**1. Introduction**

Classical swine fever (CSF) is one of the most important diseases in domestic pigs and wild boar and is notifiable to the World Organization of Animal Health (OIE). The disease is caused by the pestivirus classical swine fever virus (CSFV) which belongs to the virus family *Flaviviridae* [1]. An outbreak with CSF is associated with enormous economic losses due to the high mortality of the disease itself, as well as due to the widely conducted stamping out policy, which implies large-scale preventive culling of pigs along with transport and trade restrictions. Within the European Union, the last sporadic outbreaks of CSF occurred in Lithuania in 2009 and 2011, as well as in Latvia in 2012 up to 2015 [2]. However, CSF is still endemic in the feral and domestic pig population in many countries of Asia, Africa, South America, and the Caribbean. The recent emergence of CSF in Japan after 26 years absence provides an illustrative example for the continuous risk of reintroduction of the disease in CSF-free countries [3,4]. Maintenance of solid knowledge of the clinical disease and a rapid diagnosis of a CSF outbreak represent indispensable prerequisites for effective control of CSF. To keep diagnostic protocols up to date, constant verification and validation of the diagnostic methods is of great importance and

includes the exchange of reference material for testing. In addition, the participation of the national reference laboratories in inter-laboratory comparison tests is obligatory, which implies the shipment of infectious sample material for testing. Not all national reference laboratories have the permission to work with infectious viruses. Therefore, those laboratories have to designate another official laboratory to perform diagnostic analysis of virus positive sample material. Furthermore, some institutions have a strict separation between serological (virus-free) and virological working departments. Laboratories or working departments that are not allowed to work with infectious sample material can receive samples that are tested negative for CSFV genome. In general, such samples are taken from animals at a late time point after infection and exhibit a high antibody concentration. However, these samples have been obtained from pigs that were infected with CSFV and despite appropriate measures and testing of this material, residual virus contents or contaminations cannot be ruled out in general. In order to minimize the risk of virus contamination at the consignee's premises, especially for those laboratories that are not allowed to work with infectious virus, and to comply with legal shipping regulations, the present study aimed to establish a pragmatic approach for reliable CSFV inactivation in serum samples. Usually, complement inactivation (incubation at 56 ◦C for 30 min) of serum samples is performed prior to diagnostic analysis. However, this treatment results in a reduced viral titer of only about one log10 and is consequently insufficient to inactivate CSFV in serum samples because of virus protecting abilities of porcine serum at 56 ◦C and ambient temperatures [5]. For virus inactivation by heating, higher temperatures would be required. According to the European Commission (EC) legislation 2002/99/EC heating temperatures of 70 ◦C are necessary for elimination of infectious CSFV. However, due to denaturation of antibodies at such high temperatures, incubation of serum samples at 70 ◦C would severely impair the detection of antibodies in these samples. Previous studies have shown that a combination of heat treatment and the addition of the detergent Tween20 to serum samples results in inactivation of enveloped viruses and that these samples can be used for further serological analysis by antibody ELISAs without impairing the qualitative results. For Rift Valley fever virus as well as for Ebola virus (species *Zaire ebolavirus*), an incubation of the serum samples at 56 ◦C for 1 h in the presence of 0.5% Tween20 (*v*/*v*) is sufficient for virus inactivation [6–8]. A lower concentration of Tween20 (0.05%) resulted in inactivation of West Nile virus after an incubation at 56 ◦C for 1 h [9].

Considering the fact that complementary inactivation through heating (56 ◦C for 30 min) is routinely applied for serum samples prior to CSFV diagnosis, the basic concept of this study was to combine this heat treatment with the dilution of the serum sample in a detergent containing buffer [phosphate buffered saline(PBS)-Tween20] in order to provide a method for virus inactivation in serum samples without impairing the detection of antibodies against CSFV by diagnostic assays described within the EU legislation [10].

#### **2. Results**
