Newcastle Disease Research Articles

Pathogenicity of Newcastle Disease Virus Isolated from Ethiopia

Newcastle disease represents an enormous problem for African breeders, and country-specific fears and economic exchanges complicate its elimination and control. Slow laboratory diagnosis creates additional delays during initial epidemic discovery. To address these concerns, researchers undertook a study in Ethiopia to examine the impact of the circulating Newcastle disease virus on chicken farms. The aim of the study is to identify the virus from likely outbreak cases, confirm its pathogenicity, and evaluate its effects on vaccinated and uninfected chicks Forty chicks were separated into 02 groups and exposed to 02 virus samples at the National Veterinary Institute in Bishoftu, Ethiopia. The isolated viruses were classified as velogenic, with the BOG strains scoring 1.67 and the HAR strains rating 1.55. Immunized chicks demonstrated high herd immunity, while single immunization failure was observed. In the challenged group, the death rate was 55%, with all 20 unvaccinated chicks dying from the sickness, whereas just 2 out of 20 vaccinated chicks were afflicted. Statistical analysis showed that the virus produced general inflammation, which was more common in the BOG group (87.50%) than in the HAR group (12.50%). Vaccination significantly reduced clinical signs and gross lesions, such as irritation in multiple organs. The study showed the persistence of velogenic Newcastle disease virus strains in Ethiopia and the importance of vaccination. The findings have improved our understanding of the disease's physiopathology and provided vital information for responsible authorities to implement early intervention techniques.

Viral pathogen surveillance in wastewater using metagenomic approaches: obstacles and prospects

Abstract Background Wastewater monitoring offers an important tool for pathogen detection and tracking within large populations at a minimized cost and labor. Untargeted metagenomic approaches for the detection and sequencing of viruses still face limitations. In this study we tested the application of the sequence-independent single-primer amplification (SISPA) protocol in conjunction with nanopore sequencing to the study of viromes in wastewater samples. Methods Influent wastewater was collected from four Wastewater Treatment Plants located in the Lisbon region and Alentejo, Portugal in 2019 and 2023. Samples were processed with different methods including filtration, flocculation, Nanotraps or absorvent tampons. We applied the SISPA protocol, that includes a random amplification step, followed by nanopore sequencing on flow cells R9.4.1. We used the CZ ID (https://czid.org) pipeline for metagenomic analysis and taxonomic reporting. Results A large heterogeneity was found between samples in sequencing output (ranging from 17 thousand to 12 million reads) and the proportion of sequence reads classified as viral (ranging from 0.06% to 4% per sample). Viral taxa with higher number of reads were Picornavirales, dsDNA and ssDNA phages, Mimiviridae and Circoviridae. Known viral pathogens included Rotavirus A, Orthopoxvirus, Human betaherpesvirus and Avian orthoavulavirus. Conclusions Metagenomic studies of environmental samples generally exhibit a low proportion of viral reads, even after viral enrichment. A substantial sequencing effort is thus required to achieve a comprehensive virome characterization. As alternative, a more targeted approach, e.g. to target families of viruses of interest, may be considered. Funding: Fundação para a Ciência e Tecnologia (FCT): GHTM - UID/04413/2020; LA-REAL - LA/P/0117/2020; https://doi.org/10.54499/CEECINST/00102/2018/CP1567/CT0040 to SGS. Research council of the Vrije Universiteit Brussel (OZR-VUB): OZR3863BOF to PL. Key messages • Enrichment for viruses in metagenomic approaches remain challenging in very complex samples as are influent wastewaters. • Virome characterization still necessitates high sequencing efforts but may be used as a complementary environmental survey tool integrated with more targeted approaches.

Synergistic Anti-Tumor Effects of Newcastle Disease Virus and Doxorubicin: Evidence from A Murine Breast Cancer Model

BackgroundDespite the recent advances in the diagnosis and treatment of breast cancer, triple-negative breast cancer (TNBC) remains a clinical challenge due to its aggressive nature and resistance to conventional therapies. Virotherapy has emerged as a promising cancer treatment strategy, leveraging the ability of viruses to specifically target and replicate in cancerous cells. This study evaluated the oncolytic potential of a combined therapeutic strategy, utilizing Newcastle disease virus (NDV) and Doxorubicin hydrochloride (Dox) both in vitro and in vivo. MethodsThe in vitro experiments involved exposing human and mouse TNBC cell lines (MDA-MB-231 and 4T1, respectively) to NDV and Dox, individually or in combination. Cell viability assays and flow cytometry analyses were conducted to assess the synergistic effects of NDV and Dox on regulating breast cancer cell behavior in vitro. Furthermore, the immune-stimulating potential of NDV was investigated by examining its effects on dendritic cell (DC) maturation using flow cytometry and T cell proliferation. The in vitro anti-tumor effects of NDV were examined in both parental and tamoxifen-resistant cancer cells to assess its efficacy against chemoresistance. Animal models of breast cancer were treated with NDV in combination with Dox. The body weight changes, tumor volume, and survival rates of the mice were monitored throughout the study. Histopathological analyses were conducted to evaluate the potential toxic effects of the treatments. ResultsBased on the MTT results, NDV at optimal concentrations synergized the effect of Dox to reduce the viability of both MDA-MB231 and 4T1 cell lines (Isobologram combination index of less than 1). Additionally, individual treatment with NDV was able to significantly reduce the viability of patient-derived breast cancer cells, compared to the untreated control (P < 0.05) without affecting the cells of normal adjacent tissue. Furthermore, a combination of NDV and Dox significantly enhanced the percentage of early and late apoptotic cells in MDA-MB-231 (P < 0.0001) and late apoptotic cells in 4T1 (P < 0.0001), in comparison with individual treatment with these agents. Flow cytometry results showed that, compared to wild type MDA-MB-231 cells, NDV-infected MDA-MB-231 cells were better inducers of T cell proliferation and DC maturation as evidenced by increased proliferation index (P < 0.05) and elevated expression of CD1a, CD83, and CD86 (P < 0.0001), respectively. Moreover, co-treatment of both wild-type and (tamoxifen) TAM-resistant MCF-7/TAMR-1 cells with TAM and NDV significantly reduce the viability of the cancer cells (P < 0.0001). In tumor-bearing mice locally engrafted with 4T1 cells, combined treatment of NDV and Dox exhibited a marked reduction in median tumor volume compared to the control group, validating our in vitro findings on their synergistic anti-tumor effects. These findings suggest that combining NDV with Dox can effectively inhibit tumor progression and has the potential to reduce the dose, and consequently the toxic side-effects, of Dox in breast cancer therapy.

The heterologous expression of novel recombinant protein composed of HN and F moieties of Newcastle disease virus and immunogenicity evaluation in mouse model.

The rapid spread of Newcastle disease (ND), driven by extensive commercial exchange in the poultry industry, necessitates urgent preventive measures. Although effective vaccines against the Newcastle disease virus (NDV) have been used since 1940, recent outbreaks and the limitations of current vaccines highlight the need for improved solutions. Advances in synthetic biology, reverse vaccinology, molecular biology, and recombinant DNA technology over the past 20 years have led to the development of recombinant vaccines, which offer enhanced protection and broader immunogenic coverage against NDV. This study aimed to express the immunogenic domains of Hemagglutinin Neuraminidase (HN) and Fusion (F) glycoproteins, linked to the heat-labile enterotoxin B subunit (LTB) bio-adjuvant, to develop an effective and reliable recombinant vaccine for NDV. In this study, the L(HN)2F protein, composed of the LTB bio-adjuvant and the immunogenic regions of the doubled Hemagglutinin Neuraminidase (HN-HN) and Fusion (F) epitope, was expressed in Escherichia coli. Subcutaneous injection was used to evaluate the humoral immune response in mice and the result was compared with B1 vaccine. The induction of strong humoral immune responses proved the strong immunoreactivity of the recombinant protein. The IgG elicited by the recombinant proteins was comparable to that of the commercial B1 vaccine against NDV, indicating its potential as a viable candidate for further development and evaluation as a recombinant vaccine against NDV.

Entry of Newcastle disease virus into host cells: an interplay among viral and host factors.

Newcastle disease (ND) is a major burden for the poultry industry worldwide, especially in developing countries. The virus that causes this disease, Newcastle disease virus (NDV), is also an effective vector for the development of novel human and animal vaccines and a promising oncolytic virus for cancer therapy. The mechanism of entry of NDV into host cells is of particular interest because it has a significant impact on the infectivity, host range, and pathogenicity of the virus. Here, we present an overview of the entry of NDV into cells, focusing on the interplay among viral and host factors involved in this process. In particular, recent research revealing novel features of NDV attachment to cells, the identification of viral and cellular components that regulate binding of the virus to cells, and the emerging role of novel cellular routes of NDV entry are discussed. More importantly, some of the remaining gaps in our understanding of NDV entry and some fundamental questions for research efforts in the future are also highlighted.

Antitumor effect of the Newcastle disease virus strain NDV/Altai/pigeon/777/2010 on a model of solid Lewis carcinoma

Aim. To evaluate the efficacy of intratumoral administrations of the Newcastle disease virus strain NDV/Altai/pigeon/777/2010, to compare progression of the tumor nodes after virotherapy and to analyse pathomorphological changes in the tumor tissue in vitro and in vivo. Single intratumoral injections of the mesogenic strain of Newcastle disease virus NDV/Altai/pigeon/777/2010, isolated from a rock dove in Siberia, were done into outbred mice of the C57Bl/6 line into solid nodes of Lewis lung carcinoma that were grafted subcutaneously. Then the dynamics of tumor growth and pathomorphological changes in the tumor tissue were assessed and analyzed.It was shown that single intratumoral injections into immunocompetent C57Bl/6 mice with the mesogenic strain of Newcastle disease virus NDV/Altai/pigeon/777/2010 led to an increase in peculiar pathomorphological changes in the tumor tissue of subcutaneously grafted Lewis lung carcinoma and to a decrease in tumor growth compared to the control group of mice in vivo. A direct cytotoxic effect of the NDV strain on the Vero E6 cell line in vitro was noted.The results of this study indicate that the NDV strain NDV/Altai/pigeon/777/2010 has antitumor properties. This may enable clinical trials to prove its effectiveness as an antitumor drug.

Comparative and experimental pathology of passaged Newcastle disease virus isolates in ducks.

Although waterfowl are less susceptible to Newcastle disease (ND) virus (NDV) infection compared with chickens and turkeys, lethal ND in waterfowl has been sporadically reported. Factors underlying the high pathogenicity of certain NDV strains in waterfowl remain unclear. In ducks, the NDV 9a5b isolate shows low pathogenicity while the d5a20b isolate shows high pathogenicity. This study aimed to identify the definitive lesions that led to the lethal virulence of d5a20b by comparing the histopathology of 9a5b- or d5a20b-inoculated ducks in order to elucidate lesions related to the enhanced pathogenicity of certain NDV strains in ducks. Herein, 7-day-old ducks were intranasally inoculated with either 9a5b or d5a20b NDV strains. The neurological signs were more severe in the d5a20b-inoculated group than in the 9a5b-inoculated group. Ducks in the d5a20b-inoculated group exhibited more severe lymphoid depletion in immune organs than those in the 9a5b-inoculated group, which may have caused an immunosuppressive state in the d5a20b-inoculated ducks. Ducks in the d5a20b-inoculated group had more severe nonsuppurative encephalitis with increased NDV nucleoprotein than those in the 9a5b-inoculated group. Additionally, pancreatic necrosis, with intralesional NDV nucleoprotein, was more severe in the d5a20b-inoculated group than in the 9a5b-inoculated group. Our results showed that the immune organs, brain, and pancreas were significant targets of the NDV d5a20b infection in ducks.

Anticancer effect of the oncolytic Newcastle disease virus harboring the PTEN gene on glioblastoma.

Glioblastoma (GBM) is one of the most lethal types of human brain cancer and is characterized by rapid growth, an aggressive nature and a poor prognosis. GBM is highly heterogeneous, and often involves several genetic mutations and abnormalities. Genetic disorders or low expression of phosphatase and tensin homolog (PTEN) are associated with GBM occurrence, progression and poor prognosis of patients with GBM. However, effective delivery of PTEN for expression in GBM cells within the brain remains challenging. The aim of the present study was to develop a therapeutic strategy to restore PTEN expression in GBM cells by utilizing a recombinant Newcastle disease virus (rNDV) vector expressing the human PTEN gene (rNDV-PTEN). Methods included infection of U87-MG cells with rNDV-PTEN, followed by assessments of PTEN expression, and cell proliferation, migration and apoptosis. Additionally, an orthotopic GBM mouse model was used to evaluate the in vivo efficacy of rNDV-PTEN. Infection with recombinant rNDV-PTEN treatment increased PTEN protein expression in the cytoplasm of the U87-MG cells, reduced cell proliferation and migration, and induced apoptosis by inhibiting the AKT/mTOR signaling pathway. In the orthotopic GBM mouse model, rNDV-PTEN significantly reduced tumor size and improved survival rates. Magnetic resonance imaging and in vivo imaging analyses confirmed the targeted delivery and efficacy of rNDV-PTEN. These findings highlight the usefulness of rNDV-PTEN as a promising therapeutic agent for GBM, representing a potential advancement in treatment, especially for patients with PTEN deficiency.

Evaluation of the Efficacy of Three Newcastle Disease Vaccines Produced at the National Veterinary Institute, Bishoftu, Ethiopia, at Different Temperature Storage Conditions

Evaluation of the Efficacy of Three Newcastle Disease Vaccines Produced at the National Veterinary Institute, Bishoftu, Ethiopia, at Different Temperature Storage Conditions

Comparative Analysis of Newcastle Disease Seroprevalence in Different Breeds of Pigeon Populations in Chattogram Bangladesh

This study was performed to determine the antibody against Newcastle disease and serum protein level in pigeons of Chattogram, Bangladesh. The results were evaluated in pigeons of different groups of age, sex, and breed by applying the hemagglutination inhibition (HI) test. A total of 90 pigeons were selected for this research. Antibody titer against Newcastle Disease virus was analyzed according to the different age groups, sex, breeds and clinical signs of ND infection of pigeons by X2 test. Incase of less than 6 month age pigeons 73% (n=22) were HI titer positive whereas more than 6 months but less than 1 year 56% (n=32) were HI negative. There was no significant variation among different sex. In both male and female pigeon groups, the percentage of HI titer-positive pigeons in females and males was 51.7% and 57.4% respectively. There was significant variation among the different breeds and equal percentages of HI titer positive and negative pigeons were found in Giribaj and local breeds whereas 100% were found in King and Owl breeds and 80% were found in the Siraji breed. There was significant variation among NDV-infected and non-infected pigeons and equal percentage of HI titer negative and positive pigeons were found in ND non- infected, whereas (80.8%) were found in ND infected pigeons. A paired test was done between HI titer and total serum protein. There was a significant (t&lt;0.0) difference among the group. In the case of HI titer mean was about 23 titer and the standard error was 0.28. In the case of total serum protein mean was 4.45 and the standard error was 0.19.

Comparative Analysis of Newcastle Disease Seroprevalence in Different Breeds of Pigeon Populations in Chattogram, Bangladesh

This study was performed to determine the antibody against Newcastle disease and serum protein level in pigeons of Chattogram, Bangladesh. The results were evaluated in pigeons of different groups of age, sex, and breed by applying the hemagglutination inhibition (HI) test. A total of 90 pigeons were selected for this research. Antibody titer against Newcastle Disease virus was analyzed according to the different age groups, sex, breeds, and clinical signs of ND infection of pigeons by X2 test. In the case of fewer than 6 months of age pigeons, 73% (n=22) were HI titer positive whereas more than 6 months but less than 1 year 56% (n=32) were HI negative. There was no significant variation among the different sexes. In both male and female pigeon groups, the percentage of HI titer-positive pigeons in females and males was 51.7% and 57.4% respectively. There was significant variation among the different breeds and equal percentages of HI titer positive and negative pigeons were found in Giribaj and local breeds whereas 100% were found in King and Owl breeds and 80% were found in the Siraji breeds. There was significant variation among NDV-infected and non-infected pigeons and an equal percentage of HI titer negative and positive pigeons were found in ND non-infected, whereas (80.8%) were found in ND-infected pigeons. A paired test was done between HI titer and total serum protein. There was a significant (t&lt;0.0) difference among the groups. In the case of HI titer mean was about 23 titer and the standard error was 0.28. In the case of total serum protein mean was 4.45 and the standard error was 0.19.

Adipose tissue responds to stress-induced immunosuppression affecting immune response partially by miR-145-5p/S1PR1 pathway

Stress-induced immunosuppression (SIIS) is one of the most common problems in intensive poultry production, which can cause immunized chickens to still develop diseases and bring huge losses to production. Recently, adipose tissue, as an immunomodulatory organ, has become a hot topic of attention. However, the function and mechanism of adipose tissue involved in SIIS and its influence on the immune response are still unclear. In this study, we dynamically analyzed the correlations between the T cells migration and change of sphingosine-1-phosphate receptor 1(S1PR1) gene in adipose tissue using chicken models with different immune states, and further explored the regulatory mechanisms and application. The results showed that SIIS could significantly change the expressions of lymphocytes migration related S1PR1 gene, and SIIS could inhibit the Newcastle disease virus (NDV) immune response partially by affecting the migration and proliferation of TCRα+ T cells in adipose tissue. Moreover, the miR-145-5p/S1PR1 pathway was a potential key mechanism to regulate T cells migration in adipose tissue, and circulating miR-145-5p had potential value as a molecular marker. This research can provide innovative reference for in-depth studying the immunoregulatory function and mechanism of adipose tissue.

Newcastle disease virus enhances the antitumor efficacy of Doxorubicin in a cervical cancer mouse model

Background and aimsCervical cancer (CC) is a common cancer among women, often treated with Doxorubicin (Doxo). Research is underway to explore the use of oncolytic virus (OV) therapy as a means to improve drug efficacy and enhance the immune system’s tumor-fighting capabilities. Hence, our study purposes to evaluate the therapeutic potential of Newcastle disease virus (NDV) in increasing the antitumor efficacy of Doxo in mouse models of CC.Methods and materialsTC1 cells were administered to C57BL/6 mice (Female) in a range of 6 to 8 weeks age (n = 40) to induce tumor growth. After tumor development, four treatment groups of mice were formed. Treatment were performed through NDV, Doxo, and a combination of both in three groups of treatment twice in a one-week intervention manner, while the control group treated with PBS. Following the last treatment, half of these mice were subjected to euthanize due to the immune-response assessment, and the other half were followed up till they died naturally in a certain period of time.ResultsMice that underwent the combined treatment showed significantly improved survival rates and slower tumor progression in comparison with the control group. This combined treatment substantially elevated nitric oxide (NO) and lactate dehydrogenase (LDH) levels in the splenocytes cultures of mice bearing cervical tumors. Furthermore, combination therapy resulted in a notable elevation in TNF-α, IL-12, and IFN-γ, secretion alongside a reduction in the release of TGF-β and IL-4 within the splenocytes in counter with the treatment of just NDV or Doxo.ConclusionAccording to the findings of this study, it seems that utilizing NDV can improve the effectiveness of Doxo in a mouse model of CC, suggesting it can serve as an adjunct therapy alongside chemotherapy.

Assessment of PPMV-1 Genotype VI Virulence in Pigeons and Chickens and Protective Effectiveness of Paramyxovirus Vaccines in Pigeons.

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.

Enhanced Oncolytic Potential of Engineered Newcastle Disease Virus Lasota Strain through Modification of Its F Protein Cleavage Site.

Newcastle disease virus (NDV) is an oncolytic virus whose F protein cleavage activity is associated with viral infectivity. To explore the potential of modifying F protein cleavage activity to enhance antitumor effects, we constructed a recombinant NDV LaSota strain by replacing its F protein cleavage site with that from the mesogenic Beaudette C (BC) strain using reverse genetics techniques. The resulting virus, rLaSota-BC-RFP, demonstrated significantly enhanced infectivity and tumor cell suppression on the murine melanoma B16F10 cell, characterized by higher cytotoxicity and increased apoptosis compared to its parental strain, rLaSota-RFP. In vivo, rLaSota-BC-RFP treatment of B16F10 tumors in C57BL/6 mice resulted in significant tumor growth inhibition, improved survival rate, and induction of tumor-specific apoptosis and necrosis. Additionally, the rLaSota-BC-RFP treatment enhanced immunostimulatory effects within the tumor microenvironment (TME), characterized by increased infiltration of CD4+ and CD8+ T cells and elevated levels of antitumor immune modulator cytokines, including mouse IL-12, IFN-γ, IL-15, and TNF-α, in the rLaSota-BC-RFP-treated tumor tissues. Collectively, these findings demonstrate that the mesogenic F protein cleavage site enhances the oncolytic potential of the NDV LaSota strain, suggesting that rLaSota-BC-RFP is a promising oncolytic viral vector for gene delivery in cancer immunotherapy.

In-silico Evaluation of Moringa Oleifera Phytochemicals as Inhibitors Against Newcastle Disease Virus in Birds

Newcastle disease (ND) is a highly contagious viral infection that significantly impacts the global poultry industry, leading to substantial economic losses due to high mortality rates. Despite the availability of vaccines, the persistent threat of ND necessitates the exploration of novel therapeutic strategies. Moringa oleifera, commonly known as the "miracle tree," is renowned for its diverse pharmacological properties, including antiviral, antioxidant, and immunomodulatory effects. This study investigates the potential of Moringa oleifera phytochemicals as inhibitors of the Newcastle disease virus (NDV) through molecular docking analysis. The research focuses on identifying bioactive compounds within Moringa oleifera that exhibit strong binding affinities to the fragment of fusion protein of NDV, a critical component in the virus's ability to infect host cells. By employing in silico methods, the study predicts the efficacy of these compounds in disrupting NDV activity, offering a promising new avenue for ND treatment and prevention. The findings suggest that specific phytochemicals, such as Beta Sitosterol, catechins, and Kaempferol, demonstrate significant binding affinities, (with binding scores of -8.5 kcal/mol, -8.2 kcal/mol and -7.9 kcal/mol respectively), making them potential candidates for further development into antiviral therapies. This approach underscores the potential of Moringa oleifera in enhancing the resilience of the poultry industry against Newcastle disease, thereby contributing to improved food security and economic stability.

RAA-CRISPR/Cas12a-Mediated Rapid, Sensitive, and Onsite Detection of Newcastle Disease in Pigeons.

Pigeon Newcastle disease, caused by pigeon paramyxovirus type 1 (PPMV-1), is a significant infectious disease in pigeons that can result in substantial mortality and poses a severe threat to the pigeon industry. The rapid and accurate onsite diagnosis of pigeon disease is crucial for timely diagnosis and the implementation of effective prevention and control measures. In this study, we established a rapid detection method for PPMV-1 based on recombinase-aided amplification (RAA) and CRISPR/Cas12a. The RAA primers target the conserved regions of the L gene for preamplification in clinical nucleic acid samples, followed by CRISPR/Cas12a detection of the target gene. Visualization could be achieved by combination with a lateral flow dipstick (LFD). This method demonstrated high specificity, showing no cross-reactivity with non-PPMV-1 samples. The sensitivity of the method assessed by fluorescence analysis reached 100 copies/µL, and when it was combined with an LFD, the sensitivity was 103 copies/µL. The constructed RAA-CRISPR/Cas12a-LFD visual detection method was applied to clinical sample testing and was found to enable the rapid and accurate detection of swab samples and tissue specimens. Its sensitivity was consistent with the current gold standard, quantitative real-time PCR results. The RAA-CRISPR/Cas12a-LFD detection method we developed provides a novel approach for the rapid, simple, precise, and specific onsite diagnosis of pigeon Newcastle disease.

Voltammetric-based immunosensing of Newcastle disease virus on polyethylene glycol-containing self-assembled monolayer modified gold electrode

A voltammetric immunosensor for the detection of Newcastle disease virus (NDV) has been developed by employing polyclonal antibody targeting NDV (anti-NDV) as a bioreceptor. Anti-NDV was immobilized on polyethylene glycol (PEG)-containing self-assembled monolayer (SAM) which was activated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimidehydrochloride (EDC) and N-hydroxy succinimide (NHS) coupling on screen-printed gold electrode (SPGE). The introduction of PEG-containing SAM on the SPGE allowed the bioreceptor to covalently bound to the electrode surface whilst still providing a hydrophilic layer on the electrode which is important to greatly reduce non-specific bindings. The bioreceptor functionalized electrode was then allowed to be incubated with NDV-spiked samples. The electrode surface modification with PEG-containing SAM, immobilization of anti-NDV as bioreceptor, up to the detection of NDV were characterized electrochemically through differential pulse voltammetry (DPV) analysis in [Fe(CN)6]3- as the redox probe. Decrement of anodic current peak (Ipa) of [Fe(CN)6]3- was seen as the concentration of NDV increased from 0.156 to 20 HA μL-1 with the limit of detection (LoD) of 1.50 HA μL-1 at 3σ m-1. The detection of NDV in HA μL-1 unit in this study would ease interlaboratory interpretation as it was the same unit used in hemagglutination (HA) assay of conventional NDV diagnosis. The specificity of anti-NDV used as bioreceptor towards NDV was confirmed through western blot analysis, whilst the selectivity of the bioreceptor-functionalized electrode has been tested with allantoic fluid as the negative control in which no apparent changes of anodic peak (Ipa) has been seen. This simple, fast, and less laborious electrochemical detection method could become an alternative to the conventional method for NDV detection.

Pongamia pinnata L. seed-derived karanjin as prominent antiviral agent against Newcastle disease virus

Current study aim to explore Karanjin (Kar) isolated from the seeds of Pongamia pinnata as a potential antiviral polyphenol against Newcastle disease virus (NDV) through comprehensive investigations. For the in vitro study, plaque assays, gene and protein expression were used to analyse the inhibitory impact of Kar on NDV, where it reduced NDV replication as shown by a 13-fold suppression of the hemagglutinin-neuraminidase (HN) gene and decrease about 60% in virus activity. In ovo study showed that Kar mitigates NDV effects in chicken embryos. In silico studies involving molecular docking and molecular dynamics simulations showed strong binding between Kar and HN protein of NDV. Kar also influenced glucose metabolism, enhancing antiviral responses, as showed by the upregulation of GLUT1 and HEX genes through RT-qPCR and HPLC analyses. The study contributes valuable insights for future investigations into therapeutic applications of Kar against viral infections.

Effects of shallot (Allium ascalonicum) powder supplementation on growth, intestinal morphology, immune stimulation, and intestinal bacteria in broiler chickens

Background and Aim: Antimicrobial resistance in poultry farms is a significant global public health concern that has led farmers to explore alternative antibiotics, such as prebiotics in poultry production. This study aimed to examine the antimicrobial activity of ethanolic shallot extract (ESE) and the effects of adding shallot powder (SP) to broiler feed on broiler growth, immune response to Newcastle disease virus (NDV) vaccination, and gastrointestinal tract bacteria. Materials and Methods: We determined the antimicrobial effects of ESE against Escherichia coli O157:H7 (EOH) and Lactobacillus acidophilus TISTR 2365 (L2365) using the agar well diffusion method. We used a complete randomized design to assign 120 1-day-old Arbor Acre chicks to six groups with four replicates of five broiler chickens over 42 days. The treatment groups were as follows: T1-basal diet (B) + NDV vaccination (positive control), T2-B (negative control), T3-B + 2 g SP per kg of feed, T4-B + 2 g SP per kg of feed + NDV, T5-B + 4 g SP per kg of feed, and T6-B + 4 g SP per kg of feed + NDV. Results: The minimum inhibitory concentrations of ESE on EOH and L2365 were 62.50 and 125.00 mg/mL, respectively. The body weight gain, average daily gain, and feed conversion ratio in the 4 g SP of T5 and T6 groups were significantly improved compared with the other groups (p &lt; 0.05). The immune organ (IO) and thymus gland weights in the T4 group were significantly greater than those observed in the positive and negative control groups (p &lt; 0.05). The IO weights of the bursae of Fabricius and spleen tended to be greater in the T4 group than in the other groups. T5 group broilers had the highest ratio of villus height to crypt depth. The humoral immunity titers against NDV vaccination were improved in the SP-supplemented groups compared with the non-supplemented groups (p &gt; 0.05). SP supplementation reduced the levels of coliform (p &lt; 0.05) and E. coli in the broiler intestine by adding 4 g of SP per kg of feed. However, L2365 was more tolerant to ESE in vitro and tended to increase in line with increased SP levels. Conclusion: ESE showed strong antimicrobial activity to reduce harmful bacteria, and SP supplementation may exhibit prebiotic effects to increase broiler chicken growth, immunity, and microbial balance. Keywords: broiler chickens, food security, immune stimulation, intestinal bacteria, intestinal morphology, shallot.