Beth K. Thielen

Mouse Model of X-Linked Alport Syndrome

X-linked Alport syndrome (XLAS) is a progressive disorder of basement membranes caused by mutations in the COL4A5 gene, encoding the alpha5 chain of type IV collagen. A mouse model of this disorder was generated by targeting a human nonsense mutation, G5X, to the mouse Col4a5 gene. As predicted for a nonsense mutation, hemizygous mutant male mice are null and heterozygous carrier female mice are mosaic for alpha5(IV) chain expression. Mutant male mice and carrier female mice are viable through reproductive age and fertile. Mutant male mice died spontaneously at 6 to 34 wk of age, and carrier female mice died at 8 to 45 wk of age, manifesting proteinuria, azotemia, and progressive and manifold histologic abnormalities of the kidney glomerulus and tubulointerstitium. Ultrastructural abnormalities of the glomerular basement membrane, including lamellation and splitting, were characteristic of human XLAS. The mouse model described here recapitulates essential clinical and pathologic findings of human XLAS. With alpha5(IV) expression reflecting X-inactivation patterns, it will be especially useful in studying determinants of disease variability in the carrier state.

Innate Immune Signaling Induces High Levels of TC-specific Deaminase Activity in Primary Monocyte-derived Cells through Expression of APOBEC3A Isoforms

In HIV-1-infected individuals, G-to-A hypermutation is found in HIV-1 DNA isolated from peripheral blood mononuclear cells (PBMCs). These mutations are thought to result from editing by one or more host enzymes in the APOBEC3 (A3) family of cytidine deaminases, which act on CC (APOBEC3G) and TC (other A3 proteins) dinucleotide motifs in DNA (edited cytidine underlined). Although many A3 proteins display high levels of deaminase activity in model systems, only low levels of A3 deaminase activity have been found in primary cells examined to date. In contrast, here we report high levels of deaminase activity at TC motifs when whole PBMCs or isolated primary monocyte-derived cells were treated with interferon-α (IFNα) or IFNα-inducing toll-like receptor ligands. Induction of TC-specific deaminase activity required new transcription and translation and correlated with the appearance of two APOBEC3A (A3A) isoforms. Knockdown of A3A in monocytes with siRNA abolished TC-specific deaminase activity, confirming that A3A isoforms are responsible for all TC-specific deaminase activity observed. Both A3A isoforms appear to be enzymatically active; moreover, our mutational studies raise the possibility that the smaller isoform results from internal translational initiation. In contrast to the high levels of TC-specific activity observed in IFNα-treated monocytes, CC-specific activity remained low in PBMCs, suggesting that A3G deaminase activity is relatively inhibited, unlike that of A3A. Together, these findings suggest that deaminase activity of A3A isoforms in monocytes and macrophages may play an important role in host defense against viruses.

Childhood pneumococcal disease in Africa – A systematic review and meta-analysis of incidence, serotype distribution, and antimicrobial susceptibility

BACKGROUND Determining the incidence, disease-associated serotypes and antimicrobial susceptibility of invasive pneumococcal disease (IPD) among children in Africa is essential in order to monitor the impact of these infections prior to widespread introduction of the pneumococcal conjugate vaccine (PCV). METHODS To provide updated estimates of the incidence, serotype distribution, and antimicrobial susceptibility profile of Streptococcus pneumoniae causing disease in Africa, we performed a systematic review of articles published from 2000 to 2015 using Ovid Medline and Embase. We included prospective and surveillance studies that applied predefined diagnostic criteria. Meta-analysis for all pooled analyses was based on random-effects models. RESULTS We included 38 studies consisting of 386,880 participants in 21 countries over a total of 350,613 person-years. The pooled incidence of IPD was 62.6 (95% CI 16.9, 226.5) per 100,000 person-years, including meningitis which had a pooled incidence of 24.7 (95% CI 11.9, 51.6) per 100,000 person-years. The pooled prevalence of penicillin susceptibility was 78.1% (95% CI 61.9, 89.2). Cumulatively, PCV10 and PCV13 included 66.9% (95% CI 55.9, 76.7) and 80.6% (95% CI 66.3, 90.5) of IPD serotypes, respectively. CONCLUSIONS Our study provides an integrated and robust summary of incidence data, serotype distribution and antimicrobial susceptibility for S. pneumoniae in children ≤5years of age in Africa prior to widespread introduction of PCV on the continent. The heterogeneity of studies and wide range of incidence rates across the continent indicate that surveillance efforts should be intensified in all regions of Africa to improve the integrity of epidemiologic data, vaccine impact and cost benefit. Although the incidence of IPD in young children in Africa is substantial, currently available conjugate vaccines are estimated to cover the majority of invasive disease-causing pneumococcal serotypes. These data provide a reliable baseline from which to monitor the impact of the broad introduction of PCV.

Assembly of Immature HIV-1 Capsids Using a Cell-Free System

For many years it has been known that viral capsid proteins are capable of self-assembly, but increasing evidence over the past decade indicates that in cells HIV-1 capsid assembly occurs via a complex but transient series of steps requiring multiple viral-host interactions. To better understand the biochemistry of HIV assembly, our group established a cell-free system that faithfully reconstitutes HIV-1 Gag synthesis and post-translational events of capsid assembly using cellular extracts, albeit more slowly and less efficiently. This system allowed initial identification of interactions that occur very transiently in cells but can be tracked in the cell-free system. Analysis of the cell-free system revealed that Gag progresses sequentially through a step-wise, energy-dependent series of assembly intermediates containing cellular proteins. One of these cellular proteins, the ATPase ABCE1, has been shown to play a critical role in the assembly process. The existence of this energy-dependent assembly pathway was subsequently confirmed in cellular systems, further validating the cell-free HIV-1 capsid assembly system as an excellent tool for identifying mechanisms underlying HIV-1 capsid formation. Here we describe how to assemble immature HIV-1 capsids in a cell-free system and separate assembly intermediates by velocity sedimentation.

Detection of Influenza C Viruses Among Outpatients and Patients Hospitalized for Severe Acute Respiratory Infection, Minnesota, 2013–2016

We detected influenza C viruses mostly in children in both outpatients and inpatient surveillance populations. Our findings suggest that influenza C may be an underrecognized cause of outpatient and severe hospitalized illness in the United States.

Widespread Lichtheimia Infection in a Patient with Extensive Burns: Opportunities for Novel Antifungal Agents

The Mucorales fungi—formerly classified as the zygomycetes—are environmentally ubiquitous fungi, but generally rare causes of clinical infections. In the immunocompromised host, however, they can cause invasive, rapidly spreading infections that confer a high risk of morbidity and mortality, often despite surgical and antifungal therapy. Patients with extensive burn injuries are particularly susceptible to skin and soft-tissue infections with these organisms. Here, we present a case of Lichtheimia infection in a patient with extensive full-thickness burns that required significant and repeated surgical debridement successfully treated with isavuconazole and adjunctive topical amphotericin B washes. We also review the available literature on contemporary antifungal treatment for Lichtheimia species and related Mucorales fungi.

437 DEVELOPMENT OF A CELL-FREE SYSTEM FOR THE STUDY OF VENEZUELAN EQUINE ENCEPHALITIS VIRUS CAPSID ASSEMBLY

Venezuelan equine encephalitis is a mosquito-borne viral illness occurring primarily in parts of South and Central America. The causative agent (VEEV) is an RNA virus of the genus Alphaviridae. It is endemic in rodent populations in many areas and causes occasional epidemics in horse and human populations. Although the potential for serious illness in humans in naturally occurring outbreaks is rather low, aerosolized virus can be lethal if inhaled by susceptible individuals. Consequently, it has recently gained attention because of its potential to be used as an agent of bio-terrorism. The lack of a human vaccine and effective treatments for the disease make this virus an important subject for further study. The goal of our laboratory is to apply our cell-free virus assembly systems to the study of VEEV and to use our new knowledge of VEEV assembly to develop drugs that block this step in the viral life cycle. The cell-free system reconstitutes virus assembly using cell extracts, creating an environment that mimics the inside of target cells and allows for the manipulation of numerous variables that may have an impact on assembly. Such systems have been utilized in the past to understand assembly of poliovirus, hepadnaviruses, retroviruses, and herpesviruses. Translation machinery from either wheat germ extract or rabbit reticulocyte lysate was used to translate the VEEV capsid protein from a messenger RNA. Newly synthesized proteins were labeled with radioactive methionine during translation. Velocity sedimentation of cell-free reaction products followed by SDS-PAGE and autoradiography revealed the formation of high molecular weight complexes that are likely to represent assembled capsids. Assembly appeared to be very efficient, with greater than 90% of capsid protein present in high molecular weight complexes. Additional experiments suggested that VEEV assembled either co-translationally or soon after translation was completed. Thousand-fold dilution of VEEV transcript did not significantly reduce the percentage of capsid protein that assembled, nor did solubilization of lipid membranes with detergents. Future experiments will use mutational analyses to identify regions of the capsid protein that are necessary for capsid assembly and packaging of the viral RNA genome. These data suggest for the first time that alphavirus capsids can be assembled in a cell-free system.