MOLECULAR BASIS OF VIRULENCE IN INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS (IHNV) USING A REVERSE GENETICS APPROACH

Infectious hematopoietic necrosis virus (IHNV) is a pathogen of major economic importance to the aquaculture industry. The long-term goal of our work is to develop a safe and effective recombinant IHNV vaccine and possibly use IHNV as a virus vector to express foreign genes. To achieve this goal, the complete genome of IHNV 220-90 virulent strain was sequenced and characterized. Subsequently, a full-length cDNA clone of IHNV was generated by constructing the full length cDNA clone, between the cytomegalovirus (CMV) promoter and the autocatalytic hammerhead and hepatitis delta virus ribozymes. Transfection of a full-length plasmid, along with the supporting plasmids resulted in the recovery of infectious rIHNV-220-90. Characterization of the rIHNV-220-90 showed that its growth characteristics in tissue culture were comparable to those of the parental virus. The possible role of IHNV proteins in virulence was explored to some extent. For this, the entire genome of attenuated virus (IHNV-61) was sequenced and compared with its virulent strain. The comparative sequencing analysis studies revealed that majority of differences were located in the glycoprotein gene. The M and G genes, and the trailer region between virulent and attenuated viruses were exchanged; recombinant chimeric viruses were recovered and studied for their pathogenicity in rainbow trout. The results obtained from in vivo studies indicate that the glycoprotein plays a major role in IHNV virulence in fish, whereas the M gene and trailer region play a negligible role in virulence of IHNV. The potential of rIHNV to serve as a viral vector was explored by expressing the VP2 protein of IPNV and hemagglutinin-estrase (HE) protein of ISAV. The recovered rIHNV-VP2 and rIHNV-HE viruses stably expressed the VP2 and HE proteins respectively for at least five serial passages and showed characteristics comparable to that of the parental virus, except that there was a one-log reduction in the virus titer. These results demonstrated that the established reverse genetics system can be utilized effectively to examine the molecular determinants of virulence, pathogenesis, and new approaches for vaccine development

Molecular characterization of the Great Lakes viral hemorrhagic septicemia virus (VHSV) isolate from USA

<p>Abstract</p> <p>Background</p> <p>Viral hemorrhagic septicemia virus (VHSV) is a highly contagious viral disease of fresh and saltwater fish worldwide. VHSV caused several large scale fish kills in the Great Lakes area and has been found in 28 different host species. The emergence of VHS in the Great Lakes began with the isolation of VHSV from a diseased muskellunge (<it>Esox masquinongy</it>) caught from Lake St. Clair in 2003. VHSV is a member of the genus <it>Novirhabdovirus</it>, within the family <it>Rhabdoviridae</it>. It has a linear single-stranded, negative-sense RNA genome of approximately 11 kbp, with six genes. VHSV replicates in the cytoplasm and produces six monocistronic mRNAs. The gene order of VHSV is 3'-N-P-M-G-NV-L-5'. This study describes molecular characterization of the Great Lakes VHSV strain (MI03GL), and its phylogenetic relationships with selected European and North American isolates.</p> <p>Results</p> <p>The complete genomic sequences of VHSV-MI03GL strain was determined from cloned cDNA of six overlapping fragments, obtained by RT-PCR amplification of genomic RNA. The complete genome sequence of MI03GL comprises 11,184 nucleotides (GenBank <ext-link ext-link-id="GQ385941" ext-link-type="gen">GQ385941</ext-link>) with the gene order of 3'-N-P-M-G-NV-L-5'. These genes are separated by conserved gene junctions, with di-nucleotide gene spacers. The first 4 nucleotides at the termini of the VHSV genome are complementary and identical to other novirhadoviruses genomic termini. Sequence homology and phylogenetic analysis show that the Great Lakes virus is closely related to the Japanese strains JF00Ehi1 (96%) and KRRV9822 (95%). Among other novirhabdoviruses, VHSV shares highest sequence homology (62%) with snakehead rhabdovirus.</p> <p>Conclusion</p> <p>Phylogenetic tree obtained by comparing 48 glycoprotein gene sequences of different VHSV strains demonstrate that the Great Lakes VHSV is closely related to the North American and Japanese genotype IVa, but forms a distinct genotype IVb, which is clearly different from the three European genotypes. Molecular characterization of the Great Lakes isolate will be helpful in studying the pathogenesis of VHSV using a reverse genetics approach and developing efficient control strategies.</p

Effect of nano-polysiloxane based finishing on handle properties of jute blended fabric

Jute: polyester blended yarn has been used to develop union fabric with cotton yarn , which satisfy the desirable properties for the development of a winter garment. Due to presence of jute fibre, it deficits in surface softness. An attempt has made to apply nano-polysiloxane based finishing both in individual as well as in combination with other finishing chemicals on this fabric by conventional pad-dry-cure method in order to improve its handle property. Properties such as bending length, crease recovery angle, and surface morphology have been evaluated as per standard methods. Results show that the nano+micro-polysiloxane based finishing combination shows better improvement in the surface morphology, handle and recovery property of the fabric than other finishing combinations

Detection of NP, N3 and N7 antibodies to avian influenza virus by indirect ELISA using yeast-expressed antigens

<p>Abstract</p> <p>Background</p> <p>Avian influenza viruses, belonging to the family Orthomyxoviridae, possess distinct combinations of hemagglutinin (H) and the neuraminidase (N) surface glycoproteins. Typing of both H and N antigens is essential for the epidemiological and surveillance studies. Therefore, it is important to find a rapid, sensitive, and specific method for their assay, and ELISA can be useful for this purpose, by using recombinant proteins.</p> <p>Results</p> <p>The nucleoprotein (NP) and truncated neuraminidase subtype 3 and 7 of avian influenza virus (AIV) were expressed in <it>Saccharomyces cerevisiae </it>and used to develop an indirect enzyme-linked immunosorbent assay for antibody detection. The developed assays were evaluated with a panel of 64 chicken serum samples. The performance of NP-ELISA was compared with the commercially available ProFlok^®AIV ELISA kit. The results showed comparable agreement and sensitivity between the two tests, indicating that NP-ELISA assay can be used for screening the influenza type A antibody in AIV infected birds. The N3 and N7- ELISAs also reacted specifically to their type specific sera and did not exhibit any cross-reaction with heterologous neuraminidase subtype specific sera.</p> <p>Conclusion</p> <p>The study demonstrates the expression of the NP, N3, and N7 proteins of AIV in yeast (<it>S. cerevisiae</it>) and their application in developing an indirect ELISA for detecting NP, N3 and N7 antibodies from AIV-infected chicken sera. The described indirect ELISAs are rapid, sensitive, specific and can be used as promising tests during serological surveillance.</p

Performance of chitosan: jasmine oil microcapsule on jute fabric

Chitosan: jasmine oil based microcapsule has been prepared bycoacervation method and then applied on bleached jute fabric bypad-dry-cure method. The performance of fragrance finishedjute fabrics has been assessed by physical properties, subjectiveassessment, scanning electron microscope, percentage moistureregain (%), fourier transform infrared spectrum, durability againsthome laundering and handle property. Morphological studiesconfirm that the chitosan: jasmine oil microcapsule of 100 microndimension has formed. Performance properties show that thechitosan: jasmine oil microcapsule can be fixed on the surface ofjute fabric and the fragrance is sustained up to five homelaundering

Comparison of biopolymer finishing with functional finishing on wool fibre

Wool: cotton union fabric has been applied with chitosan biopolymer to impart shrink-proof finish and its performance is compared with two synthetic polymer finishes. Results show that chitosan forms thin film on the surface of wool fibre as in synthetic finishing polymer. The diffusion of chitosan biopolymer inside the wool fibre matrix is found to be better than synthetic polymer, which is confirmed by the cross-sectional view of finished wool fibre. It is concluded that chitosan biopolymer could be preferred over other synthetic polymer to prevent shrinkage of woollen textiles.

Effect of lac treatment on mechanical properties of jute fabric /polyester resin based biocomposite

An attempt has been made to dissolve lac in methanol / sodium hydroxide solution and to use this lac solution as a coupling agent for jute fabric. Lac treated jute fabric has been used to reinforce the unsaturated polyester resin (USP). Flexural strength and inter-laminar shear strength (ILSS) of lac modified jute/USP biocomposite have been evaluated and then compared with sodium hydroxide treated jute as well as untreated jute based biocomposites. Lac treated jute fabric shows higher flexural properties of the biocomposite than that of untreated jute fabric, which infers that lac acts as a good compatibliser between jute fibre and USP. Lac treatment on jute fabric enhances the flexural properties of biocomposite better in alkaline medium than in solvent medium. It is concluded that lac treatment can be used to improve the flexural and ILSS properties of jute / thermoset resin based biocomposite

Complete Nucleotide Analysis of the Structural Genome of the Infectious Bronchitis Virus Strain Md27 Reveals its Mosaic Nature

Infectious bronchitis virus (IBV) causes highly contagious respiratory or urogenital tract diseases in chickens. The Maryland 27(Md27) strain was first isolated in 1976 from diseased chicken flocks in the Delmarva Peninsula region. To understand the genetic diversity and phylogenetic relationship of existing strains with Md27, the complete nucleotide sequence of the 3′end coding region (∼7.2 kb) of Md27 was determined and compared with other IBV strains and coronaviruses. It has the same S-3-M-5-N-3′ gene order, as is the case of other IBV strains. The spike gene of Md27 exhibits 97% identity with the SE17 strain. There are deletions at the spike gene, non-coding region between M and 5 genes, and at the 3′ untranslated region (UTR), which is different from Ark-like strains. Phylogenetic analysis and sequence alignments demonstrate that Md27 is a chimera containing different gene segments that are most closely related to the SE17, Conn and JMK strains. This current study provides evidence for genomic mutations and intergenic recombination that have taken place in the evolution of IBV strain Md27

Functionalization of wool with L-cysteine : process characterization and assessment of antimicrobial activity and cytotoxicity

This investigation reports a new biotechnological process that uses L-cysteine (L-Cys) which provides a permanent, nontoxic and effective antimicrobial effect over wool-based materials. This process is simple and carried out via idespread exhausting methods. Typically, wool fabrics are incubated with L-Cys for 50 min at 60°C in a pH 4.8 acetate buffer solution 25 mM, under mild agitation to give a good absorption rate. The minimal inhibitory concentration (MIC) of L-Cys was evaluated by the NCCLS M07-A6 standard method, and the results showed a good antibacterial activity against S. aureus and K. pneumoniae within the range of 6.0 × 10−3 − 4.8 × 10−2 g/mL [MIC 0.6% (w/v)] and 6.0 × 10−3 − 4.8 × 10−2 g/mL [MIC 0.6% (w/v)], respectively. In addition, the antimicrobial activity of the functionalized wool was assessed by the international standard JIS 1902-2002 showing a good inhibition of bacterial growth for an L-Cys concentration of 1% over the weight of fabric, both against Staphylococcus aureus and Klebsiella pneumoniae. Moreover, the biocidal mechanism was found to be related with the increase of sulfhydryl's groups onto wool fibers, which were quantified by the Ellman's reagent (5,5′-Dithio-bis(2-nitrobenzoic acid) method. The new process is easy to perform, non toxic, preserve wool quality and is a novel biomimetic approach that uses antimicrobial amino acids and may open new avenues for the design of biomedical textiles with a broad range of applications in healthcare.Contract grant sponsor: FCT; contract grant number: PTDC/EBB-BIO/113671/2009.Contract grant sponsor: R and D Unit of Textile and Paper Materials, Faculty of Engineering, University of Beira Interior

Relationship between morphology and tensile properties of pig hair fibre

The surface and cross-sectional features of hair fibres from four different breeds of pigs has been evaluated using scanning electron microscopy. The cross-section of the pig hair is modelled into an ellipse and the elliptical features of the fibre are correlated with its tensile properties. Surface scales in pig hair are arranged in imbricate type, crenate pattern and spaced at a mean distance of 4.58±0.24μm. Overall mean eccentricity, flattening, focus, area and angular eccentricity of pig hair fibre is found to be 0.60±0.09, 0.25±0.07, 195.16±33.68μm, and 0.06±0.01mm2 and 38.24±6.61 ° respectively. The ellipticity parameters are positively correlated with tensile properties (tenacity, extensibility, initial modulus and work of rupture) of the fibre. The specific flexural rigidity is negatively correlated with the ellipticity of the fibre, suggesting that the elliptical fibres may be more flexible than the fibres with circular cross-section