smee, D.F., A. Morrison, D. Barnard, and R. Sidwell. 2002. Comparison of colorimetric, fluorometric, and visual methods for determining anti-influenza (H1N1 and H3N2) virus activities and toxicities of compounds. J. Virol. Methods.  106: 71-79.

19.  Barnard, D.L., Z.Q. Xu, V.D. Stowell, H. Yuan, D.F. Smee, R. Samy, R.W. Sidwell, M.K. Nielsen, L. Sun, H. Cao, A. Li, C. Quint, J. Diegnan, J. Crabb and M.T. Flavin.  2002.  Coumarins and pyranocoumarins, potential novel pharmacophores for inhibition of measles virus replication. Antiviral Chem. and Chemother.  13: 39-59.

20.  Zhang, N., N.M. Chen, R. Sood, K. Kalicharran, A. Fattom, R.B. Naso, D.L. Barnard, R.W. Sidwell, and R.S. Hosmane.  2002.  In vitro inhibition of the measles virus by novel ring-expanded (‘Fat’) nucleoside analogues containing the imidazo[4,5-e] [1,3]diazepine ring system.  Bioorg. & Med. Chem. Ltrs.  12: 3391-4.