PLOS Pathogens

Visceral leishmaniasis (VL) is a potentially fatal parasitic disease in which effective immunity requires sufficient inflammation to control parasites while limiting immune-mediated tissue damage. Transforming growth factor-beta (TGF{beta}) is an important regulator of immune homeostasis and has been implicated in VL, but how it directly controls parasite-specific CD4 T cell responses remains poorly understood. We used complementary transgenic mouse models with either enhanced or ablated TGF{beta} signalling in T cells during Leishmania donovani infection, combined with adoptive co-transfer of parasite-specific CD4 T cells to distinguish cell-intrinsic effects. Enhanced TGF{beta} signalling impaired hepatic parasite control and suppressed CD4 T cell immunity, reducing T helper 1 (Th1) cell differentiation, proliferation, accumulation of antigen-experienced cells, and expression of cytolytic molecules. Conversely, ablation of TGF{beta} signalling improved parasite control and promoted CD4 T cell expansion and Th1 cell differentiation, while increasing expression of cytolytic molecules and reducing interleukin-10-producing type 1 regulatory T (Tr1) cells. Adoptive co-transfer experiments confirmed that TGF{beta} directly restrained the expansion and Th1 cell differentiation of parasite-specific CD4 T cells and their acquisition of cytolytic features. Loss of signalling also impaired development of Tr1 cells and reduced expression of several chemokine receptors and co-inhibitory molecules associated with their regulatory function. However, enhanced signalling did not increase Tr1 cell development, indicating that the relationship between TGF{beta} signalling and immune regulation is not linear. TGF{beta} is a key cell-intrinsic regulator of CD4 T cell fate during experimental VL. Rather than acting solely as a general suppressor of inflammation, it calibrates the balance between protective and regulatory immunity by controlling CD4 T cell expansion, differentiation and effector function. Author summaryVisceral leishmaniasis (VL) is a potentially fatal disease caused by Leishmania parasites. The immune system must generate a strong enough response to control these parasites while preventing excessive inflammation that can damage tissues. We investigated how transforming growth factor-beta (TGF{beta}), an important regulator of immune responses, helps maintain this balance. Using mice in which signalling by TGF{beta} was either increased or removed specifically in T cells, we found that this pathway strongly influenced the development and function of CD4 T cells during infection. Increasing signalling suppressed the expansion of these cells and their development into inflammatory cells associated with parasite control. In contrast, removing signalling enhanced these responses and improved early parasite control, but also reduced the development of regulatory T cells that can limit inflammation. By studying parasite-specific T cells directly, we showed that many of these effects resulted from TGF{beta} acting within the T cells themselves. Our findings show that TGF{beta} does more than simply suppress immunity during VL. It helps determine the balance between CD4 T cell responses that control parasites and those that regulate inflammation, providing new insight into how immunity is shaped during chronic infection.

Long non-coding RNAs (lncRNAs) are a class of regulatory RNAs with lengths exceeding 200 nucleotides. The lncRNA PURPL (p53 upregulated regulator of p53 levels) is a p53-regulated gene. PURPL is an oncogenic lncRNA that represses p53 function; it is upregulated in many tumors and promotes cancer cell proliferation. Kaposi sarcoma-associated herpesvirus (KSHV) is the causative agent of all forms of Kaposi sarcoma (KS) and is tightly associated with primary effusion lymphoma (PEL). We found upregulation of PURPL in both de novo and chronically KSHV-infected cells. We found that p73 (a p53 homolog) associates with the PURPL promoter and upregulates PURPL expression. Previously, it has been shown that KSHV-encoded latency-associated nuclear antigen (LANA) stabilizes p73. We found that ectopic expression of LANA increased both p73 protein levels and PURPL expression. p73 knockdown abrogates PURPL upregulation following KSHV infection. Antisense oligonucleotides (ASO) targeting PURPL reduced PEL cell proliferation and increased acetylated p53 levels. In contrast to p53, p73 is not repressed by PURPL RNA. In summary, our study reveals how an oncogenic virus can utilize p73 to upregulate the oncogenic lncRNA PURPL as an additional way to repress p53. Moreover, we propose another mechanism by which p73 may promote tumor growth.

Aspergillus fumigatus is the most common cause of invasive aspergillosis (IA), an opportunistic pulmonary infection of immunocompromised patients that can be fatal, despite treatment with modern antifungal drugs. During infection, myeloid cells, such as neutrophils, monocytes, and monocyte-derived dendritic cells (Mo-DCs) are recruited to the lung and are critical for phagocytosing and killing A. fumigatus spores. Recent evidence has shown that intracellular crosstalk between these professional immune cells and other resident pulmonary cell types is required for an effective immune response to A. fumigatus, and that signaling via the cytokine granulocyte macrophage-colony stimulating factor (GM-CSF) is part of this intracellular communication. In this study, we sought to determine a role for GM-CSF signaling on monocytes and their progeny. We harnessed a mouse model of deletion of GM-CSF receptor {beta} chain (encoded by Csf2rb) in CCR2-expressing cells. Using this model, we found that GM-CSF signaling is required on CCR2+ cells for killing of A. fumigatus and for host survival. This observation expands the importance of GM-CSF in host defense during A. fumigatus pulmonary infection and extends its role in pathways of intracellular crosstalk that mediate effective pulmonary antifungal immunity.

Nuclear delivery of human papillomavirus (HPV) requires infected cells to undergo mitosis. Incoming HPV DNA is protected by a transport vesicle structure, which becomes unstable post-mitosis. We have previously shown that HPV genome egress takes on average 4 hours post-mitosis completion; thus, we propose that an enzymatic process is involved in degrading this transport vesicle to allow genome egress. Several phospholipase C (PLC) isoforms, including PLC{delta}3, PLC{zeta}1, and PLCL1, have been previously implicated in HPV infection in large-scale siRNA screens, and the parvovirus VP1 capsid protein has phospholipase enzymatic activity. Therefore, we hypothesized that cellular phospholipases residing in the nucleus mediate egress of the HPV genome from transport vesicles. We utilized siRNA-mediated knockdown and CRISPR/Cas9 knockout techniques to target specific PLC isoforms in both HeLa and HaCaT cell lines. We found that targeting PLC isoforms PLC{beta}1, {beta}4, {delta}3, {delta}4, and {gamma}1 significantly decreased HPV infection in both HeLa and HaCaT cells, as measured using a luciferase-based reporter assay. We also discovered a novel interaction between HPV16 minor protein L2 and PLCs {beta}1, {beta}4, {delta}1, {delta}3, and {delta}4. Furthermore, we observed that knockout of PLC{beta}4 and {delta}4 delayed egress of the HPV genome from nuclear membrane-bound vesicles after infection of HeLa cells. We propose that HPV utilizes phospholipase Cs to achieve genome egress from its protective vesicular structure after nuclear delivery.

Mammarenaviruses (MaAv) cause persistent infections in diverse rodent reservoirs worldwide and several are zoonotic pathogens with an important public-health burden in their endemic regions. Moreover, the globally distributed MaAv lymphocytic choriomeningitis virus (LCMV) is an underrecognized pathogen of clinical significance in congenital infections and immunocompromised individuals. The lack of FDA-approved vaccines or antivirals for MaAv infections underscores the urgent need for novel anti-MaAv therapeutic strategies. Neutral sphingomyelinase 2 (nSMase2) was recently identified as a host factor contributing to LCMV multiplication, and its inhibitor cambinol exhibits dose-dependent antiviral activity against LCMV but the underlying mechanisms remain undefined. Here, we show that cambinol disrupts multiple stages of the LCMV life cycle. Cambinol inhibits the pH-dependent fusion event mediated by MaAv glycoprotein, a step required for completion of virus cell entry. It also reduces viral ribonucleoprotein (vRNP)-directed genome replication and transcription and impairs the budding activity of the virus matrix Z protein. Cambinol also inhibits sirtuins 1 and 2 (Sirt-1 and Sirt-2), two NAD+-dependent protein deacetylases with pleiotropic roles in cellular metabolism and stress responses, raising the question of whether cambinol anti-LCMV activity reflects nSMase2 inhibition alone or also involves sirtuin-dependent pathways. LCMV multiplication was significantly reduced in SIRT1, but not SIRT2, knockout (KO) cells, uncovering a pro-viral role for Sirt-1 in the LCMV life cycle. Consistent with this finding, LCMV vRNP activity and production of infectious progeny were reduced in SIRT1 KO cells. These findings identify Sirt-1 as a host factor required for optimal LCMV multiplication. Sirt-1 inhibitors are in clinical development for oncological and neurological indications, raising the possibility of repurposing Sirt-1 inhibitors as host-directed antivirals (HDAs) against human pathogenic MaAv. Abstract figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/740332v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@d53c5forg.highwire.dtl.DTLVardef@16ea861org.highwire.dtl.DTLVardef@1f09addorg.highwire.dtl.DTLVardef@1472e6b_HPS_FORMAT_FIGEXP M_FIG C_FIG

The Middle East respiratory syndrome coronavirus uses DPP4/CD26 as receptor for cell entry. Soluble recombinant DPP4 can block MERS-CoV infection in cell culture and in animal models and might hold promise as antiviral. Furthermore, endogenous soluble DPP4 in plasma might reduce MERS-CoV dissemination in infected individuals. Therefore, we addressed whether and how MERS-CoV can acquire resistance against soluble DPP4 (sDPP4), employing a vesicular stomatitis virus (VSV) encoding the MERS-CoV spike (S) protein (VSV-MERS-S). Passaging of VSV-MERS-S in the presence of sDPP4 selected for viral variants with mutations L507I and L507H in the receptor binding domain (RBD) of the S protein. These mutations reduced sDPP4 binding, were compatible with robust entry into cell lines expressing high levels of DPP4 and conferred sDPP4 resistance. In contrast, entry into cell lines expressing low levels of DPP4 was reduced. Furthermore, polymorphisms L507F/R/P, which were detected in MERS-CoV sequences from patients, exhibited an even more pronounced phenotype, with L507R and L507P conferring complete sDPP4 resistance. Collectively, our results show that naturally occurring polymorphisms in the MERS-CoV S protein can confer sDPP4 resistance by reducing sDPP4 binding but might still be compatible with robust viral spread in cells and tissues expressing high levels of DPP4.

The intracellular bacterium Wolbachia pipientis alters the biology of its arthropod hosts in many ways, and can increase resistance to RNA viruses in both Drosophila and mosquitoes. Wolbachia-induced pathogen blocking has generated much interest because of its potential to restrict insect vector transmission of human diseases caused by RNA viruses. However, the molecular mechanisms by which Wolbachia affects host viral resistance are still mostly elusive. We used dilp2-3,5 mutant Drosophila, which are long-lived, but only in the presence of Wolbachia, to show that the presence of Wolbachia also increased the resistance of the mutant flies to infection with Drosophila C virus (DCV), relative both to mutants lacking Wolbachia and to wild type flies with and without Wolbachia. The insulin mutant flies had higher Wolbachia titres than wild type flies. By RNA-seq analysis of the Wolbachia transcriptome, we identified Wolbachia genes that were more strongly expressed in dilp2-3,5 mutant flies. Ankyrin-domain-containing proteins were among the most strongly up-regulated and they are predicted to be secreted effector proteins. To address their effect on host physiology, we generated 4 transgenic fly lines each with inducible expression of a different genes encoding ankyrin-domain-containing proteins. Expression of 3 of these did not cause obvious effects, but expression of WD0754 at high levels severely shortened fly survival. Interestingly, however, chronic low-level induction of WD0754 increased the resistance of the flies to DCV infection. Proteomics analysis showed a robust, tissue-specific, anti-viral response upon WD0754 induction, and identified the NFkB-like IMD pathway as a potential mediator of the antiviral activity of WD0754. Consistently, WD0754 expression activated Relish, a key downstream transcriptional mediator of IMD signalling, while loss-of Relish blocked the antiviral effect of WD0754. In summary, we identified a Wolbachia-derived ankyrin domain containing protein that modulates host immunity through the IMD pathway.

Protistan parasites of the genus Leishmania, infamous human and animal pathogens, can themselves be infected by endosymbiotic viruses, exemplified by Leishmania RNA viruses (LRVs). These viruses affect immune responses in vertebrate hosts and have been associated with adverse treatment outcomes. How parasites control replication of these viruses is not known. Intriguingly, functional RNA interference (RNAi) pathways that have been associated with antiviral responses across eukaryotes, are retained only in some Leishmania spp., including those of the subgenus Viannia. Here, we investigated effectors in the canonical RNAi response and the Piwi protein of the human pathogen L. (Viannia) guyanensis by gene ablation and identified Dicer-like 1 and Argonaute 1 proteins of the canonical RNAi pathway as critical for controlling viral RNA levels. Notably, we characterized virus-derived small interfering RNA (vsiRNA) levels and their unique properties including terminal modifications as well as, unusual for canonical Dicer cleavage, predominant perfectly matching sequence overlaps in blunt ended vsiRNA duplexes. Taken together, the data suggests that control of viral replication is directly mediated by the canonical RNAi response. This study opens the door to further investigations of antiviral RNAi in other protistan parasites and suggests that, where present, canonical RNAi is critical for such activities. Author summaryLeishmania parasites of humans and animals harbor endosymbiotic viruses, which, in some cases, have been shown to affect vertebrate immune responses and impact treatment. Thus, understanding how viral levels are controlled is critical to identify antiviral effectors, which, in turn, will allow studies on how viral levels impact parasite biology. Here, we investigated RNA interference pathways against its virus of the family Pseudototiviridae in a New World human pathogen L. guyanensis. To do that, we have produced and analyzed genetic knockouts of Dicer-like and Argonaute proteins involved in antiviral small RNA response. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=78 SRC="FIGDIR/small/743808v1_ufig1.gif" ALT="Figure 1"> View larger version (21K): org.highwire.dtl.DTLVardef@cffe40org.highwire.dtl.DTLVardef@13d4012org.highwire.dtl.DTLVardef@395e60org.highwire.dtl.DTLVardef@631fed_HPS_FORMAT_FIGEXP M_FIG C_FIG

Necrotrophic effectors (NEs) are key determinants of virulence in the necrotrophic fungal pathogen Parastagonospora nodorum that causes septoria nodorum blotch of wheat. However, targeted removal of three important NEs SnToxA, SnTox1, and SnTox3 in the mutant{Delta} toxa13 previously revealed a redundancy mechanism is triggered, whereby pathogenicity on wheat is maintained. In this study, we investigated the gene regulatory profile underpinning this phenomenon and discover that virulence is not dependent on a fixed set of dominant effectors but instead arises from a flexible, epistatic compensatory network. Although host transcriptional responses to the P. nodorum wildtype SN15 and{Delta} toxa13 infection remained largely conserved, consistent with an overlapping disease-susceptibility pathway, a significant upregulation of candidate effector genes was observed in{Delta} toxa13. This included the recently characterised NE SnTox267, and several other candidate effectors able to induce necrosis in the non-host Nicotiana benthamiana, each carrying a predicted structural fold conserved across other pathogens. We therefore provide further direct evidence that virulence is maintained in P. nodorum lacking three NEs by an epistatic and compensatory effector network, underpinned by changes in pathogen gene expression. Targeting conserved effector-mediated virulence mechanisms rather than individual host-specific gene-for-gene interactions may provide a more tractable route to host resistance.

Although many genes are associated with leprosy, a skin and nerve infection by Mycobacterium leprae, the function of most of these genes in infection remains unknown. This is partly due to a paucity of animal models that are genetically malleable and recapitulate features of the human disease. Zebrafish, a recent leprosy model, have human-like responses to M. leprae, including macrophage-mediated inflammation and neurodegeneration. We confirm this at the transcriptional level, using RNA sequencing (RNAseq) of chronic M. leprae infection of adult zebrafish. This identified regulated zebrafish orthologs of human leprosy-associated genes, including {beta}- and {gamma}-interferon, IL-6, IL-10, IL-4, and TNF superfamily members. Other regulated genes have not been previously associated with leprosy. Genes associated with tuberculoid leprosy (T-lep) were largely downregulated, while lepromatous leprosy (L-lep) genes were upregulated. Pathways relevant to leprosy, such as phagocytosis and antiviral responses, differed between early and late stages of infection. In infected rag1 mutant zebrafish, which lack T and B cells, T-lep genes were downregulated, suggesting that adaptive immunity is required for their expression. Key pathways and genes were validated by qPCR using zebrafish infection with M. marinum:PGL-1, a model pathogen that can express M. leprae genes. This system allowed for the identification of validated M. leprae-regulated genes that alter infection outcomes, by using multiplex CRISPR to simultaneously mutate many zebrafish genes. 17 genes were screened and 2 were identified that increased mortality infection: isg15 (interferon-stimulated gene 15) and serpine1 (plasminogen activator inhibitor-1). This work confirms that, in humans and zebrafish, type I interferon and plasminogen activation are required for survival of mycobacterial disease, and demonstrates that multiplex CRISPR can identify mediators of host defense in vivo.

Cathelicidins are a class of antimicrobial peptides (AMPs) that are part of the first line of defense of the innate immune system. While cathelicidin-derived peptides such as LL-37 can be directly bactericidal, Streptococcus pyogenes (Spy; Group A Streptococcus) is highly resistant to killing. Furthermore, Spy detects LL-37 through the CovRS two-component system to regulate its virulence factors. One effect of this signaling is the repression of expression of the bacterial protease SpeB. Prior work has also shown that SpeB, along with other bacterial proteases can cleave LL-37. However, it is unclear if SpeB cleavage of LL-37 impacts antimicrobial function and CovRS signaling activity. Using a genetic approach, we show that the presence SpeB did not significantly impact the killing of Spy by LL-37 relative to other known resistance factors. Furthermore, while SpeB cleaves LL-37, CovRS maintains sensitivity to LL-37 fragments. These results indicate that SpeB cleavage of LL-37 does not negatively impact virulence factor regulation in Spy.

Mareks disease virus (MDV) is an oncogenic herpesvirus that establishes latency in CD4 T cells, from which it reactivates to initiate productive replication and dissemination. To define host mechanisms governing the transition from latency to early and late lytic replication, we developed recombinant MDV expressing early RLORF4mRFP and late UL47eGFP, which was used to isolate latent (Lo; mRFP low), early lytic (Hi; mRFP high), and late lytic (DP; mRFPeGFP) populations from MDV-induced lymphoblastoid cell lines (LCLs). Two independent LCLs (Lines 62 and 82) were subjected to Illumina RNA sequencing after sorting-purified populations. Viral transcription increased progressively from Lo to Hi to DP populations; however, initiation of viral gene expression differed markedly between cell lines. During the early transition (Lo to Hi), Line 82 exhibited robust induction of 17 viral genes, whereas Line 62 showed only two differentially expressed viral genes. However, both lines converged on a highly conserved transcriptional program during the transition from latency (Lo) to late-lytic replication (DP), sharing 118 viral transcripts, most of which were structural and assembly-associated genes. Among the host transcriptional responses, Line 82 exhibited activation of TP53-associated stress signaling, chromatin remodeling factors, and RNA biogenesis pathways, consistent with a permissive cellular state. In contrast, Line 62 displayed enhanced inflammatory, metabolic, and proteostasis-associated signatures, suggesting a restrictive environment that limits early viral induction. Collectively, these findings demonstrate that MDV reactivation is a host-gated process in which the cellular state governs the initiation of the lytic switch, whereas downstream replication proceeds through a conserved viral program. IMPORTANCEThis study provides critical insights into the host-virus dynamics governing herpesvirus reactivation from latency. By developing a dual-reporter system using recombinant Mareks disease virus (MDV) in MDV-transformed lymphoblastoid cell lines (LCLs), an oncogenic herpesvirus model, reactivating cells were sorted for latent, early-lytic, and late-lytic populations from two independent LCLs. The initial transition from latency to early-lytic replication was highly variable and strongly influenced by cellular state: one line showed rapid viral gene induction linked to stress, chromatin remodeling, and permissive conditions, while the other exhibited a more restrictive, inflammatory/metabolic response. However, progression to late-lytic replication converged on a highly conserved viral transcriptional program dominated by structural genes. These findings establish that host cellular context gates the lytic switch in herpesvirus reactivation, offering a powerful framework for understanding latency control across oncogenic herpesviruses and potential therapeutic targets to prevent reactivation and tumor formation.

Arboviruses such as dengue, Zika, and chikungunya viruses cause widespread disease and continue to expand their geographical range due to climate change and vector spread. Insect-specific flaviviruses (ISFs) are promising biocontrol candidates of arboviruses, due to recent studies showing that prior infection with an ISF can reduce arbovirus replication in mosquitoes through superinfection exclusion (SIE). However, the route of infection, tissue tropism, pathogenesis and the mechanisms underlying SIE of ISFs in mosquitoes remain unclear. RNA interference (RNAi) is a potent antiviral response in insects, therefore it is expected that sequence homology between the ISF and the arbovirus will strengthen SIE. Here, we used ISF Binjari virus and a chimera containing the Zika virus structural proteins prME (BinJ-ZIKV) as a model system. Intrathoracic injection of BinJ-ZIKV in Aedes aegypti led to rapid systemic infection that excluded the midgut, subsequently blocking ZIKV dissemination from the midgut. SIE was strongest in tissues where primary-virus replication was highest. This spatial component of SIE was stronger when there was sequence homology between the ISF and arbovirus and displayed a strong 21nt siRNA response, suggesting RNAi contributed to the observed SIE. Upon oral inoculation, BinJ-ZIKV replicated efficiently in mosquitoes, was detected across multiple tissues, and saliva. BinJ-ZIKV also had higher infection establishment than BinJV at lower oral titres. SIE was observed for BinJ-ZIKV infection after oral exposure interfered with subsequent ZIKV midgut infection. Together, these findings support engineered ISF-chimeras as valuable experimental tools to dissect viral determinants of SIE and to optimize mosquito-based arbovirus interference strategies. ImportanceAnnually, over 400 million people are infected with mosquito-transmitted viruses. Insect-specific flaviviruses (ISFs) can interfere with the transmission of clinically important viruses through a phenomenon termed superinfection exclusion (SIE). However, the mechanisms of SIE remain poorly understood. Using a Binjari virus chimera expressing Zika virus (ZIKV) structural proteins, we show that SIE is highly tissue-specific, with exclusion of ZIKV only occurring at sites where the chimera actively replicates and induces the mosquito antiviral RNA interference pathway. We further demonstrate that incorporation of Zika virus prM and E proteins into the ISF backbone enhances infection of the mosquito midgut following oral exposure, which enables direct inhibition of ZIKV infection after a subsequent infectious blood meal. Together, these findings define a replication-dependent, tissue-specific mechanism of ISF-mediated protection and provide a framework for reducing mosquito-borne virus transmission through SIE.

Leishmania species are transmitted between mammals by sand flies, within which they undergo a complex developmental programme involving transitions between replicative and non-replicative life cycle forms. Recent work using single-cell transcriptomics has revealed the developmental pathways of Leishmania major in the sand fly, but it is unclear how faithfully such development is recapitulated during Leishmania culture in vitro. In addition, no work has revealed how gene expression changes across the cell cycle of replicative Leishmania forms. Using single-cell transcriptomics on unsynchronised cultures of L. major and L. mexicana, we addressed both questions. We show that in vitro L. mexicana promastigotes display the same two developmental endpoints seen in L. major cells recovered from sand flies, metacyclics and haptomonads. In contrast, L. major promastigote development in vitro does not predict the presence of leptomonads or haptomonads, although non-dividing late metacyclics form through an early metacyclic stage as parasites enter stationary phase. Using computational reconstruction of the cell cycle in procyclic promastigote forms of both Leishmania species identified hundreds of genes whose transcript levels vary as the cell cycle progresses, revealing core conserved processes between the two species and with the closely related Trypanosoma brucei. These genes allowed us to examine connections between life cycle and cell cycle progression, revealing that some developmental transitions are strongly linked to a cell cycle phase, whereas others occur asynchronously. This work provides a comprehensive atlas and marker gene list for the interlinked cell and life cycles of two laboratory-cultured Leishmania species.

Ebola virus (EBOV) disease (EVD) is a hemorrhagic disease caused by EBOV infection. EVD outcomes in pregnant women are similar to non-pregnant women however, EVD is associated with negative fetal outcomes in [~]99% of cases. There is a critical need for a tractable small animal model to study maternal/fetal transmission of EBOV. We utilized interferon /{beta} receptor knock out mice infected and authentic EBOV or the model virus, recombinant vesicular stomatitis virus encoding EBOV glycoprotein (rVSV/EBOV). Infection with either virus during late pregnancy resulted in placental infection and vertical transmission to the fetus within 2-3 days. Robust levels of maternal and fetal proinflammatory cytokines were evident by day 5 after EBOV infection. Within the placenta, trophoblasts and endothelial cells were viral antigen positive. Elimination of the endosomal receptor NPC1 in junctional zone trophoblasts reduced placental infection and virus transmission to the fetus. These studies establish an infectious model that provides EBOV trafficking and pathogenesis insights during pregnancy. TeaserThis model provides key insights into how viral trafficking and maternal immune responses drive adverse fetal outcomes during gestational Ebola virus infection.

Streptococcus pneumoniae (pneumococcus) are asymptomatic colonizers of the nasopharynx but can progress to pulmonary and systemic pathogens upon influenza A virus (IAV) infection. Polymorphonuclear cells (PMNs) are required to control bacterial numbers, but we previously found that IAV infection impairs their ability to kill S. pneumoniae. Here, using a model that allows transition of pneumococci from colonizers to disease-causing pathogens upon IAV co-infection, we examined the signaling pathways impairing PMN responses. When we investigated the effect of type I interferons (IFN) produced upon IAV infection on PMN antibacterial activity, we found that PMNs treated with IFN were unable to kill S. pneumoniae ex vivo, and that in vivo blocking of IFN receptor 1 (IFNAR1) in IAV infected mice rescued PMN antibacterial function. In exploring what controls PMN responsiveness to IFN, we examined CD73, an ectonucleotidase that is known to regulate PMN function in primary pneumococcal pneumonia. To test if there is an intersection between CD73 and IFN signaling, we examined receptor levels and IFN production in wildtype versus CD73KO mice and found no difference in IFNAR expression on PMNs or IFN[a] and IFN levels in the lungs and circulation. However, CD73KO PMNs expressed significantly lower levels of the interferon stimulated protein IFIT1. When we looked at ex vivo PMN responsiveness to IFNs, CD73KO PMNs were less responsive to IFN-mediated inhibition of antimicrobial activity. In exploring mechanisms, we found that CD73 expressing PMNs had elevated production of reactive oxygen species in response to IAV challenge, that paradoxically impaired their ability to kill S. pneumoniae. Importantly, despite similar pathogen loads in the respiratory tract, co-infected CD73KO mice cleared bacteremia and survived significantly better than wildtype controls. These findings suggest that CD73 impairs host defense against IAV/S. pneumoniae co-infection in part by sensitizing PMNs to type I IFN-mediated inhibition of antibacterial function. Author SummaryDespite available therapeutics and vaccines, secondary bacterial pneumonia following influenza A virus (IAV) infection remains a major cause of disease. A common cause of secondary bacterial pneumonia are Streptococcus pneumoniae (pneumococcus), bacteria that resides asymptomatically in the nasopharynx, but upon viral infection can transition to cause severe disease in susceptible hosts. In this study we examined how host responses change during single versus polymicrobial infections. We focused on neutrophils, which are innate immune cells that are required for effective clearance of S. pneumoniae, and proper control of IAV. We found that the immune response to IAV, mediated by type I interferons (IFN), impair the ability of neutrophils to kill bacteria. We identified an enzyme called CD73 to be required for the ability of neutrophils to respond to IFN. In exploring mechanisms, we found that IFN and CD73 result in dysregulated reactive oxygen species production by neutrophils. Importantly, this impairs the ability of the host to clear bacteria that spread from the lungs to the blood upon viral co-infection and results in overall worse host outcome. This study describes a novel interaction between CD73 and type I interferons and provides a new therapeutic target to treat secondary pneumococcal pneumonia.

Kaposis sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is an oncogenic gammaherpesvirus that causes Kaposis sarcoma and several lymphoproliferative disorders. The KSHV open reading frame (ORF) 16 encodes viral Bcl-2 (vBcl-2), which inhibits apoptosis and autophagy. vBcl-2 is required for efficient lytic replication and production of infectious progeny; however, this essential role is independent of its established functions regulating cell death. To identify host factors that may contribute to vBcl-2 function, we employed proteomic analysis of HA-vBcl-2 immunoprecipitates from lytically reactivated KSHV-infected cells. This analysis identified the BTB-Kelch protein KLHL9, a substrate-specific adaptor for Cullin 3-RING ligase (CRL3) complex, as a candidate vBcl-2-associated factor. We validated the vBcl-2-KLHL9 association by reciprocal co-immunoprecipitation from infected cells, and by ectopic expression in uninfected cells. During lytic reactivation, KLHL9 redistributed from a predominantly cytoplasmic/perinuclear pattern to a mitochondrial pattern that overlapped with HA-vBcl-2. Alanine-scanning mutagenesis mapped a KLHL9-interaction determinant to the N-terminal region of vBcl-2, and vBcl-2 mutants defective in binding failed to colocalize with KLHL9. Conversely, KLHL9 deletion analysis implicated the C-terminal Kelch-repeat region in efficient association with vBcl-2. AlphaFold modeling supported the interface regions. However, vBcl-2 did not show detectable KLHL9-dependent ubiquitination, suggesting that it is unlikely to be a substrate of a KLHL9-containing complex. Functionally, CRISPR/Cas9-mediated KLHL9 disruption reduced lytic viral protein accumulation and infectious progeny production, while re-expression of sgRNA-resistant KLHL9 partially restored these phenotypes. Together, these findings suggest that vBcl-2 may engage host CRL3 complexes during productive infection, and identify KLHL9 as a vBcl-2-associated host factor that supports efficient KSHV lytic replication. Author SummaryKaposis sarcoma-associated herpesvirus (KSHV) is a cancer-associated virus that alternates between a latent cycle and a productive lytic cycle, during which new infectious virus particles are made. The viral protein vBcl-2 is best known for regulating cell-death and autophagy pathways, but earlier studies showed that it is required for efficient virus production through additional, non-canonical functions. In this study, we identify the cellular protein KLHL9 as a host factor that associates with KSHV vBcl-2. KLHL9 normally functions as a specific adaptor in protein ubiquitination pathways that regulate the turnover, localization, or activity of target proteins through ubiquitination. We show that vBcl-2 and KLHL9 associate with one another, and that KLHL9 redistributes toward the mitochondria in cells expressing vBcl-2. Importantly, loss of KLHL9 impairs the expression of viral lytic proteins and reduces the production of new infectious viruses. This defect resembles the phenotype caused by loss of vBcl-2, suggesting that KSHV employs vBcl-2 to engage KLHL9-dependent host machinery during productive infection. Understanding this interaction may help reveal how herpesviruses reprogram infected cells to create an environment that supports virus production.

Bovine viral diarrhea virus (BVDV) remains an economically important pathogen of cattle despite widespread vaccination. A homozygous CD46-edited Gir heifer (Ginger) was previously shown to have significantly reduced susceptibility to BVDV. The edited allele contains an in-frame six amino acid substitution within the virus-binding domain of the BVDV entry receptor CD46, replacing residues G82QVLAL with A82LPTFS. Here, we investigated whether reduced BVDV susceptibility is maintained when the edited allele is inherited in the heterozygous state. Ginger was artificially inseminated with semen from an unedited Gir bull and produced a healthy heterozygous CD46-edited bull calf (Giraldo). Whole-genome sequencing confirmed the inheritance and structural integrity of Giraldo's edited allele. Compared with Ginger, Giraldo exhibited similarly reduced ex vivo BVDV susceptibility across primary fibroblasts, lymphocytes, and monocytes, despite inheriting a wild-type CD46 allele from the sire. Allele-specific CD46 RNA expression analysis demonstrated expression of both the edited and wild-type CD46 alleles. Thus, the reduced-susceptibility phenotype was not attributable to transcriptional silencing of the wild-type allele. Lentiviral complementation studies in CD46-knockout Madin-Darby bovine kidney (MDBK) cells further demonstrated that this wild-type CD46 allele was competent to support BVDV infection when expressed independently. Together, these findings indicate that the CD46 A82LPTFS allele can confer reduced BVDV susceptibility in the heterozygous state despite expression of a functional wild-type CD46 allele. This result suggests the potential to more rapidly disseminate reduced BVDV susceptibility through conventional breeding using homozygous CD46-edited sires.

Leishmania parasites must rapidly adapt to fluctuating environments to ensure survival and transmission. While acidic pH in the sand fly vector is a conserved developmental trigger, sensing mechanisms remain poorly understood. Using a barcoded protein kinase library, we screened for regulators of acid adaptation in Leishmania mexicana, identifying nine protein kinases influencing survival at low pH, including a haptomonad differentiation regulator protein kinase (HDRK1). We demonstrate that HDRK1 null mutants ({Delta}hdrk1) are predisposed to differentiate to haptomonad-like forms at low pH. While {Delta}hdrk1 mutants successfully infect the sand fly midgut, they fail to colonise the stomodeal valve, compromising transmission. Integrated transcriptomic and proteomic analyses revealed that at low pH, {Delta}hdrk1 mutants enter a low energy state reminiscent of AMPK-activated cells. We identified a second protein kinase, HDRK2, establishing a pH-dependent signalling pathway that governs the developmental fate of the parasite, directing differentiation towards either mammalian-infective metacyclic or vector-attached haptomonad stages. Finally, our screen revealed that phosphoinositide balance, regulated by the lipid kinases PI4K and PI4P5K, is vital for acid adaptation, and identified two STE transmembrane kinases as potential pH sensors. Together these findings provide a framework for how Leishmania detects and survives acid stress to coordinate its life cycle.

Porcine deltacoronavirus (PDCoV) is a globally circulating coronavirus of swine that causes severe enteric disease and mortality in neonatal piglets. Its broad host range and zoonotic potential raises concerns about continued spillover into human populations. However, the host factors that govern PDCoV infection remain largely unexplored. Identifying host factors involved in virus infection may uncover novel therapeutic targets for antiviral drug development or targets for gene editing to create resistant pigs. Here, we conducted a genome-wide CRISPR knockout (GeCKO) screen to identify host factors contributing to PDCoV infection in swine testicular (ST) cells. Small guide RNAs (sgRNAs) targeting the ANPEP gene, expressing the main receptor porcine aminopeptidase N for PDCoV, were enriched in PDCoV-resistant cells, demonstrating the validity of the screen. Moreover, we identified transmembrane protein 30A (TMEM30A), an accessory {beta}-subunit of the P4-ATPase flippase complex, as a host dependency factor for PDCoV infection along with several potential PDCoV-induced cell death regulators, including DET1, IFNAR1, RFWD2, STAT1 and TP53. We further demonstrated that PDCoV attachment and internalization into cells were significantly impaired in TMEM30A knockout ST cells, suggesting that TMEM30A is important in the early stages of the PDCoV life cycle. Because TMEM30A also contributes to SARS-CoV-2 replication, future studies are warranted to investigate its mechanistic role in the context of coronavirus infection.