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It has now been two years since the National Institute of Allergy and Infectious Diseases (NIAID) directed BEI Resources to prioritize reagents for the SARS-CoV-2 pandemic response, and BEI Resources remains committed to providing SARS-CoV-2 reagents to the research community. The BEI Resources catalog of SARS-CoV-2 reference and research reagents has expanded from the first cultured United States isolate (USA-WA1/2020; NR-52281) to more than 250 products. This includes isolates representing 13 WHO variants across 28 lineages and related reagents such as genomic RNA, proteins, inactivated virus preparations, proteins, peptide arrays and expression vectors for all 29 SARS-CoV-2 viral proteins. The newly added immunized pooled human sera are important as post-vaccination standard reference materials.¹

Recent additions to the catalog include strains of the Variant of Concern Omicron (BEI Resources NR-56461, NR-56475 and NR-56481), the dominant SARS-CoV-2 variant currently circulating in the United States.^2,3 These isolates originated in the United States and contain characteristic mutations in the Spike protein associated with increased transmission and reduced susceptibility to therapeutic agents or acquired immunity through immune evasion.^2,4

All SARS-CoV-2 variants have been analyzed by Next Generation Sequencing with respect to both the SARS-CoV-2, Wuhan-Hu-1 strain reference sequence and the original reference sequence for that isolate to confirm signature mutations. Certificates of Analysis for each stock produced include observed sequence variations noted in the Appendix section and detailed lot-specific data such as passage history and titer of each lot.

This comprehensive resource of quality-controlled reference and research reagents is made available free-of-charge to registrants supporting the development and testing of SARS-CoV-2 diagnostics, therapeutics and vaccines. For more information on the ordering process and how to register, please refer to our Frequently Answered Questions or contact Customer Care at contact@beiresources.org.

References:

1. Xiang, J., et al. "Establishment of Human Post-Vaccination SARS-CoV-2 Standard Reference Sera." medRXiv (2022). doi: 10.1101/2022.01.24.22269773. Online ahead of print.
2. "SARS-CoV-2 Variant Classifications and Definitions." Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-classifications.html. Accessed 18 February 2022.
3. "Covid Data Tracker." Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, https://covid.cdc.gov/covid-data-tracker/#variant-proportions. Accessed 18 February 2022.
4. "Omicron Variant Report." Outbreak.info, https://outbreak.info/situation-reports/omicron. Accessed 18 February 2022.

Image:  Scanning electron micrograph of a cell infected with SARS-CoV-2 UK B.1.1.7 virus particles (NIAID/CC BY 2.0)

Cell-based assays are valuable tools for SARS-CoV-2 research and offer alternative approaches for working with pathogenic viruses such as SARS-CoV-2 in a BSL-2 environment using recombinant viral systems. BEI Resources offers a variety of human and animal cell lines optimized for use in SARS-CoV-2 research through expression of the viral entry protein human angiotensin-converting enzyme 2 (hACE2) and optimized with the addition of transmembrane protease, serine 2 (TMPRSS2) or furin, which make them highly susceptible to infection.

These cell lines are ideal for a variety of SARS-CoV-2 assays such as the study of viral entry pathways and binding inhibition, evaluation of viral inhibitors, screening of antiviral compounds, determination of antibody titer, pseudotyped lentivirus production, protein detection and purification assays, neutralization assays, use as cell culture controls and more.

Cell lines undergo 21-day broth and agar sterility testing and are confirmed free of Mycoplasma contamination by Hoechst DNA stain, 14-day broth and agar culture and PCR methods. Multiplex PCR amplification of the cytochrome C oxidase I gene is performed to authenticate host cell origin. ACE2 expression is confirmed in the seed material by indirect fluorescent antibody assay.

Please refer to the Product Information Sheet and Certificate of Analysis for recommended thawing, growth and subculturing conditions and use restrictions for each cell line.

References:

1. Cong, Z., et al. "SARS-CoV-2 Spreads through Cell-to-Cell Transmission." bioRxiv (2021): doi: 10.1101/2021.06.01.446579. Online ahead of print. PubMed: 34100011.
2. Chan, S.-W., T. Shafi and R. C. Ford. "Kite-Shaped Molecules Block SARS-CoV-2 Cell Entry at a Post-Attachment Step." Viruses 13 (2021): 2306. PubMed: 34835112.
3. Crawford, K. H. D., et al. "Dynamics of Neutralizing Antibody Titers in the Months After Severe Acute Respiratory Syndrome Coronavirus 2 Infection." J. Infect. Dis. 223 (2021): 197-205. PubMed: 33535236.
4. Xia, Z., et al. "Rational Design of Hybrid SARS-CoV-2 Main Protease Inhibitors Guided by the Superimposed Cocrystal Structures with the Peptidomimetic Inhibitors GC-376, Telaprevir, and Boceprevir." ACS Pharmacol. Transl. Sci. 4 (2021): 1408-1421. PubMed: 34414360.
5. Curreli, F., et al. "Stapled Peptides Based on Human Angiotensin-Converting Enzyme 2 (ACE2) Potently Inhibit SARS-CoV-2 Infection In Vitro." mBio 11 (2020): e02451-20. PubMed: 33310780.
6. Prabhakara, C., et al. "Strategies to Target SARS-CoV-2 Entry and Infection Using Mechanisms of Inhibition by Acidification Inhibitors." PLoS Pathog. 17 (2021): e1009706. PubMed: 34252168.
7. Sievers, B. L., et al. "Antibodies Elicited by SARS-CoV-2 Infection or mRNA Vaccines Have Reduced Neutralizing Activity against Beta and Omicron Pseudoviruses." Sci. Transl. Med. (2022): doi: 10.1126/scitranslmed.abn7842. Online ahead of print. PubMed: 35025672.
8. Neerukonda, S. N., et al. "Establishment of a Well-Characterized SARS-CoV-2 Lentiviral Pseudovirus Neutralization Assay Using 293T Cells with Stable Expression of ACE2 and TMPRSS2." PLoS One 16 (2021): e0248348. PubMed: 33690649.

Image: Scanning electron microscope image of SARS-CoV-2 emerging from the surface of a cell (NIAID/CC BY 2.0)

Trypanosoma cruzi (T. cruzi) is the protozoan parasite responsible for Chagas’ disease, a debilitating illness first identified in 1909 that remains a neglected tropical disease in Central and South America where it is endemic.¹ T. cruzi has been identified through analysis of vector and climate data as a potential emerging health risk to humans in the southern United States, where the most commonly reported wildlife reservoirs are the raccoon and the Virginia opossum.^2,3

Transmission to humans occurs through triatomine insect vectors and can be passed congenitally as well as through blood infusions, organ transplantation and ingestion of contaminated food and water, and often leads to chronic conditions such as myocarditis or meningoencephalitis.^1,2,3

The BEI Resources T. cruzi catalog represents human, animal and vector isolates collected in the United States and throughout South America over a span of nearly 50 years. Many of these isolates are also available as transgenic strains, genomic DNA and monoclonal antibodies.

Recent additions of the virulent human strain Brazil are available in epimastigote and trypomastigote forms of the parasite life cycle both with and without luciferase expression, and genomic DNA.

References:

1. Bern, C. "Chagas’ Disease." N. Engl. J. Med. 373 (2015): 456-466. PubMed: 26222561.
2. Patel, J. M., et al. "Isolation, Mouse Pathogenicity, and Genotyping of Trypanosoma cruzi from an English Cocker Spaniel from Virginia, USA." Vet. Parasitol. 187 (2012): 394-398. PubMed: 22341614.
3. Garza, M., et al. "Projected Future Distributions of Vectors of Trypanosoma cruzi in North America Under Climate Change Scenarios." PLoS Negl. Trop. Dis. 8 (2014): e2818. PubMed: 24831117.

Image: Photomicrograph of four flagellated Trypanosoma cruzi parasites in a blood sample taken in 1968. (CDC)

Diversity panels deposited by the Multidrug-Resistant Organism Repository and Surveillance Network (MRSN) within the Bacterial Diseases Branch of the Walter Reed Army Institute are now available. These panels support the research and development of new antibiotics and tools to treat infections caused the opportunistic pathogens Acinetobacter baumanii (A. baumanii) and Pseudomonas aeruginosa (P. aeruginosa).

The A. baumanii MRSN diversity panel represents 70 different traditional Pasteur scheme multilocus sequence types (MLST) and contains 100 distinct alleles encompassing 32 families of antimicrobial resistance genes.¹

The P. aeruginosa MRSN diversity panel represents 91 different MLSTs and 88 antimicrobial resistance genes resulting in 71 distinct antibiotic susceptibility profiles.²

Each panel consists of 100 isolates assembled from a larger collection of clinical isolates and contains genetically distinct strains from major epidemic clones with phenotypes ranging from pan-susceptible to pan-resistant. Isolates are fully characterized using whole genome sequencing, complete with antimicrobial-resistant gene content and comprehensive susceptibility profile of clinically relevant antibiotics detailed on the individual documentation available for each isolate.

The P. aeruginosa MRSN diversity panel is available as individual isolates (BEI Resources NR-51515 through NR-51614) and a complete panel of all 100 isolates (NR-51829). The 100 isolates in the A. baumanii MRSN Diversity Panel are available as BEI Resources NR-52148 through NR-52247 and the assembled panel as BEI Resources NR-52248.

References:

1. Galac, M. R., et al. "A Diverse Panel of Clinical Acinetobacter baumannii for Research and Development." Antimicrob. Agents Chemother. 21 (2020): e00840-20. PubMed: 32718956.
2. Lebreton, F., et al. "A Panel of Diverse Pseudomonas aeruginosa Clinical Isolates for Research and Development." JAC Antimicrob. Resist. 3 (2021): dlab179. PubMed: 34909689.

Image: Antimicrobial susceptibility testing of an Acinetobacter baumannii MRSN strain. (BEI Resources)

The BEI Resources collection of SARS-CoV-2 materials includes over 40 isolates representing 7 of the 9 variants now classified by the U.S. Centers for Disease Control and Prevention (CDC) as Variants Being Monitored, as well as the Variant of Concern (VOC), Delta (also referred to as B.1.617.2 and AY lineages), which remains the dominant SARS-CoV-2 variant circulating in the United States.^1,2,3

Available Delta variant isolates (NR-55671 and NR-55672) originated in the United States and contain characteristic mutations of the Spike (S) glycoprotein, including T19R, D614G in the N3 loop of the S1 region, L452R and T478K in a key antigenic region of the receptor binding domain (RBD), P681R at the furin cleavage site, D950N in the heptad repeat region of the S2 domain, and two deletions, 156del and 157del in the N-Terminal Domain (NTD).^1,2,3

In addition to isolates, the BEI Resources catalog contains nearly 40 recombinant forms of the SARS-CoV-2 S glycoprotein and RBD, key targets for vaccine development efforts and diagnostic and surveillance measures.⁴  This expansive S protein collection includes recently added Alpha (NR-55277), Beta (NR-55278), Delta (NR-55614) and Theta (NR-55633) variants. The purity, concentration and functional activity are detailed on the individual Certificates of Analysis, along with mutations present in the S glycoprotein as compared to the SARS-CoV-2 reference sequence.

A full, up-to-date listing of all currently available and upcoming SARS-CoV-2 isolates and proteins, as well as genomic RNA, monoclonal antibodies, plasmids, peptide arrays and inactivated virus preparations, is available on the SARS-CoV-2 resource page on the BEI Resources website.

BEI Resources will continue to make available variants as they emerge, with the intent of having each variant well represented. Requests continue to be fast-tracked and prioritized, with an anticipated turnaround window of 12-to-72 hours for new SARS-CoV-2 registrations and 24-to-48 hours for shipments of new orders. For more information on the ordering process and how to register, please refer to our Frequently Answered Questions or contact Customer Care at contact@beiresources.org.

References:

1. “SARS-CoV-2 Variant Classifications and Definitions.” Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html. Accessed 26 October 2021.
2. “Covid Data Tracker.” Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, https://covid.cdc.gov/covid-data-tracker/#variant-proportions. Accessed 26 October 2021.
3. “B.1.617.2 Lineage Report.” Outbreak.info, https://outbreak.info/situation-reports/delta.
4. Winger, A. and T. Caspari. “The Spike of Concern - The Novel Variants of SARS-CoV-2.” Viruses 13 (2021): 1002. PubMed: 34071984.

Image:  Transmission electron micrograph of SARS-CoV-2 virus particles (NIAID/CC BY 2.0)

The genome of Toxoplasma gondii, the causal agent of toxoplasmosis, is readily amenable to genetic manipulation in the laboratory making it an ideal model system to study the basic biology of parasitism, such as its complex life cycle. The life cycle stages of T. gondii include sporozoites, merozoites, tachyzoites, and bradyzoites. The factors involving the stage conversion of invading tachyzoites to the bradyzoite stage contained within dormant tissue cysts and the mechanisms of cyst reactivation are not fully understood.

Recent additions to the BEI Resources protozoan catalog include two fluorescent, stage-specific, transgenic T. gondii reporter strains derived from the recombinant type I/III strain EGS, which is able to spontaneously form cysts in vitro.^1,2,3,4 Transfection of strain EGS with plasmids expressing fluorescent tags under the control of stage-specific promoters resulted in two strains, T. gondii, strain EGS SAG1-mCherry LDH2-sfGFP (NR-53930) and T. gondii, strain EGS SAG1-mCherry LDH2-sfGFP MSF-BFP (NR-53931), that allow for monitoring of parasite stage conversion both in vitro and in vivo.^5,6

NR-53930 expresses the red fluorescent protein mCherry and superfolder green fluorescent protein (sfGFP), with parasites in the bradyzoite stage positive for sfGFP and negative for mCherry; parasites in the tachyzoite stage of the life cycle are positive for mCherry and negative for sfGFP. NR-53931 has the addition of blue fluorescent protein (BFP), with merozoite-stage parasites positive for BFP and negative for sfGFP and mCherry.⁶

For more information on these strains, including specific culture conditions to induce stage conversion, please refer to the individual product documentation available on the BEI Resources website.

References:

1. Vidigal, P. V., et al. “Prenatal Toxoplasmosis Diagnosis from Amniotic Fluid by PCR.” Rev. Soc. Bras. Med. Trop. 35 (2002): 1-6. PubMed: 11873253.
2. Ferreira, A. M., et al. “Genetic Analysis of Natural Recombinant Brazilian Toxoplasma gondii Strains by Multilocus PCR-RFLP.” Infect. Genet. Evol. 6 (2006): 22-31. PubMed: 16376837.
3. Ferriera, A. M., et al. “Virulence for BALB/c Mice and Antigenic Diversity of Eight Toxoplasma gondii Strains Isolated from Animals and Humans in Brazil.” Parasite 8 (2001): 99-105. PubMed: 11474987
4. Paredes-Santos, T. C., et al. “Spontaneous Cystogenesis in vitro of a Brazilian Strain of Toxoplasma gondii.” Parasitol. Int. 62 (2013): 181-188. PubMed: 23269201.
5. Paredes-Santos, T. C., et al. “Development of Dual Fluorescent Stage Specific Reporter Strains of Toxoplasma gondii to Follow Tachyzoite and Bradyzoite Development In Vitro and In Vivo.” Microbes Infect. 18 (2016): 39-47. PubMed: 26432517.
6. Sinai, A. P., et al. “Bradyzoite and Sexual Stage Development.” In: L. M. Weiss and K. Kim, Toxoplasma gondii: The Model Apicomplexan - Perspectives and Methods (3rd ed.). Elsevier, 2020. 807-857.

Image:  Transmission electron micrograph of SARS-CoV-2 virus particles (NIAID/CC BY 2.0)

The S glycoprotein remains a primary focus of SARS-CoV-2 immunotherapy research, particularly in the screening of convalescent human plasma and sera for the presence of neutralizing antibodies. Alternative approaches for working with pathogenic viruses outside of a BSL-3 laboratory using recombinant viral systems, such as Vesicular Stomatitis Virus (VSV), have made this research more accessible.

A recombinant VSV recently deposited to BEI Resources by Dr. Kartik Chandran of Albert Einstein College of Medicine resembles SARS-CoV-2 in cellular entry and inhibition through expression of the SARS-CoV-2 S gene as the sole entry glycoprotein. rVSV-SARS-CoV-2 S (BEI Resources NR-55284) was generated by modification of a VSV antigenome, replacing its native glycoprotein with the full-length, wild-type SARS-CoV-2 Wuhan-Hu-1 S gene and enhanced green fluorescent protein (eGFP). After a plasmid-based rescue, rVSV-SARS-CoV-2 S was passaged 9 times and plaque-purified, resulting in a slowly replicating virus expressing the wild-type S gene.¹ rVSV-SARS-CoV-2 S is provided as spin-clarified cell lysate and supernatant from infected Cercopithecus aethiops kidney cells (ATCC^® CCL-81™).

rVSV-SARS-CoV-2 S is ideal for reporter-based screening of small-molecule and antibody-based inhibitors targeting the SARS-CoV-2 S glycoprotein, such as neutralizing antibodies present in convalescent human plasma and sera, as well as research targeting viral entry and its inhibition.

Please refer to the product documentation located on the BEI Resources website for detailed analysis of eGFP expression, titer determination and recommended growth conditions.

References:

1. Dieterle, M. E., et al. “A Replication-Competent Vesicular Stomatitis Virus for Studies of SARS-CoV-2 Spike-Mediated Cell Entry and Its Inhibition.” Cell Host Microbe 23 (2020): 486-496. PubMed: 32738193

Image:  Transmission electron microscopic image vesicular stomatitis virus particles. (CDC/Dr. Fred. A. Murphy)

The pseudovirus system is a useful alternative approach for pathogenic viruses such as SARS-CoV-2 outside of a BSL-3 laboratory. Two pseudotyped lentivirus plasmid kits recently deposited by the Bloom Lab at the Fred Hutchinson Research Center in Seattle, Washington, provide a method of generating non-replicative pseudotyped lentiviral particles with SARS-CoV-2 spike (S) glycoprotein in a BSL-2 environment.^1,2 These kits, NR-53816 and NR-53817, combined with HEK293T cells engineered to express the SARS-CoV-2 receptor ACE2 (NR-52511), are ideal for performing SARS-CoV-2 antibody neutralization assays in a convenient 96-well format.

Production of the pseudotyped lentiviruses is achieved through the transfection of 5 plasmids. Each kit includes: one plasmid containing a viral entry protein encoding for S glycoprotein; three helper plasmids encoding for human immunodeficiency virus Gag and Pol, Tat1b, Rev1b; two helper plasmids (Tat1b and Rev1b nonsurface proteins) for lentivirus packaging; and one lentiviral backbone plasmid encoding for synthetic firefly luciferase (Luc2) and synthetic Zoanthus sp. green fluorescent protein (ZsGreen1) for visualization.

In addition to the pseudotyped lentivirus plasmid kits, BEI Resources now offers SARS-CoV-2 spike-pseudotyped lentiviruses (NR-53818 and NR-53819) with the same characteristics as those produced with the plasmid kits. NR-53818 is pseudotyped lentivirus with a C-terminally truncated S glycoprotein, a mutation that increases the titer of viral particles produced.³ NR-53819 includes the S glycoprotein with the mutation D614G, one of the first documented S glycoprotein variants, which is more infectious than wild-type SARS-CoV-2.^4,5 Each vial of pseudotyped lentivirus is sufficient for one 96-well plate of neutralization assays

The use of the SARS-CoV-2 plasmid kits to make spike-pseudotyped lentiviruses for neutralization assays has been cited in numerous publications.6^,7,8,9 Many online resources are available supporting the production of pseudotyped lentivirus and design of neutralization assays, including up-to-date protocols maintained by the Bloom Lab, as well as basic protocols on BEI Resources product information sheets.

Please refer to the product information on the BEI Resources website for detailed information on individual kit components, use restrictions and for the limited-use, research-only licenses of Luc2 and ZsGreen.

References:

1. Crawford, K. H. D., et al. “Dynamics of Neutralizing Antibody Titers in the Months after SARS-CoV-2 Infection.” J. Infect. Dis. 223 (2021): 197-205. PubMed: 33000143.
2. Crawford, K. H. D., et al. “Protocol and Reagents for Pseudotyping Lentiviral Particles with SARS-CoV-2 Spike Protein for Neutralization Assays.” Viruses 12 (2020): E513. PubMed: 32384820.
3. Hulswit, R. J. G., C. A. M. de Haan and B. -J. Bosch. “Coronavirus Spike Protein and Tropism Changes.” Adv. Virus Res. 96 (2016): 29-57. PubMed: 27712627.
4. Wu, F., et al. “A New Coronavirus Associated with Human Respiratory Disease in China.” Nature 579 (2020): 265-269. PubMed: 32015508.
5. Klumpp-Thomas, C., et al. “Effect of D614G Spike Variant on Immunoglobulin G, M, or A Spike Seroassay Performance.” J. Infect. Dis. 223 (2021): 802-804. PubMed: 33257936.
6. Starr, T. N., et al. “Prospective Mapping of Viral Mutations that Escape Antibodies Used to Treat COVID-19.” Science 371 (2021): 850-854. PubMed: 33495308.
7. Hu, Y., et al. “Boceprevir, Calpain Inhibitors II and XII, and GC-376 have Broad-Spectrum Antiviral Activity Against Coronaviruses.” ACS Infect. Dis. 7 (2021): 586-597. PubMed: 33645977.
8. Chandrasekar, S. S., et al. “Localized and Systemic Immune Responses Against SARS-CoV-2 Following Mucosal Immunization.” Vaccines 9 (2021): 132. PubMed: 33562141.
9. Sanchez, S., et al. “Limiting the Priming Dose of a SARS-CoV-2 Vaccine Improves Virus-Specific Immunity.” bioRxiv (2021): doi: 10.1101/2021.03.31.437931. PubMed: 33821275.

Image:  Transmission electron micrograph of SARS-CoV-2 virus particles (NIAID/CC BY 2.0)

The Multidrug-Resistant Organism Repository and Surveillance Network (MRSN) Acinetobacter baumanii diversity panel consists of 100 multidrug-resistant A. baumanii strains selected from a collection of over 3,500 clinical isolates collected between 2001 and 2017 from health care facilities around the world.¹ This panel supports the research and development of new antibiotics and tools to treat infections caused by A. baumannii, an opportunistic pathogen and leading cause of infections in hospital and healthcare settings, and categorized by the World Health Organization as a priority 1 pathogen due to its increasing rates of antibiotic resistance.

The panel represents 70 different traditional Pasteur scheme multilocus sequence types, including genetically distinct isolates within the most globally prevalent clones, and contains multidrug-resistant, extensively drug-resistant and non-multidrug-resistant isolates with 100 distinct alleles encompassing 32 families of antimicrobial resistance genes. Isolates are fully characterized using whole genome sequencing complete with antibiotic susceptibility and antimicrobial-resistant gene content, ranging from pansensitive to panresistant phenotypes, and a comprehensive susceptibility profile consisting of 14 clinically relevant antibiotics.

The MRSN Acinetobacter baumanii Diversity Panel (BEI Resources NR-52148 through NR-52248) is currently in production and individual strains will be available on the BEI Resources website as they are released. The complete panel of 100 isolates will be available as BEI Resources NR-52248 later this year.

References:

1. Galac, M. R., et al. “A Diverse Panel of Clinical Acinetobacter baumannii for Research and Development.” Antimicrob. Agents Chemother. 64 (2020): e00840-20. PubMed: 32718956.

Image:  Photograph of Acinetobacter baumannii cultured on sheep blood agar medium (CDC/Todd Parker)

The African trypanosomes infect a broad range of mammals across sub-Saharan Africa and result in significant losses to domestic livestock. Subspecies of Trypanosoma brucei also cause African trypanosomiasis in humans resulting in approximately 20,000 cases per year.¹ The development of vaccines against this disease is extremely difficult due to high antigenic variation of trypanosome surface glycoproteins, and a rise in resistance to trypanocidal drugs has been documented.^2,3 Mouse models of infection and drug susceptibility are critical to understanding the mechanisms of disease and the development of new therapeutic drugs.

BEI Resources houses several isolates of T. brucei to support in vivo research on African trypanosomiasis. T. brucei subsp. gambiense, strain 348BT (NR-53513) is a recent acquisition that was isolated from a patient in the Democratic Republic of Congo before receiving treatment with melarsoprol that displays reduced sensitivity to the drug in laboratory mice.^4,5

Other African trypanosome acquisitions in BEI Resources catalog include the reference T. brucei subsp. brucei, strain TREU 667 (NR-46440), as well as a variety of clinical isolates used in drug susceptibility studies in vivo. For more information on the availability of these cultures, please visit the BEI Resources website or contact Customer Care at contact@beiresources.org.

References:

1. Brun, R., et al. “Human African Trypanosomiasis.” Lancet 375 (2010): 148-159. PubMed: 19833383.
2. Büscher, P., et al. “Human African Trypanosomiasis.” Lancet 390 (2017): 2397-2409. PubMed: 28673422.
3. “Trypanosomiasis, Human African (Sleeping Sickness).” World Health Organization, https://www.who.int/news-room/fact-sheets/detail/trypanosomiasis-human-african-(sleeping-sickness). Accessed 29 July 2021.
4. Pyana, P. P., et al. “Isolation of Trypanosoma brucei gambiense from Cured and Relapsed Sleeping Sickness Patients and Adaptation to Laboratory Mice.” PLoS Negl. Trop. Dis. 5 (2011): e1025. PubMed: 21526217.
5. Pyana, P. P., et al. “Melarsoprol Sensitivity Profile of Trypanosoma brucei gambiense Isolates from Cured and Relapsed Sleeping Sickness Patients from the Democratic Republic of Congo.” PLoS Negl. Trop. Dis. 8 (2014): e3212. PubMed: 25275572.

Image:  Photomicrograph of Trypanosoma brucei parasites in a Giemsa-stained blood sample (CDC/Blaine Mathison)

A recent review of the BEI Resources parasitology and arthropod vector research collection shows a significant increase in the availability of organisms and biomaterials supporting research of parasitic diseases.¹

Since 2010, the BEI Resources collection has expanded with integration of the Malaria Research and Reference Reagent Resource (MR4), the Schistosome Resource (SR3) and the Filariasis Research Reagent Resource (FR3) Centers, the Black Fly Research and Resource Center from the University of Georgia, and by the continued contribution of strains and reagents by the research community.

The parasitic protozoa collection now totals over 500 wild-type, transgenic, reference and clinical isolate strains representing 10 genera, along with nucleic acids, antibodies, recombinant proteins and expression vectors integral to the study of many neglected tropical diseases.

Disease vectors such as ticks, mosquitoes, triatomines, sandflies and black flies are available in a variety of life stages. Please consult the Vector Resources catalog for a complete listing of available vectors, protocols and additional information and helpful resources.

To learn more about these reagents, to suggest a new reagent, or to learn how to contribute to the collection by making a deposit, please visit the BEI Resources website or contact Customer Care at contact@BEIResources.org.

References:

1. Molestina, R. E. and T.T. Stedman. “Update on BEI Resources for Parasitology and Arthropod Vector Research.” Trends Parasitol. 36 (2020): 321-324. PubMed: 32035817

Image:  Photomicrograph of fluorescent antibody-stained Brugia malayi (CDC/A.V. Sulzer)

A selection of historical Bacillus sp. isolates derived from ATCC^® cultures have been made available to BEI registrants through the BEI Resources catalog.

This collection encompasses type strains or early isolates used to define the species, well-characterized quality control strains incorporated in biochemical identification systems and industrial applications, and represents environmental, dairy, grain and human isolates, some of which were originally isolated over 100 years ago. Several of these isolates have undergone reclassification as a result of taxonomic revision within the Bacillus genus following comparative phenotypic studies and improved genotypic analysis.¹

For more detailed information including isolate history, please refer to the product documentation on the BEI Resources website.

References:

1. Fritz, D. “Taxonomy of the Genus Bacillus and Related Genera: the Aerobic Endospore-Forming Bacteria.” Phytopathology 94 (2004): 1245-1248. PubMed: 18944461.
2. Smith, N. R., et al. “Type Cultures and Proposed Neotype Cultures of Some Species in the Genus Bacillus.” J. Gen. Microbiol. 34 (1964): 269-272. PubMed: 14135533.

Image:  Scanning electron micrograph of Bacillus cereus (M. Das Murtey, P. Ramasamy/ CC BY-SA 3.0)

The SARS-CoV-2 spike (S) glycoprotein mediates viral binding to the host angiotensin converting enzyme 2 (ACE2). This protein forms a trimer, and when bound to a host receptor allows fusion of the viral and cellular membranes.¹ Multiple SARS-CoV-2 mutations in the S glycoprotein are currently under study, in particular three mutations in the receptor binding domain (RBD), K417N, E484K and N501Y, that may have functional significance.² Structural modeling and mouse studies indicate N501Y increases S glycoprotein binding to ACE2, resulting in increased SARS-CoV-2 virulence.^3,4 In addition, the E484K mutation drives immune evasion by altering RBD conformation and has been identified in escape mutants for convalescent antisera.⁵

The SARS-CoV-2 S glycoprotein is a key target for vaccine development efforts, as well as diagnostic and surveillance measures. To support these efforts, recombinant forms of the S glycoprotein and RBD from SARS-CoV-2 variants of interest and concern are now available in the BEI Resources catalog. The purity, concentration and functional activity are detailed on the Certificates of Analysis. Please consult the product documentation located on the product web pages for specific information on each protein.

The SARS-CoV-2 S glycoproteins were contributed or manufactured by Dr. Florian Krammer and Fatima Amanat, Icahn School of Medicine at Mount Sinai, New York, New York, and Dr. Noah Sather, Center for Global Infectious Diseases, Seattle Children's Research Institute, Seattle, Washington.

References:

1. Hulswit, R. J. G., C. A. M. de Haan and B.-J. Bosch. “Coronavirus Spike Protein and Tropism Changes.” Adv. Virus Res. 96 (2016): 29-57. PubMed: 27712627.
2. Tegally, H., et al. “Emergence and Rapid Spread of a New Severe Acute Respiratory Syndrome-Related Coronavirus 2 (SARS-CoV-2) Lineage with Multiple Spike Mutations in South Africa.” medRxiv (2020). doi:10.1101/2020.12.21.20248640.
3. Gu, H., et al. “Adaptation of SARS-CoV-2 in BALB/c Mice for Testing Vaccine Efficacy.” Science 369 (2020): 1603-1607. PubMed: 32732280.
4. Leung, K., et al. “Early Transmissibility Assessment of the N501Y Mutant Strains of SARS-CoV-2 in the United Kingdom, October to November 2020.” Euro. Surveill. 26 (2021): 2002106. PubMed: 33413740.
5. Andreano, E., et al. “SARS-CoV-2 Escape in vitro from a Highly Neutralizing COVID-19 Convalescent Plasma.” bioRxiv (2020): 10.1101/2020.12.28.424451. PubMed: 33398278.

Image:  Transmission electron micrograph of a SARS-CoV-2 virus particle (NIAID/CC BY 2.0)

Thirty-five SARS-CoV-2 isolates are now available in the BEI Resources catalog. The collection includes isolates classified by the U.S. Centers for Disease Control and Prevention (CDC) as Variants of Concern and Variants of Interest, originating from the United States and internationally (Brazil, Canada, Chile, England, Germany, Hong Kong, Italy, Japan, Scotland, South Africa and Singapore).^1,2 Available and upcoming isolates represent lineages B.1.617.1, B.1.617.2, B.1.1.7, B.1.351, P.1, P.2, B.1.5, B.1.1.298, B.1.222, B.1.427, B.1.526, B.1.529, B.1.617, R.1 and C.37 (formerly B.1.1.1).

SARS-CoV-2 isolates are produced in vitro by infection of Cercopithecus aethiops kidney epithelial (Vero) cells or Homo sapiens lung adenocarcinoma (Calu-3; ATCC^® HTB-55™) cells with the deposited material and are provided frozen as spin-clarified cell lysate and supernatant. Virus propagation in Calu-3 cells have shown that the virus stocks are less prone to tissue culture adaptive changes. All SARS-CoV-2 variants have been analyzed by Next Generation Sequencing with respect to both the SARS-CoV-2, Wuhan-Hu-1 strain reference sequence and the original reference sequence for that isolate to confirm signature mutations. Certificates of Analysis for each stock produced include observed sequence variations noted in the Appendix section and detailed lot-specific data such as passage history and titer of each item lot.

BEI Resources will continue to make available variants as they emerge, with the intent to have a good representation of each variant. Requests are fast-tracked and prioritized, with an anticipated turnaround window of 12-to-72 hours for new SARS-CoV-2 registrations and 24-to-48 hours for shipments of new orders. For more information on the ordering process and how to register, please refer to our Frequently Answered Questions or contact Customer Care at contact@beiresources.org.

An up-to-date listing of all currently available and upcoming SARS-CoV-2 isolates, genomic RNA, monoclonal antibodies, plasmids, proteins, peptide arrays and inactivated virus preparations is available on the SARS-CoV-2 resource page on the BEI Resources website.

References:

1. “SARS-CoV-2 Variant Classifications and Definitions.” Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, https://www.cdc.gov/coronavirus/2019-ncov/cases-updates/variant-surveillance/variant-info.html.
2. "SARS-CoV-2 (hCoV-19) Mutation Reports." Outbreak.info, https://outbreak.info/situation-reports.

Image:  Scanning electron micrograph of an apoptotic cell infected with SARS-COV-2 virus particles (NIAID/CC BY 2.0)

Enterovirus and Acute Flaccid Myelitis Research Materials

In support of Enterovirus D68 (EV-D68) vaccine development research, BEI Resources has performed initial screening of cell lines amenable for human vaccine development. The following study assesses the suitability of cell lines for virus propagation in the presence of serum and after adaptation to serum free (SF) conditions. A guidance on how to adapt cell lines to serum free media is provided.

Enterovirus and Acute Flaccid Myelitis
Enterovirus D68 (EV-D68) has reemerged as a cause of severe respiratory infections in young children around the world and as a newly emerged neuropathogenic threat. First isolated in the United States (US) from children with serious respiratory illness in 1962¹, only sporadic clusters of EV-D68 infection were reported globally in the last decade² until 2014 when an unusually large outbreak occurred in the US. During this outbreak (and in subsequent 2016 and 2018 outbreaks), a subset of children with laboratory confirmed EV-D68 related severe respiratory illness also developed an acute flaccid myelitis (AFM). Detection of virus in the spinal fluid of AFM affected children³ is rare but recently, researchers have found new evidence linking AFM to enteroviruses due to the presence of higher levels of EV specific antibodies in cerebrospinal fluid of children with AFM compared to non-AFM controls^4,5.

BEI Resources Screening of Cell Lines for EV-D68 Propagation in Media Supplemented with Serum
Typically, EV-D68 is propagated in human rhabdomyosarcoma (RD) cells (ATCC^® CCL-136™) at 33°C. However, this cell line is not amenable for vaccine production and additionally is maintained in serum containing media. Consequently, BEI Resources has performed an initial screen of suitable cell substrates for EV-D68 vaccine development. Five cell lines were tested for EV-D68 susceptibility and production rates to assess the successful propagation using two EV-D68 isolates originally grown in RD cells that are available in BEI Resources (see Table 1 for details).

Prior to infection, the cell lines were maintained at 37°C. Viral growth media contained 2% fetal bovine serum (FBS) (ATCC^® 30-2020™) and the ATCC^® recommended base media for each cell line (Table 1). The cells were infected with each viral strain using a multiplicity of infection (MOI) of 0.01 and incubated at 33°C. When approximately 90% of the cells showed cytopathic effect (CPE), the virus was harvested by scraping the monolayer into the supernatant. The infected cell lysate was clarified by centrifugation at 2400g and the pellet underwent three freeze-thaw cycles to release the virus which was then added back to the supernatant. The viruses were titrated in RD cells by the 50% Tissue Culture Infectious Dose (TCID₅₀) method and calculated by the Reed-Muench method. If a virus infected cell line displayed no signs of CPE after an extended incubation period of 12-14 days, the absence of virus was confirmed by performing an immunofluorescence assay (IFA) with a virus specific antibody. Furthermore, they underwent two blind passages with extended incubation to confirm that the cell line did not support growth of the virus. All infected cell lines were subjected to three total serial passages with the second and third passage utilizing a 1:100 dilution of virus inoculum in media rather than MOI. For a direct comparison of virus susceptible cell lines using MOI for passage 1, virus titration was performed RD cell line (Table 1).

The cells lines A549, MRC-5 and HEK-293 T/17 support growth of both EV-D68 viral isolates. These results are comparable to the same viral isolates grown in RD cells. HEK-293 cells supported growth of US/MO/14 18947 but showed reduced virus fitness for the three passages tested and did not support growth of the US/KY/14-18953 virus. CHO cells expressing the ICAM-1 receptor was not susceptible to the two isolates used in the cell screening assessments.
 

*US/MO/14-18947 is available in the BEI Resources catalog as NR-49129 and US/KY/14-18953 as NR-49132

BEI Resources Screening of Cell Lines for EV-D68 Propagation in Serum Free Media
BEI Resources performed a second screen of cell lines under serum free conditions to assess the growth of EV-D68 . Four of the cell lines (MRC-5, A549 and HEK-293 T/17 and 293) were adapted to grow in serum free conditions by weaning serum and switching into chemically defined media with supplements . All the cell lines were grown in viral growth media (base media recommended for each cell line) supplemented with 2% FBS to serve as controls. An additional control included use of RD cells grown in base media with 2% serum. (see Table 2 for details)

Serum Free (SF) adapted cell lines were grown in appropriate growth media at 37°C and infected with the EV-D68 isolate US/MO/14-18947 at a MOI of 0.001. All cell lines were incubated at 33°C for three passages with each successive passage infected at MOI of 0.001. All control cell lines were similarly infected. At the end of each passage virus from all cell lines were titrated (see Table 2). Note that viral titers were determined by a standard assay using RD cells.
 

Infection and viral production of the EV-D68 US/MO/14-18947 virus isolate was successful over the course of three passages in serum free conditions for three cell lines, A549, MRC-5, and HEK-293 T/17, however HEK-293 did not support the growth of the virus. The CPE for HEK-293 T/17 in SF adapted cells is difficult to discern as the cells lose anchorage dependence. As a result, confirmation of viral infection in HEK-293 T/17 SF adapted cells requires IFA.

Summary of BEI Resources Cell Screening for EV-D68 Propagation
Using two EV-D68 isolates, it was observed that two cell lines A549 and MRC-5 successfully supported virus growth after adaptation to serum-free conditions for use in downstream vaccine developmental needs. Both the cell lines were found to be robust and tolerated the serum weaning process while maintaining their morphology and viability.

For guidance and protocols on serum free adaptation of cell lines A549, MRC-5, HEK-293 and HEK-293 T/17, please visit our Knowledge Base.

For information on the availability of virus isolates grown in serum free A549 adapted cells, please email Contact@BEIResources.org.
 

Coming Soon 
NR-52355        Enterovirus D68, USA/2018-23206 (produced in serum-free A549 cells)

Leishmaniasis is a neglected tropical disease caused by Leishmania parasites, which are transmitted to both humans and animals by female phlebotomine sandflies. Infections take several forms, with cutaneous leishmaniasis causing skin lesions in an estimated 700,000-to-1.2 million new cases per year, and the high mortality visceral leishmaniasis resulting in fevers, weight loss and enlargement of the spleen or liver.¹

Leishmania is divided into two subgenera, Leishmania and Viannia, with more than 30 known species of Leishmania further classified as New World (Western hemisphere) and Old World (Eastern hemisphere) species. Pathogenic species of both subgenera have been grouped into complexes based on phylogenetic analyses determined through differences in the natural history of their vertebrate hosts, vector specificity, clinical manifestations, geographical distribution and, more recently, using molecular approaches with different genetic markers. ^2,3

A recent deposit of Leishmania strains by Dr. K. P. Chang of Rosalind Franklin University has expanded the BEI Resources Leishmania collection by four new species, L. braziliensis, L.gerbilli, L. infantum and L. turanica. Strains have been sequenced for the nagt gene, allowing for inter- and/or intra-species discrimination.⁴ Please refer to the individual product documentation for more information on the nagt variant of each strain.

References:

1. “Leishmaniasis.” Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, https://www.cdc.gov/parasites/leishmaniasis.
2. Schönian, G., et al. “Molecular Epidemiology and Population Genetics in Leishmania.” Med. Microbiol. Immunol. 190 (2001): 61-63. PubMed:  11770112.
3. Marcili, A., et al. “Phylogenetic Relationships of Leishmania Species Based on Trypanosomatid Barcode (SSU rDNA) and gGAPDH Genes: Taxonomic Revision of Leishmania (L.) infantum chagasi in South America.” Infect Genet Evol. 25 (2014): 44-51. PubMed:  24747606.
4. Waki, K., et al. “Transmembrane Molecules for Phylogenetic Analyses of Pathogenic Protists: Leishmania-Specific Informative Sites in Hydrophilic Loops of Trans-Endoplasmic Reticulum N-Acetylglucosamine-1-Phosphate Transferase.” Eukaryot. Cell 6 (2007): 198-210. PubMed:  17142569.

Image:  TEM image of Leishmania braziliensis (CDC/Cynthia S. Goldsmith, Luciana Flannery)

Recent acquisitions in BEI Resources aim to contribute to research focused on understanding the mechanisms of disease and the development of novel therapeutic drugs for Trypanosome research. Such acquisitions include SSGCID clones, proteins and reference parasite strains used in genome and virulence studies. For more information on the availability of these materials please contact BEI Resources customer service.

BEI Resources is working to expand the fungal resources so that researchers have a broader range of organisms and reagents for their work.  BEI Resources currently has eight genus and species represented with several more currently going through the production pipeline.  BEI Resources is actively encouraging depositors with medically relevant strains to deposit into the repository. If you are interested in depositing, please click here.

The ISO Guide 34 accreditation by the American Association for Laboratory Accreditation (A2LA) builds upon the earlier certification for compliance with the ISO 9001:2000 standard for quality management systems received by ATCC in 2007. Biological reference materials produced under an ISO Guide 34-accredited process have confirmed identity, well-defined characteristics and an established chain of custody — qualities essential to their effectiveness as biological standards in research and development.

The genus Flavobacterium has undergone several taxonomic shifts since it was created nearly 100 years ago, beginning with the separation of a number of pathogenic species into a new genus, Chryseobacterium.¹ Further reclassification occurred with the naming of genus Elizabethkingia into which several Chryseobacterium were moved, including the pathogenic species E. meningoseptica (formerly Chryseobacterium meningosepticum; originally Flavobacterium meningosepticum) and E. miricola.² Recent genomic analysis of unidentified isolates resembling the two genera, as well as a review of the four genomospecies of Elizabethkingia, have resulted in the naming of new, novel species of Chryseobacterium and Elizabethkingia.^1,2 Chryseobacterium underwent an even more recent taxonomic reorganization following the comparison of amino acid identity among type strains, leading to the division of the Chryseobacterium genus into four different genera with Epilithonimonas, Kaistella and Halpernia gen. nov.³ Many of these reclassifications involved the renaming of historical isolates and type strains, many of which were recently deposited to BEI Resources.

Chryseobacterium spp. and Elizabethkingia spp. are Gram-negative environmental bacteria found in soil and water worldwide. Though infections in humans are rare, an average of 5-to-10 cases occur per state each year in the United States, often resulting in small healthcare-related outbreaks, such as the E. anopheles outbreak in Wisconsin in 2016.⁴

References:

1. Holmes, B., A. G. Steigerwalt and A. C. Nicholson. “DNA-DNA Hybridization Study of Strains of Chryseobacterium, Elizabethkingia and Empedobacter and of Other Usually Indole-Producing Non-Fermenters of CDC Groups IIc, IIe, IIh and IIi, Mostly from Human Clinical Sources, and Proposals of Chryseobacterium bernardetii sp. nov., Chryseobacterium carnis sp. nov., Chryseobacterium lactis sp. nov., Chryseobacterium nakagawai sp. nov. and Chryseobacterium taklimakanense comb. nov.” Int. J. Syst. Evol. Microbiol. 63 (2013):  4639-4662.  PubMed:  23934253.
2. Nicholson, A. C., et al. “Revisiting the Taxonomy of the Genus Elizabethkingia Using Whole-Genome Sequencing, Optical Mapping, and MALDI-TOF, Along with Proposal of Three Novel Elizabethkingia species: Elizabethkingia bruuniana sp. nov., Elizabethkingia ursingii sp. nov., and Elizabethkingia occulta sp. nov.” Antonie Van Leeuwenhoek 111 (2018):  55-72.  PubMed:  28856455.
3. Nicholson, A. C., et al. “Division of the genus Chryseobacterium: Observation of Discontinuities in Amino Acid Identity Values, a Possible Consequence of Major Extinction Events, Guides Transfer of Nine Species to the Genus Epilithonimonas, Eleven Species to the Genus Kaistella, and Three Species to the Genus Halpernia gen. nov., with Description of Kaistella daneshvariae sp. nov. and Epilithonimonas vandammei sp. nov. Derived from Clinical Specimens.” Int. J. Syst. Evol. Microbiol. 70 (2020):  4432-4450.  PubMed:  32735208.
4. “Elizabethkingia.”  Centers for Disease Control and Prevention, U.S. Department of Health and Human Services,  https://www.cdc.gov/elizabethkingia/index.html.

Image:  Transmission electron microscopic image of Elizabethkingia anophelis bacteria (CDC/Cynthia Goldsmith)

Candida auris is an emerging multidrug-resistant pathogenic yeast, which causes invasive infections and outbreaks in nosocomial settings resulting in high mortality. Since it was first described in 2009, C. auris has been isolated in over 30 countries on 6 continents, with the earliest known isolate from 1996 discovered during a retrospective review of unidentified yeasts.^1,2,3

C. auris has a strong phylogeographic structure comprising four distinct clades, South Asia, East Asia, South Africa and South America, separated by tens of thousands of SNPs, with smaller clusters identified in some clades.³ This high level of relatedness and low genetic diversity within clades suggests clades emerged independently and near-simultaneously in four distinct locations rather than a single spread.^1,3

Clinical isolates from a hospital in Pakistan, the site of a 2014 C. auris outbreak⁵, have been deposited to BEI Resources and represent bloodstream infections with varied antifungal resistance profiles to azole and polyene drug classes. More information about the resistance profiles of these isolates is available in the BEI Resources Antimicrobial Database and on the individual product web pages and documentation

References:

1. Forsberg, K., et al. “Candida auris:  The Recent Emergence of a Multidrug-Resistant Fungal Pathogen.” Med. Mycol. 57 (2019): 1-12.  PubMed:  30085270.
2. Kean, R. and G. Ramage. “Combined Antifungal Resistance and Biofilm Tolerance: The Global Threat of Candida auris.” mSphere 4 (2019): e00458-19.  PubMed:  31366705.
3. Lockhart, S. R., et al. “Simultaneous Emergence of Multidrug-Resistant Candida auris on 3 Continents Confirmed by Whole-Genome Sequencing and Epidemiological Analyses.” Clin. Infect. Dis. 64 (2017): 134-140. PubMed: 27988485.
4. Sayeed, M. A., et al. “Clinical Spectrum and Factors Impacting Outcome of Candida auris: A Single Center Study from Pakistan.” BMC Infect. Dis. 19 (2019): 384. PubMed:  31060514.

Image:  Medical illustration of Candida auris fungal organisms (CDC/Medical Illustrator: Stephanie Rossow)

BEI Resources is pleased to announce the upcoming availability of black flies through a partnership with the University of Georgia Black Fly Rearing and Bioassay Laboratory, which has operated the only known colony of black flies (Diptera: Simuliidae) for over 20 years. Since its establishment, the Simulium vittatum colony (Simulium vittatum cytospecies IS-7) has been used for a variety of research projects, including vector transmission studies, environmental monitoring, vector control and larval feeding studies. Rearing protocols have been designed to operate the black fly colony at maximum productivity to produce a standardized test subject and preserve colony vigor. An advantage of Simulium vittatum cytospecies IS-7 is that they can deposit their first batch of eggs without a blood meal. Consequently, no animal resources are required to maintain the colony.

The laboratory continues to conduct and collaborate in a wide range of research projects, providing all stages of the black fly life cycle to collaborating laboratories. Current research conducted in the laboratory involves larvicidal efficacy evaluations, topical repellent evaluations and growth studies related to climate change.

More information on the Simulium vittatum Black Fly Colony will be available in the BEI Resources Vector Resource Catalog and on individual product web pages and documentation.

Image:  Black Fly bite (UGA, Jena Johnson)

The extensive collection of Mycobacterium species available from BEI Resources continues to grow with the addition of M. ulcerans, the cause of a neglected tropical disease known as Buruli ulcer that causes cutaneous lesions and osteomyelitis.^1,2,3 M. ulcerans has been identified in 33 countries worldwide, most commonly in Africa, and typically occurs in wetland and tropical regions and in areas with recent environmental change.² M. ulcerans contains a virulence plasmid encoding for the production of mycolactone, a macrocyclic polyketide toxin with cytotoxic and immunosuppressive activity.

The available and upcoming isolates were collected from clinical cases of Buruli ulcer in Benin, where over 6,000 Buruli ulcer cases were reported between 2006 and 2015.⁴

References:

1. Röltgen, K., T. P. Stinear and G. Pluschke. “The Genome, Evolution and Diversity of Mycobacterium ulcerans.” Infect. Genet. Evol. 12 (2012): 522-529. PubMed: 22306192.
2. Merritt, R. W., et al. “Ecology and Transmission of Buruli Ulcer Disease: A Systematic Review.” PloS Negl.Trop. Dis. 4 (2010): e911. PubMed: 21179505.
3. Pommelet, V., et al. “Findings in Patients from Benin with Osteomyelitis and Polymerase Chain Reaction-Confirmed Mycobacterium ulcerans Infection.” Clin. Infect. Dis. 59 (2014): 1256-1264. PubMed: 25048846.
4. Degnonvi, H., et al. “Effect of Well Drilling on Buruli Ulcer Incidence in Benin: A Case-Control, Quantitative Survey.” Lancet Planet Health 3 (2019): e349-e356. PubMed: 31439316.

Image:  Mycobacterium ulcerans, strain S4018 (NR-51701) on Lowenstein-Jensen agar (BEI Resources)

Powassan virus (POWV), first described after isolation in1958 from the brain of a fatal case of encephalitis in Powassan, Ontario, Canada, is a reemerging severe neuroinvasive tick-borne disease causing human encephalitis in the United States, Canada and Russia, and the only recognized tick-borne flavivirus endemic to North America.^1,2 Though rare, infection by POWV in the United States has recently increased, with over 30 cases reported in 2017, and has a fatality rate of approximately 10%, with long-term neurological sequelae resulting in an estimated 50% of survivors.^3,4

The POWV Envelope (E) structural protein is a highly conserved structure comprising the majority of the virus surface with multiple roles in infection, including host cell receptor recognition and binding, and is divided into three domains: Domain I (DI) consisting of a central beta-barrel domain; Domain II (DII) important for dimerization and virion assembly; and Domain III (DIII) characterized by an immunoglobulin-like segment.⁴ Studies demonstrated that the flavivirus E protein DIII (EDIII) is a primary antigenic target of specific neutralizing antibodies.⁵

The EDIII protein from the 1958 lineage 1 isolate, Powassan virus, strain LB (BEI Resources NR-51181) is currently in production and coming soon to the BEI Resources catalog as NR-52391, and represents the first commercially available standard for studying such emerging tick-borne infections. NR-52391 is a recombinant form of the truncated EDIII protein containing a C-terminal hexa-histidine tag, produced in a Pichia pastoris expression system and purified by immobilized-metal affinity chromatography. The mature native full-length POWV E protein is 497 residues (GenBank: NP_775516.1).

References:

1. Hermance, M. E. and S. Thangamani. “Powassan Virus: An Emerging Arbovirus of Public Health Concern in North America.” Vector Borne Zoonotic Dis. 17 (2017): 453-462. PubMed: 28498740.
2. McLean, D. M. and W. L. Donohue. “Powassan Virus: Isolation of Virus from a Fatal Case of Encephalitis.” Can. Med. Assoc. J. 80 (1959): 708-711. PubMed: 13652010.
3. Kemenesi, G. and K. Bányai. "Tick-Borne Flaviviruses, with a Focus on Powassan Virus." Clin. Microbiol. Rev. 32 (2018): e00106-17. PubMed: 30541872.
4. “Powassan Virus Statistics & Maps.” Centers for Disease Control and Prevention, U.S. Department of Health and Human Services, https://www.cdc.gov/powassan/statistics.html. Accessed 07 August 2020.
5. Heinz, F. X. and K. Stiasny. “Flaviviruses and Their Antigenic Structure.” J. Clin. Virol. 55 (2012): 289-295. PubMed: 22999801.

Image:  The dorsal view of a female I. pacificus hard tick (CDC/James Gathany)

Apicomplexan parasites cause significant morbidity and mortality to humans and livestock animals, and share characteristic morphological features, cytoskeletal components, and mechanisms of replication, motility and invasion. Understanding these mechanisms of infection and pathogenesis requires novel tools that support and further research.

Recently described methods of conditional gene deletion in the apicomplexan parasites Toxoplasma gondii and Plasmodium falciparum, the agents of toxoplasmosis and malaria, respectively, enable reliable gene function and virulence research using the dimerizable Cre (DiCre) strategy.^1,2,3 BEI Resources now houses Toxoplasma gondii, strain RHΔku80::DiCre:T2A:CAT (NR-51627) and Plasmodium falciparum, strain NF54::DiCre (MRA-1314; Coming Soon), stable cell lines that express rapamycin-inducible Cre recombinase, thus allowing the conditional deletion of essential and non-essential parasite genes at different life cycle stages.³ These strains offer new opportunities to study genes impacting growth or stage conversion with applications in drug development and vaccination strategies.

Additional tools available from the BEI Resources catalog to support apicomplexan research include molecular standards for the development of quantitative PCR (qPCR) assays against babesiosis, an emergent tickborne parasitic disease that can also be transmitted by blood transfusion. These standards harbor the internal transcribed spacer (ITS) 1, 5.8S ribosomal RNAgene, ITS 2 regions from Babesia microti, strain GI (NR-50741) and Babesia duncani, strain WA1 (NR-50742), the most prevalent agents of babesiosis in the United States. As qPCR assays have become more commonplace in the diagnosis of parasitic infections, these standards are useful in the execution of highly specific and sensitive blood tests using droplet digital or real time PCR technologies.⁴

References:

1. Hunt, A., et al. “Differential Requirements for Cyclase-Associated Protein (CAP) in Actin-Dependent Processes of Toxoplasma gondii.” ELife 8 (2019): e50598. PubMed: 31577230.
2. Tibúrcio, M., et al. “A Novel Tool for the Generation of Conditional Knockouts to Study Gene Function across the Plasmodium falciparim Life Cycle.” mBio 10 (2019): e01170-19. PubMed: 31530668.
3. Jullien, N., et al. “Conditional Transgenesis Using Dimerizable Cre (DiCre).” PLoS One 2 (2007): e1355. PubMed: 18159238.
4. Wilson, M. et al. “Development of Droplet Digital PCR for the Detection of Babesia microti and Babesia duncani.” Exp. Parasitol. 149 (2015): 24-31. PubMed: 25500215.

Image:  Fluorescence microscopy of Toxoplasma gondii (PLoS Open-Access/Ke Hu and John M. Murray)

Incidence of tick-borne disease in the United States doubled between 2004 and 2016, and continues to rise inthe United States and in many geographic regions around the world.^1,2 Novel methods are needed to improve diagnostic assays and species-level identification, advance surveillance and epidemiological efforts and facilitate drug development of these neglected human pathogens.

The most common bacterial pathogens transmitted by ticks belong to the genus Borrelia and are the causative agents of Lyme borreliosis (Lyme disease) and relapsing fever. Borrelia spp. can be divided into two major groups affecting human health: Borrelia burgdorferi sensu lato complex containing the agents of Lyme borreliosis and Borrelia species associated with human relapsing fever.

Tick-borne relapsing fever (TBRF) occurs worldwide, often in periodic outbreaks, with specific geographic distribution in North America, Europe, Africa and Asia. While most TBRF is transmitted by soft ticks of the Argasidae family, certain species like Borrelia miyamotoi are found in hard Ixodes ticks.³ Louse-borne relapsing fever (LBRF), caused by Borrelia recurrentis, is mainly limited to endemic areas within the Horn of Africa.⁴ Introduction of LBRF intoindustrialized countries via migration underscores the importance of having effective surveillance, diagnostic and treatment tools available.⁵

The availability of different Borrelia species to researchers supports development of necessary tools to minimize the effects of the increasing incidence of tick-borne diseases. Recent additions to the BEI Resources catalog include four species of Borrelia from Dr. Gabriele Margos and Dr. Volker Fingerle of the German National Reference Center for Borrelia (NRZ). This deposit includes four species new to the BEI Resources catalog: Borrelia afzelli, Borrelia hispanica, Borrelia miyamotoi and three Borrelia recurrentis (LBRF) strains.

In vitro adaption and growth of Borrelia spirochetes can be challenging. Optimized growth conditions are detailed on the BEI Resources product information sheet for each isolate and customers are encouraged to follow these growth recommendations to ensure success of the culture and recovery from the freezing process.

References:

1. Rosenberg, R., et al. “Vital Signs: Trends in Reported Vectorborne Disease Cases – United States and Territories, 2004-2016.” MMWR Morb. Mortal. Wkly. Rep. 67 (2018): 496-501. PubMed: 29723166.
2. Chikeka, I. and J. S. Dumler. “Neglected Bacterial Zoonoses.” Clin. Microbiol. Infect. 21 (2015): 404-415. PubMed: 25964152.
3. Talagrand-Reboul, E., et al. “Relapsing Fevers: Neglected Tick-Borne Diseases.” Front. Cell. Infect. Microbiol. 8 (2018): 98. PubMed: 29670860.
4. Warrell, D. A. “Louse-Borne Relapsing Fever (Borrelia recurrentis Infection).” Epidemiol. Infect. 147 (2019): e106. PubMed: 30869050.
5. Marosevic, D., et al. “First Insights in the Variability of Borrelia recurrentis Genomes.” PLoS Negl. Trop. Dis. 11 (2017): e0005865. PubMed: 28902847.
6. Fukunaga, M., et al. “Genetic and Phenotypic Analysis of Borrelia miyamotoi sp. nov., Isolated from the Ixodid Tick Ixodes persulcatus, the Vector of Lyme Disease in Japan.” Int. J. Syst. Bacteriol. 45 (1995): 804-810. PubMed: 7547303.
7. Glöckner, G., et al. “Comparative Genome Analysis: Selection Pressure on the Borrelia VLS Cassettes is Essential for Infectivity.” BMC Genomics 7 (2006): 211. PubMed: 16914037.

Image:  Fluorescence microscopy of Borrelia hispanica, NR-51677 (BEI Resources)

C. albicans and C. glabrata are ubiquitous in the environment and commensal inhabitants of the normal flora of the oral cavity, gastrointestinal tract and skin of most healthy humans.^1,2 For the immunocompromised, however, C. albicans and C. glabrata are the two most commonly recovered pathogenic yeasts in the United States and together are responsible for approximately 70% of all cases of systemic candidiasis, with increasing occurrences of multidrug resistance.^1-4

BEI Resources houses over 50 strains of Candida spp. to support candidiasis research. Recent additions to our catalog include an azole-susceptible strain of C. glabrata (NR-51685) and an azole-resistant C. glabrata strain isolated from the same patient after 50-day azole therapy (NR-51686).⁴ Also newly available is a transgenic strain of C. albicans (NR-51634) that constitutively expresses green fluorescent protein (GFP) and red fluorescent protein via mCherry, which has been used as an in vitro system for fluorescent sorting of C. albicans with macrophages.¹

For more information about these and other Candida strains available from BEI Resources, or to contribute to our expanding catalog of biomaterials for fungal research, please contact us at Contact@BEIResources.org.

References:

1. Muñoz, J. F., et al. “Coordinated Host-Pathogen Transcriptional Dynamics Revealed Using Sorted Subpopulations and Single Macrophages Infected with Candida albicans.” Nat. Commun. 10 (2019): 1607. PubMed: 30962448.
2. Brunke, S. and B. Hube. “Two Unlike Cousins: Candida albicans and C. glabrata Infection Strategies.” Cell. Microbiol. 15 (2013): 701-708. PubMed: 23253282.
3. Hendrickson, J. A., et al. “Antifungal Resistance: A Concerning Trend for the Present and Future.” Curr. Infect. Dis. Rep. 21 (2019): 47. PubMed: 31734730.
4. Vale-Silva, L., et al. “Comparative Genomics of Two Sequential Candida glabrata Clinical Isolates.” G3 (Bethesda) 7 (2017): 2413-2426. PubMed: 28663342.

Image:  Fluorescence microscopy of transgenic Candida albicans, NR-51634 (BEI Resources)

Infections with multidrug-resistant Pseudomonas aeruginosa, one of the most common healthcare-associated infections among immunocompromised and post-surgery patients, affected an estimated 32,600 patients in the United States in 2017, resulting in nearly 3,000 deaths, and is the predominant pathogen causing lung infections in individuals with cystic fibrosis and other pulmonary disorders.^1-3 Rising rates of multidrug-resistant isolates of P. aeruginosa limit treatment options, strengthening the need for new antimicrobials and alternative tools. A new collection of P. aeruginosa strains, selected from a respository of over 3,400 clinical isolates collected worldwide between 2003 and 2017, is now available to the research community focusing on multidrug-resistant P. aeruginosa.

This unique panel of 100 highly diverse, multidrug-resistant P. aeruginosa strains from the Multidrug Resistant Organism Repository and Surveillance Network (MRSN) within Walter Reed Army Institute of Research's Bacterial Diseases Branch represents isolates from each phylogenetic group with a wide-range of well-characterized antibiotic resistance patterns. Extensive data is available for each isolate, including isolation history, whole genome sequencing, characterization by mutilocus sequence typing, a comprehensive list of antibiotic resistance genes carried, and antibiotic susceptibilities to eleven clinically relevant antibiotics commonly used to treat P. aeruginosa infections: amikacin, aztreonam, ceftazidime, ciprofloxacin, cefepime, gentamicin, imipenem, levofloxacin, meropenem, tobramycin and piperacillin/tazobactam.

The P. aeruginosa MRSN Diversity Panel is available as both a complete kit containing all 100 strains as BEI Resources NR-51829, as well as individually.

References:

1. “Multidrug-resistant Pseudomonas aeruginosa.” Centers for Disease Control and Prevention, U. S. Department of Health and Human Services, https://www.cdc.gov/drugresistance/pdf/threats-report/pseudomonas-aeruginosa-508.pdf.
2. Cabot, G., et al. “Evolution of Pseudomonas aeruginosa Antimicrobial Resistance and Fitness under Low and High Mutation Rates.” Antimicrob. Agents Chemother. 60 (2016): 1767-1778. PubMed: 26729493.
3. Pang, Z., et al. “Antibiotic Resistance in Pseudomonas aeruginosa: Mechanisms and Alternative Therapeutic Strategies.” Biotechnol. Adv. 37 (2019): 177-192. PubMed: 30500353.

Image:  Illustration of multidrug-resistant Pseudomonas aeruginosa (CDC/Medical Illustrator: Jennifer Oosthuizen)

BEI Resources is pleased to announce a new and improved website!  We heard your feedback concerning difficult searches and long checkout times…and, we invite you to explore and discover our new dynamic search package, state-of-the-art shopping cart experience and enhanced product information pages for your items on interest.

Our new search engine provides the community with a user friendly interface in which to research, search, and order reagents. We will now be using the same search and navigation features of many of the world's largest internet sites. Some of the major search features include dynamic filter searching, Intuitive Auto-Complete Text Searches, Suggested Search Terms, Dynamic Clustering and multiple sorting parameters for result sets.

Your ordering experience is also vastly improved with our new Web Services E-Commerce software.  No more waiting for your items to load to the shopping cart; with the new software, your checkout is fast and reliable. Look for all these updates today!

Multidrug-resistant (MDR) isolates of S. enterica subsp. enterica serovar Typhi (S. typhi), the causative agent of typhoid fever, prevalent in parts of Asia and Africa are often associated with the dominant H58 haplotype, which harbors an IncHI1 plasmid with multiple resistance genes to first-line drugs, including ^blaTEM-1 (ampicillin), catA1 (chloramphenicol), dfrA7, sul1, sul2 (sulfamethoxazole/trimethoprim) and strAB (streptomycin) resistance genes.¹

The recent emergence of a novel, extensively drug-resistant (XDR) S. typhi H58 clone with additional resistance to fluoroquinolones and third-generation cephalosporins has been reported in Sindh, Pakistan.^1,2 This XDR clone encodes a chromosomally located resistance region and harbors the antibiotic resistance-associated IncY plasmid p60006, specific to XDR isolates in this phylogenetic branch.¹ This plasmid encodes additional elements, including the extended-spectrum beta-lactamase (^blaCTX-M-15) and fluoroquinolone (qnrS) resistance genes, and exhibited high sequence identity to plasmids found in other enteric bacteria, particularly Escherichia coli, isolated from widely distributed geographic locations.¹

Five strains of this XDR S. typhi clone from the Pakistan outbreak have been recently added to the BEI Resources catalog, expanding our collection of bacteria with unique resistance profiles to support antimicrobial resistance research:

References:

1. Klemm, E. J., et al. “Emergence of an Extensively Drug-Resistant Salmonella enterica Serovar Typhi Clone Harboring a Promiscuous Plasmid Encoding Resistance to Fluoroquinolones and Third-Generation Cephalosporins.” MBio 9 (2018): e00105-18. PubMed: 29463654.
2. Chatham-Stephens, K., et al. “Emergence of Extensively Drug-Resistant Salmonella Typhi Infections Among Travelers to or from Pakistan - United States, 2016-2018.” MMWR Morb. Mortal. Wkly. Rep. 68 (2019): 11- 13. PubMed: 30629573.

Image:  Computer-generated image of Salmonella serotype Typhi bacteria (CDC/Medical Illustrator)

Usutu Virus (USUV) is an emerging mosquito-borne flavivirus belonging to the Japanese encephalitis virus serocomplex, with a similar transmission cycle as West Nile virus.^2,3 USUV is maintained in the environment through an enzootic cycle between several bird and mosquito species, with humans and mammals as the incidental dead-end hosts.^2,3

First isolated in 1959 from Culex neavei mosquitos in South Africa, and named after the nearby Usutu River, USUV has spread from southern Africa to Europe by way of migratory birds, thought to be the major amplifying host, where it has caused significant avian outbreaks and infections in humans and horses.^1-3 Infections in humans, while low, range from mild and asymptomatic to serious neuroinvasive diseases like encephalitis and meningoencephalitis. Co-circulation with West Nile virus, confirmed in numerous European countries in mosquito, bird and horse species, and positive serology for both viruses reported in both birds and humans, raises epidemiological issues, furthering the need for research and diagnostic tools to address USUV as a potential human pathogen in the future.^2-4

BEI Resources supports this continuing research by offering two Usutu virus isolates available in the BEI Resources catalog: ENT MP 1626 (NR-51185), isolated in 1962 from Mansonia aurites mosquitoes in Zika Forest, Uganda, and the prototype African strain SAAR 1776 (NR-51184), the first isolation of the virus in 1959 from Culex neavei mosquitos in South Africa.

References:

1. McIntosh, B. M. “Usutu (SAAr 1776); Nouvel Arbovirus du Groupe B.” Int. Cat. Arboviruses 3 (1985): 367- 374.
2. Clé, M., et al. “Usutu Virus: A New Threat?” Epidemiol. Infect. 147 (2019): e232. PubMed: 31364580.
3. Roesch, F., et al. “Usutu Virus: An Arbovirus on the Rise.” Viruses 11 (2019): E640. PubMed: 31336826.
4. Nikolay, B. “A Review of West Nile and Usutu Virus Co-Circulation in Europe: How Much Do Transmission Cycles Overlap?” Trans. R. Soc. Trop. Med. Hyg. 109 (2015): 609-618. PubMed: 26286946.

Image:  Larvae of Culex Mosquitoes (CDC/James Gathany)

Acanthamoeba spp., Balamuthia mandrillaris and Naegleria fowleri are free-living amoebae inhabiting a wide variety of environmental niches worldwide, with the potential to cause infections in both humans and animals.

Acanthamoeba spp. and B. mandrillaris are opportunistic pathogens of the central nervous system, lungs, sinuses and skin, primarily in immunocompromised patients and, in severe cases, lead to the fatal disease granulomatous amebic encephalitis.^1-3 Among individuals wearing contact lenses, Acanthamoeba spp. is the causative agent of the sight-threatening infection Acanthamoeba keratitis.³ In contrast, N. fowleri, commonly known as the “brain-eating amoeba”, is pathogenic in healthy humans, causing acute and fulminant primary amoebic meningoencephalitis, a water-borne disease of the central nervous system resulting in extensive tissue damage, inflammation and hemorrhagic necrosis.⁴ Infection with N. fowleri is primarily associated with recreational activities in freshwater lakes and reservoirs naturally heated by the sun, as well as in geothermal sources and water thermally polluted by industry.^3,4

The availability of clinical isolates is essential for the advancement of research into the biology, mechanisms of pathogenesis, immunology, antimicrobial susceptibility and molecular characteristics of these amoebae. BEI Resources maintains a collection of free-living amoeba, currently totaling 50 isolates, deposited by the Free-Living & Intestinal Amebas Laboratory, Division of Foodborne, Waterborne, and Environmental Diseases, Centers for Disease Control and Prevention. Each isolate is cataloged with the geographical location, year and source of isolation and reported genotype. Please visit the BEI Resources website for a complete list of isolates in our free-living amoeba catalog.

References:

1. Marciano-Cabral, F. and G. Cabral. ”Acanthamoeba spp. as Agents of Disease in Humans.” Clin. Microbiol. Rev. 16 (2003): 273-307. PubMed: 12692099.
2. Cope, J. R., et al. “The Epidemiology and Clinical Features of Balamuthia mandrillaris Disease in the United States, 1974-2016.” Clin. Infect. Dis. 68 (2019): 1815-1822. PubMed: 30239654.
3. Visvesvara, G. S. “Amebic Meningoencephalitides and Keratitis: Challenges in Diagnosis and Treatment.” Curr. Opin. Infect. Dis. 23 (2010): 590-594. PubMed: 20802332.
4. Siddiqui, R., et al. “Biology and Pathogenesis of Naegleria fowleri.” Acta Trop. 164 (2016): 375-394. PubMed: 27616699.

Image:  Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri (BEI Resources)

The “Asian Longhorned Tick”, Haemaphysalis longicornis, is invasive in the United States, first identified in New Jersey in 2017, and is rapidly spreading. While human pathogens have not yet been identified within H. longicornis found within the United States, the vector is known to be competent for transmission of a number of zoonotic tick-borne disease pathogens, including Borrelia, Rickettsia, Erlichia and Anaplasma species, Powassan virus and several Babesia parasites.^1-4 In China, H. longicornis is a vector of the severe fever with thrombocytopenia syndrome (SFTS) virus, an emerging phlebovirus with high mortality rates.⁵

H. longicornis is especially of agricultural concern with cattle and sheep, transmitting tropical theileriosis, a potentially fatal parasitic infection in cattle in southern Europe, Africa and Asia. In sheep, H. longicornis infestation impacts both the quality and quantity of wool. This disease host is known to be difficult to control and is unique among ticks in the ability of the females to reproduce by parthenogenesis (full development of unfertilized eggs), as well as by sexual reproduction.

As of August 1, 2019, Asian longhorned ticks have spread to nearly one quarter of the United States, affecting Arkansas, Connecticut, Delaware, Kentucky, Maryland, North Carolina, New Jersey, New York, Pennsylvania, Tennessee, Virginia and West Virginia.⁶

The newly available H. longicornis colony established from a wild-type, parthenogenic strain deposited by Dr. Michael Levin, Centers for Disease Control and Prevention, is confirmed specific-pathogen-free (SPF) by molecular screening and is available to qualified insectaries in the larval, nymph and adult life stages:

References:

1. Lee, M.-J. and Joon-Seok Chae. “Molecular Detection of Erhlichia chaffeensis and Anaplasma bovis in the Salivary Glands from Haemaphysalis longicornis Ticks.” Vector Borne Zoonotic Dis. 10 (2010): 411-413. PubMed: 19874189.
2. Meng, Z., et al. “[Detection of Co-Infection with Lyme Spirochetes and Spotted Fever Group Rickettsiae in a Group of Haemaphysalis longicornis].” Zhonghua Liu Xing Bing Xue Za Zhi [Chinese Journal of Epidemiology] 29 (2008): 1217-1220. PubMed: 19173967.
3. Hoogstraal, H. “Changing Patterns of Tickborne Diseases in Modern Society.” Annu. Rev. Entomol. 26 (1981): 75-99. PubMed: 7023373.
4. Heath, A. C. G. “Vector Competence of Haemaphysalis longicornis with Particular Reference to Blood Parasites.” Surveillance 29 (2002): 12-14. http://www.sciquest.org.nz/node/47255.
5. Yu, X. J., et al. “Fever with Thrombocytopenia Associated with a Novel Bunyavirus in China.” N. Engl. J. Med. 364 (2011): 1523-1532. PubMed: 21410387.
6. “What You Need to Know About Asian Longhorned Ticks - A New Tick in the United States.” Centers for Disease Control and Prevention, U. S. Department of Health and Human Services, //www.cdc.gov/ticks/longhorned-tick/index.html.

Image:  Adult female Haemaphysalis longicornis tick (CDC/James Gathany)

Now available are two well-characterized reference reagents for Zika virus (ZIKV) to facilitate evaluation of existing nucleic acid testing (NAT) assays and development of novel ZIKV assays. These reagents were produced by The Center for Biologics Evaluation and Research/U.S. Food and Drug Administration (CBER/FDA) from heat-inactivated (HI) ZIKV culture supernatant stock from two strains, PRVABC59 and FSS13025, which were diluted in dialyzed, defibrinated human plasma, and lyophilized. These reagents add to the expansive catalog of Zika virus materials now available from BEI Resources.

Image:  Female Aedes aegypti mosquito (CDC/James Gathany)

Coccidioidomycosis, also known as Valley fever, is an invasive fungal infection caused by the dimorphic fungi Coccidioides immitis and Coccidioides posadasii. These fungi are endemic to arid or semi-arid regions of the southwestern United Sates, Mexico, and Central and South America. Infection is acquired through inhalation of air-dispersed fungal spores termed arthroconidia. The initial or acute form of coccidioidomycosis is often mild with symptoms such as fever, cough, chest pain, and red, spotty rash. If infection is not controlled, it may progress to chronic pneumonia or even disseminate to other parts of the body including the brain, especially in immunocompromised patients. BEI Resources houses a well-characterized collection of Coccidioides strains from various origins, which have been used in studies of the ecology and distribution of different genotypes of the pathogen. Such studies are important for monitoring outbreaks, determining changes in virulence, and predicting disease progression. These strains were generously contributed by Dr. Bridget Barker, Northern Arizona University.

Image:  Colonies of Coccidioides immitis, Strain RMSCC 3476 (NR-48939) growing at 37°C on Emmons' Modification of Sabouraud agar.

BEI Resources continues to expand the catalog of luciferase-expressing parasitic protists. Recent additions include Leishmania mexicana, strain MNYC/BZ/62/M379 transfected with the luciferase gene from the sea pansy Renilla reniformis (NR-51210) and Trypanosoma cruzi, strain CL Brener expressing the thermostable red-shifted luciferase gene mutant, PpyRE9 (NR-49161). These models allow the high-throughput screening of large numbers of candidate compounds during infection in vitro and the possibility of tracking parasite distribution in vivo in laboratory mice. The expression of red-shifted luciferase in T. cruzi, in particular, results in a reporter system that is more sensitive than other bioluminescence systems previously reported. The availability of these new transgenic strains provides the researcher rapid quantification of parasite growth and simplification of the methodology for scoring inhibitor assays.

Reference:

Lewis, M.D., et al. “A New Experimental Model for Assessing Drug Efficacy Against Trypanosoma cruzi Infection Based on Highly Sensitive in vivo Imaging.” J. Biomol. Screen. 20 (2015): 36-43. PubMed: 25296657.

Image:  Trypanosoma cruzi CL Brener PpyRE9 (NR-49161) epimastigotes stained with GIEMSA (BEI Resources)

Burkholderia pseudomallei, the causative agent for melliodiosis, is an opportunistic pathogen found in soil in tropic and subtropic regions worldwide.  B. pseudomallei is classified as a category B, Tier 1 Select Agent in the United States due to a high mortality rate when untreated, and the potential for its use as a biowarfare agent through aerosol transmission. Most strains of B. pseudomallei require handling under Biosafety level 3 (BSL 3). B. pseudomallei, strain Bp82 (deltapurM) has a 109 base pair deletion within the purM open reading frame, rendering the strain avirulent in mouse and hamster models.  B. pseudomallei, strain Bp82 (deltapurM) is now an Excluded Strain from the HHS and USDA Select Agents and Toxins ­Exclusions list and can be handled under BSL 2 conditions. Strain Bp82 (deltapurM) has been proposed as a vaccine candidate and has been used in antibiotic resistance studies.  This strain was graciously deposited into BEI Resources by Dr. Herbert Schweizer, and is available as item number NR-51280.

Reference:

Propst, K. L., et al. “A Burkholderia pseudomallei deltapurM Mutant is Avirulent in Immunocompetent and Immunodeficient Animals: Candidate Strain for Exclusion from Select-Agent Lists.” Infect. Immun. 78 (2010): 3136-3143. PubMed: 20404077.

Image:  Burkholderia pseudomallei culture on sheep blood agar (CDC/ Larry Stauffer)

General Information about the Schistosoma spp. Life Cycle Training Course

Biomedical Research Institute

12111 Parklawn Drive Rockville, MD.

May 21-23, 2014

This course is designed to acquaint investigators working in the field of schistosomiasis with basic techniques and strategies for maintaining Schistosoma mansoni, S. haematobium, and S. japonicum in the laboratory, and it offers directions into making one’s research more productive.  Among the topics covered will be procedures for setting up and maintaining the complete S. mansoni/Biomphalaria glabrata life cycle in the research laboratory, an overview of schistosomiasis, the pathology of the disease, collection of adult worms and eggs from tissues, exposure of snails to miracidia, shedding snails, exposure of mice and hamsters to cercariae, and an examination of all the intramolluscan life stages.  Hands-on exercises relating to some of these topics will also be presented (ex. perfusion of mice, exposing mice/hamsters, identification of sporocysts in snails).  Since we first began offering this basic course, over 120 scientists, technicians and students have benefited from completion of the course, and have expanded their own research agendas once back in their own laboratories.

The workshop incorporates aspects of all three Schistosoma spp. life cycles.  Attendees will be exposed to aspects of maintaining S. japonicum and S. haematobium life cycles that include general snails maintenance, collection of cercariae from infected snails, and exposing mice/hamsters.  It is our hope that incorporating the other two life cycles will encourage interest in maintaining these species in attendees’ laboratories.  The three-day course will also include a lecture on NIH-NIAID parasitology and grant programs. 

We anticipate that attendees will come away with a greater appreciation of the basic biology of Schistosoma spp. parasites, greater confidence in maintaining life cycles, and they will learn strategies for optimizing life cycles in their home laboratories.  Time will be available to ask questions about life cycle maintenance and other aspects of schistosomiasis research. There is no fee for the course.  Attendees are responsible for travel and lodging expenses, but travel assistance may be available. If you have any questions about the course, please contact me at mtucker@afbr-bri.com or (301) 881-3300 x31, or Mitzi Sereno at msereno@afbr-bri.com or (301) 881-3300 x39.

Chagas’ disease caused by the protozoan parasite Trypanosoma cruzi (T. cruzi) is widespread in Central and South America infecting an estimated 12 to 14 million people, with infection often leading to chronic myocarditis resulting in high morbidity and mortality. In addition to our catalog of T. cruzi parasites and triatomine vectors, BEI Resources now offers monoclonal antibodies raised against parasite stage-specific surface antigens, which play important roles during infection. These antigens include the trypomastigote-specific antigen Ssp-1 (BEI Resources NR-50891) and the amastigote-specific surface antigen Ssp-4 (BEI Resources NR-50892). Studies have shown the presence of Ssp-4 in tissue culture supernatants of T. cruzi-infected cells and the presence of anti-Ssp4 antibodies in sera from chagasic patients. These monoclonal antibodies were generously contributed by Dr. Norma Andrews, University of Maryland.  Dr. Andrews generated these monoclonal antibodies while at the laboratory of Dr. Ruth Nussenzweig, New York University.
Image:  Immunofluorescence staining of amastigotes of Trypanosoma cruzi, strain TcVT-1 (BEI Resources NR-36630) with Monoclonal Anti-Trypanosoma cruzi, NR-50892. A, Light microscopy image of T. cruzi-infected kidney epithelial cells (ATCC^® CCL-26™). B, Immunostaining of intracellular amastigotes with NR-50892 labeled with a FITC-conjugated rabbit anti-mouse antibody. Host cell and parasite nuclei are stained with DAPI.

References:

1. Andrews, N. W., et al. “Stage-Specific Surface Antigens Expressed during the Morphogenesis of Vertebrate Forms of Trypanosoma cruzi.”  Exp. Parasitol. 64 (1987):  474-484.  PubMed:  3315736.
2. Andrews, N. W., et al. “Developmentally Regulated, Phospholipase C-Mediated Release of the Major Surface Glycoprotein of Amastigotes of Trypanosoma cruzi.”  167 (1988):  300-314.  PubMed:  3279152.

Enterobacter cloacae is part of the normal human flora and not considered a primary pathogen. Unfortunately, there is increasing incidence of nosocomial infections with E. cloacae, which frequently involve intravenous and other medical and hospital devices as well as medical solutions and disinfectants used routinely in hospitals and patient care facilities. The situation is further complicated by the ability of these bacteria to acquire resistance to antibiotics, notably carbapenems. BEI Resources has obtained a set of 15 isolates of the E. cloacae complex with defined susceptibility patterns against 16 antibiotics. This valuable set is now available to aid your studies on carbapenem-resistant Enterobacteriaceae (CRE).

Mayaro virus (MAYV) is a New World alphavirus and the etiologic agent of Mayaro fever, an acute febrile illness sometimes accompanied by severe and persistent arthritis similar to Chikungunya fever. Its transmission cycle occurs mainly through mosquito vectors, especially those of the genus Haemagogus, but Aedes spp. mosquitoes may also be competent vectors. The enzootic transmission cycle of MAYV is not fully understood, but the occurrence of relatively large outbreaks of Mayaro fever and the competence of Aedes mosquitoes for the transmission of MAYV suggest the potential for an urban human-mosquito-human transmission cycle to emerge. MAYV was first isolated in Trinidad in 1954, with sporadic outbreaks of Mayaro fever in Bolivia, Brazil, and other regions throughout Central and South America. Recently, MAYV was found in a DENV co-infected patient in Haiti¹, the first report of MAYV in the Caribbean nation, underscoring the need for MAYV inclusion in surveillance activities in these regions in addition to Dengue, Chikungunya, and Zika viruses.

Una virus (UNAV) is the closest genetic relative of MAYV that also causes a similar febrile illness. UNAV was first identified in northern Brazil in 1959. Serosurveys have identified antibodies against UNAV in birds, horses, and humans; however, the extent of viral distribution and human disease risk is unknown.

References:1. eid2211.161015

Plasmid maintenance by bacteria can be unpredictable without selective pressure. This is of particular concern during in vivo studies. To circumvent this in Staphylococcus species, the Bose Lab at The University of Kansas Medical Center has provided plasmids to BEI Resources that remain stable inStaphylococcus cells without the requirement of antibiotics during both in vitro and in vivo experiments¹. These E. coli-S. aureus shuttle vectors are based on the LAC-p01 plasmid.

Plasmid pKK22 (Accession KX085042) is designed to be used with S. aureus USA300 strains that contain LAC-p01 and will render the strains isogenic. Plasmid pKK30 (Accession KX085043) is a variant of pKK22, in which the predicted open reading frames (ORFs) that are not needed for plasmid maintenance have been deleted, and is intended for use inStaphylococcus cells that do not contain LAC-p01. Both plasmids contain a single trimethoprim resistance cassette that is functional in both E. coliand Staphylococcus. In addition, they contain the E. coli R6Kγ origin of replication and require pir+ cells for replication. DH5αλpir and the pir+dam- dcm- strain GM2163λpir are provided as a host strains, courtesy of Dr. Eric Stabb (University of Georgia)². These plasmids and host strains are now supplied individually or as a kit through BEI Resources.

References:

1. Krute, C. N., K. K. Krausz, M. A. Markiewicz, J. A. Joyner, S. Pokhrel, P. R. Hall, and J. L. Bose. 2016. "Generation of a Stable Plasmid for in vitro and in vivostudies of Staphylococcus." Applied and Environmental Microbiology 82:6859-6869. PMID 27637878

2. Dunn A. K., M. O. Martin, and E. V. Stabb. 2005. "Characterization of pES213, a Small Mobilizable Plasmid from Vibrio fischeri." Plasmid 54:114-134. PMID: 16122560

Clostridium difficile research is easier with strains from BEI Resources

Clostridium difficile (also known as Peptoclostridium difficile) is an increasingly common opportunistic pathogen that causes diarrhea and more serious intestinal conditions such as colitis. It is commonly observed in patients that have an illness that requires prolonged use of antibiotics, or can be acquired in hospitals and long-term care facilities. C. difficile infections are normally treated with antibiotics; however, this is particularly problematic due to the high levels of antibiotic resistance displayed by these bacteria. Recent studies have shown that replenishment of the normal gut microbiota provides a successful tool to control the infection with C. difficile.
BEI Resources has a large number of C. difficile strains, as well as nucleic acids, toxins, and other reagents available for your research (please click here for a list).
Among the bacterial strains, several have well characterized tcdA, tcdB, and cdtB toxin

For the benefit of researchers requiring nucleic acids from pathogens, BEI Resources offer more than 1,000 nucleic acids derived from pathogens. These reagents can be used at lower containment levels for molecular studies; to ensure their biosafety, the preparations have been safety tested for the absence of residual live organisms. Please check our website to see the wide variety viral nucleic acids extracted from various BSL-2 through BSL-4 pathogens. Notable new additions to this category of reagents include Genomic RNA from Enterovirus D68 and human metapneumovirus.

Whooping cough is a disease caused by bacteria from the genus Bordetella. Currently under widespread resurgence, whooping cough ranks globally among the 10 leading causes of childhood mortality. BEI Resources offers several isolates of Bordetella pertussis, B. bronchiseptica, and B. holmesii, along with toxins, nucleic acids, and proteins for Bordetella research. To see the full list of Bordetella reagents and strains currently available from BEI resources, please click here

Image:  Bordetella holmesii NR-43499 grown on Bordet Gengou media

Toxoplasma gondii is an obligate intracellular protozoan parasite of the phylum Apicomplexa that is the causal agent of toxoplasmosis. BEI Resources houses a comprehensive assortment of T. gondii cultures that include GFP-expressing strains; reference genome strains; genetic crosses between type strains I, II, and III; and strains used as models for parasite transmission and cyst formation. For a complete list of our Toxoplasma catalog, please contact us at Contact@BEIResources.org.

¹ Available soon. Send inquiries to Contact@BEIResources.org for availability.

BEI Resources offers 42 distinct Escherichia coli-Staphylococcus cassette-based shuttle vectors that provide all the tools you need to complete your research. These shuttle vectors are deposited as part of the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) reagents. Choose from staphylococcal low-copy-number, high-copynumber, thermosensitive, and theta-replicating plasmids. Select from erythromycin, tetracycline, chloramphenicol, kanamycin, or spectinomycin selectable markers in Staphylococcus, and ampicillin, erythromycin, kanamycin, and chloramphenicol resistance markers in E. coli. The modular design of the plasmid vector system offers flexibility and variety for expression analysis and provides the opportunity to address questions about gene dosage, complementation, and cis-trans effects. The shuttle vector system provides effective tools for studying gene regulation of staphylococci among ecosystems.

For further published details on these plasmids, please see Charpentier et al. Appl

Environ Microbiol 70(10): 6076-6085, 2004

Image:  NR-46158. Staphylococcus aureus strain 4221 containing shuttle vector pCN57, grown on TSA + 10 μg/ml erythromycin

.

Two genome sequenced isolates of Mucor circinelloides f. circinelloides (NR-49108 Strain designation 1006PhL, and NR-49117 Strain designation Mucho) are now available from BEI Resources. Mucor circinelloides f. circinelloides is surpassed only by Rhizopus species as the most common cause of mucormycosis, and is the most virulent of the Mucor circinelloides species. One isolate was obtained from the skin of a healthy individual, and the other from contaminated yogurt responsible for the 2013 Mucorales outbreak.

We have a number of expression plasmids for various Filoviral proteins for Zaire Ebolavirus and Marburgvirus. Many of the proteins have been tagged with HA or FLAG. Continue to check our catalog for new plasmid additions, such as those expressing GP, NP, VP24, VP30, VP35, and VP40 in the next few months for a complete list.

The agent of bubonic plague Yersinia pestisis a NIAID Category A Priority Pathogen and likely biothreat. In support of research and product development in this area BEI Resources released a Yersinia pestis Plasmid Detection Kit (NR-9562) in April 2008.   

This assay allows the researcher to quickly identify the presence of plasmid-encoded virulence factors pPCP1, pMT1 and pCD1 and is designed to be used for characterization purposes in conjunction with standard microbiological techniques.

The assay consists of primer sets designed to specifically detect the pPCP1, pMT1 and pCD1 plasmids using standard polymerase chain reactions and includes a species-specific positive control primer set and internal positive control template.

PCR amplification of genomic DNA from Yersinia pestis displaying a pPCP1+, pMT1+, pCD1– profile (lanes 2-4 respectively). Lanes 5 and 6 are the Yersinia pestis and internal positive controls. Lane 7 is a non-template control."

Panel A. Growth of Yersinia pestis on Congo Red media showing red/orange colonies typical of pgm+ organisms. Organisms that are confirmed pgm+ and pCD1+ are considered Select Agents.

Panel B. Bacteria lacking pgm showing distinctive variations in colony morphology and reduced coloring on Congo Red media as compared to pgm+ organisms.

For technical questions about these products or to learn more, please contact our Technical Services Department.

Registration for the BEI Resources program can be found at this link below. NARSA Strain requestors will need to register for Level 2 for continued access to the NARSA Strains. ** Please note if you are an existing NARSA registrant, BEI Resources will contact you after the Annual Meeting with specific registration instructions and paperwork to facilitate your registration.

http://www.beiresources.org/Register.aspx

Once registered with BEI Resources, researchers will have access to the full BEI Resources catalog (within the respective Biosafety Level). The catalog has over 12,000 research reagents representing more than 400 Genus and Species focusing on NIAID's Category A, B and C priority pathogen list and Emerging Infections.

Once registered with BEI Resources, ALL investigators will receive FREE shipping and handling on orders placed through BEI Resources.

Please feel free to contact BEI Resources by email: contact@beiresources.org or by phone: Toll-free telephone number: (800) 359-7370 (8:30 AM to 4:30 PM Eastern Time) with any questions or concerns.

The much awaited H7N9 Influenza virus is now available for your research needs. NR-44078 is a 6:2 reassortant virus produced using plasmid-based reverse genetics to contain the hemagglutinin and neuraminidase genes from A/Shanghai/1/2013 (H7N9) and internal genes from A/Puerto Rico/8/1934 (H1N1).

Click here to see all H7N9 reagents.

As of Wednesday, August 20th we have updated the BEI Resources and MR4 websites to further optimize your online experience; making it easier, more accessible, and hopefully a bit more pleasurable to access your desired BEI and MR4 reagents. We have rolled out a new redesigned website that we anticipate will provide a more user-friendly navigation and shopping cart experience, easier searching and reading throughout the site, and an optimal viewing experience from essentially any web accessible platform whether it is desktop, laptop, mobile phone, or tablet.

This Responsive Web Design approach suggests that design and development should respond to the user’s behavior and environment based on screen size, platform, and orientation. As more and more of our customers in the scientific community are switching to the duality of laptops and mobile devices to conduct and support their research efforts, we at BEI Resources, would like to let our customers know… We’ve heard you… and would like to thank you for your continued feedback.

This redesigned website has been engineered to automatically respond to user preferences; switching to accommodate for resolution, image size and the scripting capabilities as dictated by the device being used. We can now make images and layouts more flexible. With this design, we have essentially eliminated the need to make individual adjustments in design and development for each new mobile device produced and made available on the market.

While Responsive Design is not a complete solution, we believe this solution provides more options for our users and a more optimal online experience overall when accessing the BEI Resources and MR4 sites; from any website accessible device; mobile or otherwise.

So feel free to access the BEI Resources and MR4 sites while in the office, lab, or on-the-go with your mobile device. An option to provide feedback is available through our Online User Feedback form, provided at the completion of your online order. Tell us what you think; we look forward to hearing from you.

At MR4 and BEI Resources, “We take care of the details - so you can focus on your research.”  

BEI Resources has the MERS-CoV virus (strain EMC/2012) available for distribution to qualified laboratories.  This virus will require a BEI Level 3 registration.  Middle East Respiratory Syndrome (MERS) is viral respiratory illness first reported in Saudi Arabia in 2012. It is caused by a coronavirus called MERS-CoV. Most people who have been confirmed to have MERS-CoV infection developed severe acute respiratory illness. They had fever, cough, and shortness of breath. About half of these people died.

The catalog number for the MERS-CoV virus is NR-44260. You must be logged in with a valid BEI Level 3 Account to place an order. Please email our customer service department if you have any questions about obtaining the MERS-CoV virus.

We have available for the first time in the BEI Resources catalog, two SARS Coronaviruses (SARS-CoV) derived from the Urbani strain. NR-18925 is produced from a recombinant infectious clone, icSARS-CoV. NR-15418 is the mouse-adapted strain, Urbani v2163, developed by Dr. Barnard as a lethal model for evaluating antiviral agents (PubMed:19853271). To view a complete listing of all coronaviruses in the catalog, click here.

Chikungunya Viruses - Additional Strains Available

BEI Resources announces the addition of two novel BSL3 strains of Chikungunya virus (CHIKV), R-91142 and 182/25, to the catalog of available items. CHIKV is a mosquito-borne alphavirus that is mainly associated with acute febrile illness. Strains R-91142 and 182/25 CHIKV are of Asian origin. Strain 182/25 is the recently developed vaccine candidate and the only CHIKV strain to be tested in humans. Coming soon to the BEI catalogue is the CHIKV African prototype strain, S-27.

Click Here to view our Upcoming Events Calendar.

BEI Resources announces the addition of Sand Flies, Reduviids and Ticks for the research community.

CDC recommends handling naïve ticks from these species at ABSL2 containment in appropriate barrier protected facilities; pathogen research may require elevated ABSL containment. Check the BEI Vector Resources page for a list of other vectors and related reagents available through BEI Resources.