Outbreak In Malaysia Research Articles

Recent progress in henipavirus research

Following the discovery of two new paramyxoviruses in the 1990s, much effort has been placed on rapidly finding the reservoir hosts, characterising the genomes, identifying the viral receptors and formulating potential vaccines and therapeutic options for these viruses, Hendra and Nipah viruses caused zoonotic disease on a scale not seen before with other paramyxoviruses. Nipah virus particularly caused high morbidity and mortality in humans and high morbidity in pig populations in the first outbreak in Malaysia. Both viruses continue to pose a threat with sporadic outbreaks continuing into the 21st century. Experimental and surveillance studies identified that pteropus bats are the reservoir hosts. Research continues in an attempt to understand events that precipitated spillover of these viruses. Discovered on the cusp of the molecular technology revolution, much progress has been made in understanding these new viruses. This review endeavours to capture the depth and breadth of these recent advances.

Quantitative estimation of Nipah virus replication kinetics in vitro

BackgroundNipah virus is a zoonotic virus isolated from an outbreak in Malaysia in 1998. The virus causes infections in humans, pigs, and several other domestic animals. It has also been isolated from fruit bats. The pathogenesis of Nipah virus infection is still not well described. In the present study, Nipah virus replication kinetics were estimated from infection of African green monkey kidney cells (Vero) using the one-step SYBR® Green I-based quantitative real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) assay.ResultsThe qRT-PCR had a dynamic range of at least seven orders of magnitude and can detect Nipah virus from as low as one PFU/μL. Following initiation of infection, it was estimated that Nipah virus RNA doubles at every ~40 minutes and attained peak intracellular virus RNA level of ~8.4 log PFU/μL at about 32 hours post-infection (PI). Significant extracellular Nipah virus RNA release occurred only after 8 hours PI and the level peaked at ~7.9 log PFU/μL at 64 hours PI. The estimated rate of Nipah virus RNA released into the cell culture medium was ~0.07 log PFU/μL per hour and less than 10% of the released Nipah virus RNA was infectious.ConclusionThe SYBR® Green I-based qRT-PCR assay enabled quantitative assessment of Nipah virus RNA synthesis in Vero cells. A low rate of Nipah virus extracellular RNA release and low infectious virus yield together with extensive syncytial formation during the infection support a cell-to-cell spread mechanism for Nipah virus infection.

Phylogenetic evidence for inter-typic recombination in the emergence of human enterovirus 71 subgenotypes

BackgroundHuman enterovirus 71 (EV-71) is a common causative agent of hand, foot and mouth disease (HFMD). In recent years, the virus has caused several outbreaks with high numbers of deaths and severe neurological complications. Several new EV-71 subgenotypes were identified from these outbreaks. The mechanisms that contributed to the emergence of these subgenotypes are unknown.ResultsSix EV-71 isolates from an outbreak in Malaysia, in 1997, were sequenced completely. These isolates were identified as EV-71 subgenotypes, B3, B4 and C2. A phylogenetic tree that correlated well with the present enterovirus classification scheme was established using these full genome sequences and all other available full genome sequences of EV-71 and human enterovirus A (HEV-A). Using the 5' UTR, P2 and P3 genomic regions, however, isolates of EV-71 subgenotypes B3 and C4 segregated away from other EV-71 subgenotypes into a cluster together with coxsackievirus A16 (CV-A16/G10) and EV-71 subgenotype C2 clustered with CV-A8. Results from the similarity plot analyses supported the clustering of these isolates with other HEV-A. In contrast, at the same genomic regions, a CV-A16 isolate, Tainan5079, clustered with EV-71. This suggests that amongst EV-71 and CV-A16, only the structural genes were conserved. The 3' end of the virus genome varied and consisted of sequences highly similar to various HEV-A viruses. Numerous recombination crossover breakpoints were identified within the non-structural genes of some of these newer EV-71 subgenotypes.ConclusionPhylogenetic evidence obtained from analyses of the full genome sequence supports the possible occurrence of inter-typic recombination involving EV-71 and various HEV-A, including CV-A16, the most common causal agent of HFMD. It is suggested that these recombination events played important roles in the emergence of the various EV-71 subgenotypes.

Nipah Virus Strain Variation

To the Editor: AbuBakar et al. described strain variation in Nipah virus during the 1998–1999 outbreak in Malaysia (1). They found an isolate from pigs in Perak, as well as from a flying fox, that differed markedly from pig and human isolates from the main epidemic in southern Malaysia. AbuBakar et al. proposed that this finding indicates 2 separate spillover events from bats to pigs occurred, the first in Perak in 1998 and the second in southern Malaysia in 1999. However, investigations at the time of the outbreak showed that many pigs were moved from Perak onto southern farms in early 1999. We suggest that successive spillovers of the pig population in the north can also explain the observed strain differences between northern and southern isolates. A model from experimental studies and active farm data demonstrate that Nipah virus may have circulated repeatedly and become endemic within 1 or several large pig farms in Perak (J.R.C. Pulliam, unpub. data), which is consistent with the occurrence of human cases in Perak before the 1998–1999 outbreak. Evolution of the virus population in pigs, followed by the reintroduction in Perak of the original strain from bats and its subsequent southward movement in infected pigs, would explain observed strain differences. Models suggest that evolution of the virus within pig populations would result in lower death rates but prolonged illness. Although the pig-adapted virus strain may have circulated on both northern and southern farms, sampling biases in favor of the more virulent strain would be expected in areas of high death rates, which would explain the observed genetic relationships between sequenced isolates. We suggest that pigs be experimentally infected with the Perak strain of Nipah virus to determine whether differences exist in illness and death caused by this virus. Further sequencing of virus from archived pig samples will clarify with greater confidence whether multiple strains circulated in both regions.

Human enterovirus 71 subgenotype B3 lacks coxsackievirus A16-like neurovirulence in mice infection

BackgroundAt least three different EV-71 subgenotypes were identified from an outbreak in Malaysia in 1998. The subgenotypes C2 and B4 were associated with the severe and fatal infections, whereas the B3 virus was associated with mild to subclinical infections. The B3 virus genome sequences had ≥85% similarity at the 3' end to CV-A16. This offers opportunities to examine if there are characteristic similarities and differences in virulence between CV-A16, EV-71 B3 and EV-71 B4 and to determine if the presence of the CV-A16-liked genes in EV-71 B3 would also confer the virus with a CV-A16-liked neurovirulence in mice model infection.ResultsAnalysis of human enterovirus 71 (EV-71) subgenotype B3 genome sequences revealed that the 3D RNA polymerase and domain Z of the 3'-untranslating region RNA secondary structure had high similarity to CV-A16. Intracerebral inoculation of one-day old mice with the virus resulted in 16% of the mice showing swollen hind limbs and significantly lower weight gain in comparison to EV-71 B4-infected mice. None of the mice presented with hind leg paralysis typical in all the CV-A16 infected mice. CV-A16 genome sequences were amplified from the CV-A16-infected mice brain but no amplification was obtained from all the EV-71-inoculated mice suggesting that no replication had taken place in the suckling mice brain.ConclusionThe findings presented here suggest that EV-71 B3 viruses had CV-A16-liked non-structural gene features at the 3'-end of the genome. Their presence could have affected virulence by affecting the mice general health but was insufficient to confer the EV-71 B3 virus a CV-A16-liked neurovirulence in mice model infection.

日本の周辺国で問題となっているウイルス感染症 ４．ニパウイルス感染症

Nipah virus (NiV), emerged in Peninsular Malaysia, caused an outbreak of severe febrile encephalitis in humans and respiratory diseases in pigs between 1998 and 1999. By May of 1999, the death of 105 humans and the culling of about 1.1 million pigs were reported. Fruitbats of Pteropid species were identified as the natural reservoir hosts. The epidemiological studies suggested that NiV was introduced into pig farms by fruitbats, and was than transmitted to humans (mainly pig farmers) and other animals such as dogs, cats and horses. In 2004, NiV reappeared in Bangladesh with greater lethality. In contrast to the Malaysia case, epidemiologic characteristics of this outbreak suggested the possibility of fruitbats-to-person, or person-to-person transmission. In this article, the epidemiological comparison between two outbreaks in Malaysia and Bangladesh, and the new-trends of virological studies of NiV will be discussed.

Exotic and emerging viral encephalitides.

The exotic and emerging viral encephalitides are caused by animal or human viruses and characterised by sudden unexpected outbreaks of neurological disease, usually in tropical and sub-tropical regions, but sometimes spreading to temperate areas. Although a wide range of viruses come within this label, as this review highlights, there are common research questions as to the origin and spread of the viruses, the contribution of viral and host factors to the clinical presentations and outcome, and the possibilities for treatment and vaccination. During 2002, North America experienced the largest ever outbreak of West Nile encephalitis; a poliomyelitis-like flaccid paralysis due to West Nile virus was recognised, and transfusion-related infections were documented. Globally, Japanese encephalitis virus is the most important emerging viral encephalitis; interferon alpha was not effective against Japanese encephalitis in a double-blind placebo-controlled trial, but new chimeric vaccines are in development. Recent work suggests Japanese encephalitis virus originated in the Indonesia-Malaysia region, and spread from there. The origin of Nipah virus, which caused an encephalitis outbreak in Malaysia in 1998, is not known, but flying foxes have been identified as a natural host. Enterovirus 71 continues to cause large outbreaks of hand foot and mouth disease across Asia, associated with neurological and systemic complications; recent work has focused on the pathogenesis of these complications. Disease surveillance remains important for the early recognition and containment of encephalitis outbreaks. Detailed clinical and laboratory studies will help answer the key questions, but there is a need to ensure the results translate to real benefits for the communities affected by these diseases.

Nipah virus outbreak in Malaysia

Nipah virus, a novel paramyxovirus, closely related to Hendra virus emerged in northern part of Peninsular Malaysia in 1998. The virus caused an outbreak of severe febrile encephalitis in humans with a high mortality rate, whereas, in pigs, encephalitis and respiratory diseases but with a relatively low mortality rate. The outbreak subsequently spread to various regions of the country and Singapore in the south due to the movement of infected pigs. Nipah virus caused systemic infections in humans, pigs and other mammals. Histopathological and radiological findings were characteristic of the disease. Fruitbats of Pteropid species were identified as the natural reservoir hosts. Evidence suggested that climatic and anthropogenic driven ecological changes coupled with the location of piggeries in orchard and the design of pigsties allowed the spill-over of this novel paramyxovirus from its reservoir host into the domestic pigs and ultimately to humans and other animals.

Genetic diversity of enterovirus 71 isolated from cases of hand, foot and mouth disease in Yokohama City between 1982 and 2000

Enterovirus 71 (EV71) is known as one of the major causative agents of hand, foot and mouse disease (HFMD) and is also associated with neurological manifestations such as aseptic meningitis, polio-like paralysis and encephalitis. Recently, large HFMD outbreaks, involving severe neurological complications, have been experienced in Malaysia, Taiwan and some other countries in the Western-Pacific region. To investigate the genetic diversity of EV71 isolates in a single community in Japan, nucleotide sequences of the VP4 region of 52 EV71 isolates in Yokohama City from 1982 to 2000 were determined and the phylogenetic relationship was compared with other referential EV71 strains in Japan and in the world. There were two major genotypes of EV71 in Yokohama City through the 1980's and 1990's. Six EV71 isolates in the early 1980's in Yokohama City were closely related to those from HFMD outbreaks in Japan and from outbreaks of polio-like paralysis in Europe in the 1970's. During recent HFMD outbreaks in 1997 and 2000, two distinct genotypes of EV71 were co-circulating in Yokohama City as in HFMD outbreaks in Malaysia and Taiwan. However, the genetic diversity of EV71 in Yokohama City was not directly correlated with the severity of HFMD. The results confirmed the circulation of two distinct genotypes of EV71 over the past 20 years in Japan.

Nipah virus encephalitis outbreak in Malaysia.

Emerging infectious diseases involving zoonosis have become important global health problems. The 1998 outbreak of severe febrile encephalitis among pig farmers in Malaysia caused by a newly emergent paramyxovirus, Nipah virus, is a good example. This disease has the potential to spread to other countries through infected animals and can cause considerable economic loss. The clinical presentation includes segmental myoclonus, areflexia, hypertension, and tachycardia, and histologic evidence includes endothelial damage and vasculitis of the brain and other major organs. Magnetic resonance imaging has demonstrated the presence of discrete high-signal-intensity lesions disseminated throughout the brain. Nipah virus causes syncytial formation in Vero cells and is antigenically related to Hendra virus. The Island flying fox (Pteropus hypomelanus; the fruit bat) is a likely reservoir of this virus. The outbreak in Malaysia was controlled through the culling of >1 million pigs.

Nipah virus outbreak in Malaysia, 1998-1999

Nipah virus outbreak in Malaysia, 1998-1999

Some health implications of globalization in Thailand.

In recent years Thailand’s economy has becomeincreasingly dependent on international forces (1).Withthisexposurehavecomeadvancesinhealthcaretechnologyand improvements in living standards,aswell as increasing disparities between social groups(2) and exposure to health risks from other parts ofthe world (3, 4).Prior to 1997, when the economy was strong,there was intense competition for a share of thehealth market. Resources were invested in specu-lative markets with potential for large expansion.Private hospital beds increased from 8066 in 1982to 21 297 in 1992 and 34 973 in 1996. The numberof specialized doctors in private hospitals increased,leading to shortages in the public sector (5). Thecultureoffreeenterprisebroughtwithitanenlarged middle class, insurance coverage forhospitalization, tax incentives for private healthcare, heavy investment in advanced health technol-ogy for private sector use, and an internal ‘‘braindrain’’, at the expense of public health (5, 6).Aggressive promotion increased the demand forexpensive imported medicines and procedures (7,8). The cost of medical care for civil servants andstate employees has quadrupled in the last sevenyears, reflecting the lack of adequate governance inthe health care business sector (5, 9). Meanwhilethe share of the underprivileged in the country’soverall wealth was decreasing (4). The slump of1997, followed by devaluation of the baht, andrecession with its concomitant negative healthimpact, reflects the country’s overdependence oncheap labour and foreign investment, and conse-quent inability to control and protect its owneconomy.Direct health effectsPerhapsthemostimportant directeffectofglobaliza-tiononhealthinThailandisunequalaccesstomedicalcare by different social groups. The rise in importedsophisticated technologies has increased costs andnecessitated new training. An analysis made in 1996found that the average cost of medical care peradmission was 1558 bahts for health cardholders(rural) and 9981 bahts for civil servants (privileged), asixfold difference (10). If these facilities were treatingsimilar diseases, explanations are needed for the hugevariation. The economic gap might create demandunrelatedtoneedanddistortmarketcompetition.Theorganization of health service delivery was obscure,and there were no rules governing the payment ofproviders. Unequal access to care was reflected byunequal health status (2). Infant mortality in thepoorestregionswastwiceashighasintherichestones.Second, there are increasing problems ofenvironmental pollution. These include inadequatetreatmentofrawsewage(forinstance,intouristareas),and the notorious air pollution in Bangkok and otherbig cities(11).Environmentaldegradation anddisrup-tion of the ecosystem have led to frequent floods andchangesindisease vectorbehaviour.Theconstructionof a dam in the North-eastern region, financed by aloan from a development bank, has caused naturaldisasters affecting food production (12).Third, concerns about new infections and theresurgence of old ones have been on the rise.Internationaltradeandtravelareshapingthepatternsof epidemics. The plague scare in India had world-wide reverberations. The nipah virus outbreak inMalaysia caused concerns in Thailand (13). Choleraepidemics can inflict enormous costs on a countryandthisresultsinattemptstohidethembycallingthedisease‘‘severediarrhoea’’.Thecostsassociatedwithcontrolling HIV infection continue to rise. Fears offoot and mouth disease have affected meat con-sumption. The control of new dangers of this kindwill require global cooperation but many aspects ofcontrol have to be country-specific.Fourth, globalization has brought with itunhealthy lifestyles. Health has been damaged bythepromotionoffashionabledrugs,foodsandotherconsumer products such as tobacco, alcohol,melatonin and Viagra. Fifth and finally, globalizationbringswithitmanyconcernsabouthealthethics.Forinstance, the options for genetic manipulation andthepatentingofthetechnologieswillhavedirectandfar-reaching effects on health and social well-being.

Molecular evidence of clonality amongst Vibrio cholerae O1 biotype El Tor during an outbreak in Malaysia.

Forty-three clinical strains of V. cholerae O1 biotype E1 Tor were isolated between 3 May and 10 June 1998 during an outbreak in the metropolitan area of Kuala Lumpur and its suburbs. With the exception of three Inaba strains that were restricted to three members of a family, all the others belonged to the Ogawa serotype. The strains were analysed for clonality using ribotyping and pulsed-field gel electrophoresis (PFGE). Two ribotypes, V/B21a and B27, were identified among 40 Ogawa isolates using BglI restriction endonuclease. Ribotype V/B21a has been described previously from Taiwan and Colombia and several Asian countries while B27 has been reported among isolates from Senegal. The three Inaba strains belonged to one ribotype, designated type A, not previously reported. PFGE analysis using NotI revealed that all isolates within a ribotype had identical profiles demonstrating clonality amongst the strains. Dice coefficient analysis of the two Ogawa genotypes revealed 89% similarity on ribotype patterns and 91.3% on PFGE profiles. Ribotype V/B21a isolates were associated with cases from dispersed areas of Kuala Lumpur and its suburbs while ribotype B27 was restricted to cases from one particular area suggesting a common-source outbreak.

Molecular epidemiology and evolution of enterovirus 71 strains isolated from 1970 to 1998.

Enterovirus 71 (EV71) (genus Enterovirus, family Picornaviridae), a common cause of hand, foot, and mouth disease (HFMD), may also cause severe neurological diseases, such as encephalitis and poliomyelitis-like paralysis. To examine the genetic diversity and rate of evolution of EV71, we have determined and analyzed complete VP1 sequences (891 nucleotides) for 113 EV71 strains isolated in the United States and five other countries from 1970 to 1998. Nucleotide sequence comparisons demonstrated three distinct EV71 genotypes, designated A, B, and C. The genetic variation within genotypes (12% or fewer nucleotide differences) was less than the variation between genotypes (16.5 to 19.7%). Strains of all three genotypes were at least 94% identical to one another in deduced amino acid sequence. The EV71 prototype strain, BrCr-CA-70, isolated in California in 1970, is the sole member of genotype A. Strains isolated in the United States and Australia during the period from 1972 to 1988, a 1994 Colombian isolate, and isolates from a large HFMD outbreak in Malaysia in 1997 are all members of genotype B. Although strains of genotype B continue to circulate in other parts of the world, none have been isolated in the United States since 1988. Genotype C contains strains isolated in 1985 or later in the United States, Canada, Australia, and the Republic of China. The annual rate of evolution within both the B and C genotypes was estimated to be approximately 1.35 x 10(-2) substitutions per nucleotide and is similar to the rate observed for poliovirus. The results indicate that EV71 is a genetically diverse, rapidly evolving virus. Its worldwide circulation and potential to cause severe disease underscore the need for additional surveillance and improved methods to identify EV71 in human disease.

Molecular epidemiologic analysis of Vibrio cholerae O1 isolates by pulsed-field gel electrophoresis.

Isolates of Vibrio cholerae O1 El Tor from two well-defined cholera outbreaks in Malaysia were analyzed by using pulsed-field gel electrophoresis (PFGE). Isolates from sporadic cases occurring during the same time period were also studied. Digestion of chromosomal DNA from these isolates of V. cholerae O1 with restriction endonucleases NotI (5'-GCGGCCGC-3') and SfiI (5'-GGCCNNNN-3'), followed by PFGE, produced restriction endonuclease analysis (REA) patterns consisting of 13 to 24 bands (ranging in size from 46 to 398 kbp). Analysis of the REA patterns generated by PFGE after digestion with NotI and SfiI suggested the clonal nature and close genetic identity of the isolates obtained during each of the two outbreaks (Dice coefficient, 0.93 to 1.0). Although they had very similar REA patterns, the two outbreak clones were not identical. Isolates of V. cholerae O1 from sporadic cases, on the other hand, appeared to be much more heterogeneous (five different REA patterns detected in the five isolates tested; Dice coefficient, 0.31 to 0.81) than those obtained during the two outbreaks. We conclude that PFGE of V. cholerae O1 chromosomal DNA digested with infrequently cutting restriction endonucleases is a useful method for molecular typing of V. cholerae isolates for epidemiological purposes.

Rapid Reduction of Brown Planthoppers By Intensive Light Trapping During an Outbreak in Malaysia

Rapid Reduction of Brown Planthoppers By Intensive Light Trapping During an Outbreak in Malaysia

A shellfish-borne cholera outbreak in Malaysia

11,650 specimens were studied bacteriologically. 139 El Tor Inaba strains were isolated from the stool of suspected patients and contacts, from water of various wells and tributaries of Kelantan river, and from shellfish. 15 El Tor Inaba, 25 non-agglutinable and 10 partially-agglutinable and auto-agglutinable (rough) strains were demonstrated in edible types of shellfish. 55 non-agglutinable vibrios were isolated from stools of suspected patients, from shellfish and water, and were regarded as pathogenic. Shellfish and water were considered as predominant vehicles for the transmission of cholera in this outbreak. The short-term carrier state is emphasized and the causal relationship of cholera with carrier was found to be insignificant in this outbreak. Bacteriological significance of partially-agglutinable and auto-agglutinable (rough) El Tor vibrios is discussed.

Outbreak Of Nipah Encephalitis In Greater Faridpur District

Nipah viral encephalitis is one of the fatal re-emerging infections especially in southeast Asia. After its outbreak in Malaysia and Singapore; repeated outbreaks occurred at western part of Bangladesh especially in Faridpur region. Besides, sporadic attacks appear to occur in the country throughout the year. Here two Nipah outbreaks in greater Faridpur district in 2003 and 2004 are described along with brief review on transmission of the virus. Where the history of illness among patients are very much in favour of man to man transmission. Moreover the death of an intern doctor from Nipah encephalitis who was involved in managing such patients in Faridpur Medical College Hospital strongly suggests man to man transmission of this virus. So, aim of this review article to make the health personnel and general people be aware about man to man transmission of virus, so that they can adapt personal protection equipment (PPE) for their protection against this deadly disease. DOI: 10.3329/fmcj.v5i2.6825Faridpur Med. Coll. J. 2010;5(2):63-65