Detection and analysis of mycovirus-related RNA viruses from grape powdery mildew fungus Erysiphe necator.

The fungus, Erysiphe necator Schw., is an important plant pathogen causing powdery mildew disease in grapevines worldwide. In this study, high-throughput sequencing of double-stranded RNA extracted from the fungal tissue combined with bioinformatics was used to examine mycovirus-related sequences associated with E. necator. The results showed the presence of eight mycovirus-related sequences. Five of these sequences representing three new mycoviruses showed alignment with sequences of viruses classified in the genus Alphapartitivirus in the family Partitiviridae. Another three sequences representing three new mycoviruses showed similarity to classifiable members of the genus Mitovirus in the family Narnaviridae. These mycovirus isolates were named Erysiphe necator partitivirus 1, 2, and 3 (EnPV 1-3) and Erysiphe necator mitovirus 1, 2, and 3 (EnMV 1-3) reflecting their E. necator origin and their phylogenetic affiliation with other mycoviruses.

Surgical nuances in the separation of craniopagus twins - Our experience and a follow up of 15 years.

Craniopagus twins are conjoined twins fused at the cranium. This is the rarest anomaly seen in conjoined twins and craniopagus twins account for 2- 6% of conjoined twins. Conjoined twins are also extremely rare, with the anomaly seen in about 10-20 subjects per billion births. A female preponderance has been noted. Craniopagus twins can be classified into complete or partial, depending on whether or not they have shared dural venous sinuses. They can be further classified into angular or total depending on the alignment of the inter-twin longitudinal axis. Surgical separation of these cases can be an elective procedure or an emergency, mandated by the death of one of the twins. Surgical separation of craniopagus twins is a complex exercise needing detailed evaluation and planning. For the successful management of these twins, a multi-disciplinary approach involving neurosurgeons, plastic surgeons, anesthetists, radiologists, pediatric critical care specialists and ancillary staff is mandatory. We present a case of partially successful elective separation of partial angular craniopagus twins performed in 2002.The surviving twin was managed conservatively for a cerebrospinal fluid leak. The patient subsequently developed a pseudomeningocele, necessitating re-exploration, excision of the gliotic tissue, and repair of the dura and the overlying scalp flap. The patient has a skull defect for which cranioplasty has been deferred. The child is neurologically intact. The post-operative evaluation and the detailed periodic neurological assessment till date (with a follow up of 15 years) have been presented in this study.

Lipomyelocele with osseous dysraphic hamartoma: A rare case report.

Lipomyelocele is the most common occult spinal dysraphism. However, it can rarely be associated with aberrant tissues such as bone--making it one of the rarest of spinal dysraphism type, called as "osseous dysraphic hamartoma." The authors report such a rare case of lipomyelocele with osseous dysraphic hamartoma. CT and MRI play a complimentary role in accurate diagnosis of this rare dysraphism. Detection of aberrant bone helps plan the surgery.

A study of predictors for identification of risk of complications in patients with liver abscess.

BACKGROUND/AIMS: Liver abscess is a significant health problem in developing countries and the complications associated with it are frequently fatal. Hence identification of these complications and anticipating the same will lead to reduction in the mortality and morbidity rate. Such a work will facilitate in identifying patients with risk of complications and will allow for planning of an early intervention. The aim of this study was to identify the predictors of risk of complications in patients with liver abscess. MATERIALS AND METHODS: The data for this retrospective study was obtained from hospital records and included 100 patients diagnosed to have solitary or multiple liver abscess. Complications were defined and history, lab and radiological findings and course of treatment were observed and analyzed in correlation with occurrence of complications. A predictive scoring system was designed for 15 points by giving two points to the factors with 100% correlation and one point to other factors with strong correlation. The score was applied to a validation cohort of 114 different patients and results were noted. RESULTS: Out of the 100 cases studied 24 cases had complications of and the predictive factors included history of alcoholism (> 10 yrs), INR > 1.7 , TLC > 20000/cc and pleural effusion, while other factors had a varying degree of correlation with complication occurrence. It was observed that the new scoring system was successful in identifying patients at risk of developing complications with 100% sensitivity and 93.75% specificity. CONCLUSION: Management of liver abscess can be clearly defined by dividing patients into categories depending upon a new scoring system described in the study and intervention can be planned.

First Report of Tomato spotted wilt virus in Peppers and Tomato in the Dominican Republic.

In the Dominican Republic, green bell pepper (Capsicum annuum L.) and tomato (Solanum lycopersicum L.) are widely cultivated under protected greenhouse conditions as high value commercial crops for export. For the past 2 to 3 years, pepper and tomato have been observed in protected crop facilities in Jarabacoa and Constanza in the North Region with chlorotic and necrotic spots and rings on leaves, petioles, and stems, leaf bronzing, and tip necrosis. Fruits on symptomatic pepper and tomato plants showed concentric rings, irregular chlorotic blotches and deformation, and uneven maturation and development. Incidence on pepper and tomato was 20 to 100% and 5 to 20%, respectively. In initial tests, leaves and fruits from each of 20 symptomatic tomato and pepper plants from several greenhouse facilities were reactive in Tomato spotted wilt virus (TSWV; genus Tospovirus, family Bunyaviridae) immunostrip assays (Agdia, Inc., Elkhart, IN). Since these immunostrips are known to react with other tospoviruses, such as Tomato chlorotic spot virus (TCSV) and Groundnut ring spot virus, additional molecular diagnostic assays were conducted. Leaf and fruit samples from symptomatic plants were imprinted on nitrocellulose membrane (NCM) (2), air-dried, and sent to Washington State University for confirmatory tests. Viral nucleic acids were eluted from NCM discs (1) and subjected to reverse transcription (RT)-PCR using primers gL3637 (CCTTTAACAGTDGAAACAT) and gL4435 (CATDGCRCAAGARTGRTARACAGA) designed to amplify a portion of the L RNA segment of several tospoviruses (3). A single DNA product of ~800 bp was amplified from all samples. Amplicons from two tomato (leaf and fruit) and one pepper fruit samples were cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA). Two independent clones per amplicon were sequenced in both orientations. Sequence analyses of these clones (GenBank Accession Nos. KF 219673 to 75) showed 100% nucleotide sequence identity among themselves and 97% identity with corresponding L RNA sequences of pepper isolates of TSWV from Taiwan (HM180088) and South Korea (HM581940), 94 to 95% with tomato isolates of TSWV from South Korea (HM581934) and Hawaii (AY070218), and 89% with a tomato isolate from Indonesia (FJ177301). These results further confirm the presence of TSWV in symptomatic tomato and pepper plants. A comparison of TSWV sequences from the Dominican Republic with TSWV isolates from the United States and other countries in the Caribbean region could not be made due to the absence of corresponding sequences of the L-RNA of the virus from these countries in GenBank. TSWV-positive samples were negative for TCSV in RT-PCR, indicating the absence of this tospovirus that has been reported in the Caribbean region (data not shown). To our knowledge, this is the first confirmed report of TSWV in tomatoes and peppers in the Dominican Republic. The presence of vector thrips, Frankliniella occidentalis, on symptomatic plants was also confirmed, suggesting a role in the spread of TSWV under greenhouse conditions. Recent surveys identified some greenhouses with 100% symptomatic peppers. The presence of TSWV in tomato and pepper has important implications for the domestic and export vegetable industry in the Dominican Republic because of the broad host range of the virus (4). It is critical for commercial producers to monitor TSWV and deploy appropriate management strategies to limit virus spread. References: (1) O. J. Alabi et al. J. Virol. Methods 154:111, 2008. (2) P.-G. S. Chang et al. J. Virol. Methods 171:345, 2011. (3) F. H. Chu et al. Phytopathology 91:361, 2001. (4) G. Parrella et al. J. Plant Pathol. 85:227, 2003.

First Report of Potato virus Y in Potato in West Java, Indonesia.

Potato (Solanum tuberosum) is an important vegetable crop in Indonesia. A small survey was conducted for virus diseases in November 2011 in Lembang, West Java, as part of assessing the sanitary status of potatoes produced in farmers' fields. Among the six potato fields surveyed, one field had nearly 20% of plants displaying stunted growth with leaves showing mild chlorotic spots and reduced size of lamina. Tubers harvested from symptomatic plants showed no necrosis symptoms. Symptomatic leaves from three representative potato plants were positive for Potato virus Y (PVY) when tested with PVY-specific immunostrips (Agdia Inc., Elkhart, IN). Leaf samples from virus-positive plants were imprinted on FTA Classic Cards (Whatman International Ltd., Maidstone, UK), air dried, and shipped to Washington State University for confirmatory diagnostic tests. Total nucleic acids were eluted from FTA cards (1) and subjected to reverse transcription (RT)-PCR using primers (PVY/Y4A and PVY/Y3S) specific to the coat protein (CP) of PVY (3). Nucleic acid extracts from samples infected with PVY ordinary strain (PVYO), tuber necrosis strain (PVYNTN), tobacco veinal necrosis strains (PVYEU-N and PVYNA-N), and a recombinant strain (PVYN:O) were included as standards to validate RT-PCR assays. The approximately 480-bp DNA fragment, representing a portion of the CP, amplified in RT-PCR was cloned into pCR2.1 (Invitrogen Corp., Carlsbad, CA). DNA isolated from four independent recombinant clones was sequenced from both orientations. Pairwise comparison of these sequences (GenBank Accession Nos. KF261310 to 13) showed 100% identity among themselves and 93 to 100% identity with corresponding sequences of reference strains of PVY available in GenBank (JQ743609 to 21). To our knowledge, this study represents the first confirmed report of PVY in potato in West Java, Indonesia. Studies are in progress to assess the prevalence of PVY in other potato-growing regions of Indonesia and document the presence of different strains of the virus (2). Since the majority of farmers in Indonesia plant seed selected from their previous potato crop, there is an increased risk of primary and secondary spread of PVY through the informal seed supply system, leading to its increased significance to potato production in Indonesia. Therefore, strengthening foundation seed potato and supply chain programs will promote the production of virus-free potatoes in Indonesia. References: (1) O. J. Alabi et al. Plant Dis. 96:107, 2012. (2) A. Karasev and S. M. Gray. Am. J. Potato Res. 90:7, 2013. (3) R. P. Singh et al. J. Virol. Methods 59:189, 1996.

First Report of Impatiens necrotic spot virus (INSV) Infecting Basil (Ocimum basilicum) in the United States.

Basil (Ocimum basilicum L.), a native of India belonging to the Lamiaceae family, is an aromatic herb with distinctive aroma, and several commercial varieties are used extensively for culinary and ornamental purposes. During the summer of 2011 and 2012, potted plants of basil in a commercial greenhouse in the Richland-Kennewick area of Washington State were observed showing foliar symptoms consisting of chlorotic spots, ring spots, leaf distortion, and stem necrosis. In initial tests, extracts of symptomatic leaves were positive for Impatiens necrotic spot virus (INSV; genus Tospovirus, family Bunyaviridae), when tested with INSV immnunostrips (Agdia, Inc., Elkhart, IN). These samples were negative with immunostrips specific to Tomato spotted wilt virus (genus Tospovirus) and group-specific potyviruses. The virus from symptomatic leaves of basil was transmitted by leaf rub inoculation to Nicotiana benthamiana and Emilia sonchifolia, where it produced necrosis on inoculated leaves followed by systemic necrosis in the former and chlorotic spots and mosaic mottling in newly developed leaves in the latter. Symptomatic leaves from both host plants tested positive with INSV, but not with TSWV, immunostrips. For additional confirmation of INSV, total RNA was extracted from symptomatic leaves of basil using RNeasy Plant Minikit (Qiagen, Inc., Valencia, CA) and used for reverse transcription (RT)-PCR amplification of the nucleocapsid (N) gene using forward (5'-AGCTTAAATCAATAGTAGCA-3') and reverse (5'-AGCTTCCTCAAGAATAGGCA-3') primers. RT was carried out at 52°C for 60 min followed by denaturation at 94°C for 3 min. Subsequently, 35 cycles of PCR was carried out with each cycle consisting of 94°C for 1 min, 58°C for 45 s, and 72°C for 1 min, followed by a final extension step at 72°C for 10 min. The amplicons of about 610 nt obtained from RT-PCR were cloned into pTOPO2.1 vector (Invitrogen Corporation, Carlsbad, CA) and three independent clones were sequenced in both directions. Sequence analyses of these clones (GenBank Accession No. KC218475) showed 100% nucleotide sequence identity among themselves and 99% nucleotide sequence identity with INSV isolates from the United States (DQ523598, JX138531, and D00914) and a basil isolate (JQ724132) from Austria. These results further confirm the presence of INSV in symptomatic leaves of basil. Previously, basil has been reported to be naturally infected with TSWV in the United States (3) and INSV in Austria (2). Therefore, this study represents the first confirmed report of the virus in basil in the United States. No species of thrips vector was observed on the affected basil plants. The discovery of INSV in basil has important implications for the nursery industry due to the broad host range of the virus (1); stock plants may serve as a source of inoculum in production areas and infected plants could be distributed to homeowners. It is important for commercial nurseries to monitor for INSV to identify infected mother plants to prevent virus spread. Since more than 31 viruses belonging to 13 different genera have been reported in basil ( http://pvo.bio-mirror.cn/famly073.htm#Ocimumbasilicum ), further studies are in progress to determine if the observed symptoms on basil are only due to single infection of INSV. References: (1) M. Daughtrey et al. Plant Dis. 81:1220, 1997. (2). S. Grausgruber-Gröger. New Dis. Rep. 26:12, 2012. (3) G. E. Holcomb et al. Plant Dis. 83:966.

Pheromone-based monitoring of Pseudococcus maritimus (Hemiptera: Pseudococcidae) populations in concord grape vineyards.

The grape mealybug, Pseudococcus maritimus (Ehrhorn), is the dominant mealybug in Washington's Concord grape vineyards (Vitis labrusca L.). It is a direct pest of fruit clusters and a vector of grapevine leafroll-associated viruses. Using traps baited with the sex pheromone of Ps. maritimus, we determined the optimal trap density for monitoring Ps. maritimus, with the goal of providing a more rapid monitoring method for Ps. maritimus than visual surveys. Varying densities of pheromone-baited traps (one, four, and eight traps per 12.14 ha) were deployed in Concord vineyards to monitor Ps. maritimus seasonal phenology in 2010 and 2011. In both years, flights of adult males were detected in early May and captures peaked twice per season in mid-June and mid-August, indicating two generations each year. Trap data were analyzed using Taylor's Power Law, Iwao's patchiness regression, and the K parameter of the negative binomial model to determine optimal sample size. The formula using the K parameter provided the lowest required sample size, showing that four to eight traps per 12.14 ha were needed to provide 30% sampling precision efficiency throughout the entire season. Fewer traps were needed during flight peaks when trap capture numbers were great. Only one pheromone-baited trap per 12.14 ha was sufficient to provide Ps. maritimus flight phenology data to make informed management decisions. Species-specific pheromone-baited traps deployed for Planococcus ficus (Signoret), Pseudococcus longispinus (Targioni Tozzetti), and Pseudococcus viburni (Signoret) did not detect any of these species in the vineyards sampled.

SNP array technology: an array of hope in breast cancer research.

Breast cancer is the most common malignancy in women worldwide. The incidence of breast cancer in Malaysia is lower compared to international statistics, with peak occurrence in the age group between 50 to 59 years of age and mortality rates of 18.6%. Despite current diagnostic and prognostic methods, the outcome for individual subjects remain poor. This is in part due to breast cancers' wide genetic heterogeneity. Various platforms for genetics studies are now employed to determine the identity of these genetic abnormalities, including microarray methods like high density single-nucleotide-polymorphism (SNP) oligonucleotide arrays which combine the power of chromosomal comparative genomic hybridization (cCGH) and loss of heterozygosity (LOH) in the offering of higher-resolution mappings. These platforms and their applications in highlighting the genomic alteration frameworks manifested in breast carcinoma will be discussed.

Can the mouse model successfully predict mixed metal(loid)s bioavailability in humans from contaminated soils?

Mouse models have been employed by many scientific research groups worldwide to predict the bioavailability of metal (loid)s and other chemicals in humans. Their suitability for predicting mixed metal (loid) bioavailability has been questioned and debated for decades by many research teams. In this study soils contaminated by lead (Pb) and arsenic (As), either in the field or by spiking in the laboratory, were used in bioavailability and bioaccessibility tests. The spiked soils were aged for more than a year prior to testing to achieve steady state and eliminate soil ageing effects, as reported in previous research. The bioavailability of, firstly, Pb in the presence of As and secondly, As in the presence of Pb was determined using mice. Furthermore, bioaccessibility was determined using a range of in vitro methods: relative bioaccessibility leaching procedure (RBALP), the Unified Bioaccessibility Research Group Europe (BARGE) method (UBM) gastric and intestinal phases, and the National Institute for Public Health and the Environment (RIVM) gastric and intestinal phases. The correlations between Pb and As bioavailability and their in vitro bioaccessibility when they were present in mixtures were analysed. The results indicated that the bioavailability of Pb in mice kidney tissues significantly correlated with bioaccessibility of Pb in RBALP (p < 0.01), UBM gastric (p < 0.01) and intestinal phases (p < 0.01) and RIVM gastric phases when Pb is present in metal (loid) mixtures. Results of the current study reveal that the RBALP, and UBM gastric and intestinal phase were by far the best methods for predicting the RB of Pb when it is present in metal (loid) mixtures. Consequently, the mouse model can successfully explain the in vivo in vitro correlation (IVIVC) of Pb when it is present in metal (loid) mixtures. However, we did find that a mouse model may not be the best one to explain the IVIVC of As when it is present in metal (loid) mixtures.

Classification of COVID-19 with Belief Functions and Deep Neural Network.

At present, the entire world has suffered a lot due to the spike of COVID disease. Despite the world has been developed with so much of technology in the domain of medicine, this is a very huge challenge in all over the world. Though, there is a rapid development in medical field, those are not even sufficient to diagnose the symptoms of this COVID in earlier stage. Since the spread of this disease in all over the world, it affects the livelihood of the human. Computed Tomography (CT) images have given necessary data for the radio diagnostics to detect the COVID cases. Therefore, this paper addressed about the classification techniques to diagnose about the symptoms of this virus with the help of belief function with the support of convolution neural networks. This method initially extracts the features and correlates the features with the belief maps to decide about the classification. This research work would provide classification of more accuracy than the earlier research. Therefore, compared with the traditional deep learning method, this proposed procedure would be more efficient with desirable results achieved for accuracy as 0.87, an F1 of 0.88, and 0.95 as AUC.

The influence of biochar and black carbon on reduction and bioavailability of chromate in soils.

The widespread use of chromium (Cr) has a deleterious impact on the environment. A number of pathways, both biotic and abiotic in character, determine the fate and speciation of Cr in soils. Chromium exists in two predominant species in the environment: trivalent [(Cr(III)] and hexavalent [Cr(VI)]. Of these two forms, Cr(III) is nontoxic and is strongly bound to soil particles, whereas Cr(VI) is more toxic and soluble and readily leaches into groundwater. The toxicity of Cr(VI) can be mitigated by reducing it to Cr(III) species. The objective of this study was to examine the effect of organic carbon sources on the reduction, microbial respiration, and phytoavailability of Cr(VI) in soils. Organic carbon sources, such as black carbon (BC) and biochar, were tested for their potential in reducing Cr(VI) in acidic and alkaline contaminated soils. An alkaline soil was selected to monitor the phytotoxicity of Cr(VI) in sunflower plant. Our results showed that using BC resulted in greater reduction of Cr(VI) in soils compared with biochar. This is attributed to the differences in dissolved organic carbon and functional groups that provide electrons for the reduction of Cr(VI). When increasing levels of Cr were added to soils, both microbial respiration and plant growth decreased. The application of BC was more effective than biochar in increasing the microbial population and in mitigating the phytotoxicity of Cr(VI). The net benefit of BC emerged as an increase in plant biomass and a decrease in Cr concentration in plant tissue. Consequently, it was concluded that BC is a potential reducing amendment in mitigating Cr(VI) toxicity in soil and plants.

First Report of Potato virus Y in Potato in Tajikistan.

Potato (Solanum tuberosum L.) is widely grown as a staple food and cash crop in Tajikistan and is an important food security crop in the country. In June 2011, we conducted a survey of potatoes in farmers' fields in the Buston and Dushanbe regions (about 200 miles apart) of Tajikistan. Potato plants with stunted growth and leaves showing chlorotic spots, curling, and necrotic spots and rings were observed with the disease incidence monitored in 10 fields each in Buston and Dushanbe areas varying between 10 and 60%. Representative samples from symptomatic plants tested positive for Potato virus Y (PVY) using virus-specific immunostrips (Agdia Inc., Elkhart, IN). Leaf samples from symptomatic plants were collected from Buston and Dushanbe areas, imprinted on FTA Classic Cards (Whatman International Ltd., Maidstone, UK), air dried, and shipped to the lab at Washington State University for confirmatory diagnostic tests. Total nucleic acids were eluted from FTA cards (1) and subjected to reverse transcription (RT)-PCR with primers (PVY/Y4A and PVY/Y3S) specific to the coat protein of PVY (3). Samples infected with PVY ordinary strain (PVYO), tuber necrosis strain (PVYNTN), tobacco veinal necrosis strains (PVYEU-N and PVYNA-N), and a recombinant strain (PVYN:O) were included as references to validate RT-PCR results. A single DNA product of approximately 480 bp was amplified from potato samples that tested positive with PVY-specific immunostrips. The amplified fragments from two samples from Dushanbe and six from Buston areas were cloned separately into pCR2.1 (Invitrogen Corp., Carlsbad, CA) and two independent clones per amplicon were sequenced from both orientations. Pairwise comparison of these sequences showed 90 to 100% identity among the cloned amplicons (GenBank Accession Nos. JQ743609 to JQ743616) and 90 to 100% with corresponding nucleotide sequence of reference PVY strains (GenBank Accession Nos. JQ743617 to JQ743621). A global phylogenetic analysis of sequences revealed the presence of PVYO in both samples from Dushanbe and one sample from Buston regions and presence of PVYNTN in the remaining five samples from the Buston region. Because of the possible occurrence of mixed infections of PVY strains (2), further studies are needed to determine the presence of mixed infections of two or more strains of PVY and their specificity to potato cultivars. To our knowledge, this study represents the first confirmed report of two distinct strains of PVY in potato in Tajikistan. The occurrence of PVYNTN, a quarantine pathogen in many countries (2), warrants additional investigations to improve sanitary status of potato fields and to facilitate the availability of virus-free seed in clean plant programs for significant yield increases in Tajikistan. References: (1) O. J. Alabi et al. J. Virol. Methods 154:111, 2008. (2) S. Gray et al. Plant Dis. 94:1384, 2010. (3) R. P. Singh et al. J. Virol. Methods 59:189, 1996.

First Report of Grapevine fleck virus in Idaho Grapevines.

Idaho has a growing viticulture industry, with nearly 1,600 acres of wine grapes (Vitis vinifera L.). Production is largely concentrated in two locations, the Snake River valley, which includes Canyon County in the southwest, and the Clearwater River valley, primarily Nez Perce County in the northwest. Grapevine fleck virus (GFkV) belongs to the genus Maculavirus, family Tymoviridae, comprising positive-sense, single-stranded RNA viruses with ca. 7.6-kb genome (3). It is one of five non-mechanically transmitted viruses associated with the fleck disease complex and has been previously documented to occur in the neighboring state of Washington (2). Main sources of wine grape nursery material imported to Idaho reside in Washington or in California, and it is important to monitor virus status of the planting material brought to the state. However, no information was available on the occurrence and prevalence of GFkV in wine grapes in Idaho. During three growing seasons in 2009 through 2011, random grapevine samples were collected in 14 vineyards in Canyon, Elmore, Ada, and Nez Perce counties. A total of 434 samples were tested by one step RT-PCR using GFkV-specific primers, GFkVf: 5'-TGACCAGCCTGCTGTCTCTA-3' and GFkVr: 5'-TGGACAGGGAGGTGTAGGAG-3' designed to amplify a fragment of the GFkV capsid protein gene (1). Twenty-four samples tested positive for GFkV by RT-PCR and produced the expected 179-bp DNA fragment. These samples came from five vineyards sampled across all surveyed counties, and represented seven wine grape cultivars, including Pinot Noir, Cabernet Sauvignon, Syrah, Lemberger, Riesling, Chardonnay, Pinot Gris, and one unknown table grape cultivar. Twelve PCR products were cloned into the pGEM-T Easy plasmid vector (Promega), sequenced (numbered ID1 to 12, available upon request), and confirmed to represent fragments of the GFkV CP gene between positions 6,453 and 6,631 in the genome of GFkV isolate MT48 (GenBank Accession No. AJ309022.1). Eight of the Idaho GFkV sequences (ID2, ID3, ID7 to 11, and ID12) matched closely with other GFkV sequences from Washington State, Italy, India, and South America, showing 97 to 99% identity at the nucleotide level in pair-wise comparisons. Four GFkV sequences from Idaho (ID1 and ID4 to 6) showed only modest (90 to 92%) identity in pair-wise comparisons with GFkV sequences available in GenBank. Consequently, in phylogenetic reconstructions eight Idaho GFkV sequences clustered in the same lineage with the six GFkV sequences deposited in GenBank, and four other GFkV sequences were placed outside of this main clade. It is possible that this phylogeny of the Idaho GFkV reflects different sources of the virus-infected planting material brought to the state. In the absence of symptoms expressed in wine grape cultivars infected with GFkV, laboratory methods remain the only tool to detect the virus. To our knowledge, this is the first report of GFkV found in wine grapes in Idaho demonstrating its substantial presence in production areas. References: (1) G. Gambino and I. Gribaudo. Phytopathology 96:1223, 2006. (2) R. A. Naidu et al. Plant Dis. 94:784, 2010. (3) S. Sabanadzovic et al. J. Gen. Virol. 82:2009, 2001.

Evaluation of common operator errors in panoramic radiography in Trinidad and Tobago: a comparison of formally vs informally trained operators.

AIM: To evaluate and compare the frequency of common operator errors seen on panoramic radiographs in dental private practices and in the dental hospital (taken by informally and formally trained operators, respectively) in Trinidad and Tobago. METHOD: One thousand panoramic radiographs of patients over the age of 10 years were included in this study. These comprised 500 from the dental hospital and 500 from dental private practices. The radiographs were reviewed using standardized criteria to identify the most common operator errors. RESULTS: There were only 21 (4.2%) error free radiographs in the dental private practice sample and 29 (5.80%) in the dental hospital sample. Frequencies of specific errors were significantly higher in the dental private practice sample in each category except for "Chin tipped too low" (Chi-square p < 0.05) CONCLUSION: This study supports the need for the introduction of statutory guidelines with respect to the use of ionizing radiation in dentistry in Trinidad and Tobago and in particular, the implementation of formally assessed dedicated dental radiography training for all operators of dental X-ray equipment.

Two-port vs. three-port laparoscopic appendicectomy: A bridge to least invasive surgery.

INTRODUCTION: The conventional three-port technique for laparoscopic appendicectomy has proven its worth in the management of appendicular pathologies. From a cosmetic viewpoint, the umbilical and suprapubic port-sites are hidden by natural camouflages, the right Iliac fossa (RIF) port is the only visible external sign of surgery. The two-port technique avoids even this marker of abdominal invasion. In this study, we describe the technique of two-port laparoscopic appendicectomy (TPA) and compare it with conventional laparoscopic appendicectomy (CLA). MATERIALS AND METHODS: All patients studied underwent operation for acute appendicitis during a 6-month period. Data were collected prospectively for the TPA and retrospectively for the CLA. The TPA was performed with one 10 mm umbilical working port and one 5 mm suprapubic camera port. A hypodermic needle was introduced in the RIF to retract the appendix. The appendicular artery was controlled with diathermy or ultrasonic shears. The base was ligated with a loop knotted extracorporeally. CLA was performed via the conventional 10 mm umbilical, 5 mm suprapubic and 5 mm RIF ports. The appendicular stump was ligated with an endoloop or an intracorporeal knot. RESULTS: A total of 146 patients underwent surgery over the 6-month period for appendicitis. Out of 62 cases attempted, the TPA was successful in 51 cases, with conversion to the three-port technique in 11. The operative time, complication rates, return to work were comparable between the two groups. Patients who had TPA had a shorter postoperative stay. CONCLUSION: This is an initial experience with TPA. There is little difference in the operative time, postoperative stay and complications rates between this technique and the conventional three-port one. There is hence little to be lost and a likely benefit to be gained by performing the TPA although a randomised study is necessary.

Occurrence of Grapevine leafroll-associated virus 1 in Two Ornamental Grapevine Cultivars in Washington State.

Roger's Red, an interspecific hybrid between wild grape (Vitis californica, native to northern California) and the V. vinifera cv. Alicante Bouschet (1), and Claret Vine (V. vinifera cv. Purpurea Nana) are grown for their ornamental value in home gardens and other settings. We collected potted grapevines of Roger's Red and Claret Vine showing dull green-to-scarlet red leaves from two different retail nurseries in the Richland-Kennewick area and Prosser, WA, respectively. Since these symptoms 'mimic' grapevine leafroll disease, we tested petiole samples from four grapevines per cultivar for a panel of grapevine-infecting viruses by single-tube one-step reverse transcription (RT)-PCR (4). All samples tested positive only for Grapevine leafroll-associated virus 1 (GLRaV-1; genus Ampelovirus, family Closteroviridae). To further confirm these results, total RNA was subjected to RT-PCR to amplify a portion of the heat shock protein 70 homolog (HSP70h), coat protein duplicate 2 (CPd2), and ORF 9 (p24) of GLRaV-1. RT was performed at 52°C for 60 min, followed by 35 cycles of PCR (30 s denaturation at 94°C, 45 s annealing at 55°C, and 30 s extension at 72°C) and a 5 min final extension step at 72°C. Primers specific to HSP70h (HSP70h/416F: 5'-CAGGCGTCGTTTGTACTGTG and HSP70h/955R: 5'-TCGGACAGCGTTTAAGTTCC), CPd2 (CPd2/F: 5'-GTTACGGCCCTTTGTTTATTATGG and CPd2/R: 5'-CGACCCCTTTATTGTTTGAGTATG) and ORF 9 (p24/F: 5'-CGATGGCGTCACTTATACCTAAG and p24/R (5'-CACACCAAATTGCTAGCGATAGC) were designed based on GLRaV-1 sequence (GenBank Accession No. AF195822) to amplify 540, 398, and 633 base pair (bp) DNA fragments, respectively. To verify that the amplified products were specific to the genome of GLRaV-1, the amplicons were cloned into pCR2.1 vector (Invitrogen Corp, Carlsbad, CA) and three independent clones for each amplicon were sequenced in both directions. Pairwise comparison of HSP70h (Accession Nos. HQ833472 and HQ833473), CPd2 (Accession Nos. HQ833474 and HQ833475), and p24 (Accession Nos. HQ833476 and HQ833477) sequences from Roger's Red and Claret Vine showed 100, 96, and 99% identities, respectively, between them, and 86 to 100, 80 to 97, and 86 to 90% nucleotide sequence identities, respectively, with corresponding sequences of GLRaV-1 isolates deposited in GenBank. We further confirmed the presence of GLRaV-1 in these two ornamental grape cultivars by double antibody sandwich-ELISA using commercially available antibodies (Bioreba AG, Reinach, Germany). Previous studies have reported the presence of GLRaV-2 and -3 (1,3) and Grapevine virus A and B (2,3) in Roger's Red. To our knowledge, this study represents the first report of the occurrence of GLRaV-1 in two Vitis species distributed as ornamental grapes. It is important to prevent virus spread via the supply of virus-tested ornamental grapevines by commercial nurseries. References: (1) G. S. Dangl et al. Am. J. Enol. Vitic. 61:266, 2010. (2) D. A. Golino et al. Phytopathology (Abstr.) 99(suppl.):S44, 2009. (3) V. Klaassen et al. Online publication. doi:10.1094/PDIS-09-10-0621. Plant Dis., 2011. (4) T. A. Mekuria et al. Phytopathology (Abstr.) 99(suppl.):S83, 2009.

Dental health knowledge and attitudes of primary school teachers toward developing dental health education.

OBJECTIVE: To assess the dental health knowledge of primary school teachers, their attitudes toward the prevention of dental diseases and to identify any barriers to the implementation of oral health promotion programmes in schools. METHOD: Teachers' knowledge of the causes and prevention of dental decay and gum disease, their attitudes toward oral health and barriers to the implementation of dental health education programmes were assessed using a self-administered questionnaire. RESULTS: School teachers were generally very well informed about the causes and prevention of dental decay and gum disease. Knowledge of the appropriate management of serious dental trauma was very poor among this group although they seemed to have greater awareness of the appropriate management for less serious dental injuries. The majority of teachers demonstrated positive attitudes toward dental health and its incorporation into the school curriculum. Teachers'attitudes to their own involvement in school-based dental health education were also positive. Lack of training and resources and time within the curriculum were identified as major barriers to the implementation of a dental health education programme in primary schools. CONCLUSION: Developing teacher training programmes that include oral health knowledge and an evidence-based approach to dental health education within a school setting could enable primary school teachers to play a significant part in oral health promotion for young children in Trinidad.

A preliminary investigation of periodontal disease and diabetes in Trinidad.

OBJECTIVES: To describe periodontal disease status in diabetic patients in Trinidad. METHOD: A cross-sectional study was conducted. Patients attending a tertiary referral centre for diabetes at an out-patient clinic were invited to undergo oral examinations. The basic periodontal examination (BPE) was used to assess periodontal disease status. RESULTS: Seventy-two patients participated in the study. Mean age was 55.7 years, 54.2% were female, with 66.7% and 22.2% being of Indo-Trinidadian and Afro-Trinidadian ethnicity respectively. There were 61.1% who had not attended for dental treatment within the last year and 56.9% only attended when in pain; 15.3% had a history of cigarette smoking and 31.9% currently wore a denture. Plaque was detectable with the use of a probe in 40.3% of the 67% that underwent a BPE assessment; 38.8% were found to have advanced periodontal disease. CONCLUSION: The prevalence of periodontal disease in this sample of diabetic patients suggests that regular dental examinations, oral health education, and collaborative patient care between medical and dental practitioners should form part of the routine management of diabetic patients in Trinidad.

First Report of Grapevine fleck virus from Washington Vineyards.

Grapevine fleck virus (GFkV) is a positive-sense, single-stranded RNA virus with a genome size of 7,564 nucleotides (3). The virus is present in many grape-growing regions (1,2,4). GFkV is phloem-limited and graft transmissible, but a biological vector is not yet known (4). It causes latent infections in Vitis vinifera cultivars, but induces specific foliar symptoms in the indicator host, V. rupestris. While testing samples from wine grape cultivars, samples from cv. Chardonnay tested positive for GFkV in single-tube one-step reverse transcription (RT)-PCR assay using forward primer GFkV585F (5'-CTCAGCCTCCACCTTGCCCCGT-3') and reverse primer GFkV1117R (5'-CAATTTGGCTGGGCGAGAAGTACA-3'). The forward primer is identical to nt 585 to 606 and the reverse primer is complementary to nt 1094 to 1117 in the GFkV genome (Accession No. AJ309022) and encompass a portion of the RNA-dependent RNA polymerase. The primer pair amplified a 533-nt fragment from 15 of 37 individual grapevines in the Chardonnay block. The amplicons obtained from five grapevines were cloned individually into the pCR2.1 plasmid (Invitrogen Corp., Carlsbad, CA). Three independent clones per amplicon were sequenced in both orientations. Sequences were edited and assembled using ContigExpress project in the Vector NTI Advance 11 sequence analysis software packages (Invitrogen). Pairwise comparisons of these sequences (Accession Nos. GU372367 to GU372371) with a corresponding sequence of a GFkV isolate deposited in the GenBank (Accession No. AJ309022) showed 89 to 97% identity at the nucleotide and 95 to 98% identity at the amino acid level. To further support these results, we amplified a 714-nt fragment specific to the complete coat protein (CP) gene of GFkV from three of the five isolates sequenced above using primers GFkV-6351F (5'-CTCTCCGCCTCGTCTGATGA-3') and GFkV-7064R (5'-TCGGTTCATGACGAGGGAGT-3'). The amplicons were cloned and sequenced as described above. A comparison of these sequences (Accession Nos. GU372372 to GU372374) with the CP sequence of GFkV available in the GenBank (Accession No. AJ309022) showed 94 to 95% and 98 to 100% identity, respectively, at the nucleotide and amino acid level. ELISA with GFkV-specific antibodies (BIOREBA AG, Reinach, Switzerland) further confirmed the presence of the virus in samples that were positive in RT-PCR. ELISA results validated the data described above and confirmed the presence of GFkV in Chardonnay samples that tested positive by RT-PCR assay. Previously, GFkV was documented in grapevines in California and Missouri (2), Australia (4) and Europe (1). To our knowledge, this is the first confirmed report of the occurrence of GFkV in Washington vineyards. The results expand our current knowledge on the distribution of GFkV and help to prevent its dissemination through the supply of grapevine cuttings by 'clean' plant programs. References: (1) G. P. Martelli et al. Arch Virol. 147:1847, 2002. (2) B. N. Milkus and R. N. Goodman. Am. J. Enol. Vitic. 50:133, 1999. (3) S. Sabanadzovic et al. J. Gen. Virol 82:2009, 2001. (4) B. J. Shi et al. Ann. Appl. Biol. 142:349, 2003.