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.

Evaluation of the Presence of ASFV in Wolf Feces Collected from Areas in Poland with ASFV Persistence.

African swine fever (ASF), caused by a DNA virus (ASFV) belonging to genus Asfivirus of the Asfarviridae family, is one of the most threatening diseases of suids. During last few years, it has spread among populations of wild boars and pigs in countries of Eastern and Central Europe, causing huge economical losses. While local ASF occurrence is positively correlated with wild boar density, ecology of this species (social structure, movement behavior) constrains long-range disease transmission. Thus, it has been speculated that carnivores known for high daily movement and long-range dispersal ability, such as the wolf (Canis lupus), may be indirect ASFV vectors. To test this, we analyzed 62 wolf fecal samples for the presence of ASFV DNA, collected mostly in parts of Poland declared as ASF zones. This dataset included 20 samples confirmed to contain wild boar remains, 13 of which were collected near places where GPS-collared wolves fed on dead wild boars. All analyzed fecal samples were ASFV-negative. On the other hand, eight out of nine wild boar carcasses that were fed on by telemetrically studied wolves were positive. Thus, our results suggest that when wolves consume meat of ASFV-positive wild boars, the virus does not survive the passage through intestinal tract. Additionally, wolves may limit ASFV transmission by removing infectious carrion. We speculate that in areas where telemetric studies on large carnivores are performed, data from GPS collars could be used to enhance efficiency of carcass search, which is one of the main preventive measures to constrain ASF spread.

Bacteriophage-encoded enzymes destroying bacterial cell membranes and walls, and their potential use as antimicrobial agents.

Appearance of pathogenic bacteria resistant to most, if not all, known antibiotics is currently one of the most significant medical problems. Therefore, development of novel antibacterial therapies is crucial for efficient treatment of bacterial infections in the near future. One possible option is to employ enzymes, encoded by bacteriophages, which cause destruction of bacterial cell membranes and walls. Bacteriophages use such enzymes to destroy bacterial host cells at the final stage of their lytic development, in order to ensure effective liberation of progeny virions. Nevertheless, to use such bacteriophage-encoded proteins in medicine and/or biotechnology, it is crucial to understand details of their biological functions and biochemical properties. Therefore, in this review article, we will present and discuss our current knowledge on the processes of bacteriophage-mediated bacterial cell lysis, with special emphasis on enzymes involved in them. Regulation of timing of the lysis is also discussed. Finally, possibilities of the practical use of these enzymes as antibacterial agents will be underlined and perspectives of this aspect will be presented.

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).

Analysis of Coinfections with A/H1N1 Strain Variants among Pigs in Poland by Multitemperature Single-Strand Conformational Polymorphism.

Monitoring and control of infections are key parts of surveillance systems and epidemiological risk prevention. In the case of influenza A viruses (IAVs), which show high variability, a wide range of hosts, and a potential of reassortment between different strains, it is essential to study not only people, but also animals living in the immediate surroundings. If understated, the animals might become a source of newly formed infectious strains with a pandemic potential. Special attention should be focused on pigs, because of the receptors specific for virus strains originating from different species, localized in their respiratory tract. Pigs are prone to mixed infections and may constitute a reservoir of potentially dangerous IAV strains resulting from genetic reassortment. It has been reported that a quadruple reassortant, A(H1N1)pdm09, can be easily transmitted from humans to pigs and serve as a donor of genetic segments for new strains capable of infecting humans. Therefore, it is highly desirable to develop a simple, cost-effective, and rapid method for evaluation of IAV genetic variability. We describe a method based on multitemperature single-strand conformational polymorphism (MSSCP), using a fragment of the hemagglutinin (HA) gene, for detection of coinfections and differentiation of genetic variants of the virus, difficult to identify by conventional diagnostic.

Expression of avian influenza haemagglutinin (H5) and chicken interleukin 2 (chIL-2) under control of the ptcB promoter in Lactococcus lactis.

Gram-positive and nonpathogenic lactic acid bacteria (LAB) are considered to be promising candidates for the development of new, safe systems of heterologous protein expression. Recombinant LAB has been shown to induce specific local and systemic immune response against selected pathogens, and could be a good alternative to classical attenuated carriers. The main goal of our study was to express the avian influenza haemagglutinin (H5) and chicken interleukin 2 (chIL-2) in Lactococcus lactis. Results of this study were anticipated to lead to construction of lactococcal strain(s) with potential vaccine properties against the avian influenza A (H5N1) virus. Expression of the cloned H5 gene, its His-tagged variant and chIL-2 gene, under the control of the ptcB gene promoter was attested by RT-PCR on transcriptional level and Western or dot blot analysis on translational level, demonstrating that system can be an attractive solution for production of heterologous proteins. The results of the preliminary animal trial conducted in mice are a promising step toward development of a vaccine against avian bird flu using Lactococcus lactis cells as antigen carriers.

Native nucleic acid electrophoresis as an efficient alternative for genotyping method of influenza virus.

Influenza viruses are the worldwide major causative agents of human and animal acute respiratory infections. Some of the influenza subtypes have caused epidemics and pandemics among humans. The varieties of methods are available for the rapid isolation and identification of influenza viruses in clinical and environmental samples. Since nucleic acids amplification techniques such as RT-PCR have been adapted, fast and sensitive influenza type and subtype determination is possible. However, in some ambiguous cases other, more detailed assay might be desired. The genetic material of influenza virus is highly unstable and constantly mutates. It is known that single nucleotide polymorphisms (SNPs) results in resistance to commercially available anti-viral drugs. The genetic drift of the virus could also result in weakening of immune response to infection. Finally, in a substantial number of patients co-infection with various virus strains or types has been confirmed. Although the detection of co-infection or presence of minor genetic variants within flu-infected patients is not a routine procedure, a rapid and wide spectrum diagnostics of influenza virus infections could reveal an accurate picture of the disease and more importantly, is crucial for choosing the appropriate therapeutics and virus monitoring. Herein we present the evidences that native gel electrophoresis and MSSCP--a method based on multitemperature single strand conformation polymorphism could furnish a useful technique for minor variants, which escape discovery by conventional diagnostic assays.

Genetic diversity of hemagglutinin gene of A(H1N1)pdm09 influenza strains isolated in Taiwan and its potential impact on HA-neutralizing epitope interaction.

Pandemic influenza A(H1N1)pdm09 virus is a global health threat and between 2009-2011 it became the predominant influenza virus subtype circulating in the world. The research describes the MSSCP (Multitemperature Single Strand Conformation Polymorphism) analysis of the hemagglutinin (HA) region encompassing major neutralizing epitope in pandemic influenza isolates from Taiwan. Several genetically distinct changes appeared in isolates obtained in 2010 and 2011. The majority of changes in HA protein did not result in significant modifications, however three modifications were localized in epitope E of H1 and one was part of the interface binding antibodies BH151 and HC45 possibly making the current vaccine less effective.-Taking into account the possibility of the emergence of influenza A with antibody evading potential, the MSSCP method provides an alternative approach for detection of minor variants which escape detection by conventional Sanger sequencing.

Genes from the exo-xis region of λ and Shiga toxin-converting bacteriophages influence lysogenization and prophage induction.

The exo-xis region, present in genomes of lambdoid bacteriophages, contains highly conserved genes of largely unknown functions. In this report, using bacteriophage λ and Shiga toxin-converting bacteriophage ϕ24Β, we demonstrate that the presence of this region on a multicopy plasmid results in impaired lysogenization of Escherichia coli and delayed, while more effective, induction of prophages following stimulation by various agents (mitomycin C, hydrogen peroxide, UV irradiation). Spontaneous induction of λ and ϕ24Β prophages was also more efficient in bacteria carrying additional copies of the corresponding exo-xis region on plasmids. No significant effects of an increased copy number of genes located between exo and xis on both efficiency of adsorption on the host cells and lytic development inside the host cell of these bacteriophages were found. We conclude that genes from the exo-xis region of lambdoid bacteriophages participate in the regulation of lysogenization and prophage maintenance.

Rapid differentiation of mixed influenza A/H1N1 virus infections with seasonal and pandemic variants by multitemperature single-stranded conformational polymorphism analysis.

Mixed infections of a single host with different variants of influenza A virus are the main source of reassortants which may have unpredictable properties when they establish themselves in the human population. In this report we describe a method for rapid detection of mixed influenza virus infections with the seasonal A/H1N1 human strain and the pandemic A/H1N1/v strain which emerged in 2009 in Mexico and the United States. The influenza virus A/H1N1 variants were characterized by the multitemperature single-stranded conformational polymorphism (MSSCP) method. The MSSCP gel patterns of hemagglutinin gene fragments of pandemic A/H1N1/v and different seasonal A/H1N1 strains were easily distinguishable 2 h after completion of reverse transcription-PCR (RT-PCR). Using the MSSCP-based genotyping approach, coinfections with seasonal and pandemic variants of the A/H1N1 subtype were identified in 4 out of 23 primary samples obtained from patients that presented with influenza-like symptoms to hospitals across Poland during the 2009-2010 epidemic season. Pandemic influenza virus strain presence was confirmed in all these primary samples by real-time RT-PCR. The sensitivity level of the MSSCP-based minor genetic variant detection was 0.1%, as determined on a mixture of DNA fragments obtained from amplification of the hemagglutinin gene of seasonal and pandemic strains. The high sensitivity of the method suggests its applicability for characterization of new viral variants long before they become dominant.