• Clinical and Experimental Pediatrics
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• 제49권9호
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• pp.983-986
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• 2006

목 적 : 소아 임파선염의 흔한 원인균의 하나로 잘 알려진 Bartonella henselae는 주로 고양이 벼룩에 감염된 고양이와의 접촉을 통하여 병을 일으키는 것으로 알려졌지만, 최근에는 개도 보유 숙주임이 확인되었다. 국내의 CSD증례 보고들에서는 고양이보다 개와 접촉한 경우가 더 많은 것으로 보고되었다. 하지만 국내에서는 개나 고양이 또는 매개체인 고양이 벼룩 등에서 B. henselae가 확인된 경우가 아직 없었다. 이에 저자들은 개에게서 얻은 벼룩을 대상으로 B. henselae DNA가 존재하는지 알아보기 위하여 본 연구를 시행하였다. 방 법 : 전체 22마리의 개에서 얻은 총 42마리의 벼룩을 대상으로 연구를 시행하였다. 각각의 벼룩에서 DNA를 추출한 다음 B. henselae의 pap31 부위 유전자를 이용한 시발체를 사용하여 seminested PCR을 시행하였다. 결 과 : B. henselae DNA는 전체 22마리 중 4마리의 개(18.2%)에서 추출되었던 벼룩 42마리 중 7마리(16.7%)에서 확인되었다. PCR 결과를 확인하기 위하여 7개의 PCR 산물에 대한 염기서열 분석을 시행하였다. 염기서열 분석에 의하면 6개는 Huston-1 유전자형이었으며, 한 개는 Marseille 유전자형이었다. 결 론 : 국내 개에서 추출된 벼룩에서 Houston-1유전자형과 Marseille 유전자형을 포함한 B. henselae DNA가 존재하는 것으로 확인할 수 있었다. 국내에서 개가 CSD의 중요한 감염 경로일 가능성을 제시한 점에 의의가 있지만, 충분한 수의 고양이 및 개에서 얻은 혈청과 벼룩을 대상으로 한 추가적인 연구가 필요할 것으로 생각된다.

• 대한의생명과학회지
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• 제17권3호
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• pp.191-196
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• 2011

Noroviruses (noroV) are the major cause of nonbacterial gastroenteritis in humans worldwide. Since noroV cannot yet be cultured in vitro and their diagnosis by electron microscopy requires at least $10^6$ viral particles/g of stool a variety of molecular detection techniques represent an important step towards the detection of noroV. In the present study, we have applied real-time nucleic acid sequence-based amplification (real-time NASBA) for simultaneous detection of NoroV genogroup I (GI) and genogroup II (GII) using standard viral RNA. For real-time NASBA assay which can detected noroV GI and GII, a selective region of the genes encoding the capsid protein was used to design primers and genotype-specific molecular beacon probes. The specificity of the real-time NASBA using newly designed primers and probes were confirmed using standard viral RNA of noroV GI and GII. To determine the sensitivity of this assay, serial 10-fold dilutions of standard viral RNA of noroV GI and GII were used for reverse transcription polymerase chain reaction (RT-PCR) and real-time NASBA. The results showed that while agarose gel electrophoresis could detect RT-PCR products with 10 pg of standard viral RNA, the real-time NASBA assay could detect 100 fg of standard viral RNA. These results suggested that the real-time NASBA assay has much higher sensitivity than conventional RT-PCR assay. This assay was expected that might detect the viral RNA in the specimens which could have been false negative by RT-PCR. There were needed to perform real-time NASBA with clinical specimens for evaluating accurate sensitivity and specificity of this assay.

• 미생물학회지
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• 제45권2호
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• pp.105-111
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• 2009

노로바이러스는 급성 위장염을 일으키는 Caliciviridae 과(family)에 속하는 바이러스로 유전자형이 매우 다양하다. 본 연구에서는 노로바이러스 국내분리주의 게놈 RNA로부터 3개의 open reading frame (ORF) 모두의 염기서열을 분석하고, 유전학적 계통분석을 통하여 분자생물학적 특성을 분석하였다. 본 연구에 사용된 노로바이러스(Hu/NLV/Gunpo/2006/KO)는 바이러스성 식중독, 장염 증세를 보이는 2세 여아 가검물로부터 분리되었다. 역전사반응과 PCR 증폭을 통해서 바이러스의 게놈 RNA를 3개의 중첩되는 cDNA 단편으로 합성하였으며, 합성된 cDNA를 염기서열 분석에 직접 사용하였다. 시퀀싱 결과 Hu/NLV/Gunpo/2006/KO는 3개의 ORF (ORF1, 5,100 bp; ORF2, 1,647 bp; ORF3, 765 bp)로 구성되어 있음을 알 수 있었다. 35개의 노로바이러스 국외 분리주와 비교한 결과, ORF1은 ORF2 또는 ORF3에 비해서 상대적으로 염기의 변이율이 낮았으며, 특히 ORF2와 ORF3의 C-말단 부위에서 높은 변이율을 관찰하였다. 유전학적 계통도를 분석한 결과, Hu/NLV/Gunpo/2006/KO는 genogroup II 에 속하며, Saitama U1, Gifu'96, Mc37, Vietnam 026과 같은 클러스터를 형성하는 것을 알 수 있었다. 본 연구를 통하여 노로바이러스 Hu/NLV/Gunpo/2006/KO의 3개의 ORF 염기서열을 모두 밝힘으로써, 앞으로 노로바이러스의 검출법 개발과 유전학적 상관관계뿐 아니라, 유전자의 기능 분석과 관련된 기초연구에 중요한 기초자료를 제공할 수 있을 것으로 기대한다.

• 대한바이러스학회지
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• 제29권4호
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• pp.247-259
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• 1999

Small round structured viruses (SRSV) are the major ethological agents which can cause outbreaks of non-bacterial gastroenteritis or food poisoning both in children and adults. The classification of family Caliciviridae to which SRSV belong, is based on the genome encoding three open reading frames. The rotavirus is another major pathogen which causes diarrhea in young children. We examined stool specimens obtained from diarrheal patients in Wonju from which bacterial pathogens were not found. To detect causative viruses from stool specimens of patients, reverse transcription (RT)-polymerase chain reaction (PCR) or nested PCR using rotavirus or SRSV specific primers was performed. In this study, RT-nested PCR procedure which can amplify a 330 bp fragment derived from RNA dependent RNA polymerase (RDRP) region within ORF1 was applied for the detection of SRSV. For the detection of rotaviruses, a 877 bp fragment from the VP4 region of rotavirus genome was amplified. As a result, rotavirus was not detected while SRSV sequences were detected from one out of five specimens. The nucleotide and amino acid sequences of the Wonju isolate were compared with other 6 Korean isolates which have been isolated and sequenced in our laboratory. Sequence analysis revealed that the Wonju isolate was rather distinct from other Korean isolates: the Wonju isolate was closer to genogroup I of SRSV while other 6 Korean isolates belonged to genogroup II.

• 생명과학회지
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• 제21권6호
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• pp.845-850
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• 2011

2008년부터 2010년까지 최근 3년 동안 부산지역에서 산발적으로 발생한 급성 위장관염 환자를 대상으로 유전자를 검사한 결과 4,101건 중 426건(10.4%)에서 노로바이러스를 확인하였다. 연도별 검출현황은 2008년에 14.7%(222/1,506), 2009년에 6.9%(95/1,384), 2010년에 9.0%(109/1,211)로 나타났다. 월별 분석 결과는 2008년에는 3월에 35.7%(50/140)로 가장 높은 검출율을 보였고, 2009년 역시 3월에 21.9%(23/105)로 높게 나타났으며, 2010년에는 1월에 23.8%(29/122)로 높은 검출율을 보여 겨울절기에 노로바이러스가 유행하는 것을 알 수 있었다. 반면 매해 7-8월 여름절기에는 노로바이러스가 거의 분리되지 않았다. 연령별 로는 1세 영아군과 13-19세 중등학생군에서 각각 20.9%로 가장 높은 검출율을 나타내었으며, 2-6세 소아군에서 17.5%, 20-29세군에서 13.4%, 7-12세 초등학생군에서 12.7%, 30-39세군에서 9.1%, 0세 신생아군에서 8.7%, 50-59세군에서 7.2%, 60-69세군과 70세이상군에서 각각 6.7%, 40-49세 4.5%로 확인되었다. 노로바이러스 양성 검체 340건에서 유전자형을 분석한 결과 GI군 7종류, GII군 13종류로 총 20종류가 검출되어 다양한 유전자형의 노로바이러스들이 유행함을 알 수 있었다. 연구 결과 부산지역에서는 GI군이 21.8%(76/348), GII군이 78.2%(272/348)로 GII군이 우세하여 유행하였고, 유전자형 총 20종 중 GII.4형이 49.1%로 가장 많이 검출되었다.

• Journal of Microbiology and Biotechnology
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• 제28권12호
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• pp.2133-2140
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• 2018

Norovirus is the most common cause of acute gastroenteritis. Its pathogenesis is poorly understood owing to the difficulty of establishing viral infection in animal models. Here, post-weaning gnotobiotic pigs were infected with human norovirus genogroup II genotype 4 (HuNoV GII.4) to investigate the pathogenesis and replication of the virus. Three groups of four pigs were infected with $1{\times}10^5$, $1{\times}10^6$, or $1{\times}10^7$ genomic equivalent (GE) copies of HuNoV GII.4. Four pigs were used as negative controls. Blood and rectal swab samples were collected after viral infection, and gross legions were examined after necropsy. Diarrhea was induced in 25% and 75% of pigs infected with $1{\times}10^6$ and $1{\times}10^7$ GE copies, respectively. Viral shedding was detected in 50%, 75%, and 50% of pigs infected with $1{\times}10^5$, $1{\times}10^6$, and $1{\times}10^7$ GE copies, respectively. Viremia was detected in 25% of pigs infected with either $1{\times}10^6$ or $1{\times}10^7$ GE copies. When gross lesions of gastroenteritis were investigated, the ileum walls of the infected pigs were thinner than those of the controls. Villi atrophy and inflammatory cell infiltration were identified in the ileum of each infected pig. Viral capsid was identified in the jejunum, ileum, colon, spleen, and mesenteric lymph node. Virus replication was newly verified in the spleen and mesenteric lymph nodes by detection of negative-sense viral RNA. In conclusion, HuNoV GII.4 could induce acute gastroenteritis and replicate in the extra-intestinal lymphoid tissues in post-weaning gnotobiotic pigs. Therefore, such pigs would be a suitable animal model for studying the pathogenesis and replication of HuNoV.

• 대한수의학회지
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• 제61권3호
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• pp.23.1-23.8
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• 2021

Porcine epidemic diarrhea (PED) is characterized by acute enteritis, watery diarrhea, weight loss, dehydration, and death with high mortality in neonatal piglets. In this study, 3 virus isolates collected in Vietnam between 2016 and 2017 were successfully propagated in Vero cells at high virus titers. Sequence analysis of the full-length spike (S) gene revealed that all 3 isolates belong to genogroup 2a, which is closely related to other prevalent Asian strains. Amino acid sequence comparisons revealed 98.19% to 99.13% homology with the Vietnam isolates circulating during 2013-2015, suggesting that field PED viruses (PEDVs) evolve continuously. Experiments in animals demonstrated that antisera from guinea pigs immunized with the vaccine strain resulted in higher levels (5 log₂) of neutralizing antibody against the homologous strain, and showed a relatively lower level of neutralizing antibody against the field isolates. This finding would be helpful in choosing a PEDV strain for vaccine development.

• 대한수의학회지
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• 제63권2호
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• pp.18.1-18.8
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• 2023

Porcine epidemic diarrhea (PED) is characterized by acute enteritis, watery diarrhea, weight loss, dehydration, and death, with high mortality in neonatal piglets. In this study, 3 virus isolates collected in Vietnam between 2016 and 2017 were propagated successfully in Vero cells at high virus titers. Sequence analysis of the full-length spike (S) gene showed that all 3 isolates belong to genogroup 2b, which is closely related to other prevalent Asian strains. A comparison of the amino acid sequence revealed a 98.19% to 99.13% homology with the Vietnam isolates circulating during 2013-2015, suggesting that field PED viruses (PEDVs) are evolving continuously. Experiments in animals showed that the antisera from guinea pigs immunized with the vaccine strain resulted in higher levels (5 log₂) of neutralizing antibodies against the homologous strain and a relatively moderate level of neutralizing antibodies against the field isolates. This finding would be helpful in selecting a PEDV strain for vaccine development.

• 대한바이러스학회지
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• 제27권2호
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• pp.185-195
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• 1997

Sequence comparison of the RNA-dependent RNA polymerase of human caliciviruses (HuCVs) from Korean children with gastroenteritis revealed significant genetic variation among them. cDNA clones were produced from the HuCVs collected from pediatric population during a period of 1987-1994. The application of reverse transcription-polymerase chain reaction (RT-PCR) using primers directed to the RNA-dependent RNA polymerase region within ORF1 of Norwalk virus (NV) showed that 13.7% of HuCVs yielded PCR products of similar size to the NV prototype, NV8FIIa/68/US, with exceptions of HuCV 185/87/Korea and HuCV 1115/90/Korea. Computer analyses showed that the PCR products had a continuous protein encoding frame on the positive strand, and contained GLPSG and YGDD amino acid motifs at the predicted distance from primers. Alignment of the amino acid sequences of HuCVs with previously published sequences for Snow Mountain agent (SMA), NV, and Sapporo/82/Japan indicated that these strains can be divided into four major genogroups. There were 10 (45%) SMA-like CVs, one (4.5%) NV-like HuCVs, two (9%) Sapporo-like HuCVs, and nine (41%) unidentified HuCVs. This fourth genogroup should be investigated further. HuCV 185/87/Korea and HuCV 1115/90/Korea, Sapporo-like CVs, were genetically distinct from previously characterized HuCVs and more closely related to known animal CVs. One of the animal CV-like strain, HuCV 185/87/Korea, showed nucleotide and amino acid homology of only 67% and 73% with the prototype Sapporo/82/Japan. Further characterization of animal and human CV genomes and studies of possible cross-transmission of CVs from animals to humans are likely to be beneficial in understanding the epidemiology of HuCVs.

• 한국어병학회지
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• 제22권3호
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• pp.229-237
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• 2009

The causative agent for the tunic softness syndrome of the cultured ascidian Halocynthia roretzi from Jan 1999 to Feb 2009 was identified using virus isolation and polymerase chain reaction (PCR). The pathogenicity of the isolated virus MABV UR-1 strain was determined by experimental infection trials. The cytopathic effects was observed in CHSE-214 cell line at a level 5.1% (4/78) in normal ascidian and 1.8% in abnormal ascidian showing tunic softness syndrome signs. MABV gene was detected in 16.8% (18/107) of normal and 13.1% (5/38) of abnormal organisms by PCR. The ratio of MABV isolation and gene detection was similar level in normal and soft tunic diseased ascidian. Based on the VP2/NS junction region sequences, eight strains of virus isolated from ascidian, were included in the same genogroup with MABV which is originally isolated in wide ranges of marine fish and shellfish species. The UR-1 strain caused 60% mortality (36.5% mortality in control group) by immersion infection and 37% mortality (same mortality in control group) in injection infection indicating no significant differences in infected and control groups. These results suggest that ascidian can act as reservoir of the MABV, and this virus is not directly related with the ascidian mortality.