Utilization of Integrated Child Development Services (ICDS) and its linkages with undernutrition in India.

The Integrated Child Development Services (ICDS) programme has been the central focus of the POSHAN Abhiyaan to combat maternal and child malnutrition under the national nutrition mission in India. This paper examined the linkages between utilization of ICDS and underweight among children aged 6-59 months. The study utilized data from two recent rounds of the National Family Health Survey (NFHS-4 [2015-2016] and NFHS-5 [2019-2021]). Descriptive analyses were used to assess the change in utilization of ICDS and the prevalence of underweight at the national and state levels. Multivariable logistic regressions were performed to examine factors associated with the utilization of ICDS and underweight. Linkages between utilization of ICDS and underweight were examined using the difference-in-differences (DID) approach. Utilization of ICDS increased from 58% in 2015-2016 to 71% in 2019-2021. The prevalence of underweight decreased from 37% to 32% in the same period. Changes in ICDS utilization and underweight prevalence varied considerably across states, socioeconomic and demographic characteristics. Results from decomposition of DID models suggest that improvements in ICDS explained 9%-12% of the observed reduction in underweight children between 2016 and 2021, suggesting that ICDS made a modest but meaningful contribution in addressing undernutrition among children aged 6-59 months in this period.

Knowledge of and Self-Expressed Stigma for COVID-19 Vaccination among Pre-University Students in a College in South India: A Cross-Sectional Study.

Background: Coronavirus disease 2019 (COVID-19) is a contagious infection characterized by severe acute respiratory syndrome (SARS-CoV-2). Safe and effective vaccines are game-changers in the global vision of marking an end to the challenges posed by the COVID-19 pandemic. However, vaccine hesitancy due to perceived stigma and misinformation is a grave cause of concern. Objectives: To assess pre-university students' self-expressed stigma regarding COVID-19 vaccination and its association with their knowledge. Materials and Methods: A cross-sectional study was adopted for this research. A structured questionnaire approach was used to gather data from 384 students purposively at the selected pre-university college. The structured questionnaire consisted of three sections that explored the socio-demographic characteristics of the study participants, knowledge of vaccination, and self-expressed stigma, respectively. A total of 384 respondents took part in the study. Results: The study observes a low positive relationship (r = 0.25, P < 0.01) between knowledge and self-expressed stigma toward vaccination. Further, it was observed that participants from rural backgrounds had lower knowledge and self-expressed stigma scores than participants from urban settings. It is pertinent to note that participants with other sources of information had higher knowledge than those who used the internet, friends/peers, or newspapers. Both of the aforementioned findings are statistically significant. Conclusion: The interventions should revive trust in national health authorities, structured awareness campaigns by government agencies, and media coverage about the safety and efficacy of vaccines. In addition, it is also important to support citizens in ensuring that they have access to the right information from authentic sources in times of crisis.

Comparing trimodal therapy with radical cystectomy in muscle-invasive bladder cancer: an updated meta-analysis.

Background: We conducted this meta-analysis to compare the two muscle-invasive bladder cancer (MIBC) treatment modalities in terms of cancer-specific survival (CSS) and other outcome indicators. Method: A systematic review and meta-analysis were performed in accordance with the Preferred Reporting Items for Systematic Reviews (PRISMA) guidelines. The search was conducted using various academic databases including Scopus, PubMed, Cochrane database, EMBASE, Chinese biomedical literature database, Wan fang databases, and China National Knowledge Internet databases between 1966 and December 2023. This review protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) No. (CRD42023398977). Result: This study included a total of 54,816 patients diagnosed with bladder cancer from 14 studies, of which 6,228 patients were assigned to the trimodal therapy (TMT) group and 48,588 patients were assigned to the radical cystectomy (RC) group. Based on the results, the RC group exhibited a higher rate of survival than the TMT group [pooled hazard ratio (HR) = 1.23, 95% CI: 1.18-1.28, Z = 1.46, P < 0.001]. In terms of CSS, patients in the RC group had a longer CSS compared with those in the TMT group (pooled HR = 1.47, 95% CI: 1.29-1.67, Z = 5.893, P < 0.001). Compared with RC, TMT is significantly associated with an increased risk of both types of mortality (pooled HR: 1.30, P < 0.001). Conclusion: Overall, the findings of this meta-analysis suggest that RC treatment may be associated with improved overall survival. Moreover, it was observed that cancer-specific survival was significantly prolonged among patients in the RC group as opposed to those who received TMT. In addition, it was shown that patients who received TMT exhibited a higher risk of all-cause mortality when compared with those who underwent RC.

Hydroxytriazenes incorporating sulphonamide derivatives: evaluation of antidiabetic, antioxidant, anti-inflammatory activities, and computational study.

The existent investigation deals with synthesis, characterization, computational analysis, and biological activities of some hydroxytriazene derivatives containing sulphonamide moiety. The compounds were screened for antidiabetic, antioxidant, and anti-inflammatory activities. The antidiabetic activity was assessed using α-glucosidase and α-amylase inhibition assays with IC50 values ranging from 32.0 to 759.13 µg/mL and 157.77 to 340.47 µg/mL while standard drug acarbose showed IC50 values 12.21 and 69.74 µg/mL, respectively. The antioxidant activity was evaluated using DPPH and ABTS radical scavenging assays with IC50 value ranging from 54.01 to 912.66 µg/mL and 33.22 to 128.11 µg/mL, and standard drug ascorbic acid showed IC50 values 29.12 µg/mL and 69.13 µg/mL, respectively. Anti-inflammatory activity was investigated using the carrageenan-induced paw edema method, where percentage inhibition was up to 93.0 and 98.57 for 2 h and 4 h, respectively, and all the compounds were found to exhibit excellent anti-inflammatory activity. Moreover, prediction of activity spectra for substance and molecular docking were also performed. The PASS prediction hypothesized the potential of the compounds for anti-inflammatory activity, and docking results suggested the best binding pose for compounds 1b and 2b with the least energy value from which compounds can be considered as potent COX-2 inhibitors. Furthermore, possible interactions between hydroxytriazene analogues and the targets of antioxidant NADPH oxidase and antidiabetic human maltase-glucoamylase enzyme have been identified. The HOMO and LUMO analysis revealed charge transfer within the compounds. These findings suggested that the synthesized compounds can be potential agents for the treatment of diabetes and inflammation.

COVID-19 effect on patients with noncommunicable diseases: A narrative review.

Background and Aims: On March 11, 2020, the WHO has declared COVID-19 a global pandemic, affecting our day-to-day lives. Physical distancing and lockdown made significant obstacles to populations, particularly healthcare systems. Most healthcare workers were reallocated to COVID-19 facilities. Noncommunicable disease patients were given low priority and are at a higher risk of severe COVID-19 infection, which disrupted the treatment and disease management of these patients. This review aimed to assess the effect of COVID-19 on different types of noncommunicable diseases and the severity it may cause to patients. Methods: We have conducted a review of the literature on COVID-19 and noncommunicable diseases from December 2019 until January 2022. The search was done in PubMed and Cochrane for relevant articles using variety of searching terms. Data for study variables were extracted. At the end of the selection process, 46 papers were selected for inclusion in the literature review. Result: The result from this review found that the COVID-19 pandemic has affected the efficiency of the patient's treatment indirectly by either delaying or canceling sessions, which solidified the need to rely more on telemedicine, virtual visits, and in-home visits to improve patient education and minimize the risk of exposure to the patients. The major and most common types of noncommunicable diseases are known to be related to the severe outcomes of COVID-19 infection. It is strongly recommended to prioritize these patients for vaccinations against COVID-19 to provide them with the protection that will neutralize the risk imposed by their comorbidities. Conclusion: We recommend conducting more studies with larger population samples to further understand the role of noncommunicable diseases (NCDs) in this pandemic. However, this pandemic has also affected the efficiency of NCDs treatment indirectly by delaying or canceling sessions and others.

Small area variations in low birth weight and small size of births in India.

The states and districts are the primary focal points for policy formulation and programme intervention in India. The within-districts variation of key health indicators is not well understood and consequently underemphasised. This study aims to partition geographic variation in low birthweight (LBW) and small birth size (SBS) in India and geovisualize the distribution of small area estimates. Applying a four-level logistic regression model to the latest round of the National Family Health Survey (2015-2016) covering 640 districts within 36 states and union territories of India, the variance partitioning coefficient and precision-weighted prevalence of LBW (<2.5 kg) and SBS (mother's self-report) were estimated. For each outcome, the spatial distribution by districts of mean prevalence and small area variation (as measured by standard deviation) and the correlation between them were computed. Of the total valid sample, 17.6% (out of 193,345 children) had LBW and 12.4% (out of 253,213 children) had SBS. The small areas contributed the highest share of total geographic variance in LBW (52%) and SBS (78%). The variance of LBW attributed to small areas was unevenly distributed across the regions of India. While a strong correlation between district-wide percent and within-district standard deviation was identified in both LBW (r = 0.88) and SBS (r = 0.87), they were not necessarily concentrated in the aspirational districts. We find the necessity of precise policy attention specifically to the small areas in the districts of India with a high prevalence of LBW and SBS in programme formulation and intervention that may be beneficial to improve childbirth outcomes.

EVAP: A two-photon imaging tool to study conformational changes in endogenous Kv2 channels in live tissues.

A primary goal of molecular physiology is to understand how conformational changes of proteins affect the function of cells, tissues, and organisms. Here, we describe an imaging method for measuring the conformational changes of the voltage sensors of endogenous ion channel proteins within live tissue, without genetic modification. We synthesized GxTX-594, a variant of the peptidyl tarantula toxin guangxitoxin-1E, conjugated to a fluorophore optimal for two-photon excitation imaging through light-scattering tissue. We term this tool EVAP (Endogenous Voltage-sensor Activity Probe). GxTX-594 targets the voltage sensors of Kv2 proteins, which form potassium channels and plasma membrane-endoplasmic reticulum junctions. GxTX-594 dynamically labels Kv2 proteins on cell surfaces in response to voltage stimulation. To interpret dynamic changes in fluorescence intensity, we developed a statistical thermodynamic model that relates the conformational changes of Kv2 voltage sensors to degree of labeling. We used two-photon excitation imaging of rat brain slices to image Kv2 proteins in neurons. We found puncta of GxTX-594 on hippocampal CA1 neurons that responded to voltage stimulation and retain a voltage response roughly similar to heterologously expressed Kv2.1 protein. Our findings show that EVAP imaging methods enable the identification of conformational changes of endogenous Kv2 voltage sensors in tissue.

Constipation after surgery for anorectal malformations: Unrecognised problem until it is a problem.

BACKGROUND: Constipation is a common problem after surgery for anorectal malformations (ARMs), especially in patients having preserved rectosigmoid after pull-down surgery. Here, we present our experience with patients having constipation after surgery for ARMs and briefly discuss its management. MATERIALS AND METHODS: We retrospectively reviewed electronic operative and follow-up data from January 2015 to December 2019 of all patients having constipation after surgery for ARMs. RESULTS: A total of 77 patients were included in the study. The median age was 60 months, and thirty patients were male. Primary diagnoses were rectourethral fistula (26; 33.77%), vestibular fistula (25; 32.47%), perineal fistula/ectopic anus (18; 23.38%), rectovaginal fistula (4; 5.19%) and imperforate anus without fistula (4; 5.19%). Anal stenosis was found in 20 (25.97%) patients, posterior ledge with an adequate anal opening in 5 (6.49%), displaced anus in 4 (5.19%) but no obvious problem found in 48 (62.34%) patients. Non-operative management was successful in 75.44% (43 out of 57) of patients. Surgery was performed in 32 out of 77 patients (41.56%) with no mortality. Forty-three (72.88%) patients are continent, stayed clean, but 16 (27.19%) patients still need some sort of laxative/bowel wash/enema intermittently (Grade I/II constipation). Incidence of constipation was higher in patients operated through anterior sagittal route (27.58%) than posterior sagittal route (23.94%), but it was not statistically significant (P = 0.479). Follow-up ranged from 3 months to 5 years. CONCLUSION: Constipation is a common problem after ARMs surgery, especially in patients having preserved rectosigmoid after pull-down surgery. It is advisable to create relatively larger neoanus after pull through with early practice of neoanus dilatation, especially in redo cases. Repeated counselling, laxatives and dietary manipulations should begin at the first sign of constipation.

A Chemically Orthogonal Hole Transport Layer for Efficient Colloidal Quantum Dot Solar Cells.

Colloidal quantum dots (CQDs) are of interest in light of their solution-processing and bandgap tuning. Advances in the performance of CQD optoelectronic devices require fine control over the properties of each layer in the device materials stack. This is particularly challenging in the present best CQD solar cells, since these employ a p-type hole-transport layer (HTL) implemented using 1,2-ethanedithiol (EDT) ligand exchange on top of the CQD active layer. It is established that the high reactivity of EDT causes a severe chemical modification to the active layer that deteriorates charge extraction. By combining elemental mapping with the spatial charge collection efficiency in CQD solar cells, the key materials interface dominating the subpar performance of prior CQD PV devices is demonstrated. This motivates to develop a chemically orthogonal HTL that consists of malonic-acid-crosslinked CQDs. The new crosslinking strategy preserves the surface chemistry of the active layer beneath, and at the same time provides the needed efficient charge extraction. The new HTL enables a 1.4× increase in charge carrier diffusion length in the active layer; and as a result leads to an improvement in power conversion efficiency to 13.0% compared to EDT standard cells (12.2%).

Mixed Lead Halide Passivation of Quantum Dots.

Infrared-absorbing colloidal quantum dots (IR CQDs) are materials of interest in tandem solar cells to augment perovskite and cSi photovoltaics (PV). Today's best IR CQD solar cells rely on the use of passivation strategies based on lead iodide; however, these fail to passivate the entire surface of IR CQDs. Lead chloride passivated CQDs show improved passivation, but worse charge transport. Lead bromide passivated CQDs have higher charge mobilities, but worse passivation. Here a mixed lead-halide (MPbX) ligand exchange is introduced that enables thorough surface passivation without compromising transport. MPbX-PbS CQDs exhibit properties that exceed the best features of single lead-halide PbS CQDs: they show improved passivation (43 ± 5 meV vs 44 ± 4 meV in Stokes shift) together with higher charge transport (4 × 10-2 ± 3 × 10-3 cm2 V-1 s-1 vs 3 × 10-2 ± 3 × 10-3 cm2 V-1 s-1 in mobility). This translates into PV devices having a record IR open-circuit voltage (IR Voc ) of 0.46 ± 0.01 V while simultaneously having an external quantum efficiency of 81 ± 1%. They provide a 1.7× improvement in the power conversion efficiency of IR photons (>1.1 µm) relative to the single lead-halide controls reported herein.

Machine Learning Accelerates Discovery of Optimal Colloidal Quantum Dot Synthesis.

Colloidal quantum dots (CQDs) allow broad tuning of the bandgap across the visible and near-infrared spectral regions. Recent advances in applying CQDs in light sensing, photovoltaics, and light emission have heightened interest in achieving further synthetic improvements. In particular, improving monodispersity remains a key priority in order to improve solar cells' open-circuit voltage, decrease lasing thresholds, and improve photodetectors' noise-equivalent power. Here we utilize machine-learning-in-the-loop to learn from available experimental data, propose experimental parameters to try, and, ultimately, point to regions of synthetic parameter space that will enable record-monodispersity PbS quantum dots. The resultant studies reveal that adding a growth-slowing precursor (oleylamine) allows nucleation to prevail over growth, a strategy that enables record-large-bandgap (611 nm exciton) PbS nanoparticles with a well-defined excitonic absorption peak (half-width at half-maximum (hwhm) of 145 meV). At longer wavelengths, we also achieve improved monodispersity, with an hwhm of 55 meV at 950 nm and 24 meV at 1500 nm, compared to the best published to date values of 75 and 26 meV, respectively.

Temperature-Induced Self-Compensating Defect Traps and Gain Thresholds in Colloidal Quantum Dots.

Continuous-wave (CW) lasing was recently achieved in colloidal quantum dots (CQDs) by lowering the threshold through the introduction of biaxial strain. However, the CW laser threshold is still much higher than the femtosecond threshold. This must be addressed before electrically injected lasing can be realized. Here we investigate the relationship between threshold and temperature and find a subpicosecond recombination process that proceeds very efficiently at temperatures reached during CW excitation. We combine density functional theory and molecular dynamics simulations to explore potential candidates for such a process, and find that crystal defects having thermally vibrating energy levels can become electronic traps-i.e., they can protrude into the bandgap-when they are sufficiently distorted at higher temperatures. We find that biaxially strained CQDs, which have a lower femtosecond laser threshold than traditional CQDs, result in less heat for a given transparency/gain level and thus undergo this trapping to a lower extent. We also propose methods to tailor CQDs to avoid self-compensating defect traps.

Kv2 Ion Channels Determine the Expression and Localization of the Associated AMIGO-1 Cell Adhesion Molecule in Adult Brain Neurons.

Voltage-gated K+ (Kv) channels play important roles in regulating neuronal excitability. Kv channels comprise four principal α subunits, and transmembrane and/or cytoplasmic auxiliary subunits that modify diverse aspects of channel function. AMIGO-1, which mediates homophilic cell adhesion underlying neurite outgrowth and fasciculation during development, has recently been shown to be an auxiliary subunit of adult brain Kv2.1-containing Kv channels. We show that AMIGO-1 is extensively colocalized with both Kv2.1 and its paralog Kv2.2 in brain neurons across diverse mammals, and that in adult brain, there is no apparent population of AMIGO-1 outside of that colocalized with these Kv2 α subunits. AMIGO-1 is coclustered with Kv2 α subunits at specific plasma membrane (PM) sites associated with hypolemmal subsurface cisternae at neuronal ER:PM junctions. This distinct PM clustering of AMIGO-1 is not observed in brain neurons of mice lacking Kv2 α subunit expression. Moreover, in heterologous cells, coexpression of either Kv2.1 or Kv2.2 is sufficient to drive clustering of the otherwise uniformly expressed AMIGO-1. Kv2 α subunit coexpression also increases biosynthetic intracellular trafficking and PM expression of AMIGO-1 in heterologous cells, and analyses of Kv2.1 and Kv2.2 knockout mice show selective loss of AMIGO-1 expression and localization in neurons lacking the respective Kv2 α subunit. Together, these data suggest that in mammalian brain neurons, AMIGO-1 is exclusively associated with Kv2 α subunits, and that Kv2 α subunits are obligatory in determining the correct pattern of AMIGO-1 expression, PM trafficking and clustering.

Mutational analysis of foot and mouth disease virus nonstructural polyprotein 3AB-coding region to design a negative marker virus.

Inactivated purified whole virus vaccines are used for control of foot and mouth disease (FMD). ELISAs detecting antibodies to the nonstructural proteins (NSP), a marker of infection, are primarily used to differentiate FMD virus (FMDV) infected from vaccinated animals (DIVA). However, such DIVA assays have a limitation to their specificity since residual NSPs present in the relatively impure vaccines are suspected to induce an NSP-antibody response in the repeatedly vaccinated animals. Epitope-deleted negative marker vaccine strategy seems to have an advantage over the conventional vaccines in identifying the infected animals with accuracy. NSP 3AB contains an abundance of immunodominant B-cell epitopes of diagnostic importance. This study addresses the feasibility of producing 3AB-truncated FMDV mutant as a potential negative marker vaccine candidate. An infectious cDNA clone of FMDV serotype Asia 1 strain was used to engineer an array of deletion mutations in the established antigenic domain of 3AB. The maximum length of deletion tolerated by the virus was found to be restricted to amino acid residues 87-144 in the C-terminal half of 3A protein along with deletion of the first two copies of 3B peptide. The 3AB-truncated marker virus (Asia 1 IND 491/1997Δ3A87-1443B1,2+FLAG) demonstrated infectivity titres comparable to that of the parental virus in BHK-21 (log10 7.42 TCID50/ml) and LFBK-αVß6 (log10 8.30 TCID50/ml) cell monolayer culture. The protein fragment corresponding to the viable deletion in the 3AB region was expressed in a prokaryotic system to standardize a companion assay (3A87-1533B1,2 I-ELISA) for the negative marker virus which showed reasonably high diagnostic sensitivity (96.9%) and specificity (100% for naïve and 97.1% for uninfected vaccinated samples). The marker virus and its companion ELISA designed in this study provide a basis to devise a marker vaccine strategy for FMD control.

Sensitivity of Phacidiopycnis spp. Isolates from Pome Fruit to Six Pre- and Postharvest Fungicides.

Phacidiopycnis washingtonensis and P. pyri cause speck rot and Phacidiopycnis rot on apple and pear, respectively. Infection occurs in the orchard and remains latent, and symptoms appear after months of storage. Decay management relies on orchard sanitation and pre- and postharvest fungicides. In a 2017 survey, speck rot accounted for 6.4% of apple decay in central Washington, whereas Phacidiopycnis rot accounted for 3.9 and 6.7% of total pear decay in Washington and Oregon, respectively. Sensitivities of baseline populations of 110 P. washingtonensis and 76 P. pyri isolates collected between 2003 and 2005 to preharvest fungicides pyraclostrobin (PYRA) and boscalid (BOSC) and to postharvest fungicides thiabendazole (TBZ), fludioxonil (FDL), pyrimethanil (PYRI), and difenoconazole (DFC) were evaluated using a mycelial growth inhibition assay. Mean effective concentrations necessary to inhibit 50% growth (EC50) of P. washingtonensis were 0.1, 0.3, 0.8, 1.8, 2.1, and 4.8 µg/ml for FDL, PYRI, TBZ, DFC, PYRA, and BOSC, respectively. Respective mean EC50 values for P. pyri were 0.2, 0.6, 1.6, 1.1, 0.4, and 1.8 µg/ml. The sensitivity of exposed P. washingtonensis and P. pyri populations collected in 2017 revealed potential shifts toward BOSC and PYRA resistance. The efficacy of the six fungicides to control isolates of each pathogen with different in vitro sensitivity levels was evaluated on apple and pear fruit. FDL, DFC, and PYRI controlled both Phacidiopycnis spp. regardless of their EC50 values after 5 months of storage at 0°C in a regular atmosphere. The consistent occurrence of Phacidiopycnis spp. will require continuous monitoring and development of disease management strategies based on fungicide phenotypes and efficacy of existing fungicides assessed herein.

Climate change risks, extinction debt, and conservation implications for a threatened freshwater fish: Carmine shiner (Notropis percobromus).

Climate change is affecting many freshwater species, particularly fishes. Predictions of future climate change suggest large and deleterious effects on species with narrow dispersal abilities due to limited hydrological connectivity. In turn, this creates the potential for population isolation in thermally unsuitable habitats, leading to physiological stress, species declines or possible extirpation. The current extent of many freshwater fish species' spatio-temporal distribution patterns and their sensitivity to thermal impacts from climate change - critical information for conservation planning - are often unknown. Carmine shiner (Notropis percobromus) is an ecologically important species listed as threatened or imperilled nationally (Canada) and regionally (South Dakota, United States) due to its restricted range and sensitivity to water quality and temperature. This research aimed to determine the current distribution and spatio-temporal variability in projected suitable habitat for Carmine shiner using niche-based modeling approaches (MaxEnt, BIOCLIM, and DOMAIN models). Statistically downscaled, bias-corrected Global Circulation Models (GCMs) data was used to model the distribution of Carmine shiner in central North America for the period of 2041-2060 (2050s). Maximum mean July temperature and temperature variability were the main factors in determining Carmine shiner distribution. Patterns of projected habitat change by the 2050s suggest the spatial extent of the current distribution of Carmine shiner would shift north, with >50% of the current distribution changing with future projections based on two Representative Concentrations Pathways for CO2 emissions. Whereas the southern extent of the distribution would become unsuitable for Carmine shiner, suitable habitats are predicted to become available further north, if accessible. Importantly, the majority of habitat gains for Carmine shiner would be in areas currently inaccessible due to dispersal limitations, suggesting current populations may face an extinction debt within the next half century. These results provide evidence that Carmine shiner may be highly vulnerable to a warming climate and suggest that management actions - such as assisted migration - may be needed to mitigate impacts from climate change and ensure the long-term persistence of the species.

The carboxy-terminal half of nonstructural protein 3A is not essential for foot-and-mouth disease virus replication in cultured cell lines.

In foot-and-mouth disease (FMD)-endemic parts of the globe, control is mainly implemented by preventive vaccination with an inactivated purified vaccine. ELISAs detecting antibodies to the viral nonstructural proteins (NSP) distinguish FMD virus (FMDV)-infected animals in the vaccinated population (DIVA). However, residual NSPs present in the vaccines are suspected to be a cause of occasional false positive results, and therefore, an epitope-deleted negative marker vaccine strategy is considered a more logical option. In this study, employing a serotype Asia 1 FMDV infectious cDNA clone, it is demonstrated that while large deletions differing in size and location in the carboxy-terminal half of 3A downstream of the putative hydrophobic membrane-binding domain (deletion of residues 86-110, 101-149, 81-149 and 81-153) are tolerated by the virus without affecting its infectivity in cultured cell lines, deletions in the amino-terminal half (residues 5-54, 21-50, 21-80, 55-80 and 5-149) containing the dimerization and the transmembrane domains are deleterious to its multiplication. Most importantly, the virus could dispense with the entire carboxy-terminal half of 3A (residues 81-153) including the residues involved in the formation of the 3A-3B1 cleavage junction. The rescue of a replication-competent FMDV variant carrying the largest deletion ever in 3A (residues 81-153) and the fact that the deleted region contains a series of linear B-cell epitopes inspired us to devise an indirect ELISA based on a recombinant 3A carboxy-terminal fragment and to evaluate its potential to serve as a companion diagnostic assay for differential serosurveillance if the 3A-truncated virus is used as a marker vaccine.

Cell culture adaptation mutations in foot-and-mouth disease virus serotype A capsid proteins: implications for receptor interactions.

In this study we describe the adaptive changes fixed on the capsid of several foot-and-mouth disease virus serotype A strains during propagation in cell monolayers. Viruses passaged extensively in three cell lines (BHK-21, LFBK and IB-RS-2) consistently gained positively charged amino acids in the putative heparin-sulfate-binding pocket (VP2 ßE-ßF loop, VP1 C-terminus and VP3 ß-B knob) surrounding the fivefold symmetry axis (VP1 ßF-ßG loop) and at other discrete sites on the capsid (VP3 ßG-ßH loop, VP1 C-terminus, VP2 ßC strand and VP1 ßG-ßH loop). A lysine insertion in the VP1 ßF-ßG loop of two of the BHK-21-adapted viruses supports the biological advantage of positively charged residues acquired in cell culture. The charge transitions occurred irrespective of cell line, suggesting their possible role in ionic interaction with ubiquitous negatively charged cell-surface molecules such as glycosaminoglycans (GAG). This was supported by the ability of the cell-culture-adapted variants to replicate in the integrin-deficient, GAG-positive CHO-K1 cells and their superior fitness in competition assays compared with the lower passage viruses with WT genotypes. Substitutions fixed in the VP1 ßG-ßH loop (-3, -2 and +2 'RGD' positions) or in the structural element known to be juxtaposed against that loop (VP1 ßB-ßC loop) suggest their possible role in modulating the efficiency and specificity of interaction of the 'RGD' motif with αv-integrin receptors. The nature and location of the substitutions described in this study could be applied in the rapid cell culture adaptation of viral strains for vaccine production.

Evolution of serotype A foot-and-mouth disease virus capsid under neutralizing antibody pressure in vitro.

In this study, the Indian foot-and-mouth disease virus (FMDV) vaccine strain (A IND 40/2000) was passaged under homologous bovine convalescent serum (BCS) pressure to gain insight into the evolutionary dynamics of the antigenic sites. A considerable drop in the neutralization titres of the BCS for the isolated variants as compared to the parental population in either virus neutralization or plaque reduction neutralization test was observed. T143K substitution preceding the integrin binding 'RGD'-motif in the ßG-ßH loop of VP1 was found to be selected consistently and exclusively under immune pressure. By virtue of its location within an immunodominant site, sequence heterogeneity observed in the field viruses and residues already mapped in the neutralizing monoclonal antibody resistant mutants, position 143 in VP1 was predicted to be a critical residue of an important neutralizing epitope in serotype A FMDV. Using next-generation sequencing approach, the gradual overtaking of the originally dominating major variant by a minor one under a selective environment could be demonstrated. In the control passage regimen, VP2 E131K substitution was fixed within the heparan sulfate binding pocket probably as a result of adaptation to use alternative cellular receptors. But at the same time, these substitutions arising under selection forces other than immune pressure changed the antigenic behaviour of the virus inadvertently.