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Bovine Respiratory Disease Complex is a multifactorial disease affecting cattle worldwide resulting in high mortality and morbidity rates in the cattle farming industry. This complex is caused by multiple viral and bacterial pathogens such as Bovine Herpesvirus-1, Bovine Respiratory Syncytial Virus, Mannheimia haemolytica, and Pasteurella multocida; two of the main contributors to the initiation of this disease are Bovine Herpesvirus-1 and the bacteria, Mannheimia haemolytica. Together, these microbes co-infect immunocompromised cattle during times of increased stress and induce a severe pneumonic response along with other health complications. Research has been primarily focused on these microorganisms individually or their effect on the host, however there is a need to study them together due to the increased mortality rate associated with co-infections. In this study, we used Bovine Herpesvirus-1, Mannheimia haemolytica, Pasteurella multocida, and Bovine Respiratory Syncytial Virus to co-infect bovine tissue cultures to determine how they affect each other.

Lower respiratory tract infections, like those caused by influenza A virus (IAV) and respiratory syncytial virus (RSV), and the pneumonia they cause are a global health concern and burden. Infection and vaccination induce immune memory responses that are vital for neutralizing and resolving subsequent infections. B cells play a substantial role in preventing reinfection. They exert their effects through several functions but most appreciably via anti-viral antibody secretion. B cells, as is the case with other immune cells, possess several subsets, each with defined roles and functions. The B7 protein family surface receptors CD80 and PD-L2 have been identified as distinguishing two populations based on their presence or absence on B cells: CD80-PD-L2- (double negative, DN) and CD80+PD-L2+ (double positive, DP). In the spleen, DN cells have relatively low mutation frequencies, antigen specificity, and tend to enter the germinal center reaction. On the other hand, DP cells have a more mutated B cell receptor, increased antigen-specificity, and differentiate into antibody secreting cells upon restimulation. It remains unclear if these two populations are related, what differences, if any, they possess from each other, cellular crosstalk required for their maintenance, and their roles in immunity against IAV reinfection. Additionally, it is not known if local B cells (i.e. DN and DP) in the lung are distinct from those in the spleen or if they are a homogenous population. Our work was aimed at investigating these gaps in the field. We showed that IAV infection results in the accumulation of extravascular DP B cells in the murine lung. B cells in general appear to localize near sites of previous infection in organized lymphoid-like structures. Naphthalene-mediated club cell ablation results in the partial loss of the lung DP cell population while DN cells appear unaffected, suggesting the lung epithelium plays a role in the maintenance of the DP cells. DP cells possess more of the IAV-specific cells than the DN cells suggesting an anti-viral role. In part, the DP cells originate from DN predecessors and possess a unique V gene profile. During the immune reaction, DN cells differentiate into DP cells and, on occasion, revert. The DN cell population in the lung is largely distinct from that found in the spleen, suggesting that these cells originate from a secondary lymphoid tissue. Finally, we have engineered a recombinant RSV containing a cre-recombinase transgene that replicated with similar kinetics to the parental strain. This novel tool will allow us to examine qualities of cells previously infected with RSV and investigate any role they may play in immune maintenance and/or lung health. To conclude, we outline major differences between two populations of B cells of considerable immune interest, show how they may rely, in part, on epithelial cells in the lung, and develop a new tool to investigate these topics in other respiratory viruses of public health concern. / 2024-11-02T00:00:00Z

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Background: Respiratory syncytial virus (RSV) is the leading cause of acute lower respiratory tract infection and hospitalization in children worldwide. With no vaccine or antivirals available for the routine prevention or treatment of RSV, an effective RSV antiviral is required. Previous studies have shown that the RSV nucleocapsid complex (NC), phosphoprotein (P), and large polymerase (L) are essential for the replication and survival of RSV since they form the core of the RNA-dependent RNA polymerase (RdRp) complex. Thus, these proteins are viable targets for novel RSV antivirals. Objective: The Mahony laboratory has previously shown that 20 µM of a peptide mimetic composed of the 21 terminal amino acids of the RSV phosphoprotein (RSVP220-241) fused to an HIV-1 Tat cell penetrating peptide (CPP), a hexa-histidine (His) tag, and the Escherichia coli (E. coli) maltose binding protein carrier (MBP) molecule was sufficient to attenuate RSV A and B replication in vitro by approximately 90 and 80%, respectively. We evaluated the fusion of this His-MBP-Tat-RSVP220-241 mimetic to a more suitable carrier molecule, an albumin-binding domain (ABD), for future use in vivo. In addition, we designed a novel antiviral mimetic composed of the 30 terminal amino acids of the RSV A P protein (RSVP212-241), which are involved in binding both L polymerase and NC complexes, fused to a CPP consisting of Tat or nine arginine residues (Arg9), a His-tag, and the MBP carrier. We evaluated the activity of His-MBP-Tat-RSVP212-241, Tat-His-MBP-Tat-RSVP212-241, and His-Arg9-MBP-RSVP212-241 mimetics in vitro and hypothesized that a mimetic designed to target both L and NC interactions would be a more effective RSV antiviral than the original His-MBP-Tat-RSVP220-241 mimetic. Methods and Results: The Gateway® Cloning System was used to create expression vectors containing His-, GST-, or His-MBP-ABD-Tat-RSVP220-241 and His-MBP-Tat-RSVP212-241, whereas inverse PCR and both the In-Fusion® and Gateway® Cloning systems were used to generate expression vectors containing Tat-His-MBP-Tat-RSVP212-241 and His-Arg9-MBP-RSVP212-241. The fusion proteins were expressed, purified by affinity chromatography, and evaluated in vitro. No soluble protein was obtained for the ABD constructs. His-MBP-Tat-RSVP212-241 was toxic and not internalized by LLC-MK2 cells, whereas only 0.26 mg of Tat-His-MBP-Tat-RSVP212-241 was purified. We were able to show that His-Arg9-MBP-RSVP212-241 was non-toxic, internalized, and interacted with the RSV nucleoprotein (N) in a GST pull-down experiment. Furthermore, His-Arg9-MBP-RSVP212-241 attenuated RSV A replication and progeny production by 94.8 and 93.33% at 200 µM, respectively. We demonstrated 50.7 and 49% inhibition of RSV A replication and progeny production at 20 µM, respectively. We showed that inhibition of viral replication by 25 µM His-Arg9-MBP-RSVP212-241 was not significantly different from inhibition by 20 µM His-MBP-Tat-RSVP220-241. Thus, in this thesis we were unable to show that His-Arg9-MBP-RSVP212-241 was a more effective RSV antiviral. Conclusion: The ABD was not a suitable carrier molecule for use with our fusion protein mimetics. However, RSV P protein mimetics that target interactions with the NC complexes and L polymerase are a novel and viable antiviral strategy. We showed that a His-Arg9-MBP-RSVP212-241 mimetic was non-toxic, internalized, and interacted with the RSV N protein in vitro. Furthermore, we showed that at 200 µM this novel mimetic could attenuate RSV A replication and progeny production in vitro by 94.8 and 93.3%, respectively. Further studies are required to characterize the construct, increase its bioactivity, and identify a suitable human carrier molecule for future evaluation in vivo. / Thesis / Master of Science (MSc) / Worldwide, respiratory syncytial virus is a leading cause of lower respiratory infection and hospitalization in children. Nearly all children are infected with the virus by the young age of two. However, respiratory syncytial virus also causes a significant amount of illness and death in the elderly and in immunocompromised individuals. Furthermore, repeated infections by the virus are common throughout life in all populations. With the lack of a vaccine or treatment for this viral infection, an effective antiviral against RSV is required. In this thesis, we developed and evaluated a novel RSV antiviral therapeutic peptide that targets proteins of the viral replication machinery. Since the replication machinery is required for respiratory syncytial virus survival, we hypothesized that infection could be attenuated by preventing formation of the replication machinery. Furthermore, since small protein therapeutics are often cleared quickly from the human body, we investigated human carrier molecules that could be attached to the antiviral protein for stabilization within the body.

Introduction: le Virus Respiratoire Syncytial humain (RSV) induit un taux élevé de morbidité et de mortalité chez les enfants, les personnes immunodéprimées et les personnes âgées. Il existe un besoin urgent d'un nouveau traitement antiviral et d'un vaccin efficaces. Les cellules épithéliales des voies aériennes (AEC) sont la cible principale de RSV et constituent la première ligne de défense grâce à des mécanismes distincts, qui incluent une réponse antivirale autonome cellulaire. La protéine p62/SQSTM1 a de multiples fonctions cellulaires, y compris la séquestration spécifique de la cargaison ubiquitinée (c'est-à-dire, les protéines/organelles et les bactéries intracellulaires) pour leur clairance par autophagie. Des données publiées ont mis en évidence un rôle important de p62 dans la régulation de plusieurs virus (par exemple, le virus de la grippe et la dengue), favorisant ou restreignant sa réplication en fonction du virus. L'objectif de notre étude est de déterminer le rôle de p62 dans la régulation du cycle infectieux de RSV. Méthodes et résultats: L'analyse de l'expression de p62 dans les cellules A549 a montré que p62 est induit et phosphorylé au début de l'infection par RSV. Il est ensuite dégradé plus tardivement durant l’infection. La déplétion des niveaux de p62 a diminué l'accumulation intracellulaire des protéines virales, tandis que la relâche des virions infectieux a été augmentée. De plus, nous avons observé que la réplication de recRSV-GFP est diminuée dans des cellules exprimant de façon stable la protéine associée aux microtubules 1A/1B, chaîne légère 3 (LC3). LC3 recrute p62 et ses cargaisons à l'autophagosome pour qu'ils soient dégradés par autophagie. Des études sont actuellement en cours pour déterminer les mécanismes moléculaires, dépendant de p62, impliqués dans la régulation de la réplication de RSV. Conclusion: nos résultats mettent en évidence un rôle clé de p62 dans la réplication et la propagation de RSV. Ces études aideront à définir si p62 pourrait représenter une cible thérapeutique potentielle pour lutter contre l'infection à RSV. / Introduction: Human respiratory syncytial virus (RSV) causes a high rate of morbidity and mortality worldwide in children, immunocompromised and elderly people. There is an urgent need for effective antiviral treatments and vaccines for RSV. Airway epithelial cells (AECs) are the primary target of RSV and constitute the first line of defense through distinct mechanisms, including intrinsic antiviral responses. The p62/SQSTM1 protein has multiple cellular functions including cell signaling and sequestration of specific ubiquitinated cargo (i.e. proteins/organelles and intracellular bacteria) for autophagic degradation. The replication of several viruses has been shown to be sensitive to p62 levels. The goal of our study is to investigate the role of p62 in the regulation of RSV replication. Methods and Results: Analysis of p62 expression in A549 cells showed that p62 is induced and phosphorylated during early stages of RSV infection, followed by degradation at later times. P62 silencing diminished the intracellular accumulation of viral proteins, while causing increased release of infectious virions. Additionally, we observed that the stable expression of Microtubule-associated protein 1A/1B-light chain 3 (LC3), which recruits p62 and its cargos to the autophagosome for autophagy degradation, reduces recRSV-GFP replication. Studies are currently undertaken to determine the molecular mechanisms involved in p62-dependent regulation of RSV replication. Conclusion: Our results highlight a key role of p62 in the replication of RSV. These studies will help to define whether p62 might represent a potential therapeutic target to fight RSV infection.

Le Virus Respiratoire Syncytial (VRS) est le principal agent responsable d’infections respiratoires sévères chez les nourrissons et les veaux. Le génome du VRS est constitué d’un ARN simple brin de polarité négative qui est répliqué et transcrit par le complexe ARN-polymérase viral (RdRp). Ce complexe est composé de la nucléoprotéine N, de la polymérase L, de la phosphoprotéine P et du facteur anti-terminateur de transcription M2-1. Le but de ce travail était de mieux caractériser la structure et le fonctionnement de deux protéines du complexe RdRp: P et M2-1.M2-1 est un tétramère constitué de 4 domaines : un « doigt de zinc », un domaine d’oligomérisation hélicoïdal, une région flexible, un domaine globulaire interagissant avec l'ARN et P, et une région C-terminale désordonnée. À partir de la structure cristalline de M2-1 pleine longueur, j'ai identifié des résidus critiques sur le doigt de zinc et la région flexible pour l'activité d'anti-terminaison de transcription de M2-1.Par la suite j'ai identifié une région de P critique pour l’interaction P - M2-1 et montre qu’elle est nécessaire au recrutement de M2-1 dans des corps d’inclusion cytoplasmiques. Je montre également que la déphosphorylation de M2-1, nécessaire à la transcription virale, est modulée par un complexe formé entre P et la phosphatase cellulaire PP1.Enfin, la cyclopamine, composé chimique naturel, inhibe la réplication du VRS. Je démontre qu'une seule mutation R151K sur M2-1 est suffisante pour conférer une résistance virale à la cyclopamine. Ces données ouvrent de nouvelles perspectives pour le développement de futures thérapies contre le VRS. / Respiratory syncytial virus (RSV) is the leading cause of lower respiratory tract illness in infants and calves. The RSV genome consists of a single strand, negative-sense RNA, which is replicated and transcribed by the viral RNA-dependent RNA polymerase complex (RdRp). This complex is composed of the nucleoprotein N, the large protein L, the phosphoprotein P and the transcription anti-terminator M2-1. The aim of this work was to better characterize the structure and function of P and M2-1.M2-1 is a tetramer with 4 domains: a zinc-finger, a helical oligomerization domain, a flexible region, a RNA and P binding core domain and a C-terminal disordered region. Based on the crystal structure of the full-length M2-1 protein, I identified residues in the zinc-finger and the flexible loop critical for M2-1 antitermination activity.Then I identified a region of P critical for P – M2-1 interaction and show that it is required for the recruitment of M2-1 to cytoplasmic inclusion bodies. I also show that M2-1 dephosphorylation, which is critical for viral transcription, is modulated by a complex formed by P and the cellular phosphatase protein-1 (PP1).Finally cyclopamine, a natural chemical compound, inhibits the RSV replication. I show that a single R151K mutation in M2-1 is sufficient to confer virus resistance to cyclopamine. These data open a new avenue for the development of future therapies against RSV infection.