• Journal of Preventive Medicine and Public Health
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• v.43 no.3
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• pp.274-278
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• 2010

Objectives: This report describes the results of an investigation on an outbreak of novel influenza A (H1N1) in an English language Institute in Seoul, Korea in May 2009. Methods: In this outbreak, novel influenza A (H1N1) was confirmed in 22 of 91 trainees, trainers and staff members. The trainees and 2 staff members were isolated in an assigned facility and the rest were isolated in their homes after we discovered the first patient with novel influenza A (H1N1). After the isolation, the people in the assigned facility were educated to use N95 respirators and they received oseltamivir for prophylaxis. Results: The initial findings in this study suggest that the symptoms were mild and similar to those of seasonal influenza. The classmates and roommates of the infected patients were more likely to get infected with novel influenza A (H1N1) than the trainees who were not classmates or roommates of the patients (OR: 3.19, 95% Cl=0.91 - 11.11 for classmates and OR: 40.0, 95% Cl=7.4-215.7 for roommates). Conclusions: The public health response seems successful in terms of preventing the spread of this virus into the local community.

• Clinical and Experimental Pediatrics
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• v.46 no.10
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• pp.1024-1028
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• 2003

Influenza-associated encephalopathy is regarded as one of the major neurologic disease entities along with those of Reye syndrome, acute necrotizing encephalopathy, and myelitis which are known to be related to influenza virus, mostly type A. And it is being actively researched in Japan as it has caused a tremendous increase in the number of deaths from 1997 to 2002, but it has not been yet reported in the Korean pediatric medical community. It attacks those previously healthy children, who have not been vaccinated. Patients start with such symptoms as fever and common respiratory problems, but within 24 to 48 hours they suffer from seizures with acute mental deterioration, become worse, and suffer multiple organ failures including marked elevated transaminase levels as well as coagulopathy. It induces deaths in a couple of days after the symptoms appear or remains a serious neurologic sequelae. Confirmative diagnosis is used to demonstrate influenza viral infection. We report here a 37 month aged boy who was admitted to our hospital during the last influenza season under the diagnosis of influenza associated encephalopathy on the basis of serologic testing by hemagglutinin inhibition(HI). This is the first report confirmed by increased antibody titer of the influenza A virus in Korea.

• Korean Journal of Veterinary Service
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• v.42 no.3
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• pp.135-144
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• 2019

Low pathogenic avian influenza (LPAI; H9N2) and Newcastle disease (ND) are economically important poultry diseases in Korea. In this study, we investigated pathological features and virus distribution in the lymphoid tissues of chicks experimentally infected with H9N2 and/or ND virus. Six-weeks-old SPF chickens were divided into 4 groups, Control (C), H9N2 (E1), NDV (E2), and H9N2+NDV (E3). E1 group was challenged with 0.1 ml A/Kr/Ck/01310/01 (H9N2) $10^{5.6}$ $EID_{50}$ intranasally, E2 group was challenged with 0.5 ml KJW (NDV) $10^{5.0}{\sim}10^{6.0}$ $ELD_{50}$ intramuscularly, and E3 group was challenged with H9N2, followed 7 days later by NDV. In histopathological examination, E1 group showed depletion and necrosis in bursa of Fabricius, thymus, cecal tonsil, and spleen, whereas E2 and E3 groups were noted severe lymphocyte depletion and necrosis with destruction of lymphoid organs structures. In TUNEL assay, apoptotic bodies were detected in lymphoid organs of all experimental groups, which was most severe in E3 group. H9N2 and ND viruses were predominantly detected in cecal tonsil of E1, E2, and E3 groups by PCR and immunohistochemistry (ICH). In conclusion, co-infection of H9N2 with NDV caused severe pathologic lesions and apoptosis in lymphoid tissues compared to single infections.

• Journal of Life Science
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• v.28 no.5
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• pp.555-562
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• 2018

Swine influenza virus (SIV) causes one of the most common diseases of the pig population, and its subtypes are determined by hemagglutinin (HA) and neuraminidase (NA). Recently, the SIV subtype diagnosis has been developed. The method using antigen-antibody reaction rather than PCR was mainly used because of the large change in the ribonucleotide sequences of SIV. Here, we have developed 10 diagnostic primer sets through multi-nucleotide sequences alignment of spreaded SIV since 2008 in Korea and then optimized the reaction of the one-step RT-PCR for rapid determination of SIV subtype. In addition, specific primers were designed to early determine the pandemic SIV by detecting unique M sequences proven in highly infectious and virulent subtypes of the influenza H1N1 (pH1N1). Here, some of the SIVs spread in Korea from 2008 to 2014 have been tested to determine the subtypes and pandemic potential of SIV. All diagnostic primer sets were found to be able to accurately determine the SIV subtype and to detect the pandemic SIV. In conclusion, it was confirmed that the optimized one-step RT-PCR analysis using these primer sets is useful for rapid diagnosis of SIV subtypes. These results can be used for development of SIV subtype diagnostic kit to early detect before virulent SIV spreads do.

• Bulletin of the Korean Chemical Society
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• v.35 no.4
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• pp.1082-1086
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• 2014

A multi-channel microchip electrophoresis (MCME) method with parallel laser-induced fluorescence (LIF) detection was developed for rapid screening of H1N1 virus. The hemagglutinin (HA) and nucleocapsid protein (NP) gene of H1N1 virus were amplified using polymerase chain reaction (PCR). The amplified PCR products of the H1N1 virus DNA (HA, 116 bp and NP, 195 bp) were simultaneously detected within 25 s in three parallel channels using an expanded laser beam and a charge-coupled device camera. The parallel separations were demonstrated using a sieving gel matrix of 0.3% poly(ethylene oxide) ($M_r$ = 8,000,000) in $1{\times}$ TBE buffer (pH 8.4) with a programmed step electric field strength (PSEFS). The method was ~20 times faster than conventional slab gel electrophoresis, without any loss of resolving power or reproducibility. The proposed MCME/PSEFS assay technique provides a simple and accurate method for fast high-throughput screening of infectious virus DNA molecules under 400 bp.

• Biomedical Science Letters
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• v.17 no.4
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• pp.297-304
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• 2011

Influenza A virus of the Orthomyxoviridae family is a contagious respiratory pathogen that continues to evolve and burden in the human public health. It is able to spread efficiently from human to human and have the potential to cause pandemics with significant morbidity and mortality. It has been estimated that every year about 500 million people are infected with this virus, causing about approximately 0.25 to 0.5 million people deaths worldwide. Influenza A viruses are classified into different subtypes by antigenicity based on their hemagglutinin (HA) and neuraminidase (NA) proteins. The sudden emergence of influenza A virus subtypes and access for epidemiological analysis of this subtypes demanded a rapid development of specific diagnostic tools. Also, rapid identification of the subtypes can help to determine the antiviral treatment, because the different subtypes have a different antiviral drug resistance patterns. In this study, our aim is to detect influenza A virus subtypes by using real-time nucleic acid sequence based amplification (NASBA) which has high sensitivity and specificity through molecular beacon. Real-time NASBA is a method that able to shorten the time compare to other molecular diagnostic tools and is performed by isothermal condition. We selected major pandemic influenza A virus subtypes, H3N2 and H5N1. Three influenza A virus gene fragments such as HA, NA and matrix protein (M) gene were targeted. M gene is distinguished influenza A virus from other influenza virus. We designed specific primers and molecular beacons for HA, NA and M gene, respectively. In brief, the results showed that the specificity of the real-time NASBA was higher than reverse transcription polymerase chain reaction (RT-PCR). In addition, time to positivity (TTP) of this method was shorter than real-time PCR. This study suggests that the rapid detection of neo-appearance pandemic influenza A virus using real-time NASBA has the potential to determine the subtypes.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.08a
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• pp.273-273
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• 2013

Recently, Point-of-care (POC) testing microdevices enable to do the patient monitoring, drug screening, pathogen detection in the outside of hospital. Immunochromatographic strip (ICS) is one of the diagnostic technologies which are widely applied to POC detection. Relatively low cost, simplicity to use, easy interpretations of the diagnostic results and high stability under any circumstances are representative advantages of POC diagnosis. It would provide colorimetric results more conveniently, if the genetic analysis microsystem incorporates the ICS as a detector part. In this work, we develop a reverse transcriptase-polymerase chain reaction (RT-PCR) microfluidic device integrated with a ROSGENE strip for colorimetric influenza H1N1 virus detection. The integrated RT-PCR- ROSGENE device is consist of four functional units which are a pneumatic micropump for sample loading, 2 ${\mu}L$ volume RT-PCR chamber for target gene amplification, a resistance temperature detector (RTD) electrode for temperature control, and a ROSGENE strip for target gene detection. The device was fabricated by combining four layers: First wafer is for RTD microfabrication, the second wafer is for PCR chamber at the bottom and micropump channel on the top, the third is the monolithic PDMS, and the fourth is the manifold for micropump operation. The RT-PCR was performed with subtype specific forward and reverse primers which were labeled with Texas-red, serving as a fluorescent hapten. A biotin-dUTP was used to insert biotin moieties in the PCR amplicons, during the RT-PCR. The RT-PCR amplicons were loaded in the sample application area, and they were conjugated with Au NP-labeled hapten-antibody. The test band embedded with streptavidins captures the biotin labeled amplicons and we can see violet colorimetric signals if the target gene was amplified with the control line. The off-chip RT-PCR amplicons of the influenza H1N1 virus were analyzed with a ROSGENE strip in comparison with an agarose gel electrophoresis. The intensities of test line was proportional to the template quantity and the detection sensitivity of the strip was better than that of the agarose gel. The test band of the ROSGENE strip could be observed with only 10 copies of a RNA template by the naked eyes. For the on-chip RT-PCR-ROSGENE experiments, a RT-PCR cocktail was injected into the chamber from the inlet reservoir to the waste outlet by the micro-pump actuation. After filling without bubbles inside the chamber, a RT-PCR thermal cycling was executed for 2 hours with all the microvalves closed to isolate the PCR chamber. After thermal cycling, the RT-PCR product was delivered to the attached ROSGENE strip through the outlet reservoir. After dropping 40 ${\mu}L$ of an eluant buffer at the end of the strip, the violet test line was detected as a H1N1 virus indicator, while the negative experiment only revealed a control line and while the positive experiment a control and a test line was appeared.

• Korean Journal of Microbiology
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• v.41 no.1
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• pp.53-59
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• 2005

Influenza is an important public health problem which occurs almost every winter in temperate climates and is often associated with increased rates of hospitalization and death. In 1999, our influenza surveillance was initiated with 4 voluntary sentinel physicians and the Public Health Center. During the 2003-2004 influenza season, 124 influenza viruses were isolated from 401 clinical specimens, which were collected from patients with Influenza-like illness(ILI) in Seoul. The case definition of ILI is a case with fever more than $38^{\circ}C$ and systemic symptoms; cough, or sore throat. ILI was the highest at the 20-49 age $group(23\%)$ and the rate of virus isolation was the highest at the 7-19 age $group(50\%)$. Among 124 influenza viruses, isolates 83 were identified as A/H3N2 type and others were subtyped as influenza B viruses in 2003-2004 season. Influenza viruses were collected $39.1\%$ at Nowon-Gu, $13.5\%$ Gangnam-Gu and Seocho-Gu etc. and the isolate rate of virus had the area difference; Yongsan-Gu $66.7\%$, Gangnam-gu $50.0\%$, Nowon-Gu $39.9\%$, Kangbuk-Gu $36.8\%$, Seocho-Gu $27.8\%$, Dongjak-Gu $21.2\%$. Out of 401 individuals, 160 was vaccinated $(40\%)$ and the vaccination rate was the highest at the 20-49 age $group(32\%)$. These findings may contribute to the recommondation of the influenza vaccine formulation and the development of influenza control measure.

• Genomics & Informatics
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• v.20 no.2
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• pp.21.1-21.14
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• 2022

The influenza A viruses have high mutation rates and cause a serious health problem worldwide. Therefore, this study focused on genome characterization of the viruses isolated from Thai patients based on the next-generation sequencing technology. The nasal swabs were collected from patients with influenza-like illness in Thailand during 2017-2018. Then, the influenza A viruses were detected by reverse transcription-quantitative polymerase chain reaction and isolated by MDCK cells. The viral genomes were amplified and sequenced by Illumina MiSeq platform. Whole genome sequences were used for characterization, phylogenetic construction, mutation analysis and nucleotide diversity of the viruses. The result revealed that 90 samples were positive for the viruses including 44 of A/H1N1 and 46 of A/H3N2. Among these, 43 samples were successfully isolated and then the viral genomes of 25 samples were completely amplified. Finally, 17 whole genomes of the viruses (A/H1N1, n=12 and A/H3N2, n=5) were successfully sequenced with an average of 232,578 mapped reads and 1,720 genome coverage per sample. Phylogenetic analysis demonstrated that the A/H1N1 viruses were distinguishable from the recommended vaccine strains. However, the A/H3N2 viruses from this study were closely related to the recommended vaccine strains. The nonsynonymous mutations were found in all genes of both viruses, especially in hemagglutinin (HA) and neuraminidase (NA) genes. The nucleotide diversity analysis revealed negative selection in the PB1, PA, HA, and NA genes of the A/H1N1 viruses. High-throughput data in this study allow for genetic characterization of circulating influenza viruses which would be crucial for preparation against pandemic and epidemic outbreaks in the future.

• Journal of Microbiology and Biotechnology
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• v.24 no.12
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• pp.1719-1727
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• 2014

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.