Minute Virus of Canines NP1 Protein Interacts with the Cellular Factor CPSF6 To Regulate Viral Alternative RNA Processing.

The NP1 protein of minute virus of canines (MVC) governs production of the viral capsid proteins via its role in pre-mRNA processing. NP1 suppresses polyadenylation and cleavage at its internal site, termed the proximal polyadenylation (pA)p site, to allow accumulation of RNAs that extend into the capsid gene, and it enhances splicing of the upstream adjacent third intron, which is necessary to properly enter the capsid protein open reading frame. We find the (pA)p region to be complex. It contains redundant classical cis-acting signals necessary for the cleavage and polyadenylation reaction and splicing of the adjacent upstream third intron, as well as regions outside the classical motifs that are necessary for responding to NP1. NP1, but not processing mutants of NP1, bound to MVC RNA directly. The cellular RNA processing factor CPSF6 interacted with NP1 in transfected cells and participated with NP1 to modulate its effects. These experiments further characterize the role of NP1 in parvovirus gene expression.IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Unlike other parvoviruses, the bocavirus genus controls expression of its capsid proteins via alternative RNA processing, by both suppressing polyadenylation at an internal site, termed the proximal polyadenylation (pA)p site, and by facilitating splicing of an upstream adjacent intron. This regulation is mediated by a small genus-specific protein, NP1. Understanding the cis-acting targets of NP1, as well as the cellular factors with which it interacts, is necessary to more clearly understand this unique mode of parvovirus gene expression.

The Human Bocavirus 1 NP1 Protein Is a Multifunctional Regulator of Viral RNA Processing.

Human bocavirus 1 (HBoV1) encodes a genus-specific protein, NP1, which regulates viral alternative pre-mRNA processing. Similar to NP1 of the related bocavirus minute virus of canine (MVC), HBoV1 NP1 suppressed cleavage and polyadenylation of RNAs at the viral internal polyadenylation site (pA)p. HBoV1 (pA)p is a complex region. It contains 5 significant cleavage and polyadenylation sites, and NP1 was found to regulate only the three of these sites that are governed by canonical AAUAAA hexamer signals. HBoV1 NP1 also facilitated splicing of the upstream intron adjacent to (pA)p. Alternative polyadenylation and splicing of the upstream intron were independent of each other, functioned efficiently within an isolated transcription unit, and were responsive independent of NP1. Characterization of HBoV1 NP1 generalizes its function within the genus Bocaparvovirus, uncovers important differences, and provides important comparisons with MVC NP1 for mechanistic and evolutionary considerations.IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. The NP1 protein of human bocavirus 1 (HBoV1), similar to NP1 of the bocavirus minute virus of canine (MVC), regulates viral alternative RNA processing by both suppressing polyadenylation at an internal site, (pA)p, and facilitating splicing of an upstream adjacent intron. These effects allow both extension into the capsid gene and splicing of the viral pre-mRNA that correctly registers the capsid gene open reading frame. Characterization of HBoV1 NP1 generalizes this central mode of parvovirus gene regulation to another member of the bocavirus genus and uncovers both important similarities and differences in function compared to MVC NP1 that will be important for future comparative studies.

Suspected Iatrogenic Seeding of Oral Melanoma Secondary to Endotracheal Intubation in a Dog.

A 10 yr old castrated male pug was presented with a 3 day history of intermittent dyspnea, cough, inappetence, and inability to breathe while sleeping. He had previously received hypofractionated radiation therapy for an amelanotic oral malignant melanoma (OMM) 7 mo prior to presentation. At presentation, the dog was gasping and dyspneic. Oral examination identified the OMM on the right hard palate. Thoracic radiographs revealed an angular soft-tissue opacity within the trachea just distal to the thoracic inlet. No evidence of pulmonary metastatic disease was seen. Tracheoscopy identified a pedunculated, nonpigmented mass within the lumen of the distal trachea near the carina. Treatment options were presented to the owners and included tracheal stenting or tracheal resection and anastomosis. Because of the poor prognosis, the owners elected humane euthanasia. Postmortem examination confirmed the presence of melanoma in the distal trachea; no other sites of OMM metastasis were identified. The cause of OMM development in the distal trachea in this case is suspected to have resulted from mechanical tumor cell seeding during endotracheal tube placement for general anesthesia 7 mo prior to presentation. Despite the reported rarity of mechanical tumor seeding, this potential complication warrants consideration in dogs with OMM.

Minute Virus of Canines NP1 Protein Governs the Expression of a Subset of Essential Nonstructural Proteins via Its Role in RNA Processing.

Parvoviruses use a variety of means to control the expression of their compact genomes. The bocaparvovirus minute virus of canines (MVC) encodes a small, genus-specific protein, NP1, which governs access to the viral capsid gene via its role in alternative polyadenylation and alternative splicing of the single MVC pre-mRNA. In addition to NP1, MVC encodes five additional nonstructural proteins (NS) that share an initiation codon at the left end of the genome and which are individually encoded by alternative multiply spliced mRNAs. We found that three of these proteins were encoded by mRNAs that excise the NP1-regulated MVC intron immediately upstream of the internal polyadenylation site, (pA)p, and that generation of these proteins was thus regulated by NP1. Splicing of their progenitor mRNAs joined the amino termini of these proteins to the NP1 open reading frame, and splice site mutations that prevented their expression inhibited virus replication in a host cell-dependent manner. Thus, in addition to controlling capsid gene access, NP1 also controls the expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing.IMPORTANCE The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Minute virus of canine (MVC) is an autonomous parvovirus in the genus Bocaparvovirus It has a single promoter that generates a single pre-mRNA. NP1, a small genus-specific MVC protein, participates in the processing of this pre-mRNA and so controls capsid gene access via its role in alternative internal polyadenylation and splicing. We show that NP1 also controls the expression of three of the five identified NS proteins via its role in governing MVC pre-mRNA splicing. These NS proteins together are required for virus replication in a host cell-dependent manner.

Bilateral Malignant Seminoma With Ventricular Metastasis in a Bald Eagle ( Haliaeetus leucocephalus).

A 30-year-old bald eagle ( Haliaeetus leucocephalus) was presented with a history of hyporexia and lethargy. Results of initial hematologic testing, biochemical analysis, and fecal examination were unremarkable, and clinical signs did not resolve with supportive care and management changes. Results of echocardiography, based on auscultation of a murmur, and coelomic endoscopy, based on the presence of a soft tissue opacity on radiographs, as well as an aspergillosis panel were largely unsuccessful in determining a definitive diagnosis. Euthanasia was performed after the eagle did not recover from anesthesia after endoscopy. Necropsy results demonstrated bilateral testicular seminomas with metastases to the ventriculus. This case demonstrates an abnormal metastasis of a common reproductive tumor in an avian species.

NP1 Protein of the Bocaparvovirus Minute Virus of Canines Controls Access to the Viral Capsid Genes via Its Role in RNA Processing.

UNLABELLED: Minute virus of canines (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter that generates a single pre-mRNA processed via alternative splicing and alternative polyadenylation to produce at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another complex site, (pA)p, within the capsid-coding region. During viral infection, the mRNAs must extend through (pA)p and undergo additional splicing of the immediately upstream 3D∕3A intron to access the capsid gene. MVC NP1 is a 22-kDa nuclear phosphoprotein unique to the genus Bocaparvovirus of the Parvovirinae which we have shown governs suppression of (pA)p independently of viral genome replication. We show here that in addition to suppression of (pA)p, NP1 is also required for the excision of the MVC 3D∕3A intron, independently of its effect on alternative polyadenylation. Mutations of the arginine∕serine (SR) di-repeats within the intrinsically disordered amino terminus of NP1 are required for splicing of the capsid transcript but not suppression of polyadenylation at (pA)p. 3'-end processing of MVC mRNAs at (pA)p is critical for viral genome replication and the optimal expression of NP1 and NS1. Thus, a finely tuned balance between (pA)p suppression and usage is necessary for efficient virus replication. NP1 is the first parvovirus protein implicated in RNA processing. Its characterization reveals another way that parvoviruses govern access to their capsid protein genes, namely, at the RNA level, by regulating the essential splicing of an intron and the suppression of an internal polyadenylation site. IMPORTANCE: The Parvovirinae are small nonenveloped icosahedral viruses that are important pathogens in many animal species, including humans. Although parvoviruses have only subtle early-to-late expression shifts, they all regulate access to their capsid genes. Minute virus of canines (MVC) is an autonomous parvovirus in the genus Bocaparvovirus. It has a single promoter generating a single pre-mRNA which is processed via alternative splicing and alternative polyadenylation to generate at least 8 mRNA transcripts. MVC contains two polyadenylation sites, one at the right-hand end of the genome, (pA)d, and another, (pA)p, within the capsid-coding region. It had not been clear how the potent internal polyadenylation motif is suppressed to allow processing, export, and accumulation of the spliced capsid protein-encoding mRNAs. We show here that MVC NP1, the first parvovirus protein to be implicated in RNA processing, governs access to the MVC capsid gene by facilitating splicing and suppressing internal polyadenylation of MVC pre-mRNAs.

The adeno-associated virus type 5 small rep proteins expressed via internal translation initiation are functional.

Although precluded from using splicing to produce multiple small Rep proteins, adeno-associated virus type 5 (AAV5) generates a Rep40-like protein by alternative translation initiation at an internal AUG. A defined region upstream of the internal AUG was both required and sufficient to program internal initiation within AAV5 and may act similarly in heterologous contexts. The internally initiated AAV5 Rep40-like protein was functional and had helicase activity similar to that of AAV2 Rep40. Surprisingly, both the AAV5 Rep40-like protein and Rep52 were able to be translated from the AAV5 upstream P7-generated RNAs; however, the relative level of small to large Rep proteins was reduced compared to that of the wild type. A P19 mutant AAV5 infectious clone generated near-wild-type levels of the double-stranded monomer replicative form (mRF) replicative intermediate but reduced levels of virus, consistent with the previously defined role of Rep40-like proteins in genome encapsidation. Levels of mutant virus were dramatically reduced upon amplification.

Characterization of the nonstructural proteins of the bocavirus minute virus of canines.

We present a detailed characterization of a single-cycle infection of the bocavirus minute virus of canines (MVC) in canine WRD cells. This has allowed identification of an additional smaller NS protein that derives from an mRNA spliced within the NS gene that had not been previously reported. In addition, we have identified a role for the viral NP1 protein during infection. NP1 is required for read-through of the MVC internal polyadenylation site and, thus, access of the capsid gene by MVC mRNAs. Although the mechanism of NP1's action has not yet been fully elucidated, it represents the first parvovirus protein to be implicated directly in viral RNA processing.

Next-generation nanovaccine induces durable immunity and protects against SARS-CoV-2.

While first generation SARS-CoV-2 vaccines were effective in slowing the spread and severity of disease during the COVID-19 pandemic, there is a need for vaccines capable of inducing durable and broad immunity against emerging variants of concern. Nanoparticle-based vaccines (i.e., "nanovaccines") composed of polyanhydride nanoparticles and pentablock copolymer micelles have previously been shown to protect against respiratory pathogens, including influenza A virus, respiratory syncytial virus, and Yersinia pestis. In this work, a nanovaccine containing SARS-CoV-2 spike and nucleocapsid antigens was designed and optimized. The optimized nanovaccine induced long-lived systemic IgG antibody responses against wild-type SARS-CoV-2 virus. In addition, the nanovaccine induced antibody responses capable of neutralization and cross-reactivity to multiple SARS-CoV-2 variants (including B.1.1.529) and antigen-specific CD4+ and CD8+ T cell responses. Finally, the nanovaccine protected mice against a lethal SARS-CoV-2 challenge, setting the stage for advancing particle-based SARS-CoV-2 nanovaccines. STATEMENT OF SIGNIFICANCE: First-generation SARS-CoV-2 vaccines were effective in slowing the spread and limiting the severity of COVID-19. However, current vaccines target only one antigen of the virus (i.e., spike protein) and focus on the generation of neutralizing antibodies, which may be less effective against new, circulating strains. In this work, we demonstrated the ability of a novel nanovaccine platform, based on polyanhydride nanoparticles and pentablock copolymer micelles, to generate durable and broad immunity against SARS-CoV-2. These nanovaccines induced long-lasting (> 62 weeks) serum antibody responses which neutralized binding to ACE2 receptors and were cross-reactive to multiple SARS-CoV-2 variants. Additionally, mice immunized with the SARS-CoV-2 nanovaccine showed a significant increase of antigen-specific T cell responses in the draining lymph nodes and spleens. Together, these nanovaccine-induced immune responses contributed to the protection of mice against a lethal challenge of live SARS-CoV-2 virus, indicating that this nanovaccine platform is a promising next-generation SARS-CoV-2 vaccine.

Prostaglandin EP4 receptor mRNA expression in canine lymphoma.

Canine lymphoma (LSA) is a diverse, aggressive malignancy initiated by a variety of factors. Understanding those factors could help identify potential treatment options. Chronic inflammation drives lymphoma in human medicine and is suspected to play a role in veterinary medicine. The exact mechanisms, however, have not been elucidated. Upregulation of the cyclooxygenase enzymes, and subsequently prostaglandins, potentially play a stimulatory role. Prostaglandins work through one of four EP receptors (EP1-EP4) and the effects mediated through EP4R specifically are thought to be the primary drivers of cancer development. In human T-cell LSA, overexpression of EP4R has been found and appears to protect LSA cells from apoptosis. The role of EP4R in human B-cell LSA is more nuanced. This study aims to evaluate the mRNA expression of the EP4R gene (ptger4) in canine B-cell and T-cell LSA. Archived canine lymph nodes with histologically confirmed B-cell and T-cell LSA, and reactive lymph nodes, were evaluated for EP4R mRNA expression using a novel RNA in situ hybridization technique (RNAscope). Quantification of RNAscope signals was completed with an advanced digital pathology image analysis system (HALO). Results were reported as copy number, H-score, and percent tumour cell expression of EP4R mRNA. All reactive, B-cell LSA, and T-cell LSA lymph nodes expressed EP4R mRNA. The mRNA copy number, H-score, and percent tumour cell expression of EP4R were higher in B-cell (p < .003) and T-cell (p < .001) LSA samples compared to reactive lymph node samples. There were no differences between B-cell LSA and T-cell LSA.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Adeno-associated virus small rep proteins are modified with at least two types of polyubiquitination.

Adeno-associated virus (AAV) type 2 and 5 proteins Rep52 and Rep40 were polyubiquitinated during AAV-adenovirus type 5 (Ad5) coinfection and during transient transfection in either the presence or absence of Ad5 E4orf6 and E1b-55k. Polyubiquitination of small Rep proteins via lysine 48 (K48) linkages, normally associated with targeting of proteins for proteasomal degradation, was detected only in the presence of E4orf6. The small Rep proteins were ubiquitinated via lysine 63 (K63) following transfection in either the presence or absence of E4orf6 or following coinfection with Ad5. E4orf6/E1b-55k-dependent K48-specific polyubiquitination of small Rep proteins could be inhibited using small interfering RNA (siRNA) to cullin 5.

Meconium aspiration pneumonia and otitis media in two goat kids.

We report meconium aspiration in 2 sibling goat kids, and characterize the expected lesions of aspiration pneumonia in conjunction with the rare lesion of otitis media. Grossly, the lungs were multifocally consolidated, and there was yellow-green exudate within the middle ear. Histologically, the lung was characterized by pyogranulomatous pneumonia and foreign-body reaction around aspirated debris. Within the lumen of the middle ear, aspirated squamous cells, keratin, meconium debris, and neutrophils, without evidence of bacteria, were accompanied by a subepithelial accumulation of lymphocytes, plasma cells, and fewer macrophages. This is an especially rare phenomenon, which is thought to result from transport of meconium from the oropharynx through the auditory tube (Eustachian tube) to the middle ear.

RNAse mapping and quantitation of RNA isoforms.

The ribonuclease protection assay (RPA) has emerged as an important methodology for the detection, mapping, and quantification of RNAs. In this assay, total or cytoplasmic RNAs are hybridized to a high-specific activity antisense radioactive RNA probe synthesized by in vitro transcription from the SP6 or T7 promoter of an appropriate linearized plasmid template by the bacteriophage SP6 or T7 polymerase, respectively. The RNA hybrids are subjected to RNAse digestion and the protected products are resolved by denaturing polyacrylamide gel electrophoresis to allow detection of specific RNA fragments by subsequent autoradiography. RPAs are highly sensitive, the probes can be specifically targeted, and, when performed in probe excess, are quantitative, making them the method of choice for many analyses of RNA processing events.