Wild red deer (Cervus elaphus) do not play a role as vectors or reservoirs of SARS-CoV-2 in north-eastern Poland.

Several studies reported a high prevalence of SARS-CoV-2 among white-tailed deer in North America. Monitoring cervids in all regions to better understand SARS-CoV-2 infection and circulation in other deer populations has been urged. To evaluate deer exposure to SARS-CoV-2 in Poland, we sampled 90 reed deer individuals shot by hunters in five hunting districts in northeastern Poland. Serum and nasopharyngeal swabs were collected, and then the Immunofluorescent Assay (IFA) to detect anti-SARS-CoV-2 antibodies was performed as well as real-time PCR with reverse transcription for direct virus detection. No positive samples were detected. There is no evidence of spillover of SARS-CoV-2 from the human to deer population in Poland.

Nanobody engineering for SARS-CoV-2 neutralization and detection

In response to the ongoing SARS-CoV-2 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, nanobodies. Here, we explore the potential of nanobody engineering in the development of antivirals and diagnostic tools. Through fusion of nanobody domains that target distinct binding sites, we engineered multimodular nanobody constructs that neutralize wild-type SARS-CoV-2 and the Alpha and Delta variants with high potency, with IC50 values up to 50 pM. However, we observed a limitation in the efficacy of multimodular nanobodies against the Beta (B.1.351) and Omicron variants (B.1.1.529), underlining the importance of accounting for viral evolution in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present a novel detection assay, based on fusions of nanobodies with fragments of NanoLuc luciferase that can detect sub-nanomolar quantities of the SARS-CoV-2 spike protein in a single step. Our work showcases the potential of nanobody engineering to combat emerging infectious disease.

Serological surveillance for wild rodent infection with SARS-CoV-2 in Europe

We report serological surveillance for exposure to SARS-CoV-2 in 1,237 wild rodents and other small mammals across Europe. All samples were negative with the possible exception of one. Given the ongoing circulation of this virus in humans and potential host jumps, we suggest such surveillance be continued.

Experimental infection of mink with SARS-COV-2 Omicron (BA.1) variant leads to symptomatic disease with lung pathology and transmission

We report an experimental infection of American mink with SARS-CoV-2 Omicron variant and show that minks remain virus RNA positive for days, develop clinical signs and histopathological changes, and transmit the virus to uninfected recipients warranting further studies and preparedness.

Intranasal inhibitor blocks Omicron and other variants of SARS-CoV-2

The emergence of the SARS-CoV-2 Omicron variant capable of escaping neutralizing antibodies emphasizes the need for prophylactic strategies to complement vaccination in fighting the COVID-19 pandemic. Nasal epithelium is rich in the ACE2 receptor and important for SARS-CoV-2 transmission by supporting early viral replication before seeding to the lung1. Intranasal administration of SARS-CoV-2 neutralizing antibodies or antibody fragments has shown encouraging potential as a protective measure in animal models2-5. However, there remains a need for SARS-CoV-2 blocking agents that are more economical to produce in large scale, while less vulnerable to mutational variation in the neutralization epitopes of the viral Spike glycoprotein. Here we describe TriSb92, a highly manufacturable trimeric human nephrocystin SH3 domain-derived antibody mimetic targeted against a conserved region in the receptor-binding domain of the Spike. TriSb92 potently neutralizes SARS-CoV-2 and its variants of concern, including Delta and Omicron. Intranasal administration of a modest dose of TriSb92 (5 or 50 micrograms) as early as eight hours before the challenge with SARS-CoV-2 B.1.351 efficiently protected mice from infection. The target epitope of TriSb92 was defined by cryo-EM, which revealed triggering of a conformational shift in the Spike trimer rather than competition for ACE2 binding as the molecular basis of its strong inhibitory action. Our results highlight the potential of intranasal inhibitors in protecting susceptible individuals from SARS-CoV-2 infection, and describe a novel type of inhibitor that could be of use in addressing the challenge posed by the Omicron variant.

ClusTRace, a bioinformatic pipeline for analyzing clusters in virus phylogenies

SummarySARS-CoV-2 is the highly transmissible etiologic agent of coronavirus disease 2019 (COVID-19) and has become a global scientific and public health challenge since December 2019. Several new variants of SARS-CoV-2 have emerged globally raising concern about prevention and treatment of COVID-19. Early detection and in depth analysis of the emerging variants allowing pre-emptive alert and mitigation efforts are thus of paramount importance. Here we present ClusTRace, a novel bioinformatic pipeline for a fast and scalable analysis of sequence clusters or clades in large viral phylogenies. ClusTRace offers several high level functionalities including outlier filtering, aligning, phylogenetic tree reconstruction, cluster or clade extraction, variant calling, visualization and reporting. ClusTRace was developed as an aid for COVID-19 transmission chain tracing in Finland and the main emphasis has been on fast and unsupervised screening of phylogenies for markers of super-spreading events and other features of concern, such as high rates of cluster growth and/or accumulation of novel mutations. AvailabilityAll code is freely available from https://bitbucket.org/plyusnin/clustrace/

High prevalence of an alpha variant lineage with a premature stop codon in ORF7a in Iraq, winter 2020-2021

BackgroundSince the first reported case of coronavirus disease 2019 (COVID-19) in China, SARS-CoV-2 has been spreading worldwide. Genomic surveillance of SARS-CoV-2 has had a critical role in tracking the emergence, introduction, and spread of new variants, which may affect transmissibility, pathogenicity, and escape from infection or vaccine-induced immunity. As anticipated, the rapid increase in COVID-19 infections in Iraq in February 2021 is due to the introduction of variants of concern during the second wave of the COVID-19 pandemic. AimTo understand the molecular epidemiology of SARS-CoV-2 during the second wave in Iraq (2021), MethodWe sequenced 76 complete SARS-CoV-2 genomes using NGS technology and identified genomic mutations and proportions of circulating variants among these. Also, we performed an in silico study to predict the effect of the truncation of NS7a protein (ORF7a) on its function ResultsWe detected nine different lineages of SARS-CoV-2. The B.1.1.7 lineage was predominant (78.9%) from February to May 2021, while only one B.1.351 strain was detected. Interestingly, the phylogenetic analysis showed that multiple strains of the B.1.1.7 lineage clustered closely with those from European countries. A high frequency (88%) of stop codon mutation (NS7a Q62stop) was detected among the B.1.1.7 lineage sequences. In silico analysis of NS7a with Q62stop found that this stop codon had no significant effect on the function of NS7a. ConclusionThis work provides molecular epidemiological insights into the spread variants of SARS-CoV-2 in Iraq, which are most likely imported from Europe.

Zoonotic spillover of SARS-CoV-2: mink-adapted virus in humans

The COVID-19 pandemic caused by SARS-CoV-2 started in fall 2019. A range of different mammalian species, including farmed mink, have been confirmed as susceptible to infection with this virus. We report here the spillover of mink-adapted SARS-CoV-2 from farmed mink to humans after extensive adaptation that lasted at least 3 months. We found the presence of four mutations in the S gene (that gave rise to variant: G75V, M177T, Y453F and C1247F) and others in an isolate obtained from SARS-CoV-2 positive patient.

HaVoC, a bioinformatic pipeline for reference-based consensus assembly and lineage assignment for SARS-CoV-2 sequences

BackgroundSARS-CoV-2 related research has increased in importance worldwide since December 2019. Several new variants of SARS-CoV-2 have emerged globally, of which the most notable and concerning currently are the UK variant B.1.1.7, the South African variant B1.351 and the Brazilian variant P.1. Detecting and monitoring novel variants is essential in SARS-CoV-2 surveillance. While there are several tools for assembling virus genomes and performing lineage analyses to investigate SARS-CoV-2, each is limited to performing singular or a few functions separately. ResultsDue to the lack of publicly available pipelines, which could perform fast reference-based assemblies on raw SARS-CoV-2 sequences in addition to identifying lineages to detect variants of concern, we have developed an open source bioinformatic pipeline called HaVoC (Helsinki university Analyzer for Variants Of Concern). HaVoC can reference assemble raw sequence reads and assign the corresponding lineages to SARS-CoV-2 sequences. ConclusionsHaVoC is a pipeline utilizing several bioinformatic tools to perform multiple necessary analyses for investigating genetic variance among SARS-CoV-2 samples. The pipeline is particularly useful for those who need a more accessible and fast tool to detect and monitor the spread of SARS-CoV-2 variants of concern during local outbreaks. HaVoC is currently being used in Finland for monitoring the spread of SARS-CoV-2 variants. HaVoC user manual and source code are available at https://www.helsinki.fi/en/projects/havoc and https://bitbucket.org/auto_cov_pipeline/havoc, respectively.

Detection and molecular characterisation of SARS-CoV-2 in farmed mink (Neovision vision) in Poland

SARS-CoV-2 is the aetiological agent of COVID-19 disease and has been spreading worldwide since December 2019. The virus has been shown to infect different animal species under experimental conditions. Also, minks have been found to be susceptible to SARS-CoV-2 infection in fur farms in Europe and the USA. Here we investigated 91 individual minks from a farm located in Northern Poland. Using RT-PCR, antigen detection and NGS, we confirmed 15 animals positive for SARS-CoV-2. The result was verified by sequencing of full viral genomes, confirming SARS-CoV-2 infection in Polish mink. Country-scale monitoring conducted by veterinary inspection so far has not detected the presence of SARS-CoV-2 on other mink farms. Taking into consideration that Poland has a high level of positive diagnostic tests among its population, there is a high risk that more Polish mink farms become a source for SARS-CoV-2. Findings reported here and from other fur producing countries urge the assessment of SARS-CoV-2 prevalence in animals bred in Polish fur farms.

Real-life clinical sensitivity of SARS-CoV-2 RT-PCR test in symptomatic patients

ImportanceUnderstanding the false negative rates of SARS-CoV-2 RT-PCR testing is pivotal for the management of the COVID-19 pandemic and it has practical implications for patient management in healthcare facilities. ObjectiveTo determine the real-life clinical sensitivity of SARS-CoV-2 RT-PCR testing. DesignA retrospective study on case series from 4 March - 15 April 2020. SettingA population-based study conducted in primary and tertiary care in the Helsinki Capital Region, Finland. ParticipantsAdults who were clinically suspected of SARS-CoV-2 infection and underwent SARS-CoV-2 RT-PCR testing, and who had sufficient data for grading of clinical suspicion of COVID-19 in their medical records were eligible. All 1,194 inpatients admitted to COVID-19 cohort wards during the study period were included. The outpatient cohort of 1,814 individuals was sampled from epidemiological line lists by systematic quasi-random sampling. Altogether 83 eligible outpatients (4.6%) and 3 inpatients (0.3%) were excluded due to insufficient data for grading of clinical suspicion. ExposuresHigh clinical suspicion for COVID-19 was used as the reference standard for the RT-PCR test. Patients were considered to have high clinical suspicion of COVID-19 if the physician in charge recorded the suspicion on clinical grounds, or the patient fulfilled specifically defined clinical and exposure criteria. Main measuresSensitivity of SARS-CoV-2 RT-PCR by using manually curated clinical characteristics as the gold standard. ResultsThe study population included 1,814 outpatients (mean [SD] age, 45.4 [17.2] years; 69.1% women) and 1,194 inpatients (mean [SD] age, 63.2 [18.3] years; 45.2% women). The sensitivity (95% CI) for laboratory confirmed cases, i.e. repeatedly tested patients were as follows: 85.7% (81.5-89.1%) inpatients; 95.5% (92.2-97.5%) outpatients, 89.9% (88.2-92.1%) all. When also patients that were graded as high suspicion but never tested positive were included in the denominator, the following sensitivity values (95% CI) were observed: 67.5% (62.9-71.9%) inpatients; 34.9% (31.4-38.5%) outpatients; 47.3% (44.4-50.3%) all. Conclusions and relevanceThe clinical sensitivity of SARS-CoV-2 RT-PCR testing was only moderate at best. The relatively high false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations and when using RT-PCR as a reference for other tests. Key PointsO_ST_ABSQuestionC_ST_ABSWhat is the clinical sensitivity of SARS-CoV-2 RT-PCR test? FindingsIn this population-based retrospective study on medical records of 1,814 outpatients and 1,194 inpatients, the clinical sensitivity of SARS-CoV-2 RT-PCR was 47.3-89.9%. MeaningThe false negative rates of SARS-CoV-2 RT-PCR testing need to be accounted for in clinical decision making, epidemiological interpretations and when using RT-PCR as a reference for other tests.

Evolutionary trends of European bat lyssavirus type 2 including genetic characterization of Finnish strains of human and bat origin 24 years apart.

Among other Lyssaviruses, Daubenton's and pond-bat-related European bat lyssavirus type 2 (EBLV-2) can cause human rabies. To investigate the diversity and evolutionary trends of EBLV-2, complete genome sequences of two Finnish isolates were analysed. One originated from a human case in 1985, and the other originated from a bat in 2009. The overall nucleotide and deduced amino acid sequence identity of the two Finnish isolates were high, as well as the similarity to fully sequenced EBLV-2 strains originating from the UK and the Netherlands. In phylogenetic analysis, the EBLV-2 strains formed a monophyletic group that was separate from other bat-type lyssaviruses, with significant support. EBLV-2 shared the most recent common ancestry with Bokeloh bat lyssavirus (BBLV) and Khujan virus (KHUV). EBLV-2 showed limited diversity compared to RABV and appears to be well adapted to its host bat species. The slow tempo of viral evolution was evident in the estimations of divergence times for EBLV-2: the current diversity was estimated to have built up during the last 2000 years, and EBLV-2 diverged from KHUV about 8000 years ago. In a phylogenetic tree of partial N gene sequences, the Finnish EBLV-2 strains clustered with strains from Central Europe, supporting the hypothesis that EBLV-2 circulating in Finland might have a Central European origin. The Finnish EBLV-2 strains and a Swiss strain were estimated to have diverged from other EBLV-2 strains during the last 1000 years, and the two Finnish strains appear to have evolved from a common ancestor during the last 200 years.