Recombinant Subgroup B Human Respiratory Syncytial Virus Expressing Enhanced Green Fluorescent Protein Efficiently Replicates in Primary Human Cells and Is Virulent in Cotton Rats

Human respiratory syncytial virus (HRSV) is the most important viral cause of severe respiratory tract disease in infants. Two subgroups (A and B) have been identified, which cocirculate during, or alternate between, yearly epidemics and cause indistinguishable disease. Existing in vitro and in vivo models of HRSV focus almost exclusively on subgroup A viruses. Here, a recombinant (r) subgroup B virus (rHRSV(B05)) was generated based on a consensus genome sequence obtained directly from an unpassaged clinical specimen from a hospitalized infant. An additional transcription unit containing the gene encoding enhanced green fluorescent protein (EGFP) was introduced between the phosphoprotein and matrix genes (position 5) of the genome to generate rHRSV(B05)EGFP(5). The recombinant viruses replicated efficiently in both HEp-2 cells and in well-differentiated normal human bronchial cells grown at air-liquid interface. Intranasal infection of cotton rats (Sigmodon hispidus) resulted in high numbers of EGFP(+) cells in epithelia of the nasal septum and conchae. When administered in a relatively large inoculum volume, the virus also replicated efficiently in bronchiolar epithelial cells and spread extensively in both the upper and lower respiratory tracts. Virus replication was not observed in ciliated epithelial cells of the trachea. This is the first virulent rHRSV strain with the genetic composition of a currently circulating wild-type virus. In vivo tracking of infected cells by means of EGFP fluorescence in the absence of cytopathic changes increases the sensitivity of virus detection in HRSV pathogenesis studies.

IMPORTANCE

Virology as a discipline has depended on monitoring cytopathic effects following virus culture in vitro. However, wild-type viruses isolated from patients often do not cause significant changes to infected cells, necessitating blind passage. This can lead to genetic and phenotypic changes and the generation of high-titer, laboratory-adapted viruses with diminished virulence in animal models of disease. To address this, we determined the genome sequence of an unpassaged human respiratory syncytial virus from a sample obtained directly from an infected infant, assembled a molecular clone, and recovered a wild-type recombinant virus. Addition of a gene encoding enhanced green fluorescent protein allowed this wild-type virus to be tracked in primary human cells and living animals in the absence of significant cytopathic effects. Imaging of fluorescent cells proved to be a highly valuable tool for monitoring the spread of virus and may help improve assays for evaluating novel intervention strategies.

Characterization and Modeling of the Indoor Radio Channel at 868 MHz for a Mobile Bodyworn Wireless Personal Area Network

This letter reports the statistical characterization and modeling of the indoor radio channel for a mobile wireless personal area network operating at 868 MHz. Line of sight (LOS) and non-LOS conditions were considered for three environments: anechoic chamber, open office area and hallway. Overall, the Nakagami-m cdf best described fading for bodyworn operation in 60% of all measured channels in anechoic chamber and open office area environments. The Nakagami distribution was also found to provide a good description of Rician distributed channels which predominated in the hallway. Multipath played an important role in channel statistics with the mean recorded m value being reduced from 7.8 in the anechoic chamber to 1.3 in both the open office area and hallway.

Rarity and decline in bumblebees - A test of causes and correlates in the Irish fauna

Bees are believed to be in decline across many of the world's ecosystems. Recent studies on British bumblebees proposed alternative theories to explain declines. One study suggested that greater dietary specialization among the rarer bumblebee species makes them more susceptible to decline. A second study disputed this theory and found that declines in British bumblebees were correlated with the size of species' European ranges, leading to the suggestion that climate and habitat specialization may be better indicators of the risk of decline. Here we use a new and independent dataset based on Irish bumblebees to test the generality of these theories. We found that most of the same bumblebee species are declining across the British Isles, but that, within Ireland, a simple food-plant specialization model is inadequate to explain these declines. Furthermore, we found no evidence of a relationship between declines in Irish bumblebees and the size of species' European ranges. However, we demonstrate that the late emerging species have declined in Ireland (and in Britain), and that these species show a statistically significant westward shift to the extremity of their range, probably as a result of changing land use. Irish data support the finding that rare and declining bumblebees are later nesting species, associated with open grassy habitats. We suggest that the widespread replacement of hay with silage in the agricultural landscape, which results in earlier and more frequent mowing and a reduction in late summer wildflowers, has played a major role in bumblebee declines. (C) 2006 Elsevier Ltd. All rights reserved.

Absolute configuration of conformationally flexible cis-dihydrodiol metabolites by the method of confrontation of experimental and calculated electronic CD spectra and optical rotations

We have determined the absolute configurations of conformationally flexible cis-dihydrodiol metabolites (cis-1,2-dihydroxy-3,5-cyclohexadienes), bearing different substituents (e.g., Br, F, CF3, CN, Me) in 3- and 5-positions, by the method of confrontation of experimental and calculated electronic CD spectra and optical rotations. Convergent results were obtained by both methods in eight out of ten cases. For the difficult cases, where either conformer population and/or chiroptical properties (calculated rotational strengths of the long-wavelength Cotton effect or optical rotations) of contributing conformers remain inconclusive, the absolute configuration could still be correctly assigned based on one of the biased properties (either ECD or optical rotation). This approach appears well-suited for a broad spectrum of conformationally flexible chiral molecules.

Measurements, modeling and simulation of the off-body radio channel for the implementation of bodyworn antenna diversity at 868 MHz

This paper presents a systematic measurement campaign of diversity reception techniques for use in multiple-antenna wearable systems operating at 868 MHz. The experiments were performed using six time-synchronized bodyworn receivers and considered mobile off-body communications in an anechoic chamber, open office area and a hallway. The cross-correlation coefficient between the signal fading measured by bodyworn receivers was dependent upon the local environment and typically below 0.7. All received signal envelopes were combined in post-processing to study the potential benefits of implementing receiver diversity based upon selection combination, equal-gain and maximal-ratio combining. It is shown that, in an open office area, the 5.7 dB diversity gain obtained using a dual-branch bodyworn maximal-ratio diversity system may be further improved to 11.1 dB if a six-branch system was used. First-and second-order theoretical equations for diversity reception techniques operating in Nakagami fading conditions were used to model the postdetection combined envelopes. Maximum likelihood estimates of the Nakagami-parameter suggest that the fading conditions encountered in this study were generally less severe than Rayleigh. The paper also describes an algorithm that may be used to simulate the measured output of an M-branch diversity combiner operating in independent and identically-distributed Nakagami fading environments.

A time-domain approach to the analysis and modeling of on-body propagation characteristics using synchronized measurements at 2.45 GHz

Modeling of on-body propagation channels is of paramount importance to those wishing to evaluate radio channel performance for wearable devices in body area networks (BANs). Difficulties in modeling arise due to the highly variable channel conditions related to changes in the user's state and local environment. This study characterizes these influences by using time-series analysis to examine and model signal characteristics for on-body radio channels in user stationary and mobile scenarios in four different locations: anechoic chamber, open office area, hallway, and outdoor environment. Autocorrelation and cross-correlation functions are reported and shown to be dependent on body state and surroundings. Autoregressive (AR) transfer functions are used to perform time-series analysis and develop models for fading in various on-body links. Due to the non-Gaussian nature of the logarithmically transformed observed signal envelope in the majority of mobile user states, a simple method for reproducing the failing based on lognormal and Nakagami statistics is proposed. The validity of the AR models is evaluated using hypothesis testing, which is based on the Ljung-Box statistic, and the estimated distributional parameters of the simulator output compared with those from experimental results.

Channel characterization for single- and multiple-antenna wearable systems used for indoor body-to-body communications

In this paper, an analysis of radio channel characteristics for single- and multiple-antenna bodyworn systems for use in body-to-body communications is presented. The work was based on an extensive measurement campaign conducted at 2.45 GHz representative of an indoor sweep and search scenario for fire and rescue personnel. Using maximum-likelihood estimation in conjunction with the Akaike information criterion (AIC), five candidate probability distributions were investigated and from these the kappa - mu distribution was found to best describe small-scale fading observed in the body-to-body channels. Additional channel parameters such as autocorrelation and the cross-correlation coefficient between fading signal envelopes were also analyzed. Low cross correlation and small differences in mean signal levels between potential dual-branch diversity receivers suggested that the prospect of successfully implementing diversity in this type application is extremely good. Moreover, using selection combination, maximal ratio, and equal gain combining, up to 8.69-dB diversity gain can be made available when four spatially separated antennas are used at the receiver. Additional improvements in the combined envelopes through lower level crossing rates and fade durations at low signal levels were also observed.

An experimental investigation into the influence of user state and environment on fading characteristics in wireless body area networks at 2.45 GHz

Using seven strategically placed, time-synchronized bodyworn receivers covering the head, upper front and back torso, and the limbs, we have investigated the effect of user state: stationary or mobile and local environment: anechoic chamber, open office area and hallway upon first and second order statistics for on-body fading channels. Three candidate models were considered: Nakagami, Rice and lognormal. Using maximum likelihood estimation and the Akaike information criterion it was established that the Nakagami-m distribution best described small-scale fading for the majority of on-body channels over all the measurement scenarios. When the user was stationary, Nakagami-m parameters were found to be much greater than 1, irrespective of local surroundings. For mobile channels, Nakagami-m parameters significantly decreased, with channels in the open office area and hallway experiencing the worst fading conditions.

Characterization and modeling of on-body spatial diversity within indoor environments at 868 MHz

For the first time in the open literature we present a full characterization of the performance of receiver diversity for the on-body channels found in body area networks. The study involved three commonly encountered diversity combining schemes: selection combination (SC), maximal ratio combining (MRC) and equal gain combining (EGC). Measurements were conducted for both stationary and mobile user scenarios in an anechoic chamber and open office area environment. Achievable diversity gain for various on-body dual branch diversity receivers, consisting of horizontal and vertical spatially separated antennas, was found to be dependent upon transmitter-receive array separation, user state and level of multipath contribution from the local environment. The maximum diversity gain (6.4 dB) was observed for a horizontal two branch MRC combiner while the transmitter and receiver were on opposite sides of the body, and the user was mobile in the open office area. A novel statistical characterization of the fading experienced in on-body diversity channels is also performed using purposely derived first and second order diversity statistics for combiners operating in Nakagami fading.

An Antennas and Propagation Approach to Improving Physical Layer Performance in Wireless Body Area Networks

A combined antennas and propagation study has been undertaken with a view to directly improving link conditions for wireless body area networks. Using tissue-equivalent numerical and experimental phantoms representative of muscle tissue at 2.45 GHz, we show that the node to node [S-21] path gain performance of a new wearable integrated antenna (WIA) is up to 9 dB better than a conventional compact Printed-F antenna, both of which are suitable for integration with wireless node circuitry. Overall, the WIA performed extremely well with a measured radiation efficiency of 38% and an impedance bandwidth of 24%. Further benefits were also obtained using spatial diversity, with the WIA providing up to 7.7 dB of diversity gain for maximal ratio combining. The results also show that correlation was lower for a multipath environment leading to higher diversity gain. Furthermore, a diversity implementation with the new antenna gave up to 18 dB better performance in terms of mean power level and there was a significant improvement in level crossing rates and average fade durations when moving from a single-branch to a two-branch diversity system.

Costs of resistance: genetic correlations and potential trade-offs in an insect immune system

Theory predicts that natural selection will erode additive genetic variation in fitness-related traits. However, numerous studies have found considerable heritable variation in traits related to immune function, which should be closely linked to fitness. This could be due to trade-offs maintaining variation in these traits. We used the Egyptian cotton leafworm, Spodoptera littoralis, as a model system to examine the quantitative genetics of insect immune function. We estimated the heritabilities of several different measures of innate immunity and the genetic correlations between these immune traits and a number of life history traits. Our results provide the first evidence for a potential genetic trade-off within the insect immune system, with antibacterial activity (lysozyme-like) exhibiting a significant negative genetic correlation with haemocyte density, which itself is positively genetically correlated with both haemolymph phenoloxidase activity and cuticular melanization. We speculate on a potential trade-off between defence against parasites and predators, mediated by larval colour, and its role in maintaining genetic variation in traits under natural selection.

Widespread occurrence of the jellyfish Pelagia noctiluca in Irish coastal and shelf waters

An oceanic cruise (October 2007) revealed the widespread occurrence of Pelagia noctiluca in the NE Atlantic just prior to a major fish kill induced by P. noctiluca in Irish coastal waters.