A host species-informative internal control for molecular assessment of African swine fever virus infection rates in the African sylvatic cycle Ornithodoros vector

摘要

African swine fever virus (ASFV) infection in adult Ornithodoros porcinus(Murry 1877, sensu Walton 1979) ticks collected from warthog burrows in southernand East Africa was assessed using a duplex genomic amplification approach thatis informative with respect to the invertebrate host species and infecting sylvatic cycle virus. DNA extracted from individual ticks was used as template for thesimultaneous amplification of a C-terminal 478-bp ASFV p72 gene region and a∼313-bp fragment of the tick mitochondrial 16S rRNA gene, under optimized reactionconditions. Within-warthog burrow infection rates ranged from 0% to 43% using thisapproach, and phylogenetic analysis of 16S gene sequences revealed the presenceof three geographically discrete O. porcinus lineages, but no support for subspeciesrecognition. False negatives are precluded by the inclusion of host species-informativeprimers that ensure the DNA integrity of cytoplasmically located genome extracts.In addition, infection rate estimates are further improved as false positives arisingfrom carry-over contamination when performing a two-step nested polymerase chainreaction are negated by the one-step approach. Phylogenetic comparison of fulllength virus gene sequences with the partial C-terminal p72 gene target confirmed theepidemiological utility of the latter in a sylvatic setting. The method is therefore ofparticular value in studies assessing the prevalence and diversity of ASFV in relationto the African sylvatic tick vector and holds potential for investigating the role ofalternative tick species in virus maintenance and transmission.