MiRNA expression profiles of chicken embryonic visceral tissue infected with F48E9 or La Sota
ABSTRACT: Three cDNA libraries of mixed visceral tissues from F48E9, La Sota, or uninfected chicken embryos were constructed, and small-RNA deep sequencing was conducted to detect the expression levels of small-RNAs. Intergroup comparisons were used to identify changes in miRNA expression caused by NDV infection. La Sota affected the expression of 61 miRNAs (36 upregulated and 25 downregulated) at 36 hpi, and F48E9 infection altered the expression levels of 66 miRNAs (33 upregulated and 31 downregulated). Overall design: Using high-throughput sequencing, revealed miRNA expression differences of SPF chick embryos infected different virulence NDVs.
Project description:We attempted to characterize the transcriptome of the chicken embryo during Newcastle disease virus (NDV) infection using RNA-sequencing analysis. The cDNAs derived from Total RNA of the pooled visceral tissues infected with F48E9 or La Sota were sequenced and analysed. The collected clean reads covered about 4.02% (2,341,868 reads) of the entire F48E8 reference sequence, while only 0.02% reads (13,886) were mapped to the La Sota genome. RNA-Seq datasets from groups La Sota, F48E9 and control, were respectively mapped to 71.76%, 68.55% and 70.05% of the reference genome Galgal 4.73. Compared with the control, 2,035 and 1,604 differentially expressed genes of hosts were found responding to F48E9 and La Sota infection, respectively. GO and KEGG pathway enriched various signalling pathways with elements playing roles in enhancing or preventing viral infection, like IFP35, NMI, Mx, OAS*A, IFITM5, STAT1 and IFNβ. So far, we know that velogenic NDV made far more transcripts during infection and caused significant impact on the host, showing a large number of genes in various pathways at high levels of expression. Overall design: Using high-throughput sequencing, revealed differences of SPF chick embryos after infected different virulence NDVs.
Project description:BACKGROUND: Newcastle Disease Virus (NDV) has been considered to only infect avian species. However, one paramyxovirus named as Xiny10 was isolated from swine. The differences of Xiny10, another previous swine NDV (JL01) and vaccine strain La Sota were compared on the basis of sequences of the whole-lengthen Fusion (F) gene and biological characteristics. FINDINGS: Through serologic tests and sequence alignment, Xiny10 was proved as NDV. It has great differences with JL01 in virulence, biological characteristics, genotype and amino acid homology of F gene. The sequence alignment showed Xiny10 and La Sota both belonged to genotype II. It shared 97.3% to 98.7% identities with genotype II NDVs, which was higher than these strains from the other genotypes. CONCLUSIONS: These above data suggested that the swine virus was NDV and it might be generated from La Sota.
Project description:Thermostable Newcastle disease virus (NDV) vaccines have been widely used in areas where a "cold-chain" is not reliable. However, the molecular mechanism of NDV thermostability remains poorly understood. In this work, we constructed chimeric viruses by exchanging viral fusion (F) and/or hemagglutinin-neuraminidase (HN) genes between the heat-resistant strain HR09 and thermolabile strain La Sota utilizing a reverse genetic system. The results showed that only chimeras with HN derived from the thermostable virus exhibited a thermostable phenotype at 56°C. The hemagglutinin (HA) and neuraminidase (NA) activities of chimeras with HN derived from the HR09 strain were more thermostable than those containing HN from the La Sota strain. Then, we used molecular dynamics simulation at different temperatures (310 K and 330 K) to measure the HN protein of the La Sota strain. The conformation of an amino acid region (residues 315-375) was observed to fluctuate. Sequence alignment of the HN protein revealed that residues 315, 329, and 369 in the La Sota strain and thermostable strains differed. Whether the three amino acid substitutions affected viral thermostability was investigated. Three mutant viruses based on the thermolabile strain were generated by substituting one, two or three amino acids at positions 315, 369, and 329 in the HN protein. In comparison with the parental virus, the mutant viruses containing mutations S315P and I369V possessed higher thermostablity and HA titers, NA and fusion activities. Taken together, these data indicate that the HN gene of NDV is a major determinant of thermostability, and residues 315 and 369 have important effects on viral thermostability.
Project description:MicroRNAs regulate post-transcriptional gene expression via either translational repression or mRNA degradation. They have important roles in both viral infection and host anti-infection processes. We discovered that the miR-375 is significantly upregulated in Newcastle disease virus (NDV)-infected chicken embryonic visceral tissues using a small RNA sequencing approach. Further research revealed that the overexpression of miR-375 markedly decreases the replication of the velogenic NDV F48E9 and the lentogenic NDV La Sota by targeting the M gene of NDV in DF-1 cells. Interestingly, miR-375 has another target, ELAVL4, which regulates chicken fibrocyte cell cycle progression and decreases NDV proliferation. In addition, miR-375 can influence bystander cells by its secretion in culture medium. Our results indicated that miR-375 is an inhibitor of NDV, but can also enhance NDV growth by reducing the expression of its target ELAVL4. These results emphasize the complex roles of microRNAs in the regulation of viral infections.
Project description:Newcastle disease virus (NDV) is an enveloped RNA virus, bearing severe economic losses to the poultry industry worldwide. Previous virion proteomic studies have shown that enveloped viruses carry multiple host cellular proteins both internally and externally during their life cycle. To address whether it also occurred during NDV infection, we performed a comprehensive proteomic analysis of highly purified NDV La Sota strain particles.In addition to five viral structural proteins, we detected thirty cellular proteins associated with purified NDV La Sota particles. The identified cellular proteins comprised several functional categories, including cytoskeleton proteins, annexins, molecular chaperones, chromatin modifying proteins, enzymes-binding proteins, calcium-binding proteins and signal transduction-associated proteins. Among these, three host proteins have not been previously reported in virions of other virus families, including two signal transduction-associated proteins (syntenin and Ras small GTPase) and one tumor-associated protein (tumor protein D52). The presence of five selected cellular proteins (i.e., ?-actin, tubulin, annexin A2, heat shock protein Hsp90 and ezrin) associated with the purified NDV particles was validated by Western blot or immunogold labeling assays.The current study presented the first standard proteomic profile of NDV. The results demonstrated the incorporation of cellular proteins in NDV particles, which provides valuable information for elucidating viral infection and pathogenesis.
Project description:To investigate the roles and explore the altered expression of microRNAs (miRNAs) and mRNAs in chicken embryos in response to Newcastle disease virus (NDV) infection, deep sequencing was performed. Then, a conjoint analysis of small RNA-seq and mRNA-seq was performed to screen interactional miRNA?mRNA pairs during NDV infection. In total, 15 and 17 up- and downregulated miRNAs were identified that potentially targeted 4279 and 6080 mRNAs in NDV-infected chicken embryonic tissues, respectively; in addition, 595 upregulated and 480 downregulated mRNAs were identified. The conjoint analysis of the obtained data identified 1069 miRNA?mRNA pairs. Among these pairs, 130 pairs were related to immune or inflammatory responses. The relationship between gga-miR-203a and its target transglutaminase 2 (TGM2) was confirmed using a dual-luciferase reporter system and a real time quantitative polymerase chain reaction (RT-qPCR) assay. Overall, the discovery of miRNAs, mRNAs, and their potential pairing relationships, which may be involved in the regulation of NDV infection, will facilitate our understanding of the complex regulatory relationship between the host and the virus.
Project description:Infectious bronchitis coronavirus (IBV), Newcastle disease virus (NDV), and avian influenza virus (AIV) H9 subtype are major pathogens of chickens causing serious respiratory tract disease and heavy economic losses. To better understand the replication features of these viruses in their target organs and molecular pathogenesis of these different viruses, comparative proteomic analysis was performed to investigate the proteome changes of primary target organ during IBV, NDV, and AIV H9 infections, using 2D-DIGE followed MALDI-TOF/TOF-MS. In total, 44, 39, 41, 48, and 38 proteins were identified in the tracheal tissues of the chickens inoculated with IBV (ck/CH/LDL/97I, H120), NDV (La Sota), and AIV H9, and between ck/CH/LDL/97I and H120, respectively. Bioinformatics analysis showed that IBV, NDV, and AIV H9 induced similar core host responses involved in biosynthetic, catabolic, metabolic, signal transduction, transport, cytoskeleton organization, macromolecular complex assembly, cell death, response to stress, and immune system process. Comparative analysis of host response induced by different viruses indicated differences in protein expression changes induced by IBV, NDV, and AIV H9 may be responsible for the specific pathogenesis of these different viruses. Our result reveals specific host response to IBV, NDV, and AIVH9 infections and provides insights into the distinct pathogenic mechanisms of these avian respiratory viruses.
Project description:Although intensive vaccination programs have been implemented, Newcastle disease (ND) outbreaks, accompanied by severe economic losses, are still reported in Egypt. The genetic characterization of ND virus (NDV) strains isolated from ND-vaccinated chicken flocks provides essential information for improving ND control strategies. Therefore, here, 38 NDV strains were isolated and identified from outbreaks among vaccinated flocks of broiler chickens located in the provinces of Qena, Luxor, and Aswan of Upper Egypt during 2011-2013. The investigated broiler chicken flocks (aged 28 to 40 days) had high mortality rates of up to 80%. All NDV isolates were genetically analyzed using next-generation DNA sequencing. From these isolates, 10 representative NDV strains were selected for further genetic analyses. Phylogenetic analysis of full-length coding genes revealed that the Egyptian NDV isolates belonged to a single sub-genotype, VII.1.1. These isolates were phylogenetically distant from the vaccine strains, including La Sota or Clone 30 (genotype II), which have been commonly used to vaccinate chicken flocks. Amino acid substitution K78R was observed in the neutralizing epitopes of the F proteins; whereas several mutations were found in the neutralizing epitopes of the hemagglutinin-neuraminidase proteins, notably, E347K. Overall, our results suggested that the occurrence of neutralizing epitope variants may be one of potential reasons for ND outbreaks. Further studies are needed to determine the protective effect of current vaccines against circulating virulent NDV strains.
Project description:Newcastle disease virus (NDV) causes large losses in the global fowl industry. To better understand NDV replication and transcription cycle, quantitative detection methods for distinguishing NDV genomic RNA (gRNA), antigenomic RNA (cRNA), and messenger RNA (mRNA) in NDV-infected cells are indispensible. Three reverse transcription primers were designed to specifically target the nucleoprotein (NP) region of gRNA, cRNA, and NP mRNA, and a corresponding real-time RT-PCR assay was developed to simultaneously quantify the three types of RNAs in NDV-infected cells. This method showed very good specificity, sensitivity, and reproducibility. The detection range of the assay was between 5.5 × 10(2) and 1.1 × 10(9) copies/?L of the target gene. These methods were applied to investigate the dynamics of the gRNA, cRNA, and mRNA synthesis in NDV La Sota infected DF-1 cells. The results showed that the copy numbers of viral gRNA, cRNA, and NP mRNA all exponentially increased in the beginning. The viral RNA copy number then plateaued at 10'h postinfection and gradually decreased from 16 h postinfection. No synthesis priority was observed between replication (gRNA and cRNA amounts) and transcription (mRNA amounts) during NDV infection. However, the cRNA accumulated more rapidly than gRNA, as the cRNA copy number was three- to tenfold higher than gRNA starting from 2 h postinfection. Conclusion. A real-time RT-PCR for absolute quantitation of specific viral RNA fragments in NDV-infected cells was developed for the first time. The development of this assay will be helpful for further studies on the pathogenesis and control strategies of NDV.
Project description:Heat stress results in reduced productivity, anorexia, and mortality in chickens. The objective of the study was to identify genes and signal pathways associated with heat stress and Newcastle disease virus (NDV) infection in the liver of chickens through RNA-seq analysis, using two highly inbred chicken lines (Leghorn and Fayoumi). All birds were held in the same environment until 14 days of age. On day 14, half the birds were exposed to 38 °C with 50% relative humidity for 4 h, then 35 °C until the end of the experiment. The remaining birds were kept at 25 °C throughout the experiment. The heat-treated birds were inoculated at 21 days of age with 107 EID50 (One EID50 unit is the amount of virus that will infect 50 percent of inoculated embryos) NDV La Sota strain to investigate the effects of both heat stress and NDV infection. Physiological parameters were recorded as blood phenotypes at three stages: acute heat (AH), chronic heat (CH1), and chronic heat combined with NDV infection (CH&NDV), at 4 h, 7 days, and 10 days post-initiation of heat treatment, respectively. Our previous work revealed that the heat-resilient Fayoumi line maintained a more stable acid-base balance in their blood compared to the Leghorn line. Liver samples were harvested on both AH and CH&NDV to characterize the transcriptome profiles of these two inbred lines. Both genetic lines and treatments had large impact on the liver transcriptome. Fayoumi birds had more differentially expressed genes (DEGs) than Leghorn birds for both treatments. Metabolic and immune-related genes were on the DEG list, with Fayoumi having more immune-related DEGs than Leghorns, which was confirmed by gene functional enrichment analysis. Weighted correlation network analysis (WGCNA) indicated that the driver genes such as Solute Carrier Family genes could be very important for stabilizing the acid-base balance in Fayoumi birds during heat stress. Therefore, candidate genes such solute carrier family genes could be potential genetic targets that are regulated by Fayoumis to maintain physical hemostasis under heat stress. Differential gene expression showed that Leghorns mainly performed metabolic regulation in response to heat stress and NDV infection, while Fayoumis regulated both immune and metabolic functions. This study provides novel insights and enhances our understandings of liver response to heat stress of heat resilient and susceptible inbred chicken lines.