Dengue haemorrhagic fever: questions of pathogenesis.

The year under review has seen a remarkable proliferation of papers on dengue. Four prospective studies have been carried out across the dengue belt, many groups have been pushing at the question of pathogenesis of dengue haemorrhagic fever, and a breakthrough has been achieved in the development of a mouse model for human dengue haemorrhagic fever

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 β-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

Emerging arboviral encephalitis. Newsworthy in the West but much more common in the East.

The changing epidemiology of West Nile virus (WNV) and Japanese encephalitis virus (JEV) is tackled, with specific emphasis on the prevalence of WNV infection in the USA and Romania and JEV infection in Asia. The clinical presentation, transmission and control of WNV and JEV in the human population are briefly discussed

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

Antibodies against prM protein distinguish between previous infection with dengue and Japanese encephalitis viruses.

BACKGROUND: In Southeast Asia, dengue viruses often co-circulate with other flaviviruses such as Japanese encephalitis virus, and due to the presence of shared antigenic epitopes it is often difficult to use serological methods to distinguish between previous infections by these flaviviruses. RESULTS: Convalescent sera from 69 individuals who were known to have had dengue or Japanese encephalitis virus infection were tested by western blotting against dengue, Japanese encephalitis and West Nile virus antigens. We determined that individuals who had been infected with dengue viruses had IgG responses against the premembrane protein of dengue viruses but not Japanese encephalitis, whereas individuals who had been infected with Japanese encephalitis had IgG specific for the premembrane protein of Japanese encephalitis virus but not the dengue viruses. None reacted with the premembrane protein of West Nile virus. Using the Pearson Chi Square test, it was determined that the difference between the two groups was highly significant with a p value of <0.001. CONCLUSION: The use of flavivirus premembrane protein in seroepidemiological studies will be useful in determining what flaviviruses have circulated in a community

Dengue Virus Serotype 2 from a Sylvatic Lineage Isolated from a Patient with Dengue Hemorrhagic Fever

Dengue viruses circulate in both human and sylvatic cycles. Although dengue viruses (DENV) infecting humans can cause major epidemics and severe disease, relatively little is known about the epidemiology and etiology of sylvatic dengue viruses. A 20-year-old male developed dengue hemorrhagic fever (DHF) with thrombocytopenia (12,000/ul) and a raised hematocrit (29.5% above baseline) in January 2008 in Malaysia. Dengue virus serotype 2 was isolated from his blood on day 4 of fever. A phylogenetic analysis of the complete genome sequence revealed that this virus was a member of a sylvatic lineage of DENV-2 and most closely related to a virus isolated from a sentinel monkey in Malaysia in 1970. This is the first identification of a sylvatic DENV circulating in Asia since 1975

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

Comparative Assay on Dengue IgM-ELISA Using Reagents From 2 Sources

In order to distribute dengue IgM-capture ELISA to the peripheral laboratories, supply of key reagents should be established by local production. In this study, comparative IgM-capture ELISA was carried out on a total of 237 sera using key reagents from 2 sources. The current set consisted of suckling mouse brain (SMB)-derived dengue type 2 (D2) antigen and a D2 monoclonal antibody (MAb) supplied from the United States. While an alternative set was supplied by one of the authors, consisting of tissue culture-derived dengue antigen (TCA) and MAb established in USM. When the IgM-ELISA results were compared with those by the hemagglutination-inhibition (HI) test as a gold standard, the sensitivity was 50.0% by the current set and 75.0% by the alternative set of reagents, respectively. While the specificity was 95.2% by the current set and 90.3% by the alternative set of reagents. The results showed that the alternative set of reagents can effectively be used in dengue IgM-ELISA and is significantly more sensitive than the current set of reagents (Chi SQR=12.26, PP>0.1) at 10% risk