Shingo Inoue - Academia.edu (original) (raw)

Papers by Shingo Inoue

The Global Health Network Conference Proceedings 2022

Applied Biosafety, 2017

When properly used and adequately maintained, a biological safety cabinet (BSC) provides protecti... more When properly used and adequately maintained, a biological safety cabinet (BSC) provides protection to the laboratory worker, the products, and the environment. Knowledge about proper use of the BSC is therefore important. This study evaluated BSC usage knowledge and practices and the effect of prior experience on training outcomes. Thirteen participants randomly selected from research centers were evaluated through a structured questionnaire covering biosafety considerations involved in using a BSC as per the World Health Organization's Laboratory Biosafety Manual, third edition. The questionnaire was administered before and after the training. Training was conducted using the NUITM-KEMRI biosafety training program, enhanced to include more modules on BSCs. Differences in the participants' knowledge and practices regarding BSC usage were observed. After training, the participants were able to give correct responses. Furthermore, participants who had fewer years of experience showed the greatest improvement. Results indicate the need for proper training of laboratory workers early in their careers, coupled with close supervision, mentorship, and regular refresher training.

Antiviral Research, 2021

Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus of African origin that is transmitte... more Zika virus (ZIKV) is a re-emerging mosquito-borne flavivirus of African origin that is transmitted by Aedes mosquitoes. ZIKV was historically limited to Africa and Asia, where mild cases were reported. However, ZIKV has recently been responsible for major global outbreaks associated with a wide range of neurological complications. Since no antiviral therapy exists for ZIKV, drug discovery research for ZIKV is crucial. Intracellular lipids regulated by sterol regulatory element-binding proteins (SREBPs) are important in flavivirus pathogenesis. PF-429242 has been reported to inhibit the activity of site-1 protease (S1P), which regulates the expression of SREBP target genes. Our primary objective in this study is to elucidate the mechanism of the antiviral activity of PF-429242 against the African genotype (ZIKVMR-766) and Asian genotypes (ZIKV H/PF 2013 and ZIKV PRVABC59) using several primate-derived cell lines. The virus titer was determined via a focus-forming assay; we used flow cytometry to quantify intracellular lipids in ZIKV-infected and mock-treated cells. The PF-429242 molecule effectively suppressed ZIKV infection in neuronal cell lines; T98G, U-87MG, SK-N-SH and primary monocytes cell, indicating that PF-429242 molecule can be used therapeutically. A strong reduction in ZIKV replication was observed at 12 μM and 30 μM in in neuronal cell lines and primary monocytes, respectively. Interestingly, the inhibitory effects of the PF-429242 molecule were observed when it was tested on various ZIKV-lineage infections. Lipid quantification reveals that ZIKV increases lipogenesis in infected cells, while the exogenous addition of cholesterol effectively blocks ZIKV replication. Furthermore, the supplementation of oleic acid increases the ZIKV titer. Fenofibrate, an inhibitor of lipid droplet formation, reduces the ZIKV titer. Collectively, our results demonstrate that the development of antiviral drugs against ZIKV could be based on key regulators of lipid metabolism. In addition, this study reveals that the mechanism of the PF-429242-mediated suppression among flavivirus infections is not entirely identical. Our results warrant further evaluation of PF-429242 as a prospective antiviral drug, given the multiple advantageous properties of this compound, such as its limited toxicity, neuroprotective properties, and broad spectrum of capabilities.

International Journal of Environmental Research and Public Health, 2021

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronaviru... more Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). Real-time RT-PCR is the most commonly used method for COVID-19 diagnosis. However, serological assays are urgently needed as complementary tools to RT-PCR. Hachim et al. 2020 and Burbelo et al. 2020 demonstrated that anti-nucleocapsid(N) SARS-CoV-2 antibodies are higher and appear earlier than the spike antibodies. Additionally, cross-reactive antibodies against N protein are more prevalent than those against spike protein. We developed a less cross-reactive immunoglobulin G (IgG) indirect ELISA by using a truncated recombinant SARS-CoV-2 N protein as assay antigen. A highly conserved region of coronaviruses N protein was deleted and the protein was prepared using an E. coli protein expression system. A total of 177 samples collected from COVID-19 suspected cases and 155 negative control sera collected during the pre-COVID-19 period were applied to evaluate the...

The American Journal of Tropical Medicine and Hygiene, 2005

To demonstrate the differences of clinical features and hematologic abnormalities between dengue ... more To demonstrate the differences of clinical features and hematologic abnormalities between dengue fever (DF) and dengue hemorrhagic fever (DHF), 359 pediatric patients admitted St. Luke's Medical Center in Quezon City, between 1999 and 2001 in Metro Manila, and adjoining provinces the Philippines, with a laboratory-confirmed dengue virus infection were evaluated. One third of the patients had DHF, and most of these patients were without shock. Restlessness, epistaxis, and abdominal pain were more associated with DHF. The platelet count was significantly lower in the DHF group than in the DF group before and after defervescence. In the DHF patients, the hematocrit was significantly increased before defervescence, and decreased the day after due to administration of intravenous fluid. Coagulation abnormalities associated with most DHF patients were thrombocytopenia and an increased fibrinolysis, but not disseminated intravascular coagulation. We present recent data on readily obtained clinical and laboratory data that can be used for early diagnosis and consequently earlier appropriate treatment of dengue virus infections.

Vector-Borne and Zoonotic Diseases, 2010

To establish a new method for the diagnosis of dengue secondary infection, 187 serum samples from... more To establish a new method for the diagnosis of dengue secondary infection, 187 serum samples from the patients with dengue secondary infection, 40 serum samples from the patients with dengue primary infection, and 44 serum samples from the healthy volunteers were tested using the dengue IgG indirect enzyme-linked immunosorbent assay (DEN IgG ELISA). The results of the test were compared with those from the dengue hemagglutination inhibition (DEN HI) test, which has been recommended as the gold standard by the World Health Organization (WHO, 1997). Japanese encephalitis IgG indirect ELISA (JE IgG ELISA) was also performed to measure anti-flavivirus IgG, which cross-reacts with the Japanese encephalitis virus, to test the possibility of an alternative to DEN IgG ELISA. The results of DEN IgG and JE IgG ELISAs were highly correlated with those of the DEN HI test. In the DEN IgG ELISA, a titer of 1:29,000 was the cutoff value for the diagnosis of dengue secondary infection (91.5% accuracy [95% confidence interval, CI], 90.9% sensitivity [95%CI], and 92.9% specificity [95%CI]). A titer of 1:52,000 was the cutoff value for dengue secondary infection using JE IgG ELISA (95.6% accuracy [95%CI], 98.9% sensitivity [95%CI], and 88.1% specificity [95%CI]). In conclusion, this study confirmed that the results of both DEN IgG and JE IgG ELISAs were highly correlated with the results of DEN HI test. Thus, these ELISAs are simple, rapid, sensitive, and quantitative tests that can be used in the determination of dengue secondary infection.

Journal of Medical Virology, 2013

Viruses, 2021

Dengue fever, caused by the mosquito-borne dengue virus (DENV), has been endemic in Myanmar since... more Dengue fever, caused by the mosquito-borne dengue virus (DENV), has been endemic in Myanmar since 1970 and it has become a significant public health burden. It is crucial that circulating DENV strains are identified and monitored, and that their transmission efficiency and association with disease severity is understood. In this study, we analyzed DENV-1, DENV-2, DENV-3, and DENV-4 serotypes in 1235 serum samples collected in Myanmar between 2017 and 2019. Whole-genome sequencing of DENV-1–4 demonstrated that most DENV-1–4 strains had been circulating in Myanmar for several years. We also identified the emergence of DENV-3 genotype-I in 2017 samples, which persisted through 2018 and 2019. The emergence of the strain coincided with a period of increased DENV-3 cases and marked changes in the serotype dynamics. Nevertheless, we detected no significant differences between serum viral loads, disease severity, and infection status of individuals infected with different DENV serotypes dur...

The American Journal of Tropical Medicine and Hygiene

This study was conducted to find the burden of dengue virus (DENV) and Japanese encephalitis viru... more This study was conducted to find the burden of dengue virus (DENV) and Japanese encephalitis virus (JEV) among children under the age of 13, who presented with acute encephalitis syndrome at Mandalay Children Hospital in Myanmar in 2013. Molecular and serological investigations were performed on 123 cerebrospinal fluid (CSF) samples collected from these patients. By neutralization tests and/or virus isolation, four (3.3%) JEV-and one DENV-associated encephalitis cases (0.8%) were confirmed. Antibody titer against JEV Genotype 3 was the highest among the laboratoryconfirmed JEV cases. One strain of DENV-1 with Genotype 1 was isolated from the CSF sample of the dengue encephalitis patient; this was similar to the virus circulating in the study area and neighboring countries. This study shows that flaviviruses are important pathogens causing encephalitis in Myanmar. Active disease surveillance, vector control, and vaccination programs should be enforced to reduce the morbidity and mortality caused by flavivirus encephalitis.

Tropical medicine and health, 2018

The NUITM-KEMRI biosafety training program was developed for capacity building of new biosafety l... more The NUITM-KEMRI biosafety training program was developed for capacity building of new biosafety level three (BSL-3) laboratory users. The training program comprehensively covers biosafety and biosecurity theory and practice. Its training curriculum is based on the WHO biosafety guidelines, local biosafety standards, and ongoing biosafety level three research activities in the facility, also taking into consideration the emerging public health issues. The program's training approach enhances the participant's biosafety and biosecurity knowledge and builds their skills through the hands-on practice sessions and mentorship training. Subsequently, the trainees are able to integrate acquired knowledge and good practices into their routine laboratory procedures. This article describes implementation of the NUITM-KEMRI biosafety training program.

Virus research, Mar 2, 2018

An entomological surveillance of arboviruses was conducted in Myanmar in 2014. A total of 8357 Cu... more An entomological surveillance of arboviruses was conducted in Myanmar in 2014. A total of 8357 Culex mosquito vectors were collected in the Mandalay area and virus isolation was done by using the mosquito cell line C6/36 E2. A total of eighteen strains of Culex flavivirus (CxFV) were isolated from Cx. tritaeniorhynchus, Cx. vishnui and Cx. fuscocephala. Like other insect-specific flaviviruses, CxFV can replicate only in mosquito cells but not in mammalian cells. These CxFV strains that were isolated in Japan from mosquitoes collected in Myanmar were closely related to the Wang Thong virus detected from Cx fusocephalus in Thailand and Cx.theileri flavivirus (CTFV) isolated from Cx. theileri mosquitoes in Portugal and Turkey. They encode a single open reading frame with 3357 amino acid residues. They have the characteristics of flaviviruses and have 95.62% amino acid identity with CTFV. This is the first report of CxFV in Myanmar with the characterized viral genome. This study illustr...

The American journal of tropical medicine and hygiene, Jan 22, 2018

Zika virus (ZIKV), an emerging mosquito-borne flavivirus, causes a dengue-like infection that has... more Zika virus (ZIKV), an emerging mosquito-borne flavivirus, causes a dengue-like infection that has recently caught global attention. The infection, which also includes some birth defects, has been documented in the Americas, Pacific Islands, and some parts of Africa and Asia. There are no published reports on local ZIKV transmission in Myanmar. In this study, a total of 462 serum samples from patients and asymptomatic persons were collected in Myanmar from 2004 to 2017. They were analyzed for ZIKV infection by immunoglobulin M capture enzyme-linked immunosorbent assay (ELISA), immunoglobulin G indirect ELISA, neutralization test, real-time polymerase chain reaction (PCR), and conventional PCR. Our study confirmed ZIKV infection in 4.9% of patients with clinical dengue symptoms and in 8.6% of persons who were asymptomatic. This is the first report on ZIKV infection in Myanmar and it suggests the occurrence of ZIKV infection in two geographically distinct sites in this country since at...

Tropical Medicine and Health, 2016

Background: Japanese encephalitis (JE) is a mosquito-borne infectious disease caused by Japanese ... more Background: Japanese encephalitis (JE) is a mosquito-borne infectious disease caused by Japanese encephalitis virus (JEV). About 1-10 cases with severe central nervous system symptoms have been constantly reported every year in Japan. To clarify the mechanism of maintenance of JEV, the present study surveyed pigs for serological evidence of JEV infection and isolated JEV strains from pigs and mosquitoes in Isahaya City (Isahaya) and Goto City (Goto) in the islets of Goto in Nagasaki Prefecture from 2008 to 2014. Results: The serological survey of pigs showed the increase of IgM sero-positivity against JEV in July or August, and it was maintained until October or November in both Isahaya and Goto every year. There were 47 JEV strains isolated in Nagasaki from 2001 to 2014 including the isolates in this study, and they belonged to genotype 1. Thirty four of the isolated strains were from pigs in Isahaya and were classified under six subclusters (1-A-1, 1-A-2, 1-A-3, 1-A-4, 1-A-5, and 1-A-9). Thirteen strains were isolated from pigs and mosquitoes in Goto and were classified into three subclusters (1-A-5 (2008); 1-A-1 (2009); and 1-A-2). In the subcluster 1-A-2, three different monophyletic subgroups, 1-A-2-2 (2010), 1-A-2-3 (2011), and 1-A-2-1 (2013, 2014), appeared in Goto. Conclusions: These data strongly suggested that JEV appearance in Goto seems to depend on the frequent introduction of JEV from outside of the island and this pattern is different from what has been observed in subtropical islands in the East China Sea such as Okinawa and Taiwan, where the same populations of JEV (1-A-7 (1998-2008) in Okinawa; genotype 3 (until 2012) in Taiwan) have been maintained for a long period.

BioResearch Open Access, 2016

This study aimed to investigate the duration of humoral immune responses to dengue virus (DENV) i... more This study aimed to investigate the duration of humoral immune responses to dengue virus (DENV) infection in Japanese who experienced acute febrile illness with hemorrhagic manifestations 70 years ago, when an epidemic of dengue occurred in Nagasaki, Japan, from 1942 to 1944. A Japanese volunteer requested serological diagnosis of DENV infection in 2014 and donated blood sample to measure the antibody titer against DENV by antiflavi IgG indirect ELISA, focus reduction neutralization test, and plaque reduction neutralization test. The serum sample of the volunteer was positive in flavi IgG ELISA and it indicated primary infection. In the neutralization test, the highest neutralizing titer was ‡218 for DENV-2. We report here the existence of DENV-specific antibodies in the serum of a person after 70 years from infection. Published reports indicated that DENV-1 was responsible for the 1942-1944 outbreak in Nagasaki. However, our data suggested that DENV-2 also played a role in this Nagasaki dengue epidemic.

Clinical and vaccine immunology : CVI, Jan 15, 2016

Yellow fever (YF) is an acute haemorrhagic viral infection transmitted by mosquitoes in Africa an... more Yellow fever (YF) is an acute haemorrhagic viral infection transmitted by mosquitoes in Africa and South America. The major challenge in YF disease detection and confirmation of outbreaks in Africa is the limited availability of reference laboratories and the persistent lack of access to diagnostic tests. We used wild-type YF virus sequences to generate recombinant envelope protein in Escherichia coli expression system. Both the recombinant protein and sucrose gradient purified YF vaccine virus 17D (YF-17D) were used to immunise BALB/c mice to generate monoclonal antibodies (MAbs). Eight MAbs were established and systematically characterised by indirect ELISA, Western blot and immunofluorescence assay (IFA). The established MAbs showed strong reactivity with wild-type YF virus and recombinant protein with no detectable cross-reactivity to dengue and Japanese encephalitis viruses. Epitope mapping showed strong binding of three MAbs to amino acid positions 1-51 while two MAbs mapped t...

Tropical Medicine and Health, Sep 21, 2004

A one-step, single tube, real-time accelerated reverse transcription loop-mediated isothermal amp... more A one-step, single tube, real-time accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detecting the envelope gene of West Nile (WN) virus. The RT-LAMP assay is a novel method of gene amplification that amplifies nucleic acid with high specificity, efficiency, and rapidity under isothermal conditions with a set of six specially designed primers that recognize eight distinct sequences of the target. The whole procedure is very simple and rapid, and amplification can be obtained in less than 1 h by incubating all of the reagents in a single tube with reverse transcriptase and Bst DNA polymerase at 63°C. Detection of gene amplification could be accomplished by agarose gel electrophoresis, as well as by real-time monitoring in an inexpensive turbidimeter. When the sensitivity of the RT-LAMP assay was compared to that of conventional RT-PCR, it was found that the RT-LAMP assay demonstrated 10-fold higher sensitivity compared to RT-PCR, with a detection limit of 0.1 PFU of virus. By using real-time monitoring, 10 4 PFU of virus could be detected in as little as 17 min. The specificity of the RT-LAMP assay was validated by the absence of any cross-reaction with other, closely related, members of the Flavivirus group, followed by restriction digestion and nucleotide sequencing of the amplified product. These results indicate that the RT-LAMP assay is extremely rapid, cost-effective, highly sensitive, and specific and has potential usefulness for rapid, comprehensive WN virus surveillance along with virus isolation and/or serology.

Infection, Genetics and Evolution, 2016

In 2013 in Myanmar, dengue epidemic occurred with 20,255 cases including 84 deaths. This study ai... more In 2013 in Myanmar, dengue epidemic occurred with 20,255 cases including 84 deaths. This study aimed to determine the serological and molecular characteristics of dengue virus (DENV) infection among children with clinical diagnosis of dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS) during this period. Single acute serum samples were collected from 300 children in Mandalay Children Hospital, Mandalay, Myanmar. Out of the 300 children, 175 (58.3%) and 183 (61%) were positive for anti-dengue IgM and anti-dengue IgG, respectively. Among the IgM positives, 41 (23.4%) had primary DENV infection. Thirty-nine DENV strains (23 DENV-1, 10 DENV-2 and 6 DENV-4) were successfully isolated after inoculation of the patient serum samples onto C6/36 cells. DENV 1 was the dominant serotype in the 2013 epidemic. There was no correlation between the infecting serotypes and clinical severities. The DENV-1 strains belonged to three lineages of the genotype 1; the DENV-2 strains were of the Asian I genotype and were separated into two lineages; and DENV-4 strains belonged to the same lineage of genotype I. It is of interest to note the diversity of DENV-1 and -2 circulating in the same location during June-August 2013. These DENV isolates were genetically close (98%-100%) to the other previously reported isolates from Myanmar and its neighboring countries, namely China, Thailand, Sri Lanka, Cambodia and Vietnam. Primary DENV infection was still high among the severe dengue cases. Different serotypes of DENV were co-circulating in 2013, however, genotype shift was not observed. Additionally, amino acid mutations were detected in the study strains not seen in the previously reported strains from other countries and Myanmar. This paper provided information on the circulating serotypes for the last 15years and the recent dengue situation in Mandalay, Myanmar after 2006.

Emerging Infectious Diseases, Aug 1, 2008

Abstract We isolated and characterized a Banna virus from mosquitoes in Vietnam; 5 strains were i... more Abstract We isolated and characterized a Banna virus from mosquitoes in Vietnam; 5 strains were isolated from field-caught mosquitoes at various locations; Banna virus was previously isolated from encephalitis patients in Yunnan, China, in 1987. Together, these findings ...