| Coxsackieviruses are important human pathogens associated with a variety of diseases, which range from mild respiratory infections to severe life-threatening diseases, including aseptic meningitis, myocarditis and pancreatitis. Coxsackieviruses belong to the genus Enterovirus within the family Picornaviridae. There are 30 serotypes of coxsackieviruses, which can be classified into A groups and B groups according to pathogenic features to neonate rat and cell sensitivity. Coxsackievirus A includes A1-A24, and coxsackievirus B includes B1-B6.Coxsackievirus B3 (CVB3), one of six CVB serotypes, is a member of the genus Enterovirus within the family Picornaviridae. Since the cDNA sequence in 3′end of CVB3 was reported in 1984 by Stalhandske, the complete genome sequence of foreign strains were acquired. The genome of CVB3, like that of other enteroviruses, is a positive single-stranded RNA whose full-length is 7,400 base pairs (bp). This RNA consists of 5′-non-coding region, P1, P2, P3 regions and 3′-non-coding region.A group of reports have suggested that CVB3/20, CVB3/28, CVB3/AS and CVB3/Nancy could lead to both pancreatitis and myocarditis, CVB3/CO could only lead to pancreatitis, while CVB3/GA and CVB3/0 were non-pathogenic. Nucleotide variation in both coding-region and non-coding region may have some effect on virus toxicity, which is rarely reported in China. To clarify the relationships between different strains and virus hereditary property, we began a research to study the genomic features of China CVB3, which will help to understand the correlations between virus variation and toxicity.In the present study, I have investigated the relationship between CVB3/MKP viral stain and pancreatitis, myocarditis and type1 diabetes in mice, in order to acknowledge whether virus has a risk of developing type1 diabetes in animal. The study was performed in characterization and genotype of CVB3/MKP, all the cDNA fragments of the CVB3/MKP were amplified by RT-PCR and inserted to TA-vector. After multiple sequencing of the 7 fragments, the complete genome was obtained by assembling these sequences. The molecular characterization of CVB3/MKP was then analyzed by bioinformatics.5′NTR sequences analysis of the CVB3/MKP genes showed that the alignment of CVB3/MKP and nine CVB3 strains (CVB3/20, CVB3/28, CVB3/0, CVB3/Woodruff(CVB3/W), CVB3/AS, CVB3/Nancy, CVB3, CVB3/GA and CVB3-CO) 5′NTR sequences revealed significant nucleotide diversity among the 10 CVB3 strains. CVB3/MKP was 91.9% to 99.6% identical to CVB3/20, CVB3/28, CVB3/0, CVB3/W, CVB3-AS1, CVB3-Nancy, CVB3 stains respectively, among which 99.6%, 99.5% and 99.5% identities were present between CVB3/MKP and CVB3/20, CVB3/Nancy and CVB3/28 which can induce both pancreatitis and myocarditis. CVB3/MKP was only 83.3% and 83.6% identical to CVB3/GA and CVB3/CO. CVB3/CO is only pathogenic in pancreas and CVB3/GA is nonvirulent strain. The phylogenetic tree constructed from the sequence alignment in the 5′NTR region enabled us to establish relationships between the CVB3 strains. CVB3/MKP is very closely related to CVB3/20 and CVB3/0, CVB3/28, CVB3/Nancy strains as they form one branch of the tree.To assess the potential role that RNA secondary structure might play in determining CVB3 pancrevirulent phenotype, nt 88 181 of CVB3/MKP, CVB3/AS, CVB3/20 and CVB3/28, and nt 88 186 of CVB3/CO, which include the putative SLI II linker sequences and SLII, were analyzed and compared with avirulent strain CVB3/GA by use of the M-FOLD algorithm. Computational folding predicted a possible optimum structure for SLII region. Although CVB3/MKP has one nucleotide difference in SLII region, the predicted RNA secondary structures was nearly identical with the pancreovirulent strains CVB3/20 and CVB3/28, and similar to the pancreovirulent strain CVB3/AS. In contrast, the SLII of the pancreovirulent CVB3/CO strain which is noncardiovirulent phenotype, and the avirulent strain CVB3/GA differed significantly in its predicted structure. It is implied that the similar RNA secondary structure among CVB3/MKP, CVB3/28, CVB3/20 and CVB3/AS might play a role in determining the virulence of virus for pancreas and myocardity.Comparison of full-length CVB3/MKP nucleotide sequences show that The full-length of CVB3/MKP sequence and the CVB3/0, CVB3/28, CVB3/20, CVB3/PD, CVB3/CG, CVB3, CVB3/Nancy, CVB3/P strains from the GenBank were aligned using ClustalW(1.83). The CVB3/MKP sequence revealed relative no variation between CVB3/MKP and published CVB3 genomic sequences, also in the noncardiovirulent strain CVB3/0. However, when whole genome sequence of CVB3/MKP was clustered with other CVB3 genome sequences. The whole genomes tree demonstrated that CVB3/MKP is closely related to the pancreaovirulent strain CVB3/28 and the non-cardiovirulent strain CVB3/0 although there is less than 1% divergence between the compared CVB3 strains. The phylogenetic analysis of whole genome of CVB3/MKP showed a similar organisation in comparison to the 5′NTR tree.We then compare the amino acid sequences between CVB3/MKP and CVB3/20, CVB3/28 and CVB3/GA. The results showed that there were only 7 specific variations present in protein coding region, among which 2 sites were located in structural protein and the other 5 in non-structural protein. It was also found that there were 58 variations of amino acids between CVB3/GA and the other 3 strains. Although it wasn't known whether these variations would have any effect on the virus function, the changes of nucleotides or amino acids could contribute for the ability of CVB3/MKP in the persistant infection. Further studies should be done on how the mutations act on self-immune system during the process of pancretitis and myocarditis.In order to study the virulent effect to the pancreatic tissue and myocard tissue, CVB3/MKP was injected into mice abdominal cavity. All the experimental and control mice were sacrificed after 3 days, 7days, 21 days, 40days, 55daysafter viral infection. The weight and the blood glucose level were tested, and the pancreas and the heart were taken for histological test. The results show that the mean weights of experimental groups are close to those of normal control groups in 3days and 7days, while significantly lower in experimental groups in 21days, 40 days and 50days. Urine glucose of all the experimental and control mice were negative, while the blood glucose level of experimental groups was persistently higher than normal group. It was proposed that the deficiency of insulin results from the damage of islet after CVB3/MKP infection, which cause higher blood glucose. Persistent higher blood glucose would make higher catabolism and lower anabolic metabolism, leading to lower weight of experimental mice.The pathologic change of pancreas of rat is that amount ofβcell of islet reduces, and inflammatorycells infiltration mainly includes lymphocytes and monocytes. Vacuolation of pancreaticβcell makes lack of pancreatic islet. Pancreatic islet is broken and the structure is incomplete. The pathologic change of myocardium of rat is that the focus necrosis in myocardium and infiltration of lymphocyte and mononuclear were observed in the myocardium interstitium,and proliferation of fibrous tissue was also observed in some areas in myocard tissue. The Immunohistochemistry tests of pancreas and heart showed that the viral antigen level of experimental groups are persistently presence after 55days the viral infection. The result suggested that CVB3/MKP may persist in myocardium and pancreas, and destroy normal structure and function of both pancreas and heart.To sum up, the complete sequence of CVB3/MKP genome was acquired in this study. Through study of sequence comparison, phylogenetic tree analysis and RNA secondary prediction, CVB3/MKP was found to be closed related to CVB3/28 that is virulent to both pancreas and myocardium. Then the pancreaovirulence and myocardiovirulence of CVB3/MKP were further demonstrated in mice. The results of this study will supply primary data for the study of virus evolution and genus development, and also provide basis for the study of diseases related to CVB3/MKP. |
