bluuebunny/crossref_metadata_2025 huggingface dataset:
First Report of Brown Ring Patch Caused by Waitea circinata var. circinata on Poa annua in Virginia
Authors: Kammerer S. , Harmon P. F. , McDonald S. , Horvath B.
Published: March 2009

Brown ring patch was first described as a disease of cool-season turfgrass on creeping bentgrass (Agrostis palustris) (4) in Japan and later reported in California on annual bluegrass (Poa annua) (2). Brown ring patch symptoms were observed beginning in December 2007 through spring 2008 on 6 of 18 putting greens on a golf course in Reston, VA. Symptoms included yellow rings and patches of blighted turfgrass on the mixed stands of creeping bentgrass (A. palustris) and primarily annual bluegrass (Poa annua). Chlorosis and blight occurred predominantly on P. annua. A turfgrass sample was received from a consultant in April 2008, and disease severity on affected greens was estimated to be 40%. After incubating for 2 days in a moist chamber, Rhizoctonia-like aerial mycelia were observed. The pathogen was isolated on water agar and potato dextrose agar amended with thiophanate-methyl (100 mg/L), rifampicin (100 mg/L), and ampicillin (500 mg/L) from P. annua plants that had been surface sterilized with 70% ethanol for 15 s. Colony and sclerotia morphology were consistent with Waitea circinata var. circinata as previously described (2,4). Hyphae were stained with aniline blue and multiple nuclei were observed per cell. The teleomorph was not observed on plant material or in culture. Amplified fragments of rDNA including internal transcribed spacers from the isolate were amplified in three bacterial clones and sequenced bidirectionally (GenBank Accession Nos. FJ154894, FJ154895, and FJ154896) using primers ITS1/ITS4 (2,4). The consensus sequences matched, with 99% homology and 99% sequence overlap, isolate TRGC1.1 of W. circinata var. circinata (GenBank Accession No. DQ900586) (2). Annual bluegrass was not available for use in performing Koch's postulates, but previous studies have shown that W. circinata var. circinata is pathogenic to roughstalk bluegrass (P. trivialis) (1,3). Pots of P. trivialis cv. Cypress that were 1 week postemergence were inoculated with seven wheat grains that had been autoclaved and then infested with the isolate. Plants were incubated at 25°C in a sealed plastic bag with a moist paper towel on the bottom. Hyphae grew from the grains and colonized the grass. Individual plants began to turn chlorotic within 3 days, and more than 80% of the turf in pots was dead after 1 week. Control pots were inoculated with autoclaved wheat seed and showed no disease symptoms after 1 week. Inoculations were repeated twice more with the same results. W. circinata var. circinata was reisolated from affected plants in all replications of the test. To our knowledge, this is the first report of brown ring patch in Virginia. Additional research is needed to assess the prevalence and importance of this disease on golf course putting greens in Virginia.

References: (1) C. M. Chen et al. Plant Dis. 91:1687, 2007. (2) K. A. de la Cerda et al. Plant Dis. 91:791, 2007. (3) N. Flor et al. Plant Dis. 92:1586, 2008. (4) T. Toda et al. Plant Dis. 89:536, 2005.
First Report of Wheat streak mosaic virus on Wheat in New Zealand
Authors: Lebas B. S. M. , Ochoa-Corona F. M. , Alexander B. J. R. , Lister R. A. , Fletcher J. D. F. , Bithell S. L. , Burnip G. M.
Published: March 2009

In August of 2005, seeds of wheat (Triticum aestivum) breeding line 6065.3 tested positive for Wheat streak mosaic virus (WSMV; genus Tritimovirus) by a WSMV-specific reverse transcription (RT)-PCR assay (2). The sequence of the 200-bp amplicon (GenBank Accession No. FJ434246) was 99% identical with WSMV isolates from Turkey and the United States (GenBank Accession Nos. AF454455 and AF057533) and 96 to 97% identical to isolates from Australia (GenBank Accession Nos. DQ888801 to DQ888805 and DQ462279), which belong to the subclade D (1). As a result, an extensive survey of three cereal experimental trials and 105 commercial wheat crops grown on the South Island of New Zealand was conducted during the 2005–2006 summer to determine the distribution of WSMV. Wherever possible, only symptomatic plants were collected. Symptoms on wheat leaf samples ranged from very mild mosaic to symptomless. In total, 591 leaf samples suspected to be symptomatic were tested for WSMV by a double-antibody sandwich (DAS)-ELISA (DSMZ, Braunschweig, Germany). Of the 591 symptomatic samples, 81 tested positive. ELISA results were confirmed by RT-PCR with novel forward (WSMV-F1; 5′-TTGAGGATTTGGAGGAAGGT-3′) and reverse (WSMV-R1; 5′-GGATGTTGCCGAGTTGATTT-3′) primers designed to amplify a 391-nt fragment encoding a region of the P3 and CI proteins. Total RNA was extracted from the 81 ELISA-positive leaf samples using the Plant RNeasy Kit (Qiagen Inc., Chatsworth, CA). The expected size fragment was amplified from each of the 81 ELISA-positive samples. The positive samples represent 30 of 56 wheat cultivars (54%) collected from 28 of 108 sites (26%) sampled in the growing regions from mid-Canterbury to North Otago. These results suggest that WSMV is widespread in New Zealand both geographically and within cultivars. WSMV is transmitted by the wheat curl mite (Aceria tosichella) (3), which had not been detected in New Zealand despite repeated and targeted surveys. WSMV is of great economic importance in some countries, where the disease has been reported to cause total yield loss (3). Although WSMV is transmitted by seeds at low rates (0.1 to 0.2%) (4), it is the most likely explanation of the spread of the disease in New Zealand.

References: (1) G. I. Dwyer et al. Plant Dis. 91:164, 2007. (2) R. French and N. L. Robertson. J. Virol. Methods 49:93, 1994. (3) R. French and D. C. Stenger. Descriptions of Plant Viruses. Online publication. No. 393, 2002. (4) R. A. C. Jones et al. Plant Dis. 89:1048, 2005.
First Report of Garlic virus B and Garlic virus D in Garlic in the Pacific Northwest
Authors: Gieck S. L. , Hamm P. B. , David N. L. , Pappu H. R.
Published: March 2009

With the recent report of several viruses infecting garlic (Allium sativum L.) grown in the Pacific Northwest (1–3), studies were initiated on cloves planted in the fall of 2006 to determine the presence of additional viruses infecting plants exhibiting mosaic and/or chlorotic leaves. Cloves from symptomatic plants of the cultivar ‘Early’ from two seed production fields in Benton County, WA and two seed production fields in Morrow County, OR were tested by two-step reverse transcription (RT)-PCR using primers specific to the coat protein (CP) of the allexiviruses (4), since garlic infected with this group had similar symptoms in Asia and South America (4). Of the 87 cloves tested, 84 were positive, and four representative samples of the RT-PCR amplicons from each location were cloned and sequenced. Sequence comparisons indicated that the cloves from both locations were infected with Garlic virus D (GarV-D), also known as Japanese garlic virus (JGV), since they shared 98% identity with known isolates (GenBank Accession Nos. L388922.1, AF519572.1, and AB010303.1). In addition, sequences of isolates from the Oregon cloves shared a 96% identity with a known isolate of Garlic virus B (GarV-B; GenBank Accession No. AF543829.1). Because no antiserum specific to these viruses was available, primers specific to the CP genes of GarV-D (JGV-F2/JGV-R2 5′-GCTCACTCRGATGTGTTAGC-3′ and 5′-CGCGTGGACATAAGTTGTTG-3′) and GarV-B (GVB-F1/GVB-R2 5′-GAGGAGAACTAACGCCACAC-3′ and 5′-ACGACCTAGCTTCCTACTTG-3′) were designed and the cloves were retested by RT-PCR using these virus-specific primers. With the GarV-D specific primers, 98 and 63% of the cloves were positive from Washington and Oregon, respectively, and 52% of the cloves from Oregon were positive using the GarV-B specific primers. None of the cloves tested from Washington were positive for GarV-B. The identity of the amplicons was verified by cloning and sequencing (GarV-D, GenBank Accession No. FJ643476; GarV-B, GenBank Accession No. FJ643475). Incidence of the two viruses differed between Oregon and Washington was likely due to the expansion of the seed lots in two different locations (California and Nevada) prior to planting in 2006. With such high infection rates, studies should be conducted to determine the impact of these viruses on yield when plants are singly infected as well as in combination with the other viruses known to infect garlic in this region. These and the other viruses (1) are likely to impact yield. To our knowledge, this is the first report of GarV-D (JGV) and GarV-B in garlic in the Pacific Northwest.

References: (1) S. L. Gieck et al. Plant Dis. 91:461, 2007. (2) H. R. Pappu et al. Plant Dis. 89:205, 2005 (3) H. R. Pappu et al. Online publication. doi:10.1094/PHP-2008-0919-01-RS. Plant Health Progress, 2008. (4) T. Tsuneyoshi et al. Phytopathology 86:253, 1996.
Accelerated Degradation of Metam-Sodium in Soil and Consequences for Root-Disease Management
Authors: Triky-Dotan Shachaf , Austerweil Miriam , Steiner Bracha , Peretz-Alon Yitzhak , Katan Jaacov , Gamliel Abraham
Published: March 2009

We studied the development of accelerated degradation (AD) of methyl isothiocyanate (MITC) following repeated applications of its parent compound, metam-sodium (MS). Laboratory studies and four sets of field experiments were conducted during 2002–04 in three commercial fields in Israel. Repeated applications of MS to the three soils in the laboratory under controlled conditions demonstrated AD of MITC in some soils. In a peanut field, MS significantly reduced the incidence of Pythium pod rot and improved pod quality after a single application but its effectiveness was greatly reduced after two applications. In a second experiment, MS was significantly effective after a single application in controlling Verticillium wilt in potato but its efficacy diminished after three consecutive applications. In an additional experiment, fumigation with MS following single or double applications was more effective in reducing Verticillium wilt severity of potato compared with triple applications. Soils which did not develop AD of MITC were also recorded. Preplant MS fumigation of melon fields was effective at reducing sudden wilt following a single and two consecutive applications. Our study shows that development of AD of MITC might occur following repeated applications of MS in commercial fields. The data on MITC dissipation in soil following repeated MS applications under controlled conditions indicate the chemical's potential loss of activity under regular agricultural practices and the need for a management strategy to prevent such a development.
Evaluation of Molecular Markers for Phytophthora ramorum Detection and Identification: Testing for Specificity Using a Standardized Library of Isolates
Authors: Martin F. N. , Coffey M. D. , Zeller K. , Hamelin R. C. , Tooley P. , Garbelotto M. , Hughes K. J. D. , Kubisiak T. , Bilodeau G. J. , Levy L. , Blomquist C. , Berger P. H.
Published: March 2009

Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as β-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately identified P. ramorum in blind evaluations of 60 field samples that included examples of plant infection by 11 other Phytophthora spp. The SSCP analysis identified eight of these species, with three identified to a species group.
Highlighting Features of Spatiotemporal Spread of Powdery Mildew Epidemics in the Vineyard Using Statistical Modeling on Field Experimental Data
Authors: Calonnec A. , Cartolaro P. , Chadœuf J.
Published: March 2009

A greater understanding of the development of powdery mildew epidemics on vines would improve disease management by making assessments of the risk of invasion more accurate. We characterized the spatiotemporal spread of epidemics in the vineyard, quantified their variability, and identified the factors responsible for it. We described changes in the probability of infection of a leaf in a plot over time and as a function of distance from a source of disease. Logistic models were fitted to field data from artificially inoculated plots. The velocity of spread decreased along the row and increased in the direction of the prevailing winds. The rate of progression over time was plot dependent, and the velocity was dependent on the vigor of the vine (0.1 to 0.27 m day–1 in areas of moderate vigor and 1.1 m day–1 in areas of high vigor). When applied to a larger plot with natural primary foci, the spatiotemporal logistic model showed that the velocity and the slope of the gradient in space depended on the foci; however, the velocity remained in the same range. During the period of highest susceptibility for grape, the probability of a leaf becoming infected increased from 2.5 to 13%. Our logistic model was able to predict changes in disease over time of its extension within the plot; however, the crop heterogeneity prevented prediction of variability of disease at the vine scale.
Molecular Characterization ofCitrus tatter leaf virusHistorically Associated with Meyer Lemon Trees: Complete Genome Sequence and Development of Biologically Active In Vitro Transcripts
Authors: Tatineni Satyanarayana , Afunian Mohammad R. , Hilf Mark E. , Gowda Siddarame , Dawson William O. , Garnsey Stephen M.
Published: March 2009

Citrus tatter leaf virus isolated from Meyer lemon trees (CTLV-ML) from California and Florida induces bud union incompatibility of citrus trees grafted on the widely used trifoliate and trifoliate hybrid rootstocks. The complete genome sequence of CTLV-ML was determined to be 6,495 nucleotides (nts), with two overlapping open reading frames (ORFs) and a poly (A) tail at the 3′ end. The genome organization is similar to other capilloviruses, with ORF1 (nts 37 to 6,354) encoding a putative 242-kDa polyprotein which contains replication-associated domains plus a coat protein (CP), and ORF2 (nts 4,788 to 5,750), which is located within ORF1 in a different reading frame and encodes a putative movement protein. Although the proteins encoded by CTLV-ML possesses 84 to 96% amino acid sequence identity with strains of Apple stem grooving virus (ASGV), we observed two strikingly different regions in ORF1: variable region I (amino acids 532 to 570) and variable region II (amino acids 1,583 to 1,868), with only 15 to 18 and 56 to 62% identities, respectively, with the corresponding regions of ASGV strains. Conditions for a herbaceous systemic assay host were optimized in which the wild-type virus induced systemic infection in Phaseolus vulgaris cv. Light Red Kidney (LRK) bean plants at 19 or 22°C but not at higher temperatures. In vitro transcripts generated from full-length cDNA clones induced systemic symptoms on LRK bean plants similar to that of the wild-type virus. Replication of the recombinant virus was confirmed by hybridization of a 5′ positive-stranded RNA-specific probe to a genome-sized RNA and by reverse-transcription polymerase chain reaction.
Quantifying Wheat Yield Across the Field as a Function of Wheat Streak Mosaic Intensity: A State Space Approach
Authors: Workneh F. , Jones D. C. , Rush C. M.
Published: March 2009

Wheat streak mosaic virus (WSMV), vectored by the wheat curl mite Aceria tosichella, is one of the major limiting factors in wheat production in the Texas Panhandle. The mites are blown by wind into wheat fields from nearby volunteer wheat fields or fields supporting vegetation which harbor virulent mites. Consequently, gradients of wheat streak severity are often observed stretching from the edges of wheat fields into the center or beyond. To describe the magnitude of the spatial relationships between grain yield and wheat streak intensity across the field, studies were conducted in 2006 and 2007 in three infected fields. Wheat streak severity was quantified with reflectance measurements (remote sensing) at 555-nm wave length using a hand-held radiometer. Measurements were taken in several equally spaced 1 m2 locations along a total of eight transects and grain yield was assessed from a 0.8 m2 area of each location. State space analysis was used to describe the relationships in which yield data and reflectance values were used as dependent and explanatory variables, respectively. A structural time series model was formulated as a state space model where the unobserved components were modeled explicitly. In the analysis the state of yield at current location (d) was related to the state of wheat streak intensity either at current locations (d) or lagged locations with autoregressive values of the first order (d–1) or greater. There were significant cross-correlations between yield and wheat streak intensity up to distances of 150 m (P ≤ 0.05). Grain yield at the current position was significantly correlated with reflectance values at the same location and/or at lagged locations. The spatial aspects of the yield-reflectance relationships were best described by state space models with stochastic trends without slopes or deterministic trends with or without slopes. The models correctly predicted almost all of the observed yield values as a function of wheat streak intensity across the field within the 95% confidence interval. Results obtained in this study suggest that state space methodology can be a powerful tool in the study of plant disease spread as a function of other variables.
Predicting the Dynamics of Ascospore Maturation of Venturia pirina Based on Environmental Factors
Authors: Rossi V. , Salinari F. , Pattori E. , Giosuè, S. , Bugiani R.
Published: March 2009

Airborne ascospores of Venturia pirina were trapped at two sites in northern Italy in 2002 to 2008. The cumulative proportion of ascospores trapped at each discharge was regressed against the physiological time. The best fit (R2 = 0.90, standard error of estimates [SEest] = 0.11) was obtained using a Gompertz equation and the degree-days (>0°C) accumulated after the day on which the first ascospore of the season was trapped (biofix day), but only for the days with ≥0.2 mm rain or ≤4 hPa vapor pressure deficit (DDwet). This Italian model performed better than the models developed in Oregon, United States (R2 = 0.69, SEest = 0.16) or Victoria, Australia (R2 = 0.74, SEest = 0.18), which consider only the effect of temperature. When the Italian model was evaluated against data not used in its elaboration, it accurately predicted ascospore maturation (R2 = 0.92, SEest = 0.10). A logistic regression model was also developed to estimate the biofix for initiating the accumulation of degree-days (biofix model). The probability of the first ascospore discharge of the season increased as DDwet (calculated from 1 January) increased. Based on this model, there is low probability of the first ascospore discharge when DDwet ≤268.5 (P = 0.03) and high probability (P = 0.83) of discharge on the first day with >0.2 mm rain after such a DDwet threshold.
Carriage of both the fnbA and fnbB genes and growth at 37 °C promote FnBP-mediated biofilm development in meticillin-resistant Staphylococcus aureus clinical isolates
Authors: O'Neill Eoghan , Humphreys Hilary , O'Gara James P.
Published: March 2009

TheStaphylococcus aureusFnBPA and FnBPB proteins promote acid-induced biofilm accumulation. Meticillin-resistantS. aureus(MRSA) isolates from device-related infections with bothfnbAandfnbBproduced significantly more biofilm than isolates with either gene alone. Under mildly acidic growth conditions, FnBP-mediated biofilm andfnbAandfnbBtranscript levels were substantially higher during growth at 37 °C than at 30 °C. Thus, in addition to a lowered pH, carriage of bothfnbAandfnbBand growth at 37 °C promote MRSA biofilm development, further supporting a role for the FnBPA and FnBPB surface proteins in the pathogenesis of MRSA device-related infections.
Comparison of various antimicrobial agents as catheter lock solutions: preference for ethanol in eradication of coagulase-negative staphylococcal biofilms
Authors: Qu Yue , Istivan Taghrid S. , Daley Andrew J. , Rouch Duncan A. , Deighton Margaret A.
Published: March 2009

Coagulase-negative staphylococci (CoNS) are the main causative agents of bacteraemia in infants managed in neonatal intensive care units (NICUs). Intraluminal colonization of long-term central venous catheters by these bacteria and subsequent biofilm formation are the prerequisites of the bloodstream infections acquired in NICUs. The catheter lock technique has been used to treat catheter colonization; however, the optimum choice of antimicrobial agents and their corresponding concentrations and exposure times have not been determined. The effectiveness of catheter lock solutions (CLSs) was assessed by determining the minimal biofilm eradication concentration of antimicrobial agents against CoNS biofilms. Five conventional antibiotics (oxacillin, gentamicin, vancomycin, ciprofloxacin and rifampicin) alone or in combination, as well as ethanol, were evaluated. Ethanol was found to be superior to all of these conventional antibiotics when used as a CLS. A time–kill study and confocal laser scanning microscopy revealed that exposure to 40 % ethanol for 1 h was sufficient to kill CoNS biofilm cells. To our knowledge, this is the first in vitro study to provide solid evidence to support the rationale of using ethanol at low concentrations for a short time as a CLS, instead of using conventional antibiotics at high concentrations for a long period to treat catheter-related bloodstream infections.
Molecular characterization of the stomach microbiota in patients with gastric cancer and in controls
Authors: Dicksved Johan , Lindberg Mathilda , Rosenquist Magnus , Enroth Helena , Jansson Janet K. , Engstrand Lars
Published: March 2009

Persistent infection of the gastric mucosa by Helicobacter pylori can initiate an inflammatory cascade that progresses into atrophic gastritis, a condition associated with reduced capacity for secretion of gastric acid and an increased risk of developing gastric cancer. The role of H. pylori as an initiator of inflammation is evident but the mechanism for development into gastric cancer has not yet been proven. A reduced capacity for gastric acid secretion allows survival and proliferation of other microbes that normally are killed by the acidic environment. It has been postulated that some of these species may be involved in the development of gastric cancer; however, their identities are poorly defined. In this study, the gastric microbiota from ten patients with gastric cancer was characterized and compared with that from five dyspeptic controls using the molecular profiling approach terminal restriction fragment length polymorphism (T-RFLP), in combination with 16S rRNA gene cloning and sequencing. T-RFLP analysis revealed a complex bacterial community in the cancer patients that was not significantly different from that in the controls. Sequencing of 140 clones revealed 102 phylotypes, with representatives from five bacterial phyla (Firmicutes, Bacteroidetes, Proteobacteria, Actinobacteria and Fusobacteria). The data revealed a relatively low abundance of H. pylori and showed that the gastric cancer microbiota was instead dominated by different species of the genera Streptococcus, Lactobacillus, Veillonella and Prevotella. The respective role of these species in development of gastric cancer remains to be determined.
Development of imipenem resistance in an Aeromonas veronii biovar sobria clinical isolate recovered from a patient with cholangitis
Authors: Sánchez-Céspedes Javier , Figueras Maria José , Aspiroz Carmen , Aldea Maria José , Toledo Miguel , Alperí Anabel , Marco Francesc , Vila Jordi
Published: March 2009

Several imipenem-susceptible and -resistantAeromonas veroniibiovar sobria isolates with different morphologies and antimicrobial susceptibilities recovered from bile samples of a patient with cholangitis were analysed. These isolates belonged to the same clone and the imipenem-resistant strains showed overexpression of theimiSgene, encoding a chromosomal carbapenemase. These results should make clinicians aware of the possible emergence of multidrug-resistantA. veroniibiovar sobria, perhaps as a consequence of previous treatment of a urinary tract infection with amoxicillin plus clavulanic acid.
Multiple-hit inhibition of infection by defective interfering particles
Authors: Stauffer Thompson Kristen A. , Rempala Grzegorz A. , Yin John
Published: March 2009

Defective interfering particles (DIPs) are virus-like particles that arise during virus growth, fail to grow in the absence of virus, and replicate at the expense of virus during co-infections. The inhibitory effects of DIPs on virus growth are well established, but little is known about how DIPs influence their own growth. Here vesicular stomatitis virus (VSV) and its DIPs were used to co-infect BHK cells, and the effect of DIP dose on virus and DIP production was measured using a yield-reduction assay. The resulting dose–response data were used to fit and evaluate mathematical models that employed different assumptions. Our analysis supports a multiple-hit process where DIPs inhibit or promote virus and DIP production, depending on dose. Specifically, three regimes of co-infection were apparent: (i) low DIP – where both virus and DIPs are amplified, (ii) medium DIP – where amplification of both virus and DIPs is inhibited, and (iii) high DIP – with limited recovery of virus production and further inhibition of DIP growth. In addition, serial-passage infections enabled us to estimate the frequency of de novo DIP generation during virus amplification. Our combined experiments and models provide a means to understand better how DIPs quantitatively impact the growth of viruses and the spread of their infections.
ERK MAP kinase-activated Arf6 trafficking directs coxsackievirus type B3 into an unproductive compartment during virus host-cell entry
Authors: Marchant David , Sall Alhousseynou , Si Xiaoning , Abraham Thomas , Wu Winnie , Luo Zongshu , Petersen Tamar , Hegele Richard G. , McManus Bruce M.
Published: March 2009

Clathrin- and caveolae-mediated endocytosis have been implicated in the productive entry of many viruses into host cells. ADP-ribosylation factor 6 (Arf6)-dependent endocytosis is another endocytosis pathway that traffics from the cell surface and it is the only Arf that traffics at the plasma membrane. However, little is known about Arf6-dependent trafficking during virus entry. This study showed that coxsackievirus type B3 (CVB3) associated with decay-accelerating factor in non-polarized HeLa cells can be redirected into non-productive compartments by Arf6-dependent internalization, thus restricting infection. Overexpression of wild-type (WT) and constitutively active (CA) Arf6 in HeLa cells resulted in a 2.3- and 3.6-fold decrease in infection, respectively. A dominant-negative inhibitor of Arf6 recovered restriction of infection by WT-Arf6 and CA-Arf6. RNA interference of endogenous Arf6 resulted in a 3.3-fold increase in CVB3 titre in HeLa cells. It was shown that coxsackie–adenovirus receptor (CAR) ligation by virus or CAR-specific antibody could activate extracellular signal-regulated kinase (ERK) of the mitogen-activated protein kinase family and lead to Arf6-mediated viral restriction. In the absence of ERK activation, CVB3 internalization into early endosomes was inhibited and subsequent infection was reduced, but Arf6-mediated restriction was also abolished. In conclusion, receptor-mediated signalling enhances CVB3 entry whilst also activating non-productive pathways of virus entry; thus, virus infection is an equilibrium of productive and non-productive pathways of entry.
Alpha interferon as an adenovirus-vectored vaccine adjuvant and antiviral in Venezuelan equine encephalitis virus infection
Authors: O'Brien Lyn , Perkins Stuart , Williams Amanda , Eastaugh Lin , Phelps Amanda , Wu Josh , Phillpotts Robert
Published: March 2009

There are no widely available vaccines or antiviral drugs capable of protecting against infection with Venezuelan equine encephalitis virus (VEEV), although an adenovirus vector expressing VEEV structural proteins protects mice from challenge with VEEV and is potentially a vaccine suitable for human use. This work examines whether alpha interferon (IFN-α) could act as an adjuvant for the adenovirus-based vaccine. IFN-αwas either expressed by a plasmid linked to the adenovirus vaccine or encoded by a separate adenovirus vector administered as a mixture with the vaccine. In contrast to previous reports with other vaccines, the presence of IFN-αreduced the antibody response to VEEV. When IFN-αwas encoded by adenovirus, the lack of a VEEV-specific response was accompanied by an increase in the immune response to the adenovirus vector. IFN-αalso plays a direct role in defence against virus infection, inducing the expression of a large number of antiviral proteins. Adenovirus-delivered IFN-αprotected mice from VEEV disease when administered 24 h prior to challenge, but not when administered 6 h post-challenge, suggesting that up to 24 h is required for the development of the IFN-mediated antiviral response.
Lineages of varicella-zoster virus
Authors: McGeoch Duncan J.
Published: March 2009

Relationships among varicella-zoster virus (VZV; Human herpesvirus 3) genome sequences were examined to evaluate descent of strains, structures of lineages and incidence of recombination events. Eighteen complete, published genome sequences were aligned and 494 single nucleotide polymorphisms (SNPs) extracted, each as two alleles. At 281 SNPs, a single sequence differed from all the others. Distributions of the remaining 213 SNPs indicated that the sequences fell into five groups, which coincided with previously recognized phylogenetic groupings, termed E1, E2, J, M1 and M2. The 213-SNP set was divisible into 104 SNPs that were specific to a single group, and 109 cross-group SNPs that defined relationships among groups. This last set was evaluated by criteria of continuities in relationships between groups and breaks in such patterns, to identify crossover points and ascribe them to lineages. For the 99 cross-group SNPs in the genome's long unique region, it was seen that the E2 and M2 groups were almost completely distinct in their SNP alleles, and the E1 group was derived from a recombinant of E2 and M2. A valid phylogenetic tree could thus be constructed for the four E2 and two M2 strains. There was no substantive evidence for recombination within the E2 group or the E1 group (ten strains). The J and M1 groups each contained only one strain, and both were interpreted as having substantial distinct histories plus possible recombinant elements from the E2 and M2 lineages. The view of VZV recombination and phylogeny reached represents a major clarification of deep relationships among VZV lineages.
Molecular characterization of a novel Ljungan virus (Parechovirus; Picornaviridae) reveals a fourth genotype and indicates ancestral recombination
Authors: Tolf Conny , Gullberg Maria , Johansson E. Susanne , Tesh Robert B. , Andersson Björn , Lindberg A. Michael
Published: March 2009

Ljungan virus (LV) was discovered 20 years ago in Swedish bank voles (Myodes glareolus, previously referred to as Clethrionomys glareolus) during the search for an infectious agent causing lethal myocarditis in young athletes. To date, the genomes of four LV isolates, including the prototype 87-012 strain, have been characterized. Three of these LV strains were isolated from bank voles trapped in Sweden. Sequence analysis of an American virus (M1146), isolated from a montane vole (Microtus montanus) in western USA, indicates that this strain represents a genotype that is different from the Swedish strains. Here, we present genomic analyses of a fifth LV strain (64-7855) isolated from a southern red-backed vole (Myodes gapperi) trapped during arbovirus studies in New York state in the north-eastern USA in the 1960s. Sequence analysis of the 64-7855 genome showed an LV-like genome organization and sequence similarity to other LV strains. Genetic and phylogenetic analyses of the evolutionary relationship between the 64-7855 strain and other viruses within the family Picornaviridae, including previously published LV strains, demonstrated that the 64-7855 strain constitutes a new genotype within the LV species. Analyses also showed that different regions of the 64-7855 genome have different phylogenetic relationships with other LV strains, indicating that previous recombination events have been involved in the evolution of this virus.
Expression of hepatitis C virus (HCV) structural proteins in trans facilitates encapsidation and transmission of HCV subgenomic RNA
Authors: Adair Richard , Patel Arvind H. , Corless Lynsey , Griffin Stephen , Rowlands David J. , McCormick Christopher J.
Published: March 2009

A characteristic of many positive-strand RNA viruses is that, whilst replication of the viral genome is dependent on the expression of the majority of non-structural proteinsin cis, virus particle formation can occur when most or all of the structural proteins are co-expressedin trans. Making use of a recently identified hepatitis C virus (HCV) isolate (JFH1) that can be propagated in tissue culture, this study sought to establish whether this is also the case for hepaciviruses. Stable cell lines containing one of two bicistronic replicons derived from the JFH1 isolate were generated that expressed non-structural proteins NS3–5B or NS2–5B. Release and transmission of these replicons to naïve Huh7 cells could then be demonstrated when baculovirus transduction was used to express the HCV proteins absent from the subgenomic replicons. Transmission could be blocked by a neutralizing antibody targeted at the E2 envelope protein, consistent with this phenomenon occurring viatrans-encapsidation of replicon RNA into virus-like particles. Transmission was also dependent on expression of NS2, which was most effective at promoting virus particle formation when expressedin cison the replicon RNA compared within transvia baculovirus delivery. Density gradient analysis of the particles revealed the presence of a broad infectious peak between 1.06 and 1.11 g ml−1, comparable to that seen when propagating full-length virus in tissue culture. In summary, thetrans-encapsidation system described offers a complementary and safer approach to study HCV particle formation and transmission in tissue culture.
Acute-phase CD4+ T-cell proliferation and CD152 upregulation predict set-point virus replication in vaccinated simian–human immunodeficiency virus strain 89.6p-infected macaques
Authors: Koopman Gerrit , Mortier Daniella , Hofman Sam , Koutsoukos Marguerite , Bogers Willy M. J. M. , Wahren Britta , Voss Gerald , Heeney Jonathan L.
Published: March 2009

Human immunodeficiency virus (HIV) infection in humans and simian immunodeficiency virus (SIV) infection in macaques are accompanied by a combined early loss of CCR5 (CD195)-expressing CD4+memory T cells, loss of T-helper function and T-cell hyperactivation, which have all been associated with development of high virus load and disease progression. Here, a cohort of vaccinated simian–human immunodeficiency virus strain 89.6p (SHIV89.6p)-infected rhesus macaques, where preferential depletion of these memory T-cell subsets does not take place and CD4+T cells are relatively well maintained, was used to study the role of hyperactivation as an independent factor in the establishment of set-point virus load. In the acute phase of the infection, a transient loss of CD4+T cells, as well as strong increases in expression of proliferation and activation markers on CD4+and CD8+T cells, together with CD152 expression on CD4+T cells, were observed. Peak expression levels of these markers on CD4+T cells, but not on CD8+T cells, were correlated with high virus replication in the chronic phase of the infection. In addition, the peak expression level of these markers was correlated inversely with acute-phase, but not chronic-phase, HIV/SIV-specific gamma interferon responses. These data highlight a central role for an acute but transient CD4 decrease, as well as CD4+T-cell activation, as independent factors for prediction of set-point levels of virus replication.
Detection of typical and atypical bovine spongiform encephalopathy and scrapie prion strains by prion protein motif-grafted antibodies
Authors: Martucci Francesca , Acutis Pierluigi , Mazza Maria , Nodari Sabrina , Colussi Silvia , Corona Cristiano , Barocci Simone , Gabrielli Armando , Caramelli Maria , Casalone Cristina , Moroncini Gianluca
Published: March 2009

To evaluate further the reactivity of prion-specific monoclonal antibodies containing the 89–112 or 136–158 prion protein (PrP) polypeptides, immunoprecipitations were performed on brain extracts from Italian bovines, sheep and goats with transmissible spongiform encephalopathies. No binding of IgG 89–112 or IgG 136–158 to PrP in normal brain extracts was detected. Conversely, both reagents immunoprecipitated PrP from bovine and bovine amyloidotic spongiform encephalopathies, and from typical and atypical scrapie brain extracts. The immunoprecipitated PrP bands mirrored the Western blot (WB) profile of the different prion strains, indicating universal affinity of two independent PrP regions for disease-associated PrP conformers regardless of species source and strain properties. Immunoprecipitation with motif-grafted antibodies increased the sensitivity of conventional detection methods based on centrifugation followed by WB, which was confirmed by assay of diluted samples using both methods. These reagents or derivative molecules may thus find broad applications in prion detection and research.
High prevalence of amantadine resistance among circulating European porcine influenza A viruses
Authors: Krumbholz Andi , Schmidtke Michaela , Bergmann Silke , Motzke Susann , Bauer Katja , Stech Jürgen , Dürrwald Ralf , Wutzler Peter , Zell Roland
Published: March 2009

Genetic analysis of the M2 sequence of European porcine influenza A viruses reveals a high prevalence of amantadine resistance due to the substitution of serine 31 by asparagine in all three circulating subtypes, H1N1, H3N2 and H1N2. The M segment of all resistant strains belongs to a single genetic lineage. Whereas the first amantadine-resistant porcine strain was isolated in 1989, isolation of the last amantadine-susceptible strain dates to 1987, suggesting a displacement of amantadine-susceptible viruses by resistant strains soon after emergence of the mutation. Analysis of natural selection by codon-based tests indicates negative selection of codons 30, 31 and 34 which confer amantadine resistance. The codons 2, 11–28 and 54 of porcine and human strains exhibit differences in the patterns of substitution rates, suggesting different selection modes. Transfer of amantadine resistance by exchange of the M segment and viability of recombinant A/WSN/33 viruses with avian-like M segments raises concerns about the emergence of natural human reassortants.
‘Global worming’: first record of an epidemic of Triaenophorus crassus in a population of Arctic charr Salvelinus umbla
Authors: Achleitner D. , Gassner H. , Schabetsberger R.
Published: March 2009

In May 2005, an epidemic of the cestode Triaenophorus crassus occurred in the Salvelinus umbla population of Lake Grundlsee, an oligotrophic Austrian Alpine Lake. Based on catches with a standardized multi‐mesh gillnet survey 53% of S. umbla were infected with up to 17 cysts of T. crassus per fish. This is the first documented record of an epidemic of this tapeworm in S. umbla.
Temperature, selective mortality and early growth in the short‐lived clupeid Spratelloides gracilis
Authors: Durieux E. D. H. , Meekan M. G. , Ponton D. , Vigliola L.
Published: March 2009

Six cohorts of the silver‐stripe round herring Spratelloides gracilis, a fast‐growing and short‐lived tropical clupeid, were collected as juveniles and then as adults during austral summers from November to February in 1998–1999 and 1999–2000, using light traps in the Dampier Archipelago, Western Australia. Otolith analysis allowed backcalculation of size and growth rate at age to examine the relative influences of selective mortality and water temperature on early growth. Negative size‐selective mortality and growth‐selective mortality between the juvenile and the adult stages was found only in the cohort that was the smallest and slowest growing in the period immediately following hatching. Selective mortality preferentially removed members of this cohort that were smaller from age 0 to 15 days, and slower growing from 0 to 10 days, resulting in an elevation of size at age to, or even above, that of cohorts that had not undergone this process. Size and growth rate at 5 day age intervals in the first 20 days after hatching differed among cohorts within and between summers and were strongly and positively correlated (r2= 0·61–0·83) with water temperature.
Stunted growth of pikeperch Sander lucioperca in Lake Sahajärvi, Finland
Authors: Vinni M. , Lappalainen J. , Malinen T. , Lehtonen H.
Published: March 2009

Growth of pikeperch Sander lucioperca in the eutrophic and clay‐turbid Lake Sahajärvi, Southern Finland, was extremely slow in comparison with other lakes at similar latitudes. The most important food item in July was phantom midge larvae Chaoborus flavicans for all sizes of S. lucioperca (239–423 mm total length LT), while later, in August and September, the diet of S. lucioperca (149–407 mm LT) consisted of small (30–100 mm LT) perch Perca fluviatilis, ruffe Gymnocephalus cernuus and roach Rutilus rutilus.
Mitochondrial DNA diversity and origins of South and Central American goats
Authors: Amills M. , Ramírez O. , Tomàs A. , Badaoui B. , Marmi J. , Acosta J. , Sànchez A. , Capote J.
Published: March 2009

We have analysed the genetic diversity of South and Central American (SCA) goats by partially sequencing the mitochondrial control region of 93 individuals with a wide geographical distribution. Nucleotide and haplotype diversities reached values of 0.020 ± 0.00081 and 0.963 ± 0.0012 respectively. We have also observed a rather weak phylogeographic structure, with almost 69% of genetic variation included in the within‐breed variance component. The topology of a median‐joining network analysis including 286 European, Iberian, Atlantic and SCA mitochondrial sequences was very complex, with most of the haplotypes forming part of independent small clusters. SCA sequences showed a scattered distribution throughout the network, and clustering with Spanish and Portuguese sequences occurred only occasionally, not allowing the distinguishing of a clear Iberian signature. Conversely, we found a prominent cluster including Canarian, Chilean, Argentinian and Bolivian mitochondrial haplotypes. This result was independently confirmed by constructing a Bayesian phylogenetic tree (posterior probability of 0.97). Sharing of mitochondrial haplotypes by SCA and Canarian goats suggests that goat populations from the Atlantic archipelagos, where Spanish and Portuguese ships en route to the New World used to stow food and supplies, participated in the foundation of SCA caprine breeds.
Monophyletic origin of domestic bactrian camel (Camelus bactrianus) and its evolutionary relationship with the extant wild camel (Camelus bactrianus ferus)
Authors: Ji R. , Cui P. , Ding F. , Geng J. , Gao H. , Zhang H. , Yu J. , Hu S. , Meng H.
Published: March 2009

The evolutionary relationship between the domestic bactrian camel and the extant wild two‐humped camel and the factual origin of the domestic bactrian camel remain elusive. We determined the sequence of mitochondrial cytb gene from 21 camel samples, including 18 domestic camels (three Camelus bactrianus xinjiang, three Camelus bactrianus sunite, three Camelus bactrianus alashan, three Camelus bactrianus red, three Camelus bactrianus brown and three Camelus bactrianus normal) and three wild camels (Camelus bactrianus ferus). Our phylogenetic analyses revealed that the extant wild two‐humped camel may not share a common ancestor with the domestic bactrian camel and they are not the same subspecies at least in their maternal origins. Molecular clock analysis based on complete mitochondrial genome sequences indicated that the sub‐speciation of the two lineages had begun in the early Pleistocene, about 0.7 million years ago. According to the archaeological dating of the earliest known two‐humped camel domestication (5000–6000 years ago), we could conclude that the extant wild camel is a separate lineage but not the direct progenitor of the domestic bactrian camel. Further phylogenetic analysis suggested that the bactrian camel appeared monophyletic in evolutionary origin and that the domestic bactrian camel could originate from a single wild population. The data presented here show how conservation strategies should be implemented to protect the critically endangered wild camel, as it is the last extant form of the wild tribe Camelina.
Molecular structure in peripheral dog breeds: Portuguese native breeds as a case study
Authors: Pires A. E. , Amorim I. R. , Ginja C. , Gomes M. , Godinho I. , Simões F. , Oom M. , Petrucci‐Fonseca F. , Matos J. , Bruford M. W.
Published: March 2009

Genetic variability in purebred dogs is known to be highly structured, with differences among breeds accounting for ∼30% of the genetic variation. However, analysis of the genetic structure in non‐cosmopolitan breeds and local populations is still limited. Nine Portuguese native dog breeds, and other peripheral dog populations (five) with regional affinities, were characterized using 16 microsatellites and 225 amplified fragment length polymorphism (AFLP) markers, and the pattern of genetic differentiation was investigated. Although the level of breed differentiation detected is below that of other dog breeds, there is in most cases a correlation between breed affiliation and molecular structure. AFLP markers and Bayesian clustering methods allowed an average of 73.1% of individuals to be correctly assigned to source populations, providing robust genotypic assessment of breed affiliation. A geographical genetic structure was also detected, which suggests a limited influence of African dogs on the Iberian breeds. The sampling effect on the estimation of population structure was evaluated and there was a 2.2% decrease in genetic differentiation among breeds when working animals were included. Genetic diversity of stray dogs was also assessed and there is no evidence that they pose a threat to the preservation of the gene pool of native dog breeds.