A climate change has been reported as the most important issue related with vector borne diseases eg. Zika and Denguemainly transmitted by Aedes mosquitoes. In 2016, 24 imported Zika virus disease cases and > 300 cases of Dengue feverwere reported in the Republic of Korea (ROK). In the ROK, the control of disease vectors has been mainly carried outusing chemicals, which resulted in high insecticide resistance. Many studies and projects in Korea Center for DiseaseControl & Preventionfor overcoming the insecticide resistance of disease vectors have been carried out and some nationaldisease vector management systems for effective vector control are established throughout the ROK. In this presentation,the national vector management systems and studies will be addressed.Key words : Disease vectors, National management, the Republic of Korea, climate change

Different types of insect-borne plant viruses can modify their hosts and vectors in distinct manners. Therefore, interactionsbetween two types of viruses co-existing in a field are known to be complex to predict. Obtaining empirical data byconducting field experiments, however, requires numerous biotic and abiotic factors to be controlled, and is therefore hardto execute. Thus, we designed an individual based model to simulate the transmission pattern of two viruses, using potatoes(Solanum tuberosum) for host plant, aphids (Myzus persicae) for virus vector, potato leafroll virus and potato virus Yfor different types of plant viruses. More specifically, we aimed to investigate the effect of the following on the spreadof the plant viruses: dispersal by winged-form aphids, initial number of virus-infected seed potatoes, and indirect interactionsbetween two viruses by affecting life traits of the vectors.

Viral particles of Porcine epidemic diarrhea virus (PEDV) consist of a four structural proteins. Among them Spikeprotein mediated responsible for receptor binding and membrane fusion during viral infection and therefore the main targetof neutralizing antibodies. Virus-like particles (VLPs) are consisted of one or more viral structural proteins, and theirmorphologies closely resemble those of the native virus. VLPs have no virulence and can elicit robust immune responsesas compared with inactivated or live-attenuated virus vaccines. Thus, in this study, we tried two methods for VLP constructionin Bombyx mori, one is traditional method and the other is chimeric VLP method using the influenza matrix protein.Both methods could produce successfully PEDV VLPs.

The production of therapeutic protein from transgenic domestic animal is the major technology of biotechnology. Insulin-like growth factor-1 (IGF-1) is known to play an important role in the growth of the animal. The objective of this study is construction of knock-in vector that bovine IGF-1 gene is inserted into the exon 7 locus of β-casein gene and expressed using the gene regulatory DNA sequence of bovine β-casein gene. The knock-in vector consists of 5’ arm region (1.02 kb), bIGF-1 cDNA, CMV-EGFP, and 3’ arm region (1.81 kb). To express bIGF-1 gene as transgene, the F2A sequence was fused to the 5’ terminal of bIGF-1 gene and inserted into exon 7 of the β-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the β-casein exon 7 region to the bIGF-1 gene by DNA sequence. These knock-in vectors may help to create transgenic dairy cattle expressing bovine bIGF-1 protein in the mammary gland via the expression system of the bovine β-casein gene.

Severe combined immune deficiency (SCID) pig is very important research model for biomedical research, such as the development of humanized tissues and organs for transplantation and long-term evaluation of transplanted cancer or stem cell of human origin. FOXN1 gene encodes a transcription factor essential for the development and function of thymic epithelial cells (TECs), the primary lymphoid organ that supports T-cell development and selection. In this study, we are going to produce the FOXN1 KO SCID pigs using the Crispr/Cpf1 method. Porcine genomic DNA sequences were analyzed and the target sequences were selected using a web tool, Benchling (https://benchling.com/). The designed crDNA oligos was synthesized by the Oligonucleotide Synthesis Service (Macrogen Inc., Seoul, Korea). To generate the AsCpf1-mCherry-Puro construct, pTE4396 (#74041; Addgene, Cambridge, MA, USA) was modified by removing the NeoR/KanR sequence using BstBI and SmaI. Then, the mCherry-Puro sequence from pSicoR-Ef1a-mCh-Puro (#31845; Addgene, Cambridge, MA, USA) digested with the same restriction enzymes was inserted into the aforementioned NeoR/KanR-deleted vector. The crDNA #1 or crDNA #2 was inserted into the pTE4396 and AsCpf1-mCherry-Puro vectors in the U6 promoter region using BsmBI enzyme, respectively. The two vectors were transfected with lipofectamine 3000 (Life Technologies, Grand Island, NY, USA) and selected with puromycin and G-418 antibiotics. As a result, we established a cell line into which two vectors (pTE4396+crFOXN1#2 and AsCpf1- mCherry-Puro+ crFOXN1#1) and were inserted. Further studies are needed to characterize FOXN1 KO cell lines.

The production of feline induced pluripotent stem cells (iPSCs) can solve the problems that are related with existing unstable supply and demand of eggs as well as ethical aspects about embryonic stem cell at the same time. On the basis of excellent proliferation, it is to facilitate the researches about human disease like FIV and Allergen at the level of cells, not experimental animals. But, a lot of advanced researches are lean too much towards on the transduction using DNA type virus that have the risk of tumorigenesis during reprogramming and on the mLIF-dependent culture condition for the production of feline iPSCs. This being so, this study shows the reprogramming results using Sendai virus vector that is RNA type virus and have no the footprint after transduction. In addition, the feline iPSCs were stably cultured in bFGF-dependent culture condition during the reprogramming step and culture step. In conclusion, we found the bFGF-dependent culture condition in feline iPSCs and suggested the approach using Sendai virus vector as an alternative for reprogramming without concern about tumorigenesis. These methods can be universally applicable to not only the researches about reconstruction and conservation of feline species, but also to a lot of deep studies related with iPSCs or LIF, bFGF to find new approaches.

The purpose of this monitoring is to survey the geographical distribution of tick species using dry ice bait traps and flagging methods at each ten provinces (GangwonⅠ,GangwonⅡ, Gyeonggi, Chungbuk, Chungnam, Gyeongbuk, Gyeongnam, Jeonbuk, Jeonnam and Jeju area) and one Metropolitan area in the Republic of Korea for eight months from April through November, 2016. A total of 65,339 ixodid ticks (8,200 females, 1,988 males, 31,453 nymphs and 23,698 larvae) was collected, belonging to three genera (Haemaphysalis, Ixodes and Amblyomma). Haemaphysalis longicornis was the most commonly collected species, which is represented for 96.88% of all the collected ticks and followed by H. flava (2.69%), I. nipponensis (0.35%), A. testudinarium (0.05%) and H. japonica (0.03%) in the Republic of Korea for the study period. Haemaphysalis longicornis was a dominant species observed in these eleven areas.

The baculovirus expression vector system (BEVS) is an effective and widely used method for the production of recombinant proteins in insect cells or larvae. However, the expression efficiency of foreign proteins using the polyhedrin promoter could not obtain the protein yields observed for native polyhedrin. To enhance the production efficiency of foreign protein in baculovirus expression system, the effects of various enhancer factor were investigated by fusion expressing them with the enhanced green fluorescent protein (EGFP). As a hyper expression factor, the optimal hyper BEVS was constructed by combining Hr5 sequence, VP39 promoter and Burst sequences. Additionally, the proteins expressed by the hyper expression system was markedly increased. This study suggests a new option for higher expression of useful foreign recombinant protein using the BEVS.

The interactions of vector-virus-plant have important ecological and evolutionary implications. In this presentation, I will use the case studies on the whitefly vectors, begomoviruses, and plants to illustrate the complexity, consequences and mechanisms of this type of tripartite interactions. The interactions between begomoviruses and their whitefly vectors via their shared host plants can be mutualistic, neutral or negative depending on the species/strains of each type of the organisms involved. With regard to the mechanisms of plant-mediated positive effects of the viruses on whiteflies, three case studies indicate that suppression of jasmonic acid/salicylic acid related plant defence plays an important role. Our recent studies show that the order of arrival of the interacting vector insects and viruses on the plants may also alter the physiological feature and consequences of the interactions. Future efforts in this area should try to expand the number and diversity of case studies in order to reveal the patterns of interactions, to unravel the molecular and biochemical mechanisms of the interactions using a multidisciplinary approach, and to examine the virus-plant-vector interactions in the field and in natural plant communities.

The knock-in efficiency in the fibroblast is very important to produce transgenic domestic animal using nuclear transfer. In this research, we constructed three kinds of different knock-in vectors to study the efficiency of knock-in depending on structure of knock-in vector with different size of homologous arm on the β-casein gene locus in the somatic cells; DT-A_cEndo Knock-in vector, DT-A_tEndo Knock-in vector I, and DT-A_tEndo Knock-in vector II. The knock-in vector consists of 4.8 kb or 1.06 kb of 5’ arm region and 1.8 kb or 0.64 kb of 3’ arm region, and neomycin resistance gene(neor) as a positive selection marker gene. The cEndo Knock-in vector had 4.8 kb and 1.8 kb homologous arm. The tEndo Knock-in vector I had 1.06 kb and 0.64 kb homologous arm and tEndo Knock-in vector II had 1.06 kb and 1.8 kb homologous arm. To express endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of endostatin gene and inserted into exon 7 of the β-casein gene. The knock-in vector and TALEN were introduced into the bovine fibroblast by electroporation. The knock-in efficiencies of cEndo, tEndo I, and tEndo II vector were 4.6%, 2.2% and 4.8%, respectively. These results indicated that size of 3’ arm in the knock-in vector is important for TALEN-mediated homologous recombination in the fibroblast. In conclusion, our knock-in system may help to create transgenic dairy cattle expressing human endostatin protein via the endogenous expression system of the bovine β-casein gene in the mammary gland.

In the present study, using a MoMLV-based retrovirus vector, we successfully generated a new transgenic chicken line expressing high levels of hEPO. A replication-defective Moloney murine leukemia virus (MoMLV)-based vectors packaged with vesicular stomatitis virus G glycoprotein (VSV-G) was injected beneath the blastoderm of non-incubated chicken embryos (stage X). One rooster was mated to wild-type hens to produce 748 G1 progeny. PCR analysis of blood samples from these progeny revealed that there were seven G1 transgenic offspring, corresponding to a 0.9% germline transmission rate. Subsequently, Southern blot analysis of the genomic DNA from three G1 transgenic chickens was carried out to verify the stable genomic integration and copy number of the transgene in the genome. Quantitative analyses of the blood samples taken from G1 transgenic chickens resulted in 4,150 ～ 10,823 IU/㎖ (34.6 ～ 90.2 ㎍/㎖) of hEPO in the blood. The biological activity of the recombinant hEPO in transgenic chicken serum was comparable to its commercially available counterpart. Red blood cell numbers were more than three-fold higher in the transgenic chickens compared to the non-transgenic chickens. Successful germline transmission of the transgene was also confirmed in G2 transgenic chicks produced from crossing G1 transgenic roosters with non-transgenic hens. We confirmed that 13 transgenic chicks of 45 G2 progeny, corresponding to a 28.9% germline transmission rate. These results will help establish a useful transgenic chicken model system for studies of embryonic development and for efficient production of transgenic chickens as bioreactors. This work was supported by the Bio-industry Technology Development Program, Ministry of Agriculture, Food and Rural Affairs, Republic of Korea, and by a grant from the Next-Generation BioGreen 21 Program (No. PJ011178), Rural Development Administration, Republic of Korea.

Many transgenic domestic animals have been developed to produce therapeutic proteins in the mammary gland. However, purification of therapeutic proteins from transgenic milk are very important for productivity of recombinant protein. Development of a knock-in vector system is needed to improve production of therapeutic proteins. In this study, we are develop Knock-in vector to express human Erythropoietin protein (hEPO) using Gluthathione S-transferase (GST) fusion system on mouse β-casein exon 3 locus. The knock-in vector consisted of the 5 homologous arm (1.02 kb), GST, PreScission protease site, hEPO cDNA, BGH polyA signal, CMV-EGFP, and 3homologous arm(1.81 kb). The analysis of nucleotide and amino acid sequence revealed that GST-hEPO mRNA is probably translated with the mouse β-casein sequence and the β-casein-GST-hEPO fusion protein is probably secreted by ER-Golgi pathway. After that, the hEPO protein can be cleaved to remove the GST from the fusion protein by PreScission protease during purification of recombinant protein. This knock-in vector may help to create transgenic mouse expressing human Erythropoietin protein via the endogenous expression system of the mouse β-casein gene in the mammary gland.

The production of therapeutic proteins from transgenic animals is one of the most important successes of animal biotechnology. Endostatin is 20 KDa C-terminal fragment derived from type XVIII collagen and an endogenous inhibitor of tumor growth by inhibition of angiogenesis. In this study, we are developed knock-in vector consists of 5’ arm region (1.02 kb), human Endostatin cDNA, CMV-EGFP, and 3’ arm region (1.83 kb). To express Endostatin gene as transgene, the F2A sequence was fused to the 5’ terminal of Endostatin gene and inserted into exon 3 of the β -casein gene. If this knock-in vector is inserted into the porcine β-casein gene locus by homologous recombination, human Endostatin mRNA are expressed using the gene regulatory region of the β-casein. Also, the β-casein and Endostatin fusion protein is translated and Endostatin protein is separated by F2A self cleavage during translation. In conclusion, our knock-in vector may help to create transgenic pig expressing human Endostatin protein via the endogenous expression system of the porcine β-casein gene in the mammary gland.

Pine wilt disease (PWD) is one of the most important forest tree diseases, especially in the East Asian countries of Japan, China, and Korea. The Japanese pine sawyer, Monochamus alternatus Hope, is the PWN vector for Japanese red pine and Japanese black pine while Monochamus saltuarius Gebler is the vector for Korean white pine. Various control methods, such as aerial pesticide applications, biological control using parasitoids and fumigant such as methyl bromide are used. But the PWD still has spread. Therefore, we were selected effective aerial insecticides. Susceptibility of M. saltuarius and M. alternatus adults were investigated using 9 insecticides which are available in the market in Korea. And then, we tested them to the Apis mellifera to vertify the environmental impact.

Recombinant proteins including a polypeptide fusion partner, termed affinity tag, to facilitate the purification of the target polypeptides are widely used. However, the affinity tag must be removed from the target after the purification process. Recently, the Synechocystis sp. PCC6803 DnaB mini-intein (Ssp DnaB mini-intein) is widely used in Escherichia coli expression systems as the solution of this problem. The Ssp DnaB mini-intein can be induced simply by shifting of pH and temperature, offering a benefit to cleave a peptide bond without using a protease or chemical reagent. Although the utility of this novel tag is widely studied in E. coli, there is no report yet in baculovirus expression vector system (BEVS). In this study, we generated several recombinant baculoviruses to express foreign proteins with Ssp DnaB mini-intein. In conclusion Ssp DnaB mini-intein was good tag also in BEVS with more advantages.

As the malaria surveillance agency, the Korean Center for Control and Prevention (KCDC) has been monitoring malaria vector mosquito density and Plasmodium vivax infection since 2009. Anopheline mosquitoes were collected at 35 sites in Incheon, northern Gyeonggi and northern Gangwon Province using black light trap daily from April to October 2015. P. vivax infection of malaria vector mosquitoes tested by polymerase chain reaction (PCR). In 2015, a total of 57,926 malaria vector mosquitoes were collected among 138,119 mosquitoes. Three P. vivax positive pools were detected among 1,556 pools (13,745 individuals) and its minimum infection rate was 0.22. According to the monitoring result of malaria vector mosquitoes, early summer (June-July) could be an appropriate time for a malaria elimination campaign until September.

Alternative control agents, hopefully overcoming the present issues and problems in agrochemicals, should be considered and finally applied to the vector controls. Entomopathogenic fungi can used as one of the possible novel control agents and great considerations are given to the control of soil- or water-dwelling stages of vectors, such as mosquitoes and ticks. In our research group, the entomopathogenic fungal library has been constructed using the mealworm-based isolation system, which showed a variety of opportunities of their use in vector control. Important key production technologies including granular formulation have been developed to increase their industrialization. Some entomopathogenic fungal isolates showed high biological performance in the control of mosquitoes and ticks in field stands. R&D of down-stream process should be seriously considered as the key step.

The Asian tiger mosquito, Aedes albopictus, is considered as potential vector of Zika virus in Republic of Korea (ROK). Vector control during mosquito season is one of critical factors for decline of viral transmission. Total 14 oversea travel-associated Zika cases by mosquito bite were reported throughout ROK from January to September 2016 and vector control and monitoring at surroundings of patient’s residences was carried out during three weeks after confirmation of the virus. Although population density rates of Ae. albopictus were remarkably various according to ecological surroundings, population density of Ae. albopictus near forest was higher than urban. All captured Aedes mosquitoes were used for detection of three flavivirus, Zika, Dengue and Chikunguya, using RT-PCR and any virus was not detected. Population density of Ae. albopictus decreased > 65% on average after vector control and in one area > 95% of population density decreased. Our data might reveal that vector monitoring and control at surroundings near residences of oversea travel-associated Zika patients might effectively prevent viral transmission by mosquito bite and naturalization of the virus in ROK.

Since first reported in 1988 the Pine Wilt Disease has been established and spreaded nearly nationwide in South Korea, causing tremendous economic damage in pine forests. The pine wilt nematode (Bursaphelenchus xylophilus), causing the Pine Wilt Disease, in Korea is known to be transported by two insect vectors that are Monochamus alternatus Hope and Monochamus saltuarius Gebler. Currently the pest management strategy largely relies on pesticide application and infected tree clearing. The aim of the present study was to search for early larval parasitoids of M. alternatus using the sentinel log infested with the cerambycid eggs. Only one braconid species was collected and identified as Spathius verustus Chao. Two to ten wasp larvae attached and fed on one host larva. Parasitism rate was 55.5 to 59 % in Jinju site, while 1 to 18.1 % the other sites. Sex ratio was female-biased (female : male = 18.9 : 1). The S. verstus has potential to be use as parasitic natural enemy of M. alternatus.

Nucleotide metabolism in endothelium is variable between different species. Recent studies demonstrated that this variability could contribute coagulation dysfunction, even though organs of the alpha 1,3-galactosyltransferase gene knockout pig were transplanted into the primate. CD73 (ecto-5'-nucelotidase) is an enzyme at cell surface catalyzing the hydrolysis of adenosine triphosphate to adenosine, which plays role on a substance for anti-inflammatory and anti-coagulant. Thus, overexpression of CD73 in endothelial cells of the pig is considered as an approach to reduce coagulopathy. In this study, we constructed a human CD73 expression vector under control of porcine Icam2 promoter (pIcam2-hCD73), which is expressed specifically at endothelial cells, and of CMV promoter as a control (CMV-CD73). First, we transfected the CMV-CD73 vector into HEK293 cells, and then confirmed CD73 expression at cell surface by flow cytometry analysis. Next, we transfected the pIcma2-CD73 and CMV-CD73 vectors into primary porcine fibroblasts and endothelial cells. Consequence was that the pIcma2-CD73 vector was expressed only at the porcine endothelial cells, meaning that the pIcam2 promoter lead to endothelial cell-specific expression of CD73 in vitro. Finally, we nucleofected the pIcam2-hCD73 vector into passage 3 fibroblasts, and enforced hygromycin selection of 400mg/ml. We were able to obtain forty three colonies harboring pIcam2-CD73 to provide donor cells for transgenic cloned porcine production.