Wednesday, January 9, 2013

BVDV in commercial bovine serum...still?

by Dr. Ray Nims

One of the animal-derived materials (ADM) most commonly utilized for cell culture and for production of biologicals manufactured using cell cultures is bovine serum (most typically calf serum or fetal bovine serum). There is an inherent risk of introduction of adventitious contaminants (viruses and molllicutes) associated with the use of culture media containing serum. In fact, most of the viral contaminants that have been isolated from biologics bulk harvests (including REO type 2, Cache Valley virus, epizootic hemorrhagic disease virus, and vesivirus 2117) are believed to have been introduced via contaminated bovine serum. Another potential contaminant that may be introduced via bovine serum is the pestivirus bovine viral diarrhea virus (BVDV).
BVDV is a medium-sized (40-70 nm), enveloped, single-stranded RNA virus of the Flavivirus family. The regulatory requirements pertaining to the use of bovine materials for manufacturing biologics (9CFR113.47) contain specific instructions related to the detection of BVDV contamination. EMEA regulations require not only the testing of bovine sera for infectious BVDV, but also assessment of the presence of antibodies to BVDV. Neutralizing antibodies for BVDV are of concern since their presence could theoretically interfere with the detection of the virus in testing done for release of the serum. Although testing of bovine serum to be used for manufacturing biologicals is a regulatory expectation, experience has indicated that such testing is fraught with  false negative results. The relatively large volumes of serum that comprise a given batch are obtained by pooling large numbers of individal serum draws, and there is a chance of non-homogeneous contamination from a limited number of BVDV-infected draws.
How frequently has infectious BVDV been detected in commercially available bovine serum? What percentage of serum lots has been found to contain neutralizing antibodies to BVDV? Has BVDV genomic RNA invariably been found in bovine serum when tested by RT-PCR? These questions have been addressed by various authors over the past four decades.

Infectious BVDV continues to be detected in fetal bovine serum samples up to the present time. This reflects the fact that BVDV is distributed in cattle worldwide, subclinical infections with non-cytopathic BVDV are common in herds, and large serum pools are likely to be non-homogeneously contaminated with BVDV-infected serum. Over the past four decades, 667 lots of commercial fetal bovine serum have been examined for the presence of infectious BVDV in studies reported in the literature. Positive results have been reported for 29% of the lots examined, although the variability in frequency of detection has been quite large, as indicated by the range in the values that have been obtained in the various studies. The percentage of isolates comprising non-cytopathic BVDV has ranged from 98-100%, reflecting the continuing predominance of this variant over the cytopathic strains in cattle herds.
The frequency of detection of neutralizing anti-BVDV antibodies has ranged from 61% to 98% of fetal bovine serum lots. The overall number of commercial fetal bovine serum lots that have been evaluated for neutralizing antibodies to BVDV is 182, with antibodies being detected in 70% of these lots. 
Genomic RNA for BVDV has been detected in 79% of the 155 commercial fetal bovine serum lots that have been evaluated since 1996, when the RT-PCR methodology was initially applied to this question.

The risk of introducing infectious BVDV through contaminated FBS may be mitigated through gamma-irradiation of the FBS. BVDV is relatively sensitive to inactivation by this treatment. It is therefore unusual to detect infectious BVDV in gamma-irradiated serum, though in one case in the literature, a single lot of irradiated serum out of 9 lots tested contained infectious BVDV. This inactivation strategy used to mitigate risk of introducing infectious BVDV is not expected to reduce the frequency of detecting neturalizing anti-BVDV antibodies or genomic RNA for BVDV in the treated serum lots.

See Nims and Plavsic. The Pervasiveness of bovine viral diarrhea virus in commercial bovine serum.  BioProcessing Journal Winter 2012 /2013, 19-26 for the individual study results used to prepare the table shown above.