Quantitative Real Time PCR Detection of African Swine Fever Virus DNA from Formalin‐Fixed, Paraffin‐Embedded Tissues

African Swine Fever (ASF) is a highly contagious and fatal hemorrhagic fever in domestic pigs. ASFV is a large dsDNA virus previously grouped in the Iridoviridae family but reclassified into the Asfarviridae family in which it is the only member of the genus Asfarivirus . Moreover, ASFV is the only arthropod‐borne animal DNA virus. Natural reservoirs in wild and feral pigs exist and this virus is capable of surviving a long time in meat/tissue even following heat, putrefaction, smoking, salt curing, partial cooking, drying and cooling. These are all factors that make control and eradication of this pathogen very challenging. ASFV was first recognized in East Africa in the early 1900's and is now endemic in sub‐Saharan Africa Sardinia (Italy). ASFV spread to Portugal in 1957 and again in 1960, becoming established in the Iberian Peninsula. Subsequently, outbreaks occurred in several European countries, Cuba, the Dominican Republic, Haiti, and Brazil (1970–1980), where it was eventually eradicated but not before causing devastating impacts on the local economy and swine industry. In 2007, ASFV was reported in Georgia, then spread to Russian Federation and Eastern Europe. There is serious concern that ASFv will spread into North America. In the US, ASFV is a BSL‐3Ag Select Agent that requires high containment laboratories for diagnostic and research work. There is no effective vaccine for ASFV. Thus, the current global mitigation strategy against ASF is stringent biosurveillance with molecular and diagnostics playing a key role in prevention and control of virus spread via animals or animal products. Formalin‐fixed, paraffin‐embedded tissues (FFPET) are inactivated, i.e. non‐infectious, specimens that can be safely transported and used for molecular diagnostics and epidemiological studies. The objective of this work is to develop and evaluate a protocol for use of FFPET for the sensitive and specific detection of ASFV DNA. FFPET heart, liver, lung, spleen, tonsil, prescapular and mesenteric lymph nodes were collected from pigs experimentally infected with 4 ASFV strains (Armenia 07, E‐70, Kenya 05 and OURT 88/3). De‐paraffinization of paraffin whorls, tissue lysis with proteinase K, and reversal of nucleic acid cross‐linking in heated alkaline buffer were performed in a single tube. DNA was purified using automated magnetic bead extraction on the Taco Mini TM (GeneReach) or the BioSprint (Qiagen) systems. Real Time PCR (qPCR) for the detection of ASFv was performed using Quanta FastMix II. The ASFv copy number (CN) was calculated via a plasmid‐based standard curve. Support or Funding Information Center of Excellence for Emerging and Zoonotic Animal Disease, A Department of Homeland Security Science and Technology Center of Excellence, and the National Pork Board