KEYWORDS: African swine fever; real-time PCR; VNUA-P54, Vietnam
INTRODUCTION
African swine fever (ASF) was first described in 1921 in Kenya (Montgomery, 1921). It is a highly lethal swine disease caused by a large double-stranded DNA virus belonging to the Asfarviridaefamily (Dixon et al., 2005). African swine fever virus (ASFV) infects exclusively suids; including domestic swine, wild boars and warthogs. The disease can cause high mortality rates up to 100% resulting in large economic losses to the swine industry in affected countries due to loss of production and trade restrictions (Costard et al., 2013). ASF transmission can occur between a healthy and an infected pig through direct contact (Costard et al., 2013) or by a bite of an ASFV infected Ornithodoros soft tick (Plowright, 1977) that is responsible for maintenance of the sylvatic cycle in Africa.
Since its first discovery in Kenya, ASF has spread outside Africa twice. The first ASF outbreak in Europe was reported in Portugal in 1957 which later spread to Caribbean and South America between the 1970s and 1980s. This outbreak was completely eradicated from Europe and Americas at a great cost with the exception of Sardinia (Sánchez-Vizcaíno et al., 2015). In 2007, ASF entered Europe for the second time through Georgia (Costard et al., 2013; Rowlands et al., 2008) from where it spread to the Caucasus region and Russia (Gogin et al., 2013; Khomenko et al., 2013). Then it continued its spread into Eastern Europe and entered China in 2018 where the virus become endemic (Zhao et al., 2019). Subsequently it spread to Mongolia (Heilmann et al., 2020), Vietnam (Le et al., 2019), Cambodia, Republic of Korea (H. J. Kim et al., 2020), Laos, Philippines, Myanmar and Timor-Lester, Papua New Guinea and most recently to India (OIE). Since there is no vaccine for ASF, control of this disease relies on rapid detection and elimination of the infected animals (Oura et al., 2013). Real-time PCR is the preferred first-line diagnostic for ASF. It is highly sensitive and specific, rapid and highly scalable (Fernández‐Pinero et al., 2013; King et al., 2003). Several real-time PCR assays have been developed and validated for ASF detection and most of them target ASF p72 gene (Fernández‐Pinero et al., 2013; Tignon et al., 2011; A. Wang et al., 2020; Y. Wang et al., 2020; Zsak et al., 2005). Here we describe development of a novel real-time PCR assay (VNUA-P54) targeting a highly conserved region of the ASFV E183L gene that encodes an essential structural protein p54 (Brookes et al., 1998; Rodriguez et al., 1996).
  1. MTERIALS AND METHODS
  2. Clinical samples
One hundred and eighty-seven samples including whole blood (n=122), serum (n=12), spleen (n=37), and kidney (n=16) were collected from pigs displaying clinical signs of ASF from farms located in the different provinces in Vietnam during 2019-2020 outbreaks (Supplementary Table S4). A sample volume of 200 µl of whole blood, serum or 10% tissue homogenate were used for nucleic acid extraction using DNeasy Blood & Tissue Kit (QIAGEN, Germany) according to the manufacturer’s instructions.
Determination of analytical sensitivity and specificity
The limit of detection is defined as the highest dilution factor where 95% of the positive samples can be detected. To test the limit of detection of the VNUA-54 real-time PCR assay, 10-fold dilutions of 107, 106, 105, 104, 103, 102, and 10 HAD50/ml of VNUA/HY/ASF-1/Vietnam/2019 ASFV strain (Le et al., 2019) were generated and the real-time PCR was performed on a LightCycler® 96 Instrument (Roche, Switzerland). Additionally, the detection limit of the real-time PCR was also conducted using the known amount of DNA templates of 0.1, 1, 2, 5 and 10 copies for each reaction. Each DNA concentration was run 12 times by VNUA-54 real-time PCR assay as in the previous recommendation (Uhlig et al., 2015). To determine the diagnostic specificity of the real-time PCR assays, fifteen different ASFV reference strains representing p72 genotypes I, II and V (Supplementary Table S1) and other swine viruses including classical swine fever (Strain Vietnam/ND20/2014, GenBank accession no. MH979232), porcine reproductive and respiratory syndrome (Strain HUA/HP1963; GenBank accession no. KF699844), porcine epidemic diarrhea (Strain HUA-14PED96, GenBank accession no. KT941120), and foot and mouth disease (Strain O/VN/PT555/2018, GenBank accession no. MN379784) were used in this study.