African swine fever virus maintenance and transmission dynamics in the sylvatic Ornithodoros vector

Abstract

African swine fever (ASF) is a highly contagious and fatal haemorrhagic viral disease of domestic pigs caused by a large, DNA arbovirus with a genome ranging from 170 to 190 kbp in length, the African swine fever virus (ASFV). There is no treatment or vaccine available to combat the disease, and sporadic outbreaks of ASF have been reported from 1928 until present from within South Africa (SA). Control of the disease in SA relies on strict biosecurity measures and the establishment of a control zone that limits movement of pigs and pig products from high-risk areas. The sylvatic cycle, that involves warthogs and Ornithodoros soft ticks, plays a crucial role in the maintenance and distribution of ASFV in SA and clarification of key epidemiological factors are needed in order to enhance understanding and to assist with the formulation of more effective disease control strategies. Previous epidemiological surveys were conducted in late 1970’s and early 1980’s to determine the infection status and distribution of Ornithodoros ticks in relation to ASFV. More recently, a reassessment of ticks using more advanced techniques resulted in negative ASF results from a previously positive area, signalling the need for more extensive studies across SA, the results of which will determine the relevance of the current control zone and strategies in place in SA. A comprehensive survey to confirm the presence of Ornithodoros ticks in game parks within the control zone in SA as well as those in neighbouring Swaziland was done to determine the presence of the soft ticks and their ASFV infection status. Phylogenetic analyses based on partial C-terminal p72 gene sequences, generated for each of the virus-positive ticks revealed the presence of two additional ASF genotypes in SA, whilst characterisation of the 16S rRNA gene sequences of Ornithodoros ticks from each of the sampling sites revealed high levels of diversity (4 haplotypes) and confirmed the presence of at least three geographically distinct lineages within SA. DNA sequences generated through next generation sequencing (NGS) technologies were used to generate full-length viral genomes for two virus isolates, representative of two (genotype I and XIX) of the 23 known genotypes. One of the benefits of whole genome sequence (WGS) generation is the development of improved diagnostic approaches in infectious disease research. Sequencing of full-length viral genomes is however a difficult task due to the presence of contaminating nucleic acids of the host cell, and highly variable terminal ends for which no reference backbone data are available. In this study an enrichment technique was used to overcome host DNA interference. A long-range PCR approach targeting overlapping viral genome segments 10kbp - 20kbp in length was also evaluated across ten genotypes, with the aim of amplifying viral DNA alone. A drawback of this method is that it is only able to amplify homologous regions of genotypes that are closely related to genotype I against which the primers were designed, and thus the large variable regions at the terminal ends of the ASFV genome cannot be characterised, limiting the viability of this approach. In an attempt to better understand how ASFV adapts and changes when it cycles between the invertebrate and vertebrate host, a transmission experiment in which naturally infected Ornithodoros ticks were used to establish an infection in domestic pigs was conducted. In this transmission experiment, the virus was cycled back from the infected pigs to naïve Ornithodoros ticks, thereby emulating the transmission cycle in southern Africa in which sylvatic cycle vectors precipitate infections in domestic pigs. This cyclical vertebrateinvertebrate infection-amplification, which typically involves the warthog vertebrate host under natural settings, assists in the long-term maintenance of the virus in natural tick colonies, ensuring the central role that they play in ASFV transmission in the field. By performing the transmission experiment under laboratory conditions with domestic pigs, the changes and adaptation the virus undergoes when it cycles between its invertebrate host and vertebrate host was investigated for the first time. Results indicate minor difference between the genomes sequenced with no consistency found over the one year time frame investigated.This study gives insight in the understanding and the role that the sylvatic Ornithodoros vector plays in African swine fever virus maintenance and transmission dynamics in South Africa.