Human Hendra virus infection causes acute and relapsing encephalitis

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1 Neuropathology and Applied Neurobiology (2009), 35, doi: /j x Human Hendra virus infection causes acute and relapsing encephalitis K. T. Wong*, T. Robertson, B. B. Ong, J. W. Chong*, K. C. Yaiw*, L. F. Wang, A. J. Ansford and A. Tannenberg *Department of Pathology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia, Royal Brisbane & Women s Hospital, Queensland Health Pathology & Scientific Services, Brisbane, Queensland, and CSIRO Livestock Industries, Australian Animal Health Laboratory and Australian Biosecurity Cooperative Research Center, Geelong, Victoria, Australia K. T. Wong, T. Robertson, B. B. Ong, J. W. Chong, K. C. Yaiw, L. F. Wang, A. J. Ansford and A. Tannenberg (2009) Neuropathology and Applied Neurobiology 35, Human Hendra virus infection causes acute and relapsing encephalitis Aim: To study the pathology of two cases of human Hendra virus infection, one with no clinical encephalitis and one with relapsing encephalitis. Methods: Autopsy tissues were investigated by light microscopy, immunohistochemistry and in situ hybridization. Results: In the patient with acute pulmonary syndrome but not clinical acute encephalitis, vasculitis was found in the brain, lung, heart and kidney. Occasionally, viral antigens were demonstrated in vascular walls but multinucleated endothelial syncytia were absent. In the lung, there was severe inflammation, necrosis and viral antigens in type II pneumocytes and macrophages. The rare kidney glomerulus showed inflammation and viral antigens in capillary walls and podocytes. Discrete necrotic/vacuolar plaques in the brain parenchyma were associated with antigens and viral RNA. Brain inflammation was mild although CD68 + microglia/macrophages were significantly increased. Cytoplasmic viral inclusions and antigens and viral RNA in neurones and ependyma suggested viral replication. In the case of relapsing encephalitis, there was severe widespread meningoencephalitis characterized by neuronal loss, macrophages and other inflammatory cells, reactive blood vessels and perivascular cuffing. Antigens and viral RNA were mainly found in neurones. Vasculitis was absent in all the tissues examined. Conclusions: The case of acute Hendra virus infection demonstrated evidence of systemic infection and acute encephalitis. The case of relapsing Hendra virus encephalitis showed no signs of extraneural infection but in the brain, extensive inflammation and infected neurones were observed. Hendra virus can cause acute and relapsing encephalitis and the findings suggest that the pathology and pathogenesis are similar to Nipah virus infection. Keywords: encephalitis, Hendra virus, Henipavirus, human, infection, pathogenesis, pathology Introduction Hendra virus (HeV), an emerging paramyxovirus from the family Paramyxoviridae was first discovered in Australia in 1994 following an outbreak in horses and humans [1]. Correspondence: Kum Thong Wong, Department of Pathology, Faculty of Medicine, University of Malaya, Kuala Lumpur, Malaysia. Tel: ; Fax: ; wongkt@um.edu.my Because it shares a high degree of similarity in genome organization and sequence and other biological characteristics to another recently discovered virus, the Nipah virus (NiV), both viruses have been placed in the newly created genus Henipavirus [2,3]. There is good evidence that the natural hosts of HeV and NiV are pteropid fruit bats [4 6], whose range includes northern Australia, southeast Asia, the Indian subcontinent and eastern Africa. Hence, it was not surprising that after the first NiV outbreaks in Blackwell Publishing Ltd

2 Human Hendra virus infection 297 Table 1. Clinicopathological findings in four cases of human Hendra virus infection Case # Clinical presentation* Probable incubation period Light microscopy of autopsy tissues Positive IHC and/or ISH staining in autopsy tissues CNS tissues Non-CNS tissues Final pathological or clinical diagnosis 1 49-year-old man, presented with influenza-like illness, followed by respiratory and renal failure and cardiac irritability. Died after about 12 days of admission 6 days Blood vessels: Brain Lung Acute Hendra Vasculitis/endotheliitis in brain, lung, heart and kidney (neurones, ependyma, blood vessels) (type II pneumocytes, blood vessels) Brain: Kidney Small necrotic/vacuolar plaques in the (glomeruli, capillaries, cerebrum and cerebellum, in addition to tubules) mild meningitis and mild parenchymal inflammation Lung: Severe parenchymal inflammation and necrosis. Incidental fungal infection in the lungs was also noted Other organs: Generally mild or focal inflammation in the parenchyma of other organs infection with encephalitis 2 35-year-old man who first presented with headache, drowsiness and meningitis but after 13 months was readmitted with fever, seizures, neurological deficits and coma. Died after 25 days of admission 12 days (13 months) Brain: Brain All tissues negative Relapsing Mainly confluent geographic areas of (neurones mainly; Hendra neuronal loss, gliosis and parenchymal possibly also glial and encephalitis and perivascular inflammation were inflammatory cells) observed mainly in the cerebral cortex Other non-cns organs: No vasculitis, necrosis or significant inflammation in the lung, heart, kidney, liver and spleen 3 40-year-old man presented with influenza-like illness, myalgia, headache and vertigo of 5-day duration. Recovered apparently well after 6 weeks 5 days NA NA NA Acute Hendra infection 4 Adult female (age unknown) presented with dry cough and sore throat, cervical lymphadenopathy, fever, body aches and tiredness. Recovered and apparently well 7 days NA NA NA Acute Hendra infection *Information (age, sex, clinical presentation, incubation period) compiled from references [12 14]. IHC, immunohistochemistry; ISH, in situ hybridization; CNS, central nervous system. ISH was not performed on non-cns tissues of Case #1 due to insufficient tissues. Pathological diagnosis available for Cases # 1 and 2 only. Acute encephalitis based on our pathological evidence only; clinical encephalitis was not reported. NA = tissues not available for study.

3 298 K. T. Wong et al. Malaysia and Singapore [7 9], sporadic NiV outbreaks continued to be seen in these regions, notably in Bangladesh and India [10,11]. Although so far HeV infection has only been reported from Australia, like NiV, there is every possibility that future outbreaks may occur in other countries as well, given the wide range of fruit bats. HeV-infected horses acted as intermediate hosts for human infections [1,6]. Of the four human cases that have been reported to date (Table 1), two were fatal and all had close contact with infected horses before developing the disease [12 14]. Previous publications on human HeV infection have concentrated mainly on epidemiological, virological and clinical aspects with no detailed data on pathological features [1,3,12,14]. Moreover, the target cells of HeV in natural human infections have not been fully investigated. Studies of acute human NiV infection have demonstrated that blood vessels and a broad range of parenchymal cells were susceptible to infection [15]. The most severely involved tissues were in the central nervous system (CNS), but the lung, kidney, lymphoid and other organs were also susceptible. Two distinct clinicopathological forms of encephalitis were recognized, viz. acute and relapsed/lateonset NiV encephalitis [15,16]. Acute NiV encephalitis appears as part of the acute infection that occurs 1 2 weeks after viral exposure [17]. Pathologically, it is characterized by necrotic plaques believed to be associated with vasculitis-induced thrombosis and neuronal infection. This dual pathogenetic mechanism of tissue damage consisting of microinfarction and direct neuronal infection appears to be unique among viral encephalitides [15]. On the other hand, relapsed/late-onset NiV encephalitis usually presents several weeks after the acute infection has subsided, with new episodes of neurological manifestations. Relapsed encephalitis occurs in survivors who initially present with acute encephalitis and lateonset encephalitis is found in those with acute, mild, nonencephalitic infection earlier. Relapsed/late-onset encephalitis generally have similar clinicopathological characteristics that are distinguishable from acute NiV encephalitis [16,18]. Pathological findings suggested a true recurrence of infection rather than postinfectious encephalomyelitis as viral antigens/rna in neurones could be demonstrated. However, vasculitis and multinucleated endothelial cells or syncytia were not demonstrated [15,16]. Of the two HeV human fatalities included in this study (Table 1), Case # 1 presented clinically as an acute HeV infection characterized by a predominant pulmonary syndrome with no apparent clinical encephalitis [12,14]. However, studies of acute HeV infection in horses, guinea pigs and other animals [1,19,20] suggest that there is some similarity to acute NiV infection in that vasculitis and encephalitis can occur [15]. Hence, we hypothesize that there may be histological evidence of CNS involvement in Case # 1. In the case of relapsing HeV encephalitis (Table 1, Case # 2), it was previously reported that in addition to parenchymal inflammation and viral antigens, occasional multinucleated endothelial cells were observed in blood vessels in the brain and other organs [14]. Our objective is to undertake a more detailed study of the neuropathology of human HeV infection by studying the histopathological features, in particular CNS pathology and by investigating viral localization with immunohistochemistry (IHC) and in situ hybridization (ISH) in infected tissues. Materials and methods Formalin-fixed, paraffin-embedded autopsy tissues of various major organs were sectioned at 5 7 microns and routinely stained with haematoxylin and eosin. These organs/tissues included brain, lung, heart, kidney, liver and spleen (Table 1: Cases # 1 and 2); pancreas, thyroid, adrenal, lymph node and intestine (Case # 1 only) and spinal cord (Case # 2 only). IHC to detect HeV antigens was performed on further sections. A standard immunoperoxidase protocol was followed [21], using a primary mouse anti-hendra polyclonal antibody (1:1000 dilution; gift Figure 1. Acute Hendra virus infection: vasculitis and endotheliitis involving blood vessels in the meninges (A, arrows), brain parenchyma (D, arrows; E), lung (B, arrow) and heart (C, arrow), with intramural or subendothelial inflammatory cells. Viral antigens demonstrated in endothelial cells lining a meningeal capillary (F, arrow; inset). Inflammation in the lung (G) characterized by intra-alveolar inflammation, necrosis, macrophages and the rare multinucleated giant cell (inset, arrow). Proliferating type II pneumocytes and alveolar macrophages were positive for viral antigens (H). Kidney glomerulus with surrounding inflammation and thrombotic plug (I, arrow) and viral antigens in capillary walls, podocytes and other cells (J). A E, G, I: haematoxylin & eosin stain. F, H, J: immunohistochemistry/dab/mayer s haematoxylin. Magnification: A F, H J, G (inset): 40 obj; G: 10 obj. Bar: A F, I, J = 50 microns; G = 100 microns; G (inset), H = 25 microns.

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6 Human Hendra virus infection 301 Figure 2. Acute Hendra virus infection in the central nervous system. (A) Subtle vacuolar plaque (thin arrows) in the cerebellar molecular layer consisting of fine neuropil vacuolation with adjacent eosinophilic Purkinje cells (thick arrow). (B) Necrotic/vacuolar plaque in the dentate nucleus, showing neuronal loss, coarse neuropil vacuolation and mild inflammation. Some surviving/degenerate neurones were still present (arrows). Fine granular viral antigens in the neuropil (C) within a vacuolar plaque in the cerebellar molecular layer. (D) Viral antigens in the cytoplasm (thin arrows) and processes (thick arrow) of neurones found within or adjacent to a necrotic/vacuolar plaque in the dentate nucleus. Eosinophilic viral inclusions (E) and viral antigens (F, thick arrow) and viral RNA (G) in the cytoplasm of neurones in the hippocampus pyramidal layer. Viral protein was also detected in the nucleus (F, thin arrow). (H) Plaque-like cluster of neurones and adjacent ependymal cells (arrow) in the periaqueductal grey matter of the midbrain staining positive for viral antigens. A, B, E: haematoxylin & eosin stain; C, D, F, H: immunohistochemistry/dab/mayer s haematoxylin; G: in situ hybridization/nbt/bcip/mayer s haematoxylin. Magnification: A, B, H: 10 obj; C G: 40 obj. Bar: A D = 50 microns; E G = 25 microns, H = 100 microns. from Dr SR Zaki, CDC, Atlanta, USA), with slight modifications in that our assay utilized the ENVISION detection method (DakoCytomation, Copenhagen, Denmark). The slides were counter-stained with haematoxylin, dehydrated through serial ethanols and xylene and coverslipped before microscopic examination. IHC to CD68 to detect microglia/macrophages were performed as previously described [22]. The DNA probes were prepared from a plasmid containing the whole N gene of HeV, using a standard polymerase chain reaction (PCR) that incorporated digoxigenin-11- dutp to produce a digoxigenin-labelled DNA probe of 271 bp, as previously described [22]. The forward and reverse primers for the PCR were, respectively, 5 -gca-atggct-gac-aga-gat-ga-3 (N gene nucleotide position: , Genbank accession number: AF017149) and 5 -gct-cga-ggc-cct-att-tct-ct-3 (nucleotide position: ). The PCR conditions were 95 C for 3 min; followed by 35 cycles of 94 C for 30 s, 56 C for 1 min, 72 C for 30 s; and 72 C for 10 min, final extension. The ISH protocol used was as described previously [23] and performed on all tissues except for the non-cns tissues of Case#1(Table 1),as these tissues were insufficient for this purpose. For IHC and ISH assays, HeV-infected hamster tissues (courtesy of Dr Fabian Wild, France) served as positive controls. Negative control tissues included normal CNS tissues and CNS tissues known to be infected by Enterovirus 71 [22] and measles virus [24]. For both assays, duplicate procedures that omitted the primary antibody and probe, respectively, were performed as additional negative controls. Results Acute HeV infection (Table 1, Case # 1) The light microscopic features are summarized in the Table 1. Vasculitis and endotheliitis were observed in blood vessels in the brain, lung, kidney and heart (Figure 1A E). In contrast to perivascular cuffing, vasculitis was characterized by karyorrhexis and intramural or subendothelial inflammatory cells. However, no convincing endothelial syncytia or multinucleated giant cells were found. Occasional thrombotic plugs were detected in the pulmonary blood vessel and rarely in glomerular capillaries (Figure 1I). Viral antigens were observed in some vascular endothelium and possibly smooth muscle cells, in the brain (Figure 1F), lung and kidney (Figure 1J). Choroid plexus showed no evidence of vasculitis or virus. In the lung, there was severe parenchymal inflammation, necrosis, intra-alveolar macrophages/inflammatory cells, prominent type II pneumocyte proliferation and occasional alveolar membranes (Figure 1G). The kidney showed the rare focal glomerulitis (Figure 1I) and focal inflammation around necrotic tubules. There was evidence of emperipolesis and the occasional bizarre multinucleated giant cell in the lymph nodes. Viral antigens were demonstrated in alveolar type II pneumocytes, intraalveolar macrophages and the occasional glomeruli and tubules (Figure 1H,J). In the brain parenchyma, there were both subtle and more obvious discrete necrotic or vacuolar plaques, in the cerebellum (Figure 2A,B), the gray and white matter of the cerebral cortex (Figure 3A), corpus callosum, hippocampus, thalamus, external capsule and globus pallidus. The most obvious necrotic plaques were found in the white matter consisting of eosinophilic axonal spheroidlike material (Figure 3A). The vacuolar plaques comprised neuropil vacuolation, mild parenchymal infiltration by mononuclear inflammatory cells and mild perivascular cuffing. In addition, neuronal loss may be evident in neuronal areas (Figure 2B). In the cerebellar molecular layer, vacuolar plaques consisted of small fine vacuoles (Figure 2A). Occasional neuronal bodies at the periphery of a plaque may show cytoplasmic eosinophilia (Figure 2A). Eosinophilic cytoplasmic viral inclusions were detected in neurones in the hippocampus (pyramidal

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8 Human Hendra virus infection 303 Figure 3. Acute and relapsing Hendra encephalitis. Acute Hendra encephalitis (A D): necrotic/vacuolar plaque in cerebral white matter consisting of eosinophilic, axonal spheroid-like material (A, inset). Increased number of CD68 + microglia/macrophages in necrotic/vacuolar plaques in the cerebellar dentate nucleus (B) and cerebellar molecular layer (C) and around neurones, neuronophagia (D). Severe meningoencephalitis in relapsing Hendra encephalitis (E J): large confluent areas of inflammation in the cerebral cortex (E, arrows) consisting of perivascular cuffing, reactive blood vessels (F), parenchymal inflammation, necrosis and neuronal loss. Inflammatory cells comprised mainly of macrophages, lymphocytes and some plasma cells (G). Focal areas of viral antigens were found either within or at the edge of inflammatory lesions (H, arrows), mainly in neurones and possibly glial and inflammatory cells (I). Viral RNA was demonstrated mainly in neurone-like cells (J, arrows). A, E G: haematoxylin & eosin; B D, H, I: immunohistochemistry/dab/mayer s haematoxylin; J: in situ hybridization/nbt/bcip/mayer s haematoxylin. Magnification: A, D, G, I, J: 40 obj; B, C, F: 10 obj; E, H: 4 obj. Bar: A, D = 50 microns; A (inset), B, C, E, F, H = 100 microns, G, I, J = 25 microns. layer) (Figure 2E) and thalamus and ependymal cells. There were no nuclear inclusions detected. Overall, by light microscopy, parenchymal and meningeal inflammation and perivascular cuffing were mild and prominent microglial nodules were not observed. However, CD68 IHC showed an increased number of microglial cells/ macrophages in and around necrotic/vacuolar plaques (Figure 3B,C) and surrounding some neurones, neuronophagia (Figure 3D). Viral antigens and/or RNA were detected in the neuropil and in single or plaque-like groups of neurones (both soma and processes) in the cerebellum (dentate nucleus, molecular and granular layers), cerebral cortex, midbrain (periaqueductal gray area, substantia nigra), hippocampus (pyramidal layer, dentate gyrus, entorhinal cortex), thalamus and ependymal and subependymal cells (Figure 2C,D,F H). Positive neurones were often, but not always, found in or near necrotic/vacuolar plaques (Figure 2C,D). Conversely, not all plaque-like neuronal virus antigen positivity was associated with necrosis or prominent vacuolation (Figure 2H). Necrotic plaques in the white matter were not associated with viral antigens/rna. Relapsing HeV infection (Table 1, Case # 2) Only the CNS tissues and mainly the cerebral cortex showed evidence of inflammation although very focal areas in the pons, cerebellum and spinal cord were also inflamed. Inflammatory lesions were usually extensive and consisted of intense infiltration of macrophages, lymphocytes and some plasma cells, with prominent perivascular cuffing (Figure 3E G). There was severe neuronal loss, glial proliferation and an increased number of reactive blood vessels (Figure 3F). More discrete, plaquelike, but otherwise similar inflammatory lesions, were occasionally observed. There was no evidence of vasculitis, endothelial syncytia or thrombosis and viral inclusions were not prominent. Within inflamed areas, focal viral antigens/rna were demonstrated mainly in surviving neurones (Figure 3H J) and possibly in some glial/inflammatory cells as well. Overall, inflammation was much more extensive than viral antigens/rna. Severe meningitis was found over many areas of the cerebral cortex (Figure 3E). All the non-cns organs were uninflamed and in particular, no vasculitis, multinucleated endothelial syncytia or viral antigens/rna were detected. Discussion Pathological findings in the two cases confirmed that HeV was neuronotropic and could cause CNS infection giving rise to acute and relapsing encephalitis, respectively. In the patient with acute HeV infection without apparent clinical encephalitis (Case # 1), mild vasculitis, meningitis, parenchymal inflammation, necrotic/vacuolar plaques and neuronal infection were found in the CNS, features that have not been previously reported in acute infection. It was postulated that the characteristic and well-defined necrotic plaque described in acute NiV encephalitis is the result of a combination of neuronal infection and microinfarction following vasculitis-induced thrombosis [15]. This was because in the vicinity of necrotic plaques, vasculitis, thrombus-occluded vessels and/or infected neurones were often observed together [15]. In our case of acute HeV encephalitis, the necrotic/vacuolar plaques may be equivalent to the necrotic plaque in acute NiV encephalitis. There was evidence that the necrotic/ vacuolar plaques were associated with neuronal infection by HeV. Vasculitis and viral antigens could be demonstrated in cerebral vessels, although admittedly, these abnormal vessels are not necessarily found near the plaques. Thrombus-occluded blood vessels were also absent. Vasculitis was also noted in the lung, heart and kidney and vascular thrombi were identified in the lung and glomeruli. Hence, we think it is possible that vasculitis-induced thrombosis and microinfarction could have occurred in the

9 304 K. T. Wong et al. CNS at some stage of the acute infection. Multinucleated endothelial syncytia, a unique feature of endothelial infection, is observed in acute NiV infection but this was not observed in this HeV case. The reasons for this disparity could include the rarity of multinucleated endothelial syncytia (only found in 27% of acute NiV encephalitis) [15], a relatively mild CNS involvement, disintegration of thrombotic plugs over time or inadequate sampling. Within or near the discrete white matter necrotic plaques, there was no evidence of infection of glial or other cells whatsoever, suggesting that microinfarction may be solely involved in their pathogenesis. If this is correct, focal infarction and death of oligodendroglial cells surrounding axons may be responsible for this phenomenon. As vasculitis was so widespread, we postulate that viraemia must have occurred, spreading the infection to multiple organs; hence, acute HeV infection should be regarded as a systemic infection. Neuronal and other parenchymal cell infection was probably facilitated by vasculitic damage to the blood brain barrier and other vessels. Like meningeal vessel vasculitis, involvement of the ependyma could enable virus to enter cerebrospinal fluid and contribute to further viral spread in the CNS via this route. It is difficult to determine if clinical encephalitis was missed as a result of the patient s severe clinical condition at admission, or whether clinical encephalitis was not severe enough to be detected. Apart from blood vessels, the target cells/tissues in acute HeV infection seemed to be similar to NiV infection [15]. Like NiV, HeV was neuronotropic and to a lesser extent, ependyma were susceptible to infection. Viral inclusions, and antigens/rna in neurones and ependyma in particular, suggest that active viral replication occurs in these cells. Likewise, type II pneumocytes and alveolar macrophages may be able to support viral replication. Overall, like NiV, involvement of glial cells appears to be rare in HeV infection. The second fatal case of HeV infection with a relapse of neurological manifestations 13 months after acute disease seems to be similar to relapsed NiV encephalitis [16]. The pathological findings confirmed that the patient had recurrent infection rather than postinfectious encephalomyelitis. At variance to a previous report [14], we found no evidence of multinucleated endothelial syncytia or vasculitis in any of the organs examined, nor any evidence of viral antigens/rna in the non-cns organs. We postulate that recurrence originated from viral foci that were disseminated to the CNS by viraemia during the acute infection. After 13 months, vasculitis present initially, subsided and healed, but the virus infection recurred to spread throughout the CNS. This phenomenon has also been suggested for relapsed/late-onset NiV encephalitis [16]. Further investigations are needed to determine if the immune response and other host factors, or viral mutations were responsible for this interesting phenomenon. In the case of measles, another important human paramyxovirus, relapsing encephalitis has not been reported. However, late-onset NiV encephalitis might arguably be represented by subacute sclerosing panencephalitis and measles inclusion body encephalitis as both present months to years after the acute infection. As relapsed NiV encephalitis could occur even after 4 years or more [25], it is theoretically possible that survivors of acute HeV infection could still develop encephalitis long after 13 months. Fortunately, relapsed/late-onset NiV encephalitis is not uniformly fatal, suggesting that HeV encephalitis may not be invariably fatal as well [16]. Nonetheless, long-term follow-up of Cases # 3 and 4 (Table 1) is essential and could be invaluable to determine future disease course. Remarkably, in asymptomatic or mildly symptomatic NiV-infected patients, despite absence of neurological manifestations, brain scans may show a few discrete lesions similar to acute NiV encephalitis [26]. Acknowledgement This work was partly supported by Malaysian Government IRPA Grant ( PR0060-/04). We are grateful to Dr Sherif R Zaki, Centers for Disease Control and Prevention, Atlanta, USA for the generous gift of anti-hendra antibodies and to Dr F. Wild, INSERM, Lyon, France for the infected hamster tissues. References 1 Murray K, Selleck P, Hooper P, Hyatt A, Gould A, Gleeson L, Westbury H, Hiley L, Selvey L, Rodwell B, Ketterer P. A morbillivirus that caused fatal disease in horses and humans. 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