Six underreported viral diseases of domesticated and wild swine in Africa: Implications and perspectives.

Pig production is increasing annually in Africa as it is recognized as a significant source of income, livelihood and food security, particularly in rural communities. Understanding the circulating swine pathogens is crucial for the success of this emerging industry. Although there is extensive data available on the African swine fever virus due to its devastating impact on pig production, knowledge about the presence of other viral swine pathogens on the continent is still extremely limited. This review discusses what is currently known about six swine pathogens in Africa: classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine circovirus-2, porcine circovirus-3, porcine parvovirus-1, and pseudorabies virus. Gaps in our knowledge are identified and topics of future focus discussed.

Use of FTA card for the detection of two RNA (CSFV and SV-A) and two DNA viruses (ASFVand SuHV-1) of importance in veterinary medicine.

The FTA card has emerged as a promising alternative for nucleic acid extraction. The FTA card is a filter paper impregnated with chemicals that preserve and stabilize the genetic material present in the sample, allowing for its storage and transport at room temperature. The aim of this study was to test the card for the detection of RNA and DNA nucleic acids. Two RNA viruses (Senecavirus A and classical swine fever virus) and two DNA viruses (African swine fever virus and suid alphaherpesvirus 1) were tested, and in all cases, there was a decrease in sensitivity. The methods exhibited good repeatability and demonstrated a rapid and practical use for sample transport and nucleic acid extraction.

A case report of long-delayed diagnosis of pseudorabies virus encephalitis with endophthalmitis: lessons from metagenomic next generation sequencing.

BACKGROUND: Pseudorabies virus (PRV) was thought to only infect animals. Recent studies have shown that it can also infect human. CASE PRESENTATION: We report a case of pseudorabies virus encephalitis and endophthalmitis, diagnosed 89 days after onset, confirmed with intraocular fluid metagenomic next generation sequencing (mNGS) after the result of two cerebrospinal fluid (CSF) mNGS tests were negative. Although treatment with intravenous acyclovir, foscarnet sodium, and methylprednisolone improved the symptoms of encephalitis, significant diagnostic delay resulted in permanent visual loss. CONCLUSIONS: This case suggests that pseudorabies virus (PRV) DNA in the intraocular fluid may have a higher positivity than that in the CSF. PRV may persist in the intraocular fluid for an extended period and may thus require extended antiviral therapy. Patients with severe encephalitis and PRV should be examined with the focus on pupil reactivity and light reflex. A fundus examination should be performed in patients with a central nervous system infection, specifically, those in a comatose state, to help reduce eye disability.

Design of fusion protein for efficient preparation of cyanovirin-n and rapid enrichment of pseudorabies virus.

OBJECTIVE: Cyanovirin-N (CVN) is a cyanobacterial protein with potent neutralizing activity against enveloped virus. To achieve the economic and functional production of CVN, the CVN N-terminally fused with CL7(A mutant of the Colicin E7 Dnase) was utilized to improve the solubility and stability of CVN fusion protein (CL7-CVN). Additionally, to improve the detection limit of existing PRV diagnostic assays, CL7-CVN was used for Pseudorabies virus (PRV) enrichment from larger sample volumes. RESULTS: CVN fused with CL7 was efficiently expressed at a level of ~ 40% of the total soluble protein in E. coli by optimizing the induction conditions. Also, the stability of CVN fusion protein was enhanced, and 10 mg of CVN with a purity of ~ 99% were obtained from 1 g of cells by one-step affinity purification with the digestion of HRV 3C protease. Moreover, both purified CVN and CL7-CVN could effectively inhibit the infection of PRV to PK15 cells. Considering the bioactivity of CL7-CVN, we explored a strategy for PRV enrichment from larger samples. CONCLUSIONS: CL7 effectively promoted the soluble expression of CVN fusion protein and improved its stability, which was meaningful for its purification and application. The design of CVN fusion protein provides an efficient approach for the economical and functional production of CVN and a new strategy for PRV enrichment.

A novel paper biosensor based on Fe3O4@SiO2-NH2and MWCNTs for rapid detection of pseudorabies virus.

In this study, a novel paper biosensor based on Fe3O4@SiO2-NH2magnetic polymer microspheres and multi walled carbon nanotubes (MWCNTs) for rapid detection of pseudorabies virus (PRV) was first developed. Fe3O4@SiO2-NH2were functionalized with PRV antibody and doped in cellulose nitrate paper to fabricate the magnetic paper biosensor with good magnetic response and biocompatibility. Using MWCNTs to build conductive network of sensors, PRV antigen binds specifically to the immunomagnetic microspheres on the sensor, and the resulting immune complex changes the magnetic domain structure of the sensor and the structural gap of MWCNTs, causing the magnetic property and impedance change. TEM and EDS characterization proved that the biosensor was successfully doped with Fe3O4@SiO2-NH2and effectively recognized PRV. Under optimized conditions, the impedance variation was found to be linearly related to the logarithm value of PRV concentrations in the range of 10-1 mg ml-1, with the detection limit of 10 ng ml-1. This paper biosensor demonstrated advantages of portability, high sensitivity and specificity, providing a valuable method for early control of PRV.

Multiplex and on-site PCR detection of swine diseases based on the microfluidic chip system.

BACKGROUND: At present, the process of inspection and quarantine starts with sampling at the customs port, continues with transporting the samples to the central laboratory for inspection experiments, and ends with the inspected results being fed back to the port. This process had the risks of degradation of biological samples and generation of pathogenic microorganisms and did not meet the rapid on-site detection demand because it took a rather long time. Therefore, it is urgently needed to develop a rapid and high-throughput detection assay of pathogenic microorganisms at the customs port. The aim of this study was to develop a microfluidic chip to rapidly detect swine pathogenic microorganisms with high-throughput and higher accuracy. Moreover, this chip will decrease the risk of spreading infection during transportation. RESULTS: A series of experiments were performed to establish a microfluidic chip. The resulting data showed that the positive nucleic acid of four swine viruses were detected by using a portable and rapid microfluidic PCR system, which could achieve a on-site real-time quantitative PCR detection. Furthermore, the detection results of eight clinical samples were obtained within an hour. The lowest concentration that amplified of this microfluidic PCR detection system was as low as 1 copies/µL. The results showed that the high specificity of this chip system in disease detection played an important role in customs inspection and quarantine during customs clearance. CONCLUSION: The microfluidic PCR detection system established in this study could meet the requirement for rapid detection of samples at the customs port. This chip could avoid the risky process of transporting the samples from the sampling site to the testing lab, and drastically reduce the inspection cycle. Moreover, it would enable parallel inspections on one chip, which greatly raised the efficiency of inspection.

Pseudorabies (Aujeszky's disease) virus DNA detection in swine nasal swab and oral fluid specimens using a gB-based real-time quantitative PCR.

In this study, the detection of PRV DNA in nasal swab (n = 440) and oral fluid (n = 1,545) samples collected over time from experimentally PRV vaccinated and/or PRV inoculated pigs (n = 40) was comparatively evaluated by real-time PCR. Serum samples (n = 440) were tested by PRV gB/gE blocking ELISAs (Pseudorabies Virus gB Antibody Test Kit and Pseudorabies Virus gpI Antibody Test Kit, IDEXX Laboratories, Inc., Westbrook, ME) to monitor PRV status over time. Following exposure to a gE-deleted modified live vaccine (Ingelvac® Aujeszky MLV, Boehringer Ingelheim, Ridgefield, CT) and/or a wild-type virus (3 CR Ossabaw), PRV gB DNA was detected in oral fluid specimens in a pattern similar to that of nasal swabs. For quantitative analyses, PRV PCR quantification cycle (Cq) results were re-expressed as "efficiency standardized Cqs (ECqs)" as a function of PCR efficiency using plate-specific positive amplification controls. ROC analyses of the PRV gB PCR ECqs results showed a similar performance of the PRV gB PCR for nasal swab and oral fluid specimens (area under the ROC curve = 85 % vs 83 %) and, based on an ECq cutoff of 0.01 a diagnostic specificity of 100 % and diagnostic sensitivities for oral fluid and nasal swab specimens of 53 % (95 % CI: 43 %, 62 %) and 70 % (95 % CI: 55 %, 83 %), respectively. Thus, the results described herein demonstrated the detection of PRV gB DNA in swine oral fluid and supported the use of this specimen in PRV diagnosis and surveillance.

Latent pseudorabies virus infection in medulla oblongata from quarantined pigs.

Pseudorabies virus (PRV) is a major pathogen in pig husbandry and is also a risk to human well-being. Pigs with latent PRV infection carry the virus lifelong, and it can be activated under conducive conditions. This poses a very important challenge to the control of the virus and may even prevent its elimination. To investigate latent infection with wild-type (wt) PRV, and also infection due to the use of live attenuated vaccines on farms, 80 pigs from two large-scale swine operations were traced. At 6 months old, the quarantined pigs were slaughtered and brain samples were collected. A PCR assay targeting the gB and gE genes was developed to detect PRV DNA fragments in medulla oblongata. Five of the samples (6.3%) were gB and gE gene fragment double-positive, 60 of the samples (75%) were gB single-positive, and 15 samples (18.7%) showed double-negative. A portion of latency-associated transcripts (LATs), EP0 mRNA, were found to be present in the gB gene fragment positive samples. Furthermore, the five double-positive samples were transmitted blindly, and apparent cytopathic effects were found in three of the five samples in the fourth generation. By means of Western blotting, PCR and sequencing, two of the isolated viruses were found to be related to vaccine strain Bartha-K61. Another was closely related to domestic epidemic strains HN1201 and LA and relatively unrelated to other Asian isolates. These results suggest that the live vaccines are latently present in brains, in a manner similar to wt PRV, and this poses potential safety issues in the pig husbandry industry. Wt PRV and live vaccine viruses were found to co-exist in pigs, demonstrating that the live vaccines were unable to confer complete sterilizing immunity, which may explain outbreaks of pseudorabies on vaccinated farms.

Bilateral Necrotizing Retinitis following Encephalitis Caused by the Pseudorabies Virus Confirmed by Next-Generation Sequencing.

Purpose: The objective of this study was to report a case of bilateral necrotizing retinitis following viral encephalitis caused by the pseudorabies virus.Case report: A 49-year-old male had decreased bilateral visual acuity after the recovery of consciousness for one month. He had been in an unconsciousness status due to encephalitis for two months before the ocular symptoms developed. He was a pig slaughterer. Ocular ultrasound showed bilateral vitreous haze and retinal detachment. A vitrectomy and silicone oil tamponade were performed on the left eye. During surgery, massive periphery retinal necrosis appearing as a tattered fish net, and multiple retinal holes were observed. The pseudorabies virus was detected by next-generation sequencing in the vitreous specimen.Conclusion: The pseudorabies virus may cause bilateral necrotizing retinitis following viral encephalitis among those with close contact to pigs. Intraocular fluid provides a greater selection of samples and a longer time window for pathogenic detection.

Development of a SYBR green I-based duplex real-time PCR assay for detection of pseudorabies virus and porcine circovirus 3.

In the present study, a specific and reliable duplex SYBR green I-based quantitative real-time polymerase chain reaction assay was established to detect pseudorabies virus (PRV) and porcine circovirus 3 (PCV3) simultaneously. Viral genomes of PRV and PCV3 in one specimen were identified by their different melting temperatures with melting peaks at 87 °C and 81 °C for PRV and PCV3 respectively, whilst other non-targeted swine pathogens exhibited no fluorescent signals. The assay displayed a high degree of linearity (R2 > 0.997), and the limits of detection were 37.8 copies/µL, 30.6 copies/µL and 60 copies/µL for PRV, PCV3 and the mixture of two recombinant plasmids, respectively. It had good repeatability and reproducibility, and the coefficients of variation in intra-batch and inter-batch assays were all less than 2.0%. In this research, the duplex assay was further evaluated using 117 clinical tissue specimens from diseased pigs in the field. The results revealed the infection rates of PRV and PCV3 were 23.08% (27/117) and 55.56% (65/117) respectively, and PRV and PCV3 co-infection rate was 14.53% (17/117). The assay could be utilized as a diagnostic tool with specificity, sensitivity, and reliability for molecular epidemiological surveillance of PRV and PCV3.

Detection of pseudorabies virus antibody in swine oral fluid using a serum whole-virus indirect ELISA.

We evaluated the detection of pseudorabies virus (PRV) antibodies in swine oral fluid. Oral fluid and serum samples were obtained from 40 pigs allocated to 4 treatment groups (10 pigs/group): negative control (NC); wild-type PRV inoculation (PRV 3CR Ossabaw; hereafter PRV); PRV vaccination (Ingelvac Aujeszky MLV; Boehringer Ingelheim; hereafter MLV); and PRV vaccination followed by PRV inoculation at 21 d post-vaccination (MLV-PRV). Using a serum PRV whole-virus indirect IgG ELISA (Idexx Laboratories) adapted to the oral fluid matrix, PRV antibody was detected in oral fluid samples from treatment groups PRV, MLV, and MLV-PRV in a pattern similar to serum. Vaccination alone produced a low oral fluid antibody response (groups MLV and MLV-PRV), but a strong anamnestic response was observed following challenge with wild-type virus (group PRV). Analyses of the oral fluid PRV indirect IgG ELISA results showed good binary diagnostic performance (area under ROC curve = 93%) and excellent assay repeatability (intra-class correlation coefficient = 99.3%). The demonstrable presence of PRV antibodies in swine oral fluids suggests the possible use of oral fluids in pseudorabies surveillance.

Comparison of gE/gI- and TK/gE/gI-Gene-Deleted Pseudorabies Virus Vaccines Mediated by CRISPR/Cas9 and Cre/Lox Systems.

Pseudorabies (PR), caused by pseudorabies virus (PRV), is an acute and febrile infectious disease in swine. To eradicate PR, a more efficacious vaccine needs to be developed. Here, the gE/gI- and TK/gE/gI-gene-deleted recombinant PRV (rGXΔgE/gI and rGXΔTK/gE/gI) are constructed through CRISPR/Cas9 and Cre/Lox systems. We found that the rGXΔTK/gE/gI was safer than rGXΔgE/gI in mice. Additionally, the effects of rGXΔgE/gI and rGXΔTK/gE/gI were further evaluated in swine. The rGXΔgE/gI and rGXΔTK/gE/gI significantly increased numbers of IFN-γ-producing CD4+ and CD8+ T-cells in swine, whereas there was no difference between rGXΔgE/gI and rGXΔTK/gE/gI. Moreover, rGXΔgE/gI and rGXΔTK/gE/gI promoted a PRV-specific humoral immune response. The PRV-specific humoral immune response induced by rGXΔgE/gI was consistent with that caused by rGXΔTK/gE/gI. After the challenge, swine vaccinated with rGXΔgE/gI and rGXΔTK/gE/gI showed no clinical signs and viral shedding. However, histopathological detection revealed that rGXΔgE/gI, not rGXΔTK/gE/gI, caused pathological lesions in brain and lung tissues. In summary, these results demonstrate that the TK/gE/gI-gene-deleted recombinant PRV was safer compared with rGXΔgE/gI in swine. The data imply that the TK/gE/gI-gene-deleted recombinant PRV may be a more efficacious vaccine candidate for the prevention of PR.

Simultaneous identification of 6 pathogens causing porcine reproductive failure by using multiplex ligation-dependent probe amplification.

We developed a multiplex ligation-dependent probe amplification (MLPA) assay for the simultaneous detection of 6 clinically relevant viral pathogens causing porcine reproductive failure, that is porcine reproductive and respiratory syndrome virus (PRRSV), Japanese encephalitis virus (JEV), classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2), pseudorabies virus (PRV) and porcine parvovirus (PPV). The limits of detection for the assay varied among the 6 target organisms from 1 to 8 copies per MLPA assay. The MLPA assay was evaluated with 346 heparinized porcine umbilical cord blood specimens, and the results of the assay were compared to those of real-time PCR. The MLPA assay showed specificities and sensitivities of 99.2% and 100%, respectively, for PRRSV; 100% and 100%, respectively, for CSFV, PCV2, PRV and PPV. No sample was found to be positive for JEV by either the MLPA assay or the real-time PCR. In conclusion, the MLPA assay has comparable clinical sensitivity to that of real-time PCR assay and provides a useful tool for fast screening porcine reproductive failure-associated viruses.

Canine parvovirus and pseudorabies virus coinfection as a cause of death in a wolf (Canis lupus) from southern Italy.

Pseudorabies virus (PRV) or suid herpesvirus 1 (SHV-1) is the causative agent of Aujeszky's disease, a highly contagious viral infection which causes neurological fatal illness in mammals other than suids. Here we report a case of a young wolf (Canis lupus) of around 2 years found dead by a hunter in the province of Avellino, Campania Region. Necropsy showed pathological findings consistent with encephalitis and gastroenteritis. Organs were analysed by microbiological and molecular investigations following standard procedures to ascertain the possible cause of death. Real-time PCR revealed the presence of PRV in the brain and of canine parvovirus 2b in organs like intestine, liver, brain, kidney and pancreas. Death probably occurred very shortly after SHV-1 infection in an animal already weakened by parvovirosis.

Stability of classical swine fever virus and pseudorabies virus in animal feed ingredients exposed to transpacific shipping conditions.

Classical swine fever virus (CSFV) and pseudorabies virus (PRV) are two of the most significant trade-limiting pathogens affecting swine worldwide. Both viruses are endemic to China where millions of kilograms of feed ingredients are manufactured and subsequently imported into the United States. Although stability and oral transmission of both viruses through contaminated pork products has been demonstrated as a risk factor for transboundary spread, stability in animal feed ingredients had yet to be investigated. The objective of this study was to determine the survival of CSFV and variant PRV in 12 animal feeds and ingredients exposed to environmental conditions simulating a 37-day transpacific shipment. Virus was detected by PCR, virus isolation and nursery pig bioassay. CSFV and PRV nucleic acids were stable throughout the 37-day period in all feed matrices. Infectious CSFV was detected in two ingredients (conventional soybean meal and pork sausage casings) at 37 days post-contamination, whereas infectious PRV was detected in nine ingredients (conventional and organic soybean meal, lysine, choline, vitamin D, moist cat and dog food, dry dog food and pork sausage casings). This study demonstrates the relative stability of CSFV and PRV in different feed ingredients under shipment conditions and provides evidence that feed ingredients may represent important risk factors for the transboundary spread of these viruses.

Genetic evolution analysis of novel recombinant pseudorabies virus strain in Sichuan, China.

Pseudorabies is a disease that seriously endangers the pig industry in China. Recently, we successfully isolated a pseudorabies virus from the brain tissue of piglets at a farm in Sichuan, China, and named it the FJ62 strain. In order to understand the molecular biological characteristics of the strain, primers were designed for glycoproteins gB, gC, gD and gE, which were amplified by a polymerase chain reaction (PCR) and sequenced. After comparing the sequence with the GenBank 22 pseudorabies virus reference strains and establishing the genetic evolutionary tree, it was found that the gB gene of pseudorabies virus was highly homologous (up to 100%) with the MY-1 strain which is isolated from a wild boar in Japan (AP018925) but that homology with other strains in China was low. The gC gene was in the same branch as most of the representative strains in China, with 99.5% homology. The gD gene is in the same branch as the domestic strain LA in China (KU552118), and the homology was 99.9%. The gE gene was in the same branch as the domestic BJ/YT strain in China (KC981239), with 99.9% homology. The results showed that the FJ62 strain of the pseudorabies virus isolated here may be a variant strain of FJ62 isolated from a domestic pig after natural recombination of pseudorabies virus genotype I from wild boar and genotype II from pigs in China. There have been no similar reports in Sichuan. The discovery of the recombinant virus strain provides a reference basis for the prevention and control of pseudorabies and a design strategy for a vaccine in Sichuan, China, in the future.

Single Virus Tracking with Quantum Dots Packaged into Enveloped Viruses Using CRISPR.

Labeling viruses with high-photoluminescence quantum dots (QDs) for single virus tracking provides a visual tool to aid our understanding of viral infection mechanisms. However, efficiently labeling internal viral components without modifying the viral envelope and capsid remains a challenge, and existing strategies are not applicable to most viruses. Here, we have devised a strategy using the clustered regularly interspaced short palindromic repeats (CRISPR) imaging system to label the nucleic acids of Pseudorabies virus (PRV) with QDs. In this strategy, QDs were conjugated to viral nucleic acids with the help of nuclease-deactivated Cas9/gRNA complexes in the nuclei of living cells and then packaged into PRV during virion assembly. The processes of PRV-QD adsorption, cytoplasmic transport along microtubules, and nuclear entry were monitored in real time in both Vero and HeLa cells, demonstrating the utility and efficiency of the strategy in the study of viral infection.

Development of a revised ICC-qPCR method used for Pseudorabies virus inactivation validation study of biologically sourced materials.

To develop a precise and convenient method to evaluate the virus transmission risk of biologically sourced materials, an integrated cell culture-qPCR (ICC-qPCR) method for Pseudorabies virus (PRV) was established and revised for applications to this new field. The optimized post-infection period was found at 12-hr to achieve a reasonable detection limit (-0.25 Log10TCID50/100 µL, Logs) and a quantitative range (0.75-3.75 Logs). The results of mimic samples suggested that three 10-fold dilutions at the time of virus inoculation combined with three washes after virus absorption, and the sets of non-amplified samples as controls could efficiently eliminate the false positive signals caused by high levels of noninfectious viruses. The virus inactivation validation studies of acellular porcine corneas suggested that the logs inactivation of PRV at 12 kGy irradiation dose obtained by general ICC-qPCR, revised ICC-qPCR and cell culture were 2.49, 4.85 and 5.08, respectively. At 25 kGy, those were 2.31, 4.85 and 5.08, respectively. The results obtained by the revised ICC-qPCR were consistent with cell culture and more precise than general ICC-qPCR. Therefore, the revised ICC-qPCR proposed in this study has an application prospect in the PRV inactivation validation studies of biologically sourced materials.

Dynamics of Aujeszky's disease virus infection in wild boar in enzootic scenarios.

Aujeszky's disease (AD) virus is enzootic in Iberian wild boar, thus posing a threat to the official eradication of AD on extensive domestic pig farms in Spain. Understanding the dynamics and drivers of ADV infection in wild boar will help prevent viral transmission at the wild boar-pig interface. This study analyses the dynamics of ADV infection in wild boar and tests relevant hypotheses in order to identify drivers of ADV infection dynamics. Wild boar sera (N = 971) and oropharyngeal tonsils (TN, N = 549) collected over 11 consecutive years in south-western Spain were tested for ADV antibodies and DNA, respectively. We tested the hypotheses that population immunity modulates the risk of ADV infection (H1 ) and that detecting ADV DNA in TN is a good proxy of the annual ADV infection pressure (H2 ). This was done by building logistic regression models that were subsequently employed to test the influence of a series of host population and host individual factors-including predictors of ADV immunity in the population-on the annual risk of new ADV infections and on the presence of ADV DNA in TN. The premise of H1 was that there would be a negative association between the proportion of ADV antibody-positive wild boar in a given year and the risk of ADV infection of naïve individuals. There was, however, a positive association, and H1 was, therefore, rejected. If detecting ADV in TN had been a good indicator of ADV infection pressure, a positive association with the proportion of ADV antibody-positive wild boar would have been found. However, this was not the case and H2 was also rejected. We confirmed that ADV infection is a dynamic phenomenon. The risk of infection with ADV can change considerably between consecutive years, and these changes are positively associated with the proportion of infected wild boar in the population.

Viral safety of APOSECTM: a novel peripheral blood mononuclear cell derived-biological for regenerative medicine.

BACKGROUND: Viral reduction and inactivation of cell-derived biologicals is paramount for patients' safety and so viral reduction needs to be demonstrated to regulatory bodies in order to obtain marketing authorisation. Allogeneic human blood-derived biological medicinal products require special attention. APOSECTM, the secretome harvested from selected human blood cells, is a new biological with promising regenerative capabilities. We evaluated the effectiveness of inactivation of model viruses by methylene blue/light treatment, lyophilisation, and gamma irradiation during the manufacturing process of APOSECTM. MATERIALS AND METHODS: Samples of intermediates of APOSECTM were acquired during the manufacturing process and spiked with bovine viral diarrhoea virus (BVDV), human immunodeficiency virus type 1 (HIV-1), pseudorabies virus (PRV), hepatitis A virus (HAV), and porcine parvovirus (PPV). Viral titres were assessed with suitable cell lines. RESULTS: Methylene blue-assisted viral reduction is mainly effective against enveloped viruses: the minimum log10 reduction factors for BVDV, HIV-1, and PRV were ≥6.42, ≥6.88, and ≥6.18, respectively, with no observed residual infectivity. Viral titres of both HAV and PPV were not significantly reduced, indicating minor inactivation of non-enveloped viruses. Lyophilisation had minor effects on the viability of several enveloped model viruses. Gamma irradiation contributes to the viral safety by reduction of enveloped viruses (BVDV: ≥2.42; HIV-1: 4.53; PRV: ≥4.61) and to some degree of non-enveloped viruses as seen for HAV with a minimum log10 reduction factor of 2.92. No significant reduction could be measured for the non-enveloped virus PPV (2.60). DISCUSSION: Three manufacturing steps of APOSECTM were evaluated under Good Laboratory Practice conditions for their efficacy at reducing and inactivating potentially present viruses. It could be demonstrated that all three steps contribute to the viral safety of APOSECTM.