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Background: 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 (0.5 g/L) before and after viral challenge., Result: 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., Conclusion: We concluded that Glycyrrhiza glabra powdered extract at a dose of 0.5 g/L drinking water can effectively mitigate the debilitating effects of Newcastle disease in chickens., (© 2024. The Author(s).)

Avian paramyxovirus type 1 (APMV-1) is a virus of birds that results in a range of outcomes, from asymptomatic infections to outbreaks of systemic respiratory and neurologic disease, depending on the virus strain and the avian species affected. Humans are rarely affected; those who are predominantly experience mild conjunctivitis. We report a fatal case of neurologic disease in a 2-year-old immunocompromised child in Australia. Metagenomic sequencing and histopathology identified the causative agent as the pigeon variant of APMV-1. This diagnosis should be considered in neurologic conditions of undefined etiologies. Agnostic metagenomic sequencing methods are useful in such settings to direct diagnostic and therapeutic efforts.

Newcastle disease virus (NDV) is an avian paramyxovirus that causes major economic losses to the poultry industry around the world, with NDV pathogenicity varying due to strain virulence differences. However, the impacts of intracellular viral replication and the heterogeneity of host responses among cell types are unknown. Here, we investigated the heterogeneity of lung tissue cells in response to NDV infection in vivo and that of the chicken embryo fibroblast cell line DF-1 in response to NDV infection in vitro using single-cell RNA sequencing. We characterized the NDV target cell types in the chicken lung at the single-cell transcriptome level and classified cells into five known and two unknown cell types. The five known cell types are the targets of NDV in the lungs with virus RNA detected. Different paths of infection in the putative trajectories of NDV infection were distinguished between in vivo and in vitro , or between virulent Herts/33 strain and nonvirulent LaSota strain. Gene expression patterns and the interferon (IFN) response in different putative trajectories were demonstrated. IFN responses were elevated in vivo , especially in myeloid and endothelial cells. We distinguished the virus-infected and non-infected cells, and the Toll-like receptor signaling pathway was the main pathway after virus infection. Cell-cell communication analysis revealed the potential cell surface receptor-ligand of NDV. Our data provide a rich resource for understanding NDV pathogenesis and open the way to interventions specifically targeting infected cells. IMPORTANCE Newcastle disease virus (NDV) is an avian paramyxovirus that causes major economic losses to the poultry industry around the world, with NDV pathogenicity varying due to strain virulence differences. However, the impacts of intracellular viral replication and the heterogeneity of host responses among cell types are unknown. Here, we investigated the heterogeneity of lung tissue cells in response to NDV infection in vivo and that of the chicken embryo fibroblast cell line DF-1 in response to NDV infection in vitro using single-cell RNA sequencing. Our results open the way to interventions specifically targeting infected cells, suggest principles of virus-host interactions applicable to NDV and other similar pathogens, and highlight the potential for simultaneous single-cell measurements of both host and viral transcriptomes for delineating a comprehensive map of infection in vitro and in vivo . Therefore, this study can be a useful resource for the further investigation and understanding of NDV., Competing Interests: The authors declare no conflict of interest.

We provide a novel single restriction enzyme (RE; BsaHI) digestion approach for detecting distinct pathotypes of Newcastle disease virus (NDV). After scanning 4,000 F gene nucleotide sequences in the NCBI database, we discovered a single RE (BsaHI) digestion site in the cleavage site. APMV-I "F gene" class II-specific primer-based reverse transcriptase PCR was utilized to amplify a 535-bp fragment, which was then digested with the RE (BsaHI) for pathotyping avian NDV field isolates and pigeon paramyxovirus-1 isolates. The avirulent (lentogenic and mesogenic strains) produced 189- and 346-bp fragments, respectively, but the result in velogenic strains remained undigested with 535-bp fragments. In addition, 45 field NDV isolates and 8 vaccine strains were used to confirm the approach. The sequence-based analysis also agrees with the data obtained utilizing the single RE (BsaHI) digestion approach. The proposed technique has the potential to distinguish between avirulent and virulent strains in a short time span, making it valuable in NDV surveillance and monitoring research. IMPORTANCE The extensive use of the NDV vaccine strain and the existence of avirulent NDV strains in wild birds makes it difficult to diagnose Newcastle Disease virus (NDV). The intracerebral pathogenicity index (ICPI) and/or sequencing-based identification, which are required to determine virulent NDV, are time-consuming, costly, difficult, and cruel techniques. We evaluated 4,000 F gene nucleotide sequences and discovered a restriction enzyme (RE; BsaHI) digestion technique for detecting NDV and vaccine pathotypes in a short time span, which is cost-effective and useful for field cases as well as for large-scale NDV monitoring and surveillance. The data acquired using the single RE BsaHI digestion technique agree with the sequence-based analysis.

Newcastle disease (ND) is a highly contagious infection of many avian species, mainly chickens and turkeys, with a devastating impact on worldwide poultry production. This study was designed to examine the effect of virulent ND infection in turkey's tissues and the tissue tropism of the virus. During the previous study period, poults were inoculated at 32 days of age with 10 5 EID50 virulent Newcastle disease virus. Three poults on days 0, 1, 2, 3, 4, 6, 7, and 14 postinoculations (PI) were selected from each group. They were euthanized by intravenous sodium pentobarbital injection. After macroscopic observation, to histopathological and immunohistochemical studies, the spleen, bursa, cecal tonsils, intestine, proventriculus, lung, kidney, and brain were sampled. Clinically, the infected turkeys exhibited loss of appetite, severe depression, down on hock joint, white to greenish (sometimes bloody) diarrhea, nervous signs, and mild respiratory problems. Out of 45 birds inoculated, 9 (20%) died. Histopathological effects in lymphoid tissues included necrosis and penetration of mononuclear cells on day 4 PI, and subsequent follicular lymphoid depletion on days 6 and 8 PI was observed. Based on the immunohistochemical test, on day 3 in cecal tonsils and spleen, and on day 8 PI, all of them were positive for virus antigen. In conclusion, the NDV circulating in Iranian chicken flocks has the potential to cause severe illness in commercial turkeys., (© 2021. Sociedade Brasileira de Microbiologia.)

Newcastle disease virus (NDV) has caused significant outbreaks in South-East Asia, particularly in Indonesia in recent years. Recently emerged genotype VII NDVs (NDV-GVII) have shifted their tropism from gastrointestinal/respiratory tropism to a lymphotropic virus, invading lymphoid organs including spleen and bursa of Fabricius to cause profound lymphoid depletion. In this study, we aimed to identify candidate genes and biological pathways that contribute to the disease caused by this velogenic NDV-GVII. A transcriptomic analysis based on RNA-Seq of spleen was performed in chickens challenged with NDV-GVII and a control group. In total, 6361 genes were differentially expressed that included 3506 up-regulated genes and 2855 down-regulated genes. Real-Time PCR of ten selected genes validated the RNA-Seq results as the correlation between them is 0.98. Functional and network analysis of Differentially Expressed Genes (DEGs) showed altered regulation of ElF2 signalling, mTOR signalling, proliferation of cells of the lymphoid system, signalling by Rho family GTPases and synaptogenesis signalling in spleen. We have also identified modified expression of IFIT5, PI3K, AGT and PLP1 genes in NDV-GVII infected chickens. Our findings in activation of autophagy-mediated cell death, lymphotropic and synaptogenesis signalling pathways provide new insights into the molecular pathogenesis of this newly emerged NDV-GVII., (© 2021. The Author(s).)

Among livestock species, poultry and small ruminants are of particular importance to rural women in low- and middle-income countries, as means to generate income, provide nutritious food for the family, accumulate wealth, and confer social status. Newcastle disease (ND) and Peste des Petits Ruminants (PPR) are widespread livestock diseases of poultry and small ruminants, respectively. While both diseases are vaccine preventable, numerous constraints limit the availability of and access to livestock vaccines, especially among the most vulnerable populations in developing countries. The literature on equity and effectiveness of livestock vaccine distribution systems has emphasized many of these constraints, however a gendered analysis and deeper understanding of the vaccine system remain insufficient. This paper applies a gendered and intersectional transformational approach, or GITA, to highlight how gender and other social factors affect the provision and utilization of vaccines for ND and PPR diseases in the region of Kaffrine, Senegal. We first articulate and describe the vaccine value chains (VVCs) for these diseases in Kaffrine, and then analyze the gendered and intersectional dynamics at different nodes of the VVCs, including actors at the national level, through the regional and district levels, down to providers of animal health at community level and the livestock keepers themselves. Our findings indicate that actors' various experiences are shaped and defined mainly by rigid gender norms, location and remoteness, and to a lesser degree by other social stratifications of age, ethnicity, and livelihood. Given the significant role that gender norms play in the livestock vaccine value chains, differences according to the livestock species, regulation of vaccine administration, and vaccine distribution systems emerge as highly relevant for understanding barriers that women specifically face within the livestock vaccination system., Competing Interests: The authors have declared that no competing interests exist.

Chitosan is a non-toxic biological material, but chitosan is insoluble in water, which hinders the development and utilization of chitosan. Chitosan derivatives N -2-Hydroxypropyl trimethyl ammonium chloride ( N -2-HACC) and carboxymethyl chitosan (CMCS) with good water solubility were synthesized by our laboratory. In this study, we synthesized mesoporous SiO 2 nanoparticles by the emulsion, and then the mesoporous SiO 2 nanoparticles were modified with γ-aminopropyltriethoxysilane to synthesize aminated mesoporous SiO 2 nanoparticles; CMCS and N -2-HACC was used to cross-link the aminated mesoporous SiO 2 nanoparticles to construct SiO 2 @CMCS- N -2-HACC nanoparticles. Because the aminated mesoporous SiO 2 nanoparticles with positively charged can react with the mucous membranes, the virus enters the body mainly through mucous membranes, so Newcastle disease virus (NDV) was selected as the model drug to evaluate the performance of the SiO 2 @CMCS- N -2-HACC nanoparticles. We prepared the SiO 2 @CMCS- N -2-HACC nanoparticles loaded with inactivated NDV (NDV/SiO 2 @CMCS- N -2-HACC). The SiO 2 @CMCS- N -2-HACC nanoparticles as delivery carrier had high loading capacity, low cytotoxicity, good acid resistance and bile resistance and enteric solubility, and the structure of NDV protein encapsulated in the nano vaccine was not destroyed. In addition, the SiO 2 @CMCS- N -2-HACC nanoparticles could sustain slowly released NDV. Therefore, the SiO 2 @CMCS- N -2-HACC nanoparticles have the potential to be served as delivery vehicle for vaccine and/or drug.

Newcastle disease virus (NDV) is endemic in Bangladesh and is a major threat to commercial poultry operations. While complete fusion (F) genes are recommended for molecular characterization and classification of NDV isolates, heretofore, only partial F gene data have been available for Bangladeshi NDVs. To this end, we obtained the full-length F gene coding sequences of 11 representative NDVs isolated in Bangladesh between 2010 and 2017. In addition, one of the viruses (MK934289/chicken/Bangladesh/C161/2010) was used in an experimental infection of chickens to establish the viral pathotype and study gross and microscopic lesions. Phylogenetic analysis provided evidence that all studied Bangladeshi isolates belong to genotype XIII.2 of class II NDVs. Six of the viruses were isolated between 2010 and 2017 and grouped together with isolates from neighbouring India during 2013-2016. Another four Bangladeshi isolates (2010-2016) formed a separate monophyletic branch within XIII.2 and showed high nucleotide distance from the isolates from India and the other six Bangladeshi viruses within the sub-genotype; however, none of these groups fulfils all classification criteria to be named as a separate sub-genotype. The eleventh Bangladeshi virus studied here (C162) was genetically more distant from the remaining isolates. It out-grouped the viruses from sub-genotypes XIII.2.1 and XIII.2.2 and showed more than 9.5 % nucleotide distance from all genotype XIII sub-genotypes. This isolate may represent an NDV variant that is evolving independently from the other viruses in the region. The experimental infection in chickens revealed that the tested isolate (C161) is a velogenic viscerotropic virus. Massive haemorrhages, congestion and necrosis in different visceral organs, and lymphoid depletion in lymphoid tissues, typical for infection with velogenic NDV, were observed. Our findings demonstrate the endemic circulation of sub-genotype XIII.2 in Southcentral Asia and further genetic diversification of these viruses in Bangladesh and neighbouring India. This constant evolution of the viruses may lead to the establishment of new genetic groups in the region. Additional historical and prospective virus and surveillance data from the region and neighbouring countries will allow a more detailed epidemiological inference.

Replication of Newcastle disease virus (NDV) is regulated by various host mechanisms, but the role of the extracellular signal-regulated kinase (ERK) pathway in regulating NDV replication is an open question. In this study, the relationship between the ERK pathway and NDV replication was investigated. NDV activated the ERK signaling in chicken embryo fibroblasts at the late stage of infection, correlating to expression of viral proteins. Specific blockage of the ERK pathway activation significantly decreased the transcription and translation levels of viral genes as well as virus replication and the cytopathogenic effect caused by NDV. Our results demonstrate that activation of the ERK pathway is required for NDV replication.

Background: The frequent outbreaks of Newcastle disease virus (NDV) in Iraq pose a constant threat to commercial poultry, despite the introduction of routine vaccination programmes. Several factors, particularly stress factors and coinfections, might play a role in increasing NDV outbreaks in poultry species., Objectives: The current study was aimed to characterize an NDV isolate from an outbreak in North Baghdad, Iraq., Methods: Clinical pathogenicity of the isolate was determined experimentally in chickens. In vitro studies included cytopathological examination, as well as molecular and phylogenetic analyses., Results: Based on the clinical studies and pathogenicity indices (mean death time and intracerebral and intravenous pathogenicity indices), the isolate was characterized as velogenic (highly virulent). Reverse transcriptase polymerase chain reaction targeting the partial fusion protein gene of the NDV genome confirmed the detection. Partial sequencing of the hypervariable region of the fusion gene identified the presence of an avirulent (lentogenic) fusion protein motif (GRQGRL). Phylogenetic analysis of the new isolate along with previously known regional isolates revealed that the new isolate was related to genotype II strains. Additionally, sequence analysis indicated a distinct genetic lineage of the new isolate, which was related to some of the lineages identified in previous outbreaks in the Middle East., Conclusion: The current study offers essential information on the epidemiology, characteristics and diagnosis of NDV for disease control in Iraq. The isolate was found to belong to genotype II and possess an avirulent fusion protein motif., (© 2020 The Authors. Veterinary Medicine and Science Published by John Wiley & Sons Ltd.)

Several pigeon paramyxovirus-1 (PPMV-1) outbreaks in feral pigeons were described recently in Switzerland. The potential of PPMV-1 to induce the notifiable Newcastle disease in chickens is discussed controversially. Therefore, in order to study epidemiologically relevant parameters such as the kinetics of PPMV-1 replication and shedding as well as seroconversion after infection, chickens were infected experimentally with a Swiss PPMV-1 isolate. This generated also defined sample material for the comparison of diagnostic tests. The infectivity of the Swiss PPMV-1 isolate for chickens was demonstrated successfully by virus shedding after experimental inoculation. Our data suggest that long-lasting shedding for up to 60 days can occur in chickens infected with PPMV-1. The isolate used here was of low pathogenicity for chickens. Different quantitative reverse transcription PCR assays were evaluated with a set of Swiss PPMV-1 isolates, and various samples from experimentally infected chickens were analysed with respect to their suitability for viral RNA detection. At 14 days post-infection, virus genome was detected mainly in spleen, caecal tonsils, heart, cloacal swabs, liver, proventriculus, duodenum and kidney tissue samples. Overall, the level of virus replication was low. Not all assays used routinely in diagnostics were capable of detecting viral genome from the isolates tested. Possible explanations are the genetic divergence of PPMV-1 and the low level of viral RNA in the samples. In contrast, two methods that are not used routinely proved more suitable for virus-genome detection. Importantly, the collection of material from various different organs is recommended, in addition to the kidney and brain analysed routinely. In conclusion, this study shows that there is a need to reconsider the type of samples and the protocols used for the detection of PPMV-1 RNA in chickens.

The traditional vaccine strains, such as LaSota, do not completely prevent the shedding of NDV. An ideal vaccine which could not only prevent the clinical signs, but significantly reduce the shedding of NDV is urgently needed for the eradication of ND. In this study, an NDV isolate APMV-1/Chicken/China (SC)/PT3/2016 (hereafter referred as PT3) was identified as a class Ⅰ NDV and a lentogenic strain. The antigenic relationship between PT3 and 3 other NDV strains, including vaccine strain LaSota and 2 prevalent genotype Ⅶd and Ⅵb strains were analyzed. The protective efficacy of PT3 and LaSota against challenge with genotype Ⅶd and Ⅵb strains were assessed. The antigenic analysis result showed that 4 strains belong to the single serotype and the PT3 antiserum exhibited the highest HI titer against 3 other NDV strains. The results of protective efficacy showed that both of LaSota and PT3 could provide 100% survivability for infected chickens. However, PT3 performed better in inducing higher humoral responses and reducing virus shedding than the LaSota strain. Lentogenic strains from Class I NDV appear to be promising vaccine candidates for the control of ND, and allows for the easy discrimination of field NDV and vaccine strains., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2019 International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved.)

Newcastle disease (ND), caused by Avian orthoavulavirus 1 (AOAV-1), affects multiple avian species around the globe. Frequent disease outbreaks are not uncommon even in vaccinates despite routine vaccination and, in this regards, viruses of diverse genotypes originating from natural reservoirs (migratory waterfowls) play an important role in a disease endemic setting. Though genomic characterization of waterfowl originated viruses has been well-elucidated previously, there is a paucity of data on clinico-pathological assessment of mallard-originated sub-genotype VII.2 in commercial chickens. Hence, the current study was designed to evaluate its transmission potential, tissue tropism and micro- and macroscopic lesions in commercial broilers. Based on complete genome and complete F gene, phylogenetic analysis clustered the study isolate within genotype VII and sub-genotype VII.2 in close association with those reported previously from multiple avian species worldwide. The study strain was found to be velogenic on the basis of typical residue pattern in the F-protein cleavage site ( 112 R-RQ-K-R↓F 117 ), sever disease induction in chicken, tissue tropism and subsequent clinico-pathological characteristics. Giving a clear evidence of horizontal transmission, a 100% mortality was observed by 4 th and 6 th day post infection (dpi) in chickens challenged with the virus and those kept with the challenged birds (contact birds), respectively. The observed clinical signs, particularly the greenish diarrhea, and macroscopic lesions such as pinpoint hemorrhages in proventriculus and caecal tonsils were typical of the infection caused by an AOAV-1 in chickens. The virus exhibited a broad tissue tropism where genomic RNA corresponding to study virus was detected in all of the tissues collected from recently mortile and necropsied birds. The study concludes that mallard-originated Avian orthoavulavirus 1 is highly velogenic to commercial chicken and therefore ascertain continuous disease monitoring and surveillance of migratory/aquatic fowls to better elucidate infection epidemiology and subsequent potential impacts on commercial poultry., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Newcastle disease (ND), caused by virulent Avian avulavirus 1 (AAvV 1), affects a wide range of avian species worldwide. Recently, several AAvVs of diverse genotypes have emerged with varying genomic and residue substitutions, and subsequent clinical impact on susceptible avian species. We assessed the clinico-pathological influence of two different AAvV 1 pathotypes [wild bird originated-velogenic strain (sub-genotype VIIi, MF437287) and feral pigeon originated-mesogenic strain (sub-genotype VIm, KU885949)] in commercial broiler chickens and pigeons. The velogenic strain caused 100% mortality in both avian species while the mesogenic strain caused 0% and 30% mortality in chickens and pigeons, respectively. Both strains showed tissue tropism for multiple tissues including visceral organs; however, minor variances were observed according to host and pathotype. The observed gross and microscopic lesions were typical of AAvV 1 infection. Utilizing oropharyngeal and cloacal swabs, a comparable pattern of viral shedding was observed for both strains from each of the infected individuals of both avian species. The study concludes a varying susceptibility of chickens and pigeons to different wild bird-originated AAvV 1 pathotypes and, therefore, suggests continuous monitoring and surveillance of currently prevailing strains for effective control of the disease worldwide, particularly in disease-endemic countries.

Newcastle disease virus (NDV) causes severe infectious disease in poultry and selectively kills tumor cells, by inducing apoptosis and cytokines secretion. In this report, we study the mechanisms underlying NDV-induced apoptosis by investigating the unfolded protein response (UPR). We found that NDV infection activated all three branches of the UPR signaling (PERK-eIF2α, ATF6, and IRE1α) and triggered apoptosis, in avian cells (DF-1 and CEF) and in various human cancer cell types (HeLa, Cal27, HN13, A549, H1299, Huh7, and HepG2). Interestingly, the suppression of either apoptosis or UPR led to impaired NDV proliferation. Meanwhile, the inhibition of UPR by 4-PBA protected cells from NDV-induced apoptosis. Further study revealed that activation of PERK-eIF2α induced the expression of transcription factor CHOP, which subsequently promoted apoptosis by downregulating BCL-2/MCL-1, promoting JNK signaling and suppressing AKT signaling. In parallel, IRE1α mediated the splicing of XBP1 mRNA and resulted in the translation and nuclear translocation of XBP1s, thereby promoting the transcription of ER chaperones and components of ER-associated degradation (ERAD). Furthermore, IRE1α promoted apoptosis and cytokines secretion via the activation of JNK signaling. Knock down and overexpression studies showed that CHOP, IRE1α, XBP1, and JNK supported efficient virus proliferation. Our study demonstrates that the induction of eIF2α-CHOP-BCL-2/JNK and IRE1α-XBP1/JNK signaling cascades promote apoptosis and cytokines secretion, and these signaling cascades support NDV proliferation.

In this study, we investigated which structural proteins of pigeon paramyxovirus type 1 (PPMV-1) are responsible for its low pathogenicity in chickens. The results revealed that the pathogenicity of the virus is determined by multiple genes. The NP protein and F protein were found to have the strongest individual effect on virulence, and this effect further enhanced when the two proteins were expressed in combination. Our study highlights the influence of the NP and F proteins on the pathogenicity of PPMV-1 in chickens.

Newcastle disease virus (NDV) causes serious economic losses to the poultry industry. In our previous study, we found that NDV induced a strong innate immune response in the chicken embryo and bursa of Fabricius (BF). However, the underlying mechanisms by which NDV escapes the host innate immunity are not well-understood. The suppressor of cytokine signaling 3 (SOCS3) inhibits the type I interferon-dependent antiviral signaling pathway by utilizing a feedback loop. In this study, we analyzed the transcriptome data of the chicken embryo and BF infected with NDV and found significant upregulation of SOCS3. Next, we demonstrated that NDV infection and nonstructural V protein induced the up-regulation of SOCS3. Furthermore, we showed that overexpression of SOCS3 facilitated viral replication and reduced the expression of phosphorylation STAT1, MX1, and OASL, while inhibition of SOCS3 with siRNAs reduced virus replication and promoted the expression of phosphorylation STAT1, MX1, and OASL. Finally, we demonstrated that the MEK/ERK signaling pathway was involved in the expression of SOCS3 mediated by NDV infection and V protein transfection, and using specific inhibitor U0126 to block this signaling pathway attenuated SOCS3 expression and inhibited NDV replication through promoting the expression of type I interferon, OASL and MX1. Taken together, these data demonstrate that NDV infection and NDV nonstructural V protein activates the expression of SOCS3 at the mRNA and protein level through a mechanism dependent on the MEK/ERK signaling pathway, which benefits virus replication.

Cancer cell metastasis and its dissemination are most enigmatic and challenging aspects in the development of its therapeutics. Newcastle disease virus (NDV) is a well-studied avian paramyxovirus frequently isolated from birds and rarely from mammals. Since the first report of its oncolytic property, many NDV strains were studied for its effect in various cancer cells. In the present study, NDV strain Bareilly was characterized for its apoptotic potential and migration inhibition in human oral cancer cells. The NDV mediated apoptosis was confirmed by flow cytometry, DNA laddering, and immunoblotting. Moreover, NDV decreased the mitochondrial membrane potential suggesting an intrinsic pathway of apoptosis in oral cancer cells. NDV infection in oral cancer cells results in migration inhibition by a reduction in levels of MMP-7. MMP-7 is one of the key target genes of β-catenin. While overexpression of MMP-7 reversed the inhibitory effect of NDV mediated migration suggested its possible involvement. Wnt/β-catenin is an essential pathway for cell growth, differentiation, and metastasis. The involvement of the Wnt/β-catenin pathway in NDV infection has never been reported. Our results showed that NDV dysregulates Wnt/β-catenin by down-regulation of p-Akt and p-GSK3β leading to degradation of β-catenin. Furthermore, NDV infection leads to a reduction in cytoplasmic and nuclear levels of β-catenin. The study will provide us with a better insight into the molecular mechanism of NDV mediated oncolysis and the key cellular partners involved in the process.

The extracts of Paulownia tomentosa ( P. tomentosa ) exhibit multiple pharmacological activities. In the present study, P. tomentosa flower polysaccharides (PTFP) were extracted by water decoction and ethanol precipitation, and the immunologic modulations of PTFP against Newcastle disease (ND) vaccine was investigated in chickens. The results showed that in a certain range of concentrations, PTFP treatment can dose-dependently enhance lymphocyte proliferation. Then, 280 14-days-old chickens were randomly divided into seven groups, and vaccinated with ND vaccine except blank control (BC) group. At the first vaccination, chickens were orally administrated with PTFP at concentration ranging from 0 to 50 mg/kg once a day for 3 successive days, and the BC group was treated with physiological saline. The lymphocyte proliferation rate, serum antibody titer, and levels of interferon-γ (IFN-γ) were respectively measured on 7, 14, 21, and 28 days after the first vaccination. The results showed that PTFP at the suitable doses could significantly promote lymphocyte proliferation, enhance serum antibody titer, and improve serum IFN-γ concentrations. Taken together, these data indicated that PTFP could improve the immune efficacy against ND vaccine in chickens, and could be as the candidate of a new-type immune adjuvant., (© 2019 The Author(s).)

Limited deep studies are available in the field of early stages of pathogenesis of Newcastle disease virus (NDV) infection and tissue tropism of NDV. In this study, 24 specific pathogen free (SPF) chickens of white leghorn breed were infected with Newcastle disease (ND) by intranasal administration of 10⁵ 50% EID50/0.1 mL of velogenic NDV (vNDV). A second group of 15 chickens were kept as a control group. Chickens were monitored every day to record clinical signs. Infected chickens were euthanized by cervical dislocation at successive times, namely at hours (hrs) 2, 4, 6, 12, days 1, 2, 4, and 6 post-inoculation (pi). Whereas, control group chickens were euthanized on days 0, 1, 2, 4, and 6 pi. Tissues of brain, trachea, lung, caecal tonsil, liver, kidney, spleen, heart, proventriculus, intestine, and thymus were collected, fixed in 10% buffered formalin, embedded in paraffin, and sectioned. HS staining, immunoperoxidase staining (IPS) and in situ PCR were applied. It was concluded that at hr 2 pi, virus seemed to be inclined to trachea and respiratory tract. Meanwhile, it attacked caecal tonsils, intestine and bursa of Fabricus. While primary viraemia was ongoing, virus created footing in kidney and thymus. At hr 4 pi, proventriculus, liver, and spleen were attacked. However, at hr 6 pi, brain and heart were involved. Secondary viraemia probably started as early as hr 12 pi since all collected tissues were positive. Tissue tropism was determined in trachea, caecal tonsil, liver, bursa of Fabricius, intestine, proventriculus, lung, spleen, thymus, kidney, heart, and brain., (Copyright © 2019. Published by Elsevier Ltd.)

Autophagy triggered by glycoprotein-mediated membrane fusion has been reported for several paramyxoviruses. However, the function of HN and F glycoproteins of NDV and their role in autophagy induction have not been studied. Here, we found that co-transfection of HN and F of virulent NDV rapidly induced syncytium formation and triggered a steady state autophagy flux in adenocarcinomic human alveolar basal epithelial (A549) cells and chicken embryo fibroblast (DF-1) cells. Furthermore, we clearly identified that F and HN synergistically induced autophagosome fusion with lysosomes for subsequent degradation. The seven cleavage site mutations of F significantly decreased the autophagy induction, compared with those of wildtype virulent F. RNAi and pharmacological experiments suggested that autophagy benefitted membrane fusion and syncytium formation induced by F and HN of NDV. Activated F 1 co-operated with HN to stimulate AMPK kinase and downstream ULK1 activation to suppress mTORC1 signaling. Our data described the synergistic role of HN and F in the induction of completed autophagic flux through the activation of AMPK- mTORC1- ULK1 pathway., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Newcastle disease (ND), affecting over 250 bird species, is caused by the Newcastle disease virus (NDV). ND is one of the leading causes of morbidity and mortality in pigeons. Most studies investigating NDV in pigeons have focused on the epidemiology and pathogenicity of the virus. However, the host immune responses in pigeons infected with NDVs remains largely unclear. In this study, we investigated the host immune responses in pigeons infected with two NDV stains, a pigeon paramyxovirus type 1(PPMV-1) strain, GZH14, and a genotype II virus, KP08. Although no mortality was observed upon infection with either virus, obvious neurological effects were observed in the GZH14-infected pigeons but not in the KP08-infected pigeons. Both viruses could replicate in the examined tissues, namely brain, lung, spleen, trachea, kidney, and bursa of Fabricius. The expression level of RIG-I, IL-6, IL-1β, CCL5, and IL-8 were up-regulated by both viruses in the brain, lung and spleen at 3 and 7 days post-infection. Notably, these proinflammatory cytokines and chemokines showed more intense expression in the brain, when induced by the GZH14 strain than with the KP08 strain. These results indicate that the intense inflammatory responses induced by PPMV-1 in the brain may be a critical determinant of neurological symptoms in pigeons infected with PPMV-1. Our study provides new insight into the pathogenicity of PPMV-1 in pigeons attributable to the host immune responses., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The 3' and 5' terminal regions of Newcastle disease virus (NDV) genome are cis-acting regulatory elements involved in replication, transcription, and packaging of genomic and anti-genomic viral RNA. There are 6 different nucleotides (nts) at the 3' and 34 different nts at the 5' end of genome in the velogenic NA-1 strain and lentogenic LaSota strain, sharing 90.00% and 70.18% identity, respectively. We investigated the roles of 3' and 5' terminus in the NA-1 strain in viral replication, virulence and pathogenicity. Three NA-1 strain-based recombinant viruses (rNA-L, rNA-T, and rNA-LT) were generated using reverse genetics by either replacing the 3' leader or 5' trailer sequence of NA-1 strain or both with the corresponding sequences of the LaSota strain. Viral replication kinetics and pathogenicity of rNA-L and rNA-T were indistinguishable to that of the parental NA-1 strain, demonstrating that individual replacement or 3' or 5' terminal sequences had little influence. However, the synchronal replacement of both 3' and 5' terminal sequences resulted in decreased viral plaque size, reduced virulence and weaker pathogenicity in 2-week-old chickens. Therefore, our results suggest that the 3' and 5' terminal sequences of NDV genome could only influence the viral virulence when worked collaboratively, while separate replacement would not alter its biological characteristics., (Copyright © 2018. Published by Elsevier Ltd.)

High mobility group box 1 (HMGB1) is a key member of the "danger associated molecular patterns" (DAMPs) which plays important roles in systemic inflammation and has a pathogenic role in infectious diseases like viral or bacterial infections. Newcastle disease virus (NDV) infection is proved to cause intense inflammatory responses that result in excessive cellular apoptosis and tissue damage, but the function of HMGB1 in NDV-induced cytokine storm has not been elucidated. Here, we report that HMGB1 is a significant inflammation factor in NDV infection. HMGB1 is widely distributed in chicken tissues, and the secretion of it was induced by NDV infection both in DF-1 and A549 cells. Moreover, inhibiting the secretion of HMGB1, NDV replication was not significantly reduced, but it is involved in NDV-induced NF-κB activation and the inflammatory response. Further investigation showed that HMGB1 promotes inflammatory cytokine production through the RAGR, TLR2, and TLR4 receptors. HMGB1-RAGE interaction also takes parts in activation of ERK1/2 and JNK induced by NDV infection. Neutralizing HMGB1 in vivo, chicken viability was increased. Pathological changes and inflammatory cytokines expression were reduced under NDV infection, which further confirmed the pathogenic roles of HMGB1 in inflammatory responses. Thus, our findings show that HMGB1 contributes to the inflammatory cytokine storm induced by NDV infection, which played a critical role in viral pathogenesis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Newcastle disease (ND), caused by virulent strains of Newcastle disease virus (NDV), is a devastating disease of poultry worldwide. The pathogenesis of ND in quail is poorly documented. To characterize the ability of virulent NDV strains to replicate and cause disease in quail, groups of 14 two-week-old Japanese quail ( Coturnix japonica) were experimentally inoculated with 10 8 EID 50 (embryo infectious dose 50%) units of 1 of 4 virulent NDV strains: 2 isolated from quail ( N2, N23) and 2 from chickens ( Israel, Pakistan). At day 2 postinfection, noninfected quail (contact group) were added to each infection group to assess the efficacy of virus transmission. Tested NDV strains showed moderate pathogenicity, with highest mortality being 28% for the N2 strain and below 10% for the others. Two N2-inoculated birds showed neurological signs, such as head tremor and ataxia. Microscopic lesions were present in N2-, Israel-, and Pakistan-inoculated birds and consisted of nonsuppurative encephalitis. Contact birds showed no clinical signs or lesions. In both inoculated and contact birds, virus replication was moderate to minimal, respectively, as observed by immunohistochemistry in tissues and virus isolation from oropharyngeal and cloacal swabs. Strains originally isolated from quail resulted in higher numbers of birds shedding in the inoculation group; however, transmission appeared slightly more efficient with chicken-derived isolates. This study shows that virulent NDV strains have limited replicative potential and mild to moderate disease-inducing ability in Japanese quail.

The characteristics and risk factors of pigeon paramyxovirus type 1 (PPMV-1) infection in humans are poorly known. We performed virological, pathological, and epidemiological analyses of a Dutch case, and compared the results with those of a US case. Both infections occurred in transplant patients under immunosuppressive therapy and caused fatal respiratory failure. Both virus isolates clustered with PPMV-1, which has pigeons and doves as reservoir. Experimentally inoculated pigeons became infected and transmitted the virus to naive pigeons. Both patients were likely infected by contact with infected pigeons or doves. Given the large populations of feral pigeons with PPMV-1 infection in cities, increasing urbanization, and a higher proportion of immunocompromised individuals, the risk of severe human PPMV-1 infections may increase. We recommend testing for avian paramyxovirus type 1, including PPMV-1, in respiratory disease cases where common respiratory pathogens cannot be identified.

Pathogenesis of genotype VII Newcastle disease virus (NDV) is characterized with remarkable immunopathology in the spleen in chickens. However, the mechanism for this unique pathological phenotype is not fully understood. Previous transcriptomics data showed that genotype VII NDV JS5/05 caused a greater downregulation of extracellular matrix (ECM) genes than genotype IV virus Herts/33 in the spleen. In this study, the role of ECM in pathology of genotype VII NDV was investigated using quantitative proteomics. Pathology studies showed that JS5/05 caused severe immunopathology characterized with remarkable necrosis in the spleen, whereas Herts/33 only induced mild pathological changes. The ECM was firstly enriched from the spleens and ECM proteins of different categories were identified by LC-MS/MS. Quantitative proteomic analysis showed that JS5/05 caused a significant disruption of ECM integrity and molecular composition compared to Herts/33. Particularly, JS5/05 induced a more remarkable collagen breakdown in the spleen compared to Herts/33. Moreover, matrix metalloproteinase (MMP)-13 and -14 were significantly upregulated by JS5/05 infection. KEGG pathway analysis suggested that differential regulation of ECM proteins by JS5/05 and Herts/33 may impact pathology through different pathways. Therefore, our results suggested that MMP upregulation and consequent ECM degradation contribute to immunopathology of genotype VII NDV in the spleen., Significance: Pathogenesis of genotype VII NDV is characterized with severe immunopathology in the spleen in chickens. Elucidating the mechanism of this pathology phenotype is critical to understand pathogenesis of genotype VII NDV. Here, we present the proteomic data of an important non-cellular compartment, the extracellular matrix (ECM), in the spleen from chickens infected with genotype VII and IV NDVs. Our results suggest that significant upregulation of matrix metalloproteinases by genotype VII NDV and consequent disruption of ECM integrity and composition may be associated with immunopathology in the spleen. Moreover, ECM degradation, represented by collagen breakdown, is an important pathology event in the process of genotype VII NDV infection. Our study for the first time presents evidence of ECM regulation by NDV and adds ECM remodeling as a new manifestation for NDV pathology. Our findings also deepen the understanding of NDV pathogenesis., (Copyright © 2018. Published by Elsevier B.V.)

Infection of chickens with virulent Newcastle disease virus (NDV) is associated with severe pathology and increased morbidity and mortality. The innate immune response contributes to the pathogenicity of NDV. As professional antigen-presenting cells, dendritic cells (DCs) play a unique role in innate immunity. However, the contribution of DCs to NDV infection has not been investigated in chickens. In this study, we selected two representative NDV strains, i.e., the velogenic NDV strain Chicken/Guangdong/GM/2014 (GM) and the lentogenic NDV strain La Sota, to investigate whether NDVs could infect LPS-activated chicken bone-derived marrow DCs (mature chicken BM-DCs). We compared the viral titres and innate immune responses in mature chicken BM-DCs following infection with those strains. Both NDV strains could infect mature chicken BM-DC, but the GM strain showed stronger replication capacity than the La Sota strain in mature chicken BM-DCs. Gene expression profiling showed that MDA5, LGP2, TLR3, TLR7, IFN-α, IFN-β, IFN-γ, IL-1β, IL-6, IL-18, IL-8, CCL5, IL-10, IL-12, MHC-I, and MHC-II levels were altered in mature DCs after infection with NDVs at all evaluated times postinfection. Notably, the GM strain triggered stronger innate immune responses than the La Sota strain in chicken BM-DCs. However, both strains were able to suppress the expression of some cytokines, such as IL-6 and IFN-α, in mature chicken DCs at 24 hpi. These data provide a foundation for further investigation of the role of chicken DCs in NDV infection.

Newcastle disease (ND), characterized by visceral, respiratory, and neurological pathologies, causes heavy economic loss in the poultry industry around the globe. While significant advances have been made in effective diagnosis and vaccine development, molecular mechanisms of ND virus (NDV)-induced neuropathologies remain elusive. In this study, we report the magnitude of oxidative stress and histopathological changes induced by the virulent NDV (ZJ1 strain) and assess the impact of vitamin E in alleviating these pathologies. Comparative profiling of plasma and brains from mock and NDV-infected chicken demonstrated alterations in several oxidative stress makers such as nitric oxide, glutathione, malondialdehyde, total antioxidant capacity, glutathione S-transferase, superoxide dismutase, and catalases. While decreased levels of glutathione and total antioxidant capacity and increased concentrations of malondialdehyde and nitric oxide were observed in NDV-challenged birds at all time points, these alterations were eminent at latter time points (5 days post infection). Additionally, significant decreases in the activities of glutathione S-transferase, superoxide dismutase, and catalase were observed in the plasma and brains collected from NDV-infected chickens. Intriguingly, we observed that supplementation of vitamin E can significantly reduce the alteration of oxidative stress parameters. Under NDV infection, extensive histopathological alterations were observed in chicken brain including neural inflammation, capillary hyperemia, necrosis, and loss of prominent axons, which were reduced with the treatment of vitamin E. Taken together, our findings highlight that neurotropic NDV induces extensive tissue damage in the brain and alters plasma oxidative stress profiles. These findings also demonstrate that supplementing vitamin E ameliorates these pathologies in chickens and proposes its supplementation for NDV-induced stresses., Competing Interests: The authors declare no conflicts of interest.

This study investigated the pathological changes in the reproductive system of laying hens that lead to the poor egg production and quality in Newcastle disease (ND) and the distribution of the virus in the system. Two hundred and forty Isa-Brown pullets were divided randomly into vaccinated and unvaccinated groups (n = 120 each). The vaccinated group was given Hitchner B1 vaccine at 1 day of age, La Sota vaccine at 4 weeks of age and Komarov vaccine at 9 and 16 weeks of age. At the peak of egg production, the laying hens (32 weeks old) were assigned randomly into four groups (n = 60): VC, vaccinated with ND vaccines and inoculated intramuscularly with velogenic viscerotropic ND virus (vvNDV); VU, vaccinated unchallenged; UC, unvaccinated challenged; and UU, unvaccinated unchallenged. UC hens showed depression, diarrhoea and later torticollis. Mortality in UC hens was 90%. VC hens showed mild anorexia. The body weights of the UC hens were significantly (P <0.05) lower than those of UU hens. VC and UC hens showed a significant (P <0.05) drop in egg production. Only UC hens produced abnormal eggs and initially had swollen, oedematous, hyperaemic oviducts followed by atrophy and shortening of the reproductive tract with atresia of the ovarian follicles. The histopathological changes were of necrosis of the epithelium and secretory glands. VC hens showed mild inflammatory changes in the oviduct. Immunohistochemical labelling showed extensive presence of the virus in the ovary, infundibulum, magnum, isthmus, uterus and vagina of UC hens and in the ovary of VC hens. These changes will be the cause of serious egg production problems, especially in vaccinated layers in countries where vvNDV is enzootic., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

The aim of this project is to study the clinical signs and lesion of velogenic Newcastle disease (vND) in commercial turkeys, and also to find out if La Sota vaccination offered protection against these signs and lesions. The cockerels were included as positive controls. One hundred and twenty turkey poults and cockerels were divided into eight groups as follows: unvaccinated unchallenged turkeys (UUT), unvaccinated challenged turkeys (UCT), vaccinated unchallenged turkeys (VUT), vaccinated challenged turkeys (VCT), and along the same lines, the cockerel groups were UUC, UCC, VUC and vaccinated challenged cockerels (VCC). Vaccination was at 3 weeks of age while challenge was at 6 weeks of age. The unvaccinated turkeys and cockerels (UCT and UCC) showed different degrees of depression, diarrhoea and later paralysis at challenge. Total mortality was 100% in cockerels within 6 days, but 60% in turkeys. Similar but milder clinical signs were found in the VCC with a total mortality of 13.3%. The VCT showed mild drop in feed and water consumption, and no mortality. All the challenged groups had significant (p < 0.05) loss of weight when compared with their controls. Necropsy showed that while the UCC had severe proventricular haemorrhages, intestinal and caecal tonsil ulcers, the UCT had no digestive tract lesion. There was severe atrophy of the lymphoid organs in all the challenged groups. Histopathological sections of the bursa, spleen and thymus in all the challenged groups with special emphasis on the vaccinated and unvaccinated turkeys with mortalities of 0 and 60%, respectively, had very severe necrosis and depletion of the lymphoid tissue. Virus was isolated from the cloacal swabs. The haemagglutination inhibition antibodies were significantly higher (p < 0.05) in the challenged groups than the unchallenged. The above observations in the intestinal tracts of UCT are of diagnostic significance while the gross and microscopic lesions in the UCT and VCT show that La Sota vaccination may not protect turkeys against the destruction of the lymphoid organs by vND as earlier reported in chickens. This may lead to immunosuppression and production problems in areas where vND is enzootic.

Background/aims: Newcastle disease virus (NDV) causes a highly devastating and contagious disease in poultry, which is mainly attributed to extensive tissue damages in the digestive, respiratory and nervous systems. However, nature and dynamics of NDV-induced oxidative stresses in the intestine of chickens remain elusive., Methods: In this study, we examined the magnitude of intestinal oxidative stress and histopathological changes caused by the virulent NDV infection, and explored the protective roles of vitamin E (vit. E) in ameliorating these pathological changes. For these purposes, chickens were divided into four groups namely i) non supplemented and non-challenged (negative control, CON); ii) no supplementation of vit. E but challenged with ZJ1 (positive control, NS+CHA); iii) vit. E supplementation at the dose of 50 IU/day/Kg body weight and ZJ1 challenge (VE50+CHA); and 4) vit. E supplementation at the dose of 100 IU/day/Kg body weight and ZJ1 challenge (VE100+CHA). In all groups, we analyzed concentrations of glutathione (GSH), malondialdehyde (MDA), nitric oxide (NO), total antioxidant capacity (T-AOC), and activity of glutathione S-transferase (GST), superoxide dismutase (SOD), catalase (CAT) using biochemical methods. The virus loads were determined by quantitative RT-PCR and antibody titers by hemagglutination inhibition assays. We also examined the histopathological changes in the duodenal and jejunal mucosa at 3 and 5-day post infection (dpi) with NDV., Results: A significant elevation in the NO level was observed in NDV challenged chickens compared to the CON chickens at 2 dpi. The MDA contents were significantly increased whereas GSH was significantly decreased in NDV-challenged chickens compared to control. Furthermore, activities of GST, CAT, SOD, as well as the TOAC were markedly decreased in challenged chickens in comparison with control. Virus copy numbers were higher in NDV infected NS+CHA group compared to other groups. Severe histopathological changes including inflammation, degeneration and broken villi were observed in the intestine of NDV challenged chickens. However, all these malfunctions of antioxidant system and pathological changes in the intestine were partially or completely reversed by the vit. E supplementation., Conclusions: Our results suggest that NDV infection causes oxidative stress and histopathological changes in the duodenum and jejunum of chickens, which can be partially or fully ameliorated by supplementation of vit. E. Additionally, these findings suggest that oxidative stress contributes to the intestinal damages in NDV infected chickens. These findings will help to understand the pathogenesis of NDV and further investigation of therapeutic agents for control of Newcastle disease., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

The nucleocapsid proteins (NPs) of Newcastle disease virus (NDV) and other paramyxoviruses play an important functional role during genomic RNA replication. Our previous study showed that the NP-encoding gene significantly influenced viral replication. Here, we investigated the roles of certain amino acid residues in the NP C-terminus in viral replication and virulence. Results showed that the glutamic acid residue at position 402 (E402) in the C-terminus of the NP is critical for RNA synthesis in the NDV mini-genome system. Mutation of E402 resulted in larger viral plaques that appeared more quickly, and increased the virulence of NDV. Further study indicated that the mutant virus had increased RNA levels during the early stages of virus infection, but that RNA replication was inhibited at later time points. These findings increase our knowledge of viral replication and contribute to a more comprehensive understanding of the virulence factors associated with NDV.

The fusion (F) protein of Newcastle disease virus (NDV) affects viral infection and pathogenicity through mediating membrane fusion. Previously, we found NDV with increased fusogenic activity in which contained T458D or G459D mutation in the F protein. Here, we investigated the effects of these two mutations on viral infection, fusogenicity and pathogenicity. Syncytium formation assays indicated that T458D or G459D increased the F protein cleavage activity and enhanced cell fusion with or without the presence of HN protein. The T458D- or G459D-mutated NDV resulted in a decrease in virus replication or release from cells. The animal study showed that the pathogenicity of the mutated NDVs was attenuated in chickens. These results indicate that these two single mutations in F altered or diminished the requirement of HN for promoting membrane fusion. The increased fusogenic activity may disrupt the cellular machinery and consequently decrease the virus replication and pathogenicity in chickens., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Although vaccination has been hugely successful in protecting birds against infection by the New castle disease virus (NDV), newly-emerged highly virulent strains have been found to overcome established immune protection and threaten the poultry industry. The need to improve the immunization efficacy is, therefore, urgent. Here, we tested the potential immunostimulatory adjuvant activity of the adenoviral-expressed recombinant chicken granulocyte monocyte colony stimulating factor (rchGM-CSF) in an inactivated Newcastle Disease Virus (NDV) vaccine. 126 commercial layer chicks, divided into six groups, were first vaccinated at day 7, followed by a subsequent boost and later an intramuscular challenge at day 21 and 35 respectively. rchGM-CSF expressed by adenovirus raised NDV-specific hemagglutinin-inhibition (HI) titers from 10 to 12 (log2) and significantly upregulated the production of interferon α/β/γ (IFN-α/β/γ), interleukin-4 (IL-4) and major histocompatibility complex II (MHC-II) in spleens. Crucially, chicks inoculated with the inactivated NDV vaccine plus the rchGM-CSF adjuvant displayed only mild clinical signs, lower tissue viral loads, fewer tissue lesions, and decreased mortality and viral shedding than those in the group immunized with the vaccine alone. Our present work has demonstrated that chicken GM-CSF may act as an enhancer in the orchestration of host immune responses induced by the inactivated NDV vaccine. The molecule, expressed by an adenovirus, has the potential to be used as an immune adjuvant to improve protection by NDV vaccination., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Newcastle disease is an important disease of poultry caused by virulent strains of Newcastle disease virus (NDV). During the 1998 to 2002 outbreaks of Newcastle disease in Australia, it was observed that the mild clinical signs seen in some chickens infected with NDV did not correlate with the viruses' virulent fusion protein cleavage site motifs or standard pathogenicity indices. The pathogenicity of 2 Australian NDV isolates was evaluated in experimentally challenged chickens based on clinical evaluation, histopathology, immunohistochemistry, and molecular techniques. One of these virus isolates, Meredith/02, was shown to induce only very mild clinical signs with no mortalities in an experimental setting, in contrast to the velogenic Herts 33/56 and Texas GB isolates. This minimal pathogenicity was associated with decreased virus replication and antigen distribution in tissues. This demonstrates that the Australian Meredith/02 NDV, despite possessing a virulent fusion protein cleavage site, did not display a velogenic phenotype.

Pigeon paramyxovirus serotype 1 (PPMV-1) is a globally distributed, virulent member of the avian paramyxovirus serotype 1 serogroup that causes mortality in columbiformes and poultry. Following introduction into the United States in the mid-1980s, PPMV-1 rapidly spread causing numerous mortality events in Eurasian collared-doves ( Streptopelia decaocto) (ECDOs) and rock pigeons ( Columba livia) (ROPIs). The investigators reviewed pathological findings of 70 naturally infected, free-ranging columbiforms from 25 different mortality events in the United States. Immunohistochemistry targeting PPMV-1 nucleoprotein was used to determine the tissue distribution of the virus in a subset of 17 birds from 10 of the studied outbreaks. ECDOs (61 birds) and ROPIs (9 birds) were the only species in which PPMV-1-associated disease was confirmed by viral isolation and presence of histologic lesions. Acute to subacute tubulointerstitial nephritis and necrotizing pancreatitis were the most frequent histologic lesions, with immunolabeling of viral antigen in renal tubular epithelial cells and pancreatic acinar epithelium. Lymphoid depletion of bursa of Fabricius and spleen was common, but the presence of viral antigen in these organs was inconsistent among infected birds. Hepatocellular necrosis was occasionally present with immunolabeling of hypertrophic Kupffer cells, and immunopositive eosinophilic intracytoplasmic inclusion bodies were present in hepatocytes of 1 ECDO. Immunopositive lymphocytic choroiditis was present in 1 ECDO, while lymphocytic meningoencephalitis was frequent in ROPIs in absence of immunolabeling. This study demonstrates widespread presence of PPMV-1 antigen in association with histologic lesions, confirming the lethal potential of this virus in these particular bird species.

Newcastle disease virus (NDV) is an oncolytic virus which selectively replicates in tumor cells and exerts anti-tumor cytotoxic activity by promoting cell death. In this study, we focus on characterization of the underlying mechanisms of NDV-induced cell death in HeLa cells. We find that NDV Herts/33 strain triggers both extrinsic and intrinsic apoptosis at late infection times. The activation of NF-кB pathway and subsequent up-regulation of TNF-α/TRAIL initiates extrinsic apoptosis, leading to activation of caspase 8 and cleavage of Bid into tBid. tBid transmits the extrinsic apoptotic signals to mitochondria and mediates intrinsic apoptosis, which is hallmarked by cleavage of caspase 9. Moreover, RIP1 is cleaved into RIP1-N and RIP1-C at D324 by caspase 8, and this cleavage promotes apoptosis. Surprisingly, over expression of RIP1 reduces apoptosis and depletion of RIP1 promotes apoptosis, suggesting full length RIP1 is anti-apoptotic. Moreover, necroptosis hallmark protein MLKL is activated by phosphorylation at 12-24 h.p.i., and RIP1 regulates the level of phosphor-MLKL. Immunostaining shows that RIP1 aggregates to stress granules (SGs) at 8-24 h.p.i., and phosphor-MLKL is also recruited to SGs, instead of migrating to plasma membrane to exert its necrotic function. Immunoprecipitation study demonstrates that RIP1 bind to phosphor-MLKL, and depletion of RIP1 reduces the aggregation of MLKL to SGs, suggesting that RIP1 recruits MLKL to SGs. Altogether, NDV infection initiates extrinsic apoptosis via activation of NF-кB and secretion of TNF-α/TRAIL. Activation of caspase 8 by TNF-α/TRAIL and subsequent cleavage of Bid and RIP1 transmit the death signals to mitochondria. Meanwhile, virus subverts the host defensive necroptosis via recruiting phosphor-MLKL by RIP1 to SGs. Thus, RIP1 is a central signaling protein in regulation of apoptosis and necroptosis during NDV infection.

Newcastle disease virus (NDV) can lead to a devastating disease to various avian species including pigeons. Genotype VI NDV is a major cause of Newcastle disease (ND) in Columbiformes (i.e. pigeons and doves). Here, we analyzed the genetic diversity of genotype VI based on the complete F gene sequences of 2 pigeon-origin isolates in northeastern China in 2015, along with 238 genotype VI strains available in GenBank. The phylogenetic tree and evolutionary distances revealed that the 2 new isolates were clustered into a new sub-genotype herein proposed as VIk. Although the 2 isolates contain the 113 RQKRF 117 cleavage site, a feature generally associated with virulent NDV strains, the values of ICPI and MDT showed lentogenic characteristics. The challenge experiment demonstrated that the isolate Pigeon/China/Jilin/NG05/2015 was pathogenic to pigeons, causing lesions in multiple tissues and organs. The emergence and spread of the sub-genotype VIk viruses illustrated that the genotype VI NDV was undergoing evolutionary changes. It is necessary to pay close attention and carry out epidemiological surveillance in pigeons., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Virulent Newcastle disease virus (NDV) was reported to cause rapid depletion of chicken bursa of Fabricius. Severe pathological condition of the organ is commonly associated with high levels of virus replication, intense inflammatory response and also the degree of apoptosis. In this study, the responses of chicken bursa of Fabricius infected with two different strains of velogenic NDV, namely AF2240 and IBS002, were investigated by observing cell population changes, oxidative stress, viral replication and cytokine expression in the organ. Subsequently, apoptosis of enriched bursal IgM+ cells was determined to help us elucidate possible host pathogen relationships between the chicken bursa of Fabricius and NDV infection., Results: The depletion of IgM+ cells and infiltration of macrophages were observed to be higher in bursa infected with AF2240 as compared to IBS002. In line with the increment of the macrophage population, higher nitric oxide (NO) and malondialdehyde (MDA) contents which indicated higher oxidative stress were also detected in bursa infected with NDV AF2240. In addition, higher pro-inflammatory cytokines and chemokine gene expression such as chicken CXCLi2, IL-18 and IFN-γ were observed in AF2240 infected bursa. Depletion of IgM+ cells was further confirmed with increased cell death and apoptosis of the cells in AF2240 infected bursa as compared to IBS002. However, it was found that the viral load for NDV strain IBS002 was comparatively higher than AF2240 although the magnitude of the pro- inflammatory cytokines expression and cell apoptosis was lower than AF2240., Conclusion: The results of our study demonstrated that infection of NDV strains AF2240 and IBS002 caused apoptosis in bursa IgM+ cells and its severity was associated with increased expression of pro-inflammatory cytokines/chemokine, macrophage infiltration and oxidative stress as the infection duration was prolonged. However, of the two viruses, we observed that NDV AF2240 induced a greater magnitude of apoptosis in chicken bursa IgM+ cells in comparison to IBS002. This might be due to the high level of oxidative stress and inflammatory cytokines/chemokine as well as lower IL10 expression which subsequently led to a high rate of apoptosis in the chicken bursa of Fabricius although the detected viral load of AF2240 was lower than IBS002.

Newcastle disease (ND) is an economically important viral disease distressing poultry industry across the globe. Herein, we report the clinicopathology of sub-genotype VIIi Newcastle disease virus (NDV) isolated from peafowl in chickens. The virus isolate produced systemic infection with prominent tropism in visceral organs in chicken, confirmed on the basis of gross and microscopic lesions, and immunohistochemistry findings. The experimentally infected chickens exhibited 100% mortality with severe hemorrhagic lesions in the proventriculus and intestine, especially marked lymphocytolysis in spleen and bursa. The virus could be re-isolated from the cloacal swabs of infected chickens during 4th to 6th dpi (on 6th dpi all birds died), and all were tested positive in conventional RT-PCR. This is the first report on clinicopathology of NDV isolated from peafowl and/or sub-genotype VIIi NDV in experimentally infected chickens. Explorative epidemiological and molecular studies are suggested to screen wild peafowls and poultry flocks of the country for establishing the occurrence of this sub-genotype and opting for appropriate prevention and control strategies., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background: Newcastle disease (ND) and avian influenza subtype H9N2 (H9N2 AI) are two of the most important diseases of poultry, causing severe economic losses in the global poultry industry. Vaccination is an effective way to prevent and control the spread of ND virus (NDV) and H9N2 AI virus (AIV), but the antigenic differences between the current circulating strains and the vaccine strains might account for recent ND and H9N2 AI outbreaks in vaccinated poultry flocks., Methods: We developed an inactivated bivalent H9N2 and NDV vaccine based on the current prevalent strains of H9N2 AIV and NDV in China and evaluated its efficacy in chickens in this study., Results: The results indicated that the inactivated bivalent vaccine could induce a fast antibody response in vaccinated chickens. The hemagglutination inhibition (HI) titer in the sera increased rapidly, and the highest HI titer was observed at 4 weeks post-vaccination (wpv) with a mean titre of 8.6 log 2 for NDV and 9.5 log 2 for H9N2. Up until 15 wpv, HI titers were still detectable at a high level of over 6 log 2 . The immunized chickens showed no signs of disease after challenge at 3 wpv with the prevalent strains of NDV and H9N2 AIV isolated in 2012-2014. Moreover, viral shedding was completely inhibited in vaccinated chickens after challenge with H9N2 AIV and inhibited by at least 90% with NDV compared to the controls at 5dpc., Conclusions: Our findings suggest that the inactivated NDV and H9N2 vaccine induces a fast and strong antibody response in vaccinated chickens and is efficacious in poultry against NDVs and H9N2 AIVs.

The aim of this study was to determine the mechanism by which Newcastle disease virus (NDV) affects eggshell quality. Thirty-week-old specific pathogen free (SPF) egg-laying hens were inoculated with the velogenic genotype VIId NDV strain (infected group) or with inoculating media without virus (control group) by combined intraocular and intranasal routes. The levels of CaBP-D28k mRNA expression in the uterus, a gene related to eggshell quality, were examined by quantitative reverse transcriptase polymerase chain reaction (RT-PCR). The quality of eggshells was analyzed by scanning electron microscopy (SEM). The infected group showed a marked decline in egg production when compared to the control group. The NDV antigen was found more abundantly in the glandular epithelium of the infected hens' uteri from 1 to 15 d post-inoculation (dpi). The levels of CaBP-D28k mRNA expression in the uteri of infected hens were significantly lower than in the control hens from 3 to 15 dpi (P < 0.05). The changes in the Ca concentrations in the eggshells were consistent with the expression of CaBP-D28k mRNA in the infected hens. Ultrastructural examination of eggshells showed significantly reduced shell thickness in the infected hens from 1 to 15 dpi (P < 0.05). Furthermore, obvious changes in the structure of the external shell surface and shell membrane were detected in the infected hens compared with the control hens. In conclusion, the current study confirmed that velogenic genotype VIId NDV infection is associated with the deterioration of the eggshell quality of the laying hens., (Copyright © 2016. Published by Elsevier Inc.)

Infections of poultry with virulent strains of avian paramyxovirus 1 (APMV-1), also known as Newcastle disease viruses (NDVs), cause Newcastle disease (ND). This highly contagious disease affects poultry and many other species of birds worldwide. In countries where the disease is prevalent, constant monitoring and characterization of isolates causing outbreaks are necessary. In this study, we report the results of pathogenicity testing and phylogenetic analyses of seven NDVs isolated from several regions of Peru between 2004 and 2015. Six viruses had intracerebral pathogenicity indices (ICPIs) of between 1.75 and 1.88, corresponding to a velogenic pathotype. The remaining virus had an ICPI of 0.00, corresponding to a lentogenic pathotype. These results were consistent with amino acid sequences at the fusion protein (F) cleavage site. All velogenic isolates had the polybasic amino acid sequence 112 RRQKR↓F 117 at the F cleavage site. Phylogenetic analyses of complete F gene sequences showed that all isolates are classified in class II of APMV-1. The velogenic viruses are classified in genotype XII, while the lentogenic virus is classified in genotype II, closely related to the LaSota vaccine strain. Moreover, tree topology, bootstrap values, and genetic distances observed within genotype XII resulted in the identification of novel subgenotypes XIIa (in South America) and XIIb (in China) and possibly two clades within genotype XIIa. All velogenic Peruvian viruses belonged to subgenotype XIIa. Overall, our results confirm the presence of genotype XII in Peru and suggest that it is the prevalent genotype currently circulating in our country. The phylogenetic characterization of these isolates helps to characterize the evolution of NDV and may help with the development of vaccines specific to our regional necessities.

Newcastle disease virus (NDV) is a pathogen that most often infects poultry species. In investigating a 2014 outbreak of encephalitis and death among farmed mink (Mustela vison), we found pathological and later experimental evidence that NDV can infect and cause severe encephalitic and pneumonic disease in these animals. Our findings confirm the host range of NDV., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study, we investigated the pathogenesis of Newcastle disease virus (NDV) in the chicken kidney. Twenty-six 32-day-old specific pathogen-free chickens were intranasally inoculated with the 9a5b NDV mutant isolate. Kidney tissue samples, collected at 6 and 12 hours postinoculation (hpi) and 1, 2, 3, 5, and 10 days postinoculation (dpi), were analyzed by histopathology, immunohistochemistry (IHC), reverse transcription polymerase chain reaction (RT-PCR), and virus titration. Histopathologically, tubulointerstitial nephritis was detected in the renal cortex and predominantly in the medulla. Nephrotropism of 9a5b NDV was confirmed by IHC, RT-PCR, and virus isolation. Massive degenerative changes and infiltration of CD3-immunopositive cells accompanied replication of the 9a5b NDV isolate in chicken kidneys. In conclusion, pathological changes that were caused by NDV in chicken kidneys were similar to those caused by avian influenza virus, infectious bronchitis virus, and avian nephritis virus, and this highlights the importance of including NDV in the differential diagnosis of kidney disease in chickens.

Disease outbreak investigations were carried out in three states of Northern India namely Haryana (Rewari), Uttar Pradesh (Noida) and Delhi, where a total of 110 Indian peafowls (Pavo cristatus) showed sudden onset of nervous signs and died within a period of two weeks during June, 2012. The F (fusion) gene-based RT-PCR detection of Newcastle disease virus (NDV) in affected tissues confirmed the presence of the virus. Three NDV isolates were selected (one from each area under investigation) and further characterized. They were found to be of virulent pathotype (velogenic NDV) based on both pathogenicity assays (MDT, ICPI and IVPI) and partial F gene sequence analysis. Additionally, the phylogenetic analysis revealed that the isolates belonged to the genotype VIIi and XIII of class II avian Paramyxovirus serotype1 (APMV-1) and related closely to new emerging sub-genotypes. This is the first report regarding the presence of the fifth panzootic vNDV genotype VIIi from India. In this scenario, extensive epidemiological studies are suggested for surveillance of NDV genotypes in wild birds and poultry flocks of the country along with adopting suitable prevention and control measures.