Decolorization of sulfonated azo dye Metanil Yellow by newly isolated bacterial strains: Bacillus sp. strain AK1 and Lysinibacillus sp. strain AK2.

Two different bacterial strains capable of decolorizing a highly water soluble azo dye Metanil Yellow were isolated from dye contaminated soil sample collected from Atul Dyeing Industry, Bellary, India. The individual bacterial strains Bacillus sp. AK1 and Lysinibacillus sp. AK2 decolorized Metanil Yellow (200 mg L(-1)) completely within 27 and 12h respectively. Various parameters like pH, temperature, NaCl and initial dye concentrations were optimized to develop an economically feasible decolorization process. The maximum concentration of Metanil Yellow (1000 mg L(-1)) was decolorized by strains AK2 and AK1 within 78 and 84 h respectively. These strains could decolorize Metanil Yellow over a broad pH range 5.5-9.0; the optimum pH was 7.2. The decolorization of Metanil Yellow was most efficient at 40°C and confirmed by UV-visible spectroscopy, TLC, HPLC and GC/MS analysis. Further, both the strains showed the involvement of azoreductase in the decolorization process. Phytotoxicity studies of catabolic products of Metanil Yellow on the seeds of chick pea and pigeon pea revealed much reduction in the toxicity of metabolites as compared to the parent dye. These results indicating the effectiveness of strains AK1 and AK2 for the treatment of textile effluents containing azo dyes.

Nucleotide sequence of the 3' terminal region of the genome of four lettuce mosaic virus isolates from Greece and Yemen.

Lettuce mosaic virus (LMV) is an economically important Potyvirus causing a severe disease of commercial lettuce crops. Based on molecular data, three phylogenetic groups of isolates have previously been discriminated, reflecting their geographical origin (Western Europe-California, Greece, or Yemen). Sequence information for the entire coat protein domain was only available for one of the Western Europe-California phylogenetic group. We have now sequenced the 3' terminal region of the genome LMV-Gr4, -Gr5 and -GrB, isolates which belong to the Greek phylogenetic group and of LMV-Yar, the sole known representative of the third LMV phylogenetic group. The region sequenced encodes the last 62 amino-acids of the polymerase and the entire coat protein of the four isolates, plus the 3' non-translated region of LMV-Gr5 and -Yar. The Greek and Yemenite isolates studied are all very aggressive on lettuce, are able to overcome the resistance genes mo1(1) and mo1(2) and belong to the two phylogenetic groups which have so far been only partially characterised. As for other Potyviruses, the core and the C-terminal regions of the coat protein are highly conserved among all isolates whereas the N-terminus is more variable. No amino acid change in the coat protein or carboxy-terminal part of the polymerase could be related to the resistance-breaking properties of the isolates analysed. The sequences obtained provide the basis for the rapid typing of LMV isolates using the restriction pattern of segments of cDNA amplified by PCR.

Construction of full-length cDNA clones of lettuce mosaic virus (LMV) and the effects of intron-insertion on their viability in Escherichia coli and on their infectivity to plants.

A full length cDNA copy of the genomic RNA of lettuce mosaic virus (LMV) was constructed under the control of an enhanced CaMV 35S promoter and of the NOS terminator. This construct was found infectious when inoculated to lettuce plants. The intron II of the bean nitrite reductase gene was engineered into the LMV FL cDNA in order to relieve possible deleterious effects of viral sequences to Escherichia coli cells and to evaluate the effects of the presence of the intron on the FL cDNA infectivity. The intron-less FL cDNA was found to be as stable as its intron-containing counterpart in E. coli. Sequence analysis of progeny RNA derived from plants inoculated with the intron-containing FL cDNA demonstrated that the inserted intron was perfectly spliced out. The symptoms induced in lettuce by either the intron-less or the intro-containing constructs were identical to those caused by the wild-type virus. However a slight delay in the establishment of infection in lettuce and a more obvious lag in Nicotiana benthamiana were observed with the intron-containing FL cDNA.

Biological and molecular variability of lettuce mosaic virus isolates.

ABSTRACT Lettuce mosaic potyvirus (LMV) causes severe disease of commercial lettuce crops. LMV isolates show wide biological variability, particularly in their ability to overcome the resistance genes described in Lactuca sativa. For a better understanding of the molecular interaction between lettuce and LMV, biological and molecular characterization of a collection of 10 LMV isolates known to differ in virulence or aggressiveness was performed. The ability of these isolates to overcome the resistance genes was reevaluated under standardized conditions. To study the molecular variability of LMV, an immunocapture-reverse transcription-poly-merase chain reaction technique, coupled with direct sequencing, was used to obtain nucleotide sequence data from three short regions of the LMV genome. Clustering analysis was performed and compared to the biological properties of the 10 isolates. Three groups of LMV isolates were discriminated based on the molecular data. These groups appear to correlate with the geographic origin of the isolates rather than with their pathogenicity. Sequence comparison with California isolates clearly showed that the California isolates are related to the western European isolates, raising the possibility of past exchanges of LMV between western Europe and California.

Comparison of the complete nucleotide sequences of two isolates of lettuce mosaic virus differing in their biological properties.

The complete nucleotide sequences of the genomic RNAs of the 0 and E isolates of lettuce mosaic potyvirus (LMV) have been determined. These two isolates differ by their behavior towards two lettuce resistance genes and by their seed transmission properties. LMV-0 is unable to induce disease in lettuce carrying either one of the mol1 and mol2 recessive resistance genes, whereas LMV-E is able to induce disease in the same plants. The genomes of these two isolates are 10080 nucleotides (nt) in length, excluding the poly(A) tract, and encode polyproteins of 3255 amino acids (aa). The open reading frame is flanked by a 5' non-coding region of 103 nt and a 3' non-coding region of 212 nucleotides. Ten proteins were predicted. The P3 protein, with 377 aa, is the longest potyviral P3 protein characterized to date while the P1 protein, with 437 aa, is among the longest P1 proteins reported. Sequence comparisons between the two isolates demonstrated only limited sequence difference. The overall nucleotide and amino acid sequence identities between LMV-0 and LMV-E are 94 and 97% respectively. The greatest variability occurs in the P1 and in the variable N-terminal region of the coat protein, while the NIa protease domain, the NIb protein, the C-terminus of the helper component protease and the 3' non-coding region are extensively conserved. While this sequence analysis does not allow direct identification of determinants involved in the resistance breaking or in the seed transmissibility properties, these data are a first step towards the characterization of these determinants.

Identification of novel neutralization-inducing regions of the human T cell lymphotropic virus type I envelope glycoproteins with human HTLV-I-seropositive sera.

The humoral immune response in sera from 30 human T cell lymphotropic virus type I (HTLV-I)-positive individuals from Martinique in the French West Indies was studied. The subjects were subdivided into those suffering from TSP/HAM and those being asymptomatic. In general, TSP/HAM patient sera seemed to contain more virus-specific antibodies than did the sera from the asymptomatic subjects. Three of the 13 TSP/HAM sera and 1 of the 17 asymptomatic sera contained HTLV-I-specific IgM antibodies, whereas 6 and 5 sera, respectively, contained IgA antibodies. By correlating the ability of patient sera to inhibit HTLV-I-induced syncytia with their antibody reactivity in ELISA to 42 synthetic peptides, together corresponding to the entire envelope glycoprotein of HTLV-I, a number of putative neutralizing domains were identified. Eight synthetic peptides representing the regions with the highest coefficient of correlation between neutralizing titer and ELISA reactivity were employed to specifically adsorb potentially neutralizing antibodies, and were also used directly, without sera, in the syncytium-neutralizing test. By those techniques, three novel and two previously described domains that seemed to contain neutralizing epitopes were identified. Two of the novel neutralizing sites resided in the external glycoprotein (gp46) and were contained within amino acids 53-75 and 287-311, respectively, and one was located in the transmembrane glycoprotein (gp21) within amino acids 346-368. Our findings may have implications for the rational design of subunit vaccines for prevention of and/or alteration of the clinical outcome of HTLV-I-related diseases.

Production and characterization of a monoclonal antibody directed against HTLV-1 p19: use in a specific capture enzyme immunoassay.

An enzyme immunoassay (EIA) was developed for detection of Human T-cell Leukemia Virus antigen in culture supernatants and cell lysates. The assay used a mouse monoclonal antibody against HTLV-I p19 major core protein as capture antibody. It has a sensitivity of 1 microgram/ml of HTLV-I protein, 250 pg/ml of purified recombinant p19 and detected p19 in an 10(-2) diluted supernatant of MT2 infected cell and in a 100 MT2 cells lysate (10(6) cells taken at day 7 of culture). The assay enable us to discriminate between HTLV-I and HTLV-II antigens and is reproducibly negative for supernatants and cell lysates of uninfected cells and of HIV-1 infected cells. The assay was found to be more specific and 10 times more sensitive than the reverse transcriptase (RT) assay, and the EIA test became positive three days earlier than RT assay for the HTLV-I cell lines supernatants.

Characterization and large production of human monoclonal antibodies against the HIV-1 envelope.

Peripheral blood lymphocytes from a volunteer immunized with a recombinant vaccinia virus VSC-25 expressing the gp160 env protein of HTLV-IIIB strain and from an asymptomatic HIV-infected individual were immortalized by Epstein-Barr (EBV). Clones which secrete human monoclonal antibodies from the two individuals (DZ, IgG1, lambda and C31, IgG1, kappa) were obtained and were stable for more than 2 years. The two monoclonals were directed against the gp160 env protein of HIV, DZ directed against the gp41 and C31 directed against the gp120. C31 was group-specific, whereas DZ was directed against the HTLV-IIIB and HTLV-RF strains. The epitope recognized by DZ was mapped to the carboxy terminus of the gp41, by expression of HIV DNA fragments in a yeast system and peptide analysis. The C31 epitope was not expressed by the yeast library and not present among the peptides which were tested. Monoclonal antibodies had no inhibitory effect in an HIV-induced cell fusion assay, but DZ showed a weak neutralizing activity against the HTLV-IIIB strain. Cloned EBV-transformed cell lines were fused to a murine myeloma, which allowed the heteromyeloma to be cultivated in serum-free medium. The monoclonal antibodies were produced in large quantity in a hollow-fibre reactor at defined culture conditions and purification procedures.

High affinity human monoclonal antibodies directed against hepatitis B surface antigen.

Peripheral blood mononuclear cells from donors immunized with hepatitis B vaccine (Pasteur Hevac B) were transformed with Epstein-Barr virus. Two polyclonal cell lines, producing antibodies to hepatitis B surface antigen were established and cloned. Seven clones were isolated; they secreted between 10 and 20 micrograms/ml of HBs specific IgG1 kappa or lambda antibody with anti-HBs titer of 300-800 IU/ml. These human antibodies expressed the anti 'a' specificities and had high affinity and avidity; their potential use as reagents for hepatitis B virus detection and for passive immunotherapy is under study.