Novel phylogenetic algorithm to monitor human tropism in Egyptian H5N1-HPAIV reveals evolution toward efficient human-to-human transmission

Years of endemic infections with highly pathogenic avian influenza (HPAI) A subtype H5N1 virus in poultry and high numbers of infections in humans provide ample opportunity in Egypt for H5N1-HPAIV to develop pandemic potential. In an effort to better understand the viral determinants that facilitate human infections of the Egyptian H5N1-HPAIVvirus, we developed a new phylogenetic algorithm based on a new distance measure derived from the informational spectrum method (ISM). This new approach, which describes functional aspects of the evolution of the hemagglutinin subunit 1 (HA1), revealed a growing group G2 of H5N1-HPAIV in Egypt after 2009 that acquired new informational spectrum (IS) properties suggestive of an increased human tropism and pandemic potential. While in 2006 all viruses in Egypt belonged to the G1 group, by 2011 these viruses were virtually replaced by G2 viruses. All of the G2 viruses displayed four characteristic mutations (D43N, S120(D,N), (S,L)129Δ and I151T), three of which were previously reported to increase binding to the human receptor. Already in 2006–2008 G2 viruses were significantly (p<0.02) more often found in humans than expected from their overall prevalence and this further increased in 2009–2011 (p<0.007). Our approach also identified viruses that acquired additional mutations that we predict to further enhance their human tropism. The extensive evolution of Egyptian H5N1-HPAIV towards a preferential human tropism underlines an urgent need to closely monitor these viruses with respect to molecular determinants of virulence

Inferring stabilizing mutations from protein phylogenies : application to influenza hemagglutinin

One selection pressure shaping sequence evolution is the requirement that a protein fold with sufficient stability to perform its biological functions. We present a conceptual framework that explains how this requirement causes the probability that a particular amino acid mutation is fixed during evolution to depend on its effect on protein stability. We mathematically formalize this framework to develop a Bayesian approach for inferring the stability effects of individual mutations from homologous protein sequences of known phylogeny. This approach is able to predict published experimentally measured mutational stability effects (ΔΔG values) with an accuracy that exceeds both a state-of-the-art physicochemical modeling program and the sequence-based consensus approach. As a further test, we use our phylogenetic inference approach to predict stabilizing mutations to influenza hemagglutinin. We introduce these mutations into a temperature-sensitive influenza virus with a defect in its hemagglutinin gene and experimentally demonstrate that some of the mutations allow the virus to grow at higher temperatures. Our work therefore describes a powerful new approach for predicting stabilizing mutations that can be successfully applied even to large, complex proteins such as hemagglutinin. This approach also makes a mathematical link between phylogenetics and experimentally measurable protein properties, potentially paving the way for more accurate analyses of molecular evolution

Viral and host factors required for avian H5N1 influenza A virus replication in mammalian cells

Following the initial and sporadic emergence into humans of highly pathogenic avian H5N1 influenza A viruses in Hong Kong in 1997, we have come to realize the potential for avian influenza A viruses to be transmitted directly from birds to humans. Understanding the basic viral and cellular mechanisms that contribute to infection of mammalian species with avian influenza viruses is essential for developing prevention and control measures against possible future human pandemics. Multiple physical and functional cellular barriers can restrict influenza A virus infection in a new host species, including the cell membrane, the nuclear envelope, the nuclear environment, and innate antiviral responses. In this review, we summarize current knowledge on viral and host factors required for avian H5N1 influenza A viruses to successfully establish infections in mammalian cells. We focus on the molecular mechanisms underpinning mammalian host restrictions, as well as the adaptive mutations that are necessary for an avian influenza virus to overcome them. It is likely that many more viral and host determinants remain to be discovered, and future research in this area should provide novel and translational insights into the biology of influenza virus-host interactions

Differential contribution of PB1-F2 to the virulence of highly pathogenic H5N1 influenza A virus in mammalian and avian species

Highly pathogenic avian influenza A viruses (HPAIV) of the H5N1 subtype occasionally transmit from birds to humans and can cause severe systemic infections in both hosts. PB1-F2 is an alternative translation product of the viral PB1 segment that was initially characterized as a pro-apoptotic mitochondrial viral pathogenicity factor. A full-length PB1-F2 has been present in all human influenza pandemic virus isolates of the 20(th) century, but appears to be lost evolutionarily over time as the new virus establishes itself and circulates in the human host. In contrast, the open reading frame (ORF) for PB1-F2 is exceptionally well-conserved in avian influenza virus isolates. Here we perform a comparative study to show for the first time that PB1-F2 is a pathogenicity determinant for HPAIV (A/Viet Nam/1203/2004, VN1203 (H5N1)) in both mammals and birds. In a mammalian host, the rare N66S polymorphism in PB1-F2 that was previously described to be associated with high lethality of the 1918 influenza A virus showed increased replication and virulence of a recombinant VN1203 H5N1 virus, while deletion of the entire PB1-F2 ORF had negligible effects. Interestingly, the N66S substituted virus efficiently invades the CNS and replicates in the brain of Mx+/+ mice. In ducks deletion of PB1-F2 clearly resulted in delayed onset of clinical symptoms and systemic spreading of virus, while variations at position 66 played only a minor role in pathogenesis. These data implicate PB1-F2 as an important pathogenicity factor in ducks independent of sequence variations at position 66. Our data could explain why PB1-F2 is conserved in avian influenza virus isolates and only impacts pathogenicity in mammals when containing certain amino acid motifs such as the rare N66S polymorphism

Sequence-based prediction for vaccine strain selection and identification of antigenic variability in foot-and-mouth disease virus

Identifying when past exposure to an infectious disease will protect against newly emerging strains is central to understanding the spread and the severity of epidemics, but the prediction of viral cross-protection remains an important unsolved problem. For foot-and-mouth disease virus (FMDV) research in particular, improved methods for predicting this cross-protection are critical for predicting the severity of outbreaks within endemic settings where multiple serotypes and subtypes commonly co-circulate, as well as for deciding whether appropriate vaccine(s) exist and how much they could mitigate the effects of any outbreak. To identify antigenic relationships and their predictors, we used linear mixed effects models to account for variation in pairwise cross-neutralization titres using only viral sequences and structural data. We identified those substitutions in surface-exposed structural proteins that are correlates of loss of cross-reactivity. These allowed prediction of both the best vaccine match for any single virus and the breadth of coverage of new vaccine candidates from their capsid sequences as effectively as or better than serology. Sub-sequences chosen by the model-building process all contained sites that are known epitopes on other serotypes. Furthermore, for the SAT1 serotype, for which epitopes have never previously been identified, we provide strong evidence - by controlling for phylogenetic structure - for the presence of three epitopes across a panel of viruses and quantify the relative significance of some individual residues in determining cross-neutralization. Identifying and quantifying the importance of sites that predict viral strain cross-reactivity not just for single viruses but across entire serotypes can help in the design of vaccines with better targeting and broader coverage. These techniques can be generalized to any infectious agents where cross-reactivity assays have been carried out. As the parameterization uses pre-existing datasets, this approach quickly and cheaply increases both our understanding of antigenic relationships and our power to control disease

Construction and characterization of H5N1-recombinant fowlpox viruses co-expressing host cytokines

Possessing a large double stranded DNA genome up to 300 kb, fowlpox virus (FWPV) has been developed to express avian influenza virus (AIV) antigens since the late 1980s. A more advanced approach would be to coexpress host cytokines from such recombinants. This thesis describes the strategy to construct H5N1-recombinant FWPV (rFWPV) coexpressing chicken Interleukin 12 (IL-12) or Interleukin 15 (IL-15), and discusses the immunogenicity of the recombinants following inoculation into specific-pathogen-free (SPF) chickens. Previously cloned and sequenced cDNAs encoding full-length H5 and N1 of influenza strain A/Chicken/Malaysia/5858/2004 genes were amplified by PCR and inserted into plasmid pEFL29, under the control of a copy of the vaccinia virus p7.5 early/late promoter. The expression cassettes were recombined into the genome of the FP9 strain of FWPV at the fpv002 locus. Recombinant viruses were produced by transfection of the plasmid into chicken embryo fibroblasts (CEFs) after infection with FP9, and isolated by six fold plaque purification on CEFs using X-Gal selection. Chicken IL-12 or IL-15 genes, under control of a synthetic/hybrid poxvirus promoter, were inserted into a ‘transient dominant selection’ recombination plasmid, pPC1.X. The cytokine expression cassettes were then recombined, at the fpPC1 (fpv030) locus, into rFWPV already carrying AIV genes. Following three rounds of passage in CEFs in the presence of mycophenolic acid (MPA), recombinant viruses carrying the gpt gene were isolated. These unstable recombinants were plaque-purified in the absence of MPA until they lost the gpt gene spontaneously, verified by their failure to replicate in the presence of MPA. Recombinant proteins were successfully detected using western blotting and indirect immunofluorescence assay (IFAT). Parental and rFWPV (105 PFU) were inoculated subcutaneously into one-day-old SPF chickens. Sera from chickens immunized with rFWPV/H5 and rFWPV/H5/IL-15 demonstrated viral neutralizing activities, based on the haemagglutation inhibition (HI) test, in which reached a peak at Week 3. A competitive enzyme-linked immunosorbent (ELISA) assay detected N1-specific antibodies induced by rFWPV/N1 and rFWPV/N1/IL-12 at Weeks 4 and 5. Non-specific cellular immune responses were assessed by flow cytometric analysis to enumerate CD4+ and CD8+ T-lymphocytes in peripheral blood. Results of Experiment 2 showed chickens vaccinated with rFWPV/H5, rFWPV/H5/IL-15, rFWPV/N1 and rFWPV/N1/IL-12 demonstrated a higher increase in CD8+ than CD4+ T cell population, relative to control and chickens vaccinated with parental FWPV. Weekly weighing showed that chickens vaccinated with rFWPV/H5/IL-15 had the highest body weight compared to other groups, while the rFWPV/N1/IL-12 group showed the significantly lowest body weight. In summary, this study showed diverse immunogenicity of H5N1-rFWPV coexpressing IL-12 or IL-15. It also demonstrated a weight sparing effect of co-expressing IL-15 in rFWPV vaccines. The results provide the basis for future homologous challenge studies, using live H5N1 virus to evaluate the protective efficacy of the rFWPV vaccines

The multifunctional NS1 protein of influenza A viruses

The non-structural (NS1) protein of influenza A viruses is a non-essential virulence factor that has multiple accessory functions during viral infection. In recent years, the major role ascribed to NS1 has been its inhibition of host immune responses, especially the limitation of both interferon (IFN) production and the antiviral effects of IFN-induced proteins, such as dsRNA-dependent protein kinase R (PKR) and 2'5'-oligoadenylate synthetase (OAS)/RNase L. However, it is clear that NS1 also acts directly to modulate other important aspects of the virus replication cycle, including viral RNA replication, viral protein synthesis, and general host-cell physiology. Here, we review the current literature on this remarkably multifunctional viral protein. In the first part of this article, we summarize the basic biochemistry of NS1, in particular its synthesis, structure, and intracellular localization. We then discuss the various roles NS1 has in regulating viral replication mechanisms, host innate/adaptive immune responses, and cellular signalling pathways. We focus on the NS1-RNA and NS1-protein interactions that are fundamental to these processes, and highlight apparent strain-specific ways in which different NS1 proteins may act. In this regard, the contributions of certain NS1 functions to the pathogenicity of human and animal influenza A viruses are also discussed. Finally, we outline practical applications that future studies on NS1 may lead to, including the rational design and manufacture of influenza vaccines, the development of novel antiviral drugs, and the use of oncolytic influenza A viruses as potential anti-cancer agents.Publisher PDFPeer reviewe