Detection of adeno- and lentiviral (HIV1) contaminations on laboratory surfaces as a tool for the surveillance of biosafety standards

Abstract

Aims: As a biosafety laboratory, we survey the handling of adenovirus type 5 (Ad5) and HIV1‐derived lentivirus in contained‐use facilities in Switzerland to identify insufficiencies of the safety precautions taken by the laboratories. Methods and Results: In the past 9 years, we took 430 swab samples from various types of surfaces in research laboratories. Samples were examined for Ad5 contaminations by real‐time PCR and infectivity assay or for the presence of lentivirus (HIV1) nucleic acids by real‐time (RT) PCR. Samples collected from centrifuges did not only contain Ad5 DNA more frequently but also exhibited higher numbers of Ad5 and lentiviral (HIV1) DNA copies than swabs from any other area of sampling. Five of ten samples containing infectious Ad5 particles or lentivirus (HIV1) RNA were found in samples taken from centrifuges. Ad5 contamination rates were higher in the tube holder and lower on the inner wall of the rotor chamber in centrifuges that were fitted with aerosol tight covers compared to centrifuges without covers. Conclusions: Our results allowed the comparison of hygiene standards of different laboratories and lead to the identification of centrifuges as hotspots for contaminations. Significance and Impact of the Study: Based on our results, we propose to use the collected data as a tool for rating future swab results. Furthermore, the amount of Ad5 and HIV1‐derived lentivirus DNA could serve as an indicator of the level of good laboratory practice in contained‐use laboratories handling these viral vectors.

Washout Kinetics of Viral Vectors from Cultured Mammalian Cells

“…The pLL3.7 plasmid (lentiviral vector) (Rubinson et al, 2003) as well as the helper plasmids pMD2.G, pRSV-Rev, and pMDLg/pRRE (Dull et al, 1998) were obtained from Addgene Inc. (Cambridge, MA, USA). Lentivirus (HIV1) pLL3.7 stock solutions were prepared as described earlier (Bagutti et al, 2011) and concentrated by Lenti-X Concentrator (Clontech, Takara Bio Europe, St. Germain-en-Laye, France) according to the manufacturer's protocol. Adenoassociated virus serotype 2 (AAV2; ATCC-VR-680, [Hoggan et al, 1966]) was purchased from LGC Standards (Teddington, UK).…”

“…Ad5 infection of HEK293 and Hela cells was performed by adding Ad5ΔE1GFP stock solution (two separate viral preparations) to wells of a 12-well cell culture plate (TPP, VWR, Dietikon, Switzerland) before the cells were added, leading to a confluency of 70% (Bagutti et al, 2011). Twenty-four hours later the medium was exchanged (standard protocol) and the cells were incubated for another 3 days.…”

“…For the detection of Ad5, AAV, and vaccinia virus, DNA was extracted according to the QIAamp DNA Mini Kit protocol (Qiagen, Basel, Switzerland) in combination with the extraction robot QIAcube (Qiagen) according to the manufacturer's standard procedure. Lentiviral (HIV1)-specific nucleic acids (DNA and RNA) was extracted using the QIAamp Viral RNA Mini Kit (Qiagen) as described earlier (Bagutti et al, 2011).…”

“…The following primers and probes were used: for the detection of Ad5: Ad5fiber-F (5'-aag cta gcc ctg caa aca tca-3', at a final concentration of 100 nM), Ad5fiber-R (5'-ccc aag cta cca gtg gca gta-3', 300 nM), and Ad5fiber-FAM (FAM-5'-cct cac cac cac cga tag cag tac cct tac-3'-TAMRA, 100 nM) (Bagutti et al, 2011); for lentivirus (HIV1): HIV-1-PSS-F (5'-cgc agg act cgg ctt gct-3', 400…”

“…17, No. 4, 2012 nM), HIV-1-PSS-R (5'-gac gct ctc gca ccc at-3', 400 nM) and HIV-1-PSS-Pr (5'-FAM-ccy ctc gcc tct tgc ygt gyg crc -TAMRA-3', 150 nM) (Bagutti et al, 2011); for AAV2: AAV-WT-F (5'-agt tga act ttg gtc tct gcg tat t-3', 500nM), AAV-ITR3-R2 (5'-cat cac tag ggg ttc ctt gta gtt aat-3', 500nM), and AAV-ITR3-VIC (VIC-5'-att aac ccg cca tgc tac tta tct acg tag cc-3'-TAMRA) for the detection of a sequence covering parts of the Rep protein genes and the inverted terminal repeats (ITR) of AAV2 (GenBank ® Acc.Nr. AF043303: 4446-4525); for VVeGFP: VvI4L-F (5'-gac act ctg gca gcc gaa at-3', 500nm); VvI4L-R (5'-ctg gcg gct aga atg gca ta-3', 500 nm), and VvI4L-VIC (VIC-5'-agc agc cac ttg tac tac aca aca tcc gga-3'-TAMRA, 150 nm) for the detection of a fragment of the ribonucleotide reductase I4L (GenBank ® Acc.Nr.…”