Ultra-diluted Bryonia alba extract modulates HMOX-1 gene expression to attenuate the pathogenetic effect of SARS-CoV-2 spike protein RBD antigen.

Biochemical profile of fetal fluids in alpacas (Vicugna pacos) during pregnancy and immediate postpartum.

Rapid and sensitive time-resolved fluorescence immunochromatographic strip for detecting H10 subtype avian influenza virus

ABSTRACT Among RNA viruses, duck hepatitis A viruses (DHAVs) and duck astroviruses (DAstVs) are significant viral pathogens causing duck viral hepatitis (DVH), leading to severe economic losses in the duck industry worldwide. In August 2021, an acute and highly fatal disease affected 2-week-old Pekin ducklings on a commercial farm in Gharbia, Egypt. The ducklings displayed signs of lethargy, ataxia, and opisthotonus, accompanied by visible lesions in the liver and kidneys, as well as histopathological evidence of hepatocellular necrosis, inflammation, and degeneration of renal tubules. RT–PCR confirmed the presence of DHAV-3 but not DHAV-1. Virus isolation trials in embryonated duck eggs showed greenish allantoic fluid discolouration and embryonic deaths within 72 h. Experimental infections reproduced clinical signs and acute hepatitis in 1-week-old ducklings. Next-generation sequencing (NGS) revealed the complete genome of the DHAV-3 isolate, Du/Egy/Gharbia/2021/DHAV-3, representing the first complete genome sequencing and molecular characterization of a DHAV-3 strain from Egypt. Phylogenetic analysis revealed a high genetic similarity (92.5%) between the Egyptian DHAV-3 isolate and strains originating from China, Vietnam, and South Korea. Unexpectedly, virome sequencing identified a novel duck astrovirus, Du/Egy/Gharbia/2021/DAstV, exhibiting 93.24% nucleotide identity with the DAstV-5 JM strain from China and approximately 75% identity with other duck astroviruses. This is the first report of DAstV-5 co-infection with DHAV-3 in Egypt, highlighting the power of metagenomic approaches to detect novel pathogens. The findings expand our understanding of the molecular epidemiology and evolution of these viruses, emphasizing the need for continued surveillance and control measures in the duck industry. RESEARCH HIGHLIGHTS First complete genome sequencing of a DHAV-3 strain from Egypt using m-NGS. Discovery of a novel duck astrovirus co-infecting with DHAV-3. Phylogenetic analysis reveals cross-border transmission links with Asian strains of both DHAV-3 and DAstV-5.

In developing countries particularly in field conditions unfavorable environmental conditions, lack of availability of appropriate transport media (TM) and maintenance of cold chain during transport; sample collection, storage, and transportation is more challenging. Considering these facts, five TM out of which three laboratory-based media named phosphate-buffered saline (PBS), 50% glycerol + PBS, and normal saline (NS) and two commercially available media including viral transport medium (VTM), charcoal based viral transport medium (CVTM) were compared to protect infectivity of the H5N1 influenza virus. Spiked fecal sample and allantoic fluid with and without these TM were placed in field simulatory storage and transportation conditions and in every 12 h time interval these samples were tested for virus isolation in embryonated chicken egg inoculation and identification by HA test and RT PCR upto 7 days. Survivability of the virus was detected by calculating the percent infectivity and analysed by logistic regression analysis. NS, PBS, and CVTM, were most effective media in maintaining the integrity of the test virus. These media are easily available and economical and less complex to prepare. A significant difference was found in survivability of the virus only in VTM in between allantoic fluid and fecal sample.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-15987-6.

Background:Newcastle disease (ND) is a highly infectious disease affecting multiple avian species worldwide. Repeated ND outbreaks in Pakistan have presented substantial challenges to the poultry sector.Aim:Extensive surveillance in Pakistan over a decade has revealed a high prevalence of avian orthoavulavirus 1 (AOAV-1), highlighting the need for tailored vaccination approaches and continuous monitoring.Methods:This study presented a 10-year biosurveillance (2011–2020) of AOAV-1 across 1083 locations (including commercial farms, backyard poultry, live bird retail stalls, household birds, national zoos, and wild migratory birds), with a total of 5,145 samples (trachea and oropharyngeal swabs) and 822 blood samples collected from 18 avian species. Tracheal and swab samples were inoculated in embryonated chicken eggs, and the positive allantoic fluids were assessed for AOAV-1 prevalence using reverse-transcriptase real-time PCR (rRT-PCR) targeting the fusion (F) gene. Serum samples were evaluated using a hemagglutination inhibition (HI) test to detect AOAV-1 antibodies.Results:rRT-PCR analysis showed a prevalence of 37.41%, while seroprevalence assessed through the HI assay was 48%. Moreover, over a decade after repeated outbreaks of virulent AOAV-1 that resulted in high morbidity and mortality in Pakistan, the responsible strain was detected in vaccinated chickens across multiple commercial poultry farms, despite the existence of elevated AOAV-1-specific antibody titers (>4.6 log2).Conclusion:The observations suggest a possible role of vaccinated poultry as a reservoir of virulent AOAV-1. These findings contribute to our understanding of the high prevalence of AOAV-1 in Pakistan, emphasizing the need for customized vaccination approaches and continuous monitoring to enable effective management of AOAV-1 in avian species.

Abstract The composition of bacterial communities within the female reproductive tract has been associated with fertility status in mammals; however, limited research has explored reproductive tract microbiota in swine. The objective of this study was to analyze the abundance and diversity of bacterial communities from the mucosal surface of the vagina, cervix, uterus, and chorioallantois, and within the allantoic and amniotic fluids throughout gestation in gilts. Duroc x Landrace x Yorkshire gilts (n=38) free of physical, health or reproductive-related issues were euthanized and hysterectomized on either day 11 (n = 11; peri-implantation), 15 (n = 10; implantation), 60 (n =6; mid-gestation) or 90 (n = 6; late-gestation) of pregnancy, as well as day 15 (n = 5; cyclic) of the estrous cycle. Bacterial analyses were conducted targeting the V4 hypervariable region of the 16S rRNA gene. In day 15 cyclic gilts, genera Campylobacter, Actinobacillus, Anaerococcus, and Fusobacterium were more abundant in the vagina than in the cervix (P < 0.05). Additionally, Campylobacter, Porphyromonas, and Anaerococcus were more abundant in the vagina than in the endometrium (P < 0.05). In pregnant gilts, the genus Fusobacteria abundance was greater in the vagina than in the cervix and endometrium on days 11 (P < 0.01) and 15 (P < 0.01) of gestation. Additionally, Lactobacillus was more abundant in allantoic fluid than the endometrium on day 60 (P < 0.05). Further, Streptococcus, Campylobacter, and Actinobacillus were more abundant in the vagina than in the amniotic fluid on day 90 (P < 0.05). Alpha diversity metrics indicated lower microbial diversity in amniotic fluid relative to other tissues (P < 0.05). However, alpha diversity did not differ between cyclic and pregnant gilts (P > 0.05). Beta diversity analysis revealed distinct clustering patterns based on tissue type and stage of pregnancy. Specifically, the vagina, cervix, and endometrium clustered separately from placental fluids. Additionally, microbial communities in samples from day 15 cyclic, day 11 pregnant, and day 15 pregnant gilts clustered distinctly from days 60 and 90 of gestation (P < 0.01). These findings suggest that microbial communities within the reproductive tract are dynamic and vary by both anatomical location and stage of pregnancy or estrous cycle. The distinct clustering of microbial populations between early and late stages of gestation indicates shifts in bacterial composition potentially associated with the presence of a conceptus. Additionally, the observed differences in microbial diversity across reproductive tissues and fetal fluids highlight potential functional roles of microbiota in reproductive success.

BackgroundDuck virus hepatitis (DVH) is highly fatal disease that predominantly affects young ducklings, causing substantial losses due to its high morbidity and mortality rates. A severe outbreak occurred in young Pekin ducklings on a commercial farm in Benha, Egypt.ResultsThe affected birds exhibited neurological signs, including lethargy, ataxia, and opisthotonus, leading to a high mortality rate. The livers and kidneys of ducklings showed various degrees of gross and histopathological lesions. Virus isolation trials in embryonated duck eggs revealed the characteristic greenish discoloration of the allantoic fluid, along with hepatitis and embryonic mortality. Furthermore, RT-PCR confirmed the presence of suspected duck hepatitis A virus type 1(DHAV-1). This study presents the first complete genome sequence of DHAV-1 from Egypt using next generation sequencing (NGS). Sequence analysis revealed that DHAV-1 exhibits the characteristic genomic organization of Avihepatovirus. The whole nucleotide sequence of Du/Egy/Benha/2020/DHAV-1 showed a high similarity to viruses isolated from Hungary in 2004, with a 99.9% identity in both the complete genome and structural genes (VP0, VP3, VP1). Antigenic analysis revealed a unique escape mutation, S178Y, related to conserved antigenic determinants on VP1 of DHAV-1 isolate. The cross-neutralization assay was utilized to assess the antigenic diversity between the field strain and the locally used live attenuated vaccine strain.ConclusionThe results revealed minimal antigenic variation, highlighting the potential for immune evasion. These findings suggest that the currently administered vaccines in Egypt remain effective in controlling DHAV-1 infections. However, continuous surveillance is essential to monitor any emerging genetic or antigenic changes that could compromise vaccine efficacy in the future.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12917-025-05010-5.

Background: Hepatic injury followed by hepatic failure and death is now becoming a health-related global problem from both intentional and unintentional overdoses of acetaminophen (APAP), which is now the most used over-the-counter drug. CYP3A4 is the primary cytochrome P450 enzyme that catalyzes the oxidation of acetaminophen to the toxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). APAP overdosing activates c-jun kinase (c-JNK), which activates the mitochondrial membrane permeability transition (MPT). This cascade results in an uncontrolled systemic response. The aberrant release of cytokines IL-6, 8, 10, 1β, and TGF-β1 activates apoptosis-inducing factor (AIF) and causes centri-lobular necrosis. Methods: Initially, 70 compounds were short-listed from commonly used homeopathic medicines and considered for molecular docking with CYP3A4. Solanine-containing medicine, with the highest docking score (-11.2), was considered for this experiment. In the next phase, 14-day-old pathogen-free, fertilized chicken eggs were procured and inoculated with 100 μL of APAP (0.01 mg/ml concentration), Solanum tuberosum extracts (STE), and 70% alcohol (control) in respective groups with other control groups. After 48 hours, allantoic fluid with heart, brain, and liver tissues was preserved for analysis. Results: APAP significantly up-regulated the expressions of IL-6, IL-10, TGF-β1, and IL-1β, in addition to weight gain of the embryo. However, the STE group outperformed the APAP group in terms of embryo survival and development. In the STE group, IL-6, IL-10, and TGF-β1 were significantly balanced, showing hepato-protective effects. Histological evidence corroborated the cytokine changes, and cellular architectures were well preserved in the STE treatment group. A similar phenomenon was observed as STE inhibited APAP-induced up-regulation of c-JNK, CYP3A4, and CYP3A5 expressions. STE also showed protective effects through up-regulation of Bax, Bcl-2, and Caspase-3 expression among the challenged sets. The gene expressions corroborated the histopathological findings of the respective groups. Conclusion: STE protects against APAP-induced hepatotoxicity, but its clinical implementation in homeopathic doses should be explored.

Aim. To study some biological properties of the obtained strain of Newcastle disease virus NDV/Adygea/duck/12/2008, including the degree of virulence, and to conduct a phylogenetic study.Biological material from wild migratory birds was collected in 2008 during the hunting season. Isolation and cultivation of the isolated strains were carried out in the system of developing chicken embryos (RCE). Primary identification confirming the presence of a hemagglutinating agent in the allantoic fluid was carried out in the hemagglutination reaction (HR). Pathogenicity was assessed by MDT and ICPI methods. Sequencing, phylogenetic analysis, the genotype of the studied strain was determined.The results of studying the main biological properties of the Newcastle disease virus strain NDV/Adygea/duck/12/2008, isolated from wild migratory birds in the Southern Federal District, are presented. According to the phylogenetic study, the NDV/Adygea/duck/12/2008 strain belongs to genotype VII and genetic class 2. The MDT and ICPI virulence tests, as well as the molecular genetic study, classified the described strain as highly pathogenic.

Understanding protein and amino acid deposition in pregnant gilts is important for developing nutritional strategies that meet these demands and enhance reproductive performance. Current models, such as the NRC (2012) gestating sow model, assume a constant proportional protein and amino acid content in tissues throughout pregnancy. However, empirical data suggest that gestational tissue growth and composition change dynamically. In this study, we developed a gestation model that characterizes the dynamic changes in growth, crude protein, and amino acid deposition throughout gestation. Based on a systematized search of published data, mathematical functions were developed to estimate daily protein and amino acid deposition in key tissues, including allantoic and amniotic fluid, uterus, placenta, fetus, mammary gland, and maternal body. Our results suggest that dietary crude protein levels and amino acid profiles should be adjusted to meet metabolic demands, particularly in early gestation, where a potential nutritional deficiency was identified. Additionally, the amino acid profile of deposited protein shifts during late gestation, suggesting a changing demand for specific amino acids. These findings challenge existing models and highlight the need for adaptive dietary strategies that better align with pregnancy's biological demands. By refining protein and amino acid deposition estimates, this study provides a framework guiding future research on precision feeding, ultimately improving gilt and sow reproductive performance.

Tissue non-specific alkaline phosphatase (TNSALP) regulates postnatal phosphate homeostasis, but its role in utero-placental phosphate availability remains poorly understood. Gilts were bred and hysterectomized on Day 60 or Day 90 of gestation (n = 6/day). Phosphate was less abundant in allantoic and amniotic fluids on Day 90 compared to Day 60. TNSALP protein was immunolocalized, and enzymatic activity was quantified and localized in endometrial and chorioallantois tissues. Day had no effect on TNSALP activity in the chorioallantois. In contrast, endometrial TNSALP activity was lower on Day 90 compared to Day 60. Phosphate abundance in allantoic fluid correlated positively with endometrial TNSALP activity on Day 60 but not Day 90. TNSALP protein was abundantly expressed in the endometrium and chorioallantois on both days investigated, with localization to the endometrial, chorionic, and areolar epithelia, as well as stromal cells and endothelium. TNSALP activity was detected in the endothelium of the blood vessels in both the endometrium and chorioallantois, and on the basal surface of the endometrial glands on Day 60 but not Day 90. The endometrial stratum compactum stroma had strong TNSALP activity on Day 60. Weak TNSALP activity was present in the areolar epithelium, with a modest increase in activity on Day 90 compared to Day 60. TNSALP activity was present in the columnar chorionic epithelial cells, with an apparent decrease in activity in the chorioallantois on Day 90 compared to Day 60. These data reveal spatiotemporal changes in TNSALP localization and activity, suggesting its involvement in regulating phosphate availability at the utero-placental interface in swine.Lay SummaryPhosphate is an essential nutrient for fetal growth, but how it is managed during pregnancy is not fully understood. This study explored the role of an enzyme called tissue non-specific alkaline phosphatase (TNSALP) in regulating phosphate availability in the uterus and placenta in pigs in mid- and late pregnancy. Phosphate levels decreased in the fluids surrounding the fetus in late pregnancy. TNSALP was present in the uterus and placenta, and the amount of the enzyme varied depending on the tissue and stage of pregnancy and correlated with changes in phosphate levels. These findings suggest that TNSALP plays a key role in managing phosphate transport from the mother to the fetus in pregnancy to support fetal development.

First report of the emergence of novel sub-genotype XIII.2.3 of Newcastle disease virus in chickens from selected regions of Bangladesh.

Multi-omics analysis of yolk and allantoic fluid in chicken embryonic development and sexual differentiation

Since 2017, an infectious disease characterized by gosling gout and caused by goose astrovirus (GAstV) has affected geese in most major goose-producing regions of China. In this study, a total of 385 geese displaying gout symptoms were sampled from 12 cities in Guangdong Province, China, between 2019 and 2021. RT-PCR analysis revealed that all samples were positive for GAstV (385/385), with GAstV-II being the predominant subtype, accounting for 90.4% (348/385) of the cases. Co-infection with GAstV-I and GAstV-II was detected in 50.4% (194/385) of the samples. Additionally, different GAstV subtypes were successfully isolated using goose embryos, namely GDYJ-21-01 (GAstV-I) and GDZJ-21-01 (GAstV-II). Analysis of viral copy numbers in major pathological tissues following infection of goslings and goose embryos revealed that GDZJ strain exhibited broader tissue tropism than GDYJ strain. Compared to other tissues, GDYJ strain displayed tissue tropism exclusively in the cecal tonsils of goslings and the allantoic fluid of embryos. Structural prediction and alignment using AlphaFold 2.0 identified an α-helix in the S223-A226 region of the GDZJ VP34 protein, while a loop structure was observed in the Q235-Q237 region of the corresponding GDYJ VP34 protein. Furthermore, although the VP27 protein regions of both subtypes contained five β-sheet structures, the overall sequence similarity was relatively low, at 37.1%. This study broadens our understanding of the prevalence differences among GAstV subtypes and provides valuable insights into the development of reagents for preventing these viral infections.

Seasonal influenza continues to threaten human lives and pose a significant burden to healthcare systems and the economy, emphasizing the need for developing better influenza vaccines, diagnostics, and antiviral therapeutics. To address these challenges, we generated a library of structurally diverse sialyl glycosides containing 4‐N‐derivatized sialic acids by a highly efficient one‐pot two‐enzyme chemoenzymatic sialylation strategy. Sialosides containing 4‐azido‐substituted sialic acid were selectively cleaved by sialidases from influenza viruses, whereas sialosides containing 4‐acetamido‐modified sialic acid were resistant to sialidase cleavage. Interestingly, sialosides containing 4‐amino‐ or 4‐guanidino‐substituted sialic acid were effective inhibitors moderately or highly resistant to cleavage by influenza sialidases (also called neuraminidases). The sialosides containing the 4‐guanidino‐substituted sialic acid represent a new class of sialidase substrate analog‐based inhibitors. We took advantage of this unique property to create a ligand‐based approach for efficiently isolating influenza virions from egg allantoic fluid with high purity. Together, these compounds are versatile probes and ligands for developing new approaches to detect, profile, isolate, and characterize influenza viruses via neuraminidases on their surface.

Abstract Seasonal influenza continues to threaten human lives and pose a significant burden to healthcare systems and the economy, emphasizing the need for developing better influenza vaccines, diagnostics, and antiviral therapeutics. To address these challenges, we generated a library of structurally diverse sialyl glycosides containing 4‐N‐derivatized sialic acids by a highly efficient one‐pot two‐enzyme chemoenzymatic sialylation strategy. Sialosides containing 4‐azido‐substituted sialic acid were selectively cleaved by sialidases from influenza viruses, whereas sialosides containing 4‐acetamido‐modified sialic acid were resistant to sialidase cleavage. Interestingly, sialosides containing 4‐amino‐ or 4‐guanidino‐substituted sialic acid were effective inhibitors moderately or highly resistant to cleavage by influenza sialidases (also called neuraminidases). The sialosides containing the 4‐guanidino‐substituted sialic acid represent a new class of sialidase substrate analog‐based inhibitors. We took advantage of this unique property to create a ligand‐based approach for efficiently isolating influenza virions from egg allantoic fluid with high purity. Together, these compounds are versatile probes and ligands for developing new approaches to detect, profile, isolate, and characterize influenza viruses via neuraminidases on their surface.

Abstract Two prepartum female alpacas were referred to our institution with the presumed diagnosis of uterine torsion. In both cases, uterine torsion was not confirmed but bacterial placentitis with Escherichia coli as the aetiological agent was diagnosed. Transabdominal ultrasound confirmed the presence of a living fetus, with heart rates of 108 and 75 beats per minute, respectively. The allantoic fluid was hyperechogenic and the placenta partially detached. No signs of uterine torsion were evident. Treatment with antibiotics and NSAIDs was initiated. Female Alpaca #1 gave birth to a healthy cria on Day 5 of treatment. In female Alpaca #2, fetal heart rate had decreased on Day 2, was no longer detectable on Day 3 and 1 day later a dead cria was born. In both cases, the placenta was partially covered with purulent material. In conclusion, placentitis should be included as a differential diagnosis in prepartum alpacas and may be more frequent than previously assumed.

Newcastle Disease (ND) is a common poultry disease in Indonesia, caused by Avian Paramyxovirus serotype 1 (APMV-1). This disease is endemic and occurs throughout the year affecting various types of poultry, including commercial and backyard chickens. This study aims to compare the prevalence of the ND virus in poultry farms in Blahbatuh and Payangan Districts, Gianyar Regency. This study employed an observational method with a cross-sectional approach. Chickens showing clinical symptoms were subjected to necropsy to collect tissue samples that exhibited pathological changes. These samples were inoculated into Specific Pathogen-Free (SPF) embryonated chicken eggs for ND virus growth. After incubation, the allantoic fluid was harvested and tested using HA and HI. A positive HA test result was indicated by the formation of sand like sediment, while a positive HI test result showed a 1% erythrocyte deposit forming a dot at the bottom of the microplate well. The study results indicated that 9 out of 32 chickens (28%) tested positive for ND. Chickens sampled in Payangan Subdistrict were found to have a 1.36 times higher risk of infection compared to those sampled in Blahbatuh subdistrict. Increasing farmers awareness of the importance of regular ND vaccination and improving biosecurity like isolation cage systems is crucial to preventing the spread of the disease.

BackgroundNewcastle disease virus (NDV) is a causative agent of Newcastle disease (ND), a major infectious poultry disease associated with significant economic losses. Vaccination is usually effective at preventing the disease. However, in Ethiopia, ND is commonly detected in both unvaccinated and vaccinated chickens. In this study, we aimed to evaluate the pathogenicity of NDV isolated from both vaccinated and unvaccinated chickens, as well as to compare the antigenicity of the isolates with vaccine strains and genotyping by using the F-gene sequence.MethodsThe partial F gene sequences of all isolates and the mean death times (MDTs) of representative isolates were used to determine genotype and pathogenicity of the isolates. Antigenicities were assayed with the hemagglutinin inhibition (HI) and virus neutralization (VN) tests using antiserum against the vaccine Hitchner B1 (HB1), which is the most commonly used NDV vaccine in Ethiopia. Thermostability was evaluated by incubating infected allantoic fluid at 56 °C.ResultsOut of 231 samples tested, 10.8% (25/231) were positive for virus isolation. The F gene cleavage sites of all 25 isolates had 112RRQKRF117, a characteristic of virulent NDVs. The MDTs of representative isolates were less than 60 h, indicating highly virulent (velogenic) pathotypes. The HI test revealed significant differences between our isolates and the HB1 vaccine strain, but the VN test showed no antigenic difference. Phylogenetic analysis based on the partial F gene sequences showed that all the isolates belonged to sub-genotype VII.1.1 of genotype VII, which is closely related to NDV strains from the Middle East and Eritrea. Thermostability test showed two of the 25 isolates were thermostable.DiscussionAlthough the HI test indicates antigenic differences between the velogenic Ethiopian isolates and the HB1 vaccine, the VN test showed that the vaccine could protect infections with these isolates. Phylogenetic analysis showed that all studied isolates belong to sub-genotype VII.1.1 of genotype VII, diverging from previously reported genotype XXI in Ethiopia.ConclusionsIn Ethiopia, NDV genotype VII 1.1 is widely distributed. Since these viruses showed the same antigenicity as the HB1 vaccine in VN test, the occurrence of ND in vaccinated chickens may be due to vaccine failure caused by inadequate management or immunosuppression due to other infectious diseases.