Identification and genomic analyses of a novel endophytic actinobacterium Streptomyces endophytica sp. nov. with potential for biocontrol of yam anthracnose.

Anthracnose disease caused by Colletotrichum gloeosporioides is one of the devastating diseases of yams (Dioscorea sp.) worldwide. In this study, we aimed to isolate endophytic actinobacteria from yam plants and to evaluate their potential for the control of yam anthracnose based on bioassays and genomic analyses. A total of 116 endophytic actinomycete strains were isolated from the surface-sterilized yam tissues from a yam orchard in Hainan Province, China. In total, 23 isolates showed antagonistic activity against C. gloeosporioides. An endophytic actinomycete, designated HNM0140T, which exhibited strong antifungal activities, multiple biocontrol, and plant growth-promoting (PGP) traits was subsequently selected to colonize in the tissue-cultured seedlings of yam and was tested for its in vivo biocontrol potential on yam anthracnose. The results showed that treatment with strain HNM0140T markedly reduced the severity and incidence of yam anthracnose under greenhouse conditions. Morphological and chemotaxonomic analyses showed that strain HNM0140T was assigned to the genus Streptomyces. Phylogenetic analysis based on the 16S rRNA gene sequences indicated that strain HNM0140T formed a separate cluster together with Streptomyces lydicus ATCC 25470T (99.45%), Streptomyces chattanoogensis NRRL ISP-5002T (99.45%), and Streptomyces kronopolitis NEAU-ML8T (98.97%). The phylogenomic tree also showed that strain HNM0140T stably clustered with Streptomyces lydicus ATCC 25470T. The ANI and dDDH between strain HNM0140T and its closest related-type species were well below the recommended thresholds for species demarcation. Hence, based on the phylogenetic, genomic, and phenotypic analyses, strain HNM0140T should represent a new streptomycete species named Streptomyces endophytica sp. nov. Genomic analysis revealed that strain HNM0140T harbored 18 putative BGCs for secondary metabolites, some PGP-related genes, and several genes coding for antifungal enzymes. The presented results indicated that strain HNM0140T was a promising biocontrol agent for yam anthracnose.

Complete genome sequence of Streptomyces malaysiensis HNM0561, a marine sponge-associated actinomycete producing malaymycin and mccrearamycin E.

Streptomyces malaysiensis HNM0561 is a marine sponge-associated actinomycete with the potential to produce potential anti-androgens against prostate cancer cells, including malaymycin and mccrearamycin E. Here, we present the complete genome sequence of S. malaysiensis HNM0561, which consists of a linear chromosome of 11,656,895 bp and a circular plasmid of 32,797 bp, 9849 protein coding genes, 18 rRNA genes, 66 tRNA genes, and 191 sRNA genes. Genomic annotations revealed that 72.03% of the protein-coding genes were assigned to the COG database, among which the abundant genes were predicted to be involved in transcription, replication, carbohydrate transport and metabolism, and amino acid transport and metabolism. Forty-nine putative secondary metabolite biosynthetic gene clusters were found in the genome. Among them, the potential biosynthetic gene clusters of malaymycin and mccrearamycin E have been described respectively. The complete genome information presented here will enable us to investigate the biosynthetic mechanism of two novel structures of malaymycin and mccrearamycin E and to discover novel secondary metabolites with potential against prostate cancer cell activities.

Reticuloendotheliosis virus and avian leukosis virus subgroup J synergistically increase the accumulation of exosomal miRNAs.

BACKGROUND: Co-infection with avian leukosis virus subgroup J and reticuloendotheliosis virus induces synergistic pathogenic effects and increases mortality. However, the role of exosomal miRNAs in the molecular mechanism of the synergistic infection of the two viruses remains unknown. RESULTS: In this study, exosomal RNAs from CEF cells infected with ALV-J, REV or both at the optimal synergistic infection time were analysed by Illumina RNA deep sequencing. A total of 54 (23 upregulated and 31 downregulated) and 16 (7 upregulated and 9 downregulated) miRNAs were identified by comparing co-infection with two viruses, single-infected ALV-J and REV, respectively. Moreover, five key miRNAs, including miR-184-3p, miR-146a-3p, miR-146a-5p, miR-3538 and miR-155, were validated in both exosomes and CEF cells by qRT-PCR. GO annotation and KEGG pathway analysis of the miRNA target genes showed that the five differentially expressed miRNAs participated in virus-vector interaction, oxidative phosphorylation, energy metabolism and cell growth. CONCLUSIONS: We demonstrated that REV and ALV-J synergistically increased the accumulation of exosomal miRNAs, which sheds light on the synergistic molecular mechanism of ALV-J and REV.

Emergence of gyrovirus 3 in commercial broiler chickens with transmissible viral proventriculitis.

Gyrovirus 3 (GyV3) has been identified in faeces from children with acute gastroenteritis. However, whether GyV3 is prevalent in poultry has not been determined to date. To the best of our knowledge, this study is the first to isolate GyV3 from commercial broiler chickens with transmissible viral proventriculitis (TVP) in China. The complete genome of the virus shares 98.4% sequence identity with the FecGy strain that causes acute gastroenteritis in children. Epidemiological investigation from 2013 to 2017 revealed that the infection rate of GyV3 reached 12.5% (42/336) in commercial broiler chickens with TVP, indicating that the infection of GyV3 was ubiquitous in chickens. The emergence of GyV3 in commercial broiler chickens should be highly concerning for public health.

Synergistic Viral Replication of Marek's Disease Virus and Avian Leukosis Virus Subgroup J is Responsible for the Enhanced Pathogenicity in the Superinfection of Chickens.

Superinfection of Marek's disease virus (MDV) and avian leukosis virus subgroup J (ALV-J) causes lethal neoplasia and death in chickens. However, whether there is synergism between the two viruses in viral replication and pathogenicity has remained elusive. In this study, we found that the superinfection of MDV and ALV-J increased the viral replication of the two viruses in RNA and protein level, and synergistically promoted the expression of IL-10, IL-6, and TGF-ß in chicken embryo fibroblasts (CEF). Moreover, MDV and ALV-J protein expression in dual-infected cells detected by confocal laser scanning microscope appeared earlier in the cytoplasm and the nucleus, and caused more severe cytopathy than single infection, suggesting that synergistically increased MDV and ALV-J viral-protein biosynthesis is responsible for the severe cytopathy. In vivo, compared to the single virus infected chickens, the mortality and tumor formation rates increased significantly in MDV and ALV-J dual-infected chickens. Viral loads of MDV and ALV-J in tissues of dual-infected chickens were significantly higher than those of single-infected chickens. Histopathology observation showed that more severe inflammation and tumor cells metastases were present in dual-infected chickens. In the present study, we concluded that synergistic viral replication of MDV and ALV-J is responsible for the enhanced pathogenicity in superinfection of chickens.

Emergence of pathogenic and multiple-antibiotic-resistant Macrococcus caseolyticus in commercial broiler chickens.

Macrococcus caseolyticus is generally considered to be a non-pathogenic bacterium that does not cause human or animal diseases. However, recently, a strain of M. caseolyticus (SDLY strain) that causes high mortality rates was isolated from commercial broiler chickens in China. The main pathological changes caused by SDLY included caseous exudation in cranial cavities, inflammatory infiltration, haemorrhages and multifocal necrosis in various organs. The whole genome of the SDLY strain was sequenced and was compared with that of the non-pathogenic JCSC5402 strain of M. caseolyticus. The results showed that the SDLY strain harboured a large quantity of mutations, antibiotic resistance genes and numerous insertions and deletions of virulence genes. In particular, among the inserted genes, there is a cluster of eight connected genes associated with the synthesis of capsular polysaccharide. This cluster encodes a transferase and capsular polysaccharide synthase, promotes the formation of capsules and causes changes in pathogenicity. Electron microscopy revealed a distinct capsule surrounding the SDLY strain. The pathogenicity test showed that the SDLY strain could cause significant clinical symptoms and pathological changes in both SPF chickens and mice. In addition, these clinical symptoms and pathological changes were the same as those observed in field cases. Furthermore, the anti-microbial susceptibility test demonstrated that the SDLY strain exhibits multiple-antibiotic resistance. The emergence of pathogenic M. caseolyticus indicates that more attention should be paid to the effects of this micro-organism on both poultry and public health.

A polyclonal antibody against extracellular loops 1 of chNHE1 blocks avian leukosis virus subgroup J infection.

Avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, induces myelocytomas and other various tumors, leading to great economical losses in poultry industry. It is a great challenge to develop effective preventive methods for ALV-J control due to its antigenic variations in the variable regions of envelope. In present study, we generated a mouse polyclonal antibody targeting the first extracellular loop (ECL1) of chicken Na+/H+ exchanger isoform 1 (chNHE1), the receptor of ALV-J, to block ALV-J infection in vitro and in vivo. In ALV-J infected DF-1 cells, chNHE1 expression and the intracellular pH (pHi) were up-regulated with "wave" pattern, indicating that the disequilibrium of ALV-J infected cells associated with chNHE1. Next, we validated that ALV-J infection was significantly blocked with time dependent after treating with anti-ECL1 antibody and accordingly the pHi value were recovered, indicating the blockage of ALV-J infection did not affect Na+/H+ exchange. Furthermore, in anti-ECL1 antibody treatment chickens that infected by ALV-J, weight gain and immune organs were recovered, and viral loads were significantly decreased, and the tissue injury and inflammation were reduced significantly from 21 to 35 days of age. The study demonstrated that anti-ECL1 antibody effectively blocks ALV-J infection without affecting Na+/H+ exchange, and sheds light on a novel strategy for retroviruses control.

A novel multi-variant epitope ensemble vaccine against avian leukosis virus subgroup J.

The hypervariable antigenicity and immunosuppressive features of avian leukosis virus subgroup J (ALV-J) has led to great challenges to develop effective vaccines. Epitope vaccine will be a perspective trend. Previously, we identified a variant antigenic neutralizing epitope in hypervariable region 1 (hr1) of ALV-J, N-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-C. BLAST analysis showed that the mutation of A, E, T and H in this epitope cover 79% of all ALV-J strains. Base on this data, we designed a multi-variant epitope ensemble vaccine comprising the four mutation variants linked with glycine and serine. The recombinant multi-variant epitope gene was expressed in Escherichia coli BL21. The expressed protein of the variant multi-variant epitope gene can react with positive sera and monoclonal antibodies of ALV-J, while cannot react with ALV-J negative sera. The multi-variant epitope vaccine that conjugated Freund's adjuvant complete/incomplete showed high immunogenicity that reached the titer of 1:64,000 at 42 days post immunization and maintained the immune period for at least 126 days in SPF chickens. Further, we demonstrated that the antibody induced by the variant multi-variant ensemble epitope vaccine recognized and neutralized different ALV-J strains (NX0101, TA1, WS1, BZ1224 and BZ4). Protection experiment that was evaluated by clinical symptom, viral shedding, weight gain, gross and histopathology showed 100% chickens that inoculated the multi-epitope vaccine were well protected against ALV-J challenge. The result shows a promising multi-variant epitope ensemble vaccine against hypervariable viruses in animals.

Inhibition of avian tumor virus replication by CCCH-type zinc finger antiviral protein.

CCCH type zinc finger antiviral protein (ZAP) is a host restriction factor that inhibits the replication of a variety of viruses in mammals. However, little is known about its antiviral activity on avian tumor virus. Avian leukosis virus subgroup J (ALV-J), an oncogenic retrovirus, induces myelocytomas and various other tumors in meat and egg type chickens. Here, we identified a chicken ZAP (chZAP) that increased at early stage, and subsequently decreased after infection of ALV-J in DF-1 cells, indicating the inducible feature of the endogenous chZAP. To demonstrate the inhibitory effect on ALV-J replication by chZAP, we expressed exogenous chZAP by lentivirus based vectors in DF-1 cells that infected by ALV-J. The result showed that overexpression of chZAP significantly inhibited ALV-J replication at both mRNA level and protein level. Consequently, knockdown of endogenous chZAP by RNAi facilitated ALV-J replication in DF-1 cells. Further, we demonstrated that chZAP interacts with SU protein (encode by gp85 gene) of ALV-J in cytoplasm. Taken together, our results demonstrated that chZAP inhibits ALV-J by both mRNA and protein pathway and it may shed light on a novel antiviral approach in poultry.

Synthesis and characterization of fluorescence resonance energy transfer-based nanoprobes by coating CdTe QDs with rhodamine B in gelatin nanoparticles.

A FRET-based nanoprobe consisting of CdTe quantum dot as donor and rhodamine B as acceptor embedded in gelatin nanoparticles was constructed and the photophysics of the system was characterized. The FRET-based sensors were led to at fixed donor/acceptor distance by synthesizing via a two-step desolvation way. This approach allowed for short acceptor-donor separation and thus for high energy transfer efficiencies. The morphology of the nanoprobes was presented spherically and relatively uniform with a diameter of about 100 nm, which was characterized by transmission electron microscope. The luminescent property was proved to be stable in vitro and in living cells, indicating that the nanoprobes can be expected to be promising candidates for biological imaging studies.

Photodegradation of fleroxacin injection: different products with different concentration levels.

Photodegradation of fleroxacin is investigated in different injections and solutions. After UV irradiation, fleroxacin was degraded to afford two major products in large-volume injection (specification, 200 mg:100 ml), while degraded to afford another major product in small-volume injection (specification, 200 mg:2 ml). The photodegradation products were detected and isolated by reversed-phase HPLC. Based on the spectral data (FT-IR, MS(n), TOF-MS, (1)H/(13)C, DEPT, and 2D NMR), the structures of these products were: 8-fluoro-9-(4-methyl-piperazin-1-yl)-6-oxo-2,3-dihydro-6H-1-oxa-3a-aza-phenalene-5-carboxylic acid (impurity-I); 6-fluoro-1-(2-fluoro-ethyl)-7-(2-methylamino-ethylamino)-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (impurity-II); and 6,8-difluoro-1-(2-fluoro-ethyl)-7-(2-methylamino-ethylamino)-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid (impurity-III), respectively. Different photodegradation pathways of fleroxacin were proposed, which led to the different stability characteristics of fleroxacin in the injections. The fluorine atom at C8 is more photolabile in dilute injection, so defluorination and cyclization reactions are prone to take place, whereas photo irradiation only cause ring-opening oxidation reaction of piperazine side chain in concentrated injection.

[Study on the fluorescence resonance energy transfer between CdTe QDs and rhodamine B and its application to the determination of trace amount of copper in flos lonicerae japonicae].

The present work is to find the optimum condition for fluorescence resonance energy transfer between CdTe QDs (donor) and rhodamine B (acceptor) and form a new method to detect copper in Flos Lonicerae Japonicae. In tris-HCl buffer solution at pH 6.00, by employing cetyltrimethylammonium bromide, Cu2+ can quench the fluorescence intensity of BRB and detect the concentration of Cu2+. Results show that a linear relationship could be established between the quenched fluorescence intensity of RhB and the concentration of Cu2+ in the range of 1.3 x 10(-4) - 3.1 x 10(-2) microg x mL(-1), the limit of detection was 4.6 x 10(-5) microg x mL(-1) (r = 0.996), RSD and the average recovery was 3.5% and 103.4% (n = 5), respectively. It is concluded that the developed method was proved to be feasible to detect copper in Flos Lonicerae Japonicae.