NOD1 Is Associated With the Susceptibility of Pekin Duck Flock to Duck Hepatitis A Virus Genotype 3

Figure 1. Phenotypic differences between the Z8 and SZ7 flocks infected with DHAV-3. (A) Survival curves of populations Z8 and SZ7 used in this experiment. The figure shows the death curve for the intramuscular route of infection. (B1) Numbers of SZ7 families showing 100% survival, 100% mortality, 33.3% mortality, and 66.6% mortality. (B2) Numbers of Z8 families showing 100% survival, 100% mortality, and 11.1%-88.9% mortality. Orange represents the proportion of families with a mortality rate of 0%, green represents the proportion of families with a mortality rate of 100%, and blue represents the proportion of families with a mortality rate of >0 - <100%. (C) Blood ALT (C1), AST (C2), ALP (C3) and viral load levels (C4) in the population Z8 and SZ7 ducklings used in this experiment. The serum samples at each time point include 4 individual data points. The viral load at each time point includes 20 individual data points. ***P < 0.001. (D) The growth and reproductive performance of Z8 (n = 30) and SZ7 (n = 30), including live weight and carcass weight (D1), egg-laying rate and hatching rate (D2), chest width and keel length (D3), rate of breast meat, leg meat, sebum and abdominal fat (D4).

Figure 2. Analysis of genetic variation related to the susceptibility trait. (A) Images are from individuals of flock Z8 and flock SZ7. The dashed box shows the selection of animal materials used in this study for resequencing. (B) Manhattan plot showing the genetic effects on the susceptibility trait (observation color grade value) determined by GWAS in 110 Pekin ducks. The gray line represents the Bonferroni-corrected significance threshold (-log10 P = 9.20). (C) Regional plots of the loci ranging from 43.30 to 43.90 Mb along chromosome 2 associated with the susceptibility trait. All genotyped SNPs are color coded according to their pairwise linkage disequilibrium (LD) with the leader SNP (Chr2:43,445,923) calculated in the Pekin ducks. (D) Selective sweeps among three duck populations within the candidate region (Chr2:43.30-43.90 Mb). (E) The nucleotide diversity (π) of SZ7-S (blue line), Z8-R (green line) and Z8-S (red line) from 43.30 to 43.90 Mb on chromosome 2.

Figure 3. NOD1 is a candidate gene for the susceptibility trait. (A) Images are from individuals of flock Z8. The images in the dashed box show the selection of animal materials used in this study for transcriptome sequencing. (B) Heat map analysis classifying the gene expression patterns of DEGs. (C) Gene expression levels in candidate intervals determined by the transcriptome analysis of liver samples. Statistical analysis was performed by using Student’s t-test, and error bars indicate the standard error of the mean (SEM). The results are presented as the means ± SEM of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. (D) qPCR was used to determine the expression of NOD1 in liver tissues from Z8-R (n = 9), SZ7-S (n = 9) and Z8-S (n = 9) ducks.

Figure 4. The expression of NOD1 and downstream genes is activated by DHAV-3 infection. (A) RT-qPCR was used to quantify the expression of genes involved in NOD1 signaling pathways. (B) RT-qPCR was used to determine the expression of chemokines. (C) RT-qPCR quantification of the expression of inflammatory factors. (D) The protein expression levels of NOD1 in the livers of Z8-R and SZ7-S. Representative western blot (left panel) and quantification results from three independent experiments (right panels). (E) RT-qPCR quantification of the expression of NOD1 and the genomic copy numbers of DHAV-3 in primary liver cells from Z8-R (n = 9) and SZ7-S (n = 9). (F) RT-qPCR analysis of IL8, IFN-β, MAPK1, IFN-γ, and IL1-β mRNAs in the liver cells of Z8-R and SZ7-S ducks. (G) The expression levels of NOD1, IL8 and MAPK1 at different multiplicities of infection. Primary liver cells of SZ7-S ducks were used in this experiment.

Figure 5. NOD1 is correlated with DHAV-3 infection. (A) RT-qPCR analysis of NOD1 and DHAV-3 mRNA expression in primary liver cells. Cells of the MOCK group were sampled at 12 hpi. (B) RT-qPCR analysis of NOD1 and DHAV-3 mRNA expression in primary liver cells. Cells were treated with iE-DAP (50 μg/ml) for the indicated times. Cells of the MOCK group were sampled at 12 hpi. (C, D) The kinetics of DHAV-3 and NOD1 expression in primary liver cells. The results are presented as the means ± SD of at least three independent experiments. ***P < 0.001. (E) DHAV-3 and NOD1 were detected by the RNAscope in situ hybridization method, and RT-qPCR was used to determine the expression of NOD1 and the genomic copy numbers of DHAV-3. Statistical analysis was performed by Student’s t-test, and error bars indicate the standard error of the mean (SEM). Images were acquired with an OLYMPUS microscope. Oil objective: 40×; zoom in 1×.