Institute of Experimental and Clinical Veterinary Medicine

Kharkiv, Ukraine

Institute of Experimental and Clinical Veterinary Medicine

Kharkiv, Ukraine
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Dimitrov K.M.,U.S. Department of Agriculture | Sharma P.,U.S. Department of Agriculture | Volkening J.D.,BASE2BIO | Goraichuk I.V.,U.S. Department of Agriculture | And 8 more authors.
Virology Journal | Year: 2017

Background: Next-generation sequencing (NGS) allows ultra-deep sequencing of nucleic acids. The use of sequence-independent amplification of viral nucleic acids without utilization of target-specific primers provides advantages over traditional sequencing methods and allows detection of unsuspected variants and co-infecting agents. However, NGS is not widely used for small RNA viruses because of incorrectly perceived cost estimates and inefficient utilization of freely available bioinformatics tools. Methods: In this study, we have utilized NGS-based random sequencing of total RNA combined with barcode multiplexing of libraries to quickly, effectively and simultaneously characterize the genomic sequences of multiple avian paramyxoviruses. Thirty libraries were prepared from diagnostic samples amplified in allantoic fluids and their total RNAs were sequenced in a single flow cell on an Illumina MiSeq instrument. After digital normalization, data were assembled using the MIRA assembler within a customized workflow on the Galaxy platform. Results: Twenty-eight avian paramyxovirus 1 (APMV-1), one APMV-13, four avian influenza and two infectious bronchitis virus complete or nearly complete genome sequences were obtained from the single run. The 29 avian paramyxovirus genomes displayed 99.6% mean coverage based on bases with Phred quality scores of 30 or more. The lower and upper quartiles of sample median depth per position for those 29 samples were 2984 and 6894, respectively, indicating coverage across samples sufficient for deep variant analysis. Sample processing and library preparation took approximately 25-30 h, the sequencing run took 39 h, and processing through the Galaxy workflow took approximately 2-3 h. The cost of all steps, excluding labor, was estimated to be 106 USD per sample. Conclusions: This work describes an efficient multiplexing NGS approach, a detailed analysis workflow, and customized tools for the characterization of the genomes of RNA viruses. The combination of multiplexing NGS technology with the Galaxy workflow platform resulted in a fast, user-friendly, and cost-efficient protocol for the simultaneous characterization of multiple full-length viral genomes. Twenty-nine full-length or near-full-length APMV genomes with a high median depth were successfully sequenced out of 30 samples. The applied de novo assembly approach also allowed identification of mixed viral populations in some of the samples. © 2017 The Author(s).


Dimitrov K.M.,U.S. Department of Agriculture | Dimitrov K.M.,National Diagnostic and Research Veterinary Medicine Institute | Bolotin V.,Institute of Experimental and Clinical Veterinary Medicine | Muzyka D.,Institute of Experimental and Clinical Veterinary Medicine | And 9 more authors.
Archives of Virology | Year: 2016

Here, we report the circulation of highly related virulent Newcastle disease viruses (NDV) in Bulgaria and Ukraine from 2002 until 2013. All of these NDV isolates have the same virulence-associated cleavage site (“113RQKR↓F117”), and selected ones have intracerebral pathogenicity index values ranging from 1.61 to 1.96. These isolates are most closely related to viruses circulating in Eastern Europe, followed by viruses isolated in Asia during the same period of time. Interestingly, the majority of the viruses were isolated from backyard poultry, suggesting the possibility of a “domestic” or “urban” cycle of maintenance. The molecular characterization of the nucleotide sequence of the complete fusion protein gene of the studied viruses suggests continued circulation of virulent NDV of sub-genotype VIId in Eastern Europe, with occasional introductions from Asia. Furthermore, the high level of genetic similarity among those isolates suggests that the NDV isolates of sub-genotype VIId from Bulgaria and Ukraine may have been part of a broader epizootic process in Eastern Europe rather than separate introductions from Asia or Africa. The continuous monitoring of backyard poultry flocks for the presence of circulating virulent NDV strains will allow early identification of Newcastle disease outbreaks. © 2016 Springer-Verlag Wien (Outside the USA)


PubMed | Institute of Experimental and Clinical Veterinary Medicine, National Diagnostic and Research Veterinary Medicine Institute and U.S. Department of Agriculture
Type: Journal Article | Journal: Archives of virology | Year: 2016

Here, we report the circulation of highly related virulent Newcastle disease viruses (NDV) in Bulgaria and Ukraine from 2002 until 2013. All of these NDV isolates have the same virulence-associated cleavage site (


Ayala A.J.,University of Georgia | Ayala A.J.,Exotic and Emerging Avian Viral Diseases Research Unit | Dimitrov K.M.,Exotic and Emerging Avian Viral Diseases Research Unit | Dimitrov K.M.,National Diagnostic Research Veterinary Medical Institute | And 17 more authors.
PLoS ONE | Year: 2016

Our study demonstrates the repeated isolation of vaccine-derived Newcastle disease viruses from different species of wild birds across four continents from 1997 through 2014. The data indicate that at least 17 species from ten avian orders occupying different habitats excrete vaccine-derived Newcastle disease viruses. The most frequently reported isolates were detected among individuals in the order Columbiformes (n = 23), followed in frequency by the order Anseriformes (n = 13). Samples were isolated from both free-ranging (n = 47) and wild birds kept in captivity (n = 7). The number of recovered vaccine-derived viruses corresponded with the most widely utilized vaccines, La Sota (n = 28) and Hitchner B1 (n = 19). Other detected vaccine-derived viruses resembled the PHY-LMV2 and V4 vaccines, with five and two cases, respectively. These results and the ubiquitous and synanthropic nature of wild pigeons highlight their potential role as indicator species for the presence of Newcastle disease virus of low virulence in the environment. The reverse spillover of live agents from domestic animals to wildlife as a result of the expansion of livestock industries employing massive amounts of live virus vaccines represent an underappreciated and poorly studied effect of human activity on wildlife.


Muzyka D.,Institute of Experimental and Clinical Veterinary Medicine | Pantin-Jackwood M.,U.S. Department of Agriculture | Spackman E.,U.S. Department of Agriculture | Stegniy B.,Institute of Experimental and Clinical Veterinary Medicine | And 2 more authors.
Avian Diseases | Year: 2012

The Azov and Black Sea basins are part of the transcontinental wild bird migration routes from Northern Asia and Europe to the Mediterranean, Africa, and Southwest Asia. These regions constitute an area of transit, stops during migration, and nesting for many different bird species. From September 2010 to September 2011, a wild bird surveillance study was conducted in these regions to identify avian influenza viruses. Biological samples consisting of cloacal and tracheal swabs and fecal samples were collected from wild birds of different ecological groups, including waterfowl and sea-and land-based birds, in places of mass bird accumulations in Sivash Bay and the Utlyuksky and Molochniy estuaries. The sampling covered the following wild bird biological cycles: autumn migration, wintering, spring migration, nesting, and postnesting seasons. A total of 3634 samples were collected from 66 different species of birds. During the autumn migration, 19 hemagglutinating viruses were isolated, 14 of which were identified as low pathogenicity avian influenza (LPAI) virus subtypes H1N?, H3N8, H5N2, H7N?, H8N4, H10N7, and H11N8. From the wintering samples, 45 hemagglutinating viruses were isolated, 36 of which were identified as LPAI virus subtypes H1N1, H1N? H1N2, H4N?, H6N1, H7N3, H7N6, H7N7, H8N2, H9N2, H10N7, H10N4, H11N2, H12N2, and H15N7. Only three viruses were isolated during the spring migration, nesting, and postnesting seasons (serotypes H6, H13, and H16). The HA and NA genes were sequenced from the isolated H5 and N1 viruses, and the phylogenetic analysis revealed possible ecological connections between the Azov and Black Sea regions and Europe. The LPAI viruses were isolated mostly from mallard ducks, but also from shellducks, shovelers, teals, and white-fronted geese. The rest of the 14 hemagglutinating viruses isolated were identified as different serotypes of avian paramyxoviruses (APMV-1, APMV-4, APMV-6, and APMV-7). This information furthers our understanding of the ecology of avian influenza viruses in wild bird species.


PubMed | Friedrich Loeffler Institute, Institute of Experimental and Clinical Veterinary Medicine and U.S. Department of Agriculture
Type: Journal Article | Journal: Archives of virology | Year: 2016

Since the first detection of H15 avian influenza viruses(AIVs) in Australia in 1979, only seven H15 strains have been reported. A new H15 AIV was detected in Ukraine in 2010, carrying the unique HA-NA subtype combination H15N7. This virus replicated efficiently in chicken eggs, and antisera against it reacted strongly with the homologous antigen, but with lower titers when using the reference Australian antigen. The amino acid motifs of the HA cleavage site and receptor-binding site were different from those in the Australian viruses. The new virus, together with an H15 virus from Siberia from 2008, constitutes a new clade of H15 AIV isolates.


Muzyka D.,Institute of Experimental and Clinical Veterinary Medicine | Pantin-Jackwood M.,U.S. Department of Agriculture | Starick E.,Friedrich Loeffler Institute | Fereidouni S.,Friedrich Loeffler Institute | Fereidouni S.,University of Veterinary Medicine Vienna
Archives of Virology | Year: 2016

Since the first detection of H15 avian influenza viruses (AIVs) in Australia in 1979, only seven H15 strains have been reported. A new H15 AIV was detected in Ukraine in 2010, carrying the unique HA-NA subtype combination H15N7. This virus replicated efficiently in chicken eggs, and antisera against it reacted strongly with the homologous antigen, but with lower titers when using the reference Australian antigen. The amino acid motifs of the HA cleavage site and receptor-binding site were different from those in the Australian viruses. The new virus, together with an H15 virus from Siberia from 2008, constitutes a new clade of H15 AIV isolates. © 2015, Springer-Verlag Wien.


Brusentsov I.I.,RAS Institute of Cytology and Genetics | Katokhin A.V.,RAS Institute of Cytology and Genetics | Brusentsova I.V.,RAS Institute of Cytology and Genetics | Shekhovtsov S.V.,RAS Institute of Cytology and Genetics | And 9 more authors.
PLoS ONE | Year: 2013

Opisthorchis felineus or Siberian liver fluke is a trematode parasite (Opisthorchiidae) that infects the hepato-biliary system of humans and other mammals. Despite its public health significance, this wide-spread Eurasian species is one of the most poorly studied human liver flukes and nothing is known about its population genetic structure and demographic history. In this paper, we attempt to fill this gap for the first time and to explore the genetic diversity in O. felineus populations from Eastern Europe (Ukraine, European part of Russia), Northern Asia (Siberia) and Central Asia (Northern Kazakhstan). Analysis of marker DNA fragments from O. felineus mitochondrial cytochrome c oxidase subunit 1 and 3 (cox1, cox3) and nuclear rDNA internal transcribed spacer 1 ( ITS1) sequences revealed that genetic diversity is very low across the large geographic range of this species. Microevolutionary processes in populations of trematodes may well be influenced by their peculiar biology. Nevertheless, we suggest that lack of population genetics structure observed in O. felineus can be primarily explained by the Pleistocene glacial events and subsequent sudden population growth from a very limited group of founders. Rapid range expansion of O. felineus through Asian and European territories after severe bottleneck points to a high dispersal potential of this trematode species. © 2013 Brusentsov et al.


Limanskaya O.Y.,Ukrainian Academy of Sciences | Limanskaya O.Y.,Institute of Experimental and Clinical Veterinary Medicine | Fesenko T.N.,Ukrainian Academy of Sciences | Pokrovskiy V.A.,Ukrainian Academy of Sciences | And 4 more authors.
Biomeditsinskaya Khimiya | Year: 2012

Point mutations associated with isoniazid resistance in Mycobacterium tuberculosis (MTB) have been analyzed in codon 315 of the katG gene by conventional polymerase chain reaction (PCR) using primers containing locked nucleic acid (LNA) modified nucleotides. Purity and structure of primers containing 5 LNA monomers of 17 nucleotides in length were characterized by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) and a 17-mcr duplex formed by two complementary oligonucleotides was characterized by the method of thermal denaturation. The duplex containing five LNA monomers per each strand was characterized by a higher melting temperature than it was expected using extrapolation of theoretical calculation for nucleotide modification of one strand of the duplex. Detection of any of six possible mutations in katG codon 315 (i.e. discrimination between sensitive and resistant MTB) requires just one PCR employing a set of two primers with one LNA-modified primer; this is an important advantage of oligonucleotides containing LNA over unmodified nucleotides: employment of multiplex PCR would require up to 12 primers. Problems of control of oligonucleotide modification by LNA monomers are discussed.


Limanskaya O.Yu.,Ukrainian Academy of Sciences | Limanskaya O.Yu.,Institute of Experimental and Clinical Veterinary Medicine
Cytology and Genetics | Year: 2010

The perfect interstrand triplexes that could potentially arise in the proviral DNA of two widespread cattle retroviruses such as bovine leukemia virus (BLV) and bovine immunodeficiency virus (BIV) were determined. The fragments, which formed triplexes at acidic pH, were found in the genomes of both viruses; five fragments were found in BVL and 10 fragments in BIV. One of these fragments (it is localized in the BVL gag gene) might exist like a part of a cruciform structure. Existence of the triplexes was experimentally confirmed by visualization of supercoiled pGEMEX DNA with the use of atomic force microscopy; six fragments with mirror symmetry, which are necessary for formation of intramolecular triplexes, were found. Triplexes represent one of the elements of the signaling mechanisms of the genome function. Maps of triplex location in the cattle retroviral genome were built. © Allerton Press, Inc., 2010.

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