Porcine parvovirus (PPV), a member of the genus Parvovirus, family Parvoviridae, is a significant causative agent in porcine reproductive failure, causing serious economic losses in the swine industry. PPV is a non-enveloped virus and its capsid is assembled from three viral proteins (VP1, VP2, and VP3). The major capsid protein, VP2 is the main target for neutralizing antibodies in PPV. When VP2 was expressed in large amounts, it assembled into virus-like particles (VLPs) similar in size and morphology to the original virions. In this study, we generated the recombinant Bombyx mori nucleopolyhedrovirus (BmNPV) to express the VP2 protein. Expression of the VP2 protein was analyzed by SDS-PAGE and Western blot. The recombinant VP2 protein of approximately 64 kDa was detected by both analyses. The formation of VLP by recombinant VP2 was confirmed through transmission electron microscopy examination. The purified VP2 protein assembled into spherical particles with diameters ranging from 20 to 22 nm.

Sacbrood disease is a viral disease on honey bee larvae Apis cerana. Diseased larvae fail to pupae and to be dead at old larvae and pre-pupae stage. Currently, there is no remedy to control sacbrood disease. In this study we conducted to observe sacbrood disease on Apis cerana colonies from June to September, 2014 at the A. cerana apiary of NAAS, and using biological measure to treat this disease. Our study results were showed that sacbrood disease infected A. cerana colonies in all months of observation. The percentage of infected colonies was from 33.3% up to 100%. Controlling sacbrood disease by requeen measure, the percentage of recovered colonies was 57.1 % while of this by cage queen measure was only 28.6 %.

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.

Nucleopolyhedrovirus (NPV), which belongs to Baculoviridae, is a rod-shaped, double-stranded circular DNA virus which infects arthropods, mostly insects. NPVs are highly species-specific, and make unique crystalline polyhedral structure made of polyhedrin protein. The NPVs do not replicate in mammalian cells, are safe to human, and can be observed the viral replication with conventional compound microscope, plus the availability of susceptible insect cell lines, therefore, the NPVs became an ideal model system to study basic virology. Also, NPVs became popular because of its applications for baculovirus expression vector system (BEVS). A foreign gene will be cloned into a shuttle vector, and introduced to the NPV chromosome to make recombinant virus. This NPV will produce the protein in culture cells or host insects under the control of the strong polyhedrin promoter. So far, the commercially available BEVS has been widely used because of its high efficiency and eukaryotic characteristics, however, the hidden bottleneck is finding new useful genes which will maximize the capability of BEVS. Since the human genome project, next generation sequencing technique (NGS) is getting useful in life science field along with the development of sequence analysis algorithms and increase of computational power. Especially, RNA-seq and de novo sequence assembly technics make discovery of new genes easier even in a non-model species with a proteomics approach, and these useful tools will be the key to catalyze the insect biotechnology.

‘급속면역금나노입자막대 (RIGS) 키트’라 명명된 RIGS 키트는 간단한 면역 흡착막대 방식으로 사용자가 빠르고 편리하게 이용할 수 있으며 토마토덤불위축바이러스 (TBSV)에 대한 현장진단을 하기 위하여 개발되었다. 토끼의 TBSV 항 혈청에서 정제된 면역글로블린G (IgG)는 단백질A 크로마토그래피법으로 정제되었으며 이후 금 나노입자와 결합하여 니트로셀룰로스막에서 진단 선을 표시하도록 고안되었다. TBSV 항체와 비특이적으로 결합하는 단백질A를 같은 진단막대에서 대조선으로 이용되었다. RIGS-TBSV 키트를 이용한 진단은 의심 식물 시료를 완충액이 들어간 플라스틱 봉지에 넣고 착즙후 진단막대기를 넣으면 5-10분 후 결과를 알 수 있도록 고안되었다. TBSV가 감염된 토마토 즙액에 RIGS 막대기를 넣고 진단한 결과 TBSV 농도에 비례하여 진단 선이 형성됨이 관찰되었으며, 이들 키트들은 TBSV와 연관되지 않은 다른 고추 바이러스들에서는 비특이적 반응이 형성되지 않았다. 이런 결과들은 RIGS-TBSV 키트가 TBSV 진단에 다른 어려운 실험이나 기술이 필요하지 않고 쉽게 병원균을 진단 할 수 있다는 것을 의미한다. 그러므로, RIGS-44 TBSV 키트는 TBSV 감염이 의심되는 식물체들의 현장 진단 뿐만 아니라 실험실에서 의 TBSV 진단에 효과적이라 할 수 있다.

Bromoviridae과 Cucumovirus속에 속하는 대표 바이러스인 오이모자이크바이러스 (Cucumber mosaic virus: CMV)는 많은 경제적으로 중요한 원예작물 및 관상식물들에 심한 손실을 초래하는 바이러스이다. 다중염기서열 비교는 현재까지 서브그룹1에 속하는 모든 CMV 계통들에서 3'말단부의 보전적 염기서열들이 존재하고 있음을 보여주었다. 이런 관찰에 기초하여, 우리는 CMV RNA3와 상동성을 가지는 162 bp 상보적 DNA를 포함하며 CMV감염에 대하여 식물 유래 RNA간섭 현상을 유도할 수 있는 도치된 반복 구조를 가지는 머리핀RNA (pIR-CMVNCR)를 발현시킬 수 있는 벡터를 제작하였다. 아그로박테리움을 이용하여 IR-CMVNCR의 일시 발현은 CMV 감염을 저해하였으며, 아그로박테리움이 접종된 식물의 상엽에서는 CMV 병징이 발현되지 않았다. 또한 RT-PCR결과는 아그로박테리움이 접종된 식물의 접종엽 및 상엽에서 CMV 서브유전자 4를 포함하는 CMV RNA들이 모두 검출이 되지 않았다. CMV 유래 작은 저해 RNA들의 축적이 관찰되었으며, 이의 결과의 의미는 아그로박테리움에 의해 IRCMVNCR을 일시 발현시킨 야생담배 (Nicotiana benthamiana)의 접종엽에서 RNA 간섭 현상이 유도되어 CMV 감염을 억제시키는 것으로 판명되었다.

A 7-year-old, spayed female, domestic short hair cat showed signs of a 2-week history of chronic anorexia, depression, and severe weight loss. Upon physical examination, pyrexia, mild gingivitis, and pale mucus membranes were noted. Laboratory analysis revealed normocytic normochromic non-regenerative anemia, severe thrombocytopenia, and hypergammaglobulinemia. Serum protein electrophoresis revealed the presence of elevated alpha-2 fraction within the globulin concentration. Based on history, clinical signs, and laboratory results, systemic viral infection was strongly suspected. Reverse transcriptase polymerase chain reaction identified the presence of feline immunodeficiency virus (FIV) in the serum. Furthermore, gene sequencing revealed the virus as FIV subtype A. Treatment with anti-retroviral agents, including azidothymidine (AZT) and recombinant human interferon-alpha, was continued for 4 weeks. However, the patient’s clinical condition deteriorated, resulting in death 1 month after initiation of treatment due to progressive renal failure. Necropsy and histopathology revealed hepatic and renal necrosis with hyper-cellular bone marrow mainly comprised of myeloid precursor cells. This case report is the first to describe phylogenetic subtyping, anti-retroviral combination treatment, and clinical outcomes in an FIV-infected cat in Korea. In addition, this report suggests that treatment should be initiated during the early phase of infection that could be effective for the virus.

Rice stripe virus (RSV) is one of the serious plant pathogenic viruses for rice and mediated by small brown planthopper, Laodalphax striatellus. So far, the studies have been mainly focused on the interaction between the host plant and the virus. In this study, for better comprehension of the interactions among Rice stripe virus, rice and small brown planthopper, transcriptomes of the RSV-viruliferous (RVLS) and non-viruliferous L. striatellus (NVLS) were comparatively analysed. For this, non-viruliferous L. striatellus were collected from non-infected rice field and fed RSV-infected rice for 5 days. With the RNAs prepared from the RSV-viruliferous and the non-viruliferous small brown planthoppers, we conducted Illumina RNA sequencing (Hiseq 2000) and then two transcriptome databases were generated from RVLS and NVLS, respectively. The transcriptome of RVLS and NVLS were campared to figure out how the gene expression of the insects affected by Rice Stripe Virus. RSV-dependently regulated genes analysed from this study may have important functions in the transmission and replication of RSV.

A cell line of bovine origin was immortalized to isolate foot-and-mouth disease virus (FMDV). The immortalization was performed by infection of bovine primary epithelial cells with a recombinant retrovirus that overexpressed the human telomerase (hTERT), after primary culture of fetal bovine kidney tissue and removal of fibroblasts. After cloning the immor- talized cell line into single cells, the cloned cell lines were named JNUBK-1, JNUBK-2, JNUBK-3 and JNUBK-4, according to their characteristics. To confirm the epithelial phenotype of the cell lines JNUBK-3 and JNUBK-4, which showed stable proliferation capability over 35 generations after immortalization, the expression of cytokeratin and fibronectin was measured. Finally, the FMDV titer in the JNUBK-3 and JNUBK-4 cell lines was measured and was 800∼2,000 times higher than that of the currently used cell line IRBS-2. In conclusion, more sensitive isolation and production of FMDV became possible through the use of the immortalized JNUBK-3 and JNUBK-4 cell lines.

‘급속면역금나노입자막대 (RIGS) 키트’라 명명된 빠르며 사용자 편의 및 간단한 면역흡착막대 방식의 키트가 오이모자이크바이러스 (CMV)의 현장진단을 하기 위하여 개발되었다. 토끼의 CMV항혈청에서 정제된 면역글로블린G (IgG)는 단백질A 크로마토그래피법으로 정제되었으며 이후 금나노입자와 결합하여 니트로셀룰로스막에서 진단선 표시하도록 고안되었다.CMV항체와 비특이적으로 결합하는 단백질A를 같은 진단막대에서 대조선으로 이용되었다. RIGS-CMV 키트를 이용한 진단은 의심 식물 시료를 완충액이 들어간 플라스틱 봉지에 넣은 후 착즙 후 진단막대기를 넣으면 된다. 결과는 5-10분 후알 수 있다. CMV가 감염된 고추, 오이 및 멜론의 즙액에RIGS 막대기를 넣고 진단한 결과 CMV 농도에 비례하여 진단선이 형성됨이 관찰되었으며, 이들 키트들은 CMV와 연관되지 않은 다른 고추바이러스들에서 비특이적 반응이 형성되지 않았다. 이런 결과들은 RIGS-CMV 키트가 매우 민감하며,진단에 별 다른 실험실 기술이나 경험이 필요하지 않는 다는것을 의미한다. 그러므로, RIGS-CMV 키트는 CMV 감염이의심되는 식물체들의 현장 진단 뿐만 아니라 실험실에서의CMV 진단에 효과적이다.

Acquisition of plant viruses has various effects on physiological mechanisms in vector insects. Bemisia tabaci is the only known vector of Tomato yellow leaf curl virus (TYLCV), which is a serious virus affecting tomato cultivars. In this study, the lifespan of Q1 biotype was compared between non-viruliferous (NV) and TYLCV-viruliferous (V) whiteflies. Total lifespan from egg to adult death of NV whiteflies was 62.54 days but 10.64 days shorter in V whiteflies. We investigated the temperature susceptibility of B. tabaciby comparing mortalities as well as heat shock protein (hsp) mRNA levels between NV and V whiteflies. For this, NV and V whiteflies were exposed for either 1 or 3 h at 4, 25, and 35 °C. The mortality of V whiteflies was higher than NV ones following exposure at either 4 or 35 °C, but there was no significant difference at 25 °C. Analysis of the expression level of heat shock protein (hsp) genes using quantitative real-time PCR showed that both cold and heat shock treatments stimulated higher expression of hsps (hsp40, hsp70, and hsp90) at various rates in V whiteflies than NV ones, but there was no difference at 25 °C. All together, our results show that TYLCV acquisition accelerated the developmental rate and increased susceptibility to thermal stress in B. tabaci. Therefore, this modification may result in reduced vector longevity due to increased metabolic energy utilization. Our results provide insights into the complex interaction between vector fitness and thermal stress in relation to the acquisition and transmission of plant viruses.

Sacbrood virus (SBV) is one of the most fatal pathogens against Asian honeybee, Apis cerana. This virus cause failure of the insect larvae to pupate and death of the adult insects. This study has analyzed the host genes affected by viral infection, by comparing the expression level of host transcripts infected with or without SBV. As a first step, we sequenced the cDNA libraries of Asian honeybee by using illumina RNA sequencing. The sequences were de novo assembled to acquire honeybee transcriptome sequences. The transcriptome was annotated by the sequence comparison to known protein sequences by BLASTX and evolutionary genealogy of genes: Non-supervised Orthologous Groups (eggNOG) database with functional categories and description. By mapping the RNA-seq data to de novo assembled transcripts, we characterized the differentially expressed transcripts between SBV-infected and non-infected Asian honeybee.

Rice stripe virus disease (RSVD), one of the most serious disease of rice is mediated through the sucking by small brown planthopper, Laodalphax striatellus. So far, the studies have been mainly focused on the interaction between the host plant and the virus. In this study, for better comprehension of the interactions among the host plant, vector insect and plant-pathogenic virus, we investigated transcriptome of the vector insect and the differences between viruliferous and naïve L.striatellus. For this, naïve L. striatellus were collected from non-infected rice field and 50 L.striatellus of them were fed RSV-infected rice for 5 days. With the RSV-viruliferous and the naïve insects, we conducted Illumina RNA sequencing (Hiseq 2000) and obtained 175,243,488 and 146,031,348 reads from viruliferous and naïve L.striatellus, respectively. These reads were assembled into contigs and two transcriptome databases were generated. The transcriptome of naïve and RSV-viruliferous L. striatellus were campared to figure out up-regulated or down-regulated genes. These RSV-dependently regulated genes may have important function in the behavior of planthoppers or the transmission of RSV.

Most Korean beekeepers have moved from south to north of Korea to collect nectar from black locust (Robinia pseudoacacia) flowers for 2 months. This provided a valuable opportunity to sample bees originating from diverse areas in one location. We initiated a survey of honeybee (Apis mellifera) colonies on the blooming period of Acacia to determine the prevalence of Nosema apis and black queen cell virus (BQCV) in 2013. Nosema causes significant losses in population size of honeybees. Sixteenth hives were sampled for this study. Bees were collected on the 4th and 13th of May, 2013. Nosema spore counts ranged from zero to 1,948,333 spores per bee. The average number of nosema spores per bee was calculated to be 450,000. Approximately 94% of the apiaries examined were infected with nosema, based on the presence of spores in the flowering period of Acacia. Also nosema is thought to be associated with black queen cell virus. RT-PCR analysis shows that BQCV infection rate was 100%. This indicates that nosema and BQCV is the predominant species affecting honeybee colonies.

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.

Sacbrood virus (SBV) is one of the most serious honeybee viruses. The virus causes failure to pupate and death in both larvae and adult bees. Recently, Korean Sacbrood virus (KSBV) caused a great loss in Korean honeybee (Apis cerana) colonies. Although KSBV shows high homology with SBV strains, it has unique motifs and causes different symptoms. Therefore, a simple, sensitive and specific method for detecting KSBV is needed urgently. In this study a reverse transcription loop-mediated isothermal amplification (RT-LAMP) and a novel micro PCR-based detection method, termed ultra-rapid real-time PCR (URRT-PCR) were applied for rapid detection for korean sacbrood virus (KSBV) from honeybees (Apis cerana) infected with SBV in Korea. The LAMP could be detect the virus in RT-LAMP reactions containing 102copies of pBX-KSBV within 30 min, which was 10 times more sensitive than a RT-PCR assay. The URRT-PCR showed high sensitivities which were able to detect 10 copies in the standard assays. In the application of URRT-PCR detection to an KSBV-infected honeybee, the shortest detection time was 10 min 12 sec, including reverse transcription. In addition, these methods could be distinguished between KSBV and other closely-related SBV strains, These rapid methods were rapid molecular-based diagnostic tools and useful tool for the rapid and sensitive diagnosis of KSBV infection of honeybees.

Sacbrood virus (SBV), a causative agent of larval death in honeybees, is one of the most devastating diseases in bee industry throughout the world. Lately the Korean Sacbrood virus (KSBV) induced great losses in Korean honeybee (Apis cerana) colonies. However, there is no culture system available for honeybee viruses, including SBV, therefore, the research on honeybee viruses is practically limited until present. In this study, we investigated the growth and replication of KSBV in cell cultures. The growth of KSBV was demonstrated by RT-PCR, quantitative real-time PCR, TEM and nucleotide sequence analysis. The results demonstrated that SBVshowed the replication signals in mammalian cell lines, including Vero cells without any signs of cytopathic effect (CPE). The results of RT-PCR, quantitative real-time PCR and in vivo infection with KSBV were also indicated the replication. Phylogenetic tree analysis shows our sequence included in distinct group with other SBV strains from China and Korea. It clearly showed the differenciation between field strain and attenuated strain through cell culture. The results of present study demonstrated for the first time that SBV like other animal viruses could be adapted and attenuated in cells through the sequential passages. The sequential adaptation through cell culture could result in discrepancy of pathogenicity of virus and morphological characterization. For this reason, the present results indicated that the cell adapted SBV could be a valuable tool to study the general properties of this emerging virus, including pathogenicity in the future.

Deformed wing virus (DWV) is a serious pathogen of the honeybee, Apis mellifera L., vectored by the parasitic mite Varroa destructor. The virus is associated with wing deformity in symptomatic bees, and premature death and reduced colony performance in asymptomatic bees. In present study a novel micro PCR-based detection method, termed as ultra-rapid real-time PCR (UR-RT PCR), was developed for the fast and quantitative detection of DWV in honeybee. A specific detection primer set (DWV-UR-F3/R3) was used for the amplification of an unique 133-bp DNA fragment of DWV with a rapid real -time PCR system, GenSpector® TMC-1000, which proceed the cycling with fast heating and cooling rates and a small reaction volume. We showed that this method is able to detect DWV with DNA conditions, artificial recombinant DNA, pBX-DWV479 as well as with virus-infected honeybee samples. In application to a DWV-infected honey bee, the minimum detection time was 8 min 50 seconds under 30 cycles and 10min 11 seconds including melting temperature analysis. This optimizing detection method is one of the fastest real-time PCR-based diagnostic tools and is available to be applied to use for the detection in the field and of various persistency pathogens.

Sacbood virus (SBV) is an infectious disease, resulting in failure to pupate and death and kSBV is a disease caused perish Apis cerana of 75% in South Korea. RNA dependent RNA Polymerase(RdRP) is one of polyprotein of viral genome and an enzyme that catalyzes the replication of RNA from an RNA templates and an essential protein encoded in the genomes of all RNA containing viruses with no DNA stages. In this study, recombinant construct with RdRP partial region of kSBV was used for sequence analysis to clarify about Korean SBV. As a result it could be determined that the virus develops by infection of Korean Apis cerana called kSBV. Also, we named Apis cerana-kSBV-region to the name of the unique region of gene that kSBV has. In comparison of the RdRP region of bee RNA virus on nucleotide sequence, its sequence from same species have less variability as well as each virus species has a certainty of RdRp region. It indicated that mutations of RdRP region of each virus species is able to be a useful indicator of honeybee virus detection.

Deformed wing virus(DWV) is one of infectious disease of honey bee that is caused to wings of immature or mutation and death at last. In this study 4 kinds of polyprotein of DWV are compared and then selected 2 kinds of polyprotein, RNA-dependent RNA polymerase(RdRP) and peptidase C3G, which has relatively higher homology than others. Analysis of both RdRP region and peptidase C3G of DWV deposited in Genbank of NCBI showed 99~100% and 95~97% of homology on genomic level, respectively whereas analysis of CRPV-capsid and RNA helicase showed 86~98% and 89~98% of homology, repectively. According to the result of gene analysis, two kinds of polyprotein are constructed and analyzed the homology, resulting in RdRP and Peptidase -C3G showed about 96% and 97% of homology, respectively. It indicated that both region is able to use for generation of specific antibody for the diagnosis of Deformed Wing Virus (DWV).