Host Inflammatory Response to Mosquito Bites Enhances the Severity of Arbovirus Infection.

Aedes aegypti mosquitoes are responsible for transmitting many medically important viruses such as those that cause Zika and dengue. The inoculation of viruses into mosquito bite sites is an important and common stage of all mosquito-borne virus infections. We show, using Semliki Forest virus and Bunyamwera virus, that these viruses use this inflammatory niche to aid their replication and dissemination in vivo. Mosquito bites were characterized by an edema that retained virus at the inoculation site and an inflammatory influx of neutrophils that coordinated a localized innate immune program that inadvertently facilitated virus infection by encouraging the entry and infection of virus-permissive myeloid cells. Neutrophil depletion and therapeutic blockade of inflammasome activity suppressed inflammation and abrogated the ability of the bite to promote infection. This study identifies facets of mosquito bite inflammation that are important determinants of the subsequent systemic course and clinical outcome of virus infection.

Application of Aptamer-Based Assays to the Diagnosis of Arboviruses Important for Public Health in Brazil.

Arbovirus infections represent a global public health problem, and recent epidemics of yellow fever, dengue, and Zika have shown their critical importance in Brazil and worldwide. Whilst a major effort for vaccination programs has been in the spotlight, a number of aptamer approaches have been proposed in a complementary manner, offering the possibility of differential diagnosis between these arboviruses, which often present similar clinical symptoms, as well as the potential for a treatment option when no other alternative is available. In this review, we aim to provide a background on arbovirus, with a basic description of the main viral classes and the disease they cause, using the Brazilian context to build a comprehensive understanding of their role on a global scale. Subsequently, we offer an exhaustive revision of the diagnostic and therapeutic approaches offered by aptamers against arboviruses. We demonstrate how these promising reagents could help in the clinical diagnosis of this group of viruses, their use in a range of diagnostic formats, from biosensors to serological testing, and we give a short review on the potential approaches for novel aptamer-based antiviral treatment options against different arboviral diseases.

Host target-based approaches against arboviral diseases.

In the 20th century, socioeconomic and environmental changes facilitated the reintroduction of mosquitoes in developing cities, resulting in the reinsertion of mosquito-borne viral diseases and the dispersal of their causative agents on a worldwide scale. Recurrent outbreaks of arboviral diseases are being reported, even in regions without a previous history of arboviral disease transmission. Of note, arboviral infections represented approximately 30% of all emerging vector-borne diseases in the last decade. Therapeutic strategies against infectious viral diseases include the use of different classes of molecules that act directly on the pathogen and/or act by optimizing the host immune response. Drugs targeting the virus usually provide amelioration of symptoms by suppressing and controlling the infection. However, it is limited by the short-window of effectiveness, ineffectiveness against latent viruses, development of drug-resistant mutants and toxic side effects. Disease may also be a consequence of an excessive, uncontrolled or misplaced inflammatory response, treatments that interfere in host immune response are interesting options and can be used isolated or in combination with virus-targeted therapies. The use of host-targeted therapies requires specific knowledge regarding host immune patterns that may trigger dengue virus (DENV), chikungunya virus (CHIKV) or Zika virus (ZIKV) disease.

Emerging arboviruses in Rio Grande do Sul, Brazil: Chikungunya and Zika outbreaks, 2014-2016.

QUESTIONS INVESTIGATED: The recent emergence of arboviruses such as Chikungunya virus (CHIKV) and Zika virus (ZIKV) in Brazil has posed a threat to human health and to the country's economy. Outbreaks occur mainly in tropical areas; however, increasing number of cases have been observed in Rio Grande do Sul (RS), the Southernmost state; therefore, surveillance of these arboviruses is essential for public health measures. DESIGN: In this study, we analyzed 1276 samples from patients with clinically suspected arboviral diseases between 2014 and 2016. Demographic and clinical data were collected and described; cases of microcephaly associated with congenital infection were analyzed. ESSENTIAL FINDINGS: Results show that CHIKV and ZIKV entered RS in 2014 and 2015, respectively, with imported cases confirmed. Autochthonous infections occurred in 2016 for both viruses, with a total of 5 autochthonous cases for CHIKV and 44 for ZIKV. Most patients were older than 21 years; the main symptoms were fever, arthralgia, myalgia, and headache; rash, conjunctivitis, and pruritus were also reported in ZIKV cases. Three cases of congenital Zika syndrome were confirmed in our study, while another 20 cases of microcephaly associated with congenital infection were confirmed (10 positive for syphilis, 6 for toxoplasmosis and 4 for cytomegalovirus). MAIN CONCLUSIONS: Considering co-circulation of different arbovirus in RS, including Dengue virus, CHIKV, and ZIKV, and the presence of Aedes aegypti and Aedes albopictus in the area, surveillance of patients infected by these viruses contributes to the control and prevention of such diseases. Practical difficulties in diagnosing these infections are discussed.

Antiviral immunity of Anopheles gambiae is highly compartmentalized, with distinct roles for RNA interference and gut microbiota.

Arboviruses are transmitted by mosquitoes and other arthropods to humans and animals. The risk associated with these viruses is increasing worldwide, including new emergence in Europe and the Americas. Anopheline mosquitoes are vectors of human malaria but are believed to transmit one known arbovirus, o'nyong-nyong virus, whereas Aedes mosquitoes transmit many. Anopheles interactions with viruses have been little studied, and the initial antiviral response in the midgut has not been examined. Here, we determine the antiviral immune pathways of the Anopheles gambiae midgut, the initial site of viral infection after an infective blood meal. We compare them with the responses of the post-midgut systemic compartment, which is the site of the subsequent disseminated viral infection. Normal viral infection of the midgut requires bacterial flora and is inhibited by the activities of immune deficiency (Imd), JAK/STAT, and Leu-rich repeat immune factors. We show that the exogenous siRNA pathway, thought of as the canonical mosquito antiviral pathway, plays no detectable role in antiviral defense in the midgut but only protects later in the systemic compartment. These results alter the prevailing antiviral paradigm by describing distinct protective mechanisms in different body compartments and infection stages. Importantly, the presence of the midgut bacterial flora is required for full viral infectivity to Anopheles, in contrast to malaria infection, where the presence of the midgut bacterial flora is required for protection against infection. Thus, the enteric flora controls a reciprocal protection tradeoff in the vector for resistance to different human pathogens.

Use of Testing for West Nile Virus and Other Arboviruses.

In the United States, the most commonly diagnosed arboviral disease is West Nile virus (WNV) infection. Diagnosis is made by detecting WNV IgG or viral genomic sequences in serum or cerebrospinal fluid. To determine frequency of this testing in WNV-endemic areas, we examined the proportion of tests ordered for patients with meningitis and encephalitis at 9 hospitals in Houston, Texas, USA. We identified 751 patients (567 adults, 184 children), among whom 390 (52%) experienced illness onset during WNV season (June-October). WNV testing was ordered for 281 (37%) of the 751; results indicated acute infection for 32 (11%). Characteristics associated with WNV testing were acute focal neurologic deficits; older age; magnetic resonance imaging; empirically prescribed antiviral therapy; worse clinical outcomes: and concomitant testing for mycobacterial, fungal, or other viral infections. Testing for WNV is underutilized, and testing of patients with more severe disease raises the possibility of diagnostic bias in epidemiologic studies.

Divergent cerebrospinal fluid cytokine network induced by non-viral and different viral infections on the central nervous system.

BACKGROUND: Meningoencephalitis is one of the most common disorders of the central nervous system (CNS) worldwide. Viral meningoencephalitis differs from bacterial meningitis in several aspects. In some developing countries, bacterial meningitis has appropriate clinical management and chemotherapy is available. Virus-associated and virus not detected meningoencephalitis are treatable, however, they may cause death in a few cases. The knowledge of how mediators of inflammation can induce disease would contribute for the design of affordable therapeutic strategies, as well as to the diagnosis of virus not detected and viral meningoencephalitis. Cytokine-induced inflammation to CNS requires several factors that are not fully understood yet. METHODS: Considering this, several cytokines were measured in the cerebrospinal fluid (CSF) of patients with undiagnosed and viral meningoencephalitis, and these were correlated with cellularity in the CSF. RESULTS: The results demonstrate that an altered biochemical profile alongside increased cellularity in the cerebrospinal fluid is a feature of patients with meningoencephalitis that are not associated with the detection of virus in the CNS (P < 0.05). Moreover, HIV-positive patients (n = 10) that evolve with meningoencephalitis display a distinct biochemical/cytological profile (P < 0.05) in the cerebrospinal fluid. Meningoencephalitis brings about a prominent intrathecal cytokine storm regardless of the detection of virus as presumable etiological agent. In the case of Enterovirus infection (n = 13), meningoencephalitis elicits robust intrathecal pro-inflammatory cytokine pattern and elevated cellularity when compared to herpesvirus (n = 15) and Arbovirus (n = 5) viral infections (P < 0.05). CONCLUSION: Differences in the cytokine profile of the CSF may be unique if distinct, viral or presumably non-viral pathways initially trigger the inflammatory response in the CNS.

Rainfall and sentinel chicken seroconversions predict human cases of Murray Valley encephalitis in the north of Western Australia.

BACKGROUND: Murray Valley encephalitis virus (MVEV) is a flavivirus that occurs in Australia and New Guinea. While clinical cases are uncommon, MVEV can cause severe encephalitis with high mortality. Sentinel chicken surveillance is used at many sites around Australia to provide an early warning system for risk of human infection in areas that have low population density and geographical remoteness. MVEV in Western Australia occurs in areas of low population density and geographical remoteness, resulting in logistical challenges with surveillance systems and few human cases. While epidemiological data has suggested an association between rainfall and MVEV activity in outbreak years, it has not been quantified, and the association between rainfall and sporadic cases is less clear. In this study we analysed 22 years of sentinel chicken and human case data from Western Australia in order to evaluate the effectiveness of sentinel chicken surveillance for MVEV and assess the association between rainfall and MVEV activity. METHODS: Sentinel chicken seroconversion, human case and rainfall data from the Kimberley and Pilbara regions of Western Australia from 1990 to 2011 were analysed using negative binomial regression. Sentinel chicken seroconversion and human cases were used as dependent variables in the model. The model was then tested against sentinel chicken and rainfall data from 2012 and 2013. RESULTS: Sentinel chicken seroconversion preceded all human cases except two in March 1993. Rainfall in the prior three months was significantly associated with both sentinel chicken seroconversion and human cases across the regions of interest. Sentinel chicken seroconversion was also predictive of human cases in the models. The model predicted sentinel chicken seroconversion in the Kimberley but not in the Pilbara, where seroconversions early in 2012 were not predicted. The latter may be due to localised MVEV activity in isolated foci at dams, which do not reflect broader virus activity in the region. CONCLUSIONS: We showed that rainfall and sentinel chickens provide a useful early warning of MVEV risk to humans across endemic and epidemic areas, and that a combination of the two indicators improves the ability to assess MVEV risk and inform risk management measures.

Generating prophylactic immunity against arboviruses in vertebrates and invertebrates.

The World Health Organization recently declared a global initiative to control arboviral diseases. These are mainly caused by pathogenic flaviviruses (such as dengue, yellow fever and Zika viruses) and alphaviruses (such as chikungunya and Venezuelan equine encephalitis viruses). Vaccines represent key interventions for these viruses, with licensed human and/or veterinary vaccines being available for several members of both genera. However, a hurdle for the licensing of new vaccines is the epidemic nature of many arboviruses, which presents logistical challenges for phase III efficacy trials. Furthermore, our ability to predict or measure the post-vaccination immune responses that are sufficient for subclinical outcomes post-infection is limited. Given that arboviruses are also subject to control by the immune system of their insect vectors, several approaches are now emerging that aim to augment antiviral immunity in mosquitoes, including Wolbachia infection, transgenic mosquitoes, insect-specific viruses and paratransgenesis. In this Review, we discuss recent advances, current challenges and future prospects in exploiting both vertebrate and invertebrate immune systems for the control of flaviviral and alphaviral diseases.

Antibodies against medically relevant arthropod-borne viruses in the ubiquitous African rodent Mastomys natalensis.

Over the past decades, the number of arthropod-borne virus (arbovirus) outbreaks has increased worldwide. Knowledge regarding the sylvatic cycle (i.e., non-human hosts/environment) of arboviruses is limited, particularly in Africa, and the main hosts for virus maintenance are unknown. Previous studies have shown the presence of antibodies against certain arboviruses (i.e., chikungunya-, dengue-, and Zika virus) in African non-human primates and bats. We hypothesize that small mammals, specifically rodents, may function as amplifying hosts in anthropogenic environments. The detection of RNA of most arboviruses is complicated by the viruses' short viremic period within their hosts. An alternative to determine arbovirus hosts is by detecting antibodies, which can persist several months. Therefore, we developed a high-throughput multiplex immunoassay to detect antibodies against 15 medically relevant arboviruses. We used this assay to assess approximately 1,300 blood samples of the multimammate mouse, Mastomys natalensis from Tanzania. In 24% of the samples, we detected antibodies against at least one of the tested arboviruses, with high seroprevalences of antibodies reacting against dengue virus serotype one (7.6%) and two (8.4%), and chikungunya virus (6%). Seroprevalence was higher in females and increased with age, which could be explained by inherent immunity and behavioral differences between sexes, and the increased chance of exposure to an arbovirus with age. We evaluated whether antibodies against multiple arboviruses co-occur more often than randomly and found that this may be true for some members of the Flaviviridae and Togaviridae. In conclusion, the development of an assay against a wide diversity of medically relevant arboviruses enabled the analysis of a large sample collection of one of the most abundant African small mammals. Our findings highlight that Mastomys natalensis is involved in the transmission cycle of multiple arboviruses and provide a solid foundation to better understand the role of this ubiquitous rodent in arbovirus outbreaks.

Precision arbovirus serology with a pan-arbovirus peptidome.

Arthropod-borne viruses represent a crucial public health threat. Current arboviral serology assays are either labor intensive or incapable of distinguishing closely related viruses, and many zoonotic arboviruses that may transition to humans lack any serologic assays. In this study, we present a programmable phage display platform, ArboScan, that evaluates antibody binding to overlapping peptides that represent the proteomes of 691 human and zoonotic arboviruses. We confirm that ArboScan provides detailed antibody binding information from animal sera, human sera, and an arthropod blood meal. ArboScan identifies distinguishing features of antibody responses based on exposure history in a Colombian cohort of Zika patients. Finally, ArboScan details epitope level information that rapidly identifies candidate epitopes with potential protective significance. ArboScan thus represents a resource for characterizing human and animal arbovirus antibody responses at cohort scale.

[Immunomodulators and specific inactivated vaccines in urgent prophylaxis under experimental arboviral infection].

A reliable protective activity of the home-manufactured immunomodulators (ridostin, polyribonate glucosemuramyl-dipeptide, Mylife, and peptidoglycane-160) was detected in mice. The mice were infected with the equine eastern encephalomyelitis virus (EEEV, an alphavirus), or with the tick-borne encephalitis virus (TBEV), or the yellow fever (YF) virus (both flaviviruses). The effect of the urgent vaccination reliably increases when the vaccination is combined with the immunomodulators listed above. Under the alphavirus infection, the combined effects of the vaccine and ridostin were accompanied with increased specific humoral and cellular immune response (virus-specific antibodies and adoptive transfer of immune lymphocytes). The combined application of the specific vaccine and ridostin can be recommended for clinical trials of TBE in the foci of Infection.

[Immunotropic therapy of arbovirus infections in children].

Cytochemical indices of peripheral blood leukocytes were investigated. The results allowed to estimate the level of the disease severity and to control the immunotropic therapy efficacy. Dysbalance of the interleukocytic microbicidical systems (IMS) of neutrophils reflected the nonspecific resisistance and the state of the phagocytic system as an objective criterion of the adaptation syndrome. Association of the deficiency of the IMS components and blood serum fibronectin were considered as a predisposition of bacterial complications. Cycloferon activated the phagocyte metabolic activity and promoted elimination of the main clinical symptoms of the disease (acute infective damage of the meninges, temperature elevation, intoxication signs), thus making shorter the hospitalization terms.

[Virus infections and interferon inducers in the complex therapy].

Interferon inductors of various chemical groups, belonging to antivirals, and induction of several types of endogenous interferon in blood serum are described. Cycloferon was shown efficient in the complex treatment of chronic hepatitis C, tuberculosis in HIV-infected subjects, arbovirus diseases, influenza and acute respiratory virus infections.

ArVirInd-a database of arboviral antigenic proteins from the Indian subcontinent.

Background: Studies on antigenic proteins for arboviruses are important for providing diagnostics and vaccine development. India and its neighboring countries have a huge burden of arboviral diseases. Data mining for country-specific sequences from existing bioinformatics databases is cumbersome and time-consuming. This necessitated the development of a database of antigenic proteins from arboviruses isolated from the countries of the Indian subcontinent. Methods: Arboviral antigenic protein sequences were obtained from the NCBI and other databases. In silico antigenic characterization was performed (Epitope predictions) and data was incorporated into the database. The front end was designed and developed using HTML, CSS, and PHP. For the backend of the database, we have used MySQL. Results: A database, named ArVirInd, is created as a repository of information on curated antigenic proteins. This enlists sequences by country and year of outbreak or origin of the viral strain. For each entry, antigenic information is provided along with functional sites, etc. Researchers can search this database by virus/protein name, country, and year of collection (or in combination) as well as peptide search for epitopes. It is available publicly via the Internet at http://www.arvirind.co.in. ArVirInd will be useful in the study of immune informatics, diagnostics, and vaccinology for arboviruses.

Single-cell analysis of arthritogenic alphavirus-infected human synovial fibroblasts links low abundance of viral RNA to induction of innate immunity and arthralgia-associated gene expression.

Infection by (re-)emerging RNA arboviruses including Chikungunya virus (CHIKV) and Mayaro virus primarily cause acute febrile disease and transient polyarthralgia. However, in a significant subset of infected individuals, debilitating arthralgia persists for weeks over months up to years. The underlying immunopathogenesis of chronification of arthralgia upon primary RNA-viral infection remains unclear. Here, we analysed cell-intrinsic responses to ex vivo arthritogenic alphaviral infection of primary human synovial fibroblasts isolated from knee joints, one the most affected joint types during acute and chronic CHIKV disease. Synovial fibroblasts were susceptible and permissive to alphaviral infection. Base-line and exogenously added type I interferon (IFN) partially and potently restricted infection, respectively. RNA-seq revealed a CHIKV infection-induced transcriptional profile that comprised upregulation of expression of several hundred IFN-stimulated and arthralgia-mediating genes. Single-cell virus-inclusive RNA-seq uncovered a fine-tuned switch from induction to repression of cell-intrinsic immune responses depending on the abundance of viral RNA in an individual cell. Specifically, responses were most pronounced in cells displaying low-to-intermediate amounts of viral RNA and absence of virus-encoded, fluorescent reporter protein expression, arguing for efficient counteraction of innate immunity in cells expressing viral antagonists at sufficient quantities. In summary, cell-intrinsic sensing of viral RNA that potentially persists or replicates at low levels in synovial fibroblasts and other target cell types in vivo may contribute to the chronic arthralgia induced by alphaviral infections. Our findings might advance our understanding of the immunopathophysiology of long-term pathogenesis of RNA-viral infections.

Encephalitic Arboviruses of Africa: Emergence, Clinical Presentation and Neuropathogenesis.

Many mosquito-borne viruses (arboviruses) are endemic in Africa, contributing to systemic and neurological infections in various geographical locations on the continent. While most arboviral infections do not lead to neuroinvasive diseases of the central nervous system, neurologic diseases caused by arboviruses include flaccid paralysis, meningitis, encephalitis, myelitis, encephalomyelitis, neuritis, and post-infectious autoimmune or memory disorders. Here we review endemic members of the Flaviviridae and Togaviridae families that cause neurologic infections, their neuropathogenesis and host neuroimmunological responses in Africa. We also discuss the potential for neuroimmune responses to aide in the development of new diagnostics and therapeutics, and current knowledge gaps to be addressed by arbovirus research.

Spatial Distribution and Burden of Emerging Arboviruses in French Guiana.

Despite the health, social and economic impact of arboviruses in French Guiana, very little is known about the extent to which infection burden is shared between individuals. We conducted a large multiplexed serological survey among 2697 individuals from June to October 2017. All serum samples were tested for IgG antibodies against DENV, CHIKV, ZIKV and MAYV using a recombinant antigen-based microsphere immunoassay with a subset further evaluated through anti-ZIKV microneutralization tests. The overall DENV seroprevalence was estimated at 73.1% (70.6-75.4) in the whole territory with estimations by serotype at 68.9% for DENV-1, 38.8% for DENV-2, 42.3% for DENV-3, and 56.1% for DENV-4. The overall seroprevalence of CHIKV, ZIKV and MAYV antibodies was 20.3% (17.7-23.1), 23.3% (20.9-25.9) and 3.3% (2.7-4.1), respectively. We provide a consistent overview of the burden of emerging arboviruses in French Guiana, with useful findings for risk mapping, future prevention and control programs. The majority of the population remains susceptible to CHIKV and ZIKV, which could potentially facilitate the risk of further re-emergences. Our results underscore the need to strengthen MAYV surveillance in order to rapidly detect any substantial changes in MAYV circulation patterns.

Sphingomyelinases in a journey to combat arthropod-borne pathogen transmission.

Ixodes scapularis ticks feed on humans and other vertebrate hosts and transmit several pathogens of public health concern. Tick saliva is a complex mixture of bioactive proteins, lipids and immunomodulators, such as I. scapularis sphingomyelinase (IsSMase)-like protein, an ortholog of dermonecrotoxin SMase D found in the venom of Loxosceles spp. of spiders. IsSMase modulates the host immune response towards Th2, which suppresses Th1-mediated cytokines to facilitate pathogen transmission. Arboviruses utilize exosomes for their transmission from tick to the vertebrate host, and exosomes derived from tick saliva/salivary glands suppress C-X-C motif chemokine ligand 12 and interleukin-8 immune response(s) in human skin to delay wound healing and repair processes. IsSMase affects also viral replication and exosome biogenesis, thereby inhibiting tick-to-vertebrate host transmission of pathogenic exosomes. In this review, we elaborate on exosomes and their biogenesis as potential candidates for developing novel control measure(s) to combat tick-borne diseases. Such targets could help with the development of an efficient anti-tick vaccine for preventing the transmission of tick-borne pathogens.