본 실험에서는 고분자의 농도, 첨가제의 종류 및 함량에 따라 도프 용액을 이용하여 분리막 제조하였다. 분리막의 모폴로지는 전자주사현미경(SEM)을 통해 관찰 하였으며, 막의 모폴로지와 순수투과도의 관계를 확인 할 수 있었다. 필터화 모듈을 제조하여 수투과도 및 바이러스, 박테리아 등과 같은 미생물 제거성능을 측정하였다. 제조된 중공사막의 단면은 sponge 형태로 표명으로 갈수록 치밀한 형태를 띄는 것을 확인하였으며, 수투과도는 50-70 ml/min으로 높은 값을 나타내었으며, 필터화모듈의 경우 수투과도는 1.6 LPM의 높은 투수량을 나타내며, 박테리아와 바이러스의 제거성능은 log 6의 값의 높은 수치를 보였다.

Porcine epidemic diarrhea virus (PEDV) causes acute diarrhea and dehydration in pigs with high mortality. PEDV is belong to Coronavirus, enveloped, single-stranded, positive-sense RNA virus. PEDV particles were composed of four structure proteins such as a glycosylated peplomer (spike, S) protein, envelope (E), glycosylated membrane (M) protein, and unglycosylated RNA-binding nucleocapsid (N) protein. Many of previous studies talk about this four structure proteins have a great potential to diagnosis and prevent PEDV. In this study we investigated expression of these structure proteins using the bacterial and baculovirus expression system. In bacterial expression system, our results showed that structure proteins fused polyhedrin and intein gene were expressed higher than non-fusion structure proteins. The expressed fusion proteins were used to immune mice for generating a polyclonal antibodies. In baculovirus expression system, co-infection of insect cells with these four recombinant baculoviruses led to self-assembly of virus-like particles as demonstrated by Transmission electron microscopy. They were confirmed by western blot analysis using pre-made polyclonal antibodies. Finding in this study may provide important information for vaccine and diagnostic development.

Human hepatitis B virus (HBV) infection remains a serious public health problem worldwide, as it is one of the main risk factors for hepatocellular carcinoma (HCC). Cross-species transmission of HBV has been reported in non-human primates, and pigs may also be infected with HBV or an HBV-like agent. The purpose of this study was to demonstrate the presence of HBV antigens and anti-HBV antibodies in pig sera, providing further support for the existence of HBV or an HBV-like agent in pig populations. The HBV surface antigen (HBsAg) and HBV e antigen (HBeAg) in pig serum samples were detected using HBsAg and HBeAg ELISA Kits, respectively. Antibodies to HBsAg and the Hepatitis B core antigen (HBcAg) in serum samples were also detected using anti-HBsAg and anti-HBcAg antibody ELISA Kits, respectively. HBsAg and HBeAg were detected in 7 of 442 (1.6%) and 7 of 184 (3.8%) pig serum samples, respectively. Furthermore, antibodies specific to HBsAg and HBcAg were identified in 45 of 442 (10.2%) and 39 of 434 (9.0%) pig serum samples, respectively. However, neither HBV DNA nor antibodies to HBeAg were detected in 409 and 298 pig serum samples, respectively. HBV antigens and anti-HBV antibodies were both present in a considerable number of pig serum samples, suggesting that pigs could be infected with a variant HBV or an HBV-like agent. Further studies will be necessary to confirm cross-species infection of pigs with HBV.

Tomato ringspot virus (ToRSV)는 Group IV(+) sense ssRNA viruses, Nepovirus속으로 분류되며 식물병원성을 가진 국내 미보고 관리급 검역바이러스이다. 본 연구에서는 우리나라 검역현장에서 ToRSV를 검사하기 위한 reverse transctiption(RT)-polymerase chain reaction(PCR)과 nested PCR 진단시스템을 개발하였다. ToRSV 특이적으로 진단할 수 있는 RT-PCR은 프라이머 조합9 (F120/R20, 549 bp)와 조합31(F60/R80, 741 bp)]이며, 각각의 nested PCR 결과 439와 363 bp를 증폭할 수 있다. 한편, 본 연구에서 개발한 유전자변형-양성대조구는 실험실 오염으로 인한 거짓양성반응을 확인할 수 있다. 본 연구에서 개발한 ToRSV 검역진단시스템은 향후 식물검역에 기여할 것으로 기대된다.

The efficacy of chemical disinfectants is reduced owing to the inactivation of active ingredients after dilution. This study investigated the effect of time on the efficacy of six different disinfectants, after dilution, against avian influenza virus. When used at the recommended concentration, most disinfectants showed efficacy at a high concentrations in the presence of organic materials immediately after dilution, while sodium dichloroisocyanurate-based products, after dilution, showed reduced efficacy over time at low concentrations in the absence of organic materials. Most disinfectants were neutralized by organic materials; however, this could be compensated for by increasing the product dosage. For successful decontamination in farms, disinfectants should be used at high concentrations in accordance with the manufacturers’ recommendations. Furthermore, the presence of organic materials must be taken into consideration, and diluted disinfectant solution should be prepared no more than a day before use.

Porcine epidemic diarrhea virus (PEDV) causes porcine epidemic diarrhea (PED) and a considerable economic loss in the swine industry. In this study, the virucidal efficacy of a disinfectant composed of citric acid, benzalkonium chloride and phosphoric acid against PEDV was investigated. Virucidal efficacy was assessed as the infectivity of PEDV toward Vero cells after exposure of the virus to the disinfectant. PEDV was exposed to the disinfectant in the presence of either hard water (HW) or an organic matter suspension (OM). In HW condition, PEDV was inactivated by 600-fold dilutions of the disinfectant. In the presence of OM, the disinfectant showed virucidal activity after a 200-fold dilution. As the disinfectant possesses virucidal activity against PEDV, it should be an effective reagent to use to limit the spread of animal viral diseases.

담배가루이(Bemisia tabaci)는 외래해충으로 바이러스벡터로 작용하여, 토마토의 토마토황하잎말림병바이러스(TYLCV)를 비롯한 약 100여종의 바이러스를 매개하는 중요한 해충이다. 본 연구에서는 VIGS vector를 이용하여 담배가루이 방제를 위한 target 유전자들을 선발하기 위 해 gateway system을 이용한 담배가루이 cDNA library 제작을 시도하였다. 첫 번째 방법으로 oligo d(T) primer를 사용하였을 때, 평균 약 1 kb의 insert와 1.4×10 4 cfu의 titer를 확인하였다. 그러나 insert size가 너무 커서 적절하지 않았다. 두 번째 방법으로 attB-N25 random primer를 이 용하고, sonication을 6초 실시하여 다시 진행하였다. 그러나 확인되는 insert size는 다소 컸고, 몇몇은 insert가 너무 작아서 밴드가 확인 되지 않았으며, 1.04×10 5 cfu의 titer를 확인할 수 있었다. 세 번째 방법으로는 oligo d(T) primer를 이용하였고, sonication을 2초 실시하였다. 그 결 과 300 bp~600 bp size의 insert가 확인되었으나, electro transformation을 사용한 첫번째, 두번째 방법에 비해 heat shock transformation을 사용하여 titer가 5.2×10 2 cfu로 매우 낮은 것을 확인 할 수 있었다. 결과적으로 cDNA library를 만들 때 먼저 random primer를 사용하여 First strand를 합성하여 poly A를 제거하고, 다음으로 sonication을 1초 실시하여 300~700 bp정도의 적절한 size의 insert를 생성하고, 마지막으로 electro-transformation을 실시하여 transformation 효율을 높인다면 VIGS vector에 적합한 cDNA library를 만들 수 있을 것으로 사료된다.

The sweetpotato whitefly Bemisia tabaci (Hemiptera: Aleyrodidae) is a disastrous pest in horticultural plants worldwide. B. tabaci is a species complex including at least 24 biotypes in the world. In Korea, B-biotype has been invaded in 1998, Q-biotype in 2005 and widely spread into the country. B. tabaci is also a vector of more than 100 plant viruses, especially begomoviruses. Since 2008, Tomato yellow leaf curl virus (TYLCV) has been invaded into Korea and severely damaged tomato cultivar in all over of the country. B. tabaci is the only vector insect of TYLCV. Here we demonstrated whether TYLCV influence on the vector physiology during virus transmission. Pesticide susceptibility of whiteflies on TYLCV acquisition was determined in B. tabaci using the two-layered parafilm feeding chamber which containing 20% sugar solution including different doses of imidacloprid. Our result showed that TYLCV-viruliferous whiteflies were more susceptible to imidacloprid ingestion than non-viruliferous whiteflies. This study suggests that plant virus can manipulate the physiological conditions of vector insects.

A female wild raccoon dog was referred with a history of generalized seizure. Mild leukocytosis was noted on laboratory tests. Gross lesions included nasal hemorrhage, hemothorax, and hemorrhage in the urinary bladder with hematuria. Microscopically, interstitial and purulent bacterial pneumonia was observed in the lungs. In the cerebellum, characteristic eosinophilic intracytoplasmic and intranuclear inclusion bodies were found in Purkinje cells, and severe demyelination was observed in the cerebellar white matter. Canine distemper virus (CDV) infection was suspected and confirmed after detection of CDV nucleoprotein RNA in the cerebrum and the lungs by nested reverse transcription polymerase chain reaction (RT-PCR) and reverse transcription loop-mediated isothermal amplification (RT-LAMP). Based on the histopathological and molecular diagnostic findings, it was concluded that the raccoon dog was infected with CDV.

Kidney cells of canine embryos were separated into single cells using collagenase and dispase. Primary culture was conducted using these cells. To remove fibroblasts, these cells were treated with edetate disodium dihydrate (Na2EDDA), and pure epithelial cells were separated. Recombinant retrovirus particles that manifest teromerase were produced and inoculated into primary culture cells to produce immortalized canine cell strains (JNUCK-1 and JNUCK-2). To examine the characteristics of the produced cell strains, the growth curve, maximum cultured households, and expressed proteins (keratin) were identified. The JNUCK-1 and JNUCK-2 cell lines showed division ability until the 30th generation without growth retardation. JNUCK-1 and JNUCK-2 cell lines clearly expressed telomerase until the 25th generation. The canine distemper virus (CDV) was inoculated into the JNUCK-1 and JNUCK-2 cell lines, as well as in the Madin–Darby canine kidney (MDCK) cell line. The maximum titer of CDV from the JNUCK-1 cell strain was about 200 times higher than that from the MDCK cell strain. However, the JNUCK-2 cell strain produced a lower titer than the MDCK cell strain. We established a new canine kidney epithelial cell line (JNUCK-1) that could produce CDV with high titer.

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.

고구마 바이러스 무병묘 재배에 따른 세대 간 수량변 이를 구명하기 위하여, ‘안노베니’, ‘연황미’, ‘맛나미’ 등 3품종의 무병묘 세대(TC0, TC2, TC3) 삽수를 75×25cm 로 정식하여 흑색비닐로 멀칭재배하였다. 정식 30일째 줄기신장은 대조구인 농가묘보다 무병묘 세대에서 유의 한 증가를 보였으며, TC0에서 가장 왕성하였다. 120일째 수확기 생육은 줄기길이, 원줄기 마디수와 곁가지수는 농가묘보다 무병묘 세대에서 높았으나, 통계적 유의성은 없었다. 무병묘 세대의 지상부 생체중이 농가묘보다 유의하게 증가하였으나, 무병묘 세대간 그리고 품종간에는 유의한 차이가 없었다. 주당괴근수와 평균괴근중은 농가 묘보다 TC0와 TC1 세대에서 유의한 증가를 보였으나, TC2 세대에서는 농가묘와 차이가 없었다. 무병묘 세대의 주당괴근중은 농가묘보다 유의하게 증가하였고, 무병묘 세대간에는 TC0에서 가장 높았다. 무병묘 세대의 평균 상저수량, 상저비율과 소형 고구마(40-200g) 비율도 농가묘보다 유의한 증가를 보였다. 300g 이상 괴근비율은 TC0 세대에서 가장 낮았다. TC2 세대의 상저수량은 TC0 세대보다 유의하게 낮았고, 농가묘와도 유의한 차이가 없었다. 품종간 상저수량은 ‘맛나미’에서 가장 높았으며, ‘안노베니’, ‘연황미’ 순이었다. 따라서 무병주의 수량과 품질 유지를 위해서 농가는 3년 주기로 교체하는 것이 필요하다. 다만 교체주기는 바이러스 재감염 정도에 따 라 2-3년 주기로 실시하는 것이 바람직할 것이다.

South Korea has over 38 millions of managed honey bee (Apis cerana) colonies before 2009 years ago, which produce the highest quantity of honey in the Korea; however, almost colony (99%) were collapsed by Korean Sacbrood Virus (KSBV) in South Korea. Korean Sacbrood Virus (KSBV) is the pathogen of A. cerana Sacbrood disease, which poses a serious threat to honeybee A. cerana, and tends to cause bee colony and even the whole apiary collapse. Colony collapse of A. cerana was first reported on the Pyeong-Chang of the South Korea in 2009. Symptoms of KSBV include the rapid transmission of larval stage honeybees (A. cerana), many dead larvae found in the bottom of hive and comb. Honeybees (A. cerana) are a very important species because they provide a number of pollination services for various ecosystems in some provinces (ex. jeon-nam, jeon-buk province). They are also extremely important organisms within human society, both agriculturally and economically. The fact that a direct cause has been determined suggests that colony collapse is a complex problem with a combination of natural and anthropogenic factors. Possible instigators of colony collapse include: wax moth, viral and fungal diseases, increased population, decreased genetic diversity, climate changing and a variety of other factors. The interaction among these potential causes may be resulting in immunity loss for honeybees and the increased likelihood of collapse.

The bumblebee, Bombus terrestris, plays an important role as one of alternative pollinators since the outbreak of honeybee colony collapse disorder. Recently, pathogens and parasites such as viruses, bacteria and mites affecting the life span and fecundity of their host have been discovered in B. terrestris. In this study, in order to detect viral infection in B. terrestris, we collected B. terrestris adults and isolated total RNA for diagnostic PCR. The PCR primers specific for pathogenic viruses were newly designed and applied to gene amplification for cloning and detection. Capsid protein gene of black queen cell virus (BQCV) among examined viral genes was only successfully amplified from collected bumble bee adults and sequenced. To optimize the detection of capsid protein gene of BQCV, 4 regions in the capsid protein gene were selected and further analyzed in quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis revealed that capsid protein gene was directly detected with not more than 200 ng total RNA. This result suggests that an optimized detection via qRT-PCR can be applied for the rapid and sensitive diagnosis of BQCV infection in the field population as well as risk assessment of B. terrestris.