Catherine B. Smith

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).

Surveillance of Resistance to Adamantanes among Influenza A(H3N2) and A(H1N1) Viruses Isolated Worldwide

Our previous reports demonstrated an alarming increase in resistance to adamantanes among influenza A(H3N2) viruses isolated in 2001-2005. To continue monitoring drug resistance, we conducted a comprehensive analysis of influenza A(H3N2) and A(H1N1) viruses isolated globally in 2005-2006. The results obtained by pyrosequencing indicate that 96.4% (n=761) of A(H3N2) viruses circulating in the United States were adamantane resistant. Drug resistance has reached 100% among isolates from some Asian countries. Analysis of correlation between the appearance of drug resistance and the evolutionary pathway of the hemagglutinin (HA) gene suggests at least 2 separate introductions of resistance into circulating populations that gave rise to identifiable subclades. It also indicates that resistant A(H3N2) viruses may have emerged in Asia in late 2001. Among A(H1N1) viruses isolated worldwide, resistance reached 15.5% in 2005-2006; in the United States alone, it was 4.0%. Phylogenetic analysis of the HA and M genes indicates that the acquisition of resistance in A(H1N1) viruses can be linked to a specific genetic group and was not a result of reassortment between A(H3N2) and A(H1N1) viruses. The results of the study highlight the necessity of close monitoring of resistance to existing antivirals as wells as the need for new therapeutics.

Genetic characterization of H3N2 influenza viruses isolated from pigs in North America, 1977–1999: evidence for wholly human and reassortant virus genotypes

Since 1998, H3N2 viruses have caused epizootics of respiratory disease in pigs throughout the major swine production regions of the U.S. These outbreaks are remarkable because swine influenza in North America had previously been caused almost exclusively by H1N1 viruses. We sequenced the full-length protein coding regions of all eight RNA segments from four H3N2 viruses that we isolated from pigs in the Midwestern U.S. between March 1998 and March 1999, as well as from H3N2 viruses recovered from a piglet in Canada in January 1997 and from a pig in Colorado in 1977. Phylogenetic analyses demonstrated that the 1977 Colorado and 1997 Ontario isolates are wholly human influenza viruses. However, the viruses isolated since 1998 from pigs in the Midwestern U.S. are reassortant viruses containing hemagglutinin, neuraminidase and PB1 polymerase genes from human influenza viruses, matrix, non-structural and nucleoprotein genes from classical swine viruses, and PA and PB2 polymerase genes from avian viruses. The HA proteins of the Midwestern reassortant swine viruses can be differentiated from those of the 1995 lineage of human H3 viruses by 12 amino acid mutations in HA1. In contrast, the Sw/ONT/97 virus, which did not spread from pig-to-pig, lacks 11 of these changes.

Molecular Correlates of Influenza A H5N1 Virus Pathogenesis in Mice

ABSTRACT Highly pathogenic avian influenza A H5N1 viruses caused an outbreak of human respiratory illness in Hong Kong. Of 15 human H5N1 isolates characterized, nine displayed a high-, five a low-, and one an intermediate-pathogenicity phenotype in the BALB/c mouse model. Sequence analysis determined that five specific amino acids in four proteins correlated with pathogenicity in mice. Alone or in combination, these specific residues are the likely determinants of virulence of human H5N1 influenza viruses in this model.

Experimental Evaluation of the FluChip Diagnostic Microarray for Influenza Virus Surveillance

ABSTRACT Global surveillance of influenza is critical for improvements in disease management and is especially important for early detection, rapid intervention, and a possible reduction of the impact of an influenza pandemic. Enhanced surveillance requires rapid, robust, and inexpensive analytical techniques capable of providing a detailed analysis of influenza virus strains. Low-density oligonucleotide microarrays with highly multiplexed “signatures” for influenza viruses offer many of the desired characteristics. However, the high mutability of the influenza virus represents a design challenge. In order for an influenza virus microarray to be of utility, it must provide information for a wide range of viral strains and lineages. The design and characterization of an influenza microarray, the FluChip-55 microarray, for the relatively rapid identification of influenza A virus subtypes H1N1, H3N2, and H5N1 are described here. In this work, a small set of sequences was carefully selected to exhibit broad coverage for the influenza A and B viruses currently circulating in the human population as well as the avian A/H5N1 virus that has become enzootic in poultry in Southeast Asia and that has recently spread to Europe. A complete assay involving extraction and amplification of the viral RNA was developed and tested. In a blind study of 72 influenza virus isolates, RNA from a wide range of influenza A and B viruses was amplified, hybridized, labeled with a fluorophore, and imaged. The entire analysis time was less than 12 h. The combined results for two assays provided the absolutely correct types and subtypes for an average of 72% of the isolates, the correct type and partially correct subtype information for 13% of the isolates, the correct type only for 10% of the isolates, false-negative signals for 4% of the isolates, and false-positive signals for 1% of the isolates. In the overwhelming majority of cases in which incomplete subtyping was observed, the failure was due to the nucleic acid amplification step rather than limitations in the microarray.

Evolution of highly pathogenic H5N1 avian influenza viruses in Vietnam between 2001 and 2007

Highly pathogenic avian influenza (HPAI) H5N1 viruses have caused dramatic economic losses to the poultry industry of Vietnam and continue to pose a serious threat to public health. As of June 2008, Vietnam had reported nearly one third of worldwide laboratory confirmed human H5N1 infections. To better understand the emergence, spread and evolution of H5N1 in Vietnam we studied over 300 H5N1 avian influenza viruses isolated from Vietnam since their first detection in 2001. Our phylogenetic analyses indicated that six genetically distinct H5N1 viruses were introduced into Vietnam during the past seven years. The H5N1 lineage that evolved following the introduction in 2003 of the A/duck/Hong Kong/821/2002-like viruses, with clade 1 hemagglutinin (HA), continued to predominate in southern Vietnam as of May 2007. A virus with a clade 2.3.4 HA newly introduced into northern Vietnam in 2007, reassorted with pre-existing clade 1 viruses, resulting in the emergence of novel genotypes with neuraminidase (NA) and/or internal gene segments from clade 1 viruses. A total of nine distinct genotypes have been present in Vietnam since 2001, including five that were circulating in 2007. At least four of these genotypes appear to have originated in Vietnam and represent novel H5N1 viruses not reported elsewhere. Geographic and temporal analyses of H5N1 infection dynamics in poultry suggest that the majority of viruses containing new genes were first detected in northern Vietnam and subsequently spread to southern Vietnam after reassorting with pre-existing local viruses in northern Vietnam. Although the routes of entry and spread of H5N1 in Vietnam remain speculative, enhanced poultry import controls and virologic surveillance efforts may help curb the entry and spread of new HPAI viral genes.

Intercontinental Circulation of Human Influenza A(H1N2) Reassortant Viruses during the 2001–2002 Influenza Season

Reassortant influenza A viruses bearing the H1 subtype of hemagglutinin (HA) and the N2 subtype of neuraminidase (NA) were isolated from humans in the United States, Canada, Singapore, Malaysia, India, Oman, Egypt, and several countries in Europe during the 2001-2002 influenza season. The HAs of these H1N2 viruses were similar to that of the A/New Caledonia/20/99(H1N1) vaccine strain both antigenically and genetically, and the NAs were antigenically and genetically related to those of recent human H3N2 reference strains, such as A/Moscow/10/99(H3N2). All 6 internal genes of the H1N2 reassortants examined originated from an H3N2 virus. This article documents the first widespread circulation of H1N2 reassortants on 4 continents. The current influenza vaccine is expected to provide good protection against H1N2 viruses, because it contains the A/New Caledonia/20/99(H1N1) and A/Moscow/10/99(H3N2)-like viruses, which have H1 and N2 antigens that are similar to those of recent H1N2 viruses.

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.

Multiplex PCR Testing Detection of Higher-than-Expected Rates of Cervical Mycoplasma, Ureaplasma, and Trichomonas and Viral Agent Infections in Sexually Active Australian Women

ABSTRACT Knowing the prevalence of potential etiologic agents of nongonococcal and nonchlamydial cervicitis is important for improving the efficacy of empirical treatments for this commonly encountered condition. We describe four multiplex PCRs (mPCRs), designated VDL05, VDL06, VDL07, and VDL09, which facilitate the detection of a wide range of agents either known to be or putatively associated with cervicitis, including cytomegalovirus (CMV), enterovirus (EV), Epstein-Barr virus (EBV), varicella-zoster virus (VZV), herpes simplex virus type 1 (HSV-1), and herpes simplex virus type 2 (HSV-2) (VDL05); Ureaplasma parvum, Ureaplasma urealyticum, Mycoplasma genitalium, and Mycoplasma hominis (VDL06); Chlamydia trachomatis, Trichomonas vaginalis, Treponema pallidum, and group B streptococci (VDL07); and adenovirus species A to E (VDL09). The mPCRs were used to test 233 cervical swabs from 175 women attending a sexual-health clinic in Sydney, Australia, during 2006 and 2007. The agents detected alone or in combination in all cervical swabs (percentage of total swabs) included CMV (6.0), EV (2.1), EBV (2.6), VZV (4.7), HSV-1 (2.6), HSV-2 (0.8), HSV-2 and VZV (0.4), U. parvum (57.0), U. urealyticum (6.1), M. genitalium (1.3), M. hominis (13.7), C. trachomatis (0.4), T. vaginalis (3.4), and group B streptococci (0.4). Adenovirus species A to E and T. pallidum were not detected. These assays are adaptable for routine diagnostic laboratories and provide an opportunity to measure the true prevalence of microorganisms potentially associated with cervicitis and other genital infections.

Robust Sequence Selection Method Used To Develop the FluChip Diagnostic Microarray for Influenza Virus

ABSTRACT DNA microarrays have proven to be powerful tools for gene expression analyses and are becoming increasingly attractive for diagnostic applications, e.g., for virus identification and subtyping. The selection of appropriate sequences for use on a microarray poses a challenge, particularly for highly mutable organisms such as influenza viruses, human immunodeficiency viruses, and hepatitis C viruses. The goal of this work was to develop an efficient method for mining large databases in order to identify regions of conservation in the influenza virus genome. From these regions of conservation, capture and label sequences capable of discriminating between different viral types and subtypes were selected. The salient features of the method were the use of phylogenetic trees for data reduction and the selection of a relatively small number of capture and label sequences capable of identifying a broad spectrum of influenza viruses. A detailed experimental evaluation of the selected sequences is described in a companion paper. The software is freely available under the General Public License at http://www.colorado.edu/chemistry/RGHP/software/ .