Experimental smallpox in chimpanzees

Bulktdn of the World Health Organizaton, 57 (4): 637-641 (1979) Experimental smallpox in chimpanzees S. S. KALTER,1 A. R. RODRIGUEZ,2 L. B. CUMMINS,3 R. L. HEBERLING,4 & S. 0. FOSTER5 In an attempt to prepare highly specific antiserum to variola virus, a chimpanzee was inoculated with a virulent human strain of this virus. Three uninoculated chimpanzees were housed in the same room; two of these developed clinical disease with seroconversion, while the third developed no evidence of infection and no antibody. The three animals that became ill also developed antibody to vaccinia and monkeypox viruses. Human contacts during the study and following a break in containment showed no evidence of infection as determined by serological tests and lack of clinical disease. Global eradication of smallpox has been a goal of the biomedical community for the past ten years. This achievement is apparently now within sight (1, 2), but, according to WHO criteria, it will take at least two years after detection of the last patient before global eradication can be certified (3). Out- breaks of exanthematous diseases, especially those due to poxviruses, will continue to need specific etiological diagnosis. For these investigations, highly specific antisera for variola virus would be desirable. An antiserum for these purposes might be prepared from human or other animal sera by repeated adsorption with orthopoxviruses to eliminate cross- reacting antibodies. Alternatively, it might be poss- ible to prepare an antivariola serum of high titre and specificity by inoculation of a nonhuman primate (for example, a chimpanzee). This report describes the inoculation and infection of a chimpanzee with variola virus, with the inten- tion of obtaining specific variola antiserum, and the subsequent contact infection of 2 out of 3 other chimpanzees housed in the same room. During the experiment an unexpected potentially dangerous biohazard occurred, but fortunately, smallpox did 1 Director, Department of Microbiology and Infectious Disea- ses, Southwest Foundation for Research and Education, San Antonio, TX 78284, USA. 2Associate Foundation Scientist, Department of Microbiology and Infectious Diseases. 3Associate Foundation Veterinarian, Department of Micro- biology and Infectious Diseases. 4Foundation Scientist, Department of Microbiology and Infec- tious Diseases. I Medical Officer, Research and Development Division, Bureau of Smallpox Eradication, Center for Disease Control, US Depart- ment of Health, Education, and Welfare, Atlanta, GA 30333, USA. not spread to the community. However, the incident emphasized the importance of maintaining maxi- mum security in laboratories working with variola virus. MATERIALS AND METHODS Chimpanzees (Pan troglodytes) Four young adults, 1 male and 3 females, were individually caged in an isolation building at a controlled temperature. The male (4 x 33) was used as the test animal. All animals were first bled and specimens (throat and rectal swabs) were collected for laboratory study. No haemagglutination inhi- bition (HI) antibody to vaccinia or monkeypox virus was detected in the initial serum samples. Virus The Zaire strain of Congo isolate No. 1 (V-70-1- 46) of variola was obtained from Dr J. H. Nakano (Center for Disease Control, Atlanta, GA, USA). The inoculum was prepared by cultivation in chim- panzee lung cells (SFRE-CL 1) (4) which were grown and maintained in Eagle's medium (MEM) supplemented with 50 ml of chimpanzee (4x 33) serum per litre. The skin area (6.5 cm2) to be inoculated was first shaved, cleaned with alcohol, dried, and then scarified with a hypodermic needle. Undiluted virus suspension (1065 TCID50/O.1 ml) on a cotton tip swab was then rubbed into the area as previously described (5). Sampling On days 7, 21, 30, 39, and 77 after inoculation, blood, throat, and rectal swabs were collected for 3836 - 637 - S. S. KALTER ET AL. serological, virological, and bacteriological examin- ation from chimpanzee 4 X 33. On days 30, 39, and 77, similar specimens were collected from the other 3 chimpanzees. Approximately 100 ml of blood was taken from each animal on the last two bleedings for reference purposes. Serological procedures The antigens used were monkeypox virus, Copenhagen strain, and vaccinia virus, IHD (Inter- national Health Division) strain. Both pools were prepared at the Southwest Foundation as previously described (5). The antisera used were monkeypox virus hyperimmune serum (Rabbit No. 173) pre- pared at the Southwest Foundation and vaccinia hyperimmune serum (Rabbit Lot 4) from the Center for Disease Control. All sera were diluted 1:10 in physiological saline and heat-inactivited for use in the tests. Chicken erythrocytes, previously tested for susceptibility to haemagglutination, were used at a concentration of 0.8% in physiological saline. Stan- dard HI tests (6) were performed at room tempera- ture and read when a button had formed at the bottom of the control tube. Back-titrations of anti- gens and positive controls were included in each test. Monkeypox and vaccinia viruses were used mainly, to avoid unnecessary exposure to variola virus. One serum neutralizing test (6) was performed on the last sera taken from all 4 animals, using approximately 300 TCID50 of variola virus. Radioimmune assays (RIA) using variola virus antigen on sera obtained late in the course of the illness were performed by Dr J. H. Nakano, Center for Disease Control, Atlanta, GA, by procedures standard in that laboratory (7). Isolation of animals and environmental precautions The nature of the study required complete iso- lation of chimpanzees and strict precautions with the personnel involved (veterinarian, animal handler, two technicians). These human contacts were kept to a minimum. Housing was in an isolation hut approximately 4.8 x 6 m, located in a separate isolation area and 12 m from the other huts in the area. Each unit had its own heating and air-conditioning, with exhaust air (unfiltered) directed to the outside. Distance and sunlight were relied upon as the principal factors for reducing the risk of spread by airborne virus. Cages, walls, and floors of the building were washed daily with large amounts of chemical disinfectant. The waste was carried by an open drain to the sewer line. Each animal was individually caged. Although the cages were not touching, animals in adjoining cages could and did touch each other. Protection of personnel All personnel involved had been previously vacci- nated and had been shown to have antibody to the poxvirus group, as determined by HI tests. The staff wore coveralls while in the isolation area and wore laboratory coats, masks, head covering, and gloves while in the hut. After use, all garments were autoclaved before washing. Paper and other particu- late matter, including sponges and needles were enclosed in a bag and incinerated. RESULTS Clinical findings On the seventh day after inoculation, chimpanzee 4 x 33 had a rectal temperature of 39.4 'C. The scarified area was erythematous and had pustular lesions resembling those reported in baboons in- fected with the Harvey strain of variola (5). Four- teen days after infection, the primary lesion had very little fluid, but scrapings were taken for virus isola- tion studies. Secondary lesions were observed to be distributed over the trunk and extremities but not on the face, and the animal had a rectal temperature of 37 'C. These lesions were small (approximately 3-4 mm) and slightly pustular. Approximately 3 weeks after inoculation, chimpanzee 4 x 33 had a temperature of 37.5 'C, and all the lesions were healing. At this time (i.e., 21 days after inoculation) chimpanzee 4 x 34, which was caged to the left of chimpanzee 4 X 33, but within its reach, was noted to have lesions (small vesicular pustules) over the entire body. Chimpanzee 4 X 35, caged to the right of 4 X 33, showed no lesions, but chimpanzee 4 x 36, to the right of 4 X 35, was thought to show signs of incipient lesions. Security precautions prevented close examination of the animals. Thirty days after inoculation all 4 animals were sedated, examined, and samples were collected. The results may be seen in Table 1. Chimpanzees 4 x 33 and 4x 34 had healing lesions. However, chimpan- zee 4 x 36 was severely ill, with widespread and numerous lesions (pustules) all over the body, in- cluding the head and neck. Chimpanzee 4 x 35 still showed no evidence of clinical infection. By the 39th day, the lesions on the sick animals were either healed or healing. Chimpanzee 4x 35 still showed no signs of disease. These observations were con- 638 EXPERIMENTAL SMALLPOX IN CHIMPANZEES Table 1. Clinical and (Congo strain) serological findings on chimpanzees, following inoculation of one (4X33) with variola Clinical findings Serological findings (HI)' Day 4X33 4X34 4X35 4X36 4X33 4X34 4X35 4X36 0 - - - - <10 <10 <10 <10 7 Local - - - <10 ND ND ND pustular lesion 39.4° C 14 Dry, small - - - <10 ND ND ND lesions 370 C 21 Lesions Generalized - 40(MPV) ND ND ND healing lesions 10(VAC) 30 Lesions Lesions - Extensive 40(MPV) 40 <10 80 healing healing generalized 10(VAC) 20 <10 20 lesions 39 Healing Healing - Healing 40(MPV) 40 <10 80 20(VAC) 20 <10 40 83b 56b 25b 53b 77 Healed Healed - Healed 10(MPV) 10 <10 10 10(VAC) 10 <10 10 16c 4c ±c 2c 72b 323b l5b 11 6b :Reciprocal of serum dilution; MPV = monkeypox virus; VAC = vaccinia.b RIA titres. 'Serum neutralization test against approximately 300 TCID50 homologous (variola) virus. firmed by serological (HI and RIA) results (Table 1). The most severe form of the clinical disease was seen in chimpanzee 4 x 36, while chimpanzee 4 x 35 neither exhibited any clinical disease nor developed antibody. It would appear that chimpanzee 4X36 became infected as a result of virus aerosols. Serological findings The results are shown in Table 1. The 3 animals (4 x 33, 4 x 34, and 4 x 36) that developed clinical illness seroconverted, as evidenced by HI, SN, and RIA tests. Seventy-seven days after chimpanzee 4 x 35 had been inoculated, HI and SN titres were starting to decline. High titres were not detected by any of the 3 serological tests. Biohazard incident At the height of the infection, a break occurred in the drain leading from this isolation unit to the sewer line. Maintenance personnel, not realizing the poss- ible dangers, immediately and without consultation attempted to repair the damage. This placed several individuals in direct contact with sewage and wash- ings from the unit housing the infected chimpanzees. As soon as the scientific staff learned what had happened, immediate steps were taken to collect blood and vaccinate all contacts. Fortunately, all individuals who had been in direct contact were found to have antibody to the poxviruses by one or another serological procedure. But there were some individuals who were only remotely connected with the experiments who did not have detectable anti- body. All contacts were placed under twice daily fever surveillance for 21 days after possible exposure or 7 days after a major reaction to vaccination. No cases occurred, but one individual, an animal hand- ler, did develop a one-day fever (varying between 38 °C and 39.7 °C) during the observation period. He was immediately placed in strict quarantine and observed for 3 days, during which time his tempera- ture remained normal. Tests for active virus in the building effluent were negative. DISCUSSION One of the more interesting observations arising from this study was the difference in the severity of clinical disease in the animals. The illness varied from a relatively mild infection in the inoculated animal (4 x 33) to a severe, generalized infection in the third case (4 x 36). The reason why chimpanzee 639 S. S. KALTER ET AL. 4 X 35 did not develop clinical disease is obscure. However, among people (S. 0. Foster, unpublished data), it is not uncommon for members of a house- hold containing a smallpox patient, or other con- tacts, to fail to develop the disease. Further studies, for example testing chimpanzee 4 X 35 for suscepti- bility to smallpox virus infection, were curtailed following the break in security. The appearance of a questionably positive antibody titre against variola virus (serum neutralization) will need further study. The differences in severity might be due to the route of infection; 4x 33 had been inoculated by variolation, whereas 4 x 36 became infected via the respiratory tract. The upper respiratory route is the natural mode of infection and may result in a more severe form of disease than the mild form that often occurs after variolation. The relatively mild disease in the inoculated animal was similar clinically to that seen in baboons inoculated with the Harvey strain of smallpox virus (5). Ten passages in baboons with the Harvey strain of variola virus revealed no apparent change in virulence. The severe form of smallpox in the third chimpanzee (4 x 36), which was caused by the more virulent Congo strain, was similar to that seen in people infected with this strain. This obser- vation paralleled our findings with the Copenhagen strain of monkeypox virus when serially passaged through 10 baboons (5). During that passage series, the monkeypox virus, on cultivation in the chick embryo, assumed the plaque characteristics of vac- cinia virus, which is the possible origin of monkey- pox virus. This observation is of importance a,nd is under study. Like variola virus, vaccinia and'chimp-9 viruses in baboons showed no alterations in biologi- cal properties on serial passage. It should be noted that McConnell et al. (8) similarly reported variation in the clinical response of chimpanzees to a monkey- pox virus (Utrecht strain 65-32), along with the failure of one animal to develop clinical evidence of disease. It is not clear why the antibody response, as determined by HI tests, was so weak. Additional serological studies utilizing RIA (Table 1) confirmed the seroconversion but, again, did not show high titres to variola virus. The serological results also demonstrated the group reaction encountered when working with the orthopoxviruses. Previous associ- ation with viruses of this group was not demon- strated, as indicated by the lack of antibody in the initial serum; nevertheless, antibody to both monkeypox and vaccinia viruses developed quickly following contact with the variola virus. Thus, in order to obtain "pure" variola virus antiserum, these sera would need to be absorbed with appropriate antigens. Perhaps the most important aspect of this study was the creation of a potentially dangerous biohazard to our personnel. In retrospect, this entire episode appears inconsequential. However, at the time the implications were enormous. In spite of all precautions at an establishment well aware of biohazards associated with working with dangerous viruses, carelessness on the part of personnel not directly related to the study could have initiated an outbreak of human smallpox. All laboratory person- nel had been previously vaccinated. However, per- sons such as maintenance staff (labourers, plumbers, etc.), who were not expected to be involved, could have been exposed. Their vaccination record was regularly checked. Future studies of a similar hazatdous nature require detailed planning to in- chide the unexpected, and should take into consider- ation city, fire, and police departments, outside contractQrs, maintenance personnel, and others who may have to be brought in because of a need that is not part of the experimental protocol. ACKNOWLEDGEMENTS This study was supported in part by NIH grant RR00361 andWHO grant V4/181/38. The able assistance of Bettye E. Lynn and Lonnie C. Pleasant is acknowledged. RtSUMl, VARIOLE EXPKRIMENTALE CHEZ LES CHIMANZES Un antiserumhautement spdcifique du virus de in variole serait fort utile pour l'6tablissement d'un: diagnostic dif- ferentiel en cas de necessite. C'est dans le but d'obtenir un tel antiserum qu'a ete inoculee a un chimpanze une souche de virus variolique r6cemment isolee (souche Zaire de l'isolat Congo No 1). Au cours de l'experience, 2 des 3 autres chimpanzes enferm6s dans des cages placees dans le meme batiment d'isolement ont 6galement contracte la 640 EXPERIMENTAL SMALLPOX IN CHIMPANZEES 641. variole. C'est chez l'un de ces deux singes que la maladie a revetu la forme la plus grave, sans doute en raison du fait que l'infection a ete transmise par les voies respiratoires superieures, mais la guerison a cependant pu ultdrieure- ment etre constatee. Quant au quatrieme chimpanze, il n'a presente aucun signe de maladie clinique ou de seroconver- sion. Les serums prelev6s chez les 3 animaux atteints ont donne des resultats positifs lors des epreuves d'hemaggluti- nation et des dosages radio-immunologiques. Les titres d'anticorps observes dtaient tres faibles et tous les serums ont r6agi aussi bien au virus de fa vaccine qu'au virus du monkeypox. La preparation d'un antiserum specifique n6cessitera donc une absorption r6petee des serums avec les divers poxvirus. Une rupture du canal d'ecoulement entrainant vers les egouts les divers rejets provenant diu batiment d'isolement a amen6 le personnel d'entretien qui s'est charge de la reparation a entrer en contact avec ces matieres. Bien qu'aucun cas humain n'ait ete ainsi provoqu,la possibilite d'un accident affectant un point critique du systeme de securite a ete demontree. Toutes les categories de per- sonnel gravitant autour de lieux oiu se deroulent des expe- riences portant sur des produits biologiques dangereux doivent donc etre mises en garde contre les risques courus et plac6es sous une stricte surveillance. REFERENCES 1. HENDERSON, D. A. Smallpox eradication-the final battle. Journal of clinical pathology, 28: 843-849 (1975). 2. Weekly epidemiological record, 18: 125-130 (1978). 3. WHO Technical Report Series, No. 493, pp. 5-6, 1972 (Second report of the WHO Expert Committee on Smallpox Eraddicitioo). 4. HELm*E, R. J. ET AL. GroWth characteristics and viral susceptibility of a chimpanzee (Pan troglodytes) lung .diploid cell line, SFRE: CLI. Proceedings of the Society for Experimental Biology and Medecine, 139: 1367- 1373 (1972). 5. HEBERLING, R. L. ET AL. Poxvirus infection of the baboon (Papio cynocephalus). Bulletin of the World Health Organization, 54: 285-294 (1976). 6. KALTER, S. S. Identification and study of viruses. In: Pathology of simian primates, Part 2. Basel, Karger, 1972, pp. 382-468. 7. ZIEGLER, D. W. ET AL. Detection by radioimmunoassay of antibodies in human smallpox patients and vaccinees. Journal of clinical microbiology, 1: 311-317 (1975). 8. McCoNNELL, S. ET AL. Monkeypox: Experimental in- fection in chimpanzee (Pan satyrus) and immunization with vaccinia virus. American journal, of veterinary research, 29: 167541680 (1968).