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Abstract Roberts syndrome (RBS) is an autosomal recessive disorder with profound growth deficiency and limb reduction caused by ESCO2 loss-of-function variants. Here, we elucidate the pathogenesis of limb reduction in an Esco2 fl/fl;Prrx1-Cre Tg/0 mouse model using bulk- and single-cell-RNA-seq and gene co-expression network analyses during embryogenesis. Our results reveal morphological and vascular defects culminating in hemorrhage of mutant limbs at E12.5. Underlying this abnormal developmental progression is a pre-apoptotic, mesenchymal cell population specific to mutant limb buds enriched for p53-related signaling beginning at E9.5. We then characterize these p53-related processes of cell cycle arrest, DNA damage, cell death, and the inflammatory leukotriene signaling pathway in vivo. In utero treatment with pifithrin-α, a p53 inhibitor, rescued the hemorrhage in mutant limbs. Lastly, significant enrichments were identified among genes associated with RBS, thalidomide embryopathy, and other genetic limb reduction disorders, suggesting a common vascular etiology among these conditions.

Abstract Sero-monitoring provides context to the epidemiology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections and changes in population immunity following vaccine introduction. Here, we describe results of a cross-sectional hospital-based study of anti-spike seroprevalence in New York City (NYC) from February 2020 to July 2022, and a follow-up period from August 2023 to October 2023. Samples from 55,092 individuals, spanning five epidemiological waves were analyzed. Prevalence ratios (PR) were obtained using Poisson regression. Anti-spike antibody levels increased gradually over the first two waves, with a sharp increase during the 3rd wave coinciding with SARS-CoV-2 vaccination in NYC resulting in seroprevalence levels >90% by July 2022. Our data provide insights into the dynamic changes in immunity occurring in a large and diverse metropolitan community faced with a new viral pathogen and reflects the patterns of antibody responses as the pandemic transitions into an endemic stage.

ABSTRACT Methicillin-resistant Staphylococcus aureus (MRSA) bloodstream infection (BSI) is a serious public health concern. At times, MRSA is isolated from the blood along with other pathogens, the significance and consequences of which are not well described. This study aims to outline the clinical characteristics and outcomes of those with polymicrobial MRSA BSI compared with those with monomicrobial MRSA BSI. We conducted a retrospective case-control study of those with and without polymicrobial MRSA BSI from 2014 to 2022 at a single quaternary care center in New York City. Risk factors and outcomes for polymicrobial MRSA BSI were assessed using logistic regression analyses. Of 559 patients with MRSA BSI during the study period, 49 (9%) had polymicrobial MRSA BSI. Gram-positive Enterococcus (23%) was the most common co-pathogen. The presence of urinary (P = 0.02) and gastrointestinal (P < 0.01) devices was significantly associated with polymicrobial MRSA BSI. Polymicrobial MRSA BSI was associated with intensive care unit (ICU) admission after BSI (P = 0.01). Mortality did not differ. While polymicrobial MRSA BSI is relatively uncommon, it complicates an already complex clinical scenario of MRSA BSI.IMPORTANCEStaphylococcus aureus is a common human pathogen associated with severe disease and high mortality rates. Although clinically observed, little is known about the impact of polymicrobial staphylococcal bloodstream infection. This study evaluates polymicrobial methicillin-resistant S. aureus bloodstream infection (BSI), highlighting the increased risk of intensive care unit admission and impact on morbidity. Identifying risk factors for polymicrobial BSI, such as the presence of specific devices, can aid in early recognition and targeted interventions. Clarifying the risks and outcomes of polymicrobial infections can lead to strategies to minimize and manage these infections and explore the potential interactions between pathogens.

Abstract Human infections caused by viral pathogens trigger a complex gamut of host responses that limit disease, resolve infection, generate immunity, and contribute to severe disease or death. Here, we present experimental methods and multi-omics data capture approaches representing the global host response to infection generated from 45 individual experiments involving human viruses from the Orthomyxoviridae, Filoviridae, Flaviviridae, and Coronaviridae families. Analogous experimental designs were implemented across human or mouse host model systems, longitudinal samples were collected over defined time courses, and global multi-omics data (transcriptomics, proteomics, metabolomics, and lipidomics) were acquired by microarray, RNA sequencing, or mass spectrometry analyses. For comparison, we have included transcriptomics datasets from cells treated with type I and type II human interferon. Raw multi-omics data and metadata were deposited in public repositories, and we provide a central location linking the raw data with experimental metadata and ready-to-use, quality-controlled, statistically processed multi-omics datasets not previously available in any public repository. This compendium of infection-induced host response data for reuse will be useful for those endeavouring to understand viral disease pathophysiology and network biology.

ABSTRACTSARS-CoV-2 Omicron subvariants are still emerging and spreading worldwide. These variants contain a high number of polymorphisms in the spike (S) glycoprotein that could potentially impact their pathogenicity and transmission. We have previously shown that the S:655Y and P681H mutations enhance S protein cleavage and syncytia formation. Interestingly, these polymorphisms are present in Omicron S protein. Here, we characterized the cleavage efficiency and fusogenicity of the S protein of different Omicron sublineages. Our results showed that Omicron BA.1 subvariant is efficiently cleaved but it is poorly fusogenic compared to previous SARS-CoV-2 strains. To understand the basis of this phenotype, we generated chimeric S protein using combinations of the S1 and S2 domains from WA1, Delta and Omicron BA.1 variants. We found that the S2 domain of Omicron BA.1 hindered efficient cell–cell fusion. Interestingly, this domain only contains six unique polymorphisms never detected before in ancestral SARS-CoV-2 variants. WA1614G S proteins containing the six individuals S2 Omicron mutations were assessed for their fusogenicity and S surface expression after transfection in cells. Results showed that the S:N856K and N969K substitutions decreased syncytia formation and impacted S protein cell surface levels. However, we observed that “first-generation” Omicron sublineages that emerged subsequently, had convergently evolved to an enhanced fusogenic activity and S expression on the surface of infected cells while “second-generation” Omicron variants have highly diverged and showed lineage-specific fusogenic properties. Importantly, our findings could have potential implications in the improvement and redesign of COVID-19 vaccines.

Abstract Post-acute sequelae of SARS-CoV-2 (PASC) is a significant public health concern. We describe Patient Reported Outcomes (PROs) on 590 participants prospectively assessed from hospital admission for COVID-19 through one year after discharge. Modeling identified 4 PRO clusters based on reported deficits (minimal, physical, mental/cognitive, and multidomain), supporting heterogenous clinical presentations in PASC, with sub-phenotypes associated with female sex and distinctive comorbidities. During the acute phase of disease, a higher respiratory SARS-CoV-2 viral burden and lower Receptor Binding Domain and Spike antibody titers were associated with both the physical predominant and the multidomain deficit clusters. A lower frequency of circulating B lymphocytes by mass cytometry (CyTOF) was observed in the multidomain deficit cluster. Circulating fibroblast growth factor 21 (FGF21) was significantly elevated in the mental/cognitive predominant and the multidomain clusters. Future efforts to link PASC to acute anti-viral host responses may help to better target treatment and prevention of PASC.

Abstract The glycosylation of IgG plays a critical role during human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, activating immune cells and inducing cytokine production. However, the role of IgM N-glycosylation has not been studied during human acute viral infection. The analysis of IgM N-glycosylation from healthy controls and hospitalized coronavirus disease 2019 (COVID-19) patients reveals increased high-mannose and sialylation that correlates with COVID-19 severity. These trends are confirmed within SARS-CoV-2-specific immunoglobulin N-glycan profiles. Moreover, the degree of total IgM mannosylation and sialylation correlate significantly with markers of disease severity. We link the changes of IgM N-glycosylation with the expression of Golgi glycosyltransferases. Lastly, we observe antigen-specific IgM antibody-dependent complement deposition is elevated in severe COVID-19 patients and modulated by exoglycosidase digestion. Taken together, this work links the IgM N-glycosylation with COVID-19 severity and highlights the need to understand IgM glycosylation and downstream immune function during human disease.

Abstract Persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have been reported in immune-compromised individuals and people undergoing immune-modulatory treatments. Although intrahost evolution has been documented, direct evidence of subsequent transmission and continued stepwise adaptation is lacking. Here we describe sequential persistent SARS-CoV-2 infections in three individuals that led to the emergence, forward transmission, and continued evolution of a new Omicron sublineage, BA.1.23, over an eight-month period. The initially transmitted BA.1.23 variant encoded seven additional amino acid substitutions within the spike protein (E96D, R346T, L455W, K458M, A484V, H681R, A688V), and displayed substantial resistance to neutralization by sera from boosted and/or Omicron BA.1-infected study participants. Subsequent continued BA.1.23 replication resulted in additional substitutions in the spike protein (S254F, N448S, F456L, M458K, F981L, S982L) as well as in five other virus proteins. Our findings demonstrate not only that the Omicron BA.1 lineage can diverge further from its already exceptionally mutated genome but also that patients with persistent infections can transmit these viral variants. Thus, there is, an urgent need to implement strategies to prevent prolonged SARS-CoV-2 replication and to limit the spread of newly emerging, neutralization-resistant variants in vulnerable patients.

Summary: Background: The real-world impact of bivalent vaccines for wild type (WA.1) and Omicron variant (BA.5) is largely unknown in immunocompromised patients with Multiple Myeloma (MM). We characterize the humoral and cellular immune responses in patients with MM before and after receiving the bivalent booster, including neutralizing assays to identify patterns associated with continuing vulnerability to current variants (XBB1.16, EG5) in the current post-pandemic era. Methods: We studied the humoral and cellular immune responses before and after bivalent booster immunization in 48 MM patients. Spike binding IgG antibody levels were measured by SARS-CoV-2 spike binding ELISA and neutralization capacity was assessed by a SARS-CoV-2 multi-cycle microneutralization assays to assess inhibition of live virus. We measured spike specific T-cell function using the QuantiFERON SARS-CoV-2 (Qiagen) assay as well as flow-cytometry based T-cell. In a subset of 38 patients, high-dimensional flow cytometry was performed to identify immune cell subsets associated with lack of humoral antibodies. Findings: We find that bivalent vaccination provides significant boost in protection to the omicron variant in our MM patients, in a treatment specific manner. MM patients remain vulnerable to newer variants with mutations in the spike portion. Anti-CD38 and anti-BCMA therapies affect the immune machinery needed to produce antibodies. Interpretation: Our study highlights varying immune responses observed in MM patients after receiving bivalent COVID-19 vaccination. Specifically, a subgroup of MM patients undergoing anti-CD38 and anti-BCMA therapy experience impairment in immune cells such DCs, B cells, NK cells and TFH cells, leading to an inability to generate adequate humoral and cellular responses to vaccination. Funding: National Cancer Institute (National Institutes of Health), National Institute of Allergy and Infectious Diseases (National Institutes of Health), NCI Serological Sciences Network for COVID-19 (SeroNet) and The Icahn School of Medicine at Mount Sinai.

ABSTRACTMonkeypox (MPOX) is a zoonotic disease endemic to regions of Central/Western Africa. The geographic endemicity of MPV has expanded, broadening the human-monkeypox virus interface and its potential for spillover. Since May 2022, a large multi-country MPV outbreak with no proven links to endemic countries has originated in Europe and has rapidly expanded around the globe, setting off genomic surveillance efforts. Here, we conducted a genomic analysis of 23 MPV-infected patients from New York City during the early outbreak, assessing the phylogenetic relationship of these strains against publicly available MPV genomes. Additionally, we compared the genomic sequences of clinical isolates versus culture-passaged samples from a subset of samples. Phylogenetic analysis revealed that MPV genomes included in this study cluster within the B.1 lineage (Clade IIb), with some of the samples displaying further differentiation into five different sub-lineages of B.1. Mutational analysis revealed 55 non-synonymous polymorphisms throughout the genome, with some of these mutations located in critical regions required for viral multiplication, structural and assembly functions, as well as the target region for antiviral treatment. In addition, we identified a large majority of polymorphisms associated with GA > AA and TC > TT nucleotide replacements, suggesting the action of human APOBEC3 enzyme. A comparison between clinical isolates and cell culture-passaged samples failed to reveal any difference. Our results provide a first glance at the mutational landscape of early MPV-2022 (B.1) circulating strains in NYC.

Since late 2020, SARS-CoV-2 variants have regularly emerged with competitive and phenotypic differences from previously circulating strains, sometimes with the potential to escape from immunity produced by prior exposure and infection. The Early Detection group is one of the constituent groups of the US National Institutes of Health National Institute of Allergy and Infectious Diseases SARS-CoV-2 Assessment of Viral Evolution program. The group uses bioinformatic methods to monitor the emergence, spread, and potential phenotypic properties of emerging and circulating strains to identify the most relevant variants for experimental groups within the program to phenotypically characterize. Since April 2021, the group has prioritized variants monthly. Prioritization successes include rapidly identifying most major variants of SARS-CoV-2 and providing experimental groups within the National Institutes of Health program easy access to regularly updated information on the recent evolution and epidemiology of SARS-CoV-2 that can be used to guide phenotypic investigations.

We report the spillover of highly pathogenic avian influenza A(H5N1) into marine mammals in the northeastern United States, coincident with H5N1 in sympatric wild birds. Our data indicate monitoring both wild coastal birds and marine mammals will be critical to determine pandemic potential of influenza A viruses.

Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in hospitalized and non-hospitalized patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 SIMPLE clinical studies evaluating RDV in hospitalized participants with severe or moderate COVID-19 disease. The severe and moderate studies enrolled participants with radiologic evidence of pneumonia and a room-air oxygen saturation of ≤94% or >94%, respectively. Virology sample collection was optional in the study protocols. Sequencing and related viral load data were obtained retrospectively from participants at a subset of study sites with local sequencing capabilities (10 of 183 sites) at timepoints with detectable viral load. Among participants with both baseline and post-baseline sequencing data treated with RDV, emergent Nsp12 substitutions were observed in 4 of 19 (21%) participants in the severe study and none of the 2 participants in the moderate study. The following 5 substitutions emerged: T76I, A526V, A554V, E665K, and C697F. The substitutions T76I, A526V, A554V, and C697F had an EC50 fold change of ≤1.5 relative to the wildtype reference using a SARS-CoV-2 subgenomic replicon system, indicating no significant change in the susceptibility to RDV. The phenotyping of E665K could not be determined due to a lack of replication. These data reveal no evidence of relevant resistance emergence and further confirm the established efficacy profile of RDV with a high resistance barrier in COVID-19 patients.

Abstract Influenza B virus (IBV) strains are one of the components of seasonal influenza vaccines in both trivalent and quadrivalent formulations. The vast majority of these vaccines are produced in embryonated chickens’ eggs. While optimized backbones for vaccine production in eggs exist and are in use for influenza A viruses, no such backbones exist for IBVs, resulting in unpredictable production yields. To generate an optimal vaccine seed virus backbone, we have compiled a panel of 71 IBV strains from 1940 to present day, representing the known temporal and genetic variability of IBV circulating in humans. This panel contains strains from the B/Victoria/2/87-like lineage, B/Yamagata/16/88-like lineage and the ancestral lineage that preceded their split to provide a diverse set that would help to identify a suitable backbone which can be used in combination with hemagglutinin (HA) and neuraminidase (NA) glycoproteins from any IBV strain to be incorporated into the seasonal vaccine. We have characterized and ranked the growth profiles of the 71 IBV strains and the best performing strains were used for co-infection of eggs, followed by serial passaging to select for high-growth reassortant viruses. After serial passaging, we selected 10 clonal isolates based on their growth profiles assessed by hemagglutination and plaque-forming units. We then generated reverse genetics systems for the three clones that performed best in growth curves. The selected backbones were then used to generate different reassortant viruses with HA/NA combinations from high and low titer yielding wild type IBV. When the growth profiles of the recombinant reassortant viruses were tested, the low titer yielding HA/NA viruses with the selected backbones yielded higher titers similar to those from high titer yielding HA/NA combinations. The use of these IBV backbones with improved replication in eggs might increase yields for the influenza B virus components of seasonal influenza virus vaccines.

Antibodies that broadly inhibit influenza virus neuraminidase by binding to its active site could be therapeutic candidates, but circulating viruses have acquired a glycosylation site in that region. Here, the authors show that, while the S245N glycosylation site affects binding of tested monoclonal antibodies, protective activity in a mouse model is maintained.

Abstract A phase 1 clinical trial to test the immunogenicity of a chimeric group 1 HA (cHA) universal influenza virus vaccine targeting the conserved stalk domain of the hemagglutinin of influenza viruses was carried out. Vaccination with adjuvanted-inactivated vaccines induced high anti-stalk antibody titers. We sought to identify gene expression signatures that correlate with such induction. Messenger-RNA sequencing in whole blood was performed on the peripheral blood of 53 vaccinees. We generated longitudinal data on the peripheral blood of 53 volunteers, at early (days 3 and 7) and late (28 days) time points after priming and boosting with cHAs. Differentially expressed gene analysis showed no differences between placebo and live-attenuated vaccine groups. However, an upregulation of genes involved in innate immune responses and type I interferon signaling was found at day 3 after vaccination with inactivated adjuvanted formulations. Cell type deconvolution analysis revealed a significant enrichment for monocyte markers and different subsets of dendritic cells as mediators for optimal B cell responses and significant increase of anti-stalk antibodies in sera. A significant upregulation of immunoglobulin-related genes was only observed after administration of adjuvanted vaccines (either as primer or booster) with specific induction of anti-stalk IGVH1-69. This approach informed of specific immune signatures that correlate with robust anti-stalk antibody responses, while also helping to understand the regulation of gene expression induced by cHA proteins under different vaccine regimens.

Avian influenza viruses of the H6 subtype are prevalent in wild ducks and likely play an important role in the ecology of influenza viruses through reassortment with other avian influenza viruses. Yet, only 152 Vietnamese H6 virus sequences were available in GISAID (Global Initiative on Sharing All Influenza Data) prior to this study with the most recent sequences being from 2018. Through surveillance in Vietnamese live bird markets from 2018 to 2021, we identified 287 samples containing one or several H6 viruses and other influenza A virus subtypes, demonstrating a high rate of co-infections among birds in Vietnamese live bird markets. For the 132 H6 samples with unique influenza virus sequences, we conducted phylogenetic and genetic analyses. Most of the H6 viruses were similar to each other and closely related to other H6 viruses; however, signs of reassortment with other avian influenza viruses were evident. At the genetic level, the Vietnamese H6 viruses characterized in our study encode a single basic amino acid at the HA cleavage site, consistent with low pathogenicity in poultry. The Vietnamese H6 viruses analyzed here possess an amino acid motif in HA that confers binding to both avian- and human-type receptors on host cells, consistent with their ability to infect mammals. The frequent detection of H6 viruses in Vietnamese live bird markets, the high rate of co-infections of birds with different influenza viruses, and the dual receptor-binding specificity of these viruses warrant their close monitoring for potential infection and spread among mammals.

Abstract Background The SARS-CoV-2 virus is responsible for the COVID-19 pandemic. To better understand the evolution of SARS-CoV-2 early in the pandemic in the Province of Cordoba, Argentina, we performed a comparative genomic analysis of SARS-CoV-2 strains detected in survivors and non-survivors of COVID-19. We also carried out an epidemiological study to find a possible association between the symptoms and comorbidities of these patients with their clinical outcomes. Results A representative sampling was performed in different cities in the Province of Cordoba. Ten and nine complete SARS-CoV-2 genomes were obtained by next-generation sequencing of nasopharyngeal specimens from non-survivors and survivors, respectively. Phylogenetic and phylodynamic analyses revealed multiple introductions of the most common lineages in South America, including B.1, B.1.1.1, B.1.499, and N.3. Fifty-six mutations were identified, with 14% of those in common between the non-survivor and survivor groups. Specific SARS-CoV-2 mutations for survivors constituted 25% whereas for non-survivors they were 41% of the repertoire, indicating partial selectivity. The non-survivors’ variants showed higher diversity in 9 genes, with a majority in Nsp3, while the survivors’ variants were detected in 5 genes, with a higher incidence in the Spike protein. At least one comorbidity was present in 60% of non-survivor patients and 33% of survivors. Age 75–85 years (p = 0.018) and hospitalization (p = 0.019) were associated with non-survivor patients. Related to the most common symptoms, the prevalence of fever was similar in both groups, while dyspnea was more frequent among non-survivors and cough among survivors. Conclusions This study describes the association of clinical characteristics with the clinical outcomes of survivors and non-survivors of COVID-19 patients, and the specific mutations found in the genome sequences of SARS-CoV-2 in each patient group. Future research on the functional characterization of novel mutations should be performed to understand the role of these variations in SARS-CoV-2 pathogenesis and COVID-19 disease outcomes. These results add new genomic data to better understand the evolution of the SARS-CoV-2 variants that spread in Argentina during the first wave of the COVID-19 pandemic.

ABSTRACT Nosocomial infections caused by multidrug-resistant (MDR) Enterobacter cloacae complex (ECC) pathogens are on the rise. However, the virulence strategies employed by these pathogens remain elusive. Here, we study the interaction of ECC clinical isolates with human serum to define how this pathogen evades the antimicrobial action of complement, one of the first lines of host-mediated immune defense. We identified a small number of serum-sensitive strains, including Enterobacter hormaechei strain NR3055, which we exploited for the in vitro selection of serum-resistant clones. Comparative genomics between the serum-sensitive NR3055 strain and the isolated serum-resistant clones revealed a premature stop codon in the wzy gene of the capsular polysaccharide biosynthesis locus of NR3055. The complementation of wzy conferred serum resistance to NR3055, prevented the deposition of complement proteins on the bacterial surface, inhibited phagocytosis by human neutrophils, and rendered the bacteria virulent in a mouse model of peritonitis. Mice exposed to a nonlethal dose of encapsulated NR3055 were protected from subsequent lethal infections by encapsulated NR3055, whereas mice that were previously exposed to unencapsulated NR3055 succumbed to infection. Thus, capsule is a key immune evasion determinant for E. hormaechei, and it is a potential target for prophylactics and therapeutics to combat these increasingly MDR human pathogens. IMPORTANCE Infections caused by antimicrobial resistant bacteria are of increasing concern, especially those due to carbapenem-resistant Enterobacteriaceae pathogens. Included in this group are species of the Enterobacter cloacae complex, regarding which there is a paucity of knowledge on the infection biology of the pathogens, despite their clinical relevance. In this study, we combine techniques in comparative genomics, bacterial genetics, and diverse models of infection to establish capsule as an important mechanism of Enterobacter pathogens to resist the antibacterial activity of serum, a first line of host defense against bacterial infections. We also show that immune memory targeting the Enterobacter capsule protects against lethal infection. The further characterization of Enterobacter infection biology and the immune response to infection are needed for the development of therapies and preventative interventions targeting these highly antibiotic resistant pathogens.

Abstract Background Healthcare-associated infections pose a potentially fatal threat to patients worldwide and Staphylococcus aureus is one of the most common causes of healthcare-associated infections. S. aureus is a common commensal pathogen and a frequent cause of bacteremia, with studies demonstrating that nasal and blood isolates from single patients match more than 80% of the time. Here we report on a contemporary collection of colonizing isolates from those with methicillin-resistant S. aureus (MRSA) bloodstream infections to evaluate the diversity within hosts, and detail the clinical features associated with concomitant nasal colonization. Methods Swabs of the bilateral anterior nares were obtained from patients diagnosed with MRSA bacteremia. A single colony culture from the blood and an average of 6 colonies from the nares were evaluated for MRSA growth. For the nares cultures, we typed multiple isolates for staphylococcal protein A (spa) and derived the clonal complexes. Demographic and clinical data were obtained retrospectively from the electronic medical record system and analysed using univariate and multivariable regression models. Results Over an 11-month period, 68 patients were diagnosed with MRSA bloodstream infection, 53 were swabbed, and 37 (70%) were colonized with MRSA in the anterior nares. We performed molecular typing on 213 nasal colonies. Spa types and clonal complexes found in the blood were also detected in the nares in 95% of the cases. We also found that 11% of patients carried more than one clone of MRSA in the nares. Male sex and history of prior hospitalization within the past 90 days increased odds for MRSA colonization. Conclusion The molecular epidemiological landscape of colonization in the setting of invasive disease is diverse and defining the interplay between colonization and invasive disease is critical to combating invasive MRSA disease.

This case–control study of 25 cases with methicillin-resistant Staphylococcus aureus (MRSA) bacteremia with vancomycin minimum inhibitory concentration (MIC) ≥ 2 µg/mL and 391 controls (MIC < 2 µg/mL) characterized the clinical characteristics, treatments, and outcomes associated with elevated vancomycin MIC. Elevated vancomycin MIC was associated with baseline hemodialysis, prior MRSA colonization, and metastatic infection.

From 2011-2018, we conducted surveillance in marine mammals along the California coast for influenza A virus (IAV), frequently detecting anti-influenza antibodies and intermittently detecting IAV. In spring 2019, this pattern changed. Despite no change in surveillance intensity, we detected IAV RNA in 10 samples in March and April, mostly in nasal and rectal swabs from northern elephant seals (Mirounga angustirostris). Although virus isolation was unsuccessful, IAV sequenced from one northern elephant seal nasal swab showed close genetic identity with pandemic H1N1 IAV subclade 6B.1A.1 that was concurrently circulating in humans in the 2018/19 influenza season. This represents the first report of human A(H1N1)pdm09 IAV in northern elephant seals since 2010, suggesting IAV continues to spill over from humans to pinnipeds.

Craniofacial development depends on formation and maintenance of sutures between bones of the skull. Here the authors identify enriched expression of the hedgehog inhibitor Hhip, specifically in the mesenchyme of the murine coronal suture, and show sutural dysgenesis in Hhip−/− mutants.

ABSTRACT: Objectives: To evaluate the genomic epidemiology of SARS-CoV-2 from Venezuelan migrants living in Colombia. Methods: This study sequenced SARS-CoV-2 from 30 clinical specimens collected from Venezuelan migrants. Genomes were compared with the Wuhan reference genome to identify polymorphisms, reconstruct phylogenetic relationships and perform comparative genomic analyses. Geographic, sociodemographic and clinical data were also studied across genotypes. Results: This study demonstrated the presence of six distinct SARS-CoV-2 lineages circulating among Venezuelan migrants, as well as a close relationship between SARS-CoV-2 genomic sequences obtained from individuals living in the Venezuelan-Colombian border regions of La Guajira (Colombia) and Zulia (Venezuela). Three clusters (C-1, C-2 and C-3) were well supported by phylogenomic inference, supporting the hypothesis of three potential transmission routes across the Colombian-Venezuelan border. These genomes included point mutations previously associated with increased infectivity. A mutation (L18F) in the N-terminal domain of the spike protein that has been associated with compromised binding of neutralizing antibodies was found in 2 of 30 (6.6%) genomes. A statistically significant association was identified with symptomatology for cluster C2. Conclusion: The close phylogenetic relationships between SARS-CoV-2 genomes from Venezuelan migrants and from people living at the Venezuela-Colombian border support the importance of human movements for the spread of COVID-19 and for emerging virus variants.

Emerging and re-emerging respiratory viruses can spread rapidly and cause pandemics as demonstrated by the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. The early human immune responses to respiratory viruses are in the nasal cavity and nasopharyngeal regions. Defining biomarkers of disease trajectory at the time of a positive diagnostic test would be an important tool to facilitate decisions such as initiation of antiviral treatment. We hypothesize that nasopharyngeal tRNA profiles could be used to predict Coronavirus Disease 19 (COVID-19) severity. We carried out multiplex small RNA sequencing (MSR-seq) on residual nasopharyngeal swabs to measure simultaneously full-length tRNA abundance, tRNA modifications, and tRNA fragmentation for the human tRNA response to SARS-CoV-2 infection. We identified distinct tRNA signatures associated with mild symptoms versus severe COVID-19 manifestations requiring hospitalization. These results highlight the utility of host tRNA properties as biomarkers for the clinical outcome of SARS-CoV-2.

ABSTRACT Current influenza virus vaccines and antivirals have limitations, some of which disproportionately affect their utilization against influenza B viruses. To inform ongoing efforts to address the considerable global burden of influenza B viruses, we previously described five murine monoclonal antibodies that broadly bind conserved epitopes on the neuraminidase of influenza B viruses and protect against lethal challenge in a mouse model when delivered via intraperitoneal injection. Here, we validate the continued relevance of these antibodies by demonstrating that their protective effects extend to lethal challenge with mouse-adapted influenza B viruses recently isolated from humans. We also found that humanization of murine antibodies 1F2 and 4F11 resulted in molecules that retain the ability to protect mice from lethal challenge when administered prophylactically. Intranasal administration as an alternative route of 1F2 delivery revealed no differences in the mouse challenge model compared to intraperitoneal injection, supporting further assessment of this more targeted and convenient administration method. Lastly, we evaluated the potential for intranasal 1F2 administration initiated 1 day after infection to prevent transmission of an influenza B virus between cocaged guinea pigs. Here, we observed a 40% rate of transmission with the 1F2 antibody administered to the infected donor compared to 100% transmission with administration of an irrelevant control antibody. These data suggest that intranasal administration could be a viable route of administration for antibody therapeutics. Collectively, these findings demonstrate the potential of broad antineuraminidase antibodies as therapeutics to prevent and treat infections caused by influenza B viruses. IMPORTANCE The global health burden of influenza B viruses, especially in children, has long been underappreciated. Although two antigenically distinct influenza B virus lineages cocirculated before the coronavirus disease 2019 (COVID-19) pandemic, the commonly used trivalent seasonal vaccines contain antigens from only one influenza B virus, providing limited cross-protection against viruses of the other lineage. Additionally, studies have called into question the clinical effectiveness of the neuraminidase inhibitors that comprise the majority of available antivirals in treating influenza B virus infections. We previously described antibodies that bind broadly to neuraminidases of influenza B viruses across decades of antigenic evolution and potently protect mice against lethal challenge. Here we appraise additional factors to develop these antineuraminidase antibodies as antivirals to prevent and treat infections caused by an extensive range of influenza B viruses. In addition this work assesses recent clinical isolates belonging to the two influenza B virus lineages, finding evidence supporting the development of these antibodies for prophylactic and therapeutic use.

ABSTRACT Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants are characterized by differences in transmissibility and response to therapeutics. Therefore, discriminating among them is vital for surveillance, infection prevention, and patient care. While whole-genome sequencing (WGS) is the “gold standard” for variant identification, molecular variant panels have become increasingly available. Most, however, are based on limited targets and have not undergone comprehensive evaluation. We assessed the diagnostic performance of the highly multiplexed Agena MassARRAY SARS-CoV-2 Variant Panel v3 to identify variants in a diverse set of 391 SARS-CoV-2 clinical RNA specimens collected across our health systems in New York City, USA and Bogotá, Colombia (September 2, 2020 to March 2, 2022). We demonstrated almost perfect levels of interrater agreement between this assay and WGS for 9 of 11 variant calls (κ ≥ 0.856) and 25 of 30 targets (κ ≥ 0.820) tested on the panel. The assay had a high diagnostic sensitivity (≥93.67%) for contemporary variants (e.g., Iota, Alpha, Delta, and Omicron [BA.1 sublineage]) and a high diagnostic specificity for all 11 variants (≥96.15%) and all 30 targets (≥94.34%) tested. Moreover, we highlighted distinct target patterns that could be utilized to identify variants not yet defined on the panel, including the Omicron BA.2 and other sublineages. These findings exemplified the power of highly multiplexed diagnostic panels to accurately call variants and the potential for target result signatures to elucidate new ones. IMPORTANCE The continued circulation of SARS-CoV-2 amid limited surveillance efforts and inconsistent vaccination of populations has resulted in the emergence of variants that uniquely impact public health systems. Thus, in conjunction with functional and clinical studies, continuous detection and identification are quintessential to informing diagnostic and public health measures. Furthermore, until WGS becomes more accessible in the clinical microbiology laboratory, the ideal assay for identifying variants must be robust, provide high resolution, and be adaptable to the evolving nature of viruses like SARS-CoV-2. Here, we highlighted the diagnostic capabilities of a highly multiplexed commercial assay to identify diverse SARS-CoV-2 lineages that circulated from September 2, 2020 to March 2, 2022 among patients seeking care in our health systems. This assay demonstrated variant-specific signatures of nucleotide/amino acid polymorphisms and underscored its utility for the detection of contemporary and emerging SARS-CoV-2 variants of concern.

Summary: Background: Better understanding of the association between characteristics of patients hospitalized with coronavirus disease 2019 (COVID-19) and outcome is needed to further improve upon patient management. Methods: Immunophenotyping Assessment in a COVID-19 Cohort (IMPACC) is a prospective, observational study of 1164 patients from 20 hospitals across the United States. Disease severity was assessed using a 7-point ordinal scale based on degree of respiratory illness. Patients were prospectively surveyed for 1 year after discharge for post-acute sequalae of COVID-19 (PASC) through quarterly surveys. Demographics, comorbidities, radiographic findings, clinical laboratory values, SARS-CoV-2 PCR and serology were captured over a 28-day period. Multivariable logistic regression was performed. Findings: The median age was 59 years (interquartile range [IQR] 20); 711 (61%) were men; overall mortality was 14%, and 228 (20%) required invasive mechanical ventilation. Unsupervised clustering of ordinal score over time revealed distinct disease course trajectories. Risk factors associated with prolonged hospitalization or death by day 28 included age ≥ 65 years (odds ratio [OR], 2.01; 95% CI 1.28–3.17), Hispanic ethnicity (OR, 1.71; 95% CI 1.13–2.57), elevated baseline creatinine (OR 2.80; 95% CI 1.63– 4.80) or troponin (OR 1.89; 95% 1.03–3.47), baseline lymphopenia (OR 2.19; 95% CI 1.61–2.97), presence of infiltrate by chest imaging (OR 3.16; 95% CI 1.96–5.10), and high SARS-CoV2 viral load (OR 1.53; 95% CI 1.17–2.00). Fatal cases had the lowest ratio of SARS-CoV-2 antibody to viral load levels compared to other trajectories over time (p=0.001). 589 survivors (51%) completed at least one survey at follow-up with 305 (52%) having at least one symptom consistent with PASC, most commonly dyspnea (56% among symptomatic patients). Female sex was the only associated risk factor for PASC. Interpretation: Integration of PCR cycle threshold, and antibody values with demographics, comorbidities, and laboratory/radiographic findings identified risk factors for 28-day outcome severity, though only female sex was associated with PASC. Longitudinal clinical phenotyping offers important insights, and provides a framework for immunophenotyping for acute and long COVID-19. Funding: NIH.

We isolated 77 highly pathogenic avian influenza viruses during routine surveillance in live poultry markets in northern provinces of Vietnam from 2018 to 2021. These viruses are of the H5N6 subtype and belong to HA clades 2.3.4.4g and 2.3.4.4h. Interestingly, we did not detect viruses of clade 2.3.4.4b, which in recent years have dominated in different parts of the world. The viruses isolated in this current study do not encode major determinants of mammalian adaptation (e.g., PB2-E627K or PB1-D701N) but possess amino acid substitutions that may affect viral receptor-binding, replication, or the responses to human antiviral factors. Several of the highly pathogenic H5N6 virus samples contained other influenza viruses, providing an opportunity for reassortment. Collectively, our study demonstrates that the highly pathogenic H5 viruses circulating in Vietnam in 2018–2021 were different from those in other parts of the world, and that the Vietnamese H5 viruses continue to evolve through mutations and reassortment.

Abstract Gastrointestinal symptoms are common in COVID-19 patients but the nature of the gut immune response to SARS-CoV-2 remains poorly characterized, partly due to the difficulty of obtaining biopsy specimens from infected individuals. In lieu of tissue samples, we measured cytokines, inflammatory markers, viral RNA, microbiome composition, and antibody responses in stool samples from a cohort of 44 hospitalized COVID-19 patients. SARS-CoV-2 RNA was detected in stool of 41% of patients and more frequently in patients with diarrhea. Patients who survived had lower fecal viral RNA than those who died. Strains isolated from stool and nasopharynx of an individual were the same. Compared to uninfected controls, COVID-19 patients had higher fecal levels of IL-8 and lower levels of fecal IL-10. Stool IL-23 was higher in patients with more severe COVID-19 disease, and we found evidence of intestinal virus-specific IgA responses associated with more severe disease. We provide evidence for an ongoing humeral immune response to SARS-CoV-2 in the gastrointestinal tract, but little evidence of overt inflammation.

Matthew M. Hernandez and Ana S. Gonzalez-Reiche and colleagues report evidence of SARSCoV-2 infections in respiratory pathogen-negative nasopharyngeal specimens collected in New York, which date back to over one month before the first officially documented case in the state. The findings provide insights in to the origins of the virus in New York.

Botrytis cinerea is a necrotrophic fungus characterized mainly by its wide host range of infected plants. The deletion of the white-collar-1 gene (bcwcl1), which encodes for a blue-light receptor/transcription factor, causes a decrease in virulence, particularly when assays are conducted in the presence of light or photocycles. However, despite ample characterization, the extent of the light-modulated transcriptional responses regulated by BcWCL1 remains unknown. In this study, pathogen and pathogen:host RNA-seq analyses, conducted during non-infective in vitro plate growth and when infecting Arabidopsis thaliana leaves, respectively, informed on the global gene expression patterns after a 60 min light pulse on the wild-type B05.10 or ∆bcwcl1 B. cinerea strains. The results revealed a complex fungal photobiology, where the mutant did not react to the light pulse during its interaction with the plant. Indeed, when infecting Arabidopsis, no photoreceptor-encoding genes were upregulated upon the light pulse in the ∆bcwcl1 mutant. Differentially expressed genes (DEGs) in B. cinerea under non-infecting conditions were predominantly related to decreased energy production in response to the light pulse. In contrast, DEGs during infection significantly differ in the B05.10 strain and the ∆bcwcl1 mutant. Upon illumination at 24 h post-infection in planta, a decrease in the B. cinerea virulence-associated transcripts was observed. Accordingly, after a light pulse, biological functions associated with plant defense appear enriched among light-repressed genes in fungus-infected plants. Taken together, our results show the main transcriptomic differences between wild-type B. cinerea B05.10 and ∆bcwcl1 after a 60 min light pulse when growing saprophytically on a Petri dish and necrotrophically over A. thaliana.

Routine surveillance in live poultry markets in the northern regions of Vietnam from 2016 to 2017 resulted in the isolation of 27 highly pathogenic avian H5N1 and H5N6 viruses of 3 different clades (2.3.2.1c, 2.3.4.4f, and 2.3.4.4g). Sequence and phylogenetic analysis of these viruses revealed reassortment with various subtypes of low pathogenic avian influenza viruses. Deep-sequencing identified minor viral subpopulations encoding variants that may affect pathogenicity and sensitivity to antiviral drugs. Interestingly, mice infected with two different clade 2.3.2.1c viruses lost body weight rapidly and succumbed to virus infection, whereas mice infected with clade 2.3.4.4f or 2.3.4.4g viruses experienced non-lethal infections.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been detected in domestic and wild cats. However, little is known about natural viral infections of domestic cats, although their importance for modelling disease spread, informing strategies for managing positive human-animal relationships and disease prevention. Here, we describe the SARS-CoV-2 infection in a household of two human adults and sibling cats (one male and two females) using real-time RT–PCR, an ELISA test, viral sequencing, and virus isolation. On May 5th, 2020, the cat-owners tested positive for SARS-CoV-2. Two days later, the male cat showed mild respiratory symptoms and tested positive. Four days after the male cat, the two female cats became positive, asymptomatically. Also, one human and one cat showed antibodies against SARS-CoV-2. All cats excreted detectable SARS-CoV-2 RNA for a shorter duration than humans and viral sequences analysis confirmed human-to-cat transmission. We could not determine if cat-to-cat transmission also occurred.

Abstract Background Whole-genome sequencing (WGS) is increasingly used to map the spread of bacterial and viral pathogens in nosocomial settings. A limiting factor for more widespread adoption of WGS for hospital infection prevention practices is the availability of standardized tools for genomic epidemiology. Methods We developed the Pathogen Sequencing Phylogenomic Outbreak Toolkit (PathoSPOT) to automate integration of genomic and medical record data for rapid detection and tracing of nosocomial outbreaks. To demonstrate its capabilities, we applied PathoSPOT to complete genome surveillance data of 197 MRSA bacteremia cases from two hospitals during a 2-year period. Results PathoSPOT identified 8 clonal clusters encompassing 33 patients (16.8% of cases), none of which had been recognized by standard practices. The largest cluster corresponded to a prolonged outbreak of a hospital-associated MRSA clone among 16 adults, spanning 9 wards over a period of 21 months. Analysis of precise timeline and location data with our toolkit suggested that an initial exposure event in a single ward led to infection and long-term colonization of multiple patients, followed by transmissions to other patients during recurrent hospitalizations. Conclusions We demonstrate that PathoSPOT genomic surveillance enables the detection of complex transmission chains that are not readily apparent from epidemiological data and that contribute significantly to morbidity and mortality, enabling more effective intervention strategies.

Background: Highly efficacious vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have been developed. However, the emergence of viral variants that are more infectious than the earlier SARS-CoV-2 strains is concerning. Several of these viral variants have the potential to partially escape neutralizing antibody responses, warranting continued immune-monitoring. Methods: We used a panel of 30 post-mRNA vaccination sera to determine neutralization and RBD and spike binding activity against a number of emerging viral variants. The virus neutralization was determined using authentic SARS-CoV-2 clinical isolates in an assay format that mimics physiological conditions. Findings: We tested seven currently circulating viral variants of concern/interest, including the three Iota sublineages, Alpha (E484K), Beta, Delta and Lambda in neutralization assays. We found only small decreases in neutralization against Iota and Delta. The reduction was stronger against a sub-variant of Lambda, followed by Beta and Alpha (E484K). Lambda is currently circulating in parts of Latin America and was detected in Germany, the US and Israel. Of note, reduction in a receptor binding domain and spike binding assay that also included Gamma, Kappa and A.23.1 was negligible. Interpretation: Taken together, these findings suggest that mRNA SARS-CoV-2 vaccines may remain effective against these viral variants of concern/interest and that spike binding antibody tests likely retain specificity in the face of evolving SARS-CoV-2 diversity. Funding: This work is part of the PARIS/SPARTA studies funded by the NIAID Collaborative Influenza Vaccine Innovation Centers (CIVIC) contract 75N93019C00051. In addition, this work was also partially funded by the Centers of Excellence for Influenza Research and Surveillance (CEIRS, contract # HHSN272201400008C), the JPB Foundation, the Open Philanthropy Project (research grant 2020-215611 (5384), by anonymous donors and by the Serological Sciences Network (SeroNet) in part with Federal funds from the National Cancer Institute, National Institutes of Health, under Contract No. 75N91019D00024, Task Order No. 75N91020F00003.

Abstract In this study, we explore the circulation of low pathogenic avian influenza (LPAI) viruses in wild birds and poultry in the Netherlands. Surveillance data collected between 2006 and 2016 was used to evaluate subtype diversity, spatiotemporal distribution and genetic relationships between wild bird and poultry viruses. We observed close species-dependent associations among hemagglutinin and neuraminidase subtypes. Not all subtypes detected in wild birds were found in poultry, suggesting transmission to poultry is selective and likely depends on viral factors that determine host range restriction. Subtypes commonly detected in poultry were in wild birds most frequently detected in mallards and geese. Different temporal patterns in virus prevalence were observed between wild bird species. Virus detections in domestic ducks coincided with the prevalence peak in wild ducks, whereas virus detections in other poultry types were made throughout the year. Genetic analysis of the surface genes demonstrated that most poultry viruses were related to locally circulating wild bird viruses, but no direct spatiotemporal link was observed. Results indicate prolonged undetected virus circulation and frequent reassortment events with local and newly introduced viruses within the wild bird population. Increased knowledge on LPAI virus circulation can be used to improve surveillance strategies.

BackgroundThe SARS-CoV-2 pandemic has forced health authorities across the world to take important decisions to curtail its spread. Genomic epidemiology has emerged as a valuable tool to understand introductions and spread of the virus in a specific geographic location.Methodology/principal findingsHere, we report the sequences of 59 SARS-CoV-2 samples from inhabitants of the Colombian Amazonas department. The viral genomes were distributed in two robust clusters within the distinct GISAID clades GH and G. Spatial-temporal analyses revealed two independent introductions of SARS-CoV-2 in the region, one around April 1, 2020 associated with a local transmission, and one around April 2, 2020 associated with other South American genomes (Uruguay and Brazil). We also identified ten lineages circulating in the Amazonas department including the P.1 variant of concern (VOC).Conclusions/significanceThis study represents the first genomic epidemiology investigation of SARS-CoV-2 in one of the territories with the highest report of indigenous communities of the country. Such findings are essential to decipher viral transmission, inform on global spread and to direct implementation of infection prevention and control measures for these vulnerable populations, especially, due to the recent circulation of one of the variants of concern (P.1) associated with major transmissibility and possible reinfections.

The influenza virus neuraminidase is an emerging target for universal influenza virus vaccines. However, in contrast to influenza virus hemagglutinin, we know little about antibody epitopes and antigenic sites on the neuraminidase. Characterizing and defining these sites is aiding vaccine development and helping to understand antigenic drift of NA.

Broadly protective or universal influenza virus vaccines target viral epitopes that appear to be conserved. However, it is unclear whether the virus will be able to escape once immunological pressure is applied to these epitopes through vaccination of large proportions of the population. Studies that investigate the fitness and antigenic characteristics of viruses that escape immunological pressure on these conserved epitopes are therefore urgently needed.

We describe a case of Lemierre’s syndrome (LS) caused by a hypervirulent strain of Klebsiella pneumoniae in a 63-year-old female with hypertension, hyperlipidemia, and diabetes mellitus, who presented with right neck pain and fevers. Computerized tomography of the neck and chest revealed an occluded right internal jugular vein secondary to thrombosis and septic emboli in lungs. Blood cultures grew K. pneumoniae. The patient was treated with ampicillin-sulbactam and then transitioned to amoxicillin-clavulanate to complete a 6-week course of antibiotics, and a 3-month course of rivaroxaban. String test of the K. pneumoniae isolate was positive at 2 cm. Whole genome sequencing identified several genes associated with the hypervirulent strain, notably the genes encoding for aerobactin (iucA and iucB) and salmochelin (iroB) iron acquisition systems. LS can rarely be caused by K. pneumoniae. Clinicians should monitor for known complications, such as septic emboli in patients with LS.

Abstract Wild aquatic birds are the major reservoir of influenza A virus. Cloacal swabs and feces samples (n = 6595) were collected from 62 bird species in Argentina from 2006 to 2016 and screened for influenza A virus. Full genome sequencing of 15 influenza isolates from 6 waterfowl species revealed subtypes combinations that were previously described in South America (H1N1, H4N2, H4N6 (n = 3), H5N3, H6N2 (n = 4), and H10N7 (n = 2)), and new ones not previously identified in the region (H4N8, H7N7 and H7N9). Notably, the internal gene segments of all 15 Argentine isolates belonged to the South American lineage, showing a divergent evolution of these viruses in the Southern Hemisphere. Time-scaled phylogenies indicated that South American gene segments diverged between ~ 30 and ~ 140 years ago from the most closely related influenza lineages, which include the avian North American (PB1, HA, NA, MP, and NS-B) and Eurasian lineage (PB2), and the equine H3N8 lineage (PA, NP, and NS-A). Phylogenetic analyses of the hemagglutinin and neuraminidase gene segments of the H4, H6, and N8 subtypes revealed recent introductions and reassortment between viruses from the Northern and Southern Hemispheres in the Americas. Remarkably and despite evidence of recent hemagglutinin and neuraminidase subtype introductions, the phylogenetic composition of internal gene constellation of these influenza A viruses has remained unchanged. Considering the extended time and the number of sampled species of the current study, and the paucity of previously available data, our results contribute to a better understanding of the ecology and evolution of influenza virus in South America.

We used epidemiologic and viral genetic information to identify a case of likely reinfection in an otherwise healthy, young Marine recruit enrolled in the prospective, longitudinal COVID-19 Health Action Response for Marines (CHARM) study, and we paired these findings with serological studies. This participant had a positive RT-PCR to SARS-CoV-2 upon routine sampling on study day 7, although he was asymptomatic at that time. He cleared the infection within seven days. On study day 46, he had developed symptoms consistent with COVID-19 and tested positive by RT-PCR for SARS-CoV-2 again. Viral whole genome sequencing was conducted from nares swabs at multiple time points. The day 7 sample was determined to be lineage B.1.340, whereas both the day 46 and day 49 samples were B.1.1. The first positive result for anti-SARS-CoV-2 IgM serology was collected on day 49 and for IgG on day 91. This case appears most consistent with a reinfection event. Our investigation into this case is unique in that we compared sequence data from more than just paired specimens, and we also assayed for immune response after both the initial infection and the later reinfection. These data demonstrate that individuals who have experienced an infection with SARS-CoV-2 may fail to generate effective or long-lasting immunity, similar to endemic human beta coronaviruses.

ABSTRACT We previously attempted to establish a reporter influenza virus by inserting the gene for the Venus fluorescent protein into the NS segment of influenza A/Puerto Rico/8/34 (PR8, H1N1) virus to yield WT-Venus-PR8. Although the inserted Venus gene was deleted during serial passages of WT-Venus-PR8, we discovered that the PB2-E712D mutation stabilizes the Venus gene. Here, we explored the mechanisms by which Venus gene deletion occurs and how the polymerase mutation stabilizes the Venus gene. Deep sequencing analysis revealed that PB2-E712D does not cause an appreciable change in the mutation rate, suggesting that the stability of the Venus gene is not affected by polymerase fidelity. We found by using quantitative real-time PCR that WT-Venus-PR8 induces high-level interferon beta (IFN-β) expression. The induction of IFN-β expression seemed to result from the reduced transcription/replication efficiency of the modified NS segment in WT-Venus-PR8. In contrast, the transcription/replication efficiency of the modified NS segment was enhanced by the PB2-E712D mutation. Loss of the Venus gene in WT-Venus-PR8 appeared to be caused by internal deletions in the NS segment. Moreover, to further our understanding of the Venus stabilization mechanisms, we identified additional amino acid mutations in the virus polymerase complex that stabilize the Venus gene. We found that some of these amino acids are located near the template exit or the product exit of the viral polymerase, suggesting that these amino acids contribute to the stability of the Venus gene by affecting the binding affinity between the polymerase complex and the RNA template and product. IMPORTANCE The reverse genetics method of influenza virus generation has enabled us to generate recombinant viruses bearing modified viral proteins. Recombinant influenza viruses expressing foreign genes have become useful tools in basic research, and such viruses can be utilized as efficient virus vectors or multivalent vaccines. However, the insertion of a foreign gene into the influenza virus genome often impairs virus replication, and the inserted genes are unstable. Elucidation of the mechanisms of foreign gene stabilization will help us to establish useful recombinant influenza viruses.

Routine surveillance and surveillance in response to influenza outbreaks in avian species in Vietnam in 2009–2013 resulted in the isolation of numerous H5N1 influenza viruses of clades 1.1.2, 2.3.2.1a, 2.3.2.1b, 2.3.2.1c, and 2.3.4.1. Consistent with other studies, we found that viruses of clade 2.3.2.1c were dominant in Vietnam in 2013 and circulated in the northern, central, and southern parts of the country. Phylogenetic analysis revealed reassortment among viruses of clades 2.3.2.1a, 2.3.2.1b, and 2.3.2.1c; in contrast, no reassortment was detected between clade 2.3.2.1 viruses and viruses of clades 1.1.2 or 2.3.4.1, respectively. Deep-sequencing of 42 of the 53 isolated H5N1 viruses revealed viral subpopulations encoding variants that may affect virulence, host range, or sensitivity to antiviral compounds; virus isolates containing these subpopulations may have a higher potential to transmit and adapt to mammals. Among the viruses sequenced, a relatively high number of non-synonymous nucleotide polymorphisms was detected in a virus isolated from a barn swallow, possibly suggesting influenza virus adaption to this host.

One diagnostic feature of craniosynostosis syndromes is mandibular dysgenesis. Using three mouse models of Apert, Crouzon and Pfeiffer craniosynostosis syndromes, we investigated how embryonic development of the mandible is affected by fibroblast growth factor receptor 2 (Fgfr2) mutations. Quantitative analysis of skeletal form at birth revealed differences in mandibular morphology between mice carrying Fgfr2 mutations and their littermates that do not carry the mutations. Murine embryos with the mutations associated with Apert syndrome in humans (Fgfr2+/S252W and Fgfr2+/P253R) showed an increase in the size of the osteogenic anlagen and Meckel's cartilage (MC). Changes in the microarchitecture and mineralization of the developing mandible were visualized using histological staining. The mechanism for mandibular dysgenesis in the Apert Fgfr2+/S252W mouse resulting in the most severe phenotypic effects was further analyzed in detail and found to occur to a lesser degree in the other craniosynostosis mouse models. Laser capture microdissection and RNA-seq analysis revealed transcriptomic changes in mandibular bone at embryonic day 16.5 (E16.5), highlighting increased expression of genes related to osteoclast differentiation and dysregulated genes active in bone mineralization. Increased osteoclastic activity was corroborated by TRAP assay and in situ hybridization of Csf1r and Itgb3. Upregulated expression of Enpp1 and Ank was validated in the mandible of Fgfr2+/S252W embryos, and found to result in elevated inorganic pyrophosphate concentration. Increased proliferation of osteoblasts in the mandible and chondrocytes forming MC was identified in Fgfr2+/S252W embryos at E12.5. These findings provide evidence that FGFR2 gain-of-function mutations differentially affect cartilage formation and intramembranous ossification of dermal bone, contributing to mandibular dysmorphogenesis in craniosynostosis syndromes. This article has an associated First Person interview with the joint first authors of the paper.

Chocolate is a highly valued and palatable confectionery product. Chocolate is primarily made from the processed seeds of the tree species Theobroma cacao. Cacao cultivation is highly relevant for small-holder farmers throughout the tropics, yet its productivity remains limited by low yields and widespread pathogens. A panel of 148 improved cacao clones was assembled based on productivity and disease resistance, and phenotypic single-tree replicated clonal evaluation was performed for 8 years. Using high-density markers, the diversity of clones was expressed relative to 10 known ancestral cacao populations, and significant effects of ancestry were observed in productivity and disease resistance. Genome-wide association (GWA) was performed, and six markers were significantly associated with frosty pod disease resistance. In addition, genomic selection was performed, and consistent with the observed extensive linkage disequilibrium, high predictive ability was observed at low marker densities for all traits. Finally, quantitative trait locus mapping and differential expression analysis of two cultivars with contrasting disease phenotypes were performed to identify genes underlying frosty pod disease resistance, identifying a significant quantitative trait locus and 35 differentially expressed genes using two independent differential expression analyses. These results indicate that in breeding populations of heterozygous and recently admixed individuals, mapping approaches can be used for low complexity traits like pod color cacao, or in other species single gene disease resistance, however genomic selection for quantitative traits remains highly effective relative to mapping. Our results can help guide the breeding process for sustainable improved cacao productivity.