First report of alstroemeria mosaic virus infecting Alstroemeria in Korea.

Alstroemeria, a member of the Alstroemriaceae family, is a popular cut flower plant with a long-base life and a wide variety of flower colors. It is widely cultivated in many countries, especially in Central and South America. However, numerous viruses such as alstroemeria carlavirus (AlCV), alstroemeria mosaic virus (AlMV), cucumber mosaic virus (CMV), tomato spotted wilt virus (TSWV), alstroemeria streak virus (AlSV), and impatiens necrotic virus (INSV) can infect Alstroemeria and significantly decrease its yield (Kim, 2020). Among these viruses, AlMV is well known to cause an endemic viral disease in the Netherlands (Corine M. et al. 1992). AlMV is a member of the genus potyvirus in the family Potyviridae, one of the most widely distributed families of plant viruses. In 2021, symptomatic alstroemeria plants showing interveinal leaf streaking with elongated light green and chlorosis of leaves were identified from farms in a greenhouse in Gwangju, South Korea. Potyvirus-like particles (approximately 750-800 nm in length) were observed from sap of the symptomatic plants by electron microscope (Supplementary Fig. 1). To confirm virus infection, total RNA was extracted from an alstroemeria leaf using a Beniprep® Super Plant RNA extraction kit (IVT7005, Invirustech Co., Korea). A cDNA library was synthesized and analyzed by high throughput sequencing (HTS) using an Illumina NovaSeq6000 S4 sequencer. A total of 48,072,240 raw reads were obtained after quality filtering with FastQC. Remaining sequences were de novo assembled into contigs with a Trinity assembler. Nucleotide blast analysis of contigs against NCBI viral reference database revealed that 24 assembled contigs (> 1,000 bp) were sequences of AlMV. To confirm AlMV detection, raw reads were mapped to known AlMV complete genome (9,774 bp) using Bowtie2 program. Results showed that a total of 4,698,112 reads were mapped. A consensus sequence (9,778 bp, accession no. LC709275) was then obtained. To verify the presence of AlMV, RT-PCR assay was conducted with AlMV's CP gene-specific primers: AlMV-F (5'-CACGAGGCTGTGAAACAAGC -3') and AlMV-R (5'- CCAGGCGACACGGCTAAATA-3'). PCR products of the expected size (538 bp) were cloned, sequenced, and subjected to GenBank BLASTn search. A 538 bp partial CP sequence was used for BLAST analysis which revealed that it shared 100% identities with the consensus sequence (LC709275) and 96.99~98.76% nucleotide identities with four AlMV isolates (MK440140, NC043135, MT892648, DQ295032). Phylogenetic analysis based on partial CP sequences of representative members of potyviruses (family Potyviridae) using 1,000 bootstrap replicates based on either neighbor-joining or Kimura 2 parameter methods in MEGA-X revealed that AlMV isolate JNU-2 was grouped together with the four known AlMV isolates (Supplementary Fig. 2). To determine the incidence of AlMV in a greenhouse, 30 alstroemeria samples were collected and tested by RT-PCR. Results showed that 23 samples were positive for AlMV by PCR-gel electrophoresis and Sanger sequencing, suggesting a high incidence of AlMV infection. To the best of our knowledge, this is the first report of natural infection with AlMV in Alstroemeria in Korea. Further surveys of AlMV infection in greenhouses will help us prevent the spread of this viral disease in Alstroemeria.

Korean Ginseng Berry Fermented by Mycotoxin Non-producing Aspergillus niger and Aspergillus oryzae: Ginsenoside Analyses and Anti-proliferative Activities.

To transform ginsenosides, Korean ginseng berry (KGB) was fermented by mycotoxin non-producing Aspergillus niger and Aspergillus oryzae. Changes of ginsenoside profile and anti-proliferative activities were observed. Results showed that A. niger tended to efficiently transform protopanaxadiol (PPD) type ginsenosides such as Rb1, Rb2, Rd to compound K while A. oryzae tended to efficiently transform protopanaxatriol (PPT) type ginsenoside Re to Rh1 via Rg1. Butanol extracts of fermented KGB showed high cytotoxicity on human adenocarcinoma HT-29 cell line and hepatocellular carcinoma HepG2 cell line while that of unfermented KGB showed little. The minimum effective concentration of niger-fermented KGB was less than 2.5 µg/mL while that of oryzae-fermented KGB was about 5 µg/mL. As A. niger is more inclined to transform PPD type ginsenosides, niger-fermented KGB showed stronger anti-proliferative activity than oryzae-fermented KGB.

An RNA aptamer that binds carcinoembryonic antigen inhibits hepatic metastasis of colon cancer cells in mice.

BACKGROUND & AIMS: Carcinoembryonic antigen (CEA) is expressed by many types of cancer cells; its overexpression induces cell adhesion, increases resistance to anoikis, and promotes hepatic metastasis of colon cancer cells. The amino acid sequence PELPK in its hinge region, between the N and A1 domains, is required for migration of cancer cells to the liver. We sought to identify ligands of this domain for use in diagnosis and therapy. METHODS: We screened for RNA aptamers against the domain of CEA required for metastasis using systematic evolution of ligands by exponential enrichment. The specificity and affinity of the aptamer for CEA protein were characterized by mobility shift, uptake, and surface plasmon resonance assays. We analyzed the effects of the aptamer on metastatic properties of cells, as well as metastasis of colon cancer cells in mice. RESULTS: Using systematic evolution of ligands by exponential enrichment, we identified an RNA aptamer that bound to the PELPK sequence in CEA with high affinity and specificity. The isolated aptamer bound specifically to CEA-positive cells and inhibited interactions between CEA and heterogeneous nuclear ribonucleoprotein M4. The aptamer inhibited homotypic aggregation, migration, and invasion by CEA-positive cancer cells, but did not affect adhesion of endothelial cells. The aptamer induced colon cancer cell anoikis by interrupting the interaction between death receptor 5 and CEA. The aptamer prevented metastasis of human colon cancer cells to the livers of mice. CONCLUSIONS: An RNA aptamer that binds to the PELPK sequence in CEA inhibits its interactions with heterogeneous nuclear ribonucleoprotein M4 and death receptor 5, migration and invasion by colon cancer cells, and hepatic metastasis of colon cancer cells in mice. It promoted cancer cell anoikis and might be used to identify CEA-positive tumors in patients or be developed as an anti-cancer reagent.

Identification of Plant Viruses Infecting Pear Using RNA Sequencing.

Asian pear (Pyrus pyrifolia) is a widely cultivated and commercially important fruit crop, which is occasionally subject to severe economic losses due to latent viral infections. Thus, the aim of the present study was to examine and provide a comprehensive overview of virus populations infecting a major pear cultivar ('Singo') in Korea. From June 2017 to October 2019, leaf samples (n = 110) of pear trees from 35 orchards in five major pear-producing regions were collected and subjected to RNA sequencing. Most virus-associated contigs matched the sequences of known viruses, including apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV). However, some contigs matched the sequences of apple green crinkle-associated virus and cucumber mosaic virus. In addition, three complete or nearly complete genomes were constructed based on transcriptome data and subjected to phylogenetic analyses. Based on the number of virus-associated reads, ASGV and ASPV were identified as the dominant viruses of 'Singo.' The present study describes the virome of a major pear cultivar in Korea, and looks into the diversity of viral communities in this cultivar. This study can provide valuable information on the complexity of genetic variability of viruses infecting pear trees.

A portable detection assay for Apple stem pitting virus using reverse transcription-recombinase polymerase amplification.

A molecular diagnostic assay for the rapid, sensitive and specific detection of Apple stem pitting virus (ASPV) in infected samples, utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 42 °C and the designed target-specific primers, was developed. The RT-RPA assay was able to be used in ASPV-infected leaves, rootstocks and fruits. Sensitivity tests, using ASPV transcripts, showed that the RT-RPA with the ASPV-specific primers was more sensitive than the conventional RT-PCR, with a detection limit of 1 fg/µL of RNA. In addition, the reaction time for the amplification of ASPV was shortened to as little as 1 min. The assay was highly specific and did not give a positive reaction to other viruses infecting pears. Moreover, the amplified genomic fragment of ASPV produced by the assay could be determined within 4 min using a portable capillary gel electrophoresis system. The entire process, excluding the extraction of total RNA, could be completed in 5 min using portable equipment in the field. This is the first report of utilizing an RT-RPA assay to detect a pear tree virus and the assay could be used both in the laboratory and in the field for ASPV detection.

Rapid and Specific Detection of Apple stem grooving virus by Reverse Transcription-recombinase Polymerase Amplification.

Apple stem grooving virus (ASGV) is considered to cause the most economically important viral disease in pears in Korea. The current PCR-based methods used to diagnose ASGV are time-consuming in terms of target detection. In this study, a novel assay for specific ASGV detection that is based on reverse transcription-recombinase polymerase amplification is described. This assay has been shown to be reproducible and able to detect as little as 4.7 ng/µl of purified RNA obtained from an ASGV-infected plant. The major advantage of this assay is that the reaction for the target virus is completed in 1 min, and amplification only requires an incubation temperature of 42°C. This assay is a promising alternative method for pear breeding programs or virus-free certification laboratories.