BackgroundSevere equine asthma is a naturally occurring lung inflammatory disease of mature animals characterized by neutrophilic inflammation, bronchoconstriction, mucus hypersecretion and airway remodeling. Exacerbations are triggered by inhalation of dust and microbial components. Affected animals eventually are unable of aerobic performance. In this study transcriptomic differences between asthmatic and non-asthmatic animals in the response of the bronchial epithelium to an inhaled challenge were determined.ResultsPaired endobronchial biopsies were obtained pre- and post-challenge from asthmatic and non-asthmatic animals. The transcriptome, determined by RNA-seq and analyzed with edgeR, contained 111 genes differentially expressed (DE) after challenge between horses with and without asthma, and 81 of these were upregulated. Genes involved in neutrophil migration and activation were in central location in interaction networks, and related gene ontology terms were significantly overrepresented. Relative abundance of specific gene products as determined by immunohistochemistry was correlated with differential gene expression. Gene sets involved in neutrophil chemotaxis, immune and inflammatory response, secretion, blood coagulation and apoptosis were overrepresented among up-regulated genes, while the rhythmic process gene set was overrepresented among down-regulated genes. MMP1, IL8, TLR4 and MMP9 appeared to be the most important proteins in connecting the STRING protein network of DE genes.ConclusionsSeveral differentially expressed genes and networks in horses with asthma also contribute to human asthma, highlighting similarities between severe human adult and equine asthma. Neutrophil activation by the bronchial epithelium is suggested as the trigger of the inflammatory cascade in equine asthma, followed by epithelial injury and impaired repair and differentiation. Circadian rhythm dysregulation and the sonic Hedgehog pathway were identified as potential novel contributory factors in equine asthma.Electronic supplementary materialThe online version of this article (10.1186/s12864-017-4107-6) contains supplementary material, which is available to authorized users.

Enzootic nasal adenocarcinoma (ENA) is a contagious neoplasm of the secretory epithelial cells of the nasal mucosa of sheep and goats. It is associated with the betaretrovirus, enzootic nasal tumor virus (ENTV), but a causative relationship has yet to be demonstrated. In this study, 14-day-old lambs were experimentally infected via nebulization with cell-free tumor filtrates derived from naturally occurring cases of ENA. At 12 weeks post-infection (wpi), one of the five infected lambs developed clinical signs, including continuous nasal discharge and open mouth breathing, and was euthanized. Necropsy revealed the presence of a large bilateral tumor occupying the nasal cavity. At 45 wpi, when the study was terminated, none of the remaining infected sheep showed evidence of tumors either by computed tomography or post-mortem examination. ENTV-1 proviral DNA was detected in the nose, lung, spleen, liver and kidney of the animal with experimentally induced ENA, however there was no evidence of viral protein expression in tissues other than the nose. Density gradient analysis of virus particles purified from the experimentally induced nasal tumor revealed a peak reverse transcriptase (RT) activity at a buoyant density of 1.22 g/mL which was higher than the 1.18 g/mL density of peak RT activity of virus purified from naturally induced ENA. While the 1.22 g/mL fraction contained primarily immature unprocessed virus particles, mature virus particles with a similar morphology to naturally occurring ENA could be identified by electron microscopy. Full-length sequence analysis of the ENTV-1 genome from the experimentally induced tumor revealed very few nucleotide changes relative to the original inoculum with only one conservative amino acid change. Taken together, these results demonstrate that ENTV-1 is associated with transmissible ENA in sheep and that under experimental conditions, lethal tumors are capable of developing in as little as 12 wpi demonstrating the acutely oncogenic nature of this ovine betaretrovirus.

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To date, recombination between different strains of the avian alphaherpesvirus infectious laryngotracheitis virus (ILTV) has only been detected in field samples using full genome sequencing and sequence analysis. These previous studies have revealed that natural recombination is widespread in ILTV and have demonstrated that recombination between two attenuated ILTV vaccine strains generated highly virulent viruses that produced widespread disease within poultry flocks in Australia. In order to better understand ILTV recombination, this study developed a TaqMan single nucleotide polymorphism (SNP) genotyping assay to detect recombination between two field strains of ILTV (CSW-1 and V1-99 ILTV) under experimental conditions. Following in vivo co-inoculation of these two ILTV strains in specific pathogen free (SPF) chickens, recovered viruses were plaque purified and subjected to the SNP genotyping assay. This assay revealed ILTV recombinants in all co-inoculated chickens. In total 64/87 (74%) of the recovered viruses were recombinants and 23 different recombination patterns were detected, with some of them occurring more frequently than others. The results from this study demonstrate that the TaqMan SNP genotyping assay is a useful tool to study recombination in ILTV and also show that recombination occurs frequently during experimental co-infection with ILTV in SPF chickens. This tool, when used to assess ILTV recombination in the natural host, has the potential to greatly contribute to our understanding of alphaherpesvirus recombination.

Recombination is a feature of many alphaherpesviruses that infect people and animals. Infectious laryngotracheitis virus (ILTV; Gallid alphaherpesvirus 1) causes respiratory disease in chickens, resulting in significant production losses in poultry industries worldwide. Natural (field) ILTV recombination is widespread, particularly recombination between attenuated ILTV vaccine strains to create virulent viruses. These virulent recombinants have had a major impact on animal health. Recently, the development of a single nucleotide polymorphism (SNP) genotyping assay for ILTV has helped to understand ILTV recombination in laboratory settings. In this study, we applied this SNP genotyping assay to further examine ILTV recombination in the natural host. Following coinoculation of specific-pathogen-free chickens, we examined the resultant progeny for evidence of viral recombination and characterized the diversity of the recombinants over time. The results showed that ILTV replication and recombination are closely related and that the recombinant viral progeny are most diverse 4 days after coinoculation, which is the peak of viral replication. Further, the locations of recombination breakpoints in a selection of the recombinant progeny, and in field isolates of ILTV from different geographical regions, were examined following full-genome sequencing and used to identify recombination hot spots in the ILTV genome.IMPORTANCE Alphaherpesviruses are common causes of disease in people and animals. Recombination enables genome diversification in many different species of alphaherpesviruses, which can lead to the evolution of higher levels of viral virulence. Using the alphaherpesvirus infectious laryngotracheitis virus (ILTV), we performed coinfections in the natural host (chickens) to demonstrate high levels of virus recombination. Higher levels of diversity in the recombinant progeny coincided with the highest levels of virus replication. In the recombinant progeny, and in field isolates, recombination occurred at greater frequency in recombination hot spot regions of the virus genome. Our results suggest that control measures that aim to limit viral replication could offer the potential to limit virus recombination and thus the evolution of virulence. The development and use of vaccines that are focused on limiting virus replication, rather than vaccines that are focused more on limiting clinical disease, may be indicated in order to better control disease.

Results of this study indicated ERAV induced respiratory tract disease in seronegative ponies. However, ponies with neutralizing antibodies against ERAV did not develop clinical signs of disease when reinoculated with the virus. Therefore, immunization of ponies against ERAV could prevent respiratory tract disease attributable to that virus in such animals.

Severe equine asthma is a chronic inflammatory condition of the lower airways similar to adult-onset asthma in humans. Exacerbations are characterized by bronchial and bronchiolar neutrophilic inflammation, mucus hypersecretion and airway constriction. In this study we analyzed the gene expression response of the bronchial epithelium within groups of asthmatic and non-asthmatic animals following exposure to a dusty hay challenge. After challenge we identified 2341 and 120 differentially expressed genes in asthmatic and non-asthmatic horses, respectively. Gene set enrichment analysis of changes in gene expression after challenge identified 587 and 171 significantly enriched gene sets in asthmatic and non-asthmatic horses, respectively. Gene sets in asthmatic animals pertained, but were not limited, to cell cycle, neutrophil migration and chemotaxis, wound healing, hemostasis, coagulation, regulation of body fluid levels, and the hedgehog pathway. Furthermore, transcription factor target enrichment analysis in the asthmatic group showed that transcription factor motifs with the highest enrichment scores for up-regulated genes belonged to the E2F transcription factor family. It is postulated that engagement of hedgehog and E2F pathways in asthmatic horses promotes dysregulated cell proliferation and abnormal epithelial repair. These fundamental lesions may prevent re-establishment of homeostasis and perpetuate inflammation.

Infectious bronchitis virus (IBV) is a member of the family Coronaviridae, together with viruses such as SARS‐CoV, MERS‐CoV and SARS‐CoV‐2 (the causative agent of the COVID‐19 global pandemic). In this family of viruses, interspecies transmission has been reported, so understanding their pathobiology could lead to a better understanding of the emergence of new serotypes. IBV possesses a single‐stranded, non‐segmented RNA genome about 27.6 kb in length that encodes several non‐structural and structural proteins. Most functions of these proteins have been confirmed in IBV, but some other proposed functions have been based on research conducted on other members of the family Coronaviridae. IBV has variable tissue tropism depending on the strain, and can affect the respiratory, reproductive, or urinary tracts; however, IBV can also replicate in other organs. Additionally, the pathogenicity of IBV is also variable, with some strains causing only mild clinical signs, while infection with others results in high mortality rates in chickens. This paper extensively and comprehensibly reviews general aspects of coronaviruses and, more specifically, IBV, with emphasis on protein functions and pathogenesis. The pathogenicity of the Australian strains of IBV is also reviewed, describing the variability between the different groups of strains, from the classical to the novel and recombinant strains. Reverse genetic systems, cloning and cell culture growth techniques applicable to IBV are also reviewed.