key: cord-317355-z5tk3v3b
authors: Dunker, Susanne; Hornick, Thomas; Szczepankiewicz, Grit; Maier, Melanie; Bastl, Maximilian; Bumberger, Jan; Treudler, Regina; Liebert, Uwe G.; Simon, Jan-Christoph
title: No SARS-CoV-2 detected in air samples (pollen and particulate matter) in Leipzig during the first spread
date: 2020-10-13
journal: Sci Total Environ
DOI: 10.1016/j.scitotenv.2020.142881
sha: 
doc_id: 317355
cord_uid: z5tk3v3b

The SARS-CoV-2 pandemic co-occurred with pollen season in Europe 2020 and recent studies suggest a potential link between both. Air samples collected at our measuring station in Leipzig and purified pollen were analyzed for SARS-CoV-2 typical signals or for virus-induced cytopathic effects, to test if the virus could bind to bioaerosols and if so, whether these complexes are infectious. The results show that neither air samples nor purified pollen were infectious or could act as carrier for virus particles.

The analysis of the first pandemic phase of severe acute respiratory syndrome coronavirus 2 in Europe 2020 demonstrated that infections co-occurred with the pollen season (Brandstetter et al., 2020) . Moreover, pollen may interfere with antiviral immunity, e.g. pollen significantly diminish the epithelial response to rhinovirus infection (Gilles et al., 2020) . To date it is not known, if pollen allergy has an impact on the prevalence or severity of the SARS-CoV-2 pandemic. In China, allergic diseases, asthma, and COPD were not shown to be risk factors for SARS-CoV-2 infection (Zhang et al., 2020) , but in this region the maximum peak was reached during winter time, outside the pollen season and patients were not interviewed for any history of pollinosis.

Importantly, pollen can act as a carrier for various bacteria and moulds (Heydenreich et al., 2012; Obersteiner et al., 2016; Oteros et al., 2019) , and SARS-CoV-2 remained viable in aerosols for several hours (van Doremalen et al., 2020) . Furthermore, Morawska & Cao (2020) (Morawska and Cao, 2020) speculate that wind-induced dispersion may occur. It was speculated, if bioaerosols may serve as carriers for the virus as regions with high air pollution were more severely affected (Conticini et al., 2020) and just recently it could be demonstrated that SARS-CoV-2 RNA can be present on PM (Setti et al., 2020) . We therefore aimed at investigating whether SARS-CoV-2 can bind to pollen or other kind of particulate matter within bioaerosols sampled at our station in Leipzig and if so, whether these complexes are infectious.

J o u r n a l P r e -p r o o f Fresh pollen samples from Betula pendula, Quercus robur and Ostrya carpinifolia were collected (Tab. A1). Inflorescences were dried in paper bags at 30 C and pollen were sieved through a 70 µm filter (CellTrics, Sysmex, Norderstedt, Germany) and stored dry upon analysis. Highly purified reference material (Alnus glutinosa, Betula pendula and Corylus avellana) were purchased from Allergon AB (Ängelsholm, Sweden).

Attempts of SARS-CoV-2 isolation were made using natural environmental samples (described above, Table 1 ). Following centrifugation at 11,000 rpm (5 min/ room temperature) supernatant was removed and saved for PCR testing (CoV PCR day 0). The pellet was resuspended in DMEM supplemented with nystatin/ amphothericin (40 µg/ ml/ 50 µg/ ml), seeded onto Vero E6 cells in a 2.5 cm 2 petri dish, incubated at 37 °C/ 5 % CO 2 and observed for 5 days for the occurrence of virus-induced cytopathic effects (CPE). Negative at a final concentration of 0.2 µM, and 1 µl SSIII/Platinum Tag. Input-RNA was 5 µl, and 2.6 µl of water was added to reach a final reaction volume of 20 µl. Alternatively RdRp_SARSr-F2/R1 and RdRp_SARSr-P2/P1 were used for amplification and detection of the respective gene. RT was done at 55 °C for 10 min, followed by 3 min denaturation at 94 °C and 45 amplification cycles with denaturation for 15 sec at 94 °C, annealing for 30 sec at 58 °C and elongation for 30 sec at 60 °C. Amplification was monitored after each elongation step at 530 nm.

The pollen season in Leipzig, Germany started with a high number of hazel (Corylus sp.) pollen at the beginning of February 2020, followed by alder (Alnus sp.) one week later (Fig.   1 ). Nine days after the last higher Alnus sp. pollen peak (94 pollen/ m³), the first Covid-19 cases were documented. Overall, low case numbers were registered in Leipzig in contrast to other German cities, most likely related to an early lockdown (red bar in Fig. 1 ). The birch (Betula sp.) pollen season started during the decrease of registered Covid-19 cases in April and was followed by pollen emission from oak (Quercus sp.) and pine (Pinus sp.). Particulate matter (PM 2,5 ) concentrations were partially higher than the threshold level of 10 µg/m³ (annual mean) defined by the WHO (dotted grey line in Fig. 1) . However, the concentrations were still lower than in regions of Northern Italy (Bianconi et al., 2020; Conticini et al., 2020) or Wuhan (Ma and Kang, 2020) and could be an additional reason for low registered case numbers in Leipzig.

Air samples containing bioaerosols and particulate matter were collected starting with the first wave of infections (grey bars in Fig. 1 ). In none of these samples SARS-CoV-2 typical For a detailed analysis of a possible correlation between concentrations of the most abundant pollen, particulate matter and registered Covid-19 cases, a correlation matrix was created with R (package "PerformanceAnalytics") (Fig. 2) . The number of registered cases (green box, Fig. 2 ) was positively correlated with the pollen-concentration of Corylus sp. and negatively correlated with the concentrations of grasses (Poaceae). (Table 1) . Next, we wished to determine whether pollen can bind SARS-CoV-2 at all. To address this issue, commercially available, highly purified pollen (Alnus glutinosa, Betula pendula and Corylus avellana) were incubated in vitro with SARS-CoV-2 (3x10 4 TCID 50 in 0.5 ml DMEM, i.e. approx. 1x10 7 genome equivalents) for 1 hr. Thereafter, pollen was washed, centrifuged as indicated in the supplement before they were screened for presence of viral RNA and infectious SARS-CoV-2. Again, no SARS-CoV-2 typical signal was detected by RT-PCR or CPE at any time point (Table 1) . Moreover, we could exclude the possibility that components of the air samples interfered with virus detection since Geq of SARS-CoV-2 spiked to original samples were clearly detected by RT-PCR or CPE (Fig. A1 , Table A2 ). We are somewhat consoled by this negative finding since a positive result would have had severe implications for SARS-CoV-2 restrictions, as pollen can be transported over long distances (up to several 100 km) (Hjelmroos, 1991) .

It should be noted however, that due to a low number of registered Covid-19 cases in Leipzig, it may be difficult that a measurable virus load can be detected in the air due to low number of emitters, especially since infected persons also had to go into quarantine immediately. Therefore, a negative result for SARS-CoV-2 is not surprising for this case study, but could J o u r n a l P r e -p r o o f potentially be different in cities with much higher infection rates. Statistically, it is highly probable to find virus on pollen and particulate matter when the concentration of both is really high also meaning that there is a coalescence effect between droplets and PM. This probably confirms that the spread effect in Leipzig was essentially due to the direct contact human-to-human and not mediated by a vehicle, while in regions with higher PM load, transmission via PM could be an additional pathway (Setti et al., 2020) .

We also considered technical limitations to account for our failure to detect SARS-2 CoV-2 signals in air samples and in purified pollen preparations. Air sampling: Several authors have shown, that cyclone samplers are suitable to collect virus particles in the air (D'Arcy et al., 2014; Kim et al., 2018; Verreault et al., 2008; West and Kimber, 2015) . D`Arcy et al. (2014) (D'Arcy et al., 2014) successfully detected airborne virus particle in hospital air with a cyclone sampler, which is in accordance with the fact that this technology was originally developed to collect biological warfare agents in the air. The height of our measurement station was chosen to guarantee a representative measurement in contrast to near-ground stations which show higher variability in pollen concentrations (Rojo et al., 2019) .

Nevertheless, it would be interesting to analyze if SARS-CoV-2 signals in near-ground traps, e.g. on crowded public places can be detected. SARS-CoV-2 detection: Nucleic acids were analyzed by RT-PCR and infectivity was tested by analysis of CPE on Vero indicator cells.

We could exclude that suspended air samples or purified pollen interfered with SARS-CoV-2 replication since SARS-CoV-2 spiked to the original samples was readily detected (Fig. A1 , Table A2 ). Our in vitro incubation experiments of highly purified pollen and SARS-CoV-2 were performed in fluid suspension. However, the binding pattern between pollen and virus particles could be potentially different in the air, e.g. due to electrostatic effects. Such effects might be of interest in future studies.

In summary, this is a first study investigating the relation of pollen and SARS-CoV-2 pandemic. The results show that neither air samples nor purified pollen of different taxa were infectious or could act as a carrier for virus particles. This leaves open the possibility that pollen or particulate matter within bioaerosols may affect SARS-CoV-2 susceptibility indirectly for example by perturbing nasal or bronchial epithelial barrier function or anti-viral immunity as shown by others for rhinovirus infection (Gilles et al., 2020) .

The Leipzig e.V. as potential conflicts of interest. Note: there was no inhibition of SARS-CoV-2 replication. * SARS-CoV-2 PCR; E, RdRp and N gene, measured without prior cell cultivation, day 0 ** SARS-CoV-2 PCR; E, RdRp and N-Gen, measured after cultivation for 8 days J o u r n a l P r e -p r o o f 

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Studies were co-financed by the German Science Foundation via the iDiv-Flexpool (project 100269858). We especially want to thank the Dr. Födisch Umweltmesstechnik AG for

The author Regina Treudler indicated Sanofi Genzyme, Novartis, ALK-Abello, AbbVie, Shire, Fraunhofer Institute IZI and Hautnetz Leipzig e.V. as potential conflicts of interest.J o u r n a l P r e -p r o o f