Implications of a conserved region of bluetongue virus protein VP2 in cross-neutralisation of bluetongue virus serotypes.

Bluetongue (BT) is a vector-borne disease of ruminants caused by Bluetongue virus (BTV). Twenty-nine different serotypes of BTV are currently reported throughout the world. The main objective of this study is the development of a subunit vaccine model that could potentially be adapted to provide broad spectrum protection against multiple BTV serotypes, which the conventional vaccines fail to address. To this end, three different BTV proteins (conserved region of viral protein [VP]2, VP5 and NS1) were expressed and purified in an Escherichia coli expression system. The immunogenicity of these proteins was tested in murine models using the MontanideTM ISA 201 VG adjuvant. BALB/c mice were immunised thrice (with individual proteins and a mixture of three proteins) at two-week intervals and were monitored until Day 40 post-infection/vaccination. Protein-specific antibodies directed against the recombinant proteins were detected by indirect enzyme-linked immunosorbent assay. Neutralising antibody (Nab) titres and cross-neutralisation against a range of BTV serotypes (BTV-1, -2, -4, -5, -9, -10, -12, -16, -21, -23 and -24) were determined by serum neutralisation test. The recombinant proteins elicited higher Nab titres compared with the inactivated vaccine group, except for BTV-1, where the inactivated vaccine group elicited higher Nab titres. Additive effect of the three proteins was not observed as the Nab titres generated with a combination of conserved VP2, VP5 and NS1 was similar to those of the individual protein groups. Whilst BTV-12 could only be neutralised by serum raised against the inactivated vaccine group, BTV-5 and -24 could not be neutralised by any of the groups tested. Our cumulative data suggest that the conserved regions of VP2 (cVP2), VP5 and NS1 could play an important part in the novel vaccine design against multiple BTV serotypes. Importantly, given that VP2 was already known to elicit a serotype-specific immune response against BT, we report, for the first time, that the conserved region of VP2 has the ability to induce cross-protective immune response.

Contrasting selective patterns across the segmented genome of bluetongue virus in a global reassortment hotspot.

For segmented viruses, rapid genomic and phenotypic changes can occur through the process of reassortment, whereby co-infecting strains exchange entire segments creating novel progeny virus genotypes. However, for many viruses with segmented genomes, this process and its effect on transmission dynamics remain poorly understood. Here, we assessed the consequences of reassortment for selection on viral diversity through time using bluetongue virus (BTV), a segmented arbovirus that is the causative agent of a major disease of ruminants. We analysed ninety-two BTV genomes isolated across four decades from India, where BTV diversity, and thus opportunities for reassortment, are among the highest in the world. Our results point to frequent reassortment and segment turnover, some of which appear to be driven by selective sweeps and serial hitchhiking. Particularly, we found evidence for a recent selective sweep affecting segment 5 and its encoded NS1 protein that has allowed a single variant to essentially invade the full range of BTV genomic backgrounds and serotypes currently circulating in India. In contrast, diversifying selection was found to play an important role in maintaining genetic diversity in genes encoding outer surface proteins involved in virus interactions (VP2 and VP5, encoded by segments 2 and 6, respectively). Our results support the role of reassortment in driving rapid phenotypic change in segmented viruses and generate testable hypotheses for in vitro experiments aiming at understanding the specific mechanisms underlying differences in fitness and selection across viral genomes.

A decade of research on Bluetongue virus in Andhra Pradesh, a Southern state of India.

High sheep population density, congenial climatic conditions for Culicoides propagation, and susceptible sheep breeds may be contributing to the higher incidence of Bluetongue (BT) in Southern states of India. Sheep farming in this part of the country is nomadic in nature and BT is one of the major infectious diseases inflicting huge losses. Andhra Pradesh is one of the Southern states with high sheep population in India. Although isolation studies in this region were started in 1993, concerted efforts only began in 2002. More than 50 isolates were obtained in the last decade, and 7 Bluetongue virus (BTV) serotypes (1, 2, 9, 10, 12, 16 and 21) were isolated. Among them, BTV-10, BTV-12, and BTV-21 were reported for the first time from India and the genome analysis of these viruses revealed that BTV-10 and BTV-12 have high sequence identity with the modified live virus (MLV) vaccines used in USA and South Africa, respectively. At the same time, BTV-21 has probably originated from Southeast Asia. Furthermore, some of the BTV isolated from Europe have high sequence identity with viruses isolated from Andhra Pradesh indicating common ancestry. The analysis of different isolates involved in outbreaks revealed that more than 1 BTV serotype is involved and that mixed infections with different serotypes is not uncommon. In a limited study conducted during 2005-2009, it was observed that most of the sheep seroconverted to more than 1 serotype, which further supports circulation of multiple serotypes and mixed infections in Andhra Pradesh. Based on the virus isolation data, in this study it was observed that a few serotypes dominate for 3-4 years followed by domination of others. Continuous monitoring of circulating serotypes is essential to understand the distribution and spread BTV in endemic areas and for devising suitable control measures.

Acute antidepressant effects of intramuscular versus intravenous ketamine.

OBJECTIVE: Conventional antidepressants take two weeks before their therapeutic action begins. Recent studies have reported on the rapid antidepressant effect of ketamine when given as an intravenous (I.V.) infusion. Little is known about its intramuscular (I.M.) use in depression. Hence this study was conducted to compare the safety, tolerability and efficacy of I.M. versus. I.V. ketamine in Major Depression (ICD-10). MATERIALS AND METHODS: It was a randomized open label parallel group study in a tertiary care teaching hospital. Study sample consisted of 27 subjects having major depression divided randomly into three groups of nine subjects each. Ketamine administered to each group in the dose of 0.5 mg/kg as an I.V. infusion, as 0.5 mg/kg I.M. or 0.25 mg/kg I.M. respectively. Depression rated on the Hamilton Depression Rating Scale (HAM-D) before the injection, two hours later, the next day, and after three days. Data analyzed using the Statistical Package for Social Sciences (SPSS). RESULTS: Mean age of the sample was 36.81 years (SD 11.815). Two hours after the injection, HAM-D fell by 58.86%, 60.29% & 57.36% in each group respectively. The improvement was sustained for next three days. Adverse effects noticed were rare, of mild nature and transient, lasting less than an hour. CONCLUSIONS: Intramuscular ketamine in the dose of 0.25 mg/kg is as effective and safe as 0.5 mg/kg given either I.M. or I.V., substantially alleviating depressive symptoms within a few hours and sustained for 3 days.

DNA mismatch repair MSH2 gene-based SNP associated with different populations.

We screened for the major essential single-nucleotide polymorphism (SNP) variant that might be associated with the MSH2 gene based on the data available from three types of human tissue samples [156 lymphoblastoid cell variations (LCL), 160 epidermis, 166 fat]. An association analysis confirmed that the KCNK12 SNP variant (rs748780) was highly associated (p value 9 × 10(-4)) with the MSH2 gene for all three samples. Using SNP identification, we further found that the recognized SNP was also relevant among Hapmap populations. Techniques that display specific SNPs associated with the gene of interest or nearby genes provide more reliable genetic associations than techniques that rely on data from individual SNPs. We investigated the MSH2 gene regional linkage association with the determined SNP (rs748780), KCNK12 variant (Allele T>C) in the intronic region, in HapMap3 full dataset populations, Yoruba in Ibadan, Nigeria (YRI), Utah residents with ancestry from northern Europe (CEU), Han Chinese in Beijing, China (CHB), and a population of Mexican ancestry in Los Angeles, California (MEX). A gene-based SNP association analysis analyzes the combined impact of every variant within the gene while creating referrals to linkage disequilibrium or connections between markers. Our results indicated that among the four populations studied, this association was highest in the MEX population based on the r(2) value; a similar pattern was also observed in the other three populations. The relevant SNP rs748780 in KCNK12 is related to a superfamily of potassium channel pore-forming P-domain proteins as well as to other non-pore-forming proteins and has been shown to be relevant to neurological disorder predisposition in MEX as well as in other populations.

Atorvastatin prevents sepsis-induced downregulation of myocardial ß1-adrenoceptors and decreased cAMP response in mice.

Impaired cardiac ß-adrenoceptor signaling is an important cause of sepsis-induced myocardial depression in man and experimental animals. We examined the effect of atorvastatin (ATR) pretreatment on myocardial ß1-adrenoceptor (ß1-AR) expressions and post-receptor signaling in a mouse model of sepsis (cecal ligation and puncture [CLP]). After 20 ± 2 h of surgery, hearts were isolated for the measurement of left ventricular functions (left ventricular developed pressure, dp/dt(max) and dp/dt(min)) using Langendorff setup. Western blot was used to determine ß1-AR and G protein-coupled receptor kinase 2 protein expressions. Real-time polymerase chain reaction was done to determine ß1-AR mRNA expression. Atorvastatin prevented sepsis-induced decrease in left ventricular functions, such as left ventricular developed pressure (CLP 75.90 ± 0.53 vs. ATR 100.24 ± 1.64 mmHg), dp/dtmax (CLP 3,742 ± 71 vs. ATR 4,291 ± 88 mmHg/s), and dp/dt(min) (CLP -1,010 ± 24 vs. ATR -1,346 ± 84 mmHg/s). Associated with functional impairments, sepsis decreased both myocardial ß1-AR protein and mRNA expressions by 52% ± 9% and 62% ± 7%, respectively. However, ATR treatment of CLP mice (ATR) preserved ß1-AR protein (96% ± 11%) and mRNA (88% ± 14%) expressions comparable to sham-operated level. Furthermore, it not only attenuated sepsis-induced decrease in basal cardiac adenosine 3',5'-cyclic monophosphate content (CLP 1.30 ± 0.27 vs. ATR 6.30 ± 0.67 pmol/mg protein), but also prevented its refractoriness to dobutamine stimulation (CLP 1.72 ± 0.27 vs. ATR 10.83 ± 1.37 pmol/mg protein). Atorvastatin also inhibited sepsis-induced increase in cardiac G protein-coupled receptor kinase 2 protein expression (CLP 1.73 ± 0.18-fold vs. ATR 1.10 ± 0.18-fold), protein kinase A activity (CLP 1.12 ± 0.14 vs. ATR 0.66 ± 0.08 U/mg protein) and plasma catecholamines (CLP 138 ± 22 vs. ATR 59 ± 2 pg/mL). In conclusion, ATR seems to improve left ventricular functions in vitro through the preservation of ß(1)-AR signaling in sepsis.

Assessment of the suitability of Tinopal as an enhancing adjuvant in formulations of the insect pathogenic fungus Beauveria bassiana (Bals.) Vuillemin.

BACKGROUND: Biopesticides based on Beauveria bassiana (Bals.) Vuillemin hold great promise for the management of a wide range of insect pests. The conidia in the biopesticide formulation require an adjuvant to protect them from photoinactivation by sunlight. The suitability of Tinopal, an optical brightener used as sunscreen for baculovirus formulations, for use with B. bassiana was assessed. The aim was to study the effect of Tinopal on the growth and photoprotection of B. bassiana, and its effect on the susceptibility of insects to B. bassiana. RESULTS: Tinopal was found to have no adverse effect on the growth of B. bassiana. It was found to confer total protection (approximately 95% conidial germination at 10 g Tinopal L(-1)) from sunlight up to 3 h of exposure, and a better survival rate than controls even up to 4 h. Helicoverpa armigera Hübner larvae fed on diet with 5 g kg(-1) Tinopal were found to have reduced growth. The duration of the larval stage increased by 3-4 days in 1 and 5 g kg(-1) Tinopal treatments. Among the moths that emerged from larvae fed on diet with 5 g kg(-1) Tinopal, a significantly high number were malformed compared with controls. The larvae that were fed diet with Tinopal showed quicker and higher mortality and required a lower effective lethal dose (LC(50)) than the controls. Tinopal was found to have a synergistic effect with B. bassiana in causing insect mortality. CONCLUSIONS: Tinopal was found to be a suitable adjuvant for B. bassiana-based biopesticide formulations. It conferred tolerance to sunlight and caused stress in the insect, leading to a synergistic effect with B. bassiana.