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Reactivation from latency plays a significant role in maintaining persistent lifelong Epstein-Barr virus (EBV) infection. Mechanisms governing successful activation and progression of the EBV lytic phase are not fully understood. EBV expresses multiple viral microRNAs (miRNAs) and manipulates several cellular miRNAs to support viral infection. To gain insight into the host miRNAs regulating transitions from EBV latency into the lytic stage, we conducted a CRISPR/Cas9-based screen in EBV+ Burkitt lymphoma (BL) cells using anti-Ig antibodies to crosslink the B cell receptor (BCR) and induce reactivation. Using a gRNA library against >1500 annotated human miRNAs, we identified miR-142 as a key regulator of EBV reactivation. Genetic ablation of miR-142 enhanced levels of immediate early and early lytic gene products in infected BL cells. Ago2-PAR-CLIP experiments with reactivated cells revealed miR-142 targets related to Erk/MAPK signaling, including components directly downstream of the B cell receptor (BCR). Consistent with these findings, disruption of miR-142 enhanced SOS1 levels and Mek phosphorylation in response to surface Ig cross-linking. Effects could be rescued by inhibitors of Mek (cobimetinib) or Raf (dabrafenib). Taken together, these results show that miR-142 functionally regulates SOS1/Ras/Raf/Mek/Erk signaling initiated through the BCR and consequently, restricts EBV entry into the lytic cycle. Author summary: Epstein-Barr virus (EBV) is a common herpesvirus that infects most individuals worldwide and can persist in the body for life. Long-term viral persistence is established in part through latently infected memory B cells and periodic reactivation events which maintain viral reservoirs. To investigate functional roles for host non-coding RNAs in EBV latency and reactivation—specifically microRNAs—we conducted a genome-wide CRISPR screen in EBV-positive Burkitt's lymphoma (BL) cells. By screening >1500 human miRNAs, we identified miR-142 as a potent regulator of the lytic cycle. When miR-142 was disrupted, latently infected BL cells responded rapidly to external reactivation triggers and exhibited significantly elevated levels of lytic viral proteins. Through mechanistic studies, we found that miR-142 regulates cell signaling pathways such as the Ras/Raf/Mek/Erk pathway and can target SOS1 encoding a Ras guanine nucleotide exchange factor. Heightened EBV reactivation in miR-142 mutant cells could be reversed using chemical inhibitors to Raf, Mek, or Sos1. These findings provide novel insight into the role of miR-142 in the EBV life cycle and further unveil potential for inhibiting Sos1 as a means to suppress EBV reactivation. [ABSTRACT FROM AUTHOR]

Peste des petits ruminants virus (PPRV) is an important pathogen that seriously influences the productivity of small ruminants worldwide. PPRV is lymphotropic in nature and SLAM was identified as the primary receptor for PPRV and other Morbilliviruses. Many viruses have been demonstrated to engage extracellular vesicles (EVs) to facilitate their replication and pathogenesis. Here, we provide evidence that PPRV infection significantly induced the secretion levels of EVs from goat PBMC, and that PPRV-H protein carried in EVs can enhance SLAM receptor expression in the recipient cells via suppressing miR-218, a negative miRNA directly targeting SLAM gene. Importantly, EVs-mediated increased SLAM expression enhances PPRV infectivity as well as the expression of various cytokines related to SLAM signaling pathway in the recipient cells. Moreover, our data reveal that PPRV associate EVs rapidly entry into the recipient cells mainly through macropinocytosis pathway and cooperated with caveolin- and clathrin-mediated endocytosis. Taken together, our findings identify a new strategy by PPRV to enhance virus infection and escape innate immunity by engaging EVs pathway. Author summary: Peste des petitsruminants virus (PPRV) infection induces a transient but severe immunosuppression in the host, which threatens both small livestock and endangered susceptible wildlife populations in many countries. Despite extensive research, the mechanism underlying pathogenesis of PPRV infection remains elusive. Our data provide the first direct evidence that the EVs derived from PPRV-infected cells are involved in PPRV replication. In this study, the EVs derived from PPRV-infected goat PBMCs can enhance SLAM expression in the recipient cells, and more importantly, EVs-mediated increased SLAM expression enhances PPRV replication as well as the expression of various cytokines related to SLAM signaling pathway in the recipient cells. Taken together, our research has provided new insight into understanding the effect of EVs on PPRV replication and pathogenesis, and revealed a potential therapeutic target for antiviral intervention. [ABSTRACT FROM AUTHOR]

At the end of 2019 Wuhan witnessed an outbreak of "atypical pneumonia" that later developed into a global pandemic. Metagenomic sequencing rapidly revealed the causative agent of this outbreak to be a novel coronavirus denoted SARS-CoV-2. To provide a snapshot of the pathogens in pneumonia-associated respiratory samples from Wuhan prior to the emergence of SARS-CoV-2, we collected bronchoalveolar lavage fluid samples from 408 patients presenting with pneumonia and acute respiratory infections at the Central Hospital of Wuhan between 2016 and 2017. Unbiased total RNA sequencing was performed to reveal their "total infectome", including viruses, bacteria and fungi. We identified 35 pathogen species, comprising 13 RNA viruses, 3 DNA viruses, 16 bacteria and 3 fungi, often at high abundance and including multiple co-infections (13.5%). SARS-CoV-2 was not present. These data depict a stable core infectome comprising common respiratory pathogens such as rhinoviruses and influenza viruses, an atypical respiratory virus (EV-D68), and a single case of a sporadic zoonotic pathogen–Chlamydia psittaci. Samples from patients experiencing respiratory disease on average had higher pathogen abundance than healthy controls. Phylogenetic analyses of individual pathogens revealed multiple origins and global transmission histories, highlighting the connectedness of the Wuhan population. This study provides a comprehensive overview of the pathogens associated with acute respiratory infections and pneumonia, which were more diverse and complex than obtained using targeted PCR or qPCR approaches. These data also suggest that SARS-CoV-2 or closely related viruses were absent from Wuhan in 2016–2017. Author summary: Acute respiratory infections and pneumonia are a significant public health concern on a global scale. This was brought sharply into focus by the emergence of COVID-19 at the end of 2019 in Wuhan, China. Far less is known, however, about the total "infectomes" associated with respiratory infection and pneumonia: that is, all the viruses, bacteria and fungi that might be associated with these important human diseases. Herein, we describe total infectomes in bronchoalveolar lavage fluid samples taken from 408 patients presenting with pneumonia and acute respiratory infection in a single hospital in Wuhan between 2016 and 2017. From these samples we identified 35 pathogen species, comprising 13 RNA viruses, 3 DNA viruses, 16 bacteria and 3 fungi, often at high abundance and including multiple co-infections. We found no evidence for SARS-CoV-2. These data reveal a stable core infectome comprising common respiratory pathogens as well as their history of global transmission. [ABSTRACT FROM AUTHOR]

The hepatitis B virus (HBV) core protein (HBc) functions in multiple steps of the viral life cycle. Heteroaryldihydropyrimidine compounds (HAPs) such as Bay41-4109 are capsid protein allosteric modulators that accelerate HBc degradation and inhibit the virion secretion of HBV, specifically by misleading HBc assembly into aberrant non-capsid polymers. However, the subsequent cellular fates of these HAP-induced aberrant non-capsid polymers are not well understood. Here, we discovered that that the chaperone-binding E3 ubiquitin ligase protein STUB1 is required for the removal of Bay41-4109-induced aberrant non-capsid polymers from HepAD38 cells. Specifically, STUB1 recruits BAG3 to transport Bay41-4109-induced aberrant non-capsid polymers to the perinuclear region of cells, thereby initiating p62-mediated macroautophagy and lysosomal degradation. We also demonstrate that elevating the STUB1 level enhances the inhibitory effect of Bay41-4109 on the production of HBeAg and HBV virions in HepAD38 cells, in HBV-infected HepG2-NTCP cells, and in HBV transgenic mice. STUB1 overexpression also facilitates the inhibition of Bay41-4109 on the cccDNA formation in de novo infection of HBV. Understanding these molecular details paves the way for applying HAPs as a potentially curative regimen (or a component of a combination treatment) for eradicating HBV from hepatocytes of chronic infection patients. Author summary: Hepatitis B virus (HBV) infects more than 250 million people worldwide chronically. It is a major pathogen causing liver cirrhosis and hepatocellular carcinoma now. The HBV capsid protein (HBc) plays multiple roles in the viral life cycle, and many antivirals targeting HBc such as Heteroaryldihydropyrimidine compounds (HAPs) are under clinical trial recently. This study aimed to investigate how a HAP compound Bay41-4109 induces the degradation of HBc protein. Bay41-4109 induces aberrant non-capsid polymers, which form in complex with the chaperone-binding E3 ubiquitin ligase protein STUB1 and co-chaperone BAG3 and are transported to the perinuclear compartment. Subsequently, Bay41-4109-induced aberrant non-capsid polymers are removed by p62-mediated macroautophagy and lysosomal degradation. STUB1 overexpression accelerates Bay41-4109-induced degradation of HBc protein, and thus enhances the effect of Bay41-4109 on inhibiting secretion of HBeAg and HBV virions. When Bay41-4109 are enforced during HBV infection, de novo cccDNA formation were also negatively regulated by STUB1 overexpression. Altogether, this study provides novel mechanistic insights into developing more potent and safe HAP-based antiviral treatment. [ABSTRACT FROM AUTHOR]

The H7N9 avian influenza virus (AIV) that emerged in China have caused five waves of human infection. Further human cases have been successfully prevented since September 2017 through the use of an H7N9 vaccine in poultry. However, the H7N9 AIV has not been eradicated from poultry in China, and its evolution remains largely unexplored. In this study, we isolated 19 H7N9 AIVs during surveillance and diagnosis from February 2018 to December 2019, and genetic analysis showed that these viruses have formed two different genotypes. Animal studies indicated that the H7N9 viruses are highly lethal to chicken, cause mild infection in ducks, but have distinct pathotypes in mice. The viruses bound to avian-type receptors with high affinity, but gradually lost their ability to bind to human-type receptors. Importantly, we found that H7N9 AIVs isolated in 2019 were antigenically different from the H7N9 vaccine strain that was used for H7N9 influenza control in poultry, and that replication of these viruses cannot, therefore, be completely prevented in vaccinated chickens. We further revealed that two amino acid mutations at positions 135 and 160 in the HA protein added two glycosylation sites and facilitated the escape of the H7N9 viruses from the vaccine-induced immunity. Our study provides important insights into H7N9 virus evolution and control. Author summary: Human infection with H7N9 virus has been successfully prevented since the application of an H7N9 vaccine in poultry in September 2017 in China; however, the H7N9 virus has not been eradicated from poultry. In this study, we evaluated the genetic and biologic properties of H7N9 viruses detected in poultry in China from February 2018 to December 2019. We found that the H7N9 viruses gradually lost their binding to human-type receptors and were antigenically different from the H7N9 vaccine strain that was used in China for H7N9 influenza control in poultry. We further uncovered genetic changes that facilitate the escape of the H7N9 viruses from vaccine-induced immunity. Our study provides important insights into H7N9 virus evolution and control. [ABSTRACT FROM AUTHOR]

Nosemosis C, a Nosema disease caused by microsporidia parasite Nosema ceranae, is a significant disease burden of the European honey bee Apis mellifera which is one of the most economically important insect pollinators. Nevertheless, there is no effective treatment currently available for Nosema disease and the disease mechanisms underlying the pathological effects of N. ceranae infection in honey bees are poorly understood. Iron is an essential nutrient for growth and survival of hosts and pathogens alike. The iron tug-of-war between host and pathogen is a central battlefield at the host-pathogen interface which determines the outcome of an infection, however, has not been explored in honey bees. To fill the gap, we conducted a study to investigate the impact of N. ceranae infection on iron homeostasis in honey bees. The expression of transferrin, an iron binding and transporting protein that is one of the key players of iron homeostasis, in response to N. ceranae infection was analysed. Furthermore, the functional roles of transferrin in iron homeostasis and honey bee host immunity were characterized using an RNA interference (RNAi)-based method. The results showed that N. ceranae infection causes iron deficiency and upregulation of the A. mellifera transferrin (AmTsf) mRNA in honey bees, implying that higher expression of AmTsf allows N. ceranae to scavenge more iron from the host for its proliferation and survival. The suppressed expression levels of AmTsf via RNAi could lead to reduced N. ceranae transcription activity, alleviated iron loss, enhanced immunity, and improved survival of the infected bees. The intriguing multifunctionality of transferrin illustrated in this study is a significant contribution to the existing body of literature concerning iron homeostasis in insects. The uncovered functional role of transferrin on iron homeostasis, pathogen growth and honey bee's ability to mount immune responses may hold the key for the development of novel strategies to treat or prevent diseases in honey bees. Author summary: Nosema disease is an adult honey bee disease caused by an intracellular parasite named Nosema ceranae that lives in the digestive tract of honeybees and is linked to colony losses worldwide. Iron is an essential nutrient for both pathogenic microbes and their hosts and relies largely on an iron transporter protein, transferrin. In this study, we found that N. ceranae infection could cause iron deficiency in infected bees. We also found that N. ceranae infection led to elevated levels of transferrin, allowing N. ceranae to scavenge more iron from the host for its survival. As a result, we employed a genetic method to interfere with the production of transferrin in N. ceranae-infected honeybees. The reduction of transferrin production and activity was found to accompany reduced iron loss and to improve the survival of N. ceranae-infected bees. This study suggests a novel strategy for bee disease treatment and should be of interest to researchers, graduate students, beekeepers and policymakers worldwide. [ABSTRACT FROM AUTHOR]

Rice stripe virus (RSV, genus Tenuivirus, family Phenuiviridae) is the causal agent of rice stripe disease transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a persistent propagative manner. The midgut and salivary glands of SBPH are the first and last barriers to the viral circulation and transmission processes, respectively; however, the precise mechanisms used by RSV to cross these organs and transmit to rice plants have not been fully elucidated. We obtained the full-length cDNA sequence of L. striatellus α-tubulin 2 (LsTUB) and found that RSV infection increased the level of LsTUB in vivo. Furthermore, LsTUB was shown to co-localize with RSV nonstructural protein 3 (NS3) in vivo and bound NS3 at positions 74–76 and 80–82 in vitro. Transient gene silencing of LsTUB expression caused a significant reduction in detectable RSV loads and viral NS3 expression levels, but had no effect on NS3 silencing suppressor activity and viral replication in insect cells. However, suppression of LsTUB attenuated viral spread in the bodies of SBPHs and decreased RSV transmission rates to rice plants. Electrical penetration graphs (EPG) showed that LsTUB knockdown by RNAi did not impact SBPH feeding; therefore, the reduction in RSV transmission rates was likely caused by a decrease in viral loads inside the planthopper. These findings suggest that LsTUB mediates the passage of RSV through midgut and salivary glands and leads to successful horizontal transmission. Author summary: Over 70% of all known plant viruses are transmitted by specific arthropods, mainly including planthoppers, leafhoppers, aphids and whiteflies. Plant viruses with persistent relationships must overcome multiple barriers. Among these barriers, midgut and salivary glands are first and last barriers to the viral transmission. The interactions of host factors and virus in the insect midguts and salivary glands provide critical information regarding viral spread in the insect vector and subsequent infection of host plants. However, the precise mechanisms is still lacking. Here, we found that the specific interaction between a α-tubulin 2 of Laodelphax striatellus (SBPH) (LsTUB) and a nonstructural protein 3 (NS3) Rice stripe virus (RSV) mediates the passage of RSV through midgut and salivary glands, thereby leading to successful horizontal transmission. Our results confirm novel functions of LsTUB and NS3 in RSV transmission in insect vector. LsTUB may be a promising target for blocking horizontal transmission of RSV. These insights provide a better understanding of the interaction between plant viruses and vectors and may develop novel methods to control the systemic spread of plant viruses. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are post-transcriptional regulatory RNAs that can modulate cell signaling and play key roles in cell state transitions. Epstein-Barr virus (EBV) expresses >40 viral miRNAs that manipulate both viral and cellular gene expression patterns and contribute to reprogramming of the host environment during infection. Here, we identified a subset of EBV miRNAs that desensitize cells to B cell receptor (BCR) stimuli, and attenuate the downstream activation of NF-kappaB or AP1-dependent transcription. Bioinformatics and pathway analysis of Ago PAR-CLIP datasets identified multiple EBV miRNA targets related to BCR signal transduction, including GRB2, SOS1, MALT1, RAC1, and INPP5D, which we validated in reporter assays. BCR signaling is critical for B cell activation, proliferation, and differentiation, and for EBV, is linked to reactivation. In functional assays, we demonstrate that EBV miR-BHRF1-2-5p contributes to the growth of latently infected B cells through GRB2 regulation. We further determined that activities of EBV miR-BHRF1-2-5p, EBV miR-BART2-5p, and a cellular miRNA, miR-17-5p, directly regulate virus reactivation triggered by BCR engagement. Our findings provide mechanistic insight into some of the key miRNA interactions impacting the proliferation of latently infected B cells and importantly, governing the latent to lytic switch. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of morbidity and death. Phenol-soluble modulins (PSMs) are recently-discovered toxins with a key impact on the development of Staphylococcus aureus infections. Allelic variants of PSMs and their potential impact on pathogen success during infection have not yet been described. Here we show that the clonal complex (CC) 30 lineage, a major cause of hospital-associated sepsis and hematogenous complications, expresses an allelic variant of the PSMα3 peptide. We found that this variant, PSMα3N22Y, is characteristic of CC30 strains and has significantly reduced cytolytic and pro-inflammatory potential. Notably, CC30 strains showed reduced cytolytic and chemotactic potential toward human neutrophils, and increased hematogenous seeding in a bacteremia model, compared to strains in which the genome was altered to express non-CC30 PSMα3. Our findings describe a molecular mechanism contributing to attenuated pro-inflammatory potential in a main MRSA lineage. They suggest that reduced pathogen recognition via PSMs allows the bacteria to evade elimination by innate host defenses during bloodstream infections. Furthermore, they underscore the role of point mutations in key S. aureus toxin genes in that adaptation and the pivotal importance PSMs have in defining key S. aureus immune evasion and virulence mechanisms. [ABSTRACT FROM AUTHOR]

Israeli acute paralysis virus (IAPV) is a widespread RNA virus of honey bees that has been linked with colony losses. Here we describe the transmission, prevalence, and genetic traits of this virus, along with host transcriptional responses to infections. Further, we present RNAi-based strategies for limiting an important mechanism used by IAPV to subvert host defenses. Our study shows that IAPV is established as a persistent infection in honey bee populations, likely enabled by both horizontal and vertical transmission pathways. The phenotypic differences in pathology among different strains of IAPV found globally may be due to high levels of standing genetic variation. Microarray profiles of host responses to IAPV infection revealed that mitochondrial function is the most significantly affected biological process, suggesting that viral infection causes significant disturbance in energy-related host processes. The expression of genes involved in immune pathways in adult bees indicates that IAPV infection triggers active immune responses. The evidence that silencing an IAPV-encoded putative suppressor of RNAi reduces IAPV replication suggests a functional assignment for a particular genomic region of IAPV and closely related viruses from the Family Dicistroviridae, and indicates a novel therapeutic strategy for limiting multiple honey bee viruses simultaneously and reducing colony losses due to viral diseases. We believe that the knowledge and insights gained from this study will provide a new platform for continuing studies of the IAPV–host interactions and have positive implications for disease management that will lead to mitigation of escalating honey bee colony losses worldwide. [ABSTRACT FROM AUTHOR]

Recent steep declines in honey bee health have severely impacted the beekeeping industry, presenting new risks for agricultural commodities that depend on insect pollination. Honey bee declines could reflect increased pressures from parasites and pathogens. The incidence of the microsporidian pathogen Nosema ceranae has increased significantly in the past decade. Here we present a draft assembly (7.86 MB) of the N. ceranae genome derived from pyrosequence data, including initial gene models and genomic comparisons with other members of this highly derived fungal lineage. N. ceranae has a strongly AT-biased genome (74% A+T) and a diversity of repetitive elements, complicating the assembly. Of 2,614 predicted protein-coding sequences, we conservatively estimate that 1,366 have homologs in the microsporidian Encephalitozoon cuniculi, the most closely related published genome sequence. We identify genes conserved among microsporidia that lack clear homology outside this group, which are of special interest as potential virulence factors in this group of obligate parasites. A substantial fraction of the diminutive N. ceranae proteome consists of novel and transposable-element proteins. For a majority of well-supported gene models, a conserved sense-strand motif can be found within 15 bases upstream of the start codon; a previously uncharacterized version of this motif is also present in E. cuniculi. These comparisons provide insight into the architecture, regulation, and evolution of microsporidian genomes, and will drive investigations into honey bee-Nosema interactions.