The Induction and Characterization of Natural Porcine Interferons Alpha and Beta Hana M. Weingartl and J. Brian Derbyshire

graphy after treatment with dithiothreitol indicated a second disulphide The purpose of this study was to bond in POIFN-a which was not define optimum conditions for the essential for antiviral activity. Amino production of high concentrations of groups on the side chains of amino natural porcine interferon (POIFN)-a acids were shown to be essential for and POIFN-,B, and to characterize the the antiviral activities of POIFN-a IFNs which were produced. The and POIFN-13 since these were inducers used were Newcastle disease abrogated by acylation with benvirus (NDV), polyinosinic:poly- zoylchloride. POIFN-a was neutralcytidylic acid (poly IC), poly IC ized by antibodies against human complexed with diethylaminoethyl IFN-a, and the same antibodies dextran (poly IC-DEAEdx) and poly showed minor cross-reactivity with IC complexed with poly-L-lysine and POIFN-,8. Neither of the POIFNs was carboxymethylcellulose. The highest neutralized by antibodies against yields of POIFN-a were obtained human IFN-/3 or IFN-T. from porcine peripheral blood leukocyte (PBL) cultures induced with NDV. The concentrations of both cells RESUME and virus were critical for high yields of IFN, which were also enhanced by Cette etude visait a definir les priming. Poly IC was found to be a conditions optimisant la production relatively poor IFN inducer in PBL, in d'interferon-a et ,B porcins (INTPO) et which low yields were obtained only de les caracteriser. Les agents inducafter priming or in response to poly teurs etaient le virus de la maladie de IC-DEAEdx. POIFN-,B was prepared Newcastle (VMN), I'acide polyinosiby induction of the PK-15 cell line nique:polycytidylique (poly IC), le with poly IC or poly IC-DEAEdx. The poly IC combine au diethylaminoethyl highest yields were obtained from dextran (poly IC-DEAEdx) et le poly cultures induced 24 h after seeding, IC combine a la poly-L-lysine et au although when poly IC-DEAEdx or carboxymethyl-cellulose. La plus superinduction was used, the age of haute production de INTPO-a a ete the cells was less critical. Priming had obtenue a l'aide de cultures de little effect on the yields of POIFN-,B. leucocytes pris dans la circulation PK-15 cells induced with NDV gave (CLC) et mis en presence du VMN. La relatively low yields of IFN. Both ratio cellules: virus etait tres important POIFN-a and POIFN-,8 were classi- afin d'obtenir une haute concentration fied as type I IFN on the basis of their d'INT laquelle etait accentuee suite a resistance or susceptibility to pH 2.0, une presensibilisation. Le poly IC s'est ultracentrifugation, 560 C and trypsin revele etre un pauvre inducteur d'INT treatment. Disulphide bonds essential en CLC meme apres une presensibilifor antiviral activity were demon- sation, ou en reponse au poly ICstrated in both types of IFN by DEAEdx. L'INTPO-,B a ete obtenue reduction with 2- beta-mercaptoetha- suite aux contact de la lignee cellulaire nol, and anionic exchange chromato- PK-15 avec du poly IC ou du poly IC-

ABSTRACT

DEAEdx. Les plus hautes recoltes ont ete obtenues 24 heures apres l'ensemensement quoique lorsque le poly ICDEAEdx ou une superinduction etaient employes, l'age des cultures cellulaires devenait moins important. Une presensibilisation n'a eu que peu d'effet sur les quantites dINTPO-,3. Les cellules PK-15 mises en presence du VMN n'ont donne que des resultats metig's. LINTPO-a et sont classes comme faisant partie du type I 'a cause de leur resistance ou susceptibilite a pH 2,0, a l'ultracentrifugation, a la temperature de 560C et a la trypsine. Les ponts disulfures, essentiels a l'activite antivirale, sont presents dans les 2 types d'INT tel que demontre par reduction avec le 2,B-mercaptoethanol et par chromatographie d'echanges anioniques suite au traitement au dithiothreitol. Ces agents ont permis aussi de demontrer un autre pont disulfure sur la molecule dINTPO-a lequel n'etait pas essentiel a l'activite antivirale. Les groupes amino sur la chaine laterale d'acides amines ont ete demontres comme etant aussi essentiels a l'activite antivirale dINTPO-a et puisque l'acylation avec du chlorure de benzoyl empeche cette activite. L'INTPO-a a ete neutralise avec des anticorps diriges contre de lINT-a humain, par contre ces memes anticorps n'ont demontres que peu d'effet envers l'INTPO-13 aucun des deux INT n'a ete neutralise par des anticorps diriges contre l'INT-,8 et l'INT-T humains. (Traduit par Dr Pascal Dubreuil) INTRODUCTION Previous studies in this laboratory (1) and elsewhere (2,3) have demon-

Department of Veterinary Microbiology and Immunology, Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2WI. Reprint requests to Dr. J.B. Derbyshire. Submitted January 15, 1990.

Can J Vet Res 1990; 54: 349-354

349

strated antiviral activity in vitro of porcine interferon (POIFN)-a and POIFN-,B against a range of porcine viruses. In addition, POIFN-ai induction in newborn piglets produced a transient increase in resistance to challenge with transmissible gastroenteritis virus (TGEV) (4), as well as activation of natural killer cells (5) and modulation of the humoral immune response to vaccination with TGEV (6). In order to study further the antiviral and immune modulating activities of POIFNs, we wished to prepare POIFN-a and POIFN-,/ in sufficiently high concentrations for use in vivo. There are several reports in the literature of the production of POIFN-a in cultures of peripheral blood leukocytes (PBL) in response to induction with a range of viruses, including Sendai (7,8), influenza (9), Newcastle disease (10) and TGEV (11,12). However compared with human cells (13), the yields of IFN obtained have been relatively low. Porcine fibroblasts and epithelial cells have also produced lower yields of IFN-,B than human fibroblasts (14). The highest yields of POIFN-13 were obtained from embryonal kidney fibroblasts (15) by superinduction with polyinosinic:polycytidylic acid (poly IC) or from primed secondary pig kidney cells (3) induced with Newcastle disease virus (NDV). In earlier studies (2), a porcine kidney cell line (PK- 15) was induced with poly IC. The main objective of the present study was to develop in vitro procedures for the induction of high concentrations of POIFN-a and POIFN-,3. Newcastle disease virus, poly IC, and poly IC complexed with polycations were used as the inducers in porcine PBL and PK-15 cells, and the effects of inducer and cell concentration, and of priming and superinduction were studied. We also report some data on the physicochemical characteristics of POIFN-a and POIFN-,3, and on their antigenic relationships with human IFNs.

MATERIALS AND METHODS CELL CULTURES

The PK-15 porcine kidney and Madin-Darby bovine kidney (MDBK)

350

cell lines were obtained from the American Type Culture Collection. They were cultivated by standard procedures. Mitotic indices in PK-15 cells were determined by counting mitotic figures in at least 500 cells after staining with May-Grunwald-Giemsa (16). Peripheral blood leukocytes were separated from heparinized blood collected from healthy 3 to 8 wk old donor piglets from a specific pathogen-free herd as described (17), except that the monocyte removal step was omitted. Viability of the cells was determined by trypan blue exclusion. INTERFERON INDUCERS

The LaSota strain of NDV was cultivated in the allantoic sac of embryonated eggs and assayed by a standard hemagglutination (HA) microtest. The virus was partially purified and concentrated by ultracentrifugation at 100,000 g for 90 min through a 30% sucrose cushion in buffer containing 100 mM NaCl, 10 mM Tris pH 7.5 and 1 mM ethylenediaminetetraacetate. Poly IC (Miles Scientific), made up to 2 mg/ mL in normal saline, was reannealed by heating to 71° C for 1 h, and gradually cooled. A complex of poly IC with diethylaminoethyl dextran (poly IC-DEAEdx) was prepared by mixing equal volumes of reannealed poly IC and a stock solution in phosphate-buffered saline (PBS) of 0.25 g/mL DEAEdx (Sigma Chemical Co., molecular weight 500,000), and incubating the mixture at 37°C for 1 h. Poly IC was also complexed with poly-L-lysine and carboxymethylcellulose (poly IC-LC) as described (18). INDUCTION OF PORCINE INTERFERON-a

The method for induction with NDV was based on a previous study (19). Various concentrations of PBL were used, immediately after separation or following preincubation for 24 h or priming for 18 h with IFN at 370 C, in closed Erlenmeyer flasks under constant gentle shaking. The cultures were treated with various concentrations of NDV and incubated at 370C for 24 h. The cells were then removed by centrifugation at 400 g for 10 min, and the virus was removed by ultracentrifugation at 100,000 g for 90

min. For induction with poly IC, a similar protocol was used except that the cells were washed in PBS before and after treatment with poly IC, poly IC-DEAEdx or poly IC-LC for 1 h at 370 C, followed by incubation as above for 12 h. INDUCTION OF PORCINE

INTERFERON-:3

The methods were based on earlier studies with poly IC (2) and poly ICDEAEdx (20,21). PK-15 cells were subcultivated from two days old cultures into 25 cm2 plastic cell culture flasks (Nunclon) and used for IFN induction at various times after seeding. When priming was used, 24 h cultures were primed for 6 h, 36 h cultures for 12 h and older cultures for 24 h before induction. The monolayers were washed with PBS and treated for 1 h at 370 with 2 mL of poly IC or poly IC-DEAEdx. The inducer was removed, the cells washed again and incubated for 8 to 10 h at 370C in Eagle's minimum essential medium (EMEM) with 2% fetal bovine serum (FBS). The supernatants were then harvested and filtered through 0.22 ,um Millipore filters. For superinduction (22,23), after the induction period described above, the cell cultures were incubated at 370C for 3 h in 2 mL EMEM with 2% FBS and 0.005% cycloheximide (Sigma). This medium was then replaced by 2 mL EMEM with 2% FBS, 0.005% cycloheximide and 0.0005% actinomycin D (Sigma). After incubation for 2 h the cells were washed with PBS, and incubated for 8 to 10 h as above. For the induction of POIFN-,B with NDV, PK-15 cells were seeded into 80 cm2 flasks and cultivated for 48 h. They were then incubated for 18 or 24 h at 370 C in 10 mL of EMEM with 5% FBS and various concentrations of NDV. The supernatants were harvested, and the virus was removed by ultracentrifugation as before. INTERFERON ASSAYS

Culture supernatants were tested for antiviral activity by a plaque reduction assay, in MDBK cells for POIFN-a and in PK-15 cells for POIFN-,B, with vesicular stomatitis virus as the challenge virus (24), and human lymphoblastoid IFN-a (Lee

Biomolecular Research Laboratories) as a standard. One unit (U) of IFN was defined as the amount which caused a 50% reduction in the number of plaques. PHYSICOCHEMICAL CHARACTERIZATION OF INTERFERONS

used as a standard on each plate. After incubation for 18 h at 370 C in 5% C02, 50 ML/well of VSV containing 1.5 x 105 plaque-forming units/ mL were added. Neutralization of the antiviral activity of the IFN was indicated after incubation for a further 48 h by the presence of cytopathic effects.

Samples of POIFN-a and POIFN-

,B were characterized in accordance with the standard criteria (25) of susceptibility to trypsin and 560 C and resistance to pH 2.0 and ultracentrifugation. Two methods were used for the demonstration of disulphide bonds. Firstly, for the reduction of all disulphide bonds in the IFN molecule, samples of IFN were treated with 200 mM 2-beta mercaptoethanol (Sigma) at pH 7.4 for 24 h at 4°C (26). Antiviral activity was assayed before and after treatment. Secondly, IFN samples were treated with 0.1 mM dithiothreitol (DTT-Sigma) and dialyzed overnight against 2 mM phosphate buffer (Na2HPO4, KH2PO4) at pH 7.4. The dialyzed samples were applied to a MonoQ anionic exchange column for fast protein liquid chromatography as described (27), and eluted with 2 mM phosphate buffer containing 0.5 M NaCl. Each fraction was assayed for antiviral activity, and protein content was determined with the Bio-Rad Protein Assay Kit II. Control samples were derived from untreated IFN. For the demonstration of side chain amino groups, IFN samples were treated with 25 mM benzoylchloride (Sigma) at pH 7.4 for 2 h at 4°C, and tested for residual antiviral activity. ANTIGENIC RELATIONSHIPS OF PORCINE AND HUMAN INTERFERONS

RESULTS INDUCTION OF PORCINE INTERFERON-a

The results of the experiments in which poly IC and complexes of poly IC were used as inducers are given in Table I. Poly IC failed to induce IFN except in the cultures which were primed before induction. POIFN-a was produced in moderate amounts in PBL cultures induced with poly ICDEAEdx, but not in those induced with poly IC-LC. The results of the experiments in which NDV was used as the inducer are given in Table II. Generally higher yields of POIFN-a were obtained from the higher cell concentrations. The beneficial effect of preincubation of the PBL for 24 h was found to be associated with an increase in viable cell numbers during the preincubation period. Priming with IFN caused no

increase in cell numbers but there was an increased yield of IFN when a high concentration of PBL was used. At the conclusion of the induction period, the number of viable cells was reduced by approximately 70%, while the number of viable cells in control cultures which were not induced was reduced by only 30%. With increasing concentrations of virus, the IFN yield increased up to a certain virus concentration, beyond which the yield declined. Thus in a suspension containing 7.0 x 106 PBL/ mL, the highest IFN titer was obtained with 6,144 HA U/mL of NDV, while lower and higher concentrations of virus gave lower yields of IFN (Table II). The highest yield of IFN in these experiments was obtained from a suspension of 9.6 x 106 PBL/mL, primed with 64 U/ mL of POIFN-a for 18 h and induced with 4,915 HA U of NDV/ mL of cell suspension. INDUCTION OF PORCINE

INTERFERON-f,

The results of the experiments in which poly IC or poly IC-DEAEdx was used as the inducer are summarized in Table III. The age of the cells had a major influence on the yield of IFN, particularly in response to induction with poly IC, with the highest yields generally obtained from

TABLE I. Induction of porcine interferon-a (POIFN-a) from peripheral blood leukocytes (PBL) with polyinosinic:polycytidylic acid (poly IC) and complexes of poly IC No. of viable PBL (106/mL) Concentration Yield of of poly IC (mg/ mL) Inducer at induction POIFN-a(U/mL) 0 0.05 3.9 Poly IC 0.05 0 3.9 Poly ICa 3.9 0.05 200 Poly ICb 0 0.5 3.9 Poly IC 3.9 0.5 0 Poly ICa 0.5 150 3.4 Poly ICb 3.4 1.0 0 PolyIC 0 1.0 3.4 Polv ICa 200 3.4 1.0 Poly ICb 0 0.5 1.8 Poly IC 0 1.8 1.0 Poly IC 400 0.1 1.8 Poly IC DEAEdxc 400 1.7 0.1 Poly IC DEAEdxc 400 0.25 1.8 Poly IC DEAEdxc 200 0.5 1.8 Poly IC DEAEdxc 0 0.1 1.8 Poly ICLCd 0 0.25 1.8 Poly ICLCd 0 0.5 1.8 Poly ICLCd aPBL preincubated for 24 h before induction bPBL primed with 450 U/mL of POIFN-a for 24 h before induction cPBL preincubated, and induced with poly IC complexed with DEAE-dextran dPBL preincubated, and induced with poly IC complexed with poly-L-lysine and

Equine polyclonal antibodies against human IFN-a, IFN-,B and IFN-T were purchased from Boehringer Mannheim. Monolayers of MDBK cells in 96-well plates were used. The growth medium was removed and twofold dilutions of antihuman IFN antibody were applied across the x-axis in 100 ,L volumes, with 12 replicates of each dilution. Tenfold dilutions of the IFN sample were applied across the y-axis in 100 ,uL volumes, with seven replicates for each dilution. Human IFN-a was carboxymethylcellulose

351

TABLE II. Induction of porcine interferon-a (POIFN-a) from peripheral blood leukocytes (PBL) with Newcastle disease virus (NDV) No. of viable PBL (106/mL) Concentration at induction of NDV (HA U/mL) 2.0 186 2.0 930 4.4 2084 3277 4.4 4.4 4096 6.9 5120 6.9 6144 7.0 2048 Preincubation 7.0 4096 6144 Preincubation 7.0 Preincubation 8192 7.0 Preincubation 7.0 12,288 Preincubation 7.8 4096 Preincubation 9.6 4915 7.3 6144 Primingb Priming 9.6 4915 aPBL were preincubated for 24 h before induction bPBL were primed with 64 U/mL of POIFN-a for 18 h before induction

Pretreatment of PBL None None None None None None None Preincubationa

cells induced 24 h after seeding. When poly IC-DEAEdx or superinduction was used, the age of the cells was less critical, but superinduction never resulted in IFN yields higher than those induced by poly IC alone in the 24 h cultures. Priming had little effect on the yields of IFN. The highest yields of IFN were obtained from primed 24 h cultures induced with poly IC-DEAEdx or from unprimed 24 h cultures induced with poly IC. Cell viability was unaffected by treatment with poly IC or poly ICDEAEdx alone, but when induction was combined with priming there was some loss of viability, with viable cell counts as low as 50% of those in control cultures. Cell viability was further reduced, to approximately 10% of control values when superin-

Yield of POIFN-a (U/mL) 0 200 400 800 1600 3200 3200 1600 2400 3200 1600 800 3200 3200 3200 12,800

ANTIGENIC RELATIONSHIPS OF PORCINE AND HUMAN INTERFERONS

duction was used. The mitotic index (Table III) was highest in the PK-15 cells 36 h after seeding, and mitosis was not detected 72 h after seeding. When NDV (60 to 6,000 HA U/ mL) was used as the IFN inducer on PK-15 cells, the yields of IFN ranged from 0 to 400 U/mL, with the higher yields obtained after incubation for 24 h rather than 18 h. PHYSICOCHEMICAL CHARACTERIZATION OF PORCINE INTERFERONS

by reduction of their disulphide bonds by 2-beta-mercaptoethanol, and by acylation with benzoylchloride. Untreated POIFN-a was eluted from the MonoQ column with 0.14 M NaCl as a component of a major multiple peak of proteins, while DTTtreated POIFN-a was eluted with 0.12 M NaCl, in advance of the major peak. POIFN-,3 was eluted from the MonoQ column by a higher salt concentration (0. 19 M NaCl), and the elution patterns of untreated or DTT-treated POIFN-,3 were identical. There was no loss of antiviral activity following treatment of POIFN-a or POIFN-,B with DTT.

Relatively high cross-reactivity (80%) was demonstrated between POIFN-a and human IFN-a. Thus 1000 U of antibodies against human IFN-a neutralized 800 U of POIFN-a, and 100 U of IFN were neutralized by 125 U of antibodies. Very low crossreactivity (10%) between POIFN-f3 and human IFN-a was observed, since 100 U of POIFN-,j were neutralized by 1000 U of antibodies against human IFN-a. Antibodies against human IFN-f3 and human IFN-T failed to neutralize the antiviral activity of POIFN-a and POIFN-f3.

The antiviral activity of POIFN-a DISCUSSION and POIFN-,f was found to be resistant to pH 2.0 and ultracentrifuWhile we were able to produce gation, partially susceptible to heating and susceptible to trypsin treatment concentrations of POIFN-a and (Table IV). The antiviral activities of POIFN-,/ which were comparable to both types of IFN were also abolished those reported by other workers

TABLE III. The effects of priming and superinduction on the induction of porcine interferon-,f poly IC and poly IC-DEAEdx

(POIFN-,/) in PK-15 cells at intervals after seeding by

Mean yield of POIFN-,/ (U/ mL)a

Poly IC Hours after seeding of PK-15 cells (mitotic index) 24(0.023)

Unprimed

352

Poly IC-DEAEdx Unprimed

SuperSuperInduction induction Induction induction 400 320 1500 1500 700 320 200 200 400 200 0 100 320 0 100 1500 280 1250 0 200 320 1000 0 800 0 10 200 120 SD did not exceed ± 8.5% of the mean value for any treatment

Induction 2500

14 36(0.031) 0 48(0.003) 0 72(0.000) 0 96 (NT) 0 168 (NT) 0 240 (NT) aMeans of two to six samples per treatment; NT = not tested

Primed

Superinduction 1500 630 NT

1500 1000 800 NT

Primed

Induction 2500 320 400 400 800 400 320

TABLE IV. Physicochemical characterization of porcine interferons (POIFN)

Interferon titers (U/mL)

POIFN-f8

POIFN-a Treatment pH 2.0 Ultracentrifugation 56°C for 60 min Trypsin 2-beta-mercaptoethanol Benzoylchloride

(10,15), the yields obtained were lower than those reported for human IFNs (13,14), even though approaches which led to increased yields of human IFN, such as priming, superinduction and the complexing of poly IC with polycations, were followed. The reasons for the lower yields of porcine IFNs are not known, but the recent cloning and expression of the genes for POIFN-a (28) and POIFN-r (29) may provide the higher concentrations which are required for in vivo studies. For the induction of POIFN-a, the concentrations of the NDV inducer and of the PBL were found to be critical. The higher yields obtained after preincubation of the PBL probably resulted from an increase in the cell concentration, although spontaneous priming may have been involved since macrophages and lymphocytes have been shown to produce low titers of IFN upon cultivation (30), and a marked increase in the number of macrophages in porcine PBL cultures has been observed (15). Even higher yields of POIFN-a were obtained from PBL which were primed with IFN. Poly IC, which was tested for the first time as an IFN inducer in porcine PBL, gave relatively low yields, although it has been shown to induce IFN in human leukocytes in titers comparable to those obtained by virus induction (31). However, higher yields might have been obtained if higher concentrations of PBL had been used since the yields obtained by NDV induction were related to the cell concentration. In the present study, poly IC alone and poly IC-LC failed to induce IFN in PBL, while poly ICDEAEdx induced modest titers. This finding suggests that the critical factor for IFN induction in PBL may be associated with the cell membrane since DEAEdx increases the interac-

Control 1600 3200 3200 3200 1600 1600

Treated 1600 3200 1600 0 100 25

tion of poly IC with its receptor, facilitating uptake of poly IC by the cell (20,32). However, it is noteworthy that, while poly IC-LC failed to induce POIFN-a in vitro, it had previously been shown to be an effective inducer of IFN in pigs (18). The highest titers of POIFN-,B from PK-15 cells induced with poly IC were twice those previously reported (15). The critical factor in the induction of POIFN-/3 appeared to be the age of the PK-15 cells, and the use of priming, complexing of the poly IC with DEAEdx or superinduction, while facilitating the production of IFN from older cells, did not give higher yields than those obtained by induction of 24 h cultures with poly IC alone. Previous studies have shown that poly IC is a poor IFN inducer in aged chick embryo cells (33). The findings in the present study, supported by the mitotic indices, indicate that a particular stage of the cell cycle, most probably G2, may be critical for IFN induction. It has also been shown that in some cell cultures only a proportion of the cell population may be inducible at any given time (30), so it is possible that only a minor fraction of the PK-15 cells, which may vary with age, is capable of IFN production. The relatively low yields of POIFN-,1 from PK-15 cells induced with NDV were similar to those reported (3) from secondary pig kidney cells induced with the same virus. The effects of pH 2.0, ultracentrifugation and treatment with heat or trypsin confirmed that the preparations of POIFN-a and POIFN-,f fulfilled the usual criteria for acceptance of a viral inhibitor as a type I IFN (25). The presence of disulphide bonds in both POIFN-a and POIFN,8 was indicated by the abolition of their antiviral activity by treatment

Control 100 400 400 200 400 400

Treated 100 400 200 0 37 50

with 2-beta-mercaptoethanol. The finding of a changed elution pattern from an anionic exchange column after DTT treatment of POIFN-a, together with data on the amino acid composition of POIFN-a-1 (28), suggests that two types of disulphide bond may be present in this protein, one of which is readily accessible to reduction by DTT but not essential for its antiviral activity. Since the elution pattern of DTT-treated POIFN-,/ was unchanged, this IFN would appear to contain only a single type of disulphide bond, essential for its antiviral activity. In contrast to the present findings, inactivation of POIFN-a and POIFN-f3 by another high molecular weight reducing agent, dithioerythritol (DTE) has been reported (15), but no details were given of the concentration of DTE or the method of treatment. However, high molecular weight reducing agents have been shown by others to stabilize IFN (34), and the antiviral activities of POIFN-a and POIFN-13 on storage at 40C were found to be stabilized by DTT (35). In addition to disulphide bonds, amino groups on the side chains of amino acids appear to be essential for the antiviral activity of POIFN-a and POIFN-j3, since both IFNs were inactivated by acylation with benzoylchloride. Similar findings have been reported for chick and murine IFN-a and IFN-/3 (36). A high degree of homology between human IFN-a and POIFN-a has been reported at the genomic (28) and antigenic levels (9,15), and high crossreactivity between POIFN-a and antibodies against human IFN-a was found in the present study. The low level of neutralization of POIFN-/8 by the same antiserum confirmed similar findings by others (3,15). Antisera against human IFN-f3 or human IFN-

353

T failed to neutralize the antiviral activity of POIFN-a or POIFN-f3, but the reported demonstration of some neutralization of the cytotoxic effect of POIFN-,3 by antibodies against human IFN-/3 (35) suggests some homology between these IFNs.

9.

10.

ACKNOWLEDGMENTS The results of this study were included in a thesis submitted by the senior author to the Faculty of Graduate Studies of the University of Guelph in partial fulfilment of the requirements for the degree of Master of Science. The research was supported by the Ontario Ministry of Agriculture and Food, and by the Natural Sciences and Engineering Research Council of Canada. We acknowledge the technical assistance of Mrs. Connie E. Lesnick.

11.

12.

13.

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