Production and Storage of Virus Simulants

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.

Internal control for real-time polymerase chain reaction based on MS2 bacteriophage for RNA viruses diagnostics

BACKGROUND Real-time reverse transcription polymerase chain reaction (RT-PCR) is routinely used to detect viral infections. In Brazil, it is mandatory the use of nucleic acid tests to detect hepatitis C virus (HCV), hepatitis B virus and human immunodeficiency virus in blood banks because of the immunological window. The use of an internal control (IC) is necessary to differentiate the true negative results from those consequent from a failure in some step of the nucleic acid test. OBJECTIVES The aim of this study was the construction of virus-modified particles, based on MS2 bacteriophage, to be used as IC for the diagnosis of RNA viruses. METHODS The MS2 genome was cloned into the pET47b(+) plasmid, generating pET47b(+)-MS2. MS2-like particles were produced through the synthesis of MS2 RNA genome by T7 RNA polymerase. These particles were used as non-competitive IC in assays for RNA virus diagnostics. In addition, a competitive control for HCV diagnosis was developed by cloning a mutated HCV sequence into the MS2 replicase gene of pET47b(+)-MS2, which produces a non-propagating MS2 particle. The utility of MS2-like particles as IC was evaluated in a one-step format multiplex real-time RT-PCR for HCV detection. FINDINGS We demonstrated that both competitive and non-competitive IC could be successfully used to monitor the HCV amplification performance, including the extraction, reverse transcription, amplification and detection steps, without compromising the detection of samples with low target concentrations. In conclusion, MS2-like particles generated by this strategy proved to be useful IC for RNA virus diagnosis, with advantage that they are produced by a low cost protocol. An attractive feature of this system is that it allows the construction of a multicontrol by the insertion of sequences from more than one pathogen, increasing its applicability for diagnosing different RNA viruses.

A methodological study on testing and evaluating of filtration efficiency of canister against microbial aerosol / 中华预防医学杂志

<p><b>OBJECTIVE</b>To establish a testing and evaluating method for filtration efficiency of the canister against microbial aerosol.</p><p><b>METHODS</b>Serratia marcescens aerosol served as model of bacterial aerosol, Bacillus subtilis var niger aerosol as model of spores aerosol, bacteriophage f(2) aerosol as model of viral aerosol. Employing the microbial aerosol testing platform was established in lab, models of microbial aerosol generated artificially were sampled quantitatively by air samplers before and after filtrating by canisters, respectively. Filtration efficiency was determined by the concentration of microbial aerosol in the air sample before and after filtrating. The four canisters of 1-1, 1-2, 1-3, 1-4 were tested for the filtration efficiency against Serratia marcescens, Bacillus subtilis var niger and phage f(2) aerosol. The two canisters of 543 and 544 canisters equipped with active carbon were tested for the filtration efficiencies against Serratia marcescens aerosol.</p><p><b>RESULTS</b>The filtration efficiency of 1-1, 1-2, 1-3 canisters against Serratia marcescens, Bacillus subtilis var niger and phage f(2) aerosol was 100.000%. The filtration efficiency of 1-4 canister filtration efficiency against Bacillus subtilis var niger spores aerosol was 99.997% and efficiency of the other two aerosol was 100.000%. The filtration efficiency of the two canisters of 543 and 544 to those attached with active carbon against Serratia marcescens aerosol was 100.000%.</p><p><b>CONCLUSION</b>The testing method might be used to evaluate the protective performance of the canister against microbiological aerosol. The effect of the canisters (including those equipped with active carbon) against microbiological aerosol should be reliable.</p>