Therapeutic efficacy of small molecule inhibitors of human respiratory syncytial virus (hRSV) in neonatal lambs

Human respiratory syncytial virus (hRSV) is a common cause of respiratory infection in human infants worldwide. Direct therapeutic methods for the prevention and treatment such as vaccination and antiviral therapy are limited. In studies within this dissertation, we determined the efficacy of orally administered small molecule fusion inhibitor and replication inhibitor in hRSV-infected neonatal lambs which model hRSV infection in infants. Furthermore, we evaluated the efficacy and treatment time window for administration of a combination treatment using both compounds. We determined that daily oral administration of small molecule fusion protein inhibitor (JNJ-53718678) and replication inhibitor (JNJ-64166037) first administered to hRSV-infected within 24 hpi, reduced the viral load and lung lesion in a dose-dependent manner. The combination treatment of both hRSV fusion inhibitor and replication inhibitor given at 24 hpi decreased the amount of virus and pulmonary lesion in a more profound manner than monotherapeutic administration of each compound. Interestingly, the delayed combination treatment starting at day 3 post infection demonstrated significant reduction of viral load and pulmonary lesion compared to monotherapy administered at similar time indicating a prolonged treatment time window. The small molecule inhibitor compounds used in this study are good therapeutic candidates for future tests in human clinical trials. In addition, we were able to quantify and localize the hRSV viral RNA with RNA in situ hybridization in the infected lung tissue as well as effectively quantify the amount of RNA expression by modification of RNAscope H scoring evaluation method using an image analyzer software. A new approach for assessment of cytokine expression in lung of infected lambs was performed by creating a baseline of cytokine expression of Memphis 37 hRSV infected and non-infected lambs at 6 dpi through combining data from multiple lamb model studies

The Effect of Bone and Analytical Methods on the Assessment of Bone Mineralization Response to Dietary Phosphorus, Phytase, and Vitamin D in Nursery Pigs

Three hundred-fifty pigs (initially 26.2 ± 1.23 lb) were used to evaluate the effects of bone and analytical methods on the assessment of bone mineralization response to dietary P and vitamin D in nursery pigs. Pens of pigs (5 or 6 pigs/pen) were randomized to 6 dietary treatments in a randomized complete block design with 10 pens per treatment. Treatments were formulated to have varying levels of P, phytase, and vitamin D to provide differences in bone characteristics. After feeding diets for 28 d, eight pigs per treatment were euthanized for bone, blood, and urine analysis. The response to treatment for bone density and ash was dependent upon the bone analyzed (density × bone interaction, P = 0.044; non-defatted bone ash × bone interaction, P = 0.060; defatted bone ash × bone interaction, P = 0.068). Pigs fed 0.19% STTD P had decreased (P \u3c 0.05) bone density and ash (non-defatted and defatted) for all bones compared to 0.44% STTD P, with 0.33% STTD P generally intermediate or similar to 0.44% STTD P. Pigs fed 0.44% STTD P with no vitamin D had greater (P \u3c 0.05) non-defatted fibula ash compared to all treatments other than 0.44% STTD P with added HyD. Pigs fed the three diets with 0.44% STTD P had greater (P \u3c 0.05) defatted 2nd rib ash compared to pigs fed 0.19% STTD P or 0.33% STTD P with no phytase. In summary, bone density and ash responses varied depending on the bone analyzed. Differences in bone density and ash in response to P and vitamin D were most apparent with fibulas and 2nd ribs. The difference between bone ash procedures was more apparent than the differences between treatments. For histopathology, 10th ribs were more sensitive than 2nd ribs or fibulas for detection of lesions

The Effect of Different Bone and Analytical Methods on the Assessment of Bone Mineralization to Dietary Phosphorus, Phytase, and Vitamin D in Finishing Pigs

Eight hundred eighty-two pigs (initially 73.2 ± 0.7 lb) were used to evaluate the effects of different bones and analytical methods on the assessment of bone mineralization response to dietary P and vitamin D in growing-finishing pigs. Pens of pigs (20 pigs per pen) were randomized to 1 of 5 dietary treatments in a completely randomized design with 9 pens per treatment. Treatments were formulated to have varying levels of P, phytase, and vitamin D to potentially provide wide differences in bone characteristics. After feeding diets for 112 d, nine pigs per treatment were euthanized for bone, blood, and urine analysis. There were no significant differences for final BW, ADG, ADFI, F/G (P \u3e 0.10), or bone ash (bone ash × bone interaction, P \u3e 0.10) regardless of the ashing method. The response to treatment for bone density and bone mineral content was dependent upon the bone (density interaction, P = 0.053; mineral interaction, P = 0.078). There were no treatment differences for bone density and bone mineral content for metacarpals, fibulas, and 2nd rib (P \u3e 0.05). For 10th rib bone density, pigs fed industry levels of P and vitamin D had increased (P \u3c 0.05) bone density compared to pigs fed NRC levels with phytase, with pigs fed deficient P, NRC levels of P with no phytase, and extra 25(OH)D3 vitamin D (HyD) intermediate. Pigs fed extra vitamin D from HyD had increased (P \u3c 0.05) 10th rib bone mineral content compared to pigs fed deficient P and NRC levels of P with phytase, with pigs fed industry P and vitamin D, and NRC P with monocalcium intermediate. In summary, bone density and bone mineral content responses varied depending on the bone. The difference between bone ash procedures was more apparent than the differences between diets. Differences in bone density and mineral content in response to P and vitamin D were most apparent with the 10th ribs

Therapeutic efficacy of small molecule inhibitors of human respiratory syncytial virus (hRSV) in neonatal lambs

Human respiratory syncytial virus (hRSV) is a common cause of respiratory infection in human infants worldwide. Direct therapeutic methods for the prevention and treatment such as vaccination and antiviral therapy are limited. In studies within this dissertation, we determined the efficacy of orally administered small molecule fusion inhibitor and replication inhibitor in hRSV-infected neonatal lambs which model hRSV infection in infants. Furthermore, we evaluated the efficacy and treatment time window for administration of a combination treatment using both compounds. We determined that daily oral administration of small molecule fusion protein inhibitor (JNJ-53718678) and replication inhibitor (JNJ-64166037) first administered to hRSV-infected within 24 hpi, reduced the viral load and lung lesion in a dose-dependent manner. The combination treatment of both hRSV fusion inhibitor and replication inhibitor given at 24 hpi decreased the amount of virus and pulmonary lesion in a more profound manner than monotherapeutic administration of each compound. Interestingly, the delayed combination treatment starting at day 3 post infection demonstrated significant reduction of viral load and pulmonary lesion compared to monotherapy administered at similar time indicating a prolonged treatment time window. The small molecule inhibitor compounds used in this study are good therapeutic candidates for future tests in human clinical trials. In addition, we were able to quantify and localize the hRSV viral RNA with RNA in situ hybridization in the infected lung tissue as well as effectively quantify the amount of RNA expression by modification of RNAscope H scoring evaluation method using an image analyzer software. A new approach for assessment of cytokine expression in lung of infected lambs was performed by creating a baseline of cytokine expression of Memphis 37 hRSV infected and non-infected lambs at 6 dpi through combining data from multiple lamb model studies.</p

Streptococcus pneumoniae serotype 22F infection in respiratory syncytial virus infected neonatal lambs enhances morbidity.

Respiratory syncytial virus (RSV) is the primary cause of viral bronchiolitis resulting in hospitalization and a frequent cause of secondary respiratory bacterial infection, especially by Streptococcus pneumoniae (Spn) in infants. While murine studies have demonstrated enhanced morbidity during a viral/bacterial co-infection, human meta-studies have conflicting results. Moreover, little knowledge about the pathogenesis of emerging Spn serotype 22F, especially the co-pathologies between RSV and Spn, is known. Here, colostrum-deprived neonate lambs were divided into four groups. Two of the groups were nebulized with RSV M37, and the other two groups were mock nebulized. At day three post-RSV infection, one RSV group (RSV/Spn) and one mock-nebulized group (Spn only) were inoculated with Spn intratracheally. At day six post-RSV infection, bacterial/viral loads were assessed along with histopathology and correlated with clinical symptoms. Lambs dually infected with RSV/Spn trended with higher RSV titers, but lower Spn. Additionally, lung lesions were observed to be more frequent in the RSV/Spn group characterized by increased interalveolar wall thickness accompanied by neutrophil and lymphocyte infiltration and higher myeloperoxidase. Despite lower Spn in lungs, co-infected lambs had more significant morbidity and histopathology, which correlated with a different cytokine response. Thus, enhanced disease severity during dual infection may be due to lesion development and altered immune responses rather than bacterial counts

Detection of Actinobacillus pleuropneumoniae ApxIV toxin antibody in serum and oral fluid specimens from pigs inoculated under experimental conditions

Introduction: The prevention and control of Actinobacillus pleuropneumoniae in commercial production settings is based on serological monitoring. Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect specific antibodies against a variety of A. pleuropneumoniae antigens, including long-chain lipopolysaccharides (LPS) and the ApxIV toxin, a repeats-in-toxin (RTX) exotoxin unique to A. pleuropneumoniae and produced by all serovars. The objective of this study was to describe ApxIV antibody responses in serum and oral fluid of pigs

Detection of Actinobacillus pleuropneumoniae ApxIV toxin antibody in serum and oral fluid specimens from pigs inoculated under experimental conditions

Introduction: The prevention and control of Actinobacillus pleuropneumoniae in commercial production settings is based on serological monitoring. Enzyme-linked immunosorbent assays (ELISAs) have been developed to detect specific antibodies against a variety of A. pleuropneumoniae antigens, including long-chain lipopolysaccharides (LPS) and the ApxIV toxin, a repeats-in-toxin (RTX) exotoxin unique to A. pleuropneumoniae and produced by all serovars. The objective of this study was to describe ApxIV antibody responses in serum and oral fluid of pigs. Material and Methods: Four groups of pigs (six pigs per group) were inoculated with A. pleuropneumoniae serovars 1, 5, 7, or 12. Weekly serum samples and daily oral fluid samples were collected from individual pigs for 56 days post inoculation (DPI) and tested by LPS and ApxIV ELISAs. The ApxIV ELISA was run in three formats to detect immunlgobulins M, G, and A (IgM, IgG and IgA) while the LPS ELISA detected only IgG. Results: All pigs inoculated with A. pleuropneumoniae serovars 1 and 7 were LPS ELISA serum antibody positive from DPI 14 to 56. A transient and weak LPS ELISA antibody response was observed in pigs inoculated with serovar 5 and a single antibody positive pig was observed in serovar 12 at ≥35 DPI. Notably, ApxIV serum and oral fluid antibody responses in pig inoculated with serovars 1 and 7 reflected the patterns observed for LPS antibody, albeit with a 14 to 21 day delay. Conclusion: This work suggests that ELISAs based on ApxIV antibody detection in oral fluid samples could be effective in population monitoring for A. pleuropneumoniae.This article is published as González, Wendy, Luis G. Giménez-Lirola, Ashley Holmes, Sergio Lizano, Christa Goodell, Korakrit Poonsuk, Panchan Sitthicharoenchai, Yaxuan Sun, and Jeffrey Zimmerman. "Detection of Actinobacillus pleuropneumoniae ApxIV toxin antibody in serum and oral fluid specimens from pigs inoculated under experimental conditions." Journal of Veterinary Research 61, no. 2 (2017): 163. DOI: 10.1515/jvetres-2017-0021. Copyright 2017 W. González et al. Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0). Posted with permission

Therapeutic efficacy of a respiratory syncytial virus fusion inhibitor

Respiratory syncytial virus is a major cause of acute lower respiratory tract infection in young children, immunocompromised adults, and the elderly. Intervention with small-molecule antivirals specific for respiratory syncytial virus presents an important therapeutic opportunity, but no such compounds are approved today. Here we report the structure of JNJ-53718678 bound to respiratory syncytial virus fusion (F) protein in its prefusion conformation, and we show that the potent nanomolar activity of JNJ-53718678, as well as the preliminary structure–activity relationship and the pharmaceutical optimization strategy of the series, are consistent with the binding mode of JNJ-53718678 and other respiratory syncytial virus fusion inhibitors. Oral treatment of neonatal lambs with JNJ-53718678, or with an equally active close analog, efficiently inhibits established acute lower respiratory tract infection in the animals, even when treatment is delayed until external signs of respiratory syncytial virus illness have become visible. Together, these data suggest that JNJ-53718678 is a promising candidate for further development as a potential therapeutic in patients at risk to develop respiratory syncytial virus acute lower respiratory tract infection.This article is published as Roymans, Dirk, Sarhad S. Alnajjar, Michael B. Battles, Panchan Sitthicharoenchai, Polina Furmanova-Hollenstein, Peter Rigaux, Joke Van den Berg et al. "Therapeutic efficacy of a respiratory syncytial virus fusion inhibitor." Nature Communications 8, no. 1 (2017): 167. DOI: 10.1038/s41467-017-00170-x. Copyright 2017 The Author(s). Attribution 4.0 International (CC BY 4.0). Posted with permission

Detection and disease diagnosis trends (2017–2022) for Streptococcus suis, Glaesserella parasuis, Mycoplasma hyorhinis, Actinobacillus suis and Mycoplasma hyosynoviae at Iowa State University Veterinary Diagnostic Laboratory

Background Accurate measurement of disease associated with endemic bacterial agents in pig populations is challenging due to their commensal ecology, the lack of disease-specific antemortem diagnostic tests, and the polymicrobial nature of swine diagnostic cases. The main objective of this retrospective study was to estimate temporal patterns of agent detection and disease diagnosis for five endemic bacteria that can cause systemic disease in porcine tissue specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2017 to 2022. The study also explored the diagnostic value of specific tissue specimens for disease diagnosis, estimated the frequency of polymicrobial diagnosis, and evaluated the association between phase of pig production and disease diagnosis. Results S. suis and G. parasuis bronchopneumonia increased on average 6 and 4.3%, while S. suis endocarditis increased by 23% per year, respectively. M. hyorhinis and A. suis associated serositis increased yearly by 4.2 and 12.8%, respectively. A significant upward trend in M. hyorhinis arthritis cases was also observed. In contrast, M. hyosynoviae arthritis cases decreased by 33% average/year. Investigation into the diagnostic value of tissues showed that lungs were the most frequently submitted sample, However, the use of lung for systemic disease diagnosis requires caution due to the commensal nature of these agents in the respiratory system, compared to systemic sites that diagnosticians typically target. This study also explored associations between phase of production and specific diseases caused by each agent, showcasing the role of S. suis arthritis in suckling pigs, meningitis in early nursery and endocarditis in growing pigs, and the role of G. parasuis, A. suis, M. hyorhinis and M. hyosynoviae disease mainly in post-weaning phases. Finally, this study highlighted the high frequency of co-detection and -disease diagnosis with other infectious etiologies, such as PRRSV and IAV, demonstrating that to minimize the health impact of these endemic bacterial agents it is imperative to establish effective viral control programs. Conclusions Results from this retrospective study demonstrated significant increases in disease diagnosis for S. suis, G. parasuis, M. hyorhinis, and A. suis, and a significant decrease in detection and disease diagnosis of M. hyosynoviae. High frequencies of interactions between these endemic agents and with viral pathogens was also demonstrated. Consequently, improved control programs are needed to mitigate the adverse effect of these endemic bacterial agents on swine health and wellbeing. This includes improving diagnostic procedures, developing more effective vaccine products, fine-tuning antimicrobial approaches, and managing viral co-infections.This article is published as Silva, Ana Paula Serafini Poeta, Marcelo Almeida, Alyona Michael, Michael C. Rahe, Christopher Siepker, Drew R. Magstadt, Pablo Piñeyro et al. "Detection and disease diagnosis trends (2017–2022) for Streptococcus suis, Glaesserella parasuis, Mycoplasma hyorhinis, Actinobacillus suis and Mycoplasma hyosynoviae at Iowa State University Veterinary Diagnostic Laboratory." BMC Veterinary Research 19, no. 1 (2023): 1-20. doi: https://doi.org/10.1186/s12917-023-03807-w. Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The content of this document is not copyrighted