Synergistic adaptive mutations in the hemagglutinin and polymerase acidic protein lead to increased virulence of pandemic 2009 H1N1 influenza A virus in mice.

Influenza impressively reflects the paradigm of a viral disease in which continued evolution of the virus is of paramount importance for annual epidemics and occasional pandemics in humans. Because of the continuous threat of novel influenza outbreaks, it is essential to gather further knowledge about viral pathogenicity determinants. Here, we explored the adaptive potential of the influenza A virus subtype H1N1 variant isolate A/Hamburg/04/09 (HH/04) by sequential passaging in mice lungs. Three passages in mice lungs were sufficient to dramatically enhance pathogenicity of HH/04. Sequence analysis identified 4 nonsynonymous mutations in the third passage virus. Using reverse genetics, 3 synergistically acting mutations were defined as pathogenicity determinants, comprising 2 mutations in the hemagglutinin (HA[D222G] and HA[K163E]), whereby the HA(D222G) mutation was shown to determine receptor binding specificity and the polymerase acidic (PA) protein F35L mutation increasing polymerase activity. In conclusion, synergistic action of all 3 mutations results in a mice lethal pandemic H1N1 virus.

Improvement of H5N1 influenza vaccine viruses: influence of internal gene segments of avian and human origin on production and hemagglutinin content.

The H5N1-clade 1 influenza vaccine strain NIBRG-14 produces exceptionally low amounts of antigen, a problem recently encountered also for initial pandemic H1N1-2009 vaccine seeds. Here, we report on a strategy that may contribute to overcome this obstacle. Influenza vaccine viruses usually consist of two segments coding for the antigenic HA and NA proteins of a wild-type strain and the six residual internal gene segments of the vaccine donor strain A/PR/8/34 (PR8). To enhance the antigen yield from H5N1 vaccine virus we generated by reverse genetics a set of PR8-based reassortant viruses expressing the HA and NA segments of the prototypic strain A/Vietnam/1203/2004 and additional replacements of the internal M or PB1 genes of PR8. The reassortants were compared to the parental PR8 and H5N1 viruses in terms of growth in embryonated chicken eggs and the amount of incorporated antigenic HA protein. Compared to NIBRG-14, three out of six viruses displayed an increased replication in embryonated chicken eggs and higher HA content that was also maintained after ether/detergent extraction of virions. Electron microscopic analysis showed that the reassortment hardly affected particle shape and size. Two selected H5N1 reassortant viruses were investigated concerning their pathogenicity in ferrets and found to behave as low pathogenic as the PR8 donor strain. In conclusion, this study shows that replication and antigen content of PR8-derived H5N1 influenza vaccine viruses can be improved by incorporation of heterologous internal gene segments without compromising their attenuated character.

Measles virus modulates chemokine release and chemotactic responses of dendritic cells.

Interference with dendritic cell (DC) maturation and function is considered to be central to measles virus (MV)-induced immunosuppression. Temporally ordered production of chemokines and switches in chemokine receptor expression are essential for pathogen-driven DC maturation as they are prerequisites for chemotaxis and T cell recruitment. We found that MV infection of immature monocyte-derived DCs induced transcripts specific for CCL-1, -2, -3, -5, -17 and -22, CXCL-10 and CXCL-11, yet did not induce CXCL-8 (interleukin-8) and CCL-20 at the mRNA and protein level. Within 24 h post-infection, T cell attraction was not detectably impaired by these cells. MV infection failed to promote the switch from CCR5 to CCR7 expression and this correlated with chemotactic responses of MV-matured DC cultures to CCL-3 rather than to CCL-19. Moreover, the chemotaxis of MV-infected DCs to either chemokine was compromised, indicating that MV also interferes with this property independently of chemokine receptor modulation.

Sabotage of antiviral signaling and effectors by influenza viruses.

Vertebrate cells activate multiple signaling modules upon virus infection to eliminate the invading pathogen and to prevent the establishment of a persistent infection. A major immediate response pathway is controlled by the RNA helicases RIG-I and MDA5, which, after recognition of viral nucleic acids, signal induction of the interferon (IFN)-alpha/beta cytokine family that upregulates numerous antiviral effector proteins. Virulent viruses, in contrast, have learned during co-evolution with their hosts to manipulate or avoid this response in order to prevail in a repulsive environment. Focusing on the influenza viruses and their IFN-antagonistic NS1 proteins, we summarize recent progress in this rapidly evolving field at the intersection of virology and immunobiology involving studies of how viral pathogens induce and sabotage cellular defenses.

Measles virus targets DC-SIGN to enhance dendritic cell infection.

Dendritic cells (DCs) are involved in the pathogenesis of measles virus (MV) infection by inducing immune suppression and possibly spreading the virus from the respiratory tract to lymphatic tissues. It is becoming evident that DC function can be modulated by the involvement of different receptors in pathogen interaction. Therefore, we have investigated the relative contributions of different MV-specific receptors on DCs to MV uptake into and infection of these cells. DCs express the MV receptors CD46 and CD150, and we demonstrate that the C-type lectin DC-specific intercellular adhesion molecule 3-grabbing nonintegrin (DC-SIGN) is a novel receptor for laboratory-adapted and wild-type MV strains. The ligands for DC-SIGN are both MV glycoproteins F and H. In contrast to CD46 and CD150, DC-SIGN does not support MV entry, since DC-SIGN does not confer susceptibility when stably expressed in CHO cells. However, DC-SIGN is important for the infection of immature DCs with MV, since both attachment and infection of immature DCs with MV are blocked in the presence of DC-SIGN inhibitors. Our data demonstrate that DC-SIGN is crucial as an attachment receptor to enhance CD46/CD150-mediated infection of DCs in cis. Moreover, MV might not only target DC-SIGN to infect DCs but may also use DC-SIGN for viral transmission and immune suppression.

Impact of measles virus dendritic-cell infection on Th-cell polarization in vitro.

Interference of measles virus (MV) with dendritic-cell (DC) functions and deregulation of T-cell differentiation have been proposed to be central to the profound suppression of immune responses to secondary infections up to several weeks after the acute disease. To address the impact of MV infection on the ability of DCs to promote Th-cell differentiation, an in vitro system was used where uninfected, tumour necrosis factor alpha/interleukin (IL) 1 beta-primed DCs were co-cultured with CD45RO(-) T cells in the presence of conditioned media from MV-infected DCs primed under neutral or DC-polarizing conditions. It was found that supernatants of DCs infected with an MV vaccine strain strongly promoted Th1 differentation, whereas those obtained from wild-type MV-infected DCs generated a mixed Th1/Th0 response, irrespective of the conditions used for DC priming. Th-cell commitment in this system did not correlate with the production of IL12 p70, IL18 or IL23. Thus, a combination of these or other, as yet undefined, soluble factors is produced upon MV infection of DCs that strongly promotes Th1/Th0 differentiation.