Interferon Escape of Respiratory Syncytial Virus: Functional Analysis of Nonstructural Proteins NS1 and NS2

Respiratory syncytial virus (RSV) is recognised as the most frequent cause of severe lung infections in infants and cattle worldwide. Currently, no effective treatments are available and the development of a successful vaccine has been hampered by the fact that natural infection does not provide complete and durable protection. RSV nonstructural proteins, NS1 and NS2, are strong inhibitors of IFN α/β-production by specifically preventing interferon regulatory factor (IRF)-3 phosphorylation. However, the exact mechanisms leading to NS protein-mediated inhibition of IRF3 remain to be unravelled. One of the objectives of this study was to identify amino acid domains in the human respiratory syncytial virus (HRSV) nonstructural proteins (NS) responsible for their ability to ablate the IFN-β signalling pathway. Furthermore, I wanted to find out at which level of this signalling pathway the NS proteins exert their suppressive activity and which are their major cellular targets. HRSV strains A2 and Long differ in their ability to block interferon type I synthesis. Sequence analysis of their NS proteins revealed the presence of an amino acid residue in the NS2 protein with a potential role for RSV IFN-inhibitory functions. Two recombinant bovine respiratory syncytial (BRSV) viruses harbouring HRSV NS1 and NS2 genes were generated and tested in their ability to restrict IFN-β synthesis. These recombinant viruses differed only in the identity of the residue at position 26 of the HRSV NS2 protein: rBRSVh1/2 has a Threonine as in the Long strain, while in rBRS h1/2*T26I this amino acid was mutated into an Isoleucin similarly to A2 virus. Sets of in vitro tests revealed that IFN-β induction was impaired by rBRSVh1/2*T26I when compared to rBRSV h1/2. Analysis of the transcriptional factors (AP-1, NF-kB and IRF3) involved in the activation of IFN-β synthesis provided evidence that the inhibitory ability of rBRSVh1/2*T26I was correlated to a selective block of IRF3. The mutation (T26I) in the NS2 protein did neither effect the NF-kB activation pathway nor perturbed the IFN-resistance characteristics of the chimeric viruses. IRF3 is activated upon phosphorylation mediated by IKK-related kinases (TBK1 and IKK-ε). TBK1 was therefore cloned from a human lung cDNA library and its biological activities regarding the induction of IFN-β were compared in mock-infected and infected cells. rBRSVh1/2*T26I and HRSV A2 precluded virus-induced IRF3 activation by interfering with TBK1 functions. No direct interaction between TBK1 and NS2 protein was demonstrated indicating that the kinase TBK1 may not be the sole target involved in RSV mechanisms of evasion of the innate immune response. Recombinant IFN-inducible rabies viruses expressing HRSV Long-derived (rGFP-Ph2/1) or HRSV A2-like (rGFP-Ph2*T26I/1) NS proteins were also generated. The HRSV NS2 protein expressing an Isoleucin (NS2*T26I) at position 26 was not able to suppress IFN-β induction and to rescue the growth of the recombinant eGFP-Ph2*T26I/1 in interferon-competent cells. A low expression of the mutated NS2*T26I protein was probably the reason of this failure. In summary, these results show that the HRSV NS2 protein possesses an intrinsic IFN-β inhibitory activity, which is achieved throughout a selective inhibition of the IRF3 activation pathway. The block appears to be exerted at the level of IRF3-kinase TBK1. Interferonantagonist functions of the HRSV NS2 protein are linked to a particular amino acid motif in the N-terminus of the protein. Identification of this amino acid domain and of TBK1 as the cellular target provide a better insight of how the HRSV NS2 protein prevents the establishment of the antiviral innate immune response and therefore it might contribute to the development of an effective vaccine

Cell cycle progression or translation control is not essential for vesicular stomatitis virus oncolysis of hepatocellular carcinoma.

The intrinsic oncolytic specificity of vesicular stomatitis virus (VSV) is currently being exploited to develop alternative therapeutic strategies for hepatocellular carcinoma (HCC). Identifying key regulators in diverse transduction pathways that define VSV oncolysis in cancer cells represents a fundamental prerequisite to engineering more effective oncolytic viral vectors and adjusting combination therapies. After having identified defects in the signalling cascade of type I interferon induction, responsible for attenuated antiviral responses in human HCC cell lines, we have now investigated the role of cell proliferation and translation initiation. Cell cycle progression and translation initiation factors eIF4E and eIF2Bepsilon have been recently identified as key regulators of VSV permissiveness in T-lymphocytes and immortalized mouse embryonic fibroblasts, respectively. Here, we show that in HCC, decrease of cell proliferation by cell cycle inhibitors or siRNA-mediated reduction of G(1) cyclin-dependent kinase activities (CDK4) or cyclin D1 protein expression, do not significantly alter viral growth. Additionally, we demonstrate that translation initiation factors eIF4E and eIF2Bepsilon are negligible in sustaining VSV replication in HCC. Taken together, these results indicate that cellular proliferation and the initiation phase of cellular protein synthesis are not essential for successful VSV oncolysis of HCC. Moreover, our observations indicate the importance of cell-type specificity for VSV oncolysis, an important aspect to be considered in virotherapy applications in the future

Lead evaluation in children's lipsticks through atomic absorption spectrometry

Lead is a metal with recognized toxicity and it is known that it may be a contaminant in lipsticks. In Brazil, the Health Regulatory Agency (ANVISA) determines that the maximum limit allowed for the presence of lead in lipsticks is 20 ppm. Children are more vulnerable to lead toxicity. The objective of this study was to evaluate the presence of lead in infant lipsticks. Nineteen samples from four different brands sold in Brazil were evaluated. After sample extraction, analyses were performed by graphite furnace atomic absorption spectrometry. Lead was not detected in any of the tested lipsticks. Considering the presence of lead in adult makeup, the present study reinforces the need to use products intended for children considering kids are more vulnerable to lead toxic effects

Influenza AH1N2 Viruses, United Kingdom, 2001–02 Influenza Season

During the winter of 2001–02, influenza AH1N2 viruses were detected for the first time in humans in the U.K. The H1N2 viruses co-circulated with H3N2 viruses and a very small number of H1N1 viruses and were isolated in the community and hospitalized patients, predominantly from children <15 years of age. Characterization of H1N2 viruses indicated that they were antigenically and genetically homogeneous, deriving the hemagglutinin (HA) gene from recently circulating A/New Caledonia/20/99-like H1N1 viruses, whereas the other seven genes originated from recently circulating H3N2 viruses. Retrospective reverse transcription-polymerase chain reaction analysis of influenza A H1 viruses isolated in the U.K. during the previous winter identified a single H1N2 virus, isolated in March 2001, indicating that H1N2 viruses did not widely circulate in the U.K. before September 2001. The reassortment event is estimated to have occurred between 1999 and early 2001, and the emergence of H1N2 viruses in humans reinforces the need for frequent surveillance of circulating viruses

Phosphoinositide 3 kinase signalling may affect multiple steps during herpes simplex virus type-1 entry

Early interactions of herpes simplex virus type-1 (HSV-1) with cells lead to cytoskeletal changes facilitating filopodia formation and membrane fusion. Here, we demonstrate that phosphoinositide 3 kinase (PI3K) signalling may affect multiple steps during HSV-1 entry. An inhibitor of PI3K (LY294002) blocked HSV-1 entry and the blockage was cell-type- and gD receptor-independent. Entry inhibition was also observed with primary cultures of the human corneal fibroblasts and unrelated β- and γ-herpesviruses. Immunofluorescence analysis demonstrated that LY294002 negatively affected HSV-1-induced filopodia formation. Similar effects of the inhibitor were seen on HSV-1 glycoprotein-induced cell-to-cell fusion. Cells expressing HSV-1 glycoproteins (gB, gD, gH and gL) showed significantly less fusion with target cells in the presence of the inhibitor. Expression of a dominant-negative PI3K mutant negatively affected both entry and fusion. We also show that inhibition of PI3K signalling also affected RhoA activation required for HSV-1 entry into certain cell types

The global antigenic diversity of swine influenza A viruses.

Swine influenza presents a substantial disease burden for pig populations worldwide and poses a potential pandemic threat to humans. There is considerable diversity in both H1 and H3 influenza viruses circulating in swine due to the frequent introductions of viruses from humans and birds coupled with geographic segregation of global swine populations. Much of this diversity is characterized genetically but the antigenic diversity of these viruses is poorly understood. Critically, the antigenic diversity shapes the risk profile of swine influenza viruses in terms of their epizootic and pandemic potential. Here, using the most comprehensive set of swine influenza virus antigenic data compiled to date, we quantify the antigenic diversity of swine influenza viruses on a multi-continental scale. The substantial antigenic diversity of recently circulating viruses in different parts of the world adds complexity to the risk profiles for the movement of swine and the potential for swine-derived infections in humans.Medical Research Council Fellowship MR/K021885/1 (JF

Reassortment Patterns in Swine Influenza Viruses

Three human influenza pandemics occurred in the twentieth century, in 1918, 1957, and 1968. Influenza pandemic strains are the results of emerging viruses from non-human reservoirs to which humans have little or no immunity. At least two of these pandemic strains, in 1957 and in 1968, were the results of reassortments between human and avian viruses. Also, many cases of swine influenza viruses have reportedly infected humans, in particular, the recent H1N1 influenza virus of swine origin, isolated in Mexico and the United States. Pigs are documented to allow productive replication of human, avian, and swine influenza viruses. Thus it has been conjectured that pigs are the “mixing vessel” that create the avian-human reassortant strains, causing the human pandemics. Hence, studying the process and patterns of viral reassortment, especially in pigs, is a key to better understanding of human influenza pandemics. In the last few years, databases containing sequences of influenza A viruses, including swine viruses, collected since 1918 from diverse geographical locations, have been developed and made publicly available. In this paper, we study an ensemble of swine influenza viruses to analyze the reassortment phenomena through several statistical techniques. The reassortment patterns in swine viruses prove to be similar to the previous results found in human viruses, both in vitro and in vivo, that the surface glycoprotein coding segments reassort most often. Moreover, we find that one of the polymerase segments (PB1), reassorted in the strains responsible for the last two human pandemics, also reassorts frequently

Panorama Phylogenetic Diversity and Distribution of Type A Influenza Virus

Type A influenza virus is one of important pathogens of various animals, including humans, pigs, horses, marine mammals and birds. Currently, the viral type has been classified into 16 hemagglutinin and 9 neuraminidase subtypes, but the phylogenetic diversity and distribution within the viral type largely remain unclear from the whole view.The panorama phylogenetic trees of influenza A viruses were calculated with representative sequences selected from approximately 23000 candidates available in GenBank using web servers in NCBI and the software MEGA 4.0. Lineages and sublineages were classified according to genetic distances, topology of the phylogenetic trees and distributions of the viruses in hosts, regions and time.Here, two panorama phylogenetic trees of type A influenza virus covering all the 16 hemagglutinin subtypes and 9 neuraminidase subtypes, respectively, were generated. The trees provided us whole views and some novel information to recognize influenza A viruses including that some subtypes of avian influenza viruses are more complicated than Eurasian and North American lineages as we thought in the past. They also provide us a framework to generalize the history and explore the future of the viral circulation and evolution in different kinds of hosts. In addition, a simple and comprehensive nomenclature system for the dozens of lineages and sublineages identified within the viral type was proposed, which if universally accepted, will facilitate communications on the viral evolution, ecology and epidemiology