First report of konjac mosaic virus in Spuriopimpinella brachycarpa in Korea.

Spuriopimpinella brachycarpa Nakai (Common name, Chamnamul; family Apiaceae) is a plant whose leaves are consumed as a vegetable and used as a folk medicine in Korea (Kim et al., 2020). In February 2020, seven samples of S. brachycarpa leaf showing virus symptoms including yellowing, vein chlorosis, chlorotic lesions, and severe mottling were collected from a greenhouse in Busan, South Korea, to diagnose the potential disease (Fig. S1a, b). The disease incidence rate in the greenhouse was >10% (2,970 m2). To identify the causal virus, we analyzed leaf dip preparation and thin sections of the symptomatic leaves by transmission electron microscopy. Filamentous virus particles and pinwheel structures were observed, indicating the presence of a potyvirus (Fig. S1c, d). To confirm these results, the symptomatic leaf samples were further analyzed by reverse-transcription polymerase chain reaction (RT-PCR) using potyvirus universal primers (Table S2) and direct sequencing of the PCR products. All samples were positive for konjac mosaic virus (KoMV). To exclude the possibility of infection by multiple viruses, we performed high-throughput sequencing (HTS) on an Illumina NovaSeq 6000 system (Macrogen Inc., Seoul, South Korea). There were two contigs (9,267 and 2,851 nt) mapping to KoMV sequences. A large contig (9,267 nt; 705,967 mapped reads; mean read coverage of 11,351.4x) showed about 80% identity (93% coverage) with KoMV-F (GenBank accession no. NC_007913) isolated from Amorphophallus konjac in Japan (Nishiguchi et al., 2006). To isolate KoMV from S. brachycarpa, we mechanically inoculated leaf extracts from symptomatic samples onto Chenopodium quinoa as an assay host via three single-lesion passages, followed by propagation in Nicotiana benthamiana. In a bioassay of the KoMV isolate (KoMV-BS), we mechanically inoculated sap from infected N. benthamiana onto 31 indicator plants including Cryptotaenia japonica (Apiaceae), which is similar to S. brachycarpa (Table S3). KoMV-BS systemically induced vein chlorosis and/or leaf mottling in four Nicotiana species and C. japonica, and chlorotic local lesions in upper leaves of C. quinoa; no symptoms were observed in 25 other indicator plants. These results were confirmed by RT-PCR. Next, we obtained the complete genome sequence of KoMV-BS using HTS and 5' and 3' rapid amplification of cDNA ends, with newly designed primers (Table S2). The assembled full-length KoMV-BS genome sequence was 9,392 nt in length, excluding the poly(A) tail, and encoded a polyprotein composed of 3,060 amino acids. The sequence was deposited in GenBank (accession no. OR001914). BLAST analysis showed 84~88% and 90~98% identities at CP nucleotide and amino acid levels, respectively with the reported KoMV isolates, confirming the virus to be an isolate of KoMV (synonym; Japanese hornwort mosaic virus, zantedeschia mosaic virus) (Adams et al., 2005; Nishiguchi et al., 2006). KoMV infection was first reported in A. konjac from Japan (Shimoyama et al. 1992) and has been spread worldwide as one of the major causal agents of viral diseases in calla lily (Liao et al., 2020). To the best of our knowledge, this is the first report of KoMV infection in S. brachycarpa. To date, cucumber mosaic virus and tobacco mosaic virus have been reported to infect S. brachycarpa in Korea (Yoon et al., 2016; 2017). Our findings will be helpful for developing virus-management strategies to prevent yield and quality loss in S. brachycarpa.

First report of Ranunculus mild mosaic virus in Ranunculus asiaticus in Korea.

Ranunculus (Ranunculus asiaticus L.) is a popular ornamental plant mainly cultivated for cut flowers and flowering potted plants. In January 2021, a leaf sample of R. asiaticus that showed virus-like symptoms including mosaic, yellowing and malformation on leaves was collected from a greenhouse in Jangheung, South Korea for disease diagnosis (Fig. S1). Disease incidence was greater than 30% in the greenhouse (~1,000 m2). Transmission electron microscopy (TEM) of symptomatic leaves identified potyvirus-like filamentous virus particles of about 800 nm. To confirm the TEM results, a symptomatic leaf sample was further analyzed by reverse-transcription polymerase chain reaction (RT-PCR) using species-specific detection primers for six potyviruses that infect R. asiaticus (Sacco et al., 2018). The sample was positive only for ranunculus mild mosaic virus (RanMMV). Additional analysis of nine symptomatic R. asiaticus plants from the infected greenhouse found that all samples were positive for RanMMV. To exclude the presence of the other viruses, next generation sequencing (NGS) was carried out. Total RNA was extracted from symptomatic leaves using the RNeasy Plant Mini Kit (Qiagen, Germany) and a transcriptome library was generated using the TruSeq Stranded Total RNA LT Sample Prep kit (Illumina, San Diego, CA) acccording to the recommended protocol. NGS was performed using an Illumina NovaSeq 6000 system (Macrogen Inc., Korea). A total of 75.58 million reads were obtained, and the reads were de novo assembled to contigs using Trinity software (Grabherr et al., 2011). BLASTn and BLASTx analysis of the contigs against the NCBI viral reference database identified the assembled large contig of 9,539 nt (5,321 mapped reads, mean read coverage of 84.2 times) as RanMMV. This sequence shared 98% nt identity (99% coverage) with the RanMMV NL isolate (acc. no. LC604020) isolated from an anemone plant (A. blanda cv. Charmer) from Netherlands. To obtain the complete genome sequence, the termini sequences were determined by 5' and 3' rapid amplification of cDNA ends (RACE) methods as reported recently (Imamura et al., 2021). The assembled full-length genome sequence of RanMMV-JH is 9,574 nt in length, excluding the poly(A) tail, and encoding a polyprotein of 3,074aa. The sequence was deposited in GenBank under the accession no. OL742438. RanMMV is transmitted by aphids in a nonpersistent manner and has very narrow host range. RanMMV, one of causative agents of ranunculus mosaic disease, has been problematic in ranunculus production area of Japan (Hayahi et al., 2018; Kamikawa et al., 2022). Recently, some perennial weeds from the Ranunculaceae family (e.g. R. japonicus, R. silerifolius and R. tachiroei) are known to may act as a virus reservoir (Kamikawa et al., 2022). As R. asiaticus is cultivated by vegetative propagation, there is need to develop certification system for producing virus-free R. asiaticus. To our knowledge, this is the first report of RanMMV infection in R. asiaticus in Korea.

First report of cucurbit chlorotic yellows virus infecting cucumber in South Korea.

In October 2018, cucumber plants showing yellowing and chlorotic mottle symptoms were observed in a greenhouse in Chungbuk, South Korea. The observed symptoms were similar to those caused by cucurbit aphid-borne yellows virus (CABYV), which has been detected on cucumber plants in the region since it was reported on melon in Korea in 2015 (Lee et al 2015). To identify the potential agents causing these symptoms, 28 samples from symptomatic leaves and fruit of cucumber plants were subjected to total RNA extraction using the Plant RNA Prep Kit (Biocubesystem, Korea). Reverse transcription polymerase chain (RT-PCR) was performed on total RNA using CABYV specific primers and protocols (Kwak et al. 2018). CABYV was detected in 17 of the 28 samples, while 11 symptomatic samples tested negative. In order to identify the cause of the symptoms, RT-PCR was performed using cucurbit chlorotic yellows virus (CCYV) and cucurbit yellow stunting disorder virus (CYSDV) specific primers (Wintermantel et al. 2019). Eight of the 28 samples were positive using the CCYV specific primers while seven samples were infected with only CCYV and one contained a mixed infection of CABYV with CCYV. None of the samples tested positive for CYSDV. The expected 373 nt amplicons of CCYV were bi-directionally sequenced, and BLASTn analysis showed that the nucleotide sequences shared 98 to 100% identity with CCYV isolates from East Asia, including NC0180174 from Japan. Two pairs of primers for amplification of the complete coat protein and RNA-dependent RNA polymerase (RdRp) genes (Wintermantel et al., 2019) were used to amplify the 753bp coat protein and 1517bp RdRp genes, respectively. Amplicons of the expected sizes were obtained from a CCYV single infection and ligated into the pGEM T- Easy vector (Promega, WI, USA). Three clones from each amplicon were sequenced and aligned using Geneious Prime and found to have identical sequences (Genbank accession nos. MW033300, MW033301). The CP and RdRp sequences demonstrated 99% nucleotide and 100% amino acid identity with the respective genes and proteins of the CCYV isolates from Japan. This study documents the first report of CCYV in Korea. Since CCYV was first detected on melon in Japan, it has been reported in many other countries including those in East Asia, the Middle East, Southern Europe, North Africa, and recently in North America. CCYV has the potential to become a serious threat to production of cucurbit crops in Korea, particularly due to the increasing prevalence of the whitefly, Bemisia tabaci, in greenhouse production systems. It will be important to continue monitoring for CCYV and determine potential alternate hosts in the region to manage and prevent further spread of CCYV in Korea.

First report of Colombian datura virus in Brugmansia suaveolens in Korea.

Brugmansia suaveolens, known as angel's trumpet, is a perennial ornamental shrub in the Solanaceae with large fragrant flowers. In June 2018, a leaf sample of B. suaveolens that showed virus-like symptoms including chlorotic spots, yellowing and mottle on leaves was collected from a greenhouse in Seongnam, South Korea for disease diagnosis (Supplementary Figure S1a, b). Disease incidence in the greenhouse was greater than 80% for about 2,000 B. suaveolens plants. To identify a causal virus, transmission electron microscopy (TEM) was used to analyze symptomatic leaf samples using leaf dips and thin section methods. Filamentous virus particles and pinwheel structures were observed, indicating the presence of a potyvirus (Supplementary Figure S1c, d). To confirm the TEM results, a symptomatic leaf sample was further analyzed by reverse-transcription polymerase chain reaction (RT-PCR) using species-specific detection primers for three potyviruses that infect Brugmansia spp.: Colombian datura virus (CDV), Brugmansia mosaic virus (BruMV), and Brugmansia suaveolens mottle virus (BsMoV) (Lucinda et al, 2008; Park et al., 2014; Verma et al., 2014). The sample was positive only for CDV. CDV is transmitted by aphids in a nonpersistent manner and mechanical inoculation and can infect plants in the Solanaceae family including tomato and tobacco (Kahn and Bartels 1968; Schubert et al. 2006; Verhoeven et al. 1996) and has been designated a quarantine virus in Korea. Additional analysis of 13 symptomatic B. suaveolens plants from the infected greenhouse found that all samples except one were infected with CDV. To isolate CDV from B. suaveolens, leaf extracts from symptomatic samples were mechanically inoculated on an assay host, Nicotiana tabacum cv. BY via three single-lesion passages followed by propagation in N. benthamiana. For the bioassay of the CDV isolate (CDV-AT-Kr), sap from infected N. benthamiana was mechanically inoculated on 31 indicator plants, including B. suaveolens (Supplementary Table S2). CDV-AT-Kr induced chlorotic local lesions, necrotic local lesions, mottle, and/or mosaic systemically in 10 Nicotiana spp., and mottle and yellowing in tomato. On inoculated B. suaveolens, te mild mottle symptom was reproduced. No symptoms were observed in pepper or Datura stramonium. These results were confirmed by RT-PCR. To characterize CDV-AT-Kr genetically, the complete genome sequence of CDV-AT-Kr was obtained by RT-PCR using specific primers (Supplementary Table S3) and deposited in GenBank (accession no. MW075268). The CDV-AT-Kr RNA consists of 9,620 nt, encoding a polyprotein of 3,076 aa. BLASTn analysis showed that CDV-AT had maximum nucleotide identities of 98.9% at the complete genome level with a CDV isolate (accession no. JQ801448) from N. tabacum in the UK. To our knowledge, this is the first report of CDV infection in B. suaveolens in Korea and the second report in the world of the complete genome sequence. As B. suaveolens is cultivated by vegetative propagation, production and maintenance of virus-free, healthy B. suaveolens is needed. In addition, as new CDV hosts have been repeatedly reported (Pacifico et al., 2016; Salamon et al., 2015; Tomitaka et al., 2014; Verma et al., 2014), we are monitoring nationwide occurrence to prevent the spread of the virus to other crops.

A Plant Virus-Based Vector System for Gene Function Studies in Pepper.

While pepper (Capsicum annuum) is a highly recalcitrant species for genetic transformation studies, plant virus-based vectors can provide alternative and powerful tools for transient regulation and functional analysis of genes of interest in pepper. In this study, we established an effective virus-based vector system applicable for transient gain- and loss-of-function studies in pepper using Broad bean wilt virus2 (BBWV2). We engineered BBWV2 as a dual gene expression vector for simultaneous expression of two recombinant proteins in pepper cells. In addition, we established enhanced and stable expression of recombinant proteins from the BBWV2-based dual vector via coexpression of a heterologous viral suppressor of RNA silencing. We also developed a BBWV2-based virus-induced gene silencing (VIGS) vector, and we successfully silenced the phytoene desaturase gene (PDS) using the BBWV2-based VIGS vector in various pepper cultivars. Additionally, we optimized the BBWV2-based VIGS system in pepper by testing the efficiency of PDS gene silencing under different conditions. This BBWV2-based vector system represents a convenient approach for rapid and simple analysis of gene functions in pepper.

Potential use of newly isolated bacteriophage as a biocontrol against Acidovorax citrulli.

Acidovorax citrulli, the gram-negative bacteria that causes bacterial fruit blotch (BFB), has been responsible for huge worldwide economic losses in watermelon and melon production since 1980. No commercial cultivar resistant to BFB has been reported. Of the two reported genotypes of A. citrulli, genotype I is the main causal agent of BFB in melon and genotype II causes disease in watermelon. After the isolation of the first bacteriophage against A. citrulli (ACP17), efforts have been made to isolate bacteriophages with wider host ranges by collecting samples from watermelon, pumpkin, and cucumber. The newly isolated phage ACPWH, belonging to the Siphoviridae family, has a head size of 60 ± 5 nm and tail size of 180 ± 5 nm, and can infect 39 out of 42 A. citrulli strains. ACPWH has genome size of 42,499 and GC content of 64.44%. Coating watermelon seeds with bacteriophage ACPWH before soil inoculation with A. citrulli resulted in 96% germination and survival, compared to 13% germination of uncoated control seeds. These results suggest that phage ACPWH may be an effective and low-cost biocontrol agent against BFB.

First report of Mochi tree (Ilex integra) Anthracnose caused by Colletotrichum fioriniae in Korea.

Ilex integra, also called Mochi tree, is an woody ornamental common in Asia, particularly in Korea, China, Japan, and Taiwan. Anthracnose, caused by Colletotrichum spp., is an economically important disease worldwide, affecting both fruit and seed quality. In April 2019, symptoms of Anthracnose were observed on leaves from several Mochi trees in an urban planting in Wando-gun, South Korea. Irregularly shaped, light-to-dark brown spots of 1-4mm were observed on young leaves. The lesions coalesced as each spot enlarged, flat and black fruiting bodies (acervuli) occurred on the brown lesions. Four symptomatic leaves were collected; fractions were cut from symptomatic tissue, including healthy tissue, then were disinfected with 1% sodium hypochlorite and 70% ethanol, and placed on potato dextrose agar (PDA). After dark-incubation at 25℃ for 7 days two isolates were obtained, the fungal colonies appeared as white to light gray mycelium, then becoming dark and orange to pink on the underside. After acervuli were produced on the plate, orange-red conidial masses erupted. Conidia observed from two isolates were hyaline, 1-celled, and oblong with round to acute apices, and measured 7 to 12 × 2 to 5 µm (mean ± SD: 9.29 ±2.26 × 3.68± 1.31 µm) (n=30). Genomic DNA was extracted and multi-locus sequencing was performed with one representative isolate using the internal transcribed spacer (ITS) (White et al. 1990), actin (ACT) genes, chitin synthase 1 gene (CHS-1) (Carbone and Kohn 1999), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Calmodulin (CAL) (Weir et al. 2012) and submitted. Blast search results showed that the isolate had 100%, 98.45%, 99.74%, 100%, and 100% nucleotide sequence identity with those of C. fioriniae (MT607651, MH717601, MG666441, MN895544, MN974144) respectively (Jamin and Mateu 2008). The five sequences were deposited in NCBI GenBank (Accession No: MT457472, MT465884, MT465885, MT465886, MT465887), which were assigned to ITS, ACT, CHS-1, GAPDH, and CAL regions, respectively. Based on the morphology (Shivas and Tan 2009) and molecular characterization (Guerber et al. 2003), the isolate was identified as C. fioriniae. To confirm pathogenicity, a conidial suspension (106 conidia/ml) of the sequenced isolate was used to inoculated, young and mature leaves of a 4-year-old Mochi tree. Ten leaves of the seedling were disinfected with 70% ethanol, then were wounded with a toothpick. The conidial suspension (20 µl) was placed on the wound. The inoculated plant and control plants were tested with sterilized water and incubated at 25℃ in a moist chamber. The pathogenicity test was repeated three times. Typical spots were observed on the young leaves 2 days after inoculation, whereas they were observed on the mature leaves 7 days after inoculation. Acervuli developed on both young and mature leaves 5 and 20 days after treatment, respectively. The control plants did not show symptoms, and the fungus was re-isolated from the inoculated plant; thus, fulfilling Koch's Postulates. In Korea, C. fioriniae has been recorded as a pathogen of fruit (apple, eggplant and peach), but this is the first report of the fungus causing anthracnose on Mochi tree. The pathogen has been reported on leaves of a different Ilex species in the eastern USA (Farr and Rossman 2020). Although this new disease of I. integra is limited occurrence, C. fioriniae may be able to infect other plant species in South Korea.

First report of Tomato spotted wilt virus in Petasites japonicus in Korea.

Butterbur (Petasites japonicus [Siebold & Zucc.] Maxim.) is a perennial herb of the Asteraceae family that is cultivated for medicinal and nutritional purposes. Due to long-term vegetative propagation of virus-infected native species, the yield and quality of butterbur plants have deteriorated. Five viruses have been reported to infect this species: alfalfa mosaic virus (AMV), arabis mosaic virus (ArMV), butterbur mosaic virus (ButMV), broad bean wilt virus 2 (BBWV-2), and cucumber mosaic virus (CMV) (Ham et al. 2016; Tochihara and Tamura 1976). From 2018 to 2019, butterbur plants in four greenhouses in Nonsan, South Korea (Supplementary Figure S1a, b) were found to show virus-like symptoms such as chlorotic and necrotic ring spots, necrosis, and mild mosaic on the leaves. Disease incidence was greater than 80% in one greenhouse (~1,000 m2). To identify the causal virus, we collected 17 symptomatic butterbur leaf samples from these greenhouses and performed reverse-transcription polymerase chain reaction (RT-PCR) analysis using species-specific detection primers for the five reported viruses and tomato spotted wilt virus (TSWV) (Supplementary Table S2). RT-PCR results showed that 12 samples from three greenhouses showing necrotic ring spots and mosaic symptoms were infected with a mixture of TSWV and ButMV, whereas 5 samples from one greenhouse showing mild mosaic symptoms were infected only with ButMV. TSWV (genus Orthotospovirus, family Tospoviridae) is transmitted by thrips and causes serious damage to a wide range of economically important plants (Pappu et al. 2009). ButMV (genus Carlavirus, family Betaflexiviridae) is transmitted by aphids, as well as infected vegetative propagation material (Hashimoto et al. 2009) and is the most predominant virus in butterbur in Korea (Ham et al. 2016). To isolate TSWV from butterbur, leaf extracts from symptomatic samples were mechanically inoculated on an assay host, Chenopodium quinoa, via three single-lesion passages followed by propagation in Nicotiana tabacum cv. Samsun. Thirty different indicator plant species were used for the bioassay of the TSWV isolate (TSWV-NS-BB20) by mechanical inoculation method (Supplementary Table S3). RT-PCR analysis confirmed that TSWV-NS-BB20 induced necrotic local lesions and mosaic on Nicotiana species and ring spots and mosaic on tomatoes and peppers. Notably, TSWV-NS-BB20 reproduced necrotic local lesions and mild mosaic symptoms on butterbur plants which were infected with ButMV with no obvious symptoms. To characterize TSWV-NS-BB20 genetically, the complete genome sequences of L (8914 nt), M (4751 nt), and S (2917 nt) RNA segments were obtained by RT-PCR using specific primers for TSWV as described previously (Kwak et al., 2020). The obtained sequences were deposited in GenBank under accession nos. MT643236, MT842841, and MN854654, respectively. BLASTn analysis showed that sequences of each segment had maximum nucleotide identities of 99.0, 98.9, and 98.6% to TSWV-L, M, and S (KP008128, FM163373, and KP008129) of TSWV-LL-N.05 isolate from tomato in Spain. Since 2018, TSWV outbreaks on butterbur are observed every year and thus may act as a potential source of TSWV infection for other crops of importance to Korea, such as pepper. Owing to the butterbur vegetative propagation, the identification of TSWV infection in butterbur will be helpful for future virus management to generate virus-free materials. To our knowledge, this is the first report of TSWV infection of butterbur.

Efficacy and safety of acupuncture for functional constipation: a randomised, sham-controlled pilot trial.

BACKGROUND: The prevalence of functional constipation (FC) is 3-27%, and FC has been reported to cause discomfort in daily life and various complications. The treatment for FC depends on laxatives, and thus, effective and non-toxic alternative treatments are needed. METHODS: We conducted a randomised, sham-controlled parallel-design, pilot trial. Participants with FC were randomly assigned to either the real acupuncture (RA) or sham acupuncture (SA) group. The RA consisted of eight fixed acupuncture points (bilateral ST25, ST27, BL52 and BL25) and four additional points targeted to the individual based on Traditional Korean medicine (TKM). SA consisted of shallow acupuncture insertion at 12 non-acupuncture points. Twelve sessions were provided over 4 weeks. The outcome measures were weekly defecation frequency (DF), spontaneous complete bowel movement (SCBM), Bristol stool scale (BSS) score and constipation assessment scale (CAS) score. The participants were followed for 4 weeks after the treatment. RESULTS: Thirty participants were enrolled (15:15). The mean DF were 5.86 ± 5.62, 5.43 ± 3.39 and 5.79 ± 3.64 in the RA group and 3.73 ± 1.62, 5.00 ± 1.77 and 5.40 ± 1.96 in the SA group at weeks 1, 5, and 9, respectively. The increases in weekly SCBMs were 2.50 ± 3.86 and 2.71 ± 4.01 with RA and 2.33 ± 2.74 and 1.93 ± 2.25 with SA at weeks 5 and 9, respectively (mean difference [MD] 0.78). The BSS scores were 0.57 ± 1.72 and 1.09 ± 1.30 with RA and 0.15 ± 1.06 and 0.14 ± 0.88 with SA at weeks 5 and 9, respectively (MD 0.95). The CAS score changes were - 3.21 ± 2.91 and - 3.50 ± 3.98 with RA and - 2.67 + ±2.82 and - 2.87 ± 2.95 with SA at weeks 5 and 9, respectively. Greater improvements were observed in subgroup analysis of participants with hard stool. The numbers of participants who developed adverse events (AEs) were equal in both groups (four in each group), and the AEs were not directly related to the intervention. CONCLUSIONS: This clinical trial shows feasibility with minor modifications to the primary outcome measure and comparator. Acupuncture showed clinically meaningful improvements in terms of SCBMs occurring more than 3 times per week and in these improvements being maintained for 4 weeks after treatment completion. As this is a pilot trial, future studies are warranted to confirm the efficacy and safety. TRIAL REGISTRATION: KCT0000926 (Registered on 14 November 2013).

The complete genome sequence of a novel virus, bellflower veinal mottle virus, suggests the existence of a new genus within the family Potyviridae.

A new virus was isolated from a bellflower (Campanula takesimana) plant showing veinal mottle symptoms, and its complete genome sequence was determined. The viral genome consists of a positive-sense single-stranded RNA of 8,259 ribonucleotides. Electron microscopic observation revealed that the viral genome is packaged as a filamentous particle with an average length of approximately 760 nm. BLAST searches of protein databases showed that the encoded polyprotein has a maximum amino acid sequence identity of 34.1% (with 95% coverage) to that of the isolate AD of Chinese yam necrotic mosaic virus (CYNMV; genus Macluravirus). Phylogenetic analysis and comparison of the encoded amino acid sequences with those of other viruses demonstrated that the identified virus shows minimal sequence similarity to known viruses and should therefore be considered a member of a new genus in the family Potyviridae. The name bellflower veinal mottle virus (BVMoV) is proposed for this new virus.

Complete genome sequence of longan witches' broom-associated virus, a novel member of the family Potyviridae.

The complete genome sequence of a new virus isolated from a longan (Dimocarpus longan Lour.) plant showing witches' broom syndrome was determined. The viral genome is composed of a monopartite single-stranded RNA of 9,428 nucleotides excluding the 3' poly(A) tail and contains one large single open reading frame encoding a polyprotein of 3086 amino acids. BLAST searches of protein databases showed that the encoded polyprotein has a maximum amino acid sequence identity of 35% (with 85% coverage) to that of the isolate Minnesota of rose yellow mosaic virus (RoYMV; family Potyviridae; genus not assigned). Molecular and phylogenetic analysis of the genome and encoded protein sequences showed that the identified virus has the general features that are characteristic of members of the family Potyviridae although it has extremely low sequence similarity to known members of the family Potyviridae. The name longan witches' broom-associated virus (LWBaV) is proposed for this new virus, which may be considered a member of a new genus in the family Potyviridae.

Genetic variants of the gasdermin B gene associated with the development of aspirin-exacerbated respiratory diseases.

BACKGROUND: Aspirin-exacerbated respiratory disease (AERD) is characterized by a severe and sudden asthma attack after aspirin ingestion in patients with asthma. We studied associations with six common single nucleotide polymorphisms (SNP) of the gasdermin B gene (GSDMB). OBJECTIVE: DNA obtained from 572 patients with asthma (with AERD, n = 165; and with aspirin-tolerant asthma, n = 407) and 391 normal controls was subjected to genotyping of six SNPs of GSDMB. METHODS: An association analysis between GSDMB variants and AERD, with a fall rate of the forced expiratory volume in the first second of expiration (FEV1), was performed by using logistic and regression models. RESULTS: Two SNPs in the intron (rs870830, rs7216389) showed significant associations with AERD (minimum p = 7.00 × 10-4 in the dominant model), even after Bonferroni correction (pcorr = 0.01 for the rs870830). Regression analysis of the genetic variants with FEV1 revealed significant associations with rs870830 and the haplotype 2 (pcorr = 4.71 × 10-4 for rs870830 and pcorr = 1.14 × 10-3 for haplotype 2, respectively). CONCLUSION: We found strong associations among GSDMB polymorphisms and the presence of AERD and FEV1 in Korean patients with asthma. Our findings indicated that genetic variations of GSDMB may be associated with the development of AERD and aspirin-induced bronchospasm.

Consideration of rainwater quality parameters for drinking purposes: A case study in rural Vietnam.

Rainwater, which is used for drinking purposes near Hanoi, Vietnam, was analysed for water quality based on 1.5 years of monitoring data. In total, 23 samples were collected from different points within two rainwater harvesting systems (RWHSs). Most parameters met the standard except micro-organisms. Coliform and Escherichia coli (E. coli) were detected when the rainwater was not treated with ultraviolet (UV) light; however, analysis of rainwater after UV sterilisation showed no trace of micro-organisms. The RWHSs appear to provide drinking water of relatively good quality compared with surface water and groundwater. The superior quality of the rainwater suggests the necessity for new drinking rainwater standards because applying all of the drinking water quality standards to rainwater is highly inefficient. The traditionally implemented standards could cause more difficulties for developing countries using RWHSs installed decentralized as a source of drinking water, particularly in areas not well supplied with testing equipment, because such countries must bear the expense and time for these measures. This paper proposes the necessity of rainwater quality guideline, which could serve as a safe and cost-effective alternative to provide an access to safe drinking water.

A Phase 2a Study of Benralizumab for Patients with Eosinophilic Asthma in South Korea and Japan.

BACKGROUND: Airway eosinophils are considered to play an important role in the pathogenesis of asthma. Interleukin-5 is believed to be a key cytokine for the development, proliferation and activation of eosinophils. Benralizumab is an anti-interleukin-5 receptor α monoclonal antibody that depletes blood and airway eosinophils. We conducted a phase 2a study in South Korea and Japan to evaluate the effect of benralizumab in an East Asian population. The primary objective was to evaluate the effect of benralizumab in adults with uncontrolled eosinophilic asthma with 2-6 incidences of exacerbations in the past year using a medium/high dose of inhaled corticosteroids and long-acting ß2-agonists. METHODS: This was a multicenter, randomized, double-blind, placebo-controlled study. The subjects (n = 106) were randomized into four groups: placebo (n = 27) or benralizumab 2 mg (n = 27), 20 mg (n = 26) and 100 mg (n = 26). Benralizumab or placebo were administered subcutaneously on weeks 0 (day 1), 4, 8, 16, 24, 32 and 40. The primary endpoint was the asthma exacerbation rate at week 52. RESULTS: The asthma exacerbation rate was reduced by 33, 45 or 36% versus the placebo group when treated with 2, 20 or 100 mg of benralizumab, respectively. The percent mean change in forced expiratory volume at 1 s increased with each of the three doses in subjects treated with benralizumab. CONCLUSIONS: Benralizumab reduced asthma exacerbation and improved lung function and asthma control in adults with uncontrolled eosinophilic asthma.

Efficacy and safety evaluation of once-daily fluticasone furoate/vilanterol in Asian patients with asthma uncontrolled on a low- to mid-strength inhaled corticosteroid or low-dose inhaled corticosteroid/long-acting beta2-agonist.

BACKGROUND: Response to inhaled corticosteroid (ICS)/long-acting beta2-agonist (LABA) combinations varies across ethnic groups. OBJECTIVE: To investigate the efficacy and safety of the ICS/LABA combination fluticasone furoate/vilanterol (FF/VI) 100/25 µg in Asian patients with asthma. METHODS: A randomized (1:1), 12-week, double-blind, placebo-controlled, parallel group, multicenter phase III study of once-daily FF/VI 100/25 µg versus placebo in patients of Asian ancestry ages ≥12 years with asthma, uncontrolled on a low- to mid-strength ICS or low-dose ICS/LABA. The primary end point was the mean change from baseline in the daily evening peak expiratory flow. Secondary end points were the mean change from baseline in percentage rescue-free 24-hour periods, daily morning peak expiratory flow, percentage symptom-free 24-hour periods, Asthma Quality of Life Questionnaire score, adverse events, and severe exacerbations. RESULTS: The intent-to-treat population was 307 patients. There were significant (p < 0.001) improvements from baseline for FF/VI 100/25 µg versus placebo in evening peak expiratory flow (51.0 L/min [95% confidence interval {CI}, 42.2-59.7 L/min]) and all secondary end points (percentage rescue-free 24-hour periods 21.8% [95% CI, 14.6-29.1%]; morning peak expiratory flow 52.9 L/min [95% CI, 44.2-61.6 L/min]; percentage symptom-free 24-hour periods 15.8% [95% CI, 9.4-22.3%]; Asthma Quality of Life Questionnaire score 0.52 [95% CI, 0.28, 0.75]). On-treatment adverse events were 35% with FF/VI (n = 2 [serious]), 31% with placebo; severe exacerbations were FF/VI (n = 1), placebo (n = 7). CONCLUSIONS: In patients of Asian ancestry, once-daily FF/VI 100/25 µg produced statistically and clinically significant improvements in efficacy end points versus placebo, with a generally similar safety profile. Results were consistent with a global phase III study of FF/VI 100/25 µg. Clinicaltrials.gov NCT01498679.

Psychroserpens jangbogonensis sp. nov., a psychrophilic bacterium isolated from Antarctic marine sediment.

A Gram-staining-negative, yellow-pigmented, aerobic, rod-shaped and non-motile bacterium, PAMC 27130(T), was isolated from the marine sediment of the Ross Sea, Antarctica. The temperature, pH and NaCl tolerance ranges for growth were 4-20 °C, pH 6.0-9.0 and 0.5-5.0 % (w/v) NaCl, respectively. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain PAMC 27130(T) belonged to the genus Psychroserpens and was closely related to Psychroserpens mesophilus, Psychroserpens damuponensis and Psychroserpens burtonensis with 97.2, 94.7 and 94.2 % sequence similarities, respectively. Genomic relatedness analyses based on average nucleotide identity and genome-to-genome distance showed that strain PAMC 27130(T) could be clearly distinguished from other species of the genus Psychroserpens . The genomic DNA G+C content was 32.7 mol%. The major fatty acids (>10 %) were C20 : 4ω6c (13.2 %), iso-C15 : 0 (12.3 %), iso-C15 : 1 G (11.7 %) and iso-C15 : 0 3-OH (10.0 %). The major respiratory isoprenoid quinone was menaquinone-6 (MK-6) and the polar lipids were phosphatidylethanolamine, two unidentified aminolipids, an unidentified phospholipid, an unidentified aminophospholipid and three unidentified lipids. On the basis of genotypic and phenotypic data collected in this study, it is proposed that strain PAMC 27130(T) represents a novel species of the genus Psychroserpens, for which the name Psychroserpens jangbogonensis sp. nov. is proposed. The type strain is PAMC 27130(T) ( = KCTC 42128(T) = JCM 30228(T)).

Complete genome sequence of yacon necrotic mottle virus, a novel putative member of the genus Badnavirus.

The complete genome sequence of a previously undescribed virus isolated from a yacon plant exhibiting necrotic mottle, chlorosis, stunting, and leaf malformation symptoms in Gyeongju, Korea, was determined. The genome of this virus consists of one circular double-stranded DNA of 7661 bp in size. The genome contained four open reading frames (ORFs 1 to 4) on the plus strand that potentially encode proteins of 26, 32, 234, and 25 kDa. Protein BLAST analysis showed that ORF3, which is the largest ORF, has 45 % amino acid sequence identity (with 89 % coverage) to the ORF3 of fig badnavirus 1 (FBV-1), a recently identified badnavirus. Phylogenetic analysis provided further evidence that the virus identified in this study is probably a member of a new species in the genus Badnavirus. The name yacon necrotic mottle virus (YNMoV) is proposed for this new virus.

Complete genome sequence of bellflower vein chlorosis virus, a novel putative member of the genus Waikavirus.

The complete genome sequence of a new virus isolated from a bellflower (Campanula takesimana) plant was determined. The genome of this virus is composed of monopartite single-stranded RNA of 11,649 nucleotides in length. BLAST searches of protein databases showed that the encoded polyprotein has a maximum amino acid sequence identity of 42% (with 99% coverage) to the polyprotein of the isolate Orissa of rice tungro spherical virus (RTSV; genus Waikavirus). Phylogenetic analysis strongly supports that the identified virus is a member of a new species of the genus Waikavirus. The name bellflower vein chlorosis virus (BVCV) is proposed for this new virus.

Complete genome sequence of motherwort yellow mottle virus, a novel putative member of the genus Torradovirus.

The complete genome sequence of a new virus isolated from a motherwort plant exhibiting yellow mottle, mild mosaic, and stunting symptoms in Andong, Korea, was determined. The genome of this virus is composed of two single-stranded RNAs (7068 and 4963 nucleotides in length, respectively) carrying poly(A) tails. RNA1 contains one large open reading frame (RNA1-ORF1), while two potential ORFs (RNA2-ORF1 and RNA2-ORF2) were found in RNA2. BLAST searches of protein databases showed that RNA1-ORF1 and RNA2-ORF2 have maximum amino acid sequence identities of 53 % and 57 % to the RNA1-ORF1 and RNA2-ORF2, respectively, of lettuce necrotic leaf curl virus (LNLCV, a recently identified torradovirus). Phylogenetic analysis provided further evidence that the virus identified in this study is probably a member of a new species in the genus Torradovirus. The name "motherwort yellow mottle virus" (MYMoV) is proposed for this new virus.

Molecular genetic analysis of cucumber mosaic virus populations infecting pepper suggests unique patterns of evolution in Korea.

Studying genetic structure and diversity of viruses is important to understand the evolutionary mechanisms that generate and maintain variations in viral populations. Cucumber mosaic virus (CMV) is endemic in most pepper fields in Korea. Currently, no effective methods for control of CMV are available due to many environmental and biological factors such as the extensive evolutionary capacity of CMV. Thus, analyzing the genetic structure of CMV populations may facilitate the development of strategies for the control of CMV. In this study, 252 pepper (Capsicum annuum) samples showing virus symptoms were collected by field surveys performed throughout Korea in 2007. Reverse-transcription polymerase chain reaction analyses revealed that, in total, 165 collected samples were infected with CMV. Forty-five CMV isolates were randomly selected within each regional subpopulation and analyzed by full-genome sequencing. Analyses of genetic diversity showed that the 2b gene of CMV is under weaker purifying selection than the other genes. Based on the phylogenetic analysis of RNA1, the CMV isolates from pepper were divided into three clusters in subgroup I. Our full-genome sequence-based molecular analyses of the CMV Korean population suggest that the subpopulations of CMV have been geographically localized in pepper fields in Korea.