The most likely ecological reservoirs for SARS-CoV-2 are bats, but it is believed that the virus jumped the species barrier to humans from another intermediate animal host.

Although we have information from viruses similar to SARS-CoV-2 experts have said there is no established correlation between the variations in weather temperature and spread of COVID-19. For older flu viruses is known that they typically grow and reproduce in cold and dry conditions. They do not reproduce well if the temperature is warm. Their proliferation goes down at 30 degrees Celsius.

SARS-CoV-2 is the seventh coronavirus known to infect humans; SARS-CoV, MERS-CoV and SARS-CoV-2 can cause severe disease, whereas HKU1, NL63, OC43 and 229E are associated with mild symptoms (Corman et al., 2018). Detailed understanding of how an animal virus jumped species boundaries to infect humans so productively will help in the prevention of future zoonotic events. Forster et al. (2020) showed that the first virus genome that was sampled on 24 December 2019 already is distant from the root type according to the bat coronavirus outgroup rooting. The described core mutations have been confirmed by a variety of contributing laboratories and sequencing platforms and can be considered reliable (Forster et al., 2020). The phylogeographic patterns in the network are potentially affected by distinctive migratory histories, founder events, and sample size. Nevertheless, it would be prudent to consider the possibility that mutational variants might modulate the clinical presentation and spread of the disease.

A study done by Gundy et al. (2009) over the survival of coronaviruses in water and wastewater showed that inactivation of coronaviruses in the test water was highly dependent on water temperature. The time required for the virus titer to decrease 99.9% (T99.9) shows that in tap water, coronaviruses are inactivated faster in water at 23øC (10 days) than in water at 4øC (>100 days). Coronaviruses die off rapidly in wastewater, with T99.9 values of between 2 and 4 days. Poliovirus (virus is released through the feces of infected persons) survived longer than coronaviruses in all test waters, except the 4øC tap water.

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), was first reported in Wuhan, China in late December 2019. It is the third highly pathogenic and transmissible coronavirus after severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV) emerged in humans. The source of origin, transmission to humans and mechanisms associated with the pathogenicity of SARS-CoV-2 are not clear yet, however, its resemblance with SARS-CoV and several other bat coronaviruses.

Coronaviruses are enveloped, positive-sense single-stranded RNA viruses with a nucleocapsid of helical symmetry (protein coat) (Zulma et al., 2016). Coronaviruses are members of the subfamily “Coronavirinae” (family; Coronaviridae, 62 order; Nidovirales) that contains four genera. Alpha and beta coronaviruses are known to harm humans and animals (Khan et al., 2020). Gamma and delta coronaviruses generally infect birds, although some of them can cause infection in mammals. For human health, beta-coronaviruses are the most important group as this group contains the highly pathogenic viruses in humans including SARS-CoV-2, MERS-CoV and SARS- CoV (Cui et al., 2019; Su et al., 2016; Forni et al., 2017).

The fragments of genetic material (RNA) detected from sewage are very quickly decomposed in sewage water and will only be stable if protected by the protein coat (capsid), meaning only if the whole virus is present. Therefore, demonstrating these specific RNA fragments in sewage wastewater implies that whole virus particles were present. We currently know too little about the possible infectious activity of coronavirus (SARS-CoV-2) particles in sewage water, but from the information that we have this far it does not seem very likely that coronavirus particles in wastewater can infect people.

Wurtzer et al (2020) showed the detection of SARS-CoV-2 in Parisian treated wastewater. However, the treated wastewater effluents showed a 100 times reduction in the viral load compared to the corresponding raw wastewater samples, which is in agreement with previous work on enteric viruses (Prevost et al., 2015).

Ozone gas has been proven to kill the SARS coronavirus and since the structure of the new 2019-nCoV coronavirus is almost identical to that of the SARS coronavirus, it is relatively safe to say that it will also work on the new coronavirus though it must be noted that there is no studies to date with regards to this. Numerous families of viruses including poliovirus I and 2, human rotavruses, Norwalk virus, Parvoviruses, and Hepatitis A, B and non-A non-B are among many others that are susceptible to the virucidal actions of ozone. Ozone destroys viruses by diffusing through the protein coat into the nucleic acid core, resulting in damage of the viral RNA. At higher concentrations, ozone destroys the capsid or exterior protein shell by oxidation. Duan et al. (2003) also showed the stability of SARS coronavirus in human specimens and environment and its sensitivity to UV irradiation. The results showed that the survival abilities on the surfaces of eight different materials and in water were quite comparable, revealing reduction of infectivity after 72 to 96 h exposure. Irradiation of UV for 60 min on the virus in culture medium resulted in the destruction of viral infectivity at an undetectable level.

SARS-CoV-2 has been detected in stool of infected people and on toilet surfaces in hospitals treating COVID-19 patients. For toilets, latrines and similar sanitation systems, hygiene and cleaning are as important as ever.

Medema et al. 2020 already showed the presence of SARS-CoV-2 virus in sewage. However, wastewater already contains numerous potentially dangerous pathogens. According to current knowledge, this new virus is shed in stools, but it is questionable if it is still infectious after shedding and in sewage. The new virus is member of a group of viruses that do not have a significant waterborne transmission route. Therefore, the consensus based on current knowledge that a specifc method should not be developed and the standard protocols are enoughly protective.

There is no specific method to remove SARS-CoV-2. As SARS-CoV-2 are less robust in the sewage and water environment than other viruses, the same disinfiction method taken before should be still followed. To our knowledge, water at ambient temperatures will not rapidly inactivate the virus. The hotter the water, the faster the virus will be inactivated. If the water contains detergent or disinfectant, that greatly enhances virus inactivation. The soap will also dissociate the virus from other organic material, making it more exposed to the effect of detergents. In the study of Geller et al. (2012) we can also observe that alkaline pH’s could make coronaviruses less stable.

Coronavirus is member of a group of viruses that do not have a significant waterborne transmission route. Furthermore, corona viruses are less robust in the sewage and water environment than other viruses. Therefore, the consensus based on current knowledge that a specifc method should not be developed and the standard protocols are enoughly protective.

The operators of the WWTP sampled a 24h flow-dependent composite sample of 250 mL that was stored at 4°C during sampling. A study done by Gundy et al. (2009) over the survival of coronaviruses in water and wastewater showed that coronaviruses are inactivated faster in water at 23°C (10 days) than in water at 4°C (>100 days).

The method used for virus detection is called RT-qPCR (Reverse transcriptase quantitative polymerase chain reaction). This is the same method as is used to detect SARS-CoV-2 in patients. In this method, the RNA fragments (genes from the virus) are detected confirming the presence of SARS-CoV-2. Therefore, the method cannot be performed in situ. However, microbiologists from KWR are working on alternative methods for in situ detection in wastewater.

Our microbiology laboratory at KWR is used to do this type of molecular analysis and wastewater sampling. The encountered difficulties are inherent to any experimental research. For now, the main challenge is to improve the methods for quantification of SARS-CoV-2 and to link the results to epidemiological data.

Overall, the mutation rate of this virus is not particularly high (in line with other coronaviruses). A single patient may have several mutational strains going at the same time as the virus replicates. Constant surveillance of mutations arising and frequent comparison with primer sequences remains needed.

Several commercial parties have developed molecular biological methods based on the reverse transcriptase quantitative PCR (RT-qPCR) into assays for detection of viral genes in different environments. In the "Plus/Minus" test, samples and controls (distilled water) are measured, after which a positive or negative result is calculated based on differences in signal between the samples and controls. When applying the "Plus / Minus" test for detection of SARS-CoV-2 coronavirus genes, we observed part of the false positive and negative signals, demonstrating that the method used is valid.

The technique (RT-PCR) measures RNA fragments (genes from SARS-CoV-2 virus). Finding intact RNA fragments indicates indirectly the possible presence of the virus. This is the same technique as used by hospitals to establish COVID-19 infection. Particularly in sewage water it is highly unlikely that unprotected viruses (pieces of RNA without protein coat) would be found. In the biologically active environment of sewage, the fragments of virus-RNA would rapidly become degraded. The RNA is stable for some days when it is protected by the protein coat, meaning that it is present as whole virus particle.

The novel coronavirus was officially announced by Chinese authorities on 7 January. The complete genetic code was released for immediate public health support via the community online resource virological.org on 10 January. The genome sequences suggest presence of a virus closely related to SARS-related CoV, a species defined by the agent of the 2002/03 outbreak of SARS in humans (Peiris et al., 2003). The species also comprises a large number of viruses mostly detected in rhinolophid bats in Asia and Europe. As the sequences were publically available and it was confirmed that they were related to SARS-related CoV, a similar method detection could be used.

The method used for virus detection is called RT-PCR. This is the same method as is used to detect SARS-CoV-2 in patients. In this method, the RNA fragments (genes from the virus) are detected confirming the presence of SARS-CoV-2. This does not give additional information on its infectivity or presence of the protein coat (capsid). Medema et al. (2020) present more details about the method applied at the KWR laboratory.

Because genetic fragments are amplified through the polymerase chain reaction (PCR), RT-PCR can be consiered as a very sensitive method to demonstrate presence of SARS-CoV-2 genes. We are currently conducting further experiments to determine the extent to which the genes remain detectable in time under different (realistic wastewater) conditions, and try to link the strength of the signal to epidemiological data.

The phrase “Polymerase chain reaction” (PCR) was first used more than 30 years ago. Now, Real time PCR (RT-PCR) is a well-established method for the detection, quantification, and typing of different microbial agents in areas as diverse as clinical diagnostics, environmental microbiology and food safety.

Wastewater treatment plants (WWTP) were selected that served 2 large and 3 medium sized cities and the main airport. The operators of the WWTP sampled a 24h flow-dependent composite sample of 250 mL that was stored at 4 °C during sampling. Samples were taken in 2020: on February 5, 6 and 7 (3 weeks before the first COVID-19 case was recognized by the health surveillance system in The Netherlands), on March 4 and 5 (38 resp. 82 reported COVID-19 cases in the Netherlands, total population 17.2 million), and March 15 and 16 (1135 resp. 1413 reported COVID-19 cases in the Netherlands). As the epidemic progressed, another WWTP (Tilburg, a medium-sized city in one of the most affected areas) was included in the sampling scheme.

Details on processes applied at the different WWTPs can be requested at the respective water authorities. in general, most of the WWTPs in The Netherlands apply primary treatment (removal of solids) and secondary (biological) treatment: (typically [aerobic] active sludge) for removal of organic carbon, usually followed by tertiary treatment processes (nutrient removal) and incidentally quaternary processes for removal of organic micropollutants.

We will continue with our regular sampling of several wastewater treatment plants in the Netherlands

To better understand this outbreak, scientists are looking at comparable outbreaks like SARS and MERS. SARS, which started its spread in late 2002, shares almost 90 percent of its DNA with the current virus. The SARS outbreak started in November and lasted until July of the following year. This is the most comparable estimative.

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We also want to study other aspects, such as the density at which virus particles remain active in sewage at certain temperatures and residence times. Although studies with related viruses previously indicated that this is unlikely, this needs similar verification for the COVID-19.

KWR started the research independently but now is seeking collboration with other (Dutch) parties such as ministeries, public health authorities such as RIVM and water boards and representative organisations thereof (STOWA, UvW)

This project aims for further screening on SARS-CoV-2 presence in sewage wastewater within a selection of large communal wastewater treatment plants in The Netherlands. This will give: • trends of SARS-CoV- 2 spread of the 2020 pandemic in The Netherlands; • supportive information for evaluation of quarantine/mitigation measures; • an early warning system for when COVID-19 may be on the rise again in The Netherlands (e.g. winter 2020); • a realistic estimate of the percentage of mild COVID-19 presence (i.e., a significant proportion of COVID-19 goes undetected, since people with mild symptoms are not tested).

There are no additional risks. WHO stipulates that all diagnostic laboratory work and PCR analysis with novel coronavirus are conducted according to practices and procedures described for basic laboratory ? Biosafety Level 2 (BSL2), as detailed in the WHO Laboratory biosafety manual, 3rd edition.

It is good to point out that there are many more possible pathogens in sewage wastewater, and that it has already been demonstrated for these other pathogens that people can actually become infected. Personal protective equipment and control measures are already being used for this. It makes a lot of difference if people are aware of this and continue to apply these means and measures properly. For this matters, we should follow the advices given by the World Health Organization (WHO, 2020).

There is no evidence that water is a transmission route for the SARS-CoV-2 virus, which causes COVID-19. RNA fragments of the virus have been detected in sewage water, but they have a low probability of being infectious particles. Therefore presence of the SARS-CoV-2 virus probably poses small risks of acquiring COVID-19. Nevertheless, exposure to sewage wastewater has a (proven) risk of becoming infected by other pathogens, for which professionals working with sewage wastewater already have their (normal) personal protective equipment. Therefore RIVM and WHO (and other health services, such as US CDC) indicate that normal protection and hygiene measures are adequate. The same conclusion holds for the SARS virus (SARS-CoV) which is another coronavirus closely related to the COVID-19 virus (which is therefore called SARS-CoV-2). Inactivation experiments with SARS virus (Duan et al. 2003) revealed that the virus remains infectious in stool at 20 ºC for 4-5 days. Higher temperatures accelerate the inactivation of similar viruses (Kampf et al., 2020). Another study examined inactivation of other coronaviruses in sludge at 25 ºC and found 99.999-99.9999% (log removal rate (e.g. 99% = 2 log, 99.999% = 5 log)) inactivation in 3 weeks (Casanova et al. 2009). These authors had pre-pasteurized the sludge, which stopped the biological activity of the fermentation. Inactivation in unpasteurized sludge at 30-38 ºC is expected to occur faster. On the basis of the available knowledge it can be stated that, if infectious SARS-CoV-2 virus is present in sewage (which is already unlikely), the sludge fermentation (part of many sewage treatment processes) can completely inactivate the virus.

COVID-19 is caused by a SARS-CoV-2, a coronavirus. Coronaviruses belong to the group of viruses with an envelope (lipid membrane), which also includes the flu virus. This lipid envelope makes enveloped viruses very susceptible to routinely used disinfection methods and soap. These types of viruses are spread from person to person through the water droplets caused by coughing or sneezing, or through contact with contaminated hands or surfaces, but for these viruses there is no known transmission via the urban waste water system. Therefore, the SARS-CoV-19 virus probably does not pose any risk to acquire COVID-19, and certainly no increased risk through the wastewater system, and RIVM and WHO (and other health services, such as US CDC) all indicate that normal protection and hygiene measures are adequate when working with sewage wastewater.

Sewage water has not yet been described as a transmission route and the risk is estimated to be very low. The virus concentrations in sewage water and aerosols generated from sewage water are probably much lower than what an infected patient exhales. Examples from previous work: 1. residents near wastewater treatment plants (WWTP) have been investigated on infections by water-transmissible viruses. This did not reveal that waterborne viral diseases were occurring more frequently. 2. The strict quarantine in Asian cities has worked, while sewage and purification have not been "quarantined". So hence no indication that WWTPs or aerosols play a role.

The infection that may cause COVID-19, has airborne transmission via droplets. The rate of transmission is quite high, higher than common influenza. The preventive measurements in general for patients, healthcare workers and community are: Wash your hands often with an alcohol-based (>65%) detergent if your hands are not apparently dirty, and with soap and water if they are contaminated. Always wash your hands after contact with respiratory secretions; Avoid contact with eyes, nose and mouth; Sneeze and cough into a bent elbow or tissue and then throw it away; Wear a surgical mask if respiratory symptoms appear and wash your hands immediately afterwards ; Keep a distance of at least 1 m from patients with respiratory symptoms. More complete information could be found in World Health Organization document (WHO, 2020).