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To date, four subgroups of avian metapneumoviruses have been defined (AMPV-A, B, C and D) based on genetic and antigenic differences. The extent of infection in the three principal species (turkeys, chickens and ducks) by these subgroups is, however, not well defined. Here, a series of controlled and ethically approved experimental infections were performed in specific pathogen-free turkeys, chickens and ducks with each of the four AMPV subgroups. For subgroup C, one strain isolated from turkeys in the USA (turkey AMPV-C) and one isolated from ducks in France (duck AMPV-C) were compared. Globally, these extensive experimental trials demonstrated that AMPV-A, B, turkey C and D were well adapted to Galliformes, especially turkeys; however, chickens showed limited clinical signs and differences in seroconversion and transmission. Notably, chickens did not transmit AMPV-A to contacts and were shown for the first time to be susceptible to AMPV-D. The duck AMPV-C was well adapted to ducks; however, chickens and turkeys seroconverted and were positive by virus isolation. In addition, seroconversion of contact turkeys to duck AMPV-C demonstrated horizontal transmission of this virus in a non-palmiped species under our experimental conditions. Interestingly, in chickens and turkeys, duck AMPV-C isolation was possible despite a lack of detection of viral RNA. Likewise, the turkey AMPV-C virus was well adapted to turkeys yet was also isolated from chickens despite a lack of detection of viral RNA. These results would suggest a selection for viral genetic sequences that differ from the original strain upon adaptation to a 'non-conventional host'.

The aim of the present study was to determine whether avian metapneumovirus (aMPV)-related viruses were present in wild and synanthropic birds in Brazil. Therefore, we analysed samples from wild birds, feral pigeons and domestic chickens in order to perform a phylogenetic comparison. To detect the presence of aMPV, a nested reverse transcriptase-polymerase chain reaction was performed with the aim of amplifying a fragment of 270 bases for subtype A and 330 bases for subtype B, comprising the gene coding the G glycoprotein. Positive samples for aMPV subtypes A and B were found in seven (13.2%) different asymptomatic wild birds and pigeons (50%) that had been received at the Bosque dos Jequitibás Zoo Triage Center, Brazil. Also analysed were positive samples from 15 (12.9%) domestic chickens with swollen head syndrome from several regions of Brazil. The positive samples from wild birds, pigeons and domestic chickens clustered in two major phylogenetic groups: some with aMPV subtype A and others with subtype B. The similarity of the G fragment nucleotide sequence of aMPV isolated from chickens and synanthropic and wild avian species ranged from 100 to 97.5% (from 100 to 92.5% for the amino acids). Some positive aMPV samples, which were obtained from wild birds classified in the Orders Psittaciformes, Anseriformes and Craciformes, clustered with subtype A, and others from the Anas and Dendrocygma genera (Anseriformes Order) with subtype B. The understanding of the epizootiology of aMPV is very important, especially if this involves the participation of non-domestic bird species, which would add complexity to their control on farms and to implementation of vaccination programmes for aMPV.

Metapneumoviruses (MPVs) were first reported in avian species (aMPVs) in the late 1970s and in humans in 2001. Although aMPVs have been reported in Europe and Asia for over 20 years, the virus first appeared in the United States in 1996, leaving many to question the origin of the virus and why it proved to be a different subtype from those found elsewhere. To examine the potential role of migratory waterfowl and other wild birds in aMPV spread, our study focused on determining whether populations of wild birds have evidence of aMPV infection. Serum samples from multiple species were initially screened using a blocking enzyme-linked immunosorbent assay. Antibodies to aMPVs were identified in five of the 15 species tested: American coots, American crows, Canada geese, cattle egrets, and rock pigeons. The presence of aMPV-specific antibodies was confirmed with virus neutralization and western blot assays. Oral swabs were collected from wild bird species with the highest percentage of aMPV-seropositive serum samples: the American coots and Canada geese. From these swabs, 17 aMPV-positive samples were identified, 11 from coots and six from geese. Sequence analysis of the matrix, attachment gene and short hydrophobic genes revealed that these viruses belong to subtype C aMPV. The detection of aMPV antibodies and the presence of virus in wild birds in Georgia, South Carolina, Arkansas and Ohio demonstrates that wild birds can serve as a reservoir of subtype C aMPV, and may provide a potential mechanism to spread aMPVs to poultry in other regions of the United States and possibly to other countries in Central and South America.

Swollen head syndrome (SHS) associated with avian metapneumovirus (aMPV) subtype A or subtype B in broilers and broiler breeders has been reported worldwide. Data about pathogenesis of aMPV subtypes A and B in broilers are scarce. It has been difficult to reproduce swollen sinuses in chickens with aMPV under experimental conditions. In the field, SHS in broilers is suspected to be induced by combined infections with different respiratory pathogens. The objectives of the present study were to compare the pathogenesis of subtypes A and B aMPV in commercial broilers and to investigate the reproducibility of clinical disease. In two repeat experiments, commercial broilers free of aMPV maternal antibodies were inoculated with aMPV subtypes A and B of turkey origin. The clinical signs such as depression, coughing, nasal exudates, and frothy eyes appeared at 4 days post inoculation, followed by swelling of periorbital sinuses at 5 days post inoculation. Higher numbers of broilers showed clinical signs in subtype-B-inoculated compared with subtype-A-inoculated groups. Seroconversion to aMPV was detectable from 10 to 11 days post inoculation. The appearance of serum aMPV enzyme-linked immunosorbent assay antibodies and the clearance of the aMPV genome coincided. Subtype B aMPV showed a broader tissue distribution and longer persistence than subtype A. Histopathological changes were observed in the respiratory tract tissues of aMPV-inoculated broilers, and also in paraocular glands, such as the Harderian and lachrymal glands. Overall, our study shows that representative strains of both aMPV turkey isolates induced lesions in the respiratory tract, accompanied by swelling of infraorbital sinuses, indicating the role of aMPV as a primary pathogen for broilers.

Subtype B avian metapneumovirus (aMPV) was isolated and detected by reverse transcriptase-polymerase chain reaction (RT-PCR) in Brazilian commercial laying chicken flocks with no history of vaccination against aMPV and presenting respiratory signs and decreased egg production. RT-PCR results from samples from three affected flocks revealed that the three isolates were subtype B. Partial sequence analysis of the G glycoprotein gene confirmed that the samples belonged to subtype B and were not of the vaccine type. Comparison of nucleotide and amino acid sequences of the G gene of the three Brazilian aMPV samples with subtype B isolates from other countries revealed 95.1% to 96.1% identity. Nucleotide sequences showed 100% identity among the Brazilian subtype B samples and 95.6% identity with the subtype B vaccine strain used in Brazil. This work describes the circulation of subtype B aMPV in Brazil and discusses its importance in terms of disease epidemiology.

Although the disease associated with the avian metapneumovirus (aMPV) has been described in Israel for more than 20 years and one Israeli isolate was characterized as subtype A, this is the first study investigating the strains circulating in Israel (period 2002 to 2004). Viral RNA was detected by reverse transcription-polymerase chain reaction in 44% of the examined flocks, and one-half of the virus isolation attempts were successful. Characterization showed that 34 non-vaccinated turkey flocks had aMPV subtype B, three had aMPV subtype A and three remained undetermined. Sequence analysis of part of the attachment protein G gene (1161 base pairs) showed a relative homogeneity within the Israeli subtype B group, although they were different from the European and vaccine strains. Seven out of 10 vaccinated flocks were reverse transcription-polymerase chain reaction-positive for aMPV, and sequence analysis of 239 base pairs of the G gene revealed that field strains and not vaccine strains were recovered from these flocks. Overall, the virological examination confirmed the high prevalence of aMPV in Israel and revealed a co-circulation of two subtypes, A and B, with aMPV subtype B being the dominant subtype.

The aim of this study was to improve a reverse transcriptase (RT)-nested-polymerase chain reaction (PCR) able to differentiate avian pneumovirus (APV) subtypes A and B, and to characterize new Brazilian isolates. Representative APV strains and clinical field samples from chickens and turkey flocks were amplified in the chicken embryo-related cell line. Viral RNA was extracted from harvested cells, and submitted to cDNA synthesis. The primers utilized for RT-PCR were compatible with the G gene of both the A and B subtypes of APV, while the nested primers were subtype specific. This approach showed that three new APVs from chickens and one from turkeys were subtype A, confirmed by sequencing. This is the first report of APV isolation from turkeys in Brazil. Four other APVs were detected and classified as subtype A by RT-nested-PCR. These optimized techniques could be useful for differentiation of APV subtypes A and B, proving to be a valuable molecular epidemiological tool.