Antiviral Activity against Transmissible Gastroenteritis Virus, and Cytotoxicity, of Natural Porcine Interferons Alpha and Beta Hana M. Weingartl and J. Brian Derbyshire

I

I

RESUME

ABSTRACT

Porcine interferon (POIFN) -a preDe l'interferon porcin (INTPO)-a pared in primed peripheral blood recolte a partir de milieux de culture leukocyte cultures induced with de leucocytes stimules avec le virus Newcastle disease virus and POIFN-f de la maladie de Newcastle et de from PK-15 cell cultures induced with l'INTPO-3 rmueiii a partir de miiieux polyinosinic:polycytidylic acid were de culture de cellules PK-15 stimulees partially purified by precipitation with a I'aide d'acide polyinosinique: potassium thiocyanate and anion polycytidylique, ont ete purifies par exchange chromatography. Mean precipitation avec du thiocyanate de purification factors in terms of units potassium et par chromatographie a of POIFN per mg of protein, of 37 and echangeur d'anions. Apres purifica12 were obtained for POIFN-a and tion, 37 et 12 unitis d'INTPO et ,3 -POIFN-j. respectively. In yield reduc- par mg de proteine ont ete mesures. tion assays in swine testis and pig Dans des milieux de culture de cellules kidney cell cultures, POIFN-a and renales ou testiculaires porcines, les POIFN-3 had greater antiviral activ- INTPO et ont demontre une actiity against vesicular stomatitis virus vite antivirale plus grande contre le than against transmissible gastro- virus de la stomatite vesiculaire que enteritis virus (TGEV). The antiviral contre le virus de la gastroenterite effects were greater at higher concen- transmissible (GET) du porc. L'activite trations of interferon (IFN), and when antivirale etait plus grande lorsque la the IFN treatments were continued concentration d'INT 6tait augmentee postinfection. Porcine interferon-,B ou lorsque le traitement etait poursuivi showed greater antiviral activity apres l'infection des milieux par les against TGEV than POIFN-ax, but this virus. L'activite antivirale de l'INTPO-fl may have been partly due to cytotox- contre le virus de la GET etait supeicity. There were no major differences rieure A celle de l'INTPO-a mais cette in the antiviral activities of crude and difference pourrait etre associee a son partially purified IFN preparations. effet cytotoxique. Les preparations Both types of IFN showed antiviral brutes ou partiellement purifiees activity against TGEV in yield reduc- d'INTPO ont demontre les memes tion assays in porcine intestinal explant activites antivirales. Les deux extraits and intestinal epithelial cell cultures. d'INT ont aussi demontre une activite Crude POIFN-3 was found to be antivirale contre le virus de la GET sur rapidly cytotoxic, especially in porcine des cultures cellulaires de cellules intescells, and some fractions of partially tinales de porc. L'extrait non-purifie purified POIFN-,B were also cytotoxic. d'INTPO-j3 a rapidement provoque The cytotoxicity of POIFN-g was par- une activite cytotoxique sur les cellules tially neutralized by antibodies against d'organes porcins. Cette activite a human IFN-,3, but human IFN-,B was aussi ete notee partir de fractions not cytotoxic for porcine or bovine partiellement purifiees d'INTPO-(3. L'activit6 cytotoxique de l'INTPO-,8 a cells. a

a

partiellement ete neutraiisde par l'addition d'anticorps diriges contre l'INT-(3 humain. Par contre ce dernier ne possedait pas d'activite cytotoxique au niveau des cultures celiulaires d'origine porcine ou bovine. (Traduit par Dr Pascal Dubreuii)

INTRODUCTION Antiviral activity has been demonstrated for porcine interferon (POIFN)-a and POIFN-,B against a fairly wide range of animal viruses, including vesicular stomatitis virus (VSV), herpesviruses, influenza viruses, coronaviruses and enteroviruses (1). Among the coronaviruses tested, transmissible gastroenteritis virus (TGEV) was found to be moderately sensitive to both types of interferon (IFN). It is well established that the El glycoprotein of TGEV is a potent IFN inducer in vitro (2), and that high levels of IFN can be detected in the small intestine during lethal infections of newborn piglets with TGEV (3). High oral doses of recombinant bovine IFN, to which TGEV was sensitive in vitro, failed to protect piglets against virus challenge (4), and piglets which suckled sows whose milk contained POIFN-a also failed to resist challenge with TGEV (5). However, piglets in which POIFN-a was induced by a synthetic inducer showed a delay in onset of clinical signs when exposed to TGEV (5), although this increased resistance to challenge was attributed to activation of natural killer cells by the induced IFN, rather than to a direct antiviral effect (6).

Department of Veterinary Microbiology and Immunology, Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1. Reprint requests to Dr. J.B. Derbyshire. Submitted June 7, 1990.

Can J Vet Res 1991; 55: 143-149

143

In order to further characterize the antiviral activities of POIFN against TGEV, yield reduction assays were conducted in porcine cell cultures which were challenged with TGEV, and also for comparative purposes with VSV, which is highly sensitive to IFN. In addition yield reduction assays were conducted in porcine intestinal explant cultures challenged with TGEV, since the natural target cells for the virus in vivo are the enterocytes of the small intestine, and it is well recognized that the sensitivity of a virus to IFN is influenced by the host cell in which the virus replicates (7). Transmissible gastroenteritis virus has been cultivated previously in intestinal explants (8-10), but the effects of IFN on TGEV replication in explant cultures have not been described, although the replication of echovirus type 11 in human fetal intestinal organ cultures was inhibited by human leukocyte IFN (11). Finally, porcine intestinal epithelial cell cultures were also used in yield reduction assays. These cultures have not previously been used for virus cultivation. In all these experiments, both crude and partially purified preparations of POIFN-a and POIFN-j were used. During the course of these studies, observations were made on the cytotoxicity of POIFN, and these findings are also recorded.

3074, Becton-Dickinson Labware, Lincoln Park, New Jersey). The explants were covered with 0.5 mL of Eagle's minimum essential medium (EMEM) containing 5% FBS. The explant cultures were used for yield reduction assays after incubation for 6 h at 37°C in 5% CO2. PREPARATION OF INTERFERONS

-_w

Fig

3

,

.

.Mnlyr Wueo

cells U staine X100.

,

n

esia i

eihla

A stock of POIFN-a was prepared in primed porcine peripheral blood leukocyte cultures induced with Newcastle disease virus, and POIFN-,3 was prepared in PK-15 cell cultures induced with polyinosinic:polycytidylic acid (14). PARTIAL PURIFICATION OF INTERFERONS

The first step was precipitation with 0.5 M potassium thiocyanate (KCSNFisher Scientific, Don Mills, Ontario) as described (15). The precipitate was dissolved in 2 mM phosphate buffer, pH 7.14, treated with 0.1 mM DTT for 1 h and dialyzed overnight against phosphate buffer. For further purification, by anion-exchange chromatography on a MonoQ fast protein liquid chromatography (FPLC) column, the method of Stone-Wolff et al (16) was used, except that the starting buffer was 2 mM phosphate buffer pH 7.14. Fractions were eluted from the column with 2 mM phosphate buffer containing 0.5 M NaCl, collected into PBS with 0.1 mM DTT and titrated for antiviral activity, and for protein content by the Bradford method (17) with the Bio-Rad Protein Assay Kit II (Bio-

deprived SPF piglets, cut longitudinally and rinsed several times with 1 mM dithiothreitol (DTT - Sigma, St. Louis, Missouri) in phosphate buffered saline (PBS). The intestine was then cut into 1 mm2 explants, ten of which were placed into each well of a four-well Nunclon Delta tissue culture plate (Nunc, Rozkilde, Denmark) and incubated for 24 h at 37°C in 5% CO2 in CMRL 1066 medium with L-glutamine (Gibco/BRL, Burlington, Ontario) supplemented with 0.02%7o collagenase, 0.001%To bovine insulin (Sigma, St. Louis, Missouri) and 5% MATERIALS AND METHODS fetal bovine serum (FBS). The explants were then transferred to plates which CELL CULTURES had been pretreated by overlaying with Cell lines: The PK-1 5 porcine kidney porcine blood overnight at 4°C, then and Madin-Darby bovine kidney rinsing with PBS and sterilizing by Rad, Richmond, California). (MDBK) cell lines were obtained from ultraviolet irradiation 10 cm from a ASSAY OF INTERFERONS the American Type Culture Collection, Philips Sterilamp 782L-30 USA-K6 for and the stable swine testis (ST) line was 1 h. The explants were cultivated at Plaque reduction assay: This was conobtained from Diamond Laboratories, 370C in 5% C02, floating in ducted as described (18), in MDBK Des Moines, Iowa. The cells were CMRL 1066 medium supplemented cells for POIFN-ca and PK-1 5 cells for cultivated by standard procedures (12). with 0.00407o collagenase, 0.001 07o POIFN-,3, with VSV as the challenge and human lymphoblastoid insulin and 507o FBS. During incuba- virus, Secondary pig kidney (PK) cells: Pri- tion, IEC detached from the explants IFN-a (Lee Biomolecular Research mary cultures were prepared by disag- and attached to the serum protein Laboratories, San Diego, California) gregation of kidney tissue from coated plastic substrate. The medium as a standard. One unit (U) of IFN was newborn specific pathogen-free (SPF) was changed after seven days, and defined as the amount which reduced piglets, and subcultivated by standard after 2 wk the explants were removed. the number of plaques by 50%. methods (12) to give secondary cul- The cells were used in yield reduction tures for use in the yield reduction assays after 1-2 wk, when they had Cytopathic effect (CPE) reduction assay: This rapid microassay (19) was assays. formed monolayers (Fig. 1). used to titrate the fractions eluted from Intestinal epithelial cells (IEC): The Intestinal explants: Jejunal explants the chromatography columns during method was adapted from Quaroni were prepared as described above, and the purification of IFN by FPLC. and May (13). The jejunum was five explants were placed into each well Madin-Darby bovine kidney cells were removed from newborn, colostrum- of 24-well tissue culture plates (Falcon used for POIFN-ct and PK-15 cells for 144

POIFN-,B, with VSV as the challenge TABLE I. Partial purification of porcine interferons (POIFN) by KSCN precipitation and anion virus.

Yield reduction assay: This method (1) was used to determine the antiviral activities of POIFN-ca and POIFN-,B against TGEV and VSV in ST and PK cells, and against TGEV in intestinal explants and IEC cultures. The IFNs were used at concentrations of 100 U/mL and 40 U/mL in each assay except those using IEC, in which only 100 U/mL IFN was used because insufficient IEC cultures were available for both concentrations. The cultures were either pretreated with IFN for 18 h before infection with virus, or pretreated, challenged, and the IFN treatment continued for 48 h after infection. For virus challenge infection, 0.2 mL of virus containing 100 median cell culture infectious doses (CCID50) of the Indiana strain of VSV or the Diamond strain of TGEV which had been cultivated and titrated by microtiter infectivity assay in ST cells was added to each well. The supernatants were harvested 48 h after challenge infection and titrated by microtiter infectivity assay in ST cells. Untreated control cultures and unchallenged treated cultures were included in each test. CYTOTOXICITY ASSAYS

Macrotest: Porcine interferon samples were tested for cytotoxicity by a method based on the yield reduction assay, except that the cultures were not challenged with virus. Monolayers of ST, PK, MDBK and PK-15 cells were treated with 100 U of IFN/mL for 18 h, when the IFN was replaced with medium only and incubation continued for a further 48 h. In a second set of plates, after the 18 h initial exposure to IFN, fresh medium containing 100 U of IFN/mL was applied, and incubation continued for 48 h. At the end of incubation, the cell sheets were stained with neutral red (20) and the viability of the IFN-treated cells was expressed as a percentage of the viability of control cells which had not been treated with IFN. Microtest: Twofold dilutions of IFN were prepared in 80 ,L volumes in

96-well round-bottom microtiter plates, and 80,tL of MDBK or PK-15

exchange chromatography on a MonoQ column

Protein (mg/mL) U IFN/mL Sample 4.4 ± 0.1 1333 ± 461b Crude 2.1± 0.7 KSCN precipitate 1200 ± 400 0.07 ± 0.02 733 ± 115 MonoQ eluate 1.19 ± 0.03 POIFN-, 666 ± 231 Crude 0.73 ± 0.15 533 ± 115 KSCN precipitate 0.08 ± 0.04 533 ± 231 MonoQ eluate protein of U IFN/mg aPurification expressed in terms bAll volumes are mean ± SD of three experiments CNot applicable

Interferon POIFN-ca

cell suspension were added. After incubation for 18 h the plates were stained with 0.7507o crystal violet in 12% ethyl alcohol and 10%Mo formaldehyde for 10 min, and then washed with water. Wells which lacked a stained monolayer were recorded as positive for cytotoxicity, and one cytotoxic unit (U) was contained in the highest dilution of IFN which gave a positive result. Neutralization of cytotoxicity: Twofold dilutions of POIFN-, in 80 /AL volumes were applied along the y-axis of microtiter plates as in the microtest above. Twofold dilutions of equine antihuman IFN-a and IFN-,B polyclonal antibodies (Boehringer Mannheim Canada, Dorval, Quebec) were applied in 80 yiL volumes across the x-axis of the plates, and 80 AL of PK-15 cell suspension were added to each well. After incubation for 18 h the plates were stained with crystal violet as described above. The presence of stained monolayers indicated neutralization of the cytotoxicity of the IFN.

RESULTS PARTIAL PURIFICATION OF POIFN

Precipitation with KSCN in three experiments reduced the protein content of the IFN preparations by about 5007o, while the antiviral activity remained close to its original value (Table I). After treatment with DTT and anion exchange chromatography on a MonoQ column, POIFN-a was eluted from the column by 0.12 M NaCl, in advance of a major multiple peak of contaminating proteins, while POIFN-,B was eluted from the column by 0.19 M NaCl, immediately after a major protein peak. The mean purification factor obtained for

Logl0 U IFN/ mg protein 2.5 ± 0.1 2.7 ± 0.1 4.0 ± 0.1 2.7 ± 0.2 2.9 ± 0.2 3.8 ± 0.1

Purification factora NAc

2.16 ± 0.9 37.8 ± 13.8 NA 1.4 ± 0.1 12.6 ± 3.9

POIFN-ct was 37.8, and 12.6 for POIFN-,B (Table I). The highest specific activities after partial purification were 04 U/mg of protein for POIFNa and 103-9 U/mg of protein for POIFN-fl. ANTIVIRAL ACTIVITY OF POIFN

The results of the yield reduction assays with crude and partially purified POIFN-a in ST and PK-15 cells challenged with VSV and TGEV are given in Table II. The antiviral effect of the IFN was greater at the higher concentration and when the IFN treatment of the cells was continued postinfection. The antiviral effect was greater against VSV than TGEV, which always replicated to some extent in the IFNtreated cultures. There were no major differences in antiviral activity between the crude and partially purified preparations of POIFN-a. Porcine interferon-,B also showed greater antiviral activity against VSV than against TGEV in ST cells, but differences between the sensitivities of these viruses were less marked in PK cells, in which TGEV sometimes failed to replicate in the IFN-treated cultures (Table III). Porcine interferon-,B showed greater antiviral activity than POIFN-cx against TGEV. As for POIFN-a, continuation of the POIFN-,B treatments postinfection was usually more effective than preinfection treatment alone. The results of the yield reduction assays with TGEV in intestinal explants and IEC cultures are given in Table IV. The virus yields were lower than in ST and PK cells. There were no consistent differences in antiviral activity between POIFN-a and POIFN-,B in the explants and IEC. In the explant cultures, greater reductions in virus yield were usually obtained 145

TABLE II. Yield reduction assays of porcine interferon (POIFN)-a in swine testis (ST) and pig kidney (PK) cell cultures challenged with vesicular stomatitis (VSV) and transmissible gastroenteritis (TGEV) viruses

PK cells

ST cells Preparation Control Crude

POIFN-a Concentration (U/mL) 0 100 100 40 40

Partially purified

100 100

40 40

VSV Treatment Control Preinfection Pre- and postinfection Preinfection Pre- and postinfection

Yield 6.3a -c

Preinfection Pre- and postinfection Preinfection Pre- and postinfection

Yield reduction

NAb 6.3

TGEV Yield Yield reduction NA 5.1 2.0 3.1

VSV

Yield 6.3 0.6

Yield reduction NA 5.7

TGEV Yield reduction Yield NA 4.6 1.5 3.1

2.2

6.3 4.1

1.7 4.2

3.4 0.9

0.6 3.3

5.7 3.0

3.1 3.1

1.5 1.5

0.2

6.1

3.8

1.1

1.3

5.0

2.1

2.5

2.6

3.7

2.3

2.8

2.1

4.2

3.0

1.6

1.8 4.7

4.5 1.6

1.3

3.8 0.0

-

5.1

4.1

6.3 2.2

1.3 3.7

3.3 0.9

4.6

1.7

3.1

2.0

2.9

3.4

2.0

2.6

-

aYields of virus (log,OCCID50/0.1 mL) bNot applicable CNo viral infectivity detected

TABLE III. Yield reduction assays of porcine interferon (POIFN)-j3 in swine testis (ST) and pig kidney (PK) cell cultures challenged with vesicular stomatitis (VSV) and transmissible gastroenteritis (TGEV) viruses

ST cells

Preparation Control Crude

POIFN-, Concentration (U/mL) 0 100 100 40 40

Partially purified

100 100 40 40

aYields of virus

(log,0CCID50/0.1

VSV

Treatment Control Preinfection Pre- and postinfection Preinfection Pre- and postinfection

Yield 6.3a -c

Preinfection Pre- and postinfection Preinfection Pre- and postinfection mL)

Yield reduction NAb 6.3

PK cells

TGEV Yield Yield reduction 5.1 NA 1.3 5.8

VSV

-

Yield reduction NA 6.3

Yield 6.3

TGEV Yield Yield reduction NA 4.6 4.6

2.7

6.3 3.6

3.5

5.1 1.6

-

6.3 6.3

-

4.6 4.6

-

6.3

2.1

3.0

-

6.3

-

4.6

-

6.3

1.7

3.4

0.9

5.4

2.3

2.3

-

6.3 3.4

-

3.7

5.1 1.4

0.9 1.1

5.4 5.2

-

2.9

2.6

4.6 2.0

0.7

5.6

2.2

2.9

1.6

4.7

-

4.6

-

-

bNot applicable cNo viral infectivity detected

when the IFN treatments were continued postinfection, and the higher concentrations of IFN usually produced a greater reduction in yield than the lower concentrations. Transmissible gastroenteritis virus failed to replicate in the explants treated with 100 U of partially purified POIFN-a, but the crude POIFN-,B was more effective than the partially purified preparation, which was the least effective IFN in the explant cultures. In the untreated control explant cultures, lysis of the epithelial cells which had attached to the plastic plate was noted within 24 h of infection with TGEV, 146

while in the POIFN-treated cultures these cells remained intact. The virus failed to replicate (Table IV) in the IFN-treated IEC cultures, of which only sufficient were available to test a single concentration of IFN. These cells were lysed in the untreated controls, but they remained intact in the IFN-treated plates. CYTOTOXICITY OF POIFN

A toxic effect of POIFN on cell cultures was first observed in the experiments on partial purification, when fractions from the chromatography

columns containing high concentrations of POIFN-,B were found to be cytotoxic for PK-15 cells in the CPEreduction assay. No cytotoxicity was observed with POIFN-a in these experiments. The phenomenon was studied further in the macrotest for cytotoxicity with both types of IFN on ST, PK, PK-15 and MDBK cells, and the results are given in Table V. Porcine interferon-f was generally much more cytotoxic than POIFN-ca, and the porcine cells were more sensitive than the MDBK cells. The highest level of cytotoxicity was produced by crude

IFN-, on ST and PK cells. The cells in the cultures which showed cytotoxicity became rounded, and sometimes detached from the plates, especially after the 66 h treatments. It was also noted in the yield reduction assays in explant cultures (Table IV) that the epithelial cells attached to the plastic plates in the cultures treated with POIFN-a did not differ morphologically from those in the control plates, while in the explant cultures treated with POIFN-,B many of the epithelial cells became rounded (Fig. 2), although they remained attached to the plastic. When six samples of crude POIFN-3 were titrated for cytotoxicity by the microtest procedure in PK-15 cells, the antiviral titers were always four to eight times higher than the cytotoxicity titers (Table VI). However, when ten samples of partially purified POIFN-,B were tested in the same way, the ratios of antiviral:cytotoxic titers ranged from 1.3 to 12 in six samples, while the remaining four samples showed no detectable cytotoxicity. Sample numbers 5 and 6, and 8 and 9, of partially purified POIFN-3, were adjacent eluates from the same chromatography columns, but these showed rather widely different antiviral:cytotoxic titer ratios. Concentrations of human IFN-,B (Lee Biomolecular Research Laboratories, San Diego, California) of less than 2500 U/mL failed to cause

Fig. 2. Enterocytes in intestinal explant culture treated with 100 U/mL of porcine interferon-,8. Note rounded and refractile cells (arrows). Unstained X40.

TABLE IV. Yield reduction assays of porcine interferon (POIFN)-a and POIFN-,3 in intestinal explant and intestinal epithelial cell (IEC) cultures challenged with transmissible gastroenteritis (TGEV) virus Interferon Concentration (U/mL) Preparation 0 Control Crude 100 100 40 40

POIFN-a POIFN-a IEC Explants Explants IEC Yield Yield Yield reduction Yield Yield reduction Yield 2.6a NA 2.3 NAb 2.3 2.6 2.1 0.8 -c 0.5 1.8

Treatment Control Preinfection Pre- and post0.9 infection 2.5 Preinfection Pre- and post1.1 infection

1.7 0.1

NTd

0.1 0.8

2.5 1.8

NT

1.5

NT

0 .1

2.5

NT

2.6

NT

2.1

0.5

NT

2.6 1.5

NT NT

1.3 1.0

1.3 1.6

NT NT

2.1

NT

1.8

0.8

NT

Partially

purified

Preinfection Pre- and postinfection 1.1 40 Preinfection 40 Pre- and post0.5 infection aYields of TGEV (logl0 CCID50/0.1 mL) bNot applicable CNo viral infectivity detected dNot tested

100 100

cytotoxicity in PK-1 5 cells in the microtest procedure. In the test for neutralization of cytotoxic activity by antibodies against human IFN-a and IFN-,B, partial neutralization of the cytotoxicity of POIFN-,B was obtained with antibodies to human IFN-,B, since 1000 U of antibody neutralized 200 cytotoxic U of POIFN-,B.

DISCUSSION For the partial purification of POIFN we used the novel approach of anion exchange chromatography on FPLC, and this procedure gave specific activities of about 104 U/mg of protein, and mean purification factors of about 37 for POIFN-a and about 12 for POIFN-,B. The purification of POIFN-,B has not previously been described, and of the reports in the literature of the purification of POIFN-ca (21-23), only one (22) included data on the degree of purification, which was of the same order that we obtained. Vesicular stomatitis virus was found to be highly sensitive to both POINF-a and POIFN-,r, confirming earlier studies (reviewed in reference 1), and more sensitive than TGEV. In these experiments, TGEV seemed to be more sensitive to POIFN in cell cultures than reported previously (1), particularly to POIFN-3, although the low virus yields from the cultures treated with

POIFN-f might have been due in part to the cytotoxic effect of this IFN. Except for a single report of inhibition of the replication of echovirus type 11 by human IFN in human fetal intestinal organ cultures (11), intestinal explants and IEC have not been used previously for studies on the antiviral effects of IFN, although these cultures seem to provide a suitable model for enteric viruses with a predilection for intestinal epithelium. In the present study, the morphological observations on the cultures suggested that the enterocytes were indeed protected by the IFN treatment. The yields of virus were low from the untreated explant and IEC cultures. This may be because the epithelial cells which survived in these cultures resembled crypt cells, which are known to be relatively insensitive to TGEV compared with villous epithelial cells (24), and also because the Diamond strain of TGEV which was used was adapted to ST cell cultures, and may therefore replicate less well in intestinal epithelial cells. Nevertheless the findings are consistent with the hypothesis that the failure of TGEV to infect crypt cells in vivo might be due in part to the protection of these cells by IFN induced early in the infection. In addition to their antiviral activities, both POIFN-ae and POIFN-,B were found to affect the proliferation of porcine epithelioid cells and fibro147

TABLE V. Cytotoxicity of porcine interferons (POIFN) for ST, PK, PK-15 and MDBK cell cultures Cell viability as '70 of control cell viabilityb Duration of ST PK PK-15 treatment (h) MDBK 18 95 100 100 100 66 85 76 93 100 18 93 100 96 Partially purified a 100 66 66 64 82 67 47 98 Crude 18 58 100 66 10 21 58 80 74 Partially purified a 18 66 100 100 81 84 66 40 48 al00 U/mL POIFN used for all treatments bDetermined by neutral red staining at end of incubation

POIFNa Crude a

TABLE VI. Comparison of antiviral and cytotoxic activities of crude and partially purified porcine interferon-,B

Interferon sample number Crude 1 2 3 4 5 6

Partially purified 1 2 3 4 5 6 7

8 9 10

Antiviral titer (U/mL)

Cytotoxic titer (U/mL)

Ratio of antiviral/cytotoxic titer

400 400 1600 200 1600 40

50 100 400 25 400 100

8.0 4.0 4.0 8.0 4.0 4.0

200 1600 50 100 800 300 400 400 400 50

0 0 25 75 100 25 0 200 0 37

blasts. While the effect of POIFN-a was noticeable only in relatively high concentrations and with prolonged exposure, and could be ascribed to the well recognized antiproliferative activities of IFN (25), the cytotoxic effect of POIFN-,B was rapid. The only comparably rapid effect of IFN previously reported was also observed in porcine cells, treated with human, bovine or porcine IFN (26). The cytotoxic effect of POIFN-,f was correlated with its antiviral activity, but this was not so for partially purified POIFN-i. This difference, as well as the observed differences between the antiviral activities of crude and partially purified POIFN might be due to the removal of some subspecies of IFN during the purification process. It suggests that only certain subspecies of POIFN-, are highly cytotoxic. The partial neutralization of the cytotoxicity of POIFN-,B suggests that the cytotoxicity is a feature of the IFN 148

2.0 1.3 8.0 12.0 2.0 1.3

molecule, rather than of a contaminant, but the possibility that a toxic protein was copurified along with the IFN cannot be excluded. The high sensitivity of porcine cells to the cytotoxic effect of POIFN-, may be related to the relatively low yields of this IFN which are induced in cell cultures (14). Higher concentrations of IFN might be highly toxic to the induced cells. Much higher yields of human IFN-,B have been obtained by similar induction procedures (27), and in the present study, human IFN-,B was found to be much less toxic than

POIFN-3. ACKNOWLEDGMENTS These studies formed part of a thesis submitted by the senior author to the Faculty of Graduate Studies at the University of Guelph in partial fulfilment of the requirements for the

degree of Master of Science. The research was supported by the Natural Sciences and Engineering Research Council of Canada, and by the Ontario Ministry of Agriculture and Food. We acknowledge the technical assistance of Mrs. Connie E. Lesnick. REFERENCES 1. DERBYSHIRE JB. The interferon sensitivity of selected porcine viruses. Can J Vet Res 1989; 53: 52-55. 2. CHARLEY B, LAUDE H. Induction of alpha interferon by TGE coronavirus: role of transmembrane protein El. J Virol 1988; 62: 8-11. 3. LA BONNARDIERE C, LAUDE H. High interferon titer in newborn pig intestine dur-

ing experimentally induced viral enteritis. Infect Immun 1981; 32: 28-31. 4. MacLACHLAN NJ, ANDERSON KP. Effect of recombinant DNA-derived bovine a-I interferon on transmissible gastroenteritis virus infection in swine. Am J Vet Res 1986; 47: 1149-1152. 5. LOEWEN KG, DERBYSHIRE JB. The effect of interferon induction in parturient sows and newborn piglets on resistance to transmissible gastroenteritis. Can J Vet Res 1988; 52: 149-153. 6. LESNICK CE, DERBYSHIRE JB. Activation of natural killer cells in newborn piglets by interferon induction. Vet Immunol Immunopathol 1988; 18: 109-117. 7. STEWART WE. The Interferon System. New York: Springer-Verlag, 1979. 8. RUBENSTEIN D, TYRRELL DAJ, DERBYSHIRE JB, COLLINS AP. Growth of porcine transmissible gastroenteritis (TGE) virus in organ cultures of pig tissue. Nature 1970; 227: 1348-1349. 9. BOHAC J. Transmissible gastroenteritis virus: growth in organ culture, and agar gel precipitation reactions. PhD thesis, University of Guelph, 1973. 10. WILLIAMS PP, TARGOWSKI SP, SUBBA RAO MV. Organ culture of piglet intestinal explants: growth of viruses, incorporation of methyl-3H thymidine, and uptake of fluorescein isothiocyanate-labelled colostral cells. Microbiol Ther 1985; 15: 57-70. 11. ALBRIGHT DJ, WHALEN RA, BLACKLOW NR. Sensitivity of human foetal intestine to interferon. Nature 1974; 247: 218-220. 12. FRESHNEY RI. Culture of Animal Cells: A Manual of Basic Technique. New York: Alan R. Liss, Inc., 1987. 13. QUARONI A, MAY RJ. Establishment and characterization of intestinal epithelial cell cultures. Methods Cell Biol 1980; 21B: 403-427. 14. WEINGARTL HM, DERBYSHIRE JB. The induction and characterization of natural porcine interferons alpha and beta. Can J Vet Res 1990; 54: 349-354. 15. CANTELL K, HIRVONEN S. Large-scale production of human leukocytic interferon containing l08 units per ml. T Gen Virol 1978; 39: 541-543.

16. STONE-WOLFF DS, YIP YK, KELNER HC, LE J, HENRIKSEN-DESTEFANO D, RUBIN BY, RINDERNECHT E, AGGARWAL BB, VILCEK J. Interrelationships of human interferon gamma with lymphotoxin and monocyte cytotoxin. J Exp Med 1984; 159: 828-843. 17. BRADFORD MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976; 72: 248-254. 18. LOEWEN KG, DERBYSHIRE JB. Interferon induction with polyinosinic: polycytidylic acid in the newborn piglet. Can J Vet Res 1986; 50: 232-237. 19. FAMILLETTI PC, RUBINSTEIN S, PESTKA S. A convenient and rapid cytopathic effect inhibition assay for

20.

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Antiviral activity against transmissible gastroenteritis virus, and cytotoxicity, of natural porcine interferons alpha and beta.

Porcine interferon (POIFN)-alpha prepared in primed peripheral blood leukocyte cultures induced with Newcastle disease virus and POIFN-beta from PK-15...
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