Klaus Überla

Croup Is Associated with the Novel Coronavirus NL63

Background The clinical relevance of infections with the novel human coronavirus NL63 (HCoV-NL63) has not been investigated systematically. We therefore determined its association with disease in young children with lower respiratory tract infection (LRTI). Methods and Findings Nine hundred forty-nine samples of nasopharyngeal secretions from children under 3 y of age with LRTIs were analysed by a quantitative HCoV-NL63-specific real-time PCR. The samples had been collected from hospitalised patients and outpatients from December 1999 to October 2001 in four different regions in Germany as part of the prospective population-based PRI.DE study and analysed for RNA from respiratory viruses. Forty-nine samples (5.2%), mainly derived from the winter season, were positive for HCoV-NL63 RNA. The viral RNA was more prevalent in samples from outpatients (7.9%) than from hospitalised patients (3.2%, p = 0.003), and co-infection with either respiratory syncytial virus or parainfluenza virus 3 was observed frequently. Samples in which only HCoV-NL63 RNA could be detected had a significantly higher viral load than samples containing additional respiratory viruses (median 2.1 × 106 versus 2.7 × 102 copies/ml, p = 0.0006). A strong association with croup was apparent: 43% of the HCoV-NL63-positive patients with high HCoV-NL63 load and absence of co-infection suffered from croup, compared to 6% in the HCoV-NL63-negative group, p < 0.0001. A significantly higher fraction (17.4%) of samples from croup patients than from non-croup patients (4.2%) contained HCoV-NL63 RNA. Conclusion HCoV-NL63 infections occur frequently in young children with LRTI and show a strong association with croup, suggesting a causal relationship.

Synthetic double-stranded RNAs are adjuvants for the induction of t helper 1 and humoral immune responses to human papillomavirus in rhesus macaques

Toll-like receptor (TLR) ligands are being considered as adjuvants for the induction of antigen-specific immune responses, as in the design of vaccines. Polyriboinosinic-polyribocytoidylic acid (poly I:C), a synthetic double-stranded RNA (dsRNA), is recognized by TLR3 and other intracellular receptors. Poly ICLC is a poly I:C analogue, which has been stabilized against the serum nucleases that are present in the plasma of primates. Poly I:C12U, another analogue, is less toxic but also less stable in vivo than poly I:C, and TLR3 is essential for its recognition. To study the effects of these compounds on the induction of protein-specific immune responses in an animal model relevant to humans, rhesus macaques were immunized subcutaneously (s.c.) with keyhole limpet hemocyanin (KLH) or human papillomavirus (HPV)16 capsomeres with or without dsRNA or a control adjuvant, the TLR9 ligand CpG-C. All dsRNA compounds served as adjuvants for KLH-specific cellular immune responses, with the highest proliferative responses being observed with 2 mg/animal poly ICLC (p = 0.002) or 6 mg/animal poly I:C12U (p = 0.001) when compared with immunization with KLH alone. Notably, poly ICLC—but not CpG-C given at the same dose—also helped to induce HPV16-specific Th1 immune responses while both adjuvants supported the induction of strong anti-HPV16 L1 antibody responses as determined by ELISA and neutralization assay. In contrast, control animals injected with HPV16 capsomeres alone did not develop substantial HPV16-specific immune responses. Injection of dsRNA led to increased numbers of cells producing the T cell–activating chemokines CXCL9 and CXCL10 as detected by in situ hybridization in draining lymph nodes 18 hours after injections, and to increased serum levels of CXCL10 (p = 0.01). This was paralleled by the reduced production of the homeostatic T cell–attracting chemokine CCL21. Thus, synthetic dsRNAs induce an innate chemokine response and act as adjuvants for virus-specific Th1 and humoral immune responses in nonhuman primates.

The efficacy of DNA vaccination is enhanced in mice by targeting the encoded protein to dendritic cells

DNA vaccines promote an immune response by providing antigen-encoding DNA to the recipient, but the efficacy of such vaccines needs improving. Many approaches have considerable potential but currently induce relatively weak immune responses despite multiple high doses of DNA vaccine. Here, we asked whether targeting vaccine antigens to DCs would increase the immunity and protection that result from DNA vaccines. To determine this, we generated a DNA vaccine encoding a fusion protein comprised of the vaccine antigen and a single-chain Fv antibody (scFv) specific for the DC-restricted antigen-uptake receptor DEC205. Following vaccination of mice, the vaccine antigen was expressed selectively by DCs, which were required for the increased efficacy of MHC class I and MHC class II antigen presentation relative to a control scFv DNA vaccine. In addition, a DNA vaccine encoding an HIV gag p41-scFv DEC205 fusion protein induced 10-fold higher antibody levels and increased numbers of IFN-gamma-producing CD4+ and CD8+ T cells. After a single i.m. injection of the DNA vaccine encoding an HIV gag p41-scFv DEC205 fusion protein, mice were protected from an airway challenge with a recombinant vaccinia virus expressing the HIV gag p41, even with 1% of the dose of nontargeted DNA vaccine. The efficacy of DNA vaccines therefore may be enhanced by inclusion of sequences such as single-chain antibodies to target the antigen to DCs.

Detection of human viruses in rivers of a densly-populated area in Germany using a virus adsorption elution method optimized for PCR analyses

Transmission of viruses via surface water is a major public health concern. To determine the viral concentration in rivers of a densely-populated area in Germany, the virus adsorption elution (VIRADEL) method was optimized for downstream PCR applications. Using a high-salt alkaline phosphate buffer for elution, the median recovery efficiency from spiked 1l water samples ranged from 21.3% to 100% for JC polyomavirus, human adenovirus type 5, Echovirus 11, and norovirus genogroup I. Analyses of 41 water samples collected during the winter 2007/08 from the rivers Ruhr and Rhine yielded detection rates 97.5% for adenoviruses and human polyomavirus (JC, BK), and 90% for group A rotaviruses. Noroviruses genogroup II were detected in 31.7% of the samples and only one sample was positive for enteroviruses. Virus concentrations ranged from 9.4 to 2.3x10(4) gen.equ./l. However, the genome equivalents/liter determined for the RNA viruses and their detection frequency are only lower limits, since the concentration procedure leads to carry-over of inhibitors of the reverse transcription step. Sequence analyses of the PCR products revealed that the adenovirus and rotavirus PCRs used could cross-react with animal viruses from the respective virus families. These results suggest that detection of human polyomavirus genomes is the most sensitive and specific marker for contamination of surface water with viruses from human sewage. Although we could routinely detect nucleic acids of viral pathogens in river water by the PCR-optimized VIRADEL method, threshold levels of viral nucleic acids above which there is a risk of infection with viruses derived from human remain to be determined.

S Protein of Severe Acute Respiratory Syndrome-Associated Coronavirus Mediates Entry into Hepatoma Cell Lines and Is Targeted by Neutralizing Antibodies in Infected Patients

ABSTRACT The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) causes severe pneumonia with a fatal outcome in approximately 10% of patients. SARS-CoV is not closely related to other coronaviruses but shares a similar genome organization. Entry of coronaviruses into target cells is mediated by the viral S protein. We functionally analyzed SARS-CoV S using pseudotyped lentiviral particles (pseudotypes). The SARS-CoV S protein was found to be expressed at the cell surface upon transient transfection. Coexpression of SARS-CoV S with human immunodeficiency virus-based reporter constructs yielded viruses that were infectious for a range of cell lines. Most notably, viral pseudotypes harboring SARS-CoV S infected hepatoma cell lines but not T- and B-cell lines. Infection of the hepatoma cell line Huh-7 was also observed with replication-competent SARS-CoV, indicating that hepatocytes might be targeted by SARS-CoV in vivo. Inhibition of vacuolar acidification impaired infection by SARS-CoV S-bearing pseudotypes, indicating that S-mediated entry requires low pH. Finally, infection by SARS-CoV S pseudotypes but not by vesicular stomatitis virus G pseudotypes was efficiently inhibited by a rabbit serum raised against SARS-CoV particles and by sera from SARS patients, demonstrating that SARS-CoV S is a target for neutralizing antibodies and that such antibodies are generated in SARS-CoV-infected patients. Our results show that viral pseudotyping can be employed for the analysis of SARS-CoV S function. Moreover, we provide evidence that SARS-CoV infection might not be limited to lung tissue and can be inhibited by the humoral immune response in infected patients.

Development of a Self-Inactivating, Minimal Lentivirus Vector Based on Simian Immunodeficiency Virus

In contrast to oncoviruses, lentiviruses do not require target cell division for integration into the host genome. Lentiviral vectors can therefore expand the spectrum of target cells susceptible to retroviral gene transfer. To analyze whether vectors based on simian immunodeficiency viruses (SIVs) could be used for gene transfer, a three-plasmid vector-packaging system was developed, in which Gag-Pol and the vector itself are of SIV origin, while Env is derived either from SIV, amphotropic murine leukemia virus (MuLV), or the G glycoprotein of vesicular stomatitis virus (VSV-G). To increase the safety of the SIV vector system, a self-inactivating SIV vector was constructed. After optimization of the SIV gag-pol expression plasmid, a minimal SIV vector, which contained only SIV sequences present on the multiply spliced nef transcript, could still be produced at titers of 2 x 10(5) infectious units/ml. Growth-arrested cells could be transduced with this vector even if vif, vpr, vpx, and nef had been deleted from the packaging construct and the vector.

Evaluation of pepper mild mottle virus, human picobirnavirus and Torque teno virus as indicators of fecal contamination in river water

A reliable indicator is needed to predict and reduce the risk of infection associated with fecal contamination of surface water. Since Pepper mild mottle virus (PMMoV), human picobirnaviruses (hPBV) and Torque teno virus (TTV) have been detected at substantial levels in human feces, we explored whether detection of nucleic acids of these viruses is a suitable indicator of fecal contamination in river water. From September 2008 to December 2009, water samples (n = 111) were collected from the Ruhr and Rhine rivers and from the influents and effluents of a wastewater plant (n = 12). Quantitative real time (RT-) PCR was used to determine the abundance of PMMoV, hPBV, and TTV in comparison to human adenoviruses (HAdV) and human polyomaviruses (HPyV) that are frequently detected in surface water and were previously proposed as indicators. While PMMoV was detected in all river water samples, the other viruses were detected less frequently. The concentration of the studied viruses in positive river water ranged from 5 × 10(1) to 1.07 × 10(6) genome equivalents per liter (gen.equ./l). All wastewater samples were positive for PMMoV, HAdV and HPyV, while TTV and hPBV were detected in 6/12 and 3/12 of samples, respectively. To determine if PMMoV is specific to human-derived fecal waste, fecal samples from human (n = 20) and animal (n = 53) were also tested. In contrast to the ubiquity of PMMoV in human feces (19/20) the virus was only detected at low concentration in a minority of the animal fecal samples tested (7/15 from chicken, 1/10 from Geese and 1/6 from cows). Therefore, in this setting TTV and hPBV do not seem to be suitable indicators of fecal contamination in water. Whereas, the high excretion level and dissemination of PMMoV in human sewage and river water suggest that PMMoV could be a promising indicator of fecal pollution in surface water.

Replication Properties of Human Adenovirus In Vivo and in Cultures of Primary Cells from Different Animal Species

ABSTRACT Oncolytic adenoviruses have emerged as a promising approach for the treatment of tumors resistant to other treatment modalities. However, preclinical safety studies are hampered by the lack of a permissive nonhuman host. Screening of a panel of primary cell cultures from seven different animal species revealed that porcine cells support productive replication of human adenovirus type 5 (Ad5) nearly as efficiently as human A549 cells, while release of infectious virus by cells from other animal species tested was diminished by several orders of magnitude. Restriction of productive Ad5 replication in rodent and rabbit cells seems to act primarily at a postentry step. Replication efficiency of adenoviral vectors harboring different E1 deletions or mutations in porcine cells was similar to that in A549 cells. Side-by-side comparison of the viral load kinetics in blood of swine and mice injected with Ad5 or a replication-deficient adenoviral vector failed to provide clear evidence for virus replication in mice. In contrast, evidence suggests that adenovirus replication occurs in swine, since adenoviral late gene expression produced a 13.5-fold increase in viral load in an individual swine from day 3 to day 7 and 100-fold increase in viral DNA levels in the Ad5-infected swine compared to the animal receiving a replication-deficient adenovirus. Lung histology of Ad5-infected swine revealed a severe interstitial pneumonia. Although the results in swine are based on a small number of animals and need to be confirmed, our data strongly suggest that infection of swine with human adenovirus or oncolytic adenoviral vectors is a more appropriate animal model to study adenoviral pathogenicity or pharmacodynamic and toxicity profiles of adenoviral vectors than infection of mice.

Inhibition of early steps in the lentiviral replication cycle by cathelicidin host defense peptides.

BackgroundThe antibacterial activity of host defense peptides (HDP) is largely mediated by permeabilization of bacterial membranes. The lipid membrane of enveloped viruses might also be a target of antimicrobial peptides. Therefore, we screened a panel of naturally occurring HDPs representing different classes for inhibition of early, Env-independent steps in the HIV replication cycle. A lentiviral vector-based screening assay was used to determine the inhibitory effect of HDPs on early steps in the replication cycle and on cell metabolism.ResultsHuman LL37 and porcine Protegrin-1 specifically reduced lentiviral vector infectivity, whereas the reduction of luciferase activities observed at high concentrations of the other HDPs is primarily due to modulation of cellular activity and/ or cytotoxicity rather than antiviral activity. A retroviral vector was inhibited by LL37 and Protegrin-1 to similar extent, while no specific inhibition of adenoviral vector mediated gene transfer was observed. Specific inhibitory effects of Protegrin-1 were confirmed for wild type HIV-1.ConclusionAlthough Protegrin-1 apparently inhibits an early step in the HIV-replication cycle, cytotoxic effects might limit its use as an antiviral agent unless the specificity for the virus can be improved.

Methods to detect infectious human enteric viruses in environmental water samples

Abstract Currently, a wide range of analytical methods is available for virus detection in environmental water samples. Molecular methods such as polymerase chain reaction (PCR) and quantitative real time PCR (qPCR) have the highest sensitivity and specificity to investigate virus contamination in water, so they are the most commonly used in environmental virology. Despite great sensitivity of PCR, the main limitation is the lack of the correlation between the detected viral genome and viral infectivity, which limits conclusions regarding the significance for public health. To provide information about the infectivity of the detected viruses, cultivation on animal cell culture is the gold standard. However, cell culture infectivity assays are laborious, time consuming and costly. Also, not all viruses are able to produce cytopathic effect and viruses such as human noroviruses have no available cell line for propagation. In this brief review, we present a summary and critical evaluation of different approaches that have been recently proposed to overcome limitations of the traditional cell culture assay and PCR assay such as integrated cell culture-PCR, detection of genome integrity, detection of capsid integrity, and measurement of oxidative damages on viral capsid protein. Techniques for rapid detection of infectious viruses such as fluorescence microscopy and automated flow cytometry have also been suggested to assess virus infectivity in water samples.