Enhancing immunogenicity and antiviral protection of inactivated porcine reproductive and respiratory syndrome virus vaccine in piglets.

OBJECTIVE: Porcine interferon-γ (poIFN-γ) and porcine granulocyte-macrophage colony-stimulating factor (poGM-CSF) are multifunctional cytokines that exhibit robust antiviral activity against porcine reproductive and respiratory syndrome virus (PRRSV). In this study, the immunoadjuvant effects of recombinant poIFN-γ-poGM-CSF fusion protein in inactivated PRRSV vaccine administered to piglets were assessed. ANIMALS: Twenty-eight 4-week-old specific pathogen-free piglets. METHODS: The experimental piglets were divided into control, highly pathologic PRRSV, PRRSV killed virus vaccine (KV), poIFN-γ-poGM-CSF, KV + 1.0 mg poIFN-γ-poGM-CSF, KV + 2.0 mg poIFN-γ-poGM-CSF, and KV + 4.0 mg poIFN-γ-poGM-CSF groups. A recombinant poIFN-γ-linker-poGM-CSF fusion gene was constructed via splicing by overlap extension PCR and prepared using an Escherichia coli expression system, after which its adjuvant activity in the context of PRRSV KV administration was assessed. RESULTS: This analysis revealed the successful construction of the poIFN-γ-linker-poGM-CSF fusion gene via splicing by overlap extension PCR, with recombinant poIFN-γ-linker-poGM-CSF successfully being prepared in E coli with a plasmid vector for expressing thioredoxin fusion proteins with an enterokinase site. Importantly, the coadministration of poIFN-γ-linker-poGM-CSF and PRRSV KV significantly increased neutralizing antibody titers, accelerated viral clearance, reduced clinical symptoms, and prevented highly pathogenic PRRSV infection. CLINICAL RELEVANCE: The recombinant poIFN-γ-poGM-CSF fusion protein is a promising candidate adjuvant for use in the context of swine immunization and viral challenge.

Rainfall-runoff partitioning in small watersheds of different vegetation types in the loess area based on hydrogen and oxygen isotope tracing.

Recognizing watershed runoff process and its component sources is a prerequisite for the rational use of water resources. To elucidate the effects and quantitative contributions of various vegetation types on the components of watershed runoff, we centered on the Caijiachuan main channel watershed in Jixian, Shanxi and five sub-watersheds with distinct vegetation types. By tracking the hydrological responses to two representative rainfall events and assessing the spatiotemporal variations in hydrogen and oxygen isotope signatures, we aimed to discern disparities in the runoff processes across these sub-watersheds and pinpoint their constituent origins. The results showed that under medium rainfall condition, the contribution rates of event water to the river flow of each watershed were in an order of protected forest (94.3%) > Caijiachuan main channel (83.1%) > agro-pastoral composite (64.3%) > plantation-secondary forest (52.4%) > cropland (0.3%) > secondary forest (0.0%); under light rainfall condition, plantation-secondary forest (52.4%) > protected forest (58.5%) > cropland (40.6%) > secondary forest (15.8%) > agro-pastoral composite (12.5%) > Caijiachuan main channel (9.3%). The event water contribution rate of secondary forest and protected forest watersheds to runoff was higher than that of plantation watersheds. The secondary forests watersheds had a stronger runoff storage capacity. The event water contribution rate of protected forest and agro-pastoral composite watersheds under medium rainfall intensity condition was greater than that under light rainfall intensity condition, while the event water contribution rate of cropland, plantation-secondary forest, and secondary forest watersheds was in adverse. The event water contribution to the runoff of forested watersheds was greater than that of cropland watersheds, which may be related to the presence of silt dams at the mouth of agricultural watershed channels. This study can provide a scientific basis for the analysis of water conservation and runoff change attribution in the loess area of west Shanxi.

Porcine circovirus type 2 (PCV2) infection in Hebei Province from 2016 to 2019: a retrospective study.

Porcine circovirus type 2 (PCV2) is the primary causative agent of porcine circovirus-associated diseases in swine, the most common of which are postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). To investigate the prevalence and genetic diversity of PCV2 in Hebei Province, Northern China, from 2016 to 2019, a total of 448 suspected cases of PCV2 infection were studied, and 179 samples were positive for PCV2. A pathological and histopathological examination suggested PCV2 to be cause of the observed lesions. Phylogenetic analysis showed that four genotypes were prevalent in Hebei Province: PCV2a, 2b, 2d, and 2e. Analysis of PCV2 strains using RDP4 and SimPlot showed that there were genetic recombination events among PCV2 strains in Hebei Province. A total of 3284 serum samples were screened by ELISA, and the positive rate of PCV2 antibodies was 73.9% (2428/3284). This study provides a scientific reference for the prevention and treatment of PCV2 in Hebei Province.

Short hairpin RNAs targeting M and N genes reduce replication of porcine deltacoronavirus in ST cells.

Porcine deltacoronavirus (PDCoV) is a recently identified coronavirus that causes intestinal diseases in neonatal piglets with diarrhea, vomiting, dehydration, and post-infection mortality of 50-100%. Currently, there are no effective treatments or vaccines available to control PDCoV. To study the potential of RNA interference (RNAi) as a strategy against PDCoV infection, two short hairpin RNA (shRNA)-expressing plasmids (pGenesil-M and pGenesil-N) that targeted the M and N genes of PDCoV were constructed and transfected separately into swine testicular (ST) cells, which were then infected with PDCoV strain HB-BD. The potential of the plasmids to inhibit PDCoV replication was evaluated by cytopathic effect, virus titers, and real-time quantitative RT-PCR assay. The cytopathogenicity assays demonstrated that pGenesil-M and pGenesil-N protected ST cells against pathological changes with high specificity and efficacy. The 50% tissue culture infective dose showed that the PDCoV titers in ST cells treated with pGenesil-M and pGenesil-N were reduced 13.2- and 32.4-fold, respectively. Real-time quantitative RT-PCR also confirmed that the amount of viral RNA in cell cultures pre-transfected with pGenesil-M and pGenesil-N was reduced by 45.8 and 56.1%, respectively. This is believed to be the first report to show that shRNAs targeting the M and N genes of PDCoV exert antiviral effects in vitro, which suggests that RNAi is a promising new strategy against PDCoV infection.

Seroprevalence of porcine bocavirus in pigs in north-central China using a recombinant-NP1-protein-based indirect ELISA.

Porcine bocavirus (PBoV), which belongs the genus Bocaparvovirus, has been identified throughout the world. However, serological methods for detecting anti-PBoV antibodies are presently limited. In the present study, an indirect enzyme-linked immunosorbent assay (PBoV-rNP1 ELISA) based on a recombinant form of nucleoprotein 1 (NP1) of PBoV was established for investigating the seroprevalence of PBoV in 2025 serum specimens collected in north-central China from 2016 to 2018, and 42.3% of the samples tested positive for anti-PBoV IgG antibodies, indicating that the seroprevalence of PBoV is high in pig populations in China.

Di-(2-ethylhexyl) phthalate (DEHP)-induced hepatotoxicity in quail (Coturnix japonica) via suppression of the heat shock response.

Di-(2-ethylhexyl) phthalate (DEHP) is a widespread environmental toxicant that severely impacts agricultural production and animal and human health. Nevertheless, DEHP-induced hepatotoxicity at the molecular level in quail remains unexplored. The heat shock response (HSR), involving heat shock proteins (HSPs) and heat shock transcription factors (HSFs), is a highly conserved molecular response that is triggered by stressors, especially exposure to toxicants. To explore the DEHP-induced hepatotoxicity that occurs via regulation of HSR in birds, female quail were dosed with DEHP by oral gavage (0, 250, 500 and 1000 mg/kg) for 45 days. Based on histopathological analysis, the livers of the DEHP-treated groups exhibited structural alterations of hepatocytes, including mitochondrial swelling, derangement of hepatic plates, inflammatory cell infiltration and adipose degeneration. Ultrastructural evaluation of the livers of DEHP-treated quail revealed swollen mitochondria, partial disappearance of mitochondrial membranes and cristae, nuclear chromatin margination and nuclear condensation. The expression of HSF1 and HSF3 significantly decreased after DEHP exposure. The levels of HSPs (HSP10, HSP25, HSP27, HSP40, HSP47, HSP60, HSP70 and HSP90) were significantly downregulated in the livers of DEHP-treated quail. In this study, we concluded that DEHP exposure resulted in liver function damage and hepatotoxicity by reducing the expression of HSFs and HSPs in quail liver, which inhibited the protective effect of the HSR signaling pathway.

Di (2-ethylhexyl) phthalate (DEHP)-induced hepatotoxicity in quails (Coturnix japonica) via triggering nuclear xenobiotic receptors and modulating cytochrome P450 systems.

Di (2-ethylhexyl) phthalate (DEHP) is a widely distributed pollutant that is of great concern due to its negative health effects. However, whether DEHP exposure causes liver toxicity in birds remains unclear. To clarify the potential hepatotoxicity of DEHP, quails were exposed to 0, 250, 500 and 1000 mg/kg BW/day DEHP by gavage treatment for 45 days. The livers of DEHP-exposed quails showed histomorphological changes. DEHP exposure induced a significant increase in cytochrome P450 enzyme system (CYP450s) activity (including aniline-4-hydroxylase (AH), aminopyrine N-demethylase (APND), erythromycin N-demethylase (ERND) and NADPH-cytochrome C reductase (NCR)) and in the contents of total cytochrome P450 (CYP450) and cytochrome b5 (Cyt b5) in quail liver. DEHP exposure also influenced the expression of nuclear xenobiotic receptors (NXRs) and CYP450 isoforms in the liver. The results suggested that DEHP-induced hepatotoxicity in quail liver is associated with activation of the NXRs pathway responses and disruption of CYP450s homeostasis. This study will help to further elucidate DEHP exposure-induced liver toxicity in quails.

Atrazine-xenobiotic nuclear receptor interactions induce cardiac inflammation and endoplasmic reticulum stress in quail (Coturnix coturnix coturnix).

Atrazine (ATR) is one of the most extensively used herbicide that eventually leaches into groundwater and surface water from agricultural areas. Exposure to ATR does harm to the health of human and animals, especially the heart. However, ATR exposure caused cardiotoxicity in bird remains unclear. To evaluate ATR-exerted potential cardiotoxicity in heart, quail were exposed with 0, 50, 250, and 500 mg/kg BW/day ATR by gavage treatment for 45 days. Cardiac histopathological alternation was observed in ATR-induced quail. ATR exposure increased the Cytochrome P450s and Cytochrome b5 contents, Cytochrome P450 (CYP) enzyme system (APND, ERND, AH, and NCR) activities and the expression of CYP isoforms (CYP1B1, CYP2C18, CYP2D6, CYP3A4, CYP3A7, and CYP4B1) in quail heart. The expression of nuclear xenobiotic receptors (NXRs) was also influenced in the heart by ATR exposure. ATR exposure significantly caused the up-regulation of pro-inflammatory cytokines (TNF-α, IL-6, NF-κB, and IL-8), down-regulation of anti-inflammatory cytokines (IL-10) expression levels and increased NO content and iNOS activity. The present research provides new insights into the mechanism that ATR-induced cardiotoxicity through up-regulating the expression levels of GRP78 and XBP-1s, triggering ER stress, activating the expression of IRE1α/TRAF2/NF-κB signaling pathway related factors (IRE1α, TRAF2, IKK, and NF-κB) and inducing an inflammatory response in quail hearts. In conclusion, ATR exposure could induce cardiac inflammatory injury via activating NXRs responses, disrupting CYP homeostasis and CYP isoforms transcription, altering NO metabolism and triggering ER stress and inflammatory response by activating IRE1α/TRAF2/NF-κB signaling pathway.

Development and application of a recombinant M protein-based indirect ELISA for the detection of porcine deltacoronavirus IgG antibodies.

Porcine deltacoronavirus (PDCoV) is a recently identified coronavirus in the genus Deltacoronavirus that can cause enteric disease including diarrhea, vomiting, dehydration and mortality in neonatal piglets. Serological assays to detect anti-PDCoV antibodies are presently limited to certain laboratories and geographic regions. In this study, a recombinant M protein-based indirect enzyme-linked immunosorbent assay (PDCoV-rM ELISA) was developed and utilized to determine the prevalence of anti-PDCoV IgG in Hebei province. The PDCoV-rM ELISA showed no cross-reaction with antisera against transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhea virus (PEDV), porcine rotavirus (PRV), porcine circovirus 2 (PCV2), classical swine fever virus (CSFV) or porcine reproductive and respiratory syndrome virus (PRRSV). The diagnostic sensitivity was 90.6% and the diagnostic specificity was 93.3%. A total of 871 serum samples collected in Hebei from January 2015 to October 2016 were checked for presence of antibodies against PDCoV using the novel PDCoV-rM ELISA. Anti-PDCoV IgG antibodies were detected in 11% (96/871) of the samples and in 25% (10/40) of the investigated farms. The data suggest that PDCoV has a low seroprevalence in pig population in Hebei province, China.

[Randomized controlled trials of needle knife therapy combined with rotation traction manipulation for the treatment of cervical spondylotic radiculopathy].

OBJECTIVE: To compare the therapeutic effects between needle knife therapy combined with rotation traction manipulation and rotation traction manipulation for the treatment of cervical spondylotic radiculopathy. METHODS: From November 2013 to June 2015, 80 patients with cervical spondylotic radiculopathy meeting the inclusion criteria were divided into two groups randomly:the control group in which 39 patients were treated with rotation traction manipulation, and the treatment group in which 41 patients were treated with needle knife combined with rotation traction manipulation. The patients in the control group were treated once dayly for 2 weeks, which was 1 course. The patients in the treatment group were treated with needle knife firstly once a week for 2 weeks, which was 1 course;then were treated with the same methods as the patients in the control group. The symptoms, signs score and the therapeutic effects of the two groups before and after treatment were observed. RESULTS: After treatment, symptoms and signs scores declined in both groups(P<0.05). The results of the treatment group were better than effects in the control group(P<0.05). In the treatment group, 19 patients got an excellent result, 16 good, 5 fair and 1 bad;while in the control group, 10 patients got an excellent result, 10 good, 16 fair and 3 bad;the results of the treatment group were better than the results of the control group(P<0.01). CONCLUSIONS: Needle knife combined with rotation traction manipulation is an effective method for the treatment of cervical spondylotic radiculopathy, which is better than using manipulation method simply. Needle knife therapy has follow advantages:improving local blood circulation, reducing local content of pain substance, increasing production of substances resisting pain, opening channels and collaterals, and make body reaching new static and dynamic balance on the new foundation.

Development of an enzyme-linked immunosorbent assay for the monitoring and surveillance of antibodies to porcine epidemic diarrhea virus based on a recombinant membrane protein.

The recent dramatic increase in reported cases of porcine epidemic diarrhea (PED) in pig farms is a potential threat to the global swine industry. Therefore, the accurate diagnosis, serological monitoring, and surveillance of specific antibodies in pigs resulting from porcine epidemic diarrhea virus (PEDV) infection or vaccination would be essential in helping to control the spread of PED. We developed and validated an indirect enzyme-linked immunosorbent assay (ELISA) based on the recombinant membrane (M) protein of PEDV. To detect PEDV antibodies in eight herds, 382 serum samples were collected from sows that had been immunized with a PED vaccine, and screened using the developed ELISA in parallel with a serum neutralization (SN) assay. Of the tested samples, 276 were positive for the presence of PEDV antibodies according to both assays, while 98 were negative. An excellent agreement between the ELISA and the SN assay was observed (kappa=0.947; 95% confidence interval=0.910-0.984; McNemar's test, P=0.727). No cross-reaction was detected for the developed ELISA with other coronaviruses or other common pig pathogens. The developed ELISA could be used for serological evaluation and indirect diagnosis of PED infection.

Heterogeneity in membrane protein genes of porcine epidemic diarrhea viruses isolated in China.

Since late 2010, porcine epidemic diarrhea virus (PEDV) has been re-emerging in immunized swine herds with devastating impact in the Hebei province of China. Seven prevailing strains of PEDV were isolated from fecal samples out of piglets suffering from severe diarrhea. The M gene of the seven PEDV isolates encompasses an open reading frame of 681 nucleotides, encoding a protein of 226 amino acids. The seven PEDV isolates showed 99.4-99.9 % nucleotide sequence identity and 98.2-99.1 % deduced amino acid identity. When compared with other Chinese isolates and foreign isolates, the seven isolates showed high nucleotide identity with the Thailand isolate M-NIAH1005 (99.6-99.9 %) and Korea isolate PFF188 (99.7-100 %), but low identity with other Chinese isolates (96.6-99.1 %) and with the vaccine strain CV777 used in China (97.8-98.2 %). Phylogenetic analyses showed that all seven Chinese field isolates were grouped together in the same cluster. Although CV777 was also separated into the same cluster with the seven isolates, they were belonged to different sub-cluster. These results showed that the seven prevailing isolates in China are closely related phylogenetically to each other and have close relationships with the Korean strain PFF188 and Thailand strain M_NIAH1005. However, they differ genetically from other Chinese isolates and the vaccine strain CV777. Therefore, a more efficient vaccine strain should be chosen to prevent outbreaks of PEDV in China.