A Novel Transposon Tn7709 Harbors Multidrug Resistance Genes in a Pathogenic Aeromonas media Strain QST31.

Pathogenic Aeromonas spp. are the etiological agents of Motile Aeromonas Septicemia (MAS). This study aimed to identify the pathogen of diseased tadpoles (Quasipaa spinosa) and the antibiotic-resistance characteristics of this bacterium. A Gram-negative bacterium, named strain QST31, was isolated from the ascites of diseased tadpoles and was identified as Aeromonas media based on physiological and biochemical tests, as well as molecular identification. Artificial infection experiments showed that strain QST31 was highly virulent to tadpoles, with an LC50 of 2.56 × 107 CFU/mL. The antimicrobial susceptibility of strain QST31 was evaluated using the disk diffusion method, and the results indicated that strain QST31 was resistant to 28 antibacterial agents. In addition, the whole genome of strain QST31 was sequenced, and the presence of antimicrobial resistance genes, integron, and transposon was investigated. Genes involved in adherence, hemolysis, type II secretion system (T2SS), T6SS, iron uptake system, and quorum sensing were identified in the genome of strain QST31. More than 12 antimicrobial resistance genes were predicted in the genome of strain QST31. Interestingly, a novel Tn7709 transposon harboring sul1, aadA16, catB3, blaOXA-21, aac(6')-IIa, and tet(A) genes was identified. In conclusion, this is the first report on the isolation and identification of pathogenic A. media with multidrug resistance genes from diseased tadpoles. The results revealed that preventing and controlling aquatic animal diseases caused by multidrug resistance A. media will be a huge challenge in the future.

Effects of different regimes of low temperature on egg hatching of Dactylogyrus vastator (Monogenea: Dactylogyridae).

The development of dactylogyrids is dependent on water temperature, and their eggs fail to hatch below 5 °C. In the field, however, mean abundance of Dactylogyrus species increases and reaches a high level in winter, which suggests that infective oncomiracidia hatch from eggs in winter. Therefore, the effect of low water temperature on in vitro egg hatching of D. vastator was determined in laboratory. D. vastator hatching success was 65.3%, 62.7%, 42.6% and 22.3% when eggs were firstly incubated for 0, 7, 14 and 21 days at 5 °C and then consecutively maintained for 15 days at 20 °C. When eggs were directly incubated at 5 °C, eggs failed to hatch within one month. However, hatching success was 69.8% and 66.7%, respectively, when maintained at 5 °C after 12 and 24 h incubation at 20 °C. The results suggested that egg incubation for more than 1 week at 5 °C had significant impacts on hatching success of D. vastator subsequently maintained at 20 °C. But low temperature (5 °C) had no effect on hatching success when eggs were firstly exposed to room temperature (20 °C) for one day.

Complete genome sequence data of multidrug-resistant Stenotrophomonas sp. strain SXG-1.

Objectives A multidrug-resistant bacterium, Stenotrophomonas sp. SXG-1, was isolated from the liver of diseased hybrid sturgeon from Guizhou province, China. Methods Whole-genome sequencing was performed on the Illumina HiSeq 2500-PE125 platform with MPS (massively parallel sequencing) Illumina technology. All good quality paired reads were assembled using the SOAPdenovo into a number of scaffolds. PHI (Pathogen Host Interactions), VFDB (Virulence Factors of Pathogenic Bacteria) and ARDB (Antibiotic Resistance Genes Database) were used to analyses pathogenicity and drug resistance. Results Here we reported the complete genome sequence of Stenotrophomonas sp. SXG-1, which comprised 4534,602bp in 4077 coding sequences (CDS) with a G+C content of 66.42%. The genome contained 4 gene islands, 72 tRNAs and 13 rRNAs. According to the annotation analysis, strain SXG-1 encoded 22 genes related to the multidrug resistance. In addition to 10 genes conferring resistance to antimicrobial drugs of different classes via alternative mechanisms, 12 genes of efflux pumps were presented, 9 of which were reported for the first time in Stenotrophomonas maltophilia. Conclusion This was the first complete genome sequence of Stenotrophomonas sp. isolated from the sturgeon. The complete genome sequence of Stenotrophomonas sp. strain SXG-1 may provide insights into the mechanism of antimicrobial resistance and prevent disease.

Generation of a recombinant West Nile virus stably expressing the Gaussia luciferase for neutralization assay.

West Nile virus (WNV) is a neurotropic human pathogen that has caused increasing infected cases over recent years. There is currently no licensed vaccine or effective drug for prevention and treatment of WNV infection in humans. To facilitate antiviral drug discovery and neutralizing antibody detection, a WNV cDNA clone containing a luciferase reporter gene was constructed through incorporating Gaussia luciferase (Gluc) gene within the capsid-coding region of WNV genome. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated luciferase reporter WNV (WNV-Gluc) and the stable WNV-Gluc with high titers (>10(7)PFU/ml) was obtained through plaque purification. Luciferase activity was used to effectively quantify the viral production of WNV-Gluc. Using the reporter virus WNV-Gluc, we developed a luciferase based assay in a 12-well format for evaluating neutralizing antibodies. The reporter virus could be a powerful tool for epidemiological investigation of WNV, vaccine evaluation, antiviral drug screening, and the study of WNV replication and pathogenesis.

Development of a stable Gaussia luciferase enterovirus 71 reporter virus.

We report a stable Gaussia luciferase enterovirus 71 (Gluc-EV71) reporter virus to facilitate drug discovery. The Gluc-EV71 reporter virus was generated by engineering the Gaussia luciferase (Gluc) gene between the 5' untranslated region and VP4 gene of the EV71 genome. We could recover Gluc-EV71 after transfection of Vero cells with the cDNA clone-derived RNA. The reporter virus efficiently infects and replicates in various cell types (Vero, human rhabdomyosarcoma, and HeLa cells), producing robust luciferase activity. The Gluc-EV71 virus replicates slower than the wild-type virus in cell culture. The reporter virus is stable in maintaining the Gluc gene after five rounds of continuous passaging in Vero cells. Using known EV71 inhibitors, we demonstrate that the reporter virus can be used for antiviral testing. However, the Gluc-EV71 infection assay cannot be adapted to a homogenous format for high throughput screen, mainly due to the secreted nature of the Gluc protein and the short half-life of the Gluc luminescence signal. The Gluc-EV71 and its infection assay could be useful for antiviral drug discovery as well as for studying EV71 replication and pathogenesis.

Genetic interaction between NS4A and NS4B for replication of Japanese encephalitis virus.

Flavivirus NS4A and NS4B are important membrane proteins for viral replication that are assumed to serve as the scaffold for the formation of replication complexes. We previously demonstrated that a single Lys-to-Arg mutation at position 79 in NS4A (NS4A-K79R) significantly impaired Japanese encephalitis virus (JEV) replication. In this study, the mutant virus was subject to genetic selection to search for the potential interaction between NS4A and other viral components. Sequencing of the recovered viruses revealed that, in addition to an A97E change in NS4A itself, a Y3N compensatory mutation located in NS4B had emerged from independent selections. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that both adaptive mutations greatly restored the replication defect caused by NS4A-K79R. Our results, for the first time to our knowledge, clearly showed the genetic interaction between NS4A and NS4B, although the mechanism underlying their interaction is unknown.

Inhibition of enterovirus 71 by adenosine analog NITD008.

Enterovirus 71 (EV71) is a major viral pathogen in China and Southeast Asia. There is no clinically approved vaccine or antiviral therapy for EV71 infection. NITD008, an adenosine analog, is an inhibitor of flavivirus that blocks viral RNA synthesis. Here we report that NITD008 has potent antiviral activity against EV71. In cell culture, the compound inhibits EV71 at a 50% effective concentration of 0.67 µM and a 50% cytotoxic concentration of 119.97 µM. When administered at 5 mg/kg in an EV71 mouse model, the compound reduced viral loads in various organs and completely prevented clinical symptoms and death. To study the antiviral mechanism and drug resistance, we selected escape mutant viruses by culturing EV71 with increasing concentrations of NITD008. Resistance mutations were reproducibly mapped to the viral 3A and 3D polymerase regions. Resistance analysis with recombinant viruses demonstrated that either a 3A or a 3D mutation alone could lead to resistance to NITD008. A combination of both 3A and 3D mutations conferred higher resistance, suggesting a collaborative interplay between the 3A and 3D proteins during viral replication. The resistance results underline the importance of combination therapy required for EV71 treatment. Importance: Human enterovirus 71 (EV71) has emerged as a major cause of viral encephalitis in children worldwide, especially in the Asia-Pacific region. Vaccines and antivirals are urgently needed to prevent and treat EV71 infections. In this study, we report the in vitro and in vivo efficacy of NITD008 (an adenosine analog) as an inhibitor of EV71. The efficacy results validated the potential of nucleoside analogs as antiviral drugs for EV71 infections. Mechanistically, we showed that mutations in the viral 3A and 3D polymerases alone or in combination could confer resistance to NITD008. The resistance results suggest an intrinsic interaction between viral proteins 3A and 3D during replication, as well as the importance of combination therapy for the treatment of EV71 infections.

Recovery of a chemically synthesized Japanese encephalitis virus reveals two critical adaptive mutations in NS2B and NS4A.

A full-length genome infectious clone is a powerful tool for functional assays in virology. In this study, using a chemical synthesized complete genome of Japanese encephalitis virus (JEV) strain SA14 (GenBank accession no. U14163), we constructed a full-length genomic cDNA clone of JEV. The recovered virus from the cDNA clone replicated poorly in baby hamster kidney (BHK-21) cells and in suckling mice brain. Following serial passage in BHK-21 cells, adaptive mutations within the NS2B and NS4A proteins were recovered in the passaged viruses leading to viruses with a large-plaque phenotype. Mutagenesis analysis, using a genome-length RNA and a replicon of JEV, demonstrated that the adaptive mutations restored replication to different degrees, and the restoration efficiencies were in the order: NS2B-T102M

Development and characterization of West Nile virus replicon expressing secreted Gaussia luciferase.

We developed a Gaussia luciferase (Gluc) reporter replicon of West Nile virus (WNV) and used it to quantify viral translation and RNA replication. The advantage of the Gluc replicon is that Gaussia luciferase is secreted into the culture medium from cells transfected with Gluc replicon RNA, and the medium can be assayed directly for luciferase activity. Using a known Flavivirus inhibitor (NITD008), we demonstrated that the Gluc-WNV replicon could be used for antiviral screening. The Gluc-WNV-Rep will be useful for research in antiviral drug development programs, as well as for studying viral replication and pathogenesis of WNV.

Development and characterization of a stable eGFP enterovirus 71 for antiviral screening.

Enterovirus 71 (EV71) is one of the major causative agents for hand, foot, and mouth disease. There is currently no clinically approved vaccine or antiviral treatment for EV71 infection. To facilitate antiviral drug discovery, we developed an infectious cDNA clone of an epidemic strain of EV71 and a stable eGFP reporter EV71. The reporter virus was generated by engineering the eGFP gene between the 5' untranslated region and VP4 gene of the EV71 genome. Vero cells transfected with the cDNA clone-derived RNA generated high titers (>10(6)PFU/ml) of the eGFP reporter virus. The reporter virus was infectious to Vero cells, producing robust eGFP fluorescence signals. Compared with the wild type virus, the reporter virus replicated slower in cell culture. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. The passaged viruses maintained the eGFP gene, demonstrating the stability of the reporter virus. Using a known EV71 inhibitor, we demonstrate that the reporter virus could be used for antiviral screening. The infectious cDNA clones of the wild type virus and the eGFP reporter viruses will be useful for antiviral research as well as for studying viral replication and pathogenesis of EV71.