Experimental Infection of Domestic Pigs with African Swine Fever Virus Isolated in 2019 in Mongolia.

African swine fever (ASF) is an infectious viral disease caused by African swine fever virus (ASFV), that causes high mortality in domestic swine and wild boar (Sus scrofa). Currently, outbreaks are mitigated through strict quarantine measures and the culling of affected herds, resulting in massive economic losses to the global pork industry. In 2019, an ASFV outbreak was reported in Mongolia, describing a rapidly progressing clinical disease and gross lesions consistent with the acute form of ASF; the virus was identified as a genotype II virus. Due to the limited information on clinical disease and viral dynamics within hosts available from field observations of the Mongolian isolates, we conducted the present study to further evaluate the progression of clinical disease, virulence, and pathology of an ASFV Mongolia/2019 field isolate (ASFV-MNG19), by experimental infection of domestic pigs. Intramuscular inoculation of domestic pigs with ASFV-MNG19 resulted in clinical signs and viremia at 3 days post challenge (DPC). Clinical disease rapidly progressed, resulting in the humane euthanasia of all pigs by 7 DPC. ASFV-MNG19 infected pigs had viremic titers of 108 TCID50/mL by 5 DPC and shed virus in oral secretions late in disease, as determined from oropharyngeal swabs. Whole-genome sequencing confirmed that the ASFV-MNG19 strain used in this study was a genotype II strain highly similar to other regional strains. In conclusion, we demonstrate that ASFV-MNG19 is a virulent genotype II ASFV strain that causes acute ASF in domestic swine.

Comparison of oseltamivir and α-galactosylceramide for reducing disease and transmission in pigs infected with 2009 H1N1 pandemic influenza virus.

Influenza virus infections are a major cause of respiratory disease in humans. Neuraminidase inhibitors (NAIs) are the primary antiviral medication used to treat ongoing influenza infections. However, NAIs are not always effective for controlling virus shedding and lung inflammation. Other concerns are the emergence of NAI-resistant virus strains and the risk of side effects, which are occasionally severe. Consequently, additional anti-influenza therapies to replace or combine with NAIs are desirable. Here, we compared the efficacy of the NAI oseltamivir with the invariant natural killer T (iNKT) cell superagonist, α-galactosylceramide (α-GalCer), which induces innate immune responses that inhibit influenza virus replication in mouse models. We show that oseltamivir reduced lung lesions and lowered virus titers in the upper respiratory tract of pigs infected with A/California/04/2009 (CA04) pandemic H1N1pdm09. It also reduced virus transmission to influenza-naïve contact pigs. In contrast, α-GalCer had no impact on virus replication, lung disease, or virus transmission, even when used in combination with oseltamivir. This is significant as iNKT-cell therapy has been studied as an approach for treating humans with influenza.

Evaluating α-galactosylceramide as an adjuvant for live attenuated influenza vaccines in pigs.

Natural killer T (NKT) cells activated with the glycolipid ligand α-galactosylceramide (α-GalCer) stimulate a wide variety of immune cells that enhance vaccine-mediated immune responses. Several studies have used this approach to adjuvant inactivated and subunit influenza A virus (IAV) vaccines, including to enhance cross-protective influenza immunity. However, less is known about whether α-GalCer can enhance live attenuated influenza virus (LAIV) vaccines, which usually induce superior heterologous and heterosubtypic immunity compared to non-replicating influenza vaccines. The current study used the swine influenza challenge model to assess whether α-GalCer can enhance cross-protective immune responses elicited by a recombinant H3N2 LAIV vaccine (TX98ΔNS1) encoding a truncated NS1 protein. In one study, weaning pigs were administered the H3N2 TX98ΔNS1 LAIV vaccine with 0, 10, 50, and 100 µg/kg doses of α-GalCer, and subsequently challenged with a heterologous H3N2 virus. All treatment groups were protected from infection. However, the addition of α-GalCer appeared to suppress nasal shedding of the LAIV vaccine. In another experiment, pigs vaccinated with the H3N2 LAIV, with or without 50 µg/kg of α-GalCer, were challenged with the heterosubtypic pandemic H1N1 virus. Pigs vaccinated with the LAIV alone generated cross-reactive humoral and cellular responses which blocked virus replication in the airways, and significantly decreased virus shedding. On the other hand, combining the vaccine with α-GalCer reduced cross-protective cellular and antibody responses, and resulted in higher virus titers in respiratory tissues. These findings suggest that: (i) high doses of α-GalCer impair the replication and nasal shedding of the LAIV vaccine; and (ii) α-GalCer might interfere with heterosubtypic cross-protective immune responses. This research raise concerns that should be considered before trying to use NKT cell agonists as a possible adjuvant approach for LAIV vaccines. Supplementary Information: The online version contains supplementary material available at 10.1186/s44149-022-00051-x.

Mutations in SARS-CoV-2 variants of concern link to increased spike cleavage and virus transmission.

SARS-CoV-2 lineages have diverged into highly prevalent variants termed "variants of concern" (VOCs). Here, we characterized emerging SARS-CoV-2 spike polymorphisms in vitro and in vivo to understand their impact on transmissibility and virus pathogenicity and fitness. We demonstrate that the substitution S:655Y, represented in the gamma and omicron VOCs, enhances viral replication and spike protein cleavage. The S:655Y substitution was transmitted more efficiently than its ancestor S:655H in the hamster infection model and was able to outcompete S:655H in the hamster model and in a human primary airway system. Finally, we analyzed a set of emerging SARS-CoV-2 variants to investigate how different sets of mutations may impact spike processing. All VOCs tested exhibited increased spike cleavage and fusogenic capacity. Taken together, our study demonstrates that the spike mutations present in VOCs that become epidemiologically prevalent in humans are linked to an increase in spike processing and virus transmission.

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.

Epizootic Haemorrhagic Disease Virus Serotype 8 in Tunisia, 2021.

Epizootic haemorrhagic disease (EHD) is a Culicoides-borne viral disease caused by the epizootic haemorrhagic disease virus (EHDV) associated with clinical manifestations in domestic and wild ruminants, primarily white-tailed deer (Odocoileus virginianus) and cattle (Bos taurus). In late September 2021, EHDV was reported in cattle farms in central/western Tunisia. It rapidly spread throughout the country with more than 200 confirmed outbreaks. We applied a combination of classical and molecular techniques to characterize the causative virus as a member of the serotype EHDV-8. This is the first evidence of EHDV- 8 circulation since 1982 when the prototype EHDV-8 strain was isolated in Australia. This work highlights the urgent need for vaccines for a range of EHDV serotypes.

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

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 away from feed, 3) 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.

Reston virus causes severe respiratory disease in young domestic pigs.

Reston virus (RESTV), an ebolavirus, causes clinical disease in macaques but has yet only been associated with rare asymptomatic infections in humans. Its 2008 emergence in pigs in the Philippines raised concerns about food safety, pathogenicity, and zoonotic potential, questions that are still unanswered. Until today, the virulence of RESTV for pigs has remained elusive, with unclear pathogenicity in naturally infected animals and only one experimental study demonstrating susceptibility and evidence for shedding but no disease. Here we show that combined oropharyngeal and nasal infection of young (3- to 7-wk-old) Yorkshire cross pigs with RESTV resulted in severe respiratory disease, with most animals reaching humane endpoint within a week. RESTV-infected pigs developed severe cyanosis, tachypnea, and acute interstitial pneumonia, with RESTV shedding from oronasal mucosal membranes. Our studies indicate that RESTV should be considered a livestock pathogen with zoonotic potential.

Unaltered influenza disease outcomes in swine prophylactically treated with α-galactosylceramide.

Influenza A viruses (IAV) are a major cause of respiratory diseases in pigs. Invariant natural killer T (iNKT) cells are an innate-like T cell subset that contribute significantly to IAV resistance in mice. In the current work, we explored whether expanding and activating iNKT cells with the iNKT cell superagonist α-galactosylceramide (α-GalCer) would change the course of an IAV infection in pigs. In one study, α-GalCer was administered to pigs intramuscularly (i.m.) 9 days before infection, which systemically expanded iNKT cells. In another study, α-GalCer was administered intranasally (i.n.) 2 days before virus infection to activate mucosal iNKT cells. Despite a synergistic increase in iNKT cells when α-GalCer i.m. treated pigs were infected with IAV, neither approach reduced disease signs, lung pathology, or virus replication. Our results indicate that prophylactic use of iNKT cell agonists to prevent IAV infection is ineffective in pigs. This is significant because this type of approach has been considered for humans whose iNKT cell levels and IAV infections are more similar to those of pigs than mice.

Modulation of Immune Responses to Influenza A Virus Vaccines by Natural Killer T Cells.

Influenza A viruses (IAVs) circulate widely among different mammalian and avian hosts and sometimes give rise to zoonotic infections. Vaccination is a mainstay of IAV prevention and control. However, the efficacy of IAV vaccines is often suboptimal because of insufficient cross-protection among different IAV genotypes and subtypes as well as the inability to keep up with the rapid molecular evolution of IAV strains. Much attention is focused on improving IAV vaccine efficiency using adjuvants, which are substances that can modulate and enhance immune responses to co-administered antigens. The current review is focused on a non-traditional approach of adjuvanting IAV vaccines by therapeutically targeting the immunomodulatory functions of a rare population of innate-like T lymphocytes called invariant natural killer T (iNKT) cells. These cells bridge the innate and adaptive immune systems and are capable of stimulating a wide array of immune cells that enhance vaccine-mediated immune responses. Here we discuss the factors that influence the adjuvant effects of iNKT cells for influenza vaccines as well as the obstacles that must be overcome before this novel adjuvant approach can be considered for human or veterinary use.

What We Need to Consider During and After the SARS-CoV-2 Pandemic.

Even though extreme containment and mitigation strategies were implemented by numerous governments around the world to slow down the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the number of critically ill patients and fatalities keeps rising. This crisis has highlighted the socioeconomic disparities of health care systems within and among countries. As new CoVID policies and responses are implemented to lessen the impact of the virus, it is imperative (1) to consider additional mitigation strategies critical for the development of effective countermeasures, (2) to promote long-term policies and strict regulations of the trade of wildlife and live animal markets, and (3) to advocate for necessary funding and investments in global health, specifically for the prevention of and response to natural and manmade pandemics. This document considers some of these challenges.

Comparative evaluation of pathogenicity of three isolates of vesicular stomatitis virus (Indiana serotype) in pigs.

Vesicular stomatitis (VS) is a notifiable disease of livestock affecting cattle, horses, pigs and humans. Vesicular stomatitis virus (VSV) serotypes Indiana and New Jersey are endemic to Central America; however, they also cause sporadic and scattered outbreaks in various countries in South and North America, including the USA. In order to develop an effective experimental challenge model for VSV, we compared the pathogenicity of three VSV serotype Indiana isolates in 36 4-5 week-old pigs. Two bovine isolates of Central American origin and one equine isolate from the USA were used for the experimental infections. Each pig was inoculated with a single isolate by both the intradermal and intranasal routes. Clinical signs of VSV infection were recorded daily for 10 days post-inoculation (days p.i.). Nasal and tonsillar swab samples and blood were collected to monitor immune responses, virus replication and shedding. Post-challenge, characteristic signs of VS were observed, including vesicles on the nasal planum and coronary bands, lameness, loss of hoof walls and pyrexia. Pigs inoculated with the Central American isolates showed consistently more severe clinical signs in comparison to the pigs infected with the USA isolate. Genomic RNA was isolated from the original challenge virus stocks, sequenced and compared to VSV genomes available in GenBank. Comparative genome analysis demonstrated significant differences between the VSV isolate from the USA and the two Central American isolates. Our results indicate that the Central American isolates of VSV serotype Indiana used in this study are more virulent in swine than the USA VSV serotype Indiana isolate and represent good candidate challenge strains for future VSV studies.

Raccoons accumulate PrPSc after intracranial inoculation of the agents of chronic wasting disease or transmissible mink encephalopathy but not atypical scrapie.

Prion diseases are neurodegenerative diseases characterized by the accumulation of misfolded prion protein (PrPSc) in the brain and other tissues. Animal prion diseases include scrapie in sheep, chronic wasting disease (CWD) in cervids, and transmissible mink encephalopathy (TME) in ranch-raised mink. We investigated the susceptibility of raccoons to various prion disease agents and compared the clinicopathologic features of the resulting disease. Raccoon kits were inoculated intracranially with the agents of raccoon-passaged TME (TMERac), bovine-passaged TME (TMEBov), hamster-adapted drowsy (TMEDY) or hyper TME (TMEHY), CWD from white-tailed deer (CWDWtd) or elk (CWDElk), or atypical (Nor98) scrapie. Raccoons were euthanized when they developed clinical signs of prion disease or at study endpoint (<82 mo post-inoculation). Brain was examined for the presence of spongiform change, and disease-associated PrPSc was detected using an enzyme immunoassay, western blot, and immunohistochemistry. All raccoons inoculated with the agents of TMERac and TMEBov developed clinical disease at ~6.6 mo post-inoculation, with widespread PrPSc accumulation in central nervous system tissues. PrPSc was detected in the brain of 1 of 4 raccoons in each of the CWDWtd-, CWDElk-, and TMEHY-inoculated groups. None of the raccoons inoculated with TMEDY or atypical scrapie agents developed clinical disease or detectable PrPSc accumulation. Our results indicate that raccoons are highly susceptible to infection with raccoon- and bovine-passaged TME agents, whereas CWD isolates from white-tailed deer or elk and hamster-adapted TMEHY transmit poorly. Raccoons appear to be resistant to infection with hamster-adapted TMEDY and atypical scrapie agents.

Evaluation of a Field-Deployable Insulated Isothermal Polymerase Chain Reaction Nucleic Acid Analyzer for Influenza A Virus Detection at Swine Exhibitions.

Rapid detection of influenza A virus (IAV) at swine exhibitions, where zoonotic transmission has occurred, can allow exhibition officials to quickly implement mitigation strategies and reduce public health risk. While laboratory diagnostic methods using PCR exist, pen-side detection of IAV can reduce lag time between sample collection and results. Portable insulated isothermal PCR (RT-iiPCR) has been used for point-of-care pathogen detection in veterinary medicine. This study compared laboratory methods of real-time reverse transcription PCR (rRT-PCR) to RT-iiPCR to determine the potential effectiveness of RT-iiPCR for detection of IAV in swine in the field. Two methods of extraction (magnetic bead and spin-column) and the two PCR platforms were used in a crossover study design to detect IAV in nasal wipes of 150 individual swine from one exhibition. Magnetic bead extraction is considered the laboratory gold standard while spin-column purification is considered the field-deployable method. IAV RNA was detected in 17 samples using Mag/rRT-PCR (reference assay) and 16 samples using Mag/RT-iiPCR (Sensitivity-S 76.5%), whereas only 14 samples using Spin/rRT-PCR (S 88.2%) and 12 samples using Spin/RT-iiPCR (field method) (S 58.8%) were positive, demonstrating a reduction in detection of viral RNA using column purification. There is moderate agreement (Cohen's kappa = 0.6575) between Mag/rRT-PCR and Spin/RT-iiPCR. There is good agreement between both PCR assays when using the same method of extraction (Mag: Cohen's kappa = 0.8203, Spin: Cohen's kappa = 0.7642). RT-iiPCR requires testing of 10 more samples than the rRT-PCR to detect disease at the 95% confidence level in a population of 300 animals with a disease prevalence of 20%. In conclusion, although there is some reduction in sensitivity, RT-iiPCR used in conjunction with spin-column purification is an acceptable method of IAV in swine detection at exhibitions where it may help reduce lag time and allow for rapid control of an IAV outbreak.

Design, implementation, and interpretation of amplification studies for prion detection.

Amplification assays for transmissible spongiform encephalopathies have been in development for close to 15 years, with critical implications for the postmortem and antemortem diagnosis of human and animal prion diseases. Little has been published regarding the structured development, implementation and interpretation of experiments making use of protein misfolding cyclic amplification (PMCA) and real time quaking-induced conversion (RT-QuIC), and our goal with this Perspectives manuscript is to offer a framework which might allow for more efficient expansion of pilot studies into diagnostic trials in both human and animal subjects. This framework is made up of approaches common to diagnostic medicine, including a thorough understanding of analytical and diagnostic sensitivity and specificity, an a priori development of amplification strategy, and an effective experimental design. It is our hope that a structured framework for prion amplification assays will benefit not only experiments seeking to sensitively detect naturally-occurring cases of prion diseases and describe the pathogenesis of TSEs, but ultimately assist with future endeavors seeking to use these methods more broadly for other protein misfolding disorders, including Alzheimer's and Parkinson's disease.

Harnessing Invariant NKT Cells to Improve Influenza Vaccines: A Pig Perspective.

Invariant natural killer T (iNKT) cells are an "innate-like" T cell lineage that recognize glycolipid rather than peptide antigens by their semi-invariant T cell receptors. Because iNKT cells can stimulate an extensive array of immune responses, there is considerable interest in targeting these cells to enhance human vaccines against a wide range of microbial pathogens. However, long overlooked is the potential to harness iNKT cell antigens as vaccine adjuvants for domestic animal species that express the iNKT cell-CD1d system. In this review, we discuss the prospect of targeting porcine iNKT cells as a strategy to enhance the efficiency of swine influenza vaccines. In addition, we compare the phenotype and tissue distribution of porcine iNKT cells. Finally, we discuss the challenges that must be overcome before iNKT cell agonists can be contemplated for veterinary use in livestock.

Estimating chronic wasting disease susceptibility in cervids using real-time quaking-induced conversion.

In mammals, susceptibility to prion infection is primarily modulated by the host's cellular prion protein (PrPC) sequence. In the sheep scrapie model, a graded scale of susceptibility has been established both in vivo and in vitro based on PrPC amino acids 136, 154 and 171, leading to global breeding programmes to reduce the prevalence of scrapie in sheep. Chronic wasting disease (CWD) resistance in cervids is often characterized as decreased prevalence and/or protracted disease progression in individuals with specific alleles; at present, no PrPC allele conferring absolute resistance in cervids has been identified. To model the susceptibility of various naturally occurring and hypothetical cervid PrPC alleles in vitro, we compared the amplification rates and amyloid extension efficiencies of eight distinct CWD isolates in recombinant cervid PrPC substrates using real-time quaking-induced conversion. We hypothesized that the in vitro conversion characteristics of these isolates in cervid substrates would correlate to in vivo susceptibility - permitting susceptibility prediction for the rare alleles found in nature. We also predicted that hypothetical alleles with multiple resistance-associated codons would be more resistant to in vitro conversion than natural alleles with a single resistant codon. Our studies demonstrate that in vitro conversion metrics align with in vivo susceptibility, and that alleles with multiple amino acid substitutions, each influencing resistance independently, do not necessarily contribute additively to conversion resistance. Importantly, we found that the naturally occurring whitetail deer QGAK substrate exhibited the slowest amplification rate among those evaluated, suggesting that further investigation of this allele and its resistance in vivo is warranted.

Phenotyping and susceptibility of established porcine cells lines to African Swine Fever Virus infection and viral production.

African swine fever virus (ASFV) is a highly pathogenic, double-stranded DNA virus with a marked tropism for cells of the monocyte-macrophage lineage, affecting swine species and provoking severe economic losses and health threats. In the present study, four established porcine cell lines, IPAM-WT, IPAM-CD163, C∆2+ and WSL, were compared to porcine alveolar macrophage (PAM) in terms of surface marker phenotype, susceptibility to ASFV infection and virus production. The virulent ASFV Armenia/07, E70 or the naturally attenuated NHV/P68 strains were used as viral models. Cells expressed only low levels of specific receptors linked to the monocyte/macrophage lineage, with low levels of infection overall, with the exception of WSL, which showed more efficient production of strain NHV/P68 but not of strains E70 and Armenia/07.

Evolution of Diagnostic Tests for Chronic Wasting Disease, a Naturally Occurring Prion Disease of Cervids.

Since chronic wasting disease (CWD) was first identified nearly 50 years ago in a captive mule deer herd in the Rocky Mountains of the United States, it has slowly spread across North America through the natural and anthropogenic movement of cervids and their carcasses. As the endemic areas have expanded, so has the need for rapid, sensitive, and cost effective diagnostic tests-especially those which take advantage of samples collected antemortem. Over the past two decades, strategies have evolved from the recognition of microscopic spongiform pathology and associated immunohistochemical staining of the misfolded prion protein to enzyme-linked immunoassays capable of detecting the abnormal prion conformer in postmortem samples. In a history that parallels the diagnosis of more conventional infectious agents, both qualitative and real-time amplification assays have recently been developed to detect minute quantities of misfolded prions in a range of biological and environmental samples. With these more sensitive and semi-quantitative approaches has come a greater understanding of the pathogenesis and epidemiology of this disease in the native host. Because the molecular pathogenesis of prion protein misfolding is broadly analogous to the misfolding of other pathogenic proteins, including Aß and α-synuclein, efforts are currently underway to apply these in vitro amplification techniques towards the diagnosis of Alzheimer's disease, Parkinson's disease, and other proteinopathies. Chronic wasting disease-once a rare disease of Colorado mule deer-now represents one of the most prevalent prion diseases, and should serve as a model for the continued development and implementation of novel diagnostic strategies for protein misfolding disorders in the natural host.