Studies On Characteristics Of Different Generations Of Highly Pathogenic Porcine Reproductive And Respiratory Syndrome Virus(GD Strain) In Vivo

Porcine reproductive and respiratory syndrome virus is the main threatening pathogen to global pig-breeding industry for over twenty years since it has been found in1980s. In2006and2007, pig husbandry in the mainland of China and Vietnam had suffered prodigious loss from highly pathogenic PRRSV with the hallmark of discontinuous deletion of30amino acid in Nsp2gene. Many veterinarians applied various types of research to defense HP-PRRSV, our laboratory also carried out the development work of the attenuated live vaccine.In order to assess the attenuated effect of PRRSV GDr180strain, which is attenuated by passaging in MARC-145cells from the separated PRRSV GD strain.30SPF piglets of40-days-old were divided into three groups and inoculated with PRRSV GD strain, PRRSV GDr180strain and MEM culture fluid respectively. Then the rectal temperature and clinical characteristics of all piglets were monitored since they moved into their pens. Piglets of three groups were autopsied at3,7,14,21,28and35days post inoculation, blood, alveolar lavage fluid and tissue samples like lung, trachea, tonsil, heart, liver, spleen, kidney, duodenum, bladder, brain, testicle, ovary, submandibular lymph node, mesenteric lymph node and inguinal lymph node were collected and fixed in stationary liquid for pathological test, meanwhile, all these tissue samples were determined by fluorescent quantitative PCR for the dynamic distribution of antigen.The results showed that piglets in GD group with the characteristics of increased rectal temperature, depression, lethargy, anorexia and tachypnea, and neurological symptoms appeared before the death of piglets; piglets in GDr180group and control group were normal. Secondly, in the phase of3dpi-21dpi, the piglets in GD group appeared macroscopic lesions in many tissues and organs, lungs presented hemorrhage, congestion and consolidation in surface, lymph nodes generally showed swelling and bleeding, dusty red or white marble-like appearance in the capsule, infarction appeared in the edge of spleen, pinpoint bleeding in the mucosa of heart, kidney, trachea and bladder, mild hemorrhage and swelling in brain, but piglets in the28dpi and35dpi just presented mild swelling in lung, submandibular lymph node and inguinal lymph node, no significant changes in other tissues and organs; piglets of GDr180group only in7dpi showed mild edema in lung, and no significant changes in any other time, necropsy of piglets in the control group kept normal in whole experimental period. Moreover, histological changes showed these piglets in the GD group appeared typical interstitial pneumonia and non-suppurative encephalitis, lymph nodes presented more lymphoid follicles, deposition of large amounts of hemosiderin, accompanied with varying degrees of infiltration of mononuclear cells, white pulp was reduced in spleen, mucosal epithelium of the trachea and duodenum appeared to fall off, spongiocytes hyperplasia in brain, muscle fibers were denatured bent state; only one pig of GDr180group in7dpi revealed mild hyperplasia of macrophages and interstitial cells in lung, and no remarkable variedness in any other piglets of the experimental period; tissue slices of piglets in control group displayed ordinary microscopic structure.Results of immunohistochemical staining and fluorescent quantitative PCR reveal positive cells and viral load in the tissues of GD group are significantly higher than in the GDr180group during the trial period. Most remarkable difference appeared in the7dpi-14dpi, and the peak value of positive cells and viral load in these tissues of the two groups almost both revealed in7dpi, then gradually reduce or sustain in low level. PRRSV GD strains are widely and persistently distributed in tissue samples of SPF piglets, GDr180strains existed in the piglets with relatively lower concentration, shorter period and slower proliferation, in addition, the results of IHC revealed that GD strain and GDr180strain existed mostly in the epithelial cells and macrophages in various tissues and organs; results of tissue samples in control group by immunohistochemical staining and fluorescent quantitative PCR are both negative.To sum up, the typical pathogenic features of HP-PRRS revealed after these piglets inoculated with GD strain, and nothing abnormal appeared after these piglets inoculated with GDr180strain; in addition, both results of IHC and FQ-PCR proved that the viral load of attenuated PRRSV GDr180strain are less than GD strain in anytime of the experiment phase; furthermore, macroscopic and microscopic lesions of piglets attest that the pathogenicity of GDr180is obviously weakened and could be used as the HP-PRRS attenuated vaccine strain for clinical application.