Triple Reassortant Research Articles

Genome Stability of Pandemic Influenza A (H1N1) 2009 Based on Analysis of Hemagglutinin and Neuraminidase Genes

Influenza A virus (H1N1), which arose in 2009, constituted the fourth pandemic after the cases of 1918, 1957, and 1968. This new variant was formed by a triple reassortment, with genomic segments from swine, avian, and human influenza origins. The objective of this study was to analyze sequences of hemagglutinin (n=2038) and neuraminidase (n=1273) genes, in order to assess the extent of diversity among circulating 2009-2010 strains, estimate if these genes evolved through positive, negative, or neutral selection models of evolution during the pandemic phase, and analyze the worldwide percentage of detection of important amino acid mutations that could enhance the viral performance, such as transmissibility or resistance to drugs. A continuous surveillance by public health authorities will be critical to monitor the appearance of new influenza variants, especially in animal reservoirs such as swine and birds, in order to prevent the potential animal-human transmission of viruses with pandemic potential.

Molecular and Antigenic Characterization of Reassortant H3N2 Viruses from Turkeys with a Unique Constellation of Pandemic H1N1 Internal Genes

Triple reassortant (TR) H3N2 influenza viruses cause varying degrees of loss in egg production in breeder turkeys. In this study we characterized TR H3N2 viruses isolated from three breeder turkey farms diagnosed with a drop in egg production. The eight gene segments of the virus isolated from the first case submission (FAV-003) were all of TR H3N2 lineage. However, viruses from the two subsequent case submissions (FAV-009 and FAV-010) were unique reassortants with PB2, PA, nucleoprotein (NP) and matrix (M) gene segments from 2009 pandemic H1N1 and the remaining gene segments from TR H3N2. Phylogenetic analysis of the HA and NA genes placed the 3 virus isolates in 2 separate clades within cluster IV of TR H3N2 viruses. Birds from the latter two affected farms had been vaccinated with a H3N4 oil emulsion vaccine prior to the outbreak. The HAl subunit of the H3N4 vaccine strain had only a predicted amino acid identity of 79% with the isolate from FAV-003 and 80% for the isolates from FAV-009 and FAV-0010. By comparison, the predicted amino acid sequence identity between a prototype TR H3N2 cluster IV virus A/Sw/ON/33853/2005 and the three turkey isolates from this study was 95% while the identity between FAV-003 and FAV-009/10 isolates was 91%. When the previously identified antigenic sites A, B, C, D and E of HA1 were examined, isolates from FAV-003 and FAV-009/10 had a total of 19 and 16 amino acid substitutions respectively when compared with the H3N4 vaccine strain. These changes corresponded with the failure of the sera collected from turkeys that received this vaccine to neutralize any of the above three isolates in vitro.

Transmission of pandemic influenza H1N1 (2009) in Vietnamese swine in 2009–2010

Please cite this paper as: Trevennec et al. (2012) Transmission of pandemic influenza H1N1 (2009) in Vietnamese swine in 2009–2010. Influenza and Other Respiratory Viruses 6(5), 348–357. Background The pandemic of 2009 was caused by an H1N1 (H1N1pdm) virus of swine origin. This pandemic virus has repeatedly infected swine through reverse zoonosis, although the extent of such infection in swine remains unclear. Objective This study targets small and commercial pig producers in North Vietnam, in order to estimate the extent of H1N1pdm infection in swine and to identify the risk factors of infection. Methods Virologic and serologic surveillance of swine was carried out in 2009–2010 in pig farms (38 swabs and 1732 sera) and at a pig slaughterhouse (710 swabs and 459 sera) in North Vietnam. The sera were screened using a influenza type A‐reactive ELISA assay, and positive sera were tested using hemagglutination inhibition tests for antibody to a panel of H1‐subtype viruses representing pandemic (H1N1) 2009 (H1N1pdm), triple reassortant (TRIG), classical swine (CS), and Eurasian avian‐like (EA) swine lineages. Farm‐level risk factors were identified using a zero‐inflated negative binomial model. Results We found a maximal seroprevalence of H1N1pdm of 55·6% [95% CI: 38·1–72·1] in the slaughterhouse at the end of December 2009, 2 weeks after the peak of reported human fatalities with H1N1pdm. Farm‐level seroprevalence was 29% [95% CI: 23·2–35·7]. In seropositive farms, within‐herd seroprevalence ranged from 10 to 100%. We identified an increased risk of infection for farms that specialized in fattening and a decreased risk of infection in farms hiring external swine workers. Conclusions Our findings suggest extensive reverse‐zoonotic transmission from humans to pigs with subsequent onward transmission within pig herds.

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. Between1990 and 2010, a total of twenty seven human cases of SOIV infections have been identified in the United States. Six viruses isolated from1990 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.

Development of DIVA (differentiation of infected from vaccinated animals) vaccines utilizing heterologous NA and NS1 protein strategies for the control of triple reassortant H3N2 influenza in turkeys

Since 2003, triple reassortant (TR) swine H3N2 influenza viruses containing gene segments from human, avian, and swine origins have been detected in the U.S. turkey populations. The initial outbreak that occurred involved birds that were vaccinated with the currently available H3 swine- and avian-origin influenza vaccines. Antigenically, all turkey swine-lineage TR H3N2 isolates are closely related to each other but show little or no antigenic cross-reactivity with the avian origin or swine origin influenza vaccine strains that are currently being used in turkey operations. These results call for re-evaluation of currently available influenza vaccines being used in turkey flocks and development of more effective DIVA (differentiation of infected from vaccinated animals) vaccines. In this study, we selected one TR H3N2 strain, A/turkey/OH/313053/04 (H3N2) that showed broad cross reactivity with other recent TR turkey H3N2 isolates, and created NA- and NS-based DIVA vaccines using traditional reassortment as well as reverse genetics methods. Protective efficacy of those vaccines was determined in 2-week-old and 80-week-old breeder turkeys. The reassortant DIVA vaccines significantly reduced the presence of challenge virus in the oviduct of breeder turkeys as well as trachea and cloaca shedding of both young and old breeder turkeys, suggesting that proper vaccination could effectively prevent egg production drop and potential viral contamination of eggs in infected turkeys. Our results demonstrate that the heterologous NA and NS1 DIVA vaccines together with their corresponding serological tests could be useful for the control of TR H3N2 influenza in turkeys.

Reassortment Events among Swine Influenza A Viruses in China: Implications for the Origin of the 2009 Influenza Pandemic

That pigs may play a pivotal role in the emergence of pandemic influenza was indicated by the recent H1N1/2009 human pandemic, likely caused by a reassortant between viruses of the American triple-reassortant (TR) and Eurasian avian-like (EA) swine influenza lineages. As China has the largest human and pig populations in the world and is the only place where both TR and EA viruses have been reported to cocirculate, it is potentially the source of the H1N1/2009 pandemic virus. To examine this, the genome sequences of 405 swine influenza viruses from China were analyzed. Thirty-six TR and EA reassortant viruses were identified before and after the occurrence of the pandemic. Several of these TR-EA reassortant viruses had genotypes with most segments having the same lineage origin as the segments of the H1N1/2009 pandemic virus. However, these viruses were generated from independent reassortment events throughout our survey period and were not associated with the current pandemic. One TR-EA reassortant, which is least similar to the pandemic virus, has persisted since 2007, while all the other variants appear to be transient. Despite frequent reassortment events between TR and EA lineage viruses in China, evidence for the genesis of the 2009 pandemic virus in pigs in this region is still absent.

Compatibility of H9N2 avian influenza surface genes and 2009 pandemic H1N1 internal genes for transmission in the ferret model

In 2009, a novel H1N1 influenza (pH1N1) virus caused the first influenza pandemic in 40 y. The virus was identified as a triple reassortant between avian, swine, and human influenza viruses, highlighting the importance of reassortment in the generation of viruses with pandemic potential. Previously, we showed that a reassortant virus composed of wild-type avian H9N2 surface genes in a seasonal human H3N2 backbone could gain efficient respiratory droplet transmission in the ferret model. Here we determine the ability of the H9N2 surface genes in the context of the internal genes of a pH1N1 virus to efficiently transmit via respiratory droplets in ferrets. We generated reassorted viruses carrying the HA gene alone or in combination with the NA gene of a prototypical H9N2 virus in the background of a pH1N1 virus. Four reassortant viruses were generated, with three of them showing efficient respiratory droplet transmission. Differences in replication efficiency were observed for these viruses; however, the results clearly indicate that H9N2 avian influenza viruses and pH1N1 viruses, both of which have occasionally infected pigs, have the potential to reassort and generate novel viruses with respiratory transmission potential in mammals.

Genetic characterization of swine influenza viruses (H3N2) isolated from Minnesota in 2006–2007

Triple-reassortant (TR) H3N2 swine influenza viruses (SIV) are a major cause of respiratory disease in swine worldwide, causing considerable morbidity and mortality. Continuous surveillance of circulating SIV strains is imperative for effective control and prediction of new emerging strains with interspecies transmission potential. The current study characterized SIV isolates from commercial swine population in USA (2006-2007). Nine isolates were completely sequenced, and the molecular evolution of all gene segments was analyzed. Phylogenetic analysis of the nine H3N2 viruses indicated that these strains belonged to cluster-IV of the human/swine/avian TR genotype, grouping with H3N2 viruses of turkey origin, while forming a separate sub-lineage from those of human and avian origin strains. Ten amino acid changes were observed at the major antigenic sites of HA1 region compared to the cluster-III reference strain, with differences in glycosylation sites. All the nine strains were antigenically related to the cluster-IV turkey strain than the cluster-III reference strain. The results of this study suggest that contemporary TR H3N2 strains circulating in North America share the same genetic constellation, thus maintaining the gene pool without any further event of genetic reassortment unlike swine-origin pandemic strain A/California/04/2009/H1N1. These findings strongly support the need for continuous surveillance and monitoring of genetic changes in SIV, to identify evolving strains that might pose a threat to human or animal health.

Transmissibility of pandemic H1N1 and genetically related swine influenza viruses in ferrets

Zoonotic infections with swine influenza viruses have occurred sporadically in the past. However, sustained human-to-human transmission of a swine virus did not occur until the emergence of the 2009 pandemic H1N1 (H1N1pdm) virus. H1N1pdm possesses a unique gene combination with gene segments derived from the North America triple reassortant (PB2, PB1, PA, HA, NP, and NS) and the Eurasian avian-like (NA and M) swine influenza viruses. To identify molecular determinants that enable sustained human-to-human transmission, we compared the direct contact and aerosol transmission efficiency of the pandemic viruses with related swine influenza viruses in ferrets. The transmission potential of seasonal H3N2 [A/Wuhan/359/95 (Wuhan95)], H1N1pdm [A/California/4/09 (CA04) and A/HK/415742/09 ({type:entrez-nucleotide,attrs:{text:HK415742,term_id:915138919,term_text:HK415742}}HK415742)], and genetically related swine influenza viruses [A/sw/HK/4167/99 (H1N1) (swHK4167), A/sw/Arkansas/2976/ 02 (H1N2) (swAR2976), A/sw/HK/915/04 (H1N2) (swHK915) and A/sw/HK/201/ 10 (H1N1) (swHK201)] were studied. Ferrets were inoculated with 105 TCID50 of the virus. Naive direct contact and aerosol contact ferrets were introduced at 1day post-inoculation (dpi). Transmission was defined by detection of virus from nasal washes and/ or seroconversion. We observed direct contact transmission from inoculated donor ferrets to their cage-mates was observed for all viruses studied, albeit at different efficiency. Classical swine-like swHK4167 showed least efficient contact transmission as virus could be detected from all (3/3) direct contacts only at 6 dpi while viral shedding was detected at 4 dpi in other direct contact groups. Aerosol transmission was detected with human seasonal influenza virus Wuhan95 (2/3), H1N1pdm influenza virus CA04 (3/3), {type:entrez-nucleotide,attrs:{text:HK415742,term_id:915138919,term_text:HK415742}}HK415742 (2/3), and swine precursor virus swHK915 (1/3). Transmission of Wuhan95 or CA04 to aerosol contacts was detected at 4 or 6 dpi, while transmission of swHK915 was detected later at 8 dpi. While the swine influenza viruses studied were able to transmit via the direct contact route, only swHK915 which shares a common genetic derivation for 7 genes with H1N1pdm possessed capacity for aerosol transmission, albeit of moderate efficiency. SwHK915 differed from swine triple reassortant viruses in the origins of its M gene. It is possible that the M gene derived from Eurasian avian-like swine viruses contributes to the aerosol transmissibility of H1N1pdm influenza viruses.

Modifications in the Polymerase Genes of a Swine-Like Triple-Reassortant Influenza Virus To Generate Live Attenuated Vaccines against 2009 Pandemic H1N1 Viruses

On 11 June 2009, the World Health Organization (WHO) declared that the outbreaks caused by novel swine-origin influenza A (H1N1) virus had reached pandemic proportions. The pandemic H1N1 (H1N1pdm) virus is the predominant influenza virus strain in the human population. It has also crossed the species barriers and infected turkeys and swine in several countries. Thus, the development of a vaccine that is effective in multiple animal species is urgently needed. We have previously demonstrated that the introduction of temperature-sensitive mutations into the PB2 and PB1 genes of an avian H9N2 virus, combined with the insertion of a hemagglutinin (HA) tag in PB1, resulted in an attenuated (att) vaccine backbone for both chickens and mice. Because the new pandemic strain is a triple-reassortant (TR) virus, we chose to introduce the double attenuating modifications into a swine-like TR virus isolate, A/turkey/OH/313053/04 (H3N2) (ty/04), with the goal of producing live attenuated influenza vaccines (LAIV). This genetically modified backbone had impaired polymerase activity and restricted virus growth at elevated temperatures. In vivo characterization of two H1N1 vaccine candidates generated using the ty/04 att backbone demonstrated that this vaccine is highly attenuated in mice, as indicated by the absence of signs of disease, limited replication, and minimum histopathological alterations in the respiratory tract. A single immunization with the ty/04 att-based vaccines conferred complete protection against a lethal H1N1pdm virus infection in mice. More importantly, vaccination of pigs with a ty/04 att-H1N1 vaccine candidate resulted in sterilizing immunity upon an aggressive intratracheal challenge with the 2009 H1N1 pandemic virus. Our studies highlight the safety of the ty/04 att vaccine platform and its potential as a master donor strain for the generation of live attenuated vaccines for humans and livestock.

Genomic signature-based identification of influenza A viruses using RT-PCR/electro-spray ionization mass spectrometry (ESI-MS) technology.

BackgroundThe emergence and rapid spread of the 2009 H1N1 pandemic influenza A virus (H1N1pdm) in humans highlights the importance of enhancing the capability of existing influenza surveillance systems with tools for rapid identification of emerging and re-emerging viruses. One of the new approaches is the RT-PCR electrospray ionization mass spectrometry (RT-PCR/ESI-MS) technology, which is based on analysis of base composition (BC) of RT-PCR amplicons from influenza “core” genes. Combination of the BC signatures represents a “genomic print” of an influenza A virus.Methodology/Principal FindingsHere, 757 samples collected between 2006 and 2009 were tested, including 302 seasonal H1N1, 171 H3N2, 7 swine triple reassortants, and 277 H1N1pdm viruses. Of the 277 H1N1pdm samples, 209 were clinical specimens (throat, nasal and nasopharyngeal swabs, nasal washes, blood and sputum). BC signatures for the clinical specimen from one of the first cases of the 2009 pandemic, A/California/04/2009, confirmed it as an unusual, previously unrecognized influenza A virus, with “core” genes related to viruses of avian, human and swine origins. Subsequent analysis of additional 276 H1N1pdm samples revealed that they shared the genomic print of A/California/04/2009, which differed from those of North American swine triple reassortant viruses, seasonal H1N1 and H3N2 and other viruses tested. Moreover, this assay allowed distinction between “core” genes of co-circulating groups of seasonal H1N1, such as clades 2B, 2C, and their reassortants with dual antiviral resistance to adamantanes and oseltamivir.Conclusions/SignificanceThe RT-PCR/ESI-MS assay is a broad range influenza identification tool that can be used directly on clinical specimens for rapid and accurate detection of influenza virus genes. The assay differentiates the H1N1pdm from seasonal and other nonhuman hosts viruses. Although not a diagnostic tool, this assay demonstrates its usefulness and robustness in influenza virus surveillance and detection of novel and unusual viruses with previously unseen genomic prints.

Glycan Analysis and Influenza A Virus Infection of Primary Swine Respiratory Epithelial Cells: THE IMPORTANCE OF NeuAcα2–6 GLYCANS*

To better understand influenza virus infection of pigs, we examined primary swine respiratory epithelial cells (SRECs, the primary target cells of influenza viruses in vivo), as a model system. Glycomic profiling of SRECs by mass spectrometry revealed a diverse range of glycans terminating in sialic acid or GalαGal. In terms of sialylation, α2–6 linkage was more abundant than α2–3, and NeuAc was more abundant than NeuGc. Virus binding and infection experiments were conducted to determine functionally important glycans for influenza virus infection, with a focus on recently emerged swine viruses. Infection of SRECs with swine and human viruses resulted in different infectivity levels. Glycan microarray analysis with a high infectivity “triple reassortant” virus ((A/Swine/MN/593/99 (H3N2)) that spread widely throughout the North American swine population and a lower infectivity human virus isolated from a single pig (A/Swine/ONT/00130/97 (H3N2)) showed that both viruses bound exclusively to glycans containing NeuAcα2–6, with strong binding to sialylated polylactosamine and sialylated N-glycans. Treatment with mannosamine precursors of sialic acid (to alter NeuAc/NeuGc abundances) and linkage-specific sialidases prior to infection indicated that the influenza viruses tested preferentially utilize NeuAcα2–6-sialylated glycans to infect SRECs. Our data indicate that NeuAcα2–6-terminated polylactosamine and sialylated N-glycans are important determinants for influenza viruses to infect SRECs. As NeuAcα2–6 polylactosamine glycans play major roles in human virus infection, the importance of these receptor components in virus infection of swine cells has implications for transmission of viruses between humans and pigs and for pigs as possible adaptation hosts of novel human influenza viruses.

Analysis of synonymous codon usage bias in 09H1N1

A novel subtype of influenza A virus 09H1N1 has rapidly spread across the world. Evolutionary analyses of this virus have revealed that 09H1N1 is a triple reassortant of segments from swine, avian and human influenza viruses. In this study, we investigated factors shaping the codon usage bias of 09H1N1 and carried out cluster analysis of 60 strains of influenza A virus from different subtypes based on their codon usage bias. We discovered that more preferentially used codons of 09H1N1 are A-ended or U-ended, and the intra-genomic codon usage bias of 09H1N1 is quite low. Base composition constraint, dinucleotide biases and translational selection are the main factors influencing the codon usage bias of 09H1N1. At the genome level, we find that the codon usage bias of 09H1N1 is similar to H1N1 (A/swine/Kansas/77778/2007H1N1), H9N2 from Asia, H1N2 from Asia and North America and H3N2 from North America. Our results provide insight for understanding the processes governing evolution, regulation of gene expression, and revealing the evolution of 09H1N1.

Characterization of an H3N2 triple reassortant influenza virus with a mutation at the receptor binding domain (D190A) that occurred upon virus transmission from turkeys to pigs

The hemagglutinin (HA) protein of influenza virus mediates essential viral functions including the binding to host receptor and virus entry. It also has the antigenic sites required for virus neutralization by host antibodies. Here, we characterized an H3N2 triple reassortant (TR) influenza virus (A/turkey/Ohio/313053/04) with a mutation at the receptor binding domain (Asp190Ala) that occurred upon virus transmission from turkeys to pigs in an experimental infection study. The mutant virus replicated less efficiently than the parental virus in human, pig and turkey primary tracheal/bronchial epithelial cells, with more than 3-log10 difference in virus titer at 72 hours post infection. In addition, the mutant virus demonstrated lower binding efficiency to plasma membrane preparations from all three cell types compared to the parental virus. Antisera raised against the parental virus reacted equally to both homologous and heterlogous viruses, however, antisera raised against the mutant virus showed 4-8 folds lower reactivity to the parental virus.

Potential role of viral surface glycoproteins in the replication of H3N2 triple reassortant influenza A viruses in swine and turkeys

The H3N2 triple reassortant (TR) influenza viruses emerged in swine in 1998 and then in turkeys in 2003. It was then hypothesized that these viruses crossed the species barrier and transmitted from pigs to turkeys. In previous work we identified viruses with different transmission behavior between the two species, of which A/turkey/Ohio/313053/04 (TK04) transmitted both ways between swine and turkeys, and A/swine/North Carolina/03 (SW03) did not transmit either way between the two species. Utilizing the 12-plasmid reverse genetics (RG) system, we rescued two viruses (TK04 and SW03) with potentially different transmission behavior between pigs and turkeys. Single gene reassortants (SGR) were generated by switching the hemagglutinin (HA) or the neuraminidase (NA) genes between both viruses, and were evaluated for replication in vitro (pig and turkey tracheal/bronchial epithelial cells) and in vivo (pigs and turkeys). RG-created TK04 replicated more efficiently than SW03 in vitro and in vivo. Additionally, TK04 exhibited better binding affinity to plasma membrane preparations (PMP) from pig and turkey tracheal/bronchial epithelial cells compared to SW03. In study with SGR viruses, the HA protein was found to be essential for TK04 virus transmission amongst turkeys, but not sole factor contributing to the efficient replication of virus in turkeys and pigs. Such findings further highlight the polygenic nature of influenza virus pathogenesis.

Evidence for Amino Acid Changes in a 57-Base-Pair Region of the Highly Conserved Matrix Gene of Pandemic (H1N1) 2009 Influenza A Virus

Evidence for Amino Acid Changes in a 57-Base-Pair Region of the Highly Conserved Matrix Gene of Pandemic (H1N1) 2009 Influenza A Virus

In VitroAntiviral Activity of Favipiravir (T-705) against Drug-Resistant Influenza and 2009 A(H1N1) Viruses

Favipiravir (T-705) has previously been shown to have a potent antiviral effect against influenza virus and some other RNA viruses in both cell culture and in animal models. Currently, favipiravir is undergoing clinical evaluation for the treatment of influenza A and B virus infections. In this study, favipiravir was evaluated in vitro for its ability to inhibit the replication of a representative panel of seasonal influenza viruses, the 2009 A(H1N1) strains, and animal viruses with pandemic (pdm) potential (swine triple reassortants, H2N2, H4N2, avian H7N2, and avian H5N1), including viruses which are resistant to the currently licensed anti-influenza drugs. All viruses were tested in a plaque reduction assay with MDCK cells, and a subset was also tested in both yield reduction and focus inhibition (FI) assays. For the majority of viruses tested, favipiravir significantly inhibited plaque formation at 3.2 muM (0.5 microg/ml) (50% effective concentrations [EC(50)s] of 0.19 to 22.48 muM and 0.03 to 3.53 microg/ml), and for all viruses, with the exception of a single dually resistant 2009 A(H1N1) virus, complete inhibition of plaque formation was seen at 3.2 muM (0.5 microg/ml). Due to the 2009 pandemic and increased drug resistance in circulating seasonal influenza viruses, there is an urgent need for new drugs which target influenza. This study demonstrates that favipiravir inhibits in vitro replication of a wide range of influenza viruses, including those resistant to currently available drugs.

Identification of influenza A pandemic (H1N1) 2009 variants during the first 2009 influenza outbreak in Mexico City

Background In March 2009, public health surveillance detected increased numbers of influenza-like illness presenting to hospitals in Mexico City. The aetiological agent was subsequently determined to be a novel influenza A (H1N1) triple reassortant, which has spread worldwide. As a consequence the World Health Organisation has declared the first Influenza pandemic of the 21st century. Objectives To describe clinically and molecularly the first outbreak of influenza A pH1N1 (2009) during 1–5 May to establish a baseline of epidemiological data for pH1N1. Also, to monitor for the emergence of antiviral resistance, and mutations affecting virulence and transmissibility. Study design Samples were collected from 751 patients with influenza-like symptoms throughout Mexico City and were tested for influenza A pH1N1 (2009) using real-time PCR. In the samples that were positive for influenza A pH1N1 (2009) fragments from the haemagglutinin (H1) and neuraminidase (N1) genes were sequenced. Results A total of 203/751 (27%) patients were positive for the pandemic H1N1 (2009) virus (53% male and 47% female). The 0–12-year-old group was the most affected 85/751 (42%). Sequence analysis showed five new variants of the pandemic H1N1 (2009) virus for NA: G249E (GQ292900), M269I (GQ292892), Y274H (GQ292913), T332A (GQ292933), N344K (GQ292882), and four variants for HA: N461K (GQ293006), K505R (GQ292989), I435V (GQ292995), I527N (GQ292997). Conclusions We have provided a baseline of epidemiological data from the first outbreak of influenza A pH1N1 (2009) during 1–5 May in Mexico City. The sequencing of partial fragments of the HA and NA genes did not show the presence of previously described mutations affecting known sites of antiviral resistance in seasonal influenza A such as the H275Y (oseltamivir resistance), R293 or N295 etc.

Alternative Live-Attenuated Influenza Vaccines Based on Modifications in the Polymerase Genes Protect against Epidemic and Pandemic Flu

Human influenza is a seasonal disease associated with significant morbidity and mortality. Influenza vaccination is the most effective means for disease prevention. We have previously shown that mutations in the PB1 and PB2 genes of the live-attenuated influenza vaccine (LAIV) from the cold-adapted (ca) influenza virus A/Ann Arbor/6/60 (H2N2) could be transferred to avian influenza viruses and produce partially attenuated viruses. We also demonstrated that avian influenza viruses carrying the PB1 and PB2 mutations could be further attenuated by stably introducing a hemagglutinin (HA) epitope tag in the PB1 gene. In this work, we wanted to determine whether these modifications would also result in attenuation of a so-called triple reassortant (TR) swine influenza virus (SIV). Thus, the TR influenza A/swine/Wisconsin/14094/99 (H3N2) virus was generated by reverse genetics and subsequently mutated in the PB1 and PB2 genes. Here we show that a combination of mutations in this TR backbone results in an attenuated virus in vitro and in vivo. Furthermore, we show the potential of our TR backbone as a vaccine that provides protection against the 2009 swine-origin pandemic influenza H1N1 virus (S-OIV) when carrying the surface of a classical swine strain. We propose that the availability of alternative backbones to the conventional ca A/Ann Arbor/6/60 LAIV strain could also be useful in epidemic and pandemic influenza and should be considered for influenza vaccine development. In addition, our data provide evidence that the use of these alternative backbones could potentially circumvent the effects of original antigenic sin (OAS) in certain circumstances.

Mutability in the Matrix Gene of Novel Influenza A H1N1 Virus Detected Using a FRET Probe-Based Real-Time Reverse Transcriptase PCR Assay

Influenza A viruses are well known for their genetic diversity. They are constantly evolving through point mutations in their hemagglutinin (H) and neuraminidase (N) genes and through genetic reassortment of their segmented RNA genome (2). The current pandemic/2009/novel H1N1 influenza A virus (called novel H1N1 herein) is a product of antigenic shift resulting from triple reassortment of human, avian, and swine viruses (7). Between 19 April 2009 and November 2009, 199 countries have reported nearly 6,000 novel influenza A H1N1-related deaths to the World Health Organization (WHO) (3).