Role of the 2B4 Receptor in CD8+ T-Cell-Dependent Immune Control of Epstein-Barr Virus Infection in Mice With Reconstituted Human Immune System Components

Patients with X-linked lymphoproliferative (XLP) disease due to deficiency in the adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) are highly susceptible to one specific viral pathogen, the Epstein-Barr virus (EBV). This susceptibility might result from impaired CD8+ T-cell and natural killer cell responses to EBV infection in these patients. We demonstrate that antibody blocking of the SAP-dependent 2B4 receptor is sufficient to induce XLP-like aggravation of EBV disease in mice with reconstituted human immune system components. CD8+ T cells require 2B4 for EBV-specific immune control, because 2B4 blockade after CD8+ T-cell depletion did not further aggravate symptoms of EBV infectio

Usefulness of the GenMark ePlex RPP assay for the detection of respiratory viruses compared to the FTD21 multiplex RT-PCR

Cartridge-based multiplex panels covering numerous pathogens offer an advantage of minimal hands-on-time and short time to result to commercial RT-PCR assays. In this study, we evaluated the performance of the ePlex respiratory pathogen panel (RPP) compared to the Fast Track Diagnostics Respiratory pathogens 21 multiplex RT-PCR assay (FTD21) using 400 clinical respiratory samples. Discrepant results were further analysed by a reference nucleic acid amplification testing (NAT) and a composite reference approach was used for final interpretation. Discordant results were observed in 56 targets corresponding to 54 samples. Sensitivities and specificities were 85.5% and 99.9% for the ePlex RPP and 95.8% and 99.7% for the FTD21 system, respectively. Altogether, the ePlex RPP is a valuable tool for the rapid detection of a number of different respiratory viruses with the exception of the coronavirus family (low sensitivity ranging from 50-80%) and samples with a low pathogen load (Ct values >33)

Two Years of Viral Metagenomics in a Tertiary Diagnostics Unit: Evaluation of the First 105 Cases

Metagenomic next-generation sequencing (mNGS) can capture the full spectrum of viral pathogens in a specimen and has the potential to become an all-in-one solution for virus diagnostics. To date, clinical application is still in an early phase and limitations remain. Here, we evaluated the impact of viral mNGS for cases analyzed over two years in a tertiary diagnostics unit. High throughput mNGS was performed upon request by the treating clinician in cases where the etiology of infection remained unknown or the initial differential diagnosis was very broad. The results were compared to conventional routine testing regarding outcome and workload. In total, 163 specimens from 105 patients were sequenced. The main sample types were cerebrospinal fluid (34%), blood (33%) and throat swabs (10%). In the majority of the cases, viral encephalitis/meningitis or respiratory infection was suspected. In parallel, conventional virus diagnostic tests were performed (mean 18.5 individually probed targets/patients). mNGS detected viruses in 34 cases (32%). While often confirmatory, in multiple cases, the identified viruses were not included in the selected routine diagnostic tests. Two years of mNGS in a tertiary diagnostics unit demonstrated the advantages of a single, untargeted approach for comprehensive, rapid and efficient virus diagnostics, confirming the utility of mNGS in complementing current routine tests

MicroRNAs of Epstein-Barr Virus Attenuate T-Cell-Mediated Immune Control In Vivo

The human persistent and oncogenic Epstein-Barr virus (EBV) was one of the first viruses that were described to express viral microRNAs (miRNAs). These have been proposed to modulate many host and viral functions, but their predominant role in vivo has remained unclear. We compared recombinant EBVs expressing or lacking miRNAs during in vivo infection of mice with reconstituted human immune system components and found that miRNA-deficient EBV replicates to lower viral titers with decreased frequencies of proliferating EBV-infected B cells. In response, activated cytotoxic EBV-specific T cells expand to lower frequencies than during infection with miRNA-expressing EBV. However, when we depleted CD8$^{+}$ T cells the miRNA-deficient virus reached similar viral loads as wild-type EBV, increasing by more than 200-fold in the spleens of infected animals. Furthermore, CD8$^{+}$ T cell depletion resulted in lymphoma formation in the majority of animals after miRNA-deficient EBV infection, while no tumors emerged when CD8$^{+}$ T cells were present. Thus, miRNAs mainly serve the purpose of immune evasion from T cells in vivo and could become a therapeutic target to render EBV-associated malignancies more immunogenic.IMPORTANCE Epstein-Barr virus (EBV) infects the majority of the human population and usually persists asymptomatically within its host. Nevertheless, EBV is the causative agent for infectious mononucleosis (IM) and for lymphoproliferative disorders, including Burkitt and Hodgkin lymphomas. The immune system of the infected host is thought to prevent tumor formation in healthy virus carriers. EBV was one of the first viruses described to express miRNAs, and many host and viral targets were identified for these in vitro However, their role during EBV infection in vivo remained unclear. This work is the first to describe that EBV miRNAs mainly increase viremia and virus-associated lymphomas through dampening antigen recognition by adaptive immune responses in mice with reconstituted immune responses. Currently, there is no prophylactic or therapeutic treatment to restrict IM or EBV-associated malignancies; thus, targeting EBV miRNAs could promote immune responses and limit EBV-associated pathologies

Role of the 2B4 receptor in CD8+ T-cell-dependent immune control of Epstein-barr virus infection in mice with reconstituted human immune system components

Patients with X-linked lymphoproliferative (XLP) disease due to deficiency in the adaptor molecule signaling lymphocytic activation molecule-associated protein (SAP) are highly susceptible to one specific viral pathogen, the Epstein-Barr virus (EBV). This susceptibility might result from impaired CD8(+) T-cell and natural killer cell responses to EBV infection in these patients. We demonstrate that antibody blocking of the SAP-dependent 2B4 receptor is sufficient to induce XLP-like aggravation of EBV disease in mice with reconstituted human immune system components. CD8(+) T cells require 2B4 for EBV-specific immune control, because 2B4 blockade after CD8(+) T-cell depletion did not further aggravate symptoms of EBV infection

Evaluation of the RIDA®GENE RT-PCR assays for detection of sapovirus, astrovirus, adenovirus, and rotavirus in stool samples of adults in Switzerland

Sapovirus (SaV) and astrovirus (AstV) increasingly are recognized as cause of acute viral gastroenteritis (AGE). We evaluated the real-time RT-PCR assays RIDA®GENE SaV and viral stool panel II (RGN RT-PCR) for detection of SaV, AstV, adenovirus (AdV) F40/41 and rotavirus (RoV) in clinical stool samples (n = 69). Results were compared with reference singleplex RT-PCRs. The sensitivity for SaV, AstV and RoV are 100%, the specificity ranges from 98.1% to 100%. In 10 out of 11 AdV (all types) samples, the RGN RT-PCR for AdV F40/41 displayed negative results. Retrospectively, 196 stool specimens from adult patients previously tested negative for norovirus (NoV) were analyzed. In about 10% of NoV-negative stool samples, AdV (n = 9), RoV (n = 6), AstV (n = 3) or SaV (n = 3) were found. The RGN RT-PCR assays are useful for detection of enteric viruses other than NoV. This study emphasizes the need for further testing of NoV-negative stool samples in patients with AGE

Meningitis and epididymitis caused by Toscana virus infection imported to Switzerland diagnosed by metagenomic sequencing: a case report

BACKGROUND We report a rare case of Toscana virus infection imported into Switzerland in a 23-year old man who travelled to Imperia (Italy) 10 days before onset of symptoms. Symptoms included both meningitis and as well epididymitis. This is only the fourth case of Toscana virus reported in Switzerland. CASE PRESENTATION The patient presented with lymphocytic meningitis and scrotal pain due to epididymitis. Meningitis was initially treated with ceftriaxone. Herpes simplex, tick-borne encephalitis, enterovirus, measles, mumps, rubella and Treponema pallidum were excluded with specific polymerase chain reaction (PCR) or serology. In support of routine diagnostic PCR and serology assays, unbiased viral metagenomic sequencing was performed of cerebrospinal fluid and serum. Toscana virus infection was identified in cerebrospinal fluid and the full coding sequence could be obtained. Specific PCR in cerebrospinal fluid and blood and serology with Immunoglobulin (Ig) M and IgG against Toscana virus confirmed our diagnosis. Neurological symptoms recovered spontaneously after 5 days. CONCLUSIONS This case of Toscana virus infection highlights the benefits of unbiased metagenomic sequencing to support routine diagnostics in rare or unexpected viral infections. With increasing travel histories of patients, physicians should be aware of imported Toscana virus as the agent for viral meningitis and meningoencephalitis

Plasmacytoid dendritic cells respond to Epstein-Barr virus infection with a distinct type I interferon subtype profile

Infectious mononucleosis, caused by infection with the human gamma-herpesvirus Epstein-Barr virus (EBV), manifests with one of the strongest CD8$^{+}$ T-cell responses described in humans. The resulting T-cell memory response controls EBV infection asymptomatically in the vast majority of persistently infected individuals. Whether and how dendritic cells (DCs) contribute to the priming of this near-perfect immune control remains unclear. Here we show that of all the human DC subsets, plasmacytoid DCs (pDCs) play a central role in the detection of EBV infection in vitro and in mice with reconstituted human immune system components. pDCs respond to EBV by producing the interferon (IFN) subtypes α1, α2, α5, α7, α14, and α17. However, the virus curtails this type I IFN production with its latent EBV gene products EBNA3A and EBNA3C. The induced type I IFNs inhibit EBV entry and the proliferation of latently EBV-transformed B cells but do not influence lytic reactivation of the virus in vitro. In vivo, exogenous IFN-α14 and IFN-α17, as well as pDC expansion, delay EBV infection and the resulting CD8$^{+}$ T-cell expansion, but pDC depletion does not significantly influence EBV infection. Thus, consistent with the observation that primary immunodeficiencies compromising type I IFN responses affect only alpha- and beta-herpesvirus infections, we found that EBV elicits pDC responses that transiently suppress viral replication and attenuate CD8$^{+}$ T-cell expansion but are not required to control primary infection