Combination of immunoglobulins and natural killer cells in the context of CMV and EBV infection.

Cytomegalovirus (CMV)-specific hyperimmunoglobulin (CMV-HIG) is used to treat and prevent CMV infection in immunocompromised patients, and anti-CD20 monoclonal antibody is successfully used in the treatment for post-transplant lymphoproliferative disease caused by Epstein-Barr virus (EBV). Two immunological approaches have been suggested to further improve the control of viral reproduction in patients with active disease: first, the use of monoclonal antibodies with specificity against viral epitopes and second, coadministration of cells with the capacity to promote antibody-dependent cell-mediated cytotoxicity. Here, we have evaluated the effectiveness of these strategies in vitro (alone and in combination) with neutralization and cytotoxicity assays. Our results indicate that monoclonal antibodies (in particular SM5-1) can be as effective as CMV-HIG in neutralizing-cell-free CMV. Moreover, our data indicate that antibody-mediated elimination (either by moAb or by HIG) of EBV-infected cells can be significantly enhanced by NK cells. Using human NK cells that have been purified, cultured and expanded under GMP conditions, we were able to demonstrate that the combination of NK cells and antibodies could represent a feasible and highly effective clinical approach to achieve control of EBV infections. Especially in leukopenic patients with low numbers of ADCC-promoting cells, the combination of adoptively transferred NK cells and antiviral antibodies offers a promising strategy that should be tested in clinical trials.

Performance of the MagNA Pure 96 system for cytomegalovirus nucleic acid amplification testing in clinical samples.

This report shows the performance of MagNA Pure 96 automated nucleic acid extraction for the quantitative detection of cytomegalovirus DNA in clinical samples by real-time PCR. The obtained results demonstrate that this workflow is characterized by high sensitivity and linearity and ensures reliable, reproducible clinical diagnostics.

The broad spectrum of hantaviruses and their hosts in Central Europe.

Hantaviruses are considered to be emerging viruses due to their increasing significance as human pathogens and their cyclic reappearance during outbreaks. Central Europe is an important endemic region for hantavirus infections. Reflecting the presence of all relevant small mammals serving as reservoir hosts, close to all recognized European hantaviruses occur also in Central Europe. Important human pathogens, Puumala and Dobrava-Belgrade viruses, are present and cause hemorrhagic fever with renal syndrome of various severities. Moreover, several of the newly recognized shrew- and mole-borne hantaviruses are present. In this review, we summarize current data on molecular detection of hantaviruses in reservoir hosts as well as on molecular epidemiology of human hantavirus infections in Central Europe.

Liver transplant from a donor with occult HEV infection induced chronic hepatitis and cirrhosis in the recipient.

Acute hepatitis E virus (HEV) infection is a self-limiting symptomatic or asymptomatic disease. However, as recently observed, it can manifest itself as chronic hepatitis in patients receiving solid organ transplants as well as in patients with HIV infection or severe hematologic disorders. Here, we describe the clinical course of a 73-year-old male patient in whom HEV transmission occurred after receiving a HEV-infected liver from a donor with occult HEV infection, whereby the patient had tested negative for HEV RNA and anti-HEV antibodies shortly before explantation. Anti-HEV IgG, IgM, and HEV RNA were detected in the first tested serum sample of the liver recipient obtained 150 days after liver transplantation and remained positive (earlier samples after OLT were not available). Liver cirrhosis developed within 15 months and the patient died of septic shock. Based on phylogenetic analyses of the donor and recipient's HEV strains, we were able to prove that the occult HEV infection was transmitted via the graft.

Antiviral function and efficacy of polyvalent immunoglobulin products against CMV isolates in different human cell lines.

Primary infection and reactivation of human cytomegalovirus (CMV) remain a major problem in immunocompromised patients, frequently resulting in a life threatening CMV disease. Intravenous polyvalent (hyper)-immunoglobulins (IVIG) can be administered for therapy and prophylaxis of CMV infections. However, only limited data about the efficacy and mechanism of action of IVIG products against viral infections in vitro are available so far. In this study, the effect of IVIG on CMV infection in vitro was investigated using isolates from CMV-infected patients as well as the laboratory strains AD169 and TB40. A qualitative and quantitative comparison of five different commercially available IVIG products in different human cell lines was performed concerning their ability (1) to neutralize cell-free virus, (2) to inhibit cell-to-cell spread and cell-associated transmission and (3) to influence CMV mRNA levels. All IVIG tested exhibited a high neutralization activity in epithelial and endothelial cell cultures (50% inhibition dose <0.1 mg/ml). However, qualitative differences between the products could be demonstrated in neutralization tests using human embryonal lung fibroblasts (HELF). The IVIG products also significantly differed in their ability to inhibit cell-to-cell spread within an CMV-infected HELF monolayer displaying inhibition rates that varied between 61 and 100%. No correlation between the ability to neutralize cell-free virus and to inhibit cell-to-cell spread could be observed. The incubation with IVIG influenced the amount of CMV immediate early and late mRNA, as indicated by a significant reduction in CMV mRNA in infected epithelial cells after incubation with IVIG in a dose-dependent manner. This study suggests different antiviral functions of polyvalent IVIG and confirms their potential to inhibit a CMV infection in vitro, with profound differences between the hereby used IVIG products.

Tissue donation and virus safety: more nucleic acid amplification testing is needed.

In tissue and organ transplantation, it is of great importance to avoid the transmission of blood-borne viruses to the recipient. While serologic testing for anti-human immunodeficiency virus (HIV)-1 and -2, anti-hepatitis C virus (HCV), hepatitis B surface antigen (HBsAg), anti-hepatitis B core antigen (HBc), and Treponema pallidum infection is mandatory, there is until now in most countries no explicit demand for nucleic acid amplification testing (NAT) to detect HIV, hepatitis B virus (HBV), and HCV infection. After a review of reports in the literature on viral transmission events, tissue-specific issues, and manufacturing and inactivation procedures, we evaluated the significance of HIV, HCV, and HBV detection using NAT in donors of various types of tissues and compared our results with the experiences of blood banking organizations. There is a significant risk of HIV, HCV, and HBV transmission by musculoskeletal tissues because of their high blood content and the high donor-recipient ratio. If no effective virus inactivation procedure for musculoskeletal tissue is applied, donors should be screened using NAT for HIV, HCV, and HBV. Serologically screened cardiovascular tissue carries a very low risk of HIV, HCV, or HBV transmission. Nevertheless, because effective virus inactivation is impossible (retention of tissue morphology) and the donor-recipient ratio may be as high as 1:10, we concluded that NAT should be performed for HIV, HCV, and HBV as an additional safety measure. Although cornea allografts carry the lowest risk of transmitting HIV, HCV, and HBV owing to corneal physiology, morphology, and the epidemiology of corneal diseases, NAT for HCV should still be performed. If the NAT screening of a donor for HIV, HCV, and HBV is negative, quarantine storage of the donor tissue seems dispensable. In view of numerous synergistic effects with transfusion medicine, it would be advantageous for tissue banks to cooperate with blood bank laboratories in performing virological tests.

Steep rise in notified hantavirus infections in Germany, April 2010.

From January to April 2010, 396 hantavirus infections were notified in Germany, a considerable increase compared with previous years (mean: 83 for January-April 2004-2009) including the record-setting year, 2007 (n=232 January-April). Most patients are residents of known Puumala virus endemic areas in southern Germany. The recent increase in notified hantavirus infections is probably due to an increased population density of the main animal reservoir, the bank vole (Myodes glareolus).

[First case of nephropathia epidemica acquired in Switzerland]. / Premier cas de néphropathie epidémique endémique en Suisse.

A 43 year healthy old man complains of fever with abdominal pain, vomiting, diarrhoea, followed by the development of thrombocytopenia and acute renal failure. The laboratory tests show the presence of Hantavirus specific IgM and IgG which is confirmed by a specific test revealing Puumala serotype as responsible. The patient received a symptomatic treatment with a favourable evolution allowing discharge about ten days after the beginning of symptoms. Hantavirus are transmitted by rodents, and this patient has certainly been infected in Switzerland in the absence of travel abroad during the incubation period. This means that when confronted in Switzerland with an acute nephritis of unknown origin, a diagnosis of nephropathia epidemica must be taken into account.

The significance of distance and orientation of restriction endonuclease recognition sites in viral DNA genomes.

Studies on phage T3 and T7 have shown that these viruses avoid restriction not only by the phage-coded Ocr (and S-adenosylmethionine hydrolase) protein functions or by the complete loss of specific recognition sites for certain restriction endonucleases from their genomes, but also that there are two additional modes: resistance towards EcoP15 (which recognizes a non-symmetrical sequence) is achieved by an identical orientation of all the recognition sites in the virus genome (strand bias) and in the case of EcoRII by the extreme reduction in number and thereby greater distance between recognition sites in the genome. These observations led to the discovery that certain restriction endonucleases require the simultaneous cooperation with two DNA sites for their function, as well as to the ongoing elucidation of the molecular modes of action of these enzymes. Type II and type III enzymes display fundamentally different mechanisms of protein-DNA interaction. For EcoRII we favor a model of simultaneous binding of two DNA sites to a dimeric enzyme molecule (neighbouring sites of the same, looping, DNA molecule or sites located on different DNA molecules), while the action of EcoP15 seems to conform with a tracking-collision model of two enzyme molecules bound to inversely oriented recognition sites. In addition to podoviruses T3 and T7, strand bias of recognition sequences for different type III DNA modification-restriction enzymes is also observed in the inoviruses M13, IKE and PF3.

Abortive infection of Escherichia coli F+ cells by bacteriophage T7 requires ribosomal misreading.

The use of different precisely mapped T3/T7 recombinants strengthens the conclusion that abortive infection by T7 of F plasmid-carrying cells is due to the nucleotide sequence at the end of the T7 gene 1. Furthermore, we demonstrate that the exclusion requires suppression of ochre stop codons, a phenomenon that occurs with low frequency in wild-type cells due to ribosomal misreading. The introduction of rspL mutations in which ribosomal misreading is reduced alleviates the exclusion and the presence of ochre tRNA suppressors increases its severity.

DNA cleavage by type III restriction-modification enzyme EcoP15I is independent of spacer distance between two head to head oriented recognition sites.

The type III restriction-modification enzyme EcoP15I requires the interaction of two unmethylated, inversely oriented recognition sites 5'-CAGCAG in head to head configuration to allow an efficient DNA cleavage. It has been hypothesized that two convergent DNA-translocating enzyme-substrate complexes interact to form the active cleavage complex and that translocation is driven by ATP hydrolysis. Using a half-automated, fluorescence-based detection method, we investigated how the distance between two inversely oriented recognition sites affects DNA cleavage efficiency. We determined that EcoP15I cleaves DNA efficiently even for two adjacent head to head or tail to tail oriented target sites. Hence, DNA translocation appears not to be required for initiating DNA cleavage in these cases. Furthermore, we report here that EcoP15I is able to cleave single-site substrates. When we analyzed the interaction of EcoP15I with DNA substrates containing adjacent target sites in the presence of non-hydrolyzable ATP analogues, we found that cleavage depended on the hydrolysis of ATP. Moreover, we show that cleavage occurs at only one of the two possible cleavage positions of an interacting pair of target sequences. When EcoP15I bound to a DNA substrate containing one recognition site in the absence of ATP, we observed a 36 nucleotide DNaseI-footprint that is asymmetric on both strands. All of our footprinting experiments showed that the enzyme did not cover the region around the cleavage site. Analyzing a DNA fragment with two head to head oriented recognition sites, EcoP15I protected 27-33 nucleotides around the recognition sequence, including an additional region of 26 bp between both cleavage sites. For all DNA substrates examined, the presence of ATP caused altered footprinting patterns. We assume that the altered patterns are most likely due to a conformational change of the enzyme. Overall, our data further refine the tracking-collision model for type III restriction enzymes.

Overproduction of His-tagged EcoRII restriction endonuclease and terminally deleted mutant proteins.

EcoRII was the first restriction endonuclease (ENase) reported requiring the cooperative interaction with at least two DNA sites for activity. Using two different expression systems the enzyme could be purified and its special substrate requirements were further analyzed. At the present state of knowledge we suggest a model of simultaneous binding of two DNA sites to one dimeric enzyme molecule (see Krüger et al., FEMS Microbiol. Rev. (1995) in press).

An improved method for the detection of Dcm methylation in DNA molecules.

The intrinsic insensitivity of EcoRII recognition sites in RF DNAs of phage M13 and vector M13mp18 towards this restriction endonuclease can be overcome by adding site-specific oligodeoxyribonucleotide duplexes to the restriction sample. Since Dcm- DNA but not Dcm(+)-methylated DNA becomes susceptible under these conditions, this procedure constitutes an improvement of the Dcm methylation assay.

Restriction endonucleases functionally interacting with two DNA sites.

Simultaneous interaction with two recognition sites was found to be a precondition for DNA cleavage by certain type-II and type-III restriction endonucleases. Nevertheless, the molecular mechanisms of the protein-DNA interaction are different between members of both classes of enzymes.

A sizing artifact of DNA restriction fragments with unusually long single-stranded termini.

The restriction endonucleases (ENases) BstNI (CCATGG) and EcoRII (CCATGG) both cleave DNA at the same time sequences, but only EcoRII produces 5-nucleotide (nt) cohesive ends and is inhibited by 5-methylation of the inner cytosine. The low-Mr fragments in digests of mouse DNA made with these two ENases exhibit different mobilities during agarose-gel electrophoresis. The difference in the mobilities of the BstNI and EcoRII fragments from mouse DNA was not due to closely spaced, differentially methylated sites, or to alternate mechanisms such as circularization of the long cohesive ends of the EcoRII fragments, or to residual bound protein. Rather, it was due to the unusually long 5-nt single-stranded (ss) ends of fragments produced by EcoRII digestion, since the slower mobility of the EcoRII fragments was abolished by treatment with ss-specific nuclease. Similar mobility differences between BstNI and EcoRII fragments which could be removed by ss nuclease were also observed in digests of simian virus 40 DNA.

Oligonucleotide duplexes containing CC(A/T)GG stimulate cleavage of refractory DNA by restriction endonuclease EcoRII.

Some DNA species are resistant towards the restriction endonuclease EcoRII despite the presence of unmodified recognition sites. We show that 14 base-pair oligonucleotide duplexes containing the EcoRII recognition site 5'-CC(A/T)GG are cleaved by this enzyme and are able to stimulate EcoRII cleavage of such resistant DNA molecules (e.g. DNA of bacterial virus T3). A direct correlation between the concentration of oligonucleotide duplex molecules and the degree of EcoRII digestion of the primarily resistant DNA is observed. This indicates a stoichiometric rather than a catalytic mode of enzyme activation. An excess of DNA devoid of EcoRII sites ('non-site' DNA, e.g. MvaI-digested T7 DNA) does not interfere with the activity of EcoRII.

Interaction of wild-type and naturally occurring deleted variants of hepatitis B virus core polypeptides leads to formation of mosaic particles.

The simultaneous presence of hepatitis B virus (HBV) genomes carrying wild-type (wt) and in-frame deleted variants of the HBV core gene has been identified as a typical feature of HBV-infected renal transplant patients with severe liver disease. To investigate possible interactions of wt and deleted core polypeptides a two-vector Escherichia coli expression system ensuring their concomitant synthesis has been developed. Co-expression of wt and a mutant core lacking 17 amino acid residues (77-93) within the immunodominant region led to the formation of mosaic particles, whereas the mutant alone was incapable of self-assembly.

Mapping mutations in influenza A virus resistant to norakin.

To elucidate the mode of action of norakin against influenza A virus we sequenced the hemagglutinin gene of 11 norakin-resistant mutants. Resistance was coupled with 1-3 amino acid exchanges. The majority of mutations was localized in the HA2 polypeptide and was mostly associated with changes in charge or polarity of the amino acids. The amino acid substitutions are discussed in the context of the 3D structure of X31 hemagglutinin considered to be representative of the influenza hemagglutinins. Most of the mutations appear to destabilize the pH 7.0 structure by distorting or destroying hydrogen bonds as well as salt-bridges which are responsible for intra- and intersubunit contacts, while others destabilize the location of the fusion peptide, facilitating conformational changes in the presence of the inhibitor.

Hantavirus infections and their prevention.

Hantaviruses are rodent-borne bunyaviruses which cause haemorrhagic fever with renal syndrome and Hantavirus pulmonary syndrome in humans. This review covers the host interactions of the viruses, including the rodent reservoirs, the clinical outcome of human infections as well as the pathogenesis and laboratory diagnosis of infections. The current stage in prophylaxis and therapy of hantaviral diseases is described and different approaches in vaccine development are discussed.

Yeast-expressed hantavirus Dobrava nucleocapsid protein induces a strong, long-lasting, and highly cross-reactive immune response in mice.

In Europe, Dobrava virus (DOBV) carried by the yellow-necked field mouse Apodemus flavicollis is one of the hantaviruses that can cause severe hemorrhagic fever with renal syndrome in humans. For several hantaviruses, the nucleocapsid (N) protein has proven to be very immunogenic in humans and rodents and even can protect rodents against a virus challenge. To investigate the immunogenicity of DOBV N protein, BALB/c and C57BL/6 mice were immunized three times with a DOBV recombinant N (rN) protein expressed in yeast Saccharomyces cerevisiae together with complete Freund's, with incomplete Freund's, and without adjuvant, respectively. Mice of both strains elicited N-specific antibodies with end-point titers being as high as 1:1,000,000 in C57BL/6 mice. The antibodies induced by DOBV rN protein were highly cross-reactive to the rN proteins of hantaviruses Puumala and Hantaan. In both mice strains, DOBV rN protein induced N-specific antibodies of all IgG subclasses (IgG1, IgG2a, IgG2b, and IgG3), suggesting a mixed Th1/Th2 immune response. Taken together, yeast-expressed DOBV rN protein represents a promising vaccine candidate.