Pre-transplant crossmatch-negative donor-specific anti-HLA antibody predicts acute antibody-mediated rejection but not long-term outcomes in kidney transplantation: an analysis of the Korean Organ Transplantation Registry.

Background: Pre-transplant donor-specific anti-human leukocyte antigen antibody (HLA-DSA) is a recognized risk factor for acute antibody-mediated rejection (ABMR) and allograft failure. However, the clinical relevance of pre-transplant crossmatch (XM)-negative HLA-DSA remains unclear. Methods: We investigated the effect of XM-negative HLA-DSA on post-transplant clinical outcomes using data from the Korean Organ Transplantation Registry (KOTRY). This study included 2019 living donor kidney transplant recipients from 40 transplant centers in South Korea: 237 with HLA-DSA and 1782 without HLA-DSA. Results: ABMR developed more frequently in patients with HLA-DSA than in those without (5.5% vs. 1.5%, p<0.0001). Multivariable analysis identified HLA-DSA as a significant risk factor for ABMR (odds ratio = 3.912, 95% confidence interval = 1.831-8.360; p<0.0001). Furthermore, the presence of multiple HLA-DSAs, carrying both class I and II HLA-DSAs, or having strong HLA-DSA were associated with an increased incidence of ABMR. However, HLA-DSA did not affect long-term clinical outcomes, such as allograft function and allograft survival, patient survival, and infection-free survival. Conclusion: Pre-transplant XM-negative HLA-DSA increased the risk of ABMR but did not affect long-term allograft outcomes. HLA-incompatible kidney transplantation in the context of XM-negative HLA-DSA appears to be feasible with careful monitoring and ensuring appropriate management of any occurrence of ABMR. Furthermore, considering the characteristics of pre-transplant XM-negative HLA-DSA, the development of a more detailed and standardized desensitization protocol is warranted.

Chromosome-Scale, De Novo, Phased Genome Assemblies of Three Australian Limes: Citrus australasica, C. inodora, and C. glauca.

Huanglongbing (HLB) is a severe citrus disease worldwide. Wild Australian limes like Citrus australasica, C. inodora, and C. glauca possess beneficial HLB resistance traits. Individual trees of the three taxa were extensively used in a breeding program for over a decade to introgress resistance traits into commercial-quality citrus germplasm. We generated high-quality, phased, de novo genome assemblies of the three Australian limes using PacBio long-read sequencing. The genome assembly sizes of the primary and alternate haplotypes were determined for C. australasica (337 Mb/335 Mb), C. inodora (304 Mb/299 Mb), and C. glauca (376 Mb/379 Mb). The nine chromosome-scale scaffolds included 86-91% of the genome sequences generated. The integrity and completeness of the assembled genomes were estimated to be at 97.2-98.8%. Gene annotation studies identified 25,461 genes in C. australasica, 27,665 in C. inodora, and 30,067 in C. glauca. Genes belonging to 118 orthogroups were specific to Australian lime genomes compared to other citrus genomes analyzed. Significantly fewer canonical resistance (R) genes were found in C. inodora and C. glauca (319 and 449, respectively) compared to C. australasica (576), C. clementina (579), and C. sinensis (651). Similar patterns were observed for other gene families associated with potential HLB resistance, including Phloem protein 2 (PP2) and Callose synthase (CalS) genes predicted in the Australian lime genomes. The genomic information on Australian limes developed in the present study will help understand the genetic basis of HLB resistance.

Diversity of 'Candidatus Liberibacter asiaticus' Strains in Texas Revealed by Prophage Sequence Analyses.

Prophages/phages are important components of the genome of 'Candidatus Liberibacter asiaticus' (CLas), an unculturable alphaproteobacterium associated with citrus huanglongbing (HLB) disease. Phage variations have significant contributions to CLas strain diversity research, which provide critical information for HLB management. In this study, prophage variations among selected CLas strains from southern Texas were studied. The CLas strains were collected from three different CLas inhabitant environments: citrus leaf, citrus root, and Asian citrus psyllid (ACP), the vector of CLas. Regardless of the different habitats and time span, more than 80% of CLas strains consistently had both Type 1 and Type 2 prophages, the same prophage type profile as in CLas strains from Florida but different to those reported in California and China. Further studies were performed on prophage type diversity. Analyses on Type 1-specific PCR amplicon sequences (encoding an endolysin protein) revealed the presence of two groups: Type 1-A, clustered around prophage SC1 originating from Florida, and Type 1-B, clustered with prophage P-SGCA5-1 originating in California. Type 1-B strains were mostly from ACP of nearby citrus orchards. On the other hand, analyses on Type 2-specific PCR amplicon sequences (encoding a putative hypothetical protein) showed a single group clustering around prophage SC2 originated from Florida, although a different Type 2 prophage has been reported in California. The presence of two distinct Type 1 prophage groups suggested the possibility of two different CLas introductions in southern Texas. The results from this study provide an initial baseline of information on genomic and population diversity of CLas in Texas.

Identification of the Plant Family Caryophyllaceae in Korea Using DNA Barcoding.

Caryophyllaceae is a large angiosperm family, with many species being utilized as ornamental or medicinal plants in Korea, in addition to several endangered species that are managed by the government. In this study, we used DNA barcoding for the accurate identification of Korean Caryophyllaceae. A total of 78 taxa (n = 215) were sequenced based on three chloroplast regions (rbcL, matK, and psbA-trnH) and nuclear ribosomal internal transcribed spacers (ITS). In the neighbor-joining tree, a higher accuracy of identification was generally observed when using ITS (>73%) rather than chloroplast regions (<62%). The highest resolution was found for rbcL + ITS (77.6%), although resolution varied according to the genus. Among the genera that included two and more species, five genera (Eremogone, Minuartia, Pseudostellaria, Sagina, and Stellaria) were successfully identified. However, the species of five other genera (Cerastium, Gypsophila, Dianthus, Silene, and Spergularia) showed relatively low resolutions (0-61.1%). In the cases of Cerastium, Dianthus, and Silene, ambiguous taxonomic relationships among unidentified species may have been a factor contributing to such low resolutions. However, in contrast to these results, Gypsophila and Spergularia have been identified well in previous studies. Our findings indicate the need of taxonomic reconsideration in Korea.

First report of L1 cell adhesion molecule (L1CAM) in flat fish (Starry flounder): Molecular features and expression analysis.

In this study, the starry flounder L1 cell adhesion molecule (L1CAM) sequence was obtained using next-generation sequencing, and the integrity of the sequence was verified by cloning and sequencing. First, the amino acid sequence was predicted using the cDNA sequence, and the gene was then identified through multiple sequence alignment analysis with related sequences and phylogenetic analysis. Thus, homogeneity was confirmed. The expression level of PsL1CAM (Platichthys stellatus L1CAM) mRNA in healthy starry flounder was detected in all tissues used in the experiment, and tissue- and gene-specific expression levels were confirmed. In addition, as a result of mRNA expression analysis after artificial infection with viral hemorrhagic septicemia virus (VHSV) and Streptococcus parauberis PH0710, significant expression changes and characteristics were confirmed following infection with VHSV and S. parauberis PH0710. After artificial infection with VHSV, the expression level of PsL1CAM mRNA was significantly upregulated in almost all major tissues of the starry flounder, whereas it was significantly downregulated in mucosal-associated lymphoid tissues, such as the gills and intestine. Infection with S. parauberis PH0710 significantly upregulated the expression of PsL1CAM mRNA in almost all major tissues of the starry flounder, whereas it was significantly downregulated in the heart after infection. Our results indicate that PsL1CAM may be involved in the host immune response to starry flounders.

A Field Deployable Real-Time Loop-Mediated Isothermal Amplification Targeting Five Copy nrdB Gene for the Detection of 'Candidatus Liberibacter asiaticus' in Citrus.

Huanglongbing (HLB) is one of the most destructive diseases in citrus, which imperils the sustainability of citriculture worldwide. The presumed causal agent of HLB, 'Candidatus Liberibacter asiaticus' (CLas) is a non-culturable phloem-limited α-proteobacterium transmitted by Asian citrus psyllids (ACP, Diaphorina citri Kuwayama). A widely adopted method for HLB diagnosis is based on quantitative real-time polymerase chain reaction (qPCR). Although HLB diagnostic qPCR provides high sensitivity and good reproducibility, it is limited by time-consuming DNA preparation from plant tissue or ACP and the requirement of proper lab instruments including a thermal cycler to conduct qPCR. In an attempt to develop a quick assay that can be deployed in the field for CLas detection, we developed a real-time loop-mediated isothermal amplification (rt-LAMP) assay by targeting the CLas five copy nrdB gene. The rt-LAMP assay using various plant sample types and psyllids successfully detected the nrdB target as low as ~2.6 Log10 copies. Although the rt-LAMP assay was less sensitive than laboratory-based qPCR (detection limit ~10 copies), the data obtained with citrus leaf and bark and ACP showed that the rt-LAMP assay has >96% CLas detection rate, compared to that of laboratory-based qPCR. However, the CLas detection rate in fibrous roots was significantly decreased compared to qPCR due to low CLas titer in some root DNA sample. We also demonstrated that the rt-LAMP assay can be used with a crude leaf DNA extract which is fully deployable in the field for quick and reliable HLB screening.

Analysis of Polymeric Immunoglobulin Receptor Expression in Olive Flounder (Paralichthys olivaceus) against Viral Hemorrhagic Septicemia Virus.

Polymeric immunoglobulin receptor (pIgR) mediates the transfer of polymeric immunoglobulin to protect organisms and is one of the most important mucosal effectors. In this study, the developmental stage- and tissue-specific expression of pIgR were observed before virus inoculation in olive flounder. pIgR was gradually expressed until the formation of immune tissue, exhibiting high expression in the late juvenile period; thereafter, pIgR expression gradually decreased and exhibited high expression in the spleen and skin. Moreover, pIgR expression after viral hemorrhagic septicemia virus infection was high in the kidney and spleen tissues at high density and low at low density. The results of this study can provide a basis for future studies on breeding density, virus expression, and immune system studies in fish.

High water temperature-mediated immune gene expression of olive flounder, Paralichthys olivaceus according to pre-stimulation at high temperatures.

Increased ocean temperature due to global warming affects the health and immunity of fish. In this study, juvenile Paralichthys olivaceus were exposed to high temperature after pre-heat (Acute: Acute heat shock at 32 °C, AH-S: Acquired heat shock at 28 °C & short recovery (2 h) and heat shock at 32 °C, AH-L: acquired heat shock at 28 °C and long recovery (2 days), AH-LS: acquired heat shock at 28 °C & long (2 days) + short (2 h) recovery). Heat shock after pre-heat significantly upregulated various immune-related genes, including interleukin 8 (IL-8), c-type lysozyme (c-lys), immunoglobulin M (IgM), Toll-like receptor 3 (tlr3), major histocompatibility complex IIα (mhcIIα) and cluster of differentiation 8α (cd8α) in the liver and brain of P. olivaceus. This study showed pre-exposure to high temperatures below the critical temperature can activate fish immunity and increase tolerance to high temperatures.

The complete chloroplast genome sequence of the medicinal plant, Dracocephalum rupestre (Lamiaceae).

Dracocephalum rupestre (tribe Mentheae; Lamiaceae) is a perennial herb from Korea and China with high ornamental and medicinal value. Here, we report its complete chloroplast genome to provide insight into the phylogenetic relationships of Dracocephalum. The genome is 151,230 bp long, with two inverted repeat regions (25,643 bp each) that separate a large single-copy region (82,536 bp) and a small single-copy region (17,408 bp). It contains 133 genes that encode 88 proteins, eight rRNAs, and 37 tRNAs. The maximum-likelihood phylogenetic analysis strongly supported Dracocephalum monophyly, showing that the genus forms a sister group with the subtribe Menthinae in the tribe Mentheae.

Co-Occurrence Analysis of Citrus Root Bacterial Microbiota under Citrus Greening Disease.

Candidatus Liberibacter asiaticus (CLas) is associated with Citrus Huanglongbing (HLB), a devastating disease in the US. Previously, we conducted a two-year-long monthly HLB survey by quantitative real-time PCR using root DNA fractions prepared from 112 field grapefruit trees grafted on sour orange rootstock. Approximately 10% of the trees remained CLas-free during the entire survey period. This study conducted 16S metagenomics using the time-series root DNA fractions, monthly prepared during twenty-four consecutive months, followed by microbial co-occurrence network analysis to investigate the microbial factors contributing to the CLas-free phenotype of the aforementioned trees. Based on the HLB status and the time when the trees were first diagnosed as CLas-positive during the survey, the samples were divided into four groups, Stage H (healthy), Stage I (early), II (mid), and III (late) samples. The 16S metagenomics data using Silva 16S database v132 revealed that HLB compromised the diversity of rhizosphere microbiota. At the phylum level, Actinobacteria and Proteobacteria were the predominant bacterial phyla, comprising >93% of total bacterial phyla, irrespective of HLB status. In addition, a temporal change in the rhizosphere microbe population was observed during a two-year-long survey, from which we confirmed that some bacterial families differently responded to HLB disease status. The clustering of the bacterial co-occurrence network data revealed the presence of a subnetwork composed of Streptomycetaceae and bacterial families with plant growth-promoting activity in Stage H and III samples. These data implicated that the Streptomycetaceae subnetwork may act as a functional unit against HLB.

Lateral Flow Immunoassay Reader Technologies for Quantitative Point-of-Care Testing.

Due to the recent pandemic caused by coronavirus disease 2019 (COVID-19), the lateral flow immunoassay used for its rapid antigen test is more popular than ever before. However, the history of the lateral flow immunoassay is about 60 years old, and its original purpose of use, such as a COVID-19 rapid antigen test or a pregnancy test, was the qualitative detection of a target analyte. Recently, the demand for quantitative analysis of lateral flow immunoassays is increasing in various fields. Lateral flow immunoassays for quantitative detection using various materials and sensor technologies are being introduced, and readers for analyzing them are being developed. Quantitative analysis readers are highly anticipated for their future development in line with technological advancements such as optical, magnetic field, photothermal, and electrochemical sensors and trends such as weight reduction, miniaturization, and cost reduction of systems. In addition, the sensing, processing, and communication functions of portable personal devices such as smartphones can be used as tools for the quantitative analysis of lateral flow immunoassays. As a result, lateral flow immunoassays can efficiently achieve the goal of rapid diagnosis by point-of-care testing. Readers used for the quantification of lateral flow immunoassays were classified according to the adopted sensor technology, and the research trends in each were reviewed in this paper. The development of a quantitative analysis system was often carried out in the assay aspect, so not only the readers but also the assay development cases were reviewed if necessary. In addition, systems for quantitative analysis of COVID-19, which have recently been gaining importance, were introduced as a separate section.

Prediction of the Medicinal Mechanisms of Pinellia ternata Breitenbach, a Traditional Medicine for Gastrointestinal Motility Disorders, through Network Pharmacology.

Pinellia ternata Breitenbach (PTB) is a widely used herbal medicine in China, Japan, and South Korea. It has antiemetic, anti-inflammatory, antitussive, and sedative properties. The raw material is toxic, but can be made safer using alum solution or by boiling it for a long time. In addition, PTB seems to be effective for gastrointestinal motility disorders (GMDs), but this is yet to be conclusively proven. Herein, PTB compounds, targets, and related diseases were investigated using the traditional Chinese medical systems pharmacology database and an analysis platform. Information on target genes was confirmed using the UniProt database. Using Cytoscape 3.8.2, a network was established and GMD-related genes were searched using the Cytoscape stringApp. The effects of the PTB extract on the pacemaker potential of interstitial cells of Cajal and GMD mouse models were investigated. In total, 12 compounds were found to target 13 GMD-related genes. In animal experiments, PTB was found to better regulate pacemaker potential in vitro and inhibit GMD signs compared to control groups in vivo. Animal studies showed that the mechanism underlying the effects of PTB is closely related to gastrointestinal motility. The results obtained demonstrated that PTB offers a potential means to treat GMDs, and we suggested that the medicinal mechanism of GMDs can be explained by the relationship between 12 major components of PTB, including oleic acid, and 13 GMD-related genes.

Artificial Intelligence and Declined Guilt: Retailing Morality Comparison Between Human and AI.

Several technological developments, such as self-service technologies and artificial intelligence (AI), are disrupting the retailing industry by changing consumption and purchase habits and the overall retail experience. Although AI represents extraordinary opportunities for businesses, companies must avoid the dangers and risks associated with the adoption of such systems. Integrating perspectives from emerging research on AI, morality of machines, and norm activation, we examine how individuals morally behave toward AI agents and self-service machines. Across three studies, we demonstrate that consumers' moral concerns and behaviors differ when interacting with technologies versus humans. We show that moral intention (intention to report an error) is less likely to emerge for AI checkout and self-checkout machines compared with human checkout. In addition, moral intention decreases as people consider the machine less humanlike. We further document that the decline in morality is caused by less guilt displayed toward new technologies. The non-human nature of the interaction evokes a decreased feeling of guilt and ultimately reduces moral behavior. These findings offer insights into how technological developments influence consumer behaviors and provide guidance for businesses and retailers in understanding moral intentions related to the different types of interactions in a shopping environment.

Sensing Techniques for Organochlorides through Intermolecular Interaction with Bicyclic Amidines.

Toxic organochloride molecules are widely used in industry for various purposes. With their high volatility, the direct detection of organochlorides in environmental samples is challenging. Here, a new organochloride detection mechanism using 1,5-diazabicyclo[4.3.0]non-5-ene (DBN) is introduced to simplify a sensing method with higher detection sensitivity. Three types of organochloride compounds-trichloroethylene (TCE), dichloromethane (DCM), and dichlorodiphenyltrichloroethane (DDT)-were targeted to understand DCM conjugation chemistry by using nuclear magnetic resonance (NMR) and liquid chromatography with a mass spectrometer (LC-MS). 13C-NMR spectra and LC-MS data indicated that DBN can be labeled on these organochloride compounds by chlorine-nitrogen interaction. Furthermore, to demonstrate the organochloride sensing capability, the labeling yield and limit of detection were determined by a colorimetric assay as well as micellar electrokinetic chromatography (MEKC). The interaction with DBN was most appreciable for TCE, among other organochlorides. TCE was detected at picomolar levels, which is two orders of magnitude lower than the maximum contaminant level set by the United States Environmental Protection Agency. MEKC, in conjunction with this DBN-labeling method, enables us to develop a field-deployable sensing platform for detecting toxic organochlorides with high sensitivity.

Optical Glucose Sensor Using Pressure Sensitive Paint.

A glucose sensor is used as an essential tool for diagnosing and treating diabetic patients and controlling processes during cell culture. Since the development of an electrochemical-based glucose sensor, an optical glucose sensor has been devised to overcome its shortcomings, but this also poses a problem because it requires a complicated manufacturing process. This study aimed to develop an optical glucose sensor film that could be fabricated with a simple process using commercial pressure sensitive paints. The sensor manufacturing technology developed in this work could simplify the complex production process of the existing electrochemical or optical glucose sensors. In addition, a photometric method for glucose concentration analysis was developed using the color image of the sensor. By developing this sensor and analysis technology, the basis for glucose measurement was established that enables two-dimensional, online, and continuous measurement. The proposed sensor showed good linearity at 0-4 mM glucose in an aqueous sample solution, its limit of detection was 0.37 mM, and the response time was 2 min.

Gwakhyangjeonggi-san for irritable bowel syndrome: A protocol for systematic review and meta-analysis.

BACKGROUND: Irritable bowel syndrome (IBS) is a chronic functional bowel disorder characterized by abdominal pain or discomfort, stool irregularities, and bloating. Owing to its atypical symptoms and various mechanisms, there is no standard treatment for IBS. Gwakhyangjeonggi-san (GJS), a traditional Korean herbal medicine, has been used to treat lower intestinal abnormalities in Asia. We will systematically review randomized controlled trials (RCTs) to evaluate the efficacy and safety of GJS as a complementary treatment for IBS. METHODS AND ANALYSIS: Four English databases, namely, Medline (via PubMed), EMBASE, the Cochrane Central Register of Controlled Trials, and the Allied and Complementary Medicine Database, will be searched for entries up to May, 2021. Additional databases will include 5 Korean databases, 1 Chinese database, and 1 Japanese database. RCTs and quasi-RCTs will be searched for to assess the effectiveness and safety of GJS. The primary outcome measure will be the overall efficacy rate, and the secondary outcome will include data such as global symptom scores, IBS Quality of Life measurements, and adverse events. Data analysis will be performed using Review Manager Version 5.3, and the risk of bias will be assessed using the Cochrane Collaboration's risk-of-bias tool. The quality of the results will be evaluated using the Grading of Recommendations Assessment, Development, and Evaluation approach. CONCLUSION: This systematic review will provide evidence for the efficacy and safety of GJS for IBS. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/V93JN (https://osf.io/v93jn).

Poly(Pyridinium Salt)s Containing 2,7-Diamino-9,9'-Dioctylfluorene Moieties with Various Organic Counterions Exhibiting Both Lyotropic Liquid-Crystalline and Light-Emitting Properties.

A series of poly(pyridinium salt)s-fluorene main-chain ionic polymers with various organic counterions were synthesized by using ring-transmutation polymerization and metathesis reactions. Their chemical structures were characterized by Fourier Transform Infrared (FTIR), proton (1H), and fluorine 19 (19F) nuclear magnetic resonance (NMR) spectrometers. These polymers showed a number-average molecular weight (Mns) between 96.5 and 107.8 kg/mol and polydispersity index (PDI) in the range of 1.12-1.88. They exhibited fully-grown lyotropic phases in polar protic and aprotic solvents at different critical concentrations. Small-angle X-ray scattering for one polymer example indicates lyotropic structure formation for 60-80% solvent fraction. A lyotropic smectic phase contains 10 nm polymer platelets connected by tie molecules. The structure also incorporates a square packing motif within platelets. Thermal properties of polymers were affected by the size of counterions as determined by differential scanning calorimetry and thermogravimetric analysis measurements. Their ultraviolet-visible (UV-Vis) absorption spectra in different organic solvents were essentially identical, indicating that the closely spaced π-π* transitions occurred in their conjugated polymer structures. In contrast, the emission spectra of polymers exhibited a positive solvatochromism on changing the polarity of solvents. They emitted green lights in both polar and nonpolar organic solvents and showed blue light in the film-states, but their λem peaks were dependent on the size of the counterions. They formed aggregates in polar aprotic and protic solvents with the addition of water (v/v, 0-90%), and their λem peaks were blue shifted.

Diversity of Citrus tristeza virus Strains in the Upper Gulf Coast Area of Texas.

Citrus tristeza virus (CTV) in Texas was first reported in the 1950s and has since been sporadically reported in the residential areas in the Upper Gulf Coast region. Because the major rootstock for commercial citriculture in South Texas is sour orange, which is susceptible to CTV decline, the spread of CTV into South Texas can pose a great threat to Texas citrus industry. Thirty-six CTV-positive samples, collected during surveys conducted in the Upper Gulf Coast area of Texas from 2013 to 2018, were first analyzed by strain-specific real-time PCR (RT-PCR) targeting various regions of CTV Open reading frame (Orf) 1a and then by amplicon sequencing derived from p25 and p20 region of CTV genome. Among 36 samples, 33 were successfully genotyped by strain-specific RT-PCR and by amplicon sequencing followed by phylogenetic analysis. Variability in the detection of CTV strains was observed over a 6-year period. In 2013, T3 and T30 were the only strains detected in the Upper Gulf Coast of Texas, but in further surveys until 2018, additional strains were detected, including T36, VT, and RB. Mixed infections were also detected in 14 samples comprising about 42% of CTV samples examined in the study. Although genotyping mixed infection samples by targeting Orf 1a and full-length p25, residing in the 5' and 3' region of the CTV genome, respectively, confirmed the presence of multiple strains in these samples, incongruent genotyping data were observed. These findings suggested that the current status of CTV strain diversity in Texas Upper Gulf Coast region might have been established by multiple introductions of CTV-infected plant materials for propagation and with a potential recombination in planta.

Analysis of Tissue-Specific Interferon Regulatory Factor 3 (IRF3) Gene Expression against Viral Infection in Paralichthys olivaceus.

Interferon Regulatory Factor 3 (IRF3) is a member of interferon-regulated transcription factor family and is known to play an important role in the innate immune response against viral infections. In this study, the expression of IRF3 in different tissues, developmental stages, and stocking densities of olive flounder was investigated. The expression of IRF3 was observed to gradually increase in early-stage juvenile fish. The highest expression was observed in later-stage juvenile fish when immune tissues were formed. High IRF3 expression was observed in the muscles and the brain tissues. The expression of IRF3 was observed in fish at different stocking densities after viral hemorrhagic septicemia virus (VHSV) infection. It yielded an interesting expression pattern in the muscles and the brain tissues of fish stocked at low density. These observations can be used as basic data for the study of the expression of immune response-related genes against viruses based on stocking density and immune systems in other fish species.