A Method for Determining Sensitivities of Antiviral
Drugs in Vitro for Possible Use
as Clinical Consultation
WILLIAM E. WALKER, P H . D . , BURTON A. WAISBREN, M.D.,
AND GEORGE E. BATAYIAS,
RONALD R. MARTINS,
M.D.,
M.D.
Virus Laboratory and Department of Pathology, St. Luke's Hospital, and the Burn
Research Laboratory, St. Mary's Hospital, Milwaukee, Wisconsin 53215
ABSTRACT
Walker, William E., Waisbren, Burton A., Martins, Ronald R., and Batayias,
George E.: A method for determining sensitivities of antiviral drugs in vitro
for possible use as clinical consultation. Amer. J. Clin. Path. 56: 687-692,
1971. The availability of drugs such as cytosine arabinoside, iododeoxyuridine,
and fluorodeoxyuridine has given the clinician choices with regard to treatment of life-threatening viral diseases that are occurring with increasing frequency in immunodepressed hosts. This choice of therapy places increased
responsibility on the clinical virology laboratory to make available comparative in vitro data regarding the activity of antiviral drugs against viruses
isolated from patients. Presented in this paper are methods developed for
determining the minimal toxicity concentrations of antiviral drugs against
the tissue culture cell lines routinely used in isolating viruses. Also presented
are methods for determining the minimal inhibitory concentration of antiviral drugs against two strains of herpes simplex virus and one strain of
adenovirus. These two values give a tissue-viral susceptibility ratio for each
drug. As new drugs become available, these methods of testing in vitro may
well assume clinical importance.
FATAL VIRAL DISEASES are occurring with increasing frequency in immunodepressed
hosts. The availability of drugs such as
cytosine arabinoside, iododeoxyuridine, and
fluorodeoxyuridine has given the clinician
a choice of treatment for these viral diseases. This choice of therapy places increased responsibility on the clinical virology laboratory to make available comparative in vitro data regarding the activity of
antiviral drugs against viruses isolated from
patients.
Presented in this paper are (1) methods
developed for the determination of minimal inhibitory concentrations of antiviral
drugs against herpes virus and adenovirus;
and (2) methods developed for determining
the minimal toxicity concentrations of antiviral drugs against the tissue culture cell
lines routinely used in isolating viruses.
Received December 21, 1970; accepted for publication February 9, 1971.
Address reprint requests to Dr. Walker, Department of Pathology, St. Luke's Hospital, 2900 W.
Oklahoma Avenue, Milwaukee, Wisconsin 53215.
Materials and Methods
Tissue Culture Cells
T h e three cell lines used were rhesus
monkey kidney (RMK) and embryonic hu-
687
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10 '
10 *
10
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10 4
I0" 5
10 ••
10'
10 "
lO-**
10'°
Concentration ol lododeo*yuridine in Moles/ml
4
Standardized Inoculo CIO' T C I D 5 0 ) ol Virus in Each Tube
FIG. 1. Development of drug toxicity index (DTI)
of iododeoxyuridine (IUdR) on a low titered (10-4
TCID M ) herpes simplex virus (Type 1). MIC =
minimum inhibitory concentration of drug on virus; MTC = minimum toxicity concentration of
drug on cells. D T I for IUdR on herpes simplex
virus = M T C / M I C = 10-710-" = 102, or 100.
man lung (Wi-38), which were purchased
weekly from Grand Island Biological Co.
(GIBCO), Grand Island, New York, fully
sheeted in 16 by 100 mm. disposable tubes,
and human cervical carcinoma (HeLa),
which was prepared weekly in our laboratory. Prior to use, all tubes were refed with
maintenance media. The medium for the
Wi-38 cells consisted of Eagle's basal medium (BME, GIBCO), 2% fetal calf serum
(GIBCO), 100 units penicillin per ml., 100
mg. streptomycin per ml., and sodium bicarbonate to give a pH of 7.4. T h e medium
used for RMK cells was the same except
that calf serum was omitted. The HeLa
cells were maintained on medium No. 199
with no fetal calf serum. All tubes were
incubated without rotation at 35 C , and
no increase in cell numbers was noted in
7 days.
Virus
Five different herpes simplex isolates and
two adenovirus isolates were used. Three
of the herpesviruses were isolated from immunodepressed patients: a bone marrow
transplant recipient, a heart transplant recipient, and a patient receiving Imuran
(azathioprine) for membranous nephritis
who had developed herpes meningitis. The
oilier two herpes isolates were from vaginal
lesions in otherwise healthy patients. When
considered on the basis of typical cytopathogenicity, the three isolates from the
A. J.C.P.—Vol. 56
immunodepressed patients were Type A,
and the two from the cervical lesions were
Type B. 9 ' 1 2 Identification of all isolates
was verified serologically by the State
Health Laboratories in Madison, Wisconsin. The adenoviruses used were identified
and typed serologically by this laboratory
and the State Health Laboratory and
found to be Type 3. Both were isolated in
RMK and HeLa cells and had been found
in the throats of patients exhibiting clinical signs of an upper respiratory tract infection. None of these had been subjected to
more than three subpassages in tissue culture.
Antiviral Drugs
Five-iododeoxyuridine (IUdR) was obtained lyophilized from Calbiochem (Los
Angeles, California) and 5-fluoro-2-deoxyuridine (FUdR) from Hoffmann-La Roche,
Inc. (Nutley, New Jersey). Cytosine arabinoside (CA) was obtained from Upjohn (Kalamazoo, Michigan) under the trade name
of Cytosar. When used, the compounds
were reconstituted in the tissue culture medium appropriate for the cell line being
tested.
Drug-Tissue Culture Toxicity
T o determine the toxic effects of the
drugs on the tissue cultures, each drug was
titrated on each cell line. The maximum
toxicity concentration (MTC) was defined
as the maximum concentration of the drug
that did not visibly affect the tissue culture
cells (Fig. 1).
Titrations were performed by preparing
serial tenfold dilutions of the drugs in the
appropriate tissue culture tube. These were
then incubated at 35 C. and observed
under the microscope (X 10) each day for
the appearance of toxic effects. The degrees of cytotoxicity were rated as follows
(Fig. 2):
1+ = granularity
2+ = granularity, vacuoles, and shrinkage
3+ = shrinkage and rounding
4+ = detachment
December 1971
SENSITIVITIES OF ANTIVIRAL DRUGS IN VITRO
689
Fie. 2. Upper left: control Wi-38 cells. Upper right: 1+ cytotoxicity, characterized by granularity. Lower left: 2 + cytotoxicity, characterized by granularity and vacuoles. Lower right: 3 +
cytotoxicity, showing rounding and detachment. Not pictured: 4 + cytotoxicity, which would
be complete detachment.
Ten duplicate tests were run with each cell
line, with little or no apparent variability
between tests.
Relationship
of Virus Dose to Cell Line
Cytopathogenicity
Various inocula of virus were used to
determine the effect of virus dosage on
cytopathogenic activity. The cytopathic effect remained constant within virus strains
on the appropriate cell lines. Standard
logarithmic titrations were performed on
each virus to determine initial concentrations, which were then adjusted to dosages
that ranged from 10-' TCID r , 0 to 10"5-6
TCID 5 0 . These dosages were retitered on
the cell lines (herpesviruses 1 and 2 on
Wi-38 and adenovirus on RMK and HeLa)
rind the degree of cytopathogenicity recorded each day and rated with a 1+ to 4+
gradation system reflecting 25 to 100% in-
fectivity, respectively. Five assays with each
strain of herpesvirus and each strain of
adenovirus were done, with no obvious
variations between assays.
Drug, Tissue Culture Cell, and Virus
Interactions
One milliliter of tissue culture media
containing a known concentration of virus
was inoculated into each of 100 tubes containing tissue culture cells. The virus was
allowed to absorb for one hour and the
supernatant fluid was then decanted. One
milliliter of each serial tenfold dilution of
drug in tissue culture media was inoculated
into each of 10 virus-containing tissue culture tubes. These were incubated at 35 C.
and observed under the microscope each
day for the appearance of drug or viral
cytopathogenicity, or both. Four days was
taken as the endpoint, since no new drug
690
WALKER ET
Table 1. Maximum Toxicity Concentrations (MTC)
in Moles per ml. of Antiviral Drugs on
Tissue Culture Cells
Drugs (Moles/ml.)
Cell Line
lUdR* FUdRf
CAJ
Rhesus monkey kidnev
(RMK)
lO"2
lO"3
10"1
Human embryonic lung
(Wi-38)
lO"4
10"1
10-'
Human cervical carcinoma
(HeLa)
lO"1
10"1
10- 1
* S-iodo-2-deoxyuridine.
f 5-fluoro-2-deoxyuridine.
X Cytosine arabinoside.
A,I.C.P.—Vol.
AL.
Relationship
of Virus Dose to Cell Line
Cytopathogenicity
As would be expected, there was a direct
relationship between the viral dosage and
cell line cytopathogenicity (Fig. 3). This
relationship was maintained between assays
with very little variation, due to the initial
adjustment of virus strength within each
strain. On the basis of these studies a 10 4
TCID 5 0 of the virus was chosen as the standard inoculum for the final test because it
gave a consistent 3+ cytopathic effect in
four clays.
Viral, Cell Line
toxicity or virus cytopathic effects were apparent after this time. The minimal inhibitory concentration (MIC) was defined
as the least amount of drug that would
prevent cytopathogenicity by a standard
amount of virus (Fig. 1). T o determine
whether there was a direct relationship between viral close and antiviral activity of
the drugs, MIC was determined as described above with increasing inocula of
virus.
A third index was arbitrarily designated
as the Drug Toxicity Index (DTI). This
reflected the safety of the drug as manifested by the amount less than the cell line
toxic dose of the drug that wotdcl inhibit
the virus. For convenience this was arbitrarily designed on a log scale.
Interactions
Results of 80 tests of viral sensitivity to
the antiviral agents are summarized in
Table 2. There was no variation between
the same strains of virus tested, whereas
marked differences were found in the sensitivities of the species and types of viruses
themselves. When the distances between
the MIC's of the viruses and MTC's of the
drugs were compared, the limited data suggested that from the point of view of the
cell lines cytosine arabinoside was the safest
drug for both types of herpesvirus, whereas
adenovirus was not affected by any of the
three drugs (Table 2 and Fig. 1).
When the test was run with increasing
amounts of virus a direct relationship between viral dose and MIC was found
(Table 3).
('tint
•>! itu'i v„ut
med to mote dilutions) I
Degree
Results
56
ol
Cytopothogemcity
Drug—Tissue Culture
Toxicity
in
roui Doys
At four clays, when the maximum damage had been done, I U d R exhibited the
greatest degree of toxicity, whereas cytosine arabinoside produced very little toxicity to any of the cells (Table 1). Cytotoxicity could be reversed in all cells if the
cells were refed with drug-free media before a 2+ reaction was noted.
Undil.
10"'
lO"2
IO-!
IO"a
10"'
10"
C o n c e n t r a t i o n of Virus in T . C . I . D . 5 0
Fir.. 3. Dcgrre of rylopalhogenicity in Wi-38 cells
produced by herpes simplex virus (Type 1) in four
days. Degrees of cytopathogenicity: 1+ = foci in
25% of cell sheet, 2 + = foci in 50% of cell sheet,
3 + = foci in 75% of cell sheet, and 4 + = foci over
entile sheet.
December
JV71
SENSITIVITIES OE ANTIVIRAL DRUGS IN
691
VITRO
Table 2. Comparison of 10 4 TCIDso Concentrations of Three Viruses in Terms of Minimum
Inhibitory Concentrations (MIC) and Drug Toxicity Indices (DTI) to Three Drugs
Herpes Simplex (Type 1)
Drugs
lUdRt
EUclR§
CA'f
No.
Determinations MIC*
20
12
5
10-5
10-"
10-°
DTIf
Herpes Simplex (Type 2)
No.
Determinations MIC*
too
1,000
100,000
15
10
5
10- J
10 •«
10-«
DTIf
Adenovirus (Type 3
No.
Determinations MIC*
0
10,000
100,000
5
5
3
DTJ
10-s
10 "•
10=
o
0
0
* MfC in moles/ml.
f D T I in log unit absolute values.
% 5-iododeoxyuridine.
§ 5-fluoro-2-deoxyuridine.
II Cytosinc arabinoside.
Discussion
This study which was prompted by clinical difficulties in deciding on drug therapy
in a case of severe herpes encephalitis, indicates the practicability of obtaining laboratory data from the clinical virology laboratory which will be of potential help in the
treatment of patients. For instance, the
deaths of both the patient who received the
bone marrow transplant and the one who
received the heart were probably influenced
by the herpesvirus that had been isolated
from them ten days before their deaths.
The knowledge that now has been obtained through this study, had it been
available at that time, would have been
taken into account in the deliberations regarding treatment of the terminal infections.
The rationale of assaying the cytotoxic
potential of chemotherapeutics in tissue
culture has been investigated by others. 3 '' 3
Although this system cannot be likened to
a situation in vivo, it does provide an accurate and reproducible system for comparative testing in vitro. The MTC's derived in this study reflect the differences in
relative toxicities in antiviral drugs.
The results of this study indicate that
cytosine arabinoside is effective in vitro
against both herpesviruses but not against
adenovirus Type 3. IUdR is effective
against herpesvirus Type 1 but does not
affect Type 2. This difference may explain
the variety of results obtained in clinical
applications of IUdR, assuming no effort
was made to subtype the virus.1' 11>14 The
same situation may exist concerning FUdR
Tabic 3. Relationship Between Virus Concentration and Minimum Inhibitory
Concentration (MIC)* of Antiviral Drug
Virus Concentration in TCID50
Herpes Simplex ('type i)
Herpes Simplex (Type 2)
Adei lovirus (Ty pe 3)
Drug
10"'
1 0 -2.s
10- 4
10"'
10-2-6
10-4
10-'
JO-25
IUdR
FUdR
CA
>io- 4
10"5
10-'
10- 6
io-«
10 «
>io-4
10- 1
10-°
>io-4
10-»
10-6
>10-2
to-
>io-4
10-'
10-2
>io- 3
>io-'
>io-"
>io- 3
>io-'
10-'
10'
* MIC values given in moles/ml.
4
10
4
>10-=
>io-»
>10 '
692
WALKER ET
and herpesvirus. These results indicate that
FUdR is more effective against Type 2
than Type 1. Some investigators have reported good success with FUdR, 4 ' 6 whereas
others feel it is completely ineffectual.2 -'•10
Again, it may be a difference of subtypes.
Adenovirus did not appear to be inhibited
by either of the drugs, but this does not
mean that they are not adenovirus inhibitors. Some may be effective against only
certain subtypes and not others. 4 Also, it
must be remembered that only levels of
drug below the MTC for that cell line
were considered. It may well be that the inhibitory actions of all three drugs lie in the
ranges above the MTC's.
The lack of variability in the MIC's of
the strains of virus of each type studied,
if confirmed by further studies, may be
fortunate, since it will mean that a drug
for a particular patient may be chosen on
the basis of previous studies in vitro regarding drug interaction with the species and
type of virus that is causing an infection.
The difference between drug sensitivities
of Types 1 and 2 herpesvirus is again
worthy of mention, because it emphasizes
the need for rapid specific identification of
virus, not only in regard to species but also
in regard to type. The development of
rapid viral diagnostic laboratories has been
attempted successfully by the authors
(Amer. J. Clin. Path. Sept. 1971) and
others. 5 ' 8
T h e influence of dose on MIC is also
worthy of mention, since it means that
comparative studies between laboratories
will have to be evaluated on the basis of
the doses of virus used in the test.
T h e final place of in vitro studies of this
type will, of course, await further clinical
correlation, but if the obvious analogy of
this work with antibiotic bacterial sensitivity testing holds true, severe viral infections
AL.
A.j.C.l'.— Vol. 56
will eventually be treated in part on the
basis of activity of drugs against the specific
infecting virus in vitro.
Acknowledgments.
Patricia A. Karrels, Suzanne
M. Walter, and Mary Ann Schutten gave invaluable technical assistance.
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