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Evaluation of early African swine fever virus detection using CP204L gene encoding the p30 protein using quantitative
Ngo Thi Ngoc Tram1, Danh Cong Lai1,2, Do Thi Phuong Dung1
1Department of Infectious Diseases and Veterinary Public Health, Faculty of Animal Science and Veterinary Medicine, Nong Lam University, Ho Chi Minh City, Vietnam.
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.
Insights
Early detection of African swine fever virus (ASFV) is crucial. The study found that targeting the p30 antigen (encoded by CP204L) via quantitative polymerase chain reaction (qPCR) offers superior sensitivity and earlier detection compared to targeting the p72 antigen.
Area of Science:
- Veterinary Virology
- Molecular Diagnostics
- Animal Health
Background:
- African swine fever virus (ASFV) poses a significant threat to swine populations globally.
- ASFV genome encodes over 200 proteins, including p72 and p30, vital for viral entry and expression.
Purpose of the Study:
- To evaluate the early detection capabilities of ASFV using quantitative polymerase chain reaction (qPCR) and immunohistochemistry (IHC).
- To compare the diagnostic efficacy of targeting the B646L gene (encoding p72) versus the CP204L gene (encoding p30).
Main Methods:
- Analysis of experimentally and field-infected pig samples using qPCR and IHC.
- Collection of blood and tissue samples at various post-infection days (3, 5, 7, 9).
- Comparative analysis of p72-qPCR and p30-qPCR for ASFV detection in blood and lymphoid tissues.
Main Results:
- p30-qPCR detected ASFV earlier and with lower Ct values than p72-qPCR, particularly at 1-day post-infection.
- Lymphoid tissues (spleen, lymph nodes) showed earlier ASFV detection with p30-qPCR.
- Field outbreak data confirmed p30-qPCR's superior sensitivity and lower Ct values in blood samples.
Conclusions:
- The CP204L gene and its p30 protein enable earlier ASFV detection compared to the p72 antigen.
- p30 is a promising target for developing more sensitive ASFV antigen and antibody detection methods.
