Experimental pigeon paramyxovirus-1 infection in chicken: evaluation of infectivity, clinical and pathological manifestations and diagnostic methods.

Several pigeon paramyxovirus-1 (PPMV-1) outbreaks in feral pigeons were described recently in Switzerland. The potential of PPMV-1 to induce the notifiable Newcastle disease in chickens is discussed controversially. Therefore, in order to study epidemiologically relevant parameters such as the kinetics of PPMV-1 replication and shedding as well as seroconversion after infection, chickens were infected experimentally with a Swiss PPMV-1 isolate. This generated also defined sample material for the comparison of diagnostic tests. The infectivity of the Swiss PPMV-1 isolate for chickens was demonstrated successfully by virus shedding after experimental inoculation. Our data suggest that long-lasting shedding for up to 60 days can occur in chickens infected with PPMV-1. The isolate used here was of low pathogenicity for chickens. Different quantitative reverse transcription PCR assays were evaluated with a set of Swiss PPMV-1 isolates, and various samples from experimentally infected chickens were analysed with respect to their suitability for viral RNA detection. At 14 days post-infection, virus genome was detected mainly in spleen, caecal tonsils, heart, cloacal swabs, liver, proventriculus, duodenum and kidney tissue samples. Overall, the level of virus replication was low. Not all assays used routinely in diagnostics were capable of detecting viral genome from the isolates tested. Possible explanations are the genetic divergence of PPMV-1 and the low level of viral RNA in the samples. In contrast, two methods that are not used routinely proved more suitable for virus-genome detection. Importantly, the collection of material from various different organs is recommended, in addition to the kidney and brain analysed routinely. In conclusion, this study shows that there is a need to reconsider the type of samples and the protocols used for the detection of PPMV-1 RNA in chickens.

Comparative clinico-pathological assessment of velogenic (sub-genotype VIIi) and mesogenic (sub-genotype VIm) Avian avulavirus 1 in chickens and pigeons.

Newcastle disease (ND), caused by virulent Avian avulavirus 1 (AAvV 1), affects a wide range of avian species worldwide. Recently, several AAvVs of diverse genotypes have emerged with varying genomic and residue substitutions, and subsequent clinical impact on susceptible avian species. We assessed the clinico-pathological influence of two different AAvV 1 pathotypes [wild bird originated-velogenic strain (sub-genotype VIIi, MF437287) and feral pigeon originated-mesogenic strain (sub-genotype VIm, KU885949)] in commercial broiler chickens and pigeons. The velogenic strain caused 100% mortality in both avian species while the mesogenic strain caused 0% and 30% mortality in chickens and pigeons, respectively. Both strains showed tissue tropism for multiple tissues including visceral organs; however, minor variances were observed according to host and pathotype. The observed gross and microscopic lesions were typical of AAvV 1 infection. Utilizing oropharyngeal and cloacal swabs, a comparable pattern of viral shedding was observed for both strains from each of the infected individuals of both avian species. The study concludes a varying susceptibility of chickens and pigeons to different wild bird-originated AAvV 1 pathotypes and, therefore, suggests continuous monitoring and surveillance of currently prevailing strains for effective control of the disease worldwide, particularly in disease-endemic countries.

[Molecular-genetic characterization of Avian avulavirus 20 strains isolated from wild birds.]

INTRODUCTION: Previously unknown paramyxovirus strains were isolated from wild birds in 2013-2014 in Kazakhstan and subsequently identified as representatives of the novel Avian avulavirus 20 species. The aims and tasks were molecular genetic characterization of novel avulaviruses and investigation of their phylogenetic relationships. MATERIAL AND METHODS: Embryonated chicken eggs were inoculated with cloacal and tracheal swabs from wild birds with subsequent virus isolation. The complete nucleotide sequences of viral genomes were obtained by massive parallel sequencing with subsequent bioinformatics processing. RESULTS: By initial infection of chicken embryos with samples from 179 wild birds belonging to the Anatidae, Laridae, Scolopacidae and Charadriidae families, 19 hemagglutinating agents were isolated, and five of them were identified as representatives of new viral species. The study of their sequenced genomes revealed their similarity in size, but there was a significant genetic variability within the species. 2,640 nucleotide substitutions were identified and 273 of them were nonsynonymous, influencing the protein structure of viruses. It was shown that isolates Avian avulavirus 20/black-headed gull/Balkhash/5844/2013 and Avian avulavirus 20 /great black-headed gull/Atyrau/5541/2013 were 86% and 95% respectively identical to the previously described reference strain, indicating a significant evolutionary divergence within species. DISCUSSION: The authors suggest the existence of two independent lineages - the Caspian, represented by the reference strain Aktau/5976 and Atyrau/5541, as well as the second, geographically significantly distant Balkhash lineage. CONCLUSION: The study confirms the role of the birds of the Laridae family as the main reservoir of Avian avulavirus 20 in the avifauna that plays a key role in maintaining viruses of the genus Avulavirus in the biosphere and is a potential natural source for the emergence of new viral variants. Continuous surveillance of them in the wild is one of the most important tasks in ensuring the safety of the poultry industry.

Pathological and molecular findings of avian avulavirus type 1 outbreak in pigeons (Columba livia) of southern Brazil / Achados patológicos e moleculares da infecção por avulavirus aviário tipo 1 em pombos (Columba livia) do sul do Brasil

The Newcastle disease, caused by avian avulavirus type 1 strains (APMV-1) is an important avian disease involved into high rates of mortality and economic losses. Several outbreaks have been reported over the last 30 years in Columbiformes in different parts of the world, caused by a adapted variant strain of AAvV-1, called pigeon paramyxovirus type 1 (PPMV-1). A high mortality associated with an outbreak was analyzed in free-living pigeons (Columba livia) in a public square in Porto Alegre in Southern Brazil. A total of 24 pigeons moribund or freshly dead, within five weeks interval were submitted to necropsy, histopathological, immunohistochemical (anti-Newcastle), and RT-PCR followed by sequencing of the amplification products analysis. They presented neurological signs, non-suppurative encephalitis and encephalomyelitis, and mononuclear inflammatory infiltrate in different organs. Immunohistochemical analysis in nine pigeons tissue showed that anti-Newcastle was expressed in brain, kidney, liver and pancreas. The RT-PCR test for the M protein of Newcastle disease virus was positive in six pigeons. The differential diagnosis of Influenza, West Nile, Mycoplasma gallisepticum and Mycoplasma synoviae in all pigeons presented negative results. The sequence of amino acids in the cleavage site region of the F protein was 112RRQKRF117 classifying the strain as virulent. The phylogenetic analysis classified this virus strain into Class II and VI genotype.(AU)

A doença de Newcastle, causada por cepas de avulavirus aviário tipo 1 (AAvV-1), é uma doença de aves importante por causar altos índices de mortalidade e perdas econômicas. Vários surtos têm sido relatados ao longo de 30 anos em aves da ordem Columbiformes, em diferentes partes do mundo, causados por uma cepa variante específica de AAvV-1, denominada Pigeon paramyxovirus tipo 1 (PPMV-1). Foi analisado um surto de mortalidade em pombos domésticos (Columba livia), provenientes de uma praça pública em Porto Alegre, no Sul do Brasil. Vinte e quatro aves moribundas ou mortas foram submetidas, no intervalo de cinco semanas, ao exame de necropsia, exame histopatológico, imuno-histoquímico anti-Newcastle, RT-PCR e sequenciamento. Apresentaram sinais neurológicos, encefalite e encefalomielite não supurativas, além de infiltrado inflamatório mononuclear em diversos órgãos. Nove aves demonstraram exame imuno-histoquímico positivo em órgãos como cérebro, rim, fígado e pâncreas. Seis aves foram positivas no exame de RT-PCR para a proteína M do vírus da Doença de Newcastle. Nos exames de diagnósticos diferenciais de Influenza, West Nile, Mycoplasma gallisepticum e Mycoplasma synoviae, todas as aves testadas foram negativas. A sequência dos aminoácidos na região do sítio de clivagem da proteína foi 112RRQKRF117, classificando a cepa como virulenta. De acordo com a análise filogenética o vírus identificado foi classificado como pertencente à classe II e ao genótipo VI.(AU)

Characterization of avian paramyxovirus type 6 isolated from a Eurasian teal in the intersection of migratory flyways in Russia.

The complete genome sequence was determined for avian paramyxovirus (APMV-6) serotype 6 strain teal/Chany/455/2009, isolated from a teal (Anas crecca) in Siberia. Siberia is crossed by four major migration flyways and represents the major breeding area for many wild bird species in the Palearctic. Strain teal/Chany/455/2009 is genetically closely related to Kazakh and Chinese strains and belongs to the genetic group of duck/Hong Kong/18/199/77-like APMV-6 viruses. We show that the virus has low pathogenic potential according to genetic markers and animal model experiments.

Mutations in the fusion protein cleavage site of avian paramyxovirus serotype 2 increase cleavability and syncytium formation but do not increase viral virulence in chickens.

Avian paramyxovirus serotype 2 (APMV-2) is one of the nine serotypes of APMV, which infect a wide variety of avian species around the world. In this study, we constructed a reverse genetics system for recovery of infectious recombinant APMV-2 strain Yucaipa (APMV-2/Yuc) from cloned cDNA. The rescued recombinant virus (rAPMV-2) resembled the biological virus in growth properties in vitro and in pathogenicity in vivo. The reverse genetics system was used to analyze the role of the cleavage site of the fusion (F) protein in viral replication and pathogenesis. The cleavage site of APMV-2/Yuc (KPASR↓F) contains only a single basic residue (position -1) that matches the preferred furin cleavage site [RX(K/R)R↓]. (Underlining indicates the basic amino acids at the F protein cleavage site, and the arrow indicates the site of cleavage.) Contrary to what would be expected for this cleavage sequence, APMV-2 does not require, and is not augmented by, exogenous protease supplementation for growth in cell culture. However, it does not form syncytia, and the virus is avirulent in chickens. A total of 12 APMV-2 mutants with F protein cleavage site sequences derived from APMV serotypes 1 to 9 were generated. These sites contain from 1 to 5 basic residues. Whereas a number of these cleavage sites are associated with protease dependence and lack of syncytium formation in their respective native viruses, when transferred into the APMV-2 backbone, all of them conferred protease independence, syncytium formation, and increased replication in cell culture. Examination of selected mutants during a pulse-chase experiment demonstrated an increase in F protein cleavage compared to that for wild-type APMV-2. Despite the gains in cleavability, replication, and syncytium formation, analysis of viral pathogenicity in 9-day-old embryonated chicken eggs, 1-day-old chicks, and 2-week-old chickens showed that the F protein cleavage site mutants did not exhibit increased pathogenicity and remained avirulent. These results imply that structural features in addition to the cleavage site play a major role in the cleavability of the F protein and the activity of the cleaved protein. Furthermore, cleavage of the F protein is not a determinant of APMV-2 pathogenicity in chickens.

Pathogenesis of two strains of avian paramyxovirus serotype 2, Yucaipa and Bangor, in chickens and turkeys.

Nine serologic types of avian paramyxovirus (APMV) have been recognized. Newcastle disease virus (APMV-1) is the most extensively characterized virus, while relatively little information is available for the other APMV serotypes. In the present study, we examined the pathogenicity of two strains of APMV-2, Yucaipa and Bangor, in 9-day-old embryonated chicken eggs, 1-day-old specific-pathogen-free (SPF) chicks, and 4-wk-old SPF chickens and turkeys. The mean death time in 9-day-old embryonated chicken eggs was more than 168 hr for both strains, and their intracerebral pathogenicity index (ICPI) was zero, indicating that these viruses are nonpathogenic in chickens. When inoculated intracerebrally in 1-day-old chicks, neither strain caused disease or replicated detectably in the brain. This suggests that the zero ICPI value of APMV-2 reflects the inability of the virus to grow in neural cells. Groups of twelve 4-wk-old SPF chickens and turkeys were inoculated oculonasally with either strain, and three birds per group were euthanatized on days 2, 4, 6, and 14 postinoculation for analysis. There were no overt clinical signs of illnesses, although all birds seroconverted by day 6. The viruses were isolated predominantly from the respiratory and alimentary tracts. Immunohistochemistry studies also showed the presence of a large amount of viral antigens in epithelial linings of respiratory and alimentary tracts. There also was evidence of systemic spread even though the cleavage site of the viral fusion glycoprotein does not contain the canonical furin protease cleavage site.