Construction Of Recominant Porcine Pseudorabies Virus Expressing S Gene Of Porcine Epidemic Diarrhea Virus Chinese Variant

Since October 2010, porcine epidemic diarrhea(PED) caused by variant porcine epidemic diarrhea virus(PEDV) has been outbreaking in China and other countries. The epidemic is characterized by watery diarrhea, vomiting, dehydration, and high mortality in nursery piglets. In July 2013, PED outbroke in US for the first time and spread quickly to the whole country and other North America countries, leading to huge economic losses to pig industry all over the world. However, clinical data suggest that the conventional vaccine based on CV777 vaccine strain could not provide enough protection for vaccine pigs aganist PEDV variant infection. Thus, developing a new and effective vaccine to control PEDV variantcaused PED is necessary.Pseudorabies Virus(PRV) has been extensively used as a vector for developing genetic vaccine. Considering the first immunization of piglets for attenuated live pseudorabies vaccine virus was given within three days after birth and PED mainly causes servere consequence in nursery piglets, the recombinant pseudorabies virus expressing S gene of PEDV Chinese variant was constructed in this study for the first time. S protein of PEDV variant was expressed by the recominant pseudorabies virus successfully. The results will provide basic materials for further developing effective vaccine for prevention and control of PED and PR.1. Construction of Recombinant Pseudorabies Virus expressing enhanced green fluorescent proteinIn this experiment, two specific DNA fragments upstream and downstream of TK gene of PRV Bartha-K61 strain were amplified respectively, and then cloned into p MD-18 T vector to obtain the recominant plasmid, p TK. An expression cassette of EGFP was insert into p TK vector to generate a shuttle vector, which was then transfected into Vero cells infected with PRV Bartha-K61 strain. As a result, a recominant PRV expressing EGFP was constructed successfully on the basis of homologous recombination. The recominant PRV had the same replication dynamics with that of PRV Bartha-K61 strain. The results provide experimental basis for using the TK as insertion site to construct recominant PRV expressing S gene of PEDV variant.2. New screening method for the TK- recominant pseudorabies virus by using valaciclovirValaciclovir is an anti-herpesvirus drug, which can inhibit the replication of herpes virus at the presence of virus thymidine kinase. Basing on this, a screening method for TK-recominant pseudorabies virus was developed by using valaciclovir in this study. The maxium non-toxic concentration of valaciclovir to Vero cells was determined as 4 mg/m L. The minimum concentration of valaciclovir for inhibiting of 100TCID50 PRV Bartha-K61-induced cytopathogenic effect was determined as 3 mg/m L by plaque reduction assay and was verified by PCR. As expected, under determined condition, valaciclovir didn’t inhibit the replication of r PRV-EGFP(TK-) virus and had no effect on the EGFP expression of the recominant virus, suggesting valaciclovir can be used for screening the TK- recominant pseudorabies virus.3. Inhibition effect of valacyclovir on replication of porcine pseudorabies virus in vivoTo prove the in vivo inhibition effect of Valacyclovir on the replication of TK+ PRV in experimental mice, Mice were challenged with 1 LD50 PRV and were given different dosages of Valacyclovir orally 48 hours post-challenge. The lowest protective dose of Valacyclovir against 1 LD50 PRV challenge was determined 3 mg/mouse. Mice not given Valacyclovir developed typical neural symptoms and died, while mice given Valacyclovir remained healthy clinically and alive. Furthermore, PRV viral loads in brain and lung tissues of mice not given Valacyclovir were much higher than those of mice given Valaciclovir, and increased with time. At the same time, typical PRV pathological changes were developed in the brain and lung of mice not given Valaciclovir, The virus load and Th1 cytokine excreted by spleen T lymphocyte were decreased gradually after 72 h challenging in mice not given Valaciclovir,. Thus, the fact of valaciclovir exerting inhibition effect on the replication of TK- PRV both in vivo and in vitro, together with the previous results that valaciclovir could not inhibit the replication of TK- PRV, provide a useful back-up to conventional vaccine-based PR control measures.4. Construction of recominant PRV expressing S gene of PEDV Chinese variantThe recominant PRV expressing S protein of variant PEDV was obtained by homologous recombination and valaciclovir screening method after transfection of shuttle vector containing S1 gene of PEDV variant into PRV-infected Vero cells.PEDV S1 protein was successfully detected in recombinant PRV infected cells by Western-blotting. The research provides primary research materials for the development of new and effective vaccine against PED caused by PEDV variant.