Publications by authors named "Prasert Auewarakul"

113 Publications

Response of Severe EV71-Infected Patients to Hyperimmune Plasma Treatment: A Pilot Study.

Pathogens 2021 May 19;10(5). Epub 2021 May 19.

Center of Research Excellence in Immunoregulation, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand.

Hand, foot, and mouth disease (HFMD) is highly prevalent in East and Southeast Asia. It particularly affects children under five years of age. The most common causative agents are coxsackieviruses A6 and A16, and enterovirus A71 (EV71). The clinical presentation is usually mild and self-limited, but, in some cases, severe and fatal complications develop. To date, no specific therapy or worldwide vaccine is available. In general, viral infection invokes both antibody and cell-mediated immune responses. Passive immunity transfer can ameliorate the severe symptoms of diseases such as COVID-19, influenza, MERS, and SARS. Hyperimmune plasma (HIP) from healthy donors with high anti-EV71 neutralizing titer were used to transfuse confirmed EV71-infected children with neurological involvement (n = 6). It resulted in recovery within three days, with no neurological sequelae apparent upon examination 14 days later. Following HIP treatment, plasma chemokines were decreased, whereas anti-inflammatory and pro-inflammatory cytokines gradually increased. Interestingly, IL-6 and G-CSF levels in cerebrospinal fluid declined sharply within three days. These findings indicate that HIP has therapeutic potential for HFMD with neurological complications. However, given the small number of patients who have been treated, a larger cohort study should be undertaken. Successful outcomes would stimulate the development of anti-EV71 monoclonal antibody therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/pathogens10050625DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8161181PMC
May 2021

T cell mediated immunity against influenza H5N1 nucleoprotein, matrix and hemagglutinin derived epitopes in H5N1 survivors and non-H5N1 subjects.

PeerJ 2021 10;9:e11021. Epub 2021 Mar 10.

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok-noi, Bangkok, Thailand.

Background: Protection against the influenza virus by a specific antibody is relatively strain specific; meanwhile broader immunity may be conferred by cell-mediated immune response to the conserved epitopes across influenza virus subtypes. A universal broad-spectrum influenza vaccine which confronts not only seasonal influenza virus, but also avian influenza H5N1 virus is promising.

Methods: This study determined the specific and cross-reactive T cell responses against the highly pathogenic avian influenza A (H5N1) virus in four survivors and 33 non-H5N1 subjects including 10 H3N2 patients and 23 healthy individuals. Ex vivo IFN- ELISpot assay using overlapping peptides spanning the entire nucleoprotein (NP), matrix (M) and hemagglutinin (HA) derived from A/Thailand/1(KAN-1)/2004 (H5N1) virus was employed in adjunct with flow cytometry for determining T cell functions. Microneutralization (microNT) assay was performed to determine the status of previous H5N1 virus infection.

Results: IFN- ELISpot assay demonstrated that survivors nos. 1 and 2 had markedly higher T cell responses against H5N1 NP, M and HA epitopes than survivors nos. 3 and 4; and the magnitude of T cell responses against NP were higher than that of M and HA. Durability of the immunoreactivity persisted for as long as four years after disease onset. Upon stimulation by NP in IFN- ELISpot assay, 60% of H3N2 patients and 39% of healthy subjects exhibited a cross-reactive T cell response. The higher frequency and magnitude of responses in H3N2 patients may be due to blood collection at the convalescent phase of the patients. In H5N1 survivors, the effector peptide-specific T cells generated from bulk culture PBMCs by in vitro stimulation displayed a polyfunction by simultaneously producing IFN- and TNF-, together with upregulation of CD107a in recognition of the target cells pulsed with peptide or infected with rVac-NP virus as investigated by flow cytometry.

Conclusions: This study provides an insight into the better understanding on the homosubtypic and heterosubtypic T cell-mediated immune responses in H5N1 survivors and non-H5N1 subjects. NP is an immunodominant target of cross-recognition owing to its high conservancy. Therefore, the development of vaccine targeting the conserved NP may be a novel strategy for influenza vaccine design.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7717/peerj.11021DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955671PMC
March 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virusres.2020.198277DOI Listing
April 2021

Detection of antibodies to duck tembusu virus in human population with or without the history of contact with ducks.

Transbound Emerg Dis 2021 Jan 20. Epub 2021 Jan 20.

Faculty of Veterrinary Medicine, Kasetsart University, Bangkok, Thailand.

Duck tembusu virus (DTMUV) is an emerging duck pathogen in China and other Asian countries. It is unclear whether this emerging zoonotic infection poses a threat to humans. A previous study in 2012 showed surprisingly high rates of seropositivity and positive viral detection by RT-PCR in duck farm workers in China. To understand the nature of the threat of this emerging virus, we studied the neutralizing antibody response to a local isolate of DTMUV in an at-risk population, who were workers in duck farms and residents around farming areas in Central Thailand where DTMUV had been previously detected, and in a not-at-risk population, who were people living in the same or neighbouring province, but at a distance from the farms and who had no contact with ducks. The sera from the at-risk population showed higher anti-DTMUV neutralizing antibody titres as compared with those of the not-at-risk population. However, within the at-risk population, workers with direct contact with ducks did not show higher neutralizing titres than those without direct contact. Interestingly, some people in the not-at-risk group also displayed high neutralizing antibody titres to DTMUV. These sera were tested against other endemic Flaviviruses and showed no or low cross-reactivity suggesting the specificity of the neutralizing activity against DTMUV. These data raise a possibility of DTMUV as a potential zoonotic pathogen but the mode of transmission of the virus from ducks or other possible hosts to humans should be explored further.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/tbed.13998DOI Listing
January 2021

A modified IgG avidity assay for reliability improvement of an in-house capture ELISA to discriminate primary from secondary dengue virus infections.

J Virol Methods 2021 03 10;289:114043. Epub 2020 Dec 10.

Center for Vaccine Development, Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand. Electronic address:

Although discrimination between primary and secondary dengue infections can be performed using commercially available immunoassays or in-house tests, the evaluation of these methods is important, but is often problematic due to incomplete clinical data. In many cases, patients' sera submitted to the laboratory may not include the date of onset of illness which is necessary to discriminate primary and secondary dengue infections. This study reports improvement of an in-house capture ELISA using IgG avidity to discriminate primary and secondary dengue virus infection. Modified definition criteria were applied to characterize 99 single sera based on their IgM/IgG ratios. Regressive analysis indicated that the avidity test results (avidity index of 60 % as cutoff) for the discrimination showed good agreement (96 %) and a high correlation (r = -0.81) with those of the in-house capture ELISA (IgM/IgG ratio at 1.2 as cutoff). To further evaluate the in-house tests, 318 convalescent sera were compared with a Focus Diagnostics' anti-dengue IgM ELISA. Compared with the Focus Diagnostics system, the sensitivity of an in-house IgM determination was 83 %, whereas using both IgM and IgG capture ELISAs the sensitivity increased to 95 %.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jviromet.2020.114043DOI Listing
March 2021

Analysis of Tembusu virus infection of human cell lines and human induced pluripotent stem cell derived hepatocytes.

Virus Res 2021 01 5;292:198252. Epub 2020 Dec 5.

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand; Molecular Medicine Graduate Program, Faculty of Science, Mahidol University, Bangkok, Thailand. Electronic address:

Tembusu virus (TMUV) causes disease in poultry, especially in ducks, resulting in abnormality in egg production and with high morbidity and mortality, resulting in great loss in duck farming industry in China and Southeast Asia. Previous studies on the pathogenesis of TMUV infection have been mostly conducted in poultry, with a few studies being undertaken in mice. While TMUV does not cause disease in humans, it has been reported that antibodies against TMUV have been found in serum samples from duck farmers, and thus data on TMUV infection in humans is limited, and the pathogenesis is unclear. In this study we investigated the cell tropism and potential susceptibility of humans to TMUV using several human cell lines. The results showed that human nerve and liver cell lines were both highly susceptible and permissive, while human kidney cells were susceptible and permissive, albeit to a lower degree. In addition, human muscle cells, lung epithelial cells, B-cells, T-cells and monocytic cells were largely refractory to TMUV infection. This data suggests that liver, neuron and kidney are potential target organs during TMUV infection in humans, consistent with what has been found in animal studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virusres.2020.198252DOI Listing
January 2021

COVID-19 in dermatology practice: getting back on track.

Lasers Med Sci 2020 10 23;35(8):1871-1874. Epub 2020 Jun 23.

Department of Dermatology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Wanglang Road, Bangkok, 10700, Thailand.

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10103-020-03043-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7309206PMC
October 2020

Inhibition of human immunodeficiency virus type 1 by niclosamide through mTORC1 inhibition.

Heliyon 2020 Jun 3;6(6):e04050. Epub 2020 Jun 3.

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

Niclosamide has been known to inhibit a number of pH-dependent viruses via the neutralization of endosomal acidic pH. It has also been shown to disrupt the mTORC1 signaling pathway. The replication of many viruses requires mTORC1 activation. Here, we investigated the inhibitory activity of niclosamide against HIV-1, and determined whether mTORC1 inhibition was involved. The cytotoxicity and anti-HIV-1 activity of niclosamide were tested in TZM-bl and SupT1 cells. Niclosamide showed a dose- and time-dependent inhibitory activity against HIV-1 replication, but the inhibition did not involve the reverse transcription and transcription steps. The mechanism of mTORC1 inhibition was explored by using MHY1485, an mTORC1 activator, to reverse the mTORC1 inhibition, which could partially restore HIV-1 replication. In addition, niclosamide was found to downregulate mTORC1 via AMPK activation, resulting in a decreased phosphorylation of the downstream substrates of S6K and 4EBP1. Niclosamide could also reduce the synthesis of HIV-1 p24 protein. Likewise, MHY-1485 could partially reverse the inhibitory effect of niclosamide by increasing the phosphorylation in the mTORC1 pathway and HIV-1 viral protein synthesis. Our findings, therefore, demonstrated the antiviral mechanism of niclosamide is via the AMPK-mTORC1 pathway, which could be a common therapeutic target for various viruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.heliyon.2020.e04050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7276449PMC
June 2020

Codon usage similarity between viral and some host genes suggests a codon-specific translational regulation.

Heliyon 2020 May 8;6(5):e03915. Epub 2020 May 8.

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand.

The codon usage pattern is a specific characteristic of each species; however, the codon usage of all of the genes in a genome is not uniform. Intriguingly, most viruses have codon usage patterns that are vastly different from the optimal codon usage of their hosts. How viral genes with different codon usage patterns are efficiently expressed during a viral infection is unclear. An analysis of the similarity between viral codon usage and the codon usage of the individual genes of a host genome has never been performed. In this study, we demonstrated that the codon usage of human RNA viruses is similar to that of some human genes, especially those involved in the cell cycle. This finding was substantiated by its concordance with previous reports of an upregulation at the protein level of some of these biological processes. It therefore suggests that some suboptimal viral codon usage patterns may actually be compatible with cellular translational machineries in infected conditions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.heliyon.2020.e03915DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7205639PMC
May 2020

Influence of cellular lipid content on influenza A virus replication.

Arch Virol 2020 May 29;165(5):1151-1161. Epub 2020 Mar 29.

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, 2 Prannok Road, Bangkok, 10700, Thailand.

Influenza A virus (IAV) depends on the metabolism of its cellular host to provide energy and essential factors, including lipids, for viral replication. Previous studies have shown that fatty acids (FAs) play an important role in IAV replication and that inhibition of FA biosynthesis can diminish viral replication. However, cellular lipids can either be synthesized intracellularly or be imported from the extracellular environment. Interfering with FA import mechanisms may reduce the cellular lipid content and inhibit IAV replication. To test this hypothesis, MDCK and Detroit 562 cells were infected with IAV followed by exposure to palmitic acid and inhibitors of FA import. Replication of IAV significantly increased when infected cells were supplied with palmitic acid. This enhancement could be reduced by adding an FA import inhibitor. The addition of palmitic acid significantly increased the cellular lipid content, and this increased level was reduced by treatment with an FA import inhibitor. These results show that reducing the cellular lipid level might be an approach for IAV therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00705-020-04596-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7223680PMC
May 2020

Contact Mixing Patterns and Population Movement among Migrant Workers in an Urban Setting in Thailand.

Int J Environ Res Public Health 2020 03 26;17(7). Epub 2020 Mar 26.

Department of Tropical Hygiene, Faculty of Tropical Medicine, Mahidol University Bangkok, Bangkok 10400, Thailand.

Data relating to contact mixing patterns among humans are essential for the accurate modeling of infectious disease transmission dynamics. Here, we describe contact mixing patterns among migrant workers in urban settings in Thailand, based on a survey of 369 migrant workers of three nationalities. Respondents recorded their demographic data, including age, sex, nationality, workplace, income, and education. Each respondent chose a single day to record their contacts; this resulted in a total of more than 8300 contacts. The characteristics of contacts were recorded, including their age, sex, nationality, location of contact, and occurrence of physical contact. More than 75% of all contacts occurred among migrants aged 15 to 39 years. The contacts were highly clustered in this age group among migrant workers of all three nationalities. There were far fewer contacts between migrant workers with younger and older age groups. The pattern varied slightly among different nationalities, which was mostly dependent upon the types of jobs taken. Half of migrant workers always returned to their home country at most once a year and on a seasonal basis. The present study has helped us gain a better understanding of contact mixing patterns among migrant workers in urban settings. This information is useful both when simulating disease epidemics and for guiding optimal disease control strategies among this vulnerable section of the population.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijerph17072237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7177916PMC
March 2020

Immune response to influenza vaccination in ESRD patients undergoing hemodialysis vs. hemodiafiltration.

PLoS One 2020 3;15(2):e0227719. Epub 2020 Feb 3.

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

Background: On-line hemodiafiltration (HDF) clears more azotemic toxins compared to high-flux hemodialysis (HD). The response to vaccination is impaired in dialysis patients. We wished to determine whether the immune responses to influenza vaccine in dialysis patients treated by HDF were stronger than those treated by HD.

Materials And Methods: We conducted a prospective cohort study in chronic dialysis patients during the 2016 and 2017 influenza seasons. All participants received a single standard dose of trivalent influenza vaccine, and we studied the elicited humoral immune response by hemagglutination inhibition test, and cell-mediated immune response by enumeration of lymphocyte cellular markers and proliferation assays.

Results: We immunized 60 end-stage renal disease (ESRD) patients: 42 (70%) treated with HD and 18 patients (30%) with HDF. The median (interquartile range) age was 65.0 (55.0-74.5) years. All patients developed seroprotection to at least one influenza vaccine strain at one month post-vaccination, and did not differ between groups. By logistic regression, age was the only factor independently associated with seroconversion to all vaccine strains (odds ratio 0.89, 95% confidence interval 0.80-0.98; p = 0.022). Seroprotection to all vaccine strains was sustained for longer in patients treated with HDF, and the results remained the same after age adjustment. For cellular immune response, patients who seroconverted to all vaccine strains had higher CD38+ T cell subpopulations pre-vaccination. Patients treated by HDF had higher lymphocyte proliferation to circulating influenza A strains.

Conclusions: Seroconversion to all influenza vaccine strains was associated with age. Patients treated with HDF demonstrated seroprotection was sustained for longer compared to those treated by HD and greater lymphocyte proliferation to circulating influenza A strains. These encouraging results for HDF require confirmation in a larger dialysis population.

Trial Registration: ClinicalTrial.gov, NCT04122222.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0227719PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6996846PMC
April 2020

High correlation between Zika virus NS1 antibodies and neutralizing antibodies in selected serum samples from normal healthy Thais.

Sci Rep 2019 09 18;9(1):13498. Epub 2019 Sep 18.

Institute of Molecular Biosciences, Mahidol University, Salaya, Thailand.

Despite the widespread presence of the mosquito transmitted Zika virus (ZIKV) over much of Southeast Asia, the number of reported cases remains low. One possibility is that residents in Southeast Asia are immunologically protected, although the nature of any such protection remains unclear. This study sought to investigate the presence of antibodies directed to ZIKV NS1 protein in a selected sub-set of samples from a well characterized cohort of serum samples from normal, healthy Thais that had been previously characterized for the presence of neutralizing antibodies to ZIKV, DENV 1-4, and JEV. Because of similarities in molecular weight between the flavivirus E and NS1 proteins, an immunoblot system was established in which the NS1 antigen was not denatured, allowing detection of the dimer form of NS1, distinctly clear from the migration position of the E and NS1 monomer proteins. The results showed that antibodies to ZIKV NS1 protein were only detected in samples with ZIKV neutralizing antibodies (27/30 samples), and no sample (0/30) with a ZIKV plaque reduction neutralization test (PRNT) < 20 showed evidence of anti-ZIKV NS1 antibodies. The high correlation between the presence of ZIKV NS1 antibodies and ZIKV PRNT suggests that immunological protection against ZIKV infection in Thailand arises from prior exposure to ZIKV, and not through cross neutralization.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-019-49569-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6751300PMC
September 2019

The relationship of codon usage to the replication strategy of parvoviruses.

Arch Virol 2019 Oct 18;164(10):2479-2491. Epub 2019 Jul 18.

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand.

Codon usage is biased in most species, and the pattern of codon usage bias is specific to each species or group of closely related species. Although viruses use the host translational machinery for synthesis of their proteins, their codon usage patterns do not match those of their host. Viral codon usage is determined by a complex interplay of mutational bias, genome composition constraints, translational adaptation to the host, and host cellular innate defense. The codon usage of parvoviruses was previously shown not to be strongly biased and selective pressure was found to be a dominating factor driving codon usage. The family Parvoviridae includes the genus Dependoparvovirus, some of the members of which require a helper virus to complete their replication cycle, whereas the rest of the family can replicate without the need for helper viruses. Here, we show that difference in the replication strategy of these viruses may be an important factor determining viral codon usage. Hierarchical clustering and principal component analysis revealed that the codon usage pattern of adeno-associated viruses (AAVs) of the genus Dependoparvovirus is distinct from that of members of the other genera of vertebrate parvoviruses, and even from that of independent viruses of the genus Dependoparvovirus. Furthermore, the codon usage of human AAVs was found to be similar to that of some human adenoviruses in hierarchical clustering and principal component analysis. This suggests that the codon usage of AAVs is different from that of other parvoviruses because of their distinctive replication strategy and that their codon usage is probably driven by forces similar to those that shaped the codon usage pattern of their helper viruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00705-019-04343-5DOI Listing
October 2019

Dengue virus requires apoptosis linked gene-2-interacting protein X (ALIX) for viral propagation.

Virus Res 2019 02 29;261:65-71. Epub 2018 Dec 29.

Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamonthol Sai 4, Nakhon Pathom, Bangkok, 73170, Thailand. Electronic address:

The endosomal sorting complexes required for transport (ESCRT) pathway accessory protein apoptosis linked gene-2-interacting protein X (ALIX) has been shown to be upregulated during dengue virus (DENV) replication. Yeast-two-hybrid screens have additionally shown that ALIX interacts with DENV NS3 protein, but evaluation of the interaction through a replicon assay failed to show a functional significance to the interaction. In this study the interaction between DENV NS3 and ALIX was investigated by co-immunoprecipitation, and functional significance assessed by investigation of DENV production in ALIX expression regulated cells. The results showed that ALIX both interacted and co-localized with DENV NS3 protein and that upregulation of ALIX resulted in a significantly increased viral titer, while either siRNA or CRISPR-Cas9 mediated down regulation of ALIX significantly reduced viral production, without affecting relative DENV genome levels. These results are consistent with ALIX playing a significant role in the DENV replication cycle either during late infection or at viral egress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virusres.2018.12.015DOI Listing
February 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/acssensors.8b00885DOI Listing
January 2019

Complete Genomic Sequences of Highly Pathogenic H5N1 Avian Influenza Viruses Obtained Directly from Human Autopsy Specimens.

Microbiol Resour Announc 2018 Dec 6;7(22). Epub 2018 Dec 6.

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

The complete genomic sequences of H5N1 highly pathogenic avian influenza (HPAI) viruses were directly obtained from lung, trachea, and colon tissues and an intestinal fecal sample of a patient by using the next-generation sequencing technique. This is the first report on complete H5N1 viral genomes from human autopsy specimens.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MRA.01498-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6284082PMC
December 2018

Analysis of Zika virus neutralizing antibodies in normal healthy Thais.

Sci Rep 2018 11 21;8(1):17193. Epub 2018 Nov 21.

Institute of Molecular Biosciences, Mahidol University, Bangkok, Thailand.

Zika virus (ZIKV) infections have been reported from all over Thailand, but the number of reported cases remains low, suggesting a degree of immune protection against ZIKV infection. To address this possibility, the presence of ZIKV neutralizing antibodies was determined in serum from 135 healthy Thai adults with a plaque reduction neutralization test (PRNT), and a number of samples were subsequently analyzed for the presence of neutralizing antibodies to dengue virus (DENV) and Japanese encephalitis virus (JEV). Results showed that 70.4% (PRNT ≥ 10), 55.6 (PRNT ≥ 20) or 22.2% (PRNT ≥ 20) of the samples showed neutralizing antibodies to ZIKV. Detailed analysis showed no association between the presence of neutralizing antibodies to other flaviviruses (DENV, JEV) and the presence of ZIKV neutralizing antibodies. These results suggest that the level of ZIKV neutralizing antibodies in the Thai population is enough to dampen the transmission of the virus in Thailand.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-35643-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6249253PMC
November 2018

Genome polarity of RNA viruses reflects the different evolutionary pressures shaping codon usage.

Arch Virol 2018 Oct 9;163(10):2883-2888. Epub 2018 Jul 9.

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand.

RNA viruses are classified by their genome polarity and replication strategies. Nucleotide composition and codon usage differ among virus groups, for instance positive-sense RNA (+ssRNA) viruses have higher GC-content than the other RNA virus groups. Codon usage of +ssRNA viruses is closer to humans showing significantly higher codon adaptation index (CAI) than those of negative-sense RNA (-ssRNA), double stranded RNA (dsRNA) and retroviruses. Ambisense viruses have high CAI comparable to that of +ssRNA virus despite their lower GC content, whereas dsRNA viruses have the lowest CAI. This may provide a benefit for +ssRNA viruses as their genomes are used as mRNA. However, analyses for influence of nucleotide composition on codon usage did not show a difference between +ssRNA and -ssRNA viruses. This suggests that genome composition and hence mutational pressure remain the major pressure causing the differences in codon usage among RNA viruses with different genome types.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00705-018-3930-7DOI Listing
October 2018

Microparticle Release from Cell Lines and Its Anti-Influenza Activity.

Viral Immunol 2018 Jul/Aug;31(6):447-456. Epub 2018 May 21.

1 Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University , Bangkok, Thailand .

Microparticles (MPs) are vesicles that are released by budding from plasma membrane of living cells. Recently, the role of MPs in antiviral activity has been proposed. We investigated quantity and anti-influenza activity of MPs from human alveolar epithelial cells A549, human bronchial epithelial cells BEAS-2B, human colon adenocarcinoma cells HT-29, and the human lung fibroblast cells MRC-5. MPs were found from all four cell lines. However, anti-influenza activity against an H1N1 influenza virus was found only from MPs of A549 and BEAS-2B. BEAS-2B cell differentiation did not increase MP release. Methyl-β-cyclodextrin (MβCD) increased MP release and anti-influenza activity in HT-29 and A549. MP release increased after calcium ionophore A23187 treatment in three cell lines but only in HT-29 after forskolin treatment. These findings provide in vitro data supporting the role of MPs as an innate defense against influenza virus and as an approach to enhance the defense.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/vim.2017.0201DOI Listing
October 2018

H1N1 seasonal influenza virus evolutionary rate changed over time.

Virus Res 2018 05 30;250:43-50. Epub 2018 Mar 30.

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol Universit, 2 Prannok Road. Bangkoknoi, Bangkok, 10700, Thailand.

It was previously shown that the seasonal H1N1 influenza virus antigenic drift occurred at a slower rate than the seasonal H3N2 virus during the first decade of the 21th century. It was hypothesized that the slower antigenic evolution led to a decrease in average ages of infection, which in turn resulted in lower level of global viral circulation. It is unclear what caused the difference between the two viruses, but a plausible explanation may be related to the fact that the H1N1 virus had been in human population for much longer than the H3N2 virus. This would suggest that H1N1 antigenic drift in an earlier period may have been different from a more recent period. To test this hypothesis, we analyzed seasonal H1N1 influenza sequences during various time periods. In comparison to more recent H1N1 virus, the older H1N1 virus during the first half of the 20th century showed evidences of higher nonsynnonymous/synonymous ration (dN/dS) in its hemagglutinin (HA) gene. We compared amino acid sequence changes in the HA epitopes for each outbreak season and found that there were less changes in later years. Amino acid sequence diversity in the epitopes as measured by sequence entropy became smaller for each passing decade. These suggest that there might be some limit to the antigenic drift. The longer an influenza virus has drifted in human population, the less flexibility it may become. With less flexibility to adapt and escape the host immunity, the virus may have to rely more on younger naïve population.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virusres.2018.03.020DOI Listing
May 2018

Zika virus in Thailand.

Microbes Infect 2018 12 2;20(11-12):670-675. Epub 2018 Mar 2.

Institute of Molecular Biosciences, Mahidol University, Salaya Campus, 25/25 Phuttamonthol Sai 4, Nakhon Pathom, 73170, Thailand. Electronic address:

This review examines the historic reports of the presence of Zika virus (ZIKV) in Thailand, as well as collates such information as exists on the current situation in Thailand with regards to ZIKV. We suggest that considerable caution must be applied in interpreting early serological studies, but that ZIKV is presently circulating over much of Thailand, with increasing numbers of cases being reported.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.micinf.2018.01.007DOI Listing
December 2018

Glutathionylation of dengue and Zika NS5 proteins affects guanylyltransferase and RNA dependent RNA polymerase activities.

PLoS One 2018 22;13(2):e0193133. Epub 2018 Feb 22.

Institute of Molecular Biosciences, Mahidol University, Salaya Campus, Salaya, Thailand.

It has been estimated for dengue infection that the global population at risk is 3.5 billion people, which makes dengue an important public health problem. The causative agents of dengue are dengue viruses. For dengue virus replication, the dengue virus NS5 protein is of special importance as it has several enzyme activities important for viral replication. Previous reports of phosphorylation and SUMOylation of dengue NS5 have shown these protein modifications have important consequences for NS5 functions. In this report we identify glutathionylation, another reversible post translation modification that impacts on NS5 enzyme activity. Using dengue virus infected cells we employed specific antibodies and mass spectrometry to identify 3 cysteine residues of NS5 protein as being glutathionylated. Glutathionylation is a post translational protein modification where glutathione is covalently attached to a cysteine residue. We showed glutathionylation occurs on 3 conserved cysteine residues of dengue NS5. Then we generated two flavivirus recombinant full length proteins, dengue NS5 and Zika NS5, to characterize two of the NS5 enzyme activities, namely, guanylyltransferase and RNA-dependent RNA polymerase activities. We show glutathionylation of dengue and Zika NS5 affects enzyme activities of the two flavivirus proteins. The data suggests that glutathionylation is a general feature of the flavivirus NS5 protein and the modification has the potential to modulate several of the NS5 enzyme functions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0193133PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5823458PMC
May 2018

Codon usage of HIV regulatory genes is not determined by nucleotide composition.

Arch Virol 2018 Feb 25;163(2):337-348. Epub 2017 Oct 25.

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, 10700, Thailand.

Codon usage bias can be a result of either mutational bias or selection for translational efficiency and/or accuracy. Previous data has suggested that nucleotide composition constraint was the main determinant of HIV codon usage, and that nucleotide composition and codon usage were different between the regulatory genes, tat and rev, and other viral genes. It is not clear whether translational selection contributed to the codon usage difference and how nucleotide composition and translational selection interact to determine HIV codon usage. In this study, a model of codon bias due to GC composition with modification for the A-rich third codon position was used to calculate predicted HIV codon frequencies based on its nucleotide composition. The predicted codon usage of each gene was compared with the actual codon frequency. The predicted codon usage based on GC composition matched well with the actual codon frequencies for the structural genes (gag, pol and env). However, the codon usage of the regulatory genes (tat and rev) could not be predicted. Codon usage of the regulatory genes was also relatively unbiased showing the highest effective number of codons (ENC). Moreover, the codon adaptation index (CAI) of the regulatory genes showed better adaptation to human codons when compared to other HIV genes. Therefore, the early expressed genes responsible for regulation of the replication cycle, tat and rev, were more similar to humans in terms of codon usage and GC content than other HIV genes. This may help these genes to be expressed efficiently during the early stages of infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00705-017-3597-5DOI Listing
February 2018

Kinetics, Longevity, and Cross-Reactivity of Antineuraminidase Antibody after Natural Infection with Influenza A Viruses.

Clin Vaccine Immunol 2017 Dec 5;24(12). Epub 2017 Dec 5.

Department of Microbiology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand

The kinetics, longevity, and breadth of antibodies to influenza virus neuraminidase (NA) in archival, sequential serum/plasma samples from influenza A virus (IAV) H5N1 infection survivors and from patients infected with the 2009 pandemic IAV (H1N1) virus were determined using an enzyme-linked lectin-based assay. The reverse-genetics-derived H4N1 viruses harboring a hemagglutinin (HA) segment from A/duck/Shan Tou/461/2000 (H4N9) and an NA segment derived from either IAV H5N1 clade 1, IAV H5N1 clade 2.3.4, the 2009 pandemic IAV (H1N1) (H1N1pdm), or A/Puerto Rico/8/1934 (H1N1) virus were used as the test antigens. These serum/plasma samples were also investigated by microneutralization (MN) and/or hemagglutination inhibition (HI) assays. Neuraminidase-inhibiting (NI) antibodies against N1 NA of both homologous and heterologous viruses were observed in H5N1 survivors and H1N1pdm patients. H5N1 survivors who were never exposed to H1N1pdm virus developed NI antibodies to H1N1pdm NA. Seroconversion of NI antibodies was observed in 65% of the H1N1pdm patients at day 7 after disease onset, but an increase in titer was not observed in serum samples obtained late in infection. On the other hand, an increase in seroconversion rate with the HI assay was observed in the follow-up series of sera obtained on days 7, 14, 28, and 90 after infection. The study also showed that NI antibodies are broadly reactive, while MN and HI antibodies are more strain specific.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/CVI.00248-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5717183PMC
December 2017

Ubiquitin-Conjugating Enzyme E2 L3 is Downregulated by the Chikungunya Virus nsP2 Protease.

Proteomics Clin Appl 2018 07 16;12(4):e1700020. Epub 2017 Nov 16.

Institute of Molecular Biosciences, Mahidol University, Nakhon Pathom, Thailand.

Purpose: Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that causes chikungunya fever in humans. The CHIKV non-structural protein 2 (nsP2) is a multifunctional protein that additionally modulates the host cell to dampen the innate immune response and inhibit other cellular processes.

Experimental Design: To further investigate the interactions of nsP2 with host cells, the protease domain of CHIKV nsP2 (nsP2-pro) is transfected into Hela cells, and differential protein expression is detected by 2D polyacrylamide gel electrophoresis.

Results: A total of 21 differentially regulated (six upregulated, 15 downregulated) spots are observed, of which five are identified by mass spectrometry. The downregulation of one of the identified proteins, ubiquitin-conjugating enzyme E2 L3 (UBE2L3) is confirmed by western blotting of both nsP2-pro transfection and CHIKV natural infection, and the downregulation of UBE2L3 is additionally shown to require an enzymatically active nsP2 protease domain. Transfection of full length UBE2L3 into HEK293T/17 cells prior to CHIKV infection reduce levels of infection and E protein expression but do not alter RNA genome levels.

Conclusion: These results suggest that UBE2L3 is a cellular target of the CHIKV nsP2 protease, and this possibly mediates the pathogenesis of chikungunya fever.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/prca.201700020DOI Listing
July 2018

Microparticle and anti-influenza activity in human respiratory secretion.

PLoS One 2017 23;12(8):e0183717. Epub 2017 Aug 23.

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

Respiratory secretions, such as saliva and bronchoalveolar fluid, contain anti-influenza activity. Multiple soluble factors have been described that exert anti-influenza activity and are believed to be responsible for the anti-influenza activity in respiratory secretions. It was previously shown that a bronchial epithelial cell culture could produce exosome-like particles with anti-influenza activity. Whether such extracellular vesicles in respiratory secretions have anti-influenza activity is unknown. Therefore, we characterized bronchoalveolar lavage fluid and found microparticles, which mostly stained positive for epithelial cell markers and both α2,3- and α2,6-linked sialic acid. Microparticles were purified from bronchoalveolar lavage fluid and shown to exhibit anti-influenza activity by a hemagglutination inhibition (HI) assay and a neutralization (NT) assay. In addition, physical binding between influenza virions and microparticles was demonstrated by electron microscopy. These findings indicate that respiratory microparticles containing viral receptors can exert anti-viral activity by probably trapping viral particles. This innate mechanism may play an important role in the defense against respiratory viruses.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0183717PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5568225PMC
November 2017

Mutations in matrix protein 1 and nucleoprotein caused human-specific defects in nuclear exportation and viral assembly of an avian influenza H7N1 virus.

Virus Res 2017 06 1;238:49-62. Epub 2017 Jun 1.

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

Nuclear exportation of influenza ribonucleoprotein is a vital step in viral replication cycle. In this study a particular H7N1 (A/ostrich/Zimbabwe/222-E3/1996) virus showed exclusively nuclear localization of the viral nucleoprotein (NP) only in human cell lines but not in cell lines of other species suggesting a human-specific nuclear exportation defect. After 10 passages in human lung cells, an adapted strain (H7N1:P10) could efficiently replicate and export viral NP in human cells. Mutations in the NP and matrix M1 gene at position 297 and 227, respectively, were found to rescue the defect. While the NP mutant showed a comparable ratio of total to NP-associated negative-sense RNA in the cytoplasm as compared to the wild type, the M1 mutant showed an increase in free negative-sense RNA in the cytoplasm. These indicated that the NP mutation might cause a nuclear export defect, whereas the M1 mutation might cause a defect in ribonucleoprotein assembly step.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.virusres.2017.05.021DOI Listing
June 2017