[Preparation and identification of monoclonal antibodies against African horse sickness virus VP7].

Objective To prepare the monoclonal antibody (mAb) against the African horsefever virus (AHSV) VP7 protein and to identify it. Methods mAbs were prepared by using baculovirus expressed VP7 protein in BALB/c mice, and the effect of mAb was detected by ELISA, indirect immunofluorescence assay (IFA), and AHSV positive serum blockade. Results Four mAb strains were selected, including 20A8, 28B3, 30G8 and 47E6, among which 47E6 had the best blocking effect. Conclusion mAbs were successfully prepared against VP7 protein.

Establishment and characterization of a heart-derived cell line from goldfish (Carassius auratus).

The goldfish Carassius auratus, a freshwater fish in the family Cyprinidae, was one of the earliest fish to be domesticated for ornamental purposes. A cell line was established from goldfish heart (GH) tissue to create a biological monitoring tool for viral diseases. The GH cell line was optimally maintained at 25 °C in M199 medium supplemented with 10-20% fetal bovine serum. A chromosomal analysis indicated that the cell line remained diploid, with a mean chromosomal count of 100. In viral inoculation assays, significant cytopathic effects (CPEs) were caused by epizootic hematopoietic necrosis virus (EHNV), Andrias davidianus iridovirus (ADIV), and Bohle iridovirus (BIV) infections in the fish cells and the viral titers (average value) of EHNV, ADIV, and BIV in GH cells reached 105.0, 104.5, and 105.0 TCID50/0.1 mL, respectively, within 7 days. However, no CPE was observed in the cells infected with viral hemorrhagic septicemia virus (VHSV), infectious hematopoietic necrosis virus (IHNV), spring viremia of carp virus (SVCV), infectious pancreatic necrosis virus (IPNV), channel catfish virus (CCV), or grass carp reovirus (GCRV). These results suggest that the GH cell line is a valuable tool for studying viral pathogenesis.

Whole genome sequencing uncovers a novel IND-16 metallo-ß-lactamase from an extensively drug-resistant Chryseobacterium indologenes strain J31.

BACKGROUND: Chryseobacterium indologenes is an emerging opportunistic pathogen in hospital-acquired infection, which is intrinsically resistant to most antimicrobial agents against gram-negative bacteria. In the purpose of extending our understanding of the resistance mechanism of C. indologenes, we sequenced and analyzed the genome of an extensively antibiotic resistant C. indologenes strain, isolated from a Chinese prostate cancer patient. We also investigated the presence of antibiotic resistance genes, particularly metallo-ß-lactamase (MBL) genes, and performed a comparative genomic analysis with other Chryseobacterium species. RESULTS: 16s rRNA sequencing indicated the isolate belongs to C. indologenes. We assembled a total of 1095M bp clean-filtered reads into 171 contigs by de novo assembly. The draft genome of C. indologenes J31 consisted of 5,830,795 bp with a GC content of 36.9 %. RAST analysis revealed the genome contained 5196 coding sequences (CDSs), 28 rRNAs, 81 tRNAs and 114 pseudogenes. We detected 90 antibiotic resistance genes from different drug classes in the whole genome. Notably, a novel blaIND allele blaIND-16 was identified, which shared 99 % identity with blaIND-8 and blaIND-10. By comparing strain J31 genome to the closely four related neighbors in the genus Chryseobacterium, we identified 2634 conserved genes, and 1449 unique genes. CONCLUSIONS: In this study, we described the whole genome sequence of C. indologenes strain J31. Numerous resistance determinants were detected in the genome and might be responsible for the extensively antibiotic resistance of this strain. Comparative genomic analysis revealed the presence of considerable strain-specific genes which would contribute to the distinctive characteristics of strain J31. Our study provides the insight of the multidrug resistance mechanism in genus Chryseobacterium.

Active site analysis of sortase A from Staphylococcus simulans indicates function in cleavage of putative cell wall proteins.

Sortase mediated transpeptidation reactions play a significant role in covalent attachment of surface proteins to the cell wall of Gram-positive bacteria. Earlier studies have shown that sortase A (StrA) is required for the virulence of Staphylococci. The human pathogen Staphylococcus simulans CJ16 carries a putative sortase A (SsiStrA) encoding gene, but neither transpeptidation activity nor biochemical characteristics of SsiStrA have been investigated. Here, we identified and characterized StrA from coagulase-negative Staphylococci. SsiStrA was cloned and overexpressed in Escherichia coli BL21 in a soluble form. Size-exclusion chromatography, cross-linking and dynamic light scattering demonstrated that SsiStrA existed as monomer-dimer equilibrium in vitro. We further demonstrated that SsiStrA has sortase activity, and it recognized and cleaved the sorting motif LXPTG. H117, C180 and R193 residues were critical for enzyme activity, and calcium ions enhanced activity.