The whole genome sequence of coxsackievirus B3 MKP strain leading to myocarditis and its molecular phylogenetic analysis

Abstract

Background: In recent years, the reported infection cases by coxsackievirus (CV) have been on the rise. In order to reveal the relationship between the nucleotide and amino acid sequences and the viral virulence of the CVB3/MKP strain causing myocarditis, we initially confirmed the virulence of the strain in myocardial tissue and then carried out the whole genome sequencing of CVB3/MKP strain and performed a phylogenetic analysis among different CVB3 strains.

Methods: CVB3/MKP infected mouse model was established to check lesions of myocardial tissue in mice using immunohistochemical detection at different periods. RT-PCR analysis was used to amplify seven fragments covering the whole viral sequence and comparable analysis was performed.

Results: The immunohistochemical results showed that particles of CVB3/MKP virus persisted in the cardiac tissue and caused severe pathology. The length of whole genome sequence of CVB3/MKP strain was 7400 bp. CVB3/MKP had 99.7% and 99.6% homology in nucleotide sequence with CVB3/28 and non-virulent CVB3/0, respectively. The former can induce pancreatitis and myocarditis. The nucleotide sequence in the 5'untranslated region of CVB3/MKP strain shared 99.6% and 99.5% homology with CVB3/20 and CVB3/Nancy, respectively.

Conclusion: We confirmed in our animal experiments that CVB3/MKP had cardiotoxicity. CVB3/MKP, CVB3/28, and CVB3/0 may share evolutionary convergence and the 5'untranslated region (5'UTR) may be associated with virulence phenotype. Our findings will provide a basis for identifying the genomic determinant of viral virulence of CVB3/MKP strain and phylogenetic relationship among different CVB3 strains.

Figure 1

Cardiac muscle pathological detection of mice infected with CVB3/MKP. (A) Cardiac muscle of mice infected with CVB3/MKP at different days (H&E staining). Juvenile male Swiss mice were inoculated with virus, and at 3, 7, 21, 40, 55 days post infection, the hearts were removed, sectioned, and stained. The figure shows murine myocardium of negative control (a), and (b) to (f) represent typical histological changes in the heart after infection with CVB3/MKP on different days. (B) Immunohistochemistry staining of cardic muscle of mice infected with CVB3/MKP on different days. Anti-CVB3 monoclonal antibody was used. A large number of brown particles existed in the cytoplasm of groups (b) to (f).

Figure 2

Phylogenetic tree showing relationship between the complete genomes of CVB3/MKP strain and the other 8 CVB3 strains. CVB3/MKP is closely related to non-cardiovirulent strain CVB3/0 and cardiovirulent strain CVB3/28.

Figure 3

Phylogenetic relationship among 5′UTR sequences of 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.

Figure 4

Sequence alignment of stem loop II (SLII) in the 5′non-translation regions of CVB3/20, CVB3/MKP, CVB3/AS, CVB3/GA strains and nt 88–181 of CVB3/28, and nt 88–186 of CVB3/CO.

Figure 5

Predicted RNA secondary structures of stem loop (SL II) region of coxsackievirus B3 (CVB3). Stem loop (SL II) regions of CVB3/20,CVB3/MKP,CVB3/GA and CVB3/AS strains, nt 88–181 of CVB3/28, and nt 88–186 of CVB3/CO regions were computationally folded.