Single-cell RNA sequencing unveils the hidden powers of zebrafish kidney for 1 generating both hematopoiesis and adaptive antiviral immunity 2 3

College of Life Sciences, Key Laboratory for Cell and Gene Engineering of Zhejiang 6 Province, Zhejiang University, Hangzhou, People’s Republic of China. Laboratory 7 for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine 8 Science and Technology, Qingdao, People’s Republic of China. Division of Medical 9 Genetics and Genomics, the Children's Hospital, Zhejiang University School of 10 Medicine, Hangzhou, China. Bone Marrow Transplantation Center, the First 11 Affiliated Hospital, Zhejiang University School of Medicine, Hangzhou, China. 12

Teleost fish are a group of indispensable model organisms for comprehending the 56 evolutionary history and general principles underlying the design of the vertebrate 57 immune system, since they stand as the earliest living jawed vertebrates known as an 58 important link in vertebrate evolution 1 . Over the recent decades, the field of fish 59 immunology has made significant strides, notably hastening the identification and 60 characterization of functional genes and signaling pathways intricately associated 61 with both piscine innate and adaptive immunities. These advances stem from the 62 progress in genome projects encompassing diverse fish species, and have markedly 63 enriched our comprehension of the molecular underpinnings of fish immunity 2-4 . 64 Some breakthroughs have not only challenged established paradigms about the 65 immune system but have also unveiled novel dimensions of mammalian immunity 1,2 . 66 Thus far, it was generally accepted that teleost fish possess the fundamental 67 components of both innate and adaptive immune systems, akin to those seen in 68 humans and other mammalian counterparts 5,6 . However, our grasp of the precise 69 architectural layout, cellular coordination, and functional attributes of the fish immune 70 system, particularly the adaptive facet, remains incomplete 6,7 . Evolutionarily speaking, 71 bony fish represent the earliest living organisms endowed with a rudimentary adaptive 72 immune system encompassing basic molecular and cellular constituents. These 73 encompass immunoglobulins (Igs), antigen-specific receptors (TCR/BCR) driven by 74 the recombination-activating genes, major histocompatibility complex class I and II 75 (MHC-I/II) molecules, as well as T and B lymphocytes. These cellular elements 76 populate the primary and secondary lymphoid organs of fish, such as the thymus and 77 spleen [8][9][10] . Notwithstanding, the fish adaptive immune system is characterized by its 78 own set of specializations and distinctive attributes. Notably absent in fishes are the 79 bone marrow, histologically discernible lymph nodes, Peyer's patches, and germinal 80 centers-entities that define primary and secondary lymphoid organs in mammals 11,12 . 81 Additionally, fishes lack antibody class-switch recombination activity, albeit retaining 82 the ability to express activation-induced cytidine deaminase (AID) 13,14 . These 83 revelations underscore the substantial diversity inherent in immune systems across 84 distinct vertebrate taxonomic groups. Consequently, teleost fish emerge as invaluable 85 primitive animal models, illuminating previously uncharted events during the 86 emergence and phylogenetic progression of adaptive immune systems. Such 87 endeavors stand to elucidate the distinct principles governing the immunology of 88 ancient vertebrates, thereby furnishing scientific substantiation for a broader 89 comprehension of the evolutionary trajectory of vertebrate immune systems. 90 In light of these considerations, we have recently generated a comprehensive 91 atlas of immune cell types within the spleen of zebrafish, employing a single-cell 92 transcriptome profiling approach 15 . Our work has entailed the classification of splenic 93 leukocytes into 11 principal categories and the identification of over 50 subset 94 populations within these categories. Moreover, we have delineated the differential 95 responses of various subset cells-both innate and adaptive-to infection by the 96 spring viremia of carp virus (SVCV). Particularly noteworthy was our discovery of 97 hematopoietic stem and progenitor cells (HSPCs) existed within the spleen. This 98 observation intimates that the fish spleen may well function as a hematopoietic site, 99 transcending its established role as a secondary lymphoid organ. These insights offer 100 fresh perspectives into the intricacies and distinctive attributes of the fish immune 101 system, setting it apart from its mammalian counterparts. Seeking to attain a deeper 102 understanding of the fish immune system, our present study has undertaken a further 103 exploration of immune cell types and their functional characteristics within the kidney 104 of zebrafish. This investigation places special emphasis on the hematopoietic activity 105 of kidney HSPCs and the adaptive immune responses of kidney immune cells towards 106 viral infections. As a result, our classification efforts have yielded 13 categories of 107 cells from kidney leukocyte preparations, unearthing 59 potential subset cells 108 spanning HSPCs and immune cell-associated categories. The observed subset 109 populations manifest disparate reactions to SVCV infection. Most notably, some 110 HSPC subsets exhibit a robust proliferative response to viral infection, capable of 111 inducing trained immunity. Furthermore, we have successfully identified a 112 comprehensive array of cells-encompassing diverse antigen-presenting cells (APCs) 113 as well as effector T and B cells-integral for the full activation of adaptive immunity 114 within the kidney. These adaptive immune-associated cells display marked 115 responsiveness to SVCV infection and antigenic stimulation. Evidently, the complete 116 process of adaptive immunity unfolds within the kidney, as evidenced by the 117 substantial expansion, activation, and somatic hypermutation (SHM) of 118 antigen-specific T and B cells upon exposure to cognate antigens. These results 119 strongly affirmed the kidney's dual role as a lymphoid organ, boasting both 120 hematopoietic functionality and adaptive immune reactivity in adult fish. 121 Consequently, our findings cast the fish kidney in the role of a secondary lymphoid 122 organ, supplementing its recognized status as a primary lymphoid organ. This 123 advancement significantly improved our current comprehension of fish hematopoietic 124 and immune systems, thus advancing our insights into the multifaceted biology of the 125 kidney in ancient vertebrates. 126 127

128
Categories of immune-associated cells in kidney 129 In order to profile distinct immune-cell categories within the kidneys, leukocytes 130 were extracted from zebrafish kidney tissues. The experimental groups consisted of 131 fish treated with PBS (control), fish infected with SVCV (infected), and fish that 132 underwent both vaccination with inactivated SVCV and subsequent SVCV infection 133 (vaccinated+infected). Each sample comprised leukocytes from 60 fish and was 134 subjected to scRNA-seq using the 10× Chromium platform. Following sequencing, 135 the control group exhibited mean reads/median genes per cell of 34  Figure 1A). Moreover, specific genes were pinpointed within each cluster. Notably, 142 clear demarcation boundaries were evident for each cluster in the heatmap, while dot 143 plot and violin plot analyses indicated that a PCA-based cell separation approach was 144 indeed favorable for our investigation (Figure 1B and C, Figure 1-figure  145 supplement 2). 146 The 13 clusters were classified as neutrophils, macrophage/myeloid cells, B cells, 147 T/NK cells, and three categories of HSPCs (HSPCs 1-3), along with renal cells 148 (kidney multiciliated cells, kidney mucin cells, kidney distal tubule and proximal 149 tubule cells) (Figure 1A-C To achieve a more comprehensive understanding of kidney hematopoietic cell 193 populations, subclustering was conducted exclusively on the three HSPC clusters 194 depicted in Figure 1. This analysis unveiled 11 enrichments (designated as H0-H10) 195 (Figure 2A). To validate our outcomes, we aligned the subset-specific genes of each 196 cluster and the ensuing heatmap indicated a distinct categorization for each cluster 197 ( Figure 2B). Within this context, H0 manifested as a Nanos1 + Spi2 + phenotypic 198 cluster expressing signature genes nanos1 and spi2, alongside specific expressions of 199 sla2, csf1rb, rn7sk, sik1 and irf1b, forming a collection of potential new marker genes 200 (Figure 2A-C) Figure  225 2A-C). The endothelial progenitors were also found in the bone marrow of mouse, 226 and they are developmentally associated with a subset of CD34 + hematopoietic stem 227 cells, and can differentiate ex vivo to an endothelial phenotype 22,23 . Subsequently, 228 pseudotime trajectory analysis revealed that a significant portion of HSPCs were 229 positioned within a primary trajectory characterized by five bifurcations and five 230 stages ( Figure 3A-C). The endothelial (H9) and thrombocyte (H10) progenitors were 231 situated towards the trajectory's origin. This arrangement partially validated the 232 constructed trajectory (Figure 3C). H4 cells were positioned towards the trajectory's 233 terminus ( Figure 3C). H2 cells, along with erythrocyte (H7) and lymphoid (H8) 234 progenitors, were positioned towards the trajectory's midpoint. H0 and H3 235 populations, along with NK (H5) and myeloid (H6) progenitors, were located towards 236 both the origin and midpoint of the trajectory. Furthermore, an analysis of the 237 pseudotime dynamics of significantly changed genes in the 11 HSPC clusters revealed 238 three modules delineated by their pseudotemporal expression patterns ( Figure 3D  239 and E). In addition, RNA velocity analysis showed the differentiation potential of H0, 240 H1, H2, H3 and H4 subclusters into myeloid, thrombocyte/erythrocyte and lymphoid 241 progenitors ( Figure 3F). These findings collectively suggested that H0-H4 clusters 242 potentially represent a group of stem and early progenitors that lead to late 243 progenitors responsible for differentiation into erythroid, myeloid, and lymphocyte 244 lineages. Notably, we recently detected the presence of HSPCs in zebrafish spleen 245 tissues, indicating the spleen's role as a hematopoietic organ in fish 15 . To compare 246 HSPC characteristics between the kidney and spleen, we integrated scRNA-seq data 247 from both organs and subjected the merged dataset to cell classification. This 248 assessment demonstrated that the newly generated atlas shared overall consistency 249 with the kidney and spleen, merging proportional HSPCs and immune cells from the 250 two organs to varying extents ( Figure 4A). These observations underscored the 251 presence of substantial overlapping HSPCs and immune cell types in both the kidney 252 and spleen. Furthermore, pseudotime trajectory analysis highlighted similar cell 253 differentiation trajectories shared between the kidney and spleen (Figure 4B-E). 254 Pseudotime dynamics of significantly altered genes across all categories were 255 analyzed and organized into three modules based on their pseudotemporal expression 256 patterns ( Figure 4F and G). In aggregate, our findings reveal overall similarities in 257 HSCPs' composition and differentiation potentials between the kidney and spleen, 258 suggesting analogous functionalities in hematopoiesis and immunity across both 259 organs. Nevertheless, the precise correlation between the fish kidney and spleen 260 requires further clarification. 261 262

B cell subsets in kidney 319
Nine subclusters of B cells (designated as B0-B8) were classified through 320 unsupervised clustering of all B cells, based on their expression of distinct B-subset 321 signature genes (Figure 6A and B). B0 and B3 were designated as immature/mature 322 B cells (im/mat. B) due to their expression of zfpm1 and gata1a genes ( Figure 6A  323 and B). B1 was categorized as a canonical IgM + IgD + B subset, characterized by its 324 high expression of ighm and ighd genes. B2 and B7 were identified as pro/pre B 325 subsets, exhibiting significant expression of pclaf, pdia4, pdia6, rag1, and rag2 genes. 326 B4 likely represented a population of plasma cells that highly expressed ighm or ighz 327 ( Figure 6A and B il10 and tgfb1a/b, suggesting the potential presence of a regulatory B cell type within 334 this subcluster ( Figure 6A and B). B8 was recognized as a B1-like subset with 335 heightened immune activities, including chemokine production, based on its 336 expression of cd5, ccl33.3, ccl34b.4, nkl.4, tnfrsf18, nkl.2, sla2 and hmgn3 genes 337 ( Figure 6A and B). Interestingly, a distinct subset of IgM + β + cells was identified 338 within B8 (Figure 6A  Heterogeneity of kidney macrophage/myeloid cells 373 We categorized kidney macrophage/myeloid cells into 10 distinct subclusters 374 (designated as M0-M9) using a resolution of 0.3, as depicted in Figure 7A-C. A 375 heatmap showcasing the top ten most significant subset-specific genes for each 376 subcluster is provided in Figure 7B. M0  Neutrophils comprise the most prevalent subset of kidney leukocytes, 403 constituting 45.00% within the immune-cell categories (clusters 0, 2, and 9 in Figure  404 1), and accounting for 38.15% of the total sequenced cells in both the control and 405 SVCV-infected groups (Figure 9A and B). Subsequently, we undertook an analysis of 406 neutrophil heterogeneity. Through iterative subclustering within the neutrophil 407 category alone, we derived six distinct subgroups (designated as N0-N5) (Figure 8A

Trained immune response of HSPCs to SVCV
Numerous studies have documented the phenomenon of enhanced resistance in 545 innate immune cells such as macrophages and neutrophils upon reinfection with the 546 same or unrelated pathogens, a phenomenon termed trained immunity 39-41 . 547 Furthermore, Bacillus Calmette-Guerin (BCG) has been found to induce trained 548 immunity in hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs), 549 promoting augmented myelopoiesis at the expense of lymphopoiesis 42,43 . In this study, 550 we investigated the potential induction of trained immunity in innate immune cells 551 and HSPCs within the kidney upon stimulation by SVCV. To this end, zebrafish were 552 immunized with the SVCV vaccine for 14 days and subsequently exposed to virulent 553 SVCV for another 7 days. The outcomes demonstrated a substantial reduction in the 554 proportion of neutrophils within cluster 0 following SVCV infection. However, this 555 population exhibited minimal reduction in vaccinated zebrafish (referred to as 556 "trained zebrafish") upon the second SVCV challenge (Figure 11-figure  557 supplement 1). Furthermore, the abundance of HSPCs 1 within cluster 4 remained 558 unchanged after SVCV infection, yet experienced significant expansion during the 559 second SVCV challenge in trained zebrafish (Figure 11-figure supplement 1). 560 Notably, the H0 subset exhibited the most pronounced response to trained immunity 561 to SVCV within the HSPC categories ( Figure 11A). Specifically, the proportion of 562 H0 decreased from 27.81% in the PBS group to 19.87% in the SVCV group, then 563 rapidly surged to 41.43% in the vaccinated + SVCV group. This trend was supported 564 by the significant upregulation of numerous immune-related genes within H0 subset 565 cells of trained zebrafish ( Figure 11B). Thus, the shift in the H0 subset played a 566 pivotal role in the overall heightened activity of the HSPCs 1 category within trained 567 zebrafish. Collectively, these findings underscore an intensified immune response in 568 kidney HSPCs of zebrafish subsequent to SVCV vaccination, suggesting the 569 inducement of trained immunity in zebrafish kidney HSPCs for the generation of a 570 memory response to SVCV infection. Furthermore, the neutrophils within the kidney 571 of trained zebrafish exhibited enhanced antiviral immunity, showcasing potential 572 innate immune memory characteristics. 573 574

Infection of SVCV in kidney immune cells 575
To delineate the potential immune cells susceptible to SVCV infection, we 576 investigated the transcription of five subgenomic RNAs/genes (sgRNAs) encoding N, 577 P, M, G, and L proteins during the SVCV replication cycle across diverse kidney 578 immune-cell categories, following our recently described approach 15 . Applying an 579 unsupervised cluster detection algorithm, we identified 10 clusters characterized by 580 similar gene expression profiles. Importantly, the integration of viral sgRNA 581 sequences into the kidney scRNA-seq datasets revealed an overall preservation of the 582 composition of immune-cell categories ( Figure 12A). Subsequently, we scrutinized the transcripts of the five sgRNAs in scRNA-seq datasets from both control and 584 SVCV-infected zebrafish. As anticipated, the five sgRNAs were present in cells from 585 infected fish but absent in those from control fish, validating the effectiveness of our 586 integration method (Figure 12B). The representation of cells transcribing N-, M-, P-, 587 G-, and L-protein encoding sgRNAs exhibited proportional variation, with 588 approximately 0.15% of cells carrying sgRNA for N protein, followed by M (0.12%), 589 P (0.12%), G (0.06%), and L (0.03%) proteins, respectively ( Figure 12C). This 590 distribution aligns with prior observations, confirming the heightened prevalence of N 591 protein transcripts in SVCV-infected cells, followed by those for M, P, G, and L 592 proteins 44-46 . Refining our analysis to specific cell types, we discerned that viral 593 sgRNAs were predominantly detected in macrophage/myeloid cells within cluster 3 594 and 9. This observation strongly implies that macrophage/myeloid cells are the 595 prevailing cell types supporting productive viral replication in zebrafish kidney 596 ( Figure 12D). 597 598

Experimental assay for adaptive immunity occurring in kidney 599
The aforementioned observations underscore the immunologically responsive 600 nature of the fish kidney, revealing innate and adaptive immune activities that extend 601 beyond its previously recognized role in hematopoiesis. Among these activities, 602 adaptive immunity has traditionally been attributed to secondary lymphoid organs 603 such as the spleen and lymph nodes 9,47 . This study offers experimental confirmation 604 for the occurrence of adaptive immunity in the zebrafish kidney following viral 605 stimulation. To this end, zebrafish were exposed to inactivated SVCV (10 6 606 TCID50/fish) for 7 days, and the activation of kidney T and B cells was assessed by 607 quantifying their heightened proliferation and increased expression of CD154 and 608 CD40 costimulatory molecules through flow cytometry and RT-qPCR. As anticipated, 609 CD4 + T, CD8 + T, and IgM + B cells exhibited significant proliferation in the kidneys of 610 zebrafish stimulated with SVCV compared to control fish that received mock PBS 611 ( Figure 13A-D). In tandem, the transcriptional levels of CD154 and CD40 genes 612 displayed marked upregulation, concurrent with the proliferation of CD4 + T, CD8 + T, 613 and IgM + B cells (Figure 13E and F). Importantly, parallel activation of CD4 + T, 614 CD8 + T, and IgM + cells was also discerned in the spleens of SVCV-stimulated 615 zebrafish ( Figure 13G). These results suggested that the zebrafish kidney assumes a 616 central role in orchestrating adaptive immune responses to viral infection, a role that 617 may be comparable to that of the spleen, a well-known secondary lymphoid organ 618 crucial for initiating and advancing adaptive immunity. To substantiate this 619 perspective, we proceeded to investigate the occurrence of SHM, a hallmark event 620 driving antibody affinity maturation in adaptive humoral immunity, within kidney B 621 cells upon antigenic stimulation. We employed RT-qPCR to examine the expression of 622 the zebrafish AID gene (aicda), a pivotal initiator of SHM. As anticipated, AID 623 transcript presence was evident in the zebrafish kidney, and its expression 624 significantly increased in response to SVCV stimulation ( Figure 13H). 625 Correspondingly, SHM was identified in kidney B cells through PCR-based 626 high-throughput sequencing. Notably, the frequency of mutagenesis within V regions 627 (V4-9) of IgM genes demonstrated a substantial antigen-dependent increase (Figure  628 13I). These findings offer novel insights into the dual-functionality of the zebrafish 629 kidney, encompassing both hematopoietic and adaptive immunological 630 responsiveness. This suggests that the fish kidney operates as a unique organ 631 amalgamating the roles typically associated with primary and secondary lymphoid 632 tissues. 633 634 Discussion 635 The immune system constitutes a complex evolutionary entity marked by 636 significant heterogeneity and structural-functional diversity across various taxa. The 637 origin and phylogenetic evolution of the vertebrate immune system captivates the 638 attention of comparative immunologists, particularly concerning teleost fish due to 639 their pivotal phylogenetic standing. In human and other mammalian organisms, the 640 immune system encompasses primary and secondary lymphoid organs 11,47 , the former 641 including bone marrow and thymus, facilitating hematopoiesis, lymphocyte 642 maturation, and functional development 47,48 . Secondary lymphoid organs, including 643 lymph nodes, spleen, and sections of mucosal immune systems, trigger the activation 644 of naïve lymphocytes by antigens, thereby initiating and advancing adaptive immune 645 responses 11,49,50 . In contrast to mammals, teleost fish lack bone marrow and lymph 646 nodes. Instead, the anterior section of the fish kidney (referred to as the head kidney) 647 emerges as an operational analog of mammalian bone marrow in adult fish. 648 Accordingly, the fish kidney functions as the primary hematopoietic site, overseeing 649 the generation of circulating blood cell lineages and B cell maturation 51-53 . 650 Furthermore, the fish kidney is also thought to be an immunologically responsive 651 organ capable of eliciting immune responses against a spectrum of pathogens, 652 including bacteria, viruses, and parasites 26,27 . These findings establish the kidney's 653 pivotal role in fish immunity, although a comprehensive understanding of the cellular 654 components and their attributes within the hematopoietic process and immune 655 responses in the fish kidney remains elusive. 656 To address these gaps, we conducted a single-cell RNA sequencing (scRNA-seq) 657 analysis of kidney leukocytes in zebrafish, unraveling the spectrum of hematopoietic 658 and immune-associated cell types in adult fish. We identified 13 distinct cell 659 categories, spanning three groups of HSPCs, six cohorts of innate and adaptive 660 immune-related cells, and four renal cell types, which emerged as byproducts of 661 cellular preparation. number of fish-specific lineages, probing the relationship between these 707 uncharacterized HSPCs and fish-specific lineages remains a promising avenue of 708 exploration. 709 The spleen-an evolutionarily conserved secondary lymphoid organ spanning vertebrates 753 from fish to mammals. Consequently, our findings strongly imply that the zebrafish 754 kidney might function as a secondary lymphoid organ, fostering the generation of 755 adaptive immunity through interactions among APCs and antigen-specific T and B 756 cells. To empirically substantiate this hypothesis, we examined the occurrence of 757 antigen-stimulated adaptive immune responses in the kidney. As anticipated, the CD4 + 758 T, CD8 + T, and IgM + B cells exhibited pronounced activation in the zebrafish kidney 759 under SVCV challenge. This activation was underscored by significant proliferation 760 in CD4 + T, CD8 + T, and IgM + B cell populations, followed by upregulated 761 transcriptional expression of CD154 and CD40 genes on these cells. Furthermore, we 762 also observed the occurrence of antigen-induced expression of AID and SHM in the 763 kidney IgM + B cells, two hallmark events for the affinity maturation of antibodies in 764 adaptive humoral immunity. These results collectively establish the zebrafish kidney 765 as a distinct entity encompassing attributes of both primary and secondary lymphoid 766 organs, thus offering fresh insights into the dual functional roles of the fish kidney, 767 spanning hematopoiesis and adaptive immunity. While peripheral lymph nodes are 768 absent, the spleen has historically stood as the predominant secondary lymphoid organ 769 in fish. However, our current study introduces a novel member to the array of 770 secondary lymphoid organs. The intricate interplay between the roles of the kidney 771 and spleen in hematopoiesis and immune responses within fish warrants further 772 elucidation. Moreover, unraveling the structural foundations accommodating diverse 773 HSPCs and immune cell types and precisely orchestrating hematopoiesis and immune 774 responses within the kidney and spleen emerges as a compelling area of inquiry. 775 Addressing these queries necessitates the utilization of advanced techniques, such as 776 single-cell spatiotemporal transcriptomic analysis at the level of cross-organ 777 single-cell transcriptome profiling. 778 In summary, our study presents a comprehensive panorama of HSPCs and  779  immune cell types within the zebrafish kidney under steady-state conditions, viral  780 emergencies, and vaccine training. Our identification of a multitude of HSPCs, along 781 with developmentally-linked progenitors and immune cell types, and their 782 modifications under emergency and training scenarios, underscores the pivotal role of 783 the kidney in fish defense against viral infections. Moreover, the revelation of trained 784 immunity establishment within HSPCs holds significant implications for 785 comprehending the novel pathways and mechanisms governing trained immunity 786 formation. The paradigm shift-where the kidney, traditionally viewed as a primary 787 lymphoid organ facilitating hematopoiesis-emerges as a secondary lymphoid organ, 788 marks a pivotal transformation in understanding fish biology. Our study offers 789 insights into the intricate and heterogeneous nature of the fish immune system, 790 shedding light on the multifunctionality of the fish kidney, encompassing 791 hematopoiesis, immune defense, waste excretion, and osmoregulation-mainstays of 792 kidney function across diverse vertebrates. 793 794

Materials and methods 795
Experimental fish 796 Wild-type AB zebrafish (Danio rerio), with body weights ranging from 0.5 to 1.0 g 797 and lengths of 3-4 cm, were reared under standard laboratory conditions. The fish 798 were raised in recirculating water at a temperature of 28°C and exposed to a 12-hour 799 light/12-hour dark cycle, as previously described 65 Adult zebrafish were randomly divided into three groups, i.e. PBS-administered group 823 (control), SVCV-infected group (infected), and inactivated SVCV-vaccinated plus 824 virulent SVCV-infected group (vaccinated+infected). Each zebrafish in these groups 825 received an intraperitoneal injection of 10 μl of phosphate-buffered saline (PBS, 826 pH=7.0), 10 μl of virulent SVCV (10 3 TCID 50 /fish), or 10 μl of inactivated SVCV 827 followed by 10 μl of virulent SVCV (10 3 TCID 50 /fish). In the context of vaccination, 828 zebrafish were immunized with 10 μl of inactivated SVCV vaccine (10 5 TCID 50 /fish) 829 for two weeks, succeeded by infection with virulent SVCV (10 3 TCID 50 /fish) for an 830 additional week. Subsequently, kidney samples were collected at 7 days post-infection 831 (dpi) for the control and SVCV-infected groups and at 21 dpi for the 832 vaccinated+infected group. Equivalent quantities of tissue were pooled from each 833 group, and these samples underwent subsequent leukocyte isolation. 834 835