Recombination of variable and host range regions of glycoprotein gp85 in different avian leukosis virus subgroup K isolates.

Given the high prevalence of avian leukosis virus subgroup K (ALV-K) in chickens in China, the positive rate of ALV-K in local chickens in Henan province was investigated, and the genetic region encoding the glycoprotein gp85 of isolates from positive chickens was analyzed. The positive rate of ALV-K in local chickens in Henan was found to be 87.2% (41/47). Phylogenetic analysis of gp85 sequences revealed six clusters that differed in their host range regions (hr1 and hr2) and variable regions (vr1, vr2, and vr3). Evidence of recombination of hr1, hr2, vr1, vr2, and vr3 was observed between the different clusters. The isolate HN23LS02 appears to have obtained its hr1 and hr2 regions from separate lineages via recombination but without having a significant affect on the replication capacity of the virus.

Unveiling a Novel Antidote for Deoxynivalenol Contamination: Isolation, Identification, Whole Genome Analysis and In Vivo Safety Evaluation of Lactobacillus rhamnosus MY-1.

Deoxynivalenol (DON) is a global contaminant found in crop residues, grains, feed, and animal and human food. Biodegradation is currently the best solution for addressing DON pollution. However, efficient detoxification bacteria or enzymes that can be applied in complex matrices are lacking. The aim of this study was to isolate a DON-detoxifying probiotic strain with a high degradation rate, a good safety profile, and a clear genetic background. One hundred and eight bacterial strains were isolated from 300 samples collected from a school farm and surrounding livestock farms. A new DON-degrading strain, Lactobacillus rhamnosus MY-1 (L. rhamnosus MY-1), with a degradation rate of 93.34% after 48 h and a comprehensive degradation method, was identified. Then, MY-1 at a concentration of 1 × 108 CFU/mL was administered to mice in a chronic intoxication experiment for 28 days. The experimental group showed significantly higher weight gain and exhibited good production performance compared to the control group. The length of the ileal villi in the experimental group was significantly longer than that in the control group. The expression of pro-inflammatory cytokines decreased, while the expression of anti-inflammatory factors increased in the experimental group. Whole-genome analysis revealed that most of the MY-1 genes were involved in carbohydrate metabolism and membrane transport, with a cluster of secondary metabolite genes encoding antimicrobial properties. In summary, this study successfully identified a Lactobacillus strain with good safety performance, high DON degradation efficiency, and a clear genetic background, providing a new approach for the treatment of DON contamination.

Global distribution, cross-species transmission, and receptor binding of canine parvovirus-2: Risks and implications for humans.

For canine parvovirus -2 (CPV-2), a zoonotic virus capable of cross-species transmission in animals, the amino acid changes of capsid protein VP2 are key factors when binding to other species' transferrin receptors (TfR). CPV-2 variants can spread from felines and canines, for example, to Carnivora, Artiodactyla, and Pholidota species, and CPV-2c variants are essential to spread from Carnivora to Artiodactyla and Pholidota species in particular. In our study, a CPV-2a variant maintained a relatively stable trend, and the proportion of CPV-2c gradually rose from 1980 to 2021. The VP2 amino acid sequence analysis showed that five amino acid mutations at 426E/D, 305H/D, and 297S may be necessary for the virus to bind to different host receptors. Meanwhile, receptor-binding loop regions and amino acid sites 87 L, 93 N, 232I, and 305Y were associated with CPV-2 cross-species transmission. The homology of TfRs in different hosts infected with CPV-2 ranged from 77.2 % to 99.0 %, and from pig to feline, canine, and humans was 80.7 %, 80.4 %, and 77.2 %, respectively. The amino acid residues of TfRs involved in the viral binding in those hosts are highly conserved, which suggests that CPV-2 may be capable of pig-to-human transmission. Our analysis of the origin, evolutionary trend, cross-species transmission dynamics, and genetic characteristics of CPV-2 when binding to host receptors provides a theoretical basis for further research on CPV-2's mechanism of cross-species transmission and for establishing an early warning and monitoring mechanism for the possible threat of CPV-2 to animal-human public security.

A Novel Bacillus Velezensis for Efficient Degradation of Zearalenone.

Zearalenone (ZEN) is considered one of the most serious mycotoxins contaminating grains and their by-products, causing significant economic losses in the feed and food industries. Biodegradation pathways are currently considered the most efficient solution to remove ZEN contamination from foods. However, low degradation rates and vulnerability to environmental impacts limit the application of biodegradation pathways. Therefore, the main research objective of this article was to screen strains that can efficiently degrade ZEN and survive under harsh conditions. This study successfully isolated a new strain L9 which can efficiently degrade ZEN from 108 food ingredients. The results of sequence alignment showed that L9 is Bacillus velezensis. Meanwhile, we found that the L9 degradation rate reached 91.14% at 24 h and confirmed that the primary degradation mechanism of this strain is biodegradation. The strain exhibits resistance to high temperature, acid, and 0.3% bile salts. The results of whole-genome sequencing analysis showed that, it is possible that the strain encodes the key enzyme, such as chitinase, carboxylesterases, and lactone hydrolase, that work together to degrade ZEN. In addition, 227 unique genes in this strain are primarily involved in its replication, recombination, repair, and protective mechanisms. In summary, we successfully excavated a ZEN-degrading, genetically distinct strain of Bacillus velezensis that provides a solid foundation for the detoxification of feed and food contamination in the natural environment.

A Bacillus subtilis Strain ZJ20 with AFB1 Detoxification Ability: A Comprehensive Analysis.

As a class I carcinogen, aflatoxin can cause serious damage to various tissues and organs through oxidative stress injuries. The liver, as the target organ of AFB1, is the most seriously damaged. Biological methods are commonly used to degrade AFB1. In our study, the aflatoxin B1-degrading strain ZJ20 was screened from AFB1-contaminated feed and soil, and the degradation of AFB1 by ZJ20 was investigated. The whole genome of strain ZJ20 was analyzed, revealing the genomic complexity of strain ZJ20. The 16S rRNA analysis of strain ZJ20 showed 100% identity to Bacillus subtilis IAM 12118. Through whole gene functional annotation, it was determined that ZJ20 has high antioxidant activity and enzymatic activity; more than 100 CAZymes and 11 gene clusters are involved in the production of secondary metabolites with antimicrobial properties. In addition, B. subtilis ZJ20 was predicted to contain a cluster of genes encoding AFB1-degrading enzymes, including chitinase, laccase, lactonase, and manganese oxidase. The comprehensive analysis of B. subtilis provides a theoretical basis for the subsequent development of the biological functions of ZJ20 and the combinatorial enzyme degradation of AFB1.

Rapid construction of infectious clones for distinct Newcastle disease virus genotypes.

The reverse genetics system of the Newcastle disease virus (NDV) has provided investigators with a powerful approach to understand viral molecular biology and vaccine development. It has been impressively improved with modified strategies since its first report, but it still poses some challenges. Most noteworthy, the genome complexity and length made full-length error-free cDNA assembly the most challenging and time-consuming step of NDV rescue. In the present study, we report a rapid full-length NDV genome construction with only a two-step ligation-independent cloning (LIC) strategy, which could be applied to distinct genotypes. In this approach, the genome of NDV was divided into two segments, and the cDNA clones were generated by RT-PCR followed by LIC. Subsequently, the infectious NDVs were rescued by co-transfection of the full-length cDNA clones and supporting plasmids expressing the NP, P, and L proteins of NDV in BHK-21 cells. Compared with the conventional cloning approaches, the two-step cloning method drastically reduced the number of cloning steps and saved researchers a substantial amount of time for constructing NDV infectious clones, thus enabling a rapid rescue of different genotypes of NDVs in a matter of weeks. Therefore, this two-step LIC cloning strategy may have an application to the rapid development of NDV-vectored vaccines against emerging animal diseases and the generation of different genotypes of recombinant NDVs for cancer therapy.

Chloroquine Inhibition of Autophagy Enhanced the Anticancer Effects of Listeria monocytogenes in Melanoma.

Listeria monocytogenes has been shown to exhibit antitumor effects. However, the mechanism remains unclear. Autophagy is a cellular catabolic process that mediates the degradation of unfolded proteins and damaged organelles in the cytosol, which is a double-edged sword in tumorigenesis and treatment outcome. Tumor cells display lower levels of basal autophagic activity than normal cells. This study examined the role and molecular mechanism of autophagy in the antitumor effects induced by LM, as well as the combined antitumor effect of LM and the autophagy inhibitor chloroquine (CQ). We investigated LM-induced autophagy in B16F10 melanoma cells by real-time PCR, immunofluorescence, Western blotting, and transmission electron microscopy and found that autophagic markers were increased following the infection of tumor cells with LM. The autophagy pathway in B16F10 cells was blocked with the pharmacological autophagy inhibitor chloroquine, which led to a significant increase in intracellular bacterial multiplication in tumor cells. The combination of CQ and LM enhanced LM-mediated cancer cell death and apoptosis compared with LM infection alone. Furthermore, the combination of LM and CQ significantly inhibited tumor growth and prolonged the survival time of mice in vivo, which was associated with the increased colonization and accumulation of LM and induced more cell apoptosis in primary tumors. The data indicated that the inhibition of autophagy by CQ enhanced LM-mediated antitumor activity in vitro and in vivo and provided a novel strategy to improving the anticancer efficacy of bacterial treatment.

5'-Nucleotidase is dispensable for the growth of Salmonella Typhimurium but inhibits the bactericidal activity of macrophage extracellular traps.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a zoonotic pathogen that causes severe gastroenteritis. The 5'-nucleotidases of pathogens can dephosphorylate adenosine phosphates, boost adenosine levels and suppress the pro-inflammatory immune response. In our previous study, an extracellular nuclease, 5'-nucleotidase, was identified in the extracellular proteins of S. Typhimurium. However, the nuclease activity and the function of the 5'-nucleotidase of S. Typhimurium have not been explored. In the present study, deletion of the 5'-nucleotidase gene is dispensable for S. Typhimurium growth, even under environmental stress. Fluorescence microscopy revealed that the 5'-nucleotidase mutant induced more macrophage extracellular traps (METs) than the wild type did. Furthermore, recombinant 5'-nucleotidase protein (r5Nuc) could degrade λDNA, and the nuclease activity of r5Nuc was optimum at 37 °C and pH 6.0-7.0. The Mg2+ enhanced the nuclease activity of r5Nuc, whereas Zn2+ inhibited it. Meanwhile, deletion of the 5'-nucleotidase gene increased the bactericidal activity of METs, and r5Nuc could degrade METs and inhibit the bactericidal activity of METs. In conclusion, S. Typhimurium growth was independent of 5'-nucleotidase, but the nuclease activity of 5'-nucleotidase assisted S. Typhimurium to evade macrophage-mediated extracellular killing through degrading METs.

Probiotic Properties of Chicken-Derived Highly Adherent Lactic Acid Bacteria and Inhibition of Enteropathogenic Bacteria in Caco-2 Cells.

Lactic acid bacteria (LAB) as probiotic candidates have various beneficial functions, such as regulating gut microbiota, inhibiting intestinal pathogens, and improving gut immunity. The colonization of the intestine is a prerequisite for probiotic function. Therefore, it is necessary to screen the highly adherent LAB. In this study, the cell surface properties, such as hydrophobicity, auto-aggregation, co-aggregation, and adhesion abilities of the six chicken-derived LAB to Caco-2 cells were investigated. All six strains showed different hydrophobicity (21.18-95.27%), auto-aggregation (13.61-30.17%), co-aggregation with Escherichia coli ATCC 25922 (10.23-36.23%), and Salmonella enterica subsp. enterica serovar Typhimurium ATCC 13311 (11.71-39.35%), and adhesion to Caco-2 cells (8.57-26.37%). Pediococcus pentosaceus 2-5 and Lactobacillus reuteri L-3 were identified as the strains with strong adhesion abilities (26.37% and 21.57%, respectively). Moreover, these strains could survive in a gastric acid environment at pH 2, 3, and 4 for 3 h and in a bile salt environment at 0.1%, 0.2%, and 0.3% (w/v) concentration for 6 h. Furthermore, the cell-free supernatant of P. pentosaceus 2-5 and L. reuteri L-3 inhibited the growth of enteropathogenic bacteria and the strains inhibited the adhesion of these pathogens to Caco-2 cells. In this study, these results suggested that P. pentosaceus 2-5 and L. reuteri L-3, isolated from chicken intestines might be good probiotic candidates to be used as feed additives or delivery vehicles of biologically active substances.

Rescue of an enterotropic Newcastle disease virus strain ZM10 from cloned cDNA and stable expressing an inserted foreign gene.

BACKGROUND: Newcastle disease virus (NDV) strain ZM10, a typical enterotropic avirulent vaccine strain, has been widely used in China for chickens against Newcastle disease. To elucidate its enterotropic mechanism and develop recombiant multivalent vaccines based on it, the reverse genetics system for NDV ZM10 is an indispensable platform. RESULTS: A full-length cDNA clone of NDV ZM10 and three supporting plasmids were constructed using the ligation-independent cloning method. Recombinant NDV rZM10 was successfully rescued after these plasmids were co-transfected into BHK-21 cells. Besides, the recombinant virus rZM10-RFP encoding the red fluorescent protein was generated by inserting the RFP gene into the full-length clone of NDV between the P and M genes. These rescued viruses were genetically and biologically identical to the parental strain and showed similar growth kinetics. CONCLUSION: The recovery system of NDV ZM10 strain was established, and can be used as a foundation for research on the enterotropic mechanism and development of multivalent vaccines against viral diseases of livestock and poultry.

Research Note: Anti-inflammatory effects and antiviral activities of baicalein and chlorogenic acid against infectious bursal disease virus in embryonic eggs.

The purpose of this study was to investigate if baicalein and chlorogenic acid could inhibit the inflammatory responses induced by and protect against infectious bursal disease virus (IBDV) in chicken embryonic eggs. Nine-day-old embryonated chicken eggs were randomly divided into 3 groups of 50 eggs per group: 1) treatment with varying concentrations of baicalein, 2) treatment with varying concentrations of chlorogenic acid, or 3) left untreated as a control. Forty-eight hours after hatching, each group was inoculated with a very virulent IBDV isolate, and the survival of the embryo was monitored daily until the embryonic livers were collected 72 h after inoculation. After IBDV infection, the viral loads in the embryonic livers were evaluated using qRT-PCR, and the hepatic content of inflammatory mediators, such as histamine, interleukin 1ß (IL-1ß), tumor necrosis factor alpha (TNF-α), and nuclear factor-kappa B (NF-κB), were examined. Significant antiviral potential was demonstrated at concentrations of 108 and 215 µg/egg of baicalein and chlorogenic acid, respectively. We observed a concentration-dependent response in the antiviral properties of these chemicals. Treating the embryos with baicalein and chlorogenic acid significantly reduced histamine production. Moreover, pretreatment with baicalein and chlorogenic acid significantly inhibited NF-κB activation, and this inhibited the subsequent production of the proinflammatory cytokines TNF-α and IL-1ß in the context of IBDV infection. These findings suggest that baicalein and chlorogenic acid have anti-IBDV properties, and they may be useful in the prevention of inflammation-related diseases.

Nuclear Receptor 4A2 (NR4A2/NURR1) Regulates Autophagy and Chemoresistance in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive cancer with poor prognosis and chemotherapy with gemcitabine has limited effects and is associated with development of drug resistance. Treatment of Panc1 and MiaPaca2 pancreatic cancer cells with gemcitabine induced expression of the orphan nuclear receptor 4A2 (NURR1) and analysis of the cancer genome atlas indicated the NURR1 is overexpressed in pancreatic tumors and is a negative prognostic factor for patient survival. Results of NURR1 knockdown or treatment with the NURR1 antagonist 1,1-bis(3΄-indolyl)-1-(p-chlorophenyl)methane (C-DIM 12) demonstrated that NURR1 was pro-oncogenic in pancreatic cancer cells and regulated cancer cell and tumor growth and survival. NURR1 is induced by gemcitabine and serves as a key drug-resistance factor and is also required for gemcitabine-induced cytoprotective autophagy. NURR1 regulated genes were determined by RNA sequencing of mRNAs expressed in MiaPaCa2 cells expressing NURR1 and in CRISPR/Cas9 gene edited cells for NURR1 knockdown and KEGG enrichment analysis of the differentially expressed genes showed that autophagy was the major pathway regulated by NURR1. Moreover, NURR1 regulated expression of two major autophagic genes ATG7 and ATG12 which are also overexpressed in pancreatic tumors and like NURR1 are negative prognostic factors for patient survival. Thus, gemcitabine-induced cytoprotective autophagy is due to the NURR1 - ATG7/ATG12 axis and this can be targeted and disrupted by NURR1 antagonist C-DIM12 demonstrating the potential clinical applications for combination therapies with gemcitabine and NURR1 antagonists.

Identification and Characterization of the Nuclease Activity of the Extracellular Proteins from Salmonella enterica Serovar Typhimurium.

Pathogens have evolved an array of strategies to establish a productive infection. The extracellular proteins secreted by pathogens are one of unique mechanisms to evade the host innate immune response. Many secretory proteins transported by the bacterial secretion systems have been widely investigated in Salmonella. Certain extracellular nucleases are essential for bacterial pathogenesis. However, there is no current data available for the enzymatic properties of the proteins secreted by Salmonella. Therefore, in the present study we have identified and characterized the nuclease activity of the extracellular proteins from Salmonella enterica serovar Typhimurium. It was demonstrated that the extracellular proteins from S. Typhimurium exhibited the deoxyribonucleases activity against λDNA by agarose gel electrophoresis and agar plate diffusion method. The activity was observed at 16 °C, 37 °C and 42 °C, and found to be highest at 42 °C and inhibited at temperatures over 60 °C. The nuclease activity was stable under alkaline conditions (pH 7-10) and the optimum pH was 9.0. The nuclease activity was promoted at high ionic strength of Ba2+, Ca2+, Mg2+, and Ni2+. Nuclease zymography analysis revealed that there were four activity bands in the extracellular proteins; followed by LC-ESI/MS/MS analysis seven proteins were identified. As demonstrated by nuclease zymography, the recombinant 5'-nucleotidase protein expressed in the prokaryotic expression system displayed the DNase activity. To our knowledge, the present findings represent the first direct and unambiguous demonstration of the nuclease activity of the extracellular proteins from S. Typhimurium, and it provides an important fundamental for further investigation of the role of the extracellular proteins in pathogenicity and immune evasion.

Effect of gga-miR-155 on cell proliferation, apoptosis and invasion of Marek's disease virus (MDV) transformed cell line MSB1 by targeting RORA.

BACKGROUND: Marek's disease (MD) is caused by the oncogenic Marek's disease virus (MDV), and is a highly contagious avian infection with a complex underlying pathology that involves lymphoproliferative neoplasm formation. MicroRNAs (miRNAs) act as oncogenes or tumor suppressors in most cancers. The gga-miR-155 is downregulated in the MDV-infected chicken tissues or lymphocyte lines, although its exact role in tumorigenesis remains unclear. The aim of this study was to analyze the effects of gga-miR-155 on the proliferation, apoptosis and invasiveness of an MDV-transformed lymphocyte line MSB1 and elucidate the underlying mechanisms. RESULTS: The expression level of gga-miR-155 was manipulated in MSB1 cells using specific mimics and inhibitors. While overexpression of gga-miR-155 increased proliferation, decreased the proportion of G1 phase cells relative to that in S and G2 phases, reduced apoptosis rates and increased invasiveness. However, its downregulation had the opposite effects. Furthermore, gga-miR-155 directly targeted the RORA gene and downregulated its expression in the MSB1 cells. CONCLUSION: The gga-miR-155 promotes the proliferation and invasiveness of the MDV-transformed lymphocyte line MSB1 and inhibits apoptosis by targeting the RORA gene.

The impact of sseK2 deletion on Salmonella enterica serovar typhimurium virulence in vivo and in vitro.

BACKGROUND: Salmonella enterica is regarded as a major public health threat worldwide. Salmonella secretes the novel translocated effector protein K2 (SseK2), but it is unclear whether this protein plays a significant role in Salmonella enterica Typhimurium virulence. RESULTS: A ΔsseK2 mutant of S. Typhimurium exhibited similar growth curves, adhesion and invasive ability compared with wild-type (WT) bacteria. However, deletion of sseK2 rendered Salmonella deficient in biofilm formation and the early proliferative capacity of the ΔsseK2 mutant was significantly lower than that of the WT strain. In vivo, the LD50 (median lethal dose) of the ΔsseK2 mutant strain was increased 1.62 × 103-fold compared with the WT strain. In addition, vaccinating mice with the ΔsseK2 mutant protected them against challenge with a lethal dose of the WT strain. The ability of the ΔsseK2 mutant strain to induce systemic infection was highly attenuated compared with the WT strain, and the bacterial load in the animals' internal organs was lower when they were infected with the ΔsseK2 mutant strain than when they were infected with the WT strain. CONCLUSIONS: We conclude that sseK2 is a virulence-associated gene that plays a vital role in Salmonella virulence.

cAMP Receptor Protein of Salmonella enterica Serovar Typhimurium Modulate Glycolysis in Macrophages to Induce Cell Apoptosis.

We studied the role of glycolysis in the mechanism of cAMP receptor protein-induced macrophage cell death of Salmonella enterica serovar Typhimurium (S. Typhimurium). Cell apoptosis, caspase-3, -8, -9 enzyme activity, and pyruvic acid, lactic acid, ATP, and hexokinase (HK) contents were determined after infection of macrophages with S. Typhimurium SL1344 wild-type and a cAMP receptor protein mutant strain. While cell apoptosis, caspase-3, -8, -9 enzyme activity, lactic acid, hexokinase, and ATP levels significantly changed by infection with crp mutants compared to the wild-type strain (P < 0.05). Our data suggest that the cAMP receptor protein of S. Typhimurium can modulate macrophage death by effecting glycolysis levels. This finding may help to elucidate the mechanisms of S. Typhimurium pathogenesis.

Recombinant-attenuated Salmonella Pullorum strain expressing the hemagglutinin-neuraminidase protein of Newcastle disease virus (NDV) protects chickens against NDV and Salmonella Pullorum challenge.

Newcastle disease virus (NDV) and Salmonella Pullorum have significant damaging effects on the poultry industry, but no previous vaccine can protect poultry effectively. In this study, a recombinant-attenuated S. Pullorum strain secreting the NDV hemagglutinin-neuraminidase (HN) protein, C79-13ΔcrpΔasd (pYA-HN), was constructed by using the suicide plasmid pREasd-mediated bacteria homologous recombination method to form a new bivalent vaccine candidate against Newcastle disease (ND) and S. Pullorum disease (PD). The effect of this vaccine candidate was compared with those of the NDV LaSota and C79-13ΔcrpΔasd (pYA) strains. The serum hemagglutination inhibition antibody titers, serum immunoglobulin G (IgG) antibodies, secretory IgA, and stimulation index in lymphocyte proliferation were increased significantly more (p < 0.01) in chickens inoculated with C79-13ΔcrpΔasd (pYA-HN) than with C79-13ΔcrpΔasd (pYA) but were not significantly increased compared with the chickens immunized with the LaSota live vaccine (p > 0.05). Moreover, the novel strain provides 60% and 80% protective efficacy against the NDV virulent strain F48E9 and the S. Pullorum virulent strain C79-13. In summary, in this study, a recombinant-attenuated S. Pullorum strain secreting NDV HN protein was constructed. The generation of the S. Pullorum C79-13ΔcrpΔasd (pYA-HN) strain provides a foundation for the development of an effective living-vector double vaccine against ND and PD.

Putative roles as oncogene or tumour suppressor of the Mid-clustered microRNAs in Gallid alphaherpesvirus 2 (GaHV2) induced Marek's disease lymphomagenesis.

In the last decade, numerous microRNAs (miRNAs) have been identified in diverse virus families, particularly in herpesviruses. Gallid alphaherpesvirus 2 (GaHV2) is a representative oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in its natural hosts, namely Marek's disease (MD). In the GaHV2 genome there are 26 mature miRNAs derived from 14 precursors assembled into three clusters, namely the Meq-cluster, Mid-cluster and LAT-cluster. Several GaHV2 miRNAs, especially those in the Meq-cluster (e.g. miR-M4-5p), have been demonstrated to be critical in MD pathogenesis and/or tumorigenesis. Interestingly the downstream Mid-cluster is regulated and transcribed by the same promoter as the Meq-cluster in the latent phase of the infection, but the role of these Mid-clustered miRNAs in GaHV2 biology remains unclear. We have generated the deletion mutants of the Mid-cluster and of its associated individual miRNAs in GX0101 virus, a very virulent GaHV2 strain, and demonstrated that the Mid-clustered miRNAs are not essential for virus replication. Using GaHV2-infected chickens as an animal model, we found that, compared with parental GX0101 virus, the individual deletion of miR-M31 decreased the mortality and gross tumour incidence of infected chickens while the deletion individually of miR-M1 or miR-M11 unexpectedly increased viral pathogenicity or oncogenicity, similarly to the deletion of the entire Mid-cluster region. More importantly, our data further confirm that miR-M11-5p, the miR-M11-derived mature miRNA, targets the viral oncogene meq and suppresses its expression in GaHV2 infection. We report here that members of the Mid-clustered miRNAs, miR-M31-3p and miR-M11-5p, potentially act either as oncogene or tumour suppressor in MD lymphomagenesis.

Occurrence of canine parvovirus in dogs from Henan province of China in 2009-2014.

BACKGROUND: There is no information concerning the genotype of Canine parvovirus (CPV) currently circulating in Henan province, China. Therefore, the aim of the present study was to provide insights into the epidemiology and molecular characterization of CPV circulating in Henan province from 2009 to 2014. RESULTS: Nineteen thousand nine hundred seven dogs from pet hospitals in the cities of Luoyang, Anyang, Jiaozuo, Sanmenxia, Xinxiang, Zhengzhou in Henan province between 2009 and 2014 were investigated. Over the 6-year period, 1169 CPV-positive cases were identified and the morbidity of CPV infection ranged from 4.16 to 8.06 %, although morbidity was not significant (P > 0.05) between 2009 and 2014. Factors associated with morbidity included sampling season, dog age, breed, vaccination status, and sex. CPV co-infection with coccidium (10.00 %), canine distemper virus (4.79 %), hookworm (2.40 %), canine coronavirus (1.11 %), roundworm (1.03 %), tapeworm (0.17 %) and Babesia spp. (0.09 %) were observed. The new CPV-2a variant was more prevalent than the new CPV-2b variant in Henan province. CPV 2c was not observed in this study. CONCLUSIONS: The epidemiology of CPV infection and identification of the circulating genotypes in Henan province, China from 2009 to 2014 determined that the new CPV-2a variant was more prevalent.

[Construction and characterization of type III secretion system of attenuated Salmonella typhimurium].

In order to develop a recombinant attenuated Salmonella typhimurium as oral live vaccine vector, we constructed recombinant plasmid pYA-sopENt100 by replacing the trc promoter with the sopE promoter and secretion signal sequence sopENt100 of Salmonella typhimurium on the basis of plasmid pYA3493. Then, the complementary plasmid pYA-sopENt100 was transformed into ΔcrpΔasdSL1344 by electroporation to generate attenuated Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100). We further characterized ΔcrpΔasdSL1344 (pYA-sopENt100). We also constructed a recombinant strain ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) that harbored the reporter gene-enhanced green fluorescent protein (egfp) gene. Vero cells were infected with ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) and the ability of delivery foreign antigens was tested via Western blotting analysis. The results of PCR, enzyme digestion and sequencing showed that the ΔcrpΔasdSL1344 (pYA-sopENt100) type III secretion system was constructed successfully. The serotype of ΔcrpΔasdSL1344 (pYA-sopENt100) was identical to ΔcrpΔasdSL1344 and SL1344. Compared with wild strain SL1344, the biochemical characteristics of ΔcrpΔasdSL1344 (pYA-sopENt100) had obvious change, but it was basically the same with ΔcrpΔasdSL1344. The growth speed was much slower than that of the wild strain SL1344. The chicken virulence test (LD50) showed that the virulence of ΔcrpΔasdSL1344 (pYA-sopENt100) was 7×104 times lower than SL1344. In addition, we observed the 37 kDa SopENt100-egfp protein in the cultured supernatant of ΔcrpΔasdSL1344 (pYA-sopENt100-egfp) strain by Western blotting analysis. However, both the 37 kDa SopENt100-egfp protein and 27 kDa EGFP protein were detected in ΔcrpΔasdSL1344 (pYA-sopENt100-egfp)-infected Vero cells. These results demonstrated that the recombinant Salmonella typhimurium type III secretion system ΔcrpΔasdSL1344 (pYA-sopENt100) was successfully constructed, and it should be used as a live vaccine vector for expressing foreign genes.