Next-Generation Sequencing on Insectivorous Bat Guano: An Accurate Tool to Identify Arthropod Viruses of Potential Agricultural Concern.

Viruses belonging to the Dicistroviridae family have attracted a great deal of attention from scientists owing to their negative impact on agricultural economics, as well as their recent identification as potential aetiological agents of febrile illness in human patients. On the other hand, some Dicistroviruses are also studied for their potential biopesticide properties. To date, Dicistrovirus characterized in African mainland remain scarce. By using High-Throughput Sequencing technology on insectivorous bat faeces (Hipposideros Caffer) sampled in a cave used by humans to collect bat guano (bat manure) as fertilizer in Zimbabwe, we characterized the full-length sequences of three Dicistrovirus belonging to the Cripavirus and Aparavirus genus: Big Sioux River Virus-Like (BSRV-Like), Acute Bee Paralysis Virus (ABPV), and Aphid Lethal Paralysis Virus (ALPV). Phylogenetic analyses of ORF-1 and ORF-2 genes showed a complex evolutionary history between BSRV and close viruses, as well as for the Aparavirus genus. Herewith, we provide the first evidence of the presence of Dicistrovirus in Zimbabwe and highlight the need to further document the impact of such viruses on crops, as well as in beekeeping activities in Zimbabwe which represent a crucial source of income for Zimbabwean people.

Differential Susceptibility and Innate Immune Response of Aedes aegypti and Aedes albopictus to the Haitian Strain of the Mayaro Virus.

Mayaro (MAYV) is an emerging arthropod-borne virus belonging to the Alphavirus genus of the Togaviridae family. Although forest-dwelling Haemagogus mosquitoes have been considered as its main vector, the virus has also been detected in circulating Aedes ssp mosquitoes. Here we assess the susceptibility of Aedes aegypti and Aedes albopictus to infection with MAYV and their innate immune response at an early stage of infection. Aedes albopictus was more susceptible to infection with MAYV than Ae. aegypti. Analysis of transcript levels of twenty immunity-related genes by real-time PCR in the midgut of both mosquitoes infected with MAYV revealed increased expression of several immune genes, including CLIP-domain serine proteases, the anti-microbial peptides defensin A, E, cecropin E, and the virus inducible gene. The regulation of certain genes appeared to be Aedes species-dependent. Infection of Ae. aegypti with MAYV resulted in increased levels of myeloid differentiation2-related lipid recognition protein (ML26A) transcripts, as compared to Ae. albopictus. Increased expression levels of thio-ester-containing protein 22 (TEP22) and Niemann-Pick type C1 (NPC1) gene transcripts were observed in infected Ae. albopictus, but not Ae. aegypti. The differences in these gene expression levels during MAYV infection could explain the variation in susceptibility observed in both mosquito species.

Mayaro Virus Infects Human Chondrocytes and Induces the Expression of Arthritis-Related Genes Associated with Joint Degradation.

Mayaro virus (MAYV) is an emerging arthritogenic alphavirus belonging to the Togaviridae family. Infection leads to a dengue-like illness accompanied by severe polyarthralgia. However, the molecular and cellular mechanisms of arthritis as a result of MAYV infection remain poorly understood. In the present study, we assess the susceptibility of human chondrocytes (HC), fibroblast-like synoviocytes and osteoblasts that are the major cell types involved in osteoarthritis, to infection with MAYV. We show that these cells are highly permissive to MAYV infection and that viral RNA copy number and viral titers increase over time in infected cells. Knowing that HC are the primary cells in articular cartilage and are essential for maintaining the cartilaginous matrix, gene expression studies were conducted in MAYV-infected primary HC using polymerase chain reaction (PCR) arrays. The infection of the latter cells resulted in an induction in the expression of several matrix metalloproteinases (MMP) including MMP1, MMP7, MMP8, MMP10, MMP13, MMP14 and MMP15 which could be involved in the destruction of articular cartilage. Infected HC were also found to express significantly increased levels of various IFN-stimulated genes and arthritogenic mediators such as TNF-α and IL-6. In conclusion, MAYV-infected primary HC overexpress arthritis-related genes, which may contribute to joint degradation and pathogenesis.

Zika virus differentially infects human neural progenitor cells according to their state of differentiation and dysregulates neurogenesis through the Notch pathway.

Zika virus (ZIKV) is a mosquito-borne Flavivirus that causes Zika disease with particular neurological complications, including Guillain-Barré Syndrome and congenital microcephaly. Although ZIKV has been shown to directly infect human neural progenitor cells (hNPCs), thereby decreasing their viability and growth, it is as yet unknown which of the cellular pathways involved in the disruption of neurogenesis are affected following ZIKV infection. By comparing the effect of two ZIKV strains in vitro on hNPCs, the differentiation process of the latter cells was found to lead to a decreased susceptibility to infection and cell death induced by each of the ZIKV strains, which was associated with an earlier and stronger antiviral innate immune response in infected, differentiated hNPCs, as compared to undifferentiated cells. Moreover, ZIKV modulated, both in hNPCs and in vivo in fetal brain in an experimental mouse model, the expression of the Notch pathway which is involved in cellular proliferation, apoptosis and differentiation during neurogenesis. These results show that the differentiation state of hNPCs is a significant factor contributing to the outcome of ZIKV infection and furthermore suggest that ZIKV infection might initiate early activation of the Notch pathway resulting in an abnormal differentiation process, implicated in ZIKV-induced brain injury.

SAMHD1 Enhances Chikungunya and Zika Virus Replication in Human Skin Fibroblasts.

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are emerging arboviruses that pose a worldwide threat to human health. Currently, neither vaccine nor antiviral treatment to control their infections is available. As the skin is a major viral entry site for arboviruses in the human host, we determined the global proteomic profile of CHIKV and ZIKV infections in human skin fibroblasts using Stable Isotope Labelling by Amino acids in Cell culture (SILAC)-based mass-spectrometry analysis. We show that the expression of the interferon-stimulated proteins MX1, IFIT1, IFIT3 and ISG15, as well as expression of defense response proteins DDX58, STAT1, OAS3, EIF2AK2 and SAMHD1 was significantly up-regulated in these cells upon infection with either virus. Exogenous expression of IFITs proteins markedly inhibited CHIKV and ZIKV replication which, accordingly, was restored following the abrogation of IFIT1 or IFIT3. Overexpression of SAMHD1 in cutaneous cells, or pretreatment of cells with the virus-like particles containing SAMHD1 restriction factor Vpx, resulted in a strong increase or inhibition, respectively, of both CHIKV and ZIKV replication. Moreover, silencing of SAMHD1 by specific SAMHD1-siRNA resulted in a marked decrease of viral RNA levels. Together, these results suggest that IFITs are involved in the restriction of replication of CHIKV and ZIKV and provide, as yet unreported, evidence for a proviral role of SAMHD1 in arbovirus infection of human skin cells.

Zika virus infection modulates the metabolomic profile of microglial cells.

Zika virus (ZIKV) is an emerging arbovirus of the Flaviviridae family. Although infection with ZIKV generally leads to mild disease, its recent emergence in the Americas has been associated with an increase in the development of the Guillain-Barré syndrome in adults, as well as with neurological complications, in particular congenital microcephaly, in new-borns. To date, little information is available on neuroinflammation induced by ZIKV, notably in microglial cells in the context of their metabolic activity, a series of chemical transformations that are essential for their growth, reproduction, structural maintenance and environmental responses. Therefore, in the present study we investigated the metabolomic profile of ZIKV-infected microglia. Microglial cells were exposed to ZIKV at different time points and were analyzed by a Liquid Chromatography-High Resolution mass spectrometry-based metabolomic approach. The results show that ZIKV infection in microglia leads to modulation of the expression of numerous metabolites, including lysophospholipids, particulary Lysophosphatidylcholine, and phospholipids such as Phosphatidylcholine, Phosphatidylserine, Ceramide and Sphingomyelin, and carboxylicic acids as Undecanedioic and Dodecanedioic acid. Some of these metabolites are involved in neuronal differentiation, regulation of apoptosis, virion architecture and viral replication. ZIKV infection was associated with concomitant secretion of inflammatory mediators linked with central nervous system inflammation such as IL-6, TNF-α, IL-1ß, iNOS and NO. It also resulted in the upregulation of the expression of the gene encoding CX3CR1, a chemokine receptor known to regulate functional synapse plasticity and signaling between microglial cells. These findings highlight an important role for microglia and their metabolites in the process of neuroinflammation that occurs during ZIKV pathogenesis.

Circulation of Alphacoronavirus, Betacoronavirus and Paramyxovirus in Hipposideros bat species in Zimbabwe.

Bats carry a great diversity of zoonotic viruses with a high-impact on human health and livestock. Since the emergence of new coronaviruses and paramyxoviruses in humans (e.g. Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Nipah virus), numerous studies clearly established that bats can maintain some of these viruses. Improving our understanding on the role of bats in the epidemiology of the pathogens they harbour is necessary to prevent cross-species spill over along the wild/domestic/human gradient. In this study, we screened bat faecal samples for the presence of Coronavirus and Paramyxovirus in two caves frequently visited by local people to collect manure and/or to hunt bats in Zimbabwe. We amplified partial RNA-dependent RNA polymerase genes of Alpha and Betacoronavirus together with the partial polymerase gene of Paramyxovirus. Identified coronaviruses were related to pathogenic human strains and the paramyxovirus belonged to the recently described Jeilongvirus genus. Our results highlighted the importance of monitoring virus circulation in wildlife, especially bats, in the context of intense human-wildlife interfaces in order to strengthen prevention measures among local populations and to implement sentinel surveillance in sites with high zoonotic diseases transmission potential.

Aedes Aegypti saliva enhances chikungunya virus replication in human skin fibroblasts via inhibition of the type I interferon signaling pathway.

Chikungunya virus (CHIKV) transmission occurs through the bite of an infected Aedes mosquito which injects virus-containing saliva into the skin of the human host during blood feeding. In the present study, we have determined the effect of Aedes aegypti saliva on CHIKV replication in human skin fibroblasts, a major cell type for viral entry, which mimics the events that occur during natural transmission. A significant increase in the expression of viral transcripts and infectious viral particles was observed in fibroblasts infected with CHIKV in the presence of saliva, as compared with those infected with virus alone. CHIKV-infected human fibroblasts were found to express significantly increased levels of various type I IFN-responsive genes, as demonstrated by specific PCR array analysis. In contrast, the expression of these genes was markedly decreased in cells infected with CHIKV in the presence of mosquito saliva. Moreover, Western blotting analysis revealed that STAT2 and its phosphorylated form were down-regulated in the presence of mosquito saliva. Our data demonstrate for the first time the significance of Aedes aegypti saliva in promoting CHIKV infection via down-regulation of several type I IFN-responsive genes in infected human skin fibroblasts via the JAK-STAT signaling pathway.

Imipramine Inhibits Chikungunya Virus Replication in Human Skin Fibroblasts through Interference with Intracellular Cholesterol Trafficking.

Chikungunya virus (CHIKV) is an emerging arbovirus of the Togaviridae family that poses a present worldwide threat to human in the absence of any licensed vaccine or antiviral treatment to control viral infection. Here, we show that compounds interfering with intracellular cholesterol transport have the capacity to inhibit CHIKV replication in human skin fibroblasts, a major viral entry site in the human host. Pretreatment of these cells with the class II cationic amphiphilic compound U18666A, or treatment with the FDA-approved antidepressant drug imipramine resulted in a near total inhibition of viral replication and production at the highest concentration used without any cytotoxic effects. Imipramine was found to affect both the fusion and replication steps of the viral life cycle. The key contribution of cholesterol availability to the CHIKV life cycle was validated further by the use of fibroblasts from Niemann-Pick type C (NPC) patients in which the virus was unable to replicate. Interestingly, imipramine also strongly inhibited the replication of several Flaviviridae family members, including Zika, West Nile and Dengue virus. Together, these data show that this compound is a potential drug candidate for anti-arboviral treatment.

African and Asian Zika virus strains differentially induce early antiviral responses in primary human astrocytes.

ZIKA virus (ZIKV) is a newly emerging arbovirus. Since its discovery 60years ago in Uganda, it has spread throughout the Pacific, Latin America and the Caribbean, emphasizing the capacity of ZIKV to spread to non-endemic regions worldwide. Although infection with ZIKV often leads to mild disease, its recent emergence in the Americas has coincided with an increase in adults developing Guillain-Barré syndrome and neurological complications in new-borns, such as congenital microcephaly. Many questions remain unanswered regarding the complications caused by different primary isolates of ZIKV. Here, we report the permissiveness of primary human astrocytes for two clinically relevant, Asian and African ZIKV strains and show that both isolates strongly induce antiviral immune responses in these cells albeit with markedly different kinetics. This study describes for the first time the specific antiviral gene expression in infected primary human astrocytes, the major glial cells within the central nervous system.

The Montpellier Leishmania Collection, from a Laboratory Collection to a Biological Resource Center: A 39-Year-Long Story.

We report the development of a laboratory collection of Leishmania that was initiated in 1975 and, after 39 years, has become an international Biological Resource Center (BRC-Leish, Montpellier, France, BioBank No. BB-0033-00052), which includes 6353 strains belonging to 36 Leishmania taxa. This is a retrospective analysis of the technical and organizational changes that have been adopted over time to take into account the technological advances and related modifications in the collection management and quality system. The technical improvements concerned the culture and cryopreservation techniques, strain identification by isoenzymatic and molecular techniques, data computerization and quality management to meet the changes in international standards, and in the cryogenic and microbiological safety procedures. The BRC is working toward obtaining the NF-S 96-900 certification in the coming years. Our long-term expertise in Leishmania storage and typing and collection maintenance should encourage field epidemiologists and clinical practitioners in endemic countries to secure their own strain collection with the help of the French BRC-Leish.

Zika virus: epidemiology, clinical features and host-virus interactions.

Very recently, Zika virus (ZIKV) has gained a medical importance following the large-scale epidemics in South Pacific and Latin America. This paper reviews information on the epidemiology and clinical features of Zika disease with a particular emphasis on the host-virus interactions that contribute to the pathogenicity of ZIKV in humans.

Comparison of Leishmania killicki (syn. L. tropica) and Leishmania tropica Population Structure in Maghreb by Microsatellite Typing.

Leishmania (L.) killicki (syn. L. tropica), which causes cutaneous leishmaniasis in Maghreb, was recently described in this region and identified as a subpopulation of L. tropica. The present genetic analysis was conducted to explore the spatio-temporal distribution of L. killicki (syn. L. tropica) and its transmission dynamics. To better understand the evolution of this parasite, its population structure was then compared with that of L. tropica populations from Morocco. In total 198 samples including 85 L. killicki (syn. L. tropica) (from Tunisia, Algeria and Libya) and 113 L. tropica specimens (all from Morocco) were tested. Theses samples were composed of 168 Leishmania strains isolated from human skin lesions, 27 DNA samples from human skin lesion biopsies, two DNA samples from Ctenodactylus gundi bone marrow and one DNA sample from a Phlebotomus sergenti female. The sample was analyzed by using MultiLocus Enzyme Electrophoresis (MLEE) and MultiLocus Microsatellite Typing (MLMT) approaches. Analysis of the MLMT data support the hypothesis that L. killicki (syn. L. tropica) belongs to the L. tropica complex, despite its strong genetic differentiation, and that it emerged from this taxon by a founder effect. Moreover, it revealed a strong structuring in L. killicki (syn. L. tropica) between Tunisia and Algeria and within the different Tunisian regions, suggesting low dispersion of L. killicki (syn. L. tropica) in space and time. Comparison of the L. tropica (exclusively from Morocco) and L. killicki (syn. L. tropica) population structures revealed distinct genetic organizations, reflecting different epidemiological cycles.

Biology of Zika Virus Infection in Human Skin Cells.

UNLABELLED: 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.

Evolutionary history of Leishmania killicki (synonymous Leishmania tropica) and taxonomic implications.

BACKGROUND: The taxonomic status of Leishmania (L.) killicki, a parasite that causes chronic cutaneous leishmaniasis, is not well defined yet. Indeed, some researchers suggested that this taxon could be included in the L. tropica complex, whereas others considered it as a distinct phylogenetic complex. To try to solve this taxonomic issue we carried out a detailed study on the evolutionary history of L. killicki relative to L. tropica. METHODS: Thirty-five L. killicki and 25 L. tropica strains isolated from humans and originating from several countries were characterized using the MultiLocus Enzyme Electrophoresis (MLEE) and the MultiLocus Sequence Typing (MLST) approaches. RESULTS: The results of the genetic and phylogenetic analyses strongly support the hypothesis that L. killicki belongs to the L. tropica complex. Our data suggest that L. killicki emerged from a single founder event and that it evolved independently from L. tropica. However, they do not validate the hypothesis that L. killicki is a distinct complex. Therefore, we suggest naming this taxon L. killicki (synonymous L. tropica) until further epidemiological and phylogenetic studies justify the L. killicki denomination. CONCLUSIONS: This study provides taxonomic and phylogenetic information on L. killicki and improves our knowledge on the evolutionary history of this taxon.

Inflammasome signaling pathways exert antiviral effect against Chikungunya virus in human dermal fibroblasts.

Arboviruses represent an emerging threat to human. They are transmitted to vertebrates by the bite of infected arthropods. Early transmission to vertebrates is initiated by skin puncture and deposition of virus in this organ. However, events at the bite site remain largely unknown. Here, we report that Chikungunya virus (CHIKV) and West Nile virus (WNV), despite belonging to distinct viral families, elicit a common antiviral signature in primary human dermal fibroblasts, attesting for the up regulation of interferon signaling pathways and leading to an increased expression of IFN-ß, interleukins and chemokines. Remarkably, CHIKV and WNV enhance IL-1ß expression and induce maturation of caspase-1, indicating the capacity of these pathogens to elicit activation of the inflammasome program in resident skin cells. CHIKV and WNV also induce the expression of the inflammasome sensor AIM2 in dermal fibroblasts, whereas inhibition of caspase-1 and AIM2 with siRNA interferes with both CHIKV- and WNV-induced IL-1ß production by these cells. Finally, inhibition of the inflammasome via caspase-1 silencing was found to enhance CHIKV replication in dermal fibroblasts. Together, these results indicate that the skin contributes to the pro-inflammatory and anti-viral microenvironment via the activation of the inflammasome in the early stages following infection with arboviruses.