|Author:||Lo, Lek-hang Constance|
|Title:||Molecular and epidemiological aspects of dengue virus infection in Hong Kong|
Dengue -- Diagnosis.
Dengue -- China -- Hong Kong -- Epidemiology.
Hong Kong Polytechnic University -- Dissertations
|Department:||Department of Health Technology and Informatics|
|Pages:||xxix, 276 p. : col. ill., col. maps ; 30 cm.|
|Abstract:||Dengue has emerged as a global public health concern and the incidence has increased dramatically in recent decades. The disease is caused by the dengue virus (DV), and currently has no specific treatment. In the present study, we investigated the molecular and epidemiological aspects of dengue virus infection. For the molecular aspects, we focussed on the development of diagnostic tools and investigation of three recombinant proteins for their potential as vaccine candidates. For the epidemiological aspects, the seroprevalence of DV among the population of Hong Kong and abundance of DV vectors among mosquitoes collected in Hong Kong were studied. Two diagnostic tools, a molecular and a serological assay for DV, have been developed. For the molecular assay, a one-step reverse transcription-polymerase chain reaction (RT-PCR) LightCycler assay was developed for rapid and simultaneous detection and typing of DV. The assay's strategy for detection was based on colour and melting temperature (Tm) multiplexing. The serotypes of DEN-1 and DEN-3 amplicons were detected by their characteristic emission generated from induced fluorescence resonance energy transfer (iFRET). The presence of DEN-2 and DEN-4 amplicons was indicated by SYBR Green I (SGI) signals at the specific amplicon Tm. The assay had a dynamic range of 10³-10⁸ plaque-forming units/L and could be performed in 2 hours. By using in-house RT-PCR cocktail replacement for the commercial reagent kit, an economical in-house assay was modified based on the same detection strategy. The running cost per reaction of the in-house assay was about one-third of that of the kit-based assay. Tm profiles and detection limits of the in-house assay were comparable to the original kit-based assay. This in-house assay was validated with clinical sera collected from Hong Kong, Mainland China and Brazil. Three recombinant fusion proteins, prM (24 kiloDalton, kDa), ED3 (32 kDa) and prM-ED3 (43 kDa), of DEN-2 were produced from the Champion TM pET SUMO protein expression system in Escherichia coli for the development of a serological assay and investigation of their potential as subunit vaccine candidates. A serological assay based on the three recombinant proteins as detection antigens was set up. This assay was evaluated with sera collected from the community in Hong Kong and clinical sera collected from Brazil. Performance of the assay was compared with the commercially-available Panbio dengue IgG indirect ELISA kit. We confirmed that this test had high specificity (>90%), but low sensitivity (<30%). Interestingly, prM as capture antigen was able to detect dengue IgG-positive sera with the highest frequency. To investigate their potential as vaccine candidates, purified recombinant proteins were administrated into rabbits for polyclonal antibody production. Four rabbits were respectively injected with prM, ED3 or prM-ED3 fusion proteins or a mixture of prM plus ED3 fusion proteins. Antisera were analysed and characterised by immunoblot and ELISA. The neutralisation potential of antisera were investigated by means of an inhibition assay to determine DEN-2 recombinant subviral particle (RSP) binding to Vero E6 cells using flow cytometry. The inhibition of RSP binding to Vero E6 cells was indicated by decreased FITC fluorescence. Individual antisera showed varied ability to inhibit RSP binding to Vero E6 cells from 5.0-24.7%. Antiserum against prM-ED3 chimeric protein showed the strongest inhibition of RSP binding among the four tested.|
Two epidemiological aspects were investigated in the present study. For vector epidemiology study, a total of 1888 mosquitoes, of which 31.4% (593) were Aedes albopictus, were collected and screened for DV by our in-house RT-PCR assay and a conventional nested RT-PCR; however, DV was not detected. No PCR inhibitor was detected from the pooled mosquito materials spiked with DV RNA except mosquito pool Jan 2008. For the seroepidemiology study, a total of 685 subjects were recruited. The overall prevalence of DV was 1.61%, which was much lower than that reported by other nearby Asian countries. It was observed that seropositivity was significantly associated with increased risk for subjects who were not born and did not grown up locally in Hong Kong (P<0.01, OR 18.00, CI 2.18-148.36). Individuals with an active travel history and who had visited some areas of Eastern Asia (P=0.07, OR 5.60, CI 0.68-46.03) or Southeast Asia (P=0.04, OR 4.27, CI 0.984-18.55) in the past 12 months were more likely to be dengue seropositive. In conclusion, a rapid and economical one-step RT-PCR LightCycler assay for the detection and typing DV (DEN-1 to-4) was developed and validated. Three recombinant proteins of DEN-2 were generated. A serological assay based on the three recombinant proteins as detection antigens was set up and validated with serum samples. Polyclonal antisera were raised from rabbits and their neutralising potentials against DEN-2 RSPs were demonstrated. No DV was detected from mosquitoes collected in the present study and the seroprevalence of DV was very low among subjects recruited in Hong Kong.
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