Correction to: In-vitro assessment of cutaneous immune responses to aedes mosquito salivary gland extract and dengue virus in Cambodian individuals

Dengue virus (DENV) poses a global health threat, affecting millions individuals annually with no specific therapy and limited vaccines. Mosquitoes, mainly Aedes aegypti and Aedes albopictus worldwide, transmit DENV through their saliva during blood meals. In this study, we aimed to understand how Aedes mosquito saliva modulate skin immune responses during DENV infection in individuals living in mosquito-endemic regions. To accomplish this, we dissociated skin cells from Cambodian volunteers and incubated them with salivary gland extract (SGE) from three different mosquito strains: Ae. aegypti USDA strain, Ae. aegypti and Ae. albopictus wild type (WT) in the presence/absence of DENV. We observed notable alterations in skin immune cell phenotypes subsequent to exposure to Aedes salivary gland extract (SGE). Specifically, exposure lead to an increase in the frequency of macrophages expressing chemokine receptor CCR2, and neutrophils expressing CD69. Additionally, we noted a substantial increase in the percentage of macrophages that became infected with DENV in the presence of Aedes SGE. Differences in cellular responses were observed when Aedes SGE of three distinct mosquito strains were compared. Our findings deepen the understanding of mosquito saliva's role in DENV infection and skin immune responses in individuals regularly exposed to mosquito bites. This study provides insights into skin immune cell dynamics that could guide strategies to mitigate DENV transmission and other arbovirus diseases.

[This corrects the article DOI: 10.1093/oxfimm/iqae003.].

Flaviviruses include the most prevalent and medically challenging viruses. Persistent infection with flaviviruses of epithelial cells and hepatocytes that do not undergo cell death is common. Here, we report that, in epithelial cells, up-regulation of autophagy following flavivirus infection markedly enhances virus replication and that one flavivirus gene, NS4A, uniquely determines the up-regulation of autophagy. Dengue-2 and Modoc (a murine flavivirus) kill primary murine macrophages but protect epithelial cells and fibroblasts against death provoked by several insults. The flavivirus-induced protection derives from the up-regulation of autophagy, as up-regulation of autophagy by starvation or inactivation of mammalian target of rapamycin also protects the cells against insult, whereas inhibition of autophagy via inactivation of PI3K nullifies the protection conferred by flavivirus. Inhibition of autophagy also limits replication of both Dengue-2 and Modoc virus in epithelial cells. Expression of flavivirus NS4A is sufficient to induce PI3K-dependent autophagy and to protect cells against death; expression of other viral genes, including NS2A and NS4B, fails to protect cells against several stressors. Flavivirus NS4A protein induces autophagy in epithelial cells and thus protects them from death during infection. As autophagy is vital to flavivirus replication in these cells, NS4A is therefore also identified as a critical determinant of flavivirus replication.

Dengue virus (DENV), the etiological agent of dengue fever, is transmitted to the human host during blood uptake by an infective mosquito. Infection of vector salivary glands and further injection of infectious saliva into the human host are key events of the DENV transmission cycle. However, the molecular mechanisms of DENV entry into the mosquito salivary glands have not been clearly identified. Otherwise, although it was demonstrated for other vector-transmitted pathogens that insect salivary components may interact with host immune agents and impact the establishment of infection, the role of mosquito saliva on DENV infection in human has been only poorly documented. To identify salivary gland molecules which might interact with DENV at these key steps of transmission cycle, we investigated the presence of proteins able to bind DENV in salivary gland extracts (SGE) from two mosquito species. Using virus overlay protein binding assay, we detected several proteins able to bind DENV in SGE from Aedes aegypti (L.) and Aedes polynesiensis (Marks). The present findings pave the way for the identification of proteins mediating DENV attachment or entry into mosquito salivary glands, and of saliva-secreted proteins those might be bound to the virus at the earliest step of human infection. The present findings might contribute to the identification of new targets for anti-dengue strategies.

Aedes albopictus as well as Aedes aegypti is an important vector of chikungunya and dengue viruses. Electron microscopic observations on the salivary glands of Ae. albopictus infected with chikungunya virus were performed in comparing with those of Ae. aegypti infected with dengue virus. No virus budding from the cell surface of the chikungunya-infected mosquito's salivary glands was found as shown in dengue-infected ones, in contrast to the findings of the mammalian cells such as Vero, KB, IMR, J-111 and BHK-21 cells infected with chikungunya and/or dengue virus(es).

Mosquito Salivary Gland Extracts Induce EBV-Infected NK Cell Oncogenesis Via CD4+ T Cells in Patients with Hypersensitivity to Mosquito Bites

Arbovirus epidemics are raging in tropical areas. Dengue virus (DENV), dengue shock syndrome (DSS), and dengue hemorrhagic fever (DHF) affect millions of individuals every year and cause significant mortality in Latin America, Africa, and Asia. Chikungunya virus (CHIKV) has caused recurrent epidemics in the Indian subcontinent and recent epidemics in Reunion and other islands in the Indian Ocean, with recent detection in areas of Europe. This issue of TRANFUSION includes two articles on the detection of DENV RNA in blood donors from epidemic areas, one article on the clearance of spiked DENV in the course of the manufacture of plasma derivatives, and one article on statistical modeling of transfusion risk during an epidemic of mosquito-borne CHIKV. The surprising seriousness of recurring epidemics of West Nile virus (WNV) in North America has heightened concerns about the potential for introduction and similar epidemic spread of other arbovirus infections in the United States. Dengue has received particular attention since cases have been recognized in the United States at the border between Texas and Mexico.1 The mosquito species that transmit DENV, Aedes egypti and Aedes albopictus, are present in the Southern and Southeastern parts of the United States, raising the specter of significant spread within the United States if the virus is introduced and efficiently spreads. Does dengue represent a risk to the safety of the US blood supply? Transmission by transfusion (TT) is often difficult to evaluate in the midst of an epidemic because the infection could have been acquired through a mosquito bite, through a transfusion, or even through a needle stick. Doubts about TT of WNV were eliminated when the virus was transmitted to multiple recipients of organs and, subsequently, multiple recipients of blood components obtained from the blood donors determined to be viremic.2 Despite the recognition of millions of cases of DENV infection and disease every year, there are very few published reports of transfusion transmission. One report was submitted to the public health agency of Hong Kong and, although accessible online, has not been published in a peer-reviewed journal. The other report describes a recent cluster of TT-DENV in Singapore, which is currently in press (P.A. Tambyah, National University of Singapore, personal communication, 2008). There are also reports of transmission by needle sticks and one case associated with a marrow transplant in Puerto Rico (references are included in the articles being published). Considering the available information, should precautionary measures be considered to prevent TT-DENV in the United States or even more important in countries in which DENV epidemics are occurring at expanding rates? What are the data required for decisions to implement measures that mitigate this potential risk? The articles published in the current issue of TRANSFUSION contribute data that will be needed for policy decisions that may be required in the not so distant future. Linnen and coworkers3 used a transcription-mediated amplification (TMA) assay for detection of DENV RNA to screen 13,372 specimens collected from blood donors in Honduras, Brazil, and Australia, countries with ongoing seasonal dengue epidemics. They identified 9 donors in Honduras who were repeat reactive for DENV RNA by TMA. Eight were confirmed by a polymerase chain reaction (PCR) assay that identified three different DENV serotypes, DENV-1, -2, and -4. Infectious virus could be recovered from four of these donors. Three samples from Brazil were repeatedly reactive on TMA. One was typed as DENV-1 and the other as DENV-3. None of the Australian specimens was repeatedly reactive. Samples were also tested by enzyme-linked immunosorbent assay for immunoglobulin M and immunoglobulin G antibodies to DENV and by plaque reduction neutralization assay to determine the existence and serotype of prior DENV infections in the viremic donors. Five of the donors in Honduras and one of the donors in Brazil appeared to have secondary DENV infections. The authors discuss the implications of reinfection with heterotypic subtypes because of the increased risk of DHF in these individuals. The study thus documented the presence of asymptomatic viremic donors in Honduras and Brazil who could theoretically transmit the virus to blood recipients. Mohammed and colleagues4 screened specimens from 16,521 blood donations made during an 11-week period to the American Red Cross in Puerto Rico, starting 2 weeks after the peak of dengue activity at the end of 2005. Twelve were DENV RNA repeatedly reactive using the same TMA assay used by Linnen and coworkers; 5 of the 12 were reactive in a pool of 16 (the operational pool size for WNV using TMA), and 4 of the 5 were positive on PCR. The four PCR-positive samples had viral loads ranging from 2000 to 80 million copies per mL, of which 3 were typed as DENV-2 and 1 as DENV-3. Infectious virus was recovered from all three of these donors by either mosquito inoculation or cell culture. Interestingly, the authors indicated that some of the 77,000 units of whole blood collected in Puerto Rico are exported to the continental United States each year, implying that these imported units could potentially transmit DENV to continental US recipients. A total of 722 cases of dengue have been reported in Puerto Rico in the first 6 weeks of 2008, so such transmissions could be occurring on an ongoing basis. With regard to growing concerns over potential TT-CHIKV, Brouard and colleagues5 used statistical sampling methods to estimate the risk of viremic blood donations during the CHIKV epidemic that raged through Reunion Island in the Indian Ocean from 2005 to 2007. They placed the peak risk at 1500 viremic donors per 100,000 donations between January and March 2006. Similar to DENV, there were no reports of TT despite estimates that more than 300,000 people were infected by CHIKV during this period, some of whom undoubtedly donated blood during the presymptomatic viremic phase of infection. The French government promptly suspended whole-blood collections in Reunion Island during this epidemic and provided the blood needs from the mainland. Platelet collections by apheresis continued locally, but the collected products were subjected to a process of pathogen inactivation. The statistical approach used by Brouard and coworkers5 is identical to that used by Biggerstaff and Petersen6 when they estimated the risk of transmission of WNV by transfusion during the 1999 epidemic in Queens, New York City, in a prescient article published in TRANSFUSION in August 2002. There had been no reports of TT of WNV at that time. However, 2002 was the year in which WNV TT was demonstrated and the year with the highest number of documented TT cases in North America. Blood donor screening by nucleic acid testing (NAT) for WNV was implemented in July 2003 and is viewed as a great success with interdiction of thousands of viremic blood components. Finally, Xie and colleagues7 address concerns over risk of arbovirus transmission by plasma derivatives by evaluating clearance of spiked DENV serotype 2 as a result of fractionation and processing of therapeutic plasma proteins. They concluded that the multiplicity of steps, including treatment by solvent detergent, cumulatively reduced the risk of viral transmission by albumin by more than 10 log and for immunoglobulins by more than 14 log. The article speaks to the robustness of viral inactivation processes currently utilized by the plasma industry and lessens concerns about the transmission of these viruses by plasma derivatives. These results are quite similar to those that have been observed with WNV that led the FDA to the conclusion that plasma destined for further manufacture does not need to be screened for WNV. What is the reason why the number of reports of TT of DENV and CHIKV are so few? There are many differences between these viruses and WNV, but they do not clearly explain the lack of TT reports for DENV and CHIKV. For instance: WNV infects a large number of birds and mammals. Birds are highly efficient amplification vectors, presenting very high levels of viremia. Many species of mosquitoes that transmit WNV bite both animals and humans. DENV amplification occurs in the salivary glands of A. egypti and A. albopictus. The mosquito transmits DENV from human to human in densely populated areas. There may be differences between WNV, DENV, and CHIKV in the number of days with asymptomatic viremia during which an infectious donation could be given and the titers of infectious virus during this period; for WNV, the period of preseroconversion viremia is 7 days but the levels of viremia are higher for DENV or CHIKV. DENV and CHIKV epidemics currently occur primarily in developing countries. Large numbers of individuals are affected simultaneously, overwhelming hospital emergency rooms, making impossible accurate anamnesis, physical examination, and appropriate reporting. The environment is not conducive to clinical studies, even observational, that could adequately document case reports, let alone estimate rates of TT. This is likely the main reason why TT infections are not recognized. In epidemic regions, blood is diverted to the many cases with DHF and DSS. Thus, many of the patients that receive blood transfusions during the height of the epidemic are already infected with dengue. Postponement of other hospital activities like elective surgeries reduces the opportunity of transmission of infection to naïve patients by transfusions. Lookback is rarely performed in developing countries because of limited resources. If TT of DENV or CHIKV is documented to occur at “significant” rates, it will be important to establish whether disease penetrance is more or less serious than disease transmission from other vector-borne viruses. This issue is of particular interest with respect to DENV, since the more serious hemorrhagic syndromes are thought to be related to high-level anamnestic immune responses. It is possible that TT dengue in immunosuppressed patients will result in no symptoms or mild disease. There are reports suggesting that dengue infections in recipients of kidney transplants are milder than dengue infections of healthy individuals.8, 9 The authors of these reports speculate that immunosuppression interferes with the patient's immune response to the virus and reduces the opportunity for development of DHF. This clinical course is the opposite of what is observed with WNV infection. Immunosuppressed individuals like the elderly and recipients of organ transplants appear to be more susceptible to meningoencephalitis than young patients. It will be important to determine the risk of serious disease associated with TT of dengue and CHIKV because TT of these viruses almost certainly represents a very small proportion of the total infections during an epidemic (as is true of WNV). The value of implementation of donor screening or other high-cost prevention measures to protect blood safety would require careful consideration taking into account prevalence of viremia in donors, transmission rates, and disease penetrance in infected recipients. The availability of a potential donor screening assay for DENV using the same platform as the assays for WNV RNA is welcome and reassuring. It will also be wise to address issues related to potential testing algorithms in future studies, including the definition of a positive specimen, the need for minipool or individual-donation NAT, the evaluation and implications of antibody status of donors, the development of confirmatory assays, and the value of determining DENV serotype. The authors of the studies performed in Honduras and in Puerto Rico have indicated that further studies will be performed in an attempt to better document TT of dengue and estimate the actual risk. The need for a DENV assay and the benefits of introduction of blood donor screening are dependent on the outcomes of these studies. Finally, we hope that public health authorities, regulatory agencies, blood banking organizations, transfusing clinicians, and patient groups continue to support development of technologies that may be useful for pathogen reduction of cellular components. Pathogen reduction is a more generic and proactive approach to the risks associated with arboviruses, precluding the need for implementation of donor screening assays. The example of clearance by plasma fractionation and inactivation procedures is remarkable and should encourage further pursuit of methods applicable to cellular components. It would address DENV, CHIKV, and other reemerging agents like yellow fever virus that is reappearing in South America both in wild monkeys and in humans, with several reported human deaths, as well as other viral agents yet to be identified.10

Dengue virus (DENV) is a continuing global threat that puts half of the world's population at risk for infection. This mosquito-transmitted virus is endemic in over 100 countries. When a mosquito takes a bloodmeal, virus is deposited into the epidermal and dermal layers of human skin, infecting a variety of permissive cells, including keratinocytes, Langerhans cells, macrophages, dermal dendritic cells, fibroblasts, and mast cells. In response to infection, the skin deploys an array of defense mechanisms to inhibit viral replication and prevent dissemination. Antimicrobial peptides, pattern recognition receptors, and cytokines induce a signaling cascade to increase transcription and translation of pro-inflammatory and antiviral genes. Paradoxically, this inflammatory environment recruits skin-resident mononuclear cells that become infected and migrate out of the skin, spreading virus throughout the host. The details of the viral-host interactions in the cutaneous microenvironment remain unclear, partly due to the limited body of research focusing on DENV in human skin. This review will summarize the functional role of human skin, the cutaneous innate immune response to DENV, the contribution of the arthropod vector, and the models used to study DENV interactions in the cutaneous environment.

Functional characterization of a serine protease inhibitor modulated in the infection of the Aedes aegypti with dengue virus

Despite numerous efforts to identify the molecular and cellular effectors of the adaptive immunity that induce a long-lasting immunity against dengue or Zika virus infection, the specific mechanisms underlying such protective immunity remain largely unknown. One of the major challenges lies in the high level of dengue virus (DENV) seroprevalence in areas where Zika virus (ZIKV) is circulating. In the context of such a pre-existing DENV immunity that can exacerbate ZIKV infection and disease, and given the lack of appropriate treatment for ZIKV infection, there is an urgent need to develop an efficient vaccine against DENV and ZIKV. Notably, whereas several ZIKV vaccine candidates are currently in clinical trials, all these vaccine candidates have been designed to induce neutralizing antibodies as the primary mechanism of immune protection. Given the difficulty to elicit simultaneously high levels of neutralizing antibodies against the different DENV serotypes, and the potential impact of pre-existing subneutralizing antibodies induced upon DENV infection or vaccination on ZIKV infection and disease, additional or alternative strategies to enhance vaccine efficacy, through T cell immunity, are now being considered. In this review, we summarize recent discoveries about cross-reactive B and T cell responses against DENV and ZIKV and propose guidelines for the development of safe and efficient T cell vaccines targeting both viruses.

RIG-I is a cytosolic sensor critically involved in the activation of the innate immune response to RNA virus infection. In the present study, we evaluated the inhibitory effect of a RIG-I agonist on the replication of two emerging arthropod-borne viral pathogens, dengue virus (DENV) and chikungunya virus (CHIKV), for which no therapeutic options currently exist. We demonstrate that when a low, noncytotoxic dose of an optimized 5'triphosphorylated RNA (5'pppRNA) molecule was administered, RIG-I stimulation generated a robust antiviral response against these two viruses. Strikingly, 5'pppRNA treatment before or after challenge with DENV or CHIKV provided protection against infection. In primary human monocytes and monocyte-derived dendritic cells, the RIG-I agonist blocked both primary infection and antibody-dependent enhancement of DENV infection. The protective response against DENV and CHIKV induced by 5'pppRNA was dependent on an intact RIG-I/MAVS/TBK1/IRF3 axis and was largely independent of the type I IFN response. Altogether, this in vitro analysis of the antiviral efficacy of 5'pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV. DENV and CHIKV are two reemerging mosquito-borne viruses for which no therapeutic options currently exist. Both viruses overlap geographically in tropical regions of the world, produce similar fever-like symptoms, and are difficult to diagnose. This study investigated the inhibitory effect of a RIG-I agonist on the replication of these two viruses. RIG-I stimulation using 5'pppRNA before or after DENV or CHIKV infection generated a protective antiviral response against both pathogens in immune and nonimmune cells; interestingly, the protective response against the viruses was largely independent of the classical type I interferon response. The antiviral efficacy of 5'pppRNA highlights the therapeutic potential of RIG-I agonists against emerging viruses such as DENV and CHIKV.

IntroductionZika virus (ZIKV) and dengue virus (DENV) co‐circulated during latest outbreaks in Brazil, hence, it is important to evaluate the host cross‐reactive immune responses to these viruses. So far, little is known about human T cell responses to ZIKV and no reports detail adaptive immune responses during DENV/ZIKV coinfection.MethodsHere, we studied T cells responses in well‐characterized groups of DENV, ZIKV, or DENV/ZIKV infected patients and DENV‐exposed healthy donors. We evaluated chemokine receptors expression and single/multifunctional frequencies of IFNγ, TNF, and IL2‐producing T cells during these infections. Even without antigenic stimulation, it was possible to detect chemokine receptors and IFNγ, TNF, and IL2‐producing T cells from all individuals by flow cytometry. Additionally, PBMCs’ IFNγ response to DENV NS1 protein and to polyclonal stimuli was evaluated by ELISPOT.ResultsDENV and ZIKV infections and DENV/ZIKV coinfections similarly induced expression of CCR5, CX3CR1, and CXCR3 on CD4 and CD8 T cells. DENV/ZIKV coinfection decreased the ability of CD4+ T cells to produce IFNγ+, TNF+, TNF + IFNγ+, and TNF + IL2+, compared to DENV and ZIKV infections. A higher magnitude of IFNγ response to DENV NS1 was found in donors with a history of dengue infection, however, a hyporesponsiveness was found in acute DENV, ZIKV, or DENV/ZIKV infected patients, even previously infected with DENV.ConclusionTherefore, we emphasize the potential impact of coinfection on the immune response from human hosts, mainly in areas where DENV and ZIKV cocirculate.

Call to Action for Dengue Vaccine Failure

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.

Dengue virus (DENV) and Zika virus (ZIKV) belong to the same viral family, the Flaviviridae. They cause recurring threats to the public health systems of tropical countries such as Brazil. The primary Brazilian vector of both viruses is the mosquito Aedes aegypti. After the mosquito ingests a blood meal from an infected person, the viruses infect and replicate in the midgut, disseminate to secondary tissues and reach the salivary gland (SG), where they are ready to be transmitted to a vertebrate host. It is thought that the intrinsic discrepancies among mosquitoes could affect their ability to deal with viral infections. This study confirms that the DENV and ZIKV infection patterns of nine Ae. aegypti field populations found in geographically separate health districts of an endemic Brazilian city vary. We analyzed the infection rate, disseminated infection, vector competence, and viral load through quantitative PCR. Mosquitoes were challenged using the membrane-feeding assay technique and were tested seven and fourteen days post-infection (early and late infection phases, respectively). The infection responses varied among the Ae. aegypti populations for both flaviviruses in the two infection phases. There was no similarity between DENV and ZIKV vector competencies or viral loads. According to the results of our study, the risk of viral transmission overtime after infection either increases or remains unaltered in ZIKV infected vectors. However, the risk may increase, decrease, or remain unaltered in DENV-infected vectors depending on the mosquito population. For both flaviviruses, the viral load persisted in the body even until the late infection phase. In contrast to DENV, the ZIKV accumulated in the SG over time in all the mosquito populations. These findings are novel and may help direct the development of control strategies to fight dengue and Zika outbreaks in endemic regions, and provide a warning about the importance of understanding mosquito responses to arboviral infections.