Isolation And Identification Of BVDV-1 And Bvdv-2 Strains And The Effect Of NS5A On The IRES-Medicated Translation

Bovine viral diarrhea virus（BVDV）poses a threat to cattle industry in China.Focusing on biological and genetic features benefits us for estimating the potential risk of BVDV in cattle industry.At present,BVDV mainly relies on vaccine immunization,and there is no effective prevention and treatment method,so,it is necessary to have a deeper understanding of the virus infection and replication mechanism.The non-structural NS5A can bind 5’-UTR of the virus and interact with 3’-UTR to induce oxidative stress,to regulate the replication of the virus genome.BVDV 5’-UTR has no cap structure,and the translation of m RNA depends on the internal ribosome entry site（IRES）,which can start translation as a ribosome binding site.In this study,a total of676 serum samples from newborn calves in 4 provinces（376 samples from Ningxia province,100 samples from Shanxi province,100 samples from Shandong province,100 samples from Inner Mongolia）were collected and identified by RT-PCR and ELISA.The results showed that the positive rates of BVDV in four provinces were12.7%（48/376）,4%（4/100）,2%（2/100）and 1%（1/100）,the number（%）of serum sample with antibodies for BVDV in Ningxia,Shaanxi,Shandong,and Inner Mongolia were 9.04%（34/376）,4%（4/100）,16%（16/100）,and 12%（12/100）,respectively.Serum samples of positive by RT-PCR were inoculated with MDBK cells for virus isolation.Finally,two CP BVDV strains were successfully isolated and named“21SD-16”and“22Anhui-7”respectively.The whole genome sequences of“21SD-16”and“22Anhui-7”were obtained by designing the whole genome primers and amplifying the gene sequence.Phylogenetic tree analysis showed that homology between“21SD-16”and BVDV-1“Bega-like”were high,（93.4%）,however,“22Anhui-7”and BVDV-2“GS2018”were the highest,（99.7%.）.Further,the TCID₅₀ was calculated in“21SD-16”and“22Anhui-7”,and the results indicated that it is 10^6.2TCID₅₀/0.1m L and 10^8.3TCID₅₀/0.1m L respectively.Then,analyzing the Antigenicity and structural characteristics of BVDV-1 and BVDV-2 NS5A,primers were designed and synthesized according to the NS5A gene sequence of BVDV-1 and BVDV-2.Then,The NS5A gene was inserted into the pc MV-c-flag vector,termed as pc MV-c-flag-NS5A.And constructed the double-report vector pcDNA3.1（+）-EGFP-IRES-N^pro-flag with a BVDV IRES synchronous action element.Through western-blot analysis,the results showed that both BVDV-1 and BVDV-2 non-structural protein NS5A inhibited IRES mediated translation.Providing new experimental basis for further elucidating the molecular mechanism of BVDV replication and proliferation,as well as technical support and basic data for preventing and controlling BVD.