MOLECULAR AND BIOLOGICAL CHARACTERIZATION OF PORCINE EPIDEMIC DIARRHEA (PED) VIRUS ISOLATES DETECTED IN UKRAINE
Anna Pastyria*1, Iryna Sobko1, Daria Berezhna2, Oleksii Nechypurenko1
1Center of Veterinary Diagnostics, 2Taras Shevchenko National University of Kyiv, Kiev, Ukraine
Introduction: Porcine epidemic diarrhea virus (PEDV) is a member of the family Coronaviridae that causes severe diarrhea, vomiting, and dehydratation. PEDV can infect pigs of any age, however high mortality rates are commonly observed in newborn piglets. This virus was first observed in Belgium in 1971, and during the next few years PEDV spread across the Europe. By 2013 disease was believed to be restricted to Asian countries. However, later that year some outbreaks of infection were reported in the USA and Europe. Also in summer of 2014 outbreak was registered in Ukraine. The aim of the study was to analyze the molecular and biological features of PEDV isolates detected in Ukraine.
Materials and Methods: Viral RNA was extracted using «MagVetTM» Universal Purification Kit. Reverse transcription was performed with a commercial kit REVERTA-L (AmpliSens). Detection of the virus was carried out with an in-house nested RT-PCR kit. Full-length genome sequencing of detected virus isolate was performed by UK Animal and Plant Health Agency (Karr, 2014). The sequence was compared with other strain of PEDV from the USA, Asia and Europe. Phylogenetic analysis was done by using MEGA 6 software. The tree was constructed by using the neighbor-joining method. The intestinal tissues of infected pigs were analyzed to investigate the pathomorphological changes induced by PEDV. Tissue fixation was performed in 10% buffered formalin and ambedded in paraffin. Samples were stained with hematoxylin and eosin.
Results: 623 samples of intestines, faeces and rectal swabs from pigs with symptoms of epidemic diarrhea were analyzed during 2014-2015. 125 samples were positive for PEDV. Microscopic intestinal lesions such as vacuolation and necrosis of enterocytes were observed. Adhesion of the apical parts of villi was noted. It was also found infiltrations of lymphocytes and macrophages and hemorrhage in the lamina propria of the villi and submucosa. Such lesions could be caused by bacterial coinfection. Phylogenetic analyses of full genome of PEDV isolate detected in Ukraine showed the highest similarity with PEDV strains from USA and Mexico (GenBank accession no. KJ645707.1, KJ645700.1, KJ645708.1, KG643697.1). The nucleotide identity was 99,7%. The nucleotide identity with European and Asian PEDV strains was much lower.
Conclusion: Since the outbreak of PED in Ukraine we detected 125 positive samples of PEDV in pathological material with the use of in-house nested RT-PCR kit. Specific histopathological changes were characterized. Phylogenetic analyses showed high similarity with strains from USA and Mexico.
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