• Genomics & Informatics
• /
• v.20 no.2
• /
• pp.21.1-21.14
• /
• 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
• /
• v.24 no.12
• /
• pp.1719-1727
• /
• 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.

• Proceedings of the Korea Contents Association Conference
• /
• 2014.11a
• /
• pp.57-58
• /
• 2014

인플루엔자 A 바이러스의 아형인 H5N1은 고병원성으로 조류 독감을 일으킨다. H5N1 바이러스는 원래 조류끼리만 감염되는 독감이고, 사람에게는 전염되지 않는다고 알려져 있었으나, 2003년에 베트남과 중국을 시작으로 현재까지 168명의 사망자가 기록되고 있다. 그러나 현재 시판되고 있는 진단키트(Rapid diagnostic kits)들은 H5N1 에 특이적인 것이 아니라 influenza A virus 모두를 진단한다. 따라서 influenza 감염여부는 확인 할 수 있지만, 이것이 H5N1 인지는 확인 할 수가 없다. H5N1은 전염성이 강하기 때문에 빠르게 진단하여 감염조류를 살 처분 하여야 더 많은 경제적 손실을 줄일 수 있다. 따라서 H5N1 에만 특이적인 epitope를 네트워크 기반으로 예측하여 진단제에 응용할 수 있도록 하고자 한다. 각 서열 정보는 Openflu (http://openflu. vital-it.ch/browse.php)에서 얻었다. H5N1은 H1N1에서 유래되었기 때문에 두 subtype의 차이점을 알아보고자 TCOFFEE에서 multiple sequence alignment를 수행한 결과 N-terminal 부분이 상이하였다. 상이한 H5N1의 N-terminal 부분이 H5N1 virus에 감염된 모든 host에서 존재하는지 알아보기 위해 host가 사람인 경우와 조류인 경우를 TCOFFEE에서 alignment 하였다. 그 결과 H5N1의 N-terminal 부분은 사람과 조류에서 보존적이었다. 따라서 H5N1의 N-terminal이 다른 subtype과 유사하지 않고 H5에만 특이적이기 때문에 진단키트 제작을 위한 epitope로 사용할 수 있을 것으로 기대된다.

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

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

• Journal of Ginseng Research
• /
• v.36 no.4
• /
• pp.396-402
• /
• 2012

Vaccination is the main strategy for preventing influenza infection. However, vaccine efficacy is influenced by several factors, including age and health status. The efficacy of the influenza vaccine is much lower (17% to 53%) in individuals over 65 yr of age compared with young adults (70% to 90%). Therefore, increasing vaccine efficacy remains a challenge for the influenza vaccine field. In this study, we investigated the impact of supplementing vaccination with the dietary intake of Korean red ginseng (RG) extract and RG saponin. Mice were immunized two times intranasally with inactivated influenza A (H1N1) virus. Mice received RG extract or RG saponin orally for 14 d prior to the primary immunization. After the primary immunization, mice continued to receive RG extract or RG saponin until the secondary immunization. Mice vaccinated in combination with dietary intake of RG extract and RG saponin showed elevated serum anti-influenza A virus IgG titers and improved survival rates in lethal influenza A virus infection: 56% and 63% of mice receiving RG extract or RG saponin survived, respectively, while 38% of mice that only received the vaccine survived. Moreover, mice receiving RG extract supplementation recovered their body weight more quickly than those not receiving RG extract supplementation. We propose that the dietary intake of RG extract and RG saponin enhances the vaccine-induced immune response and aids in providing protection against influenza virus infection.

• Proceedings of the Microbiological Society of Korea Conference
• /
• 2003.05a
• /
• pp.87-89
• /
• 2003

• Clinical and Experimental Pediatrics
• /
• v.53 no.1
• /
• pp.56-66
• /
• 2010

Purpose : Although oseltamivir is widely used for treatment of influenza, few clinical studies of its efficacy and resistance have been performed in Korea. We evaluated the safety, side effects, and efficacy of oseltamivir treatment in Korean pediatric patients. Methods : We analyzed 321 children diagnosed with influenza at Busan St. Mary's Medical Center, Korea, between January 2008 and June 2008 (first study period) and November 2008 and January 2009 (second study period). Patients were divided into two groups: those receiving oseltamivir treatment for 5 days and those receiving only symptomatic treatment. We investigated clinical symptoms, side effects, and resistance to oseltamivir. We also identified influenza strains and evaluated resistance to oseltamivir using an influenza virus culture. Results : One hundred eighty-six patients were assigned to the treatment group, and 135 were assigned to the control group. The treatment group showed shorter admission duration (4.4 days) compared with controls (5.0 days) (P =0.000) and had fewer lower respiratory tract complications compared with controls (P <0.05). No significant statistical difference in the virus antigenic type was observed between the groups. In the first study period, virus culture showed influenza B (41.7% vs. 49.6%), A/H3N2 (7.9% vs. 8.4%), and A/H1N1 (9.4% vs. 6.5%). In the second study period, only A/H1N1 (55.3% vs. 50.0%) was isolated, except for one case of A (H3N2) in the treatment group. No differences in short- and long-term side effects, including neuropsychologic side effects, were noted between groups. There was no resistance to oseltamivir before or after treatment in the first study period. Conclusion : Based on our results, we suggest that osetalmivir therapy in pediatric patients is effective.

• Korean Journal of Veterinary Research
• /
• v.44 no.3
• /
• pp.449-454
• /
• 2004

Ninety pigs under the age of 120-day-old requested at the diagnostic laboratory of animal diseases in Cheju National University were evaluated for the prevalence of tissue antigen and serum antibody to swine influenza virus (SIV). For histopathologic examination there was sampled at the consolidated area in cranioventral or dorsocaudal lobes of lungs. Lung tissues from all pigs were tested for the antigen of SIV type A by immunohistochemistry (IHC). Sera from 56 pigs were used for the antibody detection to SIV type A (subtype H1N1 and H3N2) by haemagglutinin inhibition test. Pneumonic lesions were observed in 72 cases (80%) of 90 pigs. Broncho-interstitial or interstitial pneumonia were more prevalent than suppurative or fibrinous bronchopneumonia. According to HI test, 46.4% of the tested sera showed seropositive. Positive sera were consisted with 5.3% for SIV H1N1, 28.6% for SIV H3N2, and 12.5% for both subtype to be tested, respectively. SIV antigens were detected in 51 cases(56.6%) of 90 pigs. Most SIV antigens were presented in the epithelium of the bronchi and bronchiole. Necrotizing bronchitis or bronchiolitis were observed in 28(31.1%) cases of all inspected pigs. These results suggested that SIV might be an important role to induce swine pneumonia in Jeju. Also IHC was very useful for the detection of SIV in the lung.

• Korean Journal of Microbiology
• /
• v.43 no.1
• /
• pp.23-30
• /
• 2007

The most rapid Real-time PCR based detection method for Avian influenza A virus (AIV) subtype H5N1 was developed. The target DNA sequence in this study was deduced from H5N1 subtype-specific 387 bp partial gene of hemagglutinin, and was synthesized by using PCR-based gene synthesis on the ground of safety. Real-Time PCR was performed by $GenSpector^{TM}$ using microchip-based, total $1{\mu}l$ of reaction mixture with extremely short time in each steps in PCR. The detection including PCR-amplication and analysis of melting temperature was totally completed within 13 min. The H5N1-specific 189 bp PCR product was correctly amplified until 2.4 molecules of hemagglutinin gene as minimum of templates. This kind of PCR was designated as Quick Real-Time PCR in this study and it could be applied to detect not only AIV H5N1, but also other pathogens using PCR-based detection.