Screening of various aquatic animals for the presence of adenoviruses and hepesviruses

Szakdolgozati munkám témája halak és más vízi állatok (kagylók, bolharákok) PCR-rel történő szűrése volt adenovírusok (AdV) és herpeszvírusok (HV) kimutatása céljából. A hazai halpopulációk, illetve a vizekben a kórokozókat passzívan akkumuláló szervezetek fertőzöttségének mértékét kívántuk felmérni, de új AdV-ok és HV-ok felismerésére is számítottunk. Időközben a laboratóriumunkban (és ezzel egy időben az Egyesült Államokban) az eddig ismertektől jelentősen eltérő leszármazási vonalhoz tartozó AdV-okat mutattak ki ékszerteknősökben. Ennek az új AdV-nak ismételt megtalálása céljából a későbbiekben teknősökre is kiterjesztettem vizsgálataimat. A minták egy része a laboratórium korábbi gyűjtéseiből származott. A gerinctelenek és teknősök mintáit, és a halminták nagyobb részét én szereztem be. Mindkét víruscsalád tagjainak kimutatására olyan PCR-t használtam, amelynek során a vírusok DNS-függő DNS polimeráz génjének egy szakaszát lehet felsokszorozni. HV-ra a 333 vizsgált minta mindegyikét negatívnak találtam. Az AdV-ra vizsgált 570 minta közül is csak az ékszerteknősök között volt pozitív. A véletlenszerűen gyűjtött, 44 mintából 12 (27%) bizonyult pozitívnak. A szekvenálás és filogenetikai elemzés alapján megállapítottam, hogy az újnak látszó AdV a hazai (budapesti) ékszerteknős populációban viszonylag gyakran előfordul. Az új AdV változatait 5 sárgafülű és 7 vörösfülű ékszerteknős mintájában találtam meg. A PCR termékek szekvenálása 8 tiszta és 4 kevert szekvenciát eredményezett. A további AdV gének kimutatására tervezett PCR-ek közül csupán egy működött, amellyel a hexon génből nyertünk ki rövid szakaszt 2 mintából. A DNS polimeráz származtatott aminosav szekvenciája 5 különböző, gazdafüggetlen variáns meglétét mutatta, míg a hexon részleges szekvenciája az egyes mintákban azonos volt. A fertőzött állatok 2 hulla kivételével egészséges, jó kondícióban lévő egyedek voltak, így a vírus esetleges kórokozó képessége nem tisztázott. A két génszakasszal végzett törzsfa-rekonstrukciók megerősítették, hogy az új AdV-ok az Adenoviridae családon belül önálló csoportot képeznek, és nemzetség szintű elkülönítésük is indokolt lehet. Ehhez további vizsgálatok szükségesek. Az új AdV leszármazási vonal valószínűleg megfelel a teknősökkel együtt fejlődött AdV csoportnak, amit az is alátámaszt, hogy DNS-ük G+C tartalmát kiegyensúlyozottnak (50% körülinek) találtam.The topic of my diploma work was the screening of fishes and some other aquatic animals for the presence of adenoviruses (AdV) and herpesviruses (HV) by PCR. Our aim was to assess the prevalence of these viruses in the domestic fish populations and in some other organisms that can accumulate the viruses passively. The recognition of several new AdVs and HVs was also expected. During the time of my PCR survey, a new AdV was discovered in a yellow-bellied (YB) slider (Trachemys scripta scripta) in our laboratory, and almost simultaneously in a red-eared (RE) slider (T. s. elegans) in the USA. The preliminary phylogenetic analysis implied that the new slider AdVs represent a new lineage that is significantly different from the approved AdV genera known to date. To find this novel AdV again, I have expanded the collection of samples to Hungarian slider colonies. Part of the samples was from the collection of the laboratory, whereas the majority of them including every slider and invertebrate (mussels, amphipods) sample were acquired by me. For the detection of members of each virus families, a PCR with primers targeting the gene of the respective DNA-dependent DNA polymerase was used. After having screened 570 (367 fish, 156 invertebrates and 47 turtles) samples, only slider turtles were found to be positive for AdV. From the randomly collected 44 samples 12 (27%) were positive with sequencing and phylogenetic calculations. None of the 333 samples were positive for HVs. Slightly divergent AdV sequences were detected in 5 YB and 7 RE sliders. From two samples, a small fragment of the hexon gene was also amplified successfully. The sequence of the PCR products was clean in 8 cases (representing 5 variants), while the other 4 samples seemed to contain more than one type of AdVs. The positive animals with two exceptions were healthy-looking live adults, thus the pathogenic role of the new AdV remains unclear. Phylogeny reconstructions with either partial gene sequence, confirmed the separation of a monophyletic clade within the family Adenoviridae. The new AdV lineage will probably merit the rank of a new genus, but further data are needed. The balanced base composition (with a G+C content of around 50%) suggest that this new AdV lineage has co-evolved most likely with the testudinoid turtles

Diversity of adenoviruses occurring in turtles (Autoreferatum and literature review)

SUMMARY Background: Adenoviruses (AdVs) are common infectious agents of different vertebrates worldwide. The members of the family Adenoviridae are currently classified into five genera, of which Mastadenovirus and Aviadenovirus contain the usually stenoxen pathogens of mammals and birds, respectively. Members of the other two genera, Atadenovirus and Siadenovirus, seem to be euryxen and capable of infecting representatives of several distant taxa of vertebrates, both of them including birds. The genus Ichtadenovirus encompasses the single known fish AdV originating from the white sturgeon (Acipenser transmontanus).Objectives: By reviewing their own results and the international literature, the authors summarize the current knowledge about the AdV infections of turtles.Materials and Methods: Organ samples and cloacal swabs, collected from dead or live fresh water turtles, were processed for DNA extraction and screened by broad-spectrum nested PCRs. The sequences of the PCR products were used in phylogeny reconstructions.Results and Discussion: DNA fragments (of approx. 320 bp and 470 bp) were amplified and sequenced from the DNA-dependent DNA polymerase and the hexon genes, respectively. The presence of a novel type AdV was demonstrated in 14 (29%) of the 48 samples examined. In some cases, the PCR products, obtained from the DNA polymerase gene, proved to be polymorphic. By molecular cloning, several different AdV genotypes were revealed in certain animals. The hexon PCR was positive in 2 samples only, yielding identical sequences. The phylogeny reconstructions, based on the deduced amino acid sequences, showed that the novel turtle adenoviruses form a separate cluster that merits the establishment of a new genus within the family Adenoviridae. The authors proposed to name this genus Testadenovirus. Similar viruses, also belonging to the new AdV lineage, have been detected in four additional turtle species from the superfamily Testudinoidea. The genetic relationships of the AdVs seemed to be congruent with those assumed for the respective turtle species

Partial genetic characterisation of a novel alloherpesvirus detected by PCR in a farmed wels catfish (Silurus glanis)

By a broad-range PCR, we detected a novel herpesvirus (HV) in the specimen of a wels catfish (Silurus glanis) presenting disseminated, carp pox-like dermal lesions all over its body. The sequence analysis of the 463-bp PCR product from the viral DNA polymerase gene indicated the presence of a hitherto unknown virus, a putative member of the family Alloherpesviridae in the sample. Another PCR, targeting the terminase gene of fish HVs, provided an additional genomic fragment of over 1,000 bp. Surprisingly, the sequence of a co-amplified, off-target PCR product revealed its origin from a putative gene homologous to ORF87 and ORF45 of cyprinid HVs and anguillid herpesvirus 1 (AngHV-1), respectively. With specific primers, designed according to the genomic maps of the cyprinid and anguillid HVs, a genomic fragment of 15 kb was also amplified and sequenced by primer walking. In phylogeny inferences, based on several genes, the putative wels catfish HV clustered closest to various cyprinid HVs or to AngHV-1. The novel virus, named as silurid herpesvirus 2, represents a distinct species in the genus Cyprinivirus. However, its association with the skin disease remains unclear

In silico tissue-distribution of human Rho family GTPase activating proteins

Rho family small GTPases are involved in the spatio-temporal regulation of several physiological processes. They operate as molecular switches based on their GTP- or GDP-bound state. Their GTPase activator proteins (Rho/Rac GAPs) are able to increase the GTP hydrolysis of small GTPases, which turns them to an inactive state. This regulatory step is a key element of signal termination. According to the human genome project the potential number of Rho family GAPs is approximately 70. Despite their significant role in cellular signaling our knowledge on their expression pattern is quite incomplete. In this study we tried to reveal the tissue-distribution of Rho/Rac GAPs based on expressed sequence tag (EST) database from healthy and tumor tissues and microarray experiments. Our accumulated data sets can provide important starting information for future research. However, the nomenclature of Rho family GAPs is quite heterogeneous. Therefore we collected the available names, abbreviations and aliases of human Rho/Rac GAPs in a useful nomenclature table. A phylogenetic tree and domain structure of 65 human RhoGAPs are also presented

Partial characterization of a new adenovirus lineage discovered in testudinoid turtles

In the USA and in Hungary, almost simultaneously, adenoviruses of a putative novel lineage were detected by PCR and sequencing in turtles belonging to four different species (including two subspecies) of the superfamily Testudinoidea. In the USA, partial sequence of the adenoviral DNA-dependent DNA polymerase was obtained from samples of a captive pancake tortoise (Malacochersus tornieri), four eastern box turtles (Terrapene carolina carolina) and two red-eared sliders (Trachemys scripta elegans). In Hungary, several individuals of the latter subspecies as well as some yellow-bellied sliders (T. s. scripta) were found to harbor identical, or closely related, putative new adenoviruses. From numerous attempts to amplify any other genomic fragment by PCR, only a nested method was successful, in which a 476-bp fragment of the hexon gene could be obtained from several samples. In phylogeny reconstructions, based on either DNA polymerase or hexon partial sequences, the putative new adenoviruses formed a clade distinct from the five accepted genera of the family Adenoviridae. Three viral sub-clades corresponding to the three host genera (Malacochersus, Terrapene, Trachemys) were observed. Attempts to isolate the new adenoviruses on turtle heart (TH-1) cells were unsuccessful. Targeted PCR screening of live and dead specimens revealed a prevalence of approximately 25% in small shelter colonies of red-eared and yellow-bellied sliders in Hungary. The potential pathology of these viruses needs further investigation; clinically healthy sliders were found to shed the viral DNA in detectable amounts. Based on the phylogenetic distance, the new adenovirus lineage seems to merit the rank of a novel genus