Dengue outbreaks and the geographic distribution of dengue vectors in Taiwan: A 20-year epidemiological analysis.

Dengue fever is an important mosquito-borne viral infectious disease that mostly occurs in tropical and subtropical areas of the world. According to epidemiological data from the Center for Disease Control of Taiwan, more than 98.62% of outbreaks of indigenous total dengue cases were reported in the southern part of Taiwan. Southern Taiwan is an aggregate area encompassing Tainan, Kaohsiung, and Pingtung, all of which are located below the Tropic of Cancer (23º35'N). With a few exceptions, dengue outbreaks mainly occur in southern Taiwan which is highly associated or overlaps with the prevalence of Aedes aegypti. A.aegypti is presumed to be absent from the northern part of Taiwan, while Aedes albopictus breeds in areas throughout the island. According a collection of 20 years of epidemiological data from Taiwan, the inability of A. aegypti to survive the winter weather in northern Taiwan may account for its restricted geographical distribution and that of dengue outbreaks it transmits. A.aegypti, unlike temperate strains of A. albopictus, lacks embryonic diapause signaled by a short photoperiod which thus reduces its cold-hardiness. Therefore it is intolerant of low temperatures that frequently accompany rains and unable to survive during winter in the northern part of Taiwan.

A novel p53 paralogue mediates antioxidant defense of mosquito cells to survive dengue virus replication.

Mosquito cells allow dengue viruses (DENVs) to undergo replication without causing serious deleterious effects on the cells, leading to advantages for dissemination to other cells. Despite this, increased accumulation of reactive oxygen species (ROS) is usually detected in C6/36 cells with DENV2 infection as shown in mammalian cells. Uniquely, oxidative stress caused by the ROS is alleviated by eliciting antioxidant defense which leads to protection of mosquito cells from the infection. In the present study, a novel p53 paralogue (p53-2) was identified and proved to be regulated in C6/36 cells with DENV2 infection. With a gene-knockdown technique, p53-2 was demonstrated to transcribe catalase which plays a critical role in reducing ROS accumulation and the death rate of infected cells. Ecologically, a higher survival rate of mosquito cells is a prerequisite for prosperous production of viral progeny, allowing infected mosquitoes to remain healthy and active for virus transmission.

The p53 gene with emphasis on its paralogues in mosquitoes.

The p53 gene is highly important in human cancers, as it serves as a tumor-suppressor gene. Subsequently, two p53 homologues, i.e., p73 and p63, with high identity of amino acids were identified, leading to construction of the p53 family. The p53 gene is highly important in human cancer because it usually transcribes genes that function by causing apoptosis in mammalian cells. In contrast, p63 and p73 tend to be more important in modulating development than inducing cell death, even though they share similar protein structures. Relatively recently, p53 was also identified in mosquitoes and many other insect species. Uniquely, its structure lacks the sterile alpha motif domain which is a putative protein-protein interaction domain and exclusively exists at the C-terminal region in p73 and p63 in mammals. A phylogenetic analysis revealed that the p53 gene derived from mosquitoes is composed of two paralogues, p53-1 and p53-2. Of these, only p53-2 is responsively upregulated by dengue 2 virus (DENV2) in C6/36 cells which usually survive the infection. This indicates that the p53 gene is closely related to DENV infection in mosquito cells. The specific significance of p53-2's involvement in cell survival from virus-induced stress is described and briefly discussed in this report.

Distinct expression of interferon-induced protein with tetratricopeptide repeats (IFIT) 1/2/3 and other antiviral genes between subsets of dendritic cells induced by dengue virus 2 infection.

Dengue virus (DENV) infection is an emerging public health hazard threatening inhabitants of the tropics and sub-tropics. Dendritic cells (DCs) are one of the major targets of DENV and the initiators of the innate immune response against the virus. However, current in vitro research on the DENV-DC interaction is hampered by the low availability of ex vivo DCs and donor variation. In the current study, we attempted to develop a novel in vitro DC model using immature DCs derived from the myeloid leukaemia cell line MUTZ-3 (IMDCs) to investigate the DENV-DC interaction. The IMDCs morphologically and phenotypically resembled human immature monocyte-derived dendritic cells (IMMoDCs). However, the permissiveness of IMDCs to DENV2 was lower than that of IMMoDCs. RT-PCR arrays showed that a group of type I interferon (IFN) -inducible genes, especially IFIT1, IFITM1, and IFI27, were significantly up-regulated in IMMoDCs but not in IMDCs after DENV2 infection. Further investigation revealed that IFIT genes were spontaneously expressed at both transcriptional and protein levels in the naive IMDCs but not in the naive IMMoDCs. It is possible that the poor permissiveness of IMDCs to DENV2 was a result of the high basal levels of IFIT proteins. We conclude that the IMDC model, although less permissive to DENV2, is a useful platform for studying the suppression mechanism of DENV2 and we expand the knowledge of cellular factors that modulate DENV2 infection in the human body.

Discriminable roles of Aedes aegypti and Aedes albopictus in establishment of dengue outbreaks in Taiwan.

Aedes aegypti and Aedes albopictus were reported to be significant as vectors of dengue fever. In Taiwan, the latter is distributed throughout the island while the former appears only south of the Tropic of Cancer; i.e., 23.5°N. In the past decade, there were five outbreaks with over 1000 cases of dengue fever in Taiwan. Without exception, these outbreaks all occurred in the south where the two Aedes mosquitoes are sympartic. According to the Center for Disease Control of Taiwan, imported cases are thought to provide the seeds of dengue outbreaks every year. Mostly, the number of imported cases is greater in northern island, probably due to a larger population of travelers and imported workers from endemic countries. Looking at the example in 2002, northern, central, and southern parts of Taiwan reported 28, 11, and 13 imported cases, respectively. However, 54, 21, and 5309 total cases were confirmed in the corresponding regions over the entire year, indicating a significant skew of case distributions. A hypothesis is thus inspired that the existence of Ae. aegypti is a prerequisite to initiate a dengue outbreak, while participation of Ae. albopictus expands or maintains the scale until the de novo herd immunity reaches high level.

Japanese encephalitis virus induces matrix metalloproteinase-9 expression via a ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent AP-1 pathway in rat brain astrocytes.

BACKGROUND: Japanese encephalitis virus (JEV) infection is a major cause of acute encephalopathy in children, which destroys central nervous system (CNS) cells, including astrocytes and neurons. Matrix metalloproteinase (MMP)-9 has been shown to degrade components of the basal lamina, leading to disruption of the blood-brain barrier (BBB) and to contribute to neuroinflammatory responses in many neurological diseases. However, the detailed mechanisms of JEV-induced MMP-9 expression in rat brain astrocytes (RBA-1 cells) are largely unclear. METHODS: In this study, the effect of JEV on expression of MMP-9 was determined by gelatin zymography, western blot analysis, RT-PCR, and promoter assay. The involvement of AP-1 (c-Jun and c-Fos), c-Src, PDGFR, PI3K/Akt, and MAPKs in these responses were investigated by using the selective pharmacological inhibitors and transfection with siRNAs. RESULTS: Here, we demonstrate that JEV induces expression of pro-form MMP-9 via ROS/c-Src/PDGFR/PI3K/Akt/MAPKs-dependent, AP-1 activation in RBA-1 cells. JEV-induced MMP-9 expression and promoter activity were inhibited by pretreatment with inhibitors of AP-1 (tanshinone), c-Src (PP1), PDGFR (AG1296), and PI3K (LY294002), and by transfection with siRNAs of c-Jun, c-Fos, PDGFR, and Akt. Moreover, JEV-stimulated AP-1 activation was inhibited by pretreatment with the inhibitors of c-Src, PDGFR, PI3K, and MAPKs. CONCLUSION: From these results, we conclude that JEV activates the ROS/c-Src/PDGFR/PI3K/Akt/MAPKs pathway, which in turn triggers AP-1 activation and ultimately induces MMP-9 expression in RBA-1 cells. These findings concerning JEV-induced MMP-9 expression in RBA-1 cells imply that JEV might play an important role in CNS inflammation and diseases.

Polyglutamine-expanded ataxin-7 causes cerebellar dysfunction by inducing transcriptional dysregulation.

In the present study, we prepared an animal model of adult-onset spinocerebellar ataxia type 7 (SCA7) by generating transgenic mice expressing polyglutamine-expanded ataxin-7-Q52. Mutant ataxin-7-Q52 was expressed in brain areas implicated in SCA7 neurodegeneration, including cerebellum and inferior olivary nucleus. Ataxin-7-Q52 transgenic mice exhibited symptoms of motor dysfunction with an onset age of 7 months, and neurological phenotypes deteriorated in the following months. Ten to eleven-month-old ataxin-7-Q52 mice displayed ataxic symptoms without prominent cerebellar neuronal death, suggesting that ataxin-7-Q52 causes cerebellar malfunction and ataxia. To investigate the involvement of transcriptional dysregulation in ataxin-7-Q52-induced cerebellar dysfunction, microarray analysis and real-time RT-PCR assays were performed to identify altered cerebellar mRNA expressions of 10-11-month-old ataxin-7-Q52 transgenic mice. Ataxin-7-Q52 mice exhibited downregulated mRNA expressions of proteins involved in glutamatergic transmission, signal transduction, myelin formation, deubiquitination, axon transport, neuronal differentiation or glial functions and heat shock proteins. The involvement of transcriptional abnormality in initiating SCA7 pathological process was indicated by the finding that 6-month-old ataxin-7-Q52 transgenic mice, which did not display noticeable ataxic symptoms, exhibited dysregulated mRNA expressions. Our study suggests that polyglutamine-expanded ataxin-7-induced transcriptional dysregulation causes cerebellar dysfunction and ataxia.

Protective immunity of E. coli-synthesized NS1 protein of Japanese encephalitis virus.

Immunogenicity and protective efficacy of recombinant Japanese encephalitis virus (JEV) NS1 proteins generated using DNA vaccines and recombinant viruses have been demonstrated to induce protection in mice against a challenge of JEV at a lethal dose. The West Nile virus NS1 region expressed in E. coli is recognized by these protective monoclonal antibodies and, in this study, we compare immunogenicity and protective immunity of the E. coli-synthesized NS1 protein with another protective immunogen, the envelope domain III (ED3). Pre-challenge, detectable titers of JEV-specific neutralizing antibody were detected in the immunized mice with E. coli-synthesized ED3 protein (PRNT50 = 1:28) and the attenuated JEV strain T1P1 (PRNT50 = 1:53), but neutralizing antibodies were undetectable in the immunized mice with E. coli-synthesized NS1 protein (PRNT50 < 1:10). However, the survival rate of the NS1-immunized mice against the JEV challenge was 87.5% (7/8), showing significantly higher levels of protection than the ED3-immunized mice, 62.5% (5/8) (P = 0.041). In addition, E. coli-synthesized NS1 protein induced a significant increase of anti-NS1 IgG1 antibodies, resulting in an ELISA titer of 100,1000 in the immunized sera before lethal JEV challenge. Surviving mice challenged with the virulent JEV strain Beijing-1 showed a ten-fold or greater rise in IgG1 and IgG2b titers of anti-NS1 antibodies, implying that the Th2 cell activation might be predominantly responsible for antibody responses and mice protection.

Phenotypic changes in the Japanese encephalitis virus after one passage in Neuro-2a cells: generation of attenuated strains of the virus.

Live attenuated strains of the Japanese encephalitis (JE) virus are known to form in various cultured cells. In this study, we selected attenuated JE virus variants by passing the parent virus strains once through Neuro-2a cells, showing that the selection intensity ranged 2-1000-fold after infection for 3d. The selection of attenuated variants appeared in association with mutations on the envelope (E) glycoprotein. This is likely determined by the differential binding abilities of specific E proteins with highly sulfated glycosaminoglycans on the cell surface. A plaque-purification method showed that Neuro-2a-selected variants were usually less neurovirulent, leading to a longer survival time of intracerebrally inoculated mice. Specific selected variants (mostly with the small-plaque phenotype) were shown to be more efficient at replication in Vero cells and less virulent, particularly those with substitution of the E-138 (Glu-->Lys) residue. As a result, most, if not all, low-virulent variants were generated in a relatively short time, all which have the potential to be live-attenuated vaccine candidates of the JE virus.

Functional determinants of NS2B for activation of Japanese encephalitis virus NS3 protease.

Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus, causing severe central nerve system diseases without specific treatments. The NS2B-NS3 protease of flaviviruses mediates several cleavages on the flavivirus polyprotein, being believed to be a target for antiviral therapy. NS2B is the cofactor of the viral serine protease, correlating with stabilization and substrate recognition of the NS3 protease. In this study, we investigate the functional determinants in the JEV NS2B for the activation of the NS3 protease. Cis- and trans-cleavage assays of the deletions at the N-terminal of NS2B demonstrated that the NS2B residues Ser(46) to Ile(60) were the essential region required for both cis and trans activity of the NS3 protease. In addition, alanine substitution at the residues Trp53, Glu55, and Arg56 in NS2B significantly reduced the cis- and trans-cleavage activities of the NS3 protease. Sequence alignment and modeled structures suggested that functional determinants at the JEV NS2B residues Ser46 to Ile60, particularly in Trp53, Glu55 and Arg56 could play an important configuration required for the activity of the flavivirus NS3 protease. Our results might be useful for development of inhibitors that block the interaction between NS2B and NS3.

Cloning, purification, and nucleotide-binding traits of the catalytic subunit A of the V1VO ATPase from Aedes albopictus.

The Asian tiger mosquito, Aedes albopictus, is commonly infected by the gregarine parasite Ascogregarina taiwanensis, which develops extracellularly in the midgut of infected larvae. The intracellular trophozoites are usually confined within a parasitophorous vacuole, whose acidification is generated and controlled by the V(1)V(O) ATPase. This proton pump is driven by ATP hydrolysis, catalyzed inside the major subunit A. The subunit A encoding gene of the Aedes albopictus V(1)V(O) ATPase was cloned in pET9d1-His(3) and the recombinant protein, expressed in the Escherichia coli Rosetta 2 (DE3) strain, purified by immobilized metal affinity- and ion-exchange chromatography. The purified protein was soluble and properly folded. Analysis of secondary structure by circular dichroism spectroscopy showed that subunit A comprises 43% alpha-helix, 25% beta-sheet and 40% random coil content. The ability of subunit A of eukaryotic V-ATPases to bind ATP and/or ADP is demonstrated by photoaffinity labeling and fluorescence correlation spectroscopy (FCS). Quantitation of the FCS data indicates that the ADP-analogues bind slightly weaker to subunit A than the ATP-analogues. Tryptophan fluorescence quenching of subunit A after binding of different nucleotides provides evidence for secondary structural alterations in this subunit caused by nucleotide-binding.

Strategically examining the full-genome of dengue virus type 3 in clinical isolates reveals its mutation spectra.

BACKGROUND: Previous studies presented the quasispecies spectrum of the envelope region of dengue virus type 3 (DENV-3) from either clinical specimens or field-caught mosquitoes. However, the extent of sequence variation among full genomic sequences of DENV within infected individuals remains largely unknown. RESULTS: Instead of arbitrarily choosing one genomic region in this study, the full genomic consensus sequences of six DENV-3 isolates were used to locate four genomic regions that had a higher potential of sequence heterogeneity at capsid-premembrane (C-prM), envelope (E), nonstructural protein 3 (NS3), and NS5. The extent of sequence heterogeneity revealed by clonal sequencing was genomic region-dependent, whereas the NS3 and NS5 had lower sequence heterogeneity than C-prM and E. Interestingly, the Phylogenetic Analysis by Maximum Likelihood program (PAML) analysis supported that the domain III of E region, the most heterogeneous region analyzed, was under the influence of positive selection. CONCLUSION: This study confirmed previous reports that the most heterogeneous region of the dengue viral genome resided at the envelope region, of which the domain III was under positive selection pressure. Further studies will need to address the influence of these mutations on the overall fitness in different hosts (i.e., mosquito and human) during dengue viral transmission.

Fitness of Japanese encephalitis virus to Neuro-2a cells is determined by interactions of the viral envelope protein with highly sulfated glycosaminoglycans on the cell surface.

Genetically different subpopulations were identified and purified from Japanese Encephalitis virus (JEV). Those with small plaques (SPs; <2 mm in diameter), derived from strains of T1P1, CJN, and CC27, were more competent than those with large plaques (LPs; >5 mm in diameter) when passaged in Neuro-2a cells. Differences in amino acids between SPs and LPs from each strain were shown in the viral envelope (E) protein. The amino acid at E-306 was Glu in LP but was substituted by Lys in SP in the T1P1 strain. A similar substitution occurred at E-138 in the CJN strain. However, the amino acid was Asp in LP but was substituted by Asn in SP at E-389 in the CC27 strain. All SPs were shown to have a higher affinity to the cellular membrane when compared to LPs, and this resulted in more-efficient infection of Neuro-2a cells, suggesting that the differential fitness of JEV variants to Neuro-2a cells appeared in the early phase of infection. In addition, glycosaminoglycans (GAGs) on the surface of many mammalian cells have been demonstrated to be critical for infection by JEV, especially SP variants. The present results suggest that T1P1-SP1 viruses infected Neuro-2a cells more efficiently in spite of the sparse distribution of cell surface GAGs. We conclude that highly sulfated forms of GAGs expressed by Neuro-2a cells play an important role in selecting JEV variants with specific mutations in the E glycoprotein.

Study of sequence variation of dengue type 3 virus in naturally infected mosquitoes and human hosts: implications for transmission and evolution.

Dengue virus is an arbovirus that replicates alternately in the mosquito vector and human host. We investigated sequences of dengue type 3 virus in naturally infected Aedes aegypti mosquitoes and in eight patients from the same outbreak and reported that the extent of sequence variation seen with the mosquitoes was generally lower than that seen with the patients (mean diversity, 0.21 versus 0.38% and 0.09 versus 0.23% for the envelope [E] and capsid [C] genes, respectively). This was further verified with five experimentally infected mosquitoes (mean diversity, 0.09 and 0.10% for the E and C genes, respectively). Examination of the quasispecies structures of the E sequences of the mosquitoes and of the patients revealed that the sequences of the major variants were the same, suggesting that the major variant was transmitted. These findings support our hypothesis that mosquitoes contribute to the evolutionary conservation of dengue virus by maintaining a more homogenous viral population and a dominant variant during transmission.

Differential binding efficiency between the envelope protein of Japanese encephalitis virus variants and heparan sulfate on the cell surface.

Japanese encephalitis (JE) virus infects a number of host cells, either mosquitoes or vertebrates, in nature. The viral envelope (E) protein is known to interact with molecule(s) on the cell membrane during the early stage of virus infection. In this study, two sets of virus variants including T1P1-L4/T1P1-S1 and CJN-L1/CJN-S1 derived from two strains (T1P1 and CJN) of the JE virus were used to evaluate the effects of genomic variations on virus entry. Each set of virus variant (T1P1-L4/T1P1-S1 or CJN-L1/CJN-S1) possessed a single amino acid variation in the E protein. The variation of Glu/Lys at E-306 was found between T1P1-L4 and T1P1-S1 whereas the same variation at E-138 was seen between CJN-L1 and CJN-S1. The results showed that heparan sulfate (HS) differentially expressed on the surface of different types of host cells was essential for JE virus infection as shown in an evident difference in attachment efficiency between CHO-K1 cells and its mutant with defects in GAG biosynthesis. Furthermore, differential interaction of heparin with the envelope protein of JE virus variants implies the significance of virus mutations (especially Lys for E-138 and/or E306 in this case) that are rather likely involved in determining efficiencies of viral attachment, penetration, and eventual infection.

E/NS1 modifications of dengue 2 virus after serial passages in mammalian and/or mosquito cells.

OBJECTIVE: Dengue viruses are routinely maintained in nature by transmission cycles involving the passage of virus between humans and AEDES mosquitoes. The number of dengue virus lineages has been increasing over time. The aim of this study was to identify the genetic diversity of dengue 2 virus serially transferred in mammalian and/or mosquito cells. METHODS: The E/NS1 gene of dengue 2 virus variants derived from serial passages in Vero or C6/36 cells, or alternately in both cell systems, was amplified and sequenced in order to observe gene modification after serial passages. RESULTS: Three nucleotides (two in E and one in NS1) or two amino acids (one each in E and NS1) changed in the virus that was continuously cultured in Vero cells for 20 passages, whereas four nucleotides (two each in E and NS1) or three amino acids (one in E and two in NS1) changed in the virus cultured for 30 passages. The genome of dengue 2 virus remained stable even when the virus was serially transferred in C6/36 cells for 30 generations. However, there was one amino acid substitution (E46 I-->V) resulting from a single nucleotide change in the E region of dengue 2 virus alternately transferred in C6/36 and Vero cells for either 20 or 30 passages. In addition, dengue 2 virus obtained from serially cultured Vero cells usually replicated better when it reinfected Vero cells, reflecting its high adaptation fitness to the host cell. CONCLUSIONS: It is concluded that genetic changes of dengue 2 virus are constrained in Vero (mammalian) cells, resulting in a variety of genome-related quasispecies populations. Some populations of the virus are subsequently selected by and genetically (at least in the E/NS1 portion of the viral genome) maintained in C6/36 (mosquito) cells during replicative competition.