Mária Benkő

Consensus statement: Virus taxonomy in the age of metagenomics

The number and diversity of viral sequences that are identified in metagenomic data far exceeds that of experimentally characterized virus isolates. In a recent workshop, a panel of experts discussed the proposal that, with appropriate quality control, viruses that are known only from metagenomic data can, and should be, incorporated into the official classification scheme of the International Committee on Taxonomy of Viruses (ICTV). Although a taxonomy that is based on metagenomic sequence data alone represents a substantial departure from the traditional reliance on phenotypic properties, the development of a robust framework for sequence-based virus taxonomy is indispensable for the comprehensive characterization of the global virome. In this Consensus Statement article, we consider the rationale for why metagenomic sequence data should, and how it can, be incorporated into the ICTV taxonomy, and present proposals that have been endorsed by the Executive Committee of the ICTV.

Molecular typing of fowl adenoviruses, isolated in Hungary recently, reveals high diversity

Molecular typing of 26 recent fowl adenovirus (FAdV) isolates obtained in Eastern Hungary between 2006 and 2011 was performed. The viruses were isolated on primary chicken embryo liver cell cultures from carcasses of chickens, sent for diagnostic investigation. The isolates were subjected to a PCR to amplify a fragment from the hexon gene. Sequence analysis of the amplicons revealed the presence of all the five FAdV species in the country, among them FAdV-B, which has been barely found in Hungary before. The three strains classified as FAdV-B might represent a novel type within this species, as their nucleotide sequence identity to strain 340 (type FAdV-5) is below the commonly accepted intratype limit. Most of the strains (63%) were classified into species FAdV-E (12 strains) and FAdV-D (7 strains) in the study. Four of the isolates proved to be the mixture of two adenovirus strains belonging to two different FAdV types, in three from these four cases, these two types even belonged to two different species. Analysing the pathological findings bolsters certain established connections between some FAdV types and disease conditions. Gizzard erosion was found in connection with FAdV-1 strains only, and inclusion body hepatitis with FAdV-D and -E strains.

Novel adenoviruses and herpesviruses detected in bats

Samples from native Hungarian or captive bats were tested by PCR for the presence of adenoviruses and herpesviruses. Two novel adenoviruses from a common noctule (Nyctalus noctula) and a greater horseshoe (Rhinolophus ferrum-equinum) bat were detected. In captive Egyptian fruit bats (Rousettus aegyptiacus), DNA from two novel herpesviruses was demonstrated. Phylogenetic analysis facilitated provisional taxonomic placement of the newly detected viruses. Such analysis and the existence of unique, shared early proteins (E3 and E4) suggest that canine adenoviruses may have originated in vespertilinoid bats.

Development of Novel Adenoviral Vectors to Overcome Challenges Observed With HAdV-5-based Constructs.

Recombinant vectors based on human adenovirus serotype 5 (HAdV-5) have been extensively studied in preclinical models and clinical trials over the past two decades. However, the thorough understanding of the HAdV-5 interaction with human subjects has uncovered major concerns about its product applicability. High vector-associated toxicity and widespread preexisting immunity have been shown to significantly impede the effectiveness of HAdV-5–mediated gene transfer. It is therefore that the in-depth knowledge attained working on HAdV-5 is currently being used to develop alternative vectors. Here, we provide a comprehensive overview of data obtained in recent years disqualifying the HAdV-5 vector for systemic gene delivery as well as novel strategies being pursued to overcome the limitations observed with particular emphasis on the ongoing vectorization efforts to obtain vectors based on alternative serotypes.

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. scripta 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.

Molecular confirmation of a new herpesvirus from catfish (Ameiurus melas) by testing the performance of a novel PCR method, designed to target the DNA polymerase gene of alloherpesviruses.

A PCR method with consensus degenerate primers was developed for the detection of herpesviruses (HVs) of anamnia. Compared to previously published PCRs, targeting the DNA polymerase gene of fish HVs, the size of PCR products was more than tripled. Although broad applicability of the method could not be proven, approximately 1,600-bp fragments from HVs of white sturgeon (Acipenser transmontanus) and black bullhead (Ameiurus melas) were obtained and sequenced. Phylogenetic tree reconstructions showed both HVs to be monophyletic with the single member (ictalurid HV-1) of the genus Ictalurivirus in the new family Alloherpesviridae.

Complete sequence of raptor adenovirus 1 confirms the characteristic genome organization of siadenoviruses

Currently, the family Adenoviridae contains five genera, out of which Siadenovirus is one of the two least densely populated ones. A new member representing a new species in this genus has been detected in various birds of prey. The virus, named raptor adenovirus 1 (RAdV-1), could not be isolated, probably because no appropriate permissive cell-line was available. Partial genomic sequences, obtained by PCR and suggesting that the virus is a new siadenovirus species, have been published earlier. In the present paper, determination and analysis of the complete RAdV-1 genome are reported. This is the first complete genome sequence acquired from a non-isolated adenovirus (AdV). The sole source was a mixture of the internal organs of the diseased and dead birds. Until now, the genomic organization considered characteristic to siadenoviruses had been deduced from the detailed study of only two virus species, one of which originated from birds and the other from a frog. The present analysis of RAdV-1 confirmed the genus-specific genetic content and genomic features of siadenoviruses, and a putative novel gene was found as well. In general, AdVs and most of the AdV genera are thought to be strictly host specific. In the genus Siadenovirus, however, two virus species of rather divergent (avian and amphibian) host origin were present when the genus was found. Although by now the greatest number of known siadenoviruses infect birds, the original hosts of the genus remain unknown.

The first complete genome sequence of a non-chicken aviadenovirus, proposed to be turkey adenovirus 1 ☆

The complete genome sequence of an adenovirus, isolated from turkey and proposed to be turkey adenovirus type 1 (TAdV-1), was determined to extend our knowledge about the genome organisation and phylogeny of aviadenoviruses. The longest adenovirus genome, consisting of 45,412 bp, with the highest G+C content (of 67.55%) known to date, was found. The central part of the TAdV-1 genome has the conserved gene set and arrangement that are characteristic for every other adenovirus analysed to date. This genome core is flanked by the terminal early regions 1 and 4 (E1 and E4). Aviadenovirus-specific genus-common genes were found in these regions, each containing nine such open reading frames (ORFs). Additionally a type-specific novel ORF, designated as ORF50, was found in E4. Phylogenetic analysis as well as the presence of the genus-specific genes, splice sites and protease cleavage sites confirmed the classification of TAdV-1 in the genus Aviadenovirus. Intrageneric analyses of two genus-specific genes demonstrated the distinctness of TAdV-1 from other aviadenoviruses, thus supporting the proposal for the establishment of a new species, Turkey adenovirus B for TAdV-1.

Molecular Characterization of a Lizard Adenovirus Reveals The First Atadenovirus with two Fiber Genes, and the First Adenovirus with Either One Short or Three Long Fibers per Penton

ABSTRACT Although adenoviruses (AdVs) have been found in a wide variety of reptiles, including numerous squamate species, turtles, and crocodiles, the number of reptilian adenovirus isolates is still scarce. The only fully sequenced reptilian adenovirus, snake adenovirus 1 (SnAdV-1), belongs to the Atadenovirus genus. Recently, two new atadenoviruses were isolated from a captive Gila monster (Heloderma suspectum) and Mexican beaded lizards (Heloderma horridum). Here we report the full genomic and proteomic characterization of the latter, designated lizard adenovirus 2 (LAdV-2). The double-stranded DNA (dsDNA) genome of LAdV-2 is 32,965 bp long, with an average G+C content of 44.16%. The overall arrangement and gene content of the LAdV-2 genome were largely concordant with those in other atadenoviruses, except for four novel open reading frames (ORFs) at the right end of the genome. Phylogeny reconstructions and plesiomorphic traits shared with SnAdV-1 further supported the assignment of LAdV-2 to the Atadenovirus genus. Surprisingly, two fiber genes were found for the first time in an atadenovirus. After optimizing the production of LAdV-2 in cell culture, we determined the protein compositions of the virions. The two fiber genes produce two fiber proteins of different sizes that are incorporated into the viral particles. Interestingly, the two different fiber proteins assemble as either one short or three long fiber projections per vertex. Stoichiometry estimations indicate that the long fiber triplet is present at only one or two vertices per virion. Neither triple fibers nor a mixed number of fibers per vertex had previously been reported for adenoviruses or any other virus. IMPORTANCE Here we show that a lizard adenovirus, LAdV-2, has a penton architecture never observed before. LAdV-2 expresses two fiber proteins—one short and one long. In the virion, most vertices have one short fiber, but a few of them have three long fibers attached to the same penton base. This observation raises new intriguing questions on virus structure. How can the triple fiber attach to a pentameric vertex? What determines the number and location of each vertex type in the icosahedral particle? Since fibers are responsible for primary attachment to the host, this novel architecture also suggests a novel mode of cell entry for LAdV-2. Adenoviruses have a recognized potential in nanobiomedicine, but only a few of the more than 200 types found so far in nature have been characterized in detail. Exploring the taxonomic wealth of adenoviruses should improve our chances to successfully use them as therapeutic tools.

Adenoviruses – from pathogens to therapeutics: a report on the 10th International Adenovirus Meeting

Adenoviruses : from pathogens to therapeutics: a report on the 10th International Adenovirus Meeting