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

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

Staphylococcal nuclease A (SNA) may be used to produce bacterial ghosts for further inactivation of host bacteria and elimination of residual genetic materials. It is still controversial if SNA without signal peptide can be secreted to extracellular matrix and if fusion with other peptide is required for its function in the cytoplasm of host bacteria. To clarify this dispute, a series of temperature-inducible plasmids carrying SNA alone or SNA fused with partial sequences of λ phage cro gene (cSNA) or Mycobacterium tuberculosis urease gene (uSNA) were constructed and evaluated in Escherichia coli. Results show that the percentages of inactivated E. coli by SNA, cSNA and uSNA after 4 h of induction were 99.9%, 99.8% and 74.2%, respectively. Moreover, SNA and cSNA in the cytoplasm of host bacteria were initially detectable after 30 min of induction, whereas uSNA was after 1 h. In comparison, SNA and cSNA in culture supernatant were initially detectable 1 h later, whereas uSNA was 2 h later. The nuclease activity in the cytoplasm or supernatant was ranked as follows: SNA > cSNA > uSNA, and the activity in the supernatant was significantly lower than that in the cytoplasm. Furthermore, host genomic DNA was degraded by SNA or cSNA after 2 h of induction but not by uSNA even throughout the whole experiment. In conclusion, this study indicates that SNA, cSNA and uSNA expressed in host bacteria all have nuclease activity, the enzymes can be released to culture media, and fusion with exogenous peptide negatively reduces the nuclease activity of SNA.
Bacteriolysis ; Bacteriophage lambda ; DNA ; chemistry ; Escherichia coli ; Genetic Vectors ; Micrococcal Nuclease ; chemistry ; Plasmids ; Protein Sorting Signals

Cyptococcosis is generally caused by Cryptococcus neoformans, the opportunistic agent which has two species such as C. neoformans and C. gattii. Both C. neoformans and C. gattii species contain a number of genetically diverse subgroups that can be differentiated by various molecular typing methods. We conducted a molecular epidemiological analysis of 30 clinical isolates of the C. neoformans from cryptococcosis patients who had been hospitalized between 2008 and 2010 in medical centers located in Seoul and Busan in Korea. To determine the genetic diversity, 30 strains of C. neoformans were typed using PCR fingerprinting with the microsatellite specific primer of the phage M13 and the restriction fragment length polymorphism (RFLP) of orotidine monophosphosphate pyrophosphorylase (URA5) gene. All isolates were identified as serotype A, mating type MATa and molecular type VNI. The random amplified polymorphic DNA (RAPD) profiles obtained by using two primers revealed a single pattern. Our study shows that 30 strains of clinical C. neoformans are genetically homogeneous, with all of the isolates were molecular type VN1, serotype A, mating type MATa.
Bacteriophage M13 ; Cryptococcosis ; Cryptococcus ; Cryptococcus neoformans ; Dermatoglyphics ; DNA ; Genetic Variation ; Humans ; Korea ; Microsatellite Repeats ; Molecular Typing ; Polymerase Chain Reaction ; Polymorphism, Restriction Fragment Length ; Uridine

To construct a recombinant T7 phage expressing matrix protein 2 ectodomain (M2e) peptides of avian influenza A virus and test immunological and protective efficacy in the immunized SPF chickens. M2e gene sequence was obtained from Genbank and two copies of M2e gene were artificially synthesised, the M2e gene was then cloned into the T7 select 415-1b phage in the multiple cloning sites to construct the recombinant phage T7-M2e. The positive recombinant phage was identified by PCR and sequencing, and the expression of surface fusion protein was confirmed by SDS-PAGE and Western-blot. SPF chickens were subcutaneously injected with 1 X 10(10) pfu phage T7-M2e, sera samples were collected pre- and post-vaccination, and were tested for anti-M2e antibody by ELISA. The binding capacity of serum to virus was also examined by indirect immunofluorescence assay in virus- infected CEF. The immunized chickens were challenged with 200 EID50 of H9 type avian influenza virus and viral isolation rate was calculated to evaluate the immune protective efficacy. A recombinant T7 phage was obtained displaying M2e peptides of avian influenza A virus, and the fusion protein had favorable immunoreactivity. All chickens developed a certain amount of anti-M2e antibody which could specially bind to the viral particles. In addition, the protection efficacy of phage T7-M2e vaccine against H9 type avian influenza viruses was 4/5 (80%). These results indicate that the recombinant T7 phage displaying M2e peptides of avian influenza A virus has a great potential to be developed into a novel vaccine for the prevention of avian influenza infection.
Animals ; Antibodies, Viral ; blood ; Bacteriophage T7 ; genetics ; immunology ; metabolism ; Chickens ; Enzyme-Linked Immunosorbent Assay ; Gene Expression Regulation, Viral ; Immunization ; Influenza A virus ; genetics ; immunology ; Influenza Vaccines ; immunology ; Influenza in Birds ; immunology ; metabolism ; prevention & control ; Peptides ; genetics ; immunology ; metabolism ; Polymerase Chain Reaction ; Recombinant Fusion Proteins ; Specific Pathogen-Free Organisms ; Viral Matrix Proteins ; genetics ; immunology ; metabolism

Bacterial ghost is intact envelope of Gram-negative bacteria, which is produced by the function of the lysis gene E from bacteriophage PhiX174. The expression of the lysis gene E is usually controlled by the thermosensitive lambdapL/pR-cI857 promoter. In this study, we described a mutation (T --> C) at the ninth nucleotide of the OR2 in the lambdapR promoter of the lambdapL/pR-cI857 system by overlap PCR. The bacteriolytic assay showed that the mutation in the lambdapL/pR-cI857 system enhanced the temperature of repressing the expression of gene E up to 37 degrees C. The lysis efficiency of altered lambdapR promoter in Escherichia coli DH5a and avian pathogenic E. coli DE17 was up to 99.9%. The expanded range of temperature will benefit for the production of bacterial ghost.
Bacteriolysis ; physiology ; Bacteriophage lambda ; genetics ; Base Sequence ; Cell Membrane ; physiology ; DNA, Bacterial ; analysis ; Escherichia coli ; genetics ; growth & development ; physiology ; virology ; Gene Expression Regulation ; genetics ; Molecular Sequence Data ; Mutation ; Promoter Regions, Genetic ; genetics ; Viral Proteins ; genetics ; metabolism

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

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