Teriflunomide Inhibits JCPyV Infection and Spread in Glial Cells and Choroid Plexus Epithelial Cells.

Several classes of immunomodulators are used for treating relapsing-remitting multiple sclerosis (RRMS). Most of these disease-modifying therapies, except teriflunomide, carry the risk of progressive multifocal leukoencephalopathy (PML), a severely debilitating, often fatal virus-induced demyelinating disease. Because teriflunomide has been shown to have antiviral activity against DNA viruses, we investigated whether treatment of cells with teriflunomide inhibits infection and spread of JC polyomavirus (JCPyV), the causative agent of PML. Treatment of choroid plexus epithelial cells and astrocytes with teriflunomide reduced JCPyV infection and spread. We also used droplet digital PCR to quantify JCPyV DNA associated with extracellular vesicles isolated from RRMS patients. We detected JCPyV DNA in all patients with confirmed PML diagnosis (n = 2), and in six natalizumab-treated (n = 12), two teriflunomide-treated (n = 7), and two nonimmunomodulated (n = 2) patients. Of the 21 patients, 12 (57%) had detectable JCPyV in either plasma or serum. CSF was uniformly negative for JCPyV. Isolation of extracellular vesicles did not increase the level of detection of JCPyV DNA versus bulk unprocessed biofluid. Overall, our study demonstrated an effect of teriflunomide inhibiting JCPyV infection and spread in glial and choroid plexus epithelial cells. Larger studies using patient samples are needed to correlate these in vitro findings with patient data.

A Mechanically Transmitted DNA Mycovirus Is Targeted by the Defence Machinery of Its Host, Botrytis cinerea.

Eukaryotic circular single-stranded DNA (ssDNA) viruses were known only to infect plants and vertebrates until the discovery of the isolated DNA mycovirus from the fungus Sclerotinia sclerotiorum. Similar viral sequences were reported from several other sources and classified in ten genera within the Genomoviridae family. The current study reports two circular ssDNA mycoviruses isolated from the phytopathogen Botrytis cinerea, and their assignment to a newly created genus tentatively named Gemydayirivirus. The mycoviruses, tentatively named botrytis gemydayirivirus 1 (BGDaV1) and BGDaV2, are 1701 and 1693 nt long and encode three and two open reading frames (ORFs), respectively. Of the predicted ORFs, only ORF I, which codes for a replication initiation protein (Rep), shared identity with other proteins in GenBank. BGDaV1 is infective as cell-free purified particles and confers hypovirulence on its natural host. Investigation revealed that BGDaV1 is a target for RNA silencing and genomic DNA methylation, keeping the virus at very low titre. The discovery of BGDaV1 expands our knowledge of the diversity of genomoviruses and their interaction with fungal hosts.

Isolation of a novel adomavirus from cultured American eels, Anguilla rostrata, with haemorrhagic gill necrosis disease.

Recently, the culture of American eels (Anguilla rostrate) in China has been impacted by emergence of a disease with signs of haemorrhagic gill necrosis. The gills of diseased eels are covered with petecchia and they bleed when the operculum is pressed. In this study, a novel American eel adomavirus (AEAdoV) was isolated from the diseased eels using the eel ovary cell line (EO). The virus proliferated in the EO cells with a maximum TCID50 /ml of 106.29 ± 0.23 at 6 days post-infection. The virions were non-enveloped with a diameter of 75-85 nm and shown to be a DNA virus upon 5-iodo-2'-deoxyuridine (IDU) treatment. PCR assays showed that AEAdoV encodes a superfamily 3 helicases (S3H) replicase and shared high similarities with Anguilla marmorata adomavirus (MEAdoV). Although no clinical signs or mortality was observed among the eels injected with AEAdoV, the virus was reisolated from livers, kidneys and gills of injected eels at 35 days post-injection. Our results suggested that AEAdoV exhibited a latent infection in A. rostrata. The pathogenicity of the AEAdoV needs to be confirmed further.

Crosstalk between nucleocytoplasmic trafficking and the innate immune response to viral infection.

The nuclear pore complex is the sole gateway connecting the nucleoplasm and cytoplasm. In humans, the nuclear pore complex is one of the largest multiprotein assemblies in the cell, with a molecular mass of ∼110 MDa and consisting of 8 to 64 copies of about 34 different nuclear pore proteins, termed nucleoporins, for a total of 1000 subunits per pore. Trafficking events across the nuclear pore are mediated by nuclear transport receptors and are highly regulated. The nuclear pore complex is also used by several RNA viruses and almost all DNA viruses to access the host cell nucleoplasm for replication. Viruses hijack the nuclear pore complex, and nuclear transport receptors, to access the nucleoplasm where they replicate. In addition, the nuclear pore complex is used by the cell innate immune system, a network of signal transduction pathways that coordinates the first response to foreign invaders, including viruses and other pathogens. Several branches of this response depend on dynamic signaling events that involve the nuclear translocation of downstream signal transducers. Mounting evidence has shown that these signaling cascades, especially those steps that involve nucleocytoplasmic trafficking events, are targeted by viruses so that they can evade the innate immune system. This review summarizes how nuclear pore proteins and nuclear transport receptors contribute to the innate immune response and highlights how viruses manipulate this cellular machinery to favor infection. A comprehensive understanding of nuclear pore proteins in antiviral innate immunity will likely contribute to the development of new antiviral therapeutic strategies.

Reduction in Virulence over Time in Ostreid herpesvirus 1 (OsHV-1) Microvariants between 2011 and 2015 in Australia.

Microvariant genotypes of Ostreid herpesvirus 1 (OsHV-1) are associated with mass mortality events of Pacific oysters in many countries. The OsHV-1 microvariant (µVar) emerged in France 2008 and caused significant economic losses as it became endemic and displaced the previously dominant OsHV-1 reference genotype. Recently, considerable genotypic variation has been described for OsHV-1 microvariants, however, less is known about variation in viral phenotype. This study used an in vivo laboratory infection model to assess differences in total cumulative mortality, peak viral load, transmissibility, and dose-response for three OsHV-1 isolates obtained between 2011 and 2015 from endemic waterways in Australia. This followed field observations of apparent reductions in the severity of mass mortalities over this time. Significantly higher hazard of death and cumulative mortality were observed for an isolate obtained in 2011 compared to isolates from 2014-2015. In keeping with other studies, the hazard of death was higher in oysters challenged by injection compared to challenge by cohabitation and the mortality was higher when the initial dose was 1 × 104 OsHV-1 DNA copies per oyster injection compared to 1 × 102 DNA copies. There was no difference in the quantity of OsHV-1 DNA at time of death that could be related to isolate or dose, suggesting similar pathogenetic processes in the individual oysters that succumbed to end-stage disease. While the isolates examined in this study were biased towards pathogenic types of OsHV-1, as they were collected during disease outbreaks, the variation in virulence that was observed, when combined with prior data on subclinical infections, suggests that surveillance for low virulence genotypes of OsHV-1 would be rewarding. This may lead to new approaches to disease management which utilize controlled exposure to attenuated strains of OsHV-1.

The first detection of a novel OsHV-1 microvariant in San Diego, California, USA.

The spread, emergence, and adaptation of pathogens causing marine disease has been problematic to fisheries and aquaculture industries for the last several decades creating the need for strategic management and biosecurity practices. The Pacific oyster (Crassostrea gigas), a highly productive species globally, has been a target of disease and mortality caused by a viral pathogen, the Ostreid herpesvirus 1 (OsHV-1) and its microvariants (OsHV-1 µvars). During routine surveillance to establish health history at a shellfish aquaculture nursery system in San Diego, California, the presence of OsHV-1 in Pacific oyster juveniles was detected. Quantification of OsHV-1 in tissues of oysters revealed OsHV-1 viral loads > 106 copies/mg. We characterized and identified the OsHV-1 variant by sequencing of ORFs 4 (C2/C6) and 43 (IA1/IA2), which demonstrated that this variant is a novel OsHV-1 microvariant: OsHV-1 µvar SD. A pilot transmission study indicates that OsHV-1 µvar SD is infectious with high viral loads ~ 7.57 × 106 copies/mg detected in dead individuals. The detection of OsHV-1 µvar SD in a large port mirrors previous studies conducted in Australia where aquaculture farms and feral populations near port locations may be at a higher risk of OsHV-1 emergence. Further research is needed to understand the impacts of OsHV-1 µvar SD, such as transmission studies focusing on potential vectors and characterization of virulence as compared to other OsHV-1 µvars. To increase biosecurity of the global aquaculture industry, active and passive surveillance may be necessary to reduce spread of pathogens and make appropriate management decisions.

Proteomic approaches to investigate gammaherpesvirus biology and associated tumorigenesis.

The DNA viruses, Kaposi's sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are members of the gammaherpesvirus subfamily, a group of viruses whose infection is associated with multiple malignancies, including cancer. The primary host for these viruses is humans and, like all herpesviruses, infection with these pathogens is lifelong. Due to the persistence of gammaherpesvirus infection and the potential for cancer formation in infected individuals, there is a driving need to understand not only the biology of these viruses and how they remain undetected in host cells but also the mechanism(s) by which tumorigenesis occurs. One of the methods that has provided much insight into these processes is proteomics. Proteomics is the study of all the proteins that are encoded by a genome and allows for (i) identification of existing and novel proteins derived from a given genome, (ii) interrogation of protein-protein interactions within a system, and (iii) discovery of druggable targets for the treatment of malignancies. In this chapter, we explore how proteomics has contributed to our current understanding of gammaherpesvirus biology and their oncogenic processes, as well as the clinical applications of proteomics for the detection and treatment of gammaherpesvirus-associated cancers.

TRIM41 is required to innate antiviral response by polyubiquitinating BCL10 and recruiting NEMO.

Sensing of pathogenic nucleic acids by pattern recognition receptors (PRR) not only initiates anti-microbe defense but causes inflammatory and autoimmune diseases. E3 ubiquitin ligase(s) critical in innate response need to be further identified. Here we report that the tripartite motif-containing E3 ubiquitin ligase TRIM41 is required to innate antiviral response through facilitating pathogenic nucleic acids-triggered signaling pathway. TRIM41 deficiency impairs the production of inflammatory cytokines and type I interferons in macrophages after transfection with nucleic acid-mimics and infection with both DNA and RNA viruses. In vivo, TRIM41 deficiency leads to impaired innate response against viruses. Mechanistically, TRIM41 directly interacts with BCL10 (B cell lymphoma 10), a core component of CARD proteins-BCL10 - MALT1 (CBM) complex, and modifies the Lys63-linked polyubiquitylation of BCL10, which, in turn, hubs NEMO for activation of NF-κB and TANK-binding kinase 1 (TBK1) - interferon regulatory factor 3 (IRF3) pathways. Our study suggests that TRIM41 is the potential universal E3 ubiquitin ligase responsible for Lys63 linkage of BCL10 during innate antiviral response, adding new insight into the molecular mechanism for the control of innate antiviral response.

Reston virus causes severe respiratory disease in young domestic pigs.

Reston virus (RESTV), an ebolavirus, causes clinical disease in macaques but has yet only been associated with rare asymptomatic infections in humans. Its 2008 emergence in pigs in the Philippines raised concerns about food safety, pathogenicity, and zoonotic potential, questions that are still unanswered. Until today, the virulence of RESTV for pigs has remained elusive, with unclear pathogenicity in naturally infected animals and only one experimental study demonstrating susceptibility and evidence for shedding but no disease. Here we show that combined oropharyngeal and nasal infection of young (3- to 7-wk-old) Yorkshire cross pigs with RESTV resulted in severe respiratory disease, with most animals reaching humane endpoint within a week. RESTV-infected pigs developed severe cyanosis, tachypnea, and acute interstitial pneumonia, with RESTV shedding from oronasal mucosal membranes. Our studies indicate that RESTV should be considered a livestock pathogen with zoonotic potential.

ICTV Virus Taxonomy Profile: Redondoviridae.

Viruses in the family Redondoviridae have a circular genome of 3.0 kb with three open reading frames. The packaged genome is inferred to be single-stranded DNA by analogy to related viruses. Redondoviruses were discovered through metagenomic sequencing methods in samples from human subjects and are inferred to replicate in humans. Evidence of redondovirus infection is associated with periodontitis and critical illness, but redondoviruses have not been shown to be the causative agent of any diseases. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Redondoviridae, which is available at ictv.global/report/redondoviridae.

Recurrent evolution of high virulence in isolated populations of a DNA virus.

Hosts and viruses are constantly evolving in response to each other: as a host attempts to suppress a virus, the virus attempts to evade and suppress the host's immune system. Here, we describe the recurrent evolution of a virulent strain of a DNA virus, which infects multiple Drosophila species. Specifically, we identified two distinct viral types that differ 100-fold in viral titer in infected individuals, with similar differences observed in multiple species. Our analysis suggests that one of the viral types recurrently evolved at least four times in the past ~30,000 years, three times in Arizona and once in another geographically distinct species. This recurrent evolution may be facilitated by an effective mutation rate which increases as each prior mutation increases viral titer and effective population size. The higher titer viral type suppresses the host-immune system and an increased virulence compared to the low viral titer type.

Animals constantly evolve to protect themselves against viruses, and in turn, viruses evolve to escape their host's new defenses. As a result, genes involved in this arms' race are some of the fastest evolving in nature. A better understanding of how host-virus evolution works could help in the search for treatments for many human and animal diseases. Repetition is one of the gold standard requirements for biological experiments. Watching different groups of animals and viruses evolve under the same conditions makes it possible for researchers to work out whether certain changes are more likely than others. This is easy to do in the laboratory, where conditions can be controlled, but much more complicated to accomplish in the wild. Wild populations are rarely completely isolated, and often face different environmental conditions. One animal-virus pair for which this is not the case is made up of the fly Drosophila innubila, and its virus Drosophila innubila nudivirus. They live in the 'sky islands' of North America, patches of forests surrounded by hundreds of kilometers of desert. These islands are like natural test tubes, isolated ecosystems each with its own separate fly and virus populations and limited gene flow between populations. To understand how this virus-host pair evolves, Hill and Unckless sequenced the genomes of flies and viruses from four different populations. While the fly genomes did not show evidence of strong differences between populations, the virus genomes did. There were two distinct types of virus, one of which was a lot more effective than the other at infecting flies, possibly because it was better at blocking the fly's immune defenses. Unexpectedly, this virus type had evolved more than once, emerging separately on at least four different occasions. Hill and Unckless suggest that the natural interactions between flies with similar genomes and the virus guide evolution down the same path time and time again. This work on wild populations contributes to the understanding of the evolution of viruses and their hosts. One question left unanswered is why both types of virus (one more effective at infecting the flies and the other less so) persist in each population when one is better at blocking the fly's immune response? Future work using isolated populations like these could shed more light on the pressures that shape the evolution of viruses and their hosts, potentially helping in the study of human viruses, like HIV.

Parallel analysis of miRNAs and mRNAs suggests distinct regulatory networks in Crassostrea gigas infected by Ostreid herpesvirus 1.

BACKGROUND: Since 2008, the aquaculture production of Crassostrea gigas was heavily affected by mass mortalities associated to Ostreid herpesvirus 1 (OsHV-1) microvariants worldwide. Transcriptomic studies revealed the major antiviral pathways of the oyster immune response while other findings suggested that also small non-coding RNAs (sncRNA) such as microRNAs might act as key regulators of the oyster response against OsHV-1. To explore the explicit connection between small non-coding and protein-coding transcripts, we performed paired whole transcriptome analysis of sncRNA and messenger RNA (mRNA) in six oysters selected for different intensities of OsHV-1 infection. RESULTS: The mRNA profiles of the naturally infected oysters were mostly governed by the transcriptional activity of OsHV-1, with several differentially expressed genes mapping to the interferon, toll, apoptosis, and pro-PO pathways. In contrast, miRNA profiles suggested more complex regulatory mechanisms, with 15 differentially expressed miRNAs (DE-miRNA) pointing to a possible modulation of the host response during OsHV-1 infection. We predicted 68 interactions between DE-miRNAs and oyster 3'-UTRs, but only few of them involved antiviral genes. The sncRNA reads assigned to OsHV-1 rather resembled mRNA degradation products, suggesting the absence of genuine viral miRNAs. CONCLUSIONS: We provided data describing the miRNAome during OsHV-1 infection in C. gigas. This information can be used to understand the role of miRNAs in healthy and diseased oysters, to identify new targets for functional studies and, eventually to disentangle cause and effect relationships during viral infections in marine mollusks.

A novel nudivirus infecting the invasive demon shrimp Dikerogammarus haemobaphes (Amphipoda).

The Nudiviridae are a family of large double-stranded DNA viruses that infects the cells of the gut in invertebrates, including insects and crustaceans. The phylogenetic range of the family has recently been enhanced via the description of viruses infecting penaeid shrimp, crangonid shrimp, homarid lobsters and portunid crabs. Here we extend this by presenting the genome of another nudivirus infecting the amphipod Dikerogammarus haemobaphes. The virus, which infects cells of the host hepatopancreas, has a circular genome of 119,754 bp in length, and encodes a predicted 106 open reading frames. This novel virus encodes all the conserved nudiviral genes (sharing 57 gene homologues with other crustacean-infecting nudiviruses) but appears to lack the p6.9 gene. Phylogenetic analysis revealed that this virus branches before the other crustacean-infecting nudiviruses and shares low levels of gene/protein similarity to the Gammanudivirus genus. Comparison of gene synteny from known crustacean-infecting nudiviruses reveals conservation between Homarus gammarus nudivirus and Penaeus monodon nudivirus; however, three genomic rearrangements in this novel amphipod virus appear to break the gene synteny between this and the ones infecting lobsters and penaeid shrimp. We explore the evolutionary history and systematics of this novel virus, suggesting that it be included in the novel Epsilonnudivirus genus (Nudiviridae).

Human Neural Stem Cell Systems to Explore Pathogen-Related Neurodevelopmental and Neurodegenerative Disorders.

Building and functioning of the human brain requires the precise orchestration and execution of myriad molecular and cellular processes, across a multitude of cell types and over an extended period of time. Dysregulation of these processes affects structure and function of the brain and can lead to neurodevelopmental, neurological, or psychiatric disorders. Multiple environmental stimuli affect neural stem cells (NSCs) at several levels, thus impairing the normal human neurodevelopmental program. In this review article, we will delineate the main mechanisms of infection adopted by several neurotropic pathogens, and the selective NSC vulnerability. In particular, TORCH agents, i.e., Toxoplasma gondii, others (including Zika virus and Coxsackie virus), Rubella virus, Cytomegalovirus, and Herpes simplex virus, will be considered for their devastating effects on NSC self-renewal with the consequent neural progenitor depletion, the cellular substrate of microcephaly. Moreover, new evidence suggests that some of these agents may also affect the NSC progeny, producing long-term effects in the neuronal lineage. This is evident in the paradigmatic example of the neurodegeneration occurring in Alzheimer's disease.

Identification of candidate microRNAs from Ostreid herpesvirus-1 (OsHV-1) and their potential role in the infection of Pacific oysters (Crassostrea gigas).

Oyster production is an economic activity of great interest worldwide. Recently, oysters have been suffering significant mortalities from OsHV-1infection, which has resulted in substantial economic loses in several countries around the world. Understanding viral pathogenicity mechanisms is of central importance for the establishment of disease control measures. Thus, the present work aimed to identify and characterize miRNAs from OsHV-1 as well as to predict their target transcripts in the virus and the host. OsHV-1 genome was used for the in silico discovery of pre-miRNAs. Subsequently, viral and host target transcripts of the OsHV-1 miRNAs were predicted according to the base pairing interaction between mature miRNAs and mRNA 3' untranslated regions (UTRs). Six unique pre-miRNAs were found in different regions of the viral genome, ranging in length from 85 to 172 nucleotides. A complex network of self-regulation of viral gene expression mediated by the miRNAs was identified. These sequences also seem to have a broad ability to regulate the expression of host immune-related genes, especially those associated with pathogen recognition. Our results suggest that OsHV-1 encodes miRNAs with important functions in the infection process, inducing self-regulation of viral transcripts, as well as affecting the regulation of Pacific oyster transcripts related to immunity. Understanding the molecular basis of host-pathogen interactions can help mitigate the recurrent events of oyster mass mortalities by OsHV-1 observed worldwide.

Etiological analysis of virus, mycoplasma pneumoniae and chlamydia pneumoniae in hospitalized children with acute respiratory infections in Huzhou.

BACKGROUND: Acute respiratory infections are a common disease in children with high mortality and morbidity. Multiple pathogens can cause acute respiratory infections. A 2-year survey of hospitalized children was conducted to understand the epidemic situation, seasonal spread of pathogens and the improvement of clinical diagnosis, treatment and prevention of disease in Huzhou, China. METHODS: From September 2017 to August 2019, 3121 nasopharyngeal swabs from hospitalized children with acute respiratory infections were collected, and real-time PCR was used to detect various pathogens. Then, pathogen profiles, frequency and seasonality were analyzed. RESULTS: Of the 3121 specimens, 14.45% (451/3121) were positive for at least one pathogen. Of the single-pathogen infections, RSV (45.61%, 182/399) was the most frequent pathogen, followed by PIVs (14.79%, 59/399), ADV (14.54%, 58/399), MP (10.78%, 43/399), and IAV (5.26%, 21/399). Of the 52 coinfections, RSV + PIVs viruses were predominantly identified and accounted for 40.38% (21/52) of cases. RSV was the most frequent pathogen in all four groups. The highest positive rate of the pathogens occurred in the winter (21.26%), followed by autumn (14.98%), the summer (14.11%) and the spring (12.25%). CONCLUSION: Viruses are the main pathogens in hospitalized children with acute respiratory infections in Huzhou city, Zhejiang Province, China. Among the pathogens, RSV had the highest detection rate, and MP is also a common pathogen among children with acute respiratory infections. This study provided a better understanding of the distribution of pathogens in children of different ages and seasons, which is conducive to the development of more reasonable treatment strategies and prevention and control measures.

Exploration of the virome of the European brown shrimp (Crangon crangon).

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.

A Sustained Immune Response Supports Long-Term Antiviral Immune Priming in the Pacific Oyster, Crassostrea gigas.

Over the last decade, innate immune priming has been evidenced in many invertebrate phyla. If mechanistic models have been proposed, molecular studies aiming to substantiate these models have remained scarce. We reveal here the transcriptional signature associated with immune priming in the oyster Crassostrea gigas Oysters were fully protected against Ostreid herpesvirus 1 (OsHV-1), a major oyster pathogen, after priming with poly(I·C), which mimics viral double-stranded RNA. Global analysis through RNA sequencing of oyster and viral genes after immune priming and viral infection revealed that poly(I·C) induces a strong antiviral response that impairs OsHV-1 replication. Protection is based on a sustained upregulation of immune genes, notably genes involved in the interferon pathway and apoptosis, which control subsequent viral infection. This persistent antiviral alert state remains active over 4 months and supports antiviral protection in the long term. This acquired resistance mechanism reinforces the molecular foundations of the sustained response model of immune priming. It further opens the way to applications (pseudovaccination) to cope with a recurrent disease that causes dramatic economic losses in the shellfish farming industry worldwide.IMPORTANCE In the last decade, important discoveries have shown that resistance to reinfection can be achieved without a functional adaptive immune system, introducing the concept of innate immune memory in invertebrates. However, this field has been constrained by the limited number of molecular mechanisms evidenced to support these phenomena. Taking advantage of an invertebrate species, the Pacific oyster (Crassostrea gigas), in which we evidenced one of the longest and most effective periods of protection against viral infection observed in an invertebrate, we provide the first comprehensive transcriptomic analysis of antiviral innate immune priming. We show that priming with poly(I·C) induced a massive upregulation of immune-related genes, which control subsequent viral infection, and it was maintained for over 4 months after priming. This acquired resistant mechanism reinforces the molecular foundations of the sustained response model of immune priming. It opens the way to pseudovaccination to prevent the recurrent diseases that currently afflict economically or ecologically important invertebrates.

High resolution metagenomic characterization of complex infectomes in paediatric acute respiratory infection.

The diversity of pathogens associated with acute respiratory infection (ARI) makes diagnosis challenging. Traditional pathogen screening tests have a limited detection range and provide little additional information. We used total RNA sequencing ("meta-transcriptomics") to reveal the full spectrum of microbes associated with paediatric ARI. Throat swabs were collected from 48 paediatric ARI patients and 7 healthy controls. Samples were subjected to meta-transcriptomics to determine the presence and abundance of viral, bacterial, and eukaryotic pathogens, and to reveal mixed infections, pathogen genotypes/subtypes, evolutionary origins, epidemiological history, and antimicrobial resistance. We identified 11 RNA viruses, 4 DNA viruses, 4 species of bacteria, and 1 fungus. While most are known to cause ARIs, others, such as echovirus 6, are rarely associated with respiratory disease. Co-infection of viruses and bacteria and of multiple viruses were commonplace (9/48), with one patient harboring 5 different pathogens, and genome sequence data revealed large intra-species diversity. Expressed resistance against eight classes of antibiotic was detected, with those for MLS, Bla, Tet, Phe at relatively high abundance. In summary, we used a simple total RNA sequencing approach to reveal the complex polymicrobial infectome in ARI. This provided comprehensive and clinically informative information relevant to understanding respiratory disease.

A New Family of DNA Viruses Causing Disease in Crustaceans from Diverse Aquatic Biomes.

Panulirus argus virus 1 (PaV1) is the only known virus infecting the Caribbean spiny lobster (Panulirus argus) from the Caribbean Sea. Recently, related viruses, Dikerogammarus haemobaphes virus 1 (DhV1) and Carcinus maenas virus 1 (CmV1), have been detected in the demon shrimp (Dikerogammarus haemobaphes) and the European shore crab (Carcinus maenas), respectively, from sites in the United Kingdom. The virion morphology of these crustacean viruses is similar to that of iridoviruses. However, unlike iridoviruses and other nucleocytoplasmic large DNA viruses (NCLDVs), these viruses complete their morphogenesis in the host cell nucleus rather than in the cytoplasm. To date, these crustacean viruses have remained unclassified due to a lack of genomic data. Using an Illumina MiSeq sequencer, we sequenced the complete genomes of PaV1, CmV1, and DhV1. Comparative genome analysis shows that these crustacean virus genomes encode the 10 hallmark proteins previously described for the NCLDVs of eukaryotes, strongly suggesting that they are members of this group. With a size range of 70 to 74 kb, these are the smallest NCLDV genomes identified to date. Extensive gene loss, divergence of gene sequences, and the accumulation of low-complexity sequences reflect the extreme degradation of the genomes of these "minimal" NCLDVs rather than any direct relationship with the NCLDV ancestor. Phylogenomic analysis supports the classification of these crustacean viruses as a distinct family, "Mininucleoviridae," within the pitho-irido-Marseille branch of the NCLDVs.IMPORTANCE Recent genomic and metagenomic studies have led to a dramatic expansion of the known diversity of nucleocytoplasmic large DNA viruses (NCLDVs) of eukaryotes, which include giant viruses of protists and important pathogens of vertebrates, such as poxviruses. However, the characterization of viruses from nonmodel hosts still lags behind. We sequenced the complete genomes of three viruses infecting crustaceans, the Caribbean spiny lobster, demon shrimp, and European shore crab. These viruses have the smallest genomes among the known NCLDVs, with losses of many core genes, some of which are shared with iridoviruses. The deterioration of the transcription apparatus is compatible with microscopic and ultrastructural observations indicating that these viruses replicate in the nucleus of infected cells rather than in the cytoplasm. Phylogenomic analysis indicates that these viruses are sufficiently distinct from all other NCLDVs to justify the creation of a separate family, for which we propose the name "Mininucleoviridae" (i.e., small viruses reproducing in the cell nucleus).