JOURNAL OF PATHOLOGY, VOL.
162: 67-75 (1 990)
HISTOPATHOLOGICAL CHANGES IN SIMIAN IMMUNODEFICIENCY VIRUS INFECTION A. BASKERVILLE, A. RAMSAY*, M. P. CRANAGE, N. COOK, R.
w. COOK,
M. J. DENNIS, P. J. GREENAWAY,
P. A. KITCHIN? AND E. J. STOTT?
Public Health Laboratory Service, Centrefor Applied Microbiology and Research, Division of Pathology, Porton Down, Salisbury, Wiltshire. U.K.; *University of Southampton, Department of Pathology, Southampton General Hospital, Tremona Road, Southampton, U . K . ;and t National Institute,for Biological Standardisation and Control, South Mimms, Potters Bar, Hertfordshire. U.K. Received 5 April 1990 Accepted 5 May 1990
SUMMARY The histological lesions were studied in seven rhesus and three cynomolgus monkeys infected with simian immunodeficiency virus for periods ranging from nine weeks to 18 months. Lymphoreticular changes included hyperplasia, follicular involution and depletion, and one animal had amyloidosis of the spleen. Hyperplastic changes also took place in mucosa-associated lymphoid tissue and infiltrations occurred in the vaginal mucosa of one animal, which could be significant in sexual transmission of the infection. The range of opportunistic infections was small compared with that in human AIDS patients, although two monkeys had Pneumocystis carinii pneumonia. Enterocolitis was a common finding and brown adipose tissue was transformed into a large vacuolated type. Lesions of the central nervous system were found in five of nine monkeys, and consisted of foci of glial activity and perivascular and meningeal lymphocytic infiltration. A lymphoma involving the lumbar spinal cord developed in one animal. KEY
WORDS-Nonhuman
primates, immunodeficiency virus infection. distribution of lesions.
INTRODUCTION The establishment of the acquired immune deficiency syndrome (AIDS) in the human population has prompted the search for an experimental animal model, showing similar clinical disease and pathological lesions, in which candidate vaccines and potential antiviral agents can be assessed. The causative agent of AIDS is an exogenous retrovirus known as the 'human immunodeficiency virus' (HIV- 1). Experimentally, the only animal, apart from man, that HIV-1 has been shown to infect is the chimpanzee and this results in only limited disease manifestations.' However, a number of primate species are susceptible to infection with simian immunodeficiency virus (SIV), a lentivirus hylogenetically related to both HIV- 1 and I-IIV-~.'~This
results in immunodeficiency and a variety of pathological changes, including lymphadenopathy, enteritis and weight I O S S , ~ , ~similar to AIDS in man. A notable feature of infection with SIV is the variability in time of onset of disease, and the range of lesions produced, even in monkeys of the same species, sex and age. If experimental SIV infections are to be used as a model of human AIDS, then it is important to establish the pattern of pathological conditions produced by infection of susceptible animals with specific strains of SIV. This paper describes the pathological findings in rhesus and cynomolgus monkeys infected with SIVmac, SIVcyn or SIVsmm. MATERIALS AND METHODS Animals
Correspondence to Dr A Baskerville, Division of Pathology, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, U.K.
0022-341 7/90/090067-09 $05.00 0 1990 by John Wiley & Sons, Ltd.
Seven rhesus monkeys (Macaca mulatta) and three cynomolgus monkeys (Macaca fascicularis)
68
A. BASKERVILLE ET AL.
Table I-Simian
Monkey No.
Rhesus 68H Rhesus 44H Rhesus 85H Rhesus K33 Rhesus 19H Rhesus 27H Rhesus 32H Cyno 705B Cyno 630C Cyno 7250
immunodeficiency viruses used to infect monkeys
Sex
Virus
0
SIVmac32H SIVmac32H SIVmac32H SIVmac32H SIV32H/CBL SIVmac32H SIVmac25l SIVmac251 SIVcynl86 SIVsmm7
$2
9 8 $2
9 8
8
Virus dilution
lo-’* lo-’*
neat lo-’*
neat? neat neat neat
* = 11/88 pool of cell-free supernatant; t =low
infected with different SIV were included in this study. The rhesus monkeys (six female and one male) were bred in the United Kingdom, and at infection weighed between 2.9 and 5.0 kg and were aged between two and three years. The cynomolgus monkeys (two male and one female) were also U.K.-bred, weighed 1.9-2.1 kg and were two years old at infection. All animals were fed a commercial pelletted diet supplemented with fruit and were allowed water ad libitum. As control material, tissues from eight uninfected rhesus and two cynomolgus monkeys of similar age from the same breeding units were used.
Procedures For all infections or other procedures, monkeys were anaesthetized by intramuscular injection of ketamine hydrochloride (‘Vetalar’, Parke Davis). For blood sampling and intravenous injection of virus suspension the femoral vein was used. Animals were killed by intravenous injection of pentobarbitone sodium. Virus preparation The viruses given to individual monkeys are presented in Table I. Full details of virus preparation and methods of isolation from tissues of these monkeys are described elsewhere.“’ Autopsy and histopathology Autopsy was carried out immediately after death on those monkeys killed and not more than 12 hours
Time
Died (D)
to
or killed (K)
death
9 weeks 4 months 54months
D
D K K K K K D K K
4imonths 1 1 i months 11 months 18 months 10 months 12 months 14months
passage virus from Dr Desrosiers.
later on those which died. The following organs were taken and fixed in 10 per cent buffered neutral formalin: lungs, heart, liver, spleen, adrenals, kidneys, pancreas, stomach, ileum, caecum, colon, bladder, ovaries, uterus, testis, skeletal muscle, thymus, brown adipose tissue and mandibular, axillary, inguinal and mesenteric lymph nodes. The brain and spinal cord were taken from nine of the 10 animals. After fixation, the tissues were processed by standard methods and embedded in paraffin wax. Sections were cut at 5 pm and stained by haematoxylin and eosin. Selected sections were stained by periodic acid-Schiff (PAS), Gordon and Sweets’ method for reticulin, Verhoeff-Van Gieson, Congo Red, Martius’s Scarlet Blue, and Grocott’s silver methenamine techniques, and by the Gram, Ziehl-Nielsen and Giemsa methods. Lung sections from all animals were also stained with antiPneumocystis carinii monoclonal antibody (Dako Ltd, High Wycombe, U.K.) using the avidin-biotinperoxidase method, with diamino-benzidine as indicator.
RESULTS Necropsyjindings The macroscopic and histological lesions are summarized in Table 11. One cynomolgus monkey (630C) had a skin tumour on the chest and extensive retroperitoneal fibromatosis involving the intestines and mesenteric lymph nodes. One rhesus monkey
69
PATHOLOGY OF EXPERIMENTAL SIV INFECTION
Table If-Macroscopic and microscopic findings in SIV-infected monkeys
Weight
loss
Gingivitis
Pneumocystis carinii
Chronic gastritis
Enterocolitis
pneumonia
~~
68H 44H 85H K33
19H 27H 32H 705B
630C 7250
Lymphoma
Retroperitoneal CNS fibromatosis lesions
~
+ + + + + +
+
+ + + + + + + + +
+ + + +
+
+
+
+
+
+
+ + +
+
+
+
Table 111-Spleen and lymph node changes in SIV-infected monkeys Follicular Medullary expansion
involution,
Splenomegaly
Enlarged nodes
Hyperplasia
paracortex normal
68H 44H 85H K33
19H 27H 32H
+ +
705B 630C 7250
+ + +
+ + +
+ + + +
Atrophy
thymus
MALT hyperplasia
+ +
+ +
MALT = mucosa-associated lymphoid tissue.
(1 9H) had a tumour of the spinal cord at the level of the 4th lumbar vertebra and also deposits in the cauda equina and left kidney.
Histopathologicalfindings Lymphoreticular system-Lymph nodes showed three basic histological patterns: lymphoid hyperplasia, follicular involution and lymphoid depletion and atrophy. Similar changes were seen in the spleen in all animals. The distribution of these lesions in the different animals is shown in Table 111. In monkeys showing hyperplasia, there was massive follicular enlargement, the follicles having irregular outlines and thin mantle zones. Many follicles showed invagination of mantle zone cells into
the germinal centres (‘follicle lysis’). The paracortical regions contained many large ‘blast’ cells and mitotic activity was marked. The medullary sinuses showed some dilatation, and in one monkey (l9H) the medullary cords contained numerous plasma cells. This animal also had a lymphoma of the spinal cord. The splenic changes were similar, with enlarged irregular follicles (Fig. l), follicle lysis and active T-zones. In four monkeys the nodes showed follicular involution resembling atrophic nodes, but the paracortices were normal or expanded in size and composed mainly of small lymphocytes, with a few larger immunoblastic forms. Vascularity was pronounced, with many high endothelial venules showing lymphocyte traffic.
70
Fig. I-Spleen plasia
A. BASKERVILLE ET AL.
of rhesus 27H, showing massive follicular hyper-
Fig. 2-Atrophic lymph node of rhesus 85H, with shrunken, hyalinized germinal centre
Three monkeys showed an atrophic pattern, with germinal centres absent or shrunken, many showing hyalinization (Fig. 2 ) . The cellularity of the paracortical regions was reduced, with a proliferation of thin-walled blood vessels. Lymphoid follicles in the spleens were either absent or involuted and hyalinized, and the peri-arteriolar sheaths were reduced in size and cellularity. Amyloid deposition was widespread in the splenic follicles of rhesus 85H, confirmed by Congo Red staining and birefringence under polarized light.
Chronic gastritis was present in five monkeys, the changes consisting of focal and diffuse lymphocytic infiltration of the mucosa of both fundus and antrum, although in no case was there significant atrophy of glandular tissue. Eighty per cent of the normal uninfected monkeys showed a similar degree of chronic gastritis. Lesions in the ileum were less commonly observed, with only cynomolgus 7250 and rhesus 85H showing changes in the villi, with little inflammatory infiltration. Villi were irregular in size and shape, many were short or fused, and the lamina propria was packed with large histiocytes with abundant eosinophilic cytoplasm. Acid-fast bacilli could not be demonstrated in these cells. The tumour masses investing the intestines in cynomolgus 630C were made up of loose, highly vascular fibromatous tissue in which spindle-shaped cells predominated over lymphocytes and histiocytes-the typical appearance of retroperitoneal
Alimentary system-Lesions of varying severity were present in the intestines of most animals (Table 11). Colitis varied from a chronic mild form, with lymphocytic and plasma cell infiltration of the mucosa, to acute necrotizing inflammation with multiple deep ulcerations and crypt abscesses. Mild to moderate colitis was present in 80 per cent of normal control animals aged three years or over.
PATHOLOGY OF EXPERIMENTAL SIV INFECTION
71
Fig. 3-Lung of pneumocystis-infected cynomolgus 705B. Alveoli are filled with foamy material. Periodic acid-Schiff
Fig. &Lung of 705B stained by Grocott’s silver rnethenarnine to show Pneumocystis carinii organisms in an alveolus
fibromatosis.’ The subcutaneous chest nodule consisted of similar tissue, which was also present in the right axillary lymph node.
(19H) had a lymphoma of the spinal cord and kidney. It also had lymphocytic interstitial pneumonia, with numerous lymphoid nodules in alveolar walls throughout the lungs. Many had germinal centres and caused compression of surrounding alveoli or projected from the pleural surface. Control lung tissue showed no lesions.
Respiratory system-Lesions were present in the lungs of cynomolgus 705B, rhesus 19H and K33. The most significant and typically AIDSrelated was Pneumocystis carinii infection of 705B and K33. Irregular areas of all lobes were affected, many subpleural, and in these alveoli were filled with foamy, eosinophilic, PAS-positive material (Fig. 3). There was minimal inflammatory infiltration, but many alveoli were lined by large cuboidal cells. Staining by Grocott’s silver methenamine method demonstrated groups of Pneumocystis cysts and crescent forms (Fig. 4). However, much more extensive staining of the organisms was obtained using anti-Pneumocystis monoclonal antibody. The other monkey with respiratory tract disease
Central Nervous system-Lesions typical of AIDS encephalopathy were present in the brains of five animals, and one of these (19H) also had a lymphoma involving the spinal cord and cauda equina. In all cases, there were focal lesions throughout the grey and white matter of the cerebral cortex and brain stem, the medulla being particularly affected. A few of the lesions were clearly demarcated nodules composed of microglia and macrophages, but most were irregular zones containing microglia, macrophages, cells with large pale nuclei, degenerating neurones, disintegrating
72
A. BASKERVILLE ET AL.
rhesus 32H, with a typical nodule in the cerebral
Fig. &Vaginal epithelium, rhesus 27H, showing sub-epithelial and intra-epithelial lymphocytes
myelin and karyorrhectic nuclear fragments (Fig. 5). Occasionally, a multinucleated Langhans giant cell was present. Some blood vessels in these and other areas of the brain, leptomeninges, choroid plexus and cord had dense lymphocytic infiltrates and endothelial hypertrophy was a feature of small vessels in affected regions. Brain and cord tissue from uninfected animals had no lesions.
Adrenal glands-The only lesion was extensive calcification of the inner cortex and medulla without an inflammatory reaction in rhesus K33.
Fig. 5-Brain, cortex
Kidneys-Renal lesions were present in rhesus 19H and 27H and in cynomolgus 630C and 7250. Of these, one rhesus (19H) had a large lymphoma deposit and the other (27H) had numerous foci of lymphocytic accumulation throughout the cortex; glomerular changes were observed in the two cynomolgus monkeys. In these, large numbers of glomeruli exhibited a range of changes from mesangial hypercellularity and tuft adhesions to fibrosis, and, occasionally, sclerosis. A few tubules contained amorphous PAS-positive casts and there were PASpositive droplets in epithelial cells.
Male reproductive tract-Only three male animals were available in this study and each was sexually immature. The developing prostates and penile urethra had no lesions and seminiferous tubules showed no evidence of spermatogenesis or of cellular infiltrations. However, the testes of one cynomolgus (7250) contained several focal infiltrates of lymphocytes in the interductal connective tissue of the epididymis. Female reproductive tract-In a single monkey (rhesus 27H) there were lesions in the uterus and vagina. They consisted of numerous focal infiltrates of mixed lymphocytes in the uterine muscle, and large follicular lymphoid masses with a high mitotic rate in the subepithelial tissue of the vagina (Fig. 6).
73
PATHOLOGY OF EXPERIMENTAL SIV INFECTION
Table IV-Virus
detection and isolation from SIV-infected monkeys Post-mortem Ante-mortem
Animal 68H 44H 85H K33 19H 27H 32H 705B 630C 7250
PBL
PBL/Plasma
+* +* + + + + + + + +
+* +* + + + + -
NT C
+
Lymph node
Spleen
NT NT
NT NT
+
+ +
+ + NT + +
+
+
+
NT NT C
+ = SIV isolated; NT = Not tested: NA =Not availablc; contaminated; PBL = Peripheral blood lymphocytes. The latter closely resembled mucosa-associated lymphoid tissue and extended into the epithelium, which at some sites was reduced to two cells’ thickness. Lymphocytes were also migrating intra-epithelially and onto the surface from the follicles (Fig. 6). Miscellaneous organs-Lymphoid infiltrates were found in the parotid and submandibular salivary glands of cynomolgus 705B and 7250 and rhesus 19H and 27H, and in the bladder of rhesus 27H. Only one animal (27H) showed myocardial lesions, and in this there was a single area of fibre degeneration, necrosis and mineralization, with accompanying macrophage activity and local infiltration by small and large lymphocytes. Nuclei of fibres in the affected zone were enlarged and bizarre in shape. Brown adipose tissue-Tissue was taken from control and SIV-infected monkeys from the axilla, the anterior and posterior aspects of the neck and from the adrenal region. In the normal animal each depot of brown adipose tissue (BAT) is made up of several lobules of intensely eosinophilic cells with central, vesicular nuclei and foamy cytoplasm of very small vacuoles. In four monkeys (44H, 68H, 85H, 32H) the BAT at each location was morphologically normal, but in five animals (27H, 19H, 705B, 630C, 7250) the tissue had a loose, open structure and cells were distended by a single large vacuole and had a peripheral, flattened nucleus. In
Cerebrospinal fluid NA NA NA
+
NT
+
NT NA NA NA
NT C
* = PCR-No
culture; C = Culture
some lobules there were also perivascular accumulations of lymphocytes. In rhesus K33 the cervical BAT was normal but that of the peri-adrenal region had the open, vacuolated, poorly staining structure. Virus isolation-Virus was isolated by co-cultivation of monkey tissue or plasma with human cord-blood lymphocytes or the C8 166 human T-cell line. Isolates were confirmed as STV by reverse transcriptase activity, solid phase-gag immunoassay and polymerase chain reaction (PCR). The results are presented in Table IV. The monkeys’ serum was screened for antibodies to D-type retroviruses using a solid phase enzyme immunoassay. Rhesus sera taken before and after inoculation with SIVmac had no detectable antibodies to D-type retroviruses, but cynomolgus 630C was found to have antibody before infection with SIVcynl86. DISCUSSION The experimental infections of rhesus and cynomolgus monkeys with the SlVs reported here confirm the relevance of these animal model systems for the study of human AIDS. Comparable pathological changes to those in man occurred in the spleen, lymph nodes, brain, lungs and intestinal tract and other lesions were identified, such as those in brown fat and the female genital tract, which may be significant in the pathogenesis of AIDS.
74
A. BASKERVILLE ET AL.
Pneumocystis carinii pneumonia was detected in two experimentally SIV-infected monkeys. The infection was therefore much less common in this group of immunosuppressed monkeys than in human HIV-I infection, where it occurs in up to 85 per cent of patients." The pneumocystis-positive animals had been infected with SIV for 4 and 10 months, but had not been observed to cough or show dyspnoea. This may have been due to relatively small areas of lung being involved or because the animals were not undertaking exertion, but it does indicate that clinical diagnosis of this level of infecion in primates is extremely difficult. The study shows that a commercially available monoclonal antibody to human Pneumocystis carinii does stain the organism found in monkeys and is much more sensitive than silver impregnation methods. The lymph node changes seen in our series of cases fall into three groups, as described in rhesus monkeys by Wyand et al." In their study the most common pattern seen was follicular involution with normal or expanded paracortices. This occurred in 65 per cent of their autopsies; 10 per cent showed an atrophic picture, and 5 per cent showed hyperplastic changes. The most frequent pattern in our cases was lymphoid atrophy. Their remaining animals (20 per cent) showed a granulomatous lymphadenitis related to acid-fast bacterial infection. None of our cases showed this picture. Only one of our animals exhibited an increase in plasma cells, and it also developed a lymphoma which showed plasma cell differentiation (Ramsay et al., in preparation). The observation of massive amyloid deposition in the spleen of one animal is interesting, since it is a rare finding in human AIDS and the animal was young and had only been infected for five months. Our findings of hyperplastic changes at sites of MALT and in the thymus suggest that this may be a valuable area of study, since many of the lymphoreticular malignancies arising in HIV infection are extranodal in origin.12 Weight loss and muscle wasting were common clinical findings in this study, and, as in man,'j were usually associated with chronic diarrhoea. Two monkeys, however, became cachectic in the absence of diarrhoea. Enterocolitis occurred in nine of 10 monkeys, and bacteria isolated from these animals' intestines included Campylobacter jejuni, Yersinia enterocolitica and Shigella spp.-all pathogens found in AIDS patients. Yersinia is a highly virulent pathogen and causes necrotizing enterocolitis in monkeys. It is probable that these infections were exacerbated by the animals' immune deficient state,
though the prevalence of symptomless carriers of these organisms in the colony of origin is high. A further difficulty in interpretation is the high frequency of chronic colitis in normal rhesus and cynomolgus monkeys and in control tissues. This consists of a variable infiltration of the colonic mucosa by lymphocytes and plasma cells, a high mitotic index in crypt epithelium, and even crypt abscesses. The prevalence of lesions increases with age above three years, the age at which most monkeys in this study were inoculated. The cause of the lesions is unknown, although it is thought that some of the common intestinal bacterial pathogens, or immune-mediated responses to them, play a role. Similarly, the chronic gastritis observed in this study occurs in a high proportion of normal, symptomless rhesus monkeys and is associated with infection of the mucosa by Helicobacter py10ri.l~ The lesions were not made more severe by SIV infection. One cynomolgus monkey (630C) had typical retroperitoneal fibromatosis: preceded one month earlier by a subcutaneous chest nodule of the same type of tissue (subcutaneous fibromato~is'~). This was removed surgically, but subsequently a metastasis was found in the axillary lymph node, which was biopsied a week before euthanasia. This animal had been inoculated with SIVcynl86 but had antibody to D-type retrovirus before infection. Recent studies indicate that retroperitoneal fibromatosis is associated with infection by D-type retrovirus and not SIVI6 and it is probable that this was the cause of the condition in cynomolgus 630C. Our histological demonstration of lesions in the vagina of one SIV-infected rhesus monkey is particularly interesting. Large numbers of lymphocytes of various types were present in the vagina and formed follicles of mucosa-associated lymphoid tissue similar to that found in the mammalian bronchus and gut. The follicles extended into the epithelium, and lymphocytes were migrating through the epithelial layers to the surface. Immunolabelling and hybridization studies on lymph nodes and extra-nodal lymphoid foci in man and SIV-infected primates have shown that a proportion of the lymphocytes are infected with HIV-1 or SIV."3" The superficial location of lymphocytes in the vagina of this monkey therefore confirms the accessibility to the lumen of potentially virus-infected cells and suggests one mechanism by which heterosexual transmission of AIDS could occur. Brain lesions of the type observed in human AIDS encephalopathy were present in five of nine SIV-infected monkeys, a similar proportion to
P A T H O L O G Y OF EXPERIMENTAL SlV INFECTION
the 56 per cent reported in rhesus monkeys infected with SIVmac251 by Ringler et ~ 1 . The ' ~ lesions and their distribution corresponded closely to those recorded previously.I8 Neurological signs were not noticed in our monkeys, but this may be partly due to the difficulty of evaluating subtle behavioural disturbances in caged animals with limited scope for movement. We examined brown adipose tissue (BAT) because of its relationship to metabolic rate, energy balance and cachexia." There appear to have been no previous investigations on changes in BAT in either human AIDS or SIV-infected primates. Four of 10 infected monkeys had morphologically normal BAT, but five had BAT transformed into cells distended by single vacuoles. It is difficult to correlate this change in BAT with the animals' physical condition, for although the five monkeys with the 'open' structure were cachectic and had muscle wasting, three with normal BAT had also lost weight. Our results suggest that BAT may be involved in the metabolic changes and weight loss occurring in AIDS, but biochemical analysis of BAT and physiological studies would be necessary to define the extent of these and the mechanisms. ACKNOWLEDGEMENTS
We are grateful for the skilled assistance of Mr R. T. Raymond, Mr R. T. Moore and Miss Paula Freemantle. REFERENCES I . Fultz PN, McClure HM, Swenson RB, et a/.Persistent infection of chimpanzees with human T-lymphocytotropic virus type III/lymphadenopathy-associated virus: a potential model for acquired immunodeficiency syndrome. J Virol1986; 58: 116124. 2. King NW. Simian models of acquired immunodeficiency syndrome (AIDS): a review. Ver Parhol1986; 23: 345-353.
75
3. Daniel MD, Letvin NL, Sehgal PK, er a/.Long-term persistent infection of macaque monkeys with the simian immunodeficiency virus. J Gen VirolI987; 68: 3 183-3 189. 4. Chalifoux LV, Ringler DJ, King NW, ci al. Lymphadenopathy in macaques experimentally infected with the simian immunodeficiency virus(S1V). Amer J Path 1987; 128: 1 0 4 1 1 0 . 5 . Baskin GB, Murphey-Corb M, Watson EA, er al. Necropsy findings in rhesus monkeys experimentally infected with cultured simian immunodeficiency virus (SIV)/Deita. Vet Purh 1988; 2 5 45-67. 6 . Cranage MP, Cook N, Stott EJ, et a/. SIVmac causes a chronic infection in baboons: serial virus isolation and seroconversion. AIDS (in press). 7. Cranage MP, Cook N, Johnston P, et al. SIV infection of rhesus macaques: in viva titration of infectivity and development of an experimental vaccine. In: Horzinek M, ed. Proclnt TNO Meeting on Animal Models m AIDS. Elsevier/North Holland Biomedical Press (in press). 8. Kitchin PA, Cranage MP, Almond N , et al. Titration of SIVmac25I (32H isolate) in cynomolgus macaques for use as a challenge in vaccination studies. In : Horzinek M, ed. Proc Int TNO Mcering on Animul Models in AIDS. Elsevier/North Holland Biomedical Press (in press). 9 . Giddens WE, Tsai CC, Morton WR, et al. Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques: pathologic observations and transmission studies. Amer J Path 1985; 1 1 9 253-263. 10. Marchevsky A, Rosen MJ, Chrystal G. Pulmonary complications of AIDS: a clinicopathologic study of 70 cases. Hum Path 1985; 1 6 659-670. 11. Wyand MS, Ringler DJ, Naidu YM, er ul. Cellular localization of simian immunodeficiency virus (SIV) in lymphoid tissues. 11. In-situ hybridization. Am J Path 1989; 134: 385-393. 12. Levine AM, Gill PS, Rasheed S. Human retrovirus-associated lymphoprohferative disorders in homosexual men. In: Klein E, ed. Prog Allergy 37. Acquired Immunodejkiency Syndrome. Basel: Karger 1986; 244258. 13. Dworkin 8, Wormser GP, Rosenthal WS. Gastrointestinal manifestations of the acquired immunodeficiency syndrome: a review of 22 cilscs. . I m w .I (;os/riientero/ 19x5. 8 0 714 778 14 Ha\kerville A, Newell DG Naturnlly occurring chronic gastritis and C' pj,Ioriinfectiori in thcrhesus rnonhsy d pokntial model forgastritis
I6
17.
18.
19.
H, e r al. Subcutaneous tihromatosia asrociated with a n ;icquired immune deticiency syndrome in pig-tailed mncaquec Amcr J Path 19x5; 1 2 0 3@-37 Sti-mnhcrg K. Henvcnistc R, Arthur LO, ul. ('haracterisation of cxogennus typc D retrovirus from a tihronia o l i i macaque with simian AIDS and tihromatusia S
162: 67-75 (1 990)
HISTOPATHOLOGICAL CHANGES IN SIMIAN IMMUNODEFICIENCY VIRUS INFECTION A. BASKERVILLE, A. RAMSAY*, M. P. CRANAGE, N. COOK, R.
w. COOK,
M. J. DENNIS, P. J. GREENAWAY,
P. A. KITCHIN? AND E. J. STOTT?
Public Health Laboratory Service, Centrefor Applied Microbiology and Research, Division of Pathology, Porton Down, Salisbury, Wiltshire. U.K.; *University of Southampton, Department of Pathology, Southampton General Hospital, Tremona Road, Southampton, U . K . ;and t National Institute,for Biological Standardisation and Control, South Mimms, Potters Bar, Hertfordshire. U.K. Received 5 April 1990 Accepted 5 May 1990
SUMMARY The histological lesions were studied in seven rhesus and three cynomolgus monkeys infected with simian immunodeficiency virus for periods ranging from nine weeks to 18 months. Lymphoreticular changes included hyperplasia, follicular involution and depletion, and one animal had amyloidosis of the spleen. Hyperplastic changes also took place in mucosa-associated lymphoid tissue and infiltrations occurred in the vaginal mucosa of one animal, which could be significant in sexual transmission of the infection. The range of opportunistic infections was small compared with that in human AIDS patients, although two monkeys had Pneumocystis carinii pneumonia. Enterocolitis was a common finding and brown adipose tissue was transformed into a large vacuolated type. Lesions of the central nervous system were found in five of nine monkeys, and consisted of foci of glial activity and perivascular and meningeal lymphocytic infiltration. A lymphoma involving the lumbar spinal cord developed in one animal. KEY
WORDS-Nonhuman
primates, immunodeficiency virus infection. distribution of lesions.
INTRODUCTION The establishment of the acquired immune deficiency syndrome (AIDS) in the human population has prompted the search for an experimental animal model, showing similar clinical disease and pathological lesions, in which candidate vaccines and potential antiviral agents can be assessed. The causative agent of AIDS is an exogenous retrovirus known as the 'human immunodeficiency virus' (HIV- 1). Experimentally, the only animal, apart from man, that HIV-1 has been shown to infect is the chimpanzee and this results in only limited disease manifestations.' However, a number of primate species are susceptible to infection with simian immunodeficiency virus (SIV), a lentivirus hylogenetically related to both HIV- 1 and I-IIV-~.'~This
results in immunodeficiency and a variety of pathological changes, including lymphadenopathy, enteritis and weight I O S S , ~ , ~similar to AIDS in man. A notable feature of infection with SIV is the variability in time of onset of disease, and the range of lesions produced, even in monkeys of the same species, sex and age. If experimental SIV infections are to be used as a model of human AIDS, then it is important to establish the pattern of pathological conditions produced by infection of susceptible animals with specific strains of SIV. This paper describes the pathological findings in rhesus and cynomolgus monkeys infected with SIVmac, SIVcyn or SIVsmm. MATERIALS AND METHODS Animals
Correspondence to Dr A Baskerville, Division of Pathology, PHLS Centre for Applied Microbiology and Research, Porton Down, Salisbury, Wiltshire SP4 OJG, U.K.
0022-341 7/90/090067-09 $05.00 0 1990 by John Wiley & Sons, Ltd.
Seven rhesus monkeys (Macaca mulatta) and three cynomolgus monkeys (Macaca fascicularis)
68
A. BASKERVILLE ET AL.
Table I-Simian
Monkey No.
Rhesus 68H Rhesus 44H Rhesus 85H Rhesus K33 Rhesus 19H Rhesus 27H Rhesus 32H Cyno 705B Cyno 630C Cyno 7250
immunodeficiency viruses used to infect monkeys
Sex
Virus
0
SIVmac32H SIVmac32H SIVmac32H SIVmac32H SIV32H/CBL SIVmac32H SIVmac25l SIVmac251 SIVcynl86 SIVsmm7
$2
9 8 $2
9 8
8
Virus dilution
lo-’* lo-’*
neat lo-’*
neat? neat neat neat
* = 11/88 pool of cell-free supernatant; t =low
infected with different SIV were included in this study. The rhesus monkeys (six female and one male) were bred in the United Kingdom, and at infection weighed between 2.9 and 5.0 kg and were aged between two and three years. The cynomolgus monkeys (two male and one female) were also U.K.-bred, weighed 1.9-2.1 kg and were two years old at infection. All animals were fed a commercial pelletted diet supplemented with fruit and were allowed water ad libitum. As control material, tissues from eight uninfected rhesus and two cynomolgus monkeys of similar age from the same breeding units were used.
Procedures For all infections or other procedures, monkeys were anaesthetized by intramuscular injection of ketamine hydrochloride (‘Vetalar’, Parke Davis). For blood sampling and intravenous injection of virus suspension the femoral vein was used. Animals were killed by intravenous injection of pentobarbitone sodium. Virus preparation The viruses given to individual monkeys are presented in Table I. Full details of virus preparation and methods of isolation from tissues of these monkeys are described elsewhere.“’ Autopsy and histopathology Autopsy was carried out immediately after death on those monkeys killed and not more than 12 hours
Time
Died (D)
to
or killed (K)
death
9 weeks 4 months 54months
D
D K K K K K D K K
4imonths 1 1 i months 11 months 18 months 10 months 12 months 14months
passage virus from Dr Desrosiers.
later on those which died. The following organs were taken and fixed in 10 per cent buffered neutral formalin: lungs, heart, liver, spleen, adrenals, kidneys, pancreas, stomach, ileum, caecum, colon, bladder, ovaries, uterus, testis, skeletal muscle, thymus, brown adipose tissue and mandibular, axillary, inguinal and mesenteric lymph nodes. The brain and spinal cord were taken from nine of the 10 animals. After fixation, the tissues were processed by standard methods and embedded in paraffin wax. Sections were cut at 5 pm and stained by haematoxylin and eosin. Selected sections were stained by periodic acid-Schiff (PAS), Gordon and Sweets’ method for reticulin, Verhoeff-Van Gieson, Congo Red, Martius’s Scarlet Blue, and Grocott’s silver methenamine techniques, and by the Gram, Ziehl-Nielsen and Giemsa methods. Lung sections from all animals were also stained with antiPneumocystis carinii monoclonal antibody (Dako Ltd, High Wycombe, U.K.) using the avidin-biotinperoxidase method, with diamino-benzidine as indicator.
RESULTS Necropsyjindings The macroscopic and histological lesions are summarized in Table 11. One cynomolgus monkey (630C) had a skin tumour on the chest and extensive retroperitoneal fibromatosis involving the intestines and mesenteric lymph nodes. One rhesus monkey
69
PATHOLOGY OF EXPERIMENTAL SIV INFECTION
Table If-Macroscopic and microscopic findings in SIV-infected monkeys
Weight
loss
Gingivitis
Pneumocystis carinii
Chronic gastritis
Enterocolitis
pneumonia
~~
68H 44H 85H K33
19H 27H 32H 705B
630C 7250
Lymphoma
Retroperitoneal CNS fibromatosis lesions
~
+ + + + + +
+
+ + + + + + + + +
+ + + +
+
+
+
+
+
+
+ + +
+
+
+
Table 111-Spleen and lymph node changes in SIV-infected monkeys Follicular Medullary expansion
involution,
Splenomegaly
Enlarged nodes
Hyperplasia
paracortex normal
68H 44H 85H K33
19H 27H 32H
+ +
705B 630C 7250
+ + +
+ + +
+ + + +
Atrophy
thymus
MALT hyperplasia
+ +
+ +
MALT = mucosa-associated lymphoid tissue.
(1 9H) had a tumour of the spinal cord at the level of the 4th lumbar vertebra and also deposits in the cauda equina and left kidney.
Histopathologicalfindings Lymphoreticular system-Lymph nodes showed three basic histological patterns: lymphoid hyperplasia, follicular involution and lymphoid depletion and atrophy. Similar changes were seen in the spleen in all animals. The distribution of these lesions in the different animals is shown in Table 111. In monkeys showing hyperplasia, there was massive follicular enlargement, the follicles having irregular outlines and thin mantle zones. Many follicles showed invagination of mantle zone cells into
the germinal centres (‘follicle lysis’). The paracortical regions contained many large ‘blast’ cells and mitotic activity was marked. The medullary sinuses showed some dilatation, and in one monkey (l9H) the medullary cords contained numerous plasma cells. This animal also had a lymphoma of the spinal cord. The splenic changes were similar, with enlarged irregular follicles (Fig. l), follicle lysis and active T-zones. In four monkeys the nodes showed follicular involution resembling atrophic nodes, but the paracortices were normal or expanded in size and composed mainly of small lymphocytes, with a few larger immunoblastic forms. Vascularity was pronounced, with many high endothelial venules showing lymphocyte traffic.
70
Fig. I-Spleen plasia
A. BASKERVILLE ET AL.
of rhesus 27H, showing massive follicular hyper-
Fig. 2-Atrophic lymph node of rhesus 85H, with shrunken, hyalinized germinal centre
Three monkeys showed an atrophic pattern, with germinal centres absent or shrunken, many showing hyalinization (Fig. 2 ) . The cellularity of the paracortical regions was reduced, with a proliferation of thin-walled blood vessels. Lymphoid follicles in the spleens were either absent or involuted and hyalinized, and the peri-arteriolar sheaths were reduced in size and cellularity. Amyloid deposition was widespread in the splenic follicles of rhesus 85H, confirmed by Congo Red staining and birefringence under polarized light.
Chronic gastritis was present in five monkeys, the changes consisting of focal and diffuse lymphocytic infiltration of the mucosa of both fundus and antrum, although in no case was there significant atrophy of glandular tissue. Eighty per cent of the normal uninfected monkeys showed a similar degree of chronic gastritis. Lesions in the ileum were less commonly observed, with only cynomolgus 7250 and rhesus 85H showing changes in the villi, with little inflammatory infiltration. Villi were irregular in size and shape, many were short or fused, and the lamina propria was packed with large histiocytes with abundant eosinophilic cytoplasm. Acid-fast bacilli could not be demonstrated in these cells. The tumour masses investing the intestines in cynomolgus 630C were made up of loose, highly vascular fibromatous tissue in which spindle-shaped cells predominated over lymphocytes and histiocytes-the typical appearance of retroperitoneal
Alimentary system-Lesions of varying severity were present in the intestines of most animals (Table 11). Colitis varied from a chronic mild form, with lymphocytic and plasma cell infiltration of the mucosa, to acute necrotizing inflammation with multiple deep ulcerations and crypt abscesses. Mild to moderate colitis was present in 80 per cent of normal control animals aged three years or over.
PATHOLOGY OF EXPERIMENTAL SIV INFECTION
71
Fig. 3-Lung of pneumocystis-infected cynomolgus 705B. Alveoli are filled with foamy material. Periodic acid-Schiff
Fig. &Lung of 705B stained by Grocott’s silver rnethenarnine to show Pneumocystis carinii organisms in an alveolus
fibromatosis.’ The subcutaneous chest nodule consisted of similar tissue, which was also present in the right axillary lymph node.
(19H) had a lymphoma of the spinal cord and kidney. It also had lymphocytic interstitial pneumonia, with numerous lymphoid nodules in alveolar walls throughout the lungs. Many had germinal centres and caused compression of surrounding alveoli or projected from the pleural surface. Control lung tissue showed no lesions.
Respiratory system-Lesions were present in the lungs of cynomolgus 705B, rhesus 19H and K33. The most significant and typically AIDSrelated was Pneumocystis carinii infection of 705B and K33. Irregular areas of all lobes were affected, many subpleural, and in these alveoli were filled with foamy, eosinophilic, PAS-positive material (Fig. 3). There was minimal inflammatory infiltration, but many alveoli were lined by large cuboidal cells. Staining by Grocott’s silver methenamine method demonstrated groups of Pneumocystis cysts and crescent forms (Fig. 4). However, much more extensive staining of the organisms was obtained using anti-Pneumocystis monoclonal antibody. The other monkey with respiratory tract disease
Central Nervous system-Lesions typical of AIDS encephalopathy were present in the brains of five animals, and one of these (19H) also had a lymphoma involving the spinal cord and cauda equina. In all cases, there were focal lesions throughout the grey and white matter of the cerebral cortex and brain stem, the medulla being particularly affected. A few of the lesions were clearly demarcated nodules composed of microglia and macrophages, but most were irregular zones containing microglia, macrophages, cells with large pale nuclei, degenerating neurones, disintegrating
72
A. BASKERVILLE ET AL.
rhesus 32H, with a typical nodule in the cerebral
Fig. &Vaginal epithelium, rhesus 27H, showing sub-epithelial and intra-epithelial lymphocytes
myelin and karyorrhectic nuclear fragments (Fig. 5). Occasionally, a multinucleated Langhans giant cell was present. Some blood vessels in these and other areas of the brain, leptomeninges, choroid plexus and cord had dense lymphocytic infiltrates and endothelial hypertrophy was a feature of small vessels in affected regions. Brain and cord tissue from uninfected animals had no lesions.
Adrenal glands-The only lesion was extensive calcification of the inner cortex and medulla without an inflammatory reaction in rhesus K33.
Fig. 5-Brain, cortex
Kidneys-Renal lesions were present in rhesus 19H and 27H and in cynomolgus 630C and 7250. Of these, one rhesus (19H) had a large lymphoma deposit and the other (27H) had numerous foci of lymphocytic accumulation throughout the cortex; glomerular changes were observed in the two cynomolgus monkeys. In these, large numbers of glomeruli exhibited a range of changes from mesangial hypercellularity and tuft adhesions to fibrosis, and, occasionally, sclerosis. A few tubules contained amorphous PAS-positive casts and there were PASpositive droplets in epithelial cells.
Male reproductive tract-Only three male animals were available in this study and each was sexually immature. The developing prostates and penile urethra had no lesions and seminiferous tubules showed no evidence of spermatogenesis or of cellular infiltrations. However, the testes of one cynomolgus (7250) contained several focal infiltrates of lymphocytes in the interductal connective tissue of the epididymis. Female reproductive tract-In a single monkey (rhesus 27H) there were lesions in the uterus and vagina. They consisted of numerous focal infiltrates of mixed lymphocytes in the uterine muscle, and large follicular lymphoid masses with a high mitotic rate in the subepithelial tissue of the vagina (Fig. 6).
73
PATHOLOGY OF EXPERIMENTAL SIV INFECTION
Table IV-Virus
detection and isolation from SIV-infected monkeys Post-mortem Ante-mortem
Animal 68H 44H 85H K33 19H 27H 32H 705B 630C 7250
PBL
PBL/Plasma
+* +* + + + + + + + +
+* +* + + + + -
NT C
+
Lymph node
Spleen
NT NT
NT NT
+
+ +
+ + NT + +
+
+
+
NT NT C
+ = SIV isolated; NT = Not tested: NA =Not availablc; contaminated; PBL = Peripheral blood lymphocytes. The latter closely resembled mucosa-associated lymphoid tissue and extended into the epithelium, which at some sites was reduced to two cells’ thickness. Lymphocytes were also migrating intra-epithelially and onto the surface from the follicles (Fig. 6). Miscellaneous organs-Lymphoid infiltrates were found in the parotid and submandibular salivary glands of cynomolgus 705B and 7250 and rhesus 19H and 27H, and in the bladder of rhesus 27H. Only one animal (27H) showed myocardial lesions, and in this there was a single area of fibre degeneration, necrosis and mineralization, with accompanying macrophage activity and local infiltration by small and large lymphocytes. Nuclei of fibres in the affected zone were enlarged and bizarre in shape. Brown adipose tissue-Tissue was taken from control and SIV-infected monkeys from the axilla, the anterior and posterior aspects of the neck and from the adrenal region. In the normal animal each depot of brown adipose tissue (BAT) is made up of several lobules of intensely eosinophilic cells with central, vesicular nuclei and foamy cytoplasm of very small vacuoles. In four monkeys (44H, 68H, 85H, 32H) the BAT at each location was morphologically normal, but in five animals (27H, 19H, 705B, 630C, 7250) the tissue had a loose, open structure and cells were distended by a single large vacuole and had a peripheral, flattened nucleus. In
Cerebrospinal fluid NA NA NA
+
NT
+
NT NA NA NA
NT C
* = PCR-No
culture; C = Culture
some lobules there were also perivascular accumulations of lymphocytes. In rhesus K33 the cervical BAT was normal but that of the peri-adrenal region had the open, vacuolated, poorly staining structure. Virus isolation-Virus was isolated by co-cultivation of monkey tissue or plasma with human cord-blood lymphocytes or the C8 166 human T-cell line. Isolates were confirmed as STV by reverse transcriptase activity, solid phase-gag immunoassay and polymerase chain reaction (PCR). The results are presented in Table IV. The monkeys’ serum was screened for antibodies to D-type retroviruses using a solid phase enzyme immunoassay. Rhesus sera taken before and after inoculation with SIVmac had no detectable antibodies to D-type retroviruses, but cynomolgus 630C was found to have antibody before infection with SIVcynl86. DISCUSSION The experimental infections of rhesus and cynomolgus monkeys with the SlVs reported here confirm the relevance of these animal model systems for the study of human AIDS. Comparable pathological changes to those in man occurred in the spleen, lymph nodes, brain, lungs and intestinal tract and other lesions were identified, such as those in brown fat and the female genital tract, which may be significant in the pathogenesis of AIDS.
74
A. BASKERVILLE ET AL.
Pneumocystis carinii pneumonia was detected in two experimentally SIV-infected monkeys. The infection was therefore much less common in this group of immunosuppressed monkeys than in human HIV-I infection, where it occurs in up to 85 per cent of patients." The pneumocystis-positive animals had been infected with SIV for 4 and 10 months, but had not been observed to cough or show dyspnoea. This may have been due to relatively small areas of lung being involved or because the animals were not undertaking exertion, but it does indicate that clinical diagnosis of this level of infecion in primates is extremely difficult. The study shows that a commercially available monoclonal antibody to human Pneumocystis carinii does stain the organism found in monkeys and is much more sensitive than silver impregnation methods. The lymph node changes seen in our series of cases fall into three groups, as described in rhesus monkeys by Wyand et al." In their study the most common pattern seen was follicular involution with normal or expanded paracortices. This occurred in 65 per cent of their autopsies; 10 per cent showed an atrophic picture, and 5 per cent showed hyperplastic changes. The most frequent pattern in our cases was lymphoid atrophy. Their remaining animals (20 per cent) showed a granulomatous lymphadenitis related to acid-fast bacterial infection. None of our cases showed this picture. Only one of our animals exhibited an increase in plasma cells, and it also developed a lymphoma which showed plasma cell differentiation (Ramsay et al., in preparation). The observation of massive amyloid deposition in the spleen of one animal is interesting, since it is a rare finding in human AIDS and the animal was young and had only been infected for five months. Our findings of hyperplastic changes at sites of MALT and in the thymus suggest that this may be a valuable area of study, since many of the lymphoreticular malignancies arising in HIV infection are extranodal in origin.12 Weight loss and muscle wasting were common clinical findings in this study, and, as in man,'j were usually associated with chronic diarrhoea. Two monkeys, however, became cachectic in the absence of diarrhoea. Enterocolitis occurred in nine of 10 monkeys, and bacteria isolated from these animals' intestines included Campylobacter jejuni, Yersinia enterocolitica and Shigella spp.-all pathogens found in AIDS patients. Yersinia is a highly virulent pathogen and causes necrotizing enterocolitis in monkeys. It is probable that these infections were exacerbated by the animals' immune deficient state,
though the prevalence of symptomless carriers of these organisms in the colony of origin is high. A further difficulty in interpretation is the high frequency of chronic colitis in normal rhesus and cynomolgus monkeys and in control tissues. This consists of a variable infiltration of the colonic mucosa by lymphocytes and plasma cells, a high mitotic index in crypt epithelium, and even crypt abscesses. The prevalence of lesions increases with age above three years, the age at which most monkeys in this study were inoculated. The cause of the lesions is unknown, although it is thought that some of the common intestinal bacterial pathogens, or immune-mediated responses to them, play a role. Similarly, the chronic gastritis observed in this study occurs in a high proportion of normal, symptomless rhesus monkeys and is associated with infection of the mucosa by Helicobacter py10ri.l~ The lesions were not made more severe by SIV infection. One cynomolgus monkey (630C) had typical retroperitoneal fibromatosis: preceded one month earlier by a subcutaneous chest nodule of the same type of tissue (subcutaneous fibromato~is'~). This was removed surgically, but subsequently a metastasis was found in the axillary lymph node, which was biopsied a week before euthanasia. This animal had been inoculated with SIVcynl86 but had antibody to D-type retrovirus before infection. Recent studies indicate that retroperitoneal fibromatosis is associated with infection by D-type retrovirus and not SIVI6 and it is probable that this was the cause of the condition in cynomolgus 630C. Our histological demonstration of lesions in the vagina of one SIV-infected rhesus monkey is particularly interesting. Large numbers of lymphocytes of various types were present in the vagina and formed follicles of mucosa-associated lymphoid tissue similar to that found in the mammalian bronchus and gut. The follicles extended into the epithelium, and lymphocytes were migrating through the epithelial layers to the surface. Immunolabelling and hybridization studies on lymph nodes and extra-nodal lymphoid foci in man and SIV-infected primates have shown that a proportion of the lymphocytes are infected with HIV-1 or SIV."3" The superficial location of lymphocytes in the vagina of this monkey therefore confirms the accessibility to the lumen of potentially virus-infected cells and suggests one mechanism by which heterosexual transmission of AIDS could occur. Brain lesions of the type observed in human AIDS encephalopathy were present in five of nine SIV-infected monkeys, a similar proportion to
P A T H O L O G Y OF EXPERIMENTAL SlV INFECTION
the 56 per cent reported in rhesus monkeys infected with SIVmac251 by Ringler et ~ 1 . The ' ~ lesions and their distribution corresponded closely to those recorded previously.I8 Neurological signs were not noticed in our monkeys, but this may be partly due to the difficulty of evaluating subtle behavioural disturbances in caged animals with limited scope for movement. We examined brown adipose tissue (BAT) because of its relationship to metabolic rate, energy balance and cachexia." There appear to have been no previous investigations on changes in BAT in either human AIDS or SIV-infected primates. Four of 10 infected monkeys had morphologically normal BAT, but five had BAT transformed into cells distended by single vacuoles. It is difficult to correlate this change in BAT with the animals' physical condition, for although the five monkeys with the 'open' structure were cachectic and had muscle wasting, three with normal BAT had also lost weight. Our results suggest that BAT may be involved in the metabolic changes and weight loss occurring in AIDS, but biochemical analysis of BAT and physiological studies would be necessary to define the extent of these and the mechanisms. ACKNOWLEDGEMENTS
We are grateful for the skilled assistance of Mr R. T. Raymond, Mr R. T. Moore and Miss Paula Freemantle. REFERENCES I . Fultz PN, McClure HM, Swenson RB, et a/.Persistent infection of chimpanzees with human T-lymphocytotropic virus type III/lymphadenopathy-associated virus: a potential model for acquired immunodeficiency syndrome. J Virol1986; 58: 116124. 2. King NW. Simian models of acquired immunodeficiency syndrome (AIDS): a review. Ver Parhol1986; 23: 345-353.
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3. Daniel MD, Letvin NL, Sehgal PK, er a/.Long-term persistent infection of macaque monkeys with the simian immunodeficiency virus. J Gen VirolI987; 68: 3 183-3 189. 4. Chalifoux LV, Ringler DJ, King NW, ci al. Lymphadenopathy in macaques experimentally infected with the simian immunodeficiency virus(S1V). Amer J Path 1987; 128: 1 0 4 1 1 0 . 5 . Baskin GB, Murphey-Corb M, Watson EA, er al. Necropsy findings in rhesus monkeys experimentally infected with cultured simian immunodeficiency virus (SIV)/Deita. Vet Purh 1988; 2 5 45-67. 6 . Cranage MP, Cook N, Stott EJ, et a/. SIVmac causes a chronic infection in baboons: serial virus isolation and seroconversion. AIDS (in press). 7. Cranage MP, Cook N, Johnston P, et al. SIV infection of rhesus macaques: in viva titration of infectivity and development of an experimental vaccine. In: Horzinek M, ed. Proclnt TNO Meeting on Animal Models m AIDS. Elsevier/North Holland Biomedical Press (in press). 8. Kitchin PA, Cranage MP, Almond N , et al. Titration of SIVmac25I (32H isolate) in cynomolgus macaques for use as a challenge in vaccination studies. In : Horzinek M, ed. Proc Int TNO Mcering on Animul Models in AIDS. Elsevier/North Holland Biomedical Press (in press). 9 . Giddens WE, Tsai CC, Morton WR, et al. Retroperitoneal fibromatosis and acquired immunodeficiency syndrome in macaques: pathologic observations and transmission studies. Amer J Path 1985; 1 1 9 253-263. 10. Marchevsky A, Rosen MJ, Chrystal G. Pulmonary complications of AIDS: a clinicopathologic study of 70 cases. Hum Path 1985; 1 6 659-670. 11. Wyand MS, Ringler DJ, Naidu YM, er ul. Cellular localization of simian immunodeficiency virus (SIV) in lymphoid tissues. 11. In-situ hybridization. Am J Path 1989; 134: 385-393. 12. Levine AM, Gill PS, Rasheed S. Human retrovirus-associated lymphoprohferative disorders in homosexual men. In: Klein E, ed. Prog Allergy 37. Acquired Immunodejkiency Syndrome. Basel: Karger 1986; 244258. 13. Dworkin 8, Wormser GP, Rosenthal WS. Gastrointestinal manifestations of the acquired immunodeficiency syndrome: a review of 22 cilscs. . I m w .I (;os/riientero/ 19x5. 8 0 714 778 14 Ha\kerville A, Newell DG Naturnlly occurring chronic gastritis and C' pj,Ioriinfectiori in thcrhesus rnonhsy d pokntial model forgastritis
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H, e r al. Subcutaneous tihromatosia asrociated with a n ;icquired immune deticiency syndrome in pig-tailed mncaquec Amcr J Path 19x5; 1 2 0 3@-37 Sti-mnhcrg K. Henvcnistc R, Arthur LO, ul. ('haracterisation of cxogennus typc D retrovirus from a tihronia o l i i macaque with simian AIDS and tihromatusia S
