Mutations In Influenza Viruses Research Articles

Protection of Chickens against H9N2 Avian Influenza Isolates with a Live Vector Vaccine Expressing Influenza Hemagglutinin Gene Derived from Y280 Avian Influenza Virus.

Since the outbreak of the H9N2/Y439 avian influenza virus in 1996, the Korean poultry industry has incurred severe economic losses. A novel possibly zoonotic H9N2 virus from the Y280-like lineage (H9N2/Y280) has been prevalent in Korea since June 2020, posing a threat to the poultry sector. Rapid mutation of influenza viruses urges the development of effective vaccines against newly generated strains. Thus, we engineered a recombinant virus rHVT/Y280 to combat H9N2/Y280. We integrated the hemagglutinin (HA) gene of the H9N2/Y280 strain into the US2 region of the herpesvirus of turkeys (HVT) Fc126 vaccine strain, utilizing CRISPR/Cas9 gene-editing technology. The successful construction of rHVT/Y280 was confirmed by polymerase chain reaction and sequencing, followed by efficacy evaluation. Four-day-old specific pathogen-free chickens received the rHVT/Y280 vaccine and were challenged with the H9N2/Y280 strain A21-MRA-003 at 3 weeks post-vaccination. In 5 days, there were no gross lesions among the vaccinated chickens. The rHVT/Y280 vaccine induced strong humoral immunity and markedly reduced virus shedding, achieving 100% inhibition of virus recovery in the cecal tonsil and significantly lowering tissue viral load. Thus, HVT vector vaccines expressing HA can be used for protecting poultry against H9N2/Y280. The induction of humoral immunity by live vaccines is vital in such cases. In summary, the recombinant virus rHVT/Y280 is a promising vaccine candidate for the protection of chickens against the H9N2/Y280.

Computational peptide engineering approach for selection of the new C05 antibody-driven peptide with potency to blocking influenza a virus attachment; from in silico to in vivo

Antiviral drugs are currently used to prevent or treat viral infections like influenza A Virus (IAV). Nonetheless, annual genetic mutations of influenza viruses make them resistant to efficient treatment by current medications. Antiviral peptides have recently attracted researchers’ attention and can potentially supplant the current medications. This study aimed to design peptides against IAV propagation. For this purpose, P2 and P3 peptides were computationally designed based on the HCDR3 region of the C05 antibody (a monoclonal antibody that neutralizes influenza HA protein and inhibits the virus attachment). The synthesized peptides were tested against the influenza A virus (A/Puerto Rico/8/34 (H1N1)) in vitro, and the most efficient peptide was selected for in vivo experiments. It was shown that the designed peptide shows much more prophylactic and therapeutic effects against the virus. These findings demonstrated that the designed peptide can control the virus infection without any cytotoxicity effect. Antiviral peptide design is acknowledged as a critical tactic to manage viral infections by preventing viral binding to the host cells. Communicated by Ramaswamy H. Sarma

Broadly neutralizing antibodies recognizing different antigenic epitopes act synergistically against the influenza B virus.

The discovery of broadly neutralizing monoclonal antibodies against influenza viruses has raised hope for the successful development of new antiviral drugs. However, due to the speed and variety of mutations in influenza viruses, single-component antibodies that recognize specific epitopes are susceptible to viral escape and have limited efficacy when administration is delayed. Hence, it is necessary to develop alternative strategies with better antiviral activity. Influenza B virus infection can cause severe illness in children and the elderly. Commonly used anti-influenza drugs have low clinical efficacy against influenza B virus. In this study, we investigated the antiviral efficacy of combinations of representative monoclonal antibodies targeting different antigenic epitopes against the influenza B virus. We found that combinations of antibodies recognizing the hemagglutinin (HA) head and stem regions showed a stronger neutralizing activity than single antibodies and other antibody combinations in vitro. In addition, we found that pair-wise combinations of antibodies recognizing the HA head region, HA stem region, and neuraminidase enzyme-activated region showed superior antiviral activity than single antibodies in both mouse and ferret in vivo protection assays. Notably, these antibody combinations still displayed good antiviral efficacy when treatment was delayed. Mechanistic studies further revealed that combining antibodies recognizing different epitope regions resulted in extremely strong antibody-dependent cell-mediated cytotoxicity, which may partly explain their superior antiviral effects. Together, the findings of this study provide new avenues for the development of better antiviral drugs and vaccines against influenza viruses.

Discovery of oseltamivir-based novel PROTACs as degraders targeting neuraminidase to combat H1N1 influenza virus

Annual and sporadic influenza outbreaks pose a great threat to human health and the economy worldwide. Moreover, the frequent mutation of influenza viruses caused by antigen drift complicates the application of antiviral therapeutics. As such, there is an urgent need for novel antiviral agents to tackle the problem of insufficient efficacy of licensed drugs. Inspired by the success of the newly emerged PROTACs ( PRO teolysis TA rgeting C himeras) strategy, we report herein the design and synthesis of novel PROTAC molecules based on an oseltamivir scaffold to combat severe annual influenza outbreaks. Among these, several compounds showed good anti-H1N1 activity and efficient influenza neuraminidase (NA) degradation activity. The best compound, 8e , effectively induced influenza NA degradation in a dose-dependent manner and relied on the ubiquitin–proteasome pathway. Moreover, Compound 8e exhibited potent antiviral activity toward both wild-type H1N1 virus and an oseltamivir-resistant strain (H1N1, H274Y). A molecular docking study demonstrated that Compound 8e had good hydrogen-bonding and hydrophobic interactions with both the active sites of NA and Von Hippel-Lindau (VHL) proteins, which could effectively drive the favorable interaction of these two proteins. Thus, as the first report of a successful anti-influenza PROTAC, this proof of concept will greatly widen the application range of the PROTAC technique to antiviral drug discovery. • A series of oseltamivir derived PROTACs with diverse linkers and E3 ligands were synthesized. • The Compound 8e exhibited potent antiviral activity toward both wild-type H1N1 virus and an oseltamivir-resistant strain. • Mechanistic studies indicated that 8e degraded the NA protein through the ubiquitin-proteasome pathway. • This proof of concept work will greatly widen the application range of the PROTAC technique in antiviral drug discovery.

Mutations In Influenza Viruses and Prevention Against These Viruses

The flu originated in 1918 and started a pandemic that spread around the world, killing millions. Other than the current COVID-19 pandemic, the Flu Pandemic of the late 1910s and early 1920s has killed and infected more people than any other virus in modern world history. Since then, the flu has become a seasonal virus that has predictable times of infections. Due to modern medicine, most people are able to survive such infections, and continue on with their lives with little impact. However, as you might come to wonder either eventually or even right now, a person may be curious behind why such an “outdated virus” still continues to live on every winter season. This paper will research the mechanisms of such processes that allow these viruses to stay “alive” (because they technically are not a living thing) and prevent themselves from being eradicated by today’s medical technology. We also look into common and new preventions/ treatments that is/ can be used to eventually eradicate the flu.

Next-generation T cell-activating vaccination increases influenza virus mutation prevalence.

To determine the potential for viral adaptation to T cell responses, we probed the full influenza virus genome by next-generation sequencing directly ex vivo from infected mice, in the context of an experimental T cell–based vaccine, an H5N1-based viral vectored vaccinia vaccine Wyeth/IL-15/5Flu, versus the current standard-of-care, seasonal inactivated influenza vaccine (IIV) and unvaccinated conditions. Wyeth/IL-15/5Flu vaccination was coincident with increased mutation incidence and frequency across the influenza genome; however, mutations were not enriched within T cell epitope regions, but high allele frequency mutations within conserved hemagglutinin stem regions and PB2 mammalian adaptive mutations arose. Depletion of CD4+ and CD8+ T cell subsets led to reduced frequency of mutants in vaccinated mice; therefore, vaccine-mediated T cell responses were important drivers of virus diversification. Our findings suggest that Wyeth/IL-15/5Flu does not generate T cell escape mutants but increases stochastic events for virus adaptation by stringent bottlenecks.

STUDI EFEKTIVITAS VAKSIN INFLUENZA: UPDATED REVIEW

Influenza is caused by a rapidly mutating viruse that consists of 2 types, namely type A with the H1N1 and H3N2 genotypes and type B. Influenza caused global mortality with 250,000-500,000 death in 2009. The effectiveness of vaccines also changes regarding the mutation of influenza viruses, however, in the development and utilization of influenza vaccines should be supported by the economic status of a country. Up to now, there are many countries that have not prioritized the utilization of influenza vaccines. The target of influenza vaccination are children and adults (> 60 years old). The purpose of this review was to determine the effectiveness of influenza vaccines from various countries and categorized based on their income. This review used Medline, Elsevier, and BMC Public Health as the database with the keywords "Effectiveness" and "Influenza vaccine". Then, the articles are selected based on inclusion and exclusion criteria. Based on the initial search there are 784 articles that match the keywords, and only 13 articles met the criteria. These articles are classified based on the center of the study in order to classify based on their national income; 5 studies in high income countries, 5 studies in upper-middle income countries, 3 studies in lower-middle income countries, and 1 study in low income countries. The results showed that the administration of influenza vaccine in high income and upper-middle income countries is quite effective for type A H1N1 genotypes, where as H3N2 is less effective. In the lower-middle income countries, the utilization of vaccines with type A H3N2 genotypes was effective, however, in the low-income countries, the effectiveness of vaccines has not been justified due to the limited study of type of influenza and the administration of influenza vaccines in those countries.

Characterization of key amino acid substitutions and dynamics of the influenza virus H3N2 hemagglutinin

The annual epidemics of seasonal influenza is partly attributed to the continued virus evolution. It is challenging to evaluate the effect of influenza virus mutations on evading population immunity. In this study, we introduce a novel statistical and computational approach to measure the dynamic molecular determinants underlying epidemics using effective mutations (EMs), and account for the time of waning mutation advantage against herd immunity by measuring the effective mutation periods (EMPs). Extensive analysis is performed on the sequencing and epidemiology data of H3N2 epidemics in ten regions from season to season. We systematically identified 46 EMs in the hemagglutinin (HA) gene, in which the majority were antigenic sites. Eight EMs were located in immunosubdominant stalk domain, an important target for developing broadly reactive antibodies. The EMs might provide timely information on key substitutions for influenza vaccines antigen design. The EMP suggested that major genetic variants of H3N2 circulated in Southeast Asia for an average duration of 4.5 years (SD 2.4) compared to a significantly shorter 2.0 years (SD 1.0) in temperate regions. The proposed method bridges population epidemics and molecular characteristics of infectious diseases, and would find broad applications in various pathogens mutation estimations.

Analysis of marker substitutions in A/chicken/Astrakhan/2171-1/2020 H5N8 isolate of avian influenza virus recovered in the Astrakhan Oblast

At the end of 2020, a large-scale bird death was registered at one of the poultry farms in the Astrakhan region, the cause of which was avian influenza. Data on detection of the marker substitutions in viral proteins of avian influenza virus A/chicken/Astrakhan/2171-1/2020 isolate are presented in the paper. Type A Н5N8 avian influenza virus was identified with complex PCR-based methods in the submitted samples. Hemagglutinin gene fragment sequencing identified REKRRKR/ GLF, highly pathogenic avian influenza virus isolate-characteristic amino acid sequence of the hemagglutinin cleavage site. Phylogenetic analysis of nucleotide sequences of hemagglutinin gene segment (848–1105 bp ORF) allowed A/chicken/Astrakhan/2171-1/2020 H5N8 isolate to be classified to highly pathogenic avian influenza virus genetic clade 2.3.4.4. Comparative analysis of genome segments using available databases showed that A/chicken/Astrakhan/2171-1/2020 H5N8 virus related to А/Н5 avian influenza virus isolates detected in the Russian Federation in 2016–2020. Analysis of the studied virus isolate hemagglutinin amino acid identified AIV-characteristic G225QRG228amino acids in the receptor-binding domain of the protein enabling high-affinity binding to avian epithelial cell SAα-2,3- gal receptors. Single mutations, 70G in NEP protein and 13Р in PB1 protein, out of the list of the reported influenza virus mutations affecting successful influenza virus replication in mammals were identified. No mutations affecting virus sensitivity to anti-viral medicines, rimantadin, amantadine, oseltamivir and zanamivir, were detected. The following mutations recognized as pathogenicity determinants in mice were found: 42S in the NS1 protein and 30D protein 215A in M1 protein.

Animal Models for Influenza Research: Strengths and Weaknesses.

Influenza remains one of the most significant public health threats due to its ability to cause high morbidity and mortality worldwide. Although understanding of influenza viruses has greatly increased in recent years, shortcomings remain. Additionally, the continuous mutation of influenza viruses through genetic reassortment and selection of variants that escape host immune responses can render current influenza vaccines ineffective at controlling seasonal epidemics and potential pandemics. Thus, there is a knowledge gap in the understanding of influenza viruses and a corresponding need to develop novel universal vaccines and therapeutic treatments. Investigation of viral pathogenesis, transmission mechanisms, and efficacy of influenza vaccine candidates requires animal models that can recapitulate the disease. Furthermore, the choice of animal model for each research question is crucial in order for researchers to acquire a better knowledge of influenza viruses. Herein, we reviewed the advantages and limitations of each animal model—including mice, ferrets, guinea pigs, swine, felines, canines, and non-human primates—for elucidating influenza viral pathogenesis and transmission and for evaluating therapeutic agents and vaccine efficacy.

H7N7 Avian Influenza Virus Mutation from Low to High Pathogenicity on a Layer Chicken Farm in the UK.

Avian influenza virus (AIV) subtypes H5 and H7 are capable of mutating from low to high pathogenicity strains, causing high mortality in poultry with significant economic losses globally. During 2015, two outbreaks of H7N7 low pathogenicity AIV (LPAIV) in Germany, and one each in the United Kingdom (UK) and The Netherlands occurred, as well as single outbreaks of H7N7 high pathogenicity AIV (HPAIV) in Germany and the UK. Both HPAIV outbreaks were linked to precursor H7N7 LPAIV outbreaks on the same or adjacent premises. Herein, we describe the clinical, epidemiological, and virological investigations for the H7N7 UK HPAIV outbreak on a farm with layer chickens in mixed free-range and caged units. H7N7 HPAIV was identified and isolated from clinical samples, as well as H7N7 LPAIV, which could not be isolated. Using serological and molecular evidence, we postulate how the viruses spread throughout the premises, indicating potential points of incursion and possible locations for the mutation event. Serological and mortality data suggested that the LPAIV infection preceded the HPAIV infection and afforded some clinical protection against the HPAIV. These results document the identification of a LPAIV to HPAIV mutation in nature, providing insights into factors that drive its manifestation during outbreaks.

Dynamic interactions of influenza viruses in Hong Kong during 1998-2018.

Influenza epidemics cause substantial morbidity and mortality every year worldwide. Currently, two influenza A subtypes, A(H1N1) and A(H3N2), and type B viruses co-circulate in humans and infection with one type/subtype could provide cross-protection against the others. However, it remains unclear how such ecologic competition via cross-immunity and antigenic mutations that allow immune escape impact influenza epidemic dynamics at the population level. Here we develop a comprehensive model-inference system and apply it to study the evolutionary and epidemiological dynamics of the three influenza types/subtypes in Hong Kong, a city of global public health significance for influenza epidemic and pandemic control. Utilizing long-term influenza surveillance data since 1998, we are able to estimate the strength of cross-immunity between each virus-pairs, the timing and frequency of punctuated changes in population immunity in response to antigenic mutations in influenza viruses, and key epidemiological parameters over the last 20 years including the 2009 pandemic. We find evidence of cross-immunity in all types/subtypes, with strongest cross-immunity from A(H1N1) against A(H3N2). Our results also suggest that A(H3N2) may undergo antigenic mutations in both summers and winters and thus monitoring the virus in both seasons may be important for vaccine development. Overall, our study reveals intricate epidemiological interactions and underscores the importance of simultaneous monitoring of population immunity, incidence rates, and viral genetic and antigenic changes.

Alignparse: A Python package for parsing complex features from high-throughput long-read sequencing.

Summary & PurposeAdvances in sequencing technology have made it possible to generate large numbers of long, high-accuracy sequencing reads. For instance, the new PacBio Sequel platform can generate hundreds of thousands of high-quality circular consensus sequences in a single run (Hebert et al., 2018; Rhoads & Au, 2015). Good programs exist for aligning these reads for genome assembly (Chaisson & Tesler, 2012; Li, 2018). However, these long reads can also be used for other purposes, such as sequencing PCR amplicons that contain various features of interest. For instance, PacBio circular consensus sequences have been used to identify the mutations in influenza viruses in single cells (Russell et al, 2019), or to link barcodes to gene mutants in deep mutational scanning (Matreyek et al., 2018). For such applications, the alignment of the sequences to the targets may be fairly trivial, but it is not trivial to then parse specific features of interest (such as mutations, unique molecular identifiers, cell barcodes, and flanking sequences) from these alignments.Here we describe alignparse, a Python package for parsing complex sets of features from long sequences that map to known targets. Specifically, it allows the user to provide complex target sequences in Genbank Flat File format that contain an arbitrary number of user-defined sub-sequence features (Sayers et al., 2019). It then aligns the sequencing reads to these targets and filters alignments based on whether the user-specified features are present with the desired identities (which can be set to different thresholds for different features). Finally, it parses out the sequences, mutations, and/or accuracy (sequence quality) of these features as specified by the user. The flexibility of this package therefore fulfills the need for a tool to extract and analyze complex sets of features in large numbers of long sequencing reads.

Influenza A Hemagglutinin Passage Bias Sites and Host Specificity Mutations.

Animal studies aimed at understanding influenza virus mutations that change host specificity to adapt to replication in mammalian hosts are necessarily limited in sample numbers due to high cost and safety requirements. As a safe, higher-throughput alternative, we explore the possibility of using readily available passage bias data obtained mostly from seasonal H1 and H3 influenza strains that were differentially grown in mammalian (MDCK) and avian cells (eggs). Using a statistical approach over 80,000 influenza hemagglutinin sequences with passage information, we found that passage bias sites are most commonly found in three regions: (i) the globular head domain around the receptor binding site, (ii) the region that undergoes pH-dependent structural changes and (iii) the unstructured N-terminal region harbouring the signal peptide. Passage bias sites were consistent among different passage cell types as well as between influenza A subtypes. We also find epistatic interactions of site pairs supporting the notion of host-specific dependency of mutations on virus genomic background. The sites identified from our large-scale sequence analysis substantially overlap with known host adaptation sites in the WHO H5N1 genetic changes inventory suggesting information from passage bias can provide candidate sites for host specificity changes to aid in risk assessment for emerging strains.

Mutations associated with egg adaptation of influenza A(H1N1)pdm09 virus in laboratory based surveillance in China, 2009–2016

Mutations of influenza virus associated with adaptation occurring during passage in embryonated chicken eggs could result in antigenic change or reduced vaccine effectiveness. In this study, we investigated the mutations of influenza A(H1N1)pdm09 egg isolates from the Chinese National Influenza Surveillance Network between 2009 and 2016. Thirteen mutations were identified in the hemagglutinin (HA) protein from viruses passaged in eggs, in comparing to those in cells. After scanning public database, four mutations, D127E, L191I, D222G/N and Q223R in HA1, which may alter the receptor-binding specificity, were observed frequently. Although the A(H1N1)pdm09 virus has evolved in human for nearly ten years, most egg-cultured viruses acquired one or more further mutations. Using the egg isolates for influenza surveillance requires extra caution because of these selected mutations, and their impacts on antigenicity and receptor-binding property need further evaluation. Currently, most of the influenza vaccines are produced using egg isolates, particularly in China. Thus, there is an urge to promote the establishment of an alternative influenza vaccine production platform.

Optimal Control of Heterogeneous Mutating Viruses

Different strains of influenza viruses spread in human populations during every epidemic season. As the size of an infected population increases, the virus can mutate itself and grow in strength. The traditional epidemic SIR model does not capture virus mutations and, hence, the model is not sufficient to study epidemics where the virus mutates at the same time as it spreads. In this work, we establish a novel framework to study the epidemic process with mutations of influenza viruses, which couples the SIR model with replicator dynamics used for describing virus mutations. We formulated an optimal control problem to study the optimal strategies for medical treatment and quarantine decisions. We obtained structural results for the optimal strategies and used numerical examples to corroborate our results.

Predicting Influenza Antigenicity by Matrix Completion With Antigen and Antiserum Similarity.

The rapid mutation of influenza viruses especially on the two surface proteins hemagglutinin (HA) and neuraminidase (NA) has made them capable to escape from population immunity, which has become a key challenge for influenza vaccine design. Thus, it is crucial to predict influenza antigenic evolution and identify new antigenic variants in a timely manner. However, traditional experimental methods like hemagglutination inhibition (HI) assay to select vaccine strains are time and labor-intensive, while popular computational methods are less sensitive, which presents the need for more accurate algorithms. In this study, we have proposed a novel low-rank matrix completion model MCAAS to infer antigenic distances between antigens and antisera based on partially revealed antigenic distances, virus similarity based on HA protein sequences, and vaccine similarity based on vaccine strains. The model exploits the correlations of viruses and vaccines in serological tests as well as the ability of HAs from viruses and vaccine strains in inferring influenza antigenicity. We also compared the effects of comprehensive 65 amino acids substitution matrices in predicting influenza antigenicity. As a result, we applied MCAAS into H3N2 seasonal influenza virus data. Our model achieved a 10-fold cross validation root-mean-squared error (RMSE) of 0.5982, significantly outperformed existing computational methods like antigenic cartography, AntigenMap and BMCSI. We also constructed the antigenic map and studied the association between genetic and antigenic evolution of H3N2 influenza viruses. Finally, our analyses showed that homologous structure derived amino acid substitution matrix (HSDM) is most powerful in predicting influenza antigenicity, which is consistent with previous studies.

Direct evidence of H7N7 avian influenza virus mutation from low to high virulence on a single poultry premises during an outbreak in free range chickens in the UK, 2008

H5 and H7 subtypes of low pathogenicity avian influenza viruses (LPAIVs) have the potential to evolve into highly pathogenic avian influenza viruses (HPAIVs), causing high mortality in galliforme poultry with substantial economic losses for the poultry industry. This study provides direct evidence of H7N7 LPAIV mutation to HPAIV on a single poultry premises during an outbreak that occurred in June 2008 in free range laying hens in Oxfordshire, UK. We report the first detection of a rare di-basic cleavage site (CS) motif (PEIPKKRGLF), unique to galliformes, that has previously been associated with a LPAIV phenotype. Three distinct HPAIV CS sequences (PEIPKRKKRGLF, PEIPKKKKRGLF and PEIPKKKKKKRGLF) were identified in the infected sheds suggesting molecular evolution at the outbreak premises. Further evidence for H7N7 LPAIV preceding mutation to HPAIV was derived by examining clinical signs, epidemiological descriptions and analysing laboratory results on the timing and proportions of seroconversion and virus shedding at each infected shed on the premises. In addition to describing how the outbreak was diagnosed and managed via statutory laboratory testing, phylogenetic analysis revealed reassortant events during 2006–2008 that suggested likely incursion of a wild bird origin LPAIV precursor to the H7N7 HPAIV outbreak. Identifying a precursor LPAIV is important for understanding the molecular changes and mechanisms involved in the emergence of HPAIV. This information can lead to understanding how and why only some H7 LPAIVs appear to readily mutate to HPAIV.

Semi-quantitative Influenza A population averages from a multiplex respiratory viral panel (RVP): potential for reflecting target sequence changes affecting the assay

BackgroundYearly influenza virus mutations potentially affect the performance of molecular assays, if nucleic acid changes involve the sequences in the assay. Because individual patient viral loads depend on variables such as duration of illness, specimen type, age, and immunosuppression, we examined seasonal population averages of positive tests to smooth inherent variability.MethodsWe studied the population seasonal averages of the semi-quantitative nAMPs for the influenza matrix and hemagglutinin genes in the GenMark (Carlsbad, CA) Respiratory Viral Panel assay between 3 institutions over 3 Influenza seasons.ResultsPopulation average nAMPs were strikingly consistent between separate institutions, but differed substantially between H3N2 and H1N1 seasons. In the 2012–2013 and 2014–2015 influenza seasons, matrix gene H3N2 nAMP averages were 50–70% less than those of the same assay in the 2013–2014 H1N1 season. Influenza strains representative of these seasons were grown in tissue culture and when the supernatant virus was adjusted to the same copy number using a TaqMan assay, the same relative differences were reproduced in the RVP assay. Because the sequences for the PCR and PCR product detection in the GenMark assay are proprietary, the manufacturer provided single stranded DNA matching the capture probe for the representative H3N2 (3 mismatches) and H1N1 strains (2 different mismatches). Equimolar concentrations of these synthetic DNA sequences gave average nAMP values that closely correlated with the average nAMPS of the representative strains and their respective seasonal averages.ConclusionsSeasonal averages of semi-quantitative data may provide a means to follow assay performance as a reflection of the effects of molecular drift.

A novel twelve class fluctuation test reveals higher than expected mutation rates for influenza A viruses.

Influenza virus' low replicative fidelity contributes to its capacity for rapid evolution. Clonal sequencing and fluctuation tests have suggested that the influenza virus mutation rate is 2.7 × 10-6 - 3.0 × 10-5 substitutions per nucleotide per strand copied (s/n/r). However, sequencing assays are biased toward mutations with minimal fitness impacts and fluctuation tests typically investigate only a subset of all possible single nucleotide mutations. We developed a fluctuation test based on reversion to fluorescence in a set of virally encoded mutant green fluorescent proteins, which allowed us to measure the rates of selectively neutral mutations representative of the twelve different mutation types. We measured an overall mutation rate of 1.8 × 10-4 s/n/r for PR8 (H1N1) and 2.5 × 10-4 s/n/r for Hong Kong 2014 (H3N2) and a transitional bias of 2.7-3.6. Our data suggest that each replicated genome will have an average of 2-3 mutations and highlight the importance of mutational load in influenza virus evolution.