Toxicity of a novel antifungal agent (ATB1651 gel) in Yucatan minipigs (Sus scrofa) following 4 weeks of daily dermal administration.

ATB1651 gel is an antifungal drug candidate that enhances antifungal activity through substitution of several aryl rings, alkyl chains, and methyl groups. To ensure safety of use of ATB1651 gel, assessment of its potentially toxic side effects is necessary. In this study, we examined the repeated-dose toxicity of ATB1651 gel to Yucatan minipigs (Sus scrofa) in accordance with the Good Laboratory Practice guidelines. Five doses of ATB1651 gel (0%, 0.2%, 0.5%, 1.0%, 3.0%) were administered dermally to the left and right flanks of 38 minipigs daily for 4 weeks. Mortality, clinical symptoms, dermal scores, body weights, and physiological, biochemical, pathological, and toxicokinetic analyses were performed after the treatment period. No systemic toxicological damage was observed in either male or female minipigs regardless of dose; however, dermal application of ATB1651 gel caused some skin alterations at the application sites. Specifically, erythema and eschar formation, edema, and scabs or raise spots were observed at the application site(s) in males in the 3.0% ATB1651 gel treatment group and in females at ATB1651 gel concentrations ≥ 1.0%, with dermal scores ranging from grade 1 to 2. Additionally, histopathological assay indicated infiltration of different types of inflammatory cells and the presence of pustule/crust at the application site(s) in both males and females at ATB1651 gel concentrations ≥ 0.5%. However, these changes were reversible after a 2-week recovery period and were considered a local irritation effect of ATB1651 gel. The no-observed-adverse-effect level of ATB1651 gel was 3.0% with regard to topical and systemic toxicity in both male and female minipigs. Collectively, our results imply that ATB1651 gel is a safe candidate for clinical development as an antifungal drug with a wide therapeutic window.

Casein kinase 2 complex: a central regulator of multiple pathobiological signaling pathways in Cryptococcus neoformans.

The casein kinase 2 (CK2) complex has garnered extensive attention over the past decades as a potential therapeutic target for diverse human diseases, including cancer, diabetes, and obesity, due to its pivotal roles in eukaryotic growth, differentiation, and metabolic homeostasis. While CK2 is also considered a promising antifungal target, its role in fungal pathogens remains unexplored. In this study, we investigated the functions and regulatory mechanisms of the CK2 complex in Cryptococcus neoformans, a major cause of fungal meningitis. The cryptococcal CK2 complex consists of a single catalytic subunit, Cka1, and two regulatory subunits, Ckb1 and Ckb2. Our findings show that Cka1 plays a primary role as a protein kinase, while Ckb1 and Ckb2 have major and minor regulatory functions, respectively, in growth, cell cycle control, morphogenesis, stress response, antifungal drug resistance, and virulence factor production. Interestingly, triple mutants lacking all three subunits (cka1Δ ckb1Δ ckb2Δ) exhibited more severe phenotypic defects than the cka1Δ mutant alone, suggesting that Ckb1/2 may have Cka1-independent functions. In a murine model of systemic cryptococcosis, cka1Δ and cka1Δ ckb1Δ ckb2Δ mutants showed severely reduced virulence. Transcriptomic, proteomic, and phosphoproteomic analyses further revealed that the CK2 complex controls a wide array of effector proteins involved in transcriptional regulation, cell cycle control, nutrient metabolisms, and stress responses. Most notably, CK2 disruption led to dysregulation of key signaling cascades central to C. neoformans pathogenicity, including the Hog1, Mpk1 MAPKs, cAMP/PKA, and calcium/calcineurin signaling pathways. In summary, our study provides novel insights into the multifaceted roles of the fungal CK2 complex and presents a compelling case for targeting it in the development of new antifungal drugs.IMPORTANCEThe casein kinase 2 (CK2) complex, crucial for eukaryotic growth, differentiation, and metabolic regulation, presents a promising therapeutic target for various human diseases, including cancer, diabetes, and obesity. Its potential as an antifungal target is further highlighted in this study, which explores CK2's functions in C. neoformans, a key fungal meningitis pathogen. The CK2 complex in C. neoformans, comprising the Cka1 catalytic subunit and Ckb1/2 regulatory subunits, is integral to processes like growth, cell cycle, morphogenesis, stress response, drug resistance, and virulence. Our findings of CK2's role in regulating critical signaling pathways, including Hog1, Mpk1 MAPKs, cAMP/PKA, and calcium/calcineurin, underscore its importance in C. neoformans pathogenicity. This study provides valuable insights into the fungal CK2 complex, reinforcing its potential as a target for novel antifungal drug development and pointing out a promising direction for creating new antifungal agents.

Investigation of Neuromodulatory Effect of Anodal Cerebellar Transcranial Direct Current Stimulation on the Primary Motor Cortex Using Functional Near-Infrared Spectroscopy.

Cerebellar brain inhibition (CBI), a neural connection between the cerebellum and primary motor cortex (M1), has been researched as a target pathway for neuromodulation to improve clinical outcomes in various neurological diseases. However, conflicting results of anodal cerebellar transcranial direct current stimulation (acb-tDCS) on M1 excitability indicate that additional investigation is required to examine its precise effect. This study aimed to gather evidence of the neuromodulatory effect of acb-tDCS on the M1 using functional near-infrared spectroscopy (fNIRS). Sixteen healthy participants were included in this cross-over study. Participants received real and sham acb-tDCS randomly, with a minimum 1-week washout period between them. The anode and cathode were placed on the right cerebellum and the right buccinator muscle, respectively. Stimulation lasted 20 min at an intensity of 2 mA, and fNIRS data were recorded for 42 min (including a 4-min baseline before stimulation and an 18-min post-stimulation duration) using eight channels attached bilaterally on the M1. acb-tDCS induced a significant decrease in oxyhemoglobin (HbO) concentration (inhibitory effect) in the left (contralateral) M1, whereas it induced a significant increase in HbO concentration (excitatory effect) in the right (ipsilateral) M1 compared to sham tDCS during (p < 0.05) and after stimulation (p < 0.01) in a group level analysis. At the individual level, variations in response to acb-tDCS were observed. Our findings demonstrate the neuromodulatory effects of acb-tDCS on the bilateral M1 in terms of neuronal hemodynamics.

The effect of deep brain structure modeling on transcranial direct current stimulation-induced electric fields: An in-silico study.

To study transcranial direct current stimulation (tDCS) and its effect on the brain, it could be useful to predict the distribution of the electric field induced in the brain with given tDCS parameters. As a solution, simulation with realistic computational models using magnetic resonance images (MRIs) have been widely used in the fields. With the recent advance of deep learning-based segmentation techniques of the brain, questions have been raised about if tDCS-induced electric field is affected by the deep brain structures. This study aimed to investigate the effect of the deep brain structure modeling on the induced electric field. To this end, we generated models with and without the deep brain structures by using an open MRI dataset comprising tDCS parameters, electric field simulation results and in-vivo intracranial recordings in the deep brain structures. We investigated the difference between the simulation results of the two models with a statistical analysis. Our results indicated that tDCS-induced electric fields and current flow in the brain are significantly different when the deep brain structures are considered.

Adenylyl-Sulfate Kinase (Met14)-Dependent Cysteine and Methionine Biosynthesis Pathways Contribute Distinctively to Pathobiological Processes in Cryptococcus neoformans.

Blocking of nutrient uptake and amino acid biosynthesis are considered potential targets for next-generation antifungal drugs against pathogenic fungi, including Cryptococcus neoformans. In this regard, the sulfate assimilation pathway is particularly attractive, as it is only present in eukaryotes such as plants and fungi, yet not in mammals. Here, we demonstrated that the adenylyl sulfate kinase (Met14) in the sulfate assimilation pathway is not essential yet is required for the viability of C. neoformans due to its involvement in biosynthesis of two sulfur-containing amino acids, cysteine and methionine. Met14-dependent cysteine and methionine biosynthesis was found to significantly contribute to a diverse range of pathobiological processes in C. neoformans. Met14-dependent cysteine rather than methionine biosynthesis was also found to play pivotal roles in cell growth and tolerance to environmental stresses and antifungal drugs. In contrast, the Met14-dependent methionine biosynthesis was found to be more important than cysteine biosynthesis for the production of major cryptococcal virulence factors of melanin pigments and polysaccharide capsules. Finally, we also found that despite its attenuated virulence in an insect model, Galleria mellonella, the met14Δ mutant yielded no difference in virulence in a murine model of systemic cryptococcosis. Hence, clinical inhibition of Met14-dependent amino acid biosynthetic pathways may not be advantageous for the treatment of systemic cryptococcosis. IMPORTANCE Current antifungal drugs have several limitations, such as drug resistance, severe side effects, and a narrow spectrum. Therefore, novel antifungal targets are urgently needed. To this end, fungal sulfur amino acid biosynthetic pathways are considered potential targets for development of new antifungal agents. Here, we demonstrated that Met14 in the sulfate assimilation pathway promotes growth, stress response, and virulence factor production in C. neoformans via synthesis of sulfur-containing amino acids methionine and cysteine. Met14-dependent cysteine rather than methionine synthesis was found to be critical for growth and stress responses, whereas Met14-dependent methionine synthesis was more important for the production of antiphagocytic capsules and antioxidant melanin in C. neoformans. Surprisingly, deletion of the MET14 gene was found to attenuate cryptococcal virulence in an insect model, yet not in a murine model. Collectively, our results showed that Met14-dependent cysteine and methionine biosynthesis play roles that are distinct from each other in C. neoformans. Moreover, Met14 is unlikely to be a suitable anticryptococcal drug target.

Fine-grained brain tissue segmentation for brain modeling of stroke patient.

Brain segmentation of stroke patients can facilitate brain modeling for electrical non-invasive brain stimulation, a therapy for stimulating brain function using an electric current. However, it remains challenging owing to its time-consuming, labor-dependent, and complicated pipeline. In addition, conventional tools that define lesions into one region rather than distinguishing between the stroke-affected regions and cerebrospinal fluid can lead to inaccurate treatment results. In this study, we first define a novel stroke-affected region as a detailed sub-region of the conventionally defined lesion. Subsequently, a novel comprehensive framework is proposed to segment head-brain and fine-level stroke-affected regions for normal controls and chronic stroke patients. The proposed framework consists of a time-efficient and precise deep learning-based segmentation model. The experiment results indicate that the proposed method perform better than the conventional deep learning-based segmentation model in terms of the evaluation metrics. The proposed method would be a valuable addition to brain modeling for non-invasive neuromodulation.

The novel antifungal agent AB-22 displays in vitro activity against hyphal growth and biofilm formation in Candida albicans and potency for treating systemic candidiasis.

Systemic candidiasis, which is mainly caused by Candida albicans, is a serious acute fungal infection in the clinical setting. In a previous study, we reported that compound 22h (designated as AB-22 in this study), a vinyl sulfate compound, is a fast-acting fungicidal agent against a broad spectrum of fungal pathogens. In this study, we aimed to further analyze the in vitro and in vivo efficacy of AB-22 against filamentation, biofilm formation, and virulence of C. albicans. Under in vitro hyphal growth-inducing condition, AB-22 effectively inhibited germ tube formation and hyphal growth, which are required for the initiation of biofilm formation. Indeed, AB-22 significantly suppressed C. albicans biofilm formation in a dose-dependent manner. Moreover, AB-22 treatment inhibited the normal induction of ALS3, HWP1, and ECE1, which are all required for hyphal transition in C. albicans. Furthermore, AB-22 treatment increased the survival of mice systemically infected with C. albicans. In conclusion, in addition to its fungicidal activity, AB-22 inhibits filamentation and biofilm formation in C. albicans, which could collectively contribute to its potent in vivo efficacy against systemic candidiasis.

Functional Network Changes After High-Frequency rTMS Over the Most Activated Speech-Related Area Combined With Speech Therapy in Chronic Stroke With Non-fluent Aphasia.

OBJECTIVE: High-frequency repetitive transcranial magnetic stimulation (HF-rTMS) to the lesional hemisphere requires prudence in selecting the appropriate stimulation spot. Functional near-IR spectroscopy (fNIRS) can be used in both selecting the stimulation spot and assessing the changes of the brain network. This study aimed to evaluate the effect of HF-rTMS on the most activated spot identified with fNIRS and assess the changes of brain functional network in the patients with poststroke aphasia. METHODS: A total of five patients received HF-rTMS to the most activated area on the lesional hemisphere, followed by 30 min of speech therapy for 10 days. The Korean version of the Western aphasia battery (K-WAB) and fNIRS evaluation were done 1 day before the treatment, 1 day and 1 month after the last treatment session. Changes of K-WAB and paired cortical interaction and brain network analysis using graph theory were assessed. RESULTS: Aphasia quotient in K-WAB significantly increased after the treatment (P = 0.043). The correlation analysis of cortical interactions showed increased connectivity between language production and processing areas. Clustering coefficients of the left hemisphere were increased over a sparsity range between 0.45 and 0.58 (0.015 < p < 0.031), whereas the clustering coefficients of the right hemisphere, decreased over a sparsity range 0.15-0.87 (0.063 < p < 0.095). The global efficiency became lower over a network sparsity range between 0.47 and 0.75 (0.015 < p < 0.063). CONCLUSION: Improvement of language function and changes of corticocortical interaction between language-related cortical areas were observed after HF-rTMS on the most activated area identified by fNIRS with combined speech therapy in the patients with poststroke aphasia.

Feasibility study of immersive virtual prism adaptation therapy with depth-sensing camera using functional near-infrared spectroscopy in healthy adults.

Prism Adaptation (PA) is used to alleviate spatial neglect. We combined immersive virtual reality with a depth-sensing camera to develop virtual prism adaptation therapy (VPAT), which block external visual cues and easily quantify and monitor errors than conventional PA. We conducted a feasibility study to investigate whether VPAT can induce behavioral adaptations by measuring after-effect and identifying which cortical areas were most significantly activated during VPAT using functional near-infrared spectroscopy (fNIRS). Fourteen healthy subjects participated in this study. The experiment consisted of four sequential phases (pre-VPAT, VPAT-10°, VPAT-20°, and post-VPAT). To compare the most significantly activated cortical areas during pointing in different phases against pointing during the pre-VPAT phase, we analyzed changes in oxyhemoglobin concentration using fNIRS during pointing. The pointing errors of the virtual hand deviated to the right-side during early pointing blocks in the VPAT-10° and VPAT-20° phases. There was a left-side deviation of the real hand to the target in the post-VPAT phase, demonstrating after-effect. The most significantly activated channels during pointing tasks were located in the right hemisphere, and possible corresponding cortical areas included the dorsolateral prefrontal cortex and frontal eye field. In conclusion, VPAT may induce behavioral adaptation with modulation of the dorsal attentional network.

Optimization and Evaluation of Novel Antifungal Agents for the Treatment of Fungal Infection.

Due to the increased morbidity and mortality by fungal infections and the emergence of severe antifungal resistance, there is an urgent need for new antifungal agents. Here, we screened for antifungal activity in our in-house library through the minimum inhibitory concentration test and derived two hit compounds with moderate antifungal activities. The hit compounds' antifungal activities and drug-like properties were optimized by substituting various aryl ring, alkyl chain, and methyl groups. Among the optimized compounds, 22h was the most promising candidate with good drug-like properties and exhibited potent fast-acting fungicidal antifungal effects against various fungal pathogens and synergistic antifungal activities with some known antifungal drugs. Additionally, 22h was further confirmed to disturb fungal cell wall integrity by activating multiple cell wall integrity pathways. Furthermore, 22h exerted significant antifungal efficacy in both the subcutaneous infection mouse model and ex vivo human nail infection model.

Fungal kinases and transcription factors regulating brain infection in Cryptococcus neoformans.

Cryptococcus neoformans causes fatal fungal meningoencephalitis. Here, we study the roles played by fungal kinases and transcription factors (TFs) in blood-brain barrier (BBB) crossing and brain infection in mice. We use a brain infectivity assay to screen signature-tagged mutagenesis (STM)-based libraries of mutants defective in kinases and TFs, generated in the C. neoformans H99 strain. We also monitor in vivo transcription profiles of kinases and TFs during host infection using NanoString technology. These analyses identify signalling components involved in BBB adhesion and crossing, or survival in the brain parenchyma. The TFs Pdr802, Hob1, and Sre1 are required for infection under all the conditions tested here. Hob1 controls the expression of several factors involved in brain infection, including inositol transporters, a metalloprotease, PDR802, and SRE1. However, Hob1 is dispensable for most cellular functions in Cryptococcus deuterogattii R265, a strain that does not target the brain during infection. Our results indicate that Hob1 is a master regulator of brain infectivity in C. neoformans.

Requirement for Head Magnetic Resonance Imaging in Children Who Present to the Emergency Department With Acute Nontraumatic Visual Disturbance.

OBJECTIVES: This study aimed to investigate the clinical features and head magnetic resonance imaging (MRI) findings in children who presented to the emergency department with acute nontraumatic visual disturbance and to study related clinical factors for discovering positive lesions on head MRI. METHODS: We performed a retrospective study of 1-month to 15-year-old children who underwent head MRI as an evaluation for acute nontraumatic visual disturbance as a chief complaint in our pediatric emergency department between March 2010 and March 2015. The symptoms of visual disturbance were blurred vision, diplopia, loss of vision, and visual hallucination. Head MRI findings were considered positive when lesions could explain the symptoms. RESULTS: We identified 39 patients (25 with blurred vision, 9 with diplopia, 3 with loss of vision, and 2 with visual hallucination) with a mean age of 8.35 ± 4.06 years. Positive head MRI findings were identified in 13 patients (33.3%). Brain tumors were most common (53.8%), followed by optic nerve inflammations (23.1%), congenital brain lesions (15.4%), and hypertensive encephalopathy (7.7%). Compared with the negative head MRI group, the positive head MRI group showed significantly less transient visual disturbance (duration <1 hour to complete recovery) (P = 0.001), more limited eye movement (P = 0.003), and more pupillary abnormalities (P = 0.030). CONCLUSIONS: We suggest performing urgent head MRI in children with acute nontraumatic visual disturbance if the symptoms last longer than 1 hour without complete recovery and are accompanied by limited eye movement or pupillary abnormality.

A multichannel-near-infrared-spectroscopy-triggered robotic hand rehabilitation system for stroke patients.

There is a demand for a new neurorehabilitation modality with a brain-computer interface for stroke patients with insufficient or no remaining hand motor function. We previously developed a robotic hand rehabilitation system triggered by multichannel near-infrared spectroscopy (NIRS) to address this demand. In a preliminary prototype system, a robotic hand orthosis, providing one degree-of-freedom motion for a hand's closing and opening, is triggered by a wireless command from a NIRS system, capturing a subject's motor cortex activation. To examine the feasibility of the prototype, we conducted a preliminary test involving six neurologically intact participants. The test comprised a series of evaluations for two aspects of neurorehabilitation training in a real-time manner: classification accuracy and execution time. The effects of classification-related factors, namely the algorithm, signal type, and number of NIRS channels, were investigated. In the comparison of algorithms, linear discrimination analysis performed better than the support vector machine in terms of both accuracy and training time. The oxyhemoglobin versus deoxyhemoglobin comparison revealed that the two concentrations almost equally contribute to the hand motion estimation. The relationship between the number of NIRS channels and accuracy indicated that a certain number of channels are needed and suggested a need for a method of selecting informative channels. The computation time of 5.84 ms was acceptable for our purpose. Overall, the preliminary prototype showed sufficient feasibility for further development and clinical testing with stroke patients.

Registration using 3D-printed rigid templates outperforms manually scanned surface matching in image-guided temporal bone surgery.

PURPOSE: Image-guided surgery (IGS) for otological procedures requires minimal invasiveness and a high degree of accuracy. We have recently developed a noninvasive registration method, the Surface Template-Assisted Marker Positioning (STAMP) method, which uses a rigid template of the surface of the temporal bone. However, the STAMP method is not applicable when the bony surface is not exposed, such as in endoscopic surgery. Thus, we extended our research to apply the STAMP method onto the skin and tested its feasibility in this study. METHODS: We designed a phantom made of a rigid box and soft material for the study. The target registration error (TRE) was measured at preset measuring points in the phantom. We modified the STAMP method to be applicable for use on the skin around the ears (S-STAMP). The same phantom was also registered using the conventional, manually scanned surface matching method. We compared the TRE after the different registration methods. RESULTS: The TRE after the S-STAMP registration method was significantly smaller than that of the conventional surface matching method at all error measurement points in the phantom. However, the TRE after the S-STAMP registration method was significantly larger than that of paired point registration using invasive fiducial markers. CONCLUSIONS: The S-STAMP method using a rigid template on the soft surface yields a significantly smaller TRE than that of conventional, manually scanned surface matching registration. This strategy provides an alternative option to improve the accuracy of IGS without loading patients with additional invasive procedures.

Children Experiencing First-Time or Prolonged Febrile Seizure Are Prone to Stress Hyperglycemia.

The risk factors and clinical implications of stress hyperglycemia in children with febrile seizure remain uncertain. Among 479 children with febrile seizure, the prevalence of the stress hyperglycemia (blood glucose concentration ≥ 150 mg/dL) was 10.0%. Stress hyperglycemia group included larger proportion of first-time febrile seizure, prolonged febrile seizure, and smaller proportion of short febrile seizure in comparison with the non-stress hyperglycemia group. Stress hyperglycemia group demonstrated a lower pH and higher lactate levels than the non-stress hyperglycemia group. Multivariate analysis revealed that first-time febrile seizure (aOR = 3.741, P = .004) and prolonged febrile seizure (aOR = 12.855, P < .001) were significant risk factors for stress hyperglycemia. The rate of early febrile seizure recurrence in the emergency department was not different between the groups. These findings suggest that children experiencing first-time or prolonged febrile seizure are prone to stress hyperglycemia, and this can be related to febrile seizure severity. However, stress hyperglycemia is not predictive of early febrile seizure recurrence in the emergency department.

Missed clavicle fractures on anterior-posterior views of skull X-rays: a retrospective, observational, and descriptive study.

OBJECTIVE: The clavicle is almost always seen in skull X-rays of infants. The objectives of this study were to determine how often the clavicle and clavicle fractures are visible but missed on the skull anterior-posterior view (skull AP) of infants and which factors are associated with missing the diagnosis. METHODS: We retrospectively studied patients aged 1 year or younger who had a skull AP taken for any injury survey at a single urban, academic hospital between April 1999 and July 2012. Outcomes of interest were the numbers and percentages of visible clavicles; clavicle fractures, including missed ones on skull AP; and the factors associated with missing the diagnosis of a clavicle fracture. RESULTS: Both clavicles were visible in 734 patients (89.6%). Of these, 10 patients (1.4%) had confirmed clavicle fractures, and 6 patients (0.8%) had fractures that were missed at presentation. Although we tried to determine the factors that might be associated with missed diagnoses, including age <6 months, male sex, blocking by guardian's hands, associated skull fractures, and mechanism of injury, none was significantly associated with missed clavicle fractures. CONCLUSION: The clavicles were recognizable on skull X-rays in most cases. Therefore, one should check the clavicles when reading skull X-rays.

Phylogenetic and recombination analysis of tomato spotted wilt virus.

Tomato spotted wilt virus (TSWV) severely damages and reduces the yield of many economically important plants worldwide. In this study, we determined the whole-genome sequences of 10 TSWV isolates recently identified from various regions and hosts in Korea. Phylogenetic analysis of these 10 isolates as well as the three previously sequenced isolates indicated that the 13 Korean TSWV isolates could be divided into two groups reflecting either two different origins or divergences of Korean TSWV isolates. In addition, the complete nucleotide sequences for the 13 Korean TSWV isolates along with previously sequenced TSWV RNA segments from Korea and other countries were subjected to phylogenetic and recombination analysis. The phylogenetic analysis indicated that both the RNA L and RNA M segments of most Korean isolates might have originated in Western Europe and North America but that the RNA S segments for all Korean isolates might have originated in China and Japan. Recombination analysis identified a total of 12 recombination events among all isolates and segments and five recombination events among the 13 Korea isolates; among the five recombinants from Korea, three contained the whole RNA L segment, suggesting reassortment rather than recombination. Our analyses provide evidence that both recombination and reassortment have contributed to the molecular diversity of TSWV.

Highly Specific Detection of Five Exotic Quarantine Plant Viruses using RT-PCR.

To detect five plant viruses (Beet black scorch virus, Beet necrotic yellow vein virus, Eggplant mottled dwarf virus, Pelargonium zonate spot virus, and Rice yellow mottle virus) for quarantine purposes, we designed 15 RT-PCR primer sets. Primer design was based on the nucleotide sequence of the coat protein gene, which is highly conserved within species. All but one primer set successfully amplified the targets, and gradient PCRs indicated that the optimal temperature for the 14 useful primer sets was 51.9°C. Some primer sets worked well regardless of annealing temperature while others required a very specific annealing temperature. A primer specificity test using plant total RNAs and cDNAs of other plant virus-infected samples demonstrated that the designed primer sets were highly specific and generated reproducible results. The newly developed RT-PCR primer sets would be useful for quarantine inspections aimed at preventing the entry of exotic plant viruses into Korea.

Complete genome sequences of three tomato spotted wilt virus isolates from tomato and pepper plants in Korea and their phylogenetic relationship to other TSWV isolates.

Tomato spotted wilt virus (TSWV) infects numerous host plants and has three genome segments, called L, M and S. Here, we report the complete genome sequences of three Korean TSWV isolates (TSWV-1 to -3) infecting tomato and pepper plants. Although the nucleotide sequence of TSWV-1 genome isolated from tomato is very different from those of TSWV-2 and TSWV-3 isolated from pepper, the deduced amino acid sequences of the five TSWV genes are highly conserved among all three TSWV isolates. In phylogenetic analysis, deduced RdRp protein sequences of TSWV-2 and TSWV-3 were clustered together with two previously reported isolates from Japan and Korea, while TSWV-1 grouped together with a Hawaiian isolate. A phylogenetic tree based on N protein sequences, however, revealed four distinct groups of TSWV isolates, and all three Korean isolates belonged to group II, together with many other isolates, mostly from Europe and Asia. Interestingly, most American isolates grouped together as group I. Together, these results suggested that these newly identified TSWV isolates might have originated from an Asian ancestor and undergone divergence upon infecting different host plants.