Pol-miR-150 regulates anti-bacterial and viral infection in Japanese flounder (Paralichthys olivaceus) via the lysosomal protein LMP2L.

MiR-150 is a microRNA (miRNA) present in a number of teleost species, but its target and regulation mechanism are unknown. Similarly, lysosome membrane protein 2-like (LMP2L) is a gene identified in fish but with unknown function. In this study, we examined the regulation mechanism and function of flounder miR-150 (named pol-miR-150) and its target gene LMP2L (named PoLMP2L) in association with bacterial and viral infection. We found that pol-miR-150 expression was not only modulated by the bacterial pathogen Streptococcus iniae but also by the viral pathogen megalocytivirus. Pol-miR-150 targeted PoLMP2L by binding to the 3'-untranslated region (3'-UTR) of PoLMP2L and inhibited PoLMP2L expression in vitro and in vivo. PoLMP2L is a member of the CD36 superfamily of scavenger receptors and homologous to but phylogenetically distinct from lysosomal integral membrane protein type 2 (LIMP2). PoLMP2L was localized mainly in the lysosomes and expressed in multiple organs of flounder. In vivo knockdown and overexpression of PoLMP2L enhanced and suppressed, respectively, S. iniae dissemination in flounder tissues, whereas in vivo knockdown and overexpression of pol-miR-150 produced the opposite effects on S. iniae dissemination. In addition, pol-miR-150 knockdown also significantly inhibited the replication of megalocytivirus. The results of this study revealed the regulation mechanism and immune functions of fish miR-150 and LMP2L, and indicated that LMP2L and miR-150 play an important role in the antimicrobial immunity of fish.

White spot syndrome virus (WSSV) infection impacts intestinal microbiota composition and function in Litopenaeus vannamei.

Intestinal microbiota homeostasis is crucial to the health of host. Pathogen invasion results in dynamics of microbiota composition and structure, disrupting their function in maintaining host health. WSSV is the most prevalent viral pathogen and is able to cause extremely high mortality in Litopenaeus vannamei. However, the changes of intestinal microbiota induced by WSSV are yet to be elucidated. In this study, we analyzed and compared the microbiota of healthy and WSSV-challenged shrimp intestines. Though the richness and diversity of microbiota was barely affected by WSSV, the abundance of predominant phyla like Proteobacteria and Fusobacteria were upregulated significantly, while Bacteroidetes and Tenericutes were significantly decreased in WSSV-infected shrimps. At the genus level, significant increase was observed in Photobacterium, Propionigenium and Arcobacter, as well as significant decrease in Candidatus Bacilloplasma and Flavobacterium in WSSV-infected shrimps. Additionally, metagenomic predictions by PICRUSt suggested that the altered microbiota was mainly related to metabolism, human diseases, genetic information processing, environmental information processing and cellular processes. These results suggested that the invasion of WSSV could impact intestinal microbiota composition and function in L. vannamei.

Protective responses of two paralogs of granulocyte colony stimulating factor (GCSF) in rock bream, Oplegnathus fasciatus during bacterial and viral infection.

Granulocyte colony stimulating factor (GCSF) has a key role in the production of neutrophilic granulocytes during normal hematopoietic development and release of neutrophils into the blood circulation. In this study we have identified and characterized two paralogs of GCSF (RbGCSF) in rock bream. Although RbGCSF-1 and RbGCSF-2 share low sequence conservation, its domains and protein structure still share significant similarity. Basal levels of RbGCSF-1 gene expression was high in peripheral blood leukocytes (PBLs), spleen and intestine whereas the RbGCSF-2 was highly expressed in PBLs and kidney, of healthy animals. A significant induction of RbGCSFs were observed after the challenge with Streptococcus iniae in kidney, spleen and gills during initial hours of infection. Whereas Edwarsiella tarda infection caused a reasonable expression in kidney. Red seabream iridovirus caused induction of RbGCSF-1 transcription only in gills during initial hours. This higher expression of RbGCSF in early hours may be its response to induce emergency hematopoiesis, due to shortage of neutrophils to combat the surge in pathogens. The difference in induction of RbGCSF paralogs during infection may constitute to its different roles or overlapping functions.

Fish TRIM8 exerts antiviral roles through regulation of the proinflammatory factors and interferon signaling.

The tripartite motif (TRIM)-containing proteins usually exert important regulatory roles during multiple biological processes. TRIM8 has been demonstrated to be a RING domain-containing E3 ubiquitin ligase which plays critical roles in inflammation and cancer. In this study, a TRIM8 homolog from grouper, Epinephelus coioides (EcTRIM8) was cloned, and its effects on fish virus replication were investigated. The full-length EcTRIM8 cDNA encoded a polypeptide of 568 amino acids with 92% identity to TRIM8 homolog from large yellow croaker (Larimichthys crocea). Sequence alignment analysis indicated that EcTRIM8 contained conserved RING finger, B-box and coiled-coil domain. Expression patterns analysis showed that EcTRIM8 was predominant in kidney, gill, fin, liver, spleen and brain. After challenging with Singapore grouper iridovirus (SGIV) or polyinosin-polycytidylic acid (poly I:C), the EcTRIM8 transcript was significantly increased at the early stage of injection. Under fluorescence microscopy, we observed different distribution patterns of EcTRIM8 in grouper spleen (GS) cells, including punctate fluorescence evenly situated throughout the cytoplasm and bright aggregates. The ectopic expression of EcTRIM8 in vitro significantly inhibited the replication of SGIV and red spotted grouper nervous necrosis virus (RGNNV), evidenced by the obvious reduction in the severity of cytopathic effect (CPE) and the significant decrease in viral gene transcription and protein synthesis. Moreover, the transcription of the proinflammatory factors and interferon related immune factors were differently regulated by EcTRIM8 during SGIV or RGNNV infection. In addition, overexpression of EcTRIM8 significantly increased the transcription of interferon regulator factor 3 (IRF3) and IRF7, and enhanced IRF3 or IRF7 induced interferon-stimulated response element (ISRE) promoter activity. Together, our results firstly demonstrated that fish TRIM8 could exert antiviral function through the regulation of the expression of proinflammatory cytokines and interferon related transcription factors in response to fish viruses.

Molecular characterization and expression analysis of nine CC chemokines in half-smooth tongue sole, Cynoglossus semilaevis.

Chemokines are a large, diverse group of small cytokines that can be classified into several families, including the CC chemokine family, which plays a pivotal role in host defense by inducing leukocyte chemotaxis under physiological and inflammatory conditions. Here we studied 9 CC chemokines from half-smooth tongue sole (Cynoglossus semilaevis). Phylogenetic analysis divided these chemokines into four groups. The tissue specific expression patterns of the 9 chemokines under normal physiological conditions varied much, with most chemokines highly expressed in immune organs, while some other chemokines showing high expression levels in non-immune organs. In addition, the 9 chemokines exhibited similar or distinctly different expression profiles in response to the challenge of virus and intracellular and extracellular bacterial pathogens. These results indicate that in tongue sole, CC chemokines may be involved in different immune responses as homeostatic or inflammatory chemokines.

TLR7 is required for optimal immune defense against bacterial infection in tongue sole (Cynoglossus semilaevis).

In mammals as well as in teleost, toll-like receptor 7 (TLR7) is known to be involved in antiviral immunity by recognizing viral RNA. However, the antibacterial potential of fish TLR7 is unclear. In this study, we analyzed the TLR7 of tongue sole (Cynoglossus semilaevis), CsTLR7, and examined its potential involvement in antibacterial immunity. CsTLR7 is composed of 1052 amino acid residues and shares 64.0%-75.9% overall sequence identities with known teleost TLR7. CsTLR7 possesses a toll/interleukin-1 receptor domain and six leucine-rich repeats. Constitutive expression of CsTLR7 occurred in relatively high levels in kidney, spleen and liver. Bacterial infection upregulated CsTLR7 expression, whereas viral infection downregulated CsTLR7 expression. Knockdown of CsTLR7 significantly enhanced bacterial dissemination in the tissues of tongue sole. Treatment of tongue sole with the imidazoquinoline compound R848 (TLR7 activator) and the endosomal acidification inhibitor chloroquine (TLR7 inhibitor) caused enhanced and reduced resistance against bacterial infection respectively. These results indicate that CsTLR7 plays an essential role in the antibacterial immunity of tongue sole.

Identification of torque teno virus in culture-negative endophthalmitis by representational deep DNA sequencing.

PURPOSE: To test the hypothesis that uncultured organisms may be present in cases of culture-negative endophthalmitis by use of deep DNA sequencing of vitreous biopsies. DESIGN: Single-center, consecutive, prospective, observational study. PARTICIPANTS: Aqueous or vitreous biopsies from 21 consecutive patients presenting with presumed infectious endophthalmitis and 7 vitreous samples from patients undergoing surgery for noninfectious retinal disorders. METHODS: Traditional bacterial and fungal culture, 16S quantitative polymerase chain reaction (qPCR), and a representational deep-sequencing method (biome representational in silico karyotyping [BRiSK]) were applied in parallel to samples to identify DNA sequences corresponding to potential pathogens. MAIN OUTCOME MEASURES: Presence of potential pathogen DNA in ocular samples. RESULTS: Zero of 7 control eyes undergoing routine vitreous surgery yielded positive results for bacteria or virus by culture or 16S polymerase chain reaction (PCR). A total of 14 of the 21 samples (66.7%) from eyes harboring suspected infectious endophthalmitis were culture-positive, the most common being Staphylococcal and Streptococcal species. There was good agreement among culture, 16S bacterial PCR, and BRiSK methodologies for culture-positive cases (Fleiss' kappa of 0.621). 16S PCR did not yield a recognizable pathogen sequence in any culture-negative sample, whereas BRiSK suggested the presence of Streptococcus in 1 culture-negative sample. With the use of BRiSK, 57.1% of culture-positive and 100% of culture-negative samples demonstrated the presence of torque teno virus (TTV) sequences, compared with none in the controls (P=0.0005, Fisher exact test). The presence of TTV viral DNA was confirmed in 7 cases by qPCR. No other known viruses or potential pathogens were identified in these samples. CONCLUSIONS: Culture, 16S qPCR, and BRiSK provide complementary information in presumed infectious endophthalmitis. The majority of culture-negative endophthalmitis samples did not contain significant levels of bacterial DNA. "Culture negativity" does not seem to be due to failure of growth of fastidious bacteria. The small DNA virus TTV was unexpectedly found in all culture-negative samples and some culture-positive samples. This study cannot distinguish whether TTV is a direct intraocular pathogen, an adjuvant for inflammation, a general marker of inflammation, or a commensal virus but provides a testable hypothesis for a pathogenic mechanism in culture-negative endophthalmitis.

[Relationship of gastric microflora and neopterin content in gastric juice in children with chronic gastroduodenitis].

The study included 71 children with chronic gastroduodenitis aged 5-17 years. PCR DNA was determined by the presence of Helicobacter pylori, human papilloma virus high cancer risk 16, 18 types (HPV), herpes simplex 1 and type 2 (HSV), cytomegalovirus (CMV), fungi, Candida in gastric juice and biopsy specimens of gastric mucosa and duodenal. Viruses were detected in 14% of patients, an association of microorganisms - in 20% of the children, the isolated H. pylori infection - 18%. The relationship between the composition of microflora in the gastroduodenal region and neopterin levels in gastric juice with a distinct increase in its value in the presence of viruses.

Two amphibian diseases, chytridiomycosis and ranaviral disease, are now globally notifiable to the World Organization for Animal Health (OIE): an assessment.

The global trade in amphibians entails the transport of tens of millions of live animals each year. In addition to the impact harvesting wild animals can have on amphibian populations, there is mounting evidence that the emerging pathogens Batrachochytrium dendrobatidis and ranaviruses, the aetiological agents of chytridiomycosis and ranaviral disease, respectively, are spread through this trade. The link between these pathogens and amphibian declines and extinctions suggests that the epidemiological impact of the trade is significant and may negatively affect conservation and trade economics. Here we present a brief assessment of the volume of the global trade in live amphibians, the risk of individuals harboring infection, and information on the recent listing by the World Organization for Animal Health (OIE) of chytridiomycosis and ranaviral disease in the OIE Aquatic Animal Health Code. This listing made chytridiomycosis and ranaviral disease internationally notifiable diseases and thus subject to OIE standards, which aim to assure the sanitary safety of international trade in live amphibians and their products.

Opportunistic infections after renal transplantation.

Opportunistic infection is a serious clinical complication in patients receiving immunosuppressive therapy after kidney transplantation. This article deals with some of the possible infectious agents that were recently encountered at our transplantation centre in Düsseldorf, Germany. Opportunistic organsims such as human herpesviruses 6-8, polyomavirus, parvovirus B19, varicella zoster virus, Nocardia and Listeria monocytogenes are rare but severe complications that are presented in this overview. As a result of the use of new immunosuppresive drugs like tacrolimus and mycophenolate mofetil these infections are now seen more frequently, so they should always be included in differential diagnostic considerations. New diagnostic procedures and new treatment strategies should allow early detection and successful treatment of opportunistic infections in the majority of kidney transplant recipients.

Viral skin infections: diagnosis and treatment considerations.

Skin lesions are prominent features of many viral diseases. In some instances, characteristic skin lesions suggest a specific viral illness, the diagnosis of which can be quickly established by appropriate procedures. In addition to clinical manifestations, laboratory methods including virus isolation are used to diagnose viral infections. In viral diseases, prophylaxis has proved more successful than the specific treatment of established infection. However, recent progress in molecular biology has facilitated the development of new vaccines and new drugs to treat viral infections.

Comparison of European systemic piscine and amphibian iridoviruses with epizootic haematopoietic necrosis virus and frog virus 3.

Iridovirus-like agents isolated from systemic infected fish (Silurus glanis, SFIR; Ictalurus melas, CFIR I, CFIR II, CFIR III) and from frogs (Rana esculenta, REIR) in Europe, Epizootic Haematopoietic Necrosis Virus (EHNV) isolated in Australia from redfin perch (Perca fluviatilis), and Frog Virus 3 (FV 3) isolated from frogs (Rana pipiens) in the USA were investigated by electron microscopy, polypeptide composition, immunofluorescence, restriction endonuclease digestion, Southern-blot hybridization and polymerase chain reaction (PCR) amplification. All virus isolates proved to be similar in morphology and in size and reacted with EHNV polyclonal antiserum in the immunofluorescence. Whilst DNA restriction profiles of the European piscine isolates cleaved by BamH I were similar, they differed clearly from those of EHNV, REIR and FV 3. Southern-blot analysis of viral BamH I digested DNA using an EHNV DNA probe revealed cross-hybridization with DNA of the investigated iridoviruses. Using a set of primers designed for an open reading frame of the EHNV genome, PCR products of about 250 bp were obtained with the DNA of systemic piscine and amphibian iridoviruses. The data suggest that the systemic piscine and amphibian iridoviruses should be regarded as members of the the genus Ranavirus within the family Iridoviridae.