Veterinary Immunology and lmmunopathology, 35 (1992) 95-119

95

Elsevier Science Publishers B.V., Amsterdam

Interaction of acute feline herpesvirus-1 and chronic feline immunodeficiency virus infections in experimentally infected specific pathogen free cats Gerhard H. Reubel a, Jeanne W. George a, Jeffrey E. Badough a, Joanne Higgins ~, Chris K. G r a n t b and Niels C. Pedersen a

aDepartment of Medicine, School of Veterinary Medicine, Universityof California, Davis, CA 95616, USA bDepartrnent of Immunology and Retrovirus Research, PacificNorthwest Research Foundation, Seattle, WA 98122, USA

ABSTRACT Reubel, G.H., George, J.W., Barlough, J.E., Higgins, J., Grant, C.K. and Pedersen, N.C., 1992. Interaction of acute feline herpesvirus-1 and chronic feline immunodeficiency virus infections in experimentally infected specific pathogen free cats. Vet. lmmunol. Immunopathol., 35: 95-119. Cats with or without chronic feline immunodeficiency virus (FIV) infection were exposed to feline herpesvirus, type 1 (FHV-1). FIV infected cats became sicker than non-FIV infected cats and required more supportive treatment. However, there were no differences in the length of their illness or in the levels and duration of FHV-1 shedding. FHV-1 infection caused a transient neutrophilia at Day 7 with a rapid return to preinfection levels. The neutrophilia coincided with a transient lymphopenia that was accompanied by a decline in both CD4 + and CD8 + Tqymphocytes. A brief decrease in the CD4 + / C D 8 + T-lymphocyte ratio occurred at Day 14 in both FIV infected and non-infected cats. This decrease was mainly the result of an absolute and transient increase in CD8 + T-lymphocytes. CD4 + and CD8 + T-lymphocyte numbers and CD4 + / C D 8 + T-lymphocyte ratios returned to baseline within 4-8 weeks in both FIV infected and non-infected cats. FIV infected cats produced less FHV- 1 neutralizing antibodies during the first 3 weeks of infection than non-FIV infected animals. The IgM FHV-1 antibody response was depressed in FIV infected cats whereas the IgG antibody response was unaffected. FHV-1 infection evoked a comparable transient loss of lymphocyte blastogenie responses to concanavalin A and pokeweed mitogen in both FIV infected and non-infected cats. However, response to pokeweed mitogen took longer to return to normal in FIV infected animals. Lymphocytes from FIV infected cats had a greater and more sustained proliferative response to FHV1 antigen than non-FIV infected cats. The ongoing IgG antibody response to FIV was not affected by FHV-1 infection. ABBREVIATIONS CAT, chloramphenicol acetyltransferase; Con A, concanavalin A; CPE, cytopathic effect; CrFK,

Correspondence to: Gerhard H. Reubel, Department of Medicine, School of Veterinary Medicine, University of California, Davis, CA 95616, USA. Tel: (916 ) 752 7470.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00

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Crandell feline kidney (cells); FHV-1, feline herpesvirus type 1; FITC, fluorescein isothiocyanate; FIV, feline immunodeficiency virus; HIV, human immunodeficiency virus; LTR, long terminal repeat; PWM, pokeweed mitogen; SIV, simian immunodeficiency virus; TCIDso, tissue culture infectious dose, 50%.

INTRODUCTION

The role of infectious or non-infectious cofactors in the progression of immunodeficiency in human immunodeficiency virus (HIV)-infected people is unknown. Some researchers believe that the decline in immune function is solely time-dependent (Jason et al., 1989) while according to others a number of cofactors might serve either to delay or accelerate the development of immunodeficiency. These cofactors include age at time of infection (Eyster et al., 1987), genetic predisposition (Mann et al., 1988), the use of drugs (Schechter et al., 1985; Winkelstein et al., 1987) or other incidental diseases (Quinn et al., 1987; Watkins et al., 1988; Webster, 1991 ). Unfortunately, it is difficult to study the role of cofactors in HIV infection of man, especially of the more common incidental infectious diseases. This is thus one area wherein animal models could be of great value. A number of lentivirus infections of animals exist, but only two have been shown to cause acquired immunodeficiency syndrome (AIDS )-like disease: simian immunodeficiency virus (SIV) infection of macaque monkeys and feline immunodeficiency virus (FIV) infection of domestic cats (Baskin et al., 1988; Pedersen et al., 1989 ). FIV infection of cats may be an appropriate model to study the role of incidental infectious diseases in lentivirus disease; cats are available in a specific pathogen free (SPF) state and can easily be housed in pathogen free quarters in comparatively large numbers. The common infectious diseases of domestic cats have also been very well studied under natural and experimental conditions (Pedersen, 1988 ). The study reported herein is one part of a long-term multistage experiment dealing with the effect of common infectious diseases on the rate of progression of immunodeficiency in FIV infected cats. The cats were infected as adolescents with the Petaluma strain of FIV and were kept in strict pathogen free facilities. FIV infected cats underwent a primary and transient illness 68 weeks after inoculation (Pedersen et al., 1990; Yamamoto et al., 1989). About 1 year after infection, the FIV infected cats and a group of their noninfected cohorts were infected with Toxoplasma gondii (Lappin et al., 1991 ). The present study involves a second common incidental infectious disease agent of cats, feline herpesvirus, type 1 (FHV-1). FHV- 1 is an alphaherpesvirus that causes severe upper respiratory diseases (rhinitis, conjunctivitis, pharyngitis, tracheitis ), and at times pneumonia, in domestic cats (Povey, 1979 ). Clinical signs normally resolve in 7-10 days in older cats. In common with herpesvirus infections of other species of animals

FELINE HERPES AND IMMUNODEFICIENCY VIRUS INFECTIONS

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and of man, FHV-1 can persist indefinitely in the trigeminal ganglia and related nerves, creating a chronic carder state. The carder state is usually latent, although active virus shedding and occasional disease recurrence can follow periods of stress (Gaskell and Goddard, 1984). Many cats naturally infected with FIV suffer from chronic upper respiratory disease (Yamamoto et al., 1989) and FHV-1 has been associated with some of these cases. The authors have also observed one FIV infected cat with widely disseminated herpesvirus infection (L. Lowenstine and N.C. Pedersen, personal communication, 1989). Feline herpesvirus infection may also be an appropriate model to study the role of various herpesvirus interactions in people with AIDS. Human cytomegalovirus, herpes simplex virus, Epstein-Barr virus and human herpesvirus type 6, have all been associated either in vivo or in vitro with HIV infection of man and may act as cofactors in the progression of AIDS (Ostrove et al., 1987; Tremblay et al., 1989; Laurence, 1990; Okubo and Yasunaga, 1990; Cardgan et al., 1990; Webster, 1991 ). The objective of the present communication is to describe the effect of chronic FIV infection on acute FHV-1 disease in terms of disease severity, virus shedding, hematology and immunity. The effect of acute FHV- 1 infection on the antibody response to FIV will also be described. MATERIALS AND METHODS

Experimental animals SPF domestic cats were obtained from the breeding colony of the Feline Retrovirus Research Laboratory of the School of Veterinary Medicine, University of California, Davis, and were housed in small groups in infectious disease isolation facilities provided by the Animal Resources Service at the same institution.

Experimental design A total of 60 age- and sex-matched cats were selected for the study and each was assigned to one of four experimental groups. Ten eats were not exposed to any infectious agent and served as naive controls. Thirty cats were inoculated with the Petaluma isolate of FIV at an average age of 10 months, 18 months before the initiation of the present study (Pedersen et al., 1990; Yamamoto et al., 1989). Ten of these FIV infected eats were not exposed to any additional infection and served as FIV infected control. Twenty naive cats were infected with FHV-1, as were 20 previously FIV infected cats. FIV infection was confirmed by serial testing for serum antibody to FIV (O'Connor et al., 1989).

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G.H. REUBEL ET AL.

Feline herpesvirus-1 infection Cats in the FHV-1 and FIV/FHV-1 groups were inoculated with 10 6.6 TCIDso 0.4 ml -l of FHV-1, strain C27 (ATCC VR 636) by administering two drops of virus-containing cell culture medium into both eyes and nostrils (0.1 ml each). Cats in the naive and FIV alone groups received an equivalent amount of cell culture medium without virus.

Clinical scoring Clinical signs were monitored for 14 days after FHV- 1 infection and scored daily, using a modified scoring method described elsewhere (Povey et al., 1980) (Table 1 ). In addition, rectal temperature was recorded daily for 14 days after challenge. TABLE 1 Scoring system for clinical signs following infection with FHV- 1 Clinical signs

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FELINE HERPES AND IMMUNODEFICIENCY VIRUS INFECTIONS

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Clinical treatment In order to palliate clinical signs resulting from FHV-1 infection (secondary bacterial infections and dehydration), severely affected cats were given amoxiciUin (20 nag kg -1 daily) and lactated Ringer's solution (200 ml per cat per day, subcutaneously) for varying numbers of days.

Cells and media For virus isolation, titration and evaluation of neutralizing antibodies, Crandell feline kidney (CrFK) cells were used as substrate. Cells were grown in a 1 : 1 dilution of Leibovitz's L 15 medium and Earle's minimum essential medium containing 10% fetal calf serum, 100 IU penicillin m l - l, 10/lg streptomycin m l - 1 and 5/tg gentamicin m l - i.

Isolation and quantification of FHV-1 Samples of conjunctival and oral secretions were obtained at different time points after challenge using sterile cotton swabs. After transfer into sterile tubes containing 1.0 ml of cell culture medium with 300 IU penicillin m1-1, 30 #g streptomycin ml-1 and 15 #g gentamicin ml-1, swabs were stored at - 7 0 ° C for processing. CrFK cells were grown to confluence in 24-well cell culture plates and inoculated in duplicate with 200/d of each swab sample. Cytopathic effect (CPE) was monitored for 5 days; CPE typical for FHV-1 (distinct plaques, ballooning cells, cell lysis) in at least one of two wells was considered a positive result. To determine virus titers of positive samples, ten-fold dilutions were prepared in cell culture medium. Quadruplicate samples of each dilution (50/zl) were inoculated onto CrFK cells grown in 96well microtiter plates. Virus titers were evaluated according to the method of Reed and Muench, ( 1938 ).

Virus neutralization assay FHV- 1 neutralizing antibody titers in heat-inactivated serum samples (30 min, 56°C) of all cats in the FHV-1 and FIV/FHV-1 infected groups were determined. Twofold dilutions of the serum were mixed in a 96-well microtiter plate with equal volumes of FHV-1, strain C27 ( 10-100 TCIDso m l - l ) and incubated for 90 rain at 37°C. Then 100 gl of the virus-serum mixtures were transferred to corresponding wells of a 96-well microtiter plate containing confluent monolayers of CrFK cells. Plates were monitored for CPE for 5 days. Antibody titers were expressed as the reciprocal of the highest serum dilution completely neutralizing CPE.

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G.H. REUBEL ET AL.

Immunofluorescence assay Specific IgG and IgM responses to FHV-1 were evaluated in sera from five cats each in the FHV-1 and FIV/FHV-1 infected groups. Confluent monolayers of CrFK cells were cultured in eight-chamber slides and infected with 1000 TCID5o ml-~ of FHV-1. The cells were fixed in acetone for 30 min at room temperature when approximately 25 virus-induced plaques per well were present. The slides were covered with twofold dilutions of the sera and incubated at room temperature for 60 min. Thereafter, they were incubated with mouse monoclonal antibodies specific for feline IgG or feline IgM (Pacific Northwestern Research Foundation, Seattle, WA) and subsequently stained with FITC-conjugated anti-mouse-IgG (Kirkegaard and Perry, Gaithersburg, M D ) . Serum from the naive group cats served as a control. Antibody titers were expressed as the reciprocal of the highest serum dilution that resulted in specific fluorescence of FHV-1 induced plaques.

FIV antibody ELISA FIV specific IgG antibody titers were determined in sera from ten FIV infected and ten F I V / F H V - 1 infected cats at different time points after FHV- 1 infection. Microtiter plates were coated with 0.2/tg of sucrose purified FIV, strain Petaluma, per well, tenfold dilutions of the sera added and the plates incubated for 1 h at 37°C. Biotin-labeled rabbit anti-cat IgG was used as secondary antibody for 30 rain at 37°C. Thereafter, horseradish peroxidase labeled streptavidin was added and the plates incubated for 20 min at 37 °C. Tetramethyl benzidine served as substrate for the enzymatic reaction which was stopped after approximately 3 m i n with 1 N H2SO4. The optical density (OD) was measured with an automatic plate reader at a test wavelength of 495 n m against a reference wavelength of 590 nm. Serum from an SPF cat was used as blank. Antibody titers were expressed as the reciprocal of the serum dilution that would produce an absorbance reading equal to 50% of that of a 1 : 100 dilution of a FIV antibody positive control serum.

Complete blood counts Complete blood counts included hemoglobin, mean corpuscle volume, mean corpuscle hemoglobin, total leukocyte count and differentials. Electronic counts were determined by impedance using a System 9000 cell counter (Baker Instruments). Differential leukocyte counts were obtained from Wright-Giemsa stained blood smears using standard methods (Jain, 1986). In order to determine whether peripheral blood neutrophil counts increased in proportion to the degree of FHV-1 illness, the absolute blood neutrophil counts on Day 7 post infection were divided by the sum of the clinical

FELINE HERPES AND IMMUNODEFICIENCY VIRUS INFECTIONS

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score. This ratio was a measure of the cat's ability to mount a neutrophil response proportionate to the disease severity.

Enumeration of CD4 + and CD8 + feline T-lymphocytes Murine monoclonal antibodies to feline C D 4 + (Ackley et al., 1990) and feline CD8 + (Klotz and Cooper, 1986) T-lymphocyte surface markers were used for T-lymphocyte subset enumeration by flow cytometric analysis (Barlough et al., 1991 ).

Lymphocyte blastogenes& assay This whole blood microassay was a modification of standard procedures described elsewhere and was performed in 96-well flat-bottomed microtiter plates (Pauly et al., 1973; Gregory et al., 1987; Barlough et al., 1991 ). Heparinized blood ( 10 #1) was incubated in a total volume of 200 #1 per well at 37°C in 5% CO2 with either pokeweed mitogen (PWM) (3/tg m1-1 ), concanavalin A (Con A) (6/2g m l - 1), FHV- 1 antigen ( 1 : 10 dilution ) or complete RPMI (RPMI supplemented with 10% bovine fetal serum, 2 mM Lglutamine, 1% non-essential amino acids, 100 IU penicillin m l - 1, and 10 #g streptomycin m l - 1) as unstimulated controls reflecting spontaneous proliferation. FHV- 1 antigen was prepared by heat inactivation (30 rain, 56 ° C) of CrFK cell culture supernatant containing 107.0TCIDso of FHV-1 ml-1. Complete RPMI served as diluent for the assay. The cultures were labeled on Day 4 with 1 #Ci per well [3H]thymidine (New England Nuclear) and 24 h later were harvested on filtermats with a 6 well Skatron cell harvester. [ 3H ] thymidine incorporation into DNA was measured with a Tri-Carb 2000 liquid scintillation counter (United Technologies/Packard). Each of the four treatments (PWM, Con A, FHV-1 and cell culture medium) was replicated three times for each blood sample. Mean values were calculated for use in statistical analysis. Lymphocyte-adjusted values were obtained by dividing mean counts m i n - 1per culture by the total numbers oflymphocytes/11-1 blood of corresponding cats multiplied with the dilution factor (0.1), which resulted in mean counts m i n - 1 per cell.

Statistical analys& Differences in mean values between groups were evaluated for statistical significance (P_< 0.05) by Student's t test (two-tailed) and by ANOVA for repeated measures (Armitage and Berry, 1987). Data were assembled on a computerized spreadsheet program (Excel, Microsoft, Richmond, WA) and statistical calculations performed by a statistics package (Statview SE + Graphics, Abacus Concepts, Berkeley, CA).

102

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RESULTS

Clinical observations

Clinical signs of FHV-1 infection appeared 3 days post exposure in a similar proportion of FIV infected and non-infected cats (Fig. l ( a ) ) . Fever, sneezing, and serous ocular and nasal discharges were the most prominent clinical abnormalities. The FIV infected cats were more ill than the non-FIV infected cats from Day 4 post infection onward. Most of the FIV infected cats were partially or completely anorectic, 13 of 20 had pharyngitis a n d / o r rhinitis, and six of 20 became dehydrated. In comparison, eight of 20 of the nonFIV infected cats had pharyngitis a n d / o r rhinitis and three of 20 became dehydrated. Nine of 20 cats in the FIV infected group manifested signs of pneumonia (dyspnea, coughing, high fever and marked depression ) compared with one of 20 cats in the non-FIV infected group. Gingivitis and oral ulceration were observed in two of 20 FIV infected cats and in one of 20 of the non-FIV infected animals. Acute keratitis manifested by blepharospasm occurred in 50% of the cats in both FIV infected and non-infected groups; three of 20 of the FIV infected cats and none of the non-FIV infected cats developed corneal ulcers that healed within 10 days. No clinical signs of illness were observed among the two non-FHV-1 exposed control groups (naive cats and cats infected solely with FIV). FHV-1 infection was associated with a biphasic elevation of the rectal temperature on Days 3 and 7 post inoculation. The mean rectal temperature was significantly greater in FIV infected cats on Days 2, 4-7 and 11 post infection than in non-FIV infected animals (Fig. 1 ( c ) ) . Eleven of 20 cats in the FIV infected group had rectal temperatures that exceeded 40 °C at some point in their disease compared with five of 20 non-FIV infected cats. The mean rectal temperatures returned to preinfection levels in both groups of cats by Days 12-14 post infection. Based on the scoring system (Table 1 ), cats with pre-existing FIV infection were significantly (P_< 0.01 ) more ill than n o n - H V infected cats between Days 4 and 10 post infection (Fig. 1 ( a ) ) . Cats with clinical severity scores greater than 10 usually had fevers over 40 ° C. The comparative severity of the FHV-1 induced illness among FIV infected and non-FIV infected groups of cats was mirrored by the types and frequency of supportive treatment that were administered to treat dehydration and secondary bacterial infections. Seventeen of 20 FIV infected cats were treated a total of 73 times over a 9 day period. This was compared with 11/20 nonFIV infected cats that were treated 34 times over a 4 day period (Fig. 1 ( b ) ) .

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TABLE 2 Isolation of FHV-1 from oral and ocular secretions of FHV-1 and FIV/FHV-I infected cats following experimental FHV- 1 infection Days post infection

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Virus shedding F H V - 1 w a s r e c o v e r e d m o r e f r e q u e n t l y f r o m o c u l a r t h a n oral s e c r e t i o n s in b o t h F I V i n f e c t e d a n d n o n - F I V i n f e c t e d cats; 3 4 . 1 % a n d 3 2 . 8 % o f t h e t o t a l

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oral swabs taken from the FIV infected and non-FIV infected groups, respectively, yielded FHV- 1, compared with 43.5% and 43.5% of the respective ocular swabs. The proportion of cats that shed virus was similar between the two groups, except on Days 11 and 14 post infection. Eleven of 20 FIV infected cats were shedding virus on Day 11 post infection compared with four of 20 of the non-FIV infected cats; 12/20 and eight of 20 of the cats in the two respective groups were shedding FHV- 1 on Day 14. Virus shedding ceased by Day 35 post infection in all of the FIV infected and in 19/20 of the non-FIV infected cats (one non-FIV infected cat did not cease shedding until after Day 42 post infection) (Table 2). The titers of virus present in ocular and oral secretions were essentially identical in the two groups of animals for the duration of illness, with more virus present in ocular compared with oral secretions (Figs. 2 (a) and 2 (b)). Virus shedding peaked at Day 7 post infection, dropped slightly, and then levelled offuntil Day 28 post infection. Virus shedding ceased abruptly after that time. Shedding of FHV- 1 was never observed in the two non-FHV- 1 infected control groups. Antibody responses to FHV-1

Antibodies that neutralized FHV-1 in vitro were detectable as early as 14 days post infection in both FIV infected and non-FIV infected groups of cats. The mean FHV-1 neutralizing antibody titers were significantly lower ( P < 0.05) in FIV infected eats for the first 21 days post infection and then reached levels similar to those of non-FIV infected animals (Fig. 3 (a)). The peak of the FHV-1 neutralizing antibody responses occurred in both FHV-1 exposed groups of cats around 8 weeks post infection. FHV-1 neutralizing antibodies were never detectable in sera from the two non-FHV-I infected control groups. Immunofluorescence antibodies to FHV-1 that were of both IgM and IgG classes appeared in the serum 14 days post infection. The mean IgG antibody response against FHV-1 was not significantly different between FIV infected and non-FIV infected cats (Fig. 3 ( b ) ) . However, the FHV-1 specific IgM antibody response was significantly depressed in FIV infected compared with non-FIV infected cats during the entire 2 month post infection observation period (Fig. 3 ( c ) ) . Antibodies to whole FHV-1 were not observed in sera collected from the two non-FHV- 1 control groups of cats. Antibody responses to F I V

IgG specific antibodies to FIV were detected in sera from all FIV infected cats. Mean FIV antibody titers in sera from FIV alone infected cats ranged from 1 : 893 to 1:960 (Fig. 4 (a)). In the FIV/FHV-1 infected cats, mean FIV antibody titers were 1 : 1355 on Day 0, 1:733 on Week 2, 1:725 on Week 3,

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and 1:836 on Week 4 post FHV-1 infection (Fig. 4(a) ).There was no statistically significant difference in the mean titers of the two groups at any time point.

Hematologicchanges The mean preinfection blood levels of neutrophils were lower in the two FIV infected than in the two non-FIV infected groups of cats, but the decrease was only significant for the FIV infected group that was subsequently infected with FHV-1 (Fig. 5 (a)). Neutrophilia was observed on Day 7 following FHV1 infection in both FIV infected and non-FIV infected animals. There was little increase in band neutrophils, with no difference in their numbers found in circulation during this neutrophilia. Toxic changes were found in neutrophils of the sickest cats in both groups. The ratio of the absolute blood neutrophil count to total clinical score on Day 7 post infection was significantly lower in FIV/FHV- 1 infected than in non-FIV infected cats ( P = 0.008). The ratio for the FIV/FHV-1 infected cats was 0.26_+0.03 vs 0.39_+0.04

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(mean + SEM) for the non-FIV infected cats. The mean blood neutrophil counts returned to their respective baseline levels in both FIV infected and non-FIV infected group by Days 21-28 following FHV-1 exposure. The mean blood neutrophil counts remained unchanged during the entire course of the study for the two non-FHV- 1 inoculated control groups of cats. Preinfection mean blood lymphocyte counts were similar in naive, FIV alone and FIV/FHV-1 infection groups (Fig. 5 ( b ) ) . However, cats in the FHV- 1 infection group had significantly higher mean preinfection blood lymphocyte counts. A pronounced blood lymphopenia was observed on Day 7

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respond better to the specific antigen, the differences were of borderline significance (P= 0.052). Blood lymphocytes from the two non-FHV- 1 infected control groups failed to respond to specific antigen during the course of the study. The in vitro blastogenesis response of blood lymphocytes to Con A and

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0.5

0.0

0

7

14

2'1

2~1 35

42

49

.~0

7

1~1 21

28

35

42

49

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days post FHV-1 infection Fig. 8. (a) PWM induced; ( b ) Con A induced proliferation (mean counts min- 1per cell + SEM ) of peripheral blood lymphocytes of naive ( O ) , FIV infected ( • ) , FI-IV-1 infected ( A ) and FIV/FHV-1 infected cats ( • ). *Days with significant difference ( P < 0.05 ).

P W M was highest before FHV-1 infection in the naive control group of cats. The two groups of cats with pre-existent FIV infections had significantly lower responses to P W M prior to infection with FHV-1 and throughout the entire experimental period (Fig. 8 (a)). Responses to Con A tended to be more similar among the four groups of cats.

0.0

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Blood lymphocyte blastogenesis responses to the non-specific mitogens Con A and PWM were markedly decreased at Day 7 following FHV-1 exposure in both FIV infected and non-FIV infected groups and rapidly returned to baseline levels (Figs. 8 (a) and 8 (b)). The lymphocyte responsiveness to Con A was significantly lower for the FIV infected compared with non-FIV infected cats only at Day 7 following inoculation, while PWM responsiveness of the FIV infected animals remained significantly depressed compared with nonFIV infected cats for 3 weeks. DISCUSSION

Cats with pre-existing and outwardly asymptomatic FIV infection became significantly more ill after FHV-1 infection than cats that were not FIV infected. The results of this study confirmed those of similar experiments with different common feline pathogens. Cats that were in the primary stage of experimental FIV infection became more ill after being artificially infected with feline calicivirus and had diminished antibody responses (Dawson et al., 1991 ). FIV infected cats also became ticker after being infected with Chlamydia psittaci, vat. felis than non-FIV infected cats (O'Dair ct al., 1991 ). Although FIV infected cats developed a more severe disease following FHV1 infection, the pattern of illness was different than what was expected. It was anticipated that FIV infected cats would not only become more ill, but would remain sicker for a longer period of time, shed more FHV-1, have an extended period of FHV-1 carriage, and develop some of the chronic manifestations of FHV-1-related disease, e.g. chronic rhinitis, chronic conjunctivitis or chronic keratitis. However, most of the differences in FHV-1-related illness between FIV infected and non-FIV infected cats were observed in the acute rather than chronic stages of the illness, and manifestations of chronic FHV-1 related signs were not observed at all. Reasons for the increased severity of the acute FHV-1 induced disease in the FIV infected cats were not precisely determined. However, several hematologic and immunologic differences were noted between FIV infected and non-FIV infected animals. These differences appeared in neutrophil and lymphocyte numbers, lymphocyte subsets, lymphocyte blastogenesis responses, and primary antibody responses to FHV-1. FIV infected cats had significantly lower blood neutrophil counts prior to FHV-1 infection compared with non-FIV infected animals. Neutropenia is documented in clinical reports of people infected with HIV (Costello, 1988; Mir et al., 1989) and of cats infected with FIV (Shelton et al., 1989, 1990), but sequential neutrophil counts in response to FIV infection have not been monitored so far. A pronounced neutrophilia was observed at Day 7 following FHV-1 infection. Although the FIV infected cats started out with lower mean blood neutrophils, they were able to mount a mean neutrophil response

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of equal magnitude to that of the non-FIV infected cats. However, individual FIV infected cats that developed the most severe clinical signs of FHV-l induced illness were usually the cats that had the lowest neutrophil counts before FHV-1 infection, the cats that were least able to m o u n t a neutrophilia during infection, and the cats that produced the lowest titers of neutralizing antibodies. Evidently the intrinsic factors that decrease blood neutrophil counts in FIV infected cats do not necessarily prevent increased production a n d / o r release of neutrophils during times of inflammation. After the neutrophilia subsided, the mean blood neutrophil counts of cats with pre-existing FIV infection declined to their lower than normal preinfection levels. FIV infected cats had significantly lower total blood lymphocyte counts than non-FIV infected cats prior to infection with FHV-1. An absolute lymphopenia occurred following infection with FHV-1. However, the decrease in lymphocyte numbers in FIV infected and non-FIV infected cats paralleled each other and at the lowest point both groups were equally lymphopenic. The marked drop in lymphocyte counts of the cats infected with FHV-1 on Day 7 post infection may have reflected the stress of disease or, more likely, a recruitment of lymphocytes from the blood to lymphoid organs as a result of generalized i m m u n e response (Hall and Morris, 1963 ). The FIV infected cats had significantly lower CD4 + T-lymphocytes at the start of the infection than did non-FIV infected cats, consistent with the CD4 + T-lymphocyte depletion in people with HIV infection (Costello, 1988 ) and in FIV infected cats (Barlough et al., 1991; Torten et al., 1991 ). Infection with FHV-1 induced an acute drop in the numbers of both CD4 + and CD8 ÷ T-lymphocytes in both FIV infected and non-FIV infected cats that was most apparent on Day 7 post infection, coinciding with the absolute lymphopenia. The CD4 + / C D 8 ÷ T-lymphocyte ratios were unchanged at this time indicating that the lymphopenia was the result of an equal loss of both CD4 + and C D 8 ÷ T-lymphocytes from the blood. On Day 14 post infection, when the total blood lymphocyte count had returned to normal, there was a significant but transient drop in the C D 4 + / C D 8 ÷ T-lymphocyte ratios in both FIV infected and non-FIV infected cats. The decrease in the ratios was a consequence of an approximate twofold increase over preinfection levels of CD8 + T-lymphocytes; CD4 ÷ T-lymphocytes had returned to baseline values. Day 14 of the infection was just prior to the time that humoral i m m u n e responses were levelling off, and it would be tempting to attribute the absolute increase in CD8 ÷ T-lymphocytes at this time to normal immunoregulatory mechanisms. The absolute C D 4 + and CD8 + T-lymphocyte counts and the C D 4 + / CD8 ÷ T-lymphocyte ratios at the end of the study in both FIV infected and non-FIV infected groups of cats were not significantly different from their preinfection levels. Thus, it appeared that FHV-1 infection did not have a long-term deleterious effect on the levels of C D 4 + T-lymphocytes in FIV infected cats.

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Long-term experimentally FIV infected cats develop marked cellular immune dysfunctions such as loss of absolute CD4 + T-lymphocyte numbers and suppression of cell mediated immunity as measured by mitogenic lymphocyte proliferation (Taniguchi et al., 1990; Barlough et al., 1991; Torten et al., 1991 ). A similar reduction of PWM and Con A responsiveness was observed in both FIV infected groups prior to the herpesvirus challenge indicating that the FIV infected cats in the study were already becoming immunocompromised. Transient perturbations of mitogenic lymphocyte blastogenesis occur frequently in infectious diseases of normal cats (Gaskell and Povey, 1982; Tham and Studdert, 1987 ). The intensity and duration of this immune dysfunction, however, were significantly more prominent in the FIV infected than in the non-FIV infected cats. FHV-1 infection appeared to cause an increase in activated blood mononuclear cells, as evidenced by the peak of spontaneous lymphocyte proliferation observed in non-mitogen treated lymphocyte cultures taken on post infection Day 7. This spontaneous proliferation was more prominent in the FIV infected than non-FIV infected cats. In contrast to the suppressed non-specific mitogen response, antigen specific lymphocyte reactivity was higher in the FIV infected than in the nonFIV infected cats, although it was not flamboyant in either group. The relatively poor FHV-1 antigen specific response in both groups exposed to FHV1 is in agreement with previous reports (Cocker et al., 1986; Tham and Studdert, 1987) and paralleled the spontaneous proliferation. FIV infected cats also have had a greater Toxoplasma gondii antigen specific blastogenesis response than non-FIV infected cats (Lappin et al., 1991 ). The most pronounced immunologic differences between FIV infected and non-FIV infected cats were in primary virus specific antibody responses. The IgM antibody responses to FHV-1 were significantly lower in FIV infected cats, while the FHV-1 specific IgG responses were unaffected. Subtle abnormalities in primary humoral immune responses to synthetic antigens have already been recognized in experimentally FIV infected cats (Torten et al., 1991 ). This antibody defect involves mainly T-lymphocyte dependent antigens and becomes more pronounced with time after infection and CD4 + Tlymphocyte losses. Although chronic FIV infection enhanced the acute FHV-1 induced disease, there was no evidence that the FHV-1 infection or immunity affected the underlying FIV infection. The acute FHV-1 infection did not change the antibody response to FIV. This could be considered as an indirect evidence that the generalized immunologic stimulation brought about by acute FHV-1 infection did not enhance replication of FIV. This was surprising because it is known that coinfection with FHV-1 will upregulate chloramphenicol acetyltransferase (CAT) expression by a FIV-LTR-CAT construct in an in vitro cell transfection assay (E.E. Sparger, Davis, personal communication, 1991 ).

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Human herpesvirus genes will also upregulate HIV in vitro (Laurence, 1990). However, preliminary studies failed to show upregulation of either FIV or FHV-1 replication by coinfection of cells with the two viruses in vitro (E.E. Sparger, personal communication, 1991 ). This corroborated what was observed in vivo in this study. There is no evidence up to this point that common feline infectious diseases accelerate the demise of the immune system in cats chronically infected with FIV. Non-specific immune activation has been theorized to switch on virus production in latently infected lymphocytes and to lead to cell death; increased lymphocyte destruction would then lead to an accelerated decline in specific subsets of T-lymphocytes and increased immunodeficiency (Zagury et al., 1986; Margolick et al., 1987 ). In spite of a vigorous clinical course of disease, and an apparent pronounced immune response to the causative agent, the immune system of the cats infected with FIV for 18 months still performed adequately in a qualitative, if not quantitative, manner. The degree of CD4 + T-lymphocyte depletion was also not enhanced by FHV-1 infection. However, the cats reported in this study have only experienced two sequential infections, toxoplasmosis and herpesvirus infection. It is thus possible that more numerous, more frequent, or more severe immune stimuli could accelerate the normal, time related decline in C D 4 + T-lymphocyte numbers or other immune functions that have already been observed in cats kept isolated from all pathogens (Barlough et al., 1991; Torten et al., 1991 ). ACKNOWLEDGMENTS

This work was supported by Public Health Service grants A1-25802-05 and CA-50179-03 to N.C.P. and 2RO 1 AI-26120-04 to C.K.G. from the National Institutes of Health. We thank Pro-Visions, Checkerboard Square, St. Louis, MO, for the supply of cat food. We are very grateful to Nancy Delemus, Kim Floyd-Hawkins, Jenni Del Carlo, Diane Hoffmann, Renan Acevedo, Peter Hill and Dr. Dorette Reubel for expert technical assistance. We also thank Dr. Gordon Theilen, Department of Surgery, School of Veterinary Medicine, University of California, Davis, in whose laboratory a portion of this research was performed.

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