# Real-Time PCR Confirms Infection with _Lagovirus europaeus_
## 1 Introduction  
_Lagovirus (L.) europaeus_ GL.1 and GL.2 is an etiological agent that causes a severe and highly lethal disease--RHD (rabbit hemorrhagic disease) [1]. The problems posed by the spread of this virus around the globe concern not only economic considerations, in the case of countries whose economies are based on the rabbit industry, but also the negative impact of the virus on the environment [2, 3]. In addition, the interest in _L. europaeus_ is influenced by the fact that it is a model in the study of the variability of a whole range of RNA viruses, which are common pathogens in animals. The basic element necessary to start fighting any pathogen is its effective diagnosis. Nowadays, the most respected molecular methods are definitely leading the way. One of the most commonly used methods is the real-time polymerase chain reaction technique--real-time PCR [4]. This method is one of many modifications of the classical PCR reaction and is characterized by the use of a fluorescent dye that allows the analysis of product growth by following the quantitative measurement of the excited fluorescence [4]. In this method, a logarithmic increase in the level of fluorescence is considered a positive result, which determines the increase in the product [4]. Thanks to this method, it is possible to detect small amounts of viral particles, which would be unattainable in the case of classical PCR. An additional advantage is that the method enables both quantitative and qualitative analysis of the material, which makes it possible to significantly shorten and simplify the procedure [4]. _L. europaeus_ is a virus belonging to the genus _Lagovirus_ within the family Caliciviridae, which is formed by non-enveloped ssRNA (+) viruses [5]. Within the GI genogroup, the GI.1 and GI.2 genotypes are distinguished, with four variants (a-d) observed in GI.1 [6]. The first mentions of the _L. europaeus_ GL.1 virus appeared in 1984 in China [7]. Due to the fact that the virus was characterized by a high level of mortality and a significant pace of expansion, its range became global in a relatively short time [1]. In 1997, the _L. europaeus_ GI.1a virus was identified in Italy. Its antigenic features were significantly different from the previously isolated strains, and on this basis, it was considered the first antigenicvariant of rabbit hemorrhagic disease virus [8]. The presence of this pathogen was then confirmed in many places around the world, where it usually tended to displace strains belonging to other genogroups from the environment [9]. The appearance of the new virus _L. europaeus_ GI.2 was first recorded in France in 2010, among both individuals vaccinated and not vaccinated against _L. europaeus_ GI.1, showing symptoms typical of RHD [10]. In a short time, this factor was recognized in many countries, where it usually tended to displace strains belonging to the GI.1 genogroup from the environment [2]. A single virus particle is a non-enveloped structure of 35-40 nm. Viral ssRNA (+) is presented as two overlapping reading frames (ORF), and its size is approximately 7.5 kb [1]. The virion also shows the presence of 2.2 kb subgenomic RNA [1]. The ORF1 frame is located at the 5' end and encodes a 257 kDa polyprotein, from which the proteins VP60, P16, P23, and P29 are formed as a result of the action of proteases. On the other hand, the ORF2 frame is at the 3' end and encodes the VP10 protein [1]. The viral capsid has 90 dimers of the VP60 protein, forming 32 wells. This protein consists of two domains called S and P. Additionally, the P domain is divided into the P1 subdomain and the P2 subdomain, which forms the most advanced part of the capsid [1]. Moreover, six regions (A-F) have been described that show exceptionally strong genetic variation among the isolated strains [11]. It was described that the F region influences the ability of the strains to hemagglutinin, as well as the C and E regions, which are assigned the highest degree of genetic variability.