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Mutations in the 5' NTR and the Non-Structural Protein 3A of the Coxsackievirus B3 Selectively Attenuate Myocarditogenicity.

Massilamany C, Gangaplara A, Basavalingappa RH, Rajasekaran RA, Vu H, Riethoven JJ, Steffen D, Pattnaik AK, Reddy J - PLoS ONE (2015)

Bottom Line: The 5' non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3.We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced.We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.

ABSTRACT
The 5' non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3. Previous studies have reported many nucleotide (nt) sequence differences in the Nancy strain of the virus, including changes in the 5' NTR with varying degrees of disease severity. In our studies of CVB3-induced myocarditis, we sought to generate an infectious clone of the virus for routine in vivo experimentation. By determining the viral nt sequence, we identified three new nt substitutions in the clone that differed from the parental virus strain: C97U in the 5' NTR; a silent mutation, A4327G, in non-structural protein 2C; and C5088U (resulting in P1449L amino acid change) in non-structural protein 3A of the virus leading us to evaluate the role of these changes in the virulence properties of the virus. We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced. We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone. The viral titers obtained from all the clones were comparable, but the virions derived from the third clone induced myocarditis comparable to that induced by wild type virus; however, the pancreatitis-inducing ability remained unaltered, suggesting that the mutations described above selectively influence myocarditogenicity. Because the accumulation of mutations during passages is a continuous process in RNA viruses, it is possible that CVB3 viruses containing such altered nts may evolve naturally, thus favoring their survival in the environment.

No MeSH data available.


Related in: MedlinePlus

Infectivity of viruses derived from various infectious cDNA clones.(A) Detection of infectivity by immunofluorescence technique. Vero cells grown in monolayers on coverslips were cultured further in the medium alone or infected with EMEM containing CVB3 Wt, pBRCVB3, pBRCVB3/97, pBRCVB3/4327_5088, or pBRCVB3/97_4327_5088 in 12-well plates. After 12 hours of incubation at 37°C, cells were fixed, incubated with anti-CVB3 serum followed by secondary FITC-conjugated IgG to detect the viral antigens. The coverslips were washed and mounted and examined by Laser Scanning Confocal Microscope. Top panel: phase-contrast images. Bottom panel: immunofluorescence images. Original magnification: 60x. (B) Comparison of viral titers. Monolayers of Vero cells were grown to 80% confluence in 12-well plates. Triplicate wells were infected with MOI of 0.1 or 3 of P2 viruses of CVB3 Wt, pBRCVB3, pBRCVB3/97, pBRCVB3/4327_5088, or pBRCVB3/97_4327_5088. After noting the CPE, the respective viral supernatants were harvested and the viral titers (TCID50) were determined by Spearman-Karber method. Mean ± SEM values are shown (n = 3).
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pone.0131052.g003: Infectivity of viruses derived from various infectious cDNA clones.(A) Detection of infectivity by immunofluorescence technique. Vero cells grown in monolayers on coverslips were cultured further in the medium alone or infected with EMEM containing CVB3 Wt, pBRCVB3, pBRCVB3/97, pBRCVB3/4327_5088, or pBRCVB3/97_4327_5088 in 12-well plates. After 12 hours of incubation at 37°C, cells were fixed, incubated with anti-CVB3 serum followed by secondary FITC-conjugated IgG to detect the viral antigens. The coverslips were washed and mounted and examined by Laser Scanning Confocal Microscope. Top panel: phase-contrast images. Bottom panel: immunofluorescence images. Original magnification: 60x. (B) Comparison of viral titers. Monolayers of Vero cells were grown to 80% confluence in 12-well plates. Triplicate wells were infected with MOI of 0.1 or 3 of P2 viruses of CVB3 Wt, pBRCVB3, pBRCVB3/97, pBRCVB3/4327_5088, or pBRCVB3/97_4327_5088. After noting the CPE, the respective viral supernatants were harvested and the viral titers (TCID50) were determined by Spearman-Karber method. Mean ± SEM values are shown (n = 3).

Mentions: To test this hypothesis, we derived three new infectious cDNA clones using pBRCVB3 as a backbone by reverting the mutations (S1 Fig). These include pBRCVB3/97 (U97C), pBRCVB3/4327_5088 (G4327A and U5088C), and pBRCVB3/97_4327_5088 (U97C, G4327A and U5088C). First, we recovered the infectious viruses from all the clones, and after passaging twice in Vero cells, ascertained the expression of viral proteins by immunofluorescence using Wt virus as a positive control, and media containing uninfected cells as negative controls. Fig 3A shows that Vero cells infected with the Wt virus and the clone-derived viruses exhibited viral protein expression, but not in medium control, suggesting that all viral variants retained the infectivity similar to the Wt virus. We next determined the viral titers at MOIs of 0.1 and 3, and the data revealed no significant differences between groups except that the titers obtained from pBRCVB3/97_4327_5088 virus tended to be lower than those obtained from the Wt virus on day 4, only at MOI of 3 (Fig 3B). Together, the data suggest that the nt variations present in the three new infectious clones do not adversely affect viral recovery and their corresponding viruses retain viral infectivity.


Mutations in the 5' NTR and the Non-Structural Protein 3A of the Coxsackievirus B3 Selectively Attenuate Myocarditogenicity.

Massilamany C, Gangaplara A, Basavalingappa RH, Rajasekaran RA, Vu H, Riethoven JJ, Steffen D, Pattnaik AK, Reddy J - PLoS ONE (2015)

Infectivity of viruses derived from various infectious cDNA clones.(A) Detection of infectivity by immunofluorescence technique. Vero cells grown in monolayers on coverslips were cultured further in the medium alone or infected with EMEM containing CVB3 Wt, pBRCVB3, pBRCVB3/97, pBRCVB3/4327_5088, or pBRCVB3/97_4327_5088 in 12-well plates. After 12 hours of incubation at 37°C, cells were fixed, incubated with anti-CVB3 serum followed by secondary FITC-conjugated IgG to detect the viral antigens. The coverslips were washed and mounted and examined by Laser Scanning Confocal Microscope. Top panel: phase-contrast images. Bottom panel: immunofluorescence images. Original magnification: 60x. (B) Comparison of viral titers. Monolayers of Vero cells were grown to 80% confluence in 12-well plates. Triplicate wells were infected with MOI of 0.1 or 3 of P2 viruses of CVB3 Wt, pBRCVB3, pBRCVB3/97, pBRCVB3/4327_5088, or pBRCVB3/97_4327_5088. After noting the CPE, the respective viral supernatants were harvested and the viral titers (TCID50) were determined by Spearman-Karber method. Mean ± SEM values are shown (n = 3).
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pone.0131052.g003: Infectivity of viruses derived from various infectious cDNA clones.(A) Detection of infectivity by immunofluorescence technique. Vero cells grown in monolayers on coverslips were cultured further in the medium alone or infected with EMEM containing CVB3 Wt, pBRCVB3, pBRCVB3/97, pBRCVB3/4327_5088, or pBRCVB3/97_4327_5088 in 12-well plates. After 12 hours of incubation at 37°C, cells were fixed, incubated with anti-CVB3 serum followed by secondary FITC-conjugated IgG to detect the viral antigens. The coverslips were washed and mounted and examined by Laser Scanning Confocal Microscope. Top panel: phase-contrast images. Bottom panel: immunofluorescence images. Original magnification: 60x. (B) Comparison of viral titers. Monolayers of Vero cells were grown to 80% confluence in 12-well plates. Triplicate wells were infected with MOI of 0.1 or 3 of P2 viruses of CVB3 Wt, pBRCVB3, pBRCVB3/97, pBRCVB3/4327_5088, or pBRCVB3/97_4327_5088. After noting the CPE, the respective viral supernatants were harvested and the viral titers (TCID50) were determined by Spearman-Karber method. Mean ± SEM values are shown (n = 3).
Mentions: To test this hypothesis, we derived three new infectious cDNA clones using pBRCVB3 as a backbone by reverting the mutations (S1 Fig). These include pBRCVB3/97 (U97C), pBRCVB3/4327_5088 (G4327A and U5088C), and pBRCVB3/97_4327_5088 (U97C, G4327A and U5088C). First, we recovered the infectious viruses from all the clones, and after passaging twice in Vero cells, ascertained the expression of viral proteins by immunofluorescence using Wt virus as a positive control, and media containing uninfected cells as negative controls. Fig 3A shows that Vero cells infected with the Wt virus and the clone-derived viruses exhibited viral protein expression, but not in medium control, suggesting that all viral variants retained the infectivity similar to the Wt virus. We next determined the viral titers at MOIs of 0.1 and 3, and the data revealed no significant differences between groups except that the titers obtained from pBRCVB3/97_4327_5088 virus tended to be lower than those obtained from the Wt virus on day 4, only at MOI of 3 (Fig 3B). Together, the data suggest that the nt variations present in the three new infectious clones do not adversely affect viral recovery and their corresponding viruses retain viral infectivity.

Bottom Line: The 5' non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3.We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced.We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone.

View Article: PubMed Central - PubMed

Affiliation: School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, Nebraska, United States of America.

ABSTRACT
The 5' non-translated region (NTR) is an important molecular determinant that controls replication and virulence of coxsackievirus B (CVB)3. Previous studies have reported many nucleotide (nt) sequence differences in the Nancy strain of the virus, including changes in the 5' NTR with varying degrees of disease severity. In our studies of CVB3-induced myocarditis, we sought to generate an infectious clone of the virus for routine in vivo experimentation. By determining the viral nt sequence, we identified three new nt substitutions in the clone that differed from the parental virus strain: C97U in the 5' NTR; a silent mutation, A4327G, in non-structural protein 2C; and C5088U (resulting in P1449L amino acid change) in non-structural protein 3A of the virus leading us to evaluate the role of these changes in the virulence properties of the virus. We noted that the disease-inducing ability of the infectious clone-derived virus in three mouse strains was restricted to pancreatitis alone, and the incidence and severity of myocarditis were significantly reduced. We then reversed the mutations by creating three new clones, representing 1) U97C; 2) G4327A and U5088C; and 3) their combination together in the third clone. The viral titers obtained from all the clones were comparable, but the virions derived from the third clone induced myocarditis comparable to that induced by wild type virus; however, the pancreatitis-inducing ability remained unaltered, suggesting that the mutations described above selectively influence myocarditogenicity. Because the accumulation of mutations during passages is a continuous process in RNA viruses, it is possible that CVB3 viruses containing such altered nts may evolve naturally, thus favoring their survival in the environment.

No MeSH data available.


Related in: MedlinePlus