Tetracistronic Minigenomes Elucidate a Functional Promoter for Ghana Virus and Unveils Cedar Virus Replicase Promiscuity for all Henipaviruses.

Batborne henipaviruses, such as Nipah virus and Hendra virus, represent a major threat to global health due to their propensity for spillover, severe pathogenicity, and high mortality rate in human hosts. Coupled with the absence of approved vaccines or therapeutics, work with the prototypical species and uncharacterized, emergent species is restricted to high biocontainment facilities. There is a scarcity of such specialized spaces for research, and often the scope and capacity of research which can be conducted at BSL-4 is limited. Therefore, there is a pressing need for innovative life-cycle modeling systems to enable comprehensive research within lower biocontainment settings. This work showcases tetracistronic, transcription and replication competent minigenomes for Nipah virus, Hendra virus, Cedar virus, and Ghana virus, which encode viral proteins facilitating budding, fusion, and receptor binding. We validate the functionality of all encoded viral proteins and demonstrate a variety of applications to interrogate the viral life cycle. Notably, we found that the Cedar virus replicase exhibits remarkable promiscuity, efficiently rescuing minigenomes from all tested henipaviruses. We also apply this technology to GhV, an emergent species which has so far not been isolated in culture. We demonstrate that the reported sequence of GhV is incomplete, but that this missing sequence can be substituted with analogous sequences from other henipaviruses. Use of our GhV system establishes the functionality of the GhV replicase and identifies two antivirals which are highly efficacious against the GhV polymerase. Author Summary: Henipaviruses, such as the prototypical Nipah virus and Hendra virus, are recognized as significant global health threats due to their high mortality rates and lack of effective vaccines or therapeutics. Due to the requirement for high biocontainment facilities, the scope of research which may be conducted on henipaviruses is limited. To address this challenge, we developed innovative tetracistronic, transcription and replication competent minigenomes for Nipah virus, Hendra virus, Cedar virus, as well as for the emergent species, Ghana virus. We demonstrate that these systems replicate key aspects of the viral life cycle, such as budding, fusion, and receptor binding, and are safe for use in lower biocontainment settings. Importantly, application of this system to Ghana virus revealed that its known sequence is incomplete; however, substituting the missing sequences with those from other henipaviruses allowed us to overcome this challenge. We demonstrate that the Ghana virus replicative machinery is functional and identify two orally-efficacious antivirals effective against it. Further, we compare the compatibility of divergent henipavirus replicases with heterotypic viral genetic elements, providing valuable insights for how these species have evolved. Our research offers a versatile system for life-cycle modeling of highly pathogenic henipaviruses at low biocontainment.

De novo antibody discovery in human blood from full-length single B cell transcriptomics and matching haplotyped-resolved germline assemblies.

Immunoglobulin (IGH, IGK, IGL) loci in the human genome are highly polymorphic regions that encode the building blocks of the light and heavy chain IG proteins that dimerize to form antibodies. The processes of V(D)J recombination and somatic hypermutation in B cells are responsible for creating an enormous reservoir of highly specific antibodies capable of binding a vast array of possible antigens. However, the antibody repertoire is fundamentally limited by the set of variable (V), diversity (D), and joining (J) alleles present in the germline IG loci. To better understand how the germline IG haplotypes contribute to the expressed antibody repertoire, we combined genome sequencing of the germline IG loci with single-cell transcriptome sequencing of B cells from the same donor. Sequencing and assembly of the germline IG loci captured the IGH locus in a single fully-phased contig where the maternal and paternal contributions to the germline V, D, and J repertoire can be fully resolved. The B cells were collected following a measles, mumps, and rubella (MMR) vaccination, resulting in a population of cells that were activated in response to this specific immune challenge. Single-cell, full-length transcriptome sequencing of these B cells resulted in whole transcriptome characterization of each cell, as well as highly-accurate consensus sequences for the somatically rearranged and hypermutated light and heavy chain IG transcripts. A subset of antibodies synthesized based on their consensus heavy and light chain transcript sequences demonstrated binding to measles antigens and neutralization of measles live virus.

Structure-guided mutagenesis of Henipavirus receptor-binding proteins reveals molecular determinants of receptor usage and antibody-binding epitopes.

Nipah virus (NiV) is a highly lethal, zoonotic Henipavirus (HNV) that causes respiratory and neurological signs and symptoms in humans. Similar to other paramyxoviruses, HNVs mediate entry into host cells through the concerted actions of two surface glycoproteins: a receptor-binding protein (RBP) that mediates attachment and a fusion glycoprotein (F) that triggers fusion in an RBP-dependent manner. NiV uses ephrin-B2 (EFNB2) and ephrin-B3 (EFNB3) as entry receptors. Ghana virus (GhV), a novel HNV identified in a Ghanaian bat, uses EFNB2 but not EFNB3. In this study, we employ a structure-informed approach to identify receptor-interfacing residues and systematically introduce GhV-RBP residues into a NiV-RBP backbone to uncover the molecular determinants of EFNB3 usage. We reveal two regions that severely impair EFNB3 binding by NiV-RBP and EFNB3-mediated entry by NiV pseudotyped viral particles. Further analyses uncovered two-point mutations (NiVN557SGhV and NiVY581TGhV) pivotal for this phenotype. Moreover, we identify NiV interaction with Y120 of EFNB3 as important for the usage of this receptor. Beyond these EFNB3-related findings, we reveal two domains that restrict GhV binding of EFNB2, confirm the HNV-head as an immunodominant target for polyclonal and monoclonal antibodies, and describe putative epitopes for GhV- and NiV-specific monoclonal antibodies. Cumulatively, the work presented here generates useful reagents and tools that shed insight to residues important for NiV usage of EFNB3, reveal regions critical for GhV binding of EFNB2, and describe putative HNV antibody-binding epitopes. IMPORTANCE: Hendra virus and Nipah virus (NiV) are lethal, zoonotic Henipaviruses (HNVs) that cause respiratory and neurological clinical features in humans. Since their initial outbreaks in the 1990s, several novel HNVs have been discovered worldwide, including Ghana virus. Additionally, there is serological evidence of zoonotic transmission, lending way to concerns about future outbreaks. HNV infection of cells is mediated by the receptor-binding protein (RBP) and the Fusion protein (F). The work presented here identifies NiV RBP amino acids important for the usage of ephrin-B3 (EFNB3), a receptor highly expressed in neurons and predicted to be important for neurological clinical features caused by NiV. This study also characterizes epitopes recognized by antibodies against divergent HNV RBPs. Together, this sheds insight to amino acids critical for HNV receptor usage and antibody binding, which is valuable for future studies investigating determinants of viral pathogenesis and developing antibody therapies.

Functional properties of measles virus proteins derived from a subacute sclerosing panencephalitis patient who received repeated remdesivir treatments.

Subacute sclerosing panencephalitis (SSPE) is a rare but fatal late neurological complication of measles, caused by persistent measles virus (MeV) infection of the central nervous system. There are no drugs approved for the treatment of SSPE. Here, we followed the clinical progression of a 5-year-old SSPE patient after treatment with the nucleoside analog remdesivir, conducted a post-mortem evaluation of the patient's brain, and characterized the MeV detected in the brain. The quality of life of the patient transiently improved after the first two courses of remdesivir, but a third course had no further clinical effect, and the patient eventually succumbed to his condition. Post-mortem evaluation of the brain displayed histopathological changes including loss of neurons and demyelination paired with abundant presence of MeV RNA-positive cells throughout the brain. Next-generation sequencing of RNA isolated from the brain revealed a complete MeV genome with mutations that are typically detected in SSPE, characterized by a hypermutated M gene. Additional mutations were detected in the polymerase (L) gene, which were not associated with resistance to remdesivir. Functional characterization showed that mutations in the F gene led to a hyperfusogenic phenotype predominantly mediated by N465I. Additionally, recombinant wild-type-based MeV with the SSPE-F gene or the F gene with the N465I mutation was no longer lymphotropic but instead efficiently disseminated in neural cultures. Altogether, this case encourages further investigation of remdesivir as a potential treatment of SSPE and highlights the necessity to functionally understand SSPE-causing MeV.IMPORTANCEMeasles virus (MeV) causes acute, systemic disease and remains an important cause of morbidity and mortality in humans. Despite the lack of known entry receptors in the brain, MeV can persistently infect the brain causing the rare but fatal neurological disorder subacute sclerosing panencephalitis (SSPE). SSPE-causing MeVs are characterized by a hypermutated genome and a hyperfusogenic F protein that facilitates the rapid spread of MeV throughout the brain. No treatment against SSPE is available, but the nucleoside analog remdesivir was recently demonstrated to be effective against MeV in vitro. We show that treatment of an SSPE patient with remdesivir led to transient clinical improvement and did not induce viral escape mutants, encouraging the future use of remdesivir in SSPE patients. Functional characterization of the viral proteins sheds light on the shared properties of SSPE-causing MeVs and further contributes to understanding how those viruses cause disease.

An in vitro experimental pipeline to characterize the epitope of a SARS-CoV-2 neutralizing antibody.

IMPORTANCE: The COVID-19 pandemic remains a significant public health concern for the global population; the development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread so long as they recognize and interact with circulating variants. The epitope and binding specificity of a neutralizing anti-SARS-CoV-2 Spike receptor-binding domain antibody clone against many SARS-CoV-2 variants of concern were characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis and VSV-spike neutralization studies. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

Reverse Genetics Systems for the De Novo Rescue of Diverse Members of Paramyxoviridae.

Paramyxoviruses place significant burdens on both human and wildlife health; while some paramyxoviruses are established within human populations, others circulate within diverse animal reservoirs. Concerningly, bat-borne paramyxoviruses have spilled over into humans with increasing frequency in recent years, resulting in severe disease. The risk of future zoonotic outbreaks, as well as the persistence of paramyxoviruses that currently circulate within humans, highlights the need for efficient tools through which to interrogate paramyxovirus biology. Reverse genetics systems provide scientists with the ability to rescue paramyxoviruses de novo, offering versatile tools for implementation in both research and public health settings. Reverse genetics systems have greatly improved over the past 30 years, with several key innovations optimizing the success of paramyxovirus rescue. Here, we describe the significance of such advances and provide a generally applicable guide for the development and use of reverse genetics systems for the rescue of diverse members of Paramyxoviridae.

Crystal structure and solution state of the C-terminal head region of the narmovirus receptor binding protein.

IMPORTANCE: Genetically diverse paramyxoviruses are united in their presentation of a receptor-binding protein (RBP), which works in concert with the fusion protein to facilitate host-cell entry. The C-terminal head region of the paramyxoviral RBP, a primary determinant of host-cell tropism and inter-species transmission potential, forms structurally distinct classes dependent upon protein and glycan receptor specificity. Here, we reveal the architecture of the C-terminal head region of the RBPs from Nariva virus (NarV) and Mossman virus (MosV), two archetypal rodent-borne paramyxoviruses within the recently established genus Narmovirus, family Paramyxoviridae. Our analysis reveals that while narmoviruses retain the general architectural features associated with paramyxoviral RBPs, namely, a six-bladed ß-propeller fold, they lack the structural motifs associated with known receptor-mediated host-cell entry pathways. This investigation indicates that the RBPs of narmoviruses exhibit pathobiological features that are distinct from those of other paramyxoviruses.

In Vivo Imaging of Nipah Virus Infection in Small Animal Rodent Models.

In vivo imaging system (IVIS) is a powerful tool for the study of infectious diseases, providing the ability to non-invasively follow viral infection in an individual animal over time. Recombinant henipaviruses expressing bioluminescent or fluorescent reporter proteins can be used both to monitor the spatial and temporal progression of Nipah virus (NiV) infection in vivo as well as in ex vivo tissues. Virally produced luciferases react with systemically administered substrate to produce bioluminescence that can then be detected via IVIS imaging, while fluorescent reporters inherently generate detectable fluorescence without a substrate. Here we describe protocols applying bioluminescent or fluorescent reporter expressing recombinant viruses to in vivo or ex vivo imaging of NiV infection.

An in vitro experimental pipeline to characterize the binding specificity of SARS-CoV-2 neutralizing antibodies.

The coronavirus disease 2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) has led to over 760 million cases and >6.8 million deaths worldwide. We developed a panel of human neutralizing monoclonal antibodies (mAbs) targeting the SARS-CoV-2 Spike protein using Harbour H2L2 transgenic mice immunized with Spike receptor binding domain (RBD) (1). Representative antibodies from genetically-distinct families were evaluated for inhibition of replication-competent VSV expressing SARS-CoV-2 Spike (rcVSV-S) in place of VSV-G. One mAb (denoted FG-10A3) inhibited infection of all rcVSV-S variants; its therapeutically-modified version, STI-9167, inhibited infection of all tested SARS-CoV-2 variants, including Omicron BA.1 and BA.2, and limited virus proliferation in vivo (1). To characterize the binding specificity and epitope of FG-10A3, we generated mAb-resistant rcVSV-S virions and performed structural analysis of the antibody/antigen complex using cryo-EM. FG-10A3/STI-9167 is a Class 1 antibody that prevents Spike-ACE2 binding by engaging a region within the Spike receptor binding motif (RBM). Sequencing of mAb-resistant rcVSV-S virions identified F486 as a critical residue for mAb neutralization, with structural analysis revealing that both the variable heavy and light chains of STI-9167 bound the disulfide-stabilized 470-490 loop at the Spike RBD tip. Interestingly, substitutions at position 486 were later observed in emerging variants of concern BA.2.75.2 and XBB. This work provides a predictive modeling strategy to define the neutralizing capacity and limitations of mAb therapeutics against emerging SARS-CoV-2 variants. Importance: The COVID-19 pandemic remains a significant public health concern for the global population; development and characterization of therapeutics, especially ones that are broadly effective, will continue to be essential as SARS-CoV-2 variants emerge. Neutralizing monoclonal antibodies remain an effective therapeutic strategy to prevent virus infection and spread with the caveat that they interact with the circulating variants. The epitope and binding specificity of a broadly neutralizing anti-SARS-CoV-2 Spike RBD antibody clone against many SARS-CoV-2 VOC was characterized by generating antibody-resistant virions coupled with cryo-EM structural analysis. This workflow can serve to predict the efficacy of antibody therapeutics against emerging variants and inform the design of therapeutics and vaccines.

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.

Nipah Virus Bangladesh Infection Elicits Organ-Specific Innate and Inflammatory Responses in the Marmoset Model.

The common marmoset (Callithrix jacchus) is increasingly recognized as an ideal nonhuman primate (NHP) at high biocontainment due to its smaller size and relative ease of handling. Here, we evaluated the susceptibility and pathogenesis of Nipah virus Bangladesh strain (NiVB) infection in marmosets at biosafety level 4. Infection via the intranasal and intratracheal route resulted in fatal disease in all 4 infected marmosets. Three developed pulmonary edema and hemorrhage as well as multifocal hemorrhagic lymphadenopathy, while 1 recapitulated neurologic clinical manifestations and cardiomyopathy on gross pathology. Organ-specific innate and inflammatory responses were characterized by RNA sequencing in 6 different tissues from infected and control marmosets. Notably, a unique transcriptome was revealed in the brainstem of the marmoset exhibiting neurological signs. Our results provide a more comprehensive understanding of NiV pathogenesis in an accessible and novel NHP model, closely reflecting clinical disease as observed in NiV patients.

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.

Paramyxoviruses from bats: changes in receptor specificity and their role in host adaptation.

Global metagenomic surveys have revealed that bats host a diverse array of paramyxoviruses, including species from at least five major genera. An essential determinant of successful spillover is the entry of a virus into a new host. We evaluate the role of receptor usage in the zoonotic potential of bat-borne henipaviruses, morbilliviruses, pararubulaviruses, orthorubulaviruses, and jeilongviruses; successful spillover into humans depends upon compatibility of a respective viral attachment protein with its cognate receptor. We also emphasize the importance of postentry restrictions in preventing spillover. Metagenomics and characterization of newly identified paramyxoviruses have greatly improved our understanding of spillover determinants, allowing for better forecasts of which bat-borne viruses may pose the greatest risk for cross-species transmission into humans.

Immune profiles to distinguish hospitalized versus ambulatory COVID-19 cases in older patients.

A fraction of patients with COVID-19 develops severe disease requiring hospitalization, while the majority, including high-risk individuals, experience mild symptoms. Severe disease has been associated with higher levels of antibodies and inflammatory cytokines but often among patients with diverse demographics and comorbidity status. This study evaluated hospitalized vs. ambulatory patients with COVID-19 with demographic risk factors for severe COVID-19: median age of 63, >80% male, and >85% black and/or Hispanic. Sera were collected four to 243 days after symptom onset and evaluated for binding and functional antibodies as well as 48 cytokines and chemokines. SARS-CoV-2-specific antibody levels and functions were similar in ambulatory and hospitalized patients. However, a strong correlation between anti-S2 antibody levels and the other antibody parameters, along with higher IL-27 levels, was observed in hospitalized but not ambulatory cases. These data indicate that antibodies against the relatively conserved S2 spike subunit and immunoregulatory cytokines such as IL-27 are potential immune determinants of COVID-19.

Gut microbiota-derived metabolites confer protection against SARS-CoV-2 infection.

The gut microbiome is intricately coupled with immune regulation and metabolism, but its role in Coronavirus Disease 2019 (COVID-19) is not fully understood. Severe and fatal COVID-19 is characterized by poor anti-viral immunity and hypercoagulation, particularly in males. Here, we define multiple pathways by which the gut microbiome protects mammalian hosts from SARS-CoV-2 intranasal infection, both locally and systemically, via production of short-chain fatty acids (SCFAs). SCFAs reduced viral burdens in the airways and intestines by downregulating the SARS-CoV-2 entry receptor, angiotensin-converting enzyme 2 (ACE2), and enhancing adaptive immunity via GPR41 and 43 in male animals. We further identify a novel role for the gut microbiome in regulating systemic coagulation response by limiting megakaryocyte proliferation and platelet turnover via the Sh2b3-Mpl axis. Taken together, our findings have unraveled novel functions of SCFAs and fiber-fermenting gut bacteria to dampen viral entry and hypercoagulation and promote adaptive antiviral immunity.

Discovery and intranasal administration of a SARS-CoV-2 broadly acting neutralizing antibody with activity against multiple Omicron subvariants.

BACKGROUND: The continual emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern, in particular the newly emerged Omicron (B.1.1.529) variant and its BA.X lineages, has rendered ineffective a number of previously FDA emergency use authorized SARS-CoV-2 neutralizing antibody therapies. Furthermore, those approved antibodies with neutralizing activity against Omicron BA.1 are reportedly ineffective against the subset of Omicron subvariants that contain a R346K substitution, BA.1.1, and the more recently emergent BA.2, demonstrating the continued need for discovery and characterization of candidate therapeutic antibodies with the breadth and potency of neutralizing activity required to treat newly diagnosed COVID-19 linked to recently emerged variants of concern. METHODS: Following a campaign of antibody discovery based on the vaccination of Harbor H2L2 mice with defined SARS-CoV-2 spike domains, we have characterized the activity of a large collection of spike-binding antibodies and identified a lead neutralizing human IgG1 LALA antibody, STI-9167. FINDINGS: STI-9167 has potent, broad-spectrum neutralizing activity against the current SARS-COV-2 variants of concern and retained activity against each of the tested Omicron subvariants in both pseudotype and live virus neutralization assays. Furthermore, STI-9167 nAb administered intranasally or intravenously provided protection against weight loss and reduced virus lung titers to levels below the limit of quantitation in Omicron-infected K18-hACE2 transgenic mice. CONCLUSIONS: With this established activity profile, a cGMP cell line has been developed and used to produce cGMP drug product intended for intravenous or intranasal use in human clinical trials. FUNDING: Funded by CRIPT (no. 75N93021R00014), DARPA (HR0011-19-2-0020), and NCI Seronet (U54CA260560).

Measles and Nipah virus assembly: Specific lipid binding drives matrix polymerization.

Measles virus, Nipah virus, and multiple other paramyxoviruses cause disease outbreaks in humans and animals worldwide. The paramyxovirus matrix (M) protein mediates virion assembly and budding from host cell membranes. M is thus a key target for antivirals, but few high-resolution structures of paramyxovirus M are available, and we lack the clear understanding of how viral M proteins interact with membrane lipids to mediate viral assembly and egress that is needed to guide antiviral design. Here, we reveal that M proteins associate with phosphatidylserine and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] at the plasma membrane. Using x-ray crystallography, electron microscopy, and molecular dynamics, we demonstrate that PI(4,5)P2 binding induces conformational and electrostatic changes in the M protein surface that trigger membrane deformation, matrix layer polymerization, and virion assembly.

Classification of new morbillivirus and jeilongvirus sequences from bats sampled in Brazil and Malaysia.

As part of a broad One Health surveillance effort to detect novel viruses in wildlife and people, we report several paramyxovirus sequences sampled primarily from bats during 2013 and 2014 in Brazil and Malaysia, including seven from which we recovered full-length genomes. Of these, six represent the first full-length paramyxovirid genomes sequenced from the Americas, including two that are the first full-length bat morbillivirus genome sequences published to date. Our findings add to the vast number of viral sequences in public repositories, which have been increasing considerably in recent years due to the rising accessibility of metagenomics. Taxonomic classification of these sequences in the absence of phenotypic data has been a significant challenge, particularly in the subfamily Orthoparamyxovirinae, where the rate of discovery of novel sequences has been substantial. Using pairwise amino acid sequence classification (PAASC), we propose that five of these sequences belong to members of the genus Jeilongvirus and two belong to members of the genus Morbillivirus. We also highlight inconsistencies in the classification of Tupaia virus and Mòjiang virus using the same demarcation criteria and suggest reclassification of these viruses into new genera. Importantly, this study underscores the critical importance of sequence length in PAASC analysis as well as the importance of biological characteristics such as genome organization in the taxonomic classification of viral sequences.

The Impact of Evolving SARS-CoV-2 Mutations and Variants on COVID-19 Vaccines.

The emergence of several new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in recent months has raised concerns around the potential impact on ongoing vaccination programs. Data from clinical trials and real-world evidence suggest that current vaccines remain highly effective against the alpha variant (B.1.1.7), while some vaccines have reduced efficacy and effectiveness against symptomatic disease caused by the beta variant (B.1.351) and the delta variant (B.1.617.2); however, effectiveness against severe disease and hospitalization caused by delta remains high. Although data on the effectiveness of the primary regimen against omicron (B.1.1.529) are limited, booster programs using mRNA vaccines have been shown to restore protection against infection and symptomatic disease (regardless of the vaccine used for the primary regimen) and maintain high effectiveness against hospitalization. However, effectiveness against infection and symptomatic disease wanes with time after the booster dose. Studies have demonstrated reductions of varying magnitude in neutralizing activity of vaccine-elicited antibodies against a range of SARS-CoV-2 variants, with the omicron variant in particular exhibiting partial immune escape. However, evidence suggests that T-cell responses are preserved across vaccine platforms, regardless of variant of concern. Nevertheless, various mitigation strategies are under investigation to address the potential for reduced efficacy or effectiveness against current and future SARS-CoV-2 variants, including modification of vaccines for certain variants (including omicron), multivalent vaccine formulations, and different delivery mechanisms.

The IgA in milk induced by SARS-CoV-2 infection is comprised of mainly secretory antibody that is neutralizing and highly durable over time.

Approximately 10% of infants infected with SARS-CoV-2 will experience COVID-19 illness requiring advanced care. A potential mechanism to protect this population is passive immunization via the milk of a previously infected person. We and others have reported on the presence of SARS-CoV-2-specific antibodies in human milk. We now report the prevalence of SARS-CoV-2 IgA in the milk of 74 COVID-19-recovered participants, and find that 89% of samples are positive for Spike-specific IgA. In a subset of these samples, 95% exhibited robust IgA activity as determined by endpoint binding titer, with 50% considered high-titer. These IgA-positive samples were also positive for Spike-specific secretory antibody. Levels of IgA antibodies and secretory antibodies were shown to be strongly positively correlated. The secretory IgA response was dominant among the milk samples tested compared to the IgG response, which was present in 75% of samples and found to be of high-titer in only 13% of cases. Our IgA durability analysis using 28 paired samples, obtained 4-6 weeks and 4-10 months after infection, found that all samples exhibited persistently significant Spike-specific IgA, with 43% of donors exhibiting increasing IgA titers over time. Finally, COVID-19 and pre-pandemic control milk samples were tested for the presence of neutralizing antibodies; 6 of 8 COVID-19 samples exhibited neutralization of Spike-pseudotyped VSV (IC50 range, 2.39-89.4ug/mL) compared to 1 of 8 controls. IgA binding and neutralization capacities were found to be strongly positively correlated. These data are highly relevant to public health, not only in terms of the protective capacity of these antibodies for breastfed infants, but also for the potential use of such antibodies as a COVID-19 therapeutic, given that secretory IgA is highly in all mucosal compartments.