Molecular mechanism of TRIM32 in antiviral immunity in rainbow trout (Oncorhynchus mykiss).

TRIM family proteins are widely found in multicellular organisms and are involved in a wide range of life activities, and also act as crucial regulators in the antiviral natural immune response. This study aimed to reveal the molecular mechanism of rainbow trout TRIM protein in the anti-IHNV process. The results demonstrated that 99.1 % homology between the rainbow trout and the chinook salmon (Oncorhynchus tshawytscha) TRIM32. When rainbow trout were infected with IHNV, the TRIM32 was highly expressed in the gill, spleen, kidney and blood. Meanwhile, rainbow trout TRIM32 has E3 ubiquitin ligase activity and undergoes K29-linked polyubiquitination modifications dependent on the RING structural domain was determined by immunoprecipitation. TRIM32 could interact with the NV protein of IHNV and degrade NV protein through the ubiquitin-proteasome pathway, and was also able to activate NF-κB transcription, thereby inhibiting the replication of IHNV. Moreover, the results of the animal studies showed that the survival rate of rainbow trout overexpressing TRIM32 was 70.2 % which was significantly higher than that of the control group, and stimulating the body to produce high levels of IgM when the host was infected with the virus. In addition, TRIM32 can activate the NF-κB signalling pathway and participate in the antiviral natural immune response. The results of this study will help us to understand the molecular mechanism of TRIM protein resistance in rainbow trout, and provide new ideas for disease resistance breeding, vaccine development and immune formulation development in rainbow trout.

Salmonid alphavirus non-structural protein 2 is a key protein that activates the NF-κB signaling pathway to mediate inflammatory responses.

Salmonid alphavirus (SAV) infection of Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) causes pancreas disease (PD) with typical inflammatory responses, such as necrosis of the exocrine pancreas, cardiomyopathy and skeletal myopathy. However, the pathogenic mechanism underlying SAV infection is still unclear. Inflammation may cause damage to the body, but it is a defense response against infection by pathogenic microorganisms, of which nuclear factor-kappa B (NF-κB) is the main regulator. This study revealed that SAV can activate NF-κB, of which the viral nonstructural protein Nsp2 is the major activating protein. SAV activates the NF-κB signaling pathway by simultaneously up-regulating TLR3, 7, 8 and then the expression of the signaling molecule myeloid differentiation factor 88 (Myd88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). We found that Nsp2 can induce IκB degradation and p65 phosphorylation and transnucleation, and activate NF-κB downstream inflammatory cytokines. Nsp2 may simultaneously activate NF-κB through TLR3,7,8-dependent signaling pathways. Overexpression of Nsp2 can up-regulate mitochondrial antiviral signaling protein (MAVS) and then promote the expression of IFNa1 and antiviral protein Mx, which inhibits viral replication. This study shows that Nsp2 acts as a key activator protein for the NF-κB signaling pathway, which induces inflammation post-SAV infection. This study systematically analyzes the molecular mechanism of SAV activation of the NF-κB signaling pathway, and provides a theoretical basis for revealing the mechanism of innate immune response and inflammatory injury caused by SAV.

Infectious hematopoietic necrosis virus (IHNV) nucleoprotein amino acid residues affect viral virulence and immunogenicity in rainbow trout (Oncorhynchus mykiss).

This study compared the N protein sequences of genotype J with other genotypes of IHNV to select amino acid residues that may be related to the change in viral virulence. The recombinant viruses containing different mutation sites were rescued by alanine scanning mutagenesis and the reverse genetic system. The nine recombinant virus strains obtained in this work were named rIHNV-N85, rIHNV-N102, rIHNV-N146, rIHNV-N380, rIHNV-N85-102-146, rIHNV-N85-102-380, rIHNV-N85-146-380, rIHNV-N102-146-380, and rIHNV-N85-102-146-380. Pathogenicity and immunity assays were performed to determine the role of virulence sites. The result of the pathogenicity test showed that the survival rates of rIHNV-N85, rIHNV-N102, rIHNV-N85-102-146, and rIHNV-N85-102-380 groups were 52.5%, 55%, 67.5%, and 57.5%, while the survival rate of wild-type (wt) IHNV HLJ-09 group was only 10%. The replication ability of recombinant viruses with substitutions at positions 85 and 102 was significantly inhibited in vivo and in vitro. The qRT-PCR result indicated that the cytokines of IFN1, IL-8, and IL-1ß expression levels were increased in rIHNV-N85, rIHNV-N102, rIHNV-N85-102-146, and rIHNV-N85-102-380 groups. In addition, these four recombinant viruses could cause the rainbow trout to produce anti-IHNV-specific antibodies immunoglobulin M (IgM) earlier, confirming that 85 and 102 amino acid residues of N protein affected the virulence and immunogenicity of IHNV. All these results suggest that mutations of the N protein virulence sites reduce virulence while retaining immunogenicity. This also provides a new idea for studying the virulence mechanism of rhabdoviruses and preparing attenuated vaccines.

Immunological Effects of Recombinant Lactobacillus casei Expressing IHNV G Protein and Rainbow Trout (Oncorhynchus mykiss) Chemokine CK6 as an Oral Vaccine.

IHNV is a virus that infects salmonids and causes serious economic damage to the salmonid farming industry. There is no specific treatment for the disease caused by this pathogen and the main preventive measure is vaccination, but this is only possible for small groups of individuals. Therefore, it is important to investigate new oral vaccines to prevent IHNV. In this study, the CK6 chemokine protein of rainbow trout and the truncated G protein of IHNV were used to construct a secretory expression recombinant L.casei vaccine for rainbow trout. The results showed that the levels of IgM and IgT antibodies in rainbow trout reached the highest level on the 15th day after the secondary immunization, and the antibodies exhibited high inhibitory activity against viral infection. Furthermore, the expression of relevant cytokines in different tissues was detected and found to be significantly higher in the oral vaccine group than in the control group. It was also found that pPG-612-CK6-G/L.casei 393 could stimulate splenic lymphocyte proliferation and improve mucosal immunity with significant differences between the immunized and control groups. When infected with IHNV, the protection rate of pPG-612-CK6-G/L.casei 393 was 66.67% higher than that of the control group. We found that pPG-612-CK6-G/L.casei 393 expressed and secreted the rainbow trout chemokine CK6 protein and IHNV truncated G protein, retaining the original immunogenicity of rainbow trout while enhancing their survival rate. This indicates that recombinant L.casei provides a theoretical basis and rationale for the development of an oral vaccine against IHNV and has important practical implications for the protection of rainbow trout from IHNV infection.

Optimal Range of Lymphadenectomy in Pathological Stage T1 and T2 Esophageal Squamous Cell Carcinoma.

BACKGROUND: Lymph node metastasis is a primary contributor to tumor progression in esophageal squamous cell carcinoma (ESCC), and the optimal extent of lymphadenectomy during esophagectomy remains controversial. This study aimed to investigate the appropriate number of lymph nodes to be dissected in pT1-2Nany stage ESCC to achieve the best prognosis and avoid missing positive lymph nodes (PLNs). METHODS: A total of 497 patients with pT1 to pT2 esophageal cancer from two institutions were retrospectively analyzed and their surgical and pathological records were critically reviewed. Stepwise analyses were conducted by calculating a serial of hazard ratios and odd ratios to determine the optimal range of lymphadenectomy for overall survival (OS). RESULTS: The best survival outcome can be obtained when the number of lymph node examined (NLNE) is 10-18 in pT1N0 ESCC, while the NLNE should exceed 24 in pT2N0 diseases. In patients with pT1-2Nany and pT2Nany ESCC, resection of 15-25 and 24-37 lymph nodes, respectively, could provide significant added value for identifying positive nodal metastasis. When the NLNE exceeds this appropriate range, resection of extra lymph node is not helpful to improve the probability of finding PLNs. CONCLUSIONS: For ESCC patients undergoing radical esophagectomy, the optimal extent of lymphadenectomy is 15-25 for pT1Nany disease and 24-37 for pT2Nany disease.

A survey of jaagsiekte sheep retrovirus (JSRV) infection in sheep in the three northeastern provinces of China.

Ovine pulmonary adenomatosis (OPA) is caused by jaagsiekte sheep retrovirus (JSRV) and is a chronic, progressive, and infectious neoplastic lung disease in sheep, which causes significant economic losses to the sheep industry. Neither a vaccine nor serological diagnostic methods to detect OPA are available. We performed a JSRV infection survey in sheep using blood samples (n = 1,372) collected in the three northeastern provinces of China (i.e., Inner Mongolia, Heilongjiang, and Jilin) to determine JSRV infection status in sheep herds using a real-time PCR assay targeting the gag gene of JSRV. The ovine endogenous retrovirus sequence was successfully amplified in all sheep samples tested (296 from the Inner Mongolia Autonomous Region, 255 from Jilin province, and 821 from Heilongjiang province). Subsequently, we attempted to distinguish exogenous JSRV (exJSRV) and endogenous JSRV (enJSRV) infections in these JSRV-positive samples using a combination assay that identifies a ScaI restriction site in an amplified 229-bp fragment of the gag gene of JSRV and a "LHMKYXXM" motif in the cytoplasmic tail region of the JSRV envelope protein. The ScaI restriction site is present in all known oncogenic JSRVs but absent in ovine endogenous retroviruses, while the "LHMKYXXM" motif is in all known exJSRVs but not in enJSRVs. Interestingly, one JSRV strain (HH13) from Heilongjiang province contained the "LHMKYXXM" motif but not the ScaI enzyme site. Phylogenetic analysis showed that strain HH13 was closely related to strain enJSRV-21 reported in the USA, indicating that HH13 could be an exogenous virus. Our results provide valuable information for further research on the genetic evolution and pathogenesis of JSRV.

Infectious hematopoietic necrosis virus truncated G protein effect on survival, immune response, and disease resistance in rainbow trout.

The major antigenic protein of infectious hematopoietic necrosis virus (IHNV) is the surface glycoprotein G, which contains neutralizing epitopes that induce the production of immune neutralizing antibodies. In this study, the IHNV G gene sequence was truncated according to bioinformatics principles and then recombinantly expressed via an E. coli expression system. We then assessed the specific antibody immunoglobin M (IgM) levels of rainbow trout immunized with recombinant truncated G protein (emulsified with Freund's incomplete adjuvant), and showed that antibody IgM levels of immunized fish were significantly higher than in the control group (p < 0.01). The mRNA expression levels of interferon 1 (IFN1) and interleukin-8 (IL-8) were also up-regulated significantly (p < 0.01) in head kidneys and spleens of rainbow trout immunized with recombinant truncated G protein. Also, after challenge with wild-type IHNV HLJ-09 virus on Day 28, rainbow trout immunized with recombinant truncated G protein showed cumulative survival rates of 60%. These results indicate that the truncated G protein of IHNV expressed by the E. coli prokaryotic expression system can be used as a candidate immunogen for an IHNV subunit vaccine, which lays a theoretical foundation for the study of further potential IHNV subunit vaccines.

The L-domains in M and G proteins of infectious hematopoietic necrosis virus (IHNV) affect viral budding and pathogenicity.

RNA viruses including many retroviruses encode "late-domain" motifs that can interact with host proteins to mediate viral assembly and affect viral budding and pathogenicity. For IHNV, our previous studies demonstrated that the respective interactions of the L domains of IHNV with host proteins could mediate viral assembly and budding. To our knowledge, the role of L domains of the IHNV in the budding and pathogenicity has not investigated yet. In this study, we generated two recombinant IHNV strains rIHNV-M(PH>A4) and rIHNV-G(PS>A4) with mutations in the L domains (PPPH to AAAA or PSAP to AARA) of IHNV by reverse genetics and explored the effect of the mutations on budding and pathogenicity of the two recombinant viruses. The RT-qPCR results showed that the production levels of the extracellular particles of rIHNV-M(PH>A4) or rIHNV-G(PS>A4) declined significantly, compared with those of wild-type (wt) IHNV HLJ-09. Furthermore, the challenge test showed that the survival rates of juvenile rainbow trout challenged with rIHNV-M(PH>A4) or rIHNV-G(PS>A4) were 90% or 87%, respectively; however, the survivability was zero in groups challenged with wtIHNV HLJ-09 or rIHNV HLJ-09 (recombinant IHNV). Additionally, the RT-qPCR results showed that the recombinant viruses induced higher expression levels of IFN1, IL-1ß, and IL-8 compared with those induced by wtIHNV HLJ-09 as well as the ELISA results showed that fish vaccinated with recombinant viruses produced high levels of specific IgM antibodies, demonstrating that the two recombinant viruses may induce immune responses to resist infection by IHNV. Also, these results demonstrated for the first time that the L domains of the M and G proteins of IHNV could affect the budding and pathogenicity of IHNV, which may be beneficial in the prevention and control of IHNV infections in fish. Taken together, our study as the first research provides the foundation for effect of rhabdovirus L domains on viral budding and pathogenicity.

The role of infectious hematopoietic necrosis virus (IHNV) proteins in recruiting the ESCRT pathway through three ways in the host cells of fish during IHNV budding.

In cytokinetic abscission, phagophore formation, and enveloped virus budding are mediated by the endosomal sorting complex required for transport (ESCRT). Many retroviruses and RNA viruses encode "late-domain" motifs that can interact with the components of the ESCRT pathway to mediate the viral assembly and budding. However, the rhabdovirus in fish has been rarely investigated. In this study, inhibition the protein expression of the ESCRT components reduces the extracellular virion production, which preliminarily indicates that the ESCRT pathway is involved in IHNV release. The respective interactions of IHNV proteins including M, G, L protein with Nedd4, Tsg101, and Alix suggest the underlying molecular mechanism by which IHNV gets access to the ESCRT pathway. These results are the first observation that rhabdovirus in fish gains access to the ESCRT pathway through three ways of interactions between viral proteins and host proteins. In addition, the results show that IHNV is released from host cells through the ESCRT pathway. Taken together, our study provides a theoretical basis for studying the budding mechanism of IHNV.

Effects of Single and Joint Subacute Exposure of Copper and Cadmium on Heat Shock Proteins in Common Carp (Cyprinus carpio).

Copper (Cu) and cadmium (Cd) are the most common heavy metals that are easily detected in aquatic environments on a global scale. In this paper, we investigated the messenger RNA (mRNA) and protein levels of HSPs (HSP60, HSP70, and HSP90) in the liver of the common carp exposed to Cu, Cd, and a combination of both metals by real-time quantitative PCR and Western blot. The results indicated that in each exposure group, the mRNA levels of HSP60, HSP70, and HSP90 were increased significantly compared to the corresponding controls after 96 h of exposure (P < 0.05). A significant increase was observed in the HSP70 protein level in the high-dose Cu group and all of the Cd groups. Significant increases were also observed in the protein levels of HSP60 and HSP90 in the high combination group and the low combination group, respectively. These results indicated that the dynamics of HSP expression observed in the common carp support the role of HSPs as biochemical markers in response to environmental pollution and provided valuable insights into the adaptive mechanisms used by the common carp to adapt to the challenges of stressful environments.

Immunogenicity in Swine of Orally Administered Recombinant Lactobacillus plantarum Expressing Classical Swine Fever Virus E2 Protein in Conjunction with Thymosin α-1 as an Adjuvant.

Classical swine fever, caused by classical swine fever virus (CSFV), is a highly contagious disease that results in enormous economic losses in pig industries. The E2 protein is one of the main structural proteins of CSFV and is capable of inducing CSFV-neutralizing antibodies and cytotoxic T lymphocyte (CTL) activities in vivo. Thymosin α-1 (Tα1), an immune-modifier peptide, plays a very important role in the cellular immune response. In this study, genetically engineered Lactobacillus plantarum bacteria expressing CSFV E2 protein alone (L. plantarum/pYG-E2) and in combination with Tα1 (L. plantarum/pYG-E2-Tα1) were developed, and the immunogenicity of each as an oral vaccine to induce protective immunity against CSFV in pigs was evaluated. The results showed that recombinant L. plantarum/pYG-E2 and L. plantarum/pYG-E2-Tα1 were both able to effectively induce protective immune responses in pigs against CSFV infection by eliciting immunoglobulin A (IgA)-based mucosal, immunoglobulin G (IgG)-based humoral, and CTL-based cellular immune responses via oral vaccination. Significant differences (P < 0.05) in the levels of immune responses were observed between L. plantarum/pYG-E2-Tα1 and L. plantarum/pYG-E2, suggesting a better immunogenicity of L. plantarum/pYG-E2-Tα1 as a result of the Tα1 molecular adjuvant that can enhance immune responsiveness and augment specific lymphocyte functions. Our data suggest that the recombinant Lactobacillus microecological agent expressing CSFV E2 protein combined with Tα1 as an adjuvant provides a promising strategy for vaccine development against CSFV.