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.Dengue viruses are members of the genus Flavivirus of the family Flaviviridae. There are four serotypes of dengue viruses, DEN-1, DEN-2, DEN-3, and DEN-4. Over the past 20 years, epidemics caused by the four dengue viruses have emerged as one of the major public health problems in tropical and subtropical regions (4,6,18,30). Dengue virus contains a positivesense single-stranded RNA genome. Flanked by two nontranslated regions, there are three structural genes, the capsid (C), precursor membrane (PrM), and envelope (E), at the 5Ј onefourth and seven nonstructural genes at the 3Ј three-fourths (4,14).Dengue virus is transmitted to human by the bite of an infected mosquito. Aedes aegypti is the principle vector involved in the urban transmission cycle (4,10,22). After a female mosquito ingests a blood meal from a patient infected with dengue virus, viral replication is initially found in the posterior midgut of the mosquito and then in the proventriculus and in other organ systems. Dengue virus appears in the salivary gland after an extrinsic incubation period of 8 to 12 days, when the mosquito becomes capable of transmitting it to another human host (10,20,22). Following an incubation period of 3 to 14 days, the infected individuals may be asymptomatic or present a mild and self-limited illness, dengue fever (DF), or a severe and potentially life-threatening disease, dengue hemorrhage fever-dengue shock syndrome (DHF-DSS) (4, 30).The genetic stability of arboviruses that replicate alternately in the vertebrate and arthropod hosts has been well documented previously (13,27,28). In the case of DEN-3 virus, it has been reported that the amino acid similarity of the PrM/E proteins was more than 95% over a 36-year period (13). Previously, it was reported that dengue virus, like other RNA viruses, is present as a population of closely related sequences, the quasispecies, in the human host (2,3,8,16,24,25). The extent of sequence variation of dengue virus in the mosquito vector and how the quasi...

To investigate viral determinants and evolution linked to outbreak with increased severity, we examined dengue virus type 2 (DENV-2) sequences from plasma of 31 patients (14 dengue fever, 17 dengue hemorrhagic fever, DHF) continuously during the 2001 and 2002 outbreaks in southern Taiwan, in which both the total cases and proportion of DHF cases increased. Analysis of envelope (E) and full-genome sequences between viruses of the two outbreaks revealed 5 nucleotide changes in E, NS1, NS4A, and NS5 genes. None was identical to those reported in the DENV-2 outbreak in Cuba in 1997, suggesting viral determinants linked to severe outbreak are genotype dependent. Compared with previous reports of lineage turnover years apart, our findings that the 2002 viruses descended from a minor variant of the 2001 viruses in less than 6 months was novel, and may represent a mechanism of evolution of DENV from one outbreak to another.

Serologic testing remains crucial for Zika virus diagnosis. We found that urea wash in a Zika virus nonstructural protein 1 IgG ELISA distinguishes secondary dengue virus infection from Zika virus infection with previous dengue (sensitivity 87.5%, specificity 93.8%). This test will aid serodiagnosis, serosurveillance, and monitoring of Zika complications in dengue-endemic regions.

The four serotypes of dengue virus (DENV) cause one of the most important and rapidly emerging mosquito-borne viral diseases in humans. Of the currently available diagnostic tests for dengue, the reverse transcription-PCR (RT-PCR) assay is the most sensitive and specific, and so it is commonly used as the gold standard. However, the requirement of a sophisticated and expensive thermal cycler makes it very difficult to use as a point-of-care diagnostic test in resourcelimited regions where dengue is endemic. Tsai et al. (J Clin Microbiol 56:e01865-17, 2018, https://doi.org/10.1128/JCM.01865-17) report the analytical and clinical performances of a reverse transcription-insulated isothermal PCR (RT-iiPCR) assay with a portable nucleic acid analyzer for rapid detection of the four DENV serotypes; its reproducibility and complete agreement on clinical samples with the multiplex RT-PCR assay developed by the Centers for Disease Control and Prevention suggest that the dengue RT-iiPCR is a potential point-of-care test. Compared with other DENV RNA detection methods, the unique isothermal PCR design of RT-iiPCR, together with further improvements, would represent a promising new type of field-deployable diagnostic test for dengue. With an estimated 390 million new infections per year, the four dengue virus (DENV) serotypes are the leading causes of mosquito-borne viral infection and disease in humans (1, 2). While many DENV infections are clinically mild or inapparent, clinical disease ranges from a self-limited illness, dengue fever, to severe and potentially life-threatening disease, dengue hemorrhagic fever/dengue shock syndrome (1-3). The forms of the disease are classified as dengue, dengue with warming signs, and severe dengue according to the 2009 WHO revised case definition (3). There are approximately 3 billion people in more than 120 countries who are at risk of DENV infection (1, 2). Currently, no FDA-approved antiviral drug is available to treat dengue; the only licensed dengue vaccine, Dengvaxia, is recommended only for persons who have experienced previous DENV infection and not for dengue-naive individuals (4-6).Accurate and rapid laboratory diagnosis of dengue is critical for confirmation of clinically suspected cases to ensure timely management of severe dengue disease, especially in urban areas of resource-poor developing countries. Laboratory diagnosis for DENV infection includes virus isolation, reverse transcription-PCR (RT-PCR), nonstructural protein 1 (NS1) antigen detection, and serological tests such as IgM and IgG enzyme-linked immunosorbent assay (ELISA) and neutralization tests (2, 3). Each diagnostic method has its advantages and limitations; overall RT-PCR is the most sensitive and specific diagnostic test and has been commonly used as a gold standard for comparisons with other tests (reviewed in reference 7).The report by Tsai and colleagues in this month's issue of the Journal of Clinical Microbiology suggests that the reverse transcription-insulated isothermal PCR (RT-iiPCR) ass...

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