Newcastle Disease Research Articles

Development and application of quadruplex real time quantitative PCR method for differentiation of Muscovy duck parvovirus, Goose parvovirus, Duck circovirus, and Duck adenovirus 3.

Muscovy duck parvovirus (MDPV), Goose parvovirus (GPV), Duck circovirus, (DuCV) and Duck adenovirus 3 (DAdV-3) are important pathogens that cause high morbidity and mortality in ducks, causing huge economic loss for the duck industry. The present study, a quadruplex one-step real time quantitative PCR method for the detection of MDPV, GPV, DuCV, and DAdV-3 was developed. The results showed that assay had no cross-reactivity with other poultry pathogens [Duck plague virus (DPV), Duck tembusu virus (DTMUV), H6 avian influenza virus (H6 AIV), New duck reovirus (NDRV), Newcastle disease virus (NDV), H4 avian influenza virus (H4 AIV), Escherichia coli (E. coli), Muscovy duck reovirus (MDRV), Egg drop syndrome virus (EDSV), Pasteurella multocida (P. multocida)]. The sensitivity result showed that the limits of detection for MDPV, GPV, DuCV, and DAdV-3 were 10, 10, 1 and 10 copies/µl, respectively; The coefficients of variation intra- and inter-method was 1-2%; The range of linear (109 to 103 copies/µL) demonstrated the R2 values for MDPV, GPV, DuCV, and DAdV-3 as 0.9975, 0.998, 0.9964, and 0.996, respectively. The quadruplex real time quantitative PCR method efficiency was 90.30%, 101.10%, 90.72%, and 90.57% for MDPV, GPV, DuCV, and DAdV-3, respectively. 396 clinical specimens collected in some duck sausages from June 2022 to July 2023 were simultaneously detected using the established quadruplex real time quantitative PCR method and the reported assays. The detection rates for MDPV, GPV, DuCV, and DAdV-3 were 8.33% (33/396), 17.93% (71/396), 33.58% (133/396), and 29.04% (115/396), respectively. The agreement between these assays was greater than 99.56%. The developed quadruplex real-time quantitative PCR assay can accurately detect these four viruses infecting ducks, providing a rapid, sensitive, specific and accurate technique for clinical testing.

Intranasal Administration of Recombinant Newcastle Disease Virus Expressing SARS-CoV-2 Spike Protein Protects hACE2 TG Mice against Lethal SARS-CoV-2 Infection.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), emerged as a global outbreak in 2019, profoundly affecting both human health and the global economy. Various vaccine modalities were developed and commercialized to overcome this challenge, including inactivated vaccines, mRNA vaccines, adenovirus vector-based vaccines, and subunit vaccines. While intramuscular vaccines induce high IgG levels, they often fail to stimulate significant mucosal immunity in the respiratory system. We employed the Newcastle disease virus (NDV) vector expressing the spike protein of the SARS-CoV-2 Beta variant (rK148/beta-S), and evaluated the efficacy of intranasal vaccination with rK148/beta-S in K18-hACE2 transgenic mice. Intranasal vaccination with a low dose (106.0 EID50) resulted in an 86% survival rate after challenge with the SARS-CoV-2 Beta variant. Administration at a high dose (107.0 EID50) led to a reduction in lung viral load and 100% survival against the SARS-CoV-2 Beta and Delta variants. A high level of the SARS-CoV-2 spike-specific IgA was also induced in vaccinated mice lungs following the SARS-CoV-2 challenge. Our findings suggest that rK148/beta-S holds promise as an intranasal vaccine candidate that effectively induces mucosal immunity against SARS-CoV-2.

NDV targets the invasion pathway in malaria parasite through cell surface sialic acid interaction.

Merozoites utilize sialic acids on the red blood cell (RBC) cell surface to rapidly adhere to and invade the RBCs. Newcastle disease virus (NDV) displays a strong affinity toward membrane-bound sialic acids. Incubation of NDV with the malaria parasites dose-dependently reduces its cellular viability. The antiplasmodial activity of NDV is specific, as incubation with Japanese encephalitis virus, duck enteritis virus, infectious bronchitis virus, and influenza virus did not affect the parasite propagation. Interestingly, NDV is reducing more than 80% invasion when RBCs are pretreated with the virus. Removal of the RBC surface proteins or the NDV coat proteins results in disruption of the virus binding to RBC. It suggests the involvement of specific protein: ligand interaction in virus binding. We established that the virus engages with the parasitized RBCs (PRBCs) through its hemagglutinin neuraminidase (HN) protein by recognizing sialic acid-containing glycoproteins on the cell surface. Blocking of the HN protein with free sialic acid or anti-HN antibodies abolished the virus binding as well as its ability to reduce parasite growth. Interestingly, the purified HN from the virus alone could inhibit the parasite's growth in a dose-dependent manner. NDV binds strongly to knobless murine parasite strain Plasmodium yoelii and restricted the parasite growth in mice. Furthermore, the virus was found to preferentially target the PRBCs compared to normal erythrocytes. Immunolocalization studies reveal that NDV is localized on the plasma membrane as well as weakly inside the PRBC. NDV causes neither any infection nor aggregation of the human RBCs. Our findings suggest that NDV is a potential candidate for developing targeted drug delivery platforms for the Plasmodium-infected RBCs.

Cocirculation of Newcastle Disease Virus Genotypes XII and VII Along with Nonvirulent Forms Characterized in Domestic Birds from Peru.

Newcastle disease virus (NDV) is one of the most important pathogens affecting poultry, given its impact on health and production systems worldwide, despite widespread vaccination. Over the past 20 years, NDV has caused severe outbreaks of disease in Peru. These outbreaks primarily affected gamecocks and broiler chickens, with an additional reported case in commercial layers. Therefore, our objective was to identify and characterize the virus responsible for these cases in Peru. We analyzed 14 suspected clinical cases in domestic birds for NDV detection, isolation, and genetic characterization. Among these cases, seven involved gamecocks, with six genotype XII isolates and one genotype VII isolate, representing the first report of NDV genotype VII isolate from fighting roosters in Peru. Additionally, among the six cases in broiler chickens, we detected four genotype XII isolates and three genotype II isolates, including one sample containing both genotypes XII and II. Furthermore, a genotype I viral isolate was identified in a laying hen. Hence, we concluded that two divergent, highly virulent NDV genotypes, genotypes XII and VII, along with avirulent forms such as genotypes I and II are circulating among domestic birds in Peru. Genetic analysis indicates that these viruses are evolving locally within avian species and offers the basis necessary for vaccine adaptation to circulating viruses. Our results highlight the cocirculation of multiple virulent and nonvirulent NDV genotypes in domestic birds in Peru, underscoring the potential role of gamecocks as a viral source of virulent NDV strains in the country and the occurrence of outbreaks in poultry farms.

Synergistic cytotoxicity of olive leaf extract-loaded lipid nanocarriers combined with Newcastle disease virus against cervical cancer cells.

Despite recent medical progress, cervical cancer remains a major global health concern for women. Current standard treatments have limitations such as non-specific toxicity that necessitate development of safer and more effective therapeutic strategies. This research evaluated the combinatorial effects of olive leaf extract (OLE), rich in anti-cancer polyphenols, and the oncolytic Newcastle disease virus (NDV) against human cervical cancer cells. OLE was efficiently encapsulated (>94% loading) within MF59 lipid nanoparticles and nanostructured lipid carriers (NLCs; contains Precirol as NLC-P, contains Lecithin as NLC-L) to enhance stability, bioavailability, and targeted delivery. Physicochemical analysis confirmed successful encapsulation of OLE within nanoparticles smaller than 150 nm. In vitro cytotoxicity assays demonstrated significantly higher toxicity of the OLE-loaded nanoparticle formulations on HeLa cancer cells versus HDF normal cells (P<0.05). MF59 achieved the highest encapsulation efficiency, while NLC-P had the best drug release profile. NDV selectively infected and killed HeLa cells versus HDF cells. Notably, combining NDV with OLE-loaded nanoparticles led to significantly enhanced synergistic cytotoxicity against cancer cells (P<0.05), with NLC-P (OLE) and NDV producing the strongest effects. Apoptosis and cell cycle analyses confirmed the increased anti-cancer activity of the combinatorial treatment, which induced cell cycle arrest. This study provides evidence that co-delivery of OLE-loaded lipid nanoparticles and NDV potentiates anti-cancer activity against cervical cancer cells in vitro through a synergistic mechanism, warranting further development as a promising alternative cervical cancer therapy.

Microbiota but not immune modulation by a pro- and postbiotic was associated with the diet-additive interaction in broilers

This study investigated the diet-additive interactions of a Lactobacilli-based probiotic (Pro) and postbiotic (Post) on immune parameters and cecal microbiota composition, with subsequent effects on the metabolome in broilers. A completely randomized block design was employed with 2 diets [standard (SD), and challenge (CD)] and 3 additive conditions (Control, Pro, Post) involving 1,368 one-day-old male Ross 308 broilers equally distributed among 36 pens in a 42 d study. Diets were formulated to contain identical nutrient levels, with CD higher than SD in non-starch polysaccharide content by including rye and barley. Total non-specific serum Ig A, M and G concentrations were determined weekly from d14 to 35. Following vaccination, titres of specific antibodies binding Newcastle disease virus (NDV) and infectious bursal disease virus (IBDV) were measured. Microbiota composition was analyzed by 16S rRNA gene sequencing at d14 and 35, and α- and β-diversity indexes (Observed, Chao1, Bray, Jaccard) were calculated. Cecal short-chain fatty acids and the semi-polar metabolome were determined in the Control SD and all CD groups at d35. At d35, a diet-additive interaction was observed on cecal microbiota composition. Within SD, Pro and Post did not affect operational taxonomic units (OTU) abundance (adjusted-P > 0.05) and diversity indexes (P > 0.05). Within CD, Pro and Post affected the relative abundances of 37 and 44 OTUs, respectively (adjusted-P < 0.05), with Post but not Pro affecting β-diversity indexes (P = 0.041 and 0.064 for Bray and Jaccard, respectively). Within CD, Post increased cecal acetate (21%; P = 0.007) and butyrate (41%; P = 0.002) concentration and affected the concentration of 2 metabolites (adjusted-P < 0.05), while Pro affected 240 metabolites (adjusted-P < 0.05). No diet-additive interactions were observed on serum Ig (P > 0.05), except for IgM at d14 (P = 0.004). Diet composition, but not the additives, affected immune status parameters. The Pro and Post affected cecal microbiota composition only under dietary challenging conditions as previously reported for growth.

Genetic evolution of Newcastle Disease Virus sub-genotype VII.2 isolates, diagnosed from vaccinated poultry farms of Gujarat, India

Newcastle disease was suspected in 37 commercial poultry farms, including 12 layer and 25 broiler farms in four districts of Gujarat, India. Vaccination had been done in 32 (20 broilers and 12 layers) farms. Tissue samples from each farm were pooled as one sample. In egg embryo inoculation, HA-HI and PCR, respectively, 32/37, 29/37, and 24/37 samples were found positive. Pathotyping by mean death time calculation and primer combination PCR revealed velogenic NDV, which was later confirmed with the presence of the 112-RRQKR*F-117 sequence at the F protein cleavage site. Phylogenetic analysis of full F gene sequences (N=10) confirmed the presence of sub-genotype VII.2 in 9/10 sequences, and genotype II in one sample. These 9 sequences were only 0.7 to 2.6 % divergent with two VII.2 (=VIIi) sequences (HQ697254.1 chicken/Banjarmas/Indonesia and KU862293.1 Parakeet/Karachi/Pakistan) but had 2.2 to 3.6 % diversion from two VII.2 sequences (OR185447 and MZ546197) from India. Then branching was found from sequences of VIIh, VIIk (VII.2), and VIIa (VII.1.2), and then from sub-genotypes VII.1.1 and VII.1.2. Due to less than 5 % diversion, these sequences could not be qualified as new sub-genotype in evolutionary distance analysis. At the amino acid level, our sequences had aa N-T-I-A-L-T at 24–79-125–385-445–482. Whereas at the same positions, in most of the retrieved VII.2 sequences and vaccines, the sequence was S-A-V-T-Q/I- E/A. Two sequences revealed additional six and four amino acid differences,respectively.This indicates rapid continuous genetic evolution of sub-genotype VII.2 and partially explains vaccinal immunity escape.

Effect of oncolytic Newcastle disease virus harboring PTEN gene on pancreatic adenocarcinoma growth through inhibition of PI3K/mTOR signaling and promotion of apoptosis.

49 Background: Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive and intractable cancer, warranting the need for more effective therapies. Cell proliferation, progression, and severity in PDAC are highly activated owing to KRAS mutations and low PTEN expression. Methods: A recombinant Newcastle disease virus (rNDV) harboring PTEN (rNDV-PTEN) was constructed to infect PDAC cells to artificially induce PTEN expression. The cancer killing effect of the rNDV-PTEN virus was observed in an in vitro assay, and 107.0 pfu/dose(0.1 ml) of the rNDV-PTEN virus was inoculated through intravenous injection into a PANC-1 cell transplanted mouse to prove the tumor growth inhibition effect. Apoptotic signaling-related proteins were analyzed with Western blotting and qPCR assays based on the mRNA. During the treatment, biomarkers were also analyzed. Results: Factitious PTEN expression in KRAS-mutated PDAC cells following rNDV-PTEN infection lowered PI3K/AKT/mTOR signaling, induced PDAC cell death, and suppressed tumor growth. PTEN overexpression promoted apoptotic and autophagic signaling pathways in PANC-1 cells and orthotopic xenograft mice. rNDV-PTEN has substantial cancer cell-killing effects and inhibits tumor growth. rNDV-PTEN injection into animals did not elicit an immune response against rNDV and improved blood parameters, such as glucose, triglyceride, and total cholesterol levels. Conclusions: rNDV-PTEN showed a strong tumor-suppressive effect on PDAC, which was caused by an abnormally activated PI3K/AKT/mTOR signaling pathway due to KRAS mutations and PTEN loss. We also showed that rNDV-PTEN is relatively safe for use in cancer therapy. Further studies using patient-derived PDAC cells with the same genetic background, as well as more extensive animal safety tests, are required to demonstrate rNDV-PTEN as a PDAC therapeutic. However, PTEN gene-containing rNDV may be a very promising therapeutic candidate for pancreatic cancer treatment.

RT-PCR based detection and pathotypical characterization of Newcastle disease virus isolated from layer chickens in Mymensingh, Bangladesh

The Newcastle disease virus (NDV) poses a significant threat to commercial chicken operations and is prevalent in Bangladesh. This rapidly spreading viral illness has severe economic implications for the poultry industry globally. In Bangladesh, the velogenic strain of NDV is particularly widespread. This study utilized primers from the Fusion (F) and Matrix (M) genes along with haemagglutination (HA) testing and reverse transcription-polymerase chain reaction (RT-PCR) to isolate and identify NDV. Post-mortem examinations provided 12 samples (from brain, trachea, and proventriculus) taken from four birds suspected of having NDV from Trishal and Mymensingh Sadar. The samples were injected into 10-day-old embryonated chicken eggs using the chorioallantoic membrane technique. Embryos and allantoic fluid (AF) were collected at 48 and 96 hours post-infection. NDV-infected embryos showed edema and hemorrhage. Significant hemagglutination was observed in the allantoic fluid tested with HA. Through the HA test and lesion observation, four NDV isolates were identified. Pathotypic characterization of the field isolates using the Intracerebral Pathogenicity Index (ICPI) and Mean Death Time (MDT) indicated velogenic strains (ICPI: 1.5–2.0, MDT: &lt;60). RT-PCR confirmed the NDV status of all four isolates using primers for partial amplification of the F gene (839 bp) and the common sequence of the ‘F-M’ genes (970 bp). This study demonstrates that RT-PCR is an effective molecular technique for the rapid and confirmatory identification of velogenic NDV strains, particularly prevalent in Mymensingh at the field level.

A time series transcriptome profiling of host cell responses to Newcastle disease virus infection.

Newcastle disease virus (NDV), an avian paramyxovirus, causes major economic losses in the poultry industry worldwide. NDV strains are classified as avirulent, moderately virulent, or virulent according to the severity of the disease they cause. In order to gain a deeper understanding of the molecular mechanisms of virus-host interactions, we conducted Illumina HiSeq-based RNA-Seq analysis on chicken embryo fibroblast (DF1) cells during the first 24 hours of infection with NDV strain Komarov. Comparative analysis of uninfected DF1 cells versus NDV-infected DF1 cells at 6, 12, and 24h postinfection identified 462, 459, and 410 differentially expressed genes, respectively. The findings revealed an increase in the expression of genes linked to the MAPK signalling pathway in the initial stages of NDV infection. This overexpression potentially aids viral multiplication while hindering pathogen detection and subsequent immune responses from the host. Our findings provide initial insights into the early responses of DF1 cells to NDV infection.

A σC-protein-based indirect enzyme-linked immunosorbent assay for clinical detection of antiavian reovirus antibodies

Avian reovirus (ARV) is the causative agent of avian viral arthritis and causes significant economic losses to the global poultry industry. For clinical diagnosis, detecting ARV-specific antibodies is crucial. We successfully expressed the ARV-σC protein in insect cells using the baculovirus expression vector system, achieving an expression level of approximately 200 mg/L. We developed an indirect enzyme-linked immunosorbent assay (iELISA) using the ARV-σC protein as a coating antigen to detect antibodies against it. The inter-batch and intrabatch coefficients of iELISA variation were less than 10%. Its sensitivity (1:12,800 diluted in serum) was 4 times higher than that of the indirect immunofluorescence assay (IFA; 1:3200 diluted in serum), and it showed no cross-reactivity with antibodies against other common avian viruses (such as Infectious bursal disease virus, Newcastle disease virus). The practicality of the iELISA was further evaluated using clinical samples. 300 clinical sera from chickens vaccinated with the ARV attenuated vaccine and 20 SPF sera were tested using both the iELISA and the IFA, demonstrating a 100% conformity rate. In conclusion, these results suggest that the iELISA developed in this study is a rapid, sensitive, and specific method that could serve as an effective diagnostic tool for monitoring and controlling avian viral arthritis.

Molecular Cloning, Tissue Distribution and Antiviral Immune Response of Duck Src.

As a founding member of the Src family of kinases, Src has been confirmed to participate in the regulation of immune responses, integrin signaling, and motility. Ducks are usually asymptomatic carriers of RNA viruses such as Newcastle disease virus and avian influenza virus, which can be deadly to chickens. The beneficial role of Src in modulating the immune response remains largely unknown in ducks. Here, we characterized the duck Src and found that it contains a 192-base-pair 5' untranslated region, a 1602-base-pair coding region, and a 2541-base-pair 3' untranslated region, encoding 533 amino acid residues. Additionally, duSrc transcripts were significantly activated in duck tissues infected by Newcastle disease virus compared to controls. The duSrc transcripts were notably widespread in all tissues examined, and the expression level was higher in liver, blood, lung, pancreas, and thymus. Moreover, we found the expression levels of IFN-β, NF-κB, IRF3, and Src were significantly increased in DEFs after infection with 5'ppp dsRNA, but there was no significant difference before and after treatment in DF1 cells. Furthermore, overexpression of duSrc followed by stimulation with 5'ppp dsRNA led to an elevation of IFN-β levels. The SH3 and PTKc domains of duSrc contributed to promoting the activity of IFN-β and NF-κB in DEFs stimulated by 5'ppp dsRNA.

Impact of dietary arginine supplementation on immune responses and growth performance in Newcastle disease virus-infected broiler chicks.

Newcastle disease (ND) poses significant challenges within the poultry industry, leading to increased mortality rates, compromised growth, weakened immunity and elevated levels of inflammation. This study explores the potential of dietary arginine supplementation to ameliorate these adverse effects of ND, leveraging arginine's well-documented benefits in enhancing growth and immune responses. A total of 480 one-day-old male broiler chicks were meticulously categorised into eight groups, encompassing both infected and noninfected cohorts. These chicks received diets with arginine levels at 85%, 100%, 125% and 150% of recommended standards. The study entailed a comprehensive examination of clinical manifestations, growth performance metrics, haemagglutination inhibition (HI) test results, and serum concentrations of proinflammatory cytokines, adrenocorticotropic hormone (ACTH), and cortisol (CORT). The infection significantly curtailed feed consumption (p = 0.0001) and weight gain (p = 0.0001) while concurrently depressing HI titres. Additionally, infected chicks experienced an exacerbated feed conversion ratio (p = 0.0001), escalated mortality rates (p = 0.0001), and elevated serum concentrations of proinflammatory cytokines (p = 0.0001), ACTH (p = 0.0001), and CORT (p = 0.0001). Remarkably, dietary arginine supplementation effectively mitigated the adverse impacts of ND infection on growth, immune responses and proinflammatory cytokine levels. In the context of ND infection, mortality rates and inflammation surge, while growth and immunity are significantly compromised. The strategic inclusion of arginine in the diet emerges as a potent strategy to counteract the deleterious effects of ND. Supplementation with arginine at levels exceeding the conventional dietary recommendations is recommended to alleviate the detrimental consequences of ND effectively.

Evaluation of the immune responses of biological adjuvant bivalent vaccine with three different insertion modes for ND and IBD

ABSTRACT Infectious bursal disease (IBD) is a widespread problem in the poultry industry, and vaccination is the primary preventive method. However, moderately virulent vaccines may damage the bursa, necessitating the development of a safe and effective vaccine. The Newcastle disease virus (NDV) has been explored as a vector for vaccine development. In this study, reverse genetic technology was used to obtain three recombinant viruses, namely, rClone30-VP2L (P/M)-chGM-CSF (NP), rClone30-chGM-CSF (P/M)-VP2L (NP), and rClone30-VP2L-chGM-CSF (P/M). Animal experiments showed that the three biological adjuvant bivalent vaccines effectively increased anti-NDV and anti-infectious bursal disease virus (IBDV) titres, enhancing both humoral and cellular immune responses in chickens without leading to any harm. Amongst the three biological adjuvant bivalent vaccines, the rClone30-chGM-CSF (P/M)-VP2L (NP) group had higher levels of anti-NDV antibodies at 14 days after the first immunization and stimulated a greater humoral immune response in 7–10 days. While, the rClone30-VP2L (P/M)-chGM-CSF (NP) group was the most effective in producing a higher level of IBDV antibody response. In conclusion, these three vaccines can induce immune responses more rapidly and effectively, streamline production processes, be cost-effective, and provide a new avenue for the development of Newcastle disease (ND) and IBD bivalent vaccines

Detection and differentiation of low virulence and virulent Orthoavulavirus javaense using a molecular beacon with RT-LAMP

Newcastle disease (ND), an economically important disease in poultry, is caused by virulent strains of the genetically diverse Orthoavulavirus javaense (OAVJ). Laboratories rely on quantitative real-time reverse transcription PCR (qRT-PCR) to detect OAVJ and differentiate between OAVJ pathotypes. This study demonstrates that a fusion cleavage site based molecular beacon with reverse transcription loop mediated isothermal amplification (MB-RT-LAMP) assay can detect and differentiate OAVJ pathotypes in a single assay. Data show that the assay can rapidly identify diverse OAVJ genotypes with sensitivity only one log-fold lower than the current fusion qRT-PCR assay (104 copies), exhibits a high degree of specificity for OAVJ, and the molecular beacon can differentiate mesogenic/velogenic sequences from lentogenic sequences. Further, data show that a two-minute rapid lysis protocol preceding MB-RT-LAMP can detect and differentiate OAVJ RNA from both spiked samples and oropharyngeal swabs without the need for RNA isolation. As the MB-RT-LAMP assay can rapidly detect and discriminate between lentogenic and mesogenic/velogenic sequences of OAVJ within one assay, without the need for RNA isolation, and is adaptable to existing veterinary diagnostic laboratory workflow without additional equipment, this assay could be a rapid primary screening tool before qRT-PCR based validation in resource limited settings.

Isolation and Genetic Characterization of Genotype VII Velogenic Pathotype Newcastle Disease Virus from Commercial Chicken Farms in Central Ethiopia, Distinct from the Local Vaccine Strains.

Newcastle disease (ND) is caused by virulent strains of avian paramyxovirus type 1, also known as Newcastle disease virus (NDV). Despite vaccination, the frequency of reported outbreaks in Ethiopia has increased. From January to June 2022, an active outbreak investigation was conducted in six commercial chicken farms across areas of central Ethiopia to identify the circulating NDV strains. Thirty pooled tissue specimens were collected from chickens suspected of being infected with NDV. A questionnaire survey of farm owners and veterinarians was also carried out to collect information on the farms and the outbreak status. NDV was isolated using specific-pathogen-free (SPF)-embryonated chicken eggs and detected using haemagglutination and the reverse transcriptase-polymerase chain reaction (RT-PCR). The genotype and virulence of field NDV isolates were determined using phylogenetic analysis of fusion (F) protein gene sequences and the mean death time (MDT) test in SPF-embryonated chicken eggs. The questionnaire results revealed that ND caused morbidity (23.1%), mortality (16.3%), case fatality (70.8%), and significant economic losses. Eleven of thirty tissue specimens tested positive for NDV using haemagglutination and RT-PCR. The MDT testing and sequence analysis revealed the presence of virulent NDV classified as genotype VII of class II velogenic pathotype and distinct from locally used vaccine strains (genotype II). The amino acid sequences of the current virulent NDV fusion protein cleavage site motif revealed 112RRQKR↓F117, unlike the locally used avirulent vaccine strains (112GRQGR↓L117). The epidemiological data, MDT results, cleavage site sequence, and phylogenetic analysis all indicated that the present NDV isolates were virulent. The four NDV sequences were deposited in GenBank with accession numbers F gene (PP726912-15) and M gene (PP726916-19). The genetic difference between avirulent vaccine strains and circulating virulent NDV could explain the low level of protection provided by locally used vaccines. Further studies are needed to better understand the circulating NDV genotypes in different production systems.

Immune Responses in the Harderian Gland After Newcastle Disease Vaccination in Chickens with Maternal Antibodies.

Immune responses in the Harderian gland (HG) were characterized after Newcastle disease virus (NDV) LaSota ocular vaccination in antibody-naïve specific-pathogen-free (SPF) chickens and in chickens of commercial origin with NDV maternally derived antibodies (MDA). Ocular LaSota vaccination of 13-day-old white leghorn SPF chickens elicited serum antibody levels that consistently increased 15 days postvaccination, while the specific IgA response in lacrimal fluids was already detectable 10 days after vaccination. Eleven days postvaccination, the relative abundance of B cells, as well as T-helper cells (CD4+) and cytotoxic T cells (CD8+), in HGs was significantly increased, achieving maximum frequencies 16 days postvaccination. In a second experiment, chickens with MDA originating from NDV-vaccinated commercial white leghorn layer breeders, as well as white leghorn SPF chickens, were vaccinated with NDV LaSota. The LaSota virus successfully replicated in periocular tissues and in the trachea both in commercial and control SPF chickens after vaccination at 2 or 15 days of age (DOA). Vaccination at 2 DOA did not induce a serum NDV antibody response in chickens of commercial origin. In contrast, seroconversion was elicited in commercial chickens upon vaccination at 15 DOA, likely associated with waning of MDA. Unlike systemic IgG responses, vaccination at 2 or 15 DOA elicited strong specific IgA responses in lacrimal fluids in commercial chickens. The IgA response was highest 9 days after vaccination and showed a tendency to decline 15 days postvaccination. Commercial chickens vaccinated at 2 DOA showed increased B cells in HG 10 and 16 days postvaccination. The expansion of B cells in the HG in these chickens is consistent with increased IgA levels detected in lacrimal fluids. In contrast, control SPF chickens showed a more limited B-cell expansion in HG and lower IgA levels. Vaccination at 15 DOA also triggered a greater increase of B cells in HGs in commercial chickens than in control SPF chickens. The B-cell response was accompanied by T-helper (CD4+) cell expansion, occurring both in commercial and control SPF chickens. These cells expanded to a lesser extent when vaccination was performed at 2 DOA compared with vaccination at 15 DOA. CD8+ showed significant expansion irrespective of vaccination day and without differences detected between control SPF chickens and chickens with MDA. We conclude that NDV LaSota elicits vigorous humoral and cell immune responses in the HG. Furthermore, unlike the interference shown by MDA on vaccine-induced serum antibody responses, MDA do not interfere with the mucosal immune response of the HG.

PEX5-mediated modulation of apoptotic pathways in response to Newcastle disease virus infection1

Newcastle disease virus (NDV) is a highly lethal and contagious viral pathogen; it is also a potent oncolytic virus that selectively replicates in tumor cells. NDV demonstrates high replication efficiency in avian and tumor cells, causing various types of cell death, including ferroptosis, necrosis, apoptosis and autophagic cell death, with apoptosis being the most thoroughly studied. Organelles play critical and distinctive roles in the regulation and execution of apoptosis. However, the involvement of peroxisomes, an important organelle that regulates redox balance and lipid biosynthesis, in virus-induced apoptosis remains unclear. Our findings reveal that NDV infection promotes the downregulation of several peroxisome biogenesis factors (PEXs) at the mRNA level. Peroxisomal biogenesis factor 5 (PEX5), a critical peroxisomal shuttle protein, was identified to be significantly downregulated at both the mRNA and protein levels. Further, gain- and loss-of-function experiments demonstrated the negative regulation of NDV-induced apoptosis by PEX5. In addition, PEX5 inhibits NDV-induced apoptosis by regulating the anti-apoptotic protein B-cell lymphoma-2 (Bcl-2) expression. These findings reveal a novel mechanism by which NDV-induced apoptosis is modulated through the downregulation of PEXs, particularly PEX5, shedding light on the potential role of peroxisome in apoptosis regulation in response to virus infection.

Arginyltransferase 1 (ATE1)-mediated proteasomal degradation of viral haemagglutinin protein: a unique host defence mechanism.

The extensive protein production in virus-infected cells can disrupt protein homeostasis and activate various proteolytic pathways. These pathways utilize post-translational modifications (PTMs) to drive the ubiquitin-mediated proteasomal degradation of surplus proteins. Protein arginylation is the least explored PTM facilitated by arginyltransferase 1 (ATE1) enzyme. Several studies have provided evidence supporting its importance in multiple physiological processes, including ageing, stress, nerve regeneration, actin formation and embryo development. However, its function in viral pathogenesis is still unexplored. The present work utilizes Newcastle disease virus (NDV) as a model to establish the role of the ATE1 enzyme and its activity in pathogenesis. Our data indicate a rise in levels of N-arginylated cellular proteins in the infected cells. Here, we also explore the haemagglutinin-neuraminidase (HN) protein of NDV as a presumable target for arginylation. The data indicate that the administration of Arg amplifies the arginylation process, resulting in reduced stability of the HN protein. ATE1 enzyme activity inhibition and gene expression knockdown studies were also conducted to analyse modulation in HN protein levels, which further substantiated the findings. Moreover, we also observed Arg addition and probable ubiquitin modification to the HN protein, indicating engagement of the proteasomal degradation machinery. Lastly, we concluded that the enhanced levels of the ATE1 enzyme could transfer the Arg residue to the N-terminus of the HN protein, ultimately driving its proteasomal degradation.

Status and future prospects for mobile phone-enabled diagnostics in Tanzania.

Diagnosis is a key step towards the provision of medical intervention and saving lives. However, in low- and middle-income countries, diagnostic services are mainly centralized in large cities and are costly. Point of care (POC) diagnostic technologies have been developed to fill the diagnostic gap for remote areas. The linkage of POC testing onto smartphones has leveraged the ever-expanding coverage of mobile phones to enhance health services in low- and middle-income countries. Tanzania, like most other middle-income countries, is poised to adopt and deploy the use of mobile phone-enabled diagnostic devices. However, there is limited information on the situation on the ground with regard to readiness and capabilities of the veterinary and medical professionals to make use of this technology. In this study we survey awareness, digital literacy and prevalent health condition to focus on in Tanzania to guide development and future implementation of mobile phoned-enable diagnostic tools by veterinary and medical professionals. Data was collected using semi-structured questionnaire with closed and open-ended questions, guided in-depth interviews and focus group discussion administered to the participants after informed consent was obtained. A total of 305 participants from six regions of Tanzania were recruited in the study. The distribution of participants across the six regions was as follows: Kilimanjaro (37), Arusha (31), Tabora (68), Dodoma (61), Mwanza (58), and Iringa (50). Our analysis reveals that only 48.2% (126/255) of participants demonstrated significant awareness of mobile phone-enabled diagnostics. This awareness varies significantly across age groups, professions and geographical locations. Interestingly, while 97.4% of participants own and can operate a smartphone, 62% have never utilized their smartphones for health services, including disease diagnosis. Regarding prevalent health condition to focus on when developing mobile phone -enabled diagnostics tools for Tanzania; there was disparity between medical and veterinary professionals. For medical professionals the top 4 priority diseases were Malaria, Urinary Tract Infections, HIV and Diabetes, while for veterinary professionals they were Brucellosis, Anthrax, Newcastle disease and Rabies. Despite the widespread ownership of smartphones among healthcare providers (both human and animal), only a small proportion have utilized these devices for healthcare practices, with none reported for diagnostic purposes. This limited utilization may be attributed to factors such as a lack of awareness, absence of policy guidelines, limited promotion, challenges related to mobile data connectivity, and adherence to cultural practices. The majority of medical and veterinary professionals in Tanzania possess the necessary digital literacy to utilize mobile phone-enabled diagnostics and demonstrate readiness to adopt digital technologies and innovations to enhance diagnosis. However, effective implementation will require targeted training and interventions to empower them to effectively apply such innovations for disease diagnosis and other healthcare applications.