Identification of Anti-Premembrane Antibody as a Serocomplex-Specific Marker To Discriminate Zika, Dengue, and West Nile Virus Infections.

Although transmission of Zika virus (ZIKV) in the Americas has greatly declined since late 2017, recent reports of reduced risks of symptomatic Zika by prior dengue virus (DENV) infection and increased risks of severe dengue disease by previous ZIKV or DENV infection underscore a critical need for serological tests that can discriminate past ZIKV, DENV, and/or other flavivirus infections and improve our understanding of the immune interactions between these viruses and vaccine strategy in endemic regions. As serological tests for ZIKV primarily focus on envelope (E) and nonstructural protein 1 (NS1), antibodies to other ZIKV proteins have not been explored. Here, we employed Western blot analysis using antigens of 6 flaviviruses from 3 serocomplexes to investigate antibody responses following reverse transcription-PCR (RT-PCR)-confirmed ZIKV infection. Panels of 20 primary ZIKV and 20 ZIKV with previous DENV infection recognized E proteins of all 6 flaviviruses and the NS1 protein of ZIKV with some cross-reactivity to DENV. While the primary ZIKV panel recognized only the premembrane (prM) protein of ZIKV, the ZIKV with previous DENV panel recognized both ZIKV and DENV prM proteins. Analysis of antibody responses following 42 DENV and 18 West Nile virus infections revealed similar patterns of recognition by anti-E and anti-NS1 antibodies, whereas both panels recognized the prM protein of the homologous serocomplex but not others. The specificity was further supported by analysis of sequential samples. Together, these findings suggest that anti-prM antibody is a flavivirus serocomplex-specific marker and can be used to delineate current and past flavivirus infections in endemic areas. IMPORTANCE Despite a decline in Zika virus (ZIKV) transmission since late 2017, questions regarding its surveillance, potential reemergence, and interactions with other flaviviruses in regions where it is endemic remain unanswered. Recent studies have reported reduced risks of symptomatic Zika by prior dengue virus (DENV) infection and increased risks of severe dengue disease by previous ZIKV or DENV infection, highlighting a need for better serological tests to discriminate past ZIKV, DENV, and/or other flavivirus infections and improved understanding of the immune interactions and vaccine strategy for these viruses. As most serological tests for ZIKV focused on envelope and nonstructural protein 1, antibodies to other ZIKV proteins, including potentially specific antibodies, remain understudied. We employed Western blot analysis using antigens of 6 flaviviruses to study antibody responses following well-documented ZIKV, DENV, and West Nile virus infections and identified anti-premembrane antibody as a flavivirus serocomplex-specific marker to delineate current and past flavivirus infections in areas where flaviviruses are endemic.

Validation of the GeneXpert Xpress SARS-CoV-2 PCR assay using saliva as biological specimen

ABSTRACT In the pandemic, rapid and accurate detection of SARS-CoV-2 is crucial in controlling the outbreak. Recent studies have shown a high detection rate using saliva/oral fluids as specimens for laboratory detection of the virus. We intended to evaluate the test performance of the Xpert Xpress SARS-CoV-2 cartridge assay in comparison to a conventional qRT-PCR testing, using saliva as biological specimen. Forty saliva samples from symptomatic participants were collected. Conventional qRT-PCR was performed for amplification of E and RdRp genes and the Xpert Xpress SARS-CoV-2 assay amplified E and N2 genes. In the conventional assay, the median cycle threshold value of the E gene was 34.9, and of the RdRp gene was 38.3. In the Xpert Xpress assay, the median cycle threshold value of the E gene was 29.7, and of the N2 gene was 31.6. These results can allow a broaden use of molecular tests for management of COVID-19 pandemic, especially in resources-limited settings.

Saliva is a reliable, non-invasive specimen for SARS-CoV-2 detection

Abstract Background Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is the cause of Coronavirus Disease 2019 (COVID-19). Although Real Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) of respiratory specimens is the gold standard test for detection of SARS-CoV-2 infection, collecting nasopharyngeal swabs causes discomfort to patients and may represent considerable risk for healthcare workers. The use of saliva as a diagnostic sample has several advantages. Objectives The aim of this study was to validate the use of saliva as a biological sample for diagnosis of COVID-19. Methods This study was conducted at Infectious Diseases Research Laboratory (LAPI), in Salvador, Brazil. Participants presenting with signs/symptoms suggesting SARS-CoV-2 infection underwent a nasopharyngeal swab (NPS) and/or oropharyngeal swab (OPS), and saliva collection. Saliva samples were diluted in PBS, followed by RNA isolation and RT-Real Time PCR for SARS-CoV-2. Results of conventional vs saliva samples testing were compared. Statistical analyses were performed using Statistical Package for the Social Sciences software (SPSS) version 18.0. Results One hundred fifty-five participants were recruited and samples pairs of NPS/OPS and saliva were collected. The sensitivity and specificity of RT-PCR using saliva samples were 94.4% (95% CI 86.4-97.8) and 97.62% (95% CI 91.7-99.3), respectively. There was an overall high agreement (96.1%) between the two tests. Conclusions Use of self-collected saliva samples is an easy, convenient, and low-cost alternative to conventional NP swab-based molecular tests. These results may allow a broader use of molecular tests for management of COVID19 pandemic, especially in resources-limited settings.

Potent Neutralizing Human Monoclonal Antibodies Preferentially Target Mature Dengue Virus Particles: Implication for Novel Strategy for Dengue Vaccine.

The four serotypes of dengue virus (DENV) cause the most important mosquito-borne viral disease in humans. The envelope (E) protein is the major target of neutralizing antibodies and contains 3 domains (domain I [DI], DII, and DIII). Recent studies reported that human monoclonal antibodies (MAbs) recognizing DIII, the D1/DII hinge, the E-dimer epitope, or a quaternary epitope involving DI/DII/DIII are more potently neutralizing than those recognizing the fusion loop (FL) of DII. Due to inefficient cleavage of the premembrane protein, DENV suspensions consist of a mixture of mature, immature, and partially immature particles. We investigated the neutralization and binding of 22 human MAbs to DENV serotype 1 (DENV1) virions with differential maturation status. Compared with FL MAbs, DIII, DI/DII hinge, and E-dimer epitope MAbs showed higher maximum binding and avidity to mature particles relative to immature particles; this feature may contribute to the strong neutralizing potency of such MAbs. FL-specific MAbs required 57 to 87% occupancy on mature particles to achieve half-maximal neutralization (NT50), whereas the potently neutralizing MAbs achieved NT50 states at 20 to 38% occupancy. Analysis of the MAb repertoire and polyclonal sera from patients with primary DENV1 infection supports the immunodominance of cross-reactive anti-E antibodies over type-specific antibodies. After depletion with viral particles from a heterologous DENV serotype, the type-specific neutralizing antibodies remained and showed binding features shared by potent neutralizing MAbs. Taken together, these findings suggest that the use of homogeneous mature DENV particles as an immunogen may induce more potent neutralizing antibodies against DENV than the use of immature or mixed particles.IMPORTANCE With an estimated 390 million infections per year, the four serotypes of dengue virus (DENV) cause the most important mosquito-borne viral disease in humans. The dengue vaccine Dengvaxia was licensed; however, its low efficacy among dengue-naive individuals and increased risk of causing severe dengue in children highlight the need for a better understanding of the role of human antibodies in immunity against DENV. DENV suspensions contain mature, immature, and partially immature particles. We investigated the binding of 22 human monoclonal antibodies (MAbs) to the DENV envelope protein on particles with different maturation states. Potently neutralizing MAbs had higher relative maximum binding and avidity to mature particles than weakly neutralizing MAbs. This was supported by analysis of MAb repertoires and polyclonal sera from patients with primary DENV infection. Together, these findings suggest that mature particles may be the optimal form of presentation of the envelope protein to induce more potent neutralizing antibodies against DENV.

T Cell Responses to Nonstructural Protein 3 Distinguish Infections by Dengue and Zika Viruses.

The 2015-2016 Zika virus (ZIKV) epidemic in the Americas and the Caribbean demonstrated that clinical assays to detect, distinguish, and characterize immune responses to flaviviral infections are needed. ZIKV and dengue virus (DENV) are mosquito-transmitted flaviviruses sharing overlapping geographic distributions and have significant sequence similarities that can increase the potential for antibody and T cell cross-reaction. Using nonstructural protein 1-based enzyme-linked immunosorbent assays (ELISAs), we determined the serostatus of individuals living in a region of DENV and ZIKV endemicity in Brazil, identifying individuals with primary DENV (pDENV) and primary ZIKV (pZIKV), ZIKV with primary DENV (ZIKVwpDENV), and secondary DENV (sDENV) infections; the presence of pDENV and pZIKV was further confirmed by neutralization tests. Development of an enzyme-linked immunosorbent spot (ELISPOT) assay for DENV and ZIKV structural and nonstructural (NS) protein antigens enabled us to distinguish infections by these viruses based on T cell responses and to characterize those responses. We found that gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α) T cell responses to NS3 differentiated DENV and ZIKV infections with 94% sensitivity and 92% specificity. In general, we also showed that pDENV and sDENV cases and pZIKV and ZIKVwpDENV cases elicit similar T cell response patterns and that HIV-infected individuals show T cell responses that are lower than those shown by HIV-negative individuals. These results have important implications for DENV and ZIKV diagnostic and vaccine development and provide critical insights into the T cell response in individuals with multiple flaviviral infections.IMPORTANCE The potential for antibody and T cell cross-reactions to DENV and ZIKV, flaviviruses that cocirculate and can sequentially infect individuals, has complicated diagnostic and vaccine development. Our serological data show that antibodies to nonstructural protein 1 can distinguish sequential human infections by DENV and ZIKV. The development of a simple and inexpensive assay also enables the differentiation of DENV and ZIKV infections based on characterization of T cell responses. Our T cell data reveal strong response patterns that are similar in nature to those seen with individuals with one or multiple DENV infections and with individuals with only primary ZIKV infection and ZIKV-infected individuals with previous DENV exposure. The characterization of T cell responses in a serologically validated group of individuals is of relevance to the development of vaccines and immunotherapeutics against these global threats.

A new class of highly potent, broadly neutralizing antibodies isolated from viremic patients infected with dengue virus.

Dengue is a rapidly emerging, mosquito-borne viral infection, with an estimated 400 million infections occurring annually. To gain insight into dengue immunity, we characterized 145 human monoclonal antibodies (mAbs) and identified a previously unknown epitope, the envelope dimer epitope (EDE), that bridges two envelope protein subunits that make up the 90 repeating dimers on the mature virion. The mAbs to EDE were broadly reactive across the dengue serocomplex and fully neutralized virus produced in either insect cells or primary human cells, with 50% neutralization in the low picomolar range. Our results provide a path to a subunit vaccine against dengue virus and have implications for the design and monitoring of future vaccine trials in which the induction of antibody to the EDE should be prioritized.

Characterization of the ectodomain of the envelope protein of dengue virus type 4: expression, membrane association, secretion and particle formation in the absence of precursor membrane protein.

BACKGROUND: The envelope (E) of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. After biosynthesis E protein forms a heterodimer with precursor membrane (prM) protein. Recent reports of infection enhancement by anti-prM monoclonal antibodies (mAbs) suggest anti-prM responses could be potentially harmful. Previously, we studied a series of C-terminal truncation constructs expressing DENV type 4 prM/E or E proteins and found the ectodomain of E protein alone could be recognized by all 12 mAbs tested, suggesting E protein ectodomain as a potential subunit immunogen without inducing anti-prM response. The characteristics of DENV E protein ectodomain in the absence of prM protein remains largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we investigated the expression, membrane association, glycosylation pattern, secretion and particle formation of E protein ectodomain of DENV4 in the presence or absence of prM protein. E protein ectodomain associated with membrane in or beyond trans-Golgi and contained primarily complex glycans, whereas full-length E protein associated with ER membrane and contained high mannose glycans. In the absence of prM protein, E protein ectodomain can secrete as well as form particles of approximately 49 nm in diameter, as revealed by sucrose gradient ultracentrifugation with or without detergent and electron microscopy. Mutational analysis revealed that the secretion of E protein ectodomain was affected by N-linked glycosylation and could be restored by treatment with ammonia chloride. CONCLUSIONS/SIGNIFICANCE: Considering the enhancement of DENV infectivity by anti-prM antibodies, our findings provide new insights into the expression and secretion of E protein ectodomain in the absence of prM protein and contribute to future subunit vaccine design.

High-avidity and potently neutralizing cross-reactive human monoclonal antibodies derived from secondary dengue virus infection.

The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies (Abs) and vaccine development. Previous studies of human dengue-immune sera reported that a significant proportion of anti-E Abs, known as group-reactive (GR) Abs, were cross-reactive to all four DENV serotypes and to one or more other flaviviruses. Based on studies of mouse anti-E monoclonal antibodies (MAbs), GR MAbs were nonneutralizing or weakly neutralizing compared with type-specific MAbs; a GR response was thus not regarded as important for vaccine strategy. We investigated the epitopes, binding avidities, and neutralization potencies of 32 human GR anti-E MAbs. In addition to fusion loop (FL) residues in E protein domain II, human GR MAbs recognized an epitope involving both FL and bc loop residues in domain II. The neutralization potencies and binding avidities of GR MAbs derived from secondary DENV infection were stronger than those derived from primary infection. GR MAbs derived from primary DENV infection primarily blocked attachment, whereas those derived from secondary infection blocked DENV postattachment. Analysis of the repertoire of anti-E MAbs derived from patients with primary DENV infection revealed that the majority were GR, low-avidity, and weakly neutralizing MAbs, whereas those from secondary infection were primarily GR, high-avidity, and potently neutralizing MAbs. Our findings suggest that the weakly neutralizing GR anti-E Abs generated from primary DENV infection become potently neutralizing MAbs against the four serotypes after secondary infection. The observation that the dengue immune status of the host affects the quality of the cross-reactive Abs generated has implications for new strategies for DENV vaccination.

Analysis of cross-reactive antibodies recognizing the fusion loop of envelope protein and correlation with neutralizing antibody titers in Nicaraguan dengue cases.

Dengue virus (DENV) is the leading cause of arboviral diseases in humans worldwide. The envelope (E) protein of DENV is the major target of neutralizing antibodies (Abs). Previous studies have shown that a significant proportion of anti-E Abs in human serum after DENV infection recognize the highly conserved fusion loop (FL) of E protein. The role of anti-FL Abs in protection against subsequent DENV infection versus pathogenesis remains unclear. A human anti-E monoclonal Ab was used as a standard in a virion-capture ELISA to measure the concentration of anti-E Abs, [anti-E Abs], in dengue-immune sera from Nicaraguan patients collected 3, 6, 12 and 18 months post-infection. The proportion of anti-FL Abs was determined by capture ELISA using virus-like particles containing mutations in FL, and the concentration of anti-FL Abs, [anti-FL Abs], was calculated. Neutralization titers (NT50) were determined using a previously described flow cytometry-based assay. Analysis of sequential samples from 10 dengue patients revealed [anti-E Abs] and [anti-FL Abs] were higher in secondary than in primary DENV infections. While [anti-FL Abs] did not correlate with NT50 against the current infecting serotype, it correlated with NT50 against the serotypes to which patients had likely not yet been exposed ("non-exposed" serotypes) in 14 secondary DENV3 and 15 secondary DENV2 cases. These findings demonstrate the kinetics of anti-FL Abs and provide evidence that anti-FL Abs play a protective role against "non-exposed" serotypes after secondary DENV infection.

Analysis of epitopes on dengue virus envelope protein recognized by monoclonal antibodies and polyclonal human sera by a high throughput assay.

BACKGROUND: The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. METHODOLOGY/PRINCIPAL FINDINGS: We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC' loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. CONCLUSIONS/SIGNIFICANCE: Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level.

C-terminal helical domains of dengue virus type 4 E protein affect the expression/stability of prM protein and conformation of prM and E proteins.

BACKGROUND: The envelope (E) protein of dengue virus (DENV) is the major immunogen for dengue vaccine development. At the C-terminus are two α-helices (EH1 and EH2) and two transmembrane domains (ET1 and ET2). After synthesis, E protein forms a heterodimer with the precursor membrane (prM) protein, which has been shown as a chaperone for E protein and could prevent premature fusion of E protein during maturation. Recent reports of enhancement of DENV infectivity by anti-prM monoclonal antibodies (mAbs) suggest the presence of prM protein in dengue vaccine is potentially harmful. A better understanding of prM-E interaction and its effect on recognition of E and prM proteins by different antibodies would provide important information for future design of safe and effective subunit dengue vaccines. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we examined a series of C-terminal truncation constructs of DENV4 prME, E and prM. In the absence of E protein, prM protein expressed poorly. In the presence of E protein, the expression of prM protein increased in a dose-dependent manner. Radioimmunoprecipitation, sucrose gradient sedimentation and pulse-chase experiments revealed ET1 and EH2 were involved in prM-E interaction and EH2 in maintaining the stability of prM protein. Dot blot assay revealed E protein affected the recognition of prM protein by an anti-prM mAb; truncation of EH2 or EH1 affected the recognition of E protein by several anti-E mAbs, which was further verified by capture ELISA. The E protein ectodomain alone can be recognized well by all anti-E mAbs tested. CONCLUSIONS/SIGNIFICANCE: A C-terminal domain (EH2) of DENV E protein can affect the expression and stability of its chaperone prM protein. These findings not only add to our understanding of the interaction between prM and E proteins, but also suggest the ectodomain of E protein alone could be a potential subunit immunogen without inducing anti-prM response.

The helical domains of the stem region of dengue virus envelope protein are involved in both virus assembly and entry.

The envelope (E) of dengue virus (DENV) is a determinant of tropism and virulence. At the C terminus of E protein, there is a stem region containing two amphipathic α-helical domains (EH1 and EH2) and a stretch of conserved sequences in between. The crystal structure of E protein at the postfusion state suggested the involvement of the stem during the fusion; however, the critical domains or residues involved remain unknown. Site-directed mutagenesis was carried out to replace each of the stem residues at the hydrophobic face with an alanine or proline in a DENV serotype 4 (DENV4) precursor membrane (prM)/E expression construct. Most of the 15 proline mutations at either EH1 or EH2 severely affected the assembly of virus-like particles (VLPs). Radioimmunoprecipitation and membrane flotation assays revealed that EH1 mutations primarily affect prM-E heterodimerization and EH2 mutations affect the membrane binding of the stem. Introducing four proline mutations at either EH1 or EH2 into a DENV2 replicon packaging system greatly affects assembly and entry. Moreover, introducing these mutations into a DENV2 infectious clone confirmed the impairment in assembly and infectivity. Sequencing analysis of adaptive mutations in passage 5 viruses revealed a change to a leucine or wild-type residue at the original site, suggesting the importance of maintaining the helical structure. Collectively, these findings suggest that the EH1 and EH2 domains are involved in both assembly and entry steps of the DENV replication cycle; this feature, together with the high degree of sequence conservation, suggests that the stem region is a potential target of antiviral strategies.

The length of and nonhydrophobic residues in the transmembrane domain of dengue virus envelope protein are critical for its retention and assembly in the endoplasmic reticulum.

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.

Identification of a minimal peptide derived from heptad repeat (HR) 2 of spike protein of SARS-CoV and combination of HR1-derived peptides as fusion inhibitors.

The heptad repeats (HR1 and HR2) of the spike protein of SARS-CoV are highly conserved regions forming a critical 6-helix bundle during the fusion step of virus entry and are attractive targets of entry inhibitors. In this study, we report that a minimal HR2 peptide, P6 of 23-mer, can block the fusion of SARS-CoV with an IC(50) of 1.04+/-0.22 microM. This finding supports the structural prediction of the deep groove of HR1 trimer as a target for fusion inhibitors, and suggests P6 as a potential lead peptide for future drug development. Moreover, combination of an HR-1 peptide, N46, and its mutated version, N46eg, shows synergistic inhibition with an IC(50) of 1.39+/-0.05 microM and combination index of 0.75+/-0.15, suggesting a common strategy to achieve promising inhibition by HR1 peptide for other class I envelope viruses.

Similarity between coffee effects and qi-stimulating events.

AIMS: Previously, we found that qi-stimulating events exerted similar frequency-specific effects on the blood pressure pulse spectrum. Because coffee and qi induce similar stimulatory psychological responses, we aimed to determine whether they would induce similar pulse effects. Such a relationship would suggest a close linkage between the physiologic mechanisms underlying the psychostimulatory responses and vascular effects of coffee and qi. Therefore, the profound investigations into the mechanisms underlying the effects of coffee on the central nervous system and the vascular system may help to elucidate the underlying physiology mechanisms of qi. METHODS: Each test subject took three rounds of 150 mL coffee (0.1 g/kg, 0.05 g/kg, 0.05 g/kg) in a 30-minute interval. The subject's pulses were recorded at the end of each round. The changes in the test subject's pulse spectrum between before and after coffee consumption were compared with changes induced by a water placebo. RESULTS: Both coffee and qi caused the intensities of the third, sixth, and ninth harmonics of the pressure pulse spectrum to be relative peaks to their neighboring harmonics. CONCLUSION: Our results suggest that the coffee effect may be considered a qi-stimulating event, and there is a common physiologic factor determining the psychostimulatory responses of qi and coffee as well as their effects on the cardiovascular system, which results in a specific frequency pattern in the blood pulse spectrum. Adenosine, which is the main physiologic compound affected by coffee, might also be the key factor affected by qi.

Antibodies to envelope glycoprotein of dengue virus during the natural course of infection are predominantly cross-reactive and recognize epitopes containing highly conserved residues at the fusion loop of domain II.

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.

A strong endoplasmic reticulum retention signal in the stem-anchor region of envelope glycoprotein of dengue virus type 2 affects the production of virus-like particles.

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.

Incorporation of dengue virus replicon into virus-like particles by a cell line stably expressing precursor membrane and envelope proteins of dengue virus type 2.

While virus-like particles (VLPs) containing subgenomic replicons, which can transduce replicons into target cells efficiently for studying viral replication and vectors of gene therapy and vaccine, have been established for several flaviviruses, none has been reported for the four serotypes of dengue virus, the causal agent of the most important arboviral diseases in this century. In this study, we successfully established a cell line stably expressing the precursor membrane/envelope (PrM/E) proteins of dengue virus type 2 (DENV2), which can package a DENV2 replicon with deletion of PrM/E genes and produce single-round infectious VLPs. Moreover, it can package a similar replicon of different serotype, dengue virus type 4, and produce infectious chimeric VLPs. To our knowledge, this study reports for the first time replicon-containing VLPs of dengue virus. Moreover, this convenient system has potential as a valuable tool to study encapsidation of dengue virus and to develop novel chimeric VLPs containing dengue virus replicon as vaccine in the future.

Characterization of retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope glycoproteins of four serotypes of dengue viruses.

In this study, we successfully established retrovirus-based reporter viruses pseudotyped with the precursor membrane and envelope (PrM/E) proteins of each of the four serotypes of dengue viruses, which caused the most important arboviral diseases in this century. Co-sedimentation of the dengue E protein and HIV-1 core proteins by sucrose gradient analysis of the pseudotype reporter virus of dengue virus type 2, D2(HIVluc), and detection of HIV-1 core proteins by immunoprecipitation with anti-E monoclonal antibody suggested that dengue viral proteins were incorporated into the pseudotype viral particles. The infectivity in target cells, as assessed by the luciferase activity, can be inhibited by the lysosomotropic agents, suggesting a pH-dependent mechanism of entry. Amino acid substitutions of the leucine at position 107, a critical residue at the fusion loop of E protein, with lysine resulted in severe impairment in infectivity, suggesting that entry of the pseudotype reporter virus is mediated through the fusogenic properties of E protein. With more and more dengue viral sequences available from different outbreaks worldwide, this sensitive and convenient tool has the potential to facilitate molecular characterization of the PrM/E proteins of dengue field isolates.

Strategically examining the full-genome of dengue virus type 3 in clinical isolates reveals its mutation spectra.

BACKGROUND: Previous studies presented the quasispecies spectrum of the envelope region of dengue virus type 3 (DENV-3) from either clinical specimens or field-caught mosquitoes. However, the extent of sequence variation among full genomic sequences of DENV within infected individuals remains largely unknown. RESULTS: Instead of arbitrarily choosing one genomic region in this study, the full genomic consensus sequences of six DENV-3 isolates were used to locate four genomic regions that had a higher potential of sequence heterogeneity at capsid-premembrane (C-prM), envelope (E), nonstructural protein 3 (NS3), and NS5. The extent of sequence heterogeneity revealed by clonal sequencing was genomic region-dependent, whereas the NS3 and NS5 had lower sequence heterogeneity than C-prM and E. Interestingly, the Phylogenetic Analysis by Maximum Likelihood program (PAML) analysis supported that the domain III of E region, the most heterogeneous region analyzed, was under the influence of positive selection. CONCLUSION: This study confirmed previous reports that the most heterogeneous region of the dengue viral genome resided at the envelope region, of which the domain III was under positive selection pressure. Further studies will need to address the influence of these mutations on the overall fitness in different hosts (i.e., mosquito and human) during dengue viral transmission.