70 research outputs found
The genome of herpesvirus saimiri C488 which is capable of transforming human T cells
AbstractHerpesvirus saimiri (HVS), the rhadinovirus prototype, is apathogenic in the persistently infected natural host, the squirrel monkey, but causes acute T cell leukemia in other New World primate species. In contrast to subgroups A and B, only strains of HVS subgroup C such as C488 are capable of transforming primary human T cells to stable antigen-independent growth in culture. Here, we report the complete 155-kb genome sequence of the transformation-competent HVS strain C488. The A+T-rich unique L-DNA of 113,027 bp encodes at least 77 open reading frames and 5 URNAs. In addition to the viral oncogenes stp and tip, only a few genes including the transactivator orf50 and the glycoprotein orf51 are highly divergent. In a series of new primary HVS isolates, the subgroup-specific divergence of the orf50/orf51 alleles was studied. In these new isolates, the orf50/orf51 alleles of the respective subgroup segregate with the stp and/or tip oncogene alleles, which are essential for transformation
Red-Mediated Transposition and Final Release of the Mini-F Vector of a Cloned Infectious Herpesvirus Genome
Bacterial artificial chromosomes (BACs) are well-established cloning vehicles for functional genomics and for constructing targeting vectors and infectious viral DNA clones. Red-recombination-based mutagenesis techniques have enabled the manipulation of BACs in Escherichia coli without any remaining operational sequences. Here, we describe that the F-factor-derived vector sequences can be inserted into a novel position and seamlessly removed from the present location of the BAC-cloned DNA via synchronous Red-recombination in E. coli in an en passant mutagenesis-based procedure. Using this technique, the mini-F elements of a cloned infectious varicella zoster virus (VZV) genome were specifically transposed into novel positions distributed over the viral DNA to generate six different BAC variants. In comparison to the other constructs, a BAC variant with mini-F sequences directly inserted into the junction of the genomic termini resulted in highly efficient viral DNA replication-mediated spontaneous vector excision upon virus reconstitution in transfected VZV-permissive eukaryotic cells. Moreover, the derived vector-free recombinant progeny exhibited virtually indistinguishable genome properties and replication kinetics to the wild-type virus. Thus, a sequence-independent, efficient, and easy-to-apply mini-F vector transposition procedure eliminates the last hurdle to perform virtually any kind of imaginable targeted BAC modifications in E. coli. The herpesviral terminal genomic junction was identified as an optimal mini-F vector integration site for the construction of an infectious BAC, which allows the rapid generation of mutant virus without any unwanted secondary genome alterations. The novel mini-F transposition technique can be a valuable tool to optimize, repair or restructure other established BACs as well and may facilitate the development of gene therapy or vaccine vectors
The Kinocidin Interleukin-26 Shows Immediate Antimicrobial Effects Even to Multi-resistant Isolates
The cationic proinflammatory cytokine Interleukin 26 (IL-26) shows antibacterial activity and inhibits the replication of cytomegalovirus and hepatitis C virus. This study evaluates the early microbicidal activities of IL-26 against major bacterial species including multi-resistant variants and Candida albicans. Recombinant IL-26 was bacterially expressed and studied for its microbicidal effects in culture. We show that IL-26 has strong 90% bactericidal activities against Enterococcus faecalis, Enterococcus faecium, Staphylococcus aureus, and Acinetobacter baumannii. Similarly, IL-26 sensitivity was also detectable in vancomycin-resistant Enterococcus species, methicillin-resistant S. aureus, and carbapenem-resistant A. baumannii clinical isolates. Additionally, a significant, albeit weak fungicidal effect against Candida albicans was observed. Activities against Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa were not detectable. The proinflammatory cytokine and kinocidin IL-26 shows strong bactericidal activities against A. baumannii and, almost selectively, against Gram-positive bacteria
Bacterial Genetics of Large Mammalian DNA Viruses: Bacterial Artificial Chromosomes as a Prerequisite for Efficiently Studying Viral DNA Replication and Functions
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
Retinitis Due to Infections
Infections are a major cause for retinitis. Whereas Varicella-Zoster and Herpes Simplex viruses are the major reason for acute retinal necrosis, cytomegalovirus retinitis typically occurs in immunocompromised patients. Toxoplasmosis and toxocariasis are the major parasitic pathogens affecting the retina and adjacent tissues. Among the bacterial causes, tuberculosis, syphilis, and bartonellosis are discussed as retinal diseases. The emphasis is laid on the epidemiological and clinical peculiarities, the respective diagnostic procedures, and the therapeutic approaches. Moreover, global disease aspects of infectious retinitis are included
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
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
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