Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon (IFN) signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid (N) protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid (poly(I:C)) in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly(I:C)-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB (NF-κB) nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.
Active Transport, Cell Nucleus ; Animals ; Coronavirus Infections ; immunology ; veterinary ; virology ; Genes, Viral ; Host-Pathogen Interactions ; immunology ; Interferons ; antagonists & inhibitors ; biosynthesis ; genetics ; Interleukins ; antagonists & inhibitors ; biosynthesis ; genetics ; NF-kappa B ; metabolism ; Nucleocapsid Proteins ; genetics ; immunology ; physiology ; Porcine epidemic diarrhea virus ; genetics ; pathogenicity ; physiology ; Promoter Regions, Genetic ; Swine ; Swine Diseases ; immunology ; virology

Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.
Animals ; Cell Line, Tumor ; Cloning, Molecular ; DEAD Box Protein 58 ; antagonists & inhibitors ; genetics ; immunology ; Fibroblasts ; immunology ; pathology ; Gene Expression ; Hepatitis B ; genetics ; immunology ; pathology ; veterinary ; Hepatitis B Virus, Woodchuck ; Immunity, Innate ; Interferon-beta ; genetics ; immunology ; Isoelectric Point ; Kidney ; immunology ; pathology ; virology ; Liver ; immunology ; pathology ; virology ; Marmota ; genetics ; immunology ; virology ; Open Reading Frames ; Protein Domains ; RNA, Double-Stranded ; RNA, Small Interfering ; genetics ; metabolism ; Rodent Diseases ; genetics ; immunology ; pathology ; virology

Porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are very two important pathogens that have coursed huge economic losses in swine production in worldwide. In this study,a vector pCMV-TJM containing the full-length cDNA clone of PRRSV attenuated strain TJM-F92 was firstly constructed by PCR method. Then a gene sequence containing Afl II/Mlu I e restriction enzyme sites and a transcription regulatory sequence for ORF6 (TRS6) was inserted be- tween ORF7 and 3'UTR, yielding a expression vector pCMV-TJM-TRS. Subsequently, a plasmid pCMV-TJM-Cap was constructed by cloning of PCV2 ORF2 gene into the unique sites Afl II /Mlu I of pCMV- TJM-TRS plasmid DNA. Then three recombinant PRRSV, rTJM, rTJM/TRS and rTJM/Cap, were rescued by transfection of pCMV-TJM, pCMV-TJM-TRS and pCMV-TJM-Cap into Marc-145 cells, respectively,and confirmed by the genome sequence, restriction enzyme digestion, Western Blot and IFA. They all had the molecular markers which was different from the parent virus. The growth characteristics of the rescued viruses were similar to that of parent virus. rTJM/Cap could also express efficiently PCV2 Cap protein in Marc-145 cells. At passage 8, it still had PCV2 ORF2 gene which examined by RT-PCR. It indicated that the full-length cDNA clone of PRRSV attenuated strain TJM-F92 and recombinant PRRSV rTJM/Cap expressing PCV2 Cap protein were successfully constructed. It made an important foundation for studying on the pathogenic mechanisms of PRRSV and PRRSV-PCV2 vaccine in the future.
Animals ; Capsid Proteins ; genetics ; immunology ; Cell Line ; Circoviridae Infections ; veterinary ; virology ; Circovirus ; classification ; genetics ; metabolism ; Gene Expression ; Open Reading Frames ; Porcine Reproductive and Respiratory Syndrome ; virology ; Porcine respiratory and reproductive syndrome virus ; genetics ; metabolism ; Recombination, Genetic ; Swine ; Swine Diseases ; virology ; Viral Vaccines ; genetics ; immunology

During virus infection, viral RNAs and mRNAs function as blueprints for viral protein synthesis and possibly as pathogen-associated molecular patterns (PAMPs) in innate immunity. Here, considering recent research progress in microRNAs (miRNAs) and competitive endogenous RNAs (ceRNAs), we speculate that viral RNAs act as sponges and can sequester endogenous miRNAs within infected cells, thus cross-regulating the stability and translational efficiency of host mRNAs with shared miRNA response elements. This cross-talk and these reciprocal interactions between viral RNAs and host mRNAs are termed "competitive viral and host RNAs" (cvhRNAs). We further provide recent experimental evidence for the existence of cvhRNAs networks in hepatitis B virus (HBV), as well as Herpesvirus saimiri (HVS), lytic murine cytomegalovirus (MCMV) and human cytomegalovirus (HCMV) infections. In addition, the cvhRNA hypothesis also predicts possible cross-regulation between host and other viruses, such as hepatitis C virus (HCV), HIV, influenza virus, human papillomaviruses (HPV). Since the interaction between miRNAs and viral RNAs also inevitably leads to repression of viral RNA function, we speculate that virus may evolve either to employ cvhRNA networks or to avoid miRNA targeting for optimal fitness within the host. CvhRNA networks may therefore play a fundamental role in the regulation of viral replication, infection establishment, and viral pathogenesis.
Animals ; DNA Viruses ; genetics ; physiology ; Host-Pathogen Interactions ; physiology ; Humans ; MicroRNAs ; metabolism ; RNA Viruses ; genetics ; physiology ; RNA, Messenger ; metabolism ; RNA, Viral ; metabolism ; Virus Diseases ; immunology ; physiopathology ; virology ; Virus Replication

A recombinant hemagglutinin-neuraminidase (rHN) protein from Newcastle disease virus (NDV) with hemagglutination (HA) activity was expressed in Spodoptera frugiperda cells using a baculovirus expression system. The rHN protein extracted from infected cells was used as an antigen in a hemagglutination inhibition (HI) test for the detection and titration of NDV-specific antibodies present in chicken sera. The rHN antigen produced high HA titers of 2(13) per 25 microL, which were similar to those of the NDV antigen produced using chicken eggs, and it remained stable without significant loss of the HA activity for at least 12 weeks at 4degrees C. The rHN-based HI assay specifically detected NDV antibodies, but not the sera of other avian pathogens, with a specificity and sensitivity of 100% and 98.0%, respectively, in known positive and negative chicken sera (n = 430). Compared with an NDV-based HI assay, the rHN-based HI assay had a relative sensitivity and specificity of 96.1% and 95.5%, respectively, when applied to field chicken sera. The HI titers of the rHN-based HI assay were highly correlated with those in an NDV-based HI assay (r = 0.927). Overall, these results indicate that rHN protein provides a useful alternative to NDV antigen in HI assays.
Animals ; Antibodies, Viral/*blood ; Antigens, Viral/*diagnostic use/genetics/metabolism ; Baculoviridae/genetics ; Chickens ; HN Protein/*diagnostic use/genetics/metabolism ; Hemagglutination Inhibition Tests/*methods/veterinary ; Newcastle Disease/*diagnosis/immunology/virology ; Newcastle disease virus/genetics/*immunology/metabolism ; Poultry Diseases/*diagnosis/immunology/virology ; Recombinant Proteins/diagnostic use/genetics/metabolism ; Sf9 Cells ; Spodoptera

In order to detect Infectious hematopoietic necrosis virus with immunological methods, the surface glycoprotein of a recent IHNV-Sn isolated from farmed rainbow trout ( Oncorhynchus mykiss ) in China was amplified and cloned into pET27b(+) vector (designated as pET27b-G ). The expression of recombinant plasmid pET27b-G in E. coli BL21(DE3) was induced and determined by SDS-PAGE analysis. The predicted molecular weight of glycoprotein protein was approximately 55 kD and was confirmed in this study. The inclusion body of glycoprotein was treated with urea at different urea concentrations, and dialyzed into PBS buffer. Purified glycoprotein with high concentration was obtained after dialyzed in the PBS buffer. Antisera against glycoprotein were produced from immunized rabbits. The prepared antisera could react specifically with both the recombinant glycoprotein and natural glycoprotein of the IHNV-Sn isolated in the test of indirect ELISA, and the titer against the recombinant glycoprotein was 1:20,000. IFA showed that the antisera can recognize the glycoprotein located on the surface of IHNV-Sn and IHNV reference strain. These results indicated that the expressed glycoprotein was immunogenical and antigenical and could be functional as the natural IHNV glycoprotein. These results established a foundation for further study on vaccine and rapid diagnosis of IHNV.
Animals ; Antibodies, Viral ; immunology ; Escherichia coli ; genetics ; metabolism ; Female ; Fish Diseases ; immunology ; virology ; Gene Expression ; Glycoproteins ; genetics ; immunology ; Infectious hematopoietic necrosis virus ; genetics ; immunology ; Neutralization Tests ; Oncorhynchus mykiss ; Rabbits ; Rhabdoviridae Infections ; immunology ; veterinary ; virology ; Viral Proteins ; genetics ; immunology

Swine hepatitis E virus (HEV) is widespread throughout pigs in both developing and industrialized countries. This virus is an important zoonotic agent and a public concern worldwide. Infected pigs are asymptomatic, so diagnosing swine HEV relies on detection of the virus or antibodies against the virus. However, several obstacles need to be overcome for effective and practical serological diagnosis. In this study, we developed an enzyme-linked immunosorbent assay (ELISA) that used a purified recombinant capsid protein of swine HEV. The potential clinical use of this assay was evaluated by comparing it with a commercial kit (Genelabs Technologies, Diagnostics, Singapore). Results of the ELISA were highly correlated with those of the commercial kit with a sensitivity of 97% and specificity of 95%. ROC (receiving operator characteristic) analysis of the ELISA data produced a value of 0.987 (95% CI, 0.977~0.998, p < 0.01). The cut-off value for the ELISA was also determined using negative pig sera. In summary, the HEV-specific ELISA developed in the present study appears to be both practical and economical.
Animals ; Antibodies, Anti-Idiotypic/*analysis/blood/genetics ; Capsid Proteins/*genetics/metabolism ; Enzyme-Linked Immunosorbent Assay/*methods/veterinary ; Hepatitis E/diagnosis/immunology/*veterinary/virology ; Hepatitis E virus/genetics/*isolation & purification/metabolism ; Immunoglobulin G/blood/genetics ; ROC Curve ; Recombinant Proteins/genetics/metabolism ; Swine ; Swine Diseases/*diagnosis/immunology/virology

The innate immune system is essential for the initial detection of invading viruses and subsequent activation of adaptive immunity. Three types pattern recognition receptors (PRRs) in innate immune cells play a pivotal role in the first line of host defense system. PRRs include Toll-like receptors (TLRs), RIG-I-like receptors(RLRs) and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs). PRRs recognize pathogen-associated molecular patterns(PAMPs) or danger-associated molecular patterns (DAMPs) to initiate and regulate innate and adaptive immune responses. Three types PRRs have their own features in ligand recognition and cellular location. Activated PRRs deliver signals to adaptor molecules (MyD88, TRIF, IRAK, IPS-1), which act as important messengers to activate downstream kinases (IKK complex, MAPKs, TBK1, RIP-1) and transcription factors (NF-kappaB, AP-1, IRF3), which produce effected molecules including cytokines, chemokines, inflammatory enzymes, and type I interferons. This review focuses on discussing PRRs signaling pathways and achievements in this field in order to provide beneficial strategies for human life and immune diseases prevention.
Animals ; Humans ; Immunity, Innate ; Receptors, Pattern Recognition ; genetics ; immunology ; metabolism ; Signal Transduction ; Virus Diseases ; immunology ; metabolism ; virology ; Virus Physiological Phenomena

A recombinant modified vaccinia Ankara (MVA) virus expressing mature viral protein 2 (VP2) of the infectious bursal disease virus (IBDV) was constructed to develop MVA-based vaccines for poultry. We demonstrated that this recombinant virus was able to induce a specific immune response by observing the production of anti-IBDV-seroneutralizing antibodies in specific pathogen-free chickens. Besides, as the epitopes of VP2 responsible to induce IBDV-neutralizing antibodies are discontinuous, our results suggest that VP2 protein expressed from MVA-VP2 maintained the correct conformational structure. To our knowledge, this is the first report on the usefulness of MVA-based vectors for developing recombinant vaccines for poultry.
Animals ; Antibodies, Viral ; Birnaviridae Infections/prevention & control/*veterinary ; Cells, Cultured ; Chick Embryo ; Chickens ; Fibroblasts/metabolism ; Infectious bursal disease virus/*immunology ; Poultry Diseases/*prevention & control/virology ; Specific Pathogen-Free Organisms ; Vaccinia virus/*genetics/immunology/metabolism ; Viral Structural Proteins/genetics/*immunology/metabolism ; Viral Vaccines/*immunology

Interferon response is the first line of host defense against virus infection. Recent years have witnessed tremendous progress in understanding of fish innate response to virus infection, especially in fish interferon antiviral response. A line of fish genes involved in interferon antiviral response have been identified and functional studies further reveal that fish possess an IFN antiviral system similar to mammals. However, fish virus-induced interferon genes contain introns similar to mammalian type III interferon genes although they encode proteins similar to type I interferons, which makes it hard to understand the evolution of vertebrate interferon genes directly resulting in a debate on nomenclature of fish interferon genes. Actually, fish display some unique mechanisms underlying interferon antiviral response. This review documents the recent progress on fish interferon response and its molecular mechanism.
Animals ; Fish Diseases ; immunology ; virology ; Fish Proteins ; genetics ; metabolism ; Fishes ; immunology ; virology ; Gene Expression Regulation ; Interferons ; genetics ; immunology ; STAT1 Transcription Factor ; metabolism ; Virus Diseases ; immunology ; veterinary