UV-C versus small, non-enveloped viruses

By Dr. Ray Nims

Small, non-enveloped viruses (especially the circoviruses, parvoviruses, picornaviruses, caliciviruses, and polyomaviruses) and bacteriophage with similar characteristics represent a special challenge to the biologics industry.

In fact, contamination events have occurred with each of these virus families; in some cases more than once. Within the Circoviridae, the primary concern has been the porcine circovirus discovered in rotavirus vaccine. At least four contamination events involving the parvovirus, mouse minute virus, have been reported. The calicivirus vesivirus 2117 has contaminated biologics manufacturing processes on at least two occasions. The picornaviruses are a threat which has yet to be realized in the manufacturing context; but a viral safety test conducted for a biologics cell bank was the scene of a contamination with equine rhinitis A virus. The polyomavirus SV40 was found to have survived the formaldehyde inactivation used in the preparation of polio vaccines and therefore made its way into the doses of vaccine delivered to millions of individuals during the decade between 1955 and 1965 (being born in 1952, it is possible that the author received such tainted vaccine!).

The contamination events described above are, at least in part, a reflection of the ability of these viruses to withstand conditions that would lead to inactivation of other types of viruses. Inactivation strategies that are typically employed for viral safety include chemical (low pH, high pH, disinfectants, solvents, detergents, etc.) and physical (heat, irradiation, pressure, etc.) means. In a previous posting, the efficacy of gamma irradiation for inactivating various types of viruses within frozen animal serum was discussed. The use of UV-A in combination with riboflavin has also been discussed previously.

It is perhaps fortunate that the efficacies of different inactivation approaches are in many cases complementary. For instance, it appears that the efficacy of gamma irradiation for inactivation of viruses decreases as viral particle size decreases (although this relationship is not strictly linear). The outcome of this is that gamma irradiation does not appear to be particularly effective for inactivating small, non enveloped viruses from the circovirus, parvovirus, and polyomavirus families. On the other hand, ultraviolet radiation in the C range (254 nm is the most commonly employed wavelength) appears to be more effective for the inactivation of smaller viruses than for larger viruses (or bacteria).

The table below is a compilation of the UV-C inactivation constants (K, defined as the log₁₀ reduction in titer per mJ/cm² fluency) for various families of small, non-enveloped viruses. These K values represent inactivation of the viruses in a variety of matrices, ranging from water to protein-containing matrices such as albumin or complete culture medium). These results suggest that UV-C irradiation may be a viable approach for inactivating many of these problem viruses in raw materials or process intermediates used in biologics manufacture.

references for K values: * Lytle and Sagripanti 2005, ¶ Kowalski et al. 2009; † Maier 2007. The other values are the mean K values assembled by the author from the inactivation literature.

The one exception appears to be the polyomaviruses, which appear to be relatively resistant to the inactivating effects both of gamma irradiation and UV-C irradiation. This family of viruses may best be inactivated using high-temperature short-time treatment (HTST), though the efficacy of this approach for the polyomaviruses has yet to be demonstrated empirically.

Is Bovine Polyoma Virus Getting You Down?

By Dr. Ray Nims

Bovine polyomavirus (BPyV) is a double-stranded DNA virus of genus Polyomavirus. It is non-enveloped and 40-50 nm in diameter, and is a member of the same genus as SV40.

Basis of Concern. The Polyomavirus genus was so-named due to the ability of the viruses to cause tumors in susceptible host animals. Genomic sequences for the potentially oncogenic bovine polyomavirus have been detected with high frequency in bovine sera, regardless of geographic region of origin (Shuurman et al., J. Gen. Virol. 72: 2739-2745, 1991; Wang et al., New Zealand Vet. J. 53: 26-30, 2005).

Regulatory Expectations. Bovine polyomavirus is not mentioned specifically in 9CFR 113.47, Detection of extraneous viruses by the fluorescent antibody technique, as a virus of concern for raw materials of bovine origin. As a result, BPyV is not specifically probed for during most 9CFR 113.53-based raw material viral infectivity testing, and this test most likely would not be capable of detecting BPyV if present in the test material. The EMEA Note for Guidance on the use of Bovine Serum in the Manufacture of Human Biological Medicinal Products (CPMP/BWP/1793/02) states that sera users are “encouraged to apply infectivity assays for BPyV and to investigate methods for inactivation/removal of BPyV in order to limit or eliminate infectious virus from batches of serum”.

Mitigating Risk. In actual practice, the available infectivity assays for BPyV involve numerous passages using a bovine detector cell such as MDBK and are somewhat lengthy and insensitive, though more sensitive assays are under development. Cell-based infectivity testing for BPyV is not always being performed by users for each batch of bovine serum. The lack of a rapid and sensitive infectivity assay also means that viral inactivation studies for BPyV are not practically possible. While another polyomavirus such as SV40 could be used in viral inactivation/removal studies as a proxy for BPyV, in actual practice the murine parvovirus MMV (mouse minute virus) is more typically used as a worst-case model virus for such studies since it is non-enveloped and even smaller than BPyV. The few studies performed with SV40 indicate that gamma-irradiation at the dosages normally employed is not effective at inactivating this virus, as might be expected for a virus of this relatively small size (e.g., Gauvin, 2009). On the other hand, it has been shown (Wang et al., Vox Sanguinis 86: 230-238, 2004) that UVC treatment is effective in inactivating SV40. Note: since originally authoring this blog, I have come across a great number of UV-inactivation papers which indicate that polyomaviruses, and SV-40 in particular, appear to be relatively resistant to UV inactivation. The Wang et al. result may represent an outlier. I will address this in a future blog. Studies using MMV indicate that high-temperature short-time (HTST)-treatment of medium containing bovine serum is effective in inactivating this virus (Schleh et al., Biotechnol. Prog. 25: 854-860, 2009), and would by implication be effective for BPyV.

Conclusions. At the present time, infectivity screening of bovine sera for BPyV is not always being performed, and it is believed that the high frequency of detection of genomic material in bovine sera may not reflect a similarly high frequency of infectious BPyV. Risk of infection of biological products with BPyV through use of bovine-derived materials such as bovine sera may be mitigated through implementation of UVC- or HTST-treatment of media containing the sera and of viral purification processes capable of removing and inactivating an even smaller non-enveloped virus such as MMV.