By Zhichao Hu

Highlight

•Virus is indicator of environmental disturbance from pandemic prevention supplies.

•Novel Coronavirus RNA were detected in natural lake near designated hospitals.

•Trihalomethanes-induced resistance caused 21-fold rise in 2 human virus abundance.

Glucocorticoids promoted replication potential of 3 human viruses by 13.3 times.

Abstract

Due to extensive COVID-19 prevention measures, millions of tons of chemicals penetrated into natural environment. Alterations of human viruses in the environment, the neglected perceiver of environmental fluctuations, remain obscure. To decipher the interaction between human viruses and COVID-19 related chemicals, environmental samples were collected on March 2020 from surroundings of designated hospitals and receivers of wastewater treatment plant effluent in Wuhan. The virus community and chemical concentration were respectively unveiled in virtue of virome and ultra-high-performance liquid chromatography-tandem mass spectrometry. The complex relationship between virus and chemical was ulteriorly elaborated by random forest model. As an indicator, environmental viruses were corroborated to sensitively reflect the ecological disturbance originated from pandemic prevention supplies. Chemicals especially trihalomethanes restrained the virus community diversity. Confronting this adverse scenario, Human gammaherpesvirus 4 and Orf virus with resistance to trihalomethanes flourished while replication potential of Macacine alphaherpesvirus 1 ascended under glucocorticoids stress. Consequently, human viruses lurking in the environment were actuated by COVID-19 prevention chemicals, which was a constant burden to public health in this ongoing pandemic. Besides, segments of SARS-CoV-2 RNA were detected near designated hospitals, suggesting environment as a missing link in the transmission route. This research innovatively underlined the human health risk of pandemic prevention supplies from the virus – environment interaction, appealing for monitoring of environmental viruses in long term.

Graphical abstract

1. Introduction

Human viruses in the environment pose considerable effects to public health and microbial ecology. Environment matrices were gradually recognized as huge reservoirs of human viruses (Labadie et al., 2020) with the assistance of rapidly developed non-culture methods such as metagenomics and virome (Paez-Espino et al., 2016Schulz et al., 2020). In aquatic environments, gastroenteritis-related AdenovirusAstrovirus and Rotaviruses were detected in rivers, reservoirs (Wang et al., 2020), and even drinking water (Mehle et al., 2018). In terrestrial ecosystems, the prevalence of Influenza A virus was over 80% in soil matrix associated with poultry (Lau et al., 2019). Apart from causing disease, Influenza A virus threatened public health for indirectly initiating the expression of antibiotic resistance in human microbes (Zhang et al., 2020). With the raging of COVID-19 pandemic, the footprint of waterborne SARS-CoV-2 was tracked in waste water treatment plant (WWTP) systems all over the world (Carducci et al., 2020). Besides, the ubiquitous human viruses in the environment affect the microbial ecology through antagonistic or mutualistic interaction. As a consequence of antagonistic relationship (Paul et al., 2021), bacteria that grazed on Echovirus via proteolytic enzymes could outcompete their counterparts (Olive et al., 2020). Norovirus in the environment attached to pathogenic bacteria via histo-blood group antigens (Amarasiri and Sano, 2019) and heightened the bacterial adherence as well as their co-infection to eukaryotic cells (Neu and Mainou, 2020). In addition, human viruses, as the proxy of human-originated contaminants (Farkas et al., 2020), would be sensitive perceivers of environmental turbulence. During COVID-19 when anthropogenic activities to prevent the spread of pandemic tremendously altered environment, human viruses lying in the intersection of “One Health” perspective (Shaheen et al., 2022) should be elaborated to suppress the potential side effect.

Human viruses in the environment were regulated by various parameters such as temperature, relative humidity and pH (Carratalà et al., 2020). Nevertheless, the interaction between human viruses and chemicals originated from pandemic prevention supplies that were excessively used during COVID-19 remain misty. For the sake of disinfection, chlorine and quaternary ammonia surfactant containing biocidal agents were exorbitantly used in hospitals and wastewater treatment plants (WWTPs) (Elsaid et al., 2021). In Wuhan, the average usage of disinfectants in 26 WWTPs was roughly 40 t day−1 normally (Text S1) but it ascended to 84.64 t day−1 during pandemic (Wuhan Water Affairs Bureau, 2020). As a recommended adjuvant therapy for treating COVID-19 patients (Sterne et al., 2020), glucocorticoids were clinically used with a proportion of 44.5% (Guan et al., 2020). The consumption rate of glucocorticoids in Wuhan was reckoned at 44,168 mg day−1 on March 2020 (Text S2). Environment, the reservoir of human viruses, was inevitably confronted with the lash from residues and by-products derived in disinfectants and glucocorticoids. To what extent chemicals related with COVID-19 prevention i.e., chlorine, trihalomethanes, quaternary ammonium surfactants, and glucocorticoids shaped human viruses in the environment was obscure.

To delve into the interplay between chemicals from COVID-19 prevention supplies and human virus community in the environment, soil and water samples were collected from designated hospital surroundings and downstream of WWTPs in Wuhan on March 2020. Human virus community in this special period was manifested by means of metagenomics and virome. Random forest models on various taxonomic levels were executed to investigate the environment – virus interaction with total chlorine, trihalomethanes, quaternary ammonium surfactants, and glucocorticoids taken into consideration. Furthermore, mechanism underlying the bilateral relationship was explored from the dimension of virus viability represented by replication, recombination and repair genes.

2. Materials and methods

This study aimed to unravel the effect of excessively used pandemic prevention supplies on human viruses in the environment and the response of human viruses to the lash from pandemic prevention supplies. To this end, environmental samples were collected from surroundings of hotspots in Wuhan city i.e., designated hospitals and WWTPs on March 2020. Consumptions of glucocorticoids and disinfectants were calculated to measure the environmental burden from overused pandemic prevention supplies. Virome was conducted to illustrate the virus community; and UPLC-MS/MS was adopted to detect chemicals generated by pandemic prevention supplies. Random forest model was constructed to identify the interaction between human viruses and chemicals. Genes related with viability were picked out to clarify the mechanism of human viruses – pandemic prevention supplies interplay.

2.1. Sample collection

As one of the first cities that took measures to suppress COVID-19 (Zhou et al., 2020), Wuhan was typical in evaluating the ecological impact of pandemic prevention supplies. Designated hospitals for novel coronavirus pneumonia, the biggest consumer of drugs, were considered as source of glucocorticoids in the environment during COVID-19. WWTPs, the major consumer of disinfectants, linked chemicals and environment with effluent (Larsen and Wigginton, 2020). Medical wastewater from JinYinTan Hospital, HuoShenShan Hospital, and LeiShenShan Hospital was ultimately discharged to FuHe River, HanJiang River, and XunSi River after disposal of SanJinTan WWTP, CaiDian WWTP, and HuangJiaHu WWTP (Fig. 1). Hence, water and soil samples were collected at 11 sites from surroundings of designated hospitals and receivers of WWTPs (Table S1). To meet the demand of following virome analysis, 500 g of topsoil (0–10 cm) and 1.0 L surface water (∼0.5 m) were simultaneously gathered (Liu et al., 2013Hu et al., 2021). The concentration of Fe3+ was adjusted to 1 mg L-1 through adding FeCl3 solution for flocculating virus particles (John et al., 2011). Virus particles were then enriched by ultracentrifugation and 0.22 μm filtration. Soil samples and 0.22 μm membranes were stored at −80 °C for subsequent treatment.

2.2. Virome sequencing and processing

DNA and RNA were respectively extracted by PowerSoil DNA Isolation Kit (Qiagen, German) and RNeasy PowerSoil Total RNA Kit (Qiagen, German) (Wang et al., 2019). Reverse transcription reaction was conducted with PrimeScript RT Master Mix (Takara, Japan). Library construction and sequencing were carried out on Illumina HiSeq 2500 platform. To diminish ribosomal and host sequences, raw data were first processed by Trimmomatic (Bolger et al., 2014) and BWA alignment (Li and Durbin, 2009). For downstream virome analyses, clean data were assembled with Megahit v1.1.2 (Li et al., 2016) and clustered to unique contigs through CDHIT v4.7 (Fu et al., 2012). Two strategies i.e., database alignment (blast v2.9.0 + ) and hidden Markov models (Paez-Espino et al., 2016) were concurrently adopted to identify virus contigs. The RPKM (Reads Per Kilobase per Million mapped reads) value of each contig (Liu et al., 2020) was calculated to determine virus abundance. Information about virus functional genes was acquired through blastp (v2.9.0 + ) with UniProtKB/Swiss-Prot database as blueprint. Especially, zoonotic viruses and viruses that exclusively host human were categorized as human viruses in this research. A catalogue comprising sequences annotated as human viruses was constructed as human viral genomes.

2.3. Determination of chemicals from COVID-19 prevention supplies

Four types of chemicals derived from pandemic prevention supplies i.e., total chlorine, trihalomethanes represented by trichloromethane, quaternary ammonium surfactants represented by benzalkyl dimethylammonium compounds, and glucocorticoids represented by methylprednisolone were taken into consideration in this study. Prior to chemical detection, water samples and soil samples treated with ultrasonic were enriched by solid-phase extraction (SPE) (Xu et al., 2019). Subsequently, concentrations of 4 trihalomethanes, 5 quaternary ammonium surfactants, and 40 glucocorticoids were determined by ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) (Chen et al., 2021). Detailed information referring to concentration of above chemicals were exhibited in Table S2.

2.4 Estimation of COVID-19 prevention supply.

Consumptions of glucocorticoids and disinfectants were estimated in designated hospitals and WWTPs during COVID-19. According to the COVID-19 designated hospitals in Wuhan, the whole city could be divided to three regions (Fig. 1). In every region, designated hospital and WWTP would respectively be the major consumer of glucocorticoids and disinfectants. Thus, the estimated amount of glucocorticoids and disinfectants could stand for consumption of pandemic prevention supplies in each region.

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