Transcriptomic analysis of common carp anterior kidney during Cyprinid herpesvirus 3 infection: Immunoglobulin repertoire and homologue functional divergence

Abstract

Cyprinid herpesvirus 3 (CyHV-3) infects koi and common carp and causes widespread mortalities. While the virus is a significant concern for aquaculture operations in many countries, in Australia the virus may be a useful biocontrol agent for pest carp. However, carp immune responses to CyHV-3, and the molecular mechanisms underpinning resistance, are not well understood. Here we used RNA-Seq on carp during different phases of CyHV-3 infection to detect the gene expression dynamics of both host and virus simultaneously. During acute CyHV-3 infection, the carp host modified the expression of genes involved in various immune systems and detoxification pathways. Moreover, the activated pathways were skewed toward humoral immune responses, which may have been influenced by the virus itself. Many immune-related genes were duplicated in the carp genome, and often these were expressed differently across the infection phases. Of particular interest were two interleukin-10 homologues that were not expressed synchronously, suggesting neo- or sub-functionalization. The carp immunoglobulin repertoire significantly diversified during active CyHV-3 infection, which was followed by the selection of high-affinity B-cells. This is indicative of a developing adaptive immune response, and is the first attempt to use RNA-Seq to understand this process in fish during a viral infection.

Figure 1. Normalized gene expression from CyHV-3 open reading frames (ORFs).

Values were normalised to reads per kilobase of transcript per million mapped reads (RPKM) and log₁₀ transformed to enable sample comparison.

Figure 2

Similarities in carp gene expression profiles using Jensen-Shannon distances (a) and principal components (b), and significantly differently expressed carp genes in each phase (c) and common to different phases (d). In each plot the colours indicate the phase of infection. The numbers in (d) indicate significantly differently expressed genes in each phase and the overlapping segments indicate the number of genes differentially expressed in, and between, phases of infection. PC is an acronym for principal component.

Figure 3. Significantly differentially expressed carp genes that are annotated with the gene ontology term “immune response” (GO:0006955).

The genes have been further categorized into cytokines (a), major histocompatibility classes (b) or other immune system components (c). For better visualisation, raw counts have been normalised (log₂ + 1). The bars on the left side of each heatmap indicate which genes were significantly differentially expressed in each infection phase (red = acute, blue = persistent, violet = reactivation) compared to the mock controls, and the arrows indicate an up- or down-regulation. TNF is an acronym for tumor necrosis factor.

Figure 4

Genomic location, gene splicing and expression differences for interleukin-10 homologues in carp, IL-10a (cycg035130; a,d) and IL-10b (cycg006102; b,e), and the CyHV-3 homologue (c,f). Exons are indicated by yellow boxes and standardized coverage is the depth per million total mapped reads in each sample library (log₁₀ scale; a,b,c). Reads per kilobase per million mapped reads (RPKM) in d, e and f indicate the combined expression signal over the whole gene and are normalized for library and gene size. Asterisks indicate a significant difference compared to the mock infected fish (d,e).

Figure 5

Carp immunoglobulin (Ig) repertoire in different phases of infection (a), selection pressure on complementarity determining regions (CDR) and framework regions (FR) of carp Ig molecules (b), and nucleotide diversity (as measured by Shannon entropy) in a multiple alignment of carp Ig transcripts (c). Selection pressure in (b) was estimated using somatic mutation patterns and the Bayesian estimation of Antigen-driven SELectIoN (BASELINe) algorithm (Yaari et al. 2012). Variable (V), diversity (D) and joining (J) regions of the Ig transcript in (c) are annotated.