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Topic:RNA
RNA, or ribonucleic acid, is a fundamental molecule involved in various biological processes in horses, including gene expression, protein synthesis, and regulation of cellular activities. RNA plays a critical role in translating genetic information from DNA into proteins, which are essential for maintaining cellular function and overall health in horses. There are different types of RNA, such as messenger RNA (mRNA), transfer RNA (tRNA), and ribosomal RNA (rRNA), each serving distinct functions within the cell. Research on RNA in horses focuses on understanding its role in development, disease mechanisms, and potential therapeutic applications. This page compiles peer-reviewed research studies and scholarly articles that explore the structure, function, and implications of RNA in equine biology and health.
Expressed gene sequences of the equine cytokines interleukin-17 and interleukin-23. This report describes the initial cloning and characterization of the equine interleukin-17 (IL-17) expressed gene sequence from mRNA obtained from equine intestinal tissue and interleukin-23 (IL-23) expressed gene sequence from mRNA obtained from equine peripheral blood mononuclear cells. Equine IL-17 has 462 nucleotides in the translated region, determined by homology with known human and mouse sequences, and shares 84% and 75% identity, respectively. For the deduced amino acid sequences, the identity with human and mouse is 76% and 70%. Equine IL-23 has 579 nucleotides in the translated reg...
Analysis of complete genome sequence of Neorickettsia risticii: causative agent of Potomac horse fever. Neorickettsia risticii is an obligate intracellular bacterium of the trematodes and mammals. Horses develop Potomac horse fever (PHF) when they ingest aquatic insects containing encysted N. risticii-infected trematodes. The complete genome sequence of N. risticii Illinois consists of a single circular chromosome of 879 977 bp and encodes 38 RNA species and 898 proteins. Although N. risticii has limited ability to synthesize amino acids and lacks many metabolic pathways, it is capable of making major vitamins, cofactors and nucleotides. Comparison with its closely related human pathogen N. senn...
Molecular characterization and expression pattern of the equine lactate dehydrogenase A and B genes. The species-specific properties of LDH isozymes are essentially determined by M (muscle) and H (heart) subunit proteins encoded by the LDHA and LDHB genes, respectively. In the present study, we molecularly characterized the full-length equine lactate dehydrogenase A (eLDHA) and B (eLDHB) cDNAs. The eLDHA cDNA consisted of a 999-bp open reading frame (ORF), while the eLDHB and newly acquired bat LDHB consisted of a 1002-bp ORF, which is 3 bp shorter than the LDHB ORF of other registered mammals. The alignment of amino acid sequences showed that eLDHA acquired positively charged His 88 and 226,...
Small interfering RNA targeting bovine papillomavirus type 1 E2 induces apoptosis in equine sarcoid transformed fibroblasts. Equine sarcoids are skin tumours of horses caused by infection with BPV-1 or 2. Maintenance and replication of the viral genome depend upon the viral proteins E1 and E2. We examined the effects of an E2 specific siRNA on E2 and E1 viral gene expression, viral load and cell growth in BPV-1 transformed sarcoid-derived cells. Transfection with E2-siRNA caused a reduction in E2 and E1 mRNA expression as well as viral load, growth inhibition and decreased anchorage-independent growth. siRNA treated cells showed significantly higher apoptosis rates than control cells. Thus sequence specific targetin...
Effects of acute exercise and long-term exercise on total Na+,K+ -ATPase content and Na+,K+ -ATPase isoform expression profile in equine muscle. To investigate the effects of acute exercise and long-term training on Na(+),K(+)-ATPase content, mRNA isoforms, and protein concentration in equine muscle. Methods: 6 Standardbreds. Methods: Horses performed a bout of exercise on a treadmill before and after 18 weeks of combined interval and endurance training. Muscle biopsy specimens were obtained from vastus lateralis muscle (VLM) and pectoralis descendens muscle (PDM) before and after exercise. The Na(+),K(+)-ATPase content, mRNA isoforms, and protein concentrations were determined by use of [(3)H]ouabain binding, real-time PCR assay, and ...
Lineage 2 west nile virus as cause of fatal neurologic disease in horses, South Africa. Serologic evidence suggests that West Nile virus (WNV) is widely distributed in horses in southern Africa. However, because few neurologic cases have been reported, endemic lineage 2 strains were postulated to be nonpathogenic in horses. Recent evidence suggests that highly neuroinvasive lineage 2 strains exist in humans and mice. To determine whether neurologic cases are being missed in South Africa, we tested 80 serum or brain specimens from horses with unexplained fever (n = 48) and/or neurologic signs (n = 32) for WNV. From March 2007 through June 2008, using reverse transcription-PCR (RT-...
Use of serial laminar tissue collection via biopsy in conscious healthy horses. To determine the feasibility of performing serial laminar and skin biopsies on sedated horses and whether sampling affected adjacent tissues. Methods: 6 horses. Methods: Laminar tissues were harvested via biopsy through the hoof wall from healthy conscious horses via sedation and regional anesthesia. Eight specimens were collected at 4 time points during 24 hours from a single foot. Laminar biopsy specimens were harvested with a 6-mm-diameter biopsy punch after burring through the horny corium to the stratum medium. Skin biopsy specimens were collected from an area proximal to the coronary ban...
Synthesis and characterization of biologically active recombinant elk and horse FSH. The objective of this investigation was to clone and express the elk and horse common alpha-subunit and FSH beta-subunit cDNAs, and to produce recombinant FSH from both species in vitro. The RNAs extracted from elk and horse pituitary glands were reverse-transcribed and amplified by polymerase chain reaction. The cDNAs corresponding to both subunits of elk and horse were cloned into the expression vector pBudCE4.1 and transfected into CRL-9096 cells. Expression of both genes was determined in the transfected cells by Northern and Western blot analysis. Recombinant elk and horse FSH secreted in...
In silico detection and characteristics of novel microRNA genes in the Equus caballus genome using an integrated ab initio and comparative genomic approach. The importance of microRNAs at the post-transcriptional regulation level has recently been recognized in both animals and plants. We used the simple but effective sequential method of first Blasting known animal miRNAs against the horse genome and then using the located candidates to search for novel miRNAs by RNA folding method in the vicinity (+ -500 bp) of the candidates. Here, a total of 407 novel horse miRNA genes including 354 mature miRNAs were identified, of these, 75 miRNAs were grouped into 32 families based on seed sequence identity. MiRNA genes tend to be present as clusters in som...
Equine thrombospondin II and secreted protein acidic and cysteine-rich in a model of normal and pathological wound repair. Wound healing in horses is complicated, particularly when wounds are on the limb. The objectives of this study were to clone equine thrombospondin II (THBS2) and secreted protein acidic and cysteine-rich (SPARC) cDNAs and to compare the spatiotemporal expression of mRNAs and proteins during repair of body and limb wounds. These molecules were targeted in view of their potential biological contribution to angiogenesis, which is exacerbated during the repair of limb wounds in horses. Cloning was achieved by screening size-selected cDNA libraries previously derived from 7-day-old wounds. Expressi...
In vivo evolution of the gp90 gene and consistently low plasma viral load during transient immune suppression demonstrate the safety of an attenuated equine infectious anemia virus (EIAV) vaccine. To study the in vivo evolution of the attenuated Chinese equine infectious anemia virus (EIAV) vaccine, viral gp90 gene variation and virus replication in immunosuppressed hosts were investigated. The results showed that after vaccination, the gp90 gene followed an evolutionary trend of declining diversity. The trend coincided with the maturation of immunity to EIAV, and eventually, the gp90 gene became highly homologous. The sequences of these predominant quasispecies were consistently detected up to 18 months after vaccination. Furthermore, after transient immune suppression with dexamethaso...
Prostaglandin H synthase Type 2 is differentially expressed in endometrium based on pregnancy status in pony mares and responds to oxytocin and conceptus secretions in explant culture. The equine embryo must signal its presence to the uterus for pregnancy to continue to term. Mobility of the conceptus throughout the uterus is crucial for its survival, and this action presumably permits the conceptus to transmit its antiluteolytic signal to the endometrium. Studies were completed to establish whether this unidentified antiluteolytic signal targets prostaglandin G/H synthase 2 (PGHS2), a rate limiting enzyme in converting arachidonic acid to prostaglandins (PGs). In the first study, quantitative RT-PCR was used to determine the relative abundance of PGHS2 mRNA in endometrium d...
Validation of a reliable set of primer pairs for measuring gene expression by real-time quantitative RT-PCR in equine leukocytes. Quantification of gene expression using real-time reverse transcription quantitative PCR (RT-qPCR) is a reliable method to monitor cellular responses to pro-inflammatory stimuli. The main objective of this study was to validate a set of equine primer pairs that can be routinely used to monitor expression of genes that are central to inflammatory and immune responses. This paper describes the steps used to optimize and validate 29 equine primer pairs for RT-qPCR assays using SYBR Green detection. To validate these assays, monocytes were isolated from three horses and stimulated with Escherichia...
PCR detection of African horse sickness virus serogroup based on genome segment three sequence analysis. A nested reverse transcriptase (RT) polymerase chain reaction (RT-PCR), for rapid detection of African horse sickness virus (AHSV) double-stranded ribonucleic acid (dsRNA) in cell culture and tissue samples, was developed and evaluated. Using an outer pair of primers (P1 and P2), selected from genome segment three of AHSV serotype 6 (AHSV-6), the RT-PCR-based assay resulted in amplification of a 890 base pair (bp) primary PCR product. RNAs from the nine vaccine strains of AHSV, and a number of AHSV field isolates including the Central African isolates of AHSV-9 and AHSV-6, propagated in cell c...
Determination of internal control for gene expression studies in equine tissues and cell culture using quantitative RT-PCR. Quantitative reverse transcription polymerase chain reaction (RT-PCR) has become a basic, reliable and sensitive modern technique, in both biological research and clinical diagnosis, for investigation of gene expression and validation of cDNA microarray analysis. Accurate mRNA quantification using quantitative RT-PCR commonly requires data normalization through stable housekeeping genes (HKGs). Selection of HKGs for data normalization is critical for accurate mRNA quantification. Our objective was to evaluate a set of candidate HKGs as internal controls for gene expression studies using quanti...
Structural model of the Rev regulatory protein from equine infectious anemia virus. Rev is an essential regulatory protein in the equine infectious anemia virus (EIAV) and other lentiviruses, including HIV-1. It binds incompletely spliced viral mRNAs and shuttles them from the nucleus to the cytoplasm, a critical prerequisite for the production of viral structural proteins and genomic RNA. Despite its important role in production of infectious virus, the development of antiviral therapies directed against Rev has been hampered by the lack of an experimentally-determined structure of the full length protein. We have used a combined computational and biochemical approach to gen...
Interleukin-1 receptor antagonist expression in the equine endometrium during the peri-implantation period. To identify factors involved in the establishment of pregnancy in the mare, endometrium was collected from day 13 (day 0=day of ovulation) cyclic and day 13, 19, and 25 pregnant animals. From initial cDNA subtraction studies, interleukin-1 receptor antagonist (IL-1RN) mRNA was found as a candidate molecule expressed uniquely in the pregnant endometrium. Expression of IL-1RN mRNA was markedly increased in day 19 and 25 gravid endometrium. In situ hybridization analysis revealed that IL-1RN mRNA was localized to the glandular epithelium. Interleukin-1 receptor antagonist (IL-1RN) protein was fou...
Nucleotide structure and expression of equine pigment epithelium-derived factor during repair of experimentally induced wounds in horses. To clone full-length equine pigment epithelium-derived factor (PEDF) complementary DNA (cDNA) and to evaluate its temporal expression during repair of wounds in horses. Methods: 4 clinically normal 2-to 3-year-old Standardbred mares. Methods: Full-length equine PEDF cDNA was cloned by screening size-selected cDNA libraries derived from biopsy specimens obtained from the wound edge 7 days after experimental creation of a 6.25-cm(2) full-thickness wound in the skin of the lateral thoracic wall. Expression was evaluated in normal skin and in biopsy specimens obtained weekly from experimentally in...
Effective treatment of respiratory alphaherpesvirus infection using RNA interference. Equine herpesvirus type 1 (EHV-1), a member of the Alphaherpesvirinae, is spread via nasal secretions and causes respiratory disease, neurological disorders and abortions. The virus is a significant equine pathogen, but current EHV-1 vaccines are only partially protective and effective metaphylactic and therapeutic agents are not available. Small interfering RNAs (siRNA's), delivered intranasally, could prove a valuable alternative for infection control. siRNA's against two essential EHV-1 genes, encoding the viral helicase (Ori) and glycoprotein B, were evaluated for their potential to decrea...
Laminin receptor 1 is differentially expressed in thoracic and limb wounds in the horse. Healing of wounds located on the distal limbs of horses is often complicated by retarded epithelialization and the development of exuberant granulation tissue (proud flesh). Treatments that definitively resolve this pathological process are still unavailable. Molecular studies of the repair mechanism might contribute to the development of new therapeutic strategies. The study presented herein aimed to clone the full length cDNA and to study the spatio-temporal expression profile of mRNA and protein for LAMR1, previously attributed a role in wound epithelialization, during the repair of body an...
Gonadotrophin subunit and GnRH receptor gene expression in the pars distalis of the equine pituitary. In the horse, pronounced changes in fertility occur annually in response to photoperiod. However, the mechanisms regulating gonadotrophin synthesis and release in this species remain unclear. Here, we investigated the expression of gonadotrophin subunits and GnRH receptor (GnRH-R) mRNA in the pituitary glands of Thoroughbred horses during the breeding (BS) and non-breeding (NBS) season. Seasonal effects on the prevalence of gonadotrophs in the pars distalis were also examined. GnRH-R and common alpha-, LHbeta- and FSHbeta-subunit mRNA contents were determined by Northern analysis and the preva...
Assessment of the transformation of equine skin-derived fibroblasts to multinucleated skeletal myotubes following lentiviral-induced expression of equine myogenic differentiation 1. To develop a reliable method for converting cultured equine skin-derived fibroblasts into muscle cells. Methods: Equine skin-derived fibroblasts. Methods: The equine myogenic differentiation 1 (eqMyoD) genomic sequence was obtained by use of equine bacterial artificial chromosome screening and PCR sequencing. Total mRNA was extracted from foal skeletal muscle, and eqMyoD cDNA was cloned into a plasmid vector with an internal ribosomal entry site to express bicistronic eqMyoD or enhanced green fluorescent protein (EGFP). Transient expression was confirmed by immunocytochemical analysis and west...
Development of a real-time duplex TaqMan-PCR for the detection of Equine rhinitis A and B viruses in clinical specimens. Equine rhinitis A and B viruses (ERAV and ERBV) are respiratory viruses of horses belonging to the family Picornaviridae. Although these viruses are considered to cause respiratory disease in horses and are potentially infectious for humans, little is known about their prevalence and pathogenesis. Virus isolation is often unsuccessful due to their inefficient growth and lack of cytopathic effect in cell cultures. Therefore, molecular assays should be considered as the method of choice to detect infection in symptomatic or apparently healthy horses. In the present study, a novel real-time duple...
Adaptive response of equine intestinal Na+/glucose co-transporter (SGLT1) to an increase in dietary soluble carbohydrate. Experimental and epidemiological evidence suggests that consumption of hydrolyzable carbohydrate, hCHO (grain), by horses is an important risk factor for colic, a common cause of equine mortality. It is unknown whether the small intestinal capacity to digest hCHO and/or to absorb monosaccharides is limiting, or even if horses can adapt to increased carbohydrate load. We investigated changes in the brush-border membrane carbohydrate digestive enzymes and glucose absorptive capacity of horse small intestine in response to increased hCHO. Expression of the Na(+)/glucose co-transporter, SGLT1, was...
RNA interference protects horse cells in vitro from infection with Equine Arteritis Virus. Equine Arteritis Virus (EAV) belongs to the Arteriviridae and causes viral arteritis in horses. In an attempt to develop novel and save therapies against the infection it was tested whether EAV is susceptible to RNA interference (RNAi) in an equine in vitro system. Horse cells were transfected with chemically synthesized small interfering RNA oligonucleotides (siRNAs) and challenged with EAV. Application of these siRNAs led to a significant protection of the cells, and virus titers decreased drastically. siRNAs derived from DNA plasmids expressing small hairpin RNAs (shRNAs) were also effectiv...
Differential gene expression associated with postnatal equine articular cartilage maturation. Articular cartilage undergoes an important maturation process from neonate to adult that is reflected by alterations in matrix protein organization and increased heterogeneity of chondrocyte morphology. In the horse, these changes are influenced by exercise during the first five months of postnatal life. Transcriptional profiling was used to evaluate changes in articular chondrocyte gene expression during postnatal growth and development. Methods: Total RNA was isolated from the articular cartilage of neonatal (0-10 days) and adult (4-5 years) horses, subjected to one round of linear RNA ampli...
Sequence heterogeneity in the 18S rRNA gene within Theileria equi and Babesia caballi from horses in South Africa. A molecular epidemiological survey of the protozoal parasites that cause equine piroplasmosis was conducted using samples collected from horses and zebra from different geographical locations in South Africa. A total of 488 samples were tested for the presence of Theileria equi and/or Babesia caballi using the reverse line blot hybridization assay. Ten percent of the samples hybridized to the Theileria/Babesia genus-specific probe and not to the B. caballi or T. equi species-specific probes, suggesting the presence of a novel species or genotype. The small subunit of rRNA gene (18S; approximat...
Role of bacteria in the pathogenesis of recurrent uveitis in horses from the southeastern United States. To determine the role of intraocular bacteria in the pathogenesis of equine recurrent uveitis (ERU) in horses from the southeastern United States by evaluating affected eyes of horses with ERU for bacterial DNA and intraocular production of antibodies against Leptospira spp. Methods: Aqueous humor, vitreous humor, and serum samples of 24 clinically normal horses, 52 horses with ERU, and 17 horses with ocular inflammation not associated with ERU (ie, non-ERU inflammation). Methods: Ribosomal RNA quantitative PCR (real-time PCR) assay was used to detect bacterial DNA in aqueous humor and vitreou...
Effect of two synthetic peptides mimicking conserved regions of equine infectious anemia virus proteins gp90 and gp45 upon cytokine mRNA expression. Gp90 and gp45 synthetic peptides, which mimic conserved sequences of native viral proteins, are recognized by antibodies to equine infectious anemia virus (EIAV) in asymptomatic carrier horses and generate humoral and cellular responses in immunized mice. Cytokine mRNA levels were evaluated in equine peripheral blood mononuclear cells (PBMCs) after in vitro stimulation with gp90 and gp45 with the aim of determining the cytokine profile associated with the proliferative response. Stimulation index (SI) values indicate that 100 and 60% of EIAV-infected horses recognized gp90 and gp45, respective...
Evaluation of the pathogenicity of African Horsesickness (AHS) isolates in vaccinated animals. The polyvalent African Horsesickness (AHS) attenuated live vaccine (ALV) produced by Onderstepoort Biological Products (OBP) Ltd., South Africa, has been associated with some safety concerns and alleged cases of vaccine failure or vaccine-induced disease. The risk of reassortment and reversion to virulence is a common concern associated with the use of ALVs, and a phenomenon reported for viruses with segmented RNA genomes. The purpose of this study was to determine whether or not reassortment of AHS vaccine strains could result in reassortants and reversion to virulence and therefore cause AHS...
