We recently analyzed a series of H5N1 viruses isolated from healthy ducks in southern China since 1999 and found that these viruses had progressively acquired the ability to replicate and cause disease in mice. In the present study, we explored the genetic basis of this change in host range by comparing two of the viruses that are genetically similar but differ in their ability to infect mice and have different pathogenicity in mice.

Antibodies capable of neutralizing divergent influenza A viruses could form the basis of a universal vaccine. Here, from subjects enrolled in an H5N1 DNA/MIV-prime-boost influenza vaccine trial, we sorted hemagglutinin-cross reactive memory-B cells and identified three antibody classes, each capable of neutralizing diverse subtypes of group 1 and 2-influenza A viruses. Co-crystal structures with hemagglutinin revealed that each class utilized characteristic germline genes and convergent sequence motifs to recognize overlapping epitopes in the hemagglutinin stem. All six analyzed subjects had sequences from at least one multidonor class, and -in half the subjects- multidonor-class sequences were recovered from >40% of cross reactive-B cells. By contrast, these multidonor-class sequences were rare in published antibody datasets. Vaccination with a divergent hemagglutinin can thus increase the frequency of B cells encoding broad influenza A-neutralizing antibodies; we propose the sequence signature-quantified prevalence of these B cells as a metric to guide universal influenza A-immunization strategies.

Avian influenza A H5N1 viruses continue to spread globally among birds, resulting in occasional transmission of virus from infected poultry to humans. Probable human-to-human transmission has been documented rarely, but H5N1 viruses have not yet acquired the ability to transmit efficiently among humans, an essential property of a pandemic virus. The pandemics of 1957 and 1968 were caused by avian-human reassortant influenza viruses that had acquired human virus-like receptor binding properties. However, the relative contribution of human internal protein genes or other molecular changes to the efficient transmission of influenza viruses among humans remains poorly understood. Here, we report on a comparative ferret model that parallels the efficient transmission of H3N2 human viruses and the poor transmission of H5N1 avian viruses in humans. In this model, an H3N2 reassortant virus with avian virus internal protein genes exhibited efficient replication but inefficient transmission, whereas H5N1 reassortant viruses with four or six human virus internal protein genes exhibited reduced replication and no transmission. These findings indicate that the human virus H3N2 surface protein genes alone did not confer efficient transmissibility and that acquisition of human virus internal protein genes alone was insufficient for this 1997 H5N1 virus to develop pandemic capabilities, even after serial passages in a mammalian host. These results highlight the complexity of the genetic basis of influenza virus transmissibility and suggest that H5N1 viruses may require further adaptation to acquire this essential pandemic trait. transmissibility ͉ pandemic virus properties ͉ pandemic influenza ͉ animal model ͉ receptor specificity

The epidemiological success of pandemic and epidemic influenza A viruses relies on the ability to transmit efficiently from person-to-person via respiratory droplets. Respiratory droplet (RD) transmission of influenza viruses requires efficient replication and release of infectious influenza particles into the air. The 2009 pandemic H1N1 (pH1N1) virus originated by reassortment of a North American triple reassortant swine (TRS) virus with a Eurasian swine virus that contributed the neuraminidase (NA) and M gene segments. Both the TRS and Eurasian swine viruses caused sporadic infections in humans, but failed to spread from person-to-person, unlike the pH1N1 virus. We evaluated the pH1N1 and its precursor viruses in a ferret model to determine the contribution of different viral gene segments on the release of influenza virus particles into the air and on the transmissibility of the pH1N1 virus. We found that the Eurasian-origin gene segments contributed to efficient RD transmission of the pH1N1 virus likely by modulating the release of influenza viral RNA-containing particles into the air. All viruses replicated well in the upper respiratory tract of infected ferrets, suggesting that factors other than viral replication are important for the release of influenza virus particles and transmission. Our studies demonstrate that the release of influenza viral RNA-containing particles into the air correlates with increased NA activity. Additionally, the pleomorphic phenotype of the pH1N1 virus is dependent upon the Eurasian-origin gene segments, suggesting a link between transmission and virus morphology. We have demonstrated that the viruses are released into exhaled air to varying degrees and a constellation of genes influences the transmissibility of the pH1N1 virus.

Following circulation of avian influenza H5 and H7 viruses in poultry, the hemagglutinin (HA) can acquire additional glycosylation sites, and the neuraminidase (NA) stalk becomes shorter. We investigated whether these features play a role in the pathogenesis of infection in mammalian hosts. From 1996 to 2007, H5N1 viruses with a short NA stalk have become widespread in several avian species. Compared to viruses with a long-stalk NA, viruses with a short-stalk NA showed a decreased capacity to elute from red blood cells and an increased virulence in mice, but not in chickens. The presence of additional HA glycosylation sites had less of an effect on virulence than did NA stalk length. The short-stalk NA of H5N1 viruses circulating in Asia may contribute to virulence in humans.

Avian influenza A H5N1 viruses similar to those that infected humans in Hong Kong in 1997 continue to circulate in waterfowl and have reemerged in poultry in the region, raising concerns that these viruses could reappear in humans. The currently licensed trivalent inactivated influenza vaccines contain hemagglutinin (HA) and neuraminidase genes from epidemic strains in a background of internal genes derived from the vaccine donor strain, A/Puerto Rico/8/34 (PR8). Such reassortant candidate vaccine viruses are currently not licensed for the prevention of human infections by H5N1 influenza viruses. A transfectant H5N1/PR8 virus was generated by plasmid-based reverse genetics. The removal of the multibasic amino acid motif in the HA gene associated with high pathogenicity in chickens, and the new genotype of the H5N1/PR8 transfectant virus, attenuated the virus for chickens and mice without altering the antigenicity of the HA. A Formalin-inactivated vaccine prepared from this virus was immunogenic and protected mice from subsequent wild-type H5N1 virus challenge. This is the first successful attempt to develop an H5N1 vaccine seed virus resembling those used in currently licensed influenza A vaccines with properties that make it a promising candidate for further evaluation in humans.

1 Our previous studies revealed that the immunosuppressive agent, FTY720, mainly induces mitochondria-involved apoptosis in some types of cancer cells, since Bcl-2 overexpression prevents the FTY720-induction of apoptotic stimuli. Furthermore, FTY720 induces G0/G1 cell cycle arrest. The present study further examines the correlation between intracellular signaling kinases with FTY720-induced mitochondria-involved apoptosis. 2 Human T cell leukemia Jurkat was exposed to FTY720. Dephosphorylation of Akt occurred in a time-and concentration-dependent manner. FTY720 also induced Bad (Ser 136 ) and ribosomal p70S6 kinase (p70 S6k ) (Thr 389 ) dephosphorylation. 3 FTY720-induced Akt dephosphorylation was not because of Akt upstream phosphatidylinositol 3 0 -kinase (PI 3-kinase) pathway inhibition. 4 FTY720 also induced Akt dephosphorylation in human B cell leukemia BALL-1. BALL-1 cells were resistant to FTY720-induced apoptosis. 5 Okadaic acid (OA) inhibited the FTY720-induced dephosphorylation of Akt and p70 S6k , suggesting that FTY720 promotes Ser/Thr protein phosphatase (PP) activity. 6 OA partially inhibited FTY720-induced caspase-3 activation. 7 PP2A or PP2A-like phosphatase was temporarily activated in cells exposed to FTY720. In addition, FTY720 activated purified PP2A (ABC). 8 Overall, the results suggest that FTY720 activated PP2A or PP2A-like phosphatase and dephosphorylated Akt pathway factors resulting in the enhancement of apoptosis via mitochondria. British Journal of Pharmacology (2003) 138, 1303 -1312. doi:10.1038/sj.bjp.0705182 Keywords: FTY720; Akt; PKB; P70 S6k ; Bad; PP2A; apoptosis; leukemia cells Abbreviations: CDK, cyclin-dependent kinase; MAPK, mitogen-activated protein kinase; MEK, MAPK kinase; OA, okadaic acid; p70 S6k , ribosomal p70 S6 kinase; PDGF, platelet-derived growth factor; PDK, phosphoinositide-dependent kinase; PDK1, phosphoinositide-dependent kinase 1; phospho-, phosphorylated; PI(3,4)P 2 , phosphatidylinositol 3,4-diphosphate; PI(3,4,5)P 3 , phosphatidylinositol 3,4,5-triphosphate; PI 3-kinase, phosphatidylinositol 3 0 -kinase; PI(4,5)P 2 , phosphatidylinositol 4,5-diphosphate; PP, protein phosphatase; pRb, retinoblastoma protein; SGK, serum and glucocorticoid-inducible kinase; DC m , mitochondrial membrane potential

No abstract