Marek's disease virus in vaccinated poultry flocks in Turkey: its first isolation with molecular characterization.

Marek's disease (MD) is an important disease of avian species and a potential threat to the poultry industry worldwide. In this study, 16 dead commercial chickens from flocks with suspected MD were necropsied immediately after death. Pathological findings were compatible with MD, and gallid alphaherpesvirus 2 was identified in PCR of spleen samples. Virus isolation was performed in primary cell culture, and partial sequencing of the meq gene of the isolate revealed >99% nucleotide sequence identity to virulent and very virulent plus strains from a number of European countries, placing it in the same subclade of clade III as two virulent Italian strains and a very virulent plus Polish strain as well as virulent strains of geese and ducks. The data reported here indicate that a virulent strain of Marek's disease virus is circulating in Turkey and has not been stopped by the current national vaccination programme.

Bluetongue Virus NS4 Protein Is an Interferon Antagonist and a Determinant of Virus Virulence.

UNLABELLED: Bluetongue virus (BTV) is the causative agent of bluetongue, a major infectious disease of ruminants with serious consequences to both animal health and the economy. The clinical outcome of BTV infection is highly variable and dependent on a variety of factors related to both the virus and the host. In this study, we show that the BTV nonstructural protein NS4 favors viral replication in sheep, the animal species most affected by bluetongue. In addition, NS4 confers a replication advantage on the virus in interferon (IFN)-competent primary sheep endothelial cells and immortalized cell lines. We determined that in cells infected with an NS4 deletion mutant (BTV8ΔNS4), there is increased synthesis of type I IFN compared to cells infected with wild-type BTV-8. In addition, using RNA sequencing (RNA-seq), we show that NS4 modulates the host IFN response and downregulates mRNA levels of type I IFN and interferon-stimulated genes. Moreover, using reporter assays and protein synthesis assays, we show that NS4 downregulates the activities of a variety of promoters, such as the cytomegalovirus immediate-early promoter, the IFN-ß promoter, and a promoter containing interferon-stimulated response elements (ISRE). We also show that the NS4 inhibitory activity on gene expression is related to its nucleolar localization. Furthermore, NS4 does not affect mRNA splicing or cellular translation. The data obtained in this study strongly suggest that BTV NS4 is an IFN antagonist and a key determinant of viral virulence. IMPORTANCE: Bluetongue is one of the main infectious diseases of ruminants and is caused by bluetongue virus (BTV), an arthropod-borne virus transmitted from infected to susceptible animals by Culicoides biting midges. Bluetongue has a variable clinical outcome that can be related to both virus and host factors. It is therefore critical to understand the interplay between BTV and the host immune responses. In this study, we show that a nonstructural protein of BTV (NS4) is critical to counteract the innate immune response of the host. Infection of cells with a BTV mutant lacking NS4 results in increased synthesis of IFN-ß and upregulation of interferon-stimulated genes. In addition, we show that NS4 is a virulence factor for BTV by favoring viral replication in sheep, the animal species most susceptible to bluetongue.

Agricultural anaerobic digestion power plants in Ireland and Germany: policy and practice.

The process of anaerobic digestion (AD) is valued as a carbon-neutral energy source, while simultaneously treating organic waste, making it safer for disposal or use as a fertilizer on agricultural land. The AD process in many European nations, such as Germany, has grown from use of small, localized digesters to the operation of large-scale treatment facilities, which contribute significantly to national renewable energy quotas. However, these large AD plants are costly to run and demand intensive farming of energy crops for feedstock. Current policy in Germany has transitioned to support funding for smaller digesters, while also limiting the use of energy crops. AD within Ireland, as a new technology, is affected by ambiguous governmental policies concerning waste and energy. A clear governmental strategy supporting on-site AD processing of agricultural waste will significantly reduce Ireland's carbon footprint, improve the safety and bioavailability of agricultural waste, and provide an indigenous renewable energy source. © 2016 Society of Chemical Industry.

Turnover Rate of NS3 Proteins Modulates Bluetongue Virus Replication Kinetics in a Host-Specific Manner.

UNLABELLED: Bluetongue virus (BTV) is an arbovirus transmitted to livestock by midges of the Culicoides family and is the etiological agent of a hemorrhagic disease in sheep and other ruminants. In mammalian cells, BTV particles are released primarily by virus-induced cell lysis, while in insect cells they bud from the plasma membrane and establish a persistent infection. BTV possesses a ten-segmented double-stranded RNA genome, and NS3 proteins are encoded by segment 10 (Seg-10). The viral nonstructural protein 3 (NS3) plays a key role in mediating BTV egress as well as in impeding the in vitro synthesis of type I interferon in mammalian cells. In this study, we asked whether genetically distant NS3 proteins can alter BTV-host interactions. Using a reverse genetics approach, we showed that, depending on the NS3 considered, BTV replication kinetics varied in mammals but not in insects. In particular, one of the NS3 proteins analyzed harbored a proline at position 24 that leads to its rapid intracellular decay in ovine but not in Culicoides cells and to the attenuation of BTV virulence in a mouse model of disease. Overall, our data reveal that the genetic variability of Seg-10/NS3 differentially modulates BTV replication kinetics in a host-specific manner and highlight the role of the host-specific variation in NS3 protein turnover rate. IMPORTANCE: BTV is the causative agent of a severe disease transmitted between ruminants by biting midges of Culicoides species. NS3, encoded by Seg-10 of the BTV genome, fulfills key roles in BTV infection. As Seg-10 sequences from various BTV strains display genetic variability, we assessed the impact of different Seg-10 and NS3 proteins on BTV infection and host interactions. In this study, we revealed that various Seg-10/NS3 proteins alter BTV replication kinetics in mammals but not in insects. Notably, we found that NS3 protein turnover may vary in ovine but not in Culicoides cells due to a single amino acid residue that, most likely, leads to rapid and host-dependent protein degradation. Overall, this study highlights that genetically distant BTV Seg-10/NS3 influence BTV biological properties in a host-specific manner and increases our understanding of how NS3 proteins contribute to the outcome of BTV infection.

Induction and suppression of tick cell antiviral RNAi responses by tick-borne flaviviruses.

Arboviruses are transmitted by distantly related arthropod vectors such as mosquitoes (class Insecta) and ticks (class Arachnida). RNA interference (RNAi) is the major antiviral mechanism in arthropods against arboviruses. Unlike in mosquitoes, tick antiviral RNAi is not understood, although this information is important to compare arbovirus/host interactions in different classes of arbovirus vectos. Using an Ixodes scapularis-derived cell line, key Argonaute proteins involved in RNAi and the response against tick-borne Langat virus (Flaviviridae) replication were identified and phylogenetic relationships characterized. Analysis of small RNAs in infected cells showed the production of virus-derived small interfering RNAs (viRNAs), which are key molecules of the antiviral RNAi response. Importantly, viRNAs were longer (22 nucleotides) than those from other arbovirus vectors and mapped at highest frequency to the termini of the viral genome, as opposed to mosquito-borne flaviviruses. Moreover, tick-borne flaviviruses expressed subgenomic flavivirus RNAs that interfere with tick RNAi. Our results characterize the antiviral RNAi response in tick cells including phylogenetic analysis of genes encoding antiviral proteins, and viral interference with this pathway. This shows important differences in antiviral RNAi between the two major classes of arbovirus vectors, and our data broadens our understanding of arthropod antiviral RNAi.

Transcriptome analysis reveals the host response to Schmallenberg virus in bovine cells and antagonistic effects of the NSs protein.

BACKGROUND: Schmallenberg virus (SBV) is a member of the Orthobunyavirus genus (Bunyaviridae family) causing malformations and abortions in ruminants. Although, as for other members of this family/genus, the non-structural protein NSs has been shown to be an interferon antagonist, very little is known regarding the overall inhibitory effects and targets of orthobunyavirus NSs proteins on host gene expression during infection. Therefore, using RNA-seq this study describes changes to the transcriptome of primary bovine cells following infection with Schmallenberg virus (SBV) or with a mutant lacking the non-structural protein NSs (SBVdelNSs) providing a detailed comparison of the effect of NSs expression on the host cell. RESULTS: The sequence reads from all samples (uninfected cells, SBV and SBVdelNSs) assembled well to the bovine host reference genome (on average 87.43% of the reads). During infection with SBVdelNSs, 649 genes were differentially expressed compared to uninfected cells (78.7% upregulated) and many of these were known antiviral and IFN-stimulated genes. On the other hand, only nine genes were differentially expressed in SBV infected cells compared to uninfected control cells, demonstrating the strong inhibitory effect of NSs on cellular gene expression. However, the majority of the genes that were expressed during SBV infection are involved in restriction of viral replication and spread indicating that SBV does not completely manage to shutdown the host antiviral response. CONCLUSIONS: In this study we show the effects of SBV NSs on the transcriptome of infected cells as well as the cellular response to wild type SBV. Although NSs is very efficient in shutting down genes of the host innate response, a number of possible antiviral factors were identified. Thus the data from this study can serve as a base for more detailed mechanistic studies of SBV and other orthobunyaviruses.

Characterization of a second open reading frame in genome segment 10 of bluetongue virus.

Viruses have often evolved overlapping reading frames in order to maximize their coding capacity. Until recently, the segmented dsRNA genome of viruses of the Orbivirus genus was thought to be monocistronic, but the identification of the bluetongue virus (BTV) NS4 protein changed this assumption. A small ORF in segment 10, overlapping the NS3 ORF in the +1 position, is maintained in more than 300 strains of the 27 different BTV serotypes and in more than 200 strains of the phylogenetically related African horse sickness virus (AHSV). In BTV, this ORF (named S10-ORF2 in this study) encodes a putative protein 50-59 residues in length and appears to be under strong positive selection. HA- or GFP-tagged versions of S10-ORF2 expressed from transfected plasmids localized within the nucleoli of transfected cells, unless a putative nucleolar localization signal was mutated. S10-ORF2 inhibited gene expression, but not RNA translation, in transient transfection reporter assays. In both mammalian and insect cells, BTV S10-ORF2 deletion mutants (BTV8ΔS10-ORF2) displayed similar replication kinetics to wt virus. In vivo, S10-ORF2 deletion mutants were pathogenic in mouse models of disease. Although further evidence is required for S10-ORF2 expression during infection, the data presented provide an initial characterization of this ORF.

Schmallenberg virus pathogenesis, tropism and interaction with the innate immune system of the host.

Schmallenberg virus (SBV) is an emerging orthobunyavirus of ruminants associated with outbreaks of congenital malformations in aborted and stillborn animals. Since its discovery in November 2011, SBV has spread very rapidly to many European countries. Here, we developed molecular and serological tools, and an experimental in vivo model as a platform to study SBV pathogenesis, tropism and virus-host cell interactions. Using a synthetic biology approach, we developed a reverse genetics system for the rapid rescue and genetic manipulation of SBV. We showed that SBV has a wide tropism in cell culture and "synthetic" SBV replicates in vitro as efficiently as wild type virus. We developed an experimental mouse model to study SBV infection and showed that this virus replicates abundantly in neurons where it causes cerebral malacia and vacuolation of the cerebral cortex. These virus-induced acute lesions are useful in understanding the progression from vacuolation to porencephaly and extensive tissue destruction, often observed in aborted lambs and calves in naturally occurring Schmallenberg cases. Indeed, we detected high levels of SBV antigens in the neurons of the gray matter of brain and spinal cord of naturally affected lambs and calves, suggesting that muscular hypoplasia observed in SBV-infected lambs is mostly secondary to central nervous system damage. Finally, we investigated the molecular determinants of SBV virulence. Interestingly, we found a biological SBV clone that after passage in cell culture displays increased virulence in mice. We also found that a SBV deletion mutant of the non-structural NSs protein (SBVΔNSs) is less virulent in mice than wild type SBV. Attenuation of SBV virulence depends on the inability of SBVΔNSs to block IFN synthesis in virus infected cells. In conclusion, this work provides a useful experimental framework to study the biology and pathogenesis of SBV.

A systematic analysis of host factors reveals a Med23-interferon-λ regulatory axis against herpes simplex virus type 1 replication.

Herpes simplex virus type 1 (HSV-1) is a neurotropic virus causing vesicular oral or genital skin lesions, meningitis and other diseases particularly harmful in immunocompromised individuals. To comprehensively investigate the complex interaction between HSV-1 and its host we combined two genome-scale screens for host factors (HFs) involved in virus replication. A yeast two-hybrid screen for protein interactions and a RNA interference (RNAi) screen with a druggable genome small interfering RNA (siRNA) library confirmed existing and identified novel HFs which functionally influence HSV-1 infection. Bioinformatic analyses found the 358 HFs were enriched for several pathways and multi-protein complexes. Of particular interest was the identification of Med23 as a strongly anti-viral component of the largely pro-viral Mediator complex, which links specific transcription factors to RNA polymerase II. The anti-viral effect of Med23 on HSV-1 replication was confirmed in gain-of-function gene overexpression experiments, and this inhibitory effect was specific to HSV-1, as a range of other viruses including Vaccinia virus and Semliki Forest virus were unaffected by Med23 depletion. We found Med23 significantly upregulated expression of the type III interferon family (IFN-λ) at the mRNA and protein level by directly interacting with the transcription factor IRF7. The synergistic effect of Med23 and IRF7 on IFN-λ induction suggests this is the major transcription factor for IFN-λ expression. Genotypic analysis of patients suffering recurrent orofacial HSV-1 outbreaks, previously shown to be deficient in IFN-λ secretion, found a significant correlation with a single nucleotide polymorphism in the IFN-λ3 (IL28b) promoter strongly linked to Hepatitis C disease and treatment outcome. This paper describes a link between Med23 and IFN-λ, provides evidence for the crucial role of IFN-λ in HSV-1 immune control, and highlights the power of integrative genome-scale approaches to identify HFs critical for disease progression and outcome.

Comparison of detection of Borrelia burgdorferi DNA and anti-Borrelia burgdorferi antibodies in patients with erythema migrans in north-eastern Poland.

INTRODUCTION: Diagnostic methods in erythema migrans are still not standardized. AIM: To evaluate the frequency of Borrelia burgdorferi s.l. DNA presence in patients with erythema migrans (EM); to assess the polymerase chain reaction (PCR) procedure for detecting B. burgdorferi s.l. DNA in patients with the skin form of Lyme borreliosis; and to compare the results of the PCR-based method with the traditional ELISA method. MATERIAL AND METHODS: Skin biopsy and blood samples from 93 patients with EM were examined for B. burgdorferi s.l. DNA detection (PCR). Seventy-one of these patients were examined for the presence of anti-B. burgdorferi s.l. antibodies (ELISA). RESULTS: Borrelia burgdorferi s.l. DNA was detected in 48% of the skin biopsy specimens and in 2% of blood samples. Only 1 patient was PCR positive in both blood and skin samples. Seventy percent of patients whose PCR results were positive were bitten by a tick less than 14 days before. IgM anti-B. burgdorferi s.l - specific antibodies were present in the serum of 35% of patients and IgG antibodies - in 30% of patients. Seventeen percent were positive in both IgM and IgG. CONCLUSIONS: Polymerase chain reaction of skin biopsy specimens seems to be currently the most sensitive and specific test for the diagnosis of patients with EM, especially in patients with a short duration of the disease (< 14 days) but still its effectiveness is much lower than expected. Polymerase chain reaction of blood samples cannot be recommended at the present time for the routine diagnostic of patients with EM.

NSs protein of Schmallenberg virus counteracts the antiviral response of the cell by inhibiting its transcriptional machinery.

Bunyaviruses have evolved a variety of strategies to counteract the antiviral defence systems of mammalian cells. Here we show that the NSs protein of Schmallenberg virus (SBV) induces the degradation of the RPB1 subunit of RNA polymerase II and consequently inhibits global cellular protein synthesis and the antiviral response. In addition, we show that the SBV NSs protein enhances apoptosis in vitro and possibly in vivo, suggesting that this protein could be involved in SBV pathogenesis in different ways.

Phenoloxidase activity acts as a mosquito innate immune response against infection with Semliki Forest virus.

Several components of the mosquito immune system including the RNA interference (RNAi), JAK/STAT, Toll and IMD pathways have previously been implicated in controlling arbovirus infections. In contrast, the role of the phenoloxidase (PO) cascade in mosquito antiviral immunity is unknown. Here we show that conditioned medium from the Aedes albopictus-derived U4.4 cell line contains a functional PO cascade, which is activated by the bacterium Escherichia coli and the arbovirus Semliki Forest virus (SFV) (Togaviridae; Alphavirus). Production of recombinant SFV expressing the PO cascade inhibitor Egf1.0 blocked PO activity in U4.4 cell- conditioned medium, which resulted in enhanced spread of SFV. Infection of adult female Aedes aegypti by feeding mosquitoes a bloodmeal containing Egf1.0-expressing SFV increased virus replication and mosquito mortality. Collectively, these results suggest the PO cascade of mosquitoes plays an important role in immune defence against arboviruses.

Gene silencing in tick cell lines using small interfering or long double-stranded RNA.

Gene silencing by RNA interference (RNAi) is an important research tool in many areas of biology. To effectively harness the power of this technique in order to explore tick functional genomics and tick-microorganism interactions, optimised parameters for RNAi-mediated gene silencing in tick cells need to be established. Ten cell lines from four economically important ixodid tick genera (Amblyomma, Hyalomma, Ixodes and Rhipicephalus including the sub-species Boophilus) were used to examine key parameters including small interfering RNA (siRNA), double stranded RNA (dsRNA), transfection reagent and incubation time for silencing virus reporter and endogenous tick genes. Transfection reagents were essential for the uptake of siRNA whereas long dsRNA alone was taken up by most tick cell lines. Significant virus reporter protein knockdown was achieved using either siRNA or dsRNA in all the cell lines tested. Optimum conditions varied according to the cell line. Consistency between replicates and duration of incubation with dsRNA were addressed for two Ixodes scapularis cell lines; IDE8 supported more consistent and effective silencing of the endogenous gene subolesin than ISE6, and highly significant knockdown of the endogenous gene 2I1F6 in IDE8 cells was achieved within 48 h incubation with dsRNA. In summary, this study shows that gene silencing by RNAi in tick cell lines is generally more efficient with dsRNA than with siRNA but results vary between cell lines and optimal parameters need to be determined for each experimental system.

Antiviral RNA interference responses induced by Semliki Forest virus infection of mosquito cells: characterization, origin, and frequency-dependent functions of virus-derived small interfering RNAs.

RNA interference (RNAi) is an important mosquito defense mechanism against arbovirus infection. In this paper we study the processes underlying antiviral RNAi in Aedes albopictus-derived U4.4 mosquito cells infected with Semliki Forest virus (SFV) (Togaviridae; Alphavirus). The production of virus-derived small interfering RNAs (viRNAs) from viral double-stranded RNA (dsRNA) is a key event in this host response. dsRNA could be formed by RNA replication intermediates, by secondary structures in RNA genomes or antigenomes, or by both. Which of these dsRNAs is the substrate for the generation of viRNAs is a fundamental question. Here we used deep sequencing of viRNAs and bioinformatic analysis of RNA secondary structures to gain insights into the characteristics and origins of viRNAs. An asymmetric distribution of SFV-derived viRNAs with notable areas of high-level viRNA production (hot spots) and no or a low frequency of viRNA production (cold spots) along the length of the viral genome with a slight bias toward the production of genome-derived viRNAs over antigenome-derived viRNAs was observed. Bioinformatic analysis suggests that hot spots of viRNA production are rarely but not generally associated with putative secondary structures in the SFV genome, suggesting that most viRNAs are derived from replicative dsRNA. A pattern of viRNAs almost identical to those of A. albopictus cells was observed for Aedes aegypti-derived Aag2 cells, suggesting common mechanisms that lead to viRNA production. Hot-spot viRNAs were found to be significantly less efficient at mediating antiviral RNAi than cold-spot viRNAs, pointing toward a nucleic acid-based viral decoy mechanism to evade the RNAi response.

Virus particle propagation and infectivity along the respiratory tract and a case study for SARS-CoV-2.

Respiratory viruses including Respiratory Syncytial Virus, influenza virus and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cause serious and sometimes fatal disease in thousands of people annually. Understanding virus propagation dynamics within the respiratory system is critical because new insights will increase our understanding of virus pathogenesis and enable infection patterns to be more predictable in vivo, which will enhance our ability to target vaccine and drug delivery. This study presents a computational model of virus propagation within the respiratory tract network. The model includes the generation network branch structure of the respiratory tract, biophysical and infectivity properties of the virus, as well as air flow models that aid the circulation of the virus particles. As a proof of principle, the model was applied to SARS-CoV-2 by integrating data about its replication-cycle, as well as the density of Angiotensin Converting Enzyme expressing cells along the respiratory tract network. Using real-world physiological data associated with factors such as the respiratory rate, the immune response and virus load that is inhaled, the model can improve our understanding of the concentration and spatiotemporal dynamics of the virus. We collected experimental data from a number of studies and integrated them with the model in order to show in silico how the virus load propagates along the respiratory network branches.

Assessing the potential of repurposing ion channel inhibitors to treat emerging viral diseases and the role of this host factor in virus replication.

As diseases caused by new and emerging viruses continue to be a major threat to humans and animals worldwide the need for new therapeutic options intensifies. A wide variety of viruses including Influenza A virus, Human immunodeficiency virus, Middle East respiratory syndrome coronavirus and severe acute respiratory syndrome coronavirus require ion channels for efficient replication. Thus, targeting host ion channels may serve as an effective means to attenuate virus replication and help treat viral diseases. Targeting host ion channels is an attractive therapeutic option because a range of ion channel-blocking compounds already exist for the treatment of other human diseases and some of these possess in vitro and sometimes in vivo antiviral activity. Therefore, identifying the specific ion channels involved in replicative cycles could provide opportunities to repurpose these ion channel inhibitors for treating viral diseases. Furthermore, optimised methodologies for identifying effective ion channel targeting drugs and their mechanisms of action could enable rapid responses to newly emerged viruses. This review discusses the potential of ion channels as suitable drug targets to treat diseases caused by viruses by describing known ion channel targeting drugs including their antiviral activity; by summarising prior research demonstrating the requirement for host ion channels for efficient replication of some viruses; and by hypothesising about the role these drugs might play in our ongoing fight against viral diseases.

A retrospective molecular investigation of selected pigeon viruses between 2018-2021 in Turkey.

A recent first detection of pigeon aviadenovirus-1 and pigeon circovirus co-infection associated with Young Pigeon Disease Syndrome (YPDS) in a pigeon flock in Turkey, prompted a study focused on documenting the distribution of Pigeon aviadenovirus (PiAdV-1 and PiAdV-2), Pigeon circovirus (PiCV), Columbid alphaherpesvirus 1 (pigeon herpesvirus (PiHV)) and Fowl aviadenovirus (FAdV) in the country. These viruses were selected as they are associated with severe disease in pigeons across the world. A total of 192 cloacal swabs were collected from young (<1 year old) pigeons from 16 different private pigeon flocks across Turkey, between 2018 and 2021 as part of routine diagnostic sampling. PiCV genetic material was the most frequently detected 4/16 (25%), PiAdV-1 and CoHV-1 DNA were both found in one flock each, while neither PiAdV-2 and FAdV were detected in any of the studied pigeon flocks. PiCV and PiHV genetic material were both detected in the same pigeon flock's cloacal samples as a co-infection with the identification of PiHV being a first in Turkey.

A prevalence study of COVID-19 among healthcare workers in a pandemic hospital in the Samsun province of Turkey.

Among populations globally, many healthcare workers have been disproportionally impacted by the COVID-19 pandemic because of their above average exposure to people infected with SARS-CoV-2. Exposure to asymptomatic or pre-symptomatic individuals is particularly challenging, if those individuals continue to work, not knowing that they are potentially infectious. This study aimed to measure the level of asymptomatic infection in a cohort of workers in a healthcare setting in Turkey during the second major wave of infection in late 2020. Blood samples were collected and tested by electrochemiluminescence immunoassay for SARS-CoV-2 IgM and IgG antibodies. Nasal and throat swabs were performed in a subset of this cohort and RT-qPCR was used to search for the presence of SARS-CoV-2 RNA. The results showed that approximately 23% of the cohort were positive for anti-SARS-CoV-2 IgM antibodies and approximately 22% were positive for anti-SARS-CoV-2 IgG antibodies despite no reported history of COVID-19 symptoms. Just less than 30% of a subset of the group were positive for the presence of SARS-CoV-2 RNA indicating the likelihood of a current or recent infection, again despite a lack of typical COVID-19 associated symptoms. This study indicates a high rate of asymptomatic infection and highlights the need for regular testing of groups such as healthcare workers when community prevalence of disease is high and there is a desire to limit entry of virus into settings where vulnerable people may be present, because symptoms cannot be relied on as indicators of infection or infectiousness.

Rapid antigen testing for SARS-CoV-2 infection in a university setting in Ireland: Learning from a 6-week pilot study.

Objectives: With the ongoing circulation of SARS-CoV-2 in countries across the world it is essential to identify effective ways to reduce the risk of infection while allowing society to function as close to 'normal' as possible. Serial testing using rapid lateral flow antigen tests is a possible way to do this by screening populations in a targeted way, identifying infectious (both symptomatic and asymptomatic) people and removing them from circulation while infectious. To make rapid antigen testing effective, high levels of participation are important. This study was designed to evaluate the establishment of a testing programme in a university setting and assess some of the factors that impact participation in such a study among both staff and students. Study design: Observational, survey. Methods: A trial period of SARS-CoV-2 rapid testing using the Abbott Panbio rapid antigen test was set up and staff and students based in the University College Dublin Veterinary Hospital were asked to take part voluntarily for 6 weeks. Following the trial period, we used a questionnaire to evaluate satisfaction and to understand some reasons behind participation or lack thereof. Results: Overall, almost all respondents to the survey stated that they were happy with having a testing programme present in the workplace and it helped to reduce anxiety associated with COVID-19. Findings indicated that staff and students did not participate equally in the voluntary testing programme. The findings also highlighted that intrinsic motivations and extrinsic motivations for participation differ. For example, participation among staff was much higher than among students, motivational messaging focused on protecting others did not resonate with students as much as staff, convenience was a key factor driving participation in both cohorts and the pressure of being forced to miss class (if positive) close to exam time provided motivation to students to avoid testing. Conclusions: Introducing antigen testing into a workplace helped to reduce overall anxiety associated with the potential impact of COVID-19, but achieving good participation was challenging. Participation is key to a successful, campus wide antigen testing programme but reaching high levels of participation is not straightforward and can not be taken for granted. Different motivations drive participation in different cohorts and different messaging/incentivisation is needed to encourage participation in those different cohorts. The findings reported here should inform any SARS-CoV-2 testing programme that will run in these types of settings in the future.

Inactivation and Recovery of High Quality RNA From Positive SARS-CoV-2 Rapid Antigen Tests Suitable for Whole Virus Genome Sequencing.

The diagnostic protocol currently used globally to identify Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is RT-qPCR. The spread of these infections and the epidemiological imperative to describe variation across the virus genome have highlighted the importance of sequencing. SARS-CoV-2 rapid antigen diagnostic tests (RADTs) are designed to detect viral nucleocapsid protein with positive results suggestive of the presence of replicating virus and potential infectivity. In this study, we developed a protocol for recovering SARS-CoV-2 RNA from "spent" RADT devices of sufficient quality that can be used directly for whole virus genome sequencing. The experimental protocol included the spiking of RADTs at different concentrations with viable SARS-CoV-2 variant Alpha (lineage B.1.1.7), lysis for direct use or storage. The lysed suspensions were used for RNA extraction and RT-qPCR. In parallel, we also tested the stability of the viral RNA in the RADTs and the RNA extracted from the RADTs was used as a template for tiling-PCR and whole virus genome sequencing. RNA recovered from RADTs spiked with SARS-CoV-2 was detected through RT-qPCR with Ct values suitable for sequencing and the recovery from RADTs was confirmed after 7 days of storage at both 4 and 20°C. The genomic sequences obtained at each time-point aligned to the strain used for the spiking, demonstrating that sufficient SARS-CoV-2 viral genome can be readily recovered from positive-RADT devices in which the virus has been safely inactivated and genomically conserved. This protocol was applied to obtain whole virus genome sequence from RADTs ran in the field where the omicron variant was detected. The study demonstrated that viral particles of SARS-CoV-2 suitable for whole virus genome sequencing can be recovered from positive spent RADTs, extending their diagnostic utility, as a risk management tool and for epidemiology studies. In large deployment of the RADTs, positive devices could be safely stored and used as a template for sequencing allowing the rapid identification of circulating variants and to trace the source and spread of outbreaks within communities and guaranteeing public health.