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Diagnosis of Nipah virus encephalitis by electron microscopy of cerebrospinal fluid
Journal of Clinical Virology 19 (2000) 143 – 147 www.elsevier.com/locate/jcv
Diagnosis of Nipah virus encephalitis by electron microscopy of cerebrospinal fluid Vincent T.K. Chow *, Paul A. Tambyah, W.M. Yeo, M.C. Phoon, J. Howe Departments of Microbiology and Medicine, Faculty of Medicine, National Uni6ersity of Singapore, Kent Ridge 117597, Singapore Received 22 February 2000; accepted 1 June 2000
Abstract Background: between 1998 and 1999, an outbreak of potentially fatal viral encephalitis erupted among pig farm workers in West Malaysia, and later spread to Singapore where abattoir workers were afflicted. Although Japanese encephalitis virus was initially suspected, the predominant aetiologic agent was subsequently confirmed to be Nipah virus, a novel paramyxovirus related to but distinct from Hendra virus. Objecti6e: to describe a case of Nipah virus encephalitis in a pig farm worker from Malaysia. Study design: the clinical, laboratory and radiological findings of this patient were scrutinized. Special emphasis was placed on the electron microscopic analysis of the cerebrospinal fluid (CSF) specimen from this patient. Results: the neurological deficits indicative of cerebellar involvement were supported by the magnetic resonance imaging that showed prominent cerebellar and brainstem lesions. CSF examination provided further evidence of viral encephalitis. Complement fixation and/or RT-PCR assays were negative for Japanese encephalitis, herpes simplex, measles and mumps viruses. ELISA for detecting IgM and IgG antibodies against Hendra viral antigens were equivocal for the CSF specimen, and tested initially negative for the first serum sample but subsequently positive for the repeat serum sample. Transmission electron microscopy of negatively-stained preparations of CSF revealed enveloped virus-like structures fringed with surface projections as well as nucleocapsids with distinctive helical and herringbone patterns, features consistent with those of other paramyxoviruses, including Hendra virus. Conclusion: this case report reiterates the relevant and feasible role of diagnostic electron microscopy for identifying and/or classifying novel or emerging viral pathogens for which sufficiently specific and sensitive tests are lacking. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Cerebrospinal fluid; Electron microscopy; Nipah virus; Paramyxovirus; Viral encephalitis
1. Introduction Between 1998 and 1999, an outbreak of viral encephalitis involving more than 260 reported * Corresponding author. Fax: +65-7766872. E-mail address: [email protected] (V.T.K. Chow).
cases with over 100 fatalities, occurred in West Malaysia especially among pig farm workers in the states of Perak and Negri Sembilan. Although Japanese encephalitis virus was initially implicated as the main causative agent for this outbreak, epidemiological and laboratory investigations subsequently incriminated the newly recognized
1386-6532/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 0 ) 0 0 0 9 4 - 9
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Nipah virus as the predominant aetiologic pathogen (Chua et al., 1999; Farrar, 1999). The outbreak later spread to Singapore where 11 cases of Nipah virus encephalitis were described among workers in an abattoir handling pigs imported from Malaysia (Paton et al., 1999). By mid-1999, the epidemic was eventually contained, thanks to the collaborative efforts of an international team of physicians and scientists, and following the culling of over one million pigs in Malaysia. Named after the village of Kampung Baru Sungai Nipah in Negri Sembilan where it was first isolated, the Nipah virus is a member of the Paramyxo6iridae family, related to but distinct from Hendra virus that is also associated with a viral zoonosis (Yu et al., 1998). We report here the clinical features, laboratory and radiological findings of a case of Nipah virus encephalitis, and demonstrate the presence of virus-like particles consistent with a typical paramyxovirus by electron microscopic analysis of the patient’s cerebrospinal fluid (CSF) specimen. 2. Case report
2.1. Clinical and laboratory findings YBK, a 51-year-old Chinese woman was admitted to Alexandra Hospital, Singapore on 14 March, 1999. She had no past medical history of significance, but complained of a prodrome of generalized malaise for 2 – 3 weeks. In addition, she experienced difficulty in concentrating as well as a gradual decline in her mental status. On admission, she had generalized weakness and a mild headache. Her social history was remarkable in that she lived and worked in a pig farm in Negri Sembilan. Her husband mentioned that prior to her illness, she had cleaned out a barn with a large amount of bat droppings. She denied any consumption of alcohol and tobacco, and claimed no usage of nor allergy to drugs. Physical examination revealed that she was initially very drowsy and oriented to only person and time. She suffered from a right lateral gaze palsy and bilateral nystagmus. A right pronator drift, marked ataxia and mild bilateral motor weakness were also observed.
Laboratory tests showed elevations of total white blood cell count (14.5× 109/l), erythrocyte sedimentation rate (23 mm/h) and C-reactive protein (38.8 mg/l). Her haemoglobin concentration (153 g/l), platelet count (475×109/l), serum electrolytes and liver function tests were all normal. Initial CSF examination showed a white blood cell count of 107/l with lymphocytic pleocytosis, and elevated protein but decreased glucose concentrations, a picture compatible with viral encephalitis. After 2 weeks of hospitalization, a repeat CSF examination showed an increase in white blood cell count to 5.4×107/l. Titres of complement-fixing antibodies to Japanese encephalitis, herpes simplex, measles and mumps viruses were all insignificant. IgM capture and IgG ELISA for detecting IgM and IgG antibodies against Hendra viral antigens was initially negative for the patient’s serum on 23 March, 1999, but subsequently tested positive for a repeat serum sample on 9 April, 1999. The CSF specimen yielded an equivocal ELISA result. RNAs from serum and CSF samples tested negative for Japanese encephalitis virus by RT-PCR using flavivirus NS3 consensus primers (Chow et al., 1993).
2.2. Electron microscopy A CSF specimen was collected on 27 March, 1999, and prepared for electron microscopy (EM) by centrifugation at 10 000 rpm for 30 min at 4°C, and negative staining was performed on both the supernatant and pellet. Briefly, samples were placed onto the dull surface of 300-mesh carboncoated copper grids, left for 1 min, and dried with filter paper. Two percent phosphotungstic acid (pH 7) was then dropped onto the sample-loaded surface which was stained for varying times of 0.5, 1, 1.5 or 2 min. The grids were dried and viewed under a Philips CM-120 transmission electron microscope. At lower magnification were observed numerous particles resembling pleomorphic but roughly spherical viruses 50–150 nm in diameter. Higher magnification revealed enveloped virions fringed with surface projections, together with nucleocapsids with distinctive helical and herring-
V.T.K. Chow et al. / Journal of Clinical Virology 19 (2000) 143–147
bone patterns (Fig. 1). These EM features are strikingly typical of members of the Paramyxo6iridae family, including Hendra or equine morbillivirus (Ackermann and Berthiaume, 1995; Murray et al., 1995; Hyatt and Selleck, 1996).
2.3. Diagnosis and management Considering the clinical, radiological, laboratory and electron microscopic findings together
145
with the epidemiological association of the Nipah virus outbreak with pigs, a diagnosis of Nipah viral encephalitis was made. The patient was nursed in strict isolation, managed with supportive care, and treated empirically with intravenous acyclovir. She gradually improved and was discharged having minimal ataxia and dizziness. At a follow-up visit after one month, she complained of persistent dizziness but was alert and lucid. She was unable to tandem walk, displayed ataxia and
Fig. 1. Electron micrographs of negatively-stained preparations of CSF from the patient with viral encephalitis. (a) Virus-like structures of 100 nm diameter are highlighted by arrows. Magnification 100 000 × . (b – d) Individual enveloped virions fringed with surface projections at higher magnification of 135 000× . Some virus particles are partially disrupted, spilling nucleocapsids of distinctive helical and herringbone patterns.
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dysdiadochokinesia but no other cerebellar signs. Repeat magnetic resonance imaging (MRI) performed during this visit revealed an increase in white matter lesions, including more prominent cerebellar and brainstem lesions. She returned to Malaysia and suffered from a prolonged period of ataxia, but currently has only minimal symptoms. These observations are congruent with those of Lee et al. (1999) who reported that more than half of infected survivors had lingering neurological sequelae.
3. Discussion Symptoms and signs of cerebellar dysfunction in this patient match the neurological manifestations of five out of seven confirmed cases of Nipah virus encephalitis described by Lee et al. (1999), and appear to be a unique feature of this form of encephalitis. Furthermore, these clinical deficits also concur with the MRI scans that exhibited white matter lesions (Lim et al., 1999) and localized prominent lesions to the cerebellum and brainstem. The negative results of the complement fixation and RT-PCR assays excluded encephalitis caused by other viruses, particularly Japanese encephalitis virus. The ELISA detection of Nipah virusspecific antibodies relies on their cross-reactivity with related Hendra viral antigens, which may compromise its specificity and may explain the equivocal and initially negative results for the CSF and serum specimens, respectively. It is especially in these and other circumstances involving unknown or emerging viral pathogens (Tambyah, 1999) for which sufficiently specific and sensitive tests are not available, where diagnostic electron microscopy has a relevant and feasible role as exemplified by this case report. EM images of the ultrastructural morphology of novel viruses can provide clues to their identification through classification to appropriate virus families (Biel and Gelderblom, 1999). The EM observation in this case demonstrates that Nipah virus is present in the central nervous system (CNS) and causes a direct infection. This finding is corroborated by others who have iso-
lated virus (Chua et al., 1999) and shown RTPCR positivity (Paton et al., 1999) from CSF of infected patients. It is difficult to demonstrate other neurotropic viruses such as herpes simplex and enteroviruses in CSF by EM. Therefore, this case study may point to the fact that Nipah infection can cause unusually extensive damage in the CNS, culminating in high virus concentrations in the CSF. The Nipah virus shares significant nucleotide and amino acid homology with the related Hendra virus, another recently described paramyxovirus that causes potentially fatal lung or brain infections in horses and humans (Murray et al., 1995). Williamson et al. (1998) have reported that infected horses, fruit bats and cats can excrete Hendra virus in their urine and saliva. Humans acquire Hendra viral infection via close contact with infected secretions of animals such as horses, although human-to-human transmission is thought to be inefficient (Selvey et al., 1995). Interestingly, another new paramyxovirus known as Menagle virus that is infectious for pigs, humans and fruit bats has also been recently discovered (Philbey et al., 1998). It is noteworthy that being a pig farmer, this patient obviously had close contact with pigs, and even with bat droppings in her barn as divulged in her history. Analysis of Nipah viral infection in pigs, fruit bats and other animal hosts or reservoirs including the existence of persistent carrier states would help to elucidate the natural history and transmission pathways of this deadly viral affliction (Halpin et al., 1999). Furthermore, the long latency and potential for relapse of Hendra viral disease (O’Sullivan et al., 1997) also raises similar questions for Nipah virus infections. References Ackermann HW, Berthiaume L. Atlas of Virus Diagrams. Boca Raton, FL: CRC Press, 1995:50 – 2. Biel SS, Gelderblom HR. Diagnostic electron microscopy is still a timely and rewarding method. J Clin Virol 1999;13:105 – 19. Chow VTK, Seah CLK, Chan YC. Use of NS3 consensus primers for the polymerase chain reaction amplification and sequencing of dengue viruses and other flaviviruses. Arch Virol 1993;133:157 – 70.
V.T.K. Chow et al. / Journal of Clinical Virology 19 (2000) 143–147 Chua KB, Goh KJ, Wong KT, Kamarulzaman A, Tan PS, Ksiazek TG, Zaki SR, Paul G, Lam SK, Tan CT. Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet 1999;354:1257–9. Farrar JJ. Nipah-virus encephalitis — investigation of a new infection. Lancet 1999;354:1222–3. Halpin K, Young PL, Field H, Mackenzie JS. Newly discovered viruses of flying foxes. Vet Microbiol 1999;68:83–7. Hyatt AD, Selleck PW. Ultrastructure of equine morbillivirus. Virus Res 1996;43:1–15. Lee KE, Umapathi T, Tan CB, Tjia HT, Chua TS, Oh HM, Fock KM, Kurup A, Das A, Tan AK, Lee WL. The neurological manifestations of Nipah virus encephalitis, a novel paramyxovirus. Ann Neurol 1999;46:428–32. Lim CCT, Sitoh YY, Lee KE, Kurup A, Hui F. Meningoencephalitis caused by a novel paramyxovirus: an advanced MRI case report in an emerging disease. Singapore Med J 1999;40:356 – 8. 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. Science 1995;268:94–7. O’Sullivan JD, Allworth AM, Paterson DL, Snow TM, Boots R, Gleeson LJ, Gould AR, Hyatt AD, Bradfield J. Fatal encephalitis due to novel paramyxovirus transmitted from horses. Lancet 1997;349:93–5.
.
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Paton NI, Leo YS, Zaki SR, Auchus AP, Lee KE, Ling AE, Chew SK, Ang B, Rollin PE, Umapathi T, Sng I, Lee CC, Lim E, Ksiazek TG. Outbreak of Nipah-virus infection among abattoir workers in Singapore. Lancet 1999;354:1253– 6. Philbey AW, Kirkland PD, Ross AD, Davis RJ, Gleeson AB, Love RJ, Daniels PW, Gould AR, Hyatt AD. An apparently new virus (family Paramyxo6iridae) infectious for pigs, humans, and fruit bats. Emerg Infect Dis 1998;4:269 – 71. Selvey LA, Wells RM, McCormack JG, Ansford AJ, Murray K, Rogers RJ, Lavercombe PS, Selleck P, Sheridan JW. Infection of humans and horses by a newly described morbillivirus. Med J Aust 1995;162:642 – 5. Tambyah PA. The Nipah virus outbreak — a reminder. Singapore Med J 1999;40:329 – 30. Williamson MM, Hooper PT, Selleck PW, Gleeson LJ, Daniels PW, Westbury HA, Murray PK. Transmission studies of Hendra virus (equine morbillivirus) in fruit bats, horses and cats. Aust Vet J 1998;76:813 – 8. Yu M, Hansson E, Shiell B, Michalski W, Eaton BT, Wang LF. Sequence analysis of the Hendra virus nucleoprotein gene: comparison with other members of the subfamily Paramyxo6irinae. J Gen Virol 1998;79:1775 – 80.
Diagnosis of Nipah virus encephalitis by electron microscopy of cerebrospinal fluid Vincent T.K. Chow *, Paul A. Tambyah, W.M. Yeo, M.C. Phoon, J. Howe Departments of Microbiology and Medicine, Faculty of Medicine, National Uni6ersity of Singapore, Kent Ridge 117597, Singapore Received 22 February 2000; accepted 1 June 2000
Abstract Background: between 1998 and 1999, an outbreak of potentially fatal viral encephalitis erupted among pig farm workers in West Malaysia, and later spread to Singapore where abattoir workers were afflicted. Although Japanese encephalitis virus was initially suspected, the predominant aetiologic agent was subsequently confirmed to be Nipah virus, a novel paramyxovirus related to but distinct from Hendra virus. Objecti6e: to describe a case of Nipah virus encephalitis in a pig farm worker from Malaysia. Study design: the clinical, laboratory and radiological findings of this patient were scrutinized. Special emphasis was placed on the electron microscopic analysis of the cerebrospinal fluid (CSF) specimen from this patient. Results: the neurological deficits indicative of cerebellar involvement were supported by the magnetic resonance imaging that showed prominent cerebellar and brainstem lesions. CSF examination provided further evidence of viral encephalitis. Complement fixation and/or RT-PCR assays were negative for Japanese encephalitis, herpes simplex, measles and mumps viruses. ELISA for detecting IgM and IgG antibodies against Hendra viral antigens were equivocal for the CSF specimen, and tested initially negative for the first serum sample but subsequently positive for the repeat serum sample. Transmission electron microscopy of negatively-stained preparations of CSF revealed enveloped virus-like structures fringed with surface projections as well as nucleocapsids with distinctive helical and herringbone patterns, features consistent with those of other paramyxoviruses, including Hendra virus. Conclusion: this case report reiterates the relevant and feasible role of diagnostic electron microscopy for identifying and/or classifying novel or emerging viral pathogens for which sufficiently specific and sensitive tests are lacking. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Cerebrospinal fluid; Electron microscopy; Nipah virus; Paramyxovirus; Viral encephalitis
1. Introduction Between 1998 and 1999, an outbreak of viral encephalitis involving more than 260 reported * Corresponding author. Fax: +65-7766872. E-mail address: [email protected] (V.T.K. Chow).
cases with over 100 fatalities, occurred in West Malaysia especially among pig farm workers in the states of Perak and Negri Sembilan. Although Japanese encephalitis virus was initially implicated as the main causative agent for this outbreak, epidemiological and laboratory investigations subsequently incriminated the newly recognized
1386-6532/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 6 - 6 5 3 2 ( 0 0 ) 0 0 0 9 4 - 9
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V.T.K. Chow et al. / Journal of Clinical Virology 19 (2000) 143–147
Nipah virus as the predominant aetiologic pathogen (Chua et al., 1999; Farrar, 1999). The outbreak later spread to Singapore where 11 cases of Nipah virus encephalitis were described among workers in an abattoir handling pigs imported from Malaysia (Paton et al., 1999). By mid-1999, the epidemic was eventually contained, thanks to the collaborative efforts of an international team of physicians and scientists, and following the culling of over one million pigs in Malaysia. Named after the village of Kampung Baru Sungai Nipah in Negri Sembilan where it was first isolated, the Nipah virus is a member of the Paramyxo6iridae family, related to but distinct from Hendra virus that is also associated with a viral zoonosis (Yu et al., 1998). We report here the clinical features, laboratory and radiological findings of a case of Nipah virus encephalitis, and demonstrate the presence of virus-like particles consistent with a typical paramyxovirus by electron microscopic analysis of the patient’s cerebrospinal fluid (CSF) specimen. 2. Case report
2.1. Clinical and laboratory findings YBK, a 51-year-old Chinese woman was admitted to Alexandra Hospital, Singapore on 14 March, 1999. She had no past medical history of significance, but complained of a prodrome of generalized malaise for 2 – 3 weeks. In addition, she experienced difficulty in concentrating as well as a gradual decline in her mental status. On admission, she had generalized weakness and a mild headache. Her social history was remarkable in that she lived and worked in a pig farm in Negri Sembilan. Her husband mentioned that prior to her illness, she had cleaned out a barn with a large amount of bat droppings. She denied any consumption of alcohol and tobacco, and claimed no usage of nor allergy to drugs. Physical examination revealed that she was initially very drowsy and oriented to only person and time. She suffered from a right lateral gaze palsy and bilateral nystagmus. A right pronator drift, marked ataxia and mild bilateral motor weakness were also observed.
Laboratory tests showed elevations of total white blood cell count (14.5× 109/l), erythrocyte sedimentation rate (23 mm/h) and C-reactive protein (38.8 mg/l). Her haemoglobin concentration (153 g/l), platelet count (475×109/l), serum electrolytes and liver function tests were all normal. Initial CSF examination showed a white blood cell count of 107/l with lymphocytic pleocytosis, and elevated protein but decreased glucose concentrations, a picture compatible with viral encephalitis. After 2 weeks of hospitalization, a repeat CSF examination showed an increase in white blood cell count to 5.4×107/l. Titres of complement-fixing antibodies to Japanese encephalitis, herpes simplex, measles and mumps viruses were all insignificant. IgM capture and IgG ELISA for detecting IgM and IgG antibodies against Hendra viral antigens was initially negative for the patient’s serum on 23 March, 1999, but subsequently tested positive for a repeat serum sample on 9 April, 1999. The CSF specimen yielded an equivocal ELISA result. RNAs from serum and CSF samples tested negative for Japanese encephalitis virus by RT-PCR using flavivirus NS3 consensus primers (Chow et al., 1993).
2.2. Electron microscopy A CSF specimen was collected on 27 March, 1999, and prepared for electron microscopy (EM) by centrifugation at 10 000 rpm for 30 min at 4°C, and negative staining was performed on both the supernatant and pellet. Briefly, samples were placed onto the dull surface of 300-mesh carboncoated copper grids, left for 1 min, and dried with filter paper. Two percent phosphotungstic acid (pH 7) was then dropped onto the sample-loaded surface which was stained for varying times of 0.5, 1, 1.5 or 2 min. The grids were dried and viewed under a Philips CM-120 transmission electron microscope. At lower magnification were observed numerous particles resembling pleomorphic but roughly spherical viruses 50–150 nm in diameter. Higher magnification revealed enveloped virions fringed with surface projections, together with nucleocapsids with distinctive helical and herring-
V.T.K. Chow et al. / Journal of Clinical Virology 19 (2000) 143–147
bone patterns (Fig. 1). These EM features are strikingly typical of members of the Paramyxo6iridae family, including Hendra or equine morbillivirus (Ackermann and Berthiaume, 1995; Murray et al., 1995; Hyatt and Selleck, 1996).
2.3. Diagnosis and management Considering the clinical, radiological, laboratory and electron microscopic findings together
145
with the epidemiological association of the Nipah virus outbreak with pigs, a diagnosis of Nipah viral encephalitis was made. The patient was nursed in strict isolation, managed with supportive care, and treated empirically with intravenous acyclovir. She gradually improved and was discharged having minimal ataxia and dizziness. At a follow-up visit after one month, she complained of persistent dizziness but was alert and lucid. She was unable to tandem walk, displayed ataxia and
Fig. 1. Electron micrographs of negatively-stained preparations of CSF from the patient with viral encephalitis. (a) Virus-like structures of 100 nm diameter are highlighted by arrows. Magnification 100 000 × . (b – d) Individual enveloped virions fringed with surface projections at higher magnification of 135 000× . Some virus particles are partially disrupted, spilling nucleocapsids of distinctive helical and herringbone patterns.
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V.T.K. Chow et al. / Journal of Clinical Virology 19 (2000) 143–147
dysdiadochokinesia but no other cerebellar signs. Repeat magnetic resonance imaging (MRI) performed during this visit revealed an increase in white matter lesions, including more prominent cerebellar and brainstem lesions. She returned to Malaysia and suffered from a prolonged period of ataxia, but currently has only minimal symptoms. These observations are congruent with those of Lee et al. (1999) who reported that more than half of infected survivors had lingering neurological sequelae.
3. Discussion Symptoms and signs of cerebellar dysfunction in this patient match the neurological manifestations of five out of seven confirmed cases of Nipah virus encephalitis described by Lee et al. (1999), and appear to be a unique feature of this form of encephalitis. Furthermore, these clinical deficits also concur with the MRI scans that exhibited white matter lesions (Lim et al., 1999) and localized prominent lesions to the cerebellum and brainstem. The negative results of the complement fixation and RT-PCR assays excluded encephalitis caused by other viruses, particularly Japanese encephalitis virus. The ELISA detection of Nipah virusspecific antibodies relies on their cross-reactivity with related Hendra viral antigens, which may compromise its specificity and may explain the equivocal and initially negative results for the CSF and serum specimens, respectively. It is especially in these and other circumstances involving unknown or emerging viral pathogens (Tambyah, 1999) for which sufficiently specific and sensitive tests are not available, where diagnostic electron microscopy has a relevant and feasible role as exemplified by this case report. EM images of the ultrastructural morphology of novel viruses can provide clues to their identification through classification to appropriate virus families (Biel and Gelderblom, 1999). The EM observation in this case demonstrates that Nipah virus is present in the central nervous system (CNS) and causes a direct infection. This finding is corroborated by others who have iso-
lated virus (Chua et al., 1999) and shown RTPCR positivity (Paton et al., 1999) from CSF of infected patients. It is difficult to demonstrate other neurotropic viruses such as herpes simplex and enteroviruses in CSF by EM. Therefore, this case study may point to the fact that Nipah infection can cause unusually extensive damage in the CNS, culminating in high virus concentrations in the CSF. The Nipah virus shares significant nucleotide and amino acid homology with the related Hendra virus, another recently described paramyxovirus that causes potentially fatal lung or brain infections in horses and humans (Murray et al., 1995). Williamson et al. (1998) have reported that infected horses, fruit bats and cats can excrete Hendra virus in their urine and saliva. Humans acquire Hendra viral infection via close contact with infected secretions of animals such as horses, although human-to-human transmission is thought to be inefficient (Selvey et al., 1995). Interestingly, another new paramyxovirus known as Menagle virus that is infectious for pigs, humans and fruit bats has also been recently discovered (Philbey et al., 1998). It is noteworthy that being a pig farmer, this patient obviously had close contact with pigs, and even with bat droppings in her barn as divulged in her history. Analysis of Nipah viral infection in pigs, fruit bats and other animal hosts or reservoirs including the existence of persistent carrier states would help to elucidate the natural history and transmission pathways of this deadly viral affliction (Halpin et al., 1999). Furthermore, the long latency and potential for relapse of Hendra viral disease (O’Sullivan et al., 1997) also raises similar questions for Nipah virus infections. References Ackermann HW, Berthiaume L. Atlas of Virus Diagrams. Boca Raton, FL: CRC Press, 1995:50 – 2. Biel SS, Gelderblom HR. Diagnostic electron microscopy is still a timely and rewarding method. J Clin Virol 1999;13:105 – 19. Chow VTK, Seah CLK, Chan YC. Use of NS3 consensus primers for the polymerase chain reaction amplification and sequencing of dengue viruses and other flaviviruses. Arch Virol 1993;133:157 – 70.
V.T.K. Chow et al. / Journal of Clinical Virology 19 (2000) 143–147 Chua KB, Goh KJ, Wong KT, Kamarulzaman A, Tan PS, Ksiazek TG, Zaki SR, Paul G, Lam SK, Tan CT. Fatal encephalitis due to Nipah virus among pig-farmers in Malaysia. Lancet 1999;354:1257–9. Farrar JJ. Nipah-virus encephalitis — investigation of a new infection. Lancet 1999;354:1222–3. Halpin K, Young PL, Field H, Mackenzie JS. Newly discovered viruses of flying foxes. Vet Microbiol 1999;68:83–7. Hyatt AD, Selleck PW. Ultrastructure of equine morbillivirus. Virus Res 1996;43:1–15. Lee KE, Umapathi T, Tan CB, Tjia HT, Chua TS, Oh HM, Fock KM, Kurup A, Das A, Tan AK, Lee WL. The neurological manifestations of Nipah virus encephalitis, a novel paramyxovirus. Ann Neurol 1999;46:428–32. Lim CCT, Sitoh YY, Lee KE, Kurup A, Hui F. Meningoencephalitis caused by a novel paramyxovirus: an advanced MRI case report in an emerging disease. Singapore Med J 1999;40:356 – 8. 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. Science 1995;268:94–7. O’Sullivan JD, Allworth AM, Paterson DL, Snow TM, Boots R, Gleeson LJ, Gould AR, Hyatt AD, Bradfield J. Fatal encephalitis due to novel paramyxovirus transmitted from horses. Lancet 1997;349:93–5.
.
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Paton NI, Leo YS, Zaki SR, Auchus AP, Lee KE, Ling AE, Chew SK, Ang B, Rollin PE, Umapathi T, Sng I, Lee CC, Lim E, Ksiazek TG. Outbreak of Nipah-virus infection among abattoir workers in Singapore. Lancet 1999;354:1253– 6. Philbey AW, Kirkland PD, Ross AD, Davis RJ, Gleeson AB, Love RJ, Daniels PW, Gould AR, Hyatt AD. An apparently new virus (family Paramyxo6iridae) infectious for pigs, humans, and fruit bats. Emerg Infect Dis 1998;4:269 – 71. Selvey LA, Wells RM, McCormack JG, Ansford AJ, Murray K, Rogers RJ, Lavercombe PS, Selleck P, Sheridan JW. Infection of humans and horses by a newly described morbillivirus. Med J Aust 1995;162:642 – 5. Tambyah PA. The Nipah virus outbreak — a reminder. Singapore Med J 1999;40:329 – 30. Williamson MM, Hooper PT, Selleck PW, Gleeson LJ, Daniels PW, Westbury HA, Murray PK. Transmission studies of Hendra virus (equine morbillivirus) in fruit bats, horses and cats. Aust Vet J 1998;76:813 – 8. Yu M, Hansson E, Shiell B, Michalski W, Eaton BT, Wang LF. Sequence analysis of the Hendra virus nucleoprotein gene: comparison with other members of the subfamily Paramyxo6irinae. J Gen Virol 1998;79:1775 – 80.