The antibody response to the envelope (E) glycoprotein of dengue virus (DENV) is known to play a critical role in both protection from and enhancement of disease, especially after primary infection. However, the relative amounts of homologous and heterologous anti-E antibodies and their epitopes remain unclear. In this study, we examined the antibody responses to E protein as well as to precursor membrane (PrM), capsid, and nonstructural protein 1 (NS1) of four serotypes of DENV by Western blot analysis of DENV serotype 2-infected patients with different disease severity and immune status during an outbreak in southern Taiwan in 2002. Based on the early-convalescent-phase sera tested, the rates of antibody responses to PrM and NS1 proteins were significantly higher in patients with secondary infection than in those with primary infection. A blocking experiment and neutralization assay showed that more than 90% of anti-E antibodies after primary infection were cross-reactive and nonneutralizing against heterologous serotypes and that only a minor proportion were type specific, which may account for the type-specific neutralization activity. Moreover, the E-binding activity in sera of 10 patients with primary infection was greatly reduced by amino acid replacements of three fusion loop residues, tryptophan at position 101, leucine at position 107, and phenylalanine at position 108, but not by replacements of those outside the fusion loop of domain II, suggesting that the predominantly cross-reactive anti-E antibodies recognized epitopes involving the highly conserved residues at the fusion loop of domain II. These findings have implications for our understanding of the pathogenesis of dengue and for the future design of subunit vaccine against DENV as well.Dengue virus (DENV) belongs to the genus Flavivirus in the family Flaviviridae. The four serotypes of DENV (DENV1, DENV2, DENV3, and DENV4) are the leading cause of arboviral diseases in the tropical and subtropical areas (15,17,60). It has been estimated that more than 2.5 billion people in over 100 countries are at risk of infection, and more than 50 million dengue infections occur annually worldwide (15,17,60). While most DENV infections are asymptomatic or result in a self-limited illness, dengue fever (DF), some people may present with the severe and potentially life-threatening diseases dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) (15,17,60).DENV contains a positive-sense, single-stranded RNA genome of about 10.6 kilobases. Flanked by the 5Ј and 3Ј untranslated regions, the single open reading frame encodes a polyprotein precursor, which is cleaved by cellular and viral protease into three structural proteins, the capsid (C), precursor membrane (PrM), and envelope (E), as well as seven nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (34). The E protein, a glycoprotein of approximately 55 kDa, contains 12 strictly conserved cysteine residues forming six disulfide bridges and is present as a heterodimer with PrM protein before th...

Persistence of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) under current antiviral therapy is a major barrier to eradication of chronic hepatitis B (CHB). Curing CHB will require novel strategies for specific disruption of cccDNA. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is a newly developed tool for site-specific cleavage of DNA targets directed by a synthetic guide RNA (gRNA) base-paired to the target DNA sequence. To examine whether this system can cleave HBV genomes, we designed eight gRNAs against HBV of genotype A. With the HBV-specific gRNAs, the CRISPR/Cas9 system significantly reduced the production of HBV core and surface proteins in Huh-7 cells transfected with an HBV-expression vector. Among eight screened gRNAs, two effective ones were identified. Interestingly, one gRNA targeting the conserved HBV sequence acted against different genotypes. Using a hydrodynamics-HBV persistence mouse model, we further demonstrated that this system could cleave the intrahepatic HBV genome-containing plasmid and facilitate its clearance in vivo, resulting in reduction of serum surface antigen levels. These data suggest that the CRISPR/Cas9 system could disrupt the HBV-expressing templates both in vitro and in vivo, indicating its potential in eradicating persistent HBV infection.

Using reverse transcription-PCR and clonal sequencing of the dengue virus envelope gene derived from the plasma samples of six patients, we reported for the first time that dengue virus circulates as a population of closely related genomes. The extent of sequence diversity varied among patients, with the mean pairwise proportions of difference ranging from 0.21 to 1.67%. Genome-defective viruses were found in 5.8% of the total number of clones analyzed. Our findings on the quasispecies nature of dengue virus and the defective virus in vivo have implications with regard to the pathogenesis of dengue virus.

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.