Combined use of live-attenuated and inactivated influenza vaccines to enhance heterosubtypic protection.

The limited protection of current commerical vaccines necessitates the investigation of novel vaccine strategies for unpredictable outbreaks. To investigate the feasibility of using vaccines derived from Group 1 influenza A virus to induce broadly cross-reactive immune responses against multiple influenza subtypes, we tested a panel of sequential 4-dose immunization regimens in mice. Mice were treated with inactivated (seasonal H1N1, pandemic H1N1 and H5N1) and vaccinia virus-based H5N1 live-attenuated vaccines in different combinations. Mice were then challenged by viruses of either Group 1 (H1N1) or Group 2 (H3N2, H7N7) influenza virus. All studied sequential 4-dose vaccinations could induce some degrees of heterosubtypic protection in mice. Amongst all these regimens, the combined use of inactivated and live-attenuated vaccines could achieve the best heterologous protection. These results highlight the synergistic effect of combining different vaccine platforms to enhance heterosubtypic protection against influenza viruses.

Protection by universal influenza vaccine is mediated by memory CD4 T cells.

There is a diverse array of influenza viruses which circulate between different species, reassort and drift over time. Current seasonal influenza vaccines are ineffective in controlling these viruses. We have developed a novel universal vaccine which elicits robust T cell responses and protection against diverse influenza viruses in mouse and human models. Vaccine mediated protection was dependent on influenza-specific CD4+ T cells, whereby depletion of CD4+ T cells at either vaccination or challenge time points significantly reduced survival in mice. Vaccine memory CD4+ T cells were needed for early antibody production and CD8+ T cell recall responses. Furthermore, influenza-specific CD4+ T cells from vaccination manifested primarily Tfh and Th1 profiles with anti-viral cytokine production. The vaccine boosted H5-specific T cells from human PBMCs, specifically CD4+ and CD8+ T effector memory type, ensuring the vaccine was truly universal for its future application. These findings have implications for the development and optimization of T cell activating vaccines for universal immunity against influenza.

Stalking influenza by vaccination with pre-fusion headless HA mini-stem.

Inaccuracies in prediction of circulating viral strain genotypes and the possibility of novel reassortants causing a pandemic outbreak necessitate the development of an anti-influenza vaccine with increased breadth of protection and potential for rapid production and deployment. The hemagglutinin (HA) stem is a promising target for universal influenza vaccine as stem-specific antibodies have the potential to be broadly cross-reactive towards different HA subtypes. Here, we report the design of a bacterially expressed polypeptide that mimics a H5 HA stem by protein minimization to focus the antibody response towards the HA stem. The HA mini-stem folds as a trimer mimicking the HA prefusion conformation. It is resistant to thermal/chemical stress, and it binds to conformation-specific, HA stem-directed broadly neutralizing antibodies with high affinity. Mice vaccinated with the group 1 HA mini-stems are protected from morbidity and mortality against lethal challenge by both group 1 (H5 and H1) and group 2 (H3) influenza viruses, the first report of cross-group protection. Passive transfer of immune serum demonstrates the protection is mediated by stem-specific antibodies. Furthermore, antibodies induced by these HA stems have broad HA reactivity, yet they do not have antibody-dependent enhancement activity.

Generation of Live Attenuated Influenza Virus by Using Codon Usage Bias.

UNLABELLED: Seasonal influenza epidemics and occasional pandemics threaten public health worldwide. New alternative strategies for generating recombinant viruses with vaccine potential are needed. Interestingly, influenza viruses circulating in different hosts have been found to have distinct codon usage patterns, which may reflect host adaptation. We therefore hypothesized that it is possible to make a human seasonal influenza virus that is specifically attenuated in human cells but not in eggs by converting its codon usage so that it is similar to that observed from avian influenza viruses. This approach might help to generate human live attenuated viruses without affecting their yield in eggs. To test this hypothesis, over 300 silent mutations were introduced into the genome of a seasonal H1N1 influenza virus. The resultant mutant was significantly attenuated in mammalian cells and mice, yet it grew well in embryonated eggs. A single dose of intranasal vaccination induced potent innate, humoral, and cellular immune responses, and the mutant could protect mice against homologous and heterologous viral challenges. The attenuated mutant could also be used as a vaccine master donor strain by introducing hemagglutinin and neuraminidase genes derived from other strains. Thus, our approach is a successful strategy to generate attenuated viruses for future application as vaccines. IMPORTANCE: Vaccination has been one of the best protective measures in combating influenza virus infection. Current licensed influenza vaccines and their production have various limitations. Our virus attenuation strategy makes use of the codon usage biases of human and avian influenza viruses to generate a human-derived influenza virus that is attenuated in mammalian hosts. This method, however, does not affect virus replication in eggs. This makes the resultant mutants highly compatible with existing egg-based vaccine production pipelines. The viral proteins generated from the codon bias mutants are identical to the wild-type viral proteins. In addition, our massive genome-wide mutational approach further minimizes the concern over reverse mutations. The potential use of this kind of codon bias mutant as a master donor strain to generate other live attenuated viruses is also demonstrated. These findings put forward a promising live attenuated influenza vaccine generation strategy to control influenza.

Influenza A viruses with different amino acid residues at PB2-627 display distinct replication properties in vitro and in vivo: revealing the sequence plasticity of PB2-627 position.

Sequence analyses of influenza PB2 sequences indicate that the 627 position almost exclusively contains either lysine (K) or glutamic acid (E), suggesting a high sequence constraint at this genetic marker. Here, we used a site-directed random mutagenesis method to demonstrate that PB2-627 position has a high sequence plasticity. Recombinant viruses carrying various amino acid residues at this position are viable in cell cultures. These PB2-627 mutants showed various polymerase activities and replication kinetics in mammalian and avian cells as well as pathogenicity in mice. Serially passaging these mutants in MDCK cells generated some compensatory PB2 mutations that can restore polymerase activities of the PB2-627 mutants. Of these, PB2-D309N was identified as a novel one. Besides showing that influenza virus can tolerate a wide range of amino acid residues at the PB2-627 position, this study also demonstrates a potential strategy to identify novel mutations that can enhance viral polymerase.

IL-15 adjuvanted multivalent vaccinia-based universal influenza vaccine requires CD4+ T cells for heterosubtypic protection.

Current influenza vaccines are ineffective against novel viruses and the source or the strain of the next outbreak of influenza is unpredictable; therefore, establishing universal immunity by vaccination to limit the impact of influenza remains a high priority. To meet this challenge, a novel vaccine has been developed using the immunogenic live vaccinia virus as a vaccine vector, expressing multiple H5N1 viral proteins (HA, NA, M1, M2, and NP) together with IL-15 as a molecular adjuvant. Previously, this vaccine demonstrated robust sterile cross-clade protection in mice against H5 influenza viruses, and herein its use has been extended to mediate heterosubtypic immunity toward viruses from both group 1 and 2 HA lineages. The vaccine protected mice against lethal challenge by increasing survival and significantly reducing lung viral loads against the most recent human H7N9, seasonal H3N2, pandemic-2009 H1N1, and highly pathogenic H7N7 influenza A viruses. Influenza-specific antibodies elicited by the vaccine failed to neutralize heterologous viruses and were unable to confer protection by passive transfer. Importantly, heterologous influenza-specific CD4(+) and CD8(+) T-cell responses that were elicited by the vaccine were effectively recalled and amplified following viral challenge in the lungs and periphery. Selective depletion of T-cell subsets in the immunized mice revealed an important role for CD4(+) T cells in heterosubtypic protection, despite low sequence conservation among known MHC-II restricted epitopes across different influenza viruses. This study illustrates the potential utility of our multivalent Wyeth/IL-15/5Flu as a universal influenza vaccine with a correlate of protective immunity that is independent of neutralizing antibodies.

A novel molecular test for influenza B virus detection and lineage differentiation.

Contemporary influenza B viruses are classified into two groups known as Yamagata and Victoria lineages. The co-circulation of two viral lineages in recent years urges for a robust and simple diagnostic test for detecting influenza B viruses and for lineage differentiation. In this study, a SYBR green-based asymmetric PCR assay has been developed for influenza B virus detection. Apart from identifying influenza B virus, the assay contains sequence-specific probes for lineage differentiation. This allows identifying influenza B virus and detecting influenza B viral lineage in a single reaction. The test has been evaluated by a panel of respiratory specimens. Of 108 influenza B virus-positive specimens, 105 (97%) were positive in this assay. None of the negative control respiratory specimens were positive in the test (N = 60). Viral lineages of all samples that are positive in the assay (N = 105) can also be classified correctly. These results suggest that this assay has a potential for routine influenza B virus surveillance.

Long-term outcome and prognostic factors of unrelated cord blood transplantation in children with haematological malignancies: a retrospective study using the Spanish Working Party for BMT in Children (GETMON) database.

Outcomes of unrelated cord blood transplants (UCBT) were assessed in 172 consecutive children, median age 5 years (range: 0.5-18), with haematological malignancies treated at nine Spanish hospitals between February 1996 and April 2009. Data were collected from the Spanish Working Party for Blood and Marrow Transplantation in Children (GETMON) database. ALL was diagnosed in 125 patients, AML in 43 and myelodysplastic syndrome in 4. Myeloid engraftment (ANC⩾0.5 × 10(9)/L) occurred in 87.2% at a median of 22 days and was associated with the total nucleated cell (TNC) dose infused and use of a TT-containing conditioning regimen. Cumulative incidence of relapse was 20% at 1 year post transplant and 29% at 3 years, being higher in patients with a diagnosis of ALL, very high risk disease and GVHD grades 0-1. Cumulative incidence of non-relapse mortality (NRM) was 19% at 100 days post transplant and 39% at 1 year. BU-FLU-TT-ATG-conditioned patients had lower NRM. Disease-free survival (DFS) was 40% at 2 years post transplant (for patients transplanted since 2006). On multivariate analysis, TNC dose infused, AML and BU-FLU-TT-ATG-conditioning regimen increased the probability of DFS. It is of paramount importance to select cord blood units with the highest cell dose. As the BU-FLU-TT-ATG-conditioning regimen was associated with better DFS owing to lower NRM, further prospective studies testing this regimen are warranted.

The viruses of wild pigeon droppings.

Birds are frequent sources of emerging human infectious diseases. Viral particles were enriched from the feces of 51 wild urban pigeons (Columba livia) from Hong Kong and Hungary, their nucleic acids randomly amplified and then sequenced. We identified sequences from known and novel species from the viral families Circoviridae, Parvoviridae, Picornaviridae, Reoviridae, Adenovirus, Astroviridae, and Caliciviridae (listed in decreasing number of reads), as well as plant and insect viruses likely originating from consumed food. The near full genome of a new species of a proposed parvovirus genus provisionally called Aviparvovirus contained an unusually long middle ORF showing weak similarity to an ORF of unknown function from a fowl adenovirus. Picornaviruses found in both Asia and Europe that are distantly related to the turkey megrivirus and contained a highly divergent 2A1 region were named mesiviruses. All eleven segments of a novel rotavirus subgroup related to a chicken rotavirus in group G were sequenced and phylogenetically analyzed. This study provides an initial assessment of the enteric virome in the droppings of pigeons, a feral urban species with frequent human contact.

Resultados del trasplante de progenitores hematopoyéticos en hemoglobinopatías: talasemia maior y enfermedad drepanocítica / Results of hematopoietic stem cell transplantation in hemoglobinopathies: Thalassemia major and sickle cell disease

Introducción: La prevalencia de las hemoglobinopatías en nuestro medio ha aumentado como consecuencia de los flujos migratorios. La talasemia mayor cursa con anemia hemolítica crónica y necesidad de transfusiones regulares desde el año de vida. La enfermedad drepanocítica cursa con anemia, vasculopatía y daño orgánico progresivo. En ambas, la esperanza de vida está disminuida. El trasplante alogénico de progenitores hematopoyéticos es una opción de curación para estos pacientes. Pacientes: Diecisiete pacientes recibieron un trasplante alogénico de progenitores hematopoyéticos: 14 afectados de talasemia maior y 3 de enfermedad drepanocítica. Resultados: Los donantes fueron en los pacientes con talasemia mayor 9 hermanos HLA-idénticos, 2 progenitores con una diferencia antigénica HLA y 3 donantes no emparentados, y en aquellos con enfermedad drepanocítica, 3 hermanos HLA-idénticos. La fuente fue la médula ósea en todos, excepto uno. La media de edad al trasplante de progenitores hematopoyéticos fue de 6 años (intervalo: 1-16) en los niños con talasemia mayor y doce años (intervalo: 8-15) en los niños con enfermedad drepanocítica. Se confirmó injerto medular en todos los pacientes. Dos con talasemia mayor presentaron fallo de injerto secundario, precisando nuevamente soporte transfusional. Trece pacientes presentaron quimerismo completo y 2 quimerismo mixto, todos con normalización de la cifra de hemoglobina y sin requerimiento de transfusiones. Los pacientes con enfermedad drepanocítica no presentaron más episodios vasooclusivos y la funciones pulmonar y cerebral en aquellos pacientes que presentaban afectación en el momento del trasplante se estabilizaron. Tres pacientes con talasemia mayor desarrollaron enfermedad injerto contra huésped crónica y 5, hipogonadismo hipogonadotropo. Conclusiones: Nuestra experiencia confirma que el trasplante alogénico de progenitores hematopoyéticos de hermano HLA idéntico es una buena opción en el tratamiento de la talasemia mayor y la enfermedad drepanocítica. En el caso de la talasemia mayor, el trasplante de donante no emparentado es una opción terapéutica en centros especializados, ya que, a pesar de los buenos resultados presentados, la morbimortalidad de este procedimiento puede ser elevada, frente a la alternativa de un tratamiento médico no curativo pero con expectativas de supervivencia prolongada. En el caso de la enfermedad drepanocítica, el trasplante de donante no emparentado todavía está en fases preliminares de investigación (AU)

Background: The prevalence of hemoglobinopathies in Spain is increasing as a result of immigration. Thalassemia major presents with chronic hemolytic anemia that requires regular red blood cell transfusions within the first year of life. Patients with sickle cell disease suffer from chronic anemia, vasculopathy and progressive damage in almost any organ. There is decreased life expectancy in both conditions. Allogeneic hematopoietic stem cell transplantation represents the only potentially curative option. Patients: Seventeen patients (fourteen thalassemia major, and three sickle cell disease) underwent allogeneic hematopoietic stem cell transplantations. Results: In the thalassemia group, nine donors were HLA-geno-identical siblings, two were partially matched related donors (one HLA allele mismatch), and three unrelated donors. All three patients with sickle cell disease were transplanted from HLA-geno-identical siblings. The source of stem cells was bone marrow in sixteen cases. Median patient age at transplant was six years (range: 1–16) in the thalassemia group, and twelve years (range: 8–15) in the sickle cell disease group. The graft was successful in all patients. Secondary graft rejection was observed in two thalassemia patients rendering them dependent on blood transfusions. Complete chimerism was observed in thirteen patients and, although mixed chimerism occurred in two, with all of them showing normal hemoglobin levels after transplantation and not requiring further transfusion support. Patients affected by sickle cell disease did not present with new vaso-occlusive crises, and stabilization of pulmonary and neurological function was observed. Chronic graft-versus-host disease was detected in three patients affected by thalassemia, and hypogonadotrophic hypogonadism in five patients. Conclusions: We conclude that for thalassemia major and sickle cell disease, allogenic hematopoietic stem cell transplantation from HLA-geno-identical siblings offers a high probability of complication-free survival. Despite good results, morbidity and mortality associated with transplantation from unrelated donors is a risk that might be considered, in contrast to a non-curative medical treatment that offers a long term survival. For thalassemia major groups it could be an option, but not for sickle cell disease, which is still in the investigational phase (AU)

Molecular detection of human H7N9 influenza A virus causing outbreaks in China.

BACKGROUND: A novel subtype of influenza A virus (H7N9) was recently identified in humans. The virus is a reassortant of avian viruses, but these human isolates contain mutations [hemagglutinin (HA) Q226L and PB2 E627K] that might make it easier for the virus to adapt to mammalian hosts. Molecular tests for rapid detection of this virus are urgently needed. METHODS: We developed a 1-step quantitative real-time reverse-transcription PCR assay to detect the novel human H7N9 virus. The primer set was specific to the hemagglutinin (HA) gene of the H7N9 viruses currently causing the outbreak in China and had mismatches to all previously known avian or mammalian H7 HA sequences. In addition, the assay was evaluated using influenza A viruses of various genetic backgrounds and other negative controls. RESULTS: The detection limit of the assay was approximately 0.04 TCID50 (median tissue culture infective dose) per reaction. The assay specificity was high and all negative control samples, including 8 H7 viruses not closely related to the human H7N9 virus, tested negative. CONCLUSIONS: The established assay allows rapid detection of the novel human H7N9 virus, thereby allowing better pandemic preparedness.

A statistical strategy to identify recombinant viral ribonucleoprotein of avian, human, and swine influenza A viruses with elevated polymerase activity.

OBJECTIVES: Reassortment of influenza A viruses can give rise to viral ribonucleoproteins (vRNPs) with elevated polymerase activity and the previous three pandemic influenza viruses contained reassorted vRNPs of different origins. These suggest that reassorted vRNP may be one of the factors leading to a pandemic virus. In this study, we reconstituted chimeric vRNPs with three different viral strains isolated from avian, human and swine hosts. We applied a statistical strategy to identify the effect that the origin of a single vRNP protein subunit or the interactions between these subunits on polymerase activity. DESIGN: Eighty one chimeric vRNPs were reconstituted in 293T cells at different temperatures. Polymerase activity was determined by luciferase reporter assay and the results were analysed by multiway anova and other statistical methods. RESULTS: It was found that PB2, PB1, NP, PB2-PB1 interaction, PB2-PA interaction and PB1-NP interaction had significant effect on polymerase activity at 37°C and several single subunits and interactions were identified to lead to elevation of polymerase activity. Furthermore, we studied 27 out of these 81 different chimieric vRNPs in different combinations via fractional factorial design approach. Our results suggested that the approach can identify the major single subunit or interaction factors that affect the polymerase activity without the need to experimentally reproduce all possible vRNP combinations. CONCLUSIONS: Statistical approach and fractional factorial design are useful to identify the major single subunit or interaction factors that can modulate viral polymerase activity.

[Results of hematopoietic stem cell transplantation in hemoglobinopathies: thalassemia major and sickle cell disease]. / Resultados del trasplante de progenitores hematopoyéticos en hemoglobinopatías: talasemia maior y enfermedad drepanocítica.

BACKGROUND: The prevalence of hemoglobinopathies in Spain is increasing as a result of immigration. Thalassemia major presents with chronic hemolytic anemia that requires regular red blood cell transfusions within the first year of life. Patients with sickle cell disease suffer from chronic anemia, vasculopathy and progressive damage in almost any organ. There is decreased life expectancy in both conditions. Allogeneic hematopoietic stem cell transplantation represents the only potentially curative option. PATIENTS: Seventeen patients (fourteen thalassemia major, and three sickle cell disease) underwent allogeneic hematopoietic stem cell transplantations. RESULTS: In the thalassemia group, nine donors were HLA-geno-identical siblings, two were partially matched related donors (one HLA allele mismatch), and three unrelated donors. All three patients with sickle cell disease were transplanted from HLA-geno-identical siblings. The source of stem cells was bone marrow in sixteen cases. Median patient age at transplant was six years (range: 1-16) in the thalassemia group, and twelve years (range: 8-15) in the sickle cell disease group. The graft was successful in all patients. Secondary graft rejection was observed in two thalassemia patients rendering them dependent on blood transfusions. Complete chimerism was observed in thirteen patients and, although mixed chimerism occurred in two, with all of them showing normal hemoglobin levels after transplantation and not requiring further transfusion support. Patients affected by sickle cell disease did not present with new vaso-occlusive crises, and stabilization of pulmonary and neurological function was observed. Chronic graft-versus-host disease was detected in three patients affected by thalassemia, and hypogonadotrophic hypogonadism in five patients. CONCLUSIONS: We conclude that for thalassemia major and sickle cell disease, allogenic hematopoietic stem cell transplantation from HLA-geno-identical siblings offers a high probability of complication-free survival. Despite good results, morbidity and mortality associated with transplantation from unrelated donors is a risk that might be considered, in contrast to a non-curative medical treatment that offers a long term survival. For thalassemia major groups it could be an option, but not for sickle cell disease, which is still in the investigational phase.

Highly conserved protective epitopes on influenza B viruses.

Identification of broadly neutralizing antibodies against influenza A viruses has raised hopes for the development of monoclonal antibody-based immunotherapy and "universal" vaccines for influenza. However, a substantial part of the annual flu burden is caused by two cocirculating, antigenically distinct lineages of influenza B viruses. Here, we report human monoclonal antibodies, CR8033, CR8071, and CR9114, that protect mice against lethal challenge from both lineages. Antibodies CR8033 and CR8071 recognize distinct conserved epitopes in the head region of the influenza B hemagglutinin (HA), whereas CR9114 binds a conserved epitope in the HA stem and protects against lethal challenge with influenza A and B viruses. These antibodies may inform on development of monoclonal antibody-based treatments and a universal flu vaccine for all influenza A and B viruses.

Entry of influenza A Virus with a α2,6-linked sialic acid binding preference requires host fibronectin.

The receptor binding specificity of influenza A virus is one of the major determinants of viral tropism and host specificity. In general, avian viral hemagglutinin prefers to bind to α2,3-linked sialic acid, whereas the human viral hemagglutinin prefers to bind to α2,6-linked sialic acid. Here, we demonstrate that host fibronectin protein plays an important role in the life cycle of some influenza A viruses. Treating cells with anti-fibronectin antibodies or fibronectin-specific small interfering RNA can inhibit the virus replication of human H1N1 influenza A viruses. Strikingly, these inhibitory effects cannot be observed in cells infected with H5N1 viruses. By using reverse genetics techniques, we observed that the receptor binding specificity, but not the origin of the hemagglutinin subtype, is responsible for this differential inhibitory effect. Changing the binding preference of hemagglutinin from α2,6-linked sialic acid to α2,3-linked sialic acid can make the virus resistant to the anti-fibronectin antibody treatment and vice versa. Our further characterizations indicate that anti-fibronectin antibody acts on the early phase of viral replication cycle, but it has no effect on the initial binding of influenza A virus to cell surface. Our subsequent investigations further show that anti-fibronectin antibody can block the postattachment entry of influenza virus. Overall, these results indicate that the sialic acid binding preference of influenza viral hemagglutinin can modulate the preferences of viral entry pathways, suggesting that there are subtle differences between the virus entries of human and avian influenza viruses.

Viral reassortment as an information exchange between viral segments.

Viruses have an extraordinary ability to diversify and evolve. For segmented viruses, reassortment can introduce drastic genomic and phenotypic changes by allowing a direct exchange of genetic material between coinfecting strains. For instance, multiple influenza pandemics were caused by reassortments of viruses typically found in separate hosts. What is unclear, however, are the underlying mechanisms driving these events and the level of intrinsic bias in the diversity of strains that emerge from coinfection. To address this problem, previous experiments looked for correlations between segments of strains that coinfect cells in vitro. Here, we present an information theory approach as the natural mathematical framework for this question. We study, for influenza and other segmented viruses, the extent to which a virus's segments can communicate strain information across an infection and among one another. Our approach goes beyond previous association studies and quantifies how much the diversity of emerging strains is altered by patterns in reassortment, whether biases are consistent across multiple strains and cell types, and if significant information is shared among more than two segments. We apply our approach to a new experiment that examines reassortment patterns between the 2009 H1N1 pandemic and seasonal H1N1 strains, contextualizing its segmental information sharing by comparison with previously reported strain reassortments. We find evolutionary patterns across classes of experiments and previously unobserved higher-level structures. Finally, we show how this approach can be combined with virulence potentials to assess pandemic threats.

Hemagglutinin-neuraminidase balance confers respiratory-droplet transmissibility of the pandemic H1N1 influenza virus in ferrets.

A novel reassortant derived from North American triple-reassortant (TRsw) and Eurasian swine (EAsw) influenza viruses acquired sustained human-to-human transmissibility and caused the 2009 influenza pandemic. To identify molecular determinants that allowed efficient transmission of the pandemic H1N1 virus among humans, we evaluated the direct-contact and respiratory-droplet transmissibility in ferrets of representative swine influenza viruses of different lineages obtained through a 13-y surveillance program in southern China. Whereas all viruses studied were transmitted by direct contact with varying efficiency, respiratory-droplet transmissibility (albeit inefficient) was observed only in the TRsw-like A/swine/Hong Kong/915/04 (sw915) (H1N2) virus. The sw915 virus had acquired the M gene derived from EAsw and differed from the gene constellation of the pandemic H1N1 virus by the neuraminidase (NA) gene alone. Glycan array analysis showed that pandemic H1N1 virus A/HK/415742/09 (HK415742) and sw915 possess similar receptor-binding specificity and affinity for α2,6-linked sialosides. Sw915 titers in differentiated normal human bronchial epithelial cells and in ferret nasal washes were lower than those of HK415742. Introducing the NA from pandemic HK415742 into sw915 did not increase viral replication efficiency but increased respiratory-droplet transmissibility, despite a substantial amino acid difference between the two viruses. The NA of the pandemic HK415742 virus possessed significantly higher enzyme activity than that of sw915 or other swine influenza viruses. Our results suggest that a unique gene constellation and hemagglutinin-neuraminidase balance play a critical role in acquisition of efficient and sustained human-to-human transmissibility.

Rapid genotyping of swine influenza viruses.

The emergence of pandemic (H1N1) 2009 virus highlighted the need for enhanced surveillance of swine influenza viruses. We used real-time reverse-transcription PCR-based genotyping and found that this rapid and simple genotyping method may identify reassortants derived from viruses of Eurasian avian-like, triple reassortant-like, and pandemic (H1N1) 2009 virus lineages.

DNA intercalator stimulates influenza transcription and virus replication.

Influenza A virus uses its host transcription machinery to facilitate viral RNA synthesis, an event that is associated with cellular RNA polymerase II (RNAPII). In this study, various RNAPII transcription inhibitors were used to investigate the effect of RNAPII phosphorylation status on viral RNA transcription. A low concentration of DNA intercalators, such as actinomycin D (ActD), was found to stimulate viral polymerase activity and virus replication. This effect was not observed in cells treated with RNAPII kinase inhibitors. In addition, the loss of RNAPII(a) in infected cells was due to the shift of nonphosphorylated RNAPII (RNAPII(a)) to hyperphosphorylated RNAPII (RNAPII(o)).