Pathogenic microorganisms are responsible for many infectious diseases, and pathogen monitoring is important and necessary for water quality control. This study for the first time explored a multiplex quantitative real-time PCR (qPCR) technique combined with propidium monoazide (PMA) to simultaneously detect viable Legionella pneumophila, Salmonella typhimurium, and Staphylococcus aureus in one reaction from water samples. Sodium lauroyl sarcosinate (sarkosyl) was applied to enhance the dead bacterial permeability of PMA. The sensitivity of the multiplex PMA-qPCR assay achieved two colony-forming units (CFU) per reaction for L. pneumophila and three CFU per reaction for S. typhimurium and S. aureus. No PCR products were amplified from all nontarget control samples. Significantly, with comparable specificity and sensitivity, this newly invented multiplex PMA-qPCR assay took a much shorter time than did conventional culture assays when testing water samples with spiked bacteria and simulated environmental water treatment. The viable multiplex PMA-qPCR assay was further successfully applied to pathogen detection from rivers, canals, and tap water samples after simple water pretreatment.
The analysis on codon usage bias of UL24 gene of duck enteritis virus (DEV) may improve our understanding of the evolution and pathogenesis of DEV and provide a basis for understanding the relevant mechanism for biased usage of synonymous codons and for selecting appropriate expression systems to improve the expression of target genes. The codon usage bias of UL24 genes of DEV and 27 reference herpesviruses were analyzed. The results showed that codon of UL24 gene of DEV was strong bias toward the synonymous codons with A and T at the third codon position. A high level of diversity in codon usage bias existed, and the effective number of codons used in a gene plot revealed that the genetic heterogeneity in UL24 gene of herpesviruses was constrained by the G + C content. The phylogentic analysis suggested that DEV was evolutionarily closer to Alphaherpesvirinae and that there was no significant deviation in codon usage in different virus strains. There were 20 codons showing distinct usage differences between DEV and Escherichia coli, 23 between DEV and Homo sapiens, but only 16 codons between DEV and yeast. Therefore the yeast expression system may be more suitable for the expression of DEV genes.
Oncolytic herpes simplex viruses (oHSVs) have been approved for clinical usage and become more and more popular for tumor virotherapy. However, there are still many issues for the oHSVs used in clinics and clinical trials. The main issues are the limited anti-tumor effects, intratumor injection, and some side effects. To overcome such challenges, here we review the genetic engineering of the envelope glycoproteins for oHSVs to target tumors specifically, and at the same time we summarize the many neutralization antibodies against the envelope glycoproteins and align the neutralization epitopes with functional domains of the respective glycoproteins for future identification of new functions of the glycoproteins and future engineering of the epitopes to escape from host neutralization.
Compared to the UL51 gene of other alphaherpesviruses, the duck enteritis virus (DEV) UL51 gene contains ten conserved motifs and has a close evolutionary relationship with members of the genus Mardivirus. The DEV UL51 gene product was identified using a rabbit polyclonal antiserum raised against a 6-His-UL51 fusion protein expressed in Escherichia coli as a 34-kDa protein. Western blotting and RT-(real time) PCR analysis of DEV-infected cells showed that the protein was produced at the late stage of infection and that its production was highly dependent on viral DNA synthesis, suggesting that the gene should be classified as gamma2 class. Analysis of extracellular virions revealed that the protein was a component of extracellular mature DEV virions. Indirect immunofluorescence studies localized most of the protein to the juxtanuclear region. These results will provide a basis for further functional analysis of the gene.
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