Human AdV-20-42-42, a Promising Novel Adenoviral Vector for Gene Therapy and Vaccine Product Development.

Preexisting immune responses toward adenoviral vectors limit the use of a vector based on particular serotypes and its clinical applicability for gene therapy and/or vaccination. Therefore, there is a significant interest in vectorizing novel adenoviral types that have low seroprevalence in the human population. Here, we describe the discovery and vectorization of a chimeric human adenovirus, which we call HAdV-20-42-42. Full-genome sequencing revealed that this virus is closely related to human serotype 42, except for the penton base, which is derived from serotype 20. The HAdV-20-42-42 vector could be propagated stably to high titers on existing E1-complementing packaging cell lines. Receptor-binding studies revealed that the vector utilized both CAR and CD46 as receptors for cell entry. Furthermore, the HAdV-20-42-42 vector was potent in transducing human and murine cardiovascular cells and tissues, irrespective of the presence of blood coagulation factor X. In vivo characterizations demonstrate that when delivered intravenously (i.v.) in mice, HAdV-20-42-42 mainly targeted the lungs, liver, and spleen and triggered robust inflammatory immune responses. Finally, we demonstrate that potent T-cell responses against vector-delivered antigens could be induced upon intramuscular vaccination in mice. In summary, from the data obtained we conclude that HAdV-20-42-42 provides a valuable addition to the portfolio of adenoviral vectors available to develop efficacious products in the fields of gene therapy and vaccination. IMPORTANCE Adenoviral vectors are under investigation for a broad range of therapeutic indications in diverse fields, such as oncology and gene therapy, as well as for vaccination both for human and veterinary use. A wealth of data shows that preexisting immune responses may limit the use of a vector. Particularly in the current climate of global pandemic, there is a need to expand the toolbox with novel adenoviral vectors for vaccine development. Our data demonstrate that we have successfully vectorized a novel adenovirus type candidate with low seroprevalence. The cell transduction data and antigen-specific immune responses induced in vivo demonstrate that this vector is highly promising for the development of gene therapy and vaccine products.

Virus-Host Coevolution with a Focus on Animal and Human DNA Viruses.

Viruses have been infecting their host cells since the dawn of life, and this extremely long-term coevolution gave rise to some surprising consequences for the entire tree of life. It is hypothesised that viruses might have contributed to the formation of the first cellular life form, or that even the eukaryotic cell nucleus originates from an infection by a coated virus. The continuous struggle between viruses and their hosts to maintain at least a constant fitness level led to the development of an unceasing arms race, where weapons are often shuttled between the participants. In this literature review we try to give a short insight into some general consequences or traits of virus-host coevolution, and after this we zoom in to the viral clades of adenoviruses, herpesviruses, nucleo-cytoplasmic large DNA viruses, polyomaviruses and, finally, circoviruses.

Using the E4orf6-Based E3 Ubiquitin Ligase as a Tool To Analyze the Evolution of Adenoviruses.

UNLABELLED: E4orf6 proteins from all human adenoviruses form Cullin-based ubiquitin ligase complexes that, in association with E1B55K, target cellular proteins for degradation. While most are assembled with Cul5, a few utilize Cul2. BC-box motifs enable all these E4orf6 proteins to assemble ligase complexes with Elongins B and C. We also identified a Cul2-box motif used for Cul2 selection in all Cul2-based complexes. With this information, we set out to determine if other adenoviruses also possess the ability to form the ligase complex and, if so, to predict their Cullin usage. Here we report that all adenoviruses known to encode an E4orf6-like protein (mastadenoviruses and atadenoviruses) maintain the potential to form the ligase complex. We could accurately predict Cullin usage for E4orf6 products of mastadenoviruses and all but one atadenovirus. Interestingly, in nonhuman primate adenoviruses, we found a clear segregation of Cullin binding, with Cul5 utilized by viruses infecting great apes and Cul2 by Old/New World monkey viruses, suggesting that a switch from Cul2 to Cul5 binding occurred during the period when great apes diverged from monkeys. Based on the analysis of Cullin selection, we also suggest that the majority of human adenoviruses, which exhibit a broader tropism for the eye and the respiratory tract, exhibit Cul5 specificity and resemble viruses infecting great apes, whereas those that infect the gastrointestinal tract may have originated from monkey viruses that share Cul2 specificity. Finally, aviadenoviruses also appear to contain E4orf6 genes that encode proteins with a conserved XCXC motif followed by, in most cases, a BC-box motif. IMPORTANCE: Two early adenoviral proteins, E4orf6 and E1B55K, form a ubiquitin ligase complex with cellular proteins to ubiquitinate specific substrates, leading to their degradation by the proteasome. In studies with representatives of each human adenovirus species, we (and others) previously discovered that some viruses use Cul2 to form the complex, while others use Cul5. In the present study, we expanded our analyses to all sequenced adenoviruses and found that E4orf6 genes from all mast- and atadenoviruses encode proteins containing the motifs necessary to form the ligase complex. We found a clear separation in Cullin specificity between adenoviruses of great apes and Old/New World monkeys, lending support for a monkey origin for human viruses of the Human mastadenovirus A, F, and G species. We also identified previously unrecognized E4orf6 genes in the aviadenoviruses that encode proteins containing motifs permitting formation of the ubiquitin ligase.

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.

Genome analysis of four Old World monkey adenoviruses supports the proposed species classification of primate adenoviruses and reveals signs of possible homologous recombination.

Within the family Adenoviridae, presently Simian mastadenovirus A is the single species approved officially for monkey adenoviruses (AdVs), whilst the establishment of six further species (Simian mastadenovirus B to Simian mastadenovirus G) has been proposed in the last few years. We examined the genetic content and phylogenetic relationships of four Old World monkey (OWM) AdV types [namely simian AdV (SAdV)-8, -11, -16 and -19] for which it had been proposed that they should be classified into different AdV species: SAdV-11 to Human mastadenovirus G, and the other three viruses into three novel species. By full genome sequencing, we identified gene contents characteristic for the genus Mastadenovirus. Among the 36 ORFs, 2 genes of different lengths, predicted to encode the adenoviral cellular attachment protein (the fibre), were found. The E3 regions contained six genes, present in every OWM AdV, but lacked the E3 19K gene, which has seemingly appeared only in the ape (hominid) AdV lineages during evolution. For the first time in SAdVs, two other exons belonging to the gene of the so-called U exon protein were also predicted. Phylogenetic calculations, based on the fibre-1 and the major capsid protein, the hexon, implied that recombination events might have happened between different AdV species. Phylogeny inference, based on the viral DNA-dependent DNA polymerase and the penton base protein, further supported the species classification proposed earlier.

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.

UNLABELLED: 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.

Whole-genome sequencing of a green bush viper reovirus reveals a shared evolutionary history between reptilian and unusual mammalian orthoreoviruses.

In this study, we sequenced the whole genome of a reovirus isolated from a green bush viper (Atheris squamigera). The bush viper reovirus shared several features with other orthoreoviruses, including its genome organization. In phylogenetic analysis, this strain was monophyletic with Broome virus and baboon orthoreovirus, indicating that these viruses might have originated from a common ancestor. These new molecular data supplement previous information based mainly on biological properties of reptilian reoviruses, confirming their taxonomic position and broadening our knowledge of the evolution of members of the genus Orthoreovirus.

Development and use of a real-time polymerase chain reaction for the detection of group II invertebrate iridoviruses in pet lizards and prey insects.

Members of the genus Iridovirus (invertebrate iridoviruses [IIVs]) of the Iridoviridae family infect a wide range of invertebrates, mainly arthropods, but there have also been a few reports from other taxa. The cricket iridovirus described recently has been shown to infect a wide host range among insect orders and has also been described in several diseased reptiles. This virus together with the type species Chilo iridescent virus form a distinct "group II" in the genus. The aim of this study was to develop a fast and easy real-time polymerase chain reaction [quantitative (q)PCR] for the detection of these group II iridoviruses. In silico and in vitro assays demonstrated that the designed TaqMan primer-probe combination targeting a portion of the major capsid protein is specific for this group of IIVs. A sensitivity assay showed that it is able to detect as little as one copy of viral DNA. Direct comparison of cell culture isolation, nested (n)PCR, and qPCR methods has shown that PCR methods are 10(2)-10(3) more sensitive compared with the isolation method. In testing the three methods on routine diagnostic samples from lizards (n = 22) and crickets (n = 11), the nPCR and qPCR results were consistent with 19 positive lizards and 10 positive crickets, respectively, whereas isolation on cell culture detected only seven and six positives, respectively. QPCR is a fast, sensitive, and specific diagnostic method. Furthermore, it requires fewer handling steps than were previously required. It also allows the quantitation and comparison of the amounts of IIV DNA in samples.

Partial characterization of new adenoviruses found in lizards.

In the years 2011-2012, a consensus nested polymerase chain reaction was used for the detection of adenovirus (AdV) infection in reptiles. During this screening, three new AdVs were detected. One of these viruses was detected in three lizards from a group of green striped tree dragons (Japalura splendida). Another was detected in a green anole (Anolis carolinensis). A third virus was detected in a Jackson's chameleon (Chamaeleo jacksonii). Analysis of a portion of the DNA-dependent DNA polymerase genes of each of these viruses revealed that they all were different from one another and from all previously described reptilian AdVs. Phylogenetic analysis of the partial DNA polymerase gene sequence showed that all newly detected viruses clustered within the genus Atadenovirus. This is the first description of AdVs in these lizard species.

Exploring the Effectiveness of Acyclovir against Ranaviral Thymidine Kinases: Molecular Docking and Experimental Validation in a Fish Cell Line.

The effectiveness of acyclovir, a selective anti-herpesvirus agent, was tested both in silico and in vitro against two ranaviruses, namely the European catfish virus (ECV) and Frog virus 3 (FV3). ECV can cause significant losses in catfish aquaculture, while FV3 poses a risk to vulnerable amphibian populations. The genome of ranaviruses encodes thymidine kinases (TKs) similar to those of herpesviruses. Molecular docking simulations demonstrated that the acyclovir molecule can bind to the active sites of both investigated viral TKs in an orientation conducive to phosphorylation. Subsequently, the antiviral effect of acyclovir was tested in vitro in Epithelioma Papulosum Cyprini (EPC) cells with endpoint titration and qPCR. Acyclovir was used at a concentration of 800 µM, which significantly reduced the viral loads and titers of the ranaviruses. A similar reduction rate was observed with Ictalurid herpesvirus 2, which was used as a positive control virus. These promising results indicate that acyclovir might have a wider range of uses; besides its effectiveness against herpesviruses, it could also be used against ranavirus infections.

Discovery of the first sea turtle adenovirus and turtle associated circoviruses.

Turtles are an evolutionarily unique and morphologically distinctive order of reptiles, and many species are globally endangered. Although a high diversity of adenoviruses in scaled reptiles is well-documented, turtle adenoviruses remain largely understudied. To investigate their molecular diversity, we focused on the identification and characterisation of adenoviruses in turtle-derived organ, swab and egg samples. Since reptile circoviruses have been scarcely reported and no turtle circoviruses have been documented to date, we also screened our samples for circoviruses. Host-virus coevolution is a common feature of these viral families, so we aimed to investigate possible signs of this as well. Two screening projects were conducted: one on Brazilian samples collected from animals in their natural habitat, and the other on Hungarian pet shop samples. Nested PCR systems were used for the detection of adeno- and circoviruses and purified PCR products were Sanger sequenced. Phylogenetic trees for the viruses were reconstructed based on the adenoviral DNA polymerase and hexon genes, circoviral Rep genes, and for the turtle hosts based on mitochondrial cytochrome b amino acid sequences. During the screening, testadeno-, siadeno-, and circovirus strains were detected. The circovirus strains were classified into the genus Circovirus, exhibiting significant evolutionary divergence but forming a monophyletic clade within a group of fish circoviruses. The phylogenetic tree of turtles reflected their taxonomic relationships, showing a deep bifurcation between suborders and distinct monophyletic clades corresponding to families. A similar clustering pattern was observed among the testadenovirus strains in their phylogenetic tree. As a result, this screening of turtle samples revealed at least three new testadenoviruses, including the first sea turtle adenovirus, evidence of coevolution between testadenoviruses and their hosts, and the first turtle associated circoviruses. These findings underscore the need for further research on viruses in turtles, and more broadly in reptiles, to better understand their viral diversity and the evolutionary processes shaping host-virus interactions.

Detection of antibodies against paramyxoviruses in tortoises.

Sera from a total of 202 tortoises from six countries and nine species were tested for antibodies against four different reptilian paramyxoviruses (ferlaviruses, ferlaVs) by hemagglutination inhibition (HI) test. The viruses used were a tortoise PMV (tPMV) and three squamatid PMV isolates, each belonging to a different subgroup of ferlaV within the genus Ferlavirus. HI tests revealed that antibodies against ferlaVs occurred regularly in the tested samples (5.5%). One and a half percent of the tested samples have measurable antibody titers against the group A isolate, 3% had antibodies against the group B isolate, and 1% had antibodies against the group C isolate. The significantly highest number of positive reactions was detected against the tortoise isolate (5%). Most of the animals that tested positive for one of the snake isolates also tested positive in HI assays with the tortoise isolate. Of the samples from different origins, the sera from Great Britain showed the highest percentage of positive tested animals (10.3%, n = 39), followed by those from Spain (10%, n = 10), while none of the samples from Madagascar or Italy scored positive. Since in most cases animals from one country came from the same collection, this does not represent the real prevalence of ferlaV in tortoises in these countries but rather indicates that ferlaVs occur in a number of different countries and tortoise species.

Characterization of monkey adenoviruses with three fiber genes.

Although the occurrence of three fiber genes in monkey adenoviruses had already been described, the relatedness of the "extra" fibers have not yet been discussed. Here we report the genome analysis of two simian adenovirus (SAdV) serotypes from Old World monkeys and the phylogenetic analysis of the multiple fiber genes found in these and related AdVs. One of the newly sequenced serotypes (SAdV-2), isolated from a rhesus macaque (Macaca mulatta), was classified into species Human mastadenovirus G (HAdV-G), while the other serotype (SAdV-17), originating from a grivet (Chlorocebus aethiops), classified to Simian mastadenovirus F (SAdV-F). We identified unique features in the gene content of these SAdVs compared to those typical for other members of the genus Mastadenovirus. Namely, in the E1B region of SAdV-2, the 19K gene was replaced by an ITR repetition and a copy of the E4 ORF1 gene. Among the 37 genes in both SAdVs, three genes of different lengths, predicted to code for the cellular attachment proteins (the fibers), were found. These proteins exhibit high diversity. Yet, phylogenetic calculations of their conserved parts could reveal the probable evolutionary steps leading to the multiple-fibered contemporary HAdV and SAdV species. Seemingly, there existed (a) common ancestor(s) with two fiber genes for the lineages of the AdVs in species SAdV-B, -E, -F and HAdV-F, alongside a double-fibered ancestor for today's SAdV-C and HAdV-G, which later diverged into descendants forming today's species. Additionally, some HAdV-G members picked up a third fiber gene either to the left-hand or to the in-between position from the existing two. A SAdV-F progenitor also obtained a third copy to the middle, as observed in SAdV-17. The existence of three fiber genes in these contemporary AdVs brings novel possibilities for the design of optimised AdV-based vectors with potential multiple target binding abilities.

A screening of wild bird samples enhances our knowledge about the biodiversity of avian adenoviruses.

Wild birds are threatened by anthropic effects on a global scale, and their adenoviruses might contribute to their endangerment. Thus, it is important to reveal the real biodiversity of avian adenoviruses, as, unfortunately, this research topic is far from being prioritized. The turkey hemorrhagic enteritis is an economically important disease causing high mortalities, and its causative siadenoviral agent is only distantly related to other avian siadenoviruses in phylogenetic analyses. Both to enhance our knowledge about the biodiversity of wild bird adenoviruses and to possibly trace back the origin of the turkey hemorrhagic enteritis virus, numerous Hungarian wild bird samples were screened for adenoviruses using PCR, and the detected strains were typed molecularly. The screening revealed numerous new adenovirus types, several of which represent novel adenovirus species as well, in the genera Atadenovirus, Aviadenovirus and Siadenovirus.

New viruses from Lacerta monticola (Serra da Estrela, Portugal): further evidence for a new group of nucleo-cytoplasmic large deoxyriboviruses.

Lizard erythrocytic viruses (LEVs) have previously been described in Lacerta monticola from Serra da Estrela, Portugal. Like other known erythrocytic viruses of heterothermic vertebrates, these viruses have never been adapted to cell cultures and remain uncharacterized at the molecular level. In this study, we made attempts to adapt the virus to cell cultures that resulted instead in the isolation of a previously undetected Ranavirus closely related to FV3. The Ranavirus was subsequently detected by polymerase chain reaction (PCR) in the blood of infected lizards using primers for a conserved portion of the Ranavirus major capsid protein gene. Electron microscopic study of the new Ranavirus disclosed, among other features, the presence of intranuclear viruses that may be related to an unrecognized intranuclear morphogenetic process. Attempts to detect by PCR a portion of the DNA polymerase gene of the LEV in infected lizard blood were successful. The recovered sequence had 65.2/69.4% nt/aa% homology with a previously detected sequence from a snake erythrocytic virus from Florida, which is ultrastructurally different from the studied LEV. These results further support the hypothesis that erythrocytic viruses are related to one another and may represent a new group of nucleo-cytoplasmic large deoxyriboviruses.

Accumulation of a low pathogenic avian influenza virus in zebra mussels (Dreissena polymorpha).

In order to investigate the potential role of mussels as a vector of influenza A viruses, we exposed zebra mussels (Dreissena polymorpha) to natural lake water containing a low pathogenic H5N1 avian influenza virus. Mussels were kept in water containing virus for 48 hr, then transferred into fresh water for another 14 days. Virus detection in mussels and water samples was performed by quantitative real-time reverse transcriptase-PCR (qRRT-PCR) and egg culture methods. Virus uptake was detected in all of the mussel groups that were exposed to virus. Even after 14 days in fresh water, virus could still be detected in shellfish material by both qRRT-PCR and egg culture methods. The present study demonstrates that zebra mussels are capable of accumulating influenza A viruses from the surrounding water and that these viruses remain in the mussels over an extended period of time.

PCR-sequence characterization of new adenoviruses found in reptiles and the first successful isolation of a lizard adenovirus.

A consensus nested PCR was used to screen diagnostic samples from approximately 70 reptiles for the presence of adenoviruses (AdV) in the years 2006-2007. Classical virus isolation methods were also used with all samples. After adenoviruses were detected in a group of helodermatid lizards in a Danish zoo, a follow-up study was also carried out on lizards from this group (10 Mexican beaded lizards and 24 Gila monsters) over the period of a year. Adenoviruses were detected in a total of 26 lizards and snakes by PCR. The PCR amplicons from all positive animals were sequenced and the resulting polymerase gene sequences were used for phylogenetic analysis. Altogether six Agamid AdVs were amplified, with a minimal sequence variation between one another and between these and GenBank Agamid AdVs. The sequence obtained from one of the Gila monsters is identical with the GenBank Helodermatid AdV, while the sequences from the Mexican beaded lizards differ from this. In a snake collection we have detected a new AdV from an Asp viper. All of the above mentioned adenoviruses cluster in the Atadenovirus genus. However, the sequence from a new Varanid AdV detected in this study clusters outside this genus. On cell culture, viruses were isolated from three of the AdV positive helodermatid lizards (one Mexican beaded lizard and two Gila monsters) and identified as AdVs based on electron microscopy and PCR and sequencing using cell culture supernatant. This is the first report of the successful isolation of a lizard AdV.

Detection and partial characterization of herpesviruses from Egyptian tortoises (Testudo kleinmanni) imported into Italy from Libya.

A group of approximately 370 Egyptian tortoises (Testudo kleinmanni) and 36 spur-thighed tortoises (Testudo graeca) were illegally imported into Italy from Libya. Within 6 mo of their entry into Italy, all but 40 of the Egyptian tortoises had died with signs of severe stomatitis. Herpesviruses were detected from the tongues of seven Egyptian tortoises by polymerase chain reaction (PCR) and virus isolation. Sequencing of a portion of the UL39 homologue of the herpesviruses from three different tortoises demonstrated that the viruses were identical to one another and identical to a herpesvirus isolated from a Hermann's tortoise (Testudo hermanni) in Germany. This is the first description of the detection of a herpesvirus from diseased Egyptian tortoises. That these animals were imported into Europe from Libya provides circumstantial evidence for the presence of herpesviruses among tortoises in northern Africa.

Detection and Characterization of Invertebrate Iridoviruses Found in Reptiles and Prey Insects in Europe over the Past Two Decades.

Invertebrate iridoviruses (IIVs), while mostly described in a wide range of invertebrate hosts, have also been repeatedly detected in diagnostic samples from poikilothermic vertebrates including reptiles and amphibians. Since iridoviruses from invertebrate and vertebrate hosts differ strongly from one another based not only on host range but also on molecular characteristics, a series of molecular studies and bioassays were performed to characterize and compare IIVs from various hosts and evaluate their ability to infect a vertebrate host. Eight IIV isolates from reptilian and orthopteran hosts collected over a period of six years were partially sequenced. Comparison of eight genome portions (total over 14 kbp) showed that these were all very similar to one another and to an earlier described cricket IIV isolate, thus they were given the collective name lizard-cricket IV (Liz-CrIV). One isolate from a chameleon was also subjected to Illumina sequencing and almost the entire genomic sequence was obtained. Comparison of this longer genome sequence showed several differences to the most closely related IIV, Invertebrateiridovirus6 (IIV6), the type species of the genus Iridovirus, including several deletions and possible recombination sites, as well as insertions of genes of non-iridoviral origin. Three isolates from vertebrate and invertebrate hosts were also used for comparative studies on pathogenicity in crickets (Gryllusbimaculatus) at 20 and 30 °C. Finally, the chameleon isolate used for the genome sequencing studies was also used in a transmission study with bearded dragons. The transmission studies showed large variability in virus replication and pathogenicity of the three tested viruses in crickets at the two temperatures. In the infection study with bearded dragons, lizards inoculated with a Liz-CrIV did not become ill, but the virus was detected in numerous tissues by qPCR and was also isolated in cell culture from several tissues. Highest viral loads were measured in the gastro-intestinal organs and in the skin. These studies demonstrate that Liz-CrIV circulates in the pet trade in Europe. This virus is capable of infecting both invertebrates and poikilothermic vertebrates, although its involvement in disease in the latter has not been proven.

Three genetically distinct ferlaviruses have varying effects on infected corn snakes (Pantherophis guttatus).

Ferlaviruses are important pathogens in snakes and other reptiles. They cause respiratory and neurological disease in infected animals and can cause severe disease outbreaks. Isolates from this genus can be divided into four genogroups-A, B, and C, as well as a more distantly related sister group, "tortoise". Sequences from large portions (5.3 kb) of the genomes of a variety of ferlavirus isolates from genogroups A, B, and C, including the genes coding the surface glycoproteins F and HN as well as the L protein were determined and compared. In silico analyses of the glycoproteins of genogroup A, B, and C isolates were carried out. Three isolates representing these three genogroups were used in transmission studies with corn snakes (Pantherophis guttatus), and clinical signs, gross and histopathology, electronmicroscopic changes in the lungs, and isolation of bacteria from the lungs were evaluated. Analysis of the sequences supported the previous categorization of ferlaviruses into four genogroups, and criteria for definition of ferlavirus genogroups and species were established based on sequence identities (80% resp. 90%). Analysis of the ferlavirus glycoprotein models showed parallels to corresponding regions of other paramyxoviruses. The transmission studies showed clear differences in the pathogenicities of the three virus isolates used. The genogroup B isolate was the most and the group A virus the least pathogenic. Reasons for these differences were not clear based on the differences in the putative structures of their respective glycoproteins, although e.g. residue and consequential structure variation of an extended cleavage site or changes in electrostatic charges at enzyme binding sites could play a role. The presence of bacteria in the lungs of the infected animals also clearly corresponded to increased pathogenicity. This study contributes to knowledge about the structure and phylogeny of ferlaviruses and lucidly demonstrates differences in pathogenicity between strains of different genogroups.