High expression of the classical swine fever virus (CSFV) envelope protein E2 by a single amino acid mutation and its embedded in the pseudorabies virus (PRV) vector for immunization.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases in pigs. Most pig farms in China are vaccinated against these two diseases. Gene-deleted pseudorabies virus (PRV) can be used to develop promising and economical multivalent live attenuated viral vector vaccines. It has been reported that recombinant PRV can express a truncated E2 protein (1-338 aa), but it has not been reported that recombinant PRV can express a full-length E2 protein. We constructed nine groups of E2 proteins with different expression forms and found that the E2 protein could be expressed in vitro only when the transmembrane region of E2 was removed and the signal peptide was added. Analysis of the transmembrane region of E2 revealed that the high hydrophobicity of the E2 transmembrane region was the main reason for its inability to express. By mutating an amino acid to reduce the hydrophobicity of the transmembrane region, it was found that the full-length mutant of E2 (E2FL-muta3 or E2FL-muta4) could be expressed. The expressed full-length mutant E2 could also localize to the cell membrane. Mice immunized with a PRV vector vaccine expressing E2FL-muta3 or E2FL-muta4 developed specific cellular immunity to the E2 protein and stimulated higher levels of E2 antibody than mice immunized with a PRV vector expressing truncated E2. After immunizing the rabbits, the lethal challenge by PRV-ZJ2013 and the febrile response elicited by CSFV were simultaneously prevented. These results suggest that rPRV-dTK/gE-E2FL-muta4 is a promising bivalent vaccine against CSFV and PRV infections.

Recombinant pseudorabies virus (PRV) expressing stabilized E2 of classical swine fever virus (CSFV) protects against both PRV and CSFV.

Pseudorabies (PR) and classical swine fever (CSF) are economically important infectious diseases of pigs. Most pig farms in China are immunized against these two diseases. Here, we describe a stabilized E2 protein as an immunogen inserted into the PRV genome as a bivalent live virus-vectored vaccine. The E2 protein has 48 variant sites, there are 2-5 candidate amino acids per variant site, and the relative energy contribution of each amino acid to E2 energy was calculated. Combined substitutions of amino acids at the neighbor variant site (neighbor substitution) were performed to obtain the E2 protein sequence with the lowest energy (stabilized E2). Multiple amino acid substitutions at 48 variant sites were performed, and the results were consistent with neighbor substitutions. The stabilized E2 sequence was obtained, and its energy decreased by 22 Rosetta Energy Units (REUs) compared with the original sequence. After the recombinant PRV expressing stabilized E2 of CSFV was constructed, the secretion efficiency of stabilized E2 was increased by 2.97 times, and the thermal stability was increased by 10.5 times. Immunization of mice resulted in a 2-fold increase in antibody production, and a balanced antibody level against subtype 1.1 and subtype 2.1d E2 was achieved. In rabbits immunized, the lethal challenge of PRV-ZJ and the fever response induced by CSFV could be prevented simultaneously. These findings suggest that rPRV-muta/287aaE2 is a promising bivalent vaccine against CSFV and PRV infections.

Safety and protective effects of an avirulent Salmonella Gallinarum isolate as a vaccine candidate against Salmonella Gallinarum infections in young chickens.

Fowl typhoid is an important disease of chickens and turkeys, which is caused by Salmonella Gallinarum (S. Gallinarum). Vaccines with high levels of protective effects against fowl typhoid need to be developed for the poultry industry. In this study, a S. Gallinarum strain, named SG01, was isolated from a poultry farm in Mashan region of Wuxi City, China, and identified through biochemical tests and specific PCR amplifications. Then, safety evaluations of the SG01 strain were performed in young chickens. No clinical symptom including depression and diarrhea and gross lesion involved in the cardiac nodule, hepatic necrotic lesion and splenic necrotic lesion, was determined on fifteen-day-old chickens after immunization with 1 × 1010 CFU of the SG01 strain through the oral route. However, diarrhea symptoms and hepatic lesions were identified from chickens immunized with the commercial vaccine strain SG9R by the same dose and route. At 14 days post inoculation, SG01 strain was eliminated in the liver and spleen from SG01-immunized chickens, while the SG9R strain still could be identified from SG9R-immunized chickens. After challenge with the virulent S. Gallinarum strain, significant reduction of the morbidity rate was found in the SG01 immunized group (20 %) compared to the challenge group (100 %) according to signs scoring systems for clinical symptoms and gross lesions. Additionally, immunization with the SG01 strain could provide more than 8 weeks of protection periods against fowl typhoid. These results demonstrate the SG01 strain is avirulent to young chickens and might be safer compared to the SG9R strain. In addition, SG01 strain is a potential vaccine candidate against fowl typhoid in young chickens.

Heat shock protein 90 relieves heat stress damage of myocardial cells by regulating Akt and PKM2 signaling in vivo.

Heat shock protein 90 (Hsp90) is associated with resisting heat­stress injury to the heart, particularly in myocardial mitochondria. However, the mechanism underlying this effect remains unclear. The present study was based on the high expression of Hsp90 during heat stress (HS) and involved inducing higher expression of Hsp90 using aspirin in mouse hearts. Higher Hsp90 levels inhibited HS­induced myocardial damage and apoptosis, and mitochondrial dysfunction, by stimulating Akt (protein kinase B) activation and PKM2 (pyruvate kinase M2) signaling, and subsequently increasing mitochondrial Bcl­2 (B­cell lymphoma 2) levels and its phosphorylation. Functional inhibition of Hsp90 using geldanamycin verified that reducing the association of Hsp90 with Akt and PKM2 caused the functional decline of phosphorylated (p)­Akt and PKM2 that initiate Bcl­2 to move into mitochondria, where it is phosphorylated. Protection by Hsp90 was weakened by blocking Akt activation using Triciribine, which could not be recovered by normal initiation of the PKM2 pathway. Furthermore, increased Hsp70 levels induced by Akt activation in myocardial cells may flow into the blood to resist heat stress. The results provided in vivo mechanistic evidence that in myocardial cells, Hsp90 resists heat stress via separate activation of the Akt­Bcl­2 and PKM2­Bcl­2 signaling pathways, which contribute toward preserving cardiac function and mitochondrial homeostasis.

Apoptosis in response to heat stress is positively associated with heat-shock protein 90 expression in chicken myocardial cells in vitro.

To determine heat-shock protein (Hsp)90 expression is connected with cellular apoptotic response to heat stress and its mechanism, chicken (Gallus gallus) primary myocardial cells were treated with the Hsp90 promoter, aspirin, and its inhibitor, geldanamycin (GA), before heat stress. Cellular viability, heat-stressed apoptosis and reactive oxygen species level under different treatments were measured, and the expression of key proteins of the signaling pathway related to Hsp90 and their colocalization with Hsp90 were detected. The results showed that aspirin treatment increased the expression of protein kinase B (Akt), the signal transducer and activator of transcription (STAT)-3 and p-IKKα/ß and the colocalization of Akt and STAT-3 with Hsp90 during heat stress, which was accompanied by improved viability and low apoptosis. GA significantly inhibited Akt expression and p-IKKα/ß level, but not STAT-3 quantity, while the colocalization of Akt and STAT-3 with Hsp90 was weakened, followed by lower cell viability and higher apoptosis. Aspirin after GA treatment partially improved the stress response and apoptosis rate of tested cells caused by the recovery of Akt expression and colocalization, rather than the level of STAT-3 (including its co-localization with Hsp90) and p-IKKα/ß. Therefore, Hsp90 expression has a positive effect on cellular capacity to resist heat-stressed injury and apoptosis. Moreover, inhibition of Hsp90 before stress partially attenuated its positive effects.

The association of Hsp90 expression induced by aspirin with anti-stress damage in chicken myocardial cells.

The protective effect of aspirin during exposure to heat stress in broiler chickens was investigated. We assayed pathological damage, expression and distribution of Hsp90 protein and hsp90 mRNA expression in chicken heart tissues after oral administration of aspirin following exposure to high temperature for varying times. Heat stress induced increases in plasma aspartate aminotransferase, creatine kinase and lactate dehydrogenase activities while causing severe heart damage, which was characterized by granular and vacuolar degeneration, nuclear shrinkage and even myocardium fragmentation in cardiac muscle fibers. After aspirin administration, myocardial cells showed fewer pathological lesions than broilers treated with heat alone. A high positive Hsp90 signal was always detected in the nuclei of myocardial cells from broilers treated with aspirin, while in myocardial cells treated with heat alone, Hsp90 in the nuclei decreased, as did that in the cytoplasm. Aspirin induced rapid and significant synthesis of Hsp90 before and at the initial phase of heat stress, and significant expression of hsp90 mRNA was stimulated throughout the experiment when compared with cells exposed to heat stress alone. Thus, specific pre-induction of Hsp90 in cardiovascular tissue was useful for resisting heat stress damage because it produced stable damage-related enzymes and fewer pathologic changes.

Effect of transportation stress on heat shock protein 70 concentration and mRNA expression in heart and kidney tissues and serum enzyme activities and hormone concentrations of pigs.

OBJECTIVE: To determine the enzymatic and hormonal responses, heat shock protein 70 (Hsp70) production, and Hsp70 mRNA expression in heart and kidney tissues of transport-stressed pigs. ANIMALS: 24 pigs (mean weight, 20 +/- 1 kg). PROCEDURES: Pigs were randomly placed into groups of 12 each. One group was transported for 2 hours. The other group was kept under normal conditions and used as control pigs. Sera were used to detect triiodothyronine, thyroxine, and cortisol concentrations and alanine aminotransferase, aspartate aminotransferase, and creatine kinase activities. The heart and kidneys of anesthetized pigs were harvested and frozen in liquid nitrogen for quantification of Hsp70 and Hsp70 mRNA. RESULTS: No significant differences were detected in serum alanine aminotransferase activity and triiodothyronine and cortisol concentrations between groups; however, the serum creatine kinase and aspartate aminotransferase activities and thyroxine concentrations were higher in transported pigs. Densitometric readings of western blots revealed that the amount of Hsp70 in heart and kidney tissues was significantly higher in transported pigs, compared with control pigs. Results of fluorescence quantitative real-time PCR assay revealed that the Hsp70 mRNA transcription in heart tissue, but not kidney tissue, was significantly higher in transported pigs, compared with control pigs. CONCLUSIONS AND CLINICAL RELEVANCE: Transportation imposed a severe stress on pigs that was manifested as increased serum activities of aspartate aminotransferase and creatine kinase and increased amounts of Hsp70 and Hsp70 mRNA expression in heart and kidney tissues. Changes in serum enzyme activities were related to the tissue damage of transport-stressed pigs.

[Rescue of a recombinant Newcastle disease virus expressing the green fluorescent protein].

A recombinant Newcastle disease virus (NDV) expressing the green fluorescent protein (GFP) was generated by applying reverse genetics techniques. The GFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the phosphoprotein (P) and matrix protein (M) in a full-length cDNA clone of NDV Lasota vaccine strain. This plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into cells stably expressing T7 RNA polymerase. The rescued virus was first propagated in 10-day-old embryonated eggs and the allantoic fluid was used to infect primary chicken embryo fibroblasts (CEF) cells. The appearance of GFP in live infected cells confirmed further the recovery of a recombinant NDV (rNDV-GFP) expressing this reporter gene. Nine successive passages in embryonated chicken eggs were performed. Allantoic fluid samples were then titrated by a microtiter plate HA test. HA positive ailantoic fluid were used for further egg passages. All the allantoic fluid samples were titrated by end point dilutions and infected cells were examined for the presence of GFP expression. To analyze virus growth, 10-day-old embryonated SPF chicken eggs were inoculated with 1 x 10(4) EID50 rNDV or rNDV-GFP. At 24,48,72 and 96 h p.i. the allantoic fluid of inoculated eggs containing live embryos was harvested and clarified by centrifugation. Supernatants were used for titration of EID50 in 10-day-old embryonated SPF chicken eggs. rNDV and rNDV-GFP grew to similar titers (10(9) EID50/mL). In order to test the virulence of rNDV-GFP, infectious allantoic fluid of rNDV-GFP were inoculated into embryonated SPF chicken eggs at 1 x 10(6) EID50. No dead embryonated egg was found within 96 hours. The replication kinetics and pathogenicity in SPF embryonated eggs of rNDV-GFP did not differ significantly from that of the parent virus. LaSota is a widely used NDV live vaccine strain. The reverse genetic system established for this LaSota vaccine strain provided a useful platform for development of novel live viral vector vaccines in future.