Next-generation sequencing of five new avian paramyxoviruses 8 isolates from Kazakhstan indicates a low genetic evolution rate over four decades.

Five avian paramyxoviruses of serotype 8 (APMV-8) were isolated during a study monitoring wild birds in Kazakhstan in 2013 and each was further characterized. The viruses were isolated from three White-fronted geese (Anser albifrons), one Whooper swan (Cygnus cygnus), and one Little stint (Calidris minuta). Before our study, only two complete APMV-8 sequences had been reported worldwide since their discovery in the USA and Japan in the 1970s. We report the complete genome sequences of the newly detected viruses and analyze the genetic evolution of the APMV-8 viruses over four decades.

Monitoring of influenza A viruses in wild bird populations in Kazakhstan in 2002-2009.

A comprehensive influenza virus monitoring study of wild birds was carried out at important flyway resting places and wintering sites in Kazakhstan over eight years. More than 3200 birds belonging to 155 species were sampled. Nearly three-fourths of the birds belonged to the orders Anseriformes and Charadriiformes. In total, 118 hemagglutinating agents were isolated, and 95 of them were identified as influenza A viruses. The influenza viruses comprised eight different subtypes with a high prevalence of H13 and H3 viruses and also included low-pathogenic H5 viruses. The vast majority of the H13 viruses were isolated from members of the family Laridae, whereas the H3 viruses mostly originated from members of the family Anatidae, both in concordance with other monitoring studies. All virus isolates were recovered from cloacal swabs or fecal samples only. The influenza viruses were identified mainly in wetlands north of the Caspian Sea. These findings should be integrated in the design of further wild-bird-monitoring activities.

Epidemiological and Molecular Analysis of an Outbreak of Highly Pathogenic Avian Influenza H5N8 clade 2.3.4.4 in a German Zoo: Effective Disease Control with Minimal Culling.

Outbreaks of highly pathogenic avian influenza A virus (HPAIV) subtype H5N8, clade 2.3.4.4, were first reported in January 2014 from South Korea. These viruses spread rapidly to Europe and the North American continent during autumn 2014 and caused, in Germany, five outbreaks in poultry holdings until February 2015. In addition, birds kept in a zoo in north-eastern Germany were affected. Only a few individual white storks (Ciconia ciconia) showed clinical symptoms and eventually died in the course of the infection, although subsequent in-depth diagnostic investigations showed that other birds kept in the same compound of the white storks were acutely positive for or had undergone asymptomatic infection with HPAIV H5N8. An exception from culling all of the 500 remaining zoo birds was granted by the competent authority. Restriction measures included grouping the zoo birds into eight epidemiological units in which 60 birds of each unit tested repeatedly negative for H5N8. Epidemiological and phylogenetical investigations revealed that the most likely source of introduction was direct or indirect contact with infected wild birds as the white storks had access to a small pond frequented by wild mallards and other aquatic wild birds during a period of 10 days in December 2014. Median network analysis showed that the zoo bird viruses segregated into a distinct cluster of clade 2.3.4.4 with closest ties to H5N8 isolates obtained from mute swans (Cygnus olor) in Sweden in April 2015. This case demonstrates that alternatives to culling exist to rescue valuable avifaunistic collections after incursions of HPAIV.

Field study on swine influenza virus (SIV) infection in weaner pigs and sows.

OBJECTIVE: The aim of this field study was to explore the occurrence of and factors associated with the detection of swine influenza virus (SIV) by RTqPCR in weaner pigs and sows from herds with a history of respiratory or reproductive disorders. MATERIAL AND METHODS: The sample set was based on nasal swabs from 823 sows (123 submissions) and 562 weaner pigs (80 submissions). Nasal swab samples were taken and submitted by 51 veterinary practices from all over Germany. Corresponding to the pig density most of the submissions originated from the north-western part of Germany. The nasal swabs were used to detect SIV RNA by real-time RT-PCR (RTqPCR). Subtyping of SIV RNA by conventional RT-PCR and sequencing was attempted directly from clinical samples or from isolates when available. The herd characteristics, management and housing conditions of the pig herd as well as the course of the disease were collected by a telephone questionnaire with the herd attending veterinarian. RESULTS: SIV was detected by RTqPCR in 53.8% of the submissions from weaner pigs with a history of respiratory disease. Moreover SIV was detected in 10.6% of the submissions from sows. The predominant endemic subtype found in nasal swabs from sows and weaner pigs was H1N1 (60.5%) whereas subtypes H1N2 (14.0%) and H3N2 (14.0%) were detected less frequently. In addition, human pandemic H1N1 virus or reassortants thereof were found in 11.5%. CONCLUSION AND CLINICAL RELEVANCE: The results underline the significance of a SIV infection in young pigs. A significant lower detection of SIV in wea- ner pigs was associated with the vaccination of piglets against por- cine circovirus type 2 (PCV2), possibly indicating an interaction of SIV and PCV2. Most of the positive samples from sows originated from gilts, whereas only two originated from sows. An association between reproductive disorders and the detection of SIV could not be confirmed.

Consecutive natural influenza a virus infections in sentinel mallards in the evident absence of subtype-specific hemagglutination inhibiting antibodies.

Dabbling ducks, particularly Mallards (Anas platyrhynchos) have been frequently and consistently reported to play a pivotal role as a reservoir of low pathogenic avian influenza viruses (AIV). From October 2006 to November 2008, hand-raised Mallard ducks kept at a pond in an avifaunistically rich area of Southern Germany served as sentinel birds in the AIV surveillance programme in Germany. The pond was regularly visited by several species of dabbling ducks. A flock of sentinel birds, consisting of the same 16 individual birds during the whole study period, was regularly tested virologically and serologically for AIV infections. Swab samples were screened by RT-qPCR and, if positive, virus was isolated in embryonated chicken eggs. Serum samples were tested by the use of competitive ELISA and hemagglutinin inhibition (HI) assay. Sequences of full-length hemagglutinin (HA) and neuraminidase (NA) genes were phylogenetically analysed. Four episodes of infections with Eurasian-type AIV occurred in August (H6N8), October/November (H3N2, H2N3) 2007, in January (H3N2) and September (H3N8) 2008. The HA and NA genes of the H3N2 viruses of October 2007 and January 2008 were almost identical rendering the possibility of a re-introduction of that virus from the environment of the sentinel flock highly likely. The HA of the H3N8 virus of September 2008 belonged to a different cluster. As a correlate of the humoral immune response, titres of nucleocapsid protein-specific antibodies fluctuated in correlation with the course of AIV infection episodes. However, no specific systemic response of hemagglutination inhibiting antibodies could be demonstrated even if homologous viral antigens were used. Besides being useful as early indicators for the circulation of influenza viruses in a specific region, the sentinel ducks also contributed to gaining insights into the ecobiology of AIV infection in aquatic wild birds.

Differentiation of two distinct clusters among currently circulating influenza A(H1N1)v viruses, March-September 2009.

Analysis of all complete genome sequences of the pandemic influenza A(H1N1)v virus available as of 10 September 2009 revealed that two closely related but distinct clusters were circulating in most of the affected countries at the same time. The characteristic differences are located in genes encoding the two surface proteins - haemagglutinin and neuraminidase - and four internal proteins - the polymerase PB2 subunit, nucleoprotein, matrix protein M1 and the non-structural protein NS1. Phylogenetic inference was demonstrated by neighbour joining, maximum likelihood and Bayesian trees analyses of the involved genes and by tree construction of concatenated sequences.

Epidemiological and ornithological aspects of outbreaks of highly pathogenic avian influenza virus H5N1 of Asian lineage in wild birds in Germany, 2006 and 2007.

In Germany, two distinct episodes of outbreaks of highly pathogenic avian influenza virus of subtype H5N1 (HPAIV H5N1) in wild birds occurred at the beginning of 2006, and in summer 2007. High local densities of wild bird populations apparently sparked clinically detectable outbreaks. However, these remained restricted in (i) number of birds, (ii) species found to be affected, (iii) time, and (iv) location despite the presence of several hundred thousands of susceptible wild birds and further stressors (food shortage, harsh weather conditions and moulting). Northern and southern subpopulations of several migratory anseriform species can be distinguished with respect to their preference for wintering grounds in Germany. This corroborates viral genetic data by Starick et al. (2008) demonstrating the introduction of two geographically restricted virus subpopulations of Qinghai-like lineage (cluster 2.2.A and 2.2.B) into northern and southern Germany, respectively, in 2006. The incursion of virus emerging in 2007, found to be distinct from the clusters detected in 2006 (Starick et al., 2008), may have been associated with moulting movements. Intensive past-outbreak investigations with negative results of live and dead wild birds and of terrestrial scavengers excluded continued circulation of virus on a larger scale. However, persistence of virus in small pockets of local wild bird populations could not be ruled out resiliently. 1.5% of investigated sera originating from cats sampled at the epicentres of the Ruegen 2006-outbreak contained H5-antibodies. Passive monitoring was found to be highly superior to live bird surveillance when aiming at the detection of HPAIV H5N1 in wild birds (P < 0.0001).

Virological monitoring of white storks (Ciconia ciconia) for avian influenza.

Between 2003 and 2008, more than 600 white stork (Ciconia ciconia) nestlings in the German federal state of Brandenburg were ringed and examined for influenza A viruses. With the spread of highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 among wild birds in Germany in spring 2006, dead wild birds, including 88 white storks, were tested for infection with HPAIV. Furthermore, fresh fecal samples were examined by RT-PCR to monitor the occurrence of HPAIV in adult storks. While the monitoring of nestlings and adult white storks failed to yield evidence of influenza A virus infections in these birds, two storks found dead in April 2006 in the same location tested positive for HPAIV H5N1. Sequence analysis revealed that the virus isolated from one of the storks belonged to clade 2.2, which was commonly found in wild birds in the north of Germany and other European countries during the epidemic in 2006. Despite these two cases, white storks seemed to serve as neither a vector nor as a reservoir for HPAIV in Germany. The risk of white storks transmitting HPAIV to domestic poultry and humans is low.

Rapid molecular subtyping by reverse transcription polymerase chain reaction of the neuraminidase gene of avian influenza A viruses.

Accurate identification of hemagglutinin (HA) and neuraminidase (NA) subtypes of influenza A viruses is an integral part of monitoring programs targeting avian influenza viruses (AIV). Use of highly sensitive molecular screening methods such as pan influenza-specific real-time RT-PCR (rRT-PCR) yields an increasing number of samples which are positive for AIV RNA but negative by virus isolation and, therefore, require molecular, instead of serological, subtyping. We developed specific RT-PCR assays for all known nine AIV NA subtypes. Validation using 43 reference isolates from different animal species revealed good performance characteristics regarding sensitivity and specificity. On basis of serial tenfold dilution series of reference isolates a benchmark value of C(t) 32 in an M gene-specific rRT-PCR became evident below which all nine NA subtypes were readily detectable by the subtype-specific RT-PCRs. For subtypes N1, N2, N4 and N6 detection was extended to dilutions with C(t) values of up to 35. Diagnostic applicability of the whole set of conventional NA-specific RT-PCRs was evaluated by analysis of 119 different diagnostic samples from wild birds which proved to be positive for AIV by M gene-specific rRT-PCR. Diagnostic sensitivity and specificity was confirmed by sequencing NA amplicons from 41 field isolates generated from this set and by NA inhibition assays. A universal molecular HA/NA subtyping algorithm for rRT-PCR positive avian influenza virus monitoring samples is proposed which may complement classical serological subtyping of influenza A virus isolates.

Phylogenetic analyses of highly pathogenic avian influenza virus isolates from Germany in 2006 and 2007 suggest at least three separate introductions of H5N1 virus.

In spring 2006, highly pathogenic avian influenza virus (HPAIV) of subtype H5N1 was detected in Germany in 343 dead wild birds, as well as in a black swan (Cygnus atratus) kept in a zoo, three stray cats, one stone marten (Martes foina), and in a single turkey farm. In 2007 (June-July) the virus reoccurred in 96 wild birds at six geographically separate locations in the Southeast of Germany. In addition, a backyard mixed duck and goose holding was affected. Real-time RT-PCR [Hoffmann, B., Harder, T., Starick, E., Depner, K., Werner, O., Beer, M., 2007. Rapid and highly sensitive pathotyping of avian influenza A H5N1 virus by using real-time reverse transcription-PCR. J. Clin. Microbiol. 45, 600-603] and nucleotide sequencing confirmed that these H5-viruses belonged to the Qinghai lineage of HPAIV H5N1 (clade 2.2). For a more detailed analysis, the hemagglutinin and neuraminidase genes of 27 selected German H5N1 viruses isolated 2006 or 2007 and originating from different regions and animal species were sequenced and analysed phylogenetically. As a result, three closely related but distinguishable H5N1 subclades could be defined: In 2006 a 'Northern type' (subclade 2.2.2), representing virus isolates from the German federal states Mecklenburg-Western Pomerania, Schleswig-Holstein, Brandenburg, and Lower Saxony, and a 'Southern type' (subclade 2.2.1) from Baden-Württemberg and Bavaria were detected. Interestingly, representatives of both types were present in Central Germany and caused the outbreak in turkeys (subclade 2.2.2) and in a case in a tufted duck (Aythya fuligula) (subclade 2.2.1) in Saxony. Furthermore, one isolate from the South of Germany was identified as 2.2.2 and vice versa a 2.2.1-like isolate was found in Northern Germany. H5N1 viruses isolated in 2007 belonged to a third type (subclade 2.2.3) which was not detected in 2006. Our data suggest the introduction of three distinct H5N1 variants into the wild bird population of Germany. The source of these viruses and the exact time of introduction remain obscure. Based on the identification of closely related H5N1 viruses from Southern and Central Russia, a recent introduction via wild birds on winter escape from these regions, early in 2006 constitutes the most likely scenario for the 2006 outbreaks. The viruses detected in 2007 most likely represent another new incursion from an as yet unknown source.

Encephalitis in a stone marten (Martes foina) after natural infection with highly pathogenic avian influenza virus subtype H5N1.

Recent outbreaks of disease in different avian species, caused by the highly pathogenic avian influenza virus (HPAIV), have involved infection by subtype H5N1 of the virus. This virus has also crossed species barriers and infected felines and humans. Here, we report the natural infection of a stone marten (Martes foina) from an area with numerous confirmed cases of H5N1 HPAIV infection in wild birds. Histopathological examination of tissues from this animal revealed a diffuse nonsuppurative panencephalitis with perivascular cuffing, multifocal gliosis and neuronal necrosis. Additionally, focal necrosis of pancreatic acinar cells was observed. Immunohistochemically, lesions in these organs were associated with avian influenza virus antigen in neurons, glial cells and pancreatic acinar cells. Thus, the microscopical lesions and viral antigen distribution in this stone marten differs from that recently described for cats naturally and experimentally infected with the same virus subtype. This is the first report of natural infection of a mustelid with HPAIV H5N1.

Distribution of lesions and antigen of highly pathogenic avian influenza virus A/Swan/Germany/R65/06 (H5N1) in domestic cats after presumptive infection by wild birds.

In early 2006, the highly pathogenic avian influenza virus (HPAIV) H5N1 of the Asian lineage caused the death of wild aquatic birds in Northern Germany. In the mainly affected areas, a trans-species transmission of HPAIV H5N1 to mammals occurred between birds and domestic cats and 1 Stone Marten (Martes foina), respectively. Here, we report lesions and distribution of influenza virus antigen in 3 cats infected naturally with HPAIV H5N1 A/swan/Germany/R65/06. The hemagglutinin partial nucleotide sequences of the viruses were genetically closely related to a H5N1 HPAIV obtained from a dead Whooper Swan (Cygnus cygnus) of the same area. At necropsy, within the patchy dark-red and consolidated lungs, there was granulomatous pneumonia caused by Aelurostrongylus sp. Histologically, the main findings associated with influenza in all cats were bronchointerstitial pneumonia and marked random hepatic necrosis. In addition, all animals displayed lymphoid necrosis in the spleen and Peyer's patches and necrosis of the adrenal cortex. Immunohistochemically, nucleoprotein of HPAIV was present intralesionally in the lungs, liver, adrenal glands, and lymphoid tissues. Oropharyngeal swabs were shown to be suited to detect HPAIV by quantitative real-time polymerase chain reaction (RT-PCR) in these cats, despite the paucity of influenza virus antigen in the upper respiratory tract by means of immunohistochemistry. The results show that outdoor cats in areas affected by HPAIV in wild birds are at risk for lethal infection. In conclusion, hepatic necrosis was, besides bronchointerstitial pneumonia, the primary lesion, suggesting that in naturally infected cats, damage to the liver plays an important role in the pathogenesis of H5N1 influenza.

Pathology of natural infections by H5N1 highly pathogenic avian influenza virus in mute (Cygnus olor) and whooper (Cygnus cygnus) swans.

Mortality in wild aquatic birds due to infection with highly pathogenic avian influenza viruses (HPAIV) is a rare event. During the recent outbreak of highly pathogenic avian influenza in Germany, mortality due to H5N1 HPAIV was observed among mute and whooper swans as part of a rapid spread of this virus. In contrast to earlier reports, swans appeared to be highly susceptible and represented the mainly affected species. We report gross and histopathology and distribution of influenza virus antigen in mute and whooper swans that died after natural infection with H5N1 HPAIV. At necropsy, the most reliable lesions were multifocal hemorrhagic necrosis in the pancreas, pulmonary congestion and edema, and subepicardial hemorrhages. Major histologic lesions were acute pancreatic necrosis, multifocal necrotizing hepatitis, and lymphoplasmacytic encephalitis with neuronal necrosis. Adrenals displayed consistently scattered cortical and medullary necrosis. In spleen and Peyer's patches, mild lymphocyte necrosis was present. Immunohistochemical demonstration of HPAIV nucleoprotein in pancreas, adrenals, liver, and brain was strongly consistent with histologic lesions. In the brain, a large number of neurons and glial cells, especially Purkinje cells, showed immunostaining. Occasionally, ependymal cells of the spinal cord were also positive. In the lungs, influenza virus antigen was identified in a few endothelial cells but not within pneumocytes. The infection of the central nervous system supports the view that the neurotropism of H5N1 HPAIV leads to nervous disturbances with loss of orientation. More investigations are necessary to clarify the mechanisms of the final circulatory failure, lung edema, and rapid death of the swans.

Establishment of a competitive ELISA (cELISA) system for the detection of influenza A virus nucleoprotein antibodies and its application to field sera from different species.

A recombinant baculovirus (RBV) encoding the nucleoprotein (NP) of avian influenza virus (AIV) was generated and the appropriate protein was expressed in Sf9 cells. Purified recombinant NP and the NP-specific monoclonal antibody HB65 were used to establish a competitive ELISA (cELISA) system for the detection of NP-specific antibodies in sera of ducks, geese and wild birds. Tests to evaluate this method were carried out using sera of ducks experimentally infected with AIV, pre-immune duck and chicken sera, and poultry field sera, which tested negative in the haemagglutination inhibition (HI) assay, and field sera of several poultry species experimentally infected with other viruses. The evaluation of the test demonstrated a high sensitivity and specificity of this method. Tests carried out using field sera of duck and goose flocks revealed widely corresponding results obtained by HI assay and cELISA indicating that this test is applicable for flock diagnosis. Differing results were obtained for individual samples. It can be assumed that for the most part this was because of a better recognition of the conserved NP antigen by serum antibodies, although some results remained unclear.

A virological survey in migrating waders and other waterfowl in one of the most important resting sites of Germany.

Wild birds are considered a potential reservoir or a carrier of viral diseases and may therefore play a role in the epidemiology of economically important or zoonotic diseases. In 2001 and 2002, a survey with special emphasis on virus isolation in migrating waders and some other birds were conducted. In one of the most important inland resting sites for migratory waterfowl, tracheal and cloacal swabs were collected from 465 waders representing 19 different species, and 165 other birds that were not captured on purpose. A total of 42 avian viruses were isolated, 34 of these were identified as paramyxoviruses (PMVs). The majority of isolates came from waders and wild ducks, and were characterized as PMV-1. In contrast, PMV-4 was found in wild ducks only, PMV-6 was mainly detected in wader species. Four avian influenza viruses (AIVs), belonging to H4 and H3 haemagglutinin subtype, were isolated from wild duck species. Furthermore, four reo-like viruses were isolated from one particular wader species for the first time. The majority of virus positive birds were <1 year old and did not show any clinical symptoms. There was no evidence for the presence of West Nile virus in these birds. These results confirm that the restricted resting sites in Western Europe must be considered as important locations for the intra- and interspecies transmission of avian viruses.

Establishment of a new bovine leukosis virus producing cell line.

Due to the prevalence of different bovine leukosis virus (BLV) species in the cattle population in Europe, problems may arise in the serological diagnosis of BLV infections. In addition, earlier investigations demonstrated that contamination of the BLV antigen-producing cell culture systems by bovine viral diarrhea virus (BVDV) may give rise to misinterpretation of serological test results after BVDV vaccination of cattle. By co-cultivation of peripheral leukocytes of a BLV-infected cow with a permanent sheep kidney cell line, a new BLV-producing cell line named PO714 was established. This line carries a BLV provirus of the Belgian species and has been tested to be free of a variety of possibly contaminating viruses and mycoplasms. Investigations of a panel of well-characterised sera by agar gel immunodiffusion (AGID) and capture ELISA (cELISA) tests using antigen prepared from this new cell line in comparison with antigen of the well-known cell line FLK/BLV yielded comparable results. False positive results caused by BVDV cross-reactions could be eliminated when tests were carried out with antigen derived from the new cell line.

Isolation and characterization of a low-pathogenicity H7N7 influenza virus from a turkey in a small mixed free-range poultry flock in Germany.

A hemagglutinating virus was isolated from a dead turkey in a small mixed free-range flock in Southern Germany. It was identified as influenza virus type A of subtype H7N7. The pathogenicity was low. An intravenous pathogenicity index of 0.03 was recorded, and the nucleotide sequencing revealed the amino acid sequence NVPEIPKGR*GLFG at the cleavage site of the hemagglutinin. Antibodies as well as virus were detected in the affected flock. Further virus spreading to other flocks was prevented by stamping out policy. Serological monitoring of contact flocks revealed one small backyard flock of 18 hens, which was positive. This flock was also destroyed. The origin of the virus could not be identified.

Detection of H7 avian influenza virus directly from poultry specimens.

Using clinical materials from experimentally infected poultry, we established an effective method for the preparation of viral RNA directly from tissue samples and eggs. Furthermore, our type A-specific matrix reverse transcription-polymerase chain reaction (RT-PCR) test was improved, and an H7 subtype-specific nested RT-PCR, which includes the hemagglutinin cleavage site, was designed. Both RT-PCR systems proved to be as sensitive as virus isolation. In addition, the labeled H7 HA-nested PCR primers were suitable for sequencing of the PCR products. The RT-PCR amplification of viral RNA and sequencing of the PCR product allows for the sensitive and rapid differentiation between low-pathogenic and highly pathogenic avian influenza viruses.

Cattle infected with bovine leukaemia virus may not only develop persistent B-cell lymphocytosis but also persistent B-cell lymphopenia.

We investigated the distribution of B and T cells in the peripheral blood of haematologically inconspicuous (non-persistent lymphocytotic, PL-) cattle infected with the bovine leukaemia virus (BLV). Flow cytometric data were obtained from six PL- cattle and compared with six age-matched animals with persistent lymphocytosis (PL+) and five non-infected healthy controls (BLV-). In the PL- group, the percentage and number of surface immunoglobulin-positive (sIg+) B cells were significantly reduced. Whereas in BLV-cattle, about 40% of the peripheral blood lymphocytes (PBL) were sIg + and 24% were sIgM + B cells. In the PL- group, less than 20% of the PBL were sIg+ and sIgM+ B cells. Only 5% of the PBL co-expressed sIgM+ and CD5+ versus 16% in BLV-. This decrease was persistent over 3 years and predominantly affected: (i) B cells that did not express sIgM; (ii) sIgM + B cells co-expressing CD5 and CD11b; and (iii) equally both lambda- and K-type light chain B-cell subpopulations. In contrast, the number of all circulating lymphocytes, CD5- and CD11b- sIgM+ B cells and CD2+ T cells did not differ. In PL+ animals, about 75% of the PBL were sIgM+ CD5+ B cells. These cells were of polyclonal origin, as light chains of the lambda- and K-type were expressed in a ratio of 4:1 (57.7% of PBL lambda+, 14% kappa+) as in BLV- animals (33.6% of PBL lambda+, 8.7% kappa+). In PL+ cattle the absolute number of B-cells and, therefore, their relative percentage is significantly increased. For this reason, even in case of absolutely increased T-cell numbers, the relative percentage of T-cells could be lower than in normal controls. The cause for the observed B cell decrease in PL- cattle is unknown, but it can be assumed that cytotoxic T cells are involved in this B-cell lymphopenia.