• The Plant Pathology Journal
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• 제33권4호
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• pp.393-401
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• 2017

Efforts to control viral diseases in crop production include several types of physical or chemical treatments; antiviral extracts of a number of plants have also been examined to inhibit plant viral infection. However, treatments utilizing naturally selected microorganisms with activity against plant viruses are poorly documented. Here we report isolation of a soil inhabiting bacterium, Pseudomonas oleovorans strain KBPF-004 (developmental code KNF2016) which showed antiviral activity against mechanical transmission of tobamoviruses. Antiviral activity was also evaluated in seed transmission of two tobamoviruses, Pepper mild mottle virus (PMMoV) and Cucumber green mottle mosaic virus (CGMMV), by treatment of seed collected from infected pepper and watermelon, respectively. Pepper and watermelon seeds were treated with culture supernatant of P. oleovorans strain KBPF-004 or control strain ATCC 8062 before planting. Seeds germinated after treatment with water or ATCC 8062 yielded about 60% CGMMV or PMMoV positive plants, whereas < 20% of KBPF-004-treated seeds were virus-infected, a significantly reduced seed transmission rate. Furthermore, supernatant of P. oleovorans strain KBPF-004 remodeled aggregation of PMMoV 126 kDa protein and subcellular localization of movement protein in Nicotiana benthamiana, diminishing aggregation of the 126 kDa protein and essentially abolishing association of the movement protein with the microtubule network. In leaves agroinfiltrated with constructs expressing the coat protein (CP) of either PMMoV or CGMMV, less full-size CP was detected in the presence of supernatant of P. oleovorans strain KBPF-004. These changes may contribute to the antiviral effects of P. oleovorans strain KBPF-004.

• The Plant Pathology Journal
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• 제21권2호
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• pp.167-171
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• 2005

We isolated the Cucumber green mottle mosaic virus(CGMMV) particles from watermelon leaves and designated as CGMMV-HY1 as a watermelon isolate and attempted to characterize the pathogenic isolate responsible for such an epidemic in watermelon and also to monitor dominant viral isolates in greenhouse. The watermelon plants infected with CGMMV generally showed mottle mosaic, mosaic, growth stunting, necrosis and deformed fruit. The reactions of indicator plants to CGMMV-HY1 were the local lesions on Nicotiana tabacum cv. White Burley, Nicotiana tabacum cv. Samsun, and Chenopodium amaranticola, and the mosaic symptoms only on Cucumis sativus, but the CGMMV-HY1 did not infect Nicotiana sylvesytis, Datura stramonium, Chenopodium quinoa, and Petunia hybrida. Purified virus particles were rod-shaped and about 300 nm long. The coat protein (CP) of purified CGMMV-HY1 was single band with molecular weight of about 16.5 kDa which was confirmed by western blot analysis probed with monoclonal antibody of CGMMV-HY1. The genomic and subgenomic RNAs of 6.4 kb and 0.75 kb were revealed by the electrophoresis on 1.2% formaldehydedenatured agarose gel. Viral and complementary CGMMV-specific primer sets were designed for spanning the genome using previously reported CGMMV sequences. A 464bp of CP gene of CGMMV-HY1 was amplified by RT-PCR and cloned into PGEM-T easy vector. The nucleotide sequence of CP gene of CGMMV-HY1 shared 98%, 99%, and 100% identities with that of CGMMV strains W, KOM, and KW respectively. Based on these results, we identified CGMMV-HY1 as a CGMMV isolate of watermelon, a member of Tobamovirus.

• 대한수의학회지
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• 제43권2호
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• pp.239-246
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• 2003

Polymerase chain reaction (PCR) was evaluated for the early detection of Aujeszky's disease virus (ADV) DNA from virus-infected cell cultures. For the purposes, the Korean ADV NYJ1-87 was propagated in swine kidney (SK) cells and subjected to the amplification of DNA (217 bp) by PCR using sense and antisense primers specific to gp50 gene of the ADV. In detection of cell-associated viral DNA, reliable PCR conditions were determined as 30 cycles of reaction consisting 1 minute each of denaturation at $94^{\circ}C$, annealing at $55^{\circ}C$ and polymerization at $72^{\circ}C$. The PCR encountered best results with reagent mixtures of $50{\mu}l$ containing $200{\mu}M$ dNTPs, $0.2{\mu}M$ each sense and antisense primers, 1 mM $MgCl_2$ and 10% (v/v) template DNA in the final concentrations. ADV-specific DNAs were detected as early as 6, 6, and 9 hours post-infection, respectively, from lysates of the SK cells infected with ADV of $10^3$, $10^2$ and $10^1\;TCID_{50}/ml$ by this condition. In culture supernatant, the DNAs were detected from ADV of as low infectivity as $10^ {-3}\;TCID_{50}/ml$ by the reduced reagent concentrations and 30 cycles of 1 minute each of denaturation at $94^{\circ}C$ and annealing at $55^{\circ}C$, and 2 minutes of polymerization at $72^{\circ}C$. The lowest amount of detectable ADV DNA was 1 fg. In conclusion, the PCR condition established in the present study was recognized as a feasible alternative to time-consuming procedures in isolation and characterization of the virus.

• 대한수의학회지
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• 제52권4호
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• pp.223-230
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• 2012

The worldwide distribution and continuing genetic mutation of avian influenza virus (AIV) has been posed a great threat to human and animal health. A comparison of 3 isolates of AIV H9N2, A/chicken/Korea/KBNP-0028/00 (H9N2) (KBNP-0028), A/chicken/Korea/SNU8011/08 (H9N2) (SNU 8011) and an inactivated oil vaccine strain A/chicken/Korea/01310/01 (H9N2) (01310), was performed. The former 2 AIVs were isolated from field cases before and after the application of an inactivated H9N2 vaccine in 2007, respectively. The antigenic relationship, viral shedding, tissue tropism and genetic analysis were examined. The comparison of virus shedding from the cloaca and the oropharynx revealed that both isolates were more frequently isolated from the upper respiratory tract (90~100%) 1 day post inoculation (DPI) compared with isolation 5 DPI from gastrointestinal tracts (10~60%). Moreover, the isolate KBNP-0028 were recovered from all organs including bone marrow, brain and kidneys, indicating higher ability for broad tissue dissemination than that of SNU 8011. KBNP-0028 replicated earlier than other strains and with a higher titer than SNU 8011. In full-length nucleotide sequences of the NA gene and a partial sequence of the HA gene of SNU 8011, we found that there might be significant changes in tissue tropism, virus replication and genetic mutation in AIV H9N2 isolates.

• The Plant Pathology Journal
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• 제22권4호
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• pp.329-333
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• 2006

Two distinct and stable isolates of Barley mild mosaic virus(BaMMV) designated as Naju82-S(severe) and Naju82-M(mild) were obtained. These two isolates differed in their symptomatology, virus transmission characteristics and cultivar specificity at various temperature. Thus, these isolates were referred to as strains in this study. BaMMV Naju-S strain showed severe mosaic symptoms accompanied by necrosis on the infected leaves. Naju82-S strain is more virulent demonstrated by shorter incubation period and relatively high virus concentration than Naju82-M strain. Five Korean cultivars were tested for their pathogenicity to different strains based on the rate of infection. Results showed that infection rate of cultivars to both strains did not significantly differed from each other. However, under different temperatures, the pathogenicity on the two cultivars such as cultivars Hopumbori and Sessalbori were significantly affected. Hopumbori was moderately resistant to both strains at $10-12^{\circ}C$ and susceptible at $15-18^{\circ}C$. Similarly, Sessalbori was moderately resistant at $10-12^{\circ}C$ to both strains but distinctly differentiated at $15-18^{\circ}C$ wherein it was resistant to mild strain and highly susceptible to severe strain. Other cultivars including Baegdong, Jinyangbori and Neahanssalbori consistently showed susceptible reaction to both strains at varying temperatures tested in this study.

• 한국수의병리학회지
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• 제3권1호
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• pp.35-44
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• 1999

The morphologic changes of small intestinal epithelium in pigs diagnosed as porcine epidemic diarrhea(PED} by virus isolation and immunohistochemistry were studied through light microscope and transmissible electron microscope. On semi-thin section, the histologic findings showed severe villous atrophy and fusion with hyperplasia of cuboidal epithelium in the villi, inflammatory cell infiltration in lamina propria, and increased mitotic figures in the crypt. The structural changes were mostly restricted to the cytoplasm of affected absorptive epithelium of villi. 3 types of epithelial changes were found; degenerated virus-affected cells, undifferentiated cuboidal cells, and normal columnar cells. On electron microscopy, round to spherical viral particles of 50∼l00nm in diameter were found within the dilated vesicles and endoplasmic reticulums of degenerated cells, which had decreased their cytoplasmic electron density due to dilated and missing organelles(e.g. mitochondria, ERs, etc.). Microvilli were shortened and sparse, leaving denuded terminal web of the villous epithelial cells. Fat globules were often found within slightly degenerated enterocytes. On the tip of villi, severely damaged cells were exfoliated and replaced by undifferentiated cuboidal cells We found distinct ultrastructural changes in the jejunal epithelium confirming PED virus infection is involved in malabsorptive diarrhea.

• Journal of Microbiology and Biotechnology
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• 제27권11호
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• pp.2037-2043
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• 2017

The surface protein hemagglutinin (HA) mediates the attachment of influenza virus to host cells containing sialic acid and thus facilitates viral infection. Therefore, HA is considered as a good target for the development of diagnostic tools for influenza virus. Previously, we reported the isolation of single-stranded aptamers that can distinguish influenza subtype H1 from H5. In this study, we describe a method for the selective electrical detection of H1 using the isolated aptamer as a molecular probe. After immobilization of the aptamer on Si wafer, enzyme-linked immunosorbent assay (ELISA) and field emission scanning electron microscopy (FE-SEM) showed that the immobilized aptamer bound specifically to the H1 subtype but not to the H5 subtype. Assessment by cyclic voltammetry (CV) also demonstrated that the immobilized aptamer on the indium thin oxide-coated surface was specifically bound to the H1 subtype only, which was consistent with the ELISA and FE-SEM results. Further measurement of CV using various amounts of H1 subtype provided the detection limit of the immobilized aptamer, which showed that a nanomolar scale of target protein was sufficient to produce the signal. These results indicated that the selected aptamer can be an effective probe for distinguishing the subtypes of influenza viruses by monitoring current changes.

• 대한바이러스학회지
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• 제30권1호
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• pp.19-28
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• 2000

Two distinct hantaviruses have been isolated from Apodemus agrarius in 1976 and Rattus norvegicus in 1980 in Korea. Since our serosurveys conducted in 1994, a genetically distinct hantavirus from Apodemus peninsulae has been investigated. To isolate hantavirus from Apen insulae captured in Korea, the lung homogenate of seropositive Apeninsulae inoculated Vero E6 cells. Viral antigen was detected in a progressively higher percentage of cells with subsequent passage after 80 days postinoculation. The new isolate from seropositive Apodemus peninsulae was designated Suchong virus after Suchong valley located in northeastern region of South Korea. Comparing with hantaan virus 76-118 strain, Suchong virus-1, 2, 3 and 4 showed the similarity of $71.0{\sim}91.8%$ at nucleotide and $90.9{\sim}94.8%$ at amino acid sequences in 231 nucleotides region of M segment, and the similarity of $75.1{\sim}81.0%$ at nucleotide and $97.5{\sim}100%$ at amino acid sequences in 237 nucleotides of S segment.

• 대한수의학회지
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• 제33권2호
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• pp.249-254
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• 1993

설사로 폐사한 자돈의 장 절편을 이용하여 돼지 유행성 설사바이러스(PEDV), 돼지 전염성 위장염 바이러스(TGEV), 돼지 로타 바이러스(PRV)에 대한 병인학적 검사를 형광항체반응을 통하여 조사하였던 바 돼지 유행성 설사 바이러스의 감염에 의한 자돈의 폐사를 확인하였다. 간접형광항체검사시 돼지 유행성 설사 바이러스에 대한 양성반응을 보인 가검재료를 이용 Vero세포에 연속 계대한 후 plaque assay를 통하여 크로닝된 돼지 유행성 설사 바이러스 KPEDV-9주를 작성하였다. 돼지 유행성 설사 바이러스에 대한 면역혈청과 바이러스가 분리된 농장에서 채취된 돼지 혈청을 이용 돼지 유행성 설사 바이러스에 대한 구조단백성분을 분석하였던 바 88K(M.W.), 74K, 70K, 58~54K, 54~46K, 44~40K 및 33~32K에 상당하는 단백성분을 검출할 수 있었다.

• Plant Resources
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• 제4권1호
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• pp.41-47
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• 2001

This study was conducted to investigate the occurrence of Soil borne wheat mosaic virus(SbWMV) in barley fields in Korea and to examine the host pathogenicity of SbWMV. By using the ELISA test, SbWMV was detected in the six regions : Suwon, Milyang, Jinju, Youngkwang, Iksan, and Chonju. SbWMV was isolated from the two strains, Albori strain from Jinju and Eunpamil strain from Milyang. SbWMV was collected from leaves showing mosaic, yellowing and necrosis stripes. SbWMV was inoculated mechanically on 1∼1.5 leaf stages with leaf-rubbing to identify the host pathogenicity of 36 Korean barley cultivars, a wheat cultivar, two rye cultivars, three Japanese barley cultivars and Chenopodium amaranticola. Viral sympoms of inoculated leaves appeared on moulted loaves about 4 to 6 weeks of inoculation. Baegdong and Tapgolbori, infected from Albori strain and Eunpamil strain infected from Samdobori showed much higher susceptibility than C. amaranticola and C. quinoa which showed ring spots and chlorotic spots respectively. Virus particles were observed by the electron microscope. They were rod-shapes, which are bipartite, of 142 nm or 281 nm in length with 20 nm diameter on infected leaves. Specific detection and identification of SbWMV was set up using the RT-PCR. PCR fragments of SbWMV(0.5kb) were obtained by using the designed primers for SbWMV RNA 2.