Publications by authors named "Arunee Thitithanyanont"

52 Publications

Rapid and Efficient Cell-to-Cell Transmission of Avian Influenza H5N1 Virus in MDCK Cells Is Achieved by Trogocytosis.

Pathogens 2021 Apr 16;10(4). Epub 2021 Apr 16.

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

Viruses have developed direct cell-to-cell transfer strategies to enter target cells without being released to escape host immune responses and antiviral treatments. These strategies are more rapid and efficient than transmission through indirect mechanisms of viral infection between cells. Here, we demonstrate that an H5N1 influenza virus can spread via direct cell-to-cell transfer in Madin-Darby canine kidney (MDCK) cells. We compared cell-to-cell transmission of the H5N1 virus to that of a human influenza H1N1 virus. The H5N1 virus has been found to spread to recipient cells faster than the human influenza H1N1 virus. Additionally, we showed that plasma membrane exchange (trogocytosis) occurs between co-cultured infected donor cells and uninfected recipient cells early point, allowing the intercellular transfer of viral material to recipient cells. Notably, the H5N1 virus induced higher trogocytosis levels than the H1N1 virus, which could explain the faster cell-to-cell transmission rate of H5N1. Importantly, this phenomenon was also observed in A549 human lung epithelial cells, which are representative cells in the natural infection site. Altogether, our results provide evidence demonstrating that trogocytosis could be the additional mechanism utilized by the H5N1 virus for rapid and efficient cell-to-cell transmission.
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http://dx.doi.org/10.3390/pathogens10040483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8074074PMC
April 2021

Anti-SARS-CoV-2 Activity of Extract and Its Major Component Andrographolide in Human Lung Epithelial Cells and Cytotoxicity Evaluation in Major Organ Cell Representatives.

J Nat Prod 2021 04 12;84(4):1261-1270. Epub 2021 Apr 12.

Excellent Center for Drug Discovery (ECDD), Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

The coronaviruses disease 2019 (COVID-19) caused by a novel coronavirus (SARS-CoV-2) has become a major health problem, affecting more than 50 million people with over one million deaths globally. Effective antivirals are still lacking. Here, we optimized a high-content imaging platform and the plaque assay for viral output study using the legitimate model of human lung epithelial cells, Calu-3, to determine the anti-SARS-CoV-2 activity of extract and its major component, andrographolide. SARS-CoV-2 at 25TCID was able to reach the maximal infectivity of 95% in Calu-3 cells. Postinfection treatment of and andrographolide in SARS-CoV-2-infected Calu-3 cells significantly inhibited the production of infectious virions with an IC of 0.036 μg/mL and 0.034 μM, respectively, as determined by the plaque assay. The cytotoxicity profile developed over the cell line representatives of major organs, including liver (HepG2 and imHC), kidney (HK-2), intestine (Caco-2), lung (Calu-3), and brain (SH-SY5Y), showed a CC of >100 μg/mL for extract and 13.2-81.5 μM for andrographolide, respectively, corresponding to a selectivity index of over 380. In conclusion, this study provided experimental evidence in favor of and andrographolide for further development as a monotherapy or in combination with other effective drugs against SARS-CoV-2 infection.
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http://dx.doi.org/10.1021/acs.jnatprod.0c01324DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8056600PMC
April 2021

Development of Plant-Produced Recombinant ACE2-Fc Fusion Protein as a Potential Therapeutic Agent Against SARS-CoV-2.

Front Plant Sci 2020 7;11:604663. Epub 2021 Jan 7.

Research Unit for Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok, Thailand.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease (COVID-19) which has recently emerged as a potential threat to global public health. SARS-CoV-2 is the third known human coronavirus that has huge impact on the human population after SARS-CoV and MERS-CoV. Although some vaccines and therapeutic drugs are currently in clinical trials, none of them are approved for commercial use yet. As with SARS-CoV, SARS-CoV-2 utilizes angiotensin-converting enzyme 2 (ACE2) as the cell entry receptor to enter into the host cell. In this study, we have transiently produced human ACE2 fused with the Fc region of human IgG1 in and the neutralization efficacy of the plant-produced ACE2-Fc fusion protein was assessed. The recombinant ACE2-Fc fusion protein was expressed in at 100 μg/g leaf fresh weight on day 6 post-infiltration. The recombinant fusion protein showed potent binding to receptor binding domain (RBD) of SARS-CoV-2. Importantly, the plant-produced fusion protein exhibited potent anti-SARS-CoV-2 activity . Treatment with ACE2-Fc fusion protein after viral infection dramatically inhibit SARS-CoV-2 infectivity in Vero cells with an IC value of 0.84 μg/ml. Moreover, treatment with ACE2-Fc fusion protein at the pre-entry stage suppressed SARS-CoV-2 infection with an IC of 94.66 μg/ml. These findings put a spotlight on the plant-produced ACE2-Fc fusion protein as a potential therapeutic candidate against SARS-CoV-2.
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http://dx.doi.org/10.3389/fpls.2020.604663DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7874119PMC
January 2021

Repurposing of antiparasitic niclosamide to inhibit respiratory syncytial virus (RSV) replication.

Virus Res 2021 Apr 18;295:198277. Epub 2021 Jan 18.

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand. Electronic address:

Despite being an important health problem, there are only supportive care treatments for respiratory syncytial virus (RSV) infection. Thus, discovery of specific therapeutic drugs for RSV is still needed. Recently, an antiparasitic drug niclosamide has shown a broad-spectrum antiviral activity. Here, our in vitro model was used to study the antiviral effect of niclosamide on RSV and its related mechanism. Niclosamide inhibited RSV with time and dose-dependent manner. Pretreatment with submicromolar concentration of niclosamide for 6 h presented the highest anti-RSV activity of 94 % (50 % effective concentration; EC of 0.022 μM). Niclosamide efficiently blocked infection of laboratory strains and clinical isolates of both RSV-A and RSV-B in a bronchial epithelial cell line. Although a disruption of the mechanistic target of rapamycin complex 1 (mTORC1) pathway by niclosamide was previously hypothesized as a mechanism against pH-independent viruses like RSV, using a chemical mTORC1 inhibitor, temsirolimus, and a chemical mTORC1 agonist, MHY1485 (MHY), we show here that the mechanism of RSV inhibition by niclosamide was mTORC1 independent. Indeed, our data indicated that niclosamide hindered RSV infection via proapoptotic activity by a reduction of AKT prosurvival protein, activation of cleaved caspase-3 and PARP (poly ADP-ribose polymerase), and an early apoptosis induction.
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http://dx.doi.org/10.1016/j.virusres.2020.198277DOI Listing
April 2021

Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection.

Int J Mol Sci 2021 Jan 13;22(2). Epub 2021 Jan 13.

Center for Tropical Medicine and Infectious Disease, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.

DC-SIGN, a C-type lectin mainly expressed in dendritic cells (DCs), has been reported to mediate several viral infections. We previously reported that DC-SIGN mediated H5N1 influenza A virus (AIVs) infection, however, the important DC-SIGN interaction with N-glycosylation sites remain unknown. This study aims to identify the optimal DC-SIGN interacting N-glycosylation sites in HA proteins of H5N1-AIVs. Results from NetNGlyc program analyzed the H5 hemagglutinin sequences of isolates during 2004-2020, revealing that seven and two conserved N-glycosylation sites were detected in HA1 and HA2 domain, respectively. A lentivirus pseudotyped A/Vietnam/1203/04 H5N1 envelope (H5N1-PVs) was generated which displayed an abundance of HA5 proteins on the virions via immuno-electron microscope observation. Further, H5N1-PVs or reverse-genetics (H5N1-RG) strains carrying a serial N-glycosylated mutation was generated by site-directed mutagenesis assay. Human recombinant DC-SIGN (rDC-SIGN) coated ELISA showed that H5N1-PVs bound to DC-SIGN, however, mutation on the N27Q, N39Q, and N181Q significantly reduced this binding ( < 0.05). Infectivity and capture assay demonstrated that N27Q and N39Q mutations significantly ameliorated DC-SIGN mediated H5N1 infection. Furthermore, combined mutations (N27Q&N39Q) significantly waned the interaction on either H5N1-PVs or -RG infection in and in ( < 0.01). This study concludes that N27 and N39 are two essential N-glycosylation contributing to DC-SIGN mediating H5N1 infection.
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http://dx.doi.org/10.3390/ijms22020743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828482PMC
January 2021

Monoclonal Antibodies B38 and H4 Produced in Neutralize SARS-CoV-2 .

Front Plant Sci 2020 27;11:589995. Epub 2020 Nov 27.

Research Unit for Plant-produced Pharmaceuticals, Chulalongkorn University, Bangkok, Thailand.

The ongoing coronavirus disease 2019 (COVID-19) outbreak caused by novel zoonotic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was initially reported in Wuhan city, Hubei Province of China, in late December 2019. The rapid global spread of the virus calls for the urgent development of vaccines or therapeutics for human applications to combat the coronavirus infection. Monoclonal antibodies (mAbs) have been utilized as effective therapeutics for treating various infectious diseases. In the present study, we evaluated the feasibility of plant expression system for the rapid production of recently identified therapeutically suitable human anti-SARS-CoV-2 mAbs B38 and H4. Transient co-expression of heavy-chain and light-chain sequences of both the antibodies by using plant expression geminiviral vector resulted in rapid accumulation of assembled mAbs in leaves within 4 days post-infiltration. Furthermore, both the mAbs were purified from the plant crude extracts with single-step protein A affinity column chromatography. The expression level of mAb B38 and H4 was estimated to be 4 and 35 μg/g leaf fresh weight, respectively. Both plant-produced mAbs demonstrated specific binding to receptor binding domain (RBD) of SARS-CoV-2 and exhibited efficient virus neutralization activity . To the best of our knowledge, this is the first report of functional anti-SARS-CoV-2 mAbs produced in plants, which demonstrates the ability of using a plant expression system as a suitable platform for the production of effective, safe, and affordable SARS-CoV-2 mAbs to fight against the spread of this highly infectious pathogen.
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http://dx.doi.org/10.3389/fpls.2020.589995DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7728718PMC
November 2020

High-content screening of Thai medicinal plants reveals Boesenbergia rotunda extract and its component Panduratin A as anti-SARS-CoV-2 agents.

Sci Rep 2020 11 17;10(1):19963. Epub 2020 Nov 17.

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.

Since December 2019, the emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused severe pneumonia, a disease named COVID-19, that became pandemic and created an acute threat to public health. The effective therapeutics are in urgent need. Here, we developed a high-content screening for the antiviral candidates using fluorescence-based SARS-CoV-2 nucleoprotein detection in Vero E6 cells coupled with plaque reduction assay. Among 122 Thai natural products, we found that Boesenbergia rotunda extract and its phytochemical compound, panduratin A, exhibited the potent anti-SARS-CoV-2 activity. Treatment with B. rotunda extract and panduratin A after viral infection drastically suppressed SARS-CoV-2 infectivity in Vero E6 cells with IC of 3.62 μg/mL (CC = 28.06 µg/mL) and 0.81 μΜ (CC = 14.71 µM), respectively. Also, the treatment of panduratin A at the pre-entry phase inhibited SARS-CoV-2 infection with IC of 5.30 µM (CC = 43.47 µM). Our study demonstrated, for the first time, that panduratin A exerts the inhibitory effect against SARS-CoV-2 infection at both pre-entry and post-infection phases. Apart from Vero E6 cells, treatment with this compound was able to suppress viral infectivity in human airway epithelial cells. This result confirmed the potential of panduratin A as the anti-SARS-CoV-2 agent in the major target cells in human. Since B. rotunda is a culinary herb generally grown in China and Southeast Asia, its extract and the purified panduratin A may serve as the promising candidates for therapeutic purposes with economic advantage during COVID-19 situation.
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http://dx.doi.org/10.1038/s41598-020-77003-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7672115PMC
November 2020

Colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) as a visual diagnostic platform for the detection of the emerging coronavirus SARS-CoV-2.

Analyst 2021 Jan 9;146(2):471-477. Epub 2020 Nov 9.

Zenostic Co., Ltd, Bangkok 10400, Thailand.

COVID-19, caused by the infection of SARS-CoV-2, has emerged as a rapidly spreading infection. The disease has now reached the level of a global pandemic and as a result a more rapid and simple detection method is imperative to curb the spread of the virus. We aimed to develop a visual diagnostic platform for SARS-CoV-2 based on colorimetric RT-LAMP with levels of sensitivity and specificity comparable to that of commercial qRT-PCR assays. In this work, the primers were designed to target a conserved region of the RNA-dependent RNA polymerase gene (RdRp). The assay was characterized for its sensitivity and specificity, and validated with clinical specimens collected in Thailand. The developed colorimetric RT-LAMP assay could amplify the target gene and enabled visual interpretation in 60 min at 65 °C. No cross-reactivity with six other common human respiratory viruses (influenza A virus subtypes H1 and H3, influenza B virus, respiratory syncytial virus types A and B, and human metapneumovirus) and five other human coronaviruses (MERS-CoV, HKU-1, OC43, 229E and NL63) was observed. The limit of detection was 25 copies per reaction when evaluated with contrived specimens. However, the detection rate at this concentration fell to 95.8% when the incubation time was reduced from 60 to 30 min. The diagnostic performance of the developed RT-LAMP assay was evaluated in 2120 clinical specimens and compared with the commercial qRT-PCR. The results revealed high sensitivity and specificity of 95.74% and 99.95%, respectively. The overall accuracy of the RT-LAMP assay was determined to be 99.86%. In summary, our results indicate that the developed colorimetric RT-LAMP provides a simple, sensitive and reliable approach for the detection of SARS-CoV-2 in clinical samples, implying its beneficial use as a diagnostic platform for COVID-19 screening.
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http://dx.doi.org/10.1039/d0an01775bDOI Listing
January 2021

SARS-CoV-2 RNA shedding in recovered COVID-19 cases and the presence of antibodies against SARS-CoV-2 in recovered COVID-19 cases and close contacts, Thailand, April-June 2020.

PLoS One 2020 29;15(10):e0236905. Epub 2020 Oct 29.

Center of Excellence in Clinical Virology, Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand.

Coronavirus disease 2019 (COVID-19) is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Although Thailand has been fairly effective at controlling the spread of COVID-19, continued disease surveillance and information on antibody response in recovered patients and their close contacts remain necessary in the absence of approved vaccines and antivirals. Here, we examined 217 recovered COVID-19 patients to assess their viral RNA shedding and residual antibodies against SARS-CoV-2. We also evaluated antibodies in blood samples from 308 close contacts of recovered COVID-19 patients. We found that viral RNA remained detectable in 6.6% of recovered COVID-19 cases and up to 105 days. IgM, IgG, and IgA antibodies against SARS-CoV-2 were detected in 13.8%, 88.5%, and 83.4% of the recovered cases 4-12 weeks after disease onset, respectively. Higher levels of antibodies detected were associated with severe illness patients experienced while hospitalized. Fifteen of the 308 contacts (4.9%) of COVID-19 cases tested positive for IgG antibodies, suggesting probable exposure. Viral clearance and the pattern of antibody responses in infected individuals are both crucial for effectively combating SARS-CoV-2. Our study provides additional information on the natural history of this newly emerging disease related to both natural host defenses and antibody duration.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0236905PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7595404PMC
November 2020

Rapid production of SARS-CoV-2 receptor binding domain (RBD) and spike specific monoclonal antibody CR3022 in Nicotiana benthamiana.

Sci Rep 2020 10 19;10(1):17698. Epub 2020 Oct 19.

Research Unit for Plant-Produced Pharmaceuticals, Chulalongkorn University, Bangkok, Thailand.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing global outbreak of coronavirus disease (COVID-19) which is a significant threat to global public health. The rapid spread of COVID-19 necessitates the development of cost-effective technology platforms for the production of vaccines, drugs, and protein reagents for appropriate disease diagnosis and treatment. In this study, we explored the possibility of producing the receptor binding domain (RBD) of SARS-CoV-2 and an anti-SARS-CoV monoclonal antibody (mAb) CR3022 in Nicotiana benthamiana. Both RBD and mAb CR3022 were transiently produced with the highest expression level of 8 μg/g and 130 μg/g leaf fresh weight respectively at 3 days post-infiltration. The plant-produced RBD exhibited specific binding to the SARS-CoV-2 receptor, angiotensin-converting enzyme 2 (ACE2). Furthermore, the plant-produced mAb CR3022 binds to SARS-CoV-2, but fails to neutralize the virus in vitro. This is the first report showing the production of anti-SARS-CoV-2 RBD and mAb CR3022 in plants. Overall these findings provide a proof-of-concept for using plants as an expression system for the production of SARS-CoV-2 antigens and antibodies or similar other diagnostic reagents against SARS-CoV-2 rapidly, especially during epidemic or pandemic situation.
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http://dx.doi.org/10.1038/s41598-020-74904-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7573609PMC
October 2020

Trogocytosis with monocytes associated with increased α2,3 sialic acid expression on B cells during H5N1 influenza virus infection.

PLoS One 2020 18;15(9):e0239488. Epub 2020 Sep 18.

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.

The immunopathogenesis of H5N1 virus has been studied intensively since it caused cross-species infection and induced high mortality to human. We previously observed the interaction between monocytes and B cells, which increased the susceptibility of B cell to H5N1 virus infection after a co-culture. Levels of α2,3 sialic acid (avian flu receptor) were also significantly increased on B cell surface in this co-culture model with unclear explanation. In this study, we aimed to determine the possible mechanism that responded for this increase in α2,3 sialic acid on B cells. Acquisition of α2,3 SA by B cells via cell contact-dependent trogocytosis was proposed. Results showed that the lack of α2,3 SA was detected on B cell surface, and B cells acquired membrane-bound α2,3 SA molecules from monocytes in H5N1-infected co-cultures. Occurrence of membrane exchange mainly relied on H5N1 infection and cell-cell contact as opposed to a mock infection and transwell. The increase in α2,3 SA on B cell surface mediated by trogocytosis was associated with the enhanced susceptibility to H5N1 infection. These observations thus provide the evidence that H5N1 influenza virus may utilize trogocytosis to expand its cell tropism and spread to immune cells despite the lack of avian flu receptor.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0239488PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7500609PMC
November 2020

Hematopoietic stem cell transplantation from an infected SARS-CoV2 donor sibling.

Bone Marrow Transplant 2020 12 11;55(12):2359-2360. Epub 2020 Jun 11.

Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.

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http://dx.doi.org/10.1038/s41409-020-0969-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7289075PMC
December 2020

An epidemiological survey of the current status of Zika and the immune interaction between dengue and Zika infection in Southern Taiwan.

Int J Infect Dis 2020 Apr 23;93:151-159. Epub 2020 Jan 23.

Center for Tropical Medicine and Infectious Disease, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan; Department of Laboratory Medicine, Kaohsiung Medical University Hospital, Kaohsiung, 80708, Taiwan; Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan. Electronic address:

Objectives: This study was performed to examine the current status of Zika and the effects of pre-existing dengue immunity on Zika virus (ZIKV) infection in Southern Taiwan.

Methods: A phylogenetic tree was used to analyze the phylogeny of detected ZIKVs. Paired sera from dengue patients were collected for the determination of dengue and Zika infection. Plaque reduction neutralization tests (PRNT) and quantitative reverse transcription PCR (qRT-PCR) were used to determine the titers of neutralizing antibodies and viruses, respectively. An antibody-dependent enhancement (ADE) assay was used to evaluate the effect of anti-dengue antibodies on ZIKV infection.

Results: Epidemiological data indicated the continuous importation of ZIKV infection from neighboring Zika epidemic countries into Taiwan. A total of 78 dengue patients were enrolled and 21 paired serum samples were obtained. PRNT results for the 21 samples identified eight cases of primary dengue infection and 13 cases of secondary dengue infection; two samples were positive for ZIKV (MR766). Results from the ADE assay indicated that convalescent sera from primary and secondary dengue infection patients displayed significant ADE of the ZIKV infection when compared to healthy controls (p < 0.05).

Conclusions: This study suggests that pre-existing dengue immunity facilitates ZIKV infection and that the continuous importation of ZIKV infection may pose a threat to indigenous Zika emergence in Southern Taiwan.
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http://dx.doi.org/10.1016/j.ijid.2020.01.031DOI Listing
April 2020

The role of galectins in virus infection - A systemic literature review.

J Microbiol Immunol Infect 2020 Dec 30;53(6):925-935. Epub 2019 Sep 30.

Center for Tropical Medicine and Infectious Disease, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan; Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan. Electronic address:

Background: Galectins are β-Galactose binding lectins expressed in numerous cells and play multiple roles in various physiological and cellular functions. However, few information is available regarding the role of galectins in virus infections. Here, we conducted a systemic literature review to analyze the role of galectins in human virus infection.

Methods: This study uses a systematic method to identify and select eligible articles according to the PRISMA guidelines. References were selected from PubMed, Web of Science and Google Scholar database covering publication dated from August 1995 to December 2018.

Results: Results indicate that galectins play multiple roles in regulation of virus infections. Galectin-1 (Gal-1), galectin-3 (Gal-3), galectin-8 (Gal-8), and galectin-9 (Gal-9) were found as the most predominant galectins reported to participate in virus infection. The regulatory function of galectins occurs by extracellularly binding to viral glycosylated envelope proteins, interacting with ligands or receptors on immune cells, or acting intracellularly with viral or cellular components in the cytoplasm. Several galectins express either positive or negative regulatory role, while some had dual regulatory capabilities on virus propagation based on the conditions and their localization. However, limited information about the endogenous function of galectins were found. Therefore, the endogenous effects of galectins in host-virus regulation remains valuable to investigate.

Conclusions: This study offers information regarding the various roles galectins shown in viral infection and suggest that galectins can potentially be used as viral therapeutic targets or antagonists.
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http://dx.doi.org/10.1016/j.jmii.2019.09.005DOI Listing
December 2020

A clinical and epidemiological survey of the largest dengue outbreak in Southern Taiwan in 2015.

Int J Infect Dis 2019 Nov 12;88:88-99. Epub 2019 Sep 12.

Center for Tropical Medicine and Infectious Disease, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan; Department of Medical Laboratory Science and Biotechnology, Kaohsiung Medical University, Kaohsiung, 80708, Taiwan; Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung, 80708, Taiwan. Electronic address:

Objectives: This study examined the epidemiological, clinical, and immunological characteristics of the 2015 dengue outbreak in Taiwan.

Methods: Clinical data were collected from dengue fever (DF) and dengue hemorrhagic fever (DHF) patients. A phylogenetic tree was used to analyze the source of the outbreak strain. Paired plasma samples from DF/DHF patients were used for antibody-dependent enhancement (ADE) assay and cytokine multiplex biometric immunoassay to validate the immunological mechanism.

Results: This outbreak mainly occurred in two of the southern cities of Taiwan: Tainan (n=22 777; 52%) and Kaohsiung (n=19 784; 45%). A high DHF death rate was noted (34.6%). The case (DHF) and control (DF) study indicated that older age (>60 years), type II diabetes, and hypertension were risk factors correlated with the development of DHF (p< 0.0001). The phylogenetic tree results suggested that the outbreak-associated strain was dengue virus serotype 2 and cosmopolitan genotype, forming a stable cluster with the isolates from Thailand and Indonesia (bootstrap value of 99%). Cytokine analyses demonstrated that levels of interleukin (IL)-6, IL-4, IL-13, IL-1β, interferon gamma (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly higher in DHF patients compared to DF patients (p< 0.001). The ADE assay showed that diluted plasma containing preexisting dengue antibodies from DHF patients significantly enhanced dengue infection (p< 0.05).

Conclusion: The results suggest that older age, type II diabetes, hypertension, immunological cytokine dysregulation, and preexisting dengue antibodies inducing ADE infection are correlated with dengue severity. This study also indicates that the largest dengue outbreak in Taiwan might have been a result of imported DF from dengue epidemic regions.
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http://dx.doi.org/10.1016/j.ijid.2019.09.007DOI Listing
November 2019

Longitudinal study on enterovirus A71 and coxsackievirus A16 genotype/subgenotype replacements in hand, foot and mouth disease patients in Thailand, 2000-2017.

Int J Infect Dis 2019 Mar 11;80:84-91. Epub 2019 Jan 11.

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok-noi, Bangkok 10700, Thailand; Center for Research and Innovation, Faculty of Medical Technology, Mahidol University, Nakhon Pathom 73170, Thailand. Electronic address:

Background: Enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16) are the major causative agents of hand, foot and mouth disease (HFMD) worldwide, particularly in the Asia-Pacific region. Several strains have emerged, circulated, and faded out over time in recent decades. This study investigated the EV-A71 and CV-A16 circulating strains and replacement of genotypes/subgenotypes in Thailand during the years 2000-2017.

Methods: The complete VP1 regions of 92 enteroviruses obtained from 90 HFMD patients, one asymptomatic adult contact case, and one encephalitic case were sequenced and investigated for serotypes, genotypes, and subgenotypes using a phylogenetic analysis.

Results: The 92 enterovirus isolates were identified as 67 (72.8%) EV-A71 strains comprising subgenotypes B4, B5, C1, C2, C4a, C4b and C5, and 25 (27.2%) CV-A16 strains comprising subgenotypes B1a and B1b. Genotypic/subgenotypic replacements were evidenced during the study period. EV-A71 B5 and C4a have been the major circulating strains in Thailand for more than a decade, and CV-A16 B1a has been circulating for almost two decades.

Conclusions: This study provides chronological data on the molecular epidemiology of EV-A71 and CV-A16 subgenotypes in Thailand. Subgenotypic replacement frequently occurred with EV-A71, but not CV-A16. Monitoring for viral genetic and subgenotypic changes is important for molecular diagnosis, vaccine selection, and vaccine development.
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http://dx.doi.org/10.1016/j.ijid.2018.12.020DOI Listing
March 2019

Electrochemical Biosensor Based on Surface Imprinting for Zika Virus Detection in Serum.

ACS Sens 2019 01 9;4(1):69-75. Epub 2019 Jan 9.

Department of Chemistry, Faculty of Science , Kasetsart University , Bangkok 10900 , Thailand.

Zika virus (ZIKV) is a flavivirus that was first identified in 1947. Initially, the virus was of little concern for health authorities given there were very few casualties among those suffering an infection. As such, only limited studies were performed on ZIKV. Recently, the viral infection has been linked to microcephaly in infants, which has prompted a dramatic increase in scientific interest in ZIKV research, including methods to allow for rapid virus identification. In this work we report the development of a new type of ZIKV electrochemical biosensor based on surface imprinted polymers and graphene oxide composites. The biosensor was used to detect ZIKV by measuring changes in the electrical signal with changing virus concentrations in buffer and serum using standard electrochemical techniques. The detection limit of our method is similar to the detection limit of the real-time quantitative reverse transcription PCR method.
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http://dx.doi.org/10.1021/acssensors.8b00885DOI Listing
January 2019

Complete genome analysis demonstrates multiple introductions of enterovirus 71 and coxsackievirus A16 recombinant strains into Thailand during the past decade.

Emerg Microbes Infect 2018 Dec 14;7(1):214. Epub 2018 Dec 14.

Center for Research and Innovation, Faculty of Medical Technology, Mahidol University, Nakhon, Pathom, 73170, Thailand.

Hand, foot, and mouth disease (HFMD) caused by enteroviruses remains a public health threat, particularly in the Asia-Pacific region during the past two decades. Moreover, the introduction of multiple subgenotypes and the emergence of recombinant viruses is of epidemiological importance. Based on either the full genome or VP1 sequences, 32 enteroviruses (30 from HFMD patients, 1 from an encephalitic patient, and 1 from an asymptomatic contact case) isolated in Thailand between 2006 and 2014 were identified as 25 enterovirus 71 (EV71) isolates (comprising 20 B5, 1 C2, 2 C4a, and 2 C4b subgenotypes) and 7 coxsackievirus A16 (CA16) isolates (comprising 6 B1a and 1 B1b subgenotypes). The EV71 subgenotype C4b was introduced into Thailand for the first time in 2006 and was replaced by subgenotype C4a strains in 2009. Phylogenetic, similarity plot and bootscan analyses of the complete viral genomes identified 12 recombinant viruses among the 32 viral isolates. Only one EV71-B5 isolate out of 20 was a recombinant virus with one region of intratypic or intertypic recombination, while all four EV71-C4 isolates were recombinant viruses having undergone double recombination, and all seven CA16 isolates were recombinant viruses. The recombination breakpoints of these recombinants are located solely within the P2 and P3 regions. Surveillance for circulating strains and subgenotype replacement are important with respect to molecular epidemiology and the selection of the upcoming EV71 vaccine. In addition, the clinical importance of recombinant viruses needs to be further explored.
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http://dx.doi.org/10.1038/s41426-018-0215-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294798PMC
December 2018

The activation of B cells enhances DC-SIGN expression and promotes susceptibility of B cells to HPAI H5N1 infection.

Biochem Biophys Res Commun 2017 09 5;490(4):1301-1306. Epub 2017 Jul 5.

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand. Electronic address:

The interplay between highly pathogenic avian influenza (HPAI) H5N1 virus and immune cells has been extensively studied for years, as host immune components are thought to play significant roles in promoting the systemic spread of the virus and responsible for cytokine storm. Previous studies suggested that the interaction of B cells and monocytes could promote HPAI H5N1 infection by enhancing avian influenza virus receptor expression. In this study, we further investigate the relationship between the HPAI H5N1 virus, activated B cells, and DC-SIGN expression. DC-SIGN has been described as an important factor for mediating various types of viral infection. Here, we first demonstrate that HPAI H5N1 infection could induce an activation of B cells, which was associated with DC-SIGN expression. Using CD40L and recombinant IL-4 for B cell stimulation, we determined that DC-SIGN expressed on activated B cells was able to enhance its susceptibility to HPAI H5N1 infection. Our findings uncover the interplay between this H5N1 virus and B cells and provide important information in understanding how the virus overcomes our immune system, contributing to its unusual immunopathogenesis.
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http://dx.doi.org/10.1016/j.bbrc.2017.07.017DOI Listing
September 2017

An influenza A virus agglutination test using antibody-like polymers.

J Biomater Sci Polym Ed 2017 Oct 8;28(15):1786-1795. Epub 2017 Jun 8.

a Faculty of Science, Department of Chemistry , Kasetsart University , Bangkok , Thailand.

Antibodies are commonly used in diagnostic routines to identify pathogens. The testing protocols are relatively simple, requiring a certain amount of a specific antibody to detect its corresponding pathogen. Antibody functionality can be mimicked by synthesizing molecularly imprinted polymers (MIPs), i.e. polymers that can selectively recognize a given template structure. Thus, MIPs are sometimes termed 'plastic antibody (PA)'. In this study, we have synthesized new granular MIPs using influenza A virus templates by precipitation polymerization. The selective binding of influenza A to the MIP particles was assessed and subsequently contrasted with other viruses. The affinities of influenza A virus towards the MIP was estimated based on an agglutination test by measuring the amount of influenza subtypes absorbed onto the MIPs. The MIPs produced using the H1N1 template showed specific reactivity to H1N1 while those produced using H5N1 and H3N2 templates showed cross-reactivity.
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http://dx.doi.org/10.1080/09205063.2017.1338503DOI Listing
October 2017

P2Y6 receptors are involved in mediating the effect of inactivated avian influenza virus H5N1 on IL-6 & CXCL8 mRNA expression in respiratory epithelium.

PLoS One 2017 11;12(5):e0176974. Epub 2017 May 11.

Discipline of Physiology, The Bosch Institute, School of Medical Sciences, The University of Sydney, Sydney, Australia.

One of the key pathophysiologies of H5N1 infection is excessive proinflammatory cytokine response (cytokine storm) characterized by increases in IFN-β, TNF-α, IL-6, CXCL10, CCL4, CCL2 and CCL5 in the respiratory tract. H5N1-induced cytokine release can occur via an infection-independent mechanism, however, detail of the cellular signaling involved is poorly understood. To elucidate this mechanism, the effect of inactivated (β-propiolactone-treated) H5N1 on the cytokine and chemokine mRNA expression in 16HBE14o- human respiratory epithelial cells was investigated. We found that the inactivated-H5N1 increased mRNA for IL-6 and CXCL8 but not TNF-α, CCL5 or CXCL10. This effect of the inactivated-H5N1 was inhibited by sialic acid receptor inhibitor (α-2,3 sialidase), adenosine diphosphatase (apyrase), P2Y receptor (P2YR) inhibitor (suramin), P2Y6R antagonist (MRS2578), phospholipase C inhibitor (U73122), protein kinase C inhibitors (BIM and Gö6976) and cell-permeant Ca2+ chelator (BAPTA-AM). Inhibitors of MAPK signaling, including of ERK1/2 (PD98059), p38 MAPK (SB203580) and JNK (SP600125) significantly suppressed the inactivated-H5N1-induced mRNA expression of CXCL8. On the other hand, the inactivated-H5N1-induced mRNA expression of IL-6 was inhibited by SB203580, but not PD98059 or SP600125, whereas SN-50, an inhibitor of NF-κB, inhibited the effect of virus on mRNA expression of both of IL-6 and CXCL8. Taken together, our data suggest that, without infection, inactivated-H5N1 induces mRNA expression of IL-6 and CXCL8 by a mechanism, or mechanisms, requiring interaction between viral hemagglutinin and α-2,3 sialic acid receptors at the cell membrane of host cells, and involves activation of P2Y6 purinergic receptors.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0176974PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5426635PMC
September 2017

Mesenchymal Stromal Cells and Viral Infection.

Stem Cells Int 2015 29;2015:860950. Epub 2015 Jul 29.

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok 10400, Thailand.

Mesenchymal Stromal Cells (MSCs) are a subset of nonhematopoietic adult stem cells, readily isolated from various tissues and easily culture-expanded ex vivo. Intensive studies of the immune modulation and tissue regeneration over the past few years have demonstrated the great potential of MSCs for the prevention and treatment of steroid-resistant acute graft-versus-host disease (GvHD), immune-related disorders, and viral diseases. In immunocompromised individuals, the immunomodulatory activities of MSCs have raised safety concerns regarding the greater risk of primary viral infection and viral reactivation, which is a major cause of mortality after allogeneic transplantation. Moreover, high susceptibilities of MSCs to viral infections in vitro could reflect the destructive outcomes that might impair the clinical efficacy of MSCs infusion. However, the interplay between MSCs and virus is like a double-edge sword, and it also provides beneficial effects such as allowing the proliferation and function of antiviral specific effector cells instead of suppressing them, serving as an ideal tool for study of viral pathogenesis, and protecting hosts against viral challenge by using the antimicrobial activity. Here, we therefore review favorable and unfavorable consequences of MSCs and virus interaction with the highlight of safety and efficacy for applying MSCs as cell therapy.
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http://dx.doi.org/10.1155/2015/860950DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4532961PMC
August 2015

The presence of monocytes enhances the susceptibility of B cells to highly pathogenic avian influenza (HPAI) H5N1 virus possibly through the increased expression of α2,3 SA receptor.

Biochem Biophys Res Commun 2015 Aug 15;464(3):888-93. Epub 2015 Jul 15.

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand. Electronic address:

The highly pathogenic avian influenza (HPAI) H5N1 virus causes severe systemic infection in avian and mammalian species, including humans by first targeting immune cells. This subsequently renders the innate and adaptive immune responses less active, thus allowing dissemination of the virus to systemic organs. To gain insight into the pathogenesis of H5N1, this study aims to determine the susceptibility of human PBMCs to the H5N1 virus and explore the factors which influence this susceptibility. We found that PBMCs were a target of H5N1 infection, and that monocytes and B cells were populations which were clearly the most susceptible. Analysis of PBMC subpopulations showed that isolated monocytes and monocytes residing in whole PBMCs had comparable percentages of infection (28.97 ± 5.54% vs 22.23 ± 5.14%). In contrast, isolated B cells were infected to a much lower degree than B cells residing in a mixture of whole PBMCs (0.88 ± 0.34% vs 34.87 ± 4.63%). Different susceptibility levels of B cells for these tested conditions spurred us to explore the B cell-H5N1 interaction mechanisms. Here, we first demonstrated that monocytes play a crucial role in the enhancement of B cell susceptibility to H5N1 infection. Although the actual mechanism by which this enhancement occurs remains in question, α2,3-linked sialic acid (SA), known for influenza virus receptors, could be a responsible factor for the greater susceptibility of B cells, as it was highly expressed on the surface of B cells upon H5N1 infection of B cell/monocyte co-cultures. Our findings reveal some of the factors involved with the permissiveness of human immune cells to H5N1 virus and provide a better understanding of the tropism of H5N1 in immune cells.
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http://dx.doi.org/10.1016/j.bbrc.2015.07.061DOI Listing
August 2015

Tropism of avian influenza A (H5N1) virus to mesenchymal stem cells and CD34+ hematopoietic stem cells.

PLoS One 2013 10;8(12):e81805. Epub 2013 Dec 10.

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.

The presence of abnormal hematologic findings such as lymphopenia, thrombocytopenia, and pancytopenia were diagnosed in severe cases of avian influenza A H5N1. Whether direct viral dissemination to bone marrow (BM) cells causes this phenomenon remains elusive. We explore the susceptibility of the two stem cell types; hematopoietic stem cells (HSCs) and mesenchymal stromal cells (MSCs) isolated from human BM cells or cord blood, to infection with avian H5N1 viruses. For the first time, we demonstrated that the H5N1 virus could productively infect and induce cell death in both human stem cell types. In contrast, these activities were not observed upon human influenza virus infection. We also determined whether infection affects the immunomodulatory function of MSCs. We noted a consequent dysregulation of MSC-mediated immune modulation as observed by high cytokine and chemokine production in H5N1 infected MSCs and monocytes cocultures. These findings provide a better understanding of H5N1 pathogenesis in terms of broad tissue tropism and systemic spread.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0081805PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3858287PMC
September 2014

Pre-existing cross-reactive antibodies to avian influenza H5N1 and 2009 pandemic H1N1 in US military personnel.

Am J Trop Med Hyg 2014 Jan 25;90(1):149-52. Epub 2013 Nov 25.

Armed Forces Research Institute of Medical Sciences, Bangkok, Thailand; National Center for Genetic Engineering and Biotechnology, Pathum Thani, Thailand; Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand; Department of Dentistry, Chulalongkorn University, Bangkok, Thailand; Hong Kong University-Pasteur Research Center, Hong Kong.

We studied cross-reactive antibodies against avian influenza H5N1 and 2009 pandemic (p) H1N1 in 200 serum samples from US military personnel collected before the H1N1 pandemic. Assays used to measure antibodies against viral proteins involved in protection included a hemagglutination inhibition (HI) assay and a neuraminidase inhibition (NI) assay. Viral neutralization by antibodies against avian influenza H5N1 and 2009 pH1N1 was assessed by influenza (H5) pseudotyped lentiviral particle-based and H1N1 microneutralization assays. Some US military personnel had cross-neutralizing antibodies against H5N1 (14%) and 2009 pH1N1 (16.5%). The odds of having cross-neutralizing antibodies against 2009 pH1N1 were 4.4 times higher in subjects receiving more than five inactivated whole influenza virus vaccinations than those subjects with no record of vaccination. Although unclear if the result of prior vaccination or disease exposure, these pre-existing antibodies may prevent or reduce disease severity.
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http://dx.doi.org/10.4269/ajtmh.13-0151DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3886412PMC
January 2014

Self-assembled glucosamine monolayers as biomimetic receptors for detecting WGA lectin and influenza virus with a quartz crystal microbalance.

Anal Bioanal Chem 2013 Aug 29;405(20):6471-8. Epub 2013 May 29.

Department of Analytical Chemistry, University of Vienna, Vienna, Austria.

N-Acetylglucosamine (GlcNAc) is a natural ligand that interacts with the binding sites of wheat germ agglutinin (WGA) lectin. For immobilization, GlcNAc was linked to p-nitrophenol, and the nitro group was reduced and then bound to cysteine via two-step synthesis. Scanning tunneling microscopy studies revealed proper immobilization of the ligand on the gold surface of a quartz crystal microbalance (QCM) via the cysteine S-H bond as well as binding between GlcNAc and WGA. QCM measurements revealed that maximum sensitivity towards WGA can only be achieved when co-immobilizing one part ligand and 5,000 parts cysteine for steric reasons, because it allows binding of a protein monolayer on the surface. Langmuir-type treatment of the binding isotherm suggests two different binding ranges for WGA and the GlcNAc monolayer, because at concentrations of WGA below 1 μm the Gibbs energy for the binding process is one third higher than that at concentrations above this value. The same systems can be transferred to first proof-of-concept measurements with different strains of influenza A virus (H5N3, H5N1, H1N3) because GlcNAc is part of the oligosaccharide ligand responsible for the first binding step. Thus, it constitutes both a suitable tool for rapid analysis and the basis for future theoretical calculations of ligand-virus interactions.
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http://dx.doi.org/10.1007/s00216-013-7057-0DOI Listing
August 2013

Influenza A virus molecularly imprinted polymers and their application in virus sub-type classification.

J Mater Chem B 2013 Apr 11;1(16):2190-2197. Epub 2013 Mar 11.

Department of Chemistry, Faculty of Science, Kasetsart University, Chatuchak, Bangkok, Thailand.

In this work, we apply a molecular imprinting strategy as a screening protocol for different influenza A subtypes, namely H5N1, H5N3, H1N1, H1N3 and H6N1. Molecularly imprinted polymers for each of these subtypes lead to appreciable sensor characteristics on a quartz crystal microbalance leading to detection limits as low as 10 particles per ml. Selectivity studies indicate that each virus is preferably incorporated by its own MIP. Recognition in most cases is dominated by the neuraminidase residue rather than the hemagglutinin. Multivariate analysis shows that the sensor responses can be correlated with the differences in hemagglutinin and neuraminidase patterns from databases. This allows for virus subtype characterization and thus rapid screening.
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http://dx.doi.org/10.1039/c3tb00027cDOI Listing
April 2013

Involvement of GRIM-19 in apoptosis induced in H5N1 virus-infected human macrophages.

Innate Immun 2013 Dec 25;19(6):655-62. Epub 2013 Mar 25.

Department of Microbiology, Faculty of Science, Mahidol University, Bangkok, Thailand.

The fatal H5N1 infection has a high mortality rate among infected patients. The pathogenesis of H5N1 viral infection is associated with the ability of the virus to induce apoptotic cell death. However, the molecular mechanism of apoptosis induced by H5N1 remains unclear. In the present study we demonstrate that H5N1 virus is able to up-regulate the expression of gene associated with retinoid and interferon induced mortality-19 (GRIM-19) in human monocyte-derived macrophages (hMDMs). GRIM-19 has been identified as a novel gene with apoptotic effects in virus-infected cells. The percentage of apoptotic cells is significantly decreased in H5N1-infected GRIM-19 depleted hMDMs, which is also associated with a decrease of BH3-interacting domain death agonist cleavage and apoptosis-inducing factor (AIF) release to the cytosol. These results suggested the involvement of GRIM-19 in apoptosis induced by H5N1 virus. Furthermore, neutralizing-IFN-β Ab is able to suppress GRIM-19 expression in H5N1-infected cells resulting in a decrease in apoptotic cell number, indicating that IFN-β secreted by H5N1-infected hMDMs regulates GRIM-19 expression leading to apoptosis. Altogether, the results presented here provide additional insight on the regulatory mechanism of H5N1 viral-induced apoptotic cell death in hMDMs.
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http://dx.doi.org/10.1177/1753425913479149DOI Listing
December 2013