Use of the cotton leaf curl Multan alphasatellite as a silencing or expression vector.

Alphasatellites, formerly known as DNA 1, are a satellite-like components associated with begomoviruses (the family Geminiviridae) that require betasatellite for symptom induction but depend on DNA-A for systemic movement. We have converted alphasatellite into gene-silencing vector (modified alphasatellite (∆DNA 1)) by deleting its A-rich region that does not affect the replication nor the movement of the helper virus. Insertion of a transgene green florescence protein (GFP) into ∆DNA 1 resulted in the silencing g of the cognate gene in Nicotiana benthamiana. The silencing persisted for more than one and half month and was associated with the decreased level of mRNA of the target gene. This satellite-like DNA vector induced gene silencing (VIGS) promises to be applicable to other begomovirus/alphasatellite systems, thereby providing the powerful approach to gene discovery and the analysis of gene functions in malvaceous crops. Keywords: cotton; begomovirus; alphasatellite; RNAi.

Molecular characterization of a distinct monopartite begomovirus associated with betasatellites and alphasatellites infecting Pisum sativum in Nepal.

Pea (Pisum sativum) plants exhibiting leaf distortion, yellowing, stunted growth and reduction in leaf size from Rampur, Nepal were shown to be infected by a begomovirus in association with betasatellites and alphasatellites. The begomovirus associated with the disease showed only low levels of nucleotide sequence identity (<91%) to previously characterized begomoviruses. This finding indicates that the pea samples were infected with an as yet undescribed begomovirus for which the name Pea leaf distortion virus (PLDV) is proposed. Two species of betasatellite were identified in association with PLDV. One group of sequences had high (>78%) nucleotide sequence identity to isolates of Ludwigia leaf distortion betasatellite (LuLDB), and the second group had less than 78% to all other betasatellite sequences. This showed PLDV to be associated with either LuLDB or a previously undescribed betasatellite for which the name Pea leaf distortion betasatellite is proposed. Two types of alphasatellites were identified in the PLDV-infected pea plants. The first type showed high levels of sequence identity to Ageratum yellow vein alphasatellite, and the second type showed high levels of identity to isolates of Sida yellow vein China alphasatellite. These are the first begomovirus, betasatellites and alphasatellites isolated from pea.

Evaluating the suitability of maggot meal as a partial substitute of soya bean on the productive traits, digestibility indices and organoleptic properties of broiler meat.

This study was conducted to replace soya bean meal with maggot meal at difference percentages. A total of 120 one-day-old broiler chicks were divided into four groups. Four diets were formulated with maggot meal to replace soya bean meal at the rate of 0%, 10%, 20% and 30% designated as A, B, C and D respectively. Feed and water were offered ad libitum during the study which lasted for 28 days. Feed intake decreased significantly in treated groups while body weight, Feed conversion ratio and dressing percentage increased significantly in group D. Apparent metabolizable energy increased significantly in the group fed 30% maggot meal; however, no significant difference was found in dry matter, crude protein, crude fibre, ether extract, ash and nitrogen free extract between the control and treated groups. The result of sensory evaluation revealed that tenderness and juiciness increased significantly in group C and B respectively. The colour and flavour were most accepted in group B and C. The results indicate that replacement of soya bean meal at the rate of 30% in broiler feed produced most favourable results.

Identification of Tomato yellow leaf curl virus Naturally Infecting Common Bean in Japan.

Tomato yellow leaf curl virus (TYLCV; family Geminiviridae, genus Begomovirus) is a devastating pathogen vectored by the whitefly Bemisia tabaci causing significant yield losses to tomato crops in Japan since 1998 (2). So far, there has been no report of this virus infecting common bean (Phaseolus vulgaris; family Fabaceae) in Japan. But recently, TYLCV has been reported from P. vulgaris in Spain and China (1,3). P. vulgaris is a vegetable crop commonly grown during spring to summer in Japan. In the course of a study to assess virus incidence on P. vulgaris exhibiting yellowing and thickening symptoms with 20 to 30% incidence, four symptomatic and two healthy samples were collected in August, 2012 from a single mix-cropping field (where P. vulgaris was cultivated together with tomato [Solanum lycopercisum] crop and a high B. tabaci population density was observed on plants) in Komae, Japan. Meanwhile, the vector for one crop can transmit the disease to the next crop very easily. To identify possible begomovirus present in symptomatic P. vulgaris plants, total nucleic acids were extracted from plants with and without symptoms using PhytoPure Plant DNA Extration Kit (GE Amersham Biosciences, UK) and begomovirus replication was confirmed from symptomatic leaves in 3 out of 4 plants when hybridized with a specific non-radioactive probe to the coat protein region of TYLCV (5) using a Biotin DNA Labelling Kit (Fermentas). To confirm the identity of the virus detected, leaf samples were further tested by PCR using the TYLCV specific detection primers (TYF/TYR) corresponding to the V2/CP region and a DNA fragment of an expected size was obtained from the samples positive for TYLCV in Southern hybridizations (4). The PCR products were sequenced directly and these sequences showed the highest nucleotide sequence identity >99% to the TYLCV isolate AB116630 reported from Japan. Rolling circle amplification (RCA) was done on all samples to produce concatamers and with Sac I restriction enzyme an amplicon of ~2.7 kb was obtained and cloned into PUC118 vector (Takara, Japan). Multiple clones were obtained showing the same restriction pattern with different restriction enzymes, and three of them were randomly selected and sequenced by Macrogen (Japan). Sequence analysis showed all three clones were identical and one sequence (J18.11) was submitted to GenBank. This sequence (GenBank Accession No. KJ585666) was 2,774 nt long and displayed the arrangement of four ORFs (AC1, AC2, AC3, and AC4 in complementary sense) and two ORFs (AV2 and AV1) on virion sense) typical of the genome of a begomovirus. This whole genome sequence was compared with those of other reported begomoviruses and exhibited greater than 99% nt sequence identity to a previously reported TYLC sequence (JN183876). Efforts to identify the presence of an additional begomovirus components or DNA satellites either by PCR and/or RCA were negative, suggesting that this is a monopartite begomovirus and an isolate of TYLCV. This is the first report of a natural infection of TYLCV in P. vulgaris in Japan. The presence of TYLCV on P. vulgaris could therefore represent a serious threat for this valuable crop in Japan. Thus, it is important to develop an effective way to control this virus to reduce the further losses. References: (1) Y. H. Ji et al. Plant Dis. 96:1229, 2012. (2) K. Kato et al. Ann. Phytopathol. Soc. Jpn. 64:552, 1998. (3) J. Navas-Castillo et al. Plant Dis. 83:29, 1999. (4) M. S. Shahid et al. J. Phytopahol. 161:205, 2013. (5) M. S. Shahid et al. Viruses 6:189, 2014.

Complete nucleotide sequence of a monopartite Begomovirus and associated satellites infecting Carica papaya in Nepal.

Carica papaya (papaya) is a fruit crop that is cultivated mostly in kitchen gardens throughout Nepal. Leaf samples of C. papaya plants with leaf curling, vein darkening, vein thickening, and a reduction in leaf size were collected from a garden in Darai village, Rampur, Nepal in 2010. Full-length clones of a monopartite Begomovirus, a betasatellite and an alphasatellite were isolated. The complete nucleotide sequence of the Begomovirus showed the arrangement of genes typical of Old World begomoviruses with the highest nucleotide sequence identity (>99 %) to an isolate of Ageratum yellow vein virus (AYVV), confirming it as an isolate of AYVV. The complete nucleotide sequence of betasatellite showed greater than 89 % nucleotide sequence identity to an isolate of Tomato leaf curl Java betasatellite originating from Indonesian. The sequence of the alphasatellite displayed 92 % nucleotide sequence identity to Sida yellow vein China alphasatellite. This is the first identification of these components in Nepal and the first time they have been identified in papaya.

First Report of Bean common mosaic necrosis virus (BCMNV) Infecting Sweet Bean in Nepal.

Bean common mosaic necrosis virus (BCMNV; Family Potyviridae, genus Potyvirus) infects legume crops in many regions of the world. It is transmitted in a non-persistent manner by aphids and is also readily seed-transmitted (3). Sweet bean (Lablab purpureus L.) is an important legume crop widely cultivated in Nepal. In December 2010, sweet bean plants with mottle and leaf deformation, severe mosaic, necrosis, malformation of leaves and pods, downward curling of leaves, and reduction in leaf size were observed in 20 different fields with 60 to 70% incidence in Nepal. ELISA was performed by using a universal Potyvirus antiserum test kit (Agdia Inc., Elkhart, IN) on 18 symptomatic leaf samples collected from five different locations (Malepatan, Lake side Fewataal, Darai village, Pakaudi, and Rampur) of Pokhara and Chitwan provinces and 15 out of 18 samples had a positive reaction. Filamentous shaped particles similar to Potyvirus of about 690 to 720 × 10 to 12 nm were observed by electron microscopy confirming the ELISA results. To further characterize the viral isolate(s), primary leaves of some legume crops (Phaseolus vulgaris, Glycine max, Psophocarpus tetragonolobus, Vigna unguiculata, and Arachis hypogeae) and sweet bean were mechanically inoculated with sap prepared from the same leaves used for ELISA. Phaseolus vulgaris cv. Aron and G. max cv. Togenkyou plants showed necrotic spots on inoculated leaves followed by systemic necrosis and death. Psophocarpus tetragonolobus and V. unguiculata showed systemic mosaic symptoms, while A. hypogeae and sweet bean cv. Shirobhanafuji-mame showed necrotic spots and restricted veinal necrosis. Chenopodium amaranticolor and C. quinoa also showed chlorotic local lesions on inoculated leaves. For molecular identification, total RNA was isolated from 18 symptomatic plants using Trizol Reagent (Invitrogen, Carlsbad, CA). Reverse transcription (RT)-PCR was carried out using universal primer pairs that amplify the NIb-coat protein (CP) region including the 3'-untranslated regions (UTRs) of Potyvirus as described previously (1). An amplicon of approximately 1.7 kb was amplified and cloned using the pGEM-T Easy vector system (Promega, Fitchburg, WI). Two clones (GenBank Accession Nos. AB734777 and AB735585) with 99.9% sequence identity were selected for further analysis. These clones shared a maximum of 94% amino acid identity and 90% nucleotide identity in the CP region, and 93% nucleotide homology in the 3'-UTR with the 'TN1' (GenBank Accession No. U37076) strain of BCMNV isolated from Phaseolus vulgaris (2). These comparisons indicated that the viral isolates belong to the BCMNV species and are the causal agent of mosaic and necrosis observed on the sweet bean plants in Nepal. To our knowledge, this is the first report of BCMNV in Nepal and also the first report of BCMNV from sweet bean. References: (1) P. H. Berger et al. Arch. Virol. 142:1979, 1997. (2) J. Chen et al. Arch. Virol. 146:757, 2001. (3) Z. Hongying et al. Arch. Virol. 147:1257, 2002.

Screw versus staple fixation for Akin osteotomy.

INTRODUCTION: Akin osteotomies are commonly fixed with a screw or staple. Hardware-related symptoms are not uncommon. We compared the outcomes and costs of the two implants. METHODS: We evaluated 74 Akin osteotomies performed in conjunction with first metatarsal osteotomy for hallux valgus. The osteotomy was fixed with a headless compression screw in 39 cases and a staple in 35 cases. We looked at the implant-related complications, removal of metalwork, revision, non-union and cost. Pre- and postoperative hallux valgus interphalangeal (HI) angles and length of the proximal phalanx were measured. RESULTS: There was 100% union, no failure of fixation, no revision surgery and no delayed union in either group. The radiological prominence of screws was significant (p=0.02), but there was no significant difference in soft-tissue irritation (p=0.36) or removal of implants (p=0.49). Two cortical breaches (5.8%) occurred in staple fixation and 4 (10.2%) in screw fixation (not statistically significant (NS), p=0.50). The mean improvement in HI angle was 4.3° with screw fixation and 4.1° with staple fixation (NS, p=0.69). The mean shortening of the proximal phalanx was 2.5mm with screw fixation and 2.3mm with staple fixation (NS, p=0.64). The total cost was £1,925 for staple fixation and £4,290 for screw fixation. CONCLUSIONS: Staple and screw fixation are reproducible modalities with satisfactory outcomes, but screw fixation is expensive. We conclude staple fixation is a cost-effective alternative.

Association of a monopartite begomovirus and associated betasatellite with yellow vein disease of a weed host, Senna italica Mill. In Oman.

Senna italica Mill. plants exhibiting yellowing and stunting symptoms typical of begomovirus infection were collected in Oman. Molecular characterization using begomovirus and betasatellite primers in polymerase chain reaction followed by rolling circle amplification, cloning and analysis of sequences revealed the S. italica plants were infected by an isolate of Chilli leaf curl virus and tomato leaf curl betasatellite. The study describes the etiology of a yellow vein disease, identified for the first time, affecting a common weed in Oman.

Characterization of begomovirus components from a weed suggests that begomoviruses may associate with multiple distinct DNA satellites.

A begomovirus disease complex associated with Sonchus arvensis, a common weed in Pakistan was studied using cloning, nucleic acid sequencing and phylogenetic analysis. The complex associated with this weed consists of a monopartite begomovirus and several distinct betasatellites and alphasatellites. The monopartite begomovirus associated with yellow vein disease of Sonchus arvensis showed 95-99% nucleotide sequence identity with Alternanthera yellow vein virus (AlYVV) reported from China, Vietnam and India. Two betasatellites were isolated from S. arvensis: one sharing between 91.4 and 95.3% nucleotide sequence identity with isolates of Ageratum yellow leaf curl betasatellite (AYLCB), and the other sharing between 78.2 and 99.9% identity with isolates of Cotton leaf curl Multan betasatellite (CLCuMB). Two alphasatellites were identified: one was homologous to Potato leaf curl alphasatellite (PotLCuA), while the other was closely related to Hibiscus leaf curl alphasatellite (HLCuA). Thus, AlYVV in S. arvensis is associated with satellites shown previously to be associated with other begomoviruses in Pakistan. Our results suggest that monopartite begomoviruses may associate with distinct satellites that are prevalent in the region.

Molecular and biological characterization of Chilli leaf curl virus and associated betasatellite infecting Cucurbita maxima in Oman.

During a survey in February 2016, leaf curl disease symptoms were witnessed in Cucurbita maxima plants in Al-Batina commercial farm in Oman. Symptoms exhibited were characteristic of begomovirus infection as leaf curling, yellowing, and color breaking followed by mosaic pattern. The transmission electron microscopy confirmed the presence of typical twinned geminate typical of Geminate virus particles. Rolling circle amplification (RCA) was employed to characterize the unknown causal agent of C. maxima disease. In molecular identification RCA produced nearly 2.8 and 1.4 kb DNA molecules corresponding to begomovirus and satellite molecules, cloned and sequenced them. In Blast, species demarcation tool and phylogenetic analysis revealed the begomovirus and satellite isolates were determined as Chilli leaf curl virus (ChLCV) and tomato leaf curl betasatellite (ToLCB). In biological analysis by agrobacterium mediated inoculation, ChLCV displayed upward leaf curling and vein swelling symptoms in Nicotiana benthamiana plants; however, in presence of ToLCB enhanced downward leaf curling and crumpling symptoms were revealed. This study provides the first evidence that ChLCV and ToLCB caused leaf curl disease of C. maxima in Oman.

Frequent occurrence of Mungbean yellow mosaic India virus in tomato leaf curl disease affected tomato in Oman.

Next generation sequencing (NGS) of DNAs amplified by rolling circle amplification from 6 tomato (Solanum lycopersicum) plants with leaf curl symptoms identified a number of monopartite begomoviruses, including Tomato yellow leaf curl virus (TYLCV), and a betasatellite (Tomato leaf curl betasatellite [ToLCB]). Both TYLCV and ToLCB have previously been identified infecting tomato in Oman. Surprisingly the NGS results also suggested the presence of the bipartite, legume-adapted begomovirus Mungbean yellow mosaic Indian virus (MYMIV). The presence of MYMIV was confirmed by cloning and Sanger sequencing from four of the six plants. A wider analysis by PCR showed MYMIV infection of tomato in Oman to be widespread. Inoculation of plants with full-length clones showed the host range of MYMIV not to extend to Nicotiana benthamiana or tomato. Inoculation to N. benthamiana showed TYLCV to be capable of maintaining MYMIV in both the presence and absence of the betasatellite. In tomato MYMIV was only maintained by TYLCV in the presence of the betasatellite and then only at low titre and efficiency. This is the first identification of TYLCV with ToLCB and the legume adapted bipartite begomovirus MYMIV co-infecting tomato. This finding has far reaching implications. TYLCV has spread around the World from its origins in the Mediterranean/Middle East, in some instances, in live tomato planting material. The results here may suggest that begomoviruses which do not commonly infect tomato, such as MYMIV, could be spread as a passenger of TYLCV in tomato.

Aneurysms complicating pulmonary autograft procedure for aortic valve replacement.

Aneurysm formation in the left ventricular outflow tract related to the proximal end of the pulmonary autograft after the Ross procedure was present in 2 patients. Both occurred late after operation and were associated with prolapse of a leaflet of the autograft and significant regurgitation. Both were repaired with no immediate complications. There was no evidence of infection at time of operation. The probable mechanisms underlying this complication and the possibilities of avoiding it are discussed.

Results of the Ross operation in rheumatic versus non-rheumatic aortic valve disease.

BACKGROUND AND AIM OF THE STUDY: A total of 213 patients underwent the Ross operation at our institution between January 1990 and January 1999. Outcome was assessed in rheumatic (RH) patients and compared with that in patients with other etiology (non-RH). METHODS: After exclusion of 69 patients with a follow up of <18 months, the study group comprised 144 patients (119 RH, 25 non-RH). Patients were studied clinically and by echo-Doppler cardiography preoperatively, within 2 months and 6-8 months after surgery, and yearly afterwards. Preoperative assessment included age, gender, body surface area (BSA), type of aortic valve lesion and additional valve disease, left and right ventricular outflow tract (LVOT, RVOT) dimensions, and left ventricular (LV) size, function and mass. Postoperatively, patients were studied for presence and severity of autograft regurgitation, mitral regurgitation, LV size, function and mass, and incidence and timing of reoperation. RESULTS: On average, RH patients were older and had higher BSA, more aortic regurgitation than stenosis, more additional mitral valve disease (mostly regurgitation), larger LV size and poorer LV function. Mitral valve repair was performed in 24% of RH patients versus 0% of non-RH patients. Postoperatively, differences in LV size, function and mass remained present, but diminished during follow up. The autograft reoperation incidence was 22% (26/119) in RH patients versus 8% (2/25) in non-RH patients (p = NS). Preoperative predictors for reoperation in the RH group were severe concomitant mitral regurgitation (MR), followed by male gender and large indexed LVOT (all p<0.001 by discriminant analysis). CONCLUSION: Marked differences were present in patient characteristics between rheumatic and nonrheumatic patients who underwent the Ross operation. Rheumatic patients had a higher incidence of autograft reoperation. Severe concomitant MR was the most important predictor for reoperation in rheumatic patients.

A prospective study for evaluation of knee effusion after hip surgery.

Knee effusion is a common occurrence in elderly patients following hip surgery. We prospectively studied 106 patients undergoing proximal femoral surgery. These patients were examined pre-operatively, on days 4 and 8, and six weeks post operatively for the presence of an ipsilateral knee effusion. 71 patients had undergone Total Hip Arthroplasty (THA), 26 Dynamic Hip Screw (DHS) insertion and 9 had hemiarthroplasty. The incidence of pre-operative ipsilateral knee effusion was 13% (14 patients). 42% (44 patients) had an effusion at one week post operatively and 12% (13 patients) at 6 weeks. 57% (60 patients) of the study group had radiological evidence of osteoarthritis in their knees. However, the incidence of effusion in patients with and without arthritis was the same. The majority of these effusions resolved after six weeks with no residual effects.

Echocardiographic and Doppler study of patients with heatstroke and heat exhaustion.

This is a two-dimensional and Doppler echocardiographic study of the hemodynamic changes in patients with heatstroke and heat exhaustion. It demonstrates that the hemodynamic changes in severe heat exposure reflect a hyperdynamic circulation with tachycardia and high cardiac output states. Relative hypovolemia was more pronounced in patients with heatstroke compared to patients with heat exhaustion. Signs of peripheral vasoconstriction were more often present in patients with heatstroke, while patients with heat exhaustion more often demonstrated peripheral vasodilatation.