OBJECTIVES: The emergence of resistant bacteria is being increasingly reported around the world, potentially threatening millions of lives. Amongst resistant bacteria, methicillin-resistant Staphylococcus aureus (MRSA) is the most challenging to treat. This is due to emergent MRSA strains and less effective traditional antibiotic therapies to Staphylococcal infections. The use of bacteriophages (phages) against MRSA is a new, potential alternate therapy. In this study, morphology, genetic and protein structure of lytic phages against MRSA have been analysed. METHODS: Isolation of livestock and sewage bacteriophages were performed using 0.4 μm membrane filters. Plaque assays were used to determine phage quantification by double layer agar method. Pure plaques were then amplified for further characterization. Sulfate-polyacrylamide gel electrophoresis and random amplification of polymorphic DNA were run for protein evaluation, and genotyping respectively. Transmission electron microscope was also used to detect the structure and taxonomic classification of phage visually. RESULTS: Head and tail morphology of bacteriophages against MRSA were identified by transmission electron microscopy and assigned to the Siphoviridae family and the Caudovirales order. CONCLUSION: Bacteriophages are the most abundant microorganism on Earth and coexist with the bacterial population. They can destroy bacterial cells successfully and effectively. They cannot enter mammalian cells which saves the eukaryotic cells from lytic phage activity. In conclusion, phage therapy may have many potential applications in microbiology and human medicine with no side effect on eukaryotic cells.
Agar ; Bacteria ; Bacteriophages ; Caudovirales ; Classification ; DNA ; Electrophoresis ; Eukaryotic Cells ; Head ; Humans ; Livestock ; Membranes ; Methicillin Resistance ; Methicillin-Resistant Staphylococcus aureus ; Methods ; Microscopy, Electron, Scanning Transmission ; Microscopy, Electron, Transmission ; Sewage ; Siphoviridae ; Staphylococcal Infections ; Tail

To confirm the hypothesis that the high frequency sequences of high throughput sequencing are the terminal sequences of the bacteriophage genome. An adaptor of specific sequence was linked to the end of the bacteriophage T3 genomic DNA, which was then subject to high throughput sequencing; as a control, the same T3 genomic DNA without adaptor was also analyzed by high throughput sequencing. The sequencing results were examined with bioinformatics software. Similar high throughput sequencing technique was applied to analyze the genomic sequence of N4-like bacteriophage IME11. Bioinformatics study showed that the sequences tagged with adaptors were consistent with the high frequency sequences without adaptor labeling. Our analysis also indicated that the end of the T4-like phage genome had specific sequences instead of random sequences, disagreeing with the previous assertion. Evidences were provided that N4-like bacteriophage had a particular terminal sequence: the left end of the genome was unique while the right end was permuted. The high throughput sequencing technique was convenient and practical to be used to simultaneously detect the terminal sequence and the complete sequence of bacteriophage genome.
Caudovirales ; genetics ; Computational Biology ; Genome, Viral ; High-Throughput Nucleotide Sequencing ; methods

OBJECTIVETo scan the most resistable bacteriophage as an indicator in disinfection tests, and to study the resistance of bacteriophage T4, Phichi 174D, and f2 to the sodium dichloroisocyanurate (NaDCC) in laboratory.

METHODSThe virucidal activity of NaDCC against bacteriophage T4, Phichi 174D, and f2 were assessed by suspension test. The neutralizer was selected and be appraised by test of neutralizer. Bacteriophage T4, Phichi 174D, and f2 were detected and enumerated by the double-agar-layer plaque technique.

RESULTS(1) With 150 mg/L of available chlorine of NaDCC solution, within a contact time of 40 minutes, or 300 mg/L, 5 minutes, the reductions of bacteriophage T4 achieved the "disinfection" level [log(10) inactivation value or log(10) reduction value of bacteriophage T4 (log(10)No-log(10)Nt) > or = 4.00 log(10)]. (2) With 300 mg/L of available chlorine of NaDCC solution, within a contact time of 5 minutes, or 400 mg/L, 3 minutes, the reductions of bacteriophage Phichi 174D achieved the "disinfection" level. (3) With 2000 mg/L of available chlorine of NaDCC solution, within a contact time of 20 minutes, or 4000 mg/L, 5 minutes, the reductions of bacteriophage f2 might achieve the "disinfection" level.

CONCLUSIONThe order of resistance of the above three bacteriophages to NaDCC from greatest to smallest is as follows: bacteriophage f2 > bacteriophage T4 > bacteriophage Phichi 174D.

Bacteriophage T4 ; drug effects ; Bacteriophage phi X 174 ; drug effects ; Bacteriophages ; drug effects ; Disinfectants ; pharmacology ; Drug Resistance, Viral ; Sodium Hypochlorite ; pharmacology

We isolated a novel Enterobacteria phage IME08 from hospital sewage, then confirmed it was a double-stranded DNA phage by digesting its genetic material with DNase I, RNase A and several restriction endonucleases respectively. BLAST results of random fragments generated by a random PCR cloning method revealed that it belonged to T4-like virus. We subsequently determined the host recognizing genes (g37 and g38) sequence with a PCR-based "genome jumping" protocol based on highly conserved region at 5' terminus of g37 from four other T4-like Bacteriophages (T4, JS98, T2 and K3). These molecular biological methods enabled us to readily characterize the bacteriophage and efficiently determine the sequence of the genes of interest based on very limited conserved sequence information.
Bacteriophage T4 ; genetics ; isolation & purification ; Cloning, Molecular ; DNA, Viral ; genetics ; Escherichia coli ; genetics ; virology ; Genome, Viral ; genetics ; Host Specificity ; genetics ; Polymerase Chain Reaction ; methods

We have examined isolation and identification protocols for three virus simulant candidates to biological warfare agents. MS2 phage, a simulant for yellow fever virus and Hantaan virus, was propagated using as a host an E. coli strain with F pilus. MS2 phage genome was examined by reverse transcription and polymerase chain reaction (RT-PCR). Coat protein of the phage preparation was examined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and mass spectrometric analysis. Cydia pomonella granulosis virus (CpGV) is a virus simulant candidate to smallpox virus. CpGV was isolated from a commercialized CpGV pellet. In this study, we developed new isolation and identification protocols for CpGV. One disadvantage of using CpGV is that it is not easy to determine viability of the virus. Here, we have included T4 phage as an alternative. We established a high titer production protocol and developed an easy genome identification protocol that does not require purified phage DNA. Stability of these virus preparations was also examined under various storage conditions. When the virus preparations were not subjected to freeze drying, MS2 phage was most stable when it was stored in liquid nitrogen but unstable at 4℃. In contrast, T4 phage was most stable when it was stored at 4℃. CpGV was stable at −20℃ but not at 4℃. Stability during or after freeze drying was also investigated. The result showed that 70~80% MS2 survived the freeze drying process. In contrast, only about 15% of T4 phage survived during the freeze drying. CpGV was found to be degraded during freeze drying.
Bacteriophage T4 ; Bacteriophages ; Biological Warfare Agents ; DNA ; Electrophoresis ; Freeze Drying ; Genome ; Granulovirus ; Hantaan virus ; Levivirus ; Nitrogen ; Polymerase Chain Reaction ; Reverse Transcription ; Variola virus ; Yellow fever virus

BACKGROUNDTo screen for the most resistant bacteriophage as indicator in disinfection tests, the resistance of bacteriophage phi chi 174D, T4 and f2 to iodophor were observed in laboratory.

METHODSThe virucidal activity of iodophor against bacteriophage phi chi 174D, T4, and f2 were assessed by suspension test. The neutralizer is selected and appraised by testing with neutralizer. Bacteriophage phi chi 174D, T4, and f2 were detected and enumerated by the double-agar-layer plaque technique.

RESULTS(1) With 500 mg/L of available iodine of iodophor solution, within a contact time of 40 min, or 750 mg/L, 10 min, or 1000 mg/L, 5 min, the reduction of bacteriophage phi chi 174D could achieve the "disinfection" level [log10 inactivation value (LIV) or log10 reduction value (LRV) of bacteriophage phi chi 174D (log10 No-log10 Nt) was > or = 4.00 log10]. (2) With 600 mg/L of available iodine of iodophor solution, within a contact time of 40 min, or 700 mg/L, 5 min, the reductions of bacteriophage T4 could achieve the "disinfection" level. (3) With 50 mg/L of available iodine of iodophor solution, within a contact time of 10 min, or 75 mg/L, 10 min, the reductions of bacteriophage f2 could achieve the "disinfection" level.

CONCLUSIONThe order of resistance of the above three bacteriophages to iodophor from greatest to smallest is as follows: bacteriophage phi chi 174D greater than bacteriophage T4 > bacteriophage f2.

Bacteriophage T4 ; drug effects ; Bacteriophage phi X 174 ; drug effects ; Bacteriophages ; drug effects ; Disinfectants ; pharmacology ; Disinfection ; methods ; Dose-Response Relationship, Drug ; Drug Resistance, Viral ; Iodophors ; pharmacology ; Surface-Active Agents ; pharmacology ; Virus Inactivation ; drug effects

This study aimed to develop a new multiplex real-time PCR detection method for 3 species of waterborne protozoan parasites (Cryptosporidium parvum, Giardia lamblia, and Cyclospora cayetanensis) identified as major causes of traveler's diarrhea. Three target genes were specifically and simultaneously detected by the TaqMan probe method for multiple parasitic infection cases, including Cryptosporidium oocyst wall protein for C. parvum, glutamate dehydrogenase for G. lamblia, and internal transcribed spacer 1 for C. cayetanensis. Gene product 21 for bacteriophage T4 was used as an internal control DNA target for monitoring human stool DNA amplification. TaqMan probes were prepared using 4 fluorescent dyes, FAM™, HEX™, Cy5™, and CAL Fluor Red® 610 on C. parvum, G. lamblia, C. cayetanensis, and bacteriophage T4, respectively. We developed a novel primer-probe set for each parasite, a primer-probe cocktail (a mixture of primers and probes for the parasites and the internal control) for multiplex real-time PCR analysis, and a protocol for this detection method. Multiplex real-time PCR with the primer-probe cocktail successfully and specifically detected the target genes of C. parvum, G. lamblia, and C. cayetanensis in the mixed spiked human stool sample. The limit of detection for our assay was 2×10 copies for C. parvum and for C. cayetanensis, while it was 2×10³ copies for G. lamblia. We propose that the multiplex real-time PCR detection method developed here is a useful method for simultaneously diagnosing the most common causative protozoa in traveler's diarrhea.
Bacteriophage T4 ; Cryptosporidium parvum ; Cryptosporidium ; Cyclospora ; Diagnosis ; Diarrhea ; DNA ; Fluorescent Dyes ; Giardia lamblia ; Giardia ; Glutamate Dehydrogenase ; Humans ; Limit of Detection ; Methods ; Multiplex Polymerase Chain Reaction ; Oocysts ; Parasites ; Real-Time Polymerase Chain Reaction

To enhance the efficiency of the expression of target gene in eukaryotic cells, one of the strongest prokaryotic expression systems, the T7 RNA polymerase and T7 promoter, was introduced into eukaryotic cells. A duel-plasmid gene expression system of T7 bacteriophage components was developed; one containing the T7 phage RNA polymerase gene under the control of eukaryotic promoter CMV (pCMV-T7pol) and the other (pT7IRES) containing the T7 promoter and T7 terminator as well as EMCV IRES. To test the feasibility of this plasmid system for eukaryotic expression, hepatitis B virus envelop HBV preS2/S was used to construct pT7IRES-HBs. The target genes were expressed efficiently by the eukaryonized prokaryotic expression system in a variety of the cells indicating C2C12, SP2/0, NIH3T3 and BALB/c 3T3, suggesting the potential applications of the expression system in gene therapy and gene immunization.
Animals ; Bacteriophage T7 ; genetics ; Cell Line ; DNA-Directed RNA Polymerases ; genetics ; Gene Targeting ; Promoter Regions, Genetic ; genetics ; Viral Proteins ; genetics

Prospect Hill virus (PHV) is related antigenically but distinct to Hantaan virus. As a member of genus Hantavirus, PHV has three segmented RNA genome. Among these segments, Small segment(S) encodes nucleocapsid protein (NP) as structural protein and also may do functional nonstructural protein(NSs). We performed in vitro transcription to produce vRNA of PHV S genome. For the first step of in vitro transcription of S genome of PHV, the S RNA segrnent which is 1,675 nucleotides long was amplified by RT-PCR using PCR primers built according to cDNA sequence of PHV S genome. Next, a new PCR primer appended above downstream primer to T7 phage promoter sequence was reconstructed to obtain PCR product containing T7 promoter. Then another PCR was performed. Using this PCR product as the template, in vitro run-off transcription without cloning by transcriptional vector was performed to obtain viral- sense RNA transcript. Thereafter, the size of transcript was assessed by running on formaldehyde agarose gels. Since the transcription reactants contain a-S UTP, the transcript is detectable by autoradiography. The transcript was also detectable by northern hybridization with a-P dCTP- labelled PHV amplicon probe (319 bp) and the initiation start point of run-off transcription was also determined by primer extention analysis. Our data indicate that the in vitro transcript could be produced from the PCR product amplified by PCR primer containing T7 phage promoter without cloning into a phage transcription vector.
Autoradiography ; Bacteriophage T7 ; Bacteriophages ; Clone Cells ; Cloning, Organism ; DNA, Complementary ; Formaldehyde ; Gels ; Genome* ; Hantaan virus ; Hantavirus ; Nucleocapsid Proteins ; Nucleotides ; Polymerase Chain Reaction ; RNA ; Running ; Sepharose ; Uridine Triphosphate ; Viruses*

The gene encoding E7 oncoprotein of human papillomavirus type 16 was cloned and expressed in Escherichia coli, and the monoclonal antibodies (Mabs) against this expressed protein were generated. For the efficient immunization, two kind of recombinant E7 protein in fusion form were produced. One was maltose binding protein (MBP) fusion type (MBP-E7) and the other was T7 phage gene 10 product fusioa type (gene 10-E7). Immunization with these two fusion protein to mice, finally two Mabs (VD6 and IB10) were obtained. VD6 and IB10 showed reactivities with E7 protein in CaSki cell but not in HeLa by Western blot analysis. In addition, the Mab, VD6, reacted with COS-7 cell transfected with E7 gene majorly in cytoplasm by immunofluorescence test. Also VD6 could detect E7 protein in cytoplasm and nucleus of CaSki ceU by immunogold electron microscopy. Based on these results, the Mab VD6 was could be used for various E7 detection system such as Western blot analysis and immunohistochemical methods.
Animals ; Antibodies, Monoclonal* ; Bacteriophage T7 ; Blotting, Western ; Clone Cells ; COS Cells ; Cytoplasm ; Escherichia coli ; Fluorescent Antibody Technique ; Humans* ; Immunization ; Maltose-Binding Proteins ; Mice ; Microscopy, Electron