Development of a sandwich ELISA to detect virus-like-particles in enterovirus A71 vaccines.

The goal of this paper was to develop a sandwich ELISA that can detect intact human enterovirus A71 (EV-A71) virus-like particles (VLPs) in vaccines. This assay specifically detected EV-A71 viruses from different sub-genogroups as well as EV-A71 VLPs, and treatment of VLPs with high heat and low pH reduced or completely abolished detection of the VLPs suggesting that the ELISA detected assembled particles. Using a purified VLP as a reference standard, a quantitative sandwich ELISA (Q-ELISA) was established which was used to monitor the yield and purity of the VLPs during manufacturing. Coupled with immunogenicity studies, the Q-ELISA was used to evaluate the performance of the VLPs and formalin-inactivated EV-A71 vaccine. This assay has the potential to play an important role in the development of an efficient process to produce and purify the VLPs and in examining the quality of EV-A71 vaccines.

Status of research and development of vaccines for enterovirus 71.

Although outbreaks of Hand, Foot, and Mouth Disease (HFMD) in young children have long been recognized worldwide, the occurrence of rare and life-threatening neurological, respiratory, and cardiac complications has propelled this common condition into the spotlight as a major public health problem in the affected countries. Various enteroviruses cause HFMD, but the severe complications have been mostly associated with enterovirus 71 (EV71). Medical treatment is supportive and measures to interrupt transmission have been challenging to implement. Preventive vaccines could have an important clinical impact, especially among children younger than 3 years old who are most susceptible to the neurological complications. Several groups in the highly affected Asia-Pacific region are working towards vaccines against EV71 and some candidates have progressed to late-stage clinical trials with two vaccines recently reported to have been approved by the regulatory authorities in China. This report summarizes current issues and progress in the development of vaccines against EV71.

The effect of vaccination coverage and climate on Japanese encephalitis in Sarawak, Malaysia.

BACKGROUND: Japanese encephalitis (JE) is the leading cause of viral encephalitis across Asia with approximately 70,000 cases a year and 10,000 to 15,000 deaths. Because JE incidence varies widely over time, partly due to inter-annual climate variability effects on mosquito vector abundance, it becomes more complex to assess the effects of a vaccination programme since more or less climatically favourable years could also contribute to a change in incidence post-vaccination. Therefore, the objective of this study was to quantify vaccination effect on confirmed Japanese encephalitis (JE) cases in Sarawak, Malaysia after controlling for climate variability to better understand temporal dynamics of JE virus transmission and control. METHODOLOGY/PRINCIPAL FINDINGS: Monthly data on serologically confirmed JE cases were acquired from Sibu Hospital in Sarawak from 1997 to 2006. JE vaccine coverage (non-vaccine years vs. vaccine years) and meteorological predictor variables, including temperature, rainfall and the Southern Oscillation index (SOI) were tested for their association with JE cases using Poisson time series analysis and controlling for seasonality and long-term trend. Over the 10-years surveillance period, 133 confirmed JE cases were identified. There was an estimated 61% reduction in JE risk after the introduction of vaccination, when no account is taken of the effects of climate. This reduction is only approximately 45% when the effects of inter-annual variability in climate are controlled for in the model. The Poisson model indicated that rainfall (lag 1-month), minimum temperature (lag 6-months) and SOI (lag 6-months) were positively associated with JE cases. CONCLUSIONS/SIGNIFICANCE: This study provides the first improved estimate of JE reduction through vaccination by taking account of climate inter-annual variability. Our analysis confirms that vaccination has substantially reduced JE risk in Sarawak but this benefit may be overestimated if climate effects are ignored.

Enterovirus 75 encephalitis in children, southern India.

Recent outbreaks of enterovirus in Southeast Asia emphasize difficulties in diagnosis of this infection. To address this issue, we report 5 (4.7%) children infected with enterovirus 75 among 106 children with acute encephalitis syndrome during 2005-2007 in southern India. Throat swab specimens may be useful for diagnosis of enterovirus 75 infection.

Virology, epidemiology, pathogenesis, and control of enterovirus 71.

First isolated in California, USA, in 1969, enterovirus 71 (EV71) is a major public health issue across the Asia-Pacific region and beyond. The virus, which is closely related to polioviruses, mostly affects children and causes hand, foot, and mouth disease with neurological and systemic complications. Specific receptors for this virus are found on white blood cells, cells in the respiratory and gastrointestinal tract, and dendritic cells. Being an RNA virus, EV71 lacks a proofreading mechanism and is evolving rapidly, with new outbreaks occurring across Asia in regular cycles, and virus gene subgroups seem to differ in clinical epidemiological properties. The pathogenesis of the severe cardiopulmonary manifestations and the relative contributions of neurogenic pulmonary oedema, cardiac dysfunction, increased vascular permeability, and cytokine storm are controversial. Public health interventions to control outbreaks involve social distancing measures, but their effectiveness has not been fully assessed. Vaccines being developed include inactivated whole-virus, live attenuated, subviral particle, and DNA vaccines.

Clinical features, diagnosis, and management of enterovirus 71.

Although poliomyelitis has been mostly eradicated worldwide, large outbreaks of the related enterovirus 71 have been seen in Asia-Pacific countries in the past 10 years. This virus mostly affects children, manifesting as hand, foot, and mouth disease, aseptic meningitis, poliomyelitis-like acute flaccid paralysis, brainstem encephalitis, and other severe systemic disorders, including especially pulmonary oedema and cardiorespiratory collapse. Clinical predictors of severe disease include high temperature and lethargy, and lumbar puncture might reveal pleocytosis. Many diagnostic tests are available, but PCR of throat swabs and vesicle fluid, if available, is among the most efficient. Features of inflammation, particularly in the anterior horns of the spinal cord, the dorsal pons, and the medulla can be clearly seen on MRI. No established antiviral treatment is available. Intravenous immunoglobulin seems to be beneficial in severe disease, perhaps through non-specific anti-inflammatory mechanisms, but has not been tested in any formal trials. Milrinone might be helpful in patients with cardiac dysfunction.

Development of a multiplex polymerase chain reaction assay for simultaneous identification of human enterovirus 71 and coxsackievirus A16.

Human enterovirus 71 (HEV71) and coxsackievirus A16 (CVA16) are two major aetiological agents of hand, foot and mouth disease (HFMD) in children. Recently there have been several large outbreaks of HFMD in Vietnam and the Asia-Pacific region. In this study, a multiplex RT-PCR assay was developed in order to detect simultaneously HEV71, CVA16 and other human enteroviruses. Enterovirus detection was performed with a mixture of three pairs of oligonucleotide primers: one pair of published primers for amplifying all known enterovirus genomes and two new primer pairs specific for detection of the VP1 genes of HEV71 and CVA16. Enterovirus isolates, CVA16 and HEV71 strains identified previously from patients with HFMD were examined to evaluate the sensitivity and specificity of the multiplex RT-PCR assay. The assay was then applied to the direct detection of these viruses in clinical specimens obtained from HFMD cases identified at Children's Hospital Number 2, Ho Chi Minh City, Vietnam. The multiplex RT-PCR assay showed 100% specificity in screening for enteroviruses and in identifying HEV71 and CVA16. Similar results were obtained when using the multiplex RT-PCR assay to screen for enteroviruses and to identify HEV71 and CVA16 in clinical specimens obtained from HFMD cases identified at the hospital. This multiplex RT-PCR assay is a rapid, sensitive and specific assay for the diagnosis of HEV71 or CVA16 infection in cases of HFMD and is also potentially useful for molecular epidemiological investigations.

Disability after encephalitis: development and validation of a new outcome score.

OBJECTIVE: To develop a simple tool for assessing the severity of disability resulting from Japanese encephalitis and whether, as a result, a child is likely to be dependent. METHODS: A new outcome score based on a 15-item questionnaire was developed after a literature review, examination of current assessment tools, discussion with experts and a pilot study. The score was used to evaluate 100 children in Malaysia (56 Japanese encephalitis patients, 2 patients with encephalitis of unknown etiology and 42 controls) and 95 in India (36 Japanese encephalitis patients, 41 patients with encephalitis of unknown etiology and 18 controls). Inter- and intra-observer variability in the outcome score was determined and the score was compared with full clinical assessment. FINDINGS: There was good inter-observer agreement on using the new score to identify likely dependency (Kappa = 0.942 for Malaysian children; Kappa = 0.786 for Indian children) and good intra-observer agreement (Kappa = 1.000 and 0.902, respectively). In addition, agreement between the new score and clinical assessment was also good (Kappa = 0.906 and 0.762, respectively). The sensitivity and specificity of the new score for identifying children likely to be dependent were 100% and 98.4% in Malaysia and 100% and 93.8% in India. Positive and negative predictive values were 84.2% and 100% in Malaysia and 65.6% and 100% in India. CONCLUSION: The new tool for assessing disability in children after Japanese encephalitis was simple to use and scores correlated well with clinical assessment.

Formaldehyde-inactivated whole-virus vaccine protects a murine model of enterovirus 71 encephalomyelitis against disease.

Enterovirus 71 (EV71) causes childhood hand, foot, and mouth disease and neurological complications, and no vaccines or therapeutic drugs are currently available. Formaldehyde-inactivated whole-virus vaccines derived from EV71 clinical isolates and a mouse-adapted virus (MAV) were tested in a mouse model of EV71 encephalomyelitis. After only two immunizations, given to mice at 1 and 7 days of age, the MAV vaccine protected mice at 14 days of age from disease. Tissues from immunized mice were negative for virus by viral culture, reverse transcriptase PCR, immunohistochemistry analysis, and in situ hybridization. Cross-neutralizing EV71 antibodies to strains with genotypes B3, B4, and C1 to C5 generated in immunized adult mice were able to passively protect 14-day-old mice from disease.

Identification and validation of clinical predictors for the risk of neurological involvement in children with hand, foot, and mouth disease in Sarawak.

BACKGROUND: Human enterovirus 71 (HEV71) can cause Hand, foot, and mouth disease (HFMD) with neurological complications, which may rapidly progress to fulminant cardiorespiratory failure, and death. Early recognition of children at risk is the key to reduce acute mortality and morbidity. METHODS: We examined data collected through a prospective clinical study of HFMD conducted between 2000 and 2006 that included 3 distinct outbreaks of HEV71 to identify risk factors associated with neurological involvement in children with HFMD. RESULTS: Total duration of fever >or= 3 days, peak temperature >or= 38.5 degrees C and history of lethargy were identified as independent risk factors for neurological involvement (evident by CSF pleocytosis) in the analysis of 725 children admitted during the first phase of the study. When they were validated in the second phase of the study, two or more (>or= 2) risk factors were present in 162 (65%) of 250 children with CSF pleocytosis compared with 56 (30%) of 186 children with no CSF pleocytosis (OR 4.27, 95% CI2.79-6.56, p < 0.0001). The usefulness of the three risk factors in identifying children with CSF pleocytosis on hospital admission during the second phase of the study was also tested. Peak temperature >or= 38.5 degrees C and history of lethargy had the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of 28%(48/174), 89%(125/140), 76%(48/63) and 50%(125/251), respectively in predicting CSF pleocytosis in children that were seen within the first 2 days of febrile illness. For those presented on the 3rd or later day of febrile illness, the sensitivity, specificity, PPV and NPV of >or= 2 risk factors predictive of CSF pleocytosis were 75%(57/76), 59%(27/46), 75%(57/76) and 59%(27/46), respectively. CONCLUSION: Three readily elicited clinical risk factors were identified to help detect children at risk of neurological involvement. These risk factors may serve as a guide to clinicians to decide the need for hospitalization and further investigation, including cerebrospinal fluid examination, and close monitoring for disease progression in children with HFMD.

Pathologic characterization of a murine model of human enterovirus 71 encephalomyelitis.

We describe a model of Enterovirus 71 encephalomyelitis in 2-week-old mice that shares many features with the human central nervous system (CNS) disease. Mice were infected via oral and parenteral routes with a murine-adapted virus strain originally from a fatal human case. The mice succumbed to infection after 2 to 5 days. Vacuolated and normal-appearing CNS neurons showed viral RNA and antigens and virions by in situ hybridization, immunohistochemistry, and electron microscopy; inflammation was minimal. The most numerous infected neurons were in anterior horns, motor trigeminal nuclei, and brainstem reticular formation; fewer neurons in the red nucleus, lateral cerebellar nucleus, other cranial nerve nuclei, motor cortex, hypothalamus, and thalamus were infected. Other CNS regions, dorsal root, and autonomic ganglia were spared. Intramuscular-inoculated mice killed 24 to 36 hours postinfection had viral RNA and antigens in ipsilateral lumbar anterior horn cells and adjacent axons. Upper cord motor neurons, brainstem, and contralateral motor cortex neurons were infected from 48-72 hours. Viral RNA and antigens were abundant in skeletal muscle and adjacent tissues but not in other organs. The distinct, stereotypic viral distribution in this model suggests that the virus enters the CNS via peripheral motor nerves after skeletal muscle infection, and spread within the CNS involves motor and other neural pathways. This model may be useful for further studies on pathogenesis and for testing therapies.

Phylogeny of dengue viruses circulating in Jeddah, Saudi Arabia: 1994 to 2006.

The nucleotide sequence of the 240 bp E/NS1 junction of 81 dengue viruses isolated from cases in Jeddah, Saudi Arabia was determined and used to serotype the viruses. The nucleotide sequences of the complete Envelope (E) genes of 19 isolates were used for a phylogenetic analysis of the dengue viruses circulating in Saudi Arabia from 1994 to 2006. Three of the four dengue serotypes (DENV-1, DENV-2 and DENV-3) were found to circulate, often with more than one serotype in each outbreak. There was a major outbreak caused by DENV-1 and DENV-2 in 1994 while DENV-3 emerged in 1997. In the summer of 2004, all three serotypes were isolated and this gave way to an extended outbreak of DENV-1 that stretched from the summer of 2005 through early 2006. In the 1994 outbreak, the DENV-1 circulating was from the America-Africa genotype (lineage India-2) while the most recent outbreak in 2005 and 2006 was caused by a different DENV-1 strain from genotype Asia (lineage Asia-2), suggesting a re-introduction of DENV-1 a decade after the first introduction in 1994. There has been no change in the genotypes of DENV-2 (cosmopolitan genotype) and DENV-3 (genotype III) circulating since introduction in 1994 and 1997, respectively.

Of cascades and perfect storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (DHF/DSS).

The past four decades has witnessed a consolidation of the original observations made in the 1970s that dengue haemorrhagic fever (DHF) and dengue shock syndrome (DSS) have an immunological basis. Following reinfection with a dengue virus of different serotype, severe disease is linked to high levels of antibody-enhanced viral replication early in illness which is followed by a cascade of memory T-cell activation and a 'storm' of inflammatory cytokines and other chemical mediators. These compounds are released mainly from T cells, monocytes/macrophages and endothelial cells, and ultimately cause an increase in vascular permeability. The consolidation of the evidence has been largely due to several important prospective sero-epidemiological studies in areas endemic for DHF/DSS, which have shown that risk of severe disease is significantly higher in secondary dengue infections. These advances have underscored the fact that DHF/DSS pathogenesis is a complex, multifactorial process involving cocirculation of various dengue virus serotypes and the interplay of host and viral factors that influence disease severity. The continued search to define risk factors in susceptible populations must be combined with the new techniques of molecular virology and innovative approaches in vaccine design to achieve the ultimate objective of developing a safe and effective vaccine.

Epidemiologic and virologic investigation of hand, foot, and mouth disease, southern Vietnam, 2005.

During 2005, 764 children were brought to a large children's hospital in Ho Chi Minh City, Vietnam, with a diagnosis of hand, foot, and mouth disease. All enrolled children had specimens (vesicle fluid, stool, throat swab) collected for enterovirus isolation by cell culture. An enterovirus was isolated from 411 (53.8%) of the specimens: 173 (42.1%) isolates were identified as human enterovirus 71 (HEV71) and 214 (52.1%) as coxsackievirus A16. Of the identified HEV71 infections, 51 (29.5%) were complicated by acute neurologic disease and 3 (1.7%) were fatal. HEV71 was isolated throughout the year, with a period of higher prevalence in October-November. Phylogenetic analysis of 23 HEV71 isolates showed that during the first half of 2005, viruses belonging to 3 subgenogroups, C1, C4, and a previously undescribed subgenogroup, C5, cocirculated in southern Vietnam. In the second half of the year, viruses belonging to subgenogroup C5 predominated during a period of higher HEV71 activity.

Sentinel surveillance for human enterovirus 71 in Sarawak, Malaysia: lessons from the first 7 years.

BACKGROUND: A major outbreak of human enterovirus 71-associated hand, foot and mouth disease in Sarawak in 1997 marked the beginning of a series of outbreaks in the Asia Pacific region. Some of these outbreaks had unusually high numbers of fatalities and this generated much fear and anxiety in the region. METHODS: We established a sentinel surveillance programme for hand, foot and mouth disease in Sarawak, Malaysia, in March 1998, and the observations of the first 7 years are described here. Virus isolation, serotyping and genotyping were performed on throat, rectal, vesicle and other swabs. RESULTS: During this period Sarawak had two outbreaks of human enterovirus 71, in 2000 and 2003. The predominant strains circulating in the outbreaks of 1997, 2000 and 2003 were all from genogroup B, but the strains isolated during each outbreak were genetically distinct from each other. Human enterovirus 71 outbreaks occurred in a cyclical pattern every three years and Coxsackievirus A16 co-circulated with human enterovirus 71. Although vesicles were most likely to yield an isolate, this sample was not generally available from most cases and obtaining throat swabs was thus found to be the most efficient way to obtain virological information. CONCLUSION: Knowledge of the epidemiology of human enterovirus 71 transmission will allow public health personnel to predict when outbreaks might occur and to plan interventions in an effective manner in order to reduce the burden of disease.

Recombinant Newcastle Disease virus capsids displaying enterovirus 71 VP1 fragment induce a strong immune response in rabbits.

The complete VP1 protein of EV71 was truncated into six segments and fused to the C-terminal ends of full-length nucleocapsid protein (NPfl) and truncated NP (NPt; lacks 20% amino acid residues from its C-terminal end) of newcastle disease virus (NDV). Western blot analysis using anti-VP1 rabbit serum showed that the N-terminal region of the VP1 protein contains a major antigenic region. The recombinant proteins carrying the truncated VP1 protein, VP1(1-100), were expressed most efficiently in Escherichia coli as determined by Western blot analysis. Electron microscopic analysis of the purified recombinant protein, NPt-VP(1-100) revealed that it predominantly self-assembled into intact ring-like structures whereas NPfl-VP(1-100) recombinant proteins showed disrupted ring-like formations. Rabbits immunized with the purified NPt-VP(1-100) and NPfl-VP(1-100) exhibited a strong immune response against the complete VP1 protein. The antisera of these recombinant proteins also reacted positively with authentic enterovirus 71 and the closely related Coxsackievirus A16 when analyzed by an immunofluorescence assay suggesting their potential as immunological reagents for the detection of anti-enterovirus 71 antibodies in serum samples.

Two dimensional VOPBA reveals laminin receptor (LAMR1) interaction with dengue virus serotypes 1, 2 and 3.

BACKGROUND: The search for the dengue virus receptor has generated many candidates often identified only by molecular mass. The wide host range of the viruses in vitro combined with multiple approaches to identifying the receptor(s) has led to the notion that many receptors or attachment proteins may be involved and that the different dengue virus serotypes may utilize different receptors on the same cells as well as on different cell types. RESULTS: In this study we used sequential extraction of PS Clone D cell monolayers with the detergent beta-octylglucopyranoside followed by sodium deoxycholate to prepare a cell membrane-rich fraction. We then used 2 dimensional (2D) gel electrophoresis to separate the membrane proteins and applied a modified virus overlay protein binding assay (VOPBA) to show that dengue virus serotypes 1, 2 and 3 all interact with the 37 kDa/67 kDa laminin receptor (LAMR1), a common non-integrin surface protein on many cell types. CONCLUSION: At least 3 of the 4 dengue serotypes interact with the 37 kDa/67 kDa laminin receptor, LAMR1, which may be a common player in dengue virus-cell surface interaction.

Incorrect identification of recent Asian strains of Coxsackievirus A16 as human enterovirus 71: improved primers for the specific detection of human enterovirus 71 by RT PCR.

BACKGROUND: Human enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control. Recent Asian strains of Coxsackievirus A16 have changes in the VP1 gene which causes mispriming of widely used primers for human enterovirus 71 specific identification. METHODS: Local strains of Coxsackievirus A16 were sequenced in the VP4 and VP1 genes and using sequence alignment tools, an improved set of primers were designed for specific identification of human enterovirus 71. These primers were evaluated against virus isolates as well as primary clinical specimens. RESULTS: A total of 218 virus strains were tested. All 39 human enterovirus 71 isolates were positive and none of the 38 Coxsackievirus A16, 127 other enteroviruses and 14 prototype flaviviruses and adenoviruses were positive when tested with the new primers. When aliquots of primary specimens known to have yielded human enterovirus 71 were retrospectively tested, we found that within 2 months of collection of the specimens, greater than 90% were positive but that the success rate diminished rapidly to 18% after 2 years storage. CONCLUSIONS: Our new primers will be useful in rapid diagnosis of human enterovirus 71 infection, and can also be used as a screening tool in surveillance programmes for early warning of human enterovirus 71 transmission.

Molecular epidemiology of human enterovirus 71 strains and recent outbreaks in the Asia-Pacific region: comparative analysis of the VP1 and VP4 genes.

This study provides a comprehensive overview of the molecular epidemiology of human enterovirus 71 (HEV71) in the Asia-Pacific region from 1997 through 2002. Phylogenetic analysis of the VP4 and VP1 genes of recent HEV71 strains indicates that several genogroups of the virus have been circulating in the Asia-Pacific region since 1997. The first of these recent outbreaks, described in Sarawak (Malaysian Borneo) in 1997, was caused by genogroup B3. This outbreak was followed by large outbreaks in Taiwan in 1998, caused by genogroup C2, and in Perth (Western Australia) in 1999, where viruses belonging to genogroups B3 and C2 cocirculated. Singapore, Taiwan, and Sarawak had HEV71 epidemics in 2000, caused predominantly by viruses belonging to genogroup B4; however, large numbers of fatalities were observed only in Taiwan. HEV71 was identified during an epidemic of hand, foot and mouth disease in Korea; that epidemic was found to be due to viruses constituting a new genogroup, C3.