Mikhayil Hakhverdyan

Molecular Epidemiology of Bovine Coronavirus on the Basis of Comparative Analyses of the S Gene

ABSTRACT Bovine coronavirus (BCoV), a group 2 member of the genus Coronavirus in the family Coronaviridae, is an important pathogen in cattle worldwide. It causes diarrhea in adult animals (winter dysentery), as well as enteric and respiratory diseases in calves. The annual occurrence of BCoV epidemics in Sweden and Denmark led to this investigation, with the aim to deepen the knowledge of BCoV epidemiology at the molecular level. A total of 43 samples from outbreaks in both countries were used for PCR amplification and direct sequencing of a 624-nucleotide fragment of the BCoV S gene. Sequence comparison and phylogenetic studies were performed. The results showed (i) identical sequences from different animals in the same herds and from paired nasal and fecal samples, suggesting a dominant virus circulating in each herd at a given time; (ii) sequence differences among four outbreaks in different years in the same herd, indicating new introduction of virus; (iii) identical sequences in four different Danish herds in samples obtained within 2 months, implying virus transmission between herds; and (iv) that at least two different virus strains were involved in the outbreaks of BCoV in Denmark during the spring of 2003. This study presents molecular data of BCoV infections that will contribute to an increased understanding of BCoV epidemiology in cattle populations.

Evaluation of a single-tube fluorogenic RT-PCR assay for detection of bovine respiratory syncytial virus in clinical samples

n Abstractn n Bovine respiratory syncytial virus (BRSV) causes severe disease in naïve cattle of all ages and is a common pathogen in the respiratory disease complex of calves. Simplified methods for rapid BRSV diagnosis would encourage sampling during outbreaks and would consequently lead to an extended understanding of the virus. In this study, a BRSV fluorogenic reverse transcription PCR (fRT-PCR) assay, based on TaqMan principle, was developed and evaluated on a large number of clinical samples, representing various cases of natural and experimental BRSV infections. By using a single-step closed-tube format, the turn-around time was shortened drastically and results were obtained with minimal risk for cross-contamination. According to comparative analyses, the detection limit of the fRT-PCR was on the same level as that of a nested PCR and the sensitivity relatively higher than that of a conventional PCR, antigen ELISA (Ag-ELISA) and virus isolation (VI). Interspersed negative control samples, samples from healthy animals and eight symptomatically or genetically related viruses were all negative, confirming a high specificity of the assay. Taken together, the data indicated that the fRT-PCR assay can be applied to routine virus detection in clinical specimens and provides a rapid and valuable tool in BRSV research.n n

Characterization of Pisrt1/Foxl2 in Ellobius lutescens and exclusion as sex-determining genes.

The rodent Ellobius lutescens is an exceptional mammal which determines male sex constitutively without the SRY gene and, therefore, may serve as an animal model for human 46,XX female-to-male sex reversal. It was suggested that other factors of the network of sex-determining genes determine maleness in these animals. However, some sex-determining genes like SOX9 and SF1 have already been excluded by segregation analysis as primary sex-determining factors in E. lutescens. In this work, we have cloned and characterized two genes of the PIS (polled intersex syndrome) gene interval, which were reported as candidates in female-to-male sex reversal in hornless goats recently. The genes Foxl2 and Pisrt1 from that interval were identified in E. lutescens DNA and mapped to Chromosome 8. We have excluded linkage of Foxl2 and Pisrt1 loci with the sex of the animals. Hence, the involvement of this gene region in sex determination may be specific for goats and is not a general mechanism of XX sex reversal or XX male sex determination.

Improved Diagnosis for Nine Viral Diseases Considered as Notifiable By the World Organization for Animal Health

Nine viral diseases included in the World Organization for Animal Health list of notifiable diseases (former list A) were chosen for their contagiousness and high capacity of spreading to improve their diagnosis using new and emerging technologies. All the selected diseases--foot-and-mouth disease, swine vesicular disease, vesicular stomatitis, classical swine fever, African swine fever, bluetongue, African horse sickness, Newcastle disease and highly pathogenic avian influenza--are considered as transboundary diseases, which detection causes the prohibition of livestock exportation, and, thus, it leads to high economical losses. The applied diagnostic techniques can fall into two categories: (i) nucleic-acid detection, including padlock probes, real-time PCR with TaqMan, minor groove binding probes and fluorescence energy transfer reaction probes, isothermal amplification like the Cleavase/Invader assay or the loop-mediated amplification technology and the development of rapid kits for mobile PCR and (ii) antigen-antibody detection systems like simplified and more sensitive ELISA tests. Besides, internal controls have been improved for nucleic acid-detecting methods by using an RNA plant virus--Cowpea Mosaic Virus--to ensure the stability of the RNA used as a positive control in diagnostic real-time RT-PCR assays. The development of these diagnosis techniques has required the joint efforts of a European consortium in which nine diagnostic laboratories and an SME who have collaborated since 2004 within the European Union-funded Lab-on-site project. The results obtained are shown in this paper.

Equine arteritis virus induced cell death is associated with activation of the intrinsic apoptotic signalling pathway.

Equine arteritis virus (EAV) causes a respiratory and reproductive disease in horses, equine viral arteritis. Though cell death in infection with EAV is considered to occur by apoptosis, the underlying molecular mechanism has not been extensively elucidated. We investigated the expression of mRNA of pro-apoptotic and caspase genes during EAV infection in BHK21 cells, a well-established cell type for EAV replication. Using a SYBR Green real-time PCR, mRNA of p53, Bax, caspase 3 and caspase 9 were found up-regulated in a time dependent manner in EAV infected cells. Western blot analysis for caspase 3 and caspase 9 showed expression of cleaved forms of these proteins during EAV infection. In addition, a luminescence-based cell assay for caspase 3/7 activation as a hallmark in apoptosis confirmed apoptotic cell death. The findings demonstrate that cell death in EAV infected BHK21 cells results from apoptosis mediated through the intrinsic signalling pathway.

Emergence of a new rhabdovirus associated with mass mortalities in eelpout (Zoarces viviparous) in the Baltic Sea.

We report the first description of a new Rhabdoviridae tentatively named eelpout rhabdovirus (EpRV genus Perhabdovirus). This virus was associated with mass mortalities in eelpout (Zoarces viviparous, Linnaeus) along the Swedish Baltic Sea coast line in 2014. Diseased fish showed signs of central nervous system infection, and brain lesions were confirmed by histology. A cytopathogenic effect was observed in cell culture, but ELISAs for the epizootic piscine viral haemorrhagic septicaemia virus (VHSV), infectious pancreas necrosis virus (IPNV), infectious haematopoietic necrosis virus (IHNV) and spring viraemia of carp virus (SVCV) were negative. Further investigations by chloroform inactivation, indirect fluorescence antibody test and electron microscopy indicated the presence of a rhabdovirus. By deep sequencing of original tissue suspension and infected cell culture supernatant, the full viral genome was assembled and we confirmed the presence of a rhabdovirus with 59.5% nucleotide similarity to the closest relative Siniperca chuatsi rhabdovirus. The full-genome sequence of this new virus, eelpout rhabdovirus (EpRV), has been deposited in GenBank under accession number KR612230. An RT-PCR based on the L-gene sequence confirmed the presence of EpRV in sick/dead eelpout, but the virus was not found in control fish. Additional investigations to characterize the pathogenicity of EpRV are planned.

Chromosomal Evolution in Mole Voles Ellobius (Cricetidae, Rodentia): Bizarre Sex Chromosomes, Variable Autosomes and Meiosis

This study reports on extensive experimental material covering more than 30 years of studying the genetics of mole voles. Sex chromosomes of Ellobius demonstrate an extraordinary case of mammalian sex chromosomes evolution. Five species of mole voles own three types of sex chromosomes; typical for placentals: XY♂/XX♀; and atypical X0♂/X0♀; or XX♂/XX♀. Mechanisms of sex determination in all Ellobius species remain enigmatic. It was supposed that the Y chromosome was lost twice and independently in subgenera Bramus and Ellobius. Previous to the Y being lost, the X chromosome in distinct species obtained some parts of the Y chromosome, with or without Sry, and accumulated one or several copies of the Eif2s3y gene. Along with enormous variations of sex chromosomes, genes of sex determination pathway and autosomes, and five mole vole species demonstrate ability to establish different meiotic mechanisms, which stabilize their genetic systems and make it possible to overcome the evolutionary deadlocks.