Publications by authors named "Takeshi Nabeshima"

32 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

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

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

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

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

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

Japanese Encephalitis- and Dengue-Associated Acute Encephalitis Syndrome Cases in Myanmar.

Am J Trop Med Hyg 2019 03;100(3):643-646

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

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 laboratory-confirmed 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.
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http://dx.doi.org/10.4269/ajtmh.18-0530DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6402905PMC
March 2019

Mosquito arbovirus survey in selected areas of Kenya: detection of insect-specific virus.

Trop Med Health 2018 4;46:19. Epub 2018 Jun 4.

1Department of Vector Ecology and Environment, Institute of Tropical Medicine, Nagasaki University, 1-12-4 Sakamoto, Nagasaki, 852-8523 Japan.

Background: Many arboviral outbreaks have occurred in various locations in Kenya. Entomological surveys are suitable methods for revealing information about circulating arboviruses before human outbreaks are recognized. Therefore, mosquitoes were collected in Kenya to determine the distribution of arboviruses.

Methods: Various species of mosquitoes were sampled from January to July 2012 using several collection methods. Mosquito homogenates were directly tested by reverse transcription-polymerase chain reaction (RT-PCR) using various arbovirus-targeted primer pairs.

Results: We collected 12,569 mosquitoes. Although no human-related arboviruses were detected, Culex flavivirus (CxFV), an insect-specific arbovirus, was detected in 54 pools of 324 individuals collected during the rainy season. Of these 54 positive pools, 96.3% (52/54) of the mosquitoes were collected in Busia, on the border of western Kenya and Uganda. The remaining two CxFV-positive pools were collected in Mombasa and Kakamega, far from Busia. Phylogenetic analysis revealed minimal genetic diversity among the CxFVs collected in Mombasa, Kakamega, and Busia, even though these cities are in geographically different regions. Additionally, CxFV was detected in one mosquito pool collected in Mombasa during the dry season. In addition to mosquitoes, () and mosquitoes were also positive for the genus. Cell fusing agent virus was detected in one pool of . Mosquito flavivirus was detected in three pools of s.l collected in the dry and rainy seasons.

Conclusions: Although no mosquitoes were positive for human-related arbovirus, insect-specific viruses were detected in various species of mosquitoes. The heterogeneity observed in the number of CxFVs in mosquitoes in different locations in Kenya suggests that the abundance of human-related viruses might differ depending on the abundance of insect-specific viruses. We may have underestimated the circulation of any human-related arbovirus in Kenya, and the collection of larger samples may allow for determination of the presence of human-related arboviruses.
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http://dx.doi.org/10.1186/s41182-018-0095-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5987586PMC
June 2018

A single amino acid substitution in the NS4B protein of Dengue virus confers enhanced virus growth and fitness in human cells in vitro through IFN-dependent host response.

J Gen Virol 2018 08 19;99(8):1044-1057. Epub 2018 Jun 19.

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

Dengue virus (DENV) replication between mosquito and human hosts is hypothesized to be associated with viral determinants that interact in a differential manner between hosts. However, the understanding of inter-host viral determinants that drive DENV replication and growth between hosts is limited. Through the use of clinical isolates, we identified an amino acid variation of Ala, Met and Val at position 116 of DENV-1 NS4B. While the proportion of virus with the NS4B-116V variant remained constantly high in serial passages in a mosquito cell line, populations of the NS4B-116M and NS4B-116A variants became dominant after serial passages in mammalian cell lines. Using recombinant DENV-1 viruses, the Val to Ala or Met alteration at position NS4B-116 (rDENV-1-NS4B-116A and rDENV-1-NS4B-116M) resulted in enhanced virus growth in human cells in comparison to the clone with Val at NS4B-116 (rDENV-1-NS4B-116V). However, the reverse phenomenon was observed in a mosquito cell line. Additionally, in a human cell line, differential levels of IFN-α/β and IFN-stimulated gene expressions (IFIT3, IFI44L, OAS1) suggested that the enhanced viral growth was dependent on the ability of the NS4B protein to hamper host IFN response during the early phase of infection. Overall, we identified a novel and critical viral determinant at the pTMD3 of NS4B region that displayed differential effects on DENV replication and fitness in human and mosquito cell lines. Taken together, the results suggest the importance of the NS4B protein in virus replication and adaptation between hosts.
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http://dx.doi.org/10.1099/jgv.0.001092DOI Listing
August 2018

Isolation and genomic characterization of Culex flaviviruses from mosquitoes in Myanmar.

Virus Res 2018 03 4;247:120-124. Epub 2018 Feb 4.

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

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 illustrated that CxFV was circulating among the vectors of human pathogenic arboviruses in Myanmar but the impact of CxFV on other flaviviruses which are endemic in the study area still remains to be explored.
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http://dx.doi.org/10.1016/j.virusres.2018.01.007DOI Listing
March 2018

Pathogenic potential and growth kinetics of Muko virus in mice and human-derived cells.

Trop Med Health 2016 11;44:31. Epub 2016 Oct 11.

Department of Virology, Institute of Tropical Medicine (NEKKEN), 1-12-4 Sakamoto, Nagasaki, 852-8523 Japan ; Leading Graduate School Program, Nagasaki University, 1-12-4 Sakamoto, Nagasaki, 852-8523 Japan.

Background: Ticks have been long known as vectors of various pathogens, some of which can cause high fatality rates among infected individuals. Our enhanced tick surveillance around Nagasaki, Japan, led to the isolation and identification of a new strain of a recently identified , Muko virus (MUV). The orbiviruses have a wide host range, including humans, and is related to a spectrum of clinical outcomes. However, the zoonotic potential of some members of the genus, although reported, were not clearly elucidated. Hence, it is imperative to characterize newly isolated orbiviruses and investigate its ability to endanger public health.

Methods: In this study, we explored the in vivo pathogenicity of a newly isolated MUV strain (MUV-Hay) using a mouse model and demonstrated its growth kinetics in human-derived cells.

Results: Our results showed the ability of MUV-Hay to propagate in human neuronal and renal cells with some cytopathic effect. Furthermore, intracerebral inoculation of our new isolate caused high mortality in adult A129 mice.

Conclusion: Our study provided a first step to experimentally test the hypothesis, that MUV can replicate and produce cytopathic effect in human cells and demonstrate virulence in adult mice.
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http://dx.doi.org/10.1186/s41182-016-0032-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5057483PMC
October 2016

Molecular and serological epidemiology of Japanese encephalitis virus (JEV) in a remote island of western Japan: an implication of JEV migration over the East China Sea.

Trop Med Health 2016 3;44. Epub 2016 May 3.

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

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.
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http://dx.doi.org/10.1186/s41182-016-0010-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4940834PMC
July 2016

Characterization of the 2013 dengue epidemic in Myanmar with dengue virus 1 as the dominant serotype.

Infect Genet Evol 2016 09 4;43:31-7. Epub 2016 May 4.

Department of Virology, Institute of Tropical Medicine, Leading Graduate School Program, Nagasaki University, Japan. Electronic address:

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.
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http://dx.doi.org/10.1016/j.meegid.2016.04.025DOI Listing
September 2016

Tofla virus: A newly identified Nairovirus of the Crimean-Congo hemorrhagic fever group isolated from ticks in Japan.

Sci Rep 2016 Feb 11;6:20213. Epub 2016 Feb 11.

Department of Virology, 1-12-4 Sakamoto, Nagasaki, 852-8523, Japan.

Ixodid ticks transmit several important viral pathogens. We isolated a new virus (Tofla virus: TFLV) from Heamaphysalis flava and Heamaphysalis formsensis in Japan. The full-genome sequences revealed that TFLV belonged to the genus Nairovirus, family Bunyaviridae. Phylogenetic analyses and neutralization tests suggested that TFLV is closely related to the Hazara virus and that it is classified into the Crimean-Congo hemorrhagic fever group. TFLV caused lethal infection in IFNAR KO mice. The TFLV-infected mice exhibited a gastrointestinal disorder, and positron emission tomography-computed tomography images showed a significant uptake of (18)F-fluorodeoxyglucose in the intestinal tract. TFLV was able to infect and propagate in cultured cells of African green monkey-derived Vero E6 cells and human-derived SK-N-SH, T98-G and HEK-293 cells. Although TFLV infections in humans and animals are currently unknown, our findings may provide clues to understand the potential infectivity and to develop of pre-emptive countermeasures against this new tick-borne Nairovirus.
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http://dx.doi.org/10.1038/srep20213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4809068PMC
February 2016

Isolation of dengue serotype 3 virus from the cerebrospinal fluid of an encephalitis patient in Hai Phong, Vietnam in 2013.

J Clin Virol 2015 Sep 17;70:93-96. Epub 2015 Jul 17.

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan; Center of International Collaborative Research, Nagasaki University, Nagasaki, Japan. Electronic address:

Dengue encephalitis (DE) is characterized as unusual presentation of dengue infection. Despite the reports that DE accounts for only 1-5% of dengue cases, this disease tends to be increasingly reported to threaten global human health throughout dengue endemic areas particularly in Southeast Asia. The molecular information of clinically characterized, neurotropic dengue virus (DENV) in human beings is extremely scarce despite it playing an important role in deciphering the pathogenesis of dengue-related neurological cases. Here we report a case of DE caused by DENV3 genotype III in a male patient with atypical symptoms of DENV infection in Hai Phong, Vietnam in 2013. The virus isolated from the cerebrospinal fluid of this case-patient was closely related to DENV3 genotype III strains isolated from serum of two other patients, who manifested classical dengue in the same year and residing in the same area as the case-patient. It is noteworthy to mention that in 2013, DENV3 genotype III was detected for the first time in Vietnam.
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http://dx.doi.org/10.1016/j.jcv.2015.07.295DOI Listing
September 2015

A Dengue virus serotype 4-dominated outbreak in central Vietnam, 2013.

J Clin Virol 2015 May 26;66:24-6. Epub 2015 Feb 26.

Department of Virology, Institute of Tropical Medicine, Nagasaki University, Nagasaki, Japan; NIHE-Nagasaki Friendship Laboratory, Nagasaki University, Hanoi, Viet Nam. Electronic address:

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http://dx.doi.org/10.1016/j.jcv.2015.02.016DOI Listing
May 2015

Molecular Epidemiology of Dengue Viruses Co-circulating in Upper Myanmar in 2006.

Trop Med Health 2015 Mar 15;43(1):21-7. Epub 2014 Nov 15.

Department of Virology, Institute of Tropical Medicine, Nagasaki University , Japan ; Global COE Program, 21st Century COE Program, MEXT , Tokyo, Japan.

To understand the molecular epidemiology of circulating dengue viruses (DENV) in Upper Myanmar, DENV isolation was attempted by inoculating the sera of a panel of 110 serum samples onto a C6/36 mosquito cell line. The samples were collected from dengue (DEN) patients admitted at Mandalay Children's Hospital in 2006. Infected culture fluids were subjected to a RT-PCR to detect the DENV genome. Three DENV strains were isolated. This was the first DENV isolation performed either in Mandalay or in Upper Myanmar. One strain belonged to DENV serotype-3 (DENV-3), and two other strains belonged to DENV serotype-4 (DEN-4). The sequence data for the envelope gene of these strains were used in a phylogenetic comparison of DENV-3 and DENV-4 from various countries. Phylogenetic analyses revealed that this DENV-3 strain was clustered within genotype II, and the two DENV-4 strains were clustered within genotype I in each serotype. The Myanmar strains were closely related to strains from the neighboring countries of Thailand and Bangladesh. These results are important for elucidating the trends of recent and future DEN outbreaks in Myanmar.
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http://dx.doi.org/10.2149/tmh.2014-27DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4361346PMC
March 2015

The dengue virus conceals double-stranded RNA in the intracellular membrane to escape from an interferon response.

Sci Rep 2014 Dec 10;4:7395. Epub 2014 Dec 10.

Department of Virology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan.

The dengue virus (DENV) circulates between humans and mosquitoes and requires no other mammals or birds for its maintenance in nature. The virus is well-adapted to humans, as reflected by high-level viraemia in patients. To investigate its high adaptability, the DENV induction of host type-I interferon (IFN) was assessed in vitro in human-derived HeLa cells and compared with that induced by the Japanese encephalitis virus (JEV), a closely related arbovirus that generally exhibits low viraemia in humans. A sustained viral spread with a poor IFN induction was observed in the DENV-infected cells, whereas the JEV infection resulted in a self-limiting and abortive infection with a high IFN induction. There was no difference between DENV and JEV double-stranded RNA (dsRNA) as IFN inducers. Instead, the dsRNA was poorly exposed in the cytosol as late as 48 h post-infection (p.i.), despite the high level of DENV replication in the infected cells. In contrast, the JEV-derived dsRNA appeared in the cytosol as early as 24 h p.i. Our results provided evidence for the first time in DENV, that concealing dsRNA in the intracellular membrane diminishes the effect of the host defence mechanism, a strategy that differs from an active suppression of IFN activity.
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http://dx.doi.org/10.1038/srep07395DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4261170PMC
December 2014

Detection of east/central/south African genotype of chikungunya virus in Myanmar, 2010.

Emerg Infect Dis 2014 Aug;20(8):1378-81

In 2010, chikungunya virus of the East Central South African genotype was isolated from 4 children in Myanmyar who had dengue-like symptoms. Phylogenetic analysis of the E1 gene revealed that the isolates were closely related to isolates from China, Thailand, and Malaysia that harbor the A226V mutation in this gene.
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http://dx.doi.org/10.3201/eid2008.131431DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4111191PMC
August 2014

Tanay virus, a new species of virus isolated from mosquitoes in the Philippines.

J Gen Virol 2014 Jun 19;95(Pt 6):1390-1395. Epub 2014 Mar 19.

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

In 2005, we isolated a new species of virus from mosquitoes in the Philippines. The virion was elliptical in shape and had a short single projection. The virus was named Tanay virus (TANAV) after the locality in which it was found. TANAV genomic RNA was a 9562 nt+poly-A positive strand, and polycistronic. The longest ORF contained putative RNA-dependent RNA polymerase (RdRP); however, conserved short motifs in the RdRP were permuted. TANAV was phylogenetically close to Negevirus, a recently proposed taxon of viruses isolated from haemophagic insects, and to some plant viruses, such as citrus leprosis virus C, hibiscus green spot virus and blueberry necrotic ring blotch virus. In this paper, we describe TANAV and the permuted structure of its RdRP, and discuss its phylogeny together with those of plant viruses and negevirus.
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http://dx.doi.org/10.1099/vir.0.061887-0DOI Listing
June 2014

NS1' protein expression facilitates production of Japanese encephalitis virus in avian cells and embryonated chicken eggs.

J Gen Virol 2014 Feb;95(Pt 2):373-383

Department of Virology, Institute of Tropical Medicine (NEKKEN), Nagasaki University, Nagasaki, Japan.

Japanese encephalitis virus (JEV), which belongs to the genus Flavivirus of the family Flaviviridae, is a leading cause of meningo-encephalitis in Asian countries. The flavivirus non-structural protein 1 (NS1) plays a role in virus replication and in the elicitation of an immune response. The NS1' protein found among the members of the JEV subgroup is an extended form of NS1 and is generated by a -1 ribosomal frameshift. This protein is known to be involved in viral pathogenicity; however, its specific function is still unknown. Here, we describe an investigation of the molecular function of NS1' protein through the production of JEV NS1'-expressing and -non-expressing clones and their infection of avian and mammalian cells. Efficient NS1' protein expression was observed in avian cells and was found to facilitate JEV production in both avian cultured cells and embryonated chicken eggs. NS1' protein was observed to co-localize with NS5 protein and resulted in increased viral RNA levels in avian cells. These findings clearly indicate that NS1' enhances the production of JEV in avian cells and may facilitate the amplification/maintenance role of birds in the virus transmission cycle in nature.
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http://dx.doi.org/10.1099/vir.0.057968-0DOI Listing
February 2014

Survey of causative agents for acute respiratory infections among patients in Khartoum-State, Sudan, 2010-2011.

Virol J 2013 Oct 25;10:312. Epub 2013 Oct 25.

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

Background: This study was carried out to determine causative agents of acute respiratory illness of patients in Khartoum State, Sudan.

Methods: Four hundred patients experiencing respiratory infections within January-March 2010 and January-March 2011 were admitted at Khartoum Hospital and had their throat swab samples subjected to multiplex real-time RT-PCR to detect influenza viruses (including subtypes) and other viral agents. Isolation, nucleotide sequence and phylogenetic analysis on some influenza viruses based on the HA gene were done.

Results: Out of 400 patients, 66 were found to have influenza viruses (35, 27, 2, and 2 with types A, B, C, and A and B co-infections, respectively). Influenza viruses were detected in 28, 33 and 5 patients in the age groups <1, 1-10, and 11-30 years old, respectively but none in the 31-50 years old group. Out of 334 patients negative for influenza viruses, 27, 14, and 2 were positive for human respiratory syncytial virus, rhinovirus and adenovirus, respectively. Phylogenetic tree on influenza A (H1N1) pdm09 subtype shows that Sudan strains belong to the same clade and are related to those strains from several countries such as USA, Japan, Italy, United Kingdom, Germany, Russia, Greece, Denmark, Taiwan, Turkey and Kenya. Seasonal A H3 subtypes have close similarity to strains from Singapore, Brazil, Canada, Denmark, USA and Nicaragua. For influenza B, Sudan strains belong to two different clades, and just like influenza A (H1N1) pdm09 and A H3 subtypes, seem to be part of worldwide endemic population (Kenya, USA, Brazil, Russia, Taiwan and Singapore).

Conclusions: In Sudan, the existence of respiratory viruses in patients with acute respiratory infection was confirmed and characterized for the first time by using molecular techniques.
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http://dx.doi.org/10.1186/1743-422X-10-312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3831848PMC
October 2013

First isolation of dengue virus from the 2010 epidemic in Nepal.

Trop Med Health 2013 Sep 20;41(3):103-11. Epub 2013 Aug 20.

Sukra Raj Tropical and Infectious Disease Hospital, Nepal ; Everest International Clinic and Research Center, Kathmandu, Nepal.

Dengue is an emerging disease in Nepal and was first observed as an outbreak in nine lowland districts in 2006. In 2010, however, a large epidemic of dengue occurred with 4,529 suspected and 917 serologically-confirmed cases and five deaths reported in government hospitals in Nepal. The collection of demographic information was performed along with an entomological survey and clinical evaluation of the patients. A total of 280 serum samples were collected from suspected dengue patients. These samples were subjected to routine laboratory investigations and IgM-capture ELISA for dengue serological identification, and 160 acute serum samples were used for virus isolation, RT-PCR, sequencing and phylogenetic analysis. The results showed that affected patients were predominately adults, and that 10% of the cases were classified as dengue haemorrhagic fever/ dengue shock syndrome. The genetic characterization of dengue viruses isolated from patients in four major outbreak areas of Nepal suggests that the DENV-1 strain was responsible for the 2010 epidemic. Entomological studies identified Aedes aegypti in all epidemic areas. All viruses belonged to a monophyletic single clade which is phylogenetically close to Indian viruses. The dengue epidemic started in the lowlands and expanded to the highland areas. To our knowledge, this is the first dengue isolation and genetic characterization reported from Nepal.
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http://dx.doi.org/10.2149/tmh.2012-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3801155PMC
September 2013

An approach for differentiating echovirus 30 and Japanese encephalitis virus infections in acute meningitis/encephalitis: a retrospective study of 103 cases in Vietnam.

Virol J 2013 Sep 11;10:280. Epub 2013 Sep 11.

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

Background: In recent decades, Echovirus 30 (E30) and Japanese encephalitis virus (JEV) have been reported to be the common causative agents of acute meningitis among patients in South East Asia. An E30 outbreak in Vietnam in 2001-2002 gained our interest because the initial clinical diagnosis of infected patients was due to JEV infection. There are few clinical insights regarding E30 cases, and there are no reports comparing E30 and JEV acute meningitis/encephalitis cases based on clinical symptoms and case histories. We therefore aimed to identify reliable clinical methods to differentiate E30 and JEV acute meningitis/encephalitis.

Methods: A retrospective, cross-sectional study was conducted to compare E30 and JEV acute meningitis/encephalitis cases. We collected and analyzed the clinical records of 43 E30 confirmed cases (E30 group) and 60 JEV confirmed cases (JEV group). Clinical data were compared between the E30 and the JEV groups. Differences of clinical parameters were analyzed by certain statistical tests.

Results: Fever, headache, and vomiting were the most common symptoms in both the E30 and the JEV groups. Combined symptoms of headache and vomiting and the triad of symptoms of fever, headache, and vomiting were observed in more patients in the E30 group (E30 vs. JEV: 19% vs. 0%, p < 0.001; 74% vs. 27%, p < 0.001, respectively). On the other hand, strong neurological symptoms such as seizure (5% vs. 73%, p < 0.001) and altered consciousness (12% vs. 97%, p < 0.001) were manifested primarily in the JEV group. CSF leukocytosis was observed predominantly in the E30 group (80 vs. 18 cells/μL, p = 0.003), whereas decreasing CSF sugar level was observed predominantly in the JEV group (58.7 vs. 46.9 mg/dL, p < 0.001).

Conclusion: Fever, headache, vomiting, absence of neurological symptoms (seizure, altered consciousness), and presence of CSF leukocytosis are important parameters to consider in differentiating E30 from JEV cases during early infection. Then, proper measures can be adopted immediately to prevent the spread of the disease in the affected areas.
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http://dx.doi.org/10.1186/1743-422X-10-280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3847169PMC
September 2013

Discovery of the first insect nidovirus, a missing evolutionary link in the emergence of the largest RNA virus genomes.

PLoS Pathog 2011 Sep 8;7(9):e1002215. Epub 2011 Sep 8.

Department of Virology, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam.

Nidoviruses with large genomes (26.3-31.7 kb; 'large nidoviruses'), including Coronaviridae and Roniviridae, are the most complex positive-sense single-stranded RNA (ssRNA+) viruses. Based on genome size, they are far separated from all other ssRNA+ viruses (below 19.6 kb), including the distantly related Arteriviridae (12.7-15.7 kb; 'small nidoviruses'). Exceptionally for ssRNA+ viruses, large nidoviruses encode a 3'-5'exoribonuclease (ExoN) that was implicated in controlling RNA replication fidelity. Its acquisition may have given rise to the ancestor of large nidoviruses, a hypothesis for which we here provide evolutionary support using comparative genomics involving the newly discovered first insect-borne nidovirus. This Nam Dinh virus (NDiV), named after a Vietnamese province, was isolated from mosquitoes and is yet to be linked to any pathology. The genome of this enveloped 60-80 nm virus is 20,192 nt and has a nidovirus-like polycistronic organization including two large, partially overlapping open reading frames (ORF) 1a and 1b followed by several smaller 3'-proximal ORFs. Peptide sequencing assigned three virion proteins to ORFs 2a, 2b, and 3, which are expressed from two 3'-coterminal subgenomic RNAs. The NDiV ORF1a/ORF1b frameshifting signal and various replicative proteins were tentatively mapped to canonical positions in the nidovirus genome. They include six nidovirus-wide conserved replicase domains, as well as the ExoN and 2'-O-methyltransferase that are specific to large nidoviruses. NDiV ORF1b also encodes a putative N7-methyltransferase, identified in a subset of large nidoviruses, but not the uridylate-specific endonuclease that - in deviation from the current paradigm - is present exclusively in the currently known vertebrate nidoviruses. Rooted phylogenetic inference by Bayesian and Maximum Likelihood methods indicates that NDiV clusters with roniviruses and that its branch diverged from large nidoviruses early after they split from small nidoviruses. Together these characteristics identify NDiV as the prototype of a new nidovirus family and a missing link in the transition from small to large nidoviruses.
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http://dx.doi.org/10.1371/journal.ppat.1002215DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3169540PMC
September 2011

Development of a rapid and comprehensive proteomics-based arboviruses detection system.

J Virol Methods 2010 Jul 19;167(1):31-6. Epub 2010 Mar 19.

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

A rapid and comprehensive protocol, which combines simple purification and liquid chromatography-electrospray ionisation-tandem mass spectrometry (LC-ESI/MS/MS), was developed for identification of arboviruses in infected culture fluid. Using this protocol, various arboviruses were detected including uncommon viruses that were described previously as Banna virus and Yunnan orbivirus. This approach is useful for the rapid screening of viral samples that cannot be identified by conventional gene amplification or immunological methods.
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http://dx.doi.org/10.1016/j.jviromet.2010.03.006DOI Listing
July 2010

Evidence of frequent introductions of Japanese encephalitis virus from south-east Asia and continental east Asia to Japan.

J Gen Virol 2009 Apr 4;90(Pt 4):827-832. Epub 2009 Mar 4.

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

The Japanese encephalitis virus (JEV) circulating in Japan consists of viruses with multiple phylogenetic origins. Phylogenetic analysis revealed that some JEV strains have recently migrated from south-east and continental east Asian countries. One phylogenetic subcluster of the JEV strains circulating in Japan was closely related to viruses isolated in Vietnam and China's inland region while other JEV subclusters were related to viruses isolated in Shanghai, China. One virus subcluster, however, was isolated solely in Japan and was not found in any other Asian country. Therefore, our data suggests that the JEVs that have remained or are circulating in Japan include a mixture of viruses that have previously migrated from south-east and continental east Asian countries.
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http://dx.doi.org/10.1099/vir.0.007617-0DOI Listing
April 2009

Isolation and molecular characterization of Banna virus from mosquitoes, Vietnam.

Emerg Infect Dis 2008 Aug;14(8):1276-9

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

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 suggest widespread distribution of this virus throughout Southeast Asia.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2600385PMC
http://dx.doi.org/10.3201/eid1408.080100DOI Listing
August 2008