Porcine epidemic diarrhea virus (PEDV) causes an acute enteritis in pigs of all ages, often fatality for neonates. PEDV occupies an intermediate position between two well characterized members of the coronavirus group I, human coronavirus (HCoV-229E)and transmissible gastroenteritis virus (TGEV) which uses aminopeptidase N (APN), a 150 kDa protein, as their receptors. However, the receptor of the PEDV has not been identified yet. A virus overlay protein binding assay (VOPBA) was used to identify PEDV binding protein in permissive cells. The binding ability of PEDV to porcine APN (pAPN) and the effects of pAPN on infectivity of PEDV in Vero cells were also investigated. VOPBA identified a 150 kDa protein, as a putative PEDV receptor in enterocytes and swine testicle (ST) cells. Further the PEDV binding to pAPN was blocked by anti-pAPN and pAPN enhanced PEDV infectivity in Vero cells. In conclusion, these results suggested that pAPN may act as a receptor of PEDV.
Animals ; Antigens, CD13/*metabolism ; Cercopithecus aethiops ; Coronavirus/*metabolism ; Coronavirus Infections/*veterinary/virology ; Digestive System Diseases/metabolism/*veterinary/virology ; Enterocytes/enzymology/metabolism/virology ; Enzyme-Linked Immunosorbent Assay/veterinary ; Male ; Protein Binding ; Receptors, Virus/*metabolism ; Swine ; Swine Diseases/metabolism/*virology ; Vero Cells

The capability of porcine reproductive and respiratory syndrome virus (PRRSV) to be shed in semen for extended periods of time has been suggested to be a principal factor for viral transmission via insemination. In attempts to gain insights into the mechanism of PRRSV persistence in boars, tissue distribution and sites of viral infection were investigated by in situ hybridization (ISH) using digoxigenin-labeled RNA probe and the ISH results were compared with those of reverse transcription-nested polymerase chain reaction (RT-nested PCR). Animals were intranasally inoculated with 104 median tissue culture infectious dose of PRRSV VR-2332 and tissues collected at different times were examined. At day 7 postinfection, limited number of hybridization positive signals was observed in cells within or between seminiferous tubules in the testis sections while relatively abundant hybridization positive signals were observed in the brain stem and tracheobronchial lymph node. At later days of infection, hybridization positive signals were observed in cells within seminiferous tubules with much reduced frequency. Lack of agreement with the RT-nested PCR assay results in testis tissues obtained at days 14, 28, and 59 postinfection suggested that PRRSV infection in the testis may be extremely restricted, and may not necessarily constitute a major viral source in semen during extended periods of seminal shedding.
Animals ; Brain Stem/virology ; Endopeptidase K/metabolism ; *In Situ Hybridization ; Lymph Nodes/virology ; Male ; Microwaves ; Porcine Reproductive and Respiratory Syndrome/transmission/*virology ; Porcine respiratory and reproductive syndrome virus/*genetics/*isolation & purification ; RNA Probes ; Reverse Transcriptase Polymerase Chain Reaction ; Semen/virology ; Seminiferous Tubules/virology ; Sensitivity and Specificity ; Sexually Transmitted Diseases, Viral/transmission/veterinary/virology ; Swine/*virology ; Testis/virology

Ubiquitin is highly conserved 76 amino acid protein found in all eukaryotic organisms and ubiquitin-proteasome pathway (UPP) plays a very important role in regulated non-lysosomal ATP dependent protein degradation. This pathway participates in or regulates numerous cellular processes, such as selective protein degradation, cell cycle progression, apoptosis, signal transduction, transcriptional regulation, receptor control by endocytosis, immune response and the processing of antigens. Nevertheless, roles of UPP in virus infection are only beginning to be clarified. Ubiquitin homology has also been found in insect viruses. All viral ubiquitin genes encode an N-terminal ubiquitin sequence and 3-256 amino acids C-terminal peptides. Most of the residues known to be essential for ubiquitin function have been conserved in the viral variant. In Autographa californica nucleopolyhedrovirus (AcMNPV), viral ubiquitin is attached to the inner surface of budded viron membrane by a covalently linked phospholipid and is not essential for viral replication. Currently, insect viruses are the only viruses known to encode ubiquitin. However, ubiquitin also plays a role in the life cycle of other viruses. Host ubiquitin molecules have been found in some plant viruses and other animal viruses. Additionally, Africa swine fever virus (ASFV) encodes a ubiquitin-conjugating enzyme (E2) and a putative causal link between human immunodeficiency virus type 1 (HIV-1) and ubiquitin was established by showing that depletion of the intracellular pool of free ubiquitin inhibits the virus budding. Further analyses indicated that many retroviruses proteins which are required for efficient pinching off the virus bud contain a late domain. The core element of the late domain is a proline-rich motif (PPXY) which mediates the late domain to be ubiquitinated by cellular proteins. Recently, it has been shown that many retroviruses have developed mechanisms to escape the cellular immune response, to facilitate virus replication and to promote virus assembly and budding via host UPP.
African Swine Fever Virus ; metabolism ; pathogenicity ; Animals ; Humans ; Insect Viruses ; metabolism ; pathogenicity ; Proteasome Endopeptidase Complex ; metabolism ; Retroviridae ; metabolism ; pathogenicity ; Ubiquitin ; metabolism ; Ubiquitin-Protein Ligase Complexes ; metabolism ; Virus Diseases ; virology ; Viruses ; pathogenicity

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

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

Based on a pair of specific primers, a 804-bp fragment was amplified from the plasmid pT-Cap containing Cap gene of Porcine Getah Virus(PGETV) and cloned into the prokaryotic expression vector pCold I which carried the His tag, this recombinant plasmid was then determined by enzyme digestion, PCR and DNA sequencing. This recombinant plasmid pCold-Cap was transformed into E. coli Rosetta 2, and PGETV Cap fusion protein was expressed through IPTG induction. The results showed that the Cap gene obtained efficient and soluble expression in Rosetta 2 induced by 0. Immol/L IPTG under 15"C for 24h, the expression quantity was 40. 2%. The product had a molecular mass about 32. 3kD as expected. The target protein was separated in gel slices and used to immunize Balb/c mice. The polyclonal antibody with high titer against Cap protein specifically analyzed by Western blot was obtained. The successful preparation of the polyclonal antibody laid the foundation for the further study on the detection and identification of PGETV.
Alphavirus ; genetics ; immunology ; metabolism ; Alphavirus Infections ; immunology ; veterinary ; virology ; Animals ; Antibodies, Viral ; blood ; immunology ; Blotting, Western ; Capsid Proteins ; genetics ; immunology ; isolation & purification ; metabolism ; DNA Primers ; genetics ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Genetic Vectors ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Swine ; Swine Diseases ; immunology ; virology ; Zoonoses

To construct secretory expression vector of PPV NS1 gene, the fragment of PPV NS1 gene coding for major antigen region of the NS1 protein was amplified by PCR and inserted into multiple clone site of eukaryotic expression vector pPICZalpha-A. The recombinant pPICZalpha-A-NS1 plasmid was transferred into P. pastoris strain GS115 mediated by electro transform. Recombinant P. pastoris strain GS115 was induced to express the fusion protein by methanol. The expressed and purified protein was analyzed by SDS-PAGE and Western Blot. The recombinant protein was highly-expressed and showed a good immunoreactivity. The indirect ELISA method was developed for detecting antibodies against PPV by checkerboard titration assay. The result showed that the optimal concentration of coated antigen was 3.2 microg/mL and the best dilution of serum was 1 : 80. The positive cut-off value of the ELISA assay was OD450 > 0.4 and OD450 positive serum/OD450 negative serum > 2.0. Compared with HI and commercial ELISA kits, the assay revealed 94.2% and 92.1% agreement respectively. The assay demonstrates good specificity and sensitivity, and can be applied in the detection of porcine parvovirus.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; methods ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Parvoviridae Infections ; diagnosis ; immunology ; veterinary ; virology ; Parvovirus, Porcine ; genetics ; immunology ; isolation & purification ; Recombinant Proteins ; genetics ; immunology ; Swine ; Swine Diseases ; diagnosis ; immunology ; virology ; Viral Nonstructural Proteins ; genetics ; immunology

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

In order to develop a combined live vaccine that will be used to prevent against porcine parvovirus (PPV) and Pseudorabies virus (PRV) infection, the VP2 gene of PPV was inserted into the transfer vector plasmid pG to produce the recombinant plasmid pGVP2. The plasmid pGVP2 and the genome of PRV HB98 attenuated vaccine were transfected by using lipofectamine into swine testis cells for the homologous recombination. The recombinant virus rPRV-VP2 was purified by selection of green fluorescence plaques for five cycles. 6-week-old female Kunming mice were immunized intramuscularly with attenuated PRV parent HB98 strain, commercial inactivated vaccine against PPV, recombinant virus, DMEM culture solution. The injections were repeated with an equivalent dose after 2 weeks in all of the groups, and then challenged with the virulent PRV NY strain at 7 weeks after the first immunization. The recombinant virus rPRV-VP2 was successfully generated, and the recombinant virus could effectively elicite anti-PPV and PRV antibody and significant cellular immune response as indicated by anti-PPV ELISA and HI, PRV-neutralizing assay and flow cytometry. The challenge assay indicated that recombinant virus could protect the mice against the virulent PRV challenge. These results demonstrated that the recombinant virus can be a candidate recombinant vaccine strain for the prevention of PRV and PPV.
Animals ; Antibodies, Viral ; immunology ; Antigens, Viral ; administration & dosage ; genetics ; immunology ; Capsid Proteins ; administration & dosage ; genetics ; immunology ; Female ; Gene Expression ; Genetic Vectors ; genetics ; metabolism ; Herpesvirus 1, Suid ; genetics ; metabolism ; Mice ; Parvovirus, Porcine ; genetics ; immunology ; Swine ; Swine Diseases ; immunology ; prevention & control ; virology ; Viral Vaccines ; administration & dosage ; genetics ; immunology