Feline Viral Rhinotracheitis

Transcription

1 Observations on Recovery Mechanisms from Feline Viral Rhinotracheitis R. C. Wardley, B. T. Rouse and L. A. Babiuk* ABSTRACT Experiments were designed to determine immunological mechanisms responsible for controlling dissemination of feline rhinotracheitis virus in feline cell cultures. Virus infected cells could be destroyed by three mechanisms - antibody and complement mediated lysis, direct lymphocyte cytotoxicity and antibody dependent cell-mediated cytotoxicity. This latter immune parameter was mediated by both lymphocytes and macrophages and varied in extent in different cats. To ascertain the potential importance of the immunological parameters in curtailing viral spread, the time when virus infected cells could be destroyed by each component was related to the chronological events of viral replication and dissemination. Intracellular infectious virus and intracellular spread occurred at six to seven hours postinfection and extracellular spread at nine to ten hours postinfection. Antibody complement lysis and antibody dependent cell-mediated cytotoxicity occurred at six hours postinfection and direct cytotoxicity at eight hours postinfection. The relevance that these findings might have in relation to the occurrence and frequency of recrudescent disease is discussed. cellules infectees par le virus, par l'un ou l'autre des trois mecanismes suivants: la lyse par l'intermediaire des anticorps et du complement, la cytotoxicite directe des lymphocytes et la cytotoxicite par l'entremise des lymphocytes et des macrophages et dependante des anticorps. L'importance de ce dernier parametre varia d'un chat a l'autre. Afin de verifier l'importance virtuelle des parametres immunologiques dans la diminution de la dissemination du virus, on relia le temps ou les cellules infectees par le virus pouvaient etre tuees par l'une ou l'autre des composantes aux phases chronologiques de la replication et de la dissemination du virus. On constata la presence et la propagation intracellulaires de virus infectieux, de six a sept heures apres l'infection, tandis que la dissemination extra-cellulaire se produisit environ trois heures plus tard. La lyse par l'intermediaire des anticorps et du complement, de meme que la cytotoxicite par l'entremise des lymphocytes et des macrophages et dependante des anticorps, survinrent six heures apres l'infection; quant a la cytoxicite directe des lymphocytes, elle se produisit deux heures plus tard. Les auteurs commentent le rapport plausible entre leurs observations et l'occurrence, ainsi que la frequence de la recrudescence de la maladie. RESUME Cette experience visait 'a determiner les mecanismes immunologiques responsables du contr6le de la dissemination du virus de la rhinotracheite feline, dans des cultures de cellules renales de chat. II s'avera possible de tuer les *Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Saskatchewan S7N OWO. Reprint requests to Dr. B. T. Rouse. Submitted December 30, INTRODUCTION One of the major triumphs of modern medicine has been the almost total control of several viral disease agents by vaccination programmes. However, the herpesviruses as a group are probably as ubiquitous as ever and, with the possible exception of Marek's disease virus (MDV) (8), have not been adequately controlled by vaccination. The major reason for this state of affairs is because these viruses can persist in the body after primary infection. Volume 40 -July,

2 Persistence can take the form of continuous replication, as with MDV (8), or as a silent (latent presumably nonreplicating form as is assumed to occur with Herpes simplex (3) and probably feline rhinotracheitis virus (FVR) (5, R. C. Povey, personal communication). Latency, once established, seems to be permanent and animals so infected, are subject to recrudescent disease resulting from reactivation of endogenous virus. Consequently, if controls aimed at preventing primary infection are not successful, one is faced with the problem of devising programs to cure latency, prevent reactivation or reduce the severity of recrudescent disease. Of these three approaches, the latter may be the most feasible. However, before taking this approach it is necessary to identify the actual mechanism by which animals recover from FVR, determine if animals subject to frequent recrudescence have defects in one or more of the immunological components involved in recovery and finally explore immunomanipulations for their ability to enhance the activity of recovery mechanisms identified. In the present article, we have demonstrated a number of immunological mechanisms that may be responsible for controlling virus dissemination both in vitro and in vivo. CELLS MATERIALS AND METHODS Crandell feline kidney (CRFK) cells were obtained from Dr. R. C. Povey, Ontario Veterinary College, Guelph, Ontario. The cells were cultured in Eagle's base minimal essential medium (MEM) containing 10% heat-inactivated fetal calf serum (FCS). Each litre of medium was supplemented with 10 ml nonessential amino acids (Gibco, Cat. No. 114), glutamine (2 mmoles), gentamycin (25 ug,/ml), and 2.5 g of sodium bicarbonate. VIRUS Feline viral rhinotracheitis virus was obtained from Dr. J. B. Derbyshire, Ontario Veterinary College, Guelph, Ontario. Stock virus was prepared in CRFK cell mono- 258 layers as follows: One plaque forming unit (PFU) per cell of virus was allowed to adsorb for one hour at 37 C, the unadsorbed virus removed and MEM containing 2% FCS added. After incubation at 37 C for hr, all cells were rounded and approximately 50% were detached. The remaining attached cells were removed from the dishes and the entire culture fluids subjected to three freeze-thaw cycles. Cellular debris were removed by centrifugation at 1000 x g for ten min and the supernatant collected and stored in small aliquots at -70 C. This constituted stock virus. PREPARATION OF EFFECTOR CELL POPULATIONS Five ml of heparinized blood (5 IU/ml) diluted 1:2 with Puck's saline (PS) was layered onto a 3 ml volume of Ficoll-Hypaque (density of g/cm3 at 25 C: Ficoll, Pharmacia; Hypaque, sodium diatrizoate, Winthrop Labs) in a 13 x 120 mm centrifuge tube. Following centrifugation at 400 x g for 20 min at room temperature, the lymphocyte enriched interface cells were collected, washed two times in PS and resuspended in RPMI 1640 plus 10% FCS containing 25 mmoles HEPES (RPMI- HEPES). These cells constituted the peripheral blood lymphocyte (PBL) effector cell population and were either used directly in cytotoxicity assays or processed further to remove adherent and phagocytic cells. Adherent cells were removed by passing PBL through glass wool columns as previously described (10). Briefly, glass wool columns were prepared and equilibrated at 37 C for 90 min with PS + 5% FCS. PBL (2 x 108 in 2 ml) were layered onto the glass wool column, the column was sealed and reincubated at 37 C for a further 90 min. Nonadherent cells were eluted from the column using PS + 5% FCS. Effluent cells were centrifuged, washed two times in PS and resuspended at the appropriate concentration in RPMI-HEPES. This population constituted the glass wool effluent cells. Less than 1% of these cells stained with 0.005% neutral red (a marker of phagocytic cells). Phagocytic cells were removed by the carbonyl iron treatment method of Sanderson et al (11). Briefly, PBL were suspended at 3 x 106/ml in RPMI-HEPES containing 4 mg/ml of washed sonicated iron. Can. J. comp. Med.

3 The cells were incubated at 37 C in the presence of iron for min with continual inversion. The iron and iron containing cells were removed with the aid of a strong magnet. The nonphagocytic cells were washed, and resuspended at the appropriate concentrations in RPMI-HEPES. On average, 0.2% of cells stained with 0.005% neutral red. PREPARATION OF FELINE MACROPHAGES Young cats were injected intraperitoneally with 10 ml of PS containing 30 mg/ml sterile hydrolysed starch. Three days later, up to 100 ml of warm phosphate buffered saline was infused into the abdominal cavity, the abdomen massaged and the fluid aspirated into a syringe. This fluid was centrifuged and the cell pellet collected and resuspended in MEM plus 10% FCS. The cell suspension was placed in 100 mm Petri dishes (5 x 106 cells/ml) and incubated at 37 C for two hr. Nonadherent cells were removed by gentle washing and discarded. Adherent cells were recovered after trypsinization and gentle mechanical scraping with a rubber policeman. PREPARATION OF ANTISERA microtitre plates (Falcon Plastics No. 3040). Infected 51Cr labeled target cells were trypsinized at the appropriate time postinfection (PI), resuspended in RPMI- HEPES at a concentration of 2 x 105 cells/ ml and added in 100 ul aliquots to quadruplicate wells. Effector cells (100 ml volumes) were then added to each well to measure direct cytotoxicity. Similar cultures were used to measure antibody dependent cellmediated cytotoxicity (ADCC) with the exception that 20 ±l volumes of heat inactivated anti-fvr serum was also added to each well. Controls included target cells alone, target cells plus normal cat serum, target cells plus antisera, and noninfected CRFK cells plus effector cells and antisera. Plates were incubated for six hr at 37 C in a humidified atmosphere of 5% C02, centrifuged at 200 x g for three min and 50 % of the supernate removed for measurement of radioactivity in a Nuclear-Chicago gamma counter. Results were expressed as percent specific 51Cr release (SR) and were computed using the formula below: S 100 x mean CPM test - mean CPM control Releasible CPM - mean CPM control Total releasible 5'Cr was determined by measuring release from target cells in the presence of 0.5% Triton X 100. Sera were collected from cats which had recently recovered from FVR virus infections. Some sera were heat inactivated at 560C for 30 min. Sera not treated in this way were used as a source of anti-fvr antibody and complement. All sera were stored in small aliquots at -700C. CELL MEDIATED LYsIS OF VIRUS INFECTED TARGET CELLS Confluent monolayers of CRFK in 100 mm Petri dishes (6-8 x 106 cells per dish) were labelled with 100 u,ci of Na25"CrO4 (NEN, Dorval, P.Q.) in 0.7 ml of MEM. Plates were frequently rocked during the 60 min labelling period. After labelling, the monolayers were washed twice with PS and infected with FVR at a multiplicity of infection (MOI) of one. After a further incubation period of one hr, the virus was washed off and MEM plus 2% FCS added. All cytotoxicity assays were performed in ANTIBODY-COMPLEMENT LYSIS OF VIRUS INFECTED CELLS Confluent monolayers of CRFK cells were labeled with Na25"CrO4 as described above. The cells were trypsinized and dispensed in 100!il amounts into each well of a microtitre plate (50,000 cells/well). When the cells attached (six hrs), they were infected at a MOI of one. The virus was allowed to adsorb from 60 min at 37 C, the monolayers washed and replaced with fresh MEM + 4% FCS. At various time intervals PI, infected cells (in 100 ml fresh MEM) were incubated for 60 min with 25 ul heat inactivated cat serum (with anti- FVR antibody activity) or 25 utl of the same serum but not inactivated (containing antibody plus complement). The amount of radioactivity released into the medium was compared to that released by controls and was computed by the formula above. Volume 40- July,

4 X-x Ratio of inf ectious centres at x hr infectious centres at 3 hr o---o Conc. intracellular virus 107 *- *Conc. extracellular virus i 0 0).L_ 01) c a) u 0 (L) c c clc C) c 0 u 102 A D.. _ >-J HR post infection Fig. 1. Time of appearance of intracellular and extracellular infectious feline rhinotracheitis and time of spread of intracelular infectious virus. INFECTIOUS CENTRE ASSAY 260 CRFK cells grown to confluency in 35 mm Petri dishes (Falcon plastics) were infected with FVR virus at a MOI of After one hour of adsorption the unadsorbed virus was removed, monolayers washed and fresh MEM + 4% FCS containing ten neutralizing units of anti-fvr antiserum was added. At various times thereafter the cells were removed by trypsinization, washed and diluted (fourfold) in MEM + 4% FCS + FVR antiserum. These dilutions were added to confluent CRFK cells in 16 mm wells of a microtitre plate (Linbro Plastics No TC). The cells were allowed to settle without disturbance for 36 hours after which the monolayers were fixed, stained and the viral plaques enumerated (9). TIME COURSE OF VIRUS PRODUCTION CRFK cells were grown to confluency in 16 mm wells of a microtitre plate (Linbro Plastics No TC), and infected with 0.2 mls of FVR virus at a MOI of one. After adsorption at 370C for 60 min the unadsorbed virus was removed, monolayers Can. J. comp. Med.

5 TABLE I. Antibody Dependent Cell Cytotoxicity and Direct Cytotoxicity of Feline Peripheral Blood Leukocytes on Feline Rhinotracheitis Virus Infected CRFK Cellss SRb ADCCe Direct cytotoxicity PBL: target cell ratios PBL: target cell ratios PBL Expt 100:1 50:1 25:1 100:1 50:1 25:1 Source A B Cat 1...C D A Cat 2.B A Cat 3.B C A Cat 4.B C D acrfk target cells infected for 15 hr at a MOI of one bspecific release in a six hour assay. Figures are mean of quadruplicate determinations cassay performed in the presence of 1:50 dilution of heat inactivated anti-fvr serum (neutralization titre 1/30) washed three times in PS prior to addition of fresh MEM + 4% FCS. At various intervals (hourly) PI, the entire culture fluids were removed and the amount of extracellular virus present was quantitated by plaquing in microtitre plates (9). The intracellular virus was quantitated by removing the cells with a rubber policeman and subjecting the cells to three freeze thaw cycles prior to titration of the virus. spread from an infected cell to an adjacent uninfected cell by the intracellular route (1) prompted the design of experiments to determine the time when virus could spread by this route. The results in Fig. 1 demonstrate that spread by the intracellular route occurred at approximately the same time (six to seven hours) that infectious intracellular virus was detectable, i.e. about two to three hours prior to viral spread by the extracellular route. RESULTS FVR VIRUS REPLICATION AND SPREAD TO ADJACENT CELLS To determine how soon after infection infectious progeny viruses was produced, cells were infected at a MOI of one and at various times thereafter the culture fluids and cells were removed and assayed for infectious virus. The results of one such experiment, as depicted in Fig. 1, demonstrate that although infectious intracellular virus was present as early as six to seven hours PI, such virus was not released into the extracellular environment until nine to ten hours PI (three hours later). The fact that herpesviruses usually CYTOTOXICITY OF FVR INFECTED CELLS ADCC against FVR infected target cells was regularly demonstrated by PBL at effector:target cell ratios of 25:1 or higher (Table I). Although the extent of lysis varied slightly among PBL preparations from the same animal, the same general trends were observed for all cats. Thus at higher effector to target cell ratios there was a concomitant increase in specific lysis. In addition, although PBL from all cats could mediate ADCC, there may be a difference in the efficiency with which cells from some cats can mediate ADCC (i.e. the poor response of Cat #3) (Table I). All cats used in the study had been exposed to FVR, but were not excreting virus at the time of sampling. Volume 40-July,

6 o-o PBL mediated AOCC * o Antibody complement tysis x---x Mocrophoge mediated ADOCC -'*--4 Direct cytolysis (Ir) 10) / HR post infection Fig. 2. Time after infection with feline rhinotracheitis virus of CRFK cells to immune lysis by different mechanisms. The fact that ADCC could be demonstrated prompted us to determine the cell responsible for this phenomenon. Separation of PBL into various sub-populations indicated that the removal of phagocytic and/ or glass wool adherent cells did not significantly alter ADCC (Table II). Morphological examination of the glass wool effluent cells and nonphagocytic cells revealed that they were composed of approximately 98% and 95% lymphocytes respectively. Neutral red, a marker of phagocytic cells (4) stained less than 1% and 0.2 % respectively of the above populations thus 262 suggesting that the effector cell responsible for ADCC was probably of lymphoid nature. However, macrophages could also mediate ADCC since cell populations highly enriched in such cells (adherent peritoneal exudate cells) had activity (Table II). The adherent PEC were predominantly of macrophage morphology and 86-95% of the cells become darkly stained with 0.005% neutral red. Direct cytotoxicity of FVR infected cells was detectable only in some cats and on some occasions (Table I). Levels of direct cytotoxicity were always lower than ADCC. Can. J. comp. Med.

7 TABLE II. Antibodv Dependent Cell Cytotoxicity by Paritoneal Exudate Cells and Peripheral Blood Leukocytes and their Subpopulationsa SRb Effector: Target cell ratio Cell type 100:1 25:1 PBL Nonadherent PBLc Nonphagocytic PBLd PEC Adherent PECe aall cell populations taken from cat 1 bspecific 51Cr release in six hr assay from CRFK cells infected for 15 hr with FVR virus at a MOI of one. Cells sensitized with 1:50 dilution of heat inactivated anti-fvr serum. Values shown are the means of three separate experiments each done in quadruplicate 'Cells failing to adhere to glass wool dcells obtained after removal of phagocytic cells by carbonyl iron treatment ecells removed from plastic Petri dishes after adherence for two hr RELATIONSHIP OF CYTOTOXICITY TO VIRUS-HOST CELL EVENTS Having demonstrated cell mediated cytotoxicity of virus infected cells we endeavored to determine at what time PI the various cell populations could lyse FVR virus infected cells. The data in Fig. 2 indicate that PBL and macrophage mediated ADCC began at approximately six hours PI and increased rapidly thereafter. To prevent any further viral induced changes during the ADCC assay actinomycin D (2.g 'ml) was added. Previous experiments demonstrated that although this dose of actinomycin D arrested FVR replication it had no effect on ADCC. The data in Fig. 2 also demonstrate that antibody and complement mediated lysis occurred at approximately the same time (six hours) as ADCC whereas direct cell lysis did not occur until approximately two hours later (eight hours PI). DISCUSSION Feline rhinotracheitis virus causes acute disease from which animals commonly recover. However, following recovery, the virtus usually persists at some site in the body from which it can be reactivated by a plethora of circumstances to cause recrudescent disease (2, 5, 7). Consequently, although cats, following primary disease, may be immune to reinfection with extraneous virus, they are not resistant to FVR disease resulting from endogenous infection. For this reason vaccination at any time after primary infection may prove to be a futile undertaking. One suspects that persistent infections are extremely common and periodic viral shedding occurs frequently, but not all cats develop recrudescent disease (5, 7). Moreover, the severity of clinical disease is very variable (2). Such observations raise the obvious question as to what determines whether viral reactivation will result in clinical disease or silent shedding. We have adopted the working hypothesis that it is the vigor and efficiency of certain immunological components that determine whether virus reactivation results in disease or not. By inference, we suggest that disease only occurs in those circumstances in which there is a subtle defect of the recovery processes. In the present paper, we have collected data that should ultimately allow us to test this hypothesis. Thus we have identified three distinct immune defense mechanisms that could play a role in controlling viral dissemination and presumably cause recovery. Later communications will assess the respective importance of these components in causing recovery and measure whether animals subject to frequent recrudescent disease have subtle defects in one or more of these parameters. Having obtained such information, it may eventually prove possible, with judicious immunoprophylactic procedures, to correct the immunological defects. Following infection of cell cultures with FVR, free infectious virus was not released extracellularly until nine to ten hours later, even though intracellular virus was demonstrable by six to seven hours after infection. This intracellular infectious virus began to disseminate to susceptible cells by the intracellular route at six to seven hours PI, i.e. about two to three hours before extracellular viral shedding occurred. It is well known that extracellular virus can rapidly be neutralized by antibody (6) and that such neutralized virus is noninfectious. Antibody is present in persistently infected cats (7) so such extracellular viral dissemination is probably not important. We demonstrated, however, that virus infected cells could be destroyed by at least two mechanisms prior to the appearance of ex- Volume 40 -July,

8 tracellular viral shedding and it is conceivable that such destructive processess may represent important, possibly crucial, recovery mechanisms. Thus both complement mediated lysis and ADCC, as mediated by both lymphocytes and macrophages, were detectable six hours PI - i.e. at about the time that intracellular viral dissemination occurred. It needs to be shown if such components, when present from the initiation of infection, are able to destroy virus infected cells before intracellular spread occurs thereby curtailing infection. Such experiments are currently underway. We also demonstrated direct lymphocytotoxicity of virus infected cells. This, presumably T lymphocyte mediated, immune defense parameter has not been previously demonstrated in the cat. However our results lead us to suggest that the parameter may not play an important role in the recovery process since effects were variable and did not occur sufficiently early such that they killed virus infected cells before viral dissemination. However whether the T cell resistance mechanism can reinforce the humoral defense mechanism is a possibility that is in need of evaluation. We suggest that cats subject to recrudescent disease may show a decline in reactivity of one or more of these defense mechanisms prior to recrudescent disease or that the component occurs weakly or too late to adequately control viral dissemination in such cats. Consequently, upon reactivation, the virus spreads to a sufficient extent as to cause symptomatic disease. Our observations that cells from different cats were not equally effective at mediating ADCC is in need of further exploration using large numbers of cats with varying FVR disease status. Such data may not only help us better understand the feline disease but may also contribute to knowledge about a similar disease of man, recurrent labial herpes. ACKNOWLEDGMENTS Our appreciation to Lynn Franson for technical assistance. Work supported by Medical Research Council of Canada. REFERENCES 1. ALLISON, A. C. Immunity against viruses. In Scientific Basis of Medicine Annual Review. Ed. by I. Gilliland and J. Francis. pp London: The Athlone Press CRANDELL, R. A. Feline viral rhinotracheitis. Adv. vet. Sci. 17: DOCHERTY, J. J. and M. CHOPAN. The latent herpes simplex virus. Bact. Rev. 38: FILMAN, 0. J., F. R. BRAWN and W. B. DAN- LIKER. Intracellular supravital stain delocalization as an assay for antibody dependent complement mediated cell damage..j. Immun. Methods 6: GASKELL, R. M. Feline Viral Rhinotracheitis: The Carrier State. Thesis, University of Bristol POTGIETER, L. N. D. The influence of complement on the neutralization of infectious bovine rhinotracheitis virus by globulins derived from early and late bovine antisera. Can. J. comp. Med. 39: POVEY, R. C. and R. H. JOHNSON. Observations on the epidemiology and control of viral respiratory disease in cats. J. small Anim. Pract. 11: PURCHASE, H. G. Recent advances in a knowledge of Marek's disease. Adv. vet. Res. 16: ROUSE, B. T. and L. A. BABIUK. Host defense mechanisms against infectious bovine rhinotracheitis virus. II. Inhibition of plaque formnation by immune peripheral blood lymphocytes. Cell Immun. 17: ROUSE, B. T., R. C. WARDLEY and L. A. BABIUK. Antibody dependent cell-mediated cytotoxicity in the bovine-comparison of effector cell activity against heterologous erythrocyte and herpesvirus infected bovine target cells. Infection & Immunity 13: ' SANDERSON, C. J., I. A. CLARK and G. A. TAYLOR. Different effector cell types in antibodydependent cell-mediated cytotoxicity. Nature, Lond. 253: Can. J. comp. Med.