Detection of a new avian bornavirus in barn owl (Tyto alba) by pan-viral microarray.

A barn owl (Tyto alba) died with neurological signs compatible with a viral infection. After discarding other possible infections caused by circulating viruses in the area, analysis of the central nervous system using a pan-viral microarray revealed hybridization to canary bornavirus 2 (CnBV-2). Subsequent sequence analysis confirmed the presence of a virus sharing more than 83% identity with CnBV-2. Surprisingly, the new sequence corresponds to a new virus, here named Barn owl Bornavirus 1 (BoBV-1), within the Orthobornavirus serini species. Moreover, it is the first member of this species that has been detected in a non-passerine bird, indicating that Orthobornavirus serini species comprises viruses with a wider range of hosts than previously presumed. The use of this microarray has proven to be an excellent tool for viral detection in clinical samples, with capacity to detect new viral variants. This allows the diagnosis of a great range of viruses, which can cause similar disease symptoms and which identification by PCR methods might be tedious, probably unsuccessful and, in the long run, expensive. This platform is highly useful for a fast and precise viral detection, contributing to the improvement of diagnostic methods.

Rapid and Flexible RT-qPCR Surveillance Platforms To Detect SARS-CoV-2 Mutations.

Multiple mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) increase transmission, disease severity, and immune evasion and facilitate zoonotic or anthropozoonotic infections. Four such mutations, ΔH69/V70, L452R, E484K, and N501Y, occurred in the SARS-CoV-2 spike glycoprotein in combinations that allow the simultaneous detection of VOCs. Here, we present two flexible reverse transcription-quantitative PCR (RT-qPCR) platforms for small- and large-scale screening (also known as variant PCR) to detect these mutations and schemes for adapting the platforms to future mutations. The large-scale RT-qPCR platform was validated by pairwise matching of RT-qPCR results with whole-genome sequencing (WGS) consensus genomes, showing high specificity and sensitivity. Both platforms are valuable examples of complementing WGS to support the rapid detection of VOCs. Our mutational signature approach served as an important intervention measure for the Danish public health system to detect and delay the emergence of new VOCs. IMPORTANCE Denmark weathered the SARS-CoV-2 crisis with relatively low rates of infection and death. Intensive testing strategies with the aim of detecting SARS-CoV-2 in symptomatic and nonsymptomatic individuals were available by establishing a national test system called TestCenter Denmark. This testing regime included the detection of SARS-CoV-2 signature mutations, with referral to the national health system, thereby delaying outbreaks of variants of concern. Our study describes the design of the large-scale RT-qPCR platform established at TestCenter Denmark in conjunction with whole-genome sequencing to report mutations of concern to the national health system. Validation of the large-scale RT-qPCR platform using paired WGS consensus genomes showed high sensitivity and specificity. For smaller laboratories with limited infrastructure, we developed a flexible small-scale RT-qPCR platform to detect three signature mutations in a single run. The RT-qPCR platforms are important tools to support the control of the SARS-CoV-2 endemic in Denmark.

SARS-CoV-2 RNA stability in dry swabs for longer storage and transport at different temperatures.

During the current COVID-19 pandemic, different methods have been used to evaluate patients with suspected SARS-CoV-2 infection. In this study, we experimentally evaluate the ability of spiked saliva-moist swabs and spiked swabs without any transport medium to retain SARS-CoV-2 for storage and transport at different environmental settings during different incubation time periods. Our results show that at ambient temperature of 20°C, SARS-CoV-2 RNA remains stable for up to 9 days allowing a long-time span for transport and storage without compromising clinical results. Additionally, this study demonstrates that saliva-moist swabs can also be stored at -20°C and +4°C for up to 26 days without affecting RT-qPCR results. Our data are relevant for low-and middle-income countries, which have limited access to rapid refrigerated transport and storage of samples representing an economical alternative. Finally, our study demonstrates the practical and economic advantage of using swabs without transport medium.

SARS-CoV-2 detection using reverse transcription strand invasion based amplification and a portable compact size instrument.

Rapid nucleic-acid based tests that can be performed by non-professionals outside laboratory settings could help the containment of the pandemic SARS-CoV-2 virus and may potentially prevent further widespread lockdowns. Here, we present a novel compact portable detection instrument (the Egoo Health System) for extraction-free detection of SARS-CoV-2 using isothermal reverse transcription strand invasion based amplification (RT-SIBA). The SARS-CoV-2 RT-SIBA assay can be performed directly on crude oropharyngeal swabs without nucleic acid extraction with a reaction time of 30 min. The Egoo Health system uses a capsule system, which is automatically sealed tight in the Egoo instrument after applying the sample, resulting in a closed system optimal for molecular isothermal amplification. The performance of the Egoo Health System is comparable to the PCR instrument with an analytical sensitivity of 25 viral RNA copies per SARS-CoV-2 RT-SIBA reaction and a clinical sensitivity and specificity between 87.0-98.4% and 96.6-98.2% respectively.

Oral and anal carriage of Neisseria meningitidis among sexually active HIV-infected men who have sex with men in Denmark 2014-15.

INTRODUCTION: Outbreaks of invasive meningococcal disease (IMD) among men who have sex with men (MSM) caused by a hypervirulent, non-encapsulated Neisseria meningitidis (Nm) clone belonging to genogroup C have been described. We aimed to determine the oral and anal carriage rates and genogroups of Nm among MSM living with HIV. METHODS: Sexually active MSM living with HIV were included. A questionnaire, an oral wash sample and an anal swab were collected at baseline and 12 months follow-up. Identification of Nm and genogrouping was performed using real-time polymerase chain reaction analysis. RESULTS: Among 82 MSM, the Nm carriage rate was 31.7% (95% CI 21.9-42.9) at baseline. The oral carriage rate was 24.4% (95% CI 15.6-35.1) and the anal rate was 11.0% (95% CI 5.1-19.8). Non-groupable Nm were most prevalent followed by genogroup B and genogroup Y. Rates were similar at follow-up. CONCLUSION: Strains of Nm were detected in both oral washes and anal samples in our study. Our results suggest that Nm may be transmitted sexually among MSM. Non-groupable Nm were predominant in our population and no genogroup C Nm were detected.

An in-well direct lysis method for rapid detection of SARS-CoV-2 by real time RT-PCR in eSwab specimens.

BACKGROUND: Diagnostic real time reverse transcription PCR (rRT-PCR) is usually done using nucleic acid (NA) purified from the sample. In the SARS-CoV-2 pandemic reagents and utensils for NA purification has been in short supply. This has generated interest in methods that eliminate the need for NA purification. OBJECTIVES: To investigate if addition of detergent to rRT-PCR master mix (MM) enabled in-well direct lysis and detection of SARS-CoV-2 in clinical eSwab specimens. STUDY DESIGN: IGEPAL-CA-630 (IGEPAL) was added to SARS-CoV-2 MM to 0.3 % final concentration and crude sample was added directly to the PCR well containing MM. Cycle of positivity (Cp) and categorical agreement was compared in samples tested in standard rRT-PCR after NA purification and in in-well lysis, direct rRT-PCR. RESULTS: In-well lysis direct rRT-PCR detected SARS-CoV-2 in 27/30 previously SARS-CoV-2+ samples with an average bias of 3.26 cycles (95 %CI: 0.08-6.43 cycles). All 30 previously test negative samples remained negative when tested in in-well lysis, direct PCR. CONCLUSIONS: Supplementation of detergent to MM was shown to be useful for the detection of SARS CoV-2 in eSwab specimens (COPAN) by direct rRT-PCR without prior NA purification.

SARS-CoV-2 Transmission between Mink (Neovison vison) and Humans, Denmark.

Severe acute respiratory syndrome coronavirus 2 has caused a pandemic in humans. Farmed mink (Neovison vison) are also susceptible. In Denmark, this virus has spread rapidly among farmed mink, resulting in some respiratory disease. Full-length virus genome sequencing revealed novel virus variants in mink. These variants subsequently appeared within the local human community.

An alternative workflow for molecular detection of SARS-CoV-2 - escape from the NA extraction kit-shortage, Copenhagen, Denmark, March 2020.

The World Health Organization has declared COVID-19 caused by the newly discovered SARS-CoV-2 a pandemic. Due to growing demand for reagents and/or kits to extract SARS-CoV-2 RNA for subsequent RT-qPCR diagnostics, there is a worldwide risk of shortages. With a detection sensitivity of 97.4% (95% CI: 86.2-99.9%), we describe a simple, fast, alternative workflow for molecular detection of SARS-CoV-2, where samples are simply heat-processed for 5 min at 98 °C before a commonly-used RT-qPCR procedure.

New tick-borne encephalitis virus hot spot in Northern Zealand, Denmark, October 2019.

During summer 2019, three patients residing by Tisvilde Hegn, Denmark were hospitalised with tick-borne encephalitis (TBE) after tick bites. A new TBE virus (TBEV) micro-focus was identified in tick nymphs collected around a playground in Tisvilde Hegn forest. Estimated TBEV prevalence was 8%, higher than in endemic areas around Europe. Whole genome sequencing showed clustering to a TBEV strain from Norway. This is the second time TBEV is found in Ixodes ricinus outside Bornholm, Denmark.

Field samplings of Ixodes ricinus ticks from a tick-borne encephalitis virus micro-focus in Northern Zealand, Denmark.

In 2008-2009 a tick-borne encephalitis virus (TBEV) micro-focus was detected in Northern Zealand, Denmark. No new cases of TBE with an epidemiological link to Northern Zealand has been reported since. Here we undertook to investigate Ixodes ricinus ticks from this endemic micro-focus in 2016 and 2017. In addition to TBEV, I. ricinus ticks may host other pathogens that include Borrelia spp., Babesia spp., Rickettsia spp. and Neoehrlichia mikurensis, together with various endosymbiont microorganisms. To detect multiple organisms we used a metagenomics PanVirus microarray and next-generation sequencing to examine the persistence and evolution of other emerging viruses, bacteria and parasites. Here we report the rise and fall of the Danish TBEV micro-focus in Northern Zealand. However, we identify for the first time in Danish I. ricinus ticks the presence of Uukuniemi virus in addition to a tick-borne phlebovirus and a range of bacteria.

Rapid, Safe, and Simple Manual Bedside Nucleic Acid Extraction for the Detection of Virus in Whole Blood Samples.

The rapid diagnosis of an infection is essential for the outbreak management, risk containment, and patient care. We have previously shown a method for the rapid bedside inactivation of the Ebola virus during blood sampling for safe nucleic acid (NA) tests by adding a commercial lysis/binding buffer directly into the vacuum blood collection tubes. Using this bedside inactivation approach, we have developed a safe, rapid, and simplified bedside NA extraction method for the subsequent detection of a virus in lysis/binding buffer-inactivated whole blood. The NA extraction is directly performed in the blood collection tubes and requires no equipment or electricity. After the blood is collected into the lysis/binding buffer, the contents are mixed by flipping the tube by hand, and the mixture is incubated for 20 min at room temperature. Magnetic glass particles (MGPs) are added to the tube, and the contents are mixed by flipping the collection tube by hand. The MGPs are then collected on the side of the blood collection tube using a magnetic holder or a magnet and a rubber band. The MGPs are washed three times, and after the addition of elution buffer directly into the collection tube, the NAs are ready for NA tests, such as qPCR or isothermal loop amplification (LAMP), without the removal of the MGPs from the reaction. The NA extraction method is not dependent on any laboratory facilities and can easily be used anywhere (e.g., in field hospitals and hospital isolation wards). When this NA extraction method is combined with LAMP and a portable instrument, a diagnosis can be obtained within 40 min of the blood collection.

Zika Virus IgG in Infants with Microcephaly, Guinea-Bissau, 2016.

We analyzed blood samples from infants born with microcephaly and their mothers in Guinea-Bissau in 2016 for pathogens associated with birth defects. No Zika virus RNA was detected, but Zika virus IgG was highly prevalent. We recommend implementing pathogen screening of infants with congenital defects in Guinea-Bissau.

Protective effect of a polyvalent influenza DNA vaccine in pigs.

BACKGROUND: Influenza A virus in swine herds represents a major problem for the swine industry and poses a constant threat for the emergence of novel pandemic viruses and the development of more effective influenza vaccines for pigs is desired. By optimizing the vector backbone and using a needle-free delivery method, we have recently demonstrated a polyvalent influenza DNA vaccine that induces a broad immune response, including both humoral and cellular immunity. OBJECTIVES: To investigate the protection of our polyvalent influenza DNA vaccine approach in a pig challenge study. METHODS: By intradermal needle-free delivery to the skin, we immunized pigs with two different doses (500µg and 800µg) of an influenza DNA vaccine based on six genes of pandemic origin, including internally expressed matrix and nucleoprotein and externally expressed hemagglutinin and neuraminidase as previously demonstrated. Two weeks following immunization, the pigs were challenged with the 2009 pandemic H1N1 virus. RESULTS: When challenged with 2009 pandemic H1N1, 0/5 vaccinated pigs (800µg DNA) became infected whereas 5/5 unvaccinated control pigs were infected. The pigs vaccinated with the low dose (500µg DNA) were only partially protected. The DNA vaccine elicited binding-, hemagglutination inhibitory (HI) - as well as cross-reactive neutralizing antibody activity and neuraminidase inhibiting antibodies in the immunized pigs, in a dose-dependent manner. CONCLUSION: The present data, together with the previously demonstrated immunogenicity of our influenza DNA vaccine, indicate that naked DNA vaccine technology provides a strong approach for the development of improved pig vaccines, applying realistic low doses of DNA and a convenient delivery method for mass vaccination.

Screening for viral extraneous agents in live-attenuated avian vaccines by using a microbial microarray and sequencing.

The absence of extraneous agents (EA) in the raw material used for production and in finished products is one of the principal safety elements related to all medicinal products of biological origin, such as live-attenuated vaccines. The aim of this study was to investigate the applicability of the Lawrence Livermore Microbial detection array version 2 (LLMDAv2) combined with whole genome amplification and sequencing for screening for viral EAs in live-attenuated vaccines and specific pathogen-free (SPF) eggs. We detected positive microarray signals for avian endogenous retrovirus EAV-HP and several viruses belonging to the Alpharetrovirus genus in all analyzed vaccines and SPF eggs. We used a microarray probe mapping approach to evaluate the presence of intact retroviral genomes, which in addition to PCR analysis revealed that several of the positive microarray signals were most likely due to cross hybridization with the EAV-HPΔpol and ALV-E ev1, ev3 and ev6 loci sequences originating from the chicken genome. Sequencing of the vaccines on a MiSeq instrument verified the microarray findings and showed similar cross hybridization. Our results suggest that genomic microarrays and sequencing of avian attenuated vaccines may be applied in tests for EA.

Rapid Bedside Inactivation of Ebola Virus for Safe Nucleic Acid Tests.

Rapid bedside inactivation of Ebola virus would be a solution for the safety of medical and technical staff, risk containment, sample transport, and high-throughput or rapid diagnostic testing during an outbreak. We show that the commercially available Magna Pure lysis/binding buffer used for nucleic acid extraction inactivates Ebola virus. A rapid bedside inactivation method for nucleic acid tests is obtained by simply adding Magna Pure lysis/binding buffer directly into vacuum blood collection EDTA tubes using a thin needle and syringe prior to sampling. The ready-to-use inactivation vacuum tubes are stable for more than 4 months, and Ebola virus RNA is preserved in the Magna Pure lysis/binding buffer for at least 5 weeks independent of the storage temperature. We also show that Ebola virus RNA can be manually extracted from Magna Pure lysis/binding buffer-inactivated samples using the QIAamp viral RNA minikit. We present an easy and convenient method for bedside inactivation using available blood collection vacuum tubes and reagents. We propose to use this simple method for fast, safe, and easy bedside inactivation of Ebola virus for safe transport and routine nucleic acid detection.

Human pegivirus detected in a patient with severe encephalitis using a metagenomic pan-virus array.

We have used a metagenomic microarray to detect genomic RNA from human pegivirus in serum and cerebrospinal fluid from a patient suffering from severe encephalitis. No other pathogen was detected. HPgV in cerebrospinal fluid during encephalitis has never been reported before and its prevalence in cerebrospinal fluid needs further investigation.

The microbial detection array for detection of emerging viruses in clinical samples--a useful panmicrobial diagnostic tool.

Emerging viruses are usually endemic to tropical and sub-tropical regions of the world, but increased global travel, climate change and changes in lifestyle are believed to contribute to the spread of these viruses into new regions. Many of these viruses cause similar disease symptoms as other emerging viruses or common infections, making these unexpected pathogens difficult to diagnose. Broad-spectrum pathogen detection microarrays containing probes for all sequenced viruses and bacteria can provide rapid identification of viruses, guiding decisions about treatment and appropriate case management. We report a modified Whole Transcriptome Amplification (WTA) method that increases unbiased amplification, particular of RNA viruses. Using this modified WTA method, we tested the specificity and sensitivity of the Lawrence Livermore Microbial Detection Array (LLMDA) against a wide range of emerging viruses present in both non-clinical and clinical samples using two different microarray data analysis methods.

MicroRNA-29a is up-regulated in beta-cells by glucose and decreases glucose-stimulated insulin secretion.

Chronically elevated levels of glucose impair pancreatic beta-cell function while inducing beta-cell proliferation. MicroRNA-29a (miR-29a) levels are increased in several tissues in diabetic animals and mediate decreased insulin-stimulated glucose-transport of adipocytes. The aim was to investigate the impact of glucose on miR-29a levels in INS-1E beta-cells and in human islets of Langerhans and furthermore to evaluate the impact of miR-29a on beta-cell function and proliferation. Increased glucose levels up-regulated miR-29a in beta-cells and human and rat islets of Langerhans. Glucose-stimulated insulin-secretion (GSIS) of INS-1E beta-cells was decreased by forced expression of miR-29a, while depletion of endogenous miR-29a improved GSIS. Over-expression of miR-29a increased INS-1E proliferation. Thus, miR-29a up-regulation is involved in glucose-induced proliferation of beta-cells. Furthermore, as depletion of miR-29a improves beta-cell function, miR-29a is a mediator of glucose-induced beta-cell dysfunction. Glucose-induced up-regulation of miR-29a in beta-cells could be implicated in progression from impaired glucose tolerance to type 2 diabetes.

Expression and localization of microRNAs in perinatal rat pancreas: role of miR-21 in regulation of cholesterol metabolism.

OBJECTIVE: To investigate the expression of pancreatic microRNAs (miRNAs) during the period of perinatal beta-cell expansion and maturation in rats, determine the localization of these miRNAs and perform a pathway analysis with predicted target mRNAs expressed in perinatal pancreas. RESEARCH DESIGN AND METHODS: RNA was extracted from whole pancreas at embryonic day 20 (E20), on the day of birth (P0) and two days after birth (P2) and hybridized to miRNA microarrays. Differentially expressed miRNAs were verified by northern blotting and their pancreatic localization determined by in situ hybridization. Pathway analysis was done using regulated sets of mRNAs predicted as targets of the miRNAs. Possible target genes were tested using reporter-gene analysis in INS-1E cells. RESULTS: Nine miRNAs were differentially expressed perinatally, seven were confirmed to be regulated at the level of the mature miRNA. The localization studies showed endocrine localization of six of these miRNAs (miR-21, -23a, -29a, -125b-5p, -376b-3p and -451), and all were expressed in exocrine cells at one time point at least. Pathways involving metabolic processes, terpenoid and sterol metabolism were selectively affected by concomitant regulation by miRNAs and mRNAs, and Srebf1 was validated as a target of miR-21. CONCLUSIONS: The findings suggest that miRNAs are involved in the functional maturation of pancreatic exocrine and endocrine tissue following birth. Pathway analysis of target genes identify changes in sterol metabolism around birth as being selectively affected by differential miRNA expression during this period.