Early detection of SARS-CoV in throat wash and saliva suggests that these specimens are ideal for SARS diagnosis.

These findings indicate that slower rates of clearance of viral load and virus-containing immune complexes are associated with subsequent immune activation and contribute to the progression of DHF at this critical stage. Moreover, viral load on the day of defervescence can predict cases of DHF.

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...

Recombinant virus-like particles (VLPs) of flaviviruses have been shown to be produced efficiently by co-expressing the precursor membrane (PrM) and envelope (E) proteins with few exceptions, such as dengue virus type 2 (DENV2). It was reported previously that chimeric DENV2 PrM/E construct containing the stem-anchor region of E protein of Japanese encephalitis virus (JEV) produced VLPs efficiently (Chang, G. J., Hunt, A. R., Holmes, D. A., Springfield, T., Chiueh, T. S., Roehrig, J. T., and Gubler, D. J. 2003. Enhancing biosynthesis and secretion of premembrane and envelope proteins by the chimeric plasmid of dengue virus type 2 and Japanese encephalitis virus. Virology 306, 170-180.). We investigated the mechanisms involved and reported that compared with authentic DENV2 PrM/E-expressing cells, E protein in chimeric DENV2 PrM/E-expressing cells was also present in an endoglycosidase H (endo H)-resistant compartment and has shifted more to the pellets of the soluble fraction. Replacement of the transmembrane and cytoplasmic domains of CD4 with the stem-anchor of DENV2 (CD4D2) or JEV (CD4JEV) rendered the chimeric CD4 retained predominantly in the endoplasmic reticulum (ER). Flow cytometry revealed higher proportion of CD4JEV than CD4D2 expressed on the cell surface. Together, these findings suggested that the stem-anchor of DENV2 contained an ER retention signal stronger than that of JEV, which might contribute to the inefficient production of DENV2 VLPs. Moreover, co-expression of C protein can enhance the production of DENV2 VLPs, suggesting a mechanism of facilitating viral particle formation during DENV2 replication.

The morphogenesis of many enveloped viruses, in which viral nucleocapsid complex interacts with envelope (E) protein, is known to take place at various sites along the secretory pathway. How viral E protein retains in a particular intracellular organelle for assembly remains incompletely understood. In this study, we investigated determinants in the E protein of dengue virus (DENV) for its retention and assembly in the endoplasmic reticulum (ER). A chimeric experiment between CD4 and DENV precursor membrane/E constructs suggested that the transmembrane domain (TMD) of E protein contains an ER retention signal. Substitutions of three nonhydrophobic residues at the N terminus of the first helix (T1) and at either the N or C terminus of the second helix of the TMD with three hydrophobic residues, as well as an increase in the length of T1, led to the release of chimeric CD4 and E protein from the ER, suggesting that short length and certain nonhydrophobic residues of the TMD are critical for ER retention. The analysis of enveloped viruses assembled at the plasma membrane and of those assembled in the Golgi complex and ER revealed a trend of decreasing length and increasing nonhydrophobic residues of the TMD of E proteins. Taken together, these findings support a TMD-dependent sorting for viral E proteins along the secretory pathway. Moreover, similar mutations introduced into the TMD of DENV E protein resulted in the increased production of virus-like particles (VLPs), suggesting that modifications of TMD facilitate VLP production and have implications for utilizing flaviviral VLPs as serodiagnostic antigens and vaccine candidates.The assembly of many enveloped viruses is known to take place at various locations along the secretory pathway. Members of the families Orthomyxoviridae, Paramyxoviridae, Rhabdoviridae, Retroviridae, and Togaviridae, such as influenza virus, parainfluenza virus, vesicular stomatitis virus, human immunodeficiency virus, and Sindbis virus, assemble at the plasma membrane (25,30,63). Members of the families Bunyaviridae and Coronaviridae assemble in the Golgi complex and endoplasmic reticulum-Golgi intermediate compartment (ERGIC), respectively, whereas members of the family Flaviviridae assemble in the membranous structure derived from the ER (2,30,36,40). An important step in the morphogenesis of enveloped viruses involves the encounter and interaction between the viral nucleocapsid complex, which contains viral capsid (C) protein and nucleic acid derived from genome replication, and viral envelope (E) protein, as well as matrix protein in some cases at a particular organelle. As the default pathway for cellular membrane proteins goes from the ER to the plasma membrane, how the viral E protein, a membrane protein, retains in a particular intracellular organelle is critical for virus assembly.Dengue viruses (DENV) belong to the genus Flavivirus of the family Flaviviridae. There are four serotypes of DENV (DENV1 to DENV4), which are the leading cause of arboviral diseases in tropical and sub...