Assessment of TLR3 and MDA5-Mediated Immune Responses Using Knockout Quail Fibroblast Cells.

Toll-like receptor 3 (TLR3) and melanoma differentiation-associated gene 5 (MDA5) are double-stranded RNA (dsRNA)-recognizing receptors that mediate innate immune responses to virus infection. However, the roles played by these receptors in the pathogenesis of avian viruses are poorly understood. In this study, we generated TLR3 and MDA5 single knockout (SKO) and TLR3-MDA5 double knockout (DKO) quail fibroblast cells and examined dsRNA receptor-mediated innate immune responses in vitro. The knockout cells were then stimulated with a synthetic dsRNA ligand polyinosinic:polycytidylic acid [poly(I:C)] or influenza A virus. Endosomal stimulation of TLR3 by adding poly(I:C) in cell culture media or cytoplasmic stimulation of MDA5 by transfecting poly(I:C) resulted in significant increases of TLR3, MDA5, interferon (IFN) ß, and interleukin 8 gene expression levels in wild type (WT) cells. Endosomal poly(I:C) treatment induced a higher level expression of most of the genes tested in MDA5 SKO cells compared with WT cells, but not in TLR3 SKO and DKO cells. Cytoplasmic transfection of poly(I:C) led to significant upregulation of all four genes in WT, TLR3 SKO, and MDA5 SKO cells at 8 hr posttransfection and negligible gene expression changes in TLR3-MDA5 DKO cells. Upon infection with a strain of influenza virus with compromised IFN antagonistic capability, WT cells produced the highest amount of biologically active type I IFN followed by TLR3 SKO and MDA5 SKO cells. DKO cells did not produce detectable amounts of type I IFN. However, the IFN did not induce an antiviral state fast enough to block virus replication, even in WT cells under the experimental conditions employed. In summary, our data demonstrate that TLR3 and MDA5 are the key functional dsRNA receptors in quail and imply their coordinated roles in the induction of innate immune responses upon virus infection.

Evaluación de las respuestas inmunitarias mediadas por TLR3 y MDA5 utilizando células de fibroblastos de codorniz con genes eliminados. El receptor tipo Toll 3 (TLR3) y el gene 5 asociado a la diferenciación de melanoma (MDA5) son receptores de reconocimiento de ARN de doble cadena (dsRNA) que median las respuestas inmunitarias innatas a la infección por virus. Sin embargo, no se conocen bien las funciones que desempeñan estos receptores en la patogenia de los virus aviares. En este estudio, se generaron células de fibroblastos de codorniz con eliminación simple de los genes TLR3 y MDA5 (SKO) y eliminación doble de los genes TLR3-MDA5 (DKO) y se examinaron las respuestas inmunitarias innatas mediadas por el receptor de dsRNA in vitro. Posteriormente, las células con genes eliminados se estimularon con un ligando sintético de ARN de doble cadena poliinosínico: ácido policitidílico [poli (I: C)] o con el virus de la influenza A. La estimulación endosómica de TLR3 mediante la adición de poli(I: C) en medios de cultivo celular, o la estimulación citoplásmica de MDA5 mediante la transfección de poli(I: C), dieron como resultado aumentos significativos de los niveles de expresión de los genes para TLR3, MDA5, interferón (IFN) ß e interleucina 8 en células de tipo silvestre (WT). El tratamiento con poli(I: C) endosómico indujo un nivel de expresión más alto de la mayoría de los genes analizados en las células con eliminación simple del gene MDA5 en comparación con las células silvestres, pero no en las células con eliminación simple del gene TLR3 y con eliminación doble de genes. La transfección citoplásmica de poli(I: C) condujo a una regulación positiva significativa de los cuatro genes en las células silvestres, en las células con eliminación simple del gene TLR3 y en las células con eliminación simple del gene MDA5 a las ocho horas posteriores a la transfección y cambios insignificantes en la expresión de genes en las células con eliminación doble de los genes TLR3 y MDA5. Durante la infección con una cepa del virus de la influenza con una capacidad antagonista para IFN comprometida, las células silvestres produjeron la mayor cantidad de IFN de tipo I biológicamente activo, seguidas de las células con eliminación simple del gene TLR3 y de las células con eliminación simple del gene MDA5. Las células con eliminación doble de genes no produjeron cantidades detectables de IFN de tipo I. Sin embargo, el IFN no indujo un estado antiviral lo suficientemente rápido como para bloquear la replicación del virus, incluso en células silvestres bajo las condiciones experimentales empleadas. En resumen, los datos de este estudio demuestran que TLR3 y MDA5 son los receptores de ARN de doble cadena funcionales clave en la codorniz e implican sus funciones coordinadas en la inducción de respuestas inmunitarias innatas durante la infección por virus.

Assessment of two DNA extraction kits for profiling poultry respiratory microbiota from multiple sample types.

Characterization of poultry microbiota is becoming increasingly important due to the growing need for microbiome-based interventions to improve poultry health and production performance. However, the lack of standardized protocols for sampling, sample processing, DNA extraction, sequencing, and bioinformatic analysis can hinder data comparison between studies. Here, we investigated how the DNA extraction process affects microbial community compositions and diversity metrics in different chicken respiratory sample types including choanal and tracheal swabs, nasal cavity and tracheal washes, and lower respiratory lavage. We did a side-by-side comparison of the performances of Qiagen DNeasy blood and tissue (BT) and ZymoBIOMICS DNA Miniprep (ZB) kits. In general, samples extracted with the BT kit yielded higher concentrations of total DNA while those extracted with the ZB kit contained higher numbers of bacterial 16S rRNA gene copies per unit volume. Therefore, the samples were normalized to equal amounts of 16S rRNA gene copies prior to sequencing. For each sample type, all predominant bacterial taxa detected in samples extracted with one kit were present in replicate samples extracted with the other kit and did not show significant differences at the class level. However, a few differentially abundant shared taxa were observed at family and genus levels. Furthermore, between-kit differences in alpha and beta diversity metrics at the amplicon sequence variant level were statistically indistinguishable. Therefore, both kits perform similarly in terms of 16S rRNA gene-based poultry microbiome analysis for the sample types analyzed in this study.

Avian Toll-like receptor 3 isoforms and evaluation of Toll-like receptor 3-mediated immune responses using knockout quail fibroblast cells.

Toll-like receptor 3 (TLR3) induces host innate immune response on recognition of viral double-stranded RNA (dsRNA). Although several studies of avian TLR3 have been reported, none of these studies used a gene knockout (KO) model to directly assess its role in inducing the immune response and effect on other dsRNA receptors. In this study, we determined the coding sequence of quail TLR3, identified isoforms, and generated TLR3 KO quail fibroblast (QT-35) cells using a CRISPR/Cas9 system optimized for avian species. The TLR3-mediated immune response was studied by stimulating the wild-type (WT) and KO QT-35 cells with synthetic dsRNA or polyinosinic:polycytidylic acid [poly(I:C)] or infecting the cells with different RNA viruses such as influenza A virus, avian reovirus, and vesicular stomatitis virus. The direct poly(I:C) treatment significantly increased IFN-ß and IL-8 gene expression along with the cytoplasmic dsRNA receptor, melanoma differentiation-associated gene 5 (MDA5), in WT cells, whereas no changes in all corresponding genes were observed in KO cells. We further confirmed the antiviral effects of poly(I:C)-induced TLR3-mediated immunity by demonstrating significant reduction of virus titer in poly(I:C)-treated WT cells, but not in TLR3 KO cells. On virus infection, varying levels of IFN-ß, IL-8, TLR3, and MDA5 gene upregulation were observed depending on the viruses. No major differences in gene expression level were observed between WT and TLR3 KO cells, which suggests a relatively minor role of TLR3 in sensing and exerting immune response against the viruses tested in vitro. Our data show that quail TLR3 is an important endosomal dsRNA receptor responsible for regulation of type I interferon and proinflammatory cytokine, and affect the expression of MDA5, another dsRNA receptor, most likely through cytokine-mediated communication.