Construction Of Animal Model Of Coxsackie B Virus Type 1 Infectio

Objective:Coxsackievirus B1(Coxsackievirus B1,CVB1)has been circulating sporadically in the world in the past 20 years,which can cause hand,foot and mouth disease,encephalitis,myocarditis,etc.The establishment of rapid detection methods and animal models for CVB1 virus is of great significance for the clinical diagnosis of the virus,the study of virus pathogenesis,and the evaluation of drug and vaccine development.Methods:This study was divided into three parts.Part I:Establishment of CVB1 detection system:The VP4 gene fragment of CVB1 virus was used as the target gene fragment to construct recombinant vector,and the RNA standard of VP4 gene was obtained.The real-time reverse transcription polymerase chain reaction(Real-time RT-PCR)detection method was established after the probe primers were designed for this region.Part Ⅱ:An infection model of Syrian golden hamster(Mesocricetus auratus)was established.Syrian golden hamsters were infected with CVB1 virus with a titer of 107.25CCID50/mL by intraperitoneal injection and nasal instillation at a dose of 100 μL/mouse.The clinical symptoms of the animals were observed for 14 days after infection,and the virus shedding was detected..Part Ⅲ:A rhesus monkey(Macaca mulatta)model of CVB1 infection was established by intranasal instillation of CVB1 at a titer of 107.25CCID50/mL at a dose of 200 μL/monkey.The clinical symptoms of the animals were observed for 30 d after infection.Virus shedding was detected from 1 d to 30 d after infection.On the 1 d,3 d,5 d,7 d,9 d,11 d,13 d,15 d,21 d and 30 d,samples were collected for routine blood test,serum biochemical enzymes and immune cytokines detection,and tissue samples were examined for viral load,pathology and immunohistochemistry.Results:Part I:RNA standards were successfully constructed using the VP4 region of CVB1 as the target gene.The established real-time RT-PCR assay showed good specificity,sensitivity and repeatability,and could be used for rapid detection of CVB1 in animal models.Part Ⅱ:Two groups of Syrian golden hamsters infected with CVB1 virus by intraperitoneal injection or nasal instillation showed varying degrees of listlessness,decreased body temperature,and typical rash and herpes on the lips,similar to human HFMD clinical manifestations within 14 d.Viral nucleic acid could be detected in throat swabs,nasal lavage fluid and feces of both groups from 1 d to 13 d after infection,but no viral nucleic acid was detected in throat swabs,nasal lavage fluid and feces on day 14 after infection.Viral loads were detected in the heart,liver,lung,brain,olfactory bulb,submandibular gland,ovary and other tissues of the two groups of animals,and there were different degrees of pathological damage and positive virus antigen reaction by IHC.Liver function and myocardial enzymes in serum were elevated.Part Ⅲ:Rhesus monkeys infected with CVB1 developed herpes on the lips and extremities within 30 d,accompanied by diarrhea,depression,and hypothermia,which were similar to human HFMD.Viral nucleic acid could be detected in throat swabs,nasal swabs,feces and blood.The heart,spleen,pancreas,colon,olfactory bulb,pharyngeal flat,axillary lymph nodes and cervical lymph nodes could still detect viral nucleic acid and virus antigen reaction was positive by IHC on the 30st day.HE staining could observe pathological phenomena such as inflammation,hyperplasia and cell necrosis in the tissues.White blood cells,red blood cells and lymphocytes showed the characteristics of viral infection.Abnormal fluctuations of myocardial enzymes,serum enzymes of liver function and serum enzymes of kidney function were observed.The immune cytokines IL-1β,IL-4,IL-6,IL-8,IL-22,TNF-α and IFN-γ also showed varying degrees of fluctuation within 30 d after infection.Conclusions:First,the established real-time RT-PCR assay has good specificity,sensitivity and repeatability,and can be used for the detection of laboratory samples.Second,experiments have shown that both intraperitoneal injection and nasal drip can successfully infect Syrian golden hamsters to develop HFMD symptoms,but there are differences in the pathogenesis,viral infection process and related complications between the two methods of infection.According to the clinical manifestations,pathological characteristics,infection route and other aspects,the infection mode of nasal instillation can better mimic the natural respiratory tract infection.Third,3-month-old rhesus monkeys were successfully infected with CVB1 through nasal indwelling and presented with hand,foot and mouth disease and severe hand,foot and mouth disease with some organ tissue damage.The CVB1-infected rhesus monkey model can be used for human hand,foot and mouth disease and related studies.