In this study, in order to uncover the relationship between the genome and virulence of CVB3/MKP strain and the phylogenetic relationship between CVB3/MKP strain and other CVB3 strains, we first confirmed that CVB3/MKP strain caused myocarditis and performed a whole genome sequencing of the CVB3/MKP strain and compared its whole genome sequence and the 5′ non-coding region with other strains. We also predicted the RNA secondary structure of the specific function related region and compared the amino acid sequence variation in structural protein region and non-structural protein region.
We confirmed in our animal experiments that CVB3/MKP had cardiactoxicity and the myocardial tissue of mice went through a serious pathological change. This may be ascribed to the bulk replication of CVB3/MKP resulted in necrosis and rupture of myocardium and that CVB antigen triggered auoto-immunresponse via high affinity receptor binding sites of CVB3 antigen [14, 15].
The analysis of the whole genome sequence of CVB3/MKP strain revealed that CVB3/MKP shared 99.7% and 99.6% sequence identity with CVB3/28 and CVB3/0 in nucleotide sequence, respectively, and that all the three strains were within the same cluster with a close relationship in evolution. Therefore, the pathogenic strains CVB3/MKP and CVB3/28 may derive from non-pathogenic strain CVB3/0 via mutations under certain conditions. The region bearing small differences among the three strains may have an important role in the formation of viral epitopes, infection and replication of virus. Although the three CVB3 strains shared a high degree of sequence identity, they had different virulence. Therefore, it is possible that small nucleotide variations among the genome of the three CVB3 strains may enable them to have a different tissue tropism or virulence.
Sequence analysis of the 5′untranslated region of CVB3/MKP strain revealed that CVB3/MKP, CVB3/20, CVB3/0, CVB3/28 and CVB3/Nancy were significantly correlated in genetic distance. CVB3/MKP had a longer genetic distance from CVB3/CO and CVB3/GA, which is in accordance with the analysis of the whole genome sequence, further indicating that the pathogenic CVB3/MKP and CVB3/28 strains may derive from the non-pathogenic strain CVB3/0 via mutations under certain conditions. Therefore, the results further proved that the 5′untranslated region of CVB3 may be important for viral tropism and virulence. In addition, sequence identity comparison between CVB3/MKP, CVB3/GA,CVB3/20 and CVB3/28 in amino acid sequences revealed that the amino acid sequences in the coding region of the 4 strains were basically the same, but CVB3/MKP had seven unique mutations, of which, two occurred in structural protein region, five occurred in the non-structural protein region. In the structural protein coding region, the neutralizing antibody sequences in the VP2 and VP3 region of CVB3/MKP strain were substantially similar to other pathogenic CVB3strains, but the 235 amino acid in the VP2 “puff” area of CVB3/MKP strain was Glutamate (E), and the corresponding amino acid in CVB3/GA, CVB3/20 and CVB3/28 was lysine (K); the 512 amino acid in the VP3 “knob” area of CVB3/MKP strain was Valine (V), while the corresponding amino acid of CVB3/GA, CVB3/20 and CVB3/28 was Alanine (A). We also found that non-pathogenic strains CVB3/GA had 58 different amino acids from CVB3/MKP, CVB3/20 and CVB3/28. Although it is not clear whether these variations have any impact on the function of these strains, it is likely that the changes in the nucleic acid or amino acid can influence the ability of CVB3/MKP strain to produce persistent infection. It is necessary to further investigate what role(s) these mutations play in the autoimmune response during the pathogenesis of myocarditis.
In summary, we demonstrated that CVB3/MKP strain had cardiotoxicity and a very close genetic relationship with CVB3/28 strain, which can induce pancreatitis and myocarditis [8, 9], and further proved that the 5′untranslated region of CVB3 may be relevant to its cell tropism and virulence phenotypes. Our findings identified the variations in nucleotide and amino acid sequences of the CVB3/MKP strain and a phylogenetic relationship between CVB3/MKP strain and other CVB3 strains, providing a theoretical basis for the pathogenesis and prevention of CVB3-induced myocarditis.