Dengue Viruses Are Enhanced by Distinct Populations of Serotype Cross-Reactive Antibodies in Human Immune Sera

Dengue viruses (DENV) are mosquito-borne flaviviruses of global importance. DENV exist as four serotypes, DENV1-DENV4. Following a primary infection, individuals produce DENV-specific antibodies that bind only to the serotype of infection and other antibodies that cross-react with two or more serotypes. People exposed to a secondary DENV infection with another serotype are at greater risk of developing more severe forms of dengue disease. The increased risk of severe dengue in people experiencing repeat DENV infections appear to be due, at least in part, to the ability of pre-existing serotype cross-reactive antibodies to form virus-antibody complexes that can productively infect Fcγ receptor-bearing target cells. While the theory of antibody-dependent enhancement (ADE) is supported by several human and small animal model studies, the specific viral antigens and epitopes recognized by enhancing human antibodies after natural infections have not been fully defined. We used antibody-depletion techniques to remove DENV-specific antibody sub-populations from primary DENV-immune human sera. The effects of removing specific antibody populations on ADE were tested both in vitro using K562 cells and in vivo using the AG129 mouse model. Removal of serotype cross-reactive antibodies ablated enhancement of heterotypic virus infection in vitro and antibody-enhanced mortality in vivo. Further depletion studies using recombinant viral antigens showed that although the removal of DENV E-specific antibodies using recombinant E (rE) protein resulted in a partial reduction in DENV enhancement, there was a significant residual enhancement remaining. Competition ADE studies using prM-specific Fab fragments in human immune sera showed that both rE-specific and prM-specific antibodies in primary DENV-immune sera significantly contribute to enhancement of heterotypic DENV infection in vitro. Identification of the targets of DENV-enhancing antibodies should contribute to the development of safe, non-enhancing vaccines against dengue.     

The Incubation Periods of Dengue Viruses

Dengue viruses are major contributors to illness and death globally. Here we analyze the extrinsic and intrinsic incubation periods (EIP and IIP), in the mosquito and human, respectively. We identified 146 EIP observations from 8 studies and 204 IIP observations from 35 studies. These data were fitted with censored Bayesian time-to-event models. The best-fitting temperature-dependent EIP model estimated that 95% of EIPs are between 5 and 33 days at 25°C, and 2 and 15 days at 30°C, with means of 15 and 6.5 days, respectively. The mean IIP estimate was 5.9 days, with 95% expected between days 3 and 10. Differences between serotypes were not identified for either incubation period. These incubation period models should be useful in clinical diagnosis, outbreak investigation, prevention and control efforts, and mathematical modeling of dengue virus transmission.     

Induction of Neutralizing Antibodies against Four Serotypes of Dengue Viruses by MixBiEDIII,a Tetravalent Dengue Vaccine

The worldwide expansion of four serotypes of dengue virus (DENV) poses great risk to global public health. Several vaccine candidates are under development. However, none is yet available for humans. In the present study, a novel strategy to produce tetravalent DENV vaccine based on envelope protein domain III (EDIII) was proposed. Tandem EDIIIs of two serotypes (type 1–2 and type 3–4) of DENV connected by a Gly-Ser linker ((Gly₄Ser)₃) were expressed in E. coli, respectively. Then, the two bivalent recombinant EDIIIs were equally mixed to form the tetravalent vaccine candidate MixBiEDIII, and used to immunize BALB/c mice. The results showed that specific IgG and neutralizing antibodies against all four serotypes of DENV were successfully induced in the MixBiEDIII employing Freund adjuvant immunized mice. Furthermore, in the suckling mouse model, sera from mice immunized with MixBiEDIII provided significant protection against four serotypes of DENV challenge. Our data demonstrated that MixBiEDIII, as a novel form of subunit vaccine candidates, might have the potential to be further developed as a tetravalent dengue vaccine in the near future.     

白纹伊蚊和埃及伊蚊经卵传递登革病毒的研究

白纹伊蚊和埃及伊蚊通过吸食病毒液或叮吸有病毒血症的小鸡血后,能感染登革１－４型病毒,并能在蚊体内增殖,对感染雌蚊了１和子２代幼虫,雌性或雄性成虫４５５９只,分１０１批进行了病毒检测,白纹伊蚊子１代的批阳性率;登革１型为１０％（１／１０）２型为２２．２２％（２／９）３型为３３．３３％（４／１２）,４型为２８．９５％（１１／３８）登革１～４型的最低子代感染率依次主国０．２０％,０．７１％,０．７０％和     

Recovery of Dengue Viruses from Tissues of Experimentally Infected Rhesus Monkeys

A tissue explant culture technique for the recovery of dengue virus from experimentally infected monkey tissue is described and compared with tissue culture assay of tissue triturates and co-cultivation of trypsinized cells in cell cultures. The most efficient technique was one in which minced tissue was explanted in co-culture with dengue virus-susceptible LLC-MK2 monkey kidney cells. This technique shows promise of being useful for detection of virus in autopsy material from fatal dengue hemorrhagic fever cases.     

Natural Strain Variation and Antibody Neutralization of Dengue Serotype 3 Viruses

Dengue viruses (DENVs) are emerging, mosquito-borne flaviviruses which cause dengue fever and dengue hemorrhagic fever. The DENV complex consists of 4 serotypes designated DENV1-DENV4. Following natural infection with DENV, individuals develop serotype specific, neutralizing antibody responses. Monoclonal antibodies (MAbs) have been used to map neutralizing epitopes on dengue and other flaviviruses. Most serotype-specific, neutralizing MAbs bind to the lateral ridge of domain III of E protein (EDIII). It has been widely assumed that the EDIII lateral ridge epitope is conserved within each DENV serotype and a good target for vaccines. Using phylogenetic methods, we compared the amino acid sequence of 175 E proteins representing the different genotypes of DENV3 and identified a panel of surface exposed amino acids, including residues in EDIII, that are highly variant across the four DENV3 genotypes. The variable amino acids include six residues at the lateral ridge of EDIII. We used a panel of DENV3 mouse MAbs to assess the functional significance of naturally occurring amino acid variation. From the panel of antibodies, we identified three neutralizing MAbs that bound to EDIII of DENV3. Recombinant proteins and naturally occurring variant viruses were used to map the binding sites of the three MAbs. The three MAbs bound to overlapping but distinct epitopes on EDIII. Our empirical studies clearly demonstrate that the antibody binding and neutralization capacity of two MAbs was strongly influenced by naturally occurring mutations in DENV3. Our data demonstrate that the lateral ridge “type specific” epitope is not conserved between strains of DENV3. This variability should be considered when designing and evaluating DENV vaccines, especially those targeting EDIII.     

Serological Cross Reactivity between Zika and Dengue Viruses in Experimentally Infected Monkeys

正Dear Editor,Zika virus(ZIKV),a pathogen within the genus Flavivirus,family Flaviviridae brought an international public health emergency due to its association with neonatal microcephaly case(Platt and Miner 2017).Currently the most reliable diagnostic test is PCR detection of ZIKV RNA from body fluid samples.Unfortunately,the short viremia window and asymptomatic/mild infections greatly reduce the success rate of PCRs(de Vasconcelos et al.2018).     

Evolution of Fitness in Experimental Populations of Vesicular Stomatitis Virus

The evolution of fitness in experimental clonal populations of vesicular stomatitis virus (VSV) has been compared under different genetic (fitness of initial clone) and demographic (population dynamics) regimes. In spite of the high genetic heterogeneity among replicates within experiments, there is a clear effect of population dynamics on the evolution of fitness. Those populations that went through strong periodic bottlenecks showed a decreased fitness in competition experiments with wild type. Conversely, mutant populations that were transferred under the dynamics of continuous population expansions increased their fitness when compared with the same wild type. The magnitude of the observed effect depended on the fitness of the original viral clone. Thus, high fitness clones showed a larger reduction in fitness than low fitness clones under dynamics with included periodic bottleneck. In contrast, the gain in fitness was larger the lower the initial fitness of the viral clone. The quantitative genetic analysis of the trait ``fitness' in the resulting populations shows that genetic variation for the trait is positively correlated with the magnitude of the change in the same trait. The results are interpreted in terms of the operation of MULLER's ratchet and genetic drift as opposed to the appearance of beneficial mutations.     

Genetic Mapping of Specific Interactions between Aedes aegypti Mosquitoes and Dengue Viruses

Specific interactions between host genotypes and pathogen genotypes (G×G interactions) are commonly observed in invertebrate systems. Such specificity challenges our current understanding of invertebrate defenses against pathogens because it contrasts the limited discriminatory power of known invertebrate immune responses. Lack of a mechanistic explanation, however, has questioned the nature of host factors underlying G×G interactions. In this study, we aimed to determine whether G×G interactions observed between dengue viruses and their Aedes aegypti vectors in nature can be mapped to discrete loci in the mosquito genome and to document their genetic architecture. We developed an innovative genetic mapping strategy to survey G×G interactions using outbred mosquito families that were experimentally exposed to genetically distinct isolates of two dengue virus serotypes derived from human patients. Genetic loci associated with vector competence indices were detected in multiple regions of the mosquito genome. Importantly, correlation between genotype and phenotype was virus isolate-specific at several of these loci, indicating G×G interactions. The relatively high percentage of phenotypic variation explained by the markers associated with G×G interactions (ranging from 7.8% to 16.5%) is consistent with large-effect host genetic factors. Our data demonstrate that G×G interactions between dengue viruses and mosquito vectors can be assigned to physical regions of the mosquito genome, some of which have a large effect on the phenotype. This finding establishes the existence of tangible host genetic factors underlying specific interactions between invertebrates and their pathogens in a natural system. Fine mapping of the uncovered genetic loci will elucidate the molecular mechanisms of mosquito-virus specificity.     

Inferring Genome-Wide Correlations of Mutation Fitness Effects between Populations

The effect of a mutation on fitness may differ between populations depending on environmental and genetic context, but little is known about the factors that underlie such differences. To quantify genome-wide correlations in mutation fitness effects, we developed a novel concept called a joint distribution of fitness effects (DFE) between populations. We then proposed a new statistic w to measure the DFE correlation between populations. Using simulation, we showed that inferring the DFE correlation from the joint allele frequency spectrum is statistically precise and robust. Using population genomic data, we inferred DFE correlations of populations in humans, Drosophila melanogaster, and wild tomatoes. In these species, we found that the overall correlation of the joint DFE was inversely related to genetic differentiation. In humans and D. melanogaster, deleterious mutations had a lower DFE correlation than tolerated mutations, indicating a complex joint DFE. Altogether, the DFE correlation can be reliably inferred, and it offers extensive insight into the genetics of population divergence.     

Genetic Changes Accompanying Increased Fitness in Evolving Populations of Escherichia Coli

Two populations of Escherichia coli, each initiated with a single clone containing a derivative of the plasmid pBR322, were maintained for long periods in glucose-limited continuous culture. In both populations, after an extensive number of generations had elapsed, clones were isolated in which the transposon Tn3 from the plasmid had integrated into the bacterial chromosome. In both cases examined, the transpositions were shown to increase relative fitness approximately 6-7%, in the environment in which the populations were maintained. The loci of integration were mapped to approximately 13.2 min (population 1) and approximately 32.8 min (population 2).     

The Distribution of Plant Populations

In studying the distribution of plant species in sample quadratsit may be assumed that the individuals are aggregated into groupsshowing a Poisson distribution, while the number of individualsin each group follows a logarithmic distribution. The resultingcompound distribution of individuals per quadrat is the negativebinomial. Evidence is produced to show that the distributionscalculated on this basis agree with those actually obtainedin the cases examined.     

Nature and Extent of Genetic Diversity of Dengue Viruses Determined by 454 Pyrosequencing

Dengue virus (DENV) populations are characteristically highly diverse. Regular lineage extinction and replacement is an important dynamic DENV feature, and most DENV lineage turnover events are associated with increased incidence of disease. The role of genetic diversity in DENV lineage extinctions is not understood. We investigated the nature and extent of genetic diversity in the envelope (E) gene of DENV serotype 1 representing different lineages histories. A region of the DENV genome spanning the E gene was amplified and sequenced by Roche/454 pyrosequencing. The pyrosequencing results identified distinct sub-populations (haplotypes) for each DENV-1 E gene. A phylogenetic tree was constructed with the consensus DENV-1 E gene nucleotide sequences, and the sequences of each constructed haplotype showed that the haplotypes segregated with the Sanger consensus sequence of the population from which they were drawn. Haplotypes determined through pyrosequencing identified a recombinant DENV genome that could not be identified through Sanger sequencing. Nucleotide level sequence diversities of DENV-1 populations determined from SNP analysis were very low, estimated from 0.009–0.01. There were also no stop codon, frameshift or non-frameshift mutations observed in the E genes of any lineage. No significant correlations between the accumulation of deleterious mutations or increasing genetic diversity and lineage extinction were observed (p>0.5). Although our hypothesis that accumulation of deleterious mutations over time led to the extinction and replacement of DENV lineages was ultimately not supported by the data, our data does highlight the significant technical issues that must be resolved in the way in which population diversity is measured for DENV and other viruses. The results provide an insight into the within-population genetic structure and diversity of DENV-1 populations.     

Selection and Characterization of DNA Aptamers Targeting All Four Serotypes of Dengue Viruses

Dengue viruses (DENVs) are members of Flaviviridae family, which are associated with human disease. The envelope (E) protein plays an important role in viral infection. However, there is no effective antibody for clinical treatment due to antibody dependent enhancement of infection. In this study, using Systematic Evolution of Ligands by Exponential Enrichment (SELEX), we demonstrated the first aptamer (S15) that can bind to DENV-2 envelop protein domain III (ED3) with a high binding affinity. S15 was found to form a parallel quadruplex based on Quadfinder prediction, gel mobility assay and circular dichroism studies. Both the quadruplex structure and the sequence on 5’-end were necessary for the binding activity of S15. NMR titration experiments indicated that S15 bound to a highly conserved loop between β_A and β_B strands of ED3. Moreover, S15 can neutralize the infections by all four serotypes of DENVs. Our result provides a new opportunity in the development of DNA aptamers against DENVs in the future.     

High Content Screening of a Kinase-Focused Library Reveals Compounds Broadly-Active against Dengue Viruses

Dengue virus is a mosquito-borne flavivirus that has a large impact in global health. It is considered as one of the medically important arboviruses, and developing a preventive or therapeutic solution remains a top priority in the medical and scientific community. Drug discovery programs for potential dengue antivirals have increased dramatically over the last decade, largely in part to the introduction of high-throughput assays. In this study, we have developed an image-based dengue high-throughput/high-content assay (HT/HCA) using an innovative computer vision approach to screen a kinase-focused library for anti-dengue compounds. Using this dengue HT/HCA, we identified a group of compounds with a 4-(1-aminoethyl)-N-methylthiazol-2-amine as a common core structure that inhibits dengue viral infection in a human liver-derived cell line (Huh-7.5 cells). Compounds CND1201, CND1203 and CND1243 exhibited strong antiviral activities against all four dengue serotypes. Plaque reduction and time-of-addition assays suggests that these compounds interfere with the late stage of viral infection cycle. These findings demonstrate that our image-based dengue HT/HCA is a reliable tool that can be used to screen various chemical libraries for potential dengue antiviral candidates.