Epidemiological investigation and antigenic characterization of dengue virus for the development of nanobodies
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Date
2022
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Sokoine University of Agriculture
Abstract
Dengue is an important mosquito-borne viral disease of global health concerns. Vector
control and early diagnosis remain the main interventions. Lack of vaccines against
Dengue viruses (DENV) complicates the control of the disease hence increasing risk of
DENV transmission. In this thesis, a systematic review was undertaken to analyse the
prevalence of DENV infection and associated risk factors in Africa. A literature search
was done using PubMed/MEDLINE, Scopus and Embase databases to identify articles
published between 1960 and 2020. Meta-analysis was performed using a random-effect
model at a 95% confidence interval. A study on seroprevalence and risk factors of dengue
was carried out in Buhigwe, Kalambo, Kilindi, Kinondoni, Kondoa, Kyela, Mvomero, and
Ukerewe districts representing five ecological zones in Tanzania. A total of 1,818 blood
samples were collected from humans recruited from households and healthcare facilities
and tested using an enzyme-linked immunosorbent assay specific for DENV
immunoglobulin G (IgG) antibodies. During the 2019 outbreak in Tanzania, serum
samples were collected from the outpatients seeking care from health facilities in
Kinondoni and Ilala districts and tested to confirm the presence of DENV. DENV genome
sequences were generated using Nanopore MinION and analysed using NanoGalaxy tool.
The phylogenies and spatial distributions were reconstructed using maximum likelihood
method and Bayesian Evolutionary Analysis. Multi immunoinformatics tools were used to
predict B-cell epitopes on envelope protein domain III (EDIII) of DENV and the final
construct was cloned into pET-22b (+) vector and expressed in Escherichia coli.
The findings of the systematic review showed that between 1960 and 2020, 45 outbreaks
were reported in Africa, of which 17 and 16 occurred in East and West Africa,
respectively. Overall, the prevalence of DENV was 29% (95% CI: 20–39%) and 3% (95% CI: 1–5%) during the outbreak and non-outbreak periods, respectively. Old age (6/21
studies), lack of mosquito control interventions (6/21), urban residence (4/21), climate
change (3/21), and recent history of travel (3/21) were the leading risks factors.
Serological evidence from this study showed that the overall prevalence of DENV IgG
antibodies was 16.1% (n= 292) in Tanzania. The prevalence was highest in Kinondoni
district (43.8%, n= 103). Increasing age (> 28 years, p < 0.001), stagnant water bodies
around homes (p < 0.01), and piped water at home (p < 0.01) were significantly associated
with DENV seropositivity. This study reports for the first time DENV serotype 1 (DENV-
1) genotype V in Tanzania that was responsible for the 2019 outbreak in Dar es Salaam.
Spatial analysis suggests that the genotype was possibly introduced into Tanzania from a
single source in Asia. Furthermore, epitope prediction analysis revealed five continuous
epitopes at 305─320, 334─348, 320─335, 378─392, 389─400 and three conformational
epitopes at 310─312, 336─337 and 341─342 residues of envelope protein domain III
(EDIII) that can be potential targets for development of diagnostic nanobodies targeting
DENV. In conclusion, the results from a systematic review highlights and increased risk
of DENV serotype circulation in Africa during the past decade (2010─2020). The
national-wide seroprevalence study conducted in Tanzania reveals a wide circulation of
DENV in diverse ecological zones with the highest prevalence in the north-eastern zone.
This study reports for the first time DENV-1 serotype in Tanzania during the 2019
outbreak in Dar es Salaam that was likely imported from Asia.
The
possibility of future introduction of new serotypesof the virus from multiple geographic
origins due to globalization is still inevitable. It is important that surveillance and
diagnostic systems of DENV strengthened nationwide to improve early detection, clinical
management and outcomes. In addition, novel biomarkers on envelop protein domains should be explored for development of vaccines that could possibly be used for the control
of emerging variants of DENV-1 and DENV-2 strains.
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Keywords
Epidemiological investigation, Antigenic characterization, Dengue virus, Nanobodies development