• 한국식물병리학회:학술대회논문집
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• 한국식물병리학회 2003년도 정기총회 및 추계학술발표회
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• pp.117.3-118
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• 2003

We tested for the presence of double-stranded RNA (dsRNA) mycovirus in 827 Fusarium graminearum isolated from diseased barley and maize. dsRNA mycoviruses with various sizes were isolated. Of them, it was previously reported that dsRNA from DK2l isolate had pronounced morphological changes, including reduction in mycelial growth, increased to red pigmentation, reduced virulence and sporulation. (Chu et al., Appl. Environ. Microbiol. 2002). For better understanding of this hypovirulence associated with DK2l dsRNA virus, we determined the complete nucleotide sequence of dsRNA genome and named Fusarium hypovirus DK2l strain (Fhv-DK2l ). Genomic RNA of Fhv-DK2l was determined to be 6625 nucleotides in length excluding the poly (A) tail and contained three putative open reading frame. RNA-dependent RNA polymerase (RdRp) and helicase domain were expected in ORF A, 54 to 4709 nucleotide position. ORE B, 4752 to 5216 nucleotide position, and ORF C, 5475 to 6578 nucleotide position, were predicted to encode 16.7kDa and 41.3kDa protein respectively each. We could not detect any conserved domains from these two proteins. Phylogenetic analysis showed Fhv-DK2l was related to Cryphonectria hypovirus 3. Ten additional isolates were found that were infected with dsRNA mycoviruses. These mycoviruses contain 2 to 4 different segments of dsRNAs with the size range of approximately 1.7 to 10-kbp in length. The presence of dsRNAs isolates did not affect colony morphology and were transmissible through conidia and ascospore with incidence of 30-100％. These results indicate that there is genomic diversity of dsRNA mycoviruses that infect F. graminearum isolates and that impact of virus infection on host's morphology and virulence is determined by the interaction between dsRNAs and the fungal host, not by the mere presence of the dsRNAs

• 대한수의학회지
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• 제43권3호
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• pp.439-448
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• 2003

The VP2 gene of infectious bursal disease virus (IBDV) Chinju which was previously detected in Chinju, Korea was cloned and sequenced to establish the information for the development of genetically engineered vaccines and diagnostic reagents against IBDV. The nucleotide sequence of the entire Chinju VP2 gene consisted of 1,356 bases long encoding 452 amino acids in a single open reading frame (ORF). It consisted of 368 adenine (27.1%), 363 cytosine (26.8%), 339 guanine (25.0%) and 286 thymine (21.1%) residues. The predicted $M_r$ of the Chinju VP2 protein was 48 kDa, and the protein contained 13 phosphorylation sites by protein kinase C, casein kinase II or tyrosine kinase, whereas 3 asparagine-linked glycosylation sites were recognized. The nucleotide sequence of Chinju VP2 ORF had a very close phylogenetic relationship with 98-99% homology to that of the very virulent IBDVs (vvIBDVs) HK46, OKYM, D6948, UK661, UPM97/61 and BD3/99. Also, the Chinju VP2 protein revealed a very close phylogenetic relationship with 99-100% homology to that of these vvIBDVs. The Chinju VP2 protein had 100% amino acid identity in the variable region of residues 206-360 with that of the D6948, HK46, OKYM and UK661, as well as 100% identity in two hypervariable regions of residues 212-224 and 314-324 with those of the D6948, HK46, OKYM, UK661, UPM97/61 and BD3/99. The amino acid sequence of the chinju VP2 protein contained a serine-rich heptapeptide of SWSASGS as in these vvIBDVs.

• Journal of Microbiology and Biotechnology
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• 제17권11호
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• pp.1833-1840
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• 2007

Viruses in garlic plants (Allium sativum L.) have accumulated and evolved over generations, resulting in serious consequences for the garlic trade around the world. These viral epidemics are also known to be caused by aphids and eriophyid mites (Aceria tulipae) carrying Potyviruses, Carlaviruses, and Allexiviruses. However, little is known about viral epidemics in garlic plants caused by eriophyid mites. Therefore, this study investigated the infection of garlic plants with Allexiviruses by eriophyid mites. When healthy garlic plants were cocultured with eriophyid mites, the leaves of the garlic plants developed yellow mosaic strips and became distorted. In extracts from the eriophyid mites, Allexiviruses were observed using immunosorbent electron microscopy (ISEM). From an immunoblot analysis, coat proteins against an Allexivirus garlic-virus antiserum were clearly identified in purified extracts from collected viral-infected garlic plants, eriophyid mites, and garlic plants infected by eriophyid mites. A new strain of GarV-B was isolated and named GarV-B Korea isolate 1 (GarV-B1). The ORF1 and ORF2 in GarV-B1 contained a typical viral helicase, RNA-directed RNA polymerase (RdRp), and triple gene block protein (TGBp) for viral movement between cells. The newly identified GarV-B1 was phylogenetically grouped with GarV-C and GarV-X in the Allexivirus genus. All the results in this study demonstrated that eriophyid mites are a transmitter insect species for Allexiviruses.

• Asian-Australasian Journal of Animal Sciences
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• 제25권8호
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• pp.1184-1189
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• 2012

White spot syndrome virus (WSSV) is one of the major viral pathogens affecting shrimp aquaculture. Four proteins, WSSV199, WSSV 222, WSSV 249 and WSSV 403, from WSSV are predicted to encode a RING-H2 domain, which in presence of ubiquitin conjugating enzyme (E2) in shrimp can function as viral E3 ligase and modulate the host ubiquitin proteasome pathway. Modulation of host ubiquitin proteasome pathway by viral proteins is implicated in viral pathogenesis. In the present study, a time course expression profile analysis of WSSV Open Reading Frame (ORF) 199 and Penaeus monodon ubiquitin conjugating enzyme (PmUbc) was carried out at 0, 3, 6, 12, 24, 48 and 72 h post WSSV challenge by semi-quantitative RT-PCR as well as Real Time PCR. EF1${\alpha}$ was used as reference control to normalize the expression levels. A significant increase in PmUbc expression at 24 h post infection (h.p.i) was observed followed by a decline till 72 h.p.i. Expression of WSSV199 was observed at 24 h.p.i in WSSV infected P. monodon. Since the up-regulation of PmUbc was observed at 24 h.p.i where WSSV199 expression was detected, it can be speculated that these proteins might interact with host ubiquitination pathway for viral pathogenesis. However, further studies need to be carried out to unfold the molecular mechanism of interaction between host and virus to devise efficient control strategies for this chaos in the shrimp culture industry.

• Asian-Australasian Journal of Animal Sciences
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• 제31권4호
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• pp.489-498
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• 2018

Objective: Foot and mouth disease (FMD) and porcine reproductive and respiratory syndrome (PRRS) are major diseases that interrupt porcine production. Because they are viral diseases, vaccinations are of only limited effectiveness in preventing outbreaks. To establish an alternative multi-resistant strategy against FMD virus (FMDV) and PRRS virus (PRRSV), the present study introduced two genetic modification techniques to porcine cells. Methods: First, cluster of differentiation 163 (CD163), the PRRSV viral receptor, was edited with the clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 technique. The CD163 gene sequences of edited cells and control cells differed. Second, short hairpin RNA (shRNAs) were integrated into the cells. The shRNAs, targeting the 3D gene of FMDV and the open reading frame 7 (ORF7) gene of PRRSV, were transferred into fibroblasts. We also developed an in vitro shRNA verification method with a target gene expression vector. Results: shRNA activity was confirmed in vitro with vectors that expressed the 3D and ORF7 genes in the cells. Cells containing shRNAs showed lower transcript levels than cells with only the expression vectors. The shRNAs were integrated into CD163-edited cells to combine the two techniques, and the viral genes were suppressed in these cells. Conclusion: We established a multi-resistant strategy against viral diseases and an in vitro shRNA verification method.

• The Plant Pathology Journal
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• 제15권5호
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• pp.270-279
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• 1999

cDNAs complementary to the 3'-terminal regions of two potyvirus genomes were cloned and sequenced. The clone G7 contains one open reading frame (ORF) of 1,338 nucleotides and a 3' untranslated region (3'-UTR) of 403 nucleotides at the 3'-end excluding the 3'end poly(A) tail. The putative viral coat protein (CP) shows 55%-92% amino acid sequence homology to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.0 kb by Northern blot analysis. Five cDNA clones were screened out using GPV2 oligonucleotide as a probe. One of these clones, DEA72, which has a longest cDNA insert, contains one ORF of 1,459 nucleotides and a 3'-UTR of 590 nucleotides at the 3'-end excluding the 3'-end poly(A) tail. The putative viral CP shows 57%-88% amino acid sequence homologies to those of Allium potyviruses. The genome size of the virus was analyzed to be about 9.6 kb by Northern blot analysis. The results of immunoblot and Northern blot analyses suggest that almost all of the tested garlic plants showing mosaic or streak symptoms are infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus in variable degrees but rarely infected with DEA72-potyvirus in variable degrees but rarely infected with G7-potyvirus. Immunoelectron microscopy using anti-DEA72 CP antibody shows that this potyvirus is about 750 nm long and flexuous rod shaped.

• BMB Reports
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• 제34권4호
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• pp.371-378
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• 2001

The Glycoprotein D (gD) gene of the HSV-1 strain F was cloned, sequenced, recombinated into the HcNPV (Hyphantria cunea nuclear polyhedrosis virus) expression vector and expressed in insect cells. The gD gene was located in the 6.43 kb BamHI fragment of the strainF. The open reading frame (ORF) of the gD gene was 1,185 by and codes 394 amino acid residues. Recombinant baculoviruses, GD-HcNPVs, expressing the gD protein were constructed. Spodoptera frugiperda cells, infected with the recombinant virus, synthesized a matured gX-gD fusion protein with an approximate molecular weight of 54 kDa and secreted the gD proteins into the culture media by an immunoprecipitation assay The fusion gD protein was localized on the membrane of the insect cells, seen by using an immunofluorescence assay The deduced amino acid sequence presents additional characteristics compatible with the structure of a viral glycoprotein: signal peptide, putative glycosylation sites and a long C-terminal transmembrane sequence. These results indicate the utility of the HcNPV-insect cell system for producing and characterizing eukaryotic proteins.

• The Plant Pathology Journal
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• 제30권2호
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• pp.151-158
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• 2014

Three double-stranded RNAs (dsRNAs), approximately 1.85, 1.65 and 1.27 kb in size, were detected in an isolate of Cytospora sacchari from Iran. Partial nucleotide sequence revealed a 1,284 bp segment containing one ORF that potentially encodes a 405 aa protein. This protein contains conserved motifs related to RNA dependent RNA polymerases (RdRp) that showed similarity to RdRps of partitiviruses. The results indicate that these dsRNAs represent a novel Partitivirus that we tentatively designate Cytospora sacchari partitivirus (CsPV). Treatment of the fungal strain by cyclohexamide and also hyphal tip culture had no effect on removing the putative virus. Phylogenetic analysis of putative RdRp of CsPV and other partitiviruses places CsPV as a member of the genus Partitivirus in the family Partitiviridae, and clustering with Aspergillus ochraceous virus 1.

• 한국어병학회지
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• 제25권3호
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• pp.143-150
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• 2012

Infectious haematopoietic necrosis virus (IHNV) is an important fish pathogen that infects both wild and cultured salmonids. Since the first isolation of IHNV from rainbow trout and masu salmon in 1991, a series of IHN disease outbreak has been reported in Korea. In 2011, we isolated two IHNV isolates from rainbow trout cultured in Korea. The full open-reading frame (ORF) encoding the glycoprotein (G) of them were sequenced and the amino acid sequences were phylogenetically analyzed. Phylogenetic analysis of the G revealed that both IHNV isolates were grouped into an Asian genogroup containing Korean IHNV isolates and Japanese IHNV isolates. However, based on their sequence variation, they were divided into different subgroup. While one isolate was similar to other Korean isolates, the other isolate showed a high level of similarity with Japanese isolates, suggesting the possibility of influx of new IHNV strain into Korea.

• Journal of Plant Biology
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• 제39권4호
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• pp.265-271
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• 1996

Full-length cDNA for RNA2 of cucumber mosaic virus strian Kor (Kor-CMV) was cloned downstream of synthetic T7 promoter by reverse transcriptase-polymerase chain reaction (RT-PCR). The clone could generate a full-length transcript corresponding to RNA1 in size when synthesized by T7 RNA polymerase. The complete nucleotide sequence has shown that the RNA2 is composed of 3,049 nucleotides and contains one functional open reading frame (ORF) of 2,574 nucleotides encoding 2a protein. The deduced translation product of the 2,574 nucleotides contains GDD motif which is a characteristic of RNA-dependent RNA polymerase (RdRp). The amino acid sequence analysis of the 2a protein has shown that the homology is found in decreasing order with O-CMV (98.8%), Y-CMV (98.7%), Fny-CMV (98.3%), KCMV (94.9%), Ix-CMV (91.9%), and Q-CMV (74.9%). Kor-CMV is suggested to belong to subgroup Ⅰ in the aspect of nucleotide sequence homology of RNA2.