*2.1. E*ff*ect of Tween20 Concentration on Inactivation of CSFV in Serum Samples and Infectious Cell Culture Supernatants*

In the initial stage of the study, CSFV infectious cell culture supernatants of four different CSFV strains were used. The infectious cell culture supernatants showed a virus load between 1.78 <sup>×</sup> 106 to 3.16 <sup>×</sup> 106 tissue culture infection dose 50 (TCID50)/ 50 <sup>μ</sup>L after 1:2 dilution in PBS (PBS control). Incubation at 56 ◦C for 30 min resulted in a decrease of the virus titer by approximately two log units [PBS + heat (H) control]. If the infectious supernatants were diluted in PBS containing 0.05% Tween20 (final concentration of Tween20: 0.025%) and heat treatment was performed, no infectious virus was detected (Figure 1A). This was confirmed by virus isolation on two different cell lines. The samples that were diluted in PBS without heat treatment (PBS control) were tested positive by virus isolation. Generally, detection of CSFV genome was still possible, but the samples that were treated with PBS 0.05% Tween20 showed a considerable lower number of genome copies compared to the samples treated with PBS (PBS control) (Figure 2).

**Figure 1.** Classical swine fever (CSF) virus titer after sample treatment performed for virus inactivation. (**A**) Four infectious cell culture supernatants (two highly virulent strains, CSF0947 and CSF0634, as well as two moderately virulent strains, CSF0902 and CSF0104) and (**B**) four infectious serum samples were used for the experiment. The samples were 1:2 diluted in phosphate buffered saline (PBS)-Tween20 and incubated at 56 ◦C for 30 min. Two PBS controls were included; for the first, the samples were 1:2 diluted in PBS and incubated at 56 ◦C for 30 min (PBS + heat (H) control), whereas the second control was not treated with heat (PBS control). The four serum samples were treated with different Tween20 concentrations (0.05%, 0.1%, 0.3%, 0.5%, 1%, and 2%), whereas one concentration (0.05%) was used for the inactivation of infectious cell culture supernatants. Immediately after treatment, the samples were titrated. The experiment was performed in three independent runs. H = heat treatment at 56 ◦C for 30 min; T = Tween20.

However, after dilution of serum samples in PBS 0.05% Tween20 followed by heat treatment, infectious CSFV was still detectable. Accordingly, this inactivation protocol was not effective for serum samples, even though the initial virus titers in the four serum samples (ID 1, ID 4, ID 6, and ID 23) were significantly lower (2.14 <sup>×</sup> 10<sup>2</sup> to 3.77 <sup>×</sup> 104 TCID50/ 50 <sup>μ</sup>L) than the virus titers in the infectious cell culture supernatants. The calculated virus titers of the serum samples after dilution in PBS 0.05% Tween20 were comparable to the titers of the serum samples that were diluted in PBS without Tween20 (PBS + H control). Likewise, dilution of the serum in PBS 0.1% Tween20 (final concentration of Tween20: 0.05%) followed by heat treatment showed no significant differences in the decrease of the virus titer compared to the PBS control that was incubated at 56 ◦C (PBS + H control). Only after dilution in PBS 0.3% Tween20 (final concentration of Tween20: 0.15%) combined with heat treatment was the infectious virus no longer detectable (Figure 1B), which was confirmed by virus isolation on two porcine cell lines. In comparison to this, all samples that were diluted in PBS without heat treatment (PBS control) were detected positive for infectious CSFV by virus isolation. As it was shown for the infectious cell culture supernatants, CSFV genome was still detectable after treatment with Tween20. However, the number of genome copies was reduced compared to the serum samples treated with PBS (PBS control) (Figure 2). The concentration of 0.3% Tween20 had no influence on cell growth, whereas the samples diluted in PBS 1% Tween20 or PBS 2% Tween20 induced a cytopathic effect. This cytopathic effect was observed within the first two dilution steps of virus titration and did not occur after incubation of the cells with higher dilutions of the PBS 1% Tween20 and PBS 2% Tween20-treated samples (data not shown).

**Figure 2.** Classical swine fever virus (CSFV) genome detection by quantitative real-time RT-PCR (qRT-PCR) after sample treatment for inactivation of CSFV. Four infectious cell culture supernatants and four infectious serum samples were 1:2 diluted in phosphate buffered saline (PBS)-Tween20 (PBS 0.05% Tween20 for cell culture supernatant and PBS 0.3% Tween20 for serum) and incubated at 56 ◦C for 30 min (treated). As a control for viral genome load, samples were 1:2 diluted in PBS and not incubated at 56 ◦C for 30 min (PBS control). All samples were analyzed in duplicates in a CSFV-specific qRT-PCR and the mean values are shown.

#### *2.2. Validation of CSFV Inactivation*

To confirm the efficacy of the protocol for virus inactivation in serum samples and to evaluate the application of these treated samples in serological diagnostic assays [virus neutralization test (VNT) and ELISA], a panel of 33 sera was tested. The panel comprised two different sample categories. Samples of category I were tested positive for infectious CSFV whereas samples of category II were classified negative for infectious CSFV.

The serum samples were 1:2 diluted in PBS 0.3% Tween20 or PBS 0.5% Tween20 and incubated at 56 ◦C for 30 min. Immediately after treatment, CSFV positive sera of category I (*n* = 23) treated with PBS 0.3% Tween20 were subjected to virus isolation over two passages using the two porcine cell lines, porcine kidney (PK15) and swine kidney (SK6), separately. None of the 23 treated serum samples tested positive for infectious virus, neither in the undiluted approach nor in the 1:10 dilution. The positive control clearly tested positive for infectious virus confirming that the method has been performed properly. For the detection of CSFV-specific antibodies within a short time, commercial ELISAs are often used. In case a serum sample is tested positive or doubtful by the ELISA, an additional confirmatory test is performed, generally the VNT.

Previous studies have described differences in the detection of antibodies using ELISAs that are based on either E2 or Erns antigen [11]. Against this background, the samples of the present study were tested in an E2-specific (CSFV Ab ELISA; IDEXX) as well as in an Erns-specific ELISA (*pigtype*® CSF Erns AB ELISA; Indical). The results of the E2-specific ELISA showed no significant differences in the detection of antibodies between treated and non-treated samples. Also, the classification "positive for antibodies against CSFV" of samples was not negatively influenced by the treatment with PBS 0.3% Tween20 or PBS 0.5% Tween20, except for one sample (sample ID 5). However, the samples that were

treated with PBS 0.5% Tween20 showed rather higher values of blocking percentage. Four non-treated samples (ID 3, ID 6, ID 8, and ID 16) were tested doubtful for antibodies against CSFV, but positive for antibodies against CSFV after treatment with Tween20 (0.3% or 0.5%) and incubation at 56 ◦C, and one negative tested non-treated sample (ID 4) showed a doubtful result after treatment (Figure 3A).

The results obtained by the Erns antibody ELISA were different compared to the detection of antibodies against E2 (Figure 3B). One major difference was that high amounts of Erns-specific antibodies were detected in the majority of the serum samples of category I (sample IDs 1–16 and ID 23). However, several samples that were treated with Tween20 and heat showed decreased S/P values (ratio of sample OD450 to mean OD450 of the positive control) compared to the non-treated samples (S/P value difference ≤ 1). This had an impact on the classification of samples with a low antibody response. As a result, five treated samples (ID 7, ID 21, ID 27, ID 28, and ID 30) were classified as doubtful or negative for antibodies against CSFV instead of a positive result obtained by analysis of untreated samples. Sample 14 tested positive, with an S/P value very close to the cut-off (S/P value = 0.504) after treatment with PBS 0.3% Tween20. This sample was classified doubtful (S/P value = 0.459) after treatment with PBS 0.5% Tween20. However, this inter-test variance is not significant and generally observed for repetition of ELISAs. Only for one sample (ID 32) was a high difference in S/P value after treatment with PBS 0.3% Tween20 or PBS 0.5% Tween20 detected. Generally, the S/P values, which were obtained for the samples treated with PBS 0.5% Tween20 were slightly lower than the ones detected after treatment with 0.3% Tween20. These minor differences did not lead to additional difference in sample classification, except for one sample (ID 32).

As the VNT is often used for confirmation of positive or doubtful ELISA results, as well as for the differentiation between CSFV-specific and ruminant pestivirus-specific antibodies, the 33 serum samples were also tested in the VNT. The detection of virus neutralizing antibodies was performed immediately after dilution in PBS 0.3% Tween20 or PBS 0.5% Tween20 along with heat treatment. No negative effects caused by the Tween20 concentrations were detected. A low-to-moderate neutralizing antibody titer (up to 120 ND50) was detected for the samples of category I (sample ID 1–23). Sera of category II (sample ID 24–33) showed a moderate-to-high neutralizing antibody titer (>640 ND50). For most treated samples, a slight increase of the antibody titer was observed compared to the non-treated samples. However, a difference of up to three titer steps is commonly detected by repetition of VNTs and is therefore considered not significant. A threefold or higher difference in the antibody titer detected for treated and non-treated samples was not observed, except for one sample (ID 16) after treatment with PBS 0.5% Tween20 (Figure 4).

On the basis of the fact that no significant titer differences were observed for treated and non-treated samples, it can be concluded that the concentration of Tween20 (final concentration after 1:5 dilution in the VNT: 0.03% Tween20 or 0.05% Tween20) had no significant influence on inactivation of the CSFV strain CSF0104 (100 TCID50) that was used for performing the VNT.

According to the results, 11 out of the 33 serum samples were apparently hemolytic. However, this characteristic had no influence on serological analysis in VNT and ELISA using treated samples.

**Figure 3.** Detection of antibodies against Classical swine fever virus by two ELISAs. A total of 33 serum samples were treated with phosphate buffered saline (PBS) 0.3% Tween20 or PBS 0.5% Tween20 and incubated at 56 ◦C for 30 min. Afterwards, these samples were analyzed in comparison to non-treated samples in an E2-specific (**A**) and Erns-specific (**B**) ELISA.

**Figure 4.** Detection of neutralizing antibodies against Classical swine fever virus by virus neutralization test (VNT) after sample treatment. A total of 33 serum samples were treated with phosphate buffered saline (PBS) 0.3% Tween20 or PBS 0.5% Tween20 and incubated at 56 ◦C for 30 min. Afterwards, these samples were analyzed in comparison to non-treated samples in the VNT. In addition to the titers of neutralizing antibodies, titer differences between the treated and non-treated samples are depicted. A negative value for the titer difference showed a reduction of the antibody titer compared to the non-treated samples. Results for samples ID 1–15 are summarized in (**A**) and those for the sample ID 16–33 in (**B**).

#### **3. Discussion**

A rapid and safe detection of notifiable epizootic diseases, such as CSF, is of great importance. To keep the diagnostic methods up to date and to ensure a continuous efficient diagnostic performance, a regular exchange of reference material is indispensable. Therefore, reference material is often requested for verification and validation of diagnostic assays applied for detection of CSFV genomes or antibodies against CSFV. Usually, this sample material is obtained from animals that were infected with CSFV. Despite of appropriate testing [e.g., by quantitative real-time RT-PCR (qRT-PCR) or virus isolation] a contamination or residual virus in samples that were taken from animals infected with CSFV cannot be ruled out in general. For this reason, minimization of the risk of any viable CSFV in serum samples is of utmost importance, especially when the sample material is intended for laboratories that are not allowed to work with infectious viruses or that have a strict separation between serological and virological working departments. The significance of the CSFV inactivation is underlined by the fact that this topic had been already targeted before. However, former studies exclusively addressed virus inactivation in tissues or other matrices that are not relevant for serological analysis upon shipping [5,12,13]. The focus of the present study was to define a pragmatic procedure for inactivation of CSFV in serum without impairing the serological properties for subsequent antibody detection by ELISA and cell culture-based VNTs.

It has been shown for other enveloped viruses (Ebola virus and Rift Valley fever virus) that virus inactivation in serum samples can be performed by addition of the non-ionic detergent Tween20 (final concentration: 0.5%) and incubation at 56 ◦C for 1 h [6–8]. Tween20 solubilizes lipid membranes and is widely used as a component of washing buffers to prevent unspecific binding of antibodies, for example, in ELISAs and immune blots. The former studies showed that serum samples, which were treated with Tween20 and were incubated at 56 ◦C for 1 h, are suitable for antibody detection by ELISAs. However, the application in other serological assays involving cell culture systems, for example, VNT, has not been evaluated so far. VNT is one of the most important serological assays for the detection of antibodies against CSFV, particularly as a confirmatory test for positive or doubtful ELISA results, and for the differential diagnosis regarding antibodies against other closely related pestiviruses [10]. The present study showed that the use of 1% Tween20 (final concentration: 0.5%) led to virus inactivation in serum samples but cytotoxic effects were detected in cell culture. These effects can be traced back to the high Tween20 concentration. On the basis of these results, it can be concluded that samples treated with PBS 1% Tween20 cannot be used for analysis in cell culture-based techniques such as VNT. In contrast, no cytotoxic effects were observed for lower Tween20 concentrations (≤0.5% Tween20 in PBS).

Fortunately, virus inactivation was also confirmed for serum samples that were treated with a lower concentration of Tween20 (PBS 0.3% Tween20 or PBS 0.5% Tween20) without impairing the growth of tissue culture cell. Furthermore, the present study showed that serum samples, which were inactivated using PBS 0.3% Tween20 (final concentration: 0.15%) or PBS 0.5% Tween20 (final concentration: 0.25%) and heat treatment (56 ◦C for 30 min), can be used for serological assays to detect antibodies against CSFV by ELISA and VNT. One limitation of the inactivation protocol was observed for the *pigtype*® CSFV Erns AB ELISA that includes a 1:10 dilution of the sample prior to analysis. Five inactivated serum samples that were treated with PBS 0.3% Tween20 (ID 7, ID 21, ID 27, ID 28, and ID 30) were classified doubtful or negative for antibodies against CSFV, whereas the corresponding non-treated samples were tested positive. However, this finding may be tolerable because the majority of routinely applied serological assays target either E2-specific antibodies or the NS3 of CSFV (common staining method of VNT). It was shown that a dilution factor of two, which was used for the CSFV E2-specific AB ELISA and the VNT, had no significant influence on sample classification. Moreover, the present study confirmed that the immune response against the structural proteins Erns and E2 is different [11]. A high amount of antibodies against Erns was detected at an early time point after infection or in serum samples that contain virus. In comparison to this, all samples that do not contain infectious CSFV showed highly positive results in the E2-specific antibody ELISA. For the analysis of CSFV genome by quantitative real-time RT-PCR, the inactivation method is not recommended

because addition of Tween20 to the sample had a negative impact on detection of viral genomic RNA. However, the implementation of this inactivation technique was not focused on subsequent CSFV genome detection as this is considered not relevant for serological departments that are forbidden to work with infectious viruses.

Interestingly, inactivation of CSFV was dependent on the matrix. For inactivation of CSFV in cell-free supernatant, the required Tween20 concentration was considerably lower (final Tween20 concentration: 0.025%) than for CSFV inactivation in serum samples (final Tween20 concentration: 0.15%), even though the virus titers in serum samples (2.14 <sup>×</sup> 10<sup>2</sup> to 3.77 <sup>×</sup> 104 TCID50/ 50 <sup>μ</sup>L) were about 100- to 10,000-fold lower than the virus titers in tissue culture supernatants (1.78 <sup>×</sup> 106 to 3.16 <sup>×</sup> 10<sup>6</sup> TCID50/ 50 μL). A similar concentration of Tween20 was also used for inactivation of West Nile virus in cell-free supernatant [9]. It can be hypothesized that the presence of proteins in serum samples may influence the efficiency of inactivation by detergent Tween20 in lower concentrations. In this context, it has been reported that, even though CSFV can be considered as a moderately fragile virus, prolonged survival is possible under favorable conditions such as a protein-rich milieu [13]. Different results have been published with regard to the influence of the properties of the viral strain on virus stability [5,12,14]. In the present study, no differences regarding virus inactivation were detected between the highly virulent strains CSF0947 and CSF0634 and the two moderately virulent strains CSF0902 and CSF0104.

In order to prove a broad application of the inactivation technique, a serum panel has been compiled using the serum collection of the EU and OIE Reference Laboratory for CSF (Hannover, Germany). This validation panel comprised sera with different properties, for example, samples that were taken from animals infected with distinct CSFV genotypes, as well as samples containing various antibody and genome amounts. The virus titers of the serum samples used in this study are representative of those typically found after CSFV infection. The EU and OIE Reference Laboratory for CSF organizes an international inter-laboratory comparison test every year to evaluate the diagnostic methods including virus isolation and virus titration. The titer of these sera, which were taken from experimentally infected pigs, ranges between 10<sup>1</sup> and 105.5 TCID50/50 μL.

Each of the virus positive samples (*n* = 23) were completely inactivated by the suggested method. Due to the fact that virus isolation on CSFV-susceptible cells was performed immediately after sample treatment, it can be concluded that the virus inactivation resulted from the treatment itself, instead of any other possible influence of subsequent storage of the sample in PBS-Tween20. However, the volume of serum samples to be inactivated needs to be considered for the incubation time at 56 ◦C because the time for reaching the core temperature of 56 ◦C within the serum sample depends on the sample volume. Accordingly, for sample volumes > 2 ml, longer incubation times are recommended.

The present study shows that treatment with PBS-Tween20 (0.5%) and incubation at 56 ◦C for 30 min leads to complete virus inactivation. For laboratories that are not allowed to work with infectious CSFV, safety precautions must be applied before these laboratories can receive sample material that was taken from CSFV-infected animals. So far, only samples that were tested negative for CSFV genome and for infectious CSFV are shipped. As an additional safety measure, the laboratories are informed (e.g., by the corresponding material transfer agreement) that adequate disinfection and decontamination of all things that came into contact with the material are requested. The present study showed that treatment with PBS-Tween20 combined with complement inactivation represents an additional third safety margin for serum samples, which are tested negative for CSFV genome beforehand. Furthermore, this treatment represents a good alternative for laboratories that have a separation between serology (non-infectious) and virology departments. For these laboratories, the described method for CSFV inactivation offers the possibility to analyze individual serum samples first by virological methods, followed by serological analysis after sample treatment with PBS-Tween20 and heat. However, because the inactivation of an agent causing a notifiable disease is a serious issue, it is recommended to confirm virus inactivation of any sample beforehand.

In conclusion, in order to minimize the risk of CSFV contamination at the consignee's premises regarding laboratories that have no permission for working with infectious CSFV, the treatment of CSFV genome negative serum samples with Tween20 (final concentration should be 0.15%) and incubation at 56 ◦C for 30 min prior to shipment can be recommended. It is a simple, pragmatic, and cost-efficient approach for CSFV inactivation that can be performed by each laboratory as it does not require any special equipment. However, this method needs to be carefully validated in each laboratory beforehand and longer incubation times should be applied for samples containing volumes >2 ml.

### **4. Materials and Methods**

#### *4.1. Serum Samples*

In total, 33 serum samples that were taken from animals experimentally infected with CSFV were used in the present study. These samples were allocated into two different categories. Samples of category I tested positive for infectious CSFV and developed no or a low-to-moderate immune response against CSFV (*n* = 23; ID 1 to ID 23). Furthermore, 9 out of the 23 samples were visibly hemolytic (ID 2, ID 4, ID 5, ID 6, ID 9, ID 13, ID 16, ID 20, and ID 23). Category II comprised sera tested negative for infectious CSFV, but clearly positive for CSFV antibodies (*n* = 10; ID 24 to ID 33). Two of these samples were hemolytic (ID 27 and ID 31). All samples were obtained from the serum sample collection of the EU and OIE Reference Laboratory for CSF (Hannover, Germany). The corresponding animal experiments were notified by the Specialized Department of Animal Welfare Service of the Lower Saxony State Office for Consumer Protection and Food Safety (LAVES; Permit Number: LAVES AZ 08A 538) according to the German animal welfare act.

#### *4.2. Cell Culture and Viruses*

Two different cell lines, PK15 and SK6 cell line, were used in the present study. The cell line PK15 (CCLV0051) was obtained from the Collection of Cell Lines in Veterinary Medicine (CCLV, Friedrich-Loeffler-Institute, Island of Riems, Greifswald, Germany) and was cultivated in Eagle's minimum essential medium (EMEM), supplemented with fetal calf serum (FCS); 5% (for SK6 cells) or 7.5% (for PK15 cells) FCS were added to the growth medium and 10% to the virus culture medium.

SK6 cells were kindly provided by the Institute of Virology and Immunoprophylaxis, Mittelhäusern, Switzerland, and were grown in Dulbecco's modified Eagle's medium supplemented with 10% FCS. Before usage, FCS was tested for absence of pestivirus genomes and pestivirus-specific antibodies.

Two highly (CSF0947 and CSF0634) and two moderately (CSF0902 and CSF0104) virulent CSFV strains were applied for validation of virus inactivation. These virus strains derive from the virus collection at the EU and OIE Reference Laboratory for CSF in Hannover, Germany [15].
