The hallmarks of cell-cell fusion.

Cell-cell fusion is essential for fertilization and organ development. Dedicated proteins known as fusogens are responsible for mediating membrane fusion. However, until recently, these proteins either remained unidentified or were poorly understood at the mechanistic level. Here, we review how fusogens surmount multiple energy barriers to mediate cell-cell fusion. We describe how early preparatory steps bring membranes to a distance of ∼10 nm, while fusogens act in the final approach between membranes. The mechanical force exerted by cell fusogens and the accompanying lipidic rearrangements constitute the hallmarks of cell-cell fusion. Finally, we discuss the relationship between viral and eukaryotic fusogens, highlight a classification scheme regrouping a superfamily of fusogens called Fusexins, and propose new questions and avenues of enquiry.

Multiplexed kit based on Luminex technology and achievements in synthetic biology discriminates Zika, chikungunya, and dengue viruses in mosquitoes.

BACKGROUND: The global expansion of dengue (DENV), chikungunya (CHIKV), and Zika viruses (ZIKV) is having a serious impact on public health. Because these arboviruses are transmitted by the same mosquito species and co-circulate in the same area, a sensitive diagnostic assay that detects them together, with discrimination, is needed. METHODS: We present here a diagnostics panel based on reverse transcription-PCR amplification of viral RNA and an xMap Luminex architecture involving direct hybridization of PCRamplicons and virus-specific probes. Two DNA innovations ("artificially expanded genetic information systems", AEGIS, and "self-avoiding molecular recognition systems", SAMRS) increase the hybridization sensitivity on Luminex microspheres and PCR specificity of the multiplex assay compared to the standard approach (standard nucleotides). RESULTS: The diagnostics panel detects, if they are present, these viruses with a resolution of 20 genome equivalents (DENV1), or 10 (DENV3-4, CHIKV) and 80 (DENV2, ZIKV) genome equivalents per assay. It identifies ZIKV, CHIKV and DENV RNAs in a single infected mosquito, in mosquito pools comprised of 5 to 50 individuals, and mosquito saliva (ZIKV, CHIKV, and DENV2). Infected mosquitoes and saliva were also collected on a cationic surface (Q-paper), which binds mosquito and viral nucleic acids electrostatically. All samples from infected mosquitoes displayed only target-specific signals; signals from non-infected samples were at background levels. CONCLUSIONS: Our results provide an efficient and multiplex tool that may be used for surveillance of emerging mosquito-borne pathogens which aids targeted mosquito control in areas at high risk for transmission.

Seroprevalence of Dengue and Chikungunya Virus Antibodies, French Polynesia, 2014-2015.

We investigated dengue and chikungunya virus antibody seroprevalence in French Polynesia during 2014-2015. Dengue virus seroprevalence was ≈60% among schoolchildren and >83% among the general population; chikungunya virus seroprevalence was <3% before and 76% after Zika virus emergence (2013). Dengue virus herd immunity may affect Zika virus infection and pathogenesis.

Unrecognized Dengue Virus Infections in Children, Western Kenya, 2014-2015.

We detected a cluster of dengue virus infections in children in Kenya during July 2014-June 2015. Most cases were serotype 1, but we detected all 4 serotypes, including co-infections with 2 serotypes. Our findings implicate dengue as a cause of febrile illness in this population and highlight a need for robust arbovirus surveillance.

Growth characteristics and cytokine/chemokine induction profiles of dengue viruses in various cell lines.

The dengue disease varies clinically from mild fever to hemorrhagic fever up to potentially fatal shock syndrome. Such differences in manifestation of dengue virus (DENV) infection have been ascribed to intrinsic differences in DENVs. Four DENV serotypes or four dengue viruses (DENV-1 to DENV-4) have been clearly distinguished; however, only limited information is available on their biological characteristics in vitro. To shed more light on this subject, replication kinetics of all four DENVs in various cell lines and induction of 26 cytokines/chemokines were investigated. The results showed that, (i) all DENVs replicated relatively similarly in various cell lines, (ii) DENV-1 was most effective in this regard, (iii) A549 cells showed the highest virus replication rate compared to other cells, and (iv) all DENVs induced in A549 cells similar levels of several cytokines/chemokines, namely eotaxin, granulocyte colony stimulating factor (G-CSF), interferon alpha 2 (IFN-α2), interleukins 6, 8 15 (IL-6, IL-8, IL-15), and IP-10. In conclusion, this study revealed a similarity in growth characteristics and cytokine/chemokine induction profile for all four DENV serotypes in vitro.

Multi-epitope peptide vaccines targeting dengue virus serotype 2 created via immunoinformatic analysis.

The Middle East has witnessed a greater spread of infectious Dengue viruses, with serotype 2 (DENV-2) being the most prevalent form. Through this work, multi-epitope peptide vaccines against DENV-2 that target E and nonstructural (NS1) proteins were generated through an immunoinformatic approach. MHC class I and II and LBL epitopes among NS1 and envelope E proteins sequences were predicted and their antigenicity, toxicity, and allergenicity were investigated. Studies of the population coverage denoted the high prevalence of NS1 and envelope-E epitopes among different countries where DENV-2 endemic. Further, both the CTL and HTL epitopes retrieved from NS1 epitopes exhibited high conservancies' percentages with other DENV serotypes (1, 3, and 4). Three vaccine constructs were created and the expected immune responses for the constructs were estimated using C-IMMSIM and HADDOCK (against TLR 2,3,4,5, and 7). Molecular dynamics simulation for vaccine construct 2 with TLR4 denoted high binding affinity and stability of the construct with the receptor which might foretell favorable in vivo interaction and immune responses.

Dengue Fever Accompanied by Neurological Manifestations: Challenges and Treatment.

Dengue, commonly referred to as 'breakbone fever,' is a mosquito-borne arboviral infection transmitted by Aedes aegypti, featuring an average incubation period of approximately seven days. Key cytokines such as interferon-gamma (IFN-γ), tumor necrosis factor (TNF)-α, and interleukin (IL)-10 are pivotal in the pathogenesis of dengue. Travelers are particularly susceptible to contracting dengue fever, with disease severity often associated with CD8+ T cell response. Without proper hospitalization during severe cases like dengue hemorrhagic fever (DHF)/dengue shock syndrome (DSS), mortality rates can escalate to 50%. Dengue fever can lead to various complications, including neurological manifestations such as encephalopathy, encephalitis, cerebral venous thrombosis, myelitis, posterior reversible encephalopathy syndrome, strokes (both ischemic and hemorrhagic), immune-mediated neurological syndromes (such as mononeuropathy, acute transverse myelitis, Guillain-Barre syndrome, and acute disseminated encephalomyelitis), and neuromuscular complications. Treatment protocols typically involve assessing disease activity using composite indices, pursuing treatment objectives, and administering intravenous fluids according to symptomatology. Given the absence of specific antiviral treatment for dengue, supportive care, particularly hydration, remains paramount during the early stages. It is crucial to recognize that dengue viruses may contribute to the development of neurological disorders, particularly in regions where dengue is endemic. Furthermore, there is a necessity for well-defined criteria for specific neurological complications. Primary prevention strategies primarily revolve around vector control measures, which play a critical role in curtailing the spread of dengue.

Genetic Adaptation by Dengue Virus Serotype 2 to Enhance Infection of Aedes aegypti Mosquito Midguts.

Dengue viruses (DENVs), serotypes 1-4, are arthropod-borne viruses transmitted to humans by mosquitoes, primarily Aedes aegypti. The transmission cycle begins when Ae. aegypti ingest blood from a viremic human and the virus infects midgut epithelial cells. In studying viruses derived from the DENV2 infectious clone 30P-NBX, we found that when the virus was delivered to female Ae. aegypti in an infectious blood meal, the midgut infection rate (MIR) was very low. To determine if adaptive mutations in the DENV2 envelope (E) glycoprotein could be induced to increase the MIR, we serially passed 30P-NBX in Ae. aegypti midguts. After four passages, a single, non-conservative mutation in E protein domain II (DII) nucleotide position 1300 became dominant, resulting in replacement of positively-charged amino acid lysine (K) at position 122 with negatively-charged glutamic acid (E; K122E) and a significantly-enhanced MIR. Site directed mutagenesis experiments showed that reducing the positive charge of this surface-exposed region of the E protein DII correlated with improved Ae. aegypti midgut infection.

Relationship between clinical and laboratory features with infecting dengue virus serotypes in a sample of dengue suspected adult patients from 2015-2017 in Sri Lanka.

Dengue is a major viral disease affecting the tropics. Although previous research has focused on the relationship between the infecting dengue virus (DENV) serotypes and disease severity, less work has been done on the relationship between the clinical and laboratory features and the infecting DENV serotypes in Sri Lanka. We evaluated the relationship between the clinical and laboratory features and the infecting DENV serotypes in adult patients with clinically suspected dengue admitted to the Base Hospital, Mawanella, Sri Lanka from December 2015 to March 2017. Blood samples of 200 dengue suspected patients were tested for the infecting DENV serotypes using a reverse transcription polymerase chain reaction with primers targeting the envelope region of the virus. Relationship between the infecting DENV serotypes with clinical and laboratory features was assessed using Z score and paired t tests. Of the 200 patients tested, 39 (19.5%) were positive for DENV, any of the four DENV serotypes alone or in combination. The highest number of infections was noted with DENV-2 (n=18, 46.1%). Fever (P=0.000) and rash (P=0.017) were frequently noted in DENV negative patients while bleeding (P=0.012) was more frequently noted in DENV serotype positive patients. Platelet count of <100,000 µl-1 was significantly associated with DENV serotype positivity (P=0.000). Platelet count of <100,000 µl-1 (P=0.035) and haemoglobin (Hb) of >13mgdl-1 (P=0.016) were noted in 15 of the 18 DENV-2 positive patients. Clinical and laboratory features of severe dengue with bleeding manifestations, low platelet counts and high Hb were noted in DENV-2 infections.

Circulating dengue virus serotypes and vertical transmission in Aedes larvae during outbreak and inter-outbreak seasons in a high dengue risk area of Sri Lanka.

BACKGROUND: Spatial and temporal changes in the dengue incidence are associated with multiple factors, such as climate, immunity among a population against dengue viruses (DENV), circulating DENV serotypes and vertical transmission (VT) of DENV in an area at a given time. The level of VT in a specific location has epidemiological implications in terms of viral maintenance in vectors. Identification of the circulating DENV serotypes in both patients and Aedes mosquito larvae in an area may be useful for the early detection of outbreaks. We report here the results of a prospective descriptive study that was conducted to detect the levels of VT in Aedes mosquito larvae and circulating DENV serotypes in patients and Aedes mosquito larvae from December 2015 to March 2017 in an area of Sri Lanka at high risk for dengue. METHODS: A total of 200 patients with clinically suspected dengue who had been admitted to a tertiary care hospital during a dengue outbreak (3 study periods: December 2015-January 2016, June-August 2016, December 2016-January 2017) and in the inter-outbreak periods (February-May 2016 and September-November 2016) were investigated. Blood samples were drawn from the study participants to test for DENV. The houses of the study participants were visited within 7 days of admission to the hospital, and Aedes larvae were also collected within a radius of 400 m from the houses. The larvae were separately identified to species and then pooled according to each patient's identification number. Patients' sera and the Aedes larvae were tested to identify the infecting DENV serotypes using a reverse transcription PCR (RT-PCR) method. Levels of VT in Aedes mosquito larvae were also identified. RESULTS: All four DENV serotypes (DENV-1 to -4) were identified in the study area. In the early part of the study (December 2015-February 2016), DENV-3 was predominant and from April 2016 to March 2017, DENV-2 became the most predominant type. Four cases of DENV co-infections were noted during the study period in patients. Interestingly, all four DENV serotypes were detected in Aedes albopictus larvae, which was the prominent immature vectorial form identified throughout the study period in the area, showing 9.8% VT of DENV. With the exception of DENV-4, the other three DENV serotypes were identified in Aedes aegypti larvae with a VT of 8.1%. CONCLUSION: Comparatively high rates of VT of DENV was detected in Ae. albopictus and Ae. aegypti larvae. A shift in the predominant DENV serotype with simultaneous circulation of all four DENV serotypes was identified in the study area from December 2015 to March 2017.

Long-term protection against dengue viruses in mice conferred by a tetravalent DNA vaccine candidate.

The development of an effective tetravalent vaccine against dengue viruses (DENVs) has become a world priority. We previously showed that four monovalent dengue DNA vaccines expressing premembrane (prM) and envelope (E) proteins displayed effective protection against corresponding challenges in mice. Thus, to elucidate the overall immunity and persistence of the tetravalent formulation (TetraME), we evaluated the humoral and cellular immune responses as well as the long-term protection in the current study. TetraME-immunized mice displayed increased production of Th1/Th2-typed cytokines upon stimulation with heterologous DENV antigens. Moreover, high levels of tetravalent DENV antibodies and sterilized immunity were detected long-term (30 weeks after immunization). These findings provide feasible validation for the potential utility of this vaccine formulation.

Evaluation of the LIAISON XL Zika Capture IgM II for the Diagnosis of Zika Virus Infections.

The aim of this study is to evaluate the performance characteristics of the LIAISON XL Zika Capture IgM II. For this purpose we tested 128 samples obtained from recent infections caused by the Zika (ZIKV; 74 samples), dengue (DENV; 10 samples), chikungunya (CHIK V; 11 samples), rubella (RUBV; 10 samples) and measles (MeV; 10 samples) viruses, as well as human parvovirus B19 (HPVB19; 13 samples). The results of the assay under evaluation are compared with those obtained from an indirect immunofluorescence (IIF) assay, and the discrepancies are resolved by considering other laboratory results (PCR and a plaque-reduction neutralization test). The LIAISON showed excellent sensitivity (100%). The specificity (91.25%) was hampered by some false-positive results in recent dengue virus, chikungunya virus, measles virus and human parvovirus B19 infections. The method evaluated is adequate, but the low specificity makes it necessary to consider the clinical and epidemiological contexts of patients, as well as other laboratory results.

Effect of Serotype and Strain Diversity on Dengue Virus Replication in Australian Mosquito Vectors.

Dengue virus (DENV) is the most important mosquito-borne viral pathogen of humans, comprising four serotypes (DENV-1 to -4) with a myriad of genotypes and strains. The kinetics of DENV replication within the mosquito following ingestion of a blood meal influence the pathogen's ability to reach the salivary glands and thus the transmission potential. The influence of DENV serotype and strain diversity on virus kinetics in the two main vector species, Aedes aegypti and Ae. albopictus, has been poorly characterized. We tested whether DENV replication kinetics vary systematically among serotypes and strains, using Australian strains of the two vectors. Mosquitoes were blood fed with two strains per serotype, and sampled at 3, 6, 10 and 14-days post-exposure. Virus infection in mosquito bodies, and dissemination of virus to legs and wings, was detected using qRT-PCR. For both vectors, we found significant differences among serotypes in proportions of mosquitoes infected, with higher numbers for DENV-1 and -2 versus other serotypes. Consistent with this, we observed that DENV-1 and -2 generally replicated to higher RNA levels than other serotypes, particularly at earlier time points. There were no significant differences in either speed of infection or dissemination between the mosquito species. Our results suggest that DENV diversity may have important epidemiological consequences by influencing virus kinetics in mosquito vectors.

Mosquito cells persistently infected with dengue virus produce viral particles with host-dependent replication.

Dengue viruses (DENV) are important arboviruses that can establish a persistent infection in its mosquito vector Aedes. Mosquitoes have a short lifetime in nature which makes trying to study the processes that take place during persistent viral infections in vivo. Therefore, C6/36 cells have been used to study this type of infection. C6/36 cells persistently infected with DENV 2 produce virions that cannot infect BHK -21 cells. We hypothesized that the following passages in mosquito cells have a deleterious impact on DENV fitness in vertebrate cells. Here, we demonstrated that the viral particles released from persistently infected cells were infectious to mosquito but not to vertebrate cells. This host restriction occurs at the replication level and is associated with several mutations in the DENV genome. In summary, our findings provide new information about viral replication fitness in a host-dependent manner.

Dengue Mosaic Vaccines Enhance Cellular Immunity and Expand the Breadth of Neutralizing Antibody Against All Four Serotypes of Dengue Viruses in Mice.

An estimated 400 million people in the world are infected with any of the four types of dengue virus (DENV) annually. The only licensed dengue vaccine cannot effectively prevent infection with all of the four DENVs, especially in those immunologically naïve at baseline. In this study, we explored a mosaic vaccine approach, which utilizes an artificial recombinant sequence for each serotype to achieve maximum coverage of variant epitopes in the four DENVs. We determined the immunogenicity and cross-reactivity of DNA plasmids encoding individual mosaic sequences or the natural sequences in mice. We show that the mosaic vaccines, particularly those targeting DENV serotype 1 and 2, improved vaccine immunogenicity by increasing the percentage of antigen-specific IFNγ- or TNFα-secreting CD4 and CD8 T cells, and titers of neutralizing antibodies. The mosaic vaccine diversified and broadened anti-dengue T cell responses and cross-reactive neutralizing antibodies against all four serotypes. The mosaic vaccines also induced higher level of antigen-specific B cell responses. These results suggest that mosaic vaccines comprising of DENV serotype 1 and 2 variant epitopes could stimulate strong and broad immune responses against all four serotypes.

Lack of Evidence of Sylvatic Transmission of Dengue Viruses in the Amazon Rainforest Near Iquitos, Peru.

Dengue viruses (DENV) are currently responsible for more human morbidity and mortality than any other known arbovirus, and all four DENV are known to exist in sylvatic cycles that might allow these viruses to persist if the urban (Aedes aegypti) cycle could be controlled. To determine whether DENV were being maintained in a sylvatic cycle in a forested area about 14 km southwest of Iquitos, Peru, a city in which all 4 serotypes of DENV circulate, we placed 20 DENV seronegative Aotus monkeys in cages either in the canopy or near ground level for a total of 125.6 months. Despite capturing >66,000 mosquitoes in traps that collected some of the mosquitoes attracted to these monkeys, blood samples obtained once a month from each animal were tested and found to be negative by an enzyme-linked immunosorbent assay for IgM and IgG antibodies to dengue, yellow fever, Venezuelan equine encephalitis, Oropouche, and Mayaro viruses. Although all four DENV serotypes were endemic in nearby Iquitos, the findings of this study did not support a DENV sylvatic maintenance and transmission cycle in a selected area of the Amazon rainforest in northeastern Peru.

Can Complementary Prime-Boost Immunization Strategies Be an Alternative and Promising Vaccine Approach Against Dengue Virus?

Dengue is one of the most important diseases transmitted by mosquitoes. Dengvaxia®, a vaccine registered in several countries, cannot be administered to non-immune individuals and children younger than 9 years old, due to safety reasons. There are two vaccine candidates in phase 3 efficacy trials, but their registration date is completely unknown at this moment. So, the development of new vaccines or vaccine strategies continues to be a priority for the WHO. This work reviews some complementary prime-boost immunization studies against important human pathogens. Additionally, it reviews the results obtained using this regimen of immunization against dengue virus as a potential alternative approach for finding a safe and efficient vaccine. Finally, the main elements associated with this strategy are also discussed. The generation of new strategies of vaccination against dengue virus, must be directed to reduce the risk of increasing viral load through sub-neutralizing antibodies and it must be also directed to induce a polyfunctional T cell response. Complementary prime-boost immunization strategies could emerge as an interesting approach to induce solid immunity or at least to reduce viral load after natural infection, avoiding severe dengue. Subunit vaccine could be safe and attractive antigens for this strategy, especially proteins including B, and T-cells epitopes for inducing humoral and cellular immune responses, which can play an important role controlling the disease.

From Incriminating Stegomyia fasciata to Releasing Wolbachia pipientis: Australian Research on the Dengue Virus Vector, Aedes aegypti, and Development of Novel Strategies for Its Surveillance and Control.

Globally, the dengue viruses (DENVs) infect approximately 300 million people annually. Australia has a history of epidemic dengue, with outbreaks in the early decades of the twentieth century responsible for tens of thousands of cases. Seminal experiments conducted by Australian scientists during these outbreaks were the first to incriminate Aedes aegypti as a major vector of dengue viruses. One hundred years later, Australian scientists are playing a lead role in the development of surveillance and suppression strategies that target this mosquito species. Surveillance of Ae. aegypti populations and their associated dengue risk was greatly improved by understanding the contribution of key premises, key containers, and cryptic larval habitats to mosquito productivity, and, more recently, the development of novel adult traps. In terms of mosquito control, targeted indoor residual pyrethroid spraying and community-based biological control utilizing predatory copepods can significantly reduce Ae. aegypti populations. The release of Ae. aegypti transinfected with the virus-blocking bacterium, Wolbachia, provides a promising strategy for limiting DENV transmission. These diverse strategies developed by Australian scientists have the potential to alleviate the burden of dengue in the future, whether it is at the local level or as part of a country-wide program.

Comparative Evaluation of Indirect Immunofluorescence and NS-1-Based ELISA to Determine Zika Virus-Specific IgM.

Differential diagnosis of the Zika virus (ZIKV) is hampered by cross-reactivity with other flaviviruses, mainly dengue viruses. The aim of this study was to compare two commercial methods for detecting ZIKV immunoglobulin M (IgM), an indirect immunofluorescence (IIF) and an enzyme immunoassay (ELISA), using the non-structural (NS) 1 protein as an antigen, both from EuroImmun, Germany. In total, 255 serum samples were analyzed, 203 of which showed laboratory markers of ZIKV infections (PCR-positive in serum and/or in urine and/or positive or indeterminate specific IgM). When tested with IIF, 163 samples were IgM-positive, while 13 samples were indeterminate and 78 were negative. When IIF-positive samples were tested using ELISA, we found 61 positive results, 14 indeterminate results, and 88 negative results. Among the indeterminate cases tested with IIF, ELISA analysis found two positive, two indeterminate, and nine negative results. Finally, 74 of the 78 IIF-negative samples proved also to be negative using ELISA. For the calculations, all indeterminate results were considered to be positive. The agreement, sensitivity, and specificity between ELISA and IIF were 60.2%, 44.9%, and 94.9%, respectively. Overall, 101 samples showed discrepant results; these samples were finally classified on the basis of other ZIKV diagnostic approaches (PCR-positive in serum and/or in urine, IgG determinations using IIF or ELISA, and ZIKV Plaque Reduction Neutralization test-positive), when available. A final classification of 228 samples was possible; 126 of them were positive and 102 were negative. The corresponding values of agreement, sensitivity, and specificity of IIF were 86.0%, 96.8%, and 72.5%, respectively. The corresponding figures for ELISA were 81.1%, 65.9%, and 100%, respectively. The ELISA and IIF methods are both adequate approaches for detecting ZIKV-specific IgM. However, considering their respective weaknesses (low sensitivity in ELISA and low specificity in IIF), serological results must be considered jointly with other laboratory results.

Flaviviruses as a Cause of Undifferentiated Fever in Sindh Province, Pakistan: A Preliminary Report.

Arboviral diseases are expanding worldwide, yet global surveillance is often limited due to diplomatic and cultural barriers between nations. With human encroachment into new habitats, mosquito-borne viruses are also invading new areas. The actual prevalence of expanding arboviruses is unknown in Pakistan due to inappropriate diagnosis and poor testing for arboviral diseases. The primary objective of this study was to document evidence of flavivirus infections as the cause of undifferentiated fever in Pakistan. Through a cooperative effort between the USA and Pakistan, patient exposure to dengue virus (DENV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) was examined in Sindh Province for the first time in decades. Initial results from the 2015 arbovirus season consisting of a cross-sectional study of 467 patients in 5 sites, DENV NS1 antigen was identified in 63 of the screened subjects, WNV IgM antibodies in 16 patients, and JEV IgM antibodies in 32 patients. In addition, a number of practical findings were made including (1) in silico optimization of RT-PCR primers for flavivirus strains circulating in the Middle East, (2) shipping and storage of RT-PCR master mix and other reagents at ambient temperature, (3) Smart phone applications for the collection of data in areas with limited infrastructure, and (4) fast and reliable shipping for transport of reagents and specimens to and from the Middle East. Furthermore, this work is producing a group of highly trained local scientists and medical professionals disseminating modern scientific methods and more accurate diagnostic procedures to the community.