Walter Muranyi

New ecological aspects of hantavirus infection : a change of a paradigm and a challenge of prevention-a review

In the last decades a significant number of so far unknown or underestimated pathogens have emerged as fundamental health hazards of the human population despite intensive research and exceptional efforts of modern medicine to embank and eradicate infectious diseases. Almost all incidents caused by such emerging pathogens could be ascribed to agents that are zoonotic or expanded their host range and crossed species barriers. Many different factors influence the status of a pathogen to remain unnoticed or evolves into a worldwide threat. The ability of an infectious agent to adapt to changing environmental conditions and variations in human behavior, population development, nutrition, education, social, and health status are relevant factors affecting the correlation between pathogen and host. Hantaviruses belong to the emerging pathogens having gained more and more attention in the last decades. These viruses are members of the family Bunyaviridae and are grouped into a separate genus known as Hantavirus. The serotypes Hantaan (HTN), Seoul (SEO), Puumala (PUU), and Dobrava (DOB) virus predominantly cause hemorrhagic fever with renal syndrome (HFRS), a disease characterized by renal failure, hemorrhages, and shock. In the recent past, many hantavirus isolates have been identified and classified in hitherto unaffected geographic regions in the New World (North, Middle, and South America) with characteristic features affecting the lungs of infected individuals and causing an acute pulmonary syndrome. Hantavirus outbreaks in the United States of America at the beginning of the 10th decade of the last century fundamentally changed our knowledge about the appearance of the hantavirus specific clinical picture, mortality, origin, and transmission route in human beings. The hantavirus pulmonary syndrome (HPS) was first recognized in 1993 in the Four Corners Region of the United States and had a lethality of more than 50%. Although the causative virus was first termed in connection with the geographic name of its outbreak region the analysis of the individual viruses indicate that the causing virus of HPS was a genetically distinct hantavirus and consequently termed as Sin Nombre virus. Hantaviruses are distributed worldwide and are assumed to share a long time period of co-evolution with specific rodent species as their natural reservoir. The degree of relatedness between virus serotypes normally coincides with the relatedness between their respective hosts. There are no known diseases that are associated with hantavirus infections in rodents underlining the amicable relationship between virus and host developed by mutual interaction in hundreds of thousands of years. Although rodents are the major reservoir, antibodies against hantaviruses are also present in domestic and wild animals like cats, dogs, pigs, cattle, and deer. Domestic animals and rodents live jointly in a similar habitat. Therefore the transmission of hantaviruses from rodents to domestic animals seems to be possible, if the target organs, tissues, and cell parenchyma of the co-habitat domestic animals possess adequate virus receptors and are suitable for hantavirus entry and replication. The most likely incidental infection of species other than rodents as for example humans turns hantaviruses from harmless to life-threatening pathogenic agents focusing the attention on this virus group, their ecology and evolution in order to prevent the human population from a serious health risk. Much more studies on the influence of non-natural hosts on the ecology of hantaviruses are needed to understand the directions that the hantavirus evolution could pursue. At least, domestic animals that share their environmental habitat with rodents and humans particularly in areas known as high endemic hantavirus regions have to be copiously screened. Each transfer of hantaviruses from their original natural hosts to other often incidental hosts is accompanied by a change of ecology, a change of environment, a modulation of numerous factors probably influencing the pathogenicity and virulence of the virus. The new environment exerts a modified evolutionary pressure on the virus forcing it to adapt and probably to adopt a form that is much more dangerous for other host species compared to the original one.

Super-Resolution Microscopy Reveals Specific Recruitment of HIV-1 Envelope Proteins to Viral Assembly Sites Dependent on the Envelope C-Terminal Tail

The inner structural Gag proteins and the envelope (Env) glycoproteins of human immunodeficiency virus (HIV-1) traffic independently to the plasma membrane, where they assemble the nascent virion. HIV-1 carries a relatively low number of glycoproteins in its membrane, and the mechanism of Env recruitment and virus incorporation is incompletely understood. We employed dual-color super-resolution microscopy visualizing Gag assembly sites and HIV-1 Env proteins in virus-producing and in Env expressing cells. Distinctive HIV-1 Gag assembly sites were readily detected and were associated with Env clusters that always extended beyond the actual Gag assembly site and often showed enrichment at the periphery and surrounding the assembly site. Formation of these Env clusters depended on the presence of other HIV-1 proteins and on the long cytoplasmic tail (CT) of Env. CT deletion, a matrix mutation affecting Env incorporation or Env expression in the absence of other HIV-1 proteins led to much smaller Env clusters, which were not enriched at viral assembly sites. These results show that Env is recruited to HIV-1 assembly sites in a CT-dependent manner, while Env(ΔCT) appears to be randomly incorporated. The observed Env accumulation surrounding Gag assemblies, with a lower density on the actual bud, could facilitate viral spread in vivo. Keeping Env molecules on the nascent virus low may be important for escape from the humoral immune response, while cell-cell contacts mediated by surrounding Env molecules could promote HIV-1 transmission through the virological synapse.

Antifibrotic actions of mycophenolic acid

Abstract:  Mycophenolic acid (MPA) is a highly selective, non‐competitive and reversible inhibitor of the inosine monophosphate dehydrogenase (IMPDH), the rate‐limiting enzyme in the de novo biosynthesis of guanosine nucleotides. Mycophenolate mofetil (MMF, the ester prodrug of MPA) strongly inhibits both T‐ and B‐lymphocyte proliferation and has now been widely used in the prevention of acute and chronic allograft rejection. Recent evidence, however, suggests that MMF is also capable of inhibiting the proliferation of non‐immune cells. In various cell lines, e.g. smooth muscle cells, renal tubular cells, mesangial cells, and fibroblasts, MPA reduced or even abrogated proliferation in response to proliferative stimuli. In animal studies, MMF ameliorated renal lesions in immune‐mediated disease, e.g. in the Anti‐Thy 1.1 model and experimental lupus nephritis, but was also effective in non‐immune‐mediated renal damage, e.g. in the rat remnant kidney model or in a model of chronic cyclosporine nephrotoxicity in the rat. In humans, MMF reduced proteinuria in steroid‐resistant nephrotic syndrome and had beneficial effects in the prevention and treatment of chronic allograft nephropathy and calcineurin inhibitor toxicity through the reduction of immune‐ and non‐immune‐mediated renal damage. MMF is well tolerated and has proven to be a relatively safe drug. Taken together, there is a growing body of evidence pointing to therapeutic applications of MMF other than immunosuppression, in particular the prevention of fibrosis.

Quantitative microscopy of functional HIV post-entry complexes reveals association of replication with the viral capsid

The steps from HIV-1 cytoplasmic entry until integration of the reverse transcribed genome are currently enigmatic. They occur in ill-defined reverse-transcription- and pre-integration-complexes (RTC, PIC) with various host and viral proteins implicated. In this study, we report quantitative detection of functional RTC/PIC by labeling nascent DNA combined with detection of viral integrase. We show that the viral CA (capsid) protein remains associated with cytoplasmic RTC/PIC but is lost on nuclear PIC in a HeLa-derived cell line. In contrast, nuclear PIC were almost always CA-positive in primary human macrophages, indicating nuclear import of capsids or capsid-like structures. We further show that the CA-targeted inhibitor PF74 exhibits a bimodal mechanism, blocking RTC/PIC association with the host factor CPSF6 and nuclear entry at low, and abrogating reverse transcription at high concentrations. The newly developed system is ideally suited for studying retroviral post-entry events and the roles of host factors including DNA sensors and signaling molecules. DOI: http://dx.doi.org/10.7554/eLife.04114.001

A SNAP-Tagged Derivative of HIV-1—A Versatile Tool to Study Virus-Cell Interactions

Fluorescently labeled human immunodeficiency virus (HIV) derivatives, combined with the use of advanced fluorescence microscopy techniques, allow the direct visualization of dynamic events and individual steps in the viral life cycle. HIV proteins tagged with fluorescent proteins (FPs) have been successfully used for live-cell imaging analyses of HIV-cell interactions. However, FPs display limitations with respect to their physicochemical properties, and their maturation kinetics. Furthermore, several independent FP-tagged constructs have to be cloned and characterized in order to obtain spectral variations suitable for multi-color imaging setups. In contrast, the so-called SNAP-tag represents a genetically encoded non-fluorescent tag which mediates specific covalent coupling to fluorescent substrate molecules in a self-labeling reaction. Fusion of the SNAP-tag to the protein of interest allows specific labeling of the fusion protein with a variety of synthetic dyes, thereby offering enhanced flexibility for fluorescence imaging approaches. Here we describe the construction and characterization of the HIV derivative HIVSNAP, which carries the SNAP-tag as an additional domain within the viral structural polyprotein Gag. Introduction of the tag close to the C-terminus of the matrix domain of Gag did not interfere with particle assembly, release or proteolytic virus maturation. The modified virions were infectious and could be propagated in tissue culture, albeit with reduced replication capacity. Insertion of the SNAP domain within Gag allowed specific staining of the viral polyprotein in the context of virus producing cells using a SNAP reactive dye as well as the visualization of individual virions and viral budding sites by stochastic optical reconstruction microscopy. Thus, HIVSNAP represents a versatile tool which expands the possibilities for the analysis of HIV-cell interactions using live cell imaging and sub-diffraction fluorescence microscopy.

Bacterial expression of the capsid antigen domain and identification of native gag proteins in spumavirus-infected cells

A bacterial expression plasmid containing the central part of the gag gene of the human spumaretrovirus (HSRV) was constructed and expressed in E. coli. The expected protein product consisting of the complete region of the HSRV capsid antigen and part of the matrix protein was expressed in relatively large amounts. Polyclonal antisera raised against this recombinant protein were used to identify authentic gag precursors of 78 and 74 kDa and processed gag proteins of 60, 58, and 33 kDa in HSRV-infected human embryonal fibroblast cells by radioimmunoprecipitation. The recombinant antigen will be useful for the detection of antibodies against HSRV gag proteins in human sera.

Human spumaretrovirus antibody reactivity in multiple sclerosis

The role of human spumaretrovirus (HSRV) infections in the pathogenesis of multiple sclerosis (MS) was investigated with recombinant HSRV env-specific enzyme-linked immunosorbent assay. The presence of HSRV antibodies was determined in pairs of serum and cerebrospinal fluid (CSF) samples from 60 MS patients. In 7 of these patients serial serum and CSF samples were obtained in relation to the clinical activity of the disease during a period of 2 years. No increased antibody reactivity was demonstrable in the MS population compared with 14 aseptic meningitis patients, 50 blood donors and 16 healthy controls. Slightly elevated levels of antibodies were demonstrable in serum and/or CSF in 4 MS patients but also in 1 patient with aseptic meningitis, 1 blood donor and 1 child. No marked serum or CSF HSRV antibody fluctuation was observed in the MS patients followed longitudinally. Thus, this study does not support the involvement of HSRV in the pathogenesis of MS.

Characterization of a new Puumala virus genotype associated with hemorrhagic fever with renal syndrome.

Hantavirus serotype Puumala (PUUV) is the etiologic agent of Nephropathia epidemica (NE), a mild variant of Hemorrhagic Fever with Renal Syndrome (HFRS) in Europe, with lethality rates up to 1% among human patients. The serotype PUUV is composed of numerous geographically restricted strains forming a great number of phylogeographic lineages, sublineages, variants, and clusters. We describe a new, geographically and phylogenetically well-defined pathogenic PUUV sublineage in Southwest Germany (strain Heidelberg/hu) originated from an HFRS patient. The genetic analysis of PUUV strain Heidelberg/hu may contribute in future to better diagnostics, the development of vaccines, and the understanding of the factors that determine hantavirus pathogenesis.

Stable Expression of Nucleocapsid Proteins of Puumala and Hantaan Virus in Mammalian Cells

The development of an in vitro-system for the stable expression and the analysis of native hantavirus proteins using hantaviral cDNA is of particular interest. As a first step the expression of the hantavirus nucleocapsid (N) proteins in mammalian cells was studied in more detail. The cDNA of the S-RNA segment of Puumala virus strain CG-1820 and Hantaan virus strain 76–118 was used for the construction of eucaryotic expression vectors that allow the generation and selection of mammalian cells harboring and expressing the N protein genes of hantaviruses. A variety of conventional and novel expression vectors as well as different mammalian cell lines were screened. The expression of the N protein of Puumala virus using the pGRE5-1 vector in which the transcription is under control of inducible glucocorticoid responsive elements (GRE) revealed that the Puumala virus N protein can be expressed in Vero E6 cells efficiently without any detectable cell toxicity. From the variety of expression vectors tested, it was found that pCR3.1 is the vector of choice for stable expression of hantavirus N proteins. The successful establishment of different mammalian cell lines expressing considerable amounts of Puumala and Hantaan virus N protein indicates that the stable and efficient expression of this particular viral protein in the cell lines of three evolutionary distinct species (human, monkey, and mouse) is possible. The system described here represents the experimental basis for further studies of hantavirus infection, replication, and pathogenesis using a reverse genetics approach.

Fully Automated Targeted Confocal and Single-Molecule Localization Microscopy

Single-molecule localization microscopy (SMLM) enables imaging of biological structures in the nanometre range. Long measurement times are the consequence of this kind of microscopy due to the need of acquiring thousands of images. We built a setup that automatically detects target structures using confocal microscopy and images them with SMLM. Utilizing the Konstanz Information Miner (KNIME), we were able to connect a confocal microscope with an SMLM unit for targeted screening. In this process, we developed KNIME plugins to communicate with the microscope components and combined them to a workflow. Thus, measuring biological nanometre-sized structures in a sufficient number to get statistical significance becomes feasible. For proof of principle HIV-1 assembly complexes in HeLa cells derived from transfection of replication deficient viral construct were imaged by a fully automated screen.