Genus Mastadenovirus Research Articles

A Novel Mastadenovirus from Nyctalus noctula Which Represents a Distinct Evolutionary Branch of Viruses from Bats in Europe.

Bats are natural hosts of a wide variety of viruses, including adenoviruses. European bats are known to carry mastadenoviruses categorized as species B (widespread in European Vespertilionidae bats) and whose taxonomy has not been clarified. We examined fecal samples from Vespertilionidae bats (five species) captured in central Russia and found that 2/12 (16%) were positive for mastadenoviruses. The partial genome of the mastadenovirus was assembled from Pipistrellus nathusii, representing the bat adenovirus species B. The complete genome (37,915 nt) of a novel mastadenovirus was assembled from Nyctalus noctula and named BatAdV/MOW15-Nn19/Quixote. Comparative studies showed significant divergence of the Quixote genome sequence from European bat mastadenoviruses, while the only known virus showing low similarity was the isolate WA3301 from an Australian bat, and together they formed a subclade that separated from other BatAdVs. Phylogenetic and comparative analysis of the protein-coding genes provided evidence that Quixote is related to a novel species within the genus Mastadenovirus, provisionally named "K" (as the next available letter for the species). Phylogenetic analyses revealed that some earlier viruses from Western European bats, for which only partial DNA polymerase genes are known, are most likely members of the tentatively named species "K". Thus, at least two species of mastadenovirus are circulating in bats throughout Europe, from western to eastern areas.

Identification of a novel adenovirus in liver tissue sample of the Great Himalayan leaf-nosed bat (Hipposideros armiger).

Bats are important reservoirs for many zoonotic viruses. To explore and monitor potential novel viruses carried by bats, 21 liver samples of bats (Hipposideros armiger) were collected from Yunnan Province in southern China. Only one (4.8%) of all models was detected with adenovirus. The whole genome strain obtained by the viral metagenomics method combined with PCR was temporarily named YN01. The complete genome of YN01 was 37,676bp, with a G + C content of 55.20% and 28 open reading frames. Phylogenetic analysis indicated that the strain YN01 can be classified as genus Mastadenovirus and was the most similar to the adenovirus isolated from Rhinolophus sinicus in China in 2016. The analysis is needed to verify the possibility of cross-species transmission. This virological investigation has increased our understanding of the ecology of bat-borne viruses in this area and provided a reference for possible future infectious diseases.

Genetic diversity of adenovirus in neotropical bats from Brazil.

Bats can harbor a diversity of viruses, such as adenovirus. Ten different species of bat adenoviruses (BtAdV A to J) have been previousdescribedworlwide. In Brazil, BtAdV was described in three species of phyllostomid species: Artibeus lituratus, Desmodus rotundus, and Sturnira lilium. There are around 180 bat species in Brazil, with 67% inhabiting the Atlantic Forest, with few information about the circulation of BtAdV in this biome. We aimed to describe the molecular detection and the phylogenetic characterization and suggest a classification of BtAdVs circulating in bats from the Brazilian Atlantic Forest. We collected 382 oral and rectal swabs from 208 bats between 2014-2015 and 2020-2021 fromSão Paulo, Pernambuco, and Santa Catarina Brazilian states. The adenovirus detection was done by a nested PCR targeting the DNA polymerase gene, and all positive samples were sequenced by the Sanger method. The phylogenetic analyses were based on the amino acid sequences using the MEGA 7 and BEAST software. We obtained 16 positive animals (detection rate 7.7%) belonging to seven bat species: Artibeus lituratus, Carollia perspicillata, Sturnira lilium, Molossus molossus, and the first record of Phyllostomus discolor, Eptesicus diminutus, and Myotis riparius. The phylogenetic analysis based on partial amino acid sequences showed that all obtained AdV sequences belong to the Mastadenovirus genus. We observed a high genetic diversity of BtAdV and identified eleven potential BtAdV species circulating in Brazil (BtAdV K to U). Our results contribute to the epidemiological surveillance of adenovirus, increasing the knowledge about the viral diversity and the distribution of AdV in bats from the Atlantic Forest.

Detection and molecular characterisation of canine adenovirus type 1 from a fatal case of infectious canine hepatitis from India

AbstractCanine adenovirus (CAdV), a non‐enveloped, linear dsDNA virus, is a member of the genus Mastadenovirus of the family Adenoviridae. Two different serotypes of CAdVs, canine adenovirus type 1 (CAdV‐1), which causes infectious canine hepatitis (ICH), and canine adenovirus type 2 (CAdV‐2), which causes canine infectious laryngotracheitis, have been identified in dogs. In this report, we discuss a case of fatal CAdV‐1 infection in a 4‐month‐old, male, unvaccinated, Rajapalayam breed dog. Postmortem examination results were suggestive of CAdV‐1 infection, which was confirmed by histopathological examination, molecular characterisation and phylogenetic analysis. Sequence analysis of the CAdV‐1 strain showed a close similarity with the Australasian‐Europe cluster, a high identity (99.78% nucleotide and 98.91% amino acid identities) with a CAdV‐1 isolate (KU75571) from a fox in the United Kingdom, and a unique substitution (W126S) in the E3 protein encoding sequence.

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.

2197. Genomic Surveillance for Human Adenovirus Infections among Department of Defense Beneficiaries in the 2018-2019 Season

Abstract Background Human adenoviruses (family Adenoviridae, genus Mastadenovirus) (HAdV) species B, C and E cause acute respiratory illnesses (ARI). In particular, respiratory HAdV types 3, 4, 7, 11, 14, 21 and 55 were associated with ARI outbreaks in the US and in other countries. The risk of outbreaks can be effectively controlled by implementation of the live HAdV types 4 and 7 vaccines or mitigated by screening and preventive measures. Methods During the influenza season 2018 – 2019, the DoD Global Respiratory Pathogen Surveillance Program (DoDGRS) received 24,300 specimens from its surveillance network. In total, 322 (1.3%) respiratory specimens were positive for HAdV in molecular assays. HAdV samples that produced positive cytopathic effects (CPE) in subsequent viral cultivation were subjected to next-gen sequencing (NGS) for genome sequence assembly, HAdV genome typing, whole genome phylogeny, and sequence comparative analyses to identify sequence insertions, deletions and amino acid mutations. Results Out of 166 viral culture samples available for NGS, whole genome sequences were obtained for 161 isolates. A variety types of HAdV were identified, including HAdV-1 (N = 15), HAdV-2 (N = 26), HAdV-3 (N = 60), HAdV-4 (N = 13), HAdV-5 (N = 6), HAdV-6 (N = 3), HAdV-7 (N = 12), HAdV-14 (N = 10), HAdV-55 (N = 1), and HAdV-56 (N = 1). HAdV types 4, 7 and 14, which were reported causing ARI outbreaks in the US military and other congregate settings, were found in clustered cases in several US states. Comparative sequence analyses of these isolates revealed the emergence of novel genetic mutations despite the stability of adenovirus genomes. Conclusion Routine clinical respiratory pathogen diagnostics or molecular surveillance of respiratory diseases detect adenovirus without determination of HAdV type. Human adenoviruses are genetically diverse and can infect a number of tissues with severities varied from mild to fatal. Enhanced genomic surveillance of HAdV in the US and worldwide will shed light on prevalence, genetic divergence, and viral evolution of human adenoviruses and inform timely risk assessment and countermeasures. Disclosures All Authors: No reported disclosures.

Simian adenoviruses: Molecular and serological survey in monkeys and humans in Thailand

Simian adenoviruses are in the genus Mastadenovirus of the family Adenoviridae. This family is composed of non-enveloped, double-stranded DNA viruses that infect a wide range of animals. Mastadenoviruses infect mammals, including non-human primates and humans. The close genetic relatedness between simian and human adenoviruses, with its associated potential for the cross-species transmission of zoonotic adenoviruses from monkeys to humans and vice versa, poses important health concerns and thus warrants research. In this study, we performed a molecular survey of adenoviruses in monkeys in Thailand. Most of the monkeys tested here were long-tailed macaques, free-ranging in areas close to human territories across four provinces: Ratchaburi, Kanchanaburi, Lopburi, and Prachuap Khiri Khan. A few fecal samples from captive wild monkeys (a stump-tailed macaque, pig-tailed macaques, gibbons, and a leaf monkey) were also tested. Adenoviruses were detected in 33.3% (70 out of 210) of the fecal or rectal swab samples. The viruses identified in these samples included Simian adenovirus (SAdV)-A, SAdV-B, SAdV-H, Human adenovirus (HAdV)-D, HAdV-G, and a bat adenovirus species. One SAdV-B, SAdV RBR-7-10, was isolated from a long-tailed macaque fecal sample and identified by mass spectrometry. Its full hexon gene and nearly complete DNA polymerase gene were sequenced and analyzed, and the virions were imaged by transmission electron microscopy. The SAdV RBR-7-10 virus was used in a microneutralization assay to identify virus-specific antibodies in monkey plasma and human serum samples collected from the same areas in Prachuap Khiri Khan Province. We detected neutralizing antibodies against SAdV RBR-7-10 in 6.8% (n = 103) of the monkey samples but in none of the 125 human serum samples, suggesting no cross-species transmission of SAdV RBR-7-10 occurred at this study site. Nevertheless, a continuing surveillance of pathogens in monkeys is warranted to quickly identify possible emerging zoonotic outbreaks.

Genome Analyses of Ten New Ape Adenoviruses with Similarity to Human Mastadenovirus C.

The adenoviruses (AdVs) isolated from humans are taxonomically grouped in seven different species in the Mastadenovirus genus (HAdV-A through G). AdVs isolated from apes are often included in one of the human AdV species. Here we describe the sequence analyses of ten new AdVs that are related to the HAdV-C species and that were isolated from healthy western lowland gorillas, bonobos, chimpanzees, and orangutans kept in Dutch zoos. We analyzed these viruses and compared their genome sequences to those of human- and ape-derived AdV sequences in the NCBI GenBank database. Our data demonstrated that the ape-derived viruses clustering to HAdV-C are markedly distinct from the human HAdV-C species in the size and nucleotide composition (%GC) of their genome, differ in the amino-acid sequence of AdV proteins, and have longer RGD-loops in their penton-base proteins. The viruses form three well-separated clades (the human, the gorilla, and the combined group of the bonobo and chimpanzee viruses), and we propose that these should each be given species-level ranks. The Ad-lumc005 AdV isolated from orangutans was found to be very similar to the gorilla AdVs, and bootstrap inference provided evidence of recombination between the orangutan AdV and the gorilla AdVs. This suggests that this virus may not be a genuine orangutan AdV but may have been transferred from a gorilla to an orangutan host.

Complete genome sequence of a novel bat mastadenovirus C strain isolated from Rhinolophus cornutus in Japan.

Here, we report a novel bat adenovirus strain isolated from apparently healthy bats of the species Rhinolophus cornutus in Japan. The genome of the isolate was 36,506 bp in length and encoded at least 33 proteins. Phylogenetic analysis of the DNA polymerase amino acid sequence, which provides one demarcation criterion for adenoviral species, indicated that the isolate belongs to the species Bat mastadenovirus C in the genus Mastadenovirus. Most of the encoded proteins shared high sequence similarity with those of known bat adenovirus C strains detected in different species of Rhinolophus, whereas the fiber protein and some E3- and E4-related proteins shared moderate similarity, and only the large E3 protein, which contains several host immune-suppression-related motifs, showed considerably lower similarity.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00705-021-05357-8.

Изучение распространенности аденовирусов в популяциях диких парнокопытных Московской области

Adenoviruses are a group of viral pathogens that are commonly found in various vertebrates. The systematics of these viruses is based on the features of their biology and similarities in the organization of the genome. Representatives of the genera Mastadenovirus and Atadenovirus are pathogens of various ungulates, occurring in cattle and sheep, as well as in various deer species. The article presents the results of screening PCR studies aimed at detecting adenoviruses in biological material from wild mooses and roe deer shot in the Moscow region during the winter hunting seasons of 2019–2021. During PCR with electrophoretic detection of amplification products, primers were used for different parts of the adenovirus genome. Primers for amplification of the E2A fragment of the adenovirus genome made it possible to detect adenovirus DNA in the material from roe deer. The phylogenetic analysis of the nucleotide sequence confirmed that the virus belongs to adenoviruses and made it possible to attribute the detected pathogen to the genus Mastadenovirus. The phylogenetic tree showed the relationship of the detected virus with adenovirus of cattle type 3, mastadenoviruses of the white-tailed and northern salts. It is assumed that the roe deer is the owner of its own species of adenovirus. The features of diseases caused by mastadenoviruses and the problems associated with obtaining information about the complete genome and biological properties of the virus are discussed.

First Report on Detection and Molecular Characterization of Adenoviruses in the Small Indian Mongoose (Urva auropunctata).

Using a broad-range nested PCR assay targeting the DNA-dependent DNA polymerase (pol) gene, we detected adenoviruses in 17 (20.48%) out of 83 fecal samples from small Indian mongooses (Urva auropunctata) on the Caribbean island of St. Kitts. All 17 PCR amplicons were sequenced for the partial pol gene (~300 bp, hereafter referred to as Mon sequences). Fourteen of the 17 Mon sequences shared maximum homology (98.3–99.6% and 97–98.9% nucleotide (nt) and deduced amino acid (aa) sequence identities, respectively) with that of bovine adenovirus-6 (species Bovine atadenovirus E). Mongoose-associated adenovirus Mon-39 was most closely related (absolute nt and deduced aa identities) to an atadenovirus from a tropical screech owl. Mon-66 shared maximum nt and deduced aa identities of 69% and 71.4% with those of atadenoviruses from a spur-thighed tortoise and a brown anole lizard, respectively. Phylogenetically, Mon-39 and Mon-66 clustered within clades that were predominated by atadenoviruses from reptiles, indicating a reptilian origin of these viruses. Only a single mongoose-associated adenovirus, Mon-34, was related to the genus Mastadenovirus. However, phylogenetically, Mon-34 formed an isolated branch, distinct from other mastadenoviruses. Since the fecal samples were collected from apparently healthy mongooses, we could not determine whether the mongoose-associated adenoviruses infected the host. On the other hand, the phylogenetic clustering patterns of the mongoose-associated atadenoviruses pointed more towards a dietary origin of these viruses. Although the present study was based on partial pol sequences (~90 aa), sequence identities and phylogenetic analysis suggested that Mon-34, Mon-39, and Mon-66 might represent novel adenoviruses. To our knowledge, this is the first report on the detection and molecular characterization of adenoviruses from the mongoose.

RETRACTED ARTICLE: Immunoinformatic mapping of conserved regions of the human adenovirus hexon protein

Human Adenoviruses (HAdVs) are important infectious agents associated with high incidence and morbidity and mortality in immunocompromised patients, such as those undergoing allogeneic hematopoietic progenitor cell transplantation (allo-TCPH). HAdVs belong to the family Adenoviridae, genus Mastadenovirus, and are further classified into seven species (A–G) and 103 genotypes, characterized so far. The HAdV capsid consists mainly of the hexon protein, which has four highly conserved regions (CR1—4) and known for their immunogenic potential, being one of the main targets of the T and B cell-mediated anti-HAdV immune response of the present study was to perform the mapping of potentially immunogenic epitopes, located in the HAdV hexon CRs and to evaluate the HAdV infection in patients undergoing allo-TCPH. To this end, predictions of T and B cell epitopes and IFN-γ induction were performed, considering 101 HAdV genotypes with sequences available in databases. The most conserved and best-classified epitopes were then selected by the prediction programs to perform the molecular docking analysis with HLA alleles of the study population, consisting of nine adult patients undergoing allo-TCPH. It was also carried out the HAdV research by TaqMan real-time polymerase chain reaction (qPCR) in the patient's serum samples. As a result, regions containing overlapping T and B cell epitopes were obtained and, based on immunoinformatics analysis (prediction and molecular docking), two peptides with high conservation and immunogenic potential were designed. Nine patients were positive for HAdV by qPCR TaqMan, with the average viral load found in the serum samples of the study population being 6.71 × 1011 CG / mL. The genomic sequencing of the positive samples returned sequences that showed 100% similarity with sequences of the hexad protein of HAdV C, deposited in a database. The present study allowed the mapping of the main immunogenic regions located in the HAdV hexon CRs, as well as the construction of two peptides that will be used in future studies to assess the immune response of patients undergoing allo-TCPH, participants in the present study.

Molecular Detection of Human Adenovirus and Rotavirus in Feces of White-Eared Opossums.

The white-eared opossums (Didelphis albiventris) is the largest Brazilian marsupial and a great example of animal synanthropy. Considering the high potential as a carrier of viruses originating from environmental contamination, the presence of Human adenovirus (AdV) and rotavirus was investigated in the feces of rescued white-eared opossums, which were in the process of rehabilitation. The feces of 49 animals were initially investigated by immunochromatography, with three samples positive for AdV and one sample positive for rotavirus. When submitted to PCR and nested PCR, the samples of six animals were positive for AdV and three animals were positive for group A rotavirus. Two positive samples in the immunochromatographic assay were not confirmed by PCR. After sequencing and phylogenetic analysis of AdV samples, all were identified within the genus Mastadenovirus, one being HAdV-C, four HAdV-E, and one being similar to a Mastadenovirus found in primates. This is the first report of molecular confirmation of human adenovirus and rotavirus in white-eared opossums. These data could be important of anticipation some emerging diseases and their effects on ecosystems health.

Isolation and initial propagation of guinea pig adenovirus (GPAdV) in Cavia porcellus cell lines.

Background: The lack of adequate in vitro systems to isolate and propagate guinea pig adenovirus (GPAdV), a prevalent cause of respiratory illness of varaible severity in laboratory guinea pig colonies worldwide, has precluded its formal characterization to allow for the development of comprehensive diagnostic assays, and for the execution of complex pathogenesis and basic virology studies. Methods: Two strains of GPAdV were isolated in guinea pig ( Cavia porcellus) cell cultures from frozen archival infected animal tissue originated from colony outbreaks of pneumonia in Australia and the Czech Republic in 1996. Results: Commercially available guinea pig cell lines from colorectal carcinoma (GPC-16), fetal fibroblast (104-C1) and lung fibroblast (JH4 C1), and the tracheal epithelial cell line GPTEC-T developed in this study were able to support viral infection and early propagation. Sufficient viral DNA was recovered from cell cultures to PCR-amplify and obtain sequence data for the complete hexon gene and partial DNA polymerase and penton base genes. Phylogenetic analysis for the three regions of the genome provided strong evidence confirming GPAdV as a unique species in the genus Mastadenovirus. Conclusions: This study demonstrated the feasibility of propagating GPAdV in cultures of immortalized lines of GP cells of a variety of types, thus establishing a critical foundation for the development of a robust culture platform for virus stock production and titration. The generation and analysis of whole GPAdV genome sequences will provide additional data for a comprehensive description of the genetic organization of the viral genome and for a better assessment of genetic diversity between the two isolated strains.

Isolation and initial propagation of guinea pig adenovirus (GPAdV) in Cavia porcellus cell lines

Background: The lack of adequate in vitro systems to isolate and propagate guinea pig adenovirus (GPAdV), a prevalent cause of respiratory illness of varaible severity in laboratory guinea pig colonies worldwide, has precluded its formal characterization to allow for the development of comprehensive diagnostic assays, and for the execution of complex pathogenesis and basic virology studies. Methods: Two strains of GPAdV were isolated in guinea pig ( Cavia porcellus) cell cultures from frozen archival infected animal tissue originated from colony outbreaks of pneumonia in Australia and the Czech Republic in 1996. Results: Commercially available guinea pig cell lines from colorectal carcinoma (GPC-16), fetal fibroblast (104-C1) and lung fibroblast (JH4 C1), and the tracheal epithelial cell line GPTEC-T developed in this study were able to support viral infection and early propagation. Sufficient viral DNA was recovered from cell cultures to PCR-amplify and obtain sequence data for the complete hexon gene and partial DNA polymerase and penton base genes. Phylogenetic analysis for the three regions of the genome provided strong evidence confirming GPAdV as a unique species in the genus Mastadenovirus. Conclusions: This study demonstrated the feasibility of propagating GPAdV in cultures of immortalized lines of GP cells of a variety of types, thus establishing a critical foundation for the development of a robust culture platform for virus stock production and titration. The generation and analysis of whole GPAdV genome sequences will provide additional data for a comprehensive description of the genetic organization of the viral genome and for a better assessment of genetic diversity between the two isolated strains.

Full genome sequence analysis of a novel adenovirus from a captive polar bear (Ursus maritimus)

The presence of a novel adenovirus (AdV) was detected by PCR and sequencing, in the internal organs of a captive polar bear that had died in the Budapest zoo. The virus content of the samples proved to be high enough to allow for conventional Sanger sequencing on PCR-amplified genomic fragments. With this approach, the sequence of the entire genome of the putative polar bear adenovirus 1 (PBAdV-1) was obtained. Although the genome was found to be short, consisting of 27,952 base pairs merely, with a relatively balanced G + C content of 46.3 %, its organisation corresponded largely to that of a typical mastadenovirus. Every genus-common gene could be identified except that of protein IX. The short E3 region of the PBAdV-1 consisted of two novel, supposedly type-specific ORFs only, whereas no homologue of any of the E3 genes, usually conserved in mastadenoviruses, such as for example that of the 12.5 K protein, were present. In the E4 region, only the highly conserved gene of the 34 K protein was found besides two novel ORFs showing no homology to any known E4 ORFs. In silico sequence analysis revealed putative splicing donor and acceptor sites in the genes of the E1A, IVa2, DNA-dependent DNA polymerase, pTP, 33 K proteins, and also of U exon protein, all being characteristic for mastadenoviruses. Phylogenetic calculations, based on various proteins, further supported that the newly-detected PBAdV is the representative of a new species within the genus Mastadenovirus, and may represent the evolutionary lineage of adenoviruses that coevolved with carnivorans.

Two novel adenoviruses found in Cave Myotis bats (Myotis velifer) in Oklahoma.

Bats are carriers of potentially zoonotic viruses, therefore it is crucial to identify viruses currently found in bats to better understand how they are maintained in bat populations and evaluate risks for transmission to other species. Adenoviruses have been previously detected in bats throughout the world, but sampling is still limited. In this study, 30 pooled-guano samples were collected from a cave roost of Myotis velifer in Oklahoma. A portion of the DNA polymerase gene from Adenoviridae was amplified successfully in 18 M. velifer samples; however, DNA sequence was obtained from only 6 of these M. velifer samples. One was collected in October 2016, one in March 2017, and 4 in July 2017. The October and March samples contained viral DNA that was 3.1% different from each other but 33% different than the novel viral sequence found in the July 2017 samples. Phylogenetic analysis of these fragments confirmed our isolates were from the genus Mastadenovirus and had genetic diversity ranging from 20 to 50% when compared to other bat adenoviruses.

Isolation and initial propagation of guinea pig adenovirus (GPAdV) in Cavia porcellus cell lines

Background: The lack of adequate in vitro systems to isolate and propagate guinea pig adenovirus (GPAdV), a prevalent cause of respiratory illness of varaible severity in laboratory guinea pig colonies worldwide, has precluded its formal characterization to allow for the development of comprehensive diagnostic assays, and for the execution of complex pathogenesis and basic virology studies. Methods: Two strains of GPAdV were isolated in guinea pig ( Cavia porcellus) cell cultures from frozen archival infected animal tissue originated from colony outbreaks of pneumonia in Australia and the Czech Republic in 1996. Results: Commercially available guinea pig cell lines from colorectal carcinoma (GPC-16), fetal fibroblast (104-C1) and lung fibroblast (JH4 C1), and the tracheal epithelial cell line GPTEC-T developed in this study were able to support viral infection and early propagation. Sufficient viral DNA was recovered from cell cultures to PCR-amplify and obtain sequence data for the complete hexon gene and partial DNA polymerase and penton base genes. Phylogenetic analysis for the three regions of the genome provided strong evidence confirming GPAdV as a unique species in the genus Mastadenovirus. Conclusions: This study demonstrated the feasibility of propagating GPAdV in cultures of immortalized lines of GP cells of a variety of types, thus establishing a critical foundation for the development of a robust culture platform for virus stock production and titration. The generation and analysis of whole GPAdV genome sequences will provide additional data for a comprehensive description of the genetic organization of the viral genome and for a better assessment of genetic diversity between the two isolated strains.

Characterization of a novel species of adenovirus from Japanese microbat and role of CXADR as its entry factor

Recently, bat adenoviruses (BtAdVs) of genus Mastadenovirus have been isolated from various bat species, some of them displaying a wide host range in cell culture. In this study, we isolated two BtAdVs from Japanese wild microbats. While one isolate was classified as Bat mastadenovirus A, the other was phylogenetically independent of other BtAdVs. It was rather related to, but serologically different from, canine adenoviruses. We propose that the latter, isolated from Asian parti-colored bat, should be assigned to a novel species of Bat mastadenovirus. Both isolates replicated in various mammalian cell lines, implying their wide cell tropism. To gain insight into cell tropism of these BtAdVs, we investigated the coxsackievirus and adenovirus receptor (CXADR) for virus entry to the cells. We prepared CXADR-knockout canine kidney cells and found that replication of BtAdVs was significantly hampered in these cells. For confirmation, their replication in canine CXADR-addback cells was rescued to the levels with the original cells. We also found that viral replication was corrected in human or bat CXADR-transduced cells to similar levels as in canine CXADR-addback cells. These results suggest that BtAdVs were able to use several mammalian-derived CXADRs as entry factors.

Structure and N-acetylglucosamine binding of the distal domain of mouse adenovirus 2 fibre.

Murine adenovirus 2 (MAdV-2) infects cells of the mouse gastrointestinal tract. Like human adenoviruses, it is a member of the genus Mastadenovirus, family Adenoviridae. The MAdV-2 genome has a single fibre gene that expresses a 787 residue-long protein. Through analogy to other adenovirus fibre proteins, it is expected that the carboxy-terminal virus-distal head domain of the fibre is responsible for binding to the host cell, although the natural receptor is unknown. The putative head domain has little sequence identity to adenovirus fibres of known structure. In this report, we present high-resolution crystal structures of the carboxy-terminal part of the MAdV-2 fibre. The structures reveal a domain with the typical adenovirus fibre head topology and a domain containing two triple β-spiral repeats of the shaft domain. Through glycan microarray profiling, saturation transfer difference nuclear magnetic resonance spectroscopy, isothermal titration calorimetry and site-directed mutagenesis, we show that the fibre specifically binds to the monosaccharide N-acetylglucosamine (GlcNAc). The crystal structure of the complex reveals that GlcNAc binds between the AB and CD loops at the top of each of the three monomers of the MAdV-2 fibre head. However, infection competition assays show that soluble GlcNAc monosaccharide and natural GlcNAc-containing polymers do not inhibit infection by MAdV-2. Furthermore, site-directed mutation of the GlcNAc-binding residues does not prevent the inhibition of infection by soluble fibre protein. On the other hand, we show that the MAdV-2 fibre protein binds GlcNAc-containing mucin glycans, which suggests that the MAdV-2 fibre protein may play a role in viral mucin penetration in the mouse gut.