A rapid, high-throughput, viral infectivity assay using automated brightfield microscopy with machine learning.

Infectivity assays are essential for the development of viral vaccines, antiviral therapies, and the manufacture of biologicals. Traditionally, these assays take 2-7 days and require several manual processing steps after infection. We describe an automated viral infectivity assay (AVIATM), using convolutional neural networks (CNNs) and high-throughput brightfield microscopy on 96-well plates that can quantify infection phenotypes within hours, before they are manually visible, and without sample preparation. CNN models were trained on HIV, influenza A virus, coronavirus 229E, vaccinia viruses, poliovirus, and adenoviruses, which together span the four major categories of virus (DNA, RNA, enveloped, and non-enveloped). A sigmoidal function, fit between virus dilution curves and CNN predictions, results in sensitivity ranges comparable to or better than conventional plaque or TCID50 assays, and a precision of ∼10%, which is considerably better than conventional infectivity assays. Because this technology is based on sensitizing CNNs to specific phenotypes of infection, it has potential as a rapid, broad-spectrum tool for virus characterization, and potentially identification.

Effect of BIO-PLYTM, a Platelet-Rich Plasma Derived Biologic on PRRSV-2-Infected Macrophages.

Porcine Reproductive and Respiratory Syndrome (PRRS) is the one of the most devastating diseases impacting the swine industry worldwide. Control and prevention methods rely on biosafety measures and vaccination. As an RNA virus with a high rate of mutation, vaccines are only partially effective against circulating and newly emerging strains. To reduce the burden of this disease, research on alternative control methods is needed. Here, we assess the in vitro antiviral effect of a novel platelet-rich plasma-derived biologic termed BIO-PLYTM (for the BIOactive fraction of Platelet-rich plasma LYsate) from both swine and equine origin. Our results show that BIO-PLYTM significantly reduces the amount of PRRSV viral load determined by RT-qPCR and the number of infectious viral particles measured by TCID50 in infected porcine alveolar and parenchymal macrophages. This study also showed limited toxicity of BIO-PLYTM in vitro and aspects of its immunomodulatory capacity evaluating the regulation of reactive oxygen species and cytokines production in infected cells. Finally, this study presents promising data on the effect of BIO-PLYTM on other RNA viruses such as human A influenza viruses and coronavirus.

Heterologous vaccine immunogenicity, efficacy, and immune correlates of protection of a modified-live virus porcine reproductive and respiratory syndrome virus vaccine.

Although porcine reproductive and respiratory syndrome virus (PRRSV) vaccines have been available in North America for almost 30 years, many vaccines face a significant hurdle: they must provide cross-protection against the highly diverse PRRSV strains. This cross-protection, or heterologous vaccine efficacy, relies greatly on the vaccine's ability to induce a strong immune response against various strains-heterologous immunogenicity. Thus, this study investigated vaccine efficacy and immunogenicity of a modified live virus (MLV) against four heterologous type 2 PRRSV (PRRSV-2) strains. In this study, 60 pigs were divided into 10 groups. Half were MOCK-vaccinated, and the other half vaccinated with the Prevacent® PRRS MLV vaccine. Four weeks after vaccination, groups were challenged with either MOCK, or four PRRSV-2 strains from three different lineages-NC174 or NADC30 (both lineage 1), VR2332 (lineage 5), or NADC20 (lineage 8). Pre-and post-challenge, lung pathology, viral loads in both nasal swabs and sera, anti-PRRSV IgA/G, neutralizing antibodies, and the PRRSV-2 strain-specific T-cell response were evaluated. At necropsy, the lung samples were collected to assess viral loads, macroscopical and histopathological findings, and IgA levels in bronchoalveolar lavage. Lung lesions were only induced by NC174, NADC20, and NADC30; within these, vaccination resulted in lower gross and microscopic lung lesion scores of the NADC20 and NADC30 strains. All pigs became viremic and vaccinated pigs had decreased viremia upon challenge with NADC20, NADC30, and VR2332. Regarding vaccine immunogenicity, vaccination induced a strong systemic IgG response and boosted the post-challenge serum IgG levels for all strains. Furthermore, vaccination increased the number of animals with neutralizing antibodies against three of the four challenge strains-NADC20, NADC30, and VR2332. The heterologous T-cell response was also improved by vaccination: Not only did vaccination increase the induction of heterologous effector/memory CD4 T cells, but it also improved the heterologous CD4 and CD8 proliferative and/or IFN-γ response against all strains. Importantly, correlation analyses revealed that the (non-PRRSV strain-specific) serum IgG levels and the PRRSV strain-specific CD4 T-cell response were the best immune correlates of protection. Overall, the Prevacent elicited various degrees of efficacy and immunogenicity against four heterologous and phylogenetically distant strains of PRRSV-2.

The health benefits of selenium in food animals: a review.

Selenium is an essential trace mineral important for the maintenance of homeostasis in animals and humans. It evinces a strong antioxidant, anti-inflammatory and potential antimicrobial capacity. Selenium biological function is primarily achieved by its presence in selenoproteins as a form of selenocysteine. Selenium deficiency may result in an array of health disorders, affecting many organs and systems; to prevent this, dietary supplementation, mainly in the forms of organic (i.e., selenomethionine and selenocysteine) inorganic (i.e., selenate and selenite) sources is used. In pigs as well as other food animals, dietary selenium supplementation has been used for improving growth performance, immune function, and meat quality. A substantial body of knowledge demonstrates that dietary selenium supplementation is positively associated with overall animal health especially due to its immunomodulatory activity and protection from oxidative damage. Selenium also possesses potential antiviral activity and this is achieved by protecting immune cells against oxidative damage and decreasing viral replication. In this review we endeavor to combine established and novel knowledge on the beneficial effects of dietary selenium supplementation, its antioxidant and immunomodulatory actions, and the putative antimicrobial effect thereof. Furthermore, our review demonstrates the gaps in knowledge pertaining to the use of selenium as an antiviral, underscoring the need for further in vivo and in vitro studies, particularly in pigs.

Use of Crystal Violet to Improve Visual Cytopathic Effect-based Reading for Viral Titration using TCID50 Assays.

Viral titration is a key assay for virology research. The detection of cytopathic effect (CPE) via TCID50 assays and plaque-forming units (PFU) assays are the two main methods to calculate the titer of a virus stock and are often based on microscopy detection or cell staining for visualization. In the case of TCID50 assay, objective visualization is commonly based on immunocytochemical (ICC) staining of intracellular virus to calculate titers combined with visual CPE detection via microscopy. However, ICC staining is costly and time consuming. In this study, we compared visual CPE observation via microscopy, ICC staining and crystal violet staining to determine the titers of two CPE-forming viruses, Influenza A virus (IAV) of swine origin and Porcine Reproductive and Respiratory Syndrome virus (PRRSV). We show that both crystal violet and ICC staining are more accurate than visual CPE detection, presenting nearly identical levels of precision on both IAV and PRRSV. For this reason, here we present crystal violet staining as a faster and more affordable way to determine viral titrations on a TCID50 assay for CPE-forming viruses titrated in cell lines.

A Liquid Metal Mediated Metallic Coating for Antimicrobial and Antiviral Fabrics.

Fabrics are widely used in hospitals and many other settings for bedding, clothing, and face masks; however, microbial pathogens can survive on surfaces for a long time, leading to microbial transmission. Coatings of metallic particles on fabrics have been widely used to eradicate pathogens. However, current metal particle coating technologies encounter numerous issues such as nonuniformity, processing complexity, and poor adhesion. To overcome these issues, an easy-to-control and straightforward method is reported to coat a wide range of fabrics by using gallium liquid metal (LM) particles to facilitate the deposition of liquid metal copper alloy (LMCu) particles. Gallium particles coated on the fabric provide nucleation sites for forming LMCu particles at room temperature via galvanic replacement of Cu2+ ions. The LM helps promote strong adhesion of the particles to the fabric. The presence of the LMCu particles can eradicate over 99% of pathogens (including bacteria, fungi, and viruses) within 5 min, which is significantly more effective than control samples coated with only Cu. The coating remains effective over multiple usages and against contaminated droplets and aerosols, such as those encountered in facemasks. This facile coating method is promising for generating robust antibacterial, antifungal, and antiviral fabrics and surfaces.

Whole Genome or Single Genes? A Phylodynamic and Bibliometric Analysis of PRRSV.

Diversity, ecology, and evolution of viruses are commonly determined through phylogenetics, an accurate tool for the identification and study of lineages with different pathological characteristics within the same species. In the case of PRRSV, evolutionary research has divided into two main branches based on the use of a specific gene (i.e., ORF5) or whole genome sequences as the input used to produce the phylogeny. In this study, we performed a review on PRRSV phylogenetic literature and characterized the spatiotemporal trends in research of single gene vs. whole genome evolutionary approaches. Finally, using publicly available data, we produced a Bayesian phylodynamic analysis following each research branch and compared the results to determine the pros and cons of each particular approach. This study provides an exploration of the two main phylogenetic research lines applied for PRRSV evolution, as well as an example of the differences found when both methods are applied to the same database. We expect that our results will serve as a guidance for future PRRSV phylogenetic research.

The Local and Systemic Humoral Immune Response Against Homologous and Heterologous Strains of the Type 2 Porcine Reproductive and Respiratory Syndrome Virus.

The humoral immune response plays a crucial role in the combat and protection against many pathogens including the economically most important, highly prevalent, and diverse pig pathogen PRRSV - the Porcine Reproductive and Respiratory Syndrome Virus. In addition to viremia and viral shedding analyses, this study followed the local and systemic humoral immune response of pigs for 63 days upon inoculation with one of three types of Type-2 PRRSV (PRRSV-2) strains - one modified live virus (MLV) vaccine strain, and two lineage 1 PRRSV-2 strains, NC134 and NC174. The local response was analyzed by quantifying immunoglobulin (Ig)A in nasal swabs. The systemic response was studied by the quantification of IgG with ELISA and homo- and heterologous neutralizing antibodies (NAs) utilizing a novel method of flow cytometry. In all PRRSV-2 inoculated groups, viral nasal shedding started at 3 dpi, peaked between 3 and 7 days post inoculation, and was cleared at 28-35 dpi with sporadic rebounds thereafter. The local IgA response started 4-7 days after viral shedding occurred and showed a bi-phasic course with peaks at 14 dpi and at 28-35 dpi. Of note, the NC134 and NC174 strains induced a much stronger local IgA response. As reported earlier, main viremia lasted from 7 dpi to 28 dpi (NC174), 42 dpi (NC134) or until the end of the study (MLV). Similar to the local IgA response, the systemic IgG response started 4-7 days after viremia; but in contrast to viremia, serum IgG levels stayed high for all PRRSV-2 inoculated groups until the end of the study. A significant finding was that while the serum NA response in the MLV group was delayed by 28 days, serum NAs in pigs infected with our two NC134 and NC174 strains could be detected as early as 7 dpi (NC134) and 14 dpi (NC174). Compared to homologous NA responses, the NA responses against heterologous strains was strong but slightly delayed between our lineage 1 one strains or non-existent between the MLV and lineage 1 strains. This study improves our understanding of the relationship between local and systemic infections and the humoral immune response induced by PRRSV-2 infection or MLV vaccination. Our data also provide novel insights into the timeline of the development of homologous and heterologous NA levels - by both MLV vaccination or infection with two strains from the currently prevalent PRRSV-2 lineage 1.

Impact of changes of horse movement regulations on the risks of equine infectious anemia: A risk assessment approach.

Equine infectious anemia virus (EIAV) is a transboundary disease affecting a large number of equines worldwide. In this study, we assessed the transmission risk of EIAV in Rio Grande do Sul, Brazil. Serum samples from 1010 animals from 341 farms were initially analyzed using agar gel immunodiffusion to detect viral antibodies, and no antibody-positive animals were found. A risk assessment stochastic model was applied to generate the expected number of potential infections per month and to estimate the time to new infections. Our results estimated 6.5 months as the interval for new infections in the worst-case scenario. Among the variables evaluated, the number of transported animals and the test sensitivity influenced the model the most. These results were then used to revisit the impact of EIAV control regulations, which triggered a change in the diagnostic testing required for animal movement, in which the validity of a negative test for EIAV was extended from 60 to 180 days. Finally, revisiting the annual average of infected farms before and after the new regulation, the number of infected farms increased pre-implementation, and then, the number of culled animals increased, which should impact future EIAV incidence in this region. Our results demonstrated the importance of constant reviews of disease control programs and provided quantitative-based knowledge for decision-makers in official veterinary services.

A Century of Swine Influenza: Is It Really Just about the Pigs?

Influenza viruses (IV) are one of the major threats to human and animal health worldwide due to the variety of species they affect. Pigs play an important role in IV ecology as the "mixing vessel," since they can be infected by swine, avian and human IV, allowing the appearance of new subtypes. Human viruses originated in swine are known as IV of swine origin or swine influenza virus (SwIV) variants. In this study, we identified knowledge tendencies of SwIV and assessed potential bias in the literature caused by these variants. We identified the most mentioned SwIV variants and manually reviewed the literature to determine the number of publications applying the whole influenza nomenclature, a partial nomenclature, only the subtype or mixed terminology, along with the proportion of articles in which the GenBank ID number was available. We observed that the 2009 H1N1 human pandemic created an important bias in SwIV research driven by an increase in human publications on the IV of swine origin. H1N1 is the most studied subtype for swine and humans, followed by H3N2. We found differences between the nomenclatures applied, where partial classifications were slightly more common. Finally, from all the publications, only 25% stated the GenBank ID of the sequence studied. This review represents the most complete exploration of trends in SwIV knowledge to date and will serve as a guidance for future search strategies in SwIV research.

Nairobi Sheep Disease Virus: A Historical and Epidemiological Perspective.

Nairobi Sheep Disease virus (NSDv) is a zoonotic and tick-borne disease that can cause over 90% mortality in small ruminants. NSDv has historically circulated in East Africa and has recently emerged in the Asian continent. Despite efforts to control the disease, some regions, mostly in warmer climates, persistently report disease outbreaks. Consequently, it is necessary to understand how environmental tolerances and factors that influence transmission may shed light on its possible emergence in other regions. In this study, we quantified the available literature of NSDv from which occurrence data was extracted. In total, 308 locations from Uganda, Kenya, Tanzania, Somalia, India, Sri Lanka and China were coupled with landscape conditions to reconstruct the ecological conditions for NSDv circulation and identify areas of potential disease transmission risk. Our results identified areas suitable for NSDv in Ethiopia, Malawi, Zimbabwe, Southeastern China, Taiwan, and Vietnam. Unsuitable areas included Democratic Republic of Congo, Zambia, and Southern Somalia. In summary, soil moisture, livestock density, and precipitation predispose certain areas to NSDv circulation. It is critical to investigate the epidemiology of NSDv in order to promote better allocation of resources to control its spread in regions that are more at risk. This will help reduce disease impact worldwide as climate change will favor emergence of such vector-borne diseases in areas with dense small ruminant populations.

Spatial distribution and spread potential of sixteen Leptospira serovars in a subtropical region of Brazil.

Leptospirosis is a bacterial disease that represents a major problem in animal and public health due to its high prevalence and widespread distribution. This zoonotic disease is most prevalent in tropical environments where conditions favour pathogen survival. The ecological preferences of Leptospira serovars are poorly understood, limiting our knowledge of where and when outbreaks can occur, which may result in misinformed prevention and control plans. While the disease can occur consistently in time and space in tropical regions, research on the ecology of leptospirosis remains limited in subtropical regions. This research gap regarding Leptospira ecology brings public and veterinary health problems, impacting local economies. To fill this gap of knowledge, we suggest to assess geographic and ecological features among Leptospira serovars in a subtropical area of Brazil where leptospirosis is endemic to (a) highlight environmental conditions that facilitate or limit Leptospira spread and survival and (b) reconstruct its geographic distribution. An ecological niche modelling framework was used to characterize and compare Leptospira serovars in both geographic and environmental space. Our results show that despite the geographic overlap exhibited by the different serovars assessed, we found ecological divergence among their occupied ecological niches. Ecological divergences were expressed as ranges of potential distributions and environmental conditions found suitably by serovar, Sejroe being the most asymmetric (<0.15). Most important predictors for the potential distribution of most serovars were soil pH (31.7%) and landscape temperature (24.2%). Identification of environmental preferences will allow epidemiologists to better infer the presence of a serovar based on the environmental characteristics of regions rather than inferences based solely on historical epidemiological records. Including geographic and ecological ranges of serovars also may help to forecast transmission potential of Leptospira in public health and the food animal practice.

Relation of reproductive disturbance in sheep and Leptospira interrogans serovar Icterohaemorrhagiae infection: Impacts on cellular oxidation status.

This study evaluated the seropositivity of Brucella abortus and Leptospira interrogans in ewes with reproductive disturbances in southern Brazil and verified the creatine kinase (CK) activity and oxidation status via assessment of superoxide dismutase, glutathione peroxidase and glutathione transferase in serum of seropositive animals for L. interrogans serovar Icterohaemorrhagiae. For Leptospira infection 381 animals with clinical history of reproductive disturbance from Planalto Serrano de Santa Catarina (Brazil) were analyzed, showing an occurrence for L. interrogans of 20.2% from which 81.8% were seropositive for L. interrogans Icterohaemorrhagiae. Serovars Wolfii, Grippothyphosa, Bratislava, Canicola and Butembo were also identified. In the case of B. abortus, positive cases were identified by buffered acidified antigen, finding 14 positive samples, but none of them were positive after a second test (2-mercaptoethanol), showing the absence of relationship between infection with B. abortus and abortion in the tested individuals. Serum reactive oxygen species (ROS) levels and CK activity were found higher in animals positive for Leptospira infection, presenting higher titrations (1:320) than non-infected individuals. Serum glutathione peroxidase activity was higher in positive animals with titrations 1:160 and 1:320, while serum glutathione S-transferase was higher in positive individuals only for titrations 1:320. Serum superoxide dismutase showed lower activity in infected animals with titrations of 1:320. Our results show the region of Planalto Serrano de Santa Catarina with a high occurrence levels of sheep infected by L. interrogans serovar Icterohaemorrhagiae, from which animals with high titrations (1:320) present oxidative stress elicited by excessive ROS production, triggering the stimulation of antioxidant systems to counter this excess. In summary, ovine with higher titrations (1:320) present oxidative damage that can contribute to disease pathophysiology.

The Macroecology of Chemical Communication in Lizards: Do Climatic Factors Drive the Evolution of Signalling Glands?

Chemical communication plays a pivotal role in shaping sexual and ecological interactions among animals. In lizards, fundamental mechanisms of sexual selection such as female mate choice have rarely been shown to be influenced by quantitative phenotypic traits (e.g., ornaments), while chemical signals have been found to potentially influence multiple forms of sexual and social interactions, including mate choice and territoriality. Chemical signals in lizards are secreted by glands primarily located on the edge of the cloacae (precloacal glands, PG) and thighs (femoral glands), and whose interspecific and interclade number ranges from 0 to > 100. However, elucidating the factors underlying the evolution of such remarkable variation remains an elusive endeavour. Competing hypotheses suggest a dominant role for phylogenetic conservatism (i.e., species within clades share similar numbers of glands) or for natural selection (i.e., their adaptive diversification results in deviating numbers of glands from ancestors). Using the prolific Liolaemus lizard radiation from South America (where PG vary from 0 to 14), we present one of the largest-scale tests of both hypotheses to date. Based on climatic and phylogenetic modelling, we show a clear role for both phylogenetic inertia and adaptation underlying gland variation: (i) solar radiation, net primary productivity, topographic heterogeneity and precipitation range have a significant effect on PG variation, (ii) humid and cold environments tend to concentrate species with a higher number of glands, (iii) there is a strong phylogenetic signal that tends to conserve the number of PG within clades. Collectively, our study confirms that the inertia of niche conservatism can be broken down by the need of species facing different selection regimes to adjust their glands to suit the demands of their specific environments.