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.

A Receptor Integrin ß1 Promotes Infection of Avian Metapneumovirus Subgroup C by Recognizing a Viral Fusion Protein RSD Motif.

Avian metapneumovirus subgroup C (aMPV/C) causes respiratory diseases and egg dropping in chickens and turkeys, resulting in severe economic losses to the poultry industry worldwide. Integrin ß1 (ITGB1), a transmembrane cell adhesion molecule, is present in various cells and mediates numerous viral infections. Herein, we demonstrate that ITGB1 is essential for aMPV/C infection in cultured DF-1 cells, as evidenced by the inhibition of viral binding by EDTA blockade, Arg-Ser-Asp (RSD) peptide, monoclonal antibody against ITGB1, and ITGB1 short interfering (si) RNA knockdown in cultured DF-1 cells. Simulation of the binding process between the aMPV/C fusion (F) protein and avian-derived ITGB1 using molecular dynamics showed that ITGB1 may be a host factor benefiting aMPV/C attachment or internalization. The transient expression of avian ITGB1-rendered porcine and feline non-permissive cells (DQ cells and CRFK cells, respectively) is susceptible to aMPV/C infection. Kinetic replication of aMPV/C in siRNA-knockdown cells revealed that ITGB1 plays an important role in aMPV/C infection at the early stage (attachment and internalization). aMPV/C was also able to efficiently infect human non-small cell lung cancer (A549) cells. This may be a consequence of the similar structures of both metapneumovirus F protein-specific motifs (RSD for aMPV/C and RGD for human metapneumovirus) recognized by ITGB1. Overexpression of avian-derived ITGB1 and human-derived ITGB1 in A549 cells enhanced aMPV/C infectivity. Taken together, this study demonstrated that ITGB1 acts as an essential receptor for aMPV/C attachment and internalization into host cells, facilitating aMPV/C infection.

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.

First detection of avian metapneumovirus subtype C Eurasian lineage in a Eurasian wigeon (Mareca penelope) wintering in Northeastern Italy: an additional hint on the role of migrating birds in the viral epidemiology.

Avian metapneumovirus (aMPV) economically affects the global poultry industry causing respiratory and reproductive disorders. Considering the paucity of data on aMPV occurrence in European free-ranging avifauna, a molecular survey was conducted on wild birds of 23 species belonging to the orders Anseriformes, Charadriiformes or Passeriformes, captured alive and sampled in Northeast Italy as part of the national avian influenza virus (AIV) surveillance activities. A total of 492 oropharyngeal swabs, collected from 2007-2010, all AIV-negative, were screened from aMPV by subtype-specific qRT-PCR. An aMPV-C strain, named aMPV/C/IT/Wigeon/758/07, was found in a wintering young Eurasian wigeon (Mareca penelope) sampled in November 2007. The matrix, fusion, and attachment glycoprotein genes of the detected strain were subsequently amplified by specific independent RT-PCRs, then sequenced, and compared in a phylogenetic framework with known aMPV homologous sequences retrieved from GenBank. Close genetic relationships were found between the aMPV/C/IT/Wigeon/758/07 strain and subtype C Eurasian lineage strains isolated in the late 1990s in French domestic ducks, suggesting epidemiological links. Eurasian wigeons are medium/long-range migrant dabbling ducks that move along the Black Sea/Mediterranean flyway; our finding might, therefore, be related to migratory bridges between countries. To our knowledge, this is the first molecular evidence of the occurrence of aMPV subtype C in Italy and backdates the aMPV-C circulation to 2007. Moreover, the results suggest the susceptibility of Eurasian wigeons to aMPV. Broader investigations are needed to assess the role of wild ducks and the significance of the wildfowl/poultry interface in aMPV-C epidemiology.RESEARCH HIGHLIGHTSWild birds live-captured in Italy were tested for aMPV detection and characterization.aMPV-C Eurasian lineage was found for the first time in a wintering Eurasian wigeon.Migratory birds could be involved in the aMPV epidemiology.

Avian metapneumovirus subtype B vaccination in commercial broiler chicks: heterologous protection and selected host transcription responses to subtype A or B challenge.

Avian metapneumovirus (aMPV) causes respiratory disease and drops in egg production in chickens, and is routinely controlled by vaccination. However, the host's immune response to virulent challenge in vaccinated or unvaccinated broiler chickens is poorly characterized. We show that subtype B vaccination offers heterologous (subtype A challenge) and homologous (subtype B challenge) protection. Subtype B challenge caused significantly greater humoral antibody titres in vaccinated and unvaccinated chickens. In turbinate and lung tissues of unvaccinated-challenged chickens, IgA and IgY mRNA transcription was significantly up-regulated after subtype B challenge compared to subtype A. Cellular immunity (CD8-α and CD8-ß) gene transcripts were significantly up-regulated during early and later stages of infection from subtype B or subtype A, respectively. Immune gene transcriptional responses (IL-1ß, IL-6 and IL-18) were significantly up-regulated after challenge. Gene transcription results showed that mRNA expression levels of CD8-α, CD8-ß, TLR3 and IL-6, particularly in turbinate and trachea tissues, are useful parameters to include in future aMPV vaccination-challenge studies.

Avian Metapneumovirus subtype B around Europe: a phylodynamic reconstruction.

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.

Host specificity of avian metapneumoviruses.

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'.

Molecular epidemiology of respiratory viruses in commercial chicken flocks in Pakistan from 2014 through to 2016.

BACKGROUND: Viral diseases are a matter of great concern for poultry farmers in Pakistan. Multiple common viral respiratory diseases (CVRDs) cause huge economic losses in the poultry industry. The prevalence of CVRDs in many countries, including Pakistan, is not clearly understood. RESULTS: Incidences of 5 chicken respiratory viruses: avian influenza virus (AIV), Newcastle disease virus (NDV/AAVV-1), infectious bronchitis virus (IBV), avian metapneumovirus (aMPV) and infectious laryngotracheitis virus (ILTV) were assessed on commercial Pakistani farms with respiratory problems from 2014 through to 2016. While AIV and AAVV-1 were frequently detected (16 to 17% of farms), IBV and aMPV were rarely detected (in 3 to 5% of farms) and ILTV was not detected. We characterized H9 AIV of the G1 lineage, genotype VII AAVV-1, GI-13 IBV, and type B aMPV strains with very little genetic variability in the 2-year study period. Co-infections with AIV and AAVV-1 were common and wild type AAVV-1 was detected despite the use of vaccines. Control measures to limit the virus burden in chicken flocks are discussed. CONCLUSIONS: Our data shows that AIV (H9), AAVV-1, IBV and aMPV are prevalent in commercial poultry in Pakistan. Further studies are necessary to assess circulating strains, economic losses caused by infections and coinfections of these pathogens, and the costs and benefits of countermeasures. Furthermore, veterinarians and farmers should be informed of the pathogens circulating in the field and hence advised on the use of vaccines.

Role of trypsin in the replication of Avian metapneumovirus subtype C (strain MN-2a) and its entry into the Vero cells.

To understand the molecular mechanisms of Avian metapneumovirus (aMPV) and the requirements involved in the infection and fusion, trypsin treatment was done in the different stages of virus; before infection, during entry and after virus infection followed by aMPV infection. The growth kinetics of aMPV was compared in time dependent manner. The effect of trypsin was found in the later stage of aMPV infection increasing the numbers of infected cells with the significant higher titer of infectious virions to that of trypsin treated before infection, during entry and aMPV. A serine protease inhibitor reduced aMPV replication in a significant way, whereas cysteine peptidase (E-64), aspartic protease (pepstatin A), and metalloprotease (phosphoramidon) inhibitors had no effect on aMPV replication. Inoculation of aMPV on Vero cells expressing the membrane-associated protease TMPRSS2 resulted in higher virus titers than that inoculated on normal Vero cells and is statistically significant (p < 0.05). Also, an inhibitor of clathrin/caveolae-mediated endocytosis had no effect on virus progeny, indicating that aMPV does not use the endocytic pathway for entry but undergoes direct fusion. The effect of lysosomotropic agents was not significant, suggesting that aMPV does not require low-pH environment in endosomes to fuse its envelope with the plasma membrane.

Research note: Indirect evidence of avian Metapneumovirus circulation in broilers in Italy.

The clinical relevance of avian metapneumovirus (aMPV) is growing in the poultry sector, especially in broiler farming, where no vaccination is administered in Italy. Given the naïve status of the birds, a serological survey was conducted in a densely populated area of Northern Italy, to evaluate aMPV circulation. Seven farms were selected and sampled in summer/fall, then sampling was repeated in the following season (winter/spring) to assess a possible seasonal effect. In each farm, fifteen birds were blood sampled towards the end of the cycle and sera were analyzed with an ELISA test. Clinical signs were reported in 5 out of 7 farms, although all farms were positive at both sampling points, except for one, which was negative at the first sampling. The seroprevalence within farm ranged from 26.6% to 100%, and antibody titres appear to increase with age. No seasonality effect was evidenced, whereas a farm effect was more distinct. aMPV circulation appears wide in Northern Italian farms, with different clinical outcomes that could be modulated by intrinsic characteristics of the farms. In absence of vaccination, serological monitoring can be a useful tool for viral entrance monitoring, although sampling timing should be evaluated in order to spot seroconversion after late infections.

Geographical Expansion of Avian Metapneumovirus Subtype B: First Detection and Molecular Characterization of Avian Metapneumovirus Subtype B in US Poultry.

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.

Effects of G and SH Truncation on the Replication, Virulence, and Immunogenicity of Avian Metapneumovirus.

Four mutants varying the length of the G and SH genes, including a G-truncated mutant (ΔG) and three G/SH-truncated mutants (ΔSH/G-1, ΔSH/G-2, and ΔSH/G-3), were generated via serially passaging the avian metapneumovirus strain SNU21004 into the cell lines Vero E6 and DF-1 and into embryonated chicken eggs. The mutant ΔG particles resembled parental virus particles except for the variance in the density of their surface projections. G and G/SH truncation significantly affected the viral replication in chickens' tracheal ring culture and in infected chickens but not in the Vero E6 cells. In experimentally infected chickens, mutant ΔG resulted in the restriction of viral replication and the attenuation of the virulence. The mutants ΔG and ΔSH/G-1 upregulated three interleukins (IL-6, IL-12, and IL-18) and three interferons (IFNα, IFNß, and IFNγ) in infected chickens. In addition, the expression levels of innate immunity-related genes such as Mda5, Rig-I, and Lgp2, in BALB/c mice were also upregulated when compared to the parental virus. Immunologically, the mutant ΔG induced a strong, delayed humoral immune response, while the mutant ΔSH/G-1 induced no humoral immune response. Our findings indicate the potential of the mutant ΔG but not the mutant ΔSH/G-1 as a live attenuated vaccine candidate.

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.

Establishment and Application Prospect of Reverse Transcriptase Recombinase-Aided Amplification Assay for Subgroup C Avian Metapneumovirus.

Among broilers, the main pathogen that leads to swollen head syndrome (SHS) is the subgroup C avian metapneumovirus (aMPV-C). The aMPV-C infection can lead to an upsurge in the rate of soft-shell eggs, resulting in reduced egg production and seriously affecting the economy of the livestock industry. Therefore, a rapid method for aMPV-C detection needs to be invented. According to the N gene of aMPV-C, we designed the specific probe and primer and created a reverse transcription recombinase-aided amplification assay (RT-RAA) for the detection of aMPV-C. aMPV-C could be detected quickly and specifically by this method at 41 °C for 30 min. The sensitivity assay inferred that the minimum detection threshold of RT-RAA was 3.38 × 101 copies/µL. A specificity assay showed that the RT-RAA method did not cross-react with other subgroups (aMPV-A, aMPV-B, aMPV-D) or other viruses (H9N2, NDV, IBV, IBDV). Forty samples of known clinical background were tested by RT-RAA and RT-qPCR. The two approaches had a 100% correlation rate. In conclusion, this research successfully created an RT-RAA assay for aMPV-C.

Avian metapneumovirus subgroup C infection in chickens, China.

Avian metapneumovirus causes acute respiratory tract infection and reductions in egg production in various avian species. We isolated and characterized an increasingly prevalent avian metapneumovirus subgroup C strain from meat-type commercial chickens with severe respiratory signs in China. Culling of infected flocks could lead to economic consequences.

Phylogenetic and phylodynamic analyses of subtype-B metapneumovirus from chickens in Tunisia.

Swollen Head Syndrome (SHS) is an economically important viral disease of chickens caused by avian metapneumovirus (aMPV). The virus comprises 6 different subtypes (A,B,C,D, New-1 and New-2). To date, no information was available on the presence of the virus in Tunisian poultry. The present work aims to detect the presence of (aMPV) in broiler chicken in Tunisia, then to characterise the isolates in order to determine their subtype and to estimate their geographic origin of introduction. A total of 289 samples were collected, aMPV detection was detected by real time RT-PCR and molecular characterization was warried out by Sanger sequencing on the glycoprotein (G) gene. Phylogenetic analysis was carried out using Beast 2 software. Out of the 289 samples, 21 were revealed positive to aMPV. Only 2 isolates have been confirmed by sequencing analysis ; one isolate sampled in 2015 and another in 2019. Based on the partial G gene sequence, analysis of these 2 Tunisian isolates showed that they belong to subtype B. The isolate sampled in 2015, appeared to be phylogenetically related to derived vaccine strain. However, the one sampled in 2019 appeared to be a field strain. Phylodynamic analysis provided evidence that this field strain derived from a Spanish strain and probably the virus has been introduced from Spain to North Africa back in 2016. This study is the first that highlighted the circulation of (aMPV) in Tunisia. It is possible that aMPV has been circulating in Tunisia and neighboring countries without being detected. Also, multiple strains could be present and therefore multiple introductions have happened. Through this study, we shed the light on the importance of reinforcing farms biosecurity as well as virological surveillance.

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.

Vaccine Interaction and Protection against Virulent Avian Metapneumovirus (aMPV) Challenge after Combined Administration of Newcastle Disease and aMPV Live Vaccines to Day-Old Turkeys.

Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) are among the most impactful pathogens affecting the turkey industry. Since turkeys are routinely immunized against both diseases, the hatchery administration of the combined respective live vaccines would offer remarkable practical advantages. However, the compatibility of NDV and aMPV vaccines has not yet been experimentally demonstrated in this species. To address this issue, an aMPV subtype B live vaccine was administered to day-old poults either alone or in combination with one of two different ND vaccines. The birds were then challenged with a virulent aMPV subtype B strain, clinical signs were recorded and aMPV and NDV vaccine replication and humoral immune response were assessed. All results supported the absence of any interference hampering protection against aMPV, with no significant differences in terms of clinical scoring. In addition, the mean aMPV vaccine viral titers and antibody titers measured in the dual vaccinated groups were comparable or even higher than in the group vaccinated solely against aMPV. Lastly, based on the NDV viral and antibody titers, the combined aMPV and NDV vaccination does not seem to interfere with protection against NDV, although further studies involving an actual ND challenge will be necessary to fully demonstrate this hypothesis.

Effect of vaccination program on immune response, production performance, and carcass composition of Ross 308 broiler chickens.

The aim of this study was to compare the effect of 2 different vaccination programs using vaccines against infection bronchitis virus (IBV) and avian metapneumovirus (aMPV) on the humoral immune response, production performance, carcass weight and composition, and the percentage of selected internal organs. During the rearing of broiler chickens, blood was collected for serum samples from randomly selected birds from each experimental group on d 1, 21, 28, and 38. Geometric mean titer (GMT) values were determined for the aforementioned diseases using serological ELISA tests, performed during routine veterinary inspections. During rearing, body weight, mortality, and feed consumption were monitored, allowing the calculation of feed consumption per kilogram of body weight gain (FCR) and the European Production Index (EPI). At the end of rearing, 20 chickens were selected for dissection, 10 birds from each group. After slaughter, the weight of the eviscerated carcass was determined, and the percentages of carcass parts and selected internal organs in the body weight were calculated. The vaccination program exerted a significant (P<0.05) effect on body weight on days 14, 21, 28, 35 and 42, the percentage of wings in the carcass and the proportion (%) of heart, liver and spleen in body weight. ELISA showed no significant effect of the vaccination program on the anti-IBV serological response, and no negative effect of covaccination against both diseases on the anti-aMPV serological response.

Avian Metapneumovirus Subgroup C Phosphoprotein Suppresses Type I Interferon Production by Blocking Interferon Regulatory Factor 3 Nuclear Translocation.

Avian metapneumovirus subgroup C (aMPV/C) is an important pathogen that causes upper respiratory symptoms and egg production decline in turkeys and chickens. aMPV/C infection leads to inhibition of the host antiviral immune response. However, our understanding of the molecular mechanisms underlying host immune response antagonized by aMPV/C infection is limited. In this study, we demonstrated that the aMPV/C phosphoprotein (P) inhibits the IFN antiviral signaling pathway triggered by melanoma differentiation gene 5 (MDA5) and reduces interferon ß (IFN-ß) production and IFN-stimulated genes (ISGs) by targeting IFN regulatory factor 7 (IRF7) but not nuclear factor κB (NF-κB) in DF-1 cells. Moreover, we found that aMPV/C P protein only blocks the nuclear translocation of IRF3 by interacting with IRF3 in HEK-293T cells, instead of affecting IRF3 phosphorylation and inducing IRF3 degradation, which suppresses IRF3 signaling activation and results in a decrease in IFN-ß production. Collectively, these results reveal a novel mechanism by which aMPV/C infection disrupts IFN-ß production in the host. IMPORTANCE The innate immune response is the first defense line of host cells and organisms against viral infections. When RNA viruses infect cells, viral RNA induces activation of retinoic acid-induced gene I and melanoma differentiation gene 5, which initiates downstream molecules and finally produces type I interferon (IFN-I) to regulate antiviral immune responses. The mechanism for avian metapneumovirus (aMPV) modulating IFN-I production to benefit its replication remains unknown. Here, we demonstrate that phosphoprotein of aMPV subgroup C (aMPV/C) selectively inhibits the nuclear translocation of interferon regulatory 3 (IRF3), instead of affecting the expression and phosphorylation of IRF3, which finally downregulates IFN-I production. This study showed a novel mechanism for aMPV/C infection antagonizing the host IFN response.