Avian Paramyxovirus Type Research Articles

Molecular Characterization of Avian Paramyxovirus Type 1 (Newcastle Disease) Viruses Isolated from Pigeons Between 2000 and 2008 in Slovenia

Molecular Characterization of Avian Paramyxovirus Type 1 (Newcastle Disease) Viruses Isolated from Pigeons Between 2000 and 2008 in Slovenia

Infection dynamics of highly pathogenic avian influenza and virulent avian paramyxovirus type 1 viruses in chickens, turkeys and ducks

A range of virus doses were used to infect 3-week-old chickens, turkeys and ducks intranasally/intraocularly, and infection was confirmed by the detection of virus shedding from the buccal or cloacal route by analysis of swabs collected using real-time reverse transcriptase-polymerase chain reaction assays. The median infectious dose (ID50) and the median lethal dose (LD50) values for two highly pathogenic avian influenza (HPAI) viruses of H5N1 and H7N1 subtypes and one virulent Newcastle disease virus (NDV) were determined for each virus and host combination. For both HPAI viruses, turkeys were >100-fold more susceptible to infection than chickens, while both these hosts were >10-fold more susceptible to H5N1 virus than the H7N1 virus. All infected chickens and turkeys died. Ducks were also much more readily infected with the H5N1 virus (ID50≤101 median embryo infective dose [EID50]) than the H7N1 virus (ID50=104.2 EID50). However, the most notable difference between the two viruses was their virulence for ducks, with a LD50 of 103 EID50 for the H5N1 virus, but no deaths in ducks being attributed to infection with H7N1 virus even at the highest dose (106 EID50). For both HPAI virus infections of ducks, the ID50 was lower than the LD50, indicating that infected birds were able to survive and thus excrete virus over a longer period than chickens and turkeys. The NDV strain used did not appear to establish infection in ducks even at the highest dose used (106 EID50). Some turkeys challenged with 106 EID50, but not other doses, of NDV excreted virus for a number of days (ID50=104.6 EID50), but none died. In marked contrast, chickens were shown to be extremely susceptible to infection and all infected chickens died (ID50/LD50=101.9 EID50).

The Viral Replication Complex Is Associated with the Virulence of Newcastle Disease Virus

Virulent strains of Newcastle disease virus ([NDV] also known as avian paramyxovirus type 1) can be discriminated from low-virulence strains by the presence of multiple basic amino acid residues at the proteolytic cleavage site of the fusion (F) protein. However, some NDV variants isolated from pigeons (pigeon paramyxovirus type 1 [PPMV-1]) have low levels of virulence, despite the fact that their F protein cleavage sites contain a multibasic amino acid sequence and have the same functionality as that of virulent strains. To determine the molecular basis of this discrepancy, we examined the role of the internal proteins in NDV virulence. Using reverse genetics, the genes encoding the nucleoprotein (NP), phosphoprotein (P), matrix protein (M), and large polymerase protein (L) were exchanged between the nonvirulent PPMV-1 strain AV324 and the highly virulent NDV strain Herts. Recombinant viruses were evaluated for their pathogenicities and replication levels in day-old chickens, and viral genome replication and plaque sizes were examined in cell culture monolayers. We also tested the contributions of the individual NP, P, and L proteins to the activity of the viral replication complex in an in vitro replication assay. The results showed that the replication proteins of Herts are more active than those of AV324 and that the activity of the viral replication complex is directly related to virulence. Although the M protein affected viral replication in vitro, it had only a minor effect on virulence.

Complete Genome Analysis of an Avian Paramyxovirus Type 1 Strain Isolated in 1994 from an Asymptomatic Black-Headed Gull (Larus ridibundus) in Southern Sweden

Complete Genome Analysis of an Avian Paramyxovirus Type 1 Strain Isolated in 1994 from an Asymptomatic Black-Headed Gull (Larus ridibundus) in Southern Sweden

Complete Genome Analysis of an Avian Paramyxovirus Type 1 Strain Isolated in 1994 from an Asymptomatic Black-Headed Gull (Larus ridibundus) in Southern Sweden

The complete genome sequence of an avian paramyxovirus serotype 1 (APMV-1) isolated from a black-headed gull (Larus ridibundus) in Sweden was determined and compared with other APMV-1 sequences. Sequence analyses showed that this isolate consists of six genes in the order 3'-NP-P-M-F-HN-L-5', is 15,186 nucleotides long, and contains a typical, avirulent fusion protein cleavage site. It was also shown to have a hemagglutinin-neuraminidase protein with a length of 585 amino acids (aa) instead of the expected 616 aa. Phylogenetic analyses showed that the isolate belongs to genotype I, and the relationship with some other, known APMV-1 virus sequences was revealed. Waterfowl have been considered to act as a reservoir for APMV-1 and, therefore, it is important to broaden the knowledge of viruses circulating within this population.

Development of an L gene real-time reverse-transcription PCR assay for the detection of avian paramyxovirus type 1 RNA in clinical samples

A real-time reverse-transcription PCR (rRT-PCR) that targets a region of the polymerase (L) gene was developed to detect all known lineages of avian paramyxovirus type 1 (APMV-1), also known as Newcastle disease virus (NDV). A panel of 23 viruses representing the current known phylogenetic diversity of the APMV-1 population with a bias towards the more recent European strains, which had been grown in embryonated fowls' eggs, were tested. A range of positive and negative clinical samples (n = 350) provided by the National Reference Laboratory and International Reference Laboratory at VLA Weybridge were also tested. Positive clinical material included samples considered representative of lineages 3, 4 and 5 obtained from chickens, ducks, pigeons and partridges. The negative sample population was obtained from chickens, turkeys and ducks. The APMV-1 L gene rRT-PCR gave high relative sensitivity (96.05%) and specificity (98.18%) when compared with virus isolation in embryonated fowls' eggs. It is proposed that this assay could provide a first-line screening tool for the detection of APMV-1 in clinical samples.

Status of Wild Birds in Bulgarian Zoos with Regard to Orthomyxovirus and Paramyxovirus Type 1 Infections

Newcastle disease virus (NDV) and avian influenza virus (AIV) are pathogens of major economic and social importance, and the diseases they cause are often devastating, particularly in domestic poultry. Both viruses are naturally found in a wide variety of wild birds, particularly aquatic species, where asymptomatic infection typically occurs. Wild birds are therefore considered to be a natural reservoir for both viruses. Wild birds kept in captivity are in an environment that promotes transmission of infection with both influenza and Newcastle disease viruses. This report describes a survey for the detection of antibodies against Newcastle disease and avian influenza A viruses using the hemagglutination inhibition test in samples from 88 wild birds from 38 species in four Bulgarian zoos. Samples with positive results against NDV were also tested against avian paramyxovirus type 3 (APMV-3). Real-time reverse-transcriptase PCR was also performed to detect viral RNA of NDV and AIV among 127 wild birds from 57 species from the same zoos. In 13 samples from seven avian species (ten birds from the family Phasianidae, two from the family Numidae, and one from the family Columbidae), antibodies against APMV-1 were detected. Seven birds, whose sera were APMV-1 positive, had been vaccinated. The other six birds (five Phasianidae representatives and one of the Columbidae family) had no immunization history. No antibodies against both H5 and H7 AIV and against APMV-3 were detected, and no RNA of NDV and AIV were detected.

Inhibition of Newcastle disease virus replication by RNA interference targeting the matrix protein gene in chicken embryo fibroblasts

Newcastle disease (ND) is an infectious viral disease of birds caused by the Newcastle disease virus (NDV), also known as avian paramyxovirus type 1 (AMPV-1), which leads to severe economic losses in the poultry industry worldwide. In this study, the application of RNA interference (RNAi) for inhibiting the replication of NDV in cell culture by targeting the viral matrix protein gene (M) is described. Two M-specific shRNA-expressing plasmid constructs, named pS M641 and pS M827, were evaluated for antiviral activity against the NDV strain NA-1 by cytopathic effects (CPE), virus titration and real-time RT-PCR. After 36 h of infection, both pS M641 and pS M827 reduced virus titers by 79.4- and 31.6-fold, respectively, and they down-regulated mRNA expression levels of the matrix protein gene M by 94.6% and 84.8%, respectively, in chicken embryo fibroblast (CEF) cells, while only pS M641 significantly decreased CPE, compared to the control group. These results indicated that the M gene 641 and 827 sites represent potential antiviral therapy targets, and RNAi targeting of the M gene could not only represent an effective treatment in Newcastle disease but also aid as a method for studying the replication of NDV.

A diagnostic method based on MGB probes for rapid detection and simultaneous differentiation between virulent and vaccine strains of avian paramyxovirus type 1

Newcastle disease virus (NDV), also designated avian paramyxovirus type 1 (APMV-1), is a serious pathogen of poultry, causing highly contagious Newcastle disease (ND), with high morbidity or mortality, depending on the strain. Accordingly, rapid and reliable detection of APMV-1 and differentiation between vaccine and virulent strains is of crucial importance for ND diagnosis and plays an important role in effective control of the disease. In this study, two real-time reverse transcription polymerase chain reaction (real-time RT-PCR) assays using minor groove-binding (MGB) probes were developed for broad range detection and simultaneous pathotyping of APMV-1. The two assays were evaluated for their ability to detect in allantoic fluids viral RNA of all known APMV-1 lineages. Additionally, the applicability of the developed assays was assessed by the detection and pathotype prediction of APMV-1 in swabs and organs. The assays demonstrated high analytical specificity, sensitivity and good reproducibility, with coefficients of variation ranging from 0.2% to 3.9% and from 0.6% to 7.2% for intra-assay and inter-assay variability, respectively. The results indicated the suitability of both assays as a complementary method for rapid screening and typing of APMV-1.

Complete Genome Sequence of Avian Paramyxovirus (APMV) Serotype 5 Completes the Analysis of Nine APMV Serotypes and Reveals the Longest APMV Genome

BackgroundAvian paramyxoviruses (APMV) consist of nine known serotypes. The genomes of representatives of all APMV serotypes except APMV type 5 have recently been fully sequenced. Here, we report the complete genome sequence of the APMV-5 prototype strain budgerigar/Kunitachi/74.Methodology/Principal FindingsAPMV-5 Kunitachi virus is unusual in that it lacks a virion hemagglutinin and does not grow in the allantoic cavity of embryonated chicken eggs. However, the virus grew in the amniotic cavity of embryonated chicken eggs and in twelve different established cell lines and two primary cell cultures. The genome is 17,262 nucleotides (nt) long, which is the longest among members of genus Avulavirus, and encodes six non-overlapping genes in the order of 3′N-P/V/W-M-F-HN-L-5′ with intergenic regions of 4–57 nt. The genome length follows the ‘rule of six’ and contains a 55-nt leader sequence at the 3′end and a 552 nt trailer sequence at the 5′ end. The phosphoprotein (P) gene contains a conserved RNA editing site and is predicted to encode P, V, and W proteins. The cleavage site of the F protein (G-K-R-K-K-R↓F) conforms to the cleavage site motif of the ubiquitous cellular protease furin. Consistent with this, exogenous protease was not required for virus replication in vitro. However, the intracerebral pathogenicity index of APMV-5 strain Kunitachi in one-day-old chicks was found to be zero, indicating that the virus is avirulent for chickens despite the presence of a polybasic F cleavage site.Conclusions/SignificancePhylogenetic analysis of the sequences of the APVM-5 genome and proteins versus those of the other APMV serotypes showed that APMV-5 is more closely related to APMV-6 than to the other APMVs. Furthermore, these comparisons provided evidence of extensive genome-wide divergence that supports the classification of the APMVs into nine separate serotypes. The structure of the F cleavage site does not appear to be a reliable indicator of virulence among APMV serotypes 2–9. The availability of sequence information for all known APMV serotypes will facilitate studies in epidemiology and vaccinology.

Emergence of a new genetic lineage of Newcastle disease virus in West and Central Africa—Implications for diagnosis and control

Newcastle disease (ND) is an OIE listed disease caused by virulent avian paramyxovirus type 1 (APMV-1) strains, which affect many species of birds and may cause severe economic losses in the poultry sector. The disease has been officially and unofficially reported in many African countries and still remains the main poultry disease in commercial and rural chickens of Africa. Unfortunately, virological and epidemiological information concerning ND strains circulating in the Western and Central regions of Africa is extremely scarce. In the present study, sequence analysis, pathotyping and detailed genetic characterization of virulent ND strains detected in rural poultry in West and Central Africa revealed the circulation of a new genetic lineage, distinguishable from the lineages described in the Eastern and Southern parts of the continent. Several mismatches were observed in the segment of the matrix gene targeted by the primers and probe designed for the molecular detection of APMV-1, which were responsible for the false negative results in the diagnostic test conducted. Furthermore, deduced amino acid sequences of the two major antigens eliciting a protective immune response (F and HN glycoprotein) revealed protein similarities <90% if compared to some common vaccine strains. Distinct mutations located in the neutralizing epitopes were revealed, indicating the need for detailed assessment of the efficacy of the current vaccines and vaccination practices in Africa. The present investigation provides important information on the epidemiology, diagnosis and control of NDV in Africa and highlights the importance of supporting surveillance in developing countries for transboundary animal diseases.

Outbreak of New castle disease due to pigeon paramyxovirus type 1 in grey partridges (Perdix perdix) in Scotland in October 2006

In October 2006, following an initially non-statutory disease investigation affecting 12-week-old grey partridges (Perdix perdix), an outbreak of Newcastle disease due to infection with the avian paramyxovirus type 1 virus...

Development of a real-time reverse-transcription PCR for the detection and simultaneous pathotyping of Newcastle disease virus isolates using a novel probe

A real-time reverse-transcription PCR (RRT-PCR) was developed to detect and pathotype avian paramyxovirus type 1 (APMV-1), also known as Newcastle disease virus (NDV), which had been grown in embryonated fowls' eggs. Two pairs of probes, VRP1 with ARP1 and VRP2 with ARP2, each with either the 'universal base' 2' deoxyinosine incorporated or both inosines and locked nucleic acids (LNAs) incorporated, were designed to detect, respectively, a diverse range of virulent and avirulent viral templates that included the region coding for the fusion protein cleavage site. Oligonucleotide VRP1 hybridised with 76 of the 84 virulent isolates tested, while VRP2 detected 82, including 17 isolates with five or six template-probe mismatches. An alternative conventional probe, VRP3, with no inosine bases or LNAs, failed to hybridise 7 of 13 isolates, all of which tested positive with VRP2. Real-time assays with ARP1 showed that it detected 21 of the 28 avirulent isolates tested, and ARP2 detected 22/28, including one present in a mixture with virulent NDV. Neither probe was able to detect those isolates that were classified in genogroup six. All probes were specific for detecting either virulent or avirulent NDV. A specific PCR fragment of the predicted size was obtained, using the primer set designed for this study, with the 112 NDV isolates tested, including those in genogroup six. This assay demonstrates a rapid means for simultaneous detection and pathotyping of notifiable avian disease due to NDV.

Complete genome sequences of avian paramyxovirus type 8 strains goose/Delaware/1053/76 and pintail/Wakuya/20/78

Complete consensus genome sequences were determined for avian paramyxovirus type 8 (APMV-8) strains goose/Delaware/1053/76 (prototype strain) and pintail/Wakuya/20/78. The genome of each strain is 15,342 nucleotides (nt) long, which follows the “rule of six”. The genome consists of six genes in the order of 3′-N-P/V/W-M-F-HN-L-5′. The genes are flanked on either side by conserved transcription start and stop signals, and have intergenic regions ranging from 1 to 30 nt. The genome contains a 55 nt leader region at the 3′-end and a 171 nt trailer region at the 5′-end. Comparison of sequences of strains Delaware and Wakuya showed nucleotide identity of 96.8% at the genome level and amino acid identities of 99.3%, 96.5%, 98.6%, 99.4%, 98.6% and 99.1% for the predicted N, P, M, F, HN and L proteins, respectively. Both strains grew in embryonated chicken eggs and in primary chicken embryo kidney cells, and 293T cells. Both strains contained only a single basic residue at the cleavage activation site of the F protein and their efficiency of replication in vitro depended on and was augmented by, the presence of exogenous protease in most cell lines. Sequence alignment and phylogenic analysis of the predicted amino acid sequence of APMV-8 strain Delaware proteins with the cognate proteins of other available APMV serotypes showed that APMV-8 is more closely related to APMV-2 and -6 than to APMV-1, -3 and -4.

Complete sequence of the genome of avian paramyxovirus type 9 and comparison with other paramyxoviruses

The complete genome consensus sequence was determined for avian paramyxovirus (APMV) serotype 9 prototype strain PMV-9/domestic Duck/New York/22/78. The genome is 15,438 nucleotides (nt) long and encodes six non-overlapping genes in the order of 3′-N-P/V/W-M-F-HN-L-5′ with intergenic regions of 0–30 nt. The genome length follows the “rule of six” and contains a 55-nt leader sequence at the 3′ end and a 47-nt trailer sequence at the 5′ end. The cleavage site of the F protein is I-R-E-G-R-I↓F, which does not conform to the conventional cleavage site of the ubiquitous cellular protease furin. The virus required exogenous protease for in vitro replication and grew only in a few established cell lines, indicating a restricted host range. Alignment and phylogenetic analysis of the predicted amino acid sequences of APMV-9 proteins with the cognate proteins of viruses of all five genera of family Paramyxoviridae showed that APMV-9 is more closely related to APMV-1 than to other APMVs. The mean death time in embryonated chicken eggs was found to be more than 120 h, indicating APMV-9 to be avirulent for chickens.

Isolation and identification of highly pathogenic avian influenza H5N1 virus from Houbara bustards (Chlamydotis undulata macqueenii) and contact falcons

Highly pathogenic influenza virus (HPAIV) H5N1 has caused mortality and morbidity in many species of domestic and wild birds. The Houbara bustard (Chlamydotis undulata macqueenii) is a solitary bird that inhabits semi-desert regions. It is known to be susceptible to avianpox, avian paramyxovirus type 1, and low-pathogenicity avian influenza H9N2. We report an outbreak of H5N1 HPAIV in Houbara bustards, which were introduced into the Kingdom of Saudi Arabia for falconry purposes. Ninety-three per cent mortality (38 out of 41 birds) in the infected Houbara bustard flock and about 62.5% mortality (10 out of 16 birds) in falcons that came in contact with these birds were observed. Pooled cloacal and tracheal swabs from Houbara bustards as well as visceral organ homogenates collected in Houbara bustards and falcons were tested by real-time reverse transcriptase-polymerase chain reaction, and virus isolation was attempted in specific pathogen free hens’ eggs. The viruses isolated were characterized as HPAIV H5N1. Phylogenetic analysis of the haemagglutinating and Neuraminidase (NA) genes revealed that the viruses isolated from Houbara bustards and falcons were closely related to each other and to Kuwaiti H5N1 strains isolated in 2007. Interestingly, they were genetically distinguishable from the co-circulating A/H5N1 viruses in Kingdom of Saudi Arabia causing outbreaks in domestic birds. This case emphasizes the need for surveillance of this endangered species in its natural habitat.

Molecular characterization and complete genome sequence of avian paramyxovirus type 4 prototype strain duck/Hong Kong/D3/75

BackgroundAvian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds throughout the world. All APMVs, except avian metapneumovirus, are classified in the genus Avulavirus of the family Paramyxoviridae. At present, the APMVs of genus Avulavirus are divided into nine serological types (APMV 1–9). Newcastle disease virus represents APMV-1 and is the most characterized among all APMV types. Very little is known about the molecular characteristics and pathogenicity of APMV 2–9.ResultsAs a first step towards understanding the molecular genetics and pathogenicity of APMV-4, we have sequenced the complete genome of APMV-4 strain duck/Hong Kong/D3/75 and determined its pathogenicity in embryonated chicken eggs. The genome of APMV-4 is 15,054 nucleotides (nt) in length, which is consistent with the "rule of six". The genome contains six non-overlapping genes in the order 3'-N-P/V-M-F-HN-L-5'. The genes are flanked on either side by highly conserved transcription start and stop signals and have intergenic sequences varying in length from 9 to 42 nt. The genome contains a 55 nt leader region at 3' end. The 5' trailer region is 17 nt, which is the shortest in the family Paramyxoviridae. Analysis of mRNAs transcribed from the P gene showed that 35% of the transcripts were edited by insertion of one non-templated G residue at an editing site leading to production of V mRNAs. No message was detected that contained insertion of two non-templated G residues, indicating that the W mRNAs are inefficiently produced in APMV-4 infected cells. The cleavage site of the F protein (DIPQR↓F) does not conform to the preferred cleavage site of the ubiquitous intracellular protease furin. However, exogenous proteases were not required for the growth of APMV-4 in cell culture, indicating that the cleavage does not depend on a furin site.ConclusionPhylogenic analysis of the nucleotide sequences of viruses of all five genera of the family Paramyxoviridae showed that APMV-4 is more closely related to the APMVs than to other paramyxoviruses, reinforcing the classification of all APMVs in the genus Avulavirus of the family Paramyxoviridae.

Full-length genome sequence of avian paramyxovirus type 4 isolated from a mallard duck

Avian paramyxovirus (APMV) consists of nine serotypes, APMV-1 through -9, of which only APMV-1 and APMV-6 have been fully sequenced. Here, we present the complete 15,054 nt RNA genome of APMV-4 isolated from a mallard duck, which conformed to the "rule of six." The APMV-4 genome had six transcriptional units in the order 3'-NP-P/V-M-F-HN-L-5', which coded for the nucleocapsid (N), phospho- (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and large (L) proteins. Similar to APMV-1 but unlike APMV-6, APMV-4 lacked a small hydrophobic protein. The leader and trailer sequences were 55 and 17 nt in length, respectively, and the 12 nt-terminal regions of both ends of the APMV-4 genome were complementary. Using phylogenetic analysis, APMV-4 was classified as a member of the genus Avulavirus, and was more closely related to APMV-1 than to APMV-2 or APMV-6. These results may help establish the taxonomic position of Paramyxoviridae, Avulavirus members.

Complete sequence of the genome of avian paramyxovirus type 2 (strain Yucaipa) and comparison with other paramyxoviruses

The complete RNA genome sequence of avian paramyxovirus (APMV) serotype 2, strain Yucaipa isolated from chicken has been determined. With genome size of 14,904 nucleotides (nt), strain Yucaipa is consistent with the “rule of six” and is the smallest virus reported to date among the members of subfamily Paramyxovirinae. The genome contains six non-overlapping genes in the order 3′-N-P/V-M-F-HN-L-5′. The genes are flanked on either side by highly conserved transcription start and stop signals and have intergenic sequences varying in length from 3 to 23 nt. The genome contains a 55 nt leader sequence at 3′ end and a 154 nt trailer sequence at 5′ end. Alignment and phylogenetic analysis of the predicted amino acid sequences of strain Yucaipa proteins with the cognate proteins of viruses of all of the five genera of family Paramyxoviridae showed that APMV-2 strain Yucaipa is more closely related to APMV-6 than APMV-1.

Isolation and pathogenicity identification of avian paramyxovirus serotype 1 (Newcastle disease) virus from a Japanese quail flock in Iran

Newcastle disease virus (NDV) was isolated and identified for the first time from a flock of Japanese quail suspected to ND in Iran. Birds of the flock aged variously, and some of them died from the disease. Loss of appetite, weakness and decrease in egg production, diarrhea and nervous symptoms were the main clinical signs in the birds. Hemorrhagic lesions of the intestinal tracts and proventriculus were found in gross pathology investigations. Samples were taken for bacteriology, parasitology and virology. The results of parasitology and bacteriology examinations were negative. The virus was isolated only from brain samples and was identified as Avian Paramyxovirus type 1 (APMV-1) by HI test using NDV-Specific antibody. The pathogenicity of the virus was identified by mean death time (MDT) in embryonated chicken eggs, intracerebral pathogenicity index (ICPI) and intravenous pathogenicity index (IVPI) in chickens. Values 40, 1.62 and 2.31 were obtained for MDT, ICPI and IVPI respectively. These results indicate that isolated APMV-1 belongs to NDV velogenic strains with severe pathogenicity. According to these findings vaccination against NDV in quail farms of Iran is highly recommended.