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In the employment of serodiagnostic methods for the detection of orthoflavivirus infections, neutralization tests are known to be more accurate than measurements of antibody binding properties employing enzyme-linked immunosorbent assays. However, neutralization tests require infectious virus and laboratories with an appropriate level of biosafety. Single-round infectious particles (SRIPs), which encode a reporter gene instead of the viral structural protein genes, are replication incompetent and represent a safe and reliable alternative to the diagnosis of pathogenic viruses in neutralization tests. The orthoflavivirus SRIPs are produced by co-transfection of plasmids expressing virus-like particles and replicons into mammalian cell lines preferably with high transfection efficacy, such as HEK293T cells. However, certain orthoflavivirus SRIPs have limitations in their efficient expression at 37°C, which is the optimal temperature for mammalian cell growth, resulting in insufficient yields for neutralization tests. Here, we demonstrate that the production of orthoflavivirus SRIPs increases at the lower temperature of 28°C compared to 37°C. Moreover, infections with 28°C-cultured SRIPs in microneutralization tests were specifically inhibited in the presence of serum from mice infected with homologous viruses, suggesting that these SRIPs preserved their neutralizing epitopes for antibodies. Our method to produce high titer SRIPs is anticipated to promote efficient and safe SRIPs neutralization tests as a general serodiagnostic method for detecting virus-specific neutralizing antibodies against orthoflaviviruses.
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Viruses in the genus Orthohantavirus within the family Hantaviridae cause human hantavirus infections and represent a threat to public health. Hokkaido virus (HOKV), a genotype of Orthohantavirus puumalaense (Puumala virus; PUUV), was first identified in Tobetsu, Hokkaido, Japan. Although it is genetically related to the prototype of PUUV, the evolutionary pathway of HOKV is unclear. We conducted a field survey in a forest in Tobetsu in 2022 and captured 44 rodents. Complete coding genome sequences of HOKVs were obtained from five viral-RNA-positive rodents (four Myodes rufocanus bedfordiae and one Apodemus speciosus). Phylogenetic analysis revealed a close relationship between the phylogenies and geographical origins of M. rufocanus-related orthohantaviruses. Comparison of the phylogenetic trees of the S segments of orthohantaviruses and the cytochrome b genes of Myodes species suggested that Myodes-related orthohantaviruses evolved in Myodes rodent species as a result of genetic isolation and host switching.
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Evolução Molecular , Genoma Viral , Genótipo , Filogenia , Virus Puumala , Animais , Japão , Virus Puumala/genética , Virus Puumala/classificação , Arvicolinae/virologia , RNA Viral/genética , Doenças dos Roedores/virologia , Infecções por Hantavirus/virologia , Infecções por Hantavirus/veterinária , Orthohantavírus/genética , Orthohantavírus/classificaçãoRESUMO
The demand for powder X-ray diffraction analysis continues to increase in a variety of scientific fields, as the excellent beam quality of high-brightness synchrotron light sources enables the acquisition of high-quality measurement data with high intensity and angular resolution. Synchrotron powder diffraction has enabled the rapid measurement of many samples and various in situ/operando experiments in nonambient sample environments. To meet the demands for even higher throughput measurements using high-energy X-rays at SPring-8, a high-throughput and high-resolution powder diffraction system has been developed. This system is combined with six sets of two-dimensional (2D) CdTe detectors for high-energy X-rays, and various automation systems, including a system for automatic switching among large sample environmental equipment, have been developed in the third experimental hutch of the insertion device beamline BL13XU at SPring-8. In this diffractometer system, high-brilliance and high-energy X-rays ranging from 16 to 72â keV are available. The powder diffraction data measured under ambient and various nonambient conditions can be analysed using Rietveld refinement and the pair distribution function. Using the 2D CdTe detectors with variable sample-to-detector distance, three types of scan modes have been established: standard, single-step and high-resolution. A major feature is the ability to measure a whole powder pattern with millisecond resolution. Equally important, this system can measure powder diffraction data with high Q exceeding 30â Å-1 within several tens of seconds. This capability is expected to contribute significantly to new research avenues using machine learning and artificial intelligence by utilizing the large amount of data obtained from high-throughput measurements.
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The solidification of lithium-ion batteries (LIBs) by replacing liquid electrolytes with solid electrolytes enables the development of a new class of LIBs, namely all-solid-state lithium-ion batteries (ASSLIBs), with improved safety and energy density. Such battery solidification can greatly influence the properties of battery components, as exemplified by a recent report suggesting that the (dis)charge behaviour of Fe2(MoO4)3 (FMO), a promising two-phase electrode material, differs on solid electrolytes compared to liquid electrolytes. However, its underlying mechanism remains unclear. Here we examined the (de)lithiation behaviour of FMO thin films on solid electrolytes using operando synchrotron X-ray diffraction (XRD) to gain insights into the influence of the solidification on the (dis)charge mechanism of electrode materials. The XRD results revealed that FMO on solid electrolytes exhibits a monotonic peak shift over a wide capacity range, accompanied by a temporary peak broadening. This suggests that FMO possesses an expanded solid-solution reaction region and a narrower two-phase reaction region in solidified batteries compared to liquid-based LIBs. The altered (dis)charge behavior was suggested to be thermodynamically driven, as it remained largely unchanged with varying rates and under open circuit conditions. Qualitative analysis considering stress-induced variations in Gibbs free energy curves demonstrated that external stress, potentially caused by the constraint of chemo-mechanical expansion, can thermodynamically narrow the two-phase region when the chemical expansion coefficients of the two phases of FMO differ. These findings highlight the significant impact of the battery solidification on electrode material properties, emphasizing the importance of considering these unique issues in the design of ASSLIBs.
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Rab27a, a Rab family small GTPases, plays an important role in the trafficking and secretion of the intracellular proteins and has been reported to promote various viral multiplication. However, whether Rab27a is involved in West Nile virus (WNV) multiplication is unknown. This study examined the ability of Rab27a to suppress WNV multiplication. The inhibition of Rab27a expression increased viral multiplication and the intracellular levels of WNV structural proteins, E and prM proteins. Rab27a partially colocalized with E protein, mainly in the perinuclear region, while inhibition of Rab27a expression resulted in diffuse subcellular localization of E protein. In addition, some of the perinuclear E protein colocalized with the lysosomal marker LAMP1, and inhibition of lysosomal acidification increased intracellular levels of Rab27a and E proteins. These observations suggested that Rab27a inhibits WNV multiplication by inducing the degradation of viral protein in lysosomes.
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West Nile virus (WNV) causes encephalitis in human and animals. WNV is phylogenetically classified into at least five distinct genetic lineages with different pathogenicity. The pathogenesis of West Nile encephalitis is affected by ubiquitin accumulation in infected cells, but the mechanism is unknown. In this study, the association between ubiquitin accumulation and WNV pathogenicity was investigated. Ubiquitin accumulation was detected in cells infected with NY99 strain belonging to lineage-1, but not FCG and Zmq16 strains belonging to lineage-2. Substitution of the Finger and Palm sub-domains of NS5 from lineage-1 to -2 decreased ubiquitin accumulation and viral replication. Furthermore, the survival rate was increased, and viral replication and ubiquitin accumulation in the brain were attenuated, in mice inoculated with the substituted WNV compared with lineage-1 WNV. Therefore, the intracellular ubiquitin accumulation induced by the Finger and Palm sub-domains of NS5 is linked to the differences in pathogenicity among WNV lineages.
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Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Humanos , Animais , Camundongos , Vírus do Nilo Ocidental/genética , Ubiquitina , Encéfalo , Replicação Viral/genéticaRESUMO
The most conserved fusion loop (FL) domain present in the flavivirus envelope protein has been reported as a dominant epitope for cross-reactive antibodies to mosquito-borne flaviviruses (MBFVs). As a result, establishing accurate serodiagnosis for MBFV infections has been difficult as anti-FL antibodies are induced by both natural infection and following vaccination. In this study, we modified the most conserved FL domain to overcome this cross-reactivity. We showed that the FL domain of lineage I insect-specific flavivirus (ISFV) has differences in antigenicity from those of MBFVs and lineage II ISFV and determined the key amino acid residues (G106, L107, or F108), which contribute to the antigenic difference. These mutations were subsequently introduced into subviral particles (SVPs) of dengue virus type 2 (DENV2), Zika virus (ZIKV), Japanese encephalitis virus (JEV), and West Nile virus (WNV). In indirect enzyme-linked immunosorbent assays (ELISAs), these SVP mutants when used as antigens reduced the binding of cross-reactive IgG and total Ig induced by infection of ZIKV, JEV, and WNV in mice and enabled the sensitive detection of virus-specific antibodies. Furthermore, immunization of ZIKV or JEV SVP mutants provoked the production of antibodies with lower cross-reactivity to heterologous MBFV antigens compared to immunization with the wild-type SVPs in mice. This study highlights the effectiveness of introducing mutations in the FL domain in MBFV SVPs with lineage I ISFV-derived amino acids to produce SVP antigens with low cross-reactivity and demonstrates an improvement in the accuracy of indirect ELISA-based serodiagnosis for MBFV infections. KEY POINTS: ⢠The FL domain of Lineage I ISFV has a different antigenicity from that of MBFVs. ⢠Mutated SVPs reduce the binding of cross-reactive antibodies in indirect ELISAs. ⢠Inoculation of mutated SVPs induces antibodies with low cross-reactivity.
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Vírus da Encefalite Japonesa (Espécie) , Flavivirus , Vírus do Nilo Ocidental , Infecção por Zika virus , Zika virus , Animais , Camundongos , Flavivirus/genética , Zika virus/genética , Anticorpos Antivirais , Vírus do Nilo Ocidental/genética , Vírus da Encefalite Japonesa (Espécie)/genética , Mutação , Reações CruzadasRESUMO
West Nile virus (WNV) has emerged as a significant cause of viral encephalitis in humans and horses. However, the pathogenesis of the West Nile encephalitis remains unclear. Microglia are activated by WNV infection, and the pathogenic involvement of their phenotypes is controversial. In this study, we examined the diversity of microglia phenotypes caused by WNV infection by assessing various microglia markers and identified disease-associated microglia in WNV-infected mouse brain tissue. Cells positive for general microglia markers such as Iba1, P2RY12, or TMEM119 were detected in the control and WNV-infected brain tissue. The morphology of the positive cells in brain tissue infected by WNV was different from that of control brain tissue, indicating that WNV infection induced activation of microglia. The activated microglia were classified into various phenotypes by investigation of specific marker expression. Among the activated microglia, disease-associated microglia that were positive for CD11c and weakly positive for TMEM119 were detected close to the WNV-infected cells. These results indicate that WNV infection induces activation of diverse microglia phenotypes and that disease-associated microglia may be associated with the pathogenicity of WNV infection in the mouse brain.
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Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Camundongos , Animais , Humanos , Cavalos , Microglia , Encéfalo , FenótipoRESUMO
Viral protein assembly and virion budding are tightly regulated to enable the proper formation of progeny virions. At this late stage in the virus life cycle, some enveloped viruses take advantage of the host endosomal sorting complex required for transport (ESCRT) machinery, which contributes to the physiological functions of membrane modulation and abscission. Bullet-shaped viral particles are unique morphological characteristics of rhabdoviruses; however, the involvement of host factors in rhabdovirus infection and, specifically, the molecular mechanisms underlying virion formation are not fully understood. In the present study, we used a small interfering RNA (siRNA) screening approach and found that the ESCRT-I component TSG101 contributes to the propagation of rabies virus (RABV). We demonstrated that the matrix protein (M) of RABV interacts with TSG101 via the late domain containing the PY and YL motifs, which are conserved in various viral proteins. Loss of the YL motif in the RABV M or the downregulation of host TSG101 expression resulted in the intracellular aggregation of viral proteins and abnormal virus particle formation, indicating a defect in the RABV assembly and budding processes. These results indicate that the interaction of the RABV M and TSG101 is pivotal for not only the efficient budding of progeny RABV from infected cells but also for the bullet-shaped virion morphology. IMPORTANCE Enveloped viruses bud from cells with the host lipid bilayer. Generally, the membrane modulation and abscission are mediated by host ESCRT complexes. Some enveloped viruses utilize their late (L-) domain to interact with ESCRTs, which promotes viral budding. Rhabdoviruses form characteristic bullet-shaped enveloped virions, but the underlying molecular mechanisms involved remain elusive. Here, we showed that TSG101, one of the ESCRT components, supports rabies virus (RABV) budding and proliferation. TSG101 interacted with RABV matrix protein via the L-domain, and the absence of this interaction resulted in intracellular virion accumulation and distortion of the morphology of progeny virions. Our study reveals that virion formation of RABV is highly regulated by TSG101 and the virus matrix protein.
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Complexos Endossomais de Distribuição Requeridos para Transporte , Vírus da Raiva , Raiva , Humanos , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Morfogênese , Raiva/metabolismo , Vírus da Raiva/genética , Vírus da Raiva/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismo , Vírion/metabolismo , Liberação de Vírus , Linhagem Celular , AnimaisRESUMO
The chemical order and disorder of solids have a decisive influence on the material properties. There are numerous materials exhibiting chemical order/disorder of atoms with similar X-ray atomic scattering factors and similar neutron scattering lengths. It is difficult to investigate such order/disorder hidden in the data obtained from conventional diffraction methods. Herein, we quantitatively determined the Mo/Nb order in the high ion conductor Ba7Nb4MoO20 by a technique combining resonant X-ray diffraction, solid-state nuclear magnetic resonance (NMR) and first-principle calculations. NMR provided direct evidence that Mo atoms occupy only the M2 site near the intrinsically oxygen-deficient ion-conducting layer. Resonant X-ray diffraction determined the occupancy factors of Mo atoms at the M2 and other sites to be 0.50 and 0.00, respectively. These findings provide a basis for the development of ion conductors. This combined technique would open a new avenue for in-depth investigation of the hidden chemical order/disorder in materials.
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West Nile virus (WNV) is transmitted to humans and animals by a mosquito and enters the central nervous system, leading to lethal encephalitis. Reporter viruses expressing fluorescent proteins enable detection of infected cells in vitro and in vivo, facilitating evaluation of the dynamics of viral infection, and the development of diagnostic or therapeutic methods. In this study, we developed a method for production of a recombinant replication-competent WNV expressing mCherry fluorescent protein. The expression of mCherry was observed in viral antigen-positive cells in vitro and in vivo, but the growth of the reporter WNV was reduced as compared to the parental WNV. The expression of mCherry was stable during 5 passages in reporter WNV-infected culture cells. Neurological symptoms were observed in mice inoculated intracranially with the reporter WNV. The reporter WNV expressing mCherry will facilitate research into WNV replication in mouse brains.
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Febre do Nilo Ocidental , Vírus do Nilo Ocidental , Humanos , Camundongos , Animais , Vírus do Nilo Ocidental/genética , Febre do Nilo Ocidental/veterinária , Proteínas Recombinantes/genéticaRESUMO
Tick-borne encephalitis virus (TBEV) is a zoonotic virus that causes tick-borne encephalitis (TBE) in humans. Infections of Sapporo-17-Io1 (Sapporo) and Oshima 5-10 (Oshima) TBEV strains showed different pathogenic effects in mice. However, the differences between the two strains are unknown. In this study, we examined neuronal degeneration and death, and activation of glial cells in mice inoculated with each strain to investigate the pathogenesis of TBE. Viral growth was similar between Sapporo and Oshima, but neuronal degeneration and death, and activation of glial cells, was more prominent with Oshima. In human neuroblastoma cells, apoptosis and pyroptosis were not observed after TBEV infection. However, the expression of the necroptosis marker, mixed lineage kinase domain-like (MLKL) protein, was upregulated by TBEV infection, and this upregulation was more pronounced in Oshima than Sapporo infections. As necroptosis is a pro-inflammatory type of cell death, differences in necroptosis induction might be involved in the differences in neuropathogenicity of TBE.
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Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos , Animais , Encefalite Transmitida por Carrapatos/patologia , Humanos , Camundongos , Necroptose , Neurônios/patologiaRESUMO
Topochemical reactions have led to great progress in the discovery of new metastable compounds with novel chemical and physical properties. With these reactions, the overall crystal structure of the host material is generally maintained. Here we report a topochemical synthesis of a hexagonal nitride hydride, h-Ca3 CrN3 H, by heating an orthorhombic nitride, o-Ca3 CrN3 , under hydrogen at 673â K, accompanied by a rotational structural transformation. The hydrogen intercalation modifies the Ca-N rock-salt-like atomic packing in o-Ca3 CrN3 to a face-sharing octahedral chain in h-Ca3 CrN3 H, mimicking a "hinged tessellation" movement. In addition, the h-Ca3 CrN3 H exhibited stable ammonia synthesis activity when used as a catalyst.
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Hantaviruses are potentially fatal zoonotic pathogens of the family Hantaviridae. No human infection by the Hokkaido genotype of Puumala orthohantavirus (PUUV-Hok) has been reported. However, other PUUV genotypes cause hemorrhagic fever with renal syndrome (HFRS) in humans. Autophagy is a highly conserved lysosomal degradation process in eukaryotic cells that affects the replication of various viruses. In this study, we examined the role of autophagy in PUUV-Hok replication. PUUV-Hok infection induced the expression of LC3-II, an autophagosome marker, and the nucleocapsid protein (NP) of PUUV-Hok was colocalized with punctate structures of LC3. Inhibition of autophagy using an siRNA for Atg5, an autophagy-related gene, increased the replication of PUUV-Hok, whereas an autophagy inducer decreased its replication. Inhibition of lysosomal degradation increased the expression of NP and LC3-II. In summary, autophagy was induced by PUUV-Hok infection, which inhibited PUUV-Hok replication in a manner related to the degradation of the NP in lysosomes.
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Febre Hemorrágica com Síndrome Renal , Orthohantavírus , Virus Puumala , Autofagia , Genótipo , Orthohantavírus/genética , Humanos , Proteínas do Nucleocapsídeo/genética , Virus Puumala/genéticaRESUMO
In dairy cows, low fertility caused by summer heat stress continues into the cooler autumn season. This can be caused by impaired oocyte quality in small growing follicles during summer. Here, we subjected oocyte-cumulus-granulosa complexes (OCGCs) derived from early antral follicles (0.5-1 mm) to in vitro growth (IVG) culture under two different temperature settings (the control and heat shock groups), and evaluated effects of heat exposure on growth and developmental competence of oocytes, factors affecting the developmental competence of oocytes (steroidogenesis of granulosa cells, oxidative stress in oocytes, and cell-to-cell communication between oocytes and somatic cells). Oocyte diameters after culture were smaller in the heat shock group. Although nuclear maturation and cleavage rates were similar between the groups, blastocyst rates were lower in the heat shock group (0.0%) than in the control group (27.7%), and reduced glutathione (GSH) levels in oocytes were lower in the heat shock group. Supplementation of cysteine, which stimulates GSH synthesis, increased GSH level and improved blastocyst rate of heat shocked oocytes (27.9%). These results suggest that heat exposure impairs the growth and developmental competence of oocytes in early antral follicles through GSH depletion, which can induce low fertility during summer and the following autumn.
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Oócitos , Folículo Ovariano , Animais , Blastocisto , Bovinos , Feminino , Células da Granulosa , Resposta ao Choque TérmicoRESUMO
Autophagy is a lysosomal degradative pathway responsible for recycling cytosolic proteins and organelles and also functions as an innate defense mechanism that host cells use against viral infection. While many viruses have evolved mechanisms to antagonize the antiviral effects of the autophagy pathway, others subvert autophagy to facilitate replication. For flaviviruses, both the positive and negative role of autophagy in virus replication has been reported. The interplay between autophagy and tick-borne encephalitis virus (TBEV) in innate immune cells is largely unknown. Here we report the relationship between an autophagy and TBEV replication in mouse macrophage cell line PMJ2-R using Hypr strain of TBEV. First, we examined the effect of Hypr infection on the autophagy pathway. We detected a mild and a temporary increase of autophagy marker LC3-II in Hypr-infected cells. The role of autophagy in TBEV replication was evaluated in autophagy related gene 5 (Atg5) knockdown cells (shAtg5). Our results showed that during an early stage of Hypr infection the viral titers were increased, while later on, at 72 hpi, the titers have declined in shAtg5 cells compared to control. Moreover, the higher number of virus-positive cells was observed in shAtg5 cells in early stage of infection and correlated with enhanced virus entry. Finally, we found an increased production of IFN-ß in Hypr-infected shAtg5 cells in comparison to control at 48 and 72 hpi implicating that autophagy restricts the amount of IFN produced by TBEV-infected macrophages. To conclude, in mouse macrophages TBEV replication is controlled by autophagy in time dependent manner, having temporally an antiviral and then a pro-viral role during infection. Our study points out to a delicate and complex involvement of autophagy machinery at level of virus entry and IFN-ß production when controlling TBEV infection.
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Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos , Animais , Antivirais/metabolismo , Autofagia , Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/genética , Interferon beta/genética , Interferon beta/metabolismo , Macrófagos/metabolismo , Camundongos , Replicação ViralRESUMO
Tick-borne encephalitis virus (TBEV) is a zoonotic virus that causes encephalitis in humans. Various deletions have been reported in a variable region of the 3' untranslated region of the TBEV genome. This study analyzed the role of a Y-shaped secondary structure in the pathogenicity of TBEV by using reverse genetics. Deletion of the structure increased the mortality rate of virus-infected mice but did not affect the virus multiplication in cultured cells and organs. The results indicate that the secondary structure is involved in the regulation of TBEV pathogenesis.
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Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos , Animais , Vírus da Encefalite Transmitidos por Carrapatos/genética , Encefalite Transmitida por Carrapatos/genética , Encefalite Transmitida por Carrapatos/patologia , Genômica , Camundongos , Conformação de Ácido Nucleico , RNA , VirulênciaRESUMO
Tick-borne encephalitis virus (TBEV) is a zoonotic virus belonging to the genus Flavivirus of the family Flaviviridae, causing meningitis or meningoencephalitis in humans. TBEV is widely distributed across the Eurasian northern regions, including Japan. Dogs have been reported to be sentinel hosts of TBEV in endemic areas, but studies of ticks infesting dogs are limited in Japan. This study isolated a novel TBEV strain from a tick (Ixodes ovatus) collected on a dog from central Hokkaido. Whole-genome sequencing revealed that the isolated strain belonged to the Far Eastern subtype of TBEV and was classified under a different subcluster of other Japanese isolates. Nanporo-18-44 showed growth properties similar to those of Oshima 5-10 both in vitro and in vivo. The pathogenicity of both viruses was similar in mice infected intracerebrally, however they showed a distinct distribution in the infected neurons of the mouse brain. Our results suggest that infections of humans and animals by unknown strains of TBEV exist in other areas of Japan. Further surveys including those conducted outside of Hokkaido, are required to elucidate the epidemiological risk of TBEV in Japan.
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Vírus da Encefalite Transmitidos por Carrapatos , Encefalite Transmitida por Carrapatos , Ixodes , Animais , Cães , Encefalite Transmitida por Carrapatos/epidemiologia , Encefalite Transmitida por Carrapatos/veterinária , Japão/epidemiologia , Camundongos , VirulênciaRESUMO
Nucleic acid test (NAT), most typically quantitative PCR, is one of the standard methods for species specific flavivirus diagnosis. Semi-comprehensive NATs such as pan-flavivirus PCR which covers genus Flavivirus are also available; however, further specification by sequencing is required for species level differentiation. In this study, a semi-comprehensive detection system that allows species differentiation of flaviviruses was developed by integration of the pan-flavivirus PCR and Nanopore sequencing. In addition, a multiplexing method was established by adding index sequences through the PCR with a streamlined bioinformatics pipeline. This enables defining cut-off values for observed read counts. In the laboratory setting, this approach allowed the detection of up to nine different flaviviruses. Using clinical samples collected in Vietnam and Brazil, seven different flaviviruses were also detected. When compared to a commercial NAT, the sensitivity and specificity of our system were 66.7% and 95.4%, respectively. Conversely, when compared to our system, the sensitivity and specificity of the commercial NAT were 57.1% and 96.9%, respectively. In addition, Nanopore sequencing detected more positive samples (n = 8) compared to the commercial NAT (n = 6). Collectively, our study has established a semi-comprehensive sequencing-based diagnostic system for the detection of flaviviruses at extremely affordable costs, considerable sensitivity, and only requires simple experimental methods.