Natthanej Luplertlop

Biology of Zika Virus Infection in Human Skin Cells

ABSTRACT Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family, which includes dengue, West Nile, yellow fever, and Japanese encephalitis viruses, that causes a mosquito-borne disease transmitted by the Aedes genus, with recent outbreaks in the South Pacific. Here we examine the importance of human skin in the entry of ZIKV and its contribution to the induction of antiviral immune responses. We show that human dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells are permissive to the most recent ZIKV isolate, responsible for the epidemic in French Polynesia. Several entry and/or adhesion factors, including DC-SIGN, AXL, Tyro3, and, to a lesser extent, TIM-1, permitted ZIKV entry, with a major role for the TAM receptor AXL. The ZIKV permissiveness of human skin fibroblasts was confirmed by the use of a neutralizing antibody and specific RNA silencing. ZIKV induced the transcription of Toll-like receptor 3 (TLR3), RIG-I, and MDA5, as well as several interferon-stimulated genes, including OAS2, ISG15, and MX1, characterized by strongly enhanced beta interferon gene expression. ZIKV was found to be sensitive to the antiviral effects of both type I and type II interferons. Finally, infection of skin fibroblasts resulted in the formation of autophagosomes, whose presence was associated with enhanced viral replication, as shown by the use of Torin 1, a chemical inducer of autophagy, and the specific autophagy inhibitor 3-methyladenine. The results presented herein permit us to gain further insight into the biology of ZIKV and to devise strategies aiming to interfere with the pathology caused by this emerging flavivirus. IMPORTANCE Zika virus (ZIKV) is an arbovirus belonging to the Flaviviridae family. Vector-mediated transmission of ZIKV is initiated when a blood-feeding female Aedes mosquito injects the virus into the skin of its mammalian host, followed by infection of permissive cells via specific receptors. Indeed, skin immune cells, including dermal fibroblasts, epidermal keratinocytes, and immature dendritic cells, were all found to be permissive to ZIKV infection. The results also show a major role for the phosphatidylserine receptor AXL as a ZIKV entry receptor and for cellular autophagy in enhancing ZIKV replication in permissive cells. ZIKV replication leads to activation of an antiviral innate immune response and the production of type I interferons in infected cells. Taken together, these results provide the first general insights into the interaction between ZIKV and its mammalian host.

Induction of a Peptide with Activity against a Broad Spectrum of Pathogens in the Aedes aegypti Salivary Gland, following Infection with Dengue Virus

The ultimate stage of the transmission of Dengue Virus (DENV) to man is strongly dependent on crosstalk between the virus and the immune system of its vector Aedes aegypti (Ae. aegypti). Infection of the mosquitos salivary glands by DENV is the final step prior to viral transmission. Therefore, in the present study, we have determined the modulatory effects of DENV infection on the immune response in this organ by carrying out a functional genomic analysis of uninfected salivary glands and salivary glands of female Ae. aegypti mosquitoes infected with DENV. We have shown that DENV infection of salivary glands strongly up-regulates the expression of genes that encode proteins involved in the vectors innate immune response, including the immune deficiency (IMD) and Toll signalling pathways, and that it induces the expression of the gene encoding a putative anti-bacterial, cecropin-like, peptide (AAEL000598). Both the chemically synthesized non-cleaved, signal peptide-containing gene product of AAEL000598, and the cleaved, mature form, were found to exert, in addition to antibacterial activity, anti-DENV and anti-Chikungunya viral activity. However, in contrast to the mature form, the immature cecropin peptide was far more effective against Chikungunya virus (CHIKV) and, furthermore, had strong anti-parasite activity as shown by its ability to kill Leishmania spp. Results from circular dichroism analysis showed that the immature form more readily adopts a helical conformation which would help it to cause membrane permeabilization, thus permitting its transfer across hydrophobic cell surfaces, which may explain the difference in the anti-pathogenic activity between the two forms. The present study underscores not only the importance of DENV-induced cecropin in the innate immune response of Ae. aegypti, but also emphasizes the broad-spectrum anti-pathogenic activity of the immature, signal peptide-containing form of this peptide.

Dengue virus replication in infected human keratinocytes leads to activation of antiviral innate immune responses

Dengue virus (DENV) infection is the most prevalent mosquito-borne viral diseases in the world. Vector-mediated transmission of DENV is initiated when a blood-feeding female Aedes mosquito injects saliva, together with the virus, into the skin of its mammalian host. Understanding the role of skin immune cells in the activation of innate immunity to DENV at the early times of infection is a critical issue that remains to be investigated. The purpose of our study was to assess the contribution of human keratinocytes as potential host cells to DENV in the activation of immune responses at the anatomical site of mosquito bite. We show that primary keratinocytes support DENV replication with the production of negative-stranded viral RNAs inside the infected cells. In the course of DENV life cycle, we observed the activation of host genes involved in the antiviral immune responses such as intracellular RNA virus sensors Toll-Like Receptor-3, Retinoic Acid Inducible Gene-I, Melanoma Differentiation Associated gene-5 and the RNA-dependent protein kinase R. DENV infection of primary keratinocytes also resulted in up-regulation of the expression of the antiviral Ribonuclease L gene, which subsequently led to enhanced production of IFN-β and IFN-γ. Depending on stages of viral replication, we observed the activation of host genes encoding the antimicrobial proteins β-defensin and RNase 7 in infected keratinocytes. Our data demonstrate for the first time the permissiveness of human epidermal keratinocytes to DENV infection. Remarkably, DENV replication in keratinocytes contributes to the establishment of antiviral innate immunity that might occur in the early times after the bite of mosquito.

Blood-feeding and immunogenic Aedes aegypti saliva proteins.

Mosquito‐transmitted pathogens pass through the insects midgut (MG) and salivary gland (SG). What occurs in these organs in response to a blood meal is poorly understood, but identifying the physiological differences between sugar‐fed and blood‐fed (BF) mosquitoes could shed light on factors important in pathogens transmission. We compared differential protein expression in the MGs and SGs of female Aedes aegypti mosquitoes after a sugar‐ or blood‐based diet. No difference was observed in the MG protein expression levels but certain SG proteins were highly expressed only in BF mosquitoes. In sugar‐fed mosquitoes, housekeeping proteins were highly expressed (especially those related to energy metabolism) and actin was up‐regulated. The immunofluorescence assay shows that there is no disruption of the SG cytoskeletal after the blood meal. We have generated for the first time the 2‐DE profiles of immunogenic Ae. aegypti SG BF‐related proteins. These new data could contribute to the understanding of the physiological processes that appear during the blood meal.

Aedes aegypti Saliva Enhances Dengue Virus Infection of Human Keratinocytes by Suppressing Innate Immune Responses

TO THE EDITOR Dengue virus (DENV) is at present the most important emerging arbovirus. The incidence of DENV infection has grown markedly around the world in recent decades because of expanding geographic distribution of the virus and their mosquito vectors, Aedes (Ae.) aegypti and Ae. albopictus. DENV is an enveloped positive single-stranded RNA virus that belongs to the Flaviviridae family. DENV can cause dengue hemorrhagic fever and dengue shock syndrome. Vector-mediated transmission of DENV is initiated when a blood-feeding female Ae. mosquito injects saliva, together with the virus, into the skin of its mammalian host. Mosquito saliva contains a repertoire of bioactive components that modulate the host’s hemostasis and immune response, thus facilitating blood feeding and pathogen transmission (reviews in Schneider and Higgs (2008) and Ribeiro et al. (2010)). It has been demonstrated that Ae. aegypyi salivary proteins are immmunosuppressive and are associated with an increase of West Nile virus in the skin (Schneider et al., 2010). Moreover, a recent comparison of cutaneous cytokines elicited in mice by Chikungunya virus revealed a reorientation of the host’s protective TH1 to a TH2 type immune response by the presence of the Ae aegypti saliva (Thangamani et al., 2010). Taken together, these and other results obtained from studies in several experimental disease models, using various arthropod-borne viruses, demonstrate that saliva is associated with an increase in virus pathogenicity (review in Schneider and Higgs (2008)). During blood feeding, an infected mosquito transmits virus in saliva that is secreted predominantly in the extravascular regions of the uppermost layer of skin, the epidermis, which consists primarily of keratinocytes (Limon-Flores et al., 2005). Whereas dendritic cells have long been considered to be the main target of DENV (Halstead and O’Rourke, 1977), we have recently demonstrated that human keratinocytes are permissive to DENV infection as well. DENV replication in infected primary human keratinocytes was found to lead to activation of antiviral innate immune responses by activating signaling pathways that converge into secretion of antimicrobial peptides (AMPs) and IFNs (Surasombatpattana et al., 2011). Therefore, we investigated the effect of Ae. aegypti salivary gland extract (SGE) on DENV infection in human keratinocytes. Human primary keratinocytes were infected with DENV in the presence or absence of Ae. aegypti SGE obtained from a single pair of salivary glands. At 6 and 24 hours post infection (h.p.i.), intracellular viral mRNA levels were quantified by real-time PCR (Figure 1). The expression levels of DENV transcripts in the infected keratinocytes were increased in the presence of SGE, as compared with DENV infection alone, although not detected in mock-infected cells. The results furthermore show that, at 6 h.p.i., the presence of SGE during the infection of keratinocytes with DENV strongly downregulated the expression of human Abbreviations: Ae., Aedes; AMP, antimicrobial peptide; DENV, dengue virus; h.p.i., hours post infection; HSV, herpes simplex virus; SGE, salivary gland extraction; SOD2, superoxide dismutase 2

Proteomic analysis of an Aedes albopictus cell line infected with Dengue serotypes 1 and 3 viruses

BackgroundProteomic analysis was performed to identify proteins regulated during infection by Dengue serotypes 1 and 3 in an Aedes albopictus cell line. The potential of these viruses to cause severe disease at primary infection is of interest although few studies have been performed with these two Dengue serotypes.ResultsThe most relevant observation of our study is the significant overexpression of proteins involved in the cellular stress response and the glycolysis pathway after 48 hours of infection. Viral infection activates the translation of some host genes, which may result in stress due to responses involving unfolded proteins.ConclusionsTherefore, the oxidation reduction and glycolytic mechanisms could participate in the antiviral response against Dengue virus. The results of our study should help to improve our knowledge of the virus-mosquito interaction at a cellular level with the aim of designing efficient strategies for the control of Dengue virus.

Fungal quorum sensing molecules: Role in fungal morphogenesis and pathogenicity

When microorganisms live together in high numbers, they need to communicate with each other. To achieve cell–cell communication, microorganisms secrete molecules called quorum‐sensing molecules (QSMs) that control their biological activities and behaviors. Fungi secrete QSMs such as farnesol, tyrosol, phenylethanol, and tryptophol. The role of QSMs in fungi has been widely studied in both yeasts and filamentous fungi, for example in Candida albicans, C. dubliniensis, Aspergillus niger, A. nidulans, and Fusarium graminearum. QSMs impact fungal morphogenesis (yeast‐to‐hypha formation) and also play a role in the germination of macroconidia. QSMs cause fungal cells to initiate programmed cell death, or apoptosis, and play a role in fungal pathogenicity. Several types of QSMs are produced during stages of biofilm development to control cell population or morphology in biofilm communities. This review article emphasizes the role of fungal QSMs, especially in fungal morphogenesis, biofilm formation, and pathogenicity. Information about QSMs may lead to improved measures for controlling fungal infection.

In Vitro Screening of 10 Edible Thai Plants for Potential Antifungal Properties

Growing rates of fungal infections and increasing resistance against standard antifungal drugs can cause serious health problems. There is, therefore, increasing interest in the potential use of medicinal plants as novel antifungal agents. This study investigates the antifungal properties of crude plant extracts from ten medicinal plant species. Crude samples were extracted using the hot water extraction process. The minimum inhibitory concentrations (MIC) and diameter zone of inhibition were determined in each extract against ten fungal strains, and fluconazole was used as a positive control. The cytotoxicity of crude extracts on in vitro human skin fibroblast (HSF) cell models was determined by MTT assay. Of the ten crude extracts, Psidium guajava L. exhibited the highest antifungal activity, diameter zone of inhibition, and percentage HSF cell viability. Although all extracts exhibited antifungal activity, Psidium guajava L. had the greatest potential for developing antifungal treatments.

Dengue and Zika viruses: lessons learned from the similarities between these Aedes mosquito-vectored arboviruses

The currently spreading arbovirus epidemic is having a severe impact on human health worldwide. The two most common flaviviruses, dengue virus (DENV) and Zika virus (ZIKV), are transmitted through the same viral vector, Aedes spp. mosquitoes. Since the discovery of DENV in 1943, this virus has been reported to cause around 390 million human infections per year, approximately 500,000 of which require hospitalization and over 20,000 of which are lethal. The present DENV epidemic is primarily concentrated in Southeast Asia. ZIKV, which was discovered in 1952, is another important arthropod-borne flavivirus. The neurotropic role of ZIKV has been reported in infected newborns with microcephaly and in adults with Guillain-Barre syndrome. Despite DENV and ZIKV sharing the same viral vector, their complex pathogenic natures are poorly understood, and the infections they cause do not have specific treatments or effective vaccines. Therefore, this review will describe what is currently known about the clinical characteristics, pathogenesis mechanisms, and transmission of these two viruses. Better understanding of the interrelationships between DENV and ZIKV will provide a useful perspective for developing an effective strategy for controlling both viruses in the future.

Inhibition of protein kinase C promotes dengue virus replication

BackgroundDengue virus (DENV) is a member of the Flaviviridae family, transmitted to human via mosquito. DENV infection is common in tropical areas and occasionally causes life-threatening symptoms. DENV contains a relatively short positive-stranded RNA genome, which encodes ten viral proteins. Thus, the viral life cycle is necessarily rely on or regulated by host factors.MethodsIn silico analyses in conjunction with in vitro kinase assay were used to study kinases that potentially phosphorylate DENV NS5. Potential kinase was inhibited or activated by a specific inhibitor (or siRNA), or an activator. Results of the inhibition and activation on viral entry/replication and host cell survival were examined.ResultsOur in silico analyses indicated that the non-structural protein 5 (NS5), especially the RNA-dependent RNA polymerase (RdRp) domain, contains conserved phosphorylation sites for protein kinase C (PKC). Phosphorylation of NS5 RdRp was further verified by PKC in vitro kinase assay. Inhibitions of PKC by a PKC-specific chemical inhibitor or siRNA suppressed NS5 phosphorylation in vivo, increased viral replication and reduced viability of the DENV-infected cells. In contrary, activation of PKC effectively suppressed intracellular viral number.ConclusionsThese results indicated that PKC may act as a restricting mechanism that modulates the DENV replication and represses the viral outburst in the host cells.