Virulent Newcastle Disease Virus Research Articles

Mitigating impact of Glycyrrhiza glabra on virulent Newcastle disease virus challenge in chickens: clinical studies, histopathological alterations and molecular docking.

Newcastle disease (ND) is widely regarded as one of the most virulent and destructive viral infections that create chaos in the poultry industry and cause widespread epidemics and consequentially debilitating economic losses on a global scale in terms of chicken products. The current experiment evaluates the protective effect of Glycyrrhiza glabra ( G. glabra) against the Newcastle disease virus (NDV) in chickens. Ninety (90) 1-day-old SPF chicks were treated according to ethical approval (BUFVTM 05-02-22) as follows (1) non-treated non-challenged control group; (2) NDV group: Challenged with genotype VII ND virus; and (3) LE/NDV group: Challenged with the virus and intermittently treated with powdered extract of G. glabra roots (LE) in drinking water (0.5g/L) before and after viral challenge. The water medication of NDV-challenged chicks has resulted in a significant decrease in the severity of clinical symptoms, morbidity, and mortality rates, as well as the quantity of virus shed, compared with the NDV group. Treatment with LE has led to a significant reduction in serum ALT and AST activities, blood glucose level, urea, and creatinine, and significant restoration of serum proteins. In addition, the treatment has resulted in a decrease in MDA and NO levels, as well as an increase in T-SOD and catalase activities compared with untreated challenged chicks. LE decreased IFN-γ and TLR-3 gene expression in comparison with the NDV group. The treated challenged birds had fewer macroscopically detectable lesions in their respiratory, digestive, and lymphoid organs than the untreated challenged birds. Microscopically, the LE/NDV group exhibited mild to moderate pathological changes in the respiratory and digestive systems as well as lymphoid tissues, in contrast to the NDV group, which exhibited severe pathological changes. Furthermore, molecular docking assessment proved the efficacy of G. glabra against viral proliferation and invasion. We concluded that Glycyrrhiza glabra powdered extract at a dose of 0.5g/L drinking water can effectively mitigate the debilitating effects of Newcastle disease in chickens.

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.

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.

Long-Term Protection against Virulent Newcastle Disease Virus (NDV) in Chickens Immunized with a Single Dose of Recombinant Turkey Herpesvirus Expressing NDV F Protein.

Newcastle disease (ND) is a significant infectious disease in poultry, causing substantial economic losses in developing countries. To control ND, chickens must be vaccinated multiple times a year. In order to develop an improved vaccine that provides long-term protection, the F gene from genotype VII NDV was inserted into the herpesvirus of turkey (HVT) vaccine virus using CRISPR/Cas9-mediated NHEJ repair and Cre/LoxP technology. The immunogenicity and protective efficacy of the resulting recombinant vaccines were evaluated through antibody assays and virus challenge experiments. Two recombinant vaccines, rHVT-005/006-F and rHVT-US2-F, were generated, both exhibiting growth rates comparable with those of HVT in vitro and consistently expressing the F protein. One-day-old specific pathogen-free (SPF) chickens immunized with 2000 PFU/bird of either rHVT-005/006-F or rHVT-US2-F developed robust humoral immunity and were completely protected against challenge with the NDV F48E8 strain at 4 weeks post-vaccination (wpv). Furthermore, a single dose of these vaccines provided sustained protection for at least 52 wpv. Our study identifies rHVT-005/006-F and rHVT-US2-F as promising ND vaccine candidates, offering long-term protection with a single administration. Moreover, HVT-005/006 demonstrates promise for accommodating additional foreign genes, facilitating the construction of multiplex vaccines.

Long Non-Coding RNA Analysis: Severe Pathogenicity in Chicken Embryonic Visceral Tissues Infected with Highly Virulent Newcastle Disease Virus-A Comparison to the Avirulent Vaccine Virus.

There are significant variations in pathogenicity among different virulent strains of the Newcastle disease virus (NDV). Virulent NDV typically induces severe pathological changes and high mortality rates in infected birds, while avirulent NDV usually results in asymptomatic infection. Currently, the understanding of the specific mechanisms underlying the differences in host pathological responses and symptoms caused by various virulent NDV strains remains limited. Long non-coding RNA (lncRNA) can participate in a range of biological processes and plays a crucial role in viral infection and replication. Therefore, this study employed RNA-Seq to investigate the transcriptional profiles of chicken embryos' visceral tissues (CEVTs) infected with either the virulent NA-1 strain or avirulent LaSota strain at 24 hpi and 36 hpi. Using bioinformatic methods, we obtained a total of 2532 lncRNAs, of which there were 52 and 85 differentially expressed lncRNAs at 24 hpi and 36 hpi, respectively. LncRNA analysis revealed that the severe pathological changes and symptoms induced by virulent NDV infection may be partially attributed to related target genes, regulated by differentially expressed lncRNAs such as MSTRG.1545.5, MSTRG.14601.6, MSTRG.7150.1, and MSTRG.4481.1. Taken together, these findings suggest that virulent NDV infection exploits the host's metabolic resources and exerts an influence on the host's metabolic processes, accompanied by excessive activation of the immune response. This impacts the growth and development of each system of CEVTs, breaches the blood-brain barrier, inflicts severe damage on the nervous system, and induces significant lesions. These observations may be attributed to variations in pathology. Consequently, novel insights were obtained into the intricate regulatory mechanisms governing NDV and host interactions. This will aid in unraveling the molecular mechanisms underlying both virulent and avirulent forms of NDV infection.

Thermostability study of virulent Newcastle disease viruses isolated in Southern Angola.

Newcastle disease (ND) is endemic in Angola. Several outbreaks of ND occurred in small backyard flocks and village chickens with high mortality in the southern provinces of the country, Cunene, Namibe and Huíla, in 2016 and 2018. In those years, 15 virulent ND virus (NDV) strains were isolated and grouped within subgenotype 2 of genotype VII (subgenotype VII.2). We now present a study on the thermostability of the isolates, aiming at the selection of the most thermostable strains that, after being genetically modified to reduce their virulence, can be adapted to the production of vaccines less dependent on cold chain and more adequate to protect native chickens against ND. Heat-inactivation kinetics of haemagglutinin (Ha) activity and infectivity (I) of the isolates were determined by incubating aliquots of virus at 56 °C for different time intervals. The two isolates from Namibe province showed a decrease in infectivity of 2 log10in ≤ 10 min, therefore belonging to the I-phenotype, but while the NB1 isolate from 2016 maintained the Ha activity up to 30 min and was classified as thermostable virus (I-Ha+), the Ha activity of the 2018 NB2 isolate decreased by 2 log2in 30 min, being classified as a thermolabile virus (I-Ha-). Of the 13 NDV isolates from Huíla province, 10 isolates were classified as thermostable, eight with phenotype I+Ha+and 2 with phenotype I-Ha+. The other three isolates from this province were classified as thermolabile viruses (I-Ha-).Contribution:This study will contribute to the control and/or eradication of Newcastle disease virus in Angola. The thermostable viral strains isolated from chickens in the country can be genetically manipulated by reverse genetic technology in order to reduce their virulence and use them as a vaccine in the remote areas of Angola.

Evaluation of the Newcastle disease virus genotype VII–mismatched vaccines in SPF chickens: A challenge efficacy study

Newcastle disease virus (NDV) strains, while falling under a single serotype, are classified into distinct genotypes. Genotype VII virulent NDVs pose a significant threat to poultry due to their association with high mortality rates and economic losses. This study aimed to evaluate the efficacy of three commercial live vaccines based on genotype II against genotype VII virulent NDV (vNDV) in specific pathogen-free (SPF) chickens. Forty one-day-old chickens were randomly divided into four groups (n = 10) and inoculated with one dose of each ND pneumotropic vaccine—B1, Clone.12IR, and La Sota—or received phosphate-buffered saline (PBS) as a control at eight days of age via eye drop. At 28 days of age (20th post-vaccination days), chickens were intramuscularly challenged with genotype VII virulent NDV (≥ 105 LD50). Serum samples were collected at 28 days of age (challenge day), 7 and 14 post-challenge days to measure NDV antibodies via the hemagglutination inhibition (HI) test. Cloacal and oropharyngeal swabs were taken on the 3rd, 5th, 7th, and 10th post-challenge days to evaluate virus shedding. Vaccinated groups exhibited significantly higher antibody titers and greater protection levels compared to the control group (P≤ 0.001). While HI antibody titer was not different at 28 and 35 days of age between vaccinated chickens, the Clone.12IR groups showed higher HI antibody titer compared to B1 at day 42 of age (9.43 vs. 7.42; P≤ 0.002). La Sota and Clone.12IR vaccines demonstrated superior protection against mortality compared to the B1 vaccine (90 %, 80% vs. 60 %, respectively) with 6.0 and 2.67 odds ratio of survivability. All three mismatched vaccines effectively curbed the shedding of virulent genotype VII NDV, with 0 % to 11 % positive cloacal samples up to the 3rd post-challenge day. These findings demonstrate that in the experimental setting, the administration of mismatched ND vaccines, particularly La Sota and Clone.12IR, confer protection against genotype VII virulent NDV and control viral shedding, which can help to develop effective vaccination strategies to mitigate the impact of vNDV outbreaks in the poultry farms.

Phylogenetic Analysis of Newcastle Disease Virus Isolated from Poultry in Live Bird Markets and Wild Waterfowl in Zambia.

Poultry production is essential to the economy and livelihood of many rural Zambian households. However, the industry is threatened by infectious diseases, particularly Newcastle disease virus (NDV) infection. Therefore, this study employed next-generation sequencing to characterise six NDV isolates from poultry in Zambia's live bird markets (LBMs) and wild waterfowl. Four NDV isolates were detected from 410 faecal samples collected from chickens in LBMs in Lusaka and two from 2851 wild birds from Lochinvar National Park. Phylogenetic analysis revealed that the four NDVs from LBM clustered in genotype VII and sub-genotype VII.2 were closely related to viruses previously isolated in Zambia and other Southern African countries, suggesting possible local and regional transboundary circulation of the virus. In contrast, the two isolates from wild birds belonged to class I viruses, genotype 1, and were closely related to isolates from Europe and Asia, suggesting the possible introduction of these viruses from Eurasia, likely through wild bird migration. The fusion gene cleavage site motif for all LBM-associated isolates was 112RRQKR|F117, indicating that the viruses are virulent, while the isolates from wild waterfowl had the typical 112ERQER|L117 avirulent motif. This study demonstrates the circulation of virulent NDV strains in LBMs and has, for the first time, characterised NDV from wild birds in Zambia. The study further provides the first whole genomes of NDV sub-genotype VII.2 and genotype 1 from Zambia and stresses the importance of surveillance and molecular analysis for monitoring the circulation of NDV genotypes and viral evolution.

Deciphering whole genome sequence of a Newcastle disease virus genotype VII.2 isolate from a commercial poultry farm in India

Here we report the isolation and complete genome sequence of virulent Newcastle disease virus (NDV) during the outbreak in 2023. NDV is isolated from the infected egg-laying vaccinated chickens of poultry farms located in Raipur district in Chhattisgarh, India. Next-generation sequencing (NGS) revealed the present NDV isolate has a genome length of 15,118 nucleotides (GenBank: OR185447). The phylogenetic tree illustrates the evolutionary relationship among NDV species and populations indicating the present NDV strain (NDV/Chicken/RPR/01/23) classified as genotype VII in class 2. NDV isolate exhibited 99.19 % and 97.53 % similarities in the nucleotide sequence in comparison with other strains of NDV/Owl/Guwahati/01/20 (GenBank: MZ546197.1) from India and chicken/Banjarmasin/010/10 (GenBank: HQ697254.1) from Indonesia. The present NDV genome is closely aligned with the NDV/Owl/Guwahati/01/20 strain and was clustered in genotype VII in class 2 suggesting possible spillover among the viral strains. The amino acid sequence at the protein Fusion protein (F) cleavage sequence 112RRQKR*F117 which is distinctive with other lentogenic vaccine NDV strain of La Sota and postulated as a major determinant of NDV virulence. Since there is limited information available on the exact cause of the abrupt death of poultry chickens located in Raipur district in Chhattisgarh, India, the present study aims to examine molecular sequencing of circulating NDV strains causing high mortality in this region. The neutralization epitopes of virulent NDV strains will expedite in development next next-generation vaccines to control circulating viral strains across the globe. The derived scientific information on genetic diversity, current taxonomic classification, and antigenic variations that exist within the genome of NDV will help in developing strain-specific vaccines and implementing strict measures.

Geographic distribution and genetic diversity of Newcastle disease virus in pigeons from Pakistan

ABSTRACT Newcastle disease virus (NDV) is among the most important viruses of poultry; however, variants also cause clinical diseases in birds of the family Columbidae. From 2014 onwards, notable mortality rates have been observed in racing and rock pigeons, with a correlation to subgenotypes XXI.1.1 and XXI.1.2 of genotype XXI in Pakistan. In this study, we performed isolation and genetic characterization of 29 pigeon-derived NDV isolates of sub-genotypes XXI.1.1, XXI.1.2 and VII.2. Phylogenetic analysis based on complete fusion gene sequences classified seven (n = 7) isolates into sub-genotype XXI.1.1 and they were genetically closely related to isolates previously characterized in Pakistan (0.05–0.43% nucleotide divergence), and in Iran, Russia, Egypt, Nigeria, Kazakhstan and Ukraine (3.75% to 6.67% nucleotide divergence) in the period from 2005–2018. In addition, nineteen (n = 19) isolates were clustered with viruses previously isolated in Pakistan (2014–2018) in sub-genotype XXI.1.2. In addition, three (n = 3) F-gene sequences assigned to subgenotype VII.2 were identified along with viruses repeatedly isolated (2011–2020) from several poultry and non-poultry avian species in Pakistan. The analysis showed that all isolates of sub-genotypes XXI.1.1 and XXI.1.2 were mesogenic (1.42–1.49) and the isolates belonging to sub-genotype VII.2 were velogenic (1.63–1.71) in nature according to the intracerebral pathogenicity index (ICPI). This study delivers a comprehensive description of the prevalence of sub-genotypes XXI.1.1 and XXI.1.2 in Pakistan since 2014. Additionally, it underscores the role of free-range pigeons as effective carriers of virulent sub-genotype VII.2 viruses, impacting both poultry and non-poultry avian species reared in open environments. RESEARCH HIGHLIGHTS Virulent NDV genotypes were repeatedly isolated from pigeons. Evidence of epidemiological links among viruses isolated from various locations. Distinct phylogenetic branches suggest separate, simultaneous evolution of NDVs. Study information could be helpful in the development of an effective vaccine.

Mitochondrial protein CHCHD10 inhibits NDV replication and reduces pathological changes

Newcastle disease (ND) is a disease that threatens the world's poultry industry, which is caused by virulent Newcastle disease virus (NDV). As its pathogenic mechanism remains not fully clear, the proteomics of NDV-infected cells were analyzed. The results revealed that coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) protein displayed a significant decrease at the late stage of NDV infection. To investigate the function of CHCHD10 in NDV infection, its expression after NDV infection was detected both in vivo and in vitro. Besides, the tissue viral loads and pathological damage of C57BL/6 mice with CHCHD10 differently expressed were also investigated. The results showed that the CHCHD10 expression was significantly decreased both in vivo and in vitro at the late stage of NDV infection. The viral loads were significantly higher in CHCHD10 silenced C57BL/6 mice, along with more severe pathological damage and vice versa.

Genetic Diversity of Genotype VII, and Virulent Newcastle Disease Virus (NDV), Determined by Cleavage Site Fusion Protein, and Heterogeneity with Commercial Vaccine Strains

Genetic Diversity of Genotype VII, and Virulent Newcastle Disease Virus (NDV), Determined by Cleavage Site Fusion Protein, and Heterogeneity with Commercial Vaccine Strains

Experimental trials to assess the immune modulatory influence of thyme and ginseng oil on NDV-vaccinated broiler chickens.

The use of traditional medicine against viral diseases in animal production has been practiced worldwide. Herbal extracts possess organic substances that would improve chicken body performance. The current study was designed to evaluate the effect of either thyme or ginseng oil in regard to their immune-modulatory, antiviral, and growth promoter properties. Two hundred and forty-one-day-old broiler chicks were allocated into eight equal groups as the following: group 1; nonvaccinated and nontreated and group 2; Newcastle disease virus (NDV) vaccinated and nontreated. Birds of groups 3 and 4 were treated with thyme oil (200 mg/l of drinking water for 12 hours/day) without or with NDV vaccination. Birds of groups 5 and 6 were treated with ginseng oil (200 mg/l of drinking water for 12 hours/day) without or with NDV vaccination. Birds of groups 7 and 8 were treated with a combination of ginseng oil (100 mg/l of drinking water) and thyme oil (100 mg/l of drinking water) for 12 hours/day. On the 35th day of life, birds in all the experimental groups were given 0.1 ml of a virulent genotype VIId NDV strain suspension containing 106.3 EID50/ml intramuscularly. Administration of ginseng and thyme oils each alone or simultaneously to birds either vaccinated or nonvaccinated elicited a significant improvement in body performance parameters. Administration of thyme and ginseng each alone or concurrently to vaccinated birds (Gp 4, 6, and 8) induced a higher hemagglutination inhibition (HI) titer of 6, 7.3, and 6.3 log2 at 21 days of age, 6.7, 7.6, and 7 log2, at 28 days of age and 7, 8, and 6.8 log2 at 35 days of age, respectively. Challenge with vNDV genotype VII led to an increase in the NDV-specific HI-Ab titers 10 days post challenge in all the experimental groups. In addition, thyme, ginseng oils, or a combination of them improved the protection from mortality in vaccinated birds; by 100%, 100%, and 90%, respectively, compared with 80% protection from mortality in vaccinated-only birds post-NDV challenge. Moreover, NDV-vaccinated birds treated either with thyme; ginseng or their combination showed negative detection of the virus in both tracheal and cloacal swabs and nonvaccinated groups that received oils showed improvement in vNDV shedding in tracheal and cloacal swabs. It could be concluded that the administration of thyme and ginseng essential oils to broilers can improve productive performance parameters, stimulate humoral immunity against, and protect from vNDV infection.

Clinico-pathological assessment of virulent Newcastle Disease Virus in ducks.

Newcastle disease (ND) is an infectious, highly contagious and lethal disease of avian species. It is considered that ducks are natural reservoir or carrier for Newcastle disease virus (NDV) and are resistant against different strains of NDV. Current study was designed to evaluate the pathogenesis of Newcastle disease in domestic ducks through histopathology, immunohistochemistry (IHC) and serum biochemical changes. For this purpose, eighty ducks were reared for 42 days and divided in two groups A and B. Ducks in group A were challenged with (NDV) at rate of 0.1 ml of ELD50 (virus titer 107.32/100µl) on second week of age, whereas Group B was control negative. Splenomegaly, atrophy of thymus and necrotic lesion in kidney were observed on 9th day of post infection. Hepatic degeneration and mononuclear cell infiltration were noticed in proventriculus and intestine in challenged ducks. Viral antigen detected in lungs, intestine, proventriculus and lymphoid organs of infected ducks through IHC. Albumin and total protein values were significantly low in infected groups A as compared to control group B. ALT, AST, and ALP values were significantly high in infected group A. On 5th and 7th day of post infection oropharyngeal swabs were negative for NDV and cloacal swabs were positive for NDV through Reverse transcriptase polymerase chain reaction. It is concluded that ducks are susceptible to NDV and virulent strain of NDV caused disease in ducks.

Protective effects of a novel chimeric virus–like particle vaccine against virulent NDV and IBDV challenge

Newcastle disease (ND) and infectious bursal disease (IBD) pose significant threats to the chicken industry, causing substantial economic losses. Currently, immunization through vaccination is the most effective strategy to prevent ND and IBD but currently used traditional vaccines, including inactivated or attenuated vaccines, face challenges in achieving a balance between immunogenicity and safety. To develop a green and efficient novel vaccine for ND and IBD, we developed a bivalent chimeric virus-like particle vaccine (ND-IBD cVLPs) displaying the ND virus (NDV) HN protein and the IBD virus (IBDV) VP2 protein based on the ND VLPs carrier platform and insect baculovirus expression system. This study aimed to evaluate the immunogenicity and protective efficacy of ND-IBD cVLPs in specific pathogen-free chickens. Chickens were immunized with 50 µg of purified ND-IBD cVLPs at 7 days old, boosted at 21 days old, and challenged at 42 days old. The results demonstrated that ND-IBD cVLPs stimulated highly effective hemagglutination inhibition antibody levels against NDV HN protein and enzyme-linked immunosorbent assay antibody levels against the IBDV VP2 protein. Furthermore, ND-IBD cVLPs provided complete protection against virulent NDV and IBDV challenges and mitigated pathological damage to the lung caused by NDV infection and the bursa of Fabricius caused by IBDV infection. These findings suggest that ND-IBD cVLPs hold promise as a safe and efficient novel vaccine candidate for the effective prevention of ND and IBD, extending the development of a foreign protein delivery platform of ND VLPs.

Virulent Newcastle disease virus genotypes V.3, VII.2, and XIII.1.1 and their coinfections with infectious bronchitis viruses and other avian pathogens in backyard chickens in Tanzania

Oropharyngeal (OP) and cloacal (CL) swabs from 2049 adult backyard chickens collected at 12 live bird markets, two each in Arusha, Dar es Salaam, Iringa, Mbeya, Morogoro and Tanga regions of Tanzania were screened for Newcastle disease virus (NDV) using reverse transcription real-time PCR (rRT-PCR). The virus was confirmed in 25.23% of the birds (n = 517; rRT-PCR CT ≤ 30), with the highest positivity rates observed in birds from Dar es Salaam region with higher prevalence during the dry season (September–November 2018) compared to the rainy season (January and April–May 2019). Next-generation sequencing of OP/CL samples of 20 out of 32 birds that had high amounts of viral RNAs (CT ≤ 25) resulted in the assembly of 18 complete and two partial genome sequences (15,192 bp and 15,045–15,190 bp in length, respectively) of NDV sub-genotypes V.3, VII.2 and XIII.1.1 (n = 1, 13 and 4 strains, respectively). Two birds had mixed NDV infections (V.3/VII.2 and VII.2/XIII.1.1), and nine were coinfected with viruses of families Astroviridae, Coronaviridae, Orthomyxoviridae, Picornaviridae, Pneumoviridae, and Reoviridae. Of the coinfecting viruses, complete genome sequences of two avastroviruses (a recombinant chicken astrovirus antigenic group-Aii and avian nephritis virus genogroup-5) and two infectious bronchitis viruses (a turkey coronavirus-like recombinant and a GI-19 virus) were determined. The fusion (F) protein F1/F2 cleavage sites of the Tanzanian NDVs have the consensus motifs 112 RRRKR↓F 117 (VII.2 strains) and 112 RRQKR↓F 117 (V.3 and XIII.1.1 strains) consistent with virulent virus; virulence was confirmed by intracerebral pathogenicity index scores of 1.66–1.88 in 1-day-old chicks using nine of the 20 isolates. Phylogenetically, the complete F-gene and full genome sequences regionally cluster the Tanzanian NDVs with, but distinctly from, other strains previously reported in eastern and southern African countries. These data contribute to the understanding of NDV epidemiology in Tanzania and the region.

Toll-like Receptor Ligands Enhance Vaccine Efficacy against a Virulent Newcastle Disease Virus Challenge in Chickens.

To enhance the efficacy of the current Newcastle disease vaccine, we have selected potential adjuvants that target well-characterized pattern recognition receptors: the toll-like receptors (TLRs). Imiquimod is a small-molecule activator of TLR7, which is a sensor of dsDNA. ODN-1826 is a mimetic of CpG DNA and ligates TLR21 (a chicken homologue of TLR9 in mammals). The activation of TLRs leads to antiviral responses, including the induction of type I interferons (IFNs). In this study, birds were vaccinated intranasally with a live LaSota strain with or without imiquimod or ODN-1826 (50 µg/bird). Two weeks after vaccination, the birds were challenged with a virulent Newcastle disease virus (chicken/CA/212676/2002). Both adjuvants (imiquimod or ODN-1826) induced higher and more uniform antibody titers among vaccinated birds compared with the live vaccine-alone group. In addition, adjuvanted vaccines demonstrated greater protective efficacy in terms of the reduction in virus-shedding titer and the number of birds shedding the challenge virus at 2 and 4 days post-challenge. A differential expression of antiviral and immune-related genes was observed among groups from tissues (Harderian gland, trachea, cecal tonsil, and spleen) collected 1 and 3 days after treatment. These results demonstrate the potential of TLR-targeted adjuvants as mucosal vaccine enhancers and warrant a further characterization of immune correlates and optimization for efficacy.

Detection and molecular characterization of virulent Newcastle disease virus (subgenotype VII.2) in broiler chickens in Northern Vietnam.

Newcastle disease (ND) is a major viral disease of poultry worldwide. However, data on the molecular characterization of Newcastle disease virus (NDV) in Vietnam are limited. This study aimed to identify the molecular characteristics of NDV strains from the vaccinated chickens farmed in Northern Vietnam. We used reverse-transcription polymerase chain reaction (PCR), sequencing and phylogenetic analysis to characterize NDV strains from vaccinated chicken farms in Northern Vietnam. Seven out of 72 (9.7%) chicken tissue samples collected from seven chicken farms in the four cities/provinces in northern Vietnam were positive for the NDV genome by PCR method. The complete sequences of the fusion (F) and hemagglutinin-neuraminidase (HN) genes of NDVs isolated in the North of Vietnam from 2021 to 2022 were further evaluated. The results indicated that all seven Vietnamese isolates obtained were reported as virulent NDV strains with the amino acid (AA) sequence of the F0 protein proteolytic cleavage site motif (112RRRKRF117). Phylogenetic analysis revealed that they were grouped with other NDV class II from subgenotype VII.2, including the two previous Vietnamese NDV (2015), the Chinese (2017), and Southern African (2013) NDV strains. In addition, some AA substitutions were observed in the neutralizing epitopes of the F and HN proteins of the current Vietnamese NDV strains. The present findings provide useful information for future studies of the evolution of NDVs and improve strategies for ND-controlling programs in Vietnam.

Study of Cellular and Humoral Immunity and Histopathology of Target Tissues Following Newcastle Clone12IR Vaccine Administration in SPF Chickens.

Newcastle disease (ND) is a highly contagious viral infection affecting poultry production in many countries. Strict biosecurity and the administration of live attenuated vaccines against the ND virus (NDV) are the main implements of controlling programs. This study evaluated the efficacy and potency of the Razi Clone12IR Newcastle vaccine in specific pathogen-free (SPF) chickens. Chickens were vaccinated with either the Razi Clone12IR vaccine (group A1, n=20) or an imported Clone vaccine (B1, n=20) in the first week of life and boosted in the second week via eye drop, while negative control chickens received PBS (C1, n=20). Half of the birds in each group were challenged with the virulent NDV strain in the third post-vaccination week (A2, B2, and C2 groups). Specific antibody responses were determined in the collected sera by the hemagglutination inhibition (HI) assay for up to eight weeks. Cell-mediated immunity (CMI) was determined by the lymphocyte proliferation assay three and six weeks after the second vaccination. Sections of the tissues and organs, including the trachea, lungs, cecal tonsils, spleen, the bursa of Fabricius, liver, and small intestine, were subjected to histopathology. The immunized groups A1 and B1 showed significantly higher HI antibody titers before the challenge than the control group. In addition, lymphocyte proliferation responses significantly increased in the peripheral blood of the vaccinated groups. After the challenge, the A2 and B2 groups conferred good protection and drastically reduced virus shedding. No main lesions were noted in the tissues or organs of the vaccinated group in histopathology. In a few cases, mild microscopic lesions were observed, including the infiltration of inflammatory cells, which was related to the effect of the vaccine virus. These results indicate that the Razi Clone12IR vaccine is safe and can be an efficient tool for NDV infections by inducing protective humoral and CMI responses.

Complete genome sequence of seven virulent Newcastle disease virus isolates of sub-genotype XIII.1.1 from Tanzania.

We report the complete genome sequences of seven virulent Newcastle disease viruses (NDVs) that were isolated from chickens from live bird markets in the Arusha, Iringa, Mbeya, and Tanga regions of Tanzania in 2012. Phylogenetic analysis revealed that all isolates belong to sub-genotype XIII.1.1.