Immunogold-Silver Staining (IGSS) of (NCP and CP) BVDV Infected Subcellular Fraction Bands

1997 ◽  
Vol 3 (S2) ◽  
pp. 155-156
Author(s):  
C.E. Hearne ◽  
D.L. Johnson ◽  
H. Van Campen
Keyword(s):  
Subcellular Fraction ◽  
Membrane Vesicles ◽  
Bovine Viral Diarrhea ◽  
Fixed Angle ◽  
Cell Monolayers ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Transmission Electron ◽  
Viral Diarrhea Virus ◽  
Bovine Turbinate

Pre-embedding immunogold-silver (IGSS) techniques are useful to localize antigens in cell monolayers and agarose embedded cell suspensions for transmission electron microscopy. Procedural centrifugations, however, present a challenge when attempting to localize antigens in subcellular fractions. Using a Beckman Airfuge Ultracentrifuge to concentrate the subcellar fraction bands and resuspending the organelles in agarose simplifies IGSS processing and resin embedding procedures.Control bovine turbinate (BT), and BT cells infected with cytopathic (cp NADL) and non-cytopathic (ncp NY-1) strains of bovine viral diarrhea virus (BVDV) were fractioned according to Bienz et al (1992). Bands containing membrane vesicles (Fig 2) were collected and each fraction band was pelleted at 169,000g for 20 min using an A-95 fixed angle rotor in a Beckman Airfuge Ultracentrifuge. Each fraction pellet was resuspended in 50μl of 30% agarose, solidified, and trimmed to < lmm. IGSS procedures were carried out according to Nanoprobes, Inc., Stoney Brook, NY, and Hearne & Van Campen (1996).

Immunogold-Silver Staining (IGSS) of Agarose Embedded (NCP) Bvdv-Infected Cell Suspensions

1996 ◽  
Vol 54 ◽  
pp. 902-903
Author(s):  
C.E. Hearne ◽  
H. Van Campen
Keyword(s):  
Silver Staining ◽  
Phosphate Buffer ◽  
Cell Suspensions ◽  
Bovine Viral Diarrhea ◽  
Require Time ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Cell Fractions ◽  
Viral Diarrhea Virus ◽  
Bovine Turbinate

The use of pre-embedding immunogold-silver staining (IGSS) in electron microscopy of cell culture monolayers has been reported by various authors and its usefulness to localize antigens has been documented. Embedding cell suspensions or cell fractions for IGSS can require time-consuming centrifugation steps. Embedding cell suspensions in agarose simplifies IGSS processing and embedding procedures.Bovine turbinate cells (BTs) were grown to confluence in 25cm3 culture flasks. BTs were infected with a non-cytopathic strain (ncp) of bovine viral diarrhea virus (BVDV) while control cells were sham inoculated. At 18 h pi, cells were detached with trypsin. All following procedures were carried out at RT. The cells were fixed for 30 min in 2.0% paraformaldehyde + 0.2% glutaraldehyde in 0.1 M. phosphate buffer, pH 7.4, and rinsed 3 times in 0.1 M phosphate buffer, pH 7.4 (each centrifuged for 10 min at 300g). The cells were resuspended in 1 ml of buffer, transferred to an eppendorf tube and centrifuged for 10 min at 300g. The excess buffer was removed, 1-2 drops of warm, molten 1.0% agarose added, and the mixture centrifuged for 1 min at 300g to pellet the cells to the tip of the tube. The solidified cell-agarose mixture was removed, cut into <0.5mm3pieces, and placed in phosphate buffer.

Prevention and elimination of bovine viral diarrhea virus infections in fetal fibroblast cells

2004 ◽  
Vol 64 (2) ◽  
pp. 113-118 ◽  
Author(s):  
M GIVENS ◽  
D STRINGFELLOW ◽  
C DYKSTRA ◽  
K RIDDELL ◽  
P GALIK ◽  
...  
Keyword(s):  
Bovine Viral Diarrhea Virus ◽  
Bovine Viral Diarrhea ◽  
Fibroblast Cells ◽  
Virus Infections ◽  
Fetal Fibroblast ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Viral Diarrhea Virus

scholarly journals Prevalence of Bovine Viral Diarrhea Virus Genotypes and Antibody against those Viral Genotypes in Fetal Bovine Serum

1998 ◽  
Vol 10 (2) ◽  
pp. 135-139 ◽  
Author(s):  
Steven R. Bolin ◽  
Julia F. Ridpath
Keyword(s):  
Bovine Serum ◽  
Fetal Bovine Serum ◽  
Neutralizing Antibody ◽  
Bovine Viral Diarrhea ◽  
Diarrhea Virus ◽  
Viral Diarrhea ◽  
Fetal Bovine ◽  
Viral Diarrhea Virus

One thousand lots of pooled fetal bovine serum (FBS) were tested for contamination with bovine viral diarrhea virus (BVDV) and/or for contamination with neutralizing antibody against BVDV. Noncytopathic or cytopathic BVDV was isolated from 203 lots of FBS. Analysis of the viral isolates identified 115 type 1 and 65 type 2 BVDV isolates. An additional 23 virus isolates were mixtures of >2 BVDV isolates and were not classified to viral genotype. Further characterization of the type 1 viruses identified 51 subgenotype 1a and 64 subgenotype 1b BVDV isolates. Viral neutralizing antibody was detected in 113 lots of FBS. Differential viral neutralization indicated that type 1 BVDV induced the antibody detected in 48 lots of FBS and type 2 BVDV induced the antibody detected in 16 lots of FBS.

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