Bovine viral diarrhea-mucosal virus with modified erns protein
A technology for bovine viral diarrhea and viruses, applied to cells modified by introducing foreign genetic material, antiviral agents, viruses/bacteriophages, etc., can solve problems such as inability to distinguish between inoculated and naturally infected animals
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Embodiment 1
[0133] Example 1: Construction and serological characterization of chimeric pestiviruses
[0134]Escherichia coli K12 GM2163[F-ara-14,leuB6,thi-1,fhuA31,lacY1,tsx-78,galK2,galT22,supE44,hisG4,rpsL136,(Str r ), xyl-5, mtl-1, dam13::Tn9 (Cam r ), dcm-6, mcrB1, hsdR2 (rk - mk - ), mcrA] containing a plasmid containing the full-length genomic cDNA of bovine viral diarrhea virus strain NADL (BVDV-NADL), which was obtained from Dr. R. Donis of the University of Nebraska.
[0135] RD cells (bovine testicular cells transformed with SV40; obtained from Dr. R. Donis) were maintained in OptiMEM supplemented with 3% horse serum, 1% non-essential amino acids (NEAA) (in modified Eagle medium ( MEM), 2 mM GlutaMax and 10 μg / ml gentamicin. BK-6 cells were obtained from Pfizer Global Manufacturing (PGM). Cells were grown in Dulbecco's Modified Eagles Medium (DMEM) supplemented with 5% horse serum or donor bovine serum (PGM), 2 mM Glutamax, and 1% antibiotics and antifungals. All media co...
Embodiment 2
[0164] Example 2. Construction and serological characterization and efficacy testing of chimeric pestivirus vaccine candidates
[0165] Type 1 BVDV strain CM5960 and type 2 BVDV strain CM53637 were obtained from Pfizer Global Manufacturing. According to the manufacturer's instructions, use MagMax TM AI / ND Viral RNA Isolation Kit (Ambion) was used to extract viral RNA. According to the manufacturer's instructions, use ThermoScript TM RT-PCR System (Invitrogen) reverse transcribed RNA to generate cDNA. Using the overlapping PCR method, by combining the E of CM5960 and CM53637 rns Gene replacement for E of pronghorn pestivirus rns Gene (P-E rns ), resulting in chimeric pestiviruses. The oligonucleotide primers used for PCR are listed in Table 1.
[0166] To construct the chimeric CM5960 / P-E rns DNA, a fragment of CM5960 cDNA between the 5' UTR and the 3' end of the C gene was amplified from CM5960 cDNA by PCR using primers Oligo B-5 and Oligo 135. Using primers Oligo 1...
Embodiment 3
[0176] Example 3. Efficacy Testing of Chimeric Pestivirus Vaccine Candidates in a Bovine Respiratory Disease Model
[0177] Healthy cattle obtained negative for BVDV were randomized into study groups and maintained under the supervision of the attending veterinarian. The test vaccines were mixed with a sterile adjuvant and administered by intramuscular (IM) or subcutaneous (SC) injection or by intranasal (IN) inoculation. The vaccine is administered in 1 or 2 doses. Two doses of the vaccine were administered at intervals of 21-28 days. Animals were then challenged with type 1 or type 2 BVDV strains 21-28 days after the last inoculation. The challenge inoculum was administered intranasally in divided doses of 4 ml (2 ml / nostril). A control group consisting of non-vaccinated, non-challenged animals and / or non-vaccinated, challenged animals was also maintained throughout the study.
[0178] Clinical parameters including rectal temperature, depression, anorexia, and diarrhea w...
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