Browsing by Author "Kinimi, Edson"

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Establishment of long-read nanopore sequencing and proficient nanobodies against peste des petits ruminants virus on the road to develop diagnostic and therapeutic tools
(Sokoine University of Agriculture, 2022) Kinimi, Edson
Peste des petits ruminants virus (PPRV) causes a highly devastating disease of sheep and goats, peste des petits ruminants (PPR), that threatens food security, animal production and the conservation of wild small ruminants. Growing body of evidence suggests that multiple wildlife and atypical host species can be infected with PPRV, posing a serological diagnostic challenge in multi-host environment. Recent studies confirmed that single- domain antigen binding fragments (nanobodies) derived from heavy-chain-only camelid antibodies and nanopore sequencing have proven to be powerful technologies for the development of cost-effective and robust therapeutic and diagnostic tools, respectively. Therefore, the main objective of this study was to generate PPRV-reactive nanobodies in order to set pace for the development of diagnostic and possible therapeutic nanobodies in the future, alongside with establishment of rapid complete genome nanopore sequencing of PPRV. Firstly, a strategy was developed to generate nanobodies against PPRV, whereby an alpaca (Vicugna pacos) was immunized with live attenuated vaccine strain (PPRV/Nigeria/75/1) to raise an affinity-matured immune response in the heavy-chain- only antibody classes. An immune nanobody library with approximately 64 million independent transformants was engineered, of which 100% contained an insert with the proper size of nanobody gene. Following phage display and in vitro affinity selection (biopanning), nine nanobodies that specifically recognise PPRV were identified on enzyme-linked immunosorbent assay. They showed superb potency in identifying rapidly PPRV, which is likely to open a new perspective in the diagnosis and possible treatment of PPRV infection. Secondly, prior to the full genome sequencing of PPRV, nanopore sequencing protocol was tested for amplification and sequencing of PPRV. With this protocol, there were no DNA fragments and nucleotide sequences in the GC-rich region between matrix (M) and fusion (F) genes at the genome position between 4,444 and 5,526. Thus, a tiling multiplex polymerase chain reaction method was developed to amplify the missing DNA fragments. Following redesigning of three pairs of overlapping long read primers and cascade of optimization, the GC rich region was successfully amplified and sequenced (accession numbers: MW580394, MW580395 and MW580396). These three pairs of primers targeting the GC-rich region were used along with other 22 pairs of primers in tiling multiplex PCR for complete PPRV genome sequencing. The resulting PCR amplicons were used for nanopore library preparation and ultimate sequencing. This method has resulted into complete genomes of PPRV, with 15,948 nucleotides long for both isolates that were produced within four hours of sequencing (Accession numbers MW960272 and MZ322753). Phylogenetic analysis of the complete genomes revealed a high nucleotide identity between 96.19 and 99.24% with lineage III PPR viruses currently circulating in East Africa indicating a common origin. The nanopore sequencing platform can be deployed to overcome PPR diagnostic and surveillance challenges, unanticipated variations in virus pathogenicity, circulation of disease in wildlife populations and to service remote and nomadic communities with challenging geographical landscapes. However, further investigations are recommended for PPRV reactive nanobodies especially on diagnostic and therapeutic applications. Once validated, these technologies have great potential for use in the field as rapid and cost-effective tools in context of planned PPR Global Control and Eradication Programme.
Mosquito diversity and febrile illness in Karagwe and Kyerwa districts, North Western Tanzania
(Sokoine University of Agriculture, 2015) Kinimi, Edson
Mosquito-borne viruses cause emerging and re-emerging infections affecting humans and animals. These diseases present themselves mainly with fever. The present study aimed at determining socio-demographic and clinical characteristics among febrile patients, and detection of selected mosquito-borne viruses circulating in Aedes aegypti and Ae. albopictus. Using a hospital-based cross-sectional descriptive study design, in total 400 febrile patients were recruited after consenting into the present study. A structured questionnaire was administered to collect socio-demographic and clinical data. The results showed that most of the febrile patients (n=400) were aged between 20-29 years (25.25 %), followed by those aged 10-19 years (23.25 %), and only 13.15% were older than 50 years. The results show that fever (100 %) was the most common symptom reported, followed by headache (68.75 %), joint aches (67.75 %), seizures (63.50 %), vomiting (61.50 %) weakness in legs (59.50 %), laboured breathing (58.50%) and the least observed symptoms were abdominal pains (41.75 %), neck stiffness (33.75 %), and rashes (33 %). Screening 22 pools of Ae. aegypti and Ae. albopictus using reverse transcription polymerase chain reaction (RT-PCR) showed the presence of Flaviviruses, Bunyaviruses and Alphaviruses. Further screening of specific viruses in Aedes mosquitoes showed the presence of Chikungunya virus. Furthermore, the risk factors for mosquito-borne viral infections were investigated in the present study. The findings of this study show that 12.75 % of patients were in contact in forests and 79 % had been bitten by day-biting mosquitoes within three months prior to sampling. Only 28.75 % of febrile patients had malaria, indicating the widespread nature of febrile illness other than malaria. It can be concluded from the results of present study that Aedes mosquitoes are infected with Chikungunya virus and that interaction between humans and forests predisposes humans to mosquito bites.