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Occurrence of Lagovirus europaeus (Rabbit Hemorrhagic Disease Virus) in Domestic Rabbits in Southwestern Poland in 2019: Case Report

ABSTRACT Lagovirus europaeus (rabbit hemorrhagic disease virus [RHDV]) is a small, nonenveloped, single-stranded RNA virus that causes a severe, highly infectious, and fatal disease in rabbits (Oryctolagus cuniculus) called rabbit hemorrhagic disease (RHD). Since its discovery in the 1980s, it has posed a very serious threat to the global rabbit industry and the rabbit population in the wild. According to data from 2005 to 2018, the occurrence of RHD has been reported or suspected in 50 countries, with more than one-half of the reports being recorded in European countries. The main aim of the study was to detect Lagovirus europaeus (RHDV) strains found in domestic rabbits that died suddenly in the city of Wrocław in southwest Poland. All animals (n = 14) tested in this study died naturally and showed macroscopic features at necropsy that indicated the possibility of death from RHD. As a result of the research, the presence of L. europaeus virus was confirmed in 8 samples of all 14 samples collected. All strains of Lagovirus europaeus isolated in the present study showed 100% nucleotide identity to L. europaeus GI.1 strain FRG and a strain isolated in New Zealand, as well as the L. europaeus GI.1a Erfurt strain. This suggests that it is likely that L. europaeus GI.2 strains have so far not displaced L. europaeus GI.1 strains from the environment in Poland. IMPORTANCE Lagovirus europaeus (RHDV) causes a severe, highly infectious, and fatal disease in rabbits called RHD. The disease is a very serious threat to the global rabbit industry and the rabbit population in the wild. The aim of the study was to detect Lagovirus europaeus (RHDV) strains in domestic rabbits that died suddenly in Poland. The presence of RHDV was confirmed in 8 samples of all 14 samples collected. This is one of the very few reports on the existence of this virus in pet rabbits in Poland.

The first mention of L. europaeus GI.1 (RHDV) was made in 1984 in Wuxi, Jiangsu Province, China (10). The disease was observed in a population of European rabbits (O. cuniculus) of the Angora breed that had been imported from the former German Democratic Republic to China for breeding purposes (2,3,10). In China, in 1 year alone, the disease contributed to the deaths of as many as 140 million domestic rabbits and spread over approximately 50,000 km 2 , causing enormous damage to the country's economy (2,16). In a very short time, the virus has spread throughout the world and is threatening the national economies of countries that, for the most part, rely on the rabbit industry for their economies (12). The disease is now established in Europe, Asia, Africa, and Australia (4,5,17). All outbreaks of RHD until 2010 were caused by L. europaeus strains of genotype GI.1 (RHDV) and its antigenic variants (GI.1a to GI.1d) (12). A new strain of L. europaeus GI.2 that was originally identified as RHDV2 (18) was confirmed in France in 2010 and, like the classic strains of RHDV, spread worldwide very rapidly (18,19). To date, no effective cure for RHD that can save infected rabbits has been invented, and the only way to fight the disease is preventive vaccination (11,20,21). Currently, several vaccines that are effective against both strains of L. europaeus are available in the world (12).
Because of its severe course, high infectivity, and high mortality rate and because of the high risk and large financial losses for the livestock industry (2,3,11), RHD is included in the list of diseases of the World Organisation for Animal Health (WOAH) (founded as the Office International des Epizooties [OIE]) and is subject to compulsory notification of every case (22). An animal suspected or confirmed to have Lagovirus europaeus is immediately euthanized (22,23). According to data from 2005 to 2018, the occurrence of RHD was reported or suspected in 50 countries, with more than one-half of the reports being from European countries (23).
The aim of the study was to detect strains of Lagovirus europaeus (RHDV) present in domestic rabbits that died suddenly in 2019 in the city of Wrocław in southwestern Poland.

RESULTS
RT-qPCR. The results of the detection of L. europaeus GI.1 and L. europaeus GI.2 viruses by reverse transcription-quantitative PCR (RT-qPCR) are presented in Table 1.
Fourteen liver tissue samples, taken directly from animals that had died naturally, were tested. All liver tissue samples were tested for L. europaeus GI.1 and L. europaeus GI.2. In the case of L. europaeus GI.1 strains, 8 positive results were obtained, from individuals WR/1, WR/2, WR/8, WR/12, WR/13, WR/14, WR/17, and WR/20. The remaining 6 animals were negative for the presence of L. europaeus GI.1. The tests for L. europaeus GI.2 did not confirm the presence of the strains sought.
Analysis of the melting temperature (T m ) values obtained for all strains showed that all results were similar, ranging from 85.79°C to 86.50°C ( Table 1). The T m values for all tested samples exclude the possibility of a false-negative result due to the formation of nonspecific reaction products. To confirm the results obtained by RT-qPCR, all tested samples were sequenced. Table 2 shows the results of the analysis for absolute quantification. The virus copy numbers were tested in 8 positive samples and are expressed as RNA copies per milligram of liver.

DISCUSSION
Lagovirus europaeus causes RHD, which is a highly fatal disease that occurs in wild and domestic rabbits and other lagomorphs (3). The disease can occur in three clinical forms, namely, hyperacute, acute, and subacute. In the hyperacute form, sudden death is observed among animals that show little or no signs of the disease. In the acute form, the symptoms are much more severe; this is also observed in the subacute form, for which, however, the survival rate is much higher and past disease determines the occurrence of natural protection against reinfection (2, 3). Disease symptoms include neurological signs (e.g., agitation, seizures, and ataxia) and gastrointestinal disturbances, but respiratory distress may also be present. The histopathological picture shows   Occurrence of RHDV in Domestic Rabbits in Poland Microbiology Spectrum multiple lesions in multiple organs but most notably acute hepatitis and splenic enlargement. Congestion, hemorrhage, and embolism due to massive DIC can be observed in many organs (2,3). All of the animals we analyzed in our study died naturally and at necropsy showed macroscopic features that indicated the possibility of death due to RHD. However, a major limitation is that thorough histopathological examinations were not performed during the procedures, and thus the description of the pathological features in the rabbits analyzed is not very accurate. In some cases, data are incomplete or there is a complete lack of information. In a study by Harcourt-Brown et al. (24), it was noted that, if a necropsy is performed 12 to 24 h after the animal's death, then the results can be misleading due to autolysis of rabbit tissues, despite the carcass being kept in a refrigerator. In the case of our work, we cannot unequivocally determine the time between the death of the animal and the performance of the necropsy.
The liver is one of the target sites for virus replication; therefore, we determined the presence of L. europaeus GI.1 and GI.2 in liver sections collected from all dead animals by real-time PCR. As a result, we confirmed the presence of the virus in 8 samples tested, with the GI.1 genotype but not the GI.2 genotype being detected in all samples. In some cases, RHD features in the liver were poorly expressed macroscopically, and so we additionally quantified the number of viral RNA copies per milligram of liver. In their study, Harcourt-Brown et al. (25) (31). In our study, we confirmed the presence of Lagovirus europaeus in 8 domestic rabbits that suddenly died in 2019 in Wrocław, in southwest Poland. Genetic studies of the isolates showed that the three isolates (WR/1, WR/2, and WR/8) were identical and clustered in the GI.1a genogroup and showed 100% nucleotide identity with the German strain Erfurt. The Lagovirus europaeus strains isolated from samples WR/12, WR/13, WR/14, WR/17, and WR/20 were identical, as in the case of the previous three strains, but were grouped in genogroup GI.1c and showed 100% nucleotide identity with the German strain FRG and a strain isolated in New Zealand. The two groups of samples (GI.1a and GI.1c) isolated in this study showed nucleotide identity of 93.9% (263/280 nucleotides).
Conclusion. The Lagovirus europaeus strains we isolated from pet rabbits showed 100% nucleotide identity with L. europaeus GI.1strain FRG and a strain isolated in New

MATERIALS AND METHODS
Samples. Tissue samples were collected in 2019 from 14 pet rabbits with suspected Lagovirus europaeus (RHDV) infection. All tissue samples were collected within the city of Wrocław in southwestern Poland. Each animal, immediately after demise, was reported and directly transported to the Veterinary Clinic Zwierzyniec (Diagnostic and Treatment Centre for Exotic Animals), where they underwent necropsy. All tissues were described in detail, and animals were given a number with the prefix WR and an identification number. For all animals, postmortem imaging showed characteristic changes indicative of Lagovirus europaeus (RHDV) infection. Table 3 shows the postmortem descriptions of the animals, along with their sex, age, and current vaccination status.
Isolation of viral RNA. Total RNA was extracted from 30 mg of each liver sample using the ExtractMe total RNA kit (Blirt S.A, Gda nsk, Poland) according to the manufacturer's instructions.
RT and cDNA synthesis. The complementary strand of DNA (cDNA) was obtained by RT with a previously prepared viral RNA template. The reaction was performed using a Transcriptor first-strand cDNA synthesis kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the protocol provided by the manufacturer. The cDNA obtained in this way was stored at 220°C until further analysis.
RT-qPCR. The RT-qPCR was performed using the previously designed primers ( Table 4). The RT-qPCR was designed and performed on a LightCycler 480 instrument II (Roche Diagnostics GmbH) using the LightCycler 480 SYBR green I master reagent kit (Roche Diagnostics GmbH) according to the manufacturer's protocol.
The trials were divided into two groups. The first group was subjected to qualitative PCR with the use of primers specific for classic RHDV strains (L. europaeus GI.1), while primers specific for RHDV2 (L. europaeus GI.2) were used for the second group. All reactions were performed in triplicate. In addition, a series of dilutions of full-length L. europaeus GI.2 transcript standards were prepared, ranging from 1 Â 10 6 copies/mL to 1 Â 10 2 copies/mL. A curve was then prepared using a LightCycler 480 instrument II (Roche Diagnostics GmbH) and used for absolute quantitative analysis to determine the copy numbers of L. europaeus GI.2.
Sequencing and phylogenetic analysis. A 320-bp fragment of the capsid protein VP60 gene region was sequenced with the primers OIE-F (59-CCTGTTACCATCACCATGCC-39) and OIE-R (59-AACCCTCCAGG TACTGGTTG-39) (32). Sequencing was performed in both orientations using the BigDye Terminator v1.1 cycle sequencing kit (Applied Biosystems, Foster City, CA, USA) and the SeqStudio genetic analyzer (Applied Biosystems). For phylogenetic analysis of the partial VP60 sequences, the maximum likelihood method was applied using MEGA7 software (33).
Data availability. The sequence data have been submitted to GenBank under accession numbers ON548890 to ON548897.