Porcine virus inactivation

Chemical disinfectants have received the most attention and many classes are able to inactivate PEDV Bowman et al. However, some chemical disinfectants are corrosive to metal surfaces, hazardous to human and animal health, and inactivated with organic material and water with high mineral content Babb et al. One commonly used alternative to chemical disinfection is thermal inactivation of virus on inanimate objects.

Within the U. However, use of heat in this setting requires specialized equipment and a prolonged period, which can be a challenge in such a high turn-over industry. The present study sought to investigate hot water as a method for rapid thermal inactivation of PEDV.

Forty milliliters of sterile distilled water were continuously mixed while heated to one of seven treatment temperatures: 4. Water at each temperature was inoculated with 1. In the same manner, each vial of heated water was sampled immediately prior to inoculation with PEDV to serve as a negative control.

All above mentioned procedures were completed in triplicate. Inoculated wells were observed daily and all samples demonstrating cytopathic effects CPE three days post-inoculation in any of the wells were classified as positive. Previously described methods were used to measure tissue culture infective dose TCID 50 for all positive samples Bowman et al. Mean TCID 50 values were calculated for each triplicate, which were subsequently log-transformed, normality visually assessed, and compared using a two-way analysis of variance ANOVA.

Infectious PEDV was recovered from all samples in the 4. Samples from treatments of Heated water was effective at rendering PEDV nonviable at all other temperature and time points Table 1. Porcine epidemic diarrhea virus survivability over time when treated with water heated to specified temperatures. Treatment with Inactivation of PEDV in heated water was accomplished using a wide range of temperature and time combinations demonstrating the potential use as an alternative disinfection method.

Most importantly the ability of heated water to rapidly inactivate PEDV in as little as 10 s would make it an asset for many areas of swine production where economic demands require great attention to time-sensitive approaches.

The importance of fomites in PEDV transmission was highlighted when contaminated flexible intermediate bulk containers were implicated as the likely method for intercontinental movement of the virus to United States U. Once the virus was introduced to the United States, PEDV spread rapidly across the country, likely through gaps in biosecurity protocols Bowman et al. Furthermore, with the ability to survive in the environment for up to days in soybean meal Dee et al.

Heated water may be useful in situations where penetration of objects with chemical disinfectants is difficult and where corrosion of the target materials e. The swine industry is reliant upon nucleic acid detection to identify the presence of PEDV in pigs and on fomites; however, this diagnostic tool does not determine if there are intact viral particles capable of causing infection. One challenge with the use of many classes of disinfectants is the inability to disrupt RNA beyond the threshold of detection, which makes it impossible to discern disinfection effectiveness from a PCR positive test result Bowman et al.

Heated water is not immune from this challenge as PEDV nucleic acid was detected via rRT-PCR in all samples in the present study, including those rendered non-infectious by their treatment. One limitation in the present study is the cyclic operating heat element which made it difficult to maintain a perfectly constant temperature over the entire min treatment period.

However, the desired treatment temperature was always verified at the time of inoculation and given the observed rapid results, we concluded the temperature fluctuations were of minimal impact.

Additionally, the first sampling time point 5 s post inoculation may not have provided sufficient time for thorough mixing prior to sample collection. It is also important to note that cell culture assay may underestimate the actual viral infectivity when compared to animal bioassays. Further studies might include observing the infectivity in neonatal pigs in comparison to weaned pigs which would validate whether hot water would be a feasible disinfectant for a swine farm Thomas et al.

Overcoming the above mentioned limitations would be essential in conducting a follow-up experiment using field conditions e. While thermal inactivation is less impacted by organic material, it could potentially serve as viral protectant during the application of hot water. While heated water may not be practical in some situations due to animal and human health risks from scalding hot water, heated water is another tool in the veterinarian's tool box to combat PEDV.

Shades of purple indicate different haplotypes of PERVs. Through screening of different anti-apoptotic strategies Fig. S9 , suggesting that there is no viral particle secreted by the modified cells.

To examine chromosomal integrity with higher resolution, we performed PERV genomic junctions sequencing on the 3 normal karyotype clones to examine potential deletions between Cas9-induced double-strand breaks DSBs.

All 21 tested junctions remained intact Fig. S11 , which indicates no Cas9-induced macrodeletions in these regions. We observed normal cell stage blastocyst structure and validated the pluripotency of inner cell mass detected by SOX2 antibody on day 6 Fig.

Despite the lower pregnancy efficiency, which is commonly observed in transgenic pig production 11 , we successfully produced putative PERV- inactivated pigs Fig.

In addition, we observed that the copy number of PERVs in the generated pigs stays close to 25, reconfirming that there is no reinfection Fig. We performed karyotyping and did not detect abnormal structural changes in the PERV-inactivated pigs Fig. We are conducting long term studies to monitor the impact of PERV-inactivation and gene editing on big animals. This picture showed the first born pig Laika at day 2 after birth. In summary, we observed in our studies that PERVs can be transmitted from pig to human cells and transmitted among human cells in vitro.

These results substantiate the risk of cross-species viral transmission in the context of xenotransplantation. Although we have focused in this paper on the applications to xenotransplantation, we envision, more generally, that the synergistic combination of CRISPR-Cas technology with anti-apoptosis treatment may also be used to enable large-scale genome engineering in primary cells for a broad range of applications, including pathway engineering and modifications of other genetic repetitive elements of biological interest.

Therefore, our data substantiates the value of PERV-inactivation for safe xenotransplantation practice. The physiological functions of endogenous retrovirus, which exists in all mammalian species, remain largely unknown. Further studies on our PERV-inactivated pigs will shed light on the endogenous retrovirus functionalities in related to the hosts. Most importantly, the PERV-inactivated pig can serve as a foundation pig strain, which can be further engineered to provide safe and effective organ and tissue resources for xenotransplantation.

We thank J. Markmann from Massachusetts General Hospital and P. Takeuchi from University College London for his insightful comments on virus interference; T. This study is mainly funded by eGenesis Inc. National Center for Biotechnology Information , U. Author manuscript; available in PMC Feb Church , 1, 2, 4, and Luhan Yang 1,.

Dong Niu 1 eGenesis Inc. Haydy George 1 eGenesis Inc. Tao Wang 1 eGenesis Inc. I-Hsiu Lee 1 eGenesis Inc. China Find articles by Yong Wang. Yinan Kan 1 eGenesis Inc. Emal Lesha 1 eGenesis Inc. Gang Wang 1 eGenesis Inc.

China Find articles by Xiaoyang Zhou. China Find articles by Hong Wei. George M. Church 1 eGenesis Inc. During the starter phase d 14 to 28, Table 5 , in which all the pigs were fed a common diet, no differences due to the previous dietary treatment were observed for weight gain, or feed intake, but for feed efficiency G:F there was a tendency for improvement in the control group.

Spray dried porcine plasma is considered an inherently safe protein for use in animal feed, as evidenced by the fact that it was the first animal protein re-authorized in Europe for use in feed for animals intended for human consumption after the use of animal proteins in feed for meat animals was banned in due to the BSE crisis.

During the manufacturing of spray dried blood products, there are several critical control points which reduce contamination and subsequent growth of pathogens in the initial collected blood. Spray-drying has been shown effective to inactivate coliform bacteria [ 13 ], as well as swine viruses, such as porcine reproductive and respiratory syndrome virus PRRSV , pseudorabies virus [ 11 ], porcine epidemic diarrhea virus [ 14 , 15 ] and swine vesicular disease virus [ 16 ].

Likewise, feeding susceptible animals diets containing high inclusion levels of commercial SDPP for a feeding duration of 3 to 4 times higher than typically used in commercial practice, have shown that SDPP is a safe protein ingredient that does not transmit PPV and porcine circovirus type 2 PCV2 [ 11 , 12 , 17 , 18 ], which are two of the more thermal and solvent resistant known viruses. However, as microbes evolve and new pathogens are discovered, it is prudent to evaluate additional technology that may further minimize risks associated with feeding biological products to animals intended for human consumption.

In case of animal plasma, there are limitations on additional safety features that can be applied without affecting the functionality of the plasma proteins. Thermal processing is well recognized to inactivate pathogens of concern in animal by-products [ 19 ], however, for plasma products, prolonged high temperature processing or chemical treatments denature proteins. Also, e-beam or gamma irradiation is not accepted by regulatory agencies in many countries or by some customers.

Ultraviolet irradiation of different food and feed products has demonstrated pathogen inactivation efficacy without causing negative effects on the nutritional or physical qualities of the treated material [ 20 , 21 ] and it is widely recognized as a safe technology to use for inactivation of microbes in food or feed products. Nevertheless, successful application of UV-C depends on the solids content and turbidity of the liquid, which can limit the penetration of UV-C light into the liquid and therefore affect efficacy [ 22 ].

Therefore, the application of a UV-C system to turbid liquids like animal plasma require a special design of the system to assure that the liquid has a turbulent flow regime and that the UV-C light is able to penetrate the liquid to be irradiated.

This system has been proven to inactivate microorganisms in turbid liquids like fruit juices and wine [ 4 , 23 ] and milk [ 24 ] and also is a system that is capable of processing large volumes of raw material that is common for the spray dried blood product business. In experiment 1, UV-C irradiation was very effective for inactivating PPV, thus indicating successful reduction of a model virus inoculated in liquid bovine plasma. Also, UV-C irradiation has proved effective against different model viruses human B19, PPV, simian virus 40, Sindbis virus, reovirus type 3, adenovirus type 5, murine parvovirus, encephalomyocarditis virus and bovine herpes virus type 2 and both Gram negative and Gram positive bacteria in human plasma products [ 25 , 26 ] and it is likely that this technology could be applicable to inactivation of emerging pathogens.

Van Wyk and Gouws [ 27 ] also demonstrated the efficiency of UV irradiation to inactivate pathogenic and heat resistant microorganisms like Bacillus spp. In a literature review by Hijnen et al. In addition, Darnell et al. Previous research has focused on the effect of microbial quality of SDP on pig performance and the benefit of gamma irradiation, or the use of chemical products such as formaldehyde, to improve the bacterial quality of SDP and the performance of weaned pig [ 30 — 32 ].

DeRouchey et al. The use of gamma irradiation is expensive and not approved in some countries or accepted by some customers from countries where this technology is permitted. Formaldehyde treatment is correlated with significant negative effects on solubility, IgG recovery, and gelling capacity and is also perceived by some customers as a practice to mask low quality raw materials.

Moreover, formaldehyde has not proved to be effective against heat and chemical resistant viruses like PPV or PCV-2 [ 33 ]. In fact, we observed a tendency of improved pig performance during the first 14 days and better performance over the whole period when pigs were fed diets with UV- irradiated plasma compared with non UV irradiated plasma diets.

These results are in agreement with the reported values indicated by DeRouchey et al. Nevertheless, it is worthy to indicate that the control plasma showed absence of Salmonella and low level of Enterobacteriaceae indicating the low prevalence of pathogenic microorganisms in commercial SDPP. Adaptation of a uniquely designed UV-C process specific for the manufacturing of spray dried plasma that does not adversely affect its beneficial functional components has merit as an additional biosafety feature to protect against unidentified or emerging pathogens that may evolve over time in biological source material.

Such redundant technologies are aligned with suggestions from international organizations that recommend biosafety procedures to minimize risks associated with use of biological products. Conceived and designed the experiments: J.

Performed the experiments: J. Analyzed the data: J. Wrote the paper: J. Browse Subject Areas? Click through the PLOS taxonomy to find articles in your field. Abstract A novel ultraviolet light irradiation UV-C, nm process was designed as an additional safety feature for manufacturing of spray dried porcine plasma SDPP. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files.

Introduction Spray dried porcine plasma SDPP is a dehydrated product obtained from blood of healthy pigs collected at slaughterhouses.

Experiment 2 Porcine plasma was derived from whole blood collected from a Spanish inspected abattoir Costa Brava, Girona, Spain. Download: PPT. Table 1. Chemical and microbial analyses of spray dried porcine plasma used in Experiment 2 as-fed basis.

Table 2. Composition of the diets used in Experiment 2, as-fed basis. Table 3. Calculated nutrient content of the diets used in Experiment 2, as fed basis. Table 4. Average Porcine Parvovirus PPV titres in inoculated bovine plasma used in Experiment 1 at each irradiation time log10 values 1. Fig 1. Porcine parvovirus virus inactivation in liquid bovine plasma after UV-C treatment. Table 5. Least squares means of productive parameters of pigs 1 fed diets with ultraviolet irradiated spray dried plasma Experiment 2.

Discussion Spray dried porcine plasma is considered an inherently safe protein for use in animal feed, as evidenced by the fact that it was the first animal protein re-authorized in Europe for use in feed for animals intended for human consumption after the use of animal proteins in feed for meat animals was banned in due to the BSE crisis. Conclusions Adaptation of a uniquely designed UV-C process specific for the manufacturing of spray dried plasma that does not adversely affect its beneficial functional components has merit as an additional biosafety feature to protect against unidentified or emerging pathogens that may evolve over time in biological source material.

Supporting Information. S1 Table. S2 Table. UV irradiation effect on PPV virus inactivation in bovine plasma.

S1 Text. Probability of minimum virus titre when no virus is detected in the sample. Author Contributions Conceived and designed the experiments: J. References 1. Use of spray-dried animal plasma in diets for weanling pigs. Pig News Inf. View Article Google Scholar 2. Torrallardona D.