The Circoviridae family of viruses represent an extreme case for small, non-enveloped viruses. We have posted previously that the latter group constitutes a high risk for manufacturers of biologicals due to the difficulty of eradicating the viruses from raw materials or from a contaminated facility. At 17-25 nm particle size, the circoviruses are among the smallest of the animal viruses. These viruses represent more of an economic threat than a threat to human health. The porcine circoviruses (PCV-1 and PCV-2) and the chicken anemia virus are well known examples of the circoviruses and these have been studied extensively due to their impact on the swine and poultry industries. So why care about the circoviruses in the biologics industry?
We begin being concerned about porcine circoviruses in the context of xenotransplantation of porcine tissues (e.g., islet cells) into humans. The worry was that a porcine circovirus might be transmitted to a patient via the porcine donor tissue. More recently, PCV genomic sequences were discovered in rotavirus vaccines manufactured by GlaxoSmithKline and Merck. The presence of the genomic material was attributed to the use of porcine trypsin during the culture of the cell substrates in which the vaccines were manufactured (see previous post).
As a result of the heightened awareness of the contamination threat posed by the porcine circoviruses, infectivity assays for these viruses are now being offered at contract testing organizations (e.g., BioReliance, MICROBIOTEST, and WuXi Apptec), for raw material testing, cleaning efficacy testing, and for evaluating the clearance of the circoviruses by purification processes. As might be expected based on our experience with other small, non-enveloped viruses, inactivation approaches that are effective against many larger non-enveloped or enveloped viruses have little efficacy for the circoviruses.
So how much do we actually know about the inactivation of circoviruses? The literature on the subject is fairly extensive for PCV-2 and for chicken anemia virus, if one is willing to spend time digging deeply. I have done the digging, and have assembled the literature into the following categories of inactivants: heat, irradiation, and disinfectants/chemicals. Keep in mind that the inactivation potential of the physical or chemical agents depends greatly upon the matrix in which the virus is present as well as the temperature and contact time with inactivant. The matrices evaluated varied for the studies described below, and the reader is directed to the individual papers for this critical detail. In addition, some variability in results may be expected because of the relative difficulty in assaying infectivity of the circoviruses.
A number of studies on the thermal stability of the circoviruses have been published. In general, it appears that 15 or more minutes of exposure to wet heat (heating of viruses spiked into solutions) at temperatures ≥80 ◦C should provide extensive inactivation (3-5 log10) of circoviruses. Dry heat (heating of freeze-dried virus and, by implication, viruses on the surfaces of coupon materials) appears to be much less effective, resulting in <1.5 log10 inactivation even at temperatures as high as 120 ◦C . The results of at least one investigator suggest that a temperature of 95 ◦C will be sufficient for high temperature short time (HTST) treatment for mitigating the risk of introducing a circovirus in a process solution.
A number of studies on the inactivation of the circoviruses by disinfectants and other chemicals have appeared in the literature, reflecting the relatively great economic threat of the circoviruses to the swine, poultry, and exotic bird industries. While many of the chemicals/disinfectants had little efficacy for inactivation of the circoviruses (as might be expected for a non-enveloped virus), certain treatments appear to have been highly effective. These included the following: a) glutaraldehyde at 1% or 2% and 10 or more minutes contact time; b) sodium hypochlorite at 6% and 10 or more minutes contact time; c) sodium hydroxide at 0.1 N or greater; d) Roccal® D Plus at 0.5% and 10 minutes contact time; e) Virkon® S at 1% and 10 minutes contact time; f) β-propiolactone at 0.4% and 24 hours contact time; and g) formaldehyde at 10% and 2 hours contact time. Variable results were obtained for the iodine-containing disinfectants. These ranged from <1.0 log10 inactivation for iodine (10%; 30 minutes contact time; 20 ◦C) to ≥5.5 log10 for Cleanap® (1%; 2 hours contact time; 37 ◦C). A third study involving an iodine-containing disinfectant, FAM®30 (Biocide30) used at 1% or 2%; 30 minutes contact time, and 10 ◦C temperature demonstrated ≥3.5 log10 inactivation.
The literature on inactivation of circoviruses by irradiation is scant, to say the least. Plavsic and Bolin showed that gamma irradiation of PCV-2-spiked fetal bovine serum at the radiation doses normally employed for serum treatment (30 and 45 kGy) resulted in ≤1.0 log10 reduction in virus titer. Gamma irradiation (at the doses normally used) appears to be relatively ineffective for inactivating the very smallest of the viruses (parvoviruses and circoviruses) in serum so this result is perhaps not surprising. One approach that offers hope for inactivation of circoviruses is ultraviolet radiation (specifically UV-C) treatment, as this technology appears to be effective for smaller non-enveloped viruses such as the parvoviruses and caliciviruses. I expect that studies to demonstrate efficacy of UV-C for inactivating circoviruses will be performed in the near future.
In summary, there is ample evidence in the literature that effective inactivation approaches exist for the circoviruses. Careful selection of inactivation technologies that is based on the body of evidence accumulated by workers in the swine and poultry industries should enable appropriate risk mitigation and facility cleaning strategies to be adopted in the biologics industry.
<This material was excerpted in part from Nims and Plavsic, Bioprocessing J, 2012; 11:4-10>
