Molecular characterization and immune response of suppressor of cytokine signaling 5b from redlip mullet (Planiliza haematocheilus): Disclosing its anti-viral potential and effect on cell proliferation.

The suppressor of cytokine signaling (SOCS) proteins family comprising eight proteins (SOCS1-7 and cytokine-inducible SH2-containing (CIS)) are classical feedback inhibitors of cytokine signaling. Although the biological role of CIS and SOCS1-3 have been extensively studied, the biological functions of SOCS4-7 remain unclear. Here, we elucidated the molecular characteristics, expression profile, immune response, anti-viral potential, and effect on cell proliferation of Phsocs5b, a member of the SOCS protein family from redlip mullet (Planiliza haematocheilus); phsocs5b comprised 1695 nucleotides. It was 564 amino acids long with a molecular weight of 62.3 kDa and a theoretical isoelectric point of 8.95. Like SOCS4-7 proteins, Phsocs5b comprised an SH2 domain, SOCS box domain, and a long N-terminal. SH2 domain is highly identical to its orthologs in other vertebrates. Phsocs5b, highly expressed in the brain tissue, was localized in the cytoplasm. Temporal changes in phsocs5b expression were observed following immune stimulation with polyinosinic: polycytidylic acid, lipopolysaccharide, and Lactococcus garvieae. In FHM cells, Phsocs5b overexpression suppressed the viral hemorrhagic septicemia virus (VHSV) infection and epidermal growth factor receptor (egfr) expression but increased the mRNA levels of pi3k, akt, pro-inflammatory cytokines (il1ß and il8), and anti-viral genes (isg15 and ifn). Overall, our findings suggest that Phsocs5b attenuates VHSV infection, either by hindering the cell entry via degradation of Egfr, enhancing pro-inflammatory cytokines and anti-viral factor production, or both. The results also indicated that Phsocs5b could directly activate Pi3k/Akt pathway by itself, thus enhancing the proliferation and migration of cells. Taken together, Phsocs5b may be considered a potential therapeutic target to enhance immune responses while positively regulating the proliferation and migration of cells.

Molecular characterization and expression analysis of B-cell lymphoma-2 protein in Amphiprion clarkii and its role in virus infections.

Amphiprion clarkii is increasingly being used as a captive-bred ornamental fish in South Korea. However, its breeding has recently been greatly hindered by destructive diseases due to pathogens. B-cell lymphoma-2 (Bcl2), a mitochondrial apoptosis regulatory gene involved in immune responses, has not been investigated in anemonefish, including A. clarkii. Herein, we aimed to annotate Bcl2 in the A. clarkii transcriptome and examined its role against virus infections. Sequence analysis indicated that Bcl2 in A. clarkii (AcBcl2) contained all four Bcl-2 homology domains. The structure of AcBcl2 closely resembled those of previously analyzed anti-apoptotic Bcl2 proteins in mammals. Expression analysis showed that the highest level of AcBcl2 was expressed in blood. AcBcl2 expression in the blood was downregulated within 24 hpi when challenged with immune stimulants poly I:C and lipopolysaccharides. AcBcl2 reduced poly I:C-induced cell death. The propagation of viral hemorrhagic septicemia virus (VHSV) was higher in the presence of AcBcl2. Cell mortality was higher in AcBcl2 when transfected cells were infected with VHSV, and a higher viral transcript was observed compared to their respective controls. In conclusion, AcBcl2 is an anti-apoptotic protein, and its activity may facilitate the propagation of VHSV.

Molecular characterization, expression profile, and antiviral activity of redlip mullet (Liza haematocheila) viperin.

Viperin is known to exhibit activity against RNA viral infection. Viral hemorrhagic septicemia virus (VHSV) is a negative-sense single-stranded RNA virus that causes severe loss in aquaculture species. Susceptible species include redlip mullets (Liza haematocheila), which has become an economically important euryhaline mugilid species in offshore aquaculture along the west coast of Korea. Although interferon-stimulated genes are suspected to act against VHSV, specific pathways or mechanisms of these antiviral actions in redlip mullets have not yet been established. In silico studies of the mullet viperin (Lhrsad2) revealed an S-adenosyl methionine binding conserved domain containing the 77CNYKCGFC84 sequence. In the tissue distribution, the highest levels of lhrsad2 expression were observed in the blood. When injected with poly(I:C), an approximately 17-fold upregulation (compared to the control) of viperin was detected in the blood after 24 h. Furthermore, non-viral immune stimuli, including Lactococcus garvieae (L. garvieae) and lipopolysaccharide (LPS), that were injected into redlip mullets were not found to induce considerable levels of viperin expression. Subcellular analysis revealed that Lhrsad2 localized to the endoplasmic reticulum (ER). To investigate Lhrsad2's antiviral effects against VHSV, cells overexpressing lhrsad2 were infected with VHSV, and then the viral titer and viral gene expression were analyzed. Both assays revealed the potential of Lhrsad2 to significantly reduce VHSV transcription and replication. In brief, the current study illustrates the remarkable ability of viperin to weaken VHSV in redlip mullet.

Identification, molecular characterization, expression analysis and wound-healing ability of multifunctional calreticulin from big-belly seahorse Hippocampus abdominalis.

Calreticulin (CRT) is a multifunctional ubiquitous protein that is widely presented in all cells in eukaryotes except erythrocytes. CRT is well known for diverse cellular functions such as endoplasmic reticulum (ER)-specialized protein quality control during protein synthesis and folding, in-vivo Ca2+ homeostasis, antigen presentation, phagocytosis, wound-healing, proliferation, adhesion, and migration of cells. In the current study, we identified CRT from Hippocampus abdominalis (HaCRT) and analyzed expression profiles and functional properties. The cDNA sequence of HaCRT was identified with an open reading frame of 1226 bp. The molecular weight of HaCRT was estimated as 49 kDa. The in-silico study revealed conserved sequence arrangements such as two CRT signature motifs (5'-KHEQSIDCGGGYVKVF-3' and 5'-LMFGPDICG-3'), triplicate repeats (5'-IKDPEAKKPEDWD-3', 5'-IPDPDDTKPEDWD-3', 5'-IPDPDAKKPDDWD-3'), signal peptide and an ER-targeting 5'-KDEL-3' sequence of HaCRT. Close sequence similarity of HaCRT was observed with Hippocampus comes from phylogenetic analysis and pairwise sequence comparison. From quantitative polymerase chain reaction (qPCR) results, HaCRT was ubiquitously distributed in all tested tissues and expression levels of HaCRT were significantly modulated in blood, liver and gill tissues after stimulation with Streptococcus iniae, Edwardsiella tarda, polyinosinic:polycytidylic acid, and lipopolysaccharides. Bacterial- and pathogen-associated molecular patterns-binding activities were observed with recombinant HaCRT (rHaCRT). The treatment of murine macrophages with rHaCRT induced the expression of immune genes, such as tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and interleukin-1ß (IL-1ß). Furthermore, rHaCRT exhibited wound-healing ability. Based on the results from the above study, we suggest that HaCRT play an indispensable role in the immunity of big-belly seahorses by recognition and elimination of pathogens as well as the tissue repairing process.

Characterization and expression analysis of rockfish (Sebastes schlegelii) myeloid differentiation factor-88 (SsMyD88) and evaluation of its ability to induce inflammatory cytokines through NF-ĸB.

Innate immunity is characterized by nonspecific, prompt reactions toward armada of antigens. Animals funnel down a repertoire of immune stimulants to activate non-selective defense mechanisms rapidly. This study was conducted to characterize the rockfish (Sebastes schlegelii) adaptor protein MyD88 (SsMyD88), which interacts with both toll-like receptors and interleukin receptors. The tissue expression of unchallenged SsMyD88 was evaluated by quantitative real time PCR (qPCR). Fish were intraperitoneally injected with immune stimulants including poly I:C, lipopolysaccharides, and Streptococcus iniae. Then, the temporal expression of SsMyD88 was analyzed. Finally, the inflammatory gene expression and downstream promoter activation were analyzed. Strongest expressions were reported in the liver, gills and spleen in unchallenged conditions. All diverse immune stimulants were found to be capable of significantly altering SsMyD88 transcription during the challenge experiment. Evaluation of downstream promoter biases by SsMyD88 found a predominant activation of NF-ĸB transcription factors when compared with the AP-1, revealing significant and substantial upregulation of major inflammatory mediators such as IL-1-ß, IL-6, iNOS, COX-2 and TNF-α. Fluorescent detection confirmed an intense production of NO and the predominant differentiation of macrophages into M1 lineage with the overexpression of SsMyD88 in vitro. These results further corroborate the role of SsMyD88 as a mediatory molecule that bridges distinct immune stimulants to induce drastic immune responses in fish.

Molecular characterization and expression analysis of rockfish (Sebastes schlegelii) viperin, and its ability to enervate RNA virus transcription and replication in vitro.

Viperin, also known as RSAD2 (Radical S-adenosyl methionine domain containing 2), is an interferon-induced endoplasmic reticulum-associated antiviral protein. Previous studies have shown that viperin levels are elevated in the presence of viral RNA, but it has rarely been characterized in marine organisms. This study was designed to functionally characterize rockfish viperin (SsVip), to examine the effects of different immune stimulants on its expression, and to determine its subcellular localization. SsVip is a 349 amino acid protein with a predicted molecular mass of 40.24 kDa. It contains an S-adenosyl l-methionine binding conserved domain with a CNYKCGFC sequence. Unchallenged tissue expression analysis using quantitative real time PCR (qPCR) revealed SsVip expression to be the highest in the blood, followed by the spleen. When challenged with poly I:C, SsVip was upregulated by approximately 60-fold in the blood after 24 h, and approximately 50-fold in the spleen after 12 h. Notable upregulation was detected throughout the poly I:C challenge experiment in both tissues. Significant expression of SsVip was detected in the blood following Streptococcus iniae and lipopolysaccharide challenge, and viral hemorrhagic septicemia virus (VHSV) gene transcription was significantly downregulated during SsVip overexpression. Furthermore, cell viability assay and virus titer quantification with the presence of SsVip revealed a significant reduction in virus replication. As with previously identified viperin counterparts, SsVip was localized in the endoplasmic reticulum. Our findings show that SsVip is an antiviral protein crucial to innate immune defense.

Prevalence of human papilloma virus and their high-risk genotypes in Sri Lankan women.

Human papilloma virus (HPV) causes cervical cancer in women and approximately 700 deaths have been reported annually in Sri Lanka due to this cancer. Despite, attempts have not been made to investigate the prevalence of HPV amongst Sri Lankan women with normal cytology. In this study, a polymerase chain reaction based assay was set up to detect HPV in both normal and abnormal cytology and the positive samples were then tested for the genotypes, HPV 16 and HPV 18 as they have been identified as the high-risk types associating with cervical cancer. Eighty-four (number = 84) clinical samples (age range 27-69) analyzed in this study indicated that the prevalence of HPV, regardless of cytological abnormalities was 15.5%, (n = 13, 95% class interval ± 7.7) while it was 100% (n = 3) for those with abnormal cytology. Association of HPV 16 and HPV 18 among the abnormal cytology was 0 and 50% (n = 1), respectively and further, the prevalence of HPV 16 and HPV 18 in women was found to be 3.6% (n = 3, 95% CI ± 4.0) and 2.4% (n = 2, 95% CI ± 3.3), respectively. Moreover, age wise prevalence analysis revealed women of the age of 35-years or more to have higher HPV prevalence. The prevalence of HPV among normal cytology is 12.3% (n = 10, 95% CI ± 7.2) which is similar to the rates in other regions of Asia (China 15.4%; India 10.43%). Finally, higher prevalence of HPV in women of the age of 35-years or more in Sri Lanka, especially with malignant types call for such age group to be screened for proper clinical intervention to be made in reducing the incident of cervical cancers. This is the first report of prevalence of HPV among women with normal cytology in Sri Lanka.