Turkey B-cell Transcriptome Profile During Turkey Hemorrhagic Enteritis Virus (THEV) Infection Highlights Upregulated Apoptosis and Breakdown Pathways That May Mediate Immunosuppression

Background: Infection with Turkey Hemorrhagic Enteritis Virus (THEV) can cause hemorrhagic enteritis, which affects turkey poults. This disease is characterized by immunosuppression (IMS) and bloody droppings. The clinical disease usually lasts only a few days but secondary opportunistic infections due to THEV-induced IMS extend the duration of illness and mortality, which exacerbates economic losses. Although an avirulent THEV strain with only subclinical disease is used as a vaccine, some immunosuppressive properties remain in this prophylactic strain. To elucidate the mechanisms mediating THEV-induced IMS, we performed the first transcriptomic analysis of a THEV infection using bulk RNA-sequencing. Methods: After infecting a turkey B-cell line with the vaccine strain, samples in triplicates were collected at 4-, 12-, 24-, and 72-hours post-infection (hpi). Total RNA was extracted, and poly-Adenylated-tailed mRNAs were sequenced. Reads were mapped to the host turkey genome after trimming and gene expression was quantified with StringTie. Differential gene expression was performed with DESeq2 followed by functional enrichment analysis with gprofiler2 and DAVID from NCBI. RT-qPCR of select genes was performed to validate the RNA-sequencing data. Results: A total of 2,343 and 3,295 differentially expressed genes (DEGs) were identified at 12 hpi and 24 hpi, respectively. At 12 hpi, 1,079 genes were upregulated and 1,264 genes downregulated, whereas 1,512 genes were upregulated and 1,783 genes downregulated at 24 hpi. The DEGs contributed to multiple biological processes including apoptosis, ER unfolded protein response, and cell maintenance. Multiple pro-apoptotic genes, including APAF1, BNIP3L, BMF, BAK1, RIPK1, and FAS were upregulated. Genes that play a role in ER stress-induced unfolded protein response including VCP, UFD1, EDEM1, EDEM3, and ATF4 were also upregulated and may contribute to apoptosis. Conclusions: Our data suggest that several biological processes and pathways including apoptosis, immune response, ER response to stress, ubiquitin-dependent protein catabolic process, and repression of essential cellular maintenance are important aspects of the host cell response to THEV infection. It is possible that interplay between multiple processes may mediate apoptosis of infected B-cells, leading to IMS. To understand the genetic basis by which THEV causes immunosuppression (IS) in infected turkeys, we performed an RNA-seq experiment to elucidate the transcriptomic profile of THEV-infected cells, highlighting key host genes, cellular/molecular processes and pathways affected during a THEV infection. We specifically focus on cellular processes that would help in elucidating THEV-induced IMS. A THEV vaccine was used to infect a turkey cell line (MDTC-RP19) and harvested at 4-, 12-, 24-, and 72-hpi in triplicates. RNA-seq was done for polyA-tailed mRNA Reads trimmed with Cutadapt were mapped to Meleagris gallopavo (turkey) genome obtained from NCBI using Hisat2. StringTie in estimation mode was used to generate normalized gene expression estimates from the BAM files for genes in the reference GTF file after which the prepDE.py3 script was used to extract read count information from the StringTie gene expression files, providing an expression-count matrix for downstream DEG analysis. DEG analysis between mock- and THEV-infected samples was performed using the very popular DESeq2. Genes with P-adjusted value less than or equal to 0.05 were considered as differentially expressed and used as input for functional enrichment (Gene ontology [GO] and KEGG pathway) analyses. GO and KEGG pathway analysis was performed using the R package gprofiler2 and DAVID (Database for Annotation, Visualization and Integrated Discovery; version 2021) online resource.