Presence, Location and Conservation of Putative G-Quadruplex Forming Sequences in Arboviruses Infecting Humans.

Guanine quadruplexes (G4s) are non-canonical nucleic acid structures formed by guanine (G)-rich tracts that assemble into a core of stacked planar tetrads. G4s are found in the human genome and in the genomes of human pathogens, where they are involved in the regulation of gene expression and genome replication. G4s have been proposed as novel pharmacological targets in humans and their exploitation for antiviral therapy is an emerging research topic. Here, we report on the presence, conservation and localization of putative G4-forming sequences (PQSs) in human arboviruses. The prediction of PQSs was performed on more than twelve thousand viral genomes, belonging to forty different arboviruses that infect humans, and revealed that the abundance of PQSs in arboviruses is not related to the genomic GC content, but depends on the type of nucleic acid that constitutes the viral genome. Positive-strand ssRNA arboviruses, especially Flaviviruses, are significantly enriched in highly conserved PQSs, located in coding sequences (CDSs) or untranslated regions (UTRs). In contrast, negative-strand ssRNA and dsRNA arboviruses contain few conserved PQSs. Our analyses also revealed the presence of bulged PQSs, accounting for 17-26% of the total predicted PQSs. The data presented highlight the presence of highly conserved PQS in human arboviruses and present non-canonical nucleic acid-structures as promising therapeutic targets in arbovirus infections.

Shuni Virus in Cases of Neurologic Disease in Humans, South Africa.

We describe Shuni virus (SHUV) detection in human neurologic disease cases in South Africa. SHUV RNA was identified in 5% of cerebrospinal fluid specimens collected during the arbovirus season from public sector hospitals. This finding suggests that SHUV may be a previously unrecognized cause of human neurologic infections in Africa.

Socioeconomic and demographic predictors of resident knowledge, attitude, and practice regarding arthropod-borne viruses in Panama.

BACKGROUND: We sought to identify if socioeconomic and demographic factors play a role in resident knowledge, attitude, and practice regarding Dengue, Chikungunya, and Zika in order to inform effective management procedures for disease prevention in Panama, a middle-income tropical country in Central America. All three are arthropod-borne viruses transmitted by Aedes mosquito vectors present in the focal region of Panama City, the largest city in Central America and an urban region of extreme socioeconomic polarization. METHODS: Between November 2017 and February 2018, we administered standardized, anonymous knowledge, attitude, and practice surveys to 263 residents split between two neighborhoods of high socioeconomic status (SES) and two neighborhoods of low SES. We then summed the knowledge, attitude, and practice scores respectively, and used linear and logistic regressions to quantify relationships with socioeconomic and demographic factors. RESULTS: Low-SES neighborhoods with high proportions of low income residents, residents over 70 years old had lower mean knowledge scores compared to other groups. Furthermore, residents in neighborhoods of low SES reported more mosquito biting relative to residents in neighborhoods of high SES, yet comparably lower level of concerns for disease transmission. Additionally, knowledge was lower for the more novel emergent threats of Chikungunya and Zika, compared to the endemic Dengue. CONCLUSION: Findings suggest that low-SES neighborhoods with high proportions of low income, low education, and elderly residents should be targeted for outreach programs designed to prevent DENV, CHIKV, or ZIKV in Panama City. These outcomes support our initial hypotheses as lower relative knowledge and fewer practices related to the prevention of Dengue, Chikungunya, and Zika were found in low-SES neighborhoods. There is also a widespread lack of adequate knowledge regarding these diseases as well as low levels of concern in areas of highly reported mosquito biting. We provide suggestions for taking neighborhood socioeconomic status and specific aspects resident health literacy and attitude into account for creating more effective outreach campaigns as both endemic and novel arthropod-borne disease rates continue to increase throughout Latin America.

Arboviruses: A Family on the Move.

Arboviruses are a diverse group of vector-borne viruses, many of whose members are the cause of significant human morbidity and mortality. Over the last 30 years, the emergence and/or resurgence of arboviruses have posed a considerable global health threat. The ongoing geographical expansion of the dengue viruses (DENV), along with the explosive outbreaks of West Nile virus (WNV), Chikungunya virus (CHIKV) and more recently, Zika virus (ZIKV) have all served as reminders that new epidemics may emerge at any time from this diversity. A clearer understanding of what mechanisms drive these dramatic changes in vector-host transmission cycles that result in the human population becoming significantly more exposed, will help to prepare us for the next emerging epidemic/pandemic. This Chapter seeks to provide a brief overview of the arboviruses, their mode of transmission and some of the known factors that drive their expansion.

Following the Yellow Brick Road.

Charles Calisher was fascinated by microorganisms from the time he was in high school. He attended Stuyvesant High School in New York City, Philadelphia College of Pharmacy and Science (now University of the Sciences) (BS), then University of Notre Dame in South Bend, Indiana (MS), and finally Georgetown University, in Washington, DC (PhD), the latter while employed at a commercial biological house. He was hired by the US Communicable Disease Center (now the Centers for Disease Control and Prevention) in Atlanta, Georgia, was transferred to its Fort Collins laboratories in 1973, and retired from there in 1992. After traveling the world a bit, Calisher joined the faculty of Colorado State University in 1993, then semiretired as professor emeritus in 2010. During all those years, he developed from a would-be virologist to an arbovirologist-epidemiologist, identifying scores of newly recognized viruses from throughout the world and helping to investigate disease outbreaks and epidemics. His interests (always primarily arboviruses but now also rodent-borne viruses and bat-borne viruses) continue to expand, and he continues to be involved in various aspects of virology and to assist and annoy journal editors and others in regard to viral taxonomy.

Reappearance of chikungunya, formerly called dengue, in the Americas.

After an absence of ≈200 years, chikungunya returned to the American tropics in 2013. The virus is maintained in a complex African zoonotic cycle but escapes into an urban cycle at 40- to 50-year intervals, causing global pandemics. In 1823, classical chikungunya, a viral exanthem in humans, occurred on Zanzibar, and in 1827, it arrived in the Caribbean and spread to North and South America. In Zanzibar, the disease was known as kidenga pepo, Swahili for a sudden cramp-like seizure caused by an evil spirit; in Cuba, it was known as dengue, a Spanish homonym of denga. During the eighteenth century, dengue (present-day chikungunya) was distinguished from breakbone fever (present-day dengue), another febrile exanthem. In the twentieth century, experiments resulted in the recovery and naming of present-day dengue viruses. In 1952, chikungunya virus was recovered during an outbreak in Tanzania, but by then, the virus had lost its original name to present-day dengue viruses.

Rational design of a multivalent vaccine targeting arthropod-borne viruses using reverse vaccinology strategies.

Viruses transmitted by arthropods, such as Dengue, Zika, and Chikungunya, represent substantial worldwide health threats, particularly in countries like India. The lack of approved vaccines and effective antiviral therapies calls for developing innovative strategies to tackle these arboviruses. In this study, we employed immunoinformatics methodologies, incorporating reverse vaccinology, to design a multivalent vaccine targeting the predominant arboviruses. Epitopes of B and T cells were recognized within the non-structural proteins of Dengue, Zika, and Chikungunya viruses. The predicted epitopes were enhanced with adjuvants ß-defensin and RS-09 to boost the vaccine's immunogenicity. Sixteen distinct vaccine candidates were constructed, each incorporating epitopes from all three viruses. FUVAC-11 emerged as the most promising vaccine candidate through molecular docking and molecular dynamics simulations, demonstrating favorable binding interactions and stability. Its effectiveness was further evaluated using computational immunological studies confirming strong immune responses. The in silico cloning performed using the pET-28a(+) plasmid facilitates the future experimental implementation of this vaccine candidate, paving the way for potential advancements in combating these significant arboviral threats. However, further in vitro and in vivo studies are warranted to confirm the results obtained in this computational study, which highlights the effectiveness of immunoinformatics and reverse vaccinology in creating vaccines against major Arboviruses, offering a promising model for developing vaccines for other vector-borne diseases and enhancing global health security.

A Brief History of the Discovery of RNA-Mediated Antiviral Immune Defenses in Vector Mosquitos.

Arthropod-borne viruses (arboviruses) persist in a natural cycle that includes infections of humans or other vertebrates and transmission between vertebrates by infected arthropods, most commonly mosquitos. Arboviruses can cause serious, sometimes fatal diseases in humans and other vertebrates but cause little pathology in their mosquito vectors. Knowledge of the interactions between mosquito vectors and the arboviruses that they transmit is an important facet of developing schemes to control transmission. Mosquito innate immune responses to virus infection modulate virus replication in the vector, and understanding the components and mechanisms of the immune response could lead to improved methods for interrupting the transmission cycle. The most important aspect of mosquito antiviral defense is the exogenous small interfering RNA (exo-siRNA) pathway, one arm of the RNA interference (RNAi) silencing response. Our research as well as that of many other groups over the past 25 years to define this pathway are reviewed here. A more recently recognized but less well-understood RNA-mediated mosquito defense against arbovirus infections, the PIWI-interacting RNA (piRNA) pathway, is also described.

Interactions of host miRNAs in the flavivirus 3´UTR genome: From bioinformatics predictions to practical approaches.

The genus Flavivirus of the Flaviviridae family includes important viruses, such as Dengue, Zika, West Nile, Japanese encephalitis, Murray Valley encephalitis, tick-borne encephalitis, Yellow fever, Saint Louis encephalitis, and Usutu viruses. They are transmitted by mosquitoes or ticks, and they can infect humans, causing fever, encephalitis, or haemorrhagic fever. The treatment resources for these diseases and the number of vaccines available are limited. It has been discovered that eukaryotic cells synthesize small RNA molecules that can bind specifically to sequences present in messenger RNAs to inhibit the translation process, thus regulating gene expression. These small RNAs have been named microRNAs, and they have an important impact on viral infections. In this review, we compiled the available information on miRNAs that can interact with the 3' untranslated region (3'UTR) of the flavivirus genome, a conserved region that is important for viral replication and translation.

Meta-transcriptomics reveals potential virus transfer between Aedes communis mosquitoes and their parasitic water mites.

Arthropods harbor a largely undocumented diversity of RNA viruses. Some arthropods, like mosquitoes, can transmit viruses to vertebrates but are themselves parasitized by other arthropod species, such as mites. Very little is known about the viruses of these ectoparasites and how they move through the host-parasite relationship. To address this, we determined the virome of both mosquitoes and the mites that feed on them. The mosquito Aedes communis is an abundant and widely distributed species in Sweden, in northern Europe. These dipterans are commonly parasitized by water mite larvae (Trombidiformes: Mideopsidae) that are hypothesized to impose negative selection pressures on the mosquito by reducing fitness. In turn, viruses are dual-host agents in the mosquito-mite interaction. We determined the RNA virus diversity of mite-free and mite-detached mosquitoes, as well as their parasitic mites, using meta-transcriptomic sequencing. Our results revealed an extensive RNA virus diversity in both mites and mosquitoes, including thirty-seven putative novel RNA viruses that cover a wide taxonomic range. Notably, a high proportion of viruses (20/37) were shared between mites and mosquitoes, while a limited number of viruses were present in a single host. Comparisons of virus composition and abundance suggest potential virus transfer between mosquitoes and mites during their symbiotic interaction. These findings shed light on virome diversity and ecology in the context of arthropod host-parasite-virus relationships.

Tracing and tracking the emergence, epidemiology and dispersal of dengue virus to Africa during the 20th century.

The four mosquito-borne dengue virus serotypes (DENV1-DENV4) cause a high burden of disease throughout the tropical and sub-tropical regions of the world. Nevertheless, their precise epidemiological history in Africa, including when and where they originated and were distributed during the 20th century, remains unclear stressing the need for One Health focused research. Accordingly, we conducted a time-scaled molecular epidemiological reconstruction using publicly available and newly sequenced dengue virus genomes of African origin representing all four serotypes to deduce the most likely temporal and spatial transmission routes of each DENV serotype from their ancestral regions to, within and from Africa. Our analyses suggest that during the 20th century, serotypes DENV1-DENV3 were introduced to Africa from South East Asia on multiple occasions. The earliest evidence recorded indicates introduction of DENV2 during the early-1940s and of DENV1 during the mid-1940s to Western Africa from South East Asia. The analysis also implies an early introduction of DENV4 during the mid-1940s to Western Africa, alongside DENV1, probably originating in South East Asia. Establishment of DENV3 in Africa appears to have occurred later in the 1960s, apparently originating from South East Asia. However, with the re-establishment of DENV in the Americas, following the cessation of the PAHO mosquito control programme during the mid-20th century, evidence of introductions of DENV1 and DENV2 from the Americas to Western Africa was also observed. The data also identify intra-regional circulation of DENV, but also inter-regional dispersal of all four serotypes within Africa, which has led to a high degree of geographical overlap among serotypes. It is also noteworthy that DENV from both Eastern and Western Africa, have been introduced into Central Africa but there is no support for the converse relationship. For serotypes DENV1-DENV3, we observed probable exports from within established African DENV clusters (≥2 sequences) primarily to Eastern and Southern Asia. Collectively, our findings support the view that all DENV serotypes, apart from DENV4, have been introduced on multiple occasions to Africa, primarily originating from South East Asia, and subsequently to neighbouring regions within Africa.

Guillain-Barré Syndrome in Suriname; Clinical Presentation and Identification of Preceding Infections.

Guillain-Barré syndrome (GBS) is associated with various types of preceding infections including Campylobacter jejuni and cytomegalovirus, but there is also an association with arthropod borne viruses (arboviruses), such as Zika virus, that are endemic in tropical regions. Here we present the clinical characteristics of 12 GBS patients from Suriname that were hospitalized between the beginning of 2016 and half 2018. Extensive diagnostic testing was performed for pathogens that are commonly associated with GBS, but also for arboviruses, in order to identify the preceding infection that might have led to GBS. With this extensive testing algorithm, we could identify a recent infection in six patients of which four of them had evidence of a recent Zika virus or dengue virus infection. These results suggest that arboviruses, specifically Zika virus but possibly also dengue virus, might be important causative agents of GBS in Suriname. Furthermore, we found that more accessibility of intravenous immunoglobulins or plasma exchange could improve the treatment of GBS in Suriname.

Investigation and Response to an Outbreak of Dengue: Island of Hawaii, 2015-2016.

OBJECTIVES: From September 2015 through March 2016, Hawaii had the largest outbreak of locally transmitted dengue since 1944. We report on the Hawaii Department of Health's (HDOH's) investigation, findings, and response to the outbreak. METHODS: We defined cases of dengue using a modified version of the Council of State and Territorial Epidemiologists' case definition for dengue virus infections. We conducted epidemiologic investigations, including interviews with case-persons, review of medical records, laboratory testing, genetic sequencing of specimens, and geographic information system (GIS) data analysis. Outbreak response included community outreach and vector-control activities. RESULTS: We identified 264 confirmed cases of dengue; illness onset dates ranged from September 11, 2015, to March 17, 2016, all with reported travel to or residence on the Island of Hawaii. Of 264 persons with confirmed dengue, 238 (90.2%) were Hawaii residents. Thirty-seven (14.0%) persons required hospitalization; no cases of severe dengue or death were reported. GIS hot-spot analysis identified a cluster of cases on the western side of the island. Established risk factors for dengue exposure included holes in window or door screens, presence of standing water, and not using insect repellent or wearing protective clothing. CONCLUSIONS: To prevent or mitigate the spread of future arboviral introductions and outbreaks, the public health response should focus on behavioral and cultural attitudes, emphasizing personal mosquito protection and mosquito control at the community level. Outbreak responses can also be enhanced through the use of advanced GIS techniques, such as hot-spot analysis, to provide situational awareness and guide response efforts.

Risks and Challenges of Arboviral Diseases in Sudan: The Urgent Need for Actions.

The risk of emergence and/or re-emergence of arthropod-borne viral (arboviral) infections is rapidly growing worldwide, particularly in Africa. The burden of arboviral infections and diseases is not well scrutinized because of the inefficient surveillance systems in endemic countries. Furthermore, the health systems are fully occupied by the burden of other co-existing febrile illnesses, especially malaria. In this review we summarize the epidemiology and risk factors associated with the major human arboviral diseases and highlight the gap in knowledge, research, and control in Sudan. Published data in English up to March 2019 were reviewed and are discussed to identify the risks and challenges for the control of arboviruses in the country. In addition, the lack of suitable diagnostic tools such as viral genome sequencing, and the urgent need for establishing a genomic database of the circulating viruses and potential sources of entry are discussed. Moreover, the research and healthcare gaps and global health threats are analyzed, and suggestions for developing strategic health policy for the prevention and control of arboviruses with focus on building the local diagnostic and research capacity and establishing an early warning surveillance system for the early detection and containment of arboviral epidemics are offered.

Low Incidence and Mortality from SARS-CoV-2 in Southern Europe. Proposal of a hypothesis for Arthropod borne Herd immunity.

SARS-CoV-2 incidence and mortality in Europe have shown wide variation. Northern Italy in particular the Lombardy region, north-eastern French regions, Switzerland and Belgium were amongst the hardest hit, while the central and southern Italian regions, all the Balkan countries from Slovenia to Greece and the Islands of Malta and Cyprus had much fewer cases and deaths per capita, and deaths per number of cases. Differences in public health measures, and health care delivery, in the author's opinion, can only partly explain the difference. The geographical distribution of Phlebotomus sand-flies and the relative distribution of arthropod borne diseases Leishmaniasis and Phlebovirus infections especially the Sicilian Sandfly fever group corresponds to most areas of low prevalence of SARS-CoV-2. A hypothesis is proposed whereby repeated arthropod or sandfly vector infection of humans by novel viruses of zoonotic origins carrying bat or mammalian RNA/DNA, such as phleboviruses may have resulted in the development of an effective evolutionary immune response to most novel zoonotic viruses such as SARS-CoV-2 by means of survival of the fittest possibly over many generations. This process probably ran in parallel and concurrent with the progressive evolution of novel coronaviruses which spread from one mammalian species to another. Other possible, but less likely mechanisms for the role of sandfly meals within a much shorter time frame may have led to, (i) previous exposure and infection of humans with the SARS-Cov-2 virus itself, or a closely related corona virus in the previous decades, or (ii) exposure of human populations to parts coronavirus protein namely either S or more likely N protein carried mechanically by arthropods, but without clinical disease causing direct immunity or (iii) by causing infection with other arthropod borne viruses which could carry bat DNA/RNA and have similar functional proteins resulting in an immediate cross-reactive immune response rather than by natural selection. The Evidence possibly supporting or disputing this hypothesis is reviewed, however the major problem with the hypothesis is that to date no coronavirus has ever been isolated from arthropods. Such a hypothesis can only be supported by research investigating the possible biological relationship of arthropods and coronaviruses where paradoxically they may be promoting immunity rather than disease.

Effects of Arbovirus Multi-Host Life Cycles on Dinucleotide and Codon Usage Patterns.

Arthropod-borne viruses (arboviruses) of vertebrates including dengue, zika, chikungunya, Rift Valley fever, and blue tongue viruses cause extensive morbidity and mortality in humans, agricultural animals, and wildlife across the globe. As obligate intercellular pathogens, arboviruses must be well adapted to the cellular and molecular environment of both their arthropod (invertebrate) and vertebrate hosts, which are vastly different due to hundreds of millions of years of separate evolution. Here we discuss the comparative pressures on arbovirus RNA genomes as a result of a dual host life cycle, focusing on pressures that do not alter amino acids. We summarize what is currently known about arboviral genetic composition, such as dinucleotide and codon usage, and how cyclical infection of vertebrate and invertebrate hosts results in different genetic profiles compared with single-host viruses. To serve as a comparison, we compile what is known about arthropod tRNA, dinucleotide, and codon usages and compare this with vertebrates. Additionally, we discuss the potential roles of genetic robustness in arboviral evolution and how it may vary from other viruses. Overall, both arthropod and vertebrate hosts influence the resulting genetic composition of arboviruses, but a great deal remains to be investigated.

Contemporary Strategies and Current Trends in Designing Antiviral Drugs against Dengue Fever via Targeting Host-Based Approaches.

Dengue virus (DENV) is an arboviral human pathogen transmitted through mosquito bite that infects an estimated ~400 million humans (~5% of the global population) annually. To date, no specific therapeutics have been developed that can prevent or treat infections resulting from this pathogen. DENV utilizes numerous host molecules and factors for transcribing the single-stranded ~11 kb positive-sense RNA genome. For example, the glycosylation machinery of the host is required for viral particles to assemble in the endoplasmic reticulum. Since a variety of host factors seem to be utilized by the pathogens, targeting these factors may result in DENV inhibitors, and will play an important role in attenuating the rapid emergence of other flaviviruses. Many experimental studies have yielded findings indicating that host factors facilitate infection, indicating that the focus should be given to targeting the processes contributing to pathogenesis along with many other immune responses. Here, we provide an extensive literature review in order to elucidate the progress made in the development of host-based approaches for DENV viral infections, focusing on host cellular mechanisms and factors responsible for viral replication, aiming to aid the potential development of host-dependent antiviral therapeutics.

Preparing clinicians for (re-)emerging arbovirus infectious diseases in Europe.

BACKGROUND: Arthropod-borne virus (Arbovirus) infections are considered an emerging threat for Europe, with an increase in cases in recent decades. The increase in global travel and trade has contributed to the introduction of vectors and viruses into new geographical areas. Tropical arboviruses such as dengue and chikungunya have re-emerged causing local, sporadic outbreaks ignited by travel-imported cases. The recent Zika virus outbreak in the Americas highlighted a need to strengthen preparedness for (re-)emerging arbovirus infections globally. AIMS: To strengthen preparedness for the early identification of (re-)emerging arbovirus outbreaks in Europe and highlight areas for research. SOURCES: An evidence review of published and grey literature together with consultations with European arbovirus experts. CONTENT: This paper presents an overview of endemic and travel-imported arboviruses of clinical significance in Europe. The overview includes syndromic presentation, risk factors for infection and risk of transmission as well as an update on treatments and vaccinations and surveillance notifications and reporting. The paper also presents predictive modelled risks of further geographical expansion of vectors and viruses. IMPLICATIONS: There are a range of arboviruses of clinical significance to Europe. There has been an increase in notifications of endemic and travel-imported arbovirus cases in recent years and an increased geographical range of vectors and viruses. The heterogeneity in surveillance reporting indicates a risk for the early identification of (re-)emerging outbreaks. The data presented show a need to strengthen preparedness for (re-)emerging arbovirus infections and a need for research into neglected arboviruses, risks of non-vector transmission and effective therapeutics and vaccinations.

Dengue fever in Czech travellers: A 10-year retrospective study in a tertiary care centre.

BACKGROUND: Dengue fever is a frequent cause of morbidity in travellers. The objective was to describe the epidemiological and clinical characteristics of dengue fever in Czech travellers. METHOD: This descriptive study includes patients with acute dengue fever diagnosed at Hospital Na Bulovce during 2004-2013. Data were collected and analysed retrospectively. RESULTS: A total of 132 patients (83 males and 49 females) of median age 33 years (IQR 29-40) were included. Diagnosis was established by NS1 antigen detection in 87/107 cases (81.3%) and/or RT-PCR in 50/72 (69.4%) and by serology in 25 cases (18.9%). Dengue was acquired in South-East Asia in 69 cases (52.3%), followed by South Asia (48 cases; 36.3%), Latin America (14; 10.6%) and Sub-Saharan Africa (1; 0.8%). The most frequent symptoms included fever, rash and headache. Initial leukocyte and lymphocyte counts were lower in patients who presented in the early phase (0-4 days), however, platelet count was lower and AST, ALT and LDH activity higher in patients with a longer symptoms duration (≥5 days). The clinical course was mostly uncomplicated. CONCLUSIONS: Dengue fever is becoming a frequent cause of fever in Czech travellers. Clinicians should be familiar with the typical clinical findings and novel diagnostic methods.

Zoonotic encephalitides caused by arboviruses: transmission and epidemiology of alphaviruses and flaviviruses.

In this review, we mainly focus on zoonotic encephalitides caused by arthropod-borne viruses (arboviruses) of the families Flaviviridae (genus Flavivirus) and Togaviridae (genus Alphavirus) that are important in both humans and domestic animals. Specifically, we will focus on alphaviruses (Eastern equine encephalitis virus, Western equine encephalitis virus, Venezuelan equine encephalitis virus) and flaviviruses (Japanese encephalitis virus and West Nile virus). Most of these viruses were originally found in tropical regions such as Africa and South America or in some regions in Asia. However, they have dispersed widely and currently cause diseases around the world. Global warming, increasing urbanization and population size in tropical regions, faster transportation and rapid spread of arthropod vectors contribute in continuous spreading of arboviruses into new geographic areas causing reemerging or resurging diseases. Most of the reemerging arboviruses also have emerged as zoonotic disease agents and created major public health issues and disease epidemics.