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The pathogenicity of avian metapneumovirus subtype C wild bird isolates in domestic turkeys



Avian metapneumovirus subtype C (aMPV/C) causes severe upper respiratory disease in turkeys. Previous report revealed the presence of aMPV/C in wild birds in the southeast regions of the U.S.


In this study, aMPV/C positive oral swabs from American coots (AC) and Canada geese (CG) were passaged three times in the respiratory tract of specific pathogen free (SPF) turkeys and used as aMPV/C P3 virus isolates in subsequent studies.


Wild bird P3 isolates showed similar growth characteristics when compared to virulent aMPV/C in chicken embryo fibroblast ( CEF) cell cultures and their glycoprotein G gene sequence was closely related to the G gene of aMPV/C Colorado reference virus. Three-day-old commercial or SPF turkeys were inoculated oculonasally with wild bird aMPV/C P3 isolates. At 5 and 7 days post-inoculation (DPI), severe clinical signs were observed in both of the AC and CG virus-exposed groups. Viral RNA was detected in tracheal swabs by reverse transcriptase polymerase chain reaction (RT-PCR). In addition, immunohistochemistry showed virus replication in the nasal turbinate and trachea. All virus-exposed turkeys developed positive antibody response by 14 DPI.


Our data demonstrate that aMPV/C wild bird isolates induced typical aMPV/C disease in the domestic turkeys.


Avian metapneumovirus (aMPV), previously known as turkey rhinotracheitis virus (TRTV), is the cause of severe respiratory infection in turkeys [14] and may also be the etiologic agent of “swollen head syndrome (SHS)” of chickens [2]. Originally identified in South Africa in the late 1970s [5], aMPV later spread to Europe, Asia, and South America [4, 611]. aMPV/C was first isolated in the United States during 1996 in Colorado [12, 13] and subsequently outbreaks were reported in Minnesota [14] where the disease has emerged as a major economic problem for the turkey industry. The virus has also spread to other states, such as North Dakota, South Dakota, Iowa, and Wisconsin [15]. aMPV is now considered a major disease threat in both turkeys and chickens in many parts of the world.

aMPV is a member of the genus Metapneumovirus in the subfamily Pneumovirinae of the family Paramyxoviridae[16]. Members of the genus Metapneumovirus contain a nonsegmented, single-stranded negative sense RNA genome with the gene order 3’-leader-N-P-M-F-M2-SH-G-L-trailer-5’ [1722]. The aMPV isolates that exist worldwide are currently classified into four subtypes, namely, subtypes A, B, C, and D. This classification is based on sequence divergence in the attachment (G) glycoprotein and the antigenic differences observed among aMPV strains. The US isolates belong to subtype C [23, 24], while the majority of aMPVs isolated in Europe, Asia, and South America belong to the subtype A or B [6, 25, 26]. aMPV/C was also isolated from farmed ducks in France in 1999 [27] and in live bird market in Korea in 2005 [28]. In 2000, it was reported that aMPVs isolated in France in 1985 belong to subtype D [25].

In the field, aMPV/C leads to mild to severe, rapidly spreading respiratory infection that can adversely affect weight gain and feed conversion. The disease is characterized by depression, coughing, nasal and ocular discharge, and swollen infraorbital sinuses with morbidity of up to 100% [29, 30]. Microscopically, inflammatory infiltrates (lymphocytes, macrophages, plasma cells, and heterophils) were detected in nasal turbinate and sinus tissues [31]. Mortality may reach 30% in cases complicated by secondary bacterial infection. Economic losses are due to mortality, a sharp drop in egg production, and increased carcass condemnation rate at slaughter because of air sacculitis [29, 31].

The U.S. outbreaks of aMPV/C infection have occurred in a seasonal pattern (spring and fall), and wild migratory birds have been implicated in the spread of the disease [32, 33]. In fact, aMPV RNA was isolated from the nasal turbinates of wild sparrows, geese, blue-winged teal, and starlings and shown to share 90 to 95% nucleotide sequence identity with viruses isolated from domestic turkeys [15, 32, 33]. The role of wild birds in the epidemiology of the disease and transmission of the virus between commercial turkey farms is not clear. Sequence analysis at Southeast Poultry Research Laboratory (SEPRL) of four virus isolates recovered from aMPV/C outbreaks in turkeys shared 95% to 99% nucleotide sequence identity and 97% to 99% predicted amino acid sequence identity with a duck virus isolated from sentinel mallard ducks [34]. These results indicate that aMPV/C isolates from turkeys and ducks share a common source and the viruses from different avian species can cross-infect other birds. In another study the potential role of migratory waterfowl and other wild birds in aMPV/C spread was also examined [35]. Those results showed the presence of antibodies to aMPV/C in American coots, American crows, Canada geese, cattle egrets, and rock pigeons. When oral swabs were assayed by using RT-PCR, aMPV RNA was detected in samples from American coots and Canada geese. Sequence analysis indicated that the wild bird viruses belonged to aMPV/C [35]. The data clearly demonstrated that wild birds can serve as a reservoir of aMPV/C. While the study showed that aMPV/C was able to replicate and induce antibody production in wild birds, the pathogenicity of the aMPV/C isolates for domestic turkeys was not determined [35]. The presence of a potentially pathogenic virus among wild birds could eventually result in the introduction of aMPV/C into poultry flocks in the U.S.

In this study, we determined the pathogenicity of wild bird aMPV/C isolates in domestic turkeys. Biological characteristics of the isolates were studied in vitro in cell cultures and by sequence analysis of the G gene. The virulence level and pathogenic properties of the wild bird viruses were demonstrated in experimentally infected SPF and commercial turkeys.


aMPV/C-P3 virus isolation from wild birds

Five out of 23 oral swabs from American coots (AC) and 7 out of 27 oral swabs from Canada geese (CG) were found aMPV/C positive by using N and G gene specific RT-PCR (data not shown). The positive swabs were pooled (aMPV/C-AC-P0 and aMPV/C-CG-P0) and subsequently passaged three times in the upper respiratory tract of SPF turkeys, turbinate and tracheal samples were processed as described in materials and methods. Virus stocks from the third turkey passage were designated as P3 viruses and titrated in CEF cultures. The titer of aMPV/C-AC-P3 and aMPV/C-CG-P3 viruses were 104.5 TCID50/ml and 105 TCID50/ml, respectively. There was no significant difference in growth characteristics and virus yield in CEF cell cultures between the wild bird P3 viruses and the prototype virulent aMPV/C-CO Tr isolate (data not shown).

The G gene of aMPV/C wild bird isolates

Sequence analysis of the attachment (G) gene from the virus positive AC-P0 pool showed the presence of mixed G gene population that contained both full-length G gene (759 nt) and variants of truncated G genes. The size of the deletion within the truncated G open reading frame (ORF) varied from 93 nt to 579 nt (Figure 1). Only one major G gene fragment was detected within the CG-P0 pool with a deletion of 582 nt (Figure 1). Following three passages in turkeys the AC-P3 virus isolate contained a dominant G gene ORF of 759 nt. This G gene possesses the same length and sequences as that of the Colorado strain reported by Lwamba et al. in 2005 [36]. There was no detectable full-length G gene in the CG-P3 virus; instead a mixed population of shorter G gene ORFs was present with a range of molecules containing deleted segments from 126 nt to 279 nt. Some of these G gene nucleotide deletions were in-frame suggesting the presence of virus variants with truncated but potentially functional G proteins.

Figure 1
figure 1

Schematic representation of aMPV/C G gene from pooled American coot (AC) and Canada goose (CG) samples before (P0) and after three serial turkey passages (P3). Nucleotide position is indicated by numbers according to GenBank submission of aMPV/C-US strain [GenBank ID: AY579780.1]. Deletions within the G gene are represented by dotted lines and mapped by nucleotide numbers. Multiple nucleotide positions indicate more than one deletion form within the same region of the aMPV/C-AC-P0 and aMPV/C-CG-P3 viruses.

Pathogenicity of wild bird isolates in turkey

In experiment 1, SPF turkeys were inoculated with aMPV/C P3 isolates via ON route. From 5 DPI, the turkey poults exposed to either AC-P3 or CG-P3 viruses exhibited typical clinical signs of aMPV/C disease, showing nasal exudates when squeezed, nasal discharge, or frothy eyes. These clinical signs were still apparent at 7 DPI but were not detectable at later times (Table 1). RT-PCR data to detect viral RNA in tracheal swabs showed that high percentage of inoculated birds became positive at 3 DPI and the number of positives gradually declined by 10 DPI. Microscopic lesions in the turbinate tissues were only observed in a few birds (one positive in each group); however, IHC staining revealed virus replication in the nasal turbinate tissues as early as 5 DPI in 100% of turkeys infected with either AC or CG viruses (Table 1, Figure 2). All birds inoculated with AC or CG viruses developed aMPV/C specific antibody response by 14 DPI, while all sera collected from control birds were negative.

Table 1 Pathogenicity of aMPV/C wild bird isolates in SPF turkeys (experiment 1)
Figure 2
figure 2

Detection of aMPV/C infected cells in the mucosal layers of nasal turbinate from virus exposed SPF turkeys at 5 DPI by IHC staining. (a) Control untreated turkeys; (b) aMPV/C-AC-P3 virus infected turkeys; (c) aMPV/C-CG-P3 virus infected turkeys.

In experiment 2, commercial turkeys were used to study the pathogenic properties of aMPV/C wild bird isolates along with turkeys infected with the challenge virus aMPV/C-CO Tr. Clinical signs, viral RNA detection and seroconversion data following inoculation are summarized in Table 2. Extensive aMPV/C disease specific clinical signs were observed in all groups between 3 and 11 DPI and 100% of the birds seroconverted by 14 DPI. Viral RNA was detectable in turkeys from each group at 3 DPI; however, at later times positives were only observed among the virulent challenge virus inoculated birds. Microscopic lesions were much more abundant in turbinate tissues than in tracheas in each experimental group and IHC also demonstrated a more pronounced virus replication in turbinates from each group when they were compared to those observed in trachea samples at 5 and 7 DPI (Table 3). Overall, both wild bird P3 isolates exhibited very similar pathogenicity in commercial turkeys when aMPV/C disease characteristics were compared to those detected following inoculation with virulent virus aMPV/C-CO Tr.

Table 2 Pathogenicity of aMPV/C wild bird isolates in commercial turkeys (experiment 2)
Table 3 Immunohistochemistry (IHC) staining and microscopic lesions in commercial turkeys exposed to aMPV/C wild bird P3 isolates (experiment 2)


In the present study, we isolated avian metapneumoviruses from wild birds samples collected in 2000 from Georgia, South Carolina and Arkansas in the U.S. Sequence analysis of virus isolates from oral swabs of American coots and Canada geese confirmed that these viruses belong to subtype C aMPV. Our data clearly demonstrate that both AC and CG wild bird viruses were fully pathogenic in SPF and commercial turkeys. Following experimental inoculation, aMPV/C specific clinical signs (with the exception at 3DPI), virus replication and shedding in the upper respiratory tract were indistinguishable in AC and CG virus infected birds from those observed in group of turkeys inoculated with virulent virus aMPV/C-CO Tr.

Wild birds have been considered as important reservoirs for other avian viruses such as avian influenza and Newcastle disease viruses [37, 38]. Previous studies in the U.S. have shown the possible involvements of wild birds in the transmission of aMPV/C viruses [15, 32, 33, 39]. Recent report revealed the presence of aMPV/C in wild birds in the southeast regions of the U.S. [35]. Those data demonstrated that wild birds can serve as a reservoir of subtype C aMPV and may provide a potential mechanism to spread aMPVs to poultry. Here, our results clearly demonstrate the possibility of virus transmission from wild birds to domestic turkeys.

Conventional avian metapneumovirus isolation methods usually include several passages of the virus in avian and mammalian cell cultures. While this technique is convenient and routinely used to isolate virus from field samples it has the potential risk that during serial passages in cell cultures the biological characteristics of the virus, including genome structure and pathogenicity could be altered. In fact, it is well documented that a number of live attenuated aMPV vaccines were produced by passage of virulent virus in cell cultures and these attenuated viruses, which had lost their pathogenicity were subsequently used as protective empirical vaccines [4043]. It had also been reported that after 15 or more Vero cell passages, the attachment G gene is deleted from the genome of aMPV/C [44]. To avoid possible genomic alterations and/or attenuation following serial virus passage in cell cultures, our studies have focused on the isolation of aMPV/C viruses from wild birds using in vivo method. Serial passages of aMPV/C in turkeys proved to be a highly efficient technique to prepare virulent challenge viruses in past studies [45, 46]. In our studies, three serial passages of RT-PCR positive wild bird oral swab samples in the respiratory tract of SPF turkeys resulted in the isolation of aMPV/C-P3 viruses without having them propagated in cell cultures. It is important to note that the AC-P3 and CG-P3 isolates exhibited similar growth characteristics in vitro in CEF cell cultures when cytopathic effect and virus yields were compared to those observed in cultures infected with virulent aMPV/C-CO Tr virus. These data indicated that using the wild bird P3 isolates in subsequent experiments in turkeys provided true and relevant information about the pathogenic properties of aMPV/C viruses carried by wild birds in nature.

aMPV G gene encodes a major structural glycoprotein that plays important role in viral attachment, pathogenicity and protective immunity [45, 47]. The G protein of aMPV isolates exhibits extensive genetic and antigenic variations that are the primary criteria for aMPV subtype classification [7, 48, 49]. The G gene in aMPV/C isolates exhibits major sequence divergence and the length of the genome reported for different aMPV/C strains isolated in the U.S. is strikingly different [32, 36, 50, 51]. Although recent studies indicated that truncation of the G genes can occur during serial passages of the virus in cell cultures or circulation of the virus in a host in the field [44, 52] the biological significance of the G gene length variation is mostly unknown. Our data provide further evidence that the G protein may play an important role in viral pathogenesis in domestic turkey. While the original pooled wild bird samples contained a mixture of virus population with mostly truncated G genes with large deletions, following three passages in turkey the P3 virus stocks contained either full-length G gene (AC-P3) or truncated G genes (CG-P3) with in-frame deletions. It is tempting to speculate that while the G gene may not play important role for aMPV/C replication in wild birds, it is required for efficient virus growth in turkeys. This hypothesis is supported by recent studies, which showed that deletion of the G gene from virulent aMPV/C viruses resulted in attenuation of the virus in turkeys [45, 47].

In conclusion, we successfully demonstrated that aMPV/C viruses carried by wild birds in the U.S. possess a potential threat to commercial turkeys. These wild bird viruses are fully virulent and capable of initiating aMPV/C disease outbreaks in susceptible flocks by direct transmission of the virus between wild birds and turkeys. This finding provides further and more definite reason of maintaining high biosecurity standard in the poultry industry.

Materials and methods

Viruses and cell cultures

The aMPV/C Colorado strain from turkeys (aMPV/CO/96/C) was obtained from the aMPV repository bank at SEPRL (USDA-ARS, Athens, GA, USA). The virus was propagated in Vero cells (ATCC, Manassas, VA, CCL-81). A virulent challenge virus stock aMPV/C-CO Tr was prepared by three consecutive in vivo passages of the aMPV/CO/96/C in turkeys as previously described [45] and was titrated in 1-week-old turkeys for 50% infective dose (ID50). Oral swabs from wild birds [American coot (AC) (n=23) and Canada goose (CG)] (n=27) were obtained from SEPRL collection and tested for aMPV/C by reverse transcriptase-polymerase chain reaction (RT-PCR) as described in previous studies [44]. Virus positive swabs from each wild bird were pooled, labeled as P0 (aMPV/C-AC-P0, n=5; aMPV/C-CG-P0, n=7), and inoculated via oculonasal (ON) route to 3-day-old SPF turkeys at a dose of 200 μl inoculum per bird. At 5 days post-inoculation (DPI), turbinate and trachea were pooled and 20% homogenates were prepared with 1x phosphate buffered saline (PBS). Two more passages were repeated in the same manner in 3-day-old SPF turkeys to obtain P3 stock virus of each wild bird isolate (aMPV/C-AC-P3; aMPV/C-CG-P3). To rule out the possibility of the presence of avian influenza virus (AIV) and Newcastle disease virus (NDV) in the P3 isolates, 9-day-old embryonating chicken eggs were inoculated with the P3 stock viruses and after three blind passages hemagglutination assays were performed from the chorioallantoic fluids as described previously [37, 45]. Both aMPV/C-AC and aMPV/C-CG P3 isolates were negative to AIV and NDV (data not shown).

Virus titration was performed as previously described [45] with some modification. Briefly, 96-well plates were seeded with SPF chicken embryo fibroblast (CEF) cells and infected with 100 μl of serial 10-fold dilutions of the viruses. After 4–5 days of incubation at 37°C, plates were fixed with 100% ethanol and indirect fluorescence assay was performed using aMPV/C hyperimmune turkey serum. Titers were calculated by the Reed and Muench method [53] and are expressed as 50% tissue culture infective dose (TCID50). Virus growth was examined in CEF cell cultures at 0.01 multiplicity of infection (MOI). At 4 days post-infection, the monolayers were harvested and virus was titrated as described above.


RNA was extracted from oral or tracheal swabs using Viral RNA Extraction Kit (Qiagen, Valencia, CA) and one-step RT-PCR was performed with a Superscript III one-step RT-PCR Kit (Invitrogen, Carlsbad, CA), following the manufacturer’s protocol. aMPV/C N gene-specific primers were designed based on gene sequence in GenBank [GenBank ID: AM293284] [45]and primers to detect aMPV/C G gene were used as previously described [44]. The sequence of aMPV/C N gene forward primer was 5’-ATGTCTCTTCAGGGGATTC-3’ and reverse primer was 5’-GCATCATTCAGCACACG-3’, the expected size of the amplicon was 650 bp.

Nucleotide sequencing

aMPV/C G gene specific RT-PCR products were separated in ethidium bromide agarose gel, fragments of expected size were excised and DNA was extracted using a Qiagen MinElute Kit (Qiagen, Valencia, CA). Purified DNA was ligated into the TOPO PCR 2.1 cloning Vector (Invitrogen, Carlsbad, CA) and was used to transform DH5α competent cell (Invitrogen, Carlsbad, CA). Colonies were incubated in LB broth (containing kanamycin) and DNA was prepared using plasmid miniprep purification kit (5 PRIME Inc., Gaithersburg, MD). Sequencing was performed with M13 universal forward and reverse primers using the Applied Biosystems-PRISM fluorescent big dye sequencing kit and the ABI 3730 automated DNA sequencer (ABI, Foster City, CA). The sequence obtained from each colony was compared and analyzed with those in the GenBank using the BLAST algorithms and DNASTAR program (Madison, WI).

Animal experimental design

SPF turkeys were obtained from the SEPRL flocks, aMPV/C antibody-free hybrid commercial turkeys were received from Prestige farm (Charlotte, NC). The birds were housed in Horsfal isolators (Federal Designs, Inc., Comer, GA) with ad libitum access to feed and water. Sera from one-day-old turkeys (n=10) were examined by aMPV/C-specific enzyme-linked immunosorbent assay (ELISA) to ensure the absence of anti-aMPV/C antibodies. All experimental procedures in turkeys were approved by the SEPRL Institutional Animal Care and Use Committee.

In experiment 1, four-day-old SPF turkeys were divided into three groups of 15 birds. Birds in group 1 were untreated and served as negative controls. Birds in groups 2 and 3 were inoculated via oronasal (ON) route with 200 μl of aMPV/C-AC-P3 (104.5 TCID50/ml) and aMPV/C-CG-P3 (105 TCID50/ml), respectively. At 3, 5, 7 and 10 DPI, tracheal swabs were collected (from all available in each group, n=4-13) and clinical signs for aMPV disease were observed as described previously [45]. Turkeys were considered clinically positive if showing nasal exudates when squeezed, nasal discharge or frothy eyes. Turbinate and trachea tissues for histopathology and immunohistochemistry (IHC) were randomly collected at 5 and 7 DPI (n=2 from controls and n=3 from inoculated) and serum to detect virus specific antibodies was collected at 14 DPI (n=7 from control and AC group; n=4 from CG group).

In experiment 2, three-day-old aMPV/C antibody free commercial turkeys were randomly divided into four groups of 25 birds and inoculated via ON routes as described in experiment 1. Birds in group 1 were untreated, birds in group 2 and 3 received aMPV/C-AC-P3 and aMPV/C-CG-P3, respectively. Each bird in group 4 was infected with 200 μl aMPV/C-CO Tr (104.6 ID50/ml) via ON route as a positive control. At 3, 5, 7 and 11 DPI, tracheal swabs were collected (n=10) and the birds were observed for recording aMPV clinical signs. Turbinate and trachea tissues for histopathology and IHC (n=4-5) were randomly collected at 5 and 7 DPI and serum samples (n=5-9) for aMPV/C specific ELISA were collected at 14 DPI.

Histopathology and immunohistochemistry

For histopathology, turbinate and trachea tissues were fixed in formalin and embedded in paraffin. Sections were sliced into 4 μm and stained with haematoxylin and eosin (HE). All HE stained sections were examined by light microscope. Turkeys were considered positive if extensive lymphoid cell infiltrations were observed in the mucosal layer of turbinate or trachea as shown in previous reports [31, 54, 55]. Virus infected cells were detected by IHC staining as previously described with some modifications [56, 57]. Briefly, paraffin embedded sections (4 μm) of turbinate or trachea tissues were deparaffinized, dehydrated followed by antigen retrieval with citrate buffer (Biogenex, San Ramon, CA). Rabbit anti-aMPV/C hyperimmune serum (kindly provided by Dr. K. Nagaraja of University of Minnesota) was used as primary antibody in1:1000 dilution. For secondary antibody, goat anti-rabbit IgG conjugated with horseradish peroxidase (HRP) (KPL, Gaithersburg, MD) was used at dilution of 1:250. The signal was developed with 3'-diaminobenzidine (DAB) Kit (Vector Laboratory, Burlingame, CA) following manufacturer’s instruction.


Anti-aMPV/C antibody response of turkeys was determined by an aMPV/C-specific IgG ELISA as previously described [45]. Briefly, turkey sera were diluted (1:100) and added to 96-well plates in triplicates, which were coated with sucrose gradient purified aMPV/CO/96/C virus as antigen. HRP conjugated goat anti-turkey IgG antibody (KPL, Gaithersburg, MD) was added as secondary antibody. A luminal-based chemiluminescent substrate (Lumiglo substrate kit; KPL, Gaithersburg, MD) was used as a developing agent. The chemiluminescence relative light unit (RLU) was measured on a Biotek Synergy HT Microtiter Plate Luminometer (Biotek Instruments, Winooski, VT). Test sera were considered positive when the mean RLU value was higher than the mean RLU plus 2 × standard deviation of the negative turkey serum pool.


The statistical analyses were done using the Fisher’s exact test (SigmaStat 2.0.3, SPSS Inc, Chicago, IL) in Experiment 2, where groups of turkeys inoculated with aMPV/C-AC-P3 or aMPV/C-CG-P3 viruses were compared to those infected with virulent aMPV/C-CO-Tr challenge virus.



American coot


Avian influenza virus


avian metapneumovirus subtype C


Chicken embryo fibroblast


Canada goose




Days post-inoculation


Enzyme-linked immunosorbent assay


Horseradish peroxidase


50% infective dose




Multiplicity of infection


Newcastle disease virus




Open reading frame


Phosphate buffer saline


Relative light unit


Ribonucleic acid


Reverse transcriptase polymerase chain reaction


Southeast Poultry Research Laboratory


Swollen head syndrome


Specific pathogen free


50% tissue culture infective dose


Turkey rhinotracheitis virus.


  1. Alexander DJ, Senne DA: Newcastle disease, other avian Paramyxoviruses, and Pneumovirus infections. In Diseases of Poultry. 12th edition. Edited by: Saif YM, Fadly AM, Glisson JR, McDougald LR, Nolan LK, Swayne DE. Ames, Iowa, USA: Blackwell Publishing; 2008:74-100.

    Google Scholar 

  2. Cook JK: Avian pneumovirus infections of turkeys and chickens. Vet J 2000, 160: 118-125.

    Article  PubMed  Google Scholar 

  3. Lwamba HC, Bennett RS, Lauer DC, Halvorson DA, Njenga MK: Characterization of avian metapneumoviruses isolated in the USA. Anim Health Res Rev 2002, 3: 107-117. 10.1079/AHRR200243

    Article  PubMed  Google Scholar 

  4. Njenga MK, Lwamba HM, Seal BS: Metapneumoviruses in birds and humans. Virus Res 2003, 91: 163-169. 10.1016/S0168-1702(02)00256-3

    Article  PubMed  Google Scholar 

  5. Buys SB, du Preez JH: A preliminary report on the isolation of a virus causing sinusitis in turkeys in South Africa and attempts to attenuate the virus. Turkeys 1980, 28: 36.

    Google Scholar 

  6. Banet-Noach C, Simanov L, Perk S: Characterization of Israeli avian metapneumovirus strains in turkeys and chickens. Avian Pathol 2005, 34: 220-226. 10.1080/03079450500112625

    Article  PubMed  Google Scholar 

  7. Cook JK: Avian rhinotracheitis. Rev Sci Tech 2000, 19: 602-613.

    PubMed  Google Scholar 

  8. D'Arce RC, Coswig LT, Almeida RS, Trevisol IM, Monteiro MC, Rossini LI, Di Fabio J, Hafez HM, Arns CW: Subtyping of new Brazilian avian metapneumovirus isolates from chickens and turkeys by reverse transcriptase-nested-polymerase chain reaction. Avian Pathol 2005, 34: 133-136. 10.1080/03079450500059180

    Article  PubMed  Google Scholar 

  9. Giraud P, Bennejean G, Guittet M, Toquin D: Turkey rhinotracheitis in France: preliminary investigations on a ciliostatic virus. Vet Rec 1986, 119: 606-607.

    PubMed  Google Scholar 

  10. McDougall JS, Cook JK: Turkey rhinotracheitis: preliminary investigations. Vet Rec 1986, 118: 206-207. 10.1136/vr.118.8.206

    Article  PubMed  Google Scholar 

  11. Tanaka M, Takuma H, Kokumai N, Oishi E, Obi T, Hiramatsu K, Shimizu Y: Turkey rhinotracheitis virus isolated from broiler chicken with swollen head syndrome in Japan. J Vet Med Sci 1995, 57: 939-941. 10.1292/jvms.57.939

    Article  PubMed  Google Scholar 

  12. Kleven SH: Report of the committee: transmissible diseases of poultry and other avian species. Proceedings of the 101st Annual Meeting of the USAnimal Health Association (pp486-491)Washington, DC, USA 1997.

    Google Scholar 

  13. Senne DA, Edson RK, Pedersen JC, Panigrahy B: Avian pneumovirus update. In 134th Annual Congress of American Veterinary Medical Association. Reno, Nevada; 1997.

    Google Scholar 

  14. Goyal SM, Chiang SJ, Dar AM, Nagaraja KV, Shaw DP, Halvorson DA, Kapur V: Isolation of avian pneumovirus from an outbreak of respiratory illness in Minnesota turkeys. J Vet Diagn Invest 2000, 12: 166-168. 10.1177/104063870001200214

    Article  PubMed  Google Scholar 

  15. Bennett RS, Nezworski J, Velayudhan BT, Nagaraja KV, Zeman DH, Dyer N, Graham T, Lauer DC, Njenga MK, Halvorson DA: Evidence of avian pneumovirus spread beyond Minnesota among wild and domestic birds in central North America. Avian Dis 2004, 48: 902-908. 10.1637/7208-051804R

    Article  PubMed  Google Scholar 

  16. Pringle CR: Virus taxonomy–San Diego 1998. ArchVirol 1998, 143: 1449-1459.

    Google Scholar 

  17. Biacchesi S, Skiadopoulos MH, Boivin G, Hanson CT, Murphy BR, Collins PL, Buchholz UJ: Genetic diversity between human metapneumovirus subgroups. Virology 2003, 315: 1-9. 10.1016/S0042-6822(03)00528-2

    Article  PubMed  Google Scholar 

  18. Ling R, Pringle CR: Turkey rhinotracheitis virus: in vivo and in vitro polypeptide synthesis. J Gen Virol 1988, 69: 917-923. 10.1099/0022-1317-69-4-917

    Article  PubMed  Google Scholar 

  19. van den Hoogen BG, Bestebroer TM, Osterhaus AD, Fouchier RA: Analysis of the genomic sequence of a human metapneumovirus. Virology 2002, 295: 119-132. 10.1006/viro.2001.1355

    Article  PubMed  Google Scholar 

  20. Yu Q, Davis PJ, Barrett T, Binns MM, Boursnell ME, Cavanagh D: Deduced amino acid sequence of the fusion glycoprotein of turkey rhinotracheitis virus has greater identity with that of human respiratory syncytial virus, a pneumovirus, than that of paramyxoviruses and morbilliviruses. J Gen Virol 1991, 72: 75-81. 10.1099/0022-1317-72-1-75

    Article  PubMed  Google Scholar 

  21. Yu Q, Davis PJ, Brown TD, Cavanagh D: Sequence and in vitro expression of the M2 gene of turkey rhinotracheitis pneumovirus. J Gen Virol 1992, 73: 1355-1363. 10.1099/0022-1317-73-6-1355

    Article  PubMed  Google Scholar 

  22. Yu Q, Davis PJ, Li J, Cavanagh D: Cloning and sequencing of the matrix protein (M) gene of turkey rhinotracheitis virus reveal a gene order different from that of respiratory syncytial virus. Virology 1992, 186: 426-434. 10.1016/0042-6822(92)90007-C

    Article  PubMed  Google Scholar 

  23. Seal BS: Matrix protein gene nucleotide and predicted amino acid sequence demonstrate that the first US avian pneumovirus isolate is distinct from European strains. Virus Res 1998, 58: 45-52. 10.1016/S0168-1702(98)00098-7

    Article  PubMed  Google Scholar 

  24. Seal BS: Avian pneumoviruses and emergence of a new type in the United States of America. Anim Health Res Rev 2000, 1: 67-72. 10.1017/S1466252300000062

    Article  PubMed  Google Scholar 

  25. Bayon-Auboyer MH, Arnauld C, Toquin D, Eterradossi N: Nucleotide sequences of the F, L and G protein genes of two non-A/non-B avian pneumoviruses (APV) reveal a novel APV subgroup. J Gen Virol 2000, 81: 2723-2733.

    Article  PubMed  Google Scholar 

  26. Chacon JL, Mizuma M, Vejarano MP, Toquin D, Eterradossi N, Patnayak DP, Goyal SM, Ferreira AJ: Avian metapneumovirus subtypes circulating in Brazilian vaccinated and nonvaccinated chicken and turkey farms. Avian Dis 2011, 55: 82-89. 10.1637/9501-081310-Reg.1

    Article  PubMed  Google Scholar 

  27. Toquin D, Guionie O, Jestin V, Zwingelstein F, Allee C, Eterradossi N: European and American subgroup C isolates of avian metapneumovirus belong to different genetic lineages. Virus Genes 2006, 32: 97-103. 10.1007/s11262-005-5850-3

    Article  PubMed  Google Scholar 

  28. Lee E, Song MS, Shin JY, Lee YM, Kim CJ, Lee YS, Kim H, Choi YK: Genetic characterization of avian metapneumovirus subtype C isolated from pheasants in a live bird market. Virus Res 2007, 128: 18-25. 10.1016/j.virusres.2007.03.029

    Article  PubMed  Google Scholar 

  29. Gulati BR, Munir S, Patnayak DP, Goyal SM, Kapur V: Detection of antibodies to U.S. isolates of avian pneumovirus by a recombinant nucleocapsid protein-based sandwich enzyme-linked immunosorbent assay. J Clin Microbiol 2001, 39: 2967-2970. 10.1128/JCM.39.8.2967-2970.2001

    Article  PubMed  PubMed Central  Google Scholar 

  30. Jirjis FE, Noll SL, Halvorson DA, Nagaraja KV, Townsend EL, Sheikh AM, Shaw DP: Avian pneumovirus infection in Minnesota turkeys: experimental reproduction of the disease. Avian Dis 2000, 44: 222-226. 10.2307/1592530

    Article  PubMed  Google Scholar 

  31. Jirjis FF, Noll SL, Halvorson DA, Nagaraja KV, Shaw DP: Pathogenesis of avian pneumovirus infection in turkeys. Vet Pathol 2002, 39: 300-310. 10.1354/vp.39-3-300

    Article  PubMed  Google Scholar 

  32. Bennett RS, LaRue R, Shaw D, Yu Q, Nagaraja KV, Halvorson DA, Njenga MK: A wild goose metapneumovirus containing a large attachment glycoprotein is avirulent but immunoprotective in domestic turkeys. J Virol 2005, 79: 14834-14842. 10.1128/JVI.79.23.14834-14842.2005

    Article  PubMed  PubMed Central  Google Scholar 

  33. Shin HJ, Njenga MK, McComb B, Halvorson DA, Nagaraja KV: Avian pneumovirus (APV) RNA from wild and sentinel birds in the United States has genetic homology with RNA from APV isolates from domestic turkeys. J Clin Microbiol 2000, 38: 4282-4284.

    PubMed  PubMed Central  Google Scholar 

  34. Shin HJ, Nagaraja KV, McComb B, Halvorson DA, Jirjis FF, Shaw DP, Seal BS, Njenga MK: Isolation of avian pneumovirus from mallard ducks that is genetically similar to viruses isolated from neighboring commercial turkeys. Virus Res 2002, 83: 207-212. 10.1016/S0168-1702(01)00402-6

    Article  PubMed  Google Scholar 

  35. Turpin EA, Stallknecht DE, Slemons RD, Zsak L, Swayne DE: Evidence of avian metapneumovirus subtype C infection of wild birds in Georgia, South Carolina, Arkansas and Ohio. USA. Avian Pathol 2008, 37: 343-351. 10.1080/03079450802068566

    Article  PubMed  Google Scholar 

  36. Lwamba HC, Alvarez R, Wise MG, Yu Q, Halvorson D, Njenga MK, Seal BS: Comparison of the full-length genome sequence of avian metapneumovirus subtype C with other paramyxoviruses. Virus Res 2005, 107: 83-92. 10.1016/j.virusres.2004.07.002

    Article  PubMed  Google Scholar 

  37. Rosenberger JK, Krauss WC, Slemons RD: Isolation of Newcastle disease and type-A influenza viruses from migratory waterfowl in the Atlantic flyway. Avian Dis 1974, 18: 610-613. 10.2307/1589019

    Article  PubMed  Google Scholar 

  38. Slemons RD, Johnson DC, Osborn JS, Hayes F: Type-A influenza viruses isolated from wild free-flying ducks in California. Avian Dis 1974, 18: 119-124. 10.2307/1589250

    Article  PubMed  Google Scholar 

  39. Bennett RS, McComb B, Shin HJ, Njenga MK, Nagaraja KV, Halvorson DA: Detection of avian pneumovirus in wild Canada (Branta canadensis) and blue-winged teal (Anas discors) geese. Avian Dis 2002, 46: 1025-1029. 10.1637/0005-2086(2002)046[1025:DOAPIW]2.0.CO;2

    Article  PubMed  Google Scholar 

  40. Cook JKA, Ellis MM, Dolby CA, Holmes HC, Finney PM, Huggins MB: A live attenuated turkey rhinotracheitis virus vaccine: 1. Stability of the attenuated strain. Avian Pathol 1989, 18: 511-522. 10.1080/03079458908418623

    Article  PubMed  Google Scholar 

  41. Cook JKA, Ellis MM, Dolby CA, Holmes HC, Finney PM, Huggins MB: A live attenuated turkey rhinotracheitis virus vaccine: 2. The use of the attenuated strain as an experimental vaccine. Avian Pathol 1989, 18: 523-534. 10.1080/03079458908418624

    Article  PubMed  Google Scholar 

  42. Williams RA, Savage CE, Jones RC: Development of a live attenuated vaccine against turkey rhinotracheitis. Avian Pathol 1991, 20: 45-55. 10.1080/03079459108418740

    Article  PubMed  Google Scholar 

  43. Williams RA, Savage CE, Jones RC: Further studies on the development of a live attenuated vaccine against turkey rhinotracheitis. Avian Pathol 1991, 20: 585-596. 10.1080/03079459108418798

    Article  PubMed  Google Scholar 

  44. Kong BW, Foster LK, Foster DN: Species-specific deletion of the viral attachment glycoprotein of avian metapneumovirus. Virus Res 2008, 132: 114-121. 10.1016/j.virusres.2007.11.006

    Article  PubMed  Google Scholar 

  45. Yu Q, Estevez C, Song M, Kapczynski D, Zsak L: Generation and biological assessment of recombinant avian metapneumovirus subgroup C (aMPV-C) viruses containing different length of the G gene. Virus Res 2010, 147: 182-188. 10.1016/j.virusres.2009.10.021

    Article  PubMed  Google Scholar 

  46. Velayudhan BT, Noll SL, Thachil AJ, Shaw DP, Goyal SM, Halvorson DA, Nagaraja KV: Development of a vaccine-challenge model for avian metapneumovirus subtype C in turkeys. Vaccine 2007, 25: 1841-1847. 10.1016/j.vaccine.2006.10.037

    Article  PubMed  Google Scholar 

  47. Govindarajan D, Kim SH, Samal SK: Contribution of the attachment G glycoprotein to pathogenicity and immunogenicity of avian metapneumovirus subgroup C. Avian Dis 2010, 54: 59-66. 10.1637/8991-071409-Reg.1

    Article  PubMed  Google Scholar 

  48. Juhasz K, Easton AJ: Extensive sequence variation in the attachment (G) protein gene of avian pneumovirus: evidence for two distinct subgroups. J Gen Virol 1994, 75: 2873-2880. 10.1099/0022-1317-75-11-2873

    Article  PubMed  Google Scholar 

  49. Patnayak DP, Sheikh AM, Gulati BR, Goyal SM: Experimental and field evaluation of a live vaccine against avian pneumovirus. Avian Pathol 2002, 31: 377-382. 10.1080/0307945022041651

    Article  PubMed  Google Scholar 

  50. Alvarez R, Lwamba HM, Kapczynski DR, Njenga MK, Seal BS: Nucleotide and predicted amino acid sequence-based analysis of the avian metapneumovirus type C cell attachment glycoprotein gene: phylogenetic analysis and molecular epidemiology of U.S. pneumoviruses. J Clin Microbiol 2003, 41: 1730-1735. 10.1128/JCM.41.4.1730-1735.2003

    Article  PubMed  PubMed Central  Google Scholar 

  51. Govindarajan D, Yunus AS, Samal SK: Complete sequence of the G glycoprotein gene of avian metapneumovirus subgroup C and identification of a divergent domain in the predicted protein. J Gen Virol 2004, 85: 3671-3675. 10.1099/vir.0.80400-0

    Article  PubMed  Google Scholar 

  52. Velayudhan BT, Yu Q, Estevez CN, Nagaraja KV, Halvorson DA: Glycoprotein gene truncation in avian metapneumovirus subtype C isolates from the United States. Virus Genes 2008, 37: 266-272. 10.1007/s11262-008-0220-6

    Article  PubMed  PubMed Central  Google Scholar 

  53. Reed LJ, Muench H: A simple method for estimating fifty percent endpoints. Am J Hyg 1938, 27: 493-497.

    Google Scholar 

  54. Cha RM, Khatri M, Sharma JM: B-cell infiltration in the respiratory mucosa of turkeys exposed to subtype C avian metapneumovirus. Avian Dis 2007, 51: 764-770. 10.1637/0005-2086(2007)51[764:BIITRM]2.0.CO;2

    Article  PubMed  Google Scholar 

  55. Chary P, Rautenschlein S, Njenga MK, Sharma JM: Pathogenic and immunosuppressive effects of avian pneumovirus in turkeys. Avian Dis 2002, 46: 153-161. 10.1637/0005-2086(2002)046[0153:PAIEOA]2.0.CO;2

    Article  PubMed  Google Scholar 

  56. Jirjis FE, Noll SL, Halvorson DA, Nagaraja KV, Shaw DP: Immunohistochemical detection of avian pneumovirus in formalin-fixed tissues. J Vet Diagn Invest 2001, 13: 13-16. 10.1177/104063870101300103

    Article  PubMed  Google Scholar 

  57. Pantin-Jackwood MJ, Spackman E, Day JM: Pathogenesis of type 2 turkey astroviruses with variant capsid genes in 2-day-old specific pathogen free poults. Avian Pathol 2008, 37: 193-201. 10.1080/03079450801932200

    Article  PubMed  Google Scholar 

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We thank Dr. K. Nagaraja of University of Minnesota for kindly providing Rabbit anti-aMPV/C antibody, Fenglan Li for her excellent technical assistance and the sequencing unit in SEPRL for outstanding support. We also thank Dr. Mary Pantin-Jackwood and Diane Smith for technical assistance for immunohistochemistry staining. This research was supported by USDA, ARS CRIS project 6612-32000-056-00D.

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Correspondence to Laszlo Zsak.

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The authors declare that they have no competing interests.

Authors’ contributions

RMC conceived and planned the study, performed the animal experiments, and co-wrote the paper. QY prepared the wild type virus stocks and co-wrote the paper. LZ coordinated the studies, supervised the animal experiments and co-wrote the paper. All authors read and approved the final manuscript.

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Cha, R.M., Yu, Q. & Zsak, L. The pathogenicity of avian metapneumovirus subtype C wild bird isolates in domestic turkeys. Virol J 10, 38 (2013).

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