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Prolonged coxsackievirus presence in intestines and faeces of infected mice
Abstracts: Posters immunoblotting showed all 10 adults and 9 newborns to be positive. All neonates showed a response to pp38, an assembly protein, 9 responded to the pp52 immediate early antigen but only 4 had pp150 tegument associated protein reactivity. Of the mothers, 8 had pp38 reactivity, 10 showed a response to the pp52 antigen and 7 to the pp150 antigen. T cell mediated immunity was assessed by measuring cytokines using a multiplex microarray assay. Levels of IFN-alpha were high in both groups (mean • SEM; neonates: 657• pg/ml, mothers: 1072• pg/ml, pNS), however neonates had significantly higher levels of IL-8 (316• vs 48• p<0.005). High IL-8 levels in the neonate suggests an inability to control viral replication as IL-8 has been shown in vitro to enhance CMV replication. Our data reveals the development of specific humoral and cellular immune responses from a nafve state in humans in early life and insight into this process is essential in understanding immunity and development of effective vaccination strategies.
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Prolonged coxsackievirus presence in intestines and faeces of infected mice
A. Vargova 1 , A. Petrovicova1 , M. Badurova 1 , J. Galama 2, S.A. Bopegamage 1 . 1SIovak Medical University, Bratislava, Slovak
Republic, 2Radboud University Medical Center, Nijmegen, The Netherlands Background: Coxsackieviruses (CV) spread by the faecal-oral route. Therefore, virus shedding is an important factor to be considered. These viruses show target organ preferences in mice, similar to those in humans. The aim of the present study was to analyse the duration of presence of virus in the intestines and faeces after experimental infection by the oral route and to investigate a relationship with virus shedding in the stool. Methods: Outbred Swiss albino mice were infected by a diabetogenic strain of CVB4 (E2). Dose 0.5ml of 1 x l 0 7 TCID 50/ml was given by oral route. Small intestines and stool pellets were collected at selected intervals ranging from day 3 p.i. to day 105 p.i. Virus titres were determined by titration on Hep-2 monolayers on microtitre plates. The samples were analysed by nested PCR. Results: Infectious virus could be detected in the small intestine up to day 28 p.i. PCR analysis of randomly selected parts of the intestine showed that viral RNA can be detected even 105 days p.i. in the intestine but also in the stool. Conclusions: Results indicate a prolonged presence of virus in the small intestines and also in the shedded faeces of the infected mice.
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Viral RNA in hearts and pancreases of coxsackievirus infected mice
J. Motusova 1 , A. Petrovicova1 , J. Galama 2, S.A. Bopegamage 1 .
1Slovak Medical University, Bratislava, Slovak Republic, 2Radboud University Medical Center, Nijmegen, The Netherlands Aim: To compare duration of presence of viral RNA in the hearts and pancreases of mice, which were either orally or intraperitoneally infected with coxsackievirus (CV) strain CVB3 (Nancy) or diabetogenic strain CVB4 (E2). Methods: Swiss albino outbred mice were infected by the oral or intraperitoneal route with 0.5ml of 1 x l 0 7 TCID 50/ml (5x106 units) CVB4 (E2) or CVB3 (Nancy). The mock infected control mice received 0.5 of PBS by the oral or intraperitoneal route. Organs of mice were collected on the selected days 3, 5, 7, 28, 49 and 105 post infection (p.i.). Part of each sample was snap frozen in liquid nitrogen and stored at -80~ RNA was extracted from the organs and the presence of viral RNA was checked in these organs by nested PCR. Results: Viral RNA was detected in the pancreases, at day 49 p.i. in infected mice after oral infection by CVB4 (E2) and at day 105 p.i. after i.p. and oral routes of infection by CVB3 (Nancy). Whereas in hearts, viral RNA was detected to day 105 p.i. in mice infected with CVB4 (E2) and CVB3 (Nancy) by both the routes of infection. Conclusion: Differences were observed in the prolonged virus presence in pancreases after infection by diabetogenic strain versus a non-diabetogenic strain. But these differences were absent when the routes of infection by each strain were compared. There was no
$15 relationship between the presence of the virus in the pancreases and hearts of the infected mice.
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Effect of three viruses and interferon on gene expression
B. Grinde 1 , C.H. Rinaldo 2. 1Norwegian Institute of Public Health, Norway, 2University of Tromse, Norway
Background and Aims: We used microarrays to measure changes in the human transcriptosome upon in vitro infection with three different viruses (BK polyomavirus, poliovirus and vaccinia virus). The purpose was partly to understand the interaction between viruses and cells, partly to look for candidate genes involved in viral defense. Interferon-alpha treatment was included for the latter purpose. Methods: Triplicate cells were synchronously infected with viruses at titers sufficient to yield primary infection of a majority of the cells. Expressed genes in infected cells was compared with none-infected cells using human oligo-based microarrays containing 35,000 reporters. Results: In most cases more genes were up-regulated than down-regulated. There was limited concordance when comparing the effect of different viruses, and even when comparing the effect of the same virus at different time-points. However, a large fraction of the genes induced by poliovirus was also induced by interferon alpha. Conclusions and Discussion: The observation that poliovirus induced many of the same genes as interferon, at condition where the cytokine inhibited viral replication, suggests that the cells did launch antiviral defense. A list of candidate genes involved in antiviral response was set up, based on: 1, genes up- (or down-) regulated by both poliovirus and interferon; 2, genes appreciably up- (or down-) regulated, preferably to high expression values and with good concordance between parallels; 3, having annotations suggestive of an antiviral role; and 4, being up- (or down-) regulated by more than one virus.
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Rapid detection of parainfluenzavirus type 3 by real time PCR
V. Moya-Suri, K. Zimmermann, G. M611er, L. G0rtler, R. Mentel.
Friedrich Loeffler Institute of Medical Microbiology, Ernst-MoritzArndt-University Greifswald, Germany Human parainfluenza virus 3 (PIV), one of the major respiratory pathogens for a long time in hospitalized patients was frequently underestimated because a sensitive timely diagnostic method was not available. 400 clinical samples from patients with respiratory infection were analyzed by RT-PCR for PIV, human metapneumovirus (hMPV) and respiratory syncytial virus (RSV). Fifty nine (14.7%) specimens were positive for PIV, 103 (25.7%) for hMPV and 125 (31.2%) for RSV. According to our results PIV is the third viral pathogen after RSV and hMPV. The seasonal occurrence for PIV was characterized by a peak in winter. Infections of hospitalized patients with PIV were often associated with severe disease like pneumonia (37.3%) and bronchitis (27.1%). A real-time RT-PCR for PIV based on primer/probe set derived from the F gene was developed. All samples that tested positive by RT-PCR for PIV were evaluated by this real-time protocol with the corresponding results. The real-time protocol developed is a sensitive and specific approach for PIV diagnostics. Since results are available in reduced time (ca. 4h) they improve patient management. Our data show the necessity to include also PIV in the diagnostic program for management of respiratory diseases.
I~
Prolonged coxsackievirus presence in intestines and faeces of infected mice
A. Vargova 1 , A. Petrovicova1 , M. Badurova 1 , J. Galama 2, S.A. Bopegamage 1 . 1SIovak Medical University, Bratislava, Slovak
Republic, 2Radboud University Medical Center, Nijmegen, The Netherlands Background: Coxsackieviruses (CV) spread by the faecal-oral route. Therefore, virus shedding is an important factor to be considered. These viruses show target organ preferences in mice, similar to those in humans. The aim of the present study was to analyse the duration of presence of virus in the intestines and faeces after experimental infection by the oral route and to investigate a relationship with virus shedding in the stool. Methods: Outbred Swiss albino mice were infected by a diabetogenic strain of CVB4 (E2). Dose 0.5ml of 1 x l 0 7 TCID 50/ml was given by oral route. Small intestines and stool pellets were collected at selected intervals ranging from day 3 p.i. to day 105 p.i. Virus titres were determined by titration on Hep-2 monolayers on microtitre plates. The samples were analysed by nested PCR. Results: Infectious virus could be detected in the small intestine up to day 28 p.i. PCR analysis of randomly selected parts of the intestine showed that viral RNA can be detected even 105 days p.i. in the intestine but also in the stool. Conclusions: Results indicate a prolonged presence of virus in the small intestines and also in the shedded faeces of the infected mice.
I~
Viral RNA in hearts and pancreases of coxsackievirus infected mice
J. Motusova 1 , A. Petrovicova1 , J. Galama 2, S.A. Bopegamage 1 .
1Slovak Medical University, Bratislava, Slovak Republic, 2Radboud University Medical Center, Nijmegen, The Netherlands Aim: To compare duration of presence of viral RNA in the hearts and pancreases of mice, which were either orally or intraperitoneally infected with coxsackievirus (CV) strain CVB3 (Nancy) or diabetogenic strain CVB4 (E2). Methods: Swiss albino outbred mice were infected by the oral or intraperitoneal route with 0.5ml of 1 x l 0 7 TCID 50/ml (5x106 units) CVB4 (E2) or CVB3 (Nancy). The mock infected control mice received 0.5 of PBS by the oral or intraperitoneal route. Organs of mice were collected on the selected days 3, 5, 7, 28, 49 and 105 post infection (p.i.). Part of each sample was snap frozen in liquid nitrogen and stored at -80~ RNA was extracted from the organs and the presence of viral RNA was checked in these organs by nested PCR. Results: Viral RNA was detected in the pancreases, at day 49 p.i. in infected mice after oral infection by CVB4 (E2) and at day 105 p.i. after i.p. and oral routes of infection by CVB3 (Nancy). Whereas in hearts, viral RNA was detected to day 105 p.i. in mice infected with CVB4 (E2) and CVB3 (Nancy) by both the routes of infection. Conclusion: Differences were observed in the prolonged virus presence in pancreases after infection by diabetogenic strain versus a non-diabetogenic strain. But these differences were absent when the routes of infection by each strain were compared. There was no
$15 relationship between the presence of the virus in the pancreases and hearts of the infected mice.
1~
Effect of three viruses and interferon on gene expression
B. Grinde 1 , C.H. Rinaldo 2. 1Norwegian Institute of Public Health, Norway, 2University of Tromse, Norway
Background and Aims: We used microarrays to measure changes in the human transcriptosome upon in vitro infection with three different viruses (BK polyomavirus, poliovirus and vaccinia virus). The purpose was partly to understand the interaction between viruses and cells, partly to look for candidate genes involved in viral defense. Interferon-alpha treatment was included for the latter purpose. Methods: Triplicate cells were synchronously infected with viruses at titers sufficient to yield primary infection of a majority of the cells. Expressed genes in infected cells was compared with none-infected cells using human oligo-based microarrays containing 35,000 reporters. Results: In most cases more genes were up-regulated than down-regulated. There was limited concordance when comparing the effect of different viruses, and even when comparing the effect of the same virus at different time-points. However, a large fraction of the genes induced by poliovirus was also induced by interferon alpha. Conclusions and Discussion: The observation that poliovirus induced many of the same genes as interferon, at condition where the cytokine inhibited viral replication, suggests that the cells did launch antiviral defense. A list of candidate genes involved in antiviral response was set up, based on: 1, genes up- (or down-) regulated by both poliovirus and interferon; 2, genes appreciably up- (or down-) regulated, preferably to high expression values and with good concordance between parallels; 3, having annotations suggestive of an antiviral role; and 4, being up- (or down-) regulated by more than one virus.
1~
Rapid detection of parainfluenzavirus type 3 by real time PCR
V. Moya-Suri, K. Zimmermann, G. M611er, L. G0rtler, R. Mentel.
Friedrich Loeffler Institute of Medical Microbiology, Ernst-MoritzArndt-University Greifswald, Germany Human parainfluenza virus 3 (PIV), one of the major respiratory pathogens for a long time in hospitalized patients was frequently underestimated because a sensitive timely diagnostic method was not available. 400 clinical samples from patients with respiratory infection were analyzed by RT-PCR for PIV, human metapneumovirus (hMPV) and respiratory syncytial virus (RSV). Fifty nine (14.7%) specimens were positive for PIV, 103 (25.7%) for hMPV and 125 (31.2%) for RSV. According to our results PIV is the third viral pathogen after RSV and hMPV. The seasonal occurrence for PIV was characterized by a peak in winter. Infections of hospitalized patients with PIV were often associated with severe disease like pneumonia (37.3%) and bronchitis (27.1%). A real-time RT-PCR for PIV based on primer/probe set derived from the F gene was developed. All samples that tested positive by RT-PCR for PIV were evaluated by this real-time protocol with the corresponding results. The real-time protocol developed is a sensitive and specific approach for PIV diagnostics. Since results are available in reduced time (ca. 4h) they improve patient management. Our data show the necessity to include also PIV in the diagnostic program for management of respiratory diseases.