Whole Blood as a Sample Matrix in Homogeneous Time-Resolved Assay-Förster Resonance Energy Transfer-Based Antibody Detection.

The protein-L-utilizing Förster resonance energy transfer (LFRET) assay enables mix-and-read antibody detection, as demonstrated for sera from patients with, e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Zika virus, and orthohantavirus infections. In this study, we compared paired serum and whole blood (WB) samples of COVID-19 patients and SARS-CoV-2 vaccine recipients. We found that LFRET also detects specific antibodies in WB samples. In 44 serum-WB pairs from patients with laboratory-confirmed COVID-19, LFRET showed a strong correlation between the sample materials. By analyzing 89 additional WB samples, totaling 133 WB samples, we found that LFRET results were moderately correlated with enzyme-linked immunosorbent assay results for samples collected 2 to 14 months after receiving COVID-19 diagnosis. However, the correlation decreased for samples >14 months after receiving a diagnosis. When comparing the WB LFRET results to neutralizing antibody titers, a strong correlation emerged for samples collected 1 to 14 months after receiving a diagnosis. This study also highlights the versatility of LFRET in detecting antibodies directly from WB samples and suggests that it could be employed for rapidly assessing antibody responses to infectious agents or vaccines.

Ancestral allele of DNA polymerase gamma modifies antiviral tolerance.

Mitochondria are critical modulators of antiviral tolerance through the release of mitochondrial RNA and DNA (mtDNA and mtRNA) fragments into the cytoplasm after infection, activating virus sensors and type-I interferon (IFN-I) response1-4. The relevance of these mechanisms for mitochondrial diseases remains understudied. Here we investigated mitochondrial recessive ataxia syndrome (MIRAS), which is caused by a common European founder mutation in DNA polymerase gamma (POLG1)5. Patients homozygous for the MIRAS variant p.W748S show exceptionally variable ages of onset and symptoms5, indicating that unknown modifying factors contribute to disease manifestation. We report that the mtDNA replicase POLG1 has a role in antiviral defence mechanisms to double-stranded DNA and positive-strand RNA virus infections (HSV-1, TBEV and SARS-CoV-2), and its p.W748S variant dampens innate immune responses. Our patient and knock-in mouse data show that p.W748S compromises mtDNA replisome stability, causing mtDNA depletion, aggravated by virus infection. Low mtDNA and mtRNA release into the cytoplasm and a slow IFN response in MIRAS offer viruses an early replicative advantage, leading to an augmented pro-inflammatory response, a subacute loss of GABAergic neurons and liver inflammation and necrosis. A population databank of around 300,000 Finnish individuals6 demonstrates enrichment of immunodeficient traits in carriers of the POLG1 p.W748S mutation. Our evidence suggests that POLG1 defects compromise antiviral tolerance, triggering epilepsy and liver disease. The finding has important implications for the mitochondrial disease spectrum, including epilepsy, ataxia and parkinsonism.

Phylogenetic Characterization of Orthohantavirus dobravaense (Dobrava Virus).

We report complete coding sequences of Orthohantavirus dobravaense (Dobrava virus) Igneada strains and phylogenetic characterization of all available complete coding sequences. Our analyses suggested separation of host-dependent lineages, followed by geographic clustering. Surveillance of orthohantaviruses using complete genomes would be useful for assessing public health threats from Dobrava virus.

Transmission of drug-resistant Mycobacterium tuberculosis isolates between Finnish- and foreign-born cases, 2014-2021: A molecular epidemiological study.

BACKGROUND: Data on the molecular epidemiology and transmission of drug-resistant Mycobacterium tuberculosis (MTB) in low-incidence settings with immigration from high-incidence settings is limited. METHOD: We included 115 drug-resistant (DR) MTB isolates with whole-genome sequencing data isolated in Finland between 2014 and 2021. Potential transmission clusters were identified using a threshold of 12 single-nucleotide polymorphisms (SNPs). Highly related clusters were identified using a threshold of 5 SNPs. RESULT: Of the 115 DR MTB isolates, 31 (27.0%) isolates were from Finnish-born cases and 84 (73.0%) were from foreign-born cases. The proportion of multidrug-resistant (MDR) MTB isolates (30/84, 35.7%) from foreign-born cases was higher than that of MDR MTB isolates from Finnish-born cases (8/31, 25.8%). Lineage 2 (40/115, 34.8%) and lineage 4 (40/115, 34.8%) were the most prevalent lineages. A total of 25 (21.7%) isolates were classified into eight potential transmission clusters (≤12 SNPs). Furthermore, five highly related clusters (≤5 SNPs) were identified, including three DR MTB isolates from Finnish-born cases and 14 DR isolates from foreign-born cases. CONCLUSION: The risk of DR MTB transmission between Finnish- and foreign-born persons is not negligible. Further research on clustering analysis in drug-susceptible MTB is worth to inform tuberculosis management and control in low-incidence settings with increasing immigration.

Nanobody engineering for SARS-CoV-2 neutralization and detection.

In response to the ongoing COVID-19 pandemic, the quest for coronavirus inhibitors has inspired research on a variety of small proteins beyond conventional antibodies, including robust single-domain antibody fragments, i.e., "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 at high potency, with IC50 values as low as 50 pM. Despite simultaneous binding to distinct epitopes, Beta and Omicron variants were more resistant to neutralization by the multimodular nanobodies, which highlights the importance of accounting for antigenic drift in the design of biologics. To further explore the applications of nanobody engineering in outbreak management, we present an 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 diseases. IMPORTANCE: Nanobodies, small protein binders derived from the camelid antibody, are highly potent inhibitors of respiratory viruses that offer several advantages over conventional antibodies as candidates for specific therapies, including high stability and low production costs. In this work, we leverage the unique properties of nanobodies and apply them as building blocks for new therapeutic and diagnostic tools. We report ultra-potent SARS-CoV-2 inhibition by engineered nanobodies comprising multiple modules in structure-guided combinations and develop nanobodies that carry signal molecules, allowing rapid detection of the SARS-CoV-2 spike protein. Our results highlight the potential of engineered nanobodies in the development of effective countermeasures, both therapeutic and diagnostic, to manage outbreaks of emerging viruses.

The α-dystroglycan N-terminus is a broad-spectrum antiviral agent against SARS-CoV-2 and enveloped viruses.

The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on α-dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of α-dystroglycan (α-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of α-DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant α-DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant α-DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.

Exposure to urban particulate matter alters responses of olfactory mucosal cells to SARS-CoV-2 infection.

Respiratory viruses have a significant impact on health, as highlighted by the COVID-19 pandemic. Exposure to air pollution can contribute to viral susceptibility and be associated with severe outcomes, as suggested by recent epidemiological studies. Furthermore, exposure to particulate matter (PM), an important constituent of air pollution, is linked to adverse effects on the brain, including cognitive decline and Alzheimer's disease (AD). The olfactory mucosa (OM), a tissue located at the rooftop of the nasal cavity, is directly exposed to inhaled air and in direct contact with the brain. Increasing evidence of OM dysfunction related to neuropathogenesis and viral infection demonstrates the importance of elucidating the interplay between viruses and air pollutants at the OM. This study examined the effects of subacute exposure to urban PM 0.2 and PM 10-2.5 on SARS-CoV-2 infection using primary human OM cells obtained from cognitively healthy individuals and individuals diagnosed with AD. OM cells were exposed to PM and subsequently infected with the SARS-CoV-2 virus in the presence of pollutants. SARS-CoV-2 entry receptors and replication, toxicological endpoints, cytokine release, oxidative stress markers, and amyloid beta levels were measured. Exposure to PM did not enhance the expression of viral entry receptors or cellular viral load in human OM cells. However, PM-exposed and SARS-CoV-2-infected cells showed alterations in cellular and immune responses when compared to cells infected only with the virus or pollutants. These changes are highly pronounced in AD OM cells. These results suggest that exposure of human OM cells to PM does not increase susceptibility to SARS-CoV-2 infection in vitro, but it can alter cellular immune responses to the virus, particularly in AD. Understanding the interplay of air pollutants and COVID-19 can provide important insight for the development of public health policies and interventions to reduce the negative influences of air pollution exposure.

Veterinarians and zoonotic pathogens, infections and diseases - questionnaire study and case series, Finland.

BACKGROUND: Veterinarians are at risk for numerous zoonotic infections. In this paper, we summarise descriptions of zoonotic infections from a questionnaire study and a series of work-related zoonotic cases, aiming to add to the knowledge on occupational zoonotic risks of veterinarians. METHODS: We collected data on zoonotic infections contracted by veterinarians in Finland in two studies:1) using a questionnaire in 2009, and 2) inviting veterinarians who had encountered an occupational zoonosis to report it in structured interviews in 2019. RESULTS AND CONCLUSIONS: In the questionnaire study in 2009, of 306 veterinarians several reported zoonotic bacterial skin infections (12%), dermatophytosis (ringworm; 4.2%), virus infections (3.9%), bacterial gastroenteritis (3.3%), other bacterial zoonoses (2.3%), and parasitic infections/infestations (2.3%). In the 2019 interviews, 16 occupational zoonosis cases were reported. Of them, seven were selected to the case series. The selected cases included Capnocytophaga canimorsus sepsis following a dog bite, cryptosporidiosis after a contact with calves, cutaneous listeriosis following calving assistance, Salmonella gastroenteritis contracted at laboratory, Trichophyton dermatophytosis after equine contact, Bacillus anthracis exposure at necropsy, and exposure to rabies through a horse bite. In four of the seven cases, the veterinarian disagreed or strongly disagreed with having had good knowledge of the zoonosis before the incident. The results from the questionnaire study and the case series illustrate the variety of zoonotic pathogens that veterinarians may encounter. There is a need to improve the occupational health of veterinarians and to increase awareness in the occupational health sector. We encourage addressing this need using a One Health approach.

Drug repurposing platform for deciphering the druggable SARS-CoV-2 interactome.

The coronavirus disease 2019 (COVID-19) pandemic has heavily challenged the global healthcare system. Despite the vaccination programs, the new virus variants are circulating. Further research is required for understanding of the biology of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and for discovery of therapeutic agents against the virus. Here, we took advantage of drug repurposing to identify if existing drugs could inhibit SARS-CoV-2 infection. We established an open high throughput platform for in vitro screening of drugs against SARS-CoV-2 infection. We screened ∼1000 drugs for their ability to inhibit SARS-CoV-2-induced cell death in the African green monkey kidney cell line (Vero-E6), analyzed how the hit compounds affect the viral N (nucleocapsid) protein expression in human cell lines using high-content microscopic imaging and analysis, determined the hit drug targets in silico, and assessed their ability to cause phospholipidosis, which can interfere with the viral replication. Duvelisib was found by in silico interaction assay as a potential drug targeting virus-host protein interactions. The predicted interaction between PARP1 and S protein, affected by Duvelisib, was further validated by immunoprecipitation. Our results represent a rapidly applicable platform for drug repurposing and evaluation of the new emerging viruses' responses to the drugs. Further in silico studies help us to discover the druggable host pathways involved in the infectious cycle of SARS-CoV-2.

Serological and molecular screening of arenaviruses in suspected tick-borne encephalitis cases in Finland.

Lymphocytic choriomeningitis virus (LCMV) is one of the arenaviruses infecting humans. LCMV infections have been reported worldwide in humans with varying levels of severity. To detect arenavirus RNA and LCMV-reactive antibodies in different geographical regions of Finland, we screened human serum and cerebrospinal fluid (CSF) samples, taken from suspected tick-borne encephalitis (TBE) cases, using reverse transcriptase polymerase chain reaction (RT-PCR) and immunofluorescence assay (IFA). No arenavirus nucleic acids were detected, and the overall LCMV seroprevalence was 4.5%. No seroconversions were detected in paired serum samples. The highest seroprevalence (5.2%) was detected among individuals of age group III (40-59 years), followed by age group I (under-20-year-olds, 4.9%), while the lowest seroprevalence (3.8%) was found in age group IV (60 years or older). A lower LCMV seroprevalence in older age groups may suggest waning of immunity over time. The observation of a higher seroprevalence in the younger age group and the decreasing population size of the main reservoir host, the house mouse, may suggest exposure to another LCMV-like virus in Finland.

Mucosal-Associated Invariant T Cells are not susceptible in vitro to SARS-CoV-2 infection but accumulate into the lungs of COVID-19 patients.

Prolonged T cell lymphopenia is common in COVID-19, caused by SARS-CoV-2. While the mechanisms of lymphopenia during COVID-19 remain elusive, it is especially pronounced in a specialized innate-like T cell population called Mucosal Associated Invariant T cells (MAITs). MAITs has been suggested to express Angiotensin-Converting Enzyme 2 (ACE2), which is the well-known cellular receptor for SARS-CoV-2. However, it is still unclear if SARS-CoV-2 can infect or affect MAIT cells directly. In this study, we performed multicolor flow cytometry on peripheral blood mononuclear cells obtained from COVID-19 patients to assess the frequencies of CD8+Vα7.2+CD161+ MAIT subsets at acute and convalescent disease phases. The susceptibility of MAITs and T cells to direct exposure by SARS-CoV-2 was analysed using cells isolated from healthy donor buffy coats by viability assays, virus-specific RT-PCR, and flow cytometry. In situ lung immunofluorescence was used to evaluate retention of T cells, especially MAIT cells, in lung tissues during acute COVID-19. Our study confirms previous reports indicating that circulating MAITs are activated, and their frequency is declined in patients with acute SARS-CoV-2 infection, whereas an accumulation of MAITs and T cells was seen in the lung tissue of individuals with fatal COVID-19. However, despite a fraction of MAITs found to express ACE2, no evidence for the susceptibility of MAITs for direct infection or activation by SARS-CoV-2 particles was observed. Thus, their activation and decline in the circulation is most likely explained by indirect mechanisms involving other immune cells and cytokine-induced pro-inflammatory environment but not by direct exposure to viral particles at the infection site.

Long COVID-associated symptoms prevalent in both SARS-CoV-2 positive and negative individuals: A prospective follow-up study.

Background: Research into persistent symptoms among SARS-CoV-2-positive i.e. CoV(+) patients mostly focuses on hospitalized individuals. Our prospective follow-up study compares long COVID-associated symptoms among laboratory-confirmed CoV(+) and SARS-CoV-2 negative [CoV(-)] individuals. Methods: SARS-CoV-2 RT-PCR-tested volunteers were recruited into four cohorts: 1) CoV(+) outpatients, 2) CoV(-) outpatients, 3) CoV(+) intensive care unit (ICU) inpatients, and 4) CoV(+) non-ICU inpatients. Neutralizing antibodies were assessed and questionnaires filled in at enrolment and days 90-120, 121-180, 181-270, 271-365, and 365-533. Results: Of the 1326 participants, 1191 were CoV(+): 46 ICU, 123 non-ICU, and 1022 outpatients; 135 were CoV(-) outpatient controls. Both CoV(+) outpatients and CoV(-) controls showed high overall symptom rates at all time points. More prevalent among CoV(+) than CoV(-) outpatients were only impaired olfaction and taste; many others proved more frequent for CoV(-) participants. At ≥181 days, fatigue, dyspnoea, various neuropsychological symptoms and several others were recorded more often for CoV(+) inpatients than outpatients. Conclusions: Long COVID-associated symptoms were more frequent among hospitalized than non-hospitalized CoV(+) participants. As for outpatients, only impaired olfaction and taste showed higher rates in the CoV(+) group; some symptoms proved even more common among those CoV(-). Besides suggesting low long COVID prevalences for outpatients, our results highlight the weight of negative controls.

SARS-CoV-2 infections among pregnant women, 2020, Finland-Cross-testing of neutralization assays.

We studied the development of the severe acute respiratory syndrome-related coronavirus (SARS-CoV-2) pandemic in southern Finland in 2020 and evaluated the performance of two surrogate immunoassays for the detection of neutralizing antibodies (NAbs). The data set consisted of 12 000 retrospectively collected samples from pregnant women in their first trimester throughout 2020. All the samples were initially screened for immunoglobulin G (IgG) with SARS-CoV-2 spike antibody assay (EIM-S1, Euroimmun) followed by confirmation with nucleocapsid antibody assay (Architect SARS-CoV-2, Abbott). Samples that were reactive (positive or borderline) with both assays were subjected to testing with commercial surrogate immunoassays of NeutraLISA (EIM) and cPassTM (GenScript Biotech Corporation) by using pseudoneutralization assay (PNAbA) as a golden standard. No seropositive cases were detected between January and March. Between April and December, IgG (EIM-S1 and Abbott positive) and NAb (PNAbA positive) seroprevalences were between 0.4% and 1.4%. NeutraLISA showed 90% and cPass 55% concordant results with PNAbA among PNAbA negative samples and 49% and 92% among PNAbA positive samples giving NeutraLISA better specificity but lower sensitivity than cPass. To conclude, seroprevalence in pregnant women reflected that of the general population but the variability of the performance of serological protocols needs to be taken into account in inter-study comparison.

Equine dermatitis outbreak associated with parapoxvirus.

Parapoxviruses (PPV) cause skin and mucous membrane lesions in several animal species, and of the five recognized PPVs, at least three are zoonotic. Equine PPV (EqPPV) is the sixth one initially described in humans in the United States and later in a severely sick horse in Finland in 2013-2015. In 2021-2022, a large-scale pustulo-vesicular pastern dermatitis outbreak occurred in horses all over Finland. This study aimed at analysing the outbreak, identifying and describing the causative agent, describing clinical signs, and searching for risk factors. EqPPV was identified as a probable causative agent and co-infections with several potentially pathogenic and zoonotic bacteria were observed. Histopathologically, suppurative and ulcerative dermatitis was diagnosed. Due to the lack of specific tests for this virus, we developed a novel diagnostic EqPPV-PCR with sensitivity of 10 copies/reaction. Based on a large proportion of the genome sequenced directly from clinical samples, very little variation was detected between the sequences of the case from 2013 and the cases from 2021 to 2022. Based on an epidemiological survey, the main risk factor for pastern dermatitis was having racehorses. Approximately one third of the horses at each affected stable got clinical dermatitis, manifesting as severe skin lesions. Skin lesions were also occasionally reported in humans, indicating potential zoonotic transmission. Case stables commonly reported attendance at race events before acquiring the disease. Survey also identified differences in practises between case and control stables. Taken together, these results enable a better preparedness, diagnostics, and guidelines for future outbreaks.

Human-derived air-liquid interface cultures decipher Alzheimer's disease-SARS-CoV-2 crosstalk in the olfactory mucosa.

BACKGROUND: The neurological effects of the coronavirus disease of 2019 (COVID-19) raise concerns about potential long-term consequences, such as an increased risk of Alzheimer's disease (AD). Neuroinflammation and other AD-associated pathologies are also suggested to increase the risk of serious SARS-CoV-2 infection. Anosmia is a common neurological symptom reported in COVID-19 and in early AD. The olfactory mucosa (OM) is important for the perception of smell and a proposed site of viral entry to the brain. However, little is known about SARS-CoV-2 infection at the OM of individuals with AD. METHODS: To address this gap, we established a 3D in vitro model of the OM from primary cells derived from cognitively healthy and AD individuals. We cultured the cells at the air-liquid interface (ALI) to study SARS-CoV-2 infection under controlled experimental conditions. Primary OM cells in ALI expressed angiotensin-converting enzyme 2 (ACE-2), neuropilin-1 (NRP-1), and several other known SARS-CoV-2 receptor and were highly vulnerable to infection. Infection was determined by secreted viral RNA content and confirmed with SARS-CoV-2 nucleocapsid protein (NP) in the infected cells by immunocytochemistry. Differential responses of healthy and AD individuals-derived OM cells to SARS-CoV-2 were determined by RNA sequencing. RESULTS: Results indicate that cells derived from cognitively healthy donors and individuals with AD do not differ in susceptibility to infection with the wild-type SARS-CoV-2 virus. However, transcriptomic signatures in cells from individuals with AD are highly distinct. Specifically, the cells from AD patients that were infected with the virus showed increased levels of oxidative stress, desensitized inflammation and immune responses, and alterations to genes associated with olfaction. These results imply that individuals with AD may be at a greater risk of experiencing severe outcomes from the infection, potentially driven by pre-existing neuroinflammation. CONCLUSIONS: The study sheds light on the interplay between AD pathology and SARS-CoV-2 infection. Altered transcriptomic signatures in AD cells may contribute to unique symptoms and a more severe disease course, with a notable involvement of neuroinflammation. Furthermore, the research emphasizes the need for targeted interventions to enhance outcomes for AD patients with viral infection. The study is crucial to better comprehend the relationship between AD, COVID-19, and anosmia. It highlights the importance of ongoing research to develop more effective treatments for those at high risk of severe SARS-CoV-2 infection.

External quality assessment of orthohantavirus and lymphocytic choriomeningitis virus molecular detection and serology in Europe, 2021.

BackgroundRodent-borne viruses such as orthohantaviruses and arenaviruses cause considerable disease burden with regional and temporal differences in incidence and clinical awareness. Therefore, it is important to regularly evaluate laboratory diagnostic capabilities, e.g. by external quality assessments (EQA).AimWe wished to evaluate the performance and diagnostic capability of European expert laboratories to detect orthohantaviruses and lymphocytic choriomeningitis virus (LCMV) and human antibody response towards orthohantaviruses.MethodsWe conducted an EQA in 2021; molecular panels consisted of 12 samples, including different orthohantaviruses (Seoul, Dobrava-Belgrade (DOBV), Puumala (PUUV) and Hantaan orthohantavirus), LCMV and negative controls. Serological panels consisted of six human serum samples reactive to PUUV, DOBV or negative to orthohantaviruses. The EQA was sent to 25 laboratories in 20 countries.ResultsThe accuracy of molecular detection of orthohantaviruses varied (50‒67%, average 62%) among 16 participating laboratories, while LCMV samples were successfully detected in all 11 participating laboratories (91-100%, average 96%). The accuracy of serological diagnosis of acute and past orthohantavirus infections was on average 95% among 20 participating laboratories and 82% in 19 laboratories, respectively. A variety of methods was used, with predominance of in-house assays for molecular tests, and commercial assays for serological ones.ConclusionSerology, the most common tool to diagnose acute orthohantavirus infections, had a high accuracy in this EQA. The molecular detection of orthohantaviruses needs improvement while LCMV detection (performed in fewer laboratories) had 95% accuracy. Further EQAs are recommended to be performed periodically to monitor improvements and challenges in the diagnostics of rodent-borne diseases.

Validation of an antigenic site targeted by monoclonal antibodies against Puumala virus.

Identification of B-cell epitopes facilitates the development of vaccines, therapeutic antibodies and diagnostic tools. Previously, the binding site of the bank vole monoclonal antibody (mAb) 4G2 against Puumala virus (PUUV, an orthohantavirus in the Hantaviridae family of the Bunyavirales order) was predicted using a combination of methods, including pepscan, phage-display, and site-directed mutagenesis of vesicular stomatitis virus (VSV) particles pseudotyped with Gn and Gc glycoproteins from PUUV. These techniques led to the identification of the neutralization escape mutation F915A. To our surprise, a recent crystal structure of PUUV Gc in complex with Fab 4G2 revealed that residue F915 is distal from epitope of mAb 4G2. To clarify this issue and explore potential explanations for the inconsistency, we designed a mutagenesis experiment to probe the 4G2 epitope, with three PUUV pseudoviruses carrying amino acid changes E725A, S944F, and S946F, located within the structure-based 4G2 epitope on the Gc. These amino acid changes were able to convey neutralization escape from 4G2, and S944F and S946F also conveyed escape from neutralization by human mAb 1C9. Furthermore, our mapping of all the known neutralization evasion sites from hantaviral Gcs onto PUUV Gc revealed that over 60 % of these sites reside within or close to the epitope of mAb 4G2, indicating that this region may represent a crucial area targeted by neutralizing antibodies against PUUV, and to a lesser extent, other hantaviruses. The identification of this site of vulnerability could guide the creation of subunit vaccines against PUUV and other hantaviruses in the future.

Image-based and machine learning-guided multiplexed serology test for SARS-CoV-2.

We present a miniaturized immunofluorescence assay (mini-IFA) for measuring antibody response in patient blood samples. The method utilizes machine learning-guided image analysis and enables simultaneous measurement of immunoglobulin M (IgM), IgA, and IgG responses against different viral antigens in an automated and high-throughput manner. The assay relies on antigens expressed through transfection, enabling use at a low biosafety level and fast adaptation to emerging pathogens. Using severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as the model pathogen, we demonstrate that this method allows differentiation between vaccine-induced and infection-induced antibody responses. Additionally, we established a dedicated web page for quantitative visualization of sample-specific results and their distribution, comparing them with controls and other samples. Our results provide a proof of concept for the approach, demonstrating fast and accurate measurement of antibody responses in a research setup with prospects for clinical diagnostics.

Molecular detection of pathogens from ticks collected from dogs and cats at veterinary clinics in Finland.

BACKGROUND: Ticks carry microbes, some of which are pathogenic for humans and animals. To assess this One Health challenge, 342 ticks were collected from pet dogs and cats at 10 veterinary clinics in Finland as part of the European project "Protect Our Future Too". METHODS: The tick species were identified, and ticks were screened with quantitative PCR (qPCR) for tick-borne pathogens, including Borrelia burgdorferi sensu lato, Borrelia miyamotoi, Ehrlichia canis, Anaplasma spp., Candidatus Neoehrlichia mikurensis, tick-borne encephalitis virus (TBEV), and Babesia spp. For comparison, a subset of tick DNA (20 qPCR-positive samples) was analysed with 16S next-generation sequencing (NGS). RESULTS: Most ticks were Ixodes ricinus (289, 84.5%), followed by Ixodes persulcatus (51, 14.9%). One hybrid tick (I. ricinus/I. persulcatus, 0.3%) and one Rhipicephalus sanguineus tick (0.3%) were identified. We found one or more of the analysed pathogens in 17% (59/342) of the ticks. The most prevalent pathogen was B. burgdorferi s.l. (36, 10.5%), followed by Anaplasma phagocytophilum (12, 3.5%), B. miyamotoi (5, 1.5%), Babesia venatorum (4, 1.2%), and TBEV (1, 0.3%). Candidatus Neoehrlichia mikurensis DNA was amplified from three (0.9%) ticks. Ehrlichia canis was not detected. In the 16S NGS, six samples produced enough reads for the analysis. In these six samples, we confirmed all the positive qPCR findings of Borrelia spp. and Ca. N. mikurensis. CONCLUSIONS: The high prevalence of pathogenic microorganisms in the ticks of this study emphasizes the importance of awareness of ticks and tick-borne diseases and prevention. Furthermore, the results show that veterinary surveillance can facilitate early detection of tick-borne pathogens and new tick species and draw attention to possible co-infections that should be considered both in symptomatic humans and animals after tick bites.

Partial Genome Characterization of Novel Parapoxvirus in Horse, Finland.

We report a sequencing protocol and 121-kb poxvirus sequence from a clinical sample from a horse in Finland with dermatitis. Based on phylogenetic analyses, the virus is a novel parapoxvirus associated with a recent epidemic; previous data suggest zoonotic potential. Increased awareness of this virus and specific diagnostic protocols are needed.