Abstract Background Viral hemorrhagic septicemia virus (VHSV) is a highly pathogenic virus that poses a significant threat to the health of diverse marine species. Among these, trout species, particularly rainbow trout (Oncorhynchus mykiss), are highly susceptible. This study evaluated the effects of VHSV infection on the biometric traits of rainbow trout and investigated the molecular mechanisms associated with the disease. Results Biometric traits of fish were collected and documented weekly during the fourth and fifth weeks of the experiment. A statistically significant difference in body weight was observed in the fifth week, particularly between the control group and the groups injected with either physiological saline or the virus. Additionally, body length-related attributes showed significant variation across all treatment groups within the designated timeframe. RNA was extracted from spleen tissue of the group injected with high doses of physiological saline and the group injected with high doses of the virus using the TRIzol protocol. Differential gene expression analysis revealed 1,726 genes with significant differences between the two groups. Several key immune-related genes were identified, including TLR2, TLR7, TLR8, TLR22, IRF5, IRF6, IRF7, IRF8, IRF10, IL11a, IL12B, IL1b, IL7R, ILR1 II, HSP90B1, HSP47, TNF-α, TRF3, SPRY1, CASP3, FN1, GAPDH, and IgGFc-binding proteins. Network-based analysis of differentially expressed genes was conducted using the GeneMANIA module in Cytoscape, and metabolic pathways were identified through the DAVID database. The results highlighted the involvement of key pathways, including the Toll-like receptor pathway, p53 signaling pathway, PPAR signaling pathway, and the cell cycle, in the infected group. Validation tests for selected upregulated (EPCAM, APOC2 and XDD4) and downregulated (TLR7, XDH, and TSPAN36) candidate genes, were conducted using qRT-PCR. The qPCR results showed a strong and statistically significant correlation with the RNA-seq data, confirming the reliability of the findings. Conclusions VHSV significantly impacts the growth of rainbow trout, affecting both body length and gene expression. This study underscores the substantial economic risks posed by the virus and the absence of an effective cure, highlighting the importance of preventative measures. Additionally, potential resistance genes and pathways were identified through RNA sequencing, providing valuable insights for improving trout breeding programs. Supplementary Information The online version contains supplementary material available at 10.1186/s12864-025-11300-x. Keywords: Rainbow trout, RNA-seq, Gene network, Viral hemorrhagic septicemia virus Background The first isolation of Viral Hemorrhagic Septicemia Virus (VHSV) from European rainbow trout (Oncorhynchus mykiss) was reported by [[30]1]. Since then, the virus has been identified in over 82 different fish species worldwide [[31]2, [32]3]. Infectious diseases are widespread in marine ecosystems, causing devastating economic losses in the aquaculture industry and posing significant threats to fisheries [[33]4, [34]5]. Viral infections account for 25% of common marine diseases and impact 49% of fish species. The high mortality rates of wild fish in large lakes due to viral diseases have drawn considerable attention from both researchers and governments to investigate the underlying causes and identify the infectious agents responsible [[35]4]. Viral hemorrhagic septicemia (VHS) is classified as a major aquatic disease by the World Organization for Animal Health (OIE). Despite extensive efforts, no effective treatment exists to control its spread [[36]6]. VHSV affects a wide range of marine species, with rainbow trout (Oncorhynchus mykiss) being particularly susceptible [[37]7]. A member of the Rhabdoviridae family and Novirhabdovirus genus, VHSV possesses a negative-strand RNA genome approximately 12 kbp in length. This genome encodes five structural proteins: nucleoprotein (N), phosphoprotein (P), matrix protein (M), glycoprotein (G), and RNA polymerase (L) as well as a nonstructural protein (NV) [[38]8, [39]9]. Numerous studies have investigated the molecular mechanisms and key genes associated with VHSV infection and resistance [[40]5, [41]7, [42]10–[43]14]. Research on rainbow trout (Oncorhynchus mykiss) has examined both resistant and susceptible lines, resulting in the identification and precise mapping of quantitative trait loci (QTL). These studies highlight the critical role of interferon and interferon-stimulated genes in enhancing the innate immunity of rainbow trout, thereby strengthening their defenses against VHSV. In particular, cell lines A22 and B57 infected with inactivated VHSV exhibited heightened resistance responses. Additionally, the identification of genes associated with innate immunity located near QTLs in the rainbow trout (Oncorhynchus mykiss) genome provides further evidence supporting these findings [[44]5]. Wild strains of viral hemorrhagic septicemia virus (VHSV) are associated with higher mortality rates in farmed salmon (Salmo salar), whereas strains isolated from marine fish have shown reduced disease severity in rainbow trout (Oncorhynchus mykiss) [[45]7]. Comprehensive genomic analyses of various marine and freshwater species suggest that a small number of amino acids may significantly influence the severity of VHSV infections in rainbow trout (Oncorhynchus mykiss) [[46]7]. Recent studies have identified specific genetic markers within the NV and N proteins that are associated with virulence. These findings underscore the genetic diversity of VHSV and its potential impact on disease severity and outcomes in aquaculture species [[47]1]. The advent of RNA-seq technology has significantly advanced transcriptional studies in various fish species, including zebrafish (Danio rerio), channel catfish (Ictalurus punctatus), European bass (Dicentrarchus labrax), and rainbow trout (Oncorhynchus mykiss) [[48]13–[49]15]. By analyzing fish transcripts before and after exposure to challenges, researchers can identify genes and pathways associated with immune responses. Furthermore, RNA-seq facilitates the identification and selection of disease-resistant fish and enables the examination of gene expression in different tissues under varying conditions [[50]14]. The relationship between hosts and parasites or viruses is complex and warrants further investigation. Studying specific genes and mechanisms involved in disease resistance provides valuable insights for developing enhanced strategies to combat infections [[51]16]. Fish resistance to pathogens, including viruses and bacteria, is influenced by genetic factors [[52]17]. Analyzing the expression of immunity-related genes in response to pathogens provides valuable insights into identifying genes that confer resistance to aquatic diseases. Additionally, exploring immune genes within the context of vaccine development holds significant potential [[53]18]. Immunological studies on rainbow trout (Oncorhynchus mykiss) are particularly important due to their critical role in food production and their direct contributions to agriculture and industry [[54]19]. Evaluating biometric parameters in rainbow trout (Oncorhynchus mykiss) during VHSV infection provides critical insights into the impact of viral pathogens on fish physiology and health [[55]20]. Biometric data, such as body weight, total length, fork length, and standard length, are essential in fisheries science as indicators of physiological status, health, immune responses, and disease resistance [[56]21]. Body condition, a key marker of fish health, reflects energy allocation and overall physiological status [[57]22, [58]23]. While morphometric indices are commonly used to assess body condition in relation to energy reserves, they also correlate with chronic stress, parasite load, and oxidative stress [[59]24–[60]26]. Monitoring these biometric metrics allows researchers to evaluate the severity of infections like VHSV and their effects on critical traits associated with immunity and disease resistance, thereby deepening the understanding of fish health [[61]20]. Combining biometric evaluations with transcriptomic analyses provides a comprehensive perspective on VHSV, enabling more precise interventions and the discovery of novel genetic markers for selective breeding programs. This integrated approach contributes to the development of disease-resistant trout strains, enhancing the sustainability and economic viability of aquaculture [[62]20]. This study investigated the effects of VHSV on rainbow trout (Oncorhynchus mykiss), focusing on its economic impact and the critical role of genetic mechanisms in disease resistance. The primary objective was to evaluate fish biometric traits, including growth, standard length, fork length, and total length, during VHSV infection. Genome-wide expression profiling using high-throughput sequencing was conducted to track gene expression changes, coupled with pathway enrichment analysis to identify virus-affected pathways. The study aimed to pinpoint specific genes and signaling pathways altered by VHSV, providing potential targets for breeding programs to enhance disease resistance. By identifying interventions to mitigate the effects of VHSV on rainbow trout (Oncorhynchus mykiss) populations, this research supports the development of selective breeding strategies to improve disease resistance and ensure the sustainability of aquaculture practices. Methods Location and experimental design This study was conducted at the Aquaculture Laboratory Technology Incubator Center, Islamic Azad University, Roudehen Branch, located in Roudehen county, Tehran Province, Iran. A total of 310 fish, each weighing between 86 and 97 g, were obtained from a VHSV-free industrial fish farm in Alashtar County, Lorestan Province, Iran, and subsequently transported to Roudehen, Tehran Province. The fish were housed in 18 open rectangular fiberglass tanks, each with dimensions of 0.92 m in width, 0.96 m in length, and 1.2 m in depth, providing a volume of 1.058 m^3 per tank. The stocking density was maintained at 1.7 kg /m^3, with 17 fish held in 1,058 L of water, corresponding to a cultivation density of 1.5 kg/m^3 [[63]20]. Fish maintenance A total of 310 fish were housed in tanks and fed a commercial diet three times daily (Simia, Iran). The required amount of food was calculated based on the live weight of the fish, using the feeding guidelines provided on the food packaging. Meals were administered in the morning, noon, and evening. Tank cleaning and siphoning were performed twice daily. Before the start of the experiments, the fish underwent a two-week acclimation period [[64]20]. The experiment utilized a closed water system with automatic flow every 3 h. The total duration of the study, including the acclimation period and the completion of tissue sampling, was one month [[65]19, [66]20]. Physical and chemical quality of water conditions The water used in this investigation was sourced from a pit and stored in a 5,000-L fiberglass tank for 8 h prior to use. During the farming period, key water quality parameters, including temperature, pH, salinity, dissolved oxygen, and ammonium levels, were measured weekly using the KARIZAB Kit (Karizab, Iran; [67]http://www.karizab.com). The water conditions were maintained at consistent levels throughout the study: temperature at 10 °C, pH at 7.5, salinity at 3 ppt, and dissolved oxygen at 10.2 mg/L [[68]20]. Virus preparation The VHSV strain used in this study belongs to genotype Ia. It was cultured at the National Reference Laboratory for Applied Studies and Diagnosis, affiliated with the Veterinary Organization of Alborz Province in Karaj, Iran. The virus was prepared for the study following the methods described in references [[69]27, [70]28].