Koi herpesvirus encodes and expresses a functional interleukin-10

Abstract

Koi herpesvirus (KHV) (species Cyprinid herpesvirus 3) ORF134 was shown to transcribe a spliced transcript encoding a 179-amino-acid (aa) interleukin-10 (IL-10) homolog (khvIL-10) in koi fin (KF-1) cells. Pairwise sequence alignment indicated that the expressed product shares 25% identity with carp IL-10, 22 to 24% identity with mammalian (including primate) IL-10s, and 19.1% identity with European eel herpesvirus IL-10 (ahvIL-10). In phylogenetic analyses, khvIL-10 fell in a divergent position from all host IL-10 sequences, indicating extensive structural divergence following capture from the host. In KHV-infected fish, khvIL-10 transcripts were observed to be highly expressed during the acute and reactivation phases but to be expressed at very low levels during low-temperature-induced persistence. Similarly, KHV early (helicase [Hel] and DNA polymerase [DNAP]) and late (intercapsomeric triplex protein [ITP] and major capsid protein [MCP]) genes were also expressed at high levels during the acute and reactivation phases, but only low-level expression of the ITP gene was detected during the persistent phase. Injection of khvIL-10 mRNA into zebrafish (Danio rerio) embryos increased the number of lysozyme-positive cells to a similar degree as zebrafish IL-10. Downregulation of the IL-10 receptor long chain (IL-10R1) using a specific morpholino abrogated the response to both khvIL-10 and zebrafish IL-10 transcripts, indicating that, despite the structural divergence, khvIL-10 functions via this receptor. This is the first report describing the characteristics of a functional viral IL-10 gene in the Alloherpesviridae.

Fig 1

Targeted knockdown of the zebrafish il-10r1 gene using a splice site-blocking morpholino. (A) Sequence targeted by the il-10r1 morpholino. The splice site structure of the zebrafish il-10r1 gene, showing exons (rectangles) and intervening introns (chevrons) with the noncoding regions shaded black, is shown. The il-10r1 splice site-blocking morpholino targets the exon 4/intron 4 splice-site (red line), with the target sequence on the pre-mRNA (gray) and morpholino sequence (red) shown. The exon sequence is in uppercase and the intron sequence in lowercase. (B) Effectiveness of the il-10r1 morpholino in vivo, showing RT-PCR of RNA extracted from 56-hpf zebrafish embryos injected with il-10r1 splice site-blocking or control morpholinos, as indicated, including samples not treated with reverse transcriptase (RT-ve). The black arrowhead indicates the wild-type transcript in the standard control embryos that is robustly ablated in il-10r1 morpholino-injected embryos, with alternate splicing products indicated with red arrowheads.

Fig 2

PCR and RT-PCR analysis of the khvIL-10 gene and comparison of splicing patterns of host and viral IL-10 genes. (A) Predicted structure of KHV ORF134 and locations of primer binding sites. (B) Agarose gel electrophoresis of products amplified from DNA and total RNA extracted from KHV-infected cells using PCR primer sets F-R and Fb-R. Lanes 1, KHV U.S. strain F9850; lanes 2, United Kingdom strain G406; lanes 3, Indonesian strain C07; lanes 4, mock infected; lanes M, 100-bp DNA ladder. (C) Structures of host IL-10 and viral IL-10 genes. Introns (white boxes) and exons (black boxes) are indicated, and exon numbers for human IL-10 are provided.

Fig 3

Phylogenetic tree inferred by using host- and virus-encoded IL-10 proteins. Numbers on the branches are bootstrap values for 1,000 replicates expressed as percentage. Virus-encoded IL-10s are shown in bold. Clusters of host (piscine, amphibian, avian, and mammalian) IL-10 genes and eight (A to H) putatively independent host gene capture events are indicated. The systematic assignment of fish species at the level of taxonomic order is also indicated.

Fig 4

Expression of the khvIL-10 gene (A, C, and E) and carp IL-10 gene (B, D, and F) in carp tissues during the acute, persistent, and reactivation phases of KHV infection. Transcripts were quantified using a TaqMan qRT-PCR assay based on comparative threshold cycle (ΔΔCT) and normalized against expression of the 18S rRNA gene. Data are presented as the means ± standard errors for four individual fish per group. Asterisks indicate significant differences (P < 0.05) in expression between acute/reactivation and persistent phases of KHV infection. Note the difference in scale for KHV versus carp genes.

Fig 5

Expression of KHV early (helicase and DNA polymerase [DNAP]) and late (intercapsomeric triplex protein [ITP] and major capsid protein [MCP]) genes in carp tissues during the acute, persistent, and reactivation phases of KHV infection. Transcripts were quantified using a TaqMan qRT-PCR assay based on comparative threshold cycle (ΔΔCT) and normalized against expression of the 18S rRNA gene. Data are presented as the means ± standard errors for four individual fish per group. Asterisks indicate significant differences (P < 0.05) in expression between acute/reactivation and persistent phases of KHV infection.

Fig 6

Functional conservation between khvIL-10 and Danio rerio IL-10. (A to D) Both khvIL-10 and drIL-10 increase embryonic leukocyte numbers. Embryos were injected with standard control morpholino (StdCon) alone (A) or coinjected with mRNA encoding either khvIL-10 (B) or Danio rerio IL-10 (drIL-10) (C) and subjected to WISH using the pan-leukocytic marker (lyz). The number of individual blue-stained lyz⁺ cells was quantified by manual counting (D), showing the mean (dashed red line), 95% confidence interval (red lines), and level of statistical significance (P < 0.01, *). (E to H) Both khvIL-10 and drIL-10 require IL-10R1. Embryos were injected with il-10r1SSMo alone (E) or coinjected with either khvIL-10 (F) or dril-10 (G), analyzed by WISH using lyz, and quantified (H), as described above.