The exon junction complex (EJC) has roles in mRNA export and cytoplasmic quality control. However, the EJC is recruited to pre-mRNA by the spliceosome prior to the completion of splicing. When splicing is cotranscriptional, the EJC is deposited on nascent RNA early during synthesis, raising the question of whether the EJC regulates downstream RNA processing. Here we show, using long-read sequencing, that degron-mediated depletion of EJC component EIF4A3 leads to skipping of neighboring pairs of two or more exons on the same mRNA molecule. These data suggest that the entire "exon block" requires the EJC for inclusion. Introns flanking EJC-dependent exon blocks were longer and spliced after internal introns. In our working model, block exons are first spliced together to form a larger EJC-marked exon that promotes surrounding splicing events. Strikingly, analysis of 480 RNA binding protein knockdowns across two different human cell lines revealed block exons that are dependent on other splicing factors, indicating that coordinated splicing of adjacent exons is a general mechanism, of which the EJC is the dominant regulator. Cell type-specific coordinated splicing of adjacent exon pairs has been observed before. Here we identify the EJC as the main protein factor massively regulating this novel splicing mechanism in trans.

Nipah virus (NiV) is a zoonotic paramyxovirus with pandemic potential, for which no licensed vaccines or therapeutics for human use are available. The nucleoside analog 4'-fluorouridine (4'-FlU, EIDD-2749) has broad activity against RNA viruses including in vitro activity against the Malaysia and Bangladesh clades of NiV (NiV-M and NiV-B, respectively). Here, we report the pharmacokinetic profile of orally administered 4'-FlU in Syrian Golden hamsters and its efficacy against NiV-B. 4'-FlU was orally bioavailable and provided sustained exposure of its bioactive anabolite 4'-FlU 5'-triphosphate (4'-FlU-TP) in the brain. A 7-day treatment course after NiV infection delayed time to death. Extending treatment to 21-28 days reduced viremia, incidence of lung disease, lung lesion severity, inflammation, and mortality. Lesions, viral antigen, or viral RNA could be detected in some survivors, suggesting persistent infections. Sequence analysis of NiV populations showed nonsynonymous single-nucleotide variants (SNVs) in some brain specimens from 4'-FlU-treated animals. Amino acid changes occurred in the nucleocapsid, the polymerase, or the phosphoprotein. It is currently unclear whether they reduce viral susceptibility to treatment or impact virulence but caused a slight increase in 4'-FlU potency compared to the genetic parent virus in vitro. However, none of these SNVs increased viral fitness in human brain astrocytes. This study established proof-of-concept for efficacious oral treatment of lethal NiV infection with a small-molecule nucleoside analog inhibitor in a relevant animal model.

Henipaviruses (HNVs) like Nipah virus (NiV) and Hendra virus (HeV) represent severe zoonotic threats. Ghana virus (GhV), identified in 2012, is the only African bat henipavirus with a near-complete genome assembly. However, without isolates in culture, GhV biology, pathogenicity, and zoonotic potential remain poorly understood. Using reverse genetics, we recovered a full-length infectious clone of GhV at BSL-4 following rational reconstruction of its incomplete 3 ' leader and modification of a non-canonical transcriptional initiation site. GhV demonstrated restricted receptor tropism (ephrin-B2 but not ephrin-B3) and distinct innate immune antagonism. Replication was attenuated in primary human cells but was enhanced in bat cells. In Syrian golden hamsters, GhV infection caused no disease or mortality. Furthermore, a chimeric NiV encoding the GhV receptor-binding protein was completely attenuated in vivo, implicating ephrin-B3 receptor usage as a critical determinant of HNV pathogenesis. These findings elucidate GhV zoonotic potential and inform strategies for virus surveillance and control.

BACKGROUND:Despite widespread vaccination, mumps outbreaks continue to occur, raising concerns about waning immunity and strain-specific differences in neutralizing antibody responses. METHODS:We evaluated seroprevalence, total antibody levels, and neutralization capacity against the vaccine (JL5) and outbreak (genotype G) mumps virus (MuV) strains from 187 US blood donors born between 1928 and 1993. Associations with age, sex, and donation site, were analyzed. RESULTS:MuV IgG seropositivity was 84.5% and did not differ by age, sex or blood donation site. However, ELISA index values increased with age, with individuals ≥60 years exhibiting higher total MuV antibody levels than donors ≤30 years, primarily driven by males. For most donors, neutralization was consistently higher against JL5 than genotype G, and ELISA index values positively correlated with neutralization titers for both strains. Age positively associated with genotype G neutralization but not JL5, with a significant increase in the genotype G:JL5 neutralization ratio with age (p<0.0001). Males exhibited significantly higher neutralization titers than females against both strains, with older males exhibiting greater neutralization against Genotype G than both age-matched females and younger males. CONCLUSIONS:Our study suggests that antibody quantity alone does not fully predict mumps protection, and factors such as sex may contribute to functional differences in immunity. Our findings highlight potential gaps in protection against circulating strains, possibly contributing to ongoing outbreaks despite high vaccination coverage. The age-dependent increase in total MuV antibodies, which correlates with genotype G neutralization, supports that cumulative antigenic exposure may shape the breadth of MuV-specific immunity over time.

CD4(+) T cells are central regulators of adaptive immune responses, and their depletion following HIV infection leads to AIDS. HIV Nef and Gag weaken CD4(+) T cells by disrupting the actin cytoskeleton, which leads to impaired cell migration and immune synapse formation. Several mechanisms of cortical actin disruption have been proposed; however, a unifying and detailed mechanism has remained elusive. This study investigates how HIV alters the actin cytoskeleton of primary CD4(+) T cells, aiming to reconcile disparate mechanisms reported in the literature. Using a multi-modal approach combining ultrastructural microscopy, time-lapse imaging, small-molecule inhibitors, and proteomics, we identified potential actin regulators affected by HIV. Two distinct lamellipodial abnormalities were observed following infection, both of which are indicative of ARP2/3 inhibition. These morphologies were highly dependent on Nef in vitro and in vivo. Direct chemical inhibition of ARP2/3 recapitulated the lamellipodial defects in Nef-expressing cells. Proteomics of primary cells indicates ARP2/3 inhibition at the lamellipodia of infected T cells may occur through Nef-mediated inhibition of the WAVE2 complex, specifically via inhibitory phosphorylation of WASF2. Together with previous reports of WAVE2 regulation in HIV infection, these results support HIV Nef as a major disruptor of cortical actin through WAVE2-ARP2/3 inhibition at the lamellipodia, providing a novel mechanism of HIV-mediated CD4+ T-cell dysfunction and depletion. IMPORTANCE CD4(+) T cells migrate throughout the body and form immune synapses to carry out their functions. Both of these actions require dynamic actin structures, which are disrupted by HIV proteins. Our study suggests that a key HIV protein, Nef, might disrupt a vital internal cellular machinery that helps immune cells move and function properly. Our microscopic and proteomics studies suggest a new model in which Nef inhibits a large protein complex at the front of migrating T cells. Restoring this cytoskeletal dysfunction may be key to restoring CD4(+) T-cell survival and function, which may improve adaptive immune responses during HIV infection.

The Nipah and Hendra viruses (NiV and HeV, respectively) are highly pathogenic, with case fatality rates of 40 to 75%, representing substantial public health threats. Although one monoclonal antibody (mAb), mAb102.4, has advanced through phase 1 clinical trials, there remains a critical need for approved therapeutic options against these henipaviruses (HNVs). Development of human mAbs has been constrained by limited access to convalescent patient samples. Here, we describe human mAbs derived from transgenic humanized mice that cross-neutralize extant NiV and HeV strains by binding to their fusion protein (F) or receptor binding protein (RBP). Deep mutational scanning and functional studies demonstrated that the anti-RBP mAb (8G3) targets the receptor binding site and requires multiple simultaneous mutations for escape. Sequence analysis of our anti-F mAbs identified a clonally expanded VH3-33 family with evidence of somatic hypermutation, yielding high-affinity antibodies. Cryo-electron microscopy revealed that our most potent F antibody (2A1) recognizes a conserved quaternary epitope spanning two protomers in trimeric prefusion NiV-F and stabilized, rather than displaced, a key glycan shield, distinguishing it from previously described antibodies targeting this region. The 8G3 and 2A1 mAbs exhibited additive neutralization when combined and provided complete protection against lethal NiV challenge in hamsters when administered individually or as a cocktail, even when treatment was delayed. Using a pseudovirus system, we show that this dual-targeting approach was resilient against a suite of escape mutants compared with monotherapy. Our findings establish a candidate therapeutic strategy that minimizes development of resistance, providing a foundation for next-generation countermeasures against emerging HNVs.

Background/Objectives: We previously developed a low-cost vaccine based on Newcastle disease virus expressing a stabilized pre-fusion spike of SARS-CoV-2 (NDV-HXP-S), which has shown safety and immunogenicity in pre-clinical and clinical studies. Due to the emergence of immune-evasive variants and the need to protect vulnerable populations, we evaluated adjuvanted NDV-HXP-S vaccine formulations to enhance and broaden immune responses. Methods: We tested the antibody responses of mice immunized intramuscularly with an inactivated NDV-HXP-S vaccine adjuvanted with AddaVax, AddaS03, Alhydrogel adjuvant 2% (Alum), or Quil-A. Results: AddaVax, AddaS03, and Alum induced the strongest IgG responses to the ancestral spike protein, boosted cross-reactive antibodies against both S1 and S2 subunits, and elicited high cross-neutralizing titers. Conclusions: The present results highlight the critical role of adjuvant selection in shaping both the magnitude and breadth of the immune response induced by the NDV-HXP-S vaccine. AddaVax, AddaS03, and Alum stand out as promising candidates to enhance NDV-HXP-S vaccine immunogenicity, with potential applications in booster strategies against SARS-CoV-2, enabling dose sparing and reducing costs.

Orf virus (ORFV) is a globally distributed zoonotic parapoxvirus that causes a highly contagious mucocutaneous disease in small ruminants. Despite the urgent demand for vaccination-based control, no licensed vaccines are currently available universally. In this study, we generated two recombinant Sendai virus (SeV) vectors expressing ORFV 011 (rSeV-GFP-B2L) and ORFV 059 (rSeV-GFP-059) genes and evaluated their ability to stimulate antiviral responses in vitro. Following the transduction, we assessed transgene expression, innate immune activation, induction of interferon-stimulated genes (A3Z1, OBST2, SAMHD1), and antiviral activity. Both vectors significantly upregulated pattern recognition receptors (TLRs, RIG-I) and type I interferon (IFN-β) genes, with rSeV-GFP-059 inducing the strongest response. Remarkably, OBST2 was robustly upregulated, suggesting a potential role in restricting ORFV replication. Antiviral activity assays revealed a marked reduction in ORFV DNA copies and a mild decrease in ORFV RNA transcription in rSeV-GFP-059-transduced cells, particularly at later time points, accompanied by complete abrogation of the typical cytopathic effect. Collectively, these results demonstrate that SeV-based vectors, particularly rSeV-GFP-059, efficiently prime antiviral immunity and suppress ORFV replication, establishing a promising platform for further in vivo vaccine evaluation in sheep.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nonstructural protein 15 (Nsp15) is a conserved uridine-specific endoribonuclease (EndoU) and is implicated in innate immune evasion, yet its precise molecular mechanism remains incompletely understood. Here, we demonstrate that Nsp15 limits antiviral innate immune responses in part by downregulating the cGAS-STING pathway. To investigate how Nsp15 antagonizes host innate immune responses, we engineered recombinant SARS-CoV-2 bearing WT or EndoU-inactive Nsp15 (H234A). Compared with the WT virus, the Nsp15-H234A mutant exhibited a 2-log decrease in peak viral titres in interferon (IFN)-competent A549-ACE2 cells, but not in their STAT1 knockout counterparts. This attenuation was partially reversed by STING knockout or STING inhibitors, highlighting STING's involvement in Nsp15-driven immune evasion. Transcriptomic analyses revealed the upregulation of IFNs and IFN-stimulated genes in cells infected with the Nsp15-H234A mutant virus. Notably, cGAS and STING transcripts and proteins were suppressed during WT but not mutant virus infections - even in STAT1-deficient cells - suggesting that Nsp15 contributes directly to their downregulation. In vitro, Nsp15 targeted cGAS and STING transcripts in an EndoU activity-dependent manner, thereby reducing cGAS-STING-driven IFN-β and NF-κB reporter activation. Infection of Syrian hamsters confirmed that the Nsp15-H234A mutant virus replicated to lower titres in respiratory tissues and elicited stronger expression of innate immune-related genes. Collectively, these findings define a key strategy by which SARS-CoV-2 Nsp15 subverts the cGAS-STING pathway to facilitate viral replication and immune evasion and underscore Nsp15 EndoU activity as a potential target for future coronavirus antiviral or vaccine design.

Human cytomegalovirus (HCMV) genetic manipulation traditionally relies on bacterial artificial chromosome (BAC) recombineering, necessitated by its large ~236 kb genome. This approach is limited by the scarcity of HCMV strains engineered into BACs and often requires the deletion of ‘non-essential’ genes to accommodate the BAC cassette. We developed a novel approach using temperature-sensitive Sendai virus (SeV) vectors to deliver CRISPR/Cas9 for targeted HCMV genome editing without these constraints. This system achieves high editing efficiency (80–90%) in fibroblasts, epithelial cells and endothelial cells without BAC intermediates. As proof of principle, we targeted the HCMV (TB40/E strain) pentamer complex (PC) genes UL128 and UL130, crucial for viral entry into non-fibroblast cells. Edited viruses showed significantly reduced infectivity in epithelial cells, confirming functional disruption of the PC. Plaque purification yielded isogenic clones with phenotypes comparable to AD169, a naturally PC-deficient strain. Furthermore, multiplexed editing created precise 663 bp deletions in over 60% of viral genomes. Importantly, this method enables HCMV editing in physiologically relevant cell types without fibroblast passaging, which typically introduces mutations. This SeV-Cas9 system represents a significant advancement for studying HCMV biology in diverse cell types.

Zoonotic paramyxoviruses, including the highly pathogenic henipaviruses (HNVs), pose significant risks to global health due to their high mortality rates, potential for human-to-human transmission, and lack of approved countermeasures. Recent metagenomic surveys have uncovered an extensive diversity of HNVs and related paramyxoviruses circulating in wildlife, the majority of which remain uncharacterized due to the dearth of viral isolates. In lieu of viral isolates, reverse genetics systems offer an approach to derive infectious clones de novo in the laboratory, facilitating research into the biology, zoonotic potential, and pathogenicity of novel HNVs. This chapter explores the methodologies and applications of reverse genetics systems for novel HNVs, including considerations for virus sequence validation, full-length virus recovery, and the development of platforms such as minigenomes, replicons, and virus replicon particles. Such biologically-contained life cycle modeling systems enable research to be conducted at lower biocontainment, and provide accessible tools through which to investigate HNV biology. This work demonstrates the versatility of reverse genetics systems in advancing our understanding of high-consequence pathogens, enabling the proactive development of vaccines, antivirals, and diagnostic tools. By integrating these methodologies within a framework of biosafety and biosecurity, researchers can better prepare for and respond to future zoonotic threats.

In April 2024, following the annual International Committee on Taxonomy of Viruses (ICTV) ratification vote on newly proposed taxa, the phylum Negarnaviricota was expanded by 1 new order, 1 new family, 6 new subfamilies, 34 new genera and 270 new species. One class, two orders and six species were renamed. Seven families and 12 genera were moved; ten species were renamed and moved; and nine species were abolished. This article presents the updated taxonomy of Negarnaviricota as currently accepted by the ICTV, providing an essential annual update on the classification of members of this phylum that deepen understandings of their evolution, and supports critical public health measures for virus identification and tracking.

Small ruminant lentiviruses (SRLV) are responsible for significant economic losses in sheep and goat farming; however, effective vaccination strategies remain unavailable. This study evaluated the immunogenicity, safety, and protective efficacy of a recombinant Sendai virus vector (SeV) expressing SRLV gag-P25 (rSeV-GFP-P25) in lambs. Twenty-one SRLV-negative lambs were divided into three groups and inoculated intranasally thrice with culture medium (group 1); SeV-GFP (group 2) or rSeV-GFP-P25 (group 3). Lambs were challenged with homologous SRLV at 16 weeks post-first immunization. Clinical and hematological parameters, antibody responses, SRLV viral loads in peripheral blood mononuclear cells (PBMCs) and target tissues, histopathological and histomorphometric analyses, assisted with artificial intelligence, of interstitial pneumonia were assessed. No clinicopathological alterations were observed, except for a transient temperature increase in group 3 post-first immunization. Group 2 showed mild SeV-neutralizing antibodies, while rSeV-GFP-P25 (group 3) induced negligible SRLV-specific antibody responses. Group 3 exhibited higher SRLV DNA copies in PBMCs but lower in most SRLV target tissues compared to control groups, with no SRLV DNA detected in spleen and bone marrow. Histomorphometry revealed reduced alveolar septal thickening in group 3, indicating partial protection against early SRLV-associated interstitial pneumonia. These results warrant further investigation into cellular immunity and long-term protection.

Recognition of foreign RNA is critical for the innate immune response to viruses. Interferon (IFN)-induced proteins with tetratricopeptide repeats (IFIT) 2 and 3 are highly upregulated following viral infection, but mechanistic insight into their antiviral role is lacking. Here we demonstrate that short 5' untranslated regions (UTRs), a characteristic of many viral mRNAs, can serve as a molecular pattern for innate immune recognition via IFIT2 and IFIT3. Structure determination of the IFIT2-IFIT3 complex at 3.2 Å using cryo-EM reveals a domain-swapped heterodimer that is required for recognition of the viral mRNA 5' end, translation inhibition and antiviral activity. Critically, viral or host 5' UTR lengths less than 50 nucleotides are necessary and sufficient to enable translation inhibition by the IFIT2-IFIT3 complex. Accordingly, diverse viruses whose mRNAs contain short 5' UTRs, such as vesicular stomatitis virus and parainfluenza virus 3, are sensitive to IFIT2-IFIT3-mediated antiviral activity. Our work thus reveals a pattern of antiviral nucleic acid immune recognition that takes advantage of the inherent constraints on viral genome size.

Understanding the breadth and functional profile of T cell responses is crucial for assessing their role in immune surveillance of emerging SARS-CoV-2 variants. Sampling healthy individuals, we profiled the kinetics and polyfunctionality of T cell immunity elicited by mRNA vaccination. Modeling of anti-spike T cell responses against ancestral and variant strains suggested epitope immunodominance and cross-reactivity as major predictive determinants of T cell immunity. To identify immunodominant epitopes, we comprehensively mapped CD4+ and CD8+ T cell epitopes within non-spike proteins using samples from convalescent patients. We found that immunodominant epitopes mainly resided within regions that were minimally disrupted by emerging mutations. Conservation analysis across human coronaviruses and in silico alanine scanning highlighted the functional importance of mutationally constrained immunodominant regions. Collectively, these findings identify immunodominant T cell epitopes across the SARS-CoV-2 proteome that may enhance immune surveillance against emerging variants and inform next-generation vaccine designs providing broader and more durable protection.

Mumps virus (MuV) remains active worldwide, despite high vaccine coverage. MuV is thought to infect the upper respiratory tract before disseminating to other organs; however, the early cellular targets of MuV in vivo are unknown. We generated a GFP-tagged vaccine strain (JL5) of MuV to infect leukocytic cell lines and found that replication was greatest in monocytes. Infection of PBMCs also showed that both JL5 and a circulating strain of MuV (Iowa 2006; genotype G), preferentially infected monocytes. Monocyte-derived macrophages showed high susceptibility to MuV, with genotype G infecting macrophages to a greater extent. While mice are generally resistant to MuV infection, we inoculated immunocompetent Rosa26-tdTomato mice intranasally with a GFP and Cre recombinase tagged MuV to determine whether monocytes/macrophages are important targets in vivo. We observed a small population of tdtomato+ cells within the lungs and found they were primarily alveolar macrophages (AMs). We validated these findings by infecting murine AMs isolated from Rosa26-tdTomato mice with MuV. While MuV could enter AMs (tdTomato+), only a small subset expressed GFP, suggesting that inhibition in murine cells occurs post-entry. We then isolated cells from human bronchioalveolar lavage fluid and infected them with MuV ex vivo, finding the majority of GFP+ cells were AMs. These findings highlight the high susceptibility of AMs and provide a basis for early MuV pathogenesis and subsequent dissemination. Supported by: U19AI171403, PR192188, R01AI188431, T32GM146636 Viral Immunology (VIR)

Small ruminant lentiviruses (SRLV) cause multisystemic chronic inflammatory disease and significant economic losses in sheep and goats worldwide. However, no vaccines or therapies are currently available. In this study, a recombinant Sendai virus (SeV) vector encoding the SRLV gag-P25 gene (rSeV-GFP-P25) from the EV1 strain was generated using In-FUSION cloning and rescued using the SeV reverse genetic system. Transgene expression and stimulation of innate immunity and interferon-stimulated genes (ovine A3Z1, OBST2 and SAMHD1) were evaluated in ovine skin fibroblasts (OSF) transduced with SeV-GFP and rSeV-GFP-P25. Additionally, to characterize the effect of the SRLV restriction in transduced OSF, the SRLV DNA load was quantified at different times post-transduction and post-infection with strain EV1. Using immunohistochemistry and image analysis, transgene expression and tissue distribution of recombinant P25 were studied in two lambs inoculated intranasally, one with rSeV-GFP-P25 and the other with SeV-GFP. rSeV-GFP-P25 induced efficient and transient transgene expression in vitro and in vivo. Furthermore, OSF transduced with rSeV-GFP-P25 presented upregulation of TLR2, TLR3, TLR6, TLR7, RIG-I, MyD88 and IFN-β, whereas SeV-GFP did not induce TLR6 or IFN-β upregulation. Among the interferon-stimulated genes, OBST2 was significantly upregulated after transduction with rSeV-GFP-P25 compared with the empty vector. SRLV restriction gradually increased and persisted after transduction with SeV-GFP and rSeV-GFP-P25, with OSF transduced three times showing cumulative restriction. Forty-eight hours post-inoculation in vivo, marked P25 expression was observed in ciliated epithelial cells and submucosal macrophages/dendritic cells of the nasal mucosa. This study reinforces the important role of the innate immune response in controlling SRLV infection and suggests that rSeV-GFP-P25 is a potential vaccine candidate against SRLV.

Morbilliviruses, including measles virus (MV), canine distemper virus (CDV), peste des petits ruminants virus, and cetacean morbillivirus pose a significant threat to humans and animals. While the host range of morbilliviruses is generally well-defined, cross-species transmission events with significant mortality have also been reported. Their entry into immune cells, the primary targets of morbilliviruses, relies on the signaling lymphocytic activation molecule (SLAM), also known as SLAMF1 or CD150. In this study, we hypothesize that the ability of morbilliviruses to utilize heterologous SLAM receptors stems from evolutionarily conserved structural determinants within the SLAM protein and that minimal genetic changes in the viral receptor-binding H protein can enable adaptation to novel hosts. To test this, we systematically assessed SLAM utilization and adaptation by diverse morbilliviruses. We found that most morbilliviruses efficiently utilize SLAM from multiple host species, including Myotis bat SLAM, but not human SLAM. Only MV could efficiently utilize human SLAM. Additionally, unlike other morbilliviruses, MV utilized Myotis bat SLAM inefficiently. As an example of morbillivirus adaptation to non-host animal SLAM, we conducted an MV adaptation experiment with Myotis bat SLAM. We demonstrated that MV readily adapted to utilize Myotis bat SLAM by acquiring a single N187Y mutation in its hemagglutinin protein. Notably, hypothetical ancestral SLAMs acted as universal receptors for all morbilliviruses. These results reinforced that morbillivirus receptor usage is primarily supported by evolutionarily conserved structural features of SLAM, highlighting a molecular basis that enables morbilliviruses to rapidly adapt to diverse animal SLAMs.

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 results 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 demonstrate binding to measles antigens and neutralization of authentic measles virus.

Formerly a common childhood pathogen, mumps virus (MuV) remains active worldwide, despite relatively high vaccine coverage. MuV is thought to infect the upper respiratory tract before disseminating to other organs; however, the early cellular targets of MuV in vivo are unknown. To address this, we generated a green fluorescent protein (GFP)-tagged vaccine strain (JL5) of MuV to infect leukocytic cell lines and found that replication was greatest in monocytes. Infection of peripheral blood mononuclear cells (PBMCs) also showed that both JL5 and a circulating strain of MuV (Iowa 2006; genotype G), preferentially infected monocytes. Further, monocyte-derived macrophages showed high susceptibility to MuV, with genotype G infecting macrophages to a much greater extent. While mice are generally resistant to MuV infection, we inoculated immunocompetent Rosa26-tdTomato mice intranasally with a GFP and Cre recombinase tagged MuV to determine whether monocytes/macrophages are important targets in vivo. We observed a small population of tdTomato + cells within the lungs, which included epithelial cells; however, the vast majority were alveolar macrophages (AMs). To validate these findings, we infected murine AMs isolated from Rosa26-tdTomato mice with the GFP and Cre recombinase tagged MuV and found that while MuV could enter AMs, as determined by tdTomato positivity, only a small percentage of these expressed GFP, suggesting that inhibition in murine cells occurs postentry. To translate these findings, we infected cells from human bronchoalveolar lavage fluid with MuV and found that most infected cells were AMs. These findings highlight the high susceptibility of AMs and provide a basis for early MuV pathogenesis and subsequent dissemination.