The continued emergence of deadly human coronaviruses from animal reservoirs highlights the need for pan-coronavirus interventions for effective pandemic preparedness. Here, using linking B cell receptor to antigen specificity through sequencing (LIBRA-seq), we report a panel of 50 coronavirus antibodies isolated from human B cells. Of these, 54043-5 was shown to bind the S2 subunit of spike proteins from alpha-, beta-, and deltacoronaviruses. A cryoelectron microscopy (cryo-EM) structure of 54043-5 bound to the prefusion S2 subunit of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike defined an epitope at the apex of S2 that is highly conserved among betacoronaviruses. Although non-neutralizing, 54043-5 induced Fc-dependent antiviral responses in vitro, including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). In murine SARS-CoV-2 challenge studies, protection against disease was observed after introduction of Leu234Ala, Leu235Ala, and Pro329Gly (LALA-PG) substitutions in the Fc region of 54043-5. Together, these data provide new insights into the protective mechanisms of non-neutralizing antibodies and define a broadly conserved epitope within the S2 subunit.

Candida auris is an easily transmissible yeast with resistance to different antifungal compounds. Outbreaks of C. auris are mostly observed in intensive care units. To take adequate measures during an outbreak, it is essential to understand the transmission route, which requires isolate genotyping. In 2019, a short tandem repeat (STR) genotyping analysis was developed for C. auris. To determine the discriminatory power of this method, we performed STR analysis of 171 isolates with known whole-genome sequencing (WGS) data using Illumina reads, and we compared their resolutions. We found that STR analysis separated the 171 isolates into four clades (clades I to IV), as was also seen with WGS analysis. Then, to improve the separation of isolates in clade IV, the STR assay was optimized by the addition of 2 STR markers. With this improved STR assay, a total of 32 different genotypes were identified, while all isolates with differences of >50 single-nucleotide polymorphisms (SNPs) were separated by at least 1 STR marker. Altogether, we optimized and validated the C. auris STR panel for clades I to IV and established its discriminatory power, compared to WGS SNP analysis using Illumina reads. IMPORTANCE The emerging fungal pathogen Candida auris poses a threat to public health, mainly causing outbreaks in intensive care units. Genotyping is essential for investigating potential outbreaks and preventing further spread. Previously, we developed a STR genotyping scheme for rapid and high-resolution genotyping, and WGS SNP outcomes for some isolates were compared to STR data. Here, we compared WGS SNP and STR outcomes for a larger sample cohort. Also, we optimized the resolution of this typing scheme with the addition of 2 STR markers. Altogether, we validated and optimized this rapid, reliable, and high-resolution typing scheme for C. auris.

Workers in the oil and gas industry are at risk for exposure to a number of physical and chemical hazards at the workplace. Chemical hazard risks include inhalation of crude oil or its volatile components. While several studies have investigated the neurotoxic effects of volatile hydrocarbons, in general, there is a paucity of studies assessing the neurotoxicity of crude oil vapor (COV). Consequent to the 2010 Deepwater Horizon (DWH) oil spill, there is growing concern about the short- and long-term health effects of exposure to COV. NIOSH surveys suggested that the DWH oil spill cleanup workers experienced neurological symptoms, including depression and mood disorders, but the health effects apart from oil dispersants were difficult to discern. To investigate the potential neurological risks of COV, male Sprague-Dawley rats were exposed by whole-body inhalation to COV (300ppm; Macondo surrogate crude oil) following an acute (6h/d1 d) or sub-chronic (6h/d4 d/wk.4 wks) exposure regimen. At 1, 28 or 90 d post-exposure, norepinephrine (NE), epinephrine (EPI), dopamine (DA) and serotonin (5-HT) were evaluated as neurotransmitter imbalances are associated with psychosocial-, motor- and cognitive- disorders. Sub-chronic COV exposure caused significant reductions in NE, EPI and DA in the dopaminergic brain regions, striatum (STR) and midbrain (MB), and a large increase in 5-HT in the STR. Further, sub-chronic exposure to COV caused upregulation of synaptic and Parkinson's disease-related proteins in the STR and MB. Whether such effects will lead to neurodegenerative outcomes remain to be investigated.

BACKGROUND: The cross-contamination of cell lines in culture is a persistent problem. Genetically modified L20B (Mouse) and RD (Human Rhabdomyosarcoma) cell lines are commonly used in poliovirus research, surveillance, and diagnostics. Cross-contamination between these cell lines leads to unreproducible results and unreliable surveillance data, negatively affecting public health. The gold standard method for cell authentication is Short Tandem Repeats analysis, which is time-consuming and expensive. The disadvantage of STR is limited detection of interspecies contamination. METHODS: This assay targets the mitochondrial cytochrome c oxidase subunit I (MTCO1) gene, a highly conserved and emergent DNA barcode region for detection of cross-contamination in RD and L20B cell lines. The MagNA Pure Compact instrument and ABI 7500 Fast Dx Real-time PCR systems were used for DNA extraction and to perform real-time PCR respectively. RESULTS: The newly developed assay is very sensitive with a limit of detection of 100 RD cells/1 million L20B/mL. The amplification efficiency and R(2)-value were 102.26% and 0.9969 respectively. We evaluated specificity of the assay with five human and four mouse cell lines, as well as monkey and rat cell lines. The assay showed no cross-reactivity with genomic DNA from human, mouse, rat, or monkey cell lines. The analytical sensitivity was also evaluated by spiking varying amounts of RD cells (0.001% - 10%) into L20B cells. There was no difference in C(T) values when running single-plex or duplex PCR reactions with similar experimental conditions. CONCLUSIONS: We have developed and validated a TaqMan real-time PCR assay, a sensitive method for the detection of cross-contamination of RD and L20B cell lines.

In this study we compared nine Shiga toxin (Stx)-producing Escherichia coli O157:H7 patient isolates for Stx levels, stx-phage insertion site(s), and pathogenicity in a streptomycin (Str)-treated mouse model. The strains encoded stx 2a, stx 1a and stx 2a, or stx 2a and stx 2c. All of the strains elaborated 10(5)-10(6) cytotoxic doses 50% (CD50) into the supernatant after growth in vitro as measured on Vero cells, and showed variable levels of increased toxin production after growth with sub-inhibitory levels of ciprofloxacin (Cip). The stx 2a+stx 2c+ isolates were 90-100% lethal for Str-treated BALB/c mice, though one isolate, JH2013, had a delayed time-to-death. The stx 2a+ isolate was avirulent. Both an stx 2a and a recA deletion mutant of one of the stx 2a+stx 2c+ strains, JH2010, exhibited at least a three-log decrease in cytotoxicity in vitro and both were avirulent in the mice. Stool from Str-treated mice infected with the highly virulent isolates were 10- to 100-fold more cytotoxic than feces from mice infected with the clinical isolate, JH2012, that made only Stx2a. Taken together these findings demonstrate that the stx 2a-phage from JH2010 induces to higher levels in vivo than does the phage from JH2012. The stx 1a+stx 2a+ clinical isolates were avirulent and neutralization of Stx1 in stool from mice infected with those strains indicated that the toxin produced in vivo was primarily Stx1a. Treatment of mice infected with Stx1a+Stx2a+ isolates with Cip resulted in an increase in Stx2a production in vivo and lethality in the mice. Our data suggest that high levels of Stx2a in stool are predictive of virulence in mice.

Nipah virus is a highly lethal zoonotic pathogen found in Southeast Asia that has caused human encephalitis outbreaks with 40%-70% mortality. NiV encodes its own RNA-dependent RNA polymerase within the large protein, L. Efficient polymerase activity requires the phosphoprotein, P, which tethers L to its template, the viral nucleocapsid. P is a multifunctional protein with modular domains. The central P multimerization domain is composed of a long, tetrameric coiled coil. We investigated the importance of structural features found in this domain for polymerase function using a newly constructed NiV bicistronic minigenome assay. We identified a conserved basic patch and central kink in the coiled coil that are important for polymerase function, with R555 being absolutely essential. This basic patch and central kink are conserved in the related human pathogens measles and mumps viruses, suggesting that this mechanism may be conserved.

Many veterans of the 1991 Persian Gulf War (GW) returned with a chronic multisymptom illness that has been termed Gulf War Illness (GWI). Previous GWI studies have suggested that exposure to acetylcholinesterase inhibitors (AChEIs) in theater, such as sarin and/or pesticides, may have contributed to the symptomatology of GWI. Additionally, concomitant high physiological stress experienced during the war may have contributed to the initiation of the GWI phenotype. While inhibition of AChE leading to accumulation of acetylcholine (ACh) will activate the cholinergic anti-inflammatory pathway, the signature symptomatology of GWI has been shown to be associated with neuroinflammation. To investigate the relationship between ACh and neuroinflammation in discrete brain regions, we used our previously established mouse model of GWI, which combines an exposure to a high physiological stress mimic, corticosterone (CORT), with GW-relevant AChEIs. The AChEIs used in this study were diisopropyl fluorophosphate (DFP), chlorpyrifos oxon (CPO), and physostigmine (PHY). After AChEI exposure, ACh concentrations for cortex (CTX), hippocampus (HIP), and striatum (STR) were determined using hydrophilic interaction liquid chromatography (HILIC) with ultra-performance liquid chromatography (UPLC)-tandem-mass spectrometry (MS/MS). CORT pretreatment ameliorated the DFP-induced ACh increase in HIP and STR, but not CTX. CORT pretreatment did not significantly alter ACh levels for CPO and PHY. Further analysis of STR neuroinflammatory biomarkers revealed an exacerbated CORT+AChEI response, which does not correspond to measured brain ACh. By utilizing this new analytical method for discrete brain region analysis of ACh, this work suggests the exacerbated neuroinflammatory effects in our mouse model of GWI are not driven by the accumulation of brain region-specific ACh.

BACKGROUND: Crimean-Congo hemorrhagic fever (CCHF) is the most geographically widespread tick-borne viral infection. Outbreaks of CCHF in sub-Saharan Africa are largely undetected and thus under-reported. On November 9, 2015, the National Viral Hemorrhagic Fever Laboratory at the Uganda Virus Research Institute received an alert for a suspect VHF case in a 33-year-old male who presented with VHF compatible signs and symptoms at Mengo Hospital in Kampala. METHODS: A blood sample from the suspect patient was tested by RT-PCR for CCHF and found positive. Serological testing on sequential blood specimens collected from this patient showed increasing anti-CCHFV IgM antibody titers, confirming recent infection. Repeat sampling of the confirmed case post recovery showed high titers for anti-CCHFV-specific IgG. An epidemiological outbreak investigation was initiated following the initial RT-PCR positive detection to identify any additional suspect cases. RESULTS: Only a single acute case of CCHF was detected from this outbreak. No additional acute CCHF cases were identified following field investigations. Environmental investigations collected 53 tick samples, with only 1, a Boophilus decoloratus, having detectable CCHFV RNA by RT-PCR. Full-length genomic sequencing on a viral isolate from the index human case showed the virus to be related to the DRC (Africa 2) lineage. CONCLUSIONS: This is the fourth confirmed CCHF outbreak in Uganda within 2 years after more than 50 years of no reported human CCHF cases in this country. Our investigations reaffirm the endemicity of CCHFV in Uganda, and shows that exposure to ticks poses a significant risk for human infection. These findings also reflect the importance of having an established national VHF surveillance system and diagnostic capacity in a developing country like Uganda, in order to identify the first cases of VHF outbreaks and rapidly respond to reduce secondary cases. Additional efforts should focus on implementing effective tick control methods and investigating the circulation of CCHFV throughout the country.

Legionella pneumophila was first recognized as a cause of severe and potentially fatal pneumonia during a large-scale outbreak of Legionnaires' disease (LD) at a Pennsylvania veterans' convention in Philadelphia, 1976. The ensuing investigation and recovery of four clinical isolates launched the fields of Legionella epidemiology and scientific research. Only one of the original isolates, "Philadelphia-1", has been widely distributed or extensively studied. Here we describe the whole-genome sequencing (WGS), complete assembly, and comparative analysis of all Philadelphia LD strains recovered from that investigation, along with L. pneumophila isolates sharing the Philadelphia sequence type (ST36). Analyses revealed that the 1976 outbreak was due to multiple serogroup 1 strains within the same genetic lineage, differentiated by an actively mobilized, self-replicating episome that is shared with L. pneumophila str. Paris, and two large, horizontally-transferred genomic loci, among other polymorphisms. We also found a completely unassociated ST36 strain that displayed remarkable genetic similarity to the historical Philadelphia isolates. This similar strain implies the presence of a potential clonal population, and suggests important implications may exist for considering epidemiological context when interpreting phylogenetic relationships among outbreak-associated isolates. Additional extensive archival research identified the Philadelphia isolate associated with a non-Legionnaire case of "Broad Street pneumonia", and provided new historical and genetic insights into the 1976 epidemic. This retrospective analysis has underscored the utility of fully-assembled WGS data for Legionella outbreak investigations, highlighting the increased resolution that comes from long-read sequencing and a sequence type-matched genomic data set.

Occupational exposure to welding fumes (WF) is thought to cause Parkinson's disease (PD)-like neurological dysfunction. An apprehension that WF may accelerate the onset of PD also exists. Identifying reliable biomarkers of exposure and neurotoxicity are therefore critical for biomonitoring and neurological risk characterization of WF exposure. Manganese (Mn) in welding consumables is considered the causative factor for the neurological deficits seen in welders. Hence, we sought to determine if Mn accumulation in blood or nail clippings can be a marker for adverse exposure and neurotoxicity. To model this, rats were exposed by intratracheal instillation to dissolved or suspended fume components collected from gas metal arc-mild steel (GMA-MS) or manual metal arc-hard surfacing (MMA-HS) welding. Trace element analysis revealed selective Mn accumulation in dopaminergic brain areas, striatum (STR) and midbrain (MB), following exposure to the two fumes. This caused dopaminergic abnormality as evidenced by loss of striatal tyrosine hydroxylase (Th; 25-32% decrease) and Parkinson disease (autosomal recessive, early onset) 7 (Park7; 25-46% decrease) proteins. While blood Mn was not detectable, Mn levels in nails strongly correlated with the pattern of Mn accumulation in the striatum (R(2)=0.9386) and midbrain (R(2)=0.9332). Exposure to manganese chloride (MnCl(2)) caused similar Mn accumulation in STR, MB and nail. Our findings suggest that nail Mn has the potential to be a sensitive and reliable biomarker for long-term Mn exposure and associated neurotoxicity. The non-invasive means by which nail clippings can be collected, stored, and transported with relative ease, make it an attractive surrogate for biomonitoring WF exposures in occupational settings.

BACKGROUND: Buccal cell collection is a convenient DNA collection method; however, little attention has been given to the quality of DNA obtained from pediatric populations. The purpose of this study was to determine the effect of a modified cytobrush collection method on the yield and quality of infant buccal DNA collected as part of a population-based case-control study of birth defects. METHODS: Cytobrushes were collected from infants, mothers, and fathers using a standard collection method in 1997 to 2003 and a modified protocol that allows air-drying of the cytobrushes after collection from 2003 to the present. Yield and quality of DNA from 1057 cytobrushes was assessed by quantitative PCR and short tandem repeat (STR) genotyping, respectively. RESULTS: Air-dried cytobrushes from infants had higher median DNA yields (1300 ng) and STR completion rates (99.5%) than standard collection method cytobrushes (60 ng and 59.5%, respectively). A subset of DNA aliquots was genotyped for six single nucleotide polymorphisms (SNPs). Aliquots from both collection methods that passed the quality protocol (DNA concentration >1 ng/mul, and successful amplification of ≥1 STR) had high genotype completion rates (99-100%). The median DNA yield following whole genome amplification was more than twofold higher for air-dried than standard collection specimens (p < 0.001). CONCLUSION: Yield and quality of buccal DNA collected from infants are improved by using a method that incorporates air-drying; however, DNA collected by both methods is suitable for genotyping if stringent quality control procedures are instituted. These findings may be helpful for future epidemiologic studies of birth defects and other adverse pediatric outcomes. Birth Defects Research (Part A) 2011. (c) 2011 Wiley-Liss, Inc.

Short tandem repeat (STR) analysis has emerged as the method of choice for testing to resolve specimen source contamination and identity problems that arise in surgical pathology. We studied a series of consecutive cases referred for STR typing during a 5-year period to document the usefulness of the approach and to describe the broadening scope of testing. The series demonstrates that STR-based typing can be applied in virtually any setting in which specimen source confirmation is requested, that STR-based typing is informative in 92% of cases, but that exceptions occasionally arise that complicate test interpretation. The series also demonstrates that in addition to traditional uses of STR typing, testing is now performed in the absence of any direct indication that a specimen mix-up or contamination may have occurred, namely, when the pathologic findings are unexpected or the clinical setting is atypical. The case series underscores the ability of STR testing to detect errors that cannot be captured by current laboratory protocols, a finding that has important implications for patient safety.