Detection and identification of potentially infectious gastrointestinal and respiratory viruses at workplaces of wastewater treatment plants with viability qPCR/RT-qPCR

This study aimed to qualitatively and quantitatively assess the prevalence of the most common respiratory and gastrointestinal viruses in the air, surface swab, and influent/effluent samples collected in wastewater treatment plants (WWTPs). Application of qPCR/RT-qPCR (quantitative polymerase chain reaction/reverse-transcription quantitative polymerase chain reaction) assays combined with PMA (propidium monoazide) dye pretreatment allowed detecting the potentially infectious and disintegrated viral particles in collected samples. In the air at workplaces in WWTPs, the most frequent isolation with the highest concentrations (reaching up to 103 gc/m3 of potentially infectious intact viral particles) were observed in case of adenoviruses (AdVs) and rotaviruses (RoVs), followed by noroviruses (NoVs). Viruses were significantly more often detected in the air samples collected with Coriolis μ impinger, than with MAS-100NT impactor. The temperature negatively (Spearman correlation: –1 < R < 0; p < 0.05), while RH (relative humidity) positively (0 < R < 1; p < 0.05) affected airborne concentrations of potentially infectious viral particles. In turn, the predominant viruses on studied surfaces were RoVs and noroviruses GII (NoV GII) with concentrations of potentially infectious virions up to 104 gc/100 cm2. In the cases of SARS-CoV-2 and presumptive SARS-CoV-2 or other coronaviruses, their concentrations reached up to 103 gc/100 cm2. The contamination level of steel surfaces in WWTPs was similar to this on plastic ones. This study revealed that the qualitative and quantitative characteristics of respiratory and gastrointestinal viruses at workplaces in WWTPs is important for proper exposure assessment and needs to be included in risk management in occupational environment with high abundance of microbial pollutants derived from wastewater.

www.nature.com/scientificreports/ A single-stage MAS-100NT impactor operates by aspirating the air through a 400-hole perforated inlet plate onto a Petri dish containing biological collection media. Each time, the air samples were collected for 20 min at a flow rate of 100 L/min on standard Petri dishes filled with the bi-phase medium consisting of solid phase mycoplasma base agar (MBA, Oxoid Ltd., Basingstoke, UK) covered with thin layer liquid-phase VTM to maximize the potential of viral particle recovery 39,43 . After each sampling session, an impactor inlet was cleaned and disinfected with isopropyl alcohol. After collection, the samples were transported to laboratory within 12 h where they were stored in − 80 °C until further analysis 44 . Surface swab sampling. In total, 54 swab samples were collected from stainless steel and plastic surfaces (machine valves, machine handles, hatch handles, machine controllers, handrails) with sterile polyester fibertipped swabs (Deltaswab PurFlock Ultra ViCUM, Deltalab, Barcelona, Spain) prewetted in 0.9% saline solution, which ensures the most effective recovery of viruses from nonporous fomites 45,46 (Table 1).
Wastewater samples. Fifteen wastewater influent and the same number of effluent samples were collected (each of them into a sterile 1000 mL glass container) and kept in 4 °C for less than 24 h until further analysis. Laboratory analysis. Aerosol, surface swab and wastewater samples. All liquid media with air samples were concentrated by ultrafiltration using Amicon Ultra-15 (molecular weight cut-off 30 kDa) centrifugal filter device (Merck Millipore Ltd., Livingston, UK) at 3200 × g for 20 min in 4 °C 41,47 . Centrifugal concentration step was repeated until the entire volume of the sample passed through the filter. The concentrated samples (400 µL) were intended for further analysis. In turn, the swab shafts of swab samples were cut off, then placed into 400 µL of 1 × PBS (pH = 7.2) and vortexed thoroughly using a programmable rotator-mixer (Multi RS-60, Biosan, Riga, Latvia) at 800 rpm for 15 min. Influent and effluent wastewater samples were centrifuged at 4500 × g for 5 min in 4 °C and each obtained supernatant was concentrated as described above 47 .
PMA dye pretreatment. All processed samples were divided into two equal aliquots (200 µL). The first one was intended for direct viral DNA/RNA isolation, the second one for PMA dye pretreatment allowing detection of potentially infectious viral particles. In this case, the samples were treated with PMAxx Dye (20 mM in H 2 O; Biotium, Inc., Hayward, USA) for a final concentration of 60 µM 48 . Tubes were gently mixed by inverting several times and then incubated in the dark for 15 min at room temperature with rotation at 200 rpm. The treated samples were exposed to 40 W LED light with a wavelength of 460 nm for 15 min using a photo-activation system (PMA-Lite LED Photolysis Device; Biotum Inc.). Quantitative PCR/reverse-transcription quantitative PCR (qPCR/RT-qPCR) and viability quantitative PCR/viability reverse-transcription quantitative PCR (v-qPCR/v-RT-qPCR) assays. Both qPCR/v-qPCR (for DNA viruses) and RT-qPCR/v-RT-qPCR (for RNA viruses) were performed using CFX96 real-time PCR thermocycler (Bio-Rad, Hercules, USA). The detection of AdVs, HBoV, RoVs, NoVs, IAV, and SARS-CoV-2 were carried out with Adenovirus, Bocavirus, Rotavirus, Norovirus (GI and GII), Influenza A, and SARS-CoV-2 VIASURE Real Time PCR Detection Kits (all: CerTest Biotec S.L., Zaragoza, Spain), respectively, according to procedures recommended by the manufacturer. The applied PCR kits have a detection limit of ≥ 10 RNA/DNA copies per reaction. www.nature.com/scientificreports/ The target genes employed for PCR-based detection and identification of viruses represent conserved regions with the hexon gene for AdVs, the NSP3 gene for RoVs, the ORF1-ORF2 junction for NoV genogroup I (GI) and NoV genogroup II (GII), the M1 gene for IAV, the ORF1ab and N genes for SARS-CoV-2.
The cycling conditions for DNA viruses were as follows: polymerase activation at 95 °C for 2 min, then 45 cycles of denaturation at 95 °C for 10 s, and annealing at 60 °C for 50 s. In case of RNA viruses, the reverse transcription at 45 °C for 15 min was followed by initial denaturation at 95 °C for 2 min, then 45 cycles of denaturation at 95 °C for 10 s, and annealing at 60 °C for 50 s. According to the manufacturer's procedure, the fluorogenic data were collected through the FAM, ROX, and HEX channels. Both negative and positive controls, purchased from CerTest Biotec, were included in each run. All samples were tested in duplicates.
All qPCR/RT-qPCR and v-qPCR/v-RT-qPCR data were collected and quantification cycles (Cq) were calculated using CFX96 manager software (Bio-Rad). According to the manufacturer's instruction, the samples with Cq ≤ 40 for AdVs, HBoV, NoV GI, NoV GII, RoVs, and IAV as well as with Cq ≤ 38 for SARS-CoV-2 were considered as positive. In case of SARS-CoV-2, if only N gene target was positive, the interpretation was presumably positive for SARS-CoV-2 and the differentiation of SARS-CoV-2 from other coronaviruses, including animal ones, requires further analysis. The negative samples and the samples with Cq > 40 were reanalyzed after tenfold dilution to evaluate the possible presence of inhibitors. Quantification analyses were performed based on standard curves, obtained by amplification of positive control tenfold dilutions (standard from 1 × 10 1 to 1 × 10 7 gene copies/reaction), and log RNA/DNA copies were plotted against Cq value. All standard curves had efficiencies between 90 and 110% and r 2 above 0.98.
To minimize the potential contamination, all analytical steps were performed in separate rooms, including RNA/DNA isolation, preparation of reagents, sample preparation, and amplification. All analyzes were carried out using the sterile RNase/DNase-free filter pipette tips only. The obtained results were expressed as the number of viral genome copies per 1 m 3 of the air (gc/m 3 ), per 100 cm 2 of tested surfaces (gc/100 cm 2 ), and per 1 L of influent and effluent wastewater (gc/L).
Temperature and relative humidity. During sampling, the temperature and relative humidity of the air were measured using portable thermo-hygrometer (Omniport 20; E + E Elektronik GmbH, Engerwitzdorf, Austria).
Statistical analysis. The obtained results were statistically analyzed with Shapiro-Wilk, Fisher Exact, Kruskal-Wallis and Mann-Whitney test as well as Spearman's rank correlation coefficient using STATISTICA data analysis software system, version 7.1 (StatSoft Inc., Tulsa, USA). Probability values at p below 0.05 were considered statistically significant.

Results
Presence of viruses in the air, surface, and wastewater samples. The performed qPCR/RT-qPCRbased studies revealed the presence of gastrointestinal and respiratory viral nucleic acids in the air, on surface as well as in influent and effluent wastewater samples. In general, the most commonly detected nucleic acids indicated presence of AdV, RoV, and NoV GII. The most prevalent in the air were AdVs, on surfaces RoVs and NoV GII, in influent samples AdVs and NoV GII, and in effluent samples NoV GII (Table 2).
Taking into account bioaerosol sampling devices, the use of Coriolis μ impinger allowed to detect two types of DNA (AdVs, HBoV) and four types of RNA viruses (NoV GI, NoV GII, RoVs, and presumptive SARS-CoV-2 or other coronaviruses), while MAS-100NT one type of DNA (AdVs) and two types of RNA viruses (RoVs and presumptive SARS-CoV-2 or other coronaviruses). Among the air samples collected with Coriolis μ impinger, 46.2% were AdV positive, 34.6% RoV positive, 23.1% NoV GII positive, 15.4% NoV GI positive, 11.5% presumptive SARS-CoV-2 or other coronaviruses positive, and 7.7% HBoV positive, while in case of MAS-100NT impactor, 30.8% were AdV positive, and equally 19.2% RoV positive and presumptive SARS-CoV-2 or other coronaviruses positive. Viruses were significantly more often detected in the air samples collected with Coriolis μ impinger, than in samples gathered with MAS-100NT impactor (Fisher Exact test: p = 0.001).
Application of v-qPCR/v-RT-qPCR method revealed occurrence of potentially infectious intact viral particles. Percentages of samples containing potentially infectious viruses among all positive samples are presented in Table 2. In case of bioaerosol, the percentage of samples containing potentially infectious viruses among total positive samples ranged from 37.5% (for presumptive SARS-CoV-2 positive/other coronaviruses) to 75% (AdVs, NoV GI) and from 60% (for presumptive SARS-CoV-2 positive/other coronaviruses) to 87.5% (AdVs) for Coriolis μ and MAS-100NT samplers, respectively. Potentially infectious viruses were more often detected in bioaerosol collected with Coriolis μ impinger than with MAS-100NT impactor (Fisher Exact test: p = 0.033). In surface swabs, potentially infectious viruses were present from 33.3% (SARS-CoV-2) to 95.7% (AdVs) of positive samples and were more often detected on steel surfaces; however, this difference was not statistically   www.nature.com/scientificreports/ samplers, respectively (Table 3). In turn, the concentration of potentially infectious viruses revealed by v-qPCR/ v-RT-qPCR did not exceed 10 3 gc/m 3 for Coriolis μ impinger and 10 2 gc/m 3 for MAS-100NT impactor. Adenoviruses, rotaviruses and presumptive SARS-CoV-2 or other coronaviruses were the only agents detected with both samplers. The concentrations of these viruses were generally higher in samples collected with Coriolis μ impinger, especially in case of AdVs and presumptive SARS-CoV-2 or other coronaviruses (Mann-Whitney tests: p = 0.047 and p = 0.003, respectively). Regarding potentially infectious viruses, their significantly higher levels were noted for presumptive SARS-CoV-2 or other coronaviruses in Coriolis μ samples (Mann-Whitney test: p = 0.004).

Influence of temperature and relative humidity of the air on concentration of viruses.
For the examined sampling sites, the air temperature ranged from 13.9 °C in wastewater pumping section to 26.4 °C in dewatering/thickening sludge section. The highest relative humidity (RH) of the air was observed in wastewater pumping Sect. (64.3%), while the lowest within grit chamber Sect. (32.9%) ( Table 5). Statistical analysis showed that temperature negatively influenced the concentrations of all tested potentially infectious airborne viruses (Spearman correlation -in all cases: R = -0.536 to -0.951 at p < 0.05). The strongest negative correlations were observed for RoVs (R = -0.951 at p = 0.000) and AdVs (R = -0.924 at p = 0.000). Contrary to temperature, the RH positively correlated with the concentrations of all tested potentially infectious airborne viruses (in all cases: R = 0.710 to 0.747 at p < 0.05).
Quantitative analysis of DNA/RNA viruses in surface swab samples. The number of viruses detected in surface swab samples varied between 10 3 -10 6 gc/100 cm 2 . The use of v-qPCR/v-RT-qPCR revealed that the concentration of potentially infectious intact viral particles on surfaces vary from 10 1 gc/100 cm 2 to 10 4 gc/100 cm 2 ( Table 6). The highest concentrations of potentially infectious viruses were detected on steel surfaces for RoVs with the mean value of 5.37 × 10 4 gc/100 cm 2 (range 1.21 × 10 3 -2.04 × 10 4 ) and on plastic surfaces for NoV GII with mean value of 1.41 × 10 4 gc/100 cm 2 (range 2.04 × 10 2 -1.09 × 10 5 ). The average concentrations of potentially infectious AdVs, HBoV, NoV GI, and NoV GII on steel surfaces did not exceed 10 4 gc/100 cm 2 , while in the cases of SARS-CoV-2 and presumptive SARS-CoV-2 or other coronaviruses, their concentrations were below 10 3 gc/100 cm 2 . Also the average concentrations of potentially infectious HBoV, NoV GI, and RoVs on plastic surfaces did not exceed 10 4 gc/100 cm 2 , while in the case of AdVs and presumptive SARS-CoV-2 or other coronaviruses their levels were less than 10 3 gc/100 cm 2 . The comparison of potentially infectious virus   Taking into account the metal surfaces, the significant differences in concentrations of potentially infectious virions depending of sampling site were observed for AdV, HBoV, RoV and NoV GII viruses (Kruskal-Wallis tests: p = 0.03, p = 0.012, p = 0.001, and p = 0.014, respectively), while in case of plastic surfaces for AdV, NoV GII and presumptive SARS-CoV-2 viruses (Kruskal-Wallis tests: p = 0.036, p = 0.002, and p = 0.01, respectively). The sampling site dependent differences in concentrations of potentially infectious NoV GI virions were not significant for both steel and plastic surfaces ( Table 7).

Quantitative analysis of DNA/RNA viruses in influent and effluent wastewater samples.
The number of viruses in influent and effluent wastewater samples ranged between 10 4 and 10 7 gc/L and between 10 2 and 10 4 gc/L, respectively ( Table 8). The highest total concentration of viruses was observed in the cases of AdVs with the mean value of 9.84 × 10 7 gc/L (range 3.6 × 10 3 -8.45 × 10 8 ) and RoVs with the mean value of 1.14 × 10 7 gc/L (range 2.27 × 10 5 -7.68 × 10 7 ) in influent and in the case of NoV GI with the mean value of 9.29 × 10 4 gc/L (range 7.74 × 10 3 -1.42 × 10 5 ) in effluent samples.

Discussion
This study revealed that both gastrointestinal and respiratory viruses were present at workplaces in WWTPs. Their detection in influent samples indicates their wastewater-borne origin. The presence of viruses in occupational environments has been proved in several studies using PCR-based methods 1,36,37,39,49,50 ; however, it should be clearly pointed out that RT-qPCR/qPCR enables both qualitative and quantitative analyzes of viral RNA/DNA, but does not allow to assess viral infectious ability 51 . To the best of our knowledge, this is the first investigation qualitatively and quantitatively analyzing the presence of the most common gastrointestinal and respiratory viruses at workplaces in WWTPs and through the coupling of PMA dye with qPCR/RT-qPCR assays discriminating the potentially infectious and disintegrated viral particles in airborne, surface, and waterborne samples.
This study showed that both above mentioned groups of viruses were dispersed in the air at workplaces in WWTPs. Viruses, including potentially infectious ones, were significantly more often detected in the air samples collected with Coriolis μ impinger, than with MAS-100NT impactor. Coriolis μ sampler collected particles into  www.nature.com/scientificreports/ a liquid medium, while MAS-100NT device utilized bi-phase medium consisting of solid agar covered with a thin layer of liquid viral transport medium. Both samplers, however, have limitations when considering their use and induce particle loss. For single-stage impactors, bouncing of particles (when they strike the impaction surface) can lead to undersampling, destruction of collected particles, and decrease of collection efficiency. In turn, the cyclonic samplers were so far successfully used in several studies for collection of airborne viral particles 38,[52][53][54][55] . Impingers are not sensitive to overloading or undersampling as they provide generally 'gentle' particle collection. However, evaporation of the sampling liquid and reaerosolization of already trapped particles may bias the sampling results. As it was shown in this study, the strike of viral particles against the agar surface (even if it is covered with thin liquid layer) seems to be an important factor destroying viral particles in single-stage impactor. Moreover, the different sizes and airborne behaviors of tested viruses may also influence the capture efficiency of both samplers. Viruses can occur in airborne state in varied forms: as droplets that are relatively large and largely liquid (> 20 μm) or as medium (5-20 μm) and small (≤ 5 μm) size particles that may be composed of either liquid or solid materials. Fine particles can remain airborne for extended periods of time, especially if they are mostly composed of water (as the water evaporates, the viral particles become smaller in size over time) 56 . It is proved that single virus particles may exist in the air, but they tend to aggregate rapidly and/or may be 'protected' by larger particles, being adsorbed on their surfaces 40,57 . Viruses aggregated to larger  www.nature.com/scientificreports/ particles show higher survivability compared to particles, which real dimensions are close to the actual size of the virions 58,59 . Some authors indicated that the best isolation efficiency of viable intact viruses was observed in case of aggregated particles larger than 2.1 µm 58,60 . In case of NoVs and RoVs, their highest environmental concentrations in airborne state were noted for particles with diameters of > 4.5 µm and 9 µm, respectively 38,41 .
As the performed study showed the bioaerosol sampling utilizing cyclone with liquid collection medium seems to be a method of choice due to its fast collection of large air volume providing a high recovery rate for viral particles with broad spectrum of sizes. These features have high practical value, especially in occupational environments like WWTPs, where humidity conditions are very variable and other sampling methods (such as e.g. filtration or impaction) are not advisable 40,41 . At workplaces in the studied WWTPs, the most frequently isolated viruses belonged to AdV, RoV, and NoV groups, reaching up to 10 3 gc/m 3 , 10 4 gc/m 3 and 10 3 gc/m 3 of potentially infectious intact particles, respectively. It can be explained with the fact that non-enveloped viruses tend to be more stable in high RH conditions and, even being airborne, they could remain infectious for a longer period of time 1 . As it was showed the highest concentrations of airborne viruses were detected in wastewater pumping section (up to 10 4 gc/m 3 ), i.e. in the location where the lowest temperature and the highest RH were observed. For virus-containing aerosols, both these microclimate parameters have the key influence (i.e. temperature negative, while RH positive) on their survival in the air 56 . In turn, potentially infectious presumptive SARS-CoV-2 or other coronaviruses, which represent enveloped viruses, were present in the air of wastewater pumping section only, reaching concentrations at the level of 10 2 gc/m 3 . The occurrence of those viruses within described area results probably from aerosolization of raw sewage during pumping and was stabilized by high RH (64.3%) and www.nature.com/scientificreports/ low temperature (13.9 °C). Such picture is consistent with observations by Morris et al. 61 , who found that coronaviruses, including SARS-CoV-2, survive better in low temperature (about 10 °C) and at high RH (over 60%). Viruses from AdV, RoV, and NoV groups were detected in WWTPs by different research teams with the concentrations reaching 10 6 gc/m 3 for AdVs, 10 7 gc/m 3 for RoVs, 10 3 gc/m 3 for NoV GI, and 10 2 gc/m 3 for NoV GII 1,38,62 . The results obtained in this study indicated that the total virus and potentially infectious virion concentrations of AdVs and RoVs were lower and did not exceed 10 4 and 10 3 gc/m 3 , respectively; however, for both NoV genotypes were on the same levels. There is lack of information regarding the concentrations of coronaviruses in the air of WWTPs; however, airborne transmission of SARS viruses with droplets containing wastewater is very probable 63,64 . This study revealed that the concentrations of potentially infectious presumptive SARS-CoV-2 or other coronaviruses reached the level of 9 × 10 2 gc/m 3 in wastewater pumping section; however, no cases of COVID-19 among WWTP workers were observed. Hence, either the identified viruses were animal coronaviruses not harmful for humans or detected particles, even though they were intact, they lost infectious abilities.
In this study, gastrointestinal and respiratory viruses were also detected on surfaces at workplaces in WWTPs, reaching in case of potentially infectious intact viral particles the concentrations of 10 5 gc/100 cm 2 . Many studies have documented the possibility of virus transfer from hands to the surfaces of touched objects and back, which may play an important role in spreading of viral infections 42,[65][66][67] . The persistence and stability of viruses vary and depend on many biological (e.g. the type of virus, the presence of microorganisms that can show protective effects against drying and disinfectants) and environmental (e.g. temperature, RH, sunlight exposure, composition of colonized medium) factors 56,68 .
Viruses may remain infectious for extended period of time after deposition on objects. For example, the persistence of clinically relevant viruses on dry inanimate surfaces may range from 3 to 96 h for SARS associated virus and other coronaviruses, from 8 h to 7 days for NoVs, from 1 to 2 days for influenza virus, from 6 days to 2 months for RoVs, and from 7 days to 3 months for AdVs 69,70 .
According to Abad et al. 71 , viruses usually survive longer on non-porous surfaces compared to porous ones. In the present study, the swab samples from non-porous steel and plastic surfaces were analyzed.
It was found that viral particles, especially HBoV, NoV GII and SARS-CoV-2, were more often detected on steel surfaces than on plastic ones. This observation can be explained by the prolonged persistence of viruses on steel and other metal surfaces, which can be up to 120 days 72 . However, taking into account potentially infectious viral particle concentrations, it was found that both these inanimate surfaces at workplaces in WWTPs were contaminated to the same degree. The only exception in this case was noticed for HBoV. Its significantly higher concentrations on plastic than on steel surfaces could result from both the extreme stability of this pathogen and its high resistance to disinfectants 72 . The only exception was noticed for HBoV. Its significantly higher concentrations on plastic than on steel surfaces could be explained by both the extreme stability of this pathogen and its high resistance to disinfectants 73 .
The potentially infectious intact viral particles were frequently detected among virus-positive surface swab samples with the highest (above 90%) prevalence of AdVs, RoVs, and NoV GII. The DNA viruses, like AdVs, are usually more resistant to degradation than RNA viruses; however, RoVs and NoVs has been shown to tolerate a wide range of harsh environmental conditions like the presence of free chlorine, chemical disinfectants, extreme temperatures and humidities 74,75 . Thus, special measures should be applied to all surfaces and equipment having a direct contact with wastewater to remove viral contamination (e.g. the use of proper disinfectants degrading both RNA and DNA viruses). As some authors indicate that there are no direct relationships between sensitivity to UV light and the virion size, type of nucleic acid or presence/absence of the envelope, the diverse resistance of viruses to UV radiation should be taken into account, when UV light is intended to be used for inactivation of infectious viruses in WWTP environment 76 .
The abundance and diversity of pathogenic viruses, including potentially infectious intact viral particles in influent and effluent wastewater, result in their prevalence at workplaces in WWTPs. The concentrations of potentially infectious viral particles in influent wastewater reached up to 10 7 gc/L for AdVs, 10 6 gc/L for NoV GII and RoVs, 10 5 gc/L for HBoV, IAV, and presumptive SARS-CoV-2 or other coronaviruses, and 10 3 gc/L for SARS-CoV-2. Potentially infectious viruses were also detected in effluent samples in concentrations ranged from 10 2 gc/L for presumptive SARS-CoV-2 or other coronaviruses to 10 4 gc/L for HBoV and NoV GI. The obtained results were consistent with the data gathered by Corpuz et al. 36 . The presence of viruses in wastewater, and thus at workplaces of WWTPs, is closely related to the prevalence of these pathogens in the population. In this study, IAV was not detected in the majority of influent samples, suggesting that the community of the area served by the selected WWTPs was free of this pathogen. An incidentally detected positive sample among analyzed influents suggests that this sample may have been contaminated with bird feces, which are the natural reservoirs of IAV. The lack of IAV nucleic acids (noted in WWTPs and effluent samples) also suggests that IAV is susceptible to environmental degradation 26 .
The hitherto performed evaluations show that the application of PCR-based methods allows to detect both infectious and disintegrated non-infectious viral particles. Although coupling of PMA with qPCR and RT-qPCR has been successfully applied to distinguish infectious and inactivated viral particles in river water, raw manure, soil and food samples, there is lack of information about possible application of these methods in work environment research 31,34,35,48,76,77 . The results of this study showed that the application of v-qPCR/v-RT-qPCR allowed to discriminate potentially infectious intact viral particles and disintegrated virions in the air, surface, and wastewater samples. The 'classic' PCR methods are not able to discriminate between potentially infectious and non-infectious viral particles, which can lead to an overestimation of the target viruses. Hence, the positive results obtained in this way should be taken with precautions. Potentially infectious viral particle concentrations detected with v-qPCR/v-RT-qPCR in this study were usually lower (about one to three orders of magnitude) than the concentrations of total viral particles detected with q-PCR/RT-qPCR methods. The latter mentioned methods give more reliable information regarding actual contamination of sampling sites with potentially harmful viral www.nature.com/scientificreports/ particles. The application of v-qPCR/v-RT-qPCR allows eliminating the number of damaged viral particles from the results and providing information about potentially infectious intact viruses only. On the other hand, not all intact viral particles remain infectious in the environment. Hence, the concentrations of potentially infectious viruses detected with v-qPCR/v-RT-qPCR may be overestimated and further in vitro investigations are needed to define their real infectivity and subsequent real influence on workers' health. Moreover, as some authors point to biological activity of non-infectious viral particles, which may induce response of host cells, their adverse role for human health should not be neglected 78 . This study confirmed that WWTP workers were exposed to airborne viral particles, to viruses deposited on surfaces as well as present in influent and effluent wastewater samples. Such massive exposure may lead to the appearance of different infections 79 . Numerous studies have reported that gastrointestinal and respiratory symptoms (e.g. nausea, vomiting, cough, diarrhea, and fever) were observed more frequently among WWTP workers than in general population and may result from exposure to viruses during occupational activities 1,80 . Some viruses (like AdVs) cause generally mild respiratory tract infections, which are self-limiting and generally asymptomatic despite the virologic and serologic proof of infection 81 .
For SARS-CoV-2 viruses, there is evidence (albeit limited) that the minimum infectious dose in humans is greater than 100 particles 82 . In turn, inhalation of infectious dose of RoVs (which is below 100 viral particles) or NoVs (which is about 10 viral particles) may end up in adverse health outcomes 38,83 . According to Musher 82 , inhalation of 5 human adenovirus particles may cause disease in susceptible individuals and even the possibility of infections due to the inhalation of gastrointestinal viruses (with subsequent deglutition of virions deposited within oral cavity) cannot be also excluded. Although, it is difficult to directly compare the infectious doses expressed in viral genome copies with those given in the number of viral particles as these two measures are not equivalent to each other, the researchers have been constantly looking for such links to facilitate the exposure assessment to viruses and evaluate their influence on human health [84][85][86] .

Conclusions
Both gastrointestinal and respiratory viruses were present in the air and on surfaces at workplaces as well as in influent and effluent samples from WWTPs and as such may pose an occupational risk for workers. The most frequently isolated viruses, with the highest concentrations reaching up to 10 3 gc/m 3 and up to 10 4 gc/100 cm 2 of potentially infectious intact virions, were AdVs, RoVs, and NoVs. In the same time, potentially infectious viral particles of SARS-CoV-2 and presumptive SARS-CoV-2 or other coronaviruses were detected in concentrations up to 10 2 gc/m 3 and 10 2 gc/100 cm 2 . Although, the most contaminated area was in general the wastewater pumping section, the potentially infectious viruses occurred within all workplaces involved in wastewater treatment processes. Hence, the risk of infection increases especially in situations where personal hygiene is inadequate (e.g. hand washing is not proper and not enough frequent, there is a lack of personal preventive measure within the areas where bioaerosol forming process are present, eating or drinking at the workplace etc.). To reduce the probability of virus transmission, efficient cleaning procedures degrading viral particles for frequently touched surfaces and objects should be introduced and the use of personal protective equipment, especially within areas where bioaerosol particles are aerosolized, should be mandatory. In this context, both identification and quantification of potentially infectious viruses in WWTPs and other occupational environments with high abundance of microbial contaminants are an important part of safety work management and proper health risk assessment. The application of v-qPCR/v-RT-qPCR represents a big step forward in analysis of viruses in different environmental matrices allowing better interpreting the workplace exposure to these emerging pollutants and should be included in the monitoring procedures for occupational biohazards.