Generation and Genetic Stability of a PolX and 5′ MGF-Deficient African Swine Fever Virus Mutant for Vaccine Development

The African swine fever virus (ASFV) causes fatal disease in pigs and is currently spreading globally. Commercially safe vaccines are urgently required. Aiming to generate a novel live attenuated vaccine (LAV), a recombinant ASFV was generated by deleting the viral O174L (PolX) gene. However, during in vitro generation, an additional spontaneous deletion of genes belonging to the multigene families (MGF) occurred, creating a mixture of two viruses, namely, Arm-ΔPolX and Arm-ΔPolX-ΔMGF. This mixture was used to inoculate pigs in a low and high dose to assess the viral dynamics of both populations in vivo. Although the Arm-ΔPolX population was a much lower proportion of the inoculum, in the high-dose immunized animals, it was the only resulting viral population, while Arm-ΔPolX-ΔMGF only appeared in low-dose immunized animals, revealing the role of deleted MGFs in ASFV fitness in vivo. Furthermore, animals in the low-dose group survived inoculation, whereas animals in the high-dose group died, suggesting that the lack of MGF and PolX genes, and not the PolX gene alone, led to attenuation. The two recombinant viruses were individually isolated and inoculated into piglets, confirming this hypothesis. However, immunization with the Arm-ΔPolX-ΔMGF virus did not induce protection against challenge with the virulent parental ASFV strain. This study demonstrates that deletion of the PolX gene alone neither leads to attenuation nor induces an increased mutation rate in vivo

Urban semiosis: Creative industries and the clash of systems

© The Author(s) 2014. This article has two aims. The first is to make the case that the ‘universe of the mind’ imagined by Yuri Lotman may be considered as a foundational model for cultural evolution (population-wide, dynamic, autopoietic, self-organising adaptation to changing environments). The second aim is to take forward a model of culture derived from Lotman’s work – a model I call ‘the clash of systems’ – in order to apply it to creative industries research. Such a move has the salutary effect of putting the ‘universe of the mind’ literally in its place. That place, now, predominantly, is in the city. Thus, the article uses Lotman’s model of the semiosphere to link different complex systems, principally the semiosphere with that of the city, in order to explore the productive potential of encounters – clashes – between different systems. Applying these insights to the field of creative industries research, the article proposes that creative culture in the globalised, urban and web-connected era can be characterised as ‘urban semiosis’

Effect of mixing and feed batch sequencing on the prevalence and distribution of African swine fever virus in swine feed

It is critical to have methods that can detect and mitigate the risk of African swine fever virus (ASFV) in potentially contaminated feed or ingredients bound for the United States. The purpose of this work was to evaluate feed batch sequencing as a mitigation technique for ASFV contamination in a feed mill, and to determine if a feed sampling method could identify ASFV following experimental inoculation. Batches of feed were manufactured in a BSL-3Ag room at Kansas State University's Biosafety Research Institute in Manhattan, Kansas. First, the pilot feed manufacturing system mixed, conveyed, and discharged an ASFV-free diet. Next, a diet was manufactured using the same equipment, but contained feed inoculated with ASFV for final concentration of 5.6 × 104 TCID50/g. Then, four subsequent ASFV-free batches of feed were manufactured. After discharging each batch into a collection container, 10 samples were collected in a double ‘X’ pattern. Samples were analysed using a qPCR assay for ASFV p72 gene then the cycle threshold (Ct) and Log10 genomic copy number (CN)/g of feed were determined. The qPCR Ct values (p < .0001) and the Log10 genomic CN/g (p < .0001) content of feed samples were impacted based on the batch of feed. Feed samples obtained after manufacturing the ASFV-contaminated diet contained the greatest amounts of ASFV p72 DNA across all criteria (p < .05). Quantity of ASFV p72 DNA decreased sequentially as additional batches of feed were manufactured, but was still detectable after batch sequence 4. This subsampling method was able to identify ASFV genetic material in feed samples using p72 qPCR. In summary, sequencing batches of feed decreases concentration of ASFV contamination in feed, but does not eliminate it. Bulk ingredients can be accurately evaluated for ASFV contamination by collecting 10 subsamples using the sampling method described herein. Future research is needed to evaluate if different mitigation techniques can reduce ASFV feed contamination

Detection of African Swine Fever Virus in Feed and Feed Mill Environment Following Extended Storage

7 Pág.One way to mitigate risk of feed-based pathogens for swine diets is to quarantine feed ingredients before inclusion in complete diets. Data have been generated evaluating the stability of swine viruses in ingredients, but the stability of African swine fever virus (ASFV) in feed or in a feed manufacturing environment has not been well characterized. Therefore, this study aimed to determine the stability of ASFV DNA in swine feed and on mill surfaces over time. A pilot-scale feed mill was used to manufacture six sequential batches of feed consisting of a batch of ASFV-free feed, followed by a batch inoculated with ASFV (final concentration = 5.6 × 104 TCID50/g), and then four subsequent ASFV-free batches. After each batch, 10 feed samples were aseptically collected in a double “X” pattern. During feed manufacturing, 24 steel coupons were placed on the floor of the manufacturing area and allowed to collect dust during feed manufacturing. Once feed manufacturing was completed, feed samples and steel coupons were stored at room temperature. Three of each were randomly selected from storage on 3, 7, 14, 28, 60, 90, and 180 days after feed manufacturing and analyzed for ASFV DNA. For feed samples, there was evidence of a batch × day interaction (P ¼ 0:023) for the quantification of genomic copies/g of feed, indicating that the amount of ASFV DNA present was impacted by both the batch of feed and days held at room temperature. There were no differences of genomic copies/g in early batches, but quantity of detectable ASFV decreased with increasing storage time. In Batches 4–6, the greatest quantity of ASFV DNA was detected on the day of feed manufacturing. The lowest quantity was detected on Day 7 for Batch 4, Day 60 for Batch 5, and at 28 and 180 days for Batch 6. There was no evidence of ASFV degradation on environmental discs across holding times (P ¼ 0:433). In conclusion, the quarantining of feed may help reduce but not eliminate the presence of ASFV DNA in feed over time. Importantly, ASFV DNA was detectable on feed manufacturing surfaces for at least 180 days with no overt evidence of reduction, highlighting the importance of bioexclusion of ASFV within feed manufacturing facilities and the need for thorough/effective decontamination and other mitigation processes in affected areas.Funding for this work was obtained from the NBAF Transition Funds from the State of Kansas and by the National Pork Board (Award #20-018), the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases under grant number HSHQDC 16-A-B0006, and the AMP Core of the NIGMS COBRE Center on Emerging and Zoonotic Infectious Diseases (CEZID) under award number P20GM13044.Peer reviewe

Prevalence and Distribution of African Swine Fever Virus in Swine Feed After Mixing and Feed Batch Sequencing

As the United States maintains trade with countries where African swine fever virus (ASFV) is endemic, it is critical to have methods that can detect and mitigate the risk of ASFV in potentially contaminated feed or ingredients. Therefore, the objectives of this study were to 1) evaluate feed batch sequencing as a mitigation technique for ASFV contamination in a feed mill, and 2) determine if a feed sampling method could identify ASFV following experimental inoculation. Batches of feed were manufactured in a BSL-3Ag room at Kansas State University’s Biosafety Research Institute in Manhattan, KS. First, the pilot feed manufacturing system mixed, conveyed, and discharged an ASFV-free diet. Next, a diet was manufactured using the same equipment, but contained feed inoculated with ASFV for a final concentration of 5.6 × 104 TCID50/g. Then, four subsequent ASFV-free batches of feed were manufactured. After discharging each batch into a biohazard tote, 10 samples were collected in a double ‘X’ pattern. Samples were analyzed using a qPCR assay specific for the ASFV p72 gene to determine the cycle threshold (Ct) and log10 genomic copy number (CN)/g of feed. Batch of feed affected the qPCR Ct values (P \u3c 0.0001) and the log10 genomic CN/g (P \u3c 0.0001) content of feed. Feed samples obtained after manufacturing the ASFV-contaminated diet contained the greatest (P \u3c 0.05) amounts of ASFV p72 DNA across all criteria. Quantity of ASFV p72 DNA decreased sequentially as additional batches of initially ASFV-free feed were manufactured, but it was still detectable after batch sequence 4, suggesting cross contamination between batches. This subsampling method was able to identify ASFV genetic material in feed samples using the PCR assay specific for the ASFV p72 gene. In summary, sequencing batches of feed decreases concentration of ASFV contamination in feed, but does not eliminate it. Bulk ingredients or feed can be accurately evaluated for ASFV contamination by collecting 10 evenly distributed subsamples, representing 0.05% of the volume of the container, using the sampling method described herein

Evaluating the distribution of African swine fever virus within a feed mill environment following manufacture of inoculated feed

11 Pág. Centro de Investigación en Sanidad Animal (CISA)It is critical to understand the role feed manufacturing may have regarding potential African swine fever virus (ASFV) transmission, especially given the evidence that feed and/or ingredients may be potential vectors. The objective of the study was to evaluate the distribution of ASFV in a feed mill following manufacture of contaminated feed. To accomplish this, a pilot-scale feed mill consisting of a mixer, bucket elevator, and spouting was constructed in a BSL-3Ag facility. First, a batch of ASFV-free feed was manufactured, followed by a batch of feed that had an ASFV-contaminated ingredient added to feed, which was then mixed and discharged from the equipment. Subsequently, four additional ASFV-free batches of feed were manufactured using the same equipment. Environmental swabs from 18 locations within the BSL-3Ag room were collected after each batch of feed was discharged. The locations of the swabs were categorized into four zones: 1) feed contact surface, 2) non-feed contact surface 1 meter from feed, and 4) transient surfaces. Environmental swabs were analyzed using a qPCR specific for the ASFV p72 gene and reported as genomic copy number (CN)/mL of environmental swab processing buffer. Genomic copies were transformed with a log10 function for statistical analysis. There was no evidence of a zone × batch interaction for log10 genomic CN/mL (P = 0.625) or cycle threshold (Ct) value (P = 0.608). Sampling zone impacted the log10 p72 genomic CN/mL (P < 0.0001) and Ct values (P < 0.0001), with a greater amount of viral genome detected on transient surfaces compared to other surfaces (P < 0.05). This study illustrates that once ASFV enters the feed mill environment it becomes widespread and movement of people can significantly contribute to the spread of ASFV in a feed mill environment.Funding for this work was obtained from the NBAF Transition Funds from the state of Kansas (JAR), the National Pork Board under award number 20-018 (CKJ), the Department of Homeland Security Center of Excellence for Emerging and Zoonotic Animal Diseases under grant number HSHQDC 16-A-B0006 (JAR), and the AMP Core of the NIGMS COBRE Center on Emerging and Zoonotic Infectious Diseases (CEZID) under award number P20GM13044 (JAR)Peer reviewe

Analysis of two methods of isometric muscle contractions during the anti-G straining maneuver

This study investigated the difference in Mean Arterial Pressure (MAP) and Cardiac Output (CO) between two methods of isometric muscle contractions during the Anti-G Straining Maneuver (AGSM). 12 subjects (ages 18 to 38 yrs, height 176.8 +/- 7.4 cm, body mass 78.8 +/- 15.6 kg, percent body fat 14.3 +/- 6.6%) participated in the study. The study was a one-way within-subject design with test conditions counterbalanced. Two methods of isometric muscle contractions lasting 30 seconds each were assessed; an isometric push contraction and an isometric muscle tensing contraction. The dependent parameters were MAP and CO. The average MAP during the push contraction was 123 mmHg, SD +/- 11 and for tense was 118 mmHg, SD +/- 8. CO was 7.6 L/min, SD +/- 1.6 for push and 7.9 L/min, SD +/- 2.0 for tense method. Dependent t-tests revealed t(11) = 1.517, p = 0.157 for MAP and t(11) = 0.875, p = 0.400 for CO. This study demonstrated that the two methods of isometric muscle contractions were not statistically different with regards to MAP and CO. Therefore, both forms of isometric contractions may be potentially useful when performing the muscle contraction portion of the AGSM

Human Tumour Immune Evasion via TGF-β Blocks NK Cell Activation but Not Survival Allowing Therapeutic Restoration of Anti-Tumour Activity

Immune evasion is now recognized as a key feature of cancer progression. In animal models, the activity of cytotoxic lymphocytes is suppressed in the tumour microenvironment by the immunosuppressive cytokine, Transforming Growth Factor (TGF)-β. Release from TGF-β-mediated inhibition restores anti-tumour immunity, suggesting a therapeutic strategy for human cancer. We demonstrate that human natural killer (NK) cells are inhibited in a TGF-β dependent manner following chronic contact-dependent interactions with tumour cells in vitro. In vivo, NK cell inhibition was localised to the human tumour microenvironment and primary ovarian tumours conferred TGF-β dependent inhibition upon autologous NK cells ex vivo. TGF-β antagonized the interleukin (IL)-15 induced proliferation and gene expression associated with NK cell activation, inhibiting the expression of both NK cell activation receptor molecules and components of the cytotoxic apparatus. Interleukin-15 also promotes NK cell survival and IL-15 excluded the pro-apoptotic transcription factor FOXO3 from the nucleus. However, this IL-15 mediated pathway was unaffected by TGF-β treatment, allowing NK cell survival. This suggested that NK cells in the tumour microenvironment might have their activity restored by TGF-β blockade and both anti-TGF-β antibodies and a small molecule inhibitor of TGF-β signalling restored the effector function of NK cells inhibited by autologous tumour cells. Thus, TGF-β blunts NK cell activation within the human tumour microenvironment but this evasion mechanism can be therapeutically targeted, boosting anti-tumour immunity

Association of cetuximab with adverse pulmonary events in cancer patients: a comprehensive review

Compounds derived from biologic sources, or biologicals, are increasingly utilized as therapeutic agents in malignancy. Development of anti-cancer targeted therapies from biologics is increasingly being utilized. Cetuximab, a chimeric monoclonal antibody, is one such anti-cancer targeted therapeutic that has shown efficacy in quelling the rate of patient decline in colorectal, head/neck, and non-small cell lung cancer. However, due to the relatively recent addition of biologic compounds to the therapeutic arsenal, information related to adverse reactions is less well known than those seen in traditional chemotherapeutics. Dermatologic reactions have been demonstrated as the most frequent side effect cited during cetuximab therapy for malignancy; however, other effects may lead to greater morbidity. In general, pulmonary complications of therapeutics can lead to significant morbidity and mortality. The purpose of this review is to compile the various pulmonary side effects seen in patients treated with cetuximab for various malignancies, and to compare the incidence of these adverse reactions to standard therapies