Dengue Virus Nucleic Acid Research Articles

A specific and low background nucleic acids sensing strategy based on rolling circle amplification coupled with a magnetic DNA machine.

We propose a universal fluorescence method for detection of nucleic acids based on rolling circle amplification (RCA) combined with a magnetic DNA machine and using dengue virus nucleic acids as an example target. RCA specifically amplifies the target and yields a large number of initiators employing heat-labile double-stranded DNase. The magnetic DNA machine produces a fluorescence signal and eliminates background noise. This method achieved a wide linear range, promising recovery and ultrahigh recognition specificity for one-base mismatches, and indicates the potential application of this sensing strategy in the clinical diagnosis of nucleic acids of pathogens.

Electrochemical biosensing of DENV nucleic acid amplified with triplet nanostructure-mediated dendritic hybridization chain reaction

A universal and highly sensitive electrochemical biosensing strategy was developed by using a triplet nanostructure-mediated dendritic hybridization chain reaction (HCR) for the analysis of Dengue virus (DENV) nucleic acid. A locked dsDNA was firstly recognized by the target DENV nucleic acid fragment to release the initiator, which was then captured by a capture DNA modified gold electrode to initiate the HCR in the presence of biotin labeled substrates A and B, and two helpers. After the recognition reactions of the captured initiator with substrate A and then helper 1, one DNA structure was formed to react with substrate B, which produced a triplet nanostructure to trigger the dendritic HCR by the toehold of the hybridization structure. After the dendritic product was bound with avidin labeled horseradish peroxidase (avidin-HRP), an amperometric signal could be obtained to achieve ultrasensitive electrochemical detection of DENV. The non-enzymatic isothermal amplification was driven only by entropy. The stable three-dimensional dendritic nanostructure with rigid double helix could assure the excellent performance of the designed electrochemical biosensing strategy. The proposed method showed a detection range of 1.6–1000 pM with a detection limit of 188 fM and ability to distinguish single-base mutation. By changing the recognition sequence of the initiator, the detection of different DENV nucleic acid fragments could be achieved with the same performance. Thus, this method had good extendability for other nucleic acids, providing a promising candidate for nucleic acid detection in early clinical diagnosis.

Epidemiological and virus molecular characterization of dengue fever outbreak in Hunan province, 2018

Objective: To analyze the epidemiological and etiological characteristics of a dengue fever outbreak in Hunan province in 2018. Methods: Real-time PCR assay was performed for the laboratory diagnosis of 8 suspected dengue fever cases. Etiological surveillance was performed in 186 suspected dengue fever cases and fever cases who had close contacts with dengue fever patients. C6/36 cells was used for the virus isolation from acute phase serum. By sequencing the full length of E genes of 15 dengue virus strains, phylogenetic analysis was performed based on the sequences obtained, including reference sequences from the NCBI GenBank database, the serotypes and gene subtypes of the virus were analyzed to trace the possible source of transmission. An emergency monitoring of vector density and a retrospective survey of sero-epidemiology in healthy population were conducted in the epidemic area. Results: In the serum samples of 8 suspected patients, 6 were dengue virus RNA positive, and 4 were NS1 antigen positive. In 186 suspected patients, 96 were dengue virus nucleic acid, NS1 antigen or antibody positive in etiological test. A total of 64 dengue virus strains were isolated. The phylogenetic analysis showed that all the dengue virus strains belonged to type 2, which might be from Guangdong or Zhejiang provinces. The Bretub index was up to 65, indicating an extremely high risk of transmission. The positive rate of the dengue virus IgG antibody was 0.53%(2/377) in retrospective survey of 377 healthy people. Conclusion: The field epidemiologic and the molecular genetics analyses showed the outbreak of dengue fever in Hunan in 2018 was caused by imported cases and dengue virus 2.

A Label-Free Impedimetric Genosensor for the Nucleic Acid Amplification-Free Detection of Extracted RNA of Dengue Virus.

Developing rapid and sensitive diagnostic methods for dengue virus (DENV) infection is of prime priority because DENV infection is the most prevalent mosquito-borne viral disease. This work proposes an electrochemical impedance spectroscopy (EIS)-based genosensor for the label-free and nucleic acid amplification-free detection of extracted DENV RNA intended for a sensitive diagnosis of DENV infection. A concentration ratio of 0.04 mM 6-mercaptohexanoic acid (MHA) to 1 mM 6-mercapto-1-hexanol (MCH) was selected to modify thin-film gold electrodes as a link to control the coverage of self-designed probe DNA (pDNA) at a density of 4.5 ± 0.4 × 1011 pDNA/cm2. The pDNA/MHA/MCH-modified genosensors are proven to improve the hybridization efficiency of a synthetic 160-mer target DNA (160mtDNA) with a 140-mer electrode side overhang as compared to other MHA/MCH ratio-modified genosensors. The MHA(0.04 mM)/MCH(1 mM)-modified genosensors also present good hybridization efficiency with the extracted DENV serotype 1 (DENV1) RNA samples, having the same electrode side overhangs with the 160mtDNA, showing a low detection limit of 20 plaque forming units (PFU)/mL, a linear range of 102–105 PFU/mL and good selectivity for DENV1. The pDNA density-controlled method has great promise to construct sensitive genosensors based on the hybridization of extracted DENV nucleic acids.

Clinical analysis of 78 children with dengue fever

Objective To explore the clinical characteristics of children with dengue fever (DF) hospitalized in Guangzhou in 2014, and to raise clinician′s level of understanding of dengue fever in children. Methods Clinical data of 78 children hospitalized with DF in the Department of Infectious Diseases, Guangzhou Women and Children′s Medical Center were retrospectively analyzed. Chi-square was used for discontinuous variables, and t test was used for continuous variabbles. Results The 78 cases aged 27 days to 14 years old, with median of 5 years old. Fifty cases (64.1%) were male, and 28 cases (35.9%) were female. Epidemic areas had gathering trend, mainly in central urban area. Major clinical manifestations were fever (100.00%), rash (82.05%), myalgia/fatigue (28.21%), but without diarrhea, jaundice, hematemesis or hematochezia. Laboratory tests suggested leukopenia (80.77%) and thrombocytopenia (82.05%), abnormal blood coagulation function with prolonged APTT (57.69%), and abnormal liver function (47.44%). Etiology examinations showed 66 cases of children had dengue virus nucleic acid detected 1-10 days after onset, with the positive rate of 89.19% (66/74). A total of 48 cases had IgM positive, with the positive rate of 81.36% (48/59). IgM began to appear as early as the first day of disease onset, and the average period was (5.5±0.8) days. Dengue virus type 1 was the main type. Conclusions In 2014, dengue virus type 1 is the main strain causing dengue fever in children in Guangzhou. Fever, rash, leukopenia, thrombocytopenia, clotting disorders and liver function damage are the main clinical features. No serious or fatal cases are reported, and the prognosis is good. Key words: Dengue; Child; Retrospective studies; Clinical characteristics

Circulating dengue virus serotypes in Bangladesh from 2013 to 2016.

To identify the circulating serotype(s) of dengue viruses in Bangladesh, a retrospective molecular identification was performed on stored serum samples of dengue surveillance during the period of 2013-2016. Real time RT-PCR was performed on serum samples collected from the patients with less than 5days fever for detection of dengue virus nucleic acid. The samples, positive for dengue PCR were further analyzed for serotypes by real time RT-PCR. The overall prevalence of dengue virus infection was varied among 13-42% in study years with a single peak flanked by April to September. Among the four dengue serotypes DEN1 and DEN2 were in the circulation in three metropolitan cities with sequential emergence of DEN1 where DEN2 was persisted constantly during the study period. Persistence of all four serotypes in the neighboring country makes Bangladesh vulnerable for devastating secondary infection by introduction of new serotype(s) other than currently circulating viruses in the country. Thus continuous virological surveillance is crucial for early warning of emergence of new serotype in the circulation and public health preparedness.

Comparison of reverse-transcriptase qPCR and droplet digital PCR for the quantification of dengue virus nucleic acid

Polymerase chain reaction (PCR) is an important molecular biology technique for in vitro amplification of nucleic acids. Reverse transcriptase quantitative PCR (RT-qPCR) and more recently reverse transcriptase digital droplet PCR (RT-ddPCR) have been developed for the quantification of nucleic acids. We developed an RT-ddPCR assay for the quantification of attenuated dengue virus serotype 2 nucleic acid and compared it with a routine RT-qPCR assay. While the routine RT-qPCR assay targets the NS5 gene, the E gene was selected for the optimization of the RT-ddPCR assay conditions. The specificity of the assay was demonstrated using the attenuated dengue virus serotype 2 alone and in the presence of the other three dengue serotypes. The results from both assays for 25 samples of the attenuated dengue virus serotype 2 were found to be comparable, with an R2 from the linear regression analysis of >0.98. A major advantage of the RT-ddPCR assay is that it allows quantification of nucleic acid, without the need of a standard curve. RT-ddPCR can be implemented for the absolute quantification of dengue vaccine virus nucleic acid during the vaccine manufacturing process.

NS 1 lasts longer than the dengue virus nucleic acid in the clinically suspected patients with dengue fever and dengue haemorrhagic fever.

The objectives of the study were to compare the DENV NS1 and nucleic acid positivity in dengue fever (DF) and dengue haemorrhagic fever (DHF) patients and to evaluate the usefulness of these parameters in severe dengue. Blood samples were collected from 91 patients with DF or DHF (fever days 3-9) from Gampaha and Negombo General Hospitals, Sri Lanka and tested for DENV NS1 antigen and nucleic acid. On fever day 3, 67% of the DF and 33% of the DHF patients had DENV nucleic acid whereas 50% of the DF and 67% of the DHF patients had NS1. On fever day 4, 67% of the DF and 100% of the DHF patients had DENV nucleic acid whereas 83% of the DF and DHF patients had NS1. From fever day 5 in the DHF group, DENV nucleic acid declined but NS1 remained detectable even on day 9 (p<0.0001). NS1 positivity did not correlate with the decrease in the WBC count in DF and DHF patients (p>0.05) whereas NS1 positivity significantly correlated with the decrease in the platelet count in DF and DHF patients (p<0.0001). NS1 is a useful marker to detect DENV infection even in the latter stages of DHF.

No Evidence of Dengue Virus Infections in Several Species of Bats Captured in Central and Southern Mexico.

Bats are reservoirs for viruses with zoonotic potential in the Americas, and scattered evidence exists suggesting that bats may act as reservoirs for dengue virus (DENV). To explore further the role of bats as part of DENV sylvatic cycles, 240 bats of 18 species were captured in 2 states of Mexico with contrasting ecological characteristics but concurrent DENV activity in humans. RT-PCR analysis of RNA extracted from liver or spleen tissue from de bats failed to show evidence for the presence of DENV nucleic acids in these organs. In addition, plasma assayed by plaque reduction neutralization test showed no evidence of neutralizing anti-DENV antibodies. These results suggest that American bats may not be reservoirs or amplification host for DENV infection.

Epidemiological and etiological characteristics of dengue fever in Shenzhen in 2014

Objective To investigate the epidemiological characteristics of dengue fever in Shenzhen city in 2014 and to analyze the evolutional characteristics of the epidemic dengue virus (DENV) strains in order to provide scientific guidelines for the prevention and control of dengue fever. Methods Descriptive epidemiological analysis was used to analyze the prevalence of dengue fever in Shenzhen city in 2014. Immunochromatography and real-time PCR were performed to detect the specific antibodies (IgM and IgG) and DENV nucleic acids in serum samples collected from suspected cases of dengue fever. Serum samples collected from the patients at early stage of dengue fever were used to infect the C6/36 cell line for further isolation of DENV strains. The types of isolated DENV strains were determined by using real-time PCR. E genes of the isolated DENV strains were amplified by RT-PCR and then sequenced. DNAStar and Clustslx (1.83) softwares were used to analyze the homology between DENV strains isolated in Shenzhen and other areas. A phylogenetic tree based on the sequences of E genes of Shenzhen strains and other sequences of DENV reference strains downloaded from GenBank was constructed for further analysis. Results A total of 454 cases of dengue fever were reported in Shenzhen in 2014 with a male to female ratio of 1.43∶1. Local patients accounted for 76.21% and the rest 23.79% were imported cases mainly from Southeast Asian and surrounding cities. There were 441 cases reported from September to November, accounting for 97.14% of all reported cases. Most of the infected subjects were aged 20 to 50, accounting for 76.73%. Of the 270 samples positive for DENV nucleic acids, strains of DENV-1, DENV-2, DENV-3 and DENV-4 accounted for 87.41%, 8.89%, 0.37% and 2.22%, respectively. The phylogenetic tree analysis revealed that the DENV-1 strains belonged to two genotypes, which were genotypeⅠ and genotype Ⅴ. The DENV strains of genotypeⅠ were highly similar to the epidemic strain isolated in Shenzhen in 2010 and the genotype Ⅴ strains were first reported in Shenzhen. The homology analysis of the nucleotides of E genes showed that minor differences in the nucleotide sequences were found between DENV-2 strains. All of the DENV-2 strains belonged to the genotype Ⅳ as indicated by the phylogenic tree. Conclusion There were 454 cases of dengue fever (including both local and imported cases) reported in Shenzhen city in 2014, reaching an all-time high. DENV-1 was the predominant pathogen in combination with an increased infection of DENV-2. This study indicated that the prevalent DENV strains might be imported from Southeast countries and neighboring cities. Further researches should be conducted to analyze whether dengue fever is endemic in Shenzhen City. Key words: Dengue virus; Epidemiological characteristics; E gene; Evolutional analysis

Characterization of dengue virus infections in a sample of patients suggests unique clinical, immunological, and virological profiles that impact on the diagnosis of dengue and dengue hemorrhagic fever.

Dengue virus (DENV) infections are increasing with respect to incidence and severity in the Central Province of Sri Lanka. The objective of this study was to define the clinical, immunological, and virological profiles of patients admitted to the General Hospital, Kandy with clinically apparent dengue. Demographic, clinical, hematological parameters, liver enzymes (ALT and AST), and blood samples were collected from 292 patients with fever <5 days post onset and fulfilling the WHO criteria for the diagnosis of dengue fever/dengue hemorrhagic fever (DF/DHF). Samples were analyzed for, anti-DENV IgM, IgG, and DENV nucleic acid. Myalgia was the commonest complaint by 65% of the patients. Packed cell volume was >45% in 27% of the patients while 42.12% had reduced platelets and 62.67% had reduced white blood cell counts. In contrast to other studies, positive tourniquet test (PTT) and petechiae were not major indicators of DENV infection or severity of the disease. Clinical profiles were significantly different between DF and DHF/DSS and showed many similarities to that reported elsewhere. Altogether, 43 patients (14.73%) were viremic as detected by RT-PCR; 181 patients (62%) were positive for anti-DENV IgM, and 245 (84%) patients were positive for anti-DENV IgG. In combination, anti-DENV IgM and RT-PCR assays detected 224 (77.5%) of DENV infected cases, thus improving the DENV diagnosis rate. Hence, the diagnostic utility of PTT, anti-DENV IgM/IgG serology, or RT-PCR used alone in the early phase of illness is low in Sri Lanka but the diagnostic value can be improved by a combination of serology and RT-PCR. J. Med. Virol. 88:1703-1710, 2016. © 2016 Wiley Periodicals, Inc.

An autochthonous outbreak of dengue type 1 in Tokyo, Japan 2014

An outbreak of autochthonous dengue fever was reported in August 2014, with cases suspected mainly from Yoyogi Park in Tokyo. This is the first epidemic of dengue fever in Japan since 1945. From August to October 2014, the following measures were taken to control the outbreak: 1) risk communication and information sharing; 2) active case finding; 3) vector surveillance in affected sites; and 4) laboratory testing. We also reviewed the surveillance data as reported to the National Epidemiological Surveillance of Infectious Diseases during the 44 epidemiological weeks. results: An official dengue fever call center was set up temporarily for the general public and 3,005 calls were received. The Tokyo Metropolitan Government issued 39 press releases regarding patients and nine related to dengue virus (DENV) detection and vector control activities for the media. Confirmed autochthonous dengue fever cases were reported between the 35th and 44th epidemiological weeks. Out of 160 cases of outbreak, 108 (67.5%) confirmed cases were reported in Tokyo. The estimated illness onset dates were between August 9 and October 7, and estimated dates of infections were between August 3 and October 3, 2014. The data suggest that the infective mosquitoes had already been present in Yoyogi Park at the end of July 2014. During the weekly vector surveillance at Yoyogi Park, a total of 1,152 adult mosquitoes, of which 856 (73.3%) were Aedes mosquitoes, were collected over 11 weeks by a light trap with dry ice. DENV was detected from adult Aedes mosquito samples collected on the 2nd, 9th, and 16th of September, 2014. Serum samples from 240 suspected cases were examined at the Tokyo Metropolitan Institute of Public Health, and 78 were positive for the DENV NS1 antigen, DENV-specific IgM antibody, or DENV nucleic acid with reverse transcriptase polymerase chain reaction (RT-PCR) (NS1: 66 cases; IgM: 50 cases; PCR: 57 cases). Genetic analysis of DENV-positive serum and mosquito samples found all to be categorized as DENV-serotype 1 (gene type I). Phylogenetic analysis of the envelope protein genome sequence from patients and mosquitoes in Tokyo revealed more than 99% similarity with each other and with the strain from the first outbreak-associated patient in Saitama. Measures important for control of infectious disease epidemic were learned during this recent indigenous dengue outbreak in Tokyo. It also highlighted the importance of preparedness for epidemics of indigenous or imported infectious diseases, especially in light of the fact that Tokyo is in preparation for the Olympic and Paralympic Games in 2020.

Molecular epidemiology of dengue fever cases imported into Romania between 2008 and 2013.

Dengue fever is the commonest arthropod-borne infection worldwide. In recent years, rapid growth in global air travel has resulted in a considerable increase in the incidence of imported cases. In Romania it is now the second most frequent cause for hospitalization (after malaria) in patients arriving from tropical regions. Serological and molecular diagnostics were applied to samples obtained between 2008 and 2013 from travelers with suspected dengue. Molecular typing was performed by RT-PCR followed by sequencing of the E-NS1 junction. Twelve of 37 suspected cases were confirmed and three remained probable. The infections were acquired in endemic regions in Asia, Africa and in Europe (Madeira Island). Dengue virus nucleic acid was detected and sequenced in nine cases. Phylogenetic analysis indicated that the viruses were of genotypes I and V of serotype 1, cosmopolitan genotype of serotype 2 and genotypes I and III of serotype 3. Romanian tourists traveling to dengue-endemic countries are at risk of acquiring dengue infection. Appropriate prevention measures prior to travel and upon return should be taken, particularly as the dengue secondary vector Aedes albopictus is now established in Bucharest.

Aedes aegyptiMosquitoes at Nonresidential Sites Might be Related to Transmission of Dengue Virus in Monterrey, Northeastern Mexico

Traditionally the major risk environment for transmission of dengue virus has been assumed to be households. In Mexico, dengue outbreaks continue year after year despite intense control efforts. Nonresidential sites (public and private spaces) infested with Aedes aegypti (L.) were evaluated. In total, 141 nonresidential sites were sampled for the presence of potential and active oviposition sites and adult mosquitoes. Eighty percent of the sites were oviposition sites; Ae. aegypti adults were recovered at 94.7% of nonresidential sites. Most female Ae. aegypti, 21.6 and 10.4, were at schools and recreational sites, respectively. Chi-squared indicated no significant differences in the dengue vector to categories of sample sites (X2 = 17.76, df = 9, P = 0.38). Indoor-use patterns of adult mosquitoes indicated bathrooms and classrooms were preferred resting sites. Reverse transcription polymerase chain reaction (RT-PCR) assay did not identify dengue virus nucleic acids from a group of 221 pools containing 1,521 female Ae. aegypti. Daytime human activities; e.g., school and work, synchronize with the bimodal biting pattern of Ae. aegypti, increasing the chance of transferring dengue virus.

Highly Sensitive Detection of Dengue Virus Nucleic Acid in Samples from Clinically Ill Patients

Dengue virus (DENV) is a major cause of febrile illness and hemorrhagic fever in tropical and subtropical regions. Typically, patients presenting with acute dengue disease are viremic but may not have yet developed detectable titers of antibody. Therefore, early diagnosis depends mostly on detection of viral components, such as the RNA. To define the potential use of transcription-mediated amplification (TMA) DENV RNA as a diagnostic tool, we first compared its analytic sensitivity using a routine real-time reverse transcription (RT)-PCR and found that TMA is approximately 10 to 100 times more sensitive. In addition, we tested acute-phase serum samples (<5 days post-symptom onset) submitted as part of laboratory-based surveillance in Puerto Rico and determined that among patients with serologically confirmed dengue infection, TMA detected DENV RNA in almost 80% of serum specimens that were negative by the RT-PCR test used for diagnosis and in all specimens with positive RT-PCR results. We conclude that TMA is a highly sensitive method which can detect DENV RNA in approximately 89% of clinical, acute-phase serum specimens.

Detection of dengue viral RNA in Aedes aegypti (Diptera: Culicidae) exposed to sticky lures using reverse-transcriptase polymerase chain reaction.

Active surveillance for dengue (DEN) virus infected mosquitoes can be an effective way to predict the risk of dengue infection in a given area. However, doing so may pose logistical problems if mosquitoes must be kept alive or frozen fresh to detect DEN virus. In an attempt to simplify mosquito processing, we evaluated the usefulness of a sticky lure and a seminested reverse-transcriptase polymerase chain reaction assay (RT-PCR) for detecting DEN virus RNA under laboratory conditions using experimentally infected Aedes aegypti (L.) mosquitoes. In the first experiment, 40 male mosquitoes were inoculated with 0.13 microl of a 10(4) pfu/ml DEN-2 stock solution. After a 7-d incubation period, the mosquitoes were applied to the sticky lure and kept at room temperatures of 23-30 degrees C. Following 7, 10, 14, and 28 d application, 10 mosquitoes each were removed from the lure, pooled, and assayed for virus. DEN virus nucleic acid was clearly detectable in all pools up to 28 d after death. A second study evaluated sensitivity and specificity using one, two, and five DEN-infected mosquitoes removed after 7,10, 14, 21, and 30 d application and tested by RT-PCR. All four DEN serotypes were individually inoculated in mosquitoes and evaluated using the same procedures as experiment 1. The four serotypes were detectable in as few as one mosquito 30 d after applications to the lure with no evidence of cross-reactivity. The combination of sticky lures and RT-PCR show promise for mosquito and dengue virus surveillance and warrant further evaluation.