Integrative study of chicken lung transcriptome to understand the host immune response during Newcastle disease virus challenge.

Introduction: Newcastle disease is one of the significant issues in the poultry industry, having catastrophic effects worldwide. The lung is one of the essential organs which harbours Bronchus-associated lymphoid tissue and plays a vital role in the immune response. Leghorn and Fayoumi breeds are known to have differences in resistance to Newcastle disease. Along with genes and long non-coding RNAs (lncRNAs) are also known to regulate various biological pathways through gene regulation. Methods: This study analysed the lung transcriptome data and identified the role of genes and long non-coding RNAs in differential immune resistance. The computational pipeline, FHSpipe, as used in our previous studies on analysis of harderian gland and trachea transcriptome was used to identify genes and lncRNAs. This was followed by differential expression analysis, functional annotation of genes and lncRNAs, identification of transcription factors, microRNAs and finally validation using qRT-PCR. Results and discussion: A total of 8219 novel lncRNAs were identified. Of them, 1263 lncRNAs and 281 genes were differentially expressed. About 66 genes were annotated with either an immune-related GO term or pathway, and 12 were annotated with both. In challenge and breed-based analysis, most of these genes were upregulated in Fayoumi compared to Leghorn, and in timepoint-based analysis, Leghorn challenge chicken showed downregulation between time points. A similar trend was observed in the expression of lncRNAs. Co-expression analysis has revealed several lncRNAs co-expressing with immune genes with a positive correlation. Several genes annotated with non-immune pathways, including metabolism, signal transduction, transport of small molecules, extracellular matrix organization, developmental biology and cellular processes, were also impacted. With this, we can understand that Fayoumi chicken showed upregulated immune genes and positive cis-lncRNAs during both the non-challenged and NDV-challenge conditions, even without viral transcripts in the tissue. This finding shows that these immune-annotated genes and coexpressing cis-lncRNAs play a significant role in Fayoumi being comparatively resistant to NDV compared to Leghorn. Our study affirms and expands upon the outcomes of previous studies and highlights the crucial role of lncRNAs during the immune response to NDV. Conclusion: This analysis clearly shows the differences in the gene expression patterns and lncRNA co-expression with the genes between Leghorn and Fayoumi, indicating that the lncRNAs and co-expressing genes might potentially have a role in differentiating these breeds. We hypothesise that these genes and lncRNAs play a vital role in the higher resistance of Fayoumi to NDV than Leghorn. This study can pave the way for future studies to unravel the biological mechanism behind the regulation of immune-related genes.

In-silico analysis of cattle blood transcriptome to identify lncRNAs and their role during bovine tuberculosis.

Long noncoding RNAs (lncRNAs) are RNA molecules with a length greater than 200 nucleotides that do not code for functional proteins. Although, genes play a vital role in immune response against a disease, it is less known that lncRNAs also contribute through gene regulation. Bovine tuberculosis is a significant zoonotic disease caused by Mycobacterium bovis (M. bovis) in cattle. Here, we report the in-silico analysis of the publicly available transcriptomic data of calves infected with M. bovis. A total of 51,812 lncRNAs were extracted across all the samples. A total of 216 genes and 260 lncRNAs were found to be differentially expressed across all the 4 conditions-infected vs uninfected at 8- and 20-week post-infection (WPI), 8 vs 20-WPI of both infected and uninfected. Gene Ontology and Functional annotation showed that 8 DEGs were annotated with immune system GOs and 2 DEGs with REACTOME immune system pathways. Co-expression analysis of DElncRNAs with DEGs revealed the involvement of lncRNAs with the genes annotated with Immune related GOs and pathways. Overall, our study sheds light on the dynamic transcriptomic changes in response to M. bovis infection, particularly highlighting the involvement of lncRNAs with immune-related genes. The identified immune pathways and gene-lncRNA interactions offer valuable insights for further research in understanding host-pathogen interactions and potential avenues for genetic improvement strategies in cattle.

Breed and timepoint-based analysis of chicken harderian gland transcriptome during Newcastle disease virus challenge.

Introduction: Newcastle disease is a highly infectious disease caused by the Newcastle Disease Virus (NDV) and has a devastating financial impact on the global chicken industry. It was previously established that Leghorn and Fayoumi breeds of chicken exhibit variable resistance against NDV infection. The harderian gland is the less studied tissue of the chicken, known to play an essential role in the immune response. Methods: Our previous study, we reported differential gene expression and long noncoding RNAs (lncRNAs) between challenged and non-challenged chickens in the Harderian gland transcriptomic data. Now, we report the analysis of the same data studying the differential expression patterns between Leghorn and Fayoumi and between different timepoints during disease. First, the pipeline FHSpipe was used for identification of lncRNAs, followed by differential expression analysis by edgeR (GLM), functional annotation by OmicsBox, co-expression analysis using WGCNA and finally validation of selected lncRNAs and co-expressing genes using qRT-PCR. Results: Here, we observed that Leghorn showed a higher number of upregulated immune-related genes than Fayoumi in timepoint-based analysis, especially during the initial stages. Surprisingly, Fayoumi, being comparatively resistant, showed little difference between challenged and non-challenged conditions and different time points of the challenge. The breed-based analysis, which compared Leghorn with Fayoumi in both challenged and non-challenged conditions separately, identified several immune-related genes and positive co-expressing cis lncRNAs to be upregulated in Fayoumi when compared to Leghorn in both challenged and non-challenged conditions. Discussion: The current study shows that Leghorn, being comparatively more susceptible to NDV than Fayoumi, showed several immune-related genes and positive co-expressing cis lncRNAs upregulated in challenged Leghorn when compared to non-challenged Leghorn and also in different timepoints during challenge. While, breed-based analysis showed that there were more upregulated immune genes and positive cis-lncRNAs in Fayoumi than Leghorn. This result clearly shows that the differences in the expression of genes annotated with immune-related GO terms and pathways, i.e., immune-related genes and the co-expressing cis-lncRNAs between Leghorn and Fayoumi, and their role in the presence of differences in the resistance of Leghorn and Fayoumi chicken against NDV. Conclusion: These immune-genes and cis-lncRNAs could play a role in Fayoumi being comparatively more resistant to NDV than Leghorn. Our study elucidated the importance of lncRNAs during the host defense against NDV infection, paving the way for future research on the mechanisms governing the genetic improvement of chicken breeds.

Integrated analysis of genes and long non-coding RNAs in trachea transcriptome to decipher the host response during Newcastle disease challenge in different breeds of chicken.

Newcastle disease is a highly infectious economically devastating disease caused by Newcastle disease Virus in Chicken (Gallus gallus). Leghorn and Fayoumi are two breeds which show differential resistance patterns towards NDV. This study aims to identify the differentially expressed genes and lncRNAs during NDV challenge which could play a potential role in this differential resistance pattern. A total of 552 genes and 1580 lncRNAs were found to be differentially expressing. Of them, 52 genes were annotated with both Immune related pathways and Gene ontologies. We found that most of these genes were upregulated in Leghorn between normal and challenged chicken but several were down regulated between different timepoints after NDV challenge, while Fayoumi showed no such downregulation. We also observed that higher number of positively correlating lncRNAs was found to be downregulated along with these genes. This shows that although Leghorn is showing higher number of differentially expressed genes in challenged than in non-challenged, most of them were downregulated during the disease between different timepoints. With this we hypothesize that the downregulation of immune related genes and co-expressing lncRNAs could play a significant role behind the Leghorn being comparatively susceptible breed than Fayoumi. The computational pipeline is available at https://github.com/Venky2804/FHSpipe.

Identification and differential expression of long non-coding RNAs and their association with XIST gene during early embryonic developmental stages of Bos taurus.

X-chromosomes inactivation (XCI) is a phenomenon that aims to equalize the dosage of X-linked gene products between XY males and XX females in mammals. XIST gene is the master regulator of X chromosome inactivation during early embryonic developmental stages of Bos taurus. Biological molecule such as lncRNA plays significant role in the control of XCI, by RNA-based regulatory mechanisms and are non-coding regions of the genome. In our study, using in-silico transcriptome data analysis approach, we analysed RNA-seq data of E35, E39 and E43 samples from bovine genital ridges of early embryonic stages, and identified lncRNA transcripts. More than 7 lakh lncRNA transcripts were identified. Further, our study identified DE-lncRNAs and genes between male and female and studied their co-expression. More than four thousand differentially expressed lncRNAs identified. The co-expression and RT-PCR study in the result showed that there exists an association between the XIST and DE-lncRNAs in early embryonic gonads of bovine at E35. In this study, the association between DE-lncRNAs and XIST gene indicates, the potentially important role of DE-lncRNAs during embryo development in bovine. In conclusion, this study shows there exist an interplay between genes and lncRNAs at transcriptome level of bovine during early embryonic days.

In silico prediction of the animal susceptibility and virtual screening of natural compounds against SARS-CoV-2: Molecular dynamics simulation based analysis.

COVID-19 is an infectious disease caused by the SARS-CoV-2 virus. It has six open reading frames (orf1ab, orf3a, orf6, orf7a, orf8, and orf10), a spike protein, a membrane protein, an envelope small membrane protein, and a nucleocapsid protein, out of which, orf1ab is the largest ORF coding different important non-structural proteins. In this study, an effort was made to evaluate the susceptibility of different animals against SARS-CoV-2 by analyzing the interactions of Spike and ACE2 proteins of the animals and propose a list of potential natural compounds binding to orf1ab of SARS-CoV-2. Here, we analyzed structural interactions between spike proteins of SARS-CoV-2 and the ACE2 receptor of 16 different hosts. A simulation for 50 ns was performed on these complexes. Based on post-simulation analysis, Chelonia mydas was found to have a more stable complex, while Bubalus bubalis, Aquila chrysaetos chrysaetos, Crocodylus porosus, and Loxodonta africana were found to have the least stable complexes with more fluctuations than all other organisms. Apart from that, we performed domain assignment of orf1ab of SARS-CoV-2 and identified 14 distinct domains. Out of these, Domain 3 (DNA/RNA polymerases) was selected as a target, as it showed no similarities with host proteomes and was validated in silico. Then, the top 10 molecules were selected from the virtual screening of ∼1.8 lakh molecules from the ZINC database, based on binding energy, and validated for ADME and toxicological properties. Three molecules were selected and analyzed further. The structural analysis showed that these molecules were residing within the pocket of the receptor. Finally, a simulation for 200 ns was performed on complexes with three selected molecules. Based on post-simulation analysis (RMSD, RMSF, Rg, SASA, and energies), the molecule ZINC000103666966 was found as the most suitable inhibitory compound against Domain 3. As this is an in silico prediction, further experimental studies could unravel the potential of the proposed molecule against SARS-CoV-2.

Transcriptomic analysis to infer key molecular players involved during host response to NDV challenge in Gallus gallus (Leghorn & Fayoumi).

Long non-coding RNAs (lncRNAs) are the transcripts of length longer than 200 nucleotides. They are involved in the regulation of various biological activities. Leghorn and Fayoumi breeds of Gallus gallus were known to be having differential resistance against Newcastle Disease Virus (NDV) infection. Differentially expressed genes which were thought to be involved in this pattern of resistance were already studied. Here we report the analysis of the transcriptomic data of Harderian gland of Gallus gallus for studying the lncRNAs involved in regulation of these genes. Using bioinformatics approaches, a total of 37,411 lncRNAs were extracted and 359 lncRNAs were differentially expressing. Functional annotation using co-expression analysis revealed the involvement of lncRNAs in the regulation of various pathways. We also identified 1232 quantitative trait loci (QTLs) associated with the genes interacting with lncRNA. Additionally, we identified the role of lncRNAs as putative micro RNA precursors, and the interaction of differentially expressed Genes with transcription factors and micro RNAs. Our study revealed the role of lncRNAs during host response against NDV infection which would facilitate future experiments in unravelling regulatory mechanisms of development in the genetic improvement of the susceptible breeds of Gallus gallus.

Elucidation of conformational diversity of druggable enzymes and classification of chemical modulators based on inhibitor-bound structures.

Communicated by Ramaswamy H. Sarma.