Roland Zell

DNA mismatch-repair in Escherichia coli counteracting the hydrolytic deamination of 5-methyl-cytosine residues

Derivatives of phage M13 were constructed and used for the in vitro preparation of heteroduplex DNA molecules containing base/base mismatches that mimick DNA lesions caused by hydrolytic deamination of 5‐meC residues in Escherichia coli DNA (i.e. they carry a T/G mismatch in the special sequence context provided by the recognition site ‐CCA/TGG‐of the Dcm‐methyltransferase). Upon introduction of these heteroduplex DNAs into CaCl2‐treated E. coli cells, the mismatches are efficiently repaired with high bias in favour of the DNA strand containing the mismatched guanine residue. This special DNA mismatch‐repair operates on fully dam‐methylated DNA and is independent of gene mutH. It thus fulfills the salient requirements of a repair pathway responsible for counteracting the spontaneous hydrolytic deamination of 5‐meC in vivo. The repair efficiency is boosted by a 5‐methyl group present on the cytosine residue at the next‐nearest position to the 5′ side of the mismatched guanine. The repair is severely impaired in host strains carrying a mutation in any of the three loci dcm, mutL and mutS.

Apoptosis in Coxsackievirus B3-Caused Diseases: Interaction between the Capsid Protein VP2 and the Proapoptotic Protein Siva

ABSTRACT Coxsackievirus B3 (CVB3) is a common factor in human myocarditis. Apoptotic events are present in CVB3-induced disease, but it is unclear how CVB3 is involved in apoptosis and which viral proteins may induce the apoptotic pathway. In this report we demonstrate that the human and murine proapoptotic protein Siva specifically interact with the CVB3 capsid protein VP2. Furthermore, the transcription of Siva is strongly induced in tissue of CVB3-infected mice and is present in the same area which is positively stained for apoptosis, CD27, and CD70. It has been proposed that Siva is involved in the CD27/CD70-transduced apoptosis. Therefore, we suggest a molecular mechanism through which apoptotic events contributes to CVB3-caused pathogenesis.

Low-Level Expression of a Mutant Coxsackieviral cDNA Induces a Myocytopathic Effect in Culture: An Approach to the Study of Enteroviral Persistence in Cardiac Myocytes

BACKGROUND Enteroviral ribonucleic acids have been identified in heart muscle of a subset of patients with myocarditis and dilated cardiomyopathy as well as in a mouse model of persistent coxsackievirus B3 (CVB3) infection, suggesting that persistent viral infection along with activation of an immune response may contribute to the pathogenesis of ongoing cardiac disease and dilated cardiomyopathy in certain patients. It is still not known whether persistence of the viral genome contributes to the pathogenesis of dilated cardiomyopathy. METHODS AND RESULTS To determine whether low-level enteroviral gene expression similar to that observed with viral persistence can induce myocytopathic effects without formation of infectious virus progeny, the full-length infectious cDNA copy of CVB3 was mutated at the VP0 maturation cleavage site. This prevented formation of infectious virus progeny. In myocytes transfected with this mutated cDNA copy of the viral genome, both positive- and negative-strand viral RNAs were detected, demonstrating that there was replication of the viral genome by the RNA-dependent RNA polymerase. The level of viral protein expression was found to be below limits of detection by conventional methods of protein detection, thus resembling restricted virus replication. Nonetheless, the CVB3 mutant was found to induce a cytopathic effect in transfected myocytes, which was demonstrated by inhibition of cotransfected MLC-2v luciferase reporter activity and an increase in release of lactate dehydrogenase from transfected cells. CONCLUSIONS This study demonstrates that restricted replication of enteroviral genomes in myocytes in a pattern similar to that observed in hearts with persistent viral infection can induce myocytopathic effects without generation of infectious virus progeny.

Porcine Teschoviruses Comprise at Least Eleven Distinct Serotypes: Molecular and Evolutionary Aspects

ABSTRACT Nucleotide sequencing and phylogenetic analysis of 10 recognized prototype strains of the porcine enterovirus (PEV) cytopathic effect (CPE) group I reveals a close relationship of the viral genomes to the previously sequenced strain F65, supporting the concept of a reclassification of this virus group into a new picornavirus genus. Also, nucleotide sequences of the polyprotein-encoding genome region or the P1 region of 28 historic strains and recent field isolates were determined. The data suggest that several closely related but antigenically and molecular distinct serotypes constitute one species within the proposed genus Teschovirus. Based on sequence data and serological data, we propose a new serotype with strain Dresden as prototype. This hitherto unrecognized serotype is closely related to porcine teschovirus 1 (PTV-1, former PEV-1), but induces type-specific neutralizing antibodies. Sequencing of field isolates collected from animals presenting with neurological disorders prove that other serotypes than PTV-1 may also cause polioencephalomyelitis of swine.

Prevalence of hepatitis E virus-specific antibodies in humans with occupational exposure to pigs

Due to the increasing number of non-travel-associated hepatitis E virus (HEV) infections observed in several industrialised countries including Germany, there is a substantial interest in the characterisation of risk factors and transmission routes relevant to autochthonous HEV infections. Autochthonous cases are believed to be the result of a zoonotic HEV transmission from pigs, wild boars and deer. Recently, a high prevalence of HEV-specific antibodies in the German domestic pig population has been demonstrated. Thus, one may assume a higher prevalence of HEV-specific antibodies in humans with occupational exposure to pigs. In this study, sera obtained from 24 slaughterers, 14 meat inspectors, 46 pig farmers and 22 veterinarians were tested for the presence of HEV-specific antibodies using a line immunoassay. For comparison, sera obtained from 116 age- and gender-matched blood donors were also included. Twenty eight per cent (28.3%; 30/106) of the swine-exposed humans and 15.5% (18/116) of the blood donors without contact to pigs exhibited IgG-antibodies determined as reactive (i.e. borderline or positive) against HEV. Thus, an increased risk of HEV infection in humans occupationally exposed to pigs and particularly for slaughterers (41.7%; 10/24) was demonstrated.

Global climate change and the emergence/re-emergence of infectious diseases

Variation in the incidence of vector-borne diseases is associated with extreme weather events and annual changes in weather conditions. Moreover, it is assumed that global warming might lead to an increase of infectious disease outbreaks. While a number of reports link disease outbreaks to single weather events, the El Niño/Southern Oscillation and other large-scale climate fluctuations, no report unequivocally associates vector-borne diseases with increased temperature and the environmental changes expected to accompany it. The complexity of not yet fully understood pathogen transmission dynamics with numerous variables might be an explanation of the problems in assessing the risk factors.

Molecular Pathogenesis of Enterovirus-Induced Myocarditis: Virus Persistence and Chronic Inflammation

In situ hybridization studies have proved that myocardial enterovirus infections are detectable in all stages of acute and chronic enterovirus-induced myocarditis as well as in some patients with end-stage dilated cardiomyopathy, suggesting the possibility of myocardial enterovirus persistence. Possible enterovirus persistence in the human heart is supported by the discovery of enterovirus persistence in different murine models of chronic myocarditis, demonstrating that coxsackievirus B3, typically a cytolytic enterovirus, is capable of evading immunological surveillance in a host-dependent fashion. Progress is currently being made in unraveling the molecular mechanisms of enterovirus persistence, the diversity of host and virus genetics and their impact on the nature and severity of the disease. Apart from providing an etiologic diagnosis, there are therapeutic implications from the in situ demonstration of myocardial enterovirus infection. Evaluation of specific antiviral agents, for example interferons, may lead to the development of new therapeutic strategies capable of providing protection against myocardial enterovirus infection.

Pandemic Influenza A Viruses Escape from Restriction by Human MxA through Adaptive Mutations in the Nucleoprotein

The interferon-induced dynamin-like MxA GTPase restricts the replication of influenza A viruses. We identified adaptive mutations in the nucleoprotein (NP) of pandemic strains A/Brevig Mission/1/1918 (1918) and A/Hamburg/4/2009 (pH1N1) that confer MxA resistance. These resistance-associated amino acids in NP differ between the two strains but form a similar discrete surface-exposed cluster in the body domain of NP, indicating that MxA resistance evolved independently. The 1918 cluster was conserved in all descendent strains of seasonal influenza viruses. Introduction of this cluster into the NP of the MxA-sensitive influenza virus A/Thailand/1(KAN-1)/04 (H5N1) resulted in a gain of MxA resistance coupled with a decrease in viral replication fitness. Conversely, introduction of MxA-sensitive amino acids into pH1N1 NP enhanced viral growth in Mx-negative cells. We conclude that human MxA represents a barrier against zoonotic introduction of avian influenza viruses and that adaptive mutations in the viral NP should be carefully monitored.

Sequencing of Porcine Enterovirus Groups II and III Reveals Unique Features of Both Virus Groups

ABSTRACT The molecular classification of the porcine enterovirus (PEV) groups II and III was investigated. The sequence of the almost complete PEV-8 (group II) genome reveals that this virus has unique L and 2A gene regions. A reclassification of this group into a new picornavirus genus is suggested. PEV group III viruses are typical enteroviruses. They differ from other enteroviruses by a prolonged stem-loop D of the 5′-cloverleaf structure.

Phylogenetics, evolution, and medical importance of polyomaviruses

The increasing frequency of tissue transplantation, recent progress in the development and application of immunomodulators, and the depressingly high number of AIDS patients worldwide have placed human polyomaviruses, a group of pathogens that can become reactivated under the status of immunosuppression, suddenly in the spotlight. Since the first description of a polyomavirus a half-century ago in 1953, a multiplicity of human and animal polyomaviruses have been discovered. After reviewing the history of research into this group, with a special focus is made on the clinical importance of human polyomaviruses, we conclude by elucidating the phylogenetic relationships and thus evolutionary history of these viruses. Our phylogenetic analyses are based on all available putative polyomavirus species as well as including all subtypes, subgroups, and (sub)lineages of the human BK and JC polyomaviruses. Finally, we reveal that the hypothesis of a strict codivergence of polyomaviruses with their respective hosts does not represent a realistic assumption in light of phylogenetic findings presented here.