Protease activation mutants elicit protective immunity against highly pathogenic avian influenza viruses of subtype H7 in chickens and mice.

Protease activation mutants of the highly pathogenic avian influenza virus A/FPV/Rostock/34 (H7N1) have been generated that are fully dependent on the presence of trypsin for growth in cell culture. Unlike wild-type virus, the mutants do not induce systemic infection in chicken embryos and show low pathogenicity in both chicken embryos and adult chickens. Inactivated vaccines prepared from the mutants protected chickens and mice very efficiently against infection with highly pathogenic wild-type virus in a cross-reactive manner. The potential of these mutants to be used as veterinary and prepandemic vaccines will be discussed.

Isolation and characterization of avian paramyxovirus type 3b from farmed Namibian ostriches (Struthio camelus f. dom.).

Meat and skin from farmed ostriches are valuable products for European consumers. The EU regulations require that ostrich products deamed for export need to come from ostriches that are free of antibodies against Newcastle disease virus (avian paramxovirus type 1, aPMV-1). After the detection of antibodies against aPMV-1 in one of five ostrich farms in Namibia, attempts were made to isolate the causative virus. No aPMV-1 but an avian paramyxovirus type 3 (aPMV-3) was isolated from five pharyngeal/cloacal swabs of clinically healthy farmed Namibian ostriches. Subtype determination proved that all isolates are members of the subtype aPMV-3 of psittacine bird origin and were designated as aPMV-3b. In the haemagglutination inhibition test, the aPMV-3b isolates cross-reacted with aPMV-1. This allows the conclusion that the antibodies originally detected in sera of the ostriches are due to the cross-reaction with aPMV-3b, rather than to an infection with aPMV-1.To our knowledge, this is the first description of the occurrence of aPMV-3b in farmed ostriches.

Avian influenza virus monitoring in wintering waterbirds in Iran, 2003-2007.

BACKGROUND: Virological, molecular and serological studies were carried out to determine the status of infections with avian influenza viruses (AIV) in different species of wild waterbirds in Iran during 2003-2007. Samples were collected from 1146 birds representing 45 different species with the majority of samples originating from ducks, coots and shorebirds. Samples originated from 6 different provinces representative for the 15 most important wintering sites of migratory waterbirds in Iran. RESULTS: Overall, AIV were detected in approximately 3.4% of the samples. However, prevalence was higher (up to 8.3%) at selected locations and for certain species. No highly pathogenic avian influenza, including H5N1 was detected. A total of 35 AIVs were detected from cloacal or oropharyngeal swab samples. These positive samples originated mainly from Mallards and Common Teals.Of 711 serum samples tested for AIV antibodies, 345 (48.5%) were positive by using a nucleoprotein-specific competitive ELISA (NP-C-ELISA). Ducks including Mallard, Common Teal, Common Pochard, Northern Shoveler and Eurasian Wigeon revealed the highest antibody prevalence ranging from 44 to 75%. CONCLUSION: Results of these investigations provide important information about the prevalence of LPAIV in wild birds in Iran, especially wetlands around the Caspian Sea which represent an important wintering site for migratory water birds. Mallard and Common Teal exhibited the highest number of positives in virological and serological investigations: 43% and 26% virological positive cases and 24% and 46% serological positive reactions, respectively. These two species may play an important role in the ecology and perpetuation of influenza viruses in this region. In addition, it could be shown that both oropharyngeal and cloacal swab samples contribute to the detection of positive birds, and neither should be neglected.

Influenza report

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Minute excretion of highly pathogenic avian influenza virus A/chicken/Indonesia/2003 (H5N1) from experimentally infected domestic pigeons (Columbia livia) and lack of transmission to sentinel chickens.

Five out of sixteen domestic pigeons, inoculated oculo-nasally with a high dose of highly pathogenic avian influenza virus A/chicken/Indonesia/2003 (H5N1), developed clinical signs and neurological lesions leading to death of three pigeons 5-7 days after inoculation [Klopfleisch, R., Werner, O., Mundt, E., Harder, T. & Teifke, J. P. (2006). Vet Pathol 43, 463-470]. H5N1 virus was recovered from all organs sampled from two apparently healthy pigeons at 3 days post-infection and from the three pigeons which died spontaneously. All surviving birds shed virus via the oropharynx and the cloaca at minimal titres and seroconverted. Sentinel chickens reared in direct contact to the pigeons neither developed clinical signs nor seroconverted to the H5N1 virus.

Molecular analysis of highly pathogenic avian influenza virus of subtype H5N1 isolated from wild birds and mammals in northern Germany.

Analysis of the full-length sequences of all eight segments of the German wild-bird H5N1 highly pathogenic avian influenza virus index isolate, A/Cygnus cygnus/Germany/R65/2006, and an H5N1 isolate from a cat (A/cat/Germany/R606/2006) obtained during an outbreak in February 2006 revealed a very high similarity between these two sequences. One amino acid substitution in the PA gene, encoding a protein involved in virus RNA replication, and one amino acid substitution in the haemagglutinin (HA) protein were observed. Phylogenetic analyses of the HA and neuraminidase nucleotide sequences showed that avian influenza H5N1 isolates from the Astrakhan region located in southern Russia were the closest relatives. Reassortment events could be excluded in comparison with other 'Qinghai-like' H5N1 viruses. In addition, an H5N1 isolate originating from a single outbreak in poultry in Germany was found to be related closely to the H5N1 viruses circulating at that time in the wild-bird population.

Rapid and highly sensitive pathotyping of avian influenza A H5N1 virus by using real-time reverse transcription-PCR.

Rapid typing of the pathogenicity of avian influenza A viruses (AIV) of subtypes H5 and H7 is crucial to initiate adequate protective measures preventing the spread of highly pathogenic AIV (HPAIV). Here, a new real-time reverse transcription-PCR assay which enables sensitive and specific detection and cleavage site analysis of HPAIV H5N1 of the Qinghai lineage is described.

Enhancement of pathogenicity of Newcastle disease virus by alteration of specific amino acid residues in the surface glycoproteins F and HN.

Recombinant viruses were rescued after site-specific mutagenesis of a full-length clone of the lentogenic Newcastle disease virus (NDV) strain Clone 30. To assess the contribution of different amino acids to virulence, specific alterations were introduced into the fusion (F) protein and in the hemagglutinin-neuraminidase (HN) protein based on sequence comparison between NDV strains of different virulence. Modification of the proteolytic cleavage site in the F protein to a polybasic motif increased the intracerebral pathogenicity index (ICPI) from 0.0 to 1.28. Moreover, the additional exchange of amino acid 123 of the HN protein from tryptophan to cysteine in combination with alteration of amino acid 27 of the F protein from cysteine to arginine increased the ICPI to 1.5. The HN mutation visibly altered conformation of the protein, resulting in the formation of disulfide-linked HN dimers that may indicate that this HN conformation is beneficial for the virulent phenotype.

Third genome size category of avian paramyxovirus serotype 1 (Newcastle disease virus) and evolutionary implications.

The goal of the study was to establish if there was a relationship between molecular patterns and virus evolution. Therefore the complete genome sequence of two distinct apathogenic Newcastle disease virus (NDV) strains was determined and a third genome size category, containing 15,198 nucleotides, was recognized. Phylogenetic analysis revealed that two major separations resulting in three genome size categories occurred during the history of NDV. An ancient division in the primordial reservoir (wild waterbird species) led to two basal sister clades, class I and II, with genome sizes 15,198 (due to a 12 nucleotide insert in the phosphoprotein gene) and 15,186 nucleotides, respectively. Ancestors of only class II viruses colonized chicken populations and subsequently converted to virulent forms. These took place more than once and resulted in an early lineage [including genotypes I-IV and H33(W)] with genome size of 15,186 nucleotides. A second division occurred in the 20th century in the secondary (chicken) host. This gave rise to the branching-off of a clade (including recent genotypes V-VIII consisting of only pathogenic viruses) with the concomitant insertion of six nucleotides into the 5' non-coding region of the nucleoprotein gene thereby increasing the genome size to 15,192 nucleotides.

[Control and eradication strategies for classic fowl plague in Germany and the European Union]. / Bekämpfungsstrategie bei Klassischer Geflügelpest in Deutschland und der Europäischen Union.

The huge potential economic impact of highly pathogenic avian influenza (HPAI) substantiates specific and rigorous legal regulations worldwide. According to the O.I.E. Terrestrial Animal Health Code fowl plague is a notifiable disease. International trading activities concerning poultry and poultry products originating from countries with active HPAI are rigorously restricted. In EU member states directive 92/40/EEC subsumes measures against fowl plague and has been transferred into German legislation by the "Geflügelpest-Verordnung". These acts specify that vaccination against HPAI is principally prohibited. The aim of all sanctions is the extinction of disease and the eradication of the causative agent. However, HPAI viruses, exclusively belonging to subtypes H5 and H7, can re-emerge de novo from progenitor viruses of low pathogenicity which are perpetuated in the wild bird population. An outbreak of HPAI requires prompt action by a stamping out strategy. Fast and accurate diagnosis, a strict stand-still and the culling of affected flocks are at the basis of success. In areas with a high density of poultry holdings preemptive culling and creation of buffer zones, devoid of susceptible poultry, may be neccessary. In these cases emergency vaccinations can be considered as a supportive measure in order to limit mass culling. Vaccinations on merely prophylactic grounds, not being connected to acute outbreaks, should be avoided beware of selective pressures on the virus leading to antigenic drift and escape of vaccine-induced immunity. Instead, high standard biosecurity measures, particularly limiting direct and indirect contacts with wild birds, should be maintained.

[Influenza virus infections in migrating waterfowl: results of a two year study in Germany]. / Influenzavirus-Infektionen bei migrierenden Wasservögeln: Ergebnisse einer zweijährigen Studie in Deutschland.

In order to determine the actual prevalence of avian influenza viruses (AIV) in wild birds in Germany, extensive surveillance studies were carried out between March 2003 and January 2005. More than 3.000 samples of 79 different species of wild birds (migratory and resident birds) were taken and 1.151 established pools investigated. Samples came from 80 different regions of Germany. Forty AIV isolates representing 14 combinations of eight different hemagglutinin and eight neuraminidase subtypes, among them H5 and H7, were identified. All H5 and H7 isolates were found to be of low pathogenicity. The overall incidence of the investigated pools based on virus isolation was 3,5 % for AIV, with considerable variability noted among species, season and location. All AIV were isolated from birds sampled in autumn. Most of the AIV isolates came from the resting or wintering areas of mallards breeding far north. This study adds to the understanding of the ecology of influenza viruses in wild birds and empahsizes the constant need for surveillance in times of an ongoing and expanding epidemic of highly pathogenic AI.

[Classic fowl plague--a review]. / Klassische Geflügelpest--Eine Ubersicht.

Highly pathogenic avian influenza (HPAI) represents a severe form of generalized avian influenza which is characterized by a rapid and severe course of disease and a very high mortality. All poultry species are susceptible. Turkeys and chickens are most vulnerable. There are no pathognomonic symptoms or specific pathological alterations. The disease is caused by avian influenza virus strains of the subtypes H5 or H7. These viruses arise spontaneously from apathogenic progenitors by insertional mutation in the HA gene. Until recently, outbreaks of HPAI were rare events, however, they have been found to cause increasing losses over the past few years. Since 2003, a widespread occurrence of HPAI has been registered in southeast Asia, and some countries are endemically infected with HPAIV strain H5N1. In six countries this virus has also caused fatal human infections. This has sparked fears that this agent may be the progenitor of a new pandemic influenza virus. During summer 2005 the disease has slowly spread westward. Isolated outbreaks have been reported from Kazakhstan, Russia, Romania, Turkey, Croatia and Ukraine. Migratory birds have been tentatively accused for spreading the infection along their flyways.

The "Friedrich-Loeffler-Institut": past, present and future of research in infectious diseases of animals.

The Friedrich-Loeffler-Institut, founded in 1910 by Friedrich Loeffler, the discoverer of the first animal virus, foot-and-mouth disease virus, is the oldest virological research facility in the world. Beyond viruses, its area of competence has significantly expanded since its foundation and now also covers bacterial, parasitic and prion diseases of livestock, poultry and aquatic animals. Presently located at four sites within Germany (Insel Riems, Jena,Tübingen,Wusterhausen) the tasks of the institute as delineated in the Animal Disease Act encompass research on infectious animal diseases including zoonoses, import/export examinations, epidemiological studies in case of outbreaks of notifiable animal diseases, acting as reference laboratory for notifiable animal diseases and nationwide quality management of diagnosis of notifiable animal diseases. It is obliged to publish and maintain up-to-date diagnostic regimes for notifiable animal diseases, and it publishes a yearly report on animal health in Germany. With the increasing importance of infectious diseases of animals, in particular those potentially harmful to man (zoonoses), the Friedrich-Loeffler-Institut will be moving into new facilities including laboratories and animal facilities up to the highest biosafety level at its main site Insel Riems on the occasion of its 100th anniversary.

[Laboratory diagnosis of avian influenza by reverse transcription (RT)-PCR]. / Labordiagnose der Aviären Influenza mittels Reverser Transkription (RT)-PCR.

RT-PCR assays which amplify conserved regions of the influenza A virus gene are useful tools for the rapid and specific detection of infections of poultry with avian influenza virus (AIV) and for the investigation of large numbers of samples, e.g. within the framework of surveillance programs. Here, we present findings on the efficiency and on the limits of an RT-PCR assay which amplifies a part of the matrix protein gene. Sensitivity and specificity of the method were increased by the additional use of nested PCR. Parameters which may have an essential influence on the detection limit are outlined and discussed. A major focus of the study is the detection of AIV RNA from organ samples and swabs.

[Conventional and alternative housing systems for poultry--point of view of infectious disease medicine]. / Konventionelle und alternative Haltungssysteme für Geflügel--Infektionsmedizinische Gesichtspunkte.

The use of conventional battery cages for hens will be prohibited in Germany in 2007. Only few studies, however, have considered the differences between battery cages and alternative systems with regard to infectious diseases. The existing gaps in the current knowledge need to be closed by research and measures must be developed that will prevent the spread of viral, bacterial, and parasitic infections in alternative poultry housing systems. With regard to virus infections, avian influenza requires particular attention. Since wild birds, particularly anseriformes, represent a reservoir for avian influenza viruses, free-ranging poultry is much more at risk of infection than birds in closed hen-houses. Appropriate measures must prevent direct contact with wild birds and transmission via contaminated water, feed, or equipment. Several bacterial infections of poultry represent zoonoses. Salmonella and Campylobacter are considered as particularly important. To avoid a potential increase in the risk of infection for consumers due to poultry keeping systems that might favour infections with bacterial zoonotic agents, there is a special need for research in this area. With regard to parasitic infections, coccidioses may cause problems in alternative poultry housing systems, and lead to considerable economic consequences. The epidemiological situation concerning infections with Histomonas meleagridis needs to be analysed. Since all compounds that had been used for prophylactic or therapeutic purposes in the past have been banned, there is a need to develop new drugs which are safe for animals and humans.

Deletion of the non-essential UL0 gene of infectious laryngotracheitis (ILT) virus leads to attenuation in chickens, and UL0 mutants expressing influenza virus haemagglutinin (H7) protect against ILT and fowl plague.

Infectious laryngotracheitis virus (ILTV), a member of the Alphaherpesvirinae, possesses several unique genes. One of them, UL0, encodes an abundantly expressed protein that accumulates in the nuclei of ILTV-infected cells. This study demonstrates that this protein is dispensable for in vitro virus replication and that UL0 deletion mutants exhibit only minor growth defects in cultured cells. The UL0 gene locus of ILTV was also used for insertion of foreign DNA sequences encoding enhanced GFP or haemagglutinin (HA), subtype H7, of a highly pathogenic avian influenza virus under the control of the human cytomegalovirus immediate-early gene promoter. Expression of foreign proteins was shown by (immuno)fluorescence tests and Western blot analyses. After experimental infection of chickens, UL0 deletion mutants proved to be attenuated when compared to both parental wild-type ILTV and an UL0 rescue mutant. Nevertheless, all animals immunized with UL0-negative ILTV were protected from clinical disease after subsequent infection with virulent ILTV. Furthermore, all animals immunized with HA-expressing ILTV survived a lethal challenge with H7 subtype avian influenza virus with minimal clinical signs. Thus, an UL0-negative and HA-expressing ILTV recombinant may be used as a bivalent live virus vaccine against ILT and fowl plague. Unlike inactivated influenza virus vaccines, HA-expressing ILTV recombinants should be suitable for mass application and would also permit serological discrimination between vaccinated and virus-infected animals in the field.

Contribution of the length of the HN protein and the sequence of the F protein cleavage site to Newcastle disease virus pathogenicity.

Newcastle disease virus (NDV) possesses two envelope spike glycoproteins: the haemagglutinin-neuraminidase (HN) protein and the fusion (F) protein. The HN protein, which is responsible for virus attachment to sialic acid-containing receptors, varies in length due to differences in the sizes of the ORFs. An HN protein precursor of 616 aa has been found in avirulent but not in virulent NDV strains, whereas an HN protein of 571 aa can be detected in highly virulent strains only. An HN protein of 577 aa is present in virulent and avirulent strains. The F protein, which mediates virus-cell fusion, requires proteolytic activation at an internal cleavage site, whose amino acid composition determines cleavability by various proteases. Here, the functional significance of the length of the HN protein in combination with F protein cleavage sites typical for virulent (velogenic and mesogenic) or avirulent (lentogenic) strains was investigated. To this end, site-directed mutagenesis was used to construct recombinant NDV on the basis of an infectious clone of the lentogenic vaccine virus Clone-30. Only recombinant NDV expressing an F protein with a multibasic cleavage site typical of virulent strains was able to spread efficiently in cell culture, irrespective of the size of the HN protein. Moreover, as determined by the intracerebral pathogenicity index (ICPI) in 1-day-old, specific-pathogen-free chickens, pathogenicity was influenced by the cleavability of the F protein and not by the length of the HN protein. The maximum ICPI value obtained for these recombinants was 1.3, as compared to a possible maximum of 2. This demonstrates that the modifications introduced did not result in the conversion of the lentogenic Clone-30 to a velogenic strain with an ICPI value of >1.5 and suggests the involvement of additional virulence determinants that contribute to the pathogenicity of NDV.

Phylogenetic analysis reveals extensive evolution of avian paramyxovirus type 1 strains of pigeons (Columba livia) and suggests multiple species transmission.

Partial sequence and residue substitution analyses of the fusion protein gene were performed for 68 strains of avian paramyxovirus type 1 of pigeons (PPMV-1), an antigenic variant of Newcastle disease virus (NDV) of chickens, derived from 16 countries between 1978 and 2002. The majority of isolates clustered into a single genetic lineage, termed VIb/1, within genotype VI of NDV strains of chickens, whereas a small number of isolates that originated in Croatia after 1995, grouped in a highly diverged lineage, termed VIb/2, indicating a separate host-switching event from that of VIb/1 strains. Four distinct subgroups of lineage VIb/1, Iraqi (IQ), early European (EU/ea), North American (NA) and recent European (EU/re) have emerged and circulated in the past decades. Subgroup EU/ea and NA strains were responsible for the main streams of infection in the 1980s, while EU/re viruses for infections in the 1990s. The degree of genetic diversity of viruses in the early phase of the epizootic suggested a prolonged infection period of the pigeon-type viruses prior to the emergence of the disease in the early 1980s. Shared derived character analysis showed a close genetic relationship to Sudanese viruses from the mid-1970, suggesting that PPMV-1 viruses could be of African origin.

Occurrence of genotypes IV, V, VI and VIIa in Newcastle disease outbreaks in Germany between 1939 and 1995.

Forty-five velogenic Newcastle disease virus strains isolated in Germany between 1939 and 1995 were analysed by restriction enzyme digestion and sequencing to shed light on the relationships of past epizootics. Viruses derived from the period prior to 1970 belonged to a clade (IVea) of genotype IV comprising the earliest isolates from Europe, and could be isolated until the late seventies from poultry. Essex'70-like viruses, the prototype of genotype V, were already present at the beginning of the 1970-74 epizootic and in sporadic cases thereafter, indicating that these Newcastle disease outbreaks started in Western Europe. A genotype VI (subtype VIc) isolate was obtained in the early 1980s from a single outbreak in poultry. Outbreaks between 1993-95 were again part of a Western European epizootic caused by a genotype VIIa virus that was prevalent in the Far East.

Characterization of a recombinant Newcastle disease virus expressing the green fluorescent protein.

A recombinant Newcastle disease virus (NDV) expressing the green fluorescent protein (GFP) was generated by applying reverse genetics techniques. The GFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the fusion (F)- and hemagglutinin-neuraminidase genes in a full-length cDNA clone of NDV. This plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into cells stably expressing T7 RNA polymerase. The rescued virus was first propagated in embryonated eggs and the allantoic fluid was used to infect cells. Northern blot analysis of RNA isolated from infected cells demonstrated the proper transcription of the introduced GFP-mRNA. The appearance of GFP in live infected cells confirmed further the recovery of a recombinant NDV (rNDVGFP1) expressing the reporter gene. The expression of the heterologous gene was maintained stably for at least five passages in embryonated eggs. The replication kinetics in embryonated eggs and pathogenicity in chickens of rNDVGFP1 did not differ significantly from that of the parent virus. Using GFP autofluorescence, virus infected cells could be tracked easily in native preparations, organ explants and primary tracheal cell cultures. Taken together, these data demonstrate the use of GFP-expressing recombinant NDV for analysis of NDV dissemination and pathogenesis and indicate the potential usefulness of NDV as a vaccine vector.