The renewed interest in porcine coronaviruses and the role of disinfectants

Thursday, October 15, 2020


The renewed interest in porcine coronaviruses and the role of disinfectants


Vilofoss and Sanphar

 


Introduction


Coronaviruses (CoVs) are found in a wide variety of animals, in which they can cause respiratory, enteric, hepatic, and neurological diseases of varying severity. CoVs are positive‐sense RNA viruses that belong to the subfamily Orthocoronavirinae, in the family Coronaviridae, order Nidovirales. CoVsare further divided into four genera, α‐CoVs, β‐CoVs, γ‐CoVs, and δ‐CoVs.Due to the unique mechanism of viral replication, CoVs have a high frequency of recombination. Their tendency for recombination and the inherently high mutation rates in RNA viruses may allow them to adapt to new hosts and ecological niches (1, 2).


CoVs have recently received a high level of global attention due to the discovery of a novel, highly pathogenic human coronavirus in China in December 2019. Similar to humans, much remains unexplored for porcine coronaviruses due to their nature of seasonality. Swine are susceptible to several CoVs including the α‐CoVs transmissible gastroenteritis virus (TGEV), porcine epidemic diarrhoea virus (PEDV), and the recently emerging swine acute diarrhoea syndrome coronavirus (SADS‐CoV) (3, 4) which like SARS‐CoV or MERS‐CoV appears to have originated from bats. Besides these α‐CoVs, swine are also susceptible to a β‐CoV, porcine hemagglutinating encephalomyelitis virus (PHEV) (5), and a δ‐CoV, porcine deltacoronavirus (PDCoV) (6). Interestingly, swine and humans both have their own range of CoVs with no apparent overlap; CoVs in humans mainly target the respiratory tract whereas CoVs in swine mainly target the enteric system. (In swine, there is only a single CoV associated with respiratory infections, PRCV, which is a spike deletion mutant of the enteric TGEV.)


Enteric CoVs


Specifically for the porcine enteric disease PED, it was first reported in Asia in 1982 as causing high mortality in neonatal piglets and has since had a huge economic impact on the Asian pork industry. Then in May 2013, PED suddenly appeared in the United States and rapidly spread across the country, as well as to Canada and Mexico, resulting in the losses of more than eight million newborn piglets in the US alone during a one-year epidemic period.


One of the most important measures for prevention and control of porcine enteric diseases such as PED and TGEV is strict biosecurity that stops the entry of the viruses into swine farms by minimising introduction of any material or any person, which could be in contact with the viruses. To achieve this, it is important to apply disinfection throughout the production period to gain economic benefit. It is also critical to choose a disinfectant which is harmless to animals, and can be applied in the presence of animals so as to be long effective.


Accordingly, the University of Minnesota conducted tests on the virucidal efficacies of a disinfectant against PEDV and TGEV.


Challenge virus: PEDV


Product: Stalosan F powder.
Exposure temperature: Ambient room temperature (approx. 25oC).
Virus growth medium: Minimum Essential Medium (MEM) with Earle's Salts supplemented with L-
glutamine, antibiotics, 10% TPB (tryptone phosphate broth) and 3.0 µg/mL trypsin
Virus titration on: Vero-81 cells (African green monkey kidney cells).
Specific aim: To determine the virucidal efficacy of 3 doses of powder Stalosan F against PEDV in a
surface test for contact times of 5 min, 10 min, 60 min, and 6 hrs.


Test procedure:


1. A suspension of PEDV will be applied to wells of 24-well microtiter plates @100 µL of PEDV/well.
2. After the virus inoculum is dry, a dose of powder Stalosan F will be sprinkled on four virus- containing wells making sure to cover all of the virus inoculum with Stalosan F. Two other doses will be applied to eight other virus-containing wells.
3. Beef extract powder will be applied to 12 wells to serve as negative control; four wells each will be used to mimic the three doses of Stalosan F.
4. Surviving virus from pairs of wells (one experimental and one control) will be eluted in 300µL of eluent (3% beef extract solution in 0.05M glycine, pH 7.2) after 5 min, 10 min, 60 min, and 6 hrs of contact time.
5. The eluted samples will be tested for any surviving virus by preparing serial 10-fold dilutions of all eluates and inoculating them in Vero cells contained in 96-well microtiter plates. Three wells will be used per dilution.
6. The inoculated cells will be incubated at 37⁰C and 5% CO2 for up to eight days. The plates will be examined daily under an inverted microscope for the appearance of virus-induced cytopathic effects (CPE).
7. The highest dilution of the sample showing positive CPE will be considered as the endpoint.
8. Virus titres in various samples will be calculated by the Karber method(1931).
9. Comparison of virus titres in control and experimental wells will indicate the amount and per cent of virus inactivated after a given contact time.


Results:


1. An experiment was conducted with 10 mg, 50 mg and 100 mg of Stalosan F powder using the above protocol. All samples from various doses of Stalosan F and time points were cytotoxic to Vero cells while the virus titres in the control wells ranged from 104.83to 105.16per ml. The 10 mg sample was cytotoxic at 10-1dilution while 50 mg and 100 mg samples were cytotoxic at up to 102dilution. Due to cytotoxicity, no CPE could be detected in Stalosan F samples and hence this experiment was abandoned.
2. We then tried the suspension test by mixing 5, 10, and 15 mg of Stalosan F with 100 µL aliquots of the virus itself. Dilutions were made after a contact time of 5 minutes. All samples were highly toxic to cells. So, this approach was also abandoned.
3. Finally, surface test was tried once more using 10 mg and 20 mg of Stalosan F powder with four different contact times (5, 10, and 60 minutes and 6 hours). The difference in this experiment was that, after 90 minutes of incubation with different dilutions of the samples, the inoculated cells were washed twice with PBS (phosphate buffered saline) followed by incubation at 37oC. This approach was successful; no CPE was visible in any of the sample. This experiment was done in triplicate and the results are shown in Tables 1-3.
 

Table 1. Effect of Stalosan F powder on PEDV (Experiment 1)

 


Table 2. Effect of Stalosan F powder on PEDV (Experiment 2)

 


 

Table 3. Effect of Stalosan F powder on PEDV (Experiment 3)

 


Conclusion: Stalosan F powder, when applied at 10mg/2cm2and 20mg/2cm2surface area, was able to inactivate >99.97% of PEDV within 5 minutes.


Statistics: Conducting test between the mean value of control and Stalosan F at different time points revealed significant decline in the virus titre due to Stalosan F 10mg and 20mg application (P<0.05).
 

 

Challenge Virus: TGEV


Product: Stalosan F powder.
Exposure temperature: Ambient room temperature (approx. 25oC).
Virus growth medium: Minimum Essential Medium (MEM) with Earle's Salts supplemented with L-
glutamine, antibiotics, 10% TPB (tryptone phosphate broth) and 3.0 µg/mL trypsin
Virus titration on: ST (swine testicular) cells.
Specific Aim: To determine the virucidal efficacy of 2 doses of powder Stalosan F against TGEV in
a surface test for contact times of 5 min, 10 min, 60 min, and 6 hrs.


Test procedure:


1. A suspension of TGEV was applied to wells of 24-well microtiter plates @40 µL of TGEV/well.
2. After the virus inoculum is dry, a dose of powder Stalosan F was sprinkled on four virus-containing wells making sure to cover all of the virus inoculum with Stalosan F. Two other doses were applied to eight other virus-containing wells.
3. Beef extract powder was applied to 12 wells to serve as negative control; four wells each were used to mimic the three doses of Stalosan F.
4. Surviving virus from pairs of wells (one experimental and one control) were eluted in 360µL of eluent (3% beef extract solution in 0.05M glycine, pH 7.2) after 5 min, 10 min, 60 min, and 6 hrs of contact time.
5. The eluted samples were tested for any surviving virus by preparing serial 10-fold dilutions of all eluates and inoculating them in MDCK cells contained in 96-well microtiter plates after washing two times with HANKS solution. Three wells were used per dilution.
6. The inoculated cells were incubated at 37⁰C and 5% CO2 for up to two days. The plates were examined daily under an inverted microscope for the appearance of virus-induced cytopathic effects (CPE).
7. The highest dilution of the sample showing positive CPE was considered as the endpoint.
8. Virus titre in various samples were calculated by the Karber method(1931).
9. Comparison of virus titres in control and experimental wells indicated the amount and per cent of virus inactivated after a given contact time.


Results:


1. Surface test was tried using 10 mg and 20 mg of Stalosan F powder with four different contact times (5, 10, and 60 minutes and 6 hours). For each experiment the inoculated cells were washed twice with HANKS solution after 90 minutes of incubation with different dilutions of the samples, and then followed by incubation at 37°C. This approach was successful to avoid the cytotoxic effect of the sanitizer itself on MDCK cells. The 90 min was chosen because it enough for the survived virus to attach with the hosting cells if it presents.
2. This experiment was done in triplicate and the results are shown in Tables 1-3.
3. As shown in the tables, there is a reduction in virus titre in the control wells after 6 hrs. This is attributed to the low survivability of the virus under dry conditions. This noticed reduction showed minor decrease in the calculated percentage of the virus titre reduction of the sanitiser.
 
Table 1. Effect of Stalosan F powder on TGEV (Experiment 1)

 


Table 2. Effect of Stalosan F powder on TGEV (Experiment 2)

 


Table 3. Effect of Stalosan F powder on TGEV (Experiment 3)

 

 

Conclusion: Stalosan F powder, when applied at 10mg/2cm2and 20mg/2cm2 surface area, was able to inactivate ≥99.99% of TGEV within 5 minutes.

 

 

Figure 1: Effect of Stalosan F powder on TEGV at a dose of 10mg/2cm2
 

 

Figure 2: Effect of Stalosan F powder on TEGV at a dose of 20 mg/2cm2

 

Summary and conclusion


Vilofoss' Stalosan F is a mineral-based powder disinfectant suitable for hygiene applications in swine farms, in the presence of the animals. Stalosan F is suitable for application in the presence of organic matter which provides physical protection to pathogens and inhibit contact between pathogens and conventional disinfectants (CDC, 2008), making it otherwise impossible to disinfect surfaces covered with organic matter. It is therefore a broad-spectrum disinfectant that controls the growth of pathogens (fungal, bacterial, parasitic and viral) including enteric CoVs like PEDV and TGEV.


According to the research team in charge of the PEDV trial design in cooperation with the University of Minnesota, it is "significant that Stalosan F appears to have a particularly good effect, as it almost completely eliminated the PEDV in trials. According to the trials it is 99.9% effective."


Danish veterinarian Pia Conradsen, from Svinevet, adds, "In addition to the fact that infected herds need to be immunised, step two is then to reduce infection pressure. This is where the results from the Stalosan F tests are immensely interesting: until now we did not have any effective agents against it."

 

 

For more of the article, please click here.


Article made possible through the contribution of Vilofoss and Sanphar