Sprague-Dawley Rats에 있어서 Brucella abortus Biotype 1의 실험적 감염에 관한 연구

Mst. Minara Khatun 전북대학교 대학원 2010 국내박사

Brucella abortus (B. abortus) is an intracellular bacterium. Upon invasion into the host it is taken up by the phagocytic cells and undergoes proliferation and disseminates into the organs of the reticuloendothelial system such as spleen, liver, kidney and bone marrow. B. abortus causes hematological abnormalities in humans such as pancytopenia through bone marrow suppression and splenomegaly. Bacterial as well as hematological profiles have not been studied in rats after experimental infection with B. abortus. In chapter 1, the bacterial and hematological profiles in SD rats were measured during acute and subacute brucellosis caused by B. abortus biotype 1. A total of 40 SD rats received an intraperitoneal dose of 1 × 1010 CFU of B. abortus biotype 1 isolated from cattle in Korea. 4 rats were used as uninfected control rats (0). Blood, spleen, liver and kidney were collected from the 4 randomly selected rats at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90 and 120 days after infection for bacteriological and hematological examinations. The persistence of B. abortus in blood was noted for short time, up to 5 weeks of infection. Bacterial persistence was recorded in the spleen throughout the experiment. All of the infected rats manifested splenomegaly. Hematological analysis revealed significant decreased of WBC, RBC, PLT, HGB and HCT in the infected rats in compared to uninfected control rats (p < 0.05). Analysis of blood films of acutely and subacutely infected rats by WG stain revealed abnormal shapes of RBC, with extensive to moderate infiltration of the lymphocytes, neutrophils and macrophages. These changes of hematological parameter are the clear indication of pancytopenia. In chapter 2, the IgG specific immune responses as well as antigen recognition were investigated during the acute and subacute stages of B. abortus biotype 1 infection in SD rats. The IgG specific immune responses in sera were measured at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90 and 120 days after infection against LPS, WCA, OMP, PP, CP and CBP by IELISA. The IgG specific antibody response was detectable at 3 days after infection against these antigens. The peak serum IgG antibody titers were recorded against LPS, CP and PP at 28 days after infection. On the other hand, the highest serum IgG antibody titers were recorded at 42 days after infection against WCA and CBP. The highest serum IgG antibody titer was recorded at 90 days after infection only against OMP. Data of this study indicated that LPS, WCA, PP, CP and CBP of B. abortus could be useful for diagnosis of acute and subacute brucellosis in SD rat model. On the contrary, OMP of B. abortus could be useful for differential diagnosis of subacute brucellosis. Recognition of immunodominant antigens in WCA, OMP, PP, CP and CBP of B. abortus were performed by WB assay using infected rat sera collected at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90 and 120 days after infection. WB assay of the sera revealed a wide array of protein bands between molecular weight of 10 and 98 kDa for WCA, 13 and 95 kDa for OMP, 20 and 65 kDa for PP, 12 and 85 kDa for CP and 19 and 125 kDa for CBP. Proteins bands of approximately 10, 13, 20, 24, 46 and 76 kDa for WCA; 28, 35, 39, 85, and 95 for OMP; 20, 30, 40, 43, 46 and 65 kDa for PP; 12, 23, 68 and 85 for CP; 125, 105, 82, 66, 54, 46, 32, 24, 22, 21 and 19 kDa for CBP were intensively recognized by the sera of infected rats during the course of infection. These antigens should be considered useful for the diagnosis of B. abortus infection. In chapter 3, total serum level of IgA, IgG1, IgG2a against LPS, WCA, OMP, PP, CP and CBP of B. abortus as well as in vivo and in vitro production of INF- γ and IL-10 against CBP were measured in the SD rats at 0, 3, 7, 14, 21, 28, 35, 42, 60, 90 and 120 days after infection with B. abortus biotype 1 by ELISA. A very low level and short persistence of IgA antibody were recorded in this experiment. The low level and short persistence of IgA antibody suggest that this antibody isotype might not be protective against brucellosis in rats. Both Th1 and Th2 specific immune responses were recorded in this study with the production of IgG2a and IgG1 antibody isotypes, respectively. A significant dominant IgG2a antibody response over IgG1 responses were recorded throughout the experiment (p < 0.001) against LPS and OMP. A mixed Th1 and Th2 dominant immune responses mediated by IgG2a and IgG1 antibody isotypes were observed against WCA, PP, CP, and CBP. Data of this study suggest that IgG2a dominant responses in the early stages of disease play the main role in conferring protection against brucellosis and with the progress of disease IgG1 dominant responses were elicited. The INF-γ and IL-10 levels in the sera of the infected rats were detectable by ELISA at 3 days after infection. A statistically significant higher serum level of INF-γ was recorded at 3 and 7 days after infection (p < 0.001) as compared to the IL-10 level. Statistically significant higher serum IL-10 levels were recorded at 21 and 28 days after infection as compared to the serum INF-γ levels (p < 0.001). The INF-γ levels in the spleen supernatant were significantly increased at 7 and 14 days after infection as compared to the IL-10 (p < 0.001). Then IL-10 responses were found to be significantly increased at 21, 28, 35 and 42 days after infection as compared to INF-γ (p < 0.001). The in vivo and in vitro cytokine profiles in this study indicated that B. abortus infection in rats initially elicit Th1 dominant immune response followed by Th2 dominant immune response. Brucella abortus는 세포 내에서 증식 세균으로서 숙주에 침투 시 대식세포에게 섭식되고 비장, 간, 신장, 골수와 같은 세망내피계의 기관에서 증식한다. B. abortus는 골수 기능 억제와 비장 비대증을 통해 혈구 감소증과 같은 혈액 이상 현상을 유발한다. Sprague-Dawley Rats(흰쥐)에 있어서의 B. abortus 의 실험적 인공 감염 후 혈액학적 측면과 세균학적 측면에서 많은 연구가 되어 있지 않다. 제 1장에서는 B. abortus biotype 1의 감염 후 급성과 아급성 증상의 흰쥐에서 혈액학적 및 세균학적 측면에서 연구하였다. 40마리의 흰쥐를 소에서 분리한 B. abortus biotype 1을 1×1010 CFU로 복강 내 접종하였다. 4마리는 감염시키지 않은 대조군으로 사용하였다. 세균학적 및 혈액학적 검사를 위해서 혈액, 비장, 간, 신장을 감염 후 0, 3, 7, 14, 21, 28, 35, 42, 60, 90 그리고 120일째에 무작위로 각각 4두씩 희생시켜 가검물을 채취하였다. 혈액 내 B. abortus의 감염은 5주 정도에서만 관찰되었다. 그러나 비장에서 전 실험기간 관찰되었다. 감염시킨 모든 흰쥐는 비장 비대증을 보였다. 혈액학적 분석 결과 감염시키지 않은 대조군에 비해 감염된 흰쥐에서 백혈구, 적혈구, 혈소판, 헤모글로빈, 적혈구 용적율이 모두 유의성 있게 감소하였다(p < 0.05). Wright-Giemsa 염색으로 급성, 아급성으로 감염된 흰쥐의 혈액 도말 검사결과 정상크기를 벗어난 적혈구, 림프구, 중성구 그리고 대식세포의 과도한 침윤이 관찰되었다. 혈액학적 지표의 이러한 변화는 범백혈구감소증을 명백하게 나타냈다. 소와 사람에서 브루셀라병을 제거하기 위해, 쥐와 같은 자유로운 야생동물의 조기 진단이 필수적이다. 2장에서는 B. abortus biotype 1으로 감염된 흰쥐의 급성, 아급성 단계 동안 IgG 특이적 면역 반응을 관찰하였다. 혈청에서 IgG 특이적 항체 반응을 LPS, WCA, OMP, PP, CP 그리고 CBP을 항원으로한 iELISA를 감염 후 0, 3, 7, 14, 21, 28, 35, 42, 60, 90 그리고 120 일에 측정하였다. 감염 3일 후에 있어서의 항원들에 대한 IgG 특이적 항체반응을 보였다. IgG 항체 역가는 CBP에 대서 감염 후 3일에 최고 량이 관찰되었다. LPS, CP와 PP의 감염에 대한 혈청 내 IgG 가장 높은 항체 역가는 감염 28일 후에 관찰되었다. 반면 WCA와 CBP의 경우는 42일에 가장 높은 IgG 항체 역가를 나타냈다. OMP의 경우 90일에 가장 높은 항체 역가를 보였다. 이러한 결과는 B. abortus 의 LPS, WCA, CP, PP, CBP들은 흰쥐 모델에서 급성과 아급성 감염의 진단으로서 유용한 결과를 나타냈다. 이와는 달리, B. abortus 의 OMP는 아급성 감염시의 차별적인 진단으로 이용할 수 있을 것으로 보인다. B. abortus 의 WCA, OMP, CP, PP, CBP으로 감염 후 0, 3, 7, 14, 21, 28, 35, 42, 60, 90, 120일에 채취한 흰쥐의 혈청을 이용하여 Western blot을 실시하였다. 혈청의 WB 결과 WCA는 13~95KDa, OMP는 13~95KDa, PP는 15~65KDa, CP는 12~85KDa, CBP의 경우 19~125KDa의 분자량 사이의 단백질 밴드에서 다양하게 관찰되었다. WCA는 10, 13, 20, 24, 46, 76 kDa; OMP 는 28, 35, 39, 85, 95 kDa; PP는 20, 30, 40, 43, 46 , 65 kDa; CP는 12, 23, 68 , 85 kDa 그리고 CBP는 125, 105, 82, 66, 54, 46, 32, 24, 22, 21, 19 kDa 의 단백질 밴드는 감염된 흰쥐의 혈청에 반응하였다. 이들 항원은 B. abortus 감염의 진단에 유용할 것으로 사료된다. Brucella abortus 에 대한 면역은 항원 특이적 T 세포 활성화, CD4+ 와CD8+ T 세포, 체액성 면역 반응을 포함한다. Th1 반응은 IgG2a 생성을 자극하고 Th2 반응은 IgG1의 생성을 촉진한다. Brucella 감염에 대한 방어적인 면역 반응은 흰쥐 모델에서 연구되어 있지 않다. 3장에서는 ELISA을 이용하여 B. abortus biotype 1의 감염 후 흰쥐에 0, 3, 7, 14, 21, 28, 35, 42, 60, 90 및 120일 령에 CBP에 대한 INF-γ 와 IL-10의 in vivo와 in vitro 에서의 생성과 B. abortus 의 LPS, WCA, OMP, PP, CP , CBP에 대한 IgA, IgG1, IgG2a를 혈청에서 측정하였다. 본 실험에서 IgA 항체의 짧은 지속성과 낮은 수치를 확인하였다. IgA 항체의 낮은 수준과 짧은 지속성은 rat에서 이러한 항체 isotype이 브루셀라병으로부터 보호하지 않음을 의미한다. 본 연구에서 Th1 과 Th2 특이 면역 반응 모두 각각 IgG1 와IgG2a 항체를 생성하였다. 실험기간 동안 LPS, WC 그리고 OMP에 대하여 IgG1 반응 보다 IgG2a 항체반응이 우세하게 나타냈다.(p < 0.001). IgG2a 과IgG1항체에 의해 매개되어진 Th1과 Th2의 면역 반응이 CP, PP 그리고 CBP에서 발견되었다. 연구 결과 질병의 초기 단계에서 IgG2a 면역 반응이 브루셀라병에 대해 주된 보호역할을 수행하고 질병 진행 시 IgG1 면역반응이 유도됨을 확인하였다. 본 연구에서 감염된 흰쥐의 혈청내 INF-γ 과 IL-10의 수준은 감염 후 3일째 효소 항체법으로 확인 할 수 있었다. 감염 후 3일과 7일에서 IL-10 에 비해 INF-γ의 높은 혈청내 수준이 통계적으로 유의하게 확인되었다(p > 0.001). 감염 후 21일 과 28일에는 혈청 내 INF-γ 수준과 비교하여 높은 IL-10 수준이 통계적으로 유의하게 나타났다(p < 0.001). 비장의 상층액에서 INF-γ 수준은 IL-10에 비해 감염 후 7일과 14일에 유의하게 증가하였다 (p < 0.001). INF-γ 와 비교하여 볼 때 IL-10 반응은 감염 후 21, 28, 35, 42일 령에 높게 나타났다 (p < 0.001). 본 연구에서 in vivo와 in vitro 사이토카인 특성은 흰쥐에서 B. abortus 감염 시 초기에는 Th1 면역반응이 야기되고 Th2 면역반응이 뒤이어 유발되는 사실을 규명할 수 있었다.

Infection-inducible secretory catalase mediates essential host antioxidant defense system during microbial infection in Drosophila innate immunity : 초파리의 선천성 면역 반응 중 미생물 감염 시 필수적으로 작용하는 숙주 항산화 시스템에서 iCat 의 역할

하은미 이화여자대학교 대학원 2003 국내박사

살균성 활성 산소의 생성은 미생물 감염 시 가장 빠르게 반응하는 선천성 면역 중의 하나이다. 그러나 감염에 의해서 유도되는 과도한 활성산소의 양은 주위의 숙주 세포에도 매우 해롭다. 그러므로 감염에 의해 조절되는 항산화 시스템을 통한 활성산소의 효소적 제거는 숙주를 효과적으로 보호하기 위해 필수적이다. 지금까지 여러 항산화 시스템은 잘 연구되어 왔지만 감염 시 그들의 생체 기능에 대한 연구는 아직 분명하게 밝혀져 있지 않다. 본 논문에서, 초파리 Toll 수용체에 의해 조절되는 한 유전자가 감염 시 전사 유도되는 세포의 분비성 catalase이라는 것을 생화학적 방법으로 증명하였으며, 이 유전자를 inducible Catalase (iCat) 라고 명명하였다. iCat-RNAi 형질전환 초파리를 이용한 생체 기능 연구를 통해, iCat 이 미생물 감염 시 숙주의 생존에 필수적인 항산화 시스템에 관여한다 라는 것을 증명하였다. 특히 iCat-RNAi 형질전환 초파리는 미생물이 감염된 먹이의 자연적 섭식에 의해서도 현저하게 높은 치사율을 보였으나 NF-κB/relish 돌연변이 초파리는 이와 같은 방식의 미생물 감염에 저항력을 가졌다. 그뿐만 아니라, iCat-RNAi 형질전환 초파리는 다양한 발생적 결함을 보였다. 결론적으로 iCat에 의한 항산화 작용은 초파리의 선천성 면역 반응뿐만 아니라 발생과정에도 관여하는 중요한 생리학적 작용이라는 것을 생체 내 에서 증명하였다. The de novo production of bactericidal reactive oxygen species (ROS) is one of the most immediate host innate immune responses during microbial infection. However, excessive amounts of infection-induced ROS are also deleterious to surrounding host cells. The enzymatic removal of ROS by an infection-controlled antioxidant system is hence indispensable for efficient host protection. Although many antioxidant enzymes have been well documented, their exact in vivo functions during infection are poorly known. Here, we show that a Drosophila Toll-controlled gene has been biochemically characterized as an infection-inducible secretory catalase, and thus was referred as inducible Catalase (iCat). Our in vivo functional study with iCat-knock down flies demonstrates that iCat mediates a key antioxidant defense system, which is essential for the host survival against septic injury. Strikingly, flies carrying iCat-RNAi show high lethality following natural mode of infection by feeding with microbe-contaminated foods, whereas NF-κB relish mutant flies are resistant to such infection. Furthermore, flies carrying iCat-RNAi show multiple developmental defects. Altogether, these results imply that down-regulation of ROS by iCat is a critical physiological process during innate immune response as well as development in Drosophila.

119구급대원의 감염관리에 대한 인지도와 수행도 관계 연구

윤형완 전북대학교 보건대학원 2007 국내석사

In order to protect Rescue 119 workers exposed on the spot from potential infection, this study identified their awareness and practices of infection control so that it could help preventing them from infection and also provide basic materials necessary for pre-hospital infection control. This study applied questionnaire survey to total 215 Rescue 119 workers at fire stations in Jeonbuk province, Jeonnam province and Gwangju city from July 14 to Sept. 14, 2006 for the benefit of data collection. The questionnaire about possible associations between awareness and practices of infection control consisted of total 46 times across 6 categories such as washing hands during emergency activities; fluid therapy and injection; respirator maintenance; individual hygienics; disinfectant supplies and equipments maintenance; and control of infectious wastes. And collected data were processed using SPSS statistic program to analyze frequency and percentage, mean and standard deviation, Pearson's correlation coefficient, t-test and one-way ANOVA. As a result, this study came to the following conclusions: In terms of awareness about infection control, our respondents showed highest awareness about infectious waste control, and also showed highest level of practices in washing hands during emergency activities. Throughout all domains, awareness means were higher than practice means. In particular, infectious waste control was the domain of significant differences between awareness and practices. In terms of associations between awareness and individual characteristics, it was found that female rescue worker group and hospital/general hospital career group(before joining the Rescue 119) showed significantly higher awareness on statistic level. In regard to associations between individual characteristics and practices, it was found that female rescue worker group showed higher level of practices than male group on statistic level. This study also analyzed correlations between rescue workers' awareness and practice of infection control. As a result, it was found that the higher awareness was in correlations with the higher practices across all 6 domains including washing hands. In addition, the higher awareness of a questionnaire item was in significantly positive correlations with the higher practice of other items. However, our respondents showed high awareness about anti-infection, but low practices in reality. This indicates necessity of devising possible solutions to improve the practices as much as awareness. Especially, it was noted that major reasons for insufficient practices of infection control guideline come from unhabituated practices and lack of supports for infection-preventing supplies and protective device(mask, etc). Hence, it is necessary to provide more infection-preventing supplies for local rescue workers sufficiently, in parallel with steady habituation of infection control. Furthermore, it is required to manage and study infection control policies even at pre-hospital step in efforts for effective infection control, education and activities.

Human papillomavirus infection in the south korean women

고아라 고려대학교 대학원 2014 국내석사

Since the year of 2001, over 35896 tissue samples were collected from hospitals throughout South Korea. In this study, we investigated the prevalence and distribution of HPV infections in South Korean women. The collected samples were tested for HPV infection (16 genotypes of high-risk HPV and 8 genotypes of low-risk HPV) using an HPV DNA diagnostic kit ( MyGene Co. Seoul, Korea). The prevalence of HPV was 24.1% for all types combined, 12.9% for high-risk, 3.1% for low-risk, 3.9% for multi-infection, and 4.2% for unknown-risk types. In recent years, the major high-risk HPV genotypes were numbers16, 58, 53, 52, and 18 in South Korean women and their prevalence in total HPV infection were 19.30 %, 11.25 %, 8.35 %, 7.95 %, and 7.65%, respectively. The major low-risk HPV genotypes were 70, 54, 6, and 11 in South Korean women and their prevalence in total HPV infection were 7.85 %, 4.00 %, 3.00 % and 1.70 %, respectively. An epidemiological study of HPV infections in South Korean female population revealed a distinct pattern of infection. Twenty-four different genotypes were evaluated; of which genotypes number 16 and 58 were the major high-risk HPV genotypes. In addition, a few rare genotypes (number 53 and 52) were shown to be prevalent in South Korea, which suggests that the future targets for HPV protection and management in South Korea are different than those of the other countries. Further preventive and therapeutic steps to manage HPV infections in South Korea should consider the changing HPV environment.

Controlling Bacterial Super-Infection during Influenza

Rich, Helen Eva Adams University of Pittsburgh ProQuest Dissertations & 2019 해외박사(DDOD)

Bacterial super-infection during influenza increases morbidity and mortality from the viral infection. Current therapies for influenza and bacterial super-infection are limited and inadequate, and rising multi-drug resistance in bacteria underscores the need for new therapies. Administration of an engineered antimicrobial peptide, WLBU2, reduced methicillin-resistant Staphylococcus aureus burden during pulmonary bacterial infection, but failed to reduce S. aureus burden during influenza super-infection. Immunopathology is a strong driver of mortality in bacterial super-infection during influenza, so I investigated the underlying dysfunction in antibacterial immunity caused by preceding influenza. Type I interferon induced in response to influenza infection had been previously shown to be a broad suppressor of antibacterial immunity. Type III interferons had been described much more recently, and were shown to be produced in response to influenza at even higher levels than type I interferon. Thus, I interrogated the role of type III interferon in the pathogenesis of bacterial super-infection during influenza. To do this, I compared super-infection outcomes between wild-type mice and mice lacking IFNλ3, one of the two murine type III interferons. However, total IFNλ was not markedly reduced in these mice, so I instead decided to test the effect of IFNλ treatment during influenza on outcomes of bacterial super-infection, as it had been recently published that IFNλ treatment ameliorated morbidity and mortality from influenza. It had also been recently shown that mice lacking the type III interferon receptor had increased burden in S. aureus super-infection during influenza, which mimicked data from mice lacking the type I interferon receptor. Adenoviral overexpression of IFNλ3 one day prior to bacterial super-infection induced neutrophil dysregulation, with lower binding and uptake of both S. aureus and Streptococcus pneumoniae in the lungs of mice during influenza super-infection with each of these bacteria. These results suggest that, while new therapeutics for bacterial super-infection during influenza need to be discovered, neither IFNλ nor WLBU2 should be considered. WLBU2 may be effective for bacterial pneumonia alone, but IFNλ should not be used as a treatment for influenza due to the high risk of mortality and morbidity from bacterial super-infection.

Identification of novel Sca-1 expressing innate immune cells that mediate pathogenesis of bacterial infection

Park, Minyoung Sungkyunkwan university 2021 국내박사

Identification of Gr-1+Sca-1+ myeloid cells that play pathogenic roles in bacterial infection Extreme pathophysiological stressors like tumor or rampant bacterial infection induce expansion of otherwise infrequent leukocyte populations. Here I discovered a novel CD11b+Gr-1+ myeloid cell population that expresses stem cell antigen-1 (Sca-1) induced upon experimental infection with Staphylococcus aureus (S. aureus). Although CD11b+Gr-1+Sca-1+ cells have impaired migratory capacity and superoxide anion producing activity, they secrete increased levels of several cytokines and chemokines compared to Sca-1- counterparts. The generation of CD11b+Gr-1+Sca-1+ cells is dependent on IFNγ in vivo, and in vitro stimulation of bone marrow cells or granulocyte macrophage progenitors (GMPs) with IFNγ generated CD11b+Gr-1+Sca-1+ cells. Depletion of CD11b+Gr-1+Sca-1+ cells by administrating anti-Sca-1 antibody strongly increased survival rates in a S. aureus infection model by reducing organ damage and inflammatory cytokines. However, adoptive transfer of CD11b+Gr-1+Sca-1+ cells decreased survival rates by worsening the pathogenesis of S. aureus infection. Taken together, I discovered a novel pathogenic CD11b+Gr-1+Sca-1+ population that plays an essential role in mortality during bacterial infection. 세균 감염 모델에서 병원성에 중요한 역할을 하는 새롭게 발견된 CD11b+Gr-1+Sca-1+ 세포의 규명 체내에서 종양이 생기거나 세균 등에 심각하게 감염이 되면 정상상태에서 존재하는 면역세포들과는 다른 특성을 가지는 새로운 골수성 세포들의 군집이 증가하게 된다. 이번에 새롭게 밝히게 된 Gr-1+ 골수 세포는 세균 감염 모델에서 특이적으로 Stem cell antigen-1 (Sca-1)의 발현여부에 따라 나뉜다. 특히 세균 감염 상황에서 새롭게 생성되는 CD11b+Gr-1+Sca-1+ 세포는 전구세포로부터 분화가 완료된 세포로 생각됨에도 불구하고 조혈모세포의 지표인 Sca-1을 발현하고 있다는 점에 있어서 흥미로운 발견이다. CD11b+Gr-1+Sca-1+ 세포는 CD11b+Gr-1+Sca-1- 세포에 비해 특정 케모카인을 인지하여 이동하는 주화성 이주와 세균에 대한 방어 기전 중 하나인 활성산소 발생 능력은 떨어지지만 염증 반응에 중요한 사이토카인과 케모카인의 발현양이 많은 것이 특징이다. CD11b+Gr-1+Sca-1+ 세포는 골수에 존재하는 줄기세포의 일종인 GMP (Granulocyte macrophage progenitor)로부터 만들어지게 되는데, 이 때 필요한 인자가 IFNγ이다. 세균 감염 상황에서 감염 부위로 이주해온 호중구가 분비하는 IFNγ에 의해 GMP로부터 CD11b+Gr-1+Sca-1+ 세포가 만들어지게 된다. 세균 감염 모델 내에서 CD11b+Gr-1+Sca-1+ 세포의 기능을 알아보기 위해 Sca-1에 결합하여 세포를 제거하는 항체를 이용하여 확인한 결과 CD11b+Gr-1+Sca-1+ 세포가 없을 경우에 감염 모델의 생존율이 증가하고 다양한 병리학적 검사결과에서 치료효과를 보였으며 염증성 사이토카인의 감소와 함께 조직 손상정도도 완화됨을 확인하였다. 한편, CD11b+Gr-1+Sca-1- 세포와 CD11b+Gr-1+Sca-1+ 세포를 분리한 후, 각각 세균 감염 모델 내에 주입하여 생존율 및 병리학적 결과들을 비교해 보았다. 그 결과 CD11b+Gr-1+Sca-1+ 세포를 주입한 경우에 감염 모델 내에서 사망률을 증가시키는 것을 확인함으로써, CD11b+Gr-1+Sca-1+ 세포가 세균 감염의 병리를 매개하는 세포임을 밝힐 수 있었다. Identification of Sca-1+ macrophages that modulate pathogenic progress in LPS-induced sepsis model Macrophages are important innate immune cells that play crucial roles in inflammatory responses. Accumulating evidence has demonstrated macrophage heterogeneity based on biomarkers, functions, and localization. Here, I report a novel stem cell antigen-1 (Sca-1)-positive macrophage population induced in the pathological conditions caused by lipopolysaccharide (LPS). Sca-1 is only upregulated in macrophages but not in monocytes and neutrophils upon LPS injection. Sca-1+ macrophages develop from resident peritoneal macrophages. LPS-induced Sca-1+ macrophage generation was partly blocked by anti-IFN-γ antibody, suggesting a role of IFN-γ in the process. LPS-stimulated production of IL-6, TNF-α, and CCL2 is significantly lower in Sca-1+ macrophages compared to their counterpart Sca-1- macrophages. Depletion of Sca-1+ macrophages using anti-Sca-1 antibody significantly increased survival rate and reduced lung and kidney damage in an LPS-induced sepsis model. Taken together, I discovered a novel population of Sca-1+ macrophages in LPS-induced septic conditions. 패혈증 모델에서 병원성 진행과정을 조절하는 새롭게 발견된 Sca-1+ 대식 세포의 규명 대식 세포는 염증 반응에 있어서 중요한 역할을 하는 선천성 면역 세포이다. 지난 연구들은 바이오 마커, 기능, 거주하는 위치 등에 따라 대식 세포의 이질성을 입증해왔다. 이번에 LPS에 의한 병리적 환경에서 지난 연구들과는 다르게 줄기 세포항원(Sca-1)을 발현하는 새로운 타입의 대식 세포를 발견하게 되었다. Sca-1은 LPS를 주사하였을 때, 호중구나 단핵구에서는 발현되지 않고 대식 세포에서만 발현이 증가되었다. 대식 세포는 염증 상황에서 골수 세포의 단핵구로부터 분화되기도 하는데, Sca-1+ 대식 세포는 복강 내에 거주하는 대식 세포로부터 형성됨을 확인하였다. LPS에 의해 형성된 Sca-1+ 대식 세포는 IFN-γ 항체에 의해 형성이 저해되었고 이는 Sca-1+ 대식 세포의 형성과정에 IFN-γ가 기여한다는 것을 의미한다. Sca-1 발현에 의해 이질성이 구분되는 대식 세포 중에서 LPS에 의해 자극된 Sca-1+ 대식 세포는 Sca-1- 대식 세포에 비해 사이토카인 종류인 IL-6, TNF-α와 케모카인 종류인 CCL2의 분비가 더 적었다. 실제 패혈증 환경에서 Sca-1- 와 Sca-1+ 대식 세포의 기능 차이를 확인하기 위해 LPS에 의한 패혈증 모델에서 Sca-1 항체를 이용하여 Sca-1+ 대식 세포를 결핍 시켰을 때, 모델의 생존율이 높아지고 폐와 신장의 장기 손상이 감소하였다. 이를 통해 Sca-1+ 대식 세포가 LPS에 의해 유도된 패혈증 환경에서 새롭게 형성되는 세포이며, 병원성 진행과정을 조절한다는 것을 밝힐 수 있었다.

A Study on Modeling HIV Infection Dynamics

김효은 서울대학교 대학원 2018 국내박사

A focus of this thesis is to develop a mathematical modeling approach to analyze the clinical data of Human immunodeficiency virus(HIV) acute infection. From the several studies, a remarkable stability of the HIV latent reservoir is detected despite the long-term treatment and advances in anti–retroviral therapy, and it has been recognized as a major barrier to HIV cure. We analyze several nonlinear mathematical models including the one that contains latent reservoir effect which provides consecutive viral replication and derive reproductive number (R0) which is a key index on HIV dynamics. For a quantitative analysis, we estimated parameters best describe time-series viral load measurements, obtained from published clinical study. We implement an efficient estimation method for the relevant parameters and numerical algorithm to solve the HIV infection dynamics. By using a nonlinear least square method for parameter estimation, analysis on the sensitivity parameters are performed for each model. In addition, we can obtain the total contribution of the reservoir processes to the productively infected T lymphocyte cells is also examined. We also propose a new model for HIV infection dynamics. There has been some researches that some influencing fractions on the dynamics of blood flow have been associated with the severity of HIV infection. In order to explain the rheological behavior of HIV infection in T lymphocyte populations we attempt to modify Latent cell model with fractional order differentiation of order α ∈ (0, 1]. The hemorheological parameters and fractional-order derivative in HIV system embody essential features of influencing fractions on the dynamics of blood flow associated with the severity of HIV infection. We show that the modified model has non-negative, bounded solutions and stable equilibrium points. Optimal fractional order and kinetic parameters are estimated by using the nonlinear weighted least-square method, the Levenberg-Marquardt algorithm, and Adams-type predictor-corrector method is employed for the numerical solution. The numerical results confirm that a value of fractional order (α) representing the rheological behavior in plasma is significantly related with a density of lymphocyte population.

Phenotyping and characterization of dendritic cells during chronic virus infection

유승보 Graduate School, Yonsei University 2023 국내박사

Rheumatoid arthritis (RA) is one of the autoimmune diseases accompanied by chronic inflammation. Some risk factors to induce RA have been discovered, however, because of the complication of causes, symptoms and immune responses, definite mechanism of this disorder has not been completely understood. In this study, we firstly established a protocol for preparation of inflamed tissues to investigate infiltrated immune cells in collagen-induced arthritis (CIA) mice which has been widely used as an experimental model for RA. In sequence, we investigated immune cell populations in inflamed tissues and lymphoid organs focusing on identifying subsets of those immune cells. Moreover, we observed modulated hematopoiesis in the bone-marrow (BM) of CIA established mice. In inflamed footpad tissue, infiltrated total immune cells were significantly increased depending on disease severity and the most expanded population in frequency and number was CD14+ monocytes. Of interest, as the arthritis deteriorated, the frequency and number of monocytes expressing CD38, CD64, I-A/E, Ly-6/E and podoplanin (PDPN) were notably increased. Besides, CD11c+ CXCR3+ non-canonical B cell populations was expanded in its frequency and number depending on exacerbation of arthritis. We also investigated immune cells in lymphoid organs such as popliteal lymph node (pLN) and spleen (SP). In pLN, increased number and frequency of B cells were observed. We could not detect CD11c+ CXCR3+ B cell population in pLN, but CD20+ CD38- B cell population was conspicuously increased upon raised CIA score. Moreover, CD4+ T cell subsets expressing ICOS, PD-1 and CCR5 which could be considered as peripheral helper T (TPH) cells were also increased. These distribution patterns of B and T cells were similarly observed in SP. By the way, in the BM, hematopoiesis was restrained in CIA mice resulting in accumulation of stem cells and highly reduced population was common lymphoid progenitor cells. Consequently, frequency and number of lymphoid lineage cells such as T, B, NK cells were decreased whereas non-T, B, NK cell population was relatively increased in the BM. Our findings demonstrate that generation, infiltration and accumulation of non-canonical immune cells with occurrence of autoimmune arthritis thus, our study would be helpful to understand immune cell responses in RA and find a new treatment for autoimmune diseases by regulating migration, infiltration or responses of immune cells. Chronic virus infection impairs systemic immune in the host, however, the mechanism for dysfunction of immune cells in chronic virus infection has been incompletely understood. In this study, we studied the lineage differentiation of hematopoietic stem cells (HSCs) during chronic virus infection to elucidate the changes in dendritic cell (DC) differentiation and subsequent impact on T cell functionality using a chronic lymphocytic choriomeningitis virus (LCMV) infection model. We first investigated the lineage differentiation of HSCs in the bone-marrow (BM) to elucidate the modulation in immune cell differentiation and determine that which immune cell population would be most susceptible to viral infection during LCMV clone 13 (CL13) infection. We found out chronic LCMV infection led to dramatically delayed immune cell differentiation, resulting in HSC accumulation and fewer progenitor cells. Highly restrained progenitor populations in their differentiation were common myeloid progenitor (CMP) and common dendritic cell progenitor (CDP). Moreover, mainly infected immune cell population to CL13 in the BM was CD11b/c+ myeloid DCs. We next characterized those myeloid DCs during CL13 infection. Of interest, CD11b/c+ myeloid DCs differentiated during chronic LCMV infection displayed a less immunogenic phenotype than DCs in naïve or acute infected mice, showing low expression of CD80, but high expression of PD-L1, B7-H4, IDO, TGF-β, and IL-10. Consequently, those CD11b+ DCs failed to generate fully differentiated effector CD8+ T cells, while promoting the induction of Foxp3+ regulatory T (Treg) cells. Furthermore, CD11b+ DCs generated during chronic virus infection could not lead to the sufficient differentiation into effector CD8+ T cells specific to newly incoming antigens. Our findings demonstrate that DCs generated from the BM during chronic virus infection could not supply fully functional effector CD8+ T cells specific to newly incoming antigens as well as persistent antigens themselves, suggesting a potential cause for functional alterations in the T cell immune response during chronic virus infection.

간호중증도와 내과계중환자실의 병원감염과의 관계

이경희 울산대학교 산업대학원 2009 국내석사

중환자실에서의 병원감염은 일반병동보다 높은 발생률을 보이며 환자 자신의 질병의 중증도와 관련이 있다고 보고되고 있다. 또한 빈번한 치료적 행위와 의식 수준의 저하와 관련이 있어 환자의 중증도를 예측하면 병원감염에 고위험 집단인 중환자를 관리하는데 도움을 줄 수 있다. 이런 예측도구로서 APACHE, Severity-of-illness system, SAPS (Simplified Acute Physiology Score), TISS (Therapeutic Intervention Scoring System) 등이 사용되고 있으나 병원감염과의 관계는 아직까지 정립되어 있지 않은 실정이다. 간호중증도 분류도구는 환자가 제공받는 간호의 양과 간호사의 업무를 양적 개념으로 분류한 도구로서 중증도 점수가 높을수록 환자에게 제공되는 직접간호시간이 증가하는 양의 상관관계를 보인다. 직접간호시간의 증가 할수록 환자에게 접촉하는 시간이 많아져 병원 감염에 노출 될 위험성이 증가한다고 할 수 있다. 따라서 본 연구는 환자의 생리적 변수를 근거로 한 기존의 환자 분류도구 외에 이미 국내의 중환자실에서 보편적으로 사용하고 있는 간호중증도 분류도구를 사용하여 병원감염과의 관계를 조사하고 병원감염의 위험요소를 미리 사정함으로써 감염 발생률을 감소시키기 위한 기초자료를 제시하기 위하여 수행되었다. 서울시에 소재한 3차 의료기관의 내과계중환자실에 2007년 6월1일부터 2008년 3월 31일까지 입원한 환자 중 2일 이상 입원한 성인 환자 477명을 대상으로 한 후향적 서술적 조사연구이다. 간호활동을 8개 항목으로 구분한 간호 중증도 분류도구를 이용하여 매일 0시에 전날의 간호중증도를 조사하고, 병원감염은 연구 대상 병원의 감염관리실에서 자료를 받았으며 최초 병원감염 발생일과 감염종류로 정의하였다. 병원감염률은 1000 환자-일당 감연건수로 계산하였다. 자료 분석은 SPSS - win ver. 12.0 (Chicago, IL) 통계 프로그램을 이용하였다. 연구결과는 다음과 같다. 1. 병원감염이 발생한 환자 수는 72명(15.1%)이고 발생하지 않은 환자는 405명(84.9%)이었다. 진료과별로는 호흡기내과 환자수가 263명(55.1%)으로 가장 많았으며 남자 환자가 327명(68.7%) 이었다. 2. 감염 발생건수는 연환자 1000일당 8.8건이었으며 병원감염률은 15.1%이었다. 감염의 종류는 요로감염 43건(60.0%), 패혈증 26건(34.7%), 기타 감염 5 건(5.3%)이었다. 3. 감염이 발생한 환자의 평균 나이는 62.6±14.세, 남자가 57명(78.1%)로 감염이 발생하지 않은 환자와 비교하여 통계적으로 유의한 차이가 없었다. 중환자실 평균 재실 기간은 감염군이 38.1±34.31일, 비감염군이 13.3±12.8일로 통계적으 로 유의한(p<.001) 차이를 보였다. 감염이 발생하기 전까지의 기간은 평균 22.6±19.2일로 비감염군의 평균 재실 기간인 13.3±12.8일 보다 길어 통계적으 로 유의한(p=.006 차이)를 보였다. 4. 중환자실 입실 당일, 입실 2일, 입실 3일, 퇴실 1일 전의 간호중증도를 환자의 성별, 연령, 진료과, 입실 경로 등에 따라 비교한 결과를 보면 다음과 같다. 입실 당일의 중증도는 타 중환자실을 통해 입실한 경우 105.3±18.4점, 응급실 을 통해 입실한 경우 90.2±2.4점, 병동을 통해 입실한 경우 94.7±25.3점으로 통계적으로 유의한(p<.001) 차이를 보였다. 입실 2일째는 39세 이하의 환자 군이 93.5±26.3점, 40-64세군이 96.4±25.0점, 65-74세군이 100.1±18.8점, 75세 이상이 99.4±16.2점으로 통계적으로 유의한(p=.027) 차이를 보였고, 입실 경로별에서는 병동을 통해 입실한 경우 102.±18.6점, 타 중환자실을 통해 입실한 경우 103.5±15.9점, 응급실을 통해 입실한 경우 96.3±20.5점으로 통계적으로 유의한(p=.002) 차이를 보였다. 입실 3일째는 39세 이하 군에서 91.4±23.8점, 40-64세군이 102.4±19.2점, 65-74세군이 101.5±15.7점, 75세 이상 군이 101.7±15.5점으로 통계적으로 유의한(p=.004) 차이를 보였다. 퇴실 1일 전의 간호중증도는 39세 이하 군에서 88.7±21.2점, 40-64세군이 98.4±19.4점, 65-74 세군이 97.0±20.1점, 75세 이상 군이 68.5±21.점으로 통계적으로 유의한(p=.040) 차이를 보였다. 5. 감염유무에 따른 간호중증도는 입실당일에 감염군이 98.8±21.4점, 비감염군이 95.2±23.7점으로 통계적으로 유의한 차이가 없었으며 입실 2일째에도 감염군이 103.6±18.2점, 비감염군이 99.9±18.9점으로 유의한 차이는 없었다. 입실 3일째는 감염군이 107.0±18.0점, 비감염군이 99.8±17.8점으로 통계적으로 유의한 (p=.002) 차이를 보였으며 퇴실 1일전의 간호중증도는 감염군이 104.2±19.9점,비감염군이 95.8±20.0점으로 유의한(p=.001) 차이를 보였다. 또한 감염군의 감염발생 전날 중증도 점수와 비감염군의 퇴실 1일전 간호중증도를 비교한 결과 108.0±15.3점과 95.8±19.9점으로 유의한(p<.001) 차이를 보였다. 6. 간호중증도를 군으로 분류한 결과 입실 3일째 때 3군은 감염군이 1.4%, 비감염군이 4.5%였고 4군은 감염군이 42.5%, 비감염군이 40.1%였다. 5군은 감염 68.5%, 비감염군이 54.9%였고 6군은 감염군과 비감염군 모두 2.7%로 감염군과 비감염군간에 통계적으로 유의한(p=.021) 차이를 보였다. 7. 감염 종류별 간호중증도 점수는 입실 당일에 요로감염은 100.0±21.7점, 패혈증이 98.4±22.0점, 입실 2일째는 103.4±18.6점, 패혈증이 105.1±18.2점이었고 입실 3일째는 요로감염이 106.2±19.34점, 패혈증이 109.8±16.3점으로. 감염 종류와 간호중증도 점수는 유의한 차이가 없었다. 8. 입실 경로별 간호중증도 점수는 병동을 통해 입실한 경우 입실 당일, 입실 1일, 입실 2일, 입실 3일, 퇴실 1일전에 감염군과 비감염군간에 차이가 없었다. 타 중환자실을 통해 입실한 경우에는 입실 2일째 간호중증도는 감염군이 108.37±8.0점, 비감염군이 102.7±16.7점으로 유의한(p=.018) 차이를 보였다. 응급실을 통해 입원한 경우 입실 당일 감염군이 98.9±14.1점, 비감염군이 88.8±23.2점으로 통계적으로 유의한(p=.007) 차이를 보였고, 입실 3일째는 감염군이 109.5±20.0점, 비감염군이 96.2±19.7점으로 통계적으로 유의한(p=.003) 차이를 보였으며, 퇴실 1일전은 감염군이 110.2±14.0점, 비감염군이 93.8±22.5점으로 통계적으로 유의한(p=.001)차이를 보였다. 결론적으로 병실이나 타 중환자실을 경유하여 중환자실에 입원한 환자들의 간호중증도는 병원 감염군과 비감염군간에 차이가 없었으나, 응급실을 통해 중환자실로 입실한 환자들에게서는 병원감염이 발생한 환자들의 간호중증도가 병원감염이 발생하지 않은 환자들에 비하여 높은 것으로 나타났다. It has reported that the rate of healthcare-associated infection is higher in intensive care units than the general ward and that the infection of patients has a relation to severity score of their own diseases. To predict patients' severity can help serious cases who belong to the high-risk group of healthcare-associated infection, because it is also related to frequent therapeutic cares and lowering of conscious level. Although APACHE, Severity-of-illness system, SAPS (Simplified Acute Physiology Score), and TISS (Therapeutic Intervention Scoring System) have been used as predictive tools, their relationship with healthcare-associated infection has not been clarified yet. A nursing severity scoring system is to classify given amounts and services of nursing in a quantitative respect. It shows a positive correlation that the higher the severity score, the more direct nursing time. As that results in much time to contact with patients, there are, in turn, increasing risk to be exposed to the healthcare-associated infection. Thus, this study was aimed to use nursing severity scoring system, which has generally used in intensive care unit in Korea with existing tool for patient classification based on a physiological factor, investigate the relationship with healthcare-associated infection, and assess previously its risk factor to suggest basic data for reducing the incidence rate. This study was a retrospective and descriptive research for 477 adult patients, who are in medical intensive care unit of the tertiary-care medical institution in Seoul over 2 days from June 1st, 2007 to March 31st, 2008. They were investigated in their previous nursing severity scores every midnight, using the classification tool with 8 items of nursing activities. The healthcare-associated infection data were obtained from the infection control committee and it was defined as dates of origin and infection types. The infection rate was calculated by the number of infection per 1,000 patients-days. The SPSS - win ver. 12.0 Chicago, IL. was used for data analysis. The results of this study are as follows. 1. The number of infected and uninfected patients was 72(15.1%) and 405(84.9%), respectively. By medical departments, the pulmonology had the most patients with 263(55.1%), males were 327(68.7%) among them. 2. The incidence was 8.8 cases per 1,000 patients-days and the rate was 15.1 percent. For the number of infection types, urinary tract infection was 43(60.0%), bloodstream infection was 26(34.7%), and other infections were 5(5.3%). 3. The average age of infected patients was 62.6±14.0, males were 57(78.1%), and there was no significant difference. For days of intensive care unit, infected group was 38.1±34.31 day and uninfected group was 13.3±12.8 days. It indicated some significant differences with p<.001. The average days of period before infection was 22.6±19.2 and longer than the days of uninfected group in hospital (13.3±12.8) 4. For the nursing severity score according to general characteristics on the first day, the score of cases through other intensive care unit was 105.3±18.4, through an emergency room was 90.2±2.4, and through a ward was 94.7±25.3. It was appeared to be significant differences with p<.001. On the second day, the score of the patient group under 39-year-old was 93.5±26.3, from 40 to 64-year-old group was 96.4±25.0, from 65 to 74-year-old group was 100.1±18.8, and the group over 75-year-old was 99.4±16.2. There were significant differences with p=.027. For the route of ICU admission, the score of cases through a ward was 102.0±18.6, through other intensive care unit was 103.5±15.9, and through an emergency room was 96.3±20.5. It showed very significant differences with p=.002. On the third day, the score of the patient group under 39-year-old was 91.4±23.8, from 40 to 64-year-old group was 102.4±19.2, from 65 to 74-year-old group was 101.5±15.7, and the group over 75-year-old was 101.7±15.5. There were significant differences with p=.004. For the nursing severity score on 1 day before discharge from ICU, the score of the patient group under 39-year-old was 88.7±21.2, from 40 to 64-year-old group was 98.4±19.4, from 65 to 74-year-old group was 97.0±20.1, and the group over 75-year-old was 68.5±21.3. There were significant differences with p=.040. 5. For the nursing severity score according to the existence of infection, the score of infected and uninfected group was 98.8±21.4 and 95.2±23.7, respectively, on the first day. There was no significant difference. On the second day of admission, infected and uninfected group was 103.6±18.2 and 99.9±18.9, respectively. It also showed no significant difference. On the third day, infected and uninfected group was 107.0±18.0 and 99.8±17.8, respectively. It showed a significant difference with p=.002. For the nursing severity score on 1 day before leaving, infected and uninfected group was 104.2±19.9 and 95.8±20.0, respectively. There were very significant differences with p=.001. Comparing the score of infected group on 1 day before infection with uninfected group on 1 day before discharge from ICU showed a very significant difference (p< .001) with 108.0±15.3 and 95.8±19.9, respectively. 6. The nursing severity score was classified into groups. The results showed that, on the third day, infected and uninfected group in the group 3 was 1.4 and 4.5 percent, respectively. In the group 4, infected and uninfected group was 42.5 and 40.1 percent, respectively. In the group 5, infected and uninfected group was 68.5 and 54.9 percent, respectively. In the group 6, both infected and uninfected group were 2.7 percent. There was a significant difference with p=.021. 7. For the nursing severity score according to infection types, urinary tract infection was 100.0±21.7 and bloodstream infection was 98.4±22.0 on the 1st day. On the 2nd day, the score of urinary tract infection was 103.4±18.6 and bloodstream infection was 105.1±18.2. On the 3rd day, the score of urinary tract infection was 106.2±19.34 and blood poisoning was 109.8±16.3. There was no significant difference. 8. For the nursing severity score according to routes of ICU admission, infected group was 91.5±24.5 and uninfected group was 94.6±25.5 through a ward on the 1st day. It showed no statistically significant difference. The nursing severity score of infected and uninfected group was 108.37±8.0 and 102.7±16.7, respectively, through other intensive care unit on the 2nd day. It showed a significant difference with p=-.018. In case of groups through an emergency room, infected and uninfected group was 98.9±14.1 and 88.8±23.2, respectively, it showed a significant difference with p=.007. The score of infected group was 109.5±20.0 and uninfected group was 96.2±19.7 on the 3rd day. It showed a significant difference with p=.003. The score of infected and uninfected group was 110.2±14.0 and 93.8±22.5, respectively, on 1 day before leaving. It showed a significant difference with p=.001. The conclusion was that in case of healthcare-associated infection, the nursing severity score was higher in infected group on the 3rd day and 1 day before leaving. The patients, who were through an emergency room and infected from the 1st day to the 3rd day, had a higher nursing severity score. It can be an opportunity to reduce the incidence of healthcare-associated infection by caring intensively the patient who was in intensive care unit and had a high nursing severity score on the 3rd day with stabilized nursing and treatment.

Study on infection dynamics of nervous necrosis virus (NNV) in sevenband grouper, Hyporthodus septemfasciatus

Kim, Jae-ok Chonnam national university 2019 국내박사

바이러스성 신경괴사증 (viral nervous necrosis, VNN)은 능성어 (Hyporthodus septemfasciatus)를 포함하여 전 세계 120종 이상의 다양한 담수 또는 해산 어종에서 발병하는 바이러스성 질병으로 알려져 있다. 현재까지 VNN의 질병 원인체인 신경괴사증 바이러스 (nervous necrosis virus, NNV) 검출을 위한 다양한 형태의 NNV 검출법이 개발되어왔지만, 아직 바이러스 유전자 복제 또는 감염 메커니즘에 대한 연구가 충분히 되어지지 않은 실정이다. 이에 본 연구에서는 능성어의 다양한 NNV 감염 특성을 조사한 후, 이들 결과를 토대로 NNV의 감염 메커니즘에 대해 고찰하고자 하였다. Chapter II에서는 NNV RNA1과 RNA2 유전자를 목적으로 하는 정량 역전사 중합 효소 연쇄반응 (quantitative reverse transcriptase polymerase chain reaction, qRT-PCR)을 개발하고, 이를 이용하여 감염된 능성어 체내의 기관별 NNV 유전자 분포를 조사하였다. 감염된 능성어 기관에 따른 바이러스 감염가 및 NNV 유전자 카피수는 신경조직 (뇌 및 안구)에서 높게 측정되었다. 또한 감염된 능성어의 아가미, 비장 및 신장의 바이러스 감염가 및 NNV 유전자 발현은 다른 비 신경조직보다 높게 확인되었다. 능성어 기관별 NNV RNA1의 카피수는 RNA2 및 바이러스 감염가 보다 높은 것을 확인하였으나, RNA2의 카피수는 바이러스 감염가와 유사한 것을 확인하였다. 이를 토대로 RNA1 유전자는 NNV 복제 과정에서 중요한 역할을 나타내는 것으로 사료되었다. Chapter III에서는 고정된 세포 또는 조직 슬라이드상에서 NNV 유전자를 검출하기 위해 RNA probe를 사용한 RNA in situ hybridization (RNA-ISH) 검출법을 개발하고, 이를 이용하여 감염된 능성어 뇌 조직의 다양한 부위에서 NNV 유전자의 발현 위치와 발현 분포를 조사하였다. 합성된 RNA1 및 RNA2 probe는 NNV를 접종시킨 어류 주화세포 (SSN-1)로부터 특이적으로 검출되는 것을 확인하였다. NNV 감염된 능성어의 뇌 조직에서 바이러스 유전자는 시엽, 소뇌, 척수 및 시상하부에서 발현되는 것을 확인하였다. NNV 접종된 SSN-1 세포 또는 감염된 능성어의 뇌 조직에서 NNV RNA1과 RNA2 유전자를 동시에 ISH로 검출한 결과, 동일한 부위에서 RNA1과 RNA2의 유전자가 동시 발현된 것을 관찰하였다. 또한 동시 발현된 것 이외에도 RNA1 유전자가 개별적으로 발현된 것을 확인하였다. 이상의 결과로 RNA1 유전자는 감염된 세포 또는 조직에서 과발현을 나타내는 것을 확인하였다. Chapter IV에서는 NNV에 접종된 세포 내에서의 바이러스 복제 과정을 설명하고자 하였다. 바이러스 유전자는 접종 6 시간째 RNA-ISH에 의해 RNA1 및 RNA2가 동시 발현하는 부위와 RNA1 유전자가 개별적으로 발현되는 것을 확인하였다. 반면 바이러스 단백질은 접종 24시간째부터 immunocythochemistry (ICC)에 의해 검출되는 것을 확인하였다. 이들 결과로부터 NNV는 바이러스 복제 및 단백질 조립 과정에서 RNA1 유전자가 초기에 발현되고 뒤이어 RNA2 유전자가 발현됨에 따라 바이러스 감염 단백질이 조립되는 것으로 사료되었다. 마지막으로 Chapter V에서는 수평감염상에서 NNV의 감염경로 및 어체내 바이러스의 확산을 조사하기 위해 능성어를 NNV에 침지 감염 후 다양한 조직을 시간대별로 채집하여 qRT-PCR, 바이러스 감염가, 그리고 RNA-ISH 검출법을 통해 분석하였다. 뇌 조직에서의 바이러스 감염가와 유전자 카피수는 NNV 감염 후 폐사 발생 시점 (감염 후 4일째)까지 꾸준히 증가되는 것을 관찰하였다. 반면 안구 조직에서의 바이러스 감염가는 NNV 감염 직후 매우 높게 측정되는 것을 확인하였다. 감염 후 1일째, 고정된 뇌 조직 중 대뇌와 시엽에서 RNA-ISH를 통해 바이러스 유전자가 검출되는 것을 확인하였으며, 감염 후 4일째 소뇌, 척수 및 시상하부를 포함한 모든 부위에서 바이러스 유전자를 검출되는 것을 확인하였다. 그 후 NNV 유전자 발현 검출 강도는 감염 후 21 일째까지 점진적으로 감소되어 최종적으로 시엽 및 소뇌에 존재하는 것을 확인하였다. 이들 결과로부터 NNV 초기 감염경로는 안구의 시신경 세포를 통해 뇌 조직의 시엽으로 유입이 이루어지는 것으로 판단되며, 바이러스는 유입된 시엽을 통해 뇌 조직 및 어류 체내로 확산되는 것으로 사료된다. 또한 바이러스 감염으로부터 살아남은 생존어는 NNV 지속감염의 상태를 나타내는 것으로 사료되었다. 본 연구를 통해 NNV의 체내 분포, 복제과정, 감염경로 및 확산등과 같은 바이러스 감염 메커니즘에 대한 이해를 할 수 있었으며 이러한 연구결과는 앞으로의 신경괴사증 바이러스 감염 연구에 대한 밑거름이 될 수 있을 것이다. Nervous necrosis virus (NNV) is one of the most serious fish pathogen which causes damage in > 120 species of cultured marine fishes worldwide including sevenband grouper (Hyporthodus septemfasciatus). Although a number of molecular diagnostic assays have been developed to detect or quantify NNV, virus replication status and infection mechanism have not been fully understood. The present study investigated the viral replication status, viral assembly stages, route of infection, and spread of virus in sevenband grouper in order to better understand the infection mechanism of NNV. At first, NNV infection in susceptible cultured marine fishes and the current status of available molecular diagnostics for NNV detection were briefly reviewed (Chapter I). To start with, sevenband grouper was infected with NNV by intramuscular (IM) injection and immersion challenge routes to evaluate pathoformic NNV doses (Chapter II). Afterwards, NNV genomic segments (both RNA1 and RNA2) in various tissues of NNV-infected fish were detected using qRT-PCR assay to compare the expression patterns. The minimum NNV concentration pathogenic to fish by IM injection was approximately at least 10-fold lower than that by immersion challenge route. Although the viral copies were detected in both nervous as well as non-nervous tissues, but the copy number was found to be highest in nervous tissues. The number of RNA1 copy was higher than that of RNA2 in all tested organs indicating that RNA1 was overproduced than RNA2 segment. Moreover, two NNV segments were detected and visualized in infected fish cell line and brain tissues of sevenband grouper after NNV infection, using newly developed RNA in situ hybridization (RNA-ISH) assay to support the evidence that the copy number of RNA1 is higher than that of RNA2 segment (Chapter III). RNA segments were observed clearly in diencephalon, mesencephalon and metencephalon of infected brain tissues coupled with histopathological damage. Two viral genome segments were localized in same regions and additionally RNA1 also expressed separately suggesting that RNA1 over expression may be an important factor for assembly of infectious viral particles. Also, RNA-ISH and immunocytochemistry (ICC) assays were performed in a time dependent manner on NNV-infected cells to investigate the NNV replication processes (Chapter IV). NNV genomic segments were detected by RNA-ISH at early time point post-infection (12 hpi), while NNV particles were detected later (24 hpi) by ICC. Results of qRT-PCR analysis showed that RNA1 copy number was always higher than RNA2 or NNV infectivity in all tested samples. This suggests that RNA1 is expressed at the early stage of viral replication while RNA2 is expressed later. Virions are then assembled through the initially expressed viral genomic segments. NNV trafficking sevenband grouper was investigated by dynamics of NNV RNA copy numbers and infectivity titers in various fish tissues during the course of infection using immersion challenge (Chapter V). RNA-ISH was also used to localize NNV viral genome segments in whole brain tissues during experimental infection. Infectious viral particles in eye were highest at early stage of infection, and were higher than RNA1 and RNA2 copies. By RNA-ISH, the viral segments were initially detected in optic lobe at 1 day post infection (dpi), segments were then observed at 4 to 6 dpi in all parts of brain with highly localized around the ventricle. The results showed that NNV gains entry from eye to the brain through optic nerve, and then virus replicates and spreads from ventricles in a direction to all the parts of brain, and then it spreads to other organs. Finally, we discussed the present findings on NNV distribution and infection mechanism in sevenband grouper following a natural infection route (Chapter VI).