Search

During the 2017 outbreak of severe dengue in Sri Lanka, dengue virus (DENV) serotypes 2, 3, and 4 were found to be co-circulating. Our previous study of 295 patients from the National Hospital Kandy in Sri Lanka between March 2017 and January 2018 determined that the dominant infecting serotype was DENV-2. In this study, 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 and 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. Complete genome analysis determined that DENV-3 isolates belonged to 2 different clades of genotype I and were genetically close to strains from Indonesia, China, Singapore, Malaysia, and Australia. There were 16 amino acid changes among DENV-3 isolates, and a greater number of changes were found in nonstructural proteins than in 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 and genotypes is needed to determine their effects on future outbreaks and understand the molecular epidemiology of dengue., Japanese Journal of Infectious Diseases, 74(5), pp. 443-449; 2021

Dengue virus (DENV) is one of the most prevalent neglected tropical diseases, with half of the world’s population at risk of infection. In Nepal, DENV was first reported in 2004, and its prevalence is increasing every year. The present study aimed to obtain and characterize the full-length genome sequence of DENV from the 2017 outbreak. Hospital-based surveillance was conducted in two provinces of Nepal during the outbreak. Acute-phase serum samples were collected from 141 clinically suspected dengue patients after the rainy season. By serological and molecular techniques, 37 (26.9%) and 49 (34.8%), respectively, were confirmed as dengue patients. The cosmopolitan genotype of DENV-2 was isolated from 27 laboratory-confirmed dengue patients. Genomic analysis showed many amino acid substitutions distributed mainly among the E, NS3, and NS5 genes. Phylogenetic analyses of the whole genome sequence revealed two clades (Asian and Indian) among DENV-2 isolates from Nepal. The DENV isolates from hilly and Terai areas were similar to Asian and Indian strains, respectively. Further genomic study on different DENV serotypes is warranted to understand DENV epidemics in Nepal, where there are limited scientific resources and infrastructure., Viruses, 13(8), art. no. 1444；2021

Dengue fever, caused by the mosquito-borne dengue virus (DENV), has been endemic in Myanmar since 1970 and it has become a significant public health burden. It is crucial that circulating DENV strains are identified and monitored, and that their transmission efficiency and association with disease severity is understood. In this study, we analyzed DENV-1, DENV-2, DENV-3, and DENV-4 serotypes in 1235 serum samples collected in Myanmar between 2017 and 2019. Whole-genome sequencing of DENV-1–4 demonstrated that most DENV-1–4 strains had been circulating in Myanmar for several years. We also identified the emergence of DENV-3 genotype-I in 2017 samples, which persisted through 2018 and 2019. The emergence of the strain coincided with a period of increased DENV-3 cases and marked changes in the serotype dynamics. Nevertheless, we detected no significant differences between serum viral loads, disease severity, and infection status of individuals infected with different DENV serotypes during the 3-year study. Our results not only identify the spread of a new DENV-3 genotype into Yangon, Myanmar, but also support the importance of DENV evolution in changing the epidemic dynamics in endemic regions.

Background: Dengue (DEN) is a neglected tropical disease, and surveillance of dengue virus (DENV) serotypes and genotypes is critical for the early detection of outbreaks. Risk factors for outbreaks include the emergence of new genotypes and serotype shifting. Methodology and principal findings: To understand the genomic and viral characteristics of DENV-infected patients, we conducted a cross-sectional descriptive study among pediatric patients admitted at the 550-bedded Mandalay Children Hospital during the 2018 DEN endemic season. We conducted virus isolation, serological tests, viremia level measurement, and whole-genome sequencing. Among the 202 serum samples, we detected 85 samples with DENV (46 DENV-1, 10 DENV-3, 26 DENV-4 and three multiple serotype co-infections) via reverse transcription quantitative/real-time PCR (RT-qPCR), and we obtained 49 DENV isolates (31 DENV-1, 10 DENV-3 and 8 DEN-4). We did not detect DENV-2 in this study. The viral genome levels in serum did not differ significantly among virus serotypes, infection status (primary versus secondary) and disease severity. Based on the phylogenetic analysis, we identified DENV-1 genotype-1, DENV-4 genotype-1 and DENV-3 genotype-3 and genotype-1 which was detected for the first time. Next-generation sequencing analysis revealed greater frequencies of nonsynonymous and synonymous mutations per gene in the nonstructural genes. Moreover, mutation rates were also higher among DENV-1. Conclusion/Significance: In conclusion, there was an increasing trend of DENV-3 cases during DENV endemic season in 2018 with the first detection of the genotype 1. However, DENV-1 has remained the predominant serotype in this study area since 2013, and we identified stop codon mutations in the DENV-1 genome. This report is the first to feature a complete genome analysis of the strains of DENV-3 and DENV-4 circulating among pediatric patients in Myanmar. This study highlighted the importance of annual surveillance for a better understanding of the molecular epidemiology of DENVs., PLoS ONE, 16(6), art. no. e0251314; 2021

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.

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.

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–Barre 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.

engue 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 sub-group 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., Japanese Journal of Infectious Diseases, 72(6), pp.413-419; 2019

In this study, we investigated severe fever with thrombocytopenia syndrome (SFTS) virus (SFTSV) infection in cats in Nagasaki, Japan. In total, 44 of 133 (33.1%) cats with suspected SFTS were confirmed to be infected with SFTSV. Phylogenetic analyses of SFTSV isolates from cats indicated that the main genotype in Nagasaki was J1 and that unique reassortant strains with J2 (S segment) and unclassified genotypes (M and L segments) were also present. There were no significant differences in virus growth in cell cultures or fatality in SFTSV-infected mice between the SFTSV strains that were isolated from recovered and fatal cat cases. Remarkably, SFTSV RNAs were detected in the swabs from cats, indicating that the body fluids contain SFTSV. To evaluate the risk of SFTSV infection when providing animal care, we further examined the seroprevalence of SFTSV infection in veterinarian staff members, 3 of 71 (4.2%) were seropositive for SFTSV-specific antibodies. Our results provide useful information on the possibility of using cats as sentinel animals and raised concerns of the zoonotic risk of catching SFTSV from animals.

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.

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., PLOS ONE, 15(6), art.no.e0234508; 2020

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., The American journal of tropical medicine and hygiene, 100(3), pp.643-646; 2019

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.

SUMMARYHospital-based surveillance was conducted at two widely separated regions in Myanmar during the 2015 dengue epidemic. Acute phase serum samples were collected from 332 clinically diagnosed dengue patients during the peak season of dengue cases. Viremia levels were measured by quantitative real-time PCR and plaque assays using FcγRIIA-expressing and non-FcγRIIA-expressing BHK cells to specifically determine the infectious virus particles. By serology and molecular techniques, 280/332 (84·3%) were confirmed as dengue patients. All four serotypes of dengue virus (DENV) were isolated from among 104 laboratory-confirmed patients including two cases infected with two DENV serotypes. High percentage of primary infection was noted among the severe dengue patients. Patients with primary infection or DENV IgM negative demonstrated significantly higher viral loads but there was no significant difference among the severity groups. Viremia levels among dengue patients were notably high for a long period which was assumed to support the spread of the virus by the mosquito vector during epidemic. Phylogenetic analyses of the envelope gene of the epidemic strains revealed close similarity with the strains previously isolated in Myanmar and neighboring countries. DENV-1 dominated the epidemic in 2015 and the serotype (except DENV-3) and genotype distributions were similar in both study sites.

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 18F-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.

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 Culex quinquefasciatus 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 Culex mosquitoes, Aedes (Stegomyia) and Anopheles mosquitoes were also positive for the Flavivirus genus. Cell fusing agent virus was detected in one pool of Aedes aegypti. Mosquito flavivirus was detected in three pools of Anopheles gambiae 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 Culex 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., Tropical Medicine and Health, 46(1), 19; 2018

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.

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 Orbivirus, 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., Tropical Medicine and Health, 44, 31; 2016