Helga Lussy

Emergence of Usutu virus, an African Mosquito-Borne Flavivirus of the Japanese Encephalitis Virus Group, Central Europe

During late summer 2001 in Austria, a series of deaths in several species of birds occurred, similar to the beginning of the West Nile virus (WNV) epidemic in the United States. We necropsied the dead birds and examined them by various methods; pathologic and immunohistologic investigations suggested a WNV infection. Subsequently, the virus was isolated, identified, partially sequenced, and subjected to phylogenetic analysis. The isolates exhibited 97% identity to Usutu virus (USUV), a mosquito-borne Flavivirus of the Japanese encephalitis virus group; USUV has never previously been observed outside Africa nor associated with fatal disease in animals or humans. If established in central Europe, this virus may have considerable effects on avian populations; whether USUV has the potential to cause severe human disease is unknown.

Emergence of Usutu Virus in Hungary

ABSTRACT In 2001, Usutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus serogroup related to West Nile virus and previously restricted to sub-Saharan Africa, emerged in wild and zoo birds in and around Vienna, Austria. In order to monitor the spread of the infection, a dead bird surveillance program was established in Austria and in neighboring Hungary. In Hungary, 332 dead birds belonging to 52 species were tested for USUV infection between 2003 and 2006. In the first 2 years, all birds investigated were negative. In August 2005, however, USUV was detected in organ samples of a blackbird (Turdus merula), which was found dead in Budapest, Hungary, by reverse transcription-PCR, immunohistochemistry, and in situ hybridization. In July and August 2006, a further six dead blackbirds tested positive for USUV, and the virus was isolated from organ samples of one bird. These birds were also found in urban areas of Budapest. The nearly complete genomic sequence of one Hungarian USUV strain was determined; it was found to share 99.9% identity with the strain that has been circulating in Austria since 2001. This result indicates that the USUV strain responsible for the blackbird die-off in Budapest most likely spread from Austria to Hungary instead of being independently introduced from Africa.

Tick-borne Encephalitis Virus in Horses, Austria, 2011

An unexpectedly high infection rate (26.1%) of tick-borne encephalitis virus (TBEV) was identified in a herd of 257 horses of the same breed distributed among 3 federal states in Austria. Young age (p<0.001) and male sex (p = 0.001) were positively associated with infection.

In Vitro Host-Cell Susceptibility to Usutu Virus

We investigated the susceptibility to Usutu virus (Flavivirus) of 13 permanent cell lines, 3 primary cell cultures, and chicken embryos. Vero, PK-15, and goose embryo fibroblast cells developed cytopathic effects; however, viral multiplication was detected in all mammalian cell types by immunohistochemical tests. Chicken embryo fibroblast cells and chicken embryos were resistant.

Low Usutu virus seroprevalence in four zoological gardens in central Europe

BackgroundUsutu virus (USUV), a mosquito-borne flavivirus of the Japanese encephalitis virus antigenic group, caused bird die-offs in Austria, Hungary and Switzerland between 2001 and 2009. While the zoological gardens of Vienna and Zurich recorded USUV-associated mortality in different species of birds during this period, incidences in Budapest were limited to areas outside the zoo, and in the greater Basel area avian mortality due to USUV infection was not observed at all. The objectives of this investigation were to gain insight into USUV infection dynamics in captive birds in zoos with varying degrees of virus exposure and to study differences in susceptibility to USUV of different species of birds.Results372 bird sera were collected between October 2006 and August 2007. The samples were tested in parallel by hemagglutination inhibition (HI) and 90% plaque reduction neutralization tests (PRNT-90). 8.75%, 5.3% and 6.59% of birds in the zoos of Vienna, Zurich and Basel, respectively, showed USUV-specific antibodies by PRNT-90. No antibodies to USUV were detected in birds of the Budapest zoo. The order Strigiformes (owls) exhibited the highest USUV-seroprevalence, compared to other orders of birds.ConclusionsUSUV seems not to pose an imminent threat to zoo bird populations in central Europe at the moment. Depending on a variety of especially environmental factors, however, this may change at any time in the (near) future, as experienced with West Nile virus (WNV). It is therefore strongly suggested to continue with combined WNV and USUV surveillance activities in affected areas.

Identification of Mixed Infections with Different Genotypes of Avian Bornaviruses in Psittacine Birds with Proventricular Dilatation Disease

SUMMARY. Proventricular dilatation disease (PDD) is a fatal, progressive neurological disorder of psittacine birds, which is caused by a single-stranded RNA virus, the avian bornavirus (ABV). The disease pattern includes lymphoplasmacytic inflammation of the central, peripheral and autonomic nervous system. Seven avian bornavirus genotypes have been identified during the last years. So far only monoinfections with a single genotype of ABV have been attributed to PDD cases. However, after a recent survey discovered a case of a double infection with two different ABV genotypes, this seemed to indicate the need for a more systematic search for mixed infections. Brain specimens from 21 psittacine birds affected with PDD were examined. Aim of the investigation was to generate partial ABV sequences of a part of the matrix protein (M) gene and to evaluate whether sequences of more than one ABV genotype were present. RNA was extracted, and subjected to reverse transcriptase PCR with primer pairs generating a partial sequence of the matrix protein (M) gene, followed by a cloning procedure. Ten clones per case were sequenced in order to elucidate whether sequences characteristic for one or more than one genotype were present. In 19 of 21 cases clear M gene sequences could be generated; in two cases nucleic acid amplification failed. Seven birds were infected with ABV 2 and nine with ABV 4, representing the predominant genotypes in Europe. Two cases showed a mixed infection with ABV 2 and ABV 4, and one case a mixed infection with ABV 2 and ABV 6. These results suggest that the molecular cloning method is a useful tool for distinguishing between single and multiple infection events by different ABV genotypes. RESUMEN. Nota de Investigación—Identificación de infecciones mixtas con diferentes genotipos de bornavirus aviares en aves psitácidas con la enfermedad de dilatación proventricular. La enfermedad de dilatación del proventrículo es un trastorno fatal, neurológico progresivo de las aves psitácidas, que es causada por un virus ARN de cadena simple, el bornavirus aviar. El patrón de la enfermedad incluye la inflamación linfoplasmocitaria del sistema nervioso autónomo central y periférico. Siete genotipos de bornavirus aviar se han identificado en los últimos años. Hasta el momento sólo las infecciones con un solo genotipo del bornavirus aviar se han observado en los casos de enfermedad de dilatación del proventrículo. Sin embargo, después de un estudio reciente se observó un caso de una infección doble con dos genotipos de bornavirus diferentes, por lo tanto parece ser necesaria una búsqueda más sistemática para detectar infecciones mixtas. Se examinaron las muestras cerebrales de 21 psitácidos afectados con dilatación del proventrículo. El objetivo de la investigación fue generar secuencias parciales del gene de la proteína matriz (M) de bornavirus aviares y para determinar si las secuencias de más de un genotipo de bornavirus estaban presentes. Se extrajo el ARN y se sometió a una transcripción reversa y PCR (RT-PCR) con pares de iniciadores que incluían parcialmente la secuencia del gene M, seguido por un procedimiento de clonación. Se secuenciaron diez clones por caso con el fin de dilucidar si las secuencias características para uno o más genotipos estaban presentes. En 19 de los 21 casos se generaron secuencias claras del gene M, en dos casos falló la amplificación de ácidos nucleicos. Siete aves fueron infectadas con el bornavirus número dos y nueve con el bornavirus 4, que representan a los genotipos predominantes en Europa. En dos casos se observó una infección mixta con bornavirus aviar 2 y 4, y un caso tenía una infección mixta con bornavirus 2 y 6. Estos resultados sugieren que el método de clonación molecular es una herramienta útil para distinguir entre eventos de infección simples o múltiples por los genotipos de bornavirus aviar.