Sequence and phylogenetic analysis of the RNA1 and RNA2 segments of Korean Rice stripe virus isolates and comparison with those of China and Japan

Jonson, Miranda Gilda,Choi, Hong-Soo,Kim, Jeong-Soo,Choi, Il-Ryong,Kim, Kook-Hyung Springer-Verlag 2009 Archives of virology Vol.154 No.10

Effects of Recombination on the Pathogenicity and Evolution of Pepper mottle virus

Miranda Gilda Jonson,서장균,최홍수,김정수,김국형 한국식물병리학회 2009 Plant Pathology Journal Vol.25 No.4

The analysis of the full length genome of Korean isolates of Pepper mottle virus (PepMoV) in previous study showed molecular variations and are found to be related to symptom variation and pathogenicity (Kim et al., 2009, Virus Res. 144:83-88). To fully understand the molecular variation of PepMoV in Korea, we further assessed the role of RNA recombination to biological variation and evolution of PepMoV. Full-length genome of a total of 17 Korean-PepMoV and 2 American (CA and FL) isolates were examined for possible detection of genetic recombination using different recombination detections programs and detected 5 and 8 tentative recombination events using RDP3 and Splits Tree4 programs, respectively. Interestingly, tentative recombinants detected such as isolates 57, 134 and 217 were previously identified as severe isolates and 205135 and 205136 as differentiating isolates (Kim et al., 2009, Virus Res. 144:83-88). In addition, recombination was frequently detected in the Vb isolate, the first PepMoV isolate reported in Korea, suggesting significant involvement in the evolution of PepMoV in Korea. These initial results of our recombination analyses among PepMoV isolates in Korea may serve as clues to further investigate the biological variations and evolution of PepMoV brought about by recombination.

Genetic Reassortment of Rice stripe virus RNA Segments Detected by RT-PCR Restriction Enzyme Analysis-based Method

Miranda Gilda Jonson,Sen Lian,최홍수,이관석,김창석,김국형 한국식물병리학회 2011 Plant Pathology Journal Vol.27 No.2

Our previous sequence and phylogenetic analyses of the Korean Rice stripe virus (RSV) suggested possible genetic reassortment of RNA segments, but whether this RNA variation contributed to the recent RSV outbreaks in Korea is yet unclear. To further clarify these RSVRNA segment variations, we developed a reverse transcription-polymerase reaction/restriction enzyme (RTPCR/RE) analysis-based method. We identified five REs, including DraI, EcoR1, NdeI/AseI, and SpeI, that could differentiate RSV RNA 1–4 subtypes, respectively. Our RT-PCR/RE results provided a clear pattern of RNA reassortment, i.e., different groups of isolates having their RNA segments derived from two to three different RSV ancestors, such as from Eastern and Southwestern Chinese or Japanese M and T isolates. We also found that the migratory small brown planthopper from Eastern China caught by aerial net traps that possesses RSVRNA3genotypes corresponds mainly to Eastern China,with a few for Southwestern China based on RT-PCR/RE, sequence and phylogenetic analyses, indicating that RSV populations in Eastern China may also have strong RNA variation. The development of an RE analysisbased method proved a useful epidemiological tool for rapid genotyping and identification of mixed infections by RSV strain and by different subtype.

Complete Genome Sequence of the RNAs 3 and 4 Segments of Rice stripe virus Isolates in Korea and their Phylogenetic Relationships with Japan and China Isolates

Miranda Gilda Jonson,최홍수,김정수,최일영,김국형 한국식물병리학회 2009 Plant Pathology Journal Vol.25 No.2

The complete genome sequences of RNA3 and RNA4 of the 13 different Rice stripe virus (RSV) isolates were determined and characterized in this study to address the possible causes of the recent re-emergence of RSV that affected many rice fields in Korea. The genome size of each RNA segment varied among isolates and significant differences were observed in the intergenic region. There was up to 4% average divergence in the RNA4 nucleotide sequence among 13 Korean isolates and only 1.4% in the RNA3. Phylogenetic relationships among different Korean isolates revealed that there were at least 2 types of RNA3 and 4 distinct types of RNA4 genomes present in Korea. However, Korean isolates with one type of RNA3 predominate over the other while the occurrences of the RSV Korean isolates with the 4 types of RNA4 genome were not correlated to specific geographical areas. Results further indicate that RNA4 had diverged more than RNA3 and these differences in accumulation of mutations in the individual RNA segments indicate that genetic reassortment were likely to contribute to the genetic divergence in the 13 Korean isolates. All of the Korean-RNA3 sequences except for one isolate grouped with Chinese isolates (JY and Z). In contrast, the RNA 4 sequences segregated together with either Chinese (JY and Z) and Japanese (M and T) isolates but genetic relationships of Korean isolates- RNAs 3 and 4 segments to Chinese-Y isolate were low. Altogether, these results suggest that the occurrence of mixtures of RNAs 3 and 4 genotypes in the natural population of RSV may have contributed to the sudden outbreak in Korea.

Genetic Reassortment of Rice stripe virus RNA Segments Detected by RT-PCR Restriction Enzyme Analysis-based Method

Jonson, Miranda Gilda,Lian, Sen,Choi, Hong-Soo,Lee, Gwan-Seok,Kim, Chang-Suk,Kim, Kook-Hyung The Korean Society of Plant Pathology 2011 Plant Pathology Journal Vol.27 No.2

Our previous sequence and phylogenetic analyses of the Korean Rice stripe virus (RSV) suggested possible genetic reassortment of RNA segments, but whether this RNA variation contributed to the recent RSV outbreaks in Korea is yet unclear. To further clarify these RSV-RNA segment variations, we developed a reverse transcription-polymerase reaction/restriction enzyme (RT-PCR/RE) analysis-based method. We identified five REs, including DraI, EcoR1, NdeI/AseI, and SpeI, that could differentiate RSV RNA 1-4 subtypes, respectively. Our RT-PCR/RE results provided a clear pattern of RNA reassortment, i.e., different groups of isolates having their RNA segments derived from two to three different RSV ancestors, such as from Eastern and Southwestern Chinese or Japanese M and T isolates. We also found that the migratory small brown planthopper from Eastern China caught by aerial net traps that possesses RSV-RNA3 genotypes corresponds mainly to Eastern China, with a few for Southwestern China based on RT-PCR/RE, sequence and phylogenetic analyses, indicating that RSV populations in Eastern China may also have strong RNA variation. The development of an RE analysisbased method proved a useful epidemiological tool for rapid genotyping and identification of mixed infections by RSV strain and by different subtype.

Effects of Recombination on the Pathogenicity and Evolution of Pepper mottle virus

Jonson, Miranda Gilda,Seo, Jang-Kyun,Cho, Hong-Soo,Kim, Jeong-Soo,Kim, Kook-Hyung The Korean Society of Plant Pathology 2009 Plant Pathology Journal Vol.25 No.4

The analysis of the full length genome of Korean isolates of Pepper mottle virus (PepMoV) in previous study showed molecular variations and are found to be related to symptom variation and pathogenicity (Kim et al., 2009, Virus Res. 144:83-88). To fully understand the molecular variation of PepMoV in Korea, we further assessed the role of RNA recombination to biological variation and evolution of PepMoV. Full-length genome of a total of 17 Korean-PepMoV and 2 American (CA and FL) isolates were examined for possible detection of genetic recombination using different recombination detections programs and detected 5 and 8 tentative recombination events using RDP3 and Splits Tree4 programs, respectively. Interestingly, tentative recombinants detected such as isolates 57, 134 and 217 were previously identified as severe isolates and 205135 and 205136 as differentiating isolates (Kim et al., 2009, Virus Res. 144:83-88). In addition, recombination was frequently detected in the Vb isolate, the first PepMoV isolate reported in Korea, suggesting significant involvement in the evolution of PepMoV in Korea. These initial results of our recombination analyses among PepMoV isolates in Korea may serve as clues to further investigate the biological variations and evolution of PepMoV brought about by recombination.

Complete Genome Sequence of the RNAs 3 and 4 Segments of Rice stripe virus Isolates in Korea and their Phylogenetic Relationships with Japan and China Isolates

Jonson, Miranda Gilda,Choi, Hong-Soo,Kim, Jeong-Soo,Choi, Il-Ryong,Kim, Kook-Hyung The Korean Society of Plant Pathology 2009 Plant Pathology Journal Vol.25 No.2

The complete genome sequences of RNA3 and RNA4 of the 13 different Rice stripe virus (RSV) isolates were determined and characterized in this study to address the possible causes of the recent re-emergence of RSV that affected many rice fields in Korea. The genome size of each RNA segment varied among isolates and significant differences were observed in the intergenic region. There was up to 4% average divergence in the RNA4 nucleotide sequence among 13 Korean isolates and only 1.4% in the RNA3. Phylogenetic relationships among different Korean isolates revealed that there were at least 2 types of RNA3 and 4 distinct types of RNA4 genomes present in Korea. However, Korean isolates with one type of RNA3 predominate over the other while the occurrences of the RSV Korean isolates with the 4 types of RNA4 genome were not correlated to specific geographical areas. Results further indicate that RNA4 had diverged more than RNA3 and these differences in accumulation of mutations in the individual RNA segments indicate that genetic reassortment were likely to contribute to the genetic divergence in the 13 Korean isolates. All of the Korean-RNA3 sequences except for one isolate grouped with Chinese isolates (JY and Z). In contrast, the RNA 4 sequences segregated together with either Chinese (JY and Z) and Japanese (M and T) isolates but genetic relationships of Korean isolates- RNAs 3 and 4 segments to Chinese-Y isolate were low. Altogether, these results suggest that the occurrence of mixtures of RNAs 3 and 4 genotypes in the natural population of RSV may have contributed to the sudden outbreak in Korea.

Generation of Antibodies Against Rice stripe virus Proteins Based on Recombinant Proteins and Synthetic Polypeptides

Lian, Sen,Jonson, Miranda Gilda,Cho, Won-Kyong,Choi, Hong-Soo,Je, Yeon-Ho,Kim, Kook-Hyung The Korean Society of Plant Pathology 2011 Plant Pathology Journal Vol.27 No.1

Rice stripe virus (RSV) is one of serious epidemic pathogens for rice species grown in many Asian countries. Therefore, it is necessary to produce a diagnostic detection kit applicable in fields for RSV detection. In this study, RSV proteins that were derived from recombinant proteins and synthetic polypeptides as antigens were generated and were raised in rabbits for antiserum production. Among seven proteins in RSV, genes that code for NCP and NS3 proteins were cloned and subcloned into vector carrying His-tag protein and were expressed in E. coli. Of two recombinant proteins, only anti-NCP displayed stable hybridization signals in western blot analysis. Alternately, synthetic RSV polypeptides for CP, NCP, NS3 and NSvc4 we also generated and only antibodies against CP and NCP were very effective to detect RSV in both RSV infected rice and weed plants. However, antibodies against NS3 and NSvc4 showed weak specific bands as well as strong non-specific background due to the difference of viral proteins produced in the infected leaves. In summary, the antibodies generated against RSV proteins produced in this study will be useful for various assays such as for RSV diagnostic detection, immunoprecipitation, protein purification, and western blot analysis.

Generation of Antibodies Against Rice stripe virus Proteins Based on Recombinant Proteins and Synthetic Polypeptides

Sen Lian,김국형,Miranda Gilda Jonson,조원경,최홍수,제연호 한국식물병리학회 2011 Plant Pathology Journal Vol.27 No.1

Rice stripe virus (RSV) is one of serious epidemic pathogens for rice species grown in many Asian countries. Therefore, it is necessary to produce a diagnostic detection kit applicable in fields for RSV detection. In this study, RSV proteins that were derived from recombinant proteins and synthetic polypeptides as antigens were generated and were raised in rabbits for antiserum production. Among seven proteins in RSV, genes that code for NCP and NS3 proteins were cloned and subcloned into vector carrying His-tag protein and were expressed in E. coli. Of two recombinant proteins, only anti-NCP displayed stable hybridization signals in western blot analysis. Alternately, synthetic RSV polypeptides for CP, NCP, NS3 and NSvc4 we also generated and only antibodies against CP and NCP were very effective to detect RSV in both RSV infected rice and weed plants. However, antibodies against NS3 and NSvc4 showed weak specific bands as well as strong non-specific background due to the difference of viral proteins produced in the infected leaves. In summary,the antibodies generated against RSV proteins produced in this study will be useful for various assays such as for RSV diagnostic detection, immunoprecipitation,protein purification, and western blot analysis.

Isolation and Characterization of Pepper mottle virus Infecting Tomato in Korea

Kim, Mi-Kyeong,Kwak, Hae-Ryun,Han, Jung-Heon,Ko, Sug-Ju,Lee, Su-Heon,Park, Jin-Woo,Jonson, Miranda Gilda,Kim, Kook-Hyung,Kim, Jeong-Soo,Choi, Hong-Soo,Cha, Byeong-Jin The Korean Society of Plant Pathology 2008 Plant Pathology Journal Vol.24 No.2

A peculiar virus-like disease of tomato showing yellow mosaic and necrotic spots on leaves and necrosis on veins, petioles and stems was observed at the Tomato Experimental Station (TES), Buyeo, Chungcheongnamdo, Korea. The disease incidence at TES fields ranged from 21 to 35% infecting different tomato cultivars. For this reason, to identify the virus infecting tomato and to characterize the virus based on biology, serology, cytology and at molecular level. Here, leaf samples were randomly collected from different infected tomato cultivars at TES fields and greenhouses and tested by ELISA using Pepper mottle virus (PePMoV) and Tomato mosaic virus (ToMV) antisera. Infected saps were mechanically inoculated in different host plants to test for pathogenicity, symptomatology and host ranges. Infected tissues and ultrathin sections were examined by electron microscopy. Finally, putative coat protein and 3'-untranslated region (CP/3'-UTR) fragment was amplified and cloned for sequence determination and analyzed its genetic relationship to existing PepMoV and PVY sequences at the Genbank. Results showed 69% of the samples were positive with PepMoV, 13% with ToMV and 19 % were doubly infected with PepMoV and ToMV. Symptoms greatly varied from different host plants inoculated with tomato leaf sap infected with PepMoV alone and discussed in detailed in this paper. Electron microscopy from infected tissues showed filamentous particles of 720-750nm in length, a typical morphology and size of PepMoV. In addition, cylindrical inclusion bodies, pinwheels, scrolls and laminates with masses of fibrillar inclusions were also found in ultrathin sections. Alignment of the sequences of the CP/3'-UTR revealed >96% sequence identity with PepMoV and only <61% with PVY. Taken together, all these evidences presented clearly indicated that the causal agent infecting tomato at TES was PepMoV and we designated this PepMoV infecting tomato as Tom-sd2 strain in this study.