Co-circulation of five species of dog parvoviruses and canine adenovirus type 1 among gray wolves (Canis lupus) in northern Canada.

Several viruses can infect wild carnivores but their impact on wildlife health is poorly understood. We investigated the presence, diversity and distribution of various DNA viruses in 303 wolves inhabiting a vast area of the Northwest Territories, Canada, over a period of 13 years. We found evidence for the presence of canine bufavirus (CBuV, 42.6%), canine parvovirus 2 (CPV-2, 34.0%), canine bocavirus 2 (CBoV-2, 5.0%), cachavirus (CachaV-1, 2.6%), canine adenovirus 1 (CAdV-1, 1%) and minute virus of canines (MVC, 0.3%). To our knowledge, this is the first detection of CBoV-2, MVC and CachV-1 in wild animals. We also demonstrate that CBuV and CachaV-1 were already circulating among wild animals at least 11 and 10 years, respectively, before their discoveries. Although CBuV prevalence was higher, CPV-2 was the most prevalent virus among juveniles, while CBuV infection was associated with poor nutrition conditions. Even if its prevalence was low, CachaV-1 had the highest multiple infection rate (87.5%). CadV-1 and MVC sequences were highly identical to reference strains, but we observed a high diversity among the other viruses and detected three new variants. One CPV-2 variant and one CBuV variant were endemic since the beginning of the 2000s in the entire investigated region, whereas one CBuV variant and two CBoV-2 variants were found in a more restricted area over multiple years and CachaV-1 was found only in one region. Two CPV-2 variants and one CachaV-1 variant were observed only once, indicating sporadic introductions or limited circulation. Different patterns of endemicity might indicate that viruses were introduced in the wolf population at different timepoints and that mixing between wolf packs may not be constant. Different epidemiological behaviors depend on viral factors like infectivity, transmission routes, pathogenicity and tissue-tropism, and on host factors like proximity to densely populated areas, carnivory and pack density and mixing.

Novel Divergent Members of the Kitrinoviricota Discovered through Metagenomics in the Intestinal Contents of Red-Backed Voles (Clethrionomys gapperi).

Metagenomic methods are powerful tools to investigate viral diversity in biological or environmental samples and to identify previously unknown viruses. We used RNA metagenomics to identify, in the gut of red-backed voles, the nearly complete genomes of two novel members of the Kitrinoviricota, a phylum including viruses with positive-sense ssRNA genomes encoding an RNA-directed RNA polymerase. The genome of a novel member of the Tombusviridae presented four open reading frames (ORFs); a -1 frameshift is potentially involved in generating the viral replicase. This sequence was part of a phylogenetic clade that did not include any officially classified species. The second genome presented a large ORF coding for a viral polyprotein containing the typical protein domains common to flexiviruses. The sequence clustered with currently known members of the Deltaflexiviridae. Both viruses appear to represent the first members of novel species in yet undefined genera. The identified viruses likely originated from the vole diet as members of the two viral families are known to infect plants and fungi, respectively. Investigating public databases demonstrated that a much higher richness than currently recognized exists for these two viral families, highlighting the need to update taxonomy systems and possibly also include genomes identified through metagenomics.

Identification of a Novel Gammaherpesvirus in Canada lynx (Lynx canadensis).

Gammaherpesviruses (GHVs) infect many animal species and are associated with lymphoproliferative disorders in some. Previously, we identified several novel GHVs in North American felids; however, a GHV had never been identified in Canada lynx (Lynx canadensis). We, therefore, hypothesized the existence of an unidentified GHV in lynx. Using degenerate nested and subsequently virus-specific PCR, we amplified and sequenced 3.4 kb of DNA from a novel GHV in lynx, which we named Lynx canadensis gammaherpesvirus 1 (LcaGHV1). Phylogenetic analysis determined that LcaGHV1 is a distinct GHV species belonging to the genus Percavirus. We then estimated the prevalence of LcaGHV1 in lynx by developing a PCR-based assay and detected LcaGHV1 DNA in 36% (95% CI: 22-53%) of lynx spleen DNA samples from Maine, USA and 17% (95% CI: 8-31%) from Newfoundland, Canada. The LcaGHV1 DNA sequences from Maine and Newfoundland lynx were nearly identical to each other (two nucleotide substitutions in 3.4 kb), suggesting that the unique lynx subspecies present on the island of Newfoundland (Lynx canadensis subsolanus) is infected with virus that very closely resembles virus found in mainland lynx. The potential ecologic and pathologic consequences of this novel virus for Canada lynx populations warrant further study.

New Insight Into Avian Papillomavirus Ecology and Evolution From Characterization of Novel Wild Bird Papillomaviruses.

Viruses in the family Papillomaviridae have circular dsDNA genomes of approximately 5.7-8.6 kb that are packaged within non-enveloped, icosahedral capsids. The known papillomavirus (PV) representatives infect vertebrates, and there are currently more than 130 recognized PV species in more than 50 genera. We identified 12 novel avian papillomavirus (APV) types in wild birds that could represent five distinct species and two genera. Viruses were detected in paired oropharyngeal/cloacal swabs collected from six bird species, increasing the number of avian species known to harbor PVs by 40%. A new duck PV (DuPV-3) was found in mallard and American black duck (27.6% estimated prevalence) that was monophyletic with other known DuPVs. A single viral type was identified in Atlantic puffin (PuPV-1, 9.8% estimated prevalence), while a higher genetic diversity was found in other Charadriiformes. Specifically, three types [gull PV-1 (GuPV-1), -2, and -3] were identified in two gull species (estimated prevalence of 17% and 2.6% in American herring and great black-backed gull, respectively), and seven types [kittiwake PV-1 (KiPV-1) through -7] were found in black-legged kittiwake (81.3% estimated prevalence). Significantly higher DuPV-3 circulation was observed in spring compared to fall and in adults compared to juveniles. The studied host species' tendencies to be in crowded environments likely affect infection rates and their migratory behaviors could explain the high viral diversity, illustrating how host behavior can influence viral ecology and distribution. For DuPV-3, GuPV-1, PuPV-1, and KiPV-2, we obtained the complete genomic sequences, which showed the same organization as other known APVs. Phylogenetic analyses showed evidence for virus-host co-divergence at the host taxonomic levels of family, order, and inter-order, but we also observed that host-specificity constraints are relaxed among highly related hosts as we found cross-species transmission within ducks and within gulls. Furthermore, the phylogeny of viruses infecting the Charadriiformes did not match the host phylogeny and gull viruses formed distinct monophyletic clades with kittiwake viruses, possibly reflecting past host-switching events. Considering the vast PV genotype diversity in other hosts and the large number of bird species, many more APVs likely remain to be discovered.

Benign multicystic mesothelioma arising from the tunica vaginalis.

Benign multicystic mesothelioma is an extremely rare neoplasm usually arising from the visceral mesothelium of the peritoneal cavity typically presenting in young to middle-aged women. It has been deemed an indolent tumour with a possible heritable nature, but also a possible reactive neoplasm secondary to trauma. Diagnosis relies on identifying characteristic histology of cysts separated by loose connective tissue stroma and lined by pale, flat or cuboidal mesothelial cells. A 74-year-old male presented with a painless, enlarging scrotal swelling thought to be either an epididymal cyst or hydrocele. He underwent radical orchiectomy and benign multicystic mesothelioma of the tunica vaginalis was characterized on macroscopy and microscopic examination. The potential for malignant transformation of these tumours has not been demonstrated, but they are known to recur. This infrequent presentation of a rare and poorly understood tumour with description of histologic findings may allow for improved diagnosis in other cases.

Virus discovery reveals frequent infection by diverse novel members of the Flaviviridae in wild lemurs.

Lemurs are highly endangered mammals inhabiting the forests of Madagascar. In this study, we performed virus discovery on serum samples collected from 84 wild lemurs and identified viral sequence fragments from 4 novel viruses within the family Flaviviridae, including members of the genera Hepacivirus and Pegivirus. The sifaka hepacivirus (SifHV, two genotypes) and pegivirus (SifPgV, two genotypes) were discovered in the diademed sifaka (Propithecus diadema), while other pegiviral fragments were detected in samples from the indri (Indri indri, IndPgV) and the weasel sportive lemur (Lepilemur mustelinus, LepPgV). Although data are preliminary, each viral species appeared host species-specific and frequent infection was detected (18 of 84 individuals were positive for at least one virus). The complete coding sequence and partial 5' and 3' untranslated regions (UTRs) were obtained for SifHV and its genomic organization was consistent with that of other hepaciviruses, with one unique polyprotein and highly structured UTRs. Phylogenetic analyses showed the SifHV belonged to a clade that includes several viral species identified in rodents from Asia and North America, while SifPgV and IndPgV were more closely related to pegiviral species A and C, that include viruses found in humans as well as New- and Old-World monkeys. Our results support the current proposed model of virus-host co-divergence with frequent occurrence of cross-species transmission for these genera and highlight how the discovery of more members of the Flaviviridae can help clarify the ecology and evolutionary history of these viruses. Furthermore, this knowledge is important for conservation and captive management of lemurs.

Sex-biased transcriptomic response of the reproductive axis to stress.

Stress is a well-known cause of reproductive dysfunction in many species, including birds, rodents, and humans, though males and females may respond differently. A powerful way to investigate how stress affects reproduction is by examining its effects on a biological system essential for regulating reproduction, the hypothalamic-pituitary-gonadal (HPG) axis. Often this is done by observing how a stressor affects the amount of glucocorticoids, such as cortisol or corticosterone, circulating in the blood and their relationship with a handful of known HPG-producing reproductive hormones, like testosterone and estradiol. Until now, we have lacked a full understanding of how stress affects all genomic activity of the HPG axis and how this might differ between the sexes. We leveraged a highly replicated and sex-balanced experimental approach to test how male and female rock doves (Columba livia) respond to restraint stress at the level of their transcriptome. Females exhibit increased genomic responsiveness to stress at all levels of their HPG axis as compared to males, and these responsive genes are mostly unique to females. Reasons for this may be due to fluctuations in the female endocrine environment over the reproductive cycle and/or their evolutionary history, including parental investment and the potential for maternal effects. Direct links between genome to phenome cause and effect cannot be ascertained at this stage; however, the data we report provide a vital genomic foundation on which sex-specific reproductive dysfunction and adaptation in the face of stress can be further experimentally studied, as well as novel gene targets for genetic intervention and therapy investigations.

CD24 induces changes to the surface receptors of B cell microvesicles with variable effects on their RNA and protein cargo.

The CD24 cell surface receptor promotes apoptosis in developing B cells, and we recently found that it induces B cells to release plasma membrane-derived, CD24-bearing microvesicles (MVs). Here we have performed a systematic characterization of B cell MVs released from WEHI-231 B lymphoma cells in response to CD24 stimulation. We found that B cells constitutively release MVs of approximately 120 nm, and that CD24 induces an increase in phosphatidylserine-positive MV release. RNA cargo is predominantly comprised of 5S rRNA, regardless of stimulation; however, CD24 causes a decrease in the incorporation of protein coding transcripts. The MV proteome is enriched with mitochondrial and metabolism-related proteins after CD24 stimulation; however, these changes were variable and could not be fully validated by Western blotting. CD24-bearing MVs carry Siglec-2, CD63, IgM, and, unexpectedly, Ter119, but not Siglec-G or MHC-II despite their presence on the cell surface. CD24 stimulation also induces changes in CD63 and IgM expression on MVs that is not mirrored by the changes in cell surface expression. Overall, the composition of these MVs suggests that they may be involved in releasing mitochondrial components in response to pro-apoptotic stress with changes to the surface receptors potentially altering the cell type(s) that interact with the MVs.

Analysis of the structure, evolution, and expression of CD24, an important regulator of cell fate.

BACKGROUND: CD24 is a small, glycophosphatidylinositol-anchored cell surface receptor, expressed in a variety of cells types and tissues. CD24 gene and protein expression is highly dynamic in response to cellular differentiation and stimulation in a cell-specific manner. Furthermore, CD24 interacts with a diverse collection of ligands, including cell adhesion molecules such as P-selectin, and the immune-associated siglec family of transmembrane proteins. While much is known regarding the biological roles of CD24 in regulating cell survival, death and differentiation, little is known about the evolution and organization of CD24 across species or the relationship between CD24 expression and its known ligands. RESULTS: We analyzed the organization and evolution of the CD24 gene from 56 mammalian, avian and reptilian species. We further examined the mRNA expression of CD24 and its known ligands in Mus musculus in immune cells, immunologically privileged tissues, developing brain, and developing and regenerating liver. CD24 arose prior to the reptilian-avian divergence and is conserved across many mammalian species, although we were unable to identify CD24 in marsupials or monotremes. The CD24 genomic structure is diverse between and within species, with varying numbers of exons, introns, and the presence of untranslated regions. Of note, we found no obvious criteria distinguishing CD24 genes from those annotated as CD24-like. The expression of CD24 is similarly complex, with immune cells showing dynamic changes in mRNA levels during development, while immunologically privileged and developing tissues show a high, static expression level that decreases in mature tissues. Furthermore, the expression of CD24 correlated with some but not all of its known ligands in a tissues-specific manner, suggesting that novel ligands have yet to be identified and that cell-specific ligand expression can influence CD24 function. CONCLUSIONS: We find that CD24 arose prior to the divergence of reptiles, birds and mammals. Furthermore, the most highly conserved areas of the protein are the amino acids which can be glycosylated. We also find that CD24 expression is highly tissue-specific and in many cases, not well conserved with known CD24 ligands, suggesting yet-unknown CD24-ligand interactions. Together, these data are a valuable resource for furthering studies in CD24 biology.

Automatic segmentation of microcystic macular edema in OCT.

Microcystic macular edema (MME) manifests as small, hyporeflective cystic areas within the retina. For reasons that are still largely unknown, a small proportion of patients with multiple sclerosis (MS) develop MME-predominantly in the inner nuclear layer. These cystoid spaces, denoted pseudocysts, can be imaged using optical coherence tomography (OCT) where they appear as small, discrete, low intensity areas with high contrast to the surrounding tissue. The ability to automatically segment these pseudocysts would enable a more detailed study of MME than has been previously possible. Although larger pseudocysts often appear quite clearly in the OCT images, the multi-frame averaging performed by the Spectralis scanner adds a significant amount of variability to the appearance of smaller pseudocysts. Thus, simple segmentation methods only incorporating intensity information do not perform well. In this work, we propose to use a random forest classifier to classify the MME pixels. An assortment of both intensity and spatial features are used to aid the classification. Using a cross-validation evaluation strategy with manual delineation as ground truth, our method is able to correctly identify 79% of pseudocysts with a precision of 85%. Finally, we constructed a classifier from the output of our algorithm to distinguish clinically identified MME from non-MME subjects yielding an accuracy of 92%.

Seroprevalence of feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) in shelter cats on the island of Newfoundland, Canada.

Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are retroviruses found within domestic and wild cat populations. These viruses cause severe illnesses that eventually lead to death. Housing cats communally for long periods of time makes shelters at high risk for virus transmission among cats. We tested 548 cats from 5 different sites across the island of Newfoundland for FIV and FeLV. The overall seroprevalence was 2.2% and 6.2% for FIV and FeLV, respectively. Two sites had significantly higher seroprevalence of FeLV infection than the other 3 sites. Analysis of sequences from the FeLV env gene (envelope gene) from 6 positive cats showed that 4 fell within the FeLV subtype-A, while 2 sequences were most closely related to FeLV subtype-B and endogenous feline leukemia virus (en FeLV). Varying seroprevalence and the variation in sequences at different sites demonstrate that some shelters are at greater risk of FeLV infections and recombination can occur at sites of high seroprevalence.

Le virus de l'immunodéficience féline (FIV) et le virus de la leucémie féline (FeLV) sont des rétrovirus retrouvés chez les populations de chats domestiques et sauvages. Ces virus causent des maladies sévères qui éventuellement mènent à la mort. L'hébergement de chats de façon communautaire pendant de longues périodes rend les refuges à risque élevé pour la transmission du virus parmi les chats. Nous avons testé 548 chats provenant de cinq sites différents à travers l'ile de Terre-Neuve pour FIV et FeLV. La séroprévalence globale était de 2,2 % et 6,2 % pour FIV et FeLV, respectivement. Deux sites avaient une séroprévalence significativement plus élevée d'infection par FeLV que les trois autres sites. L'analyse des séquences du gène env de FeLV (gène de l'enveloppe) provenant de six chats positifs a montré que quatre appartenaient au sous-type A de FeLV, alors que deux séquences étaient plus apparentées au sous-type B de FeLV et du virus endogène de la leucémie féline (en FeLV). Une séroprévalence variable et la variation dans les séquences à différents sites démontrent que certains refuges sont à risque plus élevé d'infections par FeLV et que de la recombinaison peut survenir aux sites avec une séroprévalence élevée.(Traduit par Docteur Serge Messier).

MRI-3D ultrasound-X-ray image fusion with electromagnetic tracking for transendocardial therapeutic injections: in-vitro validation and in-vivo feasibility.

Myocardial infarction (MI) is one of the leading causes of death in the world. Small animal studies have shown that stem-cell therapy offers dramatic functional improvement post-MI. An endomyocardial catheter injection approach to therapeutic agent delivery has been proposed to improve efficacy through increased cell retention. Accurate targeting is critical for reaching areas of greatest therapeutic potential while avoiding a life-threatening myocardial perforation. Multimodal image fusion has been proposed as a way to improve these procedures by augmenting traditional intra-operative imaging modalities with high resolution pre-procedural images. Previous approaches have suffered from a lack of real-time tissue imaging and dependence on X-ray imaging to track devices, leading to increased ionizing radiation dose. In this paper, we present a new image fusion system for catheter-based targeted delivery of therapeutic agents. The system registers real-time 3D echocardiography, magnetic resonance, X-ray, and electromagnetic sensor tracking within a single flexible framework. All system calibrations and registrations were validated and found to have target registration errors less than 5 mm in the worst case. Injection accuracy was validated in a motion enabled cardiac injection phantom, where targeting accuracy ranged from 0.57 to 3.81 mm. Clinical feasibility was demonstrated with in-vivo swine experiments, where injections were successfully made into targeted regions of the heart.

Multi-modal registration of speckle-tracked freehand 3D ultrasound to CT in the lumbar spine.

A method for registration of speckle-tracked freehand 3D ultrasound (US) to preoperative CT volumes of the spine is proposed. We register the US volume to the CT volume by creating individual US "sub-volumes", each consisting of a small section of the entire US volume. The registration proceeds incrementally from the beginning of the US volume to the end, registering every sub-volume, where each sub-volume contains overlapping images with the previous sub-volume. Each registration is performed by generating simulated US images from the CT volume. As a by-product of our registration, the significant drift error common in speckle-tracked US volumes is corrected for. Results are validated through a phantom study of plastic spine phantoms created from clinical patient CT data as well as an animal study using a lamb cadaver. Results demonstrate that we were able to successfully register a speckle-tracked US volume to CT volume in four out of five phantoms with a success rate of greater than 98%. The final error of the registered US volumes decreases by over 50 percent from the speckle tracking error to consistently below 3 mm. Studies on the lamb cadaver showed a mean registration error consistently below 2 mm.

Fast and accurate calibration of an X-ray imager to an electromagnetic tracking system for interventional cardiac procedures.

Cardiovascular disease affects millions of Americans each year. Interventional guidance systems are being developed as treatment options for some of the more delicate procedures, including targeted stem cell therapy. As advanced systems for such types of interventional guidance are being developed, electromagnetic (EM) tracking is coming in demand to perform navigation. To use this EM tracking technology, a calibration is necessary to register the tracker to the imaging system. In this paper we investigate the calibration of an X-ray imaging system to EM tracking. Two specially designed calibration phantoms have been designed for this purpose, each having a rigidly attached EM sensor. From a clinical usability point-of-view, we propose to divide this calibration problem into two steps: i) in initial calibration of the EM sensor to the phantom design using an EM tracked needle to trace out grooves in the phantom surface and ii) segmentation from X-ray images and 3D reconstruction of beads embedded in the phantom in a known geometric pattern. Combining these two steps yields and X-ray-to-EM calibration accuracy of less than 1 mm when overlaying an EM tracked needle on X-ray images.

Genome sequence and characterization of a virus (HaRNAV) related to picorna-like viruses that infects the marine toxic bloom-forming alga Heterosigma akashiwo.

Heterosigma akashiwo (Rhaphidophyceae) is a unicellular, flagellated, bloom-forming, toxic alga of ecological and economic importance. Here, we report the results of sequencing and analyzing the genome of an 8.6-kb single-stranded RNA virus (HaRNAV-SOG263) that infects H. akashiwo. Our results show that HaRNAV is related to picorna-like viruses, but does not belong within any currently defined virus family. This is based on the genome organization and sequence comparisons of putative RNA-dependent RNA polymerase (RdRp), helicase, and capsid protein sequences. The genome sequence predicts a single open reading frame (orf) encoding a polyprotein that contains conserved picorna-like protein domains, with putative nonstructural protein domains present in the N-terminus and the structural proteins in the C-terminus of the polyprotein. We have analyzed and compared the virus structural proteins from infectious and noninfectious particles. In this way, we identified structural protein cleavage sites as well as protein processing events that are presumably important for maturation of virus particles. The combination of genome structure and sequence relationships to other viruses suggests that HaRNAV is the first member of a proposed new virus family (Marnaviridae), related to picorna-like viruses.

High diversity of unknown picorna-like viruses in the sea.

Picorna-like viruses are a loosely defined group of positive-sense single-stranded RNA viruses that are major pathogens of animals, plants and insects. They include viruses that are of enormous economic and public-health concern and are responsible for animal diseases (such as poliomyelitis), plant diseases (such as sharka) and insect diseases (such as sacbrood). Viruses from the six divergent families (the Picornaviridae, Caliciviridae, Comoviridae, Sequiviridae, Dicistroviridae and Potyviridae) that comprise the picorna-like virus superfamily have the following features in common: a genome with a protein attached to the 5' end and no overlapping open reading frames, all the RNAs are translated into a polyprotein before processing, and a conserved RNA-dependent RNA polymerase (RdRp) protein. Analyses of RdRp sequences from these viruses produce phylogenies that are congruent with established picorna-like virus family assignments; hence, this gene is an excellent molecular marker for examining the diversity of picorna-like viruses in nature. Here we report, on the basis of analysis of RdRp sequences amplified from marine virus communities, that a diverse array of picorna-like viruses exists in the ocean. All of the sequences amplified were divergent from known picorna-like viruses, and fell within four monophyletic groups that probably belong to at least two new families. Moreover, we show that an isolate belonging to one of these groups is a lytic pathogen of Heterosigma akashiwo, a toxic-bloom-forming alga responsible for severe economic losses to the finfish aquaculture industry, suggesting that picorna-like viruses are important pathogens of marine phytoplankton.