Evaluation of early African swine fever virus detection using CP204L gene encoding the p30 protein using quantitative polymerase chain reaction

Abstract

Background and Aim: The African swine fever virus (ASFV), spanning 170–193 kb, contains over 200 proteins, including p72 and p30, which play crucial roles in the virus’s entry and expression. This study investigated the capability of detecting ASFV early through the analysis of genes B646L and CP204L, encoding p72 and p30 antigen proteins, by employing ASFV, diagnosis, immunohistochemistry (IHC), quantitative polymerase chain reaction (qPCR), and IHC techniques. Materials and Methods: Samples were taken from both experimentally and field-infected pigs to evaluate the effectiveness of qPCR and IHC in detecting ASFV. Twenty-two infected pigs were necropsied at 3-, 5-, 7-, and 9-day post-infection to obtain the first set of samples, collecting anticoagulated blood and tissues each time. The thymus, spleen, and lymph nodes were processed by fixing in 10% formalin, paraffin-blocking, and undergoing IHC staining. Forty anticoagulated blood samples were collected from clinically infected sows at a pig farm for the second batch of samples. Based on the lowest Ct values, three blood samples were diluted fivefold for qPCR DNA testing, and their tissues were used for both qPCR and IHC analyses. Results: At 1-day post-infection, p30-qPCR identified more ASFV-positive pigs and measured lower Ct values compared to p72-qPCR. At later time points, both methods showed similar levels of detection. ASFV was detected earlier and with lower Ct values in lymphoid tissues using p30-qPCR compared to p72-qPCR, particularly in the spleen and lymph nodes. In a field outbreak study, p30-qPCR demonstrated superior sensitivity and lower Ct values when detecting ASFV in blood samples compared to p72-qPCR. Conclusion: The early detection of the CP204L gene encoding p30 and its corresponding antigenic protein in ASFV diagnosis compared to the gene encoding p72 suggests that CP204L and p30 are promising candidates for the development of more effective antigen and antibody testing methods. Keywords: African swine fever virus, diagnosis, immunohistochemistry, quantitative polymerase chain reaction.