Characterisation of a type 1 Avian Paramyxovirus belonging to a divergent group

Newcastle disease, induced by a type 1 Avian Paramyxovirus (APMV-1), is one of the most serious poultry diseases. APMV-1 are divided into two classes based on genetic analysis: class II strains have been recovered from wild or domestic birds and include virulent and avirulent isolates whereas class I strains have been mainly isolated from wild birds and are avirulent. Within class I, a new proposed genotype has recently been reported. The only full genome strain of this group is presently characterised from the point of view of codon usage with reference to class I and class II specificities. Class-specific residues were identified on HN and F proteins that are the two major proteins involved in cell attachment and pathogenicity. Comparison of protein patterns and codon usage for this newly identified APMV-1 strain indicates it is similar to class I viruses but contains a few characteristics close to the viruses of class II. Transmission of viruses from this recently identified divergent group from wild birds to domestic birds could have a major impact on the domestic poultry industry.     

A survey for paramyxoviruses in caged birds, wild birds, and poultry in New Zealand

AIMS: To determine the presence of avian paramyxovirus (APMV) types 1, 2, and 3 in caged and wild birds, and APMV-2 and -3 in poultry in New Zealand. METHODS: Blood samples collected from caged (231) and wild birds (522) from various regions of New Zealand in 1997-99 were tested by haemagglutination inhibition (HI) test for antibodies to APMV types 1, 2, and 3. Blood samples collected from 1778 commercial poultry in 1996-99 were tested for APMV-2 and APMV-3 antibodies and the samples that reacted with APMV-3 antigen were tested for antibodies to APMV-1. Isolation of APMV was attempted from cloacal swabs collected from 116 of the caged birds and 175 of the wild birds sampled. RESULTS: Antibodies to APMV types 1, 2, and 3 were detected in 4.8, 1.7, and 2.6%, respectively, of caged bird samples. The majority of these caged birds were 'exotic' or 'fancy' poultry breeds. Amongst wild birds, 4.2% had titres to APMV-2 and over half of these were passerine birds; 1.7% of the samples had titres to APMV-1 and 0.8% to APMV-3 antigen. No virus was isolated from any of the cloacal swabs tested. Of the 1778 poultry serum samples tested, only 5 reacted with APMV-3 antigen and these were later found to be cross-reactions to APMV-1. No reactions were detected with APMV-2 antigen. CONCLUSIONS: APMV-1 is present in caged birds, wild birds, and poultry of New Zealand. There is no conclusive evidence of the presence of APMV-2 and APMV-3 in poultry or APMV-3 in wild birds. The results do not provide conclusive evidence for the presence of APMV-2 in wild birds in New Zealand.     

The characterization of low pathogenic avian influenza viruses isolated from wild birds in northern Vietnam from 2006 to 2009

Due to concerns that wild birds could possibly spread H5N1 viruses, surveillance was conducted to monitor the types of avian influenza viruses circulating among the wild birds migrating to or inhabiting in northern Vietnam from 2006 to 2009. An H5N2 virus isolated from a Eurasian woodcock had a close phylogenetic relationship to H5 viruses recently isolated in South Korea and Japan, suggesting that H5N2 has been shared between Vietnam, South Korea, and Japan. An H9N2 virus isolated from a Chinese Hwamei was closely related to two H9N2 viruses that were isolated from humans in Hong Kong in 2009, suggesting that an H9N2 strain relevant to the human isolates had been transmitted to and maintained among the wild bird population in Vietnam and South China. The results support the idea that wild bird species play a significant role in the spread and maintenance of avian influenza and that this also occurs in Vietnam.     

Genomic sequences of low-virulence avian paramyxovirus-1 (Newcastle disease virus) isolates obtained from live-bird markets in North America not related to commonly utilized commercial vaccine strains

Avian paramyxovirus 1 (APMV-1), also referred to as Newcastle disease virus (NDV), variants of low virulence were isolated from chickens, ducks and other unidentified species found in live-bird markets of the northeastern United States. These isolates were characterized as APMV-1 by the hemagglutination-inhibition (HI) assay utilizing NDV-specific polyclonal antisera. However, the isolates failed to react with a monoclonal antibody that has specificity for a wide variety of APMV-1 isolates. Although only highly virulent isolates require reporting to international regulatory agencies, the ability to correctly identify APMV-1 types is important for control and regulatory purposes. Protein gel patterns of the purified isolates resembled previously reported APMV-1 and anti-NDV polyclonal sera recognized the viral proteins. For three isolates oligonucleotide primers specific for the nucleoprotein, fusion protein and polymerase genes of NDV were utilized to synthesize cDNA using viral RNA as a template. Approximately 12kb of the genome was subsequently sequenced for the three isolates that included the nucleoprotein, phosphoprotein, matrix protein, fusion (F) protein, hemagglutinin-neuraminidase protein genes and a 5' portion of the polymerase gene. The isolates had an F protein cleavage site sequence of ERQER/LVG indicating low-virulence viruses that phylogenetically separated with other unique NDV isolates designated as a lineage 6 genotype. Additionally, a four amino acid insert was detected in the predicted phosphoprotein which complies with the "rule of six" among paramyxoviruses. These APMV-1 genotypes have not been previously reported in North America and further substantiate the heterogeneous genetic nature of these commercially important pathogens found worldwide.     

Avian paramyxovirus serotype 1 isolates from the spinal cord of parrots display a very low virulence

The spinal cord of 32 psittacines suffering from proventricular dilatation disease (PDD) was investigated. In six cases, a virus was isolated which upon electron microscopic examination revealed morphological details typical of members of the Paramyxoviridae. All isolates were subsequently characterized as avian paramyxovirus serotype 1 (APMV-1) by type-specific polyclonal antisera. According to their reactivity with APMV-1 specific monoclonal antibodies, the six isolates shared epitopes within the haemagglutinin-neuraminidase spike protein, distinct from pigeon-type paramyxoviruses and the LaSota vaccine strain. This grouping was further corroborated by properties of the haemagglutinin: all isolates showed a very thermosensitive haemagglutination activity and were rapid eluters. Virulence of the APMV-1 isolates in 1-day-old specific pathogen free (spf) chicken was very low, with intracerebral pathogenicity indices between 0 and 0.1. In embryonated spf chicken eggs, psittacine isolates replicated to high titres (10(8.6)-10(10.7) EID50/ml). However, they exhibited a reduced lethality over an observation time of 7 days (10(6.1)-10(8.3) ELD50/ml). In a haemagglutination inhibition test with parrot sera from birds with no history of APMV-1 vaccination, sera reacted preferentially with two isolates compared with APMV-1 vaccine strains LaSota and B1. The other four isolates exhibited a differentiated reaction pattern with the parrot sera, indicating an antigenic inhomogeneity. This is the first report of isolating very low virulent APMV-1 from neuronal tissue of parrots and implications for a possible role in slow progressing disease will be discussed.     

Isolation, identification, and comparison of four isolates of avian paramyxovirus serotype 2 in China

Four Yucaipa-like viruses of avian paramyxovirus serotype 2 (APMV-2) were isolated in China from the imported Gouldian Finch (Chloebia gouldiae) and broilers in 1998-2002, and were named F4, F6, F8, and NK, respectively. Examined under electron microscope, the isolates were found to be round in shape and varying in size. The results of the hemagglutination inhibition test and indirect enzyme-linked immunosorbent assay (using monoclonal antibodies) showed some differences between the isolates and the reference strain Yucaipa. The isolates derived from chickens had a closer relationship to Yucaipa virus than did those of finches. Sequence comparison of the fusion gene and the haemagglutinin-neuraminidase gene showed similar results, although the variations were lesser among APMV-2 viruses in nucleotide and amino acid sequence. By sequence comparison, it was also revealed that at the molecular level the four virus strains belong to APMV-2, and that two of the strains were isolated from the same group of imported Gouldian Finches.     

Detection of paramyxoviruses in Magellanic penguins (Spheniscus magellanicus) on the Brazilian tropical coast

Aquatic migratory birds are a major vectors by which influenza viruses and paramyxoviruses are spread in nature. Magellanic penguins (Spheniscus magellanicus) are usually present on the southern shores of South America and can swim as far as the southern coast of Brazil in winter. In 2008, however, several Magellanic penguins were observed on the northeastern coast of Brazil. Paramyxoviruses were isolated from Magellanic penguins on the Espírito Santo state coast, approximately 4000 km from their breeding colonies, although influenza viruses were not detected. Among the paramyxoviruses, five Avulavirus isolates belonging to serotype APMV-2 and the serotype APMV-10, which was proposed by Miller et al. (2010), were identified. These results highlight the risks associated with the spread of paramyxoviruses between natural to non-natural habitats by birds exhibiting unusual migration patterns, and they document for the first time the presence of the APMV-2 and APMV-10 serotypes on penguins in Brazil. The local avifauna may become infected with these viruses through close contact between migratory and resident birds. Continued surveillance of virus incidence in these migratory populations of penguins is necessary to detect and prevent the potential risks associated with these unusual migration patterns.     

Mixed paramyxovirus infection of wild and domestic birds in Israel

Five cases of dual isolations of different serotypes of avian paramyxoviruses (APMV) from domestic and wild birds are described: one case of mixed infection by APMV-1 and APMV-4 and four cases of infection by APMV-1 and APMV-2 serotypes. The double infection was proven by consecutive isolations of two viruses from allantoic fluid samples derived from single swabs after their respective treatment by antisera against each suspected virus. The finding of double APMV infection in poultry farms appears to be important for epizootiology and pathogenesis of APMV-caused diseases.     

Virological and molecular epidemiological investigations into the role of wild birds in the epidemiology of influenza A/H5N1 in central Thailand

A serological and virological surveillance program to investigate the HPAI H5N1 virus in wild bird populations was undertaken from February 2007 to October 2008. The purpose of the survey was to investigate the infection status in free ranging wild birds in Banglane district, Nakhon Pathom province, central Thailand. Samples from wild birds were collected every two months. Choanal and cloacal swabs, serum and tissue samples were collected from 421 birds comprising 44 species. Sero-prevalence of the virus tested by H5N1 serum neutralization test (using a H5N1 virus clade 1; A/chicken/Thailand/vsmu-3-BKK/2004) was 2.1% (8 out of 385 samples; 95% CI 0.7, 3.5). Species that were antibody positive included rock pigeons (Columba livia), Asian pied starling (Gracupica contra), spotted dove (Streptopelia chinensis), oriental magpie robin (Copsychus saularis), blue-tailed bee-eater (Merops philippinus), myna (Acridotheres spp.), and pond heron (Ardeola spp.). Prevalence by H5N1 virus isolation was 0.5% (2 out of 421 samples; 95% CI 0.0, 1.1); the two H5N1 virus-positive samples were from Asian pied starling (Gracupica contra) and white vented myna (Acridotheres grandis). Positive virological samples were collected in June 2007 while all positive serology samples were collected between May and August except for one sample collected in December 2007. No positive samples were collected in 2008. Molecular studies revealed that the wild bird H5N1 viruses were closely related to poultry viruses isolated in other parts of Thailand. However, there was no poultry H5N1 prevalence study performed in the study site during the time of this wild bird survey. Interpretation of source of virus isolates would include spill-over of H5N1 viruses from contaminated sources due to movement of domestic poultry and/or fomites from other areas; or infection of wild birds within the outbreak locations and then translocation by wild bird movement and interaction with wild birds inhabiting distant locations.     

Paramyxovirus type 1 infections of racing pigeons: 1 characterisation of isolated viruses

Viruses isolated from field outbreaks of disease in racing pigeons in continental Europe and Great Britain were shown to be identical by serological tests using conventional chicken antisera and mouse monoclonal antibodies. The pigeon viruses showed high levels of cross-reaction to Newcastle disease virus (NDV) in haemagglutination inhibition tests and Madin-Darby bovine kidney cells infected with pigeon virus isolates bound three out of nine mouse monoclonal antibodies prepared against NDV Ulster 2C. These results confirm their classification in the paramyxovirus type 1 serotype of avian paramyxoviruses. However, the pigeon viruses could be distinguished from more classical paramyxovirus type 1 viruses by the significantly different titres obtained in haemagglutination inhibition tests, the failure of mouse monoclonal antibodies directed against the HN1 epitope of NDV Ulster 2C to inhibit their haemagglutinating activity and a unique binding pattern seen with the nine mouse monoclonal antibodies.     

Similarity of avian paramyxovirus serotype 1 isolates of low virulence for chickens obtained from contaminated poultry vaccines and from poultry flocks

At present Denmark has the status of a 'non-vaccinating' country for Newcastle disease and its poultry population should therefore be free of antibodies to avian paramyxovirus 1 (APMV-1). Three live avian vaccines against infectious bronchitis, avian encephalomyelitis, and chick anaemia which had been found to be contaminated with APMV-1 viruses of low virulence for chickens were examined. The vaccines were produced by the same company and the affected batches had been used in Denmark in 1996/97. Furthermore, APMV-1 isolates of low virulence were obtained from three commercial broiler breeder flocks, one of which had been vaccinated with two of the contaminated vaccines. The flocks belonged to the same hatchery organisation. A comparison of viral F0 gene sequences and typing of virus isolates with a panel of monoclonal antibodies showed that the vaccine and field isolates were identical.     

Mystery swine disease in The Netherlands: the isolation of Lelystad virus.

In early 1991, the Dutch pig-industry was struck by the so-called mystery swine disease. Large-scale laboratory investigations were undertaken to search for the etiological agent. We focused on isolating viruses and mycoplasmas, and we tested paired sera of affected sows for antibodies against ten known pig viruses. The mycoplasmas M. hyosynoviae, M. hyopneumoniae, and Acholeplasma laidlawii, and the viruses encephalomyocarditis virus and porcine enterovirus types 2 and 7 were isolated from individual pigs. An unknown agent, however, was isolated from 16 of 20 piglets and from 41 of 63 sows. This agent was characterised as a virus and designated Lelystad virus. No relationship between this virus and other viruses has yet been established. Of 165 sows reportedly afflicted by the disease, 123 (75 per cent) seroconverted to Lelystad virus, whereas less than 10 per cent seroconverted to any of the other virus isolates or to the known viral pathogens. Antibodies directed against Lelystad virus were also found in pigs with mystery swine disease in England, Germany, and in the United States. We conclude that infection with Lelystad virus is the likely cause of mystery swine disease.     

Characterization of H5N1 influenza viruses isolated from migratory birds in Qinghai province of China in 2006

Avian influenza H5N1 viruses pose a significant threat to human health because of their ability to infect humans directly. In the paper, three highly pathogenic H5N1 influenza viruses were isolated from three species of migratory birds in Qinghai Province of China in 2006. The analysis of the genome sequences indicated that the three isolates shared high homology with each other (94% to 99%). Three isolates shared a common ancestor and were closest to strains isolated from Qinghai and Siberia in 2005, but distinct from poultry viruses found in Southeast Asia. In experimental infection, all three viruses were highly pathogenic to chickens and mice. The results suggest that highly pathogenic avian influenza H5N1 viruses still exist in the migratory birds and could spread to other regions with wild bird migration.     

ISOLATION OF A HAEMAGGLUTINATING ADENO-LIKE VIRUS RELATED TO VIRUS 127 FROM AN AUSTRALIAN POULTRY FLOCK WITH AN EGG DROP SYNDROME

An egg drop syndrome within Australian broiler poultry is described. The syndrome was characterised by delayed onset of laying, a lower peak in egg production and a drop in egg production shortly after reaching peak production. Antibody to virus 127 was detected in 102 of 106 fowl serums tested. Two haemagglutinating viruses were isolated from one affected flock and one was subjected to further study. It was adenovirus-like on electron-microscopic examination and haemagglutination was not inhibited by a specific antiserum to Newcastle disease virus. An antiserum was raised in White Leghorn fowl against the isolate and this antiserum was found to cross-react with virus 127, a prototype virus of Egg Drop Syndrome 76.     

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.     

Experimental infection of hamsters with avian paramyxovirus serotypes 1 to 9

ABSTRACT: Avian paramyxoviruses (APMVs) are frequently isolated from domestic and wild birds throughout the world and are separated into nine serotypes (APMV-1 to -9). Only in the case of APMV-1, the infection of non-avian species has been investigated. The APMVs presently are being considered as human vaccine vectors. In this study, we evaluated the replication and pathogenicity of all nine APMV serotypes in hamsters. The hamsters were inoculated intranasally with each virus and monitored for clinical disease, pathology, histopathology, virus replication, and seroconversion. On the basis of one or more of these criteria, each of the APMV serotypes was found to replicate in hamsters. The APMVs produced mild or inapparent clinical signs in hamsters except for APMV-9, which produced moderate disease. Gross lesions were observed over the pulmonary surface of hamsters infected with APMV-2 & -3, which showed petechial and ecchymotic hemorrhages, respectively. Replication of all of the APMVs except APMV-5 was confirmed in the nasal turbinates and lungs, indicating a tropism for the respiratory tract. Histologically, the infection resulted in lung lesions consistent with bronchointerstitial pneumonia of varying severity and nasal turbinates with blunting or loss of cilia of the epithelium lining the nasal septa. The majority of APMV-infected hamsters exhibited transient histological lesions that self resolved by 14 days post infection (dpi). All of the hamsters infected with the APMVs produced serotype-specific HI or neutralizing antibodies, confirming virus replication. Taken together, these results demonstrate that all nine known APMV serotypes are capable of replicating in hamsters with minimal disease and pathology.