Quantifying the Dynamics of Bacterial Biofilm Formation on the Surface of Soft Contact Lens Materials Using Digital Holographic Tomography to Advance Biofilm Research.

The increase in bacterial resistance to antibiotics in recent years demands innovative strategies for the detection and combating of biofilms, which are notoriously resilient. Biofilms, particularly those on contact lenses, can lead to biofilm-related infections (e.g., conjunctivitis and keratitis), posing a significant risk to patients. Non-destructive and non-contact sensing techniques are essential in addressing this threat. Digital holographic tomography emerges as a promising solution. This allows for the 3D reconstruction of the refractive index distribution in biological samples, enabling label-free visualization and the quantitative analysis of biofilms. This tool provides insight into the dynamics of biofilm formation and maturation on the surface of transparent materials. Applying digital holographic tomography for biofilm examination has the potential to advance our ability to combat the antibiotic bacterial resistance crisis. A recent study focused on characterizing biofilm formation and maturation on six soft contact lens materials (three silicone hydrogels, three hydrogels), with a particular emphasis on Staphylococcus epidermis and Pseudomonas aeruginosa, both common culprits in ocular infections. The results revealed species- and time-dependent variations in the refractive indexes and volumes of biofilms, shedding light on cell dynamics, cell death, and contact lens material-related factors. The use of digital holographic tomography enables the quantitative analysis of biofilm dynamics, providing us with a better understanding and characterization of bacterial biofilms.

Molecular detection of avian hepatitis E virus (Orthohepevirus B) in chickens, ducks, geese, and western capercaillies in Poland.

Orthohepevirus B, commonly known as avian hepatitis E virus (aHEV), causes big liver and spleen disease (BLS) or hepatitis-splenomegaly syndrome (HSS) in chickens. BLS is an emerging disease among chicken flocks in several countries around the world. In our previous studies, serology and molecular biology screening revealed that chicken flocks are widely affected by aHEV in Poland. The present study, which was conducted between 2019 and 2020, aimed to investigate the prevalence of aHEV in chicken flocks and other poultry, including ducks, geese, and turkeys. A total of 307 flocks were examined. In addition, 29 samples from captive wild birds (western capercaillies, Tetrao urogallus) were analyzed. In all the investigated poultry species, except turkeys, the nucleic acid sequence covering part of the ORF1 gene of the aHEV genome was detected (34/336 samples, 10.1%). The infection rate was found to be the highest in broiler breeder chicken flocks (14/40 samples; 35%). Phylogenetic analysis of partial ORF1 gene, which encodes helicase, revealed that the obtained sequences belonged to genotypes 2 and 4, while one belonged to genotype 3. Genotype 2 was detected for the first time in domestic geese and ducks, and genotype 4 was detected for the first time in Poland. The study demonstrated the presence of aHEV among the investigated western capercaillies, suggesting that this species is susceptible to aHEV infections and biosecurity is therefore required in western capercaillie breeding facilities.

Expression of the Heterotrimeric GP2/GP3/GP4 Spike of an Arterivirus in Mammalian Cells.

Equine arteritis virus (EAV), an enveloped positive-strand RNA virus, is an important pathogen of horses and the prototype member of the Arteiviridae family. Unlike many other enveloped viruses, which possess homotrimeric spikes, the spike responsible for cellular tropism in Arteriviruses is a heterotrimer composed of 3 glycoproteins: GP2, GP3, and GP4. Together with the hydrophobic protein E they are the minor components of virus particles. We describe the expression of all 3 minor glycoproteins, each equipped with a different tag, from a multi-cassette system in mammalian BHK-21 cells. Coprecipitation studies suggest that a rather small faction of GP2, GP3, and GP4 form dimeric or trimeric complexes. GP2, GP3, and GP4 co-localize with each other and also, albeit weaker, with the E-protein. The co-localization of GP3-HA and GP2-myc was tested with markers for ER, ERGIC, and cis-Golgi. The co-localization of GP3-HA was the same regardless of whether it was expressed alone or as a complex, whereas the transport of GP2-myc to cis-Golgi was higher when this protein was expressed as a complex. The glycosylation pattern was also independent of whether the proteins were expressed alone or together. The recombinant spike might be a tool for basic research but might also be used as a subunit vaccine for horses.

Short Beak and Dwarfism Syndrome in Ducks in Poland Caused by Novel Goose Parvovirus.

Short beak and dwarfism syndrome (SBDS), which was previously identified only in mule ducks, is now an emerging disease of Pekin ducks in China and Egypt. The disease is caused by the infection of ducks with a genetic variant of goose parvovirus-novel goose parvovirus (nGPV). In 2019, SBDS was observed for the first time in Poland in eight farms of Pekin ducks. Birds in the affected flock were found to show growth retardation and beak atrophy with tongue protrusions. Morbidity ranged between 15% and 40% (in one flock), while the mortality rate was 4-6%. Co-infection with duck circovirus, a known immunosuppressive agent, was observed in 85.7% of ducks. The complete coding regions of four isolates were sequenced and submitted to GenBank. The phylogenetic analysis revealed a close relationship of Polish viral sequences with the Chinese nGPV. Genomic sequence alignments showed 98.57-99.28% identity with the nGPV sequences obtained in China, and 96.42% identity with the classical GPV (cGPV; Derzsy's disease). The rate of amino acid mutations in comparison to cGPV and Chinese nGPV was higher in the Rep protein than in the Vp1 protein. To our knowledge, this is the first report of nGPV infection in Pekin ducks in Poland and Europe. It should be emphasized that monitoring and sequencing of waterfowl parvoviruses is important for tracking the viral genetic changes that enable adaptation to new species of waterbirds.

Production of Recombinant EAV with Tagged Structural Protein Gp3 to Study Artervirus Minor Protein Localization in Infected Cells.

Equine arteritis virus (EAV) is a prototype member of the Arterivirus family, comprising important pathogens of domestic animals. Minor glycoproteins of Arteriviruses are responsible for virus entry and cellular tropism. The experimental methods for studying minor Arterivirus proteins are limited because of the lack of antibodies and nested open reading frames (ORFs). In this study, we generated recombinant EAV with separated ORFs 3 and 4, and Gp3 carrying HA-tag (Gp3-HA). The recombinant viruses were stable on passaging and replicated in titers similar to the wild-type EAV. Gp3-HA was incorporated into the virion particles as monomers and as a Gp2/Gp3-HA/Gp4 trimer. Gp3-HA localized in ER and, to a lesser extent, in the Golgi, it also co-localized with the E protein but not with the N protein. The co-localization of Gp3-HA and the E protein with ERGIC was reduced. Moreover, EAV with Gp3-HA could become a valuable research tool for identifying host cell factors during infection and the role of Gp3 in virus attachment and entry.

Avian hepatitis E virus is widespread among chickens in Poland and belongs to genotype 2.

Big liver and spleen disease, caused by avian hepatitis E virus, has been reported in Poland, but the prevalence of the virus has not yet been investigated. In this study, 1034 serum samples from 57 breeder broiler and laying hen flocks were screened for the presence of anti-aHEV antibodies. In a random serology study, 56.1% of flocks were positive. Seroprevalence was higher in laying hen flocks than in broiler breeder flocks. Phylogenetic analysis of partial ORF1 and ORF2 sequences revealed that all Polish isolates belonged to genotype 2. This is the first time this genotype has been detected in Central Europe.

Molecular characterisation of equid alphaherpesvirus 1 strains isolated from aborted fetuses in Poland.

BACKGROUND: Equid alphaherpesvirus 1 (EHV-1) is one of the main infectious causative agents of abortion in mares and can also be associated with stillbirth, neonatal foal death, rhinopneumonitis in young horses and a neurological disorder called equine herpesvirus myeloencephalopathy (EHM). The neuropathogenicity of the virus was shown to be significantly higher in EHV-1 strains that carry a single nucleotide point (SNP) mutation in the ORF30, which encodes a catalytic subunit of viral DNA polymerase (ORF30 D752). Another gene, ORF68 is frequently used for phylogenetic analysis of EHV-1. METHODS: 27 EHV-1 strains isolated from aborted equine fetuses in Poland, collected between 1993 and 2017, were subjected to PCR targeting the open reading frames (ORFs) 30 and 68 of the EHV-1 genome. PCR products obtained were sequenced and SNPs were analyzed and compared to sequences available in GenBank. RESULTS: None of the analyzed sequences belonged to the ORF30 D752neuropathogenic genotype: all EHV-1 belonged to the non-neuropathogenic variant N752. On the basis of ORF68 sequences, the majority of EHV-1 sequences (76.9%) cannot be assigned to any of the known groups; only six sequences (23.1%) clustered within groups II and IV. CONCLUSIONS: EHV-1 strains obtained from abortion cases belong to the non-neuropathogenic genotype. Many EHV-1 ORF68 sequences have similar SNPs to those already described in Poland, but a clear geographical distribution was not observed. A single particular ORF68 sequence type was observed in strains isolated from 2001 onwards.

A new duck circovirus sequence, detected in velvet scoter (Melanitta fusca) supports great diversity among this species of virus.

BACKGROUND: The aim of this study was to investigate the presence of circoviruses in wild bird populations, in Poland. Circoviruses possess immuno-suppressive properties and might interfere with the health of wild birds. METHOD: 83 birds, which belonged to 23 species, were tested with broad-range, nested PCR. The obtained PCR products were sequenced and new primers designed, to analyse the full-length, viral genome. A phylogenetic analysis was conducted, to find any relationship to known circoviruses. RESULTS: The circovirus DNA sequence was found in 4 birds. All samples originated from the velvet scoter (Melanitta fusca) a marine duck from the Merginae sub-family. Birds which tested positive for the circovirus were found dead in fishing nets, off the Baltic coast. During post-mortem examination, carcasses of two of the scoters showed only light emaciation, while the two other birds appeared healthy. The obtained, full-length, circovirus sequence revealed 1,988 nucleotides and the presence of typical features (i.e. Cap, Rep and ORF3). Nucleotide similarity to other duck circoviruses was 84 to 86 %. Phylogenetic analysis of the complete genome and cap gene, indicated that the new circovirus is related to known duck circoviruses, especially to sub-types sometimes referred to as duck circovirus genotype 1, but not genotype 2. CONCLUSIONS: In this study, we have reported a new duck circovirus sequence detected in the velvet scoter, a species of marine duck. Sequence comparison and phylogenetic analysis of the new virus sequence support previous reports that duck circovirus (DuCV) is a species with a high degree of diversity. The viral sequence obtained from the velvet scoter suggests that DuCV may infect birds from the Anatinae sub-family. More studies are needed to prove if the velvet scoter and other marine ducks act as a reservoir for DuCV.

Membrane proteins of arterivirus particles: structure, topology, processing and function.

Arteriviruses, such as equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV), are important pathogens in veterinary medicine. Despite their limited genome size, arterivirus particles contain a multitude of membrane proteins, the Gp5/M and the Gp2/3/4 complex, the small and hydrophobic E protein and the ORF5a protein. Their function during virus entry and budding is understood only incompletely. We summarize current knowledge of their primary structure, membrane topology, (co-translational) processing and intracellular targeting to membranes of the exocytic pathway, which are the budding site. We profoundly describe experimental data that led to widely believed conceptions about the function of these proteins and also report new results about processing steps for each glycoprotein. Further, we depict the location and characteristics of epitopes in the membrane proteins since the late appearance of neutralizing antibodies may lead to persistence, a characteristic hallmark of arterivirus infection. Some molecular features of the arteriviral proteins are rare or even unique from a cell biological point of view, particularly the prevention of signal peptide cleavage by co-translational glycosylation, discovered in EAV-Gp3, and the efficient use of overlapping sequons for glycosylation. This article reviews the molecular mechanisms of these cellular processes. Based on this, we present hypotheses on the structure and variability of arteriviral membrane proteins and their role during virus entry and budding.

Signal peptide cleavage from GP3 enabled by removal of adjacent glycosylation sites does not impair replication of equine arteritis virus in cell culture, but the hydrophobic C-terminus is essential.

The disulphide-linked GP2/3/4 spike of equine arteritis virus (EAV) is essential for virus entry. We showed recently that in transfected cells carbohydrates attached adjacent to the signal peptide of GP3 inhibit cleavage. Here we confirm this unique phenomenon in recombinant viruses with disabled glycosylation sites. Surprisingly, the infectivity of EAV containing GP3 with cleaved signal peptide was not impaired and GP3 with cleaved signal peptide associates with GP2/4 in virus particles. In contrast, viruses containing GP3 with deleted hydrophobic C-terminus rapidly reverted back to wild type. The data support our model that the signal peptide is exposed to the lumen of the ER and the C-terminus peripherally attaches GP3 to membranes.

Co-translational processing of glycoprotein 3 from equine arteritis virus: N-glycosylation adjacent to the signal peptide prevents cleavage.

Signal peptide cleavage and N-glycosylation of proteins are co-translational processes, but little is known about their interplay if they compete for adjacent sites. Here we report two unique findings for processing of glycoprotein 3 of equine arteritis virus. Glycoprotein 3 (Gp3) contains an N-terminal signal peptide, which is not removed, although bioinformatics predicts cleavage with high probability. There is an overlapping sequon, NNTT, adjacent to the signal peptide that we show to be glycosylated at both asparagines. Exchanging the overlapping sequon and blocking glycosylation allows signal peptide cleavage, indicating that carbohydrate attachment inhibits processing of a potentially cleavable signal peptide. Bioinformatics analyses suggest that a similar processing scheme may exist for some cellular proteins. Membrane fractionation and secretion experiments revealed that the signal peptide of Gp3 does not act as a membrane anchor, indicating that it is completely translocated into the lumen of the endoplasmic reticulum. Membrane attachment is caused by the hydrophobic C terminus of Gp3, which, however, does not span the membrane but rather attaches the protein peripherally to endoplasmic reticulum membranes.