Guinea Pig Herpes-Like Virus Infection

Transcription

1 INF7CTION AND IMMUNITY, Mar. 1973, p Copyright 1973 American Society for Microbiology Vol. 7, No. 3 Printed in U.S.A. Guinea Pig Herpes-Like Virus Infection I. Antibody Response and Virus Persistence in Guinea Pigs After Experimental Infection K. M. LAM1 AND G. D. HSIUNG Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut, and the Veterans Administration Hospital, West Haven, Connecticut Received for publication 6 November 1972 Guinea pigs, experimentally infected with guinea pig herpes-like virus produced antibodies which were detectable by indirect hemagglutination (IHA), complement-fixation (CF), and neutralization tests. The IHA test appeared to be a more sensitive method than the CF or neutralization test for determining antibody response in guinea pigs immediately after infection, but the IHA method was not suitable for the detection of antibody in animals receiving small doses of virus. Antibody titers obtained by CF tests were generally higher than those obtained by the neutralization test, and they followed the same time course when individual aniimals were studied serially. Intracardiac produced the best antibody response in guinea pigs when compared with other routes of infection. Guinea pigs infected by the intraperitoneal, intranasal, or oral route showed rising antibody titers but the levels were low. Infectious virus was isolated from and persisted in all inoculated animals in the presence of antibody regardless of the route of. Recovery of infectious virus required cultivation or cocultivation of tissue cells containing virus. The administration of antilymphocyte sera delayed the appearance of IHA antibody but had no effect on antibodies determined by the CF- and neutralization tests. A herpes-like virus originally isolated from leukemic strain 2 guinea pigs was found to be present in a high percentage of inbred strain 2 guinea pigs (2, 6, 9) and, in a few instances, in Muta strain animals, but rarely in the randombred Hartley strain (6). Subsequently, it was found that the virus can multiply in Hartley guinea pigs after experimental infection (3, 7, 8). The virus was maintained at a stable level in the blood of inoculated guinea pigs for life after an initial period of virus multiplication. A low level of virus-neutralizing antibody was detectable in the sera of animals whose tissue cells and leukocytes carried the virus. The coexistence of infectious virus and homologous antibodv in the blood prompted our interest in further studying antibody response in Hartley guinea pigs after experimental infection. A newly developed indirect hemagglutination test (IHA), together, with the conventional complement-fixation (CF) and neutralization tests, were compared with respect to the sensitivity and reliability of these three methods for the determination of antibody 1 Present addres: Department of Virology and Epidemiology, Baylor College of Medicine, Texas Medical Center, Houston, Tex titers to herpesvirus infection. Antibody responses to by different routes with varying doses of virus and the effect of antilymphocyte serum are also presented. MATERIALS AND METHODS Cell culture. Primary cell cultures were prepared from kidney tissues of normal Hartley guinea pigs according to procedures previously described (7). Virus stock. The guinea pig herpes-like virus (GPHLV), strain Y-407, was isolated from a healthy strain 2 guinea pig and was used throughout the study. Stock virus was prepared in guinea pig kidney cell monolayers infected with GPHLV at a multiplicity of infection of approximately 1.0. After more than 75% of the cells showed cytopathic effect (CPE), the infected cultures in flasks were frozen and thawed three times and clarified by centrifugation at 600 X g to remove debris. The supernatant fluid was then harvested and frozen at -70 C until use. This virus stock had an infectivity titer of approximately 106 tissue culture infective dose (TCID)50/ml and was used for the of animals and also as antigen for the IHA, neutralizatioin, and CF tests. Source of animals. Adult random-bred Hartley guinea pigs of both sexes were purchased 426

2 VOL. 7, 1973 GUINEA PIG HERPES-LIKE VIRUS INFECTION 427 from Camm Research Institute, Inc., Wayne, N.J. Adult New Zealand white rabbits were obtained from Samolis's Rabbitry, Waterville, Conn. Animal and sampling. The virus suspension was given to guinea pigs orally or by injection into the intraperitoneal cavity or into the heart. The inoculated guinea pigs were bled prior to and at frequent intervals after the of the virus. Isolation of virus from the heparinized blood was achieved by inoculating 0.1 ml of whole blood into guinea pig kidney cell cultures. The appearance of herpesvirus CPE in the inoculated cultures was considered a positive isolation. Sera were collected from clotted blood and were used for antibody analysis as described below. IHA test. A modification of the method of Bernstein and Stewart was used (1). Guinea pig red blood cells (RBC) were washed three times with phosphate buffered saline (PBS), ph 7.4. One milliliter of washed, packed RBC was then mixed with 80 ml of 1:40,000 tannic acid, incubated in a 37 C water bath for 15 min, and sedimented by centrifugation at 600 X g. The tanned RBC were then washed once with PBS, and a 10% suspension was made in the same solution. To 1 ml of 10% tanned RBC was added 0.7 ml of GPHLV stock suspension and 2 ml of PBS (ph 6.4). The mixture was then incubated in a 37 C water bath for 20 min; this was followed by centrifugation, washing, and recentrifugation. The virus-coated RBC were suspended in 6 ml of PBS (ph 7.4) to reach a final concentration of approximately 1%. A 0.1-ml amount of the virus-coated RBC was added to tubes containing 0.5 ml of twofold serially diluted test sera. The diluent was a 2% solution of heat-inactivated normal rabbit serum in PBS. RBC and diluted sera were then mixed thoroughly and incubated at 37 C for 2 h. The highest dilution of the serum that showed agglutination of the virus-coated RBC was considered the titer of the serum tested. CF test. The standard procedure of overnight fixation of complement at 4 C was used. All test sera were heat-inactivated and diluted with Veronal-buffered solution. The virus stock was heated at 56 C for 30 min, diluted 100-fold, and used as antigen. Equal volumes of serial serum dilutions and the virus antigen were mixed and incubated at 4 C for 2 h before addition of four units of complement. The mixture stood at 4 C overnight, and sensitized sheep RBC was added 18 h later; all samples were incubated at 37 C for 30 min before the results were recorded. Sera preventing 50% or more hemolysis were considered to have CF antibody. Neutralization test. Twofold serial dilutions of sera were mixed with an equal volume of virus suspension containing approximately 100 TCID,0 for 2 to 3 h at room temperature. A 0.2-ml amount of the mixture was inoculated into replicate guinea pig kidney culture tubes and incubated at 35 C. All cultures were examined daily for 4 to 5 days until the appearance of CPE in the cultures inoculated with the challenge dose. The highest serum dilution that inhibited the CPE of the challenge dose was considered the neutralizing antibody titer to GPHLV. Production of antilymphocyte serum. The thymus glands of healthy guinea pigs were removed and trypsinized. The trypsinized cells were washed once with Hanks balanced salt solution (HBSS) and centrifuged. The cell concentration was adjusted to 107 cells/ml, and an equal volume of complete Freund adjuvant was added. Rabbits were given the thymus cell suspension intramuscularly, 1 ml on day 1, 2 ml on day 8, and 3 ml on day 14. Two weeks after the third, rabbits were exsanguinated to obtain antilymphocyte serum (ALS). The potency of the ALS was determined by a 50% reduction of leukocyte counts within 24 h after guinea pigs were inoculated with 1 ml of the rabbit ALS. Inoculation of guinea pigs with GPHLV and ALS. Each of a group of six guinea pigs was inoculated with 0.5 ml of GPHLV stock suspension intraperitoneally on day 0; three of the guinea pigs were subsequently inoculated subcutaneously with 1 ml of ALS on days 1, 7, and 14. All of the six guinea pigs were bled prior to and at weekly intervals after the administration of the virus. All sera were tested for antibody response by the three serological tests described above. RESULTS Comparison of neutralization, CF, and IHA tests. Table 1 compares the three serological tests for measuring antibody titers to GPHLV infection. A control Hartley guinea pig (HAR-5), which was negative for virus isolation from the blood, had no antibody that could be detected by any of the three tests used. In both the uninoculated but naturally infected strain 2 guinea pig (Y407-1) and the contact-infected Hartley guinea pig (LKH32), no antibody titer was detectable by the CF test, and only a low titer was detectable by the neutralization test. However, an antibody titer of 1:160 and 1:80 respectively, was obtained by the IHA method. The remaining guinea pigs were all experimentally infected with the virus. Antibody titers were demonstrable in most of these animals by all of the three methods used. The IHA titers were generally higher than the titers obtained by the CF or neutralization test. No correlation between the latter two methods was evident. The IHA test appeared to be the most sensitive among the three methods used for the detection of antibodies to GPHLV infection. Antibody response in guinea pigs inoculated orally or intrana8ally. Two groups of guinea pigs were used to study the effectiveness of intranasal. Each animal received 0.1 ml of virus suspension (Table 2). In experi-

3 428 LAM AND HSIUNG INFECT. IMMUNITY ment 1, infectious virus was first detectable in the blood of one animal, LA 118, on day 14 after and on day 17 in the remaining three animals. The IHA antibody titers first appeared in LA 115 on day 10 and in the others on day 22. Three animals were used in experiment 2, all of which harbored infectious virus in their blood when examined 49 days after. The IHA antibody titers of these three animals ranged from 1:20 to 1:320, and the neutralizing antibody titer ranged from 1:10 to 1:20. Three guinea pigs were used in experimenit 3 (Table 2); each was given 0.5 ml of the same virus TABLE 1. Comparison of neutralization, complement-fixation, and indirect hemagglutination tests in determining antibody titers to GPHLV infections no. Route of infection Antibody titers Neut. CF IHA HAR-5b None <5 <5 <5 Y407-1 Natural infection 5 < LKH 32 Contact infection <5 <5 80 LA Days post IPc ,280 LA Days post IP ,280 LA Days post IP 40 <5 1,280 LA Days post IP ,280 a Reciprocal of the highest serum dilution. b Uninoculated control. c IP, intraperitoneal. suspensioin by mouth. All of the animals were sacrificed 49 days after. Virus was isolated from the blood, spleen, thymus, and testes of LA 72 which also had an IHA antibody titer of 1:20 and a neutralizing antibody titer of 1:5. The other two animals had no detectable virus in their blood or visceral tissues, and they had no detectable antibody titers. Antibody response and virus persistence in guinea pigs inoculated by the intracardiac route. Adult Hartley guinea pigs were administered intracardiac injections of 2.7 to 5.7 log TCID5, of the virus with two animals in each group. The results obtained are represented in Fig. 1. The guinea pigs receiving 5.7 log TCID50 of the virus developed antibody detectable by the IHA test 7 days after and by neutralization test 14 days after. The IHA antibody titers gradually increased in this group until they reached a peak level in the 6th week and then started to decline. Neutralizing antibodies increased slowly, reached a high level in the 5th week, and remained at that level for the duration of the experiment. The IHA antibody in guinea pigs receiving 4.7 log TCID50 of the virus rose moderately and leveled off rapidly. The neutralizing antibodies of the same guinea pigs appeared in the 3rd week, and a maximum level in the 6th week, and remained at that level. The IHA titer in the guinea pigs receiving 3.7 log TCID50 of the virus was negligible and was not persistent. The neutralizing antibody, however, was similar to that of the guinea pigs receiving 4.7 log TCID50 of the virus. The neutralizing antibody titer in the guinea pigs inoculated with 2.7 log TCID50 of the virus was significant although minimal; a low level of IHA TABLE 2. Virus isolation and antibody response in guinea pigs inoculated with GPHLV by oral and intranasal routes Route of Amount of No. days Virus isola- Antibody titerse Expt. no i uaon inoculum Guinea pig no post in- tion from Mronrlaton (log TCID&o) oculation whole blood IHA Neut. 1 Intranasal 5.2 LA NDb LA ND LA ND LA ND 2 Intranasal 5.2 LA LA LA Oral 5.7 LA _ <10 <5 LA LA _ <10 <5 a Reciprocal dilution of serum: experiment 1 blood samples were taken on day 22 for antibody analysis and experiment 2 and 3 samples were taken on day 49. b ND, Not done.

4 VOL. 7, 1973 GUINEA PIG HERPES-LIKE VIRUS INFECTION 429,,oif NO TREATMENT ALS TREATED tso A A $ B a so I0 5 WEEKS AFTER INOCULATION 4 Virus leocule I" TCIDS0 0 Neuralization Test ndirect Hem.gtlutlio T"t F1(:. 1. Antibody response and virus persistencc in guinea pigs inoculated by the intracardiac route. antibody was oinly detectable sooii after, as in the previous group. The virus was not isolated from anly of the guinea pigs before inioculatioil, but it was recovered from the blood of all inoculated guinea pigs as early as 5 min after, except those which received 2.7 log TCID50 of virus. However, 24 h after, virus was detected only in the blood of the animals receiving 5.7 log TCID50 of the virus. On day 7, isolation of the virus was achieved in guinea pigs receiving both 5.7 and 4.7 log TCID50 of virus. By day 14, the virus was detected in the blood of all inoculated animals with an infectivity titer of 1.7 log TCID50 or higher irrespective of the initial inoculum or the presence of neutralizing or IHA (or both) antibody titers. Infectious virus in significant titers persisted in all animals studied. during the entire 12 weeks. Effiect of afntilymphocyte serum on antibody response in guinea pigs inoculated intraperitoneally. Antibody response to GPHLV in guinea pigs was noted 5 days after intraperitoneal as determined by the IHA test (Fig. 2, left column). The IHA antibody titers reached maximum levels on day 9 but disappeared in two of the three guinea pigs 4 to 6 weeks after. Neutralizing and CF antibodies in these animals appeared at about -J a: hi 13 -i 2I ICI SC so '5 < C so AAA A a t0 WEEKS AFTER VIRUS INOCULATION Inoculation of ALS b-6nou t. 0-CF IHA FIG. 2. Antibody response to GPHLV in guinea pigs with or without ALS treatment. The virus was administered intraperitoneally on day 0. Each letter (A, B, or C) represents an individual guinea pig. 9 days to 3 weeks after intraperitoneal and remained detectable throughout the experiment. The titers determined by the CF test were parallel to but generally higher than those determined by the neutralization test. In guinea pigs treated with ALS, the rise in IHA antibody was delayed until about 1 week after the ALS injections were discontinued. In addition, the IHA antibody response persisted for a longer period of time than was observed in guinea -pigs without the ALS treatment. The administration of ALS did not affect the time of appearance or the titers of CF and neutralizing antibody. Both ALS-treated and nontreated guinea pigs had about the same magnitude of antibody response to the virus at about the same time, as determined by the latter two methods. DISCUSSION The use of a newly developed IHA test enabled us to detect early antibody rises in guinea

5 430 LAM AND HSIUNG INFECT IMMUNITY pigs inoculated with GPHLV. The test appeared to be sensitive, and the titers were generally higher than those determined by CF and neutralization tests. Other investigators have shown that the IHA test can be used for the detection of antibodies to herpesviruses of man, including cytomegalovirus and herpes simplex virus (1, 4, 5). However, the results of the IHA test were noted to be inconclusive in sera obtained from animals receiving smaller doses of GPHLV (Fig. 1). One must use caution, therefore, when the IHA test is to be used as a sole criterion for the detection of antibodies to herpesvirus infection. In a previous study, we reported that Hartley guinea pigs can be infected with GPHLV by the intranasal or oral routes but the latter was a less effective method (8). These observations were confirmed in the present study both by virus isolation and antibody analysis. All intranasally inoculated animals and one of the three orally inoculated animals had measurable antibody titers and infectious virus in their blood at the time of sacrifice. In both instances, antibody titers were low compared to those in the parenterally inoculated animals. Guinea pigs infected by the intracardiac route gave the best antibody response as determined by the neutralization test. Therefore, this route might be a better method for the studies of antibody responses to GPHLV in guinea pigs than other sites of. Animals receiving 3.7 to 4.7 log TCID5o had a better response than animals receiving either a higher or lower degree of virus as determined by the neutralization test. The reason for the low neutralizing antibody titers occurring in animals receiving large doses of virus is not clear. It is possible that a virusantibody complex is formed and cannot be detected by the methods described. The injection of ALS into the guinea pig did not affect the production of CF or neutralizing antibody in response to intraperitoneal of the virus. Both ALS-treated and nontreated guinea pigs developed antibody titers of approximately the same level at about the same time. In contrast, ALS appeared to suppress IHA antibody production. The IHA titers in the ALStreated animals began to rise 1 week after the discontinuation of ALS. As a result, the appearance of IHA titers was delayed for 2 to 3 weeks in the ALS-treated animals as compared with the nonitreated onies. A rapid fall of IHA titer was observed in two of the three animals without ALS, whereas the IHA antibody titer remained high in the ALS-treated aniimals. It is nlot kniown whether the administration of ALS prevented or delayed the decline of IHA antibody titers in the treated guinea pigs since experiments were terminated 3 to 7 weeks after ALS was discontinued. It was noted that GPHLV can be readily recovered from infected guinea pigs for their life span, 12 weeks in the present study, and more than 2 years in others (unpublished observation). The virus persisted in the leukocytes and various tissues of infected guinea pigs regardless of the route of. However, the site of virus multiplication and the location of the virus persistence are still unknown although the spleen appeared to be the major organ involved (12). As is shown in part II of this study (Lam and Hsiung), persistence of viremia appeared to be greatly affected by the route of in infection with the GPHLV in a foreign host, the rabbit. The findings in the present study represent an important concept concerning persistent herpesvirus infections in man and animals. In most instances, information regarding persistent herpesvirus infection was obtained from artificial model systems; for example, human herpes simplex virus infection in mice or rabbits (10, 11). It is apparent that the present study on the natural infectious process of GPHLV in guinea pigs, together with the accompanying paper on the persistence of GPHLV in rabaits, permits us to check the validity and applicability of findings obtained from artificial model systems. ACKNOWLEDGMENTS This investigation was supported by Public Health Service grant AI from the National Institute of Allergy and Infectious Diseases, and by Veterans Administration research funds. LITERATURE CITED 1. Bernstein, M. T., and J. D. Stewart Indirect hemagglutination test for detection of antibodies to cytomegalovirus. Appl. Microbiol. 21: Bhatt, P. N., D. H. Percy, J. L. Craft, and A. M. Jonas Isolation and characterization of a herpes-like (Hsiung-Kaplow) virus from guinea pigs. J. Infect. Dis. 123: Booss, J., and G. D. Hsiung Herpes-like virus of the guinea pig: Propagation in brain tissue of guinea pigs and mice. J. Infect. Dis. 123: Fuccillo, D. A., F. L. Moder, L. W. Catalano Jr., N. M. Vincent, and J. L. Sever Herpesvirue hominia types I and II: a specific micro-indirect hemagglutination test. Proc. Soc. Exp. Biol. Med. 133: Fuccillo, D. A., F. L. Moder, R. G. Traub, S. Hensen, and J. L. Sever Micro-indirect hemagglutination test for cytomegalovirus. Appl. Microbiol. 21: Hsiung, G. D., L. S. Kaplow, and J. Booss Herpesvirus infection of guinea pigs. I. Isolation,

6 VOL. 7, 1973 GUINEA PIG HERPES-LIKE VIRUS INFECTION 431 characterization and pathogenicity. Amer. J. Epidemiol. 93: Lam, K. M., and G. D. Hsiung Herpesvirus infection of guinea pigs. II. Transplacental transmission. Amer. J. Epidemiol. 93: Lam, K. M., and G. D. Hsiung Further studies on the mode of transmission of herpeslike virus in guinea pigs. Proc. Soc. Exp. Biol. Med. 138: , Nayak, D. P Isolation and characterization of a herpesvirus from leukemic guinea pigs. J. Virol. 8: Stevens, J. G., and M. L. Cook Latent herpes simplex virus in spinal ganglia of mice. Science 173: Stevens, J. G., A. B. Nesburn, and M. L. Cook Latent herpes simplex virus from trigeminal ganglia of rabbits with recurrent eye infection. Nature N. Biol. 235: Tenser, R. B., and G. D. Hsiung Infection of thymus cells in vivo and in vitro with a guinea pig herpes-like virus and the effect of antibody on virus replication in organ culture. J. Immunol 110: