Enterovirus 71 encephalomyelitis and Japanese encephalitis can be distinguished by topographic distribution of inflammation and specific intraneuronal detection of viral antigen and RNA.

AIMS: To investigate if two important epidemic viral encephalitis in children, Enterovirus 71 (EV71) encephalomyelitis and Japanese encephalitis (JE) whose clinical and pathological features may be nonspecific and overlapping, could be distinguished. METHODS: Tissue sections from the central nervous system of infected cases were examined by light microscopy, immunohistochemistry and in situ hybridization. RESULTS: All 13 cases of EV71 encephalomyelitis collected from Asia and France invariably showed stereotyped distribution of inflammation in the spinal cord, brainstem, hypothalamus, cerebellar dentate nucleus and, to a lesser extent, cerebral cortex and meninges. Anterior pons, corpus striatum, thalamus, temporal lobe, hippocampus and cerebellar cortex were always uninflamed. In contrast, the eight JE cases studied showed inflammation involving most neuronal areas of the central nervous system, including the areas that were uninflamed in EV71 encephalomyelitis. Lesions in both infections were nonspecific, consisting of perivascular and parenchymal infiltration by inflammatory cells, oedematous/necrolytic areas, microglial nodules and neuronophagia. Viral inclusions were absent. CONCLUSIONS: Immunohistochemistry and in situ hybridization assays were useful to identify the causative virus, localizing viral antigens and RNA, respectively, almost exclusively to neurones. The stereotyped distribution of inflammatory lesions in EV71 encephalomyelitis appears to be very useful to help distinguish it from JE.

Novel multiplex oligonucleotide-conjugated bead suspension array for rapid identification of enterovirus 71 subgenogroups.

A high-throughput multiplex bead suspension array was developed for the rapid subgenogrouping of EV71 strains, based on single nucleotide polymorphisms observed within the VP1 region with a high sensitivity as low as 1 PFU. Of 33 viral isolates and 55 clinical samples, all EV71 strains were successfully detected and correctly subgenogrouped.

Complement receptor mediates enhanced flavivirus replication in macrophages.

Evidence is presented that M phi complement receptors (CR3) mediate IgM-dependent enhancement of flavivirus replication in the presence of complement. Enhancement is blocked by pretreatment of macrophages with monoclonal antibody Ml/70, which inhibits CR3 binding, but not by pretreatment with monoclonal antibody 2.4G2, which inhibits FcR binding.

Possible occurrence of a genetic bottleneck in dengue serotype 2 viruses between the 1980 and 1987 epidemic seasons in Bangkok, Thailand.

Cocirculation of two genetic subtypes of dengue serotype 2 viruses was first observed in the 1980 epidemic season in Thailand. To further delineate the evolutionary history and the contribution of these subtypes to subsequent epidemics, we determined the envelope glycoprotein gene sequence of 20 dengue serotype 2 viruses isolated from infected patients during 1987 and compared them with those derived from earlier years. Subtype IIIa strains represented the majority (18 of 19) of dengue type 2 viruses derived from Bangkok metropolitan area, whereas all three strains from a province in the northeastern region belonged to subtype IIIb, indicating uneven local distribution of dengue subtypes within the same year. Three types of sequence variation were identified in both subtypes: substitutions that were unique to individual strains; substitutions that were shared among all subtype IIIa or IIIb viruses of both the 1980 and 1987 epidemics; and those that were shared only among all subtypes IIIa or IIlb viruses of the 1987 epidemic, but were absent from the corresponding subtypes of 1980. While the first and second types of substitution were indicative of the most recent random mutations and previous mutations that had been fixed in virus populations, respectively, the third type suggested possible occurrence of a genetic bottleneck and subsequent expansion of one or a limited number of subtype IIIa strains in Bangkok between 1980 and 1987. Immunoblot analysis of intracellular NS1 antigen with anti-NS1 monoclonal antibodies also revealed antigenic heterogeneity of the NS1 protein that correlated with the subdivision based on envelope protein variation.

Development of a dot enzyme immunoassay for dengue 3: a sensitive method for the detection of antidengue antibodies.

Partially purified DEN3 virus was used as antigen in a sensitive dot enzyme immunoassay (DEIA) for the detection of antibodies to flavivirus antigens. We describe here the method used to prepare and optimise the antigen-bearing nitrocellulose membranes and present the results obtained from screening 20 acute phase sera from patients shown to have had recent dengue infections by the haemagglutination inhibition (HI) test. Sixteen pairs of acute and convalescent sera from dengue-negative patients had no detectable antibody to dengue virus by HI. These were shown to have no antibody detectable by DEIA. Sera positive for dengue antibodies by HI had DEIA titers ranging from 10 to several thousand times greater than the titers detected by HI.

Convenient and versatile DNA extraction using agarose plugs for ribotyping of problematic bacterial species.

We describe a convenient, versatile and safe method for preparing bacterial DNA for ribotyping analysis. In this method, extraction of bacterial DNA from Salmnonella typhi and Burkholderia pseudomallei. and subsequent restriction endonuclease digestion, was performed in agarose blocks/plugs thus minimizing shearing and loss of DNA, problems commonly associated with liquid phase phenol extraction. Digested DNA in the plugs was then electrophoresed directly, transferred to nylon membranes and hybridized with labeled rDNA probes in the usual manner to provide reproducible restriction patterns. This method is particularly useful for bacterial species where standard DNA extraction in the liquid phase using phenol has been problematic (e.g. B. pseudomallei) but can be used for any bacterial species. The DNA extracted within the agarose plugs can be stored for long periods and can be used in other, widely-used typing methods such as pulsed-field gel electrophoresis (PFGE) and PCR-based techniques. Embedding live cells directly in agarose plugs also minimizes the risk of exposure to these virulent human pathogens among laboratory workers.

Mumps encephalomyelitis.

A 7-year-old Indian girl developed complete paralysis of her lower limbs and acute urinary retention 10 days after suffering from mumps. Encephalomyelitis due to mumps was not suspected initially since it is a rare complication of mumps, although relatively well-documented. However, the preceding history of parotitis and the presence of mumps-specific IgM in both blood and cerebrospinal fluid led to the diagnosis. The initially severe acute neurological deficits resolved completely three months after onset of her illness. Serological investigations were helpful in diagnosing neurological complications of mumps in this case, and especially where there is no preceding parotitis.

Comparison of an IgM capture ELISA with a dot enzyme immunoassay for laboratory diagnosis of dengue virus infections.

This study describes the use of an IgM capture ELISA using cell culture derived antigens and a polyclonal rabbit antiflavivirus antisera for the detection of dengue positive cases. The IgM capture ELISA is compared with the dot enzyme immunoassay and the results are discussed in the context of dengue endemicity.

A serological study of Japanese encephalitis virus infections in northern Peninsular Malaysia.

This study describes the status of viral encephalitis in Perak, Malaysia during the year 1990. In addition, 14 cases selected from Penang and Perak during the years 1989 and 1990 are presented, with data showing titers of neutralizing antibodies against Japanese encephalitis virus (JEV) and dengue 2 virus, titers of antibodies against JEV and dengue virus antigens as determined by DEIA, and a comparison of these with the presence of IgM to JEV and dengue virus. These data show that there probably is far more viral encephalitis due to JEV in Malaysia than the national figures reflect.

Japanese encephalitis virus is an important cause of encephalitis among children in Penang.

This study was carried out to determine if Japanese encephalitis virus is an important causative agent of viral encephalitis among pediatric admissions in Penang, Malaysia. 195 children with CNS symptoms and 482 children with non-specific febrile illness admitted into the Pediatric Ward of Penang Hospital during a 16 month period were entered into the study. The presence in serum of cerebrospinal fluid (csf) of Japanese encephalitis virus (JEV) specific IgM was determined by an IgM capture ELISA and cytomegalovirus (CMV) specific IgM was determined using a commercially available kit (Behringwerke AG). It was determined that 5 of 13 children with a discharge diagnosis of viral encephalitis had JEV specific IgM in csf, indicating that 38.5% of the viral encephalitis cases was due to JEV. One of the non-JEV cases was found to have mumps virus specific IgM in csf, while no etiology was determined for the other cases. It was also determined that 4 of the 195 (2.1%) cases with CNS symptoms had IgM to CMV, suggesting CMV may be an agent of encephalopathy in children in Penang. Other viruses found to be associated with CNS symptoms in children admitted into our study were measles and herpes simplex virus. A viral etiology was confirmed for 13 or the 195 cases (6.7%). We also screened 482 non-specific febrile cases for IgM to JEV and to dengue viruses and found that 2 (0.4%) had IgM specific for JEV and 9 (1.9%) had IgM specific for dengue virus.

Screening of pig sera for antibodies to Japanese encephalitis virus using a dot enzyme immunoassay and IgM capture ELISA: comparison with the hemagglutination inhibition and plaque reduction neutralization tests.

A dot enzyme immunoassay for determination of antibodies to Japanese encephalitis virus was designed for use as a field technique for the surveillance of Japanese encephalitis virus activity among domestic pigs. The test was compared with the neutralization test and the hemagglutination inhibition test and found to be more sensitive than the hemagglutination inhibition test and comparable to the neutralization test in sensitivity but more simple to perform than either the neutralization or the hemagglutination inhibition tests. An IgM capture ELISA for the determination of JEV specific porcine IgM was also utilized to determine current infection rates in pigs. The tests which do not involve the determination of specific IgM are better used for testing sentinel animals for providing clues as to the rate of transmission of JEV among pigs. IgM tests determining acute infection are less likely to be useful unless animals are tested very frequently or if a great number of animals are tested at any one time.

IgM capture ELISA for detection of IgM antibodies to dengue virus: comparison of 2 formats using hemagglutinins and cell culture derived antigens.

The highly sensitive AFRIMS format IgM capture ELISA for the diagnosis of dengue virus infections requires the use of mouse brain derived hemagglutinins and consequently also the use of 20% acetone extracted normal human serum to eliminate high background. These reagents are not always easily available and we have thus compared the AFRIMS format with another published format which uses cell culture derived antigens (culture fluid, CF, format) in order to determine if it is reasonable to use cell culture derived antigens in situations where hemagglutinins and normal human serum are difficult to obtain. The study shows that using AFRIMS results as the reference point, the CF format described here has a sensitivity of 90% and a specificity of 96%.

A dot enzyme immunoassay for dengue 3 virus: comparison with the haemagglutination inhibition test.

A dot enzyme immunoassay (DEIA) was used to determine the levels of antibody to dengue 3 virus in the acute and convalescent sera of febrile patients with a clinical diagnosis of dengue fever or dengue haemorrhagic fever. The antibody titres were compared with titres determined by the haemagglutination inhibition (HI) test. The results of the study showed that, besides being more simple to perform, the DEIA is in order of magnitude more sensitive than the HI test. Furthermore, the data suggest that it is possible to use a single dilution as a cutoff point to predict with reasonable accuracy, if a patient has had a recent dengue infection. The DEIA test for antibodies to dengue virus is an appropriate technology highly suitable for rapid diagnosis and surveillance in developing countries.

High-titred neutralizing antibodies to human enterovirus 71 preferentially bind to the N-terminal portion of the capsid protein VP1.

Human enterovirus 71 has emerged as an important pathogen of children in the Asia Pacific region, and it may be important to consider the development of a vaccine against this virus. Human cord serum was used as a source of neutralizing antibodies to determine whether the N- or C-terminal half of the VP1 capsid protein was more likely to harbour neutralizing determinants. Cord sera from 205 individuals were tested for neutralizing antibodies against human enterovirus 71 in an indirect ELISA against recombinant VP1 antigen as well as the N- and C-terminal portions of VP1 antigen. High-titred human neutralizing antibodies were significantly more reactive with the N-terminal half of VP1 than weak or negative sera. The N-terminal half of human enterovirus 71 is likely to have important neutralizing antibody determinants and should be investigated further in vaccine development efforts.

Molecular phylogeny of modern coxsackievirus A16.

A phylogenetic analysis of VP1 and VP4 nucleotide sequences of 52 recent CVA16 strains demonstrated two distinct CVA16 genogroups, A and B, with the prototype strain being the only member of genogroup A. CVA16 G-10, the prototype strain, showed a nucleotide difference of 27.7-30.2% and 19.9-25.2% in VP1 and VP4, respectively, in relation to other CVA16 strains, which formed two separate lineages in genogroup B with nucleotide variation of less than 13.4% and less than 16.3% in VP1 and VP4, respectively. Lineage 1 strains circulating before 2000 were later displaced by lineage 2 strains.

Review on flavivirus vaccine development. Proceedings of a meeting jointly organised by the World Health Organization and the Thai Ministry of Public Health, 26-27 April 2004, Bangkok, Thailand.

In light of the continuous spread of human pathogenic flaviviruses, in particular the mosquito-transmitted species, vaccine development remains a high priority on the public health agenda. On 26-27 April 2004, a conference was held in Bangkok, Thailand, to review current status of flavivirus vaccine development and related issues, focussing on dengue (DEN) and Japanese encephalitis (JE). This event, co-sponsored by the World Health Organization (WHO) and the Thai Ministry of Public Health, reviewed the progress made with vaccine development, sero-epidemiological studies and other accompanying activities critical for vaccine development and vaccination. The considerable interest in and awareness of the flavivirus diseases and their prevention by public health decision makers, as well as the establishment of two dedicated programmes for dengue and Japanese encephalitis vaccine development raise hopes that new or improved vaccines will become available in the coming years.