Darrell R. Kapczynski

Avian metapneumovirus (aMPV) is an important respiratory pathogen of global concern in poultry. Until recently, the United States (U.S.) was considered free of aMPV following the eradication of subtype C in the early 2000s. However, in 2023–2024, both aMPV subtypes A and B were confirmed in the U.S., leading to widespread outbreaks in more than 30 states. The rapid spread of these viruses, despite enhanced biosecurity measures, highlights critical gaps in our epidemiological understanding and underscores the urgent need for active surveillance programs. Whole genome sequencing has become a valuable tool for the characterization and epidemiological investigation of viruses, and viral genome enrichment methods are essential to increase the proportion of viral genetic materials in a sample prior sequencing. In this study, we evaluated two aMPV subtype B genome enrichment strategies: the currently used combination of host and bacterial RNA depletion followed by sequence-independent single-primer amplification (HD-SISPA), and a newly developed amplicon-based tiled PCR approach (aMPVB-ATP). Samples treated with aMPVB-ATP yielded significantly higher proportion of aMPV reads (41%) following short-read next-generation sequencing (NGS) compared to those treated with HD-SISPA (1%). This improvement enabled the recovery of complete or nearly complete aMPV genomes from samples with RT-PCR cycle threshold (Ct) values below 22. The compatibility of aMPVB-ATP with long-read NGS was also assessed, showing no significant differences in genome coverage or sequencing depth compared to its performance combined with short-read platforms.

Host body temperature can define a virus’s replicative profile—influenza A viruses (IAVs) adapted to 40° to 42°C in birds are less temperature sensitive in vitro compared with human isolates adapted to 33° to 37°C. In this work, we show that avian-origin PB1 polymerase subunits enable IAV replication at elevated temperatures, including avian-origin PB1s from the 1918, 1957, and 1968 pandemic viruses. Using a model system to ensure biosafety, we show that a small increase in body temperature protects against severe disease in mice and that this protection is overcome by a febrile temperature–resistant PB1. These findings indicate that although elevated temperature itself can be a potent antiviral defense, it may not be effective against all influenza strains. These data inform both the clinical use of antipyretics and IAV surveillance efforts.

Reporte de caso- La detección negativa de metapneumovirus aviar de los subtipos A y B en huevos de reproductoras comerciales de pavos positivas al virus respalda la premisa de que la transmisión vertical no contribuye a la propagación del virus. El metapneumovirus aviar (aMPV) es una enfermedad aviar de amplia distribución mundial. La infección por el virus causa enfermedades respiratorias y contribuye a infecciones secundarias, lo que resulta en un aumento de la morbilidad y la mortalidad, con graves pérdidas económicas. El virus se clasifica en cuatro subtipos (A-D). Históricamente, Estados Unidos experimentó brotes geográficamente limitados del subtipo C en pavos a finales de la década de 1990, que finalmente se erradicaron. En 2024, se notificaron casos de los subtipos A y B de metapneumovirus aviar en parvadas comerciales de pollos y pavos, lo que marcó la primera vez que se detectaron estos subtipos en los Estados Unidos. Si bien se han detectado brotes de los subtipos A y B tanto en pollos como en pavos, los pavos parecen ser más susceptibles y presentar una mayor tasa de morbilidad y mortalidad. Actualmente, una preocupación para la industria del pavo es la posibilidad de transmisión vertical del virus de la gallina al pavipollo. En estos estudios, confirmamos la presencia de metapneumovirus aviar en el tracto respiratorio de pavos comerciales infectados y, posteriormente, se examinaron los huevos procedentes de gallinas positivas a metapneumovirus aviar mediante la detección e ARN viral que pudiera sugerir transmisión vertical del virus. Se recolectaron huevos de siete parvadas diferentes de pavos comerciales que fueron positivas para metapneumovirus aviar y se analizaron para los subtipos A y B de metapneumovirus aviar mediante PCR cuantitativa en tiempo real. Los resultados no muestran evidencia de ARN viral en ninguna de las muestras de huevo o embrión analizadas, ni en el interior ni en la superficie de la cáscara. Estos resultados proporcionan evidencia directa de que la transmisión vertical parece que no contribuye a la diseminación del virus en parvadas de pavos comerciales.

The unprecedented global spread of the highly pathogenic avian influenza (HPAI) virus in wild birds, poultry, and mammalian species has challenged our control efforts. Alternative approaches to limit avian influenza viruses (AIV) include the development of resilient or resistant chickens. Genetically resilient birds may become infected but can overcome disease, whereas resistant birds prevent virus attachment or entry and do not become infected. The most intensively studied host gene is myxovirus-resistance ( Mx ), which is expressed via the interferon pathway. Both sensitive and resistant chicken Mx genotypes have been described, but this only provides limited resilience. Acidic nuclear phosphoprotein 32 family member A ( ANP32A ) has been demonstrated as a host cofactor for AIV replication via interaction with the polymerase. Small nucleotide changes within this gene have demonstrated some promise for the establishment of disease resilience. Certain MHC-defined genetic chicken lines have demonstrated increased resilience with higher innate immune responses, but HPAI-infected birds still have high morbidity and mortality. Alternatively, gene-edited or -transgenic chickens have had some success in increasing resilience. This strategy allows flexibility to include foreign genes, modification of existing genes, or combined approaches to block critical steps in the viral life cycle. Some candidate genes include solute carrier 35A1 ( SLC35A1 ), retinoic acid-inducible gene I ( RIG-I ), and toll-like receptors 3 and 7 ( TLR3/7 ), but animal testing needs to be conducted. Furthermore, existing hurdles for technology transfer to commercial application from either naturally occurring resistance genes or foreign genes remain high and will require acceptance by both the poultry industry and consumers.

During the COVID-19 pandemic, severe acute respiratory syndrome coronavirus 2 (SC2) infection was confirmed in various animal species demonstrating a wide host range of the virus. Prior studies have shown that the ACE2 protein is the primary receptor used by the virus to gain cellular entry and begin the replication cycle. In previous studies, we demonstrated that human and various bat ACE2 proteins can be utilized by SC2 viruses for entry. Bats are a suspected natural host of SC2 because of genetic homology with other bat coronaviruses. In this work, we demonstrate that expression of ACE2 genes from the common vampire bat (CVB) (Desmodus rotundus) and the pallid bat (PB) (Antrozous pallidus), supports infection and replication of some SC2 viruses in cell culture. Two cell lines were produced, CVB-ACE2 and PB-ACE2, expressing ACE2 from these bat species along with human TMPRSS2, in a model previously established using a non-permissive chicken DF-1 cell line. Results demonstrate that the original Wuhan lineage (WA1) virus and the Delta variant were able to infect and replicate in either of the bat ACE2 cell lines. In contrast, the Lambda and Omicron variant viruses infected both cell lines, but viral titers did not increase following infection. Viral detection using immunofluorescence demonstrated abundant spike (S) protein staining for the WA1 and Delta variants but little signal for the Lambda and Omicron variants. These studies demonstrate that while ACE2 from CVB and PB can be utilized by SC2 viruses to gain entry for infection, later variants (Lambda and Omicron) replicate poorly in these cell lines. These observations suggest more efficient human adaption in later SC2 variants that become less fit for replication in other animal species.

A whole-genome shotgun sequencing (sWGS) approach was applied to chicken clinical tracheal swab samples during metagenomics investigations to identify possible microorganisms among poultry with respiratory diseases. After applying shotgun sequencing, Ornithobacterium rhinotracheale (ORT) and a putative prophage candidate were found in one of the swab samples. A multi-locus sequence typing (MLST) scheme of the ORT genome involved the adk, aroE, fumC, gdhA, pgi, and pmi genes. Antibiotic resistant analysis demonstrated tetracycline-resistan t ribosomal protection protein, tetQ, the aminoglycoside-(3)-acetyltransferase IV gene, aminoglycoside antibiotic inactivation and macrolide resistance, and the ermX gene in the ORT genome. A putative prophage candidate was predicted using Prophage Hunter and PHAST, while BLAST analyses were utilized to identify genes encoding bacteriophage proteins. Interestingly, genes encoding endolysins were detected in bacteriophage genomes. The gene products encoded in the prophage sequence were most closely related to bacteriophages in the N4-like family among the Authographiviridae in the Caudovirales. This study demonstrates the potential of sWGS for the rapid detection and characterization of etiologic agents found in clinical samples.

Genetically diverse avian influenza viruses (AIVs) are maintained in wild aquatic birds with increasingly frequent spillover into mammals, yet these represent a small proportion of the overall detections. The isolation of AIVs in marine mammals, including seals, has been reported sporadically over the last 45 years. Prior to 2016, all reports of AIVs detected in seals were of low-pathogenicity AIVs. In spite of this, the majority of reported AIV outbreaks caused fatal respiratory diseases, with harbor seals particularly susceptible to infection. The H5 clade 2.3.4.4b highly pathogenic AIV (HPAIV) was detected in seals for the first time in 2016. Recently, many cases of mass seal die-offs have occurred because of 2.3.4.4b HPAIV and are attributed to spillover from wild bird species. The potential for seal-to-seal transmission has been considered after the mass mortality of southern elephant seals off the coast of Argentina. Close contact between seals and wild birds, the rapid evolution of H5N1 AIVs, and the possibility of efficient mammal-to-mammal transmission are increasing concerns due to the potential for the establishment of a marine mammal reservoir and public health risks associated with the pandemic potential of the virus. This manuscript details the detection of AIVs in the seal population, comparing interesting features of various subtypes with an emphasis on avian-to-mammal-to-mammal transmission. Phylogenetic characterizations of the representative seal isolates were performed to demonstrate the relationships within the different virus isolates. Furthermore, we demonstrate that the reassortment events between different LPAIVs occurred before and after the viruses reached the seal population. The reassortment of viral segments plays an important role in the evolution of influenza viruses. Taken together, these data report on the 45 year history between seals and AIVs.

Genetically diverse influenza A viruses (IAV) circulates in wild aquatic birds with relatively frequent spillover (cases of single infections) to other mammals, but only a small proportion of outbreaks. The spillover of IAV to marine mammals such as seals have been reported throughout the last 45 years. Majority of these viruses when reached seals population cased pneumonia and death of infected animals. Until 2016, all avian influenza viruses found in seals were of low pathotype (LP) IAV. The H5Nx clade 2.4.4.4b highly pathogenic avian influenza viruses (HPAIV) were found in seals for the first time in 2016/2017. Our review highlights 45 years of AIV history in seals population, showing interesting features of different AIV subtypes and emphasis on avian-to-mammals transmission. Furthermore, we also included phylogenetic characterisation of the representative seal isolates.

Dendritic cells (DCs) are professional antigen-presenting cells, which are key components of the immune system and involved in early immune responses. DCs are specialized in capturing, processing, and presenting antigens to facilitate immune interactions. Chickens infected with avian influenza virus (AIV) demonstrate a wide range of clinical symptoms, based on pathogenicity of the virus. Low pathogenic avian influenza (LPAI) viruses typically induce mild clinical signs, whereas high pathogenic avian influenza (HPAI) induce more severe disease, which can lead to death. For this study, chicken bone marrow-derived DC (ckBM-DC)s were produced and infected with high and low pathogenic avian influenza viruses of H5N2 or H7N3 subtypes to characterize innate immune responses, study effect on cell morphologies, and evaluate virus replication. A strong proinflammatory response was observed at 8 hours post infection, via upregulation of chicken interleukin-1β and stimulation of the interferon response pathway. Microscopically, the DCs underwent morphological changes from classic elongated dendrites to a more general rounded shape that eventually led to cell death with the presence of scattered cellular debris. Differences in onset of morphologic changes were observed between H5 and H7 subtypes. Increases in viral titers demonstrated that both HPAI and LPAI are capable of infecting and replicating in DCs. The increase in activation of infected DCs may be indicative of a dysregulated immune response typically seen with HPAI infections.

Avian influenza virus (AIV) is a constant threat to animal health with recent global outbreaks resulting in the death of hundreds of millions of birds with spillover into mammals. Myxovirus-resistance (Mx) proteins are key mediators of the antiviral response that block virus replication. Mouse (Mu) Mx (Mx1) is a strong antiviral protein that interacts with the viral nucleoprotein to inhibit polymerase function. The ability of avian Mx1 to inhibit AIV is unclear. In these studies, Mu Mx1 was stably introduced into chicken DF1 cells to enhance the immune response against AIV. Following infection, titers of AIV were significantly decreased in cells expressing Mu Mx1. In addition, considerably less cytopathic effect (CPE) and matrix protein staining was observed in gene-edited cells expressing Mu Mx1, suggesting Mu Mx1 is broadly effective against multiple AIV subtypes. This work provides foundational studies for use of gene-editing to enhance innate disease resistance against AIV.