We report for the first time the occurrence of DWV-infected bumble bees (Bombus ignitus). For the present study, the detection of DWV virus from the female and male bumble bee was investigated in the same colony. The Deformed wing virus (DWV) of honeybee (Apis mellifera) is closely associated with characteristic wing deformities, abdominal bloating, paralysis, and rapid mortality of emerging adult bees. Using specific RT-PCR protocols for the detection of DWV followed by sequencing of the PCR products we could demonstrate that the bumble bees were indeed infected with DWV. The virus was detected from Bombus ignitus, and its partial DWV gene was cloned and sequenced. The partial DWV gene encoding the polyprotein is 711-nt of 235 amino acid residues. The deduced nucleotide sequence of the polyprotein partial gene of DWV showed 96.9%, 96.2%, 96.8%, and 96.5% homology to other structure polyprotein partial gene of DWV from insects, respectively. Phylogenetic analysis further conformed that the deduced nucleotide sequence of the polyprotein partial gene of DWV divided to the outside tree. We describe the first time that presence of Deformed wing virus(DWV) from bumble bee(Bombus terrestris) in korea using RT-PCR.

The Classical Swine Fever Virus (CSFV) is a member of the Pestivirus genus of the Flaviviridae. The genome of CSFV is a positive single-stranded RNA molecule 12.3 kb and contains a single large open reading frame (ORF). The polyprotein composed of eight nonstructural and four structural proteins (nucleocapsid protein C and three envelope glycoprotein E0, E1 and E2). E2, the most immunogenic of the CSFV glycoproteins, induces a protective immune response in swine. To determine the characteristics of the CSFV, LOM strain, we investigated the nucleotide sequence of the glycoprotein E0, E1 and E2. Comparison of the LOM with the other strains revealed nucleotide sequence identity ranging from 97 to 98%. Expression of the glycoprotein E2 was identified by SDS-PAGE and Western blot analysis using anti-CSFV E2 monoclonal antibodies in Sf21 cells. The expression levels of glycoprotein E2 were observed from day 3 and 5 days maximum. In addition, its expression efficiency by media and cell line was investigated. The result showed that High-Five cells and Grace’s insect media for Sf21 were the best conditions for the expression of the glycoprotein E2.

Aujeszky’s disease (AD), also called pseudorabies, is an infectious viral disease, caused by an alpha herpes virus and has domestic and wild pigs, as well as a wide range of domestic and wild animals, as the natural host. AD affects many countries and regions in the world, causing important economic losses, mainly due to international trade restrictions. In this study, to determine the characteristics of the Aujeszky’s disease virus (ADV), NYJ strain, which was isolated from the serum of an infected pig in 1987, we investigated the nucleotide sequence and expression of the glycoproteins gB, gC, and gD using the bBpGOZA system. We found that the glycoproteins gB, gC, and gD of NYJ consisted of 2751 bp, 1443 bp, and 1203 bp, respectively. Comparison of the NYJ with the other strains revealed nucleotide sequence identity ranging from 91.tito 99.0%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The NYJ strain was formed a distinct branch with high bootstrap support. The expression of glycoprotein gD in insect cells was characterized by SDS-PAGE and Western blotting with an anti-ADV polyclonal antibody. Glycoprotein gD of approximately 45 kDa was detected. The results of this study have implications for both the taxonomy of ADV and vaccine development.

The small brown planthopper (SBPH), Laodelphax striatelleus (Fallén), is a insect vector of Rice stripe virus (RSV) in temperate countries such as Korea, Japan, China, and Taiwan. As SBPH is able to overwinter successfully in these areas, RSV disease in subsequent rice fields has been believed to be endemic. In Korea, however, the RSV disease outbreaks have been observed mainly but not continuously at some western regions since 2001, caused a severe damage to the rice production. Although many efforts are underway to explain the outbreak phenomenon, the exact related factors are not known yet. In the meantime of the study on SBPH population dynamics in 2009, we catched unusually large numbers of SBPH adults by aerial net traps, maximally over 900, in early June at western coastal counties such as Taean, Seocheon, Buan, Sinan, and Jindo in Korea. Age distribution changes of SBPH in winter and post-winter seasons at some selected fields shows that the adults might be not related to overwintering population. The adults of overwintering population emerged from early April. Newly hatched nymphs of first generation were found from mid-May. In late May, just before the unusual catch of adults, the developmental stages of SBPH were mostly below 5th instars. This means that the big adult populations would be results of mass migration of SBPH abroad. We present also spacial distribution and host relationship of overwintering population as well as viruliferous rate changes of immigratory population.

This experiment was carried out to find out the optimum variety of com hybrids and to find out alternative crops in the rice black-streaked dwarf virus(RBSDV) prevalent area. Productivity of 4 Korean improved and 6 introduced com hybrids and RBSDV infection rate were tested for 3 years in both Cheonan(middle part of Korea) and Gochang(southern part of Korea). Percentage of RBSDV diseased plants differed depending on the hybrid and region.

The effects of the genetically modified virus-resistant pepper (line: 15, 20) and the non-GM pepper (line: 2377, 915) on the insect community in the pepper cultivation area were evaluated. Sampling was conducted using yellow sticky traps and pheromone funnel traps in Anseong and Deokso fields. Total number of insects caught on sticky trap were 3273 individuals at GM pepper and 2949 individuals at non-GM pepper in Anseong and 4357 individuals at GM and 3712 individuals at non-GM in Deokso. Total number of aphids collected on leaves were 451 and 330 individuals at GM and non-GM pepper in Anseong, respectively and 79 individuals at GM and 41 individuals at non-GM pepper in Deokso. The total number of the insect individuals caught on sticky trap was not shown significant differences between GM and non-GM pepper at Anseong and Deokso fields, respectively. Also, there were no significant differences in seasonal occurrences of aphids caught on sticky traps in GM and non-GM pepper at both fields. This work was supported financially by Biogreen21 project of Rural Development Administration (No. 20070301-034-010).

The sweet potato whitefly Bemisia tabasi is one of the most important pests of various horticultural crops. In addition, B. tabaci is a vector of many plant-pathogenic viruses and cause a serious secondary damage to crop plants. Association of plant-pathogenic virus with vector insects is known to be effective on the transmission capacity, fecundity, longevity of vectors including whiteflies. However, the interactive mechanisms between virus and vector insects are still poorly understood. Recently, a serious damage caused by virus disease together with B. tabasi emergence was identified at tomato glasshouse in Tongyoung. We detected the signals of Tomato Yellow Leaf Curl Virus (TYLCV) in tomato leaves and vector whiteflies using PCR amplification and confirmed its presence by those sequence comparison. To determine the effects of TYLCV acquisition on physiological status of vector whiteflies, transcript levels of genes that associated with metamorphosis, metabolism, stress and immune processes were compared between TYLCVinfected whiteflies and non-infected ones. Generally, the transcript levels of virus-infected whiteflies were lower than those of non-infected ones. In addition, the associations of endosymbiont levels within whiteflies were discussed in aspect of the acquisition and transmission of TYLCV.

Nationwide occurrence of Bemisia tabaci Q biotype was identified from 2005 May to 2007 Dec. in total 28 cities/counties of 9 provinces such as Goyang (Kyung-gi), Gangnung (Gang-won), Jincheon (Chung-buk), Buyeo (Chung-nam), Seongju (Kyung-buk), Geoje (Kyung-nam), Bukjeju (Jeju). Host plants of the scale of B. tabaci Q biotype were over 15 crops of tomato, sweet pepper, hot pepper, eggplant, etc. and total 12 species of weeds such as Veronica persica, Ipomoea lacunosa, Conyza sumatrensis, I. hederacea, Xanthium canadense, Humulus japonicus, Boehmeria nivea, Artemisia vulgaris, Paederia scandens, Acalypha austeralis, Brassica juncea, Rumex crispus. For molecular identifying Bemisia tabaci B and Q biotypes, and Trialeurodes vaporariorum, for which it is difficult to distinguish morphologically, sequences of mitochondrial 16S rDNA and CO I (Cytochromoxidase I) gene were analyzed and restriction enzymes were selected for biotypespecific cleaved bands. As the results, Hinf I for 16S rDNA and Vsp I for CO I gene made specific band patterns for the B and Q biotypes in gel electrophoresis. Thus these methods were able to identify those biotypes and species without DNA sequence analysis. Populations of the Q biotype were collected in each regions of Korea from 2005 to 2007, and they were genetically compared using CO I gene sequences. Thus the populations were divided by three different groups which were introduced over 3-4 times before 2007 from different population sources. Geoje and Jeju were suggested as the first regions of introduction. Especially the populations in the first introduced regions were highly homologous with the Q biotype of Japan. In addition, infection pattern of secondary symbionts in populations of the B and Q biotypes in Korea were different from the Israeli populations. Thus it is suggested that Japan is the main source of B. tabaci Q biotype introduced to Korea. In addition, populations of the both of B and Q biotype in Korea were infected by Haemiltonella, which is more effectively related to the transmission of tomato yellow leaf curl begomovirus (TYLCV). Therefore it is needed to monitor continuously if the outbreak of begomovirus vectored by B. tabaci. In this molecular phylogenetic analysis, it was shown that the population of B. tabaci Q biotype in weed plants near greenhouse was introduced to crop plants in greenhouse. Therefore we understand that weed control is important to inhibit recurrence of B. tabaci in greenhouse. Three species of begomovirus, sweet potato leaf curl virus (SPLCV), tobacco leaf curl virus (TLCV), and TYLCV, were reported after introduction of B. tabaci in Korea. Especially Korean government removed all plants in the first TYLCV-occurred greenhouse in 2008. Multiplex PCR diagnosis between TLCV and TYLCV was developed for the more rapid and accurate monitoring. TLCV and TYLCV strains occurred in Korea were highly homologous with strains of Japan. Therefore these results support our suggestion that Japan is the main source of B. tabaci Q biotype introduced to Korea.

The whitefly Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) is one of the most important agricultural pests in Japan, that causes retard of plant growth and sooty moulds through excreted honeydew by direct sucking of pholoem sap, and additionally transmits several kinds of plant virus. B. tabaci consists of more than 20 biotypes which possess different ecological or physiological characters but cannot be distinguished from others morphologically. In Japan, exotic B and Q biotypes are the common pests of vegetables, flowers and ornamental plants. B biotype, the silver-leaf whitefly, was first recorded in Aichi Prefecture, Tokai region, in 1989 and expanded its distribution to almost all part of Japan, except for the northern area, within several years. Q biotype was recently found in Hiroshima Prefecture, Chugoku region, in 2004 and is still expanding the distribution in our country. Indigenous B. tabaci biotypes also exist in the southwestern part of Japan: JpL biotype was recorded in Honshu, Shikoku, Kyushu Islands and Nauru biotype was found in Amami and Ryukyu Islands. Although the host plants of these indigenous biotypes include some agricultural crops, these insects are not important as agricultural pests. The most serious problem in vegetable cultivation caused by B. tabaci is an intensive epidemic of the tomato yellow leaf curl disease (TYLCD) which leads to a large yield loss of tomato production in green houses. TYLCD distributes worldwide and it was found in Aich and Sizuoka Prefectures, Tokai region, and Nagasaki Prefecture, Kyushu region, simultaneously in 1996. The distribution of TYLCD expanded mainly in the western part of Japan for several years after its first finding, but recently TYLCD started to occur also in the eastern part of Japan, Kanto and Tohoku regions. Tomato yellow leaf curl virus (TYLCV), a pathogen of TYLCD, is transmitted by B or Q biotype of B. tabaci in a persistent manner. Although an effective control of B. tabaci is essential for decreasing of TYLCD outbreaks in tomato green houses, it is quite difficult to control these whiteflies only by the spraying of chemically synthesized insecticides due to their insecticide resistance. Especially, Q biotype shows a high level of resistance to pyriproxyfen and neonicotinoid insecticides. To avoid the development of insecticide resistance in B. tabaci, we are trying to combine some different control methods, for example, use of a fine mesh screen to prevent the invasion of vector insects, use of the physical-coating or microbial insecticides with the chemically synthesized insecticides to prevent the reproduction of vector insects, closing and steaming of a green house at the end of tomato cultivation to kill vector insects and prevent their escape from there, as an integrated pest management (IPM) system for B. tabaci and TYLCD control. We are also breeding TYLCV resistant varieties of tomato and considering how to use these varieties effectively.

To determine the characteristics of the Korean porcine reproductive and respiratory syndrome virus (PRRSV), CA, which was isolated from the serum of an infected pig in 2006, we investigated the nucleotide sequence and expression of the structural ORFs (ORFs 2 to 7) using the bApGOZA system. We found that the structural ORFs 2 to 7 of CA consisted of 3188 nucleotides that were the same as those formed from VR-2332. Comparison of the CA with the other strains revealed nucleotide sequence identity ranging from 89.8 to 99.5%. To better understand the genetic relationships between other strains, phylogenetic analyses were performed. The CA strain was closely related to the other North American genotype strains but formed a distinct branch with high bootstrap support. Additionally, expression levels of the PRRSV proteins in Sf21 cells were strong or partially weak. The results of this study have implications for both the taxonomy of PRRSV and vaccine development.

The porcine reproductive and respiratory syndrome virus (PRRSV) has six structural proteins which encoded by ORFs 2 to 7 are designated as GP2, 3, 4, 5, M and N, repectively. In this study, we determined the expression of each protein using novel transfer vector, pBmKSK4 which has the polyhedrin promoter of BmNPV and 6xHis tag. The recombinant transfer vector was co-transfected into Bm5 cells along with bBpGOZA DNA. Recombinant virus was purified by plaque assay and amplified in Bm5 cells. Expression of each protein was identified by SDS-PAGE and Western blot analysis using anti-6xHis monoclonal antibody. The expression levels of the structural proteins in Bm5 cells were stronger than the expression system using pBacPAK9 transfer vector in Sf21 cells. As expected, GP5 was expressed at low levels from its structural properties and its toxicity for cells. In addition, each recombinant protein was purified using Ni-NTA spin columns. The ability to produce each protein in the baculovirus system indicates that these could be major candidates for the development of a vaccine against PRRSV.

Sacbrood virus(SBV) causes a fatal disease(sacbrood) of honeybee larvae, which fail to pupate, change color and shape, and finally die. The complete nucleotide sequence of SBV has recently been determined, and with these data, we now report a Reverse Transcription-PCR(RT-PCR) test for the direct, rapid, and sensitive detection of these viruses. To detect the SBV infection in Korea, we collect beekeepers from various apiaries, which the RT-PCR technique was used. And we designed SBV specific primers in conserved region of the viral genome in the GenBank database. We confirmed the SBV amplicon using cloning and sequence. Homology between determined sequences of SBV korean strain and published virus sequences were 97% in DNA sequence, and 100% in amino acid sequence. We describe the first time that presence of sacbrood virus(SBV) in Korea honey bee colonies using RT-PCR. We also developed and validated a RT-PCR assay for the detection of SBV in Korea.

Viruses of the honeybee, Apis mellifera L. are known to reside at low levels in colonies, typically showing no apparent signs of infection. Chronic paralysis virus(CBPV) is known to induce significant losses in honey bee colonies. The pathology is characterized by clusters of trembling, flightless, crawling bees and by individual bees, sometimes hairless, standing at the hive entrance. A minusstrand-specific RT-PCR was used to assess viral replication. This is the first report on the infection of CBPV in Korea. Using (-)RT-PCR, 27 apiaries in korea were screened for the honeybee viruses, with positive colonies being analysed for viral genetic diversity. We got 550-nt PCR product from CBPV genomic RNA. Nucleotide sequences were aligned to the complete CBPV genomic RNA sequence deposited in the GenBank database and was revealed 96%(AM-CBPV) identity, respectively. Sequence comparison with other CBPV and honeybee virus.

For the physiological study on environmental impact of genetically modified (GM) pepper plant on non-target but three-trophically related insect species, we investigated behavioral responses of Aphis gossypii and Aphidius colemani in Y-tube olfactometer to the cucumber mosaic virus (CMV)-resistant transgenic pepper plant (H15 GM line) expressing coat protein gene of CMV and its wild type pepper plant (untransformed, susceptible to CMV pathotype II, P2377 inbred line) in relation to CMV infection. CMV-infected plants were prepared with the 30 min of inoculation by the winged A. gossypii viruliferous or mechanical inoculation using CMV-Fny, and with molecular diagnosis using reverse transcriptase-polymerase chain reaction (RT-PCR) over 2 weeks after inoculation. In this study, time for attraction responses (attraction time) of A. gossypii were not significantly different in the pepper strain, and the virus infection of plant. However, the attraction time of A. colemani was significantly different between the GM plant and the non-GM plant. In addition, the attraction time of A. colemani to the GM plant was significantly decreased according to the CMV infection. For further study, the volatile organic compounds (VOCs) emitted by these plants were collected with an entrainment kit and analyzed by Gas Chromatography (GC) on HP-1 column. The specific VOCs related to CMV infection were detected in the GM plant over 4 weeks after inoculation of CMV in this study. Thus, it is suggested that VOCs of the GM plant in this study may be produced more as a signal attracting A. colemani in relation to CMV infection.