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.

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.

Clinical efficacy of Duyiwei capsule in treating gingivitis: A protocol of systematic review and meta-analysis.

BACKGROUND: This study will investigate the clinical efficacy of Duyiwei capsule (DYWC) for the treatment of gingivitis. METHODS: Relevant studies will be searched in PUBMED, EMBASE, Cochrane Library, WANGFANG, VIP, CBM, and CNKI from inception to the March 31, 2020 without limitations of language and publication time. All potential randomized controlled trials on the clinical efficacy of DYWC for the treatment of gingivitis will be considered. Two authors will independently perform literature selection, data collection, and study quality assessment. Any disagreements will be solved by a third author through discussion. We will utilize RevMan 5.3 software for statistical analysis. RESULTS: This study will summarize present randomized controlled trials on the efficacy and safety of DYWC for the treatment of gingivitis. CONCLUSION: The findings of this study will provide evidence to show whether DYWC is effective and safety for gingivitis.Systematic review registration: INPLASY202040199.

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.

Two novel recombinant porcine reproductive and respiratory syndrome viruses belong to sublineage 3.5 originating from sublineage 3.2.

Porcine reproductive and respiratory syndrome virus (PRRSV) is an agent of porcine reproductive and respiratory syndrome (PRRS), which causes substantial economic losses to the swine industry. PRRSV displays rapid variation, and five lineages coexist in mainland China. Lineage 3 PRRSVs emerged in mainland China in 2005 and prevailed in southern China after 2010. In the present study, two lineage 3 PRRSV strains, which are named SD110-1608 and SDWH27-1710, were isolated from northern China in 2017. To explore the characteristics and origins of the two strains, we divided lineage 3 into five sublineages (3.1-3.5) based on 146 open reading frame (ORF) 5 sequences. Both strains and the strains isolated from mainland China were classified into sublineage 3.5. Lineage 3 PRRSVs isolated from Taiwan and Hong Kong were classified into sublineages 3.1-3.3 and sublineage 3.4, respectively. Recombination analysis revealed that SD110-1608 and SDWH27-1710 were derived from recombination of QYYZ (major parent strain) and JXA1 (minor parent strain). Sequence alignment showed that SD110-1608 and SDWH27-1710 shared a 36-aa insertion in Nsp2 with QYYZ isolated from Guangdong Province in 2010. Based on the evolutionary relationship among GP2a, GP3, GP4, GP5 and N proteins between sublineages 3.2 (FJ-1) and 3.5 (FJFS), we speculated that sublineage 3.5 (mainland China) originated from sublineage 3.2 (Taiwan, China). This study provides important information regarding the classification and transmission of lineage 3 PRRSVs.

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.

Effects of silymarin on p65 NF-κB, p38 MAPK and CYP450 in LPS-induced hoof dermal inflammatory cells of dairy cows.

BACKGROUND: Laminitis is considered as one of the most important causes of hoof lameness in dairy cows, which can lead to enormous economic losses. However, the etiology and pathogenesis of laminitis have not been clarified yet. Besides, it is of great significant to find alternative herbs for the prevention and treatment of dairy hooves to avoid the antibiotic abuse. In this study, the primary hoof dermal cells of dairy cows were isolated, the inflammatory model was induced by LPS, and treated with silymarin to find whether silymarin has protective effect on the inflammatory dermal cells. The viability of dermal cells, the levels of IL-1ß and TNF-α, the degree of p65 NF-κB and p38 MAPK phosphorylation, the expressions of CYP3A4 and CYP1A1 were measured. RESULTS: Hoof dermal cells of dairy cows were successfully isolated and cultured by tissue adherent culture method. Certain concentrations of LPS can increase the levels of IL-1ß and TNF-α, promote the phosphorylation of p65 NF-κB and p38 MAPK, and inhibit the mRNA expressions of CYP3A4 and CYP1A1. The optimal concentration for LPS to establish a hoof dermal cells inflammatory model was 10 µg/mL. Certain concentrations of silymarin can markedly decrease the secretions of IL-1ß and TNF-α, inhibit the phosphorylation of p65 NF-κB and p38 MAPK, and promote the mRNA expressions of CYP3A4 and CYP1A1 in LPS-induced dermal inflammatory model. CONCLUSIONS: LPS can be used for inducing the hoof dermal cells inflammatory model of dairy cows. Silymarin has protective effects on the LPS-induced inflammatory model.

Silymarin-mediated regulation of the cell cycle and DNA damage response exerts antitumor activity in human hepatocellular carcinoma.

A novel module-search algorithm method was used to screen for potential signatures and investigate the molecular mechanisms of inhibiting hepatocellular carcinoma (HCC) growth following treatment with silymarin (SM). The modules algorithm was used to identify the modules via three major steps: i) Seed gene selection; ii) module search by seed expansion and entropy minimization; and iii) module refinement. The statistical significance of modules was computed to select the differential modules (DMs), followed by the identification of core modules using the attract method. Pathway analysis for core modules was implemented to identify the biological functions associated with the disease. Subsequently, results were verified in an independent sample set using reverse transcription polymerase chain reaction (RT-PCR). In total, 18 seed genes and 12 DMs (modules 1-12) were identified. The core modules were isolated using gene expression data. Overall, there were 4 core modules (modules 11, 5, 6 and 12). Additionally, DNA topoisomerase 2-binding protein 1 (TOPBP1), non-structural maintenance of chromosomes condensing I complex subunit H, nucleolar and spindle associated protein 1 (NUSAP1) and cell division cycle associated 3 (CDCA3) were the initial seed genes of module 11, 5, 6 and 12, respectively. Pathway results revealed that cell cycle signaling pathway was enriched by all core modules simultaneously. RT-PCR results indicated that the level of CDCA3, TOPBP1 and NUSAP1 in SM-treated HCC samples was markedly decreased compared with that in non-SM-treated HCC. No statistically significant difference between the transcriptional levels of CDCA3 in SM-treated and non-treated HCC groups was identified, although CDCA3 expression was increased in the treated group compared with the untreated group. Furthermore, although the expression level of TOPBP1 and NUSAP1 in the SM-treated group was decreased compared with that in the normal group, no significant difference was observed. From the results of the present study it can be inferred that TOPBP1, NUSAP1 and CDCA3 of the core modules may serve notable functions in SM-associated growth suppression of HCC.

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.

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.

Simultaneous analysis of amoxicillin and sulbactam in human plasma by HPLC-DAD for assessment of bioequivalence.

A simple and accurate high-performance liquid chromatography with diode array detection-based (HPLC-DAD) method has been developed and validated for simultaneous determination of amoxicillin and sulbactam in human plasma. Sample preparation was involved in protein precipitation with acetonitrile followed by one-step extraction procedure. Chromatographic separation was achieved on a C18 column with an isocratic mobile phase consisting of water (containing 30 mM potassium dihydrogen phosphate, pH 2.8) and acetonitrile. The detection wavelengths of a diode array detector were set at 210 nm for amoxicillin and sulbactam, and 263 nm for the internal standard (cefadroxil). The method was validated for linearity, accuracy, precision, and stability. The calibration curve was linear from 0.163 to 14.7 µg/mL with correlation coefficient squared of 0.9991 for amoxicillin and 0.250-15.0 µg/mL with correlation coefficient squared of 0.9988 for sulbactam using 500 µL plasma samples. The lower limit of quantification was 0.163 and 0.250 µg/mL for amoxicillin and sulbactam, respectively. The imprecisions of intra- and inter-day validations for amoxicillin and sulbactam were <11% and their accuracies (%) were within the range of 95.4-105.7%. Mean recoveries were 75.9, 72.8, and 70.0% for amoxicillin, sulbactam, and cefadroxil, respectively. The established method was successfully applied to a bioequivalence study of two combination formulations of amoxicillin and sulbactam pivoxil in healthy male volunteers.