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Background: Ticks are significant vectors of pathogens, including viruses, bacteria, and protozoa. With approximately 900 tick species worldwide, many are expanding their geographical range due to changing socioeconomic and climate factors. The Danube Delta, one of Europe's largest wetlands, is an ecosystem that, despite its ecological importance, remains understudied concerning the risk of introducing new tick-borne viruses. This region serves as a critical habitat for migratory birds, which can carry ticks over long distances, potentially introducing exotic tick species and their pathogens into the local ecosystem. Hyalomma marginatum ticks, the primary vector of Crimean-Congo hemorrhagic fever virus (CCHFV), are of particular concern due to their expanding presence in Europe and potential to spread other arboviruses. In addition to being the primary vector for CCHFV, Hyalomma sp. ticks are capable of transmitting other pathogens of medical and veterinary importance, including Dugbe virus, West Nile virus, African horse sickness virus, and Kyasanur forest disease virus. Therefore, it is essential to monitor the presence of Hyalomma sp. ticks while simultaneously surveilling arbovirus circulation in tick populations to mitigate the risk of arboviral outbreaks. Methods: In this work, we used an RNA sequencing technique to analyze the virome of H. marginatum ticks collected from the Danube Delta Biosphere Reserve, Romania, one of the major bird migration hubs from Africa to Europe. Results: Among the viral taxa detected in H. marginatum ticks, sequences belonging to Volzhskoe tick virus (VTV), Balambala tick virus (BMTV) and Bole tick virus 4 (BTV4) were identified. In addition, we report the first identification of a novel Rhabdoviridae-related virus, Hyalomma marginatum rhabdovirus (HMRV). No CCHFV or any CCHFV-related nairovirus were detected in this study. Conclusions: To summarize, detecting new viruses is essential for monitoring potential viral outbreaks. Our research expands the understanding of virus diversity in Eastern Europe, including the identification of novel viruses. This insight is crucial for monitoring viruses that may pose risks to both animal and human health, such as CCHFV. [ABSTRACT FROM AUTHOR]

The development of the human immune system lasts for several years after birth. The impact of this maturation phase on the quality of adaptive immunity and the acquisition of immunological memory after infection at a young age remains incompletely defined. Here, using an antigen-reactive T cell (ARTE) assay and multidimensional flow cytometry, we profiled circulating severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)–reactive CD3+CD4+CD154+ T cells in children and adults before infection, during infection, and 11 months after infection, stratifying children into separate age groups and adults according to disease severity. During SARS-CoV-2 infection, children younger than 5 years old displayed a lower antiviral CD4+ T cell response, whereas children older than 5 years and adults with mild disease had, quantitatively and phenotypically, comparable virus-reactive CD4+ T cell responses. Adults with severe disease mounted a response characterized by higher frequencies of virus-reactive proinflammatory and cytotoxic T cells. After SARS-CoV-2 infection, preschool-age children not only maintained neutralizing SARS-CoV-2–reactive antibodies postinfection comparable to adults but also had phenotypically distinct memory T cells displaying high inflammatory features and properties associated with migration toward inflamed sites. Moreover, preschool-age children had markedly fewer circulating virus-reactive memory B cells compared with the other cohorts. Collectively, our results reveal unique facets of antiviral immunity in humans at a young age and indicate that the maturation of adaptive responses against SARS-CoV-2 toward an adult-like profile occurs in a progressive manner. Editor's summary: The immune system continues to develop throughout childhood, and it is established that children have distinct immune responses to vaccines and infections as compared with adults. Here, Manfroi et al. characterized the immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in children. The authors found that preschool-age children had a different adaptive immune response, with a lower frequency of antiviral CD4+ T cells, compared with older children and adults; this younger cohort also developed phenotypically distinct memory T and B cell responses after recovery compared with the other cohorts. This study highlights that responses to SARS-CoV-2 infection in young children are unique and reflect the ongoing maturation of the immune system into adulthood. —Courtney Malo [ABSTRACT FROM AUTHOR]

Tick-borne pathogens are responsible for many vector-borne diseases in Europe, causing important problems for human and animal health. The composition of viral communities in ticks and their interactions with pathogens is little understood, especially in Eastern Europe, an area that represents a major hub for animal-arthropod vectors exchanges. In this study, we used metatranscriptomics to characterize the virome of 2,753 Ixodes ricinus ticks collected from France and Romania, focusing on viruses that could potentially have implications for human or animal health. Among the great viral diversity of viruses identified, we reported a novel strain of Tribec virus, an important human pathogen that was found in Romanian ticks. We detected viruses belonging to the Phenuiviridae and Nairoviridae families close to human and animal pathogens, suggesting that these viruses could constitute novel arboviruses. We used luciferase immunoprecipitation system targeting external viral proteins of viruses identified among the Sedoreoviridae, Phenuiviridae, and Nairoviridae families to screen serum samples from small ruminants' exposed to tick bites. The results suggest that part (approximately 12%, 95% CI 9.1–16.2) of the small ruminant population from Danube Delta, was exposed to viruses related to bi- or tri-segmented nairoviruses, but cross-reactive viruses could not be confirmed with certainly. The strategy developed in this study serves as a key step in predicting potential new disease outbreaks and can be readily adapted to study other reservoirs, vectors, and interfaces involving susceptible hosts. [ABSTRACT FROM AUTHOR]

Bats are a major reservoir of zoonotic viruses, including coronaviruses. Since the emergence of SARS-CoV in 2002/2003 in Asia, important efforts have been made to describe the diversity of Coronaviridae circulating in bats worldwide, leading to the discovery of the precursors of epidemic and pandemic sarbecoviruses in horseshoe bats. We investigated the viral communities infecting horseshoe bats living in Northern Vietnam, and report here the first identification of sarbecoviruses in Rhinolophus thomasi and Rhinolophus siamensis bats. Phylogenetic characterization of seven strains of Vietnamese sarbecoviruses identified at least three clusters of viruses. Recombination and cross-species transmission between bats seemed to constitute major drivers of virus evolution. Vietnamese sarbecoviruses were mainly enteric, therefore constituting a risk of spillover for guano collectors or people visiting caves. To evaluate the zoonotic potential of these viruses, we analyzed in silico and in vitro the ability of their RBDs to bind to mammalian ACE2s and concluded that these viruses are likely restricted to their bat hosts. The workflow applied here to characterize the spillover potential of novel sarbecoviruses is of major interest for each time a new virus is discovered, in order to concentrate surveillance efforts on high-risk interfaces. [ABSTRACT FROM AUTHOR]

The diversity and circulation of arboviruses are not much studied in Madagascar. The fact is that arboviral emergences are rarely detected. The existing surveillance system primarily relies on serological detection and records only a few human infections annually. The city of Mahajanga, however, experienced a confirmed dengue fever epidemic in 2020 and 2021. This study aimed to characterize and analyze the virome of mosquitoes collected in Mahajanga, near patients with dengue-like syndromes to detect known and unknown viruses as well as investigate the factors contributing to the relative low circulation of arboviruses in the area. A total of 4280 mosquitoes representing at least 12 species from the Aedes, Anopheles, and Culex genera were collected during the dry and the rainy seasons from three sites, following an urbanization gradient. The virome analysis of 2192 female mosquitoes identified a diverse range of viral families and genera and revealed different patterns that are signatures of the influence of the mosquito genus or the season of collection on the composition and abundance of the virome. Despite the absence of known human or veterinary arboviruses, the identification and characterization of viral families, genera, and species in the mosquito virome contribute to our understanding of viral ecology and diversity within mosquito populations in Madagascar. This study serves as a foundation for ongoing surveillance efforts and provides a basis for the development of preventive strategies against various mosquito-borne viral diseases, including known arboviruses. [ABSTRACT FROM AUTHOR]

Arthropod-borne viruses (arboviruses) pose a significant global health threat and are primarily transmitted by mosquitoes. In Cambodia, there are currently 290 recorded mosquito species, with at least 17 of them considered potential vectors of arboviruses to humans. Effective surveillance of virome profiles in mosquitoes from Cambodia is vital, as it could help prevent and control arbovirus diseases in a country where epidemics occur frequently. The objective of this study was to identify and characterize the viral diversity in mosquitoes collected during a one-year longitudinal study conducted in various habitats across Cambodia. For this purpose, we used a metatranscriptomics approach and detected the presence of chikungunya virus in the collected mosquitoes. Additionally, we identified viruses categorized into 26 taxa, including those known to harbor arboviruses such as Flaviviridae and Orthomyxoviridae, along with a group of viruses not yet taxonomically identified and provisionally named "unclassified viruses". Interestingly, the taxa detected varied in abundance and composition depending on the mosquito genus, with no significant influence of the collection season. Furthermore, most of the identified viruses were either closely related to viruses found exclusively in insects or represented new viruses belonging to the Rhabdoviridae and Birnaviridae families. The transmission capabilities of these novel viruses to vertebrates remain unknown. [ABSTRACT FROM AUTHOR]

The improvement of our knowledge of the virosphere, which includes unknown viruses, is a key area in virology. Metagenomics tools, which perform taxonomic assignation from high throughput sequencing datasets, are generally evaluated with datasets derived from biological samples or in silico spiked samples containing known viral sequences present in public databases, resulting in the inability to evaluate the capacity of these tools to detect novel or distant viruses. Simulating realistic evolutionary directions is therefore key to benchmark and improve these tools. Additionally, expanding current databases with realistic simulated sequences can improve the capacity of alignment-based searching strategies for finding distant viruses, which could lead to a better characterization of the "dark matter" of metagenomics data. Here, we present Virus Pop, a novel pipeline for simulating realistic protein sequences and adding new branches to a protein phylogenetic tree. The tool generates simulated sequences with substitution rate variations that are dependent on protein domains and inferred from the input dataset, allowing for a realistic representation of protein evolution. The pipeline also infers ancestral sequences corresponding to multiple internal nodes of the input data phylogenetic tree, enabling new sequences to be inserted at various points of interest in the group studied. We demonstrated that Virus Pop produces simulated sequences that closely match the structural and functional characteristics of real protein sequences, taking as an example the spike protein of sarbecoviruses. Virus Pop also succeeded at creating sequences that resemble real sequences not included in the databases, which facilitated the identification of a novel pathogenic human circovirus not included in the input database. In conclusion, Virus Pop is helpful for challenging taxonomic assignation tools and could help improve databases to better detect distant viruses. [ABSTRACT FROM AUTHOR]

Bat sarbecovirus BANAL‐236 is highly related to SARS‐CoV‐2 and infects human cells, albeit lacking the furin cleavage site in its spike protein. BANAL‐236 replicates efficiently and pauci‐symptomatically in humanized mice and in macaques, where its tropism is enteric, strongly differing from that of SARS‐CoV‐2. BANAL‐236 infection leads to protection against superinfection by a virulent strain. We find no evidence of antibodies recognizing bat sarbecoviruses in populations in close contact with bats in which the virus was identified, indicating that such spillover infections, if they occur, are rare. Six passages in humanized mice or in human intestinal cells, mimicking putative early spillover events, select adaptive mutations without appearance of a furin cleavage site and no change in virulence. Therefore, acquisition of a furin site in the spike protein is likely a pre‐spillover event that did not occur upon replication of a SARS‐CoV‐2‐like bat virus in humans or other animals. Other hypotheses regarding the origin of the SARS‐CoV‐2 should therefore be evaluated, including the presence of sarbecoviruses carrying a spike with a furin cleavage site in bats. Synopsis: A SARS‐CoV‐2‐related bat virus replicates in human models as an enteric virus and does not circulate in highly exposed human populations. Genomic sequence, tissue tropism, and pathogenicity do not evolve toward SARS‐CoV‐2 characteristics upon passaging. SARS‐CoV‐2 related bat virus BANAL‐236 replicates pauci‐symptomatically and is enterotropic in human models.BANAL‐236 infection confers protection against SARS‐CoV‐2.BANAL‐236 does not spread within human populations.Passaging of BANAL‐236 in human models does not generate a furin site and pathogenicity. [ABSTRACT FROM AUTHOR]

Tick-borne diseases are responsible for many vector-borne diseases within Europe. Recently, novel viruses belonging to a new viral family of the order Bunyavirales were discovered in numerous tick species. In this study, we used metatranscriptomics to detect the virome, including novel viruses, associated with Ixodes ricinus collected from Romania and France. A bunyavirus-like virus related to the Bronnoya virus was identified for the first time in these regions. It presents a high level of amino-acid conservation with Bronnoya-related viruses identified in I. ricinus ticks from Norway and Croatia and with the Ixodes scapularis bunyavirus isolated from a tick cell line in Japan in 2014. Phylogenetic analyses revealed that the Bronnoya viruses' sub-clade is distinct from several Bunyavirales families, suggesting that it could constitute a novel family within the order. To determine if Bronnoya viruses could constitute novel tick-borne arboviruses, a Luciferase immunoprecipitation assay for detecting antibodies in the viral glycoprotein of the Romanian Bronnoya virus was used to screen sera from small ruminants exposed to tick bites. No positive serum was detected, suggesting that this virus is probably not able to infect small ruminants. This study represents the first serological investigation of mammalian infections with a Bronnoya-like virus and an initial step in the identification of potential new emergences of tick-borne arboviruses. [ABSTRACT FROM AUTHOR]

We present Microseek, a pipeline for virus identification and discovery based on RVDB-prot, a comprehensive, curated and regularly updated database of viral proteins. Microseek analyzes metagenomic Next Generation Sequencing (mNGS) raw data by performing quality steps, de novo assembly, and by scoring the Lowest Common Ancestor (LCA) from translated reads and contigs. Microseek runs on a local computer. The outcome of the pipeline is displayed through a user-friendly and dynamic graphical interface. Based on two representative mNGS datasets derived from human tissue and plasma specimens, we illustrate how Microseek works, and we report its performances. In silico spikes of known viral sequences, but also spikes of fake Neopneumovirus viral sequences generated with variable evolutionary distances from known members of the Pneumoviridae family, were used. Results were compared to Chan Zuckerberg ID (CZ ID), a reference cloud-based mNGS pipeline. We show that Microseek reliably identifies known viral sequences and performs well for the detection of distant pseudoviral sequences, especially in complex samples such as in human plasma, while minimizing non-relevant hits. [ABSTRACT FROM AUTHOR]

Crimean-Congo hemorrhagic fever (CCHF) is a tick-borne disease that can be contracted by direct contact with viremic animals or humans. Domestic animals are accidental hosts and contribute to the spread and amplification of the virus. The main objective of this study was to provide updated information related to CCHF virus (CCHFV) infection in Southern Romania by assessing the seroprevalence of CCHF in small ruminants (sheep and goats) using a double-antigen sandwich enzyme-linked immunosorbent assay and by detection of CCHFV in engorged ticks and serum samples using real-time RT-PCR. The overall seroprevalence of CCHF in small ruminants was 37.7% (95% CI 31.7 to 43.7). No statistical seroprevalence difference was observed between the two species of ruminants (p = 0.76), but a significant difference was established between the locations (p < 0.01). No CCHFV RNA was detected in tick pools and small ruminant's sera tested by real-time RT-PCR, although the high seroprevalence to CCHFV among ruminants indicates that CCHV or a closely related virus circulates in Southern Romania. [ABSTRACT FROM AUTHOR]

Ticks are involved in the transmission of various pathogens and several tick-borne diseases cause significant problems for the health of humans and livestock. The composition of viral communities in ticks and their interactions with pathogens, is poorly understood, particularly in Eastern Europe, an area that represents a major hub for animal-arthropod vectors exchanges (e.g., via bird migrations). The aim of this study was to describe the virome of Dermacentor sp., Rhipicephalus sp. and Haemaphysalis sp. ticks collected from relatively little studied regions of Romania (Iasi and Tulcea counties) located at the intersection of various biotopes, countries and routes of migrations. We also focused the study on viruses that could potentially have relevance for human and animal health. In 2019, more than 500 ticks were collected from the vegetation and from small ruminants and analysed by high-throughput transcriptome sequencing. Among the viral communities infecting Romanian ticks, viruses belonging to the Flaviviridae, Phenuiviridae and Nairoviridae families were identified and full genomes were derived. Phylogenetic analyses placed them in clades where mammalian isolates are found, suggesting that these viruses could constitute novel arboviruses. The characterization of these communities increase the knowledge of the diversity of viruses in Eastern Europe and provides a basis for further studies about the interrelationship between ticks and tick-borne viruses. [ABSTRACT FROM AUTHOR]

The animal reservoir of SARS-CoV-2 is unknown despite reports of SARS-CoV-2-related viruses in Asian Rhinolophus bats1–4, including the closest virus from R. affinis, RaTG13 (refs. 5,6), and pangolins7–9. SARS-CoV-2 has a mosaic genome, to which different progenitors contribute. The spike sequence determines the binding affinity and accessibility of its receptor-binding domain to the cellular angiotensin-converting enzyme 2 (ACE2) receptor and is responsible for host range10–12. SARS-CoV-2 progenitor bat viruses genetically close to SARS-CoV-2 and able to enter human cells through a human ACE2 (hACE2) pathway have not yet been identified, although they would be key in understanding the origin of the epidemic. Here we show that such viruses circulate in cave bats living in the limestone karstic terrain in northern Laos, in the Indochinese peninsula. We found that the receptor-binding domains of these viruses differ from that of SARS-CoV-2 by only one or two residues at the interface with ACE2, bind more efficiently to the hACE2 protein than that of the SARS-CoV-2 strain isolated in Wuhan from early human cases, and mediate hACE2-dependent entry and replication in human cells, which is inhibited by antibodies that neutralize SARS-CoV-2. None of these bat viruses contains a furin cleavage site in the spike protein. Our findings therefore indicate that bat-borne SARS-CoV-2-like viruses that are potentially infectious for humans circulate in Rhinolophus spp. in the Indochinese peninsula.A study reports the detection and characterization of SARS-CoV-2-like viruses in Laotian cave-dwelling bats that are also demonstrated to infect human cells through the ACE2 pathway. [ABSTRACT FROM AUTHOR]

Background Human encephalitis represents a medical challenge from a diagnostic and therapeutic point of view. We investigated the cause of 2 fatal cases of encephalitis of unknown origin in immunocompromised patients. Methods Untargeted metatranscriptomics was applied on the brain tissue of 2 patients to search for pathogens (viruses, bacteria, fungi, or protozoans) without a prior hypothesis. Results Umbre arbovirus, an orthobunyavirus never previously identified in humans, was found in 2 patients. In situ hybridization and reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) showed that Umbre virus infected neurons and replicated at high titers. The virus was not detected in cerebrospinal fluid by RT-qPCR. Viral sequences related to Koongol virus, another orthobunyavirus close to Umbre virus, were found in Culex pipiens mosquitoes captured in the south of France where the patients had spent some time before the onset of symptoms, demonstrating the presence of the same clade of arboviruses in Europe and their potential public health impact. A serological survey conducted in the same area did not identify individuals positive for Umbre virus. The absence of seropositivity in the population may not reflect the actual risk of disease transmission in immunocompromised individuals. Conclusions Umbre arbovirus can cause encephalitis in immunocompromised humans and is present in Europe. [ABSTRACT FROM AUTHOR]

Elucidating seroprevalence in COVID-19: Individuals infected with SARS-CoV-2 mount an antibody response that may vary depending on the severity of the disease. Grzelak et al. assessed the amounts of anti–SARS-CoV-2 antibodies in serum samples from 491 healthy individuals before the pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. The authors developed and compared the performance of four different serological assays measuring antibody amounts and their neutralizing activity. The assays enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in asymptomatic, mildly symptomatic, and severe cases of COVID-19. It is of paramount importance to evaluate the prevalence of both asymptomatic and symptomatic cases of SARS-CoV-2 infection and their differing antibody response profiles. Here, we performed a pilot study of four serological assays to assess the amounts of anti–SARS-CoV-2 antibodies in serum samples obtained from 491 healthy individuals before the SARS-CoV-2 pandemic, 51 individuals hospitalized with COVID-19, 209 suspected cases of COVID-19 with mild symptoms, and 200 healthy blood donors. We used two ELISA assays that recognized the full-length nucleoprotein (N) or trimeric spike (S) protein ectodomain of SARS-CoV-2. In addition, we developed the S-Flow assay that recognized the S protein expressed at the cell surface using flow cytometry, and the luciferase immunoprecipitation system (LIPS) assay that recognized diverse SARS-CoV-2 antigens including the S1 domain and the carboxyl-terminal domain of N by immunoprecipitation. We obtained similar results with the four serological assays. Differences in sensitivity were attributed to the technique and the antigen used. High anti–SARS-CoV-2 antibody titers were associated with neutralization activity, which was assessed using infectious SARS-CoV-2 or lentiviral-S pseudotype virus. In hospitalized patients with COVID-19, seroconversion and virus neutralization occurred between 5 and 14 days after symptom onset, confirming previous studies. Seropositivity was detected in 32% of mildly symptomatic individuals within 15 days of symptom onset and in 3% of healthy blood donors. The four antibody assays that we used enabled a broad evaluation of SARS-CoV-2 seroprevalence and antibody profiling in different subpopulations within one region. [ABSTRACT FROM AUTHOR]

Emerging zoonoses caused by previously unknown agents are one of the most important challenges for human health because of their inherent inability to be predictable, conversely to emergences caused by previously known agents that could be targeted by routine surveillance programs. Emerging zoonotic infections either originate from increasing contacts between wildlife and human populations, or from the geographical expansion of hematophagous arthropods that act as vectors, this latter being more capable to impact large-scale human populations. While characterizing the viral communities from candidate vectors in high-risk geographical areas is a necessary initial step, the need to identify which viruses are able to spill over and those restricted to their hosts has recently emerged. We hypothesized that currently unknown tick-borne arboviruses could silently circulate in specific biotopes where mammals are highly exposed to tick bites, and implemented a strategy that combined high-throughput sequencing with broad-range serological techniques to both identify novel arboviruses and tick-specific viruses in a ticks/mammals interface in Thailand. The virome of Thai ticks belonging to the Rhipicephalus, Amblyomma, Dermacentor, Hyalomma , and Haemaphysalis genera identified numerous viruses, among which several viruses could be candidates for future emergence as regards to their phylogenetic relatedness with known tick-borne arboviruses. Luciferase immunoprecipitation system targeting external viral proteins of viruses identified among the Orthomyxoviridae, Phenuiviridae, Flaviviridae, Rhabdoviridae , and Chuviridae families was used to screen human and cattle Thai populations highly exposed to tick bites. Although no positive serum was detected for any of the six viruses selected, suggesting that these viruses are not infecting these vertebrates, or at very low prevalence (upper estimate 0.017% and 0.047% in humans and cattle, respectively), the virome of Thai ticks presents an extremely rich viral diversity, among which novel tick-borne arboviruses are probably hidden and could pose a public health concern if they emerge. The strategy developed in this pilot study, starting from the inventory of viral communities of hematophagous arthropods to end by the identification of viruses able (or likely unable) to infect vertebrates, is the first step in the prediction of putative new emergences and could easily be transposed to other reservoirs/vectors/susceptible hosts interfaces. [ABSTRACT FROM AUTHOR]

Illegal bushmeat traffic is an important threat to biodiversity conservation of several endangered species and may contribute to the emergence and spread of infectious diseases in humans. The hunting, manipulation and consumption of wildlife-based products, especially those of primate origin, may be a threat to human health; however, few studies have investigated the role of bushmeat trade and consumption as a potential source of human infections to date. In this study, we report the screening of viral pathogens in African simian game seized by French customs at Toulouse Blagnac Airport. Epifluorescence microscopy revealed the presence of virus-like particles in the samples, and further metagenomic sequencing of the DNA and RNA viromes confirmed the presence of sequences related to the Siphoviridae, Myoviridae and Podoviridae bacteriophage families; some of them infecting bacterial hosts that could be potentially pathogenic for humans. To increase the sensitivity of detection, twelve pan-generic PCRs targeting several viral zoonoses were performed, but no positive signal was detected. A large-scale inventory of bacteria, viruses and parasites is urgently needed to globally assess the risk for human health of the trade, manipulation and consumption of wildlife-related bushmeat. [ABSTRACT FROM AUTHOR]

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More than two thirds of emerging viruses are of zoonotic origin, and among them RNA viruses represent the majority. Ceratopogonidae (genus Culicoides) are well-known vectors of several viruses responsible for epizooties (bluetongue, epizootic haemorrhagic disease, etc.). They are also vectors of the only known virus infecting humans: the Oropouche virus. Female midges usually feed on a variety of hosts, leading to possible transmission of emerging viruses from animals to humans. In this context, we report here the analysis of RNA viral communities of Senegalese biting midges using next-generation sequencing techniques as a preliminary step toward the identification of potential viral biohazards. Sequencing of the RNA virome of three pools of Culicoides revealed the presence of a significant diversity of viruses infecting plants, insects and mammals. Several novel viruses were detected, including a novel Thogotovirus species, related but genetically distant from previously described tick-borne thogotoviruses. Novel rhabdoviruses were also detected, possibly constituting a novel Rhabdoviridae genus, and putatively restricted to insects. Sequences related to the major viruses transmitted by Culicoides, i.e., African horse sickness, bluetongue and epizootic haemorrhagic disease viruses were also detected. This study highlights the interest in monitoring the emergence and circulation of zoonoses and epizooties using their arthropod vectors. [ABSTRACT FROM AUTHOR]

Faustovirus, a new Asfarviridae-related giant virus, was recently isolated in Vermamoeba vermiformis, a protist found in sewage water in various geographical locations and occasionally reported in human eye infection cases. As part of a global metagenomic analysis of viral communities existing in biting midges, we report here for the first time the identification and isolation of a Faustovirus-like virus in hematophagous arthropods and its detection in their animal hosts. The DNA virome analysis of three pools of Culicoides sp., engorged female Culicoides imicola and non-engorged male/female C. imicola biting midges collected in Senegal, revealed the presence of amoeba-infecting giant viruses and, among them, a majority of sequences related to Faustovirus. Phylogenetic analyses conducted on several structural genes of Faustovirus confirmed the clustering of the arthropod-borne Faustovirus with sewage-borne Faustoviruses, with a distinct geographical clustering of Senegalese Faustovirus strains. Transmission electron microscopy identified viral particles with morphologies and diameters which were compatible with Faustovirus. The presence of infectious arthropod-borne Faustovirus was finally confirmed by successful isolation on V. vermiformis amoeba. Global proteomic analysis of biting midges identified that arthropods' blood meal originating from cattle, rodents and humans. Further screening of cattle sera and rodent tissue resulted in prevalence of Faustovirus being estimated at 38% in rodents and 14% in cattle, suggesting a possible origin of Faustovirus presence in arthropods via the ingestion of contaminated blood meal. Viral loads were the highest in rodents' urine and kidney samples, suggesting a possible excretion of viral particles into the environment. Faustovirus DNA polymerase-related sequences were also detected in more than 9 and 11% of febrile patients and healthy Senegalese human sera, respectively. Our study thus, highlights the need to investigate the role of arthropods, wildlife, and domestic animals in the lifecycle of amoeba-infecting giant viruses and, in particular, the environmental cycle of Faustovirus. [ABSTRACT FROM AUTHOR]

Background: Metagenomic analyses have been widely used in the last decade to describe viral communities in various environments or to identify the etiology of human, animal, and plant pathologies. Here, we present a simple and standardized protocol that allows for the purification and sequencing of RNA viromes from complex biological samples with an important reduction of host DNA and RNA contaminants, while preserving the infectivity of viral particles. Principal Findings: We evaluated different viral purification steps, random reverse transcriptions and sequence-independent amplifications of a pool of representative RNA viruses. Viruses remained infectious after the purification process. We then validated the protocol by sequencing the RNA virome of human body lice engorged in vitro with artificially contaminated human blood. The full genomes of the most abundant viruses absorbed by the lice during the blood meal were successfully sequenced. Interestingly, random amplifications differed in the genome coverage of segmented RNA viruses. Moreover, the majority of reads were taxonomically identified, and only 7–15% of all reads were classified as “unknown”, depending on the random amplification method. Conclusion: The protocol reported here could easily be applied to generate RNA viral metagenomes from complex biological samples of different origins. Our protocol allows further virological characterizations of the described viral communities because it preserves the infectivity of viral particles and allows for the isolation of viruses. [ABSTRACT FROM AUTHOR]

Background: Rickettsia africae is the agent of African tick bite fever, a disease transmitted by ticks in sub-Saharan Africa. In Union of the Comoros, a recent study reported the presence of a Rickettsia africae vector but no information has been provided on the circulation of the pathogenic agent in this country. Methods: To evaluate the possible circulation of Rickettsia spp. in Comorian cattle, genomic DNA was extracted from 512 ticks collected either in the Union of the Comoros or from animals imported from Tanzania and subsequently tested for Rickettsia infection by quantitative PCR. Results: Rickettsia africae was detected in 90% (60/67) of Amblyomma variegatum, 1% (1/92) of Rhipicephalus appendiculatus and 2.7% (8/296) of Rhipicephalus (Boophilus) microplus ticks collected in the Union of the Comoros, as well as in 77.14% (27/35) of Amblyomma variegatum ticks collected from imported cattle. Partial sequences of both bacterial gltA and ompA genes were used in a phylogenetic analysis revealing the presence of several haplotypes, all included within the Rickettsia africae clade. Conclusions: Our study reports the first evidence of Rickettsia africae in ticks collected from the Union of the Comoros. The data show a significant difference of infection rate of Rickettsia africae infected ticks between the Islands, with maximum rates measured in Grande Comore Island, sheltering the main entry port for live animal importation from Tanzania. The high infection levels reported herein indicate the need for an in-depth assessment of the burden of rickettsioses in the Union of the Comoros, especially among those at risk of infection, such as cattle herders. [ABSTRACT FROM AUTHOR]

Many human viral infections have a zoonotic, i.e., wild or domestic animal, origin. Several zoonotic viruses are transmitted to humans directly via contact with an animal or indirectly via exposure to the urine or feces of infected animals or the bite of a bloodsucking arthropod. If a virus is able to adapt and replicate in its new human host, human-to-human transmissions may occur, possibly resulting in an epidemic, such as the A/H1N1 flu pandemic in 2009. Thus, predicting emerging zoonotic infections is an important challenge for public health officials in the coming decades. The recent development of viral metagenomics, i.e., the characterization of the complete viral diversity isolated from an organism or an environment using high-throughput sequencing technologies, is promising for the surveillance of such diseases and can be accomplished by analyzing the viromes of selected animals and arthropods that are closely in contact with humans. In this review, we summarize our current knowledge of viral diversity within such animals (in particular blood-feeding arthropods, wildlife and domestic animals) using metagenomics and present its possible future application for the surveillance of zoonotic and arboviral diseases. [ABSTRACT FROM AUTHOR]

Viruses are the most abundant obligate intracellular entities in our body. Until recently, they were only considered to be pathogens that caused a broad array of pathologies, ranging from mild disease to deaths in the most severe cases. However, recent advances in unbiased mass sequencing techniques as well as increasing epidemiological evidence have indicated that the human body is home to diverse viral species under non-pathological conditions. Despite these studies, the description of the presumably healthy viral flora, i.e. the normal human virome, is still in its infancy regarding viral composition and dynamics. This review summarizes our current knowledge of the human virome under non-pathological conditions. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

During 2009, pandemic influenza A(H1N1)pdm09 virus affected humans on Réunion Island. Since then, the virus has sustained circulation among local swine herds, raising concerns about the potential for genetic evolution of the virus and possible retransmission back to humans of variants with increased virulence. Continuous surveillance of A(H1N1)pdm09 infection in pigs is recommended. Influenza A(H1N1)pdm09 virus, which caused the last influenza pandemic among humans, is a unique reassortant derived from swine influenza viruses of the triple reassortant swine North American lineage and the avian-like swine Eurasian lineage (1). Réunion Island, a tropical French overseas department in the southwestern Indian Ocean, was struck by the influenza pandemic during July-August 2009. The epidemic had a high attack rate in humans (estimated clinically at 12.5% and serologically at 40.0%) (2,3). A(H1N1)pdm09 virus was reported to cause a reverse zoonosis in pigs (4); thus, a long-term (2009-2011) serologic and virologic survey was designed to check for transmission of the virus to pigs on Réunion Island, where the pork industry is a key economic activity and no live pigs have been imported since 1978. At 6-month intervals, a local veterinary surveillance system conducts serologic surveillance for pathogenic swine influenza viruses (H1N1, H1N2, and H3N2) among local herds, and during the last 20 years, none have been detected. [ABSTRACT FROM AUTHOR]

Objective: The aim of the present study was to weigh up, at the community level, the respective roles played by pandemic Influenza (pH1N1) virus and co-circulating human Non-Influenza Respiratory Viruses (NIRVs) during the first wave of the 2009 pH1N1 pandemic. Methods: A population-based prospective cohort study was conducted in Reunion Island during the austral winter 2009 (weeks 30-44) that allowed identification of 125 households with at least one member who developed symptoms of Influenza-like illness (ILI). Three consecutive nasal swabs were collected from each household member (443 individuals) on day 0, 3 and 8 post-ILI report and tested for pH1N1 and 15 NIRVs by RT-PCR. Results: Two successive waves of viral infections were identified: a first wave (W33-37) when pH1N1 was dominant and co-circulated with NIRVs, sharply interrupted by a second wave (W38-44), almost exclusively composed of NIRVs, mainly human Rhinoviruses (hRV) and Coronaviruses (hCoV). Data suggest that some interference may occur between NIRVs and pH1N1 when they co-circulate within the same household, where NIRVs were more likely to infect pH1N1 negative individuals than pH1N1 positive peers (relative risk: 3.13, 95% CI: 1.80-5.46, P,0.001). Viral shedding was significantly shorter (P = 0.035) in patients who were co-infected by pH1N1 and NIRV or by two different NIRVs compared to those who were infected with only one virus, whatever this virus was (pH1N1 or NIRVs). Although intense co-circulation of NIRVs (especially hRV) likely brought pH1N1 under the detection threshold, it did not prevent spread of the pandemic Influenza virus within the susceptible population nor induction of an extensive herd immunity to it. Conclusion: Our results suggest that NIRV co-infections during Influenza epidemics may act as cofactors that contribute to shape an outbreak and modulate the attack rate. They further warrant broad spectrum studies to fully understand viral epidemics. [ABSTRACT FROM AUTHOR]

Background/Objectives: Molecular epidemiology is a powerful tool to decipher the dynamics of viral transmission, quasispecies temporal evolution and origins. Little is known about the pH1N1 molecular dynamics in general population. A prospective study (CoPanFlu-RUN) was carried out in Reunion Island to characterize pH1N1 genetic variability and molecular evolution occurring in population during the pH1N1 Influenza pandemic in 2009. Methodology: We directly amplified pH1N1 genomes from 28 different nasal swabs (26 individuals from 21 households). Fifteen strains were fully sequenced and 13 partially. This includes pairs of sequences from different members of 5 separate households; and two pairs from individuals, collected at different times. We assessed the molecular evolution of pH1N1 by genetic variability and phylogenetic analyses. Principal Findings: We found that i) Reunion pH1N1 sequences stemmed from global "clade 7" but shaped two phylogenetic sub-clades; ii) D239E mutation was identified in the hemagglutinin protein of all Reunion sequences, a mutation which has been associated elsewhere with mild-, upper-respiratory tract pH1N1 infecting strains; iii) Date estimates from molecular phylogenies predicted clade emergence some time before the first detection of pH1N1 by the epidemiological surveillance system; iv) Phylogenetic relatedness was observed between Reunion pH1N1 viruses and those from other countries in South-western Indian Ocean area; v) Quasispecies populations were observed within households and individuals of the cohort-study. Conclusions: Surveillance and/or prevention systems presently based on Influenza virus sequence variation should take into account that the majority of studies of pH1N1 Influenza generate genetic data for the HA/NA viral segments obtained from hospitalized-patients, which is potentially non-representative of the overall viral diversity within whole populations. Our observations highlight the importance of collecting unbiased data at the community level and conducting whole genome analysis to accurately understand viral dynamics. [ABSTRACT FROM AUTHOR]

Background: To date, there is little information that reflects the true extent of spread of the pH1N1/2009v influenza pandemic at the community level as infection often results in mild or no clinical symptoms. This study aimed at assessing through a prospective study, the attack rate of pH1N1/2009 virus in Reunion Island and risk factors of infection, during the 2009 season. Methodology/Principal Findings: A serosurvey was conducted during the 2009 austral winter, in the frame of a prospective population study. Pairs of sera were collected from 1687 individuals belonging to 772 households, during and after passage of the pandemic wave. Antibodies to pH1N1/2009v were titered using the hemagglutination inhibition assay (HIA) with titers $1/40 being considered positive. Seroprevalence during the first two weeks of detection of pH1N1/2009v in Reunion Island was 29.8% in people under 20 years of age, 35.6% in adults (20-59 years) and 73.3% in the elderly ($60 years) (P,0.0001). Baseline corrected cumulative incidence rates, were 42.9%, 13.9% and 0% in these age groups respectively (P,0.0001). A significant decline in antibody titers occurred soon after the passage of the epidemic wave. Seroconversion rates to pH1N1/2009 correlated negatively with age: 63.2%, 39.4% and 16.7%, in each age group respectively (P,0.0001). Seroconversion occurred in 65.2% of individuals who were seronegative at inclusion compared to 6.8% in those who were initially seropositive. Conclusions: Seroincidence of pH1N1/2009v infection was three times that estimated from clinical surveillance, indicating that almost two thirds of infections occurring at the community level have escaped medical detection. People under 20 years of age were the most affected group. Pre-epidemic titers $1/40 prevented seroconversion and are likely protective against infection. A concern was raised about the long term stability of the antibody responses. [ABSTRACT FROM AUTHOR]

Toscana virus (TOSV), an arthropodborne phlebovirus transmitted by sandflies, can cause febrile illness and meningitis. The vector of TOSV in France was unknown. We detected TOSV RNA in 2 (female Phlebotomus perniciosus) of 61 pools of sandflies captured in southeastern France. Two genotypes of TOSV were identified. [ABSTRACT FROM AUTHOR]

Clinical metagenomics is a broad-range agnostic detection method of pathogens, including novel microorganisms. A major limit is the low pathogen load compared to the high background of host nucleic acids. To overcome this issue, several solutions exist, such as applying a very high depth of sequencing, or performing a relative enrichment of viral genomes associated with capsids. At the end, the quantity of total nucleic acids is often below the concentrations recommended by the manufacturers of library kits, which necessitates to random amplify nucleic acids. Using a pool of 26 viruses representative of viral diversity, we observed a deep impact of the nature of sample (total nucleic acids versus RNA only), the reverse transcription, the random amplification and library construction method on virus recovery. We further optimized the two most promising methods and assessed their performance with fully characterized reference virus stocks. Good genome coverage and limit of detection lower than 100 or 1000 genome copies per mL of plasma, depending on the genome viral type, were obtained from a three million reads dataset. Our study reveals that optimized random amplification is a technique of choice when insufficient amounts of nucleic acid are available for direct libraries constructions. [ABSTRACT FROM AUTHOR]

The article discusses a study which examined the incidence of Leptospira infections in island wild fauna, insectivorous and frugivorous bats in Madagascar and Union of the Comoros. To obtain complementary deoxribonucleic acid (cDNA), reverse transcription and GoScript reverse transcriptase were performed. According to the study, 11 out of 12 bat species tested positive for Leptospira. It also found that infected bats were prevalent in the study sites.

To determine whether sandfly fever Sicilian virus (SFSV) is present in Algeria, we tested sandflies for phlebovirus RNA. A sequence closely related to that of SFSV was detected in a Phlebotomus ariasi sandfl y. Of 60 human serum samples, 3 contained immunoglobulin G against SFSV. These data suggest SFSV is present in Algeria. [ABSTRACT FROM AUTHOR]

Evidence for the tickborne nature of Alkhurma hemorrhagic fever virus (AHFV) is indirect because AHFV has not been detected in arthropods. One Ornithodoros savignyi tick from Saudi Arabia contained AHFV RNA. This is the first direct evidence that AHFV is a tickborne flavivirus and confirms the association between human AHFV cases and tickbite history. [ABSTRACT FROM AUTHOR]

Worldwide, emerging and re-emerging infectious diseases (EIDs) are a major burden on public and animal health. Arthropod vectors, with mosquitoes being the main contributors of global disease, transmit more than 70% of the recognized EIDs. To assess new alternatives for arthropod-borne viral diseases surveillance, and for the detection of new viruses, honey-baited Flinders Technology Associates (FTA) cards were used as sugar bait in mosquito traps during entomological surveys at the Llobregat River Delta (Catalonia, Spain). Next generation sequencing (NGS) metagenomics analysis was applied on honey-baited FTA cards, which had been exposed to field-captured mosquitoes to characterize their associated virome. Arthropod- and plant-infecting viruses governed the virome profile on FTA cards. Twelve near-complete viral genomes were successfully obtained, suggesting good quality preservation of viral RNAs. Mosquito pools linked to the FTA cards were screened for the detection of mosquito-associated viruses by specific RT-PCRs to confirm the presence of these viruses. The circulation of viruses related to Alphamesonivirus, Quaranjavirus and unclassified Bunyavirales was detected in mosquitoes, and phylogenetic analyses revealed their similarities to viruses previously reported in other continents. To the best our knowledge, our findings constitute the first distribution record of these viruses in European mosquitoes and the first hint of insect-specific viruses in mosquitoes' saliva in field conditions, demonstrating the feasibility of this approach to monitor the transmissible fraction of the mosquitoes' virome. In conclusion, this pilot viromics study on honey-baited FTA cards was shown to be a valid approach for the detection of viruses circulating in mosquitoes, thereby setting up an alternative tool for arbovirus surveillance and control programs. [ABSTRACT FROM AUTHOR]

Ticks transmit a wide variety of pathogens including bacteria, parasites and viruses. Over the last decade, numerous novel viruses have been described in arthropods, including ticks, and their characterization has provided new insights into RNA virus diversity and evolution. However, little is known about their ability to infect vertebrates. As very few studies have described the diversity of viruses present in ticks from the Caribbean, we implemented an RNA-sequencing approach on Amblyomma variegatum and Rhipicephalus microplus ticks collected from cattle in Guadeloupe and Martinique. Among the viral communities infecting Caribbean ticks, we selected four viruses belonging to the Chuviridae, Phenuiviridae and Flaviviridae families for further characterization and designing antibody screening tests. While viral prevalence in individual tick samples revealed high infection rates, suggesting a high level of exposure of Caribbean cattle to these viruses, no seropositive animals were detected. These results suggest that the Chuviridae- and Phenuiviridae-related viruses identified in the present study are more likely tick endosymbionts, raising the question of the epidemiological significance of their occurrence in ticks, especially regarding their possible impact on tick biology and vector capacity. The characterization of these viruses might open the door to new ways of preventing and controlling tick-borne diseases. [ABSTRACT FROM AUTHOR]

A letter to the editor about Toscana virus is presented.