| Encephalomyocarditis virus(EMCV)belongs to the genus Cardiovirus,within the family Picornaviridae.EMCV is a non-enveloped single-stranded positive-sense RNA virus,which can cause not only myocarditis and encephalitis,but also nervous system diseases,reproductive disorders and diabetes in several kinds of mammals.EMCV has a wide range of hosts,swine is its susceptible animal,which can cause fatal acute myocarditis in piglets and fetal death or abortion in pregnant sows,and brings great economic losses to the breeding industry of our country.However,different strains of EMCV have diverse virulence and different pathogenicity in their hosts,so it is unclear of their pathogenic mechanisms.And in the process of disease prevention,more attention should be needed to develop related biological products.In this study,the pathogenicity of porcine EMCV HLJ strain in mice was studied.After the growth characteristics of the virus were determined,five-week-old female mice were inoculated.The clinical symptoms and pathological changes of infected mice were observed.The infected mice began to show clinical symptoms at 3 d post infection(dpi),such as redness and swelling of conjunctiva,paralysis of hindlimb,tremor,rotation,and other neurological symptoms,and the mice died after the onset of symptoms.At 3 to 7 dpi,the infected mice showed that the brain was hyperemia and swollen and the heart became soft with white necrotic foci.Histopathological examination showed that congestion,hemorrhage,degeneration and necrosis of a large area of neurons,decreased number of neurons,infiltration of microglia and “vascular sheath” were found in brain of infected mice 3 to 7 dpi.And in the heart of infected mice,there appeared myocardial cells degeneration and necrosis,myocardial fibers dissolve and disappear after disintegration,and diffuse infiltration of inflammatory cells.The replication of virus in various organs and tissues was detected by real-time fluorescence quantitative PCR.The results showed that the virus reached the peak at 3-5 dpi,and the obvious higher virus load in brain and heart.The indirect immunofluorescence assay was used to detect EMCV in brain and heart of infected mice,which showed that there was more virus load in the neurons of brain and myocardial cells of heart.The replication of virus in these organs was generally correlated with the pathological lesions,respectively.Theses results demonstrated that the main target organs of EMCV HLJ strain in mice were brain and heart.At the same time,it was undertook to construct the infectious c DNA clone of EMCV HLJ strain and rescue of virus.The full-length genome of EMCV HLJ strain was amplified by RT-PCR method,and then each fragment was fused by overlap PCR method to obtain the complete c DNA.After restriction endonuclease digestion,the whole genome PCR product was ligated into p CI vector to obtain full-length c DNA infectious clone.The point mutation was introduced into primers D2-F and D1-R,which formed the new Eco R I restriction site,and used as a molecular marker of infectious clone.After that,the p CI-HLJ recombinant plasmid was transfected into BHK-21 cells,and the rescued virus was harvested and named r HLJ.The rescued virus could produce typical cytopathic effects on BHK-21 cells similar to its parent virus,such as cell shrinkage,round shedding,and formation of grape seed-like lesions.In addition,the D fragments of rescued virus were amplified and identified by Eco R I single restriction endonuclease digestion,and the genomic fragment of 5958-6401 nt containing molecular markers were amplified and sequenced.The results showed that r HLJ contained Eco R I restriction site.Indirect immunofluorescence assay showed that green fluorescence signal presented on BHK-21 cells after rescued virus inoculation,and the virus was rescued successfully.The acquisition of infectious c DNA clone of EMCV HLJ strain provides a vital material basis for the study of viral gene function and development of the genetic engineering vaccine for the disease prevention. |
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