Rebecca Garten

Antigenic and Genetic Characteristics of Swine-Origin 2009 A(H1N1) Influenza Viruses Circulating in Humans

Generation of Swine Flu As the newly emerged influenza virus starts its journey to infect the worlds human population, the genetic secrets of the 2009 outbreak of swine influenza A(H1N1) are being revealed. In extensive phylogenetic analyses, Garten et al. (p. 197, published online 22 May) confirm that of the eight elements of the virus, the basic components encoded by the hemagglutinin, nucleoprotein, and nonstructural genes originated in birds and transferred to pigs in 1918. Subsequently, these formed a triple reassortant with the RNA polymerase PB1 that transferred from birds in 1968 to humans and then to pigs in 1998, coupled with RNA polymerases PA and PB2 that transferred from birds to pigs in 1998. The neuraminidase and matrix protein genes that complete the virus came from birds and entered pigs in 1979. The analysis offers insights into drug susceptibility and virulence, as well as raising the possibility of hitherto unknown factors determining host specificity. A significant question is, what is the potential for the H1 component of the current seasonal flu vaccine to act as a booster? Apart from the need for ongoing sequencing to monitor for the emergence of new reassortants, future pig populations need to be closely monitored for emerging influenza viruses. Evolutionary analysis suggests a triple reassortant avian-to-pig origin for the 2009 influenza A(H1N1) outbreak. Since its identification in April 2009, an A(H1N1) virus containing a unique combination of gene segments from both North American and Eurasian swine lineages has continued to circulate in humans. The lack of similarity between the 2009 A(H1N1) virus and its nearest relatives indicates that its gene segments have been circulating undetected for an extended period. Its low genetic diversity suggests that the introduction into humans was a single event or multiple events of similar viruses. Molecular markers predictive of adaptation to humans are not currently present in 2009 A(H1N1) viruses, suggesting that previously unrecognized molecular determinants could be responsible for the transmission among humans. Antigenically the viruses are homogeneous and similar to North American swine A(H1N1) viruses but distinct from seasonal human A(H1N1).

Triple-Reassortant Swine Influenza A (H1) in Humans in the United States, 2005-2009

BACKGROUND Triple-reassortant swine influenza A (H1) viruses--containing genes from avian, human, and swine influenza viruses--emerged and became enzootic among pig herds in North America during the late 1990s. METHODS We report the clinical features of the first 11 sporadic cases of infection of humans with triple-reassortant swine influenza A (H1) viruses reported to the Centers for Disease Control and Prevention, occurring from December 2005 through February 2009, until just before the current epidemic of swine-origin influenza A (H1N1) among humans. These data were obtained from routine national influenza surveillance reports and from joint case investigations by public and animal health agencies. RESULTS The median age of the 11 patients was 10 years (range, 16 months to 48 years), and 4 had underlying health conditions. Nine of the patients had had exposure to pigs, five through direct contact and four through visits to a location where pigs were present but without contact. In another patient, human-to-human transmission was suspected. The range of the incubation period, from the last known exposure to the onset of symptoms, was 3 to 9 days. Among the 10 patients with known clinical symptoms, symptoms included fever (in 90%), cough (in 100%), headache (in 60%), and diarrhea (in 30%). Complete blood counts were available for four patients, revealing leukopenia in two, lymphopenia in one, and thrombocytopenia in another. Four patients were hospitalized, two of whom underwent invasive mechanical ventilation. Four patients received oseltamivir, and all 11 recovered from their illness. CONCLUSIONS From December 2005 until just before the current human epidemic of swine-origin influenza viruses, there was sporadic infection with triple-reassortant swine influenza A (H1) viruses in persons with exposure to pigs in the United States. Although all the patients recovered, severe illness of the lower respiratory tract and unusual influenza signs such as diarrhea were observed in some patients, including those who had been previously healthy.

The global circulation of seasonal influenza A (H3N2) viruses.

Antigenic and genetic analysis of the hemagglutinin of ∼13,000 human influenza A (H3N2) viruses from six continents during 2002–2007 revealed that there was continuous circulation in east and Southeast Asia (E-SE Asia) via a region-wide network of temporally overlapping epidemics and that epidemics in the temperate regions were seeded from this network each year. Seed strains generally first reached Oceania, North America, and Europe, and later South America. This evidence suggests that once A (H3N2) viruses leave E-SE Asia, they are unlikely to contribute to long-term viral evolution. If the trends observed during this period are an accurate representation of overall patterns of spread, then the antigenic characteristics of A (H3N2) viruses outside E-SE Asia may be forecast each year based on surveillance within E-SE Asia, with consequent improvements to vaccine strain selection.

Detection of Molecular Markers of Drug Resistance in 2009 Pandemic Influenza A (H1N1) Viruses by Pyrosequencing

ABSTRACT The M2 blockers amantadine and rimantadine and the neuraminidase (NA) inhibitors (NAIs) oseltamivir and zanamivir are approved by the FDA for use for the control of influenza A virus infections. The 2009 pandemic influenza A (H1N1) viruses (H1N1pdm) are reassortants that acquired M and NA gene segments from a Eurasian adamantane-resistant swine influenza virus. NAI resistance in the H1N1pdm viruses has been rare, and its occurrence is mainly limited to oseltamivir-exposed patients. The pyrosequencing assay has been proven to be a useful tool in surveillance for drug resistance in seasonal influenza A viruses. We provide a protocol which allows the detection of adamantane resistance markers as well as the I43T change, which is unique to the H1N1pdm M2 protein. The protocol also allows the detection of changes at residues V116, I117, E119, Q136, K150, D151, D199, I223, H275, and N295 in the NA, known to alter NAI drug susceptibility. We report on the detection of the first cases of the oseltamivir resistance-conferring mutation H275Y and the I223V change in viruses from the United States using the approach described in this study. Moreover, the assay permits the quick identification of the major NA group (V106/N248, I106/D248, or I106/N248) to which a pandemic virus belongs. Pyrosequencing is well suited for the detection of drug resistance markers and signature mutations in the M and NA gene segments of the pandemic H1N1 influenza viruses.

Outbreak of Variant Influenza A(H3N2) Virus in the United States

During an outbreak of H3N2v variant influenza, we identified 306 cases in ten states. Most cases reported agricultural fair attendance and/or contact with swine prior to illness. We found no evidence of efficient or sustained person-to-person transmission of H3N2v.

Dual Resistance to Adamantanes and Oseltamivir Among Seasonal Influenza A(H1N1) Viruses: 2008–2010

Two distinct genetic clades of seasonal influenza A(H1N1) viruses have cocirculated in the recent seasons: clade 2B oseltamivir-resistant and adamantane-susceptible viruses, and clade 2C viruses that are resistant to adamantanes and susceptible to oseltamivir. We tested seasonal influenza A(H1N1) viruses collected in 2008-2010 from the United States and globally for resistance to antivirals approved by the Food and Drug Administration. We report 28 viruses with both adamantane and oseltamivir (dual) resistance from 5 countries belonging to 4 distinct genotypes. Because of limited options for antiviral treatment, emergence of dual-resistant influenza viruses poses a public health concern, and their circulation needs to be closely monitored.

Continuing progress towards a unified nomenclature for the highly pathogenic H5N1 avian influenza viruses: Divergence of clade 2·2 viruses

Correspondence: Dr Ruben O. Donis, Molecular Virology and Vaccines Branch, Influenza Division, NCIRD, Centers for Disease Control & Prevention, 1600 Clifton Road, NE, MS-G16, Atlanta, GA 30333, USA. Email: rvd6@cdc.gov Dr Gavin Smith, State Key Laboratory of Emerging Infectious Diseases, Department of Microbiology, The University of Hong Kong, Hong Kong SAR, China. E-mail: gjsmith@hku.hk *WHO ⁄ OIE ⁄ FAO H5N1 Evolution Working Group members and collaborators can be found in the Appendix.

Comprehensive assessment of 2009 pandemic influenza A (H1N1) virus drug susceptibility in vitro.

BACKGROUND Antiviral drugs are an important option for managing infections caused by influenza viruses. This study assessed the drug susceptibility of 2009 pandemic influenza A (H1N1) viruses collected globally between April 2009 and January 2010. METHODS Virus isolates were tested for adamantane susceptibility, using pyrosequencing to detect the S31N marker of adamantane resistance in the M2 protein and biological assays to assess viral replication in cell culture. To assess neuraminidase (NA) inhibitor (NAI) susceptibility, virus isolates were tested in chemiluminescent NA inhibition assays and by pyrosequencing to detect the H275Y (H274Y in N2 numbering) marker of oseltamivir resistance in the NA. RESULTS With the exception of three, all viruses that were tested for adamantane susceptibility (n=3,362) were resistant to this class of drugs. All viruses tested for NAI susceptibility (n=3,359) were sensitive to two US Food and Drug Administration-approved NAIs, oseltamivir (mean ±sd 50% inhibitory concentration [IC(50)] 0.25 ±0.12 nM) and zanamivir (mean IC(50) 0.29 ±0.09 nM), except 23 (0.7%), which were resistant to oseltamivir, but sensitive to zanamivir. Oseltamivir-resistant viruses had the H275Y mutation in their NA and were detected in patients exposed to the drug through prophylaxis or treatment. NA activity of all viruses was inhibited by the NAIs peramivir, laninamivir (R-125489) and A-315675, except for H275Y variants, which exhibited approximately 100-fold reduction in peramivir susceptibility. CONCLUSIONS This report provides data regarding antiviral susceptibility of 2009 pandemic influenza A (H1N1) surveillance viruses, the majority of which were resistant to adamantanes and sensitive to NAIs. These findings provide information essential for antiviral resistance monitoring and development of novel diagnostic tests for detecting influenza antiviral resistance.

Genetic analysis and antigenic characterization of swine origin influenza viruses isolated from humans in the United States, 1990–2010

Swine influenza viruses (SIV) have been recognized as important pathogens for pigs and occasional human infections with swine origin influenza viruses (SOIV) have been reported. Between 1990 and 2010, a total of twenty seven human cases of SOIV infections have been identified in the United States. Six viruses isolated from 1990 to 1995 were recognized as classical SOIV (cSOIV) A(H1N1). After 1998, twenty-one SOIV recovered from human cases were characterized as triple reassortant (tr_SOIV) inheriting genes from classical swine, avian and human influenza viruses. Of those twenty-one tr_SOIV, thirteen were of A(H1N1), one of A(H1N2), and seven of A(H3N2) subtype. SOIV characterized were antigenically and genetically closely related to the subtypes of influenza viruses circulating in pigs but distinct from contemporary influenza viruses circulating in humans. The diversity of subtypes and genetic lineages in SOIV cases highlights the importance of continued surveillance at the animal-human interface.

FluGenome: a web tool for genotyping influenza A virus

Influenza A viruses are hosted by numerous avian and mammalian species, which have shaped their evolution into distinct lineages worldwide. The viral genome consists of eight RNA segments that are frequently exchanged between different viruses via a process known as genetic reassortment. A complete genotype nomenclature is essential to describe gene segment reassortment. Specialized bioinformatic tools to analyze reassortment are not available, which hampers progress in understanding its role in host range, virulence and transmissibility of influenza viruses. To meet this need, we have developed a nomenclature to name influenza A genotypes and implemented a web server, FluGenome (http://www.flugenome.org/), for the assignment of lineages and genotypes. FluGenome provides functions for the user to interrogate the database in different modalities and get detailed reports on lineages and genotypes. These features make FluGenome unique in its ability to automatically detect genotype differences attributable to reassortment events in influenza A virus evolution.