Isolation and Characterization of Akhmeta Virus from Wild-Caught Rodents (Apodemus spp.) in Georgia.

In 2013, a novel orthopoxvirus was detected in skin lesions of two cattle herders from the Kakheti region of Georgia (country); this virus was named Akhmeta virus. Subsequent investigation of these cases revealed that small mammals in the area had serological evidence of orthopoxvirus infections, suggesting their involvement in the maintenance of these viruses in nature. In October 2015, we began a longitudinal study assessing the natural history of orthopoxviruses in Georgia. As part of this effort, we trapped small mammals near Akhmeta (n = 176) and Gudauri (n = 110). Here, we describe the isolation and molecular characterization of Akhmeta virus from lesion material and pooled heart and lung samples collected from five wood mice (Apodemus uralensis and Apodemus flavicollis) in these two locations. The genomes of Akhmeta virus obtained from rodents group into 2 clades: one clade represented by viruses isolated from A. uralensis samples, and one clade represented by viruses isolated from A. flavicollis samples. These genomes also display several presumptive recombination events for which gene truncation and identity have been examined.IMPORTANCE Akhmeta virus is a unique Orthopoxvirus that was described in 2013 from the country of Georgia. This paper presents the first isolation of this virus from small mammal (Rodentia; Apodemus spp.) samples and the molecular characterization of those isolates. The identification of the virus in small mammals is an essential component to understanding the natural history of this virus and its transmission to human populations and could guide public health interventions in Georgia. Akhmeta virus genomes harbor evidence suggestive of recombination with a variety of other orthopoxviruses; this has implications for the evolution of orthopoxviruses, their ability to infect mammalian hosts, and their ability to adapt to novel host species.

Bioinformatics for Analysis of Poxvirus Genomes.

In recent years, there have been numerous technological advances in the field of molecular biology; these include next- and third-generation sequencing of DNA genomes and mRNA transcripts and mass spectrometry of proteins. Perhaps, however, it is genome sequencing that impacts a virologist the most. In 2017, more than 480 complete genome sequences of poxviruses have been generated, and are constantly used in many different ways by almost all molecular virologists. Matching this growth in data acquisition is an explosion of the relatively new field of bioinformatics, providing databases to store and organize this valuable/expensive data and algorithms to analyze it. For the bench virologist, access to intuitive, easy-to-use, software is often critical for performing bioinformatics-based experiments. Three common hurdles for the researcher are (1) selection, retrieval, and reformatting genomics data from large databases; (2) use of tools to compare/analyze the genomics data; and (3) display and interpretation of complex sets of results. This chapter is directed at the bench virologist and describes the software that helps overcome these obstacles, with a focus on the comparison and analysis of poxvirus genomes. Although poxvirus genomes are stored in public databases such as GenBank, this resource can be cumbersome and tedious to use if large amounts of data must to be collected. Therefore, we also highlight our Viral Orthologous Clusters database system and integrated tools that we developed specifically for the management and analysis of complete viral genomes.

Base-By-Base Version 3: New Comparative Tools for Large Virus Genomes.

Base-By-Base is a comprehensive tool for the creation and editing of multiple sequence alignments that is coded in Java and runs on multiple platforms. It can be used with gene and protein sequences as well as with large viral genomes, which themselves can contain gene annotations. This report describes new features added to Base-By-Base over the last 7 years. The two most significant additions are: (1) The recoding and inclusion of "consensus-degenerate hybrid oligonucleotide primers" (CODEHOP), a popular tool for the design of degenerate primers from a multiple sequence alignment of proteins; and (2) the ability to perform fuzzy searches within the columns of sequence data in multiple sequence alignments to determine the distribution of sequence variants among the sequences. The intuitive interface focuses on the presentation of results in easily understood visualizations and providing the ability to annotate the sequences in a multiple alignment with analytic and user data.

Complete genome sequence of a novel sea otterpox virus.

Members of the Poxviridae family are large, double-stranded DNA viruses that replicate in the cytoplasm of their host cells. The subfamily Chordopoxvirinae contains viruses that infect a wide range of vertebrates including marine mammals within the Balaenidae, Delphinidae, Mustelidae, Odobenidae, Otariidae, Phocidae, and Phocoenidae families. Recently, a novel poxvirus was found in a northern sea otter pup (Enhydra lutris kenyoni) that stranded in Alaska in 2009. The phylogenetic relationships of marine mammal poxviruses are not well established because of the lack of complete genome sequences. The current study sequenced the entire sea otterpox virus Enhydra lutris kenyoni (SOPV-ELK) genome using an Illumina MiSeq sequencer. The SOPV-ELK genome is the smallest poxvirus genome known at 127,879 bp, is 68.7% A+T content, is predicted to encode 132 proteins, and has 2546 bp inverted terminal repeats at each end. Genetic and phylogenetic analyses based on the concatenated amino acid sequences of 7 chorodopoxvirus core genes revealed the SOPV-ELK is 52.5-74.1% divergent from other known chordopoxviruses and is most similar to pteropoxvirus from Australia (PTPV-Aus). SOPV-ELK represents a new chordopoxvirus species and may belong to a novel genus. SOPV-ELK encodes eight unique genes. While the function of six predicted genes remains unknown, two genes appear to function as novel immune-modulators. SOPV-ELK-003 appears to encode a novel interleukin-18 binding protein (IL-18 BP), based on limited sequence and structural similarity to other poxviral IL-18 BPs. SOPV-ELK-035 appears to encode a novel tumor necrosis factor receptor-like (TNFR) protein that may be associated with the depression of the host's antiviral response. Additionally, SOPV-ELK-036 encodes a tumor necrosis factor-like apoptosis-inducing ligand (TRAIL) protein that has previously only been found in PTPV-Aus. The SOPV-ELK genome is the first mustelid poxvirus and only the second poxvirus from a marine mammal to be fully sequenced. Sequencing of the SOPV-ELK genome is an important step in unraveling the position of marine mammal poxviruses within the larger Poxviridae phylogenetic tree and provides the necessary sequence to develop molecular tools for future diagnostics and epidemiological studies.

Complete genomic characterisation of two novel poxviruses (WKPV and EKPV) from western and eastern grey kangaroos.

Poxviruses have previously been detected in macropods with cutaneous papillomatous lesions, however to date, no comprehensive analysis of a poxvirus from kangaroos has been performed. Here we report the genome sequences of a western grey kangaroo poxvirus (WKPV) and an eastern grey kangaroo poxvirus (EKPV), named for the host species from which they were isolated, western grey (Macropus fuliginosus) and eastern grey (Macropus giganteus) kangaroos. Poxvirus DNA from WKPV and EKPV was isolated and entire coding genome regions determined through Roche GS Junior and Illumina Miseq sequencing, respectively. Viral genomes were assembled using MIRA and SPAdes, and annotations performed using tools available from the Viral Bioinformatics Resource Centre. Histopathology and transmission electron microscopy analysis was also performed on WKPV and its associated lesions. The WKPV and EKPV genomes show 96% identity (nucleotide) to each other and phylogenetic analysis places them on a distinct branch between the established Molluscipoxvirus and Avipoxvirus genera. WKPV and EKPV are 170 kbp and 167 kbp long, containing 165 and 162 putative genes, respectively. Together, their genomes encode up to 47 novel unique hypothetical proteins, and possess virulence proteins including a major histocompatibility complex class II inhibitor, a semaphorin-like protein, a serpin, a 3-ß-hydroxysteroid dehydrogenase/δ 5→4 isomerase, and a CD200-like protein. These viruses also encode a large putative protein (WKPV-WA-039 and EKPV-SC-038) with a C-terminal domain that is structurally similar to the C-terminal domain of a cullin, suggestive of a role in the control of host ubiquitination. The relationship of these viruses to members of the Molluscipoxvirus and Avipoxvirus genera is discussed in terms of sequence similarity, gene content and nucleotide composition. A novel genus within subfamily Chordopoxvirinae is proposed to accommodate these two poxvirus species from kangaroos; we suggest the name, Thylacopoxvirus (thylaco-: [Gr.] thylakos meaning sac or pouch).

Characterization of Eptesipoxvirus, a novel poxvirus from a microchiropteran bat.

The genome of Eptesipoxvirus (EPTV) is the first poxvirus genome isolated from a microbat. The 176,688 nt sequence, which is believed to encompass the complete coding region of the virus, is 67% A+T and is predicted to encode 191 genes. 11 of these genes have no counterpart in GenBank and are therefore unique to EPTV. The presence of a distantly related ortholog of Vaccinia virus F5L in EPTV uncovered a link with fragmented F5L orthologs in Molluscum contagiosum virus/squirrelpox and clade II viruses. Consistent with the unique position of EPTV approximately mid-point between the orthopoxviruses and the clade II viruses, EPTV has 11 genes that are specific to the orthopoxviruses and 13 genes that are typical, if not exclusive, to the clade II poxviruses. This mosaic nature of EPTV blurs the distinction between the old description of the orthopoxvirus and clade II groups. Genome annotation and characterization failed to find any common virulence genes shared with the other poxvirus isolated from bat (pteropoxvirus); however, EPTV encodes 3 genes that may have been transferred to or from deerpox and squirrelpox viruses; 2 of these, a putative endothelin-like protein and a MHC class I-like protein are likely to have immunomodulatory roles.

Genomic characterization of a novel poxvirus from a flying fox: evidence for a new genus?

The carcass of an Australian little red flying fox (Pteropus scapulatus) which died following entrapment on a fence was submitted to the laboratory for Australian bat lyssavirus exclusion testing, which was negative. During post-mortem, multiple nodules were noted on the wing membranes, and therefore degenerate PCR primers targeting the poxvirus DNA polymerase gene were used to screen for poxviruses. The poxvirus PCR screen was positive and sequencing of the PCR product demonstrated very low, but significant, similarity with the DNA polymerase gene from members of the Poxviridae family. Next-generation sequencing of DNA extracted from the lesions returned a contig of 132 353 nucleotides (nt), which was further extended to produce a near full-length viral genome of 133 492 nt. Analysis of the genome revealed it to be AT-rich with inverted terminal repeats of at least 1314 nt and to contain 143 predicted genes. The genome contains a surprisingly large number (29) of genes not found in other poxviruses, one of which appears to be a homologue of the mammalian TNF-related apoptosis-inducing ligand (TRAIL) gene. Phylogenetic analysis indicates that the poxvirus described here is not closely related to any other poxvirus isolated from bats or other species, and that it likely should be placed in a new genus.