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.

Quantifying Neutralizing Antibodies in Patients with COVID-19 by a Two-Variable Generalized Additive Model.

Considering the urgent demand for faster methods to quantify neutralizing antibody titers in patients with coronavirus (CoV) disease 2019 (COVID-19), developing an analytical model or method to replace the conventional virus neutralization test (NT) is essential. Moreover, a "COVID-19 immunity passport" is currently being proposed as a certification for people who travel internationally. Therefore, an enzyme-linked immunosorbent assay (ELISA) was designed to detect severe acute respiratory syndrome CoV 2 (SARS-CoV-2)-neutralizing antibodies in serum, which is based on the binding affinity of SARS-CoV-2 viral spike protein 1 (S1) and the viral spike protein receptor-binding domain (RBD) to antibodies. The RBD is considered the major binding region of neutralizing antibodies. Furthermore, S1 covers the RBD and several other regions, which are also important for neutralizing antibody binding. In this study, we assessed 144 clinical specimens, including those from patients with PCR-confirmed SARS-CoV-2 infections and healthy donors, using both the NT and ELISA. The ELISA results analyzed by spline regression and the two-variable generalized additive model precisely reflected the NT value, and the correlation between predicted and actual NT values was as high as 0.917. Therefore, our method serves as a surrogate to quantify neutralizing antibody titer. The analytic method and platform used in this study present a new perspective for serological testing of SARS-CoV-2 infection and have clinical potential to assess vaccine efficacy. IMPORTANCE Herein, we present a new approach for serological testing for SARS-CoV-2 antibodies using innovative laboratory methods that demonstrate a combination of biology and mathematics. The traditional virus neutralization test is the gold standard method; however, it is time-consuming and poses a risk to medical personnel. Thus, there is a demand for methods that rapidly quantify neutralizing antibody titers in patients with COVID-19 or examine vaccine efficacy at a biosafety level 2 containment facility. Therefore, we used a two-variable generalized additive model to analyze the results of the enzyme-linked immunosorbent assay and found the method to serve as a surrogate to quantify neutralizing antibody titers. This methodology has potential for clinical use in assessing vaccine efficacy.

Acyl-Coenzyme A Synthetase Long-Chain Family Member 4 Is Involved in Viral Replication Organelle Formation and Facilitates Virus Replication via Ferroptosis.

Enterovirus infections can cause severe complications, such as poliomyelitis, encephalitis, myocarditis, meningitis, neurological pulmonary edema, and even death. Here, we used genome-wide CRISPR screens to gain new insight into the mechanism by which enteroviruses co-opt host pathways to potentiate replication and propagation. We found that acyl-coenzyme A synthetase long-chain family member 4 (ACSL4) is involved in viral replication organelle formation. ACSL4 is a key component of ferroptosis, an iron-dependent, nonapoptotic programmed cell death. Our results indicated that enteroviruses and coronaviruses can induce ferroptosis via ACSL4. Most importantly, ferroptosis inhibitors, including two FDA-approved drugs, rosiglitazone (ROSI; ACSL4 inhibitor) and pioglitazone (PIO; ACSL4 inhibitor), decreased the viral load of human enteroviruses and coronaviruses, suggesting that ACSL4 is a target for counteracting viral infection. IMPORTANCE We provide the first evidence for the role of ACSL4 in enterovirus replication organelle formation. Moreover, both enteroviruses and coronaviruses induce ferroptosis via ACSL4. These findings establish a novel regulatory mechanism for viral replication. The inhibition of ACSL4 by ferroptosis inhibitors can reduce viral yields of enteroviruses and coronaviruses, including SARS-CoV-2, implying that ACSL4-mediated ferroptosis is a promising therapeutic target for viral diseases.

Zinc Finger Protein ZFP36L1 Inhibits Flavivirus Infection by both 5'-3' XRN1 and 3'-5' RNA-Exosome RNA Decay Pathways.

Zinc-finger protein 36, CCCH type-like 1 (ZFP36L1), containing tandem CCCH-type zinc-finger motifs with an RNA-binding property, plays an important role in cellular RNA metabolism mainly by RNA decay pathways. Recently, we demonstrated that human ZFP36L1 has potent antiviral activity against influenza A virus infection. However, its role in the host defense response against flaviviruses has not been addressed. Here, we demonstrate that ZFP36L1 functions as a host innate defender against flaviviruses, including Japanese encephalitis virus (JEV) and dengue virus (DENV). Overexpression of ZFP36L1 reduced JEV and DENV infection, and ZFP36L1 knockdown enhanced viral replication. ZFP36L1 destabilized the JEV genome by targeting and degrading viral RNA mediated by both 5'-3' XRN1 and 3'-5' RNA-exosome RNA decay pathways. Mutation in both zinc-finger motifs of ZFP36L1 disrupted RNA-binding and antiviral activity. Furthermore, the viral RNA sequences specifically recognized by ZFP36L1 were mapped to the 3'-untranslated region of the JEV genome with the AU-rich element (AUUUA) motif. We extend the function of ZFP36L1 to host antiviral defense by directly binding and destabilizing the viral genome via recruiting cellular mRNA decay machineries. IMPORTANCE Cellular RNA-binding proteins are among the first lines of defense against various viruses, particularly RNA viruses. ZFP36L1 belongs to the CCCH-type zinc-finger protein family and has RNA-binding activity; it has been reported to bind directly to the AU-rich elements (AREs) of a subset of cellular mRNAs and then lead to mRNA decay by recruiting mRNA-degrading enzymes. However, the antiviral potential of ZFP36L1 against flaviviruses has not yet been fully demonstrated. Here, we reveal the antiviral potential of human ZFP36L1 against Japanese encephalitis virus (JEV) and dengue virus (DENV). ZFP36L1 specifically targeted the ARE motif within viral RNA and triggered the degradation of viral RNA transcripts via cellular degrading enzymes 5'-3' XRN1 and 3'-5' RNA exosome. These findings provide mechanistic insights into how human ZFP36L1 serves as a host antiviral factor to restrict flavivirus replication.

Zoonotic potential of a novel bat morbillivirus.

Bats are significant reservoir hosts for many viruses with zoonotic potential1. SARS-CoV-2, Ebola virus, and Nipah virus are examples of such viruses that have caused deadly epidemics and pandemics when spilled over from bats into human and animal populations2,3. Careful surveillance of viruses in bats is critical for identifying potential zoonotic pathogens. However, metagenomic surveys in bats often do not result in full-length viral sequences that can be used to regenerate such viruses for targeted characterization4. Here, we identify and characterize a novel morbillivirus from a vespertilionid bat species (Myotis riparius) in Brazil, which we term myotis bat morbillivirus (MBaMV). There are 7 species of morbilliviruses including measles virus (MeV), canine distemper virus (CDV) and rinderpest virus (RPV)5. All morbilliviruses cause severe disease in their natural hosts6-10, and pathogenicity is largely determined by species specific expression of canonical morbillivirus receptors, CD150/SLAMF111 and NECTIN412. MBaMV used Myotis spp CD150 much better than human and dog CD150 in fusion assays. We confirmed this using live MBaMV that was rescued by reverse genetics. Surprisingly, MBaMV replicated efficiently in primary human myeloid but not lymphoid cells. Furthermore, MBaMV replicated in human epithelial cells and used human NECTIN4 almost as well as MeV. Our results demonstrate the unusual ability of MBaMV to infect and replicate in some human cells that are critical for MeV pathogenesis and transmission. This raises the specter of zoonotic transmission of a bat morbillivirus.

Neutralizing activity of Sputnik V vaccine sera against SARS-CoV-2 variants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected at least 180 million people since its identification as the cause of the current COVID-19 pandemic. The rapid pace of vaccine development has resulted in multiple vaccines already in use worldwide. The contemporaneous emergence of SARS-CoV-2 'variants of concern' (VOC) across diverse geographic locales underscores the need to monitor the efficacy of vaccines being administered globally. All WHO designated VOC carry spike (S) polymorphisms thought to enable escape from neutralizing antibodies. Here, we characterize the neutralizing activity of post-Sputnik V vaccination sera against the ensemble of S mutations present in alpha (B.1.1.7) and beta (B.1.351) VOC. Using de novo generated replication-competent vesicular stomatitis virus expressing various SARS-CoV-2-S in place of VSV-G (rcVSV-CoV2-S), coupled with a clonal 293T-ACE2 + TMPRSS2 + cell line optimized for highly efficient S-mediated infection, we determine that only 1 out of 12 post-vaccination serum samples shows effective neutralization (IC90) of rcVSV-CoV2-S: B.1.351 at full serum strength. The same set of sera efficiently neutralize S from B.1.1.7 and exhibit only moderately reduced activity against S carrying the E484K substitution alone. Taken together, our data suggest that control of some emergent SARS-CoV-2 variants may benefit from updated vaccines.

Detection of Antibody Responses against SARS-CoV-2 in Plasma and Saliva from Vaccinated and Infected Individuals.

Antibodies (Abs) are essential for the host immune response against SARS-CoV-2, and all the vaccines developed so far have been designed to induce Abs targeting the SARS-CoV-2 spike. Many studies have examined Ab responses in the blood from vaccinated and infected individuals. However, since SARS-CoV-2 is a respiratory virus, it is also critical to understand the mucosal Ab responses at the sites of initial virus exposure. Here, we examined plasma versus saliva Ab responses in vaccinated and convalescent patients. Although saliva levels were significantly lower, a strong correlation was observed between plasma and saliva total Ig levels against all SARS-CoV-2 antigens tested. Virus-specific IgG1 responses predominated in both saliva and plasma, while a lower prevalence of IgM and IgA1 Abs was observed in saliva. Antiviral activities of plasma Abs were also studied. Neutralization titers against the initial WA1 (D614G), B.1.1.7 (alpha) and B.1.617.2 (delta) strains were similar but lower against the B.1.351 (beta) strain. Spike-specific antibody-dependent cellular phagocytosis (ADCP) activities were also detected and the levels correlated with spike-binding Ig titers. Interestingly, while neutralization and ADCP potencies of vaccinated and convalescent groups were comparable, enhanced complement deposition to spike-specific Abs was noted in vaccinated versus convalescent groups and corresponded with higher levels of IgG1 plus IgG3 among the vaccinated individuals. Altogether, this study demonstrates the detection of Ab responses after vaccination or infection in plasma and saliva that correlate significantly, although Ig isotypic differences were noted. The induced plasma Abs displayed Fab-mediated and Fc-dependent functions with comparable neutralization and ADCP potencies, but a greater capacity to activate complement was elicited upon vaccination.

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.

Detection of Antibody Responses Against SARS-CoV-2 in Plasma and Saliva From Vaccinated and Infected Individuals.

Antibodies (Abs) are essential for the host immune response against SARS-CoV-2, and all the vaccines developed so far have been designed to induce Abs targeting the SARS-CoV-2 spike. Many studies have examined Ab responses in the blood from vaccinated and infected individuals. However, since SARS-CoV-2 is a respiratory virus, it is also critical to understand the mucosal Ab responses at the sites of initial virus exposure. Here, we examined plasma versus saliva Ab responses in vaccinated and convalescent patients. Although saliva levels were significantly lower, a strong correlation was observed between plasma and saliva total Ig levels against all SARS-CoV-2 antigens tested. Virus-specific IgG1 responses predominated in both saliva and plasma, while a lower prevalence of IgM and IgA1 Abs was observed in saliva. Antiviral activities of plasma Abs were also studied. Neutralization titers against the initial WA1 (D614G), B.1.1.7 (alpha) and B.1.617.2 (delta) strains were similar but lower against the B.1.351 (beta) strain. Spike-specific antibody-dependent cellular phagocytosis (ADCP) activities were also detected and the levels correlated with spike-binding Ig titers. Interestingly, while neutralization and ADCP potencies of vaccinated and convalescent groups were comparable, enhanced complement deposition to spike-specific Abs was noted in vaccinated versus convalescent groups and corresponded with higher levels of IgG1 plus IgG3 among the vaccinated individuals. Altogether, this study demonstrates the detection of Ab responses after vaccination or infection in plasma and saliva that correlate significantly, although Ig isotypic differences were noted. The induced plasma Abs displayed Fab-mediated and Fc-dependent functions with comparable neutralization and ADCP potencies, but a greater capacity to activate complement was elicited upon vaccination.

SARS-CoV-2 proteases PLpro and 3CLpro cleave IRF3 and critical modulators of inflammatory pathways (NLRP12 and TAB1): implications for disease presentation across species.

The genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory responThe genome of SARS-CoV-2 encodes two viral proteases (NSP3/papain-like protease and NSP5/3C-like protease) that are responsible for cleaving viral polyproteins during replication. Here, we discovered new functions of the NSP3 and NSP5 proteases of SARS-CoV-2, demonstrating that they could directly cleave proteins involved in the host innate immune response. We identified 3 proteins that were specifically and selectively cleaved by NSP3 or NSP5: IRF-3, and NLRP12 and TAB1, respectively. Direct cleavage of IRF3 by NSP3 could explain the blunted Type-I IFN response seen during SARS-CoV-2 infections while NSP5 mediated cleavage of NLRP12 and TAB1 point to a molecular mechanism for enhanced production of cytokines and inflammatory response observed in COVID-19 patients. We demonstrate that in the mouse NLRP12 protein, one of the recognition site is not cleaved in our in-vitro assay. We pushed this comparative alignment of IRF-3 and NLRP12 homologs and show that the lack or presence of cognate cleavage motifs in IRF-3 and NLRP12 could contribute to the presentation of disease in cats and tigers, for example. Our findings provide an explanatory framework for indepth studies into the pathophysiology of COVID-19.

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.

Zinc finger protein ZFP36L1 inhibits influenza A virus through translational repression by targeting HA, M and NS RNA transcripts.

ZFP36L1, a CCCH-type zinc finger protein, is an RNA-binding protein that participates in controlling cellular mRNA abundance and turnover by posttranscriptional regulation. Here, we demonstrated that ZFP36L1 has an important role in host defense against influenza A virus (IAV) infection. Overexpression of ZFP36L1 reduced IAV replication via translational repression of HA, M and NS RNA segment transcripts. IAV infection upregulated cellular ZFP36L1 expression, and endogenous ZFP36L1 knockdown significantly enhanced IAV replication. ZFP36L1 directly binds to IAV NS1 mRNA in the cytoplasm and blocks the expression and function of NS1 protein. Mutation of CCCH-type zinc finger domains of ZFP36L1 lost its antiviral potential and NS1 mRNA binding. Thus, ZFP36L1 can act as a host innate defense by targeting HA, M and NS mRNA transcripts to suppress viral protein translation.

Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein.

CCCH-type zinc-finger antiviral protein (ZAP) is a host factor that restricts the infection of many viruses mainly through RNA degradation, translation inhibition and innate immune responses. So far, only one flavivirus, yellow fever virus, has been reported to be ZAP-resistant. Here, we investigated the antiviral potential of human ZAP (isoform ZAP-L and ZAP-S) against three flaviviruses, Japanese encephalitis virus (JEV), dengue virus (DENV) and Zika virus (ZIKV). Infection of JEV but not DENV or ZIKV was blocked by ZAP overexpression, and depletion of endogenous ZAP enhanced JEV replication. ZAP hampered JEV translation and targeted viral RNA for 3'-5' RNA exosome-mediated degradation. The zinc-finger motifs of ZAP were essential for RNA targeting and anti-JEV activity. JEV 3'-UTR, especially in the region with dumbbell structures and high content of CG dinucleotide, was mapped to bind ZAP and confer sensitivity to ZAP. In summary, we identified JEV as the first ZAP-sensitive flavivirus. ZAP may act as an intrinsic antiviral factor through specific RNA binding to fight against JEV infection.

Comparable fitness and transmissibility between oseltamivir-resistant pandemic 2009 and seasonal H1N1 influenza viruses with the H275Y neuraminidase mutation.

Limited antiviral compounds are available for the control of influenza, and the emergence of resistant variants would further narrow the options for defense. The H275Y neuraminidase (NA) mutation, which confers resistance to oseltamivir carboxylate, has been identified among the seasonal H1N1 and 2009 pandemic influenza viruses; however, those H275Y resistant variants demonstrated distinct epidemiological outcomes in humans. Specifically, dominance of the H275Y variant over the oseltamivir-sensitive viruses was only reported for a seasonal H1N1 variant during 2008-2009. Here, we systematically analyze the effect of the H275Y NA mutation on viral fitness and transmissibility of A(H1N1)pdm09 and seasonal H1N1 influenza viruses. The NA genes from A(H1N1)pdm09 A/California/04/09 (CA04), seasonal H1N1 A/New Caledonia/20/1999 (NewCal), and A/Brisbane/59/2007 (Brisbane) were individually introduced into the genetic background of CA04. The H275Y mutation led to reduced NA enzyme activity, an increased K(m) for 3'-sialylactose or 6'-sialylactose, and decreased infectivity in mucin-secreting human airway epithelial cells compared to the oseltamivir-sensitive wild-type counterparts. Attenuated pathogenicity in both RG-CA04(NA-H275Y) and RG-CA04 × Brisbane(NA-H275Y) viruses was observed in ferrets compared to RG-CA04 virus, although the transmissibility was minimally affected. In parallel experiments using recombinant Brisbane viruses differing by hemagglutinin and NA, comparable direct contact and respiratory droplet transmissibilities were observed among RG-NewCal(HA,NA), RG-NewCal(HA,NA-H275Y), RG-Brisbane(HA,NA-H275Y), and RG-NewCal(HA) × Brisbane(NA-H275Y) viruses. Our results demonstrate that, despite the H275Y mutation leading to a minor reduction in viral fitness, the transmission potentials of three different antigenic strains carrying this mutation were comparable in the naïve ferret model.