Screening Of A BVDV Epitope And Preparation Of Monoclonal Antibodies

Bovine Viral Diarrhea-Mucosal Disease(BVD-MD)caused by Bovine Viral Diarrhea Virus(BVDV)infection,is characterized by diarrhea and mucosal ulcers.Cattle is a BVDV natural host.In addition,it can infect more than 40 animals such as alpaca,sheep,goats,pigs,camels,deer,and mice.It is an important pathogen that causes significant economic losses to the global animal husbandry.BVDV vaccine is currently applied in many countires except China.In addition,we currently have no commercial BVDV detection kit for fast testing,which is critical for the control of this virus.In this study,we have conducted BVDV epidemiological survey on some cattle farms in 7 cities belonging to Henan Province.A total of 429 bovine serum samples were tested,the antibody levels against BVDV were detected by using commercial available ELISA kit.As a result,we found 64.34%of these serum were BVDV positive,the negative detection rate 32.63%was negative,and 3.03%of them were underdetermined.Of note BVDV vaccine is not used in these farmers.So these data suggested that BVDV infection is occurred at high levels in this region,measuers were encouraged to taken to control this virus.Since BoHV-1 infection may induced immunodepression.Consequently,it may induce subsequent infection by diverse pathognes,including BVDV,and induce bovine respiratory disease complex(BRDC),a lethal pneumonia.Numerous HDAC inhibitors widely used in clinic,and have been found to have antiviral and anti-inflammatory properties.In this study,we aimed to screen a potential drugs form the HDAC inhibitors that coud have antiviral activities against both BVDV and BoHV-1,and anti-inflmmatory effctes.We hypothesized that this drug would have potential effects in the treatment of BRDC.In this study we found sodiun phenybutyrate had antiviral activity against BoHV-1.Unexpectively,it can activate signals associated with an inflammatory response such as Erkl/2 and p38MAPK.Therefore,sodium phenybutyrate has no therapeutic value.Thus and the study on the antiviral effect of BVDV was not performed.In summary,our preliminary studies have shown that sodium phenylbutyrate is not suitable for the treatment of BRDC induced by BVDV/BoHV-1 infection.Obtaining the B cell epitopes of BVDV is critically implicated in the study of vaccine and is possible to get a epitope-specific antibody for the development of a detection method.In this study,a commercial BVDV antiserum was used to perform three rounds of panning(adsorption-elution-amplification)on a phage random display peptide library.Titrate the eluate from the third round,randomly select 100 clones for DNA sequence.Several phage clones that reacted with positive serum were obtained by sequence analysis(although some clones used for the generation of antibody detection strips have been submmited for the application of a patent,the immunological function of these epitopes obtained by the relevant clones have not been fully identified,we didn't apply for a patent.Thus all sequences from phages and viruses in this study are temporarily not released in this paper).P3 was initially considered to be an mimic epitope of BVDV.Western-blot and Dot-blot method was applied to identify that synthesized peptide could bind to the antiserum.The synthesized peptide P3 or P3 derived BVDV peptide,defined as P3-BVDV1/2,coupled to either BSA or KLH also showed binding ability to the antiserum.Using KLH-P3-BVDV1/2 as an antigen,BALA/C mice were immunized,and the serum was collected after the three immunizations.Dot-blots results indicated that these serum had reactivity to BVDV particles.In addition,a monoclonal antibody against the BVDV-1/2 antigen mimic epitope was prepared in this study.A fast and effective method for screening BVDV monoclonal antibodies was established,namely Dot-blot in well ECL.Although this method has the defect of non-specific like the traditional methods,after three consecutive rigorous screening,a monoclonal antibody against P3-BVDV1/2 was finally obtained and named 11G9D10.The light chain is Kappa and the heavy chain subtype is IgM.11G9D10 has reactivity to KLH-P3-BVDV1/2.Subsequent study suggested that 11G9D10 didn't work for the generation for an antigen-detection strip.So this mAb was not further identified.However,this study suggested that the selected epitope can induce the production of antibodies in vivo,suggesting that this epitope is implicated in vaccine research in the future.In summary,although this study failed to get an ideal antiviral drugs,to gain a valuable monoclonal antibody,but we may have a BVDV epitope that are valuable for further vaccine study in the future.In addition to BVDV serological survey results show that the virus is widely popular in Henan province.So much more work need to be done in the future to control this virus.