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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

Myocarditis severity is attenuated in mice infected with the infectious clone-derived virus, pBRCVB3.Histology. A/J mice were infected with Wt virus or the virus derived from pBRCVB3 i.p., and hearts and pancreata were harvested at termination on day 21 postinfection or as the animals died or euthanized. The tissues were examined by H and E staining. Representative sections from three individual animals infected with Wt virus or pBRCVB3 virus are shown (n = 15 to 17 mice per group), whereas the sections obtained from uninfected animals were used as controls. (A) Hearts.Top panel (control group): heart section from uninfected animal without inflammation, mineralization or necrosis; middle panels (Wt virus-infected): extensive infiltrates of macrophages and lymphocytes into the myocardium filling areas of myocardial cell loss/necrosis (Mouse 1), extensive large foci of necrosis and mineralization surrounded by inflammation consisting of macrophages and lymphocytes (Mouse 2), multiple small foci of necrosis and mineralization infiltrated by a few inflammatory cells (Mouse 3); bottom panels (pBRCVB3 virus-infected): small focal inflammatory infiltrates into myocardium (Mouse 1), a small foci of necrosis in the myocardium (Mouse 2), small foci of lymphocytic inflammation (Mouse 3). (B) Pancreata.Top panel (control group): pancreas from an uninfected animal showing intact islets; middle panels (Wt virus-infected): diffuse atrophy with scattered lymphocyte infiltrates (Mouse 1), acute necrosis, mineralization and inflammation (Mouse 2), diffuse marked atrophy with mild diffuse inflammation (Mouse 3); bottom panels (pBRCVB3 virus-infected): diffuse atrophy and lymphocytic infiltrates (Mouse 1), diffuse necrosis with pyknotic nuclei (Mouse 2), diffuse atrophy with mild inflammation (Mouse 3). Arrows indicate the lesions (N, necrosis; M, mineralization; and I, inflammation). Original magnification: 10x (hearts) and 40x (pancreata). (C) Viral titers. Hearts and pancreata obtained from the above groups on days 5, 7 and 10 postinfection were processed for determining the viral titers as described in the ‘methods’ section and the titers were compared between groups. Left panel, hearts; right panel, pancreata.
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pone.0131052.g002: Myocarditis severity is attenuated in mice infected with the infectious clone-derived virus, pBRCVB3.Histology. A/J mice were infected with Wt virus or the virus derived from pBRCVB3 i.p., and hearts and pancreata were harvested at termination on day 21 postinfection or as the animals died or euthanized. The tissues were examined by H and E staining. Representative sections from three individual animals infected with Wt virus or pBRCVB3 virus are shown (n = 15 to 17 mice per group), whereas the sections obtained from uninfected animals were used as controls. (A) Hearts.Top panel (control group): heart section from uninfected animal without inflammation, mineralization or necrosis; middle panels (Wt virus-infected): extensive infiltrates of macrophages and lymphocytes into the myocardium filling areas of myocardial cell loss/necrosis (Mouse 1), extensive large foci of necrosis and mineralization surrounded by inflammation consisting of macrophages and lymphocytes (Mouse 2), multiple small foci of necrosis and mineralization infiltrated by a few inflammatory cells (Mouse 3); bottom panels (pBRCVB3 virus-infected): small focal inflammatory infiltrates into myocardium (Mouse 1), a small foci of necrosis in the myocardium (Mouse 2), small foci of lymphocytic inflammation (Mouse 3). (B) Pancreata.Top panel (control group): pancreas from an uninfected animal showing intact islets; middle panels (Wt virus-infected): diffuse atrophy with scattered lymphocyte infiltrates (Mouse 1), acute necrosis, mineralization and inflammation (Mouse 2), diffuse marked atrophy with mild diffuse inflammation (Mouse 3); bottom panels (pBRCVB3 virus-infected): diffuse atrophy and lymphocytic infiltrates (Mouse 1), diffuse necrosis with pyknotic nuclei (Mouse 2), diffuse atrophy with mild inflammation (Mouse 3). Arrows indicate the lesions (N, necrosis; M, mineralization; and I, inflammation). Original magnification: 10x (hearts) and 40x (pancreata). (C) Viral titers. Hearts and pancreata obtained from the above groups on days 5, 7 and 10 postinfection were processed for determining the viral titers as described in the ‘methods’ section and the titers were compared between groups. Left panel, hearts; right panel, pancreata.

Mentions: To determine the virulence of pBRCVB3 virus, we first recovered the virus from the infectious clone following transfection of Vero cells with in vitro-transcribed RNA. After passaging the virus twice in cultures (similar to Wt virus), we sought to determine its pathogenicity in vivo. Groups of A/J mice were infected with 2000 TCID50/animal, and on days 5, 7, 10 or 21 postinfection, animals were euthanized; hearts and pancreata were evaluated for inflammatory changes, and the lesions were compared with uninfected animals. Unexpectedly, in animals infected with the infectious clone-derived virus, myocarditis was significantly attenuated (Table 3). As shown in Fig 2A, myocarditis in mice infected with the Wt virus showed widespread lesions containing necrosis, mineralization, and lymphocytic infiltrates as opposed to occasional lesions in pBRCVB3 virus-infected animals. Pancreatitis, however was consistently present in both groups of animals with comparable severity as indicated by atrophic changes, necrosis and inflammatory infiltrates (Fig 2B; Table 4). The finding that pBRCVB3 virus-induced pancreatitis is comparable to Wt virus suggests that the virus did reach the target tissues in infected animals. But, why then attenuated myocarditogenicity? One possible reason could be that the tropism of pBRCVB3 virus containing the nt changes (C97U, A4327G, and C5088U) might have been altered for the heart. To address this possibility, we determined the viral titers from hearts and pancreata obtained from animals infected with Wt virus or pBRCVB3 virus on days 5, 7 and 10 postinfection. The virus was found to be present in hearts from animals infected with pBRCVB3 virus similar to Wt virus-infected group as the viral titers were comparable at all the time-points postinfection (Fig 2C, left panel). Likewise, viral titers from pancreata from both the groups were also comparable except that pBRCVB3 virus could not be recovered on day 10 postinfection (Fig 2C, right panel).


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)

Myocarditis severity is attenuated in mice infected with the infectious clone-derived virus, pBRCVB3.Histology. A/J mice were infected with Wt virus or the virus derived from pBRCVB3 i.p., and hearts and pancreata were harvested at termination on day 21 postinfection or as the animals died or euthanized. The tissues were examined by H and E staining. Representative sections from three individual animals infected with Wt virus or pBRCVB3 virus are shown (n = 15 to 17 mice per group), whereas the sections obtained from uninfected animals were used as controls. (A) Hearts.Top panel (control group): heart section from uninfected animal without inflammation, mineralization or necrosis; middle panels (Wt virus-infected): extensive infiltrates of macrophages and lymphocytes into the myocardium filling areas of myocardial cell loss/necrosis (Mouse 1), extensive large foci of necrosis and mineralization surrounded by inflammation consisting of macrophages and lymphocytes (Mouse 2), multiple small foci of necrosis and mineralization infiltrated by a few inflammatory cells (Mouse 3); bottom panels (pBRCVB3 virus-infected): small focal inflammatory infiltrates into myocardium (Mouse 1), a small foci of necrosis in the myocardium (Mouse 2), small foci of lymphocytic inflammation (Mouse 3). (B) Pancreata.Top panel (control group): pancreas from an uninfected animal showing intact islets; middle panels (Wt virus-infected): diffuse atrophy with scattered lymphocyte infiltrates (Mouse 1), acute necrosis, mineralization and inflammation (Mouse 2), diffuse marked atrophy with mild diffuse inflammation (Mouse 3); bottom panels (pBRCVB3 virus-infected): diffuse atrophy and lymphocytic infiltrates (Mouse 1), diffuse necrosis with pyknotic nuclei (Mouse 2), diffuse atrophy with mild inflammation (Mouse 3). Arrows indicate the lesions (N, necrosis; M, mineralization; and I, inflammation). Original magnification: 10x (hearts) and 40x (pancreata). (C) Viral titers. Hearts and pancreata obtained from the above groups on days 5, 7 and 10 postinfection were processed for determining the viral titers as described in the ‘methods’ section and the titers were compared between groups. Left panel, hearts; right panel, pancreata.
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pone.0131052.g002: Myocarditis severity is attenuated in mice infected with the infectious clone-derived virus, pBRCVB3.Histology. A/J mice were infected with Wt virus or the virus derived from pBRCVB3 i.p., and hearts and pancreata were harvested at termination on day 21 postinfection or as the animals died or euthanized. The tissues were examined by H and E staining. Representative sections from three individual animals infected with Wt virus or pBRCVB3 virus are shown (n = 15 to 17 mice per group), whereas the sections obtained from uninfected animals were used as controls. (A) Hearts.Top panel (control group): heart section from uninfected animal without inflammation, mineralization or necrosis; middle panels (Wt virus-infected): extensive infiltrates of macrophages and lymphocytes into the myocardium filling areas of myocardial cell loss/necrosis (Mouse 1), extensive large foci of necrosis and mineralization surrounded by inflammation consisting of macrophages and lymphocytes (Mouse 2), multiple small foci of necrosis and mineralization infiltrated by a few inflammatory cells (Mouse 3); bottom panels (pBRCVB3 virus-infected): small focal inflammatory infiltrates into myocardium (Mouse 1), a small foci of necrosis in the myocardium (Mouse 2), small foci of lymphocytic inflammation (Mouse 3). (B) Pancreata.Top panel (control group): pancreas from an uninfected animal showing intact islets; middle panels (Wt virus-infected): diffuse atrophy with scattered lymphocyte infiltrates (Mouse 1), acute necrosis, mineralization and inflammation (Mouse 2), diffuse marked atrophy with mild diffuse inflammation (Mouse 3); bottom panels (pBRCVB3 virus-infected): diffuse atrophy and lymphocytic infiltrates (Mouse 1), diffuse necrosis with pyknotic nuclei (Mouse 2), diffuse atrophy with mild inflammation (Mouse 3). Arrows indicate the lesions (N, necrosis; M, mineralization; and I, inflammation). Original magnification: 10x (hearts) and 40x (pancreata). (C) Viral titers. Hearts and pancreata obtained from the above groups on days 5, 7 and 10 postinfection were processed for determining the viral titers as described in the ‘methods’ section and the titers were compared between groups. Left panel, hearts; right panel, pancreata.
Mentions: To determine the virulence of pBRCVB3 virus, we first recovered the virus from the infectious clone following transfection of Vero cells with in vitro-transcribed RNA. After passaging the virus twice in cultures (similar to Wt virus), we sought to determine its pathogenicity in vivo. Groups of A/J mice were infected with 2000 TCID50/animal, and on days 5, 7, 10 or 21 postinfection, animals were euthanized; hearts and pancreata were evaluated for inflammatory changes, and the lesions were compared with uninfected animals. Unexpectedly, in animals infected with the infectious clone-derived virus, myocarditis was significantly attenuated (Table 3). As shown in Fig 2A, myocarditis in mice infected with the Wt virus showed widespread lesions containing necrosis, mineralization, and lymphocytic infiltrates as opposed to occasional lesions in pBRCVB3 virus-infected animals. Pancreatitis, however was consistently present in both groups of animals with comparable severity as indicated by atrophic changes, necrosis and inflammatory infiltrates (Fig 2B; Table 4). The finding that pBRCVB3 virus-induced pancreatitis is comparable to Wt virus suggests that the virus did reach the target tissues in infected animals. But, why then attenuated myocarditogenicity? One possible reason could be that the tropism of pBRCVB3 virus containing the nt changes (C97U, A4327G, and C5088U) might have been altered for the heart. To address this possibility, we determined the viral titers from hearts and pancreata obtained from animals infected with Wt virus or pBRCVB3 virus on days 5, 7 and 10 postinfection. The virus was found to be present in hearts from animals infected with pBRCVB3 virus similar to Wt virus-infected group as the viral titers were comparable at all the time-points postinfection (Fig 2C, left panel). Likewise, viral titers from pancreata from both the groups were also comparable except that pBRCVB3 virus could not be recovered on day 10 postinfection (Fig 2C, right panel).

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