Diagnosis of sub-clinical coccidiosis in fast growing broiler chickens by MicroRNA profiling

Coccidiosis in broiler chickens, caused by infection with Eimeria spp. remains one of the most economically important production diseases. Development of a genetic biomarker panel of sub-clinical infection would be an important biological tool for the management of broiler flocks.We analysed expression of MicroRNAs (miRNAs) to determine the potential for these in diagnosing coccidiosis in broiler flocks. miRNA expression, in the ilea of Ross 308 broilers, was compared between chickens naturally clinically or sub-clinically infected with Eimeria maxima and Eimeria acervulina using NextSeq 500 sequencing. 50 miRNAs with greatest coefficient of variance were determined and principal component analysis showed that these miRNAs clustered within the clinical and sub-clinical groups much more closely than uninfected controls. Following false detection rate analysis and quantitative PCR we validated 3 miRNAs; Gallus gallus (gga)-miR-122-5p, gga-miR-205b and gga-miR-144-3p, which may be used to diagnose sub-clinical coccidiosis.     

Infection with street strain rabies virus induces modulation of the microRNA profile of the mouse brain

ABSTRACT: BACKGROUND: Rabies virus (RABV) causes a fatal infection of the central nervous systems (CNS) of warm-blooded animals. Once the clinical symptoms develop, rabies is almost invariably fatal. The mechanism of RABV pathogenesis remains poorly understood. Recent studies have shown that microRNA (miRNA) plays an important role in the pathogenesis of viral infections. Our recent findings have revealed that infection with laboratory-fixed rabies virus strain can induce modulation of the microRNA profile of mouse brains. However, no previous report has evaluated the miRNA expression profile of mouse brains infected with RABV street strain. RESULTS: The results of microarray analysis show that miRNA expression becomes modulated in the brains of mice infected with street RABV. Quantitative real-time PCR assay of the differentially expressed miRNAs confirmed the results of microarray assay. Functional analysis showed the differentially expressed miRNAs to be involved in many immune-related signaling pathways, such as the Jak-STAT signaling pathway, the MAPK signaling pathway, cytokine-cytokine receptor interactions, and Fc gamma R-mediated phagocytosis. The predicted expression levels of the target genes of these modulated miRNAs were found to be correlated with gene expression as measured by DNA microarray and qRT-PCR. CONCLUSION: RABV causes significant changes in the miRNA expression profiles of infected mouse brains. Predicted target genes of the differentially expression miRNAs are associated with host immune response, which may provide important information for investigation of RABV pathogenesis and therapeutic method.     

Integrated analysis of miRNA and mRNA expression profiles in response to gut microbiota depletion in the abdomens of female Bactrocera dorsalis

Insect gut microbiota has been reported to participate in regulating host multiple biological processes including metabolism and reproduction. However, the corresponding molecular mechanisms remain largely unknown. Recent studies suggest that microRNAs (miRNAs) are involved in complex interactions between the gut microbiota and the host. Here, we used next-generation sequencing technology to characterize miRNA and mRNA expression profiles and construct the miRNA–gene regulatory network in response to gut microbiota depletion in the abdomens of female Bactrocera dorsalis. A total of 3016 differentially expressed genes (DEGs) and 18 differentially expressed miRNAs (DEMs) were identified. Based on the integrated analysis of miRNA and mRNA sequencing data, 229 negatively correlated miRNA–gene pairs were identified from the miRNA–mRNA network. Gene ontology enrichment analysis indicated that DEMs could target several genes involved in the metabolic process, oxidation–reduction process, oogenesis, and insulin signaling pathway. Finally, real-time quantitative polymerase chain reaction further verified the accuracy of RNA sequencing results. In conclusion, our study provides the profiles of miRNA and mRNA expressions under antibiotics treatment and provides an insight into the roles of miRNAs and their target genes in the interaction between the gut microbiota and its host.     

The Porcine MicroRNA Transcriptome Response to Transmissible Gastroenteritis Virus Infection

Transmissible gastroenteritis virus (TGEV; Coronaviridae family) causes huge economic losses to the swine industry. MicroRNAs (miRNAs) play a regulatory role in viral infection and may be involved in the mammalian immune response. Here, we report a comprehensive analysis of host miRNA expression in TGEV-infected swine testis (ST) cells. Deep sequencing generated 3,704,353 and 2,763,665 reads from uninfected ST cells and infected ST cells, respectively. The reads were aligned to known Sus scrofa pre-miRNAs in miRBase 19, identifying 284 annotated miRNAs. Certain miRNAs were differentially regulated during TGEV infection. 59 unique miRNAs displayed significant differentially expression between the normal and TGEV-infected ST cell samples: 15 miRNAs were significantly up-regulated and 44 were significantly down-regulated. Stem-loop RT-PCR was carried out to determine the expression levels of specific miRNAs in the two samples, and the results were consistent with those of sequencing. Gene ontology enrichment analysis of host target genes demonstrated that the differentially expressed miRNAs are involved in regulatory networks, including cellular process, metabolic process, immune system process. This is the first report of the identification of ST cell miRNAs and the comprehensive analysis of the miRNA regulatory mechanism during TGEV infection, which revealed the miRNA molecular regulatory mechanisms for the viral infection, expression of viral genes and the expression of immune-related genes. The results presented here will aid research on the prevention and treatment of viral diseases.     

MicroRNA profiling in the bursae of Marek's disease virus-infected resistant and susceptible chicken lines

Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a cell-associated oncogenic alpha-herpesvirus, Marek's disease virus (MDV). Clinical signs of MD include bursal/thymic atrophy, neurologic disorders, and T cell lymphomas. MiRNAs play key roles in regulation of gene expression by targeting translational suppression or mRNA degradation. MDV encodes miRNAs that are associated with viral pathogenicity and oncogenesis. In this study, we performed miRNA sequencing in the bursal tissues, non-tumorous but viral-induced atrophied lymphoid organ, from control and infected MD-resistant and susceptible chickens at 21 days post infection. In addition to some known miRNAs, a minimum of 300 novel miRNAs were identified in each group that mapped to the chicken genome with no sequence homology to existing miRNAs in chicken miRbase. Comparative analysis identified 54 deferentially expressed miRNAs between the chicken lines that might shed light on underlying mechanism of bursal atrophy and resistance or susceptibility to MD.     

MicroRNA expression profile in RAW264.7 cells in response to Brucella melitensis infection

MicroRNA (miRNA) is small non-coding RNA with approximate 22 nt in length. Recent studies indicate that miRNAs play significant roles in pathogen-host interactions. Brucella organisms are Gram-negative facultative intracellular bacteria that cause Brucellosis. Brucella strains infect macrophages and establish chronic infection by altering host life activities including apoptosis and autophagy. Here, we report a comprehensive analysis of miRNA expression profiles in mock- and Brucella-infected RAW264.7 cells using high-throughput sequencing approach. In total, 344 unique miRNAs were co-expressed in the two libraries, in which 57 miRNAs were differentially expressed. Eight differentially expressed miRNAs with high abundance were subjected to further analysis. The GO enrichment analysis suggests that the putative target genes of these differentially expressed miRNAs are involved in apoptosis, autophagy and immune response. In particular, a total of 25 target genes are involved in regulating apoptosis and autophagy, indicating that these miRNAs may play important regulatory roles in the Brucella-host interactions. Furthermore, the interactions of miR-1981 and its target genes, Bcl-2 and Bid, were validated by luciferase assay. The results show that miR-1981 mimic up-regulated the luciferase activity of psiCHECK-2 Bcl-2 3' UTR, but the luciferase activity of psiCHECK-2 Bid 3' UTR was not changed significantly. Taken together, these data provide valuable framework on Brucella induced miRNA expression in RAW264.7 cells, and suggest that Brucella may establish chronic infection by regulating miRNA expression profile.     

Small RNA and degradome deep sequencing reveals important roles of microRNAs in cotton (Gossypium hirsutum L.) response to root-knot nematode Meloidogyne incognita infection

Investigation of cotton response to nematode infection will allow us to better understand the cotton immune defense mechanism and design a better biotechnological approach for efficiently managing pest nematodes in cotton. In this study, we firstly treated cotton by root knot nematode (RKN, Meloidogyne incognita) infections, then we employed the high throughput deep sequencing technology to sequence and genome-widely identify all miRNAs in cotton; finally, we analyzed the functions of these miRNAs in cotton response to RKN infections. A total of 266 miRNAs, including 193 known and 73 novel miRNAs, were identified by deep sequencing technology, which belong to 67 conserved and 66 novel miRNA families, respectively. A majority of identified miRNA families only contain one miRNA; however, miR482 family contains 14 members and some others contain 2–13 members. Certain miRNAs were specifically expressed in RKN-infected cotton roots and others were completely inhibited by RKN infection. A total of 50 miRNAs were differentially expressed after RKN infection, in which 28 miRNAs were up-regulated and 22 were inhibited by RKN treatment. Based on degradome sequencing, 87 gene targets were identified to be targeted by 57 miRNAs. These miRNA-targeted genes are involved in the interaction of cotton plants and nematode infection. Based on GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, 466 genes from all 636 miRNA targets were mapped to 6340 GO terms, 181 genes from 228 targets of differentially expressed miRNAs were mapped to 1588 GO terms. The GO terms were then categorized into the three main GO classes: biological processes, cellular components, and molecular functions. The targets of differentially expressed miRNAs were enriched in 43 GO terms, including 22 biological processes, 10 cellular components, and 11 molecular functions (p < 0.05). Many identified processes were associated with organism responses to the environmental stresses, including regulation of nematode larval development, response to nematode, and response to flooding. Our results will enhance the study and application of developing new cotton cultivars for nematode resistance.     

Identification of Wolbachia-responsive microRNAs in the two-spotted spider mite,Tetranychus urticae

Background

The two-spotted spider mite, Tetranychus urticae, is infected with Wolbachia, which have the ability to manipulate host reproduction and fitness. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in many biological processes such as development, reproduction and host-pathogen interactions. Although miRNA was observed to involve in Wolbachia-host interactions in the other insect systems, its roles have not been fully deciphered in the two-spotted spider mite.

Results

Small RNA libraries of infected and uninfected T. urticae for both sexes (in total four libraries) were constructed. By integrating the mRNA data originated from the same samples, the target genes of the differentially expressed miRNAs were predicted. Then, GO and pathway analyses were performed for the target genes. Comparison of libraries showed that Wolbachia infection significantly regulated 91 miRNAs in females and 20 miRNAs in males, with an overall suppression of miRNAs in Wolbachia-infected libraries. A comparison of the miRNA and mRNA data predicted that the differentially expressed miRNAs negatively regulated 90 mRNAs in females and 9 mRNAs in males. An analysis of target genes showed that Wolbachia-responsive miRNAs regulated genes with function in sphingolipid metabolism, lysosome function, apoptosis and lipid transporting in both sexes, as well as reproduction in females.

Conclusion

Comparisons of the miRNA and mRNA data can help to identify miRNAs and miRNA target genes involving in Wolbachia-host interactions. The molecular targets identified in this study should be useful in further functional studies.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1122) contains supplementary material, which is available to authorized users.     

Differential regulation of the Let-7 family of microRNAs in CD4+ T cells alters IL-10 expression

MicroRNAs (miRNAs) are ～22-nt small RNAs that are important regulators of mRNA turnover and translation. Recent studies have shown the importance of the miRNA pathway in HIV-1 infection, particularly in maintaining latency. Our initial in vitro studies demonstrated that HIV-1-infected HUT78 cells expressed significantly higher IL-10 levels compared with uninfected cultures. IL-10 plays an important role in the dysregulated cytotoxic T cell response to HIV-1, and in silico algorithms suggested that let-7 miRNAs target IL10 mRNA. In a time course experiment, we demonstrated that let-7 miRNAs fall rapidly following HIV-1 infection in HUT78 cells with concomitant rises in IL-10. To show a direct link between let-7 and IL-10, forced overexpression of let-7 miRNAs resulted in significantly reduced IL-10 levels, whereas inhibition of the function of these miRNAs increased IL-10. To demonstrate the relevance of these results, we focused our attention on CD4(+) T cells from uninfected healthy controls, chronic HIV-1-infected patients, and long-term nonprogressors. We characterized miRNA changes in CD4(+) T cells from these three groups and demonstrated that let-7 miRNAs were highly expressed in CD4(+) T cells from healthy controls and let-7 miRNAs were significantly decreased in chronic HIV-1 infected compared with both healthy controls and long-term nonprogressors. We describe a novel mechanism whereby IL-10 levels can be potentially modulated by changes to let-7 miRNAs. In HIV-1 infection, the decrease in let-7 miRNAs may result in an increase in IL-10 from CD4(+) T cells and provide the virus with an important survival advantage by manipulating the host immune response.