Background
Severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) is unlikely to be a major transfusion‐transmitted pathogen; however, convalescent plasma is a treatment option used in some ...regions. The risk of transfusion‐transmitted infections can be minimized by implementing Pathogen Inactivation (PI), such as THERAFLEX MB‐plasma and THERAFLEX UV‐Platelets systems. Here we examined the capability of these PI systems to inactivate SARS‐CoV‐2.
Study Design and Methods
SARS‐CoV‐2 spiked plasma units were treated using the THERAFLEX MB‐Plasma system in the presence of methylene blue (~0.8 μmol/L; visible light doses: 20, 40, 60, and 120 standard J/cm2). SARS‐CoV‐2 spiked platelet concentrates (PCs) were treated using the THERAFLEX UV‐platelets system (UVC doses: 0.05, 0.10, 0.15, and 0.20 standard J/cm2). Samples were taken prior to the first and after each illumination dose, and viral infectivity was assessed using an immunoplaque assay.
Results
Treatment of spiked plasma with the THERAFLEX MB‐Plasma system resulted in an average ≥5.03 log10 reduction in SARS‐CoV‐2 infectivity at one third (40 J/cm2) of the standard visible light dose. For the platelet concentrates (PCs), treatment with the THERAFLEX UV‐Platelets system resulted in an average ≥5.18 log10 reduction in SARS‐CoV‐2 infectivity at the standard UVC dose (0.2 J/cm2).
Conclusions
SARS‐CoV‐2 infectivity was reduced in plasma and platelets following treatment with the THERAFLEX MB‐Plasma and THERAFLEX UV‐Platelets systems, to the limit of detection, respectively. These PI technologies could therefore be an effective option to reduce the risk of transfusion‐transmitted emerging pathogens.
Next‐generation sequencing technologies enable the rapid identification of viral infection of diseased organisms. However, despite a consistent decrease in sequencing costs, it is difficult to ...justify their use in large‐scale surveys without a virus sequence enrichment technique. As the majority of plant viruses have an RNA genome, a common approach is to extract the double‐stranded RNA (dsRNA) replicative form, to enrich the replicating virus genetic material over the host background. The traditional dsRNA extraction is time‐consuming and labour‐intensive. We present an alternative method to enrich dsRNA from plant extracts using anti‐dsRNA monoclonal antibodies in a pull‐down assay. The extracted dsRNA can be amplified by reverse transcriptase–polymerase chain reaction and sequenced by next‐generation sequencing. In our study, we have selected three distinct plant hosts: Māori potato (Solanum tuberosum), rengarenga (Arthropodium cirratum) and broadleaved dock (Rumex obtusifolius) representing a cultivated crop, a New Zealand‐native ornamental plant and a weed, respectively. Of the sequence data obtained, 31–74% of the reads were of viral origin, and we identified five viruses including Potato virus Y and Potato virus S in potato; Turnip mosaic virus in rengarenga (a new host record); and in the dock sample Cherry leaf roll virus and a novel virus belonging to the genus Macluravirus. We believe that this new assay represents a significant opportunity to upscale virus ecology studies from environmental, primary industry and/or medical samples.
Background
Japanese encephalitis virus (JEV) is endemic to tropical areas in Asia and the Western Pacific. It can cause fatal encephalitis, although most infected individuals are asymptomatic. JEV is ...mainly transmitted to humans through the bite of an infected mosquito, but can also be transmitted through blood transfusion. To manage the potential risk of transfusion transmission, pathogen inactivation (PI) technologies, such as THERAFLEX MB‐Plasma and THERAFLEX UV‐Platelets systems, have been developed. We examined the efficacy of these two PI systems to inactivate JEV.
Study Design and Methods
Japanese encephalitis virus–spiked plasma units were treated using the THERAFLEX MB‐Plasma system (visible light doses, 20, 40, 60, and 120 standard J/cm2) in the presence of methylene blue at approximately 0.8 μmol/L and spiked platelet concentrates (PCs) were treated using the THERAFLEX UV‐Platelets system (UVC doses, 0.05, 0.10, 0.15, and 0.20 standard J/cm2). Samples were taken before the first and after each illumination dose and tested for infectivity using an immunoplaque assay.
Results
Treatment of plasma with the THERAFLEX MB‐Plasma system resulted in an average of 6.59 log reduction in JEV infectivity at one‐sixth of the standard visible light dose (20 J/cm2). For PCs, treatment with the THERAFLEX UV‐Platelet system resulted in an average of 7.02 log reduction in JEV infectivity at the standard UVC dose (0.20 J/cm2).
Conclusions
The THERAFLEX MB‐Plasma and THERAFLEX UV‐Platelets systems effectively inactivated JEV in plasma or PCs, and thus these PI technologies could be an effective option to reduce the risk of JEV transfusion transmission.
BACKGROUND
Zika virus (ZIKV) has emerged as a potential threat to transfusion safety worldwide. Pathogen inactivation is one approach to manage this risk. In this study, the efficacy of the THERAFLEX ...UV‐Platelets system and THERAFLEX MB‐Plasma system to inactivate ZIKV in platelet concentrates (PCs) and plasma was investigated.
STUDY DESIGN AND METHODS
PCs spiked with ZIKV were treated with the THERAFLEX UV‐Platelets system at 0.05, 0.10, 0.15, and 0.20 J/cm2 UVC. Plasma spiked with ZIKV was treated with the THERAFLEX MB‐Plasma system at 20, 40, 60, and 120 J/cm2 light at 630 nm with at least 0.8 µmol/L methylene blue (MB). Samples were taken before the first and after each illumination dose and tested for residual virus. For each system the level of viral reduction was determined.
RESULTS
Treatment of PCs with THERAFLEX UV‐Platelets system resulted in a mean of 5 log reduction in ZIKV infectivity at the standard UVC dose (0.20 J/cm2), with dose dependency observed with increasing UVC dose. For plasma treated with MB and visible light, ZIKV infectivity was reduced by a mean of at least 5.68 log, with residual viral infectivity reaching the detection limit of the assay at 40 J/cm2 (one‐third the standard dose).
CONCLUSIONS
Our study demonstrates that the THERAFLEX UV‐Platelets system and THERAFLEX MB‐Plasma system can reduce ZIKV infectivity in PCs and pooled plasma to the detection limit of the assays used. These findings suggest both systems have the capacity to be an effective option to manage potential ZIKV transfusion transmission risk.
Abstract
The current COVID-19 pandemic is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We demonstrate that despite the large size of the viral RNA genome (~30 kb), ...infectious full-length cDNA is readily assembled in vitro by a circular polymerase extension reaction (CPER) methodology without the need for technically demanding intermediate steps. Overlapping cDNA fragments are generated from viral RNA and assembled together with a linker fragment containing CMV promoter into a circular full-length viral cDNA in a single reaction. Transfection of the circular cDNA into mammalian cells results in the recovery of infectious SARS-CoV-2 virus that exhibits properties comparable to the parental virus in vitro and in vivo. CPER is also used to generate insect-specific Casuarina virus with ~20 kb genome and the human pathogens Ross River virus (Alphavirus) and Norovirus (Calicivirus), with the latter from a clinical sample. Additionally, reporter and mutant viruses are generated and employed to study virus replication and virus-receptor interactions.
Flaviviruses are responsible for a number of important mosquito-borne diseases of man and animals globally. The short vireamic period in infected hosts means that serological assays are often the ...diagnostic method of choice. This paper will focus on the traditional methods to diagnose flaviviral infections as well as describing the modern rapid platforms and approaches for diagnostic antigen preparation.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, OILJ, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK, VSZLJ
West Nile virus (WNV) is an important zoonotic flavivirus responsible for mild fever to severe, lethal neuroinvasive disease in humans, horses, birds, and other wildlife species. Since its discovery, ...WNV has caused multiple human and animal disease outbreaks in all continents, except Antarctica. Infections are associated with economic losses, mainly due to the cost of treatment of infected patients, control programmes, and loss of animals and animal products. The pathogenesis of WNV has been extensively investigated in natural hosts as well as in several animal models, including rodents, lagomorphs, birds, and reptiles. However, most of the proposed pathogenesis hypotheses remain contentious, and much remains to be elucidated. At the same time, the unavailability of specific antiviral treatment or effective and safe vaccines contribute to the perpetuation of the disease and regular occurrence of outbreaks in both endemic and non-endemic areas. Moreover, globalisation and climate change are also important drivers of the emergence and re-emergence of the virus and disease. Here, we give an update of the pathobiology, epidemiology, diagnostics, control, and “One Health” implications of WNV infection and disease.
Zika and chikungunya viruses have caused major epidemics and are transmitted by Aedes aegypti and/or Aedes albopictus mosquitoes. The "Sementis Copenhagen Vector" (SCV) system is a recently developed ...vaccinia-based, multiplication-defective, vaccine vector technology that allows manufacture in modified CHO cells. Herein we describe a single-vector construct SCV vaccine that encodes the structural polyprotein cassettes of both Zika and chikungunya viruses from different loci. A single vaccination of mice induces neutralizing antibodies to both viruses in wild-type and IFNAR
mice and protects against (i) chikungunya virus viremia and arthritis in wild-type mice, (ii) Zika virus viremia and fetal/placental infection in female IFNAR
mice, and (iii) Zika virus viremia and testes infection and pathology in male IFNAR
mice. To our knowledge this represents the first single-vector construct, multi-pathogen vaccine encoding large polyproteins, and offers both simplified manufacturing and formulation, and reduced "shot burden" for these often co-circulating arboviruses.
Insect-specific viruses do not replicate in vertebrate cells, but persist in mosquito populations and are highly prevalent in nature. These viruses may naturally regulate the transmission of ...pathogenic vertebrate-infecting arboviruses in co-infected mosquitoes. Following the isolation of the first Australian insect-specific flavivirus (ISF), Palm Creek virus (PCV), we investigated routes of infection and transmission of this virus in key Australian arbovirus vectors and its impact on replication and transmission of West Nile virus (WNV).
Culex annulirostris, Aedes aegypti and Aedes vigilax were exposed to PCV, and infection, replication and transmission rates in individual mosquitoes determined. To test whether the virus could be transmitted vertically, progeny reared from eggs oviposited by PCV-inoculated Cx. annulirostris were analysed for the presence of PCV. To assess whether prior infection of mosquitoes with PCV could also suppress the transmission of pathogenic flaviviruses, PCV positive or negative Cx. annulirostris were subsequently exposed to WNV.
No PCV-infected Cx. annulirostris were detected 16 days after feeding on an infectious blood meal. However, when intrathoracically inoculated with PCV, Cx. annulirostris infection rates were 100 %. Similar rates of infection were observed in Ae. aegypti (100 %) and Ae. vigilax (95 %). Notably, PCV was not detected in any saliva expectorates collected from any of these species. PCV was not detected in 1038 progeny reared from 59 PCV-infected Cx. annulirostris. After feeding on a blood meal containing 10(7) infectious units of WNV, significantly fewer PCV-infected Cx. annulirostris were infected or transmitted WNV compared to PCV negative mosquitoes. Immunohistochemistry revealed that PCV localized in the midgut epithelial cells, which are the first site of infection with WNV.
Our results indicate that PCV cannot infect Cx. annulirostris via the oral route, nor be transmitted in saliva or vertically to progeny. We also provide further evidence that prior infection with insect-specific viruses can regulate the infection and transmission of pathogenic arboviruses.
SARS-CoV-2 uses the human ACE2 (hACE2) receptor for cell attachment and entry, with mouse ACE2 (mACE2) unable to support infection. Herein we describe an ACE2-lentivirus system and illustrate its ...utility for
in vitro
and
in vivo
SARS-CoV-2 infection models. Transduction of non-permissive cell lines with hACE2 imparted replication competence, and transduction with mACE2 containing N30D, N31K, F83Y and H353K substitutions, to match hACE2, rescued SARS-CoV-2 replication. Intrapulmonary hACE2-lentivirus transduction of C57BL/6J mice permitted significant virus replication in lung epithelium. RNA-Seq and histological analyses illustrated that this model involved an acute inflammatory disease followed by resolution and tissue repair, with a transcriptomic profile similar to that seen in COVID-19 patients. hACE2-lentivirus transduction of IFNAR
-/-
and IL-28RA
-/-
mouse lungs was used to illustrate that loss of type I or III interferon responses have no significant effect on virus replication. However, their importance in driving inflammatory responses was illustrated by RNA-Seq analyses. We also demonstrate the utility of the hACE2-lentivirus transduction system for vaccine evaluation in C57BL/6J mice. The ACE2-lentivirus system thus has broad application in SARS-CoV-2 research, providing a tool for both mutagenesis studies and mouse model development.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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