Borna disease in an adult free-ranging Eurasian beaver (Castor fiber albicus).

Borna disease (BD) associated with a peracute bacterial septicaemia with Escherichia coli was diagnosed in an adult female, naturally infected, free-ranging Eurasian beaver of the subspecies Castor fiber albicus, clinically characterized by weight loss, depression, weakness and gurgled peristaltic sounds. The beaver was euthanized humanely. Necropsy and light microscopy revealed a non-purulent meningoencephalitis with typical mononuclear perivascular cuffs and parenchymal infiltrates. The diagnosis of BD was confirmed by detection of viral antigen and RNA by immunohistochemistry and reverse transcription-polymerase chain reaction (RT-PCR). The PCR product was sequenced and cluster analysis revealed a close relationship between endemic clusters in Saxony-Anhalt. This is the first report of naturally occurring BD in a free-ranging Eurasian beaver.

An Explorative Study of the Causal Pathogenesis of Green Liver Discoloration in Organically Reared Female Bronze Turkeys (Meleagris gallopavo) Considering the Infectious Risk Factors.

A recent study revealed that organically raised Bronze turkeys showed a high prevalence of green liver discoloration. This alteration is commonly associated with the Turkey Osteomyelitis Complex and potentially caused by opportunistic bacteria. Therefore, 360 organically fattened Bronze turkeys were examined post-mortem throughout two fattening trials with two examinations each to determine possible infectious risk factors and reduce disease prevalence. Clinical and pathoanatomical examinations were performed on every hen. Histopathological, bacteriological, parasitological, and virological examinations were performed on at least six hens without and, if applicable, six hens with green livers on each examination date. Overall, 9.0% of all hens had a green liver without a correlation with bacterial or parasitological findings but multiple health impairments. The discoloration correlated significantly with the detection of immunosuppressive turkey hemorrhagic enteritis virus at the early stage and macro- and histological joint/bone lesions at the late fattening stage, indicating the presence of two different predisposing pathogeneses. Flocks not being vaccinated against hemorrhagic enteritis but having a virus-positive sample showed the highest prevalence of green liver discoloration and developed worse in various parameters. In conclusion, an adequate vaccination schedule and the prevention of field infections may lead to a decreased risk of performance reduction and improved animal health.

Case Series of Disseminated Xanthogranulomatosis in Red-crowned Parakeets (Cyanoramphus novaezelandiae) with Detection of Psittacine Adenovirus 2 (PsAdV-2).

Xanthogranulomatosis is a common dermatological disease in birds. This form of inflammation, possibly associated with lipometabolic disorders, can also be seen in visceral organs, which as yet has only rarely been described in avian medicine. In general, diseases related to impaired lipid metabolism are frequently reported in avian medicine, with hepatic steatosis and atherosclerosis being the most common. In human medicine, infectious agents-especially some strains of adenovirus-were implicated in contributing to lipometabolic disorders; this has also been described for chicken. Here, a case series of six Red-crowned Parakeets (Cyanoramphus novaezelandiae) is presented, all cases being characterized by psittacine adenovirus 2 (PsAdV-2) infection with or without disseminated xanthogranulomatosis. The affected individuals were examined alive by clinical examination. Total body radiographs were taken of two birds, haematology and blood biochemistry results were achieved in one bird. The birds either died immediately after clinical presentation or within two days, two individuals were euthanized due to worsening of their clinical condition. All birds underwent a post-mortem examination. While four birds were finally diagnosed with disseminated xanthogranulomatosis, all six individuals had large eosinophilic intranuclear inclusion bodies in the epithelial cells of the collecting ducts of the kidney and tested positive for PsAdV-2. Further examinations are needed to clarify to what extent PsAdV-2 might elicit lipometabolic disease in birds, or psittacines in general, and, in particular, the Red-crowned Parakeet.

Mycobacteriosis in Various Pet and Wild Birds from Germany: Pathological Findings, Coinfections, and Characterization of Causative Mycobacteria.

A total of 50 birds diagnosed with mycobacteriosis were examined for pathomorphological lesions, coinfections, and causative agents. Mycobacterial species were identified and isolates differentiated using multilocus sequence typing (MLST) and mycobacterial interspersed repetitive-unit variable-number of tandem-repeat (MIRU-VNTR) analysis. Possible associations between mycobacterial species, pathomorphological findings, coinfections, bird orders, and husbandry conditions were evaluated statistically. Mycobacteria were isolated from 34 birds (13 of 22 Psittaciformes, 12 of 18 Passeriformes, five of six Columbiformes, and four other orders) belonging to 26 species in total. Mycobacterium genavense (Mg) was cultured from 15 birds, Mycobacterium avium subsp. avium (Maa) from 20 birds, and Mycobacterium avium subsp. hominissuis (Mah) from three birds; hence, four birds had mixed infections. About equal numbers of psittacines and passerines were infected with Ma and Mg. The genetic diversity differed; Mg isolates belonged to one MLST type, Maa to six, and Mah to three combined genotypes. Several coinfections were detected; viruses and/or endoparasites affected 44%, fungi 38%, and bacteria 29% of the birds. Pathological findings and mycobacteriosis-affected organs were independent of coinfections. Overall, gross pathological findings were more often seen in mycobacteriosis caused by Ma (95%) compared with Mg (66%). Organ distribution of mycobacteriosis was independent of the mycobacterial species. Pathomorphological changes were seen in the small intestine of 71% and the lung of 65% of the birds, suggesting oral or pulmonal ingestion of mycobacteria. There were no associations between mycobacterial species and bird orders or bird husbandry conditions. Not only Mg, but also Maa and Mah, were clearly identified as primary cause of mycobacteriosis in pet birds. IMPORTANCE In this study, the causative agents and confounding factors of mycobacteriosis in a set of pet and some wild birds from Germany were examined. Not only Mycobacterium genavense, but also M. avium subsp. avium and M. avium subsp. hominissuis, contributed to mycobacteriosis in these birds. Various coinfections did not affect the manifestation of mycobacteriosis. Due to different gross necropsy findings, however, a different pathogenicity of the two species was assumed. New strains of M. avium subsp. hominissuis originating from birds were identified and characterized, which is important for epidemiological studies and for understanding the zoonotic role of this pathogen, as the subsp. hominissuis represents an increasing public health concern. The study provides some evidence of correlation between M. avium subsp. avium genotypes and virulence which will have to be confirmed by broader studies.

Avian leukosis virus (ALV) is highly prevalent in fancy-chicken flocks in Saxony.

The current prevalence of avian leukosis virus (ALV) in fancy chickens in Germany is unknown. Therefore, 537 cloacal swabs from 50 purebred fancy-chicken flocks in Saxony were tested for the presence of the ALV p27 protein using a commercial antigen-capture ELISA. The detection rate was 28.7% at the individual-animal level and 56.0% at the flock level. Phylogenetic analysis of PCR products obtained from 22 different flocks revealed the highest similarity to ALV subtype K. When classifying breeds by their origin, ALV detection rates differed significantly. Evaluation of questionnaire data revealed no significant differences between ALV-positive and negative flocks regarding mortality.

The mycotoxin deoxynivalenol (DON) can deteriorate vaccination efficacy against porcine reproductive and respiratory syndrome virus (PRRSV) at subtoxic levels.

BACKGROUND: Feedgrain contamination with mycotoxins, including deoxynivalenol (DON, "vomitoxin") is relatively frequently encountered. Pigs are particularly sensitive to the toxicity of DON. To assess the interplay between DON and porcine reproductive and respiratory syndrome virus (PRRSV), we performed an experimental DON exposure-PRRSV vaccination-challenge infection trial. Three-week-old piglets were divided into four groups. Groups I, II and III (10 animals/group) were vaccinated with a PRRSV modified live vaccine and 2 weeks later challenged with a heterologous field strain. While group I was not supplemented with DON, animals in groups II and III received DON for 4 weeks prior to challenge infection at levels that can be encountered in pig feed, employing a low-dose or high-dose regime (group II: 40 µg DON/kg body weight per day; group III: 80 µg DON/kg body weight per day, corresponding to approx. 1 or 2 mg DON/kg feed, respectively). Eight animals (group IV; unvaccinated, not DON exposed) served as control animals for the challenge infection. RESULTS: We assessed clinical signs, virus load in serum and various organs as well as antibody titres in the animals. All vaccinated animals mounted an efficient PRRSV-specific antibody response within 2 weeks, except for 20% of the animals receiving the higher DON dose. Upon virus challenge, the vaccinated animals in group I were protected from clinical signs. Vaccinated DON-exposed animals in group II and III were protected from clinical signs to a lesser extent. Clinical signs in group III receiving the higher dose of DON were as severe as in the (unvaccinated, not DON exposed) control group IV. The animals of group III also displayed lower antibody titres compared with the animals in group I and II. CONCLUSIONS: The experimental vaccination/challenge study therefore revealed that exposure of pigs to DON for a period of 4 weeks deteriorates the efficacy of vaccination against clinical signs of PRRS.

Enhanced Antiviral Function of Magnesium Chloride-Modified Heparin on a Broad Spectrum of Viruses.

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

Usutu virus infection in aviary birds during the cold season.

The mosquito-borne flavivirus Usutu virus (USUV) is responsible for countless deaths in both resident populations and birds kept in outdoor aviaries. Since 2001, USUV outbreaks have attracted increased attention due to the rapid geographical spread of the virus and its close relationship to West Nile virus (WNV), an emerging pathogen in humans and animals. Similar to WNV, the USUV enzootic transmission cycle predominantly involves Culex spp. as vectors, whereas birds serve as amplifying reservoir hosts. In Europe, USUV-associated disease outbreaks in birds are almost exclusively described during late spring and early autumn (early April to late October). Contagiousness of virus particles excreted by infected birds has not yet been proven, so that the role of non-vector-borne transmission, as it is known for the closely related WNV, remains unclear. Here we report the diagnosis of USUV infection in 15 of 24 birds from mortality outbreaks that occurred during the cold season between late October 2018 and early April 2019, in eight different aviaries located in Germany. Detection of USUV was performed using standardized molecular biological methods and immunohistochemistry for verification of the infection. USUV infection in a parrot species, a tropical finch and two estrildid finches are reported for the first time. Further research on the occurrence of USUV infection during the cold season is key to understanding the dynamics of viral transmission as well as for a profound health risk assessment for aviary birds as well as humans.

Pigeon rotavirus A genotype G18P[17]-associated disease outbreaks after fancy pigeon shows in Germany - a case series.

OBJECTIVE: Pigeon rotavirus A (RVA) isolates of genotype G18P[17] are causing disease outbreaks and fatalities in pigeon lofts in Australia, Germany, Belgium, Denmark and USA since 2016. Most disease outbreaks have been reported from juvenile pigeons (Columba livia forma domestica). However, reports on RVA-associated disease outbreaks in fancy pigeons in connection with fancy pigeon shows in Germany are rare. MATERIAL AND METHODS: Overall 18 pigeons (16 fancy pigeons and one racing pigeon from 9 pigeon fanciers, as well as one feral pigeon from a rescue center) were sent in for routine diagnostic necropsy including histopathologic, parasitologic and microbiologic examinations. Molecular biologic examinations for detection of RVA, circovirus, Usutu virus, West Nile virus and Chlamydia psittaci were also carried out on all pigeons. An accompanying questionnaire filled in by the senders was used to generate basic information on the affected pigeon lofts. RESULTS: Disease outbreaks in juvenile and adult pigeons were reported 7-14 days after fancy pigeon shows. One fancier who had previously vaccinated his pigeons with an autogenous pigeon RVA vaccine, noted no morbidity and mortality among his pigeons and thus sent in a healthy pigeon for diagnostic purposes. Reported clinical signs in the other pigeons were regurgitation, green slimy diarrhea, anorexia, apathy and death after 24 hours. Hepatic necrosis and detection of pigeon RVA isolates of genotype G18P[17] confirmed disease outbreaks caused by pigeon RVA in all pigeons, except for the vaccinated pigeon. Besides pigeon circovirus, which was detected in 15 of 18 pigeons, all other pathogens were singular findings. CONCLUSION AND CLINICAL RELEVANCE: In disease outbreaks following fancy pigeon shows in juvenile and adult pigeons diagnostics should include pigeon RVA of genotype G18P[17].

Prevalence of pigeon rotavirus infections: animal exhibitions as a risk factor for pigeon flocks.

A total of 289 cloacal swabs from pigeons from 29 different breeders in Germany were collected. In addition, samples from pigeons exhibited at shows were collected. The detailed health status of the pigeon flocks was recorded. Samples were analysed for the presence of the recently discovered pigeon rotavirus and pigeon circovirus. Pigeon rotavirus was found in 10.3% and pigeon circoviruses was found in 65.5% of sampled pigeon lofts. The study revealed a strong relationship between the attendance of shows and the occurrence of different clinical signs. The higher prevalence of pigeon rotavirus in exhibited animals indicates that exhibitions are a risk factor for the transmission of this pathogen.

Identification of Novel Feline Paramyxoviruses in Guignas (Leopardus guigna) from Chile.

The family of paramyxoviruses has received growing attention as several new species have been identified recently, notably two different clusters in domestic cats, designated as feline morbillivirus (FeMV) and feline paramyxovirus (FPaV). Their phylogenetic origin and whether wild felids also harbor these viruses are currently unknown. Kidney samples from 35 guignas (Leopardus guigna), a wild felid from Chile, were investigated for paramyxoviruses using consensus-RT-PCR. In addition, thirteen serum samples of guignas were screened for the presence of FeMV-specific antibodies by an immunofluorescence assay (IFA). Viral RNA was detected in 31% of the kidney samples. Phylogenetic analyses revealed two well-supported clusters, related to isolates from domestic cats, rodents and bats. No significant histopathology changes were recorded in infected guignas. Serology identified two samples which were positive for FeMV-specific antibodies. Our study highlights the diversity of paramyxovirus infections in felids with special emphasis on guignas from Chile.

Monitoring of free-ranging and captive Psittacula populations in Western Europe for avian bornaviruses, circoviruses and polyomaviruses.

Avian pathogens such as bornaviruses, circoviruses and polyomaviruses are widely distributed in captive collections of psittacine birds worldwide and can cause fatal diseases. In contrast, only little is known about their presence in free-ranging psittacines and their impact on these populations. Rose-ringed parakeets (Psittacula krameri) and Alexandrine parakeets (Psittacula eupatria) are non-native to Europe, but have established stable populations in parts of Western Europe. From 2012-2017, we surveyed free-ranging populations in Germany and France as well as captive Psittacula individuals from Germany and Spain for avian bornavirus, circovirus and polyomavirus infections. Samples from two out of 469 tested free-ranging birds (0.4%; 95% confidence interval [CI-95]: 0.1-1.5%) were positive for beak and feather disease virus (BeFDV), whereas avian bornaviruses and polyomaviruses were not detected in the free-ranging populations. In contrast, avian bornaviruses and polyomaviruses, but not circoviruses were detected in captive populations. Parrot bornavirus 4 (PaBV-4) infection was detected by RT-PCR in four out of 210 captive parakeets (1.9%; CI-95: 0.7-4.8%) from four different holdings in Germany and Spain and confirmed by detection of bornavirus-reactive antibodies in two of these birds. Three out of 160 tested birds (1.9%; CI-95: 0.5-5.4%) possessed serum antibodies directed against budgerigar fledgling disease virus (BuFDV). PaBV-4 and BuFDV were also detected in several psittacines of a mixed holding in Germany, which had been in contact with free-ranging parakeets. Our results demonstrate that Psittacula parakeets are susceptible to common psittacine pathogens and their populations in Western Europe are exposed to these viruses. Nevertheless, the prevalence of avian bornaviruses, circoviruses and polyomaviruses in those populations is very low.RESEARCH HIGHLIGHTS Psittacula parakeets are susceptible to bornavirus, circovirus and polyomavirus infection.Introduced Psittacula populations in Europe have been exposed to these viruses.Nevertheless, they may be absent or present at only low levels in free-ranging Psittacula populations.Free-ranging populations in Europe pose a minor threat of transmitting these viruses to captive Psittaciformes.

Prevalence of feline immunodeficiency virus and feline leukaemia virus in domestic cats in Hungary.

OBJECTIVES: Feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) are retroviruses affecting cats worldwide. The objectives of the study were to estimate the prevalence of these retroviruses in domestic cats in Hungary and to characterise the phylogenetic relationships of FIV strains. METHODS: A total of 335 anticoagulated whole-blood samples obtained from both a healthy and ill cat population were examined for the presence of FIV and FeLV with two methods: ELISA and PCR. Statistical analysis was carried out to analyse the data obtained. Sequencing and phylogenetic analysis of partial polymerase (pol) gene sequences was performed to describe circulating FIV subtypes. RESULTS: Statistical analysis showed 11.8% and 9.9% true prevalence of FeLV and FIV, respectively, with ELISA. The apparent prevalence calculated from the PCR results were 17.3% for FeLV and 13.1% for FIV. Phylogenetic analysis of partial pol gene sequences obtained from 22 FIV strains showed that all observed Hungarian strains belonged to FIV subtype B. The strains were grouped into several monophyletic subgroups reflecting the geographic locations of the origin of the samples. The overall mean genetic similarity between the analysed strains was 98.2%. CONCLUSIONS AND RELEVANCE: We report the first thorough overview of the prevalence of FeLV and FIV in Hungary, which is relatively high, and give insight into the genetic diversity of Hungarian strains of FIV.

A New Genotype of Feline Morbillivirus Infects Primary Cells of the Lung, Kidney, Brain and Peripheral Blood.

Paramyxoviruses comprise a large number of diverse viruses which in part give rise to severe diseases in affected hosts. A new genotype of feline morbillivirus, tentatively named feline morbillivirus genotype 2 (FeMV-GT2), was isolated from urine of cats with urinary tract diseases. Whole genome sequencing showed about 78% nucleotide homology to known feline morbilliviruses. The virus was isolated in permanent cell lines of feline and simian origin. To investigate the cell tropism of FeMV-GT2 feline primary epithelial cells from the kidney, the urinary bladder and the lung, peripheral blood mononuclear cells (PBMC), as well as organotypic brain slice cultures were used for infection experiments. We demonstrate that FeMV-GT2 is able to infect renal and pulmonary epithelial cells, primary cells from the cerebrum and cerebellum, as well as immune cells in the blood, especially CD4⁺ T cells, CD20⁺ B cells and monocytes. The cats used for virus isolation shed FeMV-GT2 continuously for several months despite the presence of neutralizing antibodies in the blood. Our results point towards the necessity of increased awareness for this virus when clinical signs of the aforementioned organs are encountered in cats which cannot be explained by other etiologies.

Highly sensitive ELISA for the serological detection of murine rotavirus EDIM based on its major immunogen VP6.

Precise health monitoring of laboratory animals is a critical factor for surveillance and accuracy of animal experiments. Rotavirus epizootic diarrhea of infant mice (EDIM) leads to infections in mice that can influence animal studies, e.g., by altering the intestinal physiology. Thus, the aim of this study was establishing a highly sensitive and specific ELISA for the serological detection of EDIM infections in rodents. First, virus proteins were separated by SDS-PAGE and immunogenic proteins were visualized by immunoblotting and identified after in-gel digestion by tandem mass spectrometry. Subsequently, the major immunogen VP6 (virus protein 6) was expressed in Escherichia coli in high yields, purified by affinity chromatography, and used to establish an indirect ELISA. The diagnostic sensitivity and specificity were both above 99 % and the selectivity better than 98.7 % for animals infected by other pathogens listed by the Federation of Laboratory Animal Science Associations. Importantly, the Strep-rVP6-His-ELISA was more sensitive than a commercial virus-based ELISA and is a time- and cost-efficient complement to EDIM-specific immune-fluorescence assays. In conclusion, the assay can improve health monitoring by reducing the risk of missed EDIM infections in animal housing facilities, thereby improving animal welfare, reliability of animal studies, and protection of precious mice breeds.

Outbreak and Cocirculation of Three Different Usutu Virus Strains in Eastern Germany.

Usutu virus (USUV) is a mosquito-borne flavivirus accounting for large-scale deaths in resident bird populations. In this study, we show the introduction of USUV to Eastern Germany resulting in massive death of birds, particularly blackbirds (Turdus merula). We found that three diverse USUV lineages ("Europe 3," "Africa 2," and "Africa 3-like") circulated simultaneously. Moreover, we detected USUV in Culex pipiens in a region where no dead birds were reported, strengthening the need for mosquito monitoring to uncover the spread of arboviruses. Furthermore, phylogenetic analyses revealed that mutations accumulated, in particular, in the NS3 region within short time periods. In addition, comparison of whole-genome sequences showed that diverse isolates of the cluster "Africa 3-like" are cocirculating in Germany due to independent introduction events.

Goose Parvovirus and Circovirus Coinfections in Ornamental Ducks.

Clinical observations and diagnostic procedures carried out to elucidate the cause of high mortality in 2-8-wk-old ornamental ducks (mandarin, wood, falcated, and silver teal ducks) are described. At necropsy, ducklings showed general pallor of skeletal and heart muscles, subcutaneous gelatinous transudates, pericarditis, ascites, and severe edema and hyperemia of lungs. Histopathologic examination revealed that the most important changes were located in the crop, bursa of Fabricius, and lungs with presence of amorphic basic intracytoplasmic inclusions. No bacteria or fungi could be detected from affected organs and ascitic fluid. Viral diagnosis included molecular detection for the presence of goose parvovirus (GPV), circovirus, avian influenza, herpesviruses, paramyxovirus, reovirus, and polyomavirus. Both GPV and circovirus could be detected by real-time PCR and nested broad-spectrum PCR, respectively. Phylogenetically, full-length nucleotide sequence of GPV showed a close similarity ranging from 95.6% to 97.9% with European and Asian pathogenic GPV. On the other hand, the detected circovirus showed nucleotide identity of 90% to 98% with goose circoviruses (GoCVs). This is the first report of GoCVs and GPV in ornamental ducks. The concurrence of GPV and GoCV infections is thought to contribute to the high mortality.

Complete Genome Sequence of a Novel Avian Polyomavirus Isolated from Gouldian Finch.

A novel polyomavirus was identified in a fatally diseased Gouldian finch (Erythrura gouldiae). The new polyomavirus, strain VL 1209, was detected using a broad-spectrum nested PCR.

Discovery of new feline paramyxoviruses in domestic cats with chronic kidney disease.

Paramyxoviruses constitute a large family of enveloped RNA viruses including important pathogens in veterinary and human medicine. Recently, feline paramyxoviruses, genus morbillivirus, were detected in cats from Hong Kong and Japan. Here we describe the discovery of several new feline paramyxoviruses. Infections with these diverse viruses were detected in urine samples from cats suffering from chronic kidney disease (CKD). No viral RNA was found in cats without clinical signs of uropathy highlighting an association between feline paramyxovirus (FPaV) infections and CKD. Phylogenetic analyses of the detected viruses showed that they represent at least two different species, one of them representing the feline morbilliviruses detected previously in Hong Kong and Japan. In addition, a new FPaV was detected sharing only 73 % homology on the nucleotide level of the viral L-gene to currently known paramyxoviral species.

Differential replication properties among H9N2 avian influenza viruses of Eurasian origin.

Avian influenza H9N2 viruses have become panzootic in Eurasia causing respiratory manifestations, great economic losses and occasionally being transmitted to humans. To evaluate the replication properties and compare the different virus quantification methods, four Eurasian H9N2 viruses from different geographical origins were propagated in embryonated chicken egg (ECE) and Madin-Darby canine kidney epithelial cell systems. The ECE-grown and cell culture-grown viruses were monitored for replication kinetics based on tissue culture infectious dose (TCID50), Hemagglutination (HA) test and quantitative real time RT-PCR (qRT-PCR). The cellular morphology was analyzed using immunofluorescence (IF) and cellular ELISA was used to screen the sensitivity of the viruses to amantadine. The Eurasian wild type-H9N2 virus produced lower titers compared to the three G1-H9N2 viruses at respective time points. Detectable titers were observed earliest at 16 h post inoculation (hpi), significant morphological changes on cells were first observed at 32 hpi. Few nucleotide and amino acid substitutions were noticed in the HA, NA and NS gene sequences but none of them are related to the known conserved region that can alter pathogenesis or virulence following a single passage in cell culture. All studied H9N2 viruses were sensitive to amantadine. The G1-H9N2 viruses have higher replication capabilities compared to the European wild bird-H9N2 probably due to their specific genetic constitutions which is prerequisite for a successful vaccine candidate. Both the ECE and MDCK cell system allowed efficient replication but the ECE system is considered as the better cultivation system for H9N2 viruses in order to get maximum amounts of virus within a short time period.