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Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus group has been responsible for avian mortality in Austria since 2001. In the present study, the neuropathogenicity and neuroinvasiveness of USUV for 1-week-old suckling mice was investigated. After intraperitoneal inoculation, clinical signs like depression, disorientation, paraplegia, paralysis and coma were observed between 6 and 11 days post infection. Histologically, there was widespread neuronal apoptosis especially in the brain stem. Inflammatory infiltrates were scarce. Apoptosis was also present in white matter of cerebellum, medulla and spinal cord, and was frequently accompanied by primary demyelination. While apoptosis of neurons was clearly associated with presence of viral signals, the cause of apoptosis of white matter cells was more ambiguous. However, focal immunostaining was found in the white matter, especially in the spinal cord. As with all flaviviruses, USUV proved to be neuropathogenic for mice. In contrast to other flaviviruses, neuroinvasion occurred only in animals that were not older than 1 week at the time of inoculation. While neuronal apoptosis is a general aspect of flavivirus pathogenicity, demyelination seems to be a unique feature of USUV infection.

Here we describe the complete genome sequences of two strains of Usutu virus (USUV), a mosquito-borne member of the genus Flavivirus in the Japanese encephalitis virus (JEV) serogroup. USUV was detected in Austria in 2001 causing a high mortality rate in blackbirds; the reference strain (SAAR-1776) was isolated in 1958 from mosquitoes in South Africa and has never been associated with avian mortality. The Austrian and South African isolates exhibited 97% nucleotide and 99% amino acid identity. Phylogenetic trees were constructed displaying the genetic relationships of USUV with other members of the genus Flavivirus. When comparing USUV with other JEV serogroup viruses, the closest lineage was Murray Valley encephalitis virus (nt: 73%, aa: 82%) followed by JEV (nt: 71%, aa: 81%) and West Nile virus (nt: 68%, aa: 75%). Comparison of the genomes showed that the conserved structural elements and putative enzyme motifs were homologous in the two USUV strains and the JEV serogroup. The factors that determine the severe clinical symptoms caused by the Austrian USUV strain in Eurasian blackbirds are discussed. We also offer a possible explanation for the origins and dispersal of USUV, JEV, and MVEV out of Africa.

Equine arteritis virus (EAV) is an enveloped plus-strand RNA virus of the family Arteriviridae (order Nidovirales) that causes respiratory and reproductive disease in equids. Protective, virus-neutralizing antibodies (VNAb) elicited by infection are directed predominantly against an immunodominant region in the membrane-proximal domain of the viral envelope glycoprotein G(L), allowing recently the establishment of a sensitive peptide enzyme-linked immunosorbent assay (ELISA) based on this particular domain (J. Nugent et al., J. Virol. Methods 90:167-183, 2000). By using an infectious cDNA we have now generated, in the controlled background of a nonvirulent virus, a mutant EAV from which this immunodominant domain was deleted. This virus, EAV-G(L)Delta, replicated to normal titers in culture cells, although at a slower rate than wild-type EAV, and caused an asymptomatic infection in ponies. The antibodies induced neutralized the mutant virus efficiently in vitro but reacted poorly to wild-type EAV strains. Nevertheless, when inoculated subsequently with virulent EAV, the immunized animals, in contrast to nonvaccinated controls, were fully protected against disease; replication of the challenge virus occurred briefly at low though detectable levels. The levels of protection achieved suggest that an immune effector mechanism other than VNAb plays an important role in protection against infection. As expected, infection with EAV-G(L)Delta did not induce a measurable response in our G(L)-peptide ELISA while the challenge infection of the animals clearly did. EAV-G(L)Delta or similar mutants are therefore attractive marker vaccine candidates, enabling serological discrimination between vaccinated and wild-type virus-infected animals.

The fibre gene of the bovine adenovirus type 2 (BAdV2) subtype B was prepared for sequencing by using cloning, sub-cloning and PCR amplification techniques. The nucleotide sequence of the total fibre gene was determined, and it was found to consist of 1,647 nucleotides, coding for a polypeptide of 549 amino acids. The fibre gene regions of BAdV2 A and B subtypes were aligned. The nucleotide identity of the total fibre gene was found to be 60.5%; however, the homology showed great differences in the different subregions coding for the shaft and knob part of the fibre, and the two subtypes were almost identical in the tail subregion. Remarkable changes indicating deletion, insertion and point mutations were found in the shaft subregion when BAdV2/A and B subtypes were compared. We concluded that the differences found in the haemagglutinating activity of the two subtypes of BAdV2 can mostly be explained by the changes in the polypeptide structure of the fibre shaft.

PCR assays were developed for the direct detection of Paenibacillus larvae in honey samples and compared with isolation and biochemical characterization procedures. Different primer pairs, designed from the 16S rRNA and the metalloproteinase precursor gene regions, and different DNA extraction methods were tested and compared. The sensitivity of the reactions was evaluated by serial dilutions of DNA extracts obtained from P. larvae cultures. The specificity of the primers was assessed by analyzing related Paenibacillus and Bacillus strains isolated from honey. The PCR assays also amplified these related bacteria, but at lower sensitivity. In the next step, the PCR assays were applied to contaminated honey and other bee products originating from 15 countries. Lysozyme treatment followed by proteinase K digestion was determined to be the best DNA extraction method for P. larvae spores. The most sensitive primer pair detected P. larvae in 18 of 23 contaminated honey samples, as well as in pollen, wax, and brood. Honey specimens containing saprophyte bacilli and paenibacilli, but not P. larvae, were PCR negative. Although the isolation and biochemical identification method (BioLog) showed higher sensitivity and specificity, PCR proved to be a valuable technique for large-scale screening of honey samples for American foulbrood, especially considering its rapidity and moderate costs.

Reverse transcription-PCR assays have been established for a quick, sensitive, and specific diagnosis of acute bee paralysis virus (ABPV), a common virus of the honeybee (Apis mellifera), directly from clinical samples. A 3,071-nucleotide fragment of the ABPV genome, which includes the entire capsid polyprotein gene, was amplified from Austrian, German, Polish, and Hungarian ABPV samples and sequenced, and the sequences were compared. The alignment of a smaller fragment with ABPV sequences from the United States and the United Kingdom revealed nucleotide identity rates between 89 and 96%, respectively. Phylogenetic trees which display the molecular relationship between the viruses of different geographic origin were constructed.