The case and samples
Based on the alert by the field veterinarian, the case was a male dog exhibited same clinical findings typical for PrV. The dog owner also declared that the dog consumed raw pork offal. The dog came from Mustafakemalpaşa district of Bursa province, Turkey was presented to the veterinarian exhibiting difficulty in breathing, fever, unilateral ocular swelling and pruritis few days after consuming the raw meat. Few hours after reaching to the clinic, dog started to display stumbling and inability to stand up. The whole blood and serum samples were taken while the animal was alive, however, the dog died shortly after taking blood samples. Samples of cerebrum, cerebellum, cornu ammonis, lung, heart, lymph nodes, liver, spleen, duodenum, pancreas, kidney, salivary gland, conjunctival swab and also leukocyte and serum samples were collected at the necropsy.
After the PrV case was detected, a sampling was performed for antibody screening in hunting dogs in the region. For this purpose, blood serum was collected from 31 dogs actively used in pig hunting from Mustafakemalpaşa district where the infected dog is sheltered. Sera were obtained according to rules by national and local ethical committee (HADYEK; 2021-05/01). Blood samples were centrifuged at 1500 rpm for 10 min for serum separation and 3000 rpm for 10 min for peripheral blood mononuclear cell (PBMC) separation. Sera were heat inactivated at 56°C for 30 min and collected leukocytes re-suspended in phosphate-buffer saline (PBS), then stored at -20°C. Half part of the tissue samples were immersed in %10 neutral buffered formaldehyde and the remained part was immediately used for virological examinations without freezing. The tissue samples were homogenized in PBS and were filtrated through 0.22 µm-pore filters after centrifugation at 3000x rpm for 10 min.
The virus isolation was performed on Vero (African green monkey) and MDBK (Madin-Darby Bovine Kidney) cell lines. Supernatants of tissue homogenates (0.1ml) were inoculated and incubated for 60 min at 37 ºC in an atmosphere of 5% CO2. After incubation, DMEM without fetal calf serum were added into each well and further incubated at 37 ºC for daily examination of cytopathogenic effect (CPE) using inverted light microscope. The virus isolation process was continued for 3 passages for all the samples in order to increase viral load for subsequent characterization. Viral titers in the samples with CPE in the third passage level in Vero cell line were calculated using the Spearman-Karber method.
Specimens of brain, cerebellum, lungs, kidney, liver, spleen, heart and intestine were collected, fixed in 10% buffered formalin solution and processed routinely. The samples were embedded paraffin, sectioned at 4 µm and stained with hematoxylin and eosin.
Virus neutralization assay
The virus neutralization test (VNT) was performed for analyzing the antibody titer in diseased dog and also in serum samples from dogs possible have efficient contact to infected wild pigs in the region. Serial two-fold dilution, starting by 1:2, prepared in serum-free medium was mixed with equal volume of virus suspension with a titer of 100 TCID50 in 96-well tissue culture plate. After incubation for 120 min at 37 ºC and 5% CO2 Vero cell suspension (2x105 cell/ml) was added. The plate was observed under an inverted light microscope for cpe during 4 days post infection.
Supernatants obtained from all the tissue samples and inoculated cell cultures where cpe observed were used for virus identification by Polymerase Chain Reaction (PCR). The DNA extraction was carried out by a commercial kit (Machery & Nagel, Germany). Amplification of an 791bp fragment using primers gC-2U GTTTCCTGATTCACGCCCACGC and gC-1L GAAGGGCTCACCGAAGAGGAC  targeting a part of the gC (UL44) were performed using Dream Taq™ Hot Start PCR Master Mix (Thermo‐Fisher Sci). PCR was performed with modifications as following conditions: denaturation step for 5 min at 95 ºC, 35 cycles of 50 sec at 55 ºC, 50 sec at 60.7 ºC, 50 sec at 72 ºC and final elongation step for 5 min at 72 ºC. Sequence analysis was performed on the sample (cerebellum) which both showed the fast characteristic cpe appearance and strongest band on the gel. Obtained and References sequences (Table 1.) were edited using BioEdit soft ware and aligned with the ClustalW method. Phylogenetic analyzes inferred using Maximum-likelihood method with the Kimura two-parameter model, with 1000 replicates for bootstrap analyses, using MEGA X.