Increasing transmission of dengue virus across ecologically diverse regions of Ecuador and associated risk factors.

The distribution and intensity of viral diseases transmitted by Aedes aegypti mosquitoes, including dengue, have rapidly increased over the last century. Here, we study dengue virus (DENV) transmission across the ecologically and demographically distinct regions or Ecuador. We analyzed province-level age-stratified dengue incidence data from 2000-2019 using catalytic models to estimate the force of infection of DENV over eight decades. We found that provinces established endemic DENV transmission at different time periods. Coastal provinces with the largest and most connected cities had the earliest and highest increase in DENV transmission, starting around 1980 and continuing to the present. In contrast, remote and rural areas with reduced access, like the northern coast and the Amazon regions, experienced a rise in DENV transmission and endemicity only in the last 10 to 20 years. The newly introduced chikungunya and Zika viruses have age-specific distributions of hospital-seeking cases consistent with recent emergence across all provinces. To evaluate factors associated with geographic differences in DENV transmission potential, we modeled DENV vector risk using 11,693 Aedes aegypti presence points to the resolution of 1 hectare. In total, 56% of the population of Ecuador, including in provinces identified as having increasing DENV transmission in our models, live in areas with high risk of Aedes aegypti, with population size, trash collection, elevation, and access to water as important determinants. Our investigation serves as a case study of the changes driving the expansion of DENV and other arboviruses globally and suggest that control efforts should be expanded to semi-urban and rural areas and to historically isolated regions to counteract increasing dengue outbreaks.

Increasing transmission of dengue virus across ecologically diverse regions of Ecuador and associated risk factors.

The distribution and intensity of viral diseases transmitted by Aedes aegypti mosquitoes, including dengue, have rapidly increased over the last century. Ecuador is an interesting country to study drivers of dengue virus (DENV) transmission given it has multiple ecologically and demographically distinct regions. Here, we analyze province-level age-stratified dengue prevalence data from 2000-2019 using catalytic models to estimate the force of infection of DENV over eight decades and across provinces in Ecuador. We found that provinces established endemic DENV transmission at different time periods. Coastal provinces with the largest and most connected cities had the earliest and highest increase in DENV transmission, starting around 1980 and continuing to the present. In contrast, remote and rural areas with reduced access, like the northern coast and the Amazon regions, experienced a rise in DENV transmission and endemicity only in the last 10 to 20 years. The newly introduced chikungunya and Zika viruses have distinct age-specific prevalence distributions consistent with recent emergence across all provinces. We evaluated factors to the resolution of 1 hectare associated with geographic differences in vector suitability and arbovirus disease in the last 10 years by modeling 11,693 A aegypti presence points and 73,550 arbovirus cases. In total, 56% of the population of Ecuador lives in areas with high risk of Aedes aegypti. Most suitable provinces had hotspots for arbovirus disease risk, with population size, elevation, sewage connection, trash collection, and access to water as important determinants. Our investigation serves as a case study of the changes driving the expansion of DENV and other arboviruses globally and suggest that control efforts should be expanded to semi-urban and rural areas and to historically isolated regions to counteract increasing dengue outbreaks.

A Chikungunya Outbreak in a Dengue-endemic Region in Rural Northern Coastal Ecuador.

Dengue virus (DENV) reemerged in the Americas in the 1980s and 1990s, whereas chikungunya virus (CHIKV) emerged in 2014. Although CHIKV produced large epidemics from 2014 to 2017, dengue fever has been the prominent arboviral disease identified through passive surveillance, bringing to question the degree to which cases are misdiagnosed. To address this concern, we conducted an active household-based surveillance of arboviral-like illnesses in six rural and remote communities in northern coastal Ecuador from May 2019 to February 2020. Although passive surveillance conducted by the Ecuadorian Ministry of Health reported only DENV cases in the region, more than 70% of the arbovirus-like illnesses detected by active surveillance in our study were positive for CHIKV. These findings underline the need for active surveillance of arboviral infections with laboratory confirmation, especially in rural communities where arboviral illnesses are more likely to be underreported.

Characterization of the Type-Specific and Cross-Reactive B-Cell Responses Elicited by a Live-Attenuated Tetravalent Dengue Vaccine.

BACKGROUND: Dengue is caused by 4 antigenically distinct serotypes of dengue virus (DENV1-4). Takeda's live attenuated tetravalent dengue vaccine (TAK-003) candidate is composed of an attenuated DENV2 and chimeric viruses containing prM/E of DENV1, 3 and 4 on the DENV2 backbone. The multicolor FluoroSpot (MCF) assay enables quantitation of serotype-specific and cross-reactive individual memory B cells (MBCs) secreting DENV-specific antibodies in a polyclonal mixture. METHODS: Using the MCF assay, we determined the type-specific and cross-reactive MBC response in peripheral blood mononuclear cells collected pre- and postvaccination from 7 macaques and 15 randomly selected individuals who received TAK-003 (8 DENV seronegative and 7 DENV seropositive) in a phase 2 clinical trial in Singapore (DEN-205 study). RESULTS: Preexisting DENV-specific MBC responses were detected only in seropositive vaccine recipients at day 0. Following vaccination, both type-specific and cross-reactive MBCs to all 4 DENV serotypes were observed in all macaques and clinical trial participants. The proportion of type-specific MBCs was higher than cross-reactive MBCs and remained stable between day 30 and 360 post vaccination. CONCLUSIONS: These results demonstrate that, unlike primary or secondary natural DENV infection, tetravalent vaccination elicits tetravalent type-specific MBCs, and thus all 4 components of TAK-003 contribute to the DENV-specific MBC response following vaccination. CLINICAL TRIALS REGISTRATION: NCT02425098.

Primary and Secondary Dengue Virus Infections Elicit Similar Memory B-Cell Responses, but Breadth to Other Serotypes and Cross-Reactivity to Zika Virus Is Higher in Secondary Dengue.

BACKGROUND: The 4 antigenically distinct serotypes of dengue virus (DENV) share extensive homology with each other and with the closely related Zika flavivirus (ZIKV). The development of polyclonal memory B cells (MBCs) to the 4 DENV serotypes and ZIKV during DENV infection is not fully understood. METHODS: In this study, we analyzed polyclonal MBCs at the single-cell level from peripheral blood mononuclear cells collected ~2 weeks or 6-7 months postprimary or postsecondary DENV infection from a pediatric hospital-based study in Nicaragua using a Multi-Color FluoroSpot assay. RESULTS: Dengue virus elicits robust type-specific and cross-reactive MBC responses after primary and secondary DENV infection, with a significantly higher cross-reactive response in both. Reactivity to the infecting serotype dominated the total MBC response. Although the frequency and proportion of type-specific and cross-reactive MBCs were comparable between primary and secondary DENV infections, within the cross-reactive response, the breadth of MBC responses against different serotypes was greater after secondary DENV infection. Dengue virus infection also induced cross-reactive MBC responses recognizing ZIKV, particularly after secondary DENV infection. CONCLUSIONS: Overall, our study sheds light on the polyclonal MBC response to DENV and ZIKV in naive and DENV-preimmune subjects, with important implications for natural infections and vaccine development.

Impact of pre-existing dengue immunity on human antibody and memory B cell responses to Zika.

Little is known about enduring memory B cell (MBC) responses to Zika virus (ZIKV) and their relationship with circulating antibodies. Here we comprehensively assess MBC frequency and specificity alongside serum binding and neutralizing antibody responses to ZIKV ~2 weeks and ~8 months postinfection in 31 pediatric subjects with 0, 1 or >1 prior infections with the related dengue virus (DENV). ZIKV infection elicits a robust type-specific MBC response, and the majority of late convalescent anti-ZIKV serum neutralizing activity is attributable to ZIKV-specific antibodies. The number of prior DENV infections does not influence type-specific or cross-reactive MBC responses, although ZIKV has the highest cross-reactivity with DENV3. DENV cross-reactive MBCs expanded by ZIKV infection decline in number and proportion by late convalescence. Finally, ZIKV induces greater cross-reactivity in the MBC pool than in serum antibodies. Our data suggest immunity to DENV only modestly shapes breadth and magnitude of enduring ZIKV antibody responses.

The complete mitogenome of the invasive Japanese mud snail Batillaria attramentaria (Gastropoda: Batillariidae) from Elkhorn Slough, California, USA.

Genomic analysis of the invasive marine snail Batillaria attramentaria from Elkhorn Slough, Moss Landing, California, USA using 150 bp paired-end Illumina sequences resulted in the assembly of its complete mitogenome. The mitogenome is 16,095 bp in length and contains 2 rRNA, 13 protein-coding, and 22 tRNA genes (GenBank Accession MN557850). Gene content and organization of B. attramentaria are identical to the Turritellidae and Pachychilidae. The phylogenetic analysis of B. attramentaria resolves it in a fully supported clade with these same two families in the superfamily Cerithioidea. Nucleotide BLAST searches of the Elkhorn Slough cox1 gene of B. attramentaria yielded identical sequences from invasive populations from California and British Columbia, and native populations from northeastern and central Japan. These data show that mitogenome sequencing is a useful tool for studying the classification and phylogenetic history Cerithioidea.

ELISPOT-Based "Multi-Color FluoroSpot" to Study Type-Specific and Cross-Reactive Responses in Memory B Cells after Dengue and Zika Virus Infections.

Co-circulation and re-emergence of antigenically related viruses such as dengue (DENV), Zika (ZIKV), and yellow fever (YF) in the Americas has brought a sense of urgency in the field to further define the genesis and to more fully describe the immune response. The recent explosive epidemics of Zika in the Americas and the co-circulation of ZIKV with the phylogenetically similar DENV has raised important questions and concerns regarding the role of cross-reactive immunity in protection and potential enhancement of severity of subsequent ZIKV or DENV infections in pre-immune individuals and the safety of vaccines against both viruses in endemic populations. Antibodies are a critical part of the immune response for clearing flavivirus infections, but the role of pre-existing antibodies in protection or enhancement of subsequent infection and disease with closely related viral species and strains is still not fully understood. We have developed a novel Multi-Color FluoroSpot (MCF) assay based on our ELISPOT-derived assay, previously designated the Quad-color FluoroSpot (QCF), in order to study the development of type-specific versus cross-reactive responses within the B cell pool of Zika virus (ZIKV)- and/or dengue virus (DENV)-infected patients. The QCF is based on a panel of four fluorescent Qdots, each conjugated to a monoclonal antibody specific to one of the four DENV serotypes; now we have included a fifth color (Qdot) for ZIKV to enable analysis of the specificity versus cross-reactivity of B cell populations at a single-cell level for all four DENV serotypes and ZIKV. This novel assay allows us to analyze unique human samples from long-term studies of dengue and Zika in Nicaragua to investigate the nature of B cell/antibody responses and their role in pathogenesis and/or protection in secondary flavivirus infections and could have important implications for vaccine development for Zika and dengue.

CD14+CD16+ monocytes are the main target of Zika virus infection in peripheral blood mononuclear cells in a paediatric study in Nicaragua.

The recent Zika pandemic in the Americas is linked to congenital birth defects and Guillain-Barré syndrome. White blood cells (WBCs) play an important role in host immune responses early in arboviral infection. Infected WBCs can also function as 'Trojan horses' and carry viruses into immune-sheltered spaces, including the placenta, testes and brain. Therefore, defining which WBCs are permissive to Zika virus (ZIKV) is critical. Here, we analyse ZIKV infectivity of peripheral blood mononuclear cells (PBMCs) in vitro and from Nicaraguan Zika patients and show CD14+CD16+ monocytes are the main target of infection, with ZIKV replication detected in some dendritic cells. The frequency of CD14+ monocytes was significantly decreased, while the CD14+CD16+ monocyte population was significantly expanded during ZIKV infection compared to uninfected controls. Viral RNA was detected in PBMCs from all patients, but in serum from only a subset, suggesting PBMCs may be a reservoir for ZIKV. In Zika patients, the frequency of infected cells was lower but the percentage of infected CD14+CD16+ monocytes was significantly higher compared to dengue cases. The gene expression profile in monocytes isolated from ZIKV- and dengue virus-infected patients was comparable, except for significant differences in interferon-γ, CXCL12, XCL1, interleukin-6 and interleukin-10 levels. Thus, our study provides a detailed picture of the innate immune profile of ZIKV infection and highlights the important role of monocytes, and CD14+CD16+ monocytes in particular.

Fascitis necrotizante como complicación de apendicitis aguda: a propósito de un caso / Necrotizing fasciitis as a complication of acute appendicitis: a case report

La Fascitis necrotizante (FN) es una infección aguda del tejido subcutáneo que puede comprometer la fascia y producir compromiso sistémico y sepsis. Es una complicación extremadamente rara posterior a una apendicitis aguda. Objetivo: Demostrar la importancia de realizar el diagnóstico a tiempo y la intervención de forma precoz. Caso clínico: Presentamos el caso de un paciente que evoluciona con una Fascitis necrotizante posterior a una apendicectomía. Se realiza una intervención quirúrgica y terapia antimicrobiana precoz, con una favorable evolución. Conclusiones: La FN es una patología con escasa sintomatología y signología clínica, por lo que se debe tener un alto grado de sospecha. Un diagnóstico e intervención a tiempo logra mejorar la sobrevida de los pacientes.

Necrotizing fasciitis is characterized by a necrotic infection that rapidly spreads along the fascia and progresses to systemic sepsis. A fatal complication of acute appendicitis is extremely rare. Objective: Ilustrates the importance of early diagnosis and prompt surgical intervention. Case Report: We present a case of necrotizing fasciitis after an appendicectomy. An early intervention and antibiotic therapy allowed a favorable evolution. Conclusions: The necrotizing fasciitis has a nonspecific symptomatology and signology, so we must suspect the patology. An early diagnosis and aggressive surgical intervention can be life- saving.

Functional processing and secretion of Chikungunya virus E1 and E2 glycoproteins in insect cells.

BACKGROUND: Chikungunya virus (CHIKV) is a mosquito-borne, arthrogenic Alphavirus that causes large epidemics in Africa, South-East Asia and India. Recently, CHIKV has been transmitted to humans in Southern Europe by invading and now established Asian tiger mosquitoes. To study the processing of envelope proteins E1 and E2 and to develop a CHIKV subunit vaccine, C-terminally his-tagged E1 and E2 envelope glycoproteins were produced at high levels in insect cells with baculovirus vectors using their native signal peptides located in CHIKV 6K and E3, respectively. RESULTS: Expression in the presence of either tunicamycin or furin inhibitor showed that a substantial portion of recombinant intracellular E1 and precursor E3E2 was glycosylated, but that a smaller fraction of E3E2 was processed by furin into mature E3 and E2. Deletion of the C-terminal transmembrane domains of E1 and E2 enabled secretion of furin-cleaved, fully processed E1 and E2 subunits, which could then be efficiently purified from cell culture fluid via metal affinity chromatography. Confocal laser scanning microscopy on living baculovirus-infected Sf21 cells revealed that full-length E1 and E2 translocated to the plasma membrane, suggesting similar posttranslational processing of E1 and E2, as in a natural CHIKV infection. Baculovirus-directed expression of E1 displayed fusogenic activity as concluded from syncytia formation. CHIKV-E2 was able to induce neutralizing antibodies in rabbits. CONCLUSIONS: Chikungunya virus glycoproteins could be functionally expressed at high levels in insect cells and are properly glycosylated and cleaved by furin. The ability of purified, secreted CHIKV-E2 to induce neutralizing antibodies in rabbits underscores the potential use of E2 in a subunit vaccine to prevent CHIKV infections.