Establishment of an indirect immunofluorescence assay for the detection of African swine fever virus antibodies

African swine fever(ASF)continues to cause enormous economic loss to the global pig industry.Since there is no safe and effective vaccine,accurate and timely diagnosis of ASF is essential to implement control measures.Indirect immunofluorescence assay(IFA)is a gold standard serological method recommended by the World Organization for Animal Health(WOAH).In this study,we used primary fetal kidney cells to establish a wild boar cell line(BK2258)that supported the efficient replication of ASF virus(ASFV)SD/DY-I/21 and showed visible cytopathic effect(CPE).Moreover,using BK2258,we established a sensitive and specific IFA for ASFV antibody detection.To standardize and evaluate the performance of this assay,we used serum samples from pigs infected with the low virulent genotype I SD/DY-I/21 and genotype II HLJ/HRB1/20,and immunized with the vaccine candidate HLJ/18-7GD,field samples,and negative serum samples.The IFA reacted with the ASFV-positive sera and displayed bright fluorescence foci.There was no non-specific green fluorescence due to cellular senescence or other cell damage-causing factors.Compared to a commercial indirect enzyme-linked immunosorbent assay(iELISA),ASFV antibodies were detected 1–4 days earlier using our IFA.The detection limits of the IFA and iELISA for the same ASFV-antibody positive serum samples were 1:25,600 and 1:6,400,respectively,indicating that the IFA is more sensitive than iELISA.The newly established IFA was highly specific and did not cross-react with sera positive for six other important porcine pathogens(i.e.,Classical swine fever virus(CSFV),Porcine reproductive and respiratory syndrome virus(PRRSV),Porcme circovirus type 2(PCV2),Pseudorabies virus(PRV),Foot-and-Mouth disease virus type O(FMDV/O),and Porcine epidemic diarrhea virus(PEDV)).This study thus provides a sensitive,specific,and reliable detection method that is suitable for the serological diagnosis of ASF.

Highly sensitive serological approaches for Pepino mosaic virus detection

Pepino mosaic virus(PepMV)causes severe disease in tomato and other Solanaceous crops around globe.To effectively study and manage this viral disease,researchers need new,sensitive,and high-throughput approaches for viral detection.In this study,we purified PepMV particles from the infected Nicotiana benthamiana plants and used virions to immunize BALB/c mice to prepare hybridomas secreting anti-PepMV monoclonal antibodies(mAbs).A panel of highly specific and sensitive murine mAbs(15B2,8H6,23D11,20D9,3A6,and 8E3)could be produced through cell fusion,antibody selection,and cell cloning.Using the mAbs as the detection antibodies,we established double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA),Dot-ELISA,and Tissue print-ELISA for detecting PepMV infection in tomato plants.Resulting data on sensitivity analysis assays showed that both DAS-ELISA and Dot-ELISA can efficiently monitor the virus in PepMV-infected tissue crude extracts when diluted at 1:1310720 and 1:20480(weight/volume ratio(w/v),g/mL),respectively.Among the three methods developed,the Tissue print-ELISA was found to be the most practical detection technique.Survey results from field samples by the established serological approaches were verified by reverse transcription polymerase chain reaction(RT-PCR)and DNA sequencing,dem on strati ng all three serological methods are reliable and effective for monitoring PepMV.An ti-PepMV mAbs and the newly developed DAS-ELISA,Dot-ELISA,and Tissue print-ELISA can benefit PepMV detection and field epidemiological study,and management of this viral disease,which is already widespread in tomato plants in Yunnan Province of China.