141
7
Section of Comparative Medicine
Professor B A Lapin, Dr L A Yakovleva, Dr L V Indzhlia, Dr V Z Agrba, Dr G S Tsiripova, Dr A F Voevodin, Dr M T Ivanov and Dr A G Djatchenko (Institute ofExperimentalPathology and Therapy, USSR Academy of Medical Sciences, Sukhumi, USSR)
and at a later stage lymphadenopathy and hepatomegaly. Occasionally there was hypertrophic gingivitis and hemorrhagic diathesis. The disease was malignant lymphoma, which took two forms - severe and mild. Occasionally, there was transition from one type of disease to the other. There was no regularity in the relapse periods. In the severe type of disease relapses were longer and remissions shorter than in the mild. There were usually two or three (maximum six) relapses before fatal termination. Relapses rarely lasted more than one year. A number of experimental monkeys are still alive, so that no attempt is made at this stage to define the duration of the disease. The main symptom was always splenomegaly. Lymph nodes of 1-2 cm diameter were observed in 40°/-50% of severely affected monkeys but this developed almost always after splenomegaly. Liver enlargement occurred at the second or third relapse. A haemorrhagic diathesis was shown by small pin-point or spotted petechiT on the skin of the body and extremities. The disease was aleukaemic. There was periodic appearance of 'blast' lymphoid cells (2-5 %, rarely up to 8-10%). Thrombocytopenia was inconsiderable. There was, however, an increased number of old and degenerate megakaryocytes, and involution forms. Coagulation times were decreased. When the human donor suffered from lymphocytic leukemia, the severe type of disease appeared in 40-46% of experimental monkeys. From donors suffering from granulocytic leukTemia, the incidence of the severe form was much lower.
Transmission of Human Leukiemia to Nonhuman Primates
The development of malignant lymphoma in stumptail monkeys (Macaca arctoides), following injection of human leukaemic blood has been described in previous publications (Lapin & Yakovleva 1970, Lapin et al. 1967, Lapin, Yakovleva, Indzhiia et al. 1973, Lapin, Yakovleva, Bukaeva et al. 1973, Yakovleva 1970). The present report describes the extension of this work in which both whole blood and plasma filtrate were used; the experiments have also included injections of baboons, Papio hamadryas, which reacted similarly to the stumptails and showed cross-infection. EXPERIMENTS ON MONKEYS (Macaca arctoides)
Blood for injection into the monkeys was obtained from 32 human leukeemic patients and was used in three groups (Table 1). In all cases, injections were made parenterally, mostly intraperitoneally. The volume of human leukwmic blood injected into the monkeys at primary inoculation was 20-30 ml. The volume of plasma filtrate was the same. In monkey-tomonkey passages the infecting dose was smaller (3-6 ml). The macaques were of both sexes, mostly 2-4 years old, rarely older; in some cases newborn monkeys were used. The following controls were employed: heatinactivated human leukwemic blood, 6 monkeys; healthy human blood, 8 monkeys; heat-inactivated infected monkey blood, 12 monkeys; heatinactivated monkey spleen cells, 4 monkeys; noninfected monkey blood, 4 monkeys; noninfected monkey spleen cells, 4 monkeys. Both control and experimental monkeys were subjected to the following routine studies: clinical, heematological, bone marrow aspirates, and bone marrow and lymph node biopsies. Results (1) Primary inoculation: The incubation period was usually ten days to three months; in very mild cases it was as long as fifteen months. The course was wave-like (Fig 1), with remission and relapse periods. Some monkeys remained in good condition for long periods but always showed splenomegaly of varying degree,
(2) Passage: Passages were made by means of whole blood, cell-free material, and spleen cells. On passage, no strict correlation between the severity of the disease in the monkey donor and recipient was noted, and the type of leukTmia in the original donor did not influence this. However, material from chronic lymphocytic donors induced the severe type of disease in 50 % (11/22) monkeys, whereas the incidence of severe disease from chronic granulocytic donors was only 14%
(6/43). Malignant lymphoma resulted from injection of both filtered and unfiltered material. Spleens were grossly enlarged, the capsule smooth, with greyish nodules visible through the pulp; on section the colour was dark cherry, frequently with greyish, fine granulation. The glands involved were the cervical, mediastinal and retroperitoneal. The liver showed a mild hepatomegaly in some cases. There were sometimes greyish nidi of match-head size, especially beneath the capsule. The bone marrow was dark
142 Proc. roy. Soc Med. Volume 68 March J975
8
Table 1 Results of primary inoculation and passages in monkeys inoculated with materials from patients with various types of leuksemia
Morphological
Type of leukemia Patients Age Acute z 24 A-a 53 V-a 44 K-v 20 P-v 58 K-a 56 N-v 32 R 14 B-a 42 K 28 K-j 63 P-a 58 G 6 V-n 49
No. of
Undifferentiated Undifferentiated
Granulocytic Granulocytic Granulocytic Erythromyelotic Lymphocytic Lymphocytic Lymphocytic Undifferentiated
Monocytic Granulocytic Granulocytic Granulocytic
No. of inoculated passages I 0 8
K-ov S M K-ov I p L K-o B-ja S-ov
40 29 41 87 53 29 19 69 55 39
Type of disease Remained shortly after Mild Severe healthy inoculation 0
S5 102 26 14 2 12 3 3 3 2
0 2 1 0 0 20 10 0 1 4 0 0 1 0
74
39
43
30
0 0 0 0 0 0 0 0 6 0
0 0 2 7 2 2 0
0 0 1
5
0I 0
4 0
2 0
6
18
5
1
0 2 13 3 5 0 3 0 0 0 0
5 1
Blast crisis
8 9 2 2 2 2 12
Blast crisis
Blast crisis
1
0 2 2 0 0 0 0 3 0
43 Chronic
lymphocytic
T K E S V z U K-j
54 49 69 79 64
0
0
4
9
2
0
65f 61 70
1
4
0
1
0
0
22 Sum total
251
91
0 0 l 28 6 4
0 0 0
2
0 4 3 I 4 13 0 2 0
0
00 1
0 1
0
14
.
1 I
0 0 0 41 10 8 0 7 3 2 2 0
4
186
Chronic granulocytic
Died
menkeys
variant
5I 0
00 0 14 0 3 0
0I 0
2
7 7
0
64
49
0 0
3 47
* materials from several patients were injected
red in colour and uniform throughout. Spleen/ body weight ratio was: controls, 0.05-0.15; mild cases,
0.25-0.35;
severe cases,
0.36-0.7, with
generalized lymphadenopathy and hepatomegaly.
Histology of the Disease Produced The spleen and lymph nodes were enlarged because of neoplasia. The cells involved were usually of a mixed character; most were of large or medium size with rounded or slightly oval and relatively chromatin-poor nuclei, and a narrow cytoplasmic zone not always clearly outlined. A single nucleolus was usually present in the nucleus. Varying numbers of prolymphocytes and mature lymphocytes were regularly found at the sites of clusters of the undifferentiated blast cells. The proliferating cells in the spleen were more or
less diffusely distributed, but
sometimes it was possible to determine that their preferred sites were in the red pulp and the areas surrounding the lymphoid follicles. In the lymph nodes, the cortex immediately below the capsule was mostly involved, though in some cases abundant proliferation was observed also in the greatly thickened cords. Sometimes cell clusters consisted of immature myelocytes. Some monkeys revealed an increased number of megakaryocytes in the bone marrow, the medulla of hyperplastic lymph nodes, and rarely in the red spleen pulp. Lymph nodes in both spleen and lymphatic glands were for the most part hypoplastic. Only some
of the animals showed significant and ir-
regular hyperplasia of follicles due to the enlargement of the germinal centres. The process
thus resembled malignant lymphoma of Brill-
Symmers type.
Section of Comparative Medicine
9
143
Spleen enlargement
(cm below costal arc',,)
Fig 1 Scheme of wave-like course of malignant
lymphoma in M. arctoides,
sixth passage
no n t n s
Sometimes sclerosis and thickening of the capsule were observed in the spleen and lymph nodes. Infiltration in the affected lymph nodes extended to the capsule and adjacent fibrous tissue. In the spleen the malpighian corpuscles were affected focally, and not equally throughout the organ. Proliferation could be observed in the liver along Glisson's capsule and inside the'lobules (Fig 2A), and in the parenchyma of salivary glands, lungs (Fig 2B), kidneys and bone marrow. The Viral Agent Electron-microscopic studies of (1) plasma sediments after ultra-centrifugation, (2) leukocytes, (3) cells of haemopoietic organs, and (4) ultrathin sections of some parenchymatous organs (kidney, salivary gland and others) regularly revealed C-type viral particles (Fig 3a, b). They were found in 32 of 45 inoculated monkeys examined, most frequently in spleen, lymph nodes and bone marrow (56%, 51 %, 42% respectively), rarely in other organs. Negative staining and ultrathin section of material banded at 1.16 g/cm3 in sucrose density gradients revealed
C-type particles in 28 of 30 cases. The same procedure revealed C-type particles in spleen, lymph nodes, and peripheral blood white cell homogenates banded at 1'.16 g/cm3 after ultra-centrifugation, and less frequently in urine. The virus was characterized by the presence of reverse transcriptase and 60-70S RNA. In two cases monkeys were infected with material banded at 1.16 g/cm3 in sucrose density gradient, and developed the severe form of malignant lymphoma. Long-lived virus-producing monolayer cultures of cells were established from haemopoietic organs of sick monkeys (spleen, bone marrow (Figs 3A, B), lymph nodes, peripheral blood leukocytes) in vitro. Cells were cultivated in RPMI 1640 medium +heat-inactivated 20 % fetal calf serum. EXPERIMENTS ON BABOONS (Papio hamadryas)
Experimental Transmission Human leuklmic blood was injected parenterally to baboons of different ages, as well as into the fetus at the last month of pregnancy (Yakovleva et al. 1973, Lapin, Yakovleva, Bukaeva et al. 1973). For primary inoculation 22 monkeys, 14 of
47W
Fig 2A, round-cell infiltration in liver of M. arctoides, sixth passage. H& E. x 250. B,proliferation of malignant cells in lungparenchyma of M. arctoides, seventh passage. H& E. x 150
144 Proc. roy. Soc. Med. Volume 68 March 1975
10
Fig 3 A, C-type virus budding in spleen (M. arctoides). B, Mature C-type virus in bone marrow (M. arctoides) x 150 000
which had been kept under observation for a long period of time, were used. The disease character and duration of the incubation period were generally analogous to those observed in M. arctoides. One of 3 cases was the severe form of malignant lymphoma, as confirmed at autopsy. Proliferative skin lesions were a characteristic feature of the clinical condition. Passaging was also possible as in macaques. Twenty-one baboons were employed in three subsequent passages, and the disease picture was similar to that after primary inoculation of material from human patients. The diseased baboons also regularly revealed viral C-type particles in blood and cells of the hiemopoietic organs and kidneys.
Natural Transmission Some P. hamadryas inoculated with blood from leukemic human patients and those employed in passages were housed together with healthy baboons of the Sukhumi monkey colony. Two years after the beginning of leukaemia experiments on baboons, cases of disease and deaths from malignant lymphoma were registered also among the stock animals. Up to now over 100 cases of lymphoma have occurred in the stock animals; 49 of them have ended fatally with the picture of a generalized process (Lapin, Yakovleva, Bukaeva et al. 1973, Lapin, Yakovleva, Asanov et al. 1973, Lapin 1973). The disease in baboons as in the stumptails, was characterized by the long wave-like course and short terminal period. Splenomegaly was often pronounced and the process was, as a rule, accompanied by generalized adenopathy. The skin lesions occurred frequently
both in experimental malignant lymphoma and the naturally transmitted disease. GENERAL
Immunology Investigations of materials from sick M. arctoides and P. hamadryas and of cultured cells obtained from them have failed to reveal antigens (or corresponding antibodies) of any known mammalian oncornaviruses in Ouchterlony immunodiffusion tests, i.e. M-P MTV (Mason-Pfizer tumour virus; gS-3 (C-type oncornaviruses of mammals); murine gS-1 (C-type oncomaviruses); and gS-l of simian sarcoma virus (SSV-1). Investigation of the peripheral blood leukocytes of sick monkeys by Coon's indirect immunofluorescent test (M6ller's modification) has revealed the appearance of specific membrane antigen (Kokosha et al. 1973). Hamster and rabbit antisera to leukocytes of patients with various types of leukemia served as the immune sera. The frequency of membrane-antigen demonstration was correlated with the severity of the process. This antigen was demonstrated in 100% of clinically severe types of the disease (regardless of the leukeemic type of the donor). In the mild type of the disease it was found only occasionally. In some cases, however, specific membrane antigen was detected in inoculated monkeys showing no signs of disease.
Membrane antigen usually appeared at the
same time as the disease symptoms; more rarely it preceded the appearance of clinical signs (Lapin, Yakovleva, Bukaeva et al. 1973, Indzhiia etal. 1973).
11
Section of Comparative Medicine
Epidemiology C-type particles were found in ultrathin sections of kidneys, lungs and salivary glands, i.e. the organs most directly in contact with the external environment. Viral particles could also be revealed by negative staining of high-speed urinary sediments, and these sediments also caused the development of symptoms of the disease (splenomegaly, lymphadenopathy), when injected parenterally to healthy animals. These data, combined with the natural spread of the disease to in-contact healthy baboons (among them some not bred in Sukhumi and some of another species -P. anubis), show that horizontal transmission of the malignant lymphoma can occur among baboons, and suggest that the virus may be present in saliva, expired air and urine. Other data suggest that vertical transmission of virus from sick mother to fetus may also occur. Two cases of congenital malignant lymphoma were confirmed in newborn baboons, one of which died and was autopsied. Material from this baby has been successfully transmitted in two subsequent passages to other baboons. Similar results were obseived in experiments on M. arctoides.
Lapin B A, Yakovleva L A, Kuksova M I, Adzhigitov F I, Krivoshein Y S & Skurkovich S V (1967) Byulleten' dksperimental'noi Biologii i Meditsiny 8, 78-83 Yakovleva L A (1970) Bibliotheca hematologica 36, 761-772 Yakovieva L A, Lapin B A, Fomenko V N, Ivanov M T & Schekolodkin V F (1973) Vestnik AkademilMeditsinskikh Nauk SSSR 4,20-31
Summary The experiments described show that inoculation of two monkey species, M. arctoides and P. hamadryas, with human leukamic blood or its filtrates causes a viral disease with the characteristics of malignant lymphoma of mixed type. The disease was passed in subsequent passages. The virus was isolated and identified as an oncornavirus of C-type by its characteristic morphological appearance and buoyant density (1.16 g/cm3 in sucrose and 1.21 g/cm3 in cesium chloride), and by the presence of 60-70S RNA and RNA-dependent DNA polymerase. There is some evidence that it differs immunologically from other known oncornaviruses of mammals including primates.. It is postulated that both horizontal and vertical transmission of this oncornavirus can occur. REFERENCES Indzhiia L V, Kokosha L V & Fomenko V N (1973) Vestnik Akademii Meditsinskikh Nauk SSSR 4, 40-43 Kokosha L V, Lapin B A, Deitchman G I & Yakovleva L A (1973) Voprosy VirusologiU 5, 582-588 Lapin B A (1973) Bibliotheca hamatologica 39, 263-268 Lapin B A & Yakovieva L A (1970) Vesinik Akademii Meditsinskikh Nauk SSSR 5, 60-71 Lapin B A, Yakovleva L A, Asanov N S, Kokosha L V, Tsiripova G S, Mirvis H B & Ivanov M T (1973) Voprosy Virusologii 1, 38-44 Lapin B A, Yakovieva L A, Bukaeva I A, Indzhiia L V & Kokosha L V (1973) Fourth International Congress on Primatology, Symposia 4, 1-29 Lapin B A, Yakovieva L A, Indzhiia L V, Kuksova M I, Kokosha L V, Schekolodkin V F, Lebedev V N, Bikovski A F & Lorye Y I (1973) Vestnik Akademii Meditsinskikh Nauk SSSR 4,10-20
145
Professor K McCarthy and Dr F A Tosolini' (Department of Medical Microbiology, The University, Liverpool, L69 3BX) A Review of Primate Herpes Viruses
The last twenty years have seen an ever increasing use of subhuman primates in medical and scientific research. With very few exceptions these animals are trapped in the wild and air freighted in large batches to research laboratories throughout the world. Often they will have passed through several staging posts and thus had opportunity for contact with many other species of primates, including man, as well as with animals of lower orders. Although animals which are obviously sick are not shipped for fear of incurring financial loss through immediate outbreaks of lethal disease, the pre-export scrutiny cannot detect minor infections so that on arrival in the country of destination the condition of newly arrived monkeys is often marred by a series of minor illnesses or occasionally by outbreaks of lethal disease. Many infections by viruses of the herpes group are likely to be missed because characteristically herpes infection in the natural host is trivial or completely inapparent. The full virulence of one of these viruses may not be manifest unless the virus is accidentally transmitted to a new species. The best known example of this is the infection of man by Herpesvirus simiw (B virus). Twenty years ago only three primate herpes viruses were known: Herpesvirus hominis I ('herpes simplex virus'), Herpesvirus simik and Herpesvirus varicellw ('varicella-zoster virus'). At the present time thirty-seven primate herpes virus strains are known to exist (Table 1). Several reviews of selected primate herpes viruses have appeared in recent years (Plummer 1967, Hunt & Melendez 1969, Kalter & Heberling 1971, 1972, Hull 1968, 1973). Although Herpesvirus simiw is the only simian herpes virus known to infect man, there has been an understandable reluctance to pursue extensive investigations with the other simian herpes viruses. Thus, many "Present address: Division of Microbiology, Institute of Medical and Veterinary Science, Adelaide, South Australia 5000
7
Section of Comparative Medicine
Professor B A Lapin, Dr L A Yakovleva, Dr L V Indzhlia, Dr V Z Agrba, Dr G S Tsiripova, Dr A F Voevodin, Dr M T Ivanov and Dr A G Djatchenko (Institute ofExperimentalPathology and Therapy, USSR Academy of Medical Sciences, Sukhumi, USSR)
and at a later stage lymphadenopathy and hepatomegaly. Occasionally there was hypertrophic gingivitis and hemorrhagic diathesis. The disease was malignant lymphoma, which took two forms - severe and mild. Occasionally, there was transition from one type of disease to the other. There was no regularity in the relapse periods. In the severe type of disease relapses were longer and remissions shorter than in the mild. There were usually two or three (maximum six) relapses before fatal termination. Relapses rarely lasted more than one year. A number of experimental monkeys are still alive, so that no attempt is made at this stage to define the duration of the disease. The main symptom was always splenomegaly. Lymph nodes of 1-2 cm diameter were observed in 40°/-50% of severely affected monkeys but this developed almost always after splenomegaly. Liver enlargement occurred at the second or third relapse. A haemorrhagic diathesis was shown by small pin-point or spotted petechiT on the skin of the body and extremities. The disease was aleukaemic. There was periodic appearance of 'blast' lymphoid cells (2-5 %, rarely up to 8-10%). Thrombocytopenia was inconsiderable. There was, however, an increased number of old and degenerate megakaryocytes, and involution forms. Coagulation times were decreased. When the human donor suffered from lymphocytic leukemia, the severe type of disease appeared in 40-46% of experimental monkeys. From donors suffering from granulocytic leukTemia, the incidence of the severe form was much lower.
Transmission of Human Leukiemia to Nonhuman Primates
The development of malignant lymphoma in stumptail monkeys (Macaca arctoides), following injection of human leukaemic blood has been described in previous publications (Lapin & Yakovleva 1970, Lapin et al. 1967, Lapin, Yakovleva, Indzhiia et al. 1973, Lapin, Yakovleva, Bukaeva et al. 1973, Yakovleva 1970). The present report describes the extension of this work in which both whole blood and plasma filtrate were used; the experiments have also included injections of baboons, Papio hamadryas, which reacted similarly to the stumptails and showed cross-infection. EXPERIMENTS ON MONKEYS (Macaca arctoides)
Blood for injection into the monkeys was obtained from 32 human leukeemic patients and was used in three groups (Table 1). In all cases, injections were made parenterally, mostly intraperitoneally. The volume of human leukwmic blood injected into the monkeys at primary inoculation was 20-30 ml. The volume of plasma filtrate was the same. In monkey-tomonkey passages the infecting dose was smaller (3-6 ml). The macaques were of both sexes, mostly 2-4 years old, rarely older; in some cases newborn monkeys were used. The following controls were employed: heatinactivated human leukwemic blood, 6 monkeys; healthy human blood, 8 monkeys; heat-inactivated infected monkey blood, 12 monkeys; heatinactivated monkey spleen cells, 4 monkeys; noninfected monkey blood, 4 monkeys; noninfected monkey spleen cells, 4 monkeys. Both control and experimental monkeys were subjected to the following routine studies: clinical, heematological, bone marrow aspirates, and bone marrow and lymph node biopsies. Results (1) Primary inoculation: The incubation period was usually ten days to three months; in very mild cases it was as long as fifteen months. The course was wave-like (Fig 1), with remission and relapse periods. Some monkeys remained in good condition for long periods but always showed splenomegaly of varying degree,
(2) Passage: Passages were made by means of whole blood, cell-free material, and spleen cells. On passage, no strict correlation between the severity of the disease in the monkey donor and recipient was noted, and the type of leukTmia in the original donor did not influence this. However, material from chronic lymphocytic donors induced the severe type of disease in 50 % (11/22) monkeys, whereas the incidence of severe disease from chronic granulocytic donors was only 14%
(6/43). Malignant lymphoma resulted from injection of both filtered and unfiltered material. Spleens were grossly enlarged, the capsule smooth, with greyish nodules visible through the pulp; on section the colour was dark cherry, frequently with greyish, fine granulation. The glands involved were the cervical, mediastinal and retroperitoneal. The liver showed a mild hepatomegaly in some cases. There were sometimes greyish nidi of match-head size, especially beneath the capsule. The bone marrow was dark
142 Proc. roy. Soc Med. Volume 68 March J975
8
Table 1 Results of primary inoculation and passages in monkeys inoculated with materials from patients with various types of leuksemia
Morphological
Type of leukemia Patients Age Acute z 24 A-a 53 V-a 44 K-v 20 P-v 58 K-a 56 N-v 32 R 14 B-a 42 K 28 K-j 63 P-a 58 G 6 V-n 49
No. of
Undifferentiated Undifferentiated
Granulocytic Granulocytic Granulocytic Erythromyelotic Lymphocytic Lymphocytic Lymphocytic Undifferentiated
Monocytic Granulocytic Granulocytic Granulocytic
No. of inoculated passages I 0 8
K-ov S M K-ov I p L K-o B-ja S-ov
40 29 41 87 53 29 19 69 55 39
Type of disease Remained shortly after Mild Severe healthy inoculation 0
S5 102 26 14 2 12 3 3 3 2
0 2 1 0 0 20 10 0 1 4 0 0 1 0
74
39
43
30
0 0 0 0 0 0 0 0 6 0
0 0 2 7 2 2 0
0 0 1
5
0I 0
4 0
2 0
6
18
5
1
0 2 13 3 5 0 3 0 0 0 0
5 1
Blast crisis
8 9 2 2 2 2 12
Blast crisis
Blast crisis
1
0 2 2 0 0 0 0 3 0
43 Chronic
lymphocytic
T K E S V z U K-j
54 49 69 79 64
0
0
4
9
2
0
65f 61 70
1
4
0
1
0
0
22 Sum total
251
91
0 0 l 28 6 4
0 0 0
2
0 4 3 I 4 13 0 2 0
0
00 1
0 1
0
14
.
1 I
0 0 0 41 10 8 0 7 3 2 2 0
4
186
Chronic granulocytic
Died
menkeys
variant
5I 0
00 0 14 0 3 0
0I 0
2
7 7
0
64
49
0 0
3 47
* materials from several patients were injected
red in colour and uniform throughout. Spleen/ body weight ratio was: controls, 0.05-0.15; mild cases,
0.25-0.35;
severe cases,
0.36-0.7, with
generalized lymphadenopathy and hepatomegaly.
Histology of the Disease Produced The spleen and lymph nodes were enlarged because of neoplasia. The cells involved were usually of a mixed character; most were of large or medium size with rounded or slightly oval and relatively chromatin-poor nuclei, and a narrow cytoplasmic zone not always clearly outlined. A single nucleolus was usually present in the nucleus. Varying numbers of prolymphocytes and mature lymphocytes were regularly found at the sites of clusters of the undifferentiated blast cells. The proliferating cells in the spleen were more or
less diffusely distributed, but
sometimes it was possible to determine that their preferred sites were in the red pulp and the areas surrounding the lymphoid follicles. In the lymph nodes, the cortex immediately below the capsule was mostly involved, though in some cases abundant proliferation was observed also in the greatly thickened cords. Sometimes cell clusters consisted of immature myelocytes. Some monkeys revealed an increased number of megakaryocytes in the bone marrow, the medulla of hyperplastic lymph nodes, and rarely in the red spleen pulp. Lymph nodes in both spleen and lymphatic glands were for the most part hypoplastic. Only some
of the animals showed significant and ir-
regular hyperplasia of follicles due to the enlargement of the germinal centres. The process
thus resembled malignant lymphoma of Brill-
Symmers type.
Section of Comparative Medicine
9
143
Spleen enlargement
(cm below costal arc',,)
Fig 1 Scheme of wave-like course of malignant
lymphoma in M. arctoides,
sixth passage
no n t n s
Sometimes sclerosis and thickening of the capsule were observed in the spleen and lymph nodes. Infiltration in the affected lymph nodes extended to the capsule and adjacent fibrous tissue. In the spleen the malpighian corpuscles were affected focally, and not equally throughout the organ. Proliferation could be observed in the liver along Glisson's capsule and inside the'lobules (Fig 2A), and in the parenchyma of salivary glands, lungs (Fig 2B), kidneys and bone marrow. The Viral Agent Electron-microscopic studies of (1) plasma sediments after ultra-centrifugation, (2) leukocytes, (3) cells of haemopoietic organs, and (4) ultrathin sections of some parenchymatous organs (kidney, salivary gland and others) regularly revealed C-type viral particles (Fig 3a, b). They were found in 32 of 45 inoculated monkeys examined, most frequently in spleen, lymph nodes and bone marrow (56%, 51 %, 42% respectively), rarely in other organs. Negative staining and ultrathin section of material banded at 1.16 g/cm3 in sucrose density gradients revealed
C-type particles in 28 of 30 cases. The same procedure revealed C-type particles in spleen, lymph nodes, and peripheral blood white cell homogenates banded at 1'.16 g/cm3 after ultra-centrifugation, and less frequently in urine. The virus was characterized by the presence of reverse transcriptase and 60-70S RNA. In two cases monkeys were infected with material banded at 1.16 g/cm3 in sucrose density gradient, and developed the severe form of malignant lymphoma. Long-lived virus-producing monolayer cultures of cells were established from haemopoietic organs of sick monkeys (spleen, bone marrow (Figs 3A, B), lymph nodes, peripheral blood leukocytes) in vitro. Cells were cultivated in RPMI 1640 medium +heat-inactivated 20 % fetal calf serum. EXPERIMENTS ON BABOONS (Papio hamadryas)
Experimental Transmission Human leuklmic blood was injected parenterally to baboons of different ages, as well as into the fetus at the last month of pregnancy (Yakovleva et al. 1973, Lapin, Yakovleva, Bukaeva et al. 1973). For primary inoculation 22 monkeys, 14 of
47W
Fig 2A, round-cell infiltration in liver of M. arctoides, sixth passage. H& E. x 250. B,proliferation of malignant cells in lungparenchyma of M. arctoides, seventh passage. H& E. x 150
144 Proc. roy. Soc. Med. Volume 68 March 1975
10
Fig 3 A, C-type virus budding in spleen (M. arctoides). B, Mature C-type virus in bone marrow (M. arctoides) x 150 000
which had been kept under observation for a long period of time, were used. The disease character and duration of the incubation period were generally analogous to those observed in M. arctoides. One of 3 cases was the severe form of malignant lymphoma, as confirmed at autopsy. Proliferative skin lesions were a characteristic feature of the clinical condition. Passaging was also possible as in macaques. Twenty-one baboons were employed in three subsequent passages, and the disease picture was similar to that after primary inoculation of material from human patients. The diseased baboons also regularly revealed viral C-type particles in blood and cells of the hiemopoietic organs and kidneys.
Natural Transmission Some P. hamadryas inoculated with blood from leukemic human patients and those employed in passages were housed together with healthy baboons of the Sukhumi monkey colony. Two years after the beginning of leukaemia experiments on baboons, cases of disease and deaths from malignant lymphoma were registered also among the stock animals. Up to now over 100 cases of lymphoma have occurred in the stock animals; 49 of them have ended fatally with the picture of a generalized process (Lapin, Yakovleva, Bukaeva et al. 1973, Lapin, Yakovleva, Asanov et al. 1973, Lapin 1973). The disease in baboons as in the stumptails, was characterized by the long wave-like course and short terminal period. Splenomegaly was often pronounced and the process was, as a rule, accompanied by generalized adenopathy. The skin lesions occurred frequently
both in experimental malignant lymphoma and the naturally transmitted disease. GENERAL
Immunology Investigations of materials from sick M. arctoides and P. hamadryas and of cultured cells obtained from them have failed to reveal antigens (or corresponding antibodies) of any known mammalian oncornaviruses in Ouchterlony immunodiffusion tests, i.e. M-P MTV (Mason-Pfizer tumour virus; gS-3 (C-type oncornaviruses of mammals); murine gS-1 (C-type oncomaviruses); and gS-l of simian sarcoma virus (SSV-1). Investigation of the peripheral blood leukocytes of sick monkeys by Coon's indirect immunofluorescent test (M6ller's modification) has revealed the appearance of specific membrane antigen (Kokosha et al. 1973). Hamster and rabbit antisera to leukocytes of patients with various types of leukemia served as the immune sera. The frequency of membrane-antigen demonstration was correlated with the severity of the process. This antigen was demonstrated in 100% of clinically severe types of the disease (regardless of the leukeemic type of the donor). In the mild type of the disease it was found only occasionally. In some cases, however, specific membrane antigen was detected in inoculated monkeys showing no signs of disease.
Membrane antigen usually appeared at the
same time as the disease symptoms; more rarely it preceded the appearance of clinical signs (Lapin, Yakovleva, Bukaeva et al. 1973, Indzhiia etal. 1973).
11
Section of Comparative Medicine
Epidemiology C-type particles were found in ultrathin sections of kidneys, lungs and salivary glands, i.e. the organs most directly in contact with the external environment. Viral particles could also be revealed by negative staining of high-speed urinary sediments, and these sediments also caused the development of symptoms of the disease (splenomegaly, lymphadenopathy), when injected parenterally to healthy animals. These data, combined with the natural spread of the disease to in-contact healthy baboons (among them some not bred in Sukhumi and some of another species -P. anubis), show that horizontal transmission of the malignant lymphoma can occur among baboons, and suggest that the virus may be present in saliva, expired air and urine. Other data suggest that vertical transmission of virus from sick mother to fetus may also occur. Two cases of congenital malignant lymphoma were confirmed in newborn baboons, one of which died and was autopsied. Material from this baby has been successfully transmitted in two subsequent passages to other baboons. Similar results were obseived in experiments on M. arctoides.
Lapin B A, Yakovleva L A, Kuksova M I, Adzhigitov F I, Krivoshein Y S & Skurkovich S V (1967) Byulleten' dksperimental'noi Biologii i Meditsiny 8, 78-83 Yakovleva L A (1970) Bibliotheca hematologica 36, 761-772 Yakovieva L A, Lapin B A, Fomenko V N, Ivanov M T & Schekolodkin V F (1973) Vestnik AkademilMeditsinskikh Nauk SSSR 4,20-31
Summary The experiments described show that inoculation of two monkey species, M. arctoides and P. hamadryas, with human leukamic blood or its filtrates causes a viral disease with the characteristics of malignant lymphoma of mixed type. The disease was passed in subsequent passages. The virus was isolated and identified as an oncornavirus of C-type by its characteristic morphological appearance and buoyant density (1.16 g/cm3 in sucrose and 1.21 g/cm3 in cesium chloride), and by the presence of 60-70S RNA and RNA-dependent DNA polymerase. There is some evidence that it differs immunologically from other known oncornaviruses of mammals including primates.. It is postulated that both horizontal and vertical transmission of this oncornavirus can occur. REFERENCES Indzhiia L V, Kokosha L V & Fomenko V N (1973) Vestnik Akademii Meditsinskikh Nauk SSSR 4, 40-43 Kokosha L V, Lapin B A, Deitchman G I & Yakovleva L A (1973) Voprosy VirusologiU 5, 582-588 Lapin B A (1973) Bibliotheca hamatologica 39, 263-268 Lapin B A & Yakovieva L A (1970) Vesinik Akademii Meditsinskikh Nauk SSSR 5, 60-71 Lapin B A, Yakovleva L A, Asanov N S, Kokosha L V, Tsiripova G S, Mirvis H B & Ivanov M T (1973) Voprosy Virusologii 1, 38-44 Lapin B A, Yakovieva L A, Bukaeva I A, Indzhiia L V & Kokosha L V (1973) Fourth International Congress on Primatology, Symposia 4, 1-29 Lapin B A, Yakovieva L A, Indzhiia L V, Kuksova M I, Kokosha L V, Schekolodkin V F, Lebedev V N, Bikovski A F & Lorye Y I (1973) Vestnik Akademii Meditsinskikh Nauk SSSR 4,10-20
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Professor K McCarthy and Dr F A Tosolini' (Department of Medical Microbiology, The University, Liverpool, L69 3BX) A Review of Primate Herpes Viruses
The last twenty years have seen an ever increasing use of subhuman primates in medical and scientific research. With very few exceptions these animals are trapped in the wild and air freighted in large batches to research laboratories throughout the world. Often they will have passed through several staging posts and thus had opportunity for contact with many other species of primates, including man, as well as with animals of lower orders. Although animals which are obviously sick are not shipped for fear of incurring financial loss through immediate outbreaks of lethal disease, the pre-export scrutiny cannot detect minor infections so that on arrival in the country of destination the condition of newly arrived monkeys is often marred by a series of minor illnesses or occasionally by outbreaks of lethal disease. Many infections by viruses of the herpes group are likely to be missed because characteristically herpes infection in the natural host is trivial or completely inapparent. The full virulence of one of these viruses may not be manifest unless the virus is accidentally transmitted to a new species. The best known example of this is the infection of man by Herpesvirus simiw (B virus). Twenty years ago only three primate herpes viruses were known: Herpesvirus hominis I ('herpes simplex virus'), Herpesvirus simik and Herpesvirus varicellw ('varicella-zoster virus'). At the present time thirty-seven primate herpes virus strains are known to exist (Table 1). Several reviews of selected primate herpes viruses have appeared in recent years (Plummer 1967, Hunt & Melendez 1969, Kalter & Heberling 1971, 1972, Hull 1968, 1973). Although Herpesvirus simiw is the only simian herpes virus known to infect man, there has been an understandable reluctance to pursue extensive investigations with the other simian herpes viruses. Thus, many "Present address: Division of Microbiology, Institute of Medical and Veterinary Science, Adelaide, South Australia 5000
