Biochemical and Kinetic Characterization of Geranylgeraniol 18-Hydroxylase (CYP97C27) from Croton stellatopilosus Ohba

Geranylgeraniol 18-hydroxylase (EC 1.14.13.110) that exists solely in Croton stellatopilosus Ohba catalyses the last committed step of plaunotol biosynthetic pathways by conversion of geranylgeraniol (GGOH) to plaunotol. This enzyme and its gene are an attractive target for development of plaunotol production and its detailed biochemical properties need to be understood.   Recently, even though the gene (CYP97C27) coding for GGOH 18-hydroxylase has been identified, cloned, and expressed in Escherichia coli system, the enzyme activity has been detected mainly in the insoluble fraction (20,000 g). This means that biochemical and kinetic studies could not be undertaken. However, our previous study indicated that this enzyme activity was easily and specifically detected in the microsomal fraction (100,000 g) of a crude enzyme extract. Therefore, in this report we describe a comprehensive biochemical characterization of GGOH 18-hydroxylase activity in the microsomal fraction from C. stellatopilosus Ohba. The oxygen-dependent enzyme activity of GGOH 18-hydroxylase was inhibited by carbon monoxide and the inhibition was partially reversible upon illumination with white light. Kinetic studies of the GGOH 18-hydroxylase showed high affinity to GGOH and NADPH with apparent Km values of 0.8 and 53 µM, respectively. Furthermore, the enzyme activity was inhibited by P450 inhibitors, including ancymidol, metyrapone, miconazole, potassium cyanide and cytochrome c, with the IC50 values of 428, 65, 75, 66 and 8 µM, respectively. Based on the biochemical and kinetic characteristics, the GGOH 18-hydroxylase in the microsomal fraction is likely a P450 encoded by CYP97C27 gene as previously described.Keywords:  Plaunotol, Cytochrome P450, Enzyme Activity, Enzyme Inhibitor, MicrosomeAbbreviation: GGPP, Geranylgeranyl diphosphate; GGOH, geranylgeraniol; PMSF, phenylmethylsulfonyl fluoride; KCN, potasium cyanide; CO, carbon monoxid

Dengue diversity across spatial and temporal scales: Local structure and the effect of host population size

A fundamental mystery for dengue and other infectious pathogens is how observed patterns of cases relate to actual chains of individual transmission events. These pathways are intimately tied to the mechanisms by which strains interact and compete across spatial scales. Phylogeographic methods have been used to characterize pathogen dispersal at global and regional scales but have yielded few insights into the local spatiotemporal structure of endemic transmission. Using geolocated genotype (800 cases) and serotype (17,291 cases) data, we show that in Bangkok, Thailand, 60% of dengue cases living <200 meters apart come from the same transmission chain, as opposed to 3% of cases separated by 1 to 5 kilometers. At distances <200 meters from a case (encompassing an average of 1300 people in Bangkok), the effective number of chains is 1.7. This number rises by a factor of 7 for each 10-fold increase in the population of the "enclosed" region. This trend is observed regardless of whether population density or area increases, though increases in density over 7000 people per square kilometer do not lead to additional chains. Within Thailand these chains quickly mix, and by the next dengue season viral lineages are no longer highly spatially structured within the country. In contrast, viral flow to neighboring countries is limited. These findings are consistent with local, density-dependent transmission and implicate densely populated communities as key sources of viral diversity, with home location the focal point of transmission. These findings have important implications for targeted vector control and active surveillance

Enzyme-linked immunoassay for dengue virus IgM and IgG antibodies in serum and filter paper blood

BACKGROUND: The reproducibilty of dengue IgM and IgG ELISA was studied in serum and filter paper blood spots from Vietnamese febrile patients. METHODS: 781 pairs of acute (t0) and convalescent sera, obtained after three weeks (t3) and 161 corresponding pairs of filter paper blood spots were tested with ELISA for dengue IgG and IgM. 74 serum pairs were tested again in another laboratory with similar methods, after a mean of 252 days. RESULTS: Cases were classified as no dengue (10 %), past dengue (55%) acute primary (7%) or secondary (28%) dengue. Significant differences between the two laboratories' results were found leading to different diagnostic classification (kappa 0.46, p < 0.001). Filter paper results correlated poorly to serum values, being more variable and lower with a mean (95% CI) difference of 0.82 (0.36 to 1.28) for IgMt3, 0.94 (0.51 to 1.37) for IgGt0 and 0.26 (-0.20 to 0.71) for IgGt3. This also led to differences in diagnostic classification (kappa value 0.44, p < 0.001) The duration of storage of frozen serum and dried filter papers, sealed in nylon bags in an air-conditioned room, had no significant effect on the ELISA results. CONCLUSION: Dengue virus IgG antibodies in serum and filter papers was not affected by duration of storage, but was subject to inter-laboratory variability. Dengue virus IgM antibodies measured in serum reconstituted from blood spots on filter papers were lower than in serum, in particular in the acute phase of disease. Therefore this method limits its value for diagnostic confirmation of individual patients with dengue virus infections. However the detection of dengue virus IgG antibodies eluted from filter paper can be used for sero-prevalence cross sectional studies

Best Practices in Dengue Surveillance: A Report from the Asia-Pacific and Americas Dengue Prevention Boards

The Pediatric Dengue Vaccine Initiative organized Dengue Prevention Boards in the Asia-Pacific and the Americas regions consisting of dengue experts from endemic countries. Both Boards convened meetings to review issues in surveillance. Through presentations, facilitated discussions, and surveys, the Boards identified best practices in dengue surveillance including: (1) Dengue should be a notifiable disease in endemic countries; (2) World Health Organization regional case definitions should be consistently applied; (3) electronic reporting systems should be developed and used broadly to speed delivery of data to stakeholders; (4) minimum reporting should include incidence rates of dengue fever, dengue hemorrhagic fever, dengue shock syndrome, and dengue deaths, and hospitalization and mortality rates should be reported by age group; (5) periodic additional studies (e.g., capture/recapture) should be conducted to assess under-detection, under-reporting, and the quality of surveillance; (6) laboratory methods and protocols should be standardized; (7) national authorities should encourage laboratories to develop networks to share expertise and data; and (8) RT-PCR and virus isolation (and possibly detection of the NS1 protein) are the recommended methods for confirmation of an acute dengue infection, but are recommended only for the four days after onset of fever—after day 4, IgM-capture enzyme-linked immunosorbent assay is recommended

Comparison of the diagnostic accuracy of commercial NS1-based diagnostic tests for early dengue infection

<p>Abstract</p> <p>Background</p> <p>We compared the diagnostic accuracy and reproducibility of commercially available NS1-based dengue tests and explored factors influencing their sensitivities.</p> <p>Methods</p> <p>Paired analysis of 310 samples previously characterized as positive (n = 218) and negative (n = 92) for viral isolation and/or RT-PCR and/or IgM seroconversion. Masked samples were tested by two observers with Platelia™ Dengue NS1 Ag, second generation Pan-E™ Dengue Early ELISA, SD Dengue NS1 Ag ELISA, Dengue NS1 Ag STRIP™, and SD BIOLINE™ Dengue Duo (NS1/IgM/IgG).</p> <p>Results</p> <p>SD BIOLINE™ NS1/IgM/IgG had the highest sensitivity (80.7% 95%CI 75-85.7) with likelihood ratios of 7.4 (95%CI 4.1-13.8) and 0.21 (95%CI 0.16-0.28). The ELISA-format tests showed comparable sensitivities; all below 75%. STRIP™ and SD NS1 had even lower sensitivities (<65%). The sensitivities significantly decreased in samples taken after 3 days of fever onset, in secondary infections, viral serotypes 2 and 4, and severe dengue. Adding IgM or IgG to SD NS1 increased its sensitivity in all these situations.</p> <p>Conclusions</p> <p>The simultaneous detection of NS1/IgM/IgG would be potentially useful for dengue diagnosis in both endemic and non endemic areas. A negative result does not rule out dengue. Further studies are required to assess the performance and impact of early laboratory diagnosis of dengue in the routine clinical setting.</p

Evidence and rationale for the World Health Organization recommended standards for Japanese encephalitis surveillance

<p>Abstract</p> <p>Background</p> <p>Japanese encephalitis (JE) is the most important form of viral encephalitis in Asia. Surveillance for the disease in many countries has been limited. To improve collection of accurate surveillance data in order to increase understanding of the full impact of JE and monitor control programs, World Health Organization (WHO) Recommended Standards for JE Surveillance have been developed. To aid acceptance of the Standards, we describe the process of development, provide the supporting evidence, and explain the rationale for the recommendations made in the document.</p> <p>Methods</p> <p>A JE Core Working Group was formed in 2002 and worked on development of JE surveillance standards. A series of questions on specific topics was initially developed. A literature review was undertaken and the findings were discussed and documented. The group then prepared a draft document, with emphasis placed on the feasibility of implementation in Asian countries. A field test version of the Standards was published by WHO in January 2006. Feedback was then sought from countries that piloted the Standards and from public health professionals in forums and individual meetings to modify the Standards accordingly.</p> <p>Results</p> <p>After revisions, a final version of the JE surveillance standards was published in August 2008. The supporting information is presented here together with explanations of the rationale and levels of evidence for specific recommendations.</p> <p>Conclusion</p> <p>Provision of the supporting evidence and rationale should help to facilitate successful implementation of the JE surveillance standards in JE-endemic countries which will in turn enable better understanding of disease burden and the impact of control programs.</p

Dengue Incidence in Urban and Rural Cambodia: Results from Population-Based Active Fever Surveillance, 2006–2008

Dengue is a major public health problem in South-East Asia. Several dengue vaccine candidates are now in late-stage development and are being evaluated in clinical trials. Accurate estimates of true dengue disease burden will become an important factor in the public-health decision-making process for endemic countries once safe and effective vaccines become available. However, estimates of the true disease incidence are difficult to make, because national surveillance systems suffer from disease under-recognition and reporting. Dengue is mainly reported among children, and in some countries, such as Cambodia, the national case definition only includes hospitalized children. This study used active, community-based surveillance of febrile illness coupled with laboratory testing for DENV infection to identify cases of dengue fever in rural and urban populations. We found a high burden of dengue in young children and late adolescents in both rural and urban communities at a magnitude greater than previously described. The study also confirmed the previously observed focal nature of dengue virus transmission

Dengue: a continuing global threat.

Dengue fever and dengue haemorrhagic fever are important arthropod-borne viral diseases. Each year, there are ∼50 million dengue infections and ∼500,000 individuals are hospitalized with dengue haemorrhagic fever, mainly in Southeast Asia, the Pacific and the Americas. Illness is produced by any of the four dengue virus serotypes. A global strategy aimed at increasing the capacity for surveillance and outbreak response, changing behaviours and reducing the disease burden using integrated vector management in conjunction with early and accurate diagnosis has been advocated. Antiviral drugs and vaccines that are currently under development could also make an important contribution to dengue control in the future