Characterization and in silico proteomic analysis of C2 and C3 proteins of squash leaf curl China virus associated with pumpkin leaf curl disease in Assam, India.

The pumpkin leaf curl disease is an emerging disease of pumpkin in Assam, India. Symptomatic pumpkin leaf samples from different locations were immunologically tested using Begomovirus specific antibody. PCR with the ELISA-positive samples, using Geminivirus universal primers amplified 1.4 kb virus-specific fragments. Sequence of these amplicons showed around 95% identity with squash leaf curl China virus-[Pumpkin: Varanasi] (SLCCV-Pumpkin: Varanasi EU573715). To investigate the possible functions of the viral proteins present in the fragment, the full-length C2 and C3 genes were conceptually translated and were subjected to in silico proteomic analyses. The phylogenetic analysis of both the proteins divulged the relationship of our isolate with related viruses and isolates. Multiple sequence alignment (MSA) of the proteins revealed the presence of the known viral conserved motifs, viz., zinc-finger (ZNF) motif [36CXCX(7)CX(6)H53], the arginine-rich nuclear localization signal (NLS) motif (28RRRR31) as well as the minimal activation domain in C2 protein. In the C3 protein, the 91LKYLD95 and the replication enhancer motif (30YFK32) were found to be conserved. Finally, 3-D models of the two proteins were predicted via ab initio approach and subsequently, the models were validated. To our knowledge, this study is a pioneering attempt to construct the ab initio 3-D models of two begomoviral proteins taking a SLCCV isolate as a model. Keywords: begomovirus; ELISA; ZNF motif; NLS motif; ab initio modelling.

Insights into the control of geminiviral promoters.

Geminiviruses constitute one of the largest groups of plant viruses, having characteristic twinned geminate particles encapsidating small circular single-stranded DNA molecules. Geminiviral promoters are generally located within the intergenic region, although promoters have also been detected within the genes. Similarly, the geminivirus-associated betasatellite also harbours a promoter element for driving the expression of its only ORF. These regulatory elements of geminiviral and satellite origins have been subject of great interest to develop heterologous gene expression modules. Geminiviral promoter and regulatory elements show a complex regulation that is mediated by several host as well as viral proteins. Here, the structural and functional features of geminiviral and satellite promoters are discussed along with their regulation by plant and viral proteins. Although generalization in many cases is difficult and demands further studies, a pattern is seen to emerge on the regulation of the promoters.

Analysis of full-length sequences of two Citrus yellow mosaic badnavirus isolates infecting Citrus jambhiri (Rough Lemon) and Citrus sinensis L. Osbeck (Sweet Orange) from a nursery in India.

Citrus yellow mosaic badna virus (CMBV), a member of the Family Caulimoviridae, Genus Badnavirus is the causative agent of mosaic disease among Citrus species in southern India. Despite its reported prevalence in several citrus species, complete information on clear functional genomics or functional information of full-length genomes from all the CMBV isolates infecting citrus species are not available in publicly accessible databases. CMBV isolates from Rough Lemon and Sweet Orange collected from a nursery were cloned and sequenced. The analysis revealed high sequence homology of the two CMBV isolates with previously reported CMBV sequences implying that they represent new variants. Based on computational analysis of the predicted secondary structures, the possible functions of some CMBV proteins have been analyzed.

Efficacy of Ovsynch protocol with antiprolactin treatment for timed artificial insemination during non-breeding seasons in yaks (Poephagus grunniens L.).

An attempt was undertaken to investigate the efficacy of Ovsynch protocol for timed artificial insemination (TAI) with or without Norprolac (antiprolactin) treatment during non-breeding season (winter months) in yaks (n = 25). During non-breeding season, plasma prolactin profile has been reported high due to cold and nutritional stress. The Norprolac dose of i.m. administration was standardized for prolactin suppression. Three different doses viz. 2.5, 5.0 and 7.5 mg were attempted and the dose of 7.5 mg Norprolac i.m. per animal was found to be suitable for suppression of prolactin levels up to 30 h. Ovsynch treatment with Norprolac induced more number of oestrous symptoms per animal (4.8 vs 2.1), higher LH peak concentration (24.01 vs 16.16 ng/ml), longer duration of LH surge (6.8 vs 5.2 h) and higher conception rate (70 vs 30%) in Ovsynch plus Norprolac treated animals compared with animals treated with Ovsynch alone. Therefore, this study clearly indicates the opportunity for practical application of the Ovsynch plus Norprolac protocol for TAI in yaks during non-breeding seasons.

A Geminivirus-Satellite Complex is Associated with Leaf Deformity of Mentha (Mint) Plants in Punjab.

A widespread leaf deformity disease of mentha (mint), accompanied by whiteflies, the vectors of begomoviruses, was observed in Punjab in the last few years. The presence of begomovirus was indicated by DNA dot-blot analysis using the conserved coat protein and replication-associated protein genes of another begomovirus, Sri Lankan cassava mosaic virus (SLCMV). A DNA fragment (2.0 kb), representing a partial genomic DNA of a begomovirus, amplified from the symptomatic mentha leaves was used to design end-primers and further amplify an additional 0.9 kb fragment, representing the remaining portion of the resident viral DNA. The two sequences, assembled together (2.7 kb), showed that they represented the complete sequence of an isolate of Tomato leaf curl Karnataka virus (ToLCKV) DNA. Using universal betasatellite primers, a 1.4 kb fragment was amplified from the same sample. This cloned DNA fragment showed complete sequence identity with the previously reported Cotton leaf curl Multan betasatellite (CLCuMB). Majority of the symptomatic mentha leaf samples, collected from four districts of Punjab, showed cross-hybridization in DNA dot-blot using cloned SLCMV and CLCuMB DNA, indicating the presence of one or more begomoviruses related to SLCMV and the betasatellite, CLCuMB. The begomovirus and betasatellite could be mechanically transmitted to Nicotiana benthamiana. Whitefly transmission of the resident begomovirus could also be demonstrated on mentha. The evidence indicates the association of ToLCKV and CLCuMB, a hitherto new combination of a begomovirus and a betasatellite associated with a leaf deformity disease in mentha in Punjab.

Plasma concentrations of 13, 14-dihydro-15-keto-prostaglandin F2alpha, progesterone and cortisol during periparturient period in yaks (Poephagus grunniens L.).

An attempt was made to determine plasma concentrations of, 13, 14-dihydro-15-keto-prostaglandin F(2alpha) (PGFM), cortisol and progesterone during periparturient period in yak. Plasma PGFM level showed an increasing trend beginning day 5 prior to parturition (0.48 +/- 0.14 ng/ml) and increased steeply thereafter to reach a peak level (17.16 +/- 1.31 ng/ml) on the day of parturition. The levels, then, declined sharply on day 1 postpartum to reach 1.20 +/- 0.40 ng/ml and thereafter declined gradually over the days to reach 0.28 +/- 0.09 ng/ml on day 20 postpartum and this level was maintained with fluctuation within narrow limits thereafter till conclusion of the blood sampling on day 90 post-calving. The plasma progesterone concentration on days 7 and 6 before parturition was high (2.10 +/- 0.10 and 2.12 +/- 0.10 ng/ml, respectively). The level then decreased gradually and abrupt fall was observed 1-2 days before parturition and became basal on day of parturition (0.24 +/- 0.04 ng/ml). This basal level was maintained till the end of the blood sampling on day 90 postpartum. Plasma cortisol level showed an increasing trend beginning day 2 prior to parturition (2.36 +/- 0.65 ng/ml) and increased steeply thereafter to reach a peak level (26.65 +/- 5.28 ng/ml) on the day of parturition. The levels, then, declined gradually over the days and touched 2.36 +/- 0.25 ng/ml on day 3 postpartum and this level was maintained with fluctuation within narrow limits thereafter till day 7 post-calving.

Season of the year influences semen output and concentrations of testosterone in circulation of yaks (Poephagus grunniens L.).

Seasonal changes in plasma testosterone concentration and semen quality were evaluated in yak bulls throughout a 1-year period. Blood samples were collected every week from adult yak bulls (n=15). These blood samples were analyzed for testosterone using a highly sensitive enzyme-linked immunoassay. Ejaculates were collected from five representative bulls each week. Ejaculate volume, progressive motility, live sperm count and sperm concentrations were determined. Mean testosterone in plasma was 1.03+/-0.25 ng/ml. Concentrations of testosterone changed throughout the year (P<0.05) and were found to be highest during the winter. It was also higher during the autumn than in summer and spring (P<0.05). Mean ejaculate volume, progressive motility, live sperm count and spermatozoa concentration were 2.7+/-0.3 ml, 72.8+/-1.4%, 82.3+/-0.9% and 968+/-233 x 10(6)ml(-1), respectively. Ejaculate volume and sperm concentration were higher (P<0.05) in autumn than in other seasons. To conclude, a highly sensitive EIA for testosterone was developed and validated for yak plasma. Seasonal changes in semen quality were associated with changes in the concentration of testosterone in plasma from yak bulls.

Strategies to optimize reproductive efficiency by regulation of ovarian function in yak (Poephagus grunniens L.).

Ovulatory response to the first GnRH of Ovsynch is the critical determinant for successful synchronization of ovulation in animals. An attempt was made in this study to design a pre-Ovsynch hormonal strategy in yaks to increase the ovulatory response to the first GnRH injection of Ovsynch so that overall synchronization rate to Ovsynch could be improved. Non-lactating cyclic yak cows (n=33) were assigned to receive either no treatment before Ovsynch (control) or 0.375 mg of PGF2alpha (PreP) followed 2 d later by 10 microg of GnRH (PreG), administered 4 (G4G), 5 (G5G), or 6 (G6G) d before initiating the Ovsynch protocol. Rectal palpation was performed to assess ovulation and blood samples were collected to measure progesterone concentrations during pre-treatment, treatment and post-treatment periods. All the animals received timed AI 12 and 24h after the final GnRH of Ovsynch. Diagnoses for pregnancy were performed by rectal palpation and profiles of plasma progesterone 35 d after AI. Percentage of yak cows that ovulated in response to the first GnRH injection of Ovsynch, synchronized to Ovsynch treatment, had a functional CL at PGF2alpha of Ovsynch, had circulating concentrations of P4 at PGF2alpha of Ovsynch and likely to be pregnant after 35 d after AI, were greater in G6G and G5G compared with control, whereas G4G did not differ from controls. In addition, animals that ovulated in response to first GnRH of Ovsynch had greater response to PGF2alpha of Ovsynch and greater synchronization rate to the overall protocol than those that did not ovulate. In summary, PGF2alpha-and-GnRH-based pre-Ovsynch strategies consisting of 5 or 6-d interval between PreG and first GnRH of Ovsynch resulted in a greater ovulatory and luteolytic response to first GnRH andPGF2alpha of Ovsynch, respectively, compared with control animals. These, in turn, optimized synchronization rate to Ovsynch in yaks.

Application of sensitive enzymeimmunoassay for determination of testosterone in blood plasma of yaks (Poephagus grunniens L.).

As an alternative to radioimmunoassays, a simple, highly sensitive and quick enzymeimmunoassay (EIA) for determination of testosterone in blood plasma of yaks on microtitreplates using second antibody coating technique and testosterone-horseradish peroxidase as a label has been developed for the first time. The wells of the microtitreplate were coated with affinity-purified goat immunoglobulin (antirabbit IgG) that binds the hormone specific antibody. The EIA was carried out directly in 20 mICROl of plasma after 1:10 dilution with assay buffer. The testosterone standard curve ranged from 0.2 to 200 pg/well. The sensitivity of the assay was 0.20 pg/well. Testosterone standard curve in buffer showed parallelism with serially diluted yak plasma containing high endogenous testosterone. Intra- and inter-assay coefficients of variation (CV) determined using pooled plasma was found 5.24 and 8.54%, respectively. Recovery of known concentrations of added testosterone in charcoal stripped plasma was linear (r=0.98). For biological validation of testosterone enzymeimmunoassay, the blood samples were collected from yak cows at -2h before and thereafter at 2h interval until 24h. after gonadotropin releasing hormone (GnRH) administration. There was a rapid increase (p<0.01) of luteinizing hormone (LH) and testosterone 2 and 6h after GnRH administration. Plasma testosterone concentration in normal adult yak bulls was found to be 0.52+/-0.09 ng/ml. In conclusion, the EIA developed in this study is simple, highly sensitive, valid and sufficiently reliable method for estimation of testosterone directly in yak plasma.