Conserved methionine 165 of matrix protein contributes to the nuclear import and is essential for influenza A virus replication.

BACKGROUND: The influenza matrix protein (M1) layer under the viral membrane plays multiple roles in virus assembly and infection. N-domain and C-domain are connected by a loop region, which consists of conserved RQMV motif. METHODS: The function of the highly conserve RQMV motif in the influenza virus life cycle was investigated by site-directed mutagenesis and by rescuing mutant viruses by reverse genetics. Co-localization of M1 with nucleoprotein (NP), clustered mitochondria homolog protein (CLUH), chromosome region maintenance 1 protein (CRM1), or plasma membrane were studied by confocal microscopy. RESULTS: Mutant viruses containing an alanine substitution of R163, Q164 and V166 result in the production of the virus indistinguishable from the wild type phenotype. Single M165A substitution was lethal for rescuing infection virus and had a striking effect on the distribution of M1 and NP proteins. We have observed statistically significant reduction in distribution of both M165A (p<0,05) and NP (p<0,001) proteins to the nucleus in the cells transfected with the reverse -genetic system with mutated M1. M165A protein was co-localized with CLUH protein in the cytoplasm and around the nucleus but transport of M165-CLUH complex through the nuclear membrane was restricted. CONCLUSIONS: Our finding suggest that methionine 165 is essential for virus replication and RQMV motif is involved in the nuclear import of viral proteins.

Nest ecology of blood parasites in the European roller and its ectoparasitic carnid fly.

Haemosporidian parasites are considered the most important vector-borne parasites. However, vector identity and ecology is unknown for most such host-vector-parasite systems. In this study, we employ microscopic and molecular analyses to examine haemosporidian prevalence in a migratory, cavity-nesting bird, European roller Coracias garrulus, and its nidicolous blood-feeding ectoparasite Carnus hemapterus. This system is unique in that the ectoparasite is confined to a near-closed environment, in contrast to the free-wandering system of haematophagous dipterans such as mosquitoes. Blood film analysis confirms previous works in that Haemoproteus parasites are widely prevalent in adult rollers and belong to a single species, Haemoproteus coraciae. Leucocytozoon sp. and Trypanosoma sp. also are detected in adult rollers at low intensities with this technique. By means of molecular analysis, we report for the first time Plasmodium sp. presence in C. garrulus. Based on PCR results, Plasmodium parasites are relatively less prevalent than Haemoproteus parasites (20% vs. 31%) in rollers. In contrast, haemosporidian prevalences show the opposite trend for Carnus flies: Plasmodium sp. occurrence (62%) clearly predominates over that of Haemoproteus sp. (5%). A comparison between roller and Carnus samples reveals a significantly higher prevalence of Plasmodium sp. in Carnus samples. Insect survey and phylogenetic analysis suggest Culicoides flies as Haemoproteus sp. vectors, which appear to readily transmit the parasite in southern Spain. This study does not find support for Carnus flies to serve as biological or mechanical vectors of haemosporidians. In spite of this, nidicolous blood-feeding ectoparasites, such as carnid flies, appear as a suitable model for studies on the occurrence and temporal dynamics of avian haemosporidians such as Plasmodium sp. present at low intensities.

Synergic and antagonistic effect of small hairpin RNAs targeting the NS gene of the influenza A virus in cells and mice.

In the present study, we demonstrate the effect of individual and mixtures of shRNAs targeting the NS gene to treat an established infection of influenza A virus (IAV). We prepared 10 shRNAs targeting the NS gene of the IAV, and these shRNAs were tested individually or in mixtures 16h after infection. Our results revealed: (i) shRNA targeting the NS1 transcript decreased the virus titre up to 21% (P<0.01), (ii) shRNA targeting NEP transcript did not influence the replication of IAV in the infected cells; (iii) a mixture of shRNAs targeting the NS1 transcript was less effective than the individual shRNAs and decreased the virus titre up to 42% in vitro; (iv) a mixture of individually inactive shRNAs targeting the NEP transcript significantly inhibited the replication of IAV in vitro; (v) the activities of the individual shRNAs in vivo predominantly corresponded to their activities in vitro; (vi) a synergistic effect of the shRNAs was observed in vivo; and (vii) a shRNA targeting the region common to both the NS1 and NEP transcripts, shNS593, exhibited the strongest inhibition and reduced the virus titre up to 16.4% in vitro, prolonged the survival of the mice by three days and abolished the protective effect of other shRNAs in vivo. shRNAs inhibited influenza virus infection in a gene-specific manner. NS1 mRNA was significantly reduced in lungs treated with shRNAs and the levels of RIG-1, IFN-α, IFN-ß and IFN-γ mRNAs shRNAs were not altered.