Development of Paper-Based Fluorescent Molecularly Imprinted Polymer Sensor for Rapid Detection of Lumpy Skin Disease Virus.

Lumpy Skin Disease (LSD) is a notifiable viral disease caused by Lumpy Skin Disease virus (LSDV). It is usually associated with high economic losses, including a loss of productivity, infertility, and death. LSDV shares genetic and antigenic similarities with Sheep pox virus (SPV) and Goat pox (GPV) virus. Hence, the LSDV traditional diagnostic tools faced many limitations regarding sensitivity, specificity, and cross-reactivity. Herein, we fabricated a paper-based turn-on fluorescent Molecularly Imprinted Polymer (MIP) sensor for the rapid detection of LSDV. The LSDV-MIPs sensor showed strong fluorescent intensity signal enhancement in response to the presence of the virus within minutes. Our sensor showed a limit of detection of 101 log10 TCID50/mL. Moreover, it showed significantly higher specificity to LSDV relative to other viruses, especially SPV. To our knowledge, this is the first record of a paper-based rapid detection test for LSDV depending on fluorescent turn-on behavior.

Low Pathogenic Avian Influenza H9N2 Viruses in Morocco: Antigenic and Molecular Evolution from 2021 to 2023.

Avian influenza viruses pose significant threats to both the poultry industry and public health worldwide. Among them, the H9N2 subtype has gained substantial attention due to its high prevalence, especially in Asia, the Middle East, and Africa; its ability to reassort with other influenza viruses; and its potential to infect humans. This study presents a comprehensive phylogenetic and molecular analysis of H9N2 avian influenza viruses circulating in Morocco from 2021 to 2023. Through an active epidemiological survey, a total of 1140 samples (trachea and lungs) and oropharyngeal swabs pooled into 283 pools, collected from 205 farms located in 7 regions of Morocco known for having a high density of poultry farms, were analyzed. Various poultry farms were investigated (159 broiler farms, 24 layer farms, 10 breeder farms, and 12 turkey breeder farms). A total of 21 AI H9N2 strains were isolated, and in order to understand the molecular evolution of the H9N2 avian influenza virus, their genetic sequences were determined using the Sanger sequencing technique. Phylogenetic analysis was performed using a dataset comprising global H9N2 sequences to determine the genetic relatedness and evolutionary dynamics of the Moroccan strains. The results revealed the continued circulation and diversification of H9N2 avian influenza viruses in Morocco during the study period. Real-time RT-PCR showed a positivity rate of 35.6% (73/205), with cycle threshold values ranging from 19.2 to 34.9. The phylogenetic analysis indicated that all Moroccan strains belonged to a G1-like lineage and regrouped into two distinct clusters. Our newly detected isolates aggregated distinctly from the genotypes previously isolated in Morocco, North and West Africa, and the Middle East. This indicats the potential of virus evolution resulting from both national circulation and cross-border transmission. A high genetic diversity at both nucleotide and amino-acid levels was observed among all the strains isolated in this study, as compared to H9N2 strains isolated in Morocco since 2016, which suggests the co-circulation of genetically diverse H9N2 variants. Newly discovered mutations were detected in hemagglutinin positions 226, 227, and 193 (H3 numbering), which highlights the genetic evolution of the H9N2 AIVs. These findings contribute to our understanding of the evolution and epidemiology of H9N2 in the region and provide valuable insights for the development of effective prevention and control strategies against this emerging avian influenza subtype.

Composting: A biosecurity measure to maximize the benefit of broilers̕ litter.

Objective: This study was conducted to evaluate the effect of composting on the count of Salmonella spp., Clostridium perfringens, and New Castle virus (NDV) isolated from broilers' litter. Moreover, to verify the impact of compost thermal stress on the expression of thermal genes harbored in the isolated bacteria. Materials and Methods: The prevalence of enteric aerobic and anaerobic infections by Salmonella spp., C. perfringens, and viral infections by NDV were investigated in litter samples collected from 100 broiler flocks by conventional methods and polymerase chain reaction. Results: The samples were positive for Salmonella spp., C. perfringens, and NDV, with prevalence rates of 60%, 55%, and 30%, respectively. An experiment to study the effect of compost on the microbiological quality of litter was applied using five compost heaps with an initial average count of Salmonella typhimurium (3.2 × 105CFU CFU/gm), C. perfringens (6.4 × 105 CFU/gm), and an average titer NDV (105.5 embryo infectious dose50/gm). The microbiological count of heaps after 15 days of composting revealed a reduction in the count of S. typhimurium and C. perfringens by 4 log10 CFU/gm and 3 log10 CFU/gm, respectively. Moreover, the hemagglutinating test revealed no detection of NDV after 15 days of composting. A high degree of downregulation of expression of the thermal genes, dnaK in S. typhimurium isolates and cpe gene in C. perfringens isolates, was detected by quantitative reverse transcription PCR. Conclusion: The reduction of pathogen counts, the simplicity, and the low cost associated with composting for only 15 days advocate the recommendation for raising awareness of composting as a routine biosecurity measure to prevent the spreading of infection and promote its safe use in agribusiness.

Human Wharton's Jelly Mesenchymal Stem Cells Secretome Inhibits Human SARS-CoV-2 and Avian Infectious Bronchitis Coronaviruses.

Human SARS-CoV-2 and avian infectious bronchitis virus (IBV) are highly contagious and deadly coronaviruses, causing devastating respiratory diseases in humans and chickens. The lack of effective therapeutics exacerbates the impact of outbreaks associated with SARS-CoV-2 and IBV infections. Thus, novel drugs or therapeutic agents are highly in demand for controlling viral transmission and disease progression. Mesenchymal stem cells (MSC) secreted factors (secretome) are safe and efficient alternatives to stem cells in MSC-based therapies. This study aimed to investigate the antiviral potentials of human Wharton's jelly MSC secretome (hWJ-MSC-S) against SARS-CoV-2 and IBV infections in vitro and in ovo. The half-maximal inhibitory concentrations (IC50), cytotoxic concentration (CC50), and selective index (SI) values of hWJ-MSC-S were determined using Vero-E6 cells. The virucidal, anti-adsorption, and anti-replication antiviral mechanisms of hWJ-MSC-S were evaluated. The hWJ-MSC-S significantly inhibited infection of SARS-CoV-2 and IBV, without affecting the viability of cells and embryos. Interestingly, hWJ-MSC-S reduced viral infection by >90%, in vitro. The IC50 and SI of hWJ-MSC secretome against SARS-CoV-2 were 166.6 and 235.29 µg/mL, respectively, while for IBV, IC50 and SI were 439.9 and 89.11 µg/mL, respectively. The virucidal and anti-replication antiviral effects of hWJ-MSC-S were very prominent compared to the anti-adsorption effect. In the in ovo model, hWJ-MSC-S reduced IBV titer by >99%. Liquid chromatography-tandem mass spectrometry (LC/MS-MS) analysis of hWJ-MSC-S revealed a significant enrichment of immunomodulatory and antiviral proteins. Collectively, our results not only uncovered the antiviral potency of hWJ-MSC-S against SARS-CoV-2 and IBV, but also described the mechanism by which hWJ-MSC-S inhibits viral infection. These findings indicate that hWJ-MSC-S could be utilized in future pre-clinical and clinical studies to develop effective therapeutic approaches against human COVID-19 and avian IB respiratory diseases.

Epidemiology, Genetic Characterization, and Pathogenesis of Avian Influenza H5N8 Viruses Circulating in Northern and Southern Parts of Egypt, 2017-2019.

Highly pathogenic avian influenza (HPAI) viruses of subtype H5N8 continue to circulate, causing huge economic losses and serious impact on poultry production worldwide. Recently, HPAIV H5N8 has been spreading rapidly, and a large number of HPAI H5N8 outbreaks have been reported in Eurasia 2020-2021. In this study, we conducted an epidemiological survey of HPAI H5N8 virus at different geographical locations in Egypt from 2017 to 2019. This was followed by genetic and pathogenic studies. Our findings highlight the wide spread of HPAI H5N8 viruses in Egypt, including in 22 governorates. The genetic analyses of the hemagglutinin (HA) and neuraminidase (NA) gene segments emphasized a phylogenetic relatedness between the Egyptian HPAI H5N8 viruses and viruses of clade 2.3.4.4b recently isolated in Europe. These findings suggest that a potential back transmission of Egyptian HPAI H5N8 virus has occurred from domestic poultry in Egypt to migratory wild birds, followed by further spread to different countries. This highlights the importance of continuous epidemiological and genetic studies of AIVs at the domestic-wild bird interface.

Molecular characterization of full fusion protein (F) of Newcastle disease virus genotype VIId isolated from Egypt during 2012-2016.

AIM: The aim of this work was to study the full F gene sequence of Newcastle disease virus (NDV) in regard to pathotyping and genotyping and to study the evolution of this NDV in Egypt. MATERIALS AND METHODS: The present study was conducted using samples from seven suspected NDV flocks of vaccinated chickens during 2012-2016 from six governorates in Egypt. The NDV was successfully isolated from pathological specimens through inoculation in specific pathogen-free embryonated chicken eggs. RESULTS: Pathogenicity of the NDV isolates has been estimated through intracerebral pathogenicity index and ranged from 1.66 to 1.73 which indicates the velogenic type of NDV isolates. Pathotyping and genotyping of these isolates were done through sequencing of full-length F gene. Results indicated that the seven NDV isolates showed characteristic cleavage site motif (112RRQKRF117) for the velogenic strains of NDV. Phylogenetic analysis of the F gene clustered these isolates within Group I of genotype VIId within Israeli strains NDV/IS/2015, NDV-Ch/SD883, and most of the Middle East strains. Six of seven sequenced isolates have six potential N-linked glycosylation sites. The neutralization epitope on the five antigenic sites of fusion is conserved in all Egyptian strains of this study except NDV-KFR-B7-2012 which has a substitution at D 170 N in epitope A4. In all our strains, 10 cysteine residues are recorded, except one loss of cysteine at residue 370 in both NDV-EG-35-2014 and NDV-GHB-328F-2016. CONCLUSION: All viruses in this study have 52 amino acid substitutions within fusion gene in compared with Lasota strain that reveals importance for its antigenic and structural function. The present work highlights the important need to sequence F gene of NDV genotype VIId to investigate the evolution of this NDV in Egypt.