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Rescue of vesicular stomatitis virus from interferon-induced resistance by superinfection with vaccinia virus
66, 505-511 (1973)
VIROLOGY
Rescue
of Vesicular Resistance
Stomatitis
Virus
by Superinfection
from with
Interferon-Induced Vaccinia
Virus
I. Rescue in Cell Cultures from Different Species HARSHAD Department
of Microbiology,
R. THACORE School of Medicine,
JULIUS
AND
University
S. YOUNGNER
of Pittsburgh,
Pittsburgh,
Pennsylvania
16361
Accepted September 4, 1973 The replication of two DNA viruses, vaccinia and pseudorabies (PsRV), was not inhibited in three cell lines of rabbit origin (RK-13 and RK-1337, rabbit kidney; and RC-60, rabbit cornea) which had been pretreated with rabbit interferon. In contrast, the replication of vesicular stomatitis virus (VSV), an RNA virus, was susceptible to interferon-induced resistance in rabbit cell lines. These results reinforced the possibility that there are separate interferon-induced resistance factors for RNA and DNA viruses and that cultured rabbit cells are deficient in the synthesis of the resistance factors needed to inhibit DNA viruses. When cell cultures of rabbit origin were pretreated with homologous interferon and doubly infected with vaccinia and VSV, a variety of responses was observed. Vaccinia was able to rescue VSV from the inhibitory effects of interferon-induced resistance in two rabbit cell lines (RK-13 and RC-60), but not in RK-1337 cells. Similar experiments were carried out in mouse L cells and in primary chick embryo (CE) cell cultures; both RNA and DNA viruses were susceptible to inhibition by interferoninduced resistance in these cells. In L cells, double infection with vaccinia was able to rescue VSV; however, in CE ceII cuItures superinfection with vaccinia did not rescue VSV from the inhibitory effect of interferon. In these cells the synthetic pathways or virion factors furnished by vaccinia which are required for the rescue of VSV are sensitive to the action of interferon. INTRODUCTION
We have reported that two deoxyribonucleic acid (DNA) viruses, vaccinia and pseudorabies (PsRV) were refractory to inhibition in interferon-treated rabbit kidney cells (RK-13), whereas vesicular stomatitis virus (VSV), a ribonucleic acid (RNA) virus, was susceptible to inhibition (Youngner et al., 1972). In addition, we observed that double infection of interferon-treated RK-13 cells with vaccinia and VSV resulted in the rescue of VSV from the inhibitory action of interferon (Youngner et al., 1972; Thacore and Youngner, 1973a). The present paper reports the different sensitivities of DNA and RNA viruses to interferon when tested in other cell lines of rabbit origin. In addition, double infections with vaccinia and VSV were carried out in
mouse L cells and primary chick embryo (CE) cell cultures, in which both viruses are sensitive to inhibition by homologous interferons. These experiments were done to determine whether or not superinfection with vaccinia is able to rescue VSV from the resistance developed in these cells after pretreatment with interferon. Attempts to elucidate the mechanism by which vaecinia is able to rescue VSV from interferon-induced resistance are presented in a separate report (Thacore and Youngner, 197313). MATERIALS
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Cells. Mouse L cells (clone 929), primary chick embryo (CE) cells, and a rabbit kidney cell line (RN-13) were propagated as described earlier (Youngner et al., 1966, 1972). A second rabbit kidney cell line (RK-1337) 505
Copyright AU rights
AND
506
THACORE
AND
and a rabbit cornea cell line (RC-60) were obtained from the American Type Culture Collection, Roekville, Maryland. Eagle’s minimal essential medium (MEM) supplemented with either 10% calf serum (RK-13 and RK-1337 cells) or 10% fetal calf serum (RC-60 cells) was used as growth medium. viruses. A large-plaque mutant (4) of the Indiana strain of vesicular stomatitis virus (VSV) was propagated in L cells (Wertz and Youngner, 1970). Pseudorabies virus (PsRV) and vaccinia (strain Canada) were grown in RK-13 cells and assayed in CE cell monolayers as described previously (Youngner et al., 1972). Preparation and assay of interferon. Mouse and chicken interferons were prepared in L cells and in embryonated eggs, respectively, as described (Hallum and Youngner, 1966; Wagner, 1961). Rabbit interferon was prepared in RK-13 cells using Newcastle disease virus (NDV) as the inducer (Youngner et al., 1972). Interferon determinations were carried out in homologous cell cultures by the plaque-reduction method using VSV as the challenge virus (Youngner et al., 1966). Single-cycle virus yield reduction by interferon was measured as follows. Cell cultures were incubated in the presence or absence of interferon for 18 hr at 37”. The cultures were then infected with the appropriate virus at an input multiplicity of infection (m.o.i.) of 10, and adsorption was carried out for 2 hr at 37”. The cell cultures were washed twice to remove unadsorbed virus, refed with medium, and reincubated at 37”. After 18-20 hr, the virus yield was harvested by scraping the cells into the culture fluid, and the cell suspension was subjected to sonic oscillation prior to plaque assay on CE cell monolayers. Antisera.
High-titer
antisera
against
YOUNGNER
for IS hr at 37” in t’he presence of serial IO-fold dilutions of rabbit interferon (1000 to 0.1 unit). After removal of int,erferon, the cells were infected with either VSV, PsRV, or vaccinia at an input m.o.i. of 10, and single-cycle yield reduction tests were carried out as described above. In both cell lines, the two DNA viruses were markedly resistant to the effects of interferon (Tables 1 and 2). For example, with 1000 units of TABLE
1
SENSITIVITY OF RNA AND DNA INTERFERON IN RK-1337 CELL SINGLE-CYCLE YIELD INHIBITION
Pretreatment with interferon (units/ culture)
Virus yield (PFU/ml)
-
vsv
Vaccinia
3.4 x 106 1.4 X 106 (0.4)* 6.6 X lo5 (0.7) 5.2 X lo4 (1.8) 5.4 x 10”
5.2 X lo6 4.2 X 10” (0.1) 4.0 x 106 (0.1) 3.0 x 106
.0 0.1 1 10 loo
a Log,,
3.8 x 103 (3.0) drop in virus
I
5.6 x 107 4.8 X 10’ (0.1) 5.0 x 107 (0.1) 3.2 X lo7
(0.2)
-
(0.2)
yield.
TABLE
2
SENSITIVITY OF RNA AND DNA INTERFERON IN RC-60 CELL SINGLE-CYCLK YIELD INHIBITION
Pretreatment with interferon (units/ culture)
PsRV
2.6 x 106 1.6 X lo7 (0.5) (0.3) 8.0 x 105 1.6 X lo7 (0.5) (0.8) -I
(2.8) 1000
VIRUSES TO CULTURES: METHOD
VIRUSES CULTURES: METHOD
TO
Virus yield (PFU/ml)
vsv
Vaccinia
PsRV
VSV
and vaccinia were collected from rabbits injected with multiple doses of the appropriate virus. Specific anti-PsRV chicken serum was kindly supplied by Dr. Albert S. Kaplan, RESULTS
Sensitivity of RNA and DNA Viruses to Rabbit Interferon in RK-1337 and RC-60 Cells Cultures of RI<-1337 and RC-60 cells (2 X lo6 cells per culture) were incubated
0 0.1 1 10 106 1000
5.8 X lo6 3.8 X lo5 (0.2)Q 1.4 x 105
1.0 x zfr 1.2 x 107 (0.0) 1.4 x 10’
8.9 X lo8 8.9 X 108 (0.0) 2.2 x 108
(0.6)
(0.6)
(0.6)
7.2 X 10” (0.9) 3.0 x 104 (1.3) 4.2 X lo4 (1.1)
1.3 x 107 (0.0) 4.4 x 106 (0.3) 1.4 x 106
4.4 X 108 (0.3) 4.2 X 1Oa (0.3) 3.8 x 108 (0.4)
(1Log10 drop in virus
(0.8) yield.
RESCUE
OF VSV FROM
INTERFERON
interferon, in both cell lines vaccinia and PsRV yield was reduced by less than 1 log. In contrast, VSV yield was reduced almost 3 log by pretreatment of RK-1337 cells with 100 units of interferon (Table 1) and by more than 1 log in RC-60 cells pretreated with 100 units of interferon (Table 2). It should be noted that VSV replication in control, untreated RC-60 cells was defective, yielding less than 1 plaque-forming unit (PFU) per cell. It is evident from these results that VSV is poorly suited to testing the effect of interferon in RC-60 cells. However, the data show that, in both RK-1337 and in RC-60, the DNA viruses tested were markedly resistant to the inhibitory action of interferon, as reported previously with RKl3 cells (Youngner et al., 1972; Thacore and Youngner, 1973a). Double Infection Experiments. Rescue of VSV from Interferon-Treated Rabbit Cells In previous studies we have shown that vaccinia was able to rescue VSV from the inhibitory effect of interferon in RK-13 cells (Thacore and Youngner, 1973a; Youngner et al., 1972). It was of interest to determine whether similar rescue of VSV could be accomplished in RC-60 and RK-1337 cell lines. Monolayer cell cultures of RC-60 and RK-1337 cells were treated with 1000 units of rabbit interferon as described above. After 18 hr at 37”, groups of cultures were infected singly with either vaccinia or VSV. In case of doubly infected cultures, VSV was added simultaneously with vaccinia or at 2 or 5 hr after infection with vaccinia. Also included in this experiment were groups of RK-13 cell cultures pretreated with 1000 units of interferon, and then singly or doubly infected with VSV and vaccinia. Virus yields from infected cultures were harvested 18 hr after infection and assayed for infectivity. In the case of infected fluids which contained both VSV and vaccinia, specific immune sera were used to neutralize each of the viruses in order to be able to assay the other in CE cell monolayers by the plaque method. The plaque morphologies of the two viruses were readily distinguishable, and breakthrough of infectivity by the heterologous virus in the neutralized mixtures did not occur. Since there was no significant difference in yields obtained when VSV was
BY VACCINIA
507
added simultaneously or at 2 or 5 hr after vaccinia infection, only virus yields from cultures simultaneously infected with VSV and vaccinia are presented (Table 3). In agreement with results previously obtained with RK-13 cells (Youngner et al., 1972), double infection of control cultures with VSV and vaccinia produced approximately a Y-fold facilitation of VSV yield (group 3), whereas double infection of interferontreated ceils, resulted in a 1076-fold increase in VSV yield, compared to the yield obtained in interferon-treated cells infected with VSV alone (compare groups 4 and 6). Similar results were obtained with RC-60 cells except, as noted above, that VSV replicated poorly in control cultures (group 7). However, in the presence of vaccinia, VSV yield in control cultures was increased by more than 2 log (group 9). In interferon-treated cultures doubly infected with VSV and vaccinia, VSV yield increased by 187-fold, compared to maximally inhibited control cultures (compare groups 10 and 12). The results obtained in RK-1337 differed in several respects from those in RK-13 and RC-60 cell lines. For example, there was a decrease in VSV yield in control cultures doubly infected with VSV and vaccinia (compare groups 13 and 15) ; whereas a significant increase was observed under similar experimental conditions in RK-13 (group 3) and RC-60 (group 9) cell lines. In double infection of interferon-treated RK1337 cells there was no significant increase of VSV yield (compare groups 16 and 18). These results suggest a relation between the facilitation of VSV replication by vaccinia in control cultures of RK-13 and RC-60 cells and the increase in VSV yield in interferon-treated cultures doubly infected with VSV and vaccinia. The lack of VSV rescue from interferon-treated RK-1337 cultures superinfected with vaccinia is not understood at the present time. Experiments similar to those described above for rabbit cell lines were conducted in mouse L cells and in primary CE cell cultures in order to determine whether vaccinia, a virus which has been shown to be sensitive to homologous interferon in L and CE cells (Joklik and Merigan, 1966; Youngner et al., 1972)) is able to rescue VSV from the inhibitory effect of interferon.
508
THACORE
AND
YOUNGNER
TABLE
3
EFFECT OF PRETREATMENT WITH INTERFERON ON SINGLF: AND UOUBLE: INFECTION LINES WITH VACCINIA AND VESICULAR STOMATITIS VIRUS (VSV) Experimental group
Cell line
Pretzia;ment
Virus infection (m.0.i. = 10)
interferon (1000 units per culture)
Vaccinia
VSV
-
+ + -t +
RK-13 RK-13 RK-13 RK-13 RK-13 RK-13
+ + +
+ + + +
7 8 9 10 11 12
RC-60 RC-60 RC-60 RC-60 RC-60 RC-60
+ + +
+ + + +
13 14 15 16 17 18
RK-1337 RK-1337 RK-1337 RK-1337 RK-1337 RK-1337
+ + +
+ + + +
-
-
+ + -t + + + + +
Sensitivity of Vaccinia to Interferon in L Cells We have reported earlier that in L cells, VSV and PsRV were sensitive to the action of interferon when tested by the single-cycle yield reduction method (Youngner et al., 1972). An experiment was conducted to determine the sensitivity of vaccinia using the single-cycle yield reduction method as described above. VSV and PsRV were also tested for comparison. VSV and PsRV yields were reduced by 5.2 and 4.5 log, respectively, in cells pretreated with 1000 units of mouse interferon, confirming our previous results (Youngner et al., 1972). Under similar experimental conditions vaccinia was somewhat less sensitive to interferon; vaccinia yield was reduced by only 1.2 log in L cells pretreated with 1000 units of interferon. Double Infection Experiments in L Cells Experiments were done to determine whether VSV and PsRV could be rescued
CKLL
Virus yield (PFU/ml) Vaccinia
1 2 3 4 5 6
OF RABBIT
PFU/ml
vsv Log10 drop
PFU/ml
Log10 Fold increase over drop maximally inhibited control
2.8 x 107 1.3 x 107 1.2 x 106
1.1
2.8 x 106 3.6 X lo6
0.7 0.6
8.0 x 107 2.6 X lo4
3.0
1.2 x 107
0.4
1076
6.2 X lo5 2.4 X lo6 2.0 x 105
1.0
1.0 x 106 3.8 x 105
0.3 0.8
1.2 x 108 4.8 x 104
1.1
9.0 x 106
187
3.8 X 10’ 8.0 X 10” 4.6 X lo6
0.3
3.2 X lo6 2.2 x 106
0.4 0.6
5.0 x 106 3.4 x 104 5.0 x 104
3.0 1.4
from the inhibitory effect of interferon in L cells by superinfection with vaccinia. L cell cultures were treated with 800 units of mouse interferon or with growth medium and singly or doubly infected with VSV and vaccinia in the usual manner. In the case of doubly infected cultures, VSV was added either simultaneously with vaccinia or at 2, 3, and 5 hr after infection with vaccinia. Virus yields were harvested 18 hr after infection and assayed for infectivity (Table 4). Double infection of control cultures with vaccinia and VSV produced no significant facilitation of VSV replication (compare group 2 to 3-6), whereas vaccinia yield was significantly reduced (compare group 1 to 3-6). In interferon-treated L cells infected with VSV alone, the yield was reduced by 4.1 log (group 8). However, in interferon-treated cells doubly infected with VSV and vaccinia, VSV was rescued from the effect of interferon when VSV was added simultaneously with
RESCUE
OF VSV FROM
INTERFERON TABLE
509
BY VACCINIA
4
EFFECT OF PRETREATMENT WITH INTERFERON ON SINGLE AND DOUBLE INFECTION OF L CELLS WITH VACCINIA AND VESICULAR STOMATITIS VIRUS (VSV) -
Experiment group
-
-
Virus infection Icm.0.i. = 10)
Pretreatnnent with i nterferon
(800
units/ culture)
Vaccinia
Interval between vaccinia and VSV infection 04
vsv
T
Virus yield
vsv Log10
Log10 PFU/ml
PFU/ml
drop
drop
--
+ -
-
+ + + +
7 8
+ +
9 10 11 12
+ + + +
1 2
-
-
+
2.0 x 107 1.8 X 108 8.0 9.0 1.4 2.8
+ -
+ + + + +
+ + + +
+ + + +
4.6 2.0 5.4 4.6
-
-
lo5 105 106 lo6
1.4 1.4 1.1 0.8
1.0 x 106
1.3
x x x X
X x x X
vaccinia or at 2, 3, or 5 hr after vaccinia infection. Increases of 1880- to 5000-fold in VSV yield (groups 9-12) were recorded compared to the VSV yield in interferontreated cultures (group 8) which showed maximal inhibition of VSV. A similar experiment was carried out to determine the effect, of superinfection with vaccinia on the replication of PsRV in L cells pretreated with interferon. It was not feasible to test the ability of vaccinia to rescue PsRV since vaccinia markedly inhibited the replication of PsRV in L cells. Double Infection
Fold increase of VSV over maximally inhibited control (group 8)
Experiments in CE Cells
We reported earlier that in GE cells both VSV and vaccinia are sensitive to the resistance induced by homologous interferon (Youngner et al., 1972). The sensitivity of vaccinia to chicken interferon was significantly greater in CE cells than to mouse interferon in L cells; for example, in cells pretreated with 1000 units of homologous interferon. vaccinia was inhibited bv 1.3
106 106 105 lo5
-
1.6 2.0 1.6 1.6
5.6 3.2 4.0 2.5
X X x X
lo8 lo8 108 lo8
0.4 0.2 0.3 0.1
1.6 X lo4
4.1
3.0 8.0 7.6 8.0
0.8 0.4 0.4 0.4
x X X X
107 lo7 lo7 lo7
-
1880 5ooo 4750 54loo
log in L cells and by 2.7 log in CE cells (Youngner et al., 1972). Double infection experiments as described above for rabbit and L cells were also conducted in CE cells to determine whether vaccinia could rescue VSV from the action of interferon. Monolayers (2 X lo6 cells) were pretreated with 60 units of chicken interferon or with medium and then singly or doubly infected with VSV and vaccinia in the usual manner. Virus yields were harvested at 18 hr and assayed for infectivity in CE cell monolayers (Table 5). In control cultures without interferon simultaneously infected with VSV and vaccinia, yields of both viruses decreased by 1 log (compare groups l-3 and 2 and 3). However, when vaccinia replication was allowed to proceed for several hours before superinfection with VSV, vaccinia yield was inhibited to a lesser extent, e.g., when VSV was added to the culture 5 hr after vaccinia infection, vaccinia yield was not very different from the yield obtained from cultures sin& infected with vaccinia (compare group
510
THACORF
ANI)
YOUNGNER
TABLE
5
EFFECT OF PRETREATMIWT WITH INTERFERON ON SINGLI: AND DOUBLE INFECTION OF CHICK EMHRYO CELL CULTURH Experimental group
WITH VACCINIA AND VMICULAR
PretreatVirus infection (m.o.i. = 5) ment with interferon Vac(60 units VSV cinia per culture)
1 2
-
+
+ -
3 4 5 6
-
+ + + +
+ + + +
7 8
+ +
+
+ -
9 10 11 12
+ + + +
+ + + +
+ + + +
Interval between vaccinia andVSV infection (hr)
STOMATITIS VIRUS
(VSV)
Virus yield Vaccinia PFU/ml
VW Log10 drop
PFU/ml
Log10 Fold increase drop over maximally inhibited control (grOUP7)
6.0 X 108 2.6 X lo7 0 2 3 5
0 2 3 5
106 10” 10” 107
1.1 0.8 0.5 0.4
2.2 x 106
1.0
4.2 7.0 3.2 6.8
1.8 1.5 1.9 1.6
2.0 3.6 6.9 1.0
2 to 6). In contrast’, VW yield was inhibited by 1 log when VSV was added either simultaneously with vaccinia or 2, 3, or 5 hr after vaccinia infection (compare group 1 to 3-6). In interferon-treated cultures singly infected with VW or vaccinia, the virus yield decreased by 2.7 and 1.0 log, respectively (groups 7 and 8). No significant increase in WV yield was observed in interferon-treated cultures doubly infected with VSV and vaccinia (compare group 7 to 9-12). DISCUSSION
The present study has reinforced our previous observation (Youngner et al., 1972) that DNA viruses are refractory to inhibition in interferon-treated cell cultures of rabbit origin. In addition to the RK-13 cell line originally tested, another cell line of rabbit kidney origin (RK-1337) and a rabbit cornea cell line (RC-60) behaved similarly. The replication of two DNA viruses, vaccinia and PsRV, was not inhibited in any of the interferon-treated rabbit cell lines; in contrast, the replication of VW, an RNA virus, was inhibited in all three rabbit cell lines
x X X x
X x X x
lo6 105 lo5 105
7.2 6.8 6.0 6.6
X X X x
10’ lo7 107 107
0.9 1.0 1.0 1.0
1.2 X lo6
2.7
lo6 106 lo6 lo6
1.9 2.0 2.0 2.1
7.2 6.8 5.6 4.8
X x X X
6 6 4 4
under similar conditions. Preliminary experiments with herpes simplex virus types 1 and 2 show that these DNA viruses are also refractory to inhibition in RK13 cells pretreated with rabbit interferon (Thacore and Youngner, unpublished data). The consistent failure of interferon-induced resistance to affect the replication of DNA viruses in rabbit cell lines suggests that most, if not all, rabbit cells in culture may lack t’he ability to synthesize resistance factor(s) necessary to inhibit DNA viruses. The results provide additional support for our previous suggestion t’hat there may be separate resistance factors for RNA and DNA viruses and that cultured rabbit cells are deficient in the synthesis of sufficient resistance factors t’o inhibit the DNA viruses tested. In addition to being insensitive to the inhibitory action of interferon in rabbit cell lines, vaccinia was able to rescue VW from inhibition in doubly infected cells which had been pretreated with interferon. However, there were some differences in the behavior of the three rabbit cell lines used in these
RESCUE OF VSV FROM INTERFERON
studies. For example, vaccinia was able to rescue VSV from the inhibitory effect of interferon in the RK-13 and RC-60 cell lines, whereas
in
RK-1337
cells
there
was
no
significant rescue of VSV. Although the reasons for the lack of rescue of VSV in RK-1337 cells are not understood, a correlat’ion can be made between the ability of vaccinia (1) to facilitate the replication of VSV in rabbit cells not pretreated with interferon and (2) to rescue VSV in interferontreated rabbit cells. In RK-13 and RC-60 cell lines, both facilitation and rescue of VSV were observed, while in RK-1337 cells there was neither facilitation nor rescue of VSV in interferon-treated cells. These findings suggest that the same mechanism may be involved in the facilitation and rescue of VSV by superinfection with vaccinia. This possibility had not been favored by us in an earlier publication which dealt only with RK-13 cells (Youngner et al., 1972). By the use of mouse L cell and primary light was shed CE cell cultures, additional on the possible
mechanisms
involved
in the
rescue of VSV by superinfection with vaccinia. Homologous interferon is capable of inhibiting the replication of both RNA (vaccinia and PsRV) (VSV) and DNA viruses in these cells. In the case of L cells, superinfection with vaccinia rescued VSV in cells pretreated with mouse interferon. Since vaccinia did not rescue VSV from interferon-induced is possible that
resistance in CE cells, it in these cells the synthetic
pathways or virion products required for the rescue of VSV are sensitive to the action of interferon. Evidence has been obtained which indicates that early vaccinia DNAdependent RNA synthesis is required for the rescue of VSV in interferon-treated cell cultures (Thacore and Youngner, 1973b).
BY VACCINIA
511
ACKNOWLEDGMENTS The excellent assistance of Marion Kelly is gratefully acknowledged. This work was supported by Public Health Service research grant’ AI-06264 from the National Institute of Allergy and Infectious Diseases. REFERENCES J. V., and YOUNGNER, J. S. (1966). Quantitative aspects of inhibition of virus replication by interferon in chick embryo cell cultures. J. Bacterial. 92, 1047-1050. JOKLIK, W. K., and MERIGAN, T. C. (1966). Concerning the mechanism of action of interferon. Proc. Nat. Acad. hi. U.S. 56, 558665. THACORE, H. R., and YOUNGNXR, J. S. (1973a). Different sensitivity of ribonucleic and deoxyribonucleic acid viruses to resistance induced in rabbit cells (RK-13) by polyriboinosinic acidpolyribocytidilic acid. Infect. Zmmun. 7, 685690. THACORE, H. R., and YOUNGNER, J. S. (1973b). Rescue of vesicular stomatitis virus from interferon-induced resistance by superinfection with vaccinia virus. II. Effect of UV-inactivated vaccinia and metabolic inhibitors. Virology 56, 512-522. WAGNER, R. R. (1961). Biological studies of interferon. I. Suppression of cellular infection with Eastern equine encephalomyelitis virus. Virology 13, 323-327. WERTZ, G. W., and YOUNGNF,R, J. S. (1970). Interferon production and inhibit,ion of host synthesis in cells infected with vesicular stomatitis virus. J. Viral. 6, 476484. YOUNGNICR, J. S., SCOTT, A. W., HALLUM, J. V., and STINEBRING, W. It. (1966). Interferon production by inactivated Newcastle disease virus in cell cultures and in mice. J. Bacterial. 92, 862868. YOUNGNER, J. S., THACORE, H. R., and KELLY, M. E. (1972). Sensitivity of ribonucleic acid and deoxyribonucleic acid viruses to different species of interferon in cell cultures. J. Viral. 10, 171178.
HALLUM,
VIROLOGY
Rescue
of Vesicular Resistance
Stomatitis
Virus
by Superinfection
from with
Interferon-Induced Vaccinia
Virus
I. Rescue in Cell Cultures from Different Species HARSHAD Department
of Microbiology,
R. THACORE School of Medicine,
JULIUS
AND
University
S. YOUNGNER
of Pittsburgh,
Pittsburgh,
Pennsylvania
16361
Accepted September 4, 1973 The replication of two DNA viruses, vaccinia and pseudorabies (PsRV), was not inhibited in three cell lines of rabbit origin (RK-13 and RK-1337, rabbit kidney; and RC-60, rabbit cornea) which had been pretreated with rabbit interferon. In contrast, the replication of vesicular stomatitis virus (VSV), an RNA virus, was susceptible to interferon-induced resistance in rabbit cell lines. These results reinforced the possibility that there are separate interferon-induced resistance factors for RNA and DNA viruses and that cultured rabbit cells are deficient in the synthesis of the resistance factors needed to inhibit DNA viruses. When cell cultures of rabbit origin were pretreated with homologous interferon and doubly infected with vaccinia and VSV, a variety of responses was observed. Vaccinia was able to rescue VSV from the inhibitory effects of interferon-induced resistance in two rabbit cell lines (RK-13 and RC-60), but not in RK-1337 cells. Similar experiments were carried out in mouse L cells and in primary chick embryo (CE) cell cultures; both RNA and DNA viruses were susceptible to inhibition by interferoninduced resistance in these cells. In L cells, double infection with vaccinia was able to rescue VSV; however, in CE ceII cuItures superinfection with vaccinia did not rescue VSV from the inhibitory effect of interferon. In these cells the synthetic pathways or virion factors furnished by vaccinia which are required for the rescue of VSV are sensitive to the action of interferon. INTRODUCTION
We have reported that two deoxyribonucleic acid (DNA) viruses, vaccinia and pseudorabies (PsRV) were refractory to inhibition in interferon-treated rabbit kidney cells (RK-13), whereas vesicular stomatitis virus (VSV), a ribonucleic acid (RNA) virus, was susceptible to inhibition (Youngner et al., 1972). In addition, we observed that double infection of interferon-treated RK-13 cells with vaccinia and VSV resulted in the rescue of VSV from the inhibitory action of interferon (Youngner et al., 1972; Thacore and Youngner, 1973a). The present paper reports the different sensitivities of DNA and RNA viruses to interferon when tested in other cell lines of rabbit origin. In addition, double infections with vaccinia and VSV were carried out in
mouse L cells and primary chick embryo (CE) cell cultures, in which both viruses are sensitive to inhibition by homologous interferons. These experiments were done to determine whether or not superinfection with vaccinia is able to rescue VSV from the resistance developed in these cells after pretreatment with interferon. Attempts to elucidate the mechanism by which vaecinia is able to rescue VSV from interferon-induced resistance are presented in a separate report (Thacore and Youngner, 197313). MATERIALS
@ 1973 by Academic Press, of reproduction in any form
Inc. reserved.
METHODS
Cells. Mouse L cells (clone 929), primary chick embryo (CE) cells, and a rabbit kidney cell line (RN-13) were propagated as described earlier (Youngner et al., 1966, 1972). A second rabbit kidney cell line (RK-1337) 505
Copyright AU rights
AND
506
THACORE
AND
and a rabbit cornea cell line (RC-60) were obtained from the American Type Culture Collection, Roekville, Maryland. Eagle’s minimal essential medium (MEM) supplemented with either 10% calf serum (RK-13 and RK-1337 cells) or 10% fetal calf serum (RC-60 cells) was used as growth medium. viruses. A large-plaque mutant (4) of the Indiana strain of vesicular stomatitis virus (VSV) was propagated in L cells (Wertz and Youngner, 1970). Pseudorabies virus (PsRV) and vaccinia (strain Canada) were grown in RK-13 cells and assayed in CE cell monolayers as described previously (Youngner et al., 1972). Preparation and assay of interferon. Mouse and chicken interferons were prepared in L cells and in embryonated eggs, respectively, as described (Hallum and Youngner, 1966; Wagner, 1961). Rabbit interferon was prepared in RK-13 cells using Newcastle disease virus (NDV) as the inducer (Youngner et al., 1972). Interferon determinations were carried out in homologous cell cultures by the plaque-reduction method using VSV as the challenge virus (Youngner et al., 1966). Single-cycle virus yield reduction by interferon was measured as follows. Cell cultures were incubated in the presence or absence of interferon for 18 hr at 37”. The cultures were then infected with the appropriate virus at an input multiplicity of infection (m.o.i.) of 10, and adsorption was carried out for 2 hr at 37”. The cell cultures were washed twice to remove unadsorbed virus, refed with medium, and reincubated at 37”. After 18-20 hr, the virus yield was harvested by scraping the cells into the culture fluid, and the cell suspension was subjected to sonic oscillation prior to plaque assay on CE cell monolayers. Antisera.
High-titer
antisera
against
YOUNGNER
for IS hr at 37” in t’he presence of serial IO-fold dilutions of rabbit interferon (1000 to 0.1 unit). After removal of int,erferon, the cells were infected with either VSV, PsRV, or vaccinia at an input m.o.i. of 10, and single-cycle yield reduction tests were carried out as described above. In both cell lines, the two DNA viruses were markedly resistant to the effects of interferon (Tables 1 and 2). For example, with 1000 units of TABLE
1
SENSITIVITY OF RNA AND DNA INTERFERON IN RK-1337 CELL SINGLE-CYCLE YIELD INHIBITION
Pretreatment with interferon (units/ culture)
Virus yield (PFU/ml)
-
vsv
Vaccinia
3.4 x 106 1.4 X 106 (0.4)* 6.6 X lo5 (0.7) 5.2 X lo4 (1.8) 5.4 x 10”
5.2 X lo6 4.2 X 10” (0.1) 4.0 x 106 (0.1) 3.0 x 106
.0 0.1 1 10 loo
a Log,,
3.8 x 103 (3.0) drop in virus
I
5.6 x 107 4.8 X 10’ (0.1) 5.0 x 107 (0.1) 3.2 X lo7
(0.2)
-
(0.2)
yield.
TABLE
2
SENSITIVITY OF RNA AND DNA INTERFERON IN RC-60 CELL SINGLE-CYCLK YIELD INHIBITION
Pretreatment with interferon (units/ culture)
PsRV
2.6 x 106 1.6 X lo7 (0.5) (0.3) 8.0 x 105 1.6 X lo7 (0.5) (0.8) -I
(2.8) 1000
VIRUSES TO CULTURES: METHOD
VIRUSES CULTURES: METHOD
TO
Virus yield (PFU/ml)
vsv
Vaccinia
PsRV
VSV
and vaccinia were collected from rabbits injected with multiple doses of the appropriate virus. Specific anti-PsRV chicken serum was kindly supplied by Dr. Albert S. Kaplan, RESULTS
Sensitivity of RNA and DNA Viruses to Rabbit Interferon in RK-1337 and RC-60 Cells Cultures of RI<-1337 and RC-60 cells (2 X lo6 cells per culture) were incubated
0 0.1 1 10 106 1000
5.8 X lo6 3.8 X lo5 (0.2)Q 1.4 x 105
1.0 x zfr 1.2 x 107 (0.0) 1.4 x 10’
8.9 X lo8 8.9 X 108 (0.0) 2.2 x 108
(0.6)
(0.6)
(0.6)
7.2 X 10” (0.9) 3.0 x 104 (1.3) 4.2 X lo4 (1.1)
1.3 x 107 (0.0) 4.4 x 106 (0.3) 1.4 x 106
4.4 X 108 (0.3) 4.2 X 1Oa (0.3) 3.8 x 108 (0.4)
(1Log10 drop in virus
(0.8) yield.
RESCUE
OF VSV FROM
INTERFERON
interferon, in both cell lines vaccinia and PsRV yield was reduced by less than 1 log. In contrast, VSV yield was reduced almost 3 log by pretreatment of RK-1337 cells with 100 units of interferon (Table 1) and by more than 1 log in RC-60 cells pretreated with 100 units of interferon (Table 2). It should be noted that VSV replication in control, untreated RC-60 cells was defective, yielding less than 1 plaque-forming unit (PFU) per cell. It is evident from these results that VSV is poorly suited to testing the effect of interferon in RC-60 cells. However, the data show that, in both RK-1337 and in RC-60, the DNA viruses tested were markedly resistant to the inhibitory action of interferon, as reported previously with RKl3 cells (Youngner et al., 1972; Thacore and Youngner, 1973a). Double Infection Experiments. Rescue of VSV from Interferon-Treated Rabbit Cells In previous studies we have shown that vaccinia was able to rescue VSV from the inhibitory effect of interferon in RK-13 cells (Thacore and Youngner, 1973a; Youngner et al., 1972). It was of interest to determine whether similar rescue of VSV could be accomplished in RC-60 and RK-1337 cell lines. Monolayer cell cultures of RC-60 and RK-1337 cells were treated with 1000 units of rabbit interferon as described above. After 18 hr at 37”, groups of cultures were infected singly with either vaccinia or VSV. In case of doubly infected cultures, VSV was added simultaneously with vaccinia or at 2 or 5 hr after infection with vaccinia. Also included in this experiment were groups of RK-13 cell cultures pretreated with 1000 units of interferon, and then singly or doubly infected with VSV and vaccinia. Virus yields from infected cultures were harvested 18 hr after infection and assayed for infectivity. In the case of infected fluids which contained both VSV and vaccinia, specific immune sera were used to neutralize each of the viruses in order to be able to assay the other in CE cell monolayers by the plaque method. The plaque morphologies of the two viruses were readily distinguishable, and breakthrough of infectivity by the heterologous virus in the neutralized mixtures did not occur. Since there was no significant difference in yields obtained when VSV was
BY VACCINIA
507
added simultaneously or at 2 or 5 hr after vaccinia infection, only virus yields from cultures simultaneously infected with VSV and vaccinia are presented (Table 3). In agreement with results previously obtained with RK-13 cells (Youngner et al., 1972), double infection of control cultures with VSV and vaccinia produced approximately a Y-fold facilitation of VSV yield (group 3), whereas double infection of interferontreated ceils, resulted in a 1076-fold increase in VSV yield, compared to the yield obtained in interferon-treated cells infected with VSV alone (compare groups 4 and 6). Similar results were obtained with RC-60 cells except, as noted above, that VSV replicated poorly in control cultures (group 7). However, in the presence of vaccinia, VSV yield in control cultures was increased by more than 2 log (group 9). In interferon-treated cultures doubly infected with VSV and vaccinia, VSV yield increased by 187-fold, compared to maximally inhibited control cultures (compare groups 10 and 12). The results obtained in RK-1337 differed in several respects from those in RK-13 and RC-60 cell lines. For example, there was a decrease in VSV yield in control cultures doubly infected with VSV and vaccinia (compare groups 13 and 15) ; whereas a significant increase was observed under similar experimental conditions in RK-13 (group 3) and RC-60 (group 9) cell lines. In double infection of interferon-treated RK1337 cells there was no significant increase of VSV yield (compare groups 16 and 18). These results suggest a relation between the facilitation of VSV replication by vaccinia in control cultures of RK-13 and RC-60 cells and the increase in VSV yield in interferon-treated cultures doubly infected with VSV and vaccinia. The lack of VSV rescue from interferon-treated RK-1337 cultures superinfected with vaccinia is not understood at the present time. Experiments similar to those described above for rabbit cell lines were conducted in mouse L cells and in primary CE cell cultures in order to determine whether vaccinia, a virus which has been shown to be sensitive to homologous interferon in L and CE cells (Joklik and Merigan, 1966; Youngner et al., 1972)) is able to rescue VSV from the inhibitory effect of interferon.
508
THACORE
AND
YOUNGNER
TABLE
3
EFFECT OF PRETREATMENT WITH INTERFERON ON SINGLF: AND UOUBLE: INFECTION LINES WITH VACCINIA AND VESICULAR STOMATITIS VIRUS (VSV) Experimental group
Cell line
Pretzia;ment
Virus infection (m.0.i. = 10)
interferon (1000 units per culture)
Vaccinia
VSV
-
+ + -t +
RK-13 RK-13 RK-13 RK-13 RK-13 RK-13
+ + +
+ + + +
7 8 9 10 11 12
RC-60 RC-60 RC-60 RC-60 RC-60 RC-60
+ + +
+ + + +
13 14 15 16 17 18
RK-1337 RK-1337 RK-1337 RK-1337 RK-1337 RK-1337
+ + +
+ + + +
-
-
+ + -t + + + + +
Sensitivity of Vaccinia to Interferon in L Cells We have reported earlier that in L cells, VSV and PsRV were sensitive to the action of interferon when tested by the single-cycle yield reduction method (Youngner et al., 1972). An experiment was conducted to determine the sensitivity of vaccinia using the single-cycle yield reduction method as described above. VSV and PsRV were also tested for comparison. VSV and PsRV yields were reduced by 5.2 and 4.5 log, respectively, in cells pretreated with 1000 units of mouse interferon, confirming our previous results (Youngner et al., 1972). Under similar experimental conditions vaccinia was somewhat less sensitive to interferon; vaccinia yield was reduced by only 1.2 log in L cells pretreated with 1000 units of interferon. Double Infection Experiments in L Cells Experiments were done to determine whether VSV and PsRV could be rescued
CKLL
Virus yield (PFU/ml) Vaccinia
1 2 3 4 5 6
OF RABBIT
PFU/ml
vsv Log10 drop
PFU/ml
Log10 Fold increase over drop maximally inhibited control
2.8 x 107 1.3 x 107 1.2 x 106
1.1
2.8 x 106 3.6 X lo6
0.7 0.6
8.0 x 107 2.6 X lo4
3.0
1.2 x 107
0.4
1076
6.2 X lo5 2.4 X lo6 2.0 x 105
1.0
1.0 x 106 3.8 x 105
0.3 0.8
1.2 x 108 4.8 x 104
1.1
9.0 x 106
187
3.8 X 10’ 8.0 X 10” 4.6 X lo6
0.3
3.2 X lo6 2.2 x 106
0.4 0.6
5.0 x 106 3.4 x 104 5.0 x 104
3.0 1.4
from the inhibitory effect of interferon in L cells by superinfection with vaccinia. L cell cultures were treated with 800 units of mouse interferon or with growth medium and singly or doubly infected with VSV and vaccinia in the usual manner. In the case of doubly infected cultures, VSV was added either simultaneously with vaccinia or at 2, 3, and 5 hr after infection with vaccinia. Virus yields were harvested 18 hr after infection and assayed for infectivity (Table 4). Double infection of control cultures with vaccinia and VSV produced no significant facilitation of VSV replication (compare group 2 to 3-6), whereas vaccinia yield was significantly reduced (compare group 1 to 3-6). In interferon-treated L cells infected with VSV alone, the yield was reduced by 4.1 log (group 8). However, in interferon-treated cells doubly infected with VSV and vaccinia, VSV was rescued from the effect of interferon when VSV was added simultaneously with
RESCUE
OF VSV FROM
INTERFERON TABLE
509
BY VACCINIA
4
EFFECT OF PRETREATMENT WITH INTERFERON ON SINGLE AND DOUBLE INFECTION OF L CELLS WITH VACCINIA AND VESICULAR STOMATITIS VIRUS (VSV) -
Experiment group
-
-
Virus infection Icm.0.i. = 10)
Pretreatnnent with i nterferon
(800
units/ culture)
Vaccinia
Interval between vaccinia and VSV infection 04
vsv
T
Virus yield
vsv Log10
Log10 PFU/ml
PFU/ml
drop
drop
--
+ -
-
+ + + +
7 8
+ +
9 10 11 12
+ + + +
1 2
-
-
+
2.0 x 107 1.8 X 108 8.0 9.0 1.4 2.8
+ -
+ + + + +
+ + + +
+ + + +
4.6 2.0 5.4 4.6
-
-
lo5 105 106 lo6
1.4 1.4 1.1 0.8
1.0 x 106
1.3
x x x X
X x x X
vaccinia or at 2, 3, or 5 hr after vaccinia infection. Increases of 1880- to 5000-fold in VSV yield (groups 9-12) were recorded compared to the VSV yield in interferontreated cultures (group 8) which showed maximal inhibition of VSV. A similar experiment was carried out to determine the effect, of superinfection with vaccinia on the replication of PsRV in L cells pretreated with interferon. It was not feasible to test the ability of vaccinia to rescue PsRV since vaccinia markedly inhibited the replication of PsRV in L cells. Double Infection
Fold increase of VSV over maximally inhibited control (group 8)
Experiments in CE Cells
We reported earlier that in GE cells both VSV and vaccinia are sensitive to the resistance induced by homologous interferon (Youngner et al., 1972). The sensitivity of vaccinia to chicken interferon was significantly greater in CE cells than to mouse interferon in L cells; for example, in cells pretreated with 1000 units of homologous interferon. vaccinia was inhibited bv 1.3
106 106 105 lo5
-
1.6 2.0 1.6 1.6
5.6 3.2 4.0 2.5
X X x X
lo8 lo8 108 lo8
0.4 0.2 0.3 0.1
1.6 X lo4
4.1
3.0 8.0 7.6 8.0
0.8 0.4 0.4 0.4
x X X X
107 lo7 lo7 lo7
-
1880 5ooo 4750 54loo
log in L cells and by 2.7 log in CE cells (Youngner et al., 1972). Double infection experiments as described above for rabbit and L cells were also conducted in CE cells to determine whether vaccinia could rescue VSV from the action of interferon. Monolayers (2 X lo6 cells) were pretreated with 60 units of chicken interferon or with medium and then singly or doubly infected with VSV and vaccinia in the usual manner. Virus yields were harvested at 18 hr and assayed for infectivity in CE cell monolayers (Table 5). In control cultures without interferon simultaneously infected with VSV and vaccinia, yields of both viruses decreased by 1 log (compare groups l-3 and 2 and 3). However, when vaccinia replication was allowed to proceed for several hours before superinfection with VSV, vaccinia yield was inhibited to a lesser extent, e.g., when VSV was added to the culture 5 hr after vaccinia infection, vaccinia yield was not very different from the yield obtained from cultures sin& infected with vaccinia (compare group
510
THACORF
ANI)
YOUNGNER
TABLE
5
EFFECT OF PRETREATMIWT WITH INTERFERON ON SINGLI: AND DOUBLE INFECTION OF CHICK EMHRYO CELL CULTURH Experimental group
WITH VACCINIA AND VMICULAR
PretreatVirus infection (m.o.i. = 5) ment with interferon Vac(60 units VSV cinia per culture)
1 2
-
+
+ -
3 4 5 6
-
+ + + +
+ + + +
7 8
+ +
+
+ -
9 10 11 12
+ + + +
+ + + +
+ + + +
Interval between vaccinia andVSV infection (hr)
STOMATITIS VIRUS
(VSV)
Virus yield Vaccinia PFU/ml
VW Log10 drop
PFU/ml
Log10 Fold increase drop over maximally inhibited control (grOUP7)
6.0 X 108 2.6 X lo7 0 2 3 5
0 2 3 5
106 10” 10” 107
1.1 0.8 0.5 0.4
2.2 x 106
1.0
4.2 7.0 3.2 6.8
1.8 1.5 1.9 1.6
2.0 3.6 6.9 1.0
2 to 6). In contrast’, VW yield was inhibited by 1 log when VSV was added either simultaneously with vaccinia or 2, 3, or 5 hr after vaccinia infection (compare group 1 to 3-6). In interferon-treated cultures singly infected with VW or vaccinia, the virus yield decreased by 2.7 and 1.0 log, respectively (groups 7 and 8). No significant increase in WV yield was observed in interferon-treated cultures doubly infected with VSV and vaccinia (compare group 7 to 9-12). DISCUSSION
The present study has reinforced our previous observation (Youngner et al., 1972) that DNA viruses are refractory to inhibition in interferon-treated cell cultures of rabbit origin. In addition to the RK-13 cell line originally tested, another cell line of rabbit kidney origin (RK-1337) and a rabbit cornea cell line (RC-60) behaved similarly. The replication of two DNA viruses, vaccinia and PsRV, was not inhibited in any of the interferon-treated rabbit cell lines; in contrast, the replication of VW, an RNA virus, was inhibited in all three rabbit cell lines
x X X x
X x X x
lo6 105 lo5 105
7.2 6.8 6.0 6.6
X X X x
10’ lo7 107 107
0.9 1.0 1.0 1.0
1.2 X lo6
2.7
lo6 106 lo6 lo6
1.9 2.0 2.0 2.1
7.2 6.8 5.6 4.8
X x X X
6 6 4 4
under similar conditions. Preliminary experiments with herpes simplex virus types 1 and 2 show that these DNA viruses are also refractory to inhibition in RK13 cells pretreated with rabbit interferon (Thacore and Youngner, unpublished data). The consistent failure of interferon-induced resistance to affect the replication of DNA viruses in rabbit cell lines suggests that most, if not all, rabbit cells in culture may lack t’he ability to synthesize resistance factor(s) necessary to inhibit DNA viruses. The results provide additional support for our previous suggestion t’hat there may be separate resistance factors for RNA and DNA viruses and that cultured rabbit cells are deficient in the synthesis of sufficient resistance factors t’o inhibit the DNA viruses tested. In addition to being insensitive to the inhibitory action of interferon in rabbit cell lines, vaccinia was able to rescue VW from inhibition in doubly infected cells which had been pretreated with interferon. However, there were some differences in the behavior of the three rabbit cell lines used in these
RESCUE OF VSV FROM INTERFERON
studies. For example, vaccinia was able to rescue VSV from the inhibitory effect of interferon in the RK-13 and RC-60 cell lines, whereas
in
RK-1337
cells
there
was
no
significant rescue of VSV. Although the reasons for the lack of rescue of VSV in RK-1337 cells are not understood, a correlat’ion can be made between the ability of vaccinia (1) to facilitate the replication of VSV in rabbit cells not pretreated with interferon and (2) to rescue VSV in interferontreated rabbit cells. In RK-13 and RC-60 cell lines, both facilitation and rescue of VSV were observed, while in RK-1337 cells there was neither facilitation nor rescue of VSV in interferon-treated cells. These findings suggest that the same mechanism may be involved in the facilitation and rescue of VSV by superinfection with vaccinia. This possibility had not been favored by us in an earlier publication which dealt only with RK-13 cells (Youngner et al., 1972). By the use of mouse L cell and primary light was shed CE cell cultures, additional on the possible
mechanisms
involved
in the
rescue of VSV by superinfection with vaccinia. Homologous interferon is capable of inhibiting the replication of both RNA (vaccinia and PsRV) (VSV) and DNA viruses in these cells. In the case of L cells, superinfection with vaccinia rescued VSV in cells pretreated with mouse interferon. Since vaccinia did not rescue VSV from interferon-induced is possible that
resistance in CE cells, it in these cells the synthetic
pathways or virion products required for the rescue of VSV are sensitive to the action of interferon. Evidence has been obtained which indicates that early vaccinia DNAdependent RNA synthesis is required for the rescue of VSV in interferon-treated cell cultures (Thacore and Youngner, 1973b).
BY VACCINIA
511
ACKNOWLEDGMENTS The excellent assistance of Marion Kelly is gratefully acknowledged. This work was supported by Public Health Service research grant’ AI-06264 from the National Institute of Allergy and Infectious Diseases. REFERENCES J. V., and YOUNGNER, J. S. (1966). Quantitative aspects of inhibition of virus replication by interferon in chick embryo cell cultures. J. Bacterial. 92, 1047-1050. JOKLIK, W. K., and MERIGAN, T. C. (1966). Concerning the mechanism of action of interferon. Proc. Nat. Acad. hi. U.S. 56, 558665. THACORE, H. R., and YOUNGNXR, J. S. (1973a). Different sensitivity of ribonucleic and deoxyribonucleic acid viruses to resistance induced in rabbit cells (RK-13) by polyriboinosinic acidpolyribocytidilic acid. Infect. Zmmun. 7, 685690. THACORE, H. R., and YOUNGNER, J. S. (1973b). Rescue of vesicular stomatitis virus from interferon-induced resistance by superinfection with vaccinia virus. II. Effect of UV-inactivated vaccinia and metabolic inhibitors. Virology 56, 512-522. WAGNER, R. R. (1961). Biological studies of interferon. I. Suppression of cellular infection with Eastern equine encephalomyelitis virus. Virology 13, 323-327. WERTZ, G. W., and YOUNGNF,R, J. S. (1970). Interferon production and inhibit,ion of host synthesis in cells infected with vesicular stomatitis virus. J. Viral. 6, 476484. YOUNGNICR, J. S., SCOTT, A. W., HALLUM, J. V., and STINEBRING, W. It. (1966). Interferon production by inactivated Newcastle disease virus in cell cultures and in mice. J. Bacterial. 92, 862868. YOUNGNER, J. S., THACORE, H. R., and KELLY, M. E. (1972). Sensitivity of ribonucleic acid and deoxyribonucleic acid viruses to different species of interferon in cell cultures. J. Viral. 10, 171178.
HALLUM,