IL-2Rα KO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells.

Introduction: IL-2Rα knock out (KO) mice have been instrumental to discovering the immunoregulatory properties of IL-2Rα. While initially thought of only as a stimulatory cytokine, IL-2 and IL-2Rα KO mice revealed that this cytokine-receptor system controls immune responses through restimulation-induced cell death and by promoting the survival of T regulatory cells. Although described mostly in the context of lymphocytes, recent studies by our laboratory showed that IL-2R is expressed in smooth muscle cells. Given this finding, we sought to use IL-2Rα KO to determine the function of this receptor in vascular smooth muscle cells. Surprisingly, we found that IL-2Rα KO vascular smooth muscle cells had detectable IL-2Rα. Methods: We used multiple gene and protein-based methods to determine why IL-2Rα KO vascular smooth muscle cells exhibited IL-2Rα protein. These methods included: genomic sequencing, assessing cells and tissues for evidence of maternal microchimerism, and determining the half-life of IL-2Rα protein. Results: Our studies demonstrated the following: (1) in addition to the cell surface, IL-2Rα is localized to the nucleus; (2) the genetic deletion of IL-2Rα is intact in IL-2Rα KO mice; (3) both IL-2Rα KO and WT tissues show evidence of maternal microchimerism, the likely source of IL-2Rα (4) IL-2Rα is transmitted between cells; (5) IL-2Rα has a long half-life; and (6) nuclear IL-2Rα contributes to the regulation of cell proliferation and size. Conclusion: Our findings suggest that the phenotype of complete IL-2Rα loss is more severe than demonstrated by IL-2Rα KO mice, and that IL-2Rα plays a here-to-fore unrecognized role in regulating cell proliferation in non-lymphoid cells.

IL-2RαKO mice exhibit maternal microchimerism and reveal nuclear localization of IL-2Rα in lymphoid and non-lymphoid cells.

IL-2Rα KO mice have been instrumental to discovering the immunoregulatory properties of IL-2Rα. While initially thought of only as a stimulatory cytokine, IL-2 and IL-2Rα knock out (KO) mice revealed that this cytokine-receptor system controls immune responses through restimulation-induced cell death and by promoting the survival of T regulatory cells. Although described mostly in the context of lymphocytes, recent studies by our laboratory showed that IL-2R is expressed in smooth muscle cells. Given this finding, we sought to use IL-2Rα knock mice to determine the function of this receptor in vascular smooth muscle cells. Surprisingly, we found that IL-2Rα knock out vascular smooth muscle cells had detectable IL-2Rα. Further studies suggested that the source of IL-2Rα protein was likely maternal heterozygous cells present in KO offspring due to maternal microchimerism. Because the KO was generated by using a neomycin resistance gene insert, we treated cells with G418 and were able to eliminate the majority of IL-2Rα expressing cells. This elimination revealed that IL-2Rα KO vascular smooth muscle cells exhibited increased proliferation, decreased size, and hypodiploid DNA content when compared to wildtype cells. Our findings suggest that the phenotype of complete IL-2Rα loss is more severe than demonstrated by IL-2Rα KO mice, and that IL-2Rα plays a here-to-fore unrecognized role in regulating cell proliferation in non-lymphoid cells.

Efficient pH-universal aqueous supercapacitors enabled by an azure C-decorated N-doped graphene aerogel.

Current aqueous supercapacitors (SCs) possess the relative low energy density, and there is therefore widespread interest in cost-effective fabrication of capacitive materials with promoted specific capacitance and/or broadened voltage window. Here, a redox-active azure C-decorated N-doped graphene aerogel (AC - NGA) is fabricated using a simple hydrothermal self-assembly method through strong noncovalent π-π interaction. AC - NGA highlights an excellent charge storage performance (a high 591F g-1 gravimetric capacitance under a current density of 1.0 A g-1 and ultrahigh voltage window of 2.3 V) under pH-universal conditions. The capacitive contribution of charge storage is 91.7%, exceeding or comparable to those of the best pseudocapacitors known. Furthermore, a symmetric AC - NGA//AC - NGA device realizes high energy and power densities (15.2-60.2 Wh kg-1 at 650-23,000 W kg-1) and excellent cycling stability in acidic, neutral, and basic aqueous solutions. This work offers a cost-effective strategy to combine redox dye molecules with heteroatom-doped graphene aerogel for building green efficient pH-universal aqueous supercapacitors.

Environmental Information Disclosure, Digital Transformation, and Total Factor Productivity: Evidence from Chinese Heavy Polluting Listed Companies.

Environmental information disclosure, as a new environmental regulatory model, is important for achieving collaborative environmental pollution management and sustainable socioeconomic development. Based on the data of listed firms in China's A-share heavy pollution industry from 2009 to 2019, this paper empirically tested the impact of environmental information disclosure on the total factor productivity of enterprises and the contribution of digital transformation to this impact. An increase in the level of environmental information disclosure had a significant positive effect on the total factor productivity of enterprises. However, with the increase in digital transformation among enterprises, the effect of environmental information disclosure on total factor productivity improvement is gradually being replaced. The heterogeneity test results showed that the positive effect of environmental information disclosure on total factor productivity changed depending on property rights, firm size, and geographical location. The effect of environmental information disclosure was stronger for non-state firms, large firms, and firms located in the east-central region. Further mechanism tests showed that the effect was induced through innovation incentives and facilitated financing. The above results provide a valuable reference for a comprehensive understanding of the effect of environmental information disclosure on productivity and adjustment by the digital transformation of enterprises.

Comprehensive microRNA-seq transcriptomic profiling across 11 organs, 4 ages, and 2 sexes of Fischer 344 rats.

Rat is one of the most widely-used models in chemical safety evaluation and biomedical research. However, the knowledge about its microRNA (miRNA) expression patterns across multiple organs and various developmental stages is still limited. Here, we constructed a comprehensive rat miRNA expression BodyMap using a diverse collection of 320 RNA samples from 11 organs of both sexes of juvenile, adolescent, adult and aged Fischer 344 rats with four biological replicates per group. Following the Illumina TruSeq Small RNA protocol, an average of 5.1 million 50 bp single-end reads was generated per sample, yielding a total of 1.6 billion reads. The quality of the resulting miRNA-seq data was deemed to be high from raw sequences, mapped sequences, and biological reproducibility. Importantly, aliquots of the same RNA samples have previously been used to construct the mRNA BodyMap. The currently presented miRNA-seq dataset along with the existing mRNA-seq dataset from the same RNA samples provides a unique resource for studying the expression characteristics of existing and novel miRNAs, and for integrative analysis of miRNA-mRNA interactions, thereby facilitating better utilization of rats for biomarker discovery.

Cross-oncopanel study reveals high sensitivity and accuracy with overall analytical performance depending on genomic regions.

BACKGROUND: Targeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing. RESULTS: All panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5-20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden. CONCLUSION: This comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.

A verified genomic reference sample for assessing performance of cancer panels detecting small variants of low allele frequency.

BACKGROUND: Oncopanel genomic testing, which identifies important somatic variants, is increasingly common in medical practice and especially in clinical trials. Currently, there is a paucity of reliable genomic reference samples having a suitably large number of pre-identified variants for properly assessing oncopanel assay analytical quality and performance. The FDA-led Sequencing and Quality Control Phase 2 (SEQC2) consortium analyze ten diverse cancer cell lines individually and their pool, termed Sample A, to develop a reference sample with suitably large numbers of coding positions with known (variant) positives and negatives for properly evaluating oncopanel analytical performance. RESULTS: In reference Sample A, we identify more than 40,000 variants down to 1% allele frequency with more than 25,000 variants having less than 20% allele frequency with 1653 variants in COSMIC-related genes. This is 5-100× more than existing commercially available samples. We also identify an unprecedented number of negative positions in coding regions, allowing statistical rigor in assessing limit-of-detection, sensitivity, and precision. Over 300 loci are randomly selected and independently verified via droplet digital PCR with 100% concordance. Agilent normal reference Sample B can be admixed with Sample A to create new samples with a similar number of known variants at much lower allele frequency than what exists in Sample A natively, including known variants having allele frequency of 0.02%, a range suitable for assessing liquid biopsy panels. CONCLUSION: These new reference samples and their admixtures provide superior capability for performing oncopanel quality control, analytical accuracy, and validation for small to large oncopanels and liquid biopsy assays.

Evaluating the analytical validity of circulating tumor DNA sequencing assays for precision oncology.

Circulating tumor DNA (ctDNA) sequencing is being rapidly adopted in precision oncology, but the accuracy, sensitivity and reproducibility of ctDNA assays is poorly understood. Here we report the findings of a multi-site, cross-platform evaluation of the analytical performance of five industry-leading ctDNA assays. We evaluated each stage of the ctDNA sequencing workflow with simulations, synthetic DNA spike-in experiments and proficiency testing on standardized, cell-line-derived reference samples. Above 0.5% variant allele frequency, ctDNA mutations were detected with high sensitivity, precision and reproducibility by all five assays, whereas, below this limit, detection became unreliable and varied widely between assays, especially when input material was limited. Missed mutations (false negatives) were more common than erroneous candidates (false positives), indicating that the reliable sampling of rare ctDNA fragments is the key challenge for ctDNA assays. This comprehensive evaluation of the analytical performance of ctDNA assays serves to inform best practice guidelines and provides a resource for precision oncology.

Advancing NGS quality control to enable measurement of actionable mutations in circulating tumor DNA.

The primary objective of the FDA-led Sequencing and Quality Control Phase 2 (SEQC2) project is to develop standard analysis protocols and quality control metrics for use in DNA testing to enhance scientific research and precision medicine. This study reports a targeted next-generation sequencing (NGS) method that will enable more accurate detection of actionable mutations in circulating tumor DNA (ctDNA) clinical specimens. To accomplish this, a synthetic internal standard spike-in was designed for each actionable mutation target, suitable for use in NGS following hybrid capture enrichment and unique molecular index (UMI) or non-UMI library preparation. When mixed with contrived ctDNA reference samples, internal standards enabled calculation of technical error rate, limit of blank, and limit of detection for each variant at each nucleotide position in each sample. True-positive mutations with variant allele fraction too low for detection by current practice were detected with this method, thereby increasing sensitivity.

The B cell response to both protein and nucleic acid antigens displayed on apoptotic cells are dependent on endosomal pattern recognition receptors.

In systemic autoimmune diseases such as systemic lupus erythematosus (SLE), B cell tolerance is lost and there is a production of autoantibodies that drive pathology. The specificities of these antibodies are towards a wide range of autoantigens including proteins such as serum factors including cytokines as well as towards nucleic acids and modified glycolipids. It is known that endosomal pattern recognition receptors are involved in specific responses but if they drive specificity towards a specific group of autoantigens is not known. Here, we used syngeneic apoptotic cells alone to break B cell tolerance and investigated the antibody response in Unc93b1 mutant mice that lack signalling from the TLR3, TLR7 and TLR9 receptors. We found that specific B cell responses known from patients with SLE including antibodies towards Ro-52/60, La, cardiolipin as well as DNA were all significantly lower in the knockout mice. Thus, we found that endosomal TLR receptors were involved in break of tolerance and drive B cell responses for protein, nucleic acid and modified lipid antigens. This pinpoints these receptors as key drivers for the full range of antibody driven pathology in SLE and suggests that targeting of endosomal TLR driven responses will quench all B cell driven autoreactivity.

Endoscopic thoracic sympathicotomy for primary palmar hyperhidrosis: A retrospective multicenter study in China.

BACKGROUND: This study aimed to evaluate the clinical efficacy and safety of endoscopic thoracic sympathicotomy and to explore strategies to decrease the incidence of transfer hyperhidrosis (TH). METHODS: From January 2003 to July 2016, 10,275 patients with primary palmar hyperhidrosis underwent endoscopic thoracic sympathicotomy in 15 different institutions. We carried out a retrospective analysis of these patients who were grouped into group A, those with nonretained R2 (R2, R2-3, or R2-4 ablation), and group B, those with retained R2 (single R3 or R4 ablation). RESULTS: All procedures were performed successfully. Both hands of all patients became warm and dry immediately after endoscopic thoracic sympathicotomy. Pneumothorax occurred in 146 patients, and 39 patients had intraoperative bleeding. Follow-up was carried out from 6 months to 13 years. A total of 531 patients (5.2%) were lost to follow-up. The effective rate for primary palmar hyperhidrosis was 100%. Palmar hyperhidrosis recurred in 73 patients (0.7%). Transfer hyperhidrosis appeared in 7,678 patients (78.8%). For groups A and B, the incidence of TH was 80.4% and 78.5%, respectively (P > .05), but the incidence of grade III+IV TH in group B (1.6%) was less than that in group A (4.8%; P < .001). CONCLUSION: Endoscopic thoracic sympathicotomy is a minimally invasive, safe, and effective therapeutic method for primary palmar hyperhidrosis. Although the overall incidence of TH is high, the incidence of grade III to IV TH can be decreased by reserving R2, lowering the level of thoracic sympathicotomy, and single severing of R3 or R4.

Bioaccessibility of BDE 47 in a simulated gastrointestinal system and its metabolic transformation mechanisms in Caco-2 cells.

Polybrominated diphenyl ethers (PBDEs) have been regarded as ubiquitous environmental pollutants. However, the absorption and transformation of these compounds after ingestion are not well understood yet. In this study, the bioaccessibility and metabolic pathway of 2,2',4,4'- tetrabromodiphenyl ether (BDE47) was investigated in an in vitro digestion/Caco-2 cell. Gastric and intestinal bioaccessibilities of BDE47 in 5 kinds of spiked soil samples were ranging from 11.39 ±â€¯0.83% to 36.02 ±â€¯4.34%, and 48.24 ±â€¯3.24% to 81.52 ±â€¯6.43%, respectively. Upon exposure to differentiated Caco-2 cells for 6 h, it was found that only a small amount of BDE47 in the gastrointestinal (GI) solution could pass through Caco-2 cells, and might enter the body. Moreover, BDE47 was found to be metabolized or transformed into BDE28, BDE75, BDE37, BDE32, BDE15 and BDE8 in Caco-2 cells. The metabolic pathway could be explained by using the Becke three-parameter hybrid functional (B3Lucifer yellow CHP) in the Density Functional Theory (DFT), denoted as the values of the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) at the atoms of BDE47 and its metabolic products. The obtained results suggest that oral intake of PBDEs is associated with low bioaccessibility, but also emphasize the risks associated with oral ingestion, namely toxicity resulting from the debromination of highly brominated diphenyl ethers. Although highly brominated diphenyl ethers are known to be the least toxic PBDEs, the debrominated products in human intestinal epithelia may elicit greater than expected toxicity.

Characterization of Frog Virus 3 knockout mutants lacking putative virulence genes.

To identify ranavirus virulence genes, we engineered Frog Virus 3 (FV3) knockout (KO) mutants defective for a putative viral caspase activation and recruitment domain-containing (CARD) protein (Δ64R-FV3) and a ß-hydroxysteroid dehydrogenase homolog (Δ52L-FV3). Compared to wild type (WT) FV3, infection of Xenopus tadpoles with Δ64R- or Δ52L-FV3 resulted in significantly lower levels of mortality and viral replication. We further characterized these and two earlier KO mutants lacking the immediate-early18kDa protein (FV3-Δ18K) or the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α). All KO mutants replicated as well as WT-FV3 in non-amphibian cell lines, whereas in Xenopus A6 kidney cells replication of ΔvCARD-, ΔvßHSD- and ΔvIF-2α-FV3 was markedly reduced. Furthermore, Δ64R- and ΔvIF-2α-FV3 were more sensitive to interferon than WT and Δ18-FV3. Notably, Δ64R-, Δ18K- and ΔvIF-2α- but not Δ52L-FV3 triggered more apoptosis than WT FV3. These data suggest that vCARD (64R) and vß-HSD (52L) genes contribute to viral pathogenesis.

Activating the expression of human K-rasG12D stimulates oncogenic transformation in transgenic goat fetal fibroblast cells.

Humane use of preclinical large animal cancer models plays a critical role in understanding cancer biology and developing therapeutic treatments. Among the large animal candidates, goats have great potentials as sustainable sources for large animal cancer model development. Goats are easier to handle and cheaper to raise. The genome of the goats has been sequenced recently. It has been known that goats develop skin, adrenal cortex, breast and other types of cancers. Technically, goats are subject to somatic cell nuclear transfer more efficiently and exhibit better viability through the cloning process. Towards the development of a goat cancer model, we created a transgenic goat fetal fibroblast (GFF) cell as the donor cell for SCNT. Human mutated K-ras (hK-rasG12D) was chosen as the transgene, as it is present in 20% of cancers. Both hK-rasG12D and a herpes simplex viral thymidine kinase (HSV1-tk) reporter genes, flanked by a pair of LoxP sites, were knocked in the GFF endogenous K-ras locus through homologous recombination. Following Cre-mediated activation (with a 95% activation efficiency), hK-rasG12D and HSV1-tk were expressed in the transgenic GFF cells, evidently through the presence of corresponding mRNAs, and confirmed by HSV1-tk protein function assay. The hK-rasG12D expressing GFF cells exhibited enhanced proliferation rates and an anchorage-independent growth behavior. They were able to initiate tumor growth in athymic nude mice. In conclusion, after activating hK-rasG12D gene expression, hK-rasG12D transgenic GFF cells were transformed into tumorgenesis cells. Transgenic goats via SCNT using the above-motioned cells as the donor cells have been established.

Synergistic effect of LEP and LEPR gene polymorphism on body mass index in a Chinese population.

Both leptin (LEP) and leptin receptor (LEPR) are important in the regulation of body weight. In this study, we evaluated the individual and combined effects of a polymorphic microsatellite marker in the LEP gene 3' flanking region and two polymorphisms (Lys109Arg and Lys656Asn) of the LEPR gene on metabolic markers for obesity in a Chinese population. The genotypes of polymorphisms in LEP and LEPR gene were determined by PCR and SSCP assay in 230 simple obese subjects and 202 control subjects of Chinese population. Logistic regression analysis showed that polymorphism in LEP gene 3' flanking region was associated with waist/hip ratio (WHR) (P = 0.042). Individually, Lys109Arg variant in LEPR gene was associated with systolic blood pressure (P = 0.031) in males, and Lys656Asn variant was associated with serum triglyceride level (P = 0.026). Interestingly, only subjects that simultaneously exhibit all three polymorphisms showed a significantly elevated BMI (29.30 ± 0.85 vs 26.91 ± 1.19, P = 0.037). Taken together, our data suggest that a combination of polymorphism in the LEP gene 3' flanking region, and Lys109Arg, Lys656Asn variants in LEPR gene is associated with obesity in Chinese Han population.

Immune evasion strategies of ranaviruses and innate immune responses to these emerging pathogens.

Ranaviruses (RV, Iridoviridae) are large double-stranded DNA viruses that infect fish, amphibians and reptiles. For ecological and commercial reasons, considerable attention has been drawn to the increasing prevalence of ranaviral infections of wild populations and in aquacultural settings. Importantly, RVs appear to be capable of crossing species barriers of numerous poikilotherms, suggesting that these pathogens possess a broad host range and potent immune evasion mechanisms. Indeed, while some of the 95-100 predicted ranavirus genes encode putative evasion proteins (e.g., vIFα, vCARD), roughly two-thirds of them do not share significant sequence identity with known viral or eukaryotic genes. Accordingly, the investigation of ranaviral virulence and immune evasion strategies is promising for elucidating potential antiviral targets. In this regard, recombination-based technologies are being employed to knock out gene candidates in the best-characterized RV member, Frog Virus (FV3). Concurrently, by using animal infection models with extensively characterized immune systems, such as the African clawed frog, Xenopus laevis, it is becoming evident that components of innate immunity are at the forefront of virus-host interactions. For example, cells of the macrophage lineage represent important combatants of RV infections while themselves serving as targets for viral infection, maintenance and possibly dissemination. This review focuses on the recent advances in the understanding of the RV immune evasion strategies with emphasis on the roles of the innate immune system in ranaviral infections.

Susceptibility of Xenopus laevis tadpoles to infection by the ranavirus Frog-Virus 3 correlates with a reduced and delayed innate immune response in comparison with adult frogs.

Xenopus laevis adults mount effective immune responses to ranavirus Frog Virus 3 (FV3) infections and clear the pathogen within 2-3 weeks. In contrast, most tadpoles cannot clear FV3 and succumb to infections within a month. While larval susceptibility has been attributed to ineffective adaptive immunity, the contribution of innate immune components has not been addressed. Accordingly, we performed a comprehensive gene expression analysis on FV3-infected tadpoles and adults. In comparison to adults, leukocytes and tissues of infected tadpoles exhibited modest (10-100 time lower than adult) and delayed (3 day later than adult) increase in expression of inflammation-associated (TNF-α, IL-1ß and IFN-γ) and antiviral (Mx1) genes. In contrast, these genes were readily and robustly upregulated in tadpoles upon bacterial stimulation. Furthermore, greater proportions of larval than adult PLs were infected by FV3. Our study suggests that tadpole susceptibility to FV3 infection is partially due to poor virus-elicited innate immune responses.

Antiviral immunity in amphibians.

Although a variety of virus species can infect amphibians, diseases caused by ranaviruses ([RVs]; Iridoviridae) have become prominent, and are a major concern for biodiversity, agriculture and international trade. The relatively recent and rapid increase in prevalence of RV infections, the wide range of host species infected by RVs, the variability in host resistance among population of the same species and among different developmental stages, all suggest an important involvement of the amphibian immune system. Nevertheless, the roles of the immune system in the etiology of viral diseases in amphibians are still poorly investigated. We review here the current knowledge of antiviral immunity in amphibians, focusing on model species such as the frog Xenopus and the salamander (Ambystoma tigrinum), and on recent progress in generating tools to better understand how host immune defenses control RV infections, pathogenicity, and transmission.

The cytotoxicity of OPA-modified CdSe/ZnS core/shell quantum dots and its modulation by silibinin in human skin cells.

Quantum dots (QDs) have emerged as alternative or complementary tools to organic fluorescent dyes currently used in bioimaging. QDs hold several advantages over conventional fluorescent dyes including greater photostability and a wider range of excitation/emission wavelengths. However, recent work suggests that QDs exert deleterious effects on cellular processes which could obscure bioassay results. This study examined the toxicity of octylamine-poly(acrylic acid) (OPA) modified CdSe/ZnS quantum dots (QDs) and a pharmacological means of preventing QD-induced cell death. Cell viability and the flow cytometry were used to access the toxicity of OPA-modified CdSe/ZnS QDs to human skin cells following a 24-h exposure. It is found that concentrations leading to a 50% reduction in malignant melanoma cell viability (TC50) for two OPA-QDs (QD545 and QD605) are 102.1, 57.3 nM in A375 cells and 67.2, 55.0 nM in A375.S2 cells, respectively. Moreover, QD545 and QD605 show low cytotoxic response in HaCaT keratinocyte cells with TC50 values of 818.2 nM and 162.0 nM, respectively. Silibinin, a natural product derived from milkweed thistle, is known for its powerful antioxidant and membrane stabilizing properties. Pretreatment of cells with silibinin, significantly reduced QD-induced cell death in A375 and A375-S2 cells. These findings suggest that QD cytotoxicity is sensitive to cell types and that pretreatment with antioxidants, such as the natural product silibinin, can modulate QD-induced cytotoxicity.

Improved knockout methodology reveals that frog virus 3 mutants lacking either the 18K immediate-early gene or the truncated vIF-2alpha gene are defective for replication and growth in vivo.

To better assess the roles of frog virus 3 (FV3; genus Ranavirus, family Iridoviridae) genes in virulence and immune evasion, we have developed a reliable and efficient method to systematically knock out (KO) putative virulence genes by site-specific integration into the FV3 genome. Our approach utilizes a dual selection marker consisting of the puromycin resistance gene fused in frame with the enhanced green fluorescent protein (EGFP) reporter (Puro-EGFP cassette) under the control of the FV3 immediate-early (IE) 18K promoter. By successive rounds of selection for puromycin resistance and GFP expression, we have successfully constructed three recombinant viruses. In one, a "knock-in" mutant was created by inserting the Puro-EGFP cassette into a noncoding region of the FV3 genome (FV3-Puro/GFP). In the remaining two, KO mutants were constructed by replacement of the truncated viral homolog of eIF-2α (FV3-ΔvIF-2α) or the 18K IE gene (FV3-Δ18K) with the Puro-EGFP cassette. The specificity of recombination and the clonality of each mutant were confirmed by PCR, sequencing, and immunofluorescence microscopy. Viral replication of each recombinant in cell culture was similar to that of parental FV3; however, infection in Xenopus laevis tadpoles revealed that FV3-ΔvIF-2α and FV3-Δ18K replicated less and resulted in lower mortality than did GFP-FV3 and wild-type FV3. Our results suggest that 18K, which is conserved in all ranaviruses, and the truncated vIF-2α gene contribute to virulence. In addition, our study describes a powerful methodology that lays the foundation for the discovery of potentially new ranaviral genes involved in virulence and immune escape.