A rapid and flexible microneutralization assay for serological assessment of influenza viruses.

BACKGROUND: Serological responses from influenza vaccination or infection are typically measured by hemagglutinin inhibition (HAI) or microneutralization (MN). Both methods are limited in feasibility, standardization, and generalizability to recent strains. We developed a luciferase MN (LMN) assay that combines the advantages of the conventional MN assay with the ease of the HAI assay. METHODS: Sera were obtained from the HIVE study, a Michigan household cohort. Reverse genetics was used to generate recombinant influenza viruses expressing the hemagglutinin and neuraminidase of test strains, all other viral proteins from an A/WSN/1933 backbone, and a NanoLuc reporter. Serum neutralization of luciferase-expressing targets was quantified as a reduction in light emission from infected cells. Neutralization titers were measured for cell- and egg-adapted versions of A/Hong Kong/4801/2014 and A/Singapore/INFIMH-16-0019/2016 and compared to HAI titers against egg-grown antigens. RESULTS: Three hundred thirty-three sera were collected from 259 participants between May 2016 and July 2018. Sampled participants were 7-68 years of age, and >80% were vaccinated against influenza. HAI and LMN titers were correlated for A/Hong Kong/4801/2014 (ρ = 0.52, p ≤ 0.01) and A/Singapore/INFIMH-16-0019/2016 (ρ = 0.79, p ≤ 0.01). LMN titers were lower for cell strains compared to egg strains (A/Hong Kong/4801/2014 mean log2 fold change = -2.66, p ≤ 0.01 and A/Singapore/INFIMH-16-0019/2016 mean log2 fold change = -3.15, p ≤ 0.01). CONCLUSIONS: The LMN assay was feasible using limited sample volumes and able to differentiate small antigenic differences between egg-adapted and cell-derived strains. The correspondence of these results with the commonly used HAI confirms the utility of this assay for high-throughput studies of correlates of protection and vaccine response.

Investigating Epidemiologic and Molecular Links Between Patients With Community- and Hospital-Acquired Influenza A: 2017-2018 and 2019-2020, Michigan.

Background: Hospital-acquired influenza virus infection (HAII) can cause severe morbidity and mortality. Identifying potential transmission routes can inform prevention strategies. Methods: We identified all hospitalized patients testing positive for influenza A virus at a large, tertiary care hospital during the 2017-2018 and 2019-2020 influenza seasons. Hospital admission dates, locations of inpatient service, and clinical influenza testing information were retrieved from the electronic medical record. Time-location groups of epidemiologically linked influenza patients were defined and contained ≥1 presumed HAII case (first positive ≥48 hours after admission). Genetic relatedness within time-location groups was assessed by whole genome sequencing. Results: During the 2017-2018 season, 230 patients tested positive for influenza A(H3N2) or unsubtyped influenza A including 26 HAIIs. There were 159 influenza A(H1N1)pdm09 or unsubtyped influenza A-positive patients identified during the 2019-2020 season including 33 HAIIs. Consensus sequences were obtained for 177 (77%) and 57 (36%) of influenza A cases in 2017-2018 and 2019-2020, respectively. Among all influenza A cases, there were 10 time-location groups identified in 2017-2018 and 13 in 2019-2020; 19 of 23 groups included ≤4 patients. In 2017-2018, 6 of 10 groups had ≥2 patients with sequence data, including ≥1 HAII case. Two of 13 groups met this criteria in 2019-2020. Two time-location groups from 2017-2018 each contained 3 genetically linked cases. Conclusions: Our results suggest that HAIIs arise from outbreak transmission from nosocomial sources as well as single infections from unique community introductions.

SARS-CoV-2 Genomic Surveillance Reveals Little Spread From a Large University Campus to the Surrounding Community.

BACKGROUND: Coronavirus disease 2019 (COVID-19) has had high incidence rates at institutions of higher education (IHE) in the United States, but the transmission dynamics in these settings are poorly understood. It remains unclear to what extent IHE-associated outbreaks have contributed to transmission in nearby communities. METHODS: We implemented high-density prospective genomic surveillance to investigate these dynamics at the University of Michigan and the surrounding community during the Fall 2020 semester (August 16-November 24). We sequenced complete severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from 1659 individuals, including 468 students, representing 20% of cases in students and 25% of total cases in Washtenaw County over the study interval. RESULTS: Phylogenetic analysis identified >200 introductions into the student population, most of which were not related to other student cases. There were 2 prolonged student transmission clusters, of 115 and 73 individuals, that spanned multiple on-campus residences. Remarkably, <5% of nonstudent genomes were descended from student clusters, and viral descendants of student cases were rare during a subsequent wave of infections in the community. CONCLUSIONS: The largest outbreaks among students at the University of Michigan did not significantly contribute to the rise in community cases in Fall 2020. These results provide valuable insights into SARS-CoV-2 transmission dynamics at the regional level.

Severe Acute Respiratory Syndrome Coronavirus 2 Total and Subgenomic RNA Viral Load in Hospitalized Patients.

BACKGROUND: Previous studies demonstrated that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA can be detected for weeks after infection. The significance of this finding is unclear and, in most patients, does not represent active infection. Detection of subgenomic RNA has been proposed to represent productive infection and may be a useful marker for monitoring infectivity. METHODS: We used quantitative reverse-transcription polymerase chain reaction (RT-qPCR) to quantify total and subgenomic nucleocapsid (sgN) and envelope (sgE) transcripts in 185 SARS-CoV-2-positive nasopharyngeal swab samples collected on hospital admission and to relate to symptom duration. RESULTS: We find that all transcripts decline at the same rate; however, sgE becomes undetectable before other transcripts. The median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic compared to total RNA, suggesting that subgenomic transcript copy number is dependent on copy number of total transcripts. The mean difference between total and sgN is 16-fold and the mean difference between total and sgE is 137-fold. This relationship is constant over duration of symptoms, allowing prediction of subgenomic copy number from total copy number. CONCLUSIONS: Subgenomic RNA may be no more useful in determining infectivity than a copy number threshold determined for total RNA.

Early introductions and transmission of SARS-CoV-2 variant B.1.1.7 in the United States.

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a global public health concern because of its increased transmissibility. Over 2,500 COVID-19 cases associated with this variant have been detected in the United States (US) since December 2020, but the extent of establishment is relatively unknown. Using travel, genomic, and diagnostic data, we highlight that the primary ports of entry for B.1.1.7 in the US were in New York, California, and Florida. Furthermore, we found evidence for many independent B.1.1.7 establishments starting in early December 2020, followed by interstate spread by the end of the month. Finally, we project that B.1.1.7 will be the dominant lineage in many states by mid- to late March. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.

Temporal dynamics of SARS-CoV-2 mutation accumulation within and across infected hosts.

Analysis of SARS-CoV-2 genetic diversity within infected hosts can provide insight into the generation and spread of new viral variants and may enable high resolution inference of transmission chains. However, little is known about temporal aspects of SARS-CoV-2 intrahost diversity and the extent to which shared diversity reflects convergent evolution as opposed to transmission linkage. Here we use high depth of coverage sequencing to identify within-host genetic variants in 325 specimens from hospitalized COVID-19 patients and infected employees at a single medical center. We validated our variant calling by sequencing defined RNA mixtures and identified viral load as a critical factor in variant identification. By leveraging clinical metadata, we found that intrahost diversity is low and does not vary by time from symptom onset. This suggests that variants will only rarely rise to appreciable frequency prior to transmission. Although there was generally little shared variation across the sequenced cohort, we identified intrahost variants shared across individuals who were unlikely to be related by transmission. These variants did not precede a rise in frequency in global consensus genomes, suggesting that intrahost variants may have limited utility for predicting future lineages. These results provide important context for sequence-based inference in SARS-CoV-2 evolution and epidemiology.

SARS-CoV-2 Total and Subgenomic RNA Viral Load in Hospitalized Patients.

Understanding viral load in patients infected with SARS-CoV-2 is critical to epidemiology and infection control. Previous studies have demonstrated that SARS-CoV-2 RNA can be detected for many weeks after symptom onset. The clinical significance of this finding is unclear and, in most patients, likely does not represent active infection. There are, however, patients who shed infectious virus for weeks. Detection of subgenomic RNA transcripts expressed by SARS-CoV-2 has been proposed to represent productive infection and may be a tractable marker for monitoring infectivity. Here, we use RT-PCR to quantify total and subgenomic nucleocapsid (N) and envelope (E) transcripts in 190 SARS-CoV-2 positive samples collected on hospital admission. We relate these findings to duration of symptoms. We find that all transcripts decline at the same rate; however, subgenomic E becomes undetectable before other transcripts. In Kaplan-Meier analysis the median duration of symptoms to a negative test is 14 days for sgE and 25 days for sgN. There is a linear decline in subgenomic RNA compared to total RNA suggesting subgenomic transcript copy number is highly dependent on copy number of total transcripts. The mean difference between total N and subgenomic N is 16-fold (4.0 cycles) and the mean difference between total E and sub-genomic E is 137-fold (7.1 cycles). This relationship is constant over duration of symptoms allowing prediction of subgenomic copy number from total copy number. Although Subgenomic E is undetectable at a time that may more closely reflect the duration of infectivity, its utility in determining active infection may be no more useful than a copy number threshold determined for total transcripts.

Early introductions and community transmission of SARS-CoV-2 variant B.1.1.7 in the United States.

The emergence and spread of SARS-CoV-2 lineage B.1.1.7, first detected in the United Kingdom, has become a global public health concern because of its increased transmissibility. Over 2500 COVID-19 cases associated with this variant have been detected in the US since December 2020, but the extent of establishment is relatively unknown. Using travel, genomic, and diagnostic data, we highlight the primary ports of entry for B.1.1.7 in the US and locations of possible underreporting of B.1.1.7 cases. Furthermore, we found evidence for many independent B.1.1.7 establishments starting in early December 2020, followed by interstate spread by the end of the month. Finally, we project that B.1.1.7 will be the dominant lineage in many states by mid to late March. Thus, genomic surveillance for B.1.1.7 and other variants urgently needs to be enhanced to better inform the public health response.

Pre-existing heterosubtypic immunity provides a barrier to airborne transmission of influenza viruses.

Human-to-human transmission of influenza viruses is a serious public health threat, yet the precise role of immunity from previous infections on the susceptibility to airborne infection is still unknown. Using the ferret model, we examined the roles of exposure duration and heterosubtypic immunity on influenza transmission. We demonstrate that a 48 hour exposure is sufficient for efficient transmission of H1N1 and H3N2 viruses. To test pre-existing immunity, a gap of 8-12 weeks between primary and secondary infections was imposed to reduce innate responses and ensure robust infection of donor animals with heterosubtypic viruses. We found that pre-existing H3N2 immunity did not significantly block transmission of the 2009 H1N1pandemic (H1N1pdm09) virus to immune animals. Surprisingly, airborne transmission of seasonal H3N2 influenza strains was abrogated in recipient animals with H1N1pdm09 pre-existing immunity. This protection from natural infection with H3N2 virus was independent of neutralizing antibodies. Pre-existing immunity with influenza B virus did not block H3N2 virus transmission, indicating that the protection was likely driven by the adaptive immune response. We demonstrate that pre-existing immunity can impact susceptibility to heterologous influenza virus strains, and implicate a novel correlate of protection that can limit the spread of respiratory pathogens through the air.

Validation of N95 Filtering Facepiece Respirator Decontamination Methods Available at a Large University Hospital.

BACKGROUND: Due to unprecedented shortages in N95 filtering facepiece respirators, healthcare systems have explored N95 reprocessing. No single, full-scale reprocessing publication has reported an evaluation including multiple viruses, bacteria, and fungi along with respirator filtration and fit. METHODS: We explored reprocessing methods using new 3M 1860 N95 respirators, including moist (50%-75% relative humidity [RH]) heat (80-82°C for 30 minutes), ethylene oxide (EtO), pulsed xenon UV-C (UV-PX), hydrogen peroxide gas plasma (HPGP), and hydrogen peroxide vapor (HPV). Respirator samples were analyzed using 4 viruses (MS2, phi6, influenza A virus [IAV], murine hepatitis virus [MHV)]), 3 bacteria (Escherichia coli, Staphylococcus aureus, Geobacillus stearothermophilus spores, and vegetative bacteria), and Aspergillus niger. Different application media were tested. Decontaminated respirators were evaluated for filtration integrity and fit. RESULTS: Heat with moderate RH most effectively inactivated virus, resulting in reductions of >6.6-log10 MS2, >6.7-log10 Phi6, >2.7-log10 MHV, and >3.9-log10 IAV and prokaryotes, except for G stearothermohphilus. Hydrogen peroxide vapor was moderately effective at inactivating tested viruses, resulting in 1.5- to >4-log10 observable inactivation. Staphylococcus aureus inactivation by HPV was limited. Filtration efficiency and proper fit were maintained after 5 cycles of heat with moderate RH and HPV. Although it was effective at decontamination, HPGP resulted in decreased filtration efficiency, and EtO treatment raised toxicity concerns. Observed virus inactivation varied depending upon the application media used. CONCLUSIONS: Both moist heat and HPV are scalable N95 reprocessing options because they achieve high levels of biological indicator inactivation while maintaining respirator fit and integrity.

Temporal dynamics of SARS-CoV-2 mutation accumulation within and across infected hosts.

Analysis of SARS-CoV-2 genetic diversity within infected hosts can provide insight into the generation and spread of new viral variants and may enable high resolution inference of transmission chains. However, little is known about temporal aspects of SARS-CoV-2 intrahost diversity and the extent to which shared diversity reflects convergent evolution as opposed to transmission linkage. Here we use high depth of coverage sequencing to identify within-host genetic variants in 325 specimens from hospitalized COVID-19 patients and infected employees at a single medical center. We validated our variant calling by sequencing defined RNA mixtures and identified a viral load threshold that minimizes false positives. By leveraging clinical metadata, we found that intrahost diversity is low and does not vary by time from symptom onset. This suggests that variants will only rarely rise to appreciable frequency prior to transmission. Although there was generally little shared variation across the sequenced cohort, we identified intrahost variants shared across individuals who were unlikely to be related by transmission. These variants did not precede a rise in frequency in global consensus genomes, suggesting that intrahost variants may have limited utility for predicting future lineages. These results provide important context for sequence-based inference in SARS-CoV-2 evolution and epidemiology.

The Early Evolution of Oral Poliovirus Vaccine Is Shaped by Strong Positive Selection and Tight Transmission Bottlenecks.

The emergence of circulating vaccine-derived polioviruses through evolution of the oral polio vaccine (OPV) poses a significant obstacle to polio eradication. Understanding the early genetic changes that occur as OPV evolves and transmits is important for preventing future outbreaks. Here, we use deep sequencing to define the evolutionary trajectories of type 2 OPV in a vaccine trial. By sequencing 497 longitudinal stool samples from 271 OPV2 recipients and household contacts, we were able to examine the extent of convergent evolution in vaccinated individuals and the amount of viral diversity that is transmitted. In addition to rapid reversion of key attenuating mutations, we identify strong selection at 19 sites across the genome. We find that a tight transmission bottleneck limits the onward transmission of these early adaptive mutations. Our results highlight the distinct evolutionary dynamics of live attenuated virus vaccines and have important implications for the success of next-generation OPV.

Humidity and Deposition Solution Play a Critical Role in Virus Inactivation by Heat Treatment of N95 Respirators.

Supply shortages of N95 respirators during the coronavirus disease 2019 (COVID-19) pandemic have motivated institutions to develop feasible and effective N95 respirator reuse strategies. In particular, heat decontamination is a treatment method that scales well and can be implemented in settings with variable or limited resources. Prior studies using multiple inactivation methods, however, have often focused on a single virus under narrowly defined conditions, making it difficult to develop guiding principles for inactivating emerging or difficult-to-culture viruses. We systematically explored how temperature, humidity, and virus deposition solutions impact the inactivation of viruses deposited and dried on N95 respirator coupons. We exposed four virus surrogates across a range of structures and phylogenies, including two bacteriophages (MS2 and phi6), a mouse coronavirus (murine hepatitis virus [MHV]), and a recombinant human influenza A virus subtype H3N2 (IAV), to heat treatment for 30 min in multiple deposition solutions across several temperatures and relative humidities (RHs). We observed that elevated RH was essential for effective heat inactivation of all four viruses tested. For heat treatments between 72°C and 82°C, RHs greater than 50% resulted in a >6-log10 inactivation of bacteriophages, and RHs greater than 25% resulted in a >3.5-log10 inactivation of MHV and IAV. Furthermore, deposition of viruses in host cell culture media greatly enhanced virus inactivation by heat and humidity compared to other deposition solutions, such as phosphate-buffered saline, phosphate-buffered saline with bovine serum albumin, and human saliva. Past and future heat treatment methods must therefore explicitly account for deposition solutions as a factor that will strongly influence observed virus inactivation rates. Overall, our data set can inform the design and validation of effective heat-based decontamination strategies for N95 respirators and other porous surfaces, especially for emerging viruses that may be of immediate and future public health concern.IMPORTANCE Shortages of personal protective equipment, including N95 respirators, during the coronavirus (CoV) disease 2019 (COVID-19) pandemic have highlighted the need to develop effective decontamination strategies for their reuse. This is particularly important in health care settings for reducing exposure to respiratory viruses, like severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19. Although several treatment methods are available, a widely accessible strategy will be necessary to combat shortages on a global scale. We demonstrate that the combination of heat and humidity inactivates a range of RNA viruses, including both viral pathogens and common viral pathogen surrogates, after deposition on N95 respirators and achieves the necessary virus inactivation detailed by the U.S. Food and Drug Administration guidelines to validate N95 respirator decontamination technologies. We further demonstrate that depositing viruses onto surfaces when suspended in culture media can greatly enhance observed inactivation, adding caution to how heat and humidity treatment methods are validated.

Influenza B Viruses Exhibit Lower Within-Host Diversity than Influenza A Viruses in Human Hosts.

Influenza B virus (IBV) undergoes seasonal antigenic drift more slowly than influenza A virus, but the reasons for this difference are unclear. While the evolutionary dynamics of influenza viruses play out globally, they are fundamentally driven by mutation, reassortment, drift, and selection at the level of individual hosts. These processes have recently been described for influenza A virus, but little is known about the evolutionary dynamics of IBV during individual infections and transmission events. Here, we define the within-host evolutionary dynamics of IBV by sequencing virus populations from naturally infected individuals enrolled in a prospective, community-based cohort over 8,176 person-seasons of observation. Through analysis of high depth-of-coverage sequencing data from samples from 91 individuals with influenza B, we find that IBV accumulates lower genetic diversity than previously observed for influenza A virus during acute infections. Consistent with studies of influenza A viruses, the within-host evolution of IBVs is characterized by purifying selection and the general absence of widespread positive selection of within-host variants. Analysis of shared genetic diversity across 15 sequence-validated transmission pairs suggests that IBV experiences a tight transmission bottleneck similar to that of influenza A virus. These patterns of local-scale evolution are consistent with the lower global evolutionary rate of IBV.IMPORTANCE The evolution of influenza virus is a significant public health problem and necessitates the annual evaluation of influenza vaccine formulation to keep pace with viral escape from herd immunity. Influenza B virus is a serious health concern for children, in particular, yet remains understudied compared to influenza A virus. Influenza B virus evolves more slowly than influenza A virus, but the factors underlying this are not completely understood. We studied how the within-host diversity of influenza B virus relates to its global evolution by sequencing viruses from a community-based cohort. We found that influenza B virus populations have lower within-host genetic diversity than influenza A virus and experience a tight genetic bottleneck during transmission. Our work provides insights into the varying dynamics of influenza viruses in human infection.

Antibodies Against Egg- and Cell-Grown Influenza A(H3N2) Viruses in Adults Hospitalized During the 2017-2018 Influenza Season.

BACKGROUND: Influenza vaccine effectiveness was low in 2017-2018, yet circulating influenza A(H3N2) viruses were antigenically similar to cell-grown vaccine strains. Notably, most influenza vaccines are egg propagated. METHODS: Serum specimens were collected shortly after illness onset from 15 influenza A(H3N2) virus-infected cases and 15 uninfected hospitalized adults. Geometric mean titers against egg- and cell-grown influenza A/Hong Kong/4801/2014(H3N2) virus vaccine strains and representative circulating viruses (including A/Washington/16/2017) were determined by a microneutralization (MN) assay. Independent effects of strain-specific titers on susceptibility were estimated by logistic regression. RESULTS: MN titers against egg-grown influenza A/Hong Kong virus were significantly higher among vaccinated individuals (173 vs 41; P = 0.01). In unadjusted models, a 2-fold increase in titers against egg-grown influenza A/Hong Kong virus was not significantly protective (29% reduction; P = .09), but a similar increase in the cell-grown influenza A/Washington virus antibody titer (3C.2a2) was protective (60% reduction; P = .02). Higher egg-grown influenza A/Hong Kong virus titers were not significantly associated with infection, when adjusted for antibody titers against influenza A/Washington virus (15% reduction; P = .61). A 54% reduction in the odds of infection was observed with a 2-fold increase in titer against influenza A/Washington virus (P = not significant), adjusted for the titer against egg-grown influenza A/Hong Kong virus titer. CONCLUSION: Individuals vaccinated in 2017-2018 had high antibody titers against the egg-adapted vaccine strain and lower titers against circulating viruses. Titers against circulating but not egg-adapted strains were correlated with protection.

A speed-fidelity trade-off determines the mutation rate and virulence of an RNA virus.

Mutation rates can evolve through genetic drift, indirect selection due to genetic hitchhiking, or direct selection on the physicochemical cost of high fidelity. However, for many systems, it has been difficult to disentangle the relative impact of these forces empirically. In RNA viruses, an observed correlation between mutation rate and virulence has led many to argue that their extremely high mutation rates are advantageous because they may allow for increased adaptability. This argument has profound implications because it suggests that pathogenesis in many viral infections depends on rare or de novo mutations. Here, we present data for an alternative model whereby RNA viruses evolve high mutation rates as a byproduct of selection for increased replicative speed. We find that a poliovirus antimutator, 3DG64S, has a significant replication defect and that wild-type (WT) and 3DG64S populations have similar adaptability in 2 distinct cellular environments. Experimental evolution of 3DG64S under selection for replicative speed led to reversion and compensation of the fidelity phenotype. Mice infected with 3DG64S exhibited delayed morbidity at doses well above the lethal level, consistent with attenuation by slower growth as opposed to reduced mutational supply. Furthermore, compensation of the 3DG64S growth defect restored virulence, while compensation of the fidelity phenotype did not. Our data are consistent with the kinetic proofreading model for biosynthetic reactions and suggest that speed is more important than accuracy. In contrast with what has been suggested for many RNA viruses, we find that within-host spread is associated with viral replicative speed and not standing genetic diversity.

Epistatic Interactions within the Influenza A Virus Polymerase Complex Mediate Mutagen Resistance and Replication Fidelity.

Lethal mutagenesis is a broad-spectrum antiviral strategy that employs mutagenic nucleoside analogs to exploit the high mutation rate and low mutational tolerance of many RNA viruses. Studies of mutagen-resistant viruses have identified determinants of replicative fidelity and the importance of mutation rate to viral population dynamics. We have previously demonstrated the effective lethal mutagenesis of influenza A virus using three nucleoside analogs as well as the virus's high genetic barrier to mutagen resistance. Here, we investigate the mutagen-resistant phenotypes of mutations that were enriched in drug-treated populations. We find that PB1 T123A has higher replicative fitness than the wild type, PR8, and maintains its level of genome production during 5-fluorouracil (2,4-dihydroxy-5-fluoropyrimidine) treatment. Surprisingly, this mutagen-resistant variant also has an increased baseline rate of C-to-U and G-to-A mutations. A second drug-selected mutation, PA T97I, interacts epistatically with PB1 T123A to mediate high-level mutagen resistance, predominantly by limiting the inhibitory effect of nucleosides on polymerase activity. Consistent with the importance of epistatic interactions in the influenza virus polymerase, our data suggest that nucleoside analog resistance and replication fidelity are strain dependent. Two previously identified ribavirin {1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide} resistance mutations, PB1 V43I and PB1 D27N, do not confer drug resistance in the PR8 background, and the PR8-PB1 V43I polymerase exhibits a normal baseline mutation rate. Our results highlight the genetic complexity of the influenza A virus polymerase and demonstrate that increased replicative capacity is a mechanism by which an RNA virus can counter the negative effects of elevated mutation rates. IMPORTANCE RNA viruses exist as genetically diverse populations. This standing genetic diversity gives them the potential to adapt rapidly, evolve resistance to antiviral therapeutics, and evade immune responses. Viral mutants with altered mutation rates or mutational tolerance have provided insights into how genetic diversity arises and how it affects the behavior of RNA viruses. To this end, we identified variants within the polymerase complex of influenza virus that are able to tolerate drug-mediated increases in viral mutation rates. We find that drug resistance is highly dependent on interactions among mutations in the polymerase complex. In contrast to other viruses, influenza virus counters the effect of higher mutation rates primarily by maintaining high levels of genome replication. These findings suggest the importance of maintaining large population sizes for viruses with high mutation rates and show that multiple proteins can affect both mutation rate and genome synthesis.