Newcastle Disease Virus Isolates Research Articles

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.

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.

The role of Newcastle Disease Virus in Broiler Chickens with High Mortality of Kerman Province

The Newcastle disease virus (NDV) is a member of the paramyxoviridea family and has great significance in the poultry production industry, which spends a huge amount of money every year on prevention and economic loss caused by this disease. A wide range of symptoms, including respiratory and nervous disorders, as well as hemorrhage lesions in the digestive system are observed in this disease. This research investigated the presence of NDV in 10 poultry farms with high mortality and respiratory symptoms in Kerman province, Iran (between January 2020 to October 2020). Tissue samples were collected from mortalities of 10 flocks in different parts of Kerman province and inoculated into embryonated eggs. The NDV was detected in the allantoic fluid by polymerization of partial F gene protein. The virus was positive in the samples of 5 flocks. The results of the phylogenetic analysis also showed that the sequence of isolates was related to genotype II (three isolates) and sub-genotype VIId (two isolates) of NDVs. It was also found that the amino acid sequences of sub-genotype VIId isolates in the 113 to 116 positions were RRQKR and in the 117 positions was the presence of F (phenylalanine). The other three isolates were grouped with B1, Clone, and LaSota vaccines, and the amino acid sequence in the cleavage site included GRQGRL. The similarity between the studied isolates was 99.6%-98.4%. In this study, virulent viruses were isolated and tracked in broiler farms that were vaccinated with live and killed vaccines. It is recommended to pay more attention to designing the vaccination program.

Intranasally Delivered Adenoviral Vector Protects Chickens against Newcastle Disease Virus: Vaccine Manufacturing and Stability Assessments for Liquid and Lyophilized Formulations.

Newcastle disease (ND) remains a critical disease affecting poultry in sub-Saharan Africa. In some countries, repeated outbreaks have a major impact on local economies and food security. Recently, we developed an adenovirus-vectored vaccine encoding the Fusion protein from an Ethiopian isolate of Newcastle disease virus (NDV). The adenoviral vector was designed, and a manufacturing process was developed in the context of the Livestock Vaccine Innovation Fund initiative funded by the International Development Research Centre (IDRC) of Canada. The industrially relevant recombinant vaccine technology platform is being transferred to the National Veterinary Institute (Ethiopia) for veterinary applications. Here, a manufacturing process using HEK293SF suspension cells cultured in stirred-tank bioreactors for the vaccine production is proposed. Taking into consideration supply chain limitations, options for serum-free media selection were evaluated. A streamlined downstream process including a filtration, an ultrafiltration, and a concentration step was developed. With high volumetric yields (infectious titers up to 5 × 109 TCID50/mL) in the culture supernatant, the final formulations were prepared at 1010 TCID50/mL, either in liquid or lyophilized forms. The liquid formulation was suitable and safe for mucosal vaccination and was stable for 1 week at 37 °C. Both the liquid and lyophilized formulations were stable after 6 months of storage at 4 °C. We demonstrate that the instillation of the adenoviral vector through the nasal cavity can confer protection to chickens against a lethal challenge with NDV. Overall, a manufacturing process for the adenovirus-vectored vaccine was developed, and protective doses were determined using a convenient route of delivery. Formulation and storage conditions were established, and quality control protocols were implemented.

Isolation of newcastle disease virus from commercial broilers in district Abbottabad, Khyber Pakhtunkhwa, Pakistan

Isolation of newcastle disease virus from commercial broilers in district Abbottabad, Khyber Pakhtunkhwa, Pakistan

Isolation and Biological Characterization of Newcastle Disease Virus (NDV) Field Isolate Pigeon (Columba livia) from Live Bird Market, East Java in 2019

Background: Avian Paramyxovirus (APMV) type-1 is the leading cause of Newcastle Disease (ND) and taxonomically belongs to the family Paramyxoviridae, genus Avulavirus. Due to its high transmission rate, Newcastle Disease (ND) is included in the A list by the OIE. Purpose: To determine the biological characterize the Newcastle Disease Virus (NDV) field isolate of pigeons (Columba livia) using Mean Death Time (MDT), Intracerebral Pathogenicity Index (ICPI), and Intravenous Pathogenicity Index (IVPI). Methods: Twenty pigeon organ samples were obtained from bird markets in East Java, and one was used as a positive control (LaSota). Organs were isolated from embryonated chicken eggs, identified by the HA test, and then confirmed by the HI test. Furthermore, positive samples were tested for MDT with a 10-1-10-18 dilution (0.1 ml and observed for eight days. The ICPI test used a fresh NDV suspension (0.05 ml and was observed for eight days. The IVPI test used a dose of 0.1 ml and was observed for ten days. Result: The MDT values of isolates MB1/NDV/19, MB2/NDV/19, MB3/NDV/19, and MG1/NDV/19 were 91.2 hours, 112.8 hours, 110.4 hours, and 124,8 hours. The ICPI values of isolate MB1/NDV/19 was 0.2375, MB2/NDV/19 was 0.375, MB3/NDV/19 was 0.5375, and MG1/NDV/19 was 0.3. The IVPI value of isolate MB1/NDV/19 was 0, MB2/NDV/19 was 0, MB3/NDV/19 was 0, and MG1/NDV/19 was 0. Conclusion: All four field samples were positive for NDV as a lentogenic strain based on the MDT, ICPI, and IVPI tests.

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.

Serological evidence of Newcastle disease virus antibodies in wild birds in Zaria, Kaduna State, Nigeria.

Wild birds have been reported to be reservoirs of viral diseases of poultry, and play an epidemiological role in their maintenance and spread. A serological survey was undertaken to determine the evidence of Newcastle disease virus (NDV) antibodies in wild birds in Zaria Kaduna State, Nigeria. A total of 150 apparently healthy wild birds comprising 30 each laughing dove, speckled pigeons, cattle egrets, village weavers and African silver bills were sampled. Sera collected were analysed for the presence of antibodies against NDV and avian paramyxovirus‑2 (APMV‑2) using the haemagglutination inhibition test. The results showed an overall seroprevalence of 4% (95% CI: 2.05‑10.1) to NDV. African silver bill showed a seroprevalence of 10.0% (95% CI: 2.61‑24.9) NDV antibodies while seroprevalence of 3.3% (95% CI: 0.16‑15.4) was recorded for cattle egrets, village weavers and laughing doves respectively. No statistically significant difference existed for NDV seroprevalence (P>0.05) among the different species of wild birds. All the 150 sera tested negative for APMV‑2 antibodies. The result of this study confirmed the exposure of wild birds to NDV in the study area. Continuous surveillance with isolation and characterization of NDV in the wild birds is therefore recommended for strategic planning for control.

Efficacy of Newcastle disease LaSota vaccine-induced hemagglutination inhibition antibodies against challenges with heterologous virulent strains of genotypes VII and IX

Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), still remains one of the most important avian diseases affecting the poultry industry worldwide, despite intensive vaccination programs have been implemented in many countries. All NDV isolates characterized to date are of one serotype and classified into classes I and II, with class II being further divided into twenty-one genotypes. Antigenic and genetic diversity is observed among the different genotypes. Current commercially available vaccines belonging to genotypes I and II are genetically divergent from strains that caused ND outbreaks worldwide in the last two decades. Reports of vaccination failures on their insufficient ability to inhibit infection or virus shedding have created renewed interest in developing vaccines homologous to virulent NDV circulating in the field. In this study, after vaccination with the most widely used LaSota vaccine (genotype II), chickens with different hemagglutination inhibition (HI) antibody levels were challenged with heterologous virulent NDV strains of genotypes VII and IX to evaluate how antibody levels relate to clinical protection and infection or virus shedding. Under the experimental condition, LaSota vaccine could fully protect birds from morbidity and mortality, but higher antibody levels were required to inhibit virus shedding. The number of birds shedding virus generally tended to decrease as the HI antibody titers increase in vaccinated birds. When the HI antibody titers reached ≥ 13 log2 and ≥ 10 log2, the virus shedding from JSC0804 strain (genotype VII) and F48E8 strain (genotype IX) could be completely inhibited, respectively, but it may be difficult to ensure that all individuals reach and maintain those levels in chicken flocks vaccinated according to routine procedure. Furthermore, the virus shedding in vaccinated birds was correlated with the amino acid similarity between the vaccine and challenge strains; more similarity, less virus shedding. The results obtained highlight that stringent biosecurity measures combined with vaccination are crucial for chicken farms to maintain a virulent NDV-free status.

Comparative pathogenicity of a genotype XXI.1.2 pigeon Newcastle disease virus isolate in pigeons and chickens

Here, we performed molecular and pathogenic characterization of a Newcastle disease virus (NDV) isolate from pigeons in Bangladesh. Molecular phylogenetic analysis based on the complete fusion gene sequences classified the three study isolates into genotype XXI (sub-genotype XXI.1.2) together with recent NDV isolates obtained from pigeons in Pakistan (2014–2018). The Bayesian Markov Chain Monte Carlo analysis revealed that the ancestor of Bangladeshi pigeon NDVs and the viruses from sub-genotype XXI.1.2 existed in the late 1990s. Pathogenicity testing using mean embryo death time pathotyped the viruses as mesogenic, while all isolates carried multiple basic amino acid residues at the fusion protein cleavage site. Experimental infection of chickens and pigeons revealed no or minimum clinical signs in chickens, while a relatively high morbidity (70%) and mortality (60%) were observed in pigeons. The infected pigeons showed extensive and systemic lesions including hemorrhagic and/or vascular changes in the conjunctiva, respiratory and digestive system and brain, and atrophy in the spleen, while only mild congestion in the lungs was noticed in the inoculated chickens. Histologically, consolidation in the lungs with collapsed alveoli and edema around the blood vessels, hemorrhages in the trachea, severe hemorrhages and congestion, focal aggregation of mononuclear cells, and single hepatocellular necrosis in the liver, severe congestion, multifocal tubular degeneration, and necrosis, as well as mononuclear cell infiltration in the renal parenchyma, encephalomalacia with severe neuronal necrosis with neuronophagia were noticed in the brain in infected pigeons. In contrast, only slight congestion was found in lungs of the infected chickens. qRT-PCR revealed the replication of the virus in both pigeons and chickens; however, higher viral RNA loads were observed in oropharyngeal and cloacal swabs, respiratory tissues, and spleen of infected pigeons than the chickens. In conclusion, genotype XXI.1.2 NDVs are circulating in the pigeon population of Bangladesh since 1990s, produce high mortality in pigeons with pneumonia, hepatocellular necrosis, renal tubular degeneration, and neuronal necrosis in pigeons, and may infect chickens without overt signs of clinical disease and are likely to shed viruses via the oral or cloacal routes.

Antibody response to Newcastle disease virus recombinant fusion protein in post-vaccinated laying hens

This research was aimed to analyze antibody response in laying hens post vaccinated by Newcastle Disease Virus (NDV) recombinat Fusion (F) protein which has been succesfully expressed from the F gene of local isolates of NDV from Kulon Progo strain (0663/04/2013), Yogyakarta, Indonesia. The F gene cloned into expression vector plasmid pBT7-N-His. Two types of NDV recombinant vaccine, a concentrated and pure F recombinant protein were used for vaccination. A concentrated recombinant F protein was collected from the centrifugal ultrafiltration process and a pure recombinant F protein was collected from the electroeluted process. Recombinant F protein of NDV was successfully expressed, purified, and visualized by Sodium Dodecyl Sulphate–Polyacrylamide Gel Electrophoresis (SDS-PAGE) with Coomassie Brilliant Blue staining with a molecular weight of 28 kDa. All two types of recombinant F vaccines and a commercial live vaccine as a positive control were injected two times at 14 and 18th weeks old laying hens to analyze the antibody response in serum. In comparison with a commercial live NDV vaccine, indirect Enzyme-linked Immunosorbent Assay (ELISA) revealed that antibody responses were high in both recombinant F protein vaccinated groups. In conclusion, the recombinant F protein has the potential to be developed as a recombinant vaccine candidate to obtain a higher antibody response in laying hens compared to commercially available live NDV vaccines.

The production of Newcastle disease virus-like particles in Nicotiana benthamiana as potential vaccines.

Newcastle disease (ND) is a highly contagious viral respiratory and neurological disease that has a severe impact on poultry production worldwide. In the present study, an expression platform was established for the transient production in N.bethamiana of ND virus-like particles (VLPs) for use as vaccines against ND. The expression of the ND Fusion (F) and/or Hemagglutinin-neuraminidase (HN) proteins of a genotype VII.2 strain formed ND VLPs in planta as visualized under the transmission electron microscope, and HN-containing VLPs agglutinated chicken erythrocytes with hemagglutination (HA) titres of up to 13 log2.The immunogenicity of the partially-purified ND VLPs was confirmed in specific-pathogen-free White leghorn chickens. Birds receiving a single intramuscular immunization with 1024 HA units (10 log2) of the F/HN ND VLPs administered with 20% [v/v] Emulsigen®-P adjuvant, seroconverted after 14 days with F- and HN-specific antibodies at ELISA titres of 5705.17 and HI geometric mean titres (GMTs) of 6.2 log2, respectively. Furthermore, these ND-specific antibodies successfully inhibited viral replication in vitro of two antigenically closely-related ND virus isolates, with virus-neutralization test GMTs of 3.47 and 3.4, respectively. Plant-produced ND VLPs have great potential as antigen-matched vaccines for poultry and other avian species that are highly immunogenic, cost-effective, and facilitate prompt updating to ensure improved protection against emerging ND field viruses.

Genetic diversity of Newcastle disease viruses circulating in wild and synanthropic birds in Ukraine between 2006 and 2015.

Newcastle disease virus (NDV) infects a wide range of bird species worldwide and is of importance to the poultry industry. Although certain virus genotypes are clearly associated with wild bird species, the role of those species in the movement of viruses and the migratory routes they follow is still unclear. In this study, we performed a phylogenetic analysis of nineteen NDV sequences that were identified among 21,924 samples collected from wild and synanthropic birds from different regions of Ukraine from 2006 to 2015 and compared them with isolates from other continents. In synanthropic birds, NDV strains of genotype II, VI, VII, and XXI of class II were detected. The fusion gene sequences of these strains were similar to strains detected in birds from different geographical regions of Europe and Asia. However, it is noteworthy to mention the isolation of vaccine viruses from synanthropic birds, suggesting the possibility of their role in viral transmission from vaccinated poultry to wild birds, which may lead to the further spreading of vaccine viruses into other regions during wild bird migration. Moreover, here we present the first publicly available complete NDV F gene from a crow (genus Corvus). Additionally, our phylogenetic results indicated a possible connection of Ukrainian NDV isolates with genotype XXI strains circulating in Kazakhstan. Among strains from wild birds, NDVs of genotype 1 of class I and genotype I of class II were detected. The phylogenetic analysis highlighted the possible exchange of these NDV strains between wild waterfowl from the Azov-Black Sea region of Ukraine and waterfowl from different continents, including Europe, Asia, and Africa.

Efficient Production of Evolutionary Nanoparticle Newcastle Disease Virus

Viruses are routinely isolated from infected cells through freeze–thaw (F–T) cycles or sonication. The aim of this study was to compare different methods for efficient isolation of Newcastle disease virus (NDV) particles from BSR-T7/5 (BSR) cells. The BSR cells were infected with NDV LaSota strain, and the virus particles were isolated via F–T, sonication, sonication followed by F–T, and F–T followed by sonication. The infection and proliferation kinetics of the virus were analyzed by cytopathic observation and monitoring of hemagglutination (HA) titers. The virus isolated by sequential F–T and sonication was amplified through five passages of BSR cells, and then used to infect the HepG2 cells. The viability and apoptosis rates of the infected cells were evaluated by Cell Counting Kit-8 assay and Annexin V-FITC/PI staining respectively. We successfully obtained NDV particles from persistently infected BSR cells through all four methods, which indicated that the LaSota can effectively replicate in BSR cells. However, F–T followed by sonication was optimum in terms of separation effect. The virus particles isolated by this method still exhibited cytolytic activity against HepG2 cells. Thus, our novel method can be applied to NDV production and generation of tumor vaccines.

Identification of an additional N-glycosylation site and thermostable mutations within the hemagglutinin-neuraminidase gene of the Newcastle disease virus belonging to the VII.1.1 sub-genotype.

Newcastle disease virus (NDV) is considered one of the most devastating avian viral patho-gens affecting the avian population, and it causes a significant economic burden on the poultry industry worldwide. The study aimed to gain deeper understanding of the molecular and phylogenetic analyses of the complete hemagglutinin-neuraminidase (HN) coding region among NDV isolates. The samples were obtained from different parts of Iran from July 2017 to February 2020, were used for phylogenic analysis in this study. The results confirmed the predominance of sub-genotype VII.1.1, previously known as sub-genotype VIIL, which is circulating in commercial broiler farms of Iran. Identification of (a) an additional N-glycosylation site (NIS) at position 144; (b) mutations S315P and I369V which are related to increasing the viral thermostability; (C) cysteine residues at positions 123; (d) amino acid substitutions in the HN antigenic sites, especially the mutations I514V and E347Q, as well as the other mutant within HN binding sites of the VII.1.1 sub-genotype, suggests the idea that this new sub-genotype of NDV may possess a high level of pathogenicity and virulence compared to other NDV sub-genotypes. In conclusion, the results indicate the presence of an additional NIS at position 144, which may alter the virulence of the isolates. Furthermore, the presence of the thermostable mutations (S315P and I369V) and the other amino acid substitutions among the VII.1.1 sub-genotype isolates may have an impact on the vaccine immunity against this new NDV sub-genotype.

Introduction of a Newcastle disease virus challenge strain (sub-genotype VII.1.1) isolated in Iran.

Newcastle disease virus (NDV) sub-genotype VII.1.1 is the most common circulating NDV in Iran. In this study, a velogenic NDV isolate was plaque purified and then characterized according to Office International des Epizooties (OIE) standard protocols. The biological properties of the purified isolate named CH/RT40/IR/2011 were characterized using sequencing and phylogenetic analysis, measurement of pathogenicity indexes and challenge studies. The isolate was plaque purified on chicken embryo fibroblast cells for three rounds and then characterized using molecular and biological approaches. Phylogenetic and evolutionary distance analysis of fusion and hemagglutinin-neuraminidase genes classified the virus in sub-genotype VII.1.1. No mutation was observed in the glycosylation and neutralizing epitope sites of the fusion and hemagglutinin-neuraminidase proteins compared to other reported Iranian NDV VII.1.1 isolates. The presence of the 112RRQKRF117 motif in the fusion protein cleavage site together with mean death time, intracerebral pathogenicity index and intravenous pathogenicity index of 57 hr, 1.80 and 2.50 respectively, revealed that the RT40 isolate was a velogenic NDV. In the challenge study, all chickens were inoculated via eye drop, and intranasal route with RT40 isolate died within a week. While all chickens in the vaccinated and challenged group survived and showed no clinical signs. In conclusion, according to genetic analysis, pathotyping and challenge study, the RT40 isolate was similar to virulent NDVs in Iran and was a suitable candidate for a national standard challenge strain, vaccine trials and vaccine production in commercial levers.

The First Report of a Virulent Newcastle Disease Virus of Genotype VII.2 Causing Outbreaks in Chickens in Bangladesh.

Newcastle disease (ND) is endemic in poultry in Bangladesh. We performed genotypic and pathotypic characterization of four ND virus (NDV) isolates from recent outbreaks in broiler chickens in Bangladesh during the period of 2020-2021. Phylogenetic analysis based on the complete fusion protein gene coding sequences classified the viruses into NDV class II genotype VII.2 together with viruses from Indonesia isolated between 2014 and 2021 and a single 2020 Indian isolate. Pathogenicity testing using the intracerebral pathogenicity index in day-old chickens and mean embryo death time in embryonating chicken eggs revealed that the Bangladeshi isolates are velogenic. Inoculation of 35-day-old chickens with two NDV isolates (LT67 and N5) resulted in 100% morbidity by 3 days post inoculation (DPI), and all birds succumbed to infection by 7 DPI. Massive hemorrhages, congestion and necrotic lesions were observed in different visceral organs, which were typical for infection with a velogenic viscerotropic pathotype of NDV. At microscopic examination, tracheitis, severe pneumonia, focal proventriculitis, transmural enteritis, focal myocarditis, severe congestion and necrosis in kidneys, and lymphoid depletion in lymphoid tissues were found. Our study reports the first outbreak of the panzootic genotype VII.2 NDV in poultry in Bangladesh and documents a possible recent re-introduction of this NDV genotype from Southeast or East Asia. This study further provides viral distribution and epidemiological data that can facilitate the effective control of NDV.

Thermostability and Immunogenicity of Genotype II Avian Orthoavulavirus (AOaV-1) Isolates from Duck (Anas platyrhynchos) and Parrot (Eclectusroratus).

Newcastle disease (ND) is a highly contagious viral disease of poultry causing significant economic losses worldwide. Vaccination is considered the most reliable approach to curb the economic menace that is ND, but the thermolabile nature of Newcastle disease virus (NDV) vaccination poses a significant threat to its protective efficacy. This study aimed to profile the thermostability of NDV isolates from duck (As/Km/19/44) and parrot (As/WB/19/91) and evaluate their immunogenic potential in chicks. Fusion protein cleavage site (FPCS) and phylogenetic analysis demonstrated the lentogenic nature of both the isolates/strains and classified them as class II genotype II NDV. The characterized NDV isolates were adapted in specific-pathogen-free (SPF) chicks by serially passaging. Biological pathogenicity assessment of chicken-adapted As/Km/19/44 (PSD44C) and As/WB/19/91 (PSP91C) revealed both the isolates to be avirulent with a mean death time (MDT) of more than 90 h and an intracerebral pathogenicity index (ICPI) ranging from 0.2 to 0.4. Both of the NDV isolates displayed varied thermostability profiles. PSD44C was the most thermostable strain as compared to PSP91C and the commercially available LaSota vaccine strain. The immunogenicity of PSD44C and LaSota was significantly higher than PSP91C. Based on these results, it is concluded that NDV isolate PSD44C is more thermostable and immunogenic when administered intraocularly without any adverse effects. Therefore, PSD44C is suitable for further research and vaccine development.