Over the course of two winters, the significant decline in honey bee populations in Korea has emerged as a major social issue. This phenomenon is expected as attributed to factors such as the failure of pest control due to the pesticide resistance of the Varroa mite. This mite can transmit some viruses that infect honey bees, and these viruses are among the primary causes of the globally occurring colony collapse disorder. Traditional diagnostic methods like (RT-)PCR and ELISA are not ideal for identifying pathogens that are newly emerging or have undergone mutations. To detect any novel or mutated viruses beyond those that have been primarily diagnosed in Korea, we introduced virome analysis technology in the field of honey bees. Employing this method with high-throughput sequencing techniques, we were able to identify all existing viruses within individual or group samples. We discovered that the Lake Sinai virus, which has been reported worldwide but not in Korea, has already significantly spread within the country. Additionally, we were able to confirm the prevalence of viruses previously reported in Korea, such as the recently dominant Black Queen Cell Virus. Through this virome analysis, we can provide foundational data for determining the direction and countermeasures for virus diagnosis.

Wild birds, especially aquatic birds, are the natural reservoir of avian influenza virus (AIV), and many kinds of water body can be contaminated with feces of these birds. Seasonally, AIVs can be dissolved in the environmental water from the feces of the infected birds, and this water can be a target for viral detection and identification. In this study, we employed and tested three different filters for concentrating AIV, and it was shown that high concentration factor in terms of viral density could be achieved with viral samples diluted with natural water. Wild bird fecal samples containing low pathogenicity H5 AIVs were successfully concentrated with the adsorption and elution method using mixed cellulose esters membrane; the recovery rate of virus was 35.5 % and the concentration factor was about 50 on average. For the larger volume of water sample, we proved that an inline disposable filter with high surface area, 300 cm2, has a comparable concentration factor to the adsorption and elution method and the filter could be used in the field conveniently by being plugged into peristaltic pump. These validated methods for water sampling may be used as a supplementary for virological surveillance on wild migratory birds or during the epidemiological investigation on the environment near affected premises by AIV.

African swine fever (ASF) is a hemorrhagic viral disease of pigs requiring laboratory diagnosis for confirmation. Though tissue and blood samples are considered optimal for ASF diagnosis, collection of these samples can be laborious, time-consuming, and pose a risk of contaminating the environment. Here, we suggest an alternative non-invasive sampling method, hair plucking, for ASF diagnosis. ASF virus was detected in plucked hair samples from experimentally infected pigs. Although the sensitivity was inferior to whole blood, the results suggest that hair plucking can be an alternative method that can also improve animal welfare.

Ixodidae에 속하는 일부 참진드기들은 중증열성혈소판감소증후군(severe fever with thrombocytopenia syndrome; SFTS)을 비롯한 질병을 매개하는 공중보건학적으로 중요한 해충이다. 우리나라에서는 2013년 이후로 SFTS 환자 발생수가 지속적으로 증가하는 경향을 보이며, 경상북도는 전국적으로 2번째로 많은 SFTS 환자가 발생하는 지역이다. 본 연구에서는 2019년 경북 상주 지역의 진드기 분포를 조사하기 위해 시민의 생활 반경 주변을 도심 녹지지역, 관리 취약지역 및 농촌지역으로 구별하여 flagging 방법을 통해 진드기를 채집하였고, 채집 진드기내 SFTS 바이러스 보균 여부를 조사하였다. 채집된 진드기 수를 Collection Index (CI = tick number / 1h / 2 people)로 산출한 결과, 상주시 내 총 26개 지점에서 작은소피참진드기와 개피참진드기, 일본참진드기를 포함하는 총 CI 143의 진드기가 채집되었고, 그 중 작은소피참진드기가 96.5%(CI 138)로 우점종으로 확인되었다. 채집된 진드기의 약 92%(131 CI)는 인적이 드물고 시청이나 관계 당국의 관리가 이루어지지 않는 관리 취약지역에서 채집되었으나, 도심의 녹지지역와 농촌 지역에서는 8.4%(CI 12)의 진드기만 관찰되었다. 총 CI 143의 진드기를 26개 pooling 한 후 SFTS 바이러스 존재 여부를 조사하였으나, 모두 음성으로 확인되었다. 본 연구의 결과는 지역주민들로 하여금 진드기 매개 질병으로부터 안전한 생활을 하기 위한 권고 자료로 활용할 수 있을 것으로 기대된다.

In the case of foot-and-mouth disease (FMD), there is a great deal of impact on the national economy due to the disposal of diseases, the cost of disease control such as vaccination, reduction of productivity, and restriction of international trade of livestock products. Therefore, appropriate diagnostic methods for sensitive, accurate and rapid identification of virus serotypes are continuously required in terms of early prevention of FMD. This study was conducted to confirm the feasibility of immuno-PCR diagnostic method for the more sensitive detection of Korean FMD virus (FMDV). We synthesized a partial FMD type A viral gene. Protein antigen, monoclonal and polyclonal antibodies of FMDV were cloned, expressed and purified and then magnetic particles were attached to polyclonal antibodies and and oligomers to monoclonal antibodies for the immnuno-PCR. We confirmed the antigen-antibody and oligomer reaction using ELISA, Western blot, and real-time PCR. These results show that Korean FMDV can be detected by using difference of Ct values between positive group and negative group using immuno-PCR.. The results of this study also suggest that this technique will be the basis of the diagnosis method to detect Korean FMDV more sensitively in the future.

We monitored the population of Haemaphysalis longicornis, in Andong, Gyungbuk from April to November, 2018. Among total 2,994 ticks collected for 8 months, H. longicornis 1,677(56%), H. spp Larva 1,074(35.9%), H. flava 213(7.1%), Ixodes nipponensis 30(1.0%) were identified. In addition, considering the environment, 1,727(57.7%), 907(30.3%), 192(6.4%) and 168(5.6%) ticks were collected in the grassland, graves, copse, mountain path, respectively. In the pathogen diagnosis with PCR using SFTS virus specific primers, positive viruses were detected in H. longicornis, H. flava and H. spp from June to October. The minimum field infection rate of June, July, August, September and October were 0.4%, 0.8%, 1.2%, 0.8% and 2.3%. respectively.

Heamaphysalis longicornis is a major vector for Severe Fever with Thrombocytopenia Syndrome (SFTS) virus and the density of the vector has been increasing because of the climate change. The incidence of fatalities due to SFTS is increasing every year. In this study, to evaluate the SFTS transmission by ticks, the density of ticks mediating SFTS was monitored. Tick was collected every month from four different sites (Grass land, Mountain path, Grave, Copse) in Andong with the traps containing dry ice as CO2 attractants. Among 2,572 ticks of 3 species; H. longicornis, H. flava, and Ixodes nipponensis were most abundantly collected from April to August. H. longicornis is the richest species (92.8%), whereas Ixodes nipponensis was the least species (0.8%). The 54.5% of the sample were nymph stage and female/male ratio was 64.3%. According to the pathogene analysis, SFTS virus was detected from H. longicornis adult and larvae stages collected in July and August. In July samples, SFTS virus was detected only from grassland site, but the virus was observed in the sample from all four sites in August. For effective prevention of SFTS, the tick density should be continuously monitored based on the onset time of SFTS with the consideration of habitate, habit & life history of ticks.

West Nile Virus (WNV) is transmitted by infected mosquitoes. Vector mosquitoes usually acquire these pathogens fromfeeding on an infected host, and transmit the pathogens to a naive host during feeding events. To understand the virustransmission dynamics and to survey WNV throughout country, the present study has been conducted. We collected mosquitoesat urban parks in Seongnam, Wonju, Gunsan, Daegu, and Tongyeong using CDC light trap with Dry ice from April toSeptemper in 2017 (mosquito collecting is on going). Among collected mosquitoes, blood-fed mosquitoes were conductedblood meal identification assay and the other mosquitoes were subjected to virus detection using real-time PCR method.A total of 2,290 mosquitoes representing 6 genera and 15 species were collected. The most dominant species was Culexpipiens complex (42.1%) followed by Aedes albopictus (15.1%), Ae. vexans nipponii (14.6%), Ochlerotatus koreicus (9.8%),Cx. orientalis (6.5%), and Armigeres subalbatus (4.4%). The blood meal source were of mammal (93.3%), and birds (6.7%).So far, no WNV has been detected in any mosquitoes.

Cherry leaf roll virus(CLRV)는 group IV positive sense ssRNA viruses, Nepovirus로 분류되는 식물병원성 바이러스이다. CLRV는 체리 등 목본 및 완두 등 콩과 작물을 자연 기주로 하며, 실험적으로 약 36개 과 이상의 넓은 기주 범위를 가지고 있어 국가적, 경제적 및 농가 개인적 피해를 야기 할 가능 성이 보고되고 있다. 현재 CLRV를 검출하는 방법으로 역전사(reverse transcription; RT)-nesdted 중 합효소연쇄반응(polymerase chain reaction; PCR) 이 활용되고 있으며, 다양한 기주로부터 CLRV를 검출하기 위해서는 검출 감도, 특이성, 반응 시간, 단순성 등이 중요 요소였다. 그러나 RT-nested PCR은 두 단계로 구성되어 있어 단순하지 않고, CLRV를 검출하는데 약 10시간 이상의 반응 시간이 소 요되었다. 이번 연구에서는 등온증폭법을 이용하여 단순하고 신속하게 CLRV를 검출하는 방법을 개발하 였다. 등온증폭 반응은 RT-nested PCR과 동등한 검출 감도로 CLRV를 검출 하였다. 그러나 반응 시간 을 약 2시간 수준으로 단축하였으며, 6개 영역을 사용하는 등온증폭 프라이머의 사용으로 더욱 특이적 으로 증폭 할 수 있었다.

West Nile Virus (WNV) is transmitted by infected mosquitoes. Vector mosquitoes usually acquire these pathogens from feeding on an infected host, and transmit the pathogens to a naive host during feeding events. To understand the virus transmission dynamics and to survey WNV throughout country, the present study has been conducted. We collected mosquitoes in Jeju, Busan, Gunsan, and Incheon using CDC light trap and BG Sentinel trap from April to October in 2016. Among collected mosquitoes, blood-fed mosquitoes were conducted blood meal identification assay and the other mosquitoes were subjected to virus detection using real-time PCR method. A total of 29,603 mosquitoes representing 8 genera and 19 species were collected. The most dominant species was Culex pippins complex (35.0%) followed by Cx. bitaeniorhynchus (12.2%), Armigeres subalbatus (11.2%), Aedes albopictus (10.8%), Ae. vexans nipponii (10.3%), and Ochlerotatus dorsalis (8.4%). The blood meal source were of mammal (70.4%), birds (29.0%) and amphibian (0.6%). WNV was not detected in any mosquitoes.

Potato Virus Y (PVY) (Potyviridae: potyvirus) is one of the serious emerging virus of seed potato world-wide. It affects the seed potato by transmitting non-persistently via aphids. Here, we developed a simple PVY detection method which used the boiling technique for releasing of the viral RNA from aphid such as stylet and amplification by PVY specific primers located in the viral coat protein gene which suitable for various strains. This simplified method could save the time compared to earlier detection method due to the simplified RNA extraction step. Following this procedure, we tested this one step RT-PCR based PVY detection method by using three PVY vectoring aphid species (M. persicae, A. gossypii and M. euphorbiae) as well as other sucking type insect such as thrips (F. occidentalis). This PVY detection method is rapid, easy-to-use and suitable for large-scale testing in laboratories of seed potato.

Pelargonium zonate spot virus (PZSV)는 group IV (+) ssRNA viruses, Bromoviridae에 속하는 식물 병원체로, 일반적으로 토마토, 국화, 아티초크 및 제라늄에 감염된다. 본 연구는 검역 현장에서 PZSV를 신속하고 특이적으로 진단 할 수 있는 PCR module을 개발하는 것을 목적으로 하였다. PZSV를 검출하기 위한 RT-PCR 프라이머 선발 결과, 각각 513 및 320 bp를 증폭하는2개 조합을 선발하으며, 더욱 높은 검출감도로 검출할 수 있을 뿐아니라 RT-PCR을 검증할 수 있는 nested PCR 프라이머 조합을 개발하였다. 또한, 제한효소 Xho I 부위를 삽입한 유전자변형-양성대조구 플라스미드를 설계하여, PCR module에서 대조구로부터 오염을 검증할 수 있도록 개발하였다. 본 연구에서 개발한 PCR module은 토마토, 국화, 아티초크 및 제라늄 등에서 PZSV를 간편, 신속 및 특이적으로 검출하여, 지속적으로 식물검역에 활용할 수 있을 것으로 기대된다.

PVY (Potyviridae: potyvirus) is one of the most important potato virus affecting seed potato production and also it is transmitted non-persistently via aphids. For healthy seed potato production, a virus detection system is highly important in addition to aphid monitoring and control. To achieve this detection method, it need to fast and easy to use. About two decades ago RT-PCR based PVY detection method was developed. However that was very time consuming and has low sensitivity. Here, we developed an advanced PVY detection method which a uses the boiling extraction of the viral RNA from aphid stylet and amplification by specific primers located in the viral capsid protein gene. Therefore, it could directly synthesize cDNA of PVY viral capsid gene from extracted RNA of PVY using one-step RT-PCR method in very short time compared to previous methods due to the omission of RNA extraction step. We confirmed this PVY detection method using the two aphid species (Macrosiphum euphorbiae and Aphis gossypii) that known as PVY vectors. The efficiency of this PVY detection method was 60% to 80% from two the aphid species. Hence, this method could be potentially applied to virus free seed potato production programs.

Sacbrood virus (SBV) is one of the most destructive honey bee virus. The virus causes failure to pupate and kills honey bee larvae. The infacted larvae`s color is change to brown. At the end, honey bee colony is destructed. Recently Korean Scabrood virus(KSBV) caused a great loss of Korean honey bee(Apis cerena) colonies for short period. Therefore, We need a highly rapid diagnosis method for rapid detection of KSBV. In this study, We need amicro-scale chip-based real-time PCR system (GeneChecker®). This system was developed for rapid, specific PCR based diagnosis. This system has uncommonly fast heating and cooling system. So We was able to detecting of KSBV in Apis cerena in short time. This system needs small reaction volume(total 10ul). This volume include SsoFast™ Evagreen Supermix and serially diluted cDNA templates showed a high sensitivity of 101copies.That machine can setting each PCR stage time. A specific detection primer set (KSBV-123-F/R) was used to amplify a unique 123bp DNA fragment. This PCR assays using serially diluted cDNA templates showed a high sensitivity of 101 copies. When applied to KSBV-positve samples, the result showed high specifity. The minimum diagnosis time was 9m 47s (30cycle). The amplied positive samples appear red fluorescent color. This novel detection method could be used a PCR-based diagnositic tool (GeneChecker®). The results showed high sensitivity and specifity in short time. And this diagnosis method is expected to be applied to rapidly detect various pathogens.