Transcriptome Sequencing of the Spleen Reveals Antiviral Response Genes in Chickens Infected with CAstV

Abstract

Astrovirus infections pose a significant problem in the poultry industry, leading to multiple adverse effects such as a decreased egg production, breeding disorders, poor weight gain, and even increased mortality. The commonly observed chicken astrovirus (CAstV) was recently reported to be responsible for the “white chicks syndrome” associated with an increased embryo/chick mortality. CAstV-mediated pathogenesis in chickens occurs due to complex interactions between the infectious pathogen and the immune system. Many aspects of CAstV–chicken interactions remain unclear, and there is no information available regarding possible changes in gene expression in the chicken spleen in response to CAstV infection. We aim to investigate changes in gene expression triggered by CAstV infection. Ten 21-day-old SPF White Leghorn chickens were divided into two groups of five birds each. One group was inoculated with CAstV, and the other used as the negative control. At 4 days post infection, spleen samples were collected and immediately frozen at −70 °C for RNA isolation. We analyzed the isolated RNA, using RNA-seq to generate transcriptional profiles of the chickens’ spleens and identify differentially expressed genes (DEGs). The RNA-seq findings were verified by quantitative reverse-transcription PCR (qRT-PCR). A total of 31,959 genes was identified in response to CAstV infection. Eventually, 45 DEGs (p-value < 0.05; log2 fold change > 1) were recognized in the spleen after CAstV infection (26 upregulated DEGs and 19 downregulated DEGs). qRT-PCR performed on four genes (IFIT5, OASL, RASD1, and DDX60) confirmed the RNA-seq results. The most differentially expressed genes encode putative IFN-induced CAstV restriction factors. Most DEGs were associated with the RIG-I-like signaling pathway or more generally with an innate antiviral response (upregulated: BLEC3, CMPK2, IFIT5, OASL, DDX60, and IFI6; downregulated: SPIK5, SELENOP, HSPA2, TMEM158, RASD1, and YWHAB). The study provides a global analysis of host transcriptional changes that occur during CAstV infection in vivo and proves that, in the spleen, CAstV infection in chickens predominantly affects the cell cycle and immune signaling.

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“…One of these, DDX60 , belongs to the family of DEAD/H-box RNA helicases which participate in anti-viral innate immunity, either by acting as viral nucleic acid sensors or by regulating downstream signaling events ( Fullam and Schröder, 2013 ). In line with a role in innate immune activation, DDX60 was found to be up-regulated in tonsils and intestine of chickens following infection with avian influenza virus H9N2 ( Alqazlan et al, 2021 ), and in the spleen of chickens infected with the commonly observed chicken astrovirus CAstV ( Sajewicz-Krukowska et al, 2021 ). The other gene, ETNPPL , being highly expressed in liver and brain encodes the enzyme ethanolamine phosphate phospholyase, which specifically degrades phosphoethanolamine, which itself is necessary to generate phosphatidylethanolamine via the so-called Kennedy pathway ( Pavlovic and Bakovic, 2013 ).…”