C. Meng et al. nanobe.org OPEN ACCESS Article Nano Biomed Eng ISSN 2150-5578 http://nanobe.org Development of a Rapid and Convenient Method for Sampling Airborne Virus based on Nanopar- ticle Adsorption Chun Meng*, Yulin Xiong, Weiliang Zhuang, Hang Wang, Xian’ai Shi, Yanghao Guo Department of Bioengineering, College of Biological Science and Biotechnology, Fuzhou University, Fuzhou, Fujian 350108, China. *Corresponding authors. Email: [email protected], Tel: 86-591-22866379, Fax: 86-591-22866379 Abstract An improved aerobiological virus sampling method was developed based on adding adsorptive nanoparticles in samplers for concentrating viruses in sampling liquid buffers. The objectives of this research were to select effective adsorptive materials and optimize sampling parameters for increasing recovery of airborne viruses, such as influenza A virus or respiratory syndrome virus (RSV). Three kinds of polycation nanoparticles were evaluated for direct effects on absorption and desorption of influenza virus hemagglutinin and DNA. Chitosan particles showed good performance in absorption and desorption for both influenza virus hemagglutinin and DNA. A subsequent study evaluated the effects of collection buffer, pH and sampling time on the recovery of aerosolized viruses using a method for making direct comparisons of three treatments. The results demonstrated that various components in air-sampling collection buffer, impinger model, and sampling time, independently influenced the recovery of viruses. It was shown that adsorptive samplers with air disperser had the highest levels of sensitivity and repeatability in virus sampling. Both unspecifically adsorptive chitosan particles and specifically adsorptive particles labeled specific antibody to virus significantly enhanced recovery rate of aerosolized viruses. We succeeded to sample low level different pathogen viruses in outdoor environments with the optimized sampling system. Keywords: Aerobiological virus sampling method; Adsorption; Polycation chitosan nanoparticles; Influenza virus; Air disperser Citation: C. Meng et al. Development of a Rapid and Convenient Method for Sampling Airborne Virus based on Nanoparticle Adsorption. Nano Biomed Eng. 2010, 2(3), 171-176. DOI: 10.5101/nbe.v2i3.p171-176. 1. Introduction people worldwide will suffer from this disease and die Since the beginning of last year, the H1N1 subtype from the pandemic Influenza. of influenza A virus (2009 H1N1or SIV) has been cir- Infection by direct contact can occur when infected culating in America, and now in Asia, European coun- hosts are in close proximity with a susceptible popula- tries, causing widespread infection in Human beings. tion. Moreover, infected hosts can transmit the disease As of September 13 last year, the World Health Organ- not through direct contact but through airborne trans- ization regions had reported more than 296,471 labora- mission. Viruses, can remain infectious outside their tory-confirmed cases of 2009 H1N1 influenza virus hosts for prolonged periods of time, and this can lead to with at least 3,486 deaths (http://www.who.int/csr/don infections by indirect contact in confined or public /2009_09_18/en/ ). With the season change to fall and space, such as public transport, meeting room, bar, and winter, we will face the threat of H5N1 subtype of in- so on [1]. The probability of airborne transmission of fluenza A virus in the northern hemisphere. In a very an infectious disease can be determined by conducting long period of time, the outbreak of influenza A virus epidemiological studies and/or by directly analyzing will be a big threat to humans health, and millions of the virus content of air samples [2]. Nano Biomed. Eng. 2010, 2, 171-176 171 C. Meng et al. nanobe.org Co., Ltd, China. DEAE Sephadex A-25 was purchased Table 1. PCR primer and hydrolysis probe sequences Specificity Primer/probe Sequencea (5’–3’) from Amersham Pharmacia. Sense AGA TGA GTC TTC TAA CCG AGG TCG Here, an oxime-based chemistry method was used Influenza Antisense TGC AAA AAC ATC TTC AAG TCT CTG for covalently linking hemagglutinin antibody to poly- A virus H1 Probe FAM-TCA GGC CCC CTC AAA GCC GA-TAMRA saccharide particles. In this process, aldehyde carbo- Sense ACG TAT GAC TAT CCA CAA TAC TCA G nyls of polysaccharides are reacted with the highly nuc- Avian H5 Antisense AGA CCA GCT ACC ATG ATT GC leophilic aminooxy group to form oximes [13]. For the FAM-TCA ACA GTG GCG AGT TCC CTA GCA- Probe conjugate, the ratio (wt/wt) of polysaccha- TAMRA rides/antibody was at a range between 1:0.002 and Sense GCACCACCTCACCCAGAC 1:0.005. RSV Antisense CAGTTCCTGCGCCTTGAT Probe FAM-CCTCTGCTTGCAATCGATCCAGAC-TAMRA 2.2 Samplers and sampling methods Sense GTGCTATAAACACCAGCCTYCCA At a vacuum pressure of not more than -0.05 atm, SIV H1* Antisense CGGGATATTCCTTAATCCTGTRGC the adsorptive sampler (a 0.3 liter glass cylinder reser- FAM-CAGAATATACATCCRGTCACAATTGGARAA- Probe TAMRA voir, 0.2 m high) operated at a flow rate of 1.5 liters per FAM, 6-carboxyfluorescein; TAMRA, 6-carboxytetramethylrhodamine. minute. And a circle polyethylene tube (inner diameter: * World Health Organization. CDC protocol of real-time RT-PCR for swine 2.0mm, thickness: 0.5mm) containing full of holes on influenza A (H1N1) http://www.who.int/csr/resources/publications/s wine- the wall of part immerged under water was used as the flu/realtimeptpcr/en/index.html ). disperser. Vacuum pressure was maintained using oil- Though effective vaccines and drug treatments could less pumps and was monitored using a vacuum pres- in some degree make the prevention and control of this sure gauge. Flow rates of impingers in liters per minute disease, more effective measures for limiting a poten- were verified using a flow meter. A commercial impin- tial outbreak is identifying and controlling the source of ger model was compared in terms of the recovery of inuenza. One of the most effective prevention me- aerosolized viruses. thods is a large-scale quarantine of suspected patients. We used a similar equipment designed by Hermann But it is still not enough for controlling suspected pa- to test the recovery efficiency of air virus [15]. A 20 tients because suspected patients meant that the man liter glass reservoir was modified to allow simultane- had been likely infected with the flu virus. If we detect ous sample collection at outlet ports. High pressure the virus in the air effectively, we may manage to reach sterilization silicone tube was used to connect the sam- earlier prevention of virus transmission to susceptible pling bottles to the outlet ports. This arrangement made people. Detecting virus in air is very essential because it possible to test up to different treatments simulta- inuenza is a typical airborne transmission disease neously on the same cloud of aerosolized. caused by influenza viruses. Isolation of viruses from particles was performed Impingers direct a converged stream of environmen- with different concentration NaCl buffer (5 ml buffer tal air onto or in a liquid collection medium to recover per ml particles). airborne particles in the liquid phase of the collection system [3-10], Which can be used as virus sampling 2.3 Nucleic acid isolation and amplification with because it is more effective than filters, bubblers, or RT-PCR and real-time PCR. impactors for capturing airborne viruses [11-13]. But it The influenza virus H1N1 (a gift from Prof. Pingfan is less effective in detecting low concentration viruses Rao, Fuzhou University) DNA and membrane protein in air because of difficulty of concentrating viruses and hemagglutinin was extracted with commercial kits pur- inefficiency in trapping viruses in air. Development of chased from Sangon, China, respectively. a sensitive, quick, accurate, and comprehensive sam- A QIAGEN QIAamp Viral RNA Mini kit was used pling system for air virus detection becomes very es- for the extraction of nucleic acid from all of the sam- sential. In this study, we reported the development of a ples. The starting material for nucleic acid isolation new liquid impinger sampling system for sampling in- was 200 ml viral isolate in dilution buffer (phosphate- fluenza A virus in air based on absorptive method in buffered saline). RNA was eluted into 50 ml water. liquid. The primer sequences used for these studies are shown as Tab 1. All probes were labeled at the 5’ end 2. Materials and methods with the 6-carboxyfluorescein (FAM) reporter dye and at the 3’ end with the 6-carboxytetramethylrhodamine 2.1 Particles preparation: (TAMRA) quencher dye. The Qiagen one-step RT- Chitosan nanoparticles were prepared according to PCR kit was used with a 20 l reaction mixture accord- the procedure reported by Calvo [14] based on the io- m ing to the reference [16]. The RT step conditions for nic gelation of CS with TPP anions. all primer sets were 30 min at 50°C and 15 min at 94°C. 201-4 anion exchange resin (strong basic quarternary PCR cycling protocol was used for the matrix gene ammonium) was purchased from Huachang Polymer primer set as described in table 1. Nano Biomed. Eng. 2010, 2, 171-176 172 C. Meng et al. nanobe.org Hemagglutinin was extracted from H1N1 viruses application in the next study. It was shown that only with a membrane protein extraction Kit (Shanghai chitosan particles of the three polycation absorption Sangon, China) according to the recommendations. The resins evaluated exhibited satisfying results on the both level of hemagglutinin was detected with ELISA men- absorption and desorption of either virus DNA or he- tod [17]. Statistical analysis Data (including RSD value) magglutinin in the buffer. On the basis of the overall were analyzed using the statistical software program results, chitosan nanoparticles were selected for use in (Excel). the following work. 3. Results 100 3.1 Effect of adsorptive particles on adsorptive effi- ciency 80 Based on ion adsorption of viruses or cells with sur- % 60 face negative charges on polycations particles, we de- y r e veloped an adsorptive method through adding polyca- v o c tion particles as the adsorbent in impingers for sam- e 40 R pling viruses in air.