Editor’s Choice Articles

Superinfection exclusion (SIE) is an antagonistic interaction between identical or closely related viruses in host cells. Previous studies by us and others led to the hypothesis that SIE was elicited by one or more proteins encoded in the genomes of primary viruses. Here, we tested this hypothesis using Turnip mosaic virus (TuMV), a member of the genus Potyvirus of the family Potyviridae, with significant economic consequences. To this end, individual TuMV-encoded proteins were transiently expressed in the cells of Nicotiana benthamiana leaves, followed by challenging them with a modified TuMV expressing the green fluorescent protein (TuMV-GFP). Three days after TuMV-GFP delivery, these cells were examined for the replication-dependent expression of GFP. Cells expressing TuMV P1, HC-Pro, 6K1, CI, 6K2, NIa-VPg, NIb, or CP proteins permitted an efficient expression of GFP, suggesting that these proteins failed to block the replication of a superinfecting TuMV-GFP. By contrast, N. benthamiana cells expressing TuMV P3 or NIa-Pro did not express visible GFP fluorescence, suggesting that both of them could elicit potent SIE against TuMV-GFP. The SIE elicitor activity of P3 and NIa-Pro was further confirmed by their heterologous expression from a different potyvirus, potato virus A (PVA). Plants systemically infected with PVA variants expressing TuMV P3 or NIa-Pro blocked subsequent infection by TuMV-GFP. A +1-frameshift mutation in P3 and NIa-Pro cistrons facilitated superinfection by TuMV-GFP, suggesting that the P3 and NIa-Pro proteins, but not the RNA, are involved in SIE activity. Additionally, deletion mutagenesis identified P3 amino acids 3 to 200 of 352 and NIa-Pro amino acids 3 to 40 and 181 to 242 of 242 as essential for SIE elicitation. Collectively, our study demonstrates that TuMV encodes two spatially separated proteins that act independently to exert SIE on superinfecting TuMV. These results lay the foundation for further mechanistic interrogations of SIE in this virus. Full article

Viruses encounter numerous host factors that facilitate or suppress viral infection. Although some host factors manipulated by viruses were uncovered, we have limited knowledge of the pathways hijacked to promote viral replication and activate host defense responses. Turnip mosaic virus (TuMV) is one of the most prevalent viral pathogens in many regions of the world. Here, we employed an isobaric tag for relative and absolute quantitation (iTRAQ)-based proteomics approach to characterize cellular protein changes in the early stages of infection of Nicotiana benthamiana by wild type and replication-defective TuMV. A total of 225 differentially accumulated proteins (DAPs) were identified (182 increased and 43 decreased). Bioinformatics analysis showed that a few biological pathways were associated with TuMV infection. Four upregulated DAPs belonging to uridine diphosphate-glycosyltransferase (UGT) family members were validated by their mRNA expression profiles and their effects on TuMV infection. NbUGT91C1 or NbUGT74F1 knockdown impaired TuMV replication and increased reactive oxygen species production, whereas overexpression of either promoted TuMV replication. Overall, this comparative proteomics analysis delineates the cellular protein changes during early TuMV infection and provides new insights into the role of UGTs in the context of plant viral infection. Full article

The nucleolus and Cajal bodies (CBs) are sub-nuclear domains with well-known roles in RNA metabolism and RNA-protein assembly. However, they also participate in other important aspects of cell functioning. This study uncovers a previously unrecognised mechanism by which these bodies and their components regulate host defences against pathogen attack. We show that the CB protein coilin interacts with poly(ADP-ribose) polymerase 1 (PARP1), redistributes it to the nucleolus and modifies its function, and that these events are accompanied by substantial increases in endogenous concentrations of salicylic acid (SA), activation of SA-responsive gene expression and callose deposition leading to the restriction of tobacco rattle virus (TRV) systemic infection. Consistent with this, we also find that treatment with SA subverts the negative effect of the pharmacological PARP inhibitor 3-aminobenzamide (3AB) on plant recovery from TRV infection. Our results suggest that PARP1 could act as a key molecular actuator in the regulatory network which integrates coilin activities as a stress sensor for virus infection and SA-mediated antivirus defence. Full article

Zika virus (ZIKV) has a unique ability among flaviviruses to cross the placental barrier and infect the fetal brain causing severe abnormalities of neurodevelopment known collectively as congenital Zika syndrome. In our recent study, we demonstrated that the viral noncoding RNA (subgenomic flaviviral RNA, sfRNA) of the Zika virus induces apoptosis of neural progenitors and is required for ZIKV pathogenesis in the developing brain. Herein, we expanded on our initial findings and identified biological processes and signaling pathways affected by the production of ZIKV sfRNA in the developing brain tissue. We employed 3D brain organoids generated from induced human pluripotent stem cells (ihPSC) as an ex vivo model of viral infection in the developing brain and utilized wild type (WT) ZIKV (producing sfRNA) and mutant ZIKV (deficient in the production of sfRNA). Global transcriptome profiling by RNA-Seq revealed that the production of sfRNA affects the expression of >1000 genes. We uncovered that in addition to the activation of pro-apoptotic pathways, organoids infected with sfRNA-producing WT, but not sfRNA-deficient mutant ZIKV, which exhibited a strong down-regulation of genes involved in signaling pathways that control neuron differentiation and brain development, indicating the requirement of sfRNA for the suppression of neurodevelopment associated with the ZIKV infection. Using gene set enrichment analysis and gene network reconstruction, we demonstrated that the effect of sfRNA on pathways that control brain development occurs via crosstalk between Wnt-signaling and proapoptotic pathways. Full article

Porcine deltacoronavirus (PDCoV) is an emergent swine coronavirus which infects cells from the small intestine and induces watery diarrhea, vomiting and dehydration, causing mortality in piglets (>40%). The aim of this study was to evaluate the antigenicity and immunogenicity of the recombinant membrane protein (M) of PDCoV (rM-PDCoV), which was developed from a synthetic gene obtained after an in silico analysis with a group of 138 GenBank sequences. A 3D model and phylogenetic analysis confirmed the highly conserved M protein structure. Therefore, the synthetic gene was successfully cloned in a pETSUMO vector and transformed in E. coli BL21 (DE3). The rM-PDCoV was confirmed by SDS-PAGE and Western blot with ~37.7 kDa. The rM-PDCoV immunogenicity was evaluated in immunized (BLAB/c) mice and iELISA. The data showed increased antibodies from 7 days until 28 days (p < 0.001). The rM-PDCoV antigenicity was analyzed using pig sera samples from three states located in “El Bajío” Mexico and positive sera were determined. Our results show that PDCoV has continued circulating on pig farms in Mexico since the first report in 2019; therefore, the impact of PDCoV on the swine industry could be higher than reported in other studies. Full article

Tomato chlorosis virus (ToCV) severely threatens tomato production worldwide. P27 is known to be involved in virion assembly, but its other roles in ToCV infection are unclear. In this study, we found that removal of p27 reduced systemic infection, while ectopic expression of p27 promoted systemic infection of potato virus X in Nicotiana benthamiana. We determined that Solanum lycopersicum catalases (SlCAT) can interact with p27 in vitro and in vivo and that amino acids 73 to 77 of the N-terminus of SlCAT represent the critical region for their interaction. p27 is distributed in the cytoplasm and nucleus, and its coexpression with SlCAT1 or SlCAT2 changes its distribution in the nucleus. Furthermore, we found that silencing of SlCAT1 and SlCAT2 can promote ToCV infection. In conclusion, p27 can promote viral infection by binding directly to inhibit anti-ToCV processes mediated by SlCAT1 or SlCAT2. Full article

Recent studies have indicated that bats are hosts to diverse filoviruses. Currently, no pan-filovirus molecular assays are available that have been evaluated for the detection of all mammalian filoviruses. In this study, a two-step pan-filovirus SYBR Green real-time PCR assay targeting the nucleoprotein gene was developed for filovirus surveillance in bats. Synthetic constructs were designed as representatives of nine filovirus species and used to evaluate the assay. This assay detected all synthetic constructs included with an analytical sensitivity of 3–31.7 copies/reaction and was evaluated against the field collected samples. The assay’s performance was similar to a previously published probe based assay for detecting Ebola- and Marburgvirus. The developed pan-filovirus SYBR Green assay will allow for more affordable and sensitive detection of mammalian filoviruses in bat samples. Full article

Herpes simplex virus (HSV) requires four essential virion glycoproteins—gD, gH, gL, and gB—for virus entry and cell fusion. To initiate fusion, the receptor binding protein gD interacts with one of two major cell receptors, HVEM or nectin-1. Once gD binds to a receptor, fusion is carried out by the gH/gL heterodimer and gB. A comparison of free and receptor-bound gD crystal structures revealed that receptor binding domains are located within residues in the N-terminus and core of gD. Problematically, the C-terminus lies across and occludes these binding sites. Consequentially, the C-terminus must relocate to allow for both receptor binding and the subsequent gD interaction with the regulatory complex gH/gL. We previously constructed a disulfide bonded (K190C/A277C) protein that locked the C-terminus to the gD core. Importantly, this mutant protein bound receptor but failed to trigger fusion, effectively separating receptor binding and gH/gL interaction. Here, we show that “unlocking” gD by reducing the disulfide bond restored not only gH/gL interaction but fusion activity as well, confirming the importance of C-terminal movement in triggering the fusion cascade. We characterize these changes, showing that the C-terminus region exposed by unlocking is: (1) a gH/gL binding site; (2) contains epitopes for a group (competition community) of monoclonal antibodies (Mabs) that block gH/gL binding to gD and cell–cell fusion. Here, we generated 14 mutations within the gD C-terminus to identify residues important for the interaction with gH/gL and the key conformational changes involved in fusion. As one example, we found that gD L268N was antigenically correct in that it bound most Mabs but was impaired in fusion, exhibited compromised binding of MC14 (a Mab that blocks both gD–gH/gL interaction and fusion), and failed to bind truncated gH/gL, all events that are associated with the inhibition of C-terminus movement. We conclude that, within the C-terminus, residue 268 is essential for gH/gL binding and induction of conformational changes and serves as a flexible inflection point in the critical movement of the gD C-terminus. Full article

Hepatitis E virus (HEV) is a major cause of acute viral hepatitis globally. Genotype 1 HEV (HEV-1) is responsible for multiple outbreaks in developing countries, causing high mortality rates in pregnant women. However, studies on HEV-1 have been hindered by its poor replication in cultured cells. The JE04-1601S strain recovered from a Japanese patient with fulminant hepatitis E who contracted HEV-1 while traveling to India was serially passaged 12 times in human cell lines. The cell-culture-generated viruses (passage 12; p12) grew efficiently in human cell lines, but the replication was not fully supported in porcine cells. A full-length cDNA clone was constructed using JE04-1601S_p12 as a template. It was able to produce an infectious virus, and viral protein expression was detectable in the transfected PLC/PRF/5 cells and culture supernatants. Consistently, HEV-1 growth was also not fully supported in the cell culture of cDNA-derived JE04-1601S_p12 progenies, potentially recapitulating the narrow tropism of HEV-1 observed in vivo. The availability of an efficient cell culture system for HEV-1 and its infectious cDNA clone will be useful for studying HEV species tropism and mechanisms underlying severe hepatitis in HEV-1-infected pregnant women as well as for discovering and developing safer treatment options for this condition. Full article

Barley yellow dwarf viruses (BYDVs) are one of the most widespread and economically important plant viruses affecting many cereal crops. Growing resistant varieties remains the most promising approach to reduce the impact of BYDVs. A Recent RNA sequencing analysis has revealed potential genes that respond to BYDV infection in resistant barley genotypes. Together with a comprehensive review of the current knowledge on disease resistance in plants, we selected nine putative barley and wheat genes to investigate their involvement in resistance to BYDV-PAV infection. The target classes of genes were (i) nucleotide binding site (NBS) leucine-rich repeat (LRR), (ii) coiled-coil nucleotide-binding leucine-rich repeat (CC-NB-LRR), (iii) LRR receptor-like kinase (RLK), (iv) casein kinase, (v) protein kinase, (vi) protein phosphatase subunits and the transcription factors (TF) (vii) MYB TF, (viii) GRAS (gibberellic acid-insensitive (GAI), repressor of GAI (RGA) and scarecrow (SCR)), and (ix) the MADS-box TF family. Expression of genes was analysed for six genotypes with different levels of resistance. As in previous reports, the highest BYDV-PAV titre was found in the susceptible genotypes Graciosa in barley and Semper and SGS 27-02 in wheat, which contrast with the resistant genotypes PRS-3628 and Wysor of wheat and barley, respectively. Statistically significant changes in wheat show up-regulation of NBS-LRR, CC-NBS-LRR and RLK in the susceptible genotypes and down-regulation in the resistant genotypes in response to BYDV-PAV. Similar up-regulation of NBS-LRR, CC-NBS-LRR, RLK and MYB TF in response to BYDV-PAV was also observed in the susceptible barley genotypes. However, no significant changes in the expression of these genes were generally observed in the resistant barley genotypes, except for the down-regulation of RLK. Casein kinase and Protein phosphatase were up-regulated early, 10 days after inoculation (dai) in the susceptible wheat genotypes, while the latter was down-regulated at 30 dai in resistant genotypes. Protein kinase was down-regulated both earlier (10 dai) and later (30 dai) in the susceptible wheat genotypes, but only in the later dai in the resistant genotypes. In contrast, GRAS TF and MYB TF were up-regulated in the susceptible wheat genotypes while no significant differences in MADS TF expression was observed. Protein kinase, Casein kinase (30 dai), MYB TF and GRAS TF (10 dai) were all up-regulated in the susceptible barley genotypes. However, no significant differences were found between the resistant and susceptible barley genotypes for the Protein phosphatase and MADS FT genes. Overall, our results showed a clear differentiation of gene expression patterns in both resistant and susceptible genotypes of wheat and barley. Therefore, further research on RLK, NBS-LRR, CC-NBS-LRR, GRAS TF and MYB TF can lead to BYDV-PAV resistance in cereals. Full article

The cowpea chlorotic mottle virus (CCMV) is a plant virus explored as a nanotechnological platform. The robust self-assembly mechanism of its capsid protein allows for drug encapsulation and targeted delivery. Additionally, the capsid nanoparticle can be used as a programmable platform to display different molecular moieties. In view of future applications, efficient production and purification of plant viruses are key steps. In established protocols, the need for ultracentrifugation is a significant limitation due to cost, difficult scalability, and safety issues. In addition, the purity of the final virus isolate often remains unclear. Here, an advanced protocol for the purification of the CCMV from infected plant tissue was developed, focusing on efficiency, economy, and final purity. The protocol involves precipitation with PEG 8000, followed by affinity extraction using a novel peptide aptamer. The efficiency of the protocol was validated using size exclusion chromatography, MALDI-TOF mass spectrometry, reversed-phase HPLC, and sandwich immunoassay. Furthermore, it was demonstrated that the final eluate of the affinity column is of exceptional purity (98.4%) determined by HPLC and detection at 220 nm. The scale-up of our proposed method seems to be straightforward, which opens the way to the large-scale production of such nanomaterials. This highly improved protocol may facilitate the use and implementation of plant viruses as nanotechnological platforms for in vitro and in vivo applications. Full article

Background: This is Manuscript 1 of a two-part Manuscript of the same series. Here, we present findings from our first set of studies on the abundance and compartmentalization of blood plasma extracellular microRNAs (exmiRNAs) into extracellular particles, including blood plasma extracellular vesicles (EVs) and extracellular condensates (ECs) in the setting of untreated HIV/SIV infection. The goals of the study presented in this Manuscript 1 are to (i) assess the abundance and compartmentalization of exmiRNAs in EVs versus ECs in the healthy uninfected state, and (ii) evaluate how SIV infection may affect exmiRNA abundance and compartmentalization in these particles. Considerable effort has been devoted to studying the epigenetic control of viral infection, particularly in understanding the role of exmiRNAs as key regulators of viral pathogenesis. MicroRNA (miRNAs) are small (~20–22 nts) non-coding RNAs that regulate cellular processes through targeted mRNA degradation and/or repression of protein translation. Originally associated with the cellular microenvironment, circulating miRNAs are now known to be present in various extracellular environments, including blood serum and plasma. While in circulation, miRNAs are protected from degradation by ribonucleases through their association with lipid and protein carriers, such as lipoproteins and other extracellular particles—EVs and ECs. Functionally, miRNAs play important roles in diverse biological processes and diseases (cell proliferation, differentiation, apoptosis, stress responses, inflammation, cardiovascular diseases, cancer, aging, neurological diseases, and HIV/SIV pathogenesis). While lipoproteins and EV-associated exmiRNAs have been characterized and linked to various disease processes, the association of exmiRNAs with ECs is yet to be made. Likewise, the effect of SIV infection on the abundance and compartmentalization of exmiRNAs within extracellular particles is unclear. Literature in the EV field has suggested that most circulating miRNAs may not be associated with EVs. However, a systematic analysis of the carriers of exmiRNAs has not been conducted due to the inefficient separation of EVs from other extracellular particles, including ECs. Methods: Paired EVs and ECs were separated from EDTA blood plasma of SIV-uninfected male Indian rhesus macaques (RMs, n = 15). Additionally, paired EVs and ECs were isolated from EDTA blood plasma of combination anti-retroviral therapy (cART) naïve SIV-infected (SIV+, n = 3) RMs at two time points (1- and 5-months post infection, 1 MPI and 5 MPI). Separation of EVs and ECs was achieved with PPLC, a state-of-the-art, innovative technology equipped with gradient agarose bead sizes and a fast fraction collector that allows high-resolution separation and retrieval of preparative quantities of sub-populations of extracellular particles. Global miRNA profiles of the paired EVs and ECs were determined with RealSeq Biosciences (Santa Cruz, CA) custom sequencing platform by conducting small RNA (sRNA)-seq. The sRNA-seq data were analyzed using various bioinformatic tools. Validation of key exmiRNAs was performed using specific TaqMan microRNA stem-loop RT-qPCR assays. Results: We showed that exmiRNAs in blood plasma are not restricted to any type of extracellular particles but are associated with lipid-based carriers—EVs and non-lipid-based carriers—ECs, with a significant (~30%) proportion of the exmiRNAs being associated with ECs. In the blood plasma of uninfected RMs, a total of 315 miRNAs were associated with EVs, while 410 miRNAs were associated with ECs. A comparison of detectable miRNAs within paired EVs and ECs revealed 19 and 114 common miRNAs, respectively, detected in all 15 RMs. Let-7a-5p, Let-7c-5p, miR-26a-5p, miR-191-5p, and let-7f-5p were among the top 5 detectable miRNAs associated with EVs in that order. In ECs, miR-16-5p, miR-451, miR-191-5p, miR-27a-3p, and miR-27b-3p, in that order, were the top detectable miRNAs in ECs. miRNA-target enrichment analysis of the top 10 detected common EV and EC miRNAs identified MYC and TNPO1 as top target genes, respectively. Functional enrichment analysis of top EV- and EC-associated miRNAs identified common and distinct gene-network signatures associated with various biological and disease processes. Top EV-associated miRNAs were implicated in cytokine–cytokine receptor interactions, Th17 cell differentiation, IL-17 signaling, inflammatory bowel disease, and glioma. On the other hand, top EC-associated miRNAs were implicated in lipid and atherosclerosis, Th1 and Th2 cell differentiation, Th17 cell differentiation, and glioma. Interestingly, infection of RMs with SIV revealed that the brain-enriched miR-128-3p was longitudinally and significantly downregulated in EVs, but not ECs. This SIV-mediated decrease in miR-128-3p counts was validated by specific TaqMan microRNA stem-loop RT-qPCR assay. Remarkably, the observed SIV-mediated decrease in miR-128-3p levels in EVs from RMs agrees with publicly available EV miRNAome data by Kaddour et al., 2021, which showed that miR-128-3p levels were significantly lower in semen-derived EVs from HIV-infected men who used or did not use cocaine compared to HIV-uninfected individuals. These findings confirmed our previously reported finding and suggested that miR-128 may be a target of HIV/SIV. Conclusions: In the present study, we used sRNA sequencing to provide a holistic understanding of the repertoire of circulating exmiRNAs and their association with extracellular particles, such as EVs and ECs. Our data also showed that SIV infection altered the profile of the miRNAome of EVs and revealed that miR-128-3p may be a potential target of HIV/SIV. The significant decrease in miR-128-3p in HIV-infected humans and in SIV-infected RMs may indicate disease progression. Our study has important implications for the development of biomarker approaches for various types of cancer, cardiovascular diseases, organ injury, and HIV based on the capture and analysis of circulating exmiRNAs. Full article

Papaya sticky disease is caused by the association of a fusagra-like and an umbra-like virus, named papaya meleira virus (PMeV) and papaya meleira virus 2 (PMeV2), respectively. Both viral genomes are encapsidated in particles formed by the PMeV ORF1 product, which has the potential to encode a protein with 1563 amino acids (aa). However, the structural components of the viral capsid are unknown. To characterize the structural proteins of PMeV and PMeV2, virions were purified from Carica papaya latex. SDS-PAGE analysis of purified virus revealed two major proteins of ~40 kDa and ~55 kDa. Amino-terminal sequencing of the ~55 kDa protein and LC-MS/MS of purified virions indicated that this protein starts at aa 263 of the deduced ORF1 product as a result of either degradation or proteolytic processing. A yeast two-hybrid assay was used to identify Arabidopsis proteins interacting with two PMeV ORF1 product fragments (aa 321–670 and 961–1200). The 50S ribosomal protein L17 (AtRPL17) was identified as potentially associated with modulated translation-related proteins. In plant cells, AtRPL17 co-localized and interacted with the PMeV ORF1 fragments. These findings support the hypothesis that the interaction between PMeV/PMeV2 structural proteins and RPL17 is important for virus–host interactions. Full article

With the development of NK cell-directed therapeutic strategies, the actual effect of NK cells on the cellular SIV DNA levels of the virus in SIV-infected macaques in vivo remains unclear. In this study, five chronically SIV_mac239-infected, treatment-naïve rhesus macaques were euthanized, and the blood, spleen, pararectal/paracolonic lymph nodes (PaLNs), and axillary lymph nodes (ALNs) were collected. The distributional, phenotypic, and functional profiles of NK cells were detected by flow cytometry. The highest frequency of NK cells was found in PBMC, followed by the spleen, while only 0~0.5% were found in LNs. Peripheral NK cells also exhibited higher cytotoxic potential (CD56⁻ CD16⁺ NK subsets) and IFN-γ-producing capacity but low PD-1 and Tim-3 levels than those in the spleen and LNs. Our results demonstrated a significant positive correlation between the frequency of NK cells and the ratios of cellular SIV DNA/RNA in HLADR⁻ CD4⁺ T cells (r = 0.6806, p < 0.001) in SIV-infected macaques, despite no discrepancies in the cellular SIV DNA or RNA levels that were found among the blood, spleen, and LNs. These findings showed a profile of NK cell frequencies and NK cytotoxicity levels in different immune organs from chronically SIV_mac239-infected, treatment-naïve rhesus macaques. It was suggested that NK cell frequencies could be closely related to SIV DNA/RNA levels, which could affect the transcriptional activity of SIV proviruses. However, the cytotoxicity effect of NK cells on the latent SIV viral load in LNs could be limited due to the sparse abundance of NK cells in LNs. The development of NK cell-directed treatment approaches aiming for HIV clearance remains challenging. Full article

Mast cells (MCs) are classically associated with allergic asthma but their role in antiviral immunity is unclear. Human rhinoviruses (HRVs) are a major cause of asthma exacerbations and can infect and replicate within MCs. The primary site of HRV infection is the airway epithelium and MCs localise to this site with increasing asthma severity. The asthma susceptibility gene, IL-33, encodes an epithelial-derived cytokine released following HRV infection but its impact on MC antiviral responses has yet to be determined. In this study we investigated the global response of LAD2 MCs to IL-33 stimulation using RNA sequencing and identified genes involved in antiviral immunity. In spite of this, IL-33 treatment increased permissiveness of MCs to HRV16 infection which, from the RNA-Seq data, we attributed to upregulation of ICAM1. Flow cytometric analysis confirmed an IL-33-dependent increase in ICAM1 surface expression as well as LDLR, the receptors used by major and minor group HRVs for cellular entry. Neutralisation of ICAM1 reduced the IL-33-dependent enhancement in HRV16 replication and release in both LAD2 MCs and cord blood derived MCs. These findings demonstrate that although IL-33 induces an antiviral signature in MCs, it also upregulates the receptors for HRV entry to enhance infection. This highlights the potential for a gene-environment interaction involving IL33 and HRV in MCs to contribute to virus-induced asthma exacerbations. Full article

The interferon-induced myxovirus resistance protein A (MxA) is a potent restriction factor that prevents zoonotic infection from influenza A virus (IAV) subtype H7N9. Individuals expressing antivirally inactive MxA variants are highly susceptible to these infections. However, human-adapted IAVs have acquired specific mutations in the viral nucleoprotein (NP) that allow escape from MxA-mediated restriction but that have not been observed in MxA-sensitive, human H7N9 isolates. To date, it is unknown whether H7N9 can adapt to escape MxA-mediated restriction. To study this, we infected Rag2-knockout (Rag2^−/−) mice with a defect in T and B cell maturation carrying a human MxA transgene (MxA^tg/−Rag2^−/−). In these mice, the virus could replicate for several weeks facilitating host adaptation. In MxA^tg/−Rag2^−/−, but not in Rag2^−/− mice, the well-described mammalian adaptation E627K in the viral polymerase subunit PB2 was acquired, but no variants with MxA escape mutations in NP were detected. Utilizing reverse genetics, we could show that acquisition of PB2 E627K allowed partial evasion from MxA restriction in MxA^tg/tg mice. However, pretreatment with type I interferon decreased viral replication in these mice, suggesting that PB2 E627K is not a true MxA escape mutation. Based on these results, we speculate that it might be difficult for H7N9 to acquire MxA escape mutations in the viral NP. This is consistent with previous findings showing that MxA escape mutations cause severe attenuation of IAVs of avian origin. Full article

Background: Congenital cytomegalovirus (cCMV) infection is frequent and potentially severe. The immunobiology of cCMV infection is poorly understood, involving cytokines that could be carried within or on the surface of extracellular vesicles (EV). We investigated intra-amniotic cytokines, mediated or not by EV, in cCMV infection. Methods: Forty infected fetuses following early maternal primary infection and forty negative controls were included. Infected fetuses were classified according to severity at birth: asymptomatic, moderately or severely symptomatic. Following the capture of EV in amniotic fluid (AF), the concentrations of 38 cytokines were quantified. The association with infection and its severity was determined using univariate and multivariate analysis. A prediction analysis based on principal component analysis was conducted. Results: cCMV infection was nominally associated with an increase in six cytokines, mainly soluble (IP-10, IL-18, ITAC, and TRAIL). EV-associated IP-10 was also increased in cases of fetal infection. Severity of fetal infection was nominally associated with an increase in twelve cytokines, including five also associated with fetal infection. A pattern of specific increase in six proteins fitted severely symptomatic infection, including IL-18soluble, TRAILsoluble, CRPsoluble, TRAILsurface, MIGinternal, and RANTESinternal. Conclusion: Fetal infection and its severity are associated with an increase in pro-inflammatory cytokines involved in Th1 immune response. Full article

Hepatitis E virus (HEV) is an important public health burden worldwide, causing approximately 20 million infections and 70,000 deaths annually. The viral capsid protein is encoded by open reading frame 2 (ORF2) of the HEV genome. Most ORF2 protein present in body fluids is the glycosylated secreted form of the protein (ORF2^S). A recent study suggested that ORF2^S is not necessary for the HEV life cycle. A previously reported efficient HEV cell culture system can be used to understand the origin and life cycle of ORF2^S but is not sufficient for functional research. A more rapid and productive method for yielding ORF2^S could help to study its antigenicity and immunogenicity. In this study, the ORF2^S (tPA) expression construct was designed as a candidate tool. A set of representative anti-HEV monoclonal antibodies was further used to map the functional antigenic sites in the candidates. ORF2^S (tPA) was used to study antigenicity and immunogenicity. Indirect ELISA revealed that ORF2^S (tPA) was not antigenically identical to HEV 239 antigen (p239). The ORF2^S-specific antibodies were successfully induced in one-dose-vaccinated BALB/c mice. The ORF2^S-specific antibody response was detected in plasma from HEV-infected patients. Recombinant ORF2^S (tPA) can act as a decoy to against B cells. Altogether, our study presents a design strategy for ORF2^S expression and indicates that ORF2^S (tPA) can be used for functional and structural studies of the HEV life cycle. Full article

The COVID-19 pandemic caused by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has spurred unprecedented and concerted worldwide research to curtail and eradicate this pathogen. SARS-CoV-2 has four structural proteins: Envelope (E), Membrane (M), Nucleocapsid (N), and Spike (S), which self-assemble along with its RNA into the infectious virus by budding from intracellular lipid membranes. In this paper, we develop a model to explore the mechanisms of RNA condensation by structural proteins, protein oligomerization and cellular membrane–protein interactions that control the budding process and the ultimate virus structure. Using molecular dynamics simulations, we have deciphered how the positively charged N proteins interact and condense the very long genomic RNA resulting in its packaging by a lipid envelope decorated with structural proteins inside a host cell. Furthermore, considering the length of RNA and the size of the virus, we find that the intrinsic curvature of M proteins is essential for virus budding. While most current research has focused on the S protein, which is responsible for viral entry, and it has been motivated by the need to develop efficacious vaccines, the development of resistance through mutations in this crucial protein makes it essential to elucidate the details of the viral life cycle to identify other drug targets for future therapy. Our simulations will provide insight into the viral life cycle through the assembly of viral particles de novo and potentially identify therapeutic targets for future drug development. Full article

NAD⁺ and ADP-ribose (ADPr)-containing molecules are at the interface of virus–host conflicts across life encompassing RNA processing, restriction, lysogeny/dormancy and functional hijacking. We objectively defined the central components of the NAD⁺–ADPr networks involved in these conflicts and systematically surveyed 21,191 completely sequenced viral proteomes representative of all publicly available branches of the viral world to reconstruct a comprehensive picture of the viral NAD⁺–ADPr systems. These systems have been widely and repeatedly exploited by positive-strand RNA and DNA viruses, especially those with larger genomes and more intricate life-history strategies. We present evidence that ADP-ribosyltransferases (ARTs), ADPr-targeting Macro, NADAR and Nudix proteins are frequently packaged into virions, particularly in phages with contractile tails (Myoviruses), and deployed during infection to modify host macromolecules and counter NAD⁺-derived signals involved in viral restriction. Genes encoding NAD⁺–ADPr-utilizing domains were repeatedly exchanged between distantly related viruses, hosts and endo-parasites/symbionts, suggesting selection for them across the virus world. Contextual analysis indicates that the bacteriophage versions of ADPr-targeting domains are more likely to counter soluble ADPr derivatives, while the eukaryotic RNA viral versions might prefer macromolecular ADPr adducts. Finally, we also use comparative genomics to predict host systems involved in countering viral ADP ribosylation of host molecules. Full article

Parvovirus B19 (B19V) is a human pathogen that is the causative agent of fifth disease in children. It is also known to cause hydrops in fetuses, anemia in AIDS patients, and transient aplastic crisis in patients with sickle cell disease. The unique N-terminus of Viral Protein 1 (VP1u) of parvoviruses, including B19V, exhibits phospholipase A₂ (PLA₂) activity, which is required for endosomal escape. Presented is the structural dynamics of B19V VP1u under conditions that mimic the pHs of cell entry and endosomal trafficking to the nucleus. Using circular dichroism spectroscopy, the receptor-binding domain of B19V VP1u is shown to exhibit an α-helical fold, whereas the PLA₂ domain exhibits a probable molten globule state, both of which are pH invariant. Differential scanning calorimetry performed at endosomal pHs shows that the melting temperature (T_m) of VP1u PLA₂ domain is tuned to body temperature (37 °C) at pH 7.4. In addition, PLA₂ assays performed at temperatures ranging from 25–45 °C show both a temperature and pH-dependent change in activity. We hypothesize that VP1u PLA₂ domain differences in T_m at differing pHs have enabled the virus to “switch on/off” the phospholipase activity during capsid trafficking. Furthermore, we propose the environment of the early endosome as the optimal condition for endosomal escape leading to B19V infection. Full article

The efficacy of HIV pre-exposure prophylaxis (PrEP) is high in men who have sex with men, but much more variable in women, in a manner largely attributed to low adherence. This reduced efficacy, however, could also reflect biological factors. Transmission to women is typically via the female reproductive tract (FRT), and vaginal dysbiosis, genital inflammation, and other factors specific to the FRT mucosa can all increase transmission risk. We have demonstrated that mucosal fibroblasts from the lower and upper FRT can markedly enhance HIV infection of CD4+ T cells. Given the current testing of tenofovir disoproxil fumarate, cabotegravir, and dapivirine regimens as candidate PrEP agents for women, we set out to determine using in vitro assays whether endometrial stromal fibroblasts (eSF) isolated from the FRT can affect the anti-HIV activity of these PrEP drugs. We found that PrEP drugs exhibit significantly reduced antiviral efficacy in the presence of eSFs, not because of decreased PrEP drug availability, but rather of eSF-mediated enhancement of HIV infection. These findings suggest that drug combinations that target both the virus and infection-promoting factors in the FRT—such as mucosal fibroblasts—may be more effective than PrEP alone at preventing sexual transmission of HIV to women. Full article

TRIM7 catalyzes the ubiquitination of multiple substrates with unrelated biological functions. This cross-reactivity is at odds with the specificity usually displayed by enzymes, including ubiquitin ligases. Here we show that TRIM7′s extreme substrate promiscuity is due to a highly unusual binding mechanism, in which the PRYSPRY domain captures any ligand with a C-terminal helix that terminates in a hydrophobic residue followed by a glutamine. Many of the non-structural proteins found in RNA viruses contain C-terminal glutamines as a result of polyprotein cleavage by 3C protease. This viral processing strategy generates novel substrates for TRIM7 and explains its ability to inhibit Coxsackie virus and norovirus replication. In addition to viral proteins, cellular proteins such as glycogenin have evolved C-termini that make them a TRIM7 substrate. The ‘helix-ΦQ’ degron motif recognized by TRIM7 is reminiscent of the N-end degron system and is found in ~1% of cellular proteins. These features, together with TRIM7′s restricted tissue expression and lack of immune regulation, suggest that viral restriction may not be its physiological function. Full article

Interactions between lysyl–tRNA synthetase (LysRS) and HIV-1 Gag facilitate selective packaging of the HIV-1 reverse transcription primer, tRNA^Lys3. During HIV-1 infection, LysRS is phosphorylated at S207, released from a multi-aminoacyl–tRNA synthetase complex and packaged into progeny virions. LysRS is critical for proper targeting of tRNA^Lys3 to the primer-binding site (PBS) by specifically binding a PBS-adjacent tRNA-like element (TLE), which promotes release of the tRNA proximal to the PBS. However, whether LysRS phosphorylation plays a role in this process remains unknown. Here, we used a combination of binding assays, RNA chemical probing, and small-angle X-ray scattering to show that both wild-type (WT) and a phosphomimetic S207D LysRS mutant bind similarly to the HIV-1 genomic RNA (gRNA) 5′UTR via direct interactions with the TLE and stem loop 1 (SL1) and have a modest preference for binding dimeric gRNA. Unlike WT, S207D LysRS bound in an open conformation and increased the dynamics of both the PBS region and SL1. A new working model is proposed wherein a dimeric phosphorylated LysRS/tRNA complex binds to a gRNA dimer to facilitate tRNA primer release and placement onto the PBS. Future anti-viral strategies that prevent this host factor-gRNA interaction are envisioned. Full article

Four seasonal human coronaviruses (sHCoVs) are endemic globally (229E, NL63, OC43, and HKU1), accounting for 5–30% of human respiratory infections. However, the epidemiology and evolution of these CoVs remain understudied due to their association with mild symptomatology. Using a multigene and complete genome analysis approach, we find the evolutionary histories of sHCoVs to be highly complex, owing to frequent recombination of CoVs including within and between sHCoVs, and uncertain, due to the under sampling of non-human viruses. The recombination rate was highest for 229E and OC43 whereas substitutions per recombination event were highest in NL63 and HKU1. Depending on the gene studied, OC43 may have ungulate, canine, or rabbit CoV ancestors. 229E may have origins in a bat, camel, or an unsampled intermediate host. HKU1 had the earliest common ancestor (1809–1899) but fell into two distinct clades (genotypes A and B), possibly representing two independent transmission events from murine-origin CoVs that appear to be a single introduction due to large gaps in the sampling of CoVs in animals. In fact, genotype B was genetically more diverse than all the other sHCoVs. Finally, we found shared amino acid substitutions in multiple proteins along the non-human to sHCoV host-jump branches. The complex evolution of CoVs and their frequent host switches could benefit from continued surveillance of CoVs across non-human hosts. Full article

RIG-I is an innate sensor of RNA virus infection and its activation induces interferon-stimulated genes (ISGs). In vitro studies using human cells have demonstrated the ability of synthetic RIG-I agonists (3pRNA) to inhibit IAV replication. However, in mouse models of IAV the effectiveness of 3pRNA reported to date differs markedly between studies. Myxoma resistance (Mx)1 is an ISG protein which mediates potent anti-IAV activity, however most inbred mouse strains do not express a functional Mx1. Herein, we utilised C57BL/6 mice that do (B6.A2G-Mx1) and do not (B6-WT) express functional Mx1 to assess the ability of prophylactic 3pRNA treatment to induce ISGs and to protect against subsequent IAV infection. In vitro, 3pRNA treatment of primary lung cells from B6-WT and B6.A2G-Mx1 mice resulted in ISG induction however inhibition of IAV infection was more potent in cells from B6.A2G-Mx1 mice. In vivo, a single intravenous injection of 3pRNA resulted in ISG induction in lungs of both B6-WT and B6.A2G-Mx1 mice, however potent and long-lasting protection against subsequent IAV challenge was only observed in B6.A2G-Mx1 mice. Thus, despite broad ISG induction, expression of a functional Mx1 is critical for potent and long-lasting RIG-I agonist-mediated protection in the mouse model of IAV infection. Full article

African swine fever is an important viral disease of wild and domestic pigs. To gain further knowledge of the properties of the currently circulating African swine fever virus (ASFV), experimental infections of young pigs (approximately 8 weeks of age) and pregnant sows (infected at about 100 days of gestation) with the genotype II ASFV Georgia/2007 were performed. The inoculated young pigs developed typical clinical signs of the disease and the infection was transmitted (usually within 3–4 days) to all of the “in contact” animals that shared the same pen. Furthermore, typical pathogical lesions for ASFV infection were found at necropsy. Inoculation of pregnant sows with the same virus also produced rapid onset of disease from post-infection day three; two of the three sows died suddenly on post-infection day five, while the third was euthanized on the same day for animal welfare reasons. Following necropsy, the presence of ASFV DNA was detected in tonsils, spleen and lymph nodes of some of the fetuses, but the levels of viral DNA were much lower than in these tissues from the sows. Thus, only limited transplacental transmission occurred during the course of this experiment. These studies contribute towards further understanding about the spread of this important viral disease in domestic pigs. Full article

Continued emergence of SARS-CoV-2 variants highlights the critical need for adaptable and translational animal models for acute COVID-19. Limitations to current animal models for SARS CoV-2 (e.g., transgenic mice, non-human primates, ferrets) include subclinical to mild lower respiratory disease, divergence from clinical COVID-19 disease course, and/or the need for host genetic modifications to permit infection. We therefore established a feline model to study COVID-19 disease progression and utilized this model to evaluate infection kinetics and immunopathology of the rapidly circulating Delta variant (B.1.617.2) of SARS-CoV-2. In this study, specific-pathogen-free domestic cats (n = 24) were inoculated intranasally and/or intratracheally with SARS CoV-2 (B.1.617.2). Infected cats developed severe clinical respiratory disease and pulmonary lesions at 4- and 12-days post-infection (dpi), even at 1/10 the dose of previously studied wild-type SARS-CoV-2. Infectious virus was isolated from nasal secretions of delta-variant infected cats in high amounts at multiple timepoints, and viral antigen was co-localized in ACE2-expressing cells of the lungs (pneumocytes, vascular endothelium, peribronchial glandular epithelium) and strongly associated with severe pulmonary inflammation and vasculitis that were more pronounced than in wild-type SARS-CoV-2 infection. RNA sequencing of infected feline lung tissues identified upregulation of multiple gene pathways associated with cytokine receptor interactions, chemokine signaling, and viral protein–cytokine interactions during acute infection with SARS-CoV-2. Weighted correlation network analysis (WGCNA) of differentially expressed genes identified several distinct clusters of dysregulated hub genes that are significantly correlated with both clinical signs and lesions during acute infection. Collectively, the results of these studies help to delineate the role of domestic cats in disease transmission and response to variant emergence, establish a flexible translational model to develop strategies to prevent the spread of SARS-CoV-2, and identify potential targets for downstream therapeutic development. Full article

A hallmark of severe acute respiratory syndrome virus (SARS-CoV-2) replication is the discontinuous transcription of open reading frames (ORFs) encoding structural virus proteins. Real-time reverse transcription PCR (RT-qPCR) assays in previous publications used either single or multiplex assays for SARS-CoV-2 genomic RNA detection and a singleplex approach for subgenomic RNA detection. Although multiplex approaches often target multiple genomic RNA segments, an assay that concurrently detects genomic and subgenomic targets has been lacking. To bridge this gap, we developed two duplex one-step RT-qPCR assays that detect SARS-CoV-2 genomic ORF1a and either subgenomic spike or subgenomic ORF3a RNAs. All primers and probes for our assays were designed to bind to variants of SARS-CoV-2. In this study, our assays successfully detected SARS-CoV-2 Washington strain and delta variant isolates at various time points during the course of live virus infection in vitro. The ability to quantify subgenomic SARS-CoV-2 RNA is important, as it may indicate the presence of active replication, particularly in samples collected longitudinally. Furthermore, specific detection of genomic and subgenomic RNAs simultaneously in a single reaction increases assay efficiency, potentially leading to expedited lucidity about viral replication and pathogenesis of any variant of SARS-CoV-2. Full article

Individuals infected with the SARS-CoV-2 Delta variant, lineage B.1.617.2, exhibit faster initial infection with a higher viral load than prior variants, and pseudotyped viral particles bearing the SARS-CoV-2 Delta variant spike protein induce a faster initial infection rate of target cells compared to those bearing other SARS-CoV-2 variant spikes. Here, we show that pseudotyped viral particles bearing the Delta variant spike form unique aggregates, as evidenced by negative stain and cryogenic electron microscopy (EM), flow cytometry, and nanoparticle tracking analysis. Viral particles pseudotyped with other SARS-CoV-2 spike variants do not show aggregation by any of these criteria. The contribution to infection kinetics of the Delta spike’s unique property to aggregate is discussed with respect to recent evidence for collective infection by other viruses. Irrespective of this intriguing possibility, spike-dependent aggregation is a new functional parameter of spike-expressing viral particles to evaluate in future spike protein variants. Full article

Contraceptives such as Depo-medroxyprogesterone (DMPA) are used by an estimated 34 million women worldwide. DMPA has been associated with increased risk of several viral infections including Herpes simplex virus-2 (HSV-2) and Human immunodeficiency virus (HIV). In the current study, we used the mouse papillomavirus (MmuPV1) anogenital infection model to test two hypotheses: (1) contraceptives such as DMPA increase the susceptibility of the anogenital tract to viral infection and (2) long-term contraceptive administration induces more advanced disease at the anogenital tract. DMPA treatments of both athymic nude mice and heterozygous NU/J (Foxn1^nu/+) but ovariectomized mice led to a significantly increased viral load at the anogenital tract, suggesting that endogenous sex hormones were involved in increased viral susceptibility by DMPA treatment. Consistent with previous reports, DMPA treatment suppressed host anti-viral activities at the lower genital tract. To test the impact of long-term contraceptive treatment on the MmuPV1-infected lower genital tract, we included two other treatments in addition to DMPA: 17β-estradiol and a non-hormone based contraceptive Cilostazol (CLZ, Pletal). Viral infections were monitored monthly up to nine months post infection by qPCR. The infected vaginal and anal tissues were harvested and further examined by histological, virological, and immunological analyses. Surprisingly, we did not detect a significantly higher grade of histology in animals in the long-term DMPA and 17β-estradiol treated groups when compared to the control groups in the athymic mice we tested. Therefore, although DMPA promotes initial papillomavirus infections in the lower genital tract, the chronic administration of DMPA does not promote cancer development in the infected tissues in our mouse model. Full article

Clinical studies indicate that patients infected with SARS-CoV-2 develop hyperinflammation, which correlates with increased mortality. The SARS-CoV-2/COVID-19-dependent inflammation is thought to occur via increased cytokine production and hyperactivity of RAGE in several cell types, a phenomenon observed for other disorders and diseases. Metabolic reprogramming has been shown to contribute to inflammation and is considered a hallmark of cancer, neurodegenerative diseases, and viral infections. Malfunctioning glycolysis, which normally aims to convert glucose into pyruvate, leads to the accumulation of advanced glycation end products (AGEs). Being aberrantly generated, AGEs then bind to their receptor, RAGE, and activate several pro-inflammatory genes, such as IL-1b and IL-6, thus, increasing hypoxia and inducing senescence. Using the lung epithelial cell (BEAS-2B) line, we demonstrated that SARS-CoV-2 proteins reprogram the cellular metabolism and increase pyruvate kinase muscle isoform 2 (PKM2). This deregulation promotes the accumulation of AGEs and senescence induction. We showed the ability of the PKM2 stabilizer, Tepp-46, to reverse the observed glycolysis changes/alterations and restore this essential metabolic process. Full article

The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) pandemic has now been continuing for more than two years. The infection causes COVID-19, a disease of the respiratory and cardiovascular system of variable severity. Here, the humoral immune response of 80 COVID-19 patients from the University Hospital Frankfurt/Main, Germany, was characterized longitudinally. The SARS-CoV-2 neutralization activity of serum waned over time. The neutralizing potential of serum directed towards the human alpha-coronavirus NL-63 (NL63) also waned, indicating that no cross-priming against alpha-coronaviruses occurred. A subset of the recovered patients (n = 13) was additionally vaccinated with the mRNA vaccine Comirnaty. Vaccination increased neutralization activity against SARS-CoV-2 wild-type (WT), Delta, and Omicron, although Omicron-specific neutralization was not detectable prior to vaccination. In addition, the vaccination induced neutralizing antibodies against the more distantly related SARS-CoV-1 but not against NL63. The results indicate that although SARS-CoV-2 humoral immune responses induced by infection wane, vaccination induces a broad neutralizing activity against multiple SARS-CoVs, but not to the common cold alpha-coronavirus NL63. Full article

How human cytomegalovirus (HCMV) infection impacts the transcription of the host genome remains incompletely understood. Here, we examine the global consequences of infection of primary human foreskin fibroblasts (HFFs) on transcription by RNA polymerase I, II, and III over the course of a lytic infection using PRO-Seq. The expected rapid induction of innate immune response genes is observed with specific subsets of genes exhibiting dissimilar expression kinetics. We find minimal effects on Pol II initiation, but increased rates of the release of paused Pol II into productive elongation are detected by 24 h postinfection and pronounced at late times postinfection. Pol I transcription increases during infection and we provide evidence for a potential Pol I elongation control mechanism. Pol III transcription of tRNA genes is dramatically altered, with many induced and some repressed. All effects are partially dependent on viral genome replication, suggesting a link to viral mRNA levels and/or a viral early–late or late gene product. Changes in tRNA transcription are connected to distinct alterations in the chromatin state around tRNA genes, which were probed with high-resolution DFF-ChIP. Additionally, evidence is provided that the Pol III PIC stably contacts an upstream −1 nucleosome. Finally, we compared and contrasted our HCMV data with results from published experiments with HSV-1, EBV, KSHV, and MHV68. We report disparate effects on Pol II transcription and potentially similar effects on Pol III transcription. Full article

SARS-CoV-2, the causative agent of COVID-19, emerged in late 2019. The highly contagious B.1.617.2 (Delta) variant of concern (VOC) was first identified in October 2020 in India and subsequently disseminated worldwide, later becoming the dominant lineage in the US. Understanding the local transmission dynamics of early SARS-CoV-2 introductions may inform actionable mitigation efforts during subsequent pandemic waves. Yet, despite considerable genomic analysis of SARS-CoV-2 in the US, several gaps remain. Here, we explore the early emergence of the Delta variant in Florida, US using phylogenetic analysis of representative Florida and globally sampled genomes. We find multiple independent introductions into Florida primarily from North America and Europe, with a minority originating from Asia. These introductions led to three distinct clades that demonstrated varying relative rates of transmission and possessed five distinct substitutions that were 3–21 times more prevalent in the Florida sample as compared to the global sample. Our results underscore the benefits of routine viral genomic surveillance to monitor epidemic spread and support the need for more comprehensive genomic epidemiology studies of emerging variants. In addition, we provide a model of epidemic spread of newly emerging VOCs that can inform future public health responses. Full article

The Na⁺/taurocholate co-transporting polypeptide (NTCP, gene symbol SLC10A1) is both a physiological bile acid transporter and the high-affinity hepatic receptor for the hepatitis B and D viruses (HBV/HDV). Virus entry via endocytosis of the virus/NTCP complex involves co-factors, but this process is not fully understood. As part of the innate immunity, interferon-induced transmembrane proteins (IFITM) 1–3 have been characterized as virus entry-restricting factors for many viruses. The present study identified IFITM3 as a novel protein–protein interaction (PPI) partner of NTCP based on membrane yeast-two hybrid and co-immunoprecipitation experiments. Surprisingly, IFITM3 knockdown significantly reduced in vitro HBV infection rates of NTCP-expressing HuH7 cells and primary human hepatocytes (PHHs). In addition, HuH7-NTCP cells showed significantly lower HDV infection rates, whereas infection with influenza A virus was increased. HBV-derived myr-preS1 peptide binding to HuH7-NTCP cells was intact even under IFITM3 knockdown, suggesting that IFITM3-mediated HBV/HDV infection enhancement occurs in a step subsequent to the viral attachment to NTCP. In conclusion, IFITM3 was identified as a novel NTCP co-factor that significantly affects in vitro infection with HBV and HDV in NTCP-expressing hepatoma cells and PHHs. While there is clear evidence for a direct PPI between IFITM3 and NTCP, the specific mechanism by which this PPI facilitates the infection process remains to be identified in future studies. Full article

SARS-CoV-2 infection rapidly elicits anti-Spike antibodies whose quantity in plasma gradually declines upon resolution of symptoms. This decline is part of the evolution of an immune response leading to B cell differentiation into short-lived antibody-secreting cells or resting memory B cells. At the same time, the ongoing class switch and antibody maturation processes occurring in germinal centers lead to the selection of B cell clones secreting antibodies with higher affinity for their cognate antigen, thereby improving their functional activity. To determine whether the decline in SARS-CoV-2 antibodies is paralleled with an increase in avidity of the anti-viral antibodies produced, we developed a simple assay to measure the avidity of anti-receptor binding domain (RBD) IgG elicited by SARS-CoV-2 infection. We longitudinally followed a cohort of 29 convalescent donors with blood samples collected between 6- and 32-weeks post-symptoms onset. We observed that, while the level of antibodies declines over time, the anti-RBD avidity progressively increases and correlates with the B cell class switch. Additionally, we observed that anti-RBD avidity increased similarly after SARS-CoV-2 mRNA vaccination and after SARS-CoV-2 infection. Our results suggest that anti-RBD IgG avidity determination could be a surrogate assay for antibody affinity maturation and, thus, suitable for studying humoral responses elicited by natural infection and/or vaccination. Full article

Alphasatellites, which encode only a replication-associated protein (alpha-Rep), are frequently found to be non-essential satellite components associated with begomovirus/betasatellite complexes, and their presence can modulate disease symptoms and/or viral DNA accumulation during infection. Our previous study has shown that there are three types of alphasatellites associated with begomovirus/betasatellite complexes in Yunnan province in China and they encode three corresponding types of alpha-Rep proteins. However, the biological functions of alpha-Reps remain poorly understood. In this study, we investigated the biological functions of alpha-Reps in post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS) using 16c and 16-TGS transgenic Nicotiana benthamiana plants. Results showed that all the three types of alpha-Rep proteins were capable of suppressing the PTGS and reversing the TGS. Among them, the alpha-Rep of Y10DNA1 has the strongest PTGS and TGS suppressor activities. We also found that the alpha-Rep proteins were able to increase the accumulation of their helper virus during coinfection. These results suggest that the alpha-Reps may have a role in overcoming host defense, which provides a possible explanation for the selective advantage provided by the association of alphasatellites with begomovirus/betasatellite complexes. Full article

Corticosteroids are most commonly used to treat HTLV-1-associated myelopathy (HAM); however, their clinical efficacy has not been tested in randomized clinical trials. This randomized controlled trial included 8 and 30 HAM patients with rapidly and slowly progressing walking disabilities, respectively. Rapid progressors were assigned (1:1) to receive or not receive a 3-day course of intravenous methylprednisolone in addition to oral prednisolone therapy. Meanwhile, slow progressors were assigned (1:1) to receive oral prednisolone or placebo. The primary outcomes were a composite of ≥1-grade improvement in the Osame Motor Disability Score or ≥30% improvement in the 10 m walking time (10 mWT) at week 2 for rapid progressors and changes from baseline in 10 mWT at week 24 for slow progressors. In the rapid progressor trial, all four patients with but only one of four without intravenous methylprednisolone achieved the primary outcome (p = 0.14). In the slow progressor trial, the median changes in 10 mWT were −13.8% (95% CI: −20.1–−7.1; p < 0.001) and −6.0% (95% CI: −12.8–1.3; p = 0.10) with prednisolone and placebo, respectively (p for between-group difference = 0.12). Whereas statistical significance was not reached for the primary endpoints, the overall data indicated the benefit of corticosteroid therapy. (Registration number: UMIN000023798, UMIN000024085) Full article

Tsetse flies cause major health and economic problems as they transmit trypanosomes causing sleeping sickness in humans (Human African Trypanosomosis, HAT) and nagana in animals (African Animal Trypanosomosis, AAT). A solution to control the spread of these flies and their associated diseases is the implementation of the Sterile Insect Technique (SIT). For successful application of SIT, it is important to establish and maintain healthy insect colonies and produce flies with competitive fitness. However, mass production of tsetse is threatened by covert virus infections, such as the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV). This virus infection can switch from a covert asymptomatic to an overt symptomatic state and cause the collapse of an entire fly colony. Although the effects of GpSGHV infections can be mitigated, the presence of other covert viruses threaten tsetse mass production. Here we demonstrated the presence of two single-stranded RNA viruses isolated from Glossina morsitans morsitans originating from a colony at the Seibersdorf rearing facility. The genome organization and the phylogenetic analysis based on the RNA-dependent RNA polymerase (RdRp) revealed that the two viruses belong to the genera Iflavirus and Negevirus, respectively. The names proposed for the two viruses are Glossina morsitans morsitans iflavirus (GmmIV) and Glossina morsitans morsitans negevirus (GmmNegeV). The GmmIV genome is 9685 nucleotides long with a poly(A) tail and encodes a single polyprotein processed into structural and non-structural viral proteins. The GmmNegeV genome consists of 8140 nucleotides and contains two major overlapping open reading frames (ORF1 and ORF2). ORF1 encodes the largest protein which includes a methyltransferase domain, a ribosomal RNA methyltransferase domain, a helicase domain and a RdRp domain. In this study, a selective RT-qPCR assay to detect the presence of the negative RNA strand for both GmmIV and GmmNegeV viruses proved that both viruses replicate in G. m. morsitans. We analyzed the tissue tropism of these viruses in G. m. morsitans by RNA-FISH to decipher their mode of transmission. Our results demonstrate that both viruses can be found not only in the host’s brain and fat bodies but also in their reproductive organs, and in milk and salivary glands. These findings suggest a potential horizontal viral transmission during feeding and/or a vertically viral transmission from parent to offspring. Although the impact of GmmIV and GmmNegeV in tsetse rearing facilities is still unknown, none of the currently infected tsetse species show any signs of disease from these viruses. Full article

Background: We evaluated how plasma proteomic signatures in patients with suspected COVID-19 can unravel the pathophysiology, and determine kinetics and clinical outcome of the infection. Methods: Plasma samples from patients presenting to the emergency department (ED) with symptoms of COVID-19 were stratified into: (1) patients with suspected COVID-19 that was not confirmed (n = 44); (2) non-hospitalized patients with confirmed COVID-19 (n = 44); (3) hospitalized patients with confirmed COVID-19 (n = 53) with variable outcome; and (4) patients presenting to the ED with minor diseases unrelated to SARS-CoV-2 infection (n = 20). Besides standard of care diagnostics, 177 circulating proteins related to inflammation and cardiovascular disease were analyzed using proximity extension assay (PEA, Olink) technology. Results: Comparative proteome analysis revealed 14 distinct proteins as highly associated with SARS-CoV-2 infection and 12 proteins with subsequent hospitalization (p < 0.001). ADM, IL-6, MCP-3, TRAIL-R2, and PD-L1 were each predictive for death (AUROC curve 0.80–0.87). The consistent increase of these markers, from hospital admission to intensive care and fatality, supported the concept that these proteins are of major clinical relevance. Conclusions: We identified distinct plasma proteins linked to the presence and course of COVID-19. These plasma proteomic findings may translate to a protein fingerprint, helping to assist clinical management decisions. Full article

Once low-pathogenic avian influenza viruses (LPAIVs) of the H5 and H7 subtypes from wild birds enter into poultry species, there is the possibility of them mutating into highly pathogenic avian influenza viruses (HPAIVs), resulting in severe epizootics with up to 100% mortality. This mutation from a LPAIV to HPAIV strain is the main cause of an AIV’s major economic impact on poultry production. Although AIVs are inextricably linked to their hosts in their evolutionary history, the contribution of host-related factors in the emergence of HPAI viruses has only been marginally explored so far. In this study, transcriptomic sequencing of tracheal tissue from chickens infected with four distinct LP H7 viruses, characterized by a different history of pathogenicity evolution in the field, was implemented. Despite the inoculation of a normalized infectious dose of viruses belonging to the same subtype (H7) and pathotype (LPAI), the use of animals of the same age, sex and species as well as the identification of a comparable viral load in the target samples, the analyses revealed a heterogeneity in the gene expression profile in response to infection with each of the H7 viruses administered. Full article

Leishmania spp. are important pathogens causing a vector-borne disease with a broad range of clinical manifestations from self-healing ulcers to the life-threatening visceral forms. Presence of Leishmania RNA virus (LRV) confers survival advantage to these parasites by suppressing anti-leishmanial immunity in the vertebrate host. The two viral species, LRV1 and LRV2 infect species of the subgenera Viannia and Leishmania, respectively. In this work we investigated co-phylogenetic patterns of leishmaniae and their viruses on a small scale (LRV2 in L. major) and demonstrated their predominant coevolution, occasionally broken by intraspecific host switches. Our analysis of the two viral genes, encoding the capsid and RNA-dependent RNA polymerase (RDRP), revealed them to be under the pressure of purifying selection, which was considerably stronger for the former gene across the whole tree. The selective pressure also differs between the LRV clades and correlates with the frequency of interspecific host switches. In addition, using experimental (capsid) and predicted (RDRP) models we demonstrated that the evolutionary variability across the structure is strikingly different in these two viral proteins. Full article

All known Clostridioides difficile phages encode integrases rendering them potentially able to lyse or lysogenise bacterial strains. Here, we observed the infection of the siphovirus, CDHS-1 on a ribotype 027 strain, R20291 and determined the phage and bacterial gene expression profiles, and impacts of phage infection on bacterial physiology and pathogenicity. Using RNA-seq and RT-qPCR we analysed transcriptomic changes during early, mid-log and late phases of phage replication at an MOI of 10. The phage has a 20 min latent period, takes 80 min to lyse cells and a burst size of ~37. All phage genes are highly expressed during at least one time point. The Cro/C1-transcriptional regulator, ssDNA binding protein and helicase are expressed early, the holin is expressed during the mid-log phase and structural proteins are expressed from mid-log to late phase. Most bacterial genes, particularly the metabolism and toxin production/regulatory genes, were downregulated from early phage replication. Phage-resistant strains and lysogens showed reduced virulence during Galleria mellonella colonization as ascertained by the larval survival and expression of growth (10), reproduction (2) and infection (2) marker genes. These data suggest that phage infection both reduces colonization and negatively impacts bacterial pathogenicity, providing encouraging data to support the development of this phage for therapy to treat C. difficile infection. Full article

Major histocompatibility complex class I (MHC-I) molecules play a critical role in the host’s antiviral response by presenting virus-derived antigenic peptides to cytotoxic T lymphocytes (CTLs), enabling the clearance of virus-infected cells. Human adenoviruses evade CTL-mediated cell lysis, in part, by interfering directly with the MHC-I antigen presentation pathway through the expression of E3-19K, which binds both MHC-I and the transporter associated with antigen processing protein and sequestering MHC-I within the endoplasmic reticulum. Fowl adenoviruses have no homologues of E3-19K. Here, we show that representative virus isolates of the species Fowl aviadenovirus C, Fowl aviadenovirus D, and Fowl aviadenovirus E downregulate the cell surface expression of MHC-I in chicken hepatoma cells, resulting in 71%, 11%, and 14% of the baseline expression level, respectively, at 12 h post-infection. Furthermore, this work reports that FAdV-9 downregulates cell surface MHC-I through a minimum of two separate mechanisms—a lysosomal-independent mechanism that requires the presence of the fowl adenovirus early 1 (FE1) transcription unit located within the left terminal genomic region between nts 1 and 6131 and a lysosomal-dependent mechanism that does not require the presence of FE1. These results establish a new functional role for the FE1 transcription unit in immune evasion. These studies provide important new information about the immune evasion of FAdVs and will enhance our understanding of the pathogenesis of inclusion body hepatitis and advance the progress made in next-generation FAdV-based vectors. Full article

Men are disproportionately affected by the coronavirus disease-2019 (COVID-19), and face higher odds of severe illness and death compared to women. The vascular effects of androgen signaling and inflammatory cytokines in severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2)-mediated endothelial injury are not defined. We determined the effects of SARS-CoV-2 spike protein-mediated endothelial injury under conditions of exposure to androgen dihydrotestosterone (DHT) and tumor necrosis factor-a (TNF-α) and tested potentially therapeutic effects of mineralocorticoid receptor antagonism by spironolactone. Circulating endothelial injury markers VCAM-1 and E-selectin were measured in men and women diagnosed with COVID-19. Exposure of endothelial cells (ECs) in vitro to DHT exacerbated spike protein S1-mediated endothelial injury transcripts for the cell adhesion molecules E-selectin, VCAM-1 and ICAM-1 and anti-fibrinolytic PAI-1 (p < 0.05), and increased THP-1 monocyte adhesion to ECs (p = 0.032). Spironolactone dramatically reduced DHT+S1-induced endothelial activation. TNF-α exacerbated S1-induced EC activation, which was abrogated by pretreatment with spironolactone. Analysis from patients hospitalized with COVID-19 showed concordant higher circulating VCAM-1 and E-Selectin levels in men, compared to women. A beneficial effect of the FDA-approved drug spironolactone was observed on endothelial cells in vitro, supporting a rationale for further evaluation of mineralocorticoid antagonism as an adjunct treatment in COVID-19. Full article

African swine fever virus (ASFV), causing an OIE-notifiable viral disease of swine, is spreading over the Eurasian continent and threatening the global pig industry. Here, we conducted the first proteome analysis of ASFV-infected primary porcine monocyte-derived macrophages (moMΦ). In parallel to moMΦ isolated from different pigs, the stable porcine cell line WSL-R was infected with a recombinant of ASFV genotype IX strain “Kenya1033”. The outcome of the infections was compared via quantitative mass spectrometry (MS)-based proteome analysis. Major differences with respect to the expression of viral proteins or the host cell response were not observed. However, cell-specific expression of some individual viral proteins did occur. The observed modulations of the host proteome were mainly related to cell characteristics and function. Overall, we conclude that both infection models are suitable for use in the study of ASFV infection in vitro. Full article