Cloning and further sequence analysis of the spike gene of attenuated porcine epidemic diarrhea virus DR13

The spike (S) gene of the attenuated porcine epidemic diarrhea virus (PEDV) DR13 was cloned and sequenced to further explore the functions of wild type PEDV and attenuated PEDV. Sequencing revealed a single large ORF of 4,149 nucleotides encoding a protein of 1,382 amino acids with predicted M _r of 151 kDa. The coding region of the S gene of attenuated PEDV DR13 had 20 nucleotide changes that appeared to be significant determinants of function in that they produced changes in its predicted amino acid sequence. Notably, attenuated PEDV DR13 has previously been found to exhibit reduced pathogenicity in pigs. The regions containing these 20 nucleotide changes may therefore be crucial for PEDV pathogenicity. The attenuated PEDV DR13 S protein contains 28 Asn-Xaa-Ser/Thr sequons, 21 asparagines that are predicted to be N-glycosylated and a stretch of highly hydrophobic residues at positions 1,327–1,347, which is predicted to form an α-helix and to function as a membrane anchor. One (from N to K at 378) of the changes in the deduced amino acid sequence destroyed N-linked glycosylation sites, while another change (from N to S at 114) created a new one at a different location. These alterations in N-linked glycosylation sites reflected 3 nucleotide changes, which were related to the above-mentioned nucleotide changes and are suggested to influence the pathogenicity of attenuated PEDV DR13. Attenuated PEDV DR13 has 96.5, 96.4, 96.1, 93.9, 93.5 and 96.6% DNA sequence identities with CV777, Br1/87, JS-2004-2, Spk1, Chinju99 and parent DR13, respectively. Likewise, it shares 95.7, 95.4, 95.6, 92.0, 91.6 and 95.7% identity with those genes at the deduced amino acid sequence level. Phylogenetic analysis suggested that attenuated PEDV DR13 is closely related to CV777, Br1/87, JS-2004-2 and parent DR13, rather than to Spk1 and Chinju99 and is especially close to the Chinese PEDV strain JS-2004-2. Nucleotide sequence data reported is available in the GenBank database under the Accession Nos. DQ462404 and DQ862099.     

Adhesive strength and curing rate of marine mussel protein extracts on porcine small intestinal submucosa

An adhesive protein extracted from marine mussels (Mytilus edulis) was used to bond strips of connective tissue for the purpose of evaluating the use of curing agents to improve adhesive curing. Specifically, mussel adhesive protein solution (MAPS, 0.5mM dihydroxyphenylalanine) was applied, with or without the curing agents, to the ends of two overlapping strips of porcine small intestinal submucosa (SIS).The bond strength of this lap joint was determined after curing for 1h at room temperature (25 degrees C). The strength of joints formed using only MAPS or with only the ethyl, butyl or octyl cyanoacrylate adhesives were determined. Although joints bonded using ethyl cyanoacrylate were strongest, those using MAPS were stronger than those using butyl and octyl cyanoacrylates. The addition of 25mM solutions of the transition metal ions V5+, Fe3+ and Cr6+, which are all oxidants, increased the bond strength of the MAPS joints. The V5+ gave the strongest bonds and the Fe3+ the second strongest. In subsequent tests with V5+ and Fe3+ solutions, the bond strength increased with V5+ concentration, but it did not increase with Fe3+ concentration. Addition of 250mM V5+ gave a very strong bond.     

Epitope selection and their placement for increased virus neutralization in a novel vaccination strategy for porcine epidemic diarrhea virus utilizing the Hepatitis B virus core antigen

Porcine epidemic diarrhea virus (PEDV) is a member of the Alphacoronaviridae genus within the Coronaviridae family. It is the causative agent of porcine epidemic diarrhea, a disease that can have mortality rates as high as 100% in suckling piglets. PEDV causes severe economic loss, and has been in existence for decades. A panzootic starting in 2010 renewed interest in the development of a universal vaccine toward PEDV. This report details several design changes made to a Hepatitis B virus core antigen (HBcAg)-based recombinant vaccine strategy, and their effect in vivo. Initially, several multi-antigen vaccine candidates were able to elicit antibodies specific to three out of four B-cell epitopes inserted into the chimeric proteins. However, a lack of virus neutralization led to a redesign of the vaccines. The focus of the newly redesigned vaccines was to elicit a strong immune response to the YSNIGVCK amino acid motif from PEDV. Genetically modified new vaccine candidates were able to elicit a strong antibody (Ab) response to the YSNIGVCK epitope, which correlated with an increased ability to neutralize the CO strain of PEDV. Additionally, the location of the inserted PEDV epitopes within the vector protein was shown to affect the immune recognition toward the native HBcAg during vaccination.     

Safety of intramyocardial injection of autologous bone marrow cells to treat myocardial ischemia in pigs

OBJECTIVE: The purpose of this study is to determine the potential adverse consequences of intracardiac injections of bone marrow mononuclear cells (BMCs) to facilitate the revascularization of ischemic myocardium. BACKGROUND: Bone marrow mononuclear cells are used to treat heart failure, though there are few studies that evaluated the safety of BMC transplantation for chronic myocardial ischemia. METHODS: The pigs received coronary ameroid constrictors to induce chronic myocardial ischemia and left ventricular dysfunction. At 4 weeks, autologous BMCs were injected intramyocardially by Boston Scientific Stiletto catheter with low-dose (10(7) cells) or high-dose BMC (10(8)). Control animals received saline. Blood samples were collected for hematological and chemical indices, including cardiac enzyme levels at regular time intervals postinfarction. At 7 weeks, animals underwent electrophysiological study to evaluate the arrhythmic potential of transplanted BMC, followed by necropsy and histopathology. RESULTS: No mortalities were associated with intramyocardial delivery of BMC or saline. At Day 0, the total creatine phosphokinase (CPK) was in the normal range in all groups. All groups had significant elevations in CPK after ameroid placement, with no significant differences between groups. At 7 weeks, CPK in all groups had returned to pretreatment levels. Electrophysiological assessment revealed that one control animal had an inducible arrhythmia. No arrhythmias were induced in low- or high-dose BMC-treated pigs. There were no histopathological changes associated with BMC injection. CONCLUSION: This study showed, in a clinically relevant large-animal model, that catheter-based intramyocardial injection of autologous BMC into ischemic myocardium is safe.     

猪脂肪源间充质干细胞诱导成脂分化及Krüppel样因子2的表达

目的旨在阐明猪脂肪源间充质干细胞(AMSCs)在诱导成脂分化过程中Krüppel样因子2(KLF2)的表达模式。方法采用I型胶原酶消化法处理猪脂肪组织,经原代和传代培养分离、纯化和扩增AMSCs,用流式细胞仪检测AMSCs纯度,在倒置显微镜下用形态学和油红O染色法检测AMSCs的成脂分化,用实时定量PCR检测KLF2的表达模式,并与过氧化物酶体增殖物激活受体2(PPARγ2)的表达进行了比较。结果分离、纯化的AMSCs,其间充质干细胞表面抗原CD29、CD44和CD105表达量分别为91.7%、95.1%和95.6%,而造血干细胞表面抗原CD34表达量仅为3.39%;在诱导分化2d后,个别细胞质中出现小脂滴,且含脂滴的细胞数量和脂滴量随诱导时间的延长而增加,在诱导分化第18天成脂分化率达48.2%;在诱导分化第2天、第8天和第16天,KLF2的表达量分别为1.84±0.206、1.07±0.072和0.83±0.095,而PPARγ2 mRNA的表达量分别为3.06±0.542、13.22±0.5736和15.11±1.073。结论获得纯度较高的AMSCs,具有良好的成脂分化潜能,KLF2的表达量在成脂分化早期的前脂肪细胞阶段增加,成脂分化开始后逐步减少,KLF2作为脂肪分化负调控转录因子而发挥作用。     

Transvesical NOTES: Survival Study in Porcine Model

Background and Objectives:

The optimal access route and method for natural orifice transluminal endoscopic surgery (NOTES) has not been established. A transvesical approach, with its low rate of peritoneal contamination, is an effective clean portal of entry, but a safe urinary bladder closure has been a challenge. We developed a new technique for a safe, pure transvesical NOTES approach.

Methods:

Four female piglets were used in the study. With the pigs under anesthesia, a flexible cystoscope (15Fr) was used to make an endoscopic cystotomy; diagnostic peritoneoscopy of the abdominal quadrants was done with biopsies and hemostasis. At the end, a Vicryl loop was pushed to close the bladder incision while the incision edges were pulled inwards. The pigs were euthanized after 2 wk, and necropsies were performed.

Results:

No bowel injury was noted in any of the 4 pigs. Satisfactory bladder closure was done in 2 pigs, while a partial closure was achieved in 1 case. In the postoperative period, the pigs showed no signs of pain or distress, voided normally, and had a good appetite. On necropsy, we noted healed cystotomy incisions, no intraabdominal adhesions, and no adhesions at the site.

Conclusion:

Our new technique for endoscopic cystotomy overcomes previously reported risks for bowel injuries. Using this route gives good spatial orientation and access to all quadrants, including the pelvis. Biopsies with good hemostasis can be easily achieved. Lack of intraperitoneal changes postoperatively indicate that this procedure may be safe for humans.     

Inactivated PCV2 one shot vaccine applied in 3-week-old piglets: improvement of production parameters and interaction with maternally derived immunity

The present study describes the effects of a commercially available vaccine against Porcine circovirus type 2 (PCV2) on clinical, pathological and virological outcomes of 3-week-old piglets from two farms with a clinical history of postweaning multisystemic wasting syndrome (PMWS). The study was a controlled, double-blinded, parallel group (1:1) and randomized trial (with a negative control) involving a total of 1239 animals. The study period comprised from weaning age (time of vaccination or PBS inoculation) until the first shipment of pigs to the slaughterhouse. The vaccine product was able to reduce clinical signs, PCV2 viral load in sera and faeces, and overall mortality in nurseries and fattening units. Moreover, average daily gain was significantly higher in vaccinated versus non-vaccinated piglets during the trial period. On the other hand, it was shown that maternally derived antibodies interfered with the development of an active humoral immune response after PCV2 vaccination.     

Immunogenicity study of plant-made oral subunit vaccine against porcine reproductive and respiratory syndrome virus (PRRSV)

Currently, killed-virus and modified-live PRRSV vaccines are used to control porcine reproductive and respiratory syndrome disease (PRRS). However, very limited efficacy of killed-virus vaccines and serious safety concerns for modified-live virus vaccines demand the development of novel PRRSV vaccines. In this report, we investigated the possibility of using transgenic plants as a cost-effective and scalable system for production and delivery of a viral protein as an oral subunit vaccine against PRRSV. Corn calli were genetically engineered to produce PRRSV viral envelope-associated M protein. Both serum and intestine mucosal antigen-specific antibodies were induced by oral administration of the transgenic plant tissues to mice. In addition, serum and mucosal antibodies showed virus neutralization activity. The neutralization antibody titers after the final boost reached 6.7 in serum and 3.7 in fecal extracts, respectively. A PRRSV-specific IFN-γ response was also detected in splenocytes of vaccinated animals. These results demonstrate that transgenic corn plants are an efficient subunit vaccine production and oral delivery system for generation of both systemic and mucosal immune responses against PRRSV.     

超声引导在家猪中心静脉置管教学模型建立中的应用

目的 对比手术切开直视法与超声引导法实施中心静脉置管,探索便携式超声在家猪颈内静脉穿刺和置管中的应用.方法 雌性家猪2只,氯胺酮麻醉后实施气管插管全身麻醉.分别采用手术切开直视法和超声引导下经皮穿刺法实施颈内静脉置管,记录穿刺成功率、置管成功率、成功穿刺的平均试穿次数、完整置管用时及并发症发生情况.结果 手术切开直视法颈内静脉置管用时25 min.有8位操作者采用超声引导法,分别对左侧和右侧颈内静脉实施穿刺操作8次,完整置管操作3次.穿刺、置管成功率为100％,成功穿刺的平均试穿次数为1.9次,完整置管用时平均17 min.无并发症发生.结论 超声引导下家猪血流动力学教学模型中心静脉置管具有微创、快捷、操作简便、成功率高等优势,将在麻醉模拟教学中发挥重要的作用.