Unraveling clonal CD8 T cell expansion and identification of essential factors in γ-herpesvirus-induced lymphomagenesis.

Alcelaphine gammaherpesvirus 1 (AlHV-1) asymptomatically persists in its natural host, the wildebeest. However, cross-species transmission to cattle results in the induction of an acute and lethal peripheral T cell lymphoma-like disease (PTCL), named malignant catarrhal fever (MCF). Our previous findings demonstrated an essential role for viral genome maintenance in infected CD8+ T lymphocytes but the exact mechanism(s) leading to lymphoproliferation and MCF remained unknown. To decipher how AlHV-1 dysregulates T lymphocytes, we first examined the global phenotypic changes in circulating CD8+ T cells after experimental infection of calves. T cell receptor repertoire together with transcriptomics and epigenomics analyses demonstrated an oligoclonal expansion of infected CD8+ T cells displaying effector and exhaustion gene signatures, including GZMA, GNLY, PD-1, and TOX2 expression. Then, among viral genes expressed in infected CD8+ T cells, we uncovered A10 that encodes a transmembrane signaling protein displaying multiple tyrosine residues, with predicted ITAM and SH3 motifs. Impaired A10 expression did not affect AlHV-1 replication in vitro but rendered AlHV-1 unable to induce MCF. Furthermore, A10 was phosphorylated in T lymphocytes in vitro and affected T cell signaling. Finally, while AlHV-1 mutants expressing mutated forms of A10 devoid of ITAM or SH3 motifs (or both) were able to induce MCF, a recombinant virus expressing a mutated form of A10 unable to phosphorylate its tyrosine residues resulted in the lack of MCF and protected against a wild-type virus challenge. Thus, we could characterize the nature of this γ-herpesvirus-induced PTCL-like disease and identify an essential mechanism explaining its development.

ADAM17 targeting by human cytomegalovirus remodels the cell surface proteome to simultaneously regulate multiple immune pathways.

Human cytomegalovirus (HCMV) is a major human pathogen whose life-long persistence is enabled by its remarkable capacity to systematically subvert host immune defenses. In exploring the finding that HCMV infection up-regulates tumor necrosis factor receptor 2 (TNFR2), a ligand for the pro-inflammatory antiviral cytokine TNFα, we found that the underlying mechanism was due to targeting of the protease, A Disintegrin And Metalloproteinase 17 (ADAM17). ADAM17 is the prototype 'sheddase', a family of proteases that cleaves other membrane-bound proteins to release biologically active ectodomains into the supernatant. HCMV impaired ADAM17 surface expression through the action of two virally-encoded proteins in its UL/b' region, UL148 and UL148D. Proteomic plasma membrane profiling of cells infected with an HCMV double-deletion mutant for UL148 and UL148D with restored ADAM17 expression, combined with ADAM17 functional blockade, showed that HCMV stabilized the surface expression of 114 proteins (P < 0.05) in an ADAM17-dependent fashion. These included reported substrates of ADAM17 with established immunological functions such as TNFR2 and jagged1, but also numerous unreported host and viral targets, such as nectin1, UL8, and UL144. Regulation of TNFα-induced cytokine responses and NK inhibition during HCMV infection were dependent on this impairment of ADAM17. We therefore identify a viral immunoregulatory mechanism in which targeting a single sheddase enables broad regulation of multiple critical surface receptors, revealing a paradigm for viral-encoded immunomodulation.

A comprehensive data-driven model of cat primary visual cortex.

Knowledge integration based on the relationship between structure and function of the neural substrate is one of the main targets of neuroinformatics and data-driven computational modeling. However, the multiplicity of data sources, the diversity of benchmarks, the mixing of observables of different natures, and the necessity of a long-term, systematic approach make such a task challenging. Here we present a first snapshot of a long-term integrative modeling program designed to address this issue in the domain of the visual system: a comprehensive spiking model of cat primary visual cortex. The presented model satisfies an extensive range of anatomical, statistical and functional constraints under a wide range of visual input statistics. In the presence of physiological levels of tonic stochastic bombardment by spontaneous thalamic activity, the modeled cortical reverberations self-generate a sparse asynchronous ongoing activity that quantitatively matches a range of experimentally measured statistics. When integrating feed-forward drive elicited by a high diversity of visual contexts, the simulated network produces a realistic, quantitatively accurate interplay between visually evoked excitatory and inhibitory conductances; contrast-invariant orientation-tuning width; center surround interactions; and stimulus-dependent changes in the precision of the neural code. This integrative model offers insights into how the studied properties interact, contributing to a better understanding of visual cortical dynamics. It provides a basis for future development towards a comprehensive model of low-level perception.

A fish herpesvirus highlights functional diversities among Zα domains related to phase separation induction and A-to-Z conversion.

Zalpha (Zα) domains bind to left-handed Z-DNA and Z-RNA. The Zα domain protein family includes cellular (ADAR1, ZBP1 and PKZ) and viral (vaccinia virus E3 and cyprinid herpesvirus 3 (CyHV-3) ORF112) proteins. We studied CyHV-3 ORF112, which contains an intrinsically disordered region and a Zα domain. Genome editing of CyHV-3 indicated that the expression of only the Zα domain of ORF112 was sufficient for normal viral replication in cell culture and virulence in carp. In contrast, its deletion was lethal for the virus. These observations revealed the potential of the CyHV-3 model as a unique platform to compare the exchangeability of Zα domains expressed alone in living cells. Attempts to rescue the ORF112 deletion by a broad spectrum of cellular, viral, and artificial Zα domains showed that only those expressing Z-binding activity, the capacity to induce liquid-liquid phase separation (LLPS), and A-to-Z conversion, could rescue viral replication. For the first time, this study reports the ability of some Zα domains to induce LLPS and supports the biological relevance of dsRNA A-to-Z conversion mediated by Zα domains. This study expands the functional diversity of Zα domains and stimulates new hypotheses concerning the mechanisms of action of proteins containing Zα domains.

Human cytomegalovirus protein RL1 degrades the antiviral factor SLFN11 via recruitment of the CRL4 E3 ubiquitin ligase complex.

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of viral immune evasion, targeting intrinsic, innate, and adaptive immunity. We have employed two orthogonal multiplexed tandem mass tag-based proteomic screens to identify host proteins down-regulated by viral factors expressed during the latest phases of viral infection. This approach revealed that the HIV-1 restriction factor Schlafen-11 (SLFN11) was degraded by the poorly characterized, late-expressed HCMV protein RL1, via recruitment of the Cullin4-RING E3 Ubiquitin Ligase (CRL4) complex. SLFN11 potently restricted HCMV infection, inhibiting the formation and spread of viral plaques. Overall, we show that a restriction factor previously thought only to inhibit RNA viruses additionally restricts HCMV. We define the mechanism of viral antagonism and also describe an important resource for revealing additional molecules of importance in antiviral innate immunity and viral immune evasion.

Strain-Dependent Restriction of Human Cytomegalovirus by Zinc Finger Antiviral Proteins.

Cellular antiviral factors that recognize viral nucleic acid can inhibit virus replication. These include the zinc finger antiviral protein (ZAP), which recognizes high CpG dinucleotide content in viral RNA. Here, we investigated the ability of ZAP to inhibit the replication of human cytomegalovirus (HCMV). Depletion of ZAP or its cofactor KHNYN increased the titer of the high-passage HCMV strain AD169 but had little effect on the titer of the low-passage strain Merlin. We found no obvious difference in expression of several viral proteins between AD169 and Merlin in ZAP knockdown cells, but observed a larger increase in infectious virus in AD169 compared to Merlin in the absence of ZAP, suggesting that ZAP inhibited events late in AD169 replication. In addition, there was no clear difference in the CpG abundance of AD169 and Merlin RNAs, indicating that genomic content of the two virus strains was unlikely to be responsible for differences in their sensitivity to ZAP. Instead, we observed less ZAP expression in Merlin-infected cells late in replication compared to AD169-infected cells, which may be related to different abilities of the two virus strains to regulate interferon signaling. Therefore, there are strain-dependent differences in the sensitivity of HCMV to ZAP, and the ability of low-passage HCMV strain Merlin to evade inhibition by ZAP is likely related to its ability to regulate interferon signaling, not the CpG content of RNAs produced from its genome. IMPORTANCE Determining the function of cellular antiviral factors can inform our understanding of virus replication. The zinc finger antiviral protein (ZAP) can inhibit the replication of diverse viruses. Here, we examined ZAP interaction with the DNA virus human cytomegalovirus (HCMV). We found HCMV strain-dependent differences in the ability of ZAP to influence HCMV replication, which may be related to the interaction of HCMV strains with the type I interferon system. These observations affect our current understanding of how ZAP restricts HCMV and how HCMV interacts with the type I interferon system.

Isolation and genome sequencing of cytomegaloviruses from Natal multimammate mice (Mastomys natalensis).

Distinct cytomegaloviruses (CMVs) are widely distributed across their mammalian hosts in a highly host species-restricted pattern. To date, evidence demonstrating this has been limited largely to PCR-based approaches targeting small, conserved genomic regions, and only a few complete genomes of isolated viruses representing distinct CMV species have been sequenced. We have now combined direct isolation of infectious viruses from tissues with complete genome sequencing to provide a view of CMV diversity in a wild animal population. We targeted Natal multimammate mice (Mastomys natalensis), which are common in sub-Saharan Africa, are known to carry a variety of zoonotic pathogens, and are regarded as the primary source of Lassa virus (LASV) spillover into humans. Using transformed epithelial cells prepared from M. natalensis kidneys, we isolated CMVs from the salivary gland tissue of 14 of 37 (36 %) animals from a field study site in Mali. Genome sequencing showed that these primary isolates represent three different M. natalensis CMVs (MnatCMVs: MnatCMV1, MnatCMV2 and MnatCMV3), with some animals carrying multiple MnatCMVs or multiple strains of a single MnatCMV presumably as a result of coinfection or superinfection. Including primary isolates and plaque-purified isolates, we sequenced and annotated the genomes of two MnatCMV1 strains (derived from sequencing 14 viruses), six MnatCMV2 strains (25 viruses) and ten MnatCMV3 strains (21 viruses), totalling 18 MnatCMV strains isolated as 60 infectious viruses. Phylogenetic analysis showed that these MnatCMVs group with other murid viruses in the genus Muromegalovirus (subfamily Betaherpesvirinae, family Orthoherpesviridae), and that MnatCMV1 and MnatCMV2 are more closely related to each other than to MnatCMV3. The availability of MnatCMV isolates and the characterization of their genomes will serve as the prelude to the generation of a MnatCMV-based vaccine to target LASV in the M. natalensis reservoir.

Quantitative LC-MS study of compounds found predictive of COVID-19 severity and outcome.

INTRODUCTION: Since the beginning of the SARS-CoV-2 pandemic in December 2019 multiple metabolomics studies have proposed predictive biomarkers of infection severity and outcome. Whilst some trends have emerged, the findings remain intangible and uninformative when it comes to new patients. OBJECTIVES: In this study, we accurately quantitate a subset of compounds in patient serum that were found predictive of severity and outcome. METHODS: A targeted LC-MS method was used in 46 control and 95 acute COVID-19 patient samples to quantitate the selected metabolites. These compounds included tryptophan and its degradation products kynurenine and kynurenic acid (reflective of immune response), butyrylcarnitine and its isomer (reflective of energy metabolism) and finally 3',4'-didehydro-3'-deoxycytidine, a deoxycytidine analogue, (reflective of host viral defence response). We subsequently examine changes in those markers by disease severity and outcome relative to those of control patients' levels. RESULTS & CONCLUSION: Finally, we demonstrate the added value of the kynurenic acid/tryptophan ratio for severity and outcome prediction and highlight the viral detection potential of ddhC.

Human cytomegalovirus protein pUL36: A dual cell death pathway inhibitor.

Human cytomegalovirus (HCMV) is an important human pathogen and a paradigm of intrinsic, innate, and adaptive viral immune evasion. Here, we employed multiplexed tandem mass tag-based proteomics to characterize host proteins targeted for degradation late during HCMV infection. This approach revealed that mixed lineage kinase domain-like protein (MLKL), a key terminal mediator of cellular necroptosis, was rapidly and persistently degraded by the minimally passaged HCMV strain Merlin but not the extensively passaged strain AD169. The strain Merlin viral inhibitor of apoptosis pUL36 was necessary and sufficient both to degrade MLKL and to inhibit necroptosis. Furthermore, mutation of pUL36 Cys131 abrogated MLKL degradation and restored necroptosis. As the same residue is also required for pUL36-mediated inhibition of apoptosis by preventing proteolytic activation of procaspase-8, we define pUL36 as a multifunctional inhibitor of both apoptotic and necroptotic cell death.

ICTV Virus Taxonomy Profile: Adenoviridae 2022.

The family Adenoviridae includes non-enveloped viruses with linear dsDNA genomes of 25-48 kb and medium-sized icosahedral capsids. Adenoviruses have been discovered in vertebrates from fish to humans. The family is divided into six genera, each of which is more common in certain animal groups. The outcome of infection may vary from subclinical to lethal disease. This is a summary of the ICTV Report on the family Adenoviridae, which is available at ictv.global/report/adenoviridae.

Human Cytomegalovirus RNA2.7 Is Required for Upregulating Multiple Cellular Genes To Promote Cell Motility and Viral Spread Late in Lytic Infection.

Long noncoding RNAs (lncRNAs) are frequently associated with broad modulation of gene expression and thus provide the cell with the ability to synchronize entire metabolic processes. We used transcriptomic approaches to investigate whether the most abundant human cytomegalovirus-encoded lncRNA, RNA2.7, has this characteristic. By comparing cells infected with wild-type virus (WT) to cells infected with RNA2.7 deletion mutants, RNA2.7 was implicated in regulating a large number of cellular genes late in lytic infection. Pathway analysis indicated that >100 of these genes are associated with promoting cell movement, and the 10 most highly regulated of these were validated in further experiments. Morphological analysis and live cell tracking of WT- and RNA2.7 mutant-infected cells indicated that RNA2.7 is involved in promoting the movement and detachment of infected cells late in infection, and plaque assays using sparse cell monolayers indicated that RNA2.7 is also involved in promoting cell-to-cell spread of virus. Consistent with the observation that upregulated mRNAs are relatively A+U-rich, which is a trait associated with transcript instability, and that they are also enriched in motifs associated with mRNA instability, transcriptional inhibition experiments on WT- and RNA2.7 mutant-infected cells showed that four upregulated transcripts lived longer in the presence of RNA2.7. These findings demonstrate that RNA2.7 is required for promoting cell movement and viral spread late in infection and suggest that this may be due to general stabilization of A+U-rich transcripts. IMPORTANCE In addition to messenger RNAs (mRNAs), the human genome encodes a large number of long noncoding RNAs (lncRNAs). Many lncRNAs that have been studied in detail are associated with broad modulation of gene expression and have important biological roles. Human cytomegalovirus, which is a large, clinically important DNA virus, specifies four lncRNAs, one of which (RNA2.7) is expressed at remarkably high levels during lytic infection. Our studies show that RNA2.7 is required for upregulating a large number of human genes, about 100 of which are associated with cell movement, and for promoting the movement of infected cells and the spread of virus from one cell to another. Further bioinformatic and experimental analyses indicated that RNA2.7 may exert these effects by stabilizing mRNAs that are relatively rich in A and U nucleotides. These findings increase our knowledge of how human cytomegalovirus regulates the infected cell to promote its own success.

Alcelaphine herpesvirus 1 genes A7 and A8 regulate viral spread and are essential for malignant catarrhal fever.

Alcelaphine herpesvirus 1 (AlHV-1) is a gammaherpesvirus that is carried asymptomatically by wildebeest. Upon cross-species transmission to other ruminants, including domestic cattle, AlHV-1 induces malignant catarrhal fever (MCF), which is a fatal lymphoproliferative disease resulting from proliferation and uncontrolled activation of latently infected CD8+ T cells. Two laboratory strains of AlHV-1 are used commonly in research: C500, which is pathogenic, and WC11, which has been attenuated by long-term maintenance in cell culture. The published genome sequence of a WC11 seed stock from a German laboratory revealed the deletion of two major regions. The sequence of a WC11 seed stock used in our laboratory also bears these deletions and, in addition, the duplication of an internal sequence in the terminal region. The larger of the two deletions has resulted in the absence of gene A7 and a large portion of gene A8. These genes are positional orthologs of the Epstein-Barr virus genes encoding envelope glycoproteins gp42 and gp350, respectively, which are involved in viral propagation and switching of cell tropism. To investigate the degree to which the absence of A7 and A8 participates in WC11 attenuation, recombinant viruses lacking these individual functions were generated in C500. Using bovine nasal turbinate and embryonic lung cell lines, increased cell-free viral propagation and impaired syncytia formation were observed in the absence of A7, whereas cell-free viral spread was inhibited in the absence of A8. Therefore, A7 appears to be involved in cell-to-cell viral spread, and A8 in viral cell-free propagation. Finally, infection of rabbits with either mutant did not induce the signs of MCF or the expansion of infected CD8+ T cells. These results demonstrate that A7 and A8 are both essential for regulating viral spread and suggest that AlHV-1 requires both genes to efficiently spread in vivo and reach CD8+ T lymphocytes and induce MCF.

The latency-associated transcript locus of herpes simplex virus 1 is a virulence determinant in human skin.

Herpes simplex virus 1 (HSV-1) infects skin and mucosal epithelial cells and then travels along axons to establish latency in the neurones of sensory ganglia. Although viral gene expression is restricted during latency, the latency-associated transcript (LAT) locus encodes many RNAs, including a 2 kb intron known as the hallmark of HSV-1 latency. Here, we studied HSV-1 infection and the role of the LAT locus in human skin xenografts in vivo and in cultured explants. We sequenced the genomes of our stock of HSV-1 strain 17syn+ and seven derived viruses and found nonsynonymous mutations in many viral proteins that had no impact on skin infection. In contrast, deletions in the LAT locus severely impaired HSV-1 replication and lesion formation in skin. However, skin replication was not affected by impaired intron splicing. Moreover, although the LAT locus has been implicated in regulating gene expression in neurones, we observed only small changes in transcript levels that were unrelated to the growth defect in skin, suggesting that its functions in skin may be different from those in neurones. Thus, although the LAT locus was previously thought to be dispensable for lytic infection, we show that it is a determinant of HSV-1 virulence during lytic infection of human skin.

Human cytomegalovirus long noncoding RNA4.9 regulates viral DNA replication.

Viruses are known for their extremely compact genomes composed almost entirely of protein-coding genes. Nonetheless, four long noncoding RNAs (lncRNAs) are encoded by human cytomegalovirus (HCMV). Although these RNAs accumulate to high levels during lytic infection, their functions remain largely unknown. Here, we show that HCMV-encoded lncRNA4.9 localizes to the viral nuclear replication compartment, and that its depletion restricts viral DNA replication and viral growth. RNA4.9 is transcribed from the HCMV origin of replication (oriLyt) and forms an RNA-DNA hybrid (R-loop) through its G+C-rich 5' end, which may be important for the initiation of viral DNA replication. Furthermore, targeting the RNA4.9 promoter with CRISPR-Cas9 or genetic relocalization of oriLyt leads to reduced levels of the viral single-stranded DNA-binding protein (ssDBP), suggesting that the levels of ssDBP are coupled to the oriLyt activity. We further identified a similar, oriLyt-embedded, G+C-rich lncRNA in murine cytomegalovirus (MCMV). These results indicate that HCMV RNA4.9 plays an important role in regulating viral DNA replication, that the levels of ssDBP are coupled to the oriLyt activity, and that these regulatory features may be conserved among betaherpesviruses.

HippoUnit: A software tool for the automated testing and systematic comparison of detailed models of hippocampal neurons based on electrophysiological data.

Anatomically and biophysically detailed data-driven neuronal models have become widely used tools for understanding and predicting the behavior and function of neurons. Due to the increasing availability of experimental data from anatomical and electrophysiological measurements as well as the growing number of computational and software tools that enable accurate neuronal modeling, there are now a large number of different models of many cell types available in the literature. These models were usually built to capture a few important or interesting properties of the given neuron type, and it is often unknown how they would behave outside their original context. In addition, there is currently no simple way of quantitatively comparing different models regarding how closely they match specific experimental observations. This limits the evaluation, re-use and further development of the existing models. Further, the development of new models could also be significantly facilitated by the ability to rapidly test the behavior of model candidates against the relevant collection of experimental data. We address these problems for the representative case of the CA1 pyramidal cell of the rat hippocampus by developing an open-source Python test suite, which makes it possible to automatically and systematically test multiple properties of models by making quantitative comparisons between the models and electrophysiological data. The tests cover various aspects of somatic behavior, and signal propagation and integration in apical dendrites. To demonstrate the utility of our approach, we applied our tests to compare the behavior of several different rat hippocampal CA1 pyramidal cell models from the ModelDB database against electrophysiological data available in the literature, and evaluated how well these models match experimental observations in different domains. We also show how we employed the test suite to aid the development of models within the European Human Brain Project (HBP), and describe the integration of the tests into the validation framework developed in the HBP, with the aim of facilitating more reproducible and transparent model building in the neuroscience community.

Characterization of an alloherpesvirus from wild lake sturgeon Acipenser fulvescens in Wisconsin (USA).

In the spring of 2017, 2 adult lake sturgeon (LS) Acipenser fulvescens captured from the Wolf River, Wisconsin (USA), presented with multiple cutaneous plaques that, upon microscopic examination, indicated proliferative epidermitis. Ultrastructural examination of affected keratinocytes revealed particles in the nucleus having a morphology typical of herpesviruses. A degenerate PCR assay targeting the DNA polymerase catalytic subunit (pol) gene of large double-stranded DNA viruses generated amplicons of the anticipated size from skin samples, and sequences of amplicons confirmed the presence of a novel alloherpesvirus (lake sturgeon herpesvirus, LSHV) related to acipenserid herpesvirus 1 (AciHV1). The complete genome (202660 bp) of this virus was sequenced using a MiSeq System, and phylogenetic analyses substantiated the close relationship to AciHV1. A PCR assay targeting the LSHV DNA packaging terminase subunit 1 (ter1) gene demonstrated the presence of the virus in 39/42 skin lesion samples collected from wild LS captured in 2017-2019 and 2021 in 4/4 rivers in Wisconsin. Future efforts to isolate LSHV in cell culture would facilitate challenge studies to determine the disease potential of the virus.

ICTV Virus Taxonomy Profile: Herpesviridae 2021.

Members of the family Herpesviridae have enveloped, spherical virions with characteristic complex structures consisting of symmetrical and non-symmetrical components. The linear, double-stranded DNA genomes of 125-241 kbp contain 70-170 genes, of which 43 have been inherited from an ancestral herpesvirus. In general, herpesviruses have coevolved with and are highly adapted to their hosts, which comprise many mammalian, avian and reptilian species. Following primary infection, they are able to establish lifelong latent infection, during which there is limited viral gene expression. Severe disease is usually observed only in the foetus, the very young, the immunocompromised or following infection of an alternative host. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the family Herpesviridae, which is available at ictv.global/report/herpesviridae.

The switch between acute and persistent paramyxovirus infection caused by single amino acid substitutions in the RNA polymerase P subunit.

Paramyxoviruses can establish persistent infections both in vitro and in vivo, some of which lead to chronic disease. However, little is known about the molecular events that contribute to the establishment of persistent infections by RNA viruses. Using parainfluenza virus type 5 (PIV5) as a model we show that phosphorylation of the P protein, which is a key component of the viral RNA polymerase complex, determines whether or not viral transcription and replication becomes repressed at late times after infection. If the virus becomes repressed, persistence is established, but if not, the infected cells die. We found that single amino acid changes at various positions within the P protein switched the infection phenotype from lytic to persistent. Lytic variants replicated to higher titres in mice than persistent variants and caused greater infiltration of immune cells into infected lungs but were cleared more rapidly. We propose that during the acute phases of viral infection in vivo, lytic variants of PIV5 will be selected but, as the adaptive immune response develops, variants in which viral replication can be repressed will be selected, leading to the establishment of prolonged, persistent infections. We suggest that similar selection processes may operate for other RNA viruses.

Untargeted metabolomics of COVID-19 patient serum reveals potential prognostic markers of both severity and outcome.

INTRODUCTION: The diagnosis of COVID-19 is normally based on the qualitative detection of viral nucleic acid sequences. Properties of the host response are not measured but are key in determining outcome. Although metabolic profiles are well suited to capture host state, most metabolomics studies are either underpowered, measure only a restricted subset of metabolites, compare infected individuals against uninfected control cohorts that are not suitably matched, or do not provide a compact predictive model. OBJECTIVES: Here we provide a well-powered, untargeted metabolomics assessment of 120 COVID-19 patient samples acquired at hospital admission. The study aims to predict the patient's infection severity (i.e., mild or severe) and potential outcome (i.e., discharged or deceased). METHODS: High resolution untargeted UHPLC-MS/MS analysis was performed on patient serum using both positive and negative ionization modes. A subset of 20 intermediary metabolites predictive of severity or outcome were selected based on univariate statistical significance and a multiple predictor Bayesian logistic regression model was created. RESULTS: The predictors were selected for their relevant biological function and include deoxycytidine and ureidopropionate (indirectly reflecting viral load), kynurenine (reflecting host inflammatory response), and multiple short chain acylcarnitines (energy metabolism) among others. Currently, this approach predicts outcome and severity with a Monte Carlo cross validated area under the ROC curve of 0.792 (SD 0.09) and 0.793 (SD 0.08), respectively. A blind validation study on an additional 90 patients predicted outcome and severity at ROC AUC of 0.83 (CI 0.74-0.91) and 0.76 (CI 0.67-0.86). CONCLUSION: Prognostic tests based on the markers discussed in this paper could allow improvement in the planning of COVID-19 patient treatment.

Genome sequences of human cytomegalovirus strain TB40/E variants propagated in fibroblasts and epithelial cells.

The advent of whole genome sequencing has revealed that common laboratory strains of human cytomegalovirus (HCMV) have major genetic deficiencies resulting from serial passage in fibroblasts. In particular, tropism for epithelial and endothelial cells is lost due to mutations disrupting genes UL128, UL130, or UL131A, which encode subunits of a virion-associated pentameric complex (PC) important for viral entry into these cells but not for entry into fibroblasts. The endothelial cell-adapted strain TB40/E has a relatively intact genome and has emerged as a laboratory strain that closely resembles wild-type virus. However, several heterogeneous TB40/E stocks and cloned variants exist that display a range of sequence and tropism properties. Here, we report the use of PacBio sequencing to elucidate the genetic changes that occurred, both at the consensus level and within subpopulations, upon passaging a TB40/E stock on ARPE-19 epithelial cells. The long-read data also facilitated examination of the linkage between mutations. Consistent with inefficient ARPE-19 cell entry, at least 83% of viral genomes present before adaptation contained changes impacting PC subunits. In contrast, and consistent with the importance of the PC for entry into endothelial and epithelial cells, genomes after adaptation lacked these or additional mutations impacting PC subunits. The sequence data also revealed six single noncoding substitutions in the inverted repeat regions, single nonsynonymous substitutions in genes UL26, UL69, US28, and UL122, and a frameshift truncating gene UL141. Among the changes affecting protein-coding regions, only the one in UL122 was strongly selected. This change, resulting in a D390H substitution in the encoded protein IE2, has been previously implicated in rendering another viral protein, UL84, essential for viral replication in fibroblasts. This finding suggests that IE2, and perhaps its interactions with UL84, have important functions unique to HCMV replication in epithelial cells.