Sara B. Griesemer

Multiple reassortment events in the evolutionary history of H1N1 influenza A virus since 1918.

The H1N1 subtype of influenza A virus has caused substantial morbidity and mortality in humans, first documented in the global pandemic of 1918 and continuing to the present day. Despite this disease burden, the evolutionary history of the A/H1N1 virus is not well understood, particularly whether there is a virological basis for several notable epidemics of unusual severity in the 1940s and 1950s. Using a data set of 71 representative complete genome sequences sampled between 1918 and 2006, we show that segmental reassortment has played an important role in the genomic evolution of A/H1N1 since 1918. Specifically, we demonstrate that an A/H1N1 isolate from the 1947 epidemic acquired novel PB2 and HA genes through intra-subtype reassortment, which may explain the abrupt antigenic evolution of this virus. Similarly, the 1951 influenza epidemic may also have been associated with reassortant A/H1N1 viruses. Intra-subtype reassortment therefore appears to be a more important process in the evolution and epidemiology of H1N1 influenza A virus than previously realized.

Stochastic processes are key determinants of short-term evolution in influenza a virus.

Understanding the evolutionary dynamics of influenza A virus is central to its surveillance and control. While immune-driven antigenic drift is a key determinant of viral evolution across epidemic seasons, the evolutionary processes shaping influenza virus diversity within seasons are less clear. Here we show with a phylogenetic analysis of 413 complete genomes of human H3N2 influenza A viruses collected between 1997 and 2005 from New York State, United States, that genetic diversity is both abundant and largely generated through the seasonal importation of multiple divergent clades of the same subtype. These clades cocirculated within New York State, allowing frequent reassortment and generating genome-wide diversity. However, relatively low levels of positive selection and genetic diversity were observed at amino acid sites considered important in antigenic drift. These results indicate that adaptive evolution occurs only sporadically in influenza A virus; rather, the stochastic processes of viral migration and clade reassortment play a vital role in shaping short-term evolutionary dynamics. Thus, predicting future patterns of influenza virus evolution for vaccine strain selection is inherently complex and requires intensive surveillance, whole-genome sequencing, and phenotypic analysis.

Deep Sequencing Reveals Mixed Infection with 2009 Pandemic Influenza A (H1N1) Virus Strains and the Emergence of Oseltamivir Resistance

Mixed infections with seasonal influenza A virus strains are a common occurrence and an important source of genetic diversity. Prolonged viral shedding, as observed in immunocompromised individuals, can lead to mutational accumulation over extended periods. Recently, drug resistance was reported in immunosuppressed patients infected with the 2009 pandemic influenza A (H1N1) virus within a few days after oseltamivir treatment was initiated. To better understand the evolution and emergence of drug resistance in these circumstances, we used a deep sequencing approach to survey the viral population from an immunosuppressed patient infected with H1N1/2009 influenza and treated with neuraminidase inhibitors. This patient harbored 3 genetic variants from 2 phylogenetically distinct viral clades of pandemic H1N1/2009, strongly suggestive of mixed infection. Strikingly, one of these variants also developed drug resistance de novo in response to oseltamivir treatment. Immunocompromised individuals may, therefore, constitute an important source of genetic and phenotypic diversity, both through mixed infection and de novo mutation.

Mixed Infection and the Genesis of Influenza Virus Diversity

ABSTRACT The emergence of viral infections with potentially devastating consequences for human health is highly dependent on their underlying evolutionary dynamics. One likely scenario for an avian influenza virus, such as A/H5N1, to evolve to one capable of human-to-human transmission is through the acquisition of genetic material from the A/H1N1 or A/H3N2 subtypes already circulating in human populations. This would require that viruses of both subtypes coinfect the same cells, generating a mixed infection, and then reassort. Determining the nature and frequency of mixed infection with influenza virus is therefore central to understanding the emergence of pandemic, antigenic, and drug-resistant strains. To better understand the potential for such events, we explored patterns of intrahost genetic diversity in recently circulating strains of human influenza virus. By analyzing multiple viral genome sequences sampled from individual influenza patients we reveal a high level of mixed infection, including diverse lineages of the same influenza virus subtype, drug-resistant and -sensitive strains, those that are likely to differ in antigenicity, and even viruses of different influenza virus types (A and B). These results reveal that individuals can harbor influenza viruses that differ in major phenotypic properties, including those that are antigenically distinct and those that differ in their sensitivity to antiviral agents.

Human H3N2 Influenza Viruses Isolated from 1968 To 2012 Show Varying Preference for Receptor Substructures with No Apparent Consequences for Disease or Spread

It is generally accepted that human influenza viruses bind glycans containing sialic acid linked α2–6 to the next sugar, that avian influenza viruses bind glycans containing the α2–3 linkage, and that mutations that change the binding specificity might change the host tropism. We noted that human H3N2 viruses showed dramatic differences in their binding specificity, and so we embarked on a study of representative human H3N2 influenza viruses, isolated from 1968 to 2012, that had been isolated and minimally passaged only in mammalian cells, never in eggs. The 45 viruses were grown in MDCK cells, purified, fluorescently labeled and screened on the Consortium for Functional Glycomics Glycan Array. Viruses isolated in the same season have similar binding specificity profiles but the profiles show marked year-to-year variation. None of the 610 glycans on the array (166 sialylated glycans) bound to all viruses; the closest was Neu5Acα2–6(Galβ1–4GlcNAc)3 in either a linear or biantennary form, that bound 42 of the 45 viruses. The earliest human H3N2 viruses preferentially bound short, branched sialylated glycans while recent viruses bind better to long polylactosamine chains terminating in sialic acid. Viruses isolated in 1996, 2006, 2010 and 2012 bind glycans with α2–3 linked sialic acid; for 2006, 2010 and 2012 viruses this binding was inhibited by oseltamivir, indicating binding of α2–3 sialylated glycans by neuraminidase. More significantly, oseltamivir inhibited virus entry of 2010 and 2012 viruses into MDCK cells. All of these viruses were representative of epidemic strains that spread around the world, so all could infect and transmit between humans with high efficiency. We conclude that the year-to-year variation in receptor binding specificity is a consequence of amino acid sequence changes driven by antigenic drift, and that viruses with quite different binding specificity and avidity are equally fit to infect and transmit in the human population.

Single Hemagglutinin Mutations that Alter Both Antigenicity and Receptor Binding Avidity Influence Influenza Virus Antigenic Clustering

ABSTRACT The hemagglutination inhibition (HAI) assay is the primary measurement used for identifying antigenically novel influenza virus strains. HAI assays measure the amount of reference sera required to prevent virus binding to red blood cells. Receptor binding avidities of viral strains are not usually taken into account when interpreting these assays. Here, we created antigenic maps of human H3N2 viruses that computationally account for variation in viral receptor binding avidities. These new antigenic maps differ qualitatively from conventional antigenic maps based on HAI measurements alone. We experimentally focused on an antigenic cluster associated with a single N145K hemagglutinin (HA) substitution that occurred between 1992 and 1995. Reverse-genetics experiments demonstrated that the N145K HA mutation increases viral receptor binding avidity. Enzyme-linked immunosorbent assays (ELISA) revealed that the N145K HA mutation does not prevent antibody binding; rather, viruses possessing this mutation escape antisera in HAI assays simply by attaching to cells more efficiently. Unexpectedly, we found an asymmetric antigenic effect of the N145K HA mutation. Once H3N2 viruses acquired K145, an epitope involving amino acid 145 became antigenically dominant. Antisera raised against an H3N2 strain possessing K145 had reduced reactivity to H3N2 strains possessing N145. Thus, individual mutations in HA can influence antigenic groupings of strains by altering receptor binding avidity and by changing the dominance of antibody responses. Our results indicate that it will be important to account for variation in viral receptor binding avidity when performing antigenic analyses in order to identify genuine antigenic differences among influenza virus variants.

Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes.

High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.

Rapid, simple influenza RNA extraction from nasopharyngeal samples

This report describes the development and pre-clinical testing of a new, random-access RNA sample preparation system (TruTip) for nasopharyngeal samples. The system is based on a monolithic, porous nucleic acid binding matrix embedded within an aerosol-resistant pipette tip and can be operated with single or multi-channel pipettors. Equivalent extraction efficiencies were obtained between automated QIAcube and manual TruTip methods at 10(6) gene copies influenza A per mL nasopharyngeal aspirate. Influenza A and B amended into nasopharyngeal swabs (in viral transport medium) were detected by real-time RT-PCR at approximately 745 and 370 gene copies per extraction, respectively. RNA extraction efficiency in nasopharyngeal swabs was also comparable to that obtained on an automated QIAcube instrument over a range of input concentrations; the correlation between threshold cycles (or nucleic acid recovery) for TruTip and QIAcube-purified RNA was R(2)>0.99. Preclinical testing of TruTip on blinded nasopharyngeal swab samples resulted in 98% detection accuracy relative to a clinically validated easyMAG extraction method. The physical properties of the TruTip binding matrix and ability to customize its shape and dimensions likewise make it amenable to automation and/or fluidic integration.

Unseasonal transmission of H3N2 influenza A virus during the swine-origin H1N1 pandemic.

ABSTRACT The initial wave of swine-origin influenza A virus (pandemic H1N1/09) in the United States during the spring and summer of 2009 also resulted in an increased vigilance and sampling of seasonal influenza viruses (H1N1 and H3N2), even though they are normally characterized by very low incidence outside of the winter months. To explore the nature of virus evolution during this influenza “off-season,” we conducted a phylogenetic analysis of H1N1 and H3N2 sequences sampled during April to June 2009 in New York State. Our analysis revealed that multiple lineages of both viruses were introduced and cocirculated during this time, as is typical of influenza virus during the winter. Strikingly, however, we also found strong evidence for the presence of a large transmission chain of H3N2 viruses centered on the south-east of New York State and which continued until at least 1 June 2009. These results suggest that the unseasonal transmission of influenza A viruses may be more widespread than is usually supposed.

Integrated Amplification Microarrays for Infectious Disease Diagnostics

This overview describes microarray-based tests that combine solution-phase amplification chemistry and microarray hybridization within a single microfluidic chamber. The integrated biochemical approach improves microarray workflow for diagnostic applications by reducing the number of steps and minimizing the potential for sample or amplicon cross-contamination. Examples described herein illustrate a basic, integrated approach for DNA and RNA genomes, and a simple consumable architecture for incorporating wash steps while retaining an entirely closed system. It is anticipated that integrated microarray biochemistry will provide an opportunity to significantly reduce the complexity and cost of microarray consumables, equipment, and workflow, which in turn will enable a broader spectrum of users to exploit the intrinsic multiplexing power of microarrays for infectious disease diagnostics.