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Avian metapneumovirus (aMPV), classified within the Pneumoviridae family, wreaks havoc on poultry health. It typically causes upper respiratory tract and reproductive tract infections, mainly in turkeys, chickens, and ducks. Four subtypes of AMPV (A, B, C, D) and two unclassified subtypes have been identified, of which subtypes A and B are widely distributed across the world. In January 2024, an outbreak of severe respiratory disease occurred on turkey and chicken farms across different states in the US. Metagenomics sequencing of selected tissue and swab samples confirmed the presence of aMPV subtype B. Subsequently, all samples were screened using an aMPV subtype A and B multiplex real-time RT-PCR kit. Of the 221 farms, 124 (56%) were found to be positive for aMPV-B. All samples were negative for subtype A. Six whole genomes were assembled, five from turkeys and one from chickens; all six assembled genomes showed 99.29 to 99.98% nucleotide identity, indicating a clonal expansion event for aMPV-B within the country. In addition, all six sequences showed 97.74 to 98.58% nucleotide identity with previously reported subtype B sequences, e.g., VCO3/60616, Hungary/657/4, and BR/1890/E1/19. In comparison to these two reference strains, the study sequences showed unique 49-62 amino acid changes across the genome, with maximum changes in glycoprotein (G). One unique AA change from T (Threonine) to I (Isoleucine) at position 153 in G protein was reported only in the chicken aMPV sequence, which differentiated it from turkey sequences. The twelve unique AA changes along with change in polarity of the G protein may indicate that these unique changes played a role in the adaptation of this virus in the US poultry. This is the first documented report of aMPV subtype B in US poultry, highlighting the need for further investigations into its genotypic characterization, pathogenesis, and evolutionary dynamics.

In the present study, one hundred and sixteen partial G gene sequences of Avian metapneumovirus (aMPV) subtype B, obtained during routine diagnostics in different European Countries in the last few years (2014-2019), were analysed by sequence and phylogenetic analyses in order to draw an updated picture of the molecular characteristics of circulating strains. Nucleotide sequences were compared with other sequences of European and non-European aMPV-Bs collected prior to that period or retrieved from GenBank. Phylogenetic relationships among the aMPV-B strains, reconstructed using the maximum likelihood method implemented in MEGA X, demonstrated that aMPV-B has evolved in Europe from its first appearance, frequently displaying a clear relation with the geographic area of detection. The 40% of aMPV-B viruses analysed were classified as vaccine-derived strains, being phylogenetically related, and showing high nucleotide identity with live commercial vaccine strains licensed in Europe. The remaining 60% were classified as field strains since they clustered separately and showed a low nucleotide identity with vaccines and vaccine-derived strains. The phylogenetic tree showed that the virus has continued to evolve from its first appearance in the '80s since more recently detected strains belonged to clades phylogenetically distant from the older strains. Unlike vaccine-derived strains, field strains tended to cluster according to their geographic origin and irrespective of the host species where the viruses had been detected. In conclusion, the molecular characterization of aMPV-B and the differentiation between vaccines and field strains through G gene sequence analysis can be a useful tool towards correct diagnosis and should be routinely applied in order to better address the control strategies., (© 2020 Wiley-VCH GmbH.)

Avian Metapneumovirus (aMPV) has been recognized as a respiratory pathogen of turkey and chickens for a long time. Recently, a crescent awareness of aMPV, especially subtype B, clinical and economic impact has risen among European researchers and veterinarians. Nevertheless, the knowledge of its epidemiology and evolution is still limited. In the present study, the broadest available collection of partial G gene sequences obtained from European aMPV-B strains was analyzed using different phylodynamic and biostatistical approaches to reconstruct the viral spreading over time and the role of different hosts on its evolution. After aMPV-B introduction, approximatively in 1985 in France, the infection spread was relatively quick, involving the Western and Mediterranean Europe until the end of the 1990s, and then spreading westwards at the beginning of the new millennium, in parallel with an increase of viral population size. In the following period, a wider mixing among aMPV-B strains detected in eastern and western countries could be observed. Most of the within-country genetic heterogeneity was ascribable to single or few introduction events, followed by local circulation. This, combined with the high evolutionary rate herein demonstrated, led to the establishment of genetically and phenotypically different clusters among countries, which could affect the efficacy of natural or vaccine-induced immunity and should be accounted for when planning control measure implementation. On the contrary, while a significant strain exchange was proven among turkey, guinea fowl and chicken, no evidence of differential selective pressures or specific amino-acid mutations was observed, suggesting that no host adaptation is occurring.

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 this study was to evaluate if the exposure to Avian metapneumovirus (aMPV) and/or to Turkey hemorrhagic enteritis virus (THEV) was significant for the induction of episodes of colibacillosis in aMPV and THEV vaccinated turkeys. Colibacillosis-associated mortality was recorded and longitudinal virological studies performed in three consecutive turkey flocks reared in the same farm. aMPV and THEV diagnostic swabs and blood samples were made once a week up to 14 weeks of age. Swabs were processed by molecular techniques for viruses detection and antibody titres were evaluated. Field subtype B aMPVs were detected in all flocks at different ages of life always associated with respiratory signs and increase of colibacillosis-associated mortality. THEV has been consistently detected in all flocks since the 9th week of age. Vaccination with a single dose of the THEV commercial inactivated vaccine available in Italy seems does not protect the birds from the infection. Sequence comparison of the hexon protein of one of the THEV strains detected, and strains isolated worldwide, revealed high similarity between them. These results are consistent with the notion that the hexon protein, being the major antigenic component of the virus, is highly conserved between the strains. Results showed that field aMPV infection is directly correlated to colibacillosis-associated mortality. Less clear appears the role of THEV because the endemicity of aMPV makes difficult to evaluate its role in predisposing colibacillosis in absence of aMPV. It would be interesting to further investigate this issue through experimental trials in secure isolation conditions.

Avian metapneumovirus (AMPV) causes turkey rhinotracheitis and is associated with swollen head syndrome in chickens, which is usually accompanied by secondary infections that increase mortality. AMPVs circulating in Brazilian vaccinated and nonvaccinated commercial chicken and turkey farms were detected using a universal reverse transcriptase (RT)-PCR assay that can detect the four recognized subtypes of AMPV. The AMPV status of 228 farms with respiratory and reproductive disturbances was investigated. AMPV was detected in broiler, hen, breeder, and turkey farms from six different geographic regions of Brazil. The detected viruses were subtyped using a nested RT-PCR assay and sequence analysis of the G gene. Only subtypes A and B were detected in both vaccinated and nonvaccinated farms. AMPV-A and AMPV-B were detected in 15 and 23 farms, respectively, while both subtypes were simultaneously found in one hen farm. Both vaccine and field viruses were detected in nonvaccinated farms. In five cases, the detected subtype was different than the vaccine subtype. Field subtype B virus was detected mainly during the final years of the survey period. These viruses showed high molecular similarity (more than 96% nucleotide similarity) among themselves and formed a unique phylogenetic group, suggesting that they may have originated from a common strain. These results demonstrate the cocirculation of subtypes A and B in Brazilian commercial farms.

This study investigated the prevalence of avian metapneumovirus (aMPV) and the detection of molecular subtypes of field strains of the virus using RT-PCR in clinically healthy turkeys and those showing signs of respiratory disease. In the RT-PCR examination of 624 tracheal tissue samples collected from a local turkey abattoir, 2.9 per cent (18/624) of samples tested positive. In the examination of tracheal swab samples collected from flocks with respiratory problems, 18 of 20 samples tested positive. When the results were assessed at flock level, aMPV infection was detected in only one of the 23 clinically healthy turkey flocks, whereas all four flocks with respiratory problems were infected. Molecular typing using primers specific to the attachment glycoprotein (G) gene showed that all 36 positive samples belonged to subtype B. Partial sequence analysis of DNA samples showed 95 per cent homology between the field types and the reference strain aMPV subtype B. Whereas clinically healthy turkeys had been vaccinated with a subtype A virus vaccine, the flocks with respiratory problems had been vaccinated with a subtype B virus vaccine. Despite four blind passages of RT-PCR-positive samples on Vero and chicken embryo fibroblast cells, no cytopathic effect was detected by microscopic examination.

Avian metapneumovirus (AMPV) causes an upper respiratory tract infection in turkeys leading to serious economic losses to the turkey industry. The G glycoprotein of AMPV is known to be associated with viral attachment and pathogenesis. In this study, we determined the role of the G glycoprotein in the pathogenicity and immunogenicity of AMPV strain Colorado (AMPV/CO). Recombinant AMPV/CO lacking the G protein (rAMPV/CO-deltaG) was generated using a reverse-genetics system. The recovered rAMPV/CO-deltaG replicated slightly better than did wild-type AMPV in Vero cells. However, deletion of the G gene in AMPV resulted in attenuation of the virus in turkeys. The mutant virus induced less-severe clinical signs and a weaker immune response in turkeys than did the wild-type AMPV. Our results suggest that the G glycoprotein is an important determinant for the pathogenicity and immunogenicity of AMPV.

Human (HMPV) and avian (AMPV) metapneumoviruses are closely related viruses that cause respiratory tract illnesses in humans and birds, respectively. Although HMPV was first discovered in 2001, retrospective studies have shown that HMPV has been circulating in humans for at least 50 years. AMPV was first isolated in the 1970s, and can be classified into four subgroups, A-D. AMPV subgroup C is more closely related to HMPV than to any other AMPV subgroup, suggesting that HMPV has emerged from AMPV-C upon zoonosis. Presently, at least four genetic lineages of HMPV circulate in human populations - A1, A2, B1 and B2 - of which lineages A and B are antigenically distinct. We used a Bayesian Markov Chain Monte Carlo (MCMC) framework to determine the evolutionary and epidemiological dynamics of HMPV and AMPV-C. The rates of nucleotide substitution, relative genetic diversity and time to the most recent common ancestor (TMRCA) were estimated using large sets of sequences of the nucleoprotein, the fusion protein and attachment protein genes. The sampled genetic diversity of HMPV was found to have arisen within the past 119-133 years, with consistent results across all three genes, while the TMRCA for HMPV and AMPV-C was estimated to have existed around 200 years ago. The relative genetic diversity observed in the four HMPV lineages was low, most likely reflecting continual population bottlenecks, with only limited evidence for positive selection.

In order to detect and characterize avian metapneumovirus, organs or swabs were collected from 697 chicken and 110 turkeys from commercial farms in Southwestern Nigeria and from 107 chickens from live bird markets in Southeastern China. In Nigeria, 15% and 6% of the chicken and turkey samples, respectively, and 39% of the chicken samples from China, were positive for aMPV genome by PCR. The sequence of a 400 nt fragment of the attachment protein gene (G gene) revealed the presence of aMPV subtype A in both Nigeria and Southeastern China. Essentially identical subtype A viruses were found in both countries and were also previously reported from Brazil and the United Kingdom, suggesting a link between these countries or a common source of this subtype. In Nigeria, subtype B was also found, which may be a reflection of chicken importations from most major poultry-producing countries in Europe and Asia. In order to justify countermeasures, further studies are warranted to better understand the metapneumoviruses and their impact on poultry production.

Studies were performed to determine if mucosal vaccination with inactivated avian metapneumovirus (aMPV) subtype C protected turkey poults from clinical disease and virus replication following mucosal challenge. Decreases in clinical disease were not observed in vaccinated groups, and the vaccine failed to inhibit virus replication in the tracheas of 96% of vaccinated birds. Histopathologically, enhancement of pulmonary lesions following virus challenge was associated with birds receiving the inactivated aMPV vaccine compared to unvaccinated birds. As determined by an enzyme-linked immunosorbent assay (ELISA), all virus-challenged groups increased serum immunoglobulin (Ig) G and IgA antibody production against the virus following challenge; however, the unvaccinated aMPV-challenged group displayed the highest increases in virus-neutralizing antibody. On the basis of these results it is concluded that intranasal vaccination with inactivated aMPV does not induce protective immunity, reduce virus shedding, or result in decreased histopathologic lesions.

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.

The objectives of the present study were to investigate the pathogenesis of a recent isolate of avian metapneumovirus (aMPV) in turkeys and to evaluate the quantitative distribution of the virus in various tissues during the course of infection. Seventy 2-week-old turkey poults were divided equally into two groups. One group was inoculated with aMPV (MN 19) with a titer of 10(5.5) TCID50 oculonasally. Birds in the second group were maintained as sham-inoculated controls. Birds showed severe clinical signs in the form of copious nasal discharge, swollen sinus, conjunctivitis, and depression from 4 days postinoculation (PI) to 12 days PI. Samples from nasal turbinates, trachea, conjunctiva, Harderian gland, infraorbital sinus, lungs, liver, and spleen were collected at 1, 3, 5, 7, 9, 11, and 14 days PI. Histopathologic lesions such as a multifocal loss of cilia were prominent in nasal turbinate and were seen from 3 to 11 days PI. Immunohistochemistry revealed the presence of aMPV from 3 to 9 days PI in nasal turbinate and trachea. Viral RNA could be detected for 14 days PI from nasal turbinate and for 9 days from trachea. In situ hybridization demonstrated the presence of aMPV from 1 to 11 days PI in nasal turbinates and from 3 to 9 days PI in the trachea. Quantitative real-time polymerase chain reaction data showed the presence of a maximum amount of virus at 3 days PI in nasal turbinate and trachea. Clinically and histopathologically, the new isolate appears to be more virulent compared to the early isolates of aMPV in the United States.

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.

Avian pneumovirus (APV; officially known as turkey rhinotracheitis virus) is an emergent pathogen of birds in the USA that results in upper respiratory tract disease in turkeys. Six years after the first outbreak in the USA, the disease continues to ravage turkey flocks, primarily in the state of Minnesota. From 1997 to 2000, the industry recorded losses estimated at 15 million US dollars per annum. Researchers have developed sensitive diagnostic techniques, including the enzyme-linked immunosorbent assay and the reverse transcriptase-polymerase chain reaction. which, when used together, are highly sensitive in detecting APV outbreaks in commercial turkey flocks. Phylogenetic analysis of the nucleotide and predicted amino acid sequence of 15 US viruses isolated between 1996 and 2000 demonstrated that the US viruses are relatively homogenous but different from the European APV subgroups A and B, resulting in the classification of US isolates into subgroup C. Infectious APV was isolated from sentinel waterfowls placed close to an infected commercial turkey farm and from wild Canada geese captured in Minnesota, suggesting that free-ranging birds may be involved in the spread of APV. Current efforts to prevent and control the infection include improving management and biosecurity practices and developing attenuated live and deletion mutant vaccines capable of conferring protection.

Turkey rhinotracheitis, now commonly termed avian pneumovirus (APV) infection, is associated with serious welfare and economic problems in susceptible populations of turkeys and probably also of chickens. The infection principally affects the upper respiratory tract, although egg-laying performance may also be affected in breeding turkeys. Secondary infections exacerbate the effects of the primary virus infection. The virus persists for only a short time both in the host and in the environment and is not known to be transmitted via the egg. Highly effective vaccines are available to control APV infections, and hence good biosecurity and careful use of these vaccines should enable infection to be controlled and spread restricted. Diagnosis and surveillance are normally performed serologically using enzyme-linked immunosorbent assays (ELISAs). Several different ELISA kits are available commercially, but these give variable results and are not wholly satisfactory since interpretation of results is difficult.