An affordable detection system based on RT-LAMP and DNA-nanoprobes for avian metapneumovirus.

Airborne animal viral pathogens can rapidly spread and become a global threat, resulting in substantial socioeconomic and health consequences. To prevent and control potential epidemic outbreaks, accurate, fast, and affordable point-of-care (POC) tests are essential. As a proof-of-concept, we have developed a molecular system based on the loop-mediated isothermal amplification (LAMP) technique for avian metapneumovirus (aMPV) detection, an airborne communicable agent mainly infecting turkeys and chickens. For this purpose, a colorimetric system was obtained by coupling the LAMP technique with specific DNA-functionalized AuNPs (gold nanoparticles). The system was validated using 50 different samples (pharyngeal swabs and tracheal tissue) collected from aMPV-infected and non-infected chickens and turkeys. Viral detection can be achieved in about 60 min with the naked eye, with 100% specificity and 87.88% sensitivity for aMPV. In summary, this novel molecular detection system allows suitable virus testing in the field, with accuracy and limit of detection (LOD) values highly close to qRT-PCR-based diagnosis. Furthermore, this system can be easily scalable to a platform for the detection of other viruses, addressing the current gap in the availability of POC tests for viral detection in poultry farming. KEY POINTS: •aMPV diagnosis using RT-LAMP is achieved with high sensitivity and specificity. •Fifty field samples have been visualized using DNA-nanoprobe validation. •The developed system is a reliable, fast, and cost-effective option for POCT.

Introduction of Avian metapneumovirus subtype A to the United States: molecular insights and implications.

Avian metapneumovirus (aMPV) poses a significant threat to the poultry industry worldwide, primarily affecting turkeys and chickens. The recent detection of aMPV-A and -B subtypes in the United States marks a significant shift after a prolonged period free of aMPV following the eradication of the previously circulating subtype C. Hence, the demand for molecular diagnostic tests for aMPV has arisen due to their limited availability in the US market. In this study, we present the molecular characterization based on the complete genome sequence of aMPV subtype A, which was detected in the US for the first time. Four RT-qPCR positive samples were subjected to next-generation sequencing analysis, resulting in the assembly of one complete and one near-complete genome sequences. Phylogenetic analysis revealed that the isolated strains clustered within the aMPV-A subtype and were most closely related to recent Mexican strains. A detailed amino acid analysis identified unique mutations in the G gene of the US isolates compared to Mexican strains. Additionally, we compared the performance, cross-reactivity, and limit of detection of our revised aMPV subtype-specific RT-qPCR test with two commercial kits, demonstrating similar detection and subtyping capabilities. These findings highlight the importance of accurate diagnostic methods for disease management in the poultry industry, provide valuable insights into the epidemiology of aMPV, and underscore the need for continued vigilance and surveillance to mitigate its impact on poultry production.

Tracing the Flight: Investigating the Introduction of Avian Metapneumovirus (aMPV) A and B.

Avian metapneumovirus (aMPV) has been identified as an important cause of respiratory and reproductive disease, leading to significant productive losses worldwide. Different subtypes have been found to circulate in different regions, with aMPV-A and B posing a significant burden especially in the Old World, and aMPV-C in North America, albeit with limited exceptions of marginal economic relevance. Recently, both aMPV-A and aMPV-B have been reported in the U.S.; however, the route of introduction has not been investigated. In the present study, the potential importation pathways have been studied through phylogenetic and phylodynamic analyses based on a broad collection of partial attachment (G) protein sequences collected worldwide. aMPV-B circulating in the U.S. seems the descendant of Eastern Asian strains, which, in turn, are related to European ones. A likely introduction pathway mediated by wild bird migration through the Beringian crucible, where the East Asian and Pacific American flight paths intersect, appears likely and was previously reported for avian influenza. aMPV-A, on the other hand, showed a Mexican origin, involving strains related to Asian ones. Given the low likelihood of trade or illegal importation, the role of wild birds appears probable also in this case, since the region is covered by different flight paths directed in a North-South direction through America. Since the information on the role of wild birds in aMPV epidemiology is still scarce and scattered, considering the significant practical implications for the poultry industry demonstrated by recent U.S. outbreaks, further surveys on wild birds are encouraged.

SQSTM1 downregulates avian metapneumovirus subgroup C replication via mediating selective autophagic degradation of viral M2-2 protein.

Avian metapneumovirus subgroup C (aMPV/C), an important pathogen causing acute respiratory infection in chickens and turkeys, contributes to substantial economic losses in the poultry industry worldwide. aMPV/C has been reported to induce autophagy, which is beneficial to virus replication. Sequestosome 1 (SQSTM1/P62), a selective autophagic receptor, plays a crucial role in viral replication by clearing ubiquitinated proteins. However, the relationship between SQSTM1-mediated selective autophagy and aMPV/C replication is unclear. In this study, we found that the expression of SQSTM1 negatively regulates aMPV/C replication by reducing viral protein expression and viral titers. Further studies revealed that the interaction between SQSTM1 and aMPV/C M2-2 protein is mediated via the Phox and Bem1 (PB1) domain of the former, which recognizes a ubiquitinated lysine at position 67 of the M2-2 protein, and finally degrades M2-2 via SQSTM1-mediated selective autophagy. Collectively, our results reveal that SQSTM1 degrades M2-2 via a process of selective autophagy to suppress aMPV/C replication, thereby providing novel insights for the prevention and control of aMPV/C infection.IMPORTANCEThe selective autophagy plays an important role in virus replication. As an emerging pathogen of avian respiratory virus, clarification of the effect of SQSTM1, a selective autophagic receptor, on aMPV/C replication in host cells enables us to better understand the viral pathogenesis. Previous study showed that aMPV/C infection reduced the SQSTM1 expression accompanied by virus proliferation, but the specific regulatory mechanism between them was still unclear. In this study, we demonstrated for the first time that SQSTM1 recognizes the 67th amino acid of M2-2 protein by the interaction between them, followed by M2-2 degradation via the SQSTM1-mediated selective autophagy, and finally inhibits aMPV/C replication. This information supplies the mechanism by which SQSTM1 negatively regulates viral replication, and provides new insights for preventing and controlling aMPV/C infection.

Predominance of low pathogenic avian influenza virus H9N2 in the respiratory co-infections in broilers in Tunisia: a longitudinal field study, 2018-2020.

Respiratory diseases are a health and economic concern for poultry production worldwide. Given global economic exchanges and migratory bird flyways, respiratory viruses are likely to emerge continuously in new territories. The primary aim of this study was to investigate the major pathogens involved in respiratory disease in Tunisian broiler poultry and their epidemiology. Between 2018 and 2020, broilers farms in northeastern Tunisia were monitored, and 39 clinically diseased flocks were sampled. Samples were screened for five viral and three bacterial respiratory pathogens using a panel of real-time PCR assays. The reemergence of H9N2 low pathogenic avian influenza virus (LPAIV) in commercial poultry was reported, and the Northern and Western African GI lineage strain was typed. The infectious bronchitis virus (IBV) GI-23 lineage and the avian metapneumovirus (aMPV) subtype B also were detected for the first time in broilers in Tunisia. H9N2 LPAIV was the most detected pathogen in the flocks tested, but rarely alone, as 15 of the 16 H9N2 positive flocks were co-infected. Except for infectious laryngotracheitis virus (ILTV), all of the targeted pathogens were detected, and in 61% of the respiratory disease cases, a combination of pathogens was identified. The major combinations were H9N2 + aMPV (8/39) and H9N2 + IBV (6/39), showing the high contribution of H9N2 LPAIV to the multifactorial respiratory diseases. This field survey provided evidence of the emergence of new respiratory viruses and the complexity of respiratory disease in Tunisia. A comprehensive and continuous surveillance strategy therefore is needed to better control respiratory pathogens in Tunisia.

Serological survey of avian metapneumovirus in vaccinated and unvaccinated broiler chickens in Hong Kong.

In chickens, avian metapneumovirus (aMPV) causes the swollen head syndrome, a respiratory disease often associated with a reduction in egg production. The virus' epidemiology in East and Southeast Asia is poorly understood. An aMPV serological survey was conducted on broiler chicken farms of Hong Kong SAR to assess the seroprevalence of aMPV in unvaccinated batches and the serological status of vaccinated batches. Blood samples were collected from 53-93-day-old chickens in 24 chicken farms of Hong Kong SAR and sera were tested for aMPV antibodies by ELISA. Seroprevalence in aMPV unvaccinated birds was 80.6% (95% confidence interval (CI): 78.9-82.2) with a high variation between batches. Batch-level seroprevalence was not significantly different between birds hatched during the rainy season (74.3%, 95% CI: 64.0-84.5) and the ones hatched during the dry season (88.7%, 95% CI: 80.1-97.3, p = 0.5). The high seroprevalence and high antibody titers that are reported in this study indicate repeated exposure of broiler chickens to aMPV in Hong Kong SAR poultry farms. Based on these results, we recommend improving the surveillance of respiratory pathogens and applying appropriate prophylactic measures against aMPV such as vaccination.

Research Note: Evaluation of the cross protective efficacy of a subtype B aMPV vaccine against virulent subtype A aMPV by different administration routes.

Avian metapneumovirus (aMPV) causes respiratory and reproductive diseases in birds, including chickens. In the chicken industry, live vaccines against aMPV subtypes A and B, which are the major aMPV subtypes, are widely used to control disease caused by aMPV. In this study, we evaluated the cross protective efficacy of a live aMPV subtype B vaccine administered via 3 different routes (nasal, spray, and oral) against virulent aMPV subtype A in chickens. At 3 wk after vaccination of 1-wk-old specific-pathogen-free chickens, we measured the serological responses. On the same day, we challenged the birds with aMPV subtype A. Protection was evaluated by viral gene detection and histopathological examination at 3 and 5 days postchallenge. Although there were differences in the serological responses according to administration route, all vaccinated birds showed complete protection at 5 days postchallenge. Regardless of administration route, genome of challenge virus was not detected in vaccinated group, and there were significant differences between vaccinated birds and control group. Overall, our results demonstrated that a subtype B aMPV vaccine can provide cross protection against virulent subtype A aMPV in chickens.

Characterization of avain metapneumovirus subgroup C isolated from chickens in Beijing, China.

Avian metapneumovirus (aMPV) is an important causative agent that causes acute respiratory disease and egg-dropping in chickens and turkeys. Here, we characterized an aMPV subgroup C (aMPV/C) from 320-day-old broiler breeder chickens with severe respiratory diseases in Beijing, China, as evidenced by RT-PCR typing and confirmation of the nucleoprotein (N) gene sequence. The N gene sequence of the aMPV/C strain (designated BJ17) exhibited no deletions or insertions and possessed 94.6% to 99.6% identity to those of published aMPV/C isolates. The phylogenetic tree of the nucleotide sequences constructed using the neighbor-joining clustering method showed that the BJ17 strain formed one cluster with other aMPV/C viruses and formed one subcluster with published Chinese aMPV/C isolates regardless of Muscovy duck or chicken origins. Comparative analysis of the N proteins showed that a unique amino acid residue D at position 110 might be associated with regional distribution due to its occurrence in all the Chinese aMPV/C isolates only. Strain BJ17 was successfully isolated by cultured Vero cell passage and further inoculated in 3-wk-old specific-pathogen-free chickens for the examination of pathogenicity. Animal experimental results showed that BJ17-inoculated chickens had severe respiratory diseases and inflammatory lesions, as demonstrated by pathological changes and aMPV antigen in the nasal turbinate, tracheae, and lung tissues. These results enrich the available information regarding the epidemiology and pathogenicity of aMPV/C in chickens, which may facilitate the development of effective measures against aMPV/C infection in China.

Detection and Genome Sequence Analysis of Avian Metapneumovirus Subtype A Viruses Circulating in Commercial Chicken Flocks in Mexico.

Avian metapneumoviruses (aMPV subtypes A-D) are respiratory and reproductive pathogens of poultry. Since aMPV-A was initially reported in Mexico in 2014, there have been no additional reports of its detection in the country. Using nontargeted next-generation sequencing (NGS) of FTA card-spotted respiratory samples from commercial chickens in Mexico, seven full genome sequences of aMPV-A (lengths of 13,288-13,381 nucleotides) were de novo assembled. Additionally, complete coding sequences of genes N (n = 2), P and M (n = 7 each), F and L (n = 1 each), M2 (n = 6), SH (n = 5) and G (n = 2) were reference-based assembled from another seven samples. The Mexican isolates phylogenetically group with, but in a distinct clade separate from, other aMPV-A strains. The genome and G-gene nt sequences of the Mexican aMPVs are closest to strain UK/8544/06 (97.22-97.47% and 95.07-95.83%, respectively). Various amino acid variations distinguish the Mexican isolates from each other, and other aMPV-A strains, most of which are in the G (n = 38), F (n = 12), and L (n = 19) proteins. Using our sequence data and publicly available aMPV-A data, we revised a previously published rRT-PCR test, which resulted in different cycling and amplification conditions for aMPV-A to make it more compatible with other commonly used rRT-PCR diagnostic cycling conditions. This is the first comprehensive sequence analysis of aMPVs in Mexico and demonstrates the value of nontargeted NGS to identify pathogens where targeted virus surveillance is likely not routinely performed.

Development of simplex and multiplex RT-qPCR assays for the detection of three cryptic viruses of black-grass (Alopecurus myosuroides).

Simplex and multiplex RT-qPCR assays were developed for Alopecurus myosuroides partitivirus 1 (AMPV1), Alopecurus myosuroides partitivirus 2 (AMPV2) and Alopecurus myosuroides varicosavirus 1 (AMVV1), and compared to the existing conventional PCR assays. All assays had a high specificity and their sensitivity was increased compared to the conventional RT-PCR assays. As viral quantification is an important element in comparative experiments, the effect of high- and low-temperature drying treatments, prior to RNA extraction and analysis, was studied and optimised. AMVV1 detection was reduced by both drying treatments, but particularly by the high-temperature. AMPV1 and AMPV2 detection on the other hand was not impeded by the drying treatments, and enables standardisation of plant tissue prior to extraction, in particular for quantitative analysis.

Avian Metapneumovirus Subgroup C Induces Mitochondrial Antiviral Signaling Protein Degradation through the Ubiquitin-Proteasome Pathway.

The mitochondrial antiviral signaling (MAVS) protein, a critical adapter, links the upstream recognition of viral RNA to downstream antiviral signal transduction. However, the interaction mechanism between avian metapneumovirus subgroup C (aMPV/C) infection and MAVS remains unclear. Here, we confirmed that aMPV/C infection induced a reduction in MAVS expression in Vero cells in a dose-dependent manner, and active aMPV/C replication was required for MAVS decrease. We also found that the reduction in MAVS occurred at the post-translational level rather than at the transcriptional level. Different inhibitors were used to examine the effect of proteasome or autophagy on the regulation of MAVS. Treatment with a proteasome inhibitor MG132 effectively blocked MAVS degradation. Moreover, we demonstrated that MAVS mainly underwent K48-linked ubiquitination in the presence of MG132 in aMPV/C-infected cells, with amino acids 363, 462, and 501 of MAVS being pivotal sites in the formation of polyubiquitin chains. Finally, E3 ubiquitin ligases for MAVS degradation were screened and identified and RNF5 targeting MAVS at Lysine 363 and 462 was shown to involve in MAVS degradation in aMPV/C-infected Vero cells. Overall, these results reveal the molecular mechanism underlying aMPV/C infection-induced MAVS degradation by the ubiquitin-proteasome pathway.

Arteriovenous malformation of the trigeminal nerve root presented with venous congestive edema of the medulla oblongata and upper cervical cord: illustrative case.

BACKGROUND: Arteriovenous malformation (AVM) of the trigeminal nerve root (TNR) is a rare subtype of the lateral pontine AVM. Most of them are diagnosed when they bleed or exert trigeminal neuralgia. Venous congestive edema is a rare phenomenon caused by TNR AVMs. OBSERVATIONS: An 82-year-old man was admitted with progressive limb weakness and dysphasia. Magnetic resonance imaging (MRI) revealed extensive edema of the medulla oblongata and the upper cervical cord with signal flow void at the C3 anterior spinal cord. Vertebral angiography revealed a small nidus fed mainly by the pontine perforating arteries (PPAs). The anterior pontomesencephalic vein (AMPV) was dilated, functioning as the main drainage route. This suggests that venous hypertension triggered the brainstem and upper cervical cord edema. MRI with gadolinium enhancement showed that the nidus was located around the right TNR. Because the nidus sat extrinsically on the pial surface of the right TNR's base, microsurgical obliteration with minimum parenchymal injury was achieved. Postoperative MRI showed disappearance of the brainstem and cervical cord edema with improved clinical symptoms. LESSONS: TNR AVM is rarely associated with brainstem and upper cervical cord edema caused by venous hypertension of the congestive drainage system.

Molecular Epidemiology and Genotyping of Infectious Bronchitis Virus and Avian Metapneumovirus in Backyard and Commercial Chickens in Jimma Zone, Southwestern Ethiopia.

Poultry production plays a relevant role in the Ethiopian economy and represents a source of poverty alleviation for several social classes. Infectious diseases can therefore significantly impact the economy and welfare. Despite infectious bronchitis virus (IBV) and avian metapneumovirus (aMPV) being present, the knowledge of their epidemiology and impact is extremely limited. In the present work, a cross-sectional study based on 500 tracheal swabs collected from 50 intensive and backyard unvaccinated flocks of the Jimma Zone was performed to investigate the circulation of these viruses and molecularly characterize them. IBV and aMPV presence was tested by molecular assays, and genotyping was carried out on positive samples. Accordingly, 6% (95% CI 2.06% to 16.22%) and 8% (95% CI 3.15% to 18.84%) of flocks tested IBV and aMPV positive, respectively. Particularly, IBV 793B (GI-13) strains were detected in backyard flocks only, and identical or closely related sequences (p-distance <2%) were detected in distantly spaced flocks, suggesting relevant viral circulation. On the contrary, both backyard and intensive flocks were affected by aMPV subtype B. Potential epidemiological links associated to the importation of parental birds from foreign countries could be established. These results highlight non-negligible circulation of these viruses, warranting further epidemiological studies and the evaluation of control measure implementation.

Proteome Analysis in a Mammalian Cell line Reveals that PLK2 is Involved in Avian Metapneumovirus Type C (aMPV/C)-Induced Apoptosis.

Avian metapneumovirus subtype C (aMPV/C) causes an acute respiratory disease that has caused serious economic losses in the Chinese poultry industry. In the present study, we first explored the protein profile in aMPV/C-infected Vero cells using iTRAQ quantitative proteomics. A total of 921 of 7034 proteins were identified as significantly altered by aMPV/C infection. Three selected proteins were confirmed by Western blot analysis. Bioinformatics GO analysis revealed multiple signaling pathways involving cell cycle, endocytosis, and PI3K-Akt, mTOR, MAPK and p53 signaling pathways, which might participate in viral infection. In this analysis, we found that PLK2 expression was upregulated by aMPV/C infection and investigated whether it contributed to aMPV/C-mediated cellular dysfunction. Suppressing PLK2 attenuated aMPV/C-induced reactive oxygen species (ROS) production and p53-dependent apoptosis and reduced virus release. These results in a mammalian cell line suggest that high PLK2 expression correlates with aMPV/C-induced apoptosis and viral replication, providing new insight into the potential avian host cellular response to aMPV/C infection and antiviral targets.

Detection of avian metapneumovirus subtype A from wild birds in the State of São Paulo, Brazil / Detecção de metapneumovirus aviário subtipo A em aves silvestres no estado de São Paulo, Brasil

The present study investigated the circulation of avian metapneumovirus (aMPV) in wild birds in Brazil. To do so, 131 samples from 366 oropharyngeal or cloacal swabs collected from 18 species of birds were tested individually or in pools by RT-PCR. Samples detected by RT-PCR were selected for DNA sequencing. Thirteen (9.9%) samples were detected by the RT-PCR targeting the N gene and four out of 13 samples were sequenced. Sequencing results showed a high identity with the aMPV subtype A. Our results confirm the circulation of the aMPV subtype A in wild birds in Brazil even five years after its last detection.(AU)

O presente estudo investigou a circulação de metapneumovírus aviário em aves silvestres no Brasil. Para tanto, 131 amostras de 366 suabes orofaringeanos ou cloacais coletados de 18 espécies de aves foram testadas individualmente ou na forma de pools por RT-PCR. As amostras detectadas por RT-PCR foram selecionadas para sequenciamento. Treze (9,9%) das amostras foram detectadas por RT-PCR tendo o gene N como alvo; destas, quatro foram sequenciadas com sucesso. Resultados do sequenciamento mostraram alta identidade com o aMPV de subtipo A. Nossos resultados confirmam a circulação de aMPV subtipo A em aves silvestres no Brasil mesmo cinco anos após sua última detecção.(AU)

The small hydrophobic (SH) gene of North American turkey AMPV-C does not attenuate nor modify host tropism in recombinant European duck AMPV-C.

Subgroup C Avian Metapneumoviruses (AMPV-C) has two lineages, one mostly in turkeys and one mostly in ducks. To investigate the molecular basis of AMPV-C host tropism, a reverse genetics system for a duck AMPV-C virus was developed. A recombinant copy and a recombinant virus in which the SH protein had been exchanged for that of a turkey AMPV-C were rescued. No change in cytopathogenic effect or replication profile in vitro were observed for either virus compared to the wild type. In SPF Muscovy ducks the wild type and its recombinant copy were equally pathogenic. Exchanging the SH in the recombinant copy produced the same results. In SPF turkeys, neither recombinant virus was pathogenic, although both showed a low level of replication. Thus, from the current model, it appears that AMPV-C SH proteins derived from the different species are compatible and that turkey SH does not affect duck AMPV-C pathogenicity.

Avian metapneumovirus subgroup C induces autophagy through the ATF6 UPR pathway.

An increasing number of studies have demonstrated that macroautophagy/autophagy plays an important role in the infectious processes of diverse pathogens. However, it remains unknown whether autophagy is induced in avian metapneumovirus (aMPV)-infected host cells, and, if so, how this occurs. Here, we report that aMPV subgroup C (aMPV/C) induces autophagy in cultured cells. We demonstrated this relationship by detecting classical autophagic features, including the formation of autophagsomes, the presence of GFP-LC3 puncta and the conversation of LC3-I into LC3-II. Also, we used pharmacological regulators and siRNAs targeting ATG7 or LC3 to examine the role of autophagy in aMPV/C replication. The results showed that autophagy is required for efficient replication of aMPV/C. Moreover, infection with aMPV/C promotes autophagosome maturation and induces a complete autophagic process. Finally, the ATF6 pathway, of which one component is the unfolded protein response (UPR), becomes activated in aMPV/C-infected cells. Knockdown of ATF6 inhibited aMPV/C-induced autophagy and viral replication. Collectively, these results not only show that autophagy promotes aMPV/C replication in the cultured cells, but also reveal that the molecular mechanisms underlying aMPV/C-induced autophagy depends on regulation of the ER stress-related UPR pathway.

Detection of infectious bronchitis virus 793B, avian metapneumovirus, Mycoplasma gallisepticum and Mycoplasma synoviae in poultry in Ethiopia.

A survey was conducted into respiratory infectious diseases of poultry on a chicken breeder farm run by the Ethiopian Institute of Agricultural Research (EIAR), located in Debre Zeit, Ethiopia. Oropharyngeal swabs were collected from 117 randomly selected birds, and blood was taken from a subset of 73 of these birds. A combination of serological and molecular methods was used for detection of pathogens. For the first time in Ethiopia, we report the detection of variant infectious bronchitis virus (793B genotype), avian metapneumovirus subtype B and Mycoplasma synoviae in poultry. Mycoplasma gallisepticum was also found to be present; however, infectious laryngotracheitis virus was not detected by PCR. Newcastle disease virus (NDV) was not detected by PCR, but variable levels of anti-NDV HI antibody titres shows possible exposure to virulent strains or poor vaccine take, or both. For the burgeoning-intensive industry in Ethiopia, this study highlights several circulating infectious respiratory pathogens that can impact on poultry welfare and productivity.

Immunization with avian metapneumovirus harboring chicken Fc induces higher immune responses.

In this study, we evaluated the immune responses of avian metapneumovirus harboring chicken Fc molecule. Stable Vero cells expressing chicken Fc chimera on its surface (Vero-cFc) were established, and we confirmed that aMPV grown in Vero-cFc incorporated host derived chimera Fc into the aMPV virions. Immunization of chicken with aMPV-cFc induced higher level of antibodies and inflammatory cytokines; (Interferon (IFN)-γ and Interleukin (IL)-1ß) compared to those of aMPV. The increased levels of antibodies and inflammatory cytokines in chicken immunized with aMPV-cFc were statistically significantly (p<0.05) to that of aMPV and control. The aMPV-cFc group also generated the highest neutralizing antibody response. After challenges, chickens immunized with aMPV-cFc showed much less pathological signs in nasal turbinates and trachea so that we could confirm aMPV-cFc induced higher protection than that of aMPV. The greater ability of aMPV harboring chicken Fc to that of aMPV presented it as a possible vaccine candidate.