Identification of sheep lncRNAs related to the immune response to vaccines and aluminium adjuvants.

BACKGROUND: Long non-coding RNAs (lncRNAs) are involved in several immune processes, including the immune response to vaccination, but most of them remain uncharacterised in livestock species. The mechanism of action of aluminium adjuvants as vaccine components is neither not fully understood. RESULTS: We built a transcriptome from sheep PBMCs RNA-seq data in order to identify unannotated lncRNAs and analysed their expression patterns along protein coding genes. We found 2284 novel lncRNAs and assessed their conservation in terms of sequence and synteny. Differential expression analysis performed between animals inoculated with commercial vaccines or aluminium adjuvant alone and the co-expression analysis revealed lncRNAs related to the immune response to vaccines and adjuvants. A group of co-expressed genes enriched in cytokine signalling and production highlighted the differences between different treatments. A number of differentially expressed lncRNAs were correlated with a divergently located protein-coding gene, such as the OSM cytokine. Other lncRNAs were predicted to act as sponges of miRNAs involved in immune response regulation. CONCLUSIONS: This work enlarges the lncRNA catalogue in sheep and puts an accent on their involvement in the immune response to repetitive vaccination, providing a basis for further characterisation of the non-coding sheep transcriptome within different immune cells.

Expression analysis of lung miRNAs responding to ovine VM virus infection by RNA-seq.

BACKGROUND: MicroRNAs (miRNAs) are short endogenous, single-stranded, noncoding small RNA molecules of approximately 22 nucleotides in length. They regulate gene expression posttranscriptionally by silencing mRNA expression, thus orchestrating many physiological processes. The Small Ruminant Lentiviruses (SRLV) group includes the Visna Maedi Virus (VMV) and Caprine Arthritis Encephalitis (CAEV) viruses, which cause a disease in sheep and goats characterized by pneumonia, mastitis, arthritis and encephalitis. Their main target cells are from the monocyte/macrophage lineage. To date, there are no studies on the role of miRNAs in this viral disease. RESULTS: Using RNA-seq technology and bioinformatics analysis, the expression levels of miRNAs during different clinical stages of infection were studied. A total of 212 miRNAs were identified, of which 46 were conserved sequences in other species but found for the first time in sheep, and 12 were completely novel. Differential expression analysis comparing the uninfected and seropositive groups showed changes in several miRNAs; however, no significant differences were detected between seropositive asymptomatic and diseased sheep. The robust increase in the expression level of oar-miR-21 is consistent with its increased expression in other viral diseases. Furthermore, the target prediction of the dysregulated miRNAs revealed that they control genes involved in proliferation-related signalling pathways, such as the PI3K-Akt, AMPK and ErbB pathways. CONCLUSIONS: To the best of our knowledge, this is the first study reporting miRNA profiling in sheep in response to SRLV infection. The known functions of oar-miR-21 as a regulator of inflammation and proliferation appear to be a possible cause of the lesions caused in the sheep's lungs. This miRNA could be an indicator for the severity of the lung lesions, or a putative target for therapeutic intervention.

Evolutionary history of bovine endogenous retroviruses in the Bovidae family.

BACKGROUND: Endogenous retroviruses (ERVs) are genomic elements of retroviral origin that are present in the genomes of almost all vertebrates. In cattle, more than 13,000 elements related to ERVs have been detected, and based on the pol gene, 24 families or groups of bovine ERVs have been described. However, information about ERVs in other bovids and the presence of families of related bovine ERVs in different species of the Bovidae family is scarce. RESULTS: The 24 families of bovine ERVs previously detected in cattle (Bos taurus) were also detected in zebus (Bos indicus) and yaks (Bos grunniens). In addition, six new families, named BoERV25 to BoERV30, were detected in the three Bos species. Five more ruminant species were screened for related ERVs: 26 families were detected in these species, but four families (BoERV24, BoERV26, BoERV28 and BoERV29) were specific to cattle, zebus, yaks and buffalo. An analysis of the homology of the ERVs of cattle, zebus and yaks revealed that the level of LTR divergence was similar between ERVs from cattle and zebus but was less similar between with ERVs from cattle and yaks. In addition, purifying selection was detected in the genes and retroviral regions of clusters of ERVs of cattle, zebus and yaks. CONCLUSIONS: In this work, the 24 ERV families previously identified in cattle were also found in two other species in the Bos genus. In addition, six new bovine ERV families were detected. Based on LTR divergence, the most recently inserted families are from Class II. The divergence of the LTR, used as an indirect estimate of the ERV insertion time, seemed to be influenced by the differences in genome evolution since the divergence of the species. In addition, purifying selection could be acting on clusters of ERVs from different species.

The sheep miRNAome: Characterization and distribution of miRNAs in 21 tissues.

MicroRNAs (miRNAs) are evolutionarily conserved small non-coding RNAs that regulate gene expression. In livestock species, miRNAs also show great potential as biomarkers for animal health and product quality. In sheep, few miRNAs have been described in comparison with other livestock species or model organisms. We uniformly analyzed 172 public ovine small RNA sequencing datasets from 21 different tissues in order to predict conserved and novel miRNA precursors and profile their expression patterns. In addition to the 106 annotated sheep miRNAs, 1047 previously unannotated miRNA precursor sequences were detected and 41 % of them were assigned an orthologue from other close species. Considering expression levels, a set of miRNAs with high sequence conservation were detected in all tissues, while 733 mature miRNAs were robustly expressed in at least one tissue. 270 miRNAs showed high tissue specificity index values. Brain, male reproductive tissues and PBMCs showed the most distinct expression patterns. Strikingly, over one hundred precursors from the ruminant specific family of mir-2284/mir-2285 miRNAs were found, which were enriched in immune related tissues. This work supports the known high conservation of many miRNAs, but also highlights the potential of clade-specific innovations in ruminant evolution.

Identification and characterization of miRNAs in spleens of sheep subjected to repetitive vaccination.

Accumulative evidence has shown that short non-coding RNAs such as miRNAs can regulate the innate and adaptive immune responses. Aluminium hydroxide is a commonly used adjuvant in human and veterinary vaccines. Despite its extended use, its mechanism of action is not fully understood and very few in vivo studies have been done to enhance understanding at the molecular level. In this work, we took advantage of a previous long-term experiment in which lambs were exposed to three different treatments by parallel subcutaneous inoculations with aluminium-containing commercial vaccines, an equivalent dose of aluminium or mock injections. Spleen samples were used for miRNA-seq. A total of 46 and 16 miRNAs were found differentially expressed when animals inoculated with commercial vaccines or the adjuvant alone were compared with control animals, respectively. Some miRNAs previously related to macrophage polarization were found dysregulated exclusively by the commercial vaccine treatment but not in the aluminium inoculated animals. The dysregulated miRNAs in vaccine group let-7b-5p, miR-29a-3p, miR-27a and miR-101-3p are candidates for further research, since they may play key roles in the immune response induced by aluminium adjuvants added to vaccines. Finally, protein-protein interaction network analysis points towards leucocyte transendothelial migration as a specific mechanism in animals receiving adjuvant only.

Amino acid signatures in the Ovar-DRB1 peptide-binding pockets are associated with Ovine Pulmonary Adenocarcinoma susceptibility/resistance.

Potential relationships between amino acid motifs of various alleles of the ovine major histocompatibility complex DR (Ovar-DR) molecule and occurrence of clinical OPA caused by JSRV were investigated in a case-control study. Latxa sheep (n=132) screened for presence/absence of pulmonary OPA lesions were typed for their Ovar-DRB1 2nd exon alleles by PCR and sequence-based typing (PCR-SBT). The polymorphic amino acid residues derived from the obtained 34 DRB1 protein variants were subjected to a logistic regression-based association study. The amino acids at several positions showed significant associations with the presence/absence of pulmonary OPA lesions; some of the residues were located within the peptide binding cleft of the DRB molecule, including pockets P1, P4, P7 and P9.

Human papillomavirus (HPV) genotype 18 variants in patients with clinical manifestations of HPV related infections in Bilbao, Spain.

BACKGROUND: Human papillomavirus (HPV) variants differ in their biological and chemical properties, and therefore, may present differences in pathogenicity. Most authors classified variants based on the phylogenetic analysis of L1 region. Nevertheless, recombination in HPV samples is becoming a usual finding and thus, characterizing genetic variability in other regions should be essential. OBJECTIVES: We aimed to characterize the genetic variability of HPV 18 in 5 genomic regions: E6, E7, E4, L1 and the Upstream Regulatory Region (URR), working with both single infection and multiple HPV infection samples. Furthermore, we aimed to assess the prevalence of HPV 18 variants in our region and look for possible existence of recombination as well as analyze the relationship between these variants and the type of lesion. METHODS: From 2007 to 2010, Clinical Microbiology and Infection Control Department analyzed 44 samples which were positive for HPV 18. Genetic variability was determined in PCR products and variants were assigned to European, Asian-amerindian or African lineage. Recombination and association of variants with different types of lesion was studied. RESULTS: Genetic analysis of the regions revealed a total of 56 nucleotide variations. European, African and Asian-amerindian variants were found in 25/44 (56.8%), 10/44 (22.7%) and 5/44 (11.4%) samples, respectively. We detected the presence of recombinant variants in 2/44 (4.5%) cases. Samples taken from high-grade squamous intraepithelial lesions (H-SIL) only presented variants with specific-african substitutions. CONCLUSIONS: Multiple HPV infection, non-european HPV variants prevalence and existence of recombination are considered risk factors for HPV persistence and progression of intraepithelial abnormalities, and therefore, should be taken into consideration in order to help to design and optimize diagnostics protocols as well as improve epidemiologic studies.Our study is one of the few studies in Spain which analyses the genetic variability of HPV18 and we showed the importance of characterizing more than one genomic region in order to detect recombination and classify HPV variants properly.

Transcriptomic meta-analysis reveals unannotated long non-coding RNAs related to the immune response in sheep.

Long non-coding RNAs (lncRNAs) are involved in several biological processes, including the immune system response to pathogens and vaccines. The annotation and functional characterization of lncRNAs is more advanced in humans than in livestock species. Here, we take advantage of the increasing number of high-throughput functional experiments deposited in public databases in order to uniformly analyse, profile unannotated lncRNAs and integrate 422 ovine RNA-seq samples from the ovine immune system. We identified 12302 unannotated lncRNA genes with support from independent CAGE-seq and histone modification ChIP-seq assays. Unannotated lncRNAs showed low expression levels and sequence conservation across other mammal species. There were differences in expression levels depending on the genomic location-based lncRNA classification. Differential expression analyses between unstimulated and samples stimulated with pathogen infection or vaccination resulted in hundreds of lncRNAs with changed expression. Gene co-expression analyses revealed immune gene-enriched clusters associated with immune system activation and related to interferon signalling, antiviral response or endoplasmic reticulum stress. Besides, differential co-expression networks were constructed in order to find condition-specific relationships between coding genes and lncRNAs. Overall, using a diverse set of immune system samples and bioinformatic approaches we identify several ovine lncRNAs associated with the response to an external stimulus. These findings help in the improvement of the ovine lncRNA catalogue and provide sheep-specific evidence for the implication in the general immune response for several lncRNAs.

Gene Expression Profiling Reveals New Pathways and Genes Associated with Visna/Maedi Viral Disease.

Visna/Maedi virus (VMV) is a lentivirus that infects the cells of the monocyte/macrophage lineage in sheep, goats and wild ruminants. Infection with VMV causes a multisystemic inflammatory disorder, which includes pneumonia, encephalitis, mastitis or arthritis. The immune response to VMV infection is complex, and the infection and pathogenesis of this virus are not totally characterized yet. In this work, a gene expression microarray was used to identify the differentially expressed genes in VMV infection and disease development by comparing sheep with different serologic status and with presence of VM-characteristic clinical lesions. The expression profile analysis has revealed many interesting genes that may be associated with the viral infection process. Among them, the OXT gene appeared significantly up-regulated, so the oxytocin-secreting system could play an essential role in VM disease. Moreover, some of the most significantly enriched functions in up-regulated genes appeared the complement pathway, which (in combination with the Toll-like receptor signaling network) could compose a mechanism in the VMV pathogenesis. Identifying the host genetic factors associated with VMV infection can be applied to develop strategies for preventing infection and develop effective vaccines that lead to therapeutic treatments.

Novel circRNA discovery in sheep shows evidence of high backsplice junction conservation.

Circular RNAs (circRNAs) are covalently closed circular non-coding RNAs. Due to their structure, circRNAs are more stable and have longer half-lives than linear RNAs making them good candidates for disease biomarkers. Despite the scientific relevance of these molecules, the study of circRNAs in non-model organisms is still in its infancy. Here, we analyse total RNA-seq data to identify circRNAs in sheep from peripheral blood mononuclear cells (PBMCs) and parietal lobe cortex. Out of 2510 and 3403 circRNAs detected in parietal lobe cortex and in PBMCs, a total of 1379 novel circRNAs were discovered. Remarkably, around 63% of all detected circRNAs were found to be completely homologous to a circRNA annotated in human. Functional enrichment analysis was conducted for both tissues based on GO terms and KEGG pathways. The enriched terms suggest an important role of circRNAs from encephalon in synaptic functions and the involvement of circRNAs from PBMCs in basic immune system functions. In addition to this, we investigated the role of circRNAs in repetitive vaccination experiments via differential expression analysis and did not detect any significant relationship. At last, our results support both the miRNA sponge and the miRNA shuttle functions of CDR1-AS in sheep brain. To our knowledge, this is the first study on circRNA annotation in sheep PBMCs or parietal lobe cortex samples.