[The viral protein NS1: a major player in the pathogenesis of orthoflaviviruses]. / La protéine virale NS1 : un acteur majeur de la pathogenèse des orthoflavivirus.

Orthoflaviviruses are enveloped positive-sense RNA viruses comprising numerous human pathogens transmitted by hematophagous arthropods. This includes viruses such as dengue virus, Zika virus, and yellow fever virus. The viral nonstructural protein NS1 plays a central role in the pathogenesis and cycle of these viruses by acting in two different forms: associated with the plasma membrane (NS1m) or secreted outside the cell (NS1s). The versatility of NS1 is evident in its ability to modulate various aspects of the infectious process, from immune evasion to pathogenesis. As an intracellular protein, it disrupts many processes, interfering with signaling pathways and facilitating viral replication in concert with other viral proteins. As a secreted protein, NS1 actively participates in immune evasion, interfering with the host immune system, inhibiting the complement system, facilitating viral dissemination, and disrupting the integrity of endothelial barriers. This review primarily aims to address the role of NS1 in viral pathogenesis associated with orthoflaviviruses.

Poxvirus Immune Evasion.

Poxviruses have evolved a wide array of mechanisms to evade the immune response, and we provide an overview of the different immunomodulatory strategies. Poxviruses prevent the recognition of viral DNA that triggers the immune responses and inhibit signaling pathways within the infected cell. A unique feature of poxviruses is the production of secreted proteins that mimic cytokines and cytokine receptors, acting as decoy receptors to neutralize the activity of cytokines and chemokines. The capacity of these proteins to evade cellular immune responses by inhibiting cytokine activation is complemented by poxviruses' strategies to block natural killer cells and cytotoxic T cells, often through interfering with antigen presentation pathways. Mechanisms that target complement activation are also encoded by poxviruses. Virus-encoded proteins that target immune molecules and pathways play a major role in immune modulation, and their contribution to viral pathogenesis, facilitating virus replication or preventing immunopathology, is discussed.

Ubiquitination in viral entry and replication: Mechanisms and implications.

The ubiquitination process is a reversible posttranslational modification involved in many essential cellular functions, such as innate immunity, cell signaling, trafficking, protein stability, and protein degradation. Viruses can use the ubiquitin system to efficiently enter host cells, replicate and evade host immunity, ultimately enhancing viral pathogenesis. Emerging evidence indicates that enveloped viruses can carry free (unanchored) ubiquitin or covalently ubiquitinated viral structural proteins that can increase the efficiency of viral entry into host cells. Furthermore, viruses continuously evolve and adapt to take advantage of the host ubiquitin machinery, highlighting its importance during virus infection. This review discusses the battle between viruses and hosts, focusing on how viruses hijack the ubiquitination process at different steps of the replication cycle, with a specific emphasis on viral entry. We discuss how ubiquitination of viral proteins may affect tropism and explore emerging therapeutics strategies targeting the ubiquitin system for antiviral drug discovery.

Update on human herpesvirus 7 pathogenesis and clinical aspects as a roadmap for future research.

Human herpesvirus 7 (HHV-7) is a common virus that is associated with various human diseases including febrile syndromes, dermatological lesions, neurological defects, and transplant complications. Still, HHV-7 remains one of the least studied members of all human betaherpesviruses. In addition, HHV-7-related research is mostly confined to case reports, while in vitro or in vivo studies unraveling basic virology, transmission mechanisms, and viral pathogenesis are sparse. Here, we discuss HHV-7-related literature linking clinical syndromes to the viral life cycle, epidemiology, and viral immunopathogenesis. Based on our review, we propose a hypothetical model of HHV-7 pathogenesis inside its host. Furthermore, we identify important knowledge gaps and recommendations for future research to better understand HHV-7 diseases and improve therapeutic interventions.

Sequence polymorphisms in the reovirus σ1 attachment protein modulate encapsidation efficiency and replication in mice.

Many functions of viral attachment proteins are established, but less is known about the biological importance of viral attachment protein encapsidation efficiency. The mammalian orthoreovirus (reovirus) σ1 attachment protein forms filamentous trimers that incorporate into pentamers of the λ2 capsid protein. Reovirus strains vary in the efficiency of σ1 encapsidation onto progeny virions, which influences viral stability during entry into cells and the efficacy of tumor cell lysis. While the role of σ1 encapsidation has been evaluated in studies using cultured cells, the contribution of attachment protein encapsidation efficiency to viral infection in animals is less clear. Polymorphisms in reovirus σ1 at residues 22 and 249 have been implicated in viral dissemination in mice and susceptibility to proteolysis in the murine intestine, respectively. To determine whether these residues contribute to σ1 encapsidation efficiency, we engineered σ1 mutant viruses with single- and double-residue substitutions at sites 22 and 249. We found that substitutions at these sites alter the encapsidation of σ1 and that reoviruses encapsidating higher amounts of σ1 bind cells more avidly and have a modest replication advantage in a cell-type-specific manner relative to low σ1-encapsidating reoviruses. Furthermore, we found that a high σ1-encapsidating reovirus replicates and disseminates more efficiently in mice relative to a low σ1-encapsidating reovirus. These findings provide evidence of a relationship between viral attachment protein encapsidation efficiency and viral replication in cell culture and animal hosts. IMPORTANCE: Viral attachment proteins can serve multiple functions during viral replication, including attachment to host cells, cell entry and disassembly, and modulation of host immune responses. The relationship between viral attachment protein encapsidation efficiency and viral replication in cells and animals is poorly understood. We engineered and characterized a panel of reoviruses that differ in the capacity to encapsidate the σ1 attachment protein. We found that strains encapsidating σ1 with higher efficiency bind cells more avidly and replicate and spread more efficiently in mice relative to those encapsidating σ1 with lower efficiency. These results highlight a function for σ1 attachment protein capsid abundance in viral replication in cells and animals, which may inform future use of reovirus as an oncolytic therapeutic.

"Mpox in MSM: Tackling Stigma, Minimizing Risk Factors, Exploring Pathogenesis, and Treatment Approaches".

Mpox is a zoonotic disease caused by the monkeypox virus (MPV), primarily found in Central and West African countries. The typical presentation of the disease before the 2022 mpox outbreak includes a febrile prodrome 5-13 days post-exposure, accompanied by lymphadenopathy, malaise, headache, and muscle aches. Unexpectedly, during the 2022 outbreak, several cases of atypical presentations of the disease were reported, such as the absence of prodromal symptoms and the presence of genital skin lesions suggestive of sexual transmission. As per the World Health Organization (WHO), as of March 20, 2024, 94,707 cases of mpox were reported worldwide, resulting in 181 deaths (22 in African endemic regions and 159 in non-endemic countries). The United States Centers for Disease Control and Prevention (CDC) reports a total of 32,063 cases (33.85% of total cases globally), with 58 deaths (32.04% of global deaths) due to mpox. Person-to-person transmission of mpox can occur through respiratory droplets and sustained close contact. However, during the 2022 outbreak of mpox, a high incidence of anal and perianal lesions among MSMs indicated sexual transmission of MPV as a major route of transmission. Since MSMs are disproportionately at risk for HIV transmission, this review discusses the risk factors, transmission patterns, pathogenesis, vaccine, and treatment options for mpox among MSM and people living with HIV (PLWH). Furthermore, we provide a brief perspective on the evolution of the MPV in immunocompromised people like PLWH.

Heterologous Exchanges of Glycoprotein and Non-Virion Protein in Novirhabdoviruses: Assessment of Virulence in Yellow Perch (Perca flavescens) and Rainbow Trout (Oncorhynchus mykiss).

Infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) are rhabdoviruses in two different species belonging to the Novirhabdovirus genus. IHNV has a narrow host range restricted to trout and salmon species, and viruses in the M genogroup of IHNV have high virulence in rainbow trout (Oncorhynchus mykiss). In contrast, the VHSV genotype IVb that invaded the Great Lakes in the United States has a broad host range, with high virulence in yellow perch (Perca flavescens), but not in rainbow trout. By using reverse-genetic systems of IHNV-M and VHSV-IVb strains, we generated six IHNV:VHSV chimeric viruses in which the glycoprotein (G), non-virion-protein (NV), or both G and NV genes of IHNV-M were replaced with the analogous genes from VHSV-IVb, and vice versa. These chimeric viruses were used to challenge groups of rainbow trout and yellow perch. The parental recombinants rIHNV-M and rVHSV-IVb were highly virulent in rainbow trout and yellow perch, respectively. Parental rIHNV-M was avirulent in yellow perch, and chimeric rIHNV carrying G, NV, or G and NV genes from VHSV-IVb remained low in virulence in yellow perch. Similarly, the parental rVHSV-IVb exhibited low virulence in rainbow trout, and chimeric rVHSV with substituted G, NV, or G and NV genes from IHNV-M remained avirulent in rainbow trout. Thus, the G and NV genes of either virus were not sufficient to confer high host-specific virulence when exchanged into a heterologous species genome. Some exchanges of G and/or NV genes caused a loss of host-specific virulence, providing insights into possible roles in viral virulence or fitness, and interactions between viral proteins.

Poxvirus A51R proteins regulate microtubule stability and antagonize a cell-intrinsic antiviral response.

Numerous viruses alter host microtubule (MT) networks during infection, but how and why they induce these changes is unclear in many cases. We show that the vaccinia virus (VV)-encoded A51R protein is a MT-associated protein (MAP) that directly binds MTs and stabilizes them by both promoting their growth and preventing their depolymerization. Furthermore, we demonstrate that A51R-MT interactions are conserved across A51R proteins from multiple poxvirus genera, and highly conserved, positively charged residues in A51R proteins mediate these interactions. Strikingly, we find that viruses encoding MT interaction-deficient A51R proteins fail to suppress a reactive oxygen species (ROS)-dependent antiviral response in macrophages that leads to a block in virion morphogenesis. Moreover, A51R-MT interactions are required for VV virulence in mice. Collectively, our data show that poxviral MAP-MT interactions overcome a cell-intrinsic antiviral ROS response in macrophages that would otherwise block virus morphogenesis and replication in animals.

Human B cells and dendritic cells are susceptible and permissive to enterovirus D68 infection.

Enterovirus D68 (EV-D68) is predominantly associated with mild respiratory infections, but can also cause severe respiratory disease and extra-respiratory complications, including acute flaccid myelitis. Systemic dissemination of EV-D68 is crucial for the development of extra-respiratory diseases, but it is currently unclear how EV-D68 spreads systemically (viremia). We hypothesize that immune cells contribute to the systemic dissemination of EV-D68, as this is a mechanism commonly used by other enteroviruses. Therefore, we investigated the susceptibility and permissiveness of human primary immune cells for different EV-D68 isolates. In human peripheral blood mononuclear cells inoculated with EV-D68, only B cells were susceptible but virus replication was limited. However, in B cell-rich cultures, such as Epstein-Barr virus-transformed B-lymphoblastoid cell line (BLCL) and primary lentivirus-transduced B cells, which better represent lymphoid B cells, were productively infected. Subsequently, we showed that dendritic cells (DCs), particularly immature DCs, are susceptible and permissive for EV-D68 infection and that they can spread EV-D68 to autologous BLCL. Altogether, our findings suggest that immune cells, especially B cells and DCs, could play an important role in the pathogenesis of EV-D68 infection. Infection of these cells may contribute to systemic dissemination of EV-D68, which is an essential step toward the development of extra-respiratory complications.IMPORTANCEEnterovirus D68 (EV-D68) is an emerging respiratory virus that has caused outbreaks worldwide since 2014. EV-D68 infects primarily respiratory epithelial cells resulting in mild respiratory diseases. However, EV-D68 infection is also associated with extra-respiratory complications, including polio-like paralysis. It is unclear how EV-D68 spreads systemically and infects other organs. We hypothesized that immune cells could play a role in the extra-respiratory spread of EV-D68. We showed that EV-D68 can infect and replicate in specific immune cells, that is, B cells and dendritic cells (DCs), and that virus could be transferred from DCs to B cells. Our data reveal a potential role of immune cells in the pathogenesis of EV-D68 infection. Intervention strategies that prevent EV-D68 infection of immune cells will therefore potentially prevent systemic spread of virus and thereby severe extra-respiratory complications.

ACE2 and TMPRSS2 distribution in the respiratory tract of different animal species and its correlation with SARS-CoV-2 tissue tropism.

A wide range of animal species show variable susceptibility to SARS-CoV-2; however, host factors associated with varied susceptibility remain to be defined. Here, we examined whether susceptibility to SARS-CoV-2 and virus tropism in different animal species are dependent on the expression and distribution of the virus receptor angiotensin-converting enzyme 2 (ACE2) and the host cell factor transmembrane serine protease 2 (TMPRSS2). We cataloged the upper and lower respiratory tract of multiple animal species and humans in a tissue-specific manner and quantitatively evaluated the distribution and abundance of ACE2 and TMPRSS2 mRNA in situ. Our results show that: (i) ACE2 and TMPRSS2 mRNA are abundant in the conduction portion of the respiratory tract, (ii) ACE2 mRNA occurs at a lower abundance compared to TMPRSS2 mRNA, (iii) co-expression of ACE2-TMPRSS2 mRNAs is highest in those species with the highest susceptibility to SARS-CoV-2 infection (i.e., cats, Syrian hamsters, and white-tailed deer), and (iv) expression of ACE2 and TMPRSS2 mRNA was not altered following SARS-CoV-2 infection. Our results demonstrate that while specific regions of the respiratory tract are enriched in ACE2 and TMPRSS2 mRNAs in different animal species, this is only a partial determinant of susceptibility to SARS-CoV-2 infection.IMPORTANCESARS-CoV-2 infects a wide array of domestic and wild animals, raising concerns regarding its evolutionary dynamics in animals and potential for spillback transmission of emerging variants to humans. Hence, SARS-CoV-2 infection in animals has significant public health relevance. Host factors determining animal susceptibility to SARS-CoV-2 are vastly unknown, and their characterization is critical to further understand susceptibility and viral dynamics in animal populations and anticipate potential spillback transmission. Here, we quantitatively assessed the distribution and abundance of the two most important host factors, angiotensin-converting enzyme 2 and transmembrane serine protease 2, in the respiratory tract of various animal species and humans. Our results demonstrate that while specific regions of the respiratory tract are enriched in these two host factors, they are only partial determinants of susceptibility. Detailed analysis of additional host factors is critical for our understanding of the underlying mechanisms governing viral susceptibility and reservoir hosts.

SARS-CoV-2 ORF3a Protein as a Therapeutic Target against COVID-19 and Long-Term Post-Infection Effects.

The COVID-19 pandemic caused by SARS-CoV-2 has posed unparalleled challenges due to its rapid transmission, ability to mutate, high mortality and morbidity, and enduring health complications. Vaccines have exhibited effectiveness, but their efficacy diminishes over time while new variants continue to emerge. Antiviral medications offer a viable alternative, but their success has been inconsistent. Therefore, there remains an ongoing need to identify innovative antiviral drugs for treating COVID-19 and its post-infection complications. The ORF3a (open reading frame 3a) protein found in SARS-CoV-2, represents a promising target for antiviral treatment due to its multifaceted role in viral pathogenesis, cytokine storms, disease severity, and mortality. ORF3a contributes significantly to viral pathogenesis by facilitating viral assembly and release, essential processes in the viral life cycle, while also suppressing the body's antiviral responses, thus aiding viral replication. ORF3a also has been implicated in triggering excessive inflammation, characterized by NF-κB-mediated cytokine production, ultimately leading to apoptotic cell death and tissue damage in the lungs, kidneys, and the central nervous system. Additionally, ORF3a triggers the activation of the NLRP3 inflammasome, inciting a cytokine storm, which is a major contributor to the severity of the disease and subsequent mortality. As with the spike protein, ORF3a also undergoes mutations, and certain mutant variants correlate with heightened disease severity in COVID-19. These mutations may influence viral replication and host cellular inflammatory responses. While establishing a direct link between ORF3a and mortality is difficult, its involvement in promoting inflammation and exacerbating disease severity likely contributes to higher mortality rates in severe COVID-19 cases. This review offers a comprehensive and detailed exploration of ORF3a's potential as an innovative antiviral drug target. Additionally, we outline potential strategies for discovering and developing ORF3a inhibitor drugs to counteract its harmful effects, alleviate tissue damage, and reduce the severity of COVID-19 and its lingering complications.

Exploring associations between viral titer measurements and disease outcomes in ferrets inoculated with 125 contemporary influenza A viruses.

As use of the ferret model to study influenza A virus (IAV) pathogenicity increases, periodic assessment of data generated in this model is warranted, to identify features associated with virus replication throughout the respiratory tract and to refine future analyses. However, protocol-specific differences present between independent laboratories limit easy aggregation of virological data. We compiled viral titer and clinical data from >1,000 ferrets inoculated with 125 contemporary IAV under a consistent experimental protocol (including high- and low-pathogenicity avian, swine-origin, and human viruses, spanning H1, H2, H3, H5, H7, and H9 subtypes) and examined which meaningful and statistically supported associations were present among numerous quantitative measurements. Viral titers correlated positively between ferret nasal turbinate tissue, lung tissue, and nasal wash specimens, though the strength of the associations varied, notably regarding the particular nasal wash summary measure employed and properties of the virus itself. Use of correlation coefficients and mediation analyses further supported the interconnectedness of viral titer measurements taken at different sites throughout the respiratory tract. IAV possessing mammalian host adaptation markers in the HA and PB2 exhibited more rapid growth in the ferret upper respiratory tract early after infection, supported by quantities derived from infectious titer data to capture infection progression, compared with viruses bearing hallmarks of avian IAV. Collectively, this work identifies summary metrics most closely linked with virological and phenotypic outcomes in ferrets, supporting continued refinement of data analyzed from in vivo experimentation, notably from studies conducted to evaluate the public health risk posed by novel and emerging IAV.IMPORTANCEFerrets are frequently employed to study the pandemic potential of novel and emerging influenza A viruses. However, systematic retrospective analyses of data generated from these experiments are rarely performed, limiting our ability to identify trends in this data and explore how analyses can be refined. Using logarithmic viral titer and clinical data aggregated from one research group over 20 years, we assessed which meaningful and statistically supported associations were present among numerous quantitative measurements obtained from influenza A virus (IAV)-infected ferrets, including those capturing viral titers, infection progression, and disease severity. We identified numerous linear correlations between parameters assessing virus replication at discrete sites in vivo, including parameters capturing infection progression not frequently employed in the field, and sought to investigate the interconnected nature of these associations. This work supports continued refinement of data analyzed from in vivo experimentation, notably from studies which evaluate the public health risk posed by IAV.

Utilizing non-human primate models to combat recent COVID-19/SARS-CoV-2 and viral infectious disease outbreaks.

In recent times, global viral outbreaks and diseases, such as COVID-19 (SARS-CoV-2), Zika (ZIKV), monkeypox (MPOX), Ebola (EBOV), and Marburg (MARV), have been extensively documented. Swiftly deciphering the mechanisms underlying disease pathogenesis and devising vaccines or therapeutic interventions to curtail these outbreaks stand as paramount imperatives. Amidst these endeavors, animal models emerge as pivotal tools. Among these models, non-human primates (NHPs) hold a position of particular importance. Their proximity in evolutionary lineage and physiological resemblances to humans render them a primary model for comprehending human viral infections. This review encapsulates the pivotal role of various NHP species-such as rhesus macaques (Macaca mulatta), cynomolgus macaques (Macaca fascicularis), african green monkeys (Chlorocebus sabaeus/aethiops), pigtailed macaques (Macaca nemestrina/Macaca leonina), baboons (Papio hamadryas/Papio anubis), and common marmosets (Callithrix jacchus)-in investigations pertaining to the abovementioned viral outbreaks. These NHP models play a pivotal role in illuminating key aspects of disease dynamics, facilitating the development of effective countermeasures, and contributing significantly to our overall understanding of viral pathogenesis.

Plant virology in the 21st century in China: Recent advances and future directions.

Plant viruses are a group of intracellular pathogens that persistently threaten global food security. Significant advances in plant virology have been achieved by Chinese scientists over the last 20 years, including basic research and technologies for preventing and controlling plant viral diseases. Here, we review these milestones and advances, including the identification of new crop-infecting viruses, dissection of pathogenic mechanisms of multiple viruses, examination of multilayered interactions among viruses, their host plants, and virus-transmitting arthropod vectors, and in-depth interrogation of plant-encoded resistance and susceptibility determinants. Notably, various plant virus-based vectors have also been successfully developed for gene function studies and target gene expression in plants. We also recommend future plant virology studies in China.

Rev-Rev Response Element Activity Selection Bias at the Human Immunodeficiency Virus Transmission Bottleneck.

Background: Sexual transmission of human immunodeficiency virus (HIV) is inefficient and results in selection of viral variants based on incompletely understood factors. Functional variation in the Rev-Rev response element (RRE) regulatory axis of HIV affect replication kinetics and relative expression of viral proteins. We explored whether differences in this axis among viral isolates affect transmission fitness. Methods: HIV sequences were identified from nine female-to-male transmission pairs. Using a rapid flow cytometric assay, we analyzed Rev-RRE functional activity of primary isolates. Results: Rev-RRE activity was significantly lower in recipient viruses compared with corresponding donor viruses. In most transmission events, recipient virus Rev-RRE activity clustered at the extreme low end of the range of donor virus activity. Conclusions: These data indicate selection pressure on the Rev-RRE axis during female-to-male sexual transmission. Variation in Rev-RRE activity may permit viral adaptation to different fitness landscapes and could play an important role in HIV pathogenesis.

Replication-dead gammaherpesvirus vaccine protects against acute replication, reactivation from latency, and lethal challenge in mice.

Gammaherpesviruses (GHVs) are oncogenic viruses that establish lifelong infections and are significant causes of human morbidity and mortality. While several vaccine strategies to limit GHV infection and disease are in development, there are no FDA-approved vaccines for human GHVs. As a new approach to gammaherpesvirus vaccination, we developed and tested a replication-dead virus (RDV) platform, using murine gammaherpesvirus 68 (MHV68), a well-established mouse model for gammaherpesvirus pathogenesis studies and preclinical therapeutic evaluations. We employed codon-shuffling-based complementation to generate revertant-free RDV lacking expression of the essential replication and transactivator protein (RTA) encoded by ORF50 to arrest viral gene expression early after de novo infection. Inoculation with RDV-50.stop exposes the host to intact virion particles and leads to limited lytic gene expression in infected cells. Prime-boost vaccination of mice with RDV-50.stop elicited virus-specific neutralizing antibody and effector T cell responses in the lung and spleen. Vaccination with RDV-50.stop resulted in a near complete abolishment of virus replication in the lung 7 days post-challenge and virus reactivation from spleen 16 days post-challenge with WT MHV68. Ifnar1-/- mice, which lack the type I interferon receptor, exhibit severe disease upon infection with WT MHV68. RDV-50.stop vaccination of Ifnar1-/- mice prevented wasting and mortality upon challenge with WT MHV68. These results demonstrate that prime-boost vaccination with a GHV that is unable to undergo lytic replication offers protection against acute replication, reactivation, and severe disease upon WT virus challenge.

Unambiguous detection of SARS-CoV-2 subgenomic mRNAs with single-cell RNA sequencing.

Single-cell RNA sequencing (scRNA-Seq) studies have provided critical insight into the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19). scRNA-Seq library preparation methods and data processing workflows are generally designed for the detection and quantification of eukaryotic host mRNAs and not viral RNAs. Here, we compare different scRNA-Seq library preparation methods for their ability to quantify and detect SARS-CoV-2 RNAs with a focus on subgenomic mRNAs (sgmRNAs). We show that compared to 10X Genomics Chromium Next GEM Single Cell 3' (10X 3') libraries or 10X Genomics Chromium Next GEM Single Cell V(D)J (10X 5') libraries sequenced with standard read configurations, 10X 5' libraries sequenced with an extended length read 1 (R1) that covers both cell barcode and transcript sequence (termed "10X 5' with extended R1") increase the number of unambiguous reads spanning leader-sgmRNA junction sites. We further present a data processing workflow, single-cell coronavirus sequencing (scCoVseq), which quantifies reads unambiguously assigned to viral sgmRNAs or viral genomic RNA (gRNA). We find that combining 10X 5' with extended R1 library preparation/sequencing and scCoVseq data processing maximizes the number of viral UMIs per cell quantified by scRNA-Seq. Corresponding sgmRNA expression levels are highly correlated with expression in matched bulk RNA-Seq data sets quantified with established tools for SARS-CoV-2 analysis. Using this scRNA-Seq approach, we find that SARS-CoV-2 gene expression is highly correlated across individual infected cells, which suggests that the proportion of viral sgmRNAs remains generally consistent throughout infection. Taken together, these results and corresponding data processing workflow enable robust quantification of coronavirus sgmRNA expression at single-cell resolution, thereby supporting high-resolution studies of viral RNA processes in individual cells. IMPORTANCE Single-cell RNA sequencing (scRNA-Seq) has emerged as a valuable tool to study host-virus interactions, especially for coronavirus disease 2019 (COVID-19). Here we compare the performance of different scRNA-Seq library preparation methods and sequencing strategies to detect SARS-CoV-2 RNAs and develop a data processing workflow to quantify unambiguous sequence reads derived from SARS-CoV-2 genomic RNA and subgenomic mRNAs. After establishing a workflow that maximizes the detection of SARS-CoV-2 subgenomic mRNAs, we explore patterns of SARS-CoV-2 gene expression across cells with variable levels of total viral RNA, assess host gene expression differences between infected and bystander cells, and identify non-canonical and lowly abundant SARS-CoV-2 RNAs. The sequencing and data processing strategies developed here can enhance studies of coronavirus RNA biology at single-cell resolution and thereby contribute to our understanding of viral pathogenesis.

Controlling viral inflammatory lesions by rebalancing immune response patterns.

In this review, we discuss a variety of immune modulating approaches that could be used to counteract tissue-damaging viral immunoinflammatory lesions which typify many chronic viral infections. We make the point that in several viral infections the lesions can be largely the result of one or more aspects of the host response mediating the cell and tissue damage rather than the virus itself being directly responsible. However, within the reactive inflammatory lesions along with the pro-inflammatory participants there are also other aspects of the host response that may be acting to constrain the activity of the damaging components and are contributing to resolution. This scenario should provide the prospect of rebalancing the contributions of different host responses and hence diminish or even fully control the virus-induced lesions. We identify several aspects of the host reactions that influence the pattern of immune responsiveness and describe approaches that have been used successfully, mainly in model systems, to modulate the activity of damaging participants and which has led to lesion control. We emphasize examples where such therapies are, or could be, translated for practical use in the clinic to control inflammatory lesions caused by viral infections.

Limiting viral replication in hepatocytes alters Rift Valley fever virus disease manifestations.

Rift Valley fever virus (RVFV) causes mild to severe disease in humans and livestock. Outbreaks of RVFV have been reported throughout Africa and have spread outside Africa since 2000, calling for urgent worldwide attention to this emerging virus. RVFV directly infects the liver, and elevated transaminases are a hallmark of severe RVFV infection. However, the specific contribution of viral replication in hepatocytes to pathogenesis of RVFV remains undefined. To address this, we generated a recombinant miRNA-targeted virus, RVFVmiR-122, to limit hepatocellular replication. MicroRNAs are evolutionarily conserved non-coding RNAs that regulate mRNA expression by targeting them for degradation. RVFVmiR-122 includes an insertion of four target sequences of the liver-specific miR-122. In contrast to control RVFVmiR-184, which contains four target sequences of mosquito-specific miR-184, RVFVmiR-122 has restricted replication in vitro in primary mouse hepatocytes. RVFVmiR-122-infected C57BL/6 mice survived acute hepatitis and instead developed late-onset encephalitis. This difference in clinical outcome was eliminated in Mir-122 KO mice, confirming the specificity of the finding. Interestingly, C57BL/6 mice infected with higher doses of RVFVmiR-122 had a higher survival rate which was correlated with faster clearance of virus from the liver, suggesting a role for activation of host immunity in the phenotype. Together, our data demonstrate that miR-122 can specifically restrict the replication of RVFVmiR-122 in liver tissue both in vitro and in vivo, and this restriction alters the clinical course of disease following RVFVmiR-122 infection. IMPORTANCE Rift Valley fever virus (RVFV) is a hemorrhagic fever virus that causes outbreaks in humans and livestock throughout Africa and has spread to continents outside Africa since 2000. However, no commercial vaccine or treatment is currently available for human use against RVFV. Although the liver has been demonstrated as a key target of RVFV, the contribution of viral replication in hepatocytes to overall RVFV pathogenesis is less well defined. In this study we addressed this question by using a recombinant miRNA-targeted virus with restricted replication in hepatocytes. We gained a better understanding of how this individual cell type contributes to the development of disease caused by RVFV. Techniques used in this study provide an innovative tool to the RVFV field that could be applied to study the consequences of limited RVFV replication in other target cells.

Biophysical characterization of the cetacean morbillivirus haemagglutinin glycoprotein.

Cetacean morbillivirus (CeMV) is an enveloped, non-segmented, negative-stranded RNA virus that infects marine mammals, spreading across species and causing lethal disease outbreaks worldwide. Among the eight proteins encoded by the CeMV genome, the haemagglutinin (H) glycoprotein is responsible for the virus attachment to host cell receptors. CeMV H represents an attractive target for antiviral and diagnostic research, yet the elucidation of the molecular mechanisms underlying its role in infection and inter-species transmission was hampered thus far due to the unavailability of recombinant versions of the protein. Here we present the cloning, expression and purification of a recombinant CeMV H ectodomain (rH-ecto), providing an initial characterization of its biophysical and structural properties. Sodium dodecyl sulphate - polyacrylamide gel electrophoresis (PAGE) combined to Western blot analysis and periodic acid Schiff assay showed that CeMV rH-ecto is purifiable at homogeneity from insect cells as a secreted, soluble and glycosylated protein. Miniaturized differential scanning fluorimetry, Blue Native PAGE and size exclusion chromatography coupled to multiangle light scattering revealed that CeMV rH-ecto is globularly folded, thermally stable and exists in solution in the oligomeric states of dimer and multiple of dimers. Furthermore, negative stain electron microscopy single particle analysis allowed us to delineate a low-resolution molecular architecture of the CeMV rH-ecto dimer, which recapitulates native assemblies from other morbilliviral H proteins, such as those from measles virus and canine distemper virus. This set of experiments by orthogonal techniques validates the CeMV rH-ecto as an experimental model for future biochemical studies on its structure and functions.