TPC NEWS Winter 1990  Vol.9, No.2 (Whole Number 17)

(English Summary)

Page-3  A comment from the director of TPC

　　  Dr. Sugahara, the director of the Department of Applied Immunology of the National Institute of Health, Japan, succeeded Dr. Honjo as  the director of TPC in the last  spring.

　　  In this page,　Dr.　Sugahara expresses his　deep appreciation to Dr. Honjo's　contribution to TPC for twelve years. 　He comments on a rearrangement of some management systems since his inauguration and the outbreak of varicella-like herpesvirus infection occurred in the cynomolgus monkey colony at TPC. 　And he says that the  needs for laboratory primates in highly advanced medical science have increased, therefore, TPC should respond to the expectation from in and out of Japan.

Page-4    Breeding Topics: Nursing ability of F1-breeders

　　　  Live births of laboratory-bred F1 breeders at TPC have totaled to 486. 　The incidence of well nursing in the total number was 73.7%, however, the incidence varied with the breeders' parity: the first, second, and fourth deliveries are 64%, 82%, 89%, and 85%, respectively (Fig.1). Among the breeders of first delivery, some animals could not  hold rightly  their infants. But  as the  parity progressed, the incidence of mothers who refused to nurse their infants decreased. The incidences of refusing after the second delivery  were 17.6%, 10.6% and 11,5%.

　　  The average age of mothers at the first delivery was six. As seen in Fig.2, there was no significant relationship between the mother's age at the first delivery and nursing ability.  Figure 3 shows that the infants' birth weight and sex had no obvious effects on nursing ability.

  　　These findings are invaluable for our center where F1 animals have become the main breeders.

Page-5 Special Issue:  An outbreak of nonhuman primate varicella-like herpesvirus infection in the established breeding colony of cynomolgus monkeys

  TPC experienced an outbreak of nonhuman primate varicella-like herpesvirus infection in its cynomolgus monkey colony from November 1989 to April 1990.  Here we report on the detail of the outbreak.

Clinical findings and history of the outbreak

  The main symptom was an exanthematous rash.  Pink or red skin rashes of 2-10mm in diameter accompanying at times vesicles were observed locally or generally (Fig. 1).  Vesicle erosion and ulceration were also seen on the tongue and oral mucosa. The mortality rate in the severe cases without treatment was 68%, and death occurred on average 7.2 days after the appearance of rash.

　　  The results of hematological test and biochemical  test on serum in the clinical cases were characterized by the decrease in the number of platelet and the increase　in the value of serum transaminase (GOT, GPT), blood urea nitrogen, triglyceride and lactic dehydrogenase.

  　　The history of the outbreak is shown in Table 1.

The first case was found on November 9, 1989 in a room of the building for breeding (Room 2, Build. 1st), where cynomolgus monkeys of Philippine-origin were housed. 　The diseased was a wild-originated male, and died five days later on November 14. 　The infection in Room 2 occurred continuously, totaling up to 67 cases by February 2, 1990. On December 14, 1989, the disease spreaded into Room 3 for the monkeys of Malaysia-origin, adjacent to Room 2. Forty four cases were found by April 6, 1990. No cases were observed in the other three rooms; (Room 1 for the monkeys of Indonesia-origin, Room 4 for delivery  and nursing and Room  5 for group breeding).  Among the total  of these 111  cases, 36 cases  out of 68  were the wild-originated  animals housed in TPC for 9 to 11 years  after importation, the remaining 75 out of 187 cases were the laboratory-bred ones aged 5 to 26 years. 

　　  Thirteen animals out of these cases died in Room 2, and two died in Room 3.  The difference of mortality rate seemed to be due to the presence or absence of the BV-araU treatment; an anti-herpesviral compound was administered to more cases in Room 3 than those in Room 2.  Moreover, 31 animals with severe clinical signs in Room 2 were euthanized to prevent the spread of the infection and to clarify the cause of the disease.

 　 The morbidity rate according to sex and origin of the monkeys in Rooms 2 and 3 are shown in Table 2.

Pathological findings

  Five naturally occurring death cases were wild-originated cynomolgus monkeys  imported from the Philippines.  Their age was estimated to be 14 or more years.  Four of the five cases died within five days after the appearance of a skin rash, and the fifth on the day of appearance.

  Gross findings: Exanthema  were generalized.  Exanthema, erosion  and ulceration were seen in the oral  cavity. Hemorrhagic foci were observed  in the heart muscle, liver, spleen, adrenal grands, esophagus, stomach, intestinal mucosa and testis. 

  Histological findings:  Focal necrosis, hemorrhage and basophilic intranuclear inclusions were observed in the liver cells, spleen lymphoid follicles (Fig. 2 and 3) adrenal cortex, lymph nodes and spermatocytes.  Necrosis, intranuclear inclusions and a few vesiclular lesions were seen in the epidermis.   Herpesvirus particles were observed in the spleen and liver cells with an electron

microscope. 

  Virological findings

A.  Virus isolation and serological surveys.

  Virus isolation from bulla, feces, blood, cerebrospinal fluid and tissue homogenate of the infected animals by using Vero, HEL, and RK-13 cells was unsuccessful.  However, when the primary culture of embryonal kidney and lung cells from cynomolgus monkey (MEK and MEL cells) was used, the causative virus was successfully isolated from the fresh spleen and liver of the dead animal.  Typical CPE, which is very similar to that induced by human VZV, was observed (Figs. 4 & 5).  These infected cells were used for electron microscopic study, resulting in the identification of extra cellular heterogeneous herpesviral particles (180 mm in diameter) which is characteristic to those of VZV.  When MEK cells infected with this virus were used as the antigen for indirect immunofluorescent assay(IFA), all convalescent sera reacted to this antigen.  On the other hand, 4 out of 9 animals showed typical exanthematous disease with this virus infection had cross-reactive antibodies to HSV1 and VZV.

B. Screening of antibody against the isolated virus

  After the end of the epidemic, the sera from all the monkeys in Build. 1 were analyzed by IFA for antibodies against the isolated virus.  As shown in Table 3, the rate of sero-conversion was 35 and 39 % in Rooms 2 and 3, respectively.  The rate of asymptomatic animals in the seropositive ones was 36%.  All the animals in Rooms 1 and 4, where there was no prevalence of the disease, were seronegative.  Eight percent of the animals were positive for the antibody against the isolated virus in Room 5 where monkeys are group bred.  The analysis of the sera collected before the prevalence of the disease revealed that 6 out of 100 animals in Build. 1 had been seropositive.  These results suggest that the animal in our primate breeding colony which had been latently infected became the source of the outbreak.  Further analysis, including the improvement of the detection methods, is necessary to

elucidate the cause of the outbreak.

Prophylactic and  therapeutic effects  of an  antiviral compound  (BV-araU) against 

nonhuman primate varicella-like herpesvirus infection in cynomolgus monkeys

  Several anti-herpesviral reagents have  been developed for  the treatment of the  infectious diseases by  HSV and  VZV. We  have tried  prophylaxis and  treatment of  simian viaricella-like herpesvirus infection.

A. Prophylactic effect of BV-araU

  Eighty-three monkeys in Room 2 were grouped  into three during the prevalence of  the disease. The first (5 monkeys) and  second (20 monkeys) groups received BV-araU  intramuscularly doses of  10 mg/kg  and 2  mg/kg, respectively,  once a  day for 10  consecutive days in one course of the treatment. They received three courses with 7 days interval during  the experiment. Other  58 monkeys in  the third  group had no  prophylaxis. As a result, there was no morbidity in monkeys of the first and second  groups, whereas 13 monkeys  had an onset of  the disease in  the control group. Six  monkeys of 20 in the second group  were sero-positive against the isolated virus at  the end of  the prevalence. This  suggests that they  were infected  with the virus  before or during the epidemic of the disease, but the onset of the clinical signs in these monkeys was suppressed by BV-araU administration.

B.  Therapeutic effect of BV-araU

  During the natural outbreak of the disease, 65 monkeys showing the typical symptom were divided into two groups: a treated group with 46 monkeys and a non-treated control group with 19 monkeys.  The treated monkeys were given BV-araU intramuscularly at a dose of 10 mg/kg once a day for 10 to 14 consecutive days.  As shown in Table 4, 44 monkeys in the treated group showed recovery of the symptoms and only 2 monkeys, which had severe infection, died one and four days after starting the treatment.  Thirteen out of 19 monkeys died in the control group, (The mortality rate was 68%).

Preventive measures taken during the epidemic

  The following preventive measures were taken during the outbreak.  When the first case was found on November 9, 1989, in a cage of Room 2, Build. 1, we took standard preventive measures: we observed the monkey from the outside of the cage,  confirmed  the  health  condition   of  the  neighboring  animals,  prepared   disinfectant, caught the animal with a catching net  and put it on a working table,  recorded the clinical findings and took pictures,  returned the monkey to the cage,  disinfected the hands of the persons who handled the animal, aprons, working table,  cage rack, and filth plate. These procedures were repeated when the animal died five days later.

  On the day when the second  case was found, we held  a meeting on the preventive measures and decided to prohibit transferring the animals and carrying out breeding work in Room 2. Cleaning and feeding procedures  in Build. 1 were modified, so that  the order of room 2 became the last.

  After that, we  took new  procedures fitting  to the  new cases  found one after  another and results obtained from the examination.

  When the cause of this outbreak was judged to be a virus, there was a fear that the virus might be very dangerous for humans.  Therefore, we decided to initiate euthanasia of the diseased animals.  The animals with an apparent skin rash at the time of morning inspection were lightly anesthetized with ketamin-chloride and sacrificed by an administration of large dose of pentbarbital sodium into the abdominal cavity. The carcass was put into a thick vinyl sack and  put into a container with cover, then transferred to the incinerator.  This treatment was carried out from December 6 to 15 on 28 animals.  After that, since the virus was judged not to be hazardous to humans, we stopped performing euthanasia, and went on observing, examining and treating clinical cases.

  The preventive measures taken after November 22 '89 were (1)euthanasia of diseased animals, (2) ban on transferring and handling animals in Building 1 (3) exclusive use of safety clothes and shoes in every room. (4) increasing the number of animal technicians on holidays, (5)modifying the procedure of cleaning and feeding, (6)a strict access limit, including a restriction of access order and procedure in the

room and building, and (7) administration of an anti-herpesviral compound to the clinical cases.   The clinical case had been observed by April 6.  The antibody survey carried out from May to June 1990 revealed that there were many animals free from the infection even in the rooms where many clinical cases had been found.  Therefore, we stopped performing euthanasia.  However, the survivors with antibody against this virus have remained a potential source of another outbreak.  These animals are now under close watch.

  Since its establishment, TPC has made every effort to control infectious diseases, succeeding in producing monkeys free from B virus, measles, and some other viruses.  But this  time,  we encountered  the  outbreak of  nonhuman  primate varicella-like  herpesvirus infection with high morbidity and  mortality. We fully realized that it  is very difficult to control infectious diseases in nonhuman primates.   The serum bank, in which sera from every monkey of TPC have been stocked, was very useful to trace the antibody value at the onset of the clinical signs.

  We wish to thank the staff members of NIH Japan for their able cooperation and advice to clarify the cause of this outbreak, and Dr. Machida of Yamasa Soy Sauce Co.  Ltd. who provided BV-araU to us.

 Page-10   Special Article:  TPC and Simian viruses

  Dr. Yamanouchi, professor of the Institute of Medical Science of the University of Tokyo, TPC steering committee member, comments  on some problems of simian viruses,  including the outbreak of varicella-like virus  infection that recently occurred at TPC. 

1.Biohazard by simian viruses

  Marburg virus, B  virus and  Ebola virus  have been  known to  be very hazardous viruses classified  in Class  4 agent  according to  the criteria  for biohazardous  microorganisms.

  In 1967,  Marburg virus  infection suddenly  occurred  in some  medical research  institutions in Marburg in Germany and in Yugoslavia. The source of infection was the African green  monkeys  imported  from Africa.  This  outbreak  of  Marburg disease  frightened the medical scientists working with monkeys. The causative virus, Marburg virus, was identified. This virus was not  indigenous to the African green monkeys.  Its original host in nature is not known.

   Ebola hemorrhagic fever is as dangerous as Marburg disease.  The outbreaks of this disease in  1976 in  Sudan and  Zaire showed  very high  mortalities, 90%  and 60%,  respectively. Ebola virus is  classified into filovirus  family with Marburg virus.  Spontaneously occurring infection of this virus in monkeys had not been known until  last year  when an  outbreak of  hemorrhagic fever  occurred in  cynomolgus monkeys  imported from the Philippines  in the U.S.A.  The antibody against  Ebola virus was  found and an Ebola-like virus was isolated.  This accident has resulted in complete  stop on  macaque import  in the  U.S.A.  Airlines in  Japan have  totally suspended  handling monkeys.  Therefore, monkey  importation  into Japan  has  also completely  stopped.

  It has been gradually revealed, however, that the virus isolated from monkeys is  not identical the virus that had been epidemic in Africa. Although ten or more people were infected with  this virus from  the monkeys, none  of them  have developed the  disease. Antibodies have been found also in  the people who have no connection with  the monkeys. These evidences suggest that this virus infects humans, but may not be pathogenic to humans.  Thus, possibility of the biohazard seems to be decreased.

  Twenty-three B virus infections have  been reported so far,  almost all of which were fatal. The latest one occurred in the U.S.A. in 1987. B virus is very close to human herpes viruses, being indigenous to macaque monkeys, including cynomolgus and  rhesus monkeys. It is usually present in the nervous tissue without any effect, but when it is activated, it is shed in  saliva. B virus, therefore, is transmitted via bite.

2. Simian viruses endemic in monkeys

  Simian hemorrhagic fever virus and simian varicella-like virus are typical viruses which cause large outbreaks in monkey breeding colonies.   Simian hemorrhagic  fever  virus belongs  to  the togavirus  family.  It infects  spontaneously to African monkeys  and causes severe disease  in macaque monkeys. In  1964, two outbreaks of  simian hemorrhagic fever  occurred in the  U.S.S.R., and 62  animals died. In the same year, an outbreak of this disease in the U.S.A. resulted in the death of more than  200 monkeys. In 1967,  California Regional Primate Research  Center experienced an outbreak of  more than 500 animals. In  NIH, over 200 monkeys  underwent euthanasia when another outbreak occurred in 1972.

  Simian varicella-like virus causes a disease similar to human varicella. But it is not identical  to  varicella-zoster virus  since  it  is not  pathogenic  to human.  Several outbreaks  of this  disease have  occurred  in England  and the  U.S.A. For  example, through 1969  to 1970,  three outbreaks  occurred in  a colony  of macaque  monkeys in  the Washington  Regional Primate  Research Center.  In 1973,  the Delta  Primate Center experienced an outbreak in patas monkeys. The majority of these cases showed a mortality rate of over 50%.

  In TPC, this disease suddenly occurred in November, 1989.  Clinical and virological findings suggested the infection by a virus belonging to the simian varicella-like virus.  Stringent management techniques to isolate animal rooms and the therapy using BV-araU resulted in termination of the outbreak by this July. Antibody positive animals were also in the room where no endemic was seen.  Moreover, the antibody was found in the sera taken over 10 years ago. Although the source of the virus has not been determined yet, these evidences suggest that the virus has persisted in the original monkey colony at TPC.  

3. Simian viruses for human disease models

   Various simian virus, involving simian  varicella-like virus, can provide human disease models. Among all,  the simian retrovirus has  been especially important as  models of acquired immune deficiency syndrome (AIDS). It includes simian retrovirus/D (SRV/D) and simian  immune deficiency  virus (SIV).  SRV/D has caused  epidemics of  AIDS-like disease (SAIDS) with a high mortality rate in five primate centers in the U.S.A. It had been regarded  as the most suitable virus  for AIDS' models until SIV was identified. 

  SIV is closely related to human immune  deficiency virus (HIV), belonging to the same genus Lentiviridae. Therefore, there is a thought that HIV may have originated  from SIV. Recently, some primate centers succeeded in producing the animal models of AIDS-like disease by SIV. These  models are expected to contribute  to the study on  AIDS. 

4. Conclusion

  I have  mentioned three  aspects  of  simian  viruses, i.e.  biohazard  by simian viruses, health control against simian viruses in monkey breeding colony, and simian virus infections as human disease  models. In these cases, it  must be noticed that  monkey breeding  facilities, such  as TPC,  handle  viruses infectious  to monkeys.  Handling viruses in such facilities may be undesirable from the viewpoint of safety  first. However, the fact that virus isolation during the outbreak of varicella-like

infection in TPC contributed to prevent further epidemics, indicates the necessity of such self-defense measures. Therefore, I expect TPC, whose role is producing nonhuman primates of good quality, to tackle bravely and carefully this difficult problem.

Page-12   My visit to five research laboratories in the U.S.A.

  In March,  1990,  a  young  collaborative  scientist,  Dr.  Sankai, who  studies developmental biotechnology, went to the U.S.A. with Dr. Yoshida, senior scientist of endocrinology. Here he reports on five research laboratories he visited, focusing on the present state  of developmental biotechnology  in America,  including his happy  experiences.

1. The  Laboratory  of Radiobiology  and  Environmental Health,  the  University of California, San Francisco.  The influence of radio-chemical substances released in the environment upon animals was being studied in this laboratory.  The author met Dr. Spindle, whose study of the cultural condition of mouse embryo won international recognition, and discussed with her the many problems of developmental study that TPC faces.  Her experience and advice were very significant and helpful for TPC's further study.

1. Wisconsin Regional Primate Research Center, the University of Wisconsin.   This laboratory was  in the  state capital, Madison.  Dr. Bavister  is the most  famous researcher  in the  field of  developmental  biotechnology using  the rhesus  monkey. The author met Dr.  Boatman, a study supporter and  the life partner of Dr.  Bavister, and  had a  chance to  report and  discuss  the study  results of  TPC as projected some slides.  This center  has succeeded in  implantaion of  the in vitro fertilized embryo in rhesus monkeys. The author has considered that the main cause is the condition of the recipient.  The ideal system, in which  a number of recipients  were maintained and their hormonal level was measured constantly, has been adopted in this center. Therefore, it was  possible to select the  most suitable recipient for  embryo transfer.  The discussion with Dr. Boatman was profitable.

3. Endocrine Research Center, Michigan State University.

   The author and Dr. Yoshida were greeted at Lansing Air Port by Dr. Dukelow, world-famous researcher for developmental technology using squirrel monkeys.  Although the size of this center was not large, a number of young scientists from all over the world was studying actively.  The effects of alcohol on the reproductive ability was one of their study themes.  Toxicity test using ovarian or sperm culture systems would be attracted in the future as an alternative method to animal experimentation.  The author listened carefully to Dr.Dukelow, imaging TPC's cynomolgus monkeys.  The research on squirrel monkeys in the area of developmental technology has been fairly advanced.    Dr. Yoshida and the author will never forget the taste of tempura of the bluegill caught at Lake Michigan and served by  Dr. Dukelow.

4. Primate Field Station of the Regional Primate Research Center, the University of Washington.

Owing to the kindness of  Dr. Bowden, director of  the Regional Primate Research Center, the University of Washington, the  author could visit Primate Field Station  near Spokane.  This  field station  housed  various species  of  nonhuman primates,  including baboons. The  author met Dr.  Lohr, clinical  veterinarian, and discussed  some topics of early pregnancy diagnosis and ovary observation by using  ultra-sound.

5. The  Department of  Anatomy and  Reproductive Biology,  School of  Medicine, the University of Hawaii.

   Dr. Yanagimachi  of this  laboratory is  one of  the pioneers  in the  field of  developmental technology. He welcomed many  scientists from abroad, including three  Japanese, and was conducting research actively. The discussion with Dr. Yanagimachi  held after visiting research facilities and animal rooms , resulted in much fruitful information on sperm, ovum and even on  the newest technique of nucleus transfer of  sperm. The author thought that this information would be very helpful in research at TPC in the future.

Page-15  Report on the 13th Congress of the International Primatological Society

  The 13th Congress of the International Primatological Society (IPS) held from July 18th to 24th, 1990 in Nagoya and Kyoto, Dr. Terao reports.

    The participants of the Congress were about 500, and half of them were from abroad.  One hundred sixty-nine paper sessions, 114 poster sessions, 7 work-shops, 26 symposia, and three plenary sessions by the most famous primatologists, Dr. Jack Fooden, Dr. Morris Goodman, and  Dr. Jane Goodoll were presented during the Congress.  Many topics were discussed, and most of them were reports concerning  ecology, behavior, and psychology.  The ratio of the field of biomedical science was about 10%.

  TPC staffs presented four papers in poster sessions, two in paper sessions, four in symposium, and two in work-shops. The results of six cooperative studies with TPC were presented. There was no symposium on the subject of medical primatology except  the four symposia entitled:  The  first birth, Behavior and stress, Biotechnologies relating to human  and nonhuman  primate in  vitro fertilization,  and Naturally occurring viral infections in colonies of nonhuman primates and the effort to exclude them.  They were supposed to gratify the congress organizers. 

Page-17  Sketches from animal rooms

  Three animal  technicians,  Mr.  Ono,  Mr.  Komatuzaki  and  Mr. Ogawa  describe respectively their experiences during the outbreak of nonhuman primate varicella-like herpesvirus infection, from the viewpoint of those who handled and took care of the monkeys.