Establishment and validation of a 2D primary gill cell culture of the sevenband grouper (Hyporthodus septemfasciatus).

A 2D primary gill cell culture system of the sevenband grouper (Hyporthodus septemfasciatus) was established to validate the pathogenesis of nervous necrosis virus (NNV) as observed in previous studies. This system, developed using the double-seeded insert (DSI) technique, yielded confluent cell layers. Upon challenge with NNV in a setup containing both autoclaved salt water and L15 media in the apical compartment, viral replication akin to that anticipated based on previous studies was observed. Consequently, we advocate for the utilization of primary gill cell culture as a viable alternative to conventional methodologies for investigating host pathogen interactions.

Determination of the minimum infective dose of viral hemorrhagic septicemia virus (VHSV) in juvenile olive flounder, Paralichthys olivaceus using an immersion challenge model.

Viral hemorrhagic septicemia virus (VHSV) affects over 80 fish species, leading to viral hemorrhagic septicemia (VHS). Horizontal VHSV transmission is widely studied, with researchers utilizing various doses to establish infection models. Infected hosts shed the virus into the environment, elevating the risk of transmission to naïve fish within the same system. This study aimed to ascertain the minimum infective dose of VHSV in olive flounder (Paralichthys olivaceus). In olive flounder, the detection of VHSV within the kidney exhibited the highest infection rate on the third day among days 1, 3 and 5. Doses of 103.0 to 104.7 TCID50/ml were administered to juvenile olive flounder across three farms. Results showed resistance to infection below 103.4 TCID50/ml at 15 °C. While infection frequency varied by concentration, higher concentrations correlated with more infections. Nonetheless, viral copy numbers did not differ significantly among infected fish at varying concentrations. This study underscores the need for early VHSV management and contributes essential data for pathogenicity assessment and foundational knowledge.

Production and Characterization of Monoclonal Antibodies Against Structural Proteins of Hirame Novirhabdovirus.

Hirame novirhabdovirus (HIRRV) is a significant viral pathogen of Japanese flounder (Paralichthys olivaceus). In this study, seven monoclonal antibodies (mAbs) against HIRRV (isolate CA-9703) were produced and characterized. Three mAbs (1B3, 5G6, and 36D3) were able to recognize nucleoprotein (N) (42 kDa) and four mAbs (11-2D9, 15-1G9, 17F11, and 24-1C6) recognized matrix (M) protein (24 kDa) of HIRRV. Western blot, Enzyme-linked immunosorbent assay, and indirect fluorescent antibody technique (IFAT) results indicated that the developed mAbs were specific to HIRRV without any cross-reactivity against other different fish viruses and epithelioma papulosum cyprini cells. All the mAbs comprised IgG1 heavy chain and κ light chain except 5G6, which has a heavy chain of IgG2a class. These mAbs can be very useful in development of immunodiagnosis of HIRRV infection.

Temperature dependent cellular, and epigenetic regulatory mechanisms underlying the antiviral immunity in sevenband grouper to nervous necrosis virus infection.

Changes in the thermal optima of fish impacts changes in the physiology and immune response associated with infections. The present study showed that at suboptimal temperatures (17 °C), the host tries to evade viral infection by downregulating the inflammatory response through enhanced neuronal protection. There was significantly less abundance of IgM + B cells in the 17 °C group compared to that in the 25 °C group. An increased macrophage population (Iba1+) during the survival phase in fish challenged at 25 °C demonstrated inflammation. Optimal temperature challenge activated virus-induced senescence in brain cells, demonstrated with a heterochromatin-associated H3K9me3 histone mark. There was an abundant expression of anti-inflammatory cytokines in the brain of fish at the suboptimal challenge. Besides the cytokines, the expression of BDNF was significantly higher in the suboptimally challenged group, suggesting that its neuronal protection activity following NNV infection is mediated through TGFß. The suboptimal challenge resulted in H3k9ac displaying transcriptional competency, activation of trained immunity H3K4me3, and enrichment of H3 histone-lysine-4 monomethylation (H3K4me1), resulting in a robust re-stimulatory immune response. The observations from the H4 modifications showed that besides H4K12ac and H4K20m3, all the assayed modifications were significantly higher in suboptimal convalescent fishes. The suboptimally challenged fish acquired more methylation along cytosine residues than the optimally infected fish. Together, these observations suggest that optimal temperature results in an immune priming effect, whereas the protection enabled in suboptimal convalescent fishes is operated through epigenetically controlled trained immune functions.

NLRC3 attenuates antiviral immunity and activates inflammasome responses in primary grouper brain cells following nervous necrosis virus infection.

NLRC3 is identified as a unique regulatory NLR involved in the modulation of cellular processes and inflammatory responses. In this study, a novel Nod like receptor C3 (NLRC3) was functionally characterized from seven band grouper in the context of nervous necrosis virus infection. The grouper NLRC3 is highly conserved and homologous with other vertebrate proteins with a NACHT domain and a C-terminal leucine-rich repeat (LRR) domain and an N-terminal CARD domain. Quantitative gene expression analysis revealed the highest mRNA levels of NLRC3 were in the brain and gill followed by the spleen and kidney following NNV infection. Overexpression of NLRC3 augmented the NNV replication kinetics in primary grouper brain cells. NLRC3 attenuated the interferon responses in the cells following NNV infection by impacting the TRAF6/NF-κB activity and exhibited reduced IFN sensitivity, ISRE promoter activity, and IFN pathway gene expression. In contrast, NLRC3 expression positively regulated the inflammasome response and pro-inflammatory gene expression during NNV infection. NLRC3 negatively regulates the PI3K-mTOR axis and activated the cellular autophagic response. Delineating the complexity of NLRC3 regulation of immune response in the primary grouper brain cells following NNV infection suggests that the protein acts as a virally manipulated host factor that negatively regulated the antiviral immune response to augment the NNV replication.

Heavy oil exposure suppresses antiviral activities in Japanese flounder Paralichthys olivaceus infected with viral hemorrhagic septicemia virus (VHSV).

A combined treatment of heavy oil (HO) exposure and virus infection induces increased mortality in Japanese flounder (Paralichthys olivaceus). In this study, we addressed how HO exposure affects the immune system, especially antiviral activities, in Japanese flounder. The fish were infected with viral hemorrhagic septicemia virus (VHSV), followed by exposure to HO. We analyzed virus titers in the heart and mRNA expression in the kidney of surviving fish. The virus titers in fish exposed to heavy oil were higher than the threshold for onset. The results suggest that HO exposure may allow the replication of VHSV, leading to higher mortality in the co-treated group. Gene-expression profiling demonstrated that the expression of antiviral-activity-related genes, such as those for interferon and apoptosis induction, were lower in the co-treated group than in the group with VHSV infection only. These results helped explain the high virus titers in fish treated with both stressors. Thus, interferon production in the virus-infected cells and apoptosis induction by natural killer cells worked normally in the VHSV-infected fish without HO exposure, but these antiviral activities were slightly suppressed by HO exposure, possibly leading to extensive viral replication in the host cells and the occurrence of VHS.

Beta glucan induced immune priming protects against nervous necrosis virus infection in sevenband grouper.

In the present study, we studied the effect of ß-glucan on the activation of antiviral immune responses against nervous necrosis virus (NNV) taking into consideration the role of innate immune training. Sevenband grouper primary macrophages showed an attenuated proinflammatory response and elevated antiviral response to NNV infection. In vitro, priming of ß-glucan enhanced macrophage viability against NNV infection which is associated with the activation of sustained inflammatory cytokines gene expression. Observations were clear to understand that NLR Family CARD Domain Containing 3 (NLRC3) and caspase-1 activation and subsequent IL-1ß production were reduced in ß-glucan-primed macrophages. Subsequent markers for training including Lactate and abundance of HIF-1α were elevated in the cells following training. However, the lactate dehydrogenase (LDH) concentrations remained stable among the ß-glucan stimulated infected and uninfected groups suggesting similar macrophage health in both groups. In vivo, the NNV-infected fish primed with ß-glucan had a higher survival rate (60%) than the control NNV-infected group (40%). Our findings demonstrate that ß-glucan induced protective responses against NNV infection and studies are underway to harness its potential applicability for prime and boost vaccination strategies.

Altered expression of immune factors in sevenband grouper, Hyporthodus septemfasciatus following nervous necrosis virus challenge at optimal and suboptimal temperatures.

The nervous necrosis virus (NNV) infection is generally observed in aquafarms when the seawater temperature is higher than 24 °C and the fishes seem to be refractory to disease at suboptimal temperatures below 20 °C suggesting a role of thermoregulation in NNV pathogenesis. The present study profiled the temperature-dependent regulation of cytokines (TNF-α, IL-1ß and IFN-γ), innate antiviral factors (IFN-1, Mx, ISG-15), adaptive immune factors (CD-4, CD-8, IgM), signaling regulators (SOCS-1, SOCS-3), transcription factors (STAT-1, STAT-3) and microglial and NCC/NK specific cell markers (TMEM-119 and NCCRP-1) during NNV challenge in seven-band grouper, Hyporthodus septemfasciatus. The co-habitation challenge at 17 °C with showed a sustained expression of proinflammatory cytokines and following rechallenge with a dose of 104 TCID50/100µL/fish at optimal temperature, the survivors also exhibited a stable expression of immune factors. The 100% survival following the challenge at sub-optimal (17 °C) and rechallenge at optimal (25 °C) was due to the stable and sustained activation of the immune response. However, at 25 °C, the rechallenge displayed a priming effect with hyperactivation of the immune system evident from the immune gene expression profile. The mortality pattern observed is co-related with the cytokine storm as is evident from the gene expression profile. Whereas, neither of the adaptive immune markers was suggestive of humoral immune response in the 17 °C groups. Also, the data suggest a possible role of NK cell and microglia in mediating antiviral immune response following infection in the brain at different temperatures, where, former is beneficial in restricting viral infection with higher host tolerance.

Proteasome subunit beta type-8 from sevenband grouper negatively regulates cytokine responses by interfering NF-κB signaling upon nervous necrosis viral infection.

During viral infection, proper regulation of immune signaling is essential to ensure successful clearance of virus. Immunoproteasome is constitutively expressed and gets induced during viral infection by interferon signaling and contributes to regulate proinflammatory cytokine production and activation of the NF-κB pathway. In this study, we identified Hs-PSMB8, a member of the proteasome ß-subunits (PSMB) family, as a negative regulator of NF-κB responses during NNV infection. The transient expression of Hs-PSMB8 delayed the appearance of cytopathic effect (CPE) and showed a higher viral load. The Hs-PSMB8 interacted with NNV which was confirmed using immunocolocalization and co-IP. Overexpression of Hs-PSMB8 diminished virus induced activation of the NF-κB promoters and downregulated the activation of IL-1ß, TNFα, IL6, IL8, IFNγ expression upon NNV infection. Collectively, our results demonstrate that PSMB8 is an important regulator of NF-κB signaling during NNV infection in sevenband grouper.

Efficacy of live NNV immersion vaccine immunized at low temperature in sevenband grouper, Epinephelus septemfasciatus.

The objective of this study was to investigate safety and efficacy using a low-temperature immunization protocol with NNV in sevenband grouper, Epinephelus septemfasciatus. Further, NNV specific antibody post immunization and intramuscularly challenge was also evaluated. Immunization at low temperature resulted in a low titer virus infection in brain tissues without any clinical symptoms of infection such as sluggish behavior and/or spinning, rotating swimming being observed, and no mortality was observed. Post challenge, NNV titer NNV giving an RPS of 100 %, increased in brain tissues of naïve (non-immunized) sevenband grouper NNV giving an RPS of 100 %, with a cumulative mortality of 100 % at 25 days post-infection. No mortality or disease symptoms NNV giving an RPS of 100 %, as NNV giving and of 100 %, observed in the groups immunized at low temperature with live NNV giving an RPS of 100 %. NNV giving an RPS of 100 %. NNV specific antibody was not detected in live NNV vaccinated sevenband grouper. This is the first study that confirms that field-scale NNV immersion vaccine can protect sevenband grouper against lethal infection with NNV at natural seawater temperature under the gradually increased from 14.3-24.8 °C.

Potentiality to natural immunization inducement against VHS in olive flounder by live VHSV immersion vaccination at temperature controlled culture condition.

Viral hemorrhagic septicemia virus (VHSV) is the etiological agent of viral hemorrhagic septicemia (VHS), one of the most severe viral diseases affecting cultured olive flounder (Paralichthys olivaceus) in Far East Asia. VHS occurs during the winter or spring season when the water temperature is low (9-15 °C). In our previous study found that VHSV infection had controlled by using water temperature (above 17 °C). By using water temperature, we demonstrated optimal live VHSV immersion vaccine treatment concentration, also live VHSV immersion vaccine treatment method. We confirmed that the effective VHSV immersion treatment was 105.5 TCID50/mL at 17 °C. It was no need pretreatment before live VHSV immersion vaccination. The VHSV titer of vaccinated fish organs was under the estimated limit (<1.8 log TCID50/mL) within 3 days in 105.5 TCID50/mL live VHSV immersion at 17 °C. High survival rates were observed in live VHSV immersion with 105.5 and 107.5 TCID50/mL at 17 °C and then infected VHSV at 10 °C. VHSV specific antibody was not detected from in the surviving flounder under VHSV infection after immersion treatment with live VHSV. In addition, the potentiality of natural immunization against VHS in olive flounder was suggested by live VHSV immersion vaccine at temperature controlled fish culture condition.

Early viral uptake and host-associated immune response in the tissues of seven-band grouper following a bath challenge with nervous necrosis virus.

In the present study, early uptake of nervous necrosis virus (NNV) in the tissues (gill, brain, skin, eye, heart) and immune response associated with the uptake in the gill and brain of seven-band grouper was investigated. The gill was found to act as a primary portal of entry for NNV during the initial phase of the water-borne infection. The presence of viral genome and infectious particles was demonstrated using quantitative (qPCR, viral titer) and qualitative (ISH) approach. Initially, an increased viral uptake was noticed, but the virus got cleared from the gills at the later phase of infection. Localization in the brain was evident at the blood-brain barrier followed by the brain parenchyma in the latter stage of infection. Nectin-4, an established NNV receptor, and GHSC70 showed an up-regulated expression throughout the challenge period initially in the gill and at latter phase in brain; however, it seems that the virus does not use gill as a primary replication site but brain as a permissive tissue. Combined activity as reflected by the up-regulation of cytokine, interferon, antigen-presenting cell, and immunoglobulin genes restricts early NNV replication in gill. Observations from the present study provide a better understanding of early NNV entry and also opens a window for further elucidating the modes of NNV neuro-invasion through systemic circulation.

Juvenile olive flounder immersed in live VHSV at 17 °C and 20 °C shows resistance against VHSV infection at 10 °C.

Viral hemorrhagic septicemia (VHS) causes serious economic loss in olive flounder aquaculture industry in Korea. Water temperature is known to play a critical role in VHS disease outbreak. Here, we assessed the potential efficacy of VHSV immersion treatment in relation to resistance conferred at differential water temperatures in olive flounder. VHSV acquired resistance was compared between formalin-killed VHSV immersion treatment and live VHSV immersion treatment at three different water temperatures viz., 10 °C, 17 °C, and 20 °C. At 10 °C, cumulative mortality was around 80% in live VHSV immersed group while 30% cumulative mortality was observed in formalin-killed VHSV treated group. After 4 weeks, surviving olive flounder at 17 °C and 20 °C were challenged with VHSV at 10 °C following which the VHS outbreaks took place at host susceptible water temperature. For the pre-treated flounder at 17 °C, survival rates were 80% and 30% after challenge at 10 °C in live VHSV immersed group and formalin-killed VHSV immersed group, respectively. Whereas, the pre-treated flounder at 20 °C showed survival rate of 75% and 20% after challenge at 10 °C in live VHSV immersed group and formalin-killed VHSV immersed group, respectively. Our results propose the fact that live VHSV immersion using non-susceptible water temperature has the potential to protect olive flounder against VHSV infection. Moreover, the protective efficacy of live immersion treatment in a non-excited immune state without the use of an adjuvant combined with water temperature adjustment was investigated for the first time at 17 °C. Further studies should be targeted to explore the host-associated immune factors responsible for the protective effect and acquired resistance in olive flounder after live VHSV immersion treatment.

Functional characterization of seven-band grouper immunoglobulin like cell adhesion molecule, Nectin4 as a cellular receptor for nervous necrosis virus.

Nectin-4/PVRL4 belonging to the family of immunoglobulin-like cell adhesion molecules was identified as a potential cellular receptor for several animal viruses. Here we show that nervous necrosis virus that causes viral nervous necrosis in teleosts uses the same receptor in its life cycle. Transfection of SSN-1 cell lines with an expression vector encoding Nectin-4 rendered them to be more susceptible to NNV. Immunofluorescence microscopy on Nectin-4 expressing cells revealed that the protein interacted with NNV specifically. A virus binding assay indicated that Nectin-4 was a bonafide receptor that supported virus attachment to the host cell whereas siRNA directed against Nectin-4 blocked NNV infections in grouper primary brain cells. Results of the present study will improve our understanding of the pathogenesis of NNV infection and provide a target for the development of novel antiviral interventions in marine finfish aquaculture.

Kinetics of infectious virus and viral RNA copy number in the blood of olive flounder infected with viral hemorrhagic septicemia virus (VHSV).

Viral hemorrhagic septicemia (VHS) is a cold-water disease caused by viral hemorrhagic septicemia virus (VHSV) at an optimal temperature of 9 °C-15 °C. VHSV isolation and detection have been accomplished by using a number of diagnostic methods such as cell culture and qRT-PCR. Spleen and kidney have been reported as the main target organs of VHSV-infection; however, how VHSV spreads throughout the fish body has not been clearly studied. The purpose of this study was 1) to investigate viral titer and viral RNA copy number in the blood of VHSV-infected olive flounder at 10 °C and 13 °C; 2) to compare VHSV titer and viral RNA copy numbers in blood from fish exposed to the virus by two different challenges. VHSV titer at 10 °C was higher than at 13 °C in blood samples of injection challenged group. Whereas, similar titer was observed at 10 °C and 13 °C in the blood samples of the immersion challenged group. At 10 °C, copy numbers of VHSV-N gene in blood of immersion challenged group increased slightly in comparison to injection challenged group. At 13 °C, similar patterns were observed between the injection and immersion challenged groups. Also, higher titer and copy number were observed in fish blood compared to tested organs from our previous study. Our results indicate that VHSV genome existed in fish blood at earlier time points after infection, and the blood may contribute to the spread of the virus in whole fish body. In addition, VHSV diagnosis by qRT-PCR from fish blood samples, not requiring sacrificing the host fish can be valuable to collect the kinetic information of viral infection.

Differences of Viral Hemorrhagic Septicemia Virus Loads among Organs of Dead and Surviving Olive Flounder Infected by Intramuscular Injection and Immersion Challenge.

Viral hemorrhagic septicemia virus (VHSV) is an important viral pathogen in the culture of Olive Flounder Paralichthys olivaceus. Based on cumulative mortality, the virulence of VHSV was found to be highly different depending on challenge routes and exposure doses (using tissue culture infectious dose with 50% endpoint [TCID50]). Olive Flounder were injected with VHSV at 102.5 , 104.5 , 106.5 , and 108.5 TCID50/100 µL/fish. A second group of fish was immersed at 103.5 , 105.5 , and 107.5 TCID50/mL at 10°C for 1 h in this study. The cumulative mortality was observed at 15 d postinfection. Immersion challenge at 103.5 TCID50/mL caused no mortality, while intramuscular injection challenge resulted in high levels of mortality with all VHSV exposure doses. Overall, Olive Flounder was susceptible to VHSV, with cumulative mortality of 90% or 100% in fish intramuscularly injected with high or low doses of VHSV. The cumulative mortality was 40% and 70% at 105.5 and 107.5 TCID50/mL, respectively, in the immersion challenge group. The VHSV titration and copy numbers were estimated by TCID50 and quantitative reverse transcription PCR methods. From dead Olive Flounder, VHSV titration was consistently detected in all tested organs, ranging from 105 to 109 TCID50/mL. The VHSV titration was under the detection limit from surviving Olive Flounder, but the VHSV N gene was detected.

Elucidation of mechanism for host response to VHSV infection at varying temperatures in vitro and in vivo through proteomic analysis.

Seasonal temperature has a major influence on the infectivity of pathogens and the host immune system. Viral hemorrhagic septicemia virus (VHSV) is one such pathogen that only causes the mortality of fish at low temperatures. This study aims to discover the host defense mechanism and pathway for resistance to VHSV at higher temperatures. We first observed the VHSV infection patterns at low and higher temperatures in fathead minnow (FHM) cells (20 °C and 28 °C) and zebrafish (15 °C and 25 °C). In comparison to the 20 °C infection, FHM cells infected at 28 °C showed decreased apoptosis, increased cell viability, and reduced VHSV N gene expression. In zebrafish, infection at 25 °C caused no mortality and significantly reduced the N gene copy number in comparison to infection at 15 °C. To explore the antiviral infection mechanisms induced by high temperature in vitro and in vivo, the changes in the proteomic profile were measured through UPLC-MSE analysis. ACADL, PTPN6, TLR1, F7, A2M, and GLI2 were selected as high temperature-specific biomarkers in the FHM cell proteome; and MYH9, HPX, ANTXR1, APOA1, HBZ, and MYH7 were selected in zebrafish. Increased immune response, anticoagulation effects, and the formation of lymphocytes from hematopoietic stem cells were analyzed as functions that were commonly induced by high temperature in vitro and in vivo. Among these biomarkers, GLI2 was predicted as an upstream regulator. When treated with GANT58, a GLI-specific inhibitor, cell viability was further reduced due to GLI2 inhibition during VHSV infection at varying temperatures in FHM cells, and the mortality in zebrafish was induced earlier at the low temperature. Overall, this study discovered a new mechanism for VHSV infection in vitro and in vivo that is regulated by GLI2 protein.

Development of an in-situ hybridization assay using riboprobes for detection of viral haemorrhagic septicemia virus (VHSV) mRNAs in a cell culture model.

An in situ hybridization (RNA-ISH) assay has been developed and optimized to detect viral haemorrhagic septicemia virus (VHSV), an OIE listed piscine rhabdovirus, in infected fish cells using fathead minnow (FHM) as a model cell line. Two antisense riboprobes (RNA probes) targeting viral transcripts from a fragment of nucleoprotein (N) and glycoprotein (G) genes were generated by reverse transcription polymerase chain reaction (RT-PCR) using VHSV specific primers followed by a transcription reaction in the presence of digoxigenin dUTP. The synthesized RNA probes were able to detect viral mRNAs in formalin fixed VHSV infected FHM cells at different time points post inoculation (pi). To correlate the signal intensity, a time dependent quantitation of the viral mRNA transcript and infectivity titer was done by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and 50% tissue culture infectivity dose (TCID50), respectively, from the infected cells and culture supernatants. Further, we compared the diagnostic sensitivity of ISH assay with immunocytochemistry (ICC). Both the riboprobes used in the ISH assay detected VHSV as early as 6 hpi in the FHM cells inoculated with a multiplicity of infection (moi) of 2. Also, the signal detection in ISH was at an early stage in comparison to ICC, wherein, signal was first detected at 12 hpi. Our results clearly highlight that current ISH assay can be of value as a diagnostic tool to localize and detect VHSV in conjunction with conventional virus isolation in cell culture.

Investigation of nervous necrosis virus (NNV) replication in vitro using RNA in situ hybridization.

Nervous necrosis virus (NNV) belongs to the genus Betanodavirus of family Nodaviridae. Its genome consists of two RNA segments, RNA1 and RNA2. Several studies have investigated NNV detection by in situ hybridization (ISH), but these have typically focused on the detection of the RNA2 gene. In this study, we localized both RNA1 and RNA2 NNV segments in viral-infected cells by ISH, using labeled RNA probes (RNA-ISH). Also, immunocytochemistry (ICC) assay was carried out for localization of viral particle by targeting the coat protein. Further, viral quantification assays were performed by quantitative RT-PCR and viral infectivity (TCID50) in SSN-1 cells. Viral segments were observed by RNA-ISH at 6 h post infection (hpi), while NNV particles were detected at 24 hpi by ICC. Use of double labeling RNA-ISH revealed the co-expression of the two viral segments in the same area of the cells, while RNA1 was also detected separately. Comparison of the level of viral genomic segments and viral infectivity revealed significantly more copies of RNA1 at each time points than copies of RNA2 and greater NNV titers. The results suggest that RNA1 might be expressed in the early stages of replication, with RNA2 expressed later. The virions then assemble through initially expressed viral genomic segments. Even though infectious particles displayed very efficient packaging, the RNA1 segment was still over-produced.

Application of Concanavalin A-Linked Magnetic Beads for the Detection of Hepatitis A Virus.

Prompt and inexpensive detection of hepatitis A virus (HAV) is essential to control acute hepatitis outbreaks associated with the consumption of contaminated raw or minimally processed food. In this study, various carbohydrate-binding lectins, including concanavalin A (Con A), wheat germ agglutinin, and soybean agglutinin, were compared for their binding affinity to HAV. Con A, which showed significantly higher binding affinity than other lectins, was used to develop an alternative and affordable method to conventional antibody-linked immunomagnetic separation prior to detection of HAV using reverse transcriptase PCR. This method, Con A-linked immunomagnetic separation combined with reverse transcriptase PCR, can detect HAV at a dilution concentration of 10-4 of the virus stock (titer: 104 median tissue culture infective dose per mL), indicating that Con A could be a promising candidate for concentrating HAV.