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There are currently no antivirals available to treat infection with enterovirus A71 (EV-A71) or any other enterovirus. The extensively studied capsid binders rapidly select for drug-resistant variants. We here explore whether the combination of two direct-acting enterovirus inhibitors with a different mechanism of action may delay or prevent resistance development to the capsid binders. To that end, the in vitro dynamics of resistance development to the capsid binder pirodavir was studied either alone or in combination with a viral 2C-targeting compound (SMSK_0213), a viral 3C-protease inhibitor (rupintrivir) or a viral RNA-dependent RNA polymerase inhibitor (7DMA). We demonstrate that combining pirodavir with either rupintrivir or 7DMA delays the development of resistance to pirodavir and that no resistance to the protease or polymerase inhibitor develops. The combination of pirodavir with the 2C inhibitor results in a double-resistant virus population, where only the minority carries the resistant mutation. ispartof: ACS INFECTIOUS DISEASES vol:7 issue:10 pages:2801-2806 ispartof: location:United States status: published

Crangon crangon is economically a very important species. Recently, promising culture attempts have been made, but a major problem is the uncontrollable mortality during the grow-out phase. As of yet, the life cycle of C. crangon is not closed in captivity so wild-caught individuals are used for further rearing. Therefore, it is important to investigate the virome of C. crangon both in wild-caught animals as in cultured animals. In recent years, next-generation-sequencing (NGS) technologies have been very important in the unravelling of the virome of a wide range of environments and matrices, such as soil, sea, potable water, but also of a wide range of animal species. This will be the first report of a virome study in C. crangon using NGS in combination with the NetoVIR protocol. The near complete genomes of 16 novel viruses were described, most of which were rather distantly related to unclassified viruses or viruses belonging to the Picornavirales, Bunyavirales Nudiviridae, Parvoviridae, Flaviviridae, Hepeviridae, Tombusviridae, Narnaviridae, Nodaviridae, Sobemovirus. A difference in virome composition was observed between muscle and hepatopancreatic tissue, suggesting a distinct tissue tropism of several of these viruses. Some differences in the viral composition were noted between the cultured and wild shrimp, which could indicate that in sub-optimal aquaculture conditions some viruses become more abundant. This research showed that a plethora of unknown viruses is present in C. crangon and that more research is needed to determine which virus is potentially dangerous for the culture of C. crangon.

Culex modestus mosquitoes are considered potential transmission vectors of West Nile virus and Usutu virus. Their presence has been reported across several European countries, including one larva detected in Belgium in 2018. In this study, mosquitoes were collected in the city of Leuven and surrounding areas in the summers of 2019 and 2020. Species identification was performed based on morphological features and partial sequences of the mitochondrial cytochrome oxidase subunit I (COI) gene. The 107 mosquitoes collected in 2019 belonged to eight mosquito species, Culex pipiens (24.3%), Cx. modestus (48.6%), Cx. torrentium (0.9%), Culiseta annulata (0.9%), Culiseta morsitans (0.9%), Aedes sticticus (14.0%), Aedes cinereus (9.3%), and Anopheles plumbeus (0.9%), suggesting the presence of an established Cx. modestus population in Belgium. The collection of Cx. modestus mosquitoes at the same locations in 2020 confirmed their establishment in the region. Haplotype network analysis of the COI sequences for Cx. modestus showed that the Belgian population is rather diverse, suggesting that it may have been established in Belgium for some time. The Belgian Cx. modestus population was most closely related to populations from the United Kingdom and Germany. Characterization of the virome of the collected mosquitoes resulted in the identification of at least 33 eukaryotic viral species. Nine (nearly) complete genomes belonging to 6 viral species were identified, all of which were closely related to known viruses. In conclusion, here, we report the presence of Cx. modestus in the surrounding areas of Leuven, Belgium. As this species is considered to be a vector of several arboviruses, the implementation of vector surveillance programs to monitor this species is recommended.IMPORTANCECulex modestus mosquitoes are considered to be a potential "bridge" vector, being able to transmit pathogens between birds as well as from birds to mammals, including humans. In Belgium, this mosquito species was considered absent until the finding of one larva in 2018 and subsequent evidence of a large population in 2019 to 2020 described here. We collected mosquitoes in the summers of 2019 and 2020 in the city of Leuven and surrounding areas. The mosquito species was identified by morphological and molecular methods, demonstrating the presence of Cx. modestus in this region. The ability of mosquitoes to transmit pathogens can depend on several factors, one of them being their natural virus composition. Therefore, we identified the mosquito-specific viruses harbored by Belgian mosquitoes. As Cx. modestus is able to transmit viruses such as West Nile virus and Usutu virus, the establishment of this mosquito species may increase the risk of virus transmission in the region. It is thus advisable to implement mosquito surveillance programs to monitor this species. ispartof: MSPHERE vol:6 issue:2 ispartof: location:United States status: accepted

Our study revealed that the virome was very stable across all developmental stages of both lab-derived and field-collected Aedes albopictus. The data representing the core virome in lab A. albopictus proved the vertical transmission route of these viruses, forming a “vertically transmitted core virome.” Field mosquitoes also contained this stable vertically transmitted core virome as well as additional viruses, which probably represented “environment-derived core virome” and which therefore were less stable over time and geography. By further screening publicly available SRA viral metagenomic data sets from mosquitoes belonging to the genus Aedes, some of the identified core ISVs were shown to be present in the majority of SRAs, such as Phasi Charoen-like phasivirus and Guadeloupe mosquito virus. How these core ISVs influence the biology of the mosquito host and arbovirus infection and evolution deserves to be further explored., Aedes mosquitoes can efficiently transmit many pathogenic arboviruses, placing a great burden on public health worldwide. In addition, they also carry a number of insect-specific viruses (ISVs), and it was recently suggested that some of these ISVs might form a stable species-specific “core virome” in mosquito populations. However, little is known about such a core virome in laboratory colonies and if it is present across different developmental stages. In this study, we compared the viromes in eggs, larvae, pupae, and adults of Aedes albopictus mosquitoes collected from a lab colony and compared each to the virome of different developmental stages collected in the field. The virome in lab-derived A. albopictus was very stable across all stages, consistent with a vertical transmission route of these viruses, and formed a possible “vertically transmitted core virome.” The different stages of field-collected A. albopictus mosquitoes also contained this stable vertically transmitted core virome, as well as another set of viruses (e.g., viruses distantly related to Guadeloupe mosquito virus, Hubei virga-like virus 2, and Sarawak virus) shared by mosquitoes across different stages, which might represent an “environment-derived core virome.” To further study this core set of ISVs, we screened 48 publicly available SRA viral metagenomic data sets of mosquitoes belonging to the genus Aedes, showing that some of the identified ISVs were identified in the majority of SRAs and providing further evidence supporting the core-virome concept. IMPORTANCE Our study revealed that the virome was very stable across all developmental stages of both lab-derived and field-collected Aedes albopictus. The data representing the core virome in lab A. albopictus proved the vertical transmission route of these viruses, forming a “vertically transmitted core virome.” Field mosquitoes also contained this stable vertically transmitted core virome as well as additional viruses, which probably represented “environment-derived core virome” and which therefore were less stable over time and geography. By further screening publicly available SRA viral metagenomic data sets from mosquitoes belonging to the genus Aedes, some of the identified core ISVs were shown to be present in the majority of SRAs, such as Phasi Charoen-like phasivirus and Guadeloupe mosquito virus. How these core ISVs influence the biology of the mosquito host and arbovirus infection and evolution deserves to be further explored.

There are currently no antivirals available to treat infection with enterovirus A71 (EV-A71) or any other enterovirus. The extensively studied capsid binders select rapidly for drug-resistant variants. We here explore whether the combination of two direct-acting enterovirus inhibitors with a different mechanism of action may delay or prevent resistance development to the capsid binders. To that end, the in vitro dynamics of resistance development to the capsid binder pirodavir was studied either alone or in combination with (i) a viral 2C-targeting compound (SMSK_0213), (ii) a viral 3C-protease inhibitor (rupintrivir) or (iii) a viral RNA-dependent RNA polymerase (RdRp) inhibitor [7-deaza-2’C-methyladenosine (7DMA)]. We demonstrate that combining pirodavir with either rupintrivir or the nucleoside analogue 7DMA delays the development of resistance to pirodavir and that no resistance to the protease or polymerase inhibitor develops. The combination of pirodavir with the 2C inhibitor results in a double-resistant virus population. The deep sequencing analysis of resistant populations revealed that even though resistant mutations are present in less than 30% of the population, this still provides the resistant phenotype.

Flaviviruses encompass not only medically relevant arthropod-borne viruses (arboviruses) but also insect-specific flaviviruses (ISFs) that are presumably maintained primarily through vertical transmission in the insect host. Interestingly, ISFs are commonly found infecting important arbovirus vectors such as the mosquito Aedes aegypti. Cell-fusing agent virus (CFAV) was the first described ISF of mosquitoes more than four decades ago. Despite evidence for widespread CFAV infections in A.aegypti populations and for CFAV potential to interfere with arbovirus transmission, little is known about CFAV evolutionary history. Here, we generated six novel CFAV genome sequences by sequencing three new virus isolates and subjecting three mosquito samples to untargeted viral metagenomics. We used these new genome sequences together with published ones to perform a global phylogenetic analysis of CFAV genetic diversity. Although there was some degree of geographical clustering among CFAV sequences, there were also notable discrepancies between geography and phylogeny. In particular, CFAV sequences from Cambodia and Thailand diverged significantly, despite confirmation that A.aegypti populations from both locations are genetically close. The apparent phylogenetic discrepancy between CFAV and its A.aegypti host in Southeast Asia indicates that other factors than host population structure shape CFAV genetic diversity. ispartof: VIRUS EVOLUTION vol:6 issue:1 ispartof: location:England status: published

Vast viruses are thought to be associated with mosquitoes. Anopheles sinensis, Armigeres subalbatus, Culex quinquefasciatus, and Culex tritaeniorhynchus are very common mosquito species in China, and whether the virome structure in each species is species-specific has not been evaluated. In this study, a total of 2222 mosquitoes were collected from the same geographic location, and RNAs were sequenced using the Illumina Miseq platform. After querying to the Refseq database, a total of 3,435,781, 2,223,509, 5,727,523, and 6,387,867 paired-end reads were classified under viral sequences from An. sinensis, Ar. subalbatus, Cx. quinquefasciatus, and Cx. tritaeniorhynchus, respectively, with the highest prevalence of virus-associated reads being observed in Cx. quinquefasciatus. The metagenomic comparison analysis showed that the virus-related reads were distributed across 26 virus families, together with an unclassified group of viruses. Anelloviridae, Circoviridae, Genomoviridae, Iridoviridae, Mesoniviridae, Microviridae, Myoviridae, Parvoviridae, Phenuiviridae, and Podoviridae were the top ten significantly different viral families among the four species. Further analysis reveals that the virome is species-specific in four mosquito samples, and several viral sequences which maybe belong to novel viruses are discovered for the first time in those mosquitoes. This investigation provides a basis for a comprehensive knowledge on the mosquito virome status in China. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s12250-018-0015-4) contains supplementary material, which is available to authorized users.

Lymphatic vessels are lined by lymphatic endothelial cells (LECs), and are critical for health. However, the role of metabolism in lymphatic development has not yet been elucidated. Here we report that in transgenic mouse models, LEC-specific loss of CPT1A, a rate-controlling enzyme in fatty acid β-oxidation, impairs lymphatic development. LECs use fatty acid β-oxidation to proliferate and for epigenetic regulation of lymphatic marker expression during LEC differentiation. Mechanistically, the transcription factor PROX1 upregulates CPT1A expression, which increases acetyl coenzyme A production dependent on fatty acid β-oxidation. Acetyl coenzyme A is used by the histone acetyltransferase p300 to acetylate histones at lymphangiogenic genes. PROX1-p300 interaction facilitates preferential histone acetylation at PROX1-target genes. Through this metabolism-dependent mechanism, PROX1 mediates epigenetic changes that promote lymphangiogenesis. Notably, blockade of CPT1 enzymes inhibits injury-induced lymphangiogenesis, and replenishing acetyl coenzyme A by supplementing acetate rescues this process in vivo. ispartof: Nature vol:542 issue:7639 pages:49-+ ispartof: location:England status: published

Background Mosquitoes are the most important invertebrate viral vectors in humans and harbor a high diversity of understudied viruses, which has been shown in many mosquito virome studies in recent years. These studies generally performed metagenomics sequencing on pools of mosquitoes, without assessment of the viral diversity in individual mosquitoes. To address this issue, we applied our optimized viral metagenomics protocol (NetoVIR) to compare the virome of single and pooled Aedes aegypti and Culex quinquefasciatus mosquitoes collected from different locations in Guadeloupe, in 2016 and 2017. Results The total read number and viral reads proportion of samples containing a single mosquito have no significant difference compared with those of pools containing five mosquitoes, which proved the feasibility of using single mosquito for viral metagenomics. A comparative analysis of the virome revealed a higher abundance and more diverse eukaryotic virome in Aedes aegypti, whereas Culex quinquefasciatus harbors a richer and more diverse phageome. The majority of the identified eukaryotic viruses were mosquito-species specific. We further characterized the genomes of 11 novel eukaryotic viruses. Furthermore, qRT-PCR analyses of the six most abundant eukaryotic viruses indicated that the majority of individual mosquitoes were infected by several of the selected viruses with viral genome copies per mosquito ranging from 267 to 1.01 × 108 (median 7.5 × 106) for Ae. aegypti and 192 to 8.69 × 106 (median 4.87 × 104) for Cx. quinquefasciatus. Additionally, in Cx. quinquefasciatus, a number of phage contigs co-occurred with several marker genes of Wolbachia sp. strain wPip. Conclusions We firstly demonstrate the feasibility to use single mosquito for viral metagenomics, which can provide much more precise virome profiles of mosquito populations. Interspecific comparisons show striking differences in abundance and diversity between the viromes of Ae. aegypti and Cx. quinquefasciatus. Those two mosquito species seem to have their own relatively stable "core eukaryotic virome", which might have important implications for the competence to transmit important medically relevant arboviruses. The presence of Wolbachia in Cx. quinquefasciatus might explain (1) the lower overall viral load compared to Ae. aegypti, (2) the identification of multiple unknown phage contigs, and (3) the difference in competence for important human pathogens. How these viruses, phages, and bacteria influence the physiology and vector competence of mosquito hosts warrants further research. Electronic supplementary material The online version of this article (10.1186/s40168-019-0734-2) contains supplementary material, which is available to authorized users.

Despite the availability of diagnostic tools for different enteric viral pathogens, a large fraction of human cases of gastroenteritis remains unexplained. This could be due to pathogens not tested for or novel divergent viruses of potential animal origin. Fecal virome analyses of Cameroonians showed a very diverse group of viruses, some of which are genetically related to those identified in animals. This is the first attempt to describe the gut virome of humans from Cameroon. Therefore, the data represent a baseline for future studies on enteric viral pathogens in this area and contribute to our knowledge of the world’s virome. The studies also highlight the fact that more viruses may be associated with diarrhea than the typical known ones. Hence, it provides meaningful epidemiological information on diarrhea-related viruses in this area., Diarrhea remains one of the most common causes of deaths in children. A limited number of studies have investigated the prevalence of enteric pathogens in Cameroon, and as in many other African countries, the cause of many diarrheal episodes remains unexplained. A proportion of these unknown cases of diarrhea are likely caused by yet-unidentified viral agents, some of which could be the result of (recent) interspecies transmission from animal reservoirs, like bats. Using viral metagenomics, we screened fecal samples of 221 humans (almost all with gastroenteritis symptoms) between 0 and 89 years of age with different degrees of bat contact. We identified viruses belonging to families that are known to cause gastroenteritis such as Adenoviridae, Astroviridae, Caliciviridae, Picornaviridae, and Reoviridae. Interestingly, a mammalian orthoreovirus, picobirnaviruses, a smacovirus, and a pecovirus were also found. Although there was no evidence of interspecies transmission of the most common human gastroenteritis-related viruses (Astroviridae, Caliciviridae, and Reoviridae), the phylogenies of the identified orthoreovirus, picobirnavirus, and smacovirus indicate a genetic relatedness of these viruses identified in stools of humans and those of bats and/or other animals. These findings points out the possibility of interspecies transmission or simply a shared host of these viruses (bacterial, fungal, parasitic, …) present in both animals (bats) and humans. Further screening of bat viruses in humans or vice versa will elucidate the epidemiological potential threats of animal viruses to human health. Furthermore, this study showed a huge diversity of highly divergent novel phages, thereby expanding the existing phageome considerably. IMPORTANCE Despite the availability of diagnostic tools for different enteric viral pathogens, a large fraction of human cases of gastroenteritis remains unexplained. This could be due to pathogens not tested for or novel divergent viruses of potential animal origin. Fecal virome analyses of Cameroonians showed a very diverse group of viruses, some of which are genetically related to those identified in animals. This is the first attempt to describe the gut virome of humans from Cameroon. Therefore, the data represent a baseline for future studies on enteric viral pathogens in this area and contribute to our knowledge of the world’s virome. The studies also highlight the fact that more viruses may be associated with diarrhea than the typical known ones. Hence, it provides meaningful epidemiological information on diarrhea-related viruses in this area.

The mechanisms underlying immunomodulatory ability of mesenchymal stromal cells (MSCs) remain unknown. Recently, studies suggested that the immunomodulatory activity of MSCs is largely mediated by paracrine factors. Among which, exosome is considered to play a major role in the communication between MSCs and target tissue. The aim of our study is to investigate the effect of MSCs-derived exosome on peripheral blood mononuclear cells (PBMCs), especially T cells. We find that the MSCs-derived exosome extracted from healthy donors’ bone marrow suppressed the secretion of pro-inflammatory factor TNF-α and IL-1β, but increased the concentration of anti-inflammatory factor TGF-β during in vitro culture. In addition, exosome may induce conversion of T helper type 1 (Th1) into T helper type 2 (Th2) cells and reduced potential of T cells to differentiate into interleukin 17-producing effector T cells (Th17). Moreover, the level of regulatory T cells (Treg) and cytotoxic T lymphocyte-associated protein 4 were also increased. These results suggested that MSC-derived exosome possesses the immunomodulatory properties. However, it showed no effects on the proliferation of PBMCs or CD3+ T cells, but increases the apoptosis of them. In addition, indoleamine 2, 3-dioxygenase (IDO) was previously shown to mediate the immunoregulation of MSCs, which was increased in PBMCs co-cultured with MSCs. In our study, IDO showed no significant changes in PBMCs exposed to MSCs-derived exosome. We conclude that exosome and MSCs might differ in their immune-modulating activities and mechanisms.