• 한국수산과학회지
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• 제46권1호
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• pp.46-52
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• 2013

After an outbreak of viral disease in an aquafarm, release of virus (es) from infected fish into environmental seawater has been suspected. In the present study, we utilized a negatively charged membrane (HA type) as an efficient method for concentration and detection of fish pathogenic viruses, specifically, megalocytivirus and viral hemorrhagic septicemia virus (VHSV) present in field-collected seawater samples or inoculated into seawater artificially. Positively charged viruses adsorbed onto the negatively charged membrane and were eluted with 1 mM NaOH (pH 10.5) following rinsing with 0.5 mM $H_2SO_4$ (pH 3.0). Megalocytivirus and VHSV particles isolated using anegatively charged HA membrane from seawater inoculated with each virus at a concentration of 10 viral particles/mL were of sufficient quantity to show positive results in atwo-step PCR (or RT two-step PCR); however, despite it being negatively charged, a cellulose acetate (CA) membraneshowed negative results. In quantitative PCR, the detection limits of the HA membrane for megalocytivirus and VHSV in seawater were 1.20E+00 viral particles/mL and 1.22E+01 viralparticles/mL, respectively. The calculated mean recovery yields from 1 L seawater spiked with known concentrations of megalocytivirus and VHSV particles were 28.11% and 23.00%, respectively. The concentrate of a 1-L sample of culturing seawater from the aquatank of flounder suffering from VHSV showed clear positive results in PCR when isolated with an HA, but not a CA, membrane. Thus, viral isolation using an HA membrane is a practical and reliable method for detection of fish pathogenic viruses in seawater.

• Journal of Preventive Medicine and Public Health
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• 제7권2호
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• pp.403-415
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• 1974

The author has investigated epidemiological features of human cases of epidemic encephalitis (E. E.) in the Republic of Korea and the status of antibody requisition in pre-and post-epidemic time. And virological and serological studies with regarding the relationship of E. E. infection between human and piglet, and field survey against its vector by means of virus isolation from mosquitoes were carried out. Finally, vaccine field trial against human population has also been evaluated in order to confirm its effectiveness. The results of the studies are summarized as follows : 1. The annual incidence of reported cases during the past 25 years (1949-1973) in the Republic of Korea has shown two patterns, one was typical cyclic incidence and the other one was irregular. Annual average morbidity and mortality rate per 100,000 population were 5.7 and 2.1 and fatality rate was 34.6% in typical cyclic years. 2. With regard to the geographical distribution of E. E., the province of Jeolla-Bug-Do illustrated the highest incidence regardless of the epidemic size. 3. The main epidemic period was between mid-August and mid-September (above 90% of the total number of cases). The first case was reported in middle of July and the epidemic ceased in late of October. 4. An analysis of the age distribution of cases of E. E., has shown that above 90% of the total cases occurred in the age groups under 14 years and it was noted that about its 54% were occurred in the age groups between 5-9 years group. 5. Through the Haemagglutination Inhibition (H-I) test for the laboratory diagnosis of E. E., it was found that higher H-I antibody titer was usually detected in the convalescent phase, 15 days after onset. 6. The H-I antibody survey against 563 healthy population by age groups during the pre-epidemic season showed that 422(75%) were less than H-I titer, 1:20 and 122(21.7%) were positive H-I titer, 1:20. Among the 94 American in Seoul who had not been in E. E. endemic area previously only one person had appeared sero-conversion as a H-I titer of 1:80 after post-epidemic season. 7. The E. E. virus could be isolated from the mosquitos pools-C, tritaeniorhyncus which were caught between late July and middle August. 8. E.E. Virus was also isolated from piglet blood on early August and H-I antibody conversion was occurred mostly on middle of August. 9. H-I antibody sero-conversion rate reached to high level when vaccine purified by mouse brain tissue inoculated, showing 98.9%. Higher antibody titer was acquired when booster inoculation was performed, Four fold rise of H-I add N-T antibodies was confirmed with 93.2% and 82.1% respectively.

• Asian-Australasian Journal of Animal Sciences
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• 제33권10호
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• pp.1683-1690
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• 2020

Objective: The rapid and reliable detection of the African swine fever virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including realtime polymerase chain reaction (PCR). However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on World Organization for Animal Health (OIE) protocol for accurate detection of ASFV in field samples in Vietnam. Methods: Seven positive confirmed samples (four samples have no mismatch, and three samples contained one mutation in probe binding sites) were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE. Results: Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of cycle quantification (Cq) values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe (Cq value ranked between 33.12-35.78). Conclusion: We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam.

• 한국동물위생학회지
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• 제36권3호
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• pp.163-169
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• 2013

Both of avian influenza (AI) and Newcastle disease (ND) can cause mild to severe diease in poultry. In this study, clinical signs, macro, and micro lesions were studied. Eighteen six-week-old SPF chicks were divided into 4 groups (E1, E2, E3 and C1) and housed in different rooms of the isolation facility at CAVAC (Daejeon, Korea). The control group (C1) of 3 chicks was housed separately as uninoculated. Experimental groups (E1, E2 and E3) challenged with H9N2 and/or NDV. E1 group was challenged with 0.1 mL A/Kr/Ck/01310/01 (H9N2) $10^{5.6}$ $EID_{50}$ by intranasal, E2 group was challenged with 0.5 mL Kyojeongwon (KJW) $10^{5.0}{\sim}10^{6.0}$ $ELD_{50}$ by intramuscular, and E3 group was challenged with 0.1 mL A/Kr/Ck/01310/01 $10^{5.6}$ $EID_{50}$ by intranasal and 0.5 mL KJW $10^{5.0}{\sim}10^{6.0}$ $ELD_{50}$ by intramuscular 7 days after H9N2 challenge. In clinical signs and gross findings, E1 group showed 0% mortality, anorexia, and hemorrhage of proventriculus and thymus, E2 group showed 100% mortality within 3~5 days after challenge, anorexia, green diarrhea, hemorrhage of proventriculus, proximal esophagus and thymus, enlargement of kidney, and bronze liver, and E3 group showed 100% mortality within 24~36 hours after NDV challenge, depression, anorexia, green diarrhea, hemorrhage of proventriculus, spleen, and lung, enlargement of kidney, and reduction of thymus size and number. In histopathological examination, E1 group showed depletion and necrosis in bursa of Fabricius, thymus, and spleen, and E2 and E3 group showed severe lymphocyte depletion and necrosis with destruction of lymphoid organ structures. In conclusion, co-infection of H9N2 with ND virus causes acute disease with high mortality than single infection and the pathologic lesions were more severe.

• 대한수의학회지
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• 제36권4호
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• pp.845-858
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• 1996

The purpose of this study was to establish early diagnostic methods for the detection of Aujeszky's disease viral antigens and nucleic acid in nasal cells, and buffy coats from experimentally infected living pigs by a combination of immunocytochemistry, in situ hybridization with digoxigenin(DIG)-labled probe and electron microscopy. Forty days old piglets were inoculated intranasally with $10^{7.0}TCID_{50}$ of Aujeszky's disease virus (ADV, NYJ-1-87 strain). The viral antigens and nucleic acid of ADV were detected in nasal cells, and buffy coat for 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopical method. The results were compared with conventional methods such as a porcine Aujeszky's disease serodiagnostic(PAD) kit, neutralization test(NT) and virus isolation. 1. The viral antigens, nucleic acids and capsids of ADV were detected in nasal cells, buffy coats from 3 days to 20 days after inoculation by immunocytochemistry, in situ hybridization with DIG-labeled probe and electron microscopy, respectively. 2. When viral antigens were detected by the immunocytochemical technique, a diffuse brown deposit was observed in the nucleus and cytoplasm of nasal cells, buffy coats and PK-15 cells under a microscope. 3. DIG-labeled DNA probe was prepared by amplification of conserved sequence of recombinant ADV-gp50 clone with polymerase chain reacction. When ADV-DNA was detected by ISH with DIG-labeled probe, purplish blue pigmentation were observed in the nuclei and cytoplasms of ADV-infected cells under a microscope. Positive signals were observed in nasal cells and in the buffy coat and PK-15 cells at the first day after inoculation. 4. Where ADV-capsids were detected by transmission electron microscopical method, aggregation of capsids was observed in the nuclei and cytoplasms of nasal cells, buffy coats and PK-15 cells. The results suggested that these methods were considered as the highly sensitive and reliable tools for rapid and confirmative diagnosis of Aujeszky's disease in living pigs.

• 대한수의학회지
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• 제28권1호
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• pp.99-103
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• 1988

The first outbreak of Aujeszky's disease(AD) was identified from piggery located at the southern part of Korea in July, 1987. This piggery suffered from a significant economic loss caused by unexpected piglet mortality and reproductive failure. Etiologic viral agents were isolated from tonsil and spleen of the infected piglets, and the isolates produced a typical cytopathic effects of herpesvirus with giant cell formation when inoculated in many different cells. Subsequently the field isolates were characterized as suid herpesvirus I by cross-neutralization test and indirect fluorescence assay utilizing specific monoclonal antibody, and were proved to be a pathogenic strain of AD virus(ADV).

• The Plant Pathology Journal
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• 제25권3호
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• pp.291-293
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• 2009

The low virus titer in woody plant tissues and the presence of inhibitor compounds such as polyphenols, tannins and polysaccharides are common difficulties that compromise purification of plant viroids from their woody hosts. A simple, reliable method of RNA isolation using CF11 cellulose column on a microcentrifuge tube scale for detecting Apple scar skin viroid (ASSVd) in apple was developed. Total RNA extracted from leaf, woody bark and the fruit skin was used for reverse transcription. RT-PCR products could be detected from RNA prepared from dormant woody bark, fruit skin and fresh leaves with both the CF11 cellulose column method and NucliSens extractor in February, August and November. Meanwhile, with the RNeasy kit RT-PCR, products were detected only in leaves and not from bark or fruit skin. The PCR product, about 330 base pairs, was analyzed by agarose gel electrophoresis. The CF11 cellulose column method was effective for detecting ASSVd. The method enabled the processing of a large numbers of samples of dormant woody bark, leaf and fruit skin of apple.

• Pediatric Infection and Vaccine
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• 제20권3호
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• pp.115-122
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• 2013

Reemerging infectious diseases are infections that had decreased in incidence in the global population and were brought under control through effective health care policy such as vaccination, but more recently, began to resurge as a health problem due to many reasons. Measles, rubella, mumps and pertussis are the examples. Immunization with MMR (measles, mumps, rubella) and pertussis vaccine has contributed to marked decrease in measles, mumps, rubella and pertussis incidence worldwide. In Korea, measles and rubella almost disappeared after the introduction of 2 doses of MMR immunization schedule. Recently, these infections have been reemerging in many countries with low vaccination rates and can be introduced again in Korea. However mumps and pertussis outbreaks are reported among fully vaccinated populations. Declining vaccine effectiveness, an increased awareness and surveillance of the disease or improved laboratory diagnostic tools had been suggested as possible causes. For the clinicians, it is difficult to diagnose these reemerging infectious diseases partly because of few experience of typical cases of measles and rubella or partly because of modification of clinical symptoms and signs of infectious diseases in immunized population. In this article, the diagnosis of measles, mumps, rubella and pertussis will be reviewed in the aspects of clinical characteristics, serologic methods, virus isolation, and polymerase chain reaction.

• 농업과학연구
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• 제47권1호
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• pp.183-191
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• 2020

Lichens are composite organisms consisting of a symbiotic association of a fungus with a photosynthetic partner (the photobiont or phycobiont), usually either a green alga or cyanobacterium. According to more recent studies, the biological activities of lichens and lichen substances include an antibiotic activity, antitumor and antimutagenic activity against human immunodeficiency virus (HIV), allergenic activity, plant growth inhibitory activity, and enzyme inhibitory activity. This study screened lichen extracts with a potent in vitro antifungal activity against plant diseases caused by phytopathogenic fungi. The compounds were isolated from Stereocaulon alpinum and Sphaerophorus globosus, and their chemical structures were identified as methyl hematommate, methyl β-orsellinate, 5-hydroxyferulic acid, sphaerophorin, and 2-heptyl-4,6-dimethoxybenzoic acid by electron ionization mass spectrometry (EI-MS) and nuclear magnetic resonance (NMR) spectral analyses. In vitro disease control against Alternaria mali, Cochliobolus miyabeanus, Colletotrium gloeosporioides, and Verticillum dahliae was evaluated. And among the five compounds, only methyl hematommate was effective against A. mali, C. miyabeanus, and C. gloeosporioides. The compounds were isolated from these lichens, which have a similar biosynthetic pathway, respectively. This is the first report of these compounds being isolated from these lichens.

• 한국동물위생학회지
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• 제22권1호
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• pp.43-52
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• 1999

Presently, viral isolation in the diarrheal feces can be reached by many tools such as fluorescent antibody test(FA), negative contrast electron microscopy(NCEM), virus neutralization test, cell culture, and so on. The purpose of the study was to aimed at the establishment of simplified NCEM technique which can be efficiently applied for diarrheal feces and also the understanding on prevalence of viral-induced diarrhea in calves. One hundred fourty-seven korean indigenous calves with diarrhea were examined to their feces by the modified NCEM. Among them, 98(66.7%) were confirmed to have one or more viruses in feces. The viruses detected were identified as rotavirus(33.3%), coronavirus(16.3% ), togavirus(10.2%) and herpesvirus(0.7%). Ten cases of combined viral infection were consisted of 8 with rotavirus+coronavirus, one with rotavirus+togavlrus and one with rotavirus+herpesvirus. Dirrheal types could classified by yello-wish watery(44.9a ), blood-tinged(19.7% ), white watery(17.7% ) , brownish watery(14.3%), greenish watery(3.4%) diarrhea, respectively. Yellowish watery diarrhea(66cases) was frequently included rotavirus(31.8%), coronavirus(15.2%), and togavirus(13.6%), respectively. Consequently, these results suggest that the modified NCEM is reliable and efficient diagnostic tool for detection of viruses in the diarrheal feces and many calves rearing in Chonnam province have been exposed to some enteric viral agents mainly including rotavirus and coronavirus.