| A PRRSV was isolated from samples collected from an infected pig farm in Henan Province and was carried out isolation,identification,animal tests and completely sequenced.Then compared the histological changes caused by the isolated virus and its sequences with other strains and researched pathogenicity, genetic variation and molecular biological characteristics of PRRSV .The main research are as follows:1. The Samples collected of the lungs,kidney and lymph nodes that were treated by grind were inoculated into Marc-145 cells,and the cytopathic effect(CPE) revealed after 3 times blind passages. The PRRSV was identified by RT-PCR, and named PRRSV HLJ-09. The result of HLJ-09's TCID50 test were 10-6.5/0.1 mL .2. SPF pig challenge inoculation tests were carried out by using the virus isolated from cell cultures and tissues.Then to observe clinical reaction after challenge , recorde the temperature changes and observe the pathological changes by autopsy. The tissues collected from the pigs were observed with optical microscopes after fixing, sectioning and staining by HE,meanwhile, virions were investigated by ultrathin sectioning and electron microscope of the lung tissues. Electron microscope revealed that the mutant strain basically had no differences from the previous strains in shape and size. The pathology histologic section showed that the tissues and organs occurred typical pathological changes, such as the brain's nerve cells vacuolization and "neurotropic," phenomenon, myocarditis, renal tubular "cast", lymph nodes "Star kind degeneration "and so on. Pathological changes of tissues and organs were more serious than other reported strains ,so supposed that the reason of high mortality was severe pathological lesions caused by highly pathogenic PRRSV HLJ-09.3. RNA was extracted from the PRRSV HLJ-09 obtained by passaging the virus on Marc-145 cells. Nine DNA fragments were amplified by RT-PCR using nine sets of primers based on VR-233 and JXA1 sequence. According to the overlaps of the segments, complete nucleotide sequence of genome of PRRSV HLJ-09 were obtained by DNAStar spicing sequence of each fragment in order.The sequence was compared with other typical strains published in GenBank and draw the phylogenetic tree, meanwhile, the genetic variation and molecular characteristics of HLJ-09 were analyzed. The results showed as follows: HLJ-09 had an identity of 61.6% with the Europe strain LV, 94.8% to 98.9% with the American-type strains JXA1,CH-la,HEB1 isolated in China in 2006, and above 97.1% with the PRRSV strains isolated in China after 2006. HLJ-09 had an identity of 97.1% with JXA1 and 75.8% with BJ-4 at the amino acid level. The molecular characteristics of HLJ-09 were consistent with highly pathogenic PRRSV and HLJ-09 were consistent with the nine virulence-associated amino acid of highly pathogenic PRRSV .The genome sequence of PRRSV HLJ-09 compared with other epidemic strains in China have no significant variation .The isolated highly pathogenic strain of PRRSV HLJ-09 was carried out animal tests, completely sequenced and sequence aligned and analyzed in this study,which made us preliminary understanding the source, pathogenicity and evolutionary direction of PRRSV HLJ-09 and provided reference materials for PRRS prevention and control. Meanwhile, this study enriched PRRSV gene bank and had important significance for further researching PRRSV gene function, investigating molecular mechanism of PRRSV pathogenesis and developing diagnostic kit and vaccine. |
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