Sources, enumerations and inactivation mechanisms of four emerging viruses in aqueous phase

Owing to changing climate and deforestation (Tollefson 2020), excess or unintentional anthropogenic disposal of mutagens and teratogens in the environment have facilitated the emergence of severe infectious diseases caused by a number of pathogens, e.g., Coronavirus, Ebola virus, Zika virus, and Lassa virus, that have claimed a myriad number of innocent lives and adversely impacted the socio-economic environment. Different strains of Coronaviruses (CoV), i.e., Middle East Respiratory Syndrome CoV (MERS-CoV), Severe Acute Respiratory Syndrome CoV (SARS-CoV), and SARS-CoV-2 claimed about 1,294,815 lives out of 52,743,880 confirmed cases from 2002 to 2020 (WHO 2003; JHU 2020). Five Ebola viruses, i.e., Zaire Ebola virus (ZEBOV), Sudan virus (SUDU), Reston virus (RESTV), Tai Forest virus (TAFV), and Bundibugyo virus (BDBV), including other members of the Filoviridae family, i.e., Marburgvirus genus (MARV and RAVV) (Kuhn et al. 2010) claimed about 15,697 lives of 35,215 confirmed cases since 1967 (WHO 2020), when about 87.2% deaths occurred after 2015 through various exposure routes. Although Zika virus (ZIKV) is not extremely deadly, there were 50 fatalities reported until 2019 (Cardona-Ospina et al. 2019) and Lassa virus mortality rate is about 5,000 deaths/year (Ogbu et al. 2007). Whatever the exposure routes of these pathogens, extensive cleaning or washing of infected surfaces and wastes by a universal solvent or cleaning agent, i.e. water, can convey those pathogens in water reclamation facilities (WRF) or resource recovery facilities (RRF) or other surface water that may be later reclaimed as potable water. Thus, environmental engineers have a leading role in developing early awareness through wastewater-based epidemiology (WBE) about the occurrence of an epidemic or a pandemic.

Waste from diverse sources can exacerbate the situation in an RRF; however, inactivation of pathogens by some well-known disinfectants may manage the initial trigger of an outbreak. Careful application of disinfectants is a task of the utmost importance because of the possibility of disinfection by-products’ (DBPs) formation through excessive use of disinfectants in the system. DBPs’ formation can not only impose some extra treatment costs but also activate some chemical carcinogens in the presence of natural organic matter (NOM) in secondary or tertiary treatment levels, causing deterioration of the situation.

Moreover, enumeration of viable cells by cell culture may not be fully realized at this moment as a laboratory equipped with bio-safety level 3/4 (BSL-3/4) cabinet is not available in all facilities or laboratories. Although reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been applied to extract deoxyribonucleic acid (DNA)/ribonucleic acid (RNA) from different aliquots, viability of cells may not be confirmed from the extracted DNA/RNA. The presence of DNA/RNA can deliver a clue about the presence/absence of specific pathogens but infectiousness may, nonetheless, not be comprehended. Thus, researchers have been employing significant effort in enumeration and inactivation of pathogens causing emerging infectious diseases so that wastewater-based early alertness can be effectively developed. Although enumeration will develop awareness about the presence of certain viruses in the environment and aqueous phase, inactivation may ensure the extent of effectiveness of available disinfectants and possible prevention of dispersion of cells in both gaseous and aqueous phases.

Although it is comparatively stress-free to remove and inactivate those pathogens from a surface as few factors may influence the removal process, inactivation of those pathogens from water and wastewater, where myriad number of factors may affect the removal process, is an arduous task because some inactivation processes may not be pragmatic with presently available resources. Some published review papers are dedicated to discussing the inactivation of SARS-CoV-2, but a concrete review report on transport, dispersion, and inactivation of SARS-CoV, MARS-CoV, Ebola virus, Zika virus, and Lassa virus in aqueous phase is not available; inactivation mechanisms, the most important subject to successfully tackle an outbreak, are not elaborately discussed in those review papers. This report covers inactivation techniques of all the above-mentioned emerging viruses with richly depicted inactivation mechanisms of those viruses with possible impact of climate change on the evolution of those viruses. The main objectives of this report are to discuss the possible sources of four emerging pathogens’ contaminated wastewater, their enumeration process with currently available resources, inactivation mechanisms by disinfectants and other physicochemical inactivation techniques in aqueous phase so that quality potable water can be supplied to the end consumers and community awareness can be built on some rationales.

Filoviridae, the family of filamentous (length >1,000 nm) negative-sense single-stranded RNA virions, includes five major species of enveloped Ebola viruses (average diameter 80 nm; length 130–2,300 nm), responsible for diseases in humans and primates. Coronaviridae, the family of enveloped (coated by lipid bi-layer) positive-sense single-stranded RNA Coronaviruses, i.e., alphacoronavirus (α-CoV), betacoronavirus (β-CoV), gammacoronavirus (γ-CoV), and deltacoronavirus (Δ-CoV) (ICTV 2019), is responsible for diseases of birds, mammals, and amphibians. Among these, α-CoV and β-CoV species are basically accountable for mammal diseases. ZIKV (average diameter 40 nm), an enveloped virus from the family Flaviviridae and genus Flavivirus, with positive-sense, single-stranded RNA, can cause diseases in humans, birds, reptiles, and mammals. Lassa virus (average diameter 70–150 nm), a member of the Arenaviridae family with negative-sense, single-stranded RNA with envelope, can be transmitted from rats’ feces, urine, saliva, blood, and respiratory secretion to humans (Ogbu et al. 2007).

The first two families end up in wastewater through zoonotic spillover and oral–fecal route of transmission, and prominent sources of both families in water bodies are shown in Figure 1. Although non-enveloped viruses are more stable in water compared to enveloped viruses, enveloped RNA viruses have conspicuous survivability in water from 96-h to 720-h (Judson et al. 2015). ZIKV transmission is possible not only by mosquitoes, i.e., Aedes aegypti and Aedes albopictus (Du et al. 2019), or tick bites or zoonotic spillover but also by vertical (i.e., from mother to child) or horizontal (i.e., from one person to another) transmission, sex, blood transfusion, accidental exposure; the virus can contaminate aquatic environments or wastewater through discharging of semen, urine (0.7–220 × 10⁶ genome copies/ml) (Gourinat et al. 2015), saliva, breast milk, amniotic fluid, and cerebrospinal fluid (Du et al. 2019). Lassa virus has been detected in infected people's blood, feces, urine, vomit, semen, and saliva (Ogbu et al. 2007) that can ultimately mix with wastewater and other sources of potable water. A patient infected by Ebola virus discharges about 9 l/d body fluid where virus genome concentration ranges from 2.8 to 7.2 log₁₀ viral RNA copies/ml and Ebola virus is noticeably persistent in blood (Bibby et al. 2017). Ebola virus genome concentration in stool is 7 log₁₀ RNA copies/ml, 5–6 log₁₀ RNA copies/ml in urine, and 2–9 log₁₀ RNA copies/ml in blood (Bibby et al. 2015; Casanova & Weaver 2015). MARS-CoV genome concentration in a patient's blood and serum generally varies from 3.3 to 4.2 log₁₀ genome copies/ml and from 5 to 6 log₁₀ RNA copies/ml, respectively (Keil et al. 2016; Hindawi et al. 2018). SARS-CoV-2 genome concentration in urine, stool, saliva or sputum, wastewater influent and effluent, and primary and secondary sludge can be 52 × 10⁶ genome copies/l (Bardi & Oliaee 2021), 6.8 log₁₀ genome copies/g (La Rosa et al. 2020), 10⁴–10¹⁴ genome copies/l (Giacobbo et al. 2021), from 5 × 10⁴ to 3 × 10⁶ genome copies/l (Wurtzer et al. 2020), 7.5 × 10³–14.7 × 10³ genome copies/l (Zhang et al. 2020), and 1.17–4.02 × 10⁴ genome copies/l (Bardi & Oliaee 2021), respectively. So far, SARS-CoV-2 and its gene have been detected in different types of water, e.g., untreated wastewater in France, Australia, Japan, Spain, USA, UK, Italy, Netherlands, India, UAE, Chile, Slovenia, Czech Republic and many other countries; water contaminated by untreated or partially treated wastewater; urban river water in Ecuador; sea water in Spain, UK, Morocco, and ocean organisms (García-Ávila et al. 2020). Therefore, virus cell inactivation and DNA/RNA disintegration are equally important to achieve total eradication of these viruses in aqueous phase as ruptured virus cells will spill nucleic acid and that nucleic acid may facilitate the replication of those viruses in the environment.

All these pathogen-contaminated wastes can reach water and RRF. However, when these kinds of viruses are identified in water or wastewater, treatment plant operators should not discharge those pathogens with effluent without treatment as accidental inhalation or ingestion (WHO 2018; Yaro et al. 2021) or adsorption through cuts or sores (CDC 2021) can release those viruses into the bloodstream or gastrointestinal tract or nosocomial route and severe complexity may occur after such exposure. Moreover, meat consumption of infected mammals (i.e., bats, rats, and monkeys) and wild birds may be a possible transmission route of those viruses from animals to humans as those wild animals/birds have proven to be hosts of those viruses (Gutiérrez-Bugallo et al. 2019; Yaro et al. 2021); hence, treatment plant operators must take the utmost care to prevent a probable aquatic or gaseous phase transmission route.

Precise and accurate enumerations are very important for quick and early detection and warning to safeguard society, the environment, and eco-system against any outbreak of an epidemic and pandemic caused by emerging pathogens. For this reason, scientists have made significant efforts to develop user-friendly, cost-effective, and efficacious techniques: i.e., microscopic (e.g., scanning electron microscope (SEM); transmission electron microscope (TEM); atomic force microscope (AFM)), cultural (e.g., plating methods; most probable number (MPN); primary and continuous cell culture; cytopathogenic effect (CPE); plaque-forming unit (PFU); 50% tissue culture infectious dose (TCID₅₀)), physiological, immunological (e.g., antibody; immune-affinity chromatographic assay; enzyme-linked immunosorbent assay (ELISA); immunofluorescense assay (IFA)), and nucleic acid-based (e.g., real time-PCR; RT-qPCR; digital PCR (dPCR); integrated cell culture-PCR (ICC-PCR); gene probe) analysis to support WBE, although applications of those techniques are dependent on several aspects, i.e., purpose, species, time-frame, and available resources. Among these technologies, RT-qPCR has been used as the gold standard to detect SARS-CoV-2 infection (Chen et al. 2020). Table 1 shows different enumeration techniques for targeted pathogens in water and wastewater.

Some inactivation mechanisms of species of Ebola viruses and their surrogates are discussed below with the necessary information shown in Table 2. Casanova & Weaver (2015) evaluated the thermal inactivation rate of bacteriophage Φ6, a double-stranded RNA (ds-RNA) enveloped virus, as a surrogate of Ebola virus in wastewater at 22 °C (psychrophilic condition) and 30 °C (mesophilic condition) where inactivation rate was very fast at 30 °C compared to 22 °C. Inactivation rate was linear at 30 °C but inactivation rate was non-linear and segmented, i.e., different rates at different time periods at 22 °C; and 6–7 log₁₀ inactivation could be achieved within 3–7 days where temperature, virus concentration, virus type, strains, treatment process, holding time, and wastewater composition may be important factors in virus removal. Inactivation rate of virus in surface water was slower than that in wastewater; however, inactivation rate was faster in wastewater than in pure water. Moreover, dsRNA structure of Φ6 contributed more stability in the inactivation process compared to ssRNA Ebola virus and Coronavirus.

Bibby et al. (2015) examined the persistence of Ebola virus in sterilized wastewater where virus titer had decreased due to inactivation, particle aggregation and adsorption on solids in wastewater. All these mechanisms had decreased virus titers in aqueous phase but persistence may increase on solid surfaces if those surfaces act as a shield. Ninety per cent inactivation was identified after 2.1-d including time zero time point and 6.6-d excluding time zero time point. Inactivation was slower in wastewater compared to deionized water where 90% inactivation was observed within 1.8-d at 21 °C but the rate of inactivation was faster than that of in blood as 90% inactivation was achieved in 21-d. Persistence of Ebola virus in wastewater is lower than that of other enteric viruses, i.e., hepatitis A (>17-d), adenovirus (33-d), and poliovirus (5-d). Microbial, i.e., bacteria and protozoan activity may, however, decrease virus titer in wastewater by consuming those viruses as nutrient sources or forming different metabolites that are harmful for virus existence. Again, WHO recommended a holding time of 1 week to aid 3-log virus inactivation.

Fischer et al. (2015) witnessed ZEBOV stability in different inanimate surfaces and aqueous phase with different temperatures and relative humidities (RHs); and ZEBOV was more stable at 21 °C rather than 27 °C with ≈1-log₁₀ removal/d. ZEBOV persistence in liquid blood was longer than that in dry blood because nutrient and metabolite transfer in dry conditions are relatively tough due to hampered biochemical conditions. ZEBOV stability in non-human primate (NHP) blood (14-d) is almost similar to in human blood; and several factors, i.e., virus titer concentration, virus species, sample matrices, parameters related to weather and sterility can influence the virus infectivity and dispersion when zoonotic transformation may be a serious issue.

Disinfection characteristics for three variants of ZEBOV, i.e., 1976 Mayinga, 1995 Kikwat, and 2014 Makona were examined by Cook et al. (2016) by using 70% ethanol (EtOH), 0.05%, 0.1%, 0.5%, and 1.0% sodium hypochlorite (NaOCl) as disinfectants; and Makona variant had higher resistance than that of the other two variants regardless of temperature and RH. Disinfection efficacy was increased with increasing concentration and contact time. Cultured cell inactivation was the most reliable and standard methodology; however, RNA extraction was effectively accomplished rather than recovery of Ebola virus particles from specific media. Evolution of ZEBOV was significantly noticeable as disinfectant resistance developed with time.

Bibby et al. (2017) depicted Ebola virus inactivation in unsterilized and gamma-irradiated wastewater by NaOCl with concentrations of 0, 1, 5, and 10 mg/l and noticed rapid chlorine decay in irradiated wastewater due to the presence of significant chlorine demand in the sample when virus inactivation was very slow in that same sample. Although rapid virus inactivation was observed with a disinfectant dose of 1 mg/l in the first 20-s, no removal was observed later because of the rapid decrease of free chlorine (FC) and no virus recovery was observed for disinfectant doses of 5 and 10 mg/l. 4-log₁₀ virus removal was achieved in concentration time (Ct) value of 1.1 mg-min/l when NaOCl concentration was 5 and 10 mg/L; however, USEPA recommended value for the same removal is 3 mg-min/l. Some factors, i.e., pH, composition of wastewater, mixing, and virus aggregation can affect the inactivation process. No virus removal was detected between pH 4.3 and 6.9 but 1-log₁₀ virus removal was discovered after 193-min at pH 11.2; thus, virus can be killed at elevated pH.

Lin & Marr (2017) investigated aerosolization and removal of two surrogates, i.e., MS2 and Φ6 of ZEBOV, by toilet flushing, converging of pipes and in aeration tank, although it was noticed that 94% of virions generally remain in aqueous phase rather than in gaseous phase or in bio-solids. The number of aerosols produced was high in aeration tank (aerosol diameter: 55–60 nm) compared to toilet flushing (aerosol diameter: 25–52 nm) and pipe converging (aerosol diameter: 130–162 nm), but the volume of aerosol produced was large in toilet flushing rather than in aeration tank and pipe convergence. Aerosolization of MS2 was more efficient than Φ6 due to the absence of lipid bi-layer and small structure (25–80 nm) of MS2. Virus emission in aerosol form was observed from both aeration tank and pipe convergence, although emission from toilet flushing was very slight. However, Φ6 emission was rapid compared to MS2 because Φ6 had high absorption and thermal inactivation rates. Also, virus emission was high from aeration tank and low from pipe convergence; and aeration tank virus emission rate was ten times higher than that of from pipe convergence, where both systems had high potential of inhalation exposure to treatment facility workers. MS2 and Φ6 were used as surrogates of ZEBOV in this experiment, but the icosahedral structure of Φ6 significantly differed from the filamentous structure of ZEBOV. Aerosolization may also differ due to certain factors, i.e., virion size and shape, fluid viscosity, mixing, turbulence, wastewater composition, operation variables, environmental parameters and area open to air.

Strasser (2017) inspected the survivability of three surrogates, i.e., MS2, ΦX-174, and Φ6 of ZEBOV by thermal (i.e., 22 °C and 37 °C) and chemical (i.e., NaOCl) inactivation processes applied in a mixture of raw hospital wastewater (75%) and human fecal waste (25%). Virus survivability decreased with increasing temperature, contact time, and disinfectant dose. The virus can be protected by aggregation (i.e., 2–3 virus particles are attached together), adsorption on another virus particle or on any other particulate matter, and embedded on a surface if disinfectant is not properly mixed with the sample. Therefore, chlorine can inactivate pathogens by penetrating the outer envelope, losing and damaging cell components, oxidizing a number of amino acids in proteins and enzymes, degradation of nucleic acids, altering the structural components, and hampering ATP production process; however, resistance is a factor basically depending on virus species and defense mechanisms. Moreover, ammonia concentration in wastewater with pH varying from 8.0 to 9.3 can also facilitate the virus inactivation process, although duration of the removal process is quite lengthy, but combined chlorine with ammonia by forming mono-chloramine, di-chloramine, and tri-chloramine can effectively inactivate virus with the substantial possibility of DBPs’ formation. Furthermore, inactivation mechanisms, e.g., reactions with amino acids, namely, cysteine, cystine, methionine, and tryptophan; fragmentation of RNA and denaturing the protein structure (Jacangelo et al. 1987) have been observed because of chloramines. Required chlorine concentration can be minimized in water and wastewater by reducing turbidity, oxidizable organic matter, and chlorine demand for other inorganic compounds. Average daily inactivation was 0.12-log, 0.06-log, and 0.55-log removal at 22 °C for MS2, ΦX-174, and Φ6, respectively, and 0.2-log, 0.26-log, and 3.2-log removal at 37 °C for MS2, ΦX-174, and Φ6, respectively; where as Φ6 was most sensitive to thermal treatment as well as to chemical disinfectant. Inactivation was faster in the first 3-min for all species and inactivation rate was 1.7, 4.4, and 5.7 times faster at the temperature of 37 °C for MS2, ΦX-174, and Φ6, respectively.

Wolfe et al. (2017) compared inactivation of two ZEBOV surrogates, i.e., Escherichia coli and Φ6 by chlorine-based disinfectants from rinse water loaded with organic substances (i.e., 7.80 mg/ml bovine serum albumin, 10.92 mg/ml tryptone, 2.52 mg/ml bovine mucin) or without any organic loading (OL) where inactivation rate was high for E. coli rather than Φ6 because virus has high resistance compared to bacteria against chlorine-based disinfectants; and the presence of organic substances can slightly decrease inactivation rate for E. coli but marginally increase Φ6 removal. Stabilized and freshly generated NaOCl had a similar removal rate for both surrogates regardless of OL; however, all chlorine-based disinfectants had significant removal efficacy for bacteria and virus. Chlorine generally oxidizes the outer membrane and peptidoglycan layer of bacteria, whereas lipid membrane and capsid proteins of virus are basically ruptured in the presence of corrosive oxidizing agents.

Wood et al. (2020) studied the inactivation of two well-known surrogates, i.e., MS2 (non-enveloped outer-layer) and Phi6 (enveloped outer-layer) of ZEBOV by hydrogen peroxide vapor (HPV) from six different types of surfaces where non-enveloped viruses had high resistance to disinfectant compared to enveloped viruses and high concentration of HPV (>400 mg/l) had more inactivation efficiency than that of low HPV (≈25 mg/l). Blood was present as organic loading in the system where virus persistence was significantly increased in the presence of blood since blood acted as a shield for the virus particles or blood oxidation decreased disinfectant concentration available to inactivate viruses. However, virus inactivation was more than 3-log reduction within 2-h in the absence of blood and other factors, i.e., temperature, RH, outer-layer structure and types of surfaces can influence removal mechanisms. It is also surmised that ZEBOV may be more stable in blood compared to water. Moreover, H₂O₂ (≈17 mg/l) alone had insignificant inactivation potential (<0.1-log) against MS2; but 4-log removal was realizable by the combined action of Fe(II) and H₂O₂ within a very short time and reactive oxygen species’ (ROSs) formation had a major influence on the inactivation process where increasing H₂O₂ concentration, increasing pH, and the presence of complex forming agents had deleterious impacts on the inactivation process (Kim et al. 2010).

Warfield et al. (2007) inspected inactivation of ZEBOV by combining UV irradiation and a photo-inducible alkylating agent, named 1,5 iodonaphthylazide (INA), without damaging the antigenic function and structural integrity of cells; and no sign of virus infectivity and replication was detected in the system after treatment with UV + INA. No morphological and structural conformation change was observed under TEM after treatment, although significant distortion of structural immunogenic epitopes was identified due to crosslinks of surface glycoprotein (GP) and lipid by-layers where virus infectivity was hindered by spoiling GP fusion activity. However, INA-treated ZEBOV stimulated the adaptive response since some non-crossed antibodies emerged from denatured antigens and a strong immunogenic response can be expected from INA-treated virus. This technique is a better approach to damage virus infectivity but its impact on water and wastewater treatment should be carefully evaluated because of the presence of some mutagenic, teratogenic, and carcinogenic compounds.

Elliott et al. (1982) examined Ebola virus inactivation by gamma irradiation (⁶⁰Co) at 4 °C and −60 °C. Inactivation in the frozen state needed a greater amount of energy than that in liquid state where the synergistic impact of radiation and heat was responsible for inactivation. Moreover, this method achieved a better inactivation outcome compared to UV light.

Thus, Fenton-like reactions (electro or photo-assisted) have perceptible antimicrobial potential where DBPs’ formation, because of high H₂O₂ concentration, may be minimized due to organic oxidation, and mineralization of cell debris and remaining DBPs can be achieved. Semiconductor materials in the presence of a photon source can accelerate the degradation process but the presence of OL (except biological cell) in the aliquot can decrease the efficiency of oxidative damage of the pathogen cell and its components. Excess OL, however, is a dilemma in the Fenton-supported process: OL can act as electron source (e⁻) to form ^•OH from H₂O₂ but formed ^•OH may be consumed to oxidize other organic chemicals. A parallel decontamination process may remove various pollutants from the aqueous phase, even though recalcitrant pollutant formation may be a possibility. Minimization of total organic carbon (TOC) content will be an issue for the advanced oxidation process (AOP)-assisted disinfection process efficiency; otherwise, full potential of ROSs and H₂O₂ in pathogen inactivation may not be achieved.

During the deadly outbreaks, a significant number of studies have been conducted and some of their relevant outcomes are critically discussed below, with the removal efficacy of pathogens, e.g., SARS-CoV, SARS-CoV-2, and MARS-CoV, shown in Table 3. Lamarre & Talbot (1989) enthusiastically tried to understand the impacts of pH and temperature on HCoV-229E inactivation when infectivity of different strains of the same family can respond differently to pH and temperature as most Coronaviruses may be inactivated at acidic pH and high temperature, i.e., >25 °C. However, virus viability may not be affected at extreme pH at low temperature, i.e., 4 °C, and virus was the most stable at pH 5.0–8.0. Rapid thawing and freezing of virus particles, however, may not affect the morbidity or mortality of viruses as rapid change of temperature may not distort protein and DNA/RNA structure or hinder enzymatic activity.

Sizun et al. (2000) inspected the survival of HCoV-229E and HCoV-OC43 in suspension and dry conditions where both viruses were stable for at least 6-d in suspension; and HCoV-229E had more resistance to chemical disinfectants than that of HCoV-OC43 as ten times more concentrated disinfectant (i.e., Proviodine) was required for 50% reduction of HCoV-229E but diluted chemical disinfectants had low potency to inactivate virus. Virus persistence was short in dry conditions compared to in suspension and virus had maximum infectivity at neutral pH, whereas inactivation was increased at pH <5.0 and pH >8.0. However, destabilization of lipid bi-layer was the prominent mechanism to inactivate the virus but nucleic acid disruption, protein denaturation and sorption may be other mechanisms in the complete virus removal process.

Darnell et al. (2004) scrutinized the inactivation of SARS-CoV by different mechanisms, i.e., photo-inactivation, thermal inactivation, and chemical inactivation as all inactivation mechanisms had shown noticeable virus removal in favorable conditions. Photons from UVC, UVB, and UVA can be absorbed by DNA and RNA where two neighboring pyrimidines may be covalently bonded to form dimers by photochemical fusion which will give non-paired bases in DNA or RNA; but, UVC can more effectively catalyze this photo-inactivation mechanism than that of UVB (20–100 times slower than UVC) and UVA (weaker than UVB), although UVA can facilitate the formation of ROSs to damage bases and proteins by oxidation. Gamma irradiation (⁶⁰Co) may not be effective at low dose but high dose can slowly trigger inactivation; where several factors, i.e., irradiation time and dose, wavelength, irradiation distance, pathogen concentration, antibody of pathogen, and pathogen species can affect the removal process. Thermal inactivation can hamper enzymatic activity or denature the proteins for bases, attachment and replication; and high temperature initiated rapid inactivation of virus from solid and aqueous media while aggregation of virus may need longer heat exposure to be inactivated in/on specific media. Chemical–based inactivation by formaldehyde (HCHO) and glutaraldehyde (C₅H₈O₂) can inactivate virus at high temperatures, i.e., 25 °C and 37 °C, when formaldehyde bonds with lysine, a non-protonated amino group of amino acids, form hydroxymethlyamine which reacts with other groups, i.e., amino (−NH₂), amide (−CONH₂), guanidyl (=C = NH), phenolic (−OH), or imidazole (C₃N₂) of amino acids to develop inter- or intra-molecular crosslinks. Moreover, DNA/RNA protein crosslink, mutation of specific DNA/RNA or proteins and oxidation of membrane layer to form hydrogen peroxide (H₂O₂) from unsaturated fatty acids may be possible by UVC and far-UV irradiation. Formaldehyde can also reversibly link with RNA to block genome reading. Besides, spike proteins can suffer conformational change at different pHs (specially at pH 8.0) as this change may affect the infectious nature and attachment with the host cell. Significant inactivation can be achieved at pH 1.0, 12.0, and 14.0 at temperatures of 25 °C and 37 °C whereas no removal may be observed at neutral pH. If these inactivation routes are not observed carefully, SARS-CoV can easily enter inside the human body by ingestion/inhalation or fecal–oral route to cause severe health problems.

Han et al. (2005) perceived the potential of effective inactivation of SARS-CoV by some metal catalysts, i.e., Ag/Al₂O₃, Cu/Al₂O₃, and Al₂O₃ where virus inactivation efficiency of Ag was greater compared to Cu; but Al had little contribution in the removal process. In the presence of air, organic surfaces, i.e., bacteria and virus, cells can be oxidized and decomposed with little or no replication capability of RNA or DNA due to formation of active oxygen atoms since the electron transfer process between the atmospheric oxygen and metals’ surfaces may form those atoms; however, the inactivation process was hampered when a thick layer of microbes acted as a barrier to terminate the electron transfer process between the catalyst and molecular oxygen. Virus cell rupture and incapability of DNA/RNA replication were the obvious outcomes of the process.

Lai et al. (2005) examined the survival and inactivation of SARS-CoV on different surfaces (i.e., paper, cotton gown, disposable gown) and stools (i.e., four samples with different pHs) where virus stability in stool was minimum at 3-h (baby, pH 6–7), 6-h (normal adult, pH 7–8), 1-d (normal adult, pH 8), and 5-d (adult with diarrhea, pH 9) but 24-h on paper and cotton gown and 2-d on disposable gown when virus titer was 10⁶ TCID₅₀/ml; although, >3-log removal can be achieved within 5-min by NaOCl. Moreover, Hulkower et al. (2011) also noticed inactivation of two surrogates, i.e., murine/mouse hepatitis virus (MHV) and transmissible gastroenteritis virus (TGEV) of Coronavirus by 600 mg/l NaOCl where <1-log removal was observed after 1-min contact time.

Wang et al. (2005) studied the inactivation efficiency of two disinfectants, i.e., NaOCl and chlorine dioxide (ClO₂) for SARS-CoV, E. coli, and f₂ bacteriophage removal from dechlorinated tap water, hospital and domestic wastewater when SARS-CoV could survive in those wastewaters for 2-d at 20 °C and RNA was detectable in those systems even after 7-d; but SARS-CoV had persisted for 14-d at 4 °C. Although SARS-CoV could survive about 3-d and 17-d at 20 °C in stool and urine, respectively, because of salt in urine to support osmotic pressure; SARS-CoV could stay alive in stool and urine no more than 17-d at 4 °C. SARS-CoV was completely inactivated by chlorine with a concentration of 10 mg/l and high concentration, i.e., 40 mg/l was required to achieve the same level of inactivation by ClO₂; however, E. coli and f₂ phage could not be inactivated completely by 10 mg/l of both disinfectants so SARS-CoV was easier to inactivate compared to the other two microbes. SARS-CoV concentration was several times lower than that of E. coli and f₂ phage in the aliquot, and the aforementioned conclusion is very difficult to achieve. A number of inactivation mechanisms, e.g., reaction with amino acids: i.e., cysteine, tryptophan, and tyrosine; capsid protein alteration; protein and RNA synthesis hampering; reaction with fatty acids; and conformational change of outer layer membrane lipid and proteins to interrupt permeability (Olivieri et al. 1985) were identified. SARS-CoV inactivation was also dependent on temperature, pH, wastewater composition, RH, disinfectant type, and contact time.

Kariwa et al. (2006) explored SARS-CoV inactivation by heat (56 °C), photo (UV) and chemical agents (i.e., povidone-iodine (PVP-I), 100% acetone, 100% methanol, 3.5% paraformaldehyde, 2.5% glutaraldehyde) when significant inactivation was noticed after treatment with the above-stated inactivation agents - although antigenicity of SARS-CoV was perceived after reactivation of methanol and acetone-treated virus. However, nucleocapsid protein was also discovered after heat inactivation, indicating degradation of lipid bi-layer by thermal treatment.

Casanova et al. (2009) analyzed the survival of two surrogates, i.e., MHV and TGEV of Coronavirus in reagent-grade water, lake water, and pasteurized settled wastewater (PSWW) at temperatures of 4 and 25 °C. Declining infectivity for TGEV and MHV was about 0.6-log/week and 0.8-log/week, respectively, at 25 °C in reagent-grade water while the infectivity rate declined by about 1.5-log/week and 2-log/week, respectively, at 25 °C in PSWW, but no significant infectivity decreased at 4 °C in reagent-grade water; hence, infectivity declined more rapidly at 25 °C in all three types of water. Survival rate of virus in reagent-grade water was higher than that in PSWW and other factors, i.e., virus type, temperature, incubation time, aliquot composition, organic nutrient, pH, other microbes’ concentration, presence of proteolytic enzymes and high molecular weight chemicals may affect the removal rate and mechanisms. Although pH had no or minimal impact on virus infectivity, Coronavirus can survive for a long time in water and wastewater, and regular inspection should be practiced in plumbing and exhaust systems in residential, industrial, and office buildings.

Gundy et al. (2009) attempted to comprehend the survival of human Coronaviruses (HCoV) and polioviruses (PV) in tap water and wastewater collected from primary (filtered) and activated sludge secondary (unfiltered) effluent at 4 °C and 23 °C. A number of factors such as temperature, organic matter, solid concentration, presence of other microbes, surface science of pathogens and aerobic microorganisms can affect virus survival in water and increasing temperature of water can decrease virus survival due to denaturation of proteins and activity of extracellular enzymes. Moreover, the presence of other solvents and detergents can also help in inactivation of viruses. The survival of HCoV and PV was, therefore, the same in tap water, but in wastewater, PV can survive six times longer in both filtered and unfiltered samples. Survival of HCoV was higher in unfiltered water than that of filtered water. Again, the presence of organic matter and solids has a significant impact on virus protection but settled solids can increase virus removal from aqueous phase.

Kumar et al. (2015) examined the inactivation of MARS-CoV by gamma irradiation (⁶⁰Co) and chemical disinfectants where high irradiation dose, i.e., 2–4 Mrad can enhance the removal rate; and TRIzol or 1:1 methanol:acetone, two chemical disinfectants, can excellently remove envelope viruses without any reactivation possibility of virus cell and RNA. Cell penetration by rays and disinfectants was an important factor which depended on cell types and disinfectant concentrations or ray density; however, formation of aldehyde crosslink was not dependent on cell penetration rate. Protein denaturation and membrane estrangement were the two possible mechanisms for inactivation by TRIzol with a minimum contact time of 10-min.

Kim & Jang (2018) explored photolysis and photo-catalysis oxidation (PCO) of MS2 phage, as SARS-CoV-2 surrogate, by utilizing UVC (254 nm), VUV, or FUV (<222 nm) with TiO₂ catalyst or Pd nanoparticle-deposited TiO₂ (Pd-TiO₂) sheet catalyst where numerous factors, i.e., RH, irradiation time and rate, distance travelled by photons, type of catalysts, composition of catalysts, amount of catalysts, specific energy given, reactor types, catalyst surface area to reactor volume ratio (S/V, m²/m³), and catalyst placement can stimulate the inactivation process. Significant MS2 inactivation was observed by FUV irradiation with or without catalyst as high photon energy can easily interrupt most of the chemical bonds of proteins, enzymes, and nucleic acids to promote the virus removal process; and dissociation of oxygen and water forms ozone to further accelerate the inactivation process, although inactivation by ozone (3,537 ppb) was comparatively higher than that of UV. Low ozone dose (150 ppb) had six times less inactivation potential compared to VUV photolysis because reactive oxygen generation was less. The inactivation prospect between UV and VUV was considerably different, but catalyst-reinforced VUV + UV irradiation noticeably increased ozone formation and degradation process including other chain reactions to promote inactivation. When a catalyst was used, Pd had almost no inactivation ability so catalyst-induced inactivation was mainly supported by TiO₂ and VUV; but high S/V ratio influenced high inactivation rate. High RH, furthermore, increases inactivation rate as reactive oxygens and other toxins can be easily generated and transported into cells where any smooth transportation of those elements can be hampered at dry conditions. Besides, too-high RH, hydrophobicity and surface charge can also impede the inactivation process. Moreover, Bogdan et al. (2015) revealed the inactivation potential of different microorganisms by photocatalytic oxidation in the order of viruses > prions > Gram-negative bacteria > Gram-positive bacteria > yeasts > molds where the reverse order was observed for resistance to photocatalytic inactivation. Photocatalytic inactivation can easily and quickly damage both enveloped and non-enveloped virus cells with the inactivation order of non-enveloped virus > enveloped virus; nevertheless, the same postulate was not reinforced by all research groups. Generally, envelope, a plasma membrane, is formed by virus-derived glycoproteins and host cell-derived phospholipid bi-layers where factors, i.e., number of layers, their thickness and structural form of shielding the nucleic materials, can influence the overall cell damaging mechanisms, as impairment of capsid proteins and nucleic acid can quickly inactivate virus cells. Although these nanostructured metals have noticeable virucidal property, actual inactivation mechanisms should be investigated more meticulously to gain the precise knowledge about damaging microbes’ cells.

Ye et al. (2018) had comprehended the inactivation potential of Φ6, a surrogate of Coronavirus, by FC and UV₂₅₄ by noticing the reactions of genomes, proteins, and lipids. The inactivation of virus was quick by FC but virion had 15–30 times more resistance than that of other viruses, e.g., influenza A, MS2, or adenovirus under UV₂₅₄; although aggregation, adsorption on particles, and oxidant accumulation on virus surface were detected. Genome damage in the presence of FC may not trigger inactivation as some genome repair mechanisms may slow down the annihilation process; however, significant reactivity of genomes was observed in the disinfection process by FC and UV. Susceptibility of Φ6 was dependent on three distinct protein layers, i.e., membrane proteins (five types), nucleocapsid proteins (two types), and polymerase complex proteins (four types); vulnerability was increased when membrane protein reactions started with FC as nucleocapsid proteins and polymerase complex proteins can be easily penetrated by FC molecules but membrane proteins are not easily penetrable. These protein reactions inactivate virus by structural changes, membrane penetration, hindering solvent accessibility, interruption of binding capability with host cell or UV-sensitive amino acids, e.g., Trp (W), Tyr (Y), and Phe (F) sequences. Again, membrane lipid formed by different types of phospholipids, e.g., phosphatidylethanolamine (PE), phosphatidylglycerol (PG), or cardiolipin (CL) had no impact on the inactivation process.

Ahmed et al. (2020) applied the thermal inactivation process for SARS-CoV-2 and MHV RNA in three types of samples, i.e., untreated wastewater, autoclaved wastewater (controlled or no biological activity), and dechlorinated tap water (free from enzymatic activity). First order decay rate constant was high at a temperature of 37 °C compared to that of 4 °C in autoclaved wastewater and untreated wastewater/tap water for SARS-CoV-2 and MHV; however, SARS-CoV-2 decay rate was slow in tap water compared to untreated wastewater. Temperature effect was not dependent on matrix. RNases release was higher in autoclaved wastewater because of cell lysis at high temperature (121 °C) and pressure (15 psi); and those active enzymes assisted in RNA degradation, whereas this process was not perceptible in untreated wastewater.

Biasin et al. (2021) remarked on SARS-CoV-2 inactivation and replication inhibition by UVC irradiation because UVC photons can be absorbed by nucleic acid bases and capsid proteins to produce photo-products to damage the virus cell where electron transfer can alter the oxidizing properties of the cell. Although inactivation rate was affected by virus concentration and UVC dose, partial inactivation may promote inhibition of infection, and recovery from that harsh condition may depend on the antibody of the virus. Eickmann et al. (2020) also confirmed the inactivation of SARS-CoV, Crimean–Congo hemorrhagic fever virus (CCHFV) and Nipah virus (NiV) by short-wave UV light and visible light (with methylene blue) as both types of rays could impair the nucleic acid transcription and virus replication by forming ROSs, yet formation of ROSs and inactivation rate were increased with increasing intensity of photons. The same research group in 2018 had shown significant inactivation of MARS-CoV and ZEBOV in the same mechanisms by utilizing those two rays (Eickmann et al. 2018).

Bivins et al. (2020) witnessed the persistence of SARS-CoV-2 in water and wastewater at different temperatures, i.e., 20 °C, 50 °C, and 70 °C. Targeted virus had a faster decay rate than that for MHV and TGEV in pasteurized wastewater (WW), but targeted virus had high persistence in unpasteurized WW. Although persistence of the SARS-CoV-2 RNA signal was higher than the SARS-CoV-2 in WW, infectiousness of virus may not be confirmed by RNA signal.

Buonanno et al. (2020) introduced a far-UVC or vacuum UV (VUV) irradiation system to inactivate two airborne HCoVs (229E and OC43) as surrogates of SARS-CoV-2. 229E had better resistance to far-UVC compared to OC43 where spike glycoprotein and other proteins were impaired due to far-UVC irradiation. A myriad number of factors, i.e., structural difference, genome size, nucleic acid arrangement, irradiation rate, contact time, RH, temperature, and wavelength may influence the inactivation process; however, similar inactivation rate by UV was suggested for all types of Coronaviruses. In the context of bio-physically based principle, far-UVC had the capability to penetrate micro-/nano-sized biological materials, e.g., bacteria or viruses; but this ray cannot penetrate human skin stratum corneum (i.e., outermost layer of skin) or ocular tear layer while UVC at 254 nm can pierce both micro- and mega-level bio-materials. The actual health hazard of far-UVC, nonetheless, should be tested by further research since far-UVC can generate ROSs and hydroxyl radicals may damage chemical bonds (Hadi et al. 2020).

Chan et al. (2020) followed different factors to understand the infectivity of SARS-CoV-2 and SARS-CoV, where both viruses had shown the same level of stability in a comparable environment. SARS-CoV-2 was less stable on a surface than in a solution, but high temperature and high RH were factors for viability. Moreover, extreme pH can completely reduce the viability by <1-d and neutral or near neutral pH (i.e., 5–9) had ≤1.5-log reduction within the 1st day, although chemical disinfectants can quickly inactivate the viruses within 5-min from 0.92-log removal to 3.75-log removal.

Chen et al. (2020) depicted thermal and chemical inactivation (by TRIzol reagent, a mixture of acid phenol and guanidinium thiocyanate) of SARS-CoV-2's open reading frame (ORF-1ab) and nucleo-capsid protein (N) gene where complete inactivation of genes was achieved at a temperature of 100 °C by boiling and 121 °C by autoclave; lowest inactivation was shown by TRIzol reagent. Moreover, synergic impacts of pressure and temperature were the major contributors in the inactivation process at high temperature and chemical reagent destroyed the lipid bi-layer without affecting the integrity and stability of the protein bases of the genes.

Hasan et al. (2020) observed antimicrobial efficiency of nanostructured aluminum (Al) sheets with rough (etched) and smooth (unetched) surfaces for SARS-CoV-2, where substantial virus inactivation on a rough surface was noticed within a very short time compared to on a smooth surface; factors, i.e., virus envelope and capsid composition, virus species, nanoscale topography, mechanical and physicochemical characteristics of nanostructured materials can affect the inactivation process. Furthermore, etched Al had noteworthy polarization resistance than that of unetched Al to increase corrosion resistance, but charge benefit cannot be enjoyed for virus separation from gaseous or liquid media.

Heilingloh et al. (2020) evaluated the photo-inactivation of SARS-CoV-2 by UV irradiation and UVC had higher inactivation capacity than that of UVA in high virus titer. Inagaki et al. (2020) investigated the photo-inactivation of SARS-CoV-2 by deep UV light-emitting diode (DUV-LED) with wavelength of 280 nm, and UV irradiation generally hampers the transcription, translation, and replication of DNA and RNA by forming pyrimidine dimers in DNA and RNA. Moreover, target species, irradiation rate, light source, and environmental conditions may influence the removal rate but cytopathic effects were not detected among the cells. Generally, UVC irradiation on DNA or RNA forms different types of chemical structures, i.e., cyclobutane type dimers, namely, cyclobutane pyrimidine dimers (CPD), inter-strand (i.e., between different DNAs) photoproducts, intra-strand (i.e., within the same DNA) photoproducts, and spore photoproducts, i.e., 5,6-dihydro-5-(α-thyminyl)-thymine formation due to photo-reaction of two thymine moieties. Pyrimidine (cytosine and thymine for DNA; cytosine and uracil for RNA) dimerization is possible between adjacent bases due to photo-reaction but photoproducts’ formation may be observed between non-adjacent pyrimidines; and types of photoproducts in a double-strand DNA depend on the chemical structure of the bases as several complex isomers may be observed. Anyway, spore photoproduct can act as a DNA repair enzyme to restore the original structure of the base moieties and structural consistency of DNA/RNA (Douki et al. 2003). However, six covalent-linked dimers, i.e., thymine = thymine, cytosine = cytosine, cytosine = thymine, uracil = uracil, thymine = cytosine, and uracil = thymine can be possible because of UV irradiation on DNA/RNA (Hadi et al. 2020). Moreover, CPD formation by UVC is generally 100 times higher than that of UVB and 1,000 times greater compared to UVA when factors, e.g., wavelength, dose, and photon adsorption capability of DNA/RNA can affect the formation process, although cells have slow and incomplete CPD repair mechanisms (Perdiz et al. 2000). Increasing CPD and photoproducts’ formation may decrease cell survival rate and microbes’ sensitivity to this phenomenon should be carefully examined. Again, formation and repair capability of the defect of DNA or RNA are dependent on UV exposure pattern, photo-reactions induced by that exposure and subsequent antibody formation by virus to present a specific treatment process.

Hessling et al. (2020) monitored the effect of SARS-CoV-2 inactivation by considering different factors, i.e., temperature, RH, pH, and composition of aliquot where high protein concentration and low humidity can adversely affect virus inactivation but elevated temperature can quickly inactivate virus within pH 6–8.

Gerchman et al. (2020) investigated wavelength, i.e., 267, 279, 286, and 297 nm effect of UV-LED for surrogate HCoV inactivation and concluded that longer wavelength needs a higher irradiation dose to kill virus. Four types of proteins in different locations, i.e., spike (S), envelope (E), membrane (M), and nucleocapsid (N) responsible for viral functions, assembly and pathogenesis can be damaged by UV irradiation. In this context, Mayer et al. (2015) hypothesized several factors, i.e., pyrimidine content, dimerization potential, capsid diameter, genome length, surface charge, conformational alteration, pH, sorption surface, amino acids and their functional groups, ROS concentration, structure of capsid protein, types of oxidants and photo-catalyst for effective inactivation of different types of viruses; although capsid protein structure is the most important factor in the damaging mechanism.

Jiang et al. (2020) researched the immediate impact of heat of sub-second duration to inactivate MHV, a surrogate of major types of Coronaviruses, by a flow-through extreme heating (oil bath) and subsequent quick cooling by ice bath process where the virion particles were relatively stable at room temperature; removal rate increased with increasing temperature (>65 °C) for a very short duration. The advantages of using this kind of process are to save huge amounts of energy and to inactivate the pathogens very quickly, but infrastructure development will be a major issue.

Leung et al. (2020) radiated gamma rays to inactivate SARS-CoV-2 where ionizing radiation can inactivate virus by damaging or crosslinking DNA, RNA, and other cellular macromolecules by forming reactive hydroxyl radicals, free electrons, and unstable ions, and high radiation dose can severely destroy the DNA or RNA integrity. However, the suggested dose of <0.5 Mrad may not inactivate all pathogens in a wastewater sample so a high dose should be recommended.

Kampf (2020) discussed the inactivation of Coronaviruses by disinfecting agents on inanimate surfaces as virus particles can survive on inanimate surfaces for 9-d and application of 0.1% NaOCl, 0.5% H₂O₂, and 71% C₂H₅OH can effectively inactivate the viruses within 1-min. Moreover, iso-propanol (CH₃CHOHCH₃), formaldehyde (HCHO), glutaraldehyde (C₅H₈O₂), and povidone iodine (C₆H₉I₂NO) have potential inactivation capability from different surfaces, although elevated temperatures and physical-mechanical properties of those surfaces can affect efficiency. Kampf et al. (2020) reported the persistence of Coronaviruses on inanimate surfaces, e.g., steel (8–120 h), aluminum (2–8 h), metal (120 h), wood (96 h), paper (3–120 h), glass (96–120 h), plastic (8–216 h), PVC (120 h), silicon rubber (120 h), latex (8 h), ceramic (120 h), teflon (120 h), and disposable gowns (8 h) where 0.3– > 5.5 log₁₀ inactivation can be achieved by different biocidal agents mentioned elsewhere in this paper within 15-s to 3-d. Riddell et al. (2020) learned about the viability of SARS-CoV-2 on different common surfaces, e.g., stainless steel, polymer note, paper note, glass, cotton, and vinyl at different temperatures, i.e., 20 °C, 30 °C and 40 °C, and virus reduction was increased with increasing temperature.

Patterson et al. (2020) also inspected photo-, chemical and thermal inactivation of SARS-CoV-2 to collect downstream biological assays for other purposes, and similar inactivation mechanisms by all three processes have been discussed elsewhere in this report. Moreover, detergent discharged in wastewater may disrupt the lipid layer of envelope but ingredients of a detergent may not disturb the protein structure of the virus. Exposure rate rather than illumination time was important for UVC inactivation, although UVA had no efficiency due to limitation in cell penetration.

Coronavirus inactivation by solar radiation (notably UVC, 254 nm) was investigated by Sagripanti & Lytle (2020) by considering certain factors, i.e., temperature, humidity, sunlight intensity, location, seasons, irradiation duration and rate. UV sensitivity of SARS-CoV-2 was three times higher than that of influenza A as the genome size of influenza A was 2.2 times shorter than that of Coronavirus, which had higher inactivation rate with increasing irradiation time and duration. Virus infectivity, however, can be hampered during summer with high solar intensity while, during winter, inactivation rate may be increased with time as solar intensity is generally low at that time. Furthermore, Middle Eastern and African countries observed less severe infection compared to fatality rates in Europe. Moreover, an infected person indoors should be more contagious due to the presence of favorable factors, although a person will be exposed to fewer virus titers outdoors under high solar intensity.

Tizaoui (2020) fathomed out the virucidal efficacy of a powerful oxidant named ozone by evaluating SARS-CoV-2 virus inactivation mechanisms where ozone can react with proteins, amino acids, and lipids (polyunsaturated fatty acids) to form ROSs or reactive radicals (RCOO^•); and form lipid or protein oxidation products, i.e., hydrogen peroxide (H₂O₂), ozonides, and lipid peroxides; and change in virus integrity by damaging structural protein capsid and lipid envelope. Binding efficacy of spike S-protein and angiotensin converting enzyme 2 (ACE2) is 10 to 20 times higher for SARS-CoV-2 and ozone oxidation to S-protein can hamper this infection process. Moreover, the virus reproduction system can be impeded by damaging genome capsid and RNA. Amino acids, i.e., tryptophan, cysteine, and methionine have high reactivity with ozone, although those reactions are dependent on degree of ionization which is a function of pH; and ozone inactivation rate increases with increasing pH. Besides, fatty acids’ reactivity with ozone follows a sequence, i.e., arachidonic acid (AA) > linoleic acid (LA) > oleic acid (OA) > palmitic acid (PA). Again, the effectiveness of ozone water to inactivate SARS-CoV-2 was examined by Hu et al. (2021), and ozone (18–36 mg/l) can completely inactivate all viruses (4.0 × 10³–4.0 × 10⁴ PFU/ml) within 1-min; but ozone concentration <18 mg/l may need longer contact time to fully kill all viruses. Viable cells from 1.0 × 10² to 7.0 × 10² PFU/ml were, however, detected in aqueous samples treated by ozone concentration <9 mg/l. Although ozone gas has an adverse effect on healthy tissue, gaseous phase inactivation can also be possible by ozone gas.

Vatter et al. (2020) observed the photo-inactivation of Coronavirus surrogate bacteriophage Φ6, an enveloped dsRNA, under visible light in the presence of endogenous photosensitizers, i.e., porphyrins, flavins, or NADH (nicotinamide adenine dinucleotide (NAD) + hydrogen (H)) which can absorb visible light and, subsequently, produce ROSs to destroy virus cells. ROSs generally attack envelope, proteins, and nucleic acids of pathogens and inactivation rate can be increased with increasing photosensitizer concentration when concentration of photosensitizer and irradiation rate are negatively co-related. The visible violet or blue light had less antimicrobial effect than UV light. Some photosensitizers, i.e., riboflavin (vitamin B₂) with UV-B and amotosalen with UV-A can enhance pathogen inactivation capability at long wavelengths (Keil et al. 2016; Hindawi et al. 2018; Hadi et al. 2020), although the health hazards of those chemicals should be evaluated by long-term research as further contamination of water and wastewater will not be acceptable for end-users. However, some surrogates are used as models of Ebola virus and Coronavirus to understand the inactivation potential and mechanisms, actual inactivation of the emerging viruses depending on titer concentration, sample types and composition, disinfectant types and concentrations, cell structure of targeted pathogens, different internal (i.e., in vivo) and external (i.e., in vitro) influencing parameters, and physicochemical factors of the surrounding environment.

Hindawi et al. (2018) introduced amotosalen, a photoactive mutagen, to enhance the photolysis effectiveness of UVA where amotosalen/UVA generally expedited the formation of crosslinks in nucleic acids to impede transcription and replication and no breakdown of strands was detected, though. Moreover, no CPE, i.e., agglutination or syncytia was observed in inactivated samples after 7-d. However, this type of process was successfully used to inactivate pathogens from blood, serum, and platelets without distorting the main components. Keil et al. (2020) enhanced the photo-inactivation efficiency of SARS-CoV-2 by damaging nucleic acid in an irreversible way by employing UVB with riboflavin. Furthermore, Dunford et al. (1997) proved ROS generation by TiO₂ photo-catalysis process irradiated by UVA; subsequently, UVA can be effectively applied as a virucidal agent and trigger photogenotoxic activity of TiO₂.