Sineewanlaya Wichit

Fode Diop

Rodolphe Hamel

Loïc Talignani

Pauline Ferraris

Sylvie Cornelie

Florian Liegeois

Frédéric Thomas

Hans Yssel

Dorothée Missé

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

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Aedes Aegypti saliva enhances chikungunya virus replication in human skin fibroblasts via inhibition of the type I interferon signaling pathway Sineewanlaya Wichit a , Fodé Diop a , Rodolphe Hamel a , Loïc Talignani a , Pauline Ferraris a , Sylvie Cornelie a , Florian Liegeois a , Frédéric Thomas a , Hans Yssel b , Dorothée Missé a,⁎ a Laboratoire MIVEGEC, UMR 224 IRD/CNRS/UM1, Montpellier, France b Centre d'Immunologie et des Maladies Infectieuses, Inserm, U1135, Sorbonne Universités, UPMC, APHP Hôpital Pitié-Salpêtrière, Paris, France A B S T R A C T 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.

Chikungunya virus (CHIKV), a mosquito-borne alphavirus, has caused large outbreaks of disease throughout Asia, Africa and several islands in the Indian Ocean with recent epidemics in the Americas [START_REF] Gallian | Epidemiology of Chikungunya virus outbreaks in Guadeloupe and Martinique, 2014: an observational study in volunteer blood donors[END_REF]. CHIKV is the agent of an acute febrile illness characterized by myalgia, rash, severe joint pain and, often debilitating, complications that can persist for years [START_REF] Oviedo-Pastrana | Epidemic outbreak of Chikungunya in two neighboring towns in the Colombian Caribbean: a survival analysis[END_REF]. Transmission of CHIKV occurs through the bite of an infected Aedes (Ae.) mosquito when the virus is injected, together with mosquito saliva, into the skin of the human host. The mosquito salivary glands secrete various pharmacologically active molecules that contribute to successful blood feeding by inhibiting host hemostasis, inflammation and immune responses [START_REF] Wichit | The effects of mosquito saliva on dengue virus infectivity in humans[END_REF]. In addition, the presence of saliva during CHIKV infection has been reported to significantly suppress the expression of various inflammatory genes and the production of chemokines [START_REF] Agarwal | Mosquito saliva induced cutaneous events augment Chikungunya virus replication and disease progression[END_REF].

Human dermal fibroblasts have been reported to be susceptible to CHIKV replication in vitro [START_REF] Sourisseau | Characterization of reemerging Chikungunya virus[END_REF] and we have recently demonstrated that these cells contained the highest concentration of CHIKV antigens following viral infection via the skin [START_REF] Ekchariyawat | Inflammasome signaling pathways exert antiviral effect against Chikungunya virus in human dermal fibroblasts[END_REF]. In the present study, we have determined the effect of Ae. aegypti saliva on CHIKV replication in human skin fibroblast cell line which better mimics the events that occur during natural transmission. To this end, the latter cells were infected with CHIKV at a multiplicity of infection of 1 in the absence or presence of Ae. aegypti saliva from 3 mosquitoes (1 μg/ml) which was maintained throughout the infection. Ae. aegypti saliva had no effect on the viability of the cells (Supplementary Fig. 1). At 48 h post infection a significant increase of > 2 logs in the expression of viral transcripts, measured by real-time quantitative PCR, was observed in fibroblasts infected with CHIKV in the presence of saliva, as compared with that in cells infected with virus alone (Fig. 1A). Moreover, results from a plaque assay confirmed the strong induction in the release of infectious CHIKV particles which increased in a time-dependent manner (Fig. 1B). These data support previous observations showing that Ae. aegypti saliva has an important role in the inhibition of an antiviral immune response by the host by creating an environment that favors the replication of CHIKV [START_REF] Agarwal | Mosquito saliva induced cutaneous events augment Chikungunya virus replication and disease progression[END_REF].

Type I interferons (IFNs) are known to combat viruses during viral infections and it has been well-established that CHIKV infection elicits a type I IFN response alongside the production of other pro-inflammatory MARK cytokines [START_REF] Lum | Cellular and molecular mechanisms of Chikungunya pathogenesis[END_REF]. In the present study, CHIKV-infected human fibroblasts were found to express significantly increased levels of many type I IFN-responsive genes, as demonstrated by specific PCR array analysis. In contrast however, these genes were markedly decreased in cells infected with CHIKV in the presence of mosquito saliva (Fig. 2A). The expression of the interferon-inducible (IFI) genes IFI6, IFI16, IFI27, IFI30 and IFIH1 (MDA5), as well as STAT2, TNFSF10 (TRAIL) and PRKRA, were further validated by real-time quantitative PCR (Fig. 2B). In contrast, and used as a negative control, expression levels of the PRKRA did not change under either experimental condition in accordance with the microarray data. Moreover, immunoblotting analysis revealed that STAT2 and its phosphorylation form were downregulated in the presence of mosquito saliva (Fig. 2C). Since, mosquito saliva can down-regulate these genes expression (Supplementary Fig. 2), it is therefore reasonable to hypothesize that mosquito saliva directly affects the host immune response, at least in part, via JAK-STAT signaling pathway.

Following CHIKV infection, viral RNA is recognized by cytosolic RNA sensors that trigger the expression of various pro-inflammatory genes, resulting in an anti-viral type I IFN response. In particular, MDA5 efficiently detects dsRNAs which leads to activation of the IRF3-dependent signaling pathways and, ultimately, type I IFN production. Consequently, triggering of the canonical pathway of IFN type I signaling through JAK-STAT activation results in the assembly of the ISGF3 complex that translocates to the nucleus to activate gene transcription [START_REF] Schwartz | Biology and pathogenesis of Chikungunya virus[END_REF]. These concerted activation pathways converge to restrict CHIKV replication. Support for this notion is provided by the recent observation that alphavirus-infected cells were unable to induce a type I IFN response in the absence of MDA5 [START_REF] Akhrymuk | Both RIG-I and MDA5 detect alphavirus replication in concentration-dependent mode[END_REF]. Moreover, the enhanced TRAIL expression in CHIKV-infected fibroblasts is reminiscent of published results showing that triggering of the STAT1 signaling cascade by HIV-1 Vpr also enhanced expression of this molecule, which would presumably help to eliminate HIV-1-infected cells through TRAIL-mediated cell death [START_REF] Zahoor | HIV-1 Vpr induces interferon-stimulated genes in human monocyte-derived macrophages[END_REF]. Therefore, the decrease in the expression of these molecules by mosquito saliva is aimed to impair the host immune responses which, as a consequence, favors viral replication.

As a typical adaptive immune response does not develop immediately following infection, the innate immune system seems unable to control the early phase of CHIKV infection. Concordantly, in this study, most of down-regulated genes in skin cells challenged with saliva belong to those of innate immune response. In particular, IFI6, IFI16, IFI27 and IFI30 expression was significantly decreased in CHIKV-infected skin cells in the presence of saliva. As shown previously, increased IFI6 expression levels are associated with a strong decrease in yellow fever, Dengue type 2, as well as West Nile, virus infection and IFI6 was found to also limit HCV entry and replication in human hepatoma cells [START_REF] Meyer | Interferon-α inducible protein 6 impairs EGFR activation by CD81 and inhibits hepatitis C virus infection[END_REF]. It is of note that IFI6 and IFI27 are two related proteins belonging to the FAM14 family on the basis of sequence similarity and that are commonly and concomitantly induced by IFNs. However, unlike IFI6, the latter molecule is not known to be involved in the modulation of virus infection. Another IFI gene induced in CHIKV infected skin cells, IFI16, reportedly acts as a restriction factor for several other DNA viruses [START_REF] Orzalli | cGAS-mediated stabilization of IFI16 promotes innate signaling during herpes simplex virus infection[END_REF], whereas it also induces anti-viral inflammasome activity against KSHV and EBV [START_REF] Roy | Nuclear innate immune DNA sensor IFI16 is degraded during lytic reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV): role of IFI16 in maintenance of KSHV latency[END_REF].

To our knowledge, this is the first study showing the importance of Ae. aegypti saliva in promoting CHIKV infection via downregulation of the expression of several type I IFN-responsive genes in infected human skin fibroblasts involving at least in part the JAK-STAT signaling pathway.

Fig. 1 .

 1 Fig. 1. Ae. aegypti saliva enhances CHIKV replication in human skin fibroblasts. Cells were infected with CHIKV LR2006_OPY1 strain at a multiplicity of infection of 1 in the presence or absence of Ae. aegypti saliva. After 6, 24 and 48 h, intracellular viral RNA and infectious virus production were quantified by (A) quantitative real-time PCR and (B) plaque assay, respectively. Data are representative of three independent experiments, each performed in duplicate ± standard deviation of the mean. The Student's t-test was used to analyze the differences between sets of data. A value of p < 0.01 was considered significant. ** p-values < 0.01.

Fig. 2 .

 2 Fig. 2. Expression of genes involved in the regulation of the human type I IFN response by CHIKV in human skin fibroblasts. Cells were exposed to CHIKV in the presence or absence of saliva. (A) The expression of significantly up-or down-regulated genes involved in the human type I IFN response was measured by PCR array (PAHS-016Z; SABiosciences, Frederick, MD). The fold changes of gene expression were colorcoded from blue to red (showing downregulation to up-regulation) (B) mRNA levels of the indicated gene were validated by qRT-PCR. Results are expressed as fold change of transcripts in CHIKV-infected cells in the presence or absence of saliva, relative to those in mock-infected cells (Control). (C) Mock and infected cells were lysed and analyzed by immunoblotting against STAT2, phospho-STAT2 (Tyr690) and β-actin. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Acknowledgments

This work was supported by grants from the Agence Nationale de la Recherche (grants ANR-12-BSV3-0004-01 and ANR-14-CE14-0029). Sineewanlaya Wichit was supported by a fellowship from the Infectiopôle Sud foundation.

Appendix A. Supplementary data

Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.meegid.2017.08.032.


Rodolphe Hamel

Florian Liegeois

Sineewanlaya Wichit

Julien Pompon

Fode Diop

Loïc Talignani

Frédéric Thomas

Philippe Desprès

Hans Yssel

Dorothée Missé
email: dorothee.misse@ird.fr

Florian Li

Fod E Diop

Ed Eric Thomas

Philippe Despr

Doroth Ee Miss

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

Keywords: ZIKA, Arbovirus, Innate immunity, Epidemiology, Host-pathogen interactions, Vector

niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Zika virus (ZIKV) is a little known arbovirus that was initially identified in Uganda in 1947 in a rhesus monkey used as a sentinel during sylvatic yellow fever surveillance in the Zika forest in Uganda [START_REF] Dick | I. Isolations and serological specificity[END_REF]. The virus was first reported in humans in Uganda and in Tanzania in 1952 [START_REF] Dick | II. Pathogenicity and physical properties[END_REF]. Before 2007, ZIKV was reported as causing only sporadic human infections in tropical Africa and in some areas in Southeast Asia. Since 2007, several outbreaks have been documented across the Pacific Islands showing the viral circulation outside its previously known geographic region. Autochthonous transmission of ZIKV in South America was reported in early 2015 [START_REF] Campos | Zika virus outbreak, Bahia, Brazil[END_REF][START_REF] Zanluca | First report of autochthonous transmission of Zika virus in Brazil[END_REF]. Since the first report of Zika fever on the African continent until mid-2000, few scientific articles, mainly reporting human cases and describing potential vectors of ZIKV, have been published and, due to the absence of epidemic episodes, as well as serious clinical consequences of viral infection, the interest of the scientific community remained low. However, in 2007 the first large outbreak occurred on the island of Yap, in Micronesia, resulting in a drastic change in the outlook on the ZIKV infection and a growing interest for this newcomer to the world of arboviruses (Fig. 1). Until recently, the clinical manifestations of ZIKV infection ranged from asymptomatic infections to mild, self-limited febrile illness, similar to that of a mild dengue-like syndrome, characterized by fever, headache, muscle and joint pains, as well as a characteristic maculopapular rash reminiscent to measles. Moreover, the disease occurred mainly within a narrow equatorial belt from Africa to Asia. However, an association with neurological complications such as Guillain-Barre Syndrome and congenital microcephaly has been recently suspected [START_REF] Oehler | Zika virus infection complicated by Guillain-Barre syndrome -case report, French Polynesia[END_REF][START_REF] Oliveira Melo | Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg?[END_REF], in particular following spreading of the virus in the Americas where the vectors are present. Given the rapid worldwide spread of ZIKV and the current pandemic in Latin America and the Caribbean, it is now considered as an emerging infectious disease.

To date, the biology of ZIKV infection remains still poorly understood, as compared with other emerging arboviruses such as Yellow fever, Dengue (DENV), West Nile (WNV), Japanese encephalitis (JEV) and Chikungunya (CHIKV) viruses. Here, we review recent progress on the epidemiology and the cell biology of infection by ZIKV.

Zika virus

Zika lineages

The zoonotic ZIKV is a single-stranded positive RNA virus belonging to the flavivirus genus of Flaviviridae family. The medically important mosquito-borne flaviviruses are DENV, WNV, JEV, yellow fever virus, tick-borne encephalitis and now ZIKV [START_REF] Faye | Molecular evolution of Zika virus during its emergence in the 20(th) century[END_REF]. Phylogenetic analysis and genomic comparison of ZIKV have revealed two major lineages, Asian and African [START_REF] Haddow | Genetic characterization of Zika virus strains: geographic expansion of the Asian lineage[END_REF][START_REF] Cao-Lormeau | French Polynesia, South pacific[END_REF] (Fig. 2 A, B andC). The finding that the African lineage is subdivided in two clades suggesting two different introductions [START_REF] Faye | Molecular evolution of Zika virus during its emergence in the 20(th) century[END_REF].

Faye et al. proposed the geographical spreading of ZIKV from the MR766 original Uganda strain and referred to two independent ZIKV introductions from East to West Africa [START_REF] Faye | Molecular evolution of Zika virus during its emergence in the 20(th) century[END_REF]. In addition, it is possible that the Malaysian strain has originated from Uganda ZIKV strain in the middle of the 20th century and that the Asian lineage originates from this initial Malaysian strain.

Serological evidence of ZIKV circulation in Borneo, India, Indonesia, Malaysia, Thailand and Vietnam was reported in the early 1950's [START_REF] Haddow | Genetic characterization of Zika virus strains: geographic expansion of the Asian lineage[END_REF]. According to Faye et al., whether the Asian lineage originated from an African strain the question arises of the rapid spread in Asia. However, other hypotheses could be taken into account: i/ZIKV was introduced in Asia in the middle of 20th, ii/both ZIKV lineages have had a parallel evolution from an ancient common ancestor.

The few ZIKV strains sequenced thus far has limited phylogenetic investigation. Little is know about the relationships between African and Asian lineages and further retrospective investigation have to be done to improve the knowledge on the genetic history of ZIKV. A deep analysis of the differences between the two lineages as a result of possible mutations could help to understand the observed neuronal complications. In addition, the question concerning the natural host of the virus remains unresolved. Some studies highlight serological evidence in monkeys and other mammals [START_REF] Darwish | A seroepidemiological survey for Bunyaviridae and certain other arboviruses in Pakistan[END_REF]. Nonetheless except in two cercopithecus monkey species, no ZIKV have been successfully isolated from African and Asian wild life [START_REF] Haddow | Genetic characterization of Zika virus strains: geographic expansion of the Asian lineage[END_REF].

Epidemiology

Vector

After the first isolation of ZIKV from Aedes (Ae.) africanus in 1948, it was detected, mainly in sylvatic, Aedes genus mosquitoes, including Aedes furcifer, luteocephalus, vitattus and. apicoargenteus [START_REF] Dick | I. Isolations and serological specificity[END_REF][START_REF] Haddow | Genetic characterization of Zika virus strains: geographic expansion of the Asian lineage[END_REF][START_REF] Hayes | Zika virus outside Africa[END_REF]. In several countries, ZIKV has been isolated from the antropophilic Aedes aegypti mosquitoes [START_REF] Li | Oral susceptibility of Singapore Aedes (Stegomyia) aegypti (Linnaeus) to zZika virus[END_REF], the major vector of DENV and CHIKV around the world [START_REF] Diallo | Vectors of Chikungunya virus in Senegal: current data and transmission cycles[END_REF], which is considered to be the main vector of the ZIKV in South and South-East Asia [START_REF] Li | Oral susceptibility of Singapore Aedes (Stegomyia) aegypti (Linnaeus) to zZika virus[END_REF]. During the two major outbreaks in the Pacific Islands, Aedes hensilli and Aedes polynesiensis were also identified as potential vectors of ZIKV [START_REF] Duffy | Lanciotti Zika virus outbreak on Yap Island, Federated States Micronesia[END_REF]. More importantly, however, another major DENV and CHIKV vector Aedes albopictus, was recently found to be infected by ZIKV [START_REF] Grard | Zika virus in Gabon (Central Africa) -2007: a new threat from Aedes albopictus?[END_REF]. The competence of Ae. aegypti and Ae. albopictus to transmit ZIKV has been demonstrated in two studies carried out at in interval of nearly 50 years [START_REF] Wong | Aedes (Stegomyia) albopictus (Skuse): a potential vector of Zika virus in Singapore[END_REF]. The results of both studies pointed to a short incubation period, ranging from 7 to 10 days, during which the virus replicates in Fig. 1. Publication related to ZIKV in PubMed database. Articles published between 1950 and January 2016. Recording of publication was done using the term "Zika virus" and are reported by 10-year periods. the mosquitoes' midgut, and then spreads toward the salivary glands which is associated with high levels of transmission. Nevertheless, the superiority of either vector over the other in its capacity to optimally transmit ZIKV has yet to be determined.

Although the virus reservoir is not yet clearly identified, some authors have suggested that ZIKV might be maintained in nature either by a sylvatic cycle, involving non-human primates, or in a broad range of Aedes mosquito's species [START_REF] Grard | Genomics and evolution of Aedes-borne flaviviruses[END_REF][START_REF] Wolfe | Sylvatic transmission of arboviruses among Bornean orangutans[END_REF]. In contrast, in urban areas, the transmission would be assured by the antropophilic mosquitoes Ae. aegypti and Ae. albopictus [START_REF] Li | Oral susceptibility of Singapore Aedes (Stegomyia) aegypti (Linnaeus) to zZika virus[END_REF][START_REF] Wolfe | Sylvatic transmission of arboviruses among Bornean orangutans[END_REF]. The latter one is particulary worrisome because of its diurnal feeding and their habit to bite multiple hosts during the development cycle of their eggs which makes them very efficient at transmitting diseases. However, serological studies have demonstrated the presence of specific antiviral antibodies in various mammals, among which rodents, elephants and felines, which suggests that other reservoirs may play a role in the transmission cycle of the virus [START_REF] Haddow | Genetic characterization of Zika virus strains: geographic expansion of the Asian lineage[END_REF][START_REF] Darwish | A seroepidemiological survey for Bunyaviridae and certain other arboviruses in Pakistan[END_REF]. It should be noted that non-human primate populations are absent in many Pacific islands where the virus has been detected and the nature of the viral reservoir in this area therefore remains speculative. There is an urgent need to identify vectors and potential vectors of ZIKV in vulnerable area in order to control disease outbreaks.

Geographic distribution

Following the first isolation of ZIKV from a human in Uganda in 1952 [START_REF] Simpson | Zika virus infection in man[END_REF], sporadic cases of human infection were detected in Africa [START_REF] Macnamara | Zika virus: a report on three cases of human infection during an epidemic of jaundice in Nigeria[END_REF][START_REF] Monlun | Surveillance of the circulation of arbovirus of medical interest in the region of eastern Senegal[END_REF] and later in South East Asia [START_REF] Heang | Zika virus infection, Cambodia[END_REF] (Fig. 3). Subsequently, serological, virological and entomological studies on non-human primates, human and mosquitoes pointed to a more extensive distribution in Africa, in particular in Gabon, Ivory Cost, Burkina Faso, Sierra Leone, Cameroun, Ethiopia, Kenya, Tanzania, Somalia and Egypt, as well as in Asian countries, including Malaysia, India, the Philippines, Thailand, Vietnam, Indonesia, and Pakistan [START_REF] Darwish | A seroepidemiological survey for Bunyaviridae and certain other arboviruses in Pakistan[END_REF][START_REF] Simpson | Zika virus infection in man[END_REF]. Although ZIKV was discovered more than 60 years ago, only 14 human cases were reported before 2007 after which it emerged during fours major outbreaks in Yap Island, French Polynesia, New Caledonia and, very recently, in Brazil (Fig. 3).

The first marked outbreak was reported on Yap Island, Federated States of Micronesia, in the Pacific Ocean [START_REF] Duffy | Lanciotti Zika virus outbreak on Yap Island, Federated States Micronesia[END_REF], a little island with approximately 7000 residents. Results from a seroprevalence study estimated that 73% of the population was infected with ZIKV within 4 months, with about 80% of asymptomatic patients [START_REF] Lanciotti | Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia[END_REF], highlighting for the first time the infectious capacities of the virus and marking the starting point of its spreading across the Pacific area.

During the winter of 2013e2014, a wider outbreak developed in French Polynesian Islands affecting about 1% of the population [START_REF] Cao-Lormeau | French Polynesia, South pacific[END_REF]. Phylogenetic analysis of partial membrane/ envelop genes showed the close relationship of the French Polynesian strain to those emerging in Cambodia in 2010 and Yap Island in 2007, thus highlighting the spreading of the ZIKV Asian lineage [START_REF] Faye | Molecular evolution of Zika virus during its emergence in the 20(th) century[END_REF][START_REF] Haddow | Genetic characterization of Zika virus strains: geographic expansion of the Asian lineage[END_REF]. In the sylvatic transmission cycle, humans are considered accidental hosts. In contrast, during the outbreaks in Yap Island and French Polynesia where non human monkeys are lacking, human beings were considered as the primary amplifying host of ZIKV, as already observed for other arboviroses such as CHIKV and DENV [START_REF] Diallo | Vectors of Chikungunya virus in Senegal: current data and transmission cycles[END_REF][START_REF] Duffy | Lanciotti Zika virus outbreak on Yap Island, Federated States Micronesia[END_REF].

In 2014 and 2015, following the introduction of a ZIKV belonging to the Asian lineage in French Polynesia, an outbreak occurred in New Caledonia with about 1500 cases [START_REF] Dupont-Rouzeyrol | Co-infection with Zika and dengue viruses in 2 patients, New Caledonia[END_REF]. During the same year, the presence of ZIKV was confirmed in Eastern Island [START_REF] Tognarelli | A report on the outbreak of Zika virus on Easter Island, South Pacific, 2014[END_REF] whereas imported ZIKV cases were documented in non-endemic areas including Japan [START_REF] Kutsuna | Two cases of Zika fever imported from French Polynesia to Japan, December 2013 to[END_REF], Canada [START_REF] Fonseca | First case of Zika virus infection in a returning Canadian traveler[END_REF], Australia [START_REF] Pyke | Imported zika virus infection from the Cook islands into Australia[END_REF] and Italy [START_REF] Zammarchi | Zika virus infections imported to Italy: clinical, immunological and virological findings, and public health implications[END_REF].

In May 2015, the Brazilian Health authority reported an autochthonous presence of ZIKV in the states of Bahia and Rio Grande de Norte [START_REF] Campos | Zika virus outbreak, Bahia, Brazil[END_REF] marking the first emergence of ZIKV in the Americas. Phylogenetic analysis of the ZIKV sequences showed that the virus belonged to the Asian lineage and was closely related to the strain isolated during the outbreak on French Polynesia. As of December 2015, several Latin-American countries, including Paraguay, Venezuela, Colombia, Suriname, Panama, El Salvador, Guatemala and Mexico have reported autochthonous cases of ZIKV.

In the beginning of January 2016, more than 150 cases of human infection by ZIKV were detected in Martinique Island by the regional health agency (ARS). This most recent information demonstrates the extraordinary capacity of ZIKV to rapidly spread to non-endemic areas throughout the world where the mosquito vector is present.

Transmission

Viral transmission occurs during the blood feeding of Ae. mosquitoes. Even if mosquito bites are the main mode of transmission, some cases of non-vector-borne infection have also been reported, referred to as perinatal transmission [START_REF] Besnard | Evidence of perinatal transmission of Zika virus, French Polynesia, December 2013 and February 2014[END_REF], probably following viral crossing of the placenta or during the delivery by viraemic mothers, with mother and baby presenting the same clinical signs of the disease. Although the virus has been detected in breast milk, no evidence exists as yet about the possible transmission via this route. It is of note that before spreading of ZIKV in the Americas, infection of newborns did not result in notable clinical manifestations, other than those observed in adults. Transmission by blood transfusion has not yet been demonstrated although a potential risk cannot be excluded [START_REF] Musso | Potential for Zika virus transmission through blood transfusion demonstrated during an outbreak in French Polynesia, November 2013 to[END_REF]. During the French Polynesia outbreak, the prevalence in blood samples was 1.9%, whereas 74% were found to be asymptomatic. However, the viral load in patients during the incubation period or in asymptomatic people has not been determined. The viral genome has been detected in saliva [START_REF] Musso | Detection of Zika virus in saliva[END_REF] and urine [START_REF] Gourinat | Detection of zika virus in urine[END_REF]. This finding is of interest because the presence of viral RNA was revealed between two to three weeks after the onset of clinical symptoms, whereas, at that time, it was not yet detectable in blood samples.

Finally, one sexual transmission was reported in the USA [START_REF] Foy | Probable non-vector-borne transmission of Zika virus, Colorado, USA[END_REF] and the presence of ZIKV was demonstrated in the semen of an infected patient in French Polynesia [START_REF] Musso | Potential sexual transmission of zika virus[END_REF]. The latter transmission route has not been reported before and is rare in arboviral infection: these findings nevertheless support the hypothesis that sexual relations could potentially transmit ZIKV (http://www.cdc.gov/zika/transmission/sexual-transmi ssion.html).

Clinical features

Symptomology

Asymptomatic patients are frequent, reaching up to 80%, and they constitute a high-risk source of transmission [START_REF] Musso | Rapid spread of emerging Zika virus in the Pacific area[END_REF]. The incubation period ranges from 3 to 12 days, followed by a mild "dengue-like" syndrome for a period of 2e7 days with a broad range of symptoms, including the presence of huge maculopapular rashes, a state of mild fever and headaches, arthralgia, retro-orbital pain, conjunctivitis and edema of the extremities [START_REF] Duffy | Lanciotti Zika virus outbreak on Yap Island, Federated States Micronesia[END_REF][START_REF] Simpson | Zika virus infection in man[END_REF][START_REF] Heang | Zika virus infection, Cambodia[END_REF]. The eruption of maculopapular rashes presented by more than 90% of patients remains the main clinical symptom that characterizes ZIKV infection [START_REF] Mallet | Bilan de l' epid emie a virus Zika en Polyn esie française 2013-2014[END_REF].

While the majority of human cases were benign, during the French Polynesian epidemic several neurological complications were reported presenting Guillain-Barr e Syndrome (GBS), an autoimmune disease, due to damage to the peripheral nervous system with a loss of the myelin insulation resulting in myalgia, facial palsy and muscle dysfunction. During the French Polynesian outbreak, a patient who presented with GBS was diagnosed with Zika fever. Following this first case, about 72 cases of GBS were reported with 40 patients being seropositive for the presence of the virus and link with ZIKV infection was put forward. The incidence resulted in an unexpected increase of GBS by 20 fold [START_REF] Oehler | Zika virus infection complicated by Guillain-Barre syndrome -case report, French Polynesia[END_REF]. However, the direct relationship between the virus and GBS need to be confirmed because of co-circulation of DENV (serotype 1 and 3) and ZIKV during this outbreak. Recently, the peculiarity of the ZIKV outbreak in Brazil has shown for the first time a possible link between ZIKV infection in pregnancy and microcephaly of the fetus [START_REF] Oliveira Melo | Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg?[END_REF]. and partial E (B) genes and full length genomes (C) from sequences available on GeneBank data bases were aligned using MEGA 5 [START_REF] Tamura | MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF], with minor manual adjustments. Sites that could not be unambiguously aligned were excluded and divergent regions were excluded from subsequent analyses. Phylogenetic trees were inferred using Bayesian method implemented in Topali software. Mr Bayes ran for 500 000 generations for NS5 and partial E respectively, and with 1.000.000 generations for the complete genomes with a 10% burn-in. Spondweni virus strain SM-6 V-1 (GenBank accession number DQ859064) was used as out-group for all phylogenies.

2000 in just a few months. Transplacental ability of ZIKV has been demonstrated by the presence of viral RNA in the amniotic fluid of pregnant women with fetal microcephaly [START_REF] Oliveira Melo | Zika virus intrauterine infection causes fetal brain abnormality and microcephaly: tip of the iceberg?[END_REF]. In addition to microcephaly, the possible relation between ZIKV infection and hydrops fetalis and fetal demise has recently reported in the same region [START_REF] Sarno | Zika virus infection and stillbirths: a case of hydrops fetalis, hydranencephaly and fetal demise[END_REF]. All these data suggest a possible materno-fetal transmission. Although maternalefetal transmission has been already described in DENV and WNV, no other flavivirus is known to have teratogenic effects. Nevertheless, the microcephaly epidemic in Brazil could also be linked to any other cause, such as other infectious or environmental agents. A recent study has revealed some ocular manifestations in three infants with microcephaly with one presenting with a macular neuroretinal atrophy [START_REF] Ventura | Zika virus in Brazil and macular atrophy in a child with microcephaly[END_REF]. However, further studies are needed to better define the outcomes of ZIKV infection during pregnancy. In this respect, although there is clear evidence of an increased number of cases of microcephaly in Brazil, it has been suggested that the number of suspected cases might be overestimated because of the diagnosis relying on low-specificity screening tests and the inclusion of mostly normal children with small heads [START_REF] Villar | International standards for newborn weight, length, and head circumference by gestational age and sex: the Newborn Cross-Sectional Study of the INTERGROWTH-21st Project[END_REF] requiring a stricter application of standardized anthropometric techniques and confirmation of suspected cases by laboratory or radiological evidence. There are no specific treatments or vaccine available against ZIKV and the treatment remained only a symptomatic support. The main means to combat infection are based on vector control and bite prevention.

ZIKV detection and laboratory diagnosis

Zika diagnosis remains difficult due to the similar clinical presentation of other arbovirus infections like dengue or chikungunya. Furthermore, DENV and CHIKV co-circulate in the same geographical areas, very likely resulting in an underestimation of human cases during periods of co-epidemics [START_REF] Roth | Concurrent outbreaks of dengue, chikungunya and Zika virus infectionsan unprecedented epidemic wave of mosquito-borne viruses in the Pacific 2012-2014[END_REF]. The differential diagnosis is challenging and often patients are diagnosed initially as having DENV infection. Before the contribution of molecular biology, the diagnostic assays for ZIKV were based on virus isolation and serological methods. Although these techniques remain currently relevant, the isolation of the virus is time consuming, requiring samples from convalescent patients or those with an acute infection, whereas cross-reactions with related flaviviruses is still a major issue. Serum antibody specificity can be determined using viral neutralizing assays which require highly specialized medical laboratories and remain largely complicated in their interpretation. Moreover, the viremic period is short (up to 5 days after disease onset) which complicates the detection of antibodies [START_REF] Lanciotti | Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia[END_REF]. At present, techniques permitting serological detection by immunoflorescence or ELISA are still under development and not commercially available. To improve the diagnostics, alternative molecular biology methods were developed for a wide range of flaviviruses [START_REF] Kuno | Phylogeny of the genus Flavivirus[END_REF] including ZIKV [START_REF] Faye | One-step RT-PCR for detection of Zika virus[END_REF]. These methods are rapid, sensitive and specific, but have to be used at the onset of the disease. Specific molecular assays, targeting the NS5 region or the envelope gene for the Asian and African ZIKV strains have been published [START_REF] Faye | Molecular evolution of Zika virus during its emergence in the 20(th) century[END_REF][START_REF] Lanciotti | Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia[END_REF]. With respect to serological methods, commercial detection test do not exist. It would be of interest to develop a diagnostic method using urine samples because, during the French Polynesia outbreak, Zika virus genome RNA was detected in this corporal fluid up to 10 days after onset of disease in several patients [START_REF] Gourinat | Detection of zika virus in urine[END_REF].

The pathogenicity of ZIKV in humans

ZIKV genomic organization and replication

Characteristic of the Flavivirus genus, ZIKV has a ~11 kb positive strand RNA genome [START_REF] Kuno | Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses[END_REF] which is composed of a single open reading frame, 10,794 pb in length, flanked by two non-coding regions (Fig. 4). The genome is translated into a single large polyprotein that is subsequently cleaved into three structural proteins, envelope (E), membrane precursor (prM) and capsid (C), as well as seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5), necessary for viral replication and assembly [START_REF] Kuno | Phylogeny of the genus Flavivirus[END_REF]. The envelope protein is involved in the recognition of the receptor at the surface of the host cell and then the fusion process between the viral envelope and the intracellular membranes [START_REF] Lindenbach | Molecular biology of flaviviruses[END_REF]. The different stages of ZIKV replication have not yet been well defined, but it is thought that they are essentially similar to other members of flavivirus genus. Typically, the virus has to complete four major stages to finalize its replicative cycle: translation of genomic RNA into viral proteins, replication of viral RNA molecules, assembly of virus particles in the endoplasmic reticulum and the release of virions [46]. The virion has an icosahedral capsid enclosed by a lipid envelope with a diameter of 40e70 nm [START_REF] Hamel | Biology of Zika virus infection in human skin cells[END_REF]. ZIKV replication occurs in the cellular cytoplasm and, like all other flaviviruses, multiple relationships with cellular organelles might exist to facilitate viral replication, evasion and propagation, although the exact mechanisms remain to be determined. The identification of such host factors and the characterization of their interactions with viral RNA and proteins are important for the understanding of ZIKV replication. Comprehensive analysis of the 5 0 and 3 0 untranslated regions of ZIKV also need to be carried out as these RNA structures play a critical role in viral genome cyclization and replication.

ZIKA virus permissiveness of human cells

Arthropod-mediated transmission of arbovirus is initiated when a blood-feeding female injects the virus into the human skin (reviewed in Ref. [START_REF] Briant | Role of skin immune cells on the host susceptibility to mosquito-borne viruses[END_REF]). Like many other members of the flavivirus family, ZIKV is transmitted following the bite of Aedes mosquitoes [START_REF] Grard | Zika virus in Gabon (Central Africa) -2007: a new threat from Aedes albopictus?[END_REF][START_REF] Wong | Aedes (Stegomyia) albopictus (Skuse): a potential vector of Zika virus in Singapore[END_REF]. Different cells types, such as epidermal keratinocytes [START_REF] Surasombatpattana | Dengue virus replication in infected human keratinocytes leads to activation of antiviral innate immune responses[END_REF], dendritic cells [START_REF] Lozach | Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN)-mediated enhancement of dengue virus infection is independent of DC-SIGN internalization signals[END_REF] or neurons [START_REF] Salazar | Dengue virus type 2: protein binding and active replication in human central nervous system cells[END_REF] are known to be a target of flaviviruses. Given the knowledge on the entry route of flaviviruses, potential target cells for infection with ZIKV have been investigated. Recently, we have reported that various cells in the human skin compartment are able to support ZIKV replication [START_REF] Hamel | Biology of Zika virus infection in human skin cells[END_REF].

Following infection with ZIKV, viral replication was observed in fibroblasts, keratinocytes and immature dendritic cells (iDCs), in a time dependent manner, with a substantial percentage of infected cells as early as 24 h post infection, whereas all cells were able to produce infectious virions. These results provide strong evidence for the critical role of the skin compartment in the transmission of ZIKV and are in line with those reported previously with respect to other members of the flavivirus genus [START_REF] Ivory | Early Dengue virus infection in human skin: a cycle of inflammation and infectivity[END_REF].

The interaction between the E glycoprotein of the viral particle and cell surface receptors allows the entry of the flaviviruses into the target cells. However, despite many investigations, the key cellular receptors remain relatively unknown and their importance in viral entry have yet to be clearly established. Several of those receptors have already been reported to permit viral entry of DENV and others arboviruses [START_REF] Perera-Lecoin | Flavivirus entry receptors: an update[END_REF]. DC-SIGN which is highly expressed on iDCs and macrophages [START_REF] Marovich | Human dendritic cells as targets of dengue virus infection[END_REF] has been known for many years to permit attachment and infection by Dengue virus [START_REF] Navarro-Sanchez | Dendritic-cell-specific ICAM3-grabbing non-integrin is essential for the productive infection of human dendritic cells by mosquito-cell-derived dengue viruses[END_REF][START_REF] Lozach | Dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN)-mediated enhancement of dengue virus infection is independent of DC-SIGN internalization signals[END_REF] thereby facilitating viral dissemination.

The TIM family is constituted of three receptors, TIM-1 TIM-3 and TIM-4. TIM-1 is expressed by Th2 cells and epithelial cells, whereas Th1 cells essentially express TIM-3. The expression of TIM-4 is restricted to antigen presenting cells [START_REF] Perera-Lecoin | Flavivirus entry receptors: an update[END_REF]. TIMs receptors have different roles, such as phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, or regulation of innate and adaptive immune responses. TIM-1 and TIM-4 expression is highly regulated following infection with DENV, WNV or YFV. The importance of TIM-1 receptor in infection was demonstrated using silencing technologies or blocking antibodies [START_REF] Meertens | The TIM and TAM families of phosphatidylserine receptors mediate dengue virus entry[END_REF].

The TAM receptor family is composed of TYRO3, AXL and MER receptors that are protein tyrosine kinases contributing to the regulation of immune responses [START_REF] Lai | An extended family of protein-tyrosine kinase genes differentially expressed in the vertebrate nervous system[END_REF]. Whereas AXL and MER are widely expressed, TYRO3 expression is mainly observed in the central nervous system. TAM receptors have been recently described to mediate DENV, WNV and YFV entry, both in cell lines and primary human cells [START_REF] Meertens | The TIM and TAM families of phosphatidylserine receptors mediate dengue virus entry[END_REF].

It was recently shown that DC-SIGN, TYRO3, and especially AXL, play an important role in the entry and replication of ZIKV in human skin cells [START_REF] Hamel | Biology of Zika virus infection in human skin cells[END_REF]. Specific antibodies or the use of siRNA interference to silence receptor strongly inhibited ZIKV replication. It is important to note that, in contrast to what has been reported for DENV infection, TIM-1 and TIM-4 receptors seem to play a minor role in the entry of ZIKV in human skin cells, although a cooperative role of both TIM and TAM family members has been observed. The exact contribution of each of these receptors to ZIKV entry is still under investigation.

Innate immune response to ZIKV

Following viral infection, cells mount a range of antiviral responses in order to limit the spread of the virus, with the main defense consisting of innate and adaptive immune responses. The initial response is provided for by the production of type I interferons (IFNs) [START_REF] Schneider | Interferon-stimulated genes: a complex web of host defenses[END_REF]. Early detection of pathogen-associated molecular patterns (PAMP) expressed by the virus is sensed and mediated by pattern recognition receptors (PRRs), such as the Toll-like receptor (TLR)-family or RIG-I like receptor. Following the detection of PAMPs, the triggering of various signaling pathways not only leads to a direct secretion of IFNs, but also the expression of hundreds of Interferon-Stimulated Genes (ISGs) that integrate to induce an antiviral state of cells [START_REF] Nazmi | Role of pattern recognition receptors in flavivirus infections[END_REF]. DENV and WNV infections have been the subject of a large body of studies that show a pivotal role for RIG-I, MDA5 and TLR3 sensors in the host defense against these viruses, leading to IFN type 1 production and expression of ISGs [START_REF] Nasirudeen | RIG-I, MDA5 and TLR3 synergistically play an important role in restriction of dengue virus infection[END_REF] In a similar fashion, infection of human fibroblasts with ZIKV induces the expression of RIG-I, MDA-5 and TLR3, as well as the ISGs ISG15, OAS2 and MX1. Moreover, increased expression levels of IRF7, a transcription factor that binds to promoters on IFN type I genes, corroborates the strong induction of IFN-a and IFN-b by ZIKV-infected fibroblasts. Furthermore, the expression of certain inflammatory chemokines, such as CCL5, is also induced upon ZIKV infection. The inflammasome pathway seems to be activated as well following ZIKV infection, as shown by an increase in the expression of AIM2 and interleukin-1b transcripts [START_REF] Hamel | Biology of Zika virus infection in human skin cells[END_REF]. These results notwithstanding, the involvement of the latter pathway remains to be investigated in more detail in order to better understand the involvement of interleukin-1b in the inhibition of ZIKV replication.

Different cellular processes can be hijacked by flaviviruses to evade the cellular response and facilitate virus replication. Immediately following infection, the host establishes rapid innate immune responses, including IFN type I responses, the induction of apoptosis and autophagy, in order to overcome the viral infection review in Ref. [START_REF] Ye | Immune evasion strategies of flaviviruses[END_REF]. WNV is able to elude detection or inhibit IFN gene transcription [START_REF] Shipley | Identification of multiple RIG-I-specific pathogen associated molecular patterns within the West Nile virus genome and antigenome[END_REF][START_REF] Liu | Analysis of adaptive mutations in Kunjin virus replicon RNA reveals a novel role for the flavivirus nonstructural protein NS2A in inhibition of beta interferon promoter-driven transcription[END_REF]. In addition, several arboviruses such as DENV and CHIKV can subvert the autophagy process [START_REF] Heaton | Dengue virus and autophagy[END_REF][START_REF] Krejbich-Trotot | Chikungunya triggers an autophagic process which promotes viral replication[END_REF] in order to promote their own replication and dissemination. It is known that flaviviruses rearrange host cell membranes to create an appropriate environment for their replication with the main source of membranes being the endoplasmic reticulum [START_REF] Blazquez | Stress responses in flavivirus-infected cells: activation of unfolded protein response and autophagy[END_REF]. These rearrangements result in an activation of the unfolded protein response (UPR) and overexpress the autophagic pathway in infected cells simultaneously [START_REF] Heaton | Dengue virus-induced autophagy regulates lipid metabolism[END_REF]. The double-membrane vesicles, known as autophagosome, allow the recruitment of cytoplasmic elements, proteins and organelles and permit their degradation. Autophagy could act both positively or negatively in host immunity against pathogens [START_REF] Shelly | Autophagy is an essential component of Drosophila immunity against vesicular stomatitis virus[END_REF][START_REF] Lee | Autophagic machinery activated by dengue virus enhances virus replication[END_REF]. In the last mechanism, viruses use the autophagy pathway to facilitate their own replication.

The first evidences of autophagy in ZIKV-infected fibroblasts, were highlighted by the presence of characteristic autophagosome-like vesicles in infected fibroblasts [START_REF] Hamel | Biology of Zika virus infection in human skin cells[END_REF]. Then, the authors confirmed the induction of autophagy in infected fibroblasts by an increase in production of LC3, a cytosolic microtubule-associated molecule. The latter was colocalized with viral envelope protein suggesting that autophagocytic vacuoles are the site of viral replication. The use of an inhibitor of the autophagosome formation during the infection significantly reduces viral copy number while an inducer of autophagy increases viral replication, demonstrating that autophagy could play a major role in ZIKV immune evasion. However, the intimate molecular mechanisms remain to be further investigated.

Conclusion

The most recent ZIKV outbreaks and in particular its introduction in the Americas have resulted in a change in the appreciation of this virus and the clinical repercussions of its presence in the human host. Like other arboviruses, the rapid spreading of ZIKV is due to favorable climatic and sociodemographic conditions, including the presence of its mosquito vectors in urban concentrations with poor hygienic conditions, the increase in international travel and possibly a better adaptation of the mosquitos to humans. This situation is extremely worrisome and requires efficient surveillance systems, including sensitive diagnostic methods and vector control measures. The co-circulation of ZIKV with other medically important arboviruses, such as DENV and CHIKV, constitutes an additional challenge which complicates the comprehension of this disease. The suspected association between Zika and neurological complications such as GBS and congenital malformation needs to be confirmed in order to control and reduce the negative impacts of the infection. A thorough understanding of the molecular interactions that ZIKV establishes with the host cell during infection is also necessary to determine the targets for antiviral treatment.

Fig. 2 .

 2 Fig.2. Phylogenetic analysis of the isolated strains ZIKV. Non-concatenated nucleic acid sequences of the NS5 (A) and partial E (B) genes and full length genomes (C) from sequences available on GeneBank data bases were aligned using MEGA 5[START_REF] Tamura | MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods[END_REF], with minor manual adjustments. Sites that could not be unambiguously aligned were excluded and divergent regions were excluded from subsequent analyses. Phylogenetic trees were inferred using Bayesian method implemented in Topali software. Mr Bayes ran for 500 000 generations for NS5 and partial E respectively, and with 1.000.000 generations for the complete genomes with a 10% burn-in. Spondweni virus strain SM-6 V-1 (GenBank accession number DQ859064) was used as out-group for all phylogenies.

Fig. 3 .

 3 Fig. 3. Geographic distribution of ZIKV. Adapted from Centers for Disease Control and Prevention publication, http://www.cdc.gov/zika/geo/index.html.

R. Hamel et al. / Microbes and Infection 18 (2016) 441e449

Organization of the ZIKV genome. The genomic organization is arranged as with other flaviviruses, with an open reading frame encoding three structural proteins C, prM and E and five nonstructural proteins NS1 to NS5. The structural and nonstructural proteins are generated after proteolytic cleavage of the whole translated polyprotein from the ORF region.

Acknowledgments

This work was supported by grants from the French Research Agency "Agence Nationale de la Recherche" (ANR-12-BSV3-0004-01; ANR-14-CE14-0029).

Conflict of interest

We the authors declare no conflict of interest in writing this review.


L Delfour

A Davydova

E Despiau-Pujo
email: emilie.despiau-pujo@cea.fr

G Cunge

D B Graves

L Magaud

Cleaning graphene : a first quantum/classical molecular dynamics approach

Graphene outstanding properties created a huge interest in the condensed matter community and unprecedented fundings at the international scale in the hope of application developments. Recently, there have been several reports of incomplete removal of the polymer resists used to transfer as-grown graphene from one substrate to another, resulting in altered graphene transport properties. Finding a large-scale solution to clean graphene from adsorbed residues is highly desirable and one promising possibility would be to use hydrogen plasmas. In this spirit, we couple here quantum and classical molecular dynamics simulations to explore the kinetic energy ranges required by atomic hydrogen to selectively etch a simple residue -a CH3 group -without irreversibly damaging the graphene. For incident energies in the 2-15 eV range, the CH3 radical can be etched by forming a volatile CH4 compound which leaves the surface, either in the CH4 form or breaking into CH3+H fragments, without further defect formation. At this energy, adsorption of H atoms on graphene is possible and further annealing will be required to recover pristine graphene.

Introduction

Graphene exceptional properties open exciting opportunities in many fields, such as energy storage, electronics, photonics, coating [1]. While some of these applications can work with a defected material, many other require a nearly perfect graphene. During the last years, an intense research activity has focused on how defects can affect or alter graphene properties [2,3] and graphene cleaning is becoming a crucial issue, a mandatory step for further application development.

Defects appear during the different steps required to grow and transfer graphene or create devices. Indeed, one way to grow and obtain large-scale high quality graphene is to use metallic surfaces [4]. Then, for many applications it has to be transferred to another substrate. This is commonly performed using PMMA resist -Poly(methyl methacrylate)in order to ensure rigidity while removing the metallic substrate. After transfer, the polymer is removed by thermal annealing and the use of solvents, but full removal cannot be achieved. PMMA thermal decomposition leads to the formation of residues adsorbed on graphene and modify its properties, inducing p-doping and degrading its electronic mobility [5][6][7].

Hydrogen plasma provides one way to pattern graphene and it has been shown that, depending on the temperature, it can selectively etch ribbon edges or create holes in the basal plane [8,9]. Recently, the use of hydrogen plasmas has been proposed [10] to clean graphene from PMMA resist residues. In low-temperature plasma processing, the surfaces are exposed simultaneously to an isotropic flux of thermal radicals (e.g. H atoms in a H2 plasma) and to an anisotropic flux of energetic ions (e.g. H+), which can result in the selective etching of a material. To clean graphene surfaces in H2 plasmas, the range of kinetic energies needed by the plasma species to selectively etch the residues must be determined. One important question one has to answer is whether the impinging hydrogen species (especially ions) would create new defects in graphene and whether they can be cured or not. This is a completely new and tough issue to model from scratch. To start tackling it, we use a simple description of a PMMA residue, a methyl group, and calculate the effect of an impinging H atom arriving on the C atom of the CH3 group at normal incidence with respect to the graphene plane, as a function of its incident energy and for various surface temperatures. Modeling plasma ions impacts using fast neutral atoms at normal incidence is a reasonable approximation because in low-pressure plasma reactors, ions are accelerated through the plasma sheath perpendicularly to the surfaces, so that H+ ions reach the target sample with a normal incidence [11]. Furthermore, when a plasma ion reaches a conductive surface, it is generally assumed to be neutralized by an auger mechanism [12].

Beyond the fact that CH3 radicals can result from PMMA thermal decomposition, etching of methyl groups adsorbed on graphene is also of strong interest since high quality, single layer graphene growth can be obtained through successive cycles of growth using an organic compound gas and etching by hydrogen [13].

Following what we did for elementary hydrogen interactions with surface-clean graphene [14], we couple here quantum (QMD) and classical (CMD) molecular dynamics simulations to study the effect of energetic H atoms impacting a CH3 residue adsorbed on graphene. While quantum molecular dynamics treats in an accurate way the electronic part of the problem, it remains a heavy computational approach and thus the number and duration of calculated trajectories is limited. On the other hand, classical molecular dynamics uses an empirical potential form to model the interactions between atoms. While less accurate, it enables the sampling of a much larger number of trajectories. Coupling the two approaches allows to check further the accuracy of the empirical potential against ab initio results and validate it in a more complex case than that of Ref 14. One main difficulty here comes from the fact that the potential has to describe sp 2 as well as sp 3 bonded carbon atoms together with the C-H interaction. Once validated, this empirical potential is used to address more complex cases including longer dynamics, fine sampling of the trajectories as a function of the incident H energy, effect of surface temperature.

At 0 K, identical mechanisms are predicted by both QMD and CMD for increasing incident energy: reflection and chemical etching. However, plasma processing usually takes place at temperatures closer to 300K, or even higher, and thermal vibrations of the substrate atoms may modify the reaction probabilities for the different processes. Thus, CMD is then used to evaluate the influence of graphene temperature on the interaction mechanisms.

All simulations show that at moderate incident energy, the radical is etched. This happens through the formation of a volatile CH4 compound that leaves graphene without new defect creation. At this energy, adsorption of H atoms on graphene is possible as shown in Ref. 14 and further annealing will be required to recover pristine graphene.

The paper is organized as follows: computational details are given in part 2, the different reaction mechanisms at 0K are discussed in part 3 and surface temperature effect is presented in part 4. Conclusions are discussed in part 5.

Computational Approaches

Quantum Molecular Dynamics (QMD)

Quantum Molecular Dynamics calculations are performed using the code VASP [15,16]. A CH3 radical adsorbed at the center of the graphene cell has first been fully relaxed (Figure 1). Atomic hydrogen is then sent at normal incidence on the C atom of the CH3 radical for a chosen set of initial energies. All calculations are performed in the NVE statistical ensemble at 0K, with a time step equal to 0.1 fs. Newton's equations of motion are integrated using a Verlet algorithm [17].

We are using the Perdew-Wang generalized gradient functional (PW91) [18] and the PAW approach [19]. A 6x6x1 k-points grid is used and the supercell cell has a 24.7x21.4x18 Å 3 dimension. It contains 200 +1 C atoms and 4 H atoms. The size along the z direction is large enough so that the image layers would not interact.

Classical Molecular Dynamics (CMD)

Classical molecular dynamics (CMD) refers to a class of simulation that solves Newton's equations of motion for a system of N interacting particles, treating each atoms as a classical point and modeling the quantum effects from electrons by an interatomic potential energy function. In this work, we use the 2nd generation C-H REBO potential developed by Brenner and coworkers [20]. This potential has been widely used to model solid carbon and various hydrocarbon systems. A description of our calculation methods can be found in a former paper [14], where we tested the REBO potential against DFT calculations to study elementary processes of graphene-hydrogen interaction.

CMD simulations are performed in the microcanonical NVE ensemble. The Velocity-Verlet algorithm is used to calculate positions and velocities, with a timestep equal to 0.1 fs. The graphene surface is represented as a single-layer sheet with periodic boundary conditions along the Ox and Oy axes to mimic a semi-infinite plane. Four atoms are fixed on the edges to anchor the simulation cell. The initial configuration contains 260 atoms with a surface area of 670 Å 2 (Lx =19.67 Å, Ly = 34.08 Å). In order to model a CH3 radical absorbed on the graphene surface, a methyl group is created in the middle of the cell and allowed to relax for 100 ps to get its equilibrated structure. Atomic hydrogen with energy Ei between 0.1 eV and 25 eV is sent at normal incidence, from 3 Å above the full configuration, as presented in Figure 1. After injecting the H atom above the CH3 radical, the trajectories of all atoms are followed during ~ 1 ps. At the end of each impact, the surface reaction is observed and analyzed. The original temperature of the whole system (Tcell) is set to 0K ; the simulation cell is then quenched at 300K and 600K to study the influence of the surface temperature.

Reaction mechanisms at 0K

In the following, the impact of an incident H atom with varying initial velocities is described. Various mechanisms (H reflection, CH4 formation and desorption, CH3 etching or sputtering) are observed when the incident kinetic energy is increased. At 0K and for small and intermediate incident energy, DFT and CMD calculations predict similar mechanisms but for shifted energy ranges.

Initial configuration

The geometry at 0K of a methyl group adsorbed on graphene is shown in Figure 1, together with the notations used throughout the text. DFT and CMD fully relaxed geometries are in good agreement. The CH3 group is rotated with respect to the graphene. The length of the 3 CCH3-HCH3 bonds are identical and equal to 1.1 Å. The radical carbon atom, CCH3, is located 1.56 Å above the carbon atom belonging to the graphene surface (CGR). The graphene carbon atom, CGR, raises 0.6 Å above the graphene basal plane, stretching the bonds with its nearest carbon neighbors CGR-CNBR up to 1.51 Å. The CH3 radical chemisorption depletes the local π bonds in the graphene structure, thus CGR-CNBR bonds become weaker. It leads to a partial transition of the local bonding from sp 2 to sp 3 in agreement with other calculations [21][22][23].

A hydrogen atom with different incident energies is inserted into the system 3 Å above the CH3 radical. This distance is large enough so that in the starting configuration, no interaction exist between the incident H and the CH3 group.

Reflection

Results from CMD simulations show that for Ei < 3.7 eV, the impacting H atoms are systematically reflected from the surface without modifying the structure of the CH3 radical bound to the graphene. In order to illustrate such reaction, we consider the impact of a 1 eV H atom onto the CH3 radical. Figure 2 shows the evolution of interatomic distances as a function of time before and after the impact. The solid red curve corresponds to the distance between the impinging H atom and the CCH3 atom, the dashed blue and dotted red ones to the CCH3-CGR and CCH3-HCH3 bonds, respectively. One can see that during the impact, the H atom comes close (~ 1.6 Å) to CCH3 (the carbon atom belonging to the CH3 radical) and then gets reflected. The target radical reacts to this low energy impact with small fluctuations of its CCH3-HCH3 bond lengths around their average value of 1.1 Å. At the same time, the CCH3 atom is slightly pushed down after the H impact; thus the CCH3-CGR bond is shortened and then shows small variations around its initial length (1.56 Å). Contrary to CMD results, DFT calculations at 1eV do not show H reflection from the surface but another mechanism -the formation and desorption of a CH4 molecule. This mechanism is also obtained with CMD but for slightly higher energies [3.7-8 eV], as discussed in the next paragraph. Based on this reflection result obtained with CMD, we performed QMD calculations for decreasing incident energies and indeed found the occurrence of a reflection but at much smaller incident energy (0.07 eV). It is a first evidence of the technical interest of coupling classical and quantum molecular dynamics: using the classical approach to cover a broader range of configurations and checking interesting ones with DFT.

CH4 formation and desorption

QMD calculations predict that a hydrogen atom with an initial energy equal to 1 eV is adsorbed by the methyl group to form a volatile CH4 molecule, which then desorbs from the graphene surface. Both approaches also indicate that at the end of the simulation, neither vacancy nor defects are observed on the graphene layer, which retrieves its original geometry.

QMD calculations for an incident energy close to 2.5 eV show a very similar behavior. The amplitude of the CCH3-H distance oscillations is just much larger (1.25 Å instead of 0.5 Å). This 2.5 eV case is close to the limit where the CH4 molecule first forms and then breaks into a CH3 radical and an H atom as it is shown in the next section.

CH3 formation

For higher incident energies, another reaction mechanism leading to the methyl removal from graphene is observed with both classical and quantum approaches, but again for different energy ranges (QMD at 4 eV, CMD in the [9-11.6] eV energy range). Figure 5 shows the evolution of the distances calculated by QMD for this velocity and the simulation snapshots which can be directly compared to the ones in Figure 3. The projected atomic H comes 0.18 Å closer to CCH3 than for an incident energy equal to 1 eV. The bond is then strongly compressed and the H bounces back with an energy sufficient to break the CCH3-H bond. As observed previously, the etching of the CH3 group proceeds through an "umbrella inversion" of the three HCH3 atoms. It proceeds more slowly than previously observed for CH4, since at 37 fs CCH3 is only 2.42 Å away from CGR (3.36 Å in the previous case). The CCH3-HCH3 bonds also show slightly larger amplitude oscillations of about 0.04 Å (instead of 0.03 Å). The set of curves shown in Figure 6 presents the behavior of bond distances calculated from CMD after H has impacted with Ei = 9 eV. Here the impacting H atom comes close to the CH3 radical and then binds to it and forms a CH4 molecule. The excess kinetic energy from the impacting H is transferred to the neighbor atoms in the CH3 group and to the graphene atoms. Thus both CCH3-HCH3 and CCH3-CGR bonds experience stress and fluctuations initiating the desorption process of the newly formed CH4. As a consequence, after 10 fs, the CCH3-CGR bond stretches and breaks, allowing the volatile methane product to leave the surface. However, once CH4 has left the graphene surface, it immediately disintegrates into a single H and a CH3 group. This molecular dissociation is possible due to the excess kinetic energy carried by H, which induces the desorption of a CH4 molecule in a highly excited vibrational state (pre-dissociated). In both classical and quantum cases, the etch product is CH3 and the graphene surface is free of any defects after the methyl removal by the atomic H. Indeed for high incident H energies (> 11.6eV), CMD calculations predicts not only the reflection of the incident H (like in QMD) but also another possible reaction path: the sputtering of one or more HCH3 atoms from the CH3 radical. For example, Figure 8 shows the impact of a 20 eV H atom onto the methyl radical as calculated by CMD. In this case, due to its high kinetic energy, the impacting hydrogen is reflected very rapidly but transfers a significant part of its kinetic energy to the target CH3 radical (the velocity of the reflected H atom changes after 8 fs as shown in Figure 8). As a consequence, the three H CH3 atoms are sputtered away from CCH3 as three single H atoms, leaving the single CCH3 atom chemisorbed on the graphene basal plane. This sputtering mechanism is coherent with the QMD calculations, which indicate that most of the energy of the impact goes into vibrations of the CCH3-HCH3 bonds; however, in QMD calculations, the amount of energy transferred from the H atom to the methyl group is not large enough to sputter the HCH3 atoms. In addition, the incident H impact also initiates small fluctuations of the CGR-CCH3 bond with an amplitude of 0.11 Å.

Reflection or sputtering events at high energies

Although the mechanisms differ slightly depending on the energy, both classical and quantum results show no etching of the whole CH3 group. At 0K and for incident kinetic energies higher than 8eV in QMD (12eV in CMD), the CCH3 atom of the residue always remains bound to the graphene basal plane. This suggests that too high incident energies may not be suitable to clean graphene from simple residues.

To conclude on these calculations performed at 0K, similar H/CH3 interaction mechanisms are predicted by QMD and CMD for increasing incident energy: H reflection, CH4 formation and desorption, CH3 chemical etching and eventually HCH3 sputtering. The differences in energy ranges point back to the difficulty of describing quantitatively complex quantum phenomena with a classical potential. However, the agreement between classical and quantum approaches on the nature and sequence of mechanisms allows to further validate the classical C-H REBO potential with respect to our previous study [14].

It brings fundamental information about incident energy ranges and possible reaction mechanisms. Nevertheless, key differences have to be considered if one wants to investigate real H2 plasma process conditions. First, the graphene sample would be randomly bombarded by both (i) H/H2 neutral species impacting isotropically with thermal energies (300 K) and (ii) H + /H 2+ /H 3+ ions impacting anisotropically with higher energies (10-100 eV). Second, the graphene sample temperature is generally closer to 300K than 0K. Because thermal vibrations of the substrate carbon atoms may modify the reaction probabilities for reflection, etching or sputtering, we perform further CMD calculations of H/CH3 interaction with higher surface temperatures in the next section.

Influence of surface temperature

We present in this part results from CMD simulations for cell temperatures of 300K and 600K since it is well known that plasma treatment usually takes place at room temperatures or higher. The goal of such a study is to show how the graphene and CH3 residue temperature may modify the reaction probabilities for reflection, etching or sputtering.

Due to thermal vibrations of both the graphene lattice and the CH3 molecule, incident H species may not impact exactly on top of (or perpendicularly to) the CCH3 atom of the target residue which is also a better description of the actual system.

The CH3 radical absorbed on the graphene single layer is quenched along with the other substrate atoms to 300K and 600K (surface temperature Tcell) prior to H bombardment.

Incident H energies Ei are varied from 1 to 25 eV. For each (Ei, Tcell) combination, we perform 100 impacts at normal incidence on a refreshed CH3 residue adsorbed on graphene (i.e. returned to its initial configuration). Then we calculate statistically the probability and energy thresholds for H reflection, CH4 volatile product formation, CH3 etching or HCH3 etching from the target methyl radical. Figure 9 shows the surface reaction probabilities predicted by CMD depending on the cell temperature and the incident H energy. The colored zones remind the energy ranges found for the different processes at 0K, as previously discussed. At 0K, H reflection takes place for Ei < 3.7 eV due to the presence of a potential energy barrier of ~ 2.9 eV arising from the three H atoms belonging to the methyl radical. For Ei ranging from 3.7 to 9 eV, CH4 formation and desorption from the graphene surface occurs because H has enough energy to overcome the potential barrier and bind to the CH3 radical (but not enough to induce the fragmentation of the molecule). For higher incident H energies in the [9-11.6] eV range, CMD shows that H can etch the CH3 radical by forming a CH4 volatile molecule which then splits into a single H atom and a CH3 group, leaving the graphene surface clean and undamaged. Finally, for Ei > 12 eV, an excessive energy is transferred to the CH3 residue during the impact, leading to the partial or full sputtering of HCH3 atoms belonging to the CH3 radical (leaving a stripped C atom chemisorbed on the surface).

As shown by the solid and dotted curves (corresponding to reaction rates for graphene surfaces at 300K and 600K respectively), rising the temperature of the graphene cell has a significant effect on all mechanisms described above. We observe that increasing the surface temperature broadens the energy ranges for which each mechanism -CH4 formation, CH3 etching or HCH3 etching -can occur.

Discussion

Quantum and classical molecular dynamics studies at 0K predict the same sequence of reaction mechanisms. The thermal motion of the graphene structure at higher temperatures (300K and 600K) leads to stronger fluctuations of atomic positions, bond angles and bond lengths compared to the 0K case. As a consequence, for a same incident H energy, different reaction mechanisms can occur depending on the state/position of the CH3 radical when the atomic H collides with it. Thus, an increase in the surface temperature lowers the potential barriers required for H chemisorption on the graphene surface [14] and less energy is needed to allow CH4 desorption from a heated surface. Due to stronger vibrations of CCH3-HCH3 bonds at 300K and 600K, the energy range for which CH3 etching can be observed is also wider. The latter also explains the occurrence of HCH3 etching events along all the scanned energy range.

These calculations show that it is possible to etch CH3 residues from graphene at room and higher temperatures with atomic hydrogen in the 3-15 eV energy range. In this range, H species can chemisorb on the graphene basal plane but have a low probability to damage the graphene structure (by breaking C-C bonds or creating vacancies) [14]. It may therefore be possible to clean graphene from simple CxHy residues in low-temperature H2 plasmas without making irreversible damages to the material.

Conclusion

The interaction of an incident energetic H atom with a CH3 radical adsorbed on graphene was investigated by QMD and CMD calculations. We first simulated the elementary interaction between an incident atomic hydrogen and a CH3 radical adsorbed on a graphene layer at 0K as a function of the incident H kinetic energy. Both QMD and CMD calculations showed the possibility for chemical etching of the methyl radical from graphene without damaging the graphene basal plane. In all cases, this happens through the formation of a CH4 volatile compound that leaves the graphene surface either in the CH4 form or breaking into a CH3 group and an isolated H atom. While the energy ranges of occurrence of the mechanisms differ between QMD and CMD, both approaches give the same sequence for increasing energies: incident H reflection, CH4 formation with CH4 or CH3 + H leaving the graphene basal plane. Results only slightly differ at high energy where both approaches show a reflection of the incident H atom, but with an eventual sputtering of the HCH3 atoms belonging to the CH3 residue in the case of CMD which is not found in QMD. This last difference and the variation in the energy ranges associated to each mechanism point back to the difficulty to describe so complex phenomena and especially both sp 2 and sp 3 hybridization in carbon. Nevertheless the agreement on the sequence of mechanisms further validates the REBO potential.

Temperature and incident H energy influence on surface reactions probabilities were discussed in the last part of the article. It was shown that increasing the surface temperature broadens the energy ranges for which each mechanism can occur. Indeed the potential barriers for H adsorption are lowered and less energy is needed to allow CH4 desorption from a heated surface.

A CH3 radical absorbed on graphene is a "basic" residue representation but these results are interesting for the development of future graphene surface cleaning processes by H2 plasmas. Indeed, there is very little information regarding graphene cleaning by plasmas in the literature and as a general rule, the incident kinetic energy of radicals and ions required to selectively etch residues is unknown. Similarly, PMMA etching by H2 plasmas has not been deeply investigated. However, it was shown that downstream plasmas (in which the surface is bombarded only by low energy H radicals) can be used to clean the PMMA without damaging the graphene at high temperature (H adsorption is reversible by thermal annealing). According to our work, the contaminant carbon was probably removed as volatile CH4 under these conditions. However, we underline that, from our results, CH3 residues can be removed from the surface by low-energy ions in the 1-15 eV range without damaging the graphene irreversibly. This suggests that high density plasma sources (such as Inductively-Coupled and Electron Cyclotron Resonance plasmas) could also be advantageously used for this purpose: by contrast with downstream systems, they provide large fluxes of reactive radicals and ions and are thus expected to be much more efficient to clean graphene.

Figure 1 :

 1 Figure 1: Initial configuration used in QMD and CMD

Figure 2 :

 2 Figure 2: CMD calculation of bond length variations as a function of time, when a H atom impacts onto the CH3 radical with an incident energy of 1eV. Red solid, blue dashed and red dotted curves correspond to H-CCH3, CCH3-CGR and CCH3-HCH3 distances respectively.

Figure 3

 3 presents the evolution of the CH3 target bond lengths before and after the H impact. One can see that when the H atom comes close to the radical CCH3 atom, a bond is formed and oscillates around ~ 1.1 Å, which is the equilibrium value for C-H bonds in methane molecules. Once the CH4 molecule is formed (from 10 fs), the distance between CGR and CCH3 continuously increases, which indicates that the CH4 molecule leaves the graphene surface. At the end of the simulation time, the volatile molecule is already 3.5 Å away from the graphene surface and has thus desorbed.

Figure 3 :

 3 Figure 3 : (a) QMD calculation of bond lengths describing H-CCH3 (red), CCH3-CGR (dashed blue), CCH3-HCH3 (red dots) as a function of time, for a 1 eV incident energy. (b) Snapshots of the QMD simulation for specific times of 0 fs, 17 fs, 22.7 fs, 28.3 fs and 33.9 fs. Shown by arrows in (a).

Figure 4 :

 4 Figure 4: CMD calculation of bond lengths describing H-CCH3 (red), CCH3-CGR (dashed

Figure 5 :

 5 Figure 5 : (a) QMD calculation of relevant bond lengths describing H-CCH3 (red full line),

Figure 6 :

 6 Figure 6: CMD calculation of bond lengths as a function of time when a H atom impacts onto the CH3 radical with an incident energy equal to 9 eV. Colors are identical to the ones in Figure 5.

Eventually, if theFigure 7 :

 7 Figure 7: (a) QMD calculation of bond lengths describing H-CCH3 (red), CCH3-CGR (blue), CCH3-HCH3 (red dots) as a function of time, for a 8 eV incident energy. (b) Snapshots of the QMD simulation for specific times of 0 fs, 6.2 fs, 10 fs, 20 fs and 27.9 fs.

Figure 8 :

 8 Figure 8: CMD calculation of bond distances variation as a function of time when an H atom impacts onto the CH3 radical with an incident energy equal to 20 eV. Colors are identical to the ones in Figure 7.

Figure 9 :

 9 Figure 9 : Influence of H incident energy and graphene surface temperature on the probabilities of H reflection (black/white), CH4 formation (blue), CH3 etching (red) and HCH3 etching (green) obtained from CMD statistical calculations. The colored zones indicate the exact energy ranges for these same mechanisms at 0K. The solid and dotted curves show the probabilities for each mechanism at 300K and 600K respectively.

Acknowledgement

The authors wish to thank the Nanosciences Foundation of Grenoble in the frame of the 2010 Chairs of Excellence Program and the NANOSIM-GRAPHENE project ANR-09-016-01 for financial support. This work was partly supported by the french RENATECH network. QMD simulations have been performed at the GENCI computational facility (Grant 097015).