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Parvovirus B19: Its real disease associations
S140
Abstracts / Journal of Clinical Virology 82S (2016) S1–S142
Abstract no: 218 Presentation at ESCV 2016: Poster 242 High frequency of JC human polyomavirus in Portuguese wastewaters: A possible source for human infection F. Rodrigues 1,∗ , D. Gonc¸alves 1 , C. Luxo 2 , A.M. Matos 2 1
Laboratório de Virologia, Grupo das Biociências Clínicas e Aplicadas, Faculdade de Farmácia da Universidade de Coimbra, Portugal 2 Laboratório de Virologia, Grupo das Biociências Clínicas e Aplicadas, Faculdade de Farmácia da Universidade de Coimbra, Centro de Investigac¸ão em Engenharia dos Processos Químicos e dos Produtos da Floresta (CIEPQF), Coimbra, Portugal Introduction: JC polyomavirus (JCV) is ubiquitous among the human population worldwide. Primary infection, typically asymptomatic, normally occurs during childhood and is followed by a lifelong persistent infection. JCV is excreted in the urine of nearly half of the infected individuals, without any associated clinical symptom. Under situations of severe immunosuppression, JCV may reactivate and induces a rare but fatal demyelinating disease of the central nervous system known as Progressive Multifocal Leukoencephalopathy (PML). Despite several hypothesis have been raised, the main mode of transmission remains unknown. The high frequency of urinary excretion of JCV lead several authors to evaluate its presence in sewage wastewater systems, though, to date no data have been reported for Portuguese wastewaters. In this order, the present study represents the first comprehensive assessment of JCV in Portuguese wastewaters along with its removal efficiency by wastewater treatment plants (WWTPs), in order to add further information for a possible way of JCV transmission to occur. Materials and methods: Fifteen WWTP distributed all across Portugal and serving 26.3% of the National population were selected for the present study. Two pairs of influent (WWI) (untreated) and effluent (WWE) (treated) samples, were collected from each WWTP, in September and December 2013, making a total of 60 wastewater samples. Viruses were concentrated by ultracentrifugation as previously described [1], and detection and quantification of JCV DNA in wastewater samples was obtained by a quantitative real-time PCR protocol using a set of four amplification primers and two internal TaqMan probes, previously described [2]. Results: JCV genome was detected in 14 (93%) of the 15 evaluated WWTP, in at least one of the collected samples. Ninety per cent of the tested influent samples revealed detectable JCV DNA, at relatively high concentrations (mean = 5.48 ± 0.74 log10 GC/L). The treatment of wastewater was able to completely remove JCV genome from 14 (52%) of the 27 initial positive assessed WWI samples. In the remaining 13 initially positive WWI samples, despite no complete removal was accomplished, a decrease in JCV concentration was observed in the majority of cases. Discussion: The ubiquity of JCV infection, claims for a common route of transmission, particularly when seroepidemiological surveys point to childhood as the age for first infection to occur. The present study reveals the consistent detection of JCV genome in sewage from the different regions of Portugal. Moreover, nearly half of the WWTP were not able to completely remove the virus, which, by this manner will end up incorporating treated sewage and be distributed to the surrounding environment. Such viruses may finally contaminate food and water, which may act as vehicles for JCV transmission through oral route.
Reference [1] A. Rafique, S.C. Jiang, Genetic diversity of human polyomavirus JCPyV in Southern California wastewater, J. Water Health (2008), http://dx.doi.org/10. 2166/wh.2008.067. [2] C.F. Ryschkewitsch, P.N. Jensen, E.O. Major, Multiplex qPCR assay for ultra sensitive detection of JCV DNA with simultaneous identification of genotypes that discriminates non-virulent from virulent variants, J. Clin. Virol. 57 (2013) 243–248, http://dx.doi.org/10.1016/j.jcv.2013.03.009.
http://dx.doi.org/10.1016/j.jcv.2016.08.282 Abstract no: 282 Presentation at ESCV 2016: Poster 243 An unusual course of parvovirus B19 infection, strongly suggestive of virus reactivation U. Reber 1,∗ , S. Aldabbagh 1 , S. Pietzonka 1 , O. Moser 2,3 , A. Simon 2,4 , D. Dilloo 2 , A. Eis-Hübinger 1 1
Institute of Virology, University of Bonn Medical Center, Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany 2 Department of Pediatric Hematology and Oncology, Center for Pediatrics, University of Bonn Medical Center, Bonn, Germany 3 Division of Pediatric Hematology, Oncology & Stem Cell Transplantation, University of Aachen, Germany 4 Pediatric Oncology and Hematology, Children’s University Hospital, Homburg, Saar, Germany Because of its highly efficient replication in erythroid progenitor cells, parvovirus B19 (B19V) causes an interruption of the red cell production which may result in a more or less severe anemia. After primary infection and elimination of viremia, viral DNA persists at low levels in multiple tissues, probably for life. It is not clear whether the persisting virus is able to reactivate. We present a case of severe B19V infection in a patient with spherocytosis that was followed by a further episode of severe anemia with B19V viremia three years later. Nearly full-length nucleotide sequencing of the B19V genomes detected in blood during primary infection and after three years revealed complete identity of the genomes. Although not apodictically proven, this case demonstrates that, in rare instances, recurrence of B19V infection might be possible. http://dx.doi.org/10.1016/j.jcv.2016.08.283 Abstract no: 343 Presentation at ESCV 2016: Poster 244 Parvovirus B19: Its real disease associations A.C.M. Kroes Leiden University Medical Center, The Netherlands Parvovirus B19 (B19V or primate erythroparvovirus 1, ssDNA) is dependent on actively profilerating erythroid precursor cells for its replication. This particular tropism is determined by the lack of a viral DNA polymerase and by a receptor expressed on eythroid cells, globoside. In addition to the childhood disease erythema infectiosum, as an immunopathological consequence of infection, all relevant pathology brought about by B19V is explained by the property of wiping out erythrocyte precursor cells. A severe breakdown of red cell synthesis or aplastic crisis occurs in cases lacking recruitment from a resting population, like in sickle cell anemia and in expanding fetal erythropoiesis. In the fetus, this anemia
Abstracts / Journal of Clinical Virology 82S (2016) S1–S142
presents as fetal hydrops, without fetal blood transfusion often causing intrauterine death. A temporary arrest of erythropoiesis occurs in all infected individuals, leading to disappearance of reticulocytes. This is usually too short in duration to lead to a decline of hemoglobin levels. The initial phase of B19V infection is characterized by a brisk viremic peak of an exceptional magnitude, amounting to 1012 viral particles/ml or more. This viremia is likely the result of massive replication in the bone marrow. Several studies have found evidence of B19V DNA persisting in a wide array of tissues. This has enabled historical analysis of viral genotypes in skeletal remains. Viral persistence in tissues not known to support viral replication could be understood by less specific uptake processes during the viremic peak and subsequent cellular persistence of DNA, which is not actively degraded. Independently from these observations in the last decades some additional pathological associations of B19V infection were described. These associations were based on the detection of viral DNA in affected tissues or on the occurrence of viral DNA in blood. The most frequently reported additional association of B19V infection concerns a role in various cardiac disorders, mainly myocarditis and cardiomyopathy. As these findings were described in prestigious cardiology journals, B19V was included among the possible causes of myocarditis. From a virological point of view, this association is difficult to interpret. There is no clear evidence of viral protein expression or an immune response and there are no epidemiological clues of infection, like concomitantly occurring erythema infectiosum. In addition, the nature of cardiac disorders presumably brought about by B19V appears rather variable. For these reasons, several authors assumed that passive release of B19V DNA of any damaged tissue harboring persisting virus could well be an explanation for the findings. Still, confusion on the possible role of B19V in a number of disorders remains. Recently, in the Sanquin/LUMC research group it was found that simple enzymatic treatment of B19V DNA positive blood samples provided evidence of the existence of two different states: stable, compatible with protected viral particles or vulnerable, possibly consisting of naked DNA. If confirmed, this would enable differentiation of the B19V DNA findings into those directly related to viral replication and those possibly emerging from passive tissue release. This is essential to determine the real disease associations of this infection. In this way, the case of B19V underlines the importance of a plausible pathogenesis before assuming a causal role, solely based on the finding of viral nucleic acid. http://dx.doi.org/10.1016/j.jcv.2016.08.284 Abstract no: 51 Presentation at ESCV 2016: Poster 245 Occurrence, phase and status of human parvovirus B19 infection in patients with myalgic encephalomyelitis/chronic fatigue syndrome Santa Rasa 1,∗ , Svetlana Chapenko 1 , Angelika Krumina 2 , Zane Zazerska 1 , Modra Murovska 1 1
A. Kirchenstein Institute of Microbiology and Virology, R¯ıga Stradin¸sˇ University, Latvia 2 Department of Infectology and Dermatology, R¯ıga Stradin¸sˇ University, Latvia Introduction: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is chronic, multifactorial disease with unexplained etiology. Human parvovirus B19 (B19) is immunomodulating
S141
single-stranded DNA virus belonging to Erythrovirus genus, Parvoviridae family, Parvovirinae subfamily and is considered as possible pathogen in development of ME/CFS [1,2]. The aim of this study was to estimate frequency and activity phase of B19 infection, viral load, status of infection and clinical symptoms in patients with ME/CFS. Materials and methods: 200 patients (65% female and 35% male, mean age 38 ± 12 years) with according to CDC criteria diagnosed ME/CFS and 104 age and gender matched apparently healthy individuals were enrolled in this study. Presence of B19 specific IgM and IgG class antibodies were analysed by recomWell and recomLine Parvovirus B19 IgM and IgG kits (Mikrogen Diagnostik). B19 NS1 gene sequence was detected with nested PCR and viral load was estimated by Parvovirus B19 Real-TM Quant (Sacace Biotechnologies) real-time PCR kit. Results: B19-specific IgG class antibodies were found in 140/200 (70%) patients with ME/CFS and in 60/89 (67.4%) analysed apparently healthy individuals. None of control individuals, though 16/200 (8%) patients with ME/CFS had IgM class antibodies (p = 0.0038). Persistent B19 infection in latent phase had 24/200 (12%) patients and 8/104 (7.7%) apparently healthy individuals, whereas in active phase – 34/200 (17%) ME/CFS patients and 2/104 (1.9%) control individuals (p < 0.0001). Elevated viral load was detected in 20/58 ME/CFS patients and in none of 10 apparently healthy individuals with B19 infection (p = 0.0276). B19 viral load varied from <10 copies to median 1044 (IQR 3180-503.6) copies/106 cells. Analysing B19 specific antibody reaction patterns, results show that 29.3% of patients had infection status after infection (months), 30.7% – past infection (months to years), 12% had status infection long ago, 6.7% – after infection (weeks to months) and one patient had acute infection status. Such typical ME/CFS clinical symptoms as impaired memory, subfebrility, lymphadenopathy and multijoint pain was observed more often in patients with persistent B19 infection in active than in latent phase. In 93.3% of patients onset of symptoms has occurred 8.3 ± 1.7 months ago and in 6.7% of patients symptoms had started before 2.4 ± 0.5 years. Conclusions: Results demonstrate finding of human parvovirus B19 persistent infection in active phase significantly more frequent and with higher B19 load among patients with ME/CFS than apparently healthy individuals indicating on implication of B19 infection in pathogenesis of ME/CFS. Therefore markers of active B19 infection could be used as one of biomarkers in ME/CFS diagnostics. Association of persistent B19 infection in active phase with part of typical ME/CFS clinical symptoms shows possible B19 involvement in disease development and reactivation of B19 may be a risk factor for ME/CFS.B19 infection statuses and onset of symptoms allow suggesting the feasible role of B19 infection as a trigger factor for ME/CFS. Reference [1] A.S. Bansal, A.S. Bradley, K.N. Bishop, S. Kiani-Alikhan, B. Ford, Chronic fatigue syndrome, the immune system and viral infection, Brain Behav. Immun. 26 (1) (2012) 24–31. [2] F. Barah, S. Whiteside, S. Batista, J. Morris, Neurological aspects of human parvovirus B19 infection: a systematic review, Rev. Med. Virol. 24 (3) (2014) 154–168.
http://dx.doi.org/10.1016/j.jcv.2016.08.285
Abstracts / Journal of Clinical Virology 82S (2016) S1–S142
Abstract no: 218 Presentation at ESCV 2016: Poster 242 High frequency of JC human polyomavirus in Portuguese wastewaters: A possible source for human infection F. Rodrigues 1,∗ , D. Gonc¸alves 1 , C. Luxo 2 , A.M. Matos 2 1
Laboratório de Virologia, Grupo das Biociências Clínicas e Aplicadas, Faculdade de Farmácia da Universidade de Coimbra, Portugal 2 Laboratório de Virologia, Grupo das Biociências Clínicas e Aplicadas, Faculdade de Farmácia da Universidade de Coimbra, Centro de Investigac¸ão em Engenharia dos Processos Químicos e dos Produtos da Floresta (CIEPQF), Coimbra, Portugal Introduction: JC polyomavirus (JCV) is ubiquitous among the human population worldwide. Primary infection, typically asymptomatic, normally occurs during childhood and is followed by a lifelong persistent infection. JCV is excreted in the urine of nearly half of the infected individuals, without any associated clinical symptom. Under situations of severe immunosuppression, JCV may reactivate and induces a rare but fatal demyelinating disease of the central nervous system known as Progressive Multifocal Leukoencephalopathy (PML). Despite several hypothesis have been raised, the main mode of transmission remains unknown. The high frequency of urinary excretion of JCV lead several authors to evaluate its presence in sewage wastewater systems, though, to date no data have been reported for Portuguese wastewaters. In this order, the present study represents the first comprehensive assessment of JCV in Portuguese wastewaters along with its removal efficiency by wastewater treatment plants (WWTPs), in order to add further information for a possible way of JCV transmission to occur. Materials and methods: Fifteen WWTP distributed all across Portugal and serving 26.3% of the National population were selected for the present study. Two pairs of influent (WWI) (untreated) and effluent (WWE) (treated) samples, were collected from each WWTP, in September and December 2013, making a total of 60 wastewater samples. Viruses were concentrated by ultracentrifugation as previously described [1], and detection and quantification of JCV DNA in wastewater samples was obtained by a quantitative real-time PCR protocol using a set of four amplification primers and two internal TaqMan probes, previously described [2]. Results: JCV genome was detected in 14 (93%) of the 15 evaluated WWTP, in at least one of the collected samples. Ninety per cent of the tested influent samples revealed detectable JCV DNA, at relatively high concentrations (mean = 5.48 ± 0.74 log10 GC/L). The treatment of wastewater was able to completely remove JCV genome from 14 (52%) of the 27 initial positive assessed WWI samples. In the remaining 13 initially positive WWI samples, despite no complete removal was accomplished, a decrease in JCV concentration was observed in the majority of cases. Discussion: The ubiquity of JCV infection, claims for a common route of transmission, particularly when seroepidemiological surveys point to childhood as the age for first infection to occur. The present study reveals the consistent detection of JCV genome in sewage from the different regions of Portugal. Moreover, nearly half of the WWTP were not able to completely remove the virus, which, by this manner will end up incorporating treated sewage and be distributed to the surrounding environment. Such viruses may finally contaminate food and water, which may act as vehicles for JCV transmission through oral route.
Reference [1] A. Rafique, S.C. Jiang, Genetic diversity of human polyomavirus JCPyV in Southern California wastewater, J. Water Health (2008), http://dx.doi.org/10. 2166/wh.2008.067. [2] C.F. Ryschkewitsch, P.N. Jensen, E.O. Major, Multiplex qPCR assay for ultra sensitive detection of JCV DNA with simultaneous identification of genotypes that discriminates non-virulent from virulent variants, J. Clin. Virol. 57 (2013) 243–248, http://dx.doi.org/10.1016/j.jcv.2013.03.009.
http://dx.doi.org/10.1016/j.jcv.2016.08.282 Abstract no: 282 Presentation at ESCV 2016: Poster 243 An unusual course of parvovirus B19 infection, strongly suggestive of virus reactivation U. Reber 1,∗ , S. Aldabbagh 1 , S. Pietzonka 1 , O. Moser 2,3 , A. Simon 2,4 , D. Dilloo 2 , A. Eis-Hübinger 1 1
Institute of Virology, University of Bonn Medical Center, Bonn, Sigmund-Freud-Str. 25, D-53105 Bonn, Germany 2 Department of Pediatric Hematology and Oncology, Center for Pediatrics, University of Bonn Medical Center, Bonn, Germany 3 Division of Pediatric Hematology, Oncology & Stem Cell Transplantation, University of Aachen, Germany 4 Pediatric Oncology and Hematology, Children’s University Hospital, Homburg, Saar, Germany Because of its highly efficient replication in erythroid progenitor cells, parvovirus B19 (B19V) causes an interruption of the red cell production which may result in a more or less severe anemia. After primary infection and elimination of viremia, viral DNA persists at low levels in multiple tissues, probably for life. It is not clear whether the persisting virus is able to reactivate. We present a case of severe B19V infection in a patient with spherocytosis that was followed by a further episode of severe anemia with B19V viremia three years later. Nearly full-length nucleotide sequencing of the B19V genomes detected in blood during primary infection and after three years revealed complete identity of the genomes. Although not apodictically proven, this case demonstrates that, in rare instances, recurrence of B19V infection might be possible. http://dx.doi.org/10.1016/j.jcv.2016.08.283 Abstract no: 343 Presentation at ESCV 2016: Poster 244 Parvovirus B19: Its real disease associations A.C.M. Kroes Leiden University Medical Center, The Netherlands Parvovirus B19 (B19V or primate erythroparvovirus 1, ssDNA) is dependent on actively profilerating erythroid precursor cells for its replication. This particular tropism is determined by the lack of a viral DNA polymerase and by a receptor expressed on eythroid cells, globoside. In addition to the childhood disease erythema infectiosum, as an immunopathological consequence of infection, all relevant pathology brought about by B19V is explained by the property of wiping out erythrocyte precursor cells. A severe breakdown of red cell synthesis or aplastic crisis occurs in cases lacking recruitment from a resting population, like in sickle cell anemia and in expanding fetal erythropoiesis. In the fetus, this anemia
Abstracts / Journal of Clinical Virology 82S (2016) S1–S142
presents as fetal hydrops, without fetal blood transfusion often causing intrauterine death. A temporary arrest of erythropoiesis occurs in all infected individuals, leading to disappearance of reticulocytes. This is usually too short in duration to lead to a decline of hemoglobin levels. The initial phase of B19V infection is characterized by a brisk viremic peak of an exceptional magnitude, amounting to 1012 viral particles/ml or more. This viremia is likely the result of massive replication in the bone marrow. Several studies have found evidence of B19V DNA persisting in a wide array of tissues. This has enabled historical analysis of viral genotypes in skeletal remains. Viral persistence in tissues not known to support viral replication could be understood by less specific uptake processes during the viremic peak and subsequent cellular persistence of DNA, which is not actively degraded. Independently from these observations in the last decades some additional pathological associations of B19V infection were described. These associations were based on the detection of viral DNA in affected tissues or on the occurrence of viral DNA in blood. The most frequently reported additional association of B19V infection concerns a role in various cardiac disorders, mainly myocarditis and cardiomyopathy. As these findings were described in prestigious cardiology journals, B19V was included among the possible causes of myocarditis. From a virological point of view, this association is difficult to interpret. There is no clear evidence of viral protein expression or an immune response and there are no epidemiological clues of infection, like concomitantly occurring erythema infectiosum. In addition, the nature of cardiac disorders presumably brought about by B19V appears rather variable. For these reasons, several authors assumed that passive release of B19V DNA of any damaged tissue harboring persisting virus could well be an explanation for the findings. Still, confusion on the possible role of B19V in a number of disorders remains. Recently, in the Sanquin/LUMC research group it was found that simple enzymatic treatment of B19V DNA positive blood samples provided evidence of the existence of two different states: stable, compatible with protected viral particles or vulnerable, possibly consisting of naked DNA. If confirmed, this would enable differentiation of the B19V DNA findings into those directly related to viral replication and those possibly emerging from passive tissue release. This is essential to determine the real disease associations of this infection. In this way, the case of B19V underlines the importance of a plausible pathogenesis before assuming a causal role, solely based on the finding of viral nucleic acid. http://dx.doi.org/10.1016/j.jcv.2016.08.284 Abstract no: 51 Presentation at ESCV 2016: Poster 245 Occurrence, phase and status of human parvovirus B19 infection in patients with myalgic encephalomyelitis/chronic fatigue syndrome Santa Rasa 1,∗ , Svetlana Chapenko 1 , Angelika Krumina 2 , Zane Zazerska 1 , Modra Murovska 1 1
A. Kirchenstein Institute of Microbiology and Virology, R¯ıga Stradin¸sˇ University, Latvia 2 Department of Infectology and Dermatology, R¯ıga Stradin¸sˇ University, Latvia Introduction: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is chronic, multifactorial disease with unexplained etiology. Human parvovirus B19 (B19) is immunomodulating
S141
single-stranded DNA virus belonging to Erythrovirus genus, Parvoviridae family, Parvovirinae subfamily and is considered as possible pathogen in development of ME/CFS [1,2]. The aim of this study was to estimate frequency and activity phase of B19 infection, viral load, status of infection and clinical symptoms in patients with ME/CFS. Materials and methods: 200 patients (65% female and 35% male, mean age 38 ± 12 years) with according to CDC criteria diagnosed ME/CFS and 104 age and gender matched apparently healthy individuals were enrolled in this study. Presence of B19 specific IgM and IgG class antibodies were analysed by recomWell and recomLine Parvovirus B19 IgM and IgG kits (Mikrogen Diagnostik). B19 NS1 gene sequence was detected with nested PCR and viral load was estimated by Parvovirus B19 Real-TM Quant (Sacace Biotechnologies) real-time PCR kit. Results: B19-specific IgG class antibodies were found in 140/200 (70%) patients with ME/CFS and in 60/89 (67.4%) analysed apparently healthy individuals. None of control individuals, though 16/200 (8%) patients with ME/CFS had IgM class antibodies (p = 0.0038). Persistent B19 infection in latent phase had 24/200 (12%) patients and 8/104 (7.7%) apparently healthy individuals, whereas in active phase – 34/200 (17%) ME/CFS patients and 2/104 (1.9%) control individuals (p < 0.0001). Elevated viral load was detected in 20/58 ME/CFS patients and in none of 10 apparently healthy individuals with B19 infection (p = 0.0276). B19 viral load varied from <10 copies to median 1044 (IQR 3180-503.6) copies/106 cells. Analysing B19 specific antibody reaction patterns, results show that 29.3% of patients had infection status after infection (months), 30.7% – past infection (months to years), 12% had status infection long ago, 6.7% – after infection (weeks to months) and one patient had acute infection status. Such typical ME/CFS clinical symptoms as impaired memory, subfebrility, lymphadenopathy and multijoint pain was observed more often in patients with persistent B19 infection in active than in latent phase. In 93.3% of patients onset of symptoms has occurred 8.3 ± 1.7 months ago and in 6.7% of patients symptoms had started before 2.4 ± 0.5 years. Conclusions: Results demonstrate finding of human parvovirus B19 persistent infection in active phase significantly more frequent and with higher B19 load among patients with ME/CFS than apparently healthy individuals indicating on implication of B19 infection in pathogenesis of ME/CFS. Therefore markers of active B19 infection could be used as one of biomarkers in ME/CFS diagnostics. Association of persistent B19 infection in active phase with part of typical ME/CFS clinical symptoms shows possible B19 involvement in disease development and reactivation of B19 may be a risk factor for ME/CFS.B19 infection statuses and onset of symptoms allow suggesting the feasible role of B19 infection as a trigger factor for ME/CFS. Reference [1] A.S. Bansal, A.S. Bradley, K.N. Bishop, S. Kiani-Alikhan, B. Ford, Chronic fatigue syndrome, the immune system and viral infection, Brain Behav. Immun. 26 (1) (2012) 24–31. [2] F. Barah, S. Whiteside, S. Batista, J. Morris, Neurological aspects of human parvovirus B19 infection: a systematic review, Rev. Med. Virol. 24 (3) (2014) 154–168.
http://dx.doi.org/10.1016/j.jcv.2016.08.285