Publications by authors named "Kouichi Morita"

166 Publications

Emergence of genotype I of dengue virus serotype 3 during severe dengue epidemic in Sri Lanka, 2017.

Jpn J Infect Dis 2021 Feb 26. Epub 2021 Feb 26.

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Japan.

During the 2017 outbreak of severe dengue in Sri Lanka, dengue virus (DENV) serotypes 2, 3 and 4 were co-circulating. Based on our previous study on the 295 patients from the National Hospital Kandy in Sri Lanka between March 2017-January 2018, the dominant infecting serotype was DENV-2. Here, we aimed to characterize the DENV-3 strains from non-severe and severe dengue patients from our previous study population. Patients' clinical records and previous laboratory tests including dengue-specific nonstructural protein 1 antigen rapid test, IgM-capture and IgG enzyme-linked immunosorbent assays, were analyzed together with the present results of real-time reverse transcription polymerase chain reaction, and next-generation sequencing of DENV-3. Based on complete genome analysis, DENV-3 isolates belonged to two different clades of genotype I and were genetically close to the strains from Indonesia, China, Singapore, Malaysia and Australia. There were sixteen amino acid changes among DENV-3 isolates, and the greater number of changes was found in nonstructural than structural proteins. The emergence of DENV-3 genotype I was noted for the first time in Sri Lanka. Continuous monitoring of this newly emerged genotype and other DENV serotypes/genotypes are needed to determine their effects on future outbreaks and to understand the molecular epidemiology of dengue.
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http://dx.doi.org/10.7883/yoken.JJID.2020.854DOI Listing
February 2021

Behavioural responses and anxiety symptoms during the coronavirus disease 2019 (COVID-19) pandemic in Japan: A large scale cross-sectional study.

J Psychiatr Res 2021 04 13;136:296-305. Epub 2021 Feb 13.

Department of Epidemiology and Health Statistics, School of Public Health, Fujian Medical University, Fuzhou, 350122, Fujian Province, China; Centre for Epidemiology and Evidence-Based Practice, Department of Social and Preventive Medicine, Faculty of Medicine, University of Malaya, 50603, Kuala Lumpur, Malaysia. Electronic address:

Background: This study explored the behavioural responses and anxiety symptoms of the general adult population in Japan during the ongoing coronavirus disease 2019 (COVID-19) outbreak.

Methods: A web-based cross-sectional survey was conducted between 12th and May 13, 2020. Quota sampling was used to attain equal gender and age distributions representative of the Japanese population.

Results: A total of 4127 complete responses were analysed. Higher educational level (B = 0.045, p = 0.002) and household income (B = 0.04, p = 0.009) were associated with a higher increase in preventive measures when comparing before and after the state of emergency was declared. The highest reported social anxiety was a feeling of fear (65.6%), followed by embarrassment (43.8%), keeping infection a secret (41.3%), avoidance (41.3%), and stigma (25.5%). A total of 86.1% of the respondents reported moderate to severe anxiety. The partial least square-based structural equation modelling (PLS-SEM) revealed that being female has the greatest effect (B = 0.246, p < 0.0001) on higher current preventive measures, followed by social anxiety (B = 0.119; p = 0.001) and State-Trait Anxiety Inventory scores (B = 0.153; p < 0.001). Perceived susceptibility (B = 0.033, p = 0.020), knowing someone who have been diagnosed with COVID-19 (B = 0.097, p < 0.001), higher income (B = 0.079, p < 0.001) and educational level (B = 0.045; p = 0.004) all had a small but significant effect on influencing levels of preventive measures.

Conclusions: A moderate level of preventive practices found in this study indicates the need to encourage behavioural change to limit the spread of the coronavirus. The provision of mental health services is warranted as anxiety symptoms are prevalent.
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http://dx.doi.org/10.1016/j.jpsychires.2021.02.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7880847PMC
April 2021

5-amino levulinic acid inhibits SARS-CoV-2 infection in vitro.

Biochem Biophys Res Commun 2021 03 29;545:203-207. Epub 2021 Jan 29.

Department of Emerging Infectious Diseases, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, 852-8523, Japan; National Research Center for the Control and Prevention of Infectious Diseases, Nagasaki University, Nagasaki, 852-8521, Japan. Electronic address:

The current COVID-19 pandemic requires urgent development of effective therapeutics. 5-amino levulinic acid (5-ALA) is a naturally synthesized amino acid and has been used for multiple purposes including as an anticancer therapy and as a dietary supplement due to its high bioavailability. In this study, we demonstrated that 5-ALA treatment potently inhibited infection of SARS-CoV-2, a causative agent of COVID-19, in cell culture. The antiviral effects could be detected in both human and non-human cells, without significant cytotoxicity. Therefore, 5-ALA is worth to be further investigated as an antiviral drug candidate for COVID-19.
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http://dx.doi.org/10.1016/j.bbrc.2021.01.091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7846235PMC
March 2021

A Novel Multiplex RT-PCR Assay for Simultaneous Detection of Dengue and Chikungunya Viruses.

Int J Mol Sci 2020 Nov 5;21(21). Epub 2020 Nov 5.

Department of Integrative Biology, College of Arts and Sciences, University of South Florida, St. Petersburg, FL 33701, USA.

The goal of the study was to develop a specific, sensitive, and cost-effective molecular RT-PCR diagnostic assay for the rapid and simultaneous detection of the serotypes of dengue virus (DENV) and Chikungunya virus (CHIKV) from sera of suspected febrile patients. A single-tube, single-step multiplex RT-PCR (mRT-PCR) assay was designed for the detection of viral genomes from clinical and field samples. Specificity and sensitivity of the mRT-PCR assay were evaluated against six different combinations using two reverse transcriptases (AMV-RT and RT-Ace) and three DNA polymerases (LA-Taq, rTaq, and Tth). Among the six combinations, the AMV-RT and LA- combination was more specific and sensitive than other enzyme combinations for detecting viral genomes of DENV-1, DENV-2, DENV-3, and DENV-4 ( < 0.01), and for detecting viral genomes of CHIKV ( < 0.05). The detection limits of the mRT-PCR were 10 focus forming units (FFU) for CHIKV and 1 FFU, 20 FFU, 0.1 FFU, and 10 FFU for DENV-1, DENV-2, DENV-3, and DENV-4, respectively. The primers used for the mRT-PCR did not show any cross-reactivity among the serotypes of DENV or CHIKV. Specificity and sensitivity of the newly developed mRT-PCR were validated using serum samples collected from febrile patients during dengue outbreaks in Bangladesh. The sensitivity for serotype detection of DENV and CHIKV was superior to the virus isolation method and the antigen detection method using the Dengue NS1-Ag assay. This novel mRT-PCR method can be used for molecular epidemiological surveillance of DENV and CHIKV in epidemic and endemic countries.
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http://dx.doi.org/10.3390/ijms21218281DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7663808PMC
November 2020

Chikungunya Virus Infection in Blood Donors and Patients During Outbreak, Mandalay, Myanmar, 2019.

Emerg Infect Dis 2020 11;26(11):2741-2745

In 2019, an outbreak of chikungunya virus infection occurred in Mandalay, Myanmar, and 3.2% of blood donors and 20.5% of patients who were children were confirmed as being infected. The prevalence rate was up to 6.3% among blood donors. The East Central/South African genotype was predominantly circulating during this outbreak.
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http://dx.doi.org/10.3201/eid2611.201824DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7588511PMC
November 2020

Congenital Zika Virus Infection in a Birth Cohort in Vietnam, 2017-2018.

Am J Trop Med Hyg 2020 11;103(5):2059-2064

Department of Pediatrics, Graduate School of Biomedical Sciences, Nagasaki University, Nagasaki, Japan.

To detect congenital ZIKV infection (CZI) in a birth cohort and among high-risk neonates in Vietnam, we collected umbilical cord blood plasma samples of newly delivered babies and peripheral plasma samples of high-risk neonates in Nha Trang, central Vietnam, between July 2017 and September 2018. Samples were subjected to serological and molecular tests. Of the 2013 newly delivered babies, 21 (1%) were positive for Zika virus (ZIKV) IgM and 1,599 (79%) for IgG. Among the 21 ZIKV IgM-positives, 11 were confirmed to have CZI because their plasma samples had anti-ZIKV neutralization titers ≥ 4 times higher than those against dengue virus (DENV)-1 to 4 and Japanese encephalitis virus (JEV) and were tested for the ZIKV RNA positive by real-time reverse transcription-PCR. Therefore, the incidence of CZI in our birth cohort was approximately 0.5%. Of the 150 high-risk neonates, three (2%) and 95 (63%) were positive for ZIKV IgM and IgG antibodies, respectively. None of the three ZIKV IgM-positives had ≥ 4 times higher anti-ZIKV neutralization titers than those against DENV-1 to 4 and JEV, and were therefore considered as probable CZI. Our results indicate that CZI is not rare in Vietnam. Although those with confirmed CZI did not show apparent symptoms suspected of congenital Zika syndrome at birth, detailed examinations and follow-up studies are needed to clarify the CZI impact in Vietnam. This is the first report of CZI cases in a birth cohort in Asia.
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http://dx.doi.org/10.4269/ajtmh.20-0286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7646788PMC
November 2020

Complete genome analysis and characterization of neurotropic dengue virus 2 cosmopolitan genotype isolated from the cerebrospinal fluid of encephalitis patients.

PLoS One 2020 18;15(6):e0234508. Epub 2020 Jun 18.

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.

Dengue virus (DENV) infection remains a major public health concern in many parts of the world, including Southeast Asia and the Americas. Sri Lanka experienced its largest dengue outbreak in 2017. Neurological symptoms associated with DENV infection have increasingly been reported in both children and adults. Here, we characterize DENV type 2 (DENV-2) strains, which were isolated from cerebrospinal fluid (CSF) and/or serum of patients with dengue encephalitis. Acute serum and CSF samples from each patient were subjected to dengue-specific non-structural protein 1 (NS1) antigen test, IgM and IgG enzyme-linked immunosorbent assay (ELISA), virus isolation, conventional and real-time polymerase chain reaction (PCR), and next-generation sequencing (NGS). Among the 5 dengue encephalitis patients examined, 4 recovered and 1 died. DENV-2 strains were isolated from serum and/or CSF samples of 3 patients. The highest viral genome levels were detected in the CSF and serum of the patient who succumbed to the illness. A phylogenetic tree revealed that the DENV-2 isolates belonged to a new clade of cosmopolitan genotype and were genetically close to strains identified in China, South Korea, Singapore, Malaysia, Thailand, and the Philippines. According to the NGS analysis, greater frequencies of nonsynonymous and synonymous mutations per gene were identified in the nonstructural genes. The full genomes of serum- and CSF-derived DENV-2 from the same patient shared 99.7% similarity, indicating that the virus spread across the blood-brain barrier. This is the first report to describe neurotropic DENV-2 using whole-genome analysis and to provide the clinical, immunological, and virological characteristics of dengue encephalitis patients during a severe dengue outbreak in Sri Lanka in 2017.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0234508PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302667PMC
August 2020

A Simple and High-Throughput ELISA-Based Neutralization Assay for the Determination of Anti-Flavivirus Neutralizing Antibodies.

Vaccines (Basel) 2020 Jun 10;8(2). Epub 2020 Jun 10.

Graduate School of Biomedical Sciences, Nagasaki University, Sakamoto 1-12-4, Nagasaki 852-8523, Japan.

Mosquito-borne flavivirus infections, including dengue virus and Zika virus, are major public health threats globally. While the plaque reduction neutralization test (PRNT) is considered the gold standard for determining neutralizing antibody levels to flaviviruses, the assay is time-consuming and laborious. This study, therefore, aimed to develop an enzyme-linked immunosorbent assay (ELISA)-based microneutralization test (EMNT) for the detection of neutralizing antibodies to mosquito-borne flaviviruses. The inhibition of viral growth due to neutralizing antibodies was determined colorimetrically by using EMNT. Given the significance of Fcγ-receptors (FcγR) in antibody-mediated neutralization and antibody-dependent enhancement (ADE) of flavivirus infection, non-FcγR and FcγR-expressing cell lines were used in the EMNT to allow the detection of the sum of neutralizing and immune-enhancing antibody activity as the neutralizing titer. Using anti-flavivirus monoclonal antibodies and clinical samples, the utility of EMNT was evaluated by comparing the end-point titers of the EMNT and the PRNT. The correlation between EMNT and PRNT titers was strong, indicating that EMNT was robust and reproducible. The new EMNT assay combines the biological functional assessment of virus neutralization activity and the technical advantages of ELISA and, is simple, reliable, practical, and could be automated for high-throughput implementation in flavivirus surveillance studies and vaccine trials.
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http://dx.doi.org/10.3390/vaccines8020297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7350015PMC
June 2020

Prevalence of Zika virus neutralizing antibodies in healthy adults in Vietnam during and after the Zika virus epidemic season: a longitudinal population-based survey.

BMC Infect Dis 2020 May 11;20(1):332. Epub 2020 May 11.

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Sakamoto 1-12-4, Nagasaki, 852-8523, Japan.

Background: Between 2016 and 2019, 265 cases of Zika virus (ZIKV) infection were reported in Vietnam, predominantly in southern Vietnam. In 2016, a case of ZIKV-associated microcephaly was confirmed in the Central Highlands, and several members of the infant's family were confirmed to be infected with ZIKV. The study aims to determine the level of immunity to ZIKV in the general population of the ZIKV epidemic region.

Methods: A total of 879 serum samples were collected from 801 participants between January 2017 and July 2018, during and after the ZIKV epidemic in Vietnam. The samples were tested for anti-ZIKV immunoglobulin M (IgM) and immunoglobulin G (IgG), and anti-dengue virus (DENV) IgG antibodies using enzyme-linked immunosorbent assays (ELISA). Plaque-reduction neutralization test (PRNT) for ZIKV was performed on all samples, and for DENV on the samples that ZIKV neutralizing antibody positive.

Results: A total of 83 (10.3%) participants had anti-ZIKV IgM. Of the 83, 6 were confirmed to be ZIKV antibodies positive using PRNT and anti-ZIKV IgG ELISA. Of the 718 participants who were anti-ZIKV IgM negative, a further 3 cases were confirmed as positive for antibodies against ZIKV. Of the 9 participants with ZIKV infection, 5 lived in the same village as the infant with ZIKV-associated microcephaly and the other 4 lived in 2 neighboring communes. Repeat samples were collected from the 83 ZIKV IgM positive participants 1.5 years after the first collection. No new cases of ZIKV infection were detected. In addition, 2 of 3 participants with anti-ZIKV NS1 IgG demonstrated a 4- to 8-fold increase in ZIKV neutralizing antibody titer.

Conclusions: ZIKV was present in the area around Krong Buk, with the rate of ZIKV-specific antibodies was 1.1% in the community since at least 2016. While the low levels of circulation together with low seroprevalence suggests a limited outbreak in the region, the results also reflect on low levels of protective immunity to Zika within the population. These results provide a better understanding of the current ZIKV epidemic status in the region and demonstrate a need for implementation of more effective ZIKV infection control measures.
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http://dx.doi.org/10.1186/s12879-020-05042-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7216417PMC
May 2020

Detection of viral RNA in diverse body fluids in an SFTS patient with encephalopathy, gastrointestinal bleeding and pneumonia: a case report and literature review.

BMC Infect Dis 2020 Apr 15;20(1):281. Epub 2020 Apr 15.

Department of Respiratory Medicine, Nagasaki University Hospital, Nagasaki, Japan.

Background: Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease that commonly has a lethal course caused by the tick-borne Huaiyangshan banyang virus [former SFTS virus (SFTSV)]. The viral load in various body fluids in SFTS patients and the best infection control measure for SFTS patients have not been fully established.

Case Presentation: A 79-year-old man was bitten by a tick while working in the bamboo grove in Nagasaki Prefecture in the southwest part of Japan. Due to the occurrence of impaired consciousness, he was referred to Nagasaki University Hospital for treatment. The serum sample tested positive for SFTSV-RNA in the genome amplification assay, and he was diagnosed with SFTS. Furthermore, SFTSV-RNA was detected from the tick that had bitten the patient. He was treated with multimodal therapy, including platelet transfusion, antimicrobials, antifungals, steroids, and continuous hemodiafiltration. His respiration was assisted with mechanical ventilation. On day 5, taking the day on which he was hospitalized as day 0, serum SFTSV-RNA levels reached a peak and then decreased. However, the cerebrospinal fluid collected on day 13 was positive for SFTSV-RNA. In addition, although serum SFTSV-RNA levels decreased below the detectable level on day 16, he was diagnosed with pneumonia with computed tomography. SFTSV-RNA was detected in the bronchoalveolar lavage fluid on day 21. By day 31, he recovered consciousness completely. The pneumonia improved by day 51, but SFTSV-RNA in the sputum remained positive for approximately 4 months after disease onset. Strict countermeasures against droplet/contact infection were continuously conducted.

Conclusions: Even when SFTSV genome levels become undetectable in the serum of SFTS patients in the convalescent phase, the virus genome remains in body fluids and tissues. It may be possible that body fluids such as respiratory excretions become a source of infection to others; thus, careful infection control management is needed.
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http://dx.doi.org/10.1186/s12879-020-05012-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160946PMC
April 2020

Severe Acute Respiratory Syndrome Coronavirus 2 Shedding by Travelers, Vietnam, 2020.

Emerg Infect Dis 2020 07 21;26(7):1624-1626. Epub 2020 Jun 21.

We analyzed 2 clusters of 12 patients in Vietnam with severe acute respiratory syndrome coronavirus 2 infection during January-February 2020. Analysis indicated virus transmission from a traveler from China. One asymptomatic patient demonstrated virus shedding, indicating potential virus transmission in the absence of clinical signs and symptoms.
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http://dx.doi.org/10.3201/eid2607.200591DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7323563PMC
July 2020

An Epidemic of Dengue Virus Serotype-4 during the 2015 - 2017: the Emergence of a Novel Genotype IIa of DENV-4 in the Philippines.

Jpn J Infect Dis 2020 ;73(2):176

Department of Virology, Institute of Tropical Medicine (NEKKEN).

Volume 72 no 6, p.413-419, 2019. Page 418, Acknowledgments "We would like to thank all staff and members of the Department of Virology, NEKKEN, Nagasaki University, Japan for providing technical support and advice. Our special thanks to the staff of the Pavilion II and the Central Laboratory of San Lazaro Hospital for their kind assistance during patient recruitment and data collection. We are also very grateful for the support of the Senior Vice President and Head of Research and Biotechnology (R&B) Group of St. Luke's Medical Center, Dr. Isaac David E. Ampil II. Finally, our sincere thanks to the members of R&B's dengue research group for kindly preparing the samples to be transported to NEKKEN." should read "This research was supported by grants from the Japan Agency for Medical Research and Development (AMED) under Grant Number JP18fm0108001, JP19fm0108001 (Japan Initiative for Global Research Network on Infectious Diseases (J-GRID)), AMED Research on Emerging and Re-emerging Infectious Diseases (19fk0108035j0003) and e-ASIA Joint Research Program and; Philippine Council for Health Research and Development (PCHRD) of the Department of Science and Technology (DOST), Philippines, with partial support from the Research and Biotechnology of St. Luke's Medical Center (R&B-SLMC), Philippines (Project No. 07-024). Funders have no role in the study design, data collection, and interpretation, or the decision to submit this work for publication. We would like to thank all staff and members of the Department of Virology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Japan, for providing technical support and advice. Our special thanks to the staff of the Pavilion II and the Central Laboratory of San Lazaro Hospital for their kind assistance during patient recruitment and data collection. We are also very grateful for the support of the Senior Vice President and Head of Research and Biotechnology (R&B) Group of St. Luke's Medical Center, Dr. Isaac David E. Ampil II. Finally, our sincere thanks to the members of R&B's Dengue Research Group for kindly preparing the samples to be transported to NEKKEN."
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http://dx.doi.org/10.7883/yoken.JJID.2020.E001DOI Listing
October 2020

Clinical, Virological, and Cytokine Profiles of Children Infected with Dengue Virus during the Outbreak in Southern Vietnam in 2017.

Am J Trop Med Hyg 2020 06;102(6):1217-1225

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan.

Dengue virus (DENV) infection is a major cause of morbidity and mortality in Vietnam, and the incidence is higher and more consistent in the southern part of the country. This study investigated the circulation of DENV serotypes, viremia levels, immunological status, and cytokine levels, with disease severities among children infected in 2017 in Ho Chi Minh City, Southern Vietnam. Acute and convalescent serum samples were collected from clinically diagnosed dengue children. They were confirmed to have DENV infection by NS1 antigen, IgM and IgG ELISAs, virus isolation, and conventional and real-time RT-PCR. Measurement of 10 cytokine levels was performed in the serum samples. All the children were dengue IgM positive; 28% and 72% of them had primary and secondary DENV infections, respectively, whereas 54% of those with secondary infection were children with dengue with warning signs and with severe dengue. Any or mixed infection of the four serotypes of DENV RNA was detected in 58 children. Twenty DENV strains (DENV-1 = 16 and DENV-4 = 4) were isolated. Levels of IFN-γ, TNF-α, MCP-1, IL-10, and IL-6 were significantly higher in severe dengue cases. We report the predominance of DENV-1 over other serotypes in the 2017 dengue outbreak in Southern Vietnam. Our data showed that cytokine expressions were correlated with dengue pathogenesis and may help in identifying an effective therapeutic strategy.
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http://dx.doi.org/10.4269/ajtmh.19-0607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7253104PMC
June 2020

Complete Genome Sequence of a 2019 Novel Coronavirus (SARS-CoV-2) Strain Isolated in Nepal.

Microbiol Resour Announc 2020 Mar 12;9(11). Epub 2020 Mar 12.

School of Public Health, LKS Faculty of Medicine, The University of Hong Kong, Hong Kong Special Administrative Region, China.

A complete genome sequence was obtained for a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) strain isolated from an oropharyngeal swab specimen of a Nepalese patient with coronavirus disease 2019 (COVID-19), who had returned to Nepal after traveling to Wuhan, China.
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http://dx.doi.org/10.1128/MRA.00169-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7067954PMC
March 2020

Unusual, neurological and severe dengue manifestations during the outbreak in Sri Lanka, 2017.

J Clin Virol 2020 04 27;125:104304. Epub 2020 Feb 27.

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Japan.

Background: Sri Lanka experienced its largest dengue outbreak in 2017 with more than 185,000 dengue cases including at least 250 fatalities.

Objectives: Our study aimed to characterize the clinical, immunological and virological features of confirmed dengue patients in Sri Lanka during the outbreak in 2017 when unusual manifestations of severe dengue were observed.

Study Design: Sera from 295 patients who were admitted to Teaching Hospital Kandy, Kandy, Sri Lanka between March 2017- January 2018 were subjected to NS1 antigen, IgM and IgG ELISAs, virus isolation, conventional and real time RT-PCR and next generation sequencing.

Results: Primary and secondary infections were detected in 48.5 % and 51.5 % of the study population, respectively. Two hundred twenty five DENV strains were isolated (219 DENV-2, one DENV-3, two DENV-4, two mixed infections of DENV-2 and -3 and one mixed infection of DENV-2 and -4). Unusual and severe manifestations such as encephalitis, encephalopathy, liver failure, kidney failure, myocarditis, Guillain-Barré syndrome and multi-organ failure were noted in 44 dengue patients with 11 deaths. The viraemia levels in patients with primary infection and unusual manifestations were significantly higher compared to those in patients with secondary infection. A new clade of DENV-2 Cosmopolitan genotype strains was observed with the strains closely related to those from China, Malaysia, Indonesia, Singapore and Taiwan.

Conclusions: The new clade of DENV-2 cosmopolitan genotype observed in Sri Lanka in 2017 caused an unprecedented, severe dengue outbreak. The emergence of DENV-3 and DENV-4 in the 2017 outbreak might cause future outbreaks in Sri Lanka.
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http://dx.doi.org/10.1016/j.jcv.2020.104304DOI Listing
April 2020

Zika virus infection in asymptomatic persons in Myanmar, 2018.

Trans R Soc Trop Med Hyg 2020 06;114(6):440-447

Department of Virology, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki, Japan, PO 852-8523.

Background: Zika virus (ZIKV) is a mosquito-borne flavivirus. Outbreaks of ZIKV infection have occurred in Africa, Southeast Asia, the Pacific Islands, the Americas and the Caribbean. Although most ZIKV infections are asymptomatic, cases of neurological manifestations have been described. The aim of the present study was to identify the prevalence of ZIKV infection among the asymptomatic persons in Myanmar in 2018.

Methods: A total of 284 serum samples from apparently healthy persons were collected from Yangon, Myanmar in 2018. They were analysed for ZIKV infection by immunoglobulin M (IgM) capture enzyme-linked immunosorbent assay (ELISA), IgG indirect ELISA, 50% focus reduction neutralization test, real-time reverse transcription polymerase chain reaction (RT-PCR) and conventional RT-PCR.

Results: Of the 284 apparently healthy persons, 31.3% were positive for the presence of IgM against ZIKV and 94.3% were positive for anti-flavivirus IgG. Among the ZIKV IgM-positive samples, we confirmed ZIKV infection in 15.8% of asymptomatic persons by neutralization test and real-time RT-PCR.

Conclusions: We conclude that ZIKV infection was increasing among asymptomatic persons in the same area in Myanmar during 2018 compared with 2017. It is highly recommended to strengthen the surveillance system for ZIKV to prevent possible outbreaks.
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http://dx.doi.org/10.1093/trstmh/trz134DOI Listing
June 2020

Pathogenetic Potential Relating to Metabolic Activity in a Mouse Model of Infection with the Chikungunya Virus East/Central/South African Genotype.

Viruses 2020 02 3;12(2). Epub 2020 Feb 3.

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan.

Epidemics of the Chikungunya virus (CHIKV) from 2004 onwards were caused by the East/Central/South African (ECSA) genotype. However, the pathogenesis of the genotype infection has not been fully explained. In this study, we examined the pathogenic potential of CHIKV ECSA genotype M-30 (M-30) by comparing it with that of African genotype S-27 (S-27) in mice. Following low titer infections in type-I IFN receptor KO (A129) mice, we found that the M-30 infection caused high and acute fatality compared with the S-27 infection. M-30-infected A129 mice showed higher viral loads in their central nervous systems and peripheral organs, and increased levels of IFN-γ responses in their brains. Interestingly, M-30-infected mice did not show the hypophagia and reductions in weight which were observed in S-27-infected mice. Our observations provide a novel explanation of the pathogenic mechanisms attributed to virus proliferation, anti-type-II IFN response and metabolic activity in the CHIKV ECSA virus in mice.
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http://dx.doi.org/10.3390/v12020169DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077324PMC
February 2020

Long-term surveillance needed to detect Zika virus outbreaks in endemic regions.

Lancet Infect Dis 2020 02;20(2):168-169

WHO Collaborating Centre for Reference and Research on Tropical and Emerging Virus Diseases, Institute of Tropical Medicine, Nagasaki University, Nagasaki 852-8523, Japan. Electronic address:

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http://dx.doi.org/10.1016/S1473-3099(19)30677-2DOI Listing
February 2020

Seroprevalence of yellow fever, dengue, West Nile and chikungunya viruses in children in Teso South Sub-County, Western Kenya.

Int J Infect Dis 2020 Feb 8;91:104-110. Epub 2019 Nov 8.

Centre for Infectious and Parasitic Diseases Control Research (CIPDCR), Kenya Medical Research Institute, Busia, Kenya.

Background: Arboviruses often cause widespread morbidity in children in endemic regions. Data on the burden of arboviruses in Kenyan children are limited.

Objectives: This study was performed to determine the seroprevalence of yellow fever (YFV), dengue (DENV), West Nile (WNV), and chikungunya (CHIKV) viruses among children 1-12 years of age at two health facilities in Teso South Sub-County in Western Kenya.

Methods: In a hospital-based cross-sectional survey, a questionnaire was used to collect socio-demographic information. Serum drawn from the children was tested for IgA/IgM/IgG serocomplex antibodies to selected arboviruses using indirect ELISA and plaque reduction neutralization tests.

Results: A total of 182 (27.7%) of the 656 participants tested were positive for any arbovirus antibody. Of these, 4.4% (29/656) tested positive for YFV, 9.6% (62/649) for WNV, 5.6% (36/649) for CHIKV, 1.4% (5/368) for DENV1, 9% (59/656) for DENV2, and 19.7% (40/203) for DENV3. Neutralizing antibodies to CHIKV were found in 77.8% (42/54) of participants, to YFV in 15.8% (3/19), to DENV2 in 58% (29/50), and to WNV in 8% (1/55). Sex, age, urban residence, schooling, and lack of vaccination were associated with arbovirus exposure.

Conclusions: This study confirmed that children under 12 years of age in Teso South Sub-County are exposed to ongoing arbovirus infections early in life.
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http://dx.doi.org/10.1016/j.ijid.2019.11.004DOI Listing
February 2020

The old pharmaceutical oleoresin labdanum of Cistus creticus L. exerts pronounced in vitro anti-dengue virus activity.

J Ethnopharmacol 2020 Jul 26;257:112316. Epub 2019 Oct 26.

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Japan.

Ethnopharmacological Relevance: The haemorrhagic dengue fever affects up to 500 million patients, annually causing 20.000 deaths, with no chemotherapeutic agent available. The oleoresin labdanum of Cistus creticus L. has been established as an anti-infective agent since antiquity in Mediterranean ethnopharmacology.

Materials And Methods: We tested several extracts and fractions of labdanum - standardised on labdane-type diterpenes via GC-MS - on their activity against the dengue virus (DENV-2 strain 00st-22A) in in vitro Vero cell cultures (96-well-plates, 5 days). Preliminary experiments with a labdanum diethyl ether raw-extract did not yield measureable results due to cytotoxic effects against Vero cells. In all following experiments, cell viability was constantly checked using the MTT-test. Fractionation of this raw-extract by liquid-liquid-extraction and column-chromatography on silica-gel (gradient elution with hexane, EtOAc, CHCl, MeOH) succeeded in separating the anti-viral activity of labdanum from its cytotoxic effect.

Results: In the most active fraction GS5 at 30 μg/ml, dengue virus proliferation was 100% suppressed and cell viability was over 90%. Structural elucidation of major constituents of GS5 is currently ongoing, but thin-layer chromatography showed that this fraction is mainly dominated by manoyloxides, a class of labdane-type diterpenes with known antimicrobial activity. Claims concerning the antiviral activity of above ground parts of C. creticus have been made previously, but these generally ascribe this activity to hot water soluble polyphenols and propose an unspecific tanning effect of the viral surface proteins as the mechanism of action. However, the water soluble fraction enhanced viral proliferation.

Conclusion: We therefore describe a direct, pharmacological, antiviral activity of a diethyl ether extract of labdanum against a virulent haemorrhagic fever like dengue for the first time.
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http://dx.doi.org/10.1016/j.jep.2019.112316DOI Listing
July 2020

Development of Universal and Lineage-Specific Primer Sets for Rapid Detection of the Zika Virus (ZIKV) in Blood and Urine Samples Using One-Step Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP).

Jpn J Infect Dis 2020 Mar 31;73(2):153-156. Epub 2019 Oct 31.

Vietnam Research station, Center for Infectious Disease Research in Asia and Africa, Institute of Tropical Medicine, Nagasaki University.

Zika is a mosquito-borne disease that has been posing a significant threat to public health in recent years. The Zika virus (ZIKV), the causative agent of this disease, is classified into 2 distinct genetic lineages, namely Asian and African. While molecular nucleic acid analysis methods have been shown to be useful for the diagnosis of ZIKV infection, the development of assays based on one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) offers several advantages, such as shorter incubation times, ease of handling, and rapid detection. In this study, a universal LAMP primer set was developed to target conserved sequences of known ZIKV lineages. Additionally, the Af7462 and As1788 primer sets were designed based on LAMP-based single-nucleotide polymorphism (SNPs) typing for the specific detection of the African and Asian lineages. The developed RT-LAMP assays could specifically detect the African and Asian lineages of ZIKV, with a detection limit ranging from 0.17 FFU/mL to 2.3×10 FFU/mL. As ZIKV viremia ranges between 10 to 10 PFU/mL or 10 to 10 copies/mL, the data indicate that the viremia range of clinical samples is within the detection range of our assay. Due to the high specificity and sensitivity, as well as the ease of use of our assay, it could potentially be used for early clinical diagnosis applications.
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http://dx.doi.org/10.7883/yoken.JJID.2019.073DOI Listing
March 2020

Inapparent dengue virus infection among students in Mandalay, Myanmar.

Trans R Soc Trop Med Hyg 2020 01;114(1):57-61

Department of Medical Research, Pyin Oo Lwin Branch, Ward No (16), Near Anisakhan Airport, Pyin Oo Lwin, Mandalay Region, Myanmar, PO-05062.

Background: A school- and laboratory-based cross-sectional descriptive study was conducted to find out the burden of inapparent dengue virus (DENV) infection in Mandalay where DENV is endemic and there is circulation of all four DENV serotypes.

Methods: A total of 420 students who had no history of fever and visited the hospital within 6 months were recruited from three monastic schools. Serum samples were collected and the DENV genome was checked by conventional one-step RT-PCR and anti-DENV IgM and IgG antibodies were determined. Inapparent dengue (DEN) infection is defined as individuals who were either RT-PCR-positive or anti-DENV IgM-positive with no clinical manifestations or mild symptoms, and which are not linked to a visit to a healthcare provider.

Results: Among 420 students, 38 students (9.0%, 95% CI, 6.4 to 12.2) were confirmed as recent inapparent DEN infection. The DENV serotype-1 was detected in six students. Thirty-one out of 38 (81.6%) laboratory-confirmed inapparent DEN-infected students had primary infections and seven (18.4%) had secondary infections.

Conclusion: This study explored the prevalence of inapparent DEN infection rate in urban monastic schools in Mandalay and showed that the rate of primary infection among inapparent DENV-infected children was high.
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http://dx.doi.org/10.1093/trstmh/trz071DOI Listing
January 2020

Serial analysis of cytokine and chemokine profiles and viral load in severe fever with thrombocytopenia syndrome: Case report and review of literature.

Medicine (Baltimore) 2019 Oct;98(42):e17571

Department of Immunology and Rheumatology, Graduate School of Biomedical Sciences, Nagasaki University, Sakamoto, Nagasaki.

Rationale: Severe fever with thrombocytopenia syndrome (SFTS) is a recently recognized fatal infectious disease caused by the SFTS virus, and severe cases are complicated by the presence of hemophagocytic lymphohistiocytosis (HLH) associated with a cytokine storm. Herein, we report on serial changes of serum cytokine levels and viral load in a severe case of SFTS.

Patient Concerns: A 63-year-old Japanese woman presented with high-grade fever, abdominal pain, diarrhea, impaired consciousness, leukocytopenia, and thrombocytopenia.

Diagnosis: SFTS was diagnosed based on a positive serum test for SFTS virus RNA and electroencephalogram (EEG) findings of encephalopathy.

Interventions: The patient was treated with supportive therapy, including steroid pulse therapy (intravenous methylprednisolone 1 g/d for 3 days) for HLH and intravenous recombinant thrombomodulin 19200 U/d for 7 days for disseminated intravascular coagulation.

Outcomes: Treatment for 7 days improved both symptoms and abnormal EEG findings, and SFTS virus RNA disappeared from the serum at day 10 from the onset of symptoms. The serum cytokines and chemokines analysis during the clinical course revealed 2 distinct phases: the acute phase and the recovery phase. The cytokines and chemokines elevated in the acute phase included interleukin (IL)-6, IL-10, interferon (IFN)-α2, IFN-γ, tumor necrosis factor-α, interferon-γ-induced protein-10, and fractalkine, while the IL-1β, IL-12p40, IL-17, and vascular endothelial growth factor levels were higher in the recovery phase.

Conclusion: These observations suggest that the cytokines and chemokines elevated in the acute phase may reflect the disease severity resulted in a cytokine storm, while those in the recovery phase may be attributed to T-cell activation and differentiation.
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http://dx.doi.org/10.1097/MD.0000000000017571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6824633PMC
October 2019

Severe Fever with Thrombocytopenia Syndrome Virus RNA in Semen, Japan.

Emerg Infect Dis 2019 11;25(11):2127-2128

Severe fever with thrombocytopenia syndrome virus (SFTSV) can be transmitted between humans. We describe a case of severe fever with thrombocytopenia syndrome in which SFTSV RNA was detected in semen after its disappearance from serum. Our findings indicate possible sexual transmission of this emerging virus.
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http://dx.doi.org/10.3201/eid2511.190061DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6810197PMC
November 2019

An Epidemic of Dengue Virus Serotype-4 during the 2015 - 2017: the Emergence of a Novel Genotype IIa of DENV-4 in the Philippines.

Jpn J Infect Dis 2019 Nov 30;72(6):413-419. Epub 2019 Aug 30.

Department of Virology, Institute of Tropical Medicine (NEKKEN).

Dengue remains a major public health problem in the Philippines. In this study, we determined the circulating dengue serotypes in the Philippines during the 2015-2017 outbreaks using a total of 678 serum samples from 537 individual dengue patients. Following an increase in the number of DENV-4 patients in recent years, we conducted a comprehensive molecular and epidemiology analysis on the DENV-4 strains isolated recently in the Philippines. Two genotypes of DENV-4 have been isolated in the Philippines since 1956: GI and GIIa. The GIIa DENV strains that were isolated in the present study were closely related to a distinct group of GIIa strains that were isolated from the Philippines in 2004. A majority of the isolates of this sub-group have been identified in the Philippines, suggesting that this lineage may have been introduced in the Philippines, and evolved to form the distinct sub-group within GIIa strains. The increase in DENV-4 activity also coincided with the appearance of the GIIa subgroup and the phasing-out of the GI lineage in the Philippines. Overall, our study demonstrates a shift in DENV-4 genotype and epidemic dynamics in a hyperendemic region, suggesting the importance of DENV genetic evolution in establishing and sustaining transmission.
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http://dx.doi.org/10.7883/yoken.JJID.2019.208DOI Listing
November 2019

Evaluation of commercially available three dengue rapid diagnostic test kits for diagnosis of acute dengue virus infection at the point-of-care setting in Myanmar.

J Virol Methods 2019 11 19;273:113724. Epub 2019 Aug 19.

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Japan.

Early and accurate diagnosis of dengue virus (DENV) infection is very important and Rapid Diagnostic Test (RDT) Kits are been used as a point-of-care test to check DENV infection. A Hospital and Laboratory-based descriptive study was conducted at 550-bedded Mandalay Children Hospital in 2018. Acute-phase serum samples were collected from 202 dengue suspected patients to evaluate the efficacy of RDT Kits for the diagnosis of DENV infection. Commercially available three test kits which include: ((i) CareUs Dengue Combo, Korea, (ii) Humasis Dengue Combo, Korea and (iii) Wondfo Dengue Combo, China) were validated against WHO-based reference standard tests. 140/202 patients (69.3%) was confirmed to have DENV infection. All four serotypes of dengue viruses (57 DENV-1, 7 DENV-2, 6 DENV-3 and 10 DENV-4) were identified from 80 dengue confirmed patients and DENV-1 was the dominant serotype. Combining the NS-1 antigen and IgM antibody results from the CareUs Dengue Combo Kit gave the best sensitivity (92.1%, 95% CI 86.4%-96.0%) and specificity (75.8%, 95%CI 63.3%-85.8%). Among the three RDT Kits, the performance of CareUS Kit was better than the other two. This study explored the evidence of the usefulness of RDT Kits at the point-of-care setting for diagnosis of acute dengue infection.
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http://dx.doi.org/10.1016/j.jviromet.2019.113724DOI Listing
November 2019

Characterization of Vibrio cholerae O1 isolates responsible for cholera outbreaks in Kenya between 1975 and 2017.

Microbiol Immunol 2019 Sep 12;63(9):350-358. Epub 2019 Aug 12.

Department of Bacteriology, NUITM-KEMRI Project, Kenya Research Station, Institute of Tropical Medicine, Nagasaki University, Nairobi, Kenya.

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975-1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.
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http://dx.doi.org/10.1111/1348-0421.12731DOI Listing
September 2019