Untersuchungen zur molekularen Biologie und Diagnostik ausgewählter Enteroviren

Die Vielfalt der enteroviralen Serotypen (z.Zt. über 75 Serotypen in 8 Spezies) erfordert eine molekularbiologische Einordnung als Basis einer verfeinerten Diagnostik. In diesem Zusammenhang waren die animalen Enteroviren bisher noch weitgehend unbekannt. Dabei sind die Enteroviren des Schweins (PEVs) von besonderem Interesse, da das Schwein als potentieller Organspender im Rahmen der Xenotransplantation betrachtet wird. Molekulare Genomanalysen und Sequenzvergleiche zeigen, dass es sich bei den PEVs um eine sehr heterogene Picornavirusgruppe handelt. Die meisten Serotypen können nicht zu den Enteroviren gerechnet werden, sondern bilden zwei eigene neue Genera Teschovirus und Sapelovirus. Nur zwei Serotypen (PEV-9/-10) sind aufgrund ihrer Genomorganisation als eigene Spezies dem Genus Enterovirus zuzuordnen. Als Besonderheit weisen sie in einer für die virale Replikation bedeutsamen Subdomäne der kleeblattförmigen Struktur am 5?-Ende signifikante Unterschiede zu den humanen Enterovirusspezies auf. Ausgerechnet PEV-9 und ?10 vermehren sich auf humanen Zellinien und zeigen in diesen Zellkulturen einen deutlichen zytopathischen Effekt. Sie müssen somit als potentielle Erreger im Rahmen von Xenozoonosen bewertet werden. Deshalb wurde eine real-time RT-PCR Diagnostik auf Basis der LightCycler-Technologie entwickelt, die es erlaubt, durch Verwendung interner Standards die eingesetzten viralen cDNAs direkt zu quantifizieren. Damit ist ein erster Schritt in ein klinisches Monitoring möglicher immunsupprimierter Xenotransplantatempfänger getan

Novel ressortant swine H3N2 influenza A viruses in Germany

Analysis of 228 H3N2 swine influenza A virus isolates collected between 2003 and 2015 in Germany revealed important changes in molecular epidemiology. The data indicate that a novel reassortant, Rietberg/2014-like swine H3N2, emerged in February 2014 in Northern Germany. It is comprised of a hemagglutinin gene of seasonal H3N2 (A/Denmark/129/2005-like), a neuraminidase gene of Emmelsbuell/2009-like swine H1N2 and the internal gene cassette of pandemic H1N1 viruses. Together with Danish swine H3N2 strains of 2013–2015 with identical genome layout, the Rietberg/2014-like viruses represent a second swine H3N2 lineage which cocirculates with a variant of the Gent/1984-like swine H3N2 lineage. This variant, named Gent1984/Diepholz-like swine H3N2, has a Gent/1984-like HA and a Diepholz/2008-like NA; the origin of the internal gene cassette likely derived from avian-like swine H1N1. The first isolate of the Gent1984/Diepholz reassortant emerged in Northern Germany in September 2011 whereas the last German Gent/1984-like isolate was collected in October 2011.Peer Reviewe

Humoral immune response after different SARS-CoV-2 vaccination regimens

Results After the first vaccination, the prevalence of IgG directed against the (trimeric) SARS-CoV-2 S-protein and its receptor binding domain (RBD) varied from 55-95% (AZD1222) to 100% (BNT162b2), depending on the vaccine regimen and the SARS-CoV-2 antigen used. The booster vaccination resulted in 100% seroconversion and the occurrence of highly avid IgG, which is directed against the S-protein subunit 1 and the RBD, as well as VNA against VOC B.1.1.7, while anti-NP IgGs were not detected. The results of the three anti-SARS-CoV-2 IgG tests showed an excellent correlation to the VNA titres against this VOC. The agreement of cVNT and sVNT results was good. However, the sVNT seems to overestimate non- and weak B.1.1.7-neutralising titres. The anti-SARS-CoV-2 IgG concentrations and the B.1.1.7-neutralising titres were significantly higher after heterologous vaccination compared to the homologous AZD1222 scheme. If VOC B.1.617.2 was used as antigen, significantly lower VNA titres were measured in the cVNT, and three (33.3%) vector vaccine recipients had a VNA titre < 1:10. Conclusions Heterologous SARS-CoV-2 vaccination leads to a strong antibody response with anti-SARS-CoV-2 IgG concentrations and VNA titres at a level comparable to that of a homologous BNT162b2 vaccination scheme. Irrespective of the chosen immunisation regime, highly avid IgG antibodies can be detected just 2 weeks after the second vaccine dose indicating the development of a robust humoral immunity. The reduction in the VNA titre against VOC B.1.617.2 observed in the subgroup of 26 individuals is remarkable and confirms the immune escape of the delta variant

Development of SARS-CoV-2 Specific IgG and Virus-Neutralizing Antibodies after Infection with Variants of Concern or Vaccination

The humoral immunity after SARS-CoV-2 infection or vaccination was examined. Convalescent sera after infection with variants of concern (VOCs: B.1.1.7, n = 10; B.1.351, n = 1) and sera from 100 vaccinees (Pfizer/BioNTech, BNT162b2, n = 33; Moderna, mRNA-1273, n = 11; AstraZeneca, ChAdOx1 nCoV-19/AZD1222, n = 56) were tested for the presence of immunoglobulin G (IgG) directed against the viral spike (S)-protein, its receptor-binding domain (RBD), the nucleoprotein (N) and for virus-neutralizing antibodies (VNA). For the latter, surrogate assays (sVNT) and a Vero-cell based neutralization test (cVNT) were used. Maturity of IgG was determined by measuring the avidity in an immunoblot (IB). Past VOC infection resulted in a broad reactivity of anti-S IgG (100%), anti-RBD IgG (100%), and anti-N IgG (91%), while latter were absent in 99% of vaccinees. Starting approximately two weeks after the first vaccine dose, anti-S IgG (75-100%) and particularly anti-RBD IgG (98-100%) were detectable. After the second dose, their titers increased and were higher than in the convalescents. The sVNT showed evidence of VNA in 91% of convalescents and in 80-100%/100% after first/second vaccine dose, respectively. After the second dose, an increase in VNA titer and IgGs of high avidity were demonstrated by cVNT and IB, respectively. Re-vaccination contributes to a more robust immune response

Kinetics of Nucleo- and Spike Protein-Specific Immunoglobulin G and of Virus-Neutralizing Antibodies after SARS-CoV-2 Infection

Kinetics of neutralizing antibodies and immunoglobulin G (IgG) against the nucleo (N) or spike (S) proteins of severe acute respiratory syndrome coronavirus type2 (SARS-CoV-2) were studied in patients up to 165 days after PCR diagnosis of infection. Two immunoassays were selected out of eight IgG or total antibody tests by comparing their specificities and sensitivities. Sensitivities were calculated with convalescent sera from 26 PCR-confirmed cases, of which 76.9% had neutralizing antibodies (>1:10). Stored sera collected during the summer 2018 (N = 50) and winter seasons 2018/2019 (N = 50) were included to demonstrate the test specificities. IgG kinetics, avidities, and virus-neutralizing capacities were recorded over up to 165 days in eleven patients and five individuals from routine diagnostics. Sensitivities, specificities, and diagnostic accuracies ranged between 80.8-96.3%, 96.0-100%, and 93.7-99.2%, respectively. Nearly all results were confirmed with two different SARS-CoV-2-specific immunoblots. Six (54.4%) patients exhibited stable N-specific IgG indices over 120 days and longer; three of them developed IgG of high avidity. The S-specific IgG response was stable in ten (91.0%) patients, and eight (72.7%) had neutralizing antibodies. However, the titers were relatively low, suggesting that sustained humoral immunity is uncertain, especially after outpatient SARS-CoV-2 infection

Fast Detection of SARS-CoV-2 RNA Directly from Respiratory Samples Using a Loop-Mediated Isothermal Amplification (LAMP) Test

The availability of simple SARS-CoV-2 detection methods is crucial to contain the COVID-19 pandemic. This study examined whether a commercial LAMP assay can reliably detect SARS-CoV-2 genomes directly in respiratory samples without having to extract nucleic acids (NA) beforehand. Nasopharyngeal swabs (NPS, n = 220) were tested by real-time reverse transcription (RT)-PCR and with the LAMP assay. For RT-PCR, NA were investigated. For LAMP, NA from 26 NPS in viral transport medium (VTM) were tested. The other 194 NPS were analyzed directly without prior NA extraction (140 samples in VTM; 54 dry swab samples stirred in phosphate buffered saline). Ten NPS were tested directly by LAMP using a sous-vide cooking unit. The isothermal assay demonstrated excellent specificity (100%) but moderate sensitivity (68.8%), with a positive predictive value of 1 and a negative predictive value of 0.65 for direct testing of NPS in VTM. The use of dry swabs, even without NA extraction, improved the analytical sensitivity; up to 6% of samples showed signs of inhibition. LAMP could be performed successfully with a sous-vide cooking unit. This technique is very fast, requires little laboratory resources, and can replace rapid antigen tests or verify reactive rapid tests on-site

Performance of a Point-of-Care Test for the Rapid Detection of SARS-CoV-2 Antigen

The rapid detection of infections caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is necessary in the ongoing pandemic. Antigen-specific point-of-care tests (POCT) may be useful for this purpose. Here, such a POCT (SARS-CoV-2 NADAL® COVID-19 Ag) was compared to a laboratory-developed triplex real-time polymerase chain reaction (RT-PCR) designed for the detection of viral nucleoprotein gene and two control targets. This RT-PCR served as a reference to investigate POCT sensitivity by re-testing upper respiratory tract (URT) samples (n = 124) exhibiting different SARS-CoV-2 loads in terms of RT-PCR threshold cycle (Ct) values. The optical intensities of the antigen bands were compared to the Ct values of the RT-PCR. The infectivity of various virus loads was estimated by inoculating Vero cells with URT samples (n = 64, Ct 17-34). POCT sensitivity varied from 100% (Ct 30 were negative; among SARS-CoV-2 free samples (n = 10) no false-positives were detected. A head-to-head comparison with another POCT (Abbott, Panbio™ COVID-19 Ag Rapid Test) yielded similar results. Isolation of SARS-CoV-2 in cell-culture was successful up to a Ct value of 29. The POCT reliably detects high SARS-CoV-2 loads and rapidly identifies infectious individuals

In vitro and in vivo effects of Pelargonium sidoides DC. root extract EPs® 7630 and selected constituents against SARS-CoV-2 B.1, Delta AY.4/AY.117 and Omicron BA.2

The occurrence of immune-evasive SARS-CoV-2 strains emphasizes the importance to search for broad-acting antiviral compounds. Our previous in vitro study showed that Pelargonium sidoides DC. root extract EPs® 7630 has combined antiviral and immunomodulatory properties in SARS-CoV-2-infected human lung cells. Here we assessed in vivo effects of EPs® 7630 in SARS-CoV-2-infected hamsters, and investigated properties of EPs® 7630 and its functionally relevant constituents in context of phenotypically distinct SARS-CoV-2 variants. We show that EPs® 7630 reduced viral load early in the course of infection and displayed significant immunomodulatory properties positively modulating disease progression in hamsters. In addition, we find that EPs® 7630 differentially inhibits SARS-CoV-2 variants in nasal and bronchial human airway epithelial cells. Antiviral effects were more pronounced against Omicron BA.2 compared to B.1 and Delta, the latter two preferring TMPRSS2-mediated fusion with the plasma membrane for cell entry instead of receptor-mediated low pH-dependent endocytosis. By using SARS-CoV-2 Spike VSV-based pseudo particles (VSVpp), we confirm higher EPs® 7630 activity against Omicron Spike-VSVpp, which seems independent of the serine protease TMPRSS2, suggesting that EPs® 7630 targets endosomal entry. We identify at least two molecular constituents of EPs® 7630, i.e., (−)-epigallocatechin and taxifolin with antiviral effects on SARS-CoV-2 replication and cell entry. In summary, our study shows that EPs® 7630 ameliorates disease outcome in SARS-CoV-2-infected hamsters and has enhanced activity against Omicron, apparently by limiting late endosomal SARS-CoV-2 entry

Cocirculation of Swine H1N1 Influenza A Virus Lineages in Germany

The genome analysis of 328 H1N1 swine influenza virus isolates collected in a 13-year long-term swine influenza surveillance in Germany is reported. Viral genomes were sequenced with the Illumina next-generation sequencing technique and conventional Sanger methods. Phylogenetic analyses were conducted with Bayesian tree inference. The results indicate continued prevalence of Eurasian avian swine H1N1 but also emergence of a novel H1N1 reassortant, named Schneiderkrug/2013-like swine H1N1, with human-like hemagglutinin and avian-like neuraminidase and internal genes. Additionally, the evolution of an antigenic drift variant of A (H1N1) pdm09 was observed, named Wachtum/2014-like swine H1N1. Both variants were first isolated in northwest Germany, spread to neighboring German states and reached greater proportions of the H1N1 isolates of 2014 and 2015. The upsurge of Wachtum/2014-like swine H1N1 is of interest as this is the first documented persistent swine-to-swine spread of A (H1N1) pdm09 in Germany associated with antigenic variation. Present enzootic swine influenza viruses in Germany now include two or more co-circulating, antigenically variant viruses of each of the subtypes, H1N1 and H1N2