Non-pathogenic Strains Research Articles

Arabidopsis PUB2 and PUB4 connect signaling components of pattern-triggered immunity.

Plants use pattern recognition receptors (PRRs) to detect pathogen-associated molecular patterns (PAMPs) and activate pattern-triggered immunity (PTI). Precise regulation of information from PRRs to downstream signaling components is vital to mounting an appropriate immune response and requires dynamic interactions of these PTI components. We used transcriptome profiling, phenotypic analysis, molecular genetics, and protein-protein interaction analysis to understand the roles of the Arabidopsis plant U-box (PUB) proteins PUB2 and PUB4 in disease resistance and PTI signaling. Loss of function of both PUB2 and PUB4 diminishes the PAMP-triggered oxidative bursts and dampens mitogen-activated protein kinase signaling, resulting in a severe compromise in resistance to not only pathogenic but also nonpathogenic strains of Pseudomonas syringae. Within PUB4, the E3 ligase activity is dispensable, but the armadillo repeat region is essential and sufficient for its function in immunity. PUB2 and PUB4 interact with PTI signaling components, including FLS2, BIK1, PBL27, and RbohD, and enhance FLS2-BIK1 and BIK1-RbohD interactions. Our study reveals that PUB2 and PUB4 are critical components of plant immunity and connect PTI components to positively regulate defense responses.

Comparative genomics of the black rot pathogen Xanthomonas campestris pv. campestris and non-pathogenic co-inhabitant Xanthomonas melonis from Trinidad reveal unique pathogenicity determinants and secretion system profiles.

Black-rot disease caused by the phytopathogen Xanthomonas campestris pv. campestris (Xcc) continues to have considerable impacts on the productivity of cruciferous crops in Trinidad and Tobago and the wider Caribbean region. While the widespread occurrence of resistance of Xcc against bactericidal agrochemicals can contribute to the high disease burdens, the role of virulence and pathogenicity features of local strains on disease prevalence and severity has not been investigated yet. In the present study, a comparative genomic analysis was performed on 6 pathogenic Xcc and 4 co-isolated non-pathogenic Xanthomonas melonis (Xmel) strains from diseased crucifer plants grown in fields with heavy chemical use in Trinidad. Native isolates were grouped into two known and four newly assigned ribosomal sequence types (rST). Mobile genetic elements were identified which belonged to the IS3, IS5 family, Tn3 transposon, resolvases, and tra T4SS gene clusters. Additionally, exogenous plasmid derived sequences with origins from other bacterial species were characterised. Although several instances of genomic rearrangements were observed, native Xcc and Xmel isolates shared a significant level of structural homology with reference genomes, Xcc ATCC 33913 and Xmel CFBP4644, respectively. Complete T1SS hlyDB, T2SS, T4SS vir and T5SS xadA, yapH and estA gene clusters were identified in both species. Only Xmel strains contained a complete T6SS but no T3SS. Both species contained a complex repertoire of extracellular cell wall degrading enzymes. Native Xcc strains contained 37 T3SS and effector genes but a variable and unique profile of 8 avr, 4 xop and 1 hpa genes. Interestingly, Xmel strains contained several T3SS effectors with low similarity to references including avrXccA1 (~89%), hrpG (~73%), hrpX (~90%) and xopAZ (~87%). Furthermore, only Xmel genomes contained a CRISPR-Cas I-F array, but no lipopolysaccharide wxc gene cluster. Xmel strains were confirmed to be non-pathogenic by pathogenicity assays. The results of this study will be useful to guide future research into virulence mechanisms, agrochemical resistance, pathogenomics and the potential role of the co-isolated non-pathogenic Xanthomonas strains on Xcc infections.

Comparative analysis of mRNA transcripts of HT-29 cell line expressed in identical quantities for pathogenic E. coli strains UM146 and UM147 with control Escherichia coli Nissle 1917.

Aim of study was comparative analysis of mRNA transcripts of HT-29 cell line, expressed in identical quantities for the combination of pathogenic and non-pathogenic Escherichia coli strains. HT-29 confluent monolayers infection with two pathogenic E. coli strains UM146 and UM147 resulted in two sets of mRNA transcripts that were identical with RNA transcripts obtained for non-pathogenic one strain E. coli Nissle 1917. In this study genome-wide experiments were conducted using expression microarray-system. Only one common mRNA transcript coding for CCDC65 gene was equally expressed by HT-29 cells after incubation challenge with three different E. coli strains used. This gene and its bacterial analogue are important in the ciliary or flagellar motility, respectively. Altogether, 78 and 81 HT-29 mRNA transcripts for E. coli UM146 and E. coli UM147 had identical RNA quantity in comparison to the response obtained for non-pathogenic E. coli Nissle 1917 interactions with HT-29 monolayers. Specific analysis using REACTOME and agriGO terms enrichment data-mining tools as well as word-cloud analysis allowed for identification the most important processes characteristic during HT-29 cell line infections for each pathogenic E. coli strain used. The importance of results may contribute to recognition of those processes during bacterial infections that are identical with processes arising from human interaction with non-pathogenic strains that belong to the same bacterial species.

Seawater dilution desalination with hybrid FO-RO and UF-RO: Characterisation and assessment of pathogen removal efficacy

This study investigated the feasibility of embedding two low-energy dilution desalination processes, namely osmotic dilution desalination (ODD; the FO-RO membrane configuration) and mixing dilution desalination (MDD; the UF-RO membrane configuration), in a conventional seawater desalination approach (i.e., seawater reverse osmosis) using real seawater and recycled wastewater. The removal efficacy of bacterial indicators (Clostridium perfringens and Escherichia coli), coliphage genera Microviradae, and Human adenovirus (HAdV) was assessed in influent/effluent samples collected from each membrane configuration. Mixing dilution of the influent feed waters reduced the number of all pathogenic contaminants as well as organic/inorganic compounds. The rejection rate of total suspended solids was 35.7% in FO to 63.6% in UF respectively, whereas that of total organic carbon was 53.8% and 56.9% in FO and UF, respectively. The FO membrane in the ODD process more efficiently removed viruses (5.0 LRV) and bacterial indicators (4.2 LRV) than the UF membrane (3.7 and 3.6 LRV, respectively). Nevertheless, the different methods of detection used in our study, such as culture-based and qPCR, were found to have significant influences on the LRVs. Comparative 16S rRNA sequencing indicated that the vast majority of bacterial indicators in RO permeating from the FO-RO and UF-RO configurations were nonpathogenic strains.

Deletion of the Viral Thymidine Kinase in a Meq-Deleted Recombinant Marek's Disease Virus Reduces Lymphoid Atrophy but Is Less Protective.

Marek’s disease (MD) is a ubiquitous disease of domesticated chickens and its etiologic agent is the Gallid alphaherpesvirus 2 (GaHV-2), also known as Marek’s disease virus (MDV). MD is currently controlled by vaccination using live attenuated strains of MDV (e.g., CVI988/Rispens), non-pathogenic serotypes of MDV (GaHV-3), or non-pathogenic strains of the related Melagrid alphaherpesvirus 1 (MeHV-1). One attractive strategy for the production of new vaccine strains is a recombinant MDV attenuated by the deletion of the major viral oncogene meq. However, meq-deleted variants of MDV cause atrophy of the bursa and thymus in maternal antibody-negative chickens, and the resulting immunosuppression makes them unsuitable. Herein we detail our attempt to mitigate the lymphoid atrophy caused by meq-deleted MDV by further attenuation of the virus through ablation of the viral thymidine kinase (tk) gene. We demonstrate that ablation of the viral tk from the meq-deleted virus rMd5B40/Δmeq resulted in a virus attenuated for replication in vitro and which spared chickens from atrophy of the lymphoid organs in vivo. When the rMd5B40/Δmeq/Δtk/GFP was used as a vaccine it was protective against challenge with the vv+MDV strain 686, but the protection was less than that provided by the CVI988/Rispens vaccine.

Antibiotic resistance genes and antibiotic sensitivity in bacterial aerosols and their comparisons with known respiratory pathogens

Airborne transmission of antibiotic resistance genes (ARGs) has emerged as a global health threat, while their hosts’ resistance phenotype remains largely unknown. Here, culturable bacterial aerosol samples were collected from polluted air in Beijing using a portable high volume sampler (1000 L/min). Antimicrobial susceptibility testing was conducted to explore the antibiotic sensitivity of culturable bacterial aerosols to seven widely used antibiotics. As a comparison, ARG distribution and expression in two human respiratory pathogenic bacterial strains (Pseudomonas aeruginosa and Methicillin-resistant Staphylococcus aureus) and a non-pathogenic bacterial strain (Pseudomonas fluorescens) were also studied. Relative abundances of 39 ARG subtypes in three bacterial strains including Haemophilus influenzae were investigated by high throughput real-time qPCR platform. Surprisingly, 24.66% of the culturable bacterial aerosols in polluted air were resistant to vancomycin-the most powerful antibiotics to date, while 53.05% of airborne culturable bacteria were susceptible to ampicillin (β-lactams). Different bacteria demonstrated very different ARG distribution patterns of more or less skewed toward certain one ARG type. On the other hand, the vancomycin resistance gene vanB was detected both in H. influenza and P. fluorescens, however these bacteria did not exhibit anti-vancomycin phenotype. Furthermore, the resistance to ciprofloxacin (quinolones) was not observed in P. aeruginosa and P. fluorescens although these bacteria harbor abundant quinolone resistance gene qepA. Experimental data from this work show that air represents an important active reservoir of diverse ARGs, and some bacterial aerosols are harboring vancomycin resistance gene though not expressed. Results from human respiratory pathogens have further demonstrated similar findings. This work underscores the serious concern of airborne dissemination of ARBs including those resistant to vancomycin in polluted ambient environments, while revealing abundances and patterns of various ARGs in both pathogenic and non-pathogenic bacteria.

Investigating the role of the carbon storage regulator A (CsrA) in Leptospira spp.

Carbon Storage Regulator A (CsrA) is a well-characterized post-transcriptional global regulator that plays a critical role in response to environmental changes in many bacteria. CsrA has been reported to regulate several metabolic pathways, motility, biofilm formation, and virulence-associated genes. The role of csrA in Leptospira spp., which are able to survive in different environmental niches and infect a wide variety of reservoir hosts, has not been characterized. To investigate the role of csrA as a gene regulator in Leptospira, we generated a L. biflexa csrA deletion mutant (ΔcsrA) and csrA overexpressing Leptospira strains. The ΔcsrA L. biflexa displayed poor growth under starvation conditions. RNA sequencing revealed that in rich medium only a few genes, including the gene encoding the flagellar filament protein FlaB3, were differentially expressed in the ΔcsrA mutant. In contrast, 575 transcripts were differentially expressed when csrA was overexpressed in L. biflexa. Electrophoretic mobility shift assay (EMSA) confirmed the RNA-seq data in the ΔcsrA mutant, showing direct binding of recombinant CsrA to flaB3 mRNA. In the pathogen L. interrogans, we were not able to generate a csrA mutant. We therefore decided to overexpress csrA in L. interrogans. In contrast to the overexpressing strain of L. biflexa, the overexpressing L. interrogans strain had poor motility on soft agar. The overexpressing strain of L. interrogans also showed significant upregulation of the flagellin flaB1, flaB2, and flaB4. The interaction of L. interrogans rCsrA and flaB4 was confirmed by EMSA. Our results demonstrated that CsrA may function as a global regulator in Leptospira spp. under certain conditions that cause csrA overexpression. Interestingly, the mechanisms of action and gene targets of CsrA may be different between non-pathogenic and pathogenic Leptospira strains.

Immunity analysis against Fowl Adenovirus serotype 4 (FAdV-4) based on Fiber-2 trimer Protein with the different virulence

Since June 2015, Fowl adenovirus outbreaks have occurred in China, causing significant economic losses to poultry industry. The FAdV-4 Fiber-2 proteins could induce effective protection, but the precise mechanism of immune protection remains unknown. Here, we have compared the biological characteristics of Fiber-2 protein of the very virulent WZ strain of FAdV-4 (vvFAdV-4) with that of non-virulent ON1 strain. The sequence analysis revealed natural deletions and sequence differences between the classical non-pathogenic strain ON1 and the vvFAdV-4 isolate. These two Fiber-2 proteins successfully expressed in E. coli resemble in structure and function to the native-like trimeric protein. The trimeric structure and bioreactivity of the recombinant Fiber-2 proteins to FAdV-4 specific antibodies were characterized. The immune protection induced by Fiber-2 proteins of FAdV-4 WZ and ON1 strains were compared in SPF chickens. All birds in the WZ-Fiber-2 immunized group generated systemic specific antibodies compared with both ON1-Fiber-2 protein and PBS immunized groups. According to the results of attack mortalities, viral shedding and tissue gross lesion, the WZ Fiber-2 protein induced complete protection at a dose of 2 μg per chicken, whereas the ON1-Fiber-2 protein induced 0 protection at 3 dpc. In view of the characteristics of Fiber-2 proteins of different strains, this study can help us to further understand the mechanism of protective immunity and provide a basis for the prevention and control of FAdV-4 in chickens.

Reannotation of Mycoplasma hyopneumoniae hypothetical proteins revealed novel potential virulence factors

Mycoplasma hyopneumoniae is a bacterium that inhabits the swine respiratory tract, causing porcine enzootic pneumonia, which generates significant economic losses to the swine industry worldwide. The knowledge on M. hyopneumoniae biology and virulence have been significantly increased by genomics studies. However, around 30% of the predicted proteins remained of unknown function so far. According to the original annotation, the genome of M. hyopneumoniae 7448, a Brazilian pathogenic strain, had 693 coding DNA sequences, 244 of which were annotated as coding for hypothetical or uncharacterized proteins. Among them, there may be still several genes coding for unknown virulence factors. Therefore, this study aimed to functionally reannotate the whole set of 244 M. hyopneumoniae 7448 proteins of unknown function based on currently available database and bioinformatic tools, in order to predict novel potential virulence factors. Predictions of physicochemical properties, subcellular localization, function, overall association to virulence and antigenicity are provided. With that, 159 out of the set of 244 proteins of unknown function had a putative function associated to them, allowing identification of novel enzymes, membrane transporters, lipoproteins, DNA-binding proteins and adhesins. Furthermore, 139 proteins were generally associated to virulence, 14 of which had a function assigned and were differentially expressed between pathogenic and non-pathogenic strains of M. hyopneumoniae. Moreover, all extracellular or cytoplasmic membrane predicted proteins had putative epitopes identified. Overall, these analyses improved the functional annotation of M. hyopneumoniae 7448 genome from 65% to 87% and allowed the identification of new potential virulence factors.

Susceptibility of Pathogenic Nontyphoidal Salmonella Serovars and Avirulent Salmonella LT2 to Elevated Hydrostatic Pressure and CitricidalTM

Abstract Nontyphoidal Salmonella enterica serovars are the leading cause of foodborne hospitalization and death episodes in the United States. In a typical year, it is estimated that >1 million Americans experience illness due to foodborne nontyphoidal Salmonellosis. While the private industry and manufacturing rely heavily on results of validations studies for inactivation and decontamination of this prevalent and opportunistic pathogen of public health concern, availability of such studies conducted in realistic environments of manufacturing and processing is limited due to pathogenic nature of the organism. The current study investigated effects of elevated hydrostatic pressure and a bioactive product from grapefruit seed extract (CitricidalTM) for elimination of nontyphoidal Salmonella serovars. Additionally, sensitivity of an avirulent strain of the bacterium (Salmonella LT2) investigated relative to the pathogenic Salmonella serovars. Our results show that the avirulent strain utilized in the current study, has similar sensitivity to the treatments compared to nontyphoidal pathogenic Salmonella serovars. Thus, this non-pathogenic strain could be used interchangeably for public health microbiology hurdle validation studies as a surrogate for pathogenic Salmonella serovars. Additionally, we observed >5 log reduction of the pathogen (i.e. > 99.999%) due to treatments with elevated hydrostatic pressure at 500 MPa and at 300 MPa with presence of CitricidalTM. Our result additionally exhibits that combination of mild hydrostatic pressure and bioactive compounds such as CitricidalTM could be utilized to ensure safe and efficacious elimination of this prevalent pathogen of public health concern from common commodities.

Development of a Novel Avian Vaccine Vector Derived From the Emerging Fowl Adenovirus 4.

Severe hepatitis-hydropericardium syndrome (HHS) associated with a novel viral genotype, fowl adenovirus 4 (FAdV-4), has emerged and widely spread in China since 2015, causing severe economic losses to the poultry industry. We previously reported that the hexon gene is responsible for pathogenicity and obtained a non-pathogenic hexon-replacement rHN20 strain; however, the lack of information about the non-essential regions for virus replication limits the development of a FAdV-4 vector. This study first established an enhanced green fluorescent protein (EGFP)-indicator virus based on the FAdV-4 reverse genetic technique, effective for batch operations in the virus genome. Based on this, 10 open reading frames (ORFs) at the left end and 13 ORFs at the right end of the novel FAdV-4 genome were deleted separately and identified as non-essential genes for viral replication, providing preliminary insertion sites for foreign genes. To further improve its feasibility as a vaccine vector, seven combinations of ORFs were successfully replaced with EGFP without affecting the immunogenicity of the vector backbone. Finally, a recombinant rHN20-vvIBDV-VP2 strain, expressing the VP2 protein of very virulent infectious bursa disease virus (vvIBDV), was rescued and showed complete protection against FAdV-4 and vvIBDV. Thus, the novel FAdV-4 vector could provide sufficient protection for HHS and efficient exogenous gene delivery. Overall, our findings systemically identified 23 non-essential ORFs for FAdV-4 replication and seven foreign gene insertion regions, providing valuable information for an in-depth understanding of the novel FAdV-4 pathogenesis and development of multivalent vaccines.

Galleria mellonella infection model to evaluate pathogenic and nonpathogenic Leptospira strains

The Galleria mellonella larvae infection model is emerging as a valuable tool for studying various characteristics of infectious agents and host-pathogen interaction. This system has been widely recognized as a high throughput, ethical, and cost-effective invertebrate infection model to study the virulence and pathogenesis of various bacterial pathogens. In this study, we compared the effect of Leptospira infection in G. mellonella larvae infected with Leptospira interrogans serovar Copenhageni (pathogenic) or Leptospira biflexa serovar Patoc (saprophytic) strains. We observed significant pathologic changes such as decreased activity, complete melanization, and lower survival rate in the G. mellonella larvae infected with a pathogenic strain L. interrogans serovar Copenhageni compared to those infected with a nonpathogenic strain L. biflexa serovar Patoc. Our study demonstrates the feasibility and the potential of using G. mellonella larvae as an alternative model to study virulence mechanisms and pathogenesis of Leptospira strains. Once optimized, the G. mellonella infection model can be a potential substitute for hamsters to explore various host and pathogen-related mechanistic events in Leptospira infection.

Machine learning approaches to predict the Plant-associated phenotype of Xanthomonas strains

BackgroundThe genus Xanthomonas has long been considered to consist predominantly of plant pathogens, but over the last decade there has been an increasing number of reports on non-pathogenic and endophytic members. As Xanthomonas species are prevalent pathogens on a wide variety of important crops around the world, there is a need to distinguish between these plant-associated phenotypes. To date a large number of Xanthomonas genomes have been sequenced, which enables the application of machine learning (ML) approaches on the genome content to predict this phenotype. Until now such approaches to the pathogenomics of Xanthomonas strains have been hampered by the fragmentation of information regarding pathogenicity of individual strains over many studies. Unification of this information into a single resource was therefore considered to be an essential step.ResultsMining of 39 papers considering both plant-associated phenotypes, allowed for a phenotypic classification of 578 Xanthomonas strains. For 65 plant-pathogenic and 53 non-pathogenic strains the corresponding genomes were available and de novo annotated for the presence of Pfam protein domains used as features to train and compare three ML classification algorithms; CART, Lasso and Random Forest.ConclusionThe literature resource in combination with recursive feature extraction used in the ML classification algorithms provided further insights into the virulence enabling factors, but also highlighted domains linked to traits not present in pathogenic strains.

An inactivated vaccine based on artificial non-pathogenic fowl adenovirus 4 protects chickens against hepatitis-hydropericardium syndrome

Hepatitis-hydropericardium syndrome (HHS) in birds is mainly caused by virulent fowl adenovirus 4 (FAdV-4). A novel genotype, hypervirulent FAdV-4, emerged in 2015 with a high mortality rate ranging from 30 % to 100 % in chickens. Vaccination is an economically feasible method to control HHS. Although there have been various reports of inactivated vaccines from virulent wild-type FAdV-4 against HHS, biosafety threats of inactivated vaccines from potential pathogenic components have been presented to the poultry industry, and safer vaccines are urgently needed. A non-pathogenic recombinant FAdV-4 strain, designated as rHN20, was generated based on the hypervirulent strain in our previous study. Here, we developed a novel inactivated oil-adjuvanted vaccine derived from rHN20 strain and evaluated its immunogenicity in specific-pathogen-free chickens. Chickens subcutaneously or intramuscularly immunized with the inactivated vaccine produced high titers of neutralizing antibodies and were protected from a lethal dose of virulent wild-type FAdV-4 challenge. Collectively, an inactivated vaccine was developed, which was capable of providing full protection for chickens against HHS, and significantly reduced the potential biosafety threats.

Inhibitory effect of marine microalgae used in shrimp hatcheries on Vibrio parahaemolyticus responsible for acute hepatopancreatic necrosis disease

Marine microalgae are potential producers of antimicrobial compounds. Monospecific, vibrio-free co-cultures of marine microalgae, Chaetoceros calcitrans, Tetraselmis suecica, Nannochloropsis sp. and Thalassiosira weissflogii, at the exponential phase of growth were inoculated with two Vibrio parahaemolyticus (Vp) strains. Vp M0904, a highly virulent strain responsible for acute hepatopancreatic necrosis disease (AHPND), and Vp M0702, a non-pathogenic strain, both at 1 × 105 CFU ml−1. Microalgae and bacterial growth, total lipids and carbohydrates were obtained at 1, 2 and 3 days post inoculation with bacteria. Ethanolic (EE) and seawater extracts (SE) of C. calcitrans were also obtained to evaluate the inhibitory activity against Vp M0904. Microalgae cellular growth was not affected by any tested bacteria throughout the experiment; in contrast, Vp M0904 was significantly (p < 0.05) inhibited in co-culture with C. calcitrans and T. suecica. Meanwhile, Vp M0702 was significantly (p < 0.05) inhibited by all microalgae except for Nannochloropsis sp. Minor, non-significant variations were found in the lipids and carbohydrate metabolism of all microalgae inoculated with both strains. Low CFU ml−1 of Vp M0904 was registered when it was incubated with SE of C. calcitrans at concentrations over 75 µg ml−1, however, EE did not show any antibacterial activity. The bacteriostatic effect was dependent of microalgae type and Vibrio strain. Hydrophilic compounds of C. calcitrans cells possess antibiotic activities on the highly virulent Vp M0904 responsible for AHPND, a devastating disease for farmed shrimp around the world.

Complete Genome Sequence Data of Four Burkholderia glumae Strains Isolated from Rice Fields in the United States.

Bacterial panicle blight caused by Burkholderia glumae is a major disease in rice production worldwide. Currently, only a few whole-genome sequences of B. glumae strains isolated in the United States are available. Here, we report the complete genome sequence of four B. glumae strains, including three virulent strains (336gr-1, 411gr-6, and 957856-41-c) and the nonpathogenic strain B. glumae 257sh-1, which were isolated from rice fields in Louisiana (336gr-1, 957856-41-c, and 257sh-1) and Arkansas (411gr-6). The whole-genome sequence data of B. glumae strains will contribute to investigations of the molecular mechanism underlying bacterial pathogenicity and virulence to rice plants.[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.

Immunogenicity of Novel Live Vaccine Based on an Artificial rHN20 Strain against Emerging Fowl Adenovirus 4.

In recent years, hepatitis-hydropericardium syndrome (HHS), caused by novel fowl adenovirus 4 (FAdV-4), has caused serious economic losses to the poultry industry. Vaccines are important for preventing and controlling HHS. Current FAdV-4 vaccine research and development are mainly focuses on inactivated vaccines and relatively fewer live vaccines. We previously demonstrated that the hexon gene is the key gene responsible for the high pathogenicity of FAdV-4 and constructed a non-pathogenic chimeric virus rHN20 strain based on the emerging FAdV-4. In this study, the immunogenicity of artificially rescued rHN20 was evaluated in chickens using different routes and doses as a live vaccine. The live rHN20 vaccine induced high titers of neutralizing antibodies against FAdV-4 and fully protected the immunized chickens against a lethal dose of FAdV-4. Furthermore, immunized chickens showed no clinical symptoms or histopathological changes in the FAdV-4-targeted liver, and the viral load in the tissues of immunized chickens was significantly lower than that of chickens in the challenge control group. Collectively, the live rHN20 vaccine effectively protected our sample against FAdV-4 infection and can be considered a live vaccine candidate for preventing HHS in the poultry industry.

Implication of the nuclear trafficking of rabies virus P3 protein in viral pathogenicity.

Although the majority of viruses of the family Mononegvirales replicate exclusively in the host cell cytoplasm, many of these viruses encode proteins that traffic between the nucleus and cytoplasm, which is believed to enable accessory functions in modulating the biology of the infected host cell. Among these, the P3 protein of rabies virus localizes to the nucleus through the activity of several specific nuclear localization and nuclear export signals. The major defined functions of P3 are in evasion of interferon (IFN)-mediated antiviral responses, including through inhibition of DNA-binding by IFN-activated STAT1. P3 also localizes to nucleoli and promyelocytic leukemia (PML) nuclear bodies, and interacts with nucleolin and PML protein, indicative of several intranuclear roles. The relationship of P3 nuclear localization with pathogenicity, however, is unresolved. We report that nucleocytoplasmic localization of P3 proteins from a pathogenic RABV strain, Nishigahara (Ni) and a non-pathogenic Ni-derived strain, Ni-CE, differs significantly, with nuclear accumulation defective for Ni-CE-P3. Molecular mapping indicates that altered localization derives from a coordinated effect, including two residue substitutions that independently disable nuclear localization and augment nuclear export signals, collectively promoting nuclear exclusion. Intriguingly, this appears to relate to effects on protein conformation or regulatory mechanisms, rather than direct modification of defined trafficking signal sequences. These data provide new insights into the role of regulated nuclear trafficking of a viral protein in the pathogenicity of a virus that replicates in the cytoplasm.

An Insight Into Detection Pathways/Biosensors of Highly Infectious Coronaviruses.

The outbreak of COVID-19 pandemic and its consequences have inflicted a substantial damage on the world. In this study, it was attempted to review the recent coronaviruses appeared among the human being and their epidemic/pandemic spread throughout the world. Currently, there is an inevitable need for the establishment of a quick and easily available biosensor for tracing COVID-19 in all countries. It has been known that the incubation time of COVID-19 lasts about 14 days and 25% of the infected individuals are asymptomatic. To improve the ability to determine SARS-CoV-2 precisely and reduce the risk of eliciting false-negative results produced by mutating nature of coronaviruses, many researchers have established a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) assay using mismatch-tolerant molecular beacons as multiplex real-time RT-PCR to distinguish between pathogenic and non-pathogenic strains of coronaviruses. The possible mechanisms and pathways for the detection of coronaviruses by biosensors have been reviewed in this study.

LEGAL REGULATION OF MOST DANGEROUS MICROORGANISMS STRAINS HANDLING

The statutory basis of Ukraine on the treatment of strains of most dangerous microorganisms has been analyzed, and the terminology important for further understanding of the issue has been clarified. The aim of the paper is to study the state of legal regulation in Ukraine of relations in the field of treatment of strains of especially dangerous microorganisms, to pro- vide analysis of the current legislation of Ukraine in this area, as well as the practice of its application. Theoretical bases of legal support of legitimation of activity in this sphere, including that for the purpose of protection of biological safety have been established. The necessity of legal regulation of participants of relations concerning treatment of strains of especially dangerous microorganisms activity has been substantiated. The current state of legal regulation of relations in the field of strain management requires, given the intersectoral nature of the isssue, consideration of the creation of a single intersectoral authority for the supervision of hazardous biological objects. Such a authority may not only be a licensing authority, but also responsible for the introduction of a register of owners of hazardous biological objects that are not economic entities (for example, individuals – owners of collections of such biological objects).