Role of N-linked glycosylation in porcine reproductive and respiratory syndrome virus (PRRSV) infection.

Porcine reproductive and respiratory syndrome (PRRSV) is an enveloped single-stranded positive-sense RNA virus and one of the main pathogens that causes the most significant economical losses in the swine-producing countries. PRRSV is currently divided into two distinct species, PRRSV-1 and PRRSV-2. The PRRSV virion envelope is composed of four glycosylated membrane proteins and three non-glycosylated envelope proteins. Previous work has suggested that PRRSV-linked glycans are critical structural components for virus assembly. In addition, it has been proposed that PRRSV glycans are implicated in the interaction with host cells and critical for virus infection. In contrast, recent findings showed that removal of N-glycans from PRRSV does not influence virus infection of permissive cells. Thus, there are not sufficient evidences to indicate compellingly that N-glycans present in the PRRSV envelope play a direct function in viral infection. To gain insights into the role of N-glycosylation in PRRSV infection, we analysed the specific contribution of the envelope protein-linked N-glycans to infection of permissive cells. For this purpose, we used a novel strategy to modify envelope protein-linked N-glycans that consists of production of monoglycosylated PRRSV and viral glycoproteins with different glycan states. Our results showed that removal or alteration of N-glycans from PRRSV affected virus infection. Specifically, we found that complex N-glycans are required for an efficient infection in cell cultures. Furthermore, we found that presence of high mannose type glycans on PRRSV surface is the minimal requirement for a productive viral infection. Our findings also show that PRRSV-1 and PRRSV-2 have different requirements of N-glycan structure for an optimal infection. In addition, we demonstrated that removal of N-glycans from PRRSV does not affect viral attachment, suggesting that these carbohydrates played a major role in regulating viral entry. In agreement with these findings, by performing immunoprecipitation assays and colocalization experiments, we found that N-glycans present in the viral envelope glycoproteins are not required to bind to the essential viral receptor CD163. Finally, we found that the presence of N-glycans in CD163 is not required for PRRSV infection.

Generation of a Commercial-Scale Founder Population of Porcine Reproductive and Respiratory Syndrome Virus Resistant Pigs Using CRISPR-Cas.

Disease resistance genes in livestock provide health benefits to animals and opportunities for farmers to meet the growing demand for affordable, high-quality protein. Previously, researchers used gene editing to modify the porcine CD163 gene and demonstrated resistance to a harmful virus that causes porcine reproductive and respiratory syndrome (PRRS). To maximize potential benefits, this disease resistance trait needs to be present in commercially relevant breeding populations for multiplication and distribution of pigs. Toward this goal, a first-of-its-kind, scaled gene editing program was established to introduce a single modified CD163 allele into four genetically diverse, elite porcine lines. This effort produced healthy pigs that resisted PRRS virus infection as determined by macrophage and animal challenges. This founder population will be used for additional disease and trait testing, multiplication, and commercial distribution upon regulatory approval. Applying CRISPR-Cas to eliminate a viral disease represents a major step toward improving animal health.

Genetically modified pigs lacking CD163 PSTII-domain-coding exon 13 are completely resistant to PRRSV infection.

CD163 expressed on cell surface of porcine alveolar macrophages (PAMs) serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The extracellular portion of CD163 contains nine scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. Genomic editing of pigs to remove the entire CD163 or just the SRCR5 domain confers resistance to infection with both PRRSV-1 and PRRSV-2 viruses. By performing a mutational analysis of CD163, previous in vitro infection experiments showed resistance to PRRSV infection following deletion of exon 13 which encodes the first 12 amino acids of the 16 amino acid PSTII domain. These findings predicted that removal of exon 13 can be used as a strategy to produce gene-edited pigs fully resistant to PRRSV infection. In this study, to determine whether the deletion of exon 13 is sufficient to confer resistance of pigs to PRRSV infection, we produced pigs possessing a defined CD163 exon 13 deletion (ΔExon13 pigs) and evaluated their susceptibility to viral infection. Wild type (WT) and CD163 modified pigs, placed in the same room, were infected with PRRSV-2. The modified pigs remained PCR and serologically negative for PRRSV throughout the study; whereas the WT pigs supported PRRSV infection and showed PRRSV related pathology. Importantly, our data also suggested that removal of exon 13 did not affect the main physiological function associated with CD163 in vivo. These results demonstrate that a modification of CD163 through a precise deletion of exon 13 provides a strategy for protection against PRRSV infection.

Reprogramming viral immune evasion for a rational design of next-generation vaccines for RNA viruses.

Type I interferons (IFNs-α/ß) are antiviral cytokines that constitute the innate immunity of hosts to fight against viral infections. Recent studies, however, have revealed the pleiotropic functions of IFNs, in addition to their antiviral activities, for the priming of activation and maturation of adaptive immunity. In turn, many viruses have developed various strategies to counteract the IFN response and to evade the host immune system for their benefits. The inefficient innate immunity and delayed adaptive response fail to clear of invading viruses and negatively affect the efficacy of vaccines. A better understanding of evasion strategies will provide opportunities to revert the viral IFN antagonism. Furthermore, IFN antagonism-deficient viruses can be generated by reverse genetics technology. Such viruses can potentially serve as next-generation vaccines that can induce effective and broad-spectrum responses for both innate and adaptive immunities for various pathogens. This review describes the recent advances in developing IFN antagonism-deficient viruses, their immune evasion and attenuated phenotypes in natural host animal species, and future potential as veterinary vaccines.

Time intervals between COVID-19 cases, and more severe outcomes

A critical factor in infectious disease control is the risk of an outbreak overwhelming local healthcare capacity. The overall demand on healthcare services will depend on disease severity, but the precise timing and size of peak demand also depends on the time interval (or clinical time delay) between initial infection, and development of severe disease. A broader distribution of intervals may draw that demand out over a longer period, but have a lower peak demand. These interval distributions are therefore important in modelling trajectories of e.g. hospital admissions, given a trajectory of incidence. Conversely, as testing rates decline, an incidence trajectory may need to be inferred through the delayed, but relatively unbiased signal of hospital admissions. Healthcare demand has been extensively modelled during the COVID-19 pandemic, where localised waves of infection have imposed severe stresses on healthcare services. While the initial acute threat posed by this disease has since subsided from immunity buildup from vaccination and prior infection, prevalence remains high and waning immunity may lead to substantial pressures for years to come. In this work, then, we present a set of interval distributions, for COVID-19 cases and subsequent severe outcomes; hospital admission, ICU admission, and death. These may be used to model more realistic scenarios of hospital admissions and occupancy, given a trajectory of infections or cases. We present a method for obtaining empirical distributions using COVID-19 outcomes data from Scotland between September 2020 and January 2022 (N = 31724 hospital admissions, N = 3514 ICU admissions, N = 8306 mortalities). We present separate distributions for individual age, sex, and deprivation of residing community. We show that, while the risk of severe disease following COVID-19 infection is substantially higher for the elderly or those residing in areas of high deprivation, the length of stay shows no strong dependence, suggesting that severe outcomes are equally severe across risk groups. As Scotland and other countries move into a phase where testing is no longer abundant, these intervals may be of use for retrospective modelling of patterns of infection, given data on severe outcomes.

Spatial analysis of COVID-19 booster vaccine uptake in Scotland, and projection of future distributions

Vaccination is a critical tool for controlling infectious diseases, with its use to protect against COVID-19 being a prime example. Where a disease is highly transmissible, even a small proportion of unvaccinated individuals can have substantial implications for disease burdens and compromise efforts for control. As socio-demographic factors such as deprivation and ethnicity have been shown to influence uptake rates, identifying how vaccine uptake varies with socio-demographic indicators is a critical step for reducing vaccine hesitancy and issues of access, and identifying plausible future uptake patterns. Here, we analyse the numbers of COVID-19 vaccinations subdivided by age, gender, date, dose and geographical location. We use publicly available socio-demographic data, and use random forest models to capture patterns of uptake at high spatial resolution, with systematic variation restricted to fine spatial scale (~ 1km in urban areas). We show that uptake of first vaccine booster doses in Scotland can be used to predict with high precision the distribution of second booster doses across deprivation deciles, age and gender despite the substantially lower uptake of second boosters compared to first. This analysis shows that while age and gender have the greatest impact on the model fit, there is a substantial influence of several deprivation factors and the proportion of BAME residents. The high correlation amongst these factors also suggests that, should vaccine uptake decrease, the impact of deprivation is likely to increase, furthering the disproportionate impact of COVID-19 on individuals living in highly deprived areas. As our analysis is based solely on publicly available socio-demographic data and readily recorded vaccination uptake figures, it would be easily adaptable to analysing vaccination uptake data from countries where data recording is similar, and for aiding vaccination campaigns against other infectious diseases.

Identification of CD163 regions that are required for porcine reproductive and respiratory syndrome virus (PRRSV) infection but not for binding to viral envelope glycoproteins.

CD163, a receptor for porcine reproductive and respiratory syndrome virus (PRRSV), possesses nine scavenger receptor cysteine-rich (SRCR) and two proline-serine-threonine (PST) domains. To identify CD163 regions involved in PRRSV infection, CD163 mutants were generated. Infection experiments showed resistance to infection following deletion of the SRCR4/5 interdomain or the Exon 13 that encodes a portion of PSTII. The mutation of a pentapeptide domain in SRCR5 and SRCR7 also conferred resistance. Mutant CD163 proteins that resisted infection retained the ability to interact with GP2, GP3, GP4 and GP5 viral glycoproteins. The contribution of multiple domains to infection but not to the binding of viral glycoproteins suggests that the envelope proteins may form multiple interactions with CD163, or that receptor regions important for infection have other cellular binding partners required for PRRSV infection. Finally, we mapped the localization the anti-CD163 2A10 antibody epitope.

Effect of the host genotype at a Porcine Reproductive and Respiratory Syndrome (PRRS) resistance marker on evolution of the modified-live PRRS vaccine virus in pigs.

Porcine reproductive and respiratory syndrome; Porcine reproductive and respiratory syndrome virus; PRRS; Whole genome sequencing; Quasispecies; WUR allele.

Mutations within scavenger receptor cysteine-rich (SRCR) protein domain 5 of porcine CD163 involved in infection with porcine reproductive and respiratory syndrome virus (PRRS).

CD163, a macrophage-specific membrane scavenger receptor, serves as a cellular entry receptor for porcine reproductive and respiratory syndrome virus (PRRSV). The removal of scavenger receptor cysteine-rich (SRCR) domain 5 (SRCR5) of CD163 is sufficient to make transfected cells or genetically modified pigs resistant to PRRSV-1 and PRRSV-2 genotypes, and substitution of SRCR5 with SRCR8 from human CD163-like protein (hCD163L1) confers resistance to PRRSV-1 but not PRRSV-2 isolates. However, the specific regions within the SRCR5 polypeptide involved in PRRSV infection remain largely unknown. In this report, we performed mutational studies in order to identify which regions or amino acid sequences in the SRCR5 domain are critical for PRRSV infection. The approach used in this study was to make proline-arginine (PR) insertions along the SRCR5 polypeptide. Constructs were transfected into HEK293T cells, and then evaluated for infection with PRRSV-2 or PRRSV-1. For PRRSV-2, four PR insertions located after amino acids 8 (PR-9), 47 (PR-48), 54 (PR-55), and 99 (PR-100) had the greatest impact on infection. For PRRSV-1, insertions after amino acids 57 (PR-58) and 99 (PR-100) were critical. Computer simulations based on the crystal structure of SRCR5 showed that the mutations that affected infection localized to a similar region on the surface of the 3-D structure. Specifically, we found two surface patches that are essential for PRRSV infection. PR-58 and PR-55, which were separated by only three amino acids, had reciprocal effects on PRRSV-1 and PRRSV-2. Substitution of Glu-58 with Lys-58 reduced PRRSV-1 infection without affecting PRRSV-2, which partially explains the resistance to PRRSV-1 caused by the SRCR5 replacement with the homolog human SRCR8 previously observed. Finally, resistance to infection was observed following the disruption of any of the four conserved disulfide bonds within SRCR5. In summary, the results confirm that there are distinct differences between PRRSV-1 and PRRSV-2 on recognition of CD163; however, all mutations that affect infection locate on a similar region on the same face of SRCR5.

Disruption of anthrax toxin receptor 1 in pigs leads to a rare disease phenotype and protection from senecavirus A infection.

Senecavirus A (SVA) is a cause of vesicular disease in pigs, and infection rates are rising within the swine industry. Recently, anthrax toxin receptor 1 (ANTXR1) was revealed as the receptor for SVA in human cells. Herein, the role of ANTXR1 as a receptor for SVA in pigs was investigated by CRISPR/Cas9 genome editing. Strikingly, ANTXR1 knockout (KO) pigs exhibited features consistent with the rare disease, GAPO syndrome, in humans. Fibroblasts from wild type (WT) pigs supported replication of SVA; whereas, fibroblasts from KO pigs were resistant to infection. During an SVA challenge, clinical symptoms, including vesicular lesions, and circulating viremia were present in infected WT pigs but were absent in KO pigs. Additional ANTXR1-edited piglets were generated that were homozygous for an in-frame (IF) mutation. While IF pigs presented a GAPO phenotype similar to the KO pigs, fibroblasts showed mild infection, and circulating SVA nucleic acid was decreased in IF compared to WT pigs. Thus, this new ANTXR1 mutation resulted in decreased permissiveness of SVA in pigs. Overall, genetic disruption of ANTXR1 in pigs provides a unique model for GAPO syndrome and prevents circulating SVA infection and clinical symptoms, confirming that ANTXR1 acts as a receptor for the virus.

Thyroid hormone suppression in feeder pigs following polymicrobial or porcine reproductive and respiratory syndrome virus-2 challenge.

Thyroid hormones are powerful regulators of growth, development, and basal metabolic rate and can be dysregulated under conditions of severe stress or illness. To understand the role of these hormones in porcine disease response, serum samples were obtained from three batches of nursery-aged pigs (n = 208) exposed to a natural polymicrobial disease challenge with an array of bacterial and viral pathogens. Levels of total thyroxin (T4) and triiodothyronine (T3) assessed in sera by radioimmunoassay, decreased significantly by 14 days post-exposure (DPE). Levels of T3 partially rebounded by 48 DPE, while T4 levels remain depressed. Post-exposure T3 and T4 levels were positively correlated with acute and long-term average daily gain (ADG). Cross-sectional sampling of animals maintained at the high health source farms, showed no equivalent change in either hormone when managed under standard industrial conditions. To further elucidate the effect of porcine reproductive and respiratory syndrome virus (PRRSV)-infection on thyroid hormone levels, archived sera over 42 days post inoculation (DPI) from nursery pigs (N = 190) challenged with one of two PRRSV2 strains by the PRRS Host Genetics Consortium were similarly assessed, with animals selected in a two-by-two design, to investigate biological extremes in ADG and viral load (VL). All animals showed a similar decrease in both thyroid hormones reaching a minimum at 7 DPI and returning to near pre-challenge levels by 42 DPI. Post-challenge T3 and T4 levels were significantly greater in high ADG groups, with no significant association with VL or strain. The results of this study demonstrate porcine susceptibility to thyroid disruption in response to disease challenge and demonstrate a relationship between this response and growth performance.

Associations of natural variation in the CD163 and other candidate genes on host response of nursery pigs to porcine reproductive and respiratory syndrome virus infection.

Pigs with complete resistance to porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) have been produced by genetically knocking out the CD163 gene that encodes a receptor of the PRRSV for entry into macrophages. The objectives of this study were to evaluate associations of naturally occurring single nucleotide polymorphisms (SNPs) in the CD163 gene and in three other candidate genes (CD169, RGS16, and TRAF1) with host response to PRRSV-only infection and to PRRS vaccination and PRRSV/porcine circovirus 2b (PCV2b) coinfection. SNPs in the CD163 gene were not included on SNP genotyping panels that were used for previous genome-wide association analyses of these data. An additional objective was to identify the potential genetic interaction of variants at these four candidate genes with a mutation in the GBP5 gene that was previously identified to be associated with host response to PRRSV infection. Finally, the association of SNPs with expression level of the nearby gene was tested. Several SNPs in the CD163, CD169, and RGS16 genes were significantly associated with host response under PRRSV-only and/or PRRSV/PCV2b coinfection. The effects of all SNPs that were significant in the PRRSV-only infection trials depend on genetic background. The effects of some SNPs in the CD163, CD169, and RGS16 genes depend on genotype at the putative causative mutation in the GBP5 gene, which indicates a potential biological interaction of these genes with GBP5. In addition, genome-wide association results for the PRRSV-only infection trials revealed that SNPs located in the CDK5RAP2 or MEGF9 genes, near the TRAF1 gene, had suggestive effects on PRRS viral load, which indicates that these SNPs might contribute to PRRSV neuropathogenesis. In conclusion, natural genetic variants in the CD163, CD169, and RGS16 genes are associated with resistance to PRRSV and/or PCV2b infection and appear to interact with the resistance quantitative trait locus in the GBP5 gene. The identified SNPs can be used to select for increased natural resistance to PRRSV and/or PRRSV-PCV2b coinfection.

Analysis of the Role of N-Linked Glycosylation in Cell Surface Expression, Function, and Binding Properties of SARS-CoV-2 Receptor ACE2.

Human angiotensin I-converting enzyme 2 (hACE2) is a type I transmembrane glycoprotein that serves as the major cell entry receptor for SARS-CoV and SARS-CoV-2. The viral spike (S) protein is required for the attachment to ACE2 and subsequent virus-host cell membrane fusion. Previous work has demonstrated the presence of N-linked glycans in ACE2. N-glycosylation is implicated in many biological activities, including protein folding, protein activity, and cell surface expression of biomolecules. However, the contribution of N-glycosylation to ACE2 function is poorly understood. Here, we examined the role of N-glycosylation in the activity and localization of two species with different susceptibility to SARS-CoV-2 infection, porcine ACE2 (pACE2) and hACE2. The elimination of N-glycosylation by tunicamycin (TM) treatment, or mutagenesis, showed that N-glycosylation is critical for the proper cell surface expression of ACE2 but not for its carboxiprotease activity. Furthermore, nonglycosylable ACE2 was localized predominantly in the endoplasmic reticulum (ER) and not at the cell surface. Our data also revealed that binding of SARS-CoV or SARS-CoV-2 S protein to porcine or human ACE2 was not affected by deglycosylation of ACE2 or S proteins, suggesting that N-glycosylation does not play a role in the interaction between SARS coronaviruses and the ACE2 receptor. Impairment of hACE2 N-glycosylation decreased cell-to-cell fusion mediated by SARS-CoV S protein but not that mediated by SARS-CoV-2 S protein. Finally, we found that hACE2 N-glycosylation is required for an efficient viral entry of SARS-CoV/SARS-CoV-2 S pseudotyped viruses, which may be the result of low cell surface expression of the deglycosylated ACE2 receptor. IMPORTANCE Understanding the role of glycosylation in the virus-receptor interaction is important for developing approaches that disrupt infection. In this study, we showed that deglycosylation of both ACE2 and S had a minimal effect on the spike-ACE2 interaction. In addition, we found that the removal of N-glycans of ACE2 impaired its ability to support an efficient transduction of SARS-CoV and SARS-CoV-2 S pseudotyped viruses. Our data suggest that the role of deglycosylation of ACE2 on reducing infection is likely due to a reduced expression of the viral receptor on the cell surface. These findings offer insight into the glycan structure and function of ACE2 and potentially suggest that future antiviral therapies against coronaviruses and other coronavirus-related illnesses involving inhibition of ACE2 recruitment to the cell membrane could be developed.

Development of a Blocking Enzyme-Linked Immunosorbent Assay for Detection of Antibodies against African Swine Fever Virus.

The incursion of African swine fever virus (ASFV) into Eurasia presents a threat to the world's swine industry. Highly sensitive and specific diagnostic assays are urgently needed for rapid detection during an outbreak, post-outbreak investigation, and disease surveillance. In this study, a highly specific and repeatable blocking ELISA (bELISA) was developed using a recombinant p30 protein as the antigen combined with biotinylated mAb against p30 as the detection antibody. Initial test validation included sera from 810 uninfected animals and 106 animals experimentally inoculated with ASFV or recombinant alphavirus/adenovirus expressing p30. Receiver operating characteristic (ROC) analysis of the data calculated an optimal percentage of inhibition (PI) cutoff value of 45.92%, giving a diagnostic sensitivity of 98.11% and diagnostic specificity of 99.42%. The coefficient of variation of an internal quality control serum was 6.81% for between runs, 6.71% for within run, and 6.14% for within plate. A time course study of infected pigs showed that bELISA was able to detect seroconversion as early as 7 days post-inoculation. Taken together, these results demonstrate that bELISA can be used as an alternative serological test for detecting ASFV infection.

COVID-19 mass testing: harnessing the power of wastewater epidemiology

BackgroundCOVID-19 patients shed SARS-CoV-2 RNA in their faeces. We hypothesised that detection of SARS-CoV-2 RNA in wastewater treatment plant (WWTP) influent could be a valuable tool to assist in public health decision making. We aimed to rapidly develop and validate a scalable methodology for the detection of SARS-CoV-2 RNA in wastewater that could be implemented at a national level and to determine the relationship between the wastewater signal and COVID-19 cases in the community. MethodsWe developed a filtration-based methodology for the concentration of SARS-CoV-2 from WWTP influent and subsequent detection and quantification by RT-qPCR. This methodology was used to monitor 28 WWTPs across Scotland, serving 50% of the population. For each WWTP catchment area, we collected data describing COVID-19 cases and deaths. We quantified spatial and temporal relationships between SARS-CoV-2 RNA in wastewater and COVID-19 cases. FindingsDaily WWTP SARS-CoV-2 influent viral RNA load, calculated using daily influent flow rates, had the strongest correlation ({rho}>0.9) with COVID-19 cases within a catchment. As the incidence of COVID-19 cases within a community increased, a linear relationship emerged between cases and influent viral RNA load. There were significant differences between WWTPs in their capacity to predict case numbers based on influent viral RNA load, with the limit of detection ranging from twenty-five cases for larger plants to a single case in smaller plants. InterpretationThe levels of SARS-CoV-2 RNA in WWTP influent provide a cost-effective and unbiased measure of COVID-19 incidence within a community, indicating that national scale wastewater-based epidemiology can play a role in COVID-19 surveillance. In Scotland, wastewater testing has been expanded to cover 75% of the population, with sub-catchment sampling being used to focus surge testing. SARS-CoV-2 variant detection, assessment of vaccination on community transmission and surveillance for other infectious diseases represent promising future applications. FundingThis study was funded by project grants from the Scottish Government via the Centre of Expertise for Waters (CD2019/06) and The Natural Environment Research Councils COVID-19 Rapid Response grants (NE/V010441/1). The Roslin Institute receives strategic funding from the Biotechnology and Biological Sciences Research Council (BB/P013740/1, BBS/E/D/20002173). Sample collection and supplementary analysis was funded and undertaken by Scottish Water and the majority of the sample analysis was funded and undertaken by the Scottish Environment Protection Agency.

Recent Advances in PRRS Virus Receptors and the Targeting of Receptor-Ligand for Control.

Cellular receptors play a critical role in viral infection. At least seven cellular molecules have been identified as putative viral entry mediators for porcine reproductive and respiratory syndrome virus (PRRSV). Accumulating data indicate that among these candidates, CD163, a cysteine-rich scavenger receptor on macrophages, is the major receptor for PRRSV. This review discusses the recent advances and understanding of the entry of PRRSV into cells, viral pathogenesis in CD163 gene-edited swine, and CD163 as a potential target of receptor-ligand for the control of PRRS.

Gut microbiome associations with outcome following co-infection with porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in pigs immunized with a PRRS modified live virus vaccine.

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are two of the most significant pathogens affecting swine. Co-infections are common and result in respiratory disease and reduced weight gain in growing pigs. Although PRRS modified live virus (MLV) vaccines are widely used to decrease PRRS-associated losses, they are generally considered inadequate for disease control. The gut microbiome provides an alternative strategy to enhance vaccine efficacy and improve PRRS control. The objective of this study was to identify gut microbiome characteristics associated with improved outcome in pigs immunized with a PRRS MLV and co-challenged with PRRSV and PCV2b. Twenty-eight days after vaccination and prior to co-challenge, fecal samples were collected from an experimental population of 50 nursery pigs. At 42 days post-challenge, 20 pigs were retrospectively identified as having high or low growth outcomes during the post-challenge period. Gut microbiomes of the two outcome groups were compared using the Lawrence Livermore Microbial Detection Array (LLMDA) and 16S rDNA sequencing. High growth outcomes were associated with several gut microbiome characteristics, such as increased bacterial diversity, increased Bacteroides pectinophilus, decreased Mycoplasmataceae species diversity, higher Firmicutes:Bacteroidetes ratios, increased relative abundance of the phylum Spirochaetes, reduced relative abundance of the family Lachnospiraceae, and increased Lachnospiraceae species C6A11 and P6B14. Overall, this study identifies gut microbiomes associated with improved outcomes in PRRS vaccinated pigs following a polymicrobial respiratory challenge and provides evidence towards the gut microbiome playing a role in PRRS vaccine efficacy.

Gene expression in tonsils in swine following infection with porcine reproductive and respiratory syndrome virus.

BACKGROUND: Porcine reproductive and respiratory syndrome (PRRS) is a threat to pig production worldwide. Our objective was to understand mechanisms of persistence of PRRS virus (PRRSV) in tonsil. Transcriptome data from tonsil samples collected at 42 days post infection (dpi) were generated by RNA-seq and NanoString on 51 pigs that were selected to contrast the two PRRSV isolates used, NVSL and KS06, high and low tonsil viral level at 42 dpi, and the favorable and unfavorable genotypes at a genetic marker (WUR) for the putative PRRSV resistance gene GBP5. RESULTS: The number of differentially expressed genes (DEGs) differed markedly between models with and without accounting for cell-type enrichments (CE) in the samples that were predicted from the RNA-seq data. This indicates that differences in cell composition in tissues that consist of multiple cell types, such as tonsil, can have a large impact on observed differences in gene expression. Based on both the NanoString and the RNA-seq data, KS06-infected pigs showed greater activation, or less inhibition, of immune response in tonsils at 42 dpi than NVSL-infected pigs, with and without accounting for CE. This suggests that the NVSL virus may be better than the KS06 virus at evading host immune response and persists in tonsils by weakening, or preventing, host immune responses. Pigs with high viral levels showed larger CE of immune cells than low viral level pigs, potentially to trigger stronger immune responses. Presence of high tonsil virus was associated with a stronger immune response, especially innate immune response through interferon signaling, but these differences were not significant when accounting for CE. Genotype at WUR was associated with different effects on immune response in tonsils of pigs during the persistence stage, depending on viral isolate and tonsil viral level. CONCLUSIONS: Results of this study provide insights into the effects of PRRSV isolate, tonsil viral level, and WUR genotype on host immune response and into potential mechanisms of PRRSV persistence in tonsils that could be targeted to improve strategies to reduce viral rebreaks. Finally, to understand transcriptome responses in tissues that consist of multiple cell types, it is important to consider differences in cell composition.

Estimating the proportion of SARS-CoV-2 infections reported through diagnostic testing

The proportion of SARS-CoV-2 infections ascertained through healthcare and community testing is generally unknown and expected to vary depending on natural factors and changes in test-seeking behaviour. Here we use population surveillance data and reported daily case numbers in the United Kingdom to estimate the rate of case ascertainment. We mathematically describe the relationship between the ascertainment rate, the daily number of reported cases, population prevalence, and the sensitivity of PCR and Lateral Flow tests as a function time since exposure. Applying this model to the data, we estimate that 20-40% of SARS-CoV-2 infections in the UK were ascertained with a positive test with results varying by time and region. Cases of the Alpha variant were ascertained at a higher rate than the wild type variants circulating in the early pandemic, and higher again for the Delta variant and Omi-cron BA.1 sub-lineage, but lower for the BA.2 sub-lineage. Case ascertainment was higher in adults than in children. We further estimate the daily number of infections and compare this to mortality data to estimate that the infection fatality rate increased by a factor of 3 during the period dominated by the Alpha variant, and declined in line with the distribution of vaccines.

Mitigating the risk of African swine fever virus in feed with anti-viral chemical additives.

African swine fever (ASF) is currently considered the most significant global threat to pork production worldwide. Disease caused by the ASF virus (ASFV) results in high case fatality of pigs. Importantly, ASF is a trade-limiting disease with substantial implications on both global pork and agricultural feed commodities. ASFV is transmissible through natural consumption of contaminated swine feed and is broadly stable across a wide range of commonly imported feed ingredients and conditions. The objective of the current study was to investigate the efficacy of medium-chain fatty acid and formaldehyde-based feed additives in inactivating ASFV. Feed additives were tested in cell culture and in feed ingredients under a transoceanic shipment model. Both chemical additives reduced ASFV infectivity in a dose-dependent manner. This study provides evidence that chemical feed additives may potentially serve as mitigants for reducing the risk of ASFV introduction and transmission through feed.