Structural basis of the American mink ACE2 binding by Y453F trimeric spike glycoproteins of SARS-CoV-2.

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.

An optimized cell-based assay to assess influenza virus replication by measuring neuraminidase activity and its applications for virological surveillance.

Year-round virological characterization of circulating epidemic influenza viruses is conducted worldwide to detect the emergence of viruses that may escape pre-existing immunity or acquire resistance to antivirals. High throughput phenotypic assays are needed to complement the sequence-based analysis of circulating viruses and improve pandemic preparedness. The recent entry of a polymerase inhibitor, baloxavir, into the global market further highlighted this need. Here, we optimized a cell-based assay that considerably streamlines antiviral and antigenic testing by replacing lengthy immunostaining and imaging procedures used in current assay with measuring the enzymatic activity of nascent neuraminidase (NA) molecules expressed on the surface of virus-infected cells. For convenience, this new assay was named IRINA (Influenza Replication Inhibition Neuraminidase-based Assay). IRINA was successfully validated to assess inhibitory activity of baloxavir on virus replication by testing a large set (>150) of influenza A and B viruses, including drug resistant strains and viruses collected during 2017-2022. To test its versatility, IRINA was utilized to evaluate neutralization activity of a broadly reactive human anti-HA monoclonal antibody, FI6, and post-infection ferret antisera, as well as the inhibition of NA enzyme activity by NA inhibitors. Performance of IRINA was tested in parallel using respective conventional assays. IRINA offers an attractive alternative to current phenotypic assays, while maintaining reproducibility and high throughput capacity. Additionally, the improved turnaround time may prove to be advantageous when conducting time sensitive studies, such as investigating a new virus outbreak. This assay can meet the needs of surveillance laboratories by providing a streamlined and cost-effective approach for virus characterization.

Mutation L319Q in the PB1 Polymerase Subunit Improves Attenuation of a Candidate Live-Attenuated Influenza A Virus Vaccine.

Influenza A viruses (IAV) remain emerging threats to human public health. Live-attenuated influenza vaccines (LAIV) are one of the most effective prophylactic options to prevent disease caused by influenza infections. However, licensed LAIV remain restricted for use in 2- to 49-year-old healthy and nonpregnant people. Therefore, development of LAIV with increased safety, immunogenicity, and protective efficacy is highly desired. The U.S.-licensed LAIV is based on the master donor virus (MDV) A/Ann Arbor/6/60 H2N2 backbone, which was generated by adaptation of the virus to growth at low temperatures. Introducing the genetic signature of the U.S. MDV into the backbone of other IAV strains resulted in varying levels of attenuation. While the U.S. MDV mutations conferred an attenuated phenotype to other IAV strains, the same amino acid changes did not significantly attenuate the pandemic A/California/04/09 H1N1 (pH1N1) strain. To attenuate pH1N1, we replaced the conserved leucine at position 319 with glutamine (L319Q) in PB1 and analyzed the in vitro and in vivo properties of pH1N1 viruses containing either PB1 L319Q alone or in combination with the U.S. MDV mutations using two animal models of influenza infection and transmission, ferrets and guinea pigs. Our results demonstrated that L319Q substitution in the pH1N1 PB1 alone or in combination with the mutations of the U.S. MDV resulted in reduced pathogenicity (ferrets) and transmission (guinea pigs), and an enhanced temperature sensitive phenotype. These results demonstrate the feasibility of generating an attenuated MDV based on the backbone of a contemporary pH1N1 IAV strain. IMPORTANCE Vaccination represents the most effective strategy to reduce the impact of seasonal IAV infections. Although LAIV are superior in inducing protection and sterilizing immunity, they are not recommended for many individuals who are at high risk for severe disease. Thus, development of safer and more effective LAIV are needed. A concern with the current MDV used to generate the U.S.-licensed LAIV is that it is based on a virus isolated in 1960. Moreover, mutations that confer the temperature-sensitive, cold-adapted, and attenuated phenotype of the U.S. MDV resulted in low level of attenuation in the contemporary pandemic A/California/04/09 H1N1 (pH1N1). Here, we show that introduction of PB1 L319Q substitution, alone or in combination with the U.S. MDV mutations, resulted in pH1N1 attenuation. These findings support the development of a novel LAIV MDV based on a contemporary pH1N1 strain as a medical countermeasure against currently circulating H1N1 IAV.

Changes in Influenza and Other Respiratory Virus Activity During the COVID-19 Pandemic - United States, 2020-2021.

The COVID-19 pandemic and subsequent implementation of nonpharmaceutical interventions (e.g., cessation of global travel, mask use, physical distancing, and staying home) reduced transmission of some viral respiratory pathogens (1). In the United States, influenza activity decreased in March 2020, was historically low through the summer of 2020 (2), and remained low during October 2020-May 2021 (<0.4% of respiratory specimens with positive test results for each week of the season). Circulation of other respiratory pathogens, including respiratory syncytial virus (RSV), common human coronaviruses (HCoVs) types OC43, NL63, 229E, and HKU1, and parainfluenza viruses (PIVs) types 1-4 also decreased in early 2020 and did not increase until spring 2021. Human metapneumovirus (HMPV) circulation decreased in March 2020 and remained low through May 2021. Respiratory adenovirus (RAdV) circulated at lower levels throughout 2020 and as of early May 2021. Rhinovirus and enterovirus (RV/EV) circulation decreased in March 2020, remained low until May 2020, and then increased to near prepandemic seasonal levels. Circulation of respiratory viruses could resume at prepandemic levels after COVID-19 mitigation practices become less stringent. Clinicians should be aware of increases in some respiratory virus activity and remain vigilant for off-season increases. In addition to the use of everyday preventive actions, fall influenza vaccination campaigns are an important component of prevention as COVID-19 mitigation measures are relaxed and schools and workplaces resume in-person activities.

Limited Genetic Diversity Detected in Middle East Respiratory Syndrome-Related Coronavirus Variants Circulating in Dromedary Camels in Jordan.

Middle East respiratory syndrome-related coronavirus (MERS-CoV) is a persistent zoonotic pathogen with frequent spillover from dromedary camels to humans in the Arabian Peninsula, resulting in limited outbreaks of MERS with a high case-fatality rate. Full genome sequence data from camel-derived MERS-CoV variants show diverse lineages circulating in domestic camels with frequent recombination. More than 90% of the available full MERS-CoV genome sequences derived from camels are from just two countries, the Kingdom of Saudi Arabia (KSA) and United Arab Emirates (UAE). In this study, we employ a novel method to amplify and sequence the partial MERS-CoV genome with high sensitivity from nasal swabs of infected camels. We recovered more than 99% of the MERS-CoV genome from field-collected samples with greater than 500 TCID50 equivalent per nasal swab from camel herds sampled in Jordan in May 2016. Our subsequent analyses of 14 camel-derived MERS-CoV genomes show a striking lack of genetic diversity circulating in Jordan camels relative to MERS-CoV genome sequences derived from large camel markets in KSA and UAE. The low genetic diversity detected in Jordan camels during our study is consistent with a lack of endemic circulation in these camel herds and reflective of data from MERS outbreaks in humans dominated by nosocomial transmission following a single introduction as reported during the 2015 MERS outbreak in South Korea. Our data suggest transmission of MERS-CoV among two camel herds in Jordan in 2016 following a single introduction event.

A quantitative approach to assess influenza A virus fitness and transmission in guinea pigs.

Efforts to estimate the risk posed by potentially pandemic influenza A viruses (IAV), and to understand the mechanisms governing interspecies transmission, have been hampered by a lack of animal models that yield relevant and statistically robust measures of viral fitness. To address this gap, we monitored several quantitative measures of fitness in a guinea pig model: infectivity, magnitude of replication, kinetics of replication, efficiency of transmission, and kinetics of transmission. With the goal of identifying metrics that distinguish human- and non-human-adapted IAV we compared strains derived from humans to those circulating in swine and canine populations. Influenza A/Panama/2007/99 (H3N2), A/Netherlands/602/2009 (H1N1), A/swine/Kansas/77778/2007 (H1N1), A/swine/Spain/53207/2004 [M1 P41A] (H1N1), and A/canine/Illinois/41915/2015 (H3N2) viruses were evaluated. Our results revealed higher infectivity and faster kinetics of viral replication and transmission for human and canine strains compared to the swine viruses. Conversely, peak viral titers and efficiency of transmission were higher for human strains relative to both swine and canine IAVs. Total viral loads were comparable among all strains tested. When analyzed together, data from all strains point to peak viral load as a key driver of transmission efficiency and replication kinetics as a key driver of transmission kinetics. While the dose initiating infection did not strongly impact peak viral load, dose was found to modulate kinetics of viral replication and, in turn, timing of transmission. Taken together, our results point to peak viral load and transmission efficiency as key metrics differentiating human and non-human IAVs and suggest that high peak viral load precipitates robust transmission.ImportanceInfluenza pandemics occur when an IAV from non-human hosts enters the human population and adapts to give rise to a lineage capable of sustained transmission among humans. Despite recurring zoonotic infections involving avian or swine adapted IAVs, influenza pandemics occur infrequently because IAVs typically exhibit low fitness in a new host species. Anticipating when a zoonosis might lead to a pandemic is both critical for public health preparedness and extremely challenging. The approach to characterizing IAVs reported here is designed to aid risk assessment efforts by generating rigorous and quantitative data on viral phenotypes relevant for emergence. Our data suggest that the ability to replicate to high titers and transmit efficiently irrespective of initial dose are key characteristics distinguishing IAVs that have established sustained circulation in the human population from IAVs that circulate in non-human mammalian hosts.

Characterizing Emerging Canine H3 Influenza Viruses.

The continual emergence of novel influenza A strains from non-human hosts requires constant vigilance and the need for ongoing research to identify strains that may pose a human public health risk. Since 1999, canine H3 influenza A viruses (CIVs) have caused many thousands or millions of respiratory infections in dogs in the United States. While no human infections with CIVs have been reported to date, these viruses could pose a zoonotic risk. In these studies, the National Institutes of Allergy and Infectious Diseases (NIAID) Centers of Excellence for Influenza Research and Surveillance (CEIRS) network collaboratively demonstrated that CIVs replicated in some primary human cells and transmitted effectively in mammalian models. While people born after 1970 had little or no pre-existing humoral immunity against CIVs, the viruses were sensitive to existing antivirals and we identified a panel of H3 cross-reactive human monoclonal antibodies (hmAbs) that could have prophylactic and/or therapeutic value. Our data predict these CIVs posed a low risk to humans. Importantly, we showed that the CEIRS network could work together to provide basic research information important for characterizing emerging influenza viruses, although there were valuable lessons learned.

Incomplete influenza A virus genomes occur frequently but are readily complemented during localized viral spread.

Segmentation of viral genomes into multiple RNAs creates the potential for replication of incomplete viral genomes (IVGs). Here we use a single-cell approach to quantify influenza A virus IVGs and examine their fitness implications. We find that each segment of influenza A/Panama/2007/99 (H3N2) virus has a 58% probability of being replicated in a cell infected with a single virion. Theoretical methods predict that IVGs carry high costs in a well-mixed system, as 3.6 virions are required for replication of a full genome. Spatial structure is predicted to mitigate these costs, however, and experimental manipulations of spatial structure indicate that local spread facilitates complementation. A virus entirely dependent on co-infection was used to assess relevance of IVGs in vivo. This virus grows robustly in guinea pigs, but is less infectious and does not transmit. Thus, co-infection allows IVGs to contribute to within-host spread, but complete genomes may be critical for transmission.

Dysregulation of M segment gene expression contributes to influenza A virus host restriction.

The M segment of the 2009 pandemic influenza A virus (IAV) has been implicated in its emergence into human populations. To elucidate the genetic contributions of the M segment to host adaptation, and the underlying mechanisms, we examined a panel of isogenic viruses that carry avian- or human-derived M segments. Avian, but not human, M segments restricted viral growth and transmission in mammalian model systems, and the restricted growth correlated with increased expression of M2 relative to M1. M2 overexpression was associated with intracellular accumulation of autophagosomes, which was alleviated by interference of the viral proton channel activity by amantadine treatment. As M1 and M2 are expressed from the M mRNA through alternative splicing, we separated synonymous and non-synonymous changes that differentiate human and avian M segments and found that dysregulation of gene expression leading to M2 overexpression diminished replication, irrespective of amino acid composition of M1 or M2. Moreover, in spite of efficient replication, virus possessing a human M segment that expressed avian M2 protein at low level did not transmit efficiently. We conclude that (i) determinants of transmission reside in the IAV M2 protein, and that (ii) control of M segment gene expression is a critical aspect of IAV host adaptation needed to prevent M2-mediated dysregulation of vesicular homeostasis.

H5N8 and H7N9 packaging signals constrain HA reassortment with a seasonal H3N2 influenza A virus.

Influenza A virus (IAV) has a segmented genome, which (i) allows for exchange of gene segments in coinfected cells, termed reassortment, and (ii) necessitates a selective packaging mechanism to ensure incorporation of a complete set of segments into virus particles. Packaging signals serve as segment identifiers and enable segment-specific packaging. We have previously shown that packaging signals limit reassortment between heterologous IAV strains in a segment-dependent manner. Here, we evaluated the extent to which packaging signals prevent reassortment events that would raise concern for pandemic emergence. Specifically, we tested the compatibility of hemagglutinin (HA) packaging signals from H5N8 and H7N9 avian IAVs with a human seasonal H3N2 IAV. By evaluating reassortment outcomes, we demonstrate that HA segments carrying H5 or H7 packaging signals are significantly disfavored for incorporation into a human H3N2 virus in both cell culture and a guinea pig model. However, incorporation of the heterologous HAs was not excluded fully, and variants with heterologous HA packaging signals were detected at low levels in vivo, including in naïve contact animals. This work indicates that the likelihood of reassortment between human seasonal IAV and avian IAV is reduced by divergence in the RNA packaging signals of the HA segment. These findings offer important insight into the molecular mechanisms governing IAV emergence and inform efforts to estimate the risks posed by H7N9 and H5N8 subtype avian IAVs.

Seasonal H3N2 and 2009 Pandemic H1N1 Influenza A Viruses Reassort Efficiently but Produce Attenuated Progeny.

Reassortment of gene segments between coinfecting influenza A viruses (IAVs) facilitates viral diversification and has a significant epidemiological impact on seasonal and pandemic influenza. Since 1977, human IAVs of H1N1 and H3N2 subtypes have cocirculated with relatively few documented cases of reassortment. We evaluated the potential for viruses of the 2009 pandemic H1N1 (pH1N1) and seasonal H3N2 lineages to reassort under experimental conditions. Results of heterologous coinfections with pH1N1 and H3N2 viruses were compared to those obtained following coinfection with homologous, genetically tagged, pH1N1 viruses as a control. High genotype diversity was observed among progeny of both coinfections; however, diversity was more limited following heterologous coinfection. Pairwise analysis of genotype patterns revealed that homologous reassortment was random while heterologous reassortment was characterized by specific biases. pH1N1/H3N2 reassortant genotypes produced under single-cycle coinfection conditions showed a strong preference for homologous PB2-PA combinations and general preferences for the H3N2 NA, pH1N1 M, and H3N2 PB2 except when paired with the pH1N1 PA or NP. Multicycle coinfection results corroborated these findings and revealed an additional preference for the H3N2 HA. Segment compatibility was further investigated by measuring chimeric polymerase activity and growth of selected reassortants in human tracheobronchial epithelial cells. In guinea pigs inoculated with a mixture of viruses, parental H3N2 viruses dominated but reassortants also infected and transmitted to cage mates. Taken together, our results indicate that strong intrinsic barriers to reassortment between seasonal H3N2 and pH1N1 viruses are few but that the reassortants formed are attenuated relative to parental strains.IMPORTANCE The genome of IAV is relatively simple, comprising eight RNA segments, each of which typically encodes one or two proteins. Each viral protein carries out multiple functions in coordination with other viral components and the machinery of the cell. When two IAVs coinfect a cell, they can exchange genes through reassortment. The resultant progeny viruses often suffer fitness defects due to suboptimal interactions among divergent viral components. The genetic diversity generated through reassortment can facilitate the emergence of novel outbreak strains. Thus, it is important to understand the efficiency of reassortment and the factors that limit its potential. The research described here offers new tools for studying reassortment between two strains of interest and applies those tools to viruses of the 2009 pandemic H1N1 and seasonal H3N2 lineages, which currently cocirculate in humans and therefore have the potential to give rise to novel epidemic strains.

Endothelial cell tropism is a determinant of H5N1 pathogenesis in mammalian species.

The cellular and molecular mechanisms underpinning the unusually high virulence of highly pathogenic avian influenza H5N1 viruses in mammalian species remains unknown. Here, we investigated if the cell tropism of H5N1 virus is a determinant of enhanced virulence in mammalian species. We engineered H5N1 viruses with restricted cell tropism through the exploitation of cell type-specific microRNA expression by incorporating microRNA target sites into the viral genome. Restriction of H5N1 replication in endothelial cells via miR-126 ameliorated disease symptoms, prevented systemic viral spread and limited mortality, despite showing similar levels of peak viral replication in the lungs as compared to control virus-infected mice. Similarly, restriction of H5N1 replication in endothelial cells resulted in ameliorated disease symptoms and decreased viral spread in ferrets. Our studies demonstrate that H5N1 infection of endothelial cells results in excessive production of cytokines and reduces endothelial barrier integrity in the lungs, which culminates in vascular leakage and viral pneumonia. Importantly, our studies suggest a need for a combinational therapy that targets viral components, suppresses host immune responses, and improves endothelial barrier integrity for the treatment of highly pathogenic H5N1 virus infections.

Heterologous Packaging Signals on Segment 4, but Not Segment 6 or Segment 8, Limit Influenza A Virus Reassortment.

Influenza A virus (IAV) RNA packaging signals serve to direct the incorporation of IAV gene segments into virus particles, and this process is thought to be mediated by segment-segment interactions. These packaging signals are segment and strain specific, and as such, they have the potential to impact reassortment outcomes between different IAV strains. Our study aimed to quantify the impact of packaging signal mismatch on IAV reassortment using the human seasonal influenza A/Panama/2007/99 (H3N2) and pandemic influenza A/Netherlands/602/2009 (H1N1) viruses. Focusing on the three most divergent segments, we constructed pairs of viruses that encoded identical proteins but differed in the packaging signal regions on a single segment. We then evaluated the frequency with which segments carrying homologous versus heterologous packaging signals were incorporated into reassortant progeny viruses. We found that, when segment 4 (HA) of coinfecting parental viruses was modified, there was a significant preference for the segment containing matched packaging signals relative to the background of the virus. This preference was apparent even when the homologous HA constituted a minority of the HA segment population available in the cell for packaging. Conversely, when segment 6 (NA) or segment 8 (NS) carried modified packaging signals, there was no significant preference for homologous packaging signals. These data suggest that movement of NA and NS segments between the human H3N2 and H1N1 lineages is unlikely to be restricted by packaging signal mismatch, while movement of the HA segment would be more constrained. Our results indicate that the importance of packaging signals in IAV reassortment is segment dependent.IMPORTANCE Influenza A viruses (IAVs) can exchange genes through reassortment. This process contributes to both the highly diverse population of IAVs found in nature and the formation of novel epidemic and pandemic IAV strains. Our study sought to determine the extent to which IAV packaging signal divergence impacts reassortment between seasonal IAVs. Our knowledge in this area is lacking, and insight into the factors that influence IAV reassortment will inform and strengthen ongoing public health efforts to anticipate the emergence of new viruses. We found that the packaging signals on the HA segment, but not the NA or NS segments, restricted IAV reassortment. Thus, the packaging signals of the HA segment could be an important factor in determining the likelihood that two IAV strains of public health interest will undergo reassortment.

Host Cell Copper Transporters CTR1 and ATP7A are important for Influenza A virus replication.

BACKGROUND: The essential role of copper in eukaryotic cellular physiology is known, but has not been recognized as important in the context of influenza A virus infection. In this study, we investigated the effect of cellular copper on influenza A virus replication. METHODS: Influenza A/WSN/33 (H1N1) virus growth and macromolecule syntheses were assessed in cultured human lung cells (A549) where the copper concentration of the growth medium was modified, or expression of host genes involved in copper homeostasis was targeted by RNA interference. RESULTS: Exogenously increasing copper concentration, or chelating copper, resulted in moderate defects in viral growth. Nucleoprotein (NP) localization, neuraminidase activity assays and transmission electron microscopy did not reveal significant defects in virion assembly, morphology or release under these conditions. However, RNAi knockdown of the high-affinity copper importer CTR1 resulted in significant viral growth defects (7.3-fold reduced titer at 24 hours post-infection, p = 0.04). Knockdown of CTR1 or the trans-Golgi copper transporter ATP7A significantly reduced polymerase activity in a minigenome assay. Both copper transporters were required for authentic viral RNA synthesis and NP and matrix (M1) protein accumulation in the infected cell. CONCLUSIONS: These results demonstrate that intracellular copper regulates the influenza virus life cycle, with potentially distinct mechanisms in specific cellular compartments. These observations provide a new avenue for drug development and studies of influenza virus pathogenesis.

Drivers of airborne human-to-human pathogen transmission.

Airborne pathogens - either transmitted via aerosol or droplets - include a wide variety of highly infectious and dangerous microbes such as variola virus, measles virus, influenza A viruses, Mycobacterium tuberculosis, Streptococcus pneumoniae, and Bordetella pertussis. Emerging zoonotic pathogens, for example, MERS coronavirus, avian influenza viruses, Coxiella, and Francisella, would have pandemic potential were they to acquire efficient human-to-human transmissibility. Here, we synthesize insights from microbiological, medical, social, and economic sciences to provide known mechanisms of aerosolized transmissibility and identify knowledge gaps that limit emergency preparedness plans. In particular, we propose a framework of drivers facilitating human-to-human transmission with the airspace between individuals as an intermediate stage. The model is expected to enhance identification and risk assessment of novel pathogens.

High Prevalence of Middle East Respiratory Coronavirus in Young Dromedary Camels in Jordan.

Prevalence of Middle East respiratory syndrome coronavirus (MERS-CoV) was determined in 45 dromedary camels from two geographically separated herds in Jordan. Virus shedding was only detected in swabs obtained from the respiratory tract and primarily observed in camels younger than 3 years. MERS-CoV seroprevalence increased with age of camels. Bovine and sheep sera were seronegative. Phylogenetic analysis of partial S2 clustered the Jordanian MERS-CoV strains with contemporary MERS-CoV strains associated with nosocomial outbreaks.

Influenza Virus Reassortment Is Enhanced by Semi-infectious Particles but Can Be Suppressed by Defective Interfering Particles.

A high particle to infectivity ratio is a feature common to many RNA viruses, with ~90-99% of particles unable to initiate a productive infection under low multiplicity conditions. A recent publication by Brooke et al. revealed that, for influenza A virus (IAV), a proportion of these seemingly non-infectious particles are in fact semi-infectious. Semi-infectious (SI) particles deliver an incomplete set of viral genes to the cell, and therefore cannot support a full cycle of replication unless complemented through co-infection. In addition to SI particles, IAV populations often contain defective-interfering (DI) particles, which actively interfere with production of infectious progeny. With the aim of understanding the significance to viral evolution of these incomplete particles, we tested the hypothesis that SI and DI particles promote diversification through reassortment. Our approach combined computational simulations with experimental determination of infection, co-infection and reassortment levels following co-inoculation of cultured cells with two distinct influenza A/Panama/2007/99 (H3N2)-based viruses. Computational results predicted enhanced reassortment at a given % infection or multiplicity of infection with increasing semi-infectious particle content. Comparison of experimental data to the model indicated that the likelihood that a given segment is missing varies among the segments and that most particles fail to deliver ≥1 segment. To verify the prediction that SI particles augment reassortment, we performed co-infections using viruses exposed to low dose UV. As expected, the introduction of semi-infectious particles with UV-induced lesions enhanced reassortment. In contrast to SI particles, inclusion of DI particles in modeled virus populations could not account for observed reassortment outcomes. DI particles were furthermore found experimentally to suppress detectable reassortment, relative to that seen with standard virus stocks, most likely by interfering with production of infectious progeny from co-infected cells. These data indicate that semi-infectious particles increase the rate of reassortment and may therefore accelerate adaptive evolution of IAV.