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In response to viral infection, the host innate antiviral response is elicited to limit viral replication. Many viruses have evolved various strategies to circumvent the host antiviral response. It has been reported that matrix (M) protein of vesicular stomatitis virus (VSV) can inhibit host gene expression to evade the host innate immune response. However, the molecular mechanism remains unclear. Here, we demonstrated that VSV M protein inhibited transcription of a reporter gene transfected into BSR-T7/5 cells. To further investigate the underlying mechanism, a yeast two-hybrid screen was performed to search for host proteins that interact with the M protein. The subunit of transcription/repair factor TFIIH, p8, was identified as an M binding partner, and the interaction was validated with a GST pull-down assay and laser confocal microscopy. Through a mutagenesis analysis, we found that the p8-M interaction was impaired when I96, E156, R159 and R160 residues on M were replaced with Ala. These mutants reduced the inhibitory effect on transcription of the reporter gene. Furthermore, the transcription inhibition mediated by M was impaired when co-expressed with p8. These results indicate that the p8-M interaction plays an important role in inhibiting transcription of host genes.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurovirulent coronavirus that can cause nervous symptoms in piglets with muscle tremors, hind limb paralysis, and nystagmus. Whether some factors affect virus replication and proliferation had not been fully understood in the course of nerve damage caused by PHEV infection. In recent years, some reports suggested that miRNA might play a key regulatory role in viral infection. In this study, we found the miR-21a-5p is notably up-regulated in the brains of mice and N2a cells infected with PHEV, and it down-regulated the expression of CASK-interactive protein1 (Caskin1) by directly targeting the 3′-UTR of Caskin1 using a Dual-Luciferase reporter assay. The over-expression of miR-21a-5p or Caskin1 knockdown in the host significantly contributes to PHEV proliferation. Conversely, the silencing of miR-21a-5p by miR-21a-5p inhibitors suppressed the virus proliferation. Taken together, our results indicate that Caskin1 is the direct target gene of miR-21a-5p, and it is advantageous to virus proliferation by down-regulating Caskin1. These findings may help in the development of strategies for therapeutic applications.

Background Viruses interact with host cellular factors to construct a more favourable environment for their efficient replication. Expression of cyclophilin B (CypB), a cellular peptidyl-prolyl cis-trans isomerase (PPIase), was found to be significantly up-regulated. Recently, a number of studies have shown that CypB is important in the replication of several viruses, including Japanese encephalitis virus (JEV), hepatitis C virus (HCV) and human papillomavirus type 16 (HPV 16). However, the function of cellular CypB in ORFV replication has not yet been explored. Methods Suppression subtractive hybridization (SSH) technique was applied to identify genes differentially expressed in the ORFV-infected MDBK cells at an early phase of infection. Cellular CypB was confirmed to be significantly up-regulated by quantitative reverse transcription-PCR (qRT-PCR) analysis and Western blotting. The role of CypB in ORFV infection was further determined using Cyclosporin A (CsA) and RNA interference (RNAi). Effect of CypB gene silencing on ORFV replication by 50% tissue culture infectious dose (TCID50) assay and qRT-PCR detection. Results In the present study, CypB was found to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection. Cyclosporin A (CsA) exhibited suppressive effects on ORFV replication through the inhibition of CypB. Silencing of CypB gene inhibited the replication of ORFV in MDBK cells. In conclusion, these data suggest that CypB is critical for the efficient replication of the ORFV genome. Conclusions Cellular CypB was confirmed to be significantly up-regulated in the ORFV-infected MDBK cells at an early phase of infection, which could effectively facilitate the replication of ORFV.

In this study, we investigated an acute outbreak of porcine hemagglutinating encephalomyelitis on a farm of 127 pigs in Jilin province, China. Porcine hemagglutinating encephalomyelitis virus (PHEV) was detected in suckling and weaning pigs by RT-PCR assays. Coronavirus-like particles were observed by electron microscopy. The virus isolate was designated PHEV-JT06. The clinical signs, nervous symptoms and positive labeling of neurons in the cerebral cortex with an immunohistochemical stain in PHEV-JT06-infected BALB/c mice supported the diagnosis of PHEV infection. The five full-length PHEV-JT06 structural genes were cloned, sequenced and analyzed. Phylogenetic studies based on the nucleotide and amino acid sequences of the five genes in the outbreak showed that PHEV remained genetically stable. PHEV shares 95.3-99.3 % amino acid sequence identity with American strains (AY078417), suggesting that the Chinese isolate is most likely derived from the North American strain. Additionally, PHEV, HCoV-OC43 and BCoV were genetically close. These results may provide some insights into the genotype of the etiological agent responsible for the porcine hemagglutinating encephalomyelitis outbreak and could also provide a comparative view of the genomics of the five structural proteins of PHEV.

Orf, which is caused by orf virus (ORFV), is distributed worldwide and is endemic in most sheep- and/or goat-raising countries. RNA interference (RNAi) pathways have emerged as important regulators of virus-host cell interactions. In this study, the specific effect of RNAi on the replication of ORFV was explored. The application of RNA interference (RNAi) inhibited the replication of ORFV in cell culture by targeting the ORF025 gene of ORFV, which encodes the viral polymerase. Three small interfering RNA (siRNA) (named siRNA704, siRNA1017 and siRNA1388) were prepared by in vitro transcription. The siRNAs were evaluated for antiviral activity against the ORFV Jilin isolate by the observation of cytopathic effects (CPE), virus titration, and real-time PCR. After 48 h of infection, siRNA704, siRNA1017 and siRNA1388 reduced virus titers by 59- to 199-fold and reduced the level of viral replication by 73-89 %. These results suggest that these three siRNAs can efficiently inhibit ORFV genome replication and infectious virus production. RNAi targeting of the DNA polymerase gene is therefore potentially useful for studying the replication of ORFV and may have potential therapeutic applications.

To effectively protect against oxidative damage, an important trace element, selenium, which was initially described as a toxin and subsequently shown to be an essential antioxidant, came into researchers’ view. Meanwhile, probiotics, as nonpathogenic microorganisms that can resist host small intestinal digestion and reach the colon alive, were reported to exert a myriad of beneficial effects, including balance of intestinal microbiota, improvement of growth, enhancement of immune response and inhibition of some pathogenic bacteria. Thus, selenium-enriched probiotics, which have the advantages of both two of them, have been developed to enhance the body’s antioxidant capability and immune function and protect against various oxidative stresses. However, the potential mechanisms of selenium-enriched probiotics’ functions remain unclear. Therefore, we produced a novel kind of selenium-enriched probiotic called S-BPSR via Bacillus Paralicheniformis SR14. We investigated the antioxidant effects of S-BPSR against oxidative stress and its underlying molecular mechanisms in IPEC-J2 cells. Concretely, we found that S-BPSR had the great abilities on scavenging DPPH, superoxide anion, hydroxyl radical, and could increase cell viability and prevented lactate dehydrogenase release into the medium when prior to H2O2 exposure. H2O2-induced reactive oxygen species and malondialdehyde were remarkably suppressed by S-BPSR, which also effectively attenuated cell apoptosis. Treatment with S-BPSR also separately increased the levels of antioxidant enzymes and promoted the expression of phosphorylation of ERK and p38 MAPK signaling pathways in IPEC-J2 cells. These results suggested that this new form of selenium-enriched probiotic possessed great antioxidant property and cytoprotection and may indeed have broad applications in oxidative stress-related diseases.

A rapid and simple immunochromatography strip (ICS) test for the specific detection of vesicular stomatitis virus serotype Indiana (VSV-IND) using two distinct monoclonal antibodies MAbs (1A2 and 4C3) against the G protein of VSV-IND was developed. The MAb 1A2 was conjugated with colloidal gold, and the MAb 4C3 and goat anti-mouse IgG were sprayed onto a nitrocellulose membrane in strips at positions designated T and C, respectively. The results showed that samples of VSV-IND combined with CG-MAb 1A2, and that these complexes were captured by MAb 4C3 at the test line (T), resulting in the appearance of a purple band. When samples did not contain VSV-IND or when they contained a quantity of VSV-IND below the detection limit of the test, only the control line (C) was visible. The analysis of the sensitivity of the test demonstrated that the lowest detected quantity of VSV-IND was 1.85×10(3.0)TCID50/ml. Storage of the ICS test at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity and specificity. In clinical trials using RT-PCR as a reference test, the relative specificity and sensitivity of the ICS were determined to be 98.9% and 91.4%, respectively. Based on these results, the ICS test developed may be a suitable tool for rapid on-site testing for VSV-IND infection.

Butyrate has been used to treat different inflammatory disease with positive outcomes, the mechanisms by which butyrate exerts its anti-inflammatory effects remain largely undefined. Here we proposed a new mechanism that butyrate manipulate endogenous host defense peptides (HDPs) which contributes to the elimination of Escherichia coli O157:H7, and thus affects the alleviation of inflammation. An experiment in piglets treated with butyrate (0.2% of diets) 2 days before E. coli O157:H7 challenge was designed to investigate porcine HDP expression, inflammation and E. coli O157:H7 load in feces. The mechanisms underlying butyrate-induced HDP gene expression and the antibacterial activity and bacterial clearance of macrophage 3D4/2 cells in vitro were examined. Butyrate treatment (i) alleviated the clinical symptoms of E. coli O157:H7-induced hemolytic uremic syndrome (HUS) and the severity of intestinal inflammation; (ii) reduced the E. coli O157:H7 load in feces; (iii) significantly upregulated multiple, but not all, HDPs in vitro and in vivo via histone deacetylase (HDAC) inhibition; and (iv) enhanced the antibacterial activity and bacterial clearance of 3D4/2 cells. Our findings indicate that butyrate enhances disease resistance, promotes the clearance of E. coli O157:H7, and alleviates the clinical symptoms of HUS and inflammation, partially, by affecting HDP expression via HDAC inhibition.

Porcine hemagglutinating encephalomyelitis (PHE) is caused by the coronavirus hemagglutinating encephalomyelitis virus (PHE-CoV), and the recent, rapid spread of PHE-CoV in piglets from many countries emphasizes the urgent need for a PHE-CoV vaccine. Here we use a murine model for evaluation of the induction of humoral and cellular immune responses by inactivated and PHE-CoV DNA vaccines in order to define the immune correlates for protection against PHE-CoV. The inactivated vaccine was composed of purified PHE-CoV and aluminum hydroxide gel (alum), which was chosen as an adjuvant because of its long history of safety for human use. The PHE-CoV DNA vaccine was constructed by subcloning the S1 gene of PHE-CoV into the pVAX1 vector to create the recombinant plasmid pV-S1. Our results showed that the inactivated PHE-CoV vaccine (IPV) elicited a high level of humoral immunity, resulting in good protection efficacy against PHE-CoV challenge. The IPV induced the IgG1 subclass of serum antibodies and expression of the cytokine interleukin-4 (IL-4), suggesting that the IPV generated a predominantly Th2-type immune response. The DNA vaccine was found to mediate primarily a cellular immune response with high levels of IgG2a and the cytokines IL-2 and gamma interferon (IFN-γ). However, mice that were vaccinated twice with the DNA vaccine and boosted with the IPV could mount a sufficient neutralizing antibody response against live PHE-CoV, with little variation in IgG1 and IgG2a levels, and showed high levels of IL-2 and IL-4. This response may activate both B and T cells to mount a specific humoral and cellular immune response that could, in turn, elicit a phagocyte-mediated defense against PHE-CoV infections to achieve viral clearance.

Porcine hemagglutinating encephalomyelitis virus (PHEV), which causes porcine encephalomyelitis and is widespread among swine worldwide. RNA interference (RNAi) pathways have emerged as important regulators of virus-host cell interactions. In this study, two siRNA expression plasmids (shN1 and shN2) were generated to target two different coding regions of the nucleocapsid protein (N) of PHEV. The shRNAs were transiently transfected into a porcine kidney cell line, PK-15, to determine whether these constructs inhibited PHEV production. Our results revealed that both shRNAs were highly capable of inhibiting viral RNA genome replication, especially shN2. Next, stable transfection of shN2 was used to produce two siRNA stably expressing PK-15 cell clones (shN2-1 and shN2-2), and these two lines were infected with PHEV. The analysis of cytopathic effects (CPE) demonstrated that shN2-1 and shN2-2 were capable of protecting cells against PHEV infection with high specificity and efficiency. Furthermore, effective inhibition of viral replication persisted for up to 120 h by a TCID(50) assay. These results indicated that RNAi targeting of the N gene could facilitate studies of the specific function of viral genes associated with PHEV replication and may have potential therapeutic applications.

We investigated an acute outbreak of the cutaneous form of fowlpox among chickens in China in November 2009. Using pathologic and virologic methods, we identified a novel type of fowlpox virus that carried an integrated genomic sequence of reticuloendotheliosis virus. This highly pathogenic virus could lead to severe ecologic effects and economic losses.

Background A 5-year-old female Sichuan snub-nosed monkey died at the zoological garden from infection with coxsackievirus B3. Methods The diagnosis was made on the basis of pathologic features, immunohistochemistry, microbiological detection, and reverse transcription-polymerase chain reaction (RT-PCR). Results Histologic examination of formalin-fixed, paraffin-embedded tissues revealed a severe degree of predominantly lymphocytic infiltration of the cardiac muscle. Picornaviridae-like virions were found in the supernatants of cardiac muscle tissues homogenates, in the pericardial fluid, and in Vero cell cultures, by electron microscopy. Coxsackievirus B3 particles were detected in cardiac muscle cells by immunofluorescence. RT-PCR performed on an extract of cardiac muscle tissue revealed a DNA sequence specific for coxsackievirus B3. Conclusions This is the first report of a Sichuan snub-nosed monkey dying from a virus.

A rapid and simple lateral-flow immunochromatographic assay (LFIA) was developed for the specific detection of Orf virus (ORFV) using two distinct monoclonal antibodies (MAbs: 5A5 and 6F2) against the ORFV ORF011 protein. The MAb 5A5 was conjugated with colloidal gold, and the MAb 6F2 and goat anti-mouse IgG were sprayed onto a nitrocellulose membrane in strips at positions designated test (T) and control (C), respectively. The results showed that samples of ORFV complexed with colloidal gold-conjugated MAb 5A5, were captured by MAb 6F2 at the T line resulting in the appearance of a purple band. When samples did not contain ORFV or when they contained a quantity of ORFV below the detection limit of the test, only the C line was visible. The analysis of sensitivity of the test demonstrated that the lowest detected quantity of ORFV was 2.03×10(3.0) TCID50/ml. Storage at room temperature for 6 months did not result in the loss of performance of the LFIA test. Using loop-mediated isothermal amplification (LAMP) as a reference test, the relative specificity and sensitivity of the LFIA test were determined to be 100% and 92.1%, respectively. Based on these results, the LFIA test developed may be a suitable tool for rapid on-site testing for ORFV infection.

Antimicrobial peptides are molecules of innate immunity. Cathelicidin-BF is the first cathelicidin peptide found in reptiles. However, the immunoregulatory and epithelial barrier protective properties of C-BF have not been reported. Inflammatory bowel diseases, including ulcerative colitis and Crohn's disease, can lead to colon cancer, the third most common malignant tumor. The objective is to develop the new found cathelicidin-BF as a therapeutic to patients of ulcerative colitis. The morphology of the colon epithelium was observed by HE staining; apoptosis index and infiltration of inflammatory cells in colonic epithelium were measured by TUNEL and immunohistochemistry; the expression level of endogenous mCRAMP was analyzed by immunofluorescence; and phosphorylation of the transcription factors c-jun and NF-κB in colon were analyzed by Western blot. Our results showed that the morphology of the colon epithelium in the C-BF+DSS group was improved compared with the DSS group. Apoptosis and infiltration of inflammatory cells in colonic epithelium were also significantly attenuated in the C-BF+DSS group compared with the DSS group, and the expression level of endogenous mCRAMP in the DSS group was significantly higher than other groups. DSS-induced phosphorylation level of c-jun and NF-κB while C-BF effectively inhibited phosphorylation of NF-κB (p65). The barrier protective effect of C-BF was still excellent. In conclusion, C-BF effectively attenuated inflammation and improved disrupted barrier function. Notably, this is the first report to demonstrate that C-BF attenuates DSS-induced UC both through the regulation of intestinal immune and retention of barrier function, and the exact pathway was through NF-κB.

The severity of intestinal inflammation in mammals can be profoundly impacted by weaning stress. Cathelicidins protect intestinal homeostasis by not only directly killing bacteria but also immune regulators. Here, we investigated the effects of cathelicidin-BF (C-BF) derived from the snake venoms of Bungarus fasciatus on weaning stress and intestinal inflammation and examined the mechanisms by which C-BF modulates intestinal immune responses in weanling piglets. We found that C-BF treatment significantly increased performance and reduced the diarrheal index in weanling piglets. Serum IL-6, IL-22 and TNF-α production was decreased by C-BF treatment. We demonstrated that C-BF inhibited the expression of the inflammatory cytokines TNF-α, IL-6 and IL-8 but increased the expression of the anti-inflammatory cytokine IL-10 in the intestine. We also demonstrated that C-BF suppressed inflammation by down-regulating the nuclear factor-κB (NF-κB) signaling pathway in the intestine and in LPS-induced macrophages in vitro. However, C-BF significantly induced the phosphorylation of signal transducer and activator of transcription 1 (STAT-1) to enhance the phagocytosis of macrophages when inflammation was suppressed. In summary, our study demonstrated that C-BF suppressed intestinal inflammation by down-regulating the NF-κB signaling pathway and enhancing the phagocytosis of immune cells by activating STAT-1 during weaning.

Intestinal permeability plays a critical role in the etiopathogenesis of ulcerative colitis. Defensins, including porcine β-defensin (pBD)2, are crucial antimicrobial peptides for gut protection owing to their antibacterial and immunomodulatory activities. The purpose of this study was to investigate the protective effects of pBD2 on mucosal injury and the disruption of the epithelial barrier during the pathological process of dextran sodium sulfate (DSS)–induced colitis. The effects and mechanism of pBD2 were evaluated both using a DSS-induced C57BL/6 mouse model and, in vitro, using Caco-2 and RAW264.7 cells. DSS-induced colitis was characterized by higher disease activity index, shortened colon length, elevated activities of myeloperoxidase and eosinophil peroxidase, histologic evidence of inflammation, and increased expression levels of TNF-α, IL-6, and IL-8. pBD2 increased the expression of zonula occludens-1, zonula occludens-2, claudin-1, mucin-1, and mucin-2 mRNA and proteins, and it decreased permeability to FITC-D, as well as apoptosis, in DSS-treated mice. pBD2 also decreased inflammatory infiltrates of the colon epithelium. In Caco-2 cells, pBD2 increased transepithelial electrical resistance and mucin mRNA expression, and it decreased the permeability of FITC-D while preserving the structural integrity of the tight junctions. The effects of pBD2 appeared to be through upregulation of the expression of genes associated with tight junctions and mucins, and by suppressing DSS-induced increases in inflammation, inducible NO synthase, cyclooxygenase-2, and apoptosis. These results show that pBD2 improves DSS-induced changes in mucosal lesions and paracellular permeability, possibly by affecting the activation of NF-κB signaling. The present study demonstrates that intrarectal administration of pBD2 may be a novel preventive option for ulcerative colitis.

Casein glycomacropeptide (CGMP) is a bioactive peptide derived from milk with multiple functions. This study was aimed at evaluating the effects of CGMP as a potential feed additive on growth performance, intestinal morphology, intestinal barrier permeability and inflammatory responses of Escherichia coli K88 (E. coli K88) challenged piglets. Eighteen weaning piglets were randomly assigned to three groups. Control group and K88 challenged group received a basal diet, and CGMP treated group received the basal diet supplemented with 1% of CGMP powder. The trail lasted for 12 days, K88 was orally administered to the piglets of K88 challenged group and CGMP treated group on days 8–10. The results showed that the diet containing 1% CGMP significantly alleviated the decrease in average daily gain (P 0.05) and barrier permeability damage (P

In this study, we analyzed the global gene expression profiles in the subcutaneous fat (SAT) of Jinhua pigs and Landrace pigs at 90d. Several genes were significantly highly expressed in Jinhua pigs, including genes encoding the rate limiting enzymes in the TCA cycle, fatty acid activation, fatty acid synthesis and triglyceride synthesis. We identified a novel gene tagged by the EST sequences as public No. BF702245.1, which was named porcine FAM134B (pFAM134B) and the pFAM134B mRNA levels of SAT was significantly higher in Jinhua pigs than that in Landrace pigs at 90d (P

Objective To determine the relationship between viral kinetics and the expression patterns for different cytokines and chemokines in the serum and organs of coxsackievirus B3 (SSM-CVB3)-infected macaques over the course of infection. Methods SSM-CVB3 levels in serum and organs were measured using the Spearman–Karber 50% tissue culture infectious dose (TCID50) method. Cytokine and chemokine levels in the serum and organs were measured by indirect-ELISA. Results Low viral titers were detected in the serum samples on the first day post-inoculation (p.i.) and peaked at 6 to 10 days p.i. in the serum samples from five macaques. Serum levels of IL-1β, IL-2, IL-6, IL-12p40, IL-17α, IFN-γ, TNF-α, MCP-1 and MIP-1β were detected each day and, similar to the viral titers, peaked at 6 to 10 days. IL-10 was only detected on days 10 to 14 p.i. Additionally, higher viral titers and relative viral mRNA levels were associated with higher cytokine and chemokine levels in selected tissues from infected macaques including heart, liver, spleen, lung, kidney and brain. Conclusion The results indicate that patterns of cytokine and chemokine response are associated with viral kinetics in the serum and target organs of SSM-CVB3-infected macaques, suggesting that the changes in cytokines and chemokines could help further our understanding of the progress of CVB3 infections in clinical settings.

Objective To evaluate the pathogenicity of SSM-CVB3 in a macaque model. Methods The clinical symptoms of macaques were recorded; hematological, biochemical and histopathological evaluations were completed; viral titers and neutralization titers (NT-titers) in sera were tested; and the mRNA levels of SSM-CVB3, coxsackievirus and adenovirus receptor (CAR) and decay accelerating factor (DAF) were determined. Results After SSM-CVB3 infection, the macaques showed a lack of activity, a poor appetite, a higher body temperature, and severe diarrhea. The macaques also developed hematuria and albuminuria at 4 to 10 days post-inoculation. Virus titers (5.1–6.5 LogTCID 50 /mL) were higher at 6 to 10 days post-inoculation, and NT-titers (6.5–7.3 Log2) reached plateaus at 8 to 14 days post-inoculation. The infected macaques developed serious anemia with decreased RBC and WBC, but the percentages of LYM were increased. The levels of CK, CK-MB, AST and ALT in the sera were 84–169 U/L, 87.6–271.1 U/L, 43–87 U/L and 43–82 U/L, respectively, and all of those were higher than normal. Histological analysis showed obvious cardiac, hepatic and renal damages in the infected macaques and the mRNA contents of SSM-CVB3, CAR and DAF in the heart, liver and kidneys of infected macaques were higher ( P ). Conclusion This was the first report on experimental SSM-CVB3 infections in macaques with serious hepatic and renal damage, except for myocarditis. The information obtained from this study suggests that the SSM-CVB3 strain and this macaque model could be used for studying CVB3-induced cardiac, hepatic or renal diseases.

One coronavirus strain was isolated from brain tissues of ten piglets with evident clinical manifestations of vomiting, diarrhea and dyskinesia in Jilin province in China. Antigenic and genomic characterizations of the virus (isolate PHEV-JLsp09) were based on multiplex PCR and negative staining electron microscopy and sequence analysis of the Hemagglutinin-esterase (HE) gene. These piglets were diagnosed with Porcine hemagglutinating encephalomyelitis virus (PHEV). Necropsy was performed on the piglets. Major pathological changes included meningeal hyperemia, meningeal hemorrhage and cortical hemorrhage. Minor changes were also observed in other organs. Histopathological changes included satellitosis and neuronophagia in the cerebral cortex. Mice were infected with the isolated virus. Their histopathological changes were similar to those symptoms observed in the piglets, exhibiting typical changes for non-suppurative encephalitis. Thus, Porcine hemagglutinating encephalomyelitis virus mainly causes damage to the nervous system but also impacts other organs. This viral strain (isolate PHEV-JLsp09) found in the Siping area of Jilin Province in China is evolutionally closest to the HEV-67N stain (North American strain), indicating that this viral strain evolved from the PHEV from North America.

An immunochromatographic strip was developed for the detection of an antibody against Porcine hemagglutinating encephalomyelitis virus (PHEV). Colloidal gold–labeled rabbit anti-pig immunoglobulin G (IgG) was used as the detection reagent, and the PHEV recombinant antigens and goat anti-rabbit IgG were coated on the prototype strip and the control lines, respectively. The immunochromatographic strip was capable of specifically detecting PHEV antibodies in serum with a hemagglutination inhibition (HI) titer of 2 within 10 min. Storage of the strips at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity and specificity. Using HI as a reference test, the relative specificity and sensitivity of the immunochromatographic strip were determined to be 93.41% and 98.42%, respectively. There was a strong agreement between the results obtained by HI and the immunochromatographic strips (κ = 0.926). Additionally, there was a strong agreement between enzyme-linked immunosorbent assay and immunochromatographic strips (κ = 0.929). When the immunochromatographic strip was used for serological diagnosis of 1,117 serum samples in Jilin Province in China, the seropositivity ranged from 6.5% in the Liaoyuan District to 81.6% in the Changchun District. Furthermore, many piglets were seropositive to PHEV, indicating the possible transfer of maternal antibodies via the colostrum. Based on the high specificity, sensitivity, and stability of the immunochromatographic strip, it would be suitable for on-site detection of PHEV antibodies in order to monitor PHEV infections in an animal population.

Background The spike proteins of coronaviruses associate with cellular molecules to mediate infection of their target cells. The characterization of cellular proteins required for virus infection is essential for understanding viral life cycles and may provide cellular targets for antiviral therapies. Results We identified Neural Cell Adhesion Molecule (NCAM) as a novel interacting partner of the PHE-CoV S protein. A T7 phage display cDNA library from N2a cells was constructed, and the library was screened with the soluble PHE-CoV S glycoproteins. We used a coimmunoprecipitation assay to show that only the NCAM was a binding partner of spike protein. We found that a soluble form of anti-NCAM antibody blocked association of the PHE-CoV with N2a cells. Furthermore, double-stranded siRNA targeted against NCAM inhibited PHE-CoV infection. Conclusions A novel interaction was identified between NCAM and spike protein and this association is critical during PHE-CoV infection.

This study investigated an acute outbreak of contagious ecthyma dermatitis that occurred in November 2008 in a herd of 180 small-tailed Han sheep in the Jilin province of China. The pathological findings of this case revealed severe vascular proliferation, viral cytopathic changes in keratinocytes by vacuolar degeneration, ballooning degeneration, and eosinophilic cytoplasmic inclusions, suggesting that the disease could have been caused by an Orf virus (ORFV) infection. Immunohistochemistry, indirect immunofluorescence (IFA), and transmission electron microscopy supported the diagnosis of ORFV infection. Finally, the pathogen of the disease was identified using polymerase chain reaction (PCR) and sequences of major envelope protein genes (ORFV 011(B2L) and ORFV 059(F1L)). The full-length ORFV 011(B2L) and ORFV 059(F1L) genes were cloned and sequenced. The nucleotide and amino acid sequences of ORFV 011(B2L) and ORFV 059(F1L) genes in these outbreaks were analyzed, and their phylogenetic trees were constructed. The phylogenetic studies of ORFV 011(B2L) genes and ORFV 059(F1L) genes showed that the ORFV-Jilin province isolate clustered in different branches and was closer to the ORFV-Mukteswar 67/04 isolate and the ORFV-OV/C2 isolate, respectively. To date, there is no report on the molecular characterization of any Orf virus with other isolates around the world in mainland China. Further, the above results may provide some insight into the genotype of the etiological agent responsible for the contagious ecthyma dermatitis outbreak in the Jilin province, and could also provide a comparative view of the central coding region genomics of parapoxvirus (PPV).

Coxsackievirus B3 (CVB3) is an enterovirus in the family Picornaviridae that is significant to human health, being associated with myocarditis, aseptic meningitis, and pancreatitis, among other conditions. In addition to humans, Sichuan snub-nosed monkeys can be infected and killed by CVB3. Here, we report the first complete genome sequence of a novel coxsackievirus B3 strain, SSM-CVB3, which was isolated from a deceased Sichuan snub-nosed monkey with severe myocarditis. Our findings may aid in understanding the evolutionary characteristics and molecular pathogenesis of this virus.

Background The incidence of PHE among pigs in many countries is on the rise, and it has caused great economic losses to the pig industry. Therefore, the development of a sensitive, specific, and easily-performed assay is crucial for the rapid detection and surveillance of PHE-CoV infection and transmission. Results An immunochromatographic strip was developed for the detection of PHE-CoV. The colloidal gold-labeled MAb 4D4 was used as the detection reagent, and the MAb 1E2 and goat anti-mouse IgG coated the strip's test and control lines, respectively. The immunochromatographic strip was capable of specifically detecting PHE-CoV with a HA unit of 2 within 10 min. Storage of the strips at room temperature for 6 months or at 4°C for 12 months did not change their sensitivity or specificity. Using RT-PCR as a reference test, the relative specificity and sensitivity of the immunochromatographic strip were determined to be 100% and 97.78%, respectively. There was an excellent agreement between the results obtained by RT-PCR and the immunochromatographic strips (kappa = 0.976). Additionally, there was a strong agreement between the sandwich enzyme-linked immunosorbent assay (ELISA) and immunochromatographic strips (Kappa = 0.976). When the immunochromatographic strips were used for diagnosing PHE-CoV infection in the Jilin Province, the PHE-CoV-positive rate ranged from 61.54% in the Jilin district to 17.95% in the Songyuan district. Conclusions Based on its high specificity, sensitivity, and stability, the immunochromatographic strip would be suitable for on-site detection of PHE-CoV for surveillance and epidemiological purposes.

Background ORFV attenuated live vaccines have been the main prophylactic measure against contagious ecthyma in sheep and goats in the last decades, which play an important role in preventing the outbreak of the disease. However, the available vaccines do not induce lasting immunity in sheep and goats. On the other hand, variation in the terminal genome of Orf virus vaccine strains during cell culture adaptation may affect the efficacy of a vaccine. Currently, there are no more effective antiviral treatments available for contagious ecthyma. Results We constructed three eukaryotic expression vectors pcDNA3.1-ORFV011, pcDNA3.1-ORFV059 and pcDNA3.1-ORFV011/ORFV059 and tested their immunogenicity in mouse model. High level expression of the recombinant proteins ORFV011, ORFV059 and ORFV011/ORFV059 was confirmed by western blotting analysis and indirect fluorescence antibody (IFA) tests. The ORFV-specific antibody titers and serum IgG1/IgG2a titers, the proliferation of lymphocytes and ORFV-specific cytokines (IL-2, IL-4, IL-6, IFN-γ, and TNF-α) were examined to evaluate the immune responses of the vaccinated mice. We found that mice inoculated with pcDNA3.1-ORFV 011/ORFV059 had significantly stronger immunological responses than those inoculated with pcDNA3.1-ORFV011, pcDNA3.1-ORFV059, or pcDNA3.1-ORFV011 plus pcDNA3.1-ORFV059. Compared to other vaccine plasmids immunized groups, pcDNA3.1-ORFV011/ORFV059 immunized group enhances immunogenicity. Conclusions We concluded that DNA vaccine pcDNA3.1-ORFV011/ORFV059 expressing ORFV011 and ORFV059 chemeric-proteins can significantly improve the potency of DNA vaccination and could be served as more effective and safe approach for new vaccines against ORFV.

Background Orf is a zoonotic and epitheliotrophic contagious disease that mainly affects sheep, goats, wild ruminants, and humans with a worldwide distribution. To date, there is little information on the characterization of ORFV strains that are endemic in Mainland China. In addition, the relationship between the severity of disease and the molecular profile of ORFV strains has not been fully elucidated. Results From the recent outbreak of a sheep herd in Nongan, northeast of China, the novel orf virus (ORFV) strain NA1/11 was successfully isolated. Western blot analysis indicated that the NA1/11 strain cross reacts with monoclonal antibody A3 and infected sheep ORFV antiserum. The purified virions revealed the typical ovoid shape when observed by atomic force microscopy. To determine the genetic characteristics of the NA1/11 strain, the sequences of ORFV011 (B2L), ORFV059 (F1L), ORFV109, ORFV110 and ORFv132 (VEGF) genes were amplified and compared with reference parapoxvirus strains. Non-metric multidimensional scaling (nMDS) was performed to analyze the nucleotide similarities between different ORFV strains. Conclusions Phylogenetic analysis based on ORFV 011 nucleotide sequences showed that the NA1/11strain was closely related to Xinjiang and Gansu strains. ORFV110 and ORFV132 genes are highly variable. The results revealed that precise phylogenetic analysis might provide evidence for genetic variation and movement of circulating ORFV strains in Northeast China. In addition, nMDS analysis showed that geographic isolation and animal host are likely major factors resulting in genetic differences between ORFV strains.