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Bluetongue virus (BTV) infects almost all the domestic and wild ruminants though the clinical disease is most commonly reported in sheep and some species of deer. Goat and cattle are the most common asymptomatic reservoir of the virus. Full genome sequencing and serological characterization of the virus isolates are emphasized for understanding the phylogenetic relationship and molecular epidemiology of bluetongue (BT). In this study, we report phylogenetic and phenotypic antigenic relationship of a BTV serotype-16 (PDP2/13/Ind) recovered from an apparently healthy goat from the state of Uttarakhand, a hilly terrain of sub-Himalayan India with four other BTV-16 isolates. The full genome sequence data was analyzed and the phylogenetic relationship of the goat isolate with other BTV-16 was established. Phylogenetic analysis revealed cluster of PDP2/13/Ind along with other Indian BTV-16 isolates indicating their close ancestral relationship. A cohesive ancestral relationship, irrespective of the genome segments analyzed, was also observed between Indian and Mediterranean BTV-16. The mean substitution rate of different segments of BTV-16 isolates varied from 3.231 × 10-5 (seg-2) to 1.129 × 10-3 (seg-6) substitutions per site per year. Timescale analysis indicated that all the segments had an older ancestor. No statistically significant geographic structuring of BTV-16 isolates was observed indicating frequent gene flow. The goat isolate shares highest identity (99.5%-99.8%) with G53/ABT/HSR, a BTV-16 recovered from the western part of the country whereas high level of divergence (11.9%-33.3%) at genomic segment level was observed with a Nigerian BTV-16 (NIG1982/10). Phenotypic antigenic relationship (r) of PDP2/13/Ind with other isolate-specific hyperimmune serum (HIS) determined from serum neutralization titer was 0.672 ± 0.058 to 0.948 ± 0.09. On other hand, the calculated 'r' score was 0.636 ± 0.063 to 0.814 ± 0.201 when HIS against PDP2/13/Ind was used to neutralize the other BTV-16 isolates. The percentage antigenic similarity (R) of the PDP2/13/Ind with other BTV-16 isolates was 65.39 ± 5.38-87.67 ± 14.86. Data suggests presence of subtype antigenic variation amongst the BTV-16 isolates recovered from the goats of a geographically restricted area of the state of Uttarakhand, India.

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AIM: The aim of the study was to characterize bluetongue virus serotype 16 (BTV-16), recently isolated from different states of India. The evolutionary relationship of newly isolated BTV-16 and previously reported Indian and global BTV-16 isolates were compared using molecular analysis. MATERIALS AND METHODS: In the present study, five (n=5) BTV-16 isolates were used to amplify gene segment-2 and segment-6 encoding the outer capsid proteins VP2 and VP5, respectively. The amplified products were purified and sequenced by the Sanger sequencing method. The phylogenetic relationship and nucleotide identity of all five BTV-16 isolates were compared with previously reported Indian and global BTV-16 isolates. Nucleotide sequence data were aligned using the CLUSTAL W algorithm implemented in the MegAlign of DNASTAR program package (MegAlign 5.00, DNASTAR Inc., Madison, USA). Phylogenetic analyses were carried out using MEGA version 6.0 software with the best nucleotide substitution model. RESULTS: Phylogenetic analysis based on the VP2 and VP5 encoding genes, segregates Indian BTV-16 isolates in a distinct cluster with proximity to the Eastern topotype. Indian isolates make a monophyletic cluster with Eastern topotypes with Western topotype BTV-16 (BTV-16/NIG/AJ586694) occupying a separate cluster. Indian isolates were found to share 91.5%-97.5% and 96.5%-98.9% identity at the nucleotide and deduced amino acid (aa) level, respectively, to the global BTV-16 isolates. There is a high degree of variation with the Nigerian isolate with 27.0-27.7% and 26.0-26.9% at the nucleotide and aa sequence level, respectively. These data suggest that Indian BTV-16 isolates might have evolved separately within the Eastern BTV topotype. CONCLUSION: Phylogenetic analyses and nucleotide identity of BTV-16 isolates at the VP2 and VP5 gene encoded level indicate that isolates used in the present study might have evolved from a common Eastern topotype ancestor. The data presented in this study will be helpful for future selection of reference strains in a serological and molecular epidemiology study.

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Cotton is mostly cultivated under rain-fed conditions in India, thus faces frequent drought conditions during its life cycle. Drought being a major stress factor responsible for yield penalty, there has always been a high priority to generate knowledge on adaptation and tolerance of cotton. In the present study, four cotton varieties, JKC-770 and KC-2 (Gossypium hirsutum), and JKC-717 and RAHS-187(Gossypium herbaceum), were imposed to drought. Under drought condition, differential changes in physiological characters like net photosynthesis, transpiration, stomatal conductance, chlorophyll fluorescence, relative water content (RWC), and predawn water potential (ψ 0) showed a change. While proline, malondialdehyde (MDA), and glutathione-S-transferase (GST) content increased along with a concomitant change in the expression of their associated genes. Under moderate stress, tolerant varieties maintain lower ψ 0 probably due to higher proline content as compared to sensitive varieties. Cyclic electron flow (CEF) also plays an important role in tolerance under mild water stress in G. hirsutum varieties. CEF not only activates at high light but also initiates at a very low light intensity. Expression analysis of genes reveals that drought-tolerant varieties showed enhanced detoxifying mechanism by up-regulation of asparagine synthase (AS), glutathione-S-transferase (GST), and methyl glyoxalase (GlyI) genes under drought stress. Up-regulation of Δ(1)-pyrroline-5-carboxylase synthase (Δ(1)P5CS) enhanced accumulation of proline, an osmolyte, under drought in tolerant varieties. While the drought-sensitive varieties showed up-regulation of ethylene responsive factor (ERF) and down-regulation of WRKY70 responsible for senescence of the leaf which correlated well with the high rate of leaf fall in sensitive varieties under water stress.

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The response and the functioning of the photosynthetic machinery of cotton, Gossypium hirsutum during water stress was studied by leaf optical properties, linear (ETRII) and cyclic electron flow (CEF) and chlorophyll a fluorescence. We observed that in G. hirsutum, during water limitation, Chlorophyll b showed the best correlation with reflectance at 731 nm and is a better indicator of drought. Fv /Fm was observed to be very insensitive to mild water stress. However, during severe water stress the leaves exhibit considerable inhibition in Fv /Fm and an increase in anthocyanin levels by about 20-fold. CEF was very responsive to mild water stress. The mild drought stress caused large decrease in the ability of the leaves to utilize the light energy. Photosystem I and photosystem II is protected from photoinhibition by high CEF and nonphotochemical quenching under mild water stress. While during severe drought stress, linear electron flow showed a sharp decrease in comparison to CEF. CEF play a major role in G. hirsutum leaves during mild as well as under severe water stress condition and is thus a good indicator of water stress.

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Layered nanohybrids (LNH) are a promising nonviral system allowing controlled drug and DNA delivery. In order to test the toxicity of LNH consisting of a magnesium/aluminum core, mice were subjected to subcutaneous, intraperitoneal, and intravenous injections of these nanoparticles at three doses. Intravenous injections resulted in 8% (1 out of 12) lethality at doses 100 micro l and 200 micro l of 6.96 x 10(- 4) M solution, while all mice survived after LNH administration by any other routes. Histopathological alterations were limited to mild localized inflammatory lesions in the lungs and the dermis after intravenous and subcutaneous administration, respectively. LNH labeled with Lucifer Yellow were readily detectable in both locations by fluorescent microscopy. To test their potential for intravital imaging, LNH-Lucifer Yellow were injected into the ovarian bursa and successfully visualized by multiphoton microscopy within the ovarian surface epithelial cells. In similar experiments, the ovary and the ovarian bursa were readily detectable by magnetic resonance imaging after administration of modified LNH, where aluminum was substituted for gadolinium. Taken together, these results demonstrate minimal in vivo toxicity of LNH and illuminate their potential as multifunctional nanoscale particles suitable for combination of intravital biomedical imaging with controlled drug release.

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