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Diabetes cardiac fibrosis is associated with altered DNA methylation of fibrogenic genes; however, the underlying mechanisms remain unclear.In this study, we investigate the critical role of DNA methylation aberration-associated suppression of MTHFR in diabetes cardiac fibrosis, and the protective effects of folate on diabetes cardiac fibrosis, using cultured cells, animal models, and clinical samples.Herein, we report that DNA methylation repression of MTHFR, critically involved in diabetes cardiac fibrosis, mediates the significant protective effects of folate in a mouse model of diabetes cardiac fibrosis induced by STZ. Heart MTHFR expression was markedly suppressed in diabetes cardiac fibrosis patients and mice, accompanied by increased DNMT3A and MTHFR promoter methylation. Knockdown of DNMT3A demethylated MTHFR promoter, recovered the MTHFR loss, and alleviated the diabetes cardiac fibrosis pathology and cardiac fibroblasts pyroptosis. Mechanistically, DNMT3A epigenetically repressed MTHFR expression via methylation of the promoter. Interestingly, folate supplementation can rescue the effect of MTHFR loss in diabetes cardiac fibrosis, suggesting that inactivation of MTHFR through epigenetics is a critical mediator of diabetes cardiac fibrosis.The current study identifies that MTHFR repression due to aberrant DNMT3A elevation and subsequent MTHFR promoter hypermethylation is likely an important epigenetic feature of diabetes cardiac fibrosis, and folate supplementation protects against diabetes cardiac fibrosis.

Gestational diabetes mellitus (GDM) is the most common cause of hyperglycemia during pregnancy, and its prevalence has increased over the past decades. GDM is directly related to the recent obstetric outcomes and long-term maternal and child health, which can be greatly improved by early identification and diagnosis of GDM. However, the prediction of the disease has always been a difficult problem due to the lack of simple and practical serological markers. Despite the controversy, recent studies have identified that circulating inflammatory cells and platelets, routinely included in the obstetric blood tests, are related to the development of GDM and adverse pregnancy outcomes. In this review, we summarized the studies in this field based on the recent literature. The inflammatory cell components we included were the total number of white blood cells, neutrophils, lymphocytes, monocytes and platelets, which were routinely examined in the blood tests in pregnancy. The aim of this review is not only to enrich our understanding of the pathogenesis of GDM but also to provide evidence for the value of these novel and practical serological markers in early identification of GDM and the prevention and its adverse outcomes.

RATIONALE AND OBJECTIVES Skeletal muscle mass measurement is the most important element for diagnosing sarcopenia. MRI has an excellent soft-tissue contrast, which can non-invasively assess abdominal skeletal muscle area (SMA) as well as CT. This study aimed to assess the validity and reliability of abdominal SMA measurement by comparing CT and MRI based on the fat image of IDEAL-IQ sequence at the lumbar level mid-L3. MATERIALS AND METHODS CT and MRI images of 32 patients diagnosed with various kidney diseases were used to analyze intra-observer variability among abdominal SMA measurements. This was done to evaluate the correlation of SMA between CT and fat images of MRI. SMA images were segmented using Materialise Mimics software before quantification. Interobserver reliability and validation of measurements was evaluated by two independent investigators. Abdominal SMA reproducibility and correlation between CT and MRI were then assessed using the intraclass correlation coefficient (ICC), coefficient of variation (CV), Bland-Altman plot, and Pearson's correlation coefficient respectively. RESULTS The interobserver reliability of MRI was excellent. The CV value was 2.82% while the ICC values ranged between 0.996 and 0.999. Validity was high (CV was 1.7% and ICC ranged between 0.986 and 0.996) for measurements by MRI and CT. Bland Altman analysis revealed an average difference of 2.2% between MRI and CT. The Pearson's correlation coefficient was 0.995 (p < 0.0001). This result revealed that there was a strong correlation between the two technologies. CONCLUSION MRI exhibited good interobserver reliability and excellent agreement with CT for quantification of abdominal SMA.

Belatacept confers increased patient and graft survival in renal transplant recipients relative to calcineurin inhibitors, but is associated with an increased rate of acute rejection. Recent immunophenotypic studies comparing pre-transplant T cell phenotypes of patients who reject vs. those that remain stable on belatacept identified three potential “risky” memory T cell subsets that potentially underlie belatacept-resistant rejection: CD4(+) CD28(+) T(EM), CD8(+) CD28(null) and CD4(+) CD57(+) PD1(−) subsets. Here, we compared key phenotypic and functional aspects of these human memory T cell subsets, with the goal of identifying additional potential targets to modulate them. Results demonstrate that TIGIT, an increasingly well-appreciated immune checkpoint receptor, was expressed on all three risky memory T cell subsets in vitro and in vivo in the presence of belatacept. Co-culture of human memory CD4(+) and CD8(+) T cells with an agonistic anti-TIGIT mAb significantly increased apoptotic cell death of all three risky memory T cell subsets. Mechanistically, TIGIT-mediated apoptosis of risky memory T cells was dependent on FOXP3(+) Treg, suggesting that agonism of the TIGIT pathway increases FOXP3(+) Treg suppression of human memory T cell populations. Overall, these data suggest that TIGIT agonism could represent a new therapeutic target to inhibit belatacept-resistant rejection during transplantation.

Neodynium oxyfluoride has been applied for luminescence materials and solid electrolytes because of its structure combined advantages of rare-earth cations with F− and O2− anions. Since the neodynium oxyfluoride with tetragonal structure retains a defect structure having anion vacancies where anions transfer, it shows higher anion conductivity than that with cubic and rhombohedral structures. The feasibility of synthesizing tetragonal neodynium oxyfluoride in molten fluorides was investigated in this work. The neodynium oxyfluoride was not dissolved in molten LiF–CaF2 based on the in-situ observation using confocal scanning laser microscope, therefore neodynium oxyfluoride can be easily collected from molten fluorides. The neodynium oxyfluoride with tetragonal structure was successfully synthesized by the reaction of Nd2O3 with molten fluorides. The synthesis process was analyzed by differential scanning calorimetry combining X-ray diffraction and scanning electron microscope. The reaction model was described and the reaction rate constant was calculated. According to the mathematical description of kinetic equation, the reaction rate could be proportional to the interface difference between molten fluorides and solid Nd2O3.

Tracing pedogenic processes is fundamental for reconstructing climatic and environmental changes using loess deposits. Lithium isotopes can serve as a sensitive tracer to unravel these processes, owing to its active behaviors without effect by redox and biological reactions. This study investigated the Li contents and δ7Li values of leachate and residue ([Li]leachate, [Li]residue, δ7Lileachate and δ7Liresidue) of the upper 12.5 m Luochuan loess-paleosol sediment (since the last interglacial) on the Chinese Loess Plateau, in order to better understand pedogenic processes in loess. The [Li]leachate, varying from 0.39 to 1.97 μg/g, is mainly derived from the adsorption phase, with a significant variation in δ7Lileachate, from −6.55‰ to +12.88‰. Both [Li]leachate and δ7Lileachate variations reflect weathering and adsorption processes during different periods. The [Li]residue vary from 34.4 to 46.3 μg/g (averaging 38.4 μg/g) and their δ7Liresidue vary from −3.66‰ to +2.44‰ (averaging −0.22 ± 1.68‰), both in agreement with the upper continental crust values. At initial stage after loess deposited (stage I), high [Li]leachate but low δ7Lileachate are results of preferential adsorption of 6Li by clays and migration of 7Li with soil solution during weathering and eluviation processes. When overlying loess was deposited and experienced pedogenesis as the stage I, 7Li migrated downward and was adsorbed by clays (stage II), resulting in high [Li]leachate and δ7Lileachate in underlying loess. When loess has superimposed pedogenic and adsorption processes as the stages I and II, loess became as paleosol with highest δ7Lileachate in underlying carbonate accumulation layer. A persistent increase in δ7Lileachate indicates dominant control of post-depositional adsorptions on Li fractionation along solution migrations during weathering and eluviation of overlying deposits, and vice versa. These results highlight that variations in [Li]leachate and δ7Lileachate can be used to trace soil water migration processes and the magnitude of pedogenic processes in loess-paleosol sediment at different periods.

The minimum miscible pressure is one of the key factors to realize miscible flooding. As the minimum miscible pressure in the research area is higher than the formation fracture pressure, miscible flooding cannot be formed. To address this problem, it is necessary to find a way to reduce the minimum miscible pressure. Citric acid isopentyl ester can not only be dissolved in crude oil, but also be dissolved in carbon dioxide. Therefore, citric acid isopentyl ester was chosen to reduce the minimum miscible pressure in this research. The effect of citric acid isopentyl ester on reducing the minimum miscible pressure was measured by the method of long slim tube displacement experiment. The minimum miscible pressure in the research area was 29.6 MPa. The experimental results show that the minimum miscible pressure could decrease significantly with increasing the injected slug size of citric acid isopentyl ester, but the decrease became smaller and smaller. The optimum injected slug size of the chemical reagent is 0.003 PV. Under the condition of the slug size, the minimum miscible pressure was 24.1 MPa. The reduction was 5.5 MPa. The reduction rate was 18.58%. The research results have important guiding significance for enhancing oil recovery in the research area.

Flexible high-performance electrochemical capacitors have aroused much attention with their prospect applications in future wearable electronic devices. Herein, a new strategy for fabrication the NiV2O6 nanosphere by hydrothermal combined with subsequent room temperature liquid phase synthesis method was investigated. The introduced NH4+ during the preparation is benefit to attain the NiV2O6 architectures with high electrical conductivity, large surface area, and exclusive porous networks. When applied as active electrode materials for electrochemical energy storage device, NiV2O6 gives rise to superior specific capacity (565.5 C/g at 1 A/g) and cyclic stability (84.6% of initial capacity retention after 3000 cycles). An asymmetric supercapacitor device can reach voltagewindow of 1.6 V and supply an energy density of 24.3 Wh kg-1 at the power density 800 W kg-1 in an aqueous electrolyte and 7.8 W h kg-1 at the power density of 850 W kg-1 in a solid state gel electrolyte. The cyclic stability of flexible asymmetric supercapacitor has provided 74% capacitance retention over 3000 charge-discharge cycles. Thus, the NiV2O6 nanosphere electrode presents a great potential for flexible and transparent energy storage devices.

The widespread use of metal-air batteries for mobile applications will require reduced Pt loading and enhanced catalytic activity within their cathodes to drive the sluggish oxygen reduction reaction (ORR). Despite tremendous efforts, however, elegant bottom-up synthesis of practical nanomaterials remains highly challenging. Here, we propose “in-situ observation” as a novel descriptor to nicely bridge the adsorbates coverage and the oxygen reduction activity of Pt-based catalysts. In-situ X-ray powder diffraction characterization directly observed the ORR process and the evident change of the (111) peak intensity shows continuous oxygen adsorption on the catalyst surface. As expected, the catalyst exhibiting the greatest intensity change delivers the best oxygen reduction activity with a half-wave potential of 0.92 ​V versus reversible hydrogen electrode and an excellent bifunctional performance with a small potential difference of 0.75 ​V. Once assembled as the cathode catalyst, both aqueous and all-solid-state zinc-air battery illustrate high power density and superior cycling stability, holding great potential as a novel energy supply for wearable electronics. The cutting-edge in-situ observation is thus identified as a valid activity descriptor to guide less empirical synthesis of enhanced ORR electrocatalysts.

New advances in molecular approaches for DNA analysis have enhanced our understanding of the phylogenetic relationship of birds. The Little Owl (Athene noctua) is of great significance for the integrated management of forest diseases and control of regional pests. Here, we sequenced and annotated the 17,772 bp complete mitogenome of A. noctua. The mitogenome encoded 37 typical mitochondrial genes: 13 protein-coding genes, two ribosomal RNA genes, 22 transfer RNA, and one non-coding control region (D-loop). The organization and location of genes in the A. noctua mitogenome were consistent with those reported for other Strigidae birds. Phylogenetic relationships based on Bayesian inference and Maximum likelihood methods showed that A. noctua has close relationships with Athene brama and Glaucidium cuculoides, confirming that A. noctua belongs to the Strigidae family. The phylogenetic relationships among seven genera of the Strigidae family used in this study were: Ninox and the other six genera were far apart, Otus and the clade ((Bubo + Strix) + Asio) were clustered into one branch, and Athene and Glaucidium were clustered into one branch. This phylogenetic classification is consistent with prior taxonomic studies on the Strigidae family. Our results provide new mitogenomic data to support further phylogenetic and taxonomic studies of Strigidae.

The North China Craton (NCC) is an atypical ancient landmass that suffered lithospheric destruction. Previous studies suggest that the eastern part of the lithospheric mantle of the NCC has been thinned and modified in the Mesozoic. However, the initiation time and mechanism of the destruction remain controversial. Mafic magmatism could provide a unique window into deciphering the lithospheric mantle composition and its evolution. Here we present geochemical and geochronological data of the diamond-bearing alkaline basalts from Lan'gan, located in the southeastern margin of the NCC. Zircon U-Pb dating yielded an average age of 174 ± 14 Ma, representing the first reported Jurassic basalts in the eastern NCC. The Lan'gan basalts are enriched in light rare earth elements (LREE) and large ion lithosphile elements (LILE). Sr-Nd-Pb-Hf isotopic compositions (87Sr/86Sr(t) = 0.70646–0.70925, eNd(t) = −2.1 to −4.9, 206Pb/204Pb(t) = 17.14–18.12, 207Pb/204Pb(t) = 15.28–15.61, 208Pb/204Pb(t) = 37.82–38.67, and zircon eHf(t) = −17 to −21) are enriched compared to depleted mantle. The presence of primary amphibole indicates that the magma source of the basalts was water enriched. These observations suggest that, the lithospheric mantle of the eastern NCC were significantly refertilized, likely by slab derived fluids/melts from the Paleo-Pacific subduction. Owing to the Paleo-Pacific subduction, the lithospheric mantle of the eastern NCC were reduced in viscosity and intensity, and finally promoted partial melting in a limited scale to generate the investigated alkaline basalts. Hence, the discovery of diamond in the Lan'gan basalts demonstrates that the lithosphere of the NCC remained thick, and that large-scale destruction had not initiated in the early Jurassic beneath this region.

Transformation of dissolved organic matter (DOM) in petrochemical wastewater (PCW) treatment has rarely been studied. In this work, low- and high-salinity PCW were collected from a treatment plant and the transformations of DOM at molecular level along the treatment processes of both PCW were comparatively investigated. By using Orbitrap MS, the polar DOM constituents were categorized into five molecular classes namely saturated compounds, aliphatics, highly unsaturated and phenolic compounds (Huph), polyphenols and condensed polycyclic aromatics (Cpla). Aliphatics (58.62%) with low molecular weight (150-250 Da) and O/C (0-0.2) were dominant in raw low-salinity PCW; while Huph (65.03%) with O/C at 0.2-0.8 were rich in raw high-salinity PCW. After full-scale treatment, differentiated DOM constituents in both raw PCWs were transformed into aliphatics and Huph with O/C at 0.3-0.5. Anoxic/Oxic treatment of low-salinity system (L-A/O) removed a high fraction of aliphatics (53.05%); while Huph with low O/C (0.1-0.3) (65.68%) in the effluent of L-A/O were further mineralized by ozonation of low-salinity system (L-ozonation). In comparison, anoxic/oxic treatment of high-salinity system (H-A/O) mainly removed unsaturated Huph (34.10%) and aliphatics (30.86%). This resulted in a decrease of dissolved organic carbon as indicated via Spearman correlation. Different from L-ozonation, ozonation of high-salinity system (H-ozonation) degraded aliphatics (26.09%) and Huph (41.85%) with a relatively high O/C (0.2-1.2). After L-A/O and L-ozonation treatments, remaining saturated compounds that were originated from raw low-salinity PCW, were removed by subsequent biological aerated filter. Comparatively, after H-A/O and H-ozonation treatments, residual Huph and aliphatics which were mainly bio-derivates and ozonated intermediates, were further removed by air flotation filter. Hence, DOM transformation of different PCWs along similar treatments varied significantly. This study provides in-depth insights on DOM transformation along a full-scale PCW treatment process.

Profilins are abundant cytosolic proteins that are universally expressed in eukaryotes and that regulate actin filament elongation by binding to both monomeric actin (G-actin) and formin proteins. The atypical profilin Arabidopsis AtPRF3 has been reported to cooperate with canonical profilin isoforms in suppressing formin-mediated actin polymerization during plant innate immunity responses. AtPRF3 has a 37-amino acid-long N-terminal extension (NTE), and its suppressive effect on actin assembly is derived from enhanced interaction with the polyproline (Poly-P) of the formin AtFH1. However, the molecular mechanism remains unclear. Here, we solved the crystal structures of AtPRF3Δ22 and AtPRF3Δ37, as well as AtPRF2 apo form and in complex with AtFH1 Poly-P at 1.5-3.6 A resolutions. By combining these structures with molecular modeling, we found that AtPRF3Δ22 NTE has high plasticity, with a primary "closed" conformation that can adopt an open conformation that enables Poly-P binding. Furthermore, using molecular dynamics simulation and free-energy calculations of protein-protein binding, along with experimental validation, we show that the AtPRF3Δ22 binds to Poly-P in an adaptive manner, thereby enabling different binding modes that maintain the interaction through disordered sequences. Together, our structural and simulation results suggest that the dynamic conformational changes of the AtPRF3 NTE upon Poly-P binding modulate their interactions to fine-tune formin-mediated actin assembly.

The quantity and quality of children's digital screen media exposure is an emerging area of early childhood studies because of its strong social relevance, and this has been particularly true since the COVID-19 pandemic. The few existing parental questionnaires on children's digital screen media exposure mainly focus on monolingual children's media habits and address either the quantity or quality of children's media exposure. Inspired by the existing instruments, the current study introduces a new parental questionnaire to comprehensively assess the duration, frequency, content, design, and use of bilingual children's digital screen media exposure at home, before and since the COVID-19 pandemic. Focus group discussions and the first wave of our data collection on 141 3-6 years old Singaporean bilingual children indicate good face validity and internal consistency of the parental questionnaire. Our results reveal substantial differences in children's quantity and quality of daily digital screen media exposure, as well as the discrepancies in their digital media habits between English and their mother tongue languages, before and since the COVID-19 pandemic.

The plant Bauhinia brachycarpa Benth (BBB) has been traditionally used for treating muscle aches such as bone pain, and neuralgia for a long time, on account of its sedative and antinociceptive activities in Yunnan province of China. However, there was no experimental evidence to confirm its traditional medicinal use.The antinociceptive effect and possible mechanism of ethanolic extract of BBB on neuropathic pain was evaluated through a model of partial sciatic nerve ligation in mice.A commonly employed animal model induced by partial sciatic nerve ligation (PSNL) in mice was established in the aim of studying neuropathic pain. Ethanolic extract of BBB (1000, 500, 250 mg/kg) and pregabalin (60 mg/kg) were intragastric administrated daily for 7 days post-PSNL. Mechanical and thermal hyperalgesia were assessed throughout the experimental period. After the experiment, the levels of tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) in serum were determined by ELISA. The mRNA expressions of ionized calcium binding adaptor molecule 1 (Iba-1), CD16, CD206, arginine-1 (Arg-1), interleukin-1β (IL-1β), and inducible nitric oxide synthase (iNOS) in the spinal cord were detected by qPCR. The protein levels of Iba-1, CD16, CD206, and p38 phosphorylation in the spinal cord were detected by immunohistochemistry and western blotting. The phytochemical analysis of BBB was performed through the colorimetric test.Neuropathic pain induced by PSNL was significantly alleviated by BBB treatment, which decreased pro-inflammatory cytokines such as TNF-α, IL-1β and iNOS, increased anti-inflammatory cytokine such as IL-10 and Arg-1, and attenuated p38 phosphorylation. BBB also reduced the number of Iba-1 and CD16 positive cells, but it enhanced the number of CD206 positive cells. n-Butanol portion that was partitioned from the ethanolic extract had the highest content of total flavonoids among all the portions, and the antinociceptive effect of n-butanol portion is better than that of other portions.Our findings indicate that the antinociceptive effect of BBB is mediated by inhibiting the inflammatory response and regulating the differentiation of microglia. The antinociceptive effect of BBB was related to the content of total flavonoids.

In this study, the binding characteristics of aloe-emodin (AE) and pepsin were investigated via multiple spectroscopic and molecular docking methods. The Stern-Volmer quenching constant (Ksv) suggested that AE quenched the fluorescence intensity of pepsin via static quenching. The value of the stoichiometric binding number (n) suggested only a single binding site on pepsin for AE. The value of Gibbs’ free energy change (ΔG)

Mesoporous cobalt doped nickel vanadate hierarchical microspheres were developed via microwave-assisted approach. Interestingly, the introduce of cobalt renders smooth microspheres transforming into hierarchical microspheres with uniform size self-assembled from nanorods, which displays enhanced specific surface area. Benefitting from the unique morphology and synergistic effect of cobalt and nickel, cobalt doped nickel vanadate hierarchical microspheres exhibit significantly improved performance with a higher rate capability (73.7% at 20 times) and a higher capacitance retention (82.2% after 10000 charges and discharges) compared with pure nickel vanadate. This novel design strategy has great potential for promoting the practical use of electrochemical energy storage devices.

Alkali-activated slag was first used as cementing material in seawater coral aggregate concrete (SCAC) production in this paper. It would contribute to both the environmental protection and raw materials obtained locally in island construction far away from the mainland. However, cracks would be harmful to structural durability of SCAC especially under ocean environment. Thus, the present study is mainly aimed at the fracture properties of alkali-activated slag SCAC (ASSCAC) by virtue of three-point-bending tests. Two types of slag (Types A and B) and two types of coral aggregates, namely columnar and crushed coral coarse aggregates (CCA), are considered. Besides, two beam depths of 100 mm and 150 mm are prepared and the initial notch-to-depth ratios are set from 0.1 to 0.7 for each depth. Results show that the failure modes of all the beams are fracturing of CCAs. Then an analytical approach was proposed to predict the fracture parameters of ASSCAC. The size-independent uniaxial tensile strength and fracture toughness are obtained by using the experimental maximum fracture loads and the former proves to be the maximum tensile stress at the fictitious crack-tip. Then the correlation between the maximum fracture load and the local fracture energy at crack-tip region is obtained. The size-independent fracture energy is derived based on the comparison between the analytical and experimental maximum fracture loads. The tensile strengths of ASSCAC mixed by Type A slag and crushed CCAs are larger. But the fracture toughness and fracture energy are slightly higher when the ASSCAC is made by Type B slag.

In this study, the flow and heat transfer of non-Newtonian Al2O3-water nanofluid in Rayleigh-Benard cavity are numerically studied. Power-law model is used to describe the non-Newtonian effect of nanofluids in which the flow behavior index depends on the nanoparticle volume fraction. We present a systematic study of the Al2O3-water nanofluid for Rayleigh number of 1000 ≤ Ra ≤ 500000, cavity aspect ratio of 0.25 ≤ AR ≤ 4.0 and nanoparticle volume fraction of 0% ≤ ϕ ≤ 3%. The flow pattern inside the cavity changes complicatedly as Ra increases and the heat transfer performance of the system is strongly affected by the flow pattern. The convectional structure is influenced by both of the shear-thinning effect and the variation of effective viscosity. The average Nusselt number changes non-monotonically because of the flow pattern change under the variation of Ra and n. The consideration of Brownian motion and thermophoresis could bring about 3% increase of average Nusselt number, and the value varies slightly with different AR and ϕ . The comparison between non-Newtonian and Newtonian models shows that noticeable underestimation of heat transfer performance could be resulted by Newtonian model. The average Nusselt number of non-Newtonian model is approximately 1.5 times to that of Newtonian model, and the deviation is more pronounced with the increase of particle volume fraction.

Classical swine fever (CSF) remains an important pig disease in China, where it usually presents with mild or atypical clinical manifestations, with large scale outbreaks rarely seen. This has led to speculation about the possible circulation of viral strains of low virulence. To investigate this possibility, five field isolates within the predominant genotype 2 (2.1b, 2.1c, 2.1 h and 2.2) were evaluated and compared by experimental infection of naturally farrowed but colostrum-deprived piglets. All infected piglets displayed clinical signs, including persistent high fever, depression, anorexia, dyspnea, conjunctivitis, constipation, and hesitant gait. Typical pathological lesions, including pulmonary edema, hemorrhagic or cellulosic exudation, and swelling and hemorrhage of lymph nodes, were observed. Viremia and Erns protein expression in the blood of all infected animals were detectable from 3 to 5 days post infection (DPI), their presence correlating with the onset of fever, clinical signs and leukopenia. E2 antibody did not develop in any of the field CSFV-infected piglets during the disease course, while Erns antibody was detectable in 4–56% of infected animals at various time points. Mortalities ranged from 20 to 80% within 21 DPI, progressing to 100% by 43 DPI. Based on clinical scores and fatalities within 21 DPI, 2 of the 5 field isolates were classified as of moderate virulence and 3 of high virulence; i.e., no field isolates of low virulence were identified. The study has provided data supporting the use of these isolates as challenge viruses to evaluate the efficacy of current CSF vaccines.