[Development of a real-time RT-PCR for detection of equine influenza virus]. / Entwicklung einer real-time RT-PCR zum Nachweis von equinem Influenzavirus.

Equine influenza is a highly contagious respiratory disease in horses caused by influenza A viruses. In this work a real-time RT-PCR for fast and sensitive diagnosis of equine influenza viruses (EIV) targeting a highly conserved region of the matrix gene was developed. In addition two RT-PCR methods for the amplification of large parts of the matrix- and HA gene were adapted for molecular-epidemiological characterization of viruses. The primers of the real-time RT-PCR had homologies of 99.4% to EIV- and 97.7% to all influenza A viral sequences, whereas the minor groove binder (MGB) probe showed homologies of 99.3% and 99.6%, respectively. These high values allow application of the assay for influenza viruses in other species. Using 20 equine, 11 porcine and 2 avian samples, diagnostic suitability of the assay was confirmed. High specificity for influenza viruses was shown both experimentally and by software simulation. The assay analytical sensitivity was at 10(2)-10(3) copies of RNA and 10(0)-10(1) copies of DNA, respectively. This allows virus detection also in circumstances of minor viral shedding. All amplified EIV sequences were classified phylogenetically within the known lineages.

Genetic heterogeneity of pestiviruses of ruminants in Switzerland.

We have genetically analyzed ruminant pestiviruses. All >150 bovine viral diarrhea (BVD) viruses isolated from cattle in Switzerland belonged to genotype 1, with subgenogroups e, h, k and b found in decreasing frequency. To date, representatives of subgenogroup k have been detected in Switzerland only. Despite serological evidence of Border disease in sheep, only few Border disease viruses have been isolated, all of which belong to the novel group 3. Serological evidence suggested that pestivirus infections may occur also in wild ruminants in Switzerland but no isolates are available for analysis. In addition, we describe two pestiviruses, one a cell culture contaminant and the other isolated from a buffalo, that cluster with a recently proposed novel pestivirus species.

[Border disease in a flock of sheep]. / Border Disease in einem Schafbetrieb.

This report describes border disease in a flock of sheep in Switzerland. In April 2001, three ewes in a flock of 41 sheep gave birth to lambs that had generalized tremors and excessively hairy fleece. One of these, a three-week-old female lamb, was referred to our clinic for further diagnostic work-up. The lamb was very nervous, bleated constantly and had generalized muscle tremors, which were more pronounced in the head region. Hind end ataxia was observed, and the lamb was slow to correct its posture when the hind limbs were abducted, adducted or crossed. Blood samples were collected every six weeks to determine antibody titres to pestivirus and for virus isolation via cell culture. A skin biopsy sample was also collected and examined immunohistochemically for pestivirus antigen. Antibody titres in the first tests were suspicious and those of the second were negative. Pestivirus was identified in cell culture, and the skin biopsy sample was positive for pestivirus antigen. Blood samples were collected from all of the ewes and lambs and the buck for virus isolation via cell culture and determination of pestivirus antibody titres. Thirty-one animals were seropositive, six had borderline antibody titres and four were seronegative. Pestivirus was isolated from eight animals, which included the lamb described in this report. Of the virus-positive animals, three were seronegative, three others had borderline titres and two were seropositive. Six of the eight viruses isolated from cell culture were further characterized genetically via retrotranscription and polymerase chain reaction and subsequent sequencing. The phylogenetic analysis revealed that the causative agent was border disease virus. This is the first time that border disease virus has been isolated in Switzerland. The lamb referred to our clinic was observed for three months; it was then euthanatised and a postmortem examination was performed. Immunohistochemical examination of numerous organs revealed pestivirus antigen. The source of infection was though to be infected sheep from another flock, which shared a pasture. All antigen-positive animals were slaughtered.

Sheep persistently infected with Border disease readily transmit virus to calves seronegative to BVD virus.

Bovine viral diarrhea- and Border disease viruses of sheep belong to the highly diverse genus pestivirus of the Flaviviridae. Ruminant pestiviruses may infect a wide range of domestic and wild cloven-hooved mammals (artiodactyla). Due to its economic importance, programs to eradicate bovine viral diarrhea are a high priority in the cattle industry. By contrast, Border disease is not a target of eradication, although the Border disease virus is known to be capable of also infecting cattle. In this work, we compared single dose experimental inoculation of calves with Border disease virus with co-mingling of calves with sheep persistently infected with this virus. As indicated by seroconversion, infection was achieved only in one out of seven calves with a dose of Border disease virus that was previously shown to be successful in calves inoculated with BVD virus. By contrast, all calves kept together with persistently infected sheep readily became infected with Border disease virus. The ease of viral transmission from sheep to cattle and the antigenic similarity of bovine and ovine pestiviruses may become a problem for demonstrating freedom of BVD by serology in the cattle population.

Mammalian DNA helicase.

A forked DNA was constructed to serve as a substrate for DNA helicases. It contains features closely resembling a natural replication fork. The DNA was prepared in large amounts and was used to assay displacement activity during isolation from calf thymus DNA polymerases alpha holoenzyme. One form of DNA polymerase alpha holoenzyme is possibly involved leading strand replication at the replication fork and possesses DNA dependent ATPase activity (Ottiger, H.-P. and Hübscher, U. (1984) Proc. Natl. Acad. Sci. USA 81, 3993-3997). The enzyme can be separated from DNA polymerase alpha by velocity sedimentation in conditions of very low ionic strength and then be purified by chromatography on Sephacryl S-200 and ATP-agarose. At all stages of purification, DNA dependent ATPase and displacement activity profiles were virtually superimposable. The DNA dependent ATPase can displace a hybridized DNA fragment with a short single-stranded tail at its 3'hydroxyl end only in the presence of ATP, and this displacement relies on ATP hydrolysis. Furthermore, homogeneous single-stranded binding proteins from calf thymus as well as from other tissues cannot perform this displacement reaction. By all this token the DNA dependent ATPase appears to be a DNA helicase. It is suggested that this DNA helicase might act in concert with DNA polymerase alpha at the leading strand, possibly pushing the replication fork ahead of the polymerase.