BTN3A3 evasion promotes the zoonotic potential of influenza A viruses.

Spillover events of avian influenza A viruses (IAVs) to humans could represent the first step in a future pandemic1. Several factors that limit the transmission and replication of avian IAVs in mammals have been identified. There are several gaps in our understanding to predict which virus lineages are more likely to cross the species barrier and cause disease in humans1. Here, we identified human BTN3A3 (butyrophilin subfamily 3 member A3)2 as a potent inhibitor of avian IAVs but not human IAVs. We determined that BTN3A3 is expressed in human airways and its antiviral activity evolved in primates. We show that BTN3A3 restriction acts primarily at the early stages of the virus life cycle by inhibiting avian IAV RNA replication. We identified residue 313 in the viral nucleoprotein (NP) as the genetic determinant of BTN3A3 sensitivity (313F or, rarely, 313L in avian viruses) or evasion (313Y or 313V in human viruses). However, avian IAV serotypes, such as H7 and H9, that spilled over into humans also evade BTN3A3 restriction. In these cases, BTN3A3 evasion is due to substitutions (N, H or Q) in NP residue 52 that is adjacent to residue 313 in the NP structure3. Thus, sensitivity or resistance to BTN3A3 is another factor to consider in the risk assessment of the zoonotic potential of avian influenza viruses.

Resurrection of 2'-5'-oligoadenylate synthetase 1 (OAS1) from the ancestor of modern horseshoe bats blocks SARS-CoV-2 replication.

The prenylated form of the human 2'-5'-oligoadenylate synthetase 1 (OAS1) protein has been shown to potently inhibit the replication of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the virus responsible for the Coronavirus Disease 2019 (COVID-19) pandemic. However, the OAS1 orthologue in the horseshoe bats (superfamily Rhinolophoidea), the reservoir host of SARS-related coronaviruses (SARSr-CoVs), has lost the prenylation signal required for this antiviral activity. Herein, we used an ancestral state reconstruction approach to predict and reconstitute in vitro, the most likely OAS1 protein sequence expressed by the Rhinolophoidea common ancestor prior to its prenylation loss (RhinoCA OAS1). We exogenously expressed the ancient bat protein in vitro to show that, unlike its non-prenylated horseshoe bat descendants, RhinoCA OAS1 successfully blocks SARS-CoV-2 replication. Using protein structure predictions in combination with evolutionary hypothesis testing methods, we highlight sites under unique diversifying selection specific to OAS1's evolution in the Rhinolophoidea. These sites are located near the RNA-binding region and the C-terminal end of the protein where the prenylation signal would have been. Our results confirm that OAS1 prenylation loss at the base of the Rhinolophoidea clade ablated the ability of OAS1 to restrict SARSr-CoV replication and that subsequent evolution of the gene in these bats likely favoured an alternative function. These findings can advance our understanding of the tightly linked association between SARSr-CoVs and horseshoe bats.

HCMV-secreted glycoprotein gpUL4 inhibits TRAIL-mediated apoptosis and NK cell activation.

Human cytomegalovirus (HCMV) is a paradigm of pathogen immune evasion and sustains lifelong persistent infection in the face of exceptionally powerful host immune responses through the concerted action of multiple immune-evasins. These reduce NK cell activation by inhibiting ligands for activating receptors, expressing ligands for inhibitory receptors, or inhibiting synapse formation. However, these functions only inhibit direct interactions with the infected cell. To determine whether the virus also expresses soluble factors that could modulate NK function at a distance, we systematically screened all 170 HCMV canonical protein-coding genes. This revealed that UL4 encodes a secreted and heavily glycosylated protein (gpUL4) that is expressed with late-phase kinetics and is capable of inhibiting NK cell degranulation. Analyses of gpUL4 binding partners by mass spectrometry identified an interaction with TRAIL. gpUL4 bound TRAIL with picomolar affinity and prevented TRAIL from binding its receptor, thus acting as a TRAIL decoy receptor. TRAIL is found in both soluble and membrane-bound forms, with expression of the membrane-bound form strongly up-regulated on NK cells in response to interferon. gpUL4 inhibited apoptosis induced by soluble TRAIL, while also binding to the NK cell surface in a TRAIL-dependent manner, where it blocked NK cell degranulation and cytokine secretion. gpUL4 therefore acts as an immune-evasin by inhibiting both soluble and membrane-bound TRAIL and is a viral-encoded TRAIL decoy receptor. Interestingly, gpUL4 could also suppress NK responses to heterologous viruses, suggesting that it may act as a systemic virally encoded immunosuppressive agent.

Characterisation of the antiviral RNA interference response to Toscana virus in sand fly cells.

Toscana virus (TOSV) (Bunyavirales, Phenuiviridae, Phlebovirus, Toscana phlebovirus) and other related human pathogenic arboviruses are transmitted by phlebotomine sand flies. TOSV has been reported in nations bordering the Mediterranean Sea among other regions. Infection can result in febrile illness as well as meningitis and encephalitis. Understanding vector-arbovirus interactions is crucial to improving our knowledge of how arboviruses spread, and in this context, immune responses that control viral replication play a significant role. Extensive research has been conducted on mosquito vector immunity against arboviruses, with RNA interference (RNAi) and specifically the exogenous siRNA (exo-siRNA) pathway playing a critical role. However, the antiviral immunity of phlebotomine sand flies is less well understood. Here we were able to show that the exo-siRNA pathway is active in a Phlebotomus papatasi-derived cell line. Following TOSV infection, distinctive 21 nucleotide virus-derived small interfering RNAs (vsiRNAs) were detected. We also identified the exo-siRNA effector Ago2 in this cell line, and silencing its expression rendered the exo-siRNA pathway largely inactive. Thus, our data show that this pathway is active as an antiviral response against a sand fly transmitted bunyavirus, TOSV.

Luminescence Lifetime-Based Sensing Platform Based on Cyclometalated Iridium(III) Complexes for the Detection of Perfluorooctanoic Acid in Aqueous Samples.

Luminescence lifetimes are an attractive analytical method for detection due to its high sensitivity and stability. Iridium probes exhibit luminescence with long excited-state lifetimes, which are sensitive to the local environment. Perfluorooctanoic acid (PFOA) is listed as a chemical of high concern regarding its toxicity and is classified as a "forever chemical". In addition to strict limits on the presence of PFOA in drinking water, environmental contamination from industrial effluent or chemical spills requires rapid, simple, accurate, and cost-effective analysis in order to aid containment. Herein, we report the fabrication and function of a novel and facile luminescence sensor for PFOA based on iridium modified on gold surfaces. These surfaces were modified with lipophilic iridium complexes bearing alkyl chains, namely, IrC6 and IrC12, and Zonyl-FSA surfactant. Upon addition of PFOA, the modified surfaces IrC6-FSA@Au and IrC12-FSA @Au show the largest change in the red luminescence signal with changes in the luminescence lifetime that allow monitoring of PFOA concentrations in aqueous solutions. The platform was tested for the measurement of PFOA in aqueous samples spiked with known concentrations of PFOA and demonstrated the capacity to determine PFOA at concentrations >100 µg/L (240 nM).

The apparent interferon resistance of transmitted HIV-1 is possibly a consequence of enhanced replicative fitness.

HIV-1 transmission via sexual exposure is an inefficient process. When transmission does occur, newly infected individuals are colonized by the descendants of either a single virion or a very small number of establishing virions. These transmitted founder (TF) viruses are more interferon (IFN)-resistant than chronic control (CC) viruses present 6 months after transmission. To identify the specific molecular defences that make CC viruses more susceptible to the IFN-induced 'antiviral state', we established a single pair of fluorescent TF and CC viruses and used arrayed interferon-stimulated gene (ISG) expression screening to identify candidate antiviral effectors. However, we observed a relatively uniform ISG resistance of transmitted HIV-1, and this directed us to investigate possible underlying mechanisms. Simple simulations, where we varied a single parameter, illustrated that reduced growth rate could possibly underly apparent interferon sensitivity. To examine this possibility, we closely monitored in vitro propagation of a model TF/CC pair (closely matched in replicative fitness) over a targeted range of IFN concentrations. Fitting standard four-parameter logistic growth models, in which experimental variables were regressed against growth rate and carrying capacity, to our in vitro growth curves, further highlighted that small differences in replicative growth rates could recapitulate our in vitro observations. We reasoned that if growth rate underlies apparent interferon resistance, transmitted HIV-1 would be similarly resistant to any growth rate inhibitor. Accordingly, we show that two transmitted founder HIV-1 viruses are relatively resistant to antiretroviral drugs, while their matched chronic control viruses were more sensitive. We propose that, when present, the apparent IFN resistance of transmitted HIV-1 could possibly be explained by enhanced replicative fitness, as opposed to specific resistance to individual IFN-induced defences. However, further work is required to establish how generalisable this mechanism of relative IFN resistance might be.

A plasmid DNA-launched SARS-CoV-2 reverse genetics system and coronavirus toolkit for COVID-19 research.

The recent emergence of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the underlying cause of Coronavirus Disease 2019 (COVID-19), has led to a worldwide pandemic causing substantial morbidity, mortality, and economic devastation. In response, many laboratories have redirected attention to SARS-CoV-2, meaning there is an urgent need for tools that can be used in laboratories unaccustomed to working with coronaviruses. Here we report a range of tools for SARS-CoV-2 research. First, we describe a facile single plasmid SARS-CoV-2 reverse genetics system that is simple to genetically manipulate and can be used to rescue infectious virus through transient transfection (without in vitro transcription or additional expression plasmids). The rescue system is accompanied by our panel of SARS-CoV-2 antibodies (against nearly every viral protein), SARS-CoV-2 clinical isolates, and SARS-CoV-2 permissive cell lines, which are all openly available to the scientific community. Using these tools, we demonstrate here that the controversial ORF10 protein is expressed in infected cells. Furthermore, we show that the promising repurposed antiviral activity of apilimod is dependent on TMPRSS2 expression. Altogether, our SARS-CoV-2 toolkit, which can be directly accessed via our website at https://mrcppu-covid.bio/, constitutes a resource with considerable potential to advance COVID-19 vaccine design, drug testing, and discovery science.

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.

In silico prediction of HIV-1-host molecular interactions and their directionality.

Human immunodeficiency virus type 1 (HIV-1) continues to be a major cause of disease and premature death. As with all viruses, HIV-1 exploits a host cell to replicate. Improving our understanding of the molecular interactions between virus and human host proteins is crucial for a mechanistic understanding of virus biology, infection and host antiviral activities. This knowledge will potentially permit the identification of host molecules for targeting by drugs with antiviral properties. Here, we propose a data-driven approach for the analysis and prediction of the HIV-1 interacting proteins (VIPs) with a focus on the directionality of the interaction: host-dependency versus antiviral factors. Using support vector machine learning models and features encompassing genetic, proteomic and network properties, our results reveal some significant differences between the VIPs and non-HIV-1 interacting human proteins (non-VIPs). As assessed by comparison with the HIV-1 infection pathway data in the Reactome database (sensitivity > 90%, threshold = 0.5), we demonstrate these models have good generalization properties. We find that the 'direction' of the HIV-1-host molecular interactions is also predictable due to different characteristics of 'forward'/pro-viral versus 'backward'/pro-host proteins. Additionally, we infer the previously unknown direction of the interactions between HIV-1 and 1351 human host proteins. A web server for performing predictions is available at http://hivpre.cvr.gla.ac.uk/.

Identifying potential significant factors impacting zero-inflated proportion data.

Classical supervised methods like linear regression and decision trees are not completely adapted for identifying impacting factors on a response variable corresponding to zero-inflated proportion data (ZIPD) that are dependent, continuous and bounded. In this article we propose a within-block permutation-based methodology to identify factors (discrete or continuous) that are significantly correlated with ZIPD, we propose a performance indicator quantifying the percentage of correlation explained by the subset of significant factors, and we show how to predict the ranks of the response variables conditionally on the observation of these factors. The methodology is illustrated on simulated data and on two real data sets dealing with epidemiology. In the first data set, ZIPD correspond to probabilities of transmission of Influenza between horses. In the second data set, ZIPD correspond to probabilities that geographic entities (eg, states and countries) have the same COVID-19 mortality dynamics.

The SARS-CoV-2 Spike Protein Mutation Explorer: using an interactive application to improve the public understanding of SARS-CoV-2 variants of concern.

Due to the COVID-19 pandemic the virus responsible, SARS-CoV-2, became a source of intense interest for non-expert audiences. The viral spike protein gained particular public interest as the main target for protective immune responses, including those elicited by vaccines. The rapid evolution of SARS-CoV-2 resulted in variations in the spike that enhanced transmissibility or weakened vaccine protection. This created new variants of concern (VOCs). The emergence of VOCs was studied using viral sequence data which was shared through portals such as the online Mutation Explorer of the COVID-19 Genomics UK consortium (COG-UK/ME). This was designed for an expert audience, but the information it contained could be of general interest if suitably communicated. Visualisations, interactivity and animation can improve engagement and understanding of molecular biology topics, and so we developed a graphical educational resource, the SARS-CoV-2 Spike Protein Mutation Explorer (SSPME), which used interactive 3D molecular models and animations to explain the molecular biology underpinning VOCs. User testing showed that the SSPME had better usability and improved participant knowledge confidence and knowledge acquisition compared to COG-UK/ME. This demonstrates how interactive visualisations can be used for effective molecular biology communication, as well as improving the public understanding of SARS-CoV-2 VOCs.

Using Molecular Visualisation Techniques to Explain the Molecular Biology of SARS-CoV-2 Spike Protein Mutations to a General Audience.

Since the COVID-19 pandemic started in 2019, the virus responsible for the outbreak-SARS-CoV-2-has continued to evolve. Mutations of the virus' spike protein, the main protein driving infectivity and transmissibility, are especially concerning as they may allow the virus to improve its infectivity, transmissibility, and ability to evade the immune system. Understanding how specific molecular changes can alter the behaviour of a virus is challenging for non-experts, but this information helps us to understand the pandemic we are living through and the public health measures and interventions needed to bring it under control. In response to communication challenges arising from the COVID-19 pandemic, we recently developed an online educational application to explain the molecular biology of SARS-CoV-2 spike protein mutations to the general public. We used visualisation techniques such as 3D modelling and animation, which have been shown to be highly effective teaching tools in molecular biology, allowing the viewer to better understand protein structure, function, and dynamics. We also included interactive elements for users to learn actively by engaging with the digital content, and consequently improve information retention.This chapter presents the methodological and technological framework which we used to create this resource, the 'SARS-CoV-2 Spike Protein Mutation Explorer' (SSPME). It explains how molecular visualisation and 3D modelling software were used to develop accurate models of relevant proteins; how 3D animation software was used to accurately visualise the dynamic molecular processes of SARS-CoV-2 infection, transmission, and antibody evasion; and how game development software was used to compile the 3D models and animations into a comprehensive, informative interactive application on SARS-CoV-2 spike protein mutations. This chapter indicates how cutting-edge visualisation techniques and technologies can be used to improve science communication about complex topics in molecular biology and infection biology to the general public, something that is critical to gaining control of the continuing COVID-19 pandemic.

Absence of adaptive evolution is the main barrier against influenza emergence in horses in Asia despite frequent virus interspecies transmission from wild birds.

Virus ecology and evolution play a central role in disease emergence. However, their relative roles will vary depending on the viruses and ecosystems involved. We combined field studies, phylogenetics and experimental infections to document with unprecedented detail the stages that precede initial outbreaks during viral emergence in nature. Using serological surveys we showed that in the absence of large-scale outbreaks, horses in Mongolia are routinely exposed to and infected by avian influenza viruses (AIVs) circulating among wild birds. Some of those AIVs are genetically related to an avian-origin virus that caused an epizootic in horses in 1989. Experimental infections showed that most AIVs replicate in the equine respiratory tract without causing lesions, explaining the absence of outbreaks of disease. Our results show that AIVs infect horses but do not spread, or they infect and spread but do not cause disease. Thus, the failure of AIVs to evolve greater transmissibility and to cause disease in horses is in this case the main barrier preventing disease emergence.

M Segment-Based Minigenomes and Virus-Like Particle Assays as an Approach To Assess the Potential of Tick-Borne Phlebovirus Genome Reassortment.

Bunyaviruses have a tripartite negative-sense RNA genome. Due to the segmented nature of these viruses, if two closely related viruses coinfect the same host or vector cell, it is possible that RNA segments from either of the two parental viruses will be incorporated into progeny virions to give reassortant viruses. Little is known about the ability of tick-borne phleboviruses to reassort. The present study describes the development of minigenome assays for the tick-borne viruses Uukuniemi phlebovirus (UUKV) and Heartland phlebovirus (HRTV). We used these minigenome assays in conjunction with the existing minigenome system of severe fever with thrombocytopenia syndrome (SFTS) phlebovirus (SFTSV) to assess the abilities of viral N and L proteins to recognize, transcribe, and replicate the M segment-based minigenome of a heterologous virus. The highest minigenome activity was detected with the M segment-based minigenomes of cognate viruses. However, our findings indicate that several combinations utilizing N and L proteins of heterologous viruses resulted in M segment minigenome activity. This suggests that the M segment untranslated regions (UTRs) are recognized as functional promoters of transcription and replication by the N and L proteins of related viruses. Further, virus-like particle assays demonstrated that HRTV glycoproteins can package UUKV and SFTSV S and L segment-based minigenomes. Taken together, these results suggest that coinfection with these viruses could lead to the generation of viable reassortant progeny. Thus, the tools developed in this study could aid in understanding the role of genome reassortment in the evolution of these emerging pathogens in an experimental setting.IMPORTANCE In recent years, there has been a large expansion in the number of emerging tick-borne viruses that are assigned to the Phlebovirus genus. Bunyaviruses have a tripartite segmented genome, and infection of the same host cell by two closely related bunyaviruses can, in theory, result in eight progeny viruses with different genome segment combinations. We used genome analogues expressing reporter genes to assess the abilities of Phlebovirus nucleocapsid protein and RNA-dependent RNA polymerase to recognize the untranslated region of a genome segment of a related phlebovirus, and we used virus-like particle assays to assess whether viral glycoproteins can package genome analogues of related phleboviruses. Our results provide strong evidence that these emerging pathogens could reassort their genomes if they were to meet in nature in an infected host or vector. This reassortment process could result in viruses with new pathogenic properties.

TRIM69 Inhibits Vesicular Stomatitis Indiana Virus.

Vesicular stomatitis Indiana virus (VSIV), formerly known as vesicular stomatitis virus (VSV) Indiana (VSVIND), is a model virus that is exceptionally sensitive to the inhibitory action of interferons (IFNs). Interferons induce an antiviral state by stimulating the expression of hundreds of interferon-stimulated genes (ISGs). These ISGs can constrain viral replication, limit tissue tropism, reduce pathogenicity, and inhibit viral transmission. Since VSIV is used as a backbone for multiple oncolytic and vaccine strategies, understanding how ISGs restrict VSIV not only helps in understanding VSIV-induced pathogenesis but also helps us evaluate and understand the safety and efficacy of VSIV-based therapies. Thus, there is a need to identify and characterize the ISGs that possess anti-VSIV activity. Using arrayed ISG expression screening, we identified TRIM69 as an ISG that potently inhibits VSIV. This inhibition was highly specific as multiple viruses, including influenza A virus, HIV-1, Rift Valley fever virus, and dengue virus, were unaffected by TRIM69. Indeed, just one amino acid substitution in VSIV can govern sensitivity/resistance to TRIM69. Furthermore, TRIM69 is highly divergent in human populations and exhibits signatures of positive selection that are consistent with this gene playing a key role in antiviral immunity. We propose that TRIM69 is an IFN-induced inhibitor of VSIV and speculate that TRIM69 could be important in limiting VSIV pathogenesis and might influence the specificity and/or efficacy of vesiculovirus-based therapies.IMPORTANCE Vesicular stomatitis Indiana virus (VSIV) is a veterinary pathogen that is also used as a backbone for many oncolytic and vaccine strategies. In natural and therapeutic settings, viral infections like VSIV are sensed by the host, and as a result the host cells make proteins that can protect them from viruses. In the case of VSIV, these antiviral proteins constrain viral replication and protect most healthy tissues from virus infection. In order to understand how VSIV causes disease and how healthy tissues are protected from VSIV-based therapies, it is crucial that we identify the proteins that inhibit VSIV. Here, we show that TRIM69 is an antiviral defense that can potently and specifically block VSIV infection.

Highly Diverse Hepatitis C Strains Detected in Sub-Saharan Africa Have Unknown Susceptibility to Direct-Acting Antiviral Treatments.

The global plan to eradicate hepatitis C virus (HCV) led by the World Health Organization outlines the use of highly effective direct-acting antiviral drugs (DAAs) to achieve elimination by 2030. Identifying individuals with active disease and investigation of the breadth of diversity of the virus in sub-Saharan Africa (SSA) is essential as genotypes in this region (where very few clinical trials have been carried out) are distinct from those found in other parts of the world. We undertook a population-based, nested case-control study in Uganda and obtained additional samples from the Democratic Republic of Congo (DRC) to estimate the prevalence of HCV, assess strategies for disease detection using serological and molecular techniques, and characterize genetic diversity of the virus. Using next-generation and Sanger sequencing, we aimed to identify strains circulating in East and Central Africa. A total of 7,751 Ugandan patients were initially screened for HCV, and 20 PCR-positive samples were obtained for sequencing. Serological assays were found to vary significantly in specificity for HCV. HCV strains detected in Uganda included genotype (g) 4k, g4p, g4q, and g4s and a newly identified unassigned g7 HCV strain. Two additional unassigned g7 strains were identified in patients originating from DRC (one partial and one full open reading frame sequence). These g4 and g7 strains contain nonstructural (ns) protein 3 and 5A polymorphisms associated with resistance to DAAs in other genotypes. Clinical studies are therefore indicated to investigate treatment response in infected patients. Conclusion: Although HCV prevalence and genotypes have been well characterized in patients in well-resourced countries, clinical trials are urgently required in SSA, where highly diverse g4 and g7 strains circulate.

Fundamental properties of the mammalian innate immune system revealed by multispecies comparison of type I interferon responses.

The host innate immune response mediated by type I interferon (IFN) and the resulting up-regulation of hundreds of interferon-stimulated genes (ISGs) provide an immediate barrier to virus infection. Studies of the type I 'interferome' have mainly been carried out at a single species level, often lacking the power necessary to understand key evolutionary features of this pathway. Here, using a single experimental platform, we determined the properties of the interferomes of multiple vertebrate species and developed a webserver to mine the dataset. This approach revealed a conserved 'core' of 62 ISGs, including genes not previously associated with IFN, underscoring the ancestral functions associated with this antiviral host response. We show that gene expansion contributes to the evolution of the IFN system and that interferomes are shaped by lineage-specific pressures. Consequently, each mammal possesses a unique repertoire of ISGs, including genes common to all mammals and others unique to their specific species or phylogenetic lineages. An analysis of genes commonly down-regulated by IFN suggests that epigenetic regulation of transcription is a fundamental aspect of the IFN response. Our study provides a resource for the scientific community highlighting key paradigms of the type I IFN response.

Reorganizing the family Parvoviridae: a revised taxonomy independent of the canonical approach based on host association.

Parvoviridae, a diverse family of small single-stranded DNA viruses was established in 1975. It was divided into two subfamilies, Parvovirinae and Densovirinae, in 1993 to accommodate parvoviruses that infect vertebrate and invertebrate animals, respectively. This relatively straightforward segregation, using host association as the prime criterion for subfamily-level classification, has recently been challenged by the discovery of divergent, vertebrate-infecting parvoviruses, dubbed "chapparvoviruses", which have proven to be more closely related to viruses in certain Densovirinae genera than to members of the Parvovirinae. Viruses belonging to these genera, namely Brevi-, Hepan- and Penstyldensovirus, are responsible for the unmatched heterogeneity of the subfamily Densovirinae when compared to the Parvovirinae in matters of genome organization, protein sequence homology, and phylogeny. Another genus of Densovirinae, Ambidensovirus, has challenged traditional parvovirus classification, as it includes all newly discovered densoviruses with an ambisense genome organization, which introduces genus-level paraphyly. Lastly, current taxon definition and virus inclusion criteria have significantly limited the classification of certain long-discovered parvoviruses and impedes the classification of some potential family members discovered using high-throughput sequencing methods. Here, we present a new and updated system for parvovirus classification, which includes the introduction of a third subfamily, Hamaparvovirinae, resolves the paraphyly within genus Ambidensovirus, and introduces new genera and species into the subfamily Parvovirinae. These proposals were accepted by the ICTV in 2020 March.

Interpretation of peripheral arterial and venous Doppler waveforms: A consensus statement from the Society for Vascular Medicine and Society for Vascular Ultrasound.

This expert consensus statement on the interpretation of peripheral arterial and venous spectral Doppler waveforms was jointly commissioned by the Society for Vascular Medicine (SVM) and the Society for Vascular Ultrasound (SVU). The consensus statement proposes a standardized nomenclature for arterial and venous spectral Doppler waveforms using a framework of key major descriptors and additional modifier terms. These key major descriptors and additional modifier terms are presented alongside representative Doppler waveforms, and nomenclature tables provide context by listing previous alternate terms to be replaced by the new major descriptors and modifiers. Finally, the document reviews Doppler waveform alterations with physiologic changes and disease states, provides optimization techniques for waveform acquisition and display, and provides practical guidance for incorporating the proposed nomenclature into the final interpretation report.

Human Cytomegalovirus Genomes Sequenced Directly From Clinical Material: Variation, Multiple-Strain Infection, Recombination, and Gene Loss.

The genomic characteristics of human cytomegalovirus (HCMV) strains sequenced directly from clinical pathology samples were investigated, focusing on variation, multiple-strain infection, recombination, and gene loss. A total of 207 datasets generated in this and previous studies using target enrichment and high-throughput sequencing were analyzed, in the process enabling the determination of genome sequences for 91 strains. Key findings were that (i) it is important to monitor the quality of sequencing libraries in investigating variation; (ii) many recombinant strains have been transmitted during HCMV evolution, and some have apparently survived for thousands of years without further recombination; (iii) mutants with nonfunctional genes (pseudogenes) have been circulating and recombining for long periods and can cause congenital infection and resulting clinical sequelae; and (iv) intrahost variation in single-strain infections is much less than that in multiple-strain infections. Future population-based studies are likely to continue illuminating the evolution, epidemiology, and pathogenesis of HCMV.