Localisation of swine hepatitis E virus in experimentally infected pigs

The distribution of intravenously inoculated swine hepatitis E virus (HEV) was assessed by in situ hybridisation for a period of 50 days. Evidence of apparent clinical disease was found in only one pig in the HEV infected group. The only gross lesion observed was mildly enlarged mesenteric lymph nodes at 50 days post infection (dpi). Histopathologically, mild lymphoplasmacytic infiltration and focal hepatocellular necrotic lesions were found in HEV-infected pigs. Swine HEV nucleic acids were detected by RT-PCR in the faeces at 3 dpi in 100% of the 18 pigs infected with the virus. Thereafter, the number of positives declined.The most consistent and intense signal was found in the liver of infected animals using in situ hybridisation. The positive cells were hepatocytes, Kupffer cells, bile epithelial cells and interstitial lymphocytes. Swine HEV RNA was localised in the cytoplasm of the hepatocytes, with a slightly granular pattern of staining, but hybridisation signals were not observed in degenerative or vacuolated hepatocytes. HEV was much less frequently detected in extrahepatic tissues such as lymph nodes, tonsil, spleen and small and large intestine. It was concluded that swine HEV had replicated primarily in the hepatocytes and infection resulted in subclinical infection with minimal histopathological changes in the liver.     

Optimal enhancement of in situ hybridization for the detection of porcine circovirus 2 in formalin-fixed,paraffin-wax-embedded tissues using a combined pretreatment of thermocycler and proteinase K

Optimal enhancement of the hybridization signal was developed for the detection of porcine circovirus (PCV) 2 in formalin-fixed, paraffin-wax-embedded tissues from pigs with postweaning multisystemic wasting syndrome. The hybridization signal obtained after thermocycler pretreatment was very uniform across the section, whereas the signal obtained after either proteinase K or microwave pretreatment not only was weaker but was of variable intensity across sections. Thermocycler pretreatment combined with brief proteinase K digestion can enhance signal detection for target viral nucleic acid in formalin-fixed, paraffin-wax-embedded tissues. A strong hybridization signal was detected in the cytoplasm of macrophages and multinucleated giant cells in lymph node and spleen without background staining and morphological damage. The technical improvement results, therefore, in an identical background at the same time as an increased signal and, thus, may help detect lower levels of PCV2 DNA in formalin-fixed, paraffin-wax-embedded tissues.     

检测猪戊型肝炎病毒的荧光定量PCR方法的建立

根据GenBank中猪戊型肝炎病毒的ORF2核苷酸序列的保守区域设计合成一对特异性引物,建立了一套SYBRGreen Ⅰ荧光定量PCR检测猪源戊型肝炎病毒(swHEV)的方法,并评价了该方法的灵敏度、稳定性和特异性,同时与常规的RT-nPCR进行对比分析.结果表明,建立标准曲线的相关系数为0.998,斜率为-3.039,Ct值变异系数(CV)在0.17％～1.41％之间,有良好的稳定性.同时在检测猪群常见病中显示出很好的特异性,并且比RT-nPCR更灵敏,适合于swHEV的检测.     

Prevalence and genotyping of hepatitis E virus in swine population in Korea between 1995 and 2004: a retrospective study

Hepatitis E virus (HEV) infections have been reported in pigs throughout the world but have only recently been recorded in Korean pigs. The aim of this study was to investigate whether HEV was present in archived porcine hepatic tissues collected between 1995 and 2004 using RT-PCR and immunohistochemistry and, if so, to determine the genotype of the isolates. Swine HEV was identified in the liver tissue of 42 pigs of 388 submissions (four pigs every year on average). The isolates showed genetic homology with swine and human HEV isolates identified in the United States and Japan (92.5-97%) and phylogenetic tree analysis indicated they belonged to genotype III. The study indicates that HEV is not a newly emerging virus in Korean pigs, but a pathogen that has existed in the country since at least 1995.     

Development of polymerase chain reaction and comparison with in situ hybridization for the detection of Haemophilus parasuis in formalin-fixed, paraffin-embedded tissues

DNA extraction and nested polymerase chain reaction (PCR) were developed for the detection of Haemophilus parasuis from formalin-fixed, paraffin-embedded tissues. The results for nested PCR were compared with those determined by in situ hybridization. The optimal results obtained show that use of xylene deparaffinization, digestion with proteinase K followed by nested PCR is a reliable detection method. A distinct positive signal was detected in 20 pigs naturally infected with H. parasuis by in situ hybridization. The rate of agreement between nested PCR and in situ hybridization for the detection of H. parasuis in formalin-fixed, paraffin-embedded tissues was 100%. The nested PCR could be applied successfully to formalin-fixed, paraffin-embedded tissues for the detection of H. parasuis with bacterial isolation.