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Porcine circovirus type 2 (PCV2) is the etiological agent of post-weaning multisystemic wasting syndrome (PMWS). The original prevalent genotype, PCV2a, has been replaced by genotypes 2b and 2d in the swine population worldwide. The Rep protein is critical for viral replication. Comparison of a large number of Rep protein amino acid (aa) sequences showed that three sites distinguish genotype 2b from genotype 2d. In order to analyze the effect of exchanging the amino acids (asparagine and serine) at position 6 in the Rep proteins of PCV2b and PCV2d, two wild-type and two mutant viruses were rescued. Real-time quantitative PCR and a one-step growth curve were used to determine the viral load to assess the replication ability of the rescued viruses. The results showed that there was no significant difference in in vitro performance between the wild-type PCV2b and the mutated virus, while the mutation of PCV2d enhanced viral replication.

Porcine circovirus type 2 (PCV2), a ubiquitous pathogen that primary cause of postweaning multisystemic wasting syndrome (PMWS), had caused significant morbidity and mortality in swine populations with huge economic losses in the worldwide swine industry. Currently, looking for effective antiviral drugs for PCV2 infection remains an important works. In our study, CRISPR/Cas9 system was used to further detected the key sites of PCV2 replication. We designed 8 single guide RNAs (sgRNA) by targeting essential genes across the genome of PCV2. Western-blot(WB), Cell counting kit-8 for high-throughput sgRNA screening were applied to detect PCV2 replication levels. The results showed that Oc8, O13, O134, NQT and NPS sgRNAs can edit the PCV2 genome efficiently and inhibit PCV2 replication in PK-15 cell; H3 sgRNA cannot edit the PCV2 genome successfully; NAT sgRNA can edit the PCV2 genome efficiently to improve the PCV2 replication in PK-15 cell; O26 sgRNA can edit the PCV2 genome successfully but it is not known yet of its effect on PCV2 replication, besides the Cas9 expression had no effect on cell viability. These data suggest that CRISPR/Cas9 system targeting PCV2 essential genes may serve as a novel therapeutic agent against PCV2 infection in the future.

PCV2 belongs to the genus Circovirus, family Circoviridae, who is recognized as the causative agents of postweaning multisystemic wasting syndrome. Since being found to China in 2000, it has caused serious damage to the pig industry. In this study, we downloaded 40 PCV2 genome‐wide sequences uploaded to GenBank from 2013 to 2018 in Shandong Province, including 23 uploaded by our laboratory. Construction of a genome‐wide evolution tree using MEGA V5.0 software. Phylogenetic tree analysis indicated that the genotype of PCV2 in Shandong Province was: three genotypes coexisted (2a, 2b, 2d); among them, PCV2d has become the main genotype in the province due to its number and spread range. Amino acid sequence analysis of different genotypes of ORF2 showed that specific amino acid sites exist in different genotypes, with the most significant range of 81–160; different genotypes of PCV2 can be distinguished at the molecular level. This study found that due to the increase in infections of the PCV2d genotype in recent years, it may replace PCV2b as the dominant base in Shandong., In order to understand the genetic evolution of PCV2 in Shandong in recent years, we analyzed all PCV2 complete sequences collected from different farms in six regions in Shandong Province from 2013 to 2018. In this study, we also performed homologous comparison and amino acid site analysis on the ORF2‐encoded Cap protein sequences of 40 isolates. Some specific mutation sites may also serve as reference conditions for the genetic variation of the strain.

Phospholipase C (PLC) is a key enzyme in the cell membrane. PLC hydrolyses phosphatidylinositol 4, 5-bisphosphate (PIP2) to generateinositol 1,4, 5-triphosphate (IP3) and diacylglycerol (DAG) that regulates a variety of cellular processes. Evidence indicates the pivotal role of PLC and inositol 1,4,5-trisphosphate receptor(IP3R) in influencing Ca2+ release from the endoplasmic reticulum(ER).At the same time, the imbalance of Ca2+ will stimulate endoplasmic reticulum stress(ERS), leading to cell apoptosis. Viral infection could triggers host defense through apoptosis of the infected cells.However, it is not clear how porcine circovirus type 2 (PCV2) induces apoptosis by affecting Ca2+ homeostasis. We show here that PCV2 infection induces the increased cytoplasmic Ca2+ level and apoptosis.We also found that the ER swelling of PK-15 cells after viral infection by transmission electron microscopy. Furthemore, the activation of PLC-IP3R-Ca2+ signaling enhanced apoptosis in infected PK-15 cells. Taken together,our findings suggest that PCV2 infection trigger ERS of PK-15 cells via the PLC-IP3R-Ca2+ signaling pathway to promoted the release of intracellular Ca2+, and led to cell apoptosis.

Nowadays, adjuvant is still important for boosting immunity and improving resistance in animals. In order to boost the immunity of porcine circovirus type 2 (PCV2) DNA vaccine, CpG motifs were inserted. In this study, the dose-effect was studied, and the immunity of PCV2 DNA vaccines by recombinant open reading frame 2 (ORF2) gene and CpG motifs was evaluated. Three-week-old Changbai piglets were inoculated intramuscularly with 200 μg, 400 μg, and 800 μg DNA vaccines containing 14 and 18 CpG motifs, respectively. Average gain and rectum temperature were recorded everyday during the experiments. Blood was collected from the piglets after vaccination to detect the changes of specific antibodies, interleukin-2, and immune cells every week. Tissues were collected for histopathology and polymerase chain reaction. The results indicated that compared to those of the control piglets, all concentrations of two DNA vaccines could induce PCV2-specific antibodies. A cellular immunity test showed that PCV2-specific lymphocytes proliferated the number of TH, TC, and CD3+ positive T-cells raised in the blood of DNA vaccine immune groups. There was no distinct pathological damage and viremia occurring in pigs that were inoculated with DNA vaccines, but there was some minor pathological damage in the control group. The results demonstrated that CpG motifs as an adjuvant could boost the humoral and cellular immunity of pigs to PCV2, especially in terms of cellular immunity. Comparing two DNA vaccines that were constructed, the one containing 18 CpG motifs was more effective. This is the first report that CpG motifs as an adjuvant insert to the PCV2 DNA vaccine could boost immunity.

Porcine circovirus type 2 (PCV2), the essential causative agent of postweaning multisystemic wasting syndrome (PMWS), can be divided into distinct genotypes. Thirty-two PCV2 isolates obtained made from pigs in Shandong Province between 2005 and 2013. Complete genome sequences were obtained in three replicates for each virus isolate, and the sequences were submitted to the NCBI database. The ORF1-encoded amino acid sequences had 98.4 %-100 % identity among the 32 isolates, and there were no significant differences among the three potential glycosylation positions: aa 23-25 (NPS), aa 256-258 (NQT) and aa 286-288 (NAT). The amino acid sequences of ORF2 had 88 %-100 % identity among the 32 isolates and the potential glycosylation position in the cap protein, aa 143-145 (NYS), had no variation. Phylogenetic analysis revealed that the PCV2b/1C genetic lineage was prevalent in swine populations in Shandong Province. It also suggested that selection pressure has made the PCV2 isolates more genetically distant from current vaccine strains.

Porcine circovirus (PCV) has two potential open reading frames, ORF1 and ORF2. ORF1 is predicted to encode a replication-associated protein (Rep) essential for replication of viral DNA. In some studies, PCV1 replicated more efficiently in PK-15 cells than PCV2 was elucidated. PCV1 compared with PCV2; there is some amino acids' deficiency on Rep protein. To identify whether the above amino acids deletion affects the replication of PCV1 and PCV2, we constructed three double copy clones by overlap extension PCR. The 2PCV2(vV) clone deleted the valine of Rep protein in the backbone of PCV2 genome. The 2PCV2(dSGR) clone inserted serine, glycine and arginine of Rep protein successively in the backbone of PCV2 genome. The 2PCV2(dSGR&vV) clone inserted serine, glycine and arginine as well as deleted the valine of Rep protein in the backbone of PCV2 genome. These clones we constructed with amino acid mutations and parental DNA clones were all transfected in PK-15 cells that free of PCV contamination, and their growth characteristics in vitro were determined and compared, to evaluating the replication of the mutant infectious DNA clones. Our results showed that the double copy infectious clones with amino acid mutations could be rescued in vitro. The 2PCV2(vV) replicated more efficiently than parental viruses 2PCV2 and 2PCV1 but the replicated ability of 2PCV2(dSGR) and 2PCV2(dSGR&vV) is attenuated than parental viruses 2PCV2 and 2PCV1. We can determine the valine is the important amino acid that cause PCV1 replicated more efficiently in PK-15 cells than PCV2 primarily. These findings are benefit for exploring the mechanisms of viral replication in pigs and important implications for PCV2 vaccine development.

We conducted a serologic investigation of porcine reproductive and respiratory syndrome virus (PRRSV) in hybrid wild boar herds in China during 2008–2009. PRRSV isolates with novel genetic markers were recovered. Experimental infection of pigs indicated that hybrid wild boars are involved in the epidemiology of PRRSV.

This study was aimed at determining virulence-associated genes among Haemophilus parasuis (H. parasuis) strains, and supplying for the Kielstein-Rapp-Gabrielson serotyping scheme. The subtractive fragments, obtained through suppression subtractive hybridization and reverse Southern blot hybridization, were found to encode genes representative of 7 different functions. PCR was used to investigate the distribution of these fragments in H. parasuis strains isolated from different infection sites in pigs. Mice challenge was then used to analyze the correlationship between subtractive fragments, infection sites and bacterial virulence. Eight weeks old female BALB/c mice (10 mice/group) were inoculated intraperitoneally with 3.0 × 10(9) CFU suspension (0.5 ml/mouse) of H. parasuis strains in PBS. Results indicated that H. parasuis possessed varied virulence even among the same serovar strains. Transcription units hsdR, hsdS, gpT and ompP2, identified from the subtractive fragments, were uniformly expressed in highly virulent strains, while absent in weakly virulent strains, and demonstrated variable degrees of expression in moderately virulent strains. Moreover, H. parasuis strains, isolated from pericardium and heart blood, were all highly virulent strains, while from nasal cavity and joint were moderately or weakly virulent strains. This study indicated that fragments hsdR, hsdS, gpT and ompP2 were associated with the virulence of H. parasuis. The virulence of H. parasuis strains isolated from different infection sites was different. The current research provides a new reference for determining bacterial virulence in different H. parasuis strains.

Porcine reproductive and respiratory syndrome (PRRS) has become one of the most economically important diseases to the global pork industry. Currently, the efficacies of available commercial vaccines remain questionable: the modified live-PRRSV vaccines (MLVs) were generally effective but variable in sufficient protection, and the outcomes of inactivated-PRRSV vaccines (IVs) in the field were not very promising. In the present study, we investigated the effect of swine interleukin 4 (IL-4) on the development of virus-specific immune responses elicited by an MLV. The antibody titer against PRRSV membrane proteins in pigs elicited by MLV plus recombinant plasmid encoding IL-4 (group 3) was significantly higher than those elicited by MLV alone (group 1) and MLV plus empty plasmid (group 2) from 35 days post-inoculation (dpi). Similarly, the neutralizing efficacy of sera from group 3 was markedly enhanced compared with group 1 and group 2. In cellular immunity, the ratio of CD3⁺CD4⁺/CD3⁺CD8⁺ T lymphocyte subpopulations from group 3 monitored by flow cytometry (FCM) was significantly higher than those from group 1 and group 2 from 42 dpi to 21 days post-challenge (dpc). After viral challenge, pigs in group 3 showed significantly lower virus loads in peripheral blood measured by a real-time quantitative PCR (RT-qPCR), as compared with those in group 1 and group 2. Pigs in group 1 and group 2 had a low fever and displayed mild inappetence, lethargy, rough hair coats, and no lung lesions, while those in group 3 showed almost no clinical signs, no lung lesions. The scores of clinical signs of pigs in group 3 were significantly lower than those in both group 1 and group 2. Interestingly, the scores of lung lesions showed no significant differences among the three groups. Our results indicate that swine IL-4 markedly enhanced the protective immune response of pigs and improved the efficacy of the MLV in preventing PRRS disease.

Background Porcine circovirus type 2 (PCV2) is the primary causative agent of the emerging swine disease known as postweaning multisystemic wasting syndrome (PMWS). Nowadays, polymerase chain reaction (PCR) is still the most widespread technique in pathogen detection. Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method developed in 2000, will possibly replace PCR in the field of detection. To establish a LAMP method for rapid detection of PCV2, two pairs of primers were designed specially from the open reading frame 2 (ORF2) sequences of PCV2. A LAMP method for rapid detection of PCV2 was established. To compare with PCR, sensitivity and specificity of LAMP were evaluated using the optimized reaction system. The LAMP products could be determined by agarose gel electrophoresis or adding SYBR Green I dye. Results The amplification of LAMP could be obtained at 63°C for 60 min. The detection limit was nearly 1 copy of DNA plasmid, more sensitive than PCR. There was no cross-reaction with porcine circovirus type 1 (PCV1), porcine pseudorabies virus (PRV) and porcine parvovirus (PPV) under the same conditions. Conclusions LAMP is an useful rapid detection method with high sensitivity and specificity for PCV2.

Background Knee osteoarthritis (OA) is considered a major public health issue causing chronic disability worldwide with the increasing number of aging people. In China and increasingly worldwide, many sufferers with knee OA are using complementary and alternative medicine including herbal drug, herbal patch, acupuncture and Tuina etc., to alleviate their symptoms. However, evidence gathered from systematic reviews or randomized controlled trials (RCT) has only validated acupuncture for the management of osteoarthritic pain. Moreover, such Traditional Chinese Medicine (TCM) methods above are commonly used in an integrative way. This trial is aimed to compare the efficacy of an individually integrated TCM approach in the management of knee OA with other single treatments as parallel randomized controls. Methods/design Five teaching hospitals will participate in this randomized controlled trial. 500 participants, 100 in each hospital, will be randomly assigned to receive oral administration of a Chinese herbal drug (counter osteophytes capsule), topical use of a Chinese herbal patch (Fufnag Zijin patch), acupuncture, Tuina and the individually integrated TCM approach. The individually integrated TCM approach consists of basic treatment of oral counter osteophytes capsule, variable use of Tuina, acupuncture and a herbal patch based on the severity of the patient's symptoms. The interventions are given for a period of 4 weeks. The primary outcome measure is the self-reported total score using the Western Ontario McMaster Universities Osteoarthritis Index (WOMAC). Secondary outcome measures include patient and investigator global assessment of response to treatment, patient and investigator global assessment of OA condition, WOMAC pain, stiffness, and physical function subscales, short-form 36 (SF-36) and TCM assessment of OA condition measured by syndromes questionnaire. Mixed models and sensitivity analysis will be used for the statistical analysis. Discussion The trial is designed to test the hypothesis that an individually integrated TCM approach is more effective than four treatment modalities used separately. The major limitation of this study is lack of placebo control and of double blinding. Trial Registration Chinese Cochrane Center ChiCTR-TRC-00000176

Baseline is a very important parameter in SAR interferometry (InSAR) processing and applications, and it is crucial in InSAR system design, data processing and error analysis, and then straightly impacts the generation and quality of DEM from interferogram, so how to get the high precision baseline becomes the important problem of airborne InSAR. In this paper, a new quick method of baseline estimation was proposed, which was based on the frequency shift theory for estimating the perpendicular component of the baseline. The result is validated with the SIR-C/X-SAR data over the Tienshan area of china on October 7 and 8, 1994.

With the rapidly incremental demand in electronic devices, there is something in exploring multifunctional RF systems for reducing cost, minimizing the radar cross and probability of intercept, etc. This paper presents an integration system of radar and communication based on DSSS (direct sequence spread spectrum) technique, an attractive technique which is highly secure and robust. It utilizes different PN code to spread the spectrum of radar and communication data to avoid jamming mutually. Additionally, it uses BPM to gain better performance. The simulation over an ideal AWGN channel shows that the system can work perfectly