Porcine deltacoronavirus (PDCoV) can cause diarrhea and dehydration in newborn piglets. Here, we developed a double antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-ELISA) for detection of PDCoV by using a specific monoclonal antibody against the PDCoV N protein and an anti-PDCoV rabbit polyclonal antibody. Using DAS-ELISA, the detection limit of recombinant PDCoV N protein and virus titer were approximately 0.5 ng/mL and 10^3.0 TCID₅₀/mL, respectively. A total of 59 intestinal and 205 fecal samples were screened for the presence of PDCoV by using DAS-ELISA and reverse transcriptase real-time PCR (RT-qPCR). The coincidence rate of the DAS-ELISA and RT-qPCR was 89.8%. DAS-ELISA had a sensitivity of 80.8% and specificity of 95.6%. More importantly, the DAS-ELISA could detect the antigen of PDCoV inactivated virus, and the viral antigen concentrations remained unchanged in the inactivated virus. These results suggest that DAS-ELISA could be used for antigen detection of clinical samples and inactivated vaccines. It is a novel method for detecting PDCoV infections and evaluating the PDCoV vaccine.

African swine fever (ASF) is a highly contagious hemorrhagic viral disease of domestic and wild pigs of all breeds and ages, caused by African swine fever virus (ASFV). Due to the absence of a safe and efficacious vaccine, accurate laboratory diagnosis is critical for the control of ASF prevention. The p30 protein is immunogenic and stimulates a high level of antibody response to ASFV infection. We developed a panel of 4 monoclonal antibodies (mAbs) against p30 protein, and mAb-2B4 showed the highest percent of inhibition (PI) of 70% in the solid phase blocking ELISA (bELISA). Epitope mapping revealed the mAb-2B4 recognized the epitope of aa 12–18 of p30, which is conserved among various ASFV genotypes. Subsequently, a competitive enzyme-linked immunosorbent assay (cELISA) was established using HRP-labeled mAb-2B4. The cutoff for discrimination between 98 negative sera and 40 positive sera against ASFV was determined by plotting a receiver operating characteristic (ROC) curve. It yielded the area under the curve (AUC) of 0.998, and a diagnostic specificity of 97.96% and a sensitivity of 97.5% were achieved when the cutoff value was determined at 37.1%. Furthermore, the results showed an excellent repeatability of the established cELISA and no cross-reaction to antisera against six other pig pathogens. Additionally, the cELISA detected a titer of 1:256 in the positive standard serum. Overall, mAb-2B4 showed a conserved epitope and high ability to be inhibited by positive sera in ASFV antibody detection. The cELISA based on HRP-labeled mAb-2B4 offers an alternative to other assays for a broader diagnostic coverage of ASFV infection.

Recently, we identified a novel porcine circovirus type 2-like agent P1 isolate from swine. The present study represents the first survey of P1 prevalence in swine herds from Jiangsu, China, by using PCR targeting the complete genome of P1. Prevalences of 50% and 19% were found among 6 herds and 248 animals, respectively. The results indicate a high prevalence of P1 in China pig populations.

Porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), porcine deltacoronavirus (PDCoV), and porcine rotavirus group A (PoRVA) are recognized as major enteric viral pathogens responsible for porcine viral diarrhea. These viruses exhibit similar clinical manifestations, including vomiting, diarrhea, and dehydration, which complicate differential diagnosis. Therefore, there is an urgent need for a highly sensitive and specific diagnostic method to differentiate these pathogens. In this study, we developed a TaqMan‐based one‐step quadruplex reverse transcription real‐time PCR (RT‐qPCR) assay for the simultaneous and differential detection of these four porcine diarrhea viruses. The standard curves demonstrated correlation coefficients ( R ² ) exceeded 0.990 across a dynamic range of 10 ^7.5 – 10 ^2.5 TCID ₅₀ /mL, and amplification efficiency ranged from 90% to 110%. The limit of detection (LOD) were 10 ^1.5 TCID ₅₀ /mL. Specificity analysis showed no cross‐reactivity with other related pathogens. The assay exhibited good repeatability, with coefficients of variation (CVs) ranging from 0.15% to 1.41% for intra‐assay and 0.09% to 2.09% for inter‐assay, respectively. Finally, this method was evaluated for its practicality in the field using 348 clinical fecal samples. The positive rates of PEDV, TGEV, PDCoV, and PoRVA were 43.68%, 0.57%, 26.44%, and 33.91%, respectively. Furthermore, the coinfection rates of PEDV/PDCoV, PEDV/PoRVA, PDCoV/PoRVA, and PEDV/PDCoV/PoRVA were14.37%, 13.51%, 2.01%, and 5.75%, respectively. Compared to singleplex RT‐qPCR assays, the quadruplex method demonstrated agreement rates ranging from 99.41% to 100% in detecting these four viral pathogens. In conclusion, the developed quadruplex RT‐qPCR assay offers a reliable, sensitive, and accurate tool for the identification of four causative agents of porcine viral diarrhea, making it suitable for clinical diagnosis, disease surveillance, and epidemiological studies.

Porcine epidemic diarrhea virus (PEDV) employs multiple strategies to interact with the host and promote its replication, with nonstructural proteins playing critical roles; however, the underlying mechanisms remain largely unclear. In this study, we found that PEDV infection significantly downregulated DTX3L expression both in vivo and in vitro, whereas knockdown of DTX3L markedly enhanced viral replication. Using a DTX3L knockout (DTX3L-KO) cell model combined with transcriptomic analysis, we further demonstrated that DTX3L knockout negatively regulates interferon expression and downstream signaling. Mechanistically, PEDV Nsp8 downregulates DTX3L expression. Sequence alignment revealed that while most PEDV strains harbor an alanine (A) at position 53 of Nsp8, the G2b strain AH2012/12 has a valine (V) substitution. Notably, compared with valine-53 (Nsp8-V53), Nsp8 with alanine-53 (Nsp8-A53) had a stronger downregulatory effect on DTX3L. Using reverse genetics, we generated the recombinant virus rNsp8-V53A (the V53A substitution of Nsp8 in the AH2012/12 strain), which replicated more efficiently and downregulated DTX3L more significantly in wild-type cells than the parental rAH2012/12 strain, whereas this increase was partially abolished in DTX3L-KO cells. Collectively, our findings demonstrate that PEDV promotes replication by downregulating DTX3L expression, with alanine‑53 in Nsp8 playing a critical role in a partially DTX3L‑dependent manner. This study reveals a novel immune evasion mechanism and highlights the importance of nonstructural protein variation in PEDV evolution.