A Customizable Suite of Methods to Sequence and Annotate Cattle Antibodies.

Studying the antibody response to infection or vaccination is essential for developing more effective vaccines and therapeutics. Advances in high-throughput antibody sequencing technologies and immunoinformatic tools now allow the fast and comprehensive analysis of antibody repertoires at high resolution in any species. Here, we detail a flexible and customizable suite of methods from flow cytometry, single cell sorting, heavy and light chain amplification to antibody sequencing in cattle. These methods were used successfully, including adaptation to the 10x Genomics platform, to isolate native heavy-light chain pairs. When combined with the Ig-Sequence Multi-Species Annotation Tool, this suite represents a powerful toolkit for studying the cattle antibody response with high resolution and precision. Using three workflows, we processed 84, 96, and 8313 cattle B cells from which we sequenced 24, 31, and 4756 antibody heavy-light chain pairs, respectively. Each method has strengths and limitations in terms of the throughput, timeline, specialist equipment, and cost that are each discussed. Moreover, the principles outlined here can be applied to study antibody responses in other mammalian species.

Molecular cloning and characterisation of chicken IL-18 binding protein.

The human IL-1 receptor family is comprised of 11 membrane bound or soluble receptors and the IL-18 binding protein (IL-18BP). These receptors are dispersed across seven genomic loci, with the majority at a single locus. Direct orthologues were identified in the chicken at conserved genomic loci; however, the IL-18BP remained absent from the first four builds of the chicken genome sequence. Subsequent assemblies identified the gene at a locus syntenic with mammals; however, these predicted sequences differed between genome builds and contained multiple errors. A partial IL-18BP-like sequence in the NCBI EST database was used to clone the full-length cDNA. A splice variant, which lacks the exon that encodes part of the signal peptide, was also cloned. Human IL-18BP is differentially spliced to produce a number of variants, which are all secreted. By contrast, the spliced chicken isoform was predicted to be intracellular, and we identified similar variants with the same exon missing in a limited number of divergent vertebrate species. Mammalian and viral IL-18BPs inhibit IL-18 activity by directly binding to this cytokine. Full-length and intracellular chicken IL-18BPs were equally effective at inhibiting IL-18-mediated IFN-γ release from an avian B-cell line. Analysis of the predicted chIL-18BP protein sequence revealed two crucial residues, which account for 50% of the binding affinity between human IL-18 and IL-18BP, are conserved in the chicken and a fowlpox-encoded homologue, fpv214. This suggests specific fowlpox viruses used in humans as a vaccine vector have the potential to dampen anti-viral host immune responses.

The Characterization of chIFITMs in Avian Coronavirus Infection In Vivo, Ex Vivo and In Vitro.

The coronaviruses are a large family of enveloped RNA viruses that commonly cause gastrointestinal or respiratory illnesses in the infected host. Avian coronavirus infectious bronchitis virus (IBV) is a highly contagious respiratory pathogen of chickens that can affect the kidneys and reproductive systems resulting in bird mortality and decreased reproductivity. The interferon-inducible transmembrane (IFITM) proteins are activated in response to viral infections and represent a class of cellular restriction factors that restrict the replication of many viral pathogens. Here, we characterize the relative mRNA expression of the chicken IFITM genes in response to IBV infection, in vivo, ex vivo and in vitro using the pathogenic M41-CK strain, the nephropathogenic QX strain and the nonpathogenic Beaudette strain. In vivo we demonstrate a significant upregulation of chIFITM1, 2, 3 and 5 in M41-CK- and QX-infected trachea two days post-infection. In vitro infection with Beaudette, M41-CK and QX results in a significant upregulation of chIFITM1, 2 and 3 at 24 h post-infection. We confirmed a differential innate response following infection with distinct IBV strains and believe that our data provide new insights into the possible role of chIFITMs in early IBV infection.

Temperature Sensitivity: A Potential Method for the Generation of Vaccines against the Avian Coronavirus Infectious Bronchitis Virus.

The Gammacoronavirus infectious bronchitis virus (IBV) is a highly contagious economically important respiratory pathogen of domestic fowl. Reverse genetics allows for the molecular study of pathogenic determinants to enable rational vaccine design. The recombinant IBV (rIBV) Beau-R, a molecular clone of the apathogenic Beaudette strain, has previously been investigated as a vaccine platform. To determine tissues in which Beau-R could effectively deliver antigenic genes, an in vivo study in chickens, the natural host, was used to compare the pattern of viral dissemination of Beau-R to the pathogenic strain M41-CK. Replication of Beau-R was found to be restricted to soft tissue within the beak, whereas M41-CK was detected in beak tissue, trachea and eyelid up to seven days post infection. In vitro assays further identified that, unlike M41-CK, Beau-R could not replicate at 41 °C, the core body temperature of a chicken, but is able to replicate a 37 °C, a temperature relatable to the very upper respiratory tract. Using a panel of rIBVs with defined mutations in the structural and accessory genes, viral replication at permissive and non-permissive temperatures was investigated, identifying that the Beau-R replicase gene was a determinant of temperature sensitivity and that sub-genomic mRNA synthesis had been affected. The identification of temperature sensitive allelic lesions within the Beau-R replicase gene opens up the possibility of using this method of attenuation in other IBV strains for future vaccine development as well as a method to investigate the functions of the IBV replicase proteins.

Epigenetic modification of the pentose phosphate pathway and the IGF-axis in women with gestational diabetes mellitus.

Aim: Gestational diabetes mellitus (GDM) has been linked with adverse long-term health outcomes for the fetus and mother. These effects may be mediated by epigenetic modifications. Materials & methods: Genome-wide RNA sequencing was performed in placental tissue and maternal blood in six GDM and six non-GDM pregnancies. Promoter region DNA methylation was examined for selected genes and correlated with gene expression to examine an epigenetic modulator mechanism. Results: Reductions of mRNA expression and increases in promoter methylation were observed for G6PD in GDM women, and for genes encoding IGF-binding proteins in GDM-exposed placenta. Conclusion: GDM involves epigenetic attenuation of G6PD, which may lead to hyperglycemia and oxidative stress, and the IGF-axis, which may modulate fetal macrosomia.