We report a case of Japanese encephalitis that occurred in a woman who had spent only a few days in an area where she could have been exposed to the virus. The risks and protective efficacy of vaccination against Japanese encephalitis virus for travellers who visit endemic areas for only a short period are discussed.
In late 2020, an outbreak of Tembusu virus (TMUV)-associated disease occurred in a 45-day-old white Roman geese flock in Taiwan. Here, we present the identification and isolation of a novel goose-origin TMUV strain designated as NTU/C225/2020. The virus was successfully isolated using minimal-pathogen-free duck embryos. Phylogenetic analysis of the polyprotein gene showed that NTU/C225/2020 clustered together with the earliest isolates from Malaysia and was most closely related to the first Taiwanese TMUV strain, TP1906. Genomic analysis revealed significant amino acid variations among TMUV isolates in NS1 and NS2A protein regions. In the present study, we characterized the NTU/C225/2020 culture in duck embryos, chicken embryos, primary duck embryonated fibroblasts, and DF-1 cells. All host systems were susceptible to NTU/C225/2020 infection, with observable lesions. In addition, animal experiments showed that the intramuscular inoculation of NTU/C225/2020 resulted in growth retardation and hyperthermia in day-old chicks. Gross lesions in the infected chicks included hepatomegaly, hyperemic thymus, and splenomegaly. Viral loads and histopathological damage were displayed in various tissues of both inoculated and naïve co-housed chicks, confirming the direct chick-to-chick contact transmission of TMUV. This is the first in vivo study of a local TMUV strain in Taiwan. Our findings provide essential information for TMUV propagation and suggest a potential risk of disease outbreak in chicken populations.
Introduction: Zika infection was declared as Public Health Emergency of International Concern since year 2015. Despite of no new reported case via National Surveillance System for flavivirus, an underestimated seroprevalence might occur as the country contributes to the Asian lineage of the virus. Methods: Systematic literature search using PICO framework and PRISMA checklist across four databases for articles published from year 2013-2018 yielded 189 results, 37 articles accepted by titles following criteria were subjected to abstract screening, leaving 8 articles with clear risk proceed to full text analysis using Cochrane checklist and GRADE assessment. Results: There were four high quality articles and four low quality articles based on biases in studies. Blood product management and vac-cination are strategies strongly recommended to be implemented as Zika response while vector control and family planning are public health measures to be proposed as policy if feasible. Successful factors to improve Zika surveil-lance and management includes developing algorithm for blood product management, anti-Zika vaccine research, algorithm for new-born screening, participation of policy makers, healthcare capacity building, raising healthcare and public awareness on the infection, international funding, utilization of technology in data management and bio-logical control of vector. Conclusion: Implementation of Zika response as policy is timely, should be evidence-based and follow guidelines from WHO / CDC / FDA US after cost-effectiveness evaluation for Malaysia setting.
Duck Tembusu virus (TMUV) is a recently identified pathogenic flavivirus that causes severe egg drop and encephalitis in Chinese ducks and geese. It has been found to be most closely related to the mosquito-origin Tembusu virus and chicken Sitiawan virus reported in Malaysia. However, the ecological characteristics and the pathogenesis of duck TMUV are largely unknown. We report the construction of full-length cDNA clone of duck TMUV strain JXSP. The virus genome was reverse transcribed, amplified as seven overlapping fragments and successively ligated into the low copy number vector pWSK29 under the control of a T7 promoter. Transfection of BHK-21 cells with the transcribed RNA from the full-length cDNA clone resulted in production of highly infectious progeny virus. In vitro growth characteristics in BHK-21 cells and virulence in ducklings and BALB/c mice were similar for the rescued and parental viruses. This stable infectious cDNA clone will be a valuable tool for studying the genetic determinants of duck TMUV.
Tembusu virus (TMUV, genus Flavivirus, family Flaviviridae) was first isolated in 1955 from Culex tritaeniorhynchus mosquitoes in Kuala Lumpur, Malaysia. In April 2010, duck TMUV was first identified as the causative agent of egg-drop syndrome, characterized by a substantial decrease in egg laying and depression, growth retardation and neurological signs or death in infected egg-laying and breeder ducks, in the People's Republic of China. Since 2010, duck TMUV has spread to most of the duck-producing regions in China, including many of the coastal provinces, neighbouring regions and certain Southeast Asia areas (i.e. Thailand and Malaysia). This review describes the current understanding of the genome characteristics, host range, transmission, epidemiology, phylogenetic and immune evasion of avian-origin TMUV and the innate immune response of the host.
In Southeast Asia, dengue viruses often co-circulate with other flaviviruses such as Japanese encephalitis virus, and due to the presence of shared antigenic epitopes it is often difficult to use serological methods to distinguish between previous infections by these flaviviruses.
The ability of passage in HeLa cells to attenuate flaviviruses was investigated for three different strains of the mosquito-borne West Nile (WN) virus and two tick-borne viruses, louping-ill and Langat. One strain of WN virus, Sarawak, was attenuated 4000-fold for adult mice by intraperitoneal or intranasal challenge after six HeLa passages. The HeLa-passaged virus was also found to be antigenically different and temperature-sensitive in its growth characteristics compared with the parent. After six HeLa cell passages the Egypt 101 and Smithburn strains of WN virus lost their ability to infect monkey kidney cells and no longer killed adult mice, although inoculated animals became sick for several days. In contrast, two tick-borne flaviviruses remained as virulent for mice after six HeLa passages as the parent non-HeLa-passaged virus. Neither of the tick-borne viruses exhibited characteristics associated with temperature sensitivity. The results, therefore, indicate that the mosquito-borne, but not tick-borne, flaviviruses can be attenuated by very few passages in HeLa cells. This observation may provide a model system with which to analyse the molecular basis of attenuation and/or virulence of mosquito-borne flaviviruses.
Duck Tembusu virus (DTMUV), which is similar to other mosquito-borne flaviviruses that replicate well in most mammalian cells, is an emerging pathogenic flavivirus that has caused epidemics in egg-laying and breeding waterfowl. Immune organ defects and neurological dysfunction are the main clinical symptoms of DTMUV infection. Preinfection with DTMUV makes the virus impervious to later interferon (IFN) treatment, revealing that DTMUV has evolved some strategies to defend against host IFN-dependent antiviral responses. Immune inhibition was further confirmed by screening for DTMUV-encoded proteins, which suggested that NS2A significantly inhibited IFN-β and IFN-stimulated response element (ISRE) promoter activity in a dose-dependent manner and facilitated reinfection with duck plague virus (DPV). DTMUV NS2A was able to inhibit duck retinoic acid-inducible gene-I (RIG-I)-, and melanoma differentiation-associated gene 5 (MDA5)-, mitochondrial-localized adaptor molecules (MAVS)-, stimulator of interferon genes (STING)-, and TANK-binding kinase 1 (TBK1)-induced IFN-β transcription, but not duck TBK1- and interferon regulatory factor 7 (IRF7)-mediated effective phases of IFN response. Furthermore, we found that NS2A competed with duTBK1 in binding to duck STING (duSTING), impaired duSTING-duSTING binding, and reduced duTBK1 phosphorylation, leading to the subsequent inhibition of IFN production. Importantly, we first identified that the W164A, Y167A, and S361A mutations in duSTING significantly impaired the NS2A-duSTING interaction, which is important for NS2A-induced IFN-β inhibition. Hence, our data demonstrated that DTMUV NS2A disrupts duSTING-dependent antiviral cellular defenses by binding with duSTING, which provides a novel mechanism by which DTMUV subverts host innate immune responses. The potential interaction sites between NS2A and duSTING may be the targets of future novel antiviral therapies and vaccine development.IMPORTANCE Flavivirus infections are transmitted through mosquitos or ticks and lead to significant morbidity and mortality worldwide with a spectrum of manifestations. Infection with an emerging flavivirus, DTMUV, manifests with clinical symptoms that include lesions of the immune organs and neurological dysfunction, leading to heavy egg drop and causing serious harm to the duck industry in China, Thailand, Malaysia, and other Southeast Asian countries. Mosquito cells, bird cells, and mammalian cell lines are all susceptible to DTMUV infection. An in vivo study revealed that BALB/c mice and Kunming mice were susceptible to DTMUV after intracerebral inoculation. Moreover, there are no reports about DTMUV-related human disease, but antibodies against DTMUV and viral RNA were detected in serum samples of duck industry workers. This information implies that DTMUV has expanded its host range and may pose a threat to mammalian health. However, the pathogenesis of DTMUV is largely unclear. Our results show that NS2A strongly blocks the STING-induced signal transduction cascade by binding with STING, which subsequently blocks STING-STING binding and TBK1 phosphorylation. More importantly, the W164, Y167, or S361 residues in duSTING were identified as important interaction sites between STING and NS2A that are vital for NS2A-induced IFN production and effective phases of IFN response. Uncovering the mechanism by which DTMUV NS2A inhibits IFN in the cells of its natural hosts, ducks, will help us understand the role of NS2A in DTMUV pathogenicity.
Three novel insect-specific flaviviruses, isolated from mosquitoes collected in Peru, Malaysia (Sarawak), and the United States, are characterized. The new viruses, designated La Tina, Kampung Karu, and Long Pine Key, respectively, are antigenically and phylogenetically more similar to the mosquito-borne flavivirus pathogens, than to the classical insect-specific viruses like cell fusing agent and Culex flavivirus. The potential implications of this relationship and the possible uses of these and other arbovirus-related insect-specific flaviviruses are reviewed.
Co-infections with human immunodeficiency virus type 1 (HIV-1) and human pegivirus (HPgV) are common in hepatitis C virus (HCV)-infected individuals. However, analysis on the evolutionary dynamics and transmission network profiles of these viruses among individuals with multiple infections remains limited. A total of 228 injecting drug users (IDUs), either HCV- and/or HIV-1-infected, were recruited in Kuala Lumpur, Malaysia. HCV, HIV-1 and HPgV genes were sequenced, with epidemic growth rates assessed by the Bayesian coalescent method. Based on the sequence data, mono-, dual- and triple-infection were detected in 38.8%, 40.6% and 20.6% of the subjects, respectively. Fifteen transmission networks involving HCV (subtype 1a, 1b, 3a and 3b), HIV-1 (CRF33_01B) and HPgV (genotype 2) were identified and characterized. Genealogical estimates indicated that the predominant HCV, HIV-1 and HPgV genotypes were introduced into the IDUs population through multiple sub-epidemics that emerged as early as 1950s (HCV), 1980s (HIV-1) and 1990s (HPgV). By determining the difference in divergence times between viral lineages (ΔtMRCA), we also showed that the frequency of viral co-transmission is low among these IDUs. Despite increased access to therapy and other harm reduction interventions, the continuous emergence and coexistence of new transmission networks suggest persistent multiple viral transmissions among IDUs.
Tembusu virus (TMUV) is an important emerging arthropod-borne virus that may cause encephalitis in humans and has been isolated in regions of southeast Asia, including Malaysia, Thailand, and China. Currently, detection and identification of TMUV are limited to research laboratories, because quantitative rapid diagnostic assays for the virus do not exist. We describe the development of sensitive and specific conventional and real-time quantitative reverse transcription polymerase chain reaction assays for detecting TMUV RNA in infected cell culture supernatant and Culex tarsalis mosquitoes. We used this assay to document the replication of TMUV in Cx. tarsalis, where titers increased 1,000-fold 5 days after inoculation. These assays resulted in the detection of virus-specific RNA in the presence of copurified mosquito nucleic acids. The use of these rapid diagnostic assays may have future applications for field pathogen surveillance and may assist in early detection, diagnosis, and control of the associated arthropod-borne pathogens.
Dengue viral attacks have been reported in various parts of India in recent years. In this paper we report on our studies of the characterisation and evolutionary aspects of gene sequences of the envelope glycoprotein of the prevalent Indian dengue virus type 1. Comparison with sequences from other countries shows that the envelope genes identified in India are closely related to strains from Malaysia. From the evolutionary point of view the envelope gene sequences of this dengue virus of India for past few years show that a marked mutational shift in the nucleotide sequences of the envelope gene have taken place from around the year 2000. Also, phylogenetic relationship with other three sera of dengue virus reported in India from 2005 shows that the dengue virus 1 is more closely related to dengue viruses 3 and 4 and relatively distantly to dengue virus 2.
Viruses have spread throughout the world and cause acute illness or death among millions of people. There is a growing concern about methods to control and combat early-stage viral infections to prevent the significant public health problem. However, conventional detection methods like polymerase chain reaction (PCR) requires sample purification and are time-consuming for further clinical diagnosis. Hence, establishing a portable device for rapid detection with enhanced sensitivity and selectivity for the specific virus to prevent further spread becomes an urgent need. Many research groups are focusing on the potential of the electrochemical sensor to become a key for developing point-of-care (POC) technologies for clinical analysis because it can solve most of the limitations of conventional diagnostic methods. Herein, this review discusses the current development of electrochemical sensors for the detection of respiratory virus infections and flaviviruses over the past 10 years. Trends in future perspectives in rapid clinical detection sensors on viruses are also discussed. KEY POINTS: • Respiratory related viruses and Flavivirus are being concerned for past decades. • Important to differentiate the cross-reactivity between the virus in same family. • Electrochemical biosensor as a suitable device to detect viruses with high performance.
A new virus named Sitiawan virus (SV) was isolated from sick broiler chicks in chicken embryos. The virus replicated well with cytopathogenic effect (CPE) in the chicken B-lymphocyte cell line LSCC-BK3. The virus was an enveloped RNA virus of approximately 41 nm in size with hemagglutinating activity (HA) to goose erythrocytes. It was cross-reactive with Japanese encephalitis virus (JEV), a member of flaviviruses by HA inhibition tests but not by cross-virus neutralization tests. The cDNA fragment of NS5 gene was amplified with primers corresponding to NS5 gene of flaviviruses. The nucleotide sequences were 92% homologous to Tembusu virus, a member of the mosquito-borne virus cluster of the genus Flavivirus. In cross-neutralization tests with Tembusu virus, antiserum to SV did not neutralize Tembusu virus, and antiserum to Tembusu virus neutralized more weakly to SV than against homologous virus. These results indicate that SV is a new virus which can be differentiated serologically from Tembusu virus but is otherwise similar with respect to nucleotide sequence. The virus causes encephalitis, growth retardation, and increased blood glucose levels in inoculated chicks.
Dengue fever and its fatal complications have made a comeback since its control in the 1990’s. The Flavivirus has evolved into 4 serotypes DEN 1,2,3,4 which can be passed on by the mosquitoes for 7 generations for each serotype. This communicable disease is predominantly confined to urban areas. Quick control of the spread of the disease will prevent it from becoming an epidemic. The two species mosquitoes involved have different behaviours. The Aedes aegypti is an indoor vector which breeds in clean, clear and calm freshwater. The Aedes albopictus is an outdoor breeding mosquito which breeds in stagnant waters. Surveillance of the areas prone to outbreaks is vital. One of the roles of the entomologist is to monitor the vector for resistance to the insecticides. Localities that have been subjected to recurrent outbreaks will have vector which develop resistance to the insecticides used.
Duck Tembusu virus (DTMUV) causes substantial egg drop disease. DTMUV was first identified in China and rapidly spread to Malaysia and Thailand. The antigenicity of the DTMUV E protein has not yet been characterized. Here, we investigated antigenic sites on the E protein using the non-neutralizing monoclonal antibodies (mAbs) 1F3 and 1A5. Two minimal epitopes were mapped to (221)LD/NLPW(225) and (87)YAEYI(91) by using phage display and mutagenesis. DTMUV-positive duck sera reacted with the epitopes, thus indicating the importance of the minimal amino acids of the epitopes for antibody-epitope binding. The performance of the dot blotting assay with the corresponding positive sera indicated that YAEYI was DTMUV type-specific, whereas (221)LD/NLPW(225) was a cross-reactive epitope for West Nile virus (WNV), dengue virus (DENV), and Japanese encephalitis virus (JEV) and corresponded to conserved and variable amino acid sequences among these strains. The structure model of the E protein revealed that YAEYI and LD/NLPW were located on domain (D) II, which confirmed that DII might contain a type-specific non-neutralizing epitope. The YAEYI epitope-based antigen demonstrated its diagnostic potential by reacting with high specificity to serum samples obtained from DTMUV-infected ducks. Based on these observations, a YAEYI-based serological test could be used for DTMUV surveillance and could differentiate DTMUV infections from JEV or WNV infections. These findings provide new insights into the organization of epitopes on flavivirus E proteins that might be valuable for the development of epitope-based serological diagnostic tests for DTMUV.
To reveal whether bats serve as an amplifying host for Yokose virus (YOKV), we conducted a serological survey and experimentally infected fruit bats with YOKV isolated from microbats in Japan. YOKV belongs to the Entebbe bat virus group of vector unknown group within the genus Flavivirus and family Flaviviridae. To detect antibodies against YOKV, we developed an enzyme-linked immunosorbent assay (ELISA) using biotinylated anti-bat IgG rabbit sera. Serological surveillance was conducted with samples collected in the Philippines and the sera supplied from Malaysia. One of the 36 samples from the Philippines (2.7%) and 5 of the 26 samples from Malaysia (19%) had detectable ELISA antibodies. In the experimental infections, no clinical signs of disease were observed. Moreover, no significant viral genome amplification was detected. These findings revealed that YOKV replicates poorly in the fruit bat, suggesting that fruit bats do not seem to serve as an amplifying host for YOKV.
West Nile virus (WNV) is a zoonotic mosquito-borne flavivirus that is harbored and amplified by wild birds via the enzootic transmission cycle. Wide range of hosts are found to be susceptible to WNV infection including mammals, amphibians and reptiles across the world. Several studies have demonstrated that WNV was present in the Malaysian Orang Asli and captive birds. However, no data are available on the WNV prevalence in wild birds found in Malaysia. Therefore this study was conducted to determine the serological and molecular prevalence of WNV in wild birds in selected areas in the West Coast of Peninsular Malaysia. Two types of wild birds were screened, namely migratory and resident birds in order to explore any possibility of WNV transmission from the migratory birds to the resident birds. Thus, a cross-sectional study was conducted at the migratory birds sanctuary located in Kuala Gula, Perak and Kapar, Selangor by catching 163 migratory birds, and 97 resident birds from Kuala Gula and Parit Buntar, Perak at different time between 2016 and 2017 (Total, n = 260). Blood and oropharyngeal swabs were collected for serological and molecular analysis, respectively. Serum were screened for WNV antibodies using a commercial competitive ELISA (c-ELISA) (ID Screen® West Nile Competition Multi-species ELISA, ID VET, Montpellier, France) and cross-reactivity towards Japanese Encephalitis virus (JEV) was also carried out using the JEV-double antigen sandwich (DAS) ELISA. Oropharyngeal swabs were subjected to one-step RT-PCR to detect WNV RNA, in which positive reactions were subsequently sequenced. WNV seropositive rate of 18.71% (29/155) at 95% CI (0.131 to 0.260) and molecular prevalence of 15.2% (16/105) at 95% CI (0.092 to 0.239) were demonstrated in migratory and resident wild birds found in West Coast Malaysia. Phylogenetic analyses of the 16 WNV isolates found in this study revealed that the local strains have 99% similarity to the strains from South Africa and were clustered under lineage 2. Evidence of WNV infection in resident and migratory birds were demonstrated in this study. As a summary, intervention between migratory birds, resident birds and mosquitoes might cause the introduction and maintenance of WNV in Malaysia, however the assumption could be further proven by studying the infection dynamics in the mosquitoes present in the studied areas.
Flaviviruses are group of single stranded RNA viruses that cause severe endemic infection and epidemics on a global scale. It presents a significant health impact worldwide and the viruses have the potential to emerge and outbreak in a non-endemic geographical region. Effective vaccines for prophylaxis are only available for several flaviviruses such as Yellow Fever virus, Tick-borne Encephalitis Virus, Dengue Virus and Japanese Encephalitis Virus and there is no antiflaviviral agent being marketed. This review discusses the flavivirus genome, replication cycle, epidemiology, clinical presentation and pathogenesis upon infection. Effective humoral response is critical to confer protective immunity against flaviviruses. Hence, we have also highlighted the immune responses elicited upon infection, various diagnostic facilities available for flaviviral disease and monoclonal antibodies available to date against flavivirus infection.
Flaviviruses are potentially human pathogens that cause major epidemics worldwide. Flavivirus interacts with host cell factors to form a favourable virus replication site. Cell cytoskeletons have been observed to have close contact with flaviviruses, which expands the understanding of cytoskeleton functions during virus replication, although many detailed mechanisms are still unclear. The interactions between the virus and host cytoskeletons such as actin filaments, microtubules, and intermediate filaments have provided insight into molecular alterations during the virus infection, such as viral entry, in-cell transport, scaffold assembly, and egress. This review article focuses on the utilization of cytoskeleton by Flavivirus and the respective functions during virus replication.