Origin of the European avian-like swine influenza viruses.

The avian-like swine influenza viruses emerged in 1979 in Belgium and Germany. Thereafter, they spread through many European swine-producing countries, replaced the circulating classical swine H1N1 influenza viruses, and became endemic. Serological and subsequent molecular data indicated an avian source, but details remained obscure due to a lack of relevant avian influenza virus sequence data. Here, the origin of the European avian-like swine influenza viruses was analysed using a collection of 16 European swine H1N1 influenza viruses sampled in 1979-1981 in Germany, the Netherlands, Belgium, Italy and France, as well as several contemporaneous avian influenza viruses of various serotypes. The phylogenetic trees suggested a triple reassortant with a unique genotype constellation. Time-resolved maximum clade credibility trees indicated times to the most recent common ancestors of 34-46 years (before 2008) depending on the RNA segment and the method of tree inference.

Genetics, evolution, and the zoonotic capacity of European Swine influenza viruses.

The European swine influenza virus lineage differs genetically from the classical swine influenza viruses and the triple reassortants found in North America and Asia. The avian-like swine H1N1 viruses emerged in 1979 after an avian-to-swine transmission and spread to all major European pig-producing countries. Reassortment of these viruses with seasonal H3N2 viruses led to human-like swine H3N2 viruses which appeared in 1984. Finally, human-like swine H1N2 viruses emerged in 1994. These are triple reassortants comprising genes of avian-like H1N1, seasonal H1N1, and seasonal H3N2 viruses. All three subtypes established persistent infection chains and became prevalent in the European pig population. They successively replaced the circulating classical swine H1N1 viruses of that time and gave rise to a number of reassortant viruses including the pandemic (H1N1) 2009 virus. All three European lineages have the capacity to infect humans but zoonotic infections are benign.

Circulation of classical swine influenza virus in Europe between the wars?

The complete genomes of two swine influenza viruses from England were sequenced. Phylogenetic analysis revealed classical swine H1N1 viruses, one of which, A/swine/London, is closely related to virus strains of the early 1930s. Both strains are also antigenically related to A/swine/Iowa/15/1930, the strain originally isolated by Richard Shope. The source of A/swine/London is unknown, but its relationship to early classical swine influenza viruses suggests that the emergence of these viruses in Europe has to be antedated by 15-20 years.

Origin of the 1918 pandemic H1N1 influenza A virus as studied by codon usage patterns and phylogenetic analysis.

The pandemic of 1918 was caused by an H1N1 influenza A virus, which is a negative strand RNA virus; however, little is known about the nature of its direct ancestral strains. Here we applied a broad genetic and phylogenetic analysis of a wide range of influenza virus genes, in particular the PB1 gene, to gain information about the phylogenetic relatedness of the 1918 H1N1 virus. We compared the RNA genome of the 1918 strain to many other influenza strains of different origin by several means, including relative synonymous codon usage (RSCU), effective number of codons (ENC), and phylogenetic relationship. We found that the PB1 gene of the 1918 pandemic virus had ENC values similar to the H1N1 classical swine and human viruses, but different ENC values from avian as well as H2N2 and H3N2 human viruses. Also, according to the RSCU of the PB1 gene, the 1918 virus grouped with all human isolates and "classical" swine H1N1 viruses. The phylogenetic studies of all eight RNA gene segments of influenza A viruses may indicate that the 1918 pandemic strain originated from a H1N1 swine virus, which itself might be derived from a H1N1 avian precursor, which was separated from the bulk of other avian viruses in toto a long time ago. The high stability of the RSCU pattern of the PB1 gene indicated that the integrity of RNA structure is more important for influenza virus evolution than previously thought.

Three amino acid changes in PB1-F2 of highly pathogenic H5N1 avian influenza virus affect pathogenicity in mallard ducks.

Despite reports that the PB1-F2 protein contributes to influenza virus pathogenicity in the mouse model, little is known about its significance in avian hosts. In our previous study, the A/Vietnam/1203/04 (H5N1) wild-type virus (wtVN1203) was more lethal to mallard ducks than a reverse genetics (rg)-derived VN1203. In search of potential viral factors responsible for this discrepancy, we found that synonymous mutations (SMs) had been inadvertently introduced into three genes of the rgVN1203 (rgVN1203/SM-3). Of 11 SMs in the PB1 gene, three resided in the PB1-F2 open reading frame, caused amino acid (aa) substitutions in the PB1-F2 protein, and reduced virus lethality in mallard ducks. The wtVN1203 and recombinant viruses with repairs to these three aa's (rgVN1203/R-PB1-F2) or with repairs to all 11 SMs (rgVN1203/R-PB1) were significantly more pathogenic than rgVN1203/SM-3. In cultured cells, repairing three mutations in PB1-F2 increased viral polymerase activity and expression levels of viral RNA.

The genome of an influenza virus from a pilot whale: relation to influenza viruses of gulls and marine mammals.

Influenza virus A/whale/Maine/328B/1984 (H13N2) was isolated from a diseased pilot whale. Since only a partial sequence was available, its complete genome was sequenced and compared to the sequences of subtype H13 influenza viruses from shorebirds and various influenza viruses of marine mammals. The data reveal a rare genotype constellation with all gene segments derived of an influenza virus adapted to gulls, terns and waders. In contrast, the phylogenetic trees indicate that the majority of influenza viruses isolated from marine mammals derived from influenza viruses adapted to geese and ducks. We conclude that A/whale/Maine/328B/1984 is the first record of an infection of a marine mammal from a gull-origin influenza virus.

Introduction of silent mutations into the NP gene of influenza A viruses as a possible strategy for the creation of a live attenuated vaccine.

The nucleoprotein (NP) of influenza A virus (IAV) is associated with many different functions including host range restriction. Multiple sequence alignment analyses of 748 NP gene sequences from GenBank revealed a highly conserved region of 60 nucleotides within the ORF at the 3'-ends of the cRNA, in some codons even silent mutations were not found. This suggests that the RNA structure integrity within this region is crucial for IAV replication. To explore the impact of these conserved nucleotides for viral replication we created mutant viruses with one or more silent mutations in the respective region of the NP gene of the IAV strain A/WSN/33 (H1N1) (WSN). Assessment of viral replication of these WSN mutant viruses showed significant growth disadvantages when compared to the corresponding parental strain. On the basis of these findings we tested whether the attenuation of IAV by introduction of silent mutations into the NP gene may serve as a strategy to create a live attenuated vaccine. Mice vaccinated with the attenuated WSN mutant survived a lethal challenge dose of wild type WSN virus or the mouse adapted pandemic H1N1v strain A/Hamburg/4/2009. Thus, introduction of silent mutations in the NP of IAV is a feasible approach for a novel vaccination strategy allowing attenuation of the master strain but leaves the antigenicity of the gene product unaltered. This principle is potentially applicable for all viruses with segmented genomes.

CK2beta gene silencing increases cell susceptibility to influenza A virus infection resulting in accelerated virus entry and higher viral protein content.

BACKGROUND: Influenza A virus (IVA) exploits diverse cellular gene products to support its replication in the host. The significance of the regulatory (beta) subunit of casein kinase 2 (CK2beta) in various cellular mechanisms is well established, but less is known about its potential role in IVA replication. We studied the role of CK2beta in IVA-infected A549 human epithelial lung cells. RESULTS: Activation of CK2beta was observed in A549 cells during virus binding and internalization but appeared to be constrained as replication began. We used small interfering RNAs (siRNAs) targeting CK2beta mRNA to silence CK2beta protein expression in A549 cells without affecting expression of the CK2alpha subunit. CK2beta gene silencing led to increased virus titers, consistent with the inhibition of CK2beta during IVA replication. Notably, virus titers increased significantly when CK2beta siRNA-transfected cells were inoculated at a lower multiplicity of infection. Virus titers also increased in cells treated with a specific CK2 inhibitor but decreased in cells treated with a CK2beta stimulator. CK2beta absence did not impair nuclear export of viral ribonucleoprotein complexes (6 h and 8 h after inoculation) or viral polymerase activity (analyzed in a minigenome system). The enhancement of virus titers by CK2beta siRNA reflects increased cell susceptibility to influenza virus infection resulting in accelerated virus entry and higher viral protein content. CONCLUSION: This study demonstrates the role of cellular CK2beta protein in the viral biology. Our results are the first to demonstrate a functional link between siRNA-mediated inhibition of the CK2beta protein and regulation of influenza A virus replication in infected cells. Overall, the data suggest that expression and activation of CK2beta inhibits influenza virus replication by regulating the virus entry process and viral protein synthesis.

[Influenza pandemic planning]. / Influenza Pandemie Planung.

In this article the most important properties of influenza A viruses are described to understand influenza pandemics. There are at least three possibilities: (1) By reassortment between an avian and the prevailing human influenza A virus viruses with a new surface are created, against which no neutralizing antibodies are present in the human population. Such a virus can spread immediately worldwide. (2) Viruses, which have been present in the human population some time ago, reappear and infect the new generation, which has not been in contact with this virus before. (3) An avian influenza virus crosses the species barrier to humans and forms there a new stable lineage. In relation to pandemic planning, the first possibility can be more or less excluded, since the now-a-days human influenza A viruses have evolved so far away from their original source, the avian influenza viruses, that the formation of a well-growing and well-spreading reassortant is practically not possible anymore. Point two is a dangerous possibility, in that, e.g., a human H2N2 virus could reappear, which had disappeared in 1968 from the human population. The third possibility is at the moment the most dangerous situation, if, e.g., a highly neurotropic H5N1 virus from Southeast Asia crosses the species barrier to humans. An infection with such a pandemic virus presumably cannot be treated efficiently by antivirals. In such a situation only a rapid vaccination would be successful. In this respect in the last year important results have been obtained by using the reverse genetics. Meanwhile in about 50 countries there have been drawn up pandemic-preparedness plans.

Importance of neuraminidase active-site residues to the neuraminidase inhibitor resistance of influenza viruses.

Neuraminidase inhibitors (NAIs) are antivirals designed to target conserved residues at the neuraminidase (NA) enzyme active site in influenza A and B viruses. The conserved residues that interact with NAIs are under selective pressure, but only a few have been linked to resistance. In the A/Wuhan/359/95 (H3N2) recombinant virus background, we characterized seven charged, conserved NA residues (R118, R371, E227, R152, R224, E276, and D151) that directly interact with the NAIs but have not been reported to confer resistance to NAIs. These NA residues were replaced with amino acids that possess side chains having similar properties to maintain their original charge. The NA mutations we introduced significantly decreased NA activity compared to that of the A/Wuhan/359/95 recombinant wild-type and R292K (an NA mutation frequently reported to confer resistance) viruses, which were analyzed for comparison. However, the recombinant viruses differed in replication efficiency when we serially passaged them in vitro; the growth of the R118K and E227D viruses was most impaired. The R224K, E276D, and R371K mutations conferred resistance to both zanamivir and oseltamivir, while the D151E mutation reduced susceptibility to oseltamivir only (approximately 10-fold) and the R152K mutation did not alter susceptibility to either drug. Because the R224K mutation was genetically unstable and the emergence of the R371K mutation in the N2 subtype is statistically unlikely, our results suggest that only the E276D mutation is likely to emerge under selective pressure. The results of our study may help to optimize the design of NAIs.

Cooperation between the hemagglutinin of avian viruses and the matrix protein of human influenza A viruses.

To analyze the compatibility of avian influenza A virus hemagglutinins (HAs) and human influenza A virus matrix (M) proteins M1 and M2, we doubly infected Madin-Darby canine kidney cells with amantadine (1-aminoadamantane hydrochloride)-resistant human viruses and amantadine-sensitive avian strains. By using antisera against the human virus HAs and amantadine, we selected reassortants containing the human virus M gene and the avian virus HA gene. In our system, high virus yields and large, well-defined plaques indicated that the avian HAs and the human M gene products could cooperate effectively; low virus yields and small, turbid plaques indicated that cooperation was poor. The M gene products are among the primary components that determine the species specificities of influenza A viruses. Therefore, our system also indicated whether the avian HA genes effectively reassorted into the genome and replaced the HA gene of the prevailing human influenza A viruses. Most of the avian HAs that we tested efficiently cooperated with the M gene products of the early human A/PR/8/34 (H1N1) virus; however, the avian HAs did not effectively cooperate with the most recently isolated human virus that we tested, A/Nanchang/933/95 (H3N2). Cooperation between the avian HAs and the M proteins of the human A/Singapore/57 (H2N2) virus was moderate. These results suggest that the currently prevailing human influenza A viruses might have lost their ability to undergo antigenic shift and therefore are unable to form new pandemic viruses that contain an avian HA, a finding that is of great interest for pandemic planning.