Because intact FMDV particles (146S) are often unstable in vitro, stabilizing foot-and-mouth disease virus (FMDV) antigens remains a key challenge in studying viral charateristics. Therefore, finding optimal condition to stabilize the FMDV is essential. In this study, we investigated formulations and potentials of several stabilizers such as appropriate buffer, excipients, and storage conditions to enhance the stability of 146S. Inactivated FMDV O-Jincheon (O-JC) was dissolved in various buffer formulations, and stored at 4℃ for two months to evaluate quantity of 146S at every 2-week interval. Among phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline (HBS), and MOPS buffered saline (MBS), PBS showed more effective 146S stabilization that showed 1.3-1.6 fold higher 146S fraction than TBS, HBS, and MBS after storage for 2 weeks. However, constant dissociations of 146S were observed in all formulations at 8 weeks. Compared with other FMDVs, A22 Iraq and SAT-1, in PBS, O-JC proved to be the least stable in PBS. A variety of excipients including carbohydrate, sugar alcohol, cryo-protectant were tested for the capability in protecting O-JC from dissociation. By adding 4-8% sucrose, more than 60% of 146S fractions were maintained at 8 weeks, those were at least 1.8 fold higher than the PBS-only control. Addition of 1% β-cyclodextrin showed synergistic enhancement in O-JC stability. As the results of this study, it could be suggested that the PBS-based buffer together with 4-8% sucrose + 2% sorbitol or 2% sucrose + 2% sorbitol + 1% β-cyclodextrin could help the better stability of the O-JC in vaccine preparation.

The Japanese encephalitis virus (JEV) is a zoonotic pathogen that affects the nervous systems of humans, pigs, and horses. It has been classified into five genotypes (G1-G5) based on molecular analysis of the pre-membrane or envelope gene. In the Republic of Korea, the predominant JEV genotype has recently shifted from G3 to G1 and G5, highlighting the need for a rapid and accurate diagnostic method. In this study, we designed specific common and differential primer sets for JEV G1, G3, and G5 to detect the JEV gene. Four specific primer sets for JEV G1, G3, and G5 were used to selectively amplify the target gene. The detection limits of the common primer set for JEV G1, G3, and G5 were 100, 0.1, and 10 TCID50/reaction, respectively. The detection limits of the three differential primer sets were 1, 0.1, and 1 TCID50/reaction, respectively. No cross-reactivity was observed with non-JEV reference viruses. We successfully developed a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay to distinguish the three JEV genotypes. Our multiplex RT-PCR assay is highly sensitive and specific, providing a reliable tool for confirming JEV infection in suspected samples. Additionally, our assay can be applied to suspected mosquito samples and commercial veterinary biological products.

Severe Fever with Thrombocytopenia Syndrome (SFTS) is a newly emerging tick-borne disease caused by the SFTS virus (SFTSV), which belongs to the phlebovirus in the Bunyaviridae family. SFTSV is enveloped with a tripartite ambisense RNA genome. The L segment encodes the viral RNA-dependent RNA polymerase, the M segment encodes the two glycoproteins, Gc and Gn, and the S segment encodes the nucleoprotein (NP) and the nonstructural protein (NSs). NP participates in ribonucleoprotein (RNP) packaging and commonly detected early after infection, suggesting that the N protein is possible to be used as a target antigen for early diagnosis of SFTSV infection. In this study, we analyzed a highly immunogenic multi-epitope using GnGc and NP genes from a consensus sequence of SFTSV strain isolated from infected patients in Korea. The selected genes are constructed to the expression vector plasmid pJHL65 and the recombinant plasmid vector was transformed into the Δasd Δlon ΔcpxR Salmonella Typhimurium attenuated strain JOL912 and the expression of these antigens was verified by immunoblotting assay. We observed the significant levels of systemic IgG and mucosal IgM responses against the JOL912-derived antigen in the immunized BALB/c mice. The level of CD3+CD4+, CD3+CD8+ T lymphocyte subpopulation and TNF-α were also highly regulated in splenic T cells re-stimulated in vitro with NP and Gn/Gc multi-epitope selected antigens. Therefore, immunized mice with NP and Gn/GC multi-epitope recombinant proteins of attenuated Salmonella delivery system elicited T cell-related immune response, inducing an effective immune response. In conclusion, the attenuated Salmonella expressing NP-GnGc multi-epitopes could be a novel vaccine candidate against the SFTS virus.

This study evaluated the virucidal efficacy of a chlorine dioxide (ClO2) gas-generating fumigation disinfectant consisting of sodium chlorate solution (25% sodium chlorate) and reaction solution against avian influenza virus (AIV). After AIV suspensions had been deposited on stainless steel carriers, the 9 dried carriers were exposed to the fumigant (sodium chlorate solution: 8.5, 17, 34, 50, and 100 mL) in a 25-m³ test room for 2, 3, and 4 h, respectively. Thereafter, all carriers were submerged in a neutralizing solution (20% fetal bovine serum) to scrape off the surviving viruses, and the respective suspensions were diluted. Each diluent was inoculated into the allantoic membrane of five 10-day-old embryos. After incubation for 5 days at 37℃, AIV viability in the collected allantoic fluids was examined, and the egg infectious dose 50 (EID50) was calculated. When the carrier was exposed to ClO2 gas generated from reacting 34 mL of the fumigant for 3 h, the AIV titer reduced by more than 104.0 EID50/carrier compared to the control, which was exposed to the fumigant without inoculation of AIV suspension. In addition, the control was non-toxic to the embryos.

바이러스는 생물 의약 산업에서 다양한 응용 분야를 가지고 있다. 그들은 살충제 생산, 백신 생산, 유전자 전달, 암 치료제 등에 사용된다. 바이러스의 하류 처리는 그들의 생물학적 및 의약적 응용을 위한 필수 단계이다. 다양한 과정 중에 서 바이러스의 정제는 매우 중요하다. 막 크로마토그래피는 이 과정에서 중요한 역할을 한다. 이온 교환 막 크로마토그래피는 주로 사용되는 방법이지만 크기 배제 및 불충분한 정제에 관한 다양한 제한을 가지고 있다. 또한, 이는 인플루엔자와 같은 빠 르게 변화하는 바이러스의 균주에 적용될 수 없다. 이 검토는 막 크로마토그래피의 다양한 개선된 방법 또는 대안을 검토한 다. 이는 정제, 바이러스 회수율 및 방법의 확장성에 초점을 맞추고 있다.

The major innate immune pathways in Asian longhorned ticks, Haemaphysalis longicornis, include Toll, IMD, and JAK/STAT. In the field, H. longicornis can be infected with various pathogens including Severe Fever with Thrombocytopenia Syndrome Virus (SFTS virus), Rickettsia, Babesia and Anaplasma species. One approach to identify whether ticks are infected with pathogens is by examining the expression levels of immune response genes. To evaluate whether upregulation of immune genes from H. longicornis can serve as an indicator for pathogen infection in ticks, we first designed primer sets for Dorsal, STAT, and Relish from the H. longicornis genome. We then conducted quantitative reverse transcription PCR(qRT-PCR) on cDNA of field-collected H. longicornis and identified individuals with high expression levels in immune response genes. Subsequently, we performed digital PCR assays to determine whether selected ticks were infected with SFTS virus. Using this approach, we evaluated correlation between pathogen infection and upregulation of immune response genes in ticks. Although more experiments are needed to draw conclusions, this study suggests immune response gene-based screening methods for pathogen infected ticks from the field.

국립기상연구소의 보고에 의하면 최근 한반도의 기온 상승으로 인해 온대내륙성 기후형에 속했던 지점은 온대해양성 기후형으로, 온대해양성 기후형은 아열대습윤 기후형으로 변화하고 있다. 이러한 한반도의 기후 변화는 환경 변인에 민감한 질병 매개 곤충의 분포와 밀도 변화에 영향을 미칠 수 있어 지속적인 모니터링이 중요하다. 이 연구는 철새도래지 내 발생 및 유입될 수 있는 모기와 관련 바이러스 감염률을 확인하기 위해 충남 당진의 철새도래지에서 BG-sentinel trap 및 LED trap을 사용하여 2021년부터 2023년까지 4-11월간 월 2회 수행하 였다. 채집된 모기는 총 3,723마리로, 4속 16종을 확인하였다. 그 중 금빛숲모기 (Aedes (Aedimorphus) vexans nipponii) 가 1,711마리(45.96%)로 가장 높은 우점도를 나타냈으며, 흰줄숲모기 (A. (Stegomyia) albopictus) 와 큰검 정들모기 (Armigeres (Armigeres) subalbatus) 각각 588마리(15.79%), 빨간집모기 (Culex (Culex) pipiens pallens) 269마리(7.23%)로 나타났다. 채집된 모기의 Flavi-virus 감염 여부를 확인하기 위해 RNA 추출 및 RT-PCR을 통해 확인하였으나, 모두 음성으로 확인되었다. 이러한 연구 결과들은 기후변화에 맞추어 변화하는 감염병 매개 모기 의 발생 현황을 감시·예측하는데 유의한 자료로 활용될 수 있으며, 향후 모기 매개 질환 발생을 예측하기 위한 기초 자료로 활용될 수 있을 것으로 사료된다.

Hand, foot, and mouth disease (HFMD) is a highly contagious disease with no specific treatment. Since it is common in immunocompromised children under the age of 5, there is a need to develop a safe vaccine. Virus-like particles (VLPs) are similar structures to viruses with the lack of genetic material which makes them impossible to replicate and infect, and therefore have a high level of biological safety and are considered to have high value as vaccines. In this study, the insect virus expression system that is widely used for vaccine and drug production due to its high post-translational modification efficiency, was used to produce VLPs for Coxsackievirus type A6 and A10, which are recently reported to be the main causes of HFMD. For this purpose, the selection of promoters that can control the timing and intensity of expression of 3CD protein, which is essential for VLPs assembly but has been reported to be cytotoxic, was conducted to construct an optimal expression form for HFMD-VLP.

Mycoviruses are a group of viruses that infect filamentous fungi. While most hosts infected with mycoviruses do not show any symptoms. In some cases, mycoviruses induce various phenotypic changes include alterations in morphology, drug resistance, pathogenicity, virulence, sporulation, and growth. Entomopathogenic fungi are one of the integrated pest management agents as an alternative to conventional insecticides. Mycoviruses have the potential as supportive agents, enhancing the efficiency of the insecticidal activity of the fungi. Studies about mycoviruses themselves and their interaction with their hosts, especially entomopathogenic fungi, are needed to realize their full potential. In this work, the sequence of the dsRNA element isolated from the entomopathogenic fungus Metarhizium pinghaense 4-2 strain was determined. Through this study, we report the sequence of a dsRNA virus isolated from the Metarhizium pinghaense for the first time. In further studies, the ORF of the mycovirus that induces a phenotype change in the host will be researched.

마이코바이러스는 곰팡이를 감염시키는 바이러스로 자낭균류, 담자균류 및 난균류에서 주로 발견되며 일부 의 경우 곰팡이의 표현형에 영향을 끼치는 것으로 알려져 있다. 이번 연구에서는 대한민국 토양 샘플에서 분리된 65개의 자낭균류 및 접합균류 균주의 전체 핵산을 추출하고, 전기영동을 통해 바이러스 특이적 밴드를 스크리닝 하였다. 스크리닝 결과 65개의 균주 중에서 Tolypocladium spp. 균주 2개와 Marquandomyces marquandii 균주 1개에서 바이러스 특이적 밴드를 발견하였다. 그 후, Cellulose Chromatography를 이용하여 double-stranded RNA 를 분리하고 DNase I 및 S1 Nuclease 처리를 통해 DNA와 single-stranded RNA를 제거하여, Tolypocladium sp. 균주 1개와 Marquandomyces marquandii 균주에서 발견한 특이적 밴드가 dsRNA임을 확인하였다. 추후 virus-free isogenic line을 확보하여 virus 유무에 따른 표현형 변화를 확인하고, 마이코바이러스와 곰팡이 간의 상호작용에 관해 연구할 계획이다.

Chromosomal level of Korean Diadegma fenestrale (Jeju strain, JK-2023a) of genome assembly was achieved through a combined approach utilizing Nanopore long-read sequencing and Illumina NovaSeq short-read sequencing (approximately 217.2× coverage). The assembled genome spans 221.1 Mb, comprises 68 scaffolds, with most of the genome contained within 11 chromosomal level scaffolds. The completeness of the assembly is reflected in BUSCO assessment, with values reaching 99.6%. Scaffold N50 was 17.4 Mb, and GC % was 40%. RNAseq was performed using RNA extracted from larvae, pupae, and adults at various developmental stages (trimmed RNA-Seq data, 11.3 Gb), and a total of 13,544 genes were predicted by synthesizing the transcriptome information with the annotation information of five closely related species such as, Campoletis sonorensis (GCA_013761285.1), Venturia canescens (GCF_019457755.1), and Nasonia vitripennis (GCF_000002325.3, and GCF_009193385.2). Of these, 13,498 genes were identified by BLAST and are being further analyzed. Although the frequency of DfIV genome integration into the host’s 11 chromosomes varies from 0 to 32%, it was confirmed that all 62 DfIV genome fragments were inserted into the Hymenopteran host genome.

The small brown planthopper (SBPH), Laodelphax striatellus, is a major insect pest for the rice plants. SBPH is also a known vector of rice stripe virus (RSV), which causes severe yield losses in rice crops throughout the East Asia. RSV is persistently transmitted by SBPH and can also be transmitted to offspring through transovarial transmission. SBPH is known to migrate from China to the west coast of the Republic of Korea (ROK). The study investigated the impact of temperature on the acquisition and transmission of RSV by SBPH in ROK, which is expected to experience increased migration and emergence of SBPH due to climate change. The results revealed that the acquisition and transmission rates of RSV were higher at 27°C compared to 24°C, with rates of 100% and 78.3%, respectively. However, at 30°C, the acquisition and transmission rates of RSV was decreased. The results suggests that temperature can impact the transmission of RSV by SBPH. To investigate this further, SBPH adults were fed on RSV-infected plants and infection rates were compared across various tissues, including the head, salivary glands, midgut, Malpighian tubules, ovary, and hindgut. Results showed that at 36 hours post-infection, RSV was highly detected in the Malpighian tubules, ovary, and hindgut. At 48 hours post-infection, RSV was also detected in the thorax. These results suggest that the transmission rates of RSV in SBPH increase with temperature between 24-27°C, but decrease at 30°C, indicating that the vectorial capacity of SBPH for RSV decreases above a certain threshold.

Climate change has made outbreaks of insect-transmitted plant viruses increasingly unpredictable. Understanding spatio-temporal dynamics of insect vector migration can help forecast virus outbreaks, but the relationship is often poorly characterized. The incidence of Beet curly top virus (BCTV) was examined in 2,196 tomato fields in California from 2013-2022. In addition, we experimentally showed dispersal of the beet leafhopper, the only known vector of BCTV is negatively correlated with plant greenness, and we estimated spring migration timing using a vegetation greenness-based model. Potential environmental factors and spring migration time of beet leafhoppers were associated with BCTV incidence. We found BCTV incidence is strongly associated with spring migration timing rather than environmental factors themselves. In addition, the vegetation greenness-based model was able to accurately predict the severe BCTV outbreaks in 2013 and 2021 in California. The predictive model for spring migration time was implemented into a web-based mapping system, serving as a decision support tool for management purposes.