Thomas Bruun Rasmussen

Molecular epidemiology of current classical swine fever virus isolates of wild boar in Germany.

Classical swine fever (CSF) has caused significant economic losses in industrialized pig production, and is still present in some European countries. Recent CSF outbreaks in Europe were mainly associated with strains of genogroup 2 (subgroup 2.3). Although there are extensive datasets regarding 2.3 strains, there is very little information available on longer fragments or whole classical swine fever virus (CSFV) genomes. Furthermore, there are no detailed analyses of the molecular epidemiology of CSFV wild boar isolates available. Nevertheless, complete genome sequences are supportive in phylogenetic analyses, especially in affected wild boar populations. Here, German CSFV strains of subgroup 2.3 were fully sequenced using two different approaches: (i) a universal panel of CSFV primers that were developed to amplify the complete genome in overlapping fragments for chain-terminator sequencing; and (ii) generation of a single full-length amplicon of the CSFV genome obtained by long-range RT-PCR for deep sequencing with next-generation sequencing technology. In total, five different strains of CSFV subgroup 2.3 were completely sequenced using these newly developed protocols. The approach was used to study virus spread and evolutionary history in German wild boar. For the first time, the results of our study clearly argue for the possibility of a long-term persistence of genotype 2.3 CSFV strains in affected regions at an almost undetectable level, even after long-term oral vaccination campaigns with intensive monitoring. Hence, regional persistence in wild boar populations has to be taken into account as an important factor in the continual outbreaks in affected areas.

Design and performance evaluation of 1-by-64 multimode interference power splitter for optical communications

A 1-by-64 multimode interference power splitter in SiO/sub 2/ has been designed for use in fiber-optics communication systems. The splitter exhibits a minimum loss of 0.5 db and a uniformity of 1.7 dB at a wavelength of 1.55 /spl mu/m. The polarization sensitivity is below 0.14 dB, the reflection level below -55 dB, and the optical bandwidth 30 nm. The fabrication tolerances are /spl plusmn/0.1 mm on the length and /spl plusmn/3.5 /spl mu/m on the width of the multimode section of the splitter. In comparison with a branching-type splitter it is found that the designed device is approximately 30% shorter than the branching-type device for comparable losses. >

Strategies for differentiating infection in vaccinated animals (DIVA) for foot-and-mouth disease, classical swine fever and avian influenza

The prophylactic use of vaccines against exotic viral infections in production animals is undertaken exclusively in regions where the disease concerned is endemic. In such areas, the infection pressure is very high and so, to assure optimal protection, the most efficient vaccines are used. However, in areas considered to be free from these diseases and in which there is the possibility of only limited outbreaks, the use of Differentiation of Infected from Vaccinated Animals (DIVA) or marker vaccines allows for vaccination while still retaining the possibility of serological surveillance for the presence of infection. This literature review describes the current knowledge on the use of DIVA diagnostic strategies for three important transboundary animal diseases: foot-and-mouth disease in cloven-hoofed animals, classical swine fever in pigs and avian influenza in poultry.

Characterisation of a pestivirus isolated from persistently infected mousedeer (Tragulus javanicus)

Summary. Serum samples from the male Mousedeer A and the mother, father and sister of A were tested for bovine virus diarrhoea viruses (BVDV) by isolation, and for BVDV antibodies by blocking ELISA and homologous neutralisation test. Further, RNA was extracted and tested by RT-PCR protocol analysing the 5′-untranslated region and the E2 gene of pestivirus. The RT-PCR products were subsequently sequenced. Mousedeer A was positive in virus isolation on three occasions (days 1, 19 and 40) and by RT-PCR. The sister and mother of Mousedeer A were also found virus positive by isolation and RT-PCR. Mousedeer A, its sister and its mother, all had an antibody neutralisation titer below 10. The father of A was virus negative but was positive in the blocking antibody ELISA and had a high neutralisation antibody titer. The repeated detection of BVDV in Mousedeer A, the high amount of virus in serum, the lack of antibodies and the virus positive family members documented that the mousedeer were persistently infected with a pestivirus. The father of A probably had an acute infection resulting in antibodies to pestivirus and viral clearance. Sequence analysis and phylogenetic analysis revealed that the mousedeer pestivirus was closely related to BVDV Type 1f. The existences of persistently infected animals in non-domestic species have great implications for BVDV eradication campaigns in cattle.

Modeling of Yb/sup 3+/-sensitized Er/sup 3+/-doped silica waveguide amplifiers

A model for Yb/sup 3+/-sensitized Er/sup 3+/-doped silica waveguide amplifiers is described and numerically investigated in the small-signal regime. The amplified spontaneous emission in the ytterbium-band and the quenching process between excited erbium ions are included in the model. For pump wavelengths between 860 and 995 nm, the amplified spontaneous emission in the ytterbium-band is found to reduce both the gain and the optimum length of the amplifier significantly. The achievable gain of the Yb/sup 3+/-sensitized amplifier is found to be higher than in an Er/sup 3+/-doped silica waveguide without Yb/sup 3+/ (18 dB versus 9 dB for a pump power of 100 mW). However, it is important to optimize the Yb-concentration according to the choice of pump wavelength. >

Generation of recombinant pestiviruses using a full-genome amplification strategy

Complete genome amplification of viral RNA provides a new tool for the generation of modified viruses. We have recently reported a full-genome amplification strategy for recovery of pestiviruses (Rasmussen et al., 2008). A full-length cDNA amplicon corresponding to the Border disease virus-Gifhorn genome was generated by long RT-PCR and then RNA transcripts derived from this amplicon were used to rescue infectious virus. Here, we have now used this full-genome amplification strategy for efficient and robust amplification of three additional pestivirus strains: the vaccine strain C and the virulent Paderborn strain of Classical swine fever virus plus the CP7 strain of Bovine viral diarrhoea virus. The amplicons were cloned directly into a stable single-copy bacterial artificial chromosome generating full-length pestivirus DNAs from which infectious RNA transcripts could be also derived.

Improved Safety for Molecular Diagnosis of Classical Rabies Viruses by Use of a TaqMan Real-Time Reverse Transcription-PCR “Double Check” Strategy

ABSTRACT To improve the diagnosis of classical rabies virus with molecular methods, a validated, ready-to-use, real-time reverse transcription-PCR (RT-PCR) assay was developed. In a first step, primers and 6-carboxyfluorescien-labeled TaqMan probes specific for rabies virus were selected from the consensus sequence of the nucleoprotein gene of 203 different rabies virus sequences derived from GenBank. The selected primer-probe combination was highly specific and sensitive. During validation using a sample set of rabies virus strains from the virus archives of the Friedrich-Loeffler-Institut (FLI; Germany), the Veterinary Laboratories Agency (VLA; United Kingdom), and the DTU National Veterinary Institute (Lindholm, Denmark), covering the global diversity of rabies virus lineages, it was shown that both the newly developed assay and a previously described one had some detection failures. This was overcome by a combined assay that detected all samples as positive. In addition, the introduction of labeled positive controls (LPC) increased the diagnostic safety of the single as well as the combined assay. Based on the newly developed, alternative assay for the detection of rabies virus and the application of LPCs, an improved diagnostic sensitivity and reliability can be ascertained for postmortem and intra vitam real-time RT-PCR analyses in rabies reference laboratories.

Vertical transmission of bovine viral diarrhoea virus (BVDV) in mousedeer (Tragulus javanicus) and spread to domestic cattle

Summary.This study investigates the transmission of bovine viral diarrhoea virus (BVDV) 1f from a persistently infected (PI) lesser Malayan mousedeer to two bovine calves. Different contact routes to two calves were analysed: 1) aerosol contact between two adjacent pens without physical contact; 2) indirect contact by use of common utensils; 3) direct nose-to-nose contact for 30 seconds. One of the calves was infected either by aerosol or indirect contact. The virus sequence in 247 nucleotides in the 5′-UTR was 100% identical in mousedeer and calf.To elucidate the distribution of BVDV within the affected mousedeer family the captive population in a Zoo was analysed. The maternal line of PI animals was maintained, whereas a PI male was able to reproduce and have a non-PI calf. As a consequence of this, six female PI mousedeer were killed; subsequent autopsies did not reveal any lesions. Sequencing mousedeer BVD virus in the E2 region (420 nucleotides) through 4 generations showed only 7 mutations, which were maintained from mother to offspring.

Quantitative Multiplex Assay for Simultaneous Detection and Identification of Indiana and New Jersey Serotypes of Vesicular Stomatitis Virus

ABSTRACT In order to establish a rapid and reliable system for the detection of vesicular stomatitis virus (VSV), we developed a quantitative reverse transcription-PCR assay for the detection, quantification, and differentiation of the major serotypes, VSV Indiana and VSV New Jersey, using a closed-tube multiplex format. The detection system is based on the recently invented primer-probe energy transfer (PriProET) system. A region of the gene encoding the RNA-dependent RNA polymerase was amplified by using VSV-specific primers in the presence of two serotype-specific fluorescent probes. By incorporating nucleotide analogues in the primers, both serotypes were amplified with similar efficiencies. The generation of specific amplicons resulted in fluorescent signals for either of the two serotypes, and the specificities of the reactions were confirmed from the melting temperature profiles of the fluorescent probes. The limits of detection were found to be less than 10 50% tissue culture infective doses/ml for both serotypes. The diagnostic value of the new method was tested with clinical materials from experimentally infected pigs, and it is concluded that the method is a powerful tool for the rapid identification of VSV.

Development of a real-time RT-PCR assay based on primer–probe energy transfer for the detection of all serotypes of bluetongue virus

A real-time RT-PCR assay based on the primer-probe energy transfer (PriProET) was developed to detect all 24 serotypes of bluetongue virus (BTV). BTV causes serious disease, primarily in sheep, but in other ruminants as well. A distinguishing characteristic of the assay is its tolerance toward mutations in the probe region. Furthermore, melting curve analysis following immediately PCR confirms specific probe hybridization and can reveal mutations in the probe region by showing a difference in the melting point. The assay sensitivity was in the range of 10-100 target copies and the specificity tests showed no positive results for heterologous pathogens. The assay was tested on clinical samples from BTV 8 outbreaks in Sweden and Denmark in 2008. The lowest detection limit for that serotype, determined with PCR standards, was 57 genome copies. The assay sensitivity for some other serotypes that circulate currently in Europe was also determined. BTV 2, 4, 9 and 16 were tested on available cell culture samples and the detection limits were 109, 12, 13 and 24 copies, respectively. This assay provides an important tool for early and rapid detection of a wide range of BTV strains, including emerging strains.