Serotype-specific entry of dengue virus into liver cells: identification of the 37-kilodalton/67-kilodalton high-affinity laminin receptor as a dengue virus serotype 1 receptor

Dengue virus, the causative agent of dengue fever, dengue shock syndrome, and dengue hemorrhagic fever, infects susceptible cells by initially binding to a receptor(s) located on the host cell surface. Evidence to date suggests that receptor usage may be cell and serotype specific, and this study sought to identify dengue virus serotype 1 binding proteins on the surface of liver cells, a known target organ. By using a virus overlay protein binding assay (VOPBA), in both nondenaturing and denaturing gel systems, a putative dengue virus serotype 1 binding protein of approximately 37 kDa expressed on the surface of liver (HepG2) cells was identified. Mass spectrometry analysis identified a candidate protein, the 37/67-kDa high-affinity laminin receptor. Entry of the dengue virus serotype 1 was significantly inhibited in a dose-dependent manner by both antibodies directed against the 37/67-kDa high-affinity laminin receptor and soluble laminin. No inhibition of virus entry was seen with dengue virus serotypes 2, 3, or 4, demonstrating that the 37/67-kDa high-affinity laminin receptor is a serotype-specific receptor for dengue virus entry into liver cells.     

Phylogenetic and Molecular Clock Analysis of Dengue Serotype 1 and 3 from New Delhi,India

Dengue fever is the most prevalent arboviral disease in the tropical and sub-tropical regions of the world. The present report describes molecular detection and serotyping of dengue viruses in acute phase blood samples collected from New Delhi, India. Phylogenetic and molecular clock analysis of dengue virus serotype 1 and 3 strains were also investigated. Dengue virus infection was detected in 68.87% out of 604 samples tested by RT-PCR between 2011 & 2014. Dengue serotype 1 was detected in 25.48% samples, dengue serotype 2 in 79.56% samples and dengue serotype 3 in 11.29% samples. Dengue serotype 4 was not detected. Co-infection by more than one dengue serotype was detected in 18.26% samples. Envelope gene of 29 DENV-1 and 14 DENV-3 strains were sequenced in the study. All the DENV-1 strains grouped with the American African genotype. All DENV-3 strains were found to belong to Genotype III. Nucleotide substitution rates of dengue 1 and 3 viruses were determined in the study. Time to the most recent common ancestor (TMRCA) of dengue 1 viruses was determined to be 132 years. TMRCA of DENV-3 viruses was estimated to be 149 years. Bayesian skyline plots were constructed for Indian DENV-1 and 3 strains which showed a decrease in population size since 2005 in case of DENV- 1 strains while no change was observed in recent years in case of DENV-3 strains. The study also revealed a change in the dominating serotype in Delhi, India in recent years. The study will be helpful in formulating control strategies for the outbreaks. In addition, it will also assist in tracking the movement and evolution of this emerging virus.     

Co-Circulation of Dengue Virus Type 3 Genotypes in Vientiane Capital,Lao PDR

During the 2012 epidemic of dengue in Vientiane capital, Lao PDR, a major serotype switch from dengue 1 to 3 was observed. A molecular epidemiology study demonstrated that dengue 3 remained the predominant serotype in 2013, but also revealed the co-circulation of two genotypes, supporting the hypothesis of multiple geographic origins of dengue 3 strains circulating in Vientiane capital.     

An enzyme-linked immunosorbent assay using a chaotropic agent (sodium thiocyanate) for serotype specific reaction between crude dengue viral antigen and anti-dengue mouse antibody.

An enzyme-linked immunosorbent assay (ELISA) has been developed to detect serotype specific reaction between crude dengue viral antigen and anti-dengue mouse hyperimmunized antibody under the stringent condition in the presence of a Chaotropic agent, sodium thiocyanate (NaSCN), in the reaction mixture of antigen and antibody. Rapidly sedimenting hemagglutinin (RHA) derived from type 2 dengue virus-infected mosquito cell culture fluid reacted to the antibody for both type 2 and type 3 dengue viruses in the ELISA. In contrast, its reactivity was reduced after the addition of NaSCN in the ELISA. Soluble complement-fixing antigen (SCF) derived from type 2 dengue virus-infected mosquito cell culture fluid reacted serotype specifically to anti-dengue type 2 antibody, and was relatively stable for the NaSCN treatment in the ELISA. Anti-type 2 RHA mouse antibody reacted to both type 1 and type 2 dengue viral antigens and its reactivity was reduced after the addition of NaSCN in the ELISA. Anti-type 2 SCF antibody reacted serotype specifically to type 2 dengue viral antigen with and without NaSCN in the ELISA.     

Dengue virus-specific murine T-lymphocyte proliferation: serotype specificity and response to recombinant viral proteins.

Definition of the T-lymphocyte responses to dengue viruses should aid in the development of safe and effective vaccines and help to explain the pathophysiology of dengue hemorrhagic fever and dengue shock syndrome. In this study, we demonstrated that dengue virus-specific T lymphocytes were detected in spleen cells from dengue virus-immune mice using an in vitro proliferation assay. Following immunization with a single dose of infectious dengue virus, murine lymphocytes showed increased proliferation when incubated in the presence of viral antigens of the same serotype but not in the presence of control antigens. Depletion experiments with antibody and complement showed that the population of responding cells expressed the Thy1+ L3T4+ Lyt2- phenotype. This indicates that the predominant proliferating cells are T lymphocytes of the helper-inducer phenotype. Dengue virus-specific memory lymphocyte responses were detectable for at least 22 weeks after immunization. The response to primary infection was primarily serotype specific, with some serotype cross-reactivity present at a low level. We demonstrated that lymphocytes from mice immunized with dengue 4 virus proliferate in response to a combination of dengue 4 virus C, pre-M, E, NS1, and NS2a proteins expressed in Sf9 cells with a recombinant baculovirus, and, to a lesser extent, to the dengue 4 virus E protein alone.     

Multiple specificities in the murine CD4+ and CD8+ T-cell response to dengue virus.

The target epitopes, serotype specificity, and cytolytic function of dengue virus-specific T cells may influence their theoretical roles in protection against secondary infection as well as the immunopathogenesis of dengue hemorrhagic fever. To study these factors in an experimental system, we isolated dengue virus-specific CD4+ and CD8+ T-cell clones from dengue-2 virus-immunized BALB/c mice. The T-cell response to dengue virus in this mouse strain was heterogeneous; we identified at least five different CD4+ phenotypes and six different CD8+ phenotypes. Individual T-cell clones recognized epitopes on the dengue virus pre-M, E, NSl/NS2A, and NS3 proteins and were restricted by the I-Ad, I-Ed, Ld, and Kd antigens. Both serotype-specific and serotype-cross-reactive clones were isolated in the CD4+ and CD8+ subsets; among CD8+ clones, those that recognized the dengue virus structural proteins were serotype specific whereas those that recognized the nonstructural proteins were serotype cross-reactive. All of the CD8+ and one of five CD4+ clones lysed dengue virus-infected target cells. Using synthetic peptides, we identified an Ld-restricted epitope on the E protein (residues 331 to 339, SPCKIPFEI) and a Kd-restricted epitope on the NS3 protein (residues 296 to 310, ARGYISTRVEM GEAA). These data parallel previous findings of studies using human dengue virus-specific T-cell clones. This experimental mouse system may be useful for studying the role of the virus serotype and HLA haplotype on T-cell responses after primary dengue virus infection.     

2型登革病毒检测基因芯片的研制

利用长片断PCR扩增含几乎全长的2型登革病毒cDNA,酶切PCR扩增产物,通过建立2型登革病毒。DNA文库获取芯片探针用点样仪将探针制备成2型登革病毒检测基因芯片,杂交时采用限制性显示(Restriction Display RD)技术标记样品,Scan Array芯片扫描仪检测杂交信号．杂交结果显示,样品和2型登革病毒基因芯片杂交的敏感性强、特异性高．     

Histopathological and ultrastructural aspects of mice lungs experimentally infected with dengue virus serotype 2

Histological and ultrastructural alterations in lung tissue of BALB/c mice infected with dengue virus serotype 2 (non-neuroadapted), by intraperitoneal and intravenous routes were analyzed. Lung tissues were processed following the standard techniques for photonic and electron transmission microscopies. Histopathological and ultrastructural studies showed interstitial pneumonia, characterized by the presence of mononuclear cells. In the mouse model, the dengue virus serotype 2 seems to led to a transient inflammatory process without extensive damage to the interalveolar septa, but caused focal alterations of the blood-exchange barrier. Endothelial cells of blood capillaries exhibited phyllopodia suggesting activation by presence of dengue virus. Morphometrical analysis of mast cells showed an expressive increase of the number of these cells in peribronchiolar spaces and adjacent areas to the interalveolar septa. Alveolar macrophages showed particles dengue virus-like inside rough endoplasmic reticulum and Golgi complex, suggesting viral replication. The tissue alterations observed in our experimental model were similar to the observed in human cases of dengue fever and dengue hemorrhagic fever. Our results show that BALB/c mice are permissive host for dengue virus serotype 2 replication and therefore provides an useful model to study of morphological aspects of dengue virus infection.     

Genome sequence of the Bacteroides fragilis phage ATCC 51477-B1

Background

Co-circulation of multiple dengue virus serotypes has been reported from many parts of the world including India, however concurrent infection with more than one serotype of dengue viruses in the same individual is rarely documented. An outbreak of dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) occurred in and around Delhi in 2006. This is the first report from India with high percentage of concurrent infections with different dengue virus serotypes circulating during one outbreak.

Results

Acute phase sera from patients were tested for the presence of dengue virus RNA by RT-PCR assay. Of the 69 samples tested for dengue virus RNA, 48 (69.5%) were found to be positive. All the four dengue virus serotypes were found to be co-circulating in this outbreak with DENV-3 being the predominant serotype. In addition in 9 of 48 (19%) dengue virus positive samples, concurrent infection with more than one dengue virus serotype were identified.

Conclusion

This is the first report in which concurrent infections with different dengue virus serotypes is being reported during an outbreak from India. Delhi is now truly hyperendemic for dengue.     

A synthetic peptide analog of in silico-predicted immunogenic epitope unique to dengue virus serotype 2 NS1 antigen specifically binds immunoglobulin G antibodies raised in rabbits

Development of a serotyping-capable dengue detection test is hampered by the absence of an identified unique marker that can detect specific dengue virus (DENV) serotype. In the current commercially available antibody-capture diagnostic methods, immobilized nonstructural 1 (NS1) antigen indiscriminately binds and detects immunoglobulin M or immunoglobulin G against any serotype, thus limiting its capability to distinguish existing serotypes of dengue. Identification of dengue serotype is important because certain serotypes are associated with severe forms of dengue as well as dengue hemorrhagic fever. In this study, we aimed to identify an immunogenic epitope unique to DENV2 NS1 antigen and determine the binding specificity of its synthetic peptide mimotope to antibodies raised in animal models. Selection of a putative B-cell epitope from the reported DENV2 NS1 antigen was done using Kolaskar and Tongaonkar Antigenicity prediction, Emini surface accessibility prediction, and Parker hydrophilicity prediction available at the immune epitope database and analysis resource. Uniqueness of the B-cell epitope to DENV2 was analyzed by BLASTp. Immunogenicity of the synthetic peptide analog of the predicted immunogenic epitope was tested in rabbits. The binding specificity of the antibodies raised in animals and the synthetic peptide mimotope was tested by indirect ELISA. A synthetic peptide analog comprising the unique epitope of DENV2 located at the 170th–183rd position of DENV2 NS1 was found to be immunogenic in animal models. The antipeptide antibody produced in rabbits showed specific binding to the synthetic peptide mimotope of the predicted unique DENV2 NS1 immunogenic epitope.     

The ecological determinants of severe dengue: A Bayesian inferential model

Low socioeconomic status (SES), high temperature, and increasing rainfall patterns are associated with increased dengue case counts. However, the effect of climatic variables on individual dengue virus (DENV) serotypes and the extent to which serotype count affects the rate of severe dengue in Mexico have not been studied before. A principal components analysis was used to determine the poverty indices across Mexico. Conditional autoregressive Bayesian models were used to determine the effect of poverty and climatic variables on the rate of serotype distribution and severe dengue in Mexico. A unit increase in poverty increased the rate of DENV-1, DENV-2, DENV-3, and DENV-4 by 8.4%, 5%, 16%, and 13.8% respectively. An increase in one case attributable to DENV-1, DENV-2, DENV-3, and DENV-4 was independently associated with an increase in the rate of severe dengue by 0.02%, 0.1%, 0.03%, and 5.8% respectively. Hotspots of all DENV serotypes and severe dengue are found mostly in parts of the Northeastern, Center west, and Southeastern regions of Mexico. The association between climatic parameters predominant in the Southeast region and severe dengue leaves several states in this region at an increased risk of a higher number of severe dengue cases. Our study's results may guide policies that help allocate public health resources to the most vulnerable municipalities in Mexico.     

An in-depth analysis of original antigenic sin in dengue virus infection

The evolution of dengue viruses has resulted in four antigenically similar yet distinct serotypes. Infection with one serotype likely elicits lifelong immunity to that serotype, but generally not against the other three. Secondary or sequential infections are common, as multiple viral serotypes frequently cocirculate. Dengue infection, although frequently mild, can lead to dengue hemorrhagic fever (DHF) which can be life threatening. DHF is more common in secondary dengue infections, implying a role for the adaptive immune response in the disease. There is currently much effort toward the design and implementation of a dengue vaccine but these efforts are made more difficult by the challenge of inducing durable neutralizing immunity to all four viruses. Domain 3 of the dengue virus envelope protein (ED3) has been suggested as one such candidate because it contains neutralizing epitopes and it was originally thought that relatively few cross-reactive antibodies are directed to this domain. In this study, we performed a detailed analysis of the anti-ED3 response in a cohort of patients suffering either primary or secondary dengue infections. The results show dramatic evidence of original antigenic sin in secondary infections both in terms of binding and enhancement activity. This has important implications for dengue vaccine design because heterologous boosting is likely to maintain the immunological footprint of the first vaccination. On the basis of these findings, we propose a simple in vitro enzyme-linked immunosorbent assay (ELISA) to diagnose the original dengue infection in secondary dengue cases.     

Analysis of Murine CD8+ T-Cell Clones Specific for the Dengue Virus NS3 Protein: Flavivirus Cross-Reactivity and Influence of Infecting Serotype

Serotype-cross-reactive dengue virus-specific cytotoxic T lymphocytes (CTL) induced during a primary dengue virus infection are thought to play a role in the immunopathogenesis of dengue hemorrhagic fever (DHF) during a secondary dengue virus infection. Although there is no animal model of DHF, we previously reported that murine dengue virus-specific CTL responses are qualitatively similar to human dengue virus-specific CTL responses. We used BALB/c mice to study the specificity of the CTL response to an immunodominant epitope on the dengue virus NS3 protein. We mapped the minimal H-2K^d-restricted CTL epitope to residues 298 to 306 of the dengue type 2 virus NS3 protein. In short-term T-cell lines and clones, the predominant CD8⁺ CTL to this epitope in mice immunized with dengue type 2 virus or vaccinia virus expressing the dengue type 4 virus NS3 protein were cross-reactive with dengue type 2 or type 4 virus, while broadly serotype-cross-reactive CTL were a minority population. In dengue type 3 virus-immunized mice, the predominant CTL response to this epitope was broadly serotype cross-reactive. All of the dengue virus-specific CTL clones studied also recognized the homologous NS3 sequences of one or more closely related flaviviruses, such as Kunjin virus. The critical contact residues for the CTL clones with different specificities were mapped with peptides having single amino acid substitutions. These data demonstrate that primary dengue virus infection induces a complex population of flavivirus-cross-reactive NS3-specific CTL clones in mice and suggest that CTL responses are influenced by the viral serotype. These findings suggest an additional mechanism by which the order of sequential flavivirus infections may influence disease manifestations.     

Evolutionary Analysis of Dengue Serotype 2 Viruses Using Phylogenetic and Bayesian Methods from New Delhi,India

Dengue fever is the most important arboviral disease in the tropical and sub-tropical countries of the world. Delhi, the metropolitan capital state of India, has reported many dengue outbreaks, with the last outbreak occurring in 2013. We have recently reported predominance of dengue virus serotype 2 during 2011–2014 in Delhi. In the present study, we report molecular characterization and evolutionary analysis of dengue serotype 2 viruses which were detected in 2011–2014 in Delhi. Envelope genes of 42 DENV-2 strains were sequenced in the study. All DENV-2 strains grouped within the Cosmopolitan genotype and further clustered into three lineages; Lineage I, II and III. Lineage III replaced lineage I during dengue fever outbreak of 2013. Further, a novel mutation Thr404Ile was detected in the stem region of the envelope protein of a single DENV-2 strain in 2014. Nucleotide substitution rate and time to the most recent common ancestor were determined by molecular clock analysis using Bayesian methods. A change in effective population size of Indian DENV-2 viruses was investigated through Bayesian skyline plot. The study will be a vital road map for investigation of epidemiology and evolutionary pattern of dengue viruses in India.     

Determination of dengue virus serotypes in individual Aedes aegypti mosquitoes in Colombia

Adult Aedes aegypti mosquitoes were collected in Puerto Triunfo, central Colombia, where dengue is endemic, during a six month period. Viral infection within the head of each individual mosquito was identified by an immunofluorescent assay (IFA) using a flavivirus-specific monoclonal antibody. The dengue virus serotype, present in each flavivirus-positive specimen, was then determined in portions of the remaining thorax using IFAs with serotype-specific monoclonal antibodies. Among 2065 female Aedes aegypti collected and tested, twenty-four flavivirus-positive individuals were found (minimum infection rate 11.6%), three identified as dengue type-1 and twenty-one as dengue type-2 virus. This was consistent with the isolation of only these two serotypes of dengue virus from dengue fever patients within this town. No vertical transmission of dengue virus could be detected in 1552 male Aedes aegypti collected. This method is inexpensive, simple, rapid to perform and suitable for use in developing countries to identify and distinguish different serotypes of dengue virus in their vectors during eco-epidemiological investigations.     

Dengue virus-reactive CD8+ T cells display quantitative and qualitative differences in their response to variant epitopes of heterologous viral serotypes

Reactivation of serotype cross-reactive CD8+ memory T lymphocytes is thought to contribute to the immunopathogenesis of dengue disease during secondary infection by a heterologous serotype. Using cytokine flow cytometry, we have defined four novel HLA-A*02-restricted dengue viral epitopes recognized by up to 1.5% of circulating CD8+ T cells in four donors after primary vaccination. All four donors had the highest cytokine response to the epitope NS4b 2353. We also studied the effect of sequence differences in heterologous dengue serotypes on dengue-reactive CD8+ memory T cell cytokine and proliferative responses. The D3 variant of a different NS4b epitope 2423 and the D2 variant of the NS4a epitope 2148 induced the largest cytokine response, compared with their respective heterologous sequences in all donors regardless of the primary vaccination serotype. Stimulation with variant peptides also altered the relative frequencies of the various subsets of cells that expressed IFN-gamma, TNF-alpha, MIP-1beta, and combinations of these cytokines. These results indicate that the prior infection history of the individual as well as the serotypes of the primary and heterologous secondary viruses influence the nature of the secondary response. These differences in the effector functions of serotype cross-reactive memory T cells induced by heterologous variant epitopes, which are both quantitative and qualitative, may contribute to the clinical outcome of secondary dengue infection.     

Immunological serotype interactions and their effect on the epidemiological pattern of dengue

Long-term epidemiological data reveal multi-annual fluctuations in the incidence of dengue fever and dengue haemorrhagic fever, as well as complex cyclical behaviour in the dynamics of the four serotypes of the dengue virus. It has previously been proposed that these patterns are due to the phenomenon of the so-called antibody-dependent enhancement (ADE) among dengue serotypes, whereby viral replication is increased during secondary infection with a heterologous serotype; however, recent studies have implied that this positive reinforcement cannot account for the temporal patterns of dengue and that some form of cross-immunity or external forcing is necessary. Here, we show that ADE alone can produce the observed periodicities and desynchronized oscillations of individual serotypes if its effects are decomposed into its two possible manifestations: enhancement of susceptibility to secondary infections and increased transmissibility from individuals suffering from secondary infections. This decomposition not only lowers the level of enhancement necessary for realistic disease patterns but also reduces the risk of stochastic extinction. Furthermore, our analyses reveal a time-lagged correlation between serotype dynamics and disease incidence rates, which could have important implications for understanding the irregular pattern of dengue epidemics.     

Immunologic hypo- or non-responder in natural dengue virus infection

Serologically defined primary dengue virus infection and/or subsequent homologous serotype infection is known to be associated with less severe disease as compared with secondary subsequent heterologous serotype infection. In geographical locales of high dengue endemicity, almost all individuals in the population are infected at some point in time and should therefore are at high risk of secondary infection. Interestingly, dengue viremia in healthy blood donors whose sera apparently lack detectable levels of specific antibody to dengue viral antigens has been reported. The incidence rate of potential immunologic hypo- or non-responders following natural primary dengue virus infection in dengue endemic regions, who do become immune responders only after repeated exposure, has not been described. These are the patients who may be diagnosed as primary infection in the subsequent infection, but actually are secondary infection. This concept has important implications with regards to the hypothesis of immunological enhancement of dengue pathogenesis, which has largely been advanced based on empirical observations and/or from in vitro experimental assays. The fact that dengue naïve travelers can suffer from severe dengue upon primary exposure while visiting dengue endemic countries underscores one of the major problems in explaining the role of immune enhancement in the pathogenesis of severe dengue virus infection. This evidence suggests that the mechanism(s) leading to severe dengue may not be associated with pre-existing enhancing antibody. Consequently, we propose a new paradigm for dengue virus infection classification. These include a) patients with naïve primary infection, b) those that are serologically defined primary in dengue endemic zones and c) those who are serologically defined secondary dengue virus infection. We submit that clarity with regards to such definitions may help facilitate the delineation of the potential mechanisms of severe dengue virus infection.