Accurate Strand-Specific Quantification of Viral RNA

The presence of full-length complements of viral genomic RNA is a hallmark of RNA virus replication within an infected cell. As such, methods for detecting and measuring specific strands of viral RNA in infected cells and tissues are important in the study of RNA viruses. Strand-specific quantitative real-time PCR (ssqPCR) assays are increasingly being used for this purpose, but the accuracy of these assays depends on the assumption that the amount of cDNA measured during the quantitative PCR (qPCR) step accurately reflects amounts of a specific viral RNA strand present in the RT reaction. To specifically test this assumption, we developed multiple ssqPCR assays for the positive-strand RNA virus o'nyong-nyong (ONNV) that were based upon the most prevalent ssqPCR assay design types in the literature. We then compared various parameters of the ONNV-specific assays. We found that an assay employing standard unmodified virus-specific primers failed to discern the difference between cDNAs generated from virus specific primers and those generated through false priming. Further, we were unable to accurately measure levels of ONNV (−) strand RNA with this assay when higher levels of cDNA generated from the (+) strand were present. Taken together, these results suggest that assays of this type do not accurately quantify levels of the anti-genomic strand present during RNA virus infectious cycles. However, an assay permitting the use of a tag-specific primer was able to distinguish cDNAs transcribed from ONNV (−) strand RNA from other cDNAs present, thus allowing accurate quantification of the anti-genomic strand. We also report the sensitivities of two different detection strategies and chemistries, SYBR® Green and DNA hydrolysis probes, used with our tagged ONNV-specific ssqPCR assays. Finally, we describe development, design and validation of ssqPCR assays for chikungunya virus (CHIKV), the recent cause of large outbreaks of disease in the Indian Ocean region

Epidemiologic Relationship between Toscana Virus Infection and Leishmania infantum Due to Common Exposure to Phlebotomus perniciosus Sandfly Vector

Sand flies are recognised vectors of parasites in the genus Leishmania and a number of arthropod-borne viruses, in particular viruses within the genus Phlebovirus, family Bunyaviridae. In southern France, Toscana phlebovirus (TOSV) is recognized as a prominent cause of summer meningitis. Since Leishmania and TOSV have a common vector (Phlebotomus perniciosus), an epidemiologic link has been assumed for a long time. However, there is no scientific evidence of such a link between human leishmaniosis and phleboviral infections. To identify a possible link, we investigated the presence and distribution of antibodies against these two microorganisms (i) in individuals and (ii) at a spatial level in the city of Marseille (south-eastern France). Five hundred sera were selected randomly in the biobank of the Department of Parasitology of the Public Hospitals of Marseille. All sera were previously tested for IgG against Leishmania by Western Blotting, and TOSV IgG were detected by indirect immunofluorescence. The seropositivity rates were 21.4% for TOSV and 28% for Leishmania. Statistical analysis demonstrated that seropositivity for one pathogen was significantly associated with seropositivity to the other pathogen. This result provided the first robust evidence for the existence of an epidemiological relationship between Leishmania infantum and TOSV. Addresses of tested patients were geolocalized and integrated into Geographical Information System software, in order to test spatial relationship between the two pathogens. Spatial analysis did not allow to identify (i) specific patterns for the spatial distribution of positive serological results for TOSV or Leishmania, and (ii) a spatial relationship between Leishmania and TOSV positive serological results. This may reflect the fact that the sample studied was not powerful enough to demonstrate either a spatial clustering or co-location, i.e. that the actual risk exposure area is smaller than the mean of distance between patients in our study (245 m)

Molecular approaches to the analysis of deformed wing virus replication and pathogenesis in the honey bee, Apis mellifera

<p>Abstract</p> <p>Background</p> <p>For years, the understanding of the pathogenetic mechanisms that underlie honey bee viral diseases has been severely hindered because of the lack of a cell culture system for virus propagation. As a result, it is very imperative to develop new methods that would permit the <it>in vitro </it>pathogenesis study of honey bee viruses. The identification of virus replication is an important step towards the understanding of the pathogenesis process of viruses in their respective hosts. In the present study, we developed a strand-specific RT-PCR-based method for analysis of Deformed Wing Virus (DWV) replication in honey bees and in honey bee parasitic mites, <it>Varroa Destructor</it>.</p> <p>Results</p> <p>The results shows that the method developed in our study allows reliable identification of the virus replication and solves the problem of falsely-primed cDNA amplifications that commonly exists in the current system. Using TaqMan real-time quantitative RT-PCR incorporated with biotinylated primers and magnetic beads purification step, we characterized the replication and tissue tropism of DWV infection in honey bees. We provide evidence for DWV replication in the tissues of wings, head, thorax, legs, hemolymph, and gut of honey bees and also in Varroa mites.</p> <p>Conclusion</p> <p>The strategy reported in the present study forms a model system for studying bee virus replication, pathogenesis and immunity. This study should be a significant contribution to the goal of achieving a better understanding of virus pathogenesis in honey bees and to the design of appropriate control measures for bee populations at risk to virus infections.</p

The evolving SARS-CoV-2 epidemic in Africa: insights from rapidly expanding genomic surveillance

Investment in SARS-CoV-2 sequencing in Africa over the past year has led to a major increase in the number of sequences generated, now exceeding 100,000 genomes, used to track the pandemic on the continent. Our results show an increase in the number of African countries able to sequence domestically, and highlight that local sequencing enables faster turnaround time and more regular routine surveillance. Despite limitations of low testing proportions, findings from this genomic surveillance study underscore the heterogeneous nature of the pandemic and shed light on the distinct dispersal dynamics of Variants of Concern, particularly Alpha, Beta, Delta, and Omicron, on the continent. Sustained investment for diagnostics and genomic surveillance in Africa is needed as the virus continues to evolve, while the continent faces many emerging and re-emerging infectious disease threats. These investments are crucial for pandemic preparedness and response and will serve the health of the continent well into the 21st century

Emerging viral threats in Gabon: health capacities and response to the risk of emerging zoonotic diseases in Central Africa

Emerging infectious diseases (EID) are currently the major threat to public health worldwide and most EID events have involved zoonotic infectious agents. Central Africa in general and Gabon in particular are privileged areas for the emergence of zoonotic EIDs. Indeed, human incursions in Gabonese forests for exploitation purposes lead to intensified contacts between humans and wildlife thus generating an increased risk of emergence of zoonotic diseases. In Gabon, 51 endemic or potential endemic viral infectious diseases have been reported. Among them, 22 are of zoonotic origin and involve 12 families of viruses. The most notorious are dengue, yellow fever, ebola, marburg, Rift Valley fever and chikungunya viruses. Potential EID due to wildlife in Gabon are thereby plentiful and need to be inventoried. The Gabonese Public Health system covers geographically most of the country allowing a good access to sanitary information and efficient monitoring of emerging diseases. However, access to treatment and prevention is better in urban areas where medical structures are more developed and financial means are concentrated even though the population is equally distributed between urban and rural areas. In spite of this, Gabon could be a good field for investigating the emergence or re-emergence of zoonotic EID. Indeed Gabonese health research structures such as CIRMF, advantageously located, offer high quality researchers and facilities that study pathogens and wildlife ecology, aiming toward a better understanding of the contact and transmission mechanisms of new pathogens from wildlife to human, the emergence of zoonotic EID and the breaking of species barriers by pathogens

A year of genomic surveillance reveals how the SARS-CoV-2 pandemic unfolded in Africa

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Viperin is induced following dengue virus type-2 (DENV-2) infection and has anti-viral actions requiring the C-terminal end of viperin

The host protein viperin is an interferon stimulated gene (ISG) that is up-regulated during a number of viral infections. In this study we have shown that dengue virus type-2 (DENV-2) infection significantly induced viperin, co-incident with production of viral RNA and via a mechanism requiring retinoic acid-inducible gene I (RIG-I). Viperin did not inhibit DENV-2 entry but DENV-2 RNA and infectious virus release was inhibited in viperin expressing cells. Conversely, DENV-2 replicated to higher tires earlier in viperin shRNA expressing cells. The anti-DENV effect of viperin was mediated by residues within the C-terminal 17 amino acids of viperin and did not require the N-terminal residues, including the helix domain, leucine zipper and S-adenosylmethionine (SAM) motifs known to be involved in viperin intracellular membrane association. Viperin showed co-localisation with lipid droplet markers, and was co-localised and interacted with DENV-2 capsid (CA), NS3 and viral RNA. The ability of viperin to interact with DENV-2 NS3 was associated with its anti-viral activity, while co-localisation of viperin with lipid droplets was not. Thus, DENV-2 infection induces viperin which has anti-viral properties residing in the C-terminal region of the protein that act to restrict early DENV-2 RNA production/accumulation, potentially via interaction of viperin with DENV-2 NS3 and replication complexes. These anti-DENV-2 actions of viperin show both contrasts and similarities with other described anti-viral mechanisms of viperin action and highlight the diverse nature of this unique anti-viral host protein.Karla J. Helbig, Jillian M. Carr, Julie K. Calvert, Satiya Wati, Jennifer N. Clarke, Nicholas S. Eyre, Sumudu K. Narayana, Guillaume N. Fiches, Erin M. McCartney, Michael R. Bear

A year of genomic surveillance reveals how the SARS-CoV-2 pandemic unfolded in Africa.

The progression of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic in Africa has so far been heterogeneous, and the full impact is not yet well understood. In this study, we describe the genomic epidemiology using a dataset of 8746 genomes from 33 African countries and two overseas territories. We show that the epidemics in most countries were initiated by importations predominantly from Europe, which diminished after the early introduction of international travel restrictions. As the pandemic progressed, ongoing transmission in many countries and increasing mobility led to the emergence and spread within the continent of many variants of concern and interest, such as B.1.351, B.1.525, A.23.1, and C.1.1. Although distorted by low sampling numbers and blind spots, the findings highlight that Africa must not be left behind in the global pandemic response, otherwise it could become a source for new variants