Due to its natural influenza susceptibility, clinical signs, transmission, and similar sialic acid residue distribution, the ferret is the primary animal model for human influenza research. Antibodies generated following infection of ferrets with human influenza viruses are used in surveillance to detect antigenic drift and cross-reactivity with vaccine viruses and circulating strains. Inoculation of ferrets, with over 1,500 human clinical influenza isolates (1998-2019) resulted in lower antibody responses (HI <1:160) to 86% (387 out of 448) influenza B viruses (IBVs) compared to 2.7% (30 out of 1,094) influenza A viruses (IAVs). Here, we show that the immune responses in ferrets inoculated with IBV were delayed and reduced compared to IAV. Innate gene expression in the upper respiratory tract and blood indicated that IAV generated a strong inflammatory response, including an early activation of the interferon (IFN), whereas IBV elicited a delayed and reduced response. Serum levels of cytokines and IFNs were all much higher following IAV infection than IBV infection. Pro-inflammatory, IFN, TH1/TH2, and T-effector proteins were significantly higher in sera of IAV-infected than IBV-infected ferrets over 28 days following the challenge. Serum levels of Type-I/II/III IFNs were detected following IAV infection throughout this period, whereas Type-III IFN was only late for IBV. An early increase in IFN-lambda corresponded to gene expression following IAV infection. Reduced innate immune responses following IBV infection reflected the subsequent delayed and reduced serum antibodies. These findings may help in understanding the antibody responses in humans following influenza vaccination or infection and consideration of potential addition of innate immunomodulators to overcome low responses. IMPORTANCE: The ferret is the primary animal model for human influenza research. Using a ferret model, we studied the differences in both innate and adaptive immune responses following infection with influenza A and B viruses (IAV and IBV). Antibodies generated following infection of ferrets is used for surveillance assays to detect antigenic drift and cross-reactivity with vaccine viruses and circulating influenza strains. IAV infection of ferrets to generate these reagents resulted in a strong antibody response, but IBV infection generated weak antibody responses. In this study using influenza-infected ferrets, we found that IAV resulted in an early activation of the interferon (IFN) and pro-inflammatory response, whereas IBV showed a delay and reduction in these responses. Serum levels of IFNs and other cytokines or chemokines were much higher in ferrets following IAV infection. These reduced innate responses were reflected the subsequent delayed and reduced antibody responses to IBV in the sera. These findings may help in understanding low antibody responses in humans following influenza B vaccination and infection and may warrant the use of innate immunomodulators to overcome these weak responses.

The global spread of the highly pathogenic avian influenza (HPAI) A(H5N1) virus poses a serious pandemic threat, necessitating the swift development of effective vaccines. The success of messenger RNA (mRNA) vaccine technology in the COVID-19 pandemic, marked by its rapid development and scalability, demonstrates its potential for addressing other infectious threats, such as HPAI A(H5N1). We therefore evaluated mRNA vaccine candidates targeting panzootic influenza A(H5) clade 2.3.4.4b viruses, which have been shown to infect a range of mammalian species, including most recently being detected in dairy cattle. Ferrets were immunized with mRNA vaccines encoding either hemagglutinin alone or hemagglutinin and neuraminidase, derived from a 2.3.4.4b prototype vaccine virus recommended by the World Health Organization. Kinetics of the immune responses, as well as protection against a lethal challenge dose of A(H5N1) virus, were assessed. Two doses of mRNA vaccination elicited robust neutralizing antibody titers against a 2022 avian isolate and a 2024 human isolate. Further, mRNA vaccination conferred protection from lethal challenge, whereas all unvaccinated ferrets succumbed to infection. It also reduced viral titers in the upper and lower respiratory tracts of infected ferrets. These results underscore the effectiveness of mRNA vaccines against HPAI A(H5N1), showcasing their potential as a vaccine platform for future influenza pandemics.

CDC continues to track the evolution of SARS-CoV-2, including the Omicron variant and its descendants, using national genomic surveillance. This report summarizes U.S. trends in variant proportion estimates during May 2023-September 2024, a period when SARS-CoV-2 lineages primarily comprised descendants of Omicron variants XBB and JN.1. During summer and fall 2023, multiple descendants of XBB with immune escape substitutions emerged and reached >10% prevalence, including EG.5-like lineages by June 24, FL.1.5.1-like lineages by August 5, HV.1 lineage by September 30, and HK.3-like lineages by November 11. In winter 2023, the JN.1 variant emerged in the United States and rapidly attained predominance nationwide, representing a substantial genetic shift (>30 spike protein amino acid differences) from XBB lineages. Descendants of JN.1 subsequently circulated and reached >10% prevalence, including KQ.1-like and KP.2-like lineages by April 13, KP.3 and LB.1-like lineages by May 25, and KP.3.1.1 by July 20. Surges in COVID-19 cases occurred in winter 2024 during the shift to JN.1 predominance, as well as in summer 2023 and 2024 during circulation of multiple XBB and JN.1 descendants, respectively. The ongoing evolution of the Omicron variant highlights the importance of continued genomic surveillance to guide medical countermeasure development, including the selection of antigens for updated COVID-19 vaccines.

Despite annual vaccination, influenza B viruses (IBV) continue to cause significant morbidity and mortality in humans. We have found that IBV infection resulted in a weaker innate and adaptive immune response than influenza A viruses (IAV) in ferrets. To understand and overcome the weak immune responses to IBV in ferrets, we administered type-I or type-III interferon (IFN) to ferrets following infection or vaccination and evaluated their effects on the immune response. IFN signaling following viral infection plays an important role in the initial innate immune response and affects subsequent adaptive immune responses. In the respiratory tract, IFN lambda (IFNL) has regulatory effects on adaptive immunity indirectly through thymic stromal lymphopoietin (TSLP), which then acts on immune cells to stimulate the adaptive response. Following IBV infection or vaccination, IFN treatment (IFN-Tx) upregulated gene expression of early inflammatory responses in the upper respiratory tract and robust IFN, TSLP, and inflammatory responses in peripheral blood cells. These responses were sustained following challenge or vaccination in IFN-Tx animals. Serum IFNL and TSLP levels were enhanced in IFN-Tx animals following challenge/rechallenge over mock-Tx; however, this difference was not observed following vaccination. Antibody responses in serum of IFN-Tx animals following IBV infection or vaccination increased more quickly and to higher titers and were sustained longer than mock-Tx animals over 3 months. Following rechallenge of infected animals 3 months post treatment, antibody levels remained higher than mock-Tx. However, IFN-Tx did not have an effect on antibody responses following challenge of vaccinated animals. A strong direct correlation was found between TSLP levels and antibody responses following challenge-rechallenge and vaccination-challenge indicating it as a useful tool for predicting adaptive immune responses following IBV infection or vaccination. The effects of IFN on strengthening both innate and adaptive responses to IBV may aid in development of more effective treatments following infection and improved influenza vaccines.

Swine harbors a genetically diverse population of swine influenza A viruses (IAV-S), with demonstrated potential to transmit to the human population, causing outbreaks and pandemics. Here, we describe the development of a one-step, triplex real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay that detects and distinguishes the majority of the antigenically distinct influenza A virus hemagglutinin (HA) clades currently circulating in North American swine, including the IAV-S H1 1A.1 (α), 1A.2 (β), 1A.3 (γ), 1B.2.2 (δ1) and 1B.2.1 (δ2) clades, and the IAV-S H3 2010.1 clade. We performed an in-field test at an exhibition swine show using in-field viral concentration and RNA extraction methodologies and a portable real-time PCR instrument, and rapidly identified three distinct IAV-S clades circulating within the N.A. swine population. Portable sequencing is used to further confirm the results of the in-field test of the swine triplex assay. The IAV-S triplex rRT-PCR assay can be easily transported and used in-field to characterize circulating IAV-S clades in North America, allowing for surveillance and early detection of North American IAV-S with human outbreak and pandemic potential.

On November 13-14, 2023, the National Institute of Allergy and Infectious Diseases (NIAID) in partnership with the Task Force for Global Health, Flu Lab, the Canadian Institutes of Health Research, and the Centers for Disease Control and Prevention convened a meeting on controlled human influenza virus infection model (CHIVIM) studies to review the current research landscape of CHIVIM studies and to generate actionable next steps. Presentations and panel discussions highlighted CHIVIM use cases, regulatory and ethical considerations, innovations, networks and standardization, and the utility of using CHIVIM in vaccine development. This report summarizes the presentations, discussions, key takeaways, and future directions for innovations in CHIVIMs. Experts agreed that CHIVIM studies can be valuable for the study of influenza infection, immune response, and transmission. Furthermore, they may have utility in the development of vaccines and other medical countermeasures; however, the use of CHIVIMs to de-risk clinical development of investigational vaccines should employ a cautious approach. Endpoints in CHIVIM studies should be tailored to the specific use case. CHIVIM studies can provide useful supporting data for vaccine licensure but are not required and do not obviate the need for the conduct of field efficacy trials. Future directions in this field include the continued expansion of capacity to conduct CHIVIM studies, development of a broad panel of challenge viruses and assay reagents and standards that can be shared, streamlining of manufacturing processes, the exploration of targeted delivery of virus to the lower respiratory tract, efforts to more closely replicate natural influenza disease in CHIVIM, alignment on a definition of breadth to facilitate development of more broadly protective/universal vaccine approaches, and continued collaboration between stakeholders.

This Viewpoint summarizes the factors contributing to increased risk of severe outcomes and hospitalization associated with COVID-19 among older adults, stresses the importance of assessing COVID-19 risk before infection occurs, calls for all immunocompromised older adults to be considered for COVID-19 treatment, and details 3 recommended COVID-19 therapies. | eng

Influenza viruses continually evolve new antigenic variants, through mutations in epitopes of their major surface proteins, hemagglutinin (HA) and neuraminidase (NA). Antigenic drift potentiates the reinfection of previously infected individuals, but the contribution of this process to variability in annual epidemics is not well understood. Here, we link influenza A(H3N2) virus evolution to regional epidemic dynamics in the United States during 1997-2019. We integrate phenotypic measures of HA antigenic drift and sequence-based measures of HA and NA fitness to infer antigenic and genetic distances between viruses circulating in successive seasons. We estimate the magnitude, severity, timing, transmission rate, age-specific patterns, and subtype dominance of each regional outbreak and find that genetic distance based on broad sets of epitope sites is the strongest evolutionary predictor of A(H3N2) virus epidemiology. Increased HA and NA epitope distance between seasons correlates with larger, more intense epidemics, higher transmission, greater A(H3N2) subtype dominance, and a greater proportion of cases in adults relative to children, consistent with increased population susceptibility. Based on random forest models, A(H1N1) incidence impacts A(H3N2) epidemics to a greater extent than viral evolution, suggesting that subtype interference is a major driver of influenza A virus infection ynamics, presumably via heterosubtypic cross-immunity. | Seasonal influenza (flu) viruses cause outbreaks every winter. People infected with influenza typically develop mild respiratory symptoms. But flu infections can cause serious illness in young children, older adults and people with chronic medical conditions. Infected or vaccinated individuals develop some immunity, but the viruses evolve quickly to evade these defenses in a process called antigenic drift. As the viruses change, they can re-infect previously immune people. Scientists update the flu vaccine yearly to keep up with this antigenic drift. The immune system fights flu infections by recognizing two proteins, known as antigens, on the virus’s surface, called hemagglutinin (HA) and neuraminidase (NA). However, mutations in the genes encoding these proteins can make them unrecognizable, letting the virus slip past the immune system. Scientists would like to know how these changes affect the size, severity and timing of annual influenza outbreaks. Perofsky et al. show that tracking genetic changes in HA and NA may help improve flu season predictions. The experiments compared the severity of 22 flu seasons caused by the A(H3N2) subtype in the United States with how much HA and NA had evolved since the previous year. The A(H3N2) subtype experiences the fastest rates of antigenic drift and causes more cases and deaths than other seasonal flu viruses. Genetic changes in HA and NA were a better predictor of A(H3N2) outbreak severity than the blood tests for protective antibodies that epidemiologists traditionally use to track flu evolution. However, the prevalence of another subtype of influenza A circulating in the population, called A(H1N1), was an even better predictor of how severe A(H3N2) outbreaks would be. Perofsky et al. are the first to show that genetic changes in NA contribute to the severity of flu seasons. Previous studies suggested a link between genetic changes in HA and flu season severity, and flu vaccines include the HA protein to help the body recognize new influenza strains. The results suggest that adding the NA protein to flu vaccines may improve their effectiveness. In the future, flu forecasters may want to analyze genetic changes in both NA and HA to make their outbreak predictions. Tracking how much of the A(H1N1) subtype is circulating may also be useful for predicting the severity of A(H3N2) outbreaks. | eng

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has evolved into numerous lineages with unique spike mutations and caused multiple epidemics domestically and globally. Although COVID-19 vaccines are available, new variants with the capacity for immune evasion continue to emerge. To understand and characterize the evolution of circulating SARS-CoV-2 variants in the U.S., the Centers for Disease Control and Prevention (CDC) initiated the National SARS-CoV-2 Strain Surveillance (NS3) program and has received thousands of SARS-CoV-2 clinical specimens from across the nation as part of a genotype to phenotype characterization process. Focus reduction neutralization with various antisera was used to antigenically characterize 143 SARS-CoV-2 Delta, Mu and Omicron subvariants from selected clinical specimens received between May 2021 and February 2023, representing a total of 59 unique spike protein sequences. BA.4/5 subvariants BU.1, BQ.1.1, CR.1.1, CQ.2 and BA.4/5 + D420N + K444T; BA.2.75 subvariants BM.4.1.1, BA.2.75.2, CV.1; and recombinant Omicron variants XBF, XBB.1, XBB.1.5 showed the greatest escape from neutralizing antibodies when analyzed against post third-dose original monovalent vaccinee sera. Post fourth-dose bivalent vaccinee sera provided better protection against those subvariants, but substantial reductions in neutralization titers were still observed, especially among BA.4/5 subvariants with both an N-terminal domain (NTD) deletion and receptor binding domain (RBD) substitutions K444M + N460K and recombinant Omicron variants. This analysis demonstrated a framework for long-term systematic genotype to antigenic characterization of circulating and emerging SARS-CoV-2 variants in the U.S., which is critical to assessing their potential impact on the effectiveness of current vaccines and antigen recommendations for future updates.

Influenza B viruses (IBV) cocirculate with influenza A viruses (IAV) and cause periodic epidemics of disease, yet antibody and cellular responses following IBV infection are less well understood. Using the ferret model for antisera generation for influenza surveillance purposes, IAV resulted in robust antibody responses following infection, whereas IBV required an additional booster dose, over 85% of the time, to generate equivalent antibody titers. In this study, we utilized primary differentiated ferret nasal epithelial cells (FNECs) which were inoculated with IAV and IBV to study differences in innate immune responses which may result in differences in adaptive immune responses in the host. FNECs were inoculated with IAV (H1N1pdm09 and H3N2 subtypes) or IBV (B/Victoria and B/Yamagata lineages) and assessed for 72 h. Cells were analyzed for gene expression by quantitative real-time PCR, and apical and basolateral supernatants were assessed for virus kinetics and interferon (IFN), respectively. Similar virus kinetics were observed with IAV and IBV in FNECs. A comparison of gene expression and protein secretion profiles demonstrated that IBV-inoculated FNEC expressed delayed type-I/II IFN responses and reduced type-III IFN secretion compared to IAV-inoculated cells. Concurrently, gene expression of Thymic Stromal Lymphopoietin (TSLP), a type-III IFN-induced gene that enhances adaptive immune responses, was significantly downregulated in IBV-inoculated FNECs. Significant differences in other proinflammatory and adaptive genes were suppressed and delayed following IBV inoculation. Following IBV infection, ex vivo cell cultures derived from the ferret upper respiratory tract exhibited reduced and delayed innate responses which may contribute to reduced antibody responses in vivo.IMPORTANCEInfluenza B viruses (IBV) represent nearly one-quarter of all human influenza cases and are responsible for significant clinical and socioeconomic impacts but do not pose the same pandemic risks as influenza A viruses (IAV) and have thus received much less attention. IBV accounts for greater severity and deaths in children, and vaccine efficacy remains low. The ferret can be readily infected with human clinical isolates and demonstrates a similar course of disease and immune responses. IBV, however, generates lower antibodies in ferrets than IAV following the challenge. To determine whether differences in initial innate responses following infection may affect the development of robust adaptive immune responses, ferret respiratory tract cells were isolated, infected with IAV/IBV, and compared. Understanding the differences in the initial innate immune responses to IAV and IBV may be important in the development of more effective vaccines and interventions to generate more robust protective immune responses.

BACKGROUND: In 2019, the Louisiana Department of Health reported an early influenza B/Victoria (B/VIC) virus outbreak. METHOD: As it was an atypically large outbreak, we deployed to Louisiana to investigate it using genomics and a triplex real-time RT-PCR assay to detect three antigenically distinct B/VIC lineage variant viruses. RESULTS: The investigation indicated that B/VIC V1A.3 subclade, containing a three amino acid deletion in the hemagglutinin and known to be antigenically distinct to the B/Colorado/06/2017 vaccine virus, was the most prevalent circulating virus within the specimens evaluated (86/88 in real-time RT-PCR). CONCLUSION: This work underscores the value of portable platforms for rapid, onsite pathogen characterization.

The COVID-19 pandemic was accompanied by an unprecedented surveillance effort. The resulting data were and will continue to be critical for surveillance and control of SARS-CoV-2. However, some genomic surveillance methods experienced challenges as the virus evolved, resulting in incomplete and poor quality data. Complete and quality coverage, especially of the S-gene, is important for supporting the selection of vaccine candidates. As such, we developed a robust method to target the S-gene for amplification and sequencing. By focusing on the S-gene and imposing strict coverage and quality metrics, we hope to increase the quality of surveillance data for this continually evolving gene. Our technique is currently being deployed globally to partner laboratories, and public health representatives from 79 countries have received hands-on training and support. Expanding access to quality surveillance methods will undoubtedly lead to earlier detection of novel variants and better inform vaccine strain selection.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) enters the host cell by binding to angiotensin-converting enzyme 2 (ACE2). While evolutionarily conserved, ACE2 receptors differ across various species and differential interactions with Spike (S) glycoproteins of SARS-CoV-2 viruses impact species specificity. Reverse zoonoses led to SARS-CoV-2 outbreaks on multiple American mink (Mustela vison) farms during the pandemic and gave rise to mink-associated S substitutions known for transmissibility between mink and zoonotic transmission to humans. In this study, we used bio-layer interferometry (BLI) to discern the differences in binding affinity between multiple human and mink-derived S glycoproteins of SARS-CoV-2 and their respective ACE2 receptors. Further, we conducted a structural analysis of a mink variant S glycoprotein and American mink ACE2 (mvACE2) using cryo-electron microscopy (cryo-EM), revealing four distinct conformations. We discovered a novel intermediary conformation where the mvACE2 receptor is bound to the receptor-binding domain (RBD) of the S glycoprotein in a "down" position, approximately 34° lower than previously reported "up" RBD. Finally, we compared residue interactions in the S-ACE2 complex interface of S glycoprotein conformations with varying RBD orientations. These findings provide valuable insights into the molecular mechanisms of SARS-CoV-2 entry.

Influenza virus neuraminidase (NA) can act as a receptor-binding protein, a role commonly attributed to hemagglutinin (HA). In influenza A(H3N2) viruses, three NA amino acid residues have previously been associated with NA-mediated hemagglutination: T148, D151, and more recently, H150. These residues are part of the 150-loop of the NA monomer. Substitutions at 148 and 151 arise from virus propagation in laboratory cell cultures, whereas changes at 150 occurred during virus evolution in the human host. In this study, we examined the effect of natural amino acid polymorphism at position 150 on NA-mediated hemagglutination. Using the A/Puerto Rico/8/34 backbone, we generated a comprehensive panel of recombinant A(H3N2) viruses that have different NAs but shared an HA that displays poor binding to red blood cells (RBCs). None of the tested substitutions at 150 (C, H, L, R, and S) promoted NA-binding. However, we identified two new determinants of NA-binding, Q136K and T439R, that emerged during virus culturing. Similar to T148I, both Q136K and T439R reduced NA enzyme activity by 48-86% and inhibition (14- to 173-fold) by the NA inhibitor zanamivir. NA-binding was observed when a virus preparation contained approximately 10% of NA variants with either T148I or T439R, highlighting the benefit of using deep sequencing in virus characterization. Taken together, our findings provide new insights into the molecular mechanisms underlying the ability of NA to function as a binding protein. Information gained may aid in the design of new and improved NA-targeting antivirals.

BACKGROUND: Avian influenza (AI) virus detections occurred frequently in 2022 and continue to pose a health, economic, and food security risk. The most recent global analysis of official reports of animal outbreaks and human infections with all reportable AI viruses was published almost a decade ago. Increased or renewed reports of AI viruses, especially high pathogenicity H5N8 and H5N1 in birds and H5N1, H5N8, and H5N6 in humans globally, have established the need for a comprehensive review of current global AI virus surveillance data to assess the pandemic risk of AI viruses. OBJECTIVE: This study aims to provide an analysis of global AI animal outbreak and human case surveillance information from the last decade by describing the circulating virus subtypes, regions and temporal trends in reporting, and country characteristics associated with AI virus outbreak reporting in animals; surveillance and reporting gaps for animals and humans are identified. METHODS: We analyzed AI virus infection reports among animals and humans submitted to animal and public health authorities from January 2013 to June 2022 and compared them with reports from January 2005 to December 2012. A multivariable regression analysis was used to evaluate associations between variables of interest and reported AI virus animal outbreaks. RESULTS: From 2013 to 2022, 52.2% (95/182) of World Organisation for Animal Health (WOAH) Member Countries identified 34 AI virus subtypes during 21,249 outbreaks. The most frequently reported subtypes were high pathogenicity AI H5N1 (10,079/21,249, 47.43%) and H5N8 (6722/21,249, 31.63%). A total of 10 high pathogenicity AI and 6 low pathogenicity AI virus subtypes were reported to the WOAH for the first time during 2013-2022. AI outbreaks in animals occurred in 26 more Member Countries than reported in the previous 8 years. Decreasing World Bank income classification was significantly associated with decreases in reported AI outbreaks (P<.001-.02). Between January 2013 and June 2022, 17/194 (8.8%) World Health Organization (WHO) Member States reported 2000 human AI virus infections of 10 virus subtypes. H7N9 (1568/2000, 78.40%) and H5N1 (254/2000, 12.70%) viruses accounted for the most human infections. As many as 8 of these 17 Member States did not report a human case prior to 2013. Of 1953 human cases with available information, 74.81% (n=1461) had a known animal exposure before onset of illness. The median time from illness onset to the notification posted on the WHO event information site was 15 days (IQR 9-30 days; mean 24 days). Seasonality patterns of animal outbreaks and human infections with AI viruses were very similar, occurred year-round, and peaked during November through May. CONCLUSIONS: Our analysis suggests that AI outbreaks are more frequently reported and geographically widespread than in the past. Global surveillance gaps include inconsistent reporting from all regions and human infection reporting delays. Continued monitoring for AI virus outbreaks in animals and human infections with AI viruses is crucial for pandemic preparedness.

Coinfection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and other viruses is inevitable as the COVID-19 pandemic continues. This study aimed to evaluate cell lines commonly used in virus diagnosis and isolation for their susceptibility to SARS-CoV-2. While multiple kidney cell lines from monkeys were susceptible and permissive to SARS-CoV-2, many cell types derived from human, dog, mink, cat, mouse, or chicken were not. Analysis of MDCK cells, which are most commonly used for surveillance and study of influenza viruses, demonstrated that they were insusceptible to SARS-CoV-2 and that the cellular barrier to productive infection was due to low expression level of the angiotensin converting enzyme 2 (ACE2) receptor and lower receptor affinity to SARS-CoV-2 spike, which could be overcome by over-expression of canine ACE2 in trans. Moreover, SARS-CoV-2 cell tropism did not appear to be affected by a D614G mutation in the spike protein.Competing Interest StatementThe authors have declared no competing interest.

Defective interfering (DI) genomes restrict viral replication and induce type-I interferon. Since DI genomes have been proposed as vaccine adjuvants or therapeutic antiviral agents, it is important to understand their generation, delineate their mechanism of action, develop robust production capacities, assess their safety and in vivo longevity and determine their long-term effects. To address this, we generated a recombinant (r) canine distemper virus (CDV) from an entirely synthetic molecular clone designed using the genomic sequence from a clinical isolate obtained from a free-ranging raccoon with distemper. rCDV was serially passaged in vitro to identify DI genomes that naturally arise during rCDV replication. Defective genomes were identified by Sanger and next-generation sequencing techniques and predominant genomes were synthetically generated and cloned into T7-driven plasmids. Fully encapsidated DI particles (DIPs) were then generated using a rationally attenuated rCDV as a producer virus to drive DI genome replication. We demonstrate these DIPs interfere with rCDV replication in a dose-dependent manner in vitro. Finally, we show sustained replication of a fluorescent DIP in experimentally infected ferrets over a period of 14 days. Most importantly, DIPs were isolated from the lymphoid tissues which are a major site of CDV replication. Our established pipeline for detection, generation and assaying DIPs is transferable to highly pathogenic paramyxoviruses and will allow qualitative and quantitative assessment of the therapeutic effects of DIP administration on disease outcome.Importance Defective interfering (DI) genomes have long been considered inconvenient artifacts that suppressed viral replication in vitro. However, advances in sequencing technologies have led to DI genomes being identified in clinical samples, implicating them in disease progression and outcome. It has been suggested that DI genomes could be harnessed therapeutically. Negative strand RNA virus research has provided a rich pool of natural DI genomes over many years and they are probably the best understood in vitro. Here, we demonstrate identification, synthesis, production and experimental inoculation of novel CDV DI genomes in highly susceptible ferrets. These results provide important evidence that rationally designed and packaged DI genomes can survive the course of a wild-type virus infection.

N-glycosylation plays an important role in the structure and function of membrane and secreted proteins. The spike protein on the surface of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is heavily glycosylated and the major target for developing vaccines, therapeutic drugs and diagnostic tests. The first major SARS-CoV-2 variant carries a D614G substitution in the spike (S-D614G) that has been associated with altered conformation, enhanced ACE2 binding, and increased infectivity and transmission. In this report, we used mass spectrometry techniques to characterize and compare the N-glycosylation of the wild type (S-614D) or variant (S-614G) SARS-CoV-2 spike glycoproteins prepared under identical conditions. The data showed that half of the N-glycosylation sequons changed their distribution of glycans in the S-614G variant. The S-614G variant showed a decrease in the relative abundance of complex-type glycans (up to 45%) and an increase in oligomannose glycans (up to 33%) on all altered sequons. These changes led to a reduction in the overall complexity of the total N-glycosylation profile. All the glycosylation sites with altered patterns were in the spike head while the glycosylation of three sites in the stalk remained unchanged between S-614G and S-614D proteins.Competing Interest StatementThe authors have declared no competing interest.

The need for high-affinity, SARS-CoV-2-specific monoclonal antibodies (mAbs) is critical in the face of the global COVID-19 pandemic, as such reagents can have important diagnostic, research, and therapeutic applications. Of greatest interest is the ~300 amino acid receptor binding domain (RBD) within the S1 subunit of the spike protein because of its key interaction with the human angiotensin converting enzyme 2 (hACE2) receptor present on many cell types, especially lung epithelial cells. We report here the development and functional characterization of 29 nanomolar-affinity mouse SARS-CoV-2 mAbs created by an accelerated immunization and hybridoma screening process. Differing functions, including binding of diverse protein epitopes, viral neutralization, impact on RBD-hACE2 binding, and immunohistochemical staining of infected lung tissue, were correlated with variable gene usage and sequence.Competing Interest StatementThe authors have declared no competing interest.

The 2017-2018 North American influenza season caused more hospitalizations and deaths than any year since the 2009 H1N1 pandemic. The majority of recorded influenza infections were caused by A(H3N2) viruses, with most of the virus’s North American diversity falling into the A2 clade. Within A2, we observe a subclade which we call A2/re that rose to comprise almost 70% of A(H3N2) viruses circulating in North America by early 2018. Unlike most fast-growing clades, however, A2/re contains no amino acid substitutions in the hemagglutinin (HA) segment. Moreover, HI assays did not suggest substantial antigenic differences between A2/re viruses and viruses sampled during the 2016-2017 season. Rather, we observe that the A2/re clade was the result of a reassortment event that occurred in late 2016 or early 2017 and involved the combination of the HA and PB1 segments of an A2 virus with neuraminidase (NA) and other segments a virus from the clade A1b. The success of this clade shows the need for antigenic analysis that targets NA in addition to HA. Our results illustrate the potential for non-HA drivers of viral success and necessitate the need for more thorough tracking of full viral genomes to better understand the dynamics of influenza epidemics.

Influenza B viruses have two genetically and antigenically distinct lineages, B/Victoria/2/1987-like (VIC) and B/Yamagata/16/1988-like (YAM) viruses, that emerged in the 1980s and co-circulate annually during the influenza season. During the 2016-2017 influenza season, influenza B/VIC lineage variant viruses emerged with two (K162N163) or three (K162N163D164) amino acid (AA) deletions in the hemagglutinin protein. Hemagglutination inhibition assays demonstrate that these deletion variant influenza B/VIC viruses are antigenically distinct from each other and from the progenitor B/VIC virus that lacks the deletion. Therefore, there are currently four antigenically distinct HA proteins expressed by influenza B co-circulating: B/YAM, B/VIC V1A (no deletion), B/VIC V1A.1 (two-AA deletion), and B/VIC V1A.2 and V1A.3 (three-AA deletion). The prevalence of these viruses differs across geographic regions, making it critical to have a sensitive, rapid diagnostic assay(s) that detect and distinguish these Influenza B variant viruses during surveillance. Here, we present a real time RT-PCR assay that targets the influenza B/VIC deletion region in the HA gene and detects and distinguishes the influenza B/VIC V1A, B/VIC V1A.1, B/VIC V1A.2 and B/VIC V1A.3 variant viruses, with no cross-reactivity. This assay can be run as a multiplex reaction, allowing for increased testing efficiency and reduced cost. Coupling this assay with the CDC Human Influenza Virus Real-Time RT-PCR Diagnostic Panel Influenza B Lineage Genotyping Kit results in rapid detection and characterization of circulating influenza B viruses. Having accurate and detailed surveillance information on these distinct Influenza B variant viruses will provide insight into the prevalence and geographic distribution and could aid in vaccine recommendations.

For the first time, a complete genome of an RNA virus has been sequenced in its original form. Previously, RNA was sequenced by the chemical degradation of radiolabelled RNA, a difficult method that produced only short sequences. Instead, RNA has usually been sequenced indirectly by copying it into cDNA, which is often amplified to dsDNA by PCR and subsequently analyzed using a variety of DNA sequencing methods. We designed an adapter to short highly conserved termini of the influenza virus genome to target the (-) sense RNA into a protein nanopore on the Oxford Nanopore MinION sequencing platform. Utilizing this method and total RNA extracted from the allantoic fluid of infected chicken eggs, we demonstrate successful sequencing of the complete influenza virus genome with 100% nucleotide coverage, 99% consensus identity, and 99% of reads mapped to influenza. By utilizing the same methodology we can redesign the adapter in order to expand the targets to include viral mRNA and (+) sense cRNA, which are essential to the viral life cycle. This has the potential to identify and quantify splice variants and base modifications, which are not practically measurable with current methods.

Seasonal influenza virus A/H3N2 is a major cause of death globally. Vaccination remains the most effective preventative. Rapid mutation of hemagglutinin allows viruses to escape adaptive immunity. This antigenic drift necessitates regular vaccine updates. Effective vaccine strains need to represent H3N2 populations circulating one year after strain selection. Experts select strains based on experimental measurements of antigenic drift and predictions made by models from hemagglutinin sequences. We developed a novel influenza forecasting framework that integrates phenotypic measures of antigenic drift and functional constraint with previously published sequence-only fitness estimates. Forecasts informed by phenotypic measures of antigenic drift consistently outperformed previous sequence-only estimates, while sequence-only estimates of functional constraint surpassed more comprehensive experimentally-informed estimates. Importantly, the best models integrated estimates of both functional constraint and either antigenic drift phenotypes or recent population growth.Competing Interest StatementThe authors have declared no competing interest.

Working overnight at a large swine exhibition, we identified an influenza A virus (IAV) outbreak in swine, nanopore-sequenced 13 IAV genomes from samples collected, and in real-time, determined that these viruses posed a novel risk to humans due to genetic mismatches between the viruses and current pre-pandemic candidate vaccine viruses (CVV). We developed and used a portable IAV sequencing and analysis platform called Mia (Mobile Influenza Analysis) to complete and characterize full-length consensus genomes approximately 18 hours after unpacking the mobile lab. Swine are important animal IAV reservoirs that have given rise to pandemic viruses via zoonotic transmission. Genomic analyses of IAV in swine are critical to understanding pandemic risk of viruses in this reservoir, and characterization of viruses circulating in exhibition swine enables rapid comparison to current seasonal influenza vaccines and CVVs. The Mia system rapidly identified three genetically distinct swine IAV lineages from three subtypes: A(H1N1), A(H3N2) and A(H1N2). Additional analysis of the HA protein sequences of the A(H1N2) viruses identified &gt;30 amino acid differences between the HA1 portion of the hemagglutinin of these viruses and the most closely related pre-2009 CVV. All virus sequences were emailed to colleagues at CDC who initiated development of a synthetically derived CVV designed to provide an optimal antigenic match with the viruses detected in the exhibition. In subsequent months, this virus caused 13 infections in humans, and was the dominant variant virus in the US detected in 2018. Had this virus caused a severe outbreak or pandemic, our proactive surveillance efforts and CVV derivation would have provided an approximate 8 week time advantage for vaccine manufacturing. This is the first report of the use of field-derived nanopore sequencing data to initiate a real-time, actionable public health countermeasure.

The early Omicron lineage variants evolved and gave rise to diverging lineages that fueled the COVID-19 pandemic in 2022. Bivalent mRNA vaccines, designed to broaden protection against circulating and future variants, were authorized by the U.S. Food and Drug Administration (FDA) in August 2022 and recommended by the U.S. Centers for Disease Control and Prevention (CDC) in September 2022. The impact of bivalent vaccination on eliciting neutralizing antibodies against homologous BA.4/BA.5 viruses as well as emerging heterologous viruses needs to be analyzed. In this study, we analyze the neutralizing activity of sera collected after a third dose of vaccination (2-6 weeks post monovalent booster) or a fourth dose of vaccination (2-7 weeks post bivalent booster) against 10 predominant/recent Omicron lineage viruses including BA.1, BA.2, BA.5, BA.2.75, BA.2.75.2, BN.1, BQ.1, BQ.1.1, XBB, and XBB.1. The bivalent booster vaccination enhanced neutralizing antibody titers against all Omicron lineage viruses tested, including a 10-fold increase in neutralization of BQ.1 and BQ.1.1 viruses that predominated in the U.S. during the last two months of 2022. Overall, the data indicate the bivalent vaccine booster strengthens protection against Omicron lineage variants that evolved from BA.5 and BA.2 progenitors. Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

Recombination between SARS-CoV-2 virus variants can result in different viral properties (e.g., infectiousness or pathogenicity). In this report, we describe viruses with recombinant genomes containing signature mutations from Delta and Omicron variants. These genomes are the first evidence for a Delta-Omicron hybrid Spike protein in the United States. Copyright The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

The evolution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence of many new variant lineages that have exacerbated the COVID-19 pandemic. Some of those variants were designated as variants of concern/interest (VOC/VOI) by national or international authorities based on many factors including their potential impact on vaccines. To ascertain and rank the risk of VOCs and VOIs, we analyzed their ability to escape from vaccine-induced antibodies. The variants showed differential reductions in neutralization and replication titers by post-vaccination sera. Although the Omicron variant showed the most escape from neutralization, sera collected after a third dose of vaccine (booster sera) retained moderate neutralizing activity against that variant. Therefore, vaccination remains the most effective strategy to combat the COVID-19 pandemic.

Clade 2.3.4.4 b highly pathogenic avian influenza (HPAI) A (H5N1) viruses that are responsible for devastating outbreaks in birds and mammals pose a potential threat to public health. Here, we evaluated their susceptibility to influenza antivirals. Of 1015 sequences of HPAI A (H5N1) viruses collected in the United States during 2022, eight viruses (∼0.8%) had a molecular marker of drug resistance to an FDA-approved antiviral: three adamantane-resistant (M2-V27A), four oseltamivir-resistant (NA-H275Y), and one baloxavir-resistant (PA-I38T). Additionally, 31 viruses contained mutations that may reduce susceptibility to inhibitors of neuraminidase (NA) (n = 20) or cap-dependent endonuclease (CEN) (n = 11). A panel of 22 representative viruses was tested phenotypically. Overall, clade 2.3.4.4  b A (H5N1) viruses lacking recognized resistance mutations were susceptible to FDA-approved antivirals. Oseltamivir was least potent at inhibiting NA activity, while the investigational NA inhibitor AV5080 was most potent, including against NA mutants. A novel NA substitution T438N conferred 12-fold reduced inhibition by zanamivir, and in combination with the known marker N295S, synergistically affected susceptibility to all five NA inhibitors. In cell culture-based assays HINT and IRINA, the PA-I38T virus displayed 75- to 108-fold and 37- to 78-fold reduced susceptibility to CEN inhibitors baloxavir and investigational AV5116, respectively. Viruses with PA-I38M or PA-A37T showed 5- to 10-fold reduced susceptibilities. As HPAI A (H5N1) viruses continue to circulate and evolve, close monitoring of drug susceptibility is needed for risk assessment and to inform decisions regarding antiviral stockpiling.

CDC has used national genomic surveillance since December 2020 to monitor SARS-CoV-2 variants that have emerged throughout the COVID-19 pandemic, including the Omicron variant. This report summarizes U.S. trends in variant proportions from national genomic surveillance during January 2022-May 2023. During this period, the Omicron variant remained predominant, with various descendant lineages reaching national predominance (>50% prevalence). During the first half of 2022, BA.1.1 reached predominance by the week ending January 8, 2022, followed by BA.2 (March 26), BA.2.12.1 (May 14), and BA.5 (July 2); the predominance of each variant coincided with surges in COVID-19 cases. The latter half of 2022 was characterized by the circulation of sublineages of BA.2, BA.4, and BA.5 (e.g., BQ.1 and BQ.1.1), some of which independently acquired similar spike protein substitutions associated with immune evasion. By the end of January 2023, XBB.1.5 became predominant. As of May 13, 2023, the most common circulating lineages were XBB.1.5 (61.5%), XBB.1.9.1 (10.0%), and XBB.1.16 (9.4%); XBB.1.16 and XBB.1.16.1 (2.4%), containing the K478R substitution, and XBB.2.3 (3.2%), containing the P521S substitution, had the fastest doubling times at that point. Analytic methods for estimating variant proportions have been updated as the availability of sequencing specimens has declined. The continued evolution of Omicron lineages highlights the importance of genomic surveillance to monitor emerging variants and help guide vaccine development and use of therapeutics.

During the evolution of SARS-CoV-2 in humans a D614G substitution in the spike (S) protein emerged and became the predominant circulating variant (S-614G) of the COVID-19 pandemic (1) . However, whether the increasing prevalence of the S-614G variant represents a fitness advantage that improves replication and/or transmission in humans or is merely due to founder effects remains elusive. Here, we generated isogenic SARS-CoV-2 variants and demonstrate that the S-614G variant has (i) enhanced binding to human ACE2, (ii) increased replication in primary human bronchial and nasal airway epithelial cultures as well as in a novel human ACE2 knock-in mouse model, and (iii) markedly increased replication and transmissibility in hamster and ferret models of SARS-CoV-2 infection. Collectively, our data show that while the S-614G substitution results in subtle increases in binding and replication in vitro , it provides a real competitive advantage in vivo , particularly during the transmission bottle neck, providing an explanation for the global predominance of S-614G variant among the SARS-CoV-2 viruses currently circulating.