| Enterovirus 71 (EV71) is one of the most primary viruses of HFMD (hand, foot and mouth disease), belonging to species A in family Picornaviridae, genus Enterovirus. EV71 possess ability of neurotropism andit is an important enterovirus to cause HFMD in infants and children under 5 years old. The clinical outcomes of HFMD caused by EV71 are varied. In most cases, HFMD is a self-limiting disease, only rash or herpes are observed in hand, foot and mouth of patients, however, some EV71 infectious cases can cause central nervous symptoms (CNS), such as poliomyelitis like paralysis, aseptic meningitis, brain stem encephalitis, and even death. The full-length gene of EV71 is about 7.4 kb, which includes only one open reading fragment (ORF). The 3’-terminal and 5’-terminal of ORF are 3’UTR and 5’UTR, respectively. Up to day, the pathogenic mechanisms of EV71 are not fully clear, our previous study suggested that the replication capacity of EV71 was associated with the virulence. This study made use of reverse genetic technology to replace 2Apro between the severe strain and the mild strain, which helped us to study the function of 2Apr0 in the viral replication and virulence.Objective:1. To construct chimeric cDNA infectious clone and rescue chimeric strain SDLY 107-2A-1 using the replacement of 2Apro between EV71 different clinical phenotype strains.2. To understand the influence of 2Apro in the viral replication using the different replication capacity in vivo and in vitro between wild strains and chimeric strain.3. To understand the influence of 2Apro in the virulence using the injury of cells andsuckling mice caused by the wild strains and chimeric strain.Methods:1. Overlapping PCR and suitable primers were used to obtain the 2A gene fragments of SDLY 1, and then, the 2A gene fragments of SDLY 107 were replaced by the 2A gene fragments of SDLY 1 to construct the chimeric cDNA. The chimeric cDNA was transcribed in vitro to obtain the infectious RNA transfected to Vero cells. The infectious Vero cells were propagated for three passages to save the chimeric strain SDLY 107-2A-1 until obvious cytopathic effect (CPE) was observed.2. VP1 gene fragments of chimeric strain was amplified using primers EV71-S and EV71-A by PCR and identified by gel electrophoresis. The chimeric strain was identified by indirect immunofluorescence and Western blot using serum of ICR mouse infected SDLY 107 as primary antibody. Virus titers were detected by CCID50 and plaque experiment after the full-length gene was sequenced.3:Cells were infected with each strain of EV71 (SDLY 107, SDLY 1 and SDLY 107-2A-1) for 48h and detected different injury using by LDH assay and CCK-8 assay.4. Cells were infected with each strain of EV71 (SDLY 107, SDLY 1 and SDLY 107-2A-1) and cultured at 37℃ and 39.5℃. Culture supernatant was drawn every 12 hours after infection and the step lasted 5 days, and the samples were preserved in -80 ℃. Total viral RNAs were extracted and reverse transcribed to cDNA. The extracted RNAs were quantified by real time reverse transcription polymerase chain reaction (qRT-PCR) to detect viral growth curves in vitro and compare the difference of capacity curves of EV71 strains.5. The 1-day-old ICR suckling mice were divided into eight groups and each group was composed of eleven mice. The mice of treatment groups were infected with SDLY 107, SDLY 1 and SDLY 107-2A-1, respectively. Two of six groups were infected with each EV71 strain and other two as control. The 1-week-old ICR suckling mice were divided into five groups and each group was composed of eleven mice. The mice of treatment groups were infected with SDLY 107, SDLY 1 and SDLY 107-2A-1, respectively and each strain infected one treatment group, and the other group was as control. The mice were infected by intraperitoneal injection and the control groups were injected with normal saline. All mice were monitored daily for clinical symptoms after inoculation.6. The muscle tissues were collected every day for 11 days to extract the viral RNA, and extracted RNA was reverse transcribed to cDNA, and the replication curves in vivo were measured by qRT-PCR. Moreover, the infected mice were dissected and the lung, brain, intestine and muscle tissues were collected on day 1,5,9 for histopathological and immunohistochemical to analysis injuries of mice tissues and viral distribution.Results:1. Identification of the rescued viruses by PCR, IFA, Western blot and sequencing further confirmed the successful construction of infectious virus strains. The virus titers of SDLY 107, SDLY 1 and SDLY 107-2A-1 strains detected by plaque assay were 2.22×105PFU/ml,0.65×105PFU/ml,1.25×105PFU/ml, respectively.2. LDH assay and CCK-8 assay showed that cell injuries caused by SDLY 107-2A-1 were stronger than those caused by the SDLY 1 infected group and weaker than those by the SDLY 107 (P<0.05).3. The replication capacity of the chimeric strain SDLY 107-2A-1 was lower than SLDY 107 and higher than SDLY 1 in RD cells and Vero cells both at 37℃ and 39.5 ℃. The replication capacity of the chimeric strain was lower than the severe strain and higher than the mild strain in mice.4. The 1-day-old mice infected SDLY 107-2A-1 (the chimeric strain) showed less pathologic changes than the mice infected with SDLY 107 (the severe strain) and more obvious pathologic changes than the mice infected with SDLY 1 (the mild strain). Immunohistochemical staining showed strong reaction with EV71 specific antigen in muscle tissues of the 1-day-old mice infected with SDLY 107 (the severe strain), SDLY 1 (the mild strain) and SDLY 107-2A-1 (the chimeric strain). EV71 specific antigen discovered in brain tissues of the 1-day-old mice infected with the severe strain and the chimeric strain.Conclusions:1. The chimeric strain of EV71 (SDLY 107-2A-1) was rescued successfully, which could cause similar cytopathic effect to wild strain SDLY 107.2. The replication capacity of chimeric strain of EV71 (SDLY 107-2A-1) was different from wild strains both in vitro and in vivo, which suggested that 2Apro was responsible for viral replication. However,2Apro was not associated with temperature sensitive type of EV71.3. The chimeric strain and wild strains of EV71 caused different cell injuries and histopathological injuries, which indicated that 2Apro was a key protein in virulence.4. The chimeric strain SDLY 107-2A-1 and the severe strain SDLY 107 could propagate in brains of mice, but the mild strain SDLY antigen could not propagate in brains of mice. These results illustrated that 2Apro could not affect viral invasion into the nervous system, which suggested EV71 invaded the nervous system by an unknown mechanism with the exception of 2Apro, this further indicated the complexity of neuro-virulence mechanism. |
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