Efficient Viral Replication Research Articles

The coronavirus nsp14 exoribonuclease interface with the cofactor nsp10 is essential for efficient virus replication and enzymatic activity.

Coronaviruses (CoVs) encode non-structural proteins (nsp's) 1-16, which assemble to form replication-transcription complexes that function in viral RNA synthesis. All CoVs encode a proofreading 3'-5' exoribonuclease in non-structural protein 14 (nsp14-ExoN) that mediates proofreading and high-fidelity replication and is critical for other roles in replication and pathogenesis. The in vitro enzymatic activity of nsp14-ExoN is enhanced in the presence of the cofactor nsp10. We introduced alanine substitutions in nsp14 of murine hepatitis virus (MHV) at the nsp14-nsp10 interface and recovered mutant viruses with a range of impairments in replication and in vitro biochemical exonuclease activity. Two of these substitutions, nsp14 K7A and D8A, had impairments intermediate between wild type-MHV nsp14 and the known ExoN(-) D89A/E91A nsp14 catalytic inactivation mutant. All introduced nsp14-nsp10 interface alanine substitutions impaired in vitro exonuclease activity. Passage of the K7A and D8A mutant viruses selected second-site non-synonymous mutations in nsp14 associated with improved mutant virus replication and exonuclease activity. These results confirm the essential role of the nsp14-nsp10 interaction for efficient enzymatic activity and virus replication, identify proximal and long-distance determinants of nsp14-nsp10 interaction, and support targeting the nsp14-nsp10 interface for viral inhibition and attenuation.IMPORTANCECoronavirus replication requires assembly of a replication transcription complex composed of nsp's, including polymerase, helicase, exonuclease, capping enzymes, and non-enzymatic cofactors. The coronavirus nsp14 exoribonuclease mediates several functions in the viral life cycle including genomic and subgenomic RNA synthesis, RNA recombination, RNA proofreading and high-fidelity replication, and native resistance to many nucleoside analogs. The nsp-14 exonuclease activity in vitro requires the non-enzymatic cofactor nsp10, but the determinants and importance of the nsp14-nsp10 interactions during viral replication have not been defined. Here we show that for the coronavirus murine hepatitis virus, nsp14 residues at the nsp14-nsp10 interface are essential for efficient viral replication and in vitro exonuclease activity. These results shed new light on the requirements for protein interactions within the coronavirus replication transcription complex, and they may reveal novel non-active-site targets for virus inhibition and attenuation.

IFN- γ signaling is required for the efficient replication of murine hepatitis virus (MHV) strain JHM in the brains of infected mice.

Neurotropic viruses are a major public health concern as they can cause encephalitis and other severe brain diseases. Many of these viruses, including flaviviruses, herpesviruses, rhabdoviruses and alphaviruses enter the brain through the olfactory neuroepithelium (ONE) in the olfactory bulbs (OB). Due to the low percentage of encephalitis that occurs following these infections, it's thought that the OBs have specialized innate immune responses to eliminate viruses. Murine hepatitis virus strain JHM (JHMV) is a model coronavirus that causes severe encephalitis in mice and can access the brain through olfactory sensory neurons. We've shown that a JHMV Mac1-mutant virus, N1347A, has decreased replication and disease in the brains of mice. Here we further show that this virus replicates poorly in the OB. However, it is unknown which innate immune factors restrict N1347A replication in the OB. RNA seq analysis of infected olfactory bulbs showed that IFNγ was upregulated in the OB while IFN- β was barely detectable at 5 days post-infection. To determine if IFN-γ restricts JHMV N1347A replication, we utilized IFN-γ and IFN-γ receptor (IFN-γR) knockout (KO) mice. Surprisingly we found that JHMV WT and N1347A replicated very poorly in the OB and whole brains of both IFN-γ and IFN-γR KO mice following intranasal infection, though survival and weight loss were unaltered. Furthermore, we determined that microglia were the primary cells producing IFN-γ during the early stages of this infection. We conclude that IFN-γ is required for the efficient replication of JHMV in the brains of infected mice.

Influenza B viruses are more susceptible to high temperatures than influenza A viruses

Seasonal influenza is caused by two subtypes of influenza A virus (A/H1N1 and A/H3N2) and two lineages of influenza B viruses (B/Victoria-lineage and B/Yamagata-lineage). Seasonal influenza viruses replicate efficiently in the human upper respiratory tract, where the temperature is 33 °C. In this study, we investigated the susceptibility of seasonal influenza A and B viruses to different temperatures. We examined the differences in viral replication efficiency inside cultured cells and in infectious titre outside cultured cells at different temperatures (i.e., 33 °C, 37 °C, and 39 °C). We found that there were differences in temperature sensitivity between influenza A and B viruses, with influenza B viruses being more temperature sensitive. In addition, we found that cells cultured at 39 °C and infected with influenza B virus showed decreased expression of HA protein with receptor-binding activity on the cell surface. Our findings contribute to our understanding of the properties of seasonal influenza viruses.

Generation of a SARS-CoV-2-susceptible mouse model using adenovirus vector expressing human angiotensin-converting enzyme 2 driven by an elongation factor 1α promoter with leftward orientation

IntroductionTo analyze the molecular pathogenesis of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), a small animal model such as mice is needed: human angiotensin converting enzyme 2 (hACE2), the receptor of SARS-CoV-2, needs to be expressed in the respiratory tract of mice.MethodsWe conferred SARS-CoV-2 susceptibility in mice by using an adenoviral vector expressing hACE2 driven by an elongation factor 1α (EF1α) promoter with a leftward orientation.ResultsIn this model, severe pneumonia like human COVID-19 was observed in SARS-CoV-2-infected mice, which was confirmed by dramatic infiltration of inflammatory cells in the lung with efficient viral replication. An early circulating strain of SARS-CoV-2 caused the most severe weight loss when compared to SARS-CoV-2 variants such as Alpha, Beta and Gamma, although histopathological findings, viral replication, and cytokine expression characteristics were comparableDiscussionWe found that a distinct proteome of an early circulating strain infected lung characterized by elevated complement activation and blood coagulation, which were mild in other variants, can contribute to disease severity. Unraveling the specificity of early circulating SARS-CoV-2 strains is important in elucidating the origin of the pandemic.

SARS-CoV-2 N protein recruits G3BP to double membrane vesicles to promote translation of viral mRNAs

Ras-GTPase-activating protein SH3-domain-binding proteins (G3BP) are critical for the formation of stress granules (SGs) through their RNA- and ribosome-binding properties. SARS-CoV-2 nucleocapsid (N) protein exhibits strong binding affinity for G3BP and inhibits infection-induced SG formation soon after infection. To study the impact of the G3BP-N interaction on viral replication and pathogenesis in detail, we generated a mutant SARS-CoV-2 (RATA) that specifically lacks the G3BP-binding motif in the N protein. RATA triggers a stronger and more persistent SG response in infected cells, showing reduced replication across various cell lines, and greatly reduced pathogenesis in K18-hACE2 transgenic mice. At early times of infection, G3BP and WT N protein strongly colocalise with dsRNA and with non-structural protein 3 (nsp3), a component of the pore complex in double membrane vesicles (DMVs) from which nascent viral RNA emerges. Furthermore, G3BP-N complexes promote highly localized translation of viral mRNAs in the immediate vicinity of the DMVs and thus contribute to efficient viral gene expression and replication. In contrast, G3BP is absent from the DMVs in cells infected with RATA and translation of viral mRNAs is less efficient. This work provides a fuller understanding of the multifunctional roles of G3BP in SARS-CoV-2 infection.

In vivo HIV-1 nuclear condensates safeguard against cGAS and license reverse transcription

Entry of viral capsids into the nucleus induces the formation of biomolecular condensates called HIV-1 membraneless organelles (HIV-1-MLOs). Several questions remain about their persistence, in vivo formation, composition, and function. Our study reveals that HIV-1-MLOs persisted for several weeks in infected cells, and their abundance correlated with viral infectivity. Using an appropriate animal model, we show that HIV-1-MLOs were formed in vivo during acute infection. To explore the viral structures present within these biomolecular condensates, we used a combination of double immunogold labeling, electron microscopy and tomography, and unveiled a diverse array of viral core structures. Our functional analyses showed that HIV-1-MLOs remained stable during treatment with a reverse transcriptase inhibitor, maintaining the virus in a dormant state. Drug withdrawal restored reverse transcription, promoting efficient virus replication akin to that observed in latently infected patients on antiretroviral therapy. However, when HIV-1 MLOs were deliberately disassembled by pharmacological treatment, we observed a complete loss of viral infectivity. Our findings show that HIV-1 MLOs shield the final reverse transcription product from host immune detection.

Coronavirus nucleocapsid protein enhances the binding of p-PKCα to RACK1: Implications for inhibition of nucleocytoplasmic trafficking and suppression of the innate immune response.

The hallmark of coronavirus infection lies in its ability to evade host immune defenses, a process intricately linked to the nuclear entry of transcription factors crucial for initiating the expression of antiviral genes. Central to this evasion strategy is the manipulation of the nucleocytoplasmic trafficking system, which serves as an effective target for the virus to modulate the expression of immune response-related genes. In this investigation, we discovered that infection with the infectious bronchitis virus (IBV) dynamically impedes the nuclear translocation of several transcription factors such as IRF3, STAT1, STAT2, NF-κB p65, and the p38 MAPK, leading to compromised transcriptional induction of key antiviral genes such as IFNβ, IFITM3, and IL-8. Further examination revealed that during the infection process, components of the nuclear pore complex (NPC), particularly FG-Nups (such as NUP62, NUP153, NUP42, and TPR), undergo cytosolic dispersion from the nuclear envelope; NUP62 undergoes phosphorylation, and NUP42 exhibits a mobility shift in size. These observations suggest a disruption in nucleocytoplasmic trafficking. Screening efforts identified the IBV nucleocapsid (N) protein as the agent responsible for the cytoplasmic distribution of FG-Nups, subsequently hindering the nuclear entry of transcription factors and suppressing the expression of antiviral genes. Interactome analysis further revealed that the IBV N protein interacts with the scaffold protein RACK1, facilitating the recruitment of activated protein kinase C alpha (p-PKCα) to RACK1 and relocating the p-PKCα-RACK1 complex to the cytoplasm. These observations are conserved across diverse coronaviruses N proteins. Concurrently, the presence of both RACK1 and PKCα/β proved essential for the phosphorylation and cytoplasmic dispersion of NUP62, the suppression of antiviral cytokine expression, and efficient virus replication. These findings unveil a novel, highly effective, and evolutionarily conserved mechanism.

Seoul orthohantavirus evades innate immune activation by reservoir endothelial cells.

Pathogenic hantaviruses are maintained world-wide within wild, asymptomatic rodent reservoir hosts, with increasingly frequent human spillover infections resulting in severe hemorrhagic fever or cardio-pulmonary disease. With no approved therapeutics or vaccines, research has, until recently, focused on understanding the drivers of immune-mediated pathogenesis. An emerging body of work is now investigating the mechanisms that allow for asymptomatic, persistent infections of mammalian reservoir hosts with highly pathogenic RNA viruses. Despite limited experimental data, several hypotheses have arisen to explain limited or absent disease pathology in reservoir hosts. In this study, we directly tested two leading hypotheses: 1) that reservoir host cells induce a generally muted response to viral insults, and 2) that these viruses employ host-specific mechanisms of innate antiviral antagonism to limit immune activation in reservoir cells. We demonstrate that, in contrast to human endothelial cells which mount a robust antiviral and inflammatory response to pathogenic hantaviruses, primary Norway rat endothelial cells do not induce antiviral gene expression in response to infection with their endemic hantavirus, Seoul orthohantavirus (SEOV). Reservoir rat cells do, however, induce strong innate immune responses to exogenous stimulatory RNAs, type I interferon, and infection with Hantaan virus, a closely related hantavirus for which the rat is not a natural reservoir. We also find that SEOV-infected rat endothelial cells remain competent for immune activation induced by exogenous stimuli or subsequent viral infection. Importantly, these findings support an alternative model for asymptomatic persistence within hantavirus reservoir hosts: that efficient viral replication within reservoir host cells may prevent the exposure of critical motifs for cellular antiviral recognition and thus limits immune activation that would otherwise result in viral clearance and/or immune-mediated disease. Defining the mechanisms that allow for infection tolerance and persistence within reservoir hosts will reveal novel strategies for viral countermeasures against these highly pathogenic zoonotic threats.

Chicken ANP32A-independent replication of highly pathogenic avian influenza viruses potentially leads to mammalian adaptation-related amino acid substitutions in viral PB2 and PA proteins.

Acidic nuclear phosphoprotein 32 family member A (ANP32A) is an important host factor that supports the efficient replication of avian influenza viruses (AIVs). To develop an antiviral strategy against Gs/Gd-lineage H5 highly pathogenic avian influenza (HPAI) viruses in chickens, we established chicken ANP32-knockout (chANP32A-KO) DF-1 cells and evaluated their antiviral efficacy through in vitro validation. The replication of all HPAI viruses tested in chANP32A-KO cells was significantly lower compared to that of wild-type DF-1 cells. However, when HPAI strains A/mountain hawk-eagle/Kumamoto/1/2007 (H5N1; MHE) and A/chicken/Aichi/2/2011 (H5N1; H5Aichi) were passed in chANP32A-KO cells, mutant viruses were generated, which exhibited comparable replication levels in both chANP32A-KO and wild-type DF-1 cells. Sequence analysis revealed that mammalian-adaptive amino acid mutations PB2_D256G and PA_T97I were present in the MHE mutant virus, and the PB2_E627K mutation was identified in the H5Aichi mutant virus. These mutations have also been reported to enhance the polymerase activity of AIVs in mammalian cells; however, the minigenome assay in the present study showed that the polymerase activity of mutant viruses in chANP32A-KO cells was not restored to levels comparable to those in wild-type DF-1 cells. These findings suggest that ANP32A-independent viral replication may induce amino acid substitutions associated with mammalian adaptation in AIVs. They also imply that the high efficiency of viral replication mediated by these amino acid mutations may not result from enhanced polymerase activity but rather involve other undefined mechanisms.IMPORTANCEDuring the host-switching of avian influenza viruses (AIVs) to mammalian hosts, introducing adaptive mutations into viral proteins is essential to ensure optimal functionality through virus-host protein interactions in mammalian cells. However, the mechanisms leading to adaptive mutations in viral proteins remain unclear. Among several host proteins that promote viral growth, acidic nuclear phosphoprotein 32 family member A (ANP32A) is known to be an important factor for efficient viral replication. Here, we generated mutant highly pathogenic avian influenza viruses capable of ANP32A-independent replication in a chicken-derived cell line. We demonstrated that several amino acid mutations found in the mutant viruses correspond to those associated with the mammalian adaptation of AIVs. These results suggest that ANP32A-independent viral replication is one of the mechanisms for introducing amino acid mutations that are reportedly involved in the mammalian adaptation of AIVs.

Establishment of an Immortalized Porcine Alveolar Macrophage Cell Line That Supports Efficient Replication of Porcine Reproductive and Respiratory Syndrome Viruses.

Porcine reproductive and respiratory syndrome (PRRS), which is caused by the porcine reproductive and respiratory syndrome virus (PRRSV), has a significant impact on the global pork industry. It results in reproductive failure in sows and respiratory issues in pigs of all ages. Despite the availability of vaccines, controlling the PRRSV remains challenging, partly owing to the limitations of cell culture systems. Current methods largely rely on primary porcine alveolar macrophages (PAMs), which must be harvested from piglets and have limited proliferative capacity. Although some simian cell lines support PRRSV replication, their inability to express porcine CD163, which is a key receptor for PRRSV entry, compromises their effectiveness, because the virus replicates differently in these non-target cells. To address these issues, we established an immortalized PAM cell line, PAM-T43, using SV40 large T antigen for immortalization and porcine serum as a culture supplement. PAM-T43 cells maintain essential macrophage functions, including CD163 expression and phagocytic activity, and exhibit high sensitivity to the PRRSV, efficiently supporting viral replication. This novel cell line offers significant potential for advancing PRRSV research, particularly in vaccine development and field strain isolation, by overcoming the limitations of current systems.

Proteome profiling of polyomavirus nuclear replication centers using iPOND.

Polyomaviruses (PyVs) cause diverse diseases in a variety of mammalian hosts. During the life cycle, PyVs recruit nuclear host factors to viral genomes to facilitate replication and transcription. While host factors involved in DNA replication, DNA damage sensing and repair, and cell cycle regulation have been observed to bind PyV DNA, the complete set of viral and host proteins comprising the PyV replisome remains incompletely characterized. Here, the iPOND-MS technique (Isolation of Proteins on Nascent DNA coupled with Mass Spectrometry) was used to identify the proteome bound to murine PyV (MuPyV) DNA immediately following synthesis and 2 hours post-synthesis. Several novel MuPyV DNA interactors were identified on newly synthesized viral DNA (vDNA), including MCM complex members, DNA primase, DNA polymerase alpha, DNA ligase, and replication factor C. Though displaying partial overlap, the host and viral proteins bound to MuPyV DNA 2 hours post-synthesis lacked many of the replication proteins found on newly synthesized vDNA. These data help distinguish between the host factors critical for MuPyV DNA replication and those involved in downstream processing.IMPORTANCEPolyomaviruses are the causative agents of serious diseases in humans, including progressive multifocal leukoencephalopathy (PML), BK virus nephropathy, and Merkel cell carcinoma. The exact mechanisms by which the virus replicates, and which host cell proteins are required, are incompletely characterized. Identifying the host proteins necessary for efficient viral replication in the cell may reveal targets for downstream targets that may suppress viral replication in vivo.

A cytoplasmic form of EHMT1N methylates viral proteins to enable inclusion body maturation and efficient viral replication.

Protein lysine methyltransferases (PKMTs) methylate histone and non-histone proteins to regulate biological outcomes such as development and disease including viral infection. While PKMTs have been extensively studied for modulating the antiviral responses via host gene regulation, their role in methylation of proteins encoded by viruses and its impact on host-pathogen interactions remain poorly understood. In this study, we discovered distinct nucleo-cytoplasmic form of euchromatic histone methyltransferase 1 (EHMT1N/C), a PKMT, that phase separates into viral inclusion bodies (IBs) upon cytoplasmic RNA-virus infection (Sendai Virus). EHMT1N/C interacts with cytoplasmic EHMT2 and methylates SeV-Nucleoprotein upon infection. Elevated nucleoprotein methylation during infection correlated with coalescence of small IBs into large mature platforms for efficient replication. Inhibition of EHMT activity by pharmacological inhibitors or genetic depletion of EHMT1N/C reduced the size of IBs with a concomitant reduction in replication. Additionally, we also found that EHMT1 condensation is not restricted to SeV alone but was also seen upon pathogenic RNA viral infections caused by Chandipura and Dengue virus. Collectively, our work elucidates a new mechanism by which cytoplasmic EHMT1 acts as proviral host factor to regulate host-pathogen interaction.

PARP7i Clinical Candidate RBN-2397 Exerts Antiviral Activity by Modulating Interferon-β Associated Innate Immune Response in Macrophages.

Polyadenosine diphosphate-ribose polymerase 7 (PARP7) acts as a suppressor of the type I interferon (IFN) signaling pathway via suppressing TANK-binding protein 1 (TBK1). Research study indicates that inhibition of PARP7 could potentially regulate tumor immunity. However, the effect of PARP7 inhibition on innate antiviral immunity in macrophages as well as the underlying mechanism have not been demonstrated else well. We report herein that PARP7 inhibitor clinical candidate RBN-2397 could augment type I interferon (IFN-I) production in macrophages by elevating retinoic acid-inducible gene I (RIG-I) and stimulator of interferon genes (STING) signaling pathways. Treatment with RBN-2397 leads to increased pattern recognition ligands-induced interferon-β production in primary bone marrow-derived macrophages (BMDM) and RAW264.7 cells. Additionally, RBN-2397 suppresses viral replication efficiency in macrophages infected by vesicular stomatitis virus (VSV) and amplifies the expression of interferon-stimulated chemokine genes (ISGs). Mechanistically, RBN-2397 promotes TBK1 phosphorylation, consequently leading to the amplified activation of RIG-I and STING signaling pathways. Furthermore, RBN-2397 enhances the phosphorylation of signal transducer and activator of transcription 1 (STAT1) and STAT2 induced by IFN-α/β and the expression of chemokine genes in macrophages in response to IFN stimulation. In vivo experiments demonstrated that RBN-2397 enhances innate antiviral immunity in mice infected with VSV, resulting in increased serum IFN-β levels, reduced viral loads, and alleviated pulmonary inflammatory responses of the VSV-infected mice. In conclusion, our findings highlight the potential of RBN-2397 as a promising antiviral therapeutic agent for enhancing the IFN-relative antiviral immune defense in host.

Circadian regulation of dengue virus transmission and replication: insights into vector activity and viral dynamics.

Dengue fever, caused by dengue virus, poses a significant global health challenge, particularly in tropical regions where Aedes aegypti serves as the primary vector. The circadian clock in Aedes aegypti governs key behavioral and physiological processes, including activity patterns, feeding behaviors, and susceptibility to dengue virus infection. This article explores the influence of circadian rhythms on the mosquito's ability to transmit dengue virus, emphasizing how the circadian regulation of gene expression, immune responses, and lipid metabolism in the mosquito vector creates temporal windows that affect viral replication efficiency.

Increased viperin expression induced by avian infectious bronchitis virus inhibits viral replication by restricting cholesterol synthesis: an in vitro study

With the emergence of new variant strains resulting from high mutation rates and genome recombination, avian infectious bronchitis virus (IBV) has caused significant economic losses to the poultry industry worldwide. Little is known about the underlying mechanisms of IBV-host interactions, particularly how IBV utilizes host metabolic pathways for efficient viral replication and transmission. In the present study, the effects of the cell membrane, viral envelope membrane, and viperin-mediated cholesterol synthesis on IBV replication were explored. Our results revealed significant increase in cholesterol levels and the expression of viperin after IBV infection. Acute cholesterol depletion in the cell membrane and viral envelope membrane by treating cells with methyl-β-cyclodextrin (MβCD) obviously inhibited IBV replication; thereafter, replenishment of the cell membrane with cholesterol successfully restored viral replication, and direct addition of exogenous cholesterol to the cell membrane significantly promoted IBV infection during the early stages of infection. In addition, overexpression of viperin effectively suppressed cholesterol synthesis, as well as IBV replication, whereas knockdown of viperin (gene silencing with siRNA targeting viperin, siViperin) significantly increased IBV replication and cholesterol levels, whereas supplementation with exogenous cholesterol to viperin-transfected cells markedly restored viral replication. In conclusion, the increase in viperin induced by IBV infection plays an important role in IBV replication by affecting cholesterol production, providing a theoretical basis for understanding the pathogenesis of IBV and discovering new potential antiviral targets.

The coronavirus nsp14 exoribonuclease interface with the cofactor nsp10 is essential for efficient virus replication and enzymatic activity.

Coronavirus replication requires assembly of a replication transcription complex composed of nonstructural proteins (nsp), including polymerase, helicase, exonuclease, capping enzymes, and non-enzymatic cofactors. The coronavirus nsp14 exoribonuclease mediates several functions in the viral life cycle including genomic and subgenomic RNA synthesis, RNA recombination, RNA proofreading and high-fidelity replication, and native resistance to many nucleoside analogs. The nsp-14 exonuclease activity in vitro requires the non-enzymatic co-factor nsp10, but the determinants and importance the nsp14-10 interactions during viral replication have not been defined. Here we show that for the coronavirus murine hepatitis virus, nsp14 residues at the nsp14-10 interface are essential for efficient viral replication and in vitro exonuclease activity. These results shed new light on the requirements for protein interactions within the coronavirus replication transcription complex, and they may reveal novel non active-site targets for virus inhibition and attenuation.

A novel GFP-based strategy to quantitate cellular spatial associations in HSV-1 viral pathogenesis.

Periodic reactivation of herpes simplex virus type 1 (HSV-1) triggers immune responses that result in corneal scarring (CS), known as herpes stromal keratitis (HSK). Despite considerable research, fully understanding HSK and eliminating it remains challenging due to a lack of comprehensive analysis of HSV-1-infected immune cells in both corneas and trigeminal ganglia (TG). We engineered a recombinant HSV-1 expressing green fluorescent protein (GFP) in the virulent McKrae virus strain that does not require corneal scarification for efficient virus replication (GFP-McKrae). Next-generation sequencing (NGS) analysis, along with in vitro and in vivo assays, showed that GFP-McKrae virus was similar to WT-McKrae virus. Furthermore, corneal cells infected with GFP-McKrae were quantitatively analyzed using image mass cytometry (IMC). The single-cell reconstruction data generated cellular maps of corneas based on the expression of 25 immune cell markers in GFP-McKrae-infected mice. Corneas from mock control mice showed the presence of T cells and macrophages, whereas corneas from GFP-McKrae-infected mice on days 3 and 5 post-infection (PI) exhibited increased immune cells. Notably, on day 3 PI, increased GFP expression was observed in closely situated clusters of DCs, macrophages, and epithelial cells. By day 5 PI, macrophages and T cells became prominent. Finally, immunostaining methods detected HSV-1 or GFP and gD proteins in latently infected TG. This study presents a valuable strategy for identifying cellular spatial associations in viral pathogenesis and holds promise for future therapeutic applications.IMPORTANCEThe goal of this study was to establish quantitative approaches to analyze immune cell markers in HSV-1-infected intact corneas and trigeminal ganglia from primary and latently infected mice. This allowed us to define spatial and temporal interactions between specific immune cells and their potential roles in virus replication and latency. To accomplish this important goal, we took advantage of the utility of GFP-McKrae virus as a valuable research tool while also highlighting its potential to uncover previously unrecognized cell types that play pivotal roles in HSV-1 replication and latency. Such insights will pave the way for developing targeted therapeutic approaches to tackle HSV-1 infections more effectively.

The NS1 protein of contemporary West African Zika virus potentiates viral replication and reduces innate immune activation.

Mosquito-borne Zika virus (ZIKV) from sub-Saharan Africa has recently gained attention due to its epidemic potential and its capacity to be highly teratogenic. To improve our knowledge on currently circulating strains of African ZIKV, we conducted protein sequence alignment and identified contemporary West Africa NS1 (NS1CWA) protein as a highly conserved viral protein. Comparison of NS1CWA with the NS1 of the historical African ZIKV strain MR766 (NS1MR766), revealed seven amino acid substitutions. The effects of NS1 mutations on protein expression, virus replication, and innate immune activation were assessed in human cells using recombinant NS1 proteins and a chimeric viral clone MR766 with NS1CWA replacing NS1MR766. Our data indicated higher secretion efficiency of NS1CWA compared to NS1MR766 associated with a change in subcellular distribution. A chimeric MR766 virus with NS1CWA instead of authentic protein displayed a greater viral replication efficiency, leading to more pronounced cell death compared to parental virus. Enhanced viral growth was associated with reduced activation of innate immunity. Our data raise questions of the importance of NS1 protein in the pathogenicity of contemporary ZIKV from sub-Saharan Africa and point to differences within viral strains of African lineage.

Impact of equine herpesvirus-1 ORF15 (EUL45) on viral replication and neurovirulence

Equine herpesvirus 1 (EHV-1) causes respiratory illness, fetal loss, perinatal mortality, and myeloencephalopathy. This study investigated ORF15's impact on virus infectivity and neurovirulence. The Ab4p neurovirulent strain of EHV1 was used as a backbone to create Ab4p attB, Ab4p∆ORF15, and Ab4p∆ORF15R chimeras via BAC DNA transfection into RK-13 cells. Viral growth kinetics, plaque size, transcription, and growth were assessed in MDBK cells, mouse neurons, and fetal equine brain cells. Neurovirulence was evaluated post-intranasal inoculation into male CBA/N1 SPF mice, measuring signs, virus titers, and histopathological changes. Deletion of EUL45 (Ab4p-∆EUL45) reduced viral replication efficiency, resulting in decreased release and smaller plaques. EUL45 deletion also upregulated neighbouring genes (EUL46 and EUL44). Ab4p-∆EUL45 exhibited reduced virulence and poor growth in neural cells compared to wild-type viruses. This study sheds light on EUL45's role in EHV-1, viral replication, and regulation of EUL46 and EUL44 expression, suggesting potential as a vaccine candidate.

Viral shedding and environmental dispersion of two clade 2.3.4.4b H5 high pathogenicity avian influenza viruses in experimentally infected mule ducks: implications for environmental sampling

High pathogenicity avian influenza viruses (HPAIVs) have caused major epizootics in recent years, with devastating consequences for poultry and wildlife worldwide. Domestic and wild ducks can be highly susceptible to HPAIVs, and infection leads to efficient viral replication and massive shedding (i.e., high titres for an extended time), contributing to widespread viral dissemination. Importantly, ducks are known to shed high amounts of virus in the earliest phase of infection, but the dynamics and impact of environmental contamination on the epidemiology of HPAIV outbreaks are poorly understood. In this study, we monitored mule ducks experimentally infected with two H5N8 clade 2.3.4.4b goose/Guangdong HPAIVs sampled in France in 2016–2017 and 2020–2021 epizootics. We investigated viral shedding dynamics in the oropharynx, cloaca, conjunctiva, and feathers; bird-to-bird viral transmission; and the role of the environment in viral spread and as a source of samples for early detection and surveillance. Our findings showed that viral shedding started before the onset of clinical signs, i.e., as early as 1 day post-inoculation (dpi) or post-contact exposure, peaked at 4 dpi, and lasted for up to 14 dpi. The detection of viral RNA in aerosols, dust, and water samples mirrored viral shedding dynamics, and viral isolation from these environmental samples was successful throughout the experiment. Our results confirm that mule ducks can shed high HPAIV titres through the four excretion routes tested (oropharyngeal, cloacal, conjunctival, and feather) while being asymptomatic and that environmental sampling could be a non-invasive tool for early viral RNA detection in HPAIV-infected farms.