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In this study, a novel parvovirus (zander/M5/2015/HUN, OK236393) was detected in faecal specimens from a fish – zander or pikeperch (Sander lucioperca) – and genetically characterized using viral metagenomics and PCR methods. The NS1 and VP1 proteins of zander/M5/2015/HUN share Parvoviridae. Out of 62 faecal specimens collected from 13 freshwater fish species, three (4.8%) samples were positive by PCR for the novel parvovirus – all from zander. This is the second parvovirus detected in fish – after the disease-causing tilapia parvovirus of the subfamily Hamaparvovirinae – and it potentially represents a novel genus in the subfamily Parvovirinae.

In this study, a novel picornavirus (perchPV/M9/2015/HUN, GenBank accession no. MW590713) was detected in eight (12.9%) out of 62 faecal samples collected from three (Perca fluviatilis, Sander lucioperca, and Ameiurus melas) out of 13 freshwater fish species tested and genetically characterized using viral metagenomics and RT-PCR methods. The complete genome of perchPV/M9/2015/HUN is 7,741 nt long, excluding the poly(A) tail, and has the genome organization 5’UTRIRES-?/P1(VP0-VP3-VP1)/P2(2A1NPG↓P-2A2H-box/NC-2B-2C)/P3(3A-3BVPg-3CPro-3DPol)/3’UTR-poly(A). The P1, 2C, and 3CD proteins had 41.4%, 38.1%, and 47.3% amino acid sequence identity to the corresponding proteins of Wenling lepidotrigla picornavirus (MG600079), eel picornavirus (NC_022332), and Wenling pleuronectiformes picornavirus (MG600098), respectively, as the closest relatives in the genus Potamipivirus. PerchPV/M9/2015/HUN represents a potential novel fish-origin species in an unassigned genus in the family Picornaviridae.

In this study, a novel parvovirus (gyb-MR02/2015/HUN, MT580795) was detected in barn owls (Tyto alba) and genetically characterized using viral metagenomics and PCR methods. The NS1 and VP1 proteins of gyb-MR02/2015/HUN share only 45.4% and 50.1% amino acid sequence identity, respectively, to the corresponding proteins of peafowl parvovirus 2 (MK988620), the closest relative. Out of 11 faecal specimens from owls (six from little owls, three from barn owls, and two from long-eared owls), two barn owl samples were positive for the novel parvovirus, which is distantly related to members of the recently established genus Chaphamaparvovirus in the subfamily Hamaparvovirinae. Systematic investigation is necessary to explore the diversity of parvoviruses.

There is a considerable increase in vector-borne zoonotic diseases around the world, including Turkey, such as Crimean-Congo hemorrhagic fever (CCHF), tick borne encephalitis (TBE), Rift Valley fever (RVF), and West Nile fever (WNF), causing disease and death in humans and animals and significant economical losses. Hence, the aim of this study was to investigate the presence of CCHF virus (CCHFV) and TBE virus (TBEV) in ticks and RVF virus (RVFV) and WNF virus (WNV) in mosquitos, as well as in sheep and cattle, in the Thrace district of the Marmara region, which borders Bulgaria and Greece. Buffy-coat samples from 86 cattle and 81 sheep, as well as 563 ticks and 7390 mosquitos, were collected and examined by quantitative real-time RT-PCR for the presence of CCHFV, TBEV, RVFV, and WNV. All buffy-coat samples from cattle and sheep were negative for these viruses. Similarly, all tick samples were negative for CCHFV-RNA and TBEV-RNA. Among 245 pools representing 7390 mosquitos, only 1 pool sample was found to be positive for WNV-RNA and was confirmed by sequencing. Phylogenetic analysis revealed that it was WNV lineage-2. No RVFV-RNA was detected in the 245 mosquito pools. In conclusion, results of this study indicate that CCHFV, TBEV, and RVFV are not present in livestock and respective vectors in the Thrace district of Marmara region of Turkey, whereas WNV-RNA was found in mosquitos from this region.

The unexpected seroconversion of sentinel mice in our facility to murine T lymphotrophic virus (MTLV) positivity led to our identification of a novel murine astrovirus that we designated murine astrovirus 2 (MuAstV-2). During our investigation, MuAstV-2 was found to be a contaminant of the T helper cell line (D10. G4.1) that was used to generate the MTLV antigen that we included in the multiplex fluorometric immunoassay (MFIA) that we used for sentinel screening. We eventually determined that cross-reactivity with the astrovirus generated a positive result in the MTLV assay. A confirmatory immunofluorometric assay (IFA) using the same MTLV-infected cell line yielded a similar result. However, the use of antigen prepared from MTLV-infected neonatal mouse thymus did not reproduce a positive result, leading us to suspect that the seroreactivity we had observed was not due to infection with MTLV. A mouse antibody production test showed that mice inoculated with naïve D10. G4.1 cells and their contact sentinels tested positive for MTLV using cell-line generated antigen, but tested negative in assays using MTLV antigen produced in mice. Metagenomic analysis was subsequently used to identify MuAstV-2 in feces from 2 sentinel mice that had recently seroconverted to MTLV. Two closely related astrovirus sequences (99.6% capsid identity) were obtained and shared 95% capsid amino acid identity with the MuAstV-2 virus sequenced from the D10. G4.1 cell line. These viruses are highly divergent from previously identified murine astroviruses, displaying Prkdcem26Cd52Il2rgem26Cd22/NjuCrl (NCG) mice, which have significantly impaired adaptive and innate immune systems. Neither immunocompetent nor immunodeficient mice showed any astrovirus-associated pathology. MuAstV-2 may provide a valuable model for the study of specific aspects of astrovirus pathogenesis and virus-host interactions.

Objectives Improving immune status of people living with HIV through antiretroviral therapy (ART) may also reduce shedding of other viruses in semen. We characterized the seminal fluid virome of men with HIV and tested potential associations between viruses present and CD4 T-cell count, HIV viremia, and antiretroviral therapy (ART) status. Design and methods Metagenomics was used to enrich and sequence viral nucleic acids from the seminal fluid of 55 semen samples from 42 men living with HIV from San Francisco with a median age of 33 (IQR, 28.7-45) and median CD4 T-cell counts of 837 cells/μl (IQR, 258-1571 cells/μl). All samples were collected between 2005 and 2015, and ART status was ascertained from medical records. Results Anelloviruses, cytomegalovirus (CMV), and multiple genotypes of human papillomaviruses were detected. Participants shed from 0 to 4 distinct human viruses. Longitudinally collected seminal fluid samples showed changes in the viruses shed. Viruses were more frequently shed by individuals with detectable HIV viremia (43.7 vs. 15.4%, P = 0.042). A trend was seen for increased shedding by individuals who were not on ART (42.8 vs. 17.8%, P = 0.082) or with CD4 T-cell count less than 350 cells/μl (35.3 vs. 20%, P = 0.27). Conclusion Seminal fluid from men with HIV from San Francisco contains nucleic acids from three different DNA viral families. A greater number of viruses, particularly CMV, were shed by participants with detectable HIV viremia (18.9 vs. 0%, P = 0.022). Control of viremia through ART may lower shedding of other viruses in semen in addition to HIV.

Idiopathic chronic diarrhea (ICD) is a common clinical condition in captive rhesus macaques, claiming 33% of medical culls (i.e. deaths unrelated to research). Using viral metagenomics we characterized the eukaryotic virome in digestive tract tissues collected at necropsy from nine animals with ICD. We show the presence of multiple viruses in the Parvoviridae and Picornaviridae family. We then compared the distribution of viral reads in the stomach, duodenum, jejunum, ileum, and the proximal, transverse, and distal colons. Tissues and mucosal scraping from the same locations showed closely related results while different gut tissues from the same animal varied widely. Picornavirus reads were generally more abundant in the lower digestive tract, particularly in the descending (distal) colon. Parvoviruses were more abundant in the upper reach particularly in the stomach. In situ hydridization (ISH) of fixed tissues showed punctuated staining for both these RNA and DNA viruses in the distal colon. Parvovirus ISH staining was also detected in the stomach/duodenum/jejunum in distinct oval-shaped structures. Therefore, the location of enteric viral nucleic acid differed widely between different viral families and along the length of the digestive tract.

Zebrafish have been extensively used as a model system for research in vertebrate development and pathogen-host interactions. We describe the complete genome of a novel picornavirus identified during a viral metagenomics analysis of zebrafish gut tissue. The closest relatives of this virus showed identity of

Background Stool consistency is an important diagnostic criterion in both research and clinical medicine and is often used to define diarrheal disease. Methods We examine the pediatric enteric virome across stool consistencies to evaluate differences in richness and community composition using fecal samples collected from children aged 0 to 5 years participating in a clinical trial in the Amhara region of Ethiopia. The consistency of each sample was graded according to the modified Bristol Stool Form Scale for children (mBSFS-C) before a portion of stool was preserved for viral metagenomic analysis. Stool samples were grouped into 29 pools according to stool consistency type. Differential abundance was determined using negative-binomial modeling. Results Of 446 censused children who were eligible to participate, 317 presented for the study visit examination and 269 provided stool samples. The median age of children with stool samples was 36 months. Species richness was highest in watery-consistency stool and decreased as stool consistency became firmer (Spearman’s r = − 0.45, p = 0.013). The greatest differential abundance comparing loose or watery to formed stool was for norovirus GII (7.64, 95% CI 5.8, 9.5) followed by aichivirus A (5.93, 95% CI 4.0, 7.89) and adeno-associated virus 2 (5.81, 95%CI 3.9, 7.7). Conclusions In conclusion, we documented a difference in pediatric enteric viromes according to mBSFS-C stool consistency category, both in species richness and composition. Electronic supplementary material The online version of this article (10.1186/s12879-019-3674-3) contains supplementary material, which is available to authorized users.

Circoviruses infect vertebrates where they can result in a wide range of disease signs or in asymptomatic infections. Using viral metagenomics we analyzed a pool of five sera from four healthy and one sick horse. Sequences from parvovirus-H, equus anellovirus, and distantly related to mammalian circoviruses were recognized. PCR identified the circovirus reads as originating from a pregnant mare with fever and hepatitis. That horse’s serum was also positive by real time PCR for equine parvovirus H and negative for the flavivirus equine hepacivirus. The complete circular genome of equine circovirus 1 strain Charaf (EqCV1-Charaf) was completed using PCR and Sanger sequencing. EqCV1 replicase showed 73–74% identity to those of their closest relatives, pig circoviruses 1/2, and elk circovirus. The closest capsid proteins were from the same ungulate circoviruses with 62–63% identity. The overall nucleotide identity of 72% to its closest relative indicates that EqCV1 is a new species in the Circovirus genus, the first reported in genus Equus. Whether EqCV1 alone or in co-infections can result in disease and its prevalence in different equine populations will require further studies now facilitated using EqCV1′s genome sequence.

Bats are hosts to a large variety of viruses, including many capable of cross-species transmissions to other mammals, including humans. We characterized the virome in guano from five common bat species in 9 Northern California roosts and from a pool of 5 individual bats. Genomes belonging to 14 viral families known to infect mammals and 17 viral families infecting insects or of unknown tropism were detected. Nearly complete or complete genomes of a novel parvovirus, astrovirus, nodavirus, circular Rep-encoding single-stranded DNA (CRESS-DNA) viruses, and densoviruses, and more partial genomes of a novel alphacoronavirus and a bunyavirus were characterized. Lower numbers of reads with >90% amino acid identity to previously described calicivirus, circovirus, adenoviruses, hepatovirus, bocaparvoviruses, and polyomavirus in other bat species were also found, likely reflecting their wide distribution among different bats. Unexpectedly, a few sequence reads of canine parvovirus 2 and the recently described mouse kidney parvovirus were also detected and their presence confirmed by PCR; these possibly originated from guano contamination by carnivores and rodents. The majority of eukaryotic viral reads were highly divergent, indicating that numerous viruses still remain to be characterized, even from such a heavily investigated order as Chiroptera.IMPORTANCE Characterizing the bat virome is important for understanding viral diversity and detecting viral spillover between animal species. Using an unbiased metagenomics method, we characterize the virome in guano collected from multiple roosts of common Northern California bat species. We describe several novel viral genomes and report the detection of viruses with close relatives reported in other bat species, likely reflecting cross-species transmissions. Viral sequences from well-known carnivore and rodent parvoviruses were also detected, whose presence are likely the result of contamination from defecation and urination atop guano and which reflect the close interaction of these mammals in the wild.

An emaciated subadult free-ranging California sea lion (Csl or Zalophus californianus) died following stranding with lesions similar to 11 other stranded animals characterized by chronic disseminated granulomatous inflammation with necrotizing steatitis and vasculitis, involving visceral adipose tissues in the thoracic and peritoneal cavities. Histologically, affected tissues had extensive accumulations of macrophages with perivascular lymphocytes, plasma cells, and fewer neutrophils. Using viral metagenomics on a mesenteric lymph node six mammalian viruses were identified consisting of novel parvovirus, polyomavirus, rotavirus, anellovirus, and previously described Csl adenovirus 1 and Csl bocavirus 4. The causal or contributory role of these viruses to the gross and histologic lesions of this sea lion remains to be determined.

An unexplained outbreak of feline diarrhea and vomiting, negative for common enteric viral and bacterial pathogens, was subjected to viral metagenomics and PCR. We characterized from fecal samples the genome of a novel chapparvovirus we named fechavirus that was shed by 8/17 affected cats and identified three different feline bocaviruses shed by 9/17 cats. Also detected were nucleic acids from attenuated vaccine viruses, members of the normal feline virome, viruses found in only one or two cases, and viruses likely derived from ingested food products. Epidemiological investigation of disease signs, time of onset, and transfers of affected cats between three facilities support a possible role for this new chapparvovirus in a highly contagious feline diarrhea and vomiting disease.

An unexplained outbreak of feline diarrhea and vomiting, negative for common enteric viral and bacterial pathogens, was subjected to viral metagenomics and PCR. We characterized from fecal samples the genome of a novel chapparvovirus we named fechavirus that was shed by 8/17 affected cats and different feline bocaviruses shed by 9/17 cats. Also detected were nucleic acids from attenuated vaccine viruses, members of the normal feline virome, viruses found in only one or two cases, and viruses likely derived from ingested food products. Epidemiological investigation of disease signs, time of onset, and transfers of affected cats between three facilities support a possible role for this new chapparvovirus in a highly contagious feline diarrhea and vomiting disease.

Viral infections were investigated in American black bears (Ursus americanus) from Nevada and northern California with and without idiopathic encephalitis. Metagenomics analyses of tissue pools revealed novel viruses in the generaCircoviridae,Parvoviridae,Anelloviridae,Polyomaviridae, andPapillomaviridae. The circovirus and parvovirus were of particular interest due to their potential importance as pathogens. We characterized the genomes of these viruses and subsequently screened bears by PCR to determine their prevalence. The circovirus(Ursus americanuscircovirus, UaCV) was detected at a high prevalence (10/16, 67%), and the chaphamaparvovirus (Ursus americanusparvovirus, UaPV) was found in a single bear. We showed that UaCV is present in liver, spleen/lymph node, and brain tissue of selected cases byin situhybridization (ISH) and PCR. Infections were detected in cases of idiopathic encephalitis and in cases without inflammatory brain lesions. Infection status was not clearly correlated with disease, and the significance of these infections remains unclear. Given the known pathogenicity of a closely related mammalian circovirus, and the complex manifestations of circovirus-associated diseases, we suggest that UaCV warrants further study as a possible cause or contributor to disease in American black bears.

Viral metagenomic analysis of the liver of a black headed python (Aspidites melanocephalus) euthanized for a proliferative spinal lesion of unknown etiology yielded the first characterized genome of a reptile-infecting circovirus (black-headed python circovirus or BhPyCV). BhPyCV-specific in situ hybridization (ISH) showed that viral nucleic acids were strongly expressed in the intestinal lining and mucosa and multifocally in the liver. To investigate the presence of this virus in other snakes and its possible pathogenicity, 17 snakes in the python family with spinal disease were screened with ISH yielding a second BhP positive in intestinal tissue, and a Boelen’s python (Morelia boeleni) positive in the liver. BhPyCV specific PCR was used to screen available frozen tissues from 13 of these pythons, four additional deceased pythons with and without spinal disease, and fecal samples from 37 live snakes of multiple species with unknown disease status. PCR detected multiple positive tissues in both of the ISH positive BhP and in the feces of another two live BhP and two live annulated tree boas (Corallus annulatus). Preliminary analysis indicates this circovirus can infect BhPs where it was found in 4/5 BhPs tested (2/2 with spinal disease, 2/3 live with unknown status), Boelen’s python (1/2 with spinal disease), and annulated tree boa (2/6 live with unknown status) but was not detected in other python species with the same spinal lesions. This circovirus’ causal or contributory role in spinal disease remains speculative and not well supported by these initial data. Electronic supplementary material The online version of this article (10.1186/s13567-019-0653-z) contains supplementary material, which is available to authorized users.

The aim of this study was to investigate the clinical, pathologic, and viral etiology of horses with neurologic disorders. Twelve English Thoroughbred horses with neurologic disorders were investigated for the presence of neuropathogenic equine herpesvirus-1 (EHV-1) and Borna disease virus (BDV) by real-time polymerase chain reaction (PCR) and histopathological methods. Neuropathogenic EHV-1 was detected in the brain of two horses by real-time PCR. Borna disease virus p24 and p40 gene sequences were detected by real-time RT-PCR in the brain of a 3-year-old horse and in the blood of a 1-year-old horse. High fever, ataxia, depression, lack of coordination, and gait abnormalities were present in these horses, which died within a few days of developing neurologic signs. The BDV p24 and p40 real-time PCR products were sequenced and shown to be identical to previously reported BDV sequences. In the brain of the BDV-positive horse, hyperemia was pronounced in the parenchyma and the meninges. In addition, nonpurulent poliencephalomyelitis characterized by perivascular mononuclear cell infiltration was seen. Mononuclear and polymorphonuclear cell infiltration was also seen in the liver with necrotic perihepatitis besides subacute splenitis and glomerulonephritis and severe hyperemia in the kidney and edema and emphysema in the lung. In conclusion, neuropathogenic EHV-1 and BDV were detected in horses from Turkey using molecular detection methods. Since BDV genetic signatures were detected in Turkey for the first time, future epidemiologic studies need to be performed to investigate the range of host animals, spread, frequency, and molecular diversity of BDV; this will allow to determine the risk of this pathogen for Turkish veterinary and public health. © 2018 Elsevier Inc. 26675 The authors would like to thank to the University of Istanbul for funding this study (project no: 26675) and the Kansas Biosciences Authority. The views and conclusions contained in this document are those of the authors and should not be interpreted as necessarily representing the official policies, either explicit or implicit, of the University of Kansas Biosciences Authority.

Metagenomic analysis of whole mosquitoes allows the genetic characterization of all associated viruses, including arboviruses and insect-specific viruses, plus those in their diet or infecting their parasites. We describe here the virome in mosquitoes, primarily Culex pipiens complex, Cx. tarsalis and Cx. erythrothorax, collected in 2016 from 31 counties in California, USA. The nearly complete genomes of 56 viruses, including 32 novel genomes, some from potentially novel RNA and DNA viral families or genera, were assembled and phylogenetically analyzed, significantly expanding the known Culex-associated virome. The majority of detected viral sequences originated from single-stranded RNA viral families with members known to infect insects, plants, or from unknown hosts. These reference viral genomes will facilitate the identification of related viruses in other insect species and to monitor changes in the virome of Culex mosquito populations to define factors influencing their transmission and possible impact on their insect hosts.

While recent changes in treatment have reduced the lethality of idiopathic chronic diarrhea (ICD), this condition remains one of the most common causes of rhesus macaque deaths in non-human primate research centers. We compared the eukaryotic viromes in fecal swabs from 52 animals with ICD and 41 healthy animals. Viral metagenomics targeting virus-like particles was used to identify viruses shed by each animal. Five viruses belonging to the Picornaviridae, one to the Caliciviridae, one to the Parvoviridae, and one to the Adenoviridae families were identified. The fraction of reads matching each viral species was then used to estimate and compare viral loads in ICD cases versus healthy controls. None of the eukaryotic viruses detected in fecal swabs were strongly associated with ICD. Other potential causes of ICD are discussed.

Rodents including rats are reservoir of several pathogens capable of affecting human health. In this study, faecal and different organ specimens from free-living Norway rats (Rattus norvegicus) (N = 18) and faecal samples from laboratory rodents (rats N = 21 and mice N = 20) collected from different geographic areas in Hungary between 2017 and 2020 were investigated by viral metagenomics and conventional RT-PCR methods. The complete genome of three different RNA viruses, rat astrovirus, rat norovirus and rat hepevirus were characterized and analysed in detail. Rat norovirus was detected in faecal (17.6%, 3/17) and kidney (7.1%, 1/14) samples; rat astrovirus in faecal (23.5%, 4/17) and spleen (13.3%, 2/15) samples, and rat hepevirus in 43% to 67% the faecal, liver, kidney, lung, heart, muscle, brain and blood samples from Norway rats, respectively. Rat norovirus was also identifiable in 5% (1/21) of laboratory rats and rat astrovirus in 40% (8/20) of faecal samples from laboratory mice. Co-infections were found in 28% (5/18) wild Norway rats. The highest RNA viral load of astrovirus (1.81 × 108 copy/g) and norovirus (3.49 × 107 copy/g) were measured in faecal samples; while the highest RNA viral load of hepevirus (1.16 × 109 copy/g) was found in liver samples of Norway rats, respectively. This study confirms the wide geographic distribution and high prevalence of astrovirus, norovirus and hepevirus among wild rats in Hungary with confirmation of different organ involvement of as well as the detection of norovirus and astrovirus in laboratory rats and mice, respectively. This finding further strengthens the role of rodents in the spread of viral pathogens especially infecting human.

Six foals with interstitial pneumonia of undetermined etiology from Southern California were analyzed by viral metagenomics. Spleen, lung, and colon content samples obtained during necropsy from each animal were pooled, and nucleic acids from virus-like particles enriched for deep sequencing. The recently described equine copiparvovirus named eqcopivirus, as well as three previously uncharacterized viruses, were identified. The complete ORFs genomes of two closely related protoparvoviruses, and of a bocaparvovirus, plus the partial genome of a picornavirus were assembled. The parvoviruses were classified as members of new ungulate protoparvovirus and bocaparvovirus species in the Parvoviridae family. The picornavirus was classified as a new species in the Salivirus genus of the Picornaviridae family. Spleen, lung, and colon content samples from each foal were then tested for these viral genomes by nested PCR and RT-PCR. When present, parvoviruses were detected in both feces and spleen. The picornavirus, protoparvovirus, and eqcopivirus genomes were detected in the lungs of one animal each. Three foals were co-infected with the picornavirus and either a protoparvovirus, bocaparvovirus, or eqcopivirus. Two other foals were infected with a protoparvovirus only. No viral infection was detected in one animal. The complete ORFs of the first equine protoparvoviruses and bocaparvovirus, the partial ORF of the third equine picornavirus, and their detection in tissues of foals with interstitial pneumonia are described here. Testing the involvement of these viruses in fatal interstitial pneumonia or other equine diseases will require larger epidemiological and/or inoculation studies.

Cynomolgus macaques are common across South East Asian countries including Thailand. The National Primate Research Center of Thailand, Chulalongkorn University (NPRCT-CU) captures wild-borne cynomolgus macaque for research use. Limited information is available on the enteric viruses and possible zoonotic infections into or from cynomolgus macaques. We characterized and compare the fecal virome of two populations, healthy wild-originated captive cynomolgus macaques (n = 43) reared in NPRCT-CU and healthy wild cynomolgus macaques (n = 35). Over 90% of recognized viral sequence reads amplified from feces were from bacterial viruses. Viruses from seven families of mammalian viruses were also detected (Parvoviridae, Anelloviridae, Picornaviridae, Adenoviridae, Papillomaviridae, Herpesviridae, and Caliciviridae). The genomes of a member of a new picornavirus genus we named Mafapivirus, a primate chapparvovirus, and a circular Rep-encoding single-strand (CRESS) DNA virus were also characterized. Higher abundance of CRESS DNA viruses of unknown tropism and invertebrate-tropic ambidensovirus were detected in wild versus captive macaques likely reflecting dietary differences. Short term rearing in captivity did not have a pronounced effect on the diversity of mammalian viruses of wild cynomolgus macaques. This study is the first report of the fecal virome of cynomolgus macaques, non-human primates frequently used in biomedical research and vaccination studies.

Metagenomics was used to identify viral sequences in the plasma and CSF (cerobrospinal fluid) of 13 horses with unexplained neurological signs and in the plasma and respiratory swabs of 14 horses with unexplained respiratory signs. Equine hepacivirus and two copiparvoviruses (horse parvovirus-CSF and a novel parvovirus) were detected in plasma from neurological cases. Plasma from horses with respiratory signs contained the same two copiparvoviruses plus equine pegivirus D and respiratory swabs contained equine herpes virus 2 and 5. Based on genetic distances the novel copiparvovirus qualified as a member of a new parvovirus species we named Eqcopivirus. These samples plus another 41 plasma samples from healthy horses were tested by real-time PCRs for multiple equine parvoviruses and hepacivirus. Over half the samples tested were positive for one to three viruses with eqcopivirus DNA detected in 20.5%, equine hepacivirus RNA and equine parvovirus-H DNA in 16% each, and horse parvovirus-CSF DNA in 12% of horses. Comparing viral prevalence in plasma none of the now three genetically characterized equine parvoviruses (all in the copiparvovirus genus) was significantly associated with neurological and respiratory signs in this limited sampling.

A viral metagenomic analysis of feces from an unexplained outbreak of feline diarrhea revealed the presence of Lyon-IARC polyomavirus (LIPyV) DNA. LIPyV, whose genome was originally sequenced from swabs of human skin, was fecally shed by three out of five diarrheic cats.

The influence of living in small remote villages on the diversity of viruses in the nasal mucosa was investigated in three Colombian villages with very different levels of geographic isolation. Metagenomic analysis was used to characterize viral nucleic acids in nasal swabs from 63 apparently healthy young children. Sequences from human virus members of the families Anelloviridae, Papillomaviridae, Picornaviridae, Herpesviridae, Polyomaviridae, Adenoviridae, and Paramyxoviridae were detected in decreasing proportions of children. The number of papillomavirus infections detected was greater among Hispanic children most exposed to outside contacts, while anellovirus infections were more common in the isolated indigenous villages. The diversity of the other human viruses detected did not differ among the villages. Closely related variants of rhinovirus A or B were identified in 2 to 4 children from each village, reflecting ongoing transmission clusters. Genomes of viruses not currently known to infect humans, including members of the families Parvoviridae, Partitiviridae, Dicistroviridae, and Iflaviridae and circular Rep-encoding single-stranded DNA (CRESS-DNA) virus, were also detected in nasal swabs, possibly reflecting environmental contamination from insect, fungal, or unknown sources. Despite the high levels of geographic and cultural isolation, the overall diversity of human viruses in the nasal passages of children was not reduced in highly isolated indigenous villages, indicating ongoing exposure to globally circulating viruses. IMPORTANCE Extreme geographic and cultural isolation can still be found in some indigenous South American villages. Such isolation may be expected to limit the introduction of otherwise common and widely distributed viruses. Very small population sizes may also result in rapid local viral extinction due to a lack of seronegative subjects to maintain transmission chains for rapidly cleared viruses. We compared the viruses in the nasal passages of young children in three villages with increasing levels of geographic isolation. We found that isolation did not reduce the overall diversity of viral infections. Multiple infections with nearly identical rhinoviruses could be detected within each village, likely reflecting recent viral introductions and transmission clusters among epidemiologically linked members of these very small communities. We conclude that, despite their geographic isolation, remote indigenous villages show evidence of ongoing exposure to globally circulating viruses.

Zebrafish have been extensively used as a model system for research in vertebrate development and pathogen-host interactions. We describe the complete genome of a novel picornavirus identified during a viral metagenomics analysis of zebrafish gut tissue. The closest relatives of this virus showed identity of20% in their P1 capsids and36% in their RdRp qualifying zebrafish picornavirus-1 (ZfPV-1) as member of a novel genus with a proposed name of

Feces from dogs in an unexplained outbreak of diarrhea were analyzed by viral metagenomics revealing the genome of a novel parvovirus. The parvovirus was named cachavirus and was classified within the proposed Chapparvovirus genus. Using PCR, cachavirus DNA was detected in two of nine tested dogs from that outbreak. In order to begin to elucidate the clinical impact of this virus, 2,053 canine fecal samples were screened using real-time PCR. Stool samples from 203 healthy dogs were positive for cachavirus DNA at a rate of 1.47%, while 802 diarrhea samples collected in 2017 and 964 samples collected in 2018 were positive at rates of 4.0% and 4.66% frequencies, respectively (healthy versus 2017-2018 combined diarrhea p-value of 0.05). None of 83 bloody diarrhea samples tested positive. Viral loads were generally low with average real-time PCR Ct values of 36 in all three positive groups. The species tropism and pathogenicity of cachavirus, the first chapparvovirus reported in feces of a placental carnivore, remains to be fully determined.

Tick-borne encephalitis virus (TBEV) and West Nile virus (WNV) are mainly transmitted by arthropod vectors to vertebrate hosts including humans, resulting in fever and neurological signs. The aim of this study was to investigate the presence of antibodies to TBEV and WNV, and TBEV-RNA and WNV-RNA in Turkish children with fever and/or arthritis. For this purpose, 110 sera and buffy-coat samples were collected; sera were analyzed by indirect enzyme-linked immunosorbent assay for the presence of IgM and IgG antibodies to TBEV and WNV, and buffy-coat-derived white blood cells were analyzed by quantitative real-time RT-PCR for TBEV-RNA and WNV-RNA. IgM antibodies to TBEV were detected in five children between the ages of 3 and 7 years; no IgG antibodies to TBEV were detected. IgG antibodies to WNV were detected in two children and IgM antibodies to WNV were detected in six children, between the ages of 3 and 7 years. One of the children had IgM antibodies to WNV and to TBEV. Children who had antibodies to TBEV and WNV had fever and/or arthritis but no obvious neurological signs. Molecular diagnostic approaches revealed that neither TBEV-RNA nor WNV-RNA was present in any of the buffy-coat samples, not even in children with IgM-specific antibodies. Our serological results indicate that children in Turkey are exposed to TBEV and WNV.

The avian coronavirus-infectious bronchitis virus (AvCoV-IBV) is recognized as an important avian pathogen, and new viral variants are a continuous threat to the poultry industry worldwide. Sensitive diagnostics and efficacious vaccines are necessary to combat IBV infections in chickens. The aim of this study was to produce recombinant N protein of IBV in the baculovirus system to use in ELISA diagnostic tests in order to enable the assessment of the sero-prevalence and risk of IBV infections in chickens in Turkey. For this, the gene encoding the N protein of the Beaudette strain of IBV was expressed using a recombinant baculovirus expression system. The recombinant N protein was purified using Ni-NTA affinity chromatography. An estimated 50-kDa recombinant protein corresponding to the expected molecular weight of IBV N including the 6xHis tag was detected using an anti-His monoclonal antibody. Specific immunoreactivity of the recombinant protein was confirmed by Western blot using antiserum obtained from vaccinated and naturally infected chicken from Turkey as well as using a monoclonal antibody raised against the N protein of the IBV Massachusetts strain. The results obtained with the in-house ELISA had high agreement with a commercial ELISA. Immunoreactivity analysis using antisera in Western blotting and the in-house ELISA suggests that the recombinant IBV N protein could be broadly cross-reactive with antisera produced against different IBV strains. We conclude that the recombinant baculovirus expressed IBV N protein could serve as a useful diagnostic antigen for detection of IBV infections in chickens by ELISA. TUBITAK: Scientific and Technological Research Council of TurkeyTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [113O411]; Developing Scholars Program (DSP), Kansas State University, Office of Undergraduate Research and Creative Inquiry; DHS Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD) This study was funded by TUBITAK: Scientific and Technological Research Council of Turkey (Project No: 113O411), Developing Scholars Program (DSP), Kansas State University, Office of Undergraduate Research and Creative Inquiry; DHS Center of Excellence for Emerging and Zoonotic Animal Diseases (CEEZAD).

Introduction: The aim of the study was to investigate the presence of human papillomaviruses (HPV), mouse mammary tumour virus (MMTV), Epstein-Barr virus (EBV), and human polyomavirus BK in canine mammary tumours (CMTs) and to correlate the results of histopathological classification with the results of virological examination. Material and Methods: Eighty CMTs and ten normal canine mammary gland samples were evaluated using histopathological methods and TaqMan real-time PCR analysis. Results: The results indicated that all mammary tumours and normal mammary tissue samples were negative for HPV16 and other HPV, EBV, human polyomavirus, and human mammary tumour virus strains. Conclusion: Further studies should be performed to investigate the existence of other strains of HPV, EBV, and human polyomavirus in CMTs.

Using viral metagenomics, we characterized the mammalian virome of nasal swabs from 57 dogs with unexplained signs of respiratory infection showing mostly negative results using the IDEXX Canine Respiratory Disease RealPCR™ Panel. We identified canine parainfluenza virus 5, canine respiratory coronavirus, carnivore bocaparvovirus 3, canine circovirus and canine papillomavirus 9. Novel canine taupapillomaviruses (CPV21-23) were also identified in 3 dogs and their complete genome sequenced showing L1 nucleotide identity ranging from 68.4 to 70.3% to their closest taupapillomavirus relative. Taupapillomavirus were the only mammalian viral nucleic acids detected in two affected dogs, while a third dog was coinfected with low levels of canine parainfluenza 5. A role for these taupapillomavirues in canine respiratory disease remains to be determined. Electronic supplementary material The online version of this article (10.1007/s11262-019-01634-6) contains supplementary material, which is available to authorized users.