Microbiology Commons^™

Evolution Of Subtype C Hiv-1 Env In A Slowly Progressing Zambian Infant, Hong Zhang, Federico Hoffmann, Jun He, Xiang He, Chipepo Kankasa, Ruth Ruprecht, John T. West, Guillermo Orti, Charles Wood

Nebraska Center for Virology: Faculty Publications

Background: Given the high prevalence of mother to child infection, the development of a better understanding of African subtype C HIV-1 transmission and natural evolution is of significant importance. In this study, we genotypically and phenotypically characterized subtype C viruses isolated over a 67-month follow-up period from an in utero-infected Zambian infant. Changes in genotype and phenotype were correlated to alterations of the host humoral immune response.

Results: A comparison of baseline maternal and infant samples indicated that the infant sequences are monophyletic and contain a fraction of the diversity observed in the mother. This finding suggests that selective …

Epidemiological Characteristics Of Human Herpesvirus-8 Infection In A Large Population Of Antenatal Women In Zambia, W. Klaskala, B. P. Brayfield, C. Kankasa, G. Bhat, J. T. West, C. D. Mitchell, Charles Wood

Nebraska Center for Virology: Faculty Publications

Comprehensive data describing epidemiological characteristics of the human herpesvirus-8 or Kaposi’s sarcoma-associated herpesvirus (HHV-8 or KSHV) infection among pregnant women in a central sub-Saharan Africa are not available. This study determined virus prevalence estimates and the risk factors associated with HHV-8 infection. Cross-sectional, enrollment visit data were analyzed from a prospective cohort study of perinatal transmission of HHV-8 in Lusaka, Zambia. Exposure data were obtained via structured interview, physical examination, medical chart review, and laboratory testing. Among 3,160 antenatal women serologically screened for HHV-8 between September 1998 and October 2000, 40.2% were seropositive. The HHV-8 positive women were more likely …

Compartmentalization Of Hiv-1 In The Central Nervous System: Role Of The Choroid Plexus, Evan J. Burkala, Jun He, John T. West, Charles Wood, Carol K. Petito

Nebraska Center for Virology: Faculty Publications

Objectives: To determine the genetic and phenotypic composition of HIV-1 found in the choroid plexus (CPx) and its relationship to virus in the brain and peripheral lymphoid tissue. Design: Phenotypic and molecular comparisons of HIV-1 found in autopsy brain, CPx, and spleen tissues. Methods: HIV-1 was co-cultured from matched postmortem brain (basal ganglia), CPx, and spleen tissues of AIDS patients with and without HIV-associated encephalitis and dementia. Viral phenotypes were determined by infection of monocyte-derived macrophages, MT-2 or co-receptor-specific cell lines. Viral env and pol sequences were determined from genomic DNA isolated directly from tissues or co-cultures, and phylogenetic comparisons …

Dichotomy In Cross-Clade Reactivity And Neutralization By Hiv-1 Sera: Implications For Active And Passive Immunotherapy, Lisa A. Cavacini, Mark Duval, Ajay Patil, Charles Wood, Kanneth H. Mayer, Ruth M. Ruprecht, Marshall L. Posner

Nebraska Center for Virology: Faculty Publications

The identification of broadly reactive and cross-clade neutralizing antibodies will facilitate the development of a more universally effective vaccine for human immunodeficiency virus (HIV). Antibodies in sera from individuals infected with Clade B HIV bind native primary viral isolates, and virus binding correlates with neutralization and stable clinical disease. In this study, we quantified cross-clade antibody reactivity and neutralization by Clades B and C sera. Primary viral isolates were captured by serum IgG bound to anti-human IgG and quantitated as p24 released by lysis of captured virus. Neutralization was determined using PHA-stimulated PBMC. Clade B antibodies reacted more frequently with …

Periplasm Organization In Treponema Denticola As Studied By Cryo-Electron Tomography, Jacques Izard, C.-E. Hsieh, C. A. Mannella, Ronald J. Limberger, M. Marko

Department of Food Science and Technology: Faculty Publications

As a spirochete, the genus Treponema is one of the few major bacterial groups whose natural phylogenic relationships are evident at the level of gross phenotypic characteristics such as their morphology. Treponema spp. are highly invasive due to their unique motility in dense media, and their ability to penetrate cell layers. This feature is associated with the helical cell body and the presence of flagellar filaments in the periplasm. Treponema denticola is an oral pathogen involved in endodontic infections and periodontal diseases. The presence and quantity of T. denticola in the subgingival biofilm is correlated with the severity of periodontal …

High-Temperature Fluorescent In Situ Hybridization For Detecting Escherichia Coli In Seawater Samples, Using Rrna-Targeted Oligonucleotide Probes And Flow Cytometry, Ying Zhong Tang, Karina Yew Hoong Gin, Tok Hoon Lim

OES Faculty Publications

Fluorescence in situ hybridization (FISH) is a widely used method to detect environmental microorganisms. The standard protocol is typically conducted at a temperature of 46°C and a hybridization time of 2 or 3 h, using the fluorescence signal intensity as the sole parameter to evaluate the performance of FISH. This paper reports our results for optimizing the conditions of FISH using rRNA-targeted oligonucleotide probes and flow cytometry and the application of these protocols to the detection of Escherichia coli in seawater spiked with E. coli culture. We obtained two types of optimized protocols for FISH, which showed rapid results with …

Ridding Ships' Ballast Water Of Microorganisms, Fred C. Dobbs, Andrew Rogerson

OES Faculty Publications

( First paragraph) A complex assemblage of microorganisms exists in nearly every aquatic system on earth. In lakes and oceans, every milliliter of water contains about 10² protists (single-celled eukaryotes), 10⁶ bacteria, and 10⁷–10⁹ viruses. Therefore, billions of microorganisms inevitably enter ships’ ballast tanks during normal operations. It has been argued that microorganisms must certainly be frequent invaders of coastal ecosystems, given the high densities of bacteria and viruses in ballast water—10⁸ and 10⁹ organisms per liter, respectively (1)—their potentially high reproductive rates, broad tolerances to physical conditions, and ability to form resting …

Efficacy Of Innovative Anti-Microbial Decontamination Of Minimally Processed Vegetables, Christina Busquets I Tortosa

Masters

Chlorine has widespread use commercially as a disinfectant wash for raw vegetables. However, it is an extremely corrosive gas and it may have severe health and environmental implications. The aim of this thesis was to find safe, alternative decontamination treatments for ready-to-eat Iceberg lettuce. The efficacy of 1% acetic acid, 2% citric acid; calcium lactate concentrations of 0.5%, 1.5%, 2.5% and 3% at wash temperature of 4,25 and 50°C, ozone alone (1 mg 1-1) and in combination with calcium lactate (2.5%) were compared with chlorine as decontaminating treatments. Microbiological analyses were performed following decontamination treatments using the pour plate method …

Identification Of Proteins Involved In Chloroplast Dna Replication, Matthew G. Lassen

Theses and Dissertations

Chapter 1

Chloroplast nucleoids (ct-nucleoids) are DNA/protein complexes involved in compacting the chloroplast genome, and may play a role in regulating DNA replication. Ct-nucleoids were isolated from young soybean plants and separated by 2-D gel electrophoresis. Gel spots were excised and analyzed by MALDI-ToF mass spectrometry, resulting in several protein identifications. The proteins identified all have functions unrelated to DNA replication. While some of these proteins may be due to contamination, it is possible that some of these proteins are dual-functional, playing direct roles in the regulation of DNA replication.

Chapter 2

A 28 kDa soybean protein was isolated by …

Association Of Dc-Sign Promoter Polymorphism With Increased Risk For Parenteral, But Not Mucosal, Acquisition Of Human Immunodeficiency Virus Type 1 Infection, Maureen P. Martin, Michael M. Lederman, Holli B. Hutcheson, James J. Goedert, George W. Nelson, Yvette Van Kooyk, Roger Detels, Susan Buchbinder, Keith Hoots, David Vlahov, Stephen J. O'Brien, Mary Carrington

Biology Faculty Articles

There is considerable debate about the fundamental mechanisms that underlie and restrict acquisition of human immunodeficiency virus type 1 (HIV-1) infection. In light of recent studies demonstrating the ability of C type lectins to facilitate infection with HIV-1, we explored the potential relationship between polymorphisms in the DC-SIGN promoter and risk for acquisition of HIV-1 according to route of infection. Using samples obtained from 1,611 European-American participants at risk for parenteral (n = 713) or mucosal (n = 898) infection, we identified single-nucleotide polymorphisms in the DC-SIGN promoter using single-strand conformation polymorphism. Individuals at risk for parenterally acquired …

Enterotoxin B Subunit Lectins As Adjuvants For Improvement Of Mucosal Vaccine, Nak-Won Choi

Loma Linda University Electronic Theses, Dissertations & Projects

In comparison with whole organism vaccines, subunit vaccines may be safer for immunization but may lack sufficient immunogenicity to provide complete immunity to the pathogen. To resolve this problem, bacterial and plant enterotoxin B subunit adjuvants containing a variety of receptor-binding properties were used to enhance the immunogenicity of rotavirus subunit vaccines. Enterotoxin B subunit adjuvants were employed to enhance protection against virus infection. Pentameric cholera toxin B subunit (CTB), shiga toxin-1 B subunit (STB) and monomeric ricin toxin B subunit (RTB) molecules were genetically linked to a 90 amino acid peptide from the simian rotavirus (SA11) nonstructural protein NSP4 …

The Potential Role Of Ledgf/P75 In Prostate Cancer, Tracy Ruth Daniels

Loma Linda University Electronic Theses, Dissertations & Projects

The serum autoantibody repertoire from cancer patients is currently being exploited for the identification of tumor associated antigens (TAA) and the design of TAA arrays. Such arrays would facilitate autoantibody profiling and potentially aid in the serological diagnosis and prognosis of tumors. The goal of this study was to identify and characterize prostate-specific TAA. We observed that while the general frequency and titers of autoantibodies in PCa patients were relatively similar to those in matched controls, significant differences could be detected between the two groups in the autoantibody response to the lens epithelial-derived growth factor p75 (LEDGF/p75). LEDGF/p75 is a …

Interferon Regulatory Factor 7 Is Associated With Epstein-Barr Virus-Transformed Central Nervous System Lymphoma And Has Oncogenic Properties, Luwen Zhang, Jun Zhang, Que Lambert, Channing J. Der, Luis Del Valle, Judith Miklossy, Kamel Kahlili, You Zhou, Joseph S. Pagano

Nebraska Center for Virology: Faculty Publications

EBV transforms primary B cells, and the major EBV oncoprotein, latent membrane protein 1 (LMP-1), is required for the process. LMP-1 both induces the expression of IRF-7 and activates the IRF-7 protein by phosphorylation and nuclear translocation. Here we report that the expression of IRF-7 is increased in EBV-immortalized B lymphocytes compared with that in primary B cells. IRF-7 was phosphorylated and predominantly localized in the nucleus in the immortalized cells. The expression of IRF-7 was detected in 19 of 27 specimens of primary lymphomas of the human central nervous system by immunohistochemical analysis. The association between LMP-1 and IRF-7 …

Infectious Molecular Clone Of A Recently Transmitted Pediatric Human Immunodeficiency Virus Clade C Isolate From Africa: Evidence Of Intraclade Recombination, Ricky D. Grisson, Agnes-Laurence Chenine, Lan-Yu Yeh, Jun He, Charles Wood, Ganapati J. Bhat, Weidong Zu, Chipepo Kankasa, Ruth M. Ruprecht

Nebraska Center for Virology: Faculty Publications

Although human immunodeficiency virus type 1 (HIV-1) clade C continues to dominate the pandemic, only two infectious clade C proviral DNA clones have been described (N. Mochizuki, N. Otsuka, K. Matsuo, T. Shiino, A. Kojima, T. Kurata, K. Sakai, N. Yamamoto, S. Isomura, T. N. Dhole, Y. Takebe, M. Matsuda, and M. Tatsumi, AIDS Res. Hum. Retrovir. 15:1321–1324, 1999; T. Ndung’u, B. Renjifo, and M. Essex, J. Virol. 75:4964–4972, 2001). We have generated an infectious molecular clone of a pediatric clade C strain, HIV1084i, which was isolated from a Zambian infant infected either intrapartum or through breastfeeding. HIV1084i is an …

Electrotransformation Of Clostridium Thermocellum, Michael V. Tyurin, Sunil G. Desai, Lee R. Lynd

Dartmouth Scholarship

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 􏰍 0.5) 􏰎 105 transformants per 􏰃g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 􏰍 1.8) 􏰎 104 transformants per 􏰃g of plasmid DNA for strain ATCC 27405 and 􏰁1 􏰎 103 transformants per 􏰃g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was 􏰁50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.

Transport Of The Harmful Bloom Alga Aureococcus Anophagefferens By Oceangoing Ships And Coastal Boats, Martina A. Doblin, Linda C. Popels, Kathryn J. Coyne, David A. Hutchins, S. Craig Cary, Fred C. Dobbs

OES Faculty Publications

It is well established that cyst-forming phytoplankton species are transported in ships' ballast tanks. However, there is increasing evidence that other phytoplankton species which do not encyst are also capable of surviving ballast transit. These species have alternative modes of nutrition (hetero- or mixotrophy) and/or are able to survive long-term darkness. In our studies of no-ballast-on-board vessels arriving in the Great Lakes, we tested for the presence of the harmful algal bloom species Aureococcus anophagefferens (brown tide) in residual (i.e., unpumpable) ballast water using methods based on the PCR. During 2001, the brown tide organism was detected in 7 of …

The Latent Membrane Protein 1 Of Epstein-Barr Virus Establishes An Antiviral State Via Induction Of Interferon-Stimulated Genes, Jun Zhang, Subash C. Das, Catherine Kotalik, Asit K. Pattnaik, Luwen Zhang

Nebraska Center for Virology: Faculty Publications

Epstein-Barr virus (EBV) infection is associated with several human cancers. Latent membrane protein 1 (LMP-1) is one of the key viral proteins required for transformation of primary B cells in vitro and establishment of EBV latency. In this report, we show that LMP-1 is able to induce the expression of several interferon (IFN)-stimulated genes (ISGs) with antiviral properties such as 2’-5’ oligoadenylate synthetase (OAS), stimulated trans-acting factor of 50 kDa (STAF- 50), and ISG-15. LMP-1 inhibits vesicular stomatitis virus (VSV) replication at low multiplicity of infection (0.1 pfu/cell). The antiviral effect of LMP-1 is associated with the ability of LMP-1 …

Detection And Quantitation Of Bovine Respiratory Syncytial Virus Using Real-Time Quantitative Rt-Pcr And Quantitative Competitive Rt-Pcr Assays, Jenna Achenbaum, Christina L. Topliff, Ventzislav B. Vassilev, Ruben O. Donis, Kent M. Eskridge, Clayton L. Kelling

Nebraska Center for Virology: Faculty Publications

A single tube, fluorogenic probe-based, real-time quantitative reverse transcription-polymerase chain reaction (Q-RT-PCR) assay was developed for detection and quantitation of bovine respiratory syncytial virus (BRSV) using BioRad’s iCycler iQ™. Real-time Q-RT-PCR was compared with quantitative competitive RT-PCR (QC-RT-PCR) and viral titers. Viral mRNA levels were measured in BRSV-infected bovine turbinate cell lysate harvested at eight time points (1.5, 6, 12, 24, 36, 48, 60, 72 h) post-infection. A homologous BRSV cRNA standard was used for quantitation of the mRNA by plotting a standard curve of cycle threshold (Ct) values versus standard 10-fold dilutions of cRNA of known concentrations. Detection as …

Cd8+ T Cells Responding To Influenza Infection Reach And Persist At Higher Numbers Than Cd4+ T Cells Independently Of Precursor Frequency, Timothy J. Powell, Deborah M. Brown, Joseph A. Hollenbaugh, Tina Charbonneau, Roslyn A. Kemp, Susan L. Swain, Richard Dutton

Nebraska Center for Virology: Faculty Publications

The activation, localization, phenotypic changes, and function of CFSE-labeled naive influenza-specific CD8⁺ and CD4⁺ T cells following influenza infection were examined. Response of adoptively transferred CD8⁺ T cells was seen earliest in draining lymph node. Highly activated cells were found later in the lung, airways, and spleen, were cytolytic, and expressed IFN-γ upon restimulation. Similar amounts of division at early time points, but higher numbers of CD8⁺ T cells, were detected at 9 and 30 days postinfection after cotransfer of CD4⁺ and CD8⁺ T cells followed by infection. Transfer of much smaller numbers of …

Apobec3g Genetic Variants And Their Influence On The Progression To Aids, Ping An, Gabriela Bleiber, Priya Duggal, George Nelson, Margaret May, Bastien Mangeat, Irene Alobwede, Didier Trono, David Vlahov, Sharyne Donfield, James J. Goedert, John Phair, Susan Buchbinder, Stephen J. O'Brien, Amalio Telenti, Cheryl Winkler

Biology Faculty Articles

The cytosine deaminase APOBEC3G, in the absence of the human immunodeficiency virus type 1 (HIV-1) accessory gene HIV-1 viral infectivity factor (vif), inhibits viral replication by introducing G→A hypermutation in the newly synthesized HIV-1 DNA negative strand. We tested the hypothesis that genetic variants of APOBEC3G may modify HIV-1 transmission and disease progression. Single nucleotide polymorphisms were identified in the promoter region (three), introns (two), and exons (two). Genotypes were determined for 3,073 study participants enrolled in six HIV-AIDS prospective cohorts. One codon-changing variant, H186R in exon 4, was polymorphic in African Americans (AA) (f < 37%) and rare in European Americans (f < 3%) or Europeans (f …