Andi Krumbholz

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.

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.

High prevalence of amantadine resistance among circulating European porcine influenza A viruses

Genetic analysis of the M2 sequence of European porcine influenza A viruses reveals a high prevalence of amantadine resistance due to the substitution of serine 31 by asparagine in all three circulating subtypes, H1N1, H3N2 and H1N2. The M segment of all resistant strains belongs to a single genetic lineage. Whereas the first amantadine-resistant porcine strain was isolated in 1989, isolation of the last amantadine-susceptible strain dates to 1987, suggesting a displacement of amantadine-susceptible viruses by resistant strains soon after emergence of the mutation. Analysis of natural selection by codon-based tests indicates negative selection of codons 30, 31 and 34 which confer amantadine resistance. The codons 2, 11-28 and 54 of porcine and human strains exhibit differences in the patterns of substitution rates, suggesting different selection modes. Transfer of amantadine resistance by exchange of the M segment and viability of recombinant A/WSN/33 viruses with avian-like M segments raises concerns about the emergence of natural human reassortants.

Amantadine resistance among porcine H1N1, H1N2, and H3N2 influenza A viruses isolated in Germany between 1981 and 2001

This study was designed to gain insight into amantadine susceptibility of porcine influenza A viruses isolated in Germany between 1981 and 2001. The 12 studied H1N1, H1N2, and H3N2 porcine influenza virus strains were isolated in chicken eggs and passaged once in MDCK cells. Plaque reduction assays were applied to examine virus susceptibility to amantadine. Genotyping was used to confirm drug resistance. In the results of these antiviral studies, only 3 of the 12 isolates were shown to be amantadine-susceptible. All resistant strains contained the AA substitutions G16E, S31N, and R77Q in the membrane protein 2 (M2). Additionally, L27A was detected in two H1N1 strains. S31N and/or L27A are well-known amino acid substitutions in M2 that confer amantadine resistance. The role of the pig as an intermediate host of avian and human influenza A viruses, the possible involvement of genetic reassortment, and the high incidence of naturally amantadine-resistant porcine influenza A viruses suggest a real risk of emergence of amantadine resistant human viruses. Therefore, drug susceptibility monitoring appears to be warranted for effective application of those drugs.

Evolution of four BK virus subtypes

BK viruses (BKV) comprise four subtypes that are distinguishable by serological and molecular methods with the latter indicating up to four subgroups within subtype I. In this study, the phylogeny of all BKV subtypes was analyzed. Phylogenetic analyses of the viral structural protein VP1, concatenated sequences including the T-Ag, t-Ag, VP1 and VP2 genes and the entire coding region of BKV, each employing several tree inference methods, consistently revealed seven strongly supported clades that correlate with BKV subtypes and subgroups. In general, subtype II and III viruses were found to be sister groups, the genetic distances between which were significantly lower than those between either and the other BKV subtypes. Two benchmarks of human evolution (emergence of modern humans, 200,000 years ago; out-of-Africa migration, 100,000 years ago) were assumed for the internal calibration of BKV evolution. Utilization of either calibration point resulted in the diversification of most BKV subgroups coinciding with human radiation less than 50,000 years ago. Another approach, external calibration by linking BKV divergence to the evolution of mammals, was rejected as it advances BKV divergence prior to the emergence of modern humans.

Reassortants of the pandemic (H1N1) 2009 virus and establishment of a novel porcine H1N2 influenza virus, lineage in Germany

The incursion of pandemic (H1N1) 2009 virus (pdmH1N1) into the German pig population was investigated in a serosurvey and by virological means between June 2009 and December 2012. Analysis of 23,116 pig sera from a total of 2,666 herds revealed 224 herds that reacted with pdmH1N1 but not with the prevalent avian-like H1N1 swine influenza virus. Sixty-six pdmH1N1 strains and their reassortant derivatives (pdmH1huN2, huH3pdmN1) have been collected since November 2009. Sequencing of three pdmH1N1, 20 pdmH1huN2 and one huH3pdmN1 strains with conventional and next generation sequencing techniques and subsequent phylogenetic analyses with available sequence data revealed the emergence of five distinct reassortant genotypes in Europe. The most frequent genotype emerged at least three times independently, one of which (Papenburg lineage) established a stable infection chain and became more prevalent in pigs than pdmH1N1 in Germany.

Impact of global warming on viral diseases: what is the evidence?

Global warming is believed to induce a gradual climate change. Hence, it was predicted that tropical insects might expand their habitats thereby transmitting pathogens to humans. Although this concept is a conclusive presumption, clear evidence is still lacking—at least for viral diseases. Epidemiological data indicate that seasonality of many diseases is further influenced by strong single weather events, interannual climate phenomena, and anthropogenic factors. So far, emergence of new diseases was unlinked to global warming. Re-emergence and dispersion of diseases was correlated with translocation of pathogen-infected vectors or hosts. Coupled ocean/atmosphere circulations and ‘global change’ that also includes shifting of demographic, social, and economical conditions are important drivers of viral disease variability whereas global warming at best contributes.

Novel neuraminidase inhibitors: identification, biological evaluation and investigations of the binding mode

BACKGROUND The pathogenicity of influenza A and B viruses depends on the function of influenza neuraminidase (NA). Emerging resistant influenza A viruses of subtype H1N1 increasingly challenge the effectiveness of established NA inhibitors. Recent computational studies have indicated several weak points of NA that can be exploited for rational inhibitor design to conquer this imminent threat, such as the opening of the binding pocket due to the flexibility of the 150-, 245- and 430-loops. METHODS We employed shape-focused virtual screening based on a recently discovered lead compound, katsumadain A, to identify novel promising compounds with significant inhibitory efficacy on NA and resistance-breaking capacity on oseltamivir-resistant strains. A potential binding mode of these compounds was derived employing ligand-based techniques and protein-ligand docking using representative protein conformations selected from molecular dynamics simulations. RESULTS Five novel compounds were identified by virtual screening. Their IC(50) values, determined in chemiluminescence-based NA inhibition assays, are in the range of 0.18-17 µM. In particular, artocarpin exhibits high affinity toward three H1N1 oseltamivir-sensitive influenza A viruses. It also inhibits the NA of an oseltamivir-resistant H1N1 isolate.