Tok-herpeszvírus vizsgálata és összehasonlítása más, állategészségügyi problémákat okozó herpeszvírusokkal = Study of sturgeon herpesvirus and its comparison with other herpesviruses causing veterinary problems

A fehér tok-herpeszvírus (AciHV-2) genomjának középső szakaszát sikerült molekuláris klónozással és PCR-rel felerősíteni és DNS szekvenciáját megállapítani 68.700 bp hosszban (a feltételezett genomnak több mint az 50%-a). Ez 44 gén/ORF teljes és 3 gén részleges szekvenciája. Az ORF-ek nagy többsége méretében, irányultságában és lokalizációjába megegyezik vagy hasonló a foltos csatornaharcsa-herpeszvírus (IcHV-1) megfelelő ORF-jeivel. A különbségek: az IcHV-1 szerinti ORF 24 és 25 között egy plusz gént találtunk, amely feltehetően egy szerin-proteáz; az ORF 65-nek megfelelő gén hossza jelentősen nagyobb, mint az IcHV-1-ben. Olasz fekete törpeharcsából (Ameiurus melas) izolált herpeszvírusból mintegy 10.000 bázispár hosszú szakaszt erősítettünk fel PCR-rel és szekvenáltunk. A genom-szerveződés nagyfokú hasonlóságot mutat az IcHV-1-gyel. Szibériai tok-herpeszvírus genomból 11.000 bázispárt szekvenáltunk. E vírus nagyon közeli rokonságban áll az észak-amerikai izolátumunkkal. A talált filogenetikai rokonságok alapján tett vírustaxonómiai javaslatainkat részben elfogadta a Nemzetközi Vírustaxonómiai Bizottság. Ezüstkárászból elsőként mutattunk ki eddig csak aranyhalakból leírt Cyprinid herpesvirus 2-t. Hazánkban először mutattunk ki PCR-rel a pontyhimlő vírusát (CyHV-1). Összehasonlító célból eddig ismeretlen herpeszvírusokat mutattunk ki és jellemeztünk denevérekből, ázsiai kiskarmú vidrából, majmokból, egerészölyvből, melyek a Herpesviridae mindhárom alcsaládját képviselték. | The central part of the genome of white sturgeon herpesvirus (AciHV-2) was molecularly cloned or PCR amplified, DNA sequenced in 68,700 bp-length (more than 50% of the supposed genome length). This covers 44 full and 3 partial genes/ORFs. Most of the ORFs are identical with or similar to the corresponding ORFs of channel catfish herpesvirus (IcHV-1) in their size, orientation and localization. The differences: a plus gene between ORF 24 és 25 (IcHV-1 numbering) which is supposedly a serine protease; and the size of the ORF 65 homologue is considerably larger than in IcHV-1. An about 10,000 bp long genome fragment was amplified and sequenced from a herpesvirus isolated from black bullhead (Ameiurus melas). The genome organization showed high level similarity with that of IcHV-1. 11.000 bp was sequenced from the genome of a Siberian sturgeon herpesvirus. This virus is a close relative of our North American isolate. Our taxonomical proposal made on the basis of the identified phylogenetic relatedness has been partially accepted by the International Committee on Taxonomy of Viruses. We were first to detect cyprinid herpesvirus 2 in Silver Prussian carp which had been described earlier exclusively from goldfish. We detected carp pox virus (CyHV-1) by PCR first in Hungary. For comparative studies, we detected and characterized novel herpesviruses from bats, oriental small-clawed otter, monkeys, and buzzard, which represented all three subfamilies of Herpesvirida

Novel parvovirus from the worm lizard Trogonophis wiegmanni — First virus ever detected in amphisbaenian hosts

To explore the diversity of some DNA viruses in reptiles, a continuous screening is going on, in our laboratory, by PCR using different consensus primers designed for the detection of the most conserved genome regions of adeno-, herpes- and parvoviruses. The test material consists essentially of dead specimens collected randomly from private pet owners, local pet shops, or at occasional exotic pet fairs. Here we report the partial sequence of a putative novel parvovirus obtained from a dead checkerboard worm lizard (Trogonophis wiegmanni) that had been wild-caught in its native habitat. An in-house-developed PCR with consensus primers targeting the gene of the parvoviral capsid protein was used. Other PCRs, intended to detect certain large DNA viruses, remained negative. The sequence of the PCR product indicated the presence of a hitherto unknown parvovirus in the internal organs of the checkerboard worm lizard. In phylogeny reconstruction, the novel sequence clustered with the members of the Dependovirus genus of the Parvoririnae subfamily, closest to the branch of snake adeno-associated virus. Since we could not demonstrate the presence of a potential helper virus, the putative amphisbaenian parvovirus supposedly can replicate autonomously. This is the first virus infection ever detected in any members of the suborder Amphisbaenia, and only the third parvoviral sequence obtained from any reptilian host

Comparison of the genome of ovine adenovirus types 1 through 5 by restriction enzyme analysis and DNA hybridisation

The DNA of the prototype strains of ovine adenovirus (OAdV) 1 through 5 was analysed by restriction enzyme (RE) digestion. The RE patterns generated by Hindlll and PstI enzymes were characteristic of the examined strains. OAdV-2 and 3 resembled each other the most, and their EcoRI and Hindlll patterns seemed to be identical. Considering the number of comigrating fragments, serotypes OAdV-2, 3, 4 and 5 looked more closely related to each other than to OAdV-1. This finding was strengthened by Southern blot hybridisations probed with random Hindlll clones of OAdV-3. The estimated genome size of the examined OAdV types ranged between 31.9 and 32.8 kilobase pairs. The results supported the new genus classification of OAdVs

Insulin for topical use in wound healing: opportunities and limitations

Aims: The role of insulin in the regulation of energy metabolism, protein synthesis, proliferation, migration, secretion by keratinocytes, endothelial cells, and fibroblasts suggests that its presence is essential for wound healing (WH). The present study aims to explore the opportunities and limitations of topical insulin (TI) formulations. Methods: To obtain a complete picture of the challenges of the local insulin formulation a chronological review of previous publications in electronic databases was performed, applying data collection and selection criteria. Results: The opportunity of topically applied insulin has shown active interest over time. According to studies, regular insulin and isophane are suitable for local use, but currently there is no consensus on the appropriate concentration. Insulin can be incorporated into cutaneous liquid, semisolid, and solid dosage forms, either by itself, or by prior nano-or microencapsulation methods. The most important limiting factors to be evaluated are the stability of the peptide and the sterility of the obtained products. Conclusion: Examination of the balance of opportunities and limitations of TI formulations, it can be concluded that the range of applicable technological methods is wide. A high-quality, safe, and efficacious form of TI would have great value from a socio-economic point of view

Állati eredetű adenovírusok genetikai jellemzése az evolúciót hűen tükröző rendszertan kialakítása céljából = Genetic characterization of adenoviruses of different animal hosts to achieve an improved taxonomy, which reflects the viral evolution

A pályázat által támogatott kutatások során rendszertani szempontból különösen jelentősnek ítélt adenovírusokat vizsgáltunk. Két óvilági majom-adenovírus teljes DNS-ének bázissorrendjét meghatároztuk és analizáltuk, mert ilyen vírusokból még nem volt ismert teljes genom. Befejezés előtt áll egy liba- és egy pulyka-adenovírus teljes szekvenciájának meghatározása is. Ezzel célunk az Aviadenovirus genusba tartozó ismert vírusok számának növelése volt. Filogenetikai számításokkal igazoljuk a madár adenovírusok gazdáikkal való koevolúciójára utaló jeleket. Egy kígyó- és egy hal-adenovírus genom elemzése az evolúciós viszonyok nagy vonalainak tisztázásához szolgáltat majd hasznos adatokat. Egy újnak bizonyult siadenovírus teljes genomanalízise folyik. A vírus, mely elhullást okozott Angliában ragadozómadár tenyészetekben a Siadenovirus nemzetség harmadik tagja lehet. Valamennyi vizsgált vírus új vírusfajt is képvisel, így elemzésük eredménye várhatóan tovább erősíti vagy cáfolja az általunk kialakított kategóriák helyességét. Az adenovírusok természetben megfigyelhető diverzitásának vizsgálatára érzékeny PCR módszert dolgoztunk ki. A PCR szűrés során pozitív, új típusnak látszó adenovírusok további vizsgálatát tervezzük. | In the framework of the project, we have studied adenoviruses that seemed to have great importance from the viewpoint of taxonomy. We determined and analyzed the nucleotide sequence of the full genome of two adenovirus isolates originating from Old World monkeys, because such viruses have not been examined before. The full genome sequencing of a goose and a turkey adenovirus, both isolated in Hungary, is almost finished. The purpose of the analysis of these avian adenoviruses was to increase the number of well-characterized members of the genus Aviadenoviruses. With phylogenetic calculations, we have confirmed that aviadenoviruses have a long co-evolutionary history with their avian hosts. Full genome analysis of a snake and a fish adenovirus will help to understand the large-scale evolutionary relations within the family Adenoviridae. Full genome sequencing of a novel siadenovirus is also in progress. The virus, that has caused mortality among captive birds of prey in England, will be only the third member in the genus Siadenovirus. Every adenovirus being studied represents a novel adenovirus species, thus criteria set for the demarcation of this taxon can also be evaluated. For the detection of biodiversity of adenoviruses circulating in the nature, a very sensitive PCR method was elaborated. We plan to further characterize those novel adenovirus candidates that are being detected during screening with PCR

First detection of circovirus-like sequences in amphibians and novel putative circoviruses in fishes

The negative samples of a collection, established originally for seeking new adeno- and herpesviruses in lower vertebrates, were screened for the pres-ence of circoviruses by a consensus nested PCR targeting the gene coding for the replication-associated protein. Six fish samples representing five species, namely asp (Aspius aspius), roach (Rutilus rutilus), common bream (Abramis brama), round goby (Neogobius melanostomus) and monkey goby (Neogobius fluviatilis), as well as three frog samples were found positive for circoviral DNA. Sequence analysis of the amplicons indicated the presence of three novel putative circo-like viruses and a circovirus in Hungarian fishes and one novel circovirus in a common toad (Bufo bufo), and another one in a dead and an alive specimen of green tree frog (Litoria caerulea), respectively. In phylogeny reconstruction, the putative bream circovirus clustered together with circoviruses discovered in other cyprinid fishes recently. Three other piscine circoviral sequences appeared closest to sequences derived from different environmental samples. Surprisingly, the nucleotide sequence derived from two fish samples (a bream and a monkey goby) proved to be from porcine circovirus 2 (PCV2), almost identical to a sequence detected in Sweden previously. This is the first report on the detection of PCV2 in fish and circoviral DNA in amphibian hosts

Novel parvoviruses in reptiles and genome sequence of a lizard parvovirus shed light on Dependoparvovirus genus evolution.

Here, we report the detection and partial genome characterization of two novel reptilian parvoviruses derived from a short-tailed pygmy chameleon (Rampholeon brevicaudatus) and a corn snake (Pantherophis guttatus) along with the complete genome analysis of the first lizard parvovirus, obtained from four bearded dragons (Pogona vitticeps). Both homology searches and phylogenetic tree reconstructions demonstrated that all are members of the Dependoparvovirus genus. Even though most dependoparvoviruses replicate efficiently only in co-infections with large DNA viruses, no such agents could be detected in one of the bearded dragon samples, hence the possibility of autonomous replication was explored. The alternative ORF encoding the full assembly-activating protein (AAP), typical for the genus, could be obtained from reptilian parvoviruses for the first time, with a structure that appears to be more ancient than that of avian and mammalian parvoviruses. All three viruses were found to harbor short introns as previously observed for snake adeno-associated virus (SAAV), shorter than that of any non-reptilian dependoparvovirus. According to the phylogenetic calculations based on full non-structural protein (Rep) and AAP sequences, the monophyletic cluster of reptilian parvoviruses seems to be the most basal out of all lineages of genus Dependoparvovirus. The suspected ability for autonomous replication, results of phylogenetic tree reconstruction, intron lengths and the structure of the AAP, suggested that a single Squamata origin instead of the earlier assumed diapsid (common avian-reptilian) origin is more likely for the genus Dependoparvovirus of the Parvoviridae family

Full genome sequence of a novel circo-like virus detected in an adult European eel Anguilla anguilla showing signs of cauliflower disease.

An adult European eel Anguilla anguilla, showing typical signs of the so-called cauliflower disease, was subjected to pathological and molecular virological examinations. Samples taken from internal organs and the polypoid proliferative tissue from the mouth were examined by PCR for the detection of several viruses. Positive results were obtained with a nested PCR targeting the rep gene of circoviruses. Analysis of the partial rep sequence indicated the presence of a putative novel circovirus, but attempts to isolate it remained unsuccessful. The missing part of the genome was acquired by an inverse nested PCR with 2 specific primer pairs, designed from the newly determined rep sequence, followed by genome walking. The circular full genome was found to consist of 1378 nt (GenBank accession no. KC469701). Two oppositely oriented open reading frames (ORFs) were present, of which one was unambiguously identified as a circoviral rep gene. However, the predicted product of the other ORF, though it is a clear positional counterpart of the cap genes, showed no obvious homology to any known circoviral capsid proteins. A stem-loop-like element in the intergenic region between the 5' ends of the ORFs was also found. Phylogenetic calculations indicated that the novel virus belongs to the genus Circovirus in the family Circoviridae. The relative amount of the viral DNA in the organ samples was estimated by quantitative real-time PCR. The results suggested that the examined fish was caught in an active viremic state, although the role of this circovirus in the etiology of the cauliflower diseases could not be ascertained

Genetic diversity of species Fowl aviadenovirus D and Fowl aviadenovirus E

Complete genomes of eight reference strains representing different serotypes within species Fowl aviadenovirus D (FAdV-D) and Fowl aviadenovirus E (FAdV-E) were sequenced. The sequenced genomes of FAdV-D and FAdV-E members comprise 43,287 to 44,336 bp, and have a gene organization identical to that of an earlier sequenced FAdV-D member (strain A-2A). Highest diversity was noticed in the hexon and fiber genes and ORF19. All genomes, sequenced in this study, contain one fiber gene. Phylogenetic analyses and G+C content support the division of the genus Aviadenovirus into the currently recognized species. Our data also suggest that the strain SR48 should be considered as FAdV-11 instead of FAdV-2 and similarly the strain HG as FAdV-8b. The present results complete the list of genome sequences of reference strains representing all serotypes in species FAdV-D and FAdV-E