• Journal of Microbiology and Biotechnology
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• v.18 no.6
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• pp.1164-1169
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• 2008

We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus-negative specimens. Furthermore, the assays could detect and subtype up to $10^5$ dilution of each of the reference viruses that had an original infectivity titer of $10^6\;EID_{50}/ml$. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.

• YAKHAK HOEJI
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• v.39 no.6
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• pp.645-653
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• 1995

Dibenamine inhibited [$^{3}$H]quinuclidinyl benzilate ([$^{3}$H]QNB) binding in both concentration and incubation time-dependent manners. The $IC_{50}$/ value of dibenamine for the inhibition of the specific binding of 100 pM [$^{3}$'H]QNB following incubation of cerebral microsomes with dibenamine at 37.deg. C for 15 min was 20.mu.M. Dibenamine irreversibly decreased the binding site concentration for [$^{3}$H]QNB binding without affecting the affinity of [$^{3}$H]QNB for the muscarinic receptor. Analysis of the pirenzepine inhibition curve of [$^{3}$H]QNB binding to cerebral microsomes indicated the presence of two receptor subtypes with high(M$_{1}$ receptor, Ki=5nM) and low (M$_{2}$ receptor, Ki=160nM) affinity for pirenzepine. However, dibenamine(20.mu.M) treatment under the condition employed in these experiments caused steepening of the pirenzepine competition curve. The Ki value for pirenzepine in dibenamine treated-microsomes was approximately 120nM. suggesting a selective decrease in the number of M$_{1}$ receptor. Although dibenamine also inhibited [$^{3}$H]QNB binding to ventricular microsomes with $IC_{50}$/ value of 120.mu.M, the sensitivity for dibenamine in the ventricle was much lower than that in the cerebrum. These results indicate that dibenamine at low concentrations welectively inactivates the muscarinic M$_{1}$ receptor.

• Biomolecules & Therapeutics
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• v.2 no.4
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• pp.361-368
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• 1994

The nature of the muscarinic receptors in bovine adrenal medulla was investigated in this study. [$^3$H]Quinuclidinyl benzilate(QNB) specifically bound to a single class of muscarinic receptor with a $K_{D}$ value of about 70 pM in bovine adrenal medullary, cardiac ventricular and ileal homogenates. Pirenzepine inhibition curves of [$^3$H]QNB binding to cardiac ventricular and ileal homogenates were steep, indicating the presence of a single class of binding site for pirenzepine with a Ki value of 990 nM and 508 nM, respectively. However, pirenzepine/[$^3$H]QNB competition binding curves in adrenal medulla suggested the presence of two binding sites (Hill coefficient=0.59) with a high( $M_1$) and a low( $M_2$) affinity. Respective Ki values for pirenfepine were 16 nM and 633 nM, with 44% of total sites having a high affinity( $M_1$). Gallamine, which is selective to cardiac $M_2$-receptor, inhibited [$^3$H]QNB binding to adrenal medullary, cardiac ventricular and ileal homogenates with Ki values of 12 $\mu$M, 6 $\mu$M and 13 $\mu$M, respectively. Thus, the binding affinities of pirenzepine and gallamine for $M_2$-receptor in adrenal medulla were similar to those in ileum, which contains the $M_3$-receptor. These results indicate that the $M_1$- and $M_3$- muscarinic receptor subtypes coexist in the bovine adrenal medulla.a.

• Tuberculosis and Respiratory Diseases
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• v.68 no.4
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• pp.205-211
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• 2010

Background: Procalcitonin is a well known marker in infection that plays a role in distinguishing between bacterial and viral infections in screening. The aim of the present study was to evaluate the role of procalcitonin in differentiating between 2009 H1N1 influenza pneumonia and community acquired pneumonia of bacterial origin, or mixed bacterial origin and 2009 H1N1 influenza infection. Methods: A retrospective observational study was performed over the 6-month winter period during the 2009 H1N1 influenza pandemic. Ninety-six patient-subjects were enrolled, all of whom had been diagnosed with community acquired pneumonia in emergency department during the study period. On admission, laboratory studies were performed, which included 2009 H1N1 influenza real-time polymerase chain reaction of nasal secretions and procalcitonin on serum; the laboratory values were compared between the study groups. Receiver operating characteristic curve analyses were performed on the resulting data. Results: Compared to those with bacterial or mixed infections (n=62) and bacterial pneumonia with confirmed organisms (n=30), patients with 2009 H1N1 pneumonia (n=34) were significantly more likely to have low procalcitonin levels (p=0.008, 0.001). Using cutoff of value >0.3 ng/mL, the sensitivity and specificity of procalcitonin for detection of patients with confirmed bacterial pneumonia were 76.2% and 60.6%, respectively. A significant difference in procalcitonin was found between 2009 H1N1 pneumonia and pneumonia caused by mixed influenza viral and bacterial infections (0.15 [0.05~0.84] vs. 10.3 [0.05~22.87] ng/mL, p=0.045). Conclusion: Serum procalcitonin measurement may assist in the discrimination between pneumonia of bacterial and of 2009 H1N1 influenza origin. High values of procalcitonin suggest that bacterial infection or mixed infection of bacteria and 2009 H1N1 influenza is more likely.

• Tuberculosis and Respiratory Diseases
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• v.76 no.2
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• pp.84-87
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• 2014

We hereby observe four co-infection cases of pandemic influenza H1N1 and Mycobacterium tuberculosis with various clinical presentations. It may be prudent to consider M. tuberculosis co-infections when patients with pandemic influenza reveal unusual clinical features that do not improve despite appropriate treatments against the influenza, especially in Korea, in the endemic areas of M. tuberculosis.

• Korean Journal of Poultry Science
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• v.40 no.3
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• pp.243-252
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• 2013

Among the 16 hemagglutinin (HA) subtypes of avian influenza virus (AIV), only the H5 and H7 subtypes have caused highly pathogenic avian influenza (HPAI) in poultry. However, most H5 or H7 subtype viruses are categorized as low pathogenic avian influenza (LPAI). Some AIVs, including the H5 and H7 HPAI viruses, have shown the ability to infect humans directly. In this study, we describe the biological and molecular characterization of an H5N3 AIV (SBD/KR/KNU SYG06/06) isolated from spot-billed duck (Anas poecilorhyncha) in Korea. A phylogenetic analysis of the eight viral genes showed that the SBD/KR/KNU SYG06/06 isolate belongs to the Eurasian lineage and that the SBD/KR/KNU SYG06/06 isolate was clearly different from HPAI H5N1 strains, including human isolates and the Italian HPAI H5N2 strains. Additionally, no relationship was found between SBD/KR/KNU SYG06/06 and the Korean HPAI H5N1 isolates. The SBD/KR/ KNU SYG06/06 isolate had avian specific receptor binding site residues in the HA protein and the four C-terminal amino acids in the NS1 protein. The HA protein of the SBD/KR/KNU SYG06/06 isolate exhibited the typical LPAI motif at the cleavage site and this virus produced no cytopathic effects in MDCK cells without trypsin. Given these results, we suggest that the H5N3 AIV isolated from the spot-billed duck should be considered an LPAI virus and should have no pathogenic effect in humans.

• Korean Journal of Pharmacognosy
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• v.32 no.3 s.126
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• pp.219-225
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• 2001

The water extracts of 82 Korean medicinal herbs were examined for the binding affinity on the recombinant human muscarinic acetylcholine receptor subtype 1 $(mAChR-M_1)$ produced from the CHO (Chinese Hamster Ovary) cell line. Of those tested, the extracts of Coptidis Rhizoma, Phellodendri Cortex, Hedyotis Herba and of Terminariae Fructus were found to exhibit a significant competition with $[^3H]$ N-methyl-scopolamine for the specific binding to $mAChR-M_1$ in a dose dependent manner, respectively.

• Clinical and Experimental Pediatrics
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• v.53 no.10
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• pp.886-891
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• 2010

Purpose: This study aims to investigate the clinical characteristics of children diagnosed with the novel influenza A (H1N1) in the winter of 2009 at a single medical institution. Methods: Out of 545 confirmed cases of influenza A (H1N1) in children, using the real time RT-PCR method at Kosin University Gospel Hospital from September to December of 2009, 149 patients and their medical records were reviewed in terms of symptoms, laboratory findings, complications and transmission within a family. Results: Median age of subjects was 7 years (range: 2 months-18 years). New cases increased rapidly from September to reach a peak in November, then declined rapidly. Most frequently observed symptoms were fever (96.7%), cough (73.2%), rhinorrhea (36.9%) and sore throat (31.5%). Average body temperatures on the 1st, 2nd and 3rd hospital day were $38.75{\pm}0.65^{\circ}C$, $38.08{\pm}0.87^{\circ}C$ and $37.51{\pm}0.76^{\circ}C$, respectively. Complete blood counts and biochemical tests performed on the first admission day showed within the reference values in most cases. Of the 82 patients with simple chest radiography, 18 (22%) had pneumonic lesions; multi-focal bronchopneumonia in eleven, single or multi-segmental lobar pneumonia in five, and diffuse interstitial pneumonia in two patients. All of the 149 patients improved from their symptoms and discharged within 9 days of admission without any late complication. Conclusion: Children with 2009 pandemic influenza A (H1N1) at our single institution displayed nonspecific symptoms and laboratory findings, resembling those of common viral respiratory illnesses, and did not appear to develop more severe disease.

• Korean Journal of Clinical Laboratory Science
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• v.41 no.4
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• pp.145-152
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• 2009

Novel influenza A virus, subtype H1N1 of swine-lineage, has been transmitted rapidly to many regions of the world. Rapid detection of the virus is essential to instigate appropriate patient care and public health management and for disease surveillance. The aim of this study is to determine the prevalence of novel influenza A (H1N1) virus in Korea using reverse-transcription real time polymerase chain reaction (rRT-PCR). Novel H1N1 virus was detected in a total of 8,948 nasopharyngeal samples from patients with influenza-like illness throughout Korea from August to September 2009. RNA was extracted from $300{\mu}l$of sample using an RNA extraction kit (Zymo Research, CA, USA). In the present study, Genekam kit (Genekam, Duisburg, Germany) was used to detect novel H1N1 virus. Novel H1N1 virus was found in 1,130 samples from a total of 8,948 samples (12.6%). The highest frequency was found in 10- to 19-year-olds (M: 29.3% vs. F: 16.4%), followed by 20- to 29-year-olds (M: 17.9% vs. F: 15.4%), 40- to 49-year-olds (M: 6.5% vs. F: 8.1%), 50- to 59-year-olds (M: 6.0% vs. F: 5.5%), and 30- to 39-year-olds (M: 4.6% vs. F: 3.8%). The mean positive rate was higher in men than in women (M: 14.7% vs. F: 7.4%). Novel H1N1 virus showed the lowest prevalence in patients over 60 years old. The positive rate increased daily and showed a significant high peak in mid-September 2009. In 19 provinces of Korea, Cheonan (41.1%), Busan (37.3%), Gangneung (33.3%), Jinju (32.1%), Ulsan (24.6%), Deajeon (23.7%) areas showed high frequencies and other provinces were found less than 10% of novel H1N1 virus. Since reverse-transcription real time PCR assay is rapid, accurate, and convenient, it may assist public health laboratories in detecting novel H1N1 virus. Moreover, these data could be useful for the management of patients with influenza-like illness.

• Journal of Microbiology and Biotechnology
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• v.24 no.5
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• pp.704-713
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• 2014

The highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtype infect poultry and have also been spreading to humans. Although new antiviral drugs and vaccinations can be effective, rapid detection would be more efficient to control the outbreak of infections. In this study, a phage-display library was applied to select antibody fragments for HPAI strain A/Hubei/1/2010. As a result, three clones were selected and sequenced. A hemagglutinin inhibition assay of the three scFvs revealed that none exhibited hemagglutination inhibition activity towards the H5N1 virus, yet they showed a higher binding affinity for several HPAI H5N1 strains compared with other influenza viruses. An ELISA confirmed that the HA protein was the target of the scFvs, and the results of a protein structure simulation showed that all the selected scFvs bound to the HA2 subunit of the HA protein. In conclusion, the three selected scFVs could be useful for developing a specific detection tool for the surveillance of HPAI epidemic strains.