Inhibition of mRNA nuclear export promotes SARS-CoV-2 pathogenesis.

The nonstructural protein 1 (Nsp1) of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) is a virulence factor that targets multiple cellular pathways to inhibit host gene expression and antiviral response. However, the underlying mechanisms of the various Nsp1-mediated functions and their contributions to SARS-CoV-2 virulence remain unclear. Among the targets of Nsp1 is the mRNA (messenger ribonucleic acid) export receptor NXF1-NXT1, which mediates nuclear export of mRNAs from the nucleus to the cytoplasm. Based on Nsp1 crystal structure, we generated mutants on Nsp1 surfaces and identified an acidic N-terminal patch that is critical for interaction with NXF1-NXT1. Photoactivatable Nsp1 probe reveals the RNA Recognition Motif (RRM) domain of NXF1 as an Nsp1 N-terminal binding site. By mutating the Nsp1 N-terminal acidic patch, we identified a separation-of-function mutant of Nsp1 that retains its translation inhibitory function but substantially loses its interaction with NXF1 and reverts Nsp1-mediated mRNA export inhibition. We then generated a recombinant (r)SARS-CoV-2 mutant on the Nsp1 N-terminal acidic patch and found that this surface is key to promote NXF1 binding and inhibition of host mRNA nuclear export, viral replication, and pathogenicity in vivo. Thus, these findings provide a mechanistic understanding of Nsp1-mediated mRNA export inhibition and establish the importance of this pathway in the virulence of SARS-CoV-2.

Evasion of NKG2D-mediated cytotoxic immunity by sarbecoviruses.

SARS-CoV-2 and other sarbecoviruses continue to threaten humanity, highlighting the need to characterize common mechanisms of viral immune evasion for pandemic preparedness. Cytotoxic lymphocytes are vital for antiviral immunity and express NKG2D, an activating receptor conserved among mammals that recognizes infection-induced stress ligands (e.g., MIC-A/B). We found that SARS-CoV-2 evades NKG2D recognition by surface downregulation of MIC-A/B via shedding, observed in human lung tissue and COVID-19 patient serum. Systematic testing of SARS-CoV-2 proteins revealed that ORF6, an accessory protein uniquely conserved among sarbecoviruses, was responsible for MIC-A/B downregulation via shedding. Further investigation demonstrated that natural killer (NK) cells efficiently killed SARS-CoV-2-infected cells and limited viral spread. However, inhibition of MIC-A/B shedding with a monoclonal antibody, 7C6, further enhanced NK-cell activity toward SARS-CoV-2-infected cells. Our findings unveil a strategy employed by SARS-CoV-2 to evade cytotoxic immunity, identify the culprit immunevasin shared among sarbecoviruses, and suggest a potential novel antiviral immunotherapy.

A measles-vectored vaccine candidate expressing prefusion-stabilized SARS-CoV-2 spike protein brought to phase I/II clinical trials: protection of African green monkeys from COVID-19 disease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and is responsible for the largest human pandemic in 100 years. Thirty-four vaccines are currently approved for use worldwide, and approximately 67% of the world population has received a complete primary series of one, yet countries are dealing with new waves of infections, variant viruses continue to emerge, and breakthrough infections are frequent secondary to waning immunity. Here, we evaluate a measles virus (MV)-vectored vaccine expressing a stabilized prefusion SARS-CoV-2 spike (S) protein (MV-ATU3-S2PΔF2A; V591) with demonstrated immunogenicity in mouse models (see companion article [J. Brunet, Z. Choucha, M. Gransagne, H. Tabbal, M.-W. Ku et al., J Virol 98:e01693-23, 2024, https://doi.org/10.1128/jvi.01693-23]) in an established African green monkey model of disease. Animals were vaccinated with V591 or the control vaccine (an equivalent MV-vectored vaccine with an irrelevant antigen) intramuscularly using a prime/boost schedule, followed by challenge with an early pandemic isolate of SARS-CoV-2 at 56 days post-vaccination. Pre-challenge, only V591-vaccinated animals developed S-specific antibodies that had virus-neutralizing activity as well as S-specific T cells. Following the challenge, V591-vaccinated animals had lower infectious virus and viral (v) RNA loads in mucosal secretions and stopped shedding virus in these secretions earlier. vRNA loads were lower in these animals in respiratory and gastrointestinal tract tissues at necropsy. This correlated with a lower disease burden in the lungs as quantified by PET/CT at early and late time points post-challenge and by pathological analysis at necropsy.IMPORTANCESevere acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the largest human pandemic in 100 years. Even though vaccines are currently available, countries are dealing with new waves of infections, variant viruses continue to emerge, breakthrough infections are frequent, and vaccine hesitancy persists. This study uses a safe and effective measles vaccine as a platform for vaccination against SARS-CoV-2. The candidate vaccine was used to vaccinate African green monkeys (AGMs). All vaccinated AGMs developed robust antigen-specific immune responses. After challenge, these AGMs produced less virus in mucosal secretions, for a shorter period, and had a reduced disease burden in the lungs compared to control animals. At necropsy, lower levels of viral RNA were detected in tissue samples from vaccinated animals, and the lungs of these animals lacked the histologic hallmarks of SARS-CoV-2 disease observed exclusively in the control AGMs.

Development of a dual channel detection system for pan-genotypic simultaneous quantification of hepatitis B and delta viruses.

Hepatitis B virus (HBV) infection remains a major public health problem and, in associated co-infection with hepatitis delta virus (HDV), causes the most severe viral hepatitis and accelerated liver disease progression. As a defective satellite RNA virus, HDV can only propagate in the presence of HBV infection, which makes HBV DNA and HDV RNA the standard biomarkers for monitoring the virological response upon antiviral therapy, in co-infected patients. Although assays have been described to quantify these viral nucleic acids in circulation independently, a method for monitoring both viruses simultaneously is not available, thus hampering characterization of their complex dynamic interactions. Here, we describe the development of a dual fluorescence channel detection system for pan-genotypic, simultaneous quantification of HBV DNA and HDV RNA through a one-step quantitative PCR. The sensitivity for both HBV and HDV is about 10 copies per microliter without significant interference between these two detection targets. This assay provides reliable detection for HBV and HDV basic research in vitro and in human liver chimeric mice. Preclinical validation of this system on serum samples from patients on or off antiviral therapy also illustrates a promising application that is rapid and cost-effective in monitoring HBV and HDV viral loads simultaneously.

Myc Dysregulation in Activated Macrophages Initiates Iron-Mediated Lipid Peroxidation that Fuels Type I Interferon and Compromises TB Resistance.

A quarter of human population is infected with Mycobacterium tuberculosis, but less than 10% of those infected develop clinical, mostly pulmonary, TB. To dissect mechanisms of susceptibility in immunocompetent individuals, we developed a genetically defined sst1-susceptible mouse model that uniquely reproduces a defining feature of human TB: development of necrotic lung lesions after infection with virulent Mtb. In this study, we explored the connectivity of the sst1-regulated pathways during prolonged macrophage activation with TNF. We determined that the aberrant response of the sst1-susceptible macrophages to TNF was primarily driven by conflicting Myc and antioxidant response pathways that resulted in a coordinated failure to properly sequester intracellular iron and activate ferroptosis inhibitor enzymes. Consequently, iron-mediated lipid peroxidation fueled IFNß superinduction and sustained the Type I Interferon (IFN-I) pathway hyperactivity that locked the sst1-susceptible macrophages in a state of unresolving stress and compromised their resistance to Mtb. The accumulation of the aberrantly activated, stressed, macrophages within granuloma microenvironment led to the local failure of anti-tuberculosis immunity and tissue necrosis. Our findings suggest a novel link between metabolic dysregulation in macrophages and susceptibility to TB, offering insights into potential therapeutic targets aimed at modulating macrophage function and improving TB control.

Resolution of SARS-CoV-2 infection in human lung tissues is driven by extravascular CD163+ monocytes.

The lung-resident immune mechanisms driving resolution of SARS-CoV-2 infection in humans remain elusive. Using mice co-engrafted with a genetically matched human immune system and fetal lung xenograft (fLX), we mapped the immunological events defining resolution of SARS-CoV-2 infection in human lung tissues. Viral infection is rapidly cleared from fLX following a peak of viral replication. Acute replication results in the emergence of cell subsets enriched in viral RNA, including extravascular inflammatory monocytes (iMO) and macrophage-like T-cells, which dissipate upon infection resolution. iMO display robust antiviral responses, are transcriptomically unique among myeloid lineages, and their emergence associates with the recruitment of circulating CD4+ monocytes. Consistently, mice depleted for human CD4+ cells but not CD3+ T-cells failed to robustly clear infectious viruses and displayed signatures of chronic infection. Our findings uncover the transient differentiation of extravascular iMO from CD4+ monocytes as a major hallmark of SARS-CoV-2 infection resolution and open avenues for unravelling viral and host adaptations defining persistently active SARS-CoV-2 infection.

Fecal microbiota transplanted from old mice promotes more colonic inflammation, proliferation, and tumor formation in azoxymethane-treated A/J mice than microbiota originating from young mice.

Aging is a strong risk factor for colorectal cancer (CRC). It is well established that gut microbial dysbiosis can play a role in the etiology of CRC. Although the composition of the gut microbial community changes with age and is reported to become more pro-inflammatory, it is unclear whether such changes are also pro-tumorigenic for the colon. To address this gap, we conducted fecal microbiota transplants (FMT) from young (DY, ~6 wk) and old (DO, ~72 wk) donor mice into young (8 wk) recipient mice that were pre-treated with antibiotics. After initiating tumorigenesis with azoxymethane, recipients were maintained for 19 wk during which time they received monthly FMT boosters. Compared to recipients of young donors (RY), recipients of old donors (RO) had an approximately 3-fold higher prevalence of histologically confirmed colon tumors (15.8 vs 50%, Chi2 P = .03), approximately 2-fold higher proliferating colonocytes as well as significantly elevated colonic IL-6, IL-1ß and Tnf-α. Transcriptomics analysis of the colonic mucosa revealed a striking upregulation of mitochondria-related genes in the RO mice, a finding corroborated by increased mitochondrial abundance. Amongst the differences in fecal microbiome observed between DY and DO mice, the genera Ruminoclostridium, Lachnoclostridium and Marvinbryantia were more abundant in DY mice while the genera Bacteroides and Akkermansia were more abundant in DO mice. Amongst recipients, Ruminoclostridium and Lachnoclostridium were higher in RY mice while Bacteroides was higher in RO mice. Differences in fecal microbiota were observed between young and old mice, some of which persisted upon transplant into recipient mice. Recipients of old donors displayed significantly higher colonic proliferation, inflammation and tumor abundance compared to recipients of young donors. These findings support an etiological role for altered gut microbial communities in the increased risk for CRC with increasing age and establishes that such risk can be transmitted between individuals.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encodes several proteins that inhibit host interferon responses. Among these, ORF6 antagonizes interferon signaling by disrupting nucleocytoplasmic trafficking through interactions with the nuclear pore complex components Nup98-Rae1. However, the roles and contributions of ORF6 during physiological infection remain unexplored. We assessed the role of ORF6 during infection using recombinant viruses carrying a deletion or loss-of-function (LoF) mutation in ORF6. ORF6 plays key roles in interferon antagonism and viral pathogenesis by interfering with nuclear import and specifically the translocation of IRF and STAT transcription factors. Additionally, ORF6 inhibits cellular mRNA export, resulting in the remodeling of the host cell proteome, and regulates viral protein expression. Interestingly, the ORF6:D61L mutation that emerged in the Omicron BA.2 and BA.4 variants exhibits reduced interactions with Nup98-Rae1 and consequently impairs immune evasion. Our findings highlight the role of ORF6 in antagonizing innate immunity and emphasize the importance of studying the immune evasion strategies of SARS-CoV-2.

GL7 ligand expression defines a novel subset of CD4+ TRM cells in lungs recovered from pneumococcus.

Streptococcus pneumoniae is the most common etiology of bacterial pneumonia, one of the leading causes of death in children and the elderly worldwide. During non-lethal infections with S. pneumoniae, lymphocytes accumulate in the lungs and protect against reinfection with serotype-mismatched strains. Cluster of differentiation CD4+ resident memory T (TRM) cells are known to be crucial for this protection, but the diversity of lung CD4+ TRM cells has yet to be fully delineated. We aimed to identify unique subsets and their contributions to lung immunity. After recovery from pneumococcal infections, we identified a distinct subset of CD4+ T cells defined by the phenotype CD11ahiCD69+GL7+ in mouse lungs. Phenotypic analyses for markers of lymphocyte memory and residence demonstrated that GL7+ T cells are a subset of CD4+ TRM cells. Functional studies revealed that unlike GL7- TRM subsets that were mostly (RAR-related Orphan Receptor gamma T) RORγT+, GL7+ TRM cells exhibited higher levels of (T-box expressed in T cells) T-bet and Gata-3, corresponding with increased synthesis of interferon-γ, interleukin-13, and interleukin-5, inherent to both T helper 1 (TH1) and TH2 functions. Thus, we propose that these cells provide novel contributions during pneumococcal pneumonia, serving as important determinants of lung immunity.

Pathogenicity of Lloviu and Bombali Viruses in Type I Interferon Receptor Knockout Mice.

Type I interferon receptor knockout (IFNAR-/-) mice are not able to generate a complete innate immune response; therefore, these mice are often considered to assess the pathogenicity of emerging viruses. We infected IFNAR-/- mice with a low or high dose of Lloviu virus (LLOV) or Bombali virus (BOMV) by the intranasal (IN) or intraperitoneal (IP) route and compared virus loads at early and late time points after infection. No signs of disease and no viral RNA were detected after IN infection regardless of LLOV dose. In contrast, IP infections resulted in increased viral loads in the high-dose LLOV and BOMV groups at the early time point. The low-dose LLOV and BOMV groups achieved higher viral loads at the late time point. However, there was 100% survival in all groups and no signs of disease. In conclusion, our results indicate a limited value of the IFNAR-/- mouse model for investigation of the pathogenicity of LLOV and BOMV.

Hepatic proinflammatory myeloid phenotypes are a hallmark of Ebola virus Kikwit pathogenesis in rhesus monkeys.

The liver is an early systemic target of Ebola virus (EBOV), but characterization beyond routine histopathology and viral antigen distribution is limited. We hypothesized Ebola virus disease (EVD) systemic proinflammatory responses would be reflected in temporally altered liver myeloid phenotypes. We utilized multiplex fluorescent immunohistochemistry (mfIHC), multispectral whole slide imaging, and image analysis to quantify molecular phenotypes of myeloid cells in the liver of rhesus macaques (Macaca mulatta; n = 21) infected with EBOV Kikwit. Liver samples included uninfected controls (n = 3), 3 days postinoculation (DPI; n = 3), 4 DPI (n = 3), 5 DPI (n = 3), 6 DPI (n = 3), and terminal disease (6-8 DPI; n = 6). Alterations in hepatic macrophages occurred at ≥ 5 DPI characterized by a 1.4-fold increase in CD68+ immunoreactivity and a transition from primarily CD14-CD16+ to CD14+CD16- macrophages, with a 2.1-fold decrease in CD163 expression in terminal animals compared with uninfected controls. An increase in the neutrophil chemoattractant and alarmin S100A9 occurred within hepatic myeloid cells at 5 DPI, followed by rapid neutrophil influx at ≥ 6 DPI. An acute rise in the antiviral myxovirus resistance protein 1 (MxA) occurred at ≥ 4 DPI, with a predilection for enhanced expression in uninfected cells. Distinctive expression of major histocompatibility complex (MHC) class II was observed in hepatocytes during terminal disease. Results illustrate that EBOV causes macrophage phenotype alterations as well as neutrophil influx and prominent activation of interferon host responses in the liver. Results offer insight into potential therapeutic strategies to prevent and/or modulate the host proinflammatory response to normalize hepatic myeloid functionality.

Cell culture systems for isolation of SARS-CoV-2 clinical isolates and generation of recombinant virus.

A simple and robust cell culture system is essential for generating authentic SARS-CoV-2 stocks for evaluation of viral pathogenicity, screening of antiviral compounds, and preparation of inactivated vaccines. Evidence suggests that Vero E6, a cell line commonly used in the field to grow SARS-CoV-2, does not support efficient propagation of new viral variants and triggers rapid cell culture adaptation of the virus. We generated a panel of 17 human cell lines overexpressing SARS-CoV-2 entry factors and tested their ability to support viral infection. Two cell lines, Caco-2/AT and HuH-6/AT, demonstrated exceptional susceptibility, yielding highly concentrated virus stocks. Notably, these cell lines were more sensitive than Vero E6 cells in recovering SARS-CoV-2 from clinical specimens. Further, Caco-2/AT cells provided a robust platform for producing genetically reliable recombinant SARS-CoV-2 through a reverse genetics system. These cellular models are a valuable tool for the study of SARS-CoV-2 and its continuously emerging variants.

Mouse-Adapted SARS-CoV-2 MA10 Strain Displays Differential Pulmonary Tropism and Accelerated Viral Replication, Neurodissemination, and Pulmonary Host Responses in K18-hACE2 Mice.

Several models were developed to study the pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as the in vivo efficacy of vaccines and therapeutics. Since wild-type mice are naturally resistant to infection by ancestral SARS-CoV-2 strains, several transgenic mouse models expressing human angiotensin-converting enzyme 2 (hACE2) were developed. An alternative approach has been to develop mouse-adapted SARS-CoV-2 strains. Here, we compared the clinical progression, viral replication kinetics and dissemination, pulmonary tropism, and host innate immune response dynamics between the mouse-adapted MA10 strain and its parental strain (USA-WA1/2020) following intranasal inoculation of K18-hACE2 mice, a widely used model. Compared to its parental counterpart, the MA10 strain induced earlier clinical decline with significantly higher viral replication and earlier neurodissemination. Importantly, the MA10 strain also showed a wider tropism, with infection of bronchiolar epithelia. While both SARS-CoV-2 strains induced comparable pulmonary cytokine/chemokine responses, many proinflammatory and monocyte-recruitment chemokines, such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IP-10/CXCL10, and MCP-1/CCL2, showed an earlier peak in MA10-infected mice. Furthermore, both strains induced a similar downregulation of murine Ace2, with only a transient downregulation of Tmprss2 and no alterations in hACE2 expression. Overall, these data demonstrate that in K18-hACE2 mice, the MA10 strain has a pulmonary tropism that more closely resembles SARS-CoV-2 tropism in humans (airways and pneumocytes) than its parental strain. Its rapid replication and neurodissemination and early host pulmonary responses can have a significant impact on the clinical outcomes of infection and are, therefore, critical features to consider for study designs using these strains and mouse model. IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, is still significantly impacting health care systems around the globe. Refined animal models are needed to study SARS-CoV-2 pathogenicity as well as efficacy of vaccines and therapeutics. In line with this, thorough evaluation of animal models and virus strains/variants are paramount for standardization and meaningful comparisons. Here, we demonstrated differences in replication dynamics between the Wuhan-like USA-WA1/2020 strain and the derivative mouse-adapted MA10 strain in K18-hACE2 mice. The MA10 strain showed accelerated viral replication and neurodissemination, differential pulmonary tropism, and earlier pulmonary innate immune responses. The observed differences allow us to better refine experimental designs when considering the use of the MA10 strain in the widely utilized K18-hACE2 murine model.

Spike and nsp6 are key determinants of SARS-CoV-2 Omicron BA.1 attenuation.

The SARS-CoV-2 Omicron variant is more immune evasive and less virulent than other major viral variants that have so far been recognized1-12. The Omicron spike (S) protein, which has an unusually large number of mutations, is considered to be the main driver of these phenotypes. Here we generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron (BA.1 lineage) in the backbone of an ancestral SARS-CoV-2 isolate, and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escaped vaccine-induced humoral immunity, mainly owing to mutations in the receptor-binding motif; however, unlike naturally occurring Omicron, it efficiently replicated in cell lines and primary-like distal lung cells. Similarly, in K18-hACE2 mice, although virus bearing Omicron S caused less severe disease than the ancestral virus, its virulence was not attenuated to the level of Omicron. Further investigation showed that mutating non-structural protein 6 (nsp6) in addition to the S protein was sufficient to recapitulate the attenuated phenotype of Omicron. This indicates that although the vaccine escape of Omicron is driven by mutations in S, the pathogenicity of Omicron is determined by mutations both in and outside of the S protein.

Role of spike in the pathogenic and antigenic behavior of SARS-CoV-2 BA.1 Omicron.

The recently identified, globally predominant SARS-CoV-2 Omicron variant (BA.1) is highly transmissible, even in fully vaccinated individuals, and causes attenuated disease compared with other major viral variants recognized to date. The Omicron spike (S) protein, with an unusually large number of mutations, is considered the major driver of these phenotypes. We generated chimeric recombinant SARS-CoV-2 encoding the S gene of Omicron in the backbone of an ancestral SARS-CoV-2 isolate and compared this virus with the naturally circulating Omicron variant. The Omicron S-bearing virus robustly escapes vaccine-induced humoral immunity, mainly due to mutations in the receptor binding motif (RBM), yet unlike naturally occurring Omicron, efficiently replicates in cell lines and primary-like distal lung cells. In K18-hACE2 mice, while Omicron causes mild, non-fatal infection, the Omicron S-carrying virus inflicts severe disease with a mortality rate of 80%. This indicates that while the vaccine escape of Omicron is defined by mutations in S, major determinants of viral pathogenicity reside outside of S.

FeMV is a cathepsin-dependent unique morbillivirus infecting the kidneys of domestic cats.

Feline morbillivirus (FeMV) is a recently discovered pathogen of domestic cats and has been classified as a morbillivirus in the Paramyxovirus family. We determined the complete sequence of FeMVUS5 directly from an FeMV-positive urine sample without virus isolation or cell passage. Sequence analysis of the viral genome revealed potential divergence from characteristics of archetypal morbilliviruses. First, the virus lacks the canonical polybasic furin cleavage signal in the fusion (F) glycoprotein. Second, conserved amino acids in the hemagglutinin (H) glycoprotein used by all other morbilliviruses for binding and/or fusion activation with the cellular receptor CD150 (signaling lymphocyte activation molecule [SLAM]/F1) are absent. We show that, despite this sequence divergence, FeMV H glycoprotein uses feline CD150 as a receptor and cannot use human CD150. We demonstrate that the protease responsible for cleaving the FeMV F glycoprotein is a cathepsin, making FeMV a unique morbillivirus and more similar to the closely related zoonotic Nipah and Hendra viruses. We developed a reverse genetics system for FeMVUS5 and generated recombinant viruses expressing Venus fluorescent protein from an additional transcription unit located either between the phospho-protein (P) and matrix (M) genes or the H and large (L) genes of the genome. We used these recombinant FeMVs to establish a natural infection and demonstrate that FeMV causes an acute morbillivirus-like disease in the cat. Virus was shed in the urine and detectable in the kidneys at later time points. This opens the door for long-term studies to address the postulated role of this morbillivirus in the development of chronic kidney disease.

Impact of SARS-CoV-2 ORF6 and its variant polymorphisms on host responses and viral pathogenesis.

We and others have previously shown that the SARS-CoV-2 accessory protein ORF6 is a powerful antagonist of the interferon (IFN) signaling pathway by directly interacting with Nup98-Rae1 at the nuclear pore complex (NPC) and disrupting bidirectional nucleo-cytoplasmic trafficking. In this study, we further assessed the role of ORF6 during infection using recombinant SARS-CoV-2 viruses carrying either a deletion or a well characterized M58R loss-of-function mutation in ORF6. We show that ORF6 plays a key role in the antagonism of IFN signaling and in viral pathogenesis by interfering with karyopherin(importin)-mediated nuclear import during SARS-CoV-2 infection both in vitro , and in the Syrian golden hamster model in vivo . In addition, we found that ORF6-Nup98 interaction also contributes to inhibition of cellular mRNA export during SARS-CoV-2 infection. As a result, ORF6 expression significantly remodels the host cell proteome upon infection. Importantly, we also unravel a previously unrecognized function of ORF6 in the modulation of viral protein expression, which is independent of its function at the nuclear pore. Lastly, we characterized the ORF6 D61L mutation that recently emerged in Omicron BA.2 and BA.4 and demonstrated that it is able to disrupt ORF6 protein functions at the NPC and to impair SARS-CoV-2 innate immune evasion strategies. Importantly, the now more abundant Omicron BA.5 lacks this loss-of-function polymorphism in ORF6. Altogether, our findings not only further highlight the key role of ORF6 in the antagonism of the antiviral innate immune response, but also emphasize the importance of studying the role of non-spike mutations to better understand the mechanisms governing differential pathogenicity and immune evasion strategies of SARS-CoV-2 and its evolving variants. ONE SENTENCE SUMMARY: SARS-CoV-2 ORF6 subverts bidirectional nucleo-cytoplasmic trafficking to inhibit host gene expression and contribute to viral pathogenesis.

Molecular clones of genetically distinct hepatitis B virus genotypes reveal distinct host and drug treatment responses.

Background & Aims: HBV exhibits wide genetic diversity with at least 9 genotypes (GTs), which differ in terms of prevalence, geographic distribution, natural history, disease progression, and treatment outcome. However, differences in HBV replicative capacity, gene expression, and infective capability across different GTs remain incompletely understood. Herein, we aimed to study these crucial aspects using newly constructed infectious clones covering the major HBV GTs. Methods: The replicative capacity of infectious clones covering HBV GTs A-E was analyzed in cell lines, primary hepatocytes and humanized mice. Host responses and histopathology induced by the different HBV GTs were characterized in hydrodynamically injected mice. Differences in treatment responses to entecavir and various HBV capsid inhibitors were also quantified across the different genetically defined GTs. Results: Patient-derived HBV infectious clones replicated robustly both in vitro and in vivo. GTs A and D induce more pronounced intrahepatic and proinflammatory cytokine responses which correlated with faster viral clearance. Notably, all 5 HBV clones robustly produced viral particles following transfection into HepG2 cells, and these particles were infectious in HepG2-NTCP cells, primary human hepatocytes and human chimeric mice. Notably, GT D virus exhibited higher infectivity than GTs A, B, C and E in vitro, although it was comparable to GT A and B in the human liver chimeric mice in vivo. HBV capsid inhibitors were more readily capable of suppressing HBV GTs A, B, D and E than C. Conclusions: The infectious clones described here have broad utility as genetic tools that can mechanistically dissect intergenotypic differences in antiviral immunity and pathogenesis and aid in HBV drug development and screening. Lay summary: The hepatitis B virus (HBV) is a major contributor to human morbidity and mortality. HBV can be categorized into a number of genotypes, based on their specific genetic make-up, of which 9 are well known. We isolated and cloned the genomes of 5 of these genotypes and used them to create valuable tools for future research on this clinically important virus.

Conversion of hepatitis B virus relaxed circular to covalently closed circular DNA is supported in murine cells.

Background & Aims: HBV has a narrow host restriction, with humans and chimpanzees representing the only known natural hosts. The molecular correlates of resistance in species that are commonly used in biomedical research, such as mice, are currently incompletely understood. Expression of human NTCP (hNTCP) in mouse hepatocytes enables HBV entry, but subsequently covalently closed circular (cccDNA) does not form in most murine cells. It is unknown if this blockade in cccDNA formation is due to deficiency in repair of relaxed circular DNA (rcDNA) to cccDNA. Methods: Here, we deployed both in vivo and in vitro virological and biochemical approaches to investigate if murine cells contain a complete set of repair factors capable of converting HBV rcDNA to cccDNA. Results: We demonstrate that HBV cccDNA does form in murine cell culture or in mice when recombinant rcDNA without a protein adduct is directly introduced into cells. We further show that the murine orthologues of core components in DNA lagging strand synthesis, required for the repair of rcDNA to cccDNA in human cells, can support this crucial step in the HBV life cycle. It is worth noting that recombinant HBV rcDNA substrates, either without a protein adduct or containing neutravidin to mimic HBV polymerase, were used in our study; it remains unclear if the HBV polymerase removal processes are the same in mouse and human cells. Conclusions: Collectively, our data suggest that the HBV life cycle is blocked post entry and likely before the repair stage in mouse cells, which yields critical insights that will aid in the construction of a mouse model with inbred susceptibility to HBV infection. Lay summary: Hepatitis B virus (HBV) is only known to infect humans and chimpanzees in nature. Mouse models are often used in modeling disease pathogenesis and preclinical research to assess the efficacy and safety of interventions before they are then tested in human participants. However, because mice are not susceptible to HBV infection it is difficult to accurately model human infection (and test potential treatments) in mouse models. Herein, we have shown that mice are able to perform a key step in the HBV life cycle, tightening the net around the possible reason why HBV can not efficiently infect and replicate in mice.

Progression and Dissemination of Pulmonary Mycobacterium Avium Infection in a Susceptible Immunocompetent Mouse Model.

Pulmonary infections caused by the group of nontuberculosis mycobacteria (NTM), Mycobacterium avium complex (MAC), are a growing public health concern with incidence and mortality steadily increasing globally. Granulomatous inflammation is the hallmark of MAC lung infection, yet reliable correlates of disease progression, susceptibility, and resolution are poorly defined. Unlike widely used inbred mouse strains, mice that carry the mutant allele at the genetic locus sst1 develop human-like pulmonary tuberculosis featuring well-organized caseating granulomas. We characterized pulmonary temporospatial outcomes of intranasal and left intrabronchial M. avium spp. hominissuis (M.av) induced pneumonia in B6.Sst1S mice, which carries the sst1 mutant allele. We utilized traditional semi-quantitative histomorphological evaluation, in combination with fluorescent multiplex immunohistochemistry (fmIHC), whole slide imaging, and quantitative digital image analysis. Followingintrabronchiolar infection with the laboratory M.av strain 101, the B6.Sst1S pulmonary lesions progressed 12-16 weeks post infection (wpi), with plateauing and/or resolving disease by 21 wpi. Caseating granulomas were not observed during the study. Disease progression from 12-16 wpi was associated with increased acid-fast bacilli, area of secondary granulomatous pneumonia lesions, and Arg1+ and double positive iNOS+/Arg1+ macrophages. Compared to B6 WT, at 16 wpi, B6.Sst1S lungs exhibited an increased area of acid-fast bacilli, larger secondary lesions with greater Arg1+ and double positive iNOS+/Arg1+ macrophages, and reduced T cell density. This morphomolecular analysis of histologic correlates of disease progression in B6.Sst1S could serve as a platform for assessment of medical countermeasures against NTM infection.