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Recent outbreaks of influenza A(H5N1) have affected many mammal species. We report serologic evidence of H5N1 virus infection in horses in Mongolia. Because H3N8 equine influenza virus is endemic in many countries, horses should be monitored to prevent reassortment between equine and avian influenza viruses with unknown consequences.

Alkali-activated materials, known for their economical and eco-friendly nature, are becoming popular as a viable substitute for traditional cement-based materials in the field of construction. This research thoroughly examines the intricate rheological characteristics and compressive strength (CS) of alkali-activated mortar (AAM). It specifically explores the use of waste foundry sand (WFS) as an environmentally conscious and sustainable alternative to river sand. The addition of WFS significantly improves the CS of AAM, demonstrating 35 % enhancements at a 30 % sand replacement level. The rheological properties of customized AAM mixtures were targeted to achieve optimal use in 3D printing. The increasing replacement level of WFS in the matrix induced a non-linear reduction in yield stress (37 % decrease at 30 % replacement) accompanied by a concurrent rise in plastic viscosity (38 % increase at 30 % replacement). Considering the intricate interplay between rheological parameters and CS, AAM containing 15 % sand replacement exhibited optimal properties. Moreover, through rigorous experimentation, a comprehensive dataset of 64 samples was established by varying the amount of river sand, WFS, and water content in the mix. Machine learning models—including AdaBoost, decision tree, K-nearest neighbors, stochastic gradient descent, gradient boosting, random forest, neural network, gene expression programming, and support vector machine—were developed to predict CS, yield stress, and plastic viscosity of the AAM. Modeling errors, including MSE, RMSE, MAE, and MAPE, were falling in the acceptable limits. Specifically, most models demonstrated a high level of prediction having R2 > 0.90 across training and validation datasets. In conclusion, this study not only highlights the optimized properties of AAM but also validates the efficacy of various machine learning models in predicting crucial parameters for AAM formulation.

To meet the growing need for resilient structures in seismic and high-impact zones, accurate prediction of the response of reinforced concrete (RC) beams under impact loads is essential. Traditional methods, such as experimental testing and high fidelity finite element models, are often time consuming and resource intensive. To address these challenges, this study investigates various ensemble and non-ensemble machine learning techniques—including support vector machine, gaussian process regression (GPR), k-nearest neighbor (KNN), gene expression programming, random forest, decision tree, boosted tree, adaptive boosting tree, gradient boosting algorithm, stochastic gradient descent, and artificial neural network—for predicting the peak response of RC beams under impact loads. A set of 145 experimental data points from 12 different sources is used to train and evaluate these machine learning models. Key parameters in the data include beam width and depth, span, reinforcement ratios, concrete strength, steel yield strength, deflection, and impact characteristics. Except for KNN, all models showed satisfactory generalization capabilities with R2 values over 0.8. Statistical errors such as RMSE, a-10 index, MAE, and a-20 index are within acceptable limits. The GPR model is the most effective with R2 value of 0.95. Moreover, Shapely analysis identified beam depth, impact velocity, and beam breadth as critical factors. Overall, this study demonstrates the efficacy of machine learning in accurately predicting the behavior of RC structures under impact loads, providing valuable tools for civil engineers in design and analysis.

Abstract Background Fowl adenovirus-4 is a causative agent of hydropericardium hepatitis syndrome (HHS) in chickens and has been frequently reported from many countries. Fowl adenoviruses cause severe disease and mortality in broiler and layer breeders in Azerbaijan. Therefore, in this study, pathological lesions and the dissemination of fowl adenovirus-4 into the visceral organs of infected birds were investigated as well as molecular characterisation of detected strains. For this, liver, heart and spleen from 20 necropsied chickens originated from a broiler breeder flock and a layer breeder flock were embeded on the FTA cards and the samples were analysed for adenovirus-DNA by PCR and sequencing. Results The findings of necropsy in both broiler and layer breeder chickens were similar, and the liver was severely effected showing hepatitis, and the heart with hydropericardium lesions. The kidneys were swollen with haemorrhages and small white foci on the surface of the spleens were noted. Intestinal congestion and ecchymotic hemorrhages were also observed in some birds. Fowl adenovirus-4-DNA was detected by PCR in all collected organs of 20 birds. The sequence analysis revealed that fowl adenovirus-4 present in Azerbaijan and close similarity of the hexon genes of the adenoviruses existing in the Middle East, North America, far east and Indian subcontinent were determined by phylogenetic analysis. However, sequence diversity was detected from the adenovirus strains circulating in Europe, North and South America. Conclusions This study indicates the impact of fowl adenovirus-4 on the poultry health and production, and improved disease control and prevention strategies are necessary to reduce the HHS disease in chickens in Azerbaijan.

Avian influenza viruses (AIVs) are a major economic burden to the poultry industry and pose serious zoonotic risks, with human infections being reported every year. To date, the vaccination of birds remains the most important method for the prevention and control of AIV outbreaks. Most national vaccination strategies against AIV infection use whole virus-inactivated vaccines, which predominantly trigger a systemic antibody-mediated immune response. There are currently no studies that have examined the antibody repertoire of birds that were infected with and/or vaccinated against AIV. To this end, we evaluate the changes in the H9N2-specific IgM and IgY repertoires in chickens subjected to vaccination(s) and/or infectious challenge. We show that a large proportion of the IgM and IgY clones were shared across multiple individuals, and these public clonal responses are dependent on both the immunisation status of the birds and the specific tissue that was examined. Furthermore, the analysis revealed specific clonal expansions that are restricted to particular H9N2 immunisation regimes. These results indicate that both the nature and number of immunisations are important drivers of the antibody responses and repertoire profiles in chickens following H9N2 antigenic stimulation. We discuss how the repertoire biology of avian B-cell responses may affect the success of AIV vaccination in chickens, in particular the implications of public versus private clonal selection.

The exponential growth of communication technologies and the ubiquitous use of electronic devices raise concerns about unintended electromagnetic interference (EMI). Existing research exploring mortars incorporated with carbon fibers for EMI shielding primarily relies on empirical experiments investigating the interplay between shielding effectiveness (SE) and mixed design parameters. This research aims to establish predictive models for SE, focusing on the frequency of radiation and mortar constituents (water-to-cement ratio (W/C), fiber content, sand-to-cement ratio (S/C), and fiber aspect ratio). Employing a diverse array of machine learning algorithms, including stochastic gradient descent (SGD), gradient boosting, random forest, gene expression programming (GEP), AdaBoost, decision tree, K-nearest neighbors, and stepwise linear regression, this study aims to forecast the shielding efficacy of the mortar. Results demonstrate robust model performance, with R2 values and correlation coefficients surpassing 0.85 and 0.95, respectively, across training, testing, and validation datasets. Modeling errors, such as MSE, RMSE, MAE, and MAPE, remain within acceptable bounds for all models. The accuracy of predictions is evidenced by experimental-to-predicted SE ratios falling within the 0.5–1.5 range. Based on available data, parametric investigations conducted using the GEP-derived equation reveal a positive correlation between SE and S/C ratio, aspect ratio, fiber content, and radiation frequency, while indicating an inverse relationship with the W/C ratio. Moreover, employing Shapley additive explanation (SHAP)—a technique grounded in cooperative game theory—enhances the interpretability of SE. In summary, this study provides valuable insights into SE prediction for carbon fiber reinforced cement mortar, underscoring the efficacy of machine learning models in enhancing SE.

Benign recurrent intrahepatic cholestasis is an autosomal recessive disorder presenting with intermittent episodes of cholestatic jaundice. The initial episode of benign recurrent intrahepatic cholestasis tends to occur within the first two decades of a patient’s life. Episodes can occur unprompted but can often be precipitated by infections or pregnancy. We report an interesting case of a 13-year-old girl presented with recurrent intrahepatic cholestasis. The patient has a unique homozygous USP53 genetic mutation, the first patient to present with this mutation within the South Asian region. The patient was initially misdiagnosed as a case of autoimmune hepatitis, and when presenting to our set-up was diagnosed as a case of benign recurrent intrahepatic cholestasis. The patient has since been managed on medication and remains regular in follow-up, responding well to treatment.

Outbreaks of avian pathogens such as Newcastle disease virus, avian influenza virus, and salmonella have a major impact on economies and food security worldwide. Some pathogens also pose a significant zoonotic potential, especially avian influenza viruses. Vaccination plays a key role in controlling many poultry diseases, and there are many vaccines licenced in the United Kingdom for diseases of poultry caused by viruses, bacteria, and parasites. However, these vaccines often do not provide complete protection and can cause unwanted side effects. Several factors affect the potency of poultry vaccines, including the type of vaccination used, the mechanism of delivery, and the use of adjuvants. Advancements in technology have led to the study and development of novel vaccines and vaccine adjuvants for use in poultry. These induce stronger immune responses compared with current vaccine technology and have the potential to protect against multiple poultry diseases. This review aims to discuss the existing poultry vaccine technology; the effect of delivery mechanisms on vaccine efficacy; the use of current and novel adjuvants; the ability to target antigens to antigen-presenting cells; and the use of probiotics, multivalent vaccines, and nanotechnology to enhance the potency of poultry vaccines.

Pigeon paramyxovirus serotype 1 (PPMV-1), an antigenic and host variant of avian paramyxovirus Newcastle disease virus (NDV), primarily originating from racing pigeons, has become a global panzootic. Egypt uses both inactivated PPMV-1 and conventional NDV vaccines to protect pigeons from disease and mortality. However, the impact of prevalent strains and the effectiveness of available vaccines in pigeons in Egypt are unclear. This study investigates the virulence of PPMV-1 (Pigeon/Egypt/Sharkia-19/2015/KX580988) and evaluates available paramyxovirus vaccines in protecting pigeons against a PPMV-1 challenge. Ten-day-old specific-pathogen-free (SPF) embryonated chicken eggs infected with this strain exhibited a mean death time (MDT) of 86.4 ± 5.88 h. The intracerebral pathogenicity index (ICPI) in day-old chickens was 0.8, while pigeons experienced an ICPI of 0.96 and an intravenous pathogenicity index (IVPI) of 2.11. These findings classify the strain as virulent and velogenic. Experimental infection of pigeons with this PPMV-1 strain at 106 EID50/0.1 mL resulted in a 62.5% mortality rate, displaying nervous and enteric distress. The virus caused extensive lesions in visceral organs, with strong immunohistochemistry signals in all examined organs, indicating the systemic spread of the virus concurrent to its neurotropic and viscerotropic tropism. Furthermore, vaccination using an inactivated PPMV-1 and live NDV LaSota vaccine regimen protected 100% of pigeons against mortality, while with a single NDV LaSota vaccine, it was 62.5%. The PPMV alone or combined with NDV LaSota induced protective levels of haemagglutination inhibition (HI) antibody titres and reduced virus shedding from buccal and cloacal cavities. Based on generalised linear gamma model analysis, both PPMV-1 and NDV LaSota are antigenically comparable by HI. These findings suggest that using both inactivated PPMV-1 (G-VI) and live attenuated NDV (LaSota) vaccines is an effective prophylactic regimen for preventing and controlling PPMV-1 and NDV in pigeons, thereby reducing the risk of interspecies transmission.

The complement system is an innate immune mechanism against microbial infections. It involves a cascade of effector molecules that is activated via classical, lectin and alternative pathways. Consequently, many pathogens bind to or incorporate in their structures host negative regulators of the complement pathways as an evasion mechanism. Factor H (FH) is a negative regulator of the complement alternative pathway that protects “self” cells of the host from non-specific complement attack. FH has been shown to bind viruses including human influenza A viruses (IAVs). In addition to its involvement in the regulation of complement activation, FH has also been shown to perform a range of functions on its own including its direct interaction with pathogens. Here, we show that human FH can bind directly to IAVs of both human and avian origin, and the interaction is mediated via the IAV surface glycoprotein haemagglutinin (HA). HA bound to common pathogen binding footprints on the FH structure, complement control protein modules, CCP 5-7 and CCP 15-20. The FH binding to H1 and H3 showed that the interaction overlapped with the receptor binding site of both HAs, but the footprint was more extensive for the H3 HA than the H1 HA. The HA - FH interaction impeded the initial entry of H1N1 and H3N2 IAV strains but its impact on viral multicycle replication in human lung cells was strain-specific. The H3N2 virus binding to cells was significantly inhibited by preincubation with FH, whereas there was no alteration in replicative rate and progeny virus release for human H1N1, or avian H9N2 and H5N3 IAV strains. We have mapped the interaction between FH and IAV, the in vivo significance of which for the virus or host is yet to be elucidated.

Background . Autoimmune hepatitis (AIH) is increasingly seen in children worldwide and it is more severe in children compared to adults. This study highlights the biochemical and clinical aspect, treatment given and outcome of the disease including pediatric liver transplantation. Study . Retrospective review (2012-2022) was done in Shifa International Hospital, Islamabad. Patients under 18 years diagnosed with AIH were included. Data related to age, gender, clinical features, laboratory investigations including liver function test, liver biopsy findings and imaging modalities were included. Results . Fifteen patients were included 7 (47%) were males and 8 (53%) females. AIH type 1 was the most common type seen in 7 (46%), AIH type 2 in 5 (33%) and seronegative in 3 (20%). Jaundice was the most common symptom. Liver biopsy showed findings characteristic of AIH. Liver transplant performed in 3 patients. Conclusion . The study highlights the varied clinical presentation of AIH in Pakistani children.

Concrete structures are susceptible to cracking, which can compromise their integrity and durability. Repairing them with ordinary Portland cement (OPC) paste causes shrinkage cracks to appear in the repaired surface. Alkali-activated binders offer a promising solution for repairing such cracks. This study aims to develop an alkali-activated paste (AAP) and investigate its effectiveness in repairing concrete cracks. AAPs, featuring varying percentages (0.5%, 0.75%, 1%, 1.25%, 1.5%, and 1.75%) of polyethylene (PE) fibers, are found to exhibit characteristics such as strain hardening, multiple plane cracking in tension and flexure tests, and stress-strain softening in compression tests. AAP without PE fibers experienced catastrophic failure in tension and flexure, preventing the determination of its stress-strain relationship. Notably, AAPs with 1.25% PE fibers demonstrated the highest tensile and flexural strength, exceeding that of 0.5% PE fiber reinforced AAP by 100% in tension and 70% in flexure. While 1% PE fibers resulted in the highest compressive strength, surpassing AAP without fibers by 17%. To evaluate the repair performance of AAP, OPC cubes were cast with pre-formed cracks. These cracks were induced by placing steel plates during casting and were designed to be full and half-length with widths of 1.5 mm and 3 mm. AAP both with and without PE fibers led to a substantial improvement in compressive strength, reducing the initial strength loss of 30%-50% before repair to a diminished range of 2%-20% post-repair. The impact of PE fiber content on the compressive strength of repaired OPC cube is marginal, providing more flexibility in using AAP with any fiber percentage while still achieving effective concrete crack repair. Considering economic and environmental factors, along with observed mechanical enhancements, AAPs show promising potential for widespread use in concrete repair and related applications, contributing valuable insights to the field of sustainable construction materials.

Since the first human case in 2013, H7N9 avian influenza viruses (AIVs) have caused more than 1500 human infections with a mortality rate of approximately 40%. Despite large-scale poultry vaccination regimes across China, the H7N9 AIVs continue to persist and evolve rapidly in poultry. Recently, several strains of H7N9 AIVs have been isolated and shown the ability to escape vaccine-induced immunity. To assess the zoonotic risk of the recent H7N9 AIV isolates, we rescued viruses with hemagglutinin (HA) and neuraminidase (NA) from these H7N9 AIVs and six internal segments from PR8 virus and characterized their receptor binding, pH of fusion, thermal stability, plaque morphology and in ovo virus replication. We also assessed the cross-reactivity of the viruses with human monoclonal antibodies (mAbs) against H7N9 HA and ferret antisera against H7N9 AIV candidate vaccines. The H7N9 AIVs from the early epidemic waves had dual sialic acid receptor binding characteristics, whereas the more recent H7N9 AIVs completely lost or retained only weak human sialic acid receptor binding. Compared with the H7N9 AIVs from the first epidemic wave, the 2020/21 viruses formed larger plaques in Madin-Darby canine kidney (MDCK) cells and replicated to higher titres in ovo, demonstrating increased acid stability but reduced thermal stability. Further analysis showed that these recent H7N9 AIVs had poor cross-reactivity with the human mAbs and ferret antisera, highlighting the need to update the vaccine candidates. To conclude, the newly emerged H7N9 AIVs showed characteristics of typical AIVs, posing reduced zoonotic risk but a heightened threat for poultry.

High pathogenicity avian influenza (HPAI) H5N1 is a subtype of the influenza A virus primarily found in birds. The subtype emerged in China in 1996 and has spread globally, causing significant morbidity and mortality in birds and humans. In Cambodia, a lethal case was reported in February 2023 involving an 11-year-old girl, marking the first human HPAI H5N1 infection in the country since 2014. This research examined the zoonotic potential of the human H5N1 isolate, A/Cambodia/NPH230032/2023 (KHM/23), by assessing its receptor binding, fusion pH, HA thermal stability, and antigenicity. Results showed that KHM/23 exhibits similar receptor binding and antigenicity as the early clade 2.3.2.1c HPAI H5N1 strain, and it does not bind to human-like receptors. Despite showing limited zoonotic risk, the increased thermal stability and reduced pH of fusion in KHM/23 indicate a potential threat to poultry, emphasizing the need for vigilant monitoring.

BackgroundHuman perinatal life is characterized by a period of extraordinary change during which newborns encounter abundant environmental stimuli and exposure to potential pathogens. To meet such challenges, the neonatal immune system is equipped with unique functional characteristics that adapt to changing conditions as development progresses across the early years of life, but the molecular characteristics of such adaptations remain poorly understood. The application of single cell genomics to birth cohorts provides an opportunity to investigate changes in gene expression programs elicited downstream of innate immune activation across early life at unprecedented resolution.MethodsIn this study, we performed single cell RNA-sequencing of mononuclear cells collected from matched birth cord blood and 5-year peripheral blood samples following stimulation (18hrs) with two well-characterized innate stimuli; lipopolysaccharide (LPS) and Polyinosinic:polycytidylic acid (Poly(I:C)).ResultsWe found that the transcriptional response to LPS was constrained at birth and predominantly partitioned into classical proinflammatory gene upregulation primarily by monocytes and Interferon (IFN)-signaling gene upregulation by lymphocytes. Moreover, these responses featured substantial cell-to-cell communication which appeared markedly strengthened between birth and 5 years. In contrast, stimulation with Poly(I:C) induced a robust IFN-signalling response across all cell types identified at birth and 5 years. Analysis of gene regulatory networks revealed IRF1 and STAT1 were key drivers of the LPS-induced IFN-signaling response in lymphocytes with a potential developmental role for IRF7 regulation.ConclusionAdditionally, we observed distinct activation trajectory endpoints for monocytes derived from LPS-treated cord and 5-year blood, which was not apparent among Poly(I:C)-induced monocytes. Taken together, our findings provide new insight into the gene regulatory landscape of immune cell function between birth and 5 years and point to regulatory mechanisms relevant to future investigation of infection susceptibility in early life.

IntroductionInfectious Bursal Disease Virus (IBDV) causes immunosuppression in chickens. While B-cell destruction is the main cause of humoral immunosuppression, bursal T cells from IBDV-infected birds have been reported to inhibit the mitogenic response of splenocytes, indicating that some T cell subsets in the infected bursa have immunomodulatory activities. CD4+CD25+TGFβ+ cells have been recently described in chickens that have immunoregulatory properties and play a role in the pathogenesis of Marek’s Disease Virus.MethodsTo evaluate if CD4+CD25+TGFβ+ cells infiltrated the bursa of Fabricius (BF) following IBDV infection, and influenced the outcome of infection, birds were inoculated at either 2 days or 2 weeks of age with vaccine strain (228E), classic field strain (F52/70), or PBS (mock), and bursal cell populations were quantified by flow cytometry.ResultsBoth 228E and F52/70 led to atrophy of the BF, a significant reduction of Bu1+-B cells, and a significant increase in CD4+ and CD8α+ T cells in the BF, but only F52/70 caused suppression of immune responses to a test antigen in younger birds, and clinical signs in older birds. Virus was cleared from the BF more rapidly in younger birds than older birds. An infiltration of CD4+CD25+T cells into the BF, and elevated expression of bursal TGFβ-1+ mRNA was observed at all time points following infection, irrespective of the strain or age of the birds, but CD4+TGFβ+cells and CD4+CD25+TGFβ+ cells only appeared in the BF at 28 dpi in younger birds. In older birds, CD4+TGFβ+ cells and CD4+CD25+TGFβ+ cells were present at earlier time points, from 7dpi following 228E infection, and from 14 and 28 dpi following F52/70 infection, respectively.DiscussionOur data suggest that an earlier infiltration of CD4+TGFβ+ cells into the BF correlated with a delayed clearance of virus. However, the influx of CD4+TGFβ+ cells and CD4+CD25+TGFβ+ into the BF did not correlate with increased pathogenicity, or immunosuppression.

Background: Methylmalonic acidemia (MMA), type mut (0) is a rare type of genetic inborn error of metabolism (IEM) that is caused by aberrant malonyl-CoA mutase activity. Diagnosing IEM can be challenging due to its inherited onset and varying degrees of severity. Methods and results: In the present study, a consanguineous Pakistani family suspected of IEM was genetically analyzed using whole exome sequencing. A biallelic variant c.689 C > G (p.Thr230Arg) in MMUT was identified to be the causative factor of the disease, which helped in establishing the accurate diagnosis in the family to be MMA mut(0) type. On the basis of the genetic findings, the patient's condition was appropriately managed through a supportive nutrition plan and administration of oral L-carnitine. Conclusions: Identification of MMUT mutation through whole exome sequencing was helpful in solving the family and devising targeted management strategies. This study highlights the utility of genetic analysis in diagnosing and treating metabolic disorders like MMA in Pakistani inbred population. Highlights: In the present study, a consanguineous Pakistani family suspected of IEM was genetically analyzed using whole exome sequencing. A biallelic variant c.689 C > G (p.Thr230Arg) in MMUT was identified to be the causative factor of the disease, which helped in establishing the accurate diagnosis in the family to be MMA mut(0) type. [ABSTRACT FROM AUTHOR]

Avian influenza A virus (AIV) is a significant cause of mortality in poultry, causing substantial economic loss, particularly in developing countries, and has zoonotic potential. For example, highly pathogenic avian influenza (HPAI) viruses of the H5 subtype have been circulating in Egypt for around two decades. In the last decade, H5N1 viruses of clade 2.2.1 have been succeeded by the antigenically distinct H5N8 clade 2.3.4.4b viruses. Furthermore, H9N2 viruses co-circulate with the H5N8 viruses in Egyptian poultry. It is widely recognised that effective vaccination against IAV requires a close antigenic match between the vaccine and viruses circulating in the field. Therefore, approaches to develop cost-effective vaccines that can be rapidly adapted to local virus strains are required for developing countries such as Egypt. In this project, the haemagglutinin (HA) proteins of Egyptian H5 and H9 viruses were expressed by transient transfection of plants (Nicotiana benthamiana). The formation of virus-like particles (VLPs) was confirmed by transmission electron microscopy. Mice were immunised with four doses of either H5 or H9 VLPs with adjuvant. Antibody and cellular immune responses were measured against the corresponding recombinant protein using ELISA and enzyme-linked immunosorbent assay (ELISpot), respectively. Chickens were immunised with one dose of H5 VLPs, eliciting HA-specific antibodies measured by ELISA and a pseudotyped virus neutralisation test using a heterologous H5 HA. In conclusion, plant-based VLP vaccines have potential for producing an effective vaccine candidate within a short time at a relatively low cost.

Influenza A virus (IAV) is the primary causative agent of influenza, colloquially called the flu. Each year, it infects up to a billion people, resulting in hundreds of thousands of human deaths, and causes devastating avian outbreaks with worldwide losses worth billions of dollars. Always present is the possibility that a highly pathogenic novel subtype capable of direct human-to-human transmission will spill over into humans, causing a pandemic as devastating if not more so than the 1918 influenza pandemic. While antiviral drugs for influenza do exist, they target very few aspects of IAV replication and risk becoming obsolete due to antiviral resistance. Antivirals targeting other areas of IAV replication are needed to overcome this resistance and combat the yearly epidemics, which exact a serious toll worldwide. This review aims to summarise the key steps in the IAV replication cycle, along with highlighting areas of research that need more focus.

Abstract Maternally derived antibodies (MDAs) are important for protecting chickens against pathogens in the neonatal stage however, they often interfere with vaccine performance. Here, we investigated the effects of MDAs on a targeted antigen delivery vaccine (TADV), which is developed by conjugating H9 subtype avian influenza virus haemagglutinin (HA) antigen to single chain fragment variable (scFv) antibodies specific for the chicken antigen presenting cell receptor CD83. Groups of 1-day-old chickens carrying high levels of MDAs (MDA++) and 14-day old chickens carrying medium levels of MDAs (MDA+) were immunised with TADV (rH9HA-CD83 scFv), untargeted rH9HA or inactivated H9N2 vaccines. Immunogenicity in these vaccinated chickens was compared using haemagglutination inhibition (HI) and enzyme-linked immunosorbent assays (ELISA). The results showed that the TADV (rH9HA-CD83 scFv) induced significantly higher levels of H9HA-specific antibody titres compared to the untargeted rH9HA and inactivated H9N2 vaccines in MDA++ and MDA+ chickens. Overall, the data demonstrates immune responses induced by TADV are not affected by the MDA in chickens.

Microbial colonisation is paramount to the normal development of the immune system, particularly at mucosal sites. However, the relationships between the microbiome and the adaptive immune repertoire have mostly been explored in rodents and humans. Here, we report a high-throughput sequencing analysis of the chicken TCRβ repertoire and the influences of microbial colonisation on tissue-resident TCRβ+ cells. The results reveal that the microbiome is an important driver of TCRβ diversity in both intestinal tissues and the bursa of Fabricius, but not in the spleen. Of note, public TCRβ sequences (shared across individuals) make a substantial contribution to the repertoire. Additionally, different tissues exhibit biases in terms of their V family and J gene usage, and these effects were influenced by the gut-associated microbiome. TCRβ clonal expansions were identified in both colonised and germ-free birds, but differences between the groups were indicative of an influence of the microbiota. Together, these findings provide an insight into the avian adaptive immune system and the influence of the microbiota on the TCRβ repertoire.

Abstract T-cell activation induces context-specific gene expression programs that promote energy generation and biosynthesis, progression through the cell cycle and ultimately cell differentiation. The aim of this study was to apply the omni ATAC-seq method to characterize the landscape of chromatin changes induced by T-cell activation in mature naïve CD4+ T-cells. Using a well-established ex vivo protocol of canonical T-cell receptor signaling, we generated genome-wide chromatin maps of naïve T-cells from pediatric donors in quiescent or recently activated states. We identified thousands of individual chromatin accessibility peaks that are associated with T-cell activation, the majority of which were annotated intronic and intergenic enhancer regions. A core set of 3268 gene promoters underwent chromatin remodeling and concomitant changes in gene expression in response to activation, and were enriched in multiple pathways controlling cell cycle regulation, metabolism, inflammatory response genes and cell survival. Leukemia inhibitory factor (LIF) was among those factors that gained the highest accessibility and expression, in addition to IL2-STAT5 dependent chromatin remodeling in the T-cell activation response. Using publicly available data we found the chromatin response was far more dynamic at 24-h compared with 72-h post-activation. In total 546 associations were reproduced at both time-points with similar strength of evidence and directionality of effect. At the pathways level, the IL2-STAT5, KRAS signalling and UV response pathways were replicable at both time-points, although differentially modulated from 24 to 72 h post-activation.

Abstract The immunogenicity and protective efficacy of vaccines can be enhanced by the selective delivery of antigens to the antigen-presenting cells (APCs). In this study, H9N2 avian influenza virus haemagglutinin (HA) antigen, was targeted by fusing it to single-chain fragment variable (scFv) antibodies specific to CD83 receptor expressed on chicken APCs. We observed an increased level of IFNγ, IL6, IL1β, IL4, and CxCLi2 mRNA upon stimulation of chicken splenocytes ex vivo by CD83 scFv targeted H9HA. In addition, CD83 scFv targeted H9HA induced higher serum haemagglutinin inhibition activity and virus neutralising antibodies compared to untargeted H9HA, with induction of antibodies as early as day 6 post primary vaccination. Furthermore, chickens vaccinated with CD83 scFv targeted H9HA showed reduced H9N2 challenge virus shedding compared to untargeted H9HA. These results suggest that targeting antigens to CD83 receptors could improve the efficacy of poultry vaccines.

Avian influenza virus (AIV) subtypes H5N1 and H9N2 co-circulate in poultry in Bangladesh, causing significant bird morbidity and mortality. Despite their importance to the poultry value chain, the role of farms in spreading and maintaining AIV infections remains poorly understood in most disease-endemic settings. To address this crucial gap, we conducted a cross-sectional study between 2017 and 2019 in the Chattogram Division of Bangladesh in clinically affected and dead chickens in farms with suspected AIV infection. Viral prevalence of each subtype was approximately 10% among farms for which veterinary advice was sought, indicating high levels of virus circulation in chicken farms despite the low number of reported outbreaks. Co-circulation of both subtypes was common in farms, with our findings suggest that in the field, the co-circulation of H5N1 and H9N2 can modulate disease severity, which could facilitate an underestimated level of AIV transmission in the poultry value chain. Finally, using newly generated whole-genome sequences, we investigate the evolutionary history of a small subset of H5N1 and H9N2 viruses. Our analyses revealed that for both subtypes, the sampled viruses were genetically most closely related to other viruses isolated in Bangladesh and represented multiple independent incursions. However, due to lack of longitudinal surveillance in this region, it is difficult to ascertain whether these viruses emerged from endemic strains circulating in Bangladesh or from neighbouring countries. We also show that amino acids at putative antigenic residues underwent a distinct replacement during 2012 which coincides with the use of H5N1 vaccines.

Abstract Background Newcastle disease viruses (NDVs) can spread across continents via migratory birds. Hence, we investigated the frequency of NDV in both non-migratory and birds migrating on the Black Sea-Mediterranean flyway, in Istanbul, Turkey. Birds were trapped using nets placed around the Kucukcekmece lake Avcilar, Istanbul, in spring seasons of 2016 and 2018. In total, 297 birds belonging to 42 different species were trapped, categorized according to species and sex, and flocked oropharyngeal swabs were collected. In addition, flocked swabs were also collected from 115 mallards caught by hunters around Edirne and from 207 birds which had been treated in the Veterinary Faculty of Istanbul university-Cerrahpasa. Tissue samples were taken from dead wild birds brought by public to Veterinary Faculty. A total of 619 flocked oropharyngeal swabs were pooled into 206 samples. RNA was extracted from swabs and tissue samples. Real-time RT-PCR prob. assay was used to detect NDV-RNA in samples. Results There was no amplification in real time RT-PCR in samples taken from wild birds caught by traps. However, amplification of NDV-F gene was observed in oropharyngeal swabs taken from 2 waterfowls (Common Moorhen and Mallard), and in tissue samples taken from 2 little owls and 1 common kestrel. Sequencing and phylogenetic analyses of these 5 samples for NDV-F gene showed great similarity with NDV subgenotype VII.2 viruses. Analysis also showed that there is a high similarity with the F gene sequences previously reported from Turkey in 2012 and as well as the sequences from neighbouring countries Bulgaria and Georgia and geographically close country such as Pakistan. Although the strains found in this study are closely related, there is a relatively small degree of molecular divergence within 543 bp of F gene of the Turkish NDV isolate and strains detected in Israel, Pakistan, Iran, United Arab Emirates and Belgium. Conclusions Our findings revealed the presence of subgenotype VII.2 of NDVs in wild birds in north west of Turkey and demonstrated some degree of molecular evolution when compared to the earlier NDV-VII.2 isolate in Turkey.

Abstract Background Reverse genetics is used in many laboratories around the world and enables the creation of tailor-made influenza viruses with a desired genotype or phenotype. However, the process is not flawless, and difficulties remain during cloning of influenza gene segments into reverse genetics vectors (pHW2000, pHH21, pCAGGS). Reverse genetics begins with making cDNA copies of influenza gene segments and cloning them into bi-directional (pHW2000) or uni-directional plasmids (pHH21, pCAGGS) followed by transfection of the recombinant plasmid(s) to HEK-293 T or any other suitable cells which are permissive to transfection. However, the presence of internal restriction sites in the gene segments of many field isolates of avian influenza viruses makes the cloning process difficult, if employing conventional methods. Further, the genetic instability of influenza gene-containing plasmids in bacteria (especially Polymerase Basic 2 and Polymerase Basic 1 genes; PB2 and PB1) also leads to erroneous incorporation of bacterial genomic sequences into the influenza gene of interest. Methods Herein, we report an easy and efficient ligation and restriction enzyme independent (LREI) cloning method for cloning influenza gene segments into pHW2000 vector. The method involves amplification of megaprimers followed by PCR amplification of megaprimers using a bait plasmid, DpnI digestion and transformation. Results Hard-to-clone genes: PB2 of A/chicken/Bangladesh/23527/2014 (H9N2) and PB1 of A/chicken/Bangladesh/23527/2014 (H9N2), A/chicken/Jiangxi/02.05YGYXG023-P/2015 (H5N6) and A/Chicken/Vietnam/H7F-14-BN4–315/2014 (H9N2) were cloned into pHW2000 using our LREI method and recombinant viruses were subsequently rescued. Conclusion The LREI cloning procedure represents an alternative strategy for cloning influenza gene segments which have internal restriction sites for the enzymes used in reverse genetics. Further, the problem of genetic instability in bacteria can be alleviated by growing recombinant bacterial cultures at a lower temperature. This technique can be applied to clone any influenza gene segment using universal primers, which would help in rapid generation of influenza viruses and facilitate influenza research and vaccine development.

Many cellular genes and networks induced in human lung epithelial cells infected with the influenza virus remain uncharacterized. Here, we find that p21 levels are elevated in response to influenza A virus (IAV) infection, which is independent of p53. Silencing, pharmacological inhibition or deletion of p21 promotes virus replication in vitro and in vivo, indicating that p21 is an influenza restriction factor. Mechanistically, p21 binds to the C-terminus of IAV polymerase subunit PA and competes with PB1 to limit IAV polymerase activity. Besides, p21 promotes IRF3 activation by blocking K48-linked ubiquitination degradation of HO-1 to enhance type I interferons expression. Furthermore, a synthetic p21 peptide (amino acids 36 to 43) significantly inhibits IAV replication in vitro and in vivo. Collectively, our findings reveal that p21 restricts IAV by perturbing the viral polymerase complex and activating the host innate immune response, which may aid the design of desperately needed new antiviral therapeutics.

(1) Background: The aim of this study was to produce in-house ELISAs which can be used to determine SARS-CoV-2-specific antibody levels directed against the spike protein (S), the S1 subunit of S and the receptor binding domain (RBD) of S in SARS-CoV-2 vaccinated and infected humans. (2) Methods: Three in-house ELISAs were developed by using recombinant proteins of SARS-CoV-2, namely the S, S1 and RBD proteins. Specificity and sensitivity evaluations of these tests were performed using sera from SARS-CoV-2-infected (n = 70) and SARS-CoV-2-vaccinated (n = 222; CoronaVac vaccine) humans in Istanbul, Turkey. The analyses for the presence of SARS-CoV-2-specific antibodies were performed using the in-house ELISAs, a commercial ELISA (Abbott) and a commercial surrogate virus neutralization test (sVNT). We also analyzed archival human sera (n = 50) collected before the emergence of COVID-19 cases in Turkey. (3) Results: The sensitivity of the in-house S, S1 and RBD ELISAs was found to be 88.44, 90.17 and 95.38%, while the specificity was 72.27, 89.08 and 89.92%, respectively, when compared to the commercial SARS-CoV-2 antibody test kit. The area under curve (AUC) values were 0.777 for the in-house S ELISA, 0.926 for the S1 ELISA, and 0.959 for the RBD ELISA. The kappa values were 0.62, 0.79 and 0.86 for the S, S1 and RBD ELISAs, respectively. (4) Conclusions: The in-house S1 and RBD ELISAs developed in this study have acceptable performance characteristics in terms of sensitivity, specificity, AUC and kappa values. In particular, the RBD ELISA seems viable to determine SARS-CoV-2-specific antibody levels, both in infected and vaccinated people, and help mitigate SARS-CoV-2 outbreaks and spread.

Avian influenza viruses pose a continuous threat to both poultry and human health, with significant economic impact. The ability of viruses to reassort and jump the species barrier into mammalian hosts generates a constant pandemic threat.

Recent studies demonstrated that domestic cats can be naturally and experimentally infected with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). This study was performed to investigate the presence of SARS-CoV-2-specific antibodies within the domestic cat population in Istanbul, Turkey, before the coronavirus disease 2019 (COVID-19) and during the COVID-19 pandemic. Overall, from 155 cat sera analyzed, 26.45% (41/155) tested positive in the spike protein-ELISA (S-ELISA), 28.38% (44/155) in the receptor-binding domain-ELISA (RBD-ELISA), and 21.9% (34/155) in both, the S- and RBD-ELISAs. Twenty-seven of those were also positive for the presence of antibodies to feline coronavirus (FCoV). Among the 34 SARS-CoV-2-positive sera, three of those were positive on serum neutralization assay. Six of the 30 cats before COVID-19 and 28 of the 125 cats during COVID-19 were found to be seropositive. About 20% of ELISA-positive cats exhibited mainly respiratory, gastrointestinal, and renal signs and skin lesions. Hematocrit, hemoglobin, white blood cells, lymphocyte, and platelet numbers were low in about 30% of ELISA-positive cats. The number of neutrophils and monocytes were above normal values in about 20% of ELISA-positive cats. The liver enzyme alanine aminotransferase levels were high in 23.5% ELISA-positive cats. In conclusion, this is the first report describing antibodies specific to SARS-CoV-2 antigens (S and RBD) in cats in Istanbul, Turkey, indicating the risk for domestic cats to contract SARS-CoV-2 from owners and/or household members with COVID-19. This study and others show that COVID-19-positive pet owners should limit their contact with companion animals and that pets with respiratory signs should be monitored for SARS-CoV-2 infections.

Summary: Global deployment of an effective and safe vaccine is necessary to curtail the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we evaluated a Newcastle disease virus (NDV)-based vectored-vaccine in mice and hamsters for its immunogenicity, safety, and protective efficacy against SARS-CoV-2. Intranasal administration of recombinant (r)NDV-S vaccine expressing spike (S) protein of SARS-CoV-2 to mice induced high levels of SARS-CoV-2-specific neutralizing immunoglobulin A (IgA) and IgG2a antibodies and T-cell-mediated immunity. Hamsters immunized with two doses of vaccine showed complete protection from lung infection, inflammation, and pathological lesions following SARS-CoV-2 challenge. Importantly, administration of two doses of intranasal rNDV-S vaccine significantly reduced the SARS-CoV-2 shedding in nasal turbinate and lungs in hamsters. Collectively, intranasal vaccination has the potential to control infection at the site of inoculation, which should prevent both clinical disease and virus transmission to halt the spread of the COVID-19 pandemic.

ABSTRACTInfluenza viruses have an error-prone polymerase complex that facilitates a mutagenic environment. Antigenic mutants swiftly arise from this environment with the capacity to persist in both humans and economically important livestock even in the face of vaccination. Furthermore, influenza viruses can adjust the antigenicity of the haemagglutinin (HA) protein, the primary influenza immunogen, using one of four molecular mechanisms. Two prominent mechanisms are: (1) enhancing binding avidity of HA toward cellular receptors to outcompete antibody binding and (2) amino acid substitutions that introduce an N-linked glycan on HA that sterically block antibody binding. In this study we investigate the impact that adsorptive mutation and N-linked glycosylation have on receptor-binding, viral fitness, and antigenicity. We utilize the H9N2 A/chicken/Pakistan/SKP-827/16 virus which naturally contains HA residue T180 that we have previously shown to be an adsorptive mutant relative to virus with T180A. We find that the addition of N-linked glycans can be beneficial or deleterious to virus replication depending on the background receptor binding avidity. We also find that in some cases, an N-linked glycan can trump the effect of an avidity enhancing substitution with respect to antigenicity. Taken together these data shed light on a potential route to the generation of a virus which is “fit” and able to overcome vaccine pressure.

The Enzyme Linked Immunosorbant Assay (ELISA) test of potato leaves collected from different areas within three (3) selected divisions in Khyber Pakhtunkhwa, Pakistan was carried out in 2016. The region-wise results showed the over all incidence of potato virus Y (PVY) to be highest in all the three divisions on potato crop followed by the incidence of potato leaf roll virus (PLRV). The PVYand PLRV incidence in Malakand divisionwas noted by 62.7 and 44.6 %, respectively. In Peshawar division the incidence of PVY was 55.4% and that of the PLRV was 40.7% and the incidence of PVY and PLRV was 50.7 and 37.3%, respectively in Hazara. The field-wise results of Malakand region showed the highest incidence of PVY and PLRV in Kalam was found by 70 and 50%) followed by Miandam (62 and 46 %) and the lowest incidence was recorded in Malamjaba (56 and 38%). In Peshawar the highest incidence of PVY and PLRV wasnotedon potato crop in Warmarh (66 and 46%) followed by Regi (52 and 32%) and the lowest was noted in Pul Dheri Mehra (48 and 44%). In the Hazara potato crop in Kaghan had 62 and 54% PVY and PLRV incidence followed by Bagnotar (48 and 36%) and the lowest incidence was noted in Kalanderabad (42 and 22%), respectively.Results obtained by this study indicating that both PVY and PLRV are major viruses of potato crop in selected potato growing areas of KPK province which could pose serious threat to the potato crop, development of resistance cultivar against vector insect could be recommended. [ABSTRACT FROM AUTHOR]

Abstract Background Yellow lupin (Lupinus luteus L.) is a promising grain legume for productive and sustainable crop rotations. It has the advantages of high tolerance to soil acidity and excellent seed quality, but its current yield potential is poor, especially in low rainfall environments. Key adaptation traits such as phenology and enhanced stress tolerance are often complex and controlled by several genes. Genomic-enabled technologies may help to improve our basic understanding of these traits and to provide selective markers in breeding. However, in yellow lupin there are very limited genomic resources to support research and no published information is available on the genetic control of adaptation traits. Results We aimed to address these deficiencies by developing the first linkage map for yellow lupin and conducting quantitative trait locus (QTL) analysis of yield under well-watered (WW) and water-deficit (WT) conditions. Two next-generation sequencing marker approaches - genotyping-by-sequencing (GBS) and Diversity Array Technology (DArT) sequencing - were employed to genotype a recombinant inbred line (RIL) population developed from a bi-parental cross between wild and domesticated parents. A total of 2,458 filtered single nucleotide polymorphism (SNP) and presence / absence variation (PAV) markers were used to develop a genetic map comprising 40 linkage groups, the first reported for this species. A number of significant QTLs controlling total biomass and 100-seed weight under two water (WW and WD) regimes were found on linkage groups YL-03, YL-09 and YL-26 that together explained 9 and 28% of total phenotypic variability. QTLs associated with length of the reproductive phase and time to flower were found on YL-01, YL-21, YL-35 and YL-40 that together explained a total of 12 and 44% of total phenotypic variation. Conclusion These genomic resources and the QTL information offer significant potential for use in marker-assisted selection in yellow lupin.