10
INFECTION AND IMMUNITY, Mar. 1982, p. 1048-1057 0019-9567/82/031048-10$02.00/0 Vol. 35, No. 3 Susceptibility of Bovine Macrophages to Infectious Bovine Rhinotracheitis Virus Infectiont ANTHONY J. FORMAN,lt LORNE A. BABIUK,S.2* VIKRAM MISRA,2 AND FRANK BALDWIN3 Veterinary Infectious Disease Organization' and Departments of Veterinary Microbiology2 and Veterinary Anatomy,3 Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N OWO Received 10 July 1981/Accepted 27 October 1981 Infectious bovine rhinotracheitis virus replicated in cultured bovine alveolar macrophages (AM). However, yields of infectious virus were low, with maximum titers approximately 100 times that of the residual inoculum. Immunofluorescence and electron microscopic studies indicated that the majority of macrophages produced viral antigen, but after infection at a multiplicity of 0.1, only 4.1% of AM produced infectious centers. Virus-infected AM culture supernatants possessed interfering activity, probably due to interferon. Incubation of fresh AM with these fluids rendered them refractory to infection. Although AM from infectious bovine rhinotracheitis virus-immune and -susceptible donors were equally permissive and their susceptibility was unaltered by incubation with bacterial lipopolysaccha- ride, bovine mammary macrophages which were elicited with lipopolysaccharide became nonpermissive when further incubated for 48 h with 1 ,ug of lipopolysac- charide per ml. Under these conditions, infected mammary macrophages failed to synthesize viral DNA, and there was reduced synthesis of "late" viral polypep- tides. Macrophages are an important component of host defense against viral infection due to the roles they play in phagocytosis and killing of viruses, production of interferon, and destruc- tion of virus-infected cells, as well as in the development of immune responses. Although many viruses cannot replicate in macrophages (25), others will infect macrophages and initiate virus replicative activity. This may be manifest- ed by the synthesis of viral proteins and nucleic acid (36), elaboration of viral antigen (5, 26), production of intracellular viral capsids (29, 35), or release of infectious virus to the extracellular environment (6, 20, 28, 37). Viral infection may result in death of the macrophage or, alternative- ly, may lead to a persistent infection (20, 21, 38, 39) and pathology resulting from various degrees of immunosuppression. Macrophage permissiveness can be dependent on the age (15, 22) or the immune status (2) of the host and.on the anatomical site from which the macrophages were derived (30). Unfortu- nately, no generalization can be made for a specific family of viruses. For example, both human and mouse alveolar macrophages be- come restrictive with age of the host for herpes t Journal article no. 15 of the Veterinary Infectious Disease Organization. t Present address: Australian National Animal Health Lab- oratory, CSIRO, Geelong, Victoria 3220, Australia. simplex virus infection while remaining permis- sive for replication of cytomegalovirus (9, 37). In the case of infectious bovine rhinotracheitis (IBR) virus infection in athymic nude mice, the virus establishes a persistent infection with sub- sequent transformation of the macrophages (10). In cattle, IBR virus causes disease most com- monly in the respiratory tract but also in other organs (16). Extensive virus replication occurs, and, as with most herpesvirus infections, cell- mediated immunity is of major importance in recovery (8, 31, 32). The exact role of the macrophage in IBR virus infection and recovery is unknown. However, the virus has been impli- cated as a predisposing factor in the develop- ment of bacterial pneumonia in cattle (7, 14), and it is likely that this is mediated through an inhibition of alveolar macrophage (AM) func- tion. In view of this, we have initiated studies to determine whether IBR virus can infect bovine AM and, if so, what effect this has on AM function. This work was designed to determine the susceptibility of bovine AM to infection with IBR virus and factors which may influence the outcome. MATERIALS AND METHODS Calves. Holstein calves were obtained at birth and artificially reared in isolation. They were used be- tween the ages of 4 and 12 months. IBR-immune 1048 on August 20, 2020 by guest http://iai.asm.org/ Downloaded from

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INFECTION AND IMMUNITY, Mar. 1982, p. 1048-10570019-9567/82/031048-10$02.00/0

Vol. 35, No. 3

Susceptibility of Bovine Macrophages to Infectious BovineRhinotracheitis Virus Infectiont

ANTHONY J. FORMAN,lt LORNE A. BABIUK,S.2* VIKRAM MISRA,2 AND FRANK BALDWIN3

Veterinary Infectious Disease Organization' and Departments of Veterinary Microbiology2 and VeterinaryAnatomy,3 Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Saskatchewan,

Canada S7N OWO

Received 10 July 1981/Accepted 27 October 1981

Infectious bovine rhinotracheitis virus replicated in cultured bovine alveolarmacrophages (AM). However, yields of infectious virus were low, with maximumtiters approximately 100 times that of the residual inoculum. Immunofluorescenceand electron microscopic studies indicated that the majority of macrophagesproduced viral antigen, but after infection at a multiplicity of 0.1, only 4.1% ofAMproduced infectious centers. Virus-infected AM culture supernatants possessedinterfering activity, probably due to interferon. Incubation of fresh AM with thesefluids rendered them refractory to infection. Although AM from infectious bovinerhinotracheitis virus-immune and -susceptible donors were equally permissiveand their susceptibility was unaltered by incubation with bacterial lipopolysaccha-ride, bovine mammary macrophages which were elicited with lipopolysaccharidebecame nonpermissive when further incubated for 48 h with 1 ,ug of lipopolysac-charide per ml. Under these conditions, infected mammary macrophages failed tosynthesize viral DNA, and there was reduced synthesis of "late" viral polypep-tides.

Macrophages are an important component ofhost defense against viral infection due to theroles they play in phagocytosis and killing ofviruses, production of interferon, and destruc-tion of virus-infected cells, as well as in thedevelopment of immune responses. Althoughmany viruses cannot replicate in macrophages(25), others will infect macrophages and initiatevirus replicative activity. This may be manifest-ed by the synthesis of viral proteins and nucleicacid (36), elaboration of viral antigen (5, 26),production of intracellular viral capsids (29, 35),or release of infectious virus to the extracellularenvironment (6, 20, 28, 37). Viral infection mayresult in death of the macrophage or, alternative-ly, may lead to a persistent infection (20, 21, 38,39) and pathology resulting from various degreesof immunosuppression.Macrophage permissiveness can be dependent

on the age (15, 22) or the immune status (2) ofthe host and.on the anatomical site from whichthe macrophages were derived (30). Unfortu-nately, no generalization can be made for aspecific family of viruses. For example, bothhuman and mouse alveolar macrophages be-come restrictive with age of the host for herpes

t Journal article no. 15 of the Veterinary Infectious DiseaseOrganization.

t Present address: Australian National Animal Health Lab-oratory, CSIRO, Geelong, Victoria 3220, Australia.

simplex virus infection while remaining permis-sive for replication of cytomegalovirus (9, 37).

In the case of infectious bovine rhinotracheitis(IBR) virus infection in athymic nude mice, thevirus establishes a persistent infection with sub-sequent transformation of the macrophages (10).In cattle, IBR virus causes disease most com-monly in the respiratory tract but also in otherorgans (16). Extensive virus replication occurs,and, as with most herpesvirus infections, cell-mediated immunity is of major importance inrecovery (8, 31, 32). The exact role of themacrophage in IBR virus infection and recoveryis unknown. However, the virus has been impli-cated as a predisposing factor in the develop-ment of bacterial pneumonia in cattle (7, 14), andit is likely that this is mediated through aninhibition of alveolar macrophage (AM) func-tion.

In view of this, we have initiated studies todetermine whether IBR virus can infect bovineAM and, if so, what effect this has on AMfunction. This work was designed to determinethe susceptibility of bovine AM to infection withIBR virus and factors which may influence theoutcome.

MATERIALS AND METHODS

Calves. Holstein calves were obtained at birth andartificially reared in isolation. They were used be-tween the ages of 4 and 12 months. IBR-immune

1048

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INFECTION OF BOVINE MACROPHAGES WITH IBR VIRUS 1049

calves had been challenged by intranasal instillation ofIBR virus (Colorado-1 strain; ATCC VR-864) at least 3months before use as a source of macrophages. Theydeveloped typical clinical signs of disease and showeda serological response. IBR-susceptible calves wereperiodically monitored serologically to ensure thattheir status remained IBR negative.

Collection of macrophages and maintenance in cul-ture. AM were collected by the procedure of Wilkieand Markham (42). Calves were sedated with xylazine(Rompun; Cutter Laboratories, Mississauga, Ont.,Canada; 100 to 140 mg intravenously) and placed inleft lateral recumbency. A fiber optic endoscope waspassed via the trachea into the right diaphragmaticlobe. Lung lavage fluid contained 0.15 M saline, 5 mMglucose, 2 mM EDTA, 25 mM HEPES (N-2-hydrox-yethyl piperazine-N'-2-ethanesulfonic acid) buffer, 50,ug of gentamicin and per ml, 5 ,ug of amphotericin Bper ml, pH 7.2 (41). An initial wash was made bypassing 50 ml of fluid into the lung, of which about halfwas retrieved under mild vacuum and discarded. Thesecond wash involved injecting a further 250 ml intothe lung which was retrieved as above, and the bron-choalveolar cells present in the fluid were used forpreparation of AM cultures.The lavaged cells were filtered through four layers

of sterile gauze to remove particulate matter andmucus, centrifuged at 1,000 x g for 10 min, washedonce with Eagle minimal essential medium (MEM),and suspended in MEM with 5% fetal bovine serum(FBS). The cells were then inoculated into tissueculture plates. They were incubated for 3 h at 37°C in a5% C02-humidified atmosphere, washed twice withMEM to remove nonadherent cells, and reincubatedfor 18 to 24 h in MEM with 20% FBS.Mammary macrophages (MM) were collected 96 h

after the inoculation of a nonlactating mammary glandwith 5 ml of Escherichia coli lipopolysaccharide (LPS;0128, B12; Difco Laboratories, Detroit, Mich.) at aconcentration of 1 ,ug/ml. A 10-ml amount of MEMwas then injected into the gland, and the fluid, contain-ing cells from the lactiferous sinus, was expressedfrom the teat (40).The cells were layered onto 3 ml of Ficoll-Hypaque

(density at 25°C, 1.077 g/cm3) (Ficoll was obtainedfrom Pharmacia Fine Chemicals, Inc., Montreal, P.Q.,Canada; Hypaque was obtained from Winthrop, NewYork, N.Y.) in a round-bottom tube and centrifuged at400 x g for 20 min. The cells at the interface above theFicoll-Hypaque were aspirated, washed three times inMEM, suspended in MEM with 5% FBS, and culturedin the same manner as described for AM.

In experiments carried out to determine the effect ofLPS on macrophage permissiveness for viral replica-tion, AM and MM were washed 3 h after plating andreincubated for 48 h in MEM with either 20% endotox-in-free FBS or 20% FBS and LPS at a concentration of1 ,ug/ml. Macrophages cultured by these methods wereregarded as nonstimulated or stimulated populations,respectively.

Viral replication in macrophages. Macrophagemonolayers in 24-well cluster plates (Costar no. 3524;106 cells per ml) were washed with MEM and inoculat-ed with IBR virus at a high-input multiplicity ofinfection (approximately 10, based on PFU in Madin-Darby bovine kidney [MDBK] cells) and low-inputmultiplicity (approximately 0.1) and incubated for 1 h

at 37°C. The monolayers were then washed twice withMEM to remove unadsorbed virus, reincubated inMEM with 5% FBS, and observed microscopically forcytopathic effect. Monolayers ofMDBK cells, knownas permissive cells for IBR virus, were similarlyinfected for comparison.

Infected monolayers were harvested at varioustimes after infection by removing cells from duplicatewells with a rubber policeman and freezing the har-vested cells and fluid at -80°C. These were latertitrated for infectivity by plaque assay on MDBK cellmonolayers in microtiter plates with an antibody over-lay (32).

Fluorescent-antibody staining. Cultures were grownin two-well chamber slides (no. 4802; Lab-Tek Prod-ucts, Westmont, Ill.) and infected at various multiplic-ities in the usual manner. After infection, cells wereassessed for the presence of viral antigens by indirectimmunofluorescence. Cells were fixed with acetonefor 15 min at room temperature and overlaid withbovine anti-IBR antibody. After incubation at roomtemperature for 30 min, the antibody was removed bywashing three times with phosphate-buffered saline(pH 7.2), followed by a further 30 min of incubationwith fluorescein-conjugated rabbit anti-bovineimmunoglobulin G (Cappel Laboratories, Cochran-ville, Pa.). Slides were washed, observed, and photo-graphed with the aid of a Leitz Orthomat microscope,using transmitted UV light.

Interferon assays. Interferon levels were determinedby assaying the ability of culture fluids from virus-infected macrophages to inhibit vesicular stomatitisvirus replication in MDBK cells as described previous-ly (3). Briefly, culture fluids from virus-infected mac-rophages were incubated for 1 h with anti-IBR serumto neutralize any IBR virus present. Twofold dilutionsof the culture fluids were made in MEM plus 5% FBSand incubated with confluent MDBK cells for 24 h.The culture fluids were removed, and cultures wereinfected with 100 PFU of vesicular stomatitis virus andoverlaid with 1% methylcellulose. After 24 h themethylcellulose overlay was removed, cells were fixedand stained, and the interferon titer was determined asthe dilution that resulted in a 50% reduction in thenumber of plaques compared with the controls. Repro-ducibility was checked by including a laboratory stan-dard of bovine interferon prepared in our laboratory.

Electron microscopy. AM and MDBK cells wereexamined by electron microscopy 24 h after infectionwith IBR virus at an input multiplicity of 10. Cellswhich had become nonadherent were centrifuged at400 x g for 5 min, the supernatant was removed, andthe cells were suspended in 250 ,ul of 0.5 M glutaralde-hyde in 0.06 M sodium cacodylate buffer, pH 7.4,delivered rapidly through a 26-gauge needle. Theywere immediately pelleted again in a soft plastic micro-centrifuge tube, the blind end of the tube was cut off,and the pellet was gently washed into glutaraldehyde-cacodylate fixative. After 12 h the pellet was trans-ferred to 0.1 M sodium cacodylate buffer, pH 7.4. Thepellet was subsequently embedded in Epon-Araldite,and ultrathin sections were stained with uranyl acetateand lead citrate and examined with a Zeiss EM9Selectron microscope.

Infectious center assays. AM and MDBK cells werecultured in 35-mm-diameter wells of cluster plates(Costar no. 3506) and infected with IBR virus at an

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1050 FORMAN ET AL.

FIG. 1. IBR viral antigen in (a) AM and (b) MDBK cells 8 h after infection at a multiplicity of infection of 10.Monolayers were fixed in acetone, incubated with bovine anti-IBR serum followed by fluorescein-conjugatedanti-bovine serum, and observed under UV light. Almost all MDBK cells, but only approximately 10% of AM,showed fluorescence (x1,640).

input multiplicity of 0.1 or 0.01. After a 1-h adsorptionperiod, monolayers were washed twice with MEM andoverlaid with MEM plus 10% FBS. At various timespostinfection (p.i.), the cells were removed from themonolayers. MDBK cells were removed by mild tryp-sinization, whereas AM were removed by incubationwith 9 mM lidocaine hydrochloride (Astra ChemicalsLtd., Mississauga, Ont., Canada) in MEM plus 20%

FBS. Both populations were washed, suspended, anddiluted in MEM containing anti-IBR serum, and totalcells were enumerated. Volumes of 1 ml of variousdilutions were added to confluent MDBK cell mono-layers in 24-well cluster plates. The cells were allowedto settle undisturbed for 48 h, after which the monolay-ers were fixed and stained and viral plaques wereenumerated microscopically.

INFECT. IMMUN.

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INFECTION OF BOVINE MACROPHAGES WITH IBR VIRUS 1051

Viral polypeptide analysis by polyacrylamide gel elec-trophoresis. AM, MM, and MDBK cell monolayerswere cultured in 35-mm dishes in the usual manner,washed, and infected at a multiplicity of 10 with IBRvirus. After the virus was permitted to adsorb for 1 h at37°C, the monolayers were washed and overlaid withmethionine-free MEM (no. 79-0115; GIBCO Labora-tories, Grand Island, N.Y.) containing 1% FBS. Afterincubation for a further 6 h to permit cessation of hostprotein synthesis, 25 ,uCi of [35S]methionine (SJ204;Amersham Corp., Arlington Heights, Ill.) per ml wasadded to each culture. The cells were harvested at 20 hp.i. They were suspended in 100 ,ul of dissociationbuffer (0.06 M Tris [pH 6.8], 0.00125 M bromophenolblue, 1.25% sodium dodecyl sulfate, 12.5% glycerol,0.15 M 2-mercaptoethanol), sonicated for 10 s at apower setting of 7 in a Sonifier cell disruptor (Bioson-ics, Plainsview, N.Y.), and boiled for 2 min.Samples were electrophoresed in the presence of

sodium dodecyl sulfate through 7.5 or 10% polyacryl-amide gels as described by Laemmli (17), using a 15-cm vertical electrophoresis apparatus (Richter Scien-tific, Vancouver, B.C., Canada). Gels were dried andautoradiographed on Kodak X-OMAT-R film. Molec-ular weights of radioactive bands were determined bycomparing their Rf values with those of markers withknown molecular weights (high- and low-molecular-weight calibration kits; Pharmacia) electrophoresed onthe same gel structure.

Detection of viral nucleic acid synthesis. Bovine kid-ney cells or bovine macrophages that had been incu-bated in vitro for 48 h in the presence or absence ofLPS were infected at an input multiplicity of 10 withIBR virus or mock infected. At 6 h p.i. 10 p.Ci of[methyl-3H]thymidine (24 Ci/mmol; Amersham Corp.)per ml was added to infected cultures or 2 ,uCi of[14C]thymidine (55.7 mCi/mmol; Amersham Corp.)per ml was added to the mock-infected cultures. At 20h p.i., cells were removed with the aid of a rubberpoliceman, and infected and uninfected cultures weremixed, washed with TE buffer (0.01 M Tris, 0.001 MEDTA, pH 8.0), and suspended in 1 ml ofTNE buffer(0.01 M Tris, 0.15 M NaCl, 0.001 M EDTA, pH 8.0).Proteinase K and sodium lauryl sarkosine (SigmaChemical Co., St. Louis, Mo.) were added to 100 ,ugand 1 mg/ml, respectively, to lyse the cells. After 24 hat room temperature, the lysate was made up to adensity of 1.72 g/ml with CsCl and centrifuged for 72 hat 35,000 rpm in a Beckman type 50 Ti rotor. Thegradients were fractionated, and amounts of 14C and3H in each 0.25-ml fraction were determined with theaid of a Beckman LS8000 scintillation counter.

RESULTSIBR replication in bovine AM. (i) Immunofluo-

rescence and virus yields. Initially, we attemptedto determine whether IBR virus could infectbovine AM. Infection at an input multiplicity of10 resulted in the development of cytopathicchanges in some cells as early as 8 h p.i. Also,cells began to show cytoplasmic fluorescence atthis time (Fig. la). As time progressed, thedegree of fluorescence and cytopathology in-creased such that the entire monolayer began todetach by 16 to 20 h p.i. A comparison of therate of development offluorescence demonstrat-

ed that AM required approximately 20 h beforeabout 90% were positive, whereas MDBK cellswere all positive by 8 h p.i. (Fig. lb).

Analysis of the supernatant fluids indicatedthat infection of AM was not abortive, since by24 h p.i. there was a 100-fold increase in theamount of virus present in AM culture fluids(Fig. 2). As was the case with the immunofluo-rescence studies, maximum virus yields in AMwere achieved at a slower rate than in MDBKcells (Fig. 2). Even though macrophages couldreplicate IBR virus, the actual yield on a per-cellbasis was much lower than in MDBK cells.Thus, MDBK cells released in excess of 100PFU per cell, whereas AM release was <1 PFUper cell.

(ii) Electron microscopy. Since AM did ex-press IBR antigens as determined by immunoflu-orescence but only produced very small quanti-ties of infectious virus, attempts were made todetermine whether the infection was abortive ina high percentage of cells, possibly by virtue ofthe inability of virus to assemble into completevirions within macrophages. Examination of in-fected AM by electron microscopy clearly indi-cated that virus assembly was occurring andwhat appeared to be complete virions werebudding from the nuclear membrane (Fig. 3a), aswell as being released into the extracellularspaces (Fig. 3b and c). Furthermore, much in

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HOURS POST INFECTION

FIG. 2. Replication of IBR virus in AM (0) andMDBK cells (0) after infecting at a multiplicity ofinfection of 10.

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1052 FORMAN ET AL. INFECT. IMMUN.

-y

*1 ,.~~~~~~~e~~~-A~~~~~ W~~

C '4

*0.9 ~ ~~~~Vfs41:

A~~~~~~~~~~~~~~

*9 4 4,,~~~~3..4~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~N

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FIG. 3. Replication of IBR virus in AM. At 24 h p.i., naked virions are present in the nucleus and buddingthrough the nuclear membrane (a) and becoming enveloped (a, inset). Virus particles were present extracellularly(b) and showed typical herpesvirus morphology (c). Bars, 100 nm (a, inset, and c) and 1 ,um (b).

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INFECTION OF BOVINE MACROPHAGES WITH IBR VIRUS 1053

FIG. 4. IBR-infected MDBK cells.

excess of one virion per cell was evident in themajority ofAM, although extracellular virus wasassociated with only a small percentage of cells.A comparison of virus present in MDBK cellsdemonstrated that more virus was present with-in a cell (Fig. 4); however, this difference wasnot great enough to account for a 3-log differ-ence in titer between MDBK cells and macro-phages.

(iii) Infectious centers. Even though we hadevidence that most AM did replicate the virus,

TABLE 1. Production of infectious centers by AMand MDBK cells after infection with IBR virus

% Producing infectious centerson confluent MDBK cell mono-

Cell type MOja layers at time p.i.:

4h 24h 48h

AM 0.1 0.6 2.5 4.10.01 0.04 0.4 2.9

MDBK 0.1 8.3 1000.01 1.1 89.7

a Multiplicity of infection (MOI) of cells with IBRvirus based on PFU in MDBK cells.

we could not explain the extremely low yields ofvirus. Furthermore, if cultures were infected atlow multiplicities of infection, the virus did notspread to destroy the entire monolayer (Fig. 5).Thus, the virus produced foci within 48 h p.i.,but these foci did not progress to kill the entiremonolayer nor were new foci initiated eventhough no antibody was added. This suggestedthat most of the spread was initially from cell tocell but that even this mode of spread was haltedwithin 48 h in culture.

In an attempt to see whether the infectedmacrophages within the foci could produce in-fectious centers, we plated them on susceptibleMDBK cells. Table 1 illustrates that they couldinitiate infectious centers. However, in contrastto MDBK cells, in which almost all of the cellsproduced infectious centers by 24 h, <5% of theAM did so as late as 48 h p.i. and there was no

FIG. 5. Fluorescent foci ofIBR viral antigen in AM 48 h after infection at a multiplicity of 0.01 (x 1,640).

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1054 FORMAN ET AL.

TABLE 2. Interferon production in AM culturesinfected with IBR virus

Interferon production' at p.i.:Mola

4h 24h 48h 96h

0.1 <3b 162 162 4800.01 <3 <3 18 162

a Multiplicity of infection (MOI) of AM with IBRvirus based on PFU in MDBK cells.

b Reciprocal of dilution of AM culture supernatantcausing a 50% reduction in plaques of vesicular stoma-titis virus on MDBK cell monolayers.

increase even at 96 h p.i. These results support-ed previous studies which indicated that thenumber of permissively infected cells was limit-ed since foci did not increase in size.

Interference with spread of infection in AMcultures. The reason for limited spread of infec-tion through the macrophage monolayer was notdue to a lack of IBR virus in the culture medium.Monolayers were infected at an input multiplic-ity of 0.1, and supernatant fluid and cells weresampled separately at 24, 48, and 96 h p.i. Thevirus infectivity was distributed almost equallybetween cells and supernatant fluids at the threesampling times. At present, the only explanationwe have for the refractory nature of the culturedAM is that viral infection stimulates the produc-tion of high levels of interferon (Table 2). Sup-port for the implication of interferon in limitingIBR virus infection in AM was obtained fromexperiments in which uninfected AM cultureswere incubated for 18 h with culture fluidsharvested from macrophages 96 h p.i. and cen-trifuged at 100,000 x g for 3 h to remove IBRvirus. Inoculation of treated monolayers withIBR virus at an input multiplicity of 1.0 resultedin very little detectable viral immunofluores-cence at 24 h compared with untreatedAM, which showed fluorescence in approxi-mately 70% of cells. After 48 h there was almostno infectious virus production (an approximate-ly twofold increase above the level of the residu-al inoculum) in treated AM, compared with a>100-fold increase in untreated, control mono-layers.Although the nature of the interference was

not positively identified as interferon activity,the activity was only reduced by 50% on incuba-tion at 56°C for 1 h and was not removed bycentrifugation at 100,000 x g for 3 h.

Virus replication in immune and nonimmuneAM. AM from immune and nonimmune donorsinfected with IBR virus at an input multiplicityof 0.1 supported virus replication to similarextents (Fig. 6). Comparison of viral proteinsynthesis in AM and MDBK cells demonstratedthat all previously reported IBR structural andnonstructural polypeptides (23) were detected in

AM from both immune and nonimmune animals,although in reduced amounts when comparedwith MDBK cells (Fig. 7). The polypeptides ofVP8 and VP13 have been defined as "late"polypeptides and are only synthesized in appre-ciable amounts in cells that are synthesizingviral DNA. These polypeptides were synthe-sized in AM. In addition to all of the polypep-tides synthesized in MDBK cells, AM-infectedcultures contained an additional 45,000-daltonprotein.

Viral DNA synthesis was examined by label-ing infected cells with [3H]thymidine followedby analysis of the DNA on CsCl gradients. Eachgradient contained 14C-labeled cellular DNA asa marker. The results supported the above ex-periments which showed that virus was synthe-sized. However, the quantity of DNA producedin AM, as determined by [3H]thymidine incorpo-ration, was approximately 1/10 that produced inMDBK cells.

Effect of LPS stimulation of macrophages onIBR virus replication. Previous studies havedemonstrated that MM from immune animalswere sometimes resistant to infection with IBRvirus (unpublished data). Since the immune sta-tus of the animal did not alter the AM's ability tobe infected by virus, we assumed that the LPSused in eliciting MM may activate the macro-phages, making them refractory to infection. Toexamine whether macrophages could become

4.0

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HOURS POST INFECTION

FIG. 6. Replication of IBR virus in AM from im-mune (0) and nonimmune (0) donors. Cultures weresampled for infectivity at various times after infectionat a multiplicity of 0.1.

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INFECTION OF BOVINE MACROPHAGES WITH IBR VIRUS 1055

blocked in these stimulated macrophages. Thiswas confirmed by analysis of the viral DNAfrom these cells. Thus, no [3H]thymidine labelwas present at the density of viral DNA in LPS-stimulated MM cultures (Fig. 8).

DISCUSSIONIBR virus replication in bovine AM was dem-

onstrated by a variety of methods. This evidence

4

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ra_

FIG. 7. [35S]methionine-labeled polypeptides syn-thesized in uninfected and IBR virus-infected MDBKcells and AM and MM. MDBK cells (lanes 1 and 2),AM from immune animals (lanes 3 and 4), AM fromnonimmune animals (lanes 5 and 6), and MM fromimmune animals (lanes 7, 8, 9, and 10) were mockinfected (lanes 1, 3, 5, 7, and 9) or infected with IBRvirus (lanes 2, 4, 6, 8, and 10). After being labeled with[35S]methionine from 6 to 24 h p.i., the cells were lysedand analyzed by polyacrylamide gel electrophoresis.MM were either incubated with LPS (lanes 9 and 10)or maintained without LPS (lanes 7 and 8) for 48 hbefore infection. White arrows indicate positions ofmolecular-weight markers (white numbers = g x 103).Black numbers denote IBR virus structural polypep-tide, and black letters denote nonstructural polypep-tides. Numbers 8 and 13 have been identified as latepolypeptides (23). Black arrow indicates a polypeptideonly observed in infected macrophages.

activated to alter their ability to support IBRvirus replication, both AM and MM were cul-tured in vitro for 48 h in the presence or absenceof LPS before infection. Bacterial LPS treat-ment did not significantly alter viral DNA (Fig.8) or protein synthesis (Fig. 7) in AM. However,it did reduce the quantity of late polypeptidesVP8 and VP13 (Fig. 7, line 10) in LPS-treatedMM. This implied that viral DNA synthesis was

4

3

2

A

7,

B

12 14 16 l 20 22 24FRACTION No.

FIG. 8. DNA synthesized in LPS-treated and un-treated, IBR virus-infected AM and MM from IBR-immune animals. Infected macrophages were labeledwith [3H]thymidine from 6 h after infection. At 24 hp.i., DNA from cells was analyzed by banding in CsClgradients. Each gradient contained 14C-labeled DNAfrom uninfected cells as an internal marker. (A) Analy-sis of infected AM (0) and MM (0) without LPStreatment; (B) AM (-) and MM (0) treated with LPS.Closed arrow indicates position of viral DNA, andopen arrow indicates position of cell DNA.

L

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1056 FORMAN ET AL.

suggests a mechanism for the implication of IBRvirus in bovine respiratory disease. Cattle suc-cumbing to pneumonic pasteurellosis often haveevidence of concurrent viral infection, includingIBR virus (19), and IBR virus infection renderscattle susceptible to subsequent experimentalchallenge with Pasteurella haemolytica (14).Since it is believed that the AM is the majormechanism for pulmonary bacterial clearance(13), and if IBR virus infection of AM inhibitsmacrophage function either directly by infectionor indirectly due to immunosuppressive effects,then this could explain its predisposing effect inbovine respiratory disease. It is possible thatother sequelae of viral infection, such as re-duced mucociliary clearance or altered secretionof bactericidal factors by the bronchotrachealmucosa (27), may also contribute to increasedhost susceptibility to P. haemolytica. Thesepossibilities are presently being investigated.Although AM were susceptible to IBR virus

infection, we could not fully explain the relative-ly low yield of infectious virus in macrophageswhen compared with MDBK cells. The numberof particles assembled in infected macrophages,as seen by electron microscopy, was much inexcess of one virus particle per cell. However,the actual yield of infectious virus from AM was<1 PFU per cell (Fig. 2 and 6). Thus, mostparticles were not capable of initiating replica-tion. Since the particle/infectivity ratio in mostherpesvirus infections can approach 100:1 to1,000:1, it is possible that the ratio in AM was nodifferent than that in MDBK cells. Thus, it ispossible that AM, although they replicate thevirus, produce a smaller quantity of viral prod-uct on a per-cell basis. This is partially support-ed by the observation that [3H]thymidine incor-poration into viral DNA was 90% lower in AMthan in MDBK cells. Similarly, incorporation of[35S]methionine into cells as viral protein wasalso lower in AM. Alternatively, we could notexclude the possibility that a large proportion ofthe particles produced in AM were defective.

If defective interfering particles were releasedfrom AM, this could partially explain why in-fected foci did not increase in size with contin-ued in vitro incubation. Otherwise, we mustassume that the lack of spread of virus in AMcultures was due to the release of high levels ofinterferon (Table 2) which rendered contiguouscells refractory to infection. Although we foundthat interference to virus replication was inhibit-ed by cell-free supernatant fluids from IBR-infected AM cultures and that activity in ourvesicular stomatitis virus plaque reduction assaywas not removed by centrifugation at 100,000 xg or destroyed by heating at 56°C, we did notfully characterize the activity as being due tointerferon. Since there is controversy regarding

the sensitivity of herpesviruses to interferon (1,3, 29, 34) and macrophages are possibly refrac-tory to their own interferon (12), the identity ofthe interfering factor in our AM cultures re-quires further confirmation.The role ofAM in producing interferon in vivo

also requires investigation. It may play an im-portant role in defense, not only by preventingvirus replication but also by activating othermacrophages (4), which can then influence anti-gen presentation to lymphocytes or directly af-fect lymphocytes (11, 18).AM from immune and nonimmune donors

were equally susceptible to IBR virus infection.This finding is similar to that of Brautigam et al.(6), who found that peritoneal macrophagesfrom susceptible and resistant mice were equallypermissive for murine cytomegalovirus. Howev-er, contrasting results have been obtained byBryans (personal communication), who foundthat equine rhinopneumonitis virus could repli-cate in peripheral blood monocytes from nonim-mune horses only. Hence, even within the her-pesvirus family, there appears to be a variationin this respect.

Variation in macrophage susceptibility to viralinfection, depending on anatomical site or acti-vation, has also been recorded by a number ofworkers (6, 24, 30). Although incubation ofAMwith bacterial LPS did not alter their susceptibil-ity to IBR virus infection, it rendered MMresistant to infection. OurMM differed from AMin their site of derivation and also by virtue ofbeing elicited by inoculation of LPS into themammary gland. Whatever the reason, it is ofinterest that they were resistant, and we arecurrently investigating the circumstances underwhich bovine macrophage populations can berendered refractory to virus infection and themechanisms involved in activation of variousmacrophage populations, as well as what effectsuch activation may have on virus-cell interac-tions.

ACKNOWLEDGMENTSThe willing technical assistance of M. L. Buckley and J. M.

Heise is gratefully acknowledged.This work was supported in part by grants from the Medical

Research Council of Canada, Farming for the Future, and theSaskatchewan Homed Cattle Trust Fund.

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