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1.
PLoS One ; 17(1): e0262096, 2022.
Article in English | MEDLINE | ID: covidwho-1841139

ABSTRACT

BACKGROUND: Dengue usually progress abnormally, especially in the critical phase. The main causes of death were shock, severe bleeding and organ failure. The aim of our study was to evaluate prognostic indicators of severe dengue according to the phases of the disease progression. METHODS: A cross-sectional study was conducted from July to December 2017 at the National Hospital for Tropical Diseases and the Hospital for Tropical Diseases of Ho Chi Minh City. 326 patients, aged 6 years and over, including 99/326 patients with severe dengue and 227/326 patients with non-severe dengue, hospitalized in the first 3 days of illness, confirmed Dengue virus by the RT-PCR assay have been registered for the study. Clinical manifestations were monitored daily. The hematocrit, white blood cells, platelet, serum albumin, ALT, AST, bilirubin, prothrombin time (PT%, PTs), fibrinogen, aPTT, INR and creatinine were evaluated at two times: febrile phase and critical phase. RESULTS: Independent factors associated with severe dengue were identified on multivariate logistic regression models. During the first 3 days of the disease, the prognostic indicators were platelet count ≤ 100 G/L (OR = 2.2; 95%CI: 1.2-3.9), or serum albumin < 35 g/L (OR = 3.3; 95%CI: 1.8-6.1). From day 4-6, the indicator were AST > 400 U/L (OR = 3.0; 95%CI: 1.1-7.9), ALT > 400 U/L (OR = 6.6; 95%CI: 1.7-24.6), albumin < 35 g/L (OR = 3.0; 95%CI: 1.5-5.9), and bilirubin total >17 µmol/L (OR = 4.6; 95%CI: 2.0-10.4). CONCLUSION: To predict the risk of patients with severe dengue, prognostic laboratory indicators should be indicated consistent with the progression of the disease. During the first 3 days of illness, prognostic indicators should be platelet count, or serum albumin. From the 4th - 6th day of illness, prognostic indicators should be AST, ALT, albumin, or bilirubin total.


Subject(s)
Dengue Virus/genetics , RNA, Viral/genetics , Serum Albumin/analysis , Severe Dengue/diagnosis , Adolescent , Adult , Aged , Aged, 80 and over , Child , Cross-Sectional Studies , Disease Progression , Female , Hospitalization , Humans , Leukocyte Count , Male , Middle Aged , Platelet Count , Prognosis , Severe Dengue/blood , Severe Dengue/mortality , Thrombin Time , Vietnam , Young Adult
2.
Am J Trop Med Hyg ; 106(3): 896-899, 2022 01 24.
Article in English | MEDLINE | ID: covidwho-1649221

ABSTRACT

We provide evidence of concurrent and close sequential infections between SARS-CoV-2 and select arboviruses-namely, chikungunya virus (CHIKV); dengue viruses 1, 2, and 3 (DENV1-3), and Zika virus (ZIKV)-in patients in Guerrero, southwest Mexico, in 2020-2021. The study population consisted of 176 febrile patients with laboratory evidence of SARS-CoV-2 infection. Sera from all patients were serologically and antigenically tested for seven arboviruses known to occur in Guerrero. Eighteen patients contained CHIKV IgM, six of whom also contained CHIKV RNA. Another 16 patients contained flavivirus antigen. The flaviviruses responsible for the infections were identified by plaque reduction neutralization test as DENV1 (two patients), DENV2 (five patients), DENV3 (three patients), ZIKV (three patients), and an undetermined flavivirus (three patients). In summary, we identified patients in Guerrero, Mexico, with concurrent or recent sequential infections between SARS-CoV-2 and select arboviruses, exemplifying the importance of performing differential diagnosis in regions where these viruses cocirculate.


Subject(s)
Arboviruses , COVID-19 , Chikungunya Fever , Coinfection , Dengue Virus , Dengue , Zika Virus Infection , Zika Virus , COVID-19/epidemiology , Dengue/diagnosis , Dengue Virus/genetics , Humans , Mexico/epidemiology , SARS-CoV-2 , Zika Virus/genetics , Zika Virus Infection/epidemiology
3.
Mol Ther ; 30(5): 2058-2077, 2022 May 04.
Article in English | MEDLINE | ID: covidwho-1612108

ABSTRACT

The ongoing COVID-19 pandemic highlights the need to tackle viral variants, expand the number of antigens, and assess diverse delivery systems for vaccines against emerging viruses. In the present study, a DNA vaccine candidate was generated by combining in tandem envelope protein domain III (EDIII) of dengue virus serotypes 1-4 and a dengue virus (DENV)-2 non-structural protein 1 (NS1) protein-coding region. Each domain was designed as a serotype-specific consensus coding sequence derived from different genotypes based on the whole genome sequencing of clinical isolates in India and complemented with data from Africa. This sequence was further optimized for protein expression. In silico structural analysis of the EDIII consensus sequence revealed that epitopes are structurally conserved and immunogenic. The vaccination of mice with this construct induced pan-serotype neutralizing antibodies and antigen-specific T cell responses. Assaying intracellular interferon (IFN)-γ staining, immunoglobulin IgG2(a/c)/IgG1 ratios, and immune gene profiling suggests a strong Th1-dominant immune response. Finally, the passive transfer of immune sera protected AG129 mice challenged with a virulent, non-mouse-adapted DENV-2 strain. Our findings collectively suggest an alternative strategy for dengue vaccine design by offering a novel vaccine candidate with a possible broad-spectrum protection and a successful clinical translation either as a stand alone or in a mix and match strategy.


Subject(s)
COVID-19 , Dengue Vaccines , Dengue Virus , Dengue , Vaccines, DNA , Antibodies, Neutralizing , Antibodies, Viral , Dengue/prevention & control , Dengue Vaccines/genetics , Dengue Virus/genetics , Humans , Pandemics , Viral Envelope Proteins/genetics
4.
Infect Genet Evol ; 96: 105106, 2021 12.
Article in English | MEDLINE | ID: covidwho-1506080

ABSTRACT

Coronaviruses (especially SARS-CoV-2) are characterized by rapid mutation and wide spread. As these characteristics easily lead to global pandemics, studying the evolutionary relationship between viruses is essential for clinical diagnosis. DNA sequencing has played an important role in evolutionary analysis. Recent alignment-free methods can overcome the problems of traditional alignment-based methods, which consume both time and space. This paper proposes a novel alignment-free method called the correlation coefficient feature vector (CCFV), which defines a correlation measure of the L-step delay of a nucleotide location from its location in the original DNA sequence. The numerical feature is a 16×L-dimensional numerical vector describing the distribution characteristics of the nucleotide positions in a DNA sequence. The proposed L-step delay correlation measure is interestingly related to some types of L+1 spaced mers. Unlike traditional gene comparison, our method avoids the computational complexity of multiple sequence alignment, and hence improves the speed of sequence comparison. Our method is applied to evolutionary analysis of the common human viruses including SARS-CoV-2, Dengue virus, Hepatitis B virus, and human rhinovirus and achieves the same or even better results than alignment-based methods. Especially for SARS-CoV-2, our method also confirms that bats are potential intermediate hosts of SARS-CoV-2.


Subject(s)
Genome, Viral/genetics , Phylogeny , Sequence Analysis, DNA/methods , Coronavirus/genetics , Dengue Virus/genetics , Hepatitis B/genetics , Humans , Models, Genetic , Rhinovirus/genetics , SARS-CoV-2/genetics , Sequence Alignment
5.
J Virol ; 95(24): e0059621, 2021 11 23.
Article in English | MEDLINE | ID: covidwho-1443352

ABSTRACT

Cellular factors have important roles in all facets of the flavivirus replication cycle. Deciphering viral-host protein interactions is essential for understanding the flavivirus life cycle as well as development of effective antiviral strategies. To uncover novel host factors that are co-opted by multiple flaviviruses, a CRISPR/Cas9 genome wide knockout (KO) screen was employed to identify genes required for replication of Zika virus (ZIKV). Receptor for Activated Protein C Kinase 1 (RACK1) was identified as a novel host factor required for ZIKV replication, which was confirmed via complementary experiments. Depletion of RACK1 via siRNA demonstrated that RACK1 is important for replication of a wide range of mosquito- and tick-borne flaviviruses, including West Nile Virus (WNV), Dengue Virus (DENV), Powassan Virus (POWV) and Langat Virus (LGTV) as well as the coronavirus SARS-CoV-2, but not for YFV, EBOV, VSV or HSV. Notably, flavivirus replication was only abrogated when RACK1 expression was dampened prior to infection. Utilising a non-replicative flavivirus model, we show altered morphology of viral replication factories and reduced formation of vesicle packets (VPs) in cells lacking RACK1 expression. In addition, RACK1 interacted with NS1 protein from multiple flaviviruses; a key protein for replication complex formation. Overall, these findings reveal RACK1's crucial role to the biogenesis of pan-flavivirus replication organelles. IMPORTANCE Cellular factors are critical in all facets of viral lifecycles, where overlapping interactions between the virus and host can be exploited as possible avenues for the development of antiviral therapeutics. Using a genome-wide CRISPR knockout screening approach to identify novel cellular factors important for flavivirus replication we identified RACK1 as a pro-viral host factor for both mosquito- and tick-borne flaviviruses in addition to SARS-CoV-2. Using an innovative flavivirus protein expression system, we demonstrate for the first time the impact of the loss of RACK1 on the formation of viral replication factories known as 'vesicle packets' (VPs). In addition, we show that RACK1 can interact with numerous flavivirus NS1 proteins as a potential mechanism by which VP formation can be induced by the former.


Subject(s)
CRISPR-Cas Systems , Flavivirus/genetics , Neoplasm Proteins/genetics , Receptors for Activated C Kinase/genetics , Virus Replication , A549 Cells , Aedes , Animals , COVID-19 , Chlorocebus aethiops , Culicidae , Dengue Virus/genetics , Genome-Wide Association Study , HEK293 Cells , Host-Pathogen Interactions/genetics , Humans , RNA, Small Interfering/metabolism , RNA, Viral/metabolism , SARS-CoV-2 , Vero Cells , West Nile virus/genetics , Zika Virus/genetics , Zika Virus Infection/virology
6.
Commun Biol ; 4(1): 557, 2021 05 11.
Article in English | MEDLINE | ID: covidwho-1387494

ABSTRACT

Dengue virus (DENV) is spread from human to human through the bite of the female Aedes aegypti mosquito and leads to about 100 million clinical infections yearly. Treatment options and vaccine availability for DENV are limited. Defective interfering particles (DIPs) are considered a promising antiviral approach but infectious virus contamination has limited their development. Here, a DENV-derived DIP production cell line was developed that continuously produced DENV-free DIPs. The DIPs contained and could deliver to cells a DENV serotype 2 subgenomic defective-interfering RNA, which was originally discovered in DENV infected patients. The DIPs released into cell culture supernatant were purified and could potently inhibit replication of all DENV serotypes in cells. Antiviral therapeutics are limited for many viral infection. The DIP system described could be re-purposed to make antiviral DIPs for many other RNA viruses such as SARS-CoV-2, yellow fever, West Nile and Zika viruses.


Subject(s)
Defective Viruses , Dengue Vaccines/therapeutic use , Dengue Virus/growth & development , Dengue/prevention & control , Virus Replication , Animals , Cell Line, Tumor , Chlorocebus aethiops , Defective Viruses/genetics , Defective Viruses/metabolism , Dengue/virology , Dengue Virus/genetics , Dengue Virus/metabolism , Genes, Reporter , HEK293 Cells , Host-Pathogen Interactions , Humans , Luminescent Proteins/biosynthesis , Luminescent Proteins/genetics , RNA, Viral/biosynthesis , RNA, Viral/genetics , Vero Cells , Viral Load
7.
J Travel Med ; 28(8)2021 12 29.
Article in English | MEDLINE | ID: covidwho-1364814

ABSTRACT

BACKGROUND: In August 2020, in the context of COVID-19 pandemics, an autochthonous dengue outbreak was identified for the first time in Italy. METHODS: Following the reporting of the index case of autochthonous dengue, epidemiological investigation, vector control and substances of human origin safety measures were immediately activated, according to the national arbovirus surveillance plan. Dengue cases were followed-up with weekly visits and laboratory tests until recovery and clearance of viral RNA from blood. RESULTS: The primary dengue case was identified in a young woman, who developed fever after returning from Indonesia to northern Italy, on 27 July 2020. She spent the mandatory quarantine for COVID-19 at home with relatives, six of whom developed dengue within two weeks. Epidemiological investigation identified further five autochthonous dengue cases among people who lived or stayed near the residence of the primary case. The last case of the outbreak developed fever on 29 September 2020. Dengue cases had a mild febrile illness, except one with persistent asthenia and myalgia. DENV-1 RNA was detected in blood and/or urine in all autochthonous cases, up to 35 days after fever onset. All cases developed IgM and IgG antibodies which cross-reacted with West Nile virus (WNV) and other flaviviruses. Sequencing of the full viral genome from blood samples showed over 99% nucleotide identity with DENV-1 strains isolated in China in 2014-2015; phylogenetic analysis classified the virus within Genotype I. Entomological site inspection identified a high density of Aedes albopictus mosquitoes, which conceivably sustained local DENV-1 transmission. Aedes koreicus mosquitoes were also collected in the site. CONCLUSIONS: Areas in Europe with high density of Aedes mosquitoes should be considered at risk for dengue transmission. The presence of endemic flaviviruses, such as WNV, might pose problems in the laboratory diagnosis.


Subject(s)
Aedes , COVID-19 , Dengue Virus , Dengue , Animals , Dengue/epidemiology , Dengue Virus/genetics , Disease Outbreaks , Female , Humans , Italy/epidemiology , Mosquito Vectors , Phylogeny , SARS-CoV-2
8.
Diagn Microbiol Infect Dis ; 101(4): 115517, 2021 Dec.
Article in English | MEDLINE | ID: covidwho-1347571

ABSTRACT

Dengue and COVID-19 cocirculation presents a diagnostic conundrum for physicians evaluating patients with acute febrile illnesses, both in endemic regions and among returning travelers. We present a case of a returning traveler from Pakistan who, following repeated negative SARS-CoV-2 tests, was found to have a Dengue virus serotype 2 infection.


Subject(s)
COVID-19/diagnosis , Dengue/diagnosis , SARS-CoV-2 , Adult , COVID-19/epidemiology , California/epidemiology , Dengue/epidemiology , Dengue Virus/classification , Dengue Virus/genetics , Female , Genome, Viral , Humans , Pakistan/epidemiology , Phylogeny , Reverse Transcriptase Polymerase Chain Reaction , Serogroup , Travel
9.
Drug Discov Ther ; 15(3): 130-138, 2021.
Article in English | MEDLINE | ID: covidwho-1296656

ABSTRACT

Dengue is a life-threatening mosquito borne viral disease. We are still in the era of supportive treatment where morbidity and mortality are a major concern. Dengue infection in presence of other co-infections makes this scenario rather worse. Timely recognition and raising alarm to be intensive is the need of the hour for primary care physicians practicing in the community and indoors. This review provides a comprehensive knowledge about the recent trends of coinfection in dengue as well as their management consideration which will be particularly helpful for physicians practicing in rural and remote areas of India.


Subject(s)
Bacterial Infections/therapy , Coinfection/therapy , Dengue Virus , Malaria/therapy , Virus Diseases/therapy , Bacterial Infections/epidemiology , Coinfection/epidemiology , Dengue Virus/genetics , Dengue Virus/pathogenicity , Humans , Malaria/epidemiology , Reinfection , Serogroup , Virulence , Virus Diseases/epidemiology
10.
Viruses ; 13(6)2021 06 16.
Article in English | MEDLINE | ID: covidwho-1286940

ABSTRACT

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.


Subject(s)
Communicable Diseases, Emerging/epidemiology , Communicable Diseases, Emerging/virology , Dengue Virus/classification , Dengue Virus/genetics , Dengue/epidemiology , Dengue/virology , Genotype , Adolescent , Amino Acid Substitution , Child , Child, Preschool , Dengue/diagnosis , Dengue/history , Dengue Virus/isolation & purification , Disease Outbreaks , Genetic Variation , Genome, Viral , History, 21st Century , Humans , Myanmar , Phylogeny , Seroepidemiologic Studies , Serogroup , Whole Genome Sequencing
12.
PLoS Negl Trop Dis ; 15(5): e0009441, 2021 05.
Article in English | MEDLINE | ID: covidwho-1243839

ABSTRACT

The dengue fever epidemic in Guangzhou may have been affected by the Coronavirus Disease 2019 (COVID-19) pandemic. The number of dengue cases dropped drastically in 2020, and there have been only 2 local cases, suggesting that dengue has not become endemic in Guangzhou.


Subject(s)
COVID-19/epidemiology , Dengue/epidemiology , Dengue/prevention & control , SARS-CoV-2 , China/epidemiology , Dengue/virology , Dengue Virus/genetics , Humans , Quarantine
13.
Am J Trop Med Hyg ; 104(2): 487-489, 2020 Dec 15.
Article in English | MEDLINE | ID: covidwho-1175672

ABSTRACT

We report a 50-year-old Thai woman with recent travel to Denmark who presented with acute high-grade fever, vomiting, and myalgia for 1 day. Initial laboratory results revealed leukopenia, elevated aspartate transaminase, and elevated alanine transaminase. Chest radiograph showed no pulmonary infiltration. Reverse transcriptase-PCR (RT-PCR) of the nasopharyngeal swab detected SARS-CoV-2, and RT-PCR of the blood detected dengue virus serotype 2. COVID-19 with dengue fever co-infection was diagnosed. Her symptoms were improved with supportive treatment. Integration of clinical manifestations, history of exposure, laboratory profiles, and dynamic of disease progression assisted the physicians in precise diagnosis. Co-circulating and nonspecific presentations of dengue infection and COVID-19 challenge the healthcare system in tropical countries. To solve this threat, multi-sector strategies are required, including public health policy, development of accurate point-of-care testing, and proper prevention for both diseases.


Subject(s)
COVID-19/diagnosis , Coinfection/diagnosis , Coinfection/virology , Dengue/diagnosis , Travel , Dengue Virus/classification , Dengue Virus/genetics , Dengue Virus/isolation & purification , Female , Humans , Middle Aged , SARS-CoV-2/genetics , SARS-CoV-2/isolation & purification , Serogroup , Thailand
14.
J Virol ; 95(4)2021 01 28.
Article in English | MEDLINE | ID: covidwho-1117221

ABSTRACT

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.


Subject(s)
COVID-19/virology , Dengue Virus/isolation & purification , Dengue/virology , SARS-CoV-2/isolation & purification , A549 Cells , Animals , COVID-19/diagnosis , COVID-19/metabolism , COVID-19/pathology , Cell Line , Chlorocebus aethiops , Dengue/diagnosis , Dengue/metabolism , Dengue/pathology , Dengue Virus/genetics , Dengue Virus/metabolism , Genes, Reporter , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Nuclear Localization Signals/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Vero Cells , Viral Nonstructural Proteins/metabolism , Virus Replication
16.
Biomolecules ; 11(1)2020 12 24.
Article in English | MEDLINE | ID: covidwho-1067684

ABSTRACT

Phenolic compounds have been related to multiple biological activities, and the antiviral effect of these compounds has been demonstrated in several viral models of public health concern. In this review, we show the antiviral role of phenolic compounds against dengue virus (DENV), the most widespread arbovirus globally that, after its re-emergence, has caused multiple epidemic outbreaks, especially in the last two years. Twenty phenolic compounds with anti-DENV activity are discussed, including the multiple mechanisms of action, such as those directed against viral particles or viral proteins, host proteins or pathways related to the productive replication viral cycle and the spread of the infection.


Subject(s)
Antiviral Agents/therapeutic use , Dengue/drug therapy , Phenols/therapeutic use , Virus Replication/drug effects , Animals , Chlorocebus aethiops , Dengue/genetics , Dengue/pathology , Dengue/virology , Dengue Virus/drug effects , Dengue Virus/genetics , Dengue Virus/pathogenicity , Humans , Plant Extracts/chemistry , Plant Extracts/therapeutic use , Vero Cells/drug effects , Viral Proteins/antagonists & inhibitors , Viral Proteins/genetics
17.
Curr Opin Virol ; 43: 71-78, 2020 08.
Article in English | MEDLINE | ID: covidwho-987407

ABSTRACT

The first licensed dengue vaccine led to considerable controversy, and to date, no dengue vaccine is in widespread use. All three leading dengue vaccine candidates are live attenuated vaccines, with the main difference between them being the type of backbone and the extent of chimerization. While CYD-TDV (the first licensed dengue vaccine) does not include non-structural proteins of dengue, TAK-003 contains the dengue virus serotype 2 backbone, and the Butantan/Merck vaccine contains three full-genomes of the four dengue virus serotypes. While dengue-primed individuals can already benefit from vaccination against all four serotypes with the first licensed dengue vaccine CYD-TDV, the need for dengue-naive population has not yet been met. To improve tetravalent protection, sequential vaccination should be considered in addition to a heterologous prime-boost approach.


Subject(s)
Dengue Vaccines/immunology , Dengue Virus/immunology , Dengue/prevention & control , Animals , Dengue/immunology , Dengue/virology , Dengue Vaccines/administration & dosage , Dengue Vaccines/genetics , Dengue Virus/genetics , Dengue Virus/physiology , Drug Development , Humans
18.
Euro Surveill ; 25(36)2020 09.
Article in English | MEDLINE | ID: covidwho-976158

ABSTRACT

In August 2020, during the coronavirus disease (COVID-19) pandemic, five locally acquired cases of dengue virus type 1 were detected in a family cluster in Vicenza Province, North-East Italy where Aedes albopictus mosquitoes are endemic. The primary case was an importation from West Sumatra, Indonesia. This is the first outbreak of autochthonous dengue reported in Italy. During the COVID-19 pandemic, screening of febrile travelers from endemic countries is crucial in areas where competent vectors are present.


Subject(s)
Dengue Virus/isolation & purification , Dengue/diagnosis , Travel , Adult , Child, Preschool , Dengue/epidemiology , Dengue/immunology , Dengue/virology , Dengue Virus/genetics , Disease Outbreaks , Disease Transmission, Infectious , Female , Fever/etiology , Humans , Immunoglobulin G/blood , Immunoglobulin M/blood , Indonesia , Italy/epidemiology , Male , Middle Aged , RNA, Viral/blood , Reverse Transcriptase Polymerase Chain Reaction
19.
J Virol ; 95(4)2021 01 28.
Article in English | MEDLINE | ID: covidwho-952556

ABSTRACT

Positive-strand RNA viruses have been the etiological agents in several major disease outbreaks over the last few decades. Examples of this include flaviviruses, such as dengue virus and Zika virus, which cause millions of yearly infections around the globe, and coronaviruses, such as SARS-CoV-2, the source of the current pandemic. The severity of outbreaks caused by these viruses stresses the importance of research aimed at determining methods to limit virus spread and to curb disease severity. Such studies require molecular tools to decipher virus-host interactions and to develop effective treatments. Here, we describe the generation and characterization of a reporter system that can be used to visualize and identify cells infected with dengue virus or SARS-CoV-2. This system is based on viral protease activity that mediates cleavage and nuclear translocation of an engineered fluorescent protein stably expressed in cells. We show the suitability of this system for live cell imaging, for visualization of single infected cells, and for screening and testing of antiviral compounds. With the integrated modular building blocks, this system is easy to manipulate and can be adapted to any virus encoding a protease, thus offering a high degree of flexibility.IMPORTANCE Reporter systems are useful tools for fast and quantitative visualization of virus-infected cells within a host cell population. Here, we describe a reporter system that takes advantage of virus-encoded proteases expressed in infected cells to cleave an ER-anchored fluorescent protein fused to a nuclear localization sequence. Upon cleavage, the GFP moiety translocates to the nucleus, allowing for rapid detection of the infected cells. Using this system, we demonstrate reliable reporting activity for two major human pathogens from the Flaviviridae and the Coronaviridae families: dengue virus and SARS-CoV-2. We apply this reporter system to live cell imaging and use it for proof-of-concept to validate antiviral activity of a nucleoside analogue. This reporter system is not only an invaluable tool for the characterization of viral replication, but also for the discovery and development of antivirals that are urgently needed to halt the spread of these viruses.


Subject(s)
COVID-19/virology , Dengue Virus/isolation & purification , Dengue/virology , SARS-CoV-2/isolation & purification , A549 Cells , Animals , COVID-19/diagnosis , COVID-19/metabolism , COVID-19/pathology , Cell Line , Chlorocebus aethiops , Dengue/diagnosis , Dengue/metabolism , Dengue/pathology , Dengue Virus/genetics , Dengue Virus/metabolism , Genes, Reporter , Green Fluorescent Proteins/metabolism , HEK293 Cells , Humans , Nuclear Localization Signals/metabolism , SARS-CoV-2/genetics , SARS-CoV-2/metabolism , Vero Cells , Viral Nonstructural Proteins/metabolism , Virus Replication
20.
Sci Adv ; 6(39)2020 09.
Article in English | MEDLINE | ID: covidwho-796906

ABSTRACT

Detection of viruses is critical for controlling disease spread. Recent emerging viral threats, including Zika virus, Ebola virus, and SARS-CoV-2 responsible for coronavirus disease 2019 (COVID-19) highlight the cost and difficulty in responding rapidly. To address these challenges, we develop a platform for low-cost and rapid detection of viral RNA with DNA nanoswitches that mechanically reconfigure in response to specific viruses. Using Zika virus as a model system, we show nonenzymatic detection of viral RNA with selective and multiplexed detection between related viruses and viral strains. For clinical-level sensitivity in biological fluids, we paired the assay with sample preparation using either RNA extraction or isothermal preamplification. Our assay requires minimal laboratory infrastructure and is adaptable to other viruses, as demonstrated by quickly developing DNA nanoswitches to detect SARS-CoV-2 RNA in saliva. Further development and field implementation will improve our ability to detect emergent viral threats and ultimately limit their impact.


Subject(s)
Betacoronavirus/genetics , Coronavirus Infections/diagnosis , DNA, Single-Stranded/genetics , Electrophoresis, Agar Gel/methods , Pneumonia, Viral/diagnosis , RNA, Viral/genetics , Sequence Analysis, RNA/methods , Base Sequence , COVID-19 , Cell Line, Tumor , Coronavirus Infections/virology , Dengue/diagnosis , Dengue/virology , Dengue Virus/genetics , Electrophoresis, Agar Gel/economics , Humans , Limit of Detection , Pandemics , Pneumonia, Viral/virology , SARS-CoV-2 , Saliva/virology , Sequence Analysis, RNA/economics , Zika Virus/genetics , Zika Virus Infection/diagnosis , Zika Virus Infection/virology
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