Transcriptomic analyses of host-virus interactions during in vitro infection with wild-type and glycoprotein g-deficient (ΔgG) strains of ILTV in primary and continuous cell cultures

Abstract

Infectious laryngotracheitis (ILT) remains a significant concern for the poultry industry worldwide due to its impact on animal welfare and its substantial economic consequences. The disease is caused by the alphaherpesvirus, infectious laryngotracheitis virus (ILTV). This study investigated in vitro host-virus interactions of a glycoprotein G (gG) deletion mutant vaccine strain of ILTV (ΔgG ILTV), and its parent wild-type strain (CSW-1 ILTV). Inoculations were performed separately for the two strains of ILTV using both a primary (chicken embryonic kidney, CEK) and a continuous culture (leghorn male hepatoma, LMH) of chicken cells. Transcriptome analysis was performed at 12 hours post infection. Each cell-type displayed distinct effects on host and viral gene transcription, with a greater number of viral and host genes differentially transcribed in CEK cells and LMH cells, respectively. Both cell-types infected with either strain demonstrated enrichment of pathways related to signalling, and gene ontologies (GO) associated with chemotaxis. Infection with either strain upregulated both SOCS proteins and certain proto-oncogenes, which may contribute to prolonged viral persistence by promoting immunosuppression and preventing apoptosis, respectively. Patterns of gene transcription related to cytokines, chemokines, endosomal TLRs, and interferon responses, as well as pathways associated with histone acetylation, transport, and extracellular matrix organization were similar within each cell type, regardless of the viral strain. In CEK cells, GO terms and pathways were downregulated uniquely after CSW-1 ILTV infection, indicating a viral-strain specific effect in this cell-type. Overall, this study highlights that the observed differences in host and ILTV gene transcription in vitro were more strongly influenced by the cell-types used rather than the presence or absence of gG. This underscores the importance of cell-line selection in studying host-virus interactions and interpreting experimental results.

Fig 1. ILTV genome copy numbers in cell culture supernatant.

Stacked bar plot representing log₁₀ genome copy numbers per reaction of ILTV UL15 DNA in the supernatant samples collected after mock inoculation of LMH and CEK cells with CSW-1 ILTV or ΔgG ILTV at an MOI of 3.5. Genome copy number of samples collected at 0hpi is indicated by shaded bars and at 12hpi is indicated by clear bars. Error bars indicate mean standard deviation. P-value < 0.05 was considered statistically significant. Asterisks within the bars indicate significant differences between time points within each infection group. Asterisks across bars indicates significant differences across the same virus strain in different cell types. *p- value ≤ 0.014, ** p- value ≤ 0.041.

Fig 2. Differentially regulated host and ILTV genes.

A) and B) show UpSetR plots representing the sets of common and unique host genes that were A) upregulated (Padj < 0.01, log₂ FC ≥ 1) or B) downregulated (Padj < 0.01, log₂ FC ≤ -1) in CEK or LMH cells at 12 hours post-inoculation with CSW-1 ILTV or ΔgG ILTV (compared to mock). The horizontal bar chart on the left of (A and B) indicates the total number of host genes differentially regulated after infection in each of the systems. The vertical bar chart on the top indicates the intersection size between sets of host genes differentially regulated in one or between multiple comparisons. Black dots underneath each bar shows the culture system (left) represented by the bar above. Black dots connected by vertical lines indicate the culture systems of the comparison, where the bar above shows the number of genes in common. Heatmaps representing the ILTV genes differentially regulated between CSW-1 ILTV and ΔgG ILTV are shown in C) LMH cells and D) CEK cells, respectively. Rows represent genes and columns represent samples. Gene expression levels are represented by normalised read counts for triplicates and are reflected by the intensity of colour in the squares shared by the gene and the corresponding sample; red colour indicates upregulation (Padj < 0.01, log₂ FC ≥ 1) and blue colour indicates downregulation (Padj < 0.01, log₂ FC ≤ -1).

Fig 3. Top 5 upregulated gene ontology (GO) terms.

GO terms for biological processes, molecular functions and cellular components enriched with the genes upregulated (Padj <0.01, log₂ FC ≥ 1) in LMH and CEK cells, inoculated with CSW-1 ILTV or ΔgG ILTV at 12 hours post inoculation are represented as bubble plots. The size and colour of bubbles are proportional to the percentage of genes enriched for the GO term.

Fig 4. Top 5 downregulated gene ontology (GO) terms.

GO terms for biological processes, molecular functions and cellular components enriched with the genes downregulated (Padj < 0.01, log₂ FC ≤ - 1) in LMH and CEK cells, inoculated with CSW-1 ILTV or ΔgG ILTV at 12 hours post inoculation are represented as bubble plots. The size and colour of bubbles are proportional to the percentage of genes enriched for the GO term.

Fig 5. Upregulated Reactome pathways.

Pathways enriched (FDR < 0.05) with the genes upregulated (Padj <0.01, log₂ FC ≥ 1) in LMH and CEK cells inoculated with CSW-1 ILTV or ΔgG ILTV at 12 hours post inoculation are represented as bubble plots. The size and colour of bubbles are proportional to the percentage of genes enriched for the pathway.

Fig 6. Downregulated Reactome pathways.

Pathways enriched (FDR < 0.05) with the genes downregulated (Padj < 0.01, log₂ FC ≤ -1) in LMH and CEK cells inoculated with CSW-1 ILTV or ΔgG ILTV at 12 hours post inoculation are represented as bubble plots. The size and colour of bubbles are proportional to the percentage of genes enriched for the pathway.

Fig 7. Panther protein classes.

Clustered heat map representing the protein classes overrepresented (fold-change values > 1 at FDR < 0.05) with the genes that were A) upregulated or B) downregulated at 12 hours post inoculation of LMH or CEK cells, with CSW-1 ILTV or ΔgG ILTV (as compared to Mock). Numbers in the rows indicate the number of up- or down-regulated genes in the protein class. Intensity of colour reflects the number of genes in each class.