Mutations in the NS2B and NS3 genes affect mouse neuroinvasiveness of a Western European field strain of tick-borne encephalitis virus

An attenuated strain (263) of the tick-borne encephalitis virus, isolated from field ticks, was either serially subcultured, 5 times in mice, or at 40 degrees C in PS cells, producing 2 independent strains, 263-m5 and 263-TR with identical genomes; both strains exhibited increased plaque size, neuroinvasiveness and temperature-resistance. Sequencing revealed two unique amino acid substitutions, one mapping close to the catalytic site of the viral protease. These observations imply that virus adaptation from ticks to mammals occurs by selection of pre-existing virulent variants from the quasispecies population rather than by the emergence of new random mutations. The significance of these observations is discussed. (c) 2008 Elsevier Inc. All rights reserved.

Cell entry by enveloped viruses: redox considerations for HIV and SARS-coronavirus

For enveloped viruses, genome entry into the target cell involves two major steps: virion binding to the cell-surface receptor and fusion of the virion and cell membranes. Virus-cell membrane fusion is mediated by the virus envelope complex, and its fusogenicity is the result of an active virus-cell interaction process that induces conformation changes within the envelope. For some viruses, such as influenza, exposure to an acidic milieu within the cell during the early steps of infection triggers the necessary structural changes. However, for other pathogens which are not exposed to such environmental stress, activation of fusogenicity can result from precise thiol/disulfide rearrangements mediated by either an endogenous redox autocatalytic isomerase or a cell-associated oxidoreductase. Study of the activation of HIV envelope fusogenicity has revealed new knowledge about how redox changes within a viral envelope trigger fusion. We discuss these findings and their implication for anti-HIV therapy. In addition, to compare and contrast the situation outlined for HIV with an enveloped virus that can fuse with the cell plasma membrane independent of the redox status of its envelope protein, we review parallel data obtained on SARS coronavirus entry.

Origin and evolution of 3'UTR of flaviviruses: long direct repeats as a basis for the formation of secondary structures and their significance for virus transmission

The 3' untranslated regions (3'UTRs) of flaviviruses are reviewed and analyzed in relation to short sequences conserved as direct repeats (DRs). Previously, alignments of the 3'UTRs have been constructed for three of the four recognized flavivirus groups, namely mosquito-borne, tick-borne, and nonclassified flaviviruses (MBFV, TBFV, and NCFV, respectively). This revealed (1) six long repeat sequences (LRSs) in the 3'UTR and open-reading frame (ORF) of the TBFV, (2) duplication of the 3'UTR of the NCFV by intramolecular recombination, and (3) the possibility of a common origin for all DRs within the MBFV. We have now extended this analysis and review it in the context of all previous published analyses. This has been achieved by constructing a robust alignment between all flaviviruses using the published DRs and secondary RNA structures as "anchors" to reveal additional homologies along the 3'UTR. This approach identified nucleotide regions within the MBFV, NKV (no-known vector viruses), and NCFV 3'UTRs that are homologous to different LRSs in the TBFV 3'UTR and ORF. The analysis revealed that some of the DRs and secondary RNA structures described individually within each flavivirus group share common evolutionary origins. The 3'UTR of flaviviruses, and possibly the ORF, therefore probably evolved through multiple duplication of an RNA domain, homologous to the LRS previously identified only in the TBFV. The short DRs in all virus groups appear to represent the evolutionary remnants of these domains rather than resulting from new duplications. The relevance of these flavivirus DRs to evolution, diversity, 3'UTR enhancer function, and virus transmission is reviewed.

NS1 proteins of avian influenza A viruses can act as antagonists of the human alpha/beta interferon response

Many viruses, including human influenza A virus, have developed strategies for counteracting the host type I interferon (IFN) response. We have explored whether avian influenza viruses were less capable of combating the type I IFN response in mammalian cells, as this might be a determinant of host range restriction. A panel of avian influenza viruses isolated between 1927 and 1997 was assembled. The selected viruses showed variation in their ability to activate the expression of a reporter gene under the control of the IFN-beta promoter and in the levels of IFN induced in mammalian cells. Surprisingly, the avian NS1 proteins expressed alone or in the genetic background of a human influenza virus controlled IFN-beta induction in a manner similar to the NS1 protein of human strains. There was no direct correlation between the IFN-beta induction and replication of avian influenza viruses in human A549 cells. Nevertheless, human cells deficient in the type I IFN system showed enhanced replication of the avian viruses studied, implying that the human type I IFN response limits avian influenza viruses and can contribute to host range restriction.

Alterations in receptor binding properties of recent human influenza H3N2 viruses are associated with reduced natural killer cell lysis of infected cells

Natural killer (NK) cell recognition of influenza virus-infected cells involves hemagglutinin (HA) binding to sialic acid (SA) on activating NK receptors. SA also acts as a receptor for the binding of influenza virus to its target host cells. The SA binding properties of H3N2 influenza viruses have been observed to change during circulation in humans: recent isolates are unable to agglutinate chicken red blood cells and show reduced affinity for synthetic glycopolymers representing SA-alpha-2,3-lactose (3'SL-PAA) and SA-alpha-2,6-N-acetyl lactosamine (6'SLN-PAA) carbohydrates. Here, NK lysis of cells infected with human H3N2 influenza viruses isolated between 1969 and 2003 was analyzed. Cells infected with recent isolates (1999 to 2003) were found to be lysed less effectively than cells infected with older isolates (1969 to 1996). This change occurred concurrently with the acquisition of two new potential glycosylation site motifs in RA. Deletion of the potential glycosylation site motif at 133 to 135 in HA1 from a recent isolate partially restored the agglutination phenotype to a recombinant virus, indicating that the HA-SA interaction is inhibited by the glycosylation modification. Deletion of either of the recently acquired potential glycosylation sites from HA led to increased NK lysis of cells infected with recombinant viruses carrying modified HA. These results indicate that alterations in RA glycosylation may affect NK cell recognition of influenza virus-infected cells in addition to virus binding to host cells.

Variation in the ability of human influenza A viruses to induce and inhibit the IFN-beta pathway

We investigated the ability of a selection of human influenza A viruses, including recent clinical isolates, to induce IFN-beta production in cultured cell lines. In contrast to the well-characterized laboratory strain A/PR/8/34, several, but not all, recent isolates of H3N2 viruses resulted in moderate IFN-beta stimulation. Through the generation of recombinant viruses, we were able to show that this is not due to a loss of the ability of the NS1 genes to suppress IFN-beta induction; indeed, the NS1 genes behaved similarly with respect to their abilities to block dsRNA signaling. Interestingly, replication of A/Sydney/5/97 virus was less Susceptible to pre-treatment with IFN-alpha than the other viruses. In contrast to the universal effect on dsRNA signaling, we noted differences in the effect of NS1 proteins on expression of interferon stimulated genes and also genes induced by a distinct pathway. The majority of NS1 proteins blocked expression From both IFN-dependent and TNF-dependent promoters by an apparent post-transcriptional mechanism. The NS1 gene of A/PR/8/34 NS1 did not confer these blocks. We noted striking differences in the Cellular localization of different influenza A virus NS1 proteins during infection, which might explain differences in biological activity. (C) 2005 Elsevier Inc. All rights reserved.

Characterization of recombinant influenza B viruses with key neuraminidase inhibitor resistance mutations

Objectives and methods: An influenza B virus plasmid-based rescue system was used to introduce site-specific mutations, previously observed in neuraminidase (NA) inhibitor-resistant viruses, into the NA protein of six recombinant viruses. Three mutations observed only among in vitro selected zanamivir-resistant influenza A mutants were introduced into the B/Beijing/1/87 virus NA protein, to change residue E116 to glycine, alanine or aspartic acid. Residue E116 was also mutated to valine, a mutation found in the clinic among oseltamivir-resistant viruses. An arginine to lysine change at position 291 (292 N2 numbering) mimicked that seen frequently in influenza A N2 clinical isolates resistant to oseltamivir. Similarly, an arginine to lysine change at position 149 (152 in N2 numbering) was made to reproduce the change found in the only reported zanamivir-resistant clinical isolate of influenza B virus. In vitro selection and prolonged treatment in the clinic leads to resistance pathways that require compensatory mutations in the haemagglutinin gene, but these appear not to be important for mutants isolated from immunocompetent patients. The reverse genetics system was therefore used to generate mutants containing only the NA mutation. Results and conclusions: With the exception of a virus containing the E116G mutation, mutant viruses were attenuated to different levels in comparison with wild-type virus. This attenuation was a result of altered NA activity or stability depending on the introduced mutation. Mutant viruses displayed increased resistance to zanamivir, oseltamivir and peramivir, with certain viruses displaying cross-resistance to all three drugs.

Changes in in vitro susceptibility of influenza A H3N2 viruses to a neuraminidase inhibitor drug during evolution in the human host

Objectives: Influenza A H3N2 viruses isolated recently have characteristic receptor binding properties that may decrease susceptibility to neuraminidase inhibitor drugs. A panel of clinical isolates and recombinant viruses generated by reverse genetics were characterized and tested for susceptibility to zanamivir. Methods: Plaque reduction assays and neuraminidase enzyme inhibition assays were used to assess susceptibility to zanamivir. Receptor binding properties of the viruses were characterized by differential agglutination of red blood cells (RBCs) from different species. Sequence analysis of the haemagglutinin (HA) and neuraminidase (NA) genes was carried out. Results: Characterization of a panel of H3N2 clinical isolates from 1968 to 2000 showed a gradual decrease in agglutination of chicken and guinea pig RBCs over time, although all isolates could agglutinate turkey RBCs equally. Sequence analysis of the HA and NA genes identified mutations in conserved residues of the HA1 receptor binding site, in particular Leu-226 --> Ile-226/Val-226, and modification of potential glycosylation site motifs. This may be indicative of changes in virus binding to sialic acid (SA) receptors in recent years. Although recent isolates had reduced susceptibility to zanamivir in MDCK cell based plaque reduction assays, no difference was found in an NA enzyme-inhibition assay. Assays with recombinant isogenic viruses showed that the recent HA, but not the NA, conferred reduced susceptibility to zanamivir. Conclusion: This study demonstrates that recent clinical isolates of influenza A H3N2 virus no longer agglutinate chicken RBCs, but despite significant receptor binding changes as a result of changes in HA, there was little variation in sensitivity of the NA to zanamivir.

Alterations in receptor binding properties of recent human influenza H3N2 viruses are associated with reduced natural killer cell lysis of infected cells

Natural killer (NK) cell recognition of influenza virus-infected cells involves hemagglutinin (HA) binding to sialic acid (SA) on activating NK receptors. SA also acts as a receptor for the binding of influenza virus to its target host cells. The SA binding properties of H3N2 influenza viruses have been observed to change during circulation in humans: recent isolates are unable to agglutinate chicken red blood cells and show reduced affinity for synthetic glycopolymers representing SA-alpha-2,3-lactose (3'SL-PAA) and SA-alpha-2,6-N-acetyl lactosamine (6'SLN-PAA) carbohydrates. Here, NK lysis of cells infected with human H3N2 influenza viruses isolated between 1969 and 2003 was analyzed. Cells infected with recent isolates (1999 to 2003) were found to be lysed less effectively than cells infected with older isolates (1969 to 1996). This change occurred concurrently with the acquisition of two new potential glycosylation site motifs in RA. Deletion of the potential glycosylation site motif at 133 to 135 in HA1 from a recent isolate partially restored the agglutination phenotype to a recombinant virus, indicating that the HA-SA interaction is inhibited by the glycosylation modification. Deletion of either of the recently acquired potential glycosylation sites from HA led to increased NK lysis of cells infected with recombinant viruses carrying modified HA. These results indicate that alterations in RA glycosylation may affect NK cell recognition of influenza virus-infected cells in addition to virus binding to host cells.

A method for estimating the turbulent kinetic energy dissipation rate from a vertically pointing Doppler lidar, and independent evaluation from balloon-borne in situ measurements

A method of estimating dissipation rates from a vertically pointing Doppler lidar with high temporal and spatial resolution has been evaluated by comparison with independent measurements derived from a balloon-borne sonic anemometer. This method utilizes the variance of the mean Doppler velocity from a number of sequential samples and requires an estimate of the horizontal wind speed. The noise contribution to the variance can be estimated from the observed signal-to-noise ratio and removed where appropriate. The relative size of the noise variance to the observed variance provides a measure of the confidence in the retrieval. Comparison with in situ dissipation rates derived from the balloon-borne sonic anemometer reveal that this particular Doppler lidar is capable of retrieving dissipation rates over a range of at least three orders of magnitude. This method is most suitable for retrieval of dissipation rates within the convective well-mixed boundary layer where the scales of motion that the Doppler lidar probes remain well within the inertial subrange. Caution must be applied when estimating dissipation rates in more quiescent conditions. For the particular Doppler lidar described here, the selection of suitably short integration times will permit this method to be applicable in such situations but at the expense of accuracy in the Doppler velocity estimates. The two case studies presented here suggest that, with profiles every 4 s, reliable estimates of ϵ can be derived to within at least an order of magnitude throughout almost all of the lowest 2 km and, in the convective boundary layer, to within 50%. Increasing the integration time for individual profiles to 30 s can improve the accuracy substantially but potentially confines retrievals to within the convective boundary layer. Therefore, optimization of certain instrument parameters may be required for specific implementations.

Persistent, symptomless, systemic, and seed-borne infection of lettuce by Botrytis cinerea

Experiments are presented which show that Botrytis cinerea, the cause of gray mould disease, is often present in symptomless lettuce plants as a systemic, endophytic, infection which may arise from seed. The fungus was isolated on selective media from surface sterilized sections of roots, stem pieces and leaf discs from symptomless plants grown in a conventional glasshouse and in a spore-free air-flow provided by an isolation propagator. The presence of B. cinerea was confirmed by immuno-labelling the tissues with the Botrytis-specific monoclonal antibody BC-12.CA4. As plants grew, infection spread from the roots to stems and leaves. Surface sterilization of seeds reduced the number of infected symptomless plants. Artificial infection of seedlings with dry conidia increased the rate of infection in some experiments. Selected isolates were genetically finger-printed using microsatellite loci. This confirmed systemic spread of the inoculating isolates but showed that other isolates were also present and that single plants hosted multiple isolates. This shows that B. cinerea commonly grows in lettuce plants as an endophyte, as has already been shown for Primula. If true for other hosts, the endophytic phase may be as important a component of the species population as the aggressive necrotrophic phase.

Replication enhancer elements within the open reading frame of tick-borne encephalitis virus and their evolution within the Flavivirus genus

We provide experimental evidence of a replication enhancer element (REE) within the capsid gene of tick-borne encephalitis virus (TBEV, genus Flavivirus). Thermodynamic and phylogenetic analyses predicted that the REE folds as a long stable stem–loop (designated SL6), conserved among all tick-borne flaviviruses (TBFV). Homologous sequences and potential base pairing were found in the corresponding regions of mosquito-borne flaviviruses, but not in more genetically distant flaviviruses. To investigate the role of SL6, nucleotide substitutions were introduced which changed a conserved hexanucleotide motif, the conformation of the terminal loop and the base-paired dsRNA stacking. Substitutions were made within a TBEV reverse genetic system and recovered mutants were compared for plaque morphology, single-step replication kinetics and cytopathic effect. The greatest phenotypic changes were observed in mutants with a destabilized stem. Point mutations in the conserved hexanucleotide motif of the terminal loop caused moderate virus attenuation. However, all mutants eventually reached the titre of wild-type virus late post-infection. Thus, although not essential for growth in tissue culture, the SL6 REE acts to up-regulate virus replication. We hypothesize that this modulatory role may be important for TBEV survival in nature, where the virus circulates by non-viraemic transmission between infected and non-infected ticks, during co-feeding on local rodents.

Changes in race-specific virulence in pseudomonas syringae pv. phaseolicola are Associated with a chimeric transposable element and rare deletion events in a plasmid-borne pathogenicity island

Virulence for bean and soybean is determined by effector genes in a plasmid-borne pathogenicity island (PAI) in race 7 strain 1449B of Pseudomonas syringae pv. phaseolicola. One of the effector genes, avrPphF, confers either pathogenicity, virulence, or avirulence depending on the plant host and is absent from races 2, 3, 4, 6, and 8 of this pathogen. Analysis of cosmid clones and comparison of DNA sequences showed that the absence of avrPphF from strain 1448A is due to deletion of a continuous 9.5-kb fragment. The remainder of the PAI is well conserved in strains 1448A and 1449B. The left junction of the deleted region consists of a chimeric transposable element generated from the fusion of homologs of IS1492 from Pseudomonas putida and IS1090 from Ralstonia eutropha. The borders of the deletion were conserved in 66 P. syringae pv. phaseolicola strains isolated in different countries and representing the five races lacking avrPphF. However, six strains isolated in Spain had a 10.5-kb deletion that extended 1 kb further from the right junction. The perfect conservation of the 28-nucleotide right repeat of the IS1090 homolog in the two deletion types and in the other 47 insertions of the IS1090 homolog in the 1448A genome strongly suggests that the avrPphF deletions were mediated by the activity of the chimeric mobile element. Our data strongly support a clonal origin for the races of P. syringae pv. phaseolicola lacking avrPphF.

Balloon-borne disposable radiometer for cloud detection

A low cost, disposable instrument for measuring solar radiation during meteorological balloon flights through cloud layers is described. Using a photodiode detector and low thermal drift signal conditioning circuitry, the device showed less than 1% drift for temperatures varied from +20 °C to −35 °C. The angular response to radiation, which declined less rapidly than the cosine of the angle between the incident radiation and normal incidence, is used for cloud detection exploiting the motion of the platform. Oriented upwards, the natural motion imposed by the balloon allows cloud and clear air to be distinguished by the absence of radiation variability within cloud, where the diffuse radiation present is isotropic. The optical method employed by the solar radiation instrument has also been demonstrated to provide higher resolution measurements of cloud boundaries than relative humidity measurements alone.

Animal disease and the limits of local knowledge: dealing with ticks and tick-borne diseases in South Africa

Some proponents of local knowledge, such as Sillitoe (2010), have expressed second thoughts about its capacity to effect development on the ‘revolutionary’ scale once predicted. Our argument in this article follows a similar route. Recent research into the management of livestock in South Africa makes clear that rural African livestock farmers experience uncertainty in relation to the control of stock diseases. State provision of veterinary services has been significantly reduced over the past decade. Both white and African livestock owners are to a greater extent left to their own devices. In some areas of animal disease management, African livestock owners have recourse to tried-and-tested local remedies, which are largely plant-based. But especially in the critical sphere of tick control, efficacious treatments are less evident, and livestock owners struggle to find adequate solutions to high tickloads. This is particularly important in South Africa in the early twenty-first century because land reform and the freedom to purchase land in the post-apartheid context affords African stockowners opportunities to expand livestock holdings. Our research suggests that the limits of local knowledge in dealing with ticks is one of the central problems faced by African livestock owners. We judge this not only in relation to efficacy but also the perceptions of livestock owners themselves. While confidence and practice varies, and there is increasing resort of chemical acaricides we were struck by the uncertainty of livestock owners over the best strategies.