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Background AZD7442—a combination of 2 human, extended–half-life, SARS-CoV-2–neutralizing monoclonal antibodies (mAbs) (tixagevimab/cilgavimab)—has received US Food and Drug Administration emergency use authorization for COVID-19 prevention in immunocompromised individuals. We evaluated the effect of AZD7442 in prevention and treatment settings in Syrian hamsters challenged with SARS-CoV-2. Methods Hamsters received intraperitoneal isotype control mAb (2 mg) or AZD7442 (0.002–2 mg) 1 day before intranasal (IN) SARS-CoV-2 challenge (USA-WA1/2020; 1x105 plaque-forming units) in prevention; OR control mAb (5 mg) or AZD7442 (0.5–5 mg) 1 day after IN SARS-CoV-2 challenge in treatment. The impact of AZD7442 on lung viral RNA and pathology and AZD7442 serum levels was assessed on Days 3 and 7 post infection. Body weight was recorded daily through Day 7. Results With AZD7442 prevention, lower lung viral loads were observed compared to controls; at Day 3 post infection, lowest infectious virus titer and viral subgenomic mRNA (sgmRNA) levels were seen with doses ≥0.2 mg AZD7442. Concomitantly, increased serum levels of AZD7442 were observed. By Day 7, infectious virus titer and sgmRNA fell below the level of detection (LOD) at all doses tested. Moreover, AZD7442 at doses ≥0.2 mg protected hamsters from weight loss versus controls. Lung pathology scores (scale: 0 [normal] to 25 [most severe]) were generally dose dependent, with mean scores of < 2 for AZD7442 versus 10 for controls, indicating less SARS-CoV-2–induced inflammation and alveolar damage in hamsters given AZD7442. Lower AZD7442 doses were associated with mean pathology scores similar to controls. With AZD7442 treatment, infectious virus titers were below the LOD at Day 3 post infection and at Day 7 for sgmRNA, for all doses tested. Mean lung pathology score was Conclusion In a SARS-CoV-2 challenge model, AZD7442 administered as prevention or treatment led to significantly lower lung viral loads and improved lung pathology, without weight loss. There was also no evidence that AZD7442 mediated antibody-dependent enhancement of disease or infection. Disclosures Yueh-Ming Loo, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Patrick M. McTamney, PhD, AstraZeneca: Employee Richard Roque, B.S., AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Yingyun Cai, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds Mark T. Esser, PhD, AstraZeneca: Employee|AstraZeneca: Stocks/Bonds.

INTRODUCTION: Inactivation of biological agents and particularly select agents has come under increased scrutiny since the US Army inadvertently shipped live anthrax both inside and outside the US, leading to more stringent regulations regarding inactivation. METHODS: Formalin and Trizol® LS were used to inactivate virus samples in complex matrices. Cytotoxic chemicals were removed using either desalting or concentrating columns or through dilution using HYPERFlasks. Efficacy of inactivation was evaluated either through plaque assay or immunofluorescence assay. RESULTS: All virus samples and tissue specimens were successfully inactivated using either formalin or Trizol® LS. Both the desalting columns and concentrating columns were able to remove cytotoxic chemicals to facilitate viral amplification in controls. Dilution of cytotoxic chemicals through HYPERFlasks was also successful provided that media was changed completely within 48 hours of first cell passage. DISCUSSION: All inactivation testing demonstrates that both formalin and Trizol® LS successfully inactivate virus-infected cell lines and tissues, which is consistent with previously published literature. Each sample cleanup method has its benefits and pitfalls. Desalting columns can process the largest sample size but are also susceptible to plugging and degradation, whereas concentrating columns are not as vulnerable but can only process 5% of the sample load per run. CONCLUSION: Based on our results along with those of our colleagues, it is recommended that the regulatory authorities re-evaluate the requirements for each entity to validate well-established inactivation methods in house because there would be limited benefits despite the considerable resources required for this effort.

Airborne transmission is predicted to be a prevalent route of human exposure with SARS-CoV-2. Aside from African green monkeys, nonhuman primate models that replicate airborne transmission of SARS-CoV-2 have not been investigated. A comparative evaluation of COVID-19 in African green monkeys, rhesus macaques, and cynomolgus macaques following airborne exposure to SARS-CoV-2 was performed to determine critical disease parameters associated with disease progression, and establish correlations between primate and human COVID-19. Respiratory abnormalities and viral shedding were noted for all animals, indicating successful infection. Cynomolgus macaques developed fever, and thrombocytopenia was measured for African green monkeys and rhesus macaques. Type II pneumocyte hyperplasia and alveolar fibrosis were more frequently observed in lung tissue from cynomolgus macaques and African green monkeys. The data indicate that, in addition to African green monkeys, macaques can be successfully infected by airborne SARS-CoV-2, providing viable macaque natural transmission models for medical countermeasure evaluation.

Zika virus (ZIKV) is a mosquito-borne member of the Spondweni complex of the genus Flavivirus, family Flaviviridae. The genus Flavivirus comprises many important human pathogens such as Saint Louis encephalitis, yellow fever virus, dengue virus (DENV1–DENV4), West Nile virus, Japanese encephalitis virus, and tick-borne encephalitis virus.1 The ZIKV genome comprises a single-strand, positive-sense RNA of approximately 11 kb in length. The genome consists of a 5′ untranslated region (UTR) (∼107 nt), an open reading frame (ORF) ∼10.2 kb in length, and a 3′ UTR (∼428 nt). The single ORF encodes three structural proteins: capsid (C), premembrane/membrane (prM), envelope (E), and seven non–structural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5).1 Similar to other flaviviruses, ZIKV enters cells through receptor-mediated endocytosis, and the low endosomal pH induces fusion to release the genomic RNA into the cytoplasm. The genomic RNA is translated into a single polyprotein that is processed to facilitate replication, virion assembly, and release of mature virions. The mature ZIKV virion structure is similar to that of other flaviviruses, comprising a nucleocapsid core consisting of C protein and genomic RNA and an icosahedral shell consisting of 180 copies of E and M proteins (or prM) embedded in a host-derived lipid bilayer.2 ZIKV was first isolated from the blood of a sentinel rhesus macaque in 1947 in the Zika Forest in Uganda.3 In the following year, ZIKV was isolated from pools of Aedes africanus mosquitoes, and subsequent studies demonstrated virus transmission by Aedes aegypti to both mice and rhesus macaques.4 In the following decades (1950s–1980s), ZIKV was isolated from humans and multiple mosquito species in sub-Saharan Africa and Southeast Asia, and suggesting maintenance in both sylvatic (primarily Aedes spp. mosquitoes and nonhuman primates [NHPs]) and urban cycles (mainly A. aegypti and humans).5–19 ZIKV consists of a single serotype, with isolates comprising two geographic lineages (African and Asian) that have caused sporadic or underreported human outbreaks.20,21 However, starting in 2007, outbreaks were reported in the Yap island in the Federated States of Micronesia and in Gabon, and by 2013 ZIKV was imported from French Polynesia into northeast Brazil resulting in the largest reported epidemic to date and its subsequent expansion to many parts of Americas including mainland USA (Florida and Texas).22–27 Since the start of the 2007 epidemic, ZIKV has spread to or been detected in 84 countries and territories in the Americas, Asia, and Africa, with reported evidence of autochthonous transmission (http://apps.who.int/iris/bitstream/10665/254714/1/zikasitrep10Mar17-eng.pdf?ua=1). Most human ZIKV infections are asymptomatic.28,29 Symptomatic infections typically present as a self-limiting acute febrile illness with symptoms ranging from fever, headache, myalgia, and rash; however, severe clinical manifestations including congenital microcephaly and other fetal defects, Guillain–Barre syndrome (GBS), and death have been reported.30,31 Although ZIKV is transmitted primarily through mosquito/human cycle, the virus can also be transmitted through sexual contact and blood transfusions.32–34 Before its arrival in the Americas, ZIKV was an obscure and understudied pathogen; as a consequence, few virus isolates were accessible for research and vaccine/therapeutic development. In addition, little or no basic in vitro and/or in vivo characterization data were available on accessible isolates. In the present study, we investigated the potential phenotypic differences between low-passage African (ArD 41525) and Asian isolates (CPC-0740 and SV0127-14) in vitro and in vivo.

In 2011, the Association of Analytical Communities (AOAC) International released a list of Bacillus strains relevant to biothreat molecular detection assays. We present the complete and annotated genome assemblies for the 15 strains listed on the inclusivity panel, as well as the 20 strains listed on the exclusivity panel.

Research involving biosafety level 3 pathogens such as West Nile virus (WNV) is often limited by the limited space and technical constraints of these environments. To conduct complex analytical studies outside of high containment, robust and reliable inactivation methods are needed that maintain compatibility with downstream assays. Here we report the inactivation of WNV in spiked serum samples using a commercially available SDS-PAGE sample buffer for proteomic studies. Using this method, we demonstrate its utility by identification proteins differentially expressed in the serum of mice experimentally infected with WNV.

Diagnostics for biothreat agents require sample shipment to reference labs for diagnosis of disease; however high/fluctuating temperatures during sample transport negatively affect sample quality and results. Vitrification additives preserve sample integrity for molecular-based assay diagnostics in the absence of refrigeration by imparting whole molecule stability to a plethora of environmental insults. Therefore, we have evaluated commercially available vitrification matrices' (Biomatrica's CloneStable® and RNAStable®) ability to stabilize samples of Yersinia pestis and Venezuelan Equine Encephalitis Virus. When heated to 95°C in RNAStable®, Y. pestis had a 13-fold improvement in detection via real-time PCR compared to heated samples in buffer. VEEV, in RNAStable® at 55°C, had a ~10-fold improved detection versus heated samples in buffer. CloneStable® also preserved Y. pestis antigens for 7days after exposure to cycling temperatures. Overall, RNAStable® and CloneStable® respectively offered superior stabilization to nucleic acids and proteins in response to temperature fluctuations.

Animal models are needed to better understand the pathogenic mechanisms of Zika virus (ZIKV) and to evaluate candidate medical countermeasures. Adult mice infected with ZIKV develop a transient viremia, but do not demonstrate signs of morbidity or mortality. Mice deficient in type I or a combination of type I and type II interferon (IFN) responses are highly susceptible to ZIKV infection; however, the absence of a competent immune system limits their usefulness for studying medical countermeasures. Here we employ a murine model for ZIKV using wild-type C57BL/6 mice treated with an antibody to disrupt type I IFN signaling to study ZIKV pathogenesis. We observed 40% mortality in antibody treated mice exposed to ZIKV subcutaneously whereas mice exposed by intraperitoneal inoculation were highly susceptible incurring 100% mortality. Mice infected by both exposure routes experienced weight loss, high viremia, and severe neuropathologic changes. The most significant histopathological findings occurred in the central nervous system where lesions represent an acute to subacute encephalitis/encephalomyelitis that is characterized by neuronal death, astrogliosis, microgliosis, scattered necrotic cellular debris, and inflammatory cell infiltrates. This model of ZIKV pathogenesis will be valuable for evaluating medical countermeasures and the pathogenic mechanisms of ZIKV because it allows immune responses to be elicited in immunologically competent mice with IFN I blockade only induced at the time of infection., Author Summary Research addressing the severe clinical complications associated with ZIKV infection, including GBS and congenital ZIKV syndrome, are urgently needed. Key to this effort is development of well-characterized animal models that recapitulate human disease. Adult wild-type mice infected with ZIKV can develop viremia in some instances, but they do not emulate the disease associated with the severe congenital and adult neuropathology. Several groups have recently described type I or type II IFN-deficient murine models that are permissive for viral replication in several organs including the brain. The major limitation of these models is they utilize immunodeficient knockout mice lacking key components of the innate antiviral response. We describe the use of a lethal murine model for ZIKV where the innate response of immunocompetent mice is suppressed only at the time of infection. We show that the mice develop severe neurological disease similar to that previously demonstrated in mice deficient in the type I or II IFN response. Using this model, we provide a detailed description of the ZIKV-associated pathologic changes, which mirrors the neuropathogenic properties of ZIKV in humans. These studies provide a baseline for assessment of medical countermeasures that can prevent or treat such pathogenic effects caused by ZIKV infection.

The genus Burkholderia encompasses both pathogenic (including Burkholderia mallei and Burkholderia pseudomallei, U.S. Centers for Disease Control and Prevention Category B listed), and nonpathogenic Gram-negative bacilli. Here we present full genome sequences for a panel of 59 Burkholderia strains, selected to aid in detection assay development.

Background Research with high biocontainment pathogens such as Rift Valley fever virus (RVFV) and Lassa virus (LASV) is expensive, potentially hazardous, and limited to select institutions. Surrogate pathogens such as Punta Toro virus (PTV) for RVFV infection and Pichinde virus (PICV) for LASV infection allow research to be performed under more permissive BSL-2 conditions. Although used as infection models, PTV and PICV have no standard real-time RT-qPCR assays to detect and quantify pathogenesis. PTV is also a human pathogen, making a standardized detection assay essential for biosurveillance. Here, we developed and characterized two real-time RT-qPCR assays for PICV and PTV by optimizing assay conditions and measuring the limit of detection (LOD) and performance in multiple clinical matrices. Methods Total nucleic acid from virus-infected Vero E6 cells was used to optimize TaqMan-minor groove binder (MGB) real-time RT-qPCR assays. A 10-fold dilution series of nucleic acid was used to perform analytical experiments with 60 replicates used to confirm assay LODs. Serum and whole blood spiked with 10-fold dilutions of PTV and PICV virus were assessed as matrices in a mock clinical context. The Cq, or cycle at which the fluoresce of each sample first crosses a threshold line, was determined using the second derivative method using Roche LightCycler 480 software version 1.5.1. Digital droplet PCR (ddPCR) was utilized to quantitatively determine RNA target counts/μl for PTV and PICV. Results Optimized PTV and PICV assays had LODs of 1000 PFU/ml and 100 PFU/ml, respectively, and this LOD was confirmed in 60/60 (PTV) and 58/60 (PICV) positive replicates. Preliminary mock clinical LODs remained consistent in serum and whole blood for PTV and PICV at 1000 PFU/ml and 100 PFU/ml. An exclusivity panel showed no cross reaction with near neighbors. Conclusions PTV and PICV Taq-man MGB based real-time RT-qPCR assays developed here showed relevant sensitivity and reproducibility in samples extracted from a variety of clinical matrices. These assays will be useful as a standard by researchers for future experiments utilizing PTV and PICV as infection models, offering the ability to track infection and viral replication kinetics during research studies. Electronic supplementary material The online version of this article (doi:10.1186/s12985-016-0509-3) contains supplementary material, which is available to authorized users.

Magnetic beads offer superior impurity removal and nucleic acid selection over older extraction methods. The performances of nucleic acid extraction of biothreat agents in blood or buffer by easyMAG, MagNA Pure, EZ1 Advanced XL, and Nordiag Arrow were evaluated. All instruments showed excellent performance in blood; however, the easyMAG had the best precision and versatility.

The relationship of mucoviscosity-associated (magA) and/or regulator of mucoid phenotype (rmpA) genes to the Klebsiella pneumoniae hypermucoviscosity (HMV) phenotype has been reported. We previously demonstrated that rmpA+ K. pneumoniae can cause serious disease in African green monkeys and isolated rmpA+ and magA+ HMV K. pneumoniae from other species of non-human primates. To rapidly screen African green monkeys/non-human primates for these infections, we developed three real-time PCR assays. The first was K. pneumoniae-specific, targeting the khe gene, while the others targeted rmpA and magA. Primer Express 2 was used with the three K. pneumoniae genes to generate sequence-specific TaqMan/TaqMan-Minor Groove Binder assays. Oral/rectal swabs and necropsy samples were collected; swabs were used for routine culture and DNA extraction. K. pneumoniae colonies were identified on the Vitek 2 with DNA tested using the K. pneumoniae-specific assays. Testing of 45 African green monkeys resulted in 19 khe+ samples from 14 animals with none positive for either rmpA or magA. Of these 19 khe+ samples, five were culture-positive, but none were HMV "string test"-positive. Subsequent testing of 307 non-human primates resulted in 64 HMV K. pneumoniae isolates of which 42 were rmpA+ and 15 were magA+. Non-human primate testing at the U.S. Army Medical Research Institute of Infectious Diseases demonstrated the ability to screen both live and necropsied animals for K. pneumoniae by culture and real-time PCR to determine HMV genotype.

Burkholderia mallei , the etiologic agent of glanders, is a Gram-negative, nonmotile, facultative intracellular pathogen. Although glanders has been eradicated from many parts of the world, the threat of B. mallei being used as a weapon is very real. Here we present draft genome assemblies of 8 Burkholderia mallei strains that were isolated in Turkey.

Here, we sequenced the completed genome of Yersinia pestis EV76D and KIM 10v, two genomes used as references in assay development, to improved high-quality draft status.

In 2011, the Association of Analytical Communities (AOAC) International released a list of Bacillus strains relevant to biothreat molecular detection assays. We present the complete and annotated genome assemblies for the 15 strains listed on the inclusivity panel, as well as the 20 strains listed on the exclusivity panel.

Francisella tularensis is a highly infectious bacterium with the potential to cause high fatality rates if infections are untreated. To aid in the development of rapid and accurate detection assays, we have sequenced and annotated the genomes of 18 F. tularensis and Francisella philomiragia strains.

The genus Burkholderia encompasses both pathogenic (including Burkholderia mallei and Burkholderia pseudomallei , U.S. Centers for Disease Control and Prevention Category B listed), and nonpathogenic Gram-negative bacilli. Here we present full genome sequences for a panel of 59 Burkholderia strains, selected to aid in detection assay development.

The genus Yersinia includes three human pathogens, of which Yersinia pestis is responsible for >2,000 illnesses each year. To aid in the development of detection assays and aid further phylogenetic elucidation, we sequenced and assembled the complete genomes of 32 strains (across 9 Yersinia species).

Introduction. Live vaccine strain (LVS) Francisella tularensis is a live, attenuated investigational tularemia vaccine that has been used by the US Army for decades to protect laboratory workers. Postvaccination bacterial kinetic characteristics of LVS at the inoculation site and in the blood are unknown and, therefore, were assessed in a prospective study. LVS vaccination of laboratory workers provided the opportunity to compare culture with polymerase chain reaction (PCR) for the detection of F. tularensis in human clinical samples. Methods. Blood and skin swab samples were prospectively collected from volunteers who received the LVS tularemia vaccine at baseline (negative controls) and at 5 specified time points (days 1, 2, 7 or 8, 14 or 15, and 35 after vaccination). Bacterial culture and PCR of whole blood samples (17 volunteers) and inoculation site swabs (41 volunteers) were performed. Results. The culture and PCR results of all blood samples were negative. Results of real-time PCR from the inoculation site samples were positive for 41 (100%) of 41 volunteers on day 1, for 40 (97.6%) of 41 volunteers on day 2, for 24 (58.5%) of 41 on day 7 or 8, for 6 (16.7%) of 36 on day 14 or 15, and for 0 (0%) of 9 on day 35. Positive results of bacterial cultures of the inoculation site samples occurred significantly less frequently, compared with PCR testing, with 4 (9.8%) of 41 volunteers having positive results on day 1 ( ) and 4 P ! .001 (9.8%) of 41 on day 2 ( ); all results from subsequent days were negative. P ! .001 Conclusions. F. tularensis LVS genomic DNA was detected in the majority of samples from the inoculation site up to 1 week after LVS vaccination, with real-time PCR being more sensitive than culture. Our data suggest that bacteremia does not occur after LVS vaccination in normal, healthy human volunteers.

Efficient, rapid, and reproducible procedures for isolating high-quality DNA before PCR gene amplification are essential for the diagnostic and molecular identification of pathogenic bacteria. This study evaluated the Qiagen QIAamp DNA Mini Kit and the Schleicher and Schuell IsoCode Stix DNA isolation device for isolating nucleic acid. Buffer, serum, and whole-blood samples were spiked with Bacillus anthracis Sterne vegetative cells and Yersinia pestis , while water was spiked with B. anthracis Sterne spores. Although minimal variations in limit of detection occurred among matrices, both the IsoCode Stix extraction method and the Qiagen procedure have comparable detection limits.

Burkholderia is a genus of betaproteobacteria that includes three notable human pathogens: B. cepacia , B. pseudomallei , and B. mallei . While B. pseudomallei and B. mallei are considered potential biowarfare agents, B. cepacia infections are largely limited to cystic fibrosis patients. Here, we present 56 Burkholderia genomes from 8 distinct species.

Staphylococcus epidermidis causes a large number of catheter-related sepsis infections annually in the United States. We present the 2.54-Mbp complete genome assembly of reference strain S. epidermidis AmMS 205, including a single 37.7-kbp plasmid. The annotated assembly is available in GenBank under accession numbers CP009046 and CP009047.

We present the complete genome assembly of Streptococcus pyogenes ATCC 19615 (Rosenbach) as submitted to GenBank under accession number CP008926. This group A nonmotile β-hemolytic clinical isolate is used for quality control in a variety of commercially available tests. The assembled genome is 1.84 Mb (38.5% G+C content) and contains 1,788 coding regions.

The Delftia acidovorans 2167 (ATCC 15668, Delftia type strain) genome was sequenced into a 6-contig scaffolded assembly of 6.78-Mb. This environmental microbe, previously named to both the Comamonas and Pseudomonas genera, is an opportunistic pathogen and often the subject of phylogenetic placement debates.

We present the scaffolded genome assembly of Pseudomonas aeruginosa Boston 41501, now publicly available in GenBank (JOVK00000000) in 10 contigs placed into a single scaffold. The 6.82-Mbp genome contains 66.1% G+C content and 6,295 coding sequences, including type 4 pilus and type 3 secretion system production genes.

Shigella flexneri causes shigellosis, severe and potentially life-threatening diarrhea, and accounts for 18% of shigellosis cases in the United States. Here, we present the 4.51-Mbp genome assembly of S. flexneri ATCC 12022, a quality control and reference strain, in 10 scaffolds.

Primarily a zoonotic disease, Francisella tularensis is a fastidious intracellular pathogen and is listed as a CDC category A pathogen with notably high pathogenicity. Here we present the scaffolded genome assemblies of nine Francisella strains: eight F. tularensis and one F. philomiragia .

Generally an opportunistic pathogen in the United States, Moraxella catarrhalis has acquired resistance to multiple antibacterial/antimicrobial agents. Here, we present the complete 1.9-Mb genome of M. catarrhalis strain ATCC 25240, as deposited in NCBI under the accession number CP008804.

Bacilli are genetically and physiologically diverse, ranging from innocuous to highly pathogenic. Here, we present annotated genome assemblies for 20 strains belonging to Bacillus anthracis , B. atrophaeus , B. cereus , B. licheniformis , B. macerans , B. megaterium , B. mycoides , and B. subtilis .

We present the complete genome assembly of Escherichia coli ATCC 25922 as submitted to NCBI under accession no. CP009072. This strain was originally isolated from a clinical sample in Seattle, Washington (1946), and is often used in quality control testing. The assembled genome is 5.20 Mb (50.4% G+C content) and includes two plasmids.

Klebsiella pneumoniae is a common cause of antibiotic-resistant bacterial infections in immunocompromised individuals. Here, we present the 5.54-Mb scaffolded assembly of the type strain K. pneumoniae type strain ATCC 13883, as deposited in GenBank under accession no. JOOW00000000.

Ochrobactrum anthropi is an occasional cause of nosocomial infections; however, interest in the organism lies in its phylogenetic proximity to the genus Brucella . Here, we present the 4.9-Mb finished genome of Ochrobactrum anthropi ATCC 49687, most commonly used as an exclusionary reference organism.

The pleomorphic swarming bacilli of the genus Proteus are common human gut commensal organisms but also the causative agents of recurrent urinary tract infections and bacteremia. We sequenced and assembled the 3.99-Mbp genome of Proteus mirabilis ATCC 7002 (accession no. JOVJ00000000) and the 3.97-Mbp genome of Proteus vulgaris ATCC 49132 (accession no. JPIX00000000), both of which are commonly used reference strains.

The genus Corynebacterium is best known for the pathogen C. diphtheriae ; however, it contains mostly commensal and nonpathogenic, as well as several opportunistic, pathogens. Here, we present the 2.47-Mb scaffolded assembly of the type strain, Corynebacterium sp . ATCC 6931 (NCTC 1914), as deposited into GenBank under accession number CP008913.

A member of the normal human gut microflora, Providencia stuartii is of clinical interest due to its role in nosocomial infections of the urinary tract and because it readily acquires antibiotic resistance. Here, we present the complete genome of P. stuartii strain ATCC 33672, consisting of a 4.28-Mbp chromosome and a 48.9-kbp plasmid.

Soft-tissue infection by Pasteurella multocida in humans is usually associated with a dog- or cat-related injury, and these infections can become aggressive. We sequenced the type strain P. multocida subsp. multocida ATCC 43137 into a single closed chromosome consisting of 2,271,840 bp (40.4% G+C content), which is currently available in the NCBI GenBank under the accession number CP008918.

Yersinia spp. are animal pathogens, some of which cause human disease. We sequenced 10 Yersinia isolates (from six species: Yersinia enterocolitica , Y. fredericksenii , Y. kristensenii , Y. pestis , Y. pseudotuberculosis , and Y. ruckeri ) to high-quality draft or complete status. The genomes range in size from 3.77 to 4.94 Mbp.

Staphylococcus aureus is a major cause of bacterial infections in the United States, with high percentages of serious infections resistant to a variety of β-lactam antibiotics. Here, we present the scaffolded genome assembly into 16 contigs of S. aureus CDC73-57501 (ATCC 29247), a methicillin-resistant quality control strain.

Salmonella enterica constitutes a group of enteric pathogens with a broad host range, including humans, reptiles, and birds. S. enterica subsp. enterica is a common cause of inflammatory diarrhea in humans. We present the draft genome of S. enterica subsp. enterica serovar Enteritidis strain SEJ, including a 59-kbp plasmid.

Enterococcus faecalis is a nonmotile Gram-positive coccus, found both as a commensal organism in healthy humans and animals and as a causative agent of multiple diseases, in particular endocarditis. We sequenced the genome of E. faecalis ATCC 29212, a commonly used reference strain in laboratory studies, to complete “finished” annotated assembly (3 Mb).

We present the scaffolded genome assembly of Neisseria lactamica type strain A7515 (ATCC 23970) as submitted to NCBI under accession no. JOVI00000000. This type strain of the lactose-fermenting Neisseria species is often used in quality control testing and intra-genus phylogenetic analyses. The assembly includes four contigs placed into a single scaffold.

Brucella species are intracellular zoonotic pathogens which cause, among other pathologies, increased rates of abortion in ruminants. Human infections are generally associated with exposure to contaminated and unpasteurized dairy products; however Brucellae have been developed as bioweapons. Here we present 17 complete and 7 scaffolded genome assemblies of Brucella strains.

Two sibling species of tephritid fruit fly, Bactrocera tryoni and Bactrocera neohumeralis, are differentiated by their time of mating, which is genetically determined and requires interactions between the endogenous circadian clock and light intensity. The cryptochrome (cry) gene, a light-sensitive component of the circadian clock, was isolated in the two Bactrocera species. The putative amino acid sequence is identical in the two species. In the brain, in situ hybridization showed that cry is expressed in the lateral and dorsal regions of the central brain where PER immunostaining was also observed and in a peripheral cell cluster of the antennal lobes. Levels of cry mRNA were analyzed in whole head, brain, and antennae. In whole head, cry is abundantly and constantly expressed. However, in brain and antennae the transcript cycles in abundance, with higher levels during the day than at night, and cry transcripts are more abundant in the brain and antennae of B. neohumeralis than in that of B. tryoni. Strikingly, these results are duplicated in hybrid lines, generated by rare mating between B. tryoni and B. neohumeralis and then selected on the basis of mating time, suggesting a role for the cry gene in the mating isolation mechanism that differentiates the species.

Abstract Aims: The Cepheid GeneXpert(R) is a four-site, automated sample preparation and real-time PCR detection system. In this study, the capability of the GeneXpert(R) to isolate and detect nucleic acid from Bacillus anthracis Ames spores was assessed. Methods and Results: A four-plex, dried-down bead cartridge containing PCR reagents specific for the pXO1 and pXO2 plasmids as well as sample processing and inhibition controls was evaluated. For B. anthracis Ames spores harbouring pXO1 and pXO2, samples containing 68 CFU per ml (148 spores per ml) were positive in all four replicates. A limited cross-reactivity panel, which included closely related Bacillus species, was also tested to determine the specificity of the pXO1 and pXO2 assays. No cross-reactivity occurred. Further, B. anthracis Sterne spore samples were analysed to compare results when processed using the GeneXpert(R) to those run directly on the Cepheid SmartCycler(R) without sample processing. The GeneXpert(R) detection capability was three logs lower than the SmartCycler(R) indicating the benefit of incorporating a nucleic acid extraction procedure. Conclusions: This study demonstrates that the GeneXpert(R) is a rapid and reliable system for simultaneously detecting the B. anthracis virulence plasmids pXO1 and pXO2. Significance and Impact of the Study: The GeneXpert(R) is the only platform currently available that is capable of both nucleic acid purification and real-time PCR detection enclosed within a single system. Further, all sample manipulations are automated, thus reducing errors associated with manual processing plasmids pX01 and pX02.

Development of rapid amplification assays for the detection and identification of biological threat agents has become a focus of diagnostic efforts in recent years. The use of real-time PCR assays as diagnostic tools depends upon two critical processes. First, nucleic acid purification must provide template that is both amplifiable and free of PCR inhibitors. Second, the assays themselves must be sensitive and specific for their nucleic acid targets. A differentiation must be made between results achieved due to the lack of target nucleic acid (true negatives) and those produced due to the inability to amplify target DNA (false negatives) so confidence in negative reactions is possible. False negatives can occur when inhibitors are present in the sample being tested, especially if clinical samples such as blood are analyzed. To address the problem of detecting inhibition in purified nucleic acids, an exogenous internal positive control (IPC) based on Taqman chemistry was developed. A previously optimized assay was cloned and the primer and probe sites were mutated to produce novel sequences with no known homology to published sequence data. The IPC was sensitive to a variety of inhibitors, including hemoglobin, heparin, EDTA, humic acids, and fulvic acid. It was also equally sensitive to inhibition when labeled with either 6FAM or ROX dyes. In addition, the IPC was successfully multiplexed with agent specific assays without any loss in their sensitivity. The designed IPC assay has proven to be an effective tool for monitoring inhibitors of PCR and builds confidence in negative results obtained with agent specific assays.

In May of 2011, an enteroaggregative Escherichia coli O104:H4 strain that had acquired a Shiga toxin 2-converting phage caused a large outbreak of bloody diarrhea in Europe which was notable for its high prevalence of hemolytic uremic syndrome cases. Several studies have described the genomic inventory and phylogenies of strains associated with the outbreak and a collection of historical E. coli O104:H4 isolates using draft genome assemblies. We present the complete, closed genome sequences of an isolate from the 2011 outbreak (2011C-3493) and two isolates from cases of bloody diarrhea that occurred in the Republic of Georgia in 2009 (2009EL-2050 and 2009EL-2071). Comparative genome analysis indicates that, while the Georgian strains are the nearest neighbors to the 2011 outbreak isolates sequenced to date, structural and nucleotide-level differences are evident in the Stx2 phage genomes, the mer/tet antibiotic resistance island, and in the prophage and plasmid profiles of the strains, including a previously undescribed plasmid with homology to the pMT virulence plasmid of Yersinia pestis. In addition, multiphenotype analysis showed that 2009EL-2071 possessed higher resistance to polymyxin and membrane-disrupting agents. Finally, we show evidence by electron microscopy of the presence of a common phage morphotype among the European and Georgian strains and a second phage morphotype among the Georgian strains. The presence of at least two stx2 phage genotypes in host genetic backgrounds that may derive from a recent common ancestor of the 2011 outbreak isolates indicates that the emergence of stx2 phage-containing E. coli O104:H4 strains probably occurred more than once, or that the current outbreak isolates may be the result of a recent transfer of a new stx2 phage element into a pre-existing stx2-positive genetic background.