JOURNAL OF BONE AND MINERAL RESEARCH Volume 7, Number 7, 1992 Mary Ann LieberI. Inc., Publishers
Detection of Canine Distemper Virus in Bone Cells in the Metaphyses of Distemper-Infected Dogs ANDREW P. MEE,' DAVID M. WEBBER,' CHRISTOPHER MAY,' DAVID BENNETT,' PAUL T. SHARPE.' and DAVID C. ANDERSON3
ABSTRACT In the light of recent evidence implicating canine distemper virus (CDV) as a possible etiologic agent in Paget's disease of bone, we thought that it would be of interest to examine distemper-infected bone in the natural host. Samples from the long bones, spleen, and bladder of four distemper-infected and three uninfected dogs were examined for the presence of CDV nucleocapsid and phosphoprotein genes and the measles virus (MV) nucleocapsid gene using the technique of in situ hybridization with radioactively labeled riboprobes. Two of the four distemper-infected dogs showed strongly positive hybridization with both of the CDV probes. The signal was present in marrow cells, in osteoblasts, in osteocytes, and particularly in osteoclasts. No hybridization was seen over the cartilage cells of the growth plate, and there was a clear line of demarcation at the point of invasion of osteoclasts and vascularization. The spleen and bladder samples from infected dogs also showed positive hybridization. There was no hybridization with the MV probe in any of the distemper-infected tissue. Samples from the uninfected dogs showed no evidence of hybridization with either the CDV or MV probes. These results show that CDV can infect bone cells of the natural host and provide further support for the theory that CDV may play a role in human Paget's disease of bone.
INTRODUCTION is a chronic focal bone disorder characterized by excessive osteoclastic resorption and secondary new bone formation, which affects approximately 4% of the U.K. population over 5 5 years of age.(1,2)The natural history of the disease and evidence obtained from electron microscopic,(3-6)immunocytochemical,(7-") and molecular studies(121have led to the suggestion that it might be the result of a slow virus infection by a member of the paramyxovirus family, in which measles and respiratory syncytial virus have been most strongly implicated. However, our group has previously suggested that canine distemper virus (CDV), a morbillivirus closely related to measles,('3) might be involved in the etiology of this disease. This was based on epidemiologic evidence of increased ownership of dogs among Paget's patients compared with control^^^^^'^) and on the demonstration of
P
AGET'S DISEASE
CDV mRNA in bone biopsies of these patients by in situ hybridization. (I6) In the present study, an attempt has been made to discover if CDV is present in the bone cells of distemper-infected dogs, so that more may be learnt about the predilection of this virus for bone cells in the natural host. For this purpose, we used the technique of in situ hybridization and probes to the mRNA and genomic RNA of the nucleocapsid and phosphoprotein genes of CDV. This virus usually gains entry via the respiratory tract and then spreads to bone marrow and all lymphatic tissues of infected dogs. In dogs that fail to mount a successful immune response, the virus then spreads to the respiratory, gastrointestinal, and urogenital tract epithelia and also frequently to the central nervous systern.(l7)Dogs that are acutely infected shed virus in all secretions, whether they show clinical signs or not. There are no reports of clinical problems associated with CDV infection of bone cells, although Boyce et
IBone Disease Research Centre, Department of Cell and Structural Biology, University of Manchester, England. 'Department of Veterinary Clinical Science, Small Animal Hospital, University of Liverpool, England. 'Chinese University of Hong Kong, Prince of Wales Hospital, Department of Medicine, Shatin, New Territories, Hong Kong.
829
830
MEE ET AL.
al.,(18)in a preliminary study, reported metaphyseal lesions in both naturally and experimentally infected dogs. Proteins of CDV were demonstrated within the bone cells, particularly the osteoclasts, by indirect immunofluorescence. Clearly a more complete understanding of the distribution of this virus in bone tissue could be of importance in establishing a possible effect on bone metabolism in both the dog and in human Paget's disease. It might also be of veterinary importance in view of the fact that virtually all dogs are exposed to distemper virus, either the wild type or by vaccination with live attenuated virus, and so CDV might be responsible for one or more of the hitherto unexplained canine bone disorders.
graphic emulsion. The slides were then exposed for 7-10 days, developed and counterstained with hematoxylin and eosin, and viewed by dark- and light-field microscopy.
Controls Negative control slides consisted of hybridization with a probe transcribed from a random SP6 vector sequence, a probe to the measles virus nucleocapsid gene, the addition of hybridization solution alone, and the pretreatment of sections with RNAse. Positive control slides utilized an antisense probe to P-actin.
RESULTS MATERIALS AND METHODS
Of the four distemper-infected dogs, two (the 4- and the 9-week-old dogs) showed positive hybridization with both Tissue samples and preparation the probes to the CDV nucleocapsid and phosphoprotein Metaphyseal tissue was obtained from the long bones of genes, but not with the measles probe. When present, hyfour distemper-infected dogs and three uninfected animals bridization was found in all bone cell types, including (the distemper-infected dogs were all crossbreeds aged 4 osteoblasts and osteoclasts, but not in adjacent chondroand 9 weeks and two dogs aged 5 months; the uninfected cytes. The signal was concentrated in the growth plate redogs were an 8-week-old German Shepherd, a 5-month-old gion of the long bones (Fig. l a and b) and appeared particcrossbreed, and a 5-month-old Shar Pei), fixed in 10% ularly prominent in the multinucleated osteoclasts (Fig. Ic buffered formalin for at least 24 h, decalcified in 10% and d). However, not all the osteoclasts in any one region EDTA, and processed for routine paraffin-wax-embedded were labeled to the same extent, if at all. In every case, histology. In addition, spleen and bladder samples were both the sense and antisense CDV probes were found to obtained from both infected and uninfected animals and hybridize to a similar degree. No hybridization was seen processed in the same manner without the decalcification with the measles probe (Fig. l e and f). Tissue samples step. Sections (7 pm) were cut and mounted on organosila- from uninfected dogs showed no hybridization with any of the viral probes, although accessibility to hybridization nated slides"91 for in situ hybridization. was confirmed in all tissues by a positive reaction with a probe to p-actin. Preparation of riboprobes Bladder and spleen samples from two of the infected 35S-labeled riboprobes were generated by transcription dogs were also examined and were found to be strongly of sequences to the CDV nucleocapsid and phosphopro- positive with the CDV, but not measles, probes, confirmtein genes and the measles virus nucleocapsid gene, which ing the specificity of the reaction (Figs. 2 and 3). A partichad been subcloned into the polylinker of the vector ularly distinct pattern of hybridization was observed in the pSP72. (I6) Both sense and antisense probes were produced bladder, in which discrete epithelial cells were strongly laby using the appropriate SP6 or T7 polymerase. Probes beled (Fig. 2a and b). The addition of hybridization soluwere routinely reduced to 250 base pairs (bp) in length by tion alone or the pretreatment of sections with RNase relimited alkaline hydrolysis. sulted in only background levels of silver grains in the sections.
In situ hybridization The techniques used are those described in detail by Gordon et In brief, after bringing the sections to water, they were permeabilized by treatment with 0.2 M HCI, followed by 10 pg/ml of proteinase K at 37°C for 30 minutes. Nonspecific background was reduced by treatment with 0.25% acetic anhydride. Control slides were rendered free of RNA by treatment with 100 pg/ml of RNase for 30 minutes at 37°C. Hybridization was performed in 50% formamide and 0.3 M NaCl at 50°C overnight in a sealed, humidified container. Approximately 1.O x lo5counts of radiolabeled riboprobe was added to each tissue section. After hybridization, the slides were washed at 50°C, posttreated with RNase to remove unbound probe, and dipped in 1:l dilution of Ilford K5 autoradio-
DISCUSSION In previous studies, we have shown by in situ hybridization using the same probes as those used in this study that canine distemper virus is present in the bone cells of approximately 50% of the pagetic bone biopsies tested thus far, suggesting that the virus may be involved in the etiology of this bone disorder.('61In the present work, the same technique was used to test for the presence of CDV RNA in canine bone cells, and tissue was examined from four distemper-infected dogs and three uninfected controls. The presence of CDV in bone cells was confirmed in two of the four distemper-infected bone samples but not in the controls. The virus was concentrated in the growth plate re-
CANINE DISTEMPER VIRUS IN BONE CELLS
831
FIG. 1. Distemper-infected dog bone. Positive hybridization with the CDV-N sense probe. Growth plate (GP) region viewed by (a) light-field and (b) dark-field microscopy. Arrows indicate hybridization ( x 50). Strong positive hybridization with the CDV-N sense probe in osteoblasts (OB) and particularly in osteoclasts (OC) viewed by (c) light-field and (d) dark-field microscopy ( x 160). Similar results were seen with the CDV-N antisense probe and the CDV-P sense and antisense probes (not shown). No hybridization (background level of silver grains) with MV-N sense probe viewed by (e) light-field or (f) dark-field microscopy ( x 125).
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MEE ET AL.
FIG. 2. Distemper-infected dog bladder. Positive hybridization with the CDV-N sense probe viewed by (a) light-field and (b) dark-field microscopy. Note the distinct epithelial areas of hybridization (arrows). No hybridization (background level of silver grains) with the MV-N sense probe viewed by (c) light-field or (d) dark-field microscopy ( x 125).
gion of the long bones and was particularly prominent in the multinucleated osteoclasts. The dramatic accumulation of this virus in osteoclasts of the natural host is consistent with the hypothesis that Paget’s disease is associated with CDV infection of bone-resorbing cells. In contrast to our previous findings in human pagetic bone, in which only the antisense probes (hybridizing with mRNA) were positive, both the sense and antisense probes were found to bind strongly to the dog bone sections. Since CDV is a singlestranded RNA virus, this suggests that large amounts of viral genome are present within this tissue and may represent the difference between a chronic (Paget’s) and acute (distemper) disease, or disease in the “natural” versus the “unnatural” host. CDV sequences were detected in the bladder and spleen samples from all four dogs, so the dogs were clearly infected. The inability to detect the virus in the bone samples from two of the dogs with active distemper may indicate variability between different types of dogs in susceptibility of bone cells (although all affected dogs were crossbreeds) or, more likely, be due to differences in stage and severity of the disease. That is, the virus
had not reached the bone cells at the time of death, or the bone cells were only transiently infected. Although only four distemper-infected dogs were examined in this study, it is interesting that both of the dogs that showed positive hybridization in bone cells were only several weeks old (compared with several months old), and this may reflect the inability of the virus to infect bone cells in older dogs or may be due to the changes in the vascular supply to the metaphyses that occur as dogs mature.(20’ The distinct pattern of hybridization seen in the bladder epithelium was comparable with previous immunocytochemical studies“’) and confirms the specificity of the probes, which we previously showed on measles-infected human brain (subacute sclerosing panencephalitis) and on distemper-infected dog brain. Canine distemper is a disease of the respiratory, gastrointestinal, and nervous systems, and to our knowledge there are no clinical reports of any bone involvement in this condition, although metaphyseal lesions have been described in both naturally and experimentally infected dogs.‘18) The same group has provided further immuno-
833
CANINE DISTEMPER VIRUS IN BONE CELLS
C
FIG. 3. Distemper-infected dog spleen. Positive hybridization with the CDV-N sense probe viewed by (a) light-field and (b) dark-field microscopy. No hybridization (background level of silver grains) with the MV-N sense probe, viewed by (c) dark-field microscopy ( x 125). cytochemical evidence of clear staining of osteoclasts in distemper-infected tissue, although no mention was made of its significance.( I 1 ) Certainly, there is no canine equivalent of human Paget’s disease; this might be related to a number of factors, especially the shorter life span. However, several idiopathic canine bone diseases exist that are characterized by excessive bone remodeling, for example, the conditions of metaphyseal osteopathy,‘”) panosteiti^,"^) and craniomandibular osteopathy. Hence, the possibility of wild-type or vaccine-strain CDV causing these types of disease requires further investigation. (’‘)
ACKNOWLEDGMENTS
‘’
and Miss J ’ Brownie for We thank Mrs‘ with preparing the tissue sections and Mr. J . Denton for assistance with the photographs. This study was funded by grants from the Medical Research Council, the National Association for the Relief Of Paget’s Disease, and the Salford Paget’s Disease Appeal. This work was presented as an abstract (number 434, The distribution of canine distemper virus in bone cells of distemper infected dogs) at the ASBMR annual meeting, 1991.
REFERENCES I . Barker DJP, Clough PWL, Guyer PB, Gardner MJ 1977 Paget’s disease of bone in 14 British towns. Br Med J I:11811183. 2. Barker DJP, Chamberlain AT, Guyer PB, Gardner MJ 1980 Paget’s disease of bone: The Lancashire focus. Br Med J 280: 1105-1 107. 3. Rebel A, Malkani K, Basle M 1974 Anomalies nucleaires des osteoclasts de la maladie osseuse de Paget. Nouv Presse Med 3: 1299-1301. 4. Mills BG, Singer FR 1976 Nuclear inclusions in Paget’s disease of bone. Science 194:201-202. 5 . Gheradi G, Lo Cascio V , Bonucci E 1980 Fine structure of nuclei and cytoplasm of osteoclasts in Paget’s disease of bone. Histopathology 4:63-74.
6. Howatson AF, Fornasier VL 1982 Microfilaments associated with Paget’s disease of bone. Comparison with nucleocapsids of measles virus and respiratory syncytial virus. Intervirology 18:150-159. 7. Rebel A, Basle M, Pouplard A, Kouyomdjian S, Filmon R, Lepatezour A 1980 Viral antigens in osteoclasts from Paget’s disease of bone. Lancet 2:344-346. 8. Mills BG, Singer FR, Weiner LP, Holst PA 1981 Immunohistochemical demonstration of respiratory syncytial virus antigen in Paget’s disease of bone. Proc Natl Acad Sci USA 78: 1209- I21 3. 9. Singer FR, Mills B 1983 Evidence for a viral aetiology of Paget’s disease of bone. Clin Orthop Re1 Res 178:245-25 I . 10. Basle MF, Russell WC, Goswami KKA, Rebel A , Girauden P, Wild F, Filmon R 1985 Paramyxovirus antigens in osteoclasts from Paget’s bone tissue detected by monoclonal antibodies. J Gen Virol 66:2103-2110. 1 1 . Pringle CR, Wilkie ML. Elliot RM 1985 A survey of respiratory syncytial virus and parainfluenza virus type 3 neutralising and immunoprecipitating antibodies in relation to Paget’s disease. J Med Virol 17:377-386. 12. Bade MF, Fournier JG, Rozenblatt S, Rebel A, Bouteille M 1986 Measles virus RNA detected in Paget’s disease bone tissue by in situ hybridisation. J Gen Virol 67:907-913. 13. Barrett T , Subbarao SM, Belsham GJ. Mahy BWJ 1991 The molecular biology of the morbilliviruses. In: Kingsbury DW (ed) The Paramyxoviruses. Plenum Press, London, pp. 83102. 14. ODriscoll JB, Anderson DC 1985 Past pets and Paget’s disease. Lancet 2:919-921. 15. O’Driscoll JB, Buckler HM, Jeacock J , Anderson DC 1990 Dogs, distemper and osteitis deformans. Bone Miner 11: 209-2 16. 16. Gordon MT, Anderson DC, Sharpe P T 1991 Canine distemper virus localised in bone cells of patients with Paget’s disease. Bone 12195-201. 17. Appel M J 1987 Canine distemper virus. In: Appel MJ (ed) Virus Infections of Vertebrates. Elsevier, Amsterdam, pp. 133-159. 18. Boyce RW, Axthelm MK, Krakowka S, Weisbrode SE 1983 Metaphyseal bone lesions associated with canine distemper virus infection. Abstract, Proceedings of the Annual Meeting of the American College of Veterinary Pathologists, San Antonio, T X p. 126. 19. Rentrop M, Knapp B, Winter H, Schweizer J 1986 Amino-
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20.
21.
22.
23.
alkylsilane-treated slides as support for in situ hybridisation of keratin cDNAs to frozen sections under varying fixation and pretreatment conditions. Histochem J 18:271-276. Rhinelander FW, Wilson J W 1982 Blood supply to developing, mature and healing bone. In: Sumner-Smith G (ed) Bone in Clinical Orthopaedics. W.B. Saunders, Philadelphia, pp. 81-96. Axthelm MK. Krakowka S 1986 Immunocytochemical methods for demonstrating canine distemper virus antigen in aldehyde-fixed paraffin-embedded tissue. J Virol Methods 13: 21 5-229. GrBndalen J 1976 Metaphyseal osteopathy (hypertrophic osteodystrophy) in growing dogs. A clinical study. J Small Anim Pract 17:721-735. Bohning RH, Suter PF, Hohn BR, Marshall J 1970 Clinical and radiologic survey of canine panosteitis. J Am Vet Med ASSOC156~870-884.
24. Riser WF, Parkes LJ, Shirer J F 1967 Canine craniomandibular osteopathy. J Am Vet Radio1 SOC8:23-30.
Address reprint requests to: Dr. P.T. Sharpe Department of Cell and Structural Biology Stopford Building University of Manchester Oxford Road Manchester, MI3 9PT
Received for publication October 15, 1991; in revised form January 9, 1992; accepted February I , 1992.
Detection of Canine Distemper Virus in Bone Cells in the Metaphyses of Distemper-Infected Dogs ANDREW P. MEE,' DAVID M. WEBBER,' CHRISTOPHER MAY,' DAVID BENNETT,' PAUL T. SHARPE.' and DAVID C. ANDERSON3
ABSTRACT In the light of recent evidence implicating canine distemper virus (CDV) as a possible etiologic agent in Paget's disease of bone, we thought that it would be of interest to examine distemper-infected bone in the natural host. Samples from the long bones, spleen, and bladder of four distemper-infected and three uninfected dogs were examined for the presence of CDV nucleocapsid and phosphoprotein genes and the measles virus (MV) nucleocapsid gene using the technique of in situ hybridization with radioactively labeled riboprobes. Two of the four distemper-infected dogs showed strongly positive hybridization with both of the CDV probes. The signal was present in marrow cells, in osteoblasts, in osteocytes, and particularly in osteoclasts. No hybridization was seen over the cartilage cells of the growth plate, and there was a clear line of demarcation at the point of invasion of osteoclasts and vascularization. The spleen and bladder samples from infected dogs also showed positive hybridization. There was no hybridization with the MV probe in any of the distemper-infected tissue. Samples from the uninfected dogs showed no evidence of hybridization with either the CDV or MV probes. These results show that CDV can infect bone cells of the natural host and provide further support for the theory that CDV may play a role in human Paget's disease of bone.
INTRODUCTION is a chronic focal bone disorder characterized by excessive osteoclastic resorption and secondary new bone formation, which affects approximately 4% of the U.K. population over 5 5 years of age.(1,2)The natural history of the disease and evidence obtained from electron microscopic,(3-6)immunocytochemical,(7-") and molecular studies(121have led to the suggestion that it might be the result of a slow virus infection by a member of the paramyxovirus family, in which measles and respiratory syncytial virus have been most strongly implicated. However, our group has previously suggested that canine distemper virus (CDV), a morbillivirus closely related to measles,('3) might be involved in the etiology of this disease. This was based on epidemiologic evidence of increased ownership of dogs among Paget's patients compared with control^^^^^'^) and on the demonstration of
P
AGET'S DISEASE
CDV mRNA in bone biopsies of these patients by in situ hybridization. (I6) In the present study, an attempt has been made to discover if CDV is present in the bone cells of distemper-infected dogs, so that more may be learnt about the predilection of this virus for bone cells in the natural host. For this purpose, we used the technique of in situ hybridization and probes to the mRNA and genomic RNA of the nucleocapsid and phosphoprotein genes of CDV. This virus usually gains entry via the respiratory tract and then spreads to bone marrow and all lymphatic tissues of infected dogs. In dogs that fail to mount a successful immune response, the virus then spreads to the respiratory, gastrointestinal, and urogenital tract epithelia and also frequently to the central nervous systern.(l7)Dogs that are acutely infected shed virus in all secretions, whether they show clinical signs or not. There are no reports of clinical problems associated with CDV infection of bone cells, although Boyce et
IBone Disease Research Centre, Department of Cell and Structural Biology, University of Manchester, England. 'Department of Veterinary Clinical Science, Small Animal Hospital, University of Liverpool, England. 'Chinese University of Hong Kong, Prince of Wales Hospital, Department of Medicine, Shatin, New Territories, Hong Kong.
829
830
MEE ET AL.
al.,(18)in a preliminary study, reported metaphyseal lesions in both naturally and experimentally infected dogs. Proteins of CDV were demonstrated within the bone cells, particularly the osteoclasts, by indirect immunofluorescence. Clearly a more complete understanding of the distribution of this virus in bone tissue could be of importance in establishing a possible effect on bone metabolism in both the dog and in human Paget's disease. It might also be of veterinary importance in view of the fact that virtually all dogs are exposed to distemper virus, either the wild type or by vaccination with live attenuated virus, and so CDV might be responsible for one or more of the hitherto unexplained canine bone disorders.
graphic emulsion. The slides were then exposed for 7-10 days, developed and counterstained with hematoxylin and eosin, and viewed by dark- and light-field microscopy.
Controls Negative control slides consisted of hybridization with a probe transcribed from a random SP6 vector sequence, a probe to the measles virus nucleocapsid gene, the addition of hybridization solution alone, and the pretreatment of sections with RNAse. Positive control slides utilized an antisense probe to P-actin.
RESULTS MATERIALS AND METHODS
Of the four distemper-infected dogs, two (the 4- and the 9-week-old dogs) showed positive hybridization with both Tissue samples and preparation the probes to the CDV nucleocapsid and phosphoprotein Metaphyseal tissue was obtained from the long bones of genes, but not with the measles probe. When present, hyfour distemper-infected dogs and three uninfected animals bridization was found in all bone cell types, including (the distemper-infected dogs were all crossbreeds aged 4 osteoblasts and osteoclasts, but not in adjacent chondroand 9 weeks and two dogs aged 5 months; the uninfected cytes. The signal was concentrated in the growth plate redogs were an 8-week-old German Shepherd, a 5-month-old gion of the long bones (Fig. l a and b) and appeared particcrossbreed, and a 5-month-old Shar Pei), fixed in 10% ularly prominent in the multinucleated osteoclasts (Fig. Ic buffered formalin for at least 24 h, decalcified in 10% and d). However, not all the osteoclasts in any one region EDTA, and processed for routine paraffin-wax-embedded were labeled to the same extent, if at all. In every case, histology. In addition, spleen and bladder samples were both the sense and antisense CDV probes were found to obtained from both infected and uninfected animals and hybridize to a similar degree. No hybridization was seen processed in the same manner without the decalcification with the measles probe (Fig. l e and f). Tissue samples step. Sections (7 pm) were cut and mounted on organosila- from uninfected dogs showed no hybridization with any of the viral probes, although accessibility to hybridization nated slides"91 for in situ hybridization. was confirmed in all tissues by a positive reaction with a probe to p-actin. Preparation of riboprobes Bladder and spleen samples from two of the infected 35S-labeled riboprobes were generated by transcription dogs were also examined and were found to be strongly of sequences to the CDV nucleocapsid and phosphopro- positive with the CDV, but not measles, probes, confirmtein genes and the measles virus nucleocapsid gene, which ing the specificity of the reaction (Figs. 2 and 3). A partichad been subcloned into the polylinker of the vector ularly distinct pattern of hybridization was observed in the pSP72. (I6) Both sense and antisense probes were produced bladder, in which discrete epithelial cells were strongly laby using the appropriate SP6 or T7 polymerase. Probes beled (Fig. 2a and b). The addition of hybridization soluwere routinely reduced to 250 base pairs (bp) in length by tion alone or the pretreatment of sections with RNase relimited alkaline hydrolysis. sulted in only background levels of silver grains in the sections.
In situ hybridization The techniques used are those described in detail by Gordon et In brief, after bringing the sections to water, they were permeabilized by treatment with 0.2 M HCI, followed by 10 pg/ml of proteinase K at 37°C for 30 minutes. Nonspecific background was reduced by treatment with 0.25% acetic anhydride. Control slides were rendered free of RNA by treatment with 100 pg/ml of RNase for 30 minutes at 37°C. Hybridization was performed in 50% formamide and 0.3 M NaCl at 50°C overnight in a sealed, humidified container. Approximately 1.O x lo5counts of radiolabeled riboprobe was added to each tissue section. After hybridization, the slides were washed at 50°C, posttreated with RNase to remove unbound probe, and dipped in 1:l dilution of Ilford K5 autoradio-
DISCUSSION In previous studies, we have shown by in situ hybridization using the same probes as those used in this study that canine distemper virus is present in the bone cells of approximately 50% of the pagetic bone biopsies tested thus far, suggesting that the virus may be involved in the etiology of this bone disorder.('61In the present work, the same technique was used to test for the presence of CDV RNA in canine bone cells, and tissue was examined from four distemper-infected dogs and three uninfected controls. The presence of CDV in bone cells was confirmed in two of the four distemper-infected bone samples but not in the controls. The virus was concentrated in the growth plate re-
CANINE DISTEMPER VIRUS IN BONE CELLS
831
FIG. 1. Distemper-infected dog bone. Positive hybridization with the CDV-N sense probe. Growth plate (GP) region viewed by (a) light-field and (b) dark-field microscopy. Arrows indicate hybridization ( x 50). Strong positive hybridization with the CDV-N sense probe in osteoblasts (OB) and particularly in osteoclasts (OC) viewed by (c) light-field and (d) dark-field microscopy ( x 160). Similar results were seen with the CDV-N antisense probe and the CDV-P sense and antisense probes (not shown). No hybridization (background level of silver grains) with MV-N sense probe viewed by (e) light-field or (f) dark-field microscopy ( x 125).
832
MEE ET AL.
FIG. 2. Distemper-infected dog bladder. Positive hybridization with the CDV-N sense probe viewed by (a) light-field and (b) dark-field microscopy. Note the distinct epithelial areas of hybridization (arrows). No hybridization (background level of silver grains) with the MV-N sense probe viewed by (c) light-field or (d) dark-field microscopy ( x 125).
gion of the long bones and was particularly prominent in the multinucleated osteoclasts. The dramatic accumulation of this virus in osteoclasts of the natural host is consistent with the hypothesis that Paget’s disease is associated with CDV infection of bone-resorbing cells. In contrast to our previous findings in human pagetic bone, in which only the antisense probes (hybridizing with mRNA) were positive, both the sense and antisense probes were found to bind strongly to the dog bone sections. Since CDV is a singlestranded RNA virus, this suggests that large amounts of viral genome are present within this tissue and may represent the difference between a chronic (Paget’s) and acute (distemper) disease, or disease in the “natural” versus the “unnatural” host. CDV sequences were detected in the bladder and spleen samples from all four dogs, so the dogs were clearly infected. The inability to detect the virus in the bone samples from two of the dogs with active distemper may indicate variability between different types of dogs in susceptibility of bone cells (although all affected dogs were crossbreeds) or, more likely, be due to differences in stage and severity of the disease. That is, the virus
had not reached the bone cells at the time of death, or the bone cells were only transiently infected. Although only four distemper-infected dogs were examined in this study, it is interesting that both of the dogs that showed positive hybridization in bone cells were only several weeks old (compared with several months old), and this may reflect the inability of the virus to infect bone cells in older dogs or may be due to the changes in the vascular supply to the metaphyses that occur as dogs mature.(20’ The distinct pattern of hybridization seen in the bladder epithelium was comparable with previous immunocytochemical studies“’) and confirms the specificity of the probes, which we previously showed on measles-infected human brain (subacute sclerosing panencephalitis) and on distemper-infected dog brain. Canine distemper is a disease of the respiratory, gastrointestinal, and nervous systems, and to our knowledge there are no clinical reports of any bone involvement in this condition, although metaphyseal lesions have been described in both naturally and experimentally infected dogs.‘18) The same group has provided further immuno-
833
CANINE DISTEMPER VIRUS IN BONE CELLS
C
FIG. 3. Distemper-infected dog spleen. Positive hybridization with the CDV-N sense probe viewed by (a) light-field and (b) dark-field microscopy. No hybridization (background level of silver grains) with the MV-N sense probe, viewed by (c) dark-field microscopy ( x 125). cytochemical evidence of clear staining of osteoclasts in distemper-infected tissue, although no mention was made of its significance.( I 1 ) Certainly, there is no canine equivalent of human Paget’s disease; this might be related to a number of factors, especially the shorter life span. However, several idiopathic canine bone diseases exist that are characterized by excessive bone remodeling, for example, the conditions of metaphyseal osteopathy,‘”) panosteiti^,"^) and craniomandibular osteopathy. Hence, the possibility of wild-type or vaccine-strain CDV causing these types of disease requires further investigation. (’‘)
ACKNOWLEDGMENTS
‘’
and Miss J ’ Brownie for We thank Mrs‘ with preparing the tissue sections and Mr. J . Denton for assistance with the photographs. This study was funded by grants from the Medical Research Council, the National Association for the Relief Of Paget’s Disease, and the Salford Paget’s Disease Appeal. This work was presented as an abstract (number 434, The distribution of canine distemper virus in bone cells of distemper infected dogs) at the ASBMR annual meeting, 1991.
REFERENCES I . Barker DJP, Clough PWL, Guyer PB, Gardner MJ 1977 Paget’s disease of bone in 14 British towns. Br Med J I:11811183. 2. Barker DJP, Chamberlain AT, Guyer PB, Gardner MJ 1980 Paget’s disease of bone: The Lancashire focus. Br Med J 280: 1105-1 107. 3. Rebel A, Malkani K, Basle M 1974 Anomalies nucleaires des osteoclasts de la maladie osseuse de Paget. Nouv Presse Med 3: 1299-1301. 4. Mills BG, Singer FR 1976 Nuclear inclusions in Paget’s disease of bone. Science 194:201-202. 5 . Gheradi G, Lo Cascio V , Bonucci E 1980 Fine structure of nuclei and cytoplasm of osteoclasts in Paget’s disease of bone. Histopathology 4:63-74.
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Address reprint requests to: Dr. P.T. Sharpe Department of Cell and Structural Biology Stopford Building University of Manchester Oxford Road Manchester, MI3 9PT
Received for publication October 15, 1991; in revised form January 9, 1992; accepted February I , 1992.
