Ferroptosis of tumour neutrophils causes immune suppression in cancer.

Ferroptosis is a non-apoptotic form of regulated cell death that is triggered by the discoordination of regulatory redox mechanisms culminating in massive peroxidation of polyunsaturated phospholipids. Ferroptosis inducers have shown considerable effectiveness in killing tumour cells in vitro, yet there has been no obvious success in experimental animal models, with the notable exception of immunodeficient mice1,2. This suggests that the effect of ferroptosis on immune cells remains poorly understood. Pathologically activated neutrophils (PMNs), termed myeloid-derived suppressor cells (PMN-MDSCs), are major negative regulators of anti-tumour immunity3-5. Here we found that PMN-MDSCs in the tumour microenvironment spontaneously die by ferroptosis. Although decreasing the presence of PMN-MDSCs, ferroptosis induces the release of oxygenated lipids and limits the activity of human and mouse T cells. In immunocompetent mice, genetic and pharmacological inhibition of ferroptosis abrogates suppressive activity of PMN-MDSCs, reduces tumour progression and synergizes with immune checkpoint blockade to suppress the tumour growth. By contrast, induction of ferroptosis in immunocompetent mice promotes tumour growth. Thus, ferroptosis is a unique and targetable immunosuppressive mechanism of PMN-MDSCs in the tumour microenvironment that can be pharmacologically modulated to limit tumour progression.

Influence of Sex on Respiratory Syncytial Virus Genotype Infection Frequency and Nasopharyngeal Microbiome.

Respiratory syncytial virus (RSV) has a significant health burden in children, older adults, and the immunocompromised. However, limited effort has been made to identify emergence of new RSV genotypes' frequency of infection and how the combination of nasopharyngeal microbiome and viral genotypes impact RSV disease outcomes. In an observational cohort designed to capture the first infant RSV infection, we employed multi-omics approaches to sequence 349 RSV complete genomes and matched nasopharyngeal microbiomes, during which the 2012/2013 season was dominated by RSV-A, whereas 2013 and 2014 was dominated by RSV-B. We found non-G-72nt-duplicated RSV-A strains were more frequent in male infants (P = 0.02), whereas G-72nt-duplicated genotypes (which is ON1 lineage) were seen equally in both males and females. DESeq2 testing of the nasal microbiome showed Haemophilus was significantly more abundant in infants with RSV-A infection compared to infants with RSV-B infection (adjusted P = 0.002). In addition, the broad microbial clustering of the abundant genera was significantly associated with infant sex (P = 0.03). Overall, we show sex differences in infection by RSV genotype and host nasopharyngeal microbiome, suggesting an interaction between host genetics, virus genotype, and associated nasopharyngeal microbiome. IMPORTANCE Respiratory syncytial virus (RSV) is one of the leading causes of lower respiratory tract infections in young children and is responsible for high hospitalization rates and morbidity in infants and the elderly. To understand how the emergence of RSV viral genotypes and viral-respiratory microbiome interactions contribute to infection frequency and severity, we utilized an observational cohort designed to capture the first infant RSV infection we employed multi-omics approaches to sequence 349 RSV complete genomes and matched nasopharyngeal microbiomes. We found non-G-72nt-duplicated RSV-A genotypes were more frequent in male infants, whereas G-72nt-duplicated RSV-A strains (ON1 lineage) were seen equally in both males and females. Microbiome analysis show Haemophilus was significantly more abundant in infants with RSV-A compared to infants with RSV-B infection and the microbial clustering of the abundant genera was associated with infant sex. Overall, we show sex differences in RSV genotype-nasopharyngeal microbiome, suggesting an interaction host genetics-virus-microbiome interaction.

The soft palate is an important site of adaptation for transmissible influenza viruses.

Influenza A viruses pose a major public health threat by causing seasonal epidemics and sporadic pandemics. Their epidemiological success relies on airborne transmission from person to person; however, the viral properties governing airborne transmission of influenza A viruses are complex. Influenza A virus infection is mediated via binding of the viral haemagglutinin (HA) to terminally attached α2,3 or α2,6 sialic acids on cell surface glycoproteins. Human influenza A viruses preferentially bind α2,6-linked sialic acids whereas avian influenza A viruses bind α2,3-linked sialic acids on complex glycans on airway epithelial cells. Historically, influenza A viruses with preferential association with α2,3-linked sialic acids have not been transmitted efficiently by the airborne route in ferrets. Here we observe efficient airborne transmission of a 2009 pandemic H1N1 (H1N1pdm) virus (A/California/07/2009) engineered to preferentially bind α2,3-linked sialic acids. Airborne transmission was associated with rapid selection of virus with a change at a single HA site that conferred binding to long-chain α2,6-linked sialic acids, without loss of α2,3-linked sialic acid binding. The transmissible virus emerged in experimentally infected ferrets within 24 hours after infection and was remarkably enriched in the soft palate, where long-chain α2,6-linked sialic acids predominate on the nasopharyngeal surface. Notably, presence of long-chain α2,6-linked sialic acids is conserved in ferret, pig and human soft palate. Using a loss-of-function approach with this one virus, we demonstrate that the ferret soft palate, a tissue not normally sampled in animal models of influenza, rapidly selects for transmissible influenza A viruses with human receptor (α2,6-linked sialic acids) preference.

Multiple Introductions and Antigenic Mismatch with Vaccines May Contribute to Increased Predominance of G12P[8] Rotaviruses in the United States.

Rotavirus is the leading global cause of diarrheal mortality for unvaccinated children under 5 years of age. The outer capsid of rotavirus virions consists of VP7 and VP4 proteins, which determine viral G and P types, respectively, and are primary targets of neutralizing antibodies. Successful vaccination depends upon generating broadly protective immune responses following exposure to rotaviruses presenting a limited number of G- and P-type antigens. Vaccine introduction resulted in decreased rotavirus disease burden but also coincided with the emergence of uncommon G and P genotypes, including G12. To gain insight into the recent predominance of G12P[8] rotaviruses in the United States, we evaluated 142 complete rotavirus genome sequences and metadata from 151 clinical specimens collected in Nashville, TN, from 2011 to 2013 through the New Vaccine Surveillance Network. Circulating G12P[8] strains were found to share many segments with other locally circulating strains but to have distinct constellations. Phylogenetic analyses of G12 sequences and their geographic sources provided evidence for multiple separate introductions of G12 segments into Nashville, TN. Antigenic epitopes of VP7 proteins of G12P[8] strains circulating in Nashville, TN, differ markedly from those of vaccine strains. Fully vaccinated children were found to be infected with G12P[8] strains more frequently than with other rotavirus genotypes. Multiple introductions and significant antigenic mismatch may in part explain the recent predominance of G12P[8] strains in the United States and emphasize the need for continued monitoring of rotavirus vaccine efficacy against emerging rotavirus genotypes.IMPORTANCE Rotavirus is an important cause of childhood diarrheal disease worldwide. Two immunodominant proteins of rotavirus, VP7 and VP4, determine G and P genotypes, respectively. Recently, G12P[8] rotaviruses have become increasingly predominant. By analyzing rotavirus genome sequences from stool specimens obtained in Nashville, TN, from 2011 to 2013 and globally circulating rotaviruses, we found evidence of multiple introductions of G12 genes into the area. Based on sequence polymorphisms, VP7 proteins of these viruses are predicted to present themselves to the immune system very differently than those of vaccine strains. Many of the sick children with G12P[8] rotavirus in their diarrheal stools also were fully vaccinated. Our findings emphasize the need for continued monitoring of circulating rotaviruses and the effectiveness of the vaccines against strains with emerging G and P genotypes.

Large-Scale Complete-Genome Sequencing and Phylodynamic Analysis of Eastern Equine Encephalitis Virus Reveals Source-Sink Transmission Dynamics in the United States.

Eastern equine encephalitis virus (EEEV) has a high case-fatality rate in horses and humans, and Florida has been hypothesized to be the source of EEEV epidemics for the northeastern United States. To test this hypothesis, we sequenced complete genomes of 433 EEEV strains collected within the United States from 1934 to 2014. Phylogenetic analysis suggested EEEV evolves relatively slowly and that transmission is enzootic in Florida, characterized by higher genetic diversity and long-term local persistence. In contrast, EEEV strains in New York and Massachusetts were characterized by lower genetic diversity, multiple introductions, and shorter local persistence. Our phylogeographic analysis supported a source-sink model in which Florida is the major source of EEEV compared to the other localities sampled. In sum, this study revealed the complex epidemiological dynamics of EEEV in different geographic regions in the United States and provided general insights into the evolution and transmission of other avian mosquito-borne viruses in this region.IMPORTANCE Eastern equine encephalitis virus (EEEV) infections are severe in horses and humans on the east coast of the United States with a >90% mortality rate in horses, an ∼33% mortality rate in humans, and significant brain damage in most human survivors. However, little is known about the evolutionary characteristics of EEEV due to the lack of genome sequences. By generating large collection of publicly available complete genome sequences, this study comprehensively determined the evolution of the virus, described the epidemiological dynamics of EEEV in different states in the United States, and identified Florida as one of the major sources. These results may have important implications for the control and prevention of other mosquito-borne viruses in the Americas.

Avian Influenza Viruses in Wild Birds: Virus Evolution in a Multihost Ecosystem.

Wild ducks and gulls are the major reservoirs for avian influenza A viruses (AIVs). The mechanisms that drive AIV evolution are complex at sites where various duck and gull species from multiple flyways breed, winter, or stage. The Republic of Georgia is located at the intersection of three migratory flyways: the Central Asian flyway, the East Africa/West Asia flyway, and the Black Sea/Mediterranean flyway. For six complete study years (2010 to 2016), we collected AIV samples from various duck and gull species that breed, migrate, and overwinter in Georgia. We found a substantial subtype diversity of viruses that varied in prevalence from year to year. Low-pathogenic AIV (LPAIV) subtypes included H1N1, H2N3, H2N5, H2N7, H3N8, H4N2, H6N2, H7N3, H7N7, H9N1, H9N3, H10N4, H10N7, H11N1, H13N2, H13N6, H13N8, and H16N3, and two highly pathogenic AIVs (HPAIVs) belonging to clade 2.3.4.4, H5N5 and H5N8, were found. Whole-genome phylogenetic trees showed significant host species lineage restriction for nearly all gene segments and significant differences in observed reassortment rates, as defined by quantification of phylogenetic incongruence, and in nucleotide sequence diversity for LPAIVs among different host species. Hemagglutinin clade 2.3.4.4 H5N8 viruses, which circulated in Eurasia during 2014 and 2015, did not reassort, but analysis after their subsequent dissemination during 2016 and 2017 revealed reassortment in all gene segments except NP and NS. Some virus lineages appeared to be unrelated to AIVs in wild bird populations in other regions, with maintenance of local AIVs in Georgia, whereas other lineages showed considerable genetic interrelationships with viruses circulating in other parts of Eurasia and Africa, despite relative undersampling in the area.IMPORTANCE Waterbirds (e.g., gulls and ducks) are natural reservoirs of avian influenza viruses (AIVs) and have been shown to mediate the dispersal of AIVs at intercontinental scales during seasonal migration. The segmented genome of influenza viruses enables viral RNA from different lineages to mix or reassort when two viruses infect the same host. Such reassortant viruses have been identified in most major human influenza pandemics and several poultry outbreaks. Despite their importance, we have only recently begun to understand AIV evolution and reassortment in their natural host reservoirs. This comprehensive study illustrates AIV evolutionary dynamics within a multihost ecosystem at a stopover site where three major migratory flyways intersect. Our analysis of this ecosystem over a 6-year period provides a snapshot of how these viruses are linked to global AIV populations. Understanding the evolution of AIVs in the natural host is imperative to mitigating both the risk of incursion into domestic poultry and the potential risk to mammalian hosts, including humans.

The effective rate of influenza reassortment is limited during human infection.

We characterise the evolutionary dynamics of influenza infection described by viral sequence data collected from two challenge studies conducted in human hosts. Viral sequence data were collected at regular intervals from infected hosts. Changes in the sequence data observed across time show that the within-host evolution of the virus was driven by the reversion of variants acquired during previous passaging of the virus. Treatment of some patients with oseltamivir on the first day of infection did not lead to the emergence of drug resistance variants in patients. Using an evolutionary model, we inferred the effective rate of reassortment between viral segments, measuring the extent to which randomly chosen viruses within the host exchange genetic material. We find strong evidence that the rate of effective reassortment is low, such that genetic associations between polymorphic loci in different segments are preserved during the course of an infection in a manner not compatible with epistasis. Combining our evidence with that of previous studies we suggest that spatial heterogeneity in the viral population may reduce the extent to which reassortment is observed. Our results do not contradict previous findings of high rates of viral reassortment in vitro and in small animal studies, but indicate that in human hosts the effective rate of reassortment may be substantially more limited.

Nasopharyngeal Lactobacillus is associated with a reduced risk of childhood wheezing illnesses following acute respiratory syncytial virus infection in infancy.

BACKGROUND: Early life acute respiratory infection (ARI) with respiratory syncytial virus (RSV) has been strongly associated with the development of childhood wheezing illnesses, but the pathways underlying this association are poorly understood. OBJECTIVE: To examine the role of the nasopharyngeal microbiome in the development of childhood wheezing illnesses following RSV ARI in infancy. METHODS: We conducted a nested cohort study of 118 previously healthy, term infants with confirmed RSV ARI by RT-PCR. We used next-generation sequencing of the V4 region of the 16S ribosomal RNA gene to characterize the nasopharyngeal microbiome during RSV ARI. Our main outcome of interest was 2-year subsequent wheeze. RESULTS: Of the 118 infants, 113 (95.8%) had 2-year outcome data. Of these, 46 (40.7%) had parental report of subsequent wheeze. There was no association between the overall taxonomic composition, diversity, and richness of the nasopharyngeal microbiome during RSV ARI with the development of subsequent wheeze. However, the nasopharyngeal detection and abundance of Lactobacillus was consistently higher in infants who did not develop this outcome. Lactobacillus also ranked first among the different genera in a model distinguishing infants with and without subsequent wheeze. CONCLUSIONS: The nasopharyngeal detection and increased abundance of Lactobacillus during RSV ARI in infancy are associated with a reduced risk of childhood wheezing illnesses at age 2 years.

Poor Immunogenicity, Not Vaccine Strain Egg Adaptation, May Explain the Low H3N2 Influenza Vaccine Effectiveness in 2012-2013.

Background: Influenza vaccination aims to prevent infection by influenza virus and reduce associated morbidity and mortality; however, vaccine effectiveness (VE) can be modest, especially for subtype A(H3N2). Low VE has been attributed to mismatches between the vaccine and circulating influenza strains and to the vaccine's elicitation of protective immunity in only a subset of the population. The low H3N2 VE in the 2012-2013 season was attributed to egg-adaptive mutations that created antigenic mismatch between the actual vaccine strain (IVR-165) and both the intended vaccine strain (A/Victoria/361/2011) and the predominant circulating strains (clades 3C.2 and 3C.3). Methods: We investigated the basis of low VE in 2012-2013 by determining whether vaccinated and unvaccinated individuals were infected by different viral strains and by assessing the serologic responses to IVR-165, A/Victoria/361/2011, and 3C.2 and 3C.3 strains in an adult cohort before and after vaccination. Results: We found no significant genetic differences between the strains that infected vaccinated and unvaccinated individuals. Vaccination increased titers to A/Victoria/361/2011 and 3C.2 and 3C.3 representative strains as much as to IVR-165. These results are consistent with the hypothesis that vaccination boosted cross-reactive immune responses instead of specific responses against unique vaccine epitopes. Only approximately one-third of the cohort achieved a ≥4-fold increase in titer. Conclusions: In contrast to analyses based on ferret studies, low H3N2 VE in 2012-2013 in adults does not appear to be due to egg adaptation of the vaccine strain. Instead, low VE might have been caused by low vaccine immunogenicity in a subset of the population.

Ecosystem Interactions Underlie the Spread of Avian Influenza A Viruses with Pandemic Potential.

Despite evidence for avian influenza A virus (AIV) transmission between wild and domestic ecosystems, the roles of bird migration and poultry trade in the spread of viruses remain enigmatic. In this study, we integrate ecosystem interactions into a phylogeographic model to assess the contribution of wild and domestic hosts to AIV distribution and persistence. Analysis of globally sampled AIV datasets shows frequent two-way transmission between wild and domestic ecosystems. In general, viral flow from domestic to wild bird populations was restricted to within a geographic region. In contrast, spillover from wild to domestic populations occurred both within and between regions. Wild birds mediated long-distance dispersal at intercontinental scales whereas viral spread among poultry populations was a major driver of regional spread. Viral spread between poultry flocks frequently originated from persistent lineages circulating in regions of intensive poultry production. Our analysis of long-term surveillance data demonstrates that meaningful insights can be inferred from integrating ecosystem into phylogeographic reconstructions that may be consequential for pandemic preparedness and livestock protection.

Reassortment of Influenza A Viruses in Wild Birds in Alaska before H5 Clade 2.3.4.4 Outbreaks.

Sampling of mallards in Alaska during September 2014-April 2015 identified low pathogenic avian influenza A virus (subtypes H5N2 and H1N1) that shared ancestry with highly pathogenic reassortant H5N2 and H5N1 viruses. Molecular dating indicated reassortment soon after interhemispheric movement of H5N8 clade 2.3.4.4, suggesting genetic exchange in Alaska or surrounds before outbreaks.

The emergence and evolution of influenza A (H1α) viruses in swine in Canada and the United States.

Swine are a key reservoir host for influenza A viruses (IAVs), with the potential to cause global pandemics in humans. Gaps in surveillance in many of the world's largest swine populations impede our understanding of how novel viruses emerge and expand their spatial range in pigs. Although US swine are intensively sampled, little is known about IAV diversity in Canada's population of ~12 million pigs. By sequencing 168 viruses from multiple regions of Canada, our study reveals that IAV diversity has been underestimated in Canadian pigs for many years. Critically, a new H1 clade has emerged in Canada (H1α-3), with a two-amino acid deletion at H1 positions 146-147, that experienced rapid growth in Manitoba's swine herds during 2014-2015. H1α-3 viruses also exhibit a higher capacity to invade US swine herds, resulting in multiple recent introductions of the virus into the US Heartland following large-scale movements of pigs in this direction. From the Heartland, H1α-3 viruses have disseminated onward to both the east and west coasts of the United States, and may become established in Appalachia. These findings demonstrate how long-distance trading of live pigs facilitates the spread of IAVs, increasing viral genetic diversity and complicating pathogen control. The proliferation of novel H1α-3 viruses also highlights the need for expanded surveillance in a Canadian swine population that has long been overlooked, and may have implications for vaccine design.

Multiplex Reverse Transcription-PCR for Simultaneous Surveillance of Influenza A and B Viruses.

Influenza A and B viruses are the causative agents of annual influenza epidemics that can be severe, and influenza A viruses intermittently cause pandemics. Sequence information from influenza virus genomes is instrumental in determining mechanisms underpinning antigenic evolution and antiviral resistance. However, due to sequence diversity and the dynamics of influenza virus evolution, rapid and high-throughput sequencing of influenza viruses remains a challenge. We developed a single-reaction influenza A/B virus (FluA/B) multiplex reverse transcription-PCR (RT-PCR) method that amplifies the most critical genomic segments (hemagglutinin [HA], neuraminidase [NA], and matrix [M]) of seasonal influenza A and B viruses for next-generation sequencing, regardless of viral type, subtype, or lineage. Herein, we demonstrate that the strategy is highly sensitive and robust. The strategy was validated on thousands of seasonal influenza A and B virus-positive specimens using multiple next-generation sequencing platforms.

Reversion of Cold-Adapted Live Attenuated Influenza Vaccine into a Pathogenic Virus.

UNLABELLED: The only licensed live attenuated influenza A virus vaccines (LAIVs) in the United States (FluMist) are created using internal protein-coding gene segments from the cold-adapted temperature-sensitive master donor virus A/Ann Arbor/6/1960 and HA/NA gene segments from circulating viruses. During serial passage of A/Ann Arbor/6/1960 at low temperatures to select the desired attenuating phenotypes, multiple cold-adaptive mutations and temperature-sensitive mutations arose. A substantial amount of scientific and clinical evidence has proven that FluMist is safe and effective. Nevertheless, no study has been conducted specifically to determine if the attenuating temperature-sensitive phenotype can revert and, if so, the types of substitutions that will emerge (i.e., compensatory substitutions versus reversion of existing attenuating mutations). Serial passage of the monovalent FluMist 2009 H1N1 pandemic vaccine at increasing temperatures in vitro generated a variant that replicated efficiently at higher temperatures. Sequencing of the variant identified seven nonsynonymous mutations, PB1-E51K, PB1-I171V, PA-N350K, PA-L366I, NP-N125Y, NP-V186I, and NS2-G63E. None occurred at positions previously reported to affect the temperature sensitivity of influenza A viruses. Synthetic genomics technology was used to synthesize the whole genome of the virus, and the roles of individual mutations were characterized by assessing their effects on RNA polymerase activity and virus replication kinetics at various temperatures. The revertant also regained virulence and caused significant disease in mice, with severity comparable to that caused by a wild-type 2009 H1N1 pandemic virus. IMPORTANCE: The live attenuated influenza vaccine FluMist has been proven safe and effective and is widely used in the United States. The phenotype and genotype of the vaccine virus are believed to be very stable, and mutants that cause disease in animals or humans have never been reported. By propagating the virus under well-controlled laboratory conditions, we found that the FluMist vaccine backbone could regain virulence to cause severe disease in mice. The identification of the responsible substitutions and elucidation of the underlying mechanisms provide unique insights into the attenuation of influenza virus, which is important to basic research on vaccines, attenuation reversion, and replication. In addition, this study suggests that the safety of LAIVs should be closely monitored after mass vaccination and that novel strategies to continue to improve LAIV vaccine safety should be investigated.

Comparative In Vitro and In Vivo Studies of Porcine Rotavirus G9P[13] and Human Rotavirus Wa G1P[8].

UNLABELLED: The changing epidemiology of group A rotavirus (RV) strains in humans and swine, including emerging G9 strains, poses new challenges to current vaccines. In this study, we comparatively assessed the pathogenesis of porcine RV (PRV) G9P[13] and evaluated the short-term cross-protection between this strain and human RV (HRV) Wa G1P[8] in gnotobiotic pigs. Complete genome sequencing demonstrated that PRV G9P[13] possessed a human-like G9 VP7 genotype but shared higher overall nucleotide identity with historic PRV strains. PRV G9P[13] induced longer rectal virus shedding and RV RNAemia in pigs than HRV Wa G1P[8] and generated complete short-term cross-protection in pigs challenged with HRV or PRV, whereas HRV Wa G1P[8] induced only partial protection against PRV challenge. Moreover, PRV G9P[13] replicated more extensively in porcine monocyte-derived dendritic cells (MoDCs) than did HRV Wa G1P[8]. Cross-protection was likely not dependent on serum virus-neutralizing (VN) antibodies, as the heterologous VN antibody titers in the sera of G9P[13]-inoculated pigs were low. Thus, our results suggest that heterologous protection by the current monovalent G1P[8] HRV vaccine against emerging G9 strains should be evaluated in clinical and experimental studies to prevent further dissemination of G9 strains. Differences in the pathogenesis of these two strains may be partially attributable to their variable abilities to replicate and persist in porcine immune cells, including dendritic cells (DCs). Additional studies are needed to evaluate the emerging G9 strains as potential vaccine candidates and to test the susceptibility of various immune cells to infection by G9 and other common HRV/PRV genotypes. IMPORTANCE: The changing epidemiology of porcine and human group A rotaviruses (RVs), including emerging G9 strains, may compromise the efficacy of current vaccines. An understanding of the pathogenesis and genetic, immunological, and biological features of the new emerging RV strains will contribute to the development of new surveillance and prevention tools. Additionally, studies of cross-protection between the newly identified emerging G9 porcine RV strains and a human G1 RV vaccine strain in a susceptible host (swine) will allow evaluation of G9 strains as potential novel vaccine candidates to be included in porcine or human vaccines.

Molecular Evolution and Intraclade Recombination of Enterovirus D68 during the 2014 Outbreak in the United States.

UNLABELLED: In August 2014, an outbreak of enterovirus D68 (EV-D68) occurred in North America, causing severe respiratory disease in children. Due to a lack of complete genome sequence data, there is only a limited understanding of the molecular evolution and epidemiology of EV-D68 during this outbreak, and it is uncertain whether the differing clinical manifestations of EV-D68 infection are associated with specific viral lineages. We developed a high-throughput complete genome sequencing pipeline for EV-D68 that produced a total of 59 complete genomes from respiratory samples with a 95% success rate, including 57 genomes from Kansas City, MO, collected during the 2014 outbreak. With these data in hand, we performed phylogenetic analyses of complete genome and VP1 capsid protein sequences. Notably, we observed considerable genetic diversity among EV-D68 isolates in Kansas City, manifest as phylogenetically distinct lineages, indicative of multiple introductions of this virus into the city. In addition, we identified an intersubclade recombination event within EV-D68, the first recombinant in this virus reported to date. Finally, we found no significant association between EV-D68 genetic variation, either lineages or individual mutations, and a variety of demographic and clinical variables, suggesting that host factors likely play a major role in determining disease severity. Overall, our study revealed the complex pattern of viral evolution within a single geographic locality during a single outbreak, which has implications for the design of effective intervention and prevention strategies. IMPORTANCE: Until recently, EV-D68 was considered to be an uncommon human pathogen, associated with mild respiratory illness. However, in 2014 EV-D68 was responsible for more than 1,000 disease cases in North America, including severe respiratory illness in children and acute flaccid myelitis, raising concerns about its potential impact on public health. Despite the emergence of EV-D68, a lack of full-length genome sequences means that little is known about the molecular evolution of this virus within a single geographic locality during a single outbreak. Here, we doubled the number of publicly available complete genome sequences of EV-D68 by performing high-throughput next-generation sequencing, characterized the evolutionary history of this outbreak in detail, identified a recombination event, and investigated whether there was any correlation between the demographic and clinical characteristics of the patients and the viral variant that infected them. Overall, these results will help inform the design of intervention strategies for EV-D68.

Deep Sequencing of Influenza A Virus from a Human Challenge Study Reveals a Selective Bottleneck and Only Limited Intrahost Genetic Diversification.

Knowledge of influenza virus evolution at the point of transmission and at the intrahost level remains limited, particularly for human hosts. Here, we analyze a unique viral data set of next-generation sequencing (NGS) samples generated from a human influenza challenge study wherein 17 healthy subjects were inoculated with cell- and egg-passaged virus. Nasal wash samples collected from 7 of these subjects were successfully deep sequenced. From these, we characterized changes in the subjects' viral populations during infection and identified differences between the virus in these samples and the viral stock used to inoculate the subjects. We first calculated pairwise genetic distances between the subjects' nasal wash samples, the viral stock, and the influenza virus A/Wisconsin/67/2005 (H3N2) reference strain used to generate the stock virus. These distances revealed that considerable viral evolution occurred at various points in the human challenge study. Further quantitative analyses indicated that (i) the viral stock contained genetic variants that originated and likely were selected for during the passaging process, (ii) direct intranasal inoculation with the viral stock resulted in a selective bottleneck that reduced nonsynonymous genetic diversity in the viral hemagglutinin and nucleoprotein, and (iii) intrahost viral evolution continued over the course of infection. These intrahost evolutionary dynamics were dominated by purifying selection. Our findings indicate that rapid viral evolution can occur during acute influenza infection in otherwise healthy human hosts when the founding population size of the virus is large, as is the case with direct intranasal inoculation. IMPORTANCE: Influenza viruses circulating among humans are known to rapidly evolve over time. However, little is known about how influenza virus evolves across single transmission events and over the course of a single infection. To address these issues, we analyze influenza virus sequences from a human challenge experiment that initiated infection with a cell- and egg-passaged viral stock, which appeared to have adapted during its preparation. We find that the subjects' viral populations differ genetically from the viral stock, with subjects' viral populations having lower representation of the amino-acid-changing variants that arose during viral preparation. We also find that most of the viral evolution occurring over single infections is characterized by further decreases in the frequencies of these amino-acid-changing variants and that only limited intrahost genetic diversification through new mutations is apparent. Our findings indicate that influenza virus populations can undergo rapid genetic changes during acute human infections.

Differences in the Nasopharyngeal Microbiome During Acute Respiratory Tract Infection With Human Rhinovirus and Respiratory Syncytial Virus in Infancy.

Respiratory viruses alter the nasopharyngeal microbiome and may be associated with a distinct microbial signature. To test this hypothesis, we compared the nasopharyngeal microbiome of 135 previously healthy infants with acute respiratory infection due to human rhinovirus (HRV; n = 52) or respiratory syncytial virus (RSV; n = 83). The nasopharyngeal microbiome was assessed by sequencing the V4 region of the 16S ribosomal RNA. Respiratory viruses were identified by quantitative reverse-transcription polymerase chain reaction. We found significant differences in the overall taxonomic composition and abundance of certain bacterial genera between infants infected with HRV and those infected with RSV. Our results suggest that respiratory tract viral infections are associated with different nasopharyngeal microbial profiles.

Adaptation of pandemic H2N2 influenza A viruses in humans.

The 1957 A/H2N2 influenza virus caused an estimated 2 million fatalities during the pandemic. Since viruses of the H2 subtype continue to infect avian species and pigs, the threat of reintroduction into humans remains. To determine factors involved in the zoonotic origin of the 1957 pandemic, we performed analyses on genetic sequences of 175 newly sequenced human and avian H2N2 virus isolates and all publicly available influenza virus genomes.

Evolution of Influenza B Virus in Kuala Lumpur, Malaysia, between 1995 and 2008.

Influenza B virus causes significant disease but remains understudied in tropical regions. We sequenced 72 influenza B viruses collected in Kuala Lumpur, Malaysia, from 1995 to 2008. The predominant circulating lineage (Victoria or Yamagata) changed every 1 to 3 years, and these shifts were associated with increased incidence of influenza B. We also found poor lineage matches with recommended influenza virus vaccine strains. While most influenza B virus lineages in Malaysia were short-lived, one circulated for 3 to 4 years.