Wild red foxes (Vulpes vulpes) do not participate in SARS-CoV-2 circulation in Poland.

BACKGROUND: Biomonitoring is an essential activity for identifying possible vectors and reservoirs of pathogens and predicting potential outbreaks. Wild red foxes are present in both sylvatic and synanthropic environments, making them potential carriers of zoonotic pathogens. Experimental studies have shown that both coyotes and red foxes can transmit SARS-CoV-2. This study aimed to assess the prevalence and seroprevalence of SARS-CoV-2 in wild red foxes hunted in northern Poland. METHODS: Oral swabs, blood clots or heat tissue samples were collected from 292 red foxes hunted in northern Poland. We used both molecular (RT-PCR) and serological (IFA) approaches to detect SARS-CoV-2 infections in the sampled animals. RESULTS: We did not find any evidence of SARS-CoV-2 infection in the collected samples, using both molecular and serological methods. CONCLUSIONS: Despite foxes having frequent contact with humans, human waste, and other animals, they do not appear to participate in the circulation of the SARS-CoV-2 virus in our geographical region. Nevertheless, we believe that continuous biomonitoring should be performed to assess the SARS-CoV-2 epidemiological situation in the wild.

Hibernating vesper bats are a weak source for biomonitoring of coronaviruses.

Background: Our study explores the role of bats as reservoirs of coronaviruses. Methods: We conducted virological screening of bats hibernating in military bunkers at the Natura 2000 site "Nietoperek" in Western Poland collecting oral and anal swab samples from 138 bats across six species to apply a combination of pan-coronavirus and SARS-CoV-2 specific PCR assays. Results: Only one anal swab tested positive for coronavirus. No SARS-CoV-2 was detected in any of the samples. The low prevalence of coronavirus in the studied colony contrasts with higher rates found in other regions and may be influenced by hibernation. Conclusions: Hibernating bats may show a low prevalence of coronavirus, potentially due to the hibernation process itself. This finding indicates that hibernating bats may not be the most optimal subjects for screening zoonotic pathogens. However, biomonitoring of bats for emerging and reemerging diseases is recommended for comprehensive epidemiological insights.

Leucoma salicis nucleopolyhedrovirus (LesaNPV) genome sequence shed new light on the origin of the Alphabaculovirus orpseudotsugatae species.

LesaNPV (Leucoma salicis nucleopolyhedrovirus) is an alphabaculovirus group Ib. Potentially, it can be an eco-friendly agent to control the white satin moth Leucoma salicis population. In this study, we have established the relationship between LesaNPV and other closely related alphabaculoviruses. Environmental samples of late instar of white satin moth collected in Poland infected with baculovirus have been homogenized, polyhedra were purified and subjected to scanning and transmission electron microscopy. Viral DNA was sequenced using the Illumina platform and the whole-genome sequence was established by de novo assembly of paired reads. Genome annotation and phylogenetic analyses were performed with the use of bioinformatics tools. The genome of LesaNPV is 132 549 bp long with 154 ORFs and 54.9% GC content. Whole-genome sequencing revealed deletion of dUTPase as well as ribonucleoside reductases small and large subunits region in LesaNPV genome compared to Dasychira pudibunda nucleopolyhedrovirus (DapuNPV) and Orgyia pseudotsugata multiple nucleopolyhedrovirus (OpMNPV) where this region is complete. Phylogenetic analysis of Baculoviridae family members showed that LesaNPV is less divergent from a common ancestor than closely related species DapuNPV and OpMNPV. This is interesting because their hosts do not occur in the same area. The baculoviruses described in this manuscript are probably isolates of one species and could be assigned to recently denominated species Alphabaculovirus orpseudotsugatae, historically originating from OpMNPV. This finding could have significant implications for the classification and understanding of the phylogeographical spread of baculoviruses.

Genetic Loci of Plant Pathogenic Dickeya solani IPO 2222 Expressed in Contact with Weed-Host Bittersweet Nightshade (Solanum dulcamara L.) Plants.

Dickeya solani, belonging to the Soft Rot Pectobacteriaceae, are aggressive necrotrophs, exhibiting both a wide geographic distribution and a wide host range that includes many angiosperm orders, both dicot and monocot plants, cultivated under all climatic conditions. Little is known about the infection strategies D. solani employs to infect hosts other than potato (Solanum tuberosum L.). Our earlier study identified D. solani Tn5 mutants induced exclusively by the presence of the weed host S. dulcamara. The current study assessed the identity and virulence contribution of the selected genes mutated by the Tn5 insertions and induced by the presence of S. dulcamara. These genes encode proteins with functions linked to polyketide antibiotics and polysaccharide synthesis, membrane transport, stress response, and sugar and amino acid metabolism. Eight of these genes, encoding UvrY (GacA), tRNA guanosine transglycosylase Tgt, LPS-related WbeA, capsular biosynthesis protein VpsM, DltB alanine export protein, glycosyltransferase, putative transcription regulator YheO/PAS domain-containing protein, and a hypothetical protein, were required for virulence on S. dulcamara plants. The implications of D. solani interaction with a weed host, S. dulcamara, are discussed.

An engineered A549 cell line expressing CD13 and TMPRSS2 is permissive to clinical isolate of human coronavirus 229E.

The lack of suitable in vitro culture model has hampered research on wild-type (WT) human coronaviruses. While 3D tissue or organ cultures have been instrumental for this purpose, such models are challenging, time-consuming, expensive and require extensive cell culture adaptation and directed evolution. Consequently, high-throughput applications are beyond reach in most cases. Here we developed a robust A549 cell line permissive to a human coronavirus 229E (HCoV-229E) clinical isolate by transducing CD13 and transmembrane serine protease 2 (TMPRSS2), henceforth referred to as A549++ cells. This modification allowed for productive infection, and a more detailed analysis showed that the virus might use the TMPRSS2-dependent pathway but can still bypass this pathway using cathepsin-mediated endocytosis. Overall, our data showed that A549++ cells are permissive to HCoV-229E clinical isolate, and applicable for further studies on HCoV-229E infectiology. Moreover, this line constitutes a uniform platform for studies on multiple members of the Coronaviridae family.

Surveillance of SARS-CoV-2 Genetic Variants in the Polish Armed Forces Using Whole Genome Sequencing Analysis.

Military operations involve the global movement of personnel and equipment, increasing the risk of spreading infectious pathogens such as SARS-CoV-2. Given the continuous engagement of the Polish Armed Forces in overseas operations, an active surveillance program targeting Variants of Concern (VOC) of SARS-CoV-2 was implemented among military personnel. Screening using RT-qPCR tests was conducted on 1699 soldiers between November 2021 and May 2022. Of these, 84 SARS-CoV-2 positive samples met the criteria for whole genome sequencing analysis and variant identification. Whole genome sequencing was performed using two advanced next-generation sequencing (NGS) technologies: sequencing by synthesis and nanopore sequencing. Our analysis revealed eleven SARS-CoV-2 lineages belonging to 21K, 21L, and 21J. The predominant lineage was BA.1.1 (57% of the samples), followed by BA.1 (23%) and BA.2 (6%). Notably, all identified lineages detected in post-deployment screening tests were classified as VOC and were already present in Poland, showing the effectiveness of the Military Sanitary Inspection measures in mitigating the COVID-19 spread. Pre-departure and post-mission screening and isolation successfully prevented SARS-CoV-2 VOC exportation and importation. Proactive measures are vital in minimizing the impact of COVID-19 in military settings, emphasizing the need for continued vigilance and response strategies.

Genetic Diversity of Type A Influenza Viruses Found in Swine Herds in Northwestern Poland from 2017 to 2019: The One Health Perspective.

Influenza A viruses (IAV) are still a cause of concern for public health and veterinary services worldwide. With (-) RNA-segmented genome architecture, influenza viruses are prone to reassortment and can generate a great variety of strains, some capable of crossing interspecies barriers. Seasonal IAV strains continuously spread from humans to pigs, leading to multiple reassortation events with strains endemic to swine. Due to its high adaptability to humans, a reassortant strain based on "human-like" genes could potentially be a carrier of avian origin segments responsible for high virulence, and hence become the next pandemic strain with unseen pathogenicity. The rapid evolution of sequencing methods has provided a fast and cost-efficient way to assess the genetic diversity of IAV. In this study, we investigated the genetic diversity of swine influenza viruses (swIAVs) collected from Polish farms. A total of 376 samples were collected from 11 farms. The infection was confirmed in 112 cases. The isolates were subjected to next-generation sequencing (NGS), resulting in 93 full genome sequences. Phylogenetic analysis classified 59 isolates as genotype T (H1avN2g) and 34 isolates as genotype P (H1pdmN1pdm), all of which had an internal gene cassette (IGC) derived from the H1N1pdm09-like strain. These data are consistent with evolutionary trends in European swIAVs. The applied methodology proved to be useful in monitoring the genetic diversity of IAV at the human-animal interface.

Emergence and potential transmission route of avian influenza A (H5N1) virus in domestic cats in Poland, June 2023.

In June 2023, a fatal disease outbreak in cats occurred in Poland. Most cases tested in Poland (29 of 47) were positive for highly pathogenic avian influenza (HPAI) A (H5N1) virus. Genetic analyses revealed clade 2.3.4.4b with point mutations indicative of initial mammalian hosts adaptations. Cat viral sequences were highly similar (n = 21), suggesting a potential common infection source. To investigate possible infection routes, our group tested food samples from affected households. HPAI H5N1 virus was detected in one poultry meat sample.

Clinical presentation of 13 children with alkaptonuria.

Until now, only a few studies have focused on the early onset of symptoms of alkaptonuria (AKU) in the pediatric population. This prospective, longitudinal study is a comprehensive approach to the assessment of children with recognized AKU during childhood. The study includes data from 32 visits of 13 patients (five males, eight females; age 4-17 years) with AKU. A clinical evaluation was performed with particular attention to eye, ear, and skin pigmentation, musculoskeletal complaints, magnetic resonance imaging (MRI), and ultrasound (US) imaging abnormalities. The cognitive functioning and adaptive abilities were examined. Molecular genetic analyses were performed. The most common symptoms observed were dark urine (13/13), followed by joint pain (6/13), and dark ear wax (6/13). In 4 of 13 patients the values obtained in the KOOS-child questionnaire were below the reference values. MRI and US did not show degenerative changes in knee cartilages. One child had nephrolithiasis. Almost half of the children with AKU (5/13) presented deficits in cognitive functioning and/or adaptive abilities. The most frequent HGD variants observed in the patients were c.481G>A (p.Gly161Arg) mutation and the c.240A>T (p.His80Gln) polymorphism. The newly described allele of the HGD gene (c.948G>T, p.Val316Phe) which is potentially pathogenic was identified.

Spontaneous mutations in hlyD and tuf genes result in resistance of Dickeya solani IPO 2222 to phage ϕD5 but cause decreased bacterial fitness and virulence in planta.

Lytic bacteriophages able to infect and kill Dickeya spp. can be readily isolated from virtually all Dickeya spp. containing environments, yet little is known about the selective pressure those viruses exert on their hosts. Two spontaneous D. solani IPO 2222 mutants (0.8% of all obtained mutants), DsR34 and DsR207, resistant to infection caused by lytic phage vB_Dsol_D5 (ΦD5) were identified in this study that expressed a reduced ability to macerate potato tuber tissues compared to the wild-type, phage-susceptible D. solani IPO 2222 strain. Genome sequencing revealed that genes encoding: secretion protein HlyD (in mutant DsR34) and elongation factor Tu (EF-Tu) (in mutant DsR207) were altered in these strains. These mutations impacted the DsR34 and DsR207 proteomes. Features essential for the ecological success of these mutants in a plant environment, including their ability to use various carbon and nitrogen sources, production of plant cell wall degrading enzymes, ability to form biofilms, siderophore production, swimming and swarming motility and virulence in planta were assessed. Compared to the wild-type strain, D. solani IPO 2222, mutants DsR34 and DsR207 had a reduced ability to macerate chicory leaves and to colonize and cause symptoms in growing potato plants.

Zoonotic spill-over of SARS-CoV-2: mink-adapted virus in humans.

OBJECTIVES: This work aimed to analyse possible zoonotic spill-over of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We report the spill-over of mink-adapted SARS-CoV-2 from farmed mink to humans after adaptation that lasted at least 3 months. METHODS: Next-generation sequencing and a bioinformatic approach were applied to analyse the data. RESULTS: In an isolate obtained from an asymptomatic patient testing positive for SARS-CoV-2, we found four distinguishing mutations in the S gene that gave rise to the mink-adapted variant (G75V, M177T, Y453F, and C1247F) and others. CONCLUSIONS: Zoonotic spill-over of SARS-CoV-2 can occur from mink to human.

Humoral Response after Vaccination with Half-Dose of BNT162b2 in Subjects under 55 Years of Age.

In the context of the ongoing COVID-19 pandemic, using a half-dose schedule vaccination can help to return to normalcy in a cost-efficient manner, which is especially important for low and middle-income countries. We undertook a study to confirm that in adults up to 55 years old, the humoral response to the half-dose (15 µg, 35 participants between 18 and 55 years old) and to the recommended dose (30 µg, 155 participants) in the two-dose three-week interval schedule would be comparable. Antibody levels were measured by the Elecsys Anti-SARS-CoV-2 S assay (Roche Diagnostics, upper detection limit: 2570 BAU/mL) on the day of dose 2 of the vaccine and then 8-10 days later to assess peak response to dose 2. The difference in proportions between the participants who did and did not exceed the upper detection limit 8-10 days after dose 2 was not statistically significant (p = 0.152). We suggest that a half-dose schedule can help to achieve widespread vaccination coverage more quickly and cheaply.

Expansion of a SARS-CoV-2 Delta variant with an 872 nt deletion encompassing ORF7a, ORF7b and ORF8, Poland, July to August 2021.

Routine genomic surveillance on samples from COVID-19 patients collected in Poland during summer 2021 revealed the emergence of a SARS-CoV-2 Delta variant with a large 872 nt deletion. This change, confirmed by Sanger and deep sequencing, causes complete loss of ORF7a, ORF7b, and ORF8 genes. The index case carrying the deletion is unknown. The standard pipeline for sequencing may mask this deletion with a long stretch of N's. Effects of this deletion on phenotype or immune evasion needs further study.

Humoral Response to SARS-CoV-2 Vaccine of a Patient Receiving Methotrexate Treatment and Implications for the Need of Monitoring.

We report a case of monitoring the antibody response to the BioNTech-Pfizer vaccine of a 50-year-old female diagnosed with rheumatoid arthritis undergoing treatment with methotrexate (MTX). Antibody levels were measured 21 days after dose 1 (i.e., on the day of dose 2) and then 8, 14 and 30 days after dose 2 with Elecsys Anti-SARS-CoV-2 S assay (Roche Diagnostics). Patient showed a negative result after dose 1 and had the serum sample retested using a LIAISON® SARS-CoV-2 TrimericS IgG assay (DiaSorin), which showed a positive result. Subsequent samples were tested using both assays. Antibody levels kept increasing but at a much slower rate than in patients not receiving any immunomodulatory therapies. Other research indicates that among patients with autoimmune diseases, those receiving disease-modifying antirheumatic drugs (DMARDs) have higher COVID-19 mortality than those treated with tumor necrosis factor inhibitors (TNFis). These results indicate the need for people with autoimmune diseases to be carefully observed following vaccinations, including testing of antibody levels, and treated as potentially at risk until the effect of vaccination is confirmed. The different available vaccines should also be tested to verify their usefulness in the case of people with autoimmune diseases and those who take different immunomodulatory medications.

Severe Acute Respiratory Syndrome Coronavirus 2 in Farmed Mink (Neovison vison), Poland.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiologic agent of coronavirus disease and has been spreading worldwide since December 2019. The virus can infect different animal species under experimental conditions, and mink on fur farms in Europe and other areas are susceptible to SARS-CoV-2 infection. We investigated SARS-CoV-2 infection in 91 mink from a farm in northern Poland. Using reverse transcription PCR, antigen detection, and next-generation sequencing, we confirmed that 15 animals were positive for SARS-CoV-2. We verified this finding by sequencing full viral genomes and confirmed a virus variant that has sporadic mutations through the full genome sequence in the spike protein (G75V and C1247F). We were unable to find other SARS-CoV-2 sequences simultaneously containing these 2 mutations. Country-scale monitoring by veterinary inspection should be implemented to detect SARS-CoV-2 in other mink farms.

Usefulness of IVD Kits for the Assessment of SARS-CoV-2 Antibodies to Evaluate the Humoral Response to Vaccination.

BACKGROUND: The introduction of the vaccination against SARS-CoV-2 infection creates the need for precise tools for the quality control of vaccination procedures, detection of poor humoral response, and estimation of the achieved protection against the disease. Thus, the study aimed to compare the results of the anti-SARS-CoV-2 tests to evaluate the application of the WHO standard unitage (the binding antibody units; BAU/mL) for a measurement of response to the vaccination. METHODS: Patients undergoing vaccination against SARS-CoV-2 with Pfizer/BioNTech BNT162b2 (BNT162b2) (n = 79), referred for SARS-CoV-2 antibody measurement prior to vaccination and 21 days after dose 1, and 8, 14, and 30 days after dose 2 were included. The sera were tested with three assays: Elecsys SARS-CoV-2 S (Roche), LIAISON® SARS-CoV-2 TrimericS IgG (DiaSorin), and SARS-CoV-2 IgG II Quant (Abbott). RESULTS: The three assays showed varying correlations at different time points in the study. The overall agreement for all samples was moderate to high (ρ = 0.663-0.902). We observed the most uniform agreement for the day of dose 2 (ρ = 0.775-0.825), while it was least consistent for day 8 (ρ = -0.131-0.693) and 14 (ρ = -0.247-0.603) after dose 2. The dynamics of changes of the SARS-CoV-2 antibody levels in patients without history of prior SARS-CoV-2 infection appears homogenous based on the Roche results, more heterogenous when considering the DiaSorin results, and in between for the Abbott results. CONCLUSIONS: The results highlight the need for further work on the international standard of measurement of SARS-CoV-2 Ig, especially in the era of vaccination. The serological assays can be useful to detect IgG/IgM antibodies to assess the response to the vaccination. However, they cannot be used interchangeably. In terms of the evaluation of the immune response to the BNT162b2 vaccine, Roche and Abbott kits appear to be more useful.

High-Quality Complete Genome Resource of Pathogenic Bacterium Pectobacterium atrosepticum Strain Green1 Isolated from Potato (Solanum tuberosum L.) in Greenland.

Pectobacterium atrosepticum is a narrow-host-range, pectinolytic, plant-pathogenic bacterium causing blackleg of potato (Solanum tuberosum L.) worldwide. Till present, several P. atrosepticum genomes have been sequenced and characterized in detail; however, all of these genomes have come from P. atrosepticum isolates from plants grown in temperate zones, not from hosts cultivated under different climatic conditions. Herewith, we present the first complete, high-quality genome of the P. atrosepticum strain Green1 isolated from potato plants grown under a subarctic climate in Greenland. The genome of P. atrosepticum strain Green1 consists of one chromosome of 4,959,719 bp, with a GC content of 51% and no plasmids. The genome contains 4,531 annotated features, including 4,179 protein-coding genes, 22 ribosomal RNA genes, 70 transfer RNA genes, 8 noncoding RNA genes, 2 CRISPRs, and 126 pseudogenes. We believe that the information in this first high-quality, complete, closed genome of P. atrosepticum strains isolated from host plants grown in a subarctic agricultural region will provide resources for comparative genomic studies and for analyses targeting climatic adaptation and ecological fitness mechanisms present in P. atrosepticum.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

High-Quality Complete Genome Resource of Plant-Pathogenic Bacterium Dickeya solani IPO 2019, Isolated from Hyacinthus orientalis.

Dickeya solani is an emerging plant-pathogenic bacterium causing disease symptoms in a variety of agriculturally relevant crop species worldwide. To date, a number of D. solani genomes have been sequenced and characterized; the great majority of these genomes have, however, come from D. solani strains isolated from potato (Solanum tuberosum L.) and not from other plant hosts. Herewith, we present the first complete, high-quality genome of D. solani IPO 2019 (LMG 25990), isolated from the ornamental plant Hyacinthus orientalis. The genome of D. solani IPO 2019 consists of one chromosome of 4,919,542 bp, with a GC content of 56.2% and no plasmids. The genome contains 4,502 annotated features, 22 ribosomal RNA genes, 73 transfer RNA genes, and one CRISPR. We believe that the information on this high-quality, complete, closed genome of D. solani strain isolated from a host plant different from potato (i.e. hyacinth) will provide resources for comparative genomic studies and for analyses targeting adaptation and ecological fitness mechanisms present in Dickeya solani species.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Coding-Complete Genome Sequences of Six Influenza Type A Strains Circulating in Lithuania in the 2009-2010 Epidemic Season.

Here, we report the coding-complete genome sequences of six influenza A(H1N1) strains that were detected in Vilnius, Lithuania, among patients exhibiting influenza-like symptoms during the 2009-2010 epidemic season, within national influenza surveillance. Several mutations were found in genes encoding hemagglutinin and neuraminidase, in comparison with the A/California/07/2009 reference strain (GenBank accession numbers NC_026433 and NC_026434).