The use of microarrays for the identification of the origin of genes of avian influenza viruses in wild birds

Forty-two strains of avian influenza viruses were isolated from the wild waterfowls’ feces in the city of Moscow. These viruses, as well as reference strains and some experimental reassortants, were analyzed by microarrays. The microarrays contained 176 probes to the different segments of influenza virus genome. The microarray helps to determine 1) the hemagglutinin and neuraminidase proteins subtype; 2) the primary structure of the C-terminal sequence of the viral NS1 protein, which serves as a ligand for the PDZ domain; 3) the presence of stop codons in the reading frame of PB1-F2 as well as the N66S substitution in the PB1-F2 viral protein; 4) the presence of the polybasic site for hemagglutinin cleavage. The viruses of the H3N1, H3N6, H3N8, H4N6, H1N1, H5N3, and H11N9 subtypes were identified from the group of wild birds’ isolates. All isolates contained the ESEV sequence at the C-terminus of the NS1 protein and the full-length reading frame for the PB1-F2 protein. The replacement of N66S in PB1-F2 was found in six strains. However, the presence of the ESEV sequence (ligand of PDZ domain) in the NS1 virus protein and the N66S substitution in PB1-F2 did not lead to the pathogenicity of these viruses for mice. All isolates demonstrated high yield growth in chicken embryos and were infectious and immunogenic for mice, but did not induce any clinical symptoms.Forty-two strains of avian influenza viruses were isolated from the wild waterfowls’ feces in the city of Moscow. These viruses, as well as reference strains and some experimental reassortants, were analyzed by microarrays. The microarrays contained 176 probes to the different segments of influenza virus genome. The microarray helps to determine 1) the hemagglutinin and neuraminidase proteins subtype; 2) the primary structure of the C-terminal sequence of the viral NS1 protein, which serves as a ligand for the PDZ domain; 3) the presence of stop codons in the reading frame of PB1-F2 as well as the N66S substitution in the PB1-F2 viral protein; 4) the presence of the polybasic site for hemagglutinin cleavage. The viruses of the H3N1, H3N6, H3N8, H4N6, H1N1, H5N3, and H11N9 subtypes were identified from the group of wild birds’ isolates. All isolates contained the ESEV sequence at the C-terminus of the NS1 protein and the full-length reading frame for the PB1-F2 protein. The replacement of N66S in PB1-F2 was found in six strains. However, the presence of the ESEV sequence (ligand of PDZ domain) in the NS1 virus protein and the N66S substitution in PB1-F2 did not lead to the pathogenicity of these viruses for mice. All isolates demonstrated high yield growth in chicken embryos and were infectious and immunogenic for mice, but did not induce any clinical symptoms

FAS-dependent cell death in α-synuclein transgenic oligodendrocyte models of multiple system atrophy

Multiple system atrophy is a parkinsonian neurodegenerative disorder. It is cytopathologically characterized by accumulation of the protein p25α in cell bodies of oligodendrocytes followed by accumulation of aggregated α-synuclein in so-called glial cytoplasmic inclusions. p25α is a stimulator of α-synuclein aggregation, and coexpression of α-synuclein and p25α in the oligodendroglial OLN-t40-AS cell line causes α-synuclein aggregate-dependent toxicity. In this study, we investigated whether the FAS system is involved in α-synuclein aggregate dependent degeneration in oligodendrocytes and may play a role in multiple system atrophy. Using rat oligodendroglial OLN-t40-AS cells we demonstrate that the cytotoxicity caused by coexpressing α-synuclein and p25α relies on stimulation of the death domain receptor FAS and caspase-8 activation. Using primary oligodendrocytes derived from PLP-α-synuclein transgenic mice we demonstrate that they exist in a sensitized state expressing pro-apoptotic FAS receptor, which makes them sensitive to FAS ligand-mediated apoptosis. Immunoblot analysis shows an increase in FAS in brain extracts from multiple system atrophy cases. Immunohistochemical analysis demonstrated enhanced FAS expression in multiple system atrophy brains notably in oligodendrocytes harboring the earliest stages of glial cytoplasmic inclusion formation. Oligodendroglial FAS expression is an early hallmark of oligodendroglial pathology in multiple system atrophy that mechanistically may be coupled to α-synuclein dependent degeneration and thus represent a potential target for protective intervention

Применение микрочипов для идентификации происхождения генов вирусов гриппа диких птиц

Forty-two strains of avian influenza viruses were isolated from the wild waterfowl’s feces in the city of Moscow. These viruses as well as reference strains and some experimental reassortants were analyzed by microarrays. The used microarrays contained 176 probes to the different segments of influenza virus genome. The microarray allows to determine 1) the hemagglutinin and neuraminidase proteins subtype; 2) the primary structure of the C-terminal sequence of the viral NS1 protein, which serves as a ligand for the PDZ domain; 3) the presence of stop codons and substitution N66S in the reading frame of the viral protein PB1-F2; 4) the presence of the polybasic site for hemagglutinin cleavage. The viruses of H3N1, H3N6, H3N8, H4N6, H1N1, H5N3 and H11N9 subtypes were identified from the group of wild bird’s isolates. All isolates contained the ESEV sequence at the C-terminus of the NS1 protein and the full-length reading frame for the PB1-F2 protein. The replacement of N66S in PB1-F2 was found in six strains. However, the presence of ESEV sequence (ligand of PDZ domain) in the NS1 virus protein and the N66S substitution in PB1-F2 did not lead to the pathogenicity of these viruses for mice. All isolates demonstrated high yield growth in chicken embryos, were infectious and immunogenic for mice, but did not induce any clinical symptoms.В черте города Москвы из фекалий диких водоплавающих птиц изолировали 42 штамма вируса гриппа птиц и проанализировали их на микрочипах «Биогрипп», которые содержат 176 зондов к различным участкам генома вирусов гриппа. Микрочип позволяет определять: 1) субтип поверхностных белков гемагглютинина и нейраминидазы; 2) структуру С-концевой последовательности вирусного белка NS1, влияющую на степень ингибирования транскрипции клеточных хозяйских генов, в том числе ответственных за синтез интерферона; 3) наличие стоп-кодонов и мутацию N66S в рамке считывания вирусного белка PB1-F2, 4) наличие полиосновного сайта протеолитического расщепления гемагглютинина. Среди изолятов от диких птиц идентифицированы вирусы гриппа субтипов H3N1, H3N6, H3N8, H4N6, H1N1, H5N3 и H11N9. Все они содержали последовательность ESEV на С-конце белка NS1, полноразмерную рамку считывания для белка PB1-F2. Замена N66S в PB1-F2 обнаружена у шести штаммов. Однако такие маркеры патогенности, как последовательность ESEV (лиганд PDZ-домена) в вирусном белке NS1 и замена N66S PB1-F2 в контексте генома вирусов гриппа диких уток, не делали вирус патогенным для мышей. Все изоляты были высокоурожайны в куриных эмбрионах, инфекционны и иммуногенны для мышей, но не вызывали у этих животных клинических симптомов заболевания