Complete Nucleotide Sequence Of A Coxsackievirus B4 Strain That Establishes Infection In ICR Mice Pancreas

Coxsackievirus serotype B (CVB1-6) belongs to the species Human enterovirus-B (HEV-B), a member of the family Picornaviridae. The RNA genome of Coxsackievirus B consists of 7400 nucleotides and is composed of a 5′-untranslated region (UTR) and a 3′-UTR, and an open reading frame (ORF), encoding a polyprotein precurosor that is proteolytically cleaved to 11end products. The translation of CVB RNAs generally proceeds by a cap-independent mechanism, in which ribosomes bind directly to an internal ribosome entry site(IRES) in the 5′-UTR of the RNA. It might play an important role that viruses bind cellular receptor in the infectious cycle. CBVs can make use of at least two cell membrane proteins during their entry: coxsackievirus-adenovirus receptor (CAR) and decay-accelerating factor of the complement system (DAF). Interactions between a virus and its specific receptor are thought to be crucial factors in tissue tropism.Infections with CVB usually lead to diseases including aseptic meningitis, encephalitis, myocarditis, pancreatitis, myositis and so on. Furthermore, in several studies, coxsackieviruses B, have been associated with type 1 diabetes. There is accumulating evidence that enteroviruses, especially Coxsackie B viruses, are of etiological significance in this context. Higher frequencies of neutralizing antibodies against Coxsackievirus serotype B have been seen in type 1 diabetes patients compared with healthy controls. Coxsackievirus B-derived RNA, and IgM antibodies against other enteroviruses were found in higher frequencies in newly diagnosed patients than in controls. CVBs can replicate in humanβ-cells in vitro and affect the ability to respond to high glucose by insulin release during the infection. More direct evidence has been obtained from isolation of Coxsackievirus B(strains E2 and Tuscany), capable of causing diabetes in mice, from the pancreas of a few patients with acute-onset type 1 diabetes. In several studys, infections with strains of coxsackievirus B4 resulted in a diabetes-like syndrome in mice. These indicate that CVB4 infection has a strongly association with T1D. However, the mechanisms of this phenomenon have not been determined. It has been suggested that there might be a direct or an indirect virus cell interaction withβ-cells causing its damages.The broad spectrum of diseases associated with Coxsackievirus serotype B reflects the existence of multiple strains with various degrees of virulence, within a single serotype.Many researchers pay close attention to map genetic determinants responsible for the different tissue tropism of CVBs. It has been identified the 5′UTR as the primary region that determines a cardiovirulent phenotype,workwhich led to finer mapping within the 5′UTR, which identified a region, contained within nt88–181, as a key determinant of cardiovirulence. Similar results have been obtained using mouse-adapted strains of CVB4 to map the determinants of pancreovirulence(pancreatitis): in this model, two amino acid changes in the capsid proteins 1D and 1A play primary roles. Not all variants of Coxsackievirus B4 cause overt diabetes in human and susceptible animals. There are 111 amino acids that were different between the diabetogenic E2 and nondiabetogenic JVB strains of Coxsackievirus B4. Coxsackie B4 JVB strain was passaged in murine pancreatic beta cells to obtain a beta cell tropic variant of the Coxsackie B4 JVB virus. There were only seven amino acids that were different between the sequence of the entire genome of this variant and the nucleotide sequence of the prototype strain, and these site might play an important role in diabetogenicity of CVB4. Howere, the identification of critical sites (nucleotides or amino acids) responsible for the diabetogenicity of Coxsackie B4 virus remains to be determined now.There are few study about the relationship of the virulence phenotype of pancreas and the virulence genes, and most of them usuall tend to use the prototype strain or the recombinant chimeric strain, however, it is well known that there are strains of genetic diversification of coxsackieviruses B in different geographical district, therefore, it is useful that studying the relationship between the pathogenic phenotype of geographical restrictions of endemic strains and their virulence genetype.In this paper, ICR mice were inoculated intraperitoneally (i.p.) with a epidemic strain of coxsackievirus B4 (CVB4jlu06) in china. Mice were sacrificed in different days post-infection (p.i.), and heart, pancreas, liver, spleen, lung, kidney and blood were collected. Half of the different tissue was snap-frozen for virus titration; the remaining half was fixed for histopathological examination and immunohistochemistry. The results of the animal experiment indicated that the jlu06 strain had a particular tropism for pancreatic tissues. We observed that when the susceptible mice were infected intraperitoneally with the jlu06 virus, viremia occurred rapidly, spreading the virus to secondary replication sites of infection such as the pancreas. However, the jlu06 strain was found to be more virulent in pancreatic tissues, giving yields greater than in the other tissues tested. Moreover, the jlu06 strain virus replication continued for a longer period, with ongoing replication on day 27 in the pancreas, whereas no infectious virus was detected after day 8 in the other tissues. Immunohistochemistry to detect viral antigens revealed that viral proteins were present in the majority of pancreas tissue of infected mice and a few positive-stain regions were observed in the other tissues. Histological examination of tissues showed that loss of pancreatic acinar tissue, most of exocrine tissue and a few islets damage. The CVB4jlu06 replicated transiently in other tissue and no severe damages were observed in infected mice. The indication of the jlu06 strain in its ability to infectβ-cells derived from glucose measurements. By later stage of viral infection the serum glucose concentrations in mice infected with the jlu06 strain were significantly greater compared with the uninfected controls. Although the mechanisms ofβ-cells damages are unclear, this indicates that there might be a direct or an indirect virus cell interaction withβ-cells causing glucose intolerance. All of above, the jlu06 strain virus replication continued for a longer period in the pancreas, and induce damages in ICR mice pancreas together with dysregulation in glucose metabolism, which indicate the CVB4jlu06 might be the diabetogenicity.To determine the complete nucleotide sequence of the jlu06 strain, virus RNA was purified and amplified by RT-PCR. The amplification products were sequenced and assembled into a complete genome sequence. The sequence analysis showed that the genome of the jlu06 strain is 7,395 nucleotides (nt) long, excluding the poly(A)-tract. The 5′-UTR extends to nt 743 and was followed by an open reading frame, ranging from nucleotide 744-7292, coding for a single polyprotein of 2,183 amino acids. The polyprotein coding sequence was flanked by a 103 nt-long 3′-UTR and a poly (A) tract. The coding region was divided into 1A, 1B, 1C, 1D, 2A, 2B, 2C, 3A, 3B, 3C and 3D functional regions.Phylogenetic trees of amino acid/nt sequences from the full-length jlu06 genome together with published sequences of other members of human enterovirus type B were constructed for different genomic regions. A phylogenetic tree of the complete polyprotein showed that all strains of CVB4 are closely related to each other, and CVB4jlu06 strain is closely correlated with the strain of CVB4JVB and CVB4 Tuscany. There are 33nts and 23aas difference between CVB4jlu06 strain and CVB4JVB strain by MegAlign analysis tool, which might be responsible for the different pathogenicity between the two strains.We observed that 3nts in the 5′UTR of viral genome were the same nts between jlu06 and Tuscany, which differed from JVB strain by multiple sequence alignment between CVB4. The variation nts and mutation sites involved A136T, G137A and C546G., which were located in RNA stem-loop D (SLD) and RNA stem-loop G(SLG) respectively. SLD is the homology ligand of viral protease 3C and SLD is the major component of an internal ribosome entry site (IRES).These mutations might affect the ability of replication and refer to different strains of CVB4 exhibit different virulence phenotypes. We observed that a total of 15 out of 7,395 nts in the viral genome were changed, resulting in 10 amino acid substitutions by multiple sequence alignment between CVB4. Two of the changed amino acids were located in the capsid proteins while eight were localized to the nonstructural proteins. The two mutated amino acids in the capsid proteins were close to the outer surface of the virion, suggesting that they are involved in virus–cell interactions, possibly augmenting initiation of virus infection. On the other hand, coding mutations in the nonstructural genes might also affect virulence, involving protease 2A, proteins 2C and 3A, protease 3C and the viral polymerase 3D. Little is known about the functional significance of these changes.CVB4jlu06 and CVB4 Tuscany(isolated from the patient with T1D) have the same amino acid Ile, while the other CVB4 is Val in the position 1485 of 3A protein. Moreover, jlu06 Tuscany, E2 (isolated from the patient with T1D) and E2b (E2 variant) owned the same amino acids in 4 sites in 3A protein, which are different from the nondiabetogenic strain JVB. Further work is required to determine what role these mutations are playing in determining their virulence phenotype.Our results showed that the epidemic strain CVB4jlu06 in china express diabetogenic virulence (or pathogenic) phenotypes when studied in animal models, demonstrating that key elements of pathogenesis reside in the viral genome. Here, we have provided some variant data to genetic data base and some experimental data for further study on the characteristic of enterovirus genome and the virulence variation.