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

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

In order to investigate the occurrence of norovirus in rivers and beaches, a total of 81 samples were tested at seven sites of Oncheon stream, Suyeong river and Gwanganri beach in Busan from January to November, 2017. To improve the detection of norovirus from sea water, we applied the inorganic cation-coated filter method which showed 48.8% ± 12.2% (n=3) and 27.4% ± 6.0% (n=3) recovery yields from river water and sea water inoculated with Norovirus, respectively. Norovirus was detected in a total of four samples (4.9%), which all were GII genotype. Norovirus GII was detected in three samples at two waste water treatment plants (WWTP) outlet and one sample at about 500 meter downstream from WWTP in both the winter and spring seasons. We also monitored fecal indicator organisms, Escherichia coli (E. coli), Enterococcus and coliphages [somatic coliphages (SC), male-specific coliphages (MSC)] to analyze the potential transmission of enteritis causative agent in dry and wet days. Bacterial influences were found at the site of the WWTP effluents in the dry days and spread further to the costal beach in the wet days. But no viral influences were found in the river downstream in both dry and wet days.
Busan* ; Coliphages ; Enteritis ; Enterococcus ; Escherichia coli ; Genotype ; Korea* ; Methods ; Norovirus* ; Rivers* ; Seasons ; Seawater ; Waste Water ; Water

OBJECTIVETo evaluate the protective performance of a positive pressure bio-protective clothing against viral aerosol.

METHODSThe suspension of indicating virus phage Phi-X174 was made for viral aerosol generating in a hermetic cabin. The diameter of viral aerosol particles were measured with a aerodynamics size analyzer. By adjusting the inner humidity of the cabin, the protective efficiency of the positive pressure bio-protective clothing against viral aerosol in high and low windshield conditions was determined with Andersen six-stage air sampler sampling and plage forming unit (PFU) counting, respectively.

RESULTSThe mass median diameter of Phage Phi-X174 aerosol particles was about 0.922 µm and the background concentration is beyond 2 × 10⁴ particles/m³. The protective efficiency of the clothing against phage Phi-X174 aerosol particles was above 99.9% under different test conditions with the range of viral aerosol concentration between 0 - 23 PFU/m³. Airflow (P = 0.84), environment humidity conditions (P = 0.33) and sampling time (P = 0.07) did not affect the protective efficiency statistically.

CONCLUSIONThe positive pressure bio-protective clothing provided a relatively high efficiency against phage Phi-X174 aerosol regardless of airflow rate, environment humidity and sampling time.

Aerosols ; Bacteriophage phi X 174 ; Bioterrorism ; prevention & control ; Equipment Design ; Humidity ; Occupational Exposure ; prevention & control ; Pressure ; Protective Clothing ; Time Factors ; Virus Diseases ; prevention & control

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

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

Cryptocococcus neoformans is an encapsulated yeast that can cause life-threatening infections in immunocompromised patients. In this study, the genetic variability and epidemiological relationships of clinical and environmental isolates of C. neoformans from Busan, Korea, 2000~2005 were investigated. A total of 12 strains of C. neoformans, 7 clinical and 5 environmental isolates were analyzed by random amplified polymorphic DNA (RAPD) using three different primers and PCR-fingerprinting with a minisatellite-specific core sequence of phage M13. All strains belonged to C. neoformans serotype A and mating type MATa. Two different RAPD profiles (I and II) and a single pattern by M13 PCR-fingerprinting were identified. The major RAPD profile was pattern I (8 of 12 strains) and pattern II was identified from 2 clinical and 2 environmental strains, which clearly distinguished among isolates. Clinical strains with pattern II were isolated from the patients with HIV positive. Taken together, molecular patterns provide a good characterization of strains of C. neoformans as a heterogeneous group and epidemiological relationships in clinical and environmental strains.
Bacteriophage M13 ; Cryptococcus ; Cryptococcus neoformans ; DNA ; HIV ; Humans ; Immunocompromised Host ; Korea ; Yeasts

Avidin layer was bound on the substrate surface of Silicon wafer modified with aldehyde. The interaction between avidin and biotin was adopted for the immobilization of mouse monoclonal biotin-anti-M13 (antibody GP3)-labeled biotin. The surface was incubated in a solution containing phage M13KO7, which was trapped by the antibody GP3 with the interaction between phage M13KO7 and antibody GP3, resulting in a variation of layer thickness that was detected by imaging ellipsometry. The results showed a saturated layer of antibody GP3 with a thickness about 6.9 nm on the surface of the silicon wafer. The specific interaction between phage M13KO7 and antibody GP3 resulted in a variation of the layer thickness. The layer of phage M13KO7 bound with antibody GP3 was 17.5 nm in the concentration of 1.1 x 10(10) pfu/mL. Each variation of layer thickness corresponded to a concentration of phage M13KO7 in the range of 0.1 x 10(10) approximately 2.5 x 10(10) pfu/mL, with the sensitivity of 10(9) pfu/mL. Compared with other methods, the optical protein-chip requires only short measurement time, is label free, is a quantitative test, and can be visualized. This study could be significant on the investigation of interactions between the antibody and virus, and shows potential in the early diagnosis of virosis.
Animals ; Antibodies, Viral ; immunology ; Bacteriophage M13 ; immunology ; isolation & purification ; Mice ; Protein Array Analysis ; methods

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