Bovine respiratory coronavirus as a vaccine
A technology of vaccines and isolates, applied in the direction of viruses, vaccines, veterinary vaccines, etc., can solve the problems of not showing cell fusion pathology, and not being able to obtain wild-type BECV isolates
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Embodiment 1
[0026] Clone HRT cells to make cHRT cells
[0027] Human adenoma cells derived from ATCC (designated CCL 244) were used to generate the cHRT cells described below. The obtained cells were initially plated and maintained on RMPI 1640 supplemented with 10% horse serum, or DME or MEM with 5-10% fetal bovine serum (FBS). All studies were performed with DME or MEM containing FBS.
[0028] CCL 244 clones were obtained in 96-well tissue culture plates using standard limiting dilution techniques. This technique involves isolating HRT-18 cells from vessels containing cells grown in a trypsin / EDTA solution (containing 0.1% trypsin and 0.04% EDTA), measuring the number of viable cells, in DME plus 10% FBS or A 10-fold dilution of cells was prepared in MEM so that there was theoretically only one cell in most wells of the 96-well plate. The wells were observed microscopically to confirm that the wells actually contained a single source of cells. Some wells showed small cell clusters, ...
Embodiment 2
[0032] Uniqueness of cHRT cells compared to parental CCL 244 cells
[0033] To resolve the uniqueness of CCL 244 cell clones (cHRT), including HRT-E6, an experiment was performed to evaluate the BRCV growth characteristics of this cell compared to several other cells capable of propagating bovine virus. Cell lines inoculated with the BRCV isolate RA2R7 from cattle exhibiting respiratory disease in the South Dakota region, including MadinDarby bovine kidney (MDBK), porcine testis (ST), cat lung (FL), Bayer 9009 and HRT - E6 cells. BRCV isolates and / or their subcultures were adsorbed in serum-free medium for 60 minutes. In the absence of CPE, cultures were either directly passaged onto fresh cells or frozen and then passaged onto various cell monolayers. Each cell line was passaged 5 times. Except for HRT-E6, all cells did not exhibit CPE at any time during the experiment. Thus, cHRT cells are unique.
Embodiment 3
[0035] Uniqueness of cHRT cells compared to parental CCL 244 cells
[0036] To further demonstrate the uniqueness of cHRT cells, including HRT-E6 and HRT-18G, derived from parental CCL 244 cells, the following experiments were performed. Two BRCV isolates, RA2R7 and AZ26649, and one BECV isolate, designated 50-3 (obtained from Dr. Johannes Storz), were isolated from cattle with respiratory disease in the Arizona area, as described in Example 1 way to grow. Each isolate was then titrated onto CCL 244 cells, HRT-E6 cells or HRT-18G cells. Titrations were performed using fresh monolayers of each cell in 96-well plates according to methods known in the art. The results are listed in Table 2, and the titers are expressed as log 10 TCID 50 / mL.
[0037] Virus isolate
[0038] It is clear that cHRT cells are more capable of proliferating BRCV isolates to significantly higher titers than parental CCL 244 cells. Notably, all three of these cells proliferated BECV isolat...
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