Potent universal beta-coronavirus therapeutic activity mediated by direct respiratory administration of a Spike S2 domain-specific human neutralizing monoclonal antibody.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) marks the third novel ß-coronavirus to cause significant human mortality in the last two decades. Although vaccines are available, too few have been administered worldwide to keep the virus in check and to prevent mutations leading to immune escape. To determine if antibodies could be identified with universal coronavirus activity, plasma from convalescent subjects was screened for IgG against a stabilized pre-fusion SARS-CoV-2 spike S2 domain, which is highly conserved between human ß-coronavirus. From these subjects, several S2-specific human monoclonal antibodies (hmAbs) were developed that neutralized SARS-CoV-2 with recognition of all variants of concern (VoC) tested (Beta, Gamma, Delta, Epsilon, and Omicron). The hmAb 1249A8 emerged as the most potent and broad hmAb, able to recognize all human ß-coronavirus and neutralize SARS-CoV and MERS-CoV. 1249A8 demonstrated significant prophylactic activity in K18 hACE2 mice infected with SARS-CoV-2 lineage A and lineage B Beta, and Omicron VoC. 1249A8 delivered as a single 4 mg/kg intranasal (i.n.) dose to hamsters 12 hours following infection with SARS-CoV-2 Delta protected them from weight loss, with therapeutic activity further enhanced when combined with 1213H7, an S1-specific neutralizing hmAb. As little as 2 mg/kg of 1249A8 i.n. dose 12 hours following infection with SARS-CoV Urbani strain, protected hamsters from weight loss and significantly reduced upper and lower respiratory viral burden. These results indicate in vivo cooperativity between S1 and S2 specific neutralizing hmAbs and that potent universal coronavirus neutralizing mAbs with therapeutic potential can be induced in humans and can guide universal coronavirus vaccine development.

SARS-CoV-2 evolution in animals suggests mechanisms for rapid variant selection.

SARS-CoV-2 spillback from humans into domestic and wild animals has been well documented, and an accumulating number of studies illustrate that human-to-animal transmission is widespread in cats, mink, deer, and other species. Experimental inoculations of cats, mink, and ferrets have perpetuated transmission cycles. We sequenced full genomes of Vero cell-expanded SARS-CoV-2 inoculum and viruses recovered from cats (n = 6), dogs (n = 3), hamsters (n = 3), and a ferret (n = 1) following experimental exposure. Five nonsynonymous changes relative to the USA-WA1/2020 prototype strain were near fixation in the stock used for inoculation but had reverted to wild-type sequences at these sites in dogs, cats, and hamsters within 1- to 3-d postexposure. A total of 14 emergent variants (six in nonstructural genes, six in spike, and one each in orf8 and nucleocapsid) were detected in viruses recovered from animals. This included substitutions in spike residues H69, N501, and D614, which also vary in human lineages of concern. Even though a live virus was not cultured from dogs, substitutions in replicase genes were detected in amplified sequences. The rapid selection of SARS-CoV-2 variants in vitro and in vivo reveals residues with functional significance during host switching. These observations also illustrate the potential for spillback from animal hosts to accelerate the evolution of new viral lineages, findings of particular concern for dogs and cats living in households with COVID-19 patients. More generally, this glimpse into viral host switching reveals the unrealized rapidity and plasticity of viral evolution in experimental animal model systems.

A self-amplifying mRNA SARS-CoV-2 vaccine candidate induces safe and robust protective immunity in preclinical models.

RNA vaccines have demonstrated efficacy against SARS-CoV-2 in humans, and the technology is being leveraged for rapid emergency response. In this report, we assessed immunogenicity and, for the first time, toxicity, biodistribution, and protective efficacy in preclinical models of a two-dose self-amplifying messenger RNA (SAM) vaccine, encoding a prefusion-stabilized spike antigen of SARS-CoV-2 Wuhan-Hu-1 strain and delivered by lipid nanoparticles (LNPs). In mice, one immunization with the SAM vaccine elicited a robust spike-specific antibody response, which was further boosted by a second immunization, and effectively neutralized the matched SARS-CoV-2 Wuhan strain as well as B.1.1.7 (Alpha), B.1.351 (Beta) and B.1.617.2 (Delta) variants. High frequencies of spike-specific germinal center B, Th0/Th1 CD4, and CD8 T cell responses were observed in mice. Local tolerance, potential systemic toxicity, and biodistribution of the vaccine were characterized in rats. In hamsters, the vaccine candidate was well-tolerated, markedly reduced viral load in the upper and lower airways, and protected animals against disease in a dose-dependent manner, with no evidence of disease enhancement following SARS-CoV-2 challenge. Therefore, the SARS-CoV-2 SAM (LNP) vaccine candidate has a favorable safety profile, elicits robust protective immune responses against multiple SARS-CoV-2 variants, and has been advanced to phase 1 clinical evaluation (NCT04758962).

Experimental infection of domestic dogs and cats with SARS-CoV-2: Pathogenesis, transmission, and response to reexposure in cats.

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached nearly every country in the world with extraordinary person-to-person transmission. The most likely original source of the virus was spillover from an animal reservoir and subsequent adaptation to humans sometime during the winter of 2019 in Wuhan Province, China. Because of its genetic similarity to SARS-CoV-1, it is probable that this novel virus has a similar host range and receptor specificity. Due to concern for human-pet transmission, we investigated the susceptibility of domestic cats and dogs to infection and potential for infected cats to transmit to naive cats. We report that cats are highly susceptible to infection, with a prolonged period of oral and nasal viral shedding that is not accompanied by clinical signs, and are capable of direct contact transmission to other cats. These studies confirm that cats are susceptible to productive SARS-CoV-2 infection, but are unlikely to develop clinical disease. Further, we document that cats developed a robust neutralizing antibody response that prevented reinfection following a second viral challenge. Conversely, we found that dogs do not shed virus following infection but do seroconvert and mount an antiviral neutralizing antibody response. There is currently no evidence that cats or dogs play a significant role in human infection; however, reverse zoonosis is possible if infected owners expose their domestic pets to the virus during acute infection. Resistance to reinfection holds promise that a vaccine strategy may protect cats and, by extension, humans.

"Submergence" of Western equine encephalitis virus: Evidence of positive selection argues against genetic drift and fitness reductions.

Understanding the circumstances under which arboviruses emerge is critical for the development of targeted control and prevention strategies. This is highlighted by the emergence of chikungunya and Zika viruses in the New World. However, to comprehensively understand the ways in which viruses emerge and persist, factors influencing reductions in virus activity must also be understood. Western equine encephalitis virus (WEEV), which declined during the late 20th century in apparent enzootic circulation as well as equine and human disease incidence, provides a unique case study on how reductions in virus activity can be understood by studying evolutionary trends and mechanisms. Previously, we showed using phylogenetics that during this period of decline, six amino acid residues appeared to be positively selected. To assess more directly the effect of these mutations, we utilized reverse genetics and competition fitness assays in the enzootic host and vector (house sparrows and Culex tarsalis mosquitoes). We observed that the mutations contemporary with reductions in WEEV circulation and disease that were non-conserved with respect to amino acid properties had a positive effect on enzootic fitness. We also assessed the effects of these mutations on virulence in the Syrian-Golden hamster model in relation to a general trend of increased virulence in older isolates. However, no change effect on virulence was observed based on these mutations. Thus, while WEEV apparently underwent positive selection for infection of enzootic hosts, residues associated with mammalian virulence were likely eliminated from the population by genetic drift or negative selection. These findings suggest that ecologic factors rather than fitness for natural transmission likely caused decreased levels of enzootic WEEV circulation during the late 20th century.

Pathogen to commensal? Longitudinal within-host population dynamics, evolution, and adaptation during a chronic >16-year Burkholderia pseudomallei infection.

Although acute melioidosis is the most common outcome of Burkholderia pseudomallei infection, we have documented a case, P314, where disease severity lessened with time, and the pathogen evolved towards a commensal relationship with the host. In the current study, we used whole-genome sequencing to monitor this long-term symbiotic relationship to better understand B. pseudomallei persistence in P314's sputum despite intensive initial therapeutic regimens. We collected and sequenced 118 B. pseudomallei isolates from P314's airways over a >16-year period, and also sampled the patient's home environment, recovering six closely related B. pseudomallei isolates from the household water system. Using comparative genomics, we identified 126 SNPs in the core genome of the 124 isolates or 162 SNPs/indels when the accessory genome was included. The core SNPs were used to construct a phylogenetic tree, which demonstrated a close relationship between environmental and clinical isolates and detailed within-host evolutionary patterns. The phylogeny had little homoplasy, consistent with a strictly clonal mode of genetic inheritance. Repeated sampling revealed evidence of genetic diversification, but frequent extinctions left only one successful lineage through the first four years and two lineages after that. Overall, the evolution of this population is nonadaptive and best explained by genetic drift. However, some genetic and phenotypic changes are consistent with in situ adaptation. Using a mouse model, P314 isolates caused greatly reduced morbidity and mortality compared to the environmental isolates. Additionally, potentially adaptive phenotypes emerged and included differences in the O-antigen, capsular polysaccharide, motility, and colony morphology. The >13-year co-existence of two long-lived lineages presents interesting hypotheses that can be tested in future studies to provide additional insights into selective pressures, niche differentiation, and microbial adaptation. This unusual melioidosis case presents a rare example of the evolutionary progression towards commensalism by a highly virulent pathogen within a single human host.

Potential Use for Serosurveillance of Feral Swine to Map Risk for Anthrax Exposure, Texas, USA.

Anthrax is a disease of concern in many mammals, including humans. Management primarily consists of prevention through vaccination and tracking clinical-level observations because environmental isolation is laborious and bacterial distribution across large geographic areas difficult to confirm. Feral swine (Sus scrofa) are an invasive species with an extensive range in the southern United States that rarely succumbs to anthrax. We present evidence that feral swine might serve as biosentinels based on comparative seroprevalence in swine from historically defined anthrax-endemic and non-anthrax-endemic regions of Texas. Overall seropositivity was 43.7% (n = 478), and logistic regression revealed county endemicity status, age-class, sex, latitude, and longitude were informative for predicting antibody status. However, of these covariates, only latitude was statistically significant (ß = -0.153, p = 0.047). These results suggests anthrax exposure in swine, when paired with continuous location data, could serve as a proxy for bacterial presence in specific areas.

Peridomestic Mammal Susceptibility to Severe Acute Respiratory Syndrome Coronavirus 2 Infection.

Wild animals have been implicated as the origin of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), but it is largely unknown how the virus affects most wildlife species and if wildlife could ultimately serve as a reservoir for maintaining the virus outside the human population. We show that several common peridomestic species, including deer mice, bushy-tailed woodrats, and striped skunks, are susceptible to infection and can shed the virus in respiratory secretions. In contrast, we demonstrate that cottontail rabbits, fox squirrels, Wyoming ground squirrels, black-tailed prairie dogs, house mice, and racoons are not susceptible to SARS-CoV-2 infection. Our results expand the knowledge base of susceptible species and provide evidence that human-wildlife interactions could result in continued transmission of SARS-CoV-2.

Hammett Correlation in the Accelerated Formation of 2,3-Diphenylquinoxalines in Nebulizer Microdroplets.

The accelerated formation of 2,3-diphenylquinoxalines in microdroplets generated in a nebulizer has been investigated by competition experiments in which equimolar quantities of 1,2-phenylenediamine, C6H4(NH2)2, and a 4-substituted homologue, XC6H3(NH2)2 [X = F, Cl, Br, CH3, CH3O, CO2CH3, CF3, CN or NO2], or a 4,5-disubstituted homologue, X2C6H2(NH2)2 [X = F, Cl, Br, or CH3], compete to condense with benzil, (C6H5CO)2. Electron-donating substituents (X = CH3 and CH3O) accelerate the reaction; in contrast, electron-attracting substituents (X = F, Cl, Br and particularly CO2CH3, CN, CF3 and NO2) retard it. A structure-reactivity relationship in the form of a Hammett correlation has been found by analyzing the ratio of 2,3-diphenylquinoxaline and the corresponding substituted-2,3-diphenylquinoxaline, giving a ρ value of -0.96, thus confirming that the electron density in the aromatic ring of the phenylenediamine component is reduced in the rate-limiting step in this accelerated condensation. This correlation shows that the phenylenediamine acts as a nucleophile in the reaction.

Inactivation of severe acute respiratory syndrome coronavirus 2 in plasma and platelet products using a riboflavin and ultraviolet light-based photochemical treatment.

BACKGROUND AND OBJECTIVE: Severe acute respiratory distress syndrome coronavirus-2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), is a member of the coronavirus family. Coronavirus infections in humans are typically associated with respiratory illnesses; however, viral RNA has been isolated in serum from infected patients. Coronaviruses have been identified as a potential low-risk threat to blood safety. The Mirasol Pathogen Reduction Technology (PRT) System utilizes riboflavin and ultraviolet (UV) light to render blood-borne pathogens noninfectious, while maintaining blood product quality. Here, we report on the efficacy of riboflavin and UV light against the pandemic virus SARS-CoV-2 when tested in both plasma and platelets units. MATERIALS AND METHODS: Stock SARS-CoV-2 was grown in Vero cells and inoculated into either plasma or platelet units. Those units were then treated with riboflavin and UV light. The infectious titres of SARS-CoV-2 were determined by plaque assay using Vero cells. A total of five (n = 5) plasma and three (n = 3) platelet products were evaluated in this study. RESULTS: In both experiments, the measured titre of SARS-CoV-2 was below the limit of detection following treatment with riboflavin and UV light. The mean log reductions in the viral titres were ≥3·40 and ≥4·53 for the plasma units and platelet units, respectively. CONCLUSION: Riboflavin and UV light effectively reduced the titre of SARS-CoV-2 in both plasma and platelet products to below the limit of detection in tissue culture. The data suggest that the process would be effective in reducing the theoretical risk of transfusion transmitted SARS-CoV-2.

Delays in Obtaining Knee MRI in Pediatric Sports Medicine: Impact of Insurance Type.

BACKGROUND: Increased enrollment in government-based insurance plans has been reported. With youth sports injuries on the rise, increased ordering of advanced imaging such as magnetic resonance imaging (MRI) has occurred. This study sought to report on the impact of insurance type on access to and results of knee MRI in pediatric sports medicine patients. METHODS: A retrospective review of 178 consecutive pediatric sports medicine clinics was completed. INCLUSION CRITERIA: patients younger than 18 years, routine knee MRI ordered, sports medicine diagnosis, and insurance. Data included basic demographics, injury date, date and location (urgent care vs. clinic) of the first presentation, details of MRI ordering and approval, date and location of MRI follow-up, MRI results (negative, minor findings, major findings), and eventual treatment required. RESULTS: A total of 168 charts underwent a complete review. The patients' average age was 14±3 years and 54% (N=90) were female. Ninety-eight had government insurance and 70 had commercial insurance. The time between injury and MRI completion was significantly longer with government insurance (34 vs. 67 d, P<0.01). Government insurance had increased wait time between the first visit and MRI completion (11 vs. 40 d, P<0.001) as well as MRI order and completion (9 vs. 16.5 d, P<0.001). There was no significant difference in positive findings on MRI between insurance groups, including both major and minor findings nor in the proportion receiving eventual operative treatment. CONCLUSION: Pediatric sports medicine patients with government insurance have delays in obtaining knee MRI, despite there being no difference in the rate of positive findings and subsequent operative treatments. LEVEL OF EVIDENCE: Level III-case-control study.

Evaluation of General and Musculoskeletal Health Literacy Disparities in Pediatric Sports Injury Patient and Guardian Populations.

BACKGROUND: Increased participation of adolescents in organized sports has led to an increase in pediatric sports injury. Limited health literacy puts patients at risk for worse outcomes through decreased compliance. We aim to evaluate the extent of health literacy disparities in pediatric sports medicine populations. METHODS: Patients aged 10 to 17 years and their consenting guardians visiting clinic for treatment of a sports-related injury completed a unique questionnaire including self-reported health literacy measures and direct assessment of knowledge regarding care for musculoskeletal injuries. Statistical analysis based on socioeconomic factors and demographics was performed using t tests. RESULTS: A total of 268 patient surveys (14.37±1.94 y) and 251 guardian surveys (43.62±9.08 y) were collected. In self-reported general health literacy scores for guardians, all categories except ethnicity played a statistically significant role, with higher health literacy scores associated with higher education, use of English as the primary language at home, private insurance, and female guardians (P<0.001, <0.001, <0.001, 0.011). In contrast, age was the only factor affecting scores in the patient population (P=0.015). Among self-reported musculoskeletal health literacy and directly measured musculoskeletal literacy scores, there were significant differences in groups by age, primary language, and level of education (P=0.020, 0.003). CONCLUSIONS: Significant disparities in general and musculoskeletal health literacy exist within pediatric sports medicine populations, most notably between guardian groups. Improving disparities in health literacy for these populations may best be aimed at guardians, using medical education through verbal/written instruction in multiple languages. LEVEL OF EVIDENCE: Level IV.

Iliotibial band autograft: what size is the graft? A mathematical and cadaveric model : ITB single and double strand size.

BACKGROUND: The iliotibial band (ITB) is used in anterior cruciate ligament (ACL) reconstruction in skeletally immature patients as well as several other orthopedic reconstructions. The purpose of this study is to determine the size of the ITB as an autograft option in ACL reconstruction surgery or other orthopedic soft tissue reconstructions. METHODS: Five adult cadavers resulting in nine ITB were used. Thickness and width of the ITB were determined. Using ITB width of 15-60 mm, single and doubled graft sizes were determined using standard surgical graft size technique. Geometric calculations based on average graft thickness were used to mathematically confirm the graft size of the ITB. RESULTS: The ITB is less than 1 mm in thickness in males and females. Cadaveric measurements were less than 1 mm larger than mathematical measurements, in majority of measurements. ITB autograft can be harvested to a maximum 9 mm single-stranded graft or > 12 mm doubled graft. A minimum of 50 mm of ITB width is required to make a 8 mm graft. CONCLUSIONS: ITB is a versatile graft that can be used for a graft size up to 9 mm single strand and over 12 mm double strand. A minimum of 50 mm width of ITB is required to obtain a 8 mm-diameter autograft. To ensure appropriate graft size, surgeons should consider harvesting the maximum amount of ITB when performing ACL reconstructions in skeletally immature patients. CLINICAL RELEVANCE: Surgeons have a quick reference for the width of ITB they should harvest based on the size of graft they require for a successful surgery.

Hamstring Autograft Too Small: How Much Allograft Do You Need to Supplement to a Desired Hybrid Graft Size?

PURPOSE: To determine a simple rule for choosing supplemental allograft size for hybrid anterior cruciate ligament reconstruction using mathematical and cadaveric models. METHODS: Mathematical and cadaveric models were used to determine the rule. The mathematical model required application of the geometric Pythagorean theorem to add areas of circles. Cadaveric semitendinosus and gracilis tendons were combined in multiple quadrupled hamstring size combinations and then sized using standard surgical techniques to confirm the mathematical model. RESULTS: Geometric measurement, not simple addition, of graft diameters was required to determine the final graft size. Direct comparison of cadaveric and mathematical models showed close relations. If a final graft size of 7 mm is desired, an added diameter of all grafts of approximately 9.5 mm is needed. If a final graft size of 8 mm is desired, an added diameter of all grafts of approximately 11 mm is needed. If a final graft size of 9 mm is desired, an added diameter of all grafts of approximately 12.5 mm is needed. If a final graft size of 10 mm is desired, an added graft diameter of approximately 14 mm is needed. Cadaveric hamstring measurements were similar to the mathematical model. CONCLUSIONS: By use of mathematical and cadaveric models, simple rules for determining the additional size of allograft diameter needed to supplement undersized hamstring autograft were created. CLINICAL RELEVANCE: With the increasing availability of allograft types and sizes, surgeons currently have no guidelines on the size of allograft that is required to supplement an undersized hamstring autograft. Simple rules were created for determining the amount of allograft supplementation required for undersized hamstrings and are easily applied to clinical situations.

Defining a Safe Zone for All-Inside Lateral Meniscal Repairs in Pediatric Patients: A Magnetic Resonance Imaging Study.

PURPOSE: To establish a safe zone for all-inside meniscal fixation in pediatric patients by use of magnetic resonance imaging (MRI) measurements between the popliteal tendon (PT) and popliteal neurovascular bundle (PNVB). METHODS: Patients aged 5 to 16 years with normal or nearly normal knee MRI scans were included. They were grouped by age: group I, 5 to 7 years (n = 61); group II, 8 to 10 years (n = 59); group III, 11 to 13 years (n = 60); and group IV, 14 to 16 years (n = 70). At the level of the lateral meniscus, 2 lines starting at the lateral patellar tendon border and ending at the medial edge of the PT (D1) and the lateral edge of the PNVB (D2) were made on an axial knee MRI scan. A third line (D3) connected D1 to D2 at the meniscocapsular junction of the posterior horn of the lateral meniscus (PHLM). A fourth line (D4), derived geometrically, was parallel and 8 mm anterior to D3, simulating the anterior edge of the PHLM. RESULTS: Axial MRI scans of 250 pediatric patients (aged 5-16 years) were retrospectively reviewed. Analysis showed significant correlation between age and sex for D3 (P < .0001). For D3, there were significant differences among all age groups, except between groups III and IV. The average D3 by age group was 14.1 mm (standard deviation [SD], 3.1 mm) for group I, 15.8 mm (SD, 2.5 mm) for group II, 17.0 mm (SD, 3.3 mm) for group III, and 17.2 mm (SD, 3.1 mm) for group IV. The average D4 was 11.39 mm (SD, 2.6 mm), 13.24 mm (SD, 2.24 mm), 14.59 mm (SD, 2.89 mm), and 14.80 mm (SD, 2.79 mm), respectively. There were significant differences in D3 and D4 in male versus female patients (17.6 mm vs 15.7 mm, P < .001, and 14.9 mm vs 13.2 mm, P < .001, respectively), particularly in groups III and IV (17.0 mm vs 13.8 mm and 16.8 mm vs 13.9 mm, respectively). CONCLUSIONS: This study provides normative data of the distance between the PNVB and PT at the meniscocapsular junction (D3) and anterior edge of the PHLM (D4) with the knee in full extension. Combined with previous studies showing that the addition of knee flexion increases the distance between the meniscus and the neurovascular bundle, these data can be used by surgeons to improve the safety of PHLM repair in pediatric patients. LEVEL OF EVIDENCE: Level III, diagnostic study of nonconsecutive patients.

Yersinia pestis Survival and Replication in Potential Ameba Reservoir.

Plague ecology is characterized by sporadic epizootics, then periods of dormancy. Building evidence suggests environmentally ubiquitous amebae act as feral macrophages and hosts to many intracellular pathogens. We conducted environmental genetic surveys and laboratory co-culture infection experiments to assess whether plague bacteria were resistant to digestion by 5 environmental ameba species. First, we demonstrated that Yersinia pestis is resistant or transiently resistant to various ameba species. Second, we showed that Y. pestis survives and replicates intracellularly within Dictyostelium discoideum amebae for ˃48 hours postinfection, whereas control bacteria were destroyed in <1 hour. Finally, we found that Y. pestis resides within ameba structures synonymous with those found in infected human macrophages, for which Y. pestis is a competent pathogen. Evidence supporting amebae as potential plague reservoirs stresses the importance of recognizing pathogen-harboring amebae as threats to public health, agriculture, conservation, and biodefense.

Impaired osteocyte maturation in the pathogenesis of renal osteodystrophy.

Pediatric renal osteodystrophy is characterized by skeletal mineralization defects, but the role of osteoblast and osteocyte maturation in the pathogenesis of these defects is unknown. We evaluated markers of osteocyte maturation and programmed cell death in iliac crest biopsy samples from pediatric dialysis patients and healthy controls. We evaluated the relationship between numbers of fibroblast growth factor 23 (FGF23)-expressing osteocytes and histomorphometric parameters of skeletal mineralization. We confirmed that chronic kidney disease (CKD) causes intrinsic changes in bone cell maturation using an in vitro model of primary osteoblasts from patients with CKD and healthy controls. FGF23 co-localized with the early osteocyte marker E11/gp38, suggesting that FGF23 is a marker of early osteocyte maturation. Increased numbers of early osteocytes and decreased osteocyte apoptosis characterized CKD bone. Numbers of FGF23-expressing osteocytes were highest in patients with preserved skeletal mineralization indices, and packets of matrix surrounding FGF23-expressing osteocytes appeared to have entered secondary mineralization. Primary osteoblasts from patients with CKD retained impaired maturation and mineralization characteristics in vitro. Addition of FGF23 did not affect primary osteoblast mineralization. Thus, CKD is associated with intrinsic changes in osteoblast and osteocyte maturation, and FGF23 appears to mark a relatively early stage in osteocyte maturation. Improved control of renal osteodystrophy and FGF23 excess will require further investigation into the pathogenesis of CKD-mediated osteoblast and osteocyte maturation failure.

Development and Characterization of Recombinant Virus Generated from a New World Zika Virus Infectious Clone.

Zika virus (ZIKV; family Flaviviridae, genus Flavivirus) is a rapidly expanding global pathogen that has been associated with severe clinical manifestations, including devastating neurological disease in infants. There are currently no molecular clones of a New World ZIKV available that lack significant attenuation, hindering progress toward understanding determinants of transmission and pathogenesis. Here we report the development and characterization of a novel ZIKV reverse genetics system based on a 2015 isolate from Puerto Rico (PRVABC59). We generated a two-plasmid infectious clone system from which infectious virus was rescued that replicates in human and mosquito cells with growth kinetics representative of wild-type ZIKV. Infectious clone-derived virus initiated infection and transmission rates in Aedes aegypti mosquitoes comparable to those of the primary isolate and displayed similar pathogenesis in AG129 mice. This infectious clone system provides a valuable resource to the research community to explore ZIKV molecular biology, vaccine development, antiviral development, diagnostics, vector competence, and disease pathogenesis. IMPORTANCE: ZIKV is a rapidly spreading mosquito-borne pathogen that has been linked to Guillain-Barré syndrome in adults and congenital microcephaly in developing fetuses and infants. ZIKV can also be sexually transmitted. The viral molecular determinants of any of these phenotypes are not well understood. There is no reverse genetics system available for the current epidemic virus that will allow researchers to study ZIKV immunity, develop novel vaccines, or develop antiviral drugs. Here we provide a novel infectious clone system generated from a recent ZIKV isolated from a patient infected in Puerto Rico. This infectious clone produces virus with in vitro and in vivo characteristics similar to those of the primary isolate, providing a critical tool to study ZIKV infection and disease.

Cottontail rabbits shed clade 2.3.4.4 H5 highly pathogenic avian influenza A viruses.

During 2014-2015, clade 2.3.4.4 H5Nx highly pathogenic (HP) avian influenza A viruses (IAV) were first detected in North America and subsequently caused one of the largest agricultural emergencies in U.S. HISTORY: Recent evidence has suggested that cottontail rabbits can shed multiple IAV subtypes. We experimentally infected cottontail rabbits with three HP H5Nx IAVs. All rabbits tested shed virus on at least one day by at least one route. Cottontail rabbits appear to be an exception to the limited capacity for replication that has been previously reported for certain other mammalian species inoculated with clade 2.3.4.4 HP H5Nx avian influenza A viruses.