Marco Kaiser

High HEV presence in four different wild boar populations in East and West Germany.

Swine Hepatitis E virus (HEV) can be transmitted from pigs to humans causing hepatitis. A high prevalence of HEV in wild boar populations is reported for several European countries, but actual data for Germany are missing. During the hunting season from October to December 2007 liver, bile and blood samples were collected from wild boars in four different German regions. The samples were tested for HEV RNA by quantitative PCR (qPCR) and anti-HEV IgG antibodies by two different ELISAs and a Line immunoassay. A seroprevalence of 29.9% using ELISA and 26.2% in the Line immunoassay was determined. The seroprevalence rate varied greatly within the analyzed regions. However, qPCR analysis revealed a higher prevalence of 68.2% positive animals with regional differences. Surprisingly, also adult wild sows and wild boars were highly HEV positive by qPCR. Compared to liver and serum samples, bile samples showed a higher rate of positive qPCR results. Sequencing and phylogenetic analysis of a 969nt fragment within ORF 2 revealed that all isolates clustered within genotype 3 but differed in the subtype depending on the hunting spot. Isolates clustered within genotypes 3i, 3h, 3f and 3e. Within one population HEV isolates were closely related, but social groups of animals in close proximity might be infected with different subtypes. Two full-length genomes of subtypes 3i and 3e from two different geographic regions were generated. The wild boar is discussed as one of the main sources of human autochthonous infections in Germany.

Wild Chimpanzees Infected with 5 Plasmodium Species

Data are missing on the diversity of Plasmodium spp. infecting apes that live in their natural habitat, with limited possibility of human-mosquito-ape exchange. We surveyed Plasmodium spp. diversity in wild chimpanzees living in an undisturbed tropical rainforest habitat and found 5 species: P. malariae, P. vivax, P. ovale, P. reichenowi, and P. gaboni.

2nd International External Quality Control Assessment for the Molecular Diagnosis of Dengue Infections

Background Currently dengue viruses (DENV) pose an increasing threat to over 2.5 billion people in over 100 tropical and sub-tropical countries worldwide. International air travel is facilitating rapid global movement of DENV, increasing the risk of severe dengue epidemics by introducing different serotypes. Accurate diagnosis is critical for early initiation of preventive measures. Different reverse transcriptase PCR (RT-PCR) methods are available, which should be evaluated and standardized. Epidemiological and laboratory-based surveillance is required to monitor and guide dengue prevention and control programmes, i.e., by mosquito control or possible vaccination (as soon as an effective and safe vaccine becomes available). Objective The purpose of the external quality assurance (EQA) study described is to assess the efficiency and accuracy of dengue molecular diagnosis methods applied by expert laboratories. Study Design A panel of 12 human plasma samples was distributed and tested for DENV-specific RNA. The panel comprised 9 samples spiked with different DENV serotypes (DENV-1 to DENV-4), including 10-fold dilution series of DENV-1 and DENV-3. Two specificity controls consisted of a sample with a pool of 4 other flaviviruses and a sample with chikungunya virus. A negative control sample was also included. Results Thirty-seven laboratories (from Europe, Middle East Asia, Asia, the Americas/Caribbean, and Africa) participated in this EQA study, and reports including 46 sets of results were returned. Performance among laboratories varied according to methodologies used. Only 5 (10.9%) data sets met all criteria with optimal performance, and 4 (8.7%) with acceptable performance, while 37 (80.4%) reported results showed the need for improvement regarding accomplishment of dengue molecular diagnosis. Failures were mainly due to lack of sensitivity and the presence of false positives. Conclusions The EQA provides information on each laboratorys efficacy of RT-PCR techniques for dengue diagnosis and indicates for most laboratories an urgent need to improve sensitivity and specificity.

High prevalence of porcine Hokovirus in German wild boar populations

Porcine Hokovirus (PHoV) was recently discovered in Hong Kong. This new Parvovirus of pigs is closely related to the human Parvoviruses 4 and 5 (PARV4/5) and bovine Hokovirus (BHoV). So far, nothing is known about the presence and prevalence of PHoV in regions of the world other than Hong Kong. A study was initiated to investigate PHoV in German wild boars from five different geographical regions, using a newly established quantitative real-time PCR assay. Analysis of collected liver and serum samples revealed high overall prevalence (32.7%; 51/156) of PHoV in wild boars. The prevalence differed between the regions and increased with age. Two near full-length genomes and a large fragment for three additional isolates from different regions were sequenced and used for phylogenetic analysis. The German PHoV sequences from wild boars showed a close relationship with sequences of isolates from Hong Kong.

Development of one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses

BackgroundThe genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses.MethodsA Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively.ResultsTwo degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10–100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays.ConclusionThe assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis.

Highly sensitive detection of the group A Rotavirus using Apolipoprotein H-coated ELISA plates compared to quantitative real-time PCR

BackgroundThe principle of a capture ELISA is binding of specific capture antibodies (polyclonal or monoclonal) to the surface of a suitable 96 well plate. These immobilized antibodies are capable of specifically binding a virus present in a clinical sample. Subsequently, the captured virus is detected using a specific detection antibody. The drawback of this method is that a capture ELISA can only function for a single virus captured by the primary antibody. Human Apolipoprotein H (ApoH) or β2-glycoprotein 1 is able to poly-specifically bind viral pathogens. Replacing specific capture antibodies by ApoH should allow poly-specific capture of different viruses that subsequently could be revealed using specific detection antibodies. Thus, using a single capture ELISA format different viruses could be analysed depending on the detection antibody that is applied. In order to demonstrate that this is a valid approach we show detection of group A rotaviruses from stool samples as a proof of principle for a new method of capture ELISA that should also be applicable to other viruses.ResultsStool samples of different circulating common human and potentially zoonotic group A rotavirus strains, which were pretested in commercial EIAs and genotyped by PCR, were tested in parallel in an ApoH-ELISA set-up and by quantitative real-time PCR (qPCR). Several control samples were included in the analysis. The ApoH-ELISA was suitable for the capture of rotavirus-particles and the detection down to 1,000 infectious units (TCID50/ml). Subsets of diagnostic samples of different G- and P-types were tested positive in the ApoH-ELISA in different dilutions. Compared to the qPCR results, the analysis showed high sensitivity, specificity and low cross-reactivity for the ApoH-ELISA, which was confirmed in receiver operating characteristics (ROC) analysis.ConclusionsIn this study the development of a highly sensitive and specific capture ELISA was demonstrated by combining a poly-specific ApoH capture step with specific detection antibodies using group A rotaviruses as an example.

Diversity of parvovirus 4–like viruses in humans, chimpanzees, and monkeys in hunter–prey relationships

During 2010–2011, we investigated interspecies transmission of partetraviruses between predators (humans and chimpanzees) and their prey (colobus monkeys) in Côte d’Ivoire. Despite widespread infection in all species investigated, no interspecies transmission could be detected by PCR and genome analysis. All sequences identified formed species- or subspecies (chimpanzee)-specific clusters, which supports a co-evolution hypothesis.

Porcine Hokovirus in Domestic Pigs, Cameroon

To the Editor: Since 2005, new parvoviruses forming a novel genus of the proposed name Partetravirus, within the subfamily Parvovirinae, have been described (1). Human parvovirus 4 (PARV4) with 3 different genotypes globally infects humans (2). A related porcine virus, hokovirus (HoV or porcine partetravirus), was found in wild boar and domestic pig populations in Germany, Romania, China, and the United States, with prevalences of 12%–47%, forming 1 common genotype (3–6). Prevalence figures from sub-Saharan Africa are not available. Furthermore, no information about possibly region-associated genotypes is available for porcine HoV, although it is for human PARV4 from the same genus. We therefore used samples (collected during February–March 2012) from a study investigating hepatitis E virus (HEV) in pigs from Cameroon (7) to analyze the occurrence of porcine HoV in pigs in Africa and to determine the respective genotype.

Interactions between Hepatitis C Virus and the Human Apolipoprotein H Acute Phase Protein: A Tool for a Sensitive Detection of the Virus

The Hepatitis C virus (HCV) infection exhibits a high global prevalence frequently associated with hepatocellular carcinoma, taking years to develop. Despite the standardization of highly sensitive HCV quantitative RT-PCR (qRT-PCR) detection methods, false-negative diagnoses may be generated with current methods, mainly due to the presence of PCR inhibitors and/or low viral loads in the patient’s sample. These false-negative diagnoses impact both public health systems, in developing countries, and an in lesser extent, in developed countries, including both the risk of virus transmission during organ transplantation and/or blood transfusion and the quality of the antiviral treatment monitoring. To adopt an appropriate therapeutic strategy to improve the patient’s prognosis, it is urgent to increase the HCV detection sensitivity. Based upon previous studies on HBV, we worked on the capacity of the scavenger acute phase protein, Apolipoprotein H (ApoH) to interact with HCV. Using different approaches, including immunoassays, antibody-inhibition, oxidation, ultracentrifugation, electron microscopy and RT-PCR analyses, we demonstrated specific interactions between HCV particles and ApoH. Moreover, when using a two-step HCV detection process, including capture of HCV by ApoH-coated nanomagnetic beads and a home-made real-time HCV-RT-PCR, we confirmed the presence of HCV for all samples from a clinical collection of HCV-seropositive patients exhibiting an RT-PCR COBAS® TaqMan® HCV Test, v2.0 (COBAS)-positive result. In contrast, for HCV-seropositive patients with either low HCV-load as determined with COBAS or exhibiting HCV-negative COBAS results, the addition of the two-step ApoH-HCV-capture and HCV-detection process was able to increase the sensitivity of HCV detection or more interestingly, detect in a genotype sequence-independent manner, a high-proportion (44%) of HCV/RNA-positive among the COBAS HCV-negative patients. Thus, the immune interaction between ApoH and HCV could be used as a sample preparation tool to enrich and/or cleanse HCV patient’s samples to enhance the detection sensitivity of HCV and therefore significantly reduce the numbers of false-negative HCV diagnosis results.

Wild chimpanzees are infected with homologous types of human malaria

BackgroundDespite ongoing - and in some regions escalating -morbidity and mortality associated with malaria para-sites, evolutionary epidemiology of Plasmodium is notwell characterized. Recent studies using molecularapproaches demonstrated that wild and captive gorillasand captive bonobos and chimpanzees are infected withP. falciparum and that these apes harbor parasitesbroadly related to P. falciparum[1]. Captive chimpanzeesand bonobos had malaria parasites related to humanP. ovale and P. malariae and various monkeys and onesemi-wild chimpanzee hadP. vivax[2]. It is not clearwhether these apes harbor naturally these parasites orwhether they are transmitted from humans. Most of theexamined animals had close contact with humans, com-parable studies in wild living apes are missing. We pro-vide the first survey ofPlasmodium diversity in wildchimpanzees living in an undisturbed tropical rainforestin Africa.MethodsWe examined tissue from 16 wild West African chim-panzees that lived in the Tai National Park, Ivory Coast,where human contact with animals is limited toresearchers who access the territory only during the day.Samples were collected from animals that died primarilyfrom anthrax or respiratory disease. Generic real timePCR assay was used to detect all knownPlasmodiumspecies.Results11/16 (68%) animals tested positive. Sequence analysesof cytB and 18S rRNA genes identifiedP. malariae,P. ovale, P. vivax, P. gaboni, P. reichenowi, P. billcollinsiand P. billbraii.DiscussionPrevious examination of the role of our closest phyloge-netic relatives, the great apes, in the evolution andpersistence of human malarias has been limited by a lackof data from wild ape populations. Interpretation ofpatterns of malaria infection in captive ape populationsmust consider ample opportunities for human to apetransmission, negating the opportunity to investigate theevolutionary origins and public health-related risks ofthese parasites. Our examination of malaria parasites inwild chimpanzees demonstrates that these apes are mostlikely naturally infected with Plasmodium species homolo-gous toP. malariae, P. vivaxand P. ovale as well asP. falciparum. Whether wild great apes are the origin ofthese malaria types requires further investigation but theymay act as reservoir of infection. These results haveimportant implications for global efforts underway to era-dicate malaria in humans including vaccine developmentbased on animal variants of human parasites.