Genetic characterization of Chikungunya virus from New Delhi reveal emergence of a new molecular signature in Indian isolates

ABSTRACT: BACKGROUND: Chikungunya (CHIK) is currently endemic in South and Central India and exist as co-infections with dengue in Northern India. In 2010, New Delhi witnessed an outbreak of CHIK in the months October-December. This was the first incidence of a dominant CHIK outbreak in Delhi and prompted us to characterize the Delhi virus strains. We have also investigated the evolution of CHIK spread in India. FINDINGS: Clinical samples were subjected to RT-PCR to detect CHIK viral RNA. The PCR amplified products were sequenced and the resulting sequences were genetically analyzed. Phylogenetic analysis based on partial sequences of the structural proteins E1 and E2 revealed that the viruses in the latest outbreak exhibited ECSA lineage. Two novel mutations, E1 K211E and E2 V264A were observed in all Delhi isolates. In addition, CHIKV sequences from eight states in India were analyzed along with Delhi sequences to map the genetic diversity of CHIKV within the country. Estimates of average evolutionary divergence within states showed varying divergence among the sequences both within the states and between the states. We identified distinct molecular signatures of the different genotypes of CHIKV revealing emergence of a new signature in the New Delhi clade. Statistical analyses and construction of evolutionary path of the virus within the country revealed gradual spread of one specific strain all over the country. CONCLUSION: This study has identified unique mutations in the E1 and E2 genes and has revealed the presence of ancestral CHIKV population with maximum diversity circulating in Maharashtra. The study has further revealed the trend of CHIK spread in India since its first report in 1963 and its subsequent reappearance in 2005.     

The covalent modification and regulation of TLR8 in HEK-293 cells stimulated with imidazoquinoline agonists

The mammalian TLRs (Toll-like receptors) mediate the rapid initial immune response to pathogens through recognition of pathogen-associated molecular patterns. The pathogen pattern to which TLR8 responds is ssRNA (single-stranded RNA) commonly associated with ssRNA viruses. TLR8 also responds to small, purine-like molecules including the imidazoquinoline IRMs (immune-response modifiers). The IRMs include molecules that selectively activate TLR7, selectively activate TLR8 or non-selectively activate both TLR7 and TLR8. Using HEK-293 cells (human embryonic kidney cells) stably expressing an NF-kappaB (nuclear factor kappaB)/luciferase promoter-reporter system as a model system, we have examined the regulation of TLR8 using the non-selective TLR7/8 agonist, 3M-003. Using conservative tyrosine to phenylalanine site-directed mutation, we show that of the 13 tyrosine residues resident in the cytosolic domain of TLR8, only three appear to be critical to TLR8 signalling. Two of these, Tyr898 and Tyr904, reside in the Box 1 motif and the third, Tyr1048, lies in a YXXM putative p85-binding motif. TLR8 is tyrosine-phosphorylated following 3M-003 treatment and TLR8 signalling is inhibited by tyrosine kinase inhibitors. Treatment with 3M-003 results in the association of the p85 regulatory subunit of PI3K (phosphoinositide 3-kinase) with TLR8 and this association is inhibited by tyrosine to phenylalanine mutation of either the YXXM or Box 1 motifs. As a further consequence of activation by 3M-003, TLR8 is modified to yield both higher and lower molecular mass species. These species include a monoubiquitinated form as deduced from ubiquitin peptide sequencing by HPLC/MS/MS (tandem MS).     

Preservation of cGMP-induced relaxation of pulmonary veins of fetal lambs exposed to chronic high altitude hypoxia: role of PKG and Rho kinase

The roles of Rho kinase (ROCK) and cGMP-dependent protein kinase (PKG) in cGMP-mediated relaxation of fetal pulmonary veins exposed to chronic hypoxia (CH) were investigated. Fourth generation pulmonary veins were dissected from near-term fetuses ( approximately 140 days of gestation) delivered from ewes exposed to chronic high altitude hypoxia for approximately 110 days (CH) and from control ewes. After constriction with endothelin-1, 8-bromoguanosine 3',5'-cyclic monophosphate (8-Br-cGMP) caused a similar relaxation of both control and CH vessels. Rp-8-Br-PET-cGMPS (a PKG inhibitor) inhibited whereas Y-27632 (a ROCK inhibitor) augmented relaxation of control veins to 8-Br-cGMP. These effects were significantly diminished in CH veins. PKG protein expression and activity were greater whereas ROCK protein expression and activity were less in CH vessels compared with controls. Phosphorylation of threonine 696 (ROCK substrate) and serine 695 (PKG substrate) of the regulatory myosin phosphatase targeting subunit MYPT1 of myosin light chain (MLC) phosphatase was stimulated to a lesser extent in CH than in control veins by endothelin-1 (ROCK stimulant) and 8-Br-cGMP (PKG stimulant), respectively. The phosphorylation and dephosphorylation of MLC caused by endothelin-1 and 8-Br-cGMP, respectively, were less in CH veins than in controls. These results suggest that CH in utero upregulates PKG activity but attenuates PKG action in fetal pulmonary veins. These effects are offset by the diminished ROCK action on MYPT1 and MLC and thus lead to an unaltered response to cGMP.     

The genetic structure of taro: a comparison of RAPD and isozyme markers

Germplasm characterization and evolutionary process in viable populations are important links between the conservation and utilization of plant genetic resources. Here, an investigation is made, based on molecular and biochemical techniques for assessing and exploiting the genetic variability in germplasm characterization of taro, which would be useful in plant breeding and ex situ conservation of taro plant genetic resources. Geographical differentiation and phylogenetic relationships of Indian taro, Colocasia esculenta (L.) Schott, were analyzed by random amplified polymorphic DNA (RAPD) and isozyme of seven enzyme systems with specific reference to the Muktakeshi accession, which has been to be proved resistant to taro leaf blight caused by P. colocasiae. The significant differentiations in Indian taro cultivars were clearly demonstrated by RAPD and isozyme analysis. RAPD markers showed higher values for genetic differentiation among taro cultivars and lower coefficient of variation than those obtained from isozymes. Genetic differentiation was evident in the taro accessions collected from different regions of India. It appears that when taro cultivation was introduced to a new area, only a small fraction of genetic variability in heterogeneous taro populations was transferred, possibly causing random differentiation among locally adapted taro populations. The selected primers will be useful for future genetic analysis and provide taro breeders with a genetic basis for selection of parents for crop improvement. Polymorphic markers identified in the DNA fingerprinting study will be useful for screening a segregating population, which is being generated in our laboratory aimed at developing a taro genetic linkage map.     

Comparison of pathogenic properties of the murid gammaherpesvirus (MuHV 4) strains: a role for immunomodulatory proteins encoded by the left (5′-)end of the genome

The murid herpesvirus 4 (MuHV 4) species encompasses 7 isolates, out of which at least two (MHV-68, MHV-72) became in vitro propagated laboratory strains. Following intranasal inoculation, MuHV 4 induces an acute infectious mononucleosis-like syndrome with elevated levels of peripheral blood leukocytes, shifts in the relative proportion of lymphocytes along with the appearance of atypical mononuclear cells. At least two isolates exhibited spontaneous deletions at the left hand (5′-end) of their genome, resulting in the absence of M1, M2, M3 genes (strain MHV-72) and also of the M4 gene (strain MHV-76). Based on DNA sequence amplifications only, another two isolates (MHV-Šum and MHV-60) were shown to possess similar deletions of varying length. During latency (until 24 months post-infection), the mice infected with any MuHV 4 isolate (except MHV-76) developed lymphoproliferative disorders. The lack of tumor formation in MHV-76 infected mice was associated with persistent virus production at late post-infection intervals. In addition to careful analysis of spontaneously occurring 5′-end genome defects, our knowledge of the function of 5′-end genes relies on the behaviour of mutants with corresponding deletions and/or insertions. While M2 and M3 genes encode immune evasion proteins, M4 codes for a soluble glycopeptide acting as immunomodulator and/or immunostimulator.     

Exercise and diet enhance fat oxidation and reduce insulin resistance in older obese adults.

Older, obese, and sedentary individuals are at high risk of developing diabetes and cardiovascular disease. Exercise training improves metabolic anomalies associated with such diseases, but the effects of caloric restriction in addition to exercise in such a high-risk group are not known. Changes in body composition and metabolism during a lifestyle intervention were investigated in 23 older, obese men and women (aged 66 +/- 1 yr, body mass index 33.2 +/- 1.4 kg/m(2)) with impaired glucose tolerance. All volunteers undertook 12 wk of aerobic exercise training [5 days/wk for 60 min at 75% maximal oxygen consumption (Vo(2max))] with either normal caloric intake (eucaloric group, 1,901 +/- 277 kcal/day, n = 12) or a reduced-calorie diet (hypocaloric group, 1,307 +/- 70 kcal/day, n = 11), as dictated by nutritional counseling. Body composition (decreased fat mass; maintained fat-free mass), aerobic fitness (Vo(2max)), leptinemia, insulin sensitivity, and intramyocellular lipid accumulation (IMCL) in skeletal muscle improved in both groups (P < 0.05). Improvements in body composition, leptin, and basal fat oxidation were greater in the hypocaloric group. Following the intervention, there was a correlation between the increase in basal fat oxidation and the decrease in IMCL (r = -0.53, P = 0.04). In addition, basal fat oxidation was associated with circulating leptin after (r = 0.65, P = 0.0007) but not before the intervention (r = 0.05, P = 0.84). In conclusion, these data show that exercise training improves resting substrate oxidation and creates a metabolic milieu that appears to promote lipid utilization in skeletal muscle, thus facilitating a reversal of insulin resistance. We also demonstrate that leptin sensitivity is improved but that such a trend may rely on reducing caloric intake in addition to exercise training.     

HP1-β is required for development of the cerebral neocortex and neuromuscular junctions

HP1 proteins are thought to be modulators of chromatin organization in all mammals, yet their exact physiological function remains unknown. In a first attempt to elucidate the function of these proteins in vivo, we disrupted the murine Cbx1 gene, which encodes the HP1-β isotype, and show that the Cbx1^−/−-null mutation leads to perinatal lethality. The newborn mice succumbed to acute respiratory failure, whose likely cause is the defective development of neuromuscular junctions within the endplate of the diaphragm. We also observe aberrant cerebral cortex development in Cbx1^−/− mutant brains, which have reduced proliferation of neuronal precursors, widespread cell death, and edema. In vitro cultures of neurospheres from Cbx1^−/− mutant brains reveal a dramatic genomic instability. Our results demonstrate that HP1 proteins are not functionally redundant and that they are likely to regulate lineage-specific changes in heterochromatin organization.     

Phagostimulatory sugars enhance uptake and horizontal transfer of hexaflumuron in the western subterranean termite (Isoptera: Rhinotermitidae)

Western subterranean termite, Reticulitermes hesperus Banks (Isoptera: Rhinotermitidae), workers fed more on paper disks treated with the carbohydrates xylose, ribose, and fructose than on untreated disks. This feeding behavior of termites for certain carbohydrates was used to demonstrate an increase in the uptake and transfer of the insect growth regulator hexaflumuron among termites. The addition of 3% xylose to paper disks significantly increased the uptake of [14C]hexaflumuron and its subsequent transfer to other termites. Similarly, there was a significant increase in mortality of termites fed on paper disks treated with [14C]hexaflumuron (0.1 and 0.5%) in combination with 3% xylose for similar time periods compared with termites feeding on [14C]hexaflumuron (0.1 and 0.5%) alone. Overall percentage of mortality of termites feeding on [14C]hexaflumuron in combination with 3% xylose during the duration of the study (25-30 d) was approximately equal to 70%.     

Concentration-dependent degradation of three termiticides in soil under laboratory conditions and their bioavailability to eastern subterranean termites (Isoptera: Rhinotermitidae)

Degradation and bioavailability of imidacloprid, fipronil, and bifenthrin applied at label rates ([AI], wt:wt in soil) in the loamy soil of Nebraska were determined over a 6-mo duration. Based on the calculated half-lives of the three termiticides, it was concluded that the degradation rate was lowest when a termiticide was applied at the highest label rate. Bioassays of Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) conducted at 8, 31, 65, 90, 135, 160, and 180 d posttreatment showed an inverse relationship between the LT90 values and the variable concentrations. At day 180, exposures to all the termiticide-treated soil samples (concentration x termiticide) resulted in 100% mortality of R. flavipes workers. However, lower LT90 values were observed for termites exposed to soils treated with highest label rates even when the treated soils were aged in the lab for 6 mo. This suggested a higher bioavailability of these three termiticides when applied at higher application rates. Termite mortality was fastest for bifenthrin followed by fipronil and imidacloprid.     

Effect of red- and blue-light-emitting diodes on growth and morphogenesis of grapes

The effect of red- and blue-light-emitting diodes on shoot and root growth of Hybrid Franc grape, a rootstock cultivar, along with two other grape genotypes, Kadainou R-1 and Vitis ficifolia var. ganebu, were investigated in vitro. Plants cultured under red-light-emitting diodes produced the longest shoots with longer internodes for all genotypes. The chlorophyll content measured as SPAD value and leaf number per explant were highest on plants cultured under blue-light-emitting diodes in all the genotypes. Blue light was also responsible for a higher number of stomata in all the genotypes; however, there was no significant difference in size of stomata in all genotypes under the different light conditions tested. Different light-emitting diodes did not affect the rooting percentage of Hybrid Franc but red-light-emitting diodes gave a higher rooting percentage along with higher root numbers for the two other grape genotypes.