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Monoclonal Antibody-based Immunohistochemical Diagnosis of Malaysian Nipah Virus Infection in Pigs
J. Comp. Path. 2004, Vol. 131, 199–206
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Monoclonal Antibody-based Immunohistochemical Diagnosis of Malaysian Nipah Virus Infection in Pigs N. Tanimura, T. Imada, Y. Kashiwazaki, S. Shahirudin*, S.H. Sharifah* and A.J. Aziz* National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki, 305-0856 Japan and *Department of Veterinary Services, Veterinary Research Institute, 59 Jalan Sultan Azlan Shah, 31400 Ipoh, Perak Darul Ridzuan, Malaysia
Summary Formalin-fixed, paraffin wax-embedded tissues of three Malaysian farm pigs naturally infected with Nipah virus were used to investigate the value of anti-Nipah virus mouse monoclonal antibodies (Mabs) and rabbit polyclonal antibody for immunohistochemical diagnosis. Mabs 11F6 and 12A5 gave intense immunolabelling in lung tissue that had been fixed in 10% neutral buffered formalin for about 4 years, whereas the reactivity of Mabs 13A5 and 18C4 and polyclonal antibody was reduced significantly by long-term formalin fixation. Immunohistochemical examination of Malaysian farm pig samples with Mab 11F6 confirmed the affinity of Nipah virus for respiratory epithelium, renal glomerular and tubular epithelium, meningeal arachnoidal cells, and systemic vascular endothelium and smooth muscle. In addition, Nipah virus antigens were identified in laryngeal epithelial cells, Schwann cells of peripheral nerve fascicles in the spleen, and endothelial cells in the atrioventricular valve. The study demonstrated the value of Mabs 11F6 and 12A5 for the immunohistochemical diagnosis of Nipah virus infection in pigs. q 2004 Elsevier Ltd. All rights reserved. Keywords: Nipah virus; pig; Viral infection
Introduction Nipah virus, a zoonotic paramyxovirus, emerged in Malaysia in 1998/1999, causing fatal encephalitis in man (Goh et al., 2000) and neurological and respiratory syndromes in pigs (Nor et al., 2000). Nipah virus, together with Hendra virus, may represent a new evolutionary lineage within the subfamily Paramyxovirinae, for which the genus Henipavirus has been proposed (Wang et al., 2001). Immunohistochemical examination, which has proved to be one of the most useful methods of Nipah virus detection, can be performed on formalin-fixed tissues; it is therefore safe and has enabled retrospective investigations to be made on archival material (Daniels et al., 2001). The distribution of Nipah virus antigens in natural 0021–9975/$ - see front matter doi: 10.1016/j.jcpa.2004.03.006
and experimental infections in pigs has been investigated with rabbit polyclonal antibodies against Hendra virus and Nipah virus (Shahirudin et al., 1999; Hooper et al., 2001; Middleton et al., 2002). In the present study, both mouse monoclonal and rabbit polyclonal antibodies against Nipah virus (Imada et al., 2004) were examined for their ability to detect viral antigens in porcine field samples. The objectives were (1) to examine the effect of long-term formalin fixation on the immunohistochemical detection of Nipah virus antigens, (2) to investigate the distribution of Nipah virus antigen in field cases, and (3) to confirm the usefulness of monoclonal antibodies (Mabs) for immunohistochemical diagnosis. q 2004 Elsevier Ltd. All rights reserved.
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Materials and Methods Animals Formalin-fixed tissues of three pigs (nos 1 to 3), from the Malaysian outbreak of Nipah virus infection in 1999 (Shahirudin et al., 1999), were used in this study. Animal 1 was a piglet, and nos 2 and 3 were young pigs, but the exact ages and sex were not recorded. The pigs, which were from serologically positive farms, were killed and necropsy was performed immediately post mortem. The tissue samples examined, which had been immersed in 10% neutral buffered formalin for about 4 years, included lung, larynx, kidney, spleen, brain, heart, gastrointestinal tract and liver. After trimming, they were rinsed with running water, dehydrated through graded alcohols and xylene, and then embedded in paraffin wax by standard procedures. As a positive control, formalin-fixed, paraffin wax-embedded lung tissue from a pig (no. 4) experimentally infected with Nipah virus was provided by Dr John White (Australian Animal Health Laboratory, CSIRO, Australia). The lung tissue was collected 7 days after infection of a 6-week-old pig by the subcutaneous route (Middleton et al., 2002). Antibodies Four clones of Nipah virus Mabs (11F6, 12A5, 13A5 and 18C4) and rabbit polyclonal antibody (Imada et al., 2004) were used as primary antibodies to test the effects of long-term formalin fixation on immunohistochemical labelling. Mab 11F6 was used to examine the distribution of viral antigens in the naturally infected pigs. All Mabs were prepared from mice immunized with formalin-inactivated Nipah virus. Previous tests on virus-infected culture cells by the indirect fluorescent antibody method had shown that 11F6 and 13A5 did not cross-react with Hendra virus antigens (Imada et al., 2004). Immunohistochemistry Sections (4 mm) were mounted on silane-coated glass slides and then dewaxed in xylene and alcohol. Endogenous peroxidase activity was blocked by treating the sections with H2O2 3% in phosphate-buffered saline (PBS) for 15 min. For antigen retrieval, sections were digested with actinase E (Kaken Pharmaceutical Co., Tokyo, Japan) 0.1% in PBS at 37 8C for 20 or 180 min, or heated by microwave irradiation at maximum output (approximately 500 W) in citrate buffer (pH 6.0)
(Dako ChemMatee; DakoCytomation, Glostrup, Denmark) over four 5-min cycles. Selected sections were also heated in citrate buffer (pH 6.0) by autoclaving at 121 8C for 10 min. Primary antibodies were applied for 30 min at room temperature. The optimal dilution of the primary antibody and the optimal antigen retrieval method were previously determined by titration experiments. This step was followed by sequential application of amino-acid polymer conjugated with anti-mouse or anti-rabbit IgG antibody and peroxidase (Histofine Simple Stain; Nichirei Co., Tokyo, Japan), and diaminobenzidine chromogen. Sections were then counterstained with haematoxylin, dehydrated with alcohol and xylene, and mounted under coverslips.
Results Effect of Long-term Formalin Fixation on Immunohistochemical Examination for Nipah Virus Lung tissue from the experimentally infected positive-control pig (no. 4) showed intense immunolabelling of virus-infected cells of the bronchiolar and alveolar epithelium with all four Mabs (11F6, 12A5, 13A5 and 18C4) and with the polyclonal antibody (Table 1). When the lung tissue from naturally infected pigs (nos 1– 3) was used, Mabs 11F6 and 12A5 gave intense positive reactions. However, Mabs 13A5 and 18C4 gave no positive reactions in lung tissue from these three naturally infected pigs, despite the use of the same antigen retrieval methods (microwave; or actinase E digestion for 20 min) as those applied to the samples from pig 4. Moreover, the polyclonal antibody gave weaker immunolabelling in sections from pigs 1 – 3 than in those from pig 4 (Table 1). Antigen retrieval by autoclaving enabled Mab 13A5 to produce minimal immunolabelling in lung sections from pigs 2 and 3; and retrieval with actinase E for 180 min instead of 20 min resulted in (1) weak immunolabelling with 18C4 in pig 2, (2) weak immunolabelling with polyclonal antibody in pigs 2 and 3, and (3) intense immunolabelling with polyclonal antibody in pig 1 (Table 1). Lesions and Distribution of Nipah Virus Antigens in Naturally Infected Pigs Pig 1. Macroscopically, cross-section of the lung showed lobular consolidation, bronchial obstruction and congestion (Fig. 1). The kidneys showed severe, diffuse petechial haemorrhages over the surface and in the capsule. In cross-section
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Diagnosis of Nipah Virus Infection in Pigs Table 1 Immunohistochemical reactivity of anti-Nipah virus antibodies with formalin-fixed, paraffin wax-embedded lung tissues from pigs with natural or experimental Nipah virus infection Natural infection Antibody
Antigen retrieval method
Pig 1
Pig 2
Pig 3
Experimental infection Pig 4 (positive control)
11F6* 12A5* 13A5* 18C4* PcAb† 13A5p 18C4* PcAb†
Microwave No treatment Microwave Actinase E (20 min) Actinase E (20 min) Autoclave Actinase E (180 min) Actinase E (180 min)
þþ þþ 2 2 þ 2 2 þþ
þþ þþ 2 2 þ ^ þ þ
þþ þþ 2 2 þ ^ 2 þ
þþ þþ þþ þþ þþ þþ ^‡ ^‡
þþ, Intense (dark brown) immunolabelling; þ, weak (light brown) immunolabelling; ^ , minimal immunolabelling; 2 , no immunolabelling. *Monoclonal antibody. †Polyclonal antibody. ‡Part of the tissue section was detached from the slide by excessive digestion.
of the kidney, petechial haemorrhages were seen to be localized in the cortex (Fig. 2). No gross lesion was recognized in other organs or tissues. Nipah virus antigens were distributed systemically (Table 2). In the lung, viral antigens were detected most frequently in the bronchiolar and alveolar epithelial cells (Fig. 3), but they were also observed in bronchial epithelial cells, exudative macrophages, endothelial and smooth muscle cells of small arteries and veins (Fig. 4), and endothelial cells of lymphatic vessels. Viral antigen-positive syncytia were observed in respiratory epithelial cells, endothelial and smooth muscle cells of blood vessels, and endothelial cells of lymphatics. In general, antigen-positive cells were concentrated mainly in the bronchioles and their neighbouring alveoli (Fig. 3), and less frequently in the blood vessels and their neighbouring alveoli (Fig. 4). In the laryngeal mucosa, viral antigens were localized in the mucosal epithelial cells and in
Fig. 1. Lung (fixed with formalin), Pig 1. Lobular consolidation, bronchial obstruction and congestion in the pulmonary lobule. Bar, 1 cm.
endothelial cells of blood vessels in the lamina propria mucosa (Fig. 5). In the cortex of the kidney, antigens were present in the epithelial cells of Bowman’s capsule and renal tubules, and in the endothelial cells of small veins in the interstitial connective tissues (Fig. 6). Focal lesions containing these antigenpositive cells were accompanied by haemorrhagia per rhexin. In the renal capsule, viral antigens were detected in the endothelial cells of venules. In the medulla, they occurred in the endothelial and smooth muscle cells of the small arteries, and the endothelial cells of the small veins and the lymphatics. In the lumen of some lymphatics, antigens were also present in the macrophages. In the spleen, viral antigens were present in the endothelial and smooth muscle cells of small arteries, the endothelial cells of the splenic sinus, and the Schwann cells of the peripheral nerve fascicles (Fig. 7). In the cerebrum, antigens were detected in the arachnoid cells in the meninges, their presence
Fig. 2. Kidney (fixed with formalin), Pig 1. Petechial haemorrhages in the renal cortex. Bar, 1 cm.
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N. Tanimura et al. Table 2 Distribution of Nipah virus antigen-positive cells in naturally infected pigs
Pig no.
Immunolabelling in lung
1 2 3
þþ þ þþ þ þþ
larynx
kidney
spleen
cerebrum
heart
stomach
colon
liver
þ NE NE
þþ 2 2
þ 2 NE
þ 2 2
þ NE NE
þ NE NE
þ NE NE
2 2 2
Number of Nipah virus antigen-positive cells: þ, small; þþ , moderate; þþþ, large. NE, not examined.
Fig. 3. Lung, Pig 1. Intranuclear and intracytoplasmic Nipah virus antigen in many syncytial bronchiolar epithelial cells and a few alveolar epithelial cells. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
Fig. 4. Lung, Pig 1. Nipah virus antigen in endothelial and smooth muscle cells of a small vein and in the surrounding alveolar epithelial cells. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
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Fig. 5. Larynx, Pig 1. Nipah virus antigen in syncytial epithelial cells, endothelial cells of a small vein, and interstitial mononuclear cells. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
being accompanied by nonsuppurative meningitis (Fig. 8), consisting of lymphoid cell infiltration, oedema, and fibrin precipitation. In the heart, viral antigens occurred in syncytial endothelial cells of the left atrioventricular valve and in the smooth muscle cells of the coronary arteries. In the fundic gland region of the stomach, viral antigens were detected in the endothelial and smooth muscle cells of the small arteries in the submucosa and tunica muscularis, and in the macrophages surrounding the small arteries in the tela subserosa. In the colon, viral antigens were present in the endothelial cells of the small veins in
the submucosa and tunica muscularis, and in the endothelial and smooth muscle cells of the small arteries in the submucosa, tunica muscularis and tela subserosa (Fig. 9). These antigen-positive arteries showed necrotizing arteritis. In the liver, no antigen-positive cells were detected. Pigs 2 and 3. Macroscopically, cross-section of the lungs showed lobular consolidation and bronchial obstruction. No gross lesion was recognized in other tissue sections. In the lungs of pigs 2 and 3, the distribution of Nipah virus antigens was almost the same as that in
Fig. 6. Kidney, Pig 1. Nipah virus antigen in syncytial epithelial cells of renal tubules and in the endothelial cells of interstitial veins. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
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Fig. 7. Spleen, Pig 1. Nipah virus antigen in Schwann cells in a peripheral nerve fascicle. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
pig 1. In addition, in pig 2, antigen-positive alveolar epithelial cells and macrophages were observed at the periphery of necrotic lesions, where there was a severe infiltration of neutrophils, with haemorrhage and fibrin deposition (Fig. 10). Viral antigen was not detected in the other tissues examined (Table 2).
Discussion This immunohistochemical study demonstrated that Mabs 11F6 and 12A5 could detect Nipah
virus antigens in formalin-fixed, paraffin waxembedded tissues from pigs with natural infections, irrespective of formalin fixation for up to 4 years. In contrast, the reactivity of Mabs 13A5 and 18C4 and of polyclonal antibody was severely reduced by long-term formalin fixation. Previous Western blotting analysis suggested that 11F6 and 12A5 recognized the P and F proteins of Nipah virus, respectively, and that 13A5 and 18C4 reacted with the N protein of Nipah virus (Imada et al., 2004). However, the relationship between the viral protein recognized by each monoclonal
Fig. 8. Cerebrum, Pig 1. Nipah virus antigen in syncytial arachnoid cells of the cerebral meninges. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
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Fig. 9. Colon, Pig 1. Nipah virus antigen in syncytial endothelial cells of small veins, smooth muscle cells of small arteries, and periarterial interstitial cells in the submucosa. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
antibody and the effects of formalin fixation on immunolabelling were unknown. Leong and Leong (2002) demonstrated that tissues fixed in formaldehyde displayed a distinct and progressive loss of immunohistochemical reactivity for many antigens, frequently in proportion to the duration of exposure to the fixative. Formalin fixation causes a chemical modification in protein conformation by crosslinking, which may mask the tissue antigenicity (Shi et al., 1997). The results in Table 1 suggest that 13A5 and 18C4 recognize epitopes within the protein molecules that are modified
irreversibly by long-term formalin fixation. In contrast, the antigens recognized by 11F6 and 12A5 may have been modified in a reversible manner by such fixation. Immunohistochemical studies of natural and experimental Nipah virus infections in pigs, based on the use of rabbit polyclonal antibody, were described by Hooper et al. (2001) and Middleton et al. (2002). The results of Mab-based immunolabelling in the present study were consistent with previous findings, confirming that Nipah virus has an affinity for respiratory epithelium, renal
Fig. 10. Lung, Pig 2. Nipah virus antigen in alveolar epithelial cells and macrophages surrounding necrotic lesions in lobule. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 100 mm.
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glomerular and tubular epithelium, meningeal arachnoidal cells, and systemic vascular endothelium and smooth muscle. In the present study, novel sites of immunolabelling for Nipah virus antigen were identified in the following tissues: laryngeal epithelial cells; Schwann cells of peripheral nerve fascicles in the spleen; and endothelial cells in the atrioventricular valve. All the antigen-positive tissues showed syncytium formation. Studies of natural infection of pigs in Malaysia (Shahirudin et al., 1999; Nor et al., 2000) showed that pigs aged 1– 6 months usually had an acute febrile illness with respiratory signs ranging from rapid and laboured respiration to harsh non-productive coughing, and, especially in older pigs, neurological signs such as trembling, muscle spasms, and rear leg weakness. Similar clinical signs were also seen in experimentally induced disease (Middleton et al., 2002). Coughing might be expected to be an efficient generator of infectious droplets. The presence of Nipah virus antigen in the laryngeal epithelium suggests that coughing gives rise to infectious droplets originating from laryngeal secretions as well as from the tonsil and lower respiratory tract (Middleton et al., 2002). The significance of peripheral nerve fascicles for the spread of Nipah virus remains uncertain. Further immunohistological studies are needed to clarify the pathogenesis of Nipah virus infection in pigs of different age groups.
Acknowledgments This study was supported by a joint study project of the Japan International Cooperation Agency and the Veterinary Research Institute, Malaysian Ministry of Agriculture, on the epidemiology, pathogenesis and molecular characterization of the Nipah virus.
Goh, K. J., Tan, C. T., Chew, N. K., Tan, P. S. K., Kamarulzaman, A., Sarji, S. A., Wong, K. T., Abdullah, B. J. J., Chua, K. B. and Lam, S. K. (2000). Clinical features of Nipah virus encephalitis among pig farmers in Malaysia. New England Journal of Medicine, 342, 1229 –1235. Hooper, P., Zaki, S., Daniels, P. and Middleton, D. (2001). Comparative pathology of the diseases caused by Hendra and Nipah viruses. Microbes and Infection, 3, 315–322. Imada, T., Mohd, A. A. R., Kashiwazaki, Y., Tanimura, N., Sharifah, S. H. and Aziz, A. J. (2004). Production and characterization of monoclonal antibodies against formalin-inactivated Nipah virus isolated from the lung of a pig. Journal of Veterinary Medical Science, 66, 81–83. Leong, A. S.-Y. and Leong, F. J. W.-M. (2002). Microwavestimulated antigen retrieval - an update. Acta Histochemica et Cytochemica, 35, 367 –374. Middleton, D. J., Westbury, H. A., Morrissy, C. J., van der Heide, B. M., Russell, G. M., Braun, M. A. and Hyatt, A. D. (2002). Experimental Nipah virus infection in pigs and cats. Journal of Comparative Pathology, 126, 124–136. Nor, M. N. M., Gan, C. H. and Ong, B. L. (2000). Nipah virus infection of pigs in peninsular Malaysia. Revue Scientifique et Technique (International Office of Epizootics), 19, 160 –165. Shahirudin, S., Zamri-Saad, M., Shamshad, S., Mahani, H., Harizam, Y., Norazian, B., Daniels, P. and Aziz, A. J. (1999). A novel clinicopathological disorder associated with a zoonotic paramyxovirus infection in pigs. In: Proceedings of the National Congress on Animal Health and Production, Alor Gajah, Melaka, Malaysia, pp. 367–368. Shi, S.-R., Cote, R. J. and Taylor, C. R. (1997). Antigen retrieval immunohistochemistry: past, present, and future. Journal of Histochemistry and Cytochemistry, 45, 327–343. Wang, L.-F., Harcourt, B. H., Yu, M., Tamin, A., Rota, P. A., Bellini, W. J. and Eaton, B. T. (2001). Molecular biology of Hendra and Nipah viruses. Microbes and Infection, 3, 279–287.
References Daniels, P., Ksiazek, T. and Eaton, B. T. (2001). Laboratory diagnosis of Nipah and Hendra virus infections. Microbes and Infection, 3, 289–295.
Received; November 11th; 2003 Accepted; March 22nd; 2004
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Monoclonal Antibody-based Immunohistochemical Diagnosis of Malaysian Nipah Virus Infection in Pigs N. Tanimura, T. Imada, Y. Kashiwazaki, S. Shahirudin*, S.H. Sharifah* and A.J. Aziz* National Institute of Animal Health, 3-1-5 Kannondai, Tsukuba, Ibaraki, 305-0856 Japan and *Department of Veterinary Services, Veterinary Research Institute, 59 Jalan Sultan Azlan Shah, 31400 Ipoh, Perak Darul Ridzuan, Malaysia
Summary Formalin-fixed, paraffin wax-embedded tissues of three Malaysian farm pigs naturally infected with Nipah virus were used to investigate the value of anti-Nipah virus mouse monoclonal antibodies (Mabs) and rabbit polyclonal antibody for immunohistochemical diagnosis. Mabs 11F6 and 12A5 gave intense immunolabelling in lung tissue that had been fixed in 10% neutral buffered formalin for about 4 years, whereas the reactivity of Mabs 13A5 and 18C4 and polyclonal antibody was reduced significantly by long-term formalin fixation. Immunohistochemical examination of Malaysian farm pig samples with Mab 11F6 confirmed the affinity of Nipah virus for respiratory epithelium, renal glomerular and tubular epithelium, meningeal arachnoidal cells, and systemic vascular endothelium and smooth muscle. In addition, Nipah virus antigens were identified in laryngeal epithelial cells, Schwann cells of peripheral nerve fascicles in the spleen, and endothelial cells in the atrioventricular valve. The study demonstrated the value of Mabs 11F6 and 12A5 for the immunohistochemical diagnosis of Nipah virus infection in pigs. q 2004 Elsevier Ltd. All rights reserved. Keywords: Nipah virus; pig; Viral infection
Introduction Nipah virus, a zoonotic paramyxovirus, emerged in Malaysia in 1998/1999, causing fatal encephalitis in man (Goh et al., 2000) and neurological and respiratory syndromes in pigs (Nor et al., 2000). Nipah virus, together with Hendra virus, may represent a new evolutionary lineage within the subfamily Paramyxovirinae, for which the genus Henipavirus has been proposed (Wang et al., 2001). Immunohistochemical examination, which has proved to be one of the most useful methods of Nipah virus detection, can be performed on formalin-fixed tissues; it is therefore safe and has enabled retrospective investigations to be made on archival material (Daniels et al., 2001). The distribution of Nipah virus antigens in natural 0021–9975/$ - see front matter doi: 10.1016/j.jcpa.2004.03.006
and experimental infections in pigs has been investigated with rabbit polyclonal antibodies against Hendra virus and Nipah virus (Shahirudin et al., 1999; Hooper et al., 2001; Middleton et al., 2002). In the present study, both mouse monoclonal and rabbit polyclonal antibodies against Nipah virus (Imada et al., 2004) were examined for their ability to detect viral antigens in porcine field samples. The objectives were (1) to examine the effect of long-term formalin fixation on the immunohistochemical detection of Nipah virus antigens, (2) to investigate the distribution of Nipah virus antigen in field cases, and (3) to confirm the usefulness of monoclonal antibodies (Mabs) for immunohistochemical diagnosis. q 2004 Elsevier Ltd. All rights reserved.
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Materials and Methods Animals Formalin-fixed tissues of three pigs (nos 1 to 3), from the Malaysian outbreak of Nipah virus infection in 1999 (Shahirudin et al., 1999), were used in this study. Animal 1 was a piglet, and nos 2 and 3 were young pigs, but the exact ages and sex were not recorded. The pigs, which were from serologically positive farms, were killed and necropsy was performed immediately post mortem. The tissue samples examined, which had been immersed in 10% neutral buffered formalin for about 4 years, included lung, larynx, kidney, spleen, brain, heart, gastrointestinal tract and liver. After trimming, they were rinsed with running water, dehydrated through graded alcohols and xylene, and then embedded in paraffin wax by standard procedures. As a positive control, formalin-fixed, paraffin wax-embedded lung tissue from a pig (no. 4) experimentally infected with Nipah virus was provided by Dr John White (Australian Animal Health Laboratory, CSIRO, Australia). The lung tissue was collected 7 days after infection of a 6-week-old pig by the subcutaneous route (Middleton et al., 2002). Antibodies Four clones of Nipah virus Mabs (11F6, 12A5, 13A5 and 18C4) and rabbit polyclonal antibody (Imada et al., 2004) were used as primary antibodies to test the effects of long-term formalin fixation on immunohistochemical labelling. Mab 11F6 was used to examine the distribution of viral antigens in the naturally infected pigs. All Mabs were prepared from mice immunized with formalin-inactivated Nipah virus. Previous tests on virus-infected culture cells by the indirect fluorescent antibody method had shown that 11F6 and 13A5 did not cross-react with Hendra virus antigens (Imada et al., 2004). Immunohistochemistry Sections (4 mm) were mounted on silane-coated glass slides and then dewaxed in xylene and alcohol. Endogenous peroxidase activity was blocked by treating the sections with H2O2 3% in phosphate-buffered saline (PBS) for 15 min. For antigen retrieval, sections were digested with actinase E (Kaken Pharmaceutical Co., Tokyo, Japan) 0.1% in PBS at 37 8C for 20 or 180 min, or heated by microwave irradiation at maximum output (approximately 500 W) in citrate buffer (pH 6.0)
(Dako ChemMatee; DakoCytomation, Glostrup, Denmark) over four 5-min cycles. Selected sections were also heated in citrate buffer (pH 6.0) by autoclaving at 121 8C for 10 min. Primary antibodies were applied for 30 min at room temperature. The optimal dilution of the primary antibody and the optimal antigen retrieval method were previously determined by titration experiments. This step was followed by sequential application of amino-acid polymer conjugated with anti-mouse or anti-rabbit IgG antibody and peroxidase (Histofine Simple Stain; Nichirei Co., Tokyo, Japan), and diaminobenzidine chromogen. Sections were then counterstained with haematoxylin, dehydrated with alcohol and xylene, and mounted under coverslips.
Results Effect of Long-term Formalin Fixation on Immunohistochemical Examination for Nipah Virus Lung tissue from the experimentally infected positive-control pig (no. 4) showed intense immunolabelling of virus-infected cells of the bronchiolar and alveolar epithelium with all four Mabs (11F6, 12A5, 13A5 and 18C4) and with the polyclonal antibody (Table 1). When the lung tissue from naturally infected pigs (nos 1– 3) was used, Mabs 11F6 and 12A5 gave intense positive reactions. However, Mabs 13A5 and 18C4 gave no positive reactions in lung tissue from these three naturally infected pigs, despite the use of the same antigen retrieval methods (microwave; or actinase E digestion for 20 min) as those applied to the samples from pig 4. Moreover, the polyclonal antibody gave weaker immunolabelling in sections from pigs 1 – 3 than in those from pig 4 (Table 1). Antigen retrieval by autoclaving enabled Mab 13A5 to produce minimal immunolabelling in lung sections from pigs 2 and 3; and retrieval with actinase E for 180 min instead of 20 min resulted in (1) weak immunolabelling with 18C4 in pig 2, (2) weak immunolabelling with polyclonal antibody in pigs 2 and 3, and (3) intense immunolabelling with polyclonal antibody in pig 1 (Table 1). Lesions and Distribution of Nipah Virus Antigens in Naturally Infected Pigs Pig 1. Macroscopically, cross-section of the lung showed lobular consolidation, bronchial obstruction and congestion (Fig. 1). The kidneys showed severe, diffuse petechial haemorrhages over the surface and in the capsule. In cross-section
201
Diagnosis of Nipah Virus Infection in Pigs Table 1 Immunohistochemical reactivity of anti-Nipah virus antibodies with formalin-fixed, paraffin wax-embedded lung tissues from pigs with natural or experimental Nipah virus infection Natural infection Antibody
Antigen retrieval method
Pig 1
Pig 2
Pig 3
Experimental infection Pig 4 (positive control)
11F6* 12A5* 13A5* 18C4* PcAb† 13A5p 18C4* PcAb†
Microwave No treatment Microwave Actinase E (20 min) Actinase E (20 min) Autoclave Actinase E (180 min) Actinase E (180 min)
þþ þþ 2 2 þ 2 2 þþ
þþ þþ 2 2 þ ^ þ þ
þþ þþ 2 2 þ ^ 2 þ
þþ þþ þþ þþ þþ þþ ^‡ ^‡
þþ, Intense (dark brown) immunolabelling; þ, weak (light brown) immunolabelling; ^ , minimal immunolabelling; 2 , no immunolabelling. *Monoclonal antibody. †Polyclonal antibody. ‡Part of the tissue section was detached from the slide by excessive digestion.
of the kidney, petechial haemorrhages were seen to be localized in the cortex (Fig. 2). No gross lesion was recognized in other organs or tissues. Nipah virus antigens were distributed systemically (Table 2). In the lung, viral antigens were detected most frequently in the bronchiolar and alveolar epithelial cells (Fig. 3), but they were also observed in bronchial epithelial cells, exudative macrophages, endothelial and smooth muscle cells of small arteries and veins (Fig. 4), and endothelial cells of lymphatic vessels. Viral antigen-positive syncytia were observed in respiratory epithelial cells, endothelial and smooth muscle cells of blood vessels, and endothelial cells of lymphatics. In general, antigen-positive cells were concentrated mainly in the bronchioles and their neighbouring alveoli (Fig. 3), and less frequently in the blood vessels and their neighbouring alveoli (Fig. 4). In the laryngeal mucosa, viral antigens were localized in the mucosal epithelial cells and in
Fig. 1. Lung (fixed with formalin), Pig 1. Lobular consolidation, bronchial obstruction and congestion in the pulmonary lobule. Bar, 1 cm.
endothelial cells of blood vessels in the lamina propria mucosa (Fig. 5). In the cortex of the kidney, antigens were present in the epithelial cells of Bowman’s capsule and renal tubules, and in the endothelial cells of small veins in the interstitial connective tissues (Fig. 6). Focal lesions containing these antigenpositive cells were accompanied by haemorrhagia per rhexin. In the renal capsule, viral antigens were detected in the endothelial cells of venules. In the medulla, they occurred in the endothelial and smooth muscle cells of the small arteries, and the endothelial cells of the small veins and the lymphatics. In the lumen of some lymphatics, antigens were also present in the macrophages. In the spleen, viral antigens were present in the endothelial and smooth muscle cells of small arteries, the endothelial cells of the splenic sinus, and the Schwann cells of the peripheral nerve fascicles (Fig. 7). In the cerebrum, antigens were detected in the arachnoid cells in the meninges, their presence
Fig. 2. Kidney (fixed with formalin), Pig 1. Petechial haemorrhages in the renal cortex. Bar, 1 cm.
202
N. Tanimura et al. Table 2 Distribution of Nipah virus antigen-positive cells in naturally infected pigs
Pig no.
Immunolabelling in lung
1 2 3
þþ þ þþ þ þþ
larynx
kidney
spleen
cerebrum
heart
stomach
colon
liver
þ NE NE
þþ 2 2
þ 2 NE
þ 2 2
þ NE NE
þ NE NE
þ NE NE
2 2 2
Number of Nipah virus antigen-positive cells: þ, small; þþ , moderate; þþþ, large. NE, not examined.
Fig. 3. Lung, Pig 1. Intranuclear and intracytoplasmic Nipah virus antigen in many syncytial bronchiolar epithelial cells and a few alveolar epithelial cells. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
Fig. 4. Lung, Pig 1. Nipah virus antigen in endothelial and smooth muscle cells of a small vein and in the surrounding alveolar epithelial cells. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
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Fig. 5. Larynx, Pig 1. Nipah virus antigen in syncytial epithelial cells, endothelial cells of a small vein, and interstitial mononuclear cells. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
being accompanied by nonsuppurative meningitis (Fig. 8), consisting of lymphoid cell infiltration, oedema, and fibrin precipitation. In the heart, viral antigens occurred in syncytial endothelial cells of the left atrioventricular valve and in the smooth muscle cells of the coronary arteries. In the fundic gland region of the stomach, viral antigens were detected in the endothelial and smooth muscle cells of the small arteries in the submucosa and tunica muscularis, and in the macrophages surrounding the small arteries in the tela subserosa. In the colon, viral antigens were present in the endothelial cells of the small veins in
the submucosa and tunica muscularis, and in the endothelial and smooth muscle cells of the small arteries in the submucosa, tunica muscularis and tela subserosa (Fig. 9). These antigen-positive arteries showed necrotizing arteritis. In the liver, no antigen-positive cells were detected. Pigs 2 and 3. Macroscopically, cross-section of the lungs showed lobular consolidation and bronchial obstruction. No gross lesion was recognized in other tissue sections. In the lungs of pigs 2 and 3, the distribution of Nipah virus antigens was almost the same as that in
Fig. 6. Kidney, Pig 1. Nipah virus antigen in syncytial epithelial cells of renal tubules and in the endothelial cells of interstitial veins. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
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Fig. 7. Spleen, Pig 1. Nipah virus antigen in Schwann cells in a peripheral nerve fascicle. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
pig 1. In addition, in pig 2, antigen-positive alveolar epithelial cells and macrophages were observed at the periphery of necrotic lesions, where there was a severe infiltration of neutrophils, with haemorrhage and fibrin deposition (Fig. 10). Viral antigen was not detected in the other tissues examined (Table 2).
Discussion This immunohistochemical study demonstrated that Mabs 11F6 and 12A5 could detect Nipah
virus antigens in formalin-fixed, paraffin waxembedded tissues from pigs with natural infections, irrespective of formalin fixation for up to 4 years. In contrast, the reactivity of Mabs 13A5 and 18C4 and of polyclonal antibody was severely reduced by long-term formalin fixation. Previous Western blotting analysis suggested that 11F6 and 12A5 recognized the P and F proteins of Nipah virus, respectively, and that 13A5 and 18C4 reacted with the N protein of Nipah virus (Imada et al., 2004). However, the relationship between the viral protein recognized by each monoclonal
Fig. 8. Cerebrum, Pig 1. Nipah virus antigen in syncytial arachnoid cells of the cerebral meninges. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
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Fig. 9. Colon, Pig 1. Nipah virus antigen in syncytial endothelial cells of small veins, smooth muscle cells of small arteries, and periarterial interstitial cells in the submucosa. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 50 mm.
antibody and the effects of formalin fixation on immunolabelling were unknown. Leong and Leong (2002) demonstrated that tissues fixed in formaldehyde displayed a distinct and progressive loss of immunohistochemical reactivity for many antigens, frequently in proportion to the duration of exposure to the fixative. Formalin fixation causes a chemical modification in protein conformation by crosslinking, which may mask the tissue antigenicity (Shi et al., 1997). The results in Table 1 suggest that 13A5 and 18C4 recognize epitopes within the protein molecules that are modified
irreversibly by long-term formalin fixation. In contrast, the antigens recognized by 11F6 and 12A5 may have been modified in a reversible manner by such fixation. Immunohistochemical studies of natural and experimental Nipah virus infections in pigs, based on the use of rabbit polyclonal antibody, were described by Hooper et al. (2001) and Middleton et al. (2002). The results of Mab-based immunolabelling in the present study were consistent with previous findings, confirming that Nipah virus has an affinity for respiratory epithelium, renal
Fig. 10. Lung, Pig 2. Nipah virus antigen in alveolar epithelial cells and macrophages surrounding necrotic lesions in lobule. Indirect immunoperoxidase, haematoxylin counterstain. Bar, 100 mm.
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glomerular and tubular epithelium, meningeal arachnoidal cells, and systemic vascular endothelium and smooth muscle. In the present study, novel sites of immunolabelling for Nipah virus antigen were identified in the following tissues: laryngeal epithelial cells; Schwann cells of peripheral nerve fascicles in the spleen; and endothelial cells in the atrioventricular valve. All the antigen-positive tissues showed syncytium formation. Studies of natural infection of pigs in Malaysia (Shahirudin et al., 1999; Nor et al., 2000) showed that pigs aged 1– 6 months usually had an acute febrile illness with respiratory signs ranging from rapid and laboured respiration to harsh non-productive coughing, and, especially in older pigs, neurological signs such as trembling, muscle spasms, and rear leg weakness. Similar clinical signs were also seen in experimentally induced disease (Middleton et al., 2002). Coughing might be expected to be an efficient generator of infectious droplets. The presence of Nipah virus antigen in the laryngeal epithelium suggests that coughing gives rise to infectious droplets originating from laryngeal secretions as well as from the tonsil and lower respiratory tract (Middleton et al., 2002). The significance of peripheral nerve fascicles for the spread of Nipah virus remains uncertain. Further immunohistological studies are needed to clarify the pathogenesis of Nipah virus infection in pigs of different age groups.
Acknowledgments This study was supported by a joint study project of the Japan International Cooperation Agency and the Veterinary Research Institute, Malaysian Ministry of Agriculture, on the epidemiology, pathogenesis and molecular characterization of the Nipah virus.
Goh, K. J., Tan, C. T., Chew, N. K., Tan, P. S. K., Kamarulzaman, A., Sarji, S. A., Wong, K. T., Abdullah, B. J. J., Chua, K. B. and Lam, S. K. (2000). Clinical features of Nipah virus encephalitis among pig farmers in Malaysia. New England Journal of Medicine, 342, 1229 –1235. Hooper, P., Zaki, S., Daniels, P. and Middleton, D. (2001). Comparative pathology of the diseases caused by Hendra and Nipah viruses. Microbes and Infection, 3, 315–322. Imada, T., Mohd, A. A. R., Kashiwazaki, Y., Tanimura, N., Sharifah, S. H. and Aziz, A. J. (2004). Production and characterization of monoclonal antibodies against formalin-inactivated Nipah virus isolated from the lung of a pig. Journal of Veterinary Medical Science, 66, 81–83. Leong, A. S.-Y. and Leong, F. J. W.-M. (2002). Microwavestimulated antigen retrieval - an update. Acta Histochemica et Cytochemica, 35, 367 –374. Middleton, D. J., Westbury, H. A., Morrissy, C. J., van der Heide, B. M., Russell, G. M., Braun, M. A. and Hyatt, A. D. (2002). Experimental Nipah virus infection in pigs and cats. Journal of Comparative Pathology, 126, 124–136. Nor, M. N. M., Gan, C. H. and Ong, B. L. (2000). Nipah virus infection of pigs in peninsular Malaysia. Revue Scientifique et Technique (International Office of Epizootics), 19, 160 –165. Shahirudin, S., Zamri-Saad, M., Shamshad, S., Mahani, H., Harizam, Y., Norazian, B., Daniels, P. and Aziz, A. J. (1999). A novel clinicopathological disorder associated with a zoonotic paramyxovirus infection in pigs. In: Proceedings of the National Congress on Animal Health and Production, Alor Gajah, Melaka, Malaysia, pp. 367–368. Shi, S.-R., Cote, R. J. and Taylor, C. R. (1997). Antigen retrieval immunohistochemistry: past, present, and future. Journal of Histochemistry and Cytochemistry, 45, 327–343. Wang, L.-F., Harcourt, B. H., Yu, M., Tamin, A., Rota, P. A., Bellini, W. J. and Eaton, B. T. (2001). Molecular biology of Hendra and Nipah viruses. Microbes and Infection, 3, 279–287.
References Daniels, P., Ksiazek, T. and Eaton, B. T. (2001). Laboratory diagnosis of Nipah and Hendra virus infections. Microbes and Infection, 3, 289–295.
Received; November 11th; 2003 Accepted; March 22nd; 2004