Leprosy is a chronic infectious disease caused by Mycobacterium leprae. As with other intracellular parasites, protective immunity is dependent on T cells and cell-mediated immunity. In animal models, immunization with killed armadillo-derived M. leprae elicits strong T-cell responses, delayed-type hypersensitivity and protection against viable challenge. We have recently shown that killed M. leprae can induce delayed-type hypersensitivity in healthy human volunteers. Identification of the M. leprae antigens that are recognized by T cells and may be involved in protection has been hampered by the inability to cultivate the organism in vitro and by difficulties in antigen purification from limited quantities of armadillo-derived bacillus. Because genes for the major protein antigens of M. leprae as seen by mouse monoclonal antibodies have been isolated, it has become possible to test whether these individual antigens are recognized by T cells. We screened crude lambda gtll phage lysates of Escherichia coli containing individual M. leprae antigens using M. leprae-specific T-cell clones isolated from M. leprae-vaccinated volunteers. Using this method, we find that nearly half of the M. leprae-specific T-cell clones are stimulated to proliferate by lysates containing an epitope of a M. leprae protein of relative molecular mass 18,000 (18K).

An outbreak of nodavirus infection in turbot larvae is described with respect to histopathology, immunohistochemistry, cell culture cultivation, RT-PCR amplification and sequence analysis of the capsid protein gene RNA2. Affected turbot developed classical signs of viral encephalopathy and retinopathy (VER) with abnormal swimming behaviour and high mortality levels. In the acute stage of infection, light microscopy revealed vacuolation of the central nervous system (CNS), with positive immunohistochemical staining for nodavirus. Later in the infection, CNS lesions appeared more chronic and contained clusters of cells immunopositive for nodavirus. Bacterial overgrowth in the intestines of the fish may have provoked or influenced the course of the nodavirus infection. We were unable to propagate the virus in cell culture. While RT-PCR using primers designed to detect Atlantic halibut nodavirus gave negative results, further testing with primers complementary to a more conserved region of RNA2 resulted in amplification of a product of the expected size. The entire RNA2 segment was cloned and sequenced. Sequence alignment showed that the turbot nodavirus (TNV) was different from previously described fish nodaviruses. In addition, phylogenetic analysis based on an 823 nt region of the sequence indicated that TNV clustered outside the four established fish nodavirus genotypes, suggesting a fifth genotype within the betanodaviruses.

This review summarises the state of knowledge of both viral and bacterial diseases of Atlantic cod Gadus morhua, and their diagnosis, prophylaxis and treatment. The most important losses have been at the larval and juvenile stages, and vibriosis has long been the most important bacterial disease in cod, with Listonella (Vibrio) anguillarum dominant among pathogenic isolates. Vaccination of cod against pathogens such as L. anguillarum and Aeromonas salmonicida clearly demonstrates that the cod immune system possesses an effective memory and appropriate mechanisms sufficient for protection, at least against some diseases. Well-known viruses such as the nodavirus that causes viral encephalopathy and retinopathy (VER), infectious pancreatic necrosis virus (IPNV) and viral haemorrhagic septicaemia virus (VHSV) have been isolated from Atlantic cod and can be a potential problem under intensive rearing conditions. No commercial vaccines against nodavirus are currently available, whereas vaccines against IPNV infections based upon inactivated virus as well as IPNV recombinant antigens are available. A number of investigations of the pharmacokinetic properties of antibacterial agents in cod and their efficacy in treating bacterial infections have been reviewed.

A 1349 nucleotide fragment of the RNA2 from a nodavirus affecting Atlantic halibut Hippoglossus hippoglossus was characterised and the nuclotide sequence (accession no, AJ245641) was employed to develop an optimal reverse-transcriptase polymerase chain reaction (RT-PCR) detection assay. The sequenced part of the RNA2 of Atlantic halibut nodavirus (strain AH95NorA) was highly similar in organisation to that of the RNA2 of striped jack nervous necrosis virus (SJNNV), and comprised features common to all nodaviruses. These characteristics confirmed that the virus that causes viral encephalopathy and retinopathy (VER) in Atlantic halibut is a nodavirus. The nucleotide sequence of the 1349 nucleotide fragment of Atlantic halibut nodavirus RNA2 was 80% identical to the RNA2 of SJNNV. The T2 region (830 nucleotides) of the RNA2 of Atlantic halibut nodavirus shared 98% of the nucleotide sequence when compared with the homologous region of barfin flounder nervous necrosis virus (BFNNV), while the nucleotide sequence identity to SJNNV in this region was 76 %. Phylogenetic analysis based on the nucleotide sequences of the T4 region (421 nucleotides) of Atlantic halibut nodavirus and of other fish nodaviruses revealed a close relationship to the nodaviruses of the barfin flounder clad that have been found in other cold-water species (Pacific cod Gadus macrocephalus and barfin flounder Verasper mosen). The nucleotide sequence of the RNA2 of Atlantic halibut nodavirus included some features that differ from that of SJNNV. The ORF of the RNA2 of Atlantic halibut nodavirus lacked 6 nucleotides through a slngle deletion and a 5-nucleotide deletion, separated by 4 nucleotides. The 3'-non-encoding region contained a 21 nucleotide insert and a 3 nucleotide deletion when compared with SJNNV. In comparison with the RNA2 of SJNNV, the 3'-non-encoding region showed a nucleotide sequence identity of 84.5%. A primer set based on the Atlantic halibut nodavirus nucleotide sequence was employed in order to design an optimal RT-PCR. The detection limit of the PCR was 10 to 100 copies of plasrnid, while the detection limit of the RT-PCR assay was 100 to 1000 copies of in vitro transcribed viral RNA.

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The Nodaviridae are divided into the alphanodavirus genus, which infects insects, and the betanodavirus genus, which infects fishes. Betanodaviruses are the causative agent of viral encephalopathy and retinopathy (VER) in a number of cultivated marine fish species. The Nodaviridae are small non-enveloped RNA viruses that contain a genome consisting of 2 single-stranded positivesense RNA segments: RNA1 (3.1 kb), which encodes the viral part of the RNA-dependent RNA polymerase (RdRp); and RNA2 (1.4 kb), which encodes the capsid protein. In addition to RNA1 and RNA2, a subgenomic transcript of RNA1, RNA3, is present in infected cells. We have cloned and sequenced RNA1 from the Atlantic halibut Hippoglossus hippoglossus nodavirus (AHNV), and for the first time, the sequence of a betanodaviral subgenomic RNA3 has been determined. AHNV RNA1 was 3100 nucleotides in length and contained a main open reading frame encoding a polypeptide of 981 amino acids. Conservative motifs for RdRp were found in the deduced amino acid sequence. RNA3 was 371 nucleotides in length, and contained an open reading frame encoding a peptide of 75 amino acids corresponding to a hypothetical B2 protein, although sequence alignments with the alphanodavirus B2 proteins showed only marginal similarities. AHNV RNA replication in the fish cell-line SSN-1 (derived from striped snakehead) was analysed by Northern blot analysis, which indicated that RNA3 was synthesised in large amounts (compared to RNA1) at an early point in time post-infection.KEY WORDS: Fish nodavirus · RNA-dependent RNA polymerase · RdRp · RNA1 · Subgenomic RNA 3 Resale or republication not permitted without written consent of the publisherDis Aquat Org 58: [117][118][119][120][121][122][123][124][125] 2004 2003). B1 is translated in the same reading frame as protein A, while B2 is translated in a +1 reading frame. PaV RNA3 has the coding potential of B2 and a second, smaller open reading frame (ORF) corresponding to the C-terminal region of protein A (Johnson et al. 2000), whereas the B1 ORF is absent from BoV RNA3 (Gene Bank Accession No. AF329080;Harper 1994). The function of protein B1 is not known, while the function of the FHV B2 protein has recently been identified as a potent RNA-silencing inhibitor that renders infected plant cells or Drosophila spp. cells less resistant to the virus (Li et al. 2002).Independent of its protein encoding potential, it has been suggested that RNA3 may act as a transactivator in the replication of RNA2 (Eckerle & Ball 2002). In contrast, RNA3 synthesis is suppressed by the replication of RNA2 (Zhong & Rueckert 1993). RNA3 has not been characterized in fish nodaviruses, although Delsert et al. (1997) detected an RNA segment of 0.4 kb in sea bass Dicentrarchus labrax larvae infected with D. labrax encephalitis virus (Dl EV). Iwamoto et al. (2001) also detected a faster migrating RNA (0.4 kb) from fish cells (E-11 cell line, a cloned version of SSN-1) that had been transfected with in vitro transcribed striped-jack nervous necrosis ...

BackgroundLeishmania is a protozoan parasite that alternates its life cycle between the sand-fly vector and the mammalian host. This alternation involves environmental changes and leads the parasite to dynamic modifications in morphology, metabolism, cellular signaling and regulation of gene expression to allow for a rapid adaptation to new conditions. The L-arginine pathway in L. amazonensis is important during the parasite life cycle and interferes in the establishment and maintenance of the infection in mammalian macrophages. Host arginase is an immune-regulatory enzyme that can reduce the production of nitric oxide by activated macrophages, directing the availability of L-arginine to the polyamine pathway, resulting in parasite replication. In this work, we performed transcriptional profiling to identify differentially expressed genes in L. amazonensis wild-type (La-WT) versus L. amazonensis arginase knockout (La-arg-) promastigotes and axenic amastigotes.Methodology/Principal findingsA total of 8253 transcripts were identified in La-WT and La-arg- promastigotes and axenic amastigotes, about 60% of them codifying hypothetical proteins and 443 novel transcripts, which did not match any previously annotated genes. Our RNA-seq data revealed that 85% of genes were constitutively expressed. The comparison of transcriptome and metabolome data showed lower levels of arginase and higher levels of glutamate-5-kinase in La-WT axenic amastigotes compared to promastigotes. The absence of arginase activity in promastigotes increased the levels of pyrroline 5-carboxylate reductase, but decreased the levels of arginosuccinate synthase, pyrroline 5-carboxylate dehydrogenase, acetylornithine deacetylase and spermidine synthase transcripts levels. These observations can explain previous metabolomic data pointing to the increase of L-arginine, citrulline and L-glutamate and reduction of aspartate, proline, ornithine and putrescine. Altogether, these results indicate that arginase activity is important in Leishmania gene expression modulation during differentiation and adaptation to environmental changes. Here, we confirmed this hypothesis with the identification of differential gene expression of the enzymes involved in biosynthesis of amino acids, arginine and proline metabolism and arginine biosynthesis.Conclusions/SignificanceAll data provided information about the transcriptomic profiling and the expression levels of La-WT and La-arg- promastigotes and axenic amastigotes. These findings revealed the importance of arginase in parasite survival and differentiation, and indicated the existence of a coordinated response in the absence of arginase activity related to arginine and polyamine pathways.

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