Mcp Gene Research Articles

Immunoprotective effect of recombinant Lactobacillus plantarum expressing largemouth bass virus MCP on largemouth bass

Largemouth bass virus (LMBV) is an infectious pathogen that causes high mortality rates in largemouth bass, and outbreaks of this virus can significantly harm the aquaculture industry. Currently, no vaccine has been developed that can effectively prevent the transmission of LMBV. In this study, we constructed a recombinant Lactobacillus plantarum (L. plantarum) strain capable of expressing the MCP gene of LMBV and displaying this protein on its surface; then, we evaluated the immunoprotective effect of this recombinant bacterium on largemouth bass. Western blotting, immunofluorescence, and flow cytometry confirmed that MCP was successfully expressed and anchored on the surfaces of NC8 cells. Immunization of largemouth bass with NC8-pSIP409-pgsA'-MCP via the oral feeding route induced CD4, CD8, IL-1β, and IL-6 gene expression. In addition, NC8-pSIP409-pgsA'-MCP at different CFUs increased the survival of largemouth bass after LMBV infection; in particular, NC8-pSIP409-pgsA'-MCP (109 CFU) resulted in approximately 30% survival. NC8-pSIP409-pgsA'-MCP immunization alleviated the pathological changes in the liver and spleen, exerting a more advantageous protective effect. These data suggest that the recombinant L. plantarum strain NC8-pSIP409-pgsA'-MCP can increase the resistance of largemouth bass to LMBV infection and that this strain is a promising candidate oral vaccine for the prevention of LMBV infection.

Development and application of a quantitative real-time PCR method for detection of Decapod iridescent virus 1.

As a newly discovered virus, Decapoda iridovirus 1 (DIV1) can cause a mortality rate of up to 100% in crustaceans, leading to huge economic losses. At present, there is no effective prevention and control measures for this disease. In the present study, the specific primers targeting highly conserved regions of MCP gene were designed, and then a quantitative real-time PCR method was established. The results indicate that DIV1 quantitative real-time PCR established has good specificity and does not cross react with other pathogens including white spot syndrome virus (WSSV), infectious subcutaneous and hematopoietic necrosis virus (IHHNV) and Vibrio parahaemolyticus induced acute hepatopancreatic necrosis disease (VpAHPND). The real-time PCR was capable of detecting DIV1 DNA at a minimum concentration of 10 copies/μL within 34 cycles. The method has good repeatability, with intra group and inter group coefficients of variation both less than 2%. Thirty-two clinical samples were assessed using both the real-time PCR and conventional PCR. The results shown real-time PCR we established are more sensitive than conventional PCR. In conclusion, this method has strong specificity, stable repeatability, and high sensitivity, providing technical support for clinical diagnosis, epidemiology investigation and monitoring of DIV1.

Effect of rock bream iridovirus (RBIV) contained tissue intake on rock bream (Oplegnathus fasciatus) mortality and blood cell distribution

Rock bream (Oplegnathus fasciatus) is one of the highly priced cultured marine fish in Korea. Rock bream iridovirus (RBIV) outbreaks in aquaculture farms may involve environmental factors, co-infection with other pathogenic microorganisms and grounded (raw) fish feed. This study evaluated the effects of RBIV-containing tissue intake on mortality and oral transmission in rock bream. Virus-containing tissues administered to rock bream [50 mg (1.53 × 108/major capsid protein, MCP gene copies) to 2400 mg (7.34 × 109)] held at 23 °C lead to 100 % mortality by 27 days post administration. Interestingly, the mortality rates were not viral dose- or concentration dependent. Further, high MCP gene copy numbers were observed in the gill, liver, intestine, stomach, spleen, heart, kidney, brain and muscle tissues (viral load range of 3.03 × 106 to 4.01 × 107/mg, average viral load 1.70 × 107/mg) of dead rock bream. Moreover, a high viral load was detected in the intestine and stomach, where the virus was directly administered. This indicated that the intake of RBIV-containing tissue feed weakens the intestinal mucosal immunity and increases viral load in the intestine. Moreover, the levels of complete blood cell count (CBC) indicators, such as red blood cell (RBC), hemoglobin (HGB) and hematocrit (HCT) significantly decreased from 15 dpi with red blood cell distribution width (RDW), and white blood cells (lymphocyte, monocyte and granulocyte) significantly increased from the initial to later stage of infection. These results highlight the significance of blood-mediated indicators against RBIV infection in rock bream. We demonstrate the existence of an oral transmission route for RBIV in rock bream. Our findings indicate that pathogen-containing feed is an important risk factor for disease outbreaks in rock bream.

Detection of Acipenser European Iridovirus (AcIV-E) in Sturgeon Farms in Northern Italy between 2021-2023.

Sturgeon farming is rapidly expanding in Europe, where Italy ranks first in farmed caviar production. A major threat to sturgeon health in captivity is infection with Acipenser European Iridovirus (AcIV-E), a viral disease definitively identified in 2016. Here we present data on the occurrence of AcIV-E in 482 sturgeons (age ≤ 12 months, species of the genus Acipenser and the species Huso huso) collected from sturgeon farms in northern Italy between January 2021 and December 2023. The health status of each specimen was determined by necroscopy and virological assay. Virological analysis was performed on gill samples and real-time PCR specific to the MCP gene of the iridovirus viral capsid. Molecular analysis revealed positivity to the virus in 204 samples (42.68% of the total), while anatomopathological examination of nearly all fish with positive real-time PCR disclosed swollen abdomen, hepatic steatosis, splenomegaly, and increased gill volume. Two challenges to timely diagnosis are the absence of pathognomonic symptoms and the inability to isolate the virus on cell monolayers. Continuous and widespread health monitoring is therefore crucial for disease management and to effectively control spread of the virus.

Development of recombinant major capsid protein Vaccine and assessment of its efficacy against SRDV in similar damselfish (Pomacentrus similis)

Viral vaccines against emerging viral diseases are crucial for encouraging successful aquaculture production. In this research, an experimental recombinant major capsid protein vaccine of similar damselfish virus was prepared and examined for its efficacy in marine ornamental fish, similar damselfish (Pomacentrus similis). The MCP gene of the SRDV was amplified from the viral DNA by a specific primer set viz bamHI and XhoI- restriction sites and confirmed by agarose gel electrophoresis with a target size of 1416 bp. The gel-purified PCR product was double-digested with the said enzymes and incorporated into the pTriEx1.1 vector, which was subsequently transformed to E. coli DH5α. The plasmids of the two clones pTriEx-MCP-1416-1 and pTriEx-MCP-1416-3 were transformed to E. coli BL21 (DE-3) pLacI. A crude protein compound derived from a colony of E. coli BL21 (DE-3) with expressed MCP inserts was used to evaluate efficacy in similar damselfish by intra-peritoneal injection. After the challenge with SRDV, damselfish vaccinated with recombinant protein showed a lower protection level, while the fish vaccinated with recombinant protein supplemented Quil-A® adjuvant showed an RPS of 26%. According to RPS values recorded from the vaccinated and non-vaccinated damselfish group, the recombinant protein vaccine conferred only marginal protection against the SRDV challenge.

Development of a droplet digital PCR assay for the sensitive detection of iridovirus in Andrias davidianus.

Chinese giant salamander iridovirus (GSIV) is the first known and causative viral pathogen in Andrias davidianus. Developing a sensitive, accurate and specific assay to detect GSIV in samples is essential to prevent the further spread of the pathogen. In this study, we established a droplet digital PCR (ddPCR) assay that targeted the mcp gene of GSIV, enabling rapid and quantitative detection of the virus. We determined that the optimal annealing temperature, primer concentration and probe concentration were 57.1°C, 50 nM and 500 nM, respectively. We analysed the specificity and sensitivity of the ddPCR assay and found that five common aquatic animal viruses, including Cyprinid herpesvirus 2 (CyHV-2), infectious spleen and kidney necrosis virus (ISKNV), Koi herpesvirus (KHV) and Carp Edema Virus (CEV) displayed negative results based on this GSIV ddPCR assay. The assay can detect GSIV with the lowest detection limit of 3.7 copies per reaction. To evaluate the sensitivity and accuracy of the ddPCR assay, we tested different infected tissue samples with both the ddPCR and TaqMan real-time PCR assays. Our results showed that the ddPCR assay detected GSIV in all samples with 100% positivity, while the TaqMan real-time PCR assay detected GSIV in only 82.1% of samples. The established ddPCR method provided several advantages in detecting GISV, including high sensitivity, high precision and absolute quantification, making it a powerful tool for detection of possible and potential GSIV infection, even in samples with low viral load.

Viruses of a key coral symbiont exhibit temperature-driven productivity across a reefscape

Viruses can affect coral health by infecting their symbiotic dinoflagellate partners (Symbiodiniaceae). Yet, viral dynamics in coral colonies exposed to environmental stress have not been studied at the reef scale, particularly within individual viral lineages. We sequenced the viral major capsid protein (mcp) gene of positive-sense single-stranded RNA viruses known to infect symbiotic dinoflagellates (‘dinoRNAVs’) to analyze their dynamics in the reef-building coral, Porites lobata. We repeatedly sampled 54 colonies harboring Cladocopium C15 dinoflagellates, across three environmentally distinct reef zones (fringing reef, back reef, and forereef) around the island of Moorea, French Polynesia over a 3-year period and spanning a reef-wide thermal stress event. By the end of the sampling period, 28% (5/18) of corals in the fringing reef experienced partial mortality versus 78% (14/18) of corals in the forereef. Over 90% (50/54) of colonies had detectable dinoRNAV infections. Reef zone influenced the composition and richness of viral mcp amino acid types (‘aminotypes’), with the fringing reef containing the highest aminotype richness. The reef-wide thermal stress event significantly increased aminotype dispersion, and this pattern was strongest in the colonies that experienced partial mortality. These findings demonstrate that dinoRNAV infections respond to environmental fluctuations experienced in situ on reefs. Further, viral productivity will likely increase as ocean temperatures continue to rise, potentially impacting the foundational symbiosis underpinning coral reef ecosystems.

Evaluation of the antimicrobial mechanism of biogenic selenium nanoparticles against Pseudomonas fluorescens

Selenium nanoparticles (SeNPs) can be biosynthesized by most Lactic acid bacteria thereby converting toxic sodium into SeNPs. However, few studies have reported the antimicrobial activity of biogenic SeNPs against Pseudomonas fluorescens which are the main species of psychrotrophic bacteria in raw milk. This study reported the synthesis and characterization of SeNPs from Lactobacillus casei ZK-AS 1.1482, and the antimicrobial mechanism against P. fluorescens ATCC 13525. The synthesized SeNPs were amorphous with sizes ranging from 52 to 103 nm. Fourier transform infrared spectroscopy (FT-IR) spectra showed the presence of proteins, polysaccharides, and lipids on the surface of particles, which evidently stabilized the SeNPs structure and morphology. Energy-dispersive X-ray (EDX) analysis revealed that the nanoparticles contained selenium. In addition, the minimal inhibitory concentration (MIC) of SeNPs against P. fluorescens ATCC 13525 was 0.1 mg ml−1 and the biofilm inhibition rate was 43.52 ± 0.26%. SeNPs decreased the number of living bacteria observed by confocal laser scanning microscopy (CLSM). Meanwhile, after SeNPs treatment, the intracellular adenosine triphosphate (ATP) concentration and antioxidant enzyme activity decreased, the content of reactive oxygen species (ROS) and the malondialdehyde (MDA) content increased, and lipid peroxidation intensified. Real-time fluorescence quantitative PCR (RT-qPCR) assay showed that the expression of flgA, luxR, lapD, MCP, cheA, c-di-GMP, phoB, and pstC gene were down-regulated after SeNPs treatment. The rfbC and DegT/DnrJ/EryC1/StrS gene were significantly up-regulated, indicating that SeNPs could destroy the integrity of cell membrane and thus play an antimicrobial role. Biogenic SeNPs are expected to be developed as an efficient and novel antimicrobial agent for application in the food industry.

Development of a droplet digital PCR method for the sensitive detection and quantification of largemouth bass ranavirus.

Largemouth bass ranavirus (LMBRaV), also known as largemouth bass virus (LMBV), is a high mortality pathogen in largemouth bass. A rapid, sensitive, specific and convenient diagnosis method is an urgent requirement for the prevention of virus transmission. In the present study, a droplet digital PCR (ddPCR) method based on the major capsid protein (mcp) gene was established to detect and quantify the virus genome copy number. Oligonucleotide primers were designed based on the LMBRaV mcp gene sequence. The specificity and sensitivity of ddPCR assay were analysed. The other aquatic virus including Chinese giant salamander iridovirus (GSIV), Cyprinid herpesvirus II (CyHV-2) and infectious spleen and kidney necrosis virus could not be detected by LMBRaV ddPCR assay. The detection limit of ddPCR assay was 2 ± 0.37 copies/μl DNA sample. And this ddPCR assay had great repeatability and reproducibility. In clinical diagnosis of 50 largemouth bass, 43 positive samples were detected by ddPCR, whereas only 34 positive samples were detected by quantitative PCR (qPCR). This LMBRaV detection assay provided a specific and sensitive method for the rapid diagnosis of LMBRaV infection in largemouth bass as well as quantification of the virus load.

Detection of lymphocystis disease virus (LCDV) from yellowbar angelfish (Pomacanthus maculosus Forsskål, 1775) in Iran: Histopathological and phylogenetic analysis

Lymphocystis disease (LCD) is a benign viral disease characterized by the appearance of hypertrophic fibroblastic cells in the connective tissues of fish. In December 2021, a group of yellowbar angelfish (Pomacanthus maculosus Forsskål, 1775) which had significant mortalities (10%) and showed whitish nodules on the fins and skin, were packed in water-filled plastic bags, supplied with oxygen, and transported to the Ornamental Fish Clinic, Faculty of Veterinary Medicine, University of Tehran (Tehran, Iran). Following the clinical inspection and observation of whitish clusters of nodules mostly on the fins and skin by stereomicroscopy, wet smears of skin and fins revealed numerous hypertrophic lymphocystis cells. Histopathological study of the whitish nodules in skin and fins revealed many hypertrophic cells with thick hyaline capsules, irregular nucleus, and multiple intracytoplasmic inclusion bodies. In addition to the extension of hypertrophic lymphocystis cells' growth until the superficial muscle layer, infiltration of inflammatory cells and hemorrhage between hypertrophic cells are also observed. The hyaline capsule of hypertrophic fibroblasts stained with the PAS. Molecular characterization (PCR) confirmed the presence of Lymphocystis disease virus (LCDV) in the external nodules and the internal organs homogenate of the infected fish. Sequence analysis of the MCP gene of the detected virus (IR_LCDV_RGZIS) assigned it to genogroup 5. This is the first detection and characterization of LCDV in yellowbar angelfish (Pomacanthus maculosus) in Iran.

Infection of Infectious Spleen and Kidney Necrosis Virus and Viral Nervouse Necrosis in Seawater Fish Identifed Using Multiplex Polymerase Chain Reaction

Indonesia become a maritime country with large of ocean area and beach line with marine species diversity reaches 37% of the world’s fish. This makes Indonesia have aquaculture potential that supported by an appropriate climate. However, the challenge of viral diseases caused by Infectious Spleen and Kidney Necrosis Virus (ISKNV) and Viral Nervous Necrosis (VNN) would hamper marine aquaculture and causing huge economic losses. Therefore, it is necessary to carry out early detection methods that are efficient, fast, precise and accurate to identifying these viral disease. Here we developed a multiplex-PCR (mPCR) a method that can detect simultaneously of the ISKNV and VNN. The results of this method showed high sensitivity and specificity by using MCP and CP gene target primers to detect ISKNV and VNN, respectively. From a total of 353 samples of seawater fish examined by mPCR, positive results following of single infection of ISKNV and VNN were 14 and 2, respectively. We also found that 18 grouper fish was co-infection with these viruses. From these results, it shows that the mPCR method developed has efficiency, faster and high accuracy. This founding was possible to be applied in laboratory testing or early detection system in the field for ISKNV and VNN.

Influenza B-Associated Atypical Hemolytic Uremic Syndrome

Hemolytic uremic syndrome (HUS) triggered by influenza virus (iHUS) is rare. Influenza A infections have been described to trigger atypical HUS (aHUS) in individuals with an underlying genetic complement dysregulation. To date there are only few reports of Influenza B as a trigger of aHUS, all identified cases associated with mutations in the MCP or C3 gene, occasionally combined with other mutations. aHUS patients should be screened for all known disease-associated genes and screening should not be stopped after finding a mutation, to identify other genetic susceptibility factors influencing gene phenotype, particularly in patients with MCP or CFI mutations. Complement blockade using a monoclonal anti-C5 antibody, eculizumab, has greatly improved the outcome in recent years for certain groups of HUS. The decision on whether to treat or not with eculizumab should be made based on clinical and laboratorial evolution as well as molecular studies results. Influenza A and B are preventable through vaccination and strategies should be addressed for patients with complement gene mutations identified.

Antiviral Activities of Green Tea Components against Grouper Iridovirus Infection In Vitro and In Vivo.

(1) Background: Singapore grouper iridovirus (SGIV) can cause extensive fish deaths. Therefore, developing treatments to combat virulent SGIV is of great economic importance to address this challenge to the grouper aquaculture industry. Green tea is an important medicinal and edible plant throughout the world. In this study, we evaluated the use of green tea components against SGIV infection. (2) Methods: The safe working concentrations of green tea components were identified by cell viability detection and light microscopy. Additionally, the antiviral activity of each green tea component against SGIV infection was determined with light microscopy, an aptamer (Q5c)-based fluorescent molecular probe, and reverse transcription quantitative PCR. (3) Results: The safe working concentrations of green tea components were green tea aqueous extract (GTAE) ≤ 100 μg/mL, green tea polyphenols (TP) ≤ 10 μg/mL, epigallocatechin-3-gallate (EGCG) ≤ 12 μg/mL, (-)-epigallocatechin (EGC) ≤ 10 μg/mL, (-)-epicatechin gallate (EGC) ≤ 5 μg/mL, and (-)-epicatechin (EC) ≤ 50 μg/mL. The relative antiviral activities of the green tea components determined in terms of MCP gene expression were TP > EGCG > GTAE > ECG > EGC > EC, with inhibition rates of 99.34%, 98.31%, 98.23%, 88.62%, 73.80%, and 44.31%, respectively. The antiviral effect of aptamer-Q5c was consistent with the results of qPCR. Also, TP had an excellent antiviral effect in vitro, wherein the mortality of fish in only the SGIV-injection group and TP + SGIV-injection group were 100% and 11.67%, respectively. (4) Conclusions: In conclusion, our results suggest that green tea components have effective antiviral properties against SGIV and may be candidate agents for the effective treatment and control of SGIV infections in grouper aquaculture.

Red sea bream iridovirus infection in cage farmed Asian sea bass (Lates calcarifer): Insights into the pathology, epizootiology, and genetic diversity

Red sea bream iridovirus (RSIV) is an emerging viral pathogen having significant socio-economic and environmental impacts on marine and brackish aquaculture systems. The study documents certain interesting findings of an RSIV disease outbreak that caused 50% cumulative mortality among cage farmed Asian sea bass in India. The confirmatory diagnosis was made based on internationally approved protocols. The affected fish showed splenomegaly, anaemia, and abnormally enlarged cells within the spleen, liver, kidney, and brain. The consistent presence of two fish pathogens, viz., Vibrio alginolyticus and Vibrio fluvialis, in the liver, along with characteristic vibrio-induced pathologies were noted, indicating the possible role of these bacterial pathogens in RSIV pathogenesis during the outbreak. More strikingly, the surviving fish were found to carry the virus even after two months post-outbreak, warranting future investigations on the carrier status of RSIV survivors. Transmission electron microscopy in the survivors demonstrated atypical virus particles within the degenerated cells of the vital organs, suggesting the role of host immune response in preventing the maturation of RSIV particles in the survivors. Phylogram based on MCP and ATPase genes demonstrated the spread of the same RSIV clone across different countries, regardless of the fish species. Results also revealed the presence of two RSIV subtypes. Both subtypes were mainly distributed in marine demersal fishes of Asian countries. Presently, subtype 2 is the most frequently reported one. Subtype 1 revealed higher genetic diversity with a wider geographical spread and host range. Molecular analyses revealed that MCP and ATPase genes could be targeted in RSIV subtyping, with MCP as a better candidate. A purifying selection was observed on the MCP gene that can be applied for diagnostic protocols. In short, the study generates certain thought-provoking insights on RSIV through the analyses of a recent outbreak along with the genetic and epizootiological data of global strains. Further, the paper also forms the first report on the presence of RSIV in surviving fish post-disease outbreak in the absence of clinical signs. Altogether, the results highlight the necessity of strengthening quarantine and RSIV diagnostic measures to have sustainable aquaculture practices.

Intracellular DNA transfer events restricted to the genus Convallaria within the Asparagaceae family: Possible mechanisms and potential as genetic markers for biographical studies

Intracellular gene transfer among plant genomes is a common phenomenon. Due to their high conservation and high plastid membrane integrity, chloroplast (cp) genomes incorporate foreign genetic material very rarely. Convallaria is a small monocotyledonous genus consisting of C. keiskei, C. majalis and C. montana. Here, we characterized, analyzed and identified 3.3 and 3.7 kb of mitochondrial DNA sequences in the plastome (MCP) of C. majalis and C. montana, respectively. We identified 6 bp and 23 bp direct repeats and mitochondrial pseudogenes, with rps3, rps19 and rpl10 identified in the MCP region. Additionally, we developed novel plastid molecular genetic markers to differentiate Convallaria spp. based on 21 populations. BEAST and biogeographical analyses suggested that Convallaria separated into Eurasian and North American lineages during the middle Pliocene and originated in East Asia. Vicariance in the genus was followed by dispersal into Europe and southeastern North America. These analyses indicate that the MCP event was restricted to the genus Convallaria of Asparagaceae, in contrast to similar events that occurred in its common ancestors with other families of land plants. However, further mitochondrial and population studies are necessary to understand the integration of the MCP region and gene flow in the genus Convallaria.

The Whole Exome Analysis and Clinical Relevance in Patients with Atypical Hemolytic Uremic Syndrome

INTRODUCTION:Atypical hemolytic uremic syndrome (aHUS) is a rare, genetic, progressive, life-threatening form of thrombotic microangiopathy (TMA). A genetic mutation has been found as an explanation for constitutive complement activation in 50-60% of cases. In this abstract, we present whole exome analysis data results and their clinical relevance in 12 patients with aHUS.MATERIALS AND METHOD:We have enrolled 24 patients in this study who were diagnosed as aHUS and followed in our clinic since 2012. For exome sequencing, libraries were prepared using SeqCap EZ Human Exome Library v3.0 kit. Real Time Analysis software version with standard parameters was used for real-time image analysis and base calling. Raw sequencing data were aligned to the human genome hg19 reference (Homo sapiens (hg19) sequence) using CLC Genomic Workbench V10 (Qiagen) with standard parameters in paired-end mode. All variants were filtered according to the following criteria: not in dbSNP144 and also 1000 genome MAF <0.01, homozygous in the affected patients and heterozygous in the carrier parents, Genotype Quality (GQ) >30 and coverage (DP) >20 and, in homozygous region.FINDINGS AND RESULTS:We performed whole exome sequencing in all 24 patients but during raw data analysis 12 patients were found to be eligible for further analysis according to QC value. We didn't find any mutation in one of the patients (case 10) who had presented with diarrhea and had very good response to plasmapheresis only. We detected 32 different complement mutations in 4 different genes including CC3, CFI, CFH and membrane MCP in all other 11 patients. These mutations are summarized in the table.Four of the patients have responded to plasmapheresis and eculizumab was not needed. Of the remaining 20 patients eculizumab was aimed to be used. However, two of them died due to acute respiratory distress and thrombosis prior to eculizumab therapy. Case 9 was one of them and we found 21 different mutation including nine in CC3, five in CFH and 7 in MCF. One of our patients died in her 3rd month of eculizumab therapy (Case 2) and she had also seven mutations mostly in CFH gene. We used eculizumab in 18 of the patients and got complete response for thrombotic microangiopathy (TMA) in all. We had very good response to eculizumab in all these patients; even hemodialysis need disappeared in nine of them during eculizumab therapy. We experienced recurrence of renal failure in one patient (Case11) under eculizumab treatment in two years. She had also two different mutations in CFI gene four of the seven mutations in MCP gene and four of the 15 mutations in CC3 gene. This patient was lost due to multiple organ failure.We decided to stop eculizumab at the ninth month of treatment in two of the patients (Case 1 and Case 3) who had complete hematologic and renal recovery. Case 1 relapsed 16 months later and successfully retreated with eculizumab. She had 15 different mutations as eight mutations in CC3, six mutations in CFH, and 1mutation in MCP gene. Case 3 is still under control for three years after stopping eculizumab treatment. But as an interesting finding, this patient was 80 years old and had the heaviest mutation load with 22 different mutations (6 of the 7 mutations in MCP gene, 11 of the 15 mutation in CC3 gene, 4 of the 8 mutation in CFH gene).AHUS has been diagnosed during pregnancy in two of the patients and they had a successful delivery with healthy babies without any complication including ESRD. We also found 14 different mutations mostly in CFH gene in one of them (Case 8).DISCUSSION AND CONCLUSION:We found at least four different mutations in all patients except one case. Although MCP mutations accounts for only 15% of the aHUS cases we also found at least one MCP mutation in 91% of our cases. We found the rarest mutations in CFI gene. Although there are no clinical predictors of outcome, knowledge of the underlying genetic defect is helpful in predicting prognosis. But the patient who had the most number of mutations also had the best prognosis in our cohort. Therefore it is hard to use mutation data for management and prognosis of the syndrome. But the presence of a mutation is clearly important for the ESRD before transplantation decision and also would encourage patients to continue treatment. [Display omitted] DisclosuresYenerel:Alexion: Honoraria. Caliskan:Alexion: Honoraria. Yildiz:Alexion: Honoraria. Turkmen:Alexion: Honoraria.

Evaluation of Gilthead Seabream (Sparus aurata) Immune Response after LCDV-Sa DNA Vaccination.

Simple SummaryLymphocystis disease is the main viral pathology in gilthead seabream aquaculture. Currently, there are no treatments or vaccines to control this disease, thus our main goal was to construct a DNA vaccine that can be used in the future to stop the spread of this pathology in sea farms. The vaccine consisted of a plasmid DNA that contains a known viral gene. Once it was established that the vaccine drives the expression of the antigenic viral protein in fish, vaccination experiments were conducted to determine if the vaccinated fish become protected against the viral infection. In addition, the immune response triggered by the vaccine was also evaluated in order to understand the mechanisms underlying such protection. The obtained results showed that in vaccinated fish an activation of several genes relating to both the inflammatory process and the mucosal immunity were produced, as well as specific anti-viral antibodies. Although limited, our results deserve further investigation to assess the efficacy of the vaccine in bigger fish populations and to confirm the mode of action of the vaccine.Lymphocystis disease is the main viral pathology reported in gilthead seabream. Its etiological agent is Lymphocystis disease virus 3 (LCDV-Sa), genus Lymphocystivirus, family Iridoviridae. There are no effective treatments or vaccines for LCDV control, thus the main aim of this study was to develop a DNA vaccine, and to evaluate both the protection conferred against LCDV-Sa infection and the immune response in vaccinated fish. The vaccine was constructed by cloning the mcp gene (ORF LCDVSa062R) into pcDNA3.1/NT-GFP-TOPO. Two independent vaccination trials were conducted. In the first one, 5–7 g fish were intramuscularly injected with the vaccine (pcDNA-MCP) or the empty-plasmid, and the distribution and expression of the vaccine was investigated. Furthermore, vaccinated fish were challenged with LCDV-Sa in order to access the protective capacity of the vaccine. In the second trial, 70–100 g fish were vaccinated as specified, and the immune response was evaluated analyzing the expression of 23 immune-related genes and the production of specific antibodies. The results showed that the vaccine triggers an immune response characterized by the overexpression of genes relating to the inflammatory process, but not the innate antiviral immunity relating to type I IFN (interferon), and also induces the production of specific neutralizing antibodies, which could explain the protection against LCDV-Sa in vaccinated fish.

Development of a Microfluidics-Based Quantitative Real-Time PCR to Rapidly Identify Photobacterium damselae subsp. damselae with Different Pathogenicity by Detecting the Presence of mcp or dly Gene

As a marine bacterial pathogen, Photobacterium damselae subsp. damselae (PDD) is distributed in seawater worldwide. It can infect different animals as well as humans, even cause deaths. The highly conserved regions of PDD mcp gene on chromosome and dly gene on plasmid were selected as the target fragments to design the specific primers. Recombinant plasmid standard was prepared based on the primers. With GENECHECKER UF-150 qRT-PCR instrument as the platform, a fluorescence-based quantitative real-time PCR (qRT-PCR) method was established for the detection of PDD. This method can specifically detect PDD and distinguish the highly virulent strains. Additionally, the test results can be qualitatively judged by visualization, while the quantitative detection can be achieved through the standard curve calculation. The minimum limit of detection was 1.0×101 copies µL−1, and the detection time was less than 20 min. In summary, this new method has outstanding advantages, such as strong specificity, high sensitivity, and low site requirements. It is a rapid on-site detection technology for highly virulent PDD strains.

Tomato Chlorosis Virus Minor Coat Protein as a Novel Target To Screen Antiviral Drugs.

Minor coat protein (mCP), an important component of tomato chlorosis virus (ToCV), plays a significant role in the process of virus assembly and movement and is directly related to the virus-insect transmission. Therefore, ToCV mCP could be considered as a potent target for anti-ToCV drugs. In this study, ToCV mCP was first cloned, expressed, purified, and a novel target to screen the antiviral agents. The results showed that some antiviral compounds bound to ToCV mCP with strongly affinities in vitro, including quinazoline derivatives 4a and 4b, Ningnanmycin, and Ribavirin. Subsequently, three-dimensional-quantitative structure-activity relationship (3D-QSAR) analysis was performed based on the binding affinities, and the model indicated that 4a and 4b had indeed stronger binding effects on ToCV mCP than other quinazoline derivatives. Finally, the anti-ToCV activities of compounds 4a and 4b were evaluated by quantitative real-time polymerase chain reaction in vivo. Compounds 4a and 4b inhibited infection of ToCV in the host and as well as reduced the level of ToCV mCP gene expression. Thus, ToCV mCP can be used as a novel drug target for screening anti-ToCV agents, and the ligand-based 3D-QSAR analysis of quinazoline derivatives provided new insights into the design and optimization of novel anti-ToCV drug molecules based on ToCV mCP.

Single-walled carbon nanotubes as delivery vehicles enhance the immunoprotective effect of an immersion DNA vaccine against infectious spleen and kidney necrosis virus in mandarin fish

As a high mortality disease, Infectious spleen and kidney necrosis virus (ISKNV) can cause massive economic damage on mandarin fish farming industry in China, which seriously hindered the development of mandarin fish farming industry. In this research, SWCNTs (single-walled carbon nanotubes) as a candidate for DNA vaccine carrier was vaccinated by immersion (1, 2, 5, 10, 20 mg/L) in juvenile mandarin fish. In muscle, spleen and kidney tissues, the results showed that transcription and expression of MCP gene can be detected in pcDNA-MCP and SWCNTs-pcDNA-MCP groups after bath immunization. The immune response (immune-related genes expression, serum antibody production, enzyme activities and C3 content) was significantly enhanced in fish which vaccinated with SWCNTs-pcDNA-MCP in comparison with those vaccinated with pcDNA-MCP alone. After 14 d challenge, the RPS (relative percentage survival) can be enhanced which using SWCNTs as a carrier in SWCNTs-pcDNA-MCP (82.4%) group at 20 mg/L (the highest vaccine dose) than the naked pcDNA-MCP (54.2%) group. This study reveals that functionalized SWCNTs could be a promising immersion DNA vaccine carrier in aquaculture.