Making Sense of Opposing Research on African Swine Fever Virus Inactivation Temperatures
By: Dr. Joe Crenshaw & Dr. Yanbin Shen
With African swine fever virus (ASFV) concerns continuing to weigh heavily on the swine industry, at times it can be difficult to interpret and reconcile opposing research results. A recent example of this involves separate studies related to the temperature required to inactivate ASFV to prevent transmission.
The University of Minnesota recently released preprint data indicating two double-stranded, multi-layered membrane enveloped DNA mega-viruses, Emiliania huxleyi virus (EhV) and African swine fever virus (ASFV), were damaged by heat treatment, but remained potentially viable when exposed to temperatures up to 100°C for 20 minutes (Balestreri et al., 2023). However, in a published journal article in Pathogens, the German ASFV reference lab reports that 59°C effectively eliminated infectivity of ASFV (Carrau et al., 2023).
Why do results from these two papers have vastly different conclusions? The main difference is based on the methods used to evaluate the potential infective capacity of ASFV subjected to different heat treatments.
UMN: Viable PCR Method
UMN used a “viable” PCR method. The “viable” PCR method uses a special dye to penetrate the damaged membrane of the mega-viruses caused by heat treatment (Figure 1).
Figure 1. The basic v-PCR workflow adapted from Biotium.
The theory is that the dye can penetrate damaged membrane and bind with the DNA genome thus interfering with PCR replication, however the dye does not penetrate un-damaged membranes. Therefore, the viable PCR method described by UMN can distinguish between intact membrane virus particles versus damaged membrane virus particles, which is an advantage over standard PCR methods that only detect genome regardless of the degree of membrane damage. The discovery of limited dye penetration in both EhV and ASFV even when exposed to temperatures up to 100°C for 20 minutes is an interesting observation that needs further research, however this does not necessarily mean the virus particles are still capable of causing infection. Even if the dye does not penetrate undamaged membranes, this does not mean that the treatment did not damage virus genome rendering it incapable of further replication. For example, ultraviolet light (UV-C) exposure is known to damage genome cross-linking rendering a virus incapable of replication, but it does not necessarily damage the virus membrane.
This aspect of the viable PCR method to equate the resistance to dye penetration with the ability of the virus particle to infect an animal cell has not been scientifically validated for ASFV. In fact, in this same publication by UMN the authors also evaluated the infectivity of EhV in cell culture and exposure to 50°C and 60°C was sufficient to eliminate viral infection after 30 seconds.
German ASFV Reference Lab: Widely Used Method
The German ASFV reference lab used the historically widely accepted method to examine the infectivity of ASFV after composting infected wild boar carcasses. This approach is a scientifically validated method for evaluating ASFV infectivity using a virus isolation method with porcine peripheral blood-derived macrophages. The primary mechanism of infection by ASFV is through phagocytosis by porcine macrophage cells. Exposing ASFV to their target macrophage cells in culture provides phagocytosis conditions that mimic a natural infection. Their study showed that ASFV was no longer infectious once the compost pile temperature reached 59°C, although the ASFV genome using the standard PCR method remained present, again confirming that standard PCR methods do not distinguish between viable and non-viable virus. Considering the limited membrane damage reported by UMN, the loss of infectivity at 59°C suggests that part of the ASFV genome or key components of membranes were damaged rendering it incapable of infection. The thermal inactivation data from the German ASFV reference lab confirms previous results that 60°C for 30 minutes (Plowright and Parker, 1967) and 60°C for 15 to 20 minutes (Mazur-Panasiuk et al., 2020) was sufficient to inactivate ASFV infectivity. Also, the EU Directive 2002/99/EC recognizes that a heat treatment at a minimum temperature of 80°C is sufficient to inactivate ASFV in meat.
Potential Viable Genome vs. Infectivity of the Virus
A potential viable genome does not equal infectivity. It is well-known that DNA is very stable at temperatures below 100°C. PCR tests are dependent on the fact that DNA remains stable at 95°C. Therefore, it is not surprising that PCR tests can detect the ASFV genome even after 100°C heat treatment. Limited dye penetration even when both EhV and ASFV were exposed to temperatures up to 100°C for 20 minutes is a very interesting discovery. These results suggest that viable genome could potentially remain even with high-temperature treatment. In fact, both EhV and ASFV lost their infectivity with 50°C and 60°C heat treatments, as reported by both the UMN and the Germany ASFV reference lab paper, as well as in previous publications. Both studies demonstrated that heat treatments were effective in reducing infectivity. Other methods like viral exposure to UV-C could provide additional assurance for inactivating hardy viruses like ASFV (Blázquez et al., 2021).
Key Takeaway
Further development and scientific validation of the viable PCR method as an indicator of viable virus is required before this technology should be used to provide appropriate temperature and/or chemical treatment guidelines to inactivate ASFV.
References
Balestreri C, Schroeder D. C, Sampedro F, Marqués G, Palowski A, Urriola P. E, van de Ligt J. L. G, Yancy H. F, Shurson G. C. Unexpected thermal stability of two enveloped megaviruses, Emiliania huxleyi virus and African swine fever virus, as measured by viability PCR. 2023. Under Review. https://doi.org/10.21203/rs.3.rs-2508557/v1
Blázquez E, C Rodríguez, J Ródenas, R Rosell, J Segalés, J Pujols, J Polo. 2021. Effect of spray-drying and ultraviolet C radiation as biosafety steps for CSFV and ASFV inactivation in porcine plasma. PLoS ONE 16(4): e0249935. https://doi.org/10.1371/journal.pone.0249935
Carrau T, Malakauskas A, Masiulis M, Bušauskas P, Japertas S, Blome S, Deutschmann P, Friedrichs V, Pileviečienė S, Dietze K, Beltrán-Alcrudo D, Hóvári M, Flory GA. Composting of Wild Boar Carcasses in Lithuania Leads to Inactivation of African Swine Fever Virus in Wintertime. Pathogens. 2023; 12(2):285. https://doi.org/10.3390/pathogens12020285
Mazur-Panasiuk, N., and Wozniakowski, G. Natural inactivation of African swine fever virus in tissues: Influence of temperature and environmental conditions on virus survival. Vet. Microbiol. 2020, 242, 108609.
Plowright W, and Parker J. The stability of African swine fever virus with particular reference to heat and pH inactivation. Arch Gesamte Virusforsch 1967, 21:383-402.
Official Journal of the European Communities; European Directive 2002/99/EC; Annex III
