33 results on '"Kaith S"'
Search Results
2. The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation
- Author
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Ji-Young Park, Amal Senevirathne, Khristine Kaith S. Lloren, and John Hwa Lee
- Subjects
SFTSV ,NSs ,NP ,replication ,autophagosome ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome–lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication.
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- 2024
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3. An mRNA-Based Multiple Antigenic Gene Expression System Delivered by Engineered Salmonella for Severe Fever with Thrombocytopenia Syndrome and Assessment of Its Immunogenicity and Protection Using a Human DC-SIGN-Transduced Mouse Model
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Ji-Young Park, Chamith Hewawaduge, Chandran Sivasankar, Khristine Kaith S. Lloren, Byungkwan Oh, Mi Young So, and John Hwa Lee
- Subjects
Salmonella delivery ,vaccine ,SFTS ,viral protein ,hDC-SIGN ,Pharmacy and materia medica ,RS1-441 - Abstract
Currently, there are no commercial vaccines or therapeutics against severe fever with thrombocytopenia syndrome (SFTS) virus. This study explored an engineered Salmonella as a vaccine carrier to deliver a eukaryotic self-mRNA replicating vector, pJHL204. This vector expresses multiple SFTS virus antigenic genes for the nucleocapsid protein (NP), glycoprotein precursor (Gn/Gc), and nonstructural protein (NS) to induce host immune responses. The engineered constructs were designed and validated through 3D structure modeling. Western blot and qRT-PCR analyses of transformed HEK293T cells confirmed the delivery and expression of the vaccine antigens. Significantly, mice immunized with these constructs demonstrated a cell-mediated and humoral response as balanced Th1/Th2 immunity. The JOL2424 and JOL2425 delivering NP and Gn/Gc generated strong immunoglobulin IgG and IgM antibodies and high neutralizing titers. To further examine the immunogenicity and protection, we utilized a human DC-SIGN receptor transduced mouse model for SFTS virus infection by an adeno-associated viral vector system. Among the SFTSV antigen constructs, the construct with full-length NP and Gn/Gc and the construct with NP and selected Gn/Gc epitopes induced robust cellular and humoral immune responses. These were followed by adequate protection based on viral titer reduction and reduced histopathological lesions in the spleen and liver. In conclusion, these data indicate that recombinant attenuated Salmonella JOL2424 and JOL2425 delivering NP and Gn/Gc antigens of SFTSV are promising vaccine candidates that induce strong humoral and cellular immune responses and protection against SFTSV. Moreover, the data proved that the hDC-SIGN transduced mice as a worthy tool for immunogenicity study for SFTSV.
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- 2023
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4. The Effect of Tryptophan-to-Tyrosine Mutation at Position 61 of the Nonstructural Protein of Severe Fever with Thrombocytopenia Syndrome Virus on Viral Replication through Autophagosome Modulation.
- Author
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Park, Ji-Young, Senevirathne, Amal, Lloren, Khristine Kaith S., and Lee, John Hwa
- Subjects
FEVER ,VIRAL replication ,THROMBOCYTOPENIA ,WESTERN immunoblotting ,VIRAL proteins ,HELA cells ,THROMBOPOIETIN receptors - Abstract
In our prior investigations, we elucidated the role of the tryptophan-to-tyrosine substitution at the 61st position in the nonstructural protein NSsW61Y in diminishing the interaction between nonstructural proteins (NSs) and nucleoprotein (NP), impeding viral replication. In this study, we focused on the involvement of NSs in replication via the modulation of autophagosomes. Initially, we examined the impact of NP expression levels, a marker for replication, upon the infection of HeLa cells with severe fever thrombocytopenia syndrome virus (SFTSV), with or without the inhibition of NP binding. Western blot analysis revealed a reduction in NP levels in NSsW61Y-expressing conditions. Furthermore, the expression levels of the canonical autophagosome markers p62 and LC3 decreased in HeLa cells expressing NSsW61Y, revealing the involvement of individual viral proteins on autophagy. Subsequent experiments confirmed that NSsW61Y perturbs autophagy flux, as evidenced by reduced levels of LC3B and p62 upon treatment with chloroquine, an inhibitor of autophagosome–lysosome fusion. LysoTracker staining demonstrated a decrease in lysosomes in cells expressing the NS mutant compared to those expressing wild-type NS. We further explored the mTOR-associated regulatory pathway, a key regulator affected by NS mutant expression. The observed inhibition of replication could be linked to conformational changes in the NSs, impairing their binding to NP and altering mTOR regulation, a crucial upstream signaling component in autophagy. These findings illuminate the intricate interplay between NSsW61Y and the suppression of host autophagy machinery, which is crucial for the generation of autophagosomes to facilitate viral replication. [ABSTRACT FROM AUTHOR]
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- 2024
- Full Text
- View/download PDF
5. Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform
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Su Jeong Ahn, Yun Hee Baek, Khristine Kaith S. Lloren, Won-Suk Choi, Ju Hwan Jeong, Khristine Joy C. Antigua, Hyeok-il Kwon, Su-Jin Park, Eun-Ha Kim, Young-il Kim, Young-Jae Si, Seung Bok Hong, Kyeong Seob Shin, Sungkun Chun, Young Ki Choi, and Min-Suk Song
- Subjects
RT-LAMP ,Seasonal influenza ,Avian influenza ,Multiplex detection ,Colorimetric visualization ,Infectious and parasitic diseases ,RC109-216 - Abstract
Abstract Background In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans. Methods We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses). Results We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%). Conclusions Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.
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- 2019
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- View/download PDF
6. Correction to: Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loopmediated isothermal amplification (RT-LAMP) diagnostic platform
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Ahn, Su Jeong, Baek, Yun Hee, Lloren, Khristine Kaith S., Choi, Won-Suk, Jeong, Ju Hwan, Antigua, Khristine Joy C., Kwon, Hyeok-il, Park, Su-Jin, Kim, Eun-Ha, Kim, Young-il, Si, Young-Jae, Hong, Seung Bok, Shin, Kyeong Seob, Chun, Sungkun, Choi, Young Ki, and Song, Min-Suk
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- 2020
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7. Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform
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Ahn, Su Jeong, Baek, Yun Hee, Lloren, Khristine Kaith S., Choi, Won-Suk, Jeong, Ju Hwan, Antigua, Khristine Joy C., Kwon, Hyeok-il, Park, Su-Jin, Kim, Eun-Ha, Kim, Young-il, Si, Young-Jae, Hong, Seung Bok, Shin, Kyeong Seob, Chun, Sungkun, Choi, Young Ki, and Song, Min-Suk
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- 2019
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8. Correction to: Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loopmediated isothermal amplification (RT-LAMP) diagnostic platform
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Su Jeong Ahn, Yun Hee Baek, Khristine Kaith S. Lloren, Won-Suk Choi, Ju Hwan Jeong, Khristine Joy C. Antigua, Hyeok-il Kwon, Su-Jin Park, Eun-Ha Kim, Young-il Kim, Young-Jae Si, Seung Bok Hong, Kyeong Seob Shin, Sungkun Chun, Young Ki Choi, and Min-Suk Song
- Subjects
Infectious and parasitic diseases ,RC109-216 - Abstract
An amendment to this paper has been published and can be accessed via the original article.
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- 2020
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9. An mRNA-Based Multiple Antigenic Gene Expression System Delivered by Engineered Salmonella for Severe Fever with Thrombocytopenia Syndrome and Assessment of Its Immunogenicity and Protection Using a Human DC-SIGN-Transduced Mouse Model
- Author
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Park, Ji-Young, primary, Hewawaduge, Chamith, additional, Sivasankar, Chandran, additional, Lloren, Khristine Kaith S., additional, Oh, Byungkwan, additional, So, Mi Young, additional, and Lee, John Hwa, additional
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- 2023
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10. Development of a rapid, simple and efficient one-pot cloning method for a reverse genetics system of broad subtypes of influenza A virus
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Choi, Won-Suk, Jeong, Ju Hwan, Lloren, Khristine Kaith S., Ahn, Su Jeong, Antigua, Khristine Joy C., Kim, Young-il, Si, Young-Jae, Baek, Yun Hee, Choi, Young Ki, and Song, Min-Suk
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- 2019
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11. Salmonella-mediated oral delivery of multiple-target vaccine constructs with conserved and variable regions of SARS-CoV-2 protect against the Delta and Omicron variants in hamster
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Lloren, Khristine Kaith S., primary, Jawalagatti, Vijayakumar, additional, Hewawaduge, Chamith, additional, Sivasankar, Chandran, additional, Park, Ji-Young, additional, and Lee, John Hwa, additional
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- 2023
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12. Molecular Markers for Interspecies Transmission of Avian Influenza Viruses in Mammalian Hosts
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Khristine Kaith S. Lloren, Taehyung Lee, Jin Jung Kwon, and Min-Suk Song
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avian influenza ,interspecies transmission ,mammalian host ,molecular marker ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
In the last decade, a wide range of avian influenza viruses (AIVs) have infected various mammalian hosts and continuously threaten both human and animal health. It is a result of overcoming the inter-species barrier which is mostly associated with gene reassortment and accumulation of mutations in their gene segments. Several recent studies have shed insights into the phenotypic and genetic changes that are involved in the interspecies transmission of AIVs. These studies have a major focus on transmission from avian to mammalian species due to the high zoonotic potential of the viruses. As more mammalian species have been infected with these viruses, there is higher risk of genetic evolution of these viruses that may lead to the next human pandemic which represents and raises public health concern. Thus, understanding the mechanism of interspecies transmission and molecular determinants through which the emerging AIVs can acquire the ability to transmit to humans and other mammals is an important key in evaluating the potential risk caused by AIVs among humans. Here, we summarize previous and recent studies on molecular markers that are specifically involved in the transmission of avian-derived influenza viruses to various mammalian hosts including humans, pigs, horses, dogs, and marine mammals.
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- 2017
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13. Preclinical evaluation of the efficacy of an H5N8 vaccine candidate (IDCDC-RG43A) in mouse and ferret models for pandemic preparedness
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Khristine Kaith S. Lloren, Min-Suk Song, Won-Suk Choi, Ju Hwan Jeong, Ok-Jun Lee, Hyeok-il Kwon, Su Jeong Ahn, Eun-Ha Kim, Young Ki Choi, Jin Jung Kwon, Yun Hee Baek, Chul-Joong Kim, Young-Jae Si, Hae Jung Han, and Young-Il Kim
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Cross Protection ,medicine.medical_treatment ,030231 tropical medicine ,Drug Evaluation, Preclinical ,Hemagglutinin (influenza) ,Biology ,Antibodies, Viral ,medicine.disease_cause ,Virus ,Mice ,03 medical and health sciences ,Immunogenicity, Vaccine ,0302 clinical medicine ,Adjuvants, Immunologic ,Orthomyxoviridae Infections ,medicine ,Animals ,Influenza A Virus, H5N8 Subtype ,030212 general & internal medicine ,Pandemics ,General Veterinary ,General Immunology and Microbiology ,Immunogenicity ,Ferrets ,Public Health, Environmental and Occupational Health ,Antibody titer ,Antibodies, Neutralizing ,Virology ,Influenza A virus subtype H5N1 ,Disease Models, Animal ,Infectious Diseases ,Viral replication ,Influenza Vaccines ,biology.protein ,Molecular Medicine ,Antibody ,Adjuvant - Abstract
Because H5N1 influenza viruses continuously threaten the public health, the WHO has prepared various clades of H5N1 mock-up vaccines as one of the measures for pandemic preparedness. The recent worldwide outbreak of H5Nx virus which belongs to clade 2.3.4.4 and of which H5N6 subtype belongs and already caused human infection also increases the need of pandemic vaccine for such novel emerging viruses. In this study, we evaluated the protective efficacy and immunogenicity of an egg-based and inactivated whole-virus H5N8 (IDCDC-RG43A) developed by CDC containing HA and NA gene of the parent virus A/gyrfalcon/Washington/41088-6/2014. Mice vaccinated two times elicited low to moderate antibody titer in varying amount of antigen doses against the homologous H5N8 vaccine virus and heterologous intra–clade 2.3.4.4 H5N6 (A/Sichuan/26221/2014) virus. Mice immunized with at least 3.0 µg/dose of IDCDC-RG43A with aluminum hydroxide adjuvant were completely protected from lethal challenge with the mouse-adapted H5N8 (A/Environment/Korea/ma468/2015, maH5N8) as well as cleared the viral replication in tissues including lung, brain, spleen, and kidney. Vaccinated ferrets induced high antibody titers against clade 2.3.4.4 H5N8/H5N6 viruses and the antibody showed high cross-reactivity to clade 2.2 H5N1 but not to clade 1 and 2.3.4 viruses as measured by hemagglutinin inhibition and serum neutralization assays. Furthermore, administration of the vaccine in ferrets resulted in attenuation of clinical disease signs and virus spread to peripheral organs including lung, spleen, and kidney from high dose challenge with maH5N8 virus. The protective and immunogenic characteristic of the candidate vaccine are essential attributes to be considered for further clinical trials as a pre-pandemic vaccine for a potential pandemic virus.
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- 2019
14. Rapid and simple colorimetric detection of multiple influenza viruses infecting humans using a reverse transcriptional loop-mediated isothermal amplification (RT-LAMP) diagnostic platform
- Author
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Young-Jae Si, Hyeok-il Kwon, Young-Il Kim, Ju Hwan Jeong, Kyeong Seob Shin, Khristine Kaith S. Lloren, Khristine Joy C. Antigua, Min-Suk Song, Su-Jin Park, Sungkun Chun, Seung Bok Hong, Yun Hee Baek, Eun-Ha Kim, Young Ki Choi, Su Jeong Ahn, and Won-Suk Choi
- Subjects
0301 basic medicine ,medicine.medical_specialty ,viruses ,030106 microbiology ,Loop-mediated isothermal amplification ,Avian influenza ,Cross Reactions ,Biology ,Influenza A Virus, H7N9 Subtype ,medicine.disease_cause ,Genome ,Virus ,lcsh:Infectious and parasitic diseases ,03 medical and health sciences ,Influenza A Virus, H1N1 Subtype ,Medical microbiology ,Influenza, Human ,medicine ,Humans ,lcsh:RC109-216 ,Multiplex ,Seasonal influenza ,RT-LAMP ,Influenza A Virus, H5N1 Subtype ,Influenza A Virus, H3N2 Subtype ,Correction ,virus diseases ,Reverse Transcription ,Virology ,Reverse transcriptase ,Influenza A virus subtype H5N1 ,030104 developmental biology ,Infectious Diseases ,Technical Advance ,Parasitology ,Influenza A virus ,Colorimetric visualization ,Colorimetry ,Nucleic Acid Amplification Techniques ,Multiplex detection - Abstract
Background In addition to seasonal influenza viruses recently circulating in humans, avian influenza viruses (AIVs) of H5N1, H5N6 and H7N9 subtypes have also emerged and demonstrated human infection abilities with high mortality rates. Although influenza viral infections are usually diagnosed using viral isolation and serological/molecular analyses, the cost, accessibility, and availability of these methods may limit their utility in various settings. The objective of this study was to develop and optimized a multiplex detection system for most influenza viruses currently infecting humans. Methods We developed and optimized a multiplex detection system for most influenza viruses currently infecting humans including two type B (both Victoria lineages and Yamagata lineages), H1N1, H3N2, H5N1, H5N6, and H7N9 using Reverse Transcriptional Loop-mediated Isothermal Amplification (RT-LAMP) technology coupled with a one-pot colorimetric visualization system to facilitate direct determination of results without additional steps. We also evaluated this multiplex RT-LAMP for clinical use using a total of 135 clinical and spiked samples (91 influenza viruses and 44 other human infectious viruses). Results We achieved rapid detection of seasonal influenza viruses (H1N1, H3N2, and Type B) and avian influenza viruses (H5N1, H5N6, H5N8 and H7N9) within an hour. The assay could detect influenza viruses with high sensitivity (i.e., from 100 to 0.1 viral genome copies), comparable to conventional RT-PCR-based approaches which would typically take several hours and require expensive equipment. This assay was capable of specifically detecting each influenza virus (Type B, H1N1, H3N2, H5N1, H5N6, H5N8 and H7N9) without cross-reactivity with other subtypes of AIVs or other human infectious viruses. Furthermore, 91 clinical and spiked samples confirmed by qRT-PCR were also detected by this multiplex RT-LAMP with 98.9% agreement. It was more sensitive than one-step RT-PCR approach (92.3%). Conclusions Results of this study suggest that our multiplex RT-LAMP assay may provide a rapid, sensitive, cost-effective, and reliable diagnostic method for identifying recent influenza viruses infecting humans, especially in locations without access to large platforms or sophisticated equipment.
- Published
- 2019
15. In Vitro and In Vivo Characterization of Novel Neuraminidase Substitutions in Influenza A(H1N1)pdm09 Virus Identified Using Laninamivir-Mediated In Vitro Selection
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Min-Suk Song, Su Jeong Ahn, Won-Suk Choi, Yun Hee Baek, Young Ki Choi, Khristine Kaith S. Lloren, Jin Jung Kwon, and Ju Hwan Jeong
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0303 health sciences ,Mutation ,030306 microbiology ,viruses ,Immunology ,Mutant ,Mutagenesis (molecular biology technique) ,Biology ,medicine.disease_cause ,Microbiology ,Laninamivir ,Virology ,Virus ,03 medical and health sciences ,Viral replication ,Insect Science ,Vaccines and Antiviral Agents ,Influenza A virus ,medicine ,biology.protein ,Neuraminidase ,030304 developmental biology - Abstract
Neuraminidase (NA) inhibitors (NAIs) are widely used antiviral drugs for the treatment of humans with influenza virus infections. There have been widespread reports of NAI resistance among seasonal A(H1N1) viruses, and most have been identified in oseltamivir-exposed patients or those treated with other NAIs. Thus, monitoring and identifying NA markers conferring resistance to NAIs—particularly newly introduced treatments—are critical to the management of viral infections. Therefore, we screened and identified substitutions conferring resistance to laninamivir by enriching random mutations in the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] virus followed by deep sequencing of the laninamivir-selected variants. After the generation of single mutants possessing each identified mutation, two A(H1N1)pdm09 recombinants possessing novel NA gene substitutions (i.e., D199E and P458T) were shown to exhibit resistance to more than one NAI. Of note, mutants possessing P458T—which is located outside of the catalytic or framework residue of the NA active site—exhibited highly reduced inhibition by all four approved NAIs. Using MDCK cells, we observed that the in vitro viral replication of the two recombinants was lower than that of the wild type (WT). Additionally, in infected mice, decreased mortality and/or mean lung viral titers were observed in mutants compared with the WT. Reverse mutations to the WT were observed in lung homogenate samples from D199E-infected mice after 3 serial passages. Overall, the novel NA substitutions identified could possibly emerge in influenza A(H1N1)pdm09 viruses during laninamivir therapy and the viruses could have altered NAI susceptibility, but the compromised in vitro/in vivo viral fitness may limit viral spreading. IMPORTANCE With the widespread emergence of NAI-resistant influenza virus strains, continuous monitoring of mutations that confer antiviral resistance is needed. Laninamivir is the most recently approved NAI in several countries; few data exist related to the in vitro selection of viral mutations conferring resistance to laninamivir. Thus, we screened and identified substitutions conferring resistance to laninamivir by random mutagenesis of the NA gene of the 2009 pandemic influenza [A(H1N1)pdm09] virus strain followed by deep sequencing of the laninamivir-selected variants. We found several novel substitutions in NA (D199E and P458T) in an A(H1N1)pdm09 background which conferred resistance to NAIs and which had an impact on viral fitness. Our study highlights the importance of continued surveillance for potential antiviral-resistant variants and the development of alternative therapeutics.
- Published
- 2019
16. Development of a rapid, simple and efficient one-pot cloning method for a reverse genetics system of broad subtypes of influenza A virus
- Author
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Yun Hee Baek, Young Ki Choi, Su Jeong Ahn, Young-Il Kim, Khristine Joy C. Antigua, Ju Hwan Jeong, Khristine Kaith S. Lloren, Young-Jae Si, Min-Suk Song, and Won-Suk Choi
- Subjects
0301 basic medicine ,DNA, Complementary ,Genes, Viral ,Genetic Vectors ,lcsh:Medicine ,Computational biology ,Biology ,medicine.disease_cause ,Genome ,Polymerase Chain Reaction ,Article ,Madin Darby Canine Kidney Cells ,03 medical and health sciences ,0302 clinical medicine ,Dogs ,Influenza A virus ,medicine ,Animals ,Humans ,Vector (molecular biology) ,Cloning, Molecular ,lcsh:Science ,DNA Primers ,Cloning ,Multidisciplinary ,lcsh:R ,Reverse genetics ,Reverse transcriptase ,Reverse Genetics ,Transformation (genetics) ,030104 developmental biology ,HEK293 Cells ,Phenotype ,lcsh:Q ,Ligation ,Influenza virus ,PCR-based techniques ,030217 neurology & neurosurgery ,Plasmids - Abstract
The reverse genetics (RG) system of influenza A viruses is well established. However, the conventional sequence-dependent method for cloning influenza genome segments is time-consuming and requires multiple processes (eg. enzyme digestion and ligation) and exhibits low cloning efficiency compared to the sequence-independent cloning method. In this study, we improved influenza genome cloning into the pHW2000 vector for an RG system by incorporating a sequence-independent circular polymerase extension cloning (CPEC) approach which requires only 2 steps (reverse transcription and one-pot CPEC-PCR) and takes about 4 hours before the transformation. The specifically designed viral gene and vector primers used for CPEC-PCR have improved cloning efficiency ranging from 63.6 to 100% based on the results of gene-specific colony PCR which was additionally confirmed by enzyme digestion. We successfully cloned all genes from broad subtypes of influenza A viruses (H1-H12, N1-N9) and rescued by the RG system. Our results demonstrate that this method—one-Pot cloning for influenza A virus—was efficient in terms of required time and cloning rate. In conclusion, the novel cloning method for influenza A virus will contribute to a significant reduction in the time required for genetic studies of emerging influenza viruses.
- Published
- 2018
17. Simple, Rapid and Sensitive Portable Molecular Diagnosis of SFTS Virus Using Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP)
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Ju Hwan Jeong, Min-Suk Song, Min Ah Yu, Won-Suk Choi, Young-Il Kim, Khristine Kaith S. Lloren, Su-Jin Park, Hye Won Jeong, Yun Hee Baek, Hyeok Il Kwon, Eun-Ha Kim, Young Ki Choi, Seok Yong Kim, Khristine Joy C. Antigua, Hyo Soon Cheon, Su Jeong Ahn, and Jin Jung Kwon
- Subjects
0301 basic medicine ,Phlebovirus ,Genes, Viral ,Middle East respiratory syndrome coronavirus ,Loop-mediated isothermal amplification ,Biology ,medicine.disease_cause ,Applied Microbiology and Biotechnology ,Sensitivity and Specificity ,Virus ,Dengue fever ,Hospitals, University ,03 medical and health sciences ,Limit of Detection ,Republic of Korea ,medicine ,Humans ,SFTS virus ,General Medicine ,Nucleic acid amplification technique ,medicine.disease ,biology.organism_classification ,Virology ,030104 developmental biology ,Phlebotomus Fever ,Molecular Diagnostic Techniques ,Enterovirus ,RNA, Viral ,Colorimetry ,Nucleic Acid Amplification Techniques ,Biotechnology ,Severe fever with thrombocytopenia syndrome virus - Abstract
Recently, human infections caused by severe fever with thrombocytopenia syndrome virus (SFTSV), which can lead to fatality, have dramatically increased in East Asia. With the unavailability of vaccines or antiviral drugs to prevent and/or treat SFTSV infection, early rapid diagnosis is critical for prevention and control of the disease. Here, we report the development of a simple, rapid and sensitive portable detection method for SFTSV infection applying reverse transcription-loop mediated isothermal amplification (RT-LAMP) combined with one-pot colorimetric visualization and electro-free reaction platform. This method utilizes a pocket warmer to facilitate diagnosis in a resource-limited setting. Specific primers were designed to target the highly-conserved region of L gene of SFTSV. The detection limit of the RT-LAMP assay was approximately 100 viral genome copies from three different SFTSV strains. This assay exhibited comparable sensitivity to qRT-PCR and 10-fold more sensitivity than conventional RT-PCR, with a rapid detection time of 30 to 60 minutes. The RT-LAMP assay using SFTSV clinical specimens has demonstrated a similar detection rate to qRT-PCR and a higher detection rate compared to conventional RT-PCR. Moreover, there was no observed cross-reactive amplification of other human infectious viruses including Japanese Encephalitis Virus (JEV), Dengue, Enterovirus, Zika, Influenza and Middle East Respiratory Syndrome Coronavirus (MERS-CoV). This highly sensitive, electro- and equipment-free rapid colorimetric visualization method is feasible for resource-limited SFTSV field diagnosis.
- Published
- 2018
18. Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes
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Jungwon Hwang, Young Ki Choi, Myung Hee Kim, Ju Hwan Jeong, Khristine Kaith S. Lloren, Hee Bok Chae, Jin Jung Kwon, Won-Suk Choi, Min-Suk Song, Yun Hee Baek, Su Jeong Ahn, Hyeok-il Kwon, Elena A. Govorkova, Chul-Joong Kim, and Richard J. Webby
- Subjects
0301 basic medicine ,medicine.drug_class ,Immunology ,Acids, Carbocyclic ,Neuraminidase ,Cyclopentanes ,medicine.disease_cause ,Antiviral Agents ,Guanidines ,Microbiology ,Virus ,Madin Darby Canine Kidney Cells ,Birds ,03 medical and health sciences ,Dogs ,Oseltamivir ,Zanamivir ,Virology ,Vaccines and Antiviral Agents ,Drug Resistance, Viral ,Influenza, Human ,medicine ,Animals ,Humans ,Enzyme Inhibitors ,Gene ,biology ,Neuraminidase inhibitor ,virus diseases ,Orthomyxoviridae ,Reverse Genetics ,Reverse genetics ,Influenza A virus subtype H5N1 ,030104 developmental biology ,Amino Acid Substitution ,Mutagenesis ,Influenza in Birds ,Insect Science ,biology.protein ,Peramivir ,medicine.drug - Abstract
Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs.IMPORTANCEThe frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs.
- Published
- 2018
19. In Vitro and In Vivo Characterization of Novel Neuraminidase Substitutions in Influenza A(H1N1)pdm09 Virus Identified Using Laninamivir-Mediated In Vitro Selection
- Author
-
Lloren, Khristine Kaith S., primary, Kwon, Jin Jung, additional, Choi, Won-Suk, additional, Jeong, Ju Hwan, additional, Ahn, Su Jeong, additional, Choi, Young Ki, additional, Baek, Yun Hee, additional, and Song, Min-Suk, additional
- Published
- 2019
- Full Text
- View/download PDF
20. Preclinical evaluation of the efficacy of an H5N8 vaccine candidate (IDCDC-RG43A) in mouse and ferret models for pandemic preparedness
- Author
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Jeong, Ju Hwan, primary, Kim, Eun-Ha, additional, Lloren, Khristine Kaith S., additional, Kwon, Jin Jung, additional, Kwon, Hyeok-il, additional, Ahn, Su Jeong, additional, Kim, Young-Il, additional, Choi, Won-Suk, additional, Si, Young-Jae, additional, Lee, Ok-Jun, additional, Han, Hae Jung, additional, Baek, Yun Hee, additional, Song, Min-Suk, additional, Choi, Young Ki, additional, and Kim, Chul-Joong, additional
- Published
- 2019
- Full Text
- View/download PDF
21. Molecular Markers for Interspecies Transmission of Avian Influenza Viruses in Mammalian Hosts
- Author
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Min-Suk Song, Jin Jung Kwon, Khristine Kaith S. Lloren, and Taehyung Lee
- Subjects
0301 basic medicine ,Reassortment ,mammalian host ,Neuraminidase ,interspecies transmission ,Review ,Biology ,Influenza A Virus, H7N9 Subtype ,medicine.disease_cause ,Catalysis ,Birds ,Inorganic Chemistry ,lcsh:Chemistry ,03 medical and health sciences ,chemistry.chemical_compound ,Molecular marker ,Pandemic ,Influenza A Virus, H9N2 Subtype ,medicine ,Animals ,Humans ,Physical and Theoretical Chemistry ,Molecular Biology ,Gene ,lcsh:QH301-705.5 ,Spectroscopy ,Genetics ,molecular marker ,Influenza A Virus, H5N1 Subtype ,Mechanism (biology) ,Transmission (medicine) ,Organic Chemistry ,General Medicine ,Phenotype ,Influenza A virus subtype H5N1 ,Computer Science Applications ,Hemagglutinins ,030104 developmental biology ,chemistry ,lcsh:Biology (General) ,lcsh:QD1-999 ,Influenza in Birds ,Receptors, Virus ,avian influenza - Abstract
In the last decade, a wide range of avian influenza viruses (AIVs) have infected various mammalian hosts and continuously threaten both human and animal health. It is a result of overcoming the inter-species barrier which is mostly associated with gene reassortment and accumulation of mutations in their gene segments. Several recent studies have shed insights into the phenotypic and genetic changes that are involved in the interspecies transmission of AIVs. These studies have a major focus on transmission from avian to mammalian species due to the high zoonotic potential of the viruses. As more mammalian species have been infected with these viruses, there is higher risk of genetic evolution of these viruses that may lead to the next human pandemic which represents and raises public health concern. Thus, understanding the mechanism of interspecies transmission and molecular determinants through which the emerging AIVs can acquire the ability to transmit to humans and other mammals is an important key in evaluating the potential risk caused by AIVs among humans. Here, we summarize previous and recent studies on molecular markers that are specifically involved in the transmission of avian-derived influenza viruses to various mammalian hosts including humans, pigs, horses, dogs, and marine mammals.
- Published
- 2017
22. Simple, Rapid and Sensitive Portable Molecular Diagnosis of SFTS Virus Using Reverse Transcriptional Loop-Mediated Isothermal Amplification (RT-LAMP)
- Author
-
Baek, Yun Hee, primary, Cheon, Hyo-Soon, additional, Park, Su-Jin, additional, Lloren, Khristine Kaith S., additional, Ahn, Su Jeong, additional, Jeong, Ju Hwan, additional, Choi, Won-Suk, additional, Yu, Min-Ah, additional, Kwon, Hyeok-il, additional, Kwon, Jin-Jung, additional, Kim, Eun-Ha, additional, Kim, Young-il, additional, Antigua, Khristine Joy C., additional, Kim, Seok-Yong, additional, Jeong, Hye Won, additional, Choi, Young Ki, additional, and Song, Min-Suk, additional
- Published
- 2018
- Full Text
- View/download PDF
23. The significance of avian influenza virus mouse-adaptation and its application in characterizing the efficacy of new vaccines and therapeutic agents
- Author
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Yun Hee Baek, Khristine Kaith S. Lloren, Min-Suk Song, and Won-Suk Choi
- Subjects
0301 basic medicine ,030106 microbiology ,Virulence ,Review Article ,Biology ,medicine.disease_cause ,Virus ,Mouse adaptation ,03 medical and health sciences ,Serial passage ,Pandemic ,Influenza A virus ,medicine ,Immunology and Allergy ,Pharmacology ,Vaccination ,Public Health, Environmental and Occupational Health ,Molecular determinant ,Virology ,Influenza A virus subtype H5N1 ,030104 developmental biology ,Infectious Diseases ,Adaptation - Abstract
Due to the increased frequency of interspecies transmission of avian influenza viruses, studies designed to identify the molecular determinants that could lead to an expansion of the host range have been increased. A variety of mouse-based mammalian-adaptation studies of avian influenza viruses have provided insight into the genetic alterations of various avian influenza subtypes that may contribute to the generation of a pandemic virus. To date, the studies have focused on avian influenza subtypes H5, H6, H7, H9, and H10 which have recently caused human infection. Although mice cannot fully reflect the course of human infection with avian influenza, these mouse studies can be a useful method for investigating potential mammalian adaptive markers against newly emerging avian influenza viruses. In addition, due to the lack of appropriate vaccines against the diverse emerging influenza viruses, the generation of mouse-adapted lethal variants could contribute to the development of effective vaccines or therapeutic agents. Within this review, we will summarize studies that have demonstrated adaptations of avian influenza viruses that result in an altered pathogenicity in mice which may suggest the potential application of mouse-lethal strains in the development of influenza vaccines and/or therapeutics in preclinical studies.
- Published
- 2017
24. Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes
- Author
-
Choi, Won-Suk, primary, Jeong, Ju Hwan, additional, Kwon, Jin Jung, additional, Ahn, Su Jeong, additional, Lloren, Khristine Kaith S., additional, Kwon, Hyeok-Il, additional, Chae, Hee Bok, additional, Hwang, Jungwon, additional, Kim, Myung Hee, additional, Kim, Chul-Joong, additional, Webby, Richard J., additional, Govorkova, Elena A., additional, Choi, Young Ki, additional, Baek, Yun Hee, additional, and Song, Min-Suk, additional
- Published
- 2018
- Full Text
- View/download PDF
25. Molecular Markers for Interspecies Transmission of Avian Influenza Viruses in Mammalian Hosts
- Author
-
Lloren, Khristine Kaith S., primary, Lee, Taehyung, additional, Kwon, Jin Jung, additional, and Song, Min-Suk, additional
- Published
- 2017
- Full Text
- View/download PDF
26. S100 can be used as a tumor marker in canine mammary tumors
- Author
-
Rodel Jonathan S Vitor and Khristine Kaith S Lloren
- Subjects
Pathology ,medicine.medical_specialty ,Stromal cell ,Physiology ,Veterinary clinics ,Mammary gland ,Biology ,medicine.disease ,S100 protein ,medicine.anatomical_structure ,medicine ,Carcinoma ,Immunohistochemistry ,Distribution (pharmacology) ,General Pharmacology, Toxicology and Pharmaceutics ,Tumor marker - Abstract
Background: S100 protein has been localized in different normal and tumorous tissues. Furthermore, its use in tumor localization and identification has already been established. Aims and Objectives: The aim of this study is to determine the localization and distribution of S100 immunoreactive cells in different canine mammary tumors (CMTs). Materials and Methods: The localization of S100 protein was demonstrated in sections of ten different classifications of CMTs and one normal lactating mammary gland by immunohistochemistry. 10 CMT samples were obtained from tumor resections in different veterinary clinics and hospital in Metro Manila, Philippines, and one normal lactating mammary gland was obtained from an anatomy cadaver for comparison. Results: S100 was observed to be positive in the endothelial cells of blood vessels in all the samples which make it suitable as a marker for CMT. Other cells that have been observed to be moderate-to-high immunoreactivity are spindle-shaped cells, chondrocytes and stromal cells which may serve as tumor markers. A positive immunoreactivity on some neuroendocrine cells and epithelial cells was also observed in neuroendocrine differentiated carcinoma. Conclusion: Based on the results in 10 CMT, this study suggests that S100 immunohistochemistry can be used as a marker to confirm CMT.
- Published
- 2018
27. The significance of avian influenza virus mouse-adaptation and its application in characterizing the efficacy of new vaccines and therapeutic agents
- Author
-
Choi, Won-Suk, primary, Lloren, Khristine Kaith S., additional, Baek, Yun Hee, additional, and Song, Min-Suk, additional
- Published
- 2017
- Full Text
- View/download PDF
28. In vitro and in vivo characterization of novel neuraminidase substitutions in influenza A(H1N1)pdm09 identified using Laninamivir-mediated in vitro selection.
- Author
-
Lloren, Khristine Kaith S., Jin Jung Kwon, Won-Suk Choi, Ju Hwan Jeong, Su Jeong Ahn, Young Ki Choi, Yun Hee Baek, and Min-Suk Song
- Subjects
- *
H1N1 influenza , *INFLUENZA - Abstract
Neuraminidase inhibitors (NAIs) are widely used antiviral drugs for the treatment of humans with influenza virus infections. There have been widespread reports of NAI resistance among seasonal A(H1N1) viruses, and most have been identified in oseltamivir-exposed patients or those treated with other NAIs. Thus, monitoring and identifying NA markers conferring resistance to NAIs--particularly newly introduced treatments--is critical to manage viral infections. Therefore, we screened and identified substitutions conferring resistance to laninamivir by enriching random mutations in the NA gene of the 2009 pandemic influenza A(H1N1)pdm09] followed by deep sequencing of the laninamivir-selected variants. After the generation of single mutants possessing each identified mutation, two A(H1N1)pdm09 recombinants possessing novel NA gene substitutions (ie, D199E and P458T) were shown to confer resistance to more than one NAI. Of note, mutants possessing P458T--which is located outside of the catalytic or framework residue of the NA active site--exhibited highly reduced inhibition by all four approved NAIs. Using MDCK cells, we observed that the in vitro viral replication of the two recombinants was lower compared with WT. Additionally, in infected mice, decreased mortality and/or mean lung viral titers were observed in mutants compared with WT. Reverse mutations to WT were observed in the lung homogenate samples from D199E-infected mice after 3 serial passages. Overall, the novel NA substitutions identified which could possibly emerge in influenza A(H1N1)pdm09 during laninamivir therapy and have altered NAI susceptibility, but the compromised in vitro/in vivo viral fitness may limit viral spreading. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
29. S100 can be used as a tumor marker in canine mammary tumors.
- Author
-
Lloren, Khristine Kaith S. and Vitor II, Rodel Jonathan S.
- Subjects
MAMMARY gland tumors ,TUMOR proteins ,DOG diseases ,IMMUNOHISTOCHEMISTRY ,IMMUNE response ,CELLS - Published
- 2018
- Full Text
- View/download PDF
30. Early Cleavage of Handmade Cloned Buffalo (Bubalus bubalis ) Embryos is an Indicator of Their Developmental Competence and Quality
- Author
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Kaith, S, primary, Saini, M, additional, Raja, AK, additional, Sahare, AA, additional, Jyotsana, B, additional, Madheshiya, P, additional, Palta, P, additional, Chauhan, MS, additional, Manik, RS, additional, and Singla, SK, additional
- Published
- 2015
- Full Text
- View/download PDF
31. Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes.
- Author
-
Won-Suk Choi, Ju Hwan Jeong, Jin Jung Kwon, Su Jeong Ahn, Lloren, Khristine Kaith S., Hyeok-Il Kwon, Hee Bok Chae, Jungwon Hwang, Myung Hee Kim, Chul-Joong Kim, Webby, Richard J., Govorkova, Elena A., Young Ki Choi, Yun Hee Baek, and Min-Suk Song
- Subjects
- *
AVIAN influenza A virus , *NEURAMINIDASE , *REVERSE genetics , *MUTAGENESIS , *AMINO acids - Abstract
Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using genefragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
32. Screening for Neuraminidase Inhibitor Resistance Markers among Avian Influenza Viruses of the N4, N5, N6, and N8 Neuraminidase Subtypes.
- Author
-
Choi WS, Jeong JH, Kwon JJ, Ahn SJ, Lloren KKS, Kwon HI, Chae HB, Hwang J, Kim MH, Kim CJ, Webby RJ, Govorkova EA, Choi YK, Baek YH, and Song MS
- Subjects
- Acids, Carbocyclic, Amino Acid Substitution, Animals, Antiviral Agents pharmacology, Birds, Cyclopentanes pharmacology, Dogs, Enzyme Inhibitors, Guanidines pharmacology, Humans, Influenza in Birds drug therapy, Influenza, Human virology, Madin Darby Canine Kidney Cells, Mutagenesis, Neuraminidase chemistry, Neuraminidase classification, Orthomyxoviridae drug effects, Orthomyxoviridae genetics, Oseltamivir analogs & derivatives, Oseltamivir pharmacology, Reverse Genetics, Zanamivir pharmacology, Drug Resistance, Viral genetics, Influenza in Birds virology, Neuraminidase antagonists & inhibitors, Neuraminidase genetics, Orthomyxoviridae enzymology
- Abstract
Several subtypes of avian influenza viruses (AIVs) are emerging as novel human pathogens, and the frequency of related infections has increased in recent years. Although neuraminidase (NA) inhibitors (NAIs) are the only class of antiviral drugs available for therapeutic intervention for AIV-infected patients, studies on NAI resistance among AIVs have been limited, and markers of resistance are poorly understood. Previously, we identified unique NAI resistance substitutions in AIVs of the N3, N7, and N9 NA subtypes. Here, we report profiles of NA substitutions that confer NAI resistance in AIVs of the N4, N5, N6, and N8 NA subtypes using gene-fragmented random mutagenesis. We generated libraries of mutant influenza viruses using reverse genetics (RG) and selected resistant variants in the presence of the NAIs oseltamivir carboxylate and zanamivir in MDCK cells. In addition, two substitutions, H274Y and R292K (N2 numbering), were introduced into each NA gene for comparison. We identified 37 amino acid substitutions within the NA gene, 16 of which (4 in N4, 4 in N5, 4 in N6, and 4 in N8) conferred resistance to NAIs (oseltamivir carboxylate, zanamivir, or peramivir) as determined using a fluorescence-based NA inhibition assay. Substitutions conferring NAI resistance were mainly categorized as either novel NA subtype specific (G/N147V/I, A246V, and I427L) or previously reported in other subtypes (E119A/D/V, Q136K, E276D, R292K, and R371K). Our results demonstrate that each NA subtype possesses unique NAI resistance markers, and knowledge of these substitutions in AIVs is important in facilitating antiviral susceptibility monitoring of NAI resistance in AIVs. IMPORTANCE The frequency of human infections with avian influenza viruses (AIVs) has increased in recent years. Despite the availability of vaccines, neuraminidase inhibitors (NAIs), as the only available class of drugs for AIVs in humans, have been constantly used for treatment, leading to the inevitable emergence of drug-resistant variants. To screen for substitutions conferring NAI resistance in AIVs of N4, N5, N6, and N8 NA subtypes, random mutations within the target gene were generated, and resistant viruses were selected from mutant libraries in the presence of individual drugs. We identified 16 NA substitutions conferring NAI resistance in the tested AIV subtypes; some are novel and subtype specific, and others have been previously reported in other subtypes. Our findings will contribute to an increased and more comprehensive understanding of the mechanisms of NAI-induced inhibition of influenza virus and help lead to the development of drugs that bind to alternative interaction motifs., (Copyright © 2017 American Society for Microbiology.)
- Published
- 2017
- Full Text
- View/download PDF
33. Production of a Cloned Buffalo (Bubalus bubalis) Calf from Somatic Cells Isolated from Urine.
- Author
-
Madheshiya PK, Sahare AA, Jyotsana B, Singh KP, Saini M, Raja AK, Kaith S, Singla SK, Chauhan MS, Manik RS, and Palta P
- Subjects
- Animals, Blastocyst, Cell Separation, Ear, Female, Gene Expression, Nuclear Transfer Techniques, Skin cytology, Tail cytology, Buffaloes genetics, Cloning, Organism, Urine cytology
- Abstract
This study was aimed at isolation of cells from urine and skin on the ventral part of the tails of healthy adult female buffaloes (Bubalus bubalis), an area rarely exposed to solar radiation, establishment of the cells in culture, and their use as donor cells for production of buffalo embryos by handmade cloning (HMC). The blastocyst rate and total cell number of urine- and tail skin-derived embryos were similar to those of control embryos derived from ear skin cells; however, their apoptotic index was lower (p<0.05) than that of control blastocysts. The global level of histone H3 acetylated at lysine 9 (H3K9ac) was similar in the three types of donor cells and in urine- and tail skin-derived HMC blastocysts and in vitro-fertilized (IVF) blastocysts (controls). The global level of histone H3 trimethylated at lysine 27 (H3K27me3) in the cells was in the order (p<0.05) urine≥tail skin>ear skin-derived cells, whereas in blastocysts, it was higher (p<0.05) in urine- and tail skin-derived HMC blastocysts than that in IVF blastocysts. The expression level of CASPASE3, CASPASE9, P53, DNMT1, DNMT3a, OCT4, and NANOG, which was similar in HMC blastocysts of three the groups, was lower (p<0.05) than that in IVF blastocysts, whereas that of HDAC1 was similar among the four groups. Following transfer of urine-derived embryos (n=10) to five recipients (two embryos/recipient), one of the recipients delivered a normal calf that is now 5 weeks old.
- Published
- 2015
- Full Text
- View/download PDF
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