Japan. J. Med. Sci. Biol., 18, 151-156, 1965 NOTES CONTAMINATION OF CYNOMOLGUS MONKEY KIDNEY CELL CULTURES BY HEMAGGLUTINATING SIMIAN VIRUS (SV 5) Since the extensive use of cynomolgus monkey kidney cell cultures (MK) for virus researches, contaminations of MK with simian viruses (SV) have been serious problems in virus laboratories (Endere and Peebles, 1954; Rustigian et al., 1955; Hull et al., 1656; Hull and Minner, 1957; Hull et al., 1958). In the National Institute of Health of Japan (NIH), about ten monkeys have been used every week since 1961 and every batch of MK has been checked for the contamination of SV since 1962. The following is a part of the results of SV examination (from January 1962 to March 1962), which showed a high rate of contamination by hemagglutinating SV 5. The communication also contains some analysis about the origin of the contamination. Materials and Methods 2) Monkeys: Cynomolgus monkeys were imported from Malaya and Cambodia. They had been captured and pooled somewhere of those countries, sent to Japan in wooden or metal boxes and, immediately after arrival, transferred to the animal house of the NIH from the airport of Tokyo. They were kept there for quarantine for various periods in individual cages. 2) Preparation of MK: After various quarantine periods, usually 4 to 6 weeks, monkeys were sacrificed and nephrectomized. Cell suspensions were prepared from the kidney by routine trypsin treatment (in 0.2 % Trypsin (Difco 1 : 250) solution with stirring by a magnetic stirrer). The cells were suspended in GM (Growth medium ; Earle's BSS with 0.5 % lactalbumin hydrolysate, supplemented with 2% calf serum and an appropriate concentration of penicillin and streptomycin) and incubated at 36C for about a week, after which confluent monolayers of MK were formed. 3) Examination of MK: One or two bottles (each containing 10 ml of cell suspension with the concentration of about 3 ~ 105 cells per ml) were prepared from each batch of MK for the examination of SV. GM was removed from the confluent monolayer of MK bottles, the monolayer was washed with PBS (phosphate buffered saline, ph 7.4) and then Parker's synthetic medium 199 without any serum was added to serve as MM (maintenance medium). The cells were kept at 36C for three weeks, during which MM was changed once a week. The morphology of cells were examined microscopically every day and HA (hemagglutination) of the medium was tested every week. When any CPE (cytopathic effect) was observed or when the medium showed positive HA, the culture was frozen and then passaged to fresh MK tubes. The MK batch was regarded as contaminated with SV only when the subculture showed marked CPE or positive HA. 4) HA and HI (Hemagglutination inhibition) : To 0.5ml of each of two-fold 151
152 NOTE Vol. 18 serial dilutions of the test material, 0.5ml of 0.5 % RBC (red blood cell suspension) was added. The mixtures were kept at 4C overnight and then the endpoints of HA were read by the patterns at the bottom of the tubes. Different kinds of RBC from various animals were tested. When chicken RBC was used, the result was read after an hr at 4C. The highest dilution of the material, which showed complete HA, was taken as HA titre. Before HI, serum was inactivated at 56C for 30 min and then treated with periodate to remove non-specific inhibitor. To one volume of inactivated serum, one volume of KIO M/100 4 solution was added. The mixture was kept standing at 4C overnight and two volumes of 10% glucose solution was added to neutralize excess of KIO4. To 0.25 ml of two-fold dilutions of the treated serum, 0.25ml of virus fluid diluted to contain 4 HAU/0.25 ml was added. After an hr at 4 C, 0.5 ml of 0.5 % cynomolgus monkey or guinea pig RBC was added. The mixture was kept standing at 4 C overnight and then the results were read. The highest dilution of the serum which showed complete HI was taken as HI titer of the serum. 5) Other materials used in the experiment : HeLa, KB, HEp-2 and GMK-II (continuous cell culture obtained in our laboratory) cells were also used. SV 5, strain 21005-2WR was received from DBS, the NIH of the U. S. A. in 1961* and used as a reference strain of SV 5. Antiserum against the reference strain and an isolate (2669) of SV 5 were prepared by immunization of rabbits with the respective virus and used for the identification of the isolated strains. Results: 1. SV isolation from MK: Thirty-three out of 105 batches of MK cultures (31 %) showed SV contamination during three months from January to March 1962. All the isolates were easily detected by HA but marked CPE, which night be due to other adventitious agent, was not found during the above period, except that giant cell formations were sometimes observed in monolayers contaminated with these HA agents by close examination. 2. Characteristics and identification of isolated SV : Agglutinability of RBC from various animals was first compared. The results of the test with 8 strains out of 33 isolates are shown in Table 1. Cynomolgus monkey and guinea pig RBC were the most sensitive, human 0 and bovine RBC were less sensitive and the results with chicken RBC were variable. HA with mammalian RBC was marked after 4 hrs at 4 C and HA pattern did not change up to 24 hrs. HA with chicken RBC was observed after one hr at 4 C, then became gradually vague and difficult to read later. Although no marked CPE was observed in unstained bottle cultures, all the isolates showed giant cell formation by examination of preparations stained with Giemsa. The isolates multiplied in MK primary cultures but did not in HeLa, KB, HEp-2 or GMK-II cells. Inoculation of the isolates into the amniotic cavity of 8 day-old embryonated hen's eggs showed no evidence of virus multiplication. Multiplication of an isolate (strain 2669) and SV 5 reference strain was followed by hemagglutinin titers of the fluid in the MK bottles, inoculated with undiluted virus fluid. Hemagglutinin titers increased gradually, reached its peak (HA titer 1 : 256) about 4 days after inoculation, then declined a little and kept constant (HA titer 1 : 32) for about two weeks. The isolate and the * The authors express their appreciation to Dr. G. L. Van Hoosier, Jr. of DBS, the NIH for his kind supply of the strain.
1965 NOTE 153 Table1. Agglutinability of RBC from various animals *The figures indicate reciprocal dilution of the MK culture fluids. reference strain ran quite pararell. The day of maximum HA differed by inoculum size. Infectivity titration was rather difficult but a rough estimate showed infectivity titers of from 103.5 to 105.5 TCID50/ml. All the isolates were identified serologically, using HI test with anti-sv 5 reference strain serum and anti-isolate serum. Typical results with seven strains out of 33 isolates are shown in Table 2. Table 2. Serological identification of isolated viruses Both pre-immune sera showed no HI antibody against all the strains tested. * The figures indicate reciprocal dilution of the antisera. 3. Analysis of the origin of SV 5 contamination : Contamination of MK with SV 5 may be due to infection of monkeys with SV 5 and a high isolation rate suggested the prevalence of SV 5 infections in monkeys used in our laboratory. This was confirmed by a high rate of positive HI antibody against SV 5 in sera of monkeys used in our laboratory for about two years (Table 3). The origin of infections was further analyzed concerning the countries of origin, the lot of monkeys and the feeding period in our animal house. No difference was found in SV 5 isolation rate or in HI antibody distribution, between monkeys from Malaya (SV isolation 13 out of 40, 33 %, HI andibody positive 67 out of 84, 80 %) and from Cambodia (SV isolation 19 out of 64, 30 %, HI antibody positive 30 out of 42, 70 %). A closer analysis showed that the SV 5 isolation rate differed from a lot of monkeys to another. (Table 4). A lot refers to a group of monkeys imported at a time. This fact suggested that the infections may have occurred during the period, in which captured monkeys were pooled before ship-
154 NOTE Vol. 18 Table 3. HI antibody distribution in sera of monkeys used in NIH Table 4. Monkey lot and SV isolation ment, and the suggestion was further confirmed by the HI test of sera bled at various times, after being captured in jungles. Eleven monkeys were bled in Malaya before shipment to Japan, two days after arrival in Tokyo, after 25-50 days' feeding and finally after 60-90 days' feeding. HI titers of these sera were compared (Table 5). As shown in Table 5, HI titers were absent or very low in monkey sera bled in Malaya, but the same monkeys showed a slight but significant increase in serum HI titers when they were bled shortly after arrival in Tokyo and then a marked increase of HI antibody was observed in 25-50 days' feeding, indicating the interventation of a chance of infection of SV 5 just before rather than after arrival in Tokyo, or most probably during the shipment of monkeys. A longer feeding period in animal house seemed to increase the SV 5 isolation rate and HI antibody positive rate, but the differences were not so clear. Discussion A high incidence of SV 5 contaminations in MK cultures used in our Institute once again stressed the possible hazard of SV 5 contamination not only in researches but also
1965 NOTE 155 Table 5. Changes in HI antibody titers in monkeys in the course of time in practical problems such as vaccine preparations or epidemiological examinations. Although the present data showed the contamination of MK only by SV 5, the possibility of contamination of MK by other SV could not be excluded if MK were tested during other period. Treatment such as the addition of anti-sv 5 serum (Chanock, et al., 1961) or the addition of alminum chloride (Melnick and Wallis, 1962) has been proposed to prevent contamination by SV and they seemed to be of some value in light of the experiences in our laboratories but the results seemed to be far from elimination of SV contamination. SV contaminations are thought to be due to the natural infection of monkeys with SV and the prevention of SV infection must be the most reliable method to avoid SV contamination. The present analysis of SV 5 infection showed that the main chance of infection might exist in the care of monkeys before and during shipment. The improvement in the care of monkeys, keeping them in separate cages before and during shipment may be one of the most important measures to minimize the chance of infection of monkeys by simian viruses. Summary: During three months from January to March 1962, about 31 % of cynomolgus monkey kidney cultures prepared in our laboratory showed an evidence of contamination by hemagglutinating simian virus (SV 5). Analysis of the origin of the contamination showed the prevalence of 5V 5 infection in monkeys used in our Institute and suggested that the main chance of SV 5 infection in monkeys used in our Institute might have taken place before their arrival in Japan. The authors wish to express their appreciation to Dr. Toshio Karasawa and Miss Reiko Takayama for the preparation of MK cultures, to Dr. Shigeo Honjo and Mr. Toru Fujiwara for collecting monkey specimens, to Dr. Yasuo Kawanishi for collecting monkey specimens in Malaya, and to Dr. Isamu Tagaya for his support of the study.
156 NOTE Vol. 18 REFERENCES CHANOCK, R. M., JOHNSON, K. M., COOK, M. K., WONG, D. C. & VARGOSCO, A. (1961): The hemadsorption technique with special references to the problem of naturally occurring simian parainfluenza virus. Am. Rev. Respirat. Diseases, 83, 125-129. ENDERS, J. F. & PEEBLEȘ T. C. (1954): Propagation in tissue cultures of cytopathogenic agents from patients with measles. Proc. Soc. Exptl. Biol. Med., 86, 277-286. HULL, R. N., MINNER, J. R. & SMITH, J. W. (1956): New viral agents recovered from tissue cultures of monkey kidney cells. I. Origin and properties of cytopathogenic agents SV 1, SV 2, SV 4, SV 5, SV 6, SV 11, SV 12 and SV 15. Am. J. Hyg., 63, 204-215. HULL, R. N. & MINNER, J. R. (1957): New viral agents recovered from tissue cultures of monkey kidney cells. II. Problems of isolation and identification. Ann. N. Y. Acad. Sci., 67, 413-423. HULL, R. N., MINNER, J. R. & MASCOLI, C. C. (1958): New viral agents recover from tissue cultures of monkey kidney cells. III. Recovery of additional agents both from cultures of monkey tissues and directly from tissues and excreta. Am. J. Hyg., 68, 31-44. MELNICK, J. L. & WALLIS, C. (1962): Suppression of adventitious agents recovered in monkey tissue cultures. Texas Rep. Biol. Med., 20, 465-475. RUSTIGIAN, R., JOHNSTON, P. & REINHART, H. (1955): Infection of monkey tissue cultures with virus like agents. Proc. Soc. Exptl. Biol. Med., 88, 8-16. Department of Enteroviruses, National Institute of Health, Tokyo EIICHI YOSHIDA* HIROSHI YAMAMOTO HIROTO SHIMOJO Received : March 31st, 1965 * Present adress : Shizuoka Public Health Laborato ry, Shizuoka City., Japan.