Evaluation of a panel of therapeutic antibody clinical candidates for efficacy against SARS-CoV-2 in Syrian hamsters.

The COVID-19 pandemic spurred the rapid development of a range of therapeutic antibody treatments. As part of the US government's COVID-19 therapeutic response, a research team was assembled to support assay and animal model development to assess activity for therapeutics candidates against SARS-CoV-2. Candidate treatments included monoclonal antibodies, antibody cocktails, and products derived from blood donated by convalescent patients. Sixteen candidate antibody products were obtained directly from manufacturers and evaluated for neutralization activity against the WA-01 isolate of SARS-CoV-2. Products were further tested in the Syrian hamster model using prophylactic (-24 h) or therapeutic (+8 h) treatment approaches relative to intranasal SARS-CoV-2 exposure. In vivo assessments included daily clinical scores and body weights. Viral RNA and viable virus titers were quantified in serum and lung tissue with histopathology performed at 3d and 7d post-virus-exposure. Sham-treated, virus-exposed hamsters showed consistent clinical signs with concomitant weight loss and had detectable viral RNA and viable virus in lung tissue. Histopathologically, interstitial pneumonia with consolidation was present. Therapeutic efficacy was identified in treated hamsters by the absence or diminution of clinical scores, body weight loss, viral loads, and improved semiquantitative lung histopathology scores. This work serves as a model for the rapid, systematic in vitro and in vivo assessment of the efficacy of candidate therapeutics at various stages of clinical development. These efforts provided preclinical efficacy data for therapeutic candidates. Furthermore, these studies were invaluable for the phenotypic characterization of SARS CoV-2 disease in hamsters and of utility to the broader scientific community.

Reproductive and developmental toxicity of perfluorooctane sulfonate (PFOS) in the white-footed mouse (Peromyscus leucopus).

Concerns about per- and polyfluoroalkyl substances (PFAS) stem from their ubiquitous presence in the environment, bioaccumulation, resistance to degradation, and toxicity. Previously, toxicity data relevant to ecological risk assessment has largely been aquatic, terrestrial invertebrates, or avian in origin. In this study, repeated oral exposures of perfluorooctane sulfonate (PFOS) were administered to white-footed mice (Peromyscus leucopus) to evaluate effects on reproduction and development. Prenatal exposure to high doses of PFOS caused neonatal mortality, though growth and development were unaffected by low doses. Additionally, parental (P) generation animals exhibited increased liver:body weight, increased hepatocyte cytoplasmic vacuolization, and decreased serum thyroxine (T4) levels. Total litter loss was selected as the protective critical effect in this study resulting in a benchmark dose low (BMDL) of 0.12 mg/kg-d PFOS. Importantly, PFOS exposure has been linked to reduced adult recruitment in myriad species and at similar thresholds to this study. Similarities in critical/toxicologic effects across taxa may add confidence in risk assessments at sites with multiple taxa or environments.

Molecular analysis of the 2012 Bundibugyo virus disease outbreak.

Bundibugyo virus (BDBV) is one of four ebolaviruses known to cause disease in humans. Bundibugyo virus disease (BVD) outbreaks occurred in 2007-2008 in Bundibugyo District, Uganda, and in 2012 in Isiro, Province Orientale, Democratic Republic of the Congo. The 2012 BVD outbreak resulted in 38 laboratory-confirmed cases of human infection, 13 of whom died. However, only 4 BDBV specimens from the 2012 outbreak have been sequenced. Here, we provide BDBV sequences from seven additional patients. Analysis of the molecular epidemiology and evolutionary dynamics of the 2012 outbreak with these additional isolates challenges the current hypothesis that the outbreak was the result of a single spillover event. In addition, one patient record indicates that BDBV's initial emergence in Isiro occurred 50 days earlier than previously accepted. Collectively, this work demonstrates how retrospective sequencing can be used to elucidate outbreak origins and provide epidemiological contexts to a medically relevant pathogen.

Molecular epidemiology and glycomics of swine influenza viruses circulating in commercial swine farms in the southeastern and midwest United States.

Tracking the genetic diversity and spread of swine influenza viruses (SIVs) in commercial swine farms is central for control and to reduce the potential emergence of SIV reassortants. We analyzed the diversity of SIVs in nasal washes or oral fluids from commercial swine farms in North Carolina using influenza M qRT-PCR and hemagglutinin (HA) and neuraminidase (NA) subtyping. We found a predominance of H1 HAs and N2 NAs in the samples examined. The majority of the H1 HAs could be further classified into gamma and delta subclusters. We also identified HAs of the H1 alpha cluster, and those of human novel pandemic origin. Glycan binding profiles from a representative subset of these viruses revealed broad α2,6 sialylated glycan recognition, though some strains exhibited the ability to bind to α2,3 sialic acid. These data show that SIV surveillance can aid our understanding of viral transmission dynamics and help uncover the diversity at the human-swine interface.

Molecular epidemiology of swine influenza A viruses in the Southeastern United States, highlights regional differences in circulating strains.

Swine influenza A virus (IAV) can cause widespread respiratory disease with high morbidity, low mortality, and have a substantial economic impact to the swine industry. Swine infection may contribute to pandemic IAV given their susceptibility to both avian and human IAVs. Currently, three IAV subtypes (H1N1, H3N2 and H1N2) circulate in swine in North America frequently combining gene segments from avian or human viruses. This study investigated the prevalence of IAV in commercial swine herds. A total of 1878 oral fluid samples were collected from pigs of all ages from 201 commercial farms located in North Carolina and South Carolina. Sixty-eight oral fluid samples from 35 farms were positive by MP gene PCR with an overall IAV-positivity of 3.6%. On the herd level, the percentage of IAV positivity was 17.4%. Fifty-six viruses were subtyped, while 12 were partly subtyped or not subtyped at all. Using de novo assembly, complete sequences were obtained for 59 HA genes. The majority of IAVs subtyped had an H1 HA demonstrating a considerable prevalence over H3 viruses. Furthermore, only six out of eleven HA types were detected which has implications for the selection of vaccines used by swine producers in the region.

Comparison of Microchip Transponder and Noncontact Infrared Thermometry with Rectal Thermometry in Domestic Swine (Sus scrofa domestica).

During disease outbreaks, core temperature is a useful health metric in swine, due to the presence of pyrexia especially during the acute phase of infection. Despite technologic advances in other facets of swine production and health management, rectal thermometry continues to be the 'gold standard' for measuring core body temperature. However, for various reasons, collecting rectal temperatures can be difficult and unsafe depending on the housing modality. In addition, the delay between insertion of the rectal thermometer and obtaining a reading can affect measurement accuracy, especially when the pig requires physical restraint. Clearly safer, faster, and more accurate and precise temperature acquisition methods that necessitate minimal or no handling of swine are needed. We therefore compared rectal thermometers, subcutaneous microchips, and an inexpensive handheld infrared thermometer by measuring the core body temperature of 24 male castrated piglets at random intervals over a 5-wk period. The core body temperature (mean ± 1 SD) was 39.3±0.5 °C by rectal thermometry, 39.0±0.7 °C by microchip transponder, and 34.3±1.0 °C by infrared thermometry; these 3 values differed significantly. Although the readings obtain by using infrared thermometry were numerically lower than those from the other methods, it is arguably the safest method for assessing the core temperature of swine and showed strong relative correlation with rectal temperature.

Passive narcosis for anesthesia induction in cotton rats (Sigmodon hispidus).

Cotton rats (Sigmodon hispidus) are widely used as animal models for infectious disease and immunological research. They emulate many aspects of human disease pathogenesis, and the introduction of cotton rat-specific immunological reagents, cell lines and sequencing of relevant genes have all helped to increase the popularity of this disease model. However, the use of cotton rats is problematic owing to their propensity for aggressive responses when handled, which can lead to escape, increased stress to the animals, and bites to staff. When cotton rats are co-housed, which is recommended under current social housing guidelines, these risks are increased. Here, we describe a method of isoflurane anesthesia induction in the home cage that reduces the risk of animal escape, minimizes stress during induction, and provides additional safety for staff. The method uses inexpensive materials that are widely available and can be easily disinfected. Our method also eliminates the need for expensive and cumbersome machines traditionally used with anesthetic chambers, and uses a minimal amount of inhalant anesthetic, saving resources and protecting staff from inhalation of leaked gas.

Hypothiocyanite produced by human and rat respiratory epithelial cells inactivates extracellular H1N2 influenza A virus.

OBJECTIVE AND DESIGN: Our aim was to study whether an extracellular, oxidative antimicrobial mechanism inherent to tracheal epithelial cells is capable of inactivating influenza H1N2 virus. MATERIAL OR SUBJECTS: Epithelial cells were isolated from tracheas of male Sprague-Dawley rats. Both primary human and rat tracheobronchial epithelial cells were differentiated in air-liquid interface cultures. TREATMENT: A/swine/Illinois/02860/09 (swH1N2) influenza A virions were added to the apical side of airway cells for 1 h in the presence or absence of lactoperoxidase or thiocyanate. METHODS: Characterization of rat epithelial cells (morphology, Duox expression) occurred via western blotting, PCR, hydrogen peroxide production measurement and histology. The number of viable virions was determined by plaque assays. Statistical difference of the results was analyzed by ANOVA and Tukey's test. RESULTS: Our data show that rat tracheobronchial epithelial cells develop a differentiated, polarized monolayer with high transepithelial electrical resistance, mucin production and expression of dual oxidases. Influenza A virions are inactivated by human and rat epithelial cells via a dual oxidase-, lactoperoxidase- and thiocyanate-dependent mechanism. CONCLUSIONS: Differentiated air-liquid interface cultures of rat tracheal epithelial cells provide a novel model to study airway epithelium-influenza interactions. The dual oxidase/lactoperoxidase/thiocyanate extracellular oxidative system producing hypothiocyanite is a fast and potent anti-influenza mechanism inactivating H1N2 viruses prior to infection of the epithelium.