Survival of coronavirus in water and wastewater
The emergence of severe acute respiratory syndrome and its potential environmental transmission suggest that more information is needed on the survival of coronaviruses in water and wastewater.
The survival rates of cat infectious peritonitis virus and human coronavirus 229E were measured in filtered and unfiltered tap water (4 ℃ and 23 ℃) and waste water (23 ℃).
Under the same experimental conditions, it was compared with poliovirus type 1.
The inactivation of coronavirus in test water is highly dependent on temperature, organic matter level and the presence of antagonistic bacteria. The time required for the titer of the virus to decline 99.9% (t99.9) showed that the coronavirus was inactivated faster in 23 ℃ (10 days) water than in 4 ℃ (> 100 days) water.
The value of t99.9 was between 2 and 4 days. Except for 4 ℃ tap water, the survival time of poliovirus in all test water was longer than that of coronavirus.
The SARS outbreak involving more than 300 people in high-rise residential areas in Hong Kong in March 2003 was related to a defective sewage system (Peiris et al. 2003).
The spread of SARS may be related to water and wastewater.
The fact that SARS CoV can be replicated in the intestine (Leung et al. 2003) makes it a possible intestinal pathogen, and the incidence of diarrhea in SARS cases is between 8% and 73% (SARS Epidemiology Working Group, 2003), which has aroused people’s concern about its potential environmental transmission.
Leung et al. (2003) also reported that the production of virus culture from the small intestine of SARS patients was higher than that of lung tissue as the target organ of the virus.
Infectious viruses were isolated from the feces of SARS patients within 3 weeks after infection (Chan et al. 2004; Liu et al. 2004).
The emergence and spread of SARS indicate that more information is needed, especially the survival of coronavirus in water and wastewater.
This study compared the survival of representative coronavirus and poliovirus type 1 in tap water and wastewater.
Materials and methods
Feline infectious peritonitis virus (FIPV) (atcc-990): an enteric coronavirus of feline animals, which is propagated and detected in the CrFK (atcc-94).
Human coronavirus 229E (HCoV) (atcc-740): a respiratory virus that reproduces and analyzes in fetal lung fibroblasts, MRC-5 cell line (atcc-171).
Poliovirus 1 lsc-2ab (PV-1): a human enterovirus known to be very stable in the environment, which is reproduced and detected in the buffalo green monkey kidney (BGM) cell line.
After CPE was observed in monolayer cells, the infected cells were frozen and thawed three times at – 20 ℃ to release the virus.
Then centrifuged at 1000 g for 10 minutes to remove cell fragments, added to 9% PEG (relative molecular weight 8000) and 0.5 M sodium chloride virus suspension, and stirred overnight at 4 ℃.
After centrifugation of 10000g for 30 minutes, 0.01 M phosphate buffered saline (PBS; pH 7.4) (sigma, St. Louis, Mo) was used to resuspend to 5% of the volume of the original virus suspension.
Then the titer of coronavirus was determined and stored at – 80 ℃.
Poliovirus was further purified by extraction with equal volume of vertell XF (DuPont, Wilmington, de). After emulsification, it was centrifuged at 7500g for 15 minutes, and then the top water layer was collected for titer determination and stored at – 80 ℃.
Tap water samples were collected from cold water taps in the laboratory.
The water source is groundwater. The water quality parameters are pH 7.8, dissolved solids 297mg / L and total organic carbon 0.1mg/l. Allow the water to flow for 3 minutes before collecting the sample.
The virus survival rate was determined in unfiltered tap water and tap water with 0.2 μ m pore size filter to remove bacteria.
Tap water (30ml) was added to a sterile 50ml polypropylene centrifuge tube to reduce the virus loss attached to the container.
Add sterile sodium thiosulfate to the final concentration of 33 μ g / ml to dechlorinate the water.
After the vortex, the virus was added to each tube, and the final concentration was 105 TCID50 / ml.
Vortex the centrifuge tube again and sample immediately (zero time point). The centrifuge tube is then stored at 4 ℃ or room temperature (23 ℃).
Centrifuges stored at room temperature are covered with aluminum foil to prevent exposure to light.
After 1, 3, 6, 10, 15 and 21 days, the centrifuge tubes were sampled and frozen at – 80 ° C until they were analyzed on the cells.
The primary effluent is collected after sedimentation and the secondary effluent is collected before chlorination.
The typical water quality parameters of the primary effluent of the facility are biological oxygen demand (BOD) and 1 suspended solid (10-220mg / L).
Generally, compared with primary effluent, BOD and suspended solids in secondary effluent are reduced by 90% ~ 95%.
The effluent (30ml) was added to a sterile 50ml polypropylene centrifuge tube.
Before adding the virus, the primary effluent was filtered by a 50 microns wedge wire filter system and a 0.2 μ m filter, and the unfiltered effluent was also tested.
The secondary effluent is only tested without filtration.
The virus was then added to each centrifuge tube at a final concentration of 105 TCID50 / ml. Vortex the centrifuge tube and sample immediately (zero time point).
The centrifuge tube is then covered and held at room temperature (23 ℃). The samples were collected after 1, 2, 3, 6, 10, 15 and 21 days and frozen at – 80 ℃ until analysis.
The virus was counted on the cell culture by plaque test or TCID50 technique.
The potency of PV-1 was determined by Payment and Trudel 1993 in 6 well plastic cell culture plate.
This is a direct quantitative method with a minimum detection limit of 10 PFU / ml.
Inoculate each diluent into two holes. The titer of coronavirus, which did not form plaque in cell culture, was determined in 24 well plastic cell culture plate by tissue culture infection dose 50% technique (TCID50) (payment and trudel 1993).
This technique determines the dilution of the stock solution showing 50% CPE. The titer of TCID50 per ml was obtained by taking the objection number of dilution multiple.
The minimum detection value of this method is 3.7 viruses per ml. Inoculate each diluent into at least 8 holes. Before adding the virus, any sample from the test water must be filtered before the cell culture test to eliminate bacterial contamination.
At pH 7, 3% beef extract (Becton Dick inson, sparks, MD) was used to prepare a 0.2 μ m low protein binding millex filter (millipore, Billerica, MA) with PES membrane by blocking the possible sites of virus adsorption. All experiments were repeated three times.
Results and discussion
The factors influencing the survival of virus in water include temperature, organic matter and aerobic microorganism (John and rose 2005; Melnick and Gerba 1980; sobsey and meschke 2003).
Temperature is the most critical factor for virus survival.
Previous studies have shown that the virus survival rate decreases with the increase of temperature, which is mainly caused by protein denaturation and the increase of extracellular enzyme activity (Hurst et al. 1980; John and rose 2005).
The results of tap water research confirm that this is the case with coronavirus. By testing filtered tap water, we reduce or eliminate the impact of particulate organic matter and bacteria.
At room temperature, it only takes 10 days to reduce the coronavirus in filtered tap water by 99.9%, while at 4 ℃, it takes more than 100 days to inactivate the virus.
The survival time of PV-1 in tap water at 4 ℃ is similar to that of coronavirus, but at room temperature (23 ℃), the survival time of PV-1 in filtered water and unfiltered water is six times longer.