Establishment Of RT-PCR Assay On CSFV E2 Gene And Study On CSFV Molecular Epidemiology

Classical swine fever(CSF),caused by classical swine fever virus(CSFV) belongs to Pestivirus genus in Flaviviridae family,is a highly fatal and contagious disease causing major losses in pig populations almost worldwide.A sensitive and rapid detection method based on reverse transcription-polymerase chain reaction(RT-PCR) of CSFV E2 was designed to allow the differentiation of pestiviruses by the specific size of the amplified fragments,localization of CSFV from acute infected pigs and CSF molecular epidemiology were researched by the assay.Four pairs of specific CSFV primers in the E2 region,named F1-R1,F2-R1,F3-R1,F4-R2,are designed according to integrated arrangement and comparison of 34 CSFV,18 BVDV-â… and 6 BVDV-â…¡complete gene sequences downloaded from the GenBank,at the same time skimming through homologous part together with BVDV.A sensitive,specific CSFV RT-PCR assay is established through the selection from 4 pairs primers using RNA templates of CSFV-Shimen and the optimization of reaction components in the RT-PCR system.No cross-reactivity was observed from the samples of other related viruses including FMDV/C220,PPV09/79,PRRSV/BJ,BVDV-OregonC24,BVDV-NADL,PRV-BJ/99,PCV-2/71.The detection scope of the assay cover CSFV subgroup 1.1,2.1,2.2,2.3,and the threshold of sensitivity was 8.9 pg/uL RNA for CSFV.The total coincidence rate between the assay and the FQ-PCR used by EU Classical Swine Fever Reference Laboratory was 90.00%in conformance test.The well repeatability was demonstrated by repeated experiment.To study localization of CSFV from experimental acute infected pigs,sixteen piglets were inoculated subcutaneously with a highly virulent CSFV Shimen.Piglets were kept in isolation units and were examined daily by clinical inspection for signs of CSF,and rectum temperature determination.Two piglets were bloodletting to dead at axillary artery by random every day-postinfection (DPI),30 organ-samples,2 excrement and 2 secretion,were collected.CSFV nucleic acid and antigen were detected in these samples with infecting acute CSF by CSFV RT-PCR and immunohistochemistry.CSFV nucleic acid was detected in 7 different acute samples at 1DPI,and CSFV nucleic acid of 34 different samples was detected at 5DPI.The most detection number of CSFV nucleic acid was blood,the lymph node and lymphoid tissue related to immune response took second place,the skin and muscle were at last.The positive were detected from 3DPI to 5DPI in 2 excreta(feces and urine) and 2 secretion(nasal swab and eye secretion).Positive cells were detected from 19 of 22 organs by immunohistochemistry assay,and the coincidence rate with CSFV nucleic acid positive rate of all the antigen positive organs were higher.The skin,muscle and larynx were not detected the positive signal.These results indicate that the virus is distributed in all the organs in early infection,that the virus exhibit higher affinity to blood and organs related to immune response, that the infected piglets excrete the CSFV from 2DPI to 4DPI.A good conformance is shown between CSFV RT-PCR assay and immunohistochemistry on detection to acute sample.A conclusion is got that blood and immune tissues must be collected,jejunum and ileum are mainly collected in the whole gastrointestinal tract.Muscle and skin are not suitable for sensitive CSFV detection,only must be strictly controlled about carcass transport for prevention and control of CSF.A specific E2 fragment was amplified by the CSFV RT-PCR,sequenced,from 644 clinical specimens representing 44 epizootic sites in 18 provinces during the last decade in China. Phylogenetic relationships between the detected viruses' nucleotide sequences as well as 15 reference strains were analyzed by DNAStar biological software.In the phylogenetic tree,37 of the 73 field viruses(50.68%) were clustered into subgroupl.1 within group 1,while 32 field viruses (43.86) clustered into subgroup 2.1 and 4 field viruses(5.48%) clustered into subgroup2.2 within group 2.However,none of these viruses were members of subgroup 1.2(represented by reference strain Brescia),subgroup 2.3(represented by reference strain Alfort) and group3.All of these viruses were geographical distributed in mid-eastern china.Conclusion:A novel CSFV RT-PCR assay is established,witch can be used for diagnostic and molecular epidemiology investigation to CSFV,and we get a new regularity of CSFV distribution in infected organs.