1. Introduction
Climate change, urbanization, and increasing global population have placed considerable pressure on freshwater supplies [
1,
2,
3]. Wastewater can be used as an alternative water resource for agriculture irrigation and aquifer recharge but would first require appropriate treatment in wastewater treatment plants (WWTPs). WWTPs act as engineered barriers to treat municipal wastewater to a quality that is sufficiently safe for reuse. In most WWTPs, the final treatment step typically includes the use of chlorine as a disinfectant to reduce the biological activity of remnant pathogens present in the treated effluent [
4]. However, each pathogen reacts differently to different disinfectants, and a single disinfection strategy is rarely effective against all pathogens [
5]. For example, a WWTP utilizing chlorination as a disinfection strategy was able to inactivate human adenoviruses but not enteroviruses fully from wastewater [
6]. Additional disinfection strategies, particularly those that are low-cost and easily accessible, may have to be deployed to further inactivate remnant viral contaminants.
Solar irradiation is a freely accessible, low-cost biocidal strategy that is abundant in many tropical countries and can be used to circumvent this need. The biocidal effect of sunlight works through the effects of ultraviolet A (UV-A) and ultraviolet B (UV-B). UV-A, of wavelengths 320–400 nm, is absorbed by molecular chromophores which, in turn, generate reactive oxidative species (ROS). ROS induce damage to cellular membranes, proteins and nucleic acids, rendering viruses and other pathogens inactive. UV-B, of wavelengths 280–320 nm, functions directly through absorption by nucleic acids and proteins. UV-B can also affect pyrimidines directly, inducing mutagenic and genotoxic effects in the genomes of microbes [
7,
8].
Several studies have documented the effects of irradiation on viruses. However, the dosage required for viral inactivation varies widely with viral species, particle size, genome type, length, and polarity [
9,
10,
11,
12]. For instance, numerous studies have investigated the effects of solar irradiation on members of the
Picornaviridae family, which contain a single positively stranded RNA genome [
13]. Heaselgrave et al., reported a 4-log inactivation of polioviruses with solar irradiation ranging from 198 to 1224 J/cm
2 [
14,
15]. In contrast, Coxsackie viruses required 117–198 J/cm
2 of solar irradiation for a 4-log inactivation, while ECHO viruses required 50–60 J/cm
2 for a 2-log reduction [
15,
16].
The variation in solar intensity required to inactivate different RNA viruses within the same family shows that susceptibility of viruses to solar irradiation differs at the species level. A species within the
Picornaviridae family that has not been studied in this aspect is enterovirus 70 (EV70). These viruses are mainly transmitted by the fecal-oral route and cause gastroenteritis. However, it can cause other symptoms, which include hemorrhagic conjunctivitis, diabetes (through infection of islet cells), and central nervous system complications [
17,
18,
19,
20]. These viruses are acid and heat stable, allowing for their survival in the gastrointestinal tract but inadvertently conferring persistence in WWTPs [
21,
22]. Infectious EV70 has been detected in the effluents of several WWTPs globally [
6,
23,
24,
25]. This indicates that the existing disinfection procedures employed are not adequate to provide safe water for reuse, and there exists a need to explore the efficacy of solar irradiation as a possible additional disinfection strategy against enterovirus 70 (EV70).
In this study, EV70 was exposed to simulated sunlight irradiation for 24 h at a fluence rate of 28.67 J/cm
2/h. Aliquots of the virus were harvested at specific time points followed by determination of its infectious titer and RNA concentration. We employed a focus forming assay to overcome the inability of EV70 to replicate well in cell culture [
6,
26]. To determine if any damage was incurred on the capsid, viruses were assayed for their binding ability to Vero cells. The viral growth kinetics were also assayed by counting the foci generated over a nine-day infection period. Ten days after infection, the genomes of EV70 were sequenced. The assays revealed that irradiated viruses had inhibited replication and binding and harbored nonsynonymous nucleotide substitutions compared to dark-control viruses. Viruses suspended in a wastewater matrix also experienced a significant reduction in viral activity upon exposure to solar irradiation, albeit not as pronounced as that observed when suspended in a saline buffer. Interestingly, all of the irradiated viruses in this study failed to replicate in cell culture, providing a strong endorsement of sunlight as a low-cost natural disinfection strategy.
4. Discussion
Earlier observations of viable and infective viruses in post-treated effluent provided the main impetus for this study [
6], as their presence can complicate the reuse of reclaimed waters. To circumvent viral risks, chlorine disinfection is typically performed at the last step of a wastewater treatment process. However, chlorine works with varying effectiveness against different types of viruses [
5]. This led to the suggestion of including combinations of various disinfection processes in a single WWTP. However, retrofitting different modular units of disinfection processes may incur additional operating costs. Solar disinfection of treated wastewater was therefore studied to provide a natural, low-cost and abundant disinfection strategy to further inactivate remnant viruses present in the reclaimed waters.
Specifically, EV70 was chosen as a model organism in this study, as infectious enteroviruses were previously found after wastewater treatment in concentrations approximating the infectious dose [
6,
33]. EV70 has not been studied extensively for its susceptibility to disinfectants due to its lack of plaque-producing capability in cell culture. To overcome this hurdle, a focus-forming assay was employed, which measured viral titer by fluorescently labeling virus-infected cells with virus-specific antibodies. This technique also required shorter duration compared to a traditional plaque assay [
26].
We observed that EV70 in PBS experienced a 1.7-log reduction in infectivity after a dose of 688 J/cm
2 (
Figure 1). This is consistent with the finding that poliovirus type 2 experienced a 4-log reduction with a simulated solar irradiation of 1224 J/cm
2, which is equivalent to a 2-log reduction at approximately 612 J/cm
2 [
14]. Both EV70 and polioviruses are from the
Picornaviridae family and have similar sizes (approximately 30 nm in diameter), capsid structures and genome lengths (EV70: 7200 nt, poliovirus: 7500 nt) [
34,
35]. In contrast, other members of
Picornaviridae require differing doses of solar irradiation to achieve a similar reduction in infectivity. For example, Coxsackie viruses require approximately 58.5–99 J/cm
2, and ECHO viruses require 50–60 J/cm
2 of solar irradiation to achieve a 2-log reduction [
15,
16]. Both Coxsackie and ECHO viruses have similar sizes (28 nm and 24–30 nm, respectively) and genome lengths (approximately 7400 nt and 7500 nt, respectively) to EV70 [
36,
37,
38]. The data presented in this study agree with earlier studies that infer the need for varying solar fluence to inactivate different viral species. While the structures of viruses are generally similar within a family, species might differ in protein folding and genome secondary structure, which give rise to differences in susceptibility to solar irradiation [
39].
Picornaviridae have a positively stranded RNA genome that is directly translated by host-cell ribosomes [
13]. Here, damage to the genome was indicated by the decay in the RNA concentrations in the presence of solar irradiation (
Figure 1B). In addition, the reduction in binding capacity of L24 indicated conformational damage to the capsid, stopping it from recognizing the viral receptor on the Vero cells (
Figure 3). This reduction in binding was of a larger magnitude than the reduction in infectivity as seen in
Figure 1A at 24 h across all three matrices. Since receptor binding is the first step in a virus replication cycle, any irradiation-induced damage to the capsid could result in the inability of the capsid to recognize the receptor on host cells. Hence, this decrease in binding of L24 was most likely the primary cause for the decrease in infectious capacity.
Although EV70 with a damaged capsid may have a reduced binding capability, replication would theoretically still be possible if the interior structure of the viral particle remained undamaged. To test this, we performed a growth curve analysis, which showed that L24 was unable to replicate to similar titers as T0 or D24 even after 9 dpi in all three water matrices (
Figure 2). This was observed despite the similar multiplicity of infection between T0, D24, and L24. This information indicates an inability of solar-irradiated EV70 to replicate as effectively as wild-type or dark-control viruses. The capsids of
picornaviruses undergo a dramatic antigenic alteration before the virus uncoats [
40]. Translation is then initiated by the internal ribosomal entry sites in the 5′ untranslated region of the genome, which is composed of five stem-loops (II-VI) [
41,
42]. Viral translation is also promoted by the binding of host-cell IRES trans-acting factors, such as FBP1-3, hnRNP K and hnRNP A, which recognize the 5′ untranslated region of the viral genome [
42,
43,
44,
45]. Solar irradiation could induce structural damage to the capsid and genome of EV70, leading to reduced binding and replication capacity. Not only would a structurally damaged capsid fail to bind to the host-cell receptor, but it might fail to undergo the antigenic alteration necessary for uncoating to occur [
40]. UV irradiation promotes RNA-protein cross-linking [
46]. The formation of covalent bonds between the EV70 genome and the capsid might affect the release of the RNA out of the capsid during the uncoating process. The integrity of the cloverleaf and stem loop structure present in the 5’ untranslated region of the EV70 genome might be negatively affected by solar irradiation. A disintegration of structure in this region of the genome may prevent successful docking of the ribosome and other host-cell translation initiation factors. Lastly, owing to the structural damage to the genome, translation might not proceed as efficiently as in wild-type viruses, producing proteins which might not support viral replication.
To elucidate if mutations did indeed occur in key proteins of EV70, we sequenced the coding region of the genome. Initially, fragment PCR of viruses directly sampled after 24 h of solar irradiation was performed (data not shown). However, this did not yield sufficient concentrations of PCR amplicons for sequencing. To overcome this technical constraint, Vero cells were infected with L24 or D24 for 10 days, and the viral RNA, which had amplified in the course of the infection, was extracted and sequenced. The sequence of the L24 viral genome derived from this experiment is, hence, not a direct product of solar irradiation but was selected for 10 dpi. This genome could be viewed as an ‘escape mutant’, being the only sequence that had replicated enough to be amplified by PCR. However, this sequence was still unable to replicate as effectively as T0 or D24 (
Figure 2).
The irreproducibility of nucleotide substitutions between trials 1 and 2 for L24 in PBS indicate that solar irradiation induces mutations in a random manner. However, four out of the six mutations listed occurred in the capsid genes, which are at the 5′ end of the genome. Positions 40 and 4801 also showed mutations in two of the four irradiated samples (
Figure 4A,F). These findings might suggest that the capsid genes, as well as position 4801 in the 3C
pro gene, are more prone to mutation by solar irradiation compared to the rest of the genome.
The structure of the capsid of bovine enterovirus (BEV), a
picornavirus, has been determined [
47]. The structural proteins of BEV share 48% identity with EV70 [
48], and its tertiary structure is collinear with other enteroviruses [
49]. Comparisons with the amino acid sequence of BEV’s capsid reveal that the amino acid substitutions of EV70 listed in this study did not occur in any of the known functional motifs. However, an earlier study showed that an introduction of a single amino acid substitution at five different positions in the capsid genes resulted in a change in viral tropism [
50]. These proteins constitute the capsid and form the depression known as the ‘canyon’, which recognizes the cellular receptor DAF/CD55 for attachment to the host [
51,
52]. VP1, which is the most exposed protein of the capsid of
Picornavirus [
53], forms a hydrophobic pocket that allows for myristic acid binding [
54] and is believed to be involved in the binding of metal ions [
55,
56]. VP1 is also believed to have a role in the uncoating of the virus particle [
50]. Even though Glu50Gln in VP1 occurred in an unstructured motif (
Figure S1), the substitution might alter the charge of VP1.
EV70 with a glutamic acid instead of a lysine at position 14 of VP4 protein replicates poorly in HeLa cells [
50]. In this current study, L24 in PBS and chlorinated effluent displayed this substitution (
Figure 4), which may have accounted for the poor replication. It is likely that this mutation resulted in a change in the charge of the overall protein, as lysine is typically positive at neutral pH while glutamic acid is negatively charged. This would have resulted in poor binding of EV70 to the host cells. Similarly, even though the amino acid substitution Gly71Ser did not affect the folding of the β-sheet in VP3 (
Figure S1), the overall polarity of the protein might have been affected owing to the polar nature of serine as opposed to glycine. Both these substitutions might have synergistically affected viral function.
In addition to assessing changes in the capsid proteins, the 3C
pro protein of
Picornaviridae was also assessed since this protein displays a multitude of functions in the infected cell. Initially shown to be a protease that cleaves the functional proteins from the polyprotein precursor, 3C
pro also cleaves host-cell proteins to shut down host-cell transcription, translation, and nucleo-cytoplasmic trafficking and promote apoptosis (reviewed in [
57]). There exist four main functional domains in the 3C
pro protein: the
N-terminal domain (aa 12–13), the central domain (aa 82–86), the β-ribbon (aa 123–133) and the C-terminal domain (aa 154–156) [
58,
59,
60]. The amino acid substitution Ile47Leu occurred in between the
N-terminal domain and the central domain, an area that lacks any known function (
Figure 4). It is important to note that this substitution did not affect the integrity of the β-sheet motif of this protein (
Figure S1). However, further investigations into this amino acid substitution should be carried out to determine if the function of the 3C
pro protein is altered. If this substitution results in a change in the function of the protein, this could explain the reduced ability of L24 to replicate to high titers, as seen in cells infected with T0 and D24 (
Figure 2). This could be a result of inadequate cleavage of the viral polyprotein or inadequate suppression of host-cell factors, allowing for the host cell to overcome the viral replication machinery.
In addition to observing a significant impact on the viral infectivity and persistence due to solar irradiation, we also observed that viral inactivation occurred at a slower rate when the viruses were present in wastewater matrix. This concurs with earlier observations [
61,
62,
63]. Furthermore, out of the six nucleotide mutations found, only 2 were seen in L24 in wastewater matrices, A40G and G4698A, while L24 in PBS had 5 mutations (
Figure 4). This indicates that viruses in the wastewater are less susceptible to UV-B [
12,
16]. Effluent and chlorinated effluent wastewaters used in this study had a total organic carbon (TOC) concentration of 4.2 mg/L and 5.2 mg/L, respectively, while PBS had undetected levels of TOC, as expected (
Table S1). These organic compounds can act as radical scavengers [
64,
65], reduce light intensity [
66], or encapsulate viruses with a protective organic coating that makes them more resistant to external environmental stressors when present in wastewaters. The latter has been alluded to by the findings that non-enveloped viruses are stable in wastewaters [
67,
68]. Alternatively, the high alkalinity in wastewater might favor the reaction between bicarbonates and hydroxyl radicals formed upon solar irradiation. This reaction results in the generation of CO
3●- which reacts slower with organic molecules compared to ●O
2 radicals [
27,
69,
70].
While these reasons could explain the slower inactivation rates of EV70 in wastewater matrices, it is important to note that irradiated viruses, irrespective of matrix, all failed to propagate in cell culture (
Figure 4). This indicates that solar irradiation successfully inhibits viral replication in cell culture, preventing the generation of infectious viral progeny in all three water matrices evaluated in this study. This strongly suggests that solar irradiation modifies the replication capacity of EV70 to the point that it might not pose a significant public health threat. Although the data from this study suggest that solar irradiation may serve as a good disinfection technique, its efficacy may be lower in turbid waters due to lower solar penetration and higher light-scattering effect. Operators would also need to create a holding tank that is shallow enough to allow for proper solar penetration and irradiation. This would not be feasible in densely populated places with limited land space. Hence, the use of solar irradiation as an effective, natural, and low-cost disinfection strategy against EV70 would only be feasible for use in low-turbidity waters, presumably in permeates after membrane filtration processes, and in places unconstrained by land availability.