Construction And Characterization Of Infectious Clone Of Coxsackievirus A 10

Objective:Coxsackievirus A 10 is a member of enterovirus family.It has become one of major pathogens causing hand,foot and mouth disease in recent years.CA10 can also cause herpes angina,encephalitis,acute flaccid paralysis,neurorespiratory syndrome and even death.At present,there are no preventive vaccines and special therapeutic drugs for CA10.Previous research results suggest that the construction of infective cloning of viral genes by reverse genetics technology can save viruses,and then provide a powerful tool for further research.Therefore,this study intends to construct an infectious clone of CA10 gene to obtain the rescued virus,and then use the rescued CA10 virus to establish ICR suckling mice model,in order to provide experimental basis for the diagnosis,prevention and treatment of CA10-related diseases.Methods:1.Construction of CA10 Infectious Clone and Virus RescueThe plasmid pSVA-CA10-P148 was successfully constructed and co-transfected into 293T cells with pLVX-Puro-T7 RNA polymerase（synthesized by Huada Gene Company）.RD cells were infected with the supernatant after transfection,and the virus was harvested when the infected cells had pathological changes.The rescue virus was detected by TEM,RT-PCR,and Western Blot.When RD cells were infected,the untreated group was used as negative control and the maternal virus strain group was used as positive control.2.Construction of ICR suckling mice model with rescue virus CA10（1）Infection of one-day-old ICR suckling mice with rescue virus CA10One-day-old ICR suckling mice infected with rescue virus CA10 were divided into experimental group and negative control group,in which rescue virus CA10 was used in infected group and sterile PBS buffer was used in negative control group by intracranial injection.The body weight,clinical score and survival status of experimental animals were recorded within 21 days after injection.In the experiment,the supernatant of the brain tissue of the dying mice in the infected group was collected,and the CA10 virus was detected by RT-PCR.If it was positive,it would be used as the virus infection fluid in the second animal experiment and named CA10-ICR-BTS1.（2）Infection of one-day-old ICR suckling mice with CA10-ICR-BTS1The experimental grouping,treatment and observation indexes were the same as those of one-day-old ICR suckling mice infected with rescue virus CA10.The difference was that the experimental group used CA10-ICR-BTS1.In the experiment,the supernatant of the brain tissue of the dying mice in the infected group was collected,and the CA10 virus was detected by RT-PCR.If it was positive,it would be used as the virus infection fluid in the third animal experiment and named CA10-ICR-BTS2.（3）Infection of one-day-old ICR suckling mice with CA10-ICR-BTS2（1）CA10-ICR-BTS2 was diluted 10 times continuously(10⁰-10^-6).One-day-old ICR suckling mice were infected by intracranial injection.Each dilution was divided into a group of 8 rats,and PBS treatment group was used as negative control.The clinical manifestations,average body weight and survival status of suckling mice were observed within 21 days after injection.（2）One-day-old ICR suckling mice were infected with CA10-ICR-BTS2 at the dose of 10^-4 TCID₅₀0 20ul and observed for 21 days.The clinical manifestations,average body weight and survival status of suckling mice were observed.The brain,heart,liver,lung,intestine and limb muscles of the dying suckling mice or the surviving experimental mice were analyzed by real-time quantitative PCR,HE staining and immunohistochemistry.Results:1.Construction of infectious clone of CA10 gene and results of virus rescue.CPE appeared in RD cells infected with rescued CA10 virus.TEM showed that there were virus particles of the same size and shape in the experimental group and the control group.At the same time,CA10 virus specific gene and VP1 protein were expressed in both groups.2.Construction of ICR suckling mice model with rescue virus CA10（1）The results of rescuing ICR suckling mice infected with CA10 showed that:compared with the control group,the weight growth of infected mice was significantly affected.The average clinical score of the infected group increased significantly on the 1st to 4th day and stabilized after the 14th to 16th day,while that of the negative control group remained at 0.The survival rate of infection group was 37.5%,and that of negative control group was 100%.The results of RT-PCR showed that CA10 virus was positive in brain tissue of some infected mice.（2）The results of CA10-ICR-BTS1 infection in one-day-old ICR suckling mice showed that:compared with the control group,the weight growth of infected mice was still significantly affected.The average clinical score of the infected group increased significantly on the 1st to 2nd day and died on the 3rd day with an average clinical symptom score of 5,while that of the negative control group remained at 0.The survival rate of infection group was 0,and that of negative control group was 100%.The results of RT-PCR showed that CA10virus was positive in brain tissue of all infected mice.（3）The results of CA10-ICR-BTS2 infection in one-day-old ICR suckling mice showed that:（1）The mortality rate of suckling mice rescued from virus infection in10⁰-10^-44 groups was 100%.The survival rate of suckling mice in 10^-5 groups was25%.The average body weight of the surviving experimental mice increased slightly after 5 days of infection.The average clinical score at 5 days of infection was 4.After 16 days of infection,the average clinical score decreased to 3.75.There was no death in the 10^-6 group,and there was no significant difference in average body weight between the negative control group and the10^-66 group.The average clinical score was grade 3 at 4 dpi and decreased to 0after 17 dpi.（2）The results of 10^-4 TCID₅₀ dose CA10-ICR-BTS2 infection in one-day-old ICR suckling mice showed that:A.Real-time quantitative PCR results indicated that the average viral copies in brain,intestine,heart,limb muscles,liver and lung were 6.0×10⁵,1.1×10⁵,1.9×10⁶,8.9×10⁸,7.0×10⁶ and 1.2×10⁷（copies/mg）,respectively.The replication of rescue virus in limb muscles was significantly different from that in brain,intestine and heart（p<0.01）,as well as liver and lung（p<0.05）.B.Compared with the negative control group,HE staining results showed that muscle necrosis,broken muscle fibers and disappearance of some striations could be seen in the infected limbs of rats,and interstitial myxoid degeneration accompanied by focal inflammatory cell infiltration.Alveolar atrophy and atelectasis were found in part of the lung tissues,and interstitial widening and thickening were observed.In the intestinal tissues,some of the small intestinal villous vessels showed widening of the axial septum,marked edema,scattered intracellular edema and degeneration,and no obvious inflammatory cell infiltration.No obvious pathological changes were found in the other tissues.C.Compared with the negative control group,the results of IHC staining showed that the virus antigen was positive in the limb muscle tissue of suckling mice in the infected group and negative in the other tissues.Conclusions:1.The rescue virus CA10 has similar physical and chemical characteristics with the maternal strain.The infectious clone of CA10 gene was successfully constructed.2.Rescue of virus CA10 can lead to clinical symptoms and death of typical enterovirus-related suckling mice in laboratory animals.The results of viral load,histopathology and immunohistochemistry showed that CA10 could cause injury to many tissues of infected mice,including limb muscles,lung and intestine,and CA10 virus mainly replicated in limb muscles cells.Therefore,the suckling mice model constructed in this study laid a foundation for the subsequent research and development of CA10 vaccine and the pathogenic mechanism.