Detection and genetic characterization of sylvatic and outbreak African swine fever virus isolates in selected zones of Tanzania

Abstract

This thesis is about African swine fever (ASF), a severe disease of domestic pigs that is notifiable according to OIE. Virus strains circulating in Tanzania have been changing from time to time making surveillance studies imperative. This research aimed at understanding the African Swine Fever Virus (ASFV) setting in the sylvatic cycle devoid of domestic pigs and genetic characterization of the ASFV genotypes circulating in the domestic pig cycle by genome sequencing of the virus so as to provide information such as the relatedness of the virus strains for other studies. In the study, blood and tissue samples from outbreaks and from selected seven agro-ecological areas of Tanzania (Northern (n=58) Southern (n=18), Southern highlands (n=60), Coastal (n=30), Western (n=40), Central (n=40) and lake zone (n=56)) were screened for ASFV using antibody ELISA and PCR techniques. The study population included domestic cycle hosts (domestic pigs (n=302) and sylvatic cycle hosts (warthogs (n=9) and soft ticks (n=300)). Snowball sampling was applied for domestic pigs following outbreak notifications from the Ministry of Livestock and Fisheries for the areas that experienced outbreaks (Southern highlands, Lake Victoria, Coastal and Northern zones). Convenient sampling was implied for non-outbreak areas (Central, Western and Southern zones) and the Saadani National Park. Blood and tissue samples from domestic pigs (n=302), blood samples from warthogs (n=19) and whole ticks (n=300) from warthog burrows (n=5) were collected from the field and transported to the laboratory for analysis. Serum samples extracted from blood were analysed for the presence of ASFV antibodies. DNA was extracted from the tissues, ticks and whole blood samples for ASFV diagnosis. Representative positive samples were Sanger sequenced for genotyping and tissue cultured for whole genome sequencing. While using standard OIE recommended ASF diagnostic and genotyping primers, positive and negative controls were used in each process to ensure reliability and validity of the obtained results. ELISA results confirmed the exposure of warthogs to ASFV although none of the animals were found to be in active viremia. As opposed to warthogs, domestic pigs did not react positively to an ELISA test but they were in active viremia following a PCR test with ASFV primers. This study reported four ASFV genotypes currently present in Tanzania. Genotypes II, IX and X were reported from domestic pigs whereas genotype XV was reported from the ticks in the sylvatic cycle. This is the first study to report the presence of genotype XV from an ecosystem that has never recorded interaction with domestic pigs. The study also released the first full genome sequence of a genotype II ASFV strain from Africa (Tanzania/ Rukwa/2017/1) generated using Illumina sequencing (Deposited in the INSDC databases through the European Nucleotide Archive (ENA) under accession number LR813622). The reported strain was observed to have a 99.961 percentage identity with the updated Georgia ii 2007/1 reference isolate (FR682468.2), 99.960% identity with Polish isolate Pol16_29413_o23 (MG939586) and 99.957% identity with Chinese isolate ASFV-wbBS01 (MK645909.1). This gave a proposition on the relatedness of genotype II strains of the virus from the known geographical hotspot given that this was the most globally spread genotype. Findings from this study add an information on what was known regarding ASFV in the sylvatic cycle in the country, and as a global hotspot for ASF. The first ASF vaccine is likely to focus on genotype II as it is the most widespread genotype. This has an implication on the global efforts to control ASF while being able to follow the molecular changes of the virus from the hotspot areas. There is also a possibility that in Tanzania, ASFV is present in new areas and distributed widely than it was previously anticipated. Information on circulating genotypes and spatial distribution is paramount for devising control interventions in Tanzania.