COVID-19: HOW OZONE WORKS TOWARDS VIRUSES AND THEIR GENOME

Let’s start with an in-depth analysis of the action of ozone on viruses and their genome.

A very in-depth study on the inactivation of poliovirus by ozone and the impact of ozone on the viral genome provides us with all the data we need. ( Biomed Environ Sci, 2019; 32 (5): 324-333 doi: 10.3967 / bes2019.044)

Poliovirus (PV), the causative agent of poliomyelitis, invades the central nervous system and destroys motor nerve cells

the anterior horn of the spinal cord, resulting in paralysis of the limbs.

In rare cases, it can even cause death by paralyzing the muscles that control the throat or breathing [1].

Poliovirus (PV) was one of the most feared pathogens in industrialized countries during the 20th century, affecting hundreds of thousands of children each year through epidemics during the hot summer months.

Although highly effective vaccines exist to control polio, it remains endemic in some countries, from which spread and epidemics continue to occur around the world [2-4].

Poliovirus (PV) is an enterovirus and is mainly transmitted via the fecal-oral and oral-oral routes [1].

It can be excreted through the feces of patients, flowing into the aquatic environment and consequently spreading through the water.

The resistance shown by enteroviruses in the aquatic environment (including survival time and resistance to various purification and disinfectant measures) is radically stronger than that shown by bacteria [5].

Lukasik et al. evaluated the effectiveness levels of various disinfectants in reducing or completely eliminating vital bacteria and viruses from strawberry surfaces and reported that free chlorine concentrations of up to 300 ppm in the wash water were required to achieve disinfection [6].

Even after disinfection and contaminants are within acceptable levels in the water, viruses can sometimes still be detected, of which enteroviruses are the most common. It has been reported that an individual needs to ingest only one detectable unit of virus to develop an infection [7-10].

Using thorough disinfection methods is the only effective way to completely eradicate pathogens in the water and thus control infections; it is therefore imperative to study the effectiveness of the different water disinfection methods to combat pathogens such as Poliovirus type 1 (PV1).

Ozone has strong oxidizing properties and has been used extensively in recent years to disinfect drinking water [11-12].

The disinfection speed is very high and the ozone inactivation effect is better than that of disinfectants such as chlorine, chloramines and chlorine dioxide (ClO2); Ozone can also oxidize and degrade organic substances present in water [13-15].

The ozone remaining in the water can naturally decompose so that the dissolved oxygen content in the effluent is high, the load on the receiving water body is reduced and the water quality can be improved [16-17].

Roy et al. reported that ozone damages two of the four polypeptide chains present in the PV1 viral capsid; however, alteration of the protein coat does not significantly compromise virus adsorption or alter the integrity of the viral particles; damage to viral nucleic acids by ozone was the main reason for the inactivation of PV1 [18].

By considering the different functions of each viral nucleic acid region, the target sequences in the PV1 genome can be segregated into a 5′-non-coding region (NCR), coding region, 3′-NCR and poly (A) tail [19] .

Disinfection with ozone

The viral suspension was added to 100 mL of water sample at 20 ° C and thoroughly mixed to obtain a concentration of 106 plaque-forming units (PFU) / L.

The ozone flow rate was 0.5 L / min and the ventilation time was set to 0, 30, 60, 90, 105 and 120 s [24].

The ozone sterilized viral suspension was linked to Vero cells to detect any live viruses in the sample.

The ozone concentration in the water sample at each time point was measured with the above method and three replicates were used for each time point.

PV1 infection

The sterilized viral suspension was diluted and inoculated into a 25 cm2 cell culture flask containing a single layer of Vero cells; three replicates were used for each timepoint.

After mixing 2 × 1640 maintenance medium and 2% agar in equal volumes, it was added to a cell culture flask to cover the cells to form a solid medium and the number of plaques formed was counted after 2 days of incubation in a cell culture incubator.

The PV1 concentration was subsequently calculated from the number of plaques formed and expressed as PFU.

In the above formula, P represents the number of plaques formed, n is the number of cell culture flasks and v is the inoculum volume (mL).

RESULTS

Relationship between ozone inactivation of PV1 and antigenic lesions

The concentration of ozone in water (mg / L) increased with increasing ventilation time and the concentration became stable after saturation (Supplementary Table S4, available on www.besjournal.com).

Because ozone-depleting substances were present in the water body containing PV1, the ozone concentration in the water was 0 mg / L when ventilation was started.

 When equilibrium was reached, the ozone concentration increased with the ventilation time.

Time- and dose-dependent effects of ozone on PV1 inactivation and antigenic damage were observed.

 An increase in ozone concentration or ventilation time resulted in a decrease in the survival rate of PV1 and an increase in the degree of antigenic lesion (Figure 1).

The time it took for ozone to inactivate PV1 was very short: PV1 was completely eradicated from the ozone after disinfecting the water for 105-120 s (Supplementary Table S5, available at www.besjournal.com).

Validation of primers

Primers designed to amplify the full-length PV1 genome were validated by RT-PCR using untreated (native) PV1 RNA as a template. All the expected goals,

6 long and 3 short fragments were successfully amplified (Supplementary Figure S1, available at www.besjournal.com). Furthermore, the 6 long fragment sequences were verified by sequencing and alignment to the 7441-nt region, which was shown by BLAST to be identical to those previously published [28].

Effect of ozone on the infectivity and genomic integrity of PV1

PV1 infectivity was completely inhibited after disinfecting the water sample with ozone for 105 s.

When water samples exposed to different disinfection times were separately subjected to RT-PCR to study the PV1 genome, region-specific ozone sensitivities were revealed.

The first region lost for detection was the 1-1,297 nt region in the 5′-NCR (Figure 2). Further studies were conducted to elucidate the relationship between PV1 genome damage and changes in viral infectivity, and the results obtained showed that the 1-124 nt region was damaged (Figure 3).

With increased ozone concentration or disinfection time, other regions of the genome were damaged after 120 s of disinfection.

These results indicated that different regions of the PV1 genome have different resistance to ozone.

The 1-124 nt region was the most sensitive and its damage was consistent with the disappearance of PV1 infectivity.

The rest of the genome showed increased resistance and damage to other regions, as evident from the data presented in Table 1, was not completely consistent with the changes in PV1 infectivity.

These observations indicated that ozone-induced inactivation of PV1 occurs via damage to the 1-124 nt region in the 5′-NCR.

Figure 1. (A) Ozone-induced changes of PV1 inactivation rate. (B) Dissolved concentration curve of ozone in 100 mL distilled water and 100 mL PV1-containing water. The baseline conditions were: initial concentration of PV1 was 106 PFU / L in 100 mL water sample, ventilation flow was 0.5 L / min, and temperature was 20 ° C.

Table 1. Damage to PV1 RNA by Different Concentrations of Ozone

Ventilation	Ozone Concentration	Nucleic Acid Region (nt)
Time (s)	(mg / L)	Infectivity	1-1.297	1-124	108-679	669-1.274	939-7.441
0	0	+	+	+	+	+	+
30	0.60	+	+	+	+	+	+
60	1.08	+	+	+	+	+	+
90	1.44	+	+	+	+	+	+
105	1.44	–	–	–	+	+	+
120	1.44	–	–	–	–	–	–

Note. +, Positive result; -, Negative result in replicate experiments ( n = 3).

Figure 2. Detection of RT-PCR products in virus-containing water samples sterilized by ozone at different times. (A) The RT-PCR products of PV1 cDNA synthesized with random primers. (B) The RT-PCR products of PV1 cDNA synthesized with oligo-dT primers. Lanes 1-6 were Positive controls, correspond to the amplification products of primer set 1 to primer set 6, respectively. Lanes 7-12: Disinfection time of 105 s, lanes 13-18: Disinfection time of 120 s, lane 19 was a negative control for primer set 1. Lanes MA and MB, DL2000 DNA markers (2,000, 1,000, 750, 500, 250, and 100 bp).

Figure 3. Precise detection of RT-PCR products in virus-containing water samples sterilized by ozone at different times. (A) The RT-PCR products of PV1 cDNA in the 5′-NCR synthesized with random primers.

• The RT-PCR products of PV1 cDNA in the 5′-NCR synthesized with oligo-dT primers. Lanes 1-3 were positive controls, correspond to the amplification products of primer sets 5′-1 / 1F, 5′-2F / 5′-2R, and 5′-3 / 1R, respectively. Lanes 4-6: Disinfection time of 30 s, lanes 7-9: Disinfection time of 60 s, lanes 10-12: Disinfection time of 90 s, lanes 13-15: Disinfection time of 105 s, lanes 16-18: Disinfection time of 120 s, lane 19-21 were negative controls. Lanes MA and MB, DL2000 DNA markers (2,000, 1,000, 750, 500, 250, and 100 bp).

Fine mapping of 5 ′ -NCR sensitive targets in the PV1 genome

Spot hybridization tests reportedly have better sensitivity and the detection effect is ideal.

Probe 2 was used for spot hybridization.

The method could detect 17 PFU / mL of viral RNA (Supplementary Figure S2, available at www.besjournal.com).

Viral RNA was extracted from PV1 after disinfection using ozone for 0, 90, 105, and 120 if the spot hybridization test was used to accurately locate 5′-NCR sensitive targets in the PV1 genome.

The 5′-NCR loci corresponding to probe 3 (80-124 nt) were the most sensitive to damage caused by ozone (Figure 4).

Furthermore, the loss of integrity of this small 45 nt region was accompanied by the loss of PV1 infectivity (Supplementary Table S6, available at www.besjournal.com).

Relationship between loss of infectivity and genomic damage

PV1 genomic DNA and RNA lacking different target sequences were constructed and verified using RT-PCR (Supplementary Figure S3, available at www.besjournal.com).

Plaque formation test of the corresponding recombinant viral genomes indicated that the full-length viral genomic RNA had high infectivity (Figure 5), while the viral genome devoid of the 80-124 nt and 1-124 nt regions did not have infectivity (Figure 6).

Figure 4. Spot hybridization assay to determine ozone sensitive regions in the 5′-NCR of the PV1 genome (n = 3). PV1 RNA was cross-linked onto a membrane and hybridized with biotinylated probes targeting different regions. Step 5 corresponds to the negative control. Points 1-4 correspond to the RNA of PV1 treated with ozone for 0, 90, 105 and 120 sec. The points were detected with probes in the region P1 (1-40 nt), P2 (40-80 nt), P3 (80-124 nt).

Figure 5. Analysis of plaque-forming cells using full-length viral genomic RNA and viral particles (n = 3). (A) Negative control, no PV1 RNA was inoculated, (B) Integrated viral RNA, (C) (C) Complete viral particle (17 PFU), (D) Complete viral particle (170 PFU).

Figure 6. Testing of plaque-forming cells to determine infectivity of recombinant PV1 RNA with cleared sensitive targets (n = 3). Recombinant viral RNA was inoculated into the Vero cell culture supernatant. (A) 1-124 nt free RNA of 5′-NCR, (B) is parallel to, (C) 80-124 nt free RNA of 5′-NCR, (D) is parallel to C.

Viral nucleic acids were not detected in culture supernatants from inoculated cells infected with recombinant viruses, indicating loss of infectivity when 80-124 nt and 1-124 nt regions were missing.

Viral nucleic acids were not detected in culture supernatants from inoculated cells infected with recombinant viruses, indicating loss of infectivity when 80-124 nt and 1-124 nt regions were missing.

Ozone is an effective disinfectant and is widely used to purify drinking water [12].

In this study, we found that the effect of ozone on PV1 infectivity was both time- and dose-dependent and that PV1 could be completely eliminated after ozone disinfection for 105s.

It has been previously reported that PV1 infectivity is completely inhibited during treatment with 0.2 mg / L ClO2 for 12 min, 0.4 mg / L ClO2 for 8 min, or 0.8 mg / L ClO2 for 4 min [ 22].

This shows that ozone is more effective as a disinfectant than ClO2. When ozone is used to disinfect drinking water, the required ozone concentration is also very low.

By disinfecting water with ozone at a mass concentration of 0.4 mg / L for 4 min, effective disinfection can be achieved [29].

These results indicate that ozone has a very broad perspective as a water disinfectant.

Researchers have suggested that ozone inactivates viruses by destroying viral nucleic acids or capsid proteins. Moore and Margolin published a study in which PV1 was treated with chlorine, ClO2, ozone and UV irradiation to determine the efficacy of each disinfectant and were able to detect viral nucleic acids even after PV1 infectivity was completely eliminated.

They hypothesized that viral inactivation may be associated with damage to capsid proteins [30].

However, Roy et al. found that although ozone can destroy the PV1 capsid protein, viral infectivity still exists.

The destruction of viral nucleic acid by ozone is the main reason for the inactivation of PV1 [18].

Although most of the related studies indicate that ozone-induced inactivation of PV1 is associated with damage to the viral genome, specific sequences targeting ozone within the viral genome have not yet been identified.

Here we used RT-PCR and spot hybridization tests to identify the sites of ozone action in the 5′-NCR of the viral genome.

Our results indicated that the ozone-damaged genetic target was mainly found in the 80-124 nt region in the 5′-NCR.

Furthermore, our infection model confirmed that RNA lacking the 80-124 nt region in the 5′-NCR caused complete loss of infectivity.

The results obtained also showed that viral nucleic acids could be detected after complete elimination of the viral infection and that all detectable regions were located in the coding region of the viral genome, indicating that this region is more resistant to the effect of disinfectants.

Simonet and Gantzer also reported that 5′- and 3′-NCR of the PV1 genome appeared to be the most sensitive to ClO2 treatment [31].

In one of our previous studies, Jin et al. determined that the ClO2-damaged genetic target in 5′-NCR is mainly found in the 40-80 nt region, resulting in PV1 inactivation [22].

Regarding other enteroviruses, Jin et al. reported that ClO2 inactivates EV71 by disrupting the 1-118 nt region in the 5′-NCR [5]; Li et al. also determined that ClO2-induced loss of HAV infectivity is associated with damage to 5′-NCR [32].

The 5′-NCR of the PV1 genome is mainly associated with transcription, replication, translation and viral invasion [33].

The nucleotide sequence of this region contains a high degree of mutual complementarity between the strands, which supports the formation of hairpin or stem-loop secondary structures.

The 5′-NCR has six stem-loop domains, which represent two functional elements. Domain I is the 1-80 nt region which is a four-leaf clover structure and associated with viral nucleic acid replication [34-35].

This region is bound by polyC-binding protein and viral protease polymerase 3CD to form the ribonucleoprotein complex B [36], which catalyzes the synthesis of stranded RNA more than viruses.

Domains II-VI in the 130-610 nt region are associated with viral protein synthesis.

Defined as an internal ribosomal entry site, they direct ribosomes near the start codon for RNA translation, improving translation efficiency [33,37-38].

The secondary structure predicted by the software showed that the 80-124 nt region is free single-stranded and linked to domain II. Interconnected secondary structures can have a decisive influence on viral replication, which can be attenuated during the normal passage of the virus due to variations of some bases in the hairpin or stem secondary structure [39].

Elimination of the region of the loop structure can prevent the virus from multiplying [40].

From an energy point of view, the energy provided by ozone acts on the nucleic acid strand and since the nucleotides of the single stranded region are mainly bonded to hydrogen at a lower energy level, the energy required to break the single-stranded hydrogen bond connection is minimal.

Free single-stranded breaks between domains I and II can cause basic breaks or mutations, leading to alterations in secondary structure.

The fragmentation of these bases directly causes the deletion of domain I, causing the loss of viral infectivity.

Traditional methods for detecting viruses involving cell culture are complex and time-consuming and, therefore, are not feasible to assess the subtle genetic or molecular effects of disinfectants. Spot hybridization and RT-PCR tests are rapid and scalable, but limited in their ability to distinguish between active and inactive viruses [30].

A couple of studies involving the use of RT-PCR to determine PV1 status after treatment with UV irradiation, chlorine, HCl and NaOH reported that the viral nucleic acids detected did not correspond to the loss of viral infectivity seen in the culture. cellular [41 -42].

Similarly, Lim et al. reported that long and short template RT-PCR tests significantly underestimated the extent of virus inactivation compared to plaque formation tests [43].

As a result, most researchers felt that since RT-PCR or nucleic acid probes are unable to distinguish between inactive and active viruses, these methods may overestimate viral infectivity and are therefore not suitable for evaluation. reliable of the effects of disinfectants.

Here we hypothesized that if the inactivation of viruses by disinfectants is based on viral genomic damage, molecular biology techniques should be able to effectively evaluate the effects of disinfectants since these techniques are highly dependent on the integrity of the target nucleic acids, which are used as a template.

Previous researchers have detected only target nucleic acid fragments in the coding region of the viral genome, while our results suggest that this region hosts the strongest resistance to disinfectants. F.

furthermore, previous studies have confirmed that the target of disinfectant-induced inactivation of PV1 lies in the 5′-NCR, rather than in the coding region [31-32].

Therefore, it was assumed that if disinfectant-sensitive sequences were instead used as detection targets for inactivated viruses, it would be possible to evaluate the disinfection effect using molecular biology techniques.

The overall results of this study support this view.

In conclusion, we evaluated the mechanisms by which ozone inactivates PV1 and determined that the inactivation of PV1 by ozone is associated with damage to the viral genome, rather than the capsid protein.

 Our results also indicate that ozone-sensitive genomic targets are found within the 5′-NCR of the PV1 genome.

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