Engineering Resistance Against Viruses in Field Crops Using CRISPR- Cas9.

Food security is threatened by various biotic stresses that affect the growth and production of agricultural crops. Viral diseases have become a serious concern for crop plants as they incur huge yield losses. The enhancement of host resistance against plant viruses is a priority for the effective management of plant viral diseases. However, in the present context of the climate change scenario, plant viruses are rapidly evolving, resulting in the loss of the host resistance mechanism. Advances in genome editing techniques, such as CRISPR-Cas9 [clustered regularly interspaced palindromic repeats-CRISPR-associated 9], have been recognized as promising tools for the development of plant virus resistance. CRISPR-Cas9 genome editing tool is widely preferred due to high target specificity, simplicity, efficiency, and reproducibility. CRISPR-Cas9 based virus resistance in plants has been successfully achieved by gene targeting and cleaving the viral genome or altering the plant genome to enhance plant innate immunity. In this article, we have described the CRISPR-Cas9 system, mechanism of plant immunity against viruses and highlighted the use of the CRISPR-Cas9 system to engineer virus resistance in plants. We also discussed prospects and challenges on the use of CRISPR-Cas9-mediated plant virus resistance in crop improvement.

Lung metastasis, an incidental finding in maxilla - A case report.

The aim of this article was to present an asymptomatic lesion with insignificant clinical findings which turned out to be metastatic lesion in the jaws with primary in lung. The most common site of lung metastasis in the orofacial region is the mandible, but in our case it was seen in the maxilla. Metastases to the jaw bones occur in later stages. Hence, a careful examination of patients with jaw bone lesions is strongly suggested. Metastasis to the jaw should be considered while doing oral examination as observed in the current case because such lesions usually develop at terminal stage of cancer.

Fermentative production of rhamnolipid and purification by adsorption chromatography.

Glycolipids are one of the major classes of biosurfactants in which the rhamnolipids are best studied. The present work investigates the optimization of inoculum age and batch time for maximizing the yield of rhamnolipid from Pseudomonas aeruginosa (MTCC 2453). The yield and titer of rhamnolipids were maximum in the fermentation batch with an inoculum age of 24 hr. Batch time studies were performed on biomass production, rhamnolipid production, and sunflower oil utilization. The maximum yield of rhamnolipid was achieved at 96 hr when the culture cells were in the late exponential/early stationary phase. At optimum substrate concentration, maximum yield of 10.8 g/L was achieved. Further, downstream processing of crude rhamnolipid from broth using organic solvent extraction and subsequent purification using adsorption chromatography was done. In this study, chromatographic method was developed for purification of rhamnolipid by adsorption phenomena with more than 88.7% purity and 86.5% recovery. The present study provides new perspective on concepts involving separation by adsorption. Further antimicrobial properties and surfactant properties were studied for rhamnolipid production.

Nod2 is required for the early innate immune clearance of Acinetobacter baumannii from the lungs.

Acinetobacter baumannii (A. baumannii) is a significant cause of severe nosocomial pneumonia in immunocompromised individuals world-wide. With limited treatment options available, a better understanding of host immnity to A. baumannii infection is critical to devise alternative control strategies. Our previous study has identified that intracellular Nod1/Nod2 signaling pathway is required for the immune control of A. baumannii in airway epithelial cells in vitro. In the current study, using Nod2-/- mice and an in vivo sublethal model of pulmonary infection, we show that Nod2 contributes to the early lung defense against A. baumannii infection through reactive oxygen species (ROS)/reactive nitrogen species (RNS) production as Nod2-/- mice showed significantly reduced production of ROS/RNS in the lungs following A. baumannii infection. Consistent with the higher bacterial load, A. baumannii-induced neutrophil recruitment, cytokine/chemokine response and lung pathology was also exacerbated in Nod2-/- mice at early time points post-infection. Finally, we show that administration of Nod2 ligand muramyl dipeptide (MDP) prior to infection protected the wild- type mice from A. baumannii pulmonary challenge. Collectively, Nod2 is an important player in the early lung immunity against A. baumannii and modulating Nod2 pathway could be considered as a viable therapeutic strategy to control A. baumannii pulmonary infection.

Evaluation of different inactivation methods for high and low pathogenic avian influenza viruses in egg-fluids for antigen preparation.

In view of the emerging avian influenza (AI) viruses, it is important to study the susceptibility of AI viruses to inactivating agents for preparation of antigens and inactivated vaccines. The available information on susceptibility of both the high and low pathogenic AI viruses to different inactivating agents is inadequate and ambiguous. It has been shown that different subtypes of influenza viruses require different physical and chemical conditions for inactivation of infectivity. The present study was undertaken to evaluate the use of beta-propiolactone (BPL), formalin and ether for inactivation and its impact on antigenicity of AI viruses. A total of nine high and low pathogenic AI viruses belonging to four influenza A subtypes were included in the study. The H5N1 viruses were from the clades 2.2, 2.3.2.1 and 2.3.4. The H9N2 virus included in the study was of the G1 genotype, while the H11N1 and H4N6 viruses were from the Eurasian lineage. The viruses were treated with BPL, formalin and with ether. The confirmation of virus inactivation was performed by two serial passages of inactivated viruses in embryonated chicken eggs. The infectivity of all tested AI viruses was eliminated using 0.1% BPL and 0.1% formalin. Ether eliminated infectivity of all tested low pathogenic AI viruses; however, ether with 0.2% or 0.5% Tween-20 was required for inactivation of the highly pathogenic AI H5N1 viruses. Treatment with BPL, ether and formalin retained virus hemagglutination (HA) titers. Interestingly ether treatment resulted in significant rise in HA titers (P<0.05) of all tested AI viruses. This data demonstrated the utility of BPL, formalin and ether for the inactivation of infectivity of AI viruses used in the study for the preparation of inactivated virus antigens for research and diagnosis of AI.

Kafirin adsorption on ion-exchange resins: isotherm and kinetic studies.

Kafirin is a natural, hydrophobic and celiac safe prolamin protein obtained from sorghum seeds. Today kafirin is found to be useful in designing delayed delivery systems and coatings of pharmaceuticals and nutraceuticals where its purity is important and this can be obtained by adsorptive chromatography. This study is the first scientific insight into the isotherm and kinetic studies of kafirin adsorption on anion- and cation-exchange resins for practical applications in preparative scale chromatography. Adsorption isotherms of kafirin were determined for five anion- and two cation-exchange resins in batch systems. Isotherm parameters such as maximum binding capacity and dissociation constant were determined from Langmuir isotherm, and adsorptive capacity and affinity constant from Freundlich isotherm. Langmuir isotherm was found to fit the adsorption equilibrium data well. Batch uptake kinetics for kafirin adsorption on these resins was also carried out and critical parameters including the diffusion coefficient, film mass transfer coefficient, and Biot number for film-pore diffusion model were calculated. Both the isotherm and the kinetic parameters were considered for selection of appropriate resin for kafirin purification. UNOsphere Q (78.26 mg/ml) and Toyopearl SP-650M (57.4 mg/ml) were found to offer better kafirin binding capacities and interaction strength with excellent uptake kinetics under moderate operating conditions. With these adsorbents, film diffusion resistance was found to be major governing factor for adsorption (Bi<10 and δ<1). Based on designer objective function, UNOsphere Q was found be best adsorbent for binding of kafirin. The data presented is valuable for designing large scale preparative adsorptive chromatographic kafirin purification systems.

Suitability of specimen types for isolation of avian influenza viruses from poultry.

In view of the outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus in poultry in India, its impact on global public health and growing concerns of avian influenza (AI) viruses, surveys in wet poultry markets were conducted in the states of Maharashtra, West Bengal and Jharkhand in India during the period 2009-2012. During these surveys various types of samples from poultry were collected. During outbreaks and surveys in poultry, tracheal swabs (TS), cloacal swabs (CS), poultry drinking water (PDW) samples and fecal samples (FS) are preferred samples for AI diagnosis. The suitability of various types of poultry samples for AI virus isolation was analyzed. The parameters such as availability of specimen, ease of collection, quality of the specimen for the presence of contaminants such as organic debris or solid matter were considered for the analysis. A total of 2,405 samples were collected, which included 1,297 TS, 1,012 CS, 79 PDW, and 17 FS. Out of 2,309 TS and CS samples 1,752 samples were paired samples, collected from 876 birds. All samples were processed for virus isolation and identification. Of the 2,405 samples AI H9N2 was isolated from 199 samples (8.27 %). The virus isolation rate was significantly higher in PDW samples (21.5 %) (P < 0.05) and TS samples (12.1 %), in comparison with CS (2.3 %) (P < 0.001). Other viruses isolated were AI H4N6 and HPAI H5N1viruses; however the number of isolates of AI H4N6 and H5N1 were not sufficient for comparison. In conclusion, the PDW and TS samples were suitable for AI H9N2 virus isolation from poultry.

Avian influenza surveillance reveals presence of low pathogenic avian influenza viruses in poultry during 2009-2011 in the West Bengal State, India.

INTRODUCTION: More than 70 outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 have been reported in poultry in the western and north-eastern parts of India. Therefore, in view of the recent HPAI H5N1 outbreaks in poultry, active AI surveillance encompassing wild, resident, migratory birds and poultry was undertaken during 2009-2011 in the State of West Bengal. METHODS: A total of 5722 samples were collected from West Bengal; 3522 samples (2906 fecal droppings + 616 other environmental samples) were from migratory birds and 2200 samples [1604 tracheal, cloacal swabs, environmental samples, tissue samples + 596 blood (serum)] were from domestic ducks and poultry. All tracheal, cloacal and environmental samples were processed for virus isolation. Virus isolates were detected using hemagglutination assay and identified using hemagglutination inhibition (HI) and reverse transcriptase polymerase chain reaction (RT-PCR) assays. Sequencing and phylogenetic analysis of partial region of the hemagglutinin and neuraminidase genes was done. Intravenous pathogenicity index assays were performed in chickens to assess pathogenicity of AI virus isolates. Serum samples were tested for detection of antibodies against AI viruses using HI assay. RESULTS: A total of 57 AI H9N2, 15 AI H4N6 and 15 Newcastle Disease (NDV) viruses were isolated from chickens, from both backyard and wet poultry markets; AI H4N6 viruses were isolated from backyard chickens and domestic ducks. Characterization of AI H9N2 and H4N6 viruses revealed that they were of low pathogenicity. Domestic ducks were positive for antibodies against H5 and H7 viruses while chickens were positive for presence of antibodies against AI H9N2 and NDV. CONCLUSIONS: In the current scenario of HPAI H5N1 outbreaks in West Bengal, this report shows presence of low pathogenic AI H9N2 and H4N6 viruses in chickens and domestic ducks during the period 2009-2011. This is the first report of isolation of H4N6 from India. Antibodies against AI H5 and H7 in ducks highlight the probable role of domestic ducks in the transmission of AI viruses. Human infections of H9N2 have been reported from China and Hong Kong. This necessitates implementation of prevention and control measures to limit the spread of AI viruses.

Serologic evidence of avian influenza H9N2 and paramyxovirus type 1 infection in emus (Dromaius novaehollandiae) in India.

An avian influenza (AI) surveillance was undertaken in Maharashtra state, India during the period 2010-2011. There are no reports of AI surveillance in emus from India. A total of 202 blood samples and 467 tracheal and cloacal swabs were collected from eight emu farms. A hemagglutination inhibition (HI) assay was performed for detection of antibodies against AI H5N1, H7N1, H9N2, and avian paramyxovirus type 1 (APMV-1) viruses. A microneutralization (MN) assay was performed to confirm the presence of neutralizing antibodies against AI H9N2 and to compare with HI assays. A total of 28.2% and 28.7% of samples were positive for antibodies against AI H9N2 by HI and MN assays, respectively, using > or = 1:40 as a cut-off titer; 15.3% samples were positive for APMV-1 by HI assay using a > or = 1:10 cut-off titer. Seropositivity of AI H9N2 was nil in the grower (<1 yr) age group and highest (78%) in the breeder (2-3 yr) age group, whereas seropositivity against APMV-1 was observed in all age groups. Performance of both HI and MN assays was similar, suggesting the utility of using the MN assay along with HI assay for surveillance studies. This is the first report of the seroprevalence of AI H9N2 and APMV-1 in emus in India.

Characterization of superporous cellulose matrix for high-throughput adsorptive purification of lysozyme.

Protein purification essentially requires macroporous adsorbents matrices, which can provide high efficiency in packed bed and expanded bed (EB) even at high flow rates on account of reduced pore diffusion resistance resulting from finite intraparticle flow in the superpores. Rigid spherical superporous adsorbent beads with high carboxyl group density were prepared by crosslinking of cellulose. The matrix (diameter: 100-300 µm, mean pore size: 1-3 µm, pore volume: 57-59%, and bulk density: ∼1,438 kg/m3) could be used in packed bed as well as EB for purification of various biomolecules. Attempts were made to use indigenously developed rigid, superporous crosslinked cellulose adsorbent for high-throughput purification of lysozyme from chicken egg white's extract. A typical adsorption isotherm for lysozyme in crude was well correlated with the Langmuir isotherm model. Two maxima of binding capacity on celbeads bearing carboxymethyl (celbeads-CM) group for lysozyme were observed at pH 4.5 and 7.5. Uptake kinetics showed that the diffusivity of lysozyme was 100 times higher than conventional matrices. Such superporous matrix can be used for high-throughput purification of proteins from crude feedstocks and is reflected in leveling off of height equivalent to theoretical plate vs. flow curve after threshold velocity. Optimization of binding and elution conditions resulted in overall purification of lysozyme in a high yield and purity of 98.22 and 98.8%, respectively, with purification factor of 51.54 in a single step. The overall productivity (14.21 kg/m3 h) and specific activity (2.2×10(5) U/mg) were higher than that obtained with traditional particulate resins.