Vanessa Ditt

Lambda Interferon Renders Epithelial Cells of the Respiratory and Gastrointestinal Tracts Resistant to Viral Infections

ABSTRACT Virus-infected cells secrete a broad range of interferons (IFN) which confer resistance to yet uninfected cells by triggering the synthesis of antiviral factors. The relative contributions of the various IFN subtypes to innate immunity against virus infections remain elusive. IFN-α, IFN-β, and other type I IFN molecules signal through a common, universally expressed cell surface receptor, whereas type III IFN (IFN-λ) uses a distinct cell-type-specific receptor complex for signaling. Using mice lacking functional receptors for type I IFN, type III IFN, or both, we found that IFN-λ plays an important role in the defense against several human pathogens that infect the respiratory tract, such as influenza A virus, influenza B virus, respiratory syncytial virus, human metapneumovirus, and severe acute respiratory syndrome (SARS) coronavirus. These viruses were more pathogenic and replicated to higher titers in the lungs of mice lacking both IFN receptors than in mice with single IFN receptor defects. In contrast, Lassa fever virus, which infects via the respiratory tract but primarily replicates in the liver, was not influenced by the IFN-λ receptor defect. Careful analysis revealed that expression of functional IFN-λ receptor complexes in the lung and intestinal tract is restricted to epithelial cells and a few other, undefined cell types. Interestingly, we found that SARS coronavirus was present in feces from infected mice lacking receptors for both type I and type III IFN but not in those from mice lacking single receptors, supporting the view that IFN-λ contributes to the control of viral infections in epithelial cells of both respiratory and gastrointestinal tracts.

The SARS-Coronavirus-Host Interactome: Identification of Cyclophilins as Target for Pan-Coronavirus Inhibitors

Coronaviruses (CoVs) are important human and animal pathogens that induce fatal respiratory, gastrointestinal and neurological disease. The outbreak of the severe acute respiratory syndrome (SARS) in 2002/2003 has demonstrated human vulnerability to (Coronavirus) CoV epidemics. Neither vaccines nor therapeutics are available against human and animal CoVs. Knowledge of host cell proteins that take part in pivotal virus-host interactions could define broad-spectrum antiviral targets. In this study, we used a systems biology approach employing a genome-wide yeast-two hybrid interaction screen to identify immunopilins (PPIA, PPIB, PPIH, PPIG, FKBP1A, FKBP1B) as interaction partners of the CoV non-structural protein 1 (Nsp1). These molecules modulate the Calcineurin/NFAT pathway that plays an important role in immune cell activation. Overexpression of NSP1 and infection with live SARS-CoV strongly increased signalling through the Calcineurin/NFAT pathway and enhanced the induction of interleukin 2, compatible with late-stage immunopathogenicity and long-term cytokine dysregulation as observed in severe SARS cases. Conversely, inhibition of cyclophilins by cyclosporine A (CspA) blocked the replication of CoVs of all genera, including SARS-CoV, human CoV-229E and -NL-63, feline CoV, as well as avian infectious bronchitis virus. Non-immunosuppressive derivatives of CspA might serve as broad-range CoV inhibitors applicable against emerging CoVs as well as ubiquitous pathogens of humans and livestock.

Prospective study of Human Bocavirus (HBoV) infection in a pediatric university hospital in Germany 2005/2006

Abstract Background Human Bocavirus (HBoV), a new species of the genus parvovirus newly detected in 2005, seems to be a worldwide distributed pathogen among children with respiratory tract infection (prevalence 2%–18%). Recently published retrospective studies and one prospective birth cohort study suggest that HBoV-primary infection occurs in infants. Methods Prospective single center study over one winter season (November 2005–May 2006) with hospitalized children without age restriction using PCR-based diagnostic methods. Results HBoV DNA was detected in 11 (2.8%) of 389 nasopharyngeal aspirates from symptomatic hospitalized children (median age 9.0 months; range: 3–17 months). RSV, HMPV, HCoV, and Influenza B were detected in 13.9% (n =54), 5.1% (n =20), 2.6% (n =10), and 1.8% (n =7), respectively. There was no influenza A DNA detected in any of the specimens. The clinical diagnoses were acute wheezing (bronchitis) in four patients, radiologically confirmed pneumonia in six patients (55%) and croup syndrome in one patient. In five to six patients with pneumonia, HBoV was the only pathogen detected. While no patient had to be mechanically ventilated, 73% needed oxygen supplementation. In four (36.4%) patients at least one other viral pathogen was found (plus RSV n =3; 27.3%; Norovirus n =1; 9.1%). Conclusion HBoV causes severe respiratory tract infections in infants and young children. Its role as a copathogen and many other open questions has to be defined in further prospective studies.

Reverse genetic characterization of the natural genomic deletion in SARS-Coronavirus strain Frankfurt-1 open reading frame 7b reveals an attenuating function of the 7b protein in-vitro and in-vivo

During the outbreak of SARS in 2002/3, a prototype virus was isolated from a patient in Frankfurt/Germany (strain Frankfurt-1). As opposed to all other SARS-Coronavirus strains, Frankfurt-1 has a 45-nucleotide deletion in the transmembrane domain of its ORF 7b protein. When over-expressed in HEK 293 cells, the full-length protein but not the variant with the deletion caused interferon beta induction and cleavage of procaspase 3. To study the role of ORF 7b in the context of virus replication, we cloned a full genome cDNA copy of Frankfurt-1 in a bacterial artificial chromosome downstream of a T7 RNA polymerase promoter. Transfection of capped RNA transcribed from this construct yielded infectious virus that was indistinguishable from the original virus isolate. The presumed Frankfurt-1 ancestor with an intact ORF 7b was reconstructed. In CaCo-2 and HUH7 cells, but not in Vero cells, the variant carrying the ORF 7b deletion had a replicative advantage against the parental virus (4- and 6-fold increase of virus RNA in supernatant, respectively). This effect was neither associated with changes in the induction or secretion of type I interferon, nor with altered induction of apoptosis in cell culture. However, pretreatment of cells with interferon beta caused the deleted virus to replicate to higher titers than the parental strain (3.4-fold in Vero cells, 7.9-fold in CaCo-2 cells).In Syrian Golden Hamsters inoculated intranasally with 10e4 plaque forming units of either virus, mean titers of infectious virus and viral RNA in the lungs after 24 h were increased 23- and 94.8-fold, respectively, with the deleted virus. This difference could explain earlier observations of enhanced virulence of Frankfurt-1 in Hamsters as compared to other SARS-Coronavirus reference strains and identifies the SARS-CoV 7b protein as an attenuating factor with the SARS-Coronavirus genome. Because attenuation was focused on the early phase of infection in-vivo, ORF 7b might have contributed to the delayed accumulation of virus in patients that was suggested to have limited the spread of the SARS epidemic.

High Seroprevalence of Neutralizing Capacity against Human Metapneumovirus in All Age Groups Studied in Bonn, Germany

ABSTRACT Human metapneumovirus (hMPV) infections occur frequently despite high rates of perpetual seroprevalence for all age groups. Analyses of ∼2,000 archived, randomly selected serum samples demonstrated that neutralizing capacities remain high, with a minor decrease for individuals over 69 years of age, leading to the hypothesis that reinfections occur because humoral immune responses play minor roles in the clearance of hMPV infections.

Human HepG2 cells support respiratory syncytial virus and human metapneumovirus replication

Human metapneumovirus (hMPV) and human respiratory syncytial (RSV) virus cause mild to severe infections of the respiratory tract in all age groups. So far, several cell lines derived from respiratory tissues have been identified to support replication of both viruses. Unfortunately, titers attained during replication differ between the both viruses within one cell line despite equal infection conditions, on the one hand giving raise to the assumption that the individual susceptibility may vary in dependence of the virus, and, on the other hand, making it difficult to compare results between both viruses. Low titers may cause problems in experiments such as animal trials, in which high titers in low volumes are a prerequisite for successful experiments. The advantages are described of the use of a human cell line (normally used for hepatitis viruses research) susceptible for RSV and hMPV in which both viruses replicate to comparable and high titers. It is also shown that the cell line can also be used for applications such as cell viability tests. Cell viability tests can be used as reciprocal determination tests of viral titers and therefore offer the opportunity to replace classical virological tests such as TCID(50). The cell line can be also used for high throughput applications like drug screening, making it a useful tool for screening for antiviral compound active against RSV and hMPV.

Severe acute respiratory syndrome-coronavirus and human coronavirus-NL63: an updated overview.

Severe acute respiratory syndrome-coronavirus and human coronavirus-NL63, though both belonging to different groups of the same genus, are interesting representatives of their kind; the first one is of zoonotic origin, accounted for the pandemic in 2002/2003 that was distributed worldwide and had a mortality rate of about 10%, the other one was identified later and probably has been circulating within the population for centuries and belongs to those viruses that cause the ‘common cold’. A lot of effort has been made to investigate both viruses and to understand their differences and their similarities with regard to further zoonotic events. This review gives an overview of severe acute respiratory syndrome-coronavirus and human coronavirus-NL63, their history and the current state of research.