Állati rotavírusok összehasonlító genomvizsgálata = Comparative genomics of animal rotaviruses

A PD76364 sz. OTKA pályázatunk célja a különböző gazdafajok rotavírus fertőzéseinek epidemiológiájával kapcsolatos adatgyűjtés és az azonosított törzsek genomjának meghatározása volt. A támogatási időszak alatt 2000-nél több állati bélsármintát szűrtünk meg rotavírusra, közel 90 rotavírus törzs részleges vagy teljes genomját határoztuk meg és elemeztük a kapott adatokat az egyes törzsek rokonsági kapcsolatainak megértése érdekében. Tizenegy olyan gazdafajnál igazoltunk rotavírus fertőzést, amelynél korábban senki sem és leírtunk 17 új genotípust. Módszertani fejlesztésként hozzákezdtünk egy, a rotavírus genomok meghatározásában használható költséghatékony újgenerációs szekvenálási módszer adaptálásához. | The objectives of our OTKA research grant (PD76364) have been the collection of epidemiologic information about rotavirus infections in various host species and the determination of genome sequence of selected strains. During the research period over 2000 fecal specimens collected from a variety of animal species were screened for rotaviruses, and the partial or full-length genomes were determined and analyzed for nearly 90 strains to better understand the genetic relationship among different animal and human strains. Also, 11 new host species and 17 novel genotypes of rotaviruses were described. Moreover, the adaptation of a next generation sequencing method was started to make sequencing of full-length rotavirus genomes more cost effective

Assessing the role of Pasteurella multocida putative adhesion factors in fowl cholera

Fowl cholera caused by Pasteurella multocida is a contagious disease that affects most of the avian species and leads to significant economic losses in domestic poultry breeding worldwide. The course of infection can be either acute or chronic. In acute infections, the clinical signs are often restricted only to the observation of sudden death. In chronic infections, the spectrum of clinical signs is rather wide, and it may affect the limb joints, the comb, the oviduct, the respiratory tract, the wattles, and the subcutaneous tissue of the head. Diverse virulence traits could explain the different clinical outcomes of the infection. All bacterial infection begins with the process of colonization. Thus, adhesion to mucosal epithelial cells is an important virulence attribute of bacterial pathogens. This ability depends on the expression of adhesive molecules or structures, called adhesins. The aim of our study was to detect and characterise the putative adhesins of P. multocida, like fimA (fimbriae), hsf-1, -2 (autotransporter adhesins), pfhA (filamentous haemagglutinin), tadD (nonspecific tight adherence secretion system, secretin protein), and ptfA (subunit of type 4 fimbriae). Fifty-two field strains of P. multocida were involved in the study isolated from different avian hosts representing both forms of infection and, and type strains of somatic serotype 1, 3, and 4 were used for reference. The fimA and hsf-2 were present in all strains, but the occurrence of other genes (hsf-1, pfhA, and tadD) showed great variety. Eight combinations of virulence genes could be detected within the tested population. Notably, the tadD gene was observed hardly in any other strains than A:1 serotype which were mostly responsible for acute from of the disease and could be characterized with A allele type of ptfA as well. This result revealed that the tad locus-bearing widespread colonization island was not frequent among the avian P. multocida strains as it predicted from the published genome sequence data (originating mainly from mammals)

Gyermekkori gastroenteritisekben szerepet játszó vírusok molekuláris epidemiológiai vizsgálata = Molecular epidemiology of viruses in childhood gastroenteritis

A "Gyermekkori gastroenteritisekben szerepet játszó vírusok molekuláris epidemiológiai vizsgálata" című OTKA pályázatunkkal az volt a célunk, hogy azonosítsuk és jellemezzük a hasmenéses megbetegedések kialakításáért felelős vírusokat. Vizsgálatainkban molekuláris módszereket (vírus RNS eletroforézis, PCR és reverz-transzkripció-PCR) alkalmaztunk. Kutatásaink lefedték a már jól ismert és a gyermekkori hasmenéses kórképekben fontos szerepet játszó vírusokat (A csoportú rotavírusok, enterális adenovírusok, norovírusok) éppúgy, mint a ritka enterális vírusokat (C csoportú rotavírusok) és számos régről ismert illetve újonnan felfedezett vírust is (pl. picobirnavírus, HAdV-52, nodavírus, astrovírus), mely utóbbiak etiológiai szerepe egyelőre nem tisztázott. A gyakori vírusok molekuláris epidemiológiai vizsgálata komplex járványügyi képet és nagy (geno)típus gazdagságot mutatott; az újonnan leírt potenciális enterális kórokozók hazai jelenlétére azonban eddig nem találtunk bizonyítékot. A vizsgálatok további folytatását a virális gastroenteritisek közegészségügyi jelentősége és a megelőzésükre irányuló törekvések indokolják. | The primary aim of our OTKA research proposal entitled "Molecular epidemiology of viruses in childhood gastroenteritis" was to identify and characterize viruses which play a role in acute dehydrating diarrhea. To achieve our aims, we utilized a variety of molecular methods, such as polyacryamide gelelectrophoresis of genomic viral RNA, and, virus-specific PCR and reverse transcription-PCR assays. Our study covered both common enteric viruses (such as group A rotaviruses, enteric adenoviruses and noroviruses), rare enteric viruses (group C rotaviruses), as well as a number of known or newly described enteric viruses (eg, picobirnavirus, HAdV-52, nodavirus, or a novel astrovirus), some of which are not definitively associated with acute diarrhea. Detection and characterization of the medically important enteric viruses revealed a complex epidemiologic profile, and considerable genetic heterogeneity at the nuceic acid sequence level. Thus far, there is no evidence of circulation of the putative enteric pathogens in Hungary. Continued epidemiologic monitoring of enteric viruses is important for public health as this information facilitates the design of effective and appropriate prevention measures

Molecular analysis of adhesive substances in Pasteurella multocida

Pasteurella multocida is a member of the normal flora of mucosal surfaces and a widely distributed pathogen of many animal species. Molecular bases of pathogenesis and protective immunity against P. multocida infections are far from fully understood. Putative virulence determinants like outer membrane proteins, iron uptake and acquisition proteins, sialidases, toxins and various adhesins may play a part in these mechanisms. Adhesins have a crucial role in mediating colonization and invasion of the host. Thus, their presence on the bacterial surfaces and their morphological variability usually correlate with virulence. The aim of our study was to characterize the molecular diversity of some earlier detected adhesive appendages, like auto transporters (hsf1,2), filamentous haemagglutinin (pfhB1,2) and fimbrial structures (tight adherence macromolecular transport system, type IV pili), in P. multocida strains using with data generated by direct and next generation sequencing. The different types of adhesives have a role in various stages and sites of infection. The filamentous haemagglutinins and autotransporters are essential for initial colonization of trachea while the Tad system and P4T are involved in biofilm initiation and development promoting bacterial persistence in the lower respiratory tract. Lack of proper annotation of most P multocida genomes in the GenBank, the PM70 and multiple alignment (progressiveMauve algorithm) of other genomes (36950, NH06, 3480, X73, P1059) were used as a reference for selection of adequate genes from our assembling genomes. Exception of pfhB2, both of structural and functional genes of the above mentioned structures were detectable in all strains. The functional genes presented allele differentiations in various rates. The supporting of these results comparison of further gene sequences from different strains are needed. Detection and studying of these characteristic sequence variances we obtain useful information about structural changing of adhesins of strains possessing different adhesive features and the results yield targets for prediction of virulence

COVID-19-Associated Fungal Infections: An Urgent Need for Alternative Therapeutic Approach?

Secondary fungal infections may complicate the clinical course of patients affected by viral respiratory diseases, especially those admitted to intensive care unit. Hospitalized COVID-19 patients are at increased risk of fungal co-infections exacerbating the prognosis of disease due to misdiagnosis that often result in treatment failure and high mortality rate. COVID-19-associated fungal infections caused by predominantly Aspergillus and Candida species, and fungi of the order Mucorales have been reported from several countries to become significant challenge for healthcare system. Early diagnosis and adequate antifungal therapy is essential to improve clinical outcomes, however, drug resistance shows a rising trend highlighting the need for alternative therapeutic agents. The purpose of this review is to summarize the current knowledge on COVID-19-associated mycoses, treatment strategies and the most recent advancements in antifungal drug development focusing on peptides with antifungal activity

Comparative molecular characterization of Corynebacterium pseudotuberculosis strains isolated from Hungarian sheep and goat flocks

The genus Corynebacterium belongs to the order Actinomycetales that also includes the genera Mycobacterium, Nocardia, Streptomyces. Although this group is very heterogeneous, most of the species share particular characteristics, such as the cell wall composition and the high G+C ratio of the genomes. C. pseudotuberculosis is a Gram-positive, club-shaped rod, facultative intracellular bacteria that is commonly found in the environment as well as on the skin and mucous surfaces of animals. Based on biochemical features, the strains are classified into two biovars (ovis and equi). The strains within the biovar ovis presented negative nitrate reduction and mainly affect sheep and goats, causing caseous lymphadenitis (CL). The strains biovar equi reduce nitrate and cause ulcerative lymphangitis of the distal extremities, ventral abscesses of the thorax and abdomen, and furunculosis in horses. Occasionally, both types could generate subcutaneous abscesses or visceral organs in cattle. In addition, in rare instances, other domestic or wild animal species, and even human are affected. Due to the sale restrictions of pedigree sires, the reduction of milk, meat and woollen yields, expressive losses in reproductive efficiency, and condemnation of carcasses in slaughterhouse the caseous lymphadenitis causes significant economic losses to sheep producers worldwide. While the illness is common on the tropical areas, it occurs only sporadically in the Mediterraneum. In default of the appropriate import regulations it has spread throughout the world by the pedigree sires. The aim of our study was to molecular comparison of phenotypically characterized C. pseudotuberculosis strains have been collected form sheep and goat flocks in different areas of the country over the past twenty years (1994-2014). We opted for the multilocus sequence typing (MLST) analysis. In the absence of data from literature, we were going to use the MLST had been developed for the analysis of human pathogenic C. diphtheriae strains recently (2010). However the applied primers were C. diphtheriae specific, the developing of own system was needed. For designing of appropriate primers, we have collected and compare of sequences of 16 commonly applied housekeeping using whole genomes of C. pseudotuberculosis at the Genebank. During the examination of the individual genes, the C. pseudotuberculosis and C. diphtheriae strains proved to be distinct in any case and two biovars were found to various in some measure. Finally, the 600-800 bp long regions covering utmost single-nucleotide polymorphism (SNP) between the biovars and present identical phylogenetic topology of the entire genes of dnaK, odhA, the groL1 and infB were examined in detail. Although each of our strains were classified to C. pseudotuberculosis biovar ovis, the MLST examination demonstrated that the two biovars are separated at the level of the individual housekeeping genes. In light of the results is thought-provoking that the strains belong to ovis and the equi biovars represent the same taxonomic group despite the differences of genetic and phenotypic properties (nitrate reduction), of host spectrum (sheep, goat; horse) and of the manifestation of induced disease

Distribution of adhesion factors and their impact on the pathogenicity of bovine Pasteurella multocida strains in Bovine Respiratory Disease

Bovine Respiratory Disease (BRD) is the most common and costly disease affecting beef and dairy cattle industry all over the world. The economic losses are either direct due to lethal cases, or indirect through additional expenses such as the cost of treatment and lower weight gain and decreased carcass value. For those cattle that survive, the presence of pulmonary lesions at slaughter has been associated with significant reduction in daily weight gain. The pathogenesis of BRD is complex, involving a number of viruses, bacteria and stress factors. The stressors, combined with the impact on the immune system and viral infections, allow bacteria to invade the lungs. One of the most frequently isolated bacteriological agents is Pasteurella multocida in these cases. This organism commonly inhabits the pharynx or upper respiratory tract of most cattle. However, it is not considered as normal flora of the lungs. The inhaled bacteria that attached on the mucus of lower respiratory tract successfully using a wide-range of adhesins, under conditions of impaired pulmonary defences, replicate rapidly and cause pneumonia. The aim of our study was to detect and characterise the known putative adhesive factors within the bovine P. multocida population in Hungary. The studied 39 strains were isolated from diseased animals in different cattle herds and they were earlier characterized with traditional microbiological and general molecular methods and divided into subpopulation considered to contain strains of different virulence. The presence of ptfA (subunit of type IV fimbriae), fimA (fimbriae), hsf-1, 2 (autotransporter adhesins), tadD (putative nonspecific tight adherence protein D) and pfhA (filamentous haemagglutinin) genes were detected by PCR. The distribution of virulence factors was compared with the strains’ recorded diagnostic data. The prevalence of the different genes was various. Whereas the fimA was present in all and hsf-2, tadD, ptfA genes in most strains, the occurrence of hsf-1 and pfhA showed variability. Based on these results, seven combinations of the genes could be detected. These profiles well characterized the delineated main subpopulations. Two of them were dominant. The strains with these latter ones varied in possession of gene encoding filamentous haemagglutinin. The presence of pfhA showed significant correlation with diagnostic data. It was associated with pneumonia. For the discrepancy of virulence, the changes in structures of the virulence factors could also be responsible. Thus detailed study of them is recommended. For the additional characterization of the strains we carried out the sequencing of extended genome region included tadD gene. The sequence analysis showed low per cent but characteristic differences between the representative strains. The effect of the changing to the protein structure or function required further investigations

Complete Genome Sequence of a Variant Pyrrhula pyrrhula polyomavirus 1 Strain Isolated from White-Headed Munia (Lonchura maja)

A novel variant of finch polyomavirus has been identified and sequenced from a diseased white-headed munia (Lonchura maja)