Using single cell RNA sequencing to assess immunological responses in quails injected with porcine circovirus-like particles

Govindasamy, Lekita

Using single cell RNA sequencing to assess immunological responses in quails injected with porcine circovirus-like particles

Thesis / Dissertation

2025

Abstract

Animal models play a pivotal role in studying molecular characteristics of host responses to infection. Based on physical characteristics, Japanese quails may be suitable for use as animal models and hold many advantages compared to their model counterparts, the chicken. However, little is known about the immunological responses of Japanese quails during viral exposure. It is imperative to understand how these animals handle adverse conditions when exposed to foreign invaders, such as viruses. Detailed information regarding their innate and adaptive immune responses can be obtained by studying the transcriptome of peripheral blood mononuclear cells (PBMCs). Such information is poorly researched in these birds and can provide helpful information for future studies. This study aimed at using porcine circovirus type 2 (PCV-2) to assess the evolution of the immune response of quails using single cell RNA sequencing (scRNA-seq) and computational methods. PCV-2 has a single stranded circular DNA genome that encodes the single capsid protein (CP) as well as a replication-associated protein. The impacts of infection with PCV-2 have had devastating effects on the swine industry, causing major stress to the socio economic state of many countries, and therapies are needed. This study successfully expressed PCV-2 capsid protein in E.coli, with the formation of virus like particles (VLPs). The purified VLPs were assessed by transmission electron microscopy and groups of quails were immunised either with the VLPs or with buffer. A specific antibody response was elicited and detected in both the blood and egg samples of quails immunised with PCV-2 VLPs. For analysis of the transcriptome from the immunized quails, the 10X Genomics Chromium platform was used to isolate the PBMC samples into individual droplets, extract the mRNA from each cell, and synthesize barcoded cDNA that was then sequenced. Sequenced reads were analysed bioinformatically using software packages in R and Python. Datasets analysed were, the pooled and integrated dataset obtained from the two quail experimental groups, as well as the demultiplexed dataset that separated the pooled samples into the individual PBMC replicates. Analyses revealed 14 clusters for the pooled PBMC dataset, and 11 clusters for the demultiplexed dataset. Six cell types belonging to the innate and adaptive immune system were identified in both datasets, with macrophages being the most dominant cell type and T cells being the second largest cell type in both the PCV-2 datasets. Differential gene expression revealed genes that were uniquely upregulated in specific cell types and subtypes, while many differentially expressed genes (DEGs) were shared between cell types. The similarity in immune response between the control and PCV-2 quail groups in this study demonstrated that quails could tolerate exposure to PCV-2 capsid protein with no major harm. Based on upregulated and downregulated DEGs as well as identified molecular functions and biological processes, an innate immune response was induced with early stages of the adaptive immune response and a regulated pro-inflammatory response. Increased levels of cell differentiation and metabolism was activated during the response. These outcomes highlight the immunological response of Japanese quails to PCV-2.