Metagenomics-enabled reverse-genetics assembly and characterization of myotis bat morbillivirus.

Morbilliviruses are among the most contagious viral pathogens of mammals. Although previous metagenomic surveys have identified morbillivirus sequences in bats, full-length morbilliviruses from bats are limited. Here we characterize the myotis bat morbillivirus (MBaMV) from a bat surveillance programme in Brazil, whose full genome was recently published. We demonstrate that the fusion and receptor binding protein of MBaMV utilize bat CD150 and not human CD150, as an entry receptor in a mammalian cell line. Using reverse genetics, we produced a clone of MBaMV that infected Vero cells expressing bat CD150. Electron microscopy of MBaMV-infected cells revealed budding of pleomorphic virions, a characteristic morbillivirus feature. MBaMV replication reached 103-105 plaque-forming units ml-1 in human epithelial cell lines and was dependent on nectin-4. Infection of human macrophages also occurred, albeit 2-10-fold less efficiently than measles virus. Importantly, MBaMV is restricted by cross-neutralizing human sera elicited by measles, mumps and rubella vaccination and is inhibited by orally bioavailable polymerase inhibitors in vitro. MBaMV-encoded P/V genes did not antagonize human interferon induction. Finally, we show that MBaMV does not cause disease in Jamaican fruit bats. We conclude that, while zoonotic spillover into humans may theoretically be plausible, MBaMV replication would probably be controlled by the human immune system.

Alpha-1-antitrypsin and its variant-dependent role in COVID-19 pathogenesis.

Rationale: SARS-CoV-2 entry into host cells is facilitated by endogenous and exogenous proteases that proteolytically activate the spike glycoprotein and antiproteases inhibiting this process. Understanding the key actors in viral entry is crucial for advancing knowledge of virus tropism, pathogenesis, and potential therapeutic targets. Objectives: We aimed to investigate the role of naïve serum and alpha-1-antitrypsin (AAT) in inhibiting protease-mediated SARS-CoV-2 entry and explore the implications of AAT deficiency on susceptibility to different SARS-CoV-2 variants. Findings: Our study demonstrates that naïve serum exhibits significant inhibition of SARS-CoV-2 entry, with AAT identified as the major serum protease inhibitor potently restricting entry. Using pseudoparticles, replication-competent pseudoviruses, and authentic SARS-CoV-2, we show that AAT inhibition occurs at low concentrations compared with those in serum and bronchoalveolar tissues, suggesting physiological relevance. Furthermore, sera from subjects with an AAT-deficient genotype show reduced ability to inhibit entry of both Wuhan-Hu-1 (WT) and B.1.617.2 (Delta) but exhibit no difference in inhibiting B.1.1.529 (Omicron) entry. Conclusions: AAT may have a variant-dependent therapeutic potential against SARS-CoV-2. Our findings highlight the importance of further investigating the complex interplay between proteases, antiproteases, and spike glycoprotein activation in SARS-CoV-2 and other respiratory viruses to identify potential therapeutic targets and improve understanding of disease pathogenesis.

Human Dectin-1 deficiency impairs macrophage-mediated defense against phaeohyphomycosis.

Subcutaneous phaeohyphomycosis typically affects immunocompetent individuals following traumatic inoculation. Severe or disseminated infection can occur in CARD9 deficiency or after transplantation, but the mechanisms protecting against phaeohyphomycosis remain unclear. We evaluated a patient with progressive, refractory Corynespora cassiicola phaeohyphomycosis and found that he carried biallelic deleterious mutations in CLEC7A encoding the CARD9-coupled, ß-glucan-binding receptor, Dectin-1. The patient's PBMCs failed to produce TNF-α and IL-1ß in response to ß-glucan and/or C. cassiicola. To confirm the cellular and molecular requirements for immunity against C. cassiicola, we developed a mouse model of this infection. Mouse macrophages required Dectin-1 and CARD9 for IL-1ß and TNF-α production, which enhanced fungal killing in an interdependent manner. Deficiency of either Dectin-1 or CARD9 was associated with more severe fungal disease, recapitulating the human observation. Because these data implicated impaired Dectin-1 responses in susceptibility to phaeohyphomycosis, we evaluated 17 additional unrelated patients with severe forms of the infection. We found that 12 out of 17 carried deleterious CLEC7A mutations associated with an altered Dectin-1 extracellular C-terminal domain and impaired Dectin-1-dependent cytokine production. Thus, we show that Dectin-1 and CARD9 promote protective TNF-α- and IL-1ß-mediated macrophage defense against C. cassiicola. More broadly, we demonstrate that human Dectin-1 deficiency may contribute to susceptibility to severe phaeohyphomycosis by certain dematiaceous fungi.

The NF-κB Transcriptional Footprint Is Essential for SARS-CoV-2 Replication.

SARS-CoV-2, the etiological agent of COVID-19, is characterized by a delay in type I interferon (IFN-I)-mediated antiviral defenses alongside robust cytokine production. Here, we investigate the underlying molecular basis for this imbalance and implicate virus-mediated activation of NF-κB in the absence of other canonical IFN-I-related transcription factors. Epigenetic and single-cell transcriptomic analyses show a selective NF-κB signature that was most prominent in infected cells. Disruption of NF-κB signaling through the silencing of the NF-κB transcription factor p65 or p50 resulted in loss of virus replication that was rescued upon reconstitution. These findings could be further corroborated with the use of NF-κB inhibitors, which reduced SARS-CoV-2 replication in vitro. These data suggest that the robust cytokine production in response to SARS-CoV-2, despite a diminished IFN-I response, is the product of a dependency on NF-κB for viral replication. IMPORTANCE The COVID-19 pandemic has caused significant mortality and morbidity around the world. Although effective vaccines have been developed, large parts of the world remain unvaccinated while new SARS-CoV-2 variants keep emerging. Furthermore, despite extensive efforts and large-scale drug screenings, no fully effective antiviral treatment options have been discovered yet. Therefore, it is of the utmost importance to gain a better understanding of essential factors driving SARS-CoV-2 replication to be able to develop novel approaches to target SARS-CoV-2 biology.

Evaluating the Safety of West Nile Virus Immunity During Congenital Zika Virus Infection in Mice.

Antibody-dependent enhancement (ADE) is a phenomenon that occurs when cross-reactive antibodies generated from a previous flaviviral infection increase the pathogenesis of a related virus. Zika virus (ZIKV) is the most recent flavivirus introduced to the Western Hemisphere and has become a significant public health threat due to the unanticipated impact on the developing fetus. West Nile virus (WNV) is the primary flavivirus that circulates in North America, and we and others have shown that antibodies against WNV are cross-reactive to ZIKV. Thus, there is concern that WNV immunity could increase the risk of severe ZIKV infection, particularly during pregnancy. In this study, we examined the extent to which WNV antibodies could impact ZIKV pathogenesis in a murine pregnancy model. To test this, we passively transferred WNV antibodies into pregnant Stat2-/- mice on E6.5 prior to infection with ZIKV. Evaluation of pregnant dams showed weight loss following ZIKV infection; however, no differences in maternal weights or viral loads in the maternal brain, spleen, or spinal cord were observed in the presence of WNV antibodies. Resorption rates, and other fetal parameters, including fetal and placental size, were similarly unaffected. Further, the presence of WNV antibodies did not significantly alter the viral load or the inflammatory response in the placenta or the fetus in response to ZIKV. Our data suggest that pre-existing WNV immunity may not significantly impact the pathogenesis of ZIKV infection during pregnancy. Our findings are promising for the safety of implementing WNV vaccines in the continental US.

SARS-CoV-2 infection induces beta cell transdifferentiation.

Recent clinical data have suggested a correlation between coronavirus disease 2019 (COVID-19) and diabetes. Here, we describe the detection of SARS-CoV-2 viral antigen in pancreatic beta cells in autopsy samples from individuals with COVID-19. Single-cell RNA sequencing and immunostaining from ex vivo infections confirmed that multiple types of pancreatic islet cells were susceptible to SARS-CoV-2, eliciting a cellular stress response and the induction of chemokines. Upon SARS-CoV-2 infection, beta cells showed a lower expression of insulin and a higher expression of alpha and acinar cell markers, including glucagon and trypsin1, respectively, suggesting cellular transdifferentiation. Trajectory analysis indicated that SARS-CoV-2 induced eIF2-pathway-mediated beta cell transdifferentiation, a phenotype that could be reversed with trans-integrated stress response inhibitor (trans-ISRIB). Altogether, this study demonstrates an example of SARS-CoV-2 infection causing cell fate change, which provides further insight into the pathomechanisms of COVID-19.

An Immuno-Cardiac Model for Macrophage-Mediated Inflammation in COVID-19 Hearts.

[Figure: see text].

Quantifying Absolute Neutralization Titers against SARS-CoV-2 by a Standardized Virus Neutralization Assay Allows for Cross-Cohort Comparisons of COVID-19 Sera.

The global coronavirus disease 2019 (COVID-19) pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent-phase plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous biosafety level 3 (BSL3) conditions, which limits high-throughput screening of patient and vaccine sera. Myriad BSL2-compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making intergroup comparisons difficult. To address these limitations, we developed a standardized VNA using CoV2-S pseudotyped particles (CoV2pp) based on vesicular stomatitis virus bearing the Renilla luciferase gene in place of its G glycoprotein (VSVΔG); this assay can be robustly produced at scale and generate accurate neutralizing titers within 18 h postinfection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S enzyme-linked immunosorbent assay (ELISA) results and live-virus neutralizations in confirmed convalescent-patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n > 120). Our data (i) show that absolute 50% inhibitory concentration (absIC50), absIC80, and absIC90 values can be legitimately compared across diverse cohorts, (ii) highlight the substantial but consistent variability in neutralization potency across these cohorts, and (iii) support the use of the absIC80 as a more meaningful metric for assessing the neutralization potency of a vaccine or convalescent-phase sera. Lastly, we used our CoV2pp in a screen to identify ultrapermissive 293T clones that stably express ACE2 or ACE2 plus TMPRSS2. When these are used in combination with our CoV2pp, we can produce CoV2pp sufficient for 150,000 standardized VNAs/week.IMPORTANCE Vaccines and antibody-based therapeutics like convalescent-phase plasma therapy are premised upon inducing or transferring neutralizing antibodies that inhibit SARS-CoV-2 entry into cells. Virus neutralization assays (VNAs) for measuring neutralizing antibody titers (NATs) are an essential part of determining vaccine or therapeutic efficacy. However, such efficacy testing is limited by the inherent dangers of working with the live virus, which requires specialized high-level biocontainment facilities. We therefore developed a standardized replication-defective pseudotyped particle system that mimics the entry of live SARS-CoV-2. This tool allows for the safe and efficient measurement of NATs, determination of other forms of entry inhibition, and thorough investigation of virus entry mechanisms. Four independent labs across the globe validated our standardized VNA using diverse cohorts. We argue that a standardized and scalable assay is necessary for meaningful comparisons of the myriad of vaccines and antibody-based therapeutics becoming available. Our data provide generalizable metrics for assessing their efficacy.

Quantifying absolute neutralization titers against SARS-CoV-2 by a standardized virus neutralization assay allows for cross-cohort comparisons of COVID-19 sera.

The global COVID-19 pandemic has mobilized efforts to develop vaccines and antibody-based therapeutics, including convalescent plasma therapy, that inhibit viral entry by inducing or transferring neutralizing antibodies (nAbs) against the SARS-CoV-2 spike glycoprotein (CoV2-S). However, rigorous efficacy testing requires extensive screening with live virus under onerous BSL3 conditions which limits high throughput screening of patient and vaccine sera. Myriad BSL-2 compatible surrogate virus neutralization assays (VNAs) have been developed to overcome this barrier. Yet, there is marked variability between VNAs and how their results are presented, making inter-group comparisons difficult. To address these limitations, we developed a standardized VNA using VSVΔG-based CoV-2-S pseudotyped particles (CoV2pp) that can be robustly produced at scale and generate accurate neutralizing titers within 18 hours post-infection. Our standardized CoV2pp VNA showed a strong positive correlation with CoV2-S ELISA and live virus neutralizations in confirmed convalescent patient sera. Three independent groups subsequently validated our standardized CoV2pp VNA (n>120). Our data show that absolute (abs) IC50, IC80, and IC90 values can be legitimately compared across diverse cohorts, highlight the substantial but consistent variability in neutralization potency across these cohorts, and support the use of absIC80 as a more meaningful metric for assessing the neutralization potency of vaccine or convalescent sera. Lastly, we used our CoV2pp in a screen to identify ultra-permissive 293T clones that stably express ACE2 or ACE2+TMPRSS2. When used in combination with our CoV2pp, we can now produce CoV2pp sufficient for 150,000 standardized VNA/week.

Zika virus tropism during early infection of the testicular interstitium and its role in viral pathogenesis in the testes.

Sexual transmission and persistence of Zika virus (ZIKV) in the testes pose new challenges for controlling virus outbreaks and developing live-attenuated vaccines. It has been shown that testicular infection of ZIKV is initiated in the testicular interstitium, followed by spread of the virus in the seminiferous tubules. This leads to testicular damage and/or viral dissemination into the epididymis and eventually into semen. However, it remains unknown which cell types are targeted by ZIKV in the testicular interstitium, and what is the specific order of infectious events leading to ZIKV invasion of the seminiferous tubules. Here, we demonstrate that interstitial leukocytes expressing mir-511-3p microRNA are the initial targets of ZIKV in the testes, and infection of mir-511-3p-expressing cells in the testicular interstitium is necessary for downstream infection of the seminiferous tubules. Mir-511-3p is expressed concurrently with CD206, a marker of lineage 2 (M2) macrophages and monocyte derived dendritic cells (moDCs). Selective restriction of ZIKV infection of CD206-expressing M2 macrophages/moDCs results in the attenuation of macrophage-associated inflammatory responses in vivo and prevents the disruption of the Sertoli cell barrier in vitro. Finally, we show that targeting of viral genome for mir-511-3p significantly attenuates early ZIKV replication not only in the testes, but also in many peripheral organs, including spleen, epididymis, and pancreas. This incriminates M2 macrophages/moDCs as important targets for visceral ZIKV replication following hematogenous dissemination of the virus from the site of infection.

Zika virus envelope nanoparticle antibodies protect mice without risk of disease enhancement.

BACKGROUND: Zika virus (ZIKV), an arbovirus capable of causing neurological abnormalities, is a recognised human pathogen, for which a vaccine is required. As ZIKV antibodies can mediate antibody-dependent enhancement (ADE) of dengue virus (DENV) infection, a ZIKV vaccine must not only protect against ZIKV but must also not sensitise vaccinees to severe dengue. METHODS: The N-terminal 80% of ZIKV envelope protein (80E) was expressed in Pichia pastoris and its capacity to self-assemble into particulate structures evaluated using dynamic light scattering and electron microscopy. Antigenic integrity of the 80E protein was evaluated using ZIKV-specific monoclonal antibodies. Its immunogenicity and protective efficacy were assessed in BALB/c and C57BL/6 Stat2-/- mice, respectively. Its capacity to enhance DENV and ZIKV infection was assessed in AG129 and C57BL/6 Stat2-/- mice, respectively. FINDINGS: ZIKV-80E protein self-assembled into discrete nanoparticles (NPs), which preserved the antigenic integrity of neutralising epitopes on E domain III (EDIII) and elicited potent ZIKV-neutralising antibodies predominantly against this domain in BALB/c mice. These antibodies conferred statistically significant protection in vivo (p = 0.01, Mantel-Cox test), and did not exacerbate sub-lethal DENV-2 or ZIKV challenges in vivo. INTERPRETATION: Yeast-expressed ZIKV-80E, which forms highly immunogenic EDIII-displaying NPs, elicits ZIKV EDIII-specific antibodies capable of offering significant protection in vivo, without the potential risk of ADE upon subsequent DENV-2 or ZIKV infection. This offers a promising vaccine candidate for further development. FUNDING: This study was supported partly by ICGEB, India, and by NIAID, USA.

Dengue Virus Immunity Increases Zika Virus-Induced Damage during Pregnancy.

Zika virus (ZIKV) has recently been associated with birth defects and pregnancy loss after maternal infection. Because dengue virus (DENV) and ZIKV co-circulate, understanding the role of antibody-dependent enhancement in the context of pregnancy is critical. Here, we showed that the presence of DENV-specific antibodies in ZIKV-infected pregnant mice significantly increased placental damage, fetal growth restriction, and fetal resorption. This was associated with enhanced viral replication in the placenta that coincided with an increased frequency of infected trophoblasts. ZIKV-infected human placental tissues also showed increased replication in the presence of DENV antibodies, which was reversed by FcγR blocking antibodies. Furthermore, ZIKV-mediated fetal pathogenesis was enhanced in mice in the presence of a DENV-reactive monoclonal antibody, but not in the presence of the LALA variant, indicating a dependence on FcγR engagement. Our data suggest a possible mechanism for the recent increase in severe pregnancy outcomes after ZIKV infection in DENV-endemic areas.