key: cord-0288854-wgi3hbbe authors: Dolinski, Amanda C.; Homola, Jared J.; Jankowski, Mark D.; Robinson, John D.; Owen, Jennifer C. title: Differential gene expression reveals host factors for viral shedding variation in mallards (Anas platyrhynchos) infected with low-pathogenic avian influenza virus date: 2021-08-23 journal: bioRxiv DOI: 10.1101/2021.08.23.457327 sha: 60b71ddf7d35337de735a0e1c9ff7e4a9f85a6be doc_id: 288854 cord_uid: wgi3hbbe Intraspecific variation in pathogen shedding impacts disease transmission dynamics; therefore, understanding the host factors associated with individual variation in pathogen shedding is key to controlling and preventing outbreaks. In this study, ileum and bursa of Fabricius tissues of mallards (Anas platyrhynchos) infected with low-pathogenic avian influenza (LPAIV) were evaluated at various post-infection time points to determine genetic host factors associated with intraspecific variation in viral shedding. By analyzing transcriptome sequencing data (RNA-seq), we found that LPAIV-infected mallards do not exhibit differential gene expression compared to uninfected birds, but that gene expression was associated with viral shedding quantity early in the infection. In both tissues, immune genes were mostly up-regulated in higher shedding birds and had significant positive relationships with viral shedding. In the ileum, host genes involved in viral cell entry were down-regulated in low shedders one day post-infection (DPI), and host genes promoting viral replication were up-regulated in high shedders on two DPI. Our findings indicate that viral shedding is a key factor for gene expression differences in LPAIV-infected mallards, and the genes identified in this study could be important for understanding the molecular mechanisms driving intraspecific variation in pathogen shedding. Heterogeneity in pathogen transmission across individuals can impact the magnitude and 42 duration of a disease outbreak (1,2). Some individuals in a population are super-spreaders; that 43 is, they are disproportionately responsible for secondary transmission cases of a contagious 44 pathogen through increased contact rate and/or higher infectiousness (3). The super-spreader Due to low host RNA quality, *individuals were not included in ileum differential gene 207 expression analyses. After evaluating virus titer data, we observed that group I2 cumulative virus titer data 210 clustered into two distinct shed level groups (Fig 2) ; therefore, we also conducted differential 243 Quality of raw cDNA reads was assessed using FastQC (62) prior to trimming and filtering. Sequence regions with a quality score below 15 and sequences shorter than 40bp were removed 245 using Trimmomatic (63). FastQC was used again to verify quality and trimming. Sequences were aligned to the most recent mallard reference genome, Ensembl 247 ASM874695v1 (64), using STAR (65,66). Gene-specific read counts for bursa samples were 248 acquired with HT-seq (67) using the STAR output, then RSEM (68) was used to estimate read We performed differential gene expression analyses using the R packages "EdgeR" (73,74) and Lowly expressed genes and transcripts were filtered by requiring expression of >0.5 262 counts per million in at least 25% of the birds. A false discovery rate (FDR) corrected alpha 263 value of 0.1 and a log fold count difference (LFC) of 0.5 were required to establish differential 264 expression. These thresholds were established to allow for detection of subtle differential gene 265 expression as previously observed between LPAIV-infected and uninfected ducks (41-43). To 266 account for sex-based differences and variation in sequencing pool, sex and pool were included 267 as covariates in each analysis. Gene names (HGNC symbols) were assigned based on 268 ENSEMBL annotation information that accompanied the reference genome. KEGG pathway 269 analyses were performed using the "kegga" function of "limma". Pathways with p-values <0.05 270 were determined as over-represented (enriched) pathways of differentially expressed genes or 271 transcripts (DEG, DET) per analysis. Preliminary differential expression analysis showed that ileum and bursa samples 273 clustered based on tissue-specific gene expression (Section 1 in S2 Document). Improper tissue-274 based clustering for two individuals (1 and 72) in group C1 suggested improper sample labeling 275 may have occurred in those cases; therefore, these two individuals were removed from analyses, 276 leaving a total of 68 bursa samples and 40 ileum samples. Additionally, due to an absence of 277 differential gene expression between C1 and C29 (Section 2 in S2 Document), we combined the two control groups into one group "C" for subsequent analyses. Sample size for each group in 279 each differential expression analysis is provided in Table 1 and HSPA8 in the bursa as a protective mechanism in response to LPAIV infection. In the ileum, only one gene (S100A12) that codes for a calcium binding protein was 373 differentially expressed and had lower expression in group I1 compared to group I2 (Section 3A 374 in S2 Document). No KEGG pathways were enriched in the ileum. Genes of the immune system were differentially expressed between shed level groups 436 early in the infection in both the ileum and the bursa. In the ileum of groups I1 and I2, genes with 437 interferon pathway, cellular immunity, and apoptosis functions were up-regulated in higher 438 shedding birds for 43 out of the 47 significant comparisons (Fig 8, Table 2 ). In the bursa of 439 group I2, four interferon-stimulated genes were up-regulated in high shedders compared to low 440 shedders ( Table 3 ). The up-regulation of immune genes in higher shedding birds of I1 and I2 441 groups supports our prediction that viral shedding is closely associated with innate immunity 442 early in the infection. Table) , which is consistent with other virus titers were related to the host immune system (Fig 9) . Conversely, none of the (Table 568 3) also supports this hypothesis. With fewer receptors (SAα2,3Gal) for the virus in the ileum, 569 less virus is bound and replicated; therefore, the low viral shedding may induce a weaker innate 570 immune response. Together, these findings may suggest that the ileum is more involved than the 571 bursa in the relationship between the immune response and viral shedding variation. shedding and gene expression, we were unable to determine whether viral shedding influenced 592 gene expression or vice versa, which also makes it difficult to determine an inherent cause and 593 effect. Single nucleotide polymorphisms (SNPs) in relevant genes could be accounting for the 594 differential gene expression observed; however, the gene mapping methods we used to detect 595 gene expression did not account for SNP differences (66). A potential next research step would be to identify SNPs in observed DEGs to determine if a specific gene SNP is associated with 597 intraspecific viral shedding variation (177). For example, a specific allele identified in cattle has 598 been associated with lower proviral loads of bovine leukemia virus (178). When BLV-infected 599 cattle selected for this specific low proviral allele were introduced to uninfected dairy herds, viral 600 transmission stopped (26). We recommend that the genes identified in our current study, such as 601 interferon pathway genes DDX58, LGP2, IFIT5, IFITM2, IFI35, FOSB (Fig 10) , viral replication 602 promoting genes HSPA8, ST3GAL1, ST3GAL5, and other genes described in Tables 2, 3 , and 4, 603 be examined further by identifying SNPs, utilizing gene-knockout studies, and conducting site-604 directed mutagenesis to understand the potential for inherent genetic differences (e.g., gene 605 mutations) and associated molecular mechanisms that may contribute to viral shedding variation. The data acquired in this study should also be used to determine how the intrinsic host 620 factors associated with viral shedding contribute to pathogen transmission. 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