Japanese encephalitis virus: Biological clones from a clinical isolate quasispecies show differing neurovirulence in vitro and in a mouse model

@#The Japanese encephalitis virus (JEV), a leading cause of encephalitis, exists as quasispecies in clinical isolates. Using a limiting dilution method combined with immunohistochemistry to detect viral antigens, 10 biological clones were isolated and purified from a clinical JEV isolate (CNS138/9) derived from an autopsy brain. These biological clones were tested for neurovirulence in SK-N-MC and NIE-115 neuronal cells, and a 2-week-old, footpad-infected, JE mouse model. Nine clones were found to be neurovirulent; one clone neuroattenuated. Although further studies are needed to determine genotypic differences, if any, in these clones, the limiting dilution purification and neurovirulence testing methods described herein should be useful for phenotypic studies of quasispecies of neurotropic viruses in general, and JEV and other flaviviruses in particular.

Level 4 comprehensive epilepsy program in Malaysia, a resource-limited country

Background and Objective: There is a great challenge to establish a level 4 epilepsy care offeringcomplete evaluation for epilepsy surgery including invasive monitoring in a resource-limited country.This study aimed to report the setup of a level 4 comprehensive epilepsy program in Malaysia and theoutcome of epilepsy surgery over the past 4 years. Methods: This is a retrospective study analyzingcases with intractable epilepsy in a comprehensive epilepsy program in University Malaya MedicalCenter (UMMC), Kuala Lumpur, from January 2012 to August 2016. Results: A total of 92 caseshad comprehensive epilepsy evaluation from January 2012 till August 2016. The mean age was 35.57years old (range 15-59) and 54 (58.7%) were male. There were 17 cases having epilepsy surgeryafter stage-1 evaluation. Eleven cases had mesial temporal sclerosis and 81% achieved Engel classI surgical outcome. Six cases had lesionectomy and 60% had Engel class I outcome. A total of 16surgeries were performed after stage-2 evaluation, including invasive EEG monitoring in 9 cases.Among those with surgery performed more than 12 months from the time of data collection, 5/10(50%) achieved Engel I outcome, whereas 2 (20%) had worthwhile improvement (Engel class III)with 75% and 90% seizure reduction.Conclusion: Level 4 epilepsy care has an important role and is possible with joint multidisciplinaryeffort in a middle-income country like Malaysia despite resource limitation.

Pathological findings in a mouse model of Japanese encephalitis infected via the footpad

We have developed and characterised a mouse model of Japanese encephalitis virus (JEV) infection via footpad inoculation in order to better mimic viral transmission by mosquito bites. Two-week-old and 5-week-old mice consistently developed signs of infection such as ruffled fur, weight loss, hunchback posture, tremors, mask-like facies and occasionally, hindlimb paralysis at 4 days post infection (dpi) and 11-13 dpi, respectively. Most of the animals died within 24 to 48 hours following the onset of signs of infection, with mortalities of 100% and 33.3% in 2-week-old and 5-week-old mice, respectively. Mild meningitis and variable parenchymal inflammation with formation of microglial nodules, focal necrosis and neuronophagia, and perivascular cuffing by inflammatory cells were observed in the caudate nucleus, putamen, thalamus, cerebral cortex, brainstem, and spinal cord. Viral antigens/RNA were demonstrated by immunohistochemisty and in situ hybridization, respectively, in most of these areas as well as in the hippocampus and cerebellum, albeit more focally. The pathological findings in this mouse model were generally similar to human Japanese encephalitis (JE) and other established JE models but perhaps, compared to other JEV mouse models, it demonstrates lethal encephalitic infection more consistently. We believe that our mouse model should be useful to study the pathogenesis of JE, and for testing anti-viral drugs and vaccines

Pathological findings in a mouse model for Coxsackievirus A16 infection

Coxsackievirus A16 (CV-A16) is the leading cause of hand-foot-mouth disease (HFMD), which usually presents as mild and self-limiting symptoms in young children. Rarely, CV-A16 has been reported to cause severe and fatal neurological complications but little is known about these complications. In the present study, 1-day and 7-day old mouse models of CV-A16 were developed using a clinical strain via subcutaneous inoculation. All infected mice exhibited clinical signs of infection, including reduced mobility, limb weakness and paralysis between 3 to 6 days post-infection. Pathologically, the main organs involved were the central nervous system (CNS), skeletal muscles and brown fat. In the CNS, viral antigens as demonstrated by immunohistochemistry, were localized mainly to neurons in the brain stem and spinal cord, suggesting that CV-A16 is neurotropic although inflammation is very mild. The skeletal muscles showed necrosis and myositis due to viral infection as evidenced by the dense viral antigens. Focal viral antigens were also detected in the brown fat. These preliminary pathological findings indicate that our mouse models can be further developed to be useful models for pathogenesis studies, and vaccine and anti-viral drug evaluation.

Pathological findings in a mouse model of Japanese encephalitis infected via the footpad

We have developed and characterised a mouse model of Japanese encephalitis virus (JEV) infection via footpad inoculation in order to better mimic viral transmission by mosquito bites. Two-week-old and 5-week-old mice consistently developed signs of infection such as ruffled fur, weight loss, hunchback posture, tremors, mask-like facies and occasionally, hindlimb paralysis at 4 days post infection (dpi) and 11-13 dpi, respectively. Most of the animals died within 24 to 48 hours following the onset of signs of infection, with mortalities of 100% and 33.3% in 2-week-old and 5-week-old mice, respectively. Mild meningitis and variable parenchymal inflammation with formation of microglial nodules, focal necrosis and neuronophagia, and perivascular cuffing by inflammatory cells were observed in the caudate nucleus, putamen, thalamus, cerebral cortex, brainstem, and spinal cord. Viral antigens/RNA were demonstrated by immunohistochemisty and in situ hybridization, respectively, in most of these areas as well as in the hippocampus and cerebellum, albeit more focally. The pathological findings in this mouse model were generally similar to human Japanese encephalitis (JE) and other established JE models but perhaps, compared to other JEV mouse models, it demonstrates lethal encephalitic infection more consistently. We believe that our mouse model should be useful to study the pathogenesis of JE, and for testing anti-viral drugs and vaccines

Pathological findings in a mouse model for Coxsackievirus A16 infection

Coxsackievirus A16 (CV-A16) is the leading cause of hand-foot-mouth disease (HFMD), which usually presents as mild and self-limiting symptoms in young children. Rarely, CV-A16 has been reported to cause severe and fatal neurological complications but little is known about these complications. In the present study, 1-day and 7-day old mouse models of CV-A16 were developed using a clinical strain via subcutaneous inoculation. All infected mice exhibited clinical signs of infection, including reduced mobility, limb weakness and paralysis between 3 to 6 days post-infection. Pathologically, the main organs involved were the central nervous system (CNS), skeletal muscles and brown fat. In the CNS, viral antigens as demonstrated by immunohistochemistry, were localized mainly to neurons in the brain stem and spinal cord, suggesting that CV-A16 is neurotropic although inflammation is very mild. The skeletal muscles showed necrosis and myositis due to viral infection as evidenced by the dense viral antigens. Focal viral antigens were also detected in the brown fat. These preliminary pathological findings indicate that our mouse models can be further developed to be useful models for pathogenesis studies, and vaccine and anti-viral drug evaluation.

Single mitochondrial DNA deletions in chronic progressive external ophthalmoplegia (CPEO) and Kearns-Sayre syndrome (KSS) patients from a multiethnic Asian population

Mitochondrial DNA (mtDNA) deletions are a major cause of chronic progressive external ophthalmoplegia (CPEO) and Kearns-Sayre syndrome (KSS). We analyzed single mtDNA deletions in 11 CPEO and one KSS patients by means of Southern blot and long polymerase chain reaction (PCR) assays. The deletion sizes ranged from 3.4 kb to 6.9 kb whereas the heteroplasmy level varied from 18.8% to 85.5%. Two unique deletions sized 4320 bp and 4717 bp were found. This study represents the first genetic screen of mtDNA disorders in Malaysia, and it follows the data seen in other published reports on CPEO and KSS genetic aetiology.

The spectrum of elderly myopathies in an Asian population

Myopathies, although presenting more commonly in the younger age group, can occur and contribute signifi cantly to disability in the elderly. To describe the spectrum of elderly myopathies, we reviewed 52 elderly patients (> 65 years) from the University of Malaya Medical Centre muscle biopsy databank, constituting 6.8% of 759 adult patients (> 18 years) who underwent muscle biopsy between 1992 and 2012. Commonest were the infl ammatory myopathies (41/52, 78.8%), of which 43.9% had dermatomyositis; 23.9% polymyositis; 14.6% sporadic inclusion body myositis; 9.8% undifferentiated myositis and 2.4% overlap myositis. Seven patients (13.4%) had genetic myopathy; 2 muscular dystrophy and 5 chronic progressive external ophthalmoplegia, while 4 patients (7.7%) had drug-associated myopathy, 3 with statins. Malignancies were seen in 9.8% of infl ammatory myopathies at diagnosis. Both acquired and genetic myopathies are seen in elderly Malaysians of all ethnicities and should not be misdiagnosed as some are potentially treatable and/or associated with malignancy.

A prevalence study of single nucleotide polymorphisms in the promoter of the apolipoprotein E gene in different ethnic groups in Malaysia

Background and Objective: The promoter of the apolipoprotein E (APOE) gene is polymorphic at positions -491A/T, -427C/T and -219G/T. These single nucleotide polymorphisms may alter transcriptional activity and impact APOE expression due to differential binding of transcription factors. It has been suggested that the -491 A, -427 C and -219 T alleles are associated with a high risk of developing Alzheimer’s disease. This study aims to investigate the frequencies of APOE promoter polymorphisms in three major ethnic groups (Malay, Chinese and Indian) in Malaysia. Method: DNA was extracted from blood obtained from 290 healthy people (Malay: n= 92; Chinese: n= 105; and Indian: n= 93), and the promoter region was amplifi ed using PCR and genotyped by direct sequencing. Result: The Indian group has the lowest frequencies of - 491 A, - 427 C and - 219 T alleles (83.9%, 3.2% and 56.5%, respectively) compared to the Chinese group with the highest frequencies (97.1%, 11.9% and 67.1%, respectively). The frequencies in the Malay group were somewhere in between (94.6%, 8.2% and 61.4%, respectively). Moreover, for the - 491 and - 427 positions, the frequencies of possible genotypes viz., AA or AT or TT and CC or CT or TT, respectively, were statistically signifi cant (P < 0.05, Chi- Square Test) between the 3 ethnic groups. Conclusion: Based on the frequency of APOE promoter polymorphisms alone, the ethnic Indian may be predisposed to lower risks for AD than the Chinese or Malay.

No apparent association between beta-amyloid deposition and apolipoprotein E genotype in the non-demented aging brain in a Malaysian population

Little is known about the relationship between cerebral beta amyloid (Aβ) deposition and apolipoprotein E (ApoE) genotype in either Alzheimer disease or the aging brain in multi-ethnic Southeast Asia. We prospectively examined Aβ deposition in relation to ApoE genotype in 50 normal, non-demented, aging brains drawn from a Malaysian population, aged 52-92 years, using immunohistochemistry to detect Aβ and direct PCR sequencing for genotyping. The prevalence of Aβ deposition was 8%. There was no apparent association between Aβ deposition and possession of the ApoE ε4 allele in our cohort. Out of 4 cases with Aβ deposition, only one case was heterozygous for the ε4 allele; 3 cases did not have the ε4 allele. The Aβ deposition appears to increase with age and is more likely to be vascular-type deposition (cerebral amyloid angiopathy) rather than parenchymal deposition in the form of diffuse and neuritic plaques. A larger sample size with more cases of ApoE ε4 and Aβ deposition is needed to provide conclusive evidence for the apparent non-association between ApoE ε4 and Aβ deposition in the aging brain in our multi-ethnic local population.

The frequency of common mitochondrial DNA mutations in a cohort of Malaysian patients with specifi c mitochondrial encephalomyopathy syndromes

A cohort of Malaysian patients with clinico-pathological diagnosis of three specifi c mitochondrial encephalomyopathy syndromes comprising of mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), myoclonus epilepsy with ragged-red fi bers (MERRF) and Leigh syndrome were studied to determine the frequency of their common mitochondrial DNA mutations. The ‘hot-spot’ point mutations for MELAS, MERRF and Leigh syndrome were screened. In the absence of common point mutations, screening of large-scale deletions as well as sequencing of tRNALeu and tRNALys genes were performed. Of 22 patients studied, nine m.3243A>G mutations, four m.8344A>G mutations, one m.8993T>G mutation and one deletion were identifi ed (65% detection rate). While the m.3243A>G mutation was closely associated with MELAS, the m.8344A>G was more heterogenous, being seen in one MERFF, two isolated mitochondrial myopathies and one Leigh syndrome patient. Screening for m.8993T>G in Leigh syndrome has a low yield as unsurprisingly Leigh syndrome has considerable genetic heterogeneity.

Progressive multifocal leukoencephalopathy limited to the posterior fossa

Progressive multifocal leukoencephalopathy (PML) is a rapidly progressive demyelinating disease caused by the reactivation of JC papova virus usually in immunocompromised hosts.1 The disease is a chronic viral infection resulting in mortality within a year.2 The condition characterized by white matter changes in multiple locations of the brain is caused by destruction of the oligodendrogliocytes.2 We report a case of AIDS associated PML presenting with progressive cerebellar symptoms, with the unusual feature of imaging abnormalities limited to the posterior fossa.

Exercise induced cramps and myoglobinuria in dystrophinopathy – a report of three Malaysian patients

Dystrophinopathies commonly present as Duchenne or Becker muscular dystrophy but rare, unusual phenotypes have also been described. We have identifi ed three Malaysian boys with an unusual form of dystrophinopathy, presenting with exercise-induced cramps and myoglobinuria, but with no apparent muscle weakness. Immunohistochemistry for dystrophin and genetic analysis confi rmed the diagnosis. The frequency of this phenotype is unknown but there have been several case reports. Consistent with these reports, we also found that two of our patients had deletions in the rod domain of dystrophin, which has been suggested to be associated with this unusual manifestation

Dystrophin gene analysis in Duchenne/Becker dystrophy in a Malaysian population using multiplex polymerase chain reaction

Dystrophinopathy is the commonest form of muscular dystrophy and comprises clinically recognized forms, Duchenne dystrophy and Becker dystrophy. Mutations in the dystrophin gene which consist of large gene deletions (65%), duplications (5%) and point mutations (30%) are responsible for reducing the amount of functional dystrophin protein in skeletal muscle fi bres leading to fi bre destruction and disease. The aims of this study are to investigate the detection rate, types and distribution of large gene deletions in Malaysian dystrophinopathy patients using the multiplex polymerase chain reaction (MPCR). MPCR of 18 “hot-spot deletion” regions along the dystrophin gene was performed on DNA from 48 muscle biopsy-confi rmed cases of dystrophinopathy. A positive detection rate of 58% (28/48) was observed, where 84% (16/19) Indian, 35% (6/17) Chinese and 50% (6/12) Malay ethnic groups showed deletions in their dystrophin genes. The Malaysian Indians appear to have a higher prevalence for large gene deletions compared to the Chinese and Malays. Further analyses of 42 confi rmed positive cases (present 28 plus previous 14 cases) by MPCR showed the majority of deletions were in the mid-distal region of the dystrophin gene (81% in exons 45-60). The MPCR is a specifi c and sensitive method for confi rmation of gene deletions responsible for dystrophinopathy.

Aetiology of viral central nervous system infection, a Malaysian study

Over 100 viruses are known to cause acute viral encephalitis in human. In order to diagnose a viral central nervous system infection, various laboratory diagnosis methods have been used. In this study, we examined 220 cerebrospinal fluid samples that were received at the Diagnostic Virology Laboratory of University Malaya Medical Centre between year 2004 to 2006, by viral isolation, pathogen specific antibody ELISA, polymerase chain reaction (PCR) and Real-Time PCR. Majority of the samples were from patients <10 years old. Out of 220 samples, 3 were positive for viral isolation, 27 for PCR (inclusive for the 3 positive for viral isolation) and 39 for pathogen specific ELISA. The total positive detection rate of this study was 30%. Herpes virus was the most important aetiologic agent, responsible for 58% of infection, followed by paramyxovirus (especially measles virus) in 26% of infection, and 14% by enterovirus. Parvovirus and flavivirus were the other common viruses. Among the herpes viruses, herpes simplex and cytomegalovirus were the most common.