Different transcriptional response between susceptible and resistant common carp (Cyprinus carpio) fish hints on the mechanism of CyHV-3 disease resistance

- Cyprinid herpes virus-3 (CyHV-3) is a dsDNA virus widely hampering production of common carp (Cyprinus carpio), one of the most farmed fish species worldwide.
- To study the genetic basis and cellular pathways underlying disease resistance, RNA-Seq was used to characterize transcriptional responses of susceptible and resistant fish at day 4 after CyHV-3 infection..
- Results: In susceptible fish, over four times more differentially expressed genes were up-regulated between day 0 and 4 compared to resistant fish.
- Susceptible and resistant fish responded distinctively to infection as only 55 (9%) of the up-regulated genes were shared by these two fish types.
- 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- 1 Department of Animal Sciences, RH Smith Faculty of Agriculture, Food and Environment, The Hebrew University of Jerusalem, Rehovot, Israel Full list of author information is available at the end of the article.
- Securing a steady supply of healthy and nutritious foods for the growing human population is one of the main challenges of today and aquaculture takes a growing share in addressing this challenge [1].
- Major impediments in sustainable production and further growth of the food- fish sector are caused by a whole range of infectious diseases [2], because measures for their prevention and control under aquaculture conditions are very limited..
- Common carp (Cyprinus carpio), henceforth referred to as ‘carp’, is one of the five most produced fish species worldwide and an important food source in many heav- ily populated low-income countries.
- Out- breaks of CyHV-3 have started in the late 1990s and have been persisting since then, spreading to most envi- ronments in which carp is cultured and causing signifi- cant losses of up to 100% of the pond fish [11–13].
- However, the pro- tective response in fish might have a more complex genetic basis since the bony-fishes specific whole genome duplica- tion further contributed to the degree of complexity of the immune repertoire, especially when considering the expan- sion in the number of interleukins (ILs), Toll-like receptors.
- However, to date most of the genetic variation affecting this trait remains unknown..
- Transcriptome differences between susceptible and resistant fish.
- In a clustering analysis, based on normalized read counts of differentially expressed genes (DEGs), the three replicates of susceptible families were highly corre- lated among themselves and so were the three replicates of resistant families, thus, attesting to the high reproducibility of the data (Fig.
- comparisons indicated that transcriptomes of the four treatments were considerably distinct (Fig.
- The largest overlap (435 DEGs) was found between S0/R0 and S4/R4, which represented fish-type differences regardless of the disease infection.
- Each of the four comparisons identified both up- and down-regulated genes relative to a reference treatment (the denominator of each comparison), all with a wide.
- These changes in expression between days 0 and 4 are reflecting the response to infection, and this response differed considerably between susceptible and resistant fish in two aspects.
- Sec- ond, there was little overlap in the set of DEGs between susceptible and resistant fish.
- (n = 14) of the up- and down-regulated DEGs, respectively, were shared between these fish types (Fig.
- 0006950), which reflects the general response of the fish to the challenge conditions.
- Susceptible and resistant fish were different both in how many chemokines were up-regulated and also in which types.
- Of the six CC chemokines, the five which were up-regulated in susceptible fish were ccl19 homologs and the one in resistant fish was a different chemokine, ccl35 (Fig.
- Of the ten CXC chemokines, two.
- Of the seven non- chemokine genes in this GO term, five were up-regulated in both susceptible and resistant fish (Fig.
- Expression pattern of duplicated chemokine genes Chemokines form a large gene family with several homo- logs for many of the different family members in genomes of bony-fishes.
- a Volcano plots showing the distribution of normalized read counts as a function of Log 2 (fold-change) for DEGs in each of the four comparisons.
- Negative and positive Log 2 (fold-change) describe down-regulated and up-regulated genes, respectively, relative to the reference treatment, which is always the denominator of the title (e.g for S0/R0, R0 is the reference).
- b and c Overlap in number of genes more highly expressed at days 0 and 4 (b) in susceptible compared to resistant fish and c in resistant compared to susceptible fish.
- First, the proportion of DEGs out of the total gene copies was examined.
- 7 and cxcl8b.2 is unmapped according to the latest version of the ZFIN.org database.
- This family expansion was probably a result of the carp-specific whole genome duplication (as might be suggested by the 2 in carp:1 in ZF ratio observed for Table 1 Carp genes (cypCars) in the top enriched GO terms in susceptible fish.
- 4b) and only cypCar_00016657 was a DEG between susceptible and resistant fish.
- Some of these transcriptional dif- ferences in homologs and paralogs were DEGs between susceptible and resistant fish..
- As a reference, the significance [Log 10 (p- value)] of the test for association of markers with survival was also plotted against their genomic position (Fig.
- 5a), a ccl20 homolog (cypCar_00050111) was more highly expressed in sus- ceptible than in resistant fish at day 4.
- 00023948) was up-regulated at day4 in resistant fish..
- The expression of Interleukin-10b (il10b), the paralog of il10a, was in- duced by the infection, but similarly in susceptible and resistant fish.
- Each of the selected genes was up-regulated in response to CyHV-3 infection in one of the fish types based on the RNA-Seq analysis.
- Based on the RNA-Seq data, both genes were up- regulated DEGs at day 4 in susceptible, but not in resistant fish.
- In the RT-qPCR analysis, no induction was observed at day 2, whereas at day 8 the genes were equally induced in both susceptible and resistant fish (Fig.
- Thus, also in resistant fish ISGs are up-regulated in response to infection, but apparently at a later stage and not as part of the early induced immune response..
- ‘leukocyte migration’ (GO:0050900), which was the most enriched one in resistant fish and was also enriched in.
- 45160) to represent the main up-regulated CXC chemo- kine in resistant fish.
- 19885 (ccl19a.1) and cypCar_47570 (cxcl8/il8) were up- regulated DEGs at day 4 in susceptible, but not in resistant fish.
- Distinct transcriptional responses between susceptible and resistant fish.
- Different clades of the tree are different chemokines, colored and named after ZF gene family.
- DEGs location is displayed in the context of the QTL shape (grey line) as deduced from the significance level [Log 10 (p-value)] of the association test between markers and survival.
- Each panel refers to a different gene that was initially found to be differentially expressed between day 0 and 4 in susceptible or resistant fish in the RNA-Seq analysis.
- Comparing the RNA-Seq data between days 0 and 4 identified ccl19a1.1 (c) was up-regulated only in susceptible fish, while cxcl8b1.2 (d) and cxcl8b1.3 (e) were up-regulated only in resistant fish..
- None of the within-day comparisons between resistant and susceptible fish were statistically significant.
- level of details in fish is challenging compared to mam- mals because many of the immune system gene families had expanded during fish evolution.
- The diversification levels we identified indicated that for studying mecha- nisms in carp based on transcriptional changes, the reso- lution of analysis should be down to copy specific expression and for gene families like chemokines, a phylogenetic reference is indispensable for interpretation of the results..
- Interestingly, in contrast to susceptible fish, among the DEGs of resistant fish at day 4 post infection, IFNs and ISGs were absent.
- A few stud- ies comparing between susceptible and resistant fish identified differences in their type-I IFN mediated anti- viral responses.
- These studies potentially suggested that an earlier activation of the IFN response could improve re- sistance.
- These genes in susceptible fish were induced at day 4 and were still induced at day 8 post infection while in resistant fish induction was ob- served only later (day 8).
- This delay in the IFN temporal response correlates with the delay in virus load elevation seen in resistant fish [18].
- Distinct chemokine profiles for susceptible and resistant fish.
- The most enriched GO term in the list of up-regulated genes in response to infection of resistant fish was.
- Many more genes were up-regulated in sus- ceptible fish than in resistant fish in response to CyHV-3 infection, including more chemokines.
- However, because of the fewer DEGs, the fold-enrichment of ‘leukocyte mi- gration’ in resistant fish was higher than in susceptible, but most importantly included different genes.
- Almost each chemokine type actually comprises a gene family with multiple copies in the ZF genome, let alone in that of the common carp.
- Notably, subjected to the accuracy of the annotations from the ZF, the carp chemokines that were up-regulated in susceptible fish were different than those in resistant.
- Contrary to susceptible fish, up-regulated in resistant fish were mainly the cxcl8/il8 che- mokines, which are known to attract neutrophils [44, 52]..
- Thus, according to chemokine profile induction, different arms of the cellular immunity are elicited by susceptible and resistant fish at this stage of infection..
- Some of these chemokines were expressed specifically in resistant fish and others expressed in susceptible fish only at day 8 post infection when their mortalities start..
- Thus, this finding suggests that early activation of neutrophil is a part of the innate immune mechanism against the virus.
- Mice infected with Herpes Simplex Virus [55] or with Neurotropic mouse Hepatitis Virus [56] that were depleted of neutrophils had elevated virus loads compared to controls, indicating that in some virus infections, neutrophils can play an important role in suppression of the virus.
- To enhance our understanding of viral resistance mecha- nisms, in this study we characterized the transcriptional re- sponse to CyHV-3 infection of susceptible and resistant fish.
- Susceptible and resistant fish differed in both the mag- nitude of response as well as in sets of regulated genes.
- A different immune-related response as seen here, may pro- vide clues to why the outcome of the disease is different between susceptible and resistant fish.
- Expanding the ana- lysis to further time points and possibly more tissues might strengthen our findings and yield a more comprehensive picture of the resistance mechanisms.
- However, in addition to the specific knowledge on carp CyHV-3 resistance, already now, some insights emerged that are shared with other species in response to other diseases regarding differ- ences between susceptible and resistant fish.
- The % survival of the selected fam- ilies was determined beforehand (Fig.
- To obtain samples for RNA- Seq, 20–50 nạve fish from each of the six families, with a mean weight of 30–50 g, were infected using our estab- lished cohabitation disease challenge model [18].
- The effectiveness of the challenge was monitored also by including suscep- tible koi fish as controls.
- RNA was extracted from spleen samples of three fish of each of the six families at time points 0 and 4 (a total of 36 samples) using RNeasy mini kit (Qiagen), according to the manufacturer’s instructions.
- All 36 individual samples passed the quality level and then 12 RNA pools were created, each containing equal amounts of total RNA from three fish of the same family and time- point.
- The pooled RNA samples also passed the quality checks for quantity and integrity of the Bioanalyzer test..
- Four comparisons were analyzed to identify DEGs: between day 0 and day 4 in susceptible fish (S4/S0) and in resistant fish (R4/R0), as well as between susceptible and resistant fish at day 0 (S0/.
- Similarity between samples was evaluated by correlating the normalized expression values of all DEGs between each pair of the 12 samples and by hierarchical clustering of these correlation coefficients using central- ized and log2 transformation [61] and R Bioconductor modules [64].
- There were some differences in length and coverage between accessions, however, all these tran- scripts include a significant portion of the ORF of the genes ensuring correct gene alignments.
- DEGs from all four pairwise comparisons were considered, since some of the differences in resistance might be due basic (day 0) differences in expression, while others due to expression differences only in response to the infec- tion.
- For these QTL DEGs, absolute Log 2 (fold-change) values were plotted against their location (according to [35]) on the orthologous chromosome of the closely re- lated ZF.
- At this time, carp genomic location is uncertain due to the discontinuous state of the carp genome as- sembly.
- The significance level Log 10 (P- value) of the association of markers with survival was plotted as a function of their ZF genomic position.
- Ex- pression levels of target genes were then calculated rela- tive to the mean value of the reference gene and to the mean value of one resistant family at day 0.
- Statistical analyses for all parts of the results were done using the JMP14 software (SAS institute, NC)..
- ADF carried out all the bioinformatic analysis of the RNAseq data up to the stage of identifying differentially expressed genes.
- The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript..
- Protocols for fish reproduction, rearing, euthanasia, and tissue sampling were reviewed, approved and granted, with permission number AG from the Animal research ethics committee of the Hebrew University of Jerusalem.
- All experimental procedures followed these approved protocols in accordance with the recommendations of the review committee and in ac- cordance with national laws and regulations for ethics in animal research..
- Comparison of the resistance of selected families of common carp, Cyprinus carpio L., to koi herpesvirus: preliminary study.
- Recent duplication of the common carp (Cyprinus carpio L.) genome as revealed by analyses of microsatellite loci.
- Comparison of the exomes of common carp (Cyprinus carpio) and zebrafish (Danio rerio).
- Genome sequence and genetic diversity of the common carp, Cyprinus carpio.
- Extensive expansion and diversification of the chemokine gene family in zebrafish: identification of a novel chemokine subfamily CX.
- Neutrophil-mediated suppression of virus replication after herpes simplex virus type 1 infection of the murine cornea.
- Genetic variation in seine escapability of the common carp

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