Viral Metagenomics on Blood-Feeding Arthropods as a Tool for Human Disease Surveillance

Surveillance and monitoring of viral pathogens circulating in humans and wildlife, together with the identification of emerging infectious diseases (EIDs), are critical for the prediction of future disease outbreaks and epidemics at an early stage. It is advisable to sample a broad range of vertebrates and invertebrates at different temporospatial levels on a regular basis to detect possible candidate viruses at their natural source. However, virus surveillance systems can be expensive, costly in terms of finances and resources and inadequate for sampling sufficient numbers of different host species over space and time. Recent publications have presented the concept of a new virus surveillance system, coining the terms “flying biological syringes”, “xenosurveillance” and “vector-enabled metagenomics”. According to these novel and promising surveillance approaches, viral metagenomics on engorged mosquitoes might reflect the viral diversity of numerous mammals, birds and humans, combined in the mosquitoes’ blood meal during feeding on the host. In this review article, we summarize the literature on vector-enabled metagenomics (VEM) techniques and its application in disease surveillance in humans. Furthermore, we highlight the combination of VEM and “invertebrate-derived DNA” (iDNA) analysis to identify the host DNA within the mosquito midgut

Quantitative expression analysis of HHV-6 cell receptor CD46 on cells of human cord blood, peripheral blood and G-CSF mobilised leukapheresis cells

Human herpesvirus-6 (HHV-6) can infect blood cells and thereby may inhibit hematopoietic stem and progenitor cell expansion and differentiation. In this context, it has been discussed if early progenitor cells can be infected by HHV-6. CD46 was identified as one possible cellular surface receptor for HHV-6. The study presented here had been done to get insight into the susceptibility of various leukocyte subpopulations to HHV-6 (including early hematopoietic progenitors) by determining the amount of CD46 molecules expressed on their surfaces. Human cord blood cells, peripheral blood cells and G-CSF mobilised progenitor cells were analysed by flow cytometry. CD46 molecule number per cell was determined and compared to calibration beads conjugated with known ratio of PE per bead. Highest CD46 expression was detected on B- lymphocytes, whereas T-lymphocytes only showed about half of the amount found on B cells. Hematopoietic progenitors also carried CD46 at intermediate levels. Unexpectedly, CD46 expression on progenitors from G-CSF mobilised leukapheresis products was approximately 20% of that found on comparable cells from untreated cord blood. In conclusion, hematopoietic progenitor cells express CD46 on their surface, thereby fulfilling a basic requirement for the susceptibility of HHV-6 infection

Visualization of SARS-CoV-2 particles in naso/oropharyngeal swabs by thin section electron microscopy

Background SARS-CoV-2 replicates efficiently in the upper airways of humans and produces high loads of virus RNA and, at least in the initial phase after infection, many infectious virus particles. Studying virus ultrastructure, such as particle integrity or presence of spike proteins, and effects on their host cells in patient samples is important to understand the pathogenicity of SARS-CoV-2. Methods Suspensions from swab samples with a high load of virus RNA (Ct < 20) were sedimented by desktop ultracentrifugation and prepared for thin section electron microscopy using a novel method which is described in detail. Embedding was performed in Epon or in LR White resin using standard or rapid protocols. Thin sections were examined using transmission electron microscopy. Results Virus particles could be regularly detected in the extracellular space, embedded in a background of heterogenous material (e.g. vesicles and needle-like crystals), and within ciliated cells. Morphology (i.e. shape, size, spike density) of virus particles in the swab samples was very similar to particle morphology in cell culture. However, in some of the samples the virus particles hardly revealed spikes. Infected ciliated cells occasionally showed replication organelles, such as double-membrane vesicles. The most common cells in all samples were keratinocytes from the mucosa and bacteria. Conclusions The new method allows the ultrastructural visualization and analysis of coronavirus particles and of infected host cells from easy to collect naso/oropharyngeal patient swab samples.Peer Reviewe

Cytokine profiles of cord and adult blood leukocytes: differences in expression are due to differences in expression and activation of transcription factors

<p>Abstract</p> <p>Background</p> <p>Stem cell transplantation as therapy for hematological disorders is often hampered by severe graft-versus-host-disease. This may be reduced by umbilical cord blood transplantation, an effect that has been attributed to qualitative differences between neonatal and adult T cells. We compared levels of secreted proteins and cytokine mRNA induced in cord blood leukocytes (CBL) and adult blood leukocytes (ABL) by various stimuli.</p> <p>Results</p> <p>While interleukin-2 (IL-2) levels were similar in CBL and ABL, there was less induction of the Th1 cytokine interferon-γ in CBL. Production of the Th2 cytokines IL-4, IL-5, and IL-13 and the hematopoietic cytokine IL-3 was much lower in CBL versus ABL after T-cell receptor-mediated stimulation, whereas production of GM-CSF was comparable in the 2 cell types. The lower levels of Th1 and Th2 cytokines were maintained in CBL during a 4-day time-course study, while after 12 hours IL-3 and GM-CSF reached in CBL levels similar to those in ABL. For all cytokines except IFNγ, the IC₅₀values for inhibition by cyclosporin A were similar in ABL and CBL. In contrast, there was less expression and activation of transcription factors in CBL. Activation of NF-κB by TPA/ionomycin was detected in ABL but not CBL. Furthermore, there was less expression of the Th subset-specific transcription factors T-bet and c-maf in CBL versus ABL, whereas GATA-3 expression was similar. Expression of T-bet and c-maf correlated with expression of the Th1 and Th2 cytokines, respectively. Time course experiments revealed that T-bet expression was stimulated in both cell types, whereas c-maf and GATA-3 were induced only in ABL.</p> <p>Conclusion</p> <p>The diminished capability of CBL to synthesize cytokines is probably due to decreased activation of NF-κB, whereas differences in Th subsets are due to differences in regulation of Th lineage-specific transcriptions factors. We propose that the reduced incidence and severity of GvHD after allogeneic transplantation of umbilical CB cells is due to lesser activation of specific transcription factors and a subsequent reduction in production of certain cytokines.</p

Detection of Infectious Poxvirus Particles

To enable rapid and reliable detection of poxviruses in clinical and environmental specimens, a diagnostic approach was developed to detect <3 PFU of infectious poxvirus particles in <5 hours. This approach involved virus culture combined with real-time reverse transcription–polymerase chain reaction detection of 2 viral genes expressed immediately after infection

Evaluation of a commercial ELISA as alternative to plaque reduction neutralization test to detect neutralizing antibodies against SARS-CoV-2

High-throughput detection of neutralizing antibodies against SARS-CoV-2 presents a valuable tool for vaccine trials or investigations of population immunity. We evaluate the performance of the first commercial surrogate virus neutralization test (sVNT, GenScript Biotech) against SARS-CoV-2 plaque reduction neutralization test (PRNT) in convalescent and vaccinated individuals. We compare it to five other ELISAs, two of which are designed to detect neutralizing antibodies. In 491 pre-vaccination serum samples, sVNT missed 23.6% of PRNT-positive samples when using the manufacturer-recommended cutoff of 30% binding inhibition. Introducing an equivocal area between 15 and 35% maximized sensitivity and specificity against PRNT to 72.8–93.1% and 73.5–97.6%, respectively. The overall diagnostic performance of the other ELISAs for neutralizing antibodies was below that of sVNT. Vaccinated individuals exhibited higher antibody titers by PRNT (median 119.8, IQR 56.7–160) and binding inhibition by sVNT (median 95.7, IQR 88.1–96.8) than convalescent patients (median 49.1, IQR 20–62; median 52.9, IQR 31.2–76.2). GenScript sVNT is suitable to screen for SARS-CoV-2-neutralizing antibodies; however, to obtain accurate results, confirmatory testing by PRNT in a equivocal area is required. This equivocal area may require adaptation for use in vaccinated individuals, due to higher antibody titers.Peer Reviewe

Comparative Pathogenesis, Genomics and Phylogeography of Mousepox

Ectromelia virus (ECTV), the causative agent of mousepox, has threatened laboratory mouse colonies worldwide for almost a century. Mousepox has been valuable for the understanding of poxvirus pathogenesis and immune evasion. Here, we have monitored in parallel the pathogenesis of nine ECTVs in BALB/cJ mice and report the full-length genome sequence of eight novel ECTV isolates or strains, including the first ECTV isolated from a field mouse, ECTV-MouKre. This approach allowed us to identify several genes, absent in strains attenuated through serial passages in culture, that may play a role in virulence and a set of putative genes that may be involved in enhancing viral growth in vitro. We identified a putative strong inhibitor of the host inflammatory response in ECTV-MouKre, an isolate that did not cause local foot swelling and developed a moderate virulence. Most of the ECTVs, except ECTV-Hampstead, encode a truncated version of the P4c protein that impairs the recruitment of virions into the A-type inclusion bodies, and our data suggest that P4c may play a role in viral dissemination and transmission. This is the first comprehensive report that sheds light into the phylogenetic and geographic relationship of the worldwide outbreak dynamics for the ECTV species.Peer Reviewe

Discovery of herpesviruses in multi-infected primates using locked nucleic acids (LNA) and a bigenic PCR approach

Targeting the highly conserved herpes DNA polymerase (DPOL) gene with PCR using panherpes degenerate primers is a powerful tool to universally detect unknown herpesviruses. However, vertebrate hosts are often infected with more than one herpesvirus in the same tissue, and pan-herpes DPOL PCR often favors the amplification of one viral sequence at the expense of the others. Here we present two different technical approaches that overcome this obstacle: (i) Pan-herpes DPOL PCR is carried out in the presence of an oligonucleotide substituted with locked nucleic acids (LNA).This suppresses the amplification of a specific herpesvirus DPOL sequence by a factor of approximately 1000, thereby enabling the amplification of a second, different DPOL sequence. (ii) The less conserved glycoprotein B (gB) gene is targeted with several sets of degenerate primers that are restricted to gB genes of different herpesvirus subfamilies or genera. These techniques enable the amplification of gB and DPOL sequences of multiple viruses from a single specimen. The partial gB and DPOL sequences can be connected by long-distance PCR, producing final contiguous sequences of approximately 3.5 kbp. Such sequences include parts of two genes and therefore allow for a robust phylogenetic analysis. To illustrate this principle, six novel herpesviruses of the genera Rhadinovirus, Lymphocryptovirus and Cytomegalovirus were discovered in multi-infected samples of non-human primates and phylogenetically characterized