In situ cytokine gene expression in early stage of virulent Newcastle disease in chickens

Selected lymphoid and reproductive tissues were examined from groups of 3-week-old chickens and 62-week-old hens that were inoculated choanally and conjunctivally with 106 EID50 of a virulent Newcastle disease virus (NDV) isolate from the California 2018–2020 outbreak, and euthanized at 1, 2, and 3 days postinfection. In the 3-week-old chickens, immunohistochemistry for NDV and for T and B cell lymphocytes, as well as in situ hybridization for IL-1β, IL-6, IFN-γ, and TNF-α revealed extensive expression of IL-1β and IL-6 in lymphoid tissues, often coinciding with NDV antigen. IFN-γ was only expressed infrequently in the same lymphoid tissues, and TNF-α was rarely expressed. T-cell populations initially expanded but by day 3 their numbers were below control levels. B cells underwent a similar expansion but remained elevated in some tissues, notably spleen, cecal tonsils, and cloacal bursa. Cytokine expression in the 62-week-old hens was overall lower than in the 3-week-old birds, and there was more prolonged infiltration of both T and B cells in the older birds. The strong pro-inflammatory cytokine response in young chickens is proposed as the reason for more severe disease.

Molecular Characterization and Pathology of Virulent Newcastle Disease Virus Isolated from Broiler Chickens in District Lahore, Pakistan

Background: This study elucidated the molecular detection and pathological alterations in broiler chickens naturally infected with field circulating NDV strains along with their phylogenomic dynamics. Methods: Morbid tissue samples of diseased/dead chickens were collected from 100 poultry flocks presented to poultry disease diagnostic laboratories from September 2018 to August 2019. Samples were subjected to molecular detection of NDV along with phylogenetic analysis and subsequent gross and histopathological examination. Result: Based on RT-PCR results, the positivity of NDV was 04/100 (4%). Genetic analysis of the NDV Fusion (F) gene revealed 98.92% and 98.74% similarity with Iranian and Pakistani isolates, respectively. The evolutionary tree showed that present study isolates were placed in a clade belongs to genotype Vll sub-genotype i and l. Necropsy examinations revealed the petechial haemorrhages associated with multifocal necrosis in gastrointestinal and respiratory organs. Besides these pathological findings, amino acid sequence of F gene revealed that study isolates are having pathogenic potential similar to the velogenic strains of NDV. Based on all essential analyses, the present study concluded that the evolution and distribution of the Newcastle disease virus of various genotypes VIIi and VIIl in Pakistan are having significant pathogenic potential. Therefore, it emphasizes developing ND vaccine from indigenous strains for better protection of commercial poultry in Pakistan.

A Review on the Anti-Viral Activity of Houttuynia cordata: A Possible Herb for the Management and Treatment of COVID-19

Plants are an important source of natural products and they play a vital role in the field of medicinal chemistry and pharmaceutical science. Traditional medicines have been practiced and used for thousands of years, mostly in Asian countries, where plants are the main sources of medicine. Houttuynia cordata, a herb that belongs to the family Saururaceae, has a wide range of pharmacological activities and is used traditionally in conditions like anisolobis sores, heatstroke, lung carbuncles, malaria, scrotal abscess, tonsillitis, salammoniac poison and has also been widely accepted to possess anti-cancer, anti-oxidant, anti-hypertension, anti-inflammatory, anti-mutagenic, antibacterial, anti-viral and anti-purulent activity. Moreover, it is one of the herbs that was recognized during pandemic outbreaks, such as Severe Acute Respiratory Syndrome Coronavirus (SARS CoV) in China, virulent Newcastle Disease Virus (VNDV) in Java (Indonesia) and Newcastle (England). In this review, we briefly discuss the role of H. cordata as an anti-viral agent and the possibility of developing a dosage form against Coronavirus disease-19 (COVID-19).

Indicators of the molecular pathogenesis of virulent Newcastle disease virus in chickens revealed by transcriptomic profiling of spleen

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.

Isolation and Molecular Characterization of Newcastle Disease Virus in Layers

Background: Newcastle disease (ND) in spite of the availability of vaccines remains a constant threat to poultry producers worldwide. It is prevalent in Indian subcontinent and leads to economic losses. The present study was aimed with isolate and identify virulent Newcastle disease virus (NDV) in layer poultry from field outbreaks.Methods: Total 47 samples consisting of nasal (05), oropharyngeal (13) and cloacal swabs (11) and tissue samples consisting of trachea (07), lungs (06), larynx (05) were collected from layer birds. For isolation of NDV swab and tissue samples were inoculated in 9-11 days old embryonated eggs via allantoic cavity route. After preparing the viral inoculum, 47 suspected samples (29 swab and 18 tissue samples) were inoculated in 141 embryonated eggs to isolate the virus.Result: Out of 47 samples 10 (21.27%) samples were positive for HA activity. All the 10 isolates showing HA activity subjected to Reverse-Transcriptase PCR of F gene and 6 were found positive in RT-PCR for F1 gene. The PCR amplified product showed amplicon at 356 bp and 254 bp positive for F1 and F2 gene, respectively. On basis of F gene, 06 (50%) isolates were considered as virulent Newcastle Disease Virus. One isolate sequence was submitted at NCBI with accession MT890653 On phylogenetic analysis MT890653 designated as Class II/ genotype II/ virulent strain and had the motif 112R-R-R-K-R-F117 at the cleavage site of the fusion protein.

Thymic transcriptome analysis after Newcastle disease virus inoculation in chickens and the influence of host small RNAs on NDV replication

Newcastle disease (ND), caused by virulent Newcastle disease virus (NDV), is a contagious viral disease affecting various birds and poultry worldwide. In this project, differentially expressed (DE) circRNAs, miRNAs and mRNAs were identified by high-throughput RNA sequencing (RNA-Seq) in chicken thymus at 24, 48, 72 or 96 h post LaSota NDV vaccine injection versus pre-inoculation group. The vital terms or pathways enriched by vaccine-influenced genes were tested through KEGG and GO analysis. DE genes implicated in innate immunity were preliminarily screened out through GO, InnateDB and Reactome Pathway databases. The interaction networks of DE innate immune genes were established by STRING website. Considering the high expression of gga-miR-6631-5p across all the four time points, DE circRNAs or mRNAs with the possibility to bind to gga-miR-6631-5p were screened out. Among DE genes that had the probability to interact with gga-miR-6631-5p, 7 genes were found to be related to innate immunity. Furthermore, gga-miR-6631-5p promoted LaSota NDV replication by targeting insulin induced gene 1 (INSIG1) in DF-1 chicken fibroblast cells. Taken together, our data provided the comprehensive information about molecular responses to NDV LaSota vaccine in Chinese Partridge Shank Chickens and elucidated the vital roles of gga-miR-6631-5p/INSIG1 axis in LaSota NDV replication.

Monoclonal antibodies specific for the hemagglutinin-neuraminidase protein define neutralizing epitopes specific for Newcastle disease virus genotype 2.VII from Egypt

Background Newcastle disease is a devastating disease in poultry caused by virulent Newcastle disease virus (NDV), a paramyxovirus endemic in many regions of the world despite intensive vaccination. Phylogenetic analyses reveal ongoing evolution of the predominant circulating genotype 2.VII, and the relevance of potential antigenic drift is under discussion. To investigate variation within neutralization-sensitive epitopes within the protein responsible for receptor binding, i.e. the Hemagglutinin-Neuraminidase (HN) spike protein, we were interested in establishing genotype-specific monoclonal antibodies (MAbs). Methods An HN-enriched fraction of a gradient-purified NDV genotype 2.VII was prepared and successfully employed to induce antibodies in BalbC mice that recognize conformationally intact sites reactive by haemagglutination inhibition (HI). For subsequent screening of mouse hybridoma cultures, an NDV-ELISA was established that utilizes Concanavalin A (ConA-ELISA) coupled glycoproteins proven to present conformation-dependent epitopes. Results Six out of nine selected MAbs were able to block receptor binding as demonstrated by HI activity. One MAb recognized an epitope only present in the homologue virus, while four other MAbs showed weak reactivity to selected other genotypes. On the other hand, one broadly cross-reacting MAb reacted with all genotypes tested and resembled the reactivity profile of genotype-specific polyclonal antibody preparations that point to minor antigenic differences between tested NDV genotpyes. Conclusions These results point to the concurrent presence of variable and conserved epitopes within the HN molecule of NDV. The described protocol should help to generate MAbs against a variety of NDV strains and to enable in depth analysis of the antigenic profiles of different genotypes.

The 16S rRNA gene sequencing of gut microbiota in chickens infected with different virulent Newcastle disease virus

Background Newcastle disease virus (NDV) is pathogenic to chickens, which is characterized by dyspnea, diarrhea, nervous disorder and hemorrhages. However, the influence of different virulent NDV infection on the host gut microbiota composition is still poorly understood. In this study, twenty 21-day-old specific pathogen free chickens were inoculated with either the velogenic Herts33 NDV strain, lentogenic La Sota NDV strain or sterile phosphate buffer solution (PBS). Through 16S rRNA sequencing, the collected fecal samples of control and NDV infected chickens were examined. Results The results showed that the gut microbiota were mainly dominated by Firmicutes, Bacteroidetes and Proteobacteria in both healthy and NDV infected chickens. NDV infection altered the structure and composition of gut microbiota. As compared to PBS group, phylum Firmicutes were remarkably reduced, whereas Proteobacteria was significantly increased in velogenic NDV infected group. While the gut community structure has no significant differences between lentogenic NDV infected group and PBS group at phylum level. At genus level, Escherichia-Shigella was significantly increased in both velogenic and lentogenic NDV infected groups, but the lactobacillus was only remarkably decreased in velogenic NDV infected group. Collectively, different virulent NDV infection resulted in a different alteration of the gut microbiota in chickens, including a loss of probiotic bacteria and a expansion of pathogenic bacteria. Conclusion These results indicated that NDV with different virulence have different impact on chicken gut microbiota and may provide new insights into the intestinal pathogenesis of NDV.