| BYD1 strain characterized as a new type reovirus was isolated from the sample of a SRAS-patient by our lab in July,2003. It is the second report of reovirus-isolation twenty-one years after the first internal one in 1982. Reovirus strains involved in human diseases were isolated from infants and children, who were severely suffered with fatal meningitis, fatal pneumonia, diarrhea, or intermittent fevers. While it is a ready concept that infection with reovirus causes no any disease to the human more than inapparent infection. So, Chinese researchers seldom work on it. Nearly all the clinical and laboratory studies were reported abroad. Contributing to the isolation of BYD1 strain, Chinese researchers have been paying more attention to reovirus. Our lab has conducted a series of studies on BYD1 strain.Many reoviruses induce apoptosis in a wide variety of cultured cells in vitro, which is also well characterized to be as a main role associated with viral pathogenesis. This study will be carried out to reveal some aspects of BYD1, including the ability of apoptotic induction, the molecular mechanism of BYD1 to induce apoptosis, pathogenesis of BYD1, and relationship between pathogenesis and apoptotic induction.HeLa cell line was taken to be infected by 105TCID50 of strain BYD1. After 12 hours post infection, HeLa cells were digested with pancreatin. Then they were dyed by propidine iodide and FITC labeled Annexin-V to measure the rate of apoptotic cells by the flow cytometer. The data was analysed by student t-test. The rate of apoptotic cells is 14.32±0.71, while the one of mock infection is 4.06±0.23.t value is 26.69,lager than t0.05. BYD1 has the ability to induce apoptosis.It will take no more than 16 hrs for reovirus to replicate themselves in cytoplasm. The apoptosis of HeLa cells induced by BYD1 can be detested 12 hrs post infection. It is suggested that apoptosis-induction happens ahead of viral replication and it is participated with viral proteins but viral complication. The viral structural proteinσ1 andμ1 of reovirus were reported have close relationship with apoptosis. The outer capsid of reovirus is assembled with structural proteinsμ1,σ3, andσ1. These proteins are all involved in penetration of virions into cytoplasm. Reovirus infection is initiated by the attachment of virions to cell surface receptors via theσ1 protein. After receptor binding, virions are internalized into cells by receptormediated endocytosis. Virions undergo acid-dependent proteolytic disassembly within cellular endosomes, leading to the formation of infectious subvirion particles (ISVPs). ISVPs are characterized by the loss of outercapsid proteinσ3, a conformational change in attachment proteinσ1, and cleavage of outer-capsid proteinμ1 to form particle- associated fragmentsδandφ. Subsequent to ISVP formation theσ1 protein is shed, and theμ1 cleavage fragments undergo conformational rearrangement, yielding ISVP*s. ISVP*s are putative to penetrate endosomes and deliver transcriptionally active cores into the cytoplasm. Then viron's replication occur in cytoplasm.In order to scrutinize the molecular mechanism, the genes of full lengthσ1, full lengthμ1,μ1(1-675), andφwere selected to clone into vector pEGFP-C3 to construct recombinant plasmid pEGFP-C-σ1(1-461), pEGFP-C-μ1(1-708), pEGFP-C-μ1(1-675), and pEGFP-C-μ1(582-708). Then, these recombinant plasmids were taken to infect 293T cells, respectively. At 18h post transfection, cytopathic effects were observed and the ratios of apoptotic cells were detested. At 48h post transfection, cells were harvested to observe apoptotic morphology under the scope of electron microscope (EM). It was obviously that cytopathic effects were typical enough in cellular morphology , including indistinct boundary, cell shrinkage, and even cell debris under the scope of light microscope. Those cytopathic cells displayed fusion proteins expression under the scope of fluorescence microscopes. With the help of EM, classic apoptotic morphology of early phase, middle phase, and advanced phase were all found among the treated 293T cells. Statistic analysis of the ratios of apoptotic cells acquired from flow cytometer gave the conclusion that bothσ1 and three forms ofμ1 are certainly able to induce apoptosis.σ1 andμ1 are two determinants of BYD1 strain to induce apoptosis. It is suggested that more than one proteins of BYD1 strain posses the ability to initiate proapoptotic signals exclusively or mutually.To analyze the possible relationship among BYD1's pathology and its induced apoptosis, suckling mice in 3-day old were inoculated intracranially with BYD1 strain. Each mice were inoculated with 20μl virus culture then they were surveyed every day to find symptoms. Based on the observation, the disease course of infected mice undergo acute stage, stationary stage, and aggravation stage. The treated mice were executed at the 8th day post inoculation and dissected to get organs to make histological paraffin sections. The sections were used to carry out pathologic analysis and TUNEL detest of apoptosis in situ. Pathologic results approved that BYD1's infection had caused multiplicate tissue damages located in central nerve, heart, and lung. So, BYD1's infection has close relationship with central nerve damage, myocarditis, and pneumonia. There are bound of apoptotic cells found in hippocampus through TUNEL test. Combined with clinical symptoms and histological morphology appearance, it is reliable to draw the conclusion that apoptosis induced by BYD1 is one of direct causes contributing to tissue's damage.
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