Avian Metapneumovirus Subgroup Research Articles

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.

A Receptor Integrin β1 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 β1 (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.

Interaction of Nucleolin with the Fusion Protein of Avian Metapneumovirus Subgroup C Contributes to Viral Replication

Avian metapneumovirus subgroup C (aMPV/C) is highly pathogenic to various avian species with acute respiratory tract clinicopathology and/or drops in egg production. Nucleolin (NCL), an important nucleolar protein, has been shown to regulate multiple viral replication and serve as a functional receptor for viral entry and internalization. Whether NCL is involved in aMPV/C pathogenesis is not known. In this study, we found that aMPV/C infection altered the subcellular localization of NCL in cultured cells. siRNA-targeted NCL resulted in a remarkable decline in aMPV/C replication in Vero cells. DF-1 cells showed a similar response after CRISPR/Cas9-mediated knock out of NCL during aMPV/C infection. Conversely, NCL overexpression significantly increased aMPV/C replication. Pretreatment with AS1411-a aptamer, a guanine (G)-rich oligonucleotide that forms four-stranded structures and competitively binding to NCL, decreased aMPV/C replication and viral titers in cultured cells. Additionally, we found that the aMPV/C fusion (F) protein specifically interacts with NCL through its central domain and that AS1411 disrupts this interaction, thus inhibiting viral replication. Taken together, these results reveal that the aMPV/C F protein interacts with NCL, which is employed by aMPV/C for efficient replication, thereby highlighting the strategic potential for control and therapy of aMPV/C infection.

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.

Research Note: Detection of Avian metapneumovirus subgroup C specific antibodies in a mallard flock in Italy

Avian metapneumovirus (aMPV) is a viral pathogen that causes respiratory signs and drops in egg production most notably in turkeys but also affects chickens, ducks and other species. Four subgroups (aMPV-A, -B, -C and -D) have been characterized to date, which differ genetically, antigenically and in terms of geographic distribution and host range. Despite the rising awareness towards aMPV, many aspects of its epidemiology are still poorly understood.In this pilot study, a serological survey was carried out to assess the possible circulation of subgroups A, B, and C in intensively raised ducks in Northern Italy. A total of 220 sera from 10 Pekin duck flocks and one mallard flock were collected at slaughter and tested by subgroup-specific aMPV-A, B, and C indirect ELISA assays. None of the tested birds had been vaccinated against aMPV and no symptoms had been reported. No aMPV-A, B or C antibodies were detected in any bird of the Pekin duck flocks, whereas the entire mallard flock tested positive for aMPV-C antibodies. This is the first report of aMPV-C antibodies in ducks in Italy, where only aMPV-B has been reported to circulate in recent years. Further research efforts will be targeted towards the screening of a larger panel of samples, in the hope of obtaining positive samples from which full length genome sequences and infectious viruses can be isolated for virus characterization. Finally, the demonstration of aMPV-C specific antibodies in the serum of all sampled mallards indirectly suggests that this species is readily infected by aMPV-C and supports a possible role of wild anatids as a transmission vector of the virus.

Avian metapneumovirus subgroup C induces autophagy through the ATF6 UPR pathway

ABSTRACTAn 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.

Viral replication and lung lesions in BALB/c mice experimentally inoculated with avian metapneumovirus subgroup C isolated from chickens.

Avian metapneumovirus (aMPV) emerged as an important respiratory pathogen causing acute respiratory tract infection in avian species. Here we used a chicken aMPV subgroup C (aMPV/C) isolate to inoculate experimentally BALB/c mice and found that the aMPV/C can efficiently replicate and persist in the lungs of mice for at least 21 days with a peak viral load at day 6 postinoculation. Lung pathological changes were characterized by increased inflammatory cells. Immunochemical assay showed the presence of viral antigens in the lungs and significant upregulation of pulmonary inflammatory cytokines and chemokines including MCP-1, MIP-1α, RANTES, IL-1β, IFN-γ, and TNF-α were detected following inoculation. These results indicate for the first time that chicken aMPV/C may replicate in the lung of mice. Whether aMPV/C has potential as zoonotic pathogen, further investigation will be required.

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.

Generation and evaluation of a recombinant Newcastle disease virus expressing the glycoprotein (G) of avian metapneumovirus subgroup C as a bivalent vaccine in turkeys

Virulent strains of Newcastle disease virus (NDV) and avian metapneumovirus (aMPV) can cause serious respiratory diseases in poultry. Vaccination combined with strict biosecurity practices has been the recommendation for controlling both NDV and aMPV diseases in the field. In the present study, an NDV based, LaSota strain recombinant vaccine virus expressing the glycoprotein (G) of aMPV subgroup C (aMPV-C) was generated as a bivalent vaccine using a reverse genetics approach. The recombinant virus, rLS/aMPV-C G was slightly attenuated in vivo, yet maintained similar growth dynamics, cytopathic effects, and virus titers in vitro when compared to the parental LaSota virus. Expression of the aMPV G protein in rLS/aMPV-C G-infected cells was detected by immunofluorescence assay. Vaccination of turkeys with one dose of rLS/aMPV-C G induced moderate aMPV-C-specific immune responses and comparable NDV-specific serum antibody responses to a LaSota vaccination control. Partial protection against pathogenic aMPV-C challenge and complete protection against velogenic NDV challenge was conferred. These results suggest that the LaSota recombinant virus is a safe and effective vaccine vector and that expression of the aMPV-C G protein alone is not sufficient to provide full protection against an aMPV-C infection. Expression of other immunogenic protein(s) of the aMPV-C virus alone or in conjunction with the G protein may be needed to induce a stronger protective immunity against the aMPV-C disease.

Topology and cellular localization of the small hydrophobic protein of avian metapneumovirus

The small hydrophobic protein (SH) is a type II integral membrane protein that is packaged into virions and is only present in certain paramyxoviruses including metapneumovirus. In addition to a highly divergent primary sequence, SH proteins vary significantly in size amongst the different viruses. Human respiratory syncytial virus (HRSV) encodes the smallest SH protein consisting of only 64 amino acids, while metapneumoviruses have the longest SH protein ranging from 174 to 179 amino acids in length. Little is currently known about the cellular localization and topology of the metapneumovirus SH protein. Here we characterize for the first time metapneumovirus SH protein with respect to topology, subcellular localization, and transport using avian metapneumovirus subgroup C (AMPV-C) as a model system. We show that AMPV-C SH is an integral membrane protein with NinCout orientation located in both the plasma membrane as well as within intracellular compartments, which is similar to what has been described previously for SH proteins of other paramyxoviruses. Furthermore, we demonstrate that AMPV-C SH protein localizes in the endoplasmic reticulum (ER), Golgi, and cell surface, and is transported through ER–Golgi secretory pathway.

Deletion of the M2-2 gene from avian metapneumovirus subgroup C impairs virus replication and immunogenicity in Turkeys

The second matrix (M2) gene of avian metapneumovirus subgroup C (aMPV-C) contains two overlapping open reading frames (ORFs), encoding two putative proteins, M2-1 and M2-2. Both proteins are believed to be involved in viral RNA transcription or replication. To further characterize the function of the M2-2 protein in virus replication, the non-overlapping region of the M2-2 ORF was deleted from an infectious cDNA clone of the aMPV-C strain, and a viable virus was rescued by using reverse genetics technology. The recombinant virus, raMPV-C ΔM2-2, was characterized in vitro and in vivo. In Vero cells, raMPV-C ΔM2-2 replicated slightly less efficiently than the parental virus, 10-fold reduction at 48-h post-infection. The raMPV-C ΔM2-2 virus induced typical cytopathic effects (CPE) that were indistinguishable from those seen with the parental virus infection. In specific-pathogen-free (SPF) turkeys, raMPV-C ΔM2-2 was attenuated and caused no clinical signs of disease. Less than 20% of the inoculated birds shed detectable virus in tracheal tissue during the first 5 days post-infection, and no virus shedding was detected afterward. Forty percent of infected birds produced a weak antibody response at 14 days post-infection. Upon challenge with a virulent aMPV-C strain, more than 80% of the raMPV-C ΔM2-2-inoculated birds showed typical disease signs and virus shedding in tracheal tissue. These results suggest that the M2-2 protein of aMPV-C virus is not essential for virus replication in vitro, but is required for sufficient virus replication to maintain pathogenicity and immunogenicity in the natural host.

Contribution of the Attachment G Glycoprotein to Pathogenicity and Immunogenicity of Avian Metapneumovirus Subgroup C

Contribution of the Attachment G Glycoprotein to Pathogenicity and Immunogenicity of Avian Metapneumovirus Subgroup C

Contribution of the Attachment G Glycoprotein to Pathogenicity and Immunogenicity of Avian Metapneumovirus Subgroup C

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

Generation and biological assessment of recombinant avian metapneumovirus subgroup C (aMPV-C) viruses containing different length of the G gene

Genetic variation in length of the G gene among different avian metapneumovirus subgroup C (aMPV-C) isolates has been reported. However, its biological significance in virus replication, pathogenicity and immunity is unknown. In this study, we developed a reverse genetics system for aMPV-C and generated two Colorado (CO) strain-based recombinant viruses containing either the full-length G gene derived from a Canadian goose isolate or a C-terminally truncated G gene of the CO strain. The truncated short G (sG) gene encoded 252 amino acids (aa), which is 333 aa shorter than the full-length G (585 aa). The biological properties of these two recombinant G variants were assessed in Vero cells and in specific-pathogen-free (SPF) turkeys. In Vero cells, the short G variant displayed a similar level of growth dynamics and virus titers as the parental aMPV-CO strain, whereas the full-length G variant replicated less efficiently than the sG variant during the first 72 h post-infection. Both of the G variants induced typical cytopathic effects (CPE) that were indistinguishable from those seen with the parental aMPV-CO infection. In SPF turkeys, both of the G variants were attenuated and caused little or no disease signs, but the full-length G variant appeared to grow more readily in tracheal tissue than the sG variant during the first 5 days post-infection. Both G variants were immunogenic and induced a slightly different level of antibody response. These results demonstrated that the large portion (333 aa) of the extracellular domain of the viral attachment protein is not essential for virus viability in vitro and in vivo, but may play a role in enhancing virus attachment specificity and immunity in a natural host.

Fusion protein is the main determinant of metapneumovirus host tropism

Human metapneumovirus (HMPV) and avian metapneumovirus subgroup C (AMPV-C) infect humans and birds, respectively. This study confirmed the difference in host range in turkey poults, and analysed the contribution of the individual metapneumovirus genes to host range in an in vitro cell-culture model. Mammalian Vero-118 cells supported replication of both HMPV and AMPV-C in contrast to avian quail fibroblast (QT6) cells in which only AMPV-C replicated to high titres. Inoculation of Vero-118 and QT6 cells with recombinant HMPV in which genes were exchanged with those of AMPV-C revealed that the metapneumovirus fusion (F) protein is the main determinant for host tropism. Chimeric viruses in which polymerase complex proteins were exchanged between HMPV and AMPV-C replicated less efficiently compared with HMPV in QT6 cells. Using mini-genome systems, it was shown that exchanging these polymerase proteins resulted in reduced replication and transcription efficiency in QT6 cells. Examination of infected Vero-118 and QT6 cells revealed that viruses containing the F protein of AMPV-C yielded larger syncytia compared with viruses containing the HMPV F protein. Cell-content mixing assays revealed that the F protein of AMPV-C was more fusogenic compared with the F protein of HMPV, and that the F2 region is responsible for the difference observed between AMPV-C and HMPV F-promoted fusion in QT6 and Vero-118 cells. This study provides insight into the determinants of host tropism and membrane fusion of metapneumoviruses.

Specificity and functional interaction of the polymerase complex proteins of human and avian metapneumoviruses

Human metapneumovirus (HMPV) and avian metapneumovirus (AMPV) have a similar genome organization and protein composition, but a different host range. AMPV subgroup C (AMPV-C) is more closely related to HMPV than other AMPVs. To investigate the specificity and functional interaction of the polymerase complex proteins of human and avian metapneumoviruses, a minireplicon system was generated for AMPV-C and used in combination with minireplicon systems for HMPV lineages A1 and B1. Viral RNA-like molecules representing HMPV-A1 and -B1, AMPV-A and -C and human respiratory syncytial virus were replicated efficiently by polymerase complexes of HMPV-A1 and -B1 and AMPV-C, but not by polymerase complexes of bovine parainfluenza virus 3. Upon exchange of HMPV and AMPV-C polymerase complex components, all chimeric polymerase complexes were functional; exchange between HMPVs did not result in altered polymerase activity, whereas exchange between HMPVs and AMPV-C did. Recombinant HMPV-B1 viruses in which polymerase genes were exchanged with those of HMPV-A1 replicated with normal kinetics in vitro, whilst replacement with AMPV-C genes resulted in moderate differences in virus replication. In hamsters, recombinant HMPV-B1 viruses in which individual polymerase genes were exchanged with those of AMPV-C were attenuated, irrespective of the results obtained with minireplicon systems or in vitro replication assays. This study provides insight into the specificity and functional interaction of polymerase complex proteins of human and avian metapneumoviruses, but neither minireplicon systems nor in vitro replication kinetics were found to be predictive for attenuation in permissive animals.

Recovery of avian metapneumovirus subgroup C from cDNA: cross-recognition of avian and human metapneumovirus support proteins.

Avian metapneumovirus (AMPV) causes an acute respiratory disease in turkeys and is associated with "swollen head syndrome" in chickens, contributing to significant economic losses for the U.S. poultry industry. With a long-term goal of developing a better vaccine for controlling AMPV in the United States, we established a reverse genetics system to produce infectious AMPV of subgroup C entirely from cDNA. A cDNA clone encoding the entire 14,150-nucleotide genome of AMPV subgroup C strain Colorado (AMPV/CO) was generated by assembling five cDNA fragments between the T7 RNA polymerase promoter and the autocatalytic hepatitis delta virus ribozyme of a transcription plasmid, pBR 322. Transfection of this plasmid, along with the expression plasmids encoding the N, P, M2-1, and L proteins of AMPV/CO, into cells stably expressing T7 RNA polymerase resulted in the recovery of infectious AMPV/CO. Characterization of the recombinant AMPV/CO showed that its growth properties in tissue culture were similar to those of the parental virus. The potential of AMPV/CO to serve as a viral vector was also assessed by generating another recombinant virus, rAMPV/CO-GFP, that expressed the enhanced green fluorescent protein (GFP) as a foreign protein. Interestingly, GFP-expressing AMPV and GFP-expressing human metapneumovirus (HMPV) could be recovered using the support plasmids of either virus, denoting that the genome promoters are conserved between the two metapneumoviruses and can be cross-recognized by the polymerase complex proteins of either virus. These results indicate a close functional relationship between AMPV/CO and HMPV.

Analysis of the complete genome sequence of avian metapneumovirus subgroup C indicates that it possesses the longest genome among metapneumoviruses

Analysis of the complete genome sequence of avian metapneumovirus subgroup C indicates that it possesses the longest genome among metapneumoviruses

Complete sequence of the G glycoprotein gene of avian metapneumovirus subgroup C and identification of a divergent domain in the predicted protein

The complete nucleotide sequences of the attachment glycoprotein (G) genes of three strains of avian metapneumovirus subgroup C (AMPV-C) were determined from the viral genomic and mRNAs. The G gene of AMPV-C was 1798 nt (1015 nt longer than previously reported) and the derived polypeptide had 585 aa. The deduced amino acid sequence of the predicted G protein of AMPV-C strain Colorado (AMPV-CO) showed 21-25 % amino acid identity to the G proteins of human metapneumoviruses, but only 14-16 % amino acid identity to those of other AMPV subgroups. The predicted G protein of AMPV-CO showed 98 and 81 % amino acid identity to those of AMPV-C strains Mn-1a and Mn-2a, respectively, indicating considerable sequence variation in the G proteins of AMPV-C isolates. Comparison of the G protein sequences of AMPV-CO and Mn-2a identified a highly divergent domain (48 % amino acid identity) at aa 300-450.

Sequence analysis of the large polymerase (L) protein of the US strain of avian metapneumovirus indicates a close resemblance to that of the human metapneumovirus

The complete nucleotide sequence of the large polymerase (L) protein of the avian metapneumovirus subgroup C strain Colorado was determined. The L protein gene of avian pneumovirus Colorado isolate (APV-C) was 6173 nucleotides in length from the gene-start to the gene-end and encoded a polypeptide of 2005 amino acids in length. The length of the L protein of APV-C was exactly the same as that of human metapneumovirus (hMPV) and one amino acid longer than the L protein of APV subgroup A. The L protein of APV-C showed 80% amino acid identity with the L protein of hMPV, but only 64% amino acid identity with the L protein of APV-A. The nucleotide and deduced amino acid sequences were compared with the corresponding sequences of eleven other paramyxoviruses. All six domains characteristic of paramyxovirus L proteins were also observed in the L protein of APV-C. All the polymerase core motifs in domain III were conserved to nearly 100% in the metapneumoviruses. Similarly, the putative ATP-binding motif in domain VI was completely conserved among the metapneumoviruses and differed in length, by one intermediate residue, from other paramyxoviruses. Phylogenetic analysis of the different L proteins also revealed a closer relationship between APV-C and hMPV.