Publications by authors named "Meng Ling Moi"

65 Publications

U.S.-Japan cooperative medical sciences program: 22nd International Conference on Emerging Infectious Diseases in the Pacific Rim.

Virology 2021 03 30;555:71-77. Epub 2020 Dec 30.

National Institute of Infectious Diseases, Japan.

This review summarizes the presentations given at the 22nd International conference on Emerging Infectious Diseases in the Pacific Rim. The purpose of this annual meeting is to foster international collaborations and address important public health issues in the Asia-Pacific region. This meeting was held in Bangkok in February 2020 and focused on emerging virus infections. Unexpectedly, the SARS-CoV-2 pandemic was in the initial stages leading to a special session on COVID-19 in addition to talks on dengue, influenza, hepatitis, AIDS, Zika, chikungunya, rabies, cervical cancer and nasopharyngeal carcinoma.
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http://dx.doi.org/10.1016/j.virol.2020.12.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7870554PMC
March 2021

Characteristics of COVID-19 epidemic and control measures to curb transmission in Malaysia.

Int J Infect Dis 2020 Dec 17;101:409-411. Epub 2020 Oct 17.

Faculty of Health Sciences, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.

The first wave of COVID-19 epidemic began in late January in Malaysia and ended with a very small final size. The second wave of infections broke out in late February and grew rapidly in the first 3 weeks. Authorities in the country responded quickly with a series of control strategies collectively known as the Movement Control Order (MCO) with different levels of intensity matching the progression of the epidemic. We examined the characteristics of the second wave and discussed the key control strategies implemented in the country. In the second wave, the epidemic doubled in size every 3.8 days (95% confidence interval [CI]: 3.3, 4.5) in the first month and decayed slowly after that with a halving time of approximately 3 weeks. The time-varying reproduction number R peaked at 3.1 (95% credible interval: 2.7, 3.5) in the 3rd week, declined sharply thereafter and stayed below 1 in the last 3 weeks of April, indicating low transmissibility approximately 3 weeks after the MCO. Experience of the country suggests that adaptive triggering of distancing policies combined with a population-wide movement control measure can be effective in suppressing transmission and preventing a rebound.
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http://dx.doi.org/10.1016/j.ijid.2020.10.027DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7567666PMC
December 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

Dengue epidemic in Malaysia: urban versus rural comparison of dengue immunoglobulin G seroprevalence among Malaysian adults aged 35-74 years.

Trans R Soc Trop Med Hyg 2020 11;114(11):798-811

UKM Medical Molecular Biology Institute, Universiti Kebangsaan Malaysia, Kuala Lumpur, 56000, Malaysia.

Background: A periodic serosurvey of dengue seroprevalence is vital to determine the prevalence of dengue in countries where this disease is endemic. This study aimed to determine the prevalence of dengue immunoglobulin G (IgG) seropositivity among healthy Malaysian adults living in urban and rural areas.

Methods: A total of 2598 serum samples (1417 urban samples, 1181 rural samples) were randomly collected from adults ages 35-74 y. The presence of the dengue IgG antibody and neutralising antibodies to dengue virus (DENV) 1-4 was determined using enzyme-linked immunosorbent assay and the plaque reduction neutralisation test assay, respectively.

Results: The prevalence of dengue IgG seropositivity was 85.39% in urban areas and 83.48% in rural areas. The seropositivity increased with every 10-y increase in age. Ethnicity was associated with dengue seropositivity in urban areas but not in rural areas. The factors associated with dengue seropositivity were sex and working outdoors. In dengue IgG-positive serum samples, 98.39% of the samples had neutralising antibodies against DENV3, but only 70.97% of them had neutralising antibodies against DENV4.

Conclusion: The high seroprevalence of dengue found in urban and rural areas suggests that both urban and rural communities are vital for establishing and sustaining DENV transmission in Malaysia.
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http://dx.doi.org/10.1093/trstmh/traa056DOI Listing
November 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

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

Non-Human Primate Models of Dengue Virus Infection: A Comparison of Viremia Levels and Antibody Responses during Primary and Secondary Infection among Old World and New World Monkeys.

Pathogens 2020 Mar 27;9(4). Epub 2020 Mar 27.

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

Due to the global burden of dengue disease, a vaccine is urgently needed. One of the key points in vaccine development is the development of a robust and reliable animal model of dengue virus infection. Characteristics including the ability to sustain viral replication, demonstration of clinical signs, and immune response that resemble those of human dengue virus infection are vital in animal models. Preclinical studies in vaccine development usually include parameters such as safety evaluation, induction of viremia and antigenemia, immunogenicity, and vaccine effectiveness. Although mice have been used as a model, non-human primates have an advantage over mice because of their relative similarity to humans in their genetic composition and immune responses. This review compares the viremia kinetics and antibody responses of cynomolgus macaques (, common marmosets (, and tamarins ( and ) and summarize the perspectives and the usefulness along with challenges in dengue vaccine development.
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http://dx.doi.org/10.3390/pathogens9040247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7238212PMC
March 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

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

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

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

Neutralization Potency of Sera from Vietnamese Patients with Japanese Encephalitis (JE) against Genotypes I and V JE Viruses.

Jpn J Infect Dis 2019 Mar 31;72(2):115-117. Epub 2018 Oct 31.

Vietnam Research Station, National Institute of Hygiene and Epidemiology-Nagasaki University.

Japanese encephalitis virus (JEV) is classified into 5 genotypes (GI, GII, GIII, GIV, and GV), and the GI and GIII strains are the most widely distributed in JE endemic areas. In recent years, GV JEV has been detected in China and Korea, suggesting that GV JEV may invade other JE endemic areas, including Vietnam, and that more attention should be paid to the JEV strains circulating in these areas. In this study, we investigated the neutralization ability of the sera collected from 22 Vietnamese patients with JE who lived in northern Vietnam against the GI and GV JEV strains. In most cases, the ratios of the titer against GV to that against GI (GV:GI) were equal to or less than 1:4. However, the titer against GV JEV was equivalent (1:1) to that against GI JEV in only a few cases, and no serum had a ratio higher than 1:1. Thus, our results did not show convincing evidence that GV JEV was emerging in northern Vietnam in 2014.
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http://dx.doi.org/10.7883/yoken.JJID.2018.232DOI Listing
March 2019

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

Genotype-specific and cross-reactive neutralizing antibodies induced by dengue virus infection: detection of antibodies with different levels of neutralizing activities against homologous and heterologous genotypes of dengue virus type 2 in common marmosets (Callithrix jacchus).

Virol J 2018 03 27;15(1):51. Epub 2018 Mar 27.

National Institute of Infectious Diseases, 1-23-1 Toyama, Shinjuku, Tokyo, 162-8640, Japan.

Background: A vaccine against all four dengue virus (DENV) serotypes includes the formulation of one genotype of each serotype. Although genetic similarities among genotypes within a serotype are higher as compared to those among serotypes, differences in the immunogenicity of the included genotypes would be a critical issue in maximizing successful dengue vaccine development. Thus, we determined the neutralizing antibody responses against three genotypes of dengue virus serotype 2 (DENV-2), namely Cosmopolitan, Asian I, and Asian/American, after primary and secondary inoculation with DENV-2 in a dengue animal model, the common marmoset (Callithrix jacchus).

Methods: A total of fifty-four plasma samples were obtained from thirty-four marmosets that were inoculated with clinically-isolated DENV strains or DENV candidate vaccines, were used in this study. Plasma samples were obtained from marmosets after primary inoculation with DENV-2 infection, secondary inoculation with homologous or heterologous genotypes, and tertiary inoculation with heterologous DENV. Neutralizing antibody titers against DENV-2 (Cosmopolitan, Asian I, and Asian/American genotypes) and DENV-1 were determined using a conventional plaque reduction neutralization assay.

Results: In marmosets that were inoculated with the Cosmopolitan genotype in primary infection, neutralizing antibody neutralized 3 genotypes, and the titers to Asian I genotype were significantly higher than those to homologous Cosmopolitan genotype. After secondary DENV-2 infection with heterologous genotype (Asian I in primary and Asian/American in secondary), neutralizing antibody titers to Asian/American genotype was significantly higher than those against Cosmopolitan and Asian I genotypes. Following tertiary infection with DENV-1 following DENV-2 Asian I and Cosmopolitan genotypes, neutralizing antibody titers to Asian/American were also significantly higher than those against Cosmopolitan and Asian I genotypes.

Conclusion: The present study demonstrated that different levels of neutralizing antibodies were induced against variable DENV-2 genotypes after primary, secondary and tertiary infections, and that neutralizing antibody titers to some heterologous genotypes were higher than those to homologous genotypes within a serotype. The results indicate that heterogeneity and homogeneity of infecting genotypes influence the levels and cross-reactivity of neutralizing antibodies induced in following infections. The results also suggest that certain genotypes may possess advantage in terms of breakthrough infections against vaccination.
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http://dx.doi.org/10.1186/s12985-018-0967-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5870686PMC
March 2018

Dengue Virus Infection in during the 2014 Autochthonous Dengue Outbreak in Tokyo Metropolis, Japan.

Am J Trop Med Hyg 2018 05 15;98(5):1460-1468. Epub 2018 Mar 15.

Department of Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.

In 2014 in Japan, 162 autochthonous dengue cases were reported for the first time in nearly 70 years. Here, we report the results of the detection and isolation of dengue virus (DENV) from mosquitoes collected in Tokyo Metropolis in 2014 and 2015. The phylogenetic relationship among DENV isolates from mosquitoes and from patients based on both the entire envelope gene and whole coding sequences was evaluated. Herein, 2,298 female and 956 male mosquitoes were collected at six suspected locations of DENV infection in Tokyo Metropolis from August to October in 2014 and grouped into 124 and 35 pools, respectively, for viral genome detection and DENV isolation. Dengue virus RNA was detected using reverse transcription polymerase chain reaction and TaqMan assays from 49 female pools; 16 isolates were obtained using C6/36 and Vero cells. High minimum infection rates (11.2-66.7) persisted until mid-September. All DENV isolates belonged to the genotype I in serotype 1 (DENV-1), and its sequences demonstrated > 99% homology to the sequence of the DENV isolated from a patient in the vicinity of Tokyo Metropolis in 2014. Therefore, was a major DENV vector, and a single DENV-1 strain circulated in Tokyo Metropolis in 2014. Dengue virus was not detected from male mosquitoes in 2014 and wild larvae in April 2015. Thus, the possibility of both vertical transmission and overwintering of DENV was extremely low, even in dengue-epidemic areas. This study reports the first entomological information on a dengue outbreak in a temperate region, where no mosquitoes are distributed.
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http://dx.doi.org/10.4269/ajtmh.17-0954DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5953391PMC
May 2018

Detection of Zika Virus Infection in Myanmar.

Am J Trop Med Hyg 2018 03 18;98(3):868-871. Epub 2018 Jan 18.

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

Zika virus (ZIKV), an emerging mosquito-borne flavivirus, causes a dengue-like infection that has recently caught global attention. The infection, which also includes some birth defects, has been documented in the Americas, Pacific Islands, and some parts of Africa and Asia. There are no published reports on local ZIKV transmission in Myanmar. In this study, a total of 462 serum samples from patients and asymptomatic persons were collected in Myanmar from 2004 to 2017. They were analyzed for ZIKV infection by immunoglobulin M capture enzyme-linked immunosorbent assay (ELISA), immunoglobulin G indirect ELISA, neutralization test, real-time polymerase chain reaction (PCR), and conventional PCR. Our study confirmed ZIKV infection in 4.9% of patients with clinical dengue symptoms and in 8.6% of persons who were asymptomatic. This is the first report on ZIKV infection in Myanmar and it suggests the occurrence of ZIKV infection in two geographically distinct sites in this country since at least 2006.
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http://dx.doi.org/10.4269/ajtmh.17-0708DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5930904PMC
March 2018

Dengue virus infection-enhancement activity in neutralizing antibodies of healthy adults before dengue season as determined by using FcγR-expressing cells.

BMC Infect Dis 2018 01 10;18(1):31. Epub 2018 Jan 10.

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

Background: Antibodies are critical responses to protect the host from dengue virus(DENV) infection. Antibodies target DENV by two pathologic mechanisms: virus neutralization and infection enhancement. In dengue patients, the absence of neutralizing activity in the presence of FcγR implies that infection-enhancing activity hampers the neutralizing activity of antibodies, which could potentially lead to symptomatic presentations and severe clinical outcomes.

Methods: A total of 100 pair serum samples from adult healthy volunteers were obtained during the dengue season in Ha Noi in 2015 for evaluation of neutralizing and infection-enhancing activity. Additionally, 20 serum samples from acute secondary DENV infection patients were also used as the patient group in this study. PRNT was performed on BHK cells and FcγR-expressing BHK cell lines for all serum samples.

Results: Out of 100 residents, positive neutralizing antibodies (N.A) were found in 44.23 and 76.92% for DENV-1; 38.46 and 75% for DENV-2; 19.23 and 15.38% for DENV-3; and 1.92 and 9.62% for DENV-4 for pre and post-dengue season respectively. The percentage of post-exposure residents having positive responses against single, two, or more than three DENV serotypes were 38.46, 44.23 and 15.38%, respectively. A total of 34 residents were DENV seropositive before the dengue season and these individuals demonstrated further elevation of IgG antibodies after the dengue season. At the end of the season, 18 residents were confirmed to be new asymptomatic DENV infection cases. In both groups, N.A titers determined on BHK cells were higher than that on FcγR-expressing BHK cells. In heterotypic N.A responses, N.A titers to the infecting serotype from the samples obtained from pre-exposure group were significantly higher than those of the patient group. However, fold enhancement to the infecting serotypes from the samples in the pre-exposure group was substantially lower as compared to that of the patient group.

Conclusion: Before and after the dengue season, serum samples from healthy volunteers demonstrated high levels of neutralizing antibodies and low or absence of infection-enhancement activity. The results suggest that while infection-enhancement activity hampers neutralizing activity of antibodies, high levels of DENV neutralizing antibodies set a critical threshold in facilitating the prevention of disease progression.
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http://dx.doi.org/10.1186/s12879-017-2894-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5763606PMC
January 2018

Marmosets (Callithrix jacchus) as a non-human primate model for evaluation of candidate dengue vaccines: induction and maintenance of specific protective immunity against challenges with clinical isolates.

J Gen Virol 2017 12 21;98(12):2955-2967. Epub 2017 Nov 21.

National Institute of Infectious Diseases, Tokyo, Japan.

Dengue virus (DENV) is one of the major infectious diseases in tropical regions and approximately half of the world population is at risk of infection. Vaccines would offer an effective control measure against this disease. We previously reported on the utility of marmosets as an animal model for studying primary and secondary dengue infections. Infected marmosets consistently develop viraemia and antibody kinetics that reflect those of patients with dengue. Thus, it is important to determine the utility of marmosets as an animal model for demonstrating vaccine efficacy. In this study, marmosets were inoculated with candidate vaccine and parent strains and challenged with a clinical DENV strain. The viraemia and antibody kinetics in these marmosets were determined. Marmosets consistently develop lower viraemia with an attenuated vaccine strain. During secondary challenge, the IgM response was delayed, whereas the IgG levels rose rapidly, indicating a secondary antibody response. The neutralizing activities against the homotypic serotype were high; all marmosets were protected against viraemia following secondary inoculation. The viraemia markers and antibody responses were consistent with those of human DENV infection and vaccinees. These results demonstrate the utility of marmosets as an animal model for the study of vaccine efficacy.
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http://dx.doi.org/10.1099/jgv.0.000913DOI Listing
December 2017

Detection of Virus-Antibody Immune Complexes in Secondary Dengue Virus Infection.

Methods Mol Biol 2018 ;1604:331-337

National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku, 162-8640, Japan.

It has been reported that virus-antibody immune complexes formed during secondary dengue virus infection are associated with increased disease severity. Here, we describe the details of a plaque titration method that uses FcγR -expressing BHK cells to detect and quantify infectious virus-immune complexes and a quantitative real-time PCR method for the quantification of virus genome in patients with secondary dengue infection. These methods detect both viruses in free-form and in immune complexes in patients with dengue infection, and are useful for determining viremia levels and patterns that better reflect in vivo conditions.
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http://dx.doi.org/10.1007/978-1-4939-6981-4_25DOI Listing
June 2018

An Envelope-Modified Tetravalent Dengue Virus-Like-Particle Vaccine Has Implications for Flavivirus Vaccine Design.

J Virol 2017 12 14;91(23). Epub 2017 Nov 14.

VLP Therapeutics, Gaithersburg, Maryland, USA

Dengue viruses (DENV) infect 50 to 100 million people each year. The spread of DENV-associated infections is one of the most serious public health problems worldwide, as there is no widely available vaccine or specific therapeutic for DENV infections. To address this, we developed a novel tetravalent dengue vaccine by utilizing virus-like particles (VLPs). We created recombinant DENV1 to -4 (DENV1-4) VLPs by coexpressing precursor membrane (prM) and envelope (E) proteins, with an F108A mutation in the fusion loop structure of E to increase the production of VLPs in mammalian cells. Immunization with DENV1-4 VLPs as individual, monovalent vaccines elicited strong neutralization activity against each DENV serotype in mice. For use as a tetravalent vaccine, DENV1-4 VLPs elicited high levels of neutralization activity against all four serotypes simultaneously. The neutralization antibody responses induced by the VLPs were significantly higher than those with DNA or recombinant E protein immunization. Moreover, antibody-dependent enhancement (ADE) was not observed against any serotype at a 1:10 serum dilution. We also demonstrated that the Zika virus (ZIKV) VLP production level was enhanced by introducing the same F108A mutation into the ZIKV envelope protein. Taken together, these results suggest that our strategy for DENV VLP production is applicable to other flavivirus VLP vaccine development, due to the similarity in viral structures, and they describe the promising development of an effective tetravalent vaccine against the prevalent flavivirus. Dengue virus poses one of the most serious public health problems worldwide, and the incidence of diseases caused by the virus has increased dramatically. Despite decades of effort, there is no effective treatment against dengue. A safe and potent vaccine against dengue is still needed. We developed a novel tetravalent dengue vaccine by using virus-like particles (VLPs), which are noninfectious because they lack the viral genome. Previous attempts of other groups to use dengue VLPs resulted in generally poor yields. We found that a critical amino acid mutation in the envelope protein enhances the production of VLPs. Our tetravalent vaccine elicited potent neutralizing antibody responses against all four DENV serotypes. Our findings can also be applied to vaccine development against other flaviviruses, such as Zika virus or West Nile virus.
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http://dx.doi.org/10.1128/JVI.01181-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5686733PMC
December 2017

Zika virus infection and microcephaly in Vietnam.

Lancet Infect Dis 2017 08;17(8):805-806

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(17)30412-7DOI Listing
August 2017

Utility of Japanese encephalitis virus subgenomic replicon-based single-round infectious particles as antigens in neutralization tests for Zika virus and three other flaviviruses.

J Virol Methods 2017 05 20;243:164-171. Epub 2017 Feb 20.

BIKEN Endowed Department of Dengue Vaccine Development, Faculty of Tropical Medicine, Mahidol University,420/6 Ratchawithi Road, Ratchathewi, Bangkok 10400, Thailand(3); BIKEN Endowed Department of Dengue Vaccine Development, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka, 565-0871, Japan.

The introduction of a foreign virus into an area may cause an outbreak, as with the Zika virus (ZIKV) outbreak in the Americas. Preparedness for handling a viral outbreak involves the development of tests for the serodiagnosis of foreign virus infections. We previously established a gene-based technology to generate some flaviviral antigens useful for functional antibody assays. The technology utilizes a Japanese encephalitis virus subgenomic replicon to generate single-round infectious particles (SRIPs) that possess designed surface antigens. In the present study, we successfully expanded the capacity of SRIPs to four human-pathogenic mosquito-borne flaviviruses that could potentially be introduced from endemic to non-endemic countries: ZIKV, Sepik virus, Wesselsbron virus, and Usutu virus. Flavivirus-crossreactive monoclonal antibodies dose-dependently neutralized these SRIPs. ZIKV-SRIPs also produced antibody-dose-dependent neutralization curves equivalent to those shown by authentic ZIKV particles using sera from a Zika fever patient. The faithful expression of designed surface antigens on SRIPs will allow their use in neutralization tests to diagnose foreign flaviviral infections.
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http://dx.doi.org/10.1016/j.jviromet.2017.02.011DOI Listing
May 2017

Dengue Virus Isolation in Mosquito Aedes albopictus Captured During an Outbreak in Tokyo, 2014, by a Method Relying on Antibody-Dependent Enhancement Mechanism Using FcγR-Expressing BHK Cells.

Vector Borne Zoonotic Dis 2016 12 24;16(12):810-812. Epub 2016 Oct 24.

2 Department of Virology 1, National Institute of Infectious Diseases , Tokyo, Japan .

Dengue virus (DENV) isolation from mosquitoes is necessary for providing definitive evidence of virus circulation, and is critical for further virological characterization and determination of epidemiological characteristics. By using Aedes albopictus mosquitoes captured during an outbreak in Tokyo in 2014, we compared the DENV isolation rates of a conventional virus isolation method that uses C6/36 mosquito cells as assay cells with those of a virus isolation method that relies on an antibody-dependent enhancement (ADE) mechanism by using FcγR-expressing baby hamster kidney (BHK) cells and an antibody with ADE activity. The number of DENV genome copies and infectious virus titers in cell culture supernatant fluids of FcγR-expressing BHK cells were significantly higher than those of the C6/36 cells. In addition, DENV was isolated from a mosquito pool by using FcγR-expressing BHK cells only in the presence of infection-enhancing antibody. Infectious virus was detected in six mosquito pools only by using FcγR-expressing BHK cells. The results suggest that the method that relies on ADE mechanism by using the FcγR-expressing BHK cells and an antibody with ADE activity is useful for DENV isolation from mosquitoes caught in the field.
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http://dx.doi.org/10.1089/vbz.2016.1982DOI Listing
December 2016

Neutralizing and enhancing antibody responses to five genotypes of dengue virus type 1 (DENV-1) in DENV-1 patients.

J Gen Virol 2017 02 24;98(2):166-172. Epub 2017 Feb 24.

BIKEN Endowed Department of Dengue Vaccine Development, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.

Dengue virus (DENV) has four distinct serotypes, DENV-1-4, with four to six genotypes in each serotype. The World Health Organization recommends tetravalent formulations including one genotype of each serotype as safe and effective dengue vaccines. Here, we investigated the impact of genotype on the neutralizing antibody responses to DENV-1 in humans. Convalescent sera collected from patients with primary infection of DENV-1 were examined for neutralizing antibody against single-round infectious particles of the five DENV-1 genotypes (GI-GV). In both GI- and GIV-infected patients, their neutralizing antibody titres against the five genotypes were similar, differing ≤4-fold from the homogenotypic responses. The enhancing activities against the five genotypes were also similar in these sera. Thus, the genotype strains of DENV-1 showed no significant antigenic differences in these patients, suggesting that GI- or GIV-derived vaccine antigens should induce equivalent levels of neutralizing antibodies against all DENV-1 genotypes.
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http://dx.doi.org/10.1099/jgv.0.000669DOI Listing
February 2017

Dengue Associated Acute Encephalitis Syndrome Cases in Son La Province, Vietnam in 2014.

Jpn J Infect Dis 2017 07 31;70(4):357-361. Epub 2016 Oct 31.

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

Acute encephalitis syndrome (AES) is associated with high morbidity and mortality, and affects both children and adults. The main etiologic agent is Japanese encephalitis virus (JEV); however, there are also reports of Dengue virus (DENV) encephalitis. The objectives of this study were to determine the proportion of patients with encephalitis due to JEV during the 2014 outbreak in Son La Province in Vietnam and to explore the association of DENV in non-JEV viral encephalitis cases. Of 90 patients, 6 (6.7%) were positive for anti-JEV immunoglobulin M (IgM), 5 (5.6%) were positive for anti-DENV IgM, 30 (33.3%) were positive for both anti-JEV and anti-DENV IgM, and 56 (62.2%) were positive for flavivirus immunoglobulin G (IgG). In 5 patients with AES, who had positive anti-DENV IgM results in at least one of the paired serum samples, DENV was confirmed by neutralization testing. The incidence of JEV infection was high. There is still a need to maintain and strengthen the national JEV immunization program. This noticeable occurrence of DENV infection was not reported in Son La Province in 2013-2014. Our data suggested that in addition to JEV, DENV was also a causative agent of AES in 2014 in Son La Province, and this finding also confirmed the local occurrence of DENV infection.
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http://dx.doi.org/10.7883/yoken.JJID.2016.246DOI Listing
July 2017

Japanese encephalitis vaccine-facilitated dengue virus infection-enhancement antibody in adults.

BMC Infect Dis 2016 Oct 18;16(1):578. Epub 2016 Oct 18.

Kanagawa Prefectural Institute of Public Health, Chigasaki, Kanagawa, 253-0087, Japan.

Background: Dengue virus (DENV) and Japanese encephalitis virus (JEV) belong to the genus Flavivirus, and infection with a virus within this genus induces antibodies that are cross-reactive to other flaviviruses. Particularly in DENV infection, antibodies to DENV possess two competing activities: neutralizing activity and infection-enhancing activity. These antibody activities are considered central in modulating clinical outcomes of DENV infection. Here, we determined the neutralizing and infection-enhancing activity of DENV cross-reactive antibodies in adults before and after JE vaccination.

Methods: Participants were 77 Japanese adults who had received a single dose of inactivated Vero cell-derived JE vaccine. A total of 154 serum samples were obtained either before or approximately a month after a single dose of JE vaccination. The antibody-dependent enhancement (ADE) activity to each of four DENV serotypes and the neutralizing activities to DENV and to JEV were determined in each of the serum samples by using baby hamster kidney (BHK) cells and FcγR-expressing BHK cells.

Results: A total of 18 post-JE immunization samples demonstrated cross-reactivity to DENV in an anti-DENV IgG ELISA. DENV neutralizing antibodies were not detected after JE vaccination in this study. However, undiluted post-JE vaccination serum samples from 26 participants demonstrated monotypic and heterotypic ADE activity to DENV. ADE activity was also observed in 1:10-diluted samples from 35 of the JE vaccine recipients (35/77, 45 %).

Conclusion: In summary, JE vaccination induced DENV cross-reactive antibodies, and at sub-neutralizing levels, these DENV cross-reactive antibodies possess DENV infection-enhancement activity. The results also indicate that cross-reactivity to DENV is associated with high levels of JEV neutralizing antibodies and, the DENV cross-reactivity is further facilitated by JE vaccination.
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http://dx.doi.org/10.1186/s12879-016-1873-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5070094PMC
October 2016