• Journal of Microbiology and Biotechnology
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• 제29권12호
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• pp.2006-2013
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• 2019

The isolation of respiratory viruses, especially from clinical specimens, often shows poor efficiency with classical cell culture methods. The lack of suitable methods to generate virus particles inhibits the development of diagnostic assays, treatments, and vaccines. We compared three inoculation methods, classical cell culture, the addition of a JAK2 inhibitor AZD1480, and centrifugation-enhanced inoculation (CEI), to replicate human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV). In addition, a combined method using AZD1480 treatment and CEI was used on throat swabs to verify that this method could increase virus isolation efficiency from human clinical specimens. Both CEI and AZD1480 treatment increased HRSV and HMPV genome replication. Also, the combined method using CEI and AZD1480 treatment enhanced virus proliferation synergistically. The combined method is particularly suited for the isolation of interferon-sensitive or slowly growing viruses from human clinical specimens.

• 미생물학회지
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• 제55권1호
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• pp.25-32
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• 2019

Influenza A (H1N1) virus caused a worldwide pandemic in 2009-2010 and still remains in seasonal circulation. Continuous surveillance activities are encouraged in the post pandemic phase to watch over the trend of occurrence every year, this is better to be done by a rapid and sensitive method for its detection. This study was conducted to detect proportions of occurrence of influenza A virus (H1N1) in patients with influenza-like illness. Samples from 500 patients with influenza or influenza-like clinical presentation were tested by real-time reverse transcription polymerase chain reaction (RT-PCR) and virus tissue culture. Among the total 500 participants, 193 (38.6%) were females and 307 (61.4%) males. Seventy-one patients (14.2%) were positive for H1N1 virus infection with real-time RT-PCR while 52 (10.4%) were positive by tissue culture. Non-statistically significant relation was found between age and gender with the positivity of H1N1. Sensitivity and specificity of real-time RT-PCR was 98.08% and 95.54%, respectively, in comparison to virus isolation with accuracy 95.8%. This study showed that H1N1 virus was responsible for a good proportion of influenza during the post-pandemic period. Real-time RT-PCR provides rapidity and sensitivity for the detection of influenza A virus (H1N1) compared with virus isolation and thus it is recommended as a diagnostic tool.

• 대한바이러스학회지
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• 제26권1호
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• pp.67-76
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• 1996

The methods that make Hantavirus grow consist of inoculation into the experimental animals and cultured cells. The cultured cells, such as Vero-E6 and A549 cells, have been usually used for isolation of the virus and the animals, such as mice and rats, are used for large scale preparation of the virus so far. Furthermore, the cell can be used to maintain the virus and assay the infectivity and the animals can be used for the experiment of viral pathogenicity and challenge for assessment of vaccine. Apodemus mice, the own natural host of the virus, has been used for challenge test of Hantaan virus. However it has been pointed out to difficult handling and breeding the animal in laboratory. Therefore, we attempted to establish a new animal model for challenge test at the time of isolation of Maaji virus which is a new hantavirus similar but distinct to Hantaan virus. In suckling hamster, the titer of Maaji virus and the lethality to mice of the virus were increased gradually in the titer and lethality through passage by intracerebral (IC) inoculation. We tried to re-adapt this brain virus to lung of weanling hamster. The brain passaged virus was inoculated into weanling hamster intramuscularly. Again, the titer of the virus in lung was also increased by continuous passage of this virus. This facts could regarded as adaptation to new environment in which the virus proliferates. To identity the virus passaged in hamster with Maaji virus, both of the virus passaged in hamster brain and lung were compared with Maaji virus (MAA-I) and Hantaan virus (HTN 76-118) by means of restriction fragment length polymorphism (RFLP) and slingle strand conformation polymophism (SSCP). As a result, we conclude that Maaji virus could be adapted successfully to weanling hamster through this passage strategy. Utilizing this adapted Maaji virus strain, hamster model is able to be used for challenge test in hantaviral vaccinology and further experiments utilizing hamster system as a rather available and convenient lab animal are expected.

• 대한수의학회지
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• 제40권1호
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• pp.42-48
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• 2000

Akabane disease is transmitted through mosquitoes in cattle, sheep and goats. It shows congenital abnormalities including encephalomyetitis, hydranencephaly, neurogenic arthrogryposis, and deformed neonatal calves. Akabane viruses, 93FMX and K-9 strain, were isolated from fetal matrix of aborted cow and blood of healthy cow, respectively. S gene sequences of 93FMX and K-9 showed 100% homology with that of OBE-1 strain isolated in Japan. Based upon our sequencing data, we synthesized specific primers for PCR diagnosis. Using these primers, we were able to amplify the S gene of Akabane virus not only from the culture fluid of Vero cells but also from the brain tissue of suckling mouse inoculated with, Akabane virus. These PCR products were confirmed by Southern blot hybridization. Not only the sensitivity of PCR test was high enough to detect the viruses of $10^{1.0}TCID_{50}/ml$, but also the time for diagnosis was significantly shorter than that of the virus isolation by tissue culture method. This method was also effective for the detection of Akabane virus in the cerebrum of fetus. RT-PCR method may be used for a useful diagnostic test of the clinical cases of Akabane disease.

• 대한수의학회지
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• 제37권2호
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• pp.339-347
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• 1997

A cytopathogenic virus was isolated from the brain tissues of pig showing ataxia. The biophysical, morphological and serological assay showed that the isolate belongs to a coronavirus. The differential identification of the isolate with monoclonal antibodies against A and X sites of transmissible gastroenteritis virus indicated that the virus has a characteristics of porcine respiratory coronavirus. The RT-PCR on nucleocapsid region of TGEV also showed that the isolate has the same conserved sequence. The diverse pathogenesis of PRCV and its implication in field were discussed.

• 한국동물위생학회지
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• 제20권2호
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• pp.195-203
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• 1997

Fecal samples were collected from 47 calves with diarrhea and 12 clinically normal co-h-abitants, and tested for virus using MDBK cell cultures. Three cytopathic viruses were isolated from 8 fecal samples obtained from diarrheic calves. The isolated viruses were neutralized by bovine coronavirus hyperimmune serum In plaque reduction assay and were detected in the cytoplasm of MDBK cell by bovine coronavirus hyperimmune serum using immunofluorescence staining. The viruses agglutinated mouse erythrocytes only among the various animal erythrocytes tested and new isolates were identified as bovine coronavirus.

• 대한수의학회지
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• 제31권2호
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• pp.189-194
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• 1991

Getah virus is known as a causative agent of recognized febrile illness of horses characterized by fever, rash and edema. A serological survey indicated that hemagglutination inhibition antibody against Getah virus was detected in 34% of 464 racehorses from Korean Horse Affairs Association and 57% of 262 ponies from Cheju island, respectively. Several field strains of Getah virus isolated were from the racehorse that have been shown fever and febrile signs in 1989. The field isolates produced cytopathic effect in Vero, MA-104, BHK-21 cell cultures. Especially, they multiplied to the highest titer($10^6TCID_{50}/0.1ml$) in Vero cell cultures. When day-old mice were inoculated with field isolates by the intracerebral route, they showed a typical paralysis sign and died within seven days after inoculation. The guinea pig exhibited skin rash and edema, and died with neural signs after inoculation with the field isolates. In the cross neutralization test and indirect immunofuorescent assay, the field isolates were proved to be closely related to the Sakai strain of Getah virus antigenically.

• 생명과학회지
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• 제9권3호
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• pp.289-292
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• 1999

1998년 부산지역에서 호흡기 환자의 인후 가검물로부터 분리된 influenza 바이러스 다음과 같은 양사을 볼 수 있었다. 1.분리된 바이러스는 influenza A/Sydney/05/97-like(H3N2)형과 influenza A/Beijing/262/95-like(H1N1)형으로 동정되었다.2.바이러스분리는 4월에서 9월까지를 제외한 모든 달에서 이루어 졌으며 특히 12월에는 집중적인 양상을 보였다.3.연령별로 본 바이러스의 분리는 1세에서 68세까지 전 연령층에서 나타났으며 10세 이하가 가장 높은 분리율을 나타내었다.4.바이러스 분리 비율은 남녀가 1:1.2의 비율로 나타났다.5.분리 바이러스는 MDCK 세포에서 세포병변이 관찰 되었다.6.influenza 바이러스를 Negative staining으로 염색하여 전자현미경으로 관찰한 결과 원형의 바이러스로 in-fluenza A/Sydney/05/97-like(H3N2)는 130 nm,influenza A/Beijing/262/95-like(H1N1) 형은 145nm의 크기로 나타났다.

• 한국동물위생학회지
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• 제22권4호
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• pp.363-370
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• 1999

돼지 호흡기 생식기증 바이러스(porcine reproductive and respiratory syndrome virus: PRRSV) 감염이 의심되는 농장으로부터 수집된 혈청가검물 646개로부터 MARC-145 cell을 이용하여 PRRSV 분리를 시도한 바 MARC-145 세포단층상에 세포변성효과(cytopathic effects : CPE)를 나타내는 바이러스 36주를 분리하였다. 분리된 36주가 PRRSV인지 여부를 확인하기 위하여 PRRSV를 실험적으로 접종한 혈청을 이용하여 간접형광항체시험과 혈청중화시험을 실시한 결과 36주 모두가 PRRSV로 동정되었다. 혈청학적인 동정법과 더불어 reverse-transcription polymerase chain reaction을 이용하여 PRRSV open reading frame 5(ORF5)의 유전자를 증폭한 결과 선발된 6주 모두에서 80bp의 flanking sequencing를 포함하여 약 680bp의 ORF5의 유전자를 증폭할 수 있었다.

• Journal of Microbiology
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• 제34권2호
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• pp.124-130
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• 1996

A survey was conducted to determine the prevalance of infectious pancreatic necrosis virus (IPNV) on fish farms in Korea and the epidemiology of IPNV infection in the farmed rainbow trout. In total, 43 pools of rainbow trout with apparent signs of viral infection from five provinces were obtained and analyzed. Evident cytopathic effects, including karyopycnosis and cell destruction, were observed in CHSE (chinook samlmon embyro)-214 cells infected with the virus isolates. Of these, ten viral isolates were assumed to be IPNV based on biophysical properties. RNA analysis revealed that the isolates contained two-segmented RNA genomes, further indicating that the viral isolates are IPNV. Antigenic comparison of the IPNV isolates identified three distinct serological groups separable by the cross-neutralization test. Of the ten IPNV isolates, six could be classified as strain DRT, two as strain Ab, and two as strain VR299. We were not able to isolate new strain of IPNV or any isolate serologically similar to the standard strain Sp.poraceae and families of the Agaricales, they are genetically more related to the Polyporaceae. These results are consistent with morphological characters observed in those mushrooms. However, it is premature to conclude taxonomic status Ganoderma species in the present study employing small sample size.