• Journal of Microbiology and Biotechnology
• /
• 제31권4호
• /
• pp.529-539
• /
• 2021

The beet armyworm, Spodoptera exigua, is a serious insect pest infesting various vegetable crops. Two infectious insect viruses, baculovirus and iflavirus, are known to induce epizootics in S. exigua populations. Indeed, some laboratory colonies have appeared to be covertly infected by these viruses. Diagnostic PCR tests detected two different viruses: Spodoptera exigua multiple nucleopolyhedrosis virus (SeMNPV) and iflaviruses (SeIfV1 and SeIfV2). Viral extract from dead larvae of S. exigua could infect Sf9 cells and produce occlusion bodies (OBs). Feeding OBs to asymptomatic larvae of S. exigua caused significant viral disease. Interestingly, both SeIfV1 and SeIfV2 increased their titers at late larval stages. Sterilization of laid eggs with 1% sodium hypochloride significantly reduced SeMNPV titers and increased larval survival rate. Double-stranded RNA (dsRNA) specific to SeIfV1 or SeIfV2 significantly reduced viral titers and increased larval survival rate. To continuously feed dsRNA, a recombinant Escherichia coli HT115 expressing SeIfV1-dsRNA was constructed with an L4440 expression vector. Adding this recombinant E. coli to the artificial diet significantly reduced the SeIfV1 titer and increased larval survival. These results indicate that laboratory colony collapse of S. exigua is induced by multiple viral infections. In addition, either suppression of SeMNPV or SeIfV infection significantly increased larval survival, suggesting a cooperative pathogenicity between baculovirus and iflavirus against S. exigua.

• 현장농수산연구지
• /
• 제5권1호
• /
• pp.57-63
• /
• 2003

The bovine rotavirus(BRV), bovine coronavirus(BCV) and bovine viral diarrhea virus(BVDV) are main viruses of bovine viral diarrhea disease. These viruses could be rapidly amplified by the reverse transcriptase polymerase chain reaction(RT-PCR). This study was conducted to develop rapid and accurate diagnostic methods of these viral diseases by multiplex RT-PCR. Specific primers were designed based on the sequences reported by Chang KO et. al. (1997) and Schroeder BA, et. al. (1990), RNA were prepared from the cultured viruses, first-stranded DNAs were synthesised by reverse transcriptase. PCR were conducted to amplify specific regions of the viruses by multiplex. Three bands such as 1,062bp for BRV, 458bp for BCV, and 300bp for BVDV were successfully produced by multiplex RT-PCR. In conclusion, this result suggested that these viruses could be diagnosed rapidly and accurately by multiplex RT-PCR.

• The Plant Pathology Journal
• /
• 제30권3호
• /
• pp.316-322
• /
• 2014

Recently, a Cucumber mosaic virus (CMV) strain, named as CMV-209, was isolated from Glycine soja. In this study, symptom expression of CMV-209 was analyzed in detail in Nicotiana benthamiana by comparing with that of CMV-Fny, which is a representative strain of CMV. Using infectious cDNA clones of CMV strains 209 and Fny, symptom expression of various pseudorecombinants between these two strains were examined in the early and late infection stages. In the early infection stage, the pseudorecombinants containing Fny-RNA2 induced stunting and leaf distortion on the newly emerged leaves whereas the pseudorecombinants containing 209-RNA2 caused no obvious symptoms. In the late infection stage, the pseudorecombinants containing 209-RNA1 and Fny-RNA2 induced severe leaf distortion and stunting, while CMV-209 induced mild symptom and CMV-Fny caused typical mosaic, general stunting, and leaf distortion symptoms, indicating that RNA 2 encodes a symptom determinant(s) of CMV, which is capable of enhancing symptoms. Furthermore, our results support the possibility that natural recombination between compatible viruses can result in emergence of novel viruses causing severe damages in crop fields.

• 미생물학회지
• /
• 제38권3호
• /
• pp.186-191
• /
• 2002

장내바이러스(enterovirus),로타바이러스(rotavirus),그리고 아데노바이러스(adenovirus)등 물을 통해 전파되는 환경바이러스의 신속한 검출 및 1 차적인 분류를 위해 올리고뉴클레오티드 DNA chip을 이용한 분석 시스템의 유용성에 대하여 연구하였다. BGM 세포배양실험에서 바이러스성 세포병변효과(cytopathic effect) 양성으로 판정된 세포단층으로부터 접종배양 3 일 후 세포내 모든 RNA를 분리하여 중합효소연쇄반응과 DNA chip으로 검출여부 및 유전형을 비교 분석한 결과 세포배양에서 양성으로 판정된 10개의 시료 중 3개가 바이러스 음성으로, 7개가 바이러스 양성으로 나타났다. 중합효소연쇄반응과 DNA chip에 의해 양성으로 나타난 7개 시료의 유전형은 두 방법에 의해 모두 장내바이러스로 동정되어 DNA chip에 의한 1차 동정의 유용성도 증명하였다. 세포배양 후 전기영동분석 없이 일차적인 동정과정까지 불과 3∼4 시간 이내에 수행해낼 수 있는 DNA chip분석은 특이성, 신속성, 및 경제성을 고루 갖추어 환경바이러스 검출방법론의 새로운 영역을 구성할 것으로 사료된다.

• 한국식물병리학회:학술대회논문집
• /
• 한국식물병리학회 1994년도 Proceedings of International Symposium on BIOLOGICAL CONTROL OF PLANT DISEASES Korean Society of Plant Pathology
• /
• pp.86-102
• /
• 1994

To understand the molecular structure and pathogenesis mechanism of Korean garlic viruses, we have isolate cDNA clones for garlic viruses. The partial nucleotide sequences of 24 cDNA clones were determined and that of six clones containing poly (A) tail were compared with those of other plant viruses. One of those clones, V9 has 81.8% similarity in nucleotide sequence and 93.0% in deduced amino acid sequence, respectively, to the coat protein gene for garlic mosaic virus (GMV). Northern blot analysis with the clone V9 demonstrated that the genome of GMV is 7.8 kb long and has poly (A) tail. The anti-coat protein antibody for GMV recognizes 35 kDa polypeptide which could be the coat protein of GMV from infected garlic leaf extract or virus preparation. Clone G7 has about 62% of deduced amino acid sequence identity with the members of potyvirus group. Northern blot analysis with the clone G7 demonstrated that the genome of the potyvirus I garlic is 9.0 kb long and has poly (A) tail. The third clone, S81, shows 42% amino acid identity to the potexvirus. The other clones are under the characterization. To test the possibility of producing garlic virus resistant plant, we have designed a hairpin type ribozyme to cleave V9 RNA at the middle of the coat protein gene. From the cleavage reactions in vitro with two different sizes of RNA substrates, V9SUB (144 nucleotides) and V9 RNA (1,361 nucleotides), the ribozyme can cleave V9 sequence effectively at the predicted site. To study the activity of the ribozyme in vivo, plant transformation is in progress. Further possibilities to produce garlic virus resistant plant will be discussed.

• Journal of Microbiology and Biotechnology
• /
• 제24권7호
• /
• pp.979-986
• /
• 2014

Plant pathogenic RNA viruses are present in a variety of plant-based foods. When ingested by humans, these viruses can survive the passage through the digestive tract, and are frequently detected in human feces. Kimchi is a traditional fermented Korean food made from cabbage or vegetables, with a variety of other plant-based ingredients, including ground red pepper and garlic paste. We analyzed microbial metatranscriptome data from kimchi at five fermentation stages to identify plant RNA virus-derived sequences. We successfully identified a substantial amount of plant RNA virus sequences, especially during the early stages of fermentation: 23.47% and 16.45% of total clean reads on days 7 and 13, respectively. The most abundant plant RNA virus sequences were from pepper mild mottle virus, a major pathogen of red peppers; this constituted 95% of the total RNA virus sequences identified throughout the fermentation period. We observed distinct sequencing read-depth distributions for plant RNA virus genomes, possibly implying intrinsic and/or technical biases during the metatranscriptome generation procedure. We also identified RNA virus sequences in publicly available microbial metatranscriptome data sets. We propose that metatranscriptome data may serve as a valuable resource for RNA virus detection, and a systematic screening of the ingredients may help prevent the use of virus-infected low-quality materials for food production.

• The Plant Pathology Journal
• /
• 제20권2호
• /
• pp.142-146
• /
• 2004

Woody plant tissues contain great amounts of phenolic compounds and polysaccharides. These substances inhibit the activation of reverse transcriptase and/or Taq polymerase in RT-PCR. The commonly used multiple-step protocols using several additives to diminish polyphenolic compounds during nucleic acid extraction are time consuming and laborious. In this study, sodium sulfite was evaluated as an additive for nucleic acid extraction from woody plants and the efficiency of RT-PCR assay of commercial nucleic acid extraction kits and small-scale dsRNA extraction was compared. Sodium sulfite was used as an inhibitor against polyphenolic oxidases and its effects were compared in RNA extraction by commercial extraction kit and small-scale double-stranded RNA (dsRNA) extraction method for RT-PCR. During nucleic acid extraction, addition of 0.5%-1.5%(w/v) of sodium sulfite to lysis buffer or STE buffer resulted in lighter browning by oxidation than extracts without sodium sulfite and improved the RT-PCR detection. When commercial RNA extraction kit was used, optimal concentrations of sodium sulfite were variable according to the tested plant. However, with dsRNA as RT-PCR template, sodium sulfite 1.5% in STE buffer improved the detection efficiency of Apple chlorotic leaf spot virus (ACLSV) and Apple stem grooving virus (ASGV) in fruit trees, and reduced the unspecific amplifications signi-ficantly. Furthermore, when viruses existed at low titers in host plant, small-scale dsRNA extractions were very reliable.

• The Plant Pathology Journal
• /
• 제34권2호
• /
• pp.150-156
• /
• 2018

The genome sequences of two novel monopartite RNA viruses were identified in a common eelgrass (Zostera marina) transcriptome dataset. Sequence comparison and phylogenetic analyses revealed that these two novel viruses belong to the genus Amalgavirus in the family Amalgaviridae. They were named Zostera marina amalgavirus 1 (ZmAV1) and Zostera marina amalgavirus 2 (ZmAV2). Genomes of both ZmAV1 and ZmAV2 contain two overlapping open reading frames (ORFs). ORF1 encodes a putative replication factory matrix-like protein, while ORF2 encodes a RNA-dependent RNA polymerase (RdRp) domain. The fusion protein (ORF1+2) of ORF1 and ORF2, which mediates RNA replication, was produced using the +1 programmed ribosomal frameshifting (PRF) mechanism. The +1 PRF motif sequence, UUU_CGN, which is highly conserved among known amalgaviruses, was also found in ZmAV1 and ZmAV2. Multiple sequence alignment of the ORF1+2 fusion proteins from 24 amalgaviruses revealed that +1 PRF occurred only at three different positions within the 13-amino acid-long segment, which was surrounded by highly conserved regions on both sides. This suggested that the +1 PRF may be constrained by the structure of fusion proteins. Genome sequences of ZmAV1 and ZmAV2, which are the first viruses to be identified in common eelgrass, will serve as useful resources for studying evolution and diversity of amalgaviruses.

• 대한바이러스학회지
• /
• 제29권4호
• /
• pp.247-259
• /
• 1999

Small round structured viruses (SRSV) are the major ethological agents which can cause outbreaks of non-bacterial gastroenteritis or food poisoning both in children and adults. The classification of family Caliciviridae to which SRSV belong, is based on the genome encoding three open reading frames. The rotavirus is another major pathogen which causes diarrhea in young children. We examined stool specimens obtained from diarrheal patients in Wonju from which bacterial pathogens were not found. To detect causative viruses from stool specimens of patients, reverse transcription (RT)-polymerase chain reaction (PCR) or nested PCR using rotavirus or SRSV specific primers was performed. In this study, RT-nested PCR procedure which can amplify a 330 bp fragment derived from RNA dependent RNA polymerase (RDRP) region within ORF1 was applied for the detection of SRSV. For the detection of rotaviruses, a 877 bp fragment from the VP4 region of rotavirus genome was amplified. As a result, rotavirus was not detected while SRSV sequences were detected from one out of five specimens. The nucleotide and amino acid sequences of the Wonju isolate were compared with other 6 Korean isolates which have been isolated and sequenced in our laboratory. Sequence analysis revealed that the Wonju isolate was rather distinct from other Korean isolates: the Wonju isolate was closer to genogroup I of SRSV while other 6 Korean isolates belonged to genogroup II.

• The Plant Pathology Journal
• /
• 제35권1호
• /
• pp.32-40
• /
• 2019

Symptoms of fig mosaic disease have been noticed on leaves of fig (Ficus carica) for several decades, in Montenegro. In 2014, leaf samples were collected from trees of six fig cultivars in a plantation located in the main fig-producing area of Montenegro, to study the disease. After RNA isolation, samples were tested by RT-PCR for detection of nine fig viruses and three viroids. Four viruses were detected: fig leaf mottle-associated virus 1 (FLMaV-1), fig mosaic virus (FMV), fig mild mottle-associated-virus (FMMaV) and fig badnavirus 1 (FBV-1). Most of the viruses were present in mixed infections. The amplicons of the viruses were directly sequenced from both directions. A BLAST search of these sequences revealed sequence identities with their closest counterparts at GenBank of 92, 97, 92 and 100%, for FLMaV-1, FMV, FMMaV and FBV-1, respectively. Different responses in symptom expression due to the various virus combinations detected have been demonstrated. Variety $Su{\check{s}}ilica$ had the least symptom expression, with only one virus (FBV-1) found. Considering that the production of figs in Montenegro is increasing and has a substantial relevance in this geographic location, the results indicate that more attention should be given to improving the phytosanitary condition of fig trees in the country.