J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.71.4.552 on 1 October 2001. Downloaded from 552 J Neurol Neurosurg Psychiatry 2001;71:552–554 SHORT REPORT Late presentation of Nipah virus encephalitis and kinetics of the humoral immune response S C Wong, M H Ooi, MNLWong,PHTio,TSolomon, M J Cardosa Abstract and parents to a pig farm in Negeri Sembilan, Nipah virus is a newly discovered para- peninsular Malaysia, in 1997. Her work here myxovirus transmitted directly from pigs included feeding the pigs, artificial insemina- to humans. During a large encephalitis tion of sows, delivering piglets, and injecting outbreak in Malaysia and Singapore in sick pigs. In December 1998, when the Nipah 1998–9, most patients presented acutely. A virus outbreak reached Negeri Sembilan, her 12 year old child is described who devel- father became ill with encephalitis. In the same oped encephalitis 4 months after exposure week our patient hada2dayfebrile illness with to the virus. She was diagnosed by a new a runny nose, cough, headache, and blurred indirect IgG enzyme linked immuno- vision, which resolved spontaneously. In Janu- sorbent assay (ELISA), which is also ary 1999, after her father’s death, she returned described. The late presentation and IgG to Sarawak with her family. In February she subclass responses had similarities to had another brief episode of fever and chills subacute sclerosing panencephalitis. which resolved with paracetamol. Her mother Nipah virus should be considered in and sisters had remained well throughout. As a patients with encephalitis even months child the patient had been immunised with after their possible exposure. BCG, hepatitis B, oral polio vaccine, measles, (J Neurol Neurosurg Psychiatry 2001;71:552–554) diphtheria, pertussis, and tetanus. During the encephalitis outbreak she, along with many Keywords: Nipah virus; encephalitis; diagnosis others, was vaccinated against Japanese en- cephalitis virus (JEV). Between September 1998 and June 1999, an On examination at Sibu hospital, the patient outbreak of severe viral encephalitis occurred was apyrexial, and looked well. General in peninsular Malaysia and Singapore, caused medical examination was normal. She was fully 12 conscious, but during the examination devel- by the newly discovered Nipah virus. This http://jnnp.bmj.com/ Department of oped a further 4 minute episode of rhythmic Paediatrics, Sibu paramyxovirus, closely related to Hendra 3 Hospital, Sibu, virus, caused illness in pigs, and humans in jerking of the left leg. This limb was weak Sarawak, Malaysia close contact with pigs or their secretions, such (power grade 4/5 in all groups), reflexes were SCWong as farmers and abattoir workers. During the normal, but tone was increased in both the left M H Ooi outbreak, the diagnosis was made by virus iso- leg and left arm. She had no sensory loss, MNLWong lation from CSF, or by serological tests against although she continued to report paraesthesia of the left leg. Over the next few hours her Institute of Health and Hendra virus, because serological tests for 4 residual weakness and paraesthesia resolved. Community Medicine, Nipah virus had not yet been developed. Most on September 25, 2021 by guest. Protected copyright. Universiti Malaysia patients infected with Nipah virus presented Initial investigations including full blood Sarawak, Kota with acute encephalitis,256 but three patients count, urea, electrolytes, and liver function Samarahan, Sarawak, had a neurological relapse up to 39 days after tests, were normal, except for a slightly low Malaysia 4 sodium concentration at 132 mmol/l. Exam- P H Tio an initial mild illness. We report here a patient M J Cardosa who presented with mild Nipah encephalitis 4 ination of CSF showed a pleocytosis with 170 months after exposure to the virus; in addition cells/mm3 (100% lymphocytes), a protein con- Department of we describe the development of an indirect IgG centration of 82 mg/dl, and a glucose concen- Neurological Science, enzyme linked immunosorbent assay (ELISA) tration of 48 mg/dl. No organisms were seen University of for diagnosing Nipah virus infection, and the and bacterial culture was negative. Brain CT Liverpool, Walton was normal. Centre for Neurology IgG subclass responses. and Neurosurgery, Over the next 2 days, the patient had an Liverpool L9 7LJ, UK Case report intermittent mild pyrexia, and two brief T Solomon A 12 year old girl presented to Sibu hospital, episodes of generalised tonic-clonic seizures, Sarawak in April 1999 witha2dayhistory of which resolved without treatment. After load- Correspondence to: fever and right frontal headache, and a 5 ing with intravenous phenytoin (10 mg/kg) she Dr T Solomon [email protected] minute episode of sudden jerking movements had no further convulsions. Because of the of the left leg, which was followed by weakness CSF pleocytosis she was treated with penicillin Received 12 February 2001 and numbness of the same limb. and chloramphenicol, but she made an un- and in revised form 30 May 2001 She was born in Sarawak, on the island of eventful recovery and was discharged well, 10 Accepted 8 June 2001 Borneo, but had moved with her two sisters days after admission. At 1 week, 1 month, 6 www.jnnp.com J Neurol Neurosurg Psychiatry: first published as 10.1136/jnnp.71.4.552 on 1 October 2001. Downloaded from Nipah virus encephalitis 553 3 2 1 Adjusted OD 0 Jul 99 Jul 00 Oct 99 Apr 99 Apr 00 Jan 99 Feb 99 Jan 00 Feb 00 Dec 98 Jun 99 Dec 99 Jun 00 Sep 99 Mar 99 Mar 00 Aug 99 Aug 00 Nov 99 May 99 May 00 Month Date of exposure to Nipah virus Figure 1 Kinetics of the serum IgG response (dilution 1 in 1000) in a patient with Nipah encephalitis. She was exposed to the virus in December 1998, but developed encephalitis in April 1999. months, and 1 year follow up she remained OD490 of the control wells containing unin- well, and was working as a waitress. fected cell lysates was determined to be the adjusted OD490. The patient’s acute CSF and VIROLOGICAL INVESTIGATIONS serum samples from April were both positive The patient’s CSF and serum were negative for for IgG against Nipah virus when tested at 7 IgM against JEV and dengue viruses. Serum 1:100 dilution, with OD490 of 0.6 and 2.4 IgG was strongly positive for JEV at 1:500 respectively. The second serum sample taken 2 dilution, which was consistent with her recent weeks after admission showed that a clear IgG vaccination. Polymerase chain reaction (PCR) seroconversion had occurred (fig 1). IgG of CSF and serum was negative for enterovi- remained high 12 months after the illness. ruses and adenoviruses.8 No virus was isolated. Serum samples from her sisters and mother RNA extracted from 50 µl CSF was subjected were negative. An acute serum sample submit- to RT PCR using primers for a portion of the N ted to the Nipah virus task force in Kuala gene of Nipah virus,9 but was negative. The Lumpur was also reported to be positive for patient’s acute and convalescent samples were IgG antibodies against Hendra virus, but was investigated using an indirect IgG ELISA for negative for IgM. antibodies against Nipah virus, which we It was not possible to develop an IgM developed as follows. capture ELISA for detection of Nipah virus specific IgM due to lack of suitable contain- DEVELOPMENT OF INDIRECT IGG ELISA ment facilities for generating hyperimmune Vero E6 cells were infected with a Nipah virus antisera. We therefore used the indirect ELISA strain isolated from the pons of a fatal human to test for the presence of Nipah virus specific case in 1998. Nipah virus infected and IgG subclasses using mouse monoclonal anti- uninfected Vero cells were lysed using a hypot- bodies against IgG1, IgG2, IgG3, and IgG4 http://jnnp.bmj.com/ onic buVer containing 1% TritonX100, then (Oxoid, Baskingstoke, Hants, UK). The bound heat inactivated for 30 minutes at 56oC before monoclonal antibodies were detected using a coating microtitre plates (Maxisorp C bottom, rabbit antimouse IgG HRP (Dako, Glostrup, Nalge Nunc International, Rochester, NY, Denmark). Nipah virus specific IgG1 was USA) at 1:500 dilution. Lysates were also sub- detected in all serum specimens and in the jected to RNA extraction and the identity of CSF (table 1). IgG3 was negative in the CSF the virus was confirmed using RT PCR and and first serum sample, but was positive in serum 2 weeks later. IgG2 and IgG4 were not nucleic acid sequencing. Serum dilutions were on September 25, 2021 by guest. Protected copyright. loaded into duplicate wells coated with infected detected. cell and control lysates and incubated for 1 hour at room temperature before washing and Discussion the addition of conjugated rabbit antihuman The 1998 epidemic of encephalitis in Malaysia IgG (Dako, Glostrup, Denmark) for 1 hour. started in Ipoh, in the north, and soon spread Bound conjugate was detected using the among pig farming communities across the colorigenic substrate o-phenylenediamine and country. Being a major pig farming area, Neg- hydrogen peroxide; the reaction was stopped eri Sembilan was severely aVected. Although with 2M sulphuric acid. The absorbance was Japanese encephalitis was initially suspected, read at 490 nm wavelength with 650 nm as the the fact that pigs were also sick, and that most reference wavelength. The mean OD490 of the victims were adults in contact with pig wells containing infected lysates minus the secretions made it unlikely.10 A virus isolated from a patient’s CSF was shown to be a Table 1 Immunoglobulin G subclass determination in a patient with Nipah encephalitis paramyxovirus similar to Hendra virus, which 9 CSF April Serum April Serum May Serum October Serum August aVected humans and horses in Australia.